Modulation of p-glycoprotein function by caveolin-1 phosphorylation

p-glycoprotein (p-gp) is an ATP-binding cassette transporter and its overexpression is responsible for the acquisition of the multidrug resistance phenotype in human tumors. p-gp is localized at the blood-brain barrier and is involved in brain cytoprotection. Our previous work used immunoprecipitation to show that caveolin-1 can interact with p-gp. In this study, we provide evidence that caveolin-1 regulates p-gp transport activity in a rat brain endothelial cell line (RBE4). Down-regulation of caveolin-1 by siRNA reduced the interaction between p-gp and caveolin-1, followed by a decrease in [3H]-Taxol and [3H]-Vinblastine accumulation in RBE4 cells. The latter result showed that down-regulation of caveolin-1 enhanced p-gp transport activity. RBE4 cells were also transfected with Sarcoma in order to modulate caveolin-1 phosphorylation. Overexpression of Sarcoma, a protein tyrosine kinase, stimulated caveolin-1 phosphorylation and increased both [3H]-Taxol and [3H]-Vinblastine accumulation as well as Hoechst 33342 accumulation. Transfection of caveolin-1 inhibits p-gp transport activity. Conversely, transfection of the mutant cavY14F decreased the p-gp/caveolin-1 interaction and reduced accumulation of the two p-gp substrates. Thus, our data show that caveolin-1 regulates p-gp function through the phosphorylation state of caveolin-1 in endothelial cells from the blood-brain barrier.     

Experimental Colonization and Alteration of Orthopyroxene by the Pleomorphic Bacteria Ramlibacter tataouinensis

The colonization of orthopyroxene crystals by a pleomorphic bacterium and the mineralogical products resulting from a prolonged interaction have been studied. We used Ramlibacter tataouinensis (strain TTB310), which is an aerobic β-Proteobacterium moving over surfaces by gliding motility and whose life cycle includes rods and spherical cysts. Analysis of cultures grown on solid media with micrometer-sized pyroxene and quartz crystals scattered over the surface revealed a taxis of the bacteria toward the crystals. Given the mineralogical non-specificity of the interaction, a mechanism of elasticotaxis is inferred. After the rods had adhered to the pyroxene surface, they differentiated into cysts leading to the formation of microcolonies that were centered on a crystal grain. This suggests an original coupling between the life cycle of R. tataouinensis and the interaction with the crystals. The alteration of orthopyroxene was studied by high-resolution transmission electron microscopy at the interface between cysts and pyroxene crystals. The pyroxene surface showed an amorphous layer that was more developed than that of abiotic control samples processed under the same conditions. Moreover, chemical analyses showed that the dissolution of pyroxene was reduced in the presence of R. tataouinensis. The origin and the significance of the amorphous layers is discussed.     

Examining requirement for formation of functional Presenilin proteins and their processing events in vivo

Presenilin (Psn) is a multipass transmembrane protein that functions as the catalytic subunit of γ‐secretase for mediating intramembrane cleavage of type 1 transmembrane proteins. Normally active Psn is in the form of a heterodimer composed by its N‐terminal and C‐terminal fragments that are generated from a Presenilinase‐mediated endoproteolytic cleavage within its large cytosolic loop during assembly of the protease complex. Using the Psn forms that either bypass or disable Presenilinase‐mediated endoproteolysis, and a Psn form that has most of the large cytosolic loop deleted, we have established an in vivo system to enable investigations of Psn functional domains in Drosophila. We show that the Presenilinase‐mediated endoproteolytic event is not essential for producing Psn activity during animal development, and is regulated by integrity of the large cytosolic loop of Psn in Drosophila. The Psn transgenic flies described here could be applied to a broad range of studies on Psn functioning and its related γ‐secretase activity at any developmental stage. genesis 47:161–168, 2009. © 2009 Wiley‐Liss, Inc.     

Optimization of Drug Delivery by Drug-Eluting Stents

Drug-eluting stents (DES), which release anti-proliferative drugs into the arterial wall in a controlled manner, have drastically reduced the rate of in-stent restenosis and revolutionized the treatment of atherosclerosis. However, late stent thrombosis remains a safety concern in DES, mainly due to delayed healing of the endothelial wound inflicted during DES implantation. We present a framework to optimize DES design such that restenosis is inhibited without affecting the endothelial healing process. To this end, we have developed a computational model of fluid flow and drug transport in stented arteries and have used this model to establish a metric for quantifying DES performance. The model takes into account the multi-layered structure of the arterial wall and incorporates a reversible binding model to describe drug interaction with the cells of the arterial wall. The model is coupled to a novel optimization algorithm that allows identification of optimal DES designs. We show that optimizing the period of drug release from DES and the initial drug concentration within the coating has a drastic effect on DES performance. Paclitaxel-eluting stents perform optimally by releasing their drug either very rapidly (within a few hours) or very slowly (over periods of several months up to one year) at concentrations considerably lower than current DES. In contrast, sirolimus-eluting stents perform optimally only when drug release is slow. The results offer explanations for recent trends in the development of DES and demonstrate the potential for large improvements in DES design relative to the current state of commercial devices.     

Fatty acid oxidation by liver and muscle preparations of exhaustively exercised rats.

