The Ras inhibitors caveolin-1 and docking protein 1 activate peroxisome proliferator-activated receptor ? through spatial relocalization at helix 7 of its ligand-binding domain

Peroxisome proliferator-activated receptor ? (PPAR?) is a transcription factor that promotes differentiation and cell survival in the stomach. PPAR? upregulates and interacts with caveolin-1 (Cav1), a scaffold protein of Ras/mitogen-activated protein kinases (MAPKs). The cytoplasmic-to-nuclear localization of PPAR? is altered in gastric cancer (GC) patients, suggesting a so-far-unknown role for Cav1 in spatial regulation of PPAR? signaling. We show here that loss of Cav1 accelerated proliferation of normal stomach and GC cells in vitro and in vivo. Downregulation of Cav1 increased Ras/MAPK-dependent phosphorylation of serine 84 in PPAR? and enhanced nuclear translocation and ligand-independent transcription of PPAR? target genes. In contrast, Cav1 overexpression sequestered PPAR? in the cytosol through interaction of the Cav1 scaffolding domain (CSD) with a conserved hydrophobic motif in helix 7 of PPAR?'s ligand-binding domain. Cav1 cooperated with the endogenous Ras/MAPK inhibitor docking protein 1 (Dok1) to promote the ligand-dependent transcriptional activity of PPAR? and to inhibit cell proliferation. Ligand-activated PPAR? also reduced tumor growth and upregulated the Ras/MAPK inhibitors Cav1 and Dok1 in a murine model of GC. These results suggest a novel mechanism of PPAR? regulation by which Ras/MAPK inhibitors act as scaffold proteins that sequester and sensitize PPAR? to ligands, limiting proliferation of gastric epithelial cells.

Expression patterns of Bmi-1 and p16 significantly correlate with overall, disease-specific, and recurrence-free survival in oropharyngeal squamous cell carcinoma

BACKGROUND: The objective of this study was to link expression patterns of B-cell-specific Moloney murine leukemia virus integration site 1 (Bmi-1) and p16 to patient outcome (recurrence and survival) in a cohort of 252 patients with oral and oropharyngeal squamous cell cancer (OSCC). METHODS: Expression levels of Bmi-1 and p16 in samples from 252 patients with OSCC were evaluated immunohistochemically using the tissue microarray method. Staining intensity was determined by calculating an intensity reactivity score (IRS). Staining intensity and the localization of expression within tumor cells (nuclear or cytoplasmic) were correlated with overall, disease-specific, and recurrence-free survival. RESULTS: The majority of cancers were localized in the oropharynx (61.1%). In univariate analysis, patients who had OSCC and strong Bmi-1 expression (IRS >10) had worse outcomes compared with patients who had low and moderate Bmi-1 expression (P = .008; hazard ratio [HR], 1.82; 95% confidence interval [CI], 1.167-2.838); this correlation was also observed for atypical cytoplasmic Bmi-1 expression (P = .001; HR, 2.164; 95% CI, 1.389-3.371) and for negative p16 expression (P < .001; HR, 0.292; 95% CI, 0.178-0.477). The combination of both markers, as anticipated, had an even stronger correlation with overall survival (P < .001; HR, 8.485; 95% CI, 4.237-16.994). Multivariate analysis demonstrated significant results for patients with oropharyngeal cancers, but not for patients with oral cavity tumors: Tumor classification (P = .011; HR, 1.838; 95%CI, 1.146-2.947) and the combined marker expression patterns (P < .001; HR, 6.254; 95% CI, 2.869-13.635) were correlated with overall survival, disease-specific survival (tumor classification: P = .002; HR, 2.807; 95% CI, 1.477-5.334; combined markers: P = .002; HR, 5.386; 95% CI, 1.850-15.679), and the combined markers also were correlated with recurrence-free survival (P = .001; HR, 8.943; 95% CI, 2.562-31.220). CONCLUSIONS: Cytoplasmic Bmi-1 expression, an absence of p16 expression, and especially the combination of those 2 predictive markers were correlated negatively with disease-specific and recurrence-free survival in patients with oropharyngeal cancer. Therefore, the current results indicate that these may be applicable as predictive markers in combination with other factors to select patients for more aggressive treatment and follow-up. Cancer 2011;. © 2011 American Cancer Society.

Exploring High-resolution Magic Angle Spinning (HR-MAS) NMR Spectroscopy for Metabonomic Analysis of Apples

Classical liquid-state high-resolution (HR) NMR spectroscopy has proved a powerful tool in the metabonomic analysis of liquid food samples like fruit juices. In this paper the application of (1)H high-resolution magic angle spinning (HR-MAS) NMR spectroscopy to apple tissue is presented probing its potential for metabonomic studies. The (1)H HR-MAS NMR spectra are discussed in terms of the chemical composition of apple tissue and compared to liquid-state NMR spectra of apple juice. Differences indicate that specific metabolic changes are induced by juice preparation. The feasibility of HR-MAS NMR-based multivariate analysis is demonstrated by a study distinguishing three different apple cultivars by principal component analysis (PCA). Preliminary results are shown from subsequent studies comparing three different cultivation methods by means of PCA and partial least squares discriminant analysis (PLS-DA) of the HR-MAS NMR data. The compounds responsible for discriminating organically grown apples are discussed. Finally, an outlook of our ongoing work is given including a longitudinal study on apples.

Shear stress modulates the expression of thrombospondin-1 and CD36 in endothelial cells in vitro and during shear stress-induced angiogenesis in vivo

Binding of thrombospondin-1 (TSP-1) to the CD36 receptor inhibits angiogenesis and induces apoptosis in endothelial cells (EC). Conversely, matrix-bound TSP-1 supports vessel formation. In this study we analyzed the shear stress-dependent expression of TSP-1 and CD36 in endothelial cells in vitro and in vivo to reveal its putative role in the blood flow-induced remodelling of vascular networks. Shear stress was applied to EC using a cone-and-plate apparatus and gene expression was analyzed by RT-PCR, Northern and Western blot. Angiogenesis in skeletal muscles of prazosin-fed (50 mg/l drinking water; 4 d) mice was assessed by measuring capillary-to-fiber (C/F) ratios. Protein expression in whole muscle homogenates (WMH) or BS-1 lectin-enriched EC fractions (ECF) was analyzed by Western blot. Shear stress downregulated TSP-1 and CD36 expression in vitro in a force- and time-dependent manner sustained for at least 72 h and reversible by restoration of no-flow conditions. In vivo, shear stress-driven increase of C/F in prazosin-fed mice was associated with reduced expression of TSP-1 and CD36 in ECF, while TSP-1 expression in WMH was increased. Down-regulation of endothelial TSP-1/CD36 by shear stress suggests a mechanism for inhibition of apoptosis in perfused vessels and pruning in the absence of flow. The increase of extra-endothelial (e.g. matrix-bound) TSP-1 could support a splitting type of vessel growth.

QTc interval and resting heart rate as long-term predictors of mortality in type 1 and type 2 diabetes mellitus: a 23-year follow-up

We evaluated the association of QT interval corrected for heart rate (QT(c)) and resting heart rate (rHR) with mortality (all-causes, cardiovascular, cardiac, and ischaemic heart disease) in subjects with type 1 and type 2 diabetes.

Serum leptin and C-reactive protein levels in the physiological spontaneous menstrual cycle in reproductive age women

OBJECTIVE: Only a few studies have investigated variations of different markers for inflammatory processes during the physiological menstrual cycle. The results are conflicting, particularly concerning the correlation between the marker leptin and steroid hormones. The aim of the study was to investigate the inflammatory markers C-reactive protein (CRP) and leptin in the serum of healthy, normally ovulating women and to correlate these with each other and with the hormones of the gonadal axis. A cycle-dependence of the markers studied would imply an exact timing of the blood sampling for clinical needs. DESIGN: Observational study investigating the two inflammatory markers CRP and leptin in relation to the hormonal pattern of the gonadal axis during the normal cycle. METHODS: Ovulatory cycles of 36 healthy, young, normo-androgenic women, having a normal body mass index were evaluated. Serum concentrations of leptin and CRP, as well as of follicle-stimulating hormone, luteinising hormone, 17beta-oestradiol, progesterone, prolactin (PRL) and free testosterone