Control of intramolecular interactions between the pleckstrin homology and Dbl homology domains of Vav and Sos1 regulates Rac binding

Vav and Sos1 are Dbl family guanine nucleotide exchange factors, which activate Rho family GTPases in response to phosphatidylinositol 3-kinase products. A pleckstrin homology domain adjacent to the catalytic Dbl homology domain via an unknown mechanism mediates the effects of phosphoinositides on guanine nucleotide exchange activity. Here we tested the possibility that phosphatidylinositol 3-kinase substrates and products control an interaction between the pleckstrin homology domain and the Dbl homology domain, thereby explaining the inhibitory effects of phosphatidylinositol 3-kinase substrates and stimulatory effects of the products. Binding studies using isolated fragments of Vav and Sos indicate phosphatidylinositol 3-kinase substrate promotes the binding of the pleckstrin homology domain to the Dbl homology domain and blocks Rac binding to the DH domain, whereas phosphatidylinositol 3-kinase products disrupt the Dbl homology/pleckstrin homology interactions and permit Rac binding. Additionally, Lck phosphorylation of Vav, a known activating event, reduces the affinities between the Vav Dbl homology and pleckstrin homology domains and permits Rac binding. We also show Vav activation in cells, as monitored by phosphorylation of Vav, Vav association with phosphatidylinositol 3,4,5-trisphosphate, and Vav guanine nucleotide exchange activity, is blocked by the phosphatidylinositol 3-kinase inhibitor wortmannin. These results suggest the molecular mechanisms for activation of Vav and Sos1 require disruption of inhibitory intramolecular interactions involving the pleckstrin homology and Dbl homology domains.     

Purification and characterisation of a beta-galactosidase from Aspergillus aculeatus with activity towards (modified) exopolysaccharides from Lactococcus lactis subsp. cremoris B39 and B891

Beta-galactosidase from Aspergillus aculeatus was purified from a commercial source for its hydrolytic activity towards (modified) exopolysaccharides (EPSs) produced by Lactococcus lactis subsp. cremoris B39 and B891. The enzyme had a molecular mass of approximately 120 kDa, a pI between 5.3 and 5.7 and was optimally active at pH 5.4 and 55-60 degrees C. Based on the N-terminal amino acid sequence, the enzyme probably belongs to family 35 of the glycosyl hydrolases. The catalytic mechanism was shown to be retaining and transglycosylation products were demonstrated using lactose as a substrate. The beta-galactosidase was also characterised using its activity towards two EPSs having lactosyl side chains attached to different backbone structures. The enzyme degraded O-deacetylated EPS B891 faster than EPS B39. Furthermore, the presence of acetyl groups in EPS B891 slowed down the hydrolysing rate, but the enzyme was still able to release all terminally linked galactose.     

CD8(+) T cells secreting type 2 lymphokines are defective in protection against viral infection

Effector T cells secreting type 1 and/or type 2 lymphokines (Tc1, Tc0, Tc2) were generated in vitro from CD8(+) T cells of mice with a transgenic TCR recognizing lymphocytic choriomeningitis virus (LCMV) glycoprotein to compare their effector function in vitro and in vivo. Tc1, Tc2, and Tc0 showed similar Fas- and perforin-mediated cytotoxicity in vitro. Upon adoptive transfer, Tc2 and Tc0 effectors were less efficient than Tc1 at controlling LCMV or recombinant vaccinia virus expressing the LCMV glycoprotein in vivo. Tc2 and Tc0 had decreased surface VLA-4 density and deficient activation-induced LFA-1/ICAM-1-dependent homotypic adhesion in vitro. Therefore, the reduced antiviral activity in vivo of Tc2 and Tc0 compared with Tc1 is not due to reduced cytotoxic activity or IFN-gamma secretion but may be explained by defective homing to the target organ due to decreased expression and/or lower activity of adhesion molecules.     

Identification of a second transforming function in bovine papillomavirus type 1 E6 and the role of E6 interactions with paxillin, E6BP, and E6AP

Papillomavirus E6 oncoproteins transform mammalian cells through interaction with cellular proteins. Bovine papillomavirus type 1 E6 (BE6) interacts with three previously described cellular targets: the E6AP E3 ubiquitin ligase, the calcium-binding protein E6BP (also known as ERC-55), and paxillin, which is a focal adhesion adapter protein. BE6 interacts strongly with each of these proteins in vitro, binding to similar peptide sequences found in E6AP, E6BP, and paxillin. To determine which BE6 interactions are necessary for transformation by BE6, we used a novel selection strategy for temperature-sensitive BE6 mutants in yeast that could discriminate in their interaction between E6AP, E6BP, and paxillin. All BE6 mutants that retained transforming ability retained association with paxillin, while some mutants that were transformation positive failed to interact with E6AP or E6BP. This study demonstrates that oncogene mutants that are temperature sensitive for transformation can be selected in yeast and that the induction of anchorage-independent cell proliferation by BE6 does not require strong association of BE6 with either E6AP or E6BP. Of particular interest is the identification of a BE6 mutant that interacts strongly with the acidic charged leucine motifs of E6AP, E6BP, and paxillin but is devoid of transformation activity, thereby genetically identifying a second essential transformation function in BE6 that is independent of interaction with acidic charged leucine motifs.     

Ticagrelor Does Not Inhibit Adenosine Transport at Relevant Concentrations: A Randomized Cross-Over Study in Healthy Subjects In Vivo

Background and Purpose

In patients with myocardial infarction, ticagrelor reduces cardiovascular and sepsis-related mortality, and can cause dyspnea. It is suggested that this is caused by adenosine receptor stimulation, because in preclinical studies, ticagrelor blocks the nucleoside transporter and increases cellular ATP release. We now investigated the effects of ticagrelor on the adenosine system in humans in vivo.

Experimental Approach

In a double-blinded, placebo-controlled cross-over trial in 14 healthy subjects, we have tested whether ticagrelor (180 mg) affects adenosine- and dipyridamole-induced forearm vasodilation, as surrogates of nucleoside uptake inhibition and adenosine formation, respectively. Also, ex vivo uptake of adenosine and uridine in isolated red blood cells was measured. Primary endpoint was adenosine-induced vasodilation.

Key Results

Ticagrelor did not affect adenosine- or dipyridamole-induced forearm vasodilation. Also, ex vivo uptake of adenosine and uridine in isolated red blood cells was not affected by ticagrelor. In vitro, ticagrelor dose-dependently inhibited nucleoside uptake, but only at supra-physiological concentrations.

Conclusion and Implications

In conclusion, at relevant plasma concentration, ticagrelor does not affect adenosine transport, nor adenosine formation in healthy subjects. Therefore, it is unlikely that this mechanism is a relevant pleiotropic effect of ticagrelor.

Trial Registration

ClinicalTrials.gov NCT01996735     

Porohyperelastic finite element modeling of abdominal aortic aneurysms

Abdominal aortic aneurysm (AAA) is the gradual weakening and dilation of the infrarenal aorta. This disease is progressive, asymptomatic, and can eventually lead to rupture--a catastrophic event leading to massive internal bleeding and possibly death. The mechanical environment present in AAA is currently thought to be important in disease initiation, progression, and diagnosis. In this study, we utilize porohyperelastic (PHE) finite element models (FEMs) to investigate how such modeling can be used to better understand the local biomechanical environment in AAA. A 3D hypothetical AAA was constructed with a preferential anterior bulge assuming both the intraluminal thrombus (ILT) and the AAA wall act as porous materials. A parametric study was performed to investigate how physiologically meaningful variations in AAA wall and ILT hydraulic permeabilities affect luminal interstitial fluid velocities and wall stresses within an AAA. A corresponding hyperelastic (HE) simulation was also run in order to be able to compare stress values between PHE and HE simulations. The effect of AAA size on local interstitial fluid velocity was also investigated by simulating maximum diameters (5.5 cm, 4.5 cm, and 3.5 cm) at the baseline values of ILT and AAA wall permeability. Finally, a cyclic PHE simulation was utilized to study the variation in local fluid velocities as a result of a physiologic pulsatile blood pressure. While the ILT hydraulic permeability was found to have minimal affect on interstitial velocities, our simulations demonstrated a 28% increase and a 20% decrease in luminal interstitial fluid velocity as a result of a 1 standard deviation increase and decrease in AAA wall hydraulic permeability, respectively. Peak interstitial velocities in all simulations occurred on the luminal surface adjacent to the region of maximum diameter. These values increased with increasing AAA size. PHE simulations resulted in 19.4%, 40.1%, and 81.0% increases in peak maximum principal wall stresses in comparison to HE simulations for maximum diameters of 35 mm, 45 mm, and 55 mm, respectively. The pulsatile AAA PHE FEM demonstrated a complex interstitial fluid velocity field the direction of which alternated in to and out of the luminal layer of the ILT. The biomechanical environment within both the aneurysmal wall and the ILT is involved in AAA pathogenesis and rupture. Assuming these tissues to be porohyperelastic materials may provide additional insight into the complex solid and fluid forces acting on the cells responsible for aneurysmal remodeling and weakening.     

Specific Investigation of Sample Handling Effects on Protease Activities and Absolute Serum Concentrations of Various Putative Peptidome Cancer Biomarkers

Introduction  

In the search for novel cancer biomarkers, various proteolytically derived peptides have been proposed to exhibit cancer or cancer-type specificity. As these peptides are presumably also generated after sample collection by tumor-specific proteases, extensive investigation of the involved proteolytic processes is crucial for further research.     

Serum Zinc Concentrations in Cystic Fibrosis Patients Aged Above 4 Years: A Cross-sectional Evaluation

Aim Assess the risk of zinc (Zn) deficiency in the older cystic fibrosis (CF) population. Method Cross-sectional investigation of all CF patients above the age of 4 followed at the Ghent University center between 2002 and 2003. Data on age, weight, height z-score, pancreatic and pulmonary functions, chronic Pseudomonas infection, and CF transmembrane conductance regulator (CFTR) mutations were collected. Serum Zn, vitamins (vit) A and E, retinol-binding protein (RBP), albumin, sedimentation rate, total IgG, and cholesterol were determined. Serum Zn was compared with a local healthy control group (Van Biervliet et al., Biol Trace Elem Res 94:33–40, 2003) and with literature data (Hotz C, et al. Am J Clin Nutr 78:756–764, 2003). Results 101 patients (median age 16 years) were included. There was no difference in serum Zn concentration between CF patients and controls. In CF patients no difference in serum Zn concentration between pancreatic-sufficient or pancreatic-insufficient patients was seen. Serum Zn was not associated to nutritional status or height z-score. A significant association serum Zn to serum albumin (p < 0.0005) and to vit A (p < 0.01) was seen. No associations of serum Zn to serum vit E, RBP, cholesterol, or CFTR were present, but there is a significant association serum Zn to forced vital capacity (p < 0.01). Serum Zn was not associated to inflammatory parameters or chronic Pseudomonas infection. Conclusion Comparison of CF patients with local controls revealed no significant differences. However, because persisting steatorrhea increases Zn loss (Easley et al., J Pediatr Gastroenterol Nutr 26:136–139, 1998) and 12.6% of our population has a serum Zn below the p value of 2.5 of the NHANES II study (Hotz C, et al. Am J Clin Nutr 78:756–764, 2003), there could remain an increased risk of Zn deficiency in some CF patients. Furthermore, the association with pulmonary function needs more investigation.