The influence of exhaustive exercise on the capacity of liver and muscle of rats to oxidize fatty acids was investigated in vitro. The rate of oxidation of fatty acids by liver preparations was significantly elevated as a result of exhaustion. Concurrently, the concentrations of beta-hydroxybutyrate were elevated in the plasma of the exhausted rats, suggesting that oxidation of fatty acids was also elevated in vivo. These findings are analogous to the findings of increased oxidation of fatty acids that results from training. In muscle, oxidation of palmitate, palmitoylcarnitine and beta-hydroxybutyrate by homogenates and isolated mitochondria was depressed with exercise. Despite the decrease in the oxidative capacity of the muscle preparations, the activities of several enzymes of beta-oxidation were either increased or unchanged as a result of exercise, suggesting that the depression in fatty acid oxidation may not be related to alterations in the process of beta-oxidation. Further studies showed that oxidation of [2-(14)C]pyruvate by muscle was depressed, whereas oxidation of [1-(14)C]pyruvate was not changed as a result of exercise. These results suggest that the decrease in fatty acid oxidation may be related to aberrations in the oxidation of acetyl-CoA. The changes in fatty acid oxidation that were observed, which are at variance with what is reported to occur with training, may have resulted from increased fragility of muscle mitochondria as a result of exercise. This increased fragility may render the mitochondria more susceptible to experimental manipulations in vitro and a subsequent loss of normal function.     

Fatty acid oxidation in African-American and Caucasian women during physical activity.

The goal of this study was to determine whether differences in physical activity-related fat oxidation exist between lean and obese African-American (LAA and OAA) and lean and obese Caucasian (LC and OC) premenopausal women. Lean AA (28.4 +/- 2.8 yr, n = 7), LC (24.7 +/- 1.8 yr, n = 9), OAA (30.9 +/- 2.2 yr, n = 11), and OC (34.1 +/- 2.5 yr, n = 9) women underwent preliminary assessment of peak aerobic capacity (VO2 peak). On a subsequent testing day, participants exercised after an 8-h fast on a cycle ergometer at 15 W (approximately 40% VO2 peak) for 10 min and then for 10 min at approximately 65% VO2 peak). Fatty acid oxidation was determined using the average respiratory exchange ratio and O2 consumption during minutes 5-9 of the exercise session. Percent body fat and fat-free mass were lower (P < 0.05) in LAA (25.8 +/- 2.8% and 48.3 kg) and LC (26.4 +/- 2.0% and 45.8 +/- 1.7 kg) than in OAA (41.2 +/- 1.3% and 58.8 +/- 3.3 kg) and OC (39.3 +/- 2.7% and 58.6 kg) women. Fat oxidation among the groups was analyzed statistically using analysis of covariance with fat-free mass and VO2 peak) as covariates. During exercise at 15 W, fat oxidation was as low in LAA (0.134 +/- 0.024 g/min) as in OAA (0.144 +/- 0.026 g/min) and OC (0.156 +/- 0.020 g/min) women: all these rates of fat oxidation were lower than in LC women (0.200 +/- 0.021 g/min, P < 0.05, LC vs. all other groups). Fatty acid oxidation during higher-intensity exercise (65% VO2 peak)) was higher in LC than in OC women but was not statistically different between African-American and Caucasian groups. Fatty acid oxidation was therefore lower during low-intensity physical activity in OAA, LAA, and OC than in LC women.     

Modulation of P-glycoprotein function by sphingosine kinase-1 in brain endothelial cells

P-glycoprotein (P-gp), an ABC-transporter highly expressed in brain capillaries, protects the brain by extruding xenobiotics. However, its overexpression has also been associated with the multidrug resistance phenotype in tumors. Here, we have investigated the regulation of P-gp transport activity by sphingosine kinase 1 (SphK-1) in brain endothelial cells. We first demonstrated that SphK-1 is overexpressed in endothelial cells (EC) isolated from rat brain tumors compared with EC from normal brain. We also provide evidence that the overexpression of SphK-1 in the cerebral EC line RBE4 leads to the up-regulation of P-gp, both at the gene and protein levels, and that this modulation depends on the catalytic activity of SphK-1. Moreover, we determined the effect of sphingosine-1-phosphate (S1P), the product of SphK-1, on P-gp function. S1P strongly stimulates P-gp transport activity, without modulating its expression. Finally, we found that the S1P-mediated stimulation of P-gp activity is mediated by S1P-1 and S1P-3 receptors at the RBE4 cell surface. Altogether, these results indicate that SphK-1 and its product S1P are involved in the control of P-gp activity in RBE4 cells. Since SphK-1 is overexpressed in EC from brain tumors, these data also suggest that this kinase and its product could contribute to the acquisition and the maintenance of the multidrug resistance phenotype in brain tumor-derived endothelial cells.     

Role of Organic Acids in Sunflower Tolerance to Heavy Metals

Exposure of Helianthus annuus L. seedlings to Al³⁺, Cd²⁺ or Zn²⁺ resulted in a marked decrease of fresh and dry masses of the shoots and the roots. The increase of Al³⁺, Cd²⁺ or Zn²⁺ uptake was accompanied by a significant decrease of nitrate, phosphorus and K⁺ uptake. There was a significant increase of malic and citric acid contents in the shoots and roots of heavy metal-treated seedlings whereas the change in fumaric acid was insignificant. Al³⁺ and Zn²⁺ alone stimulated excretion of malic and citric acids to the rhizosphere. Addition of high concentrations of malic or citric acid alleviate to some extent the inhibitory effect of Al³⁺ and Zn²⁺ on plant growth. This revised version was published online in July 2006 with corrections to the Cover Date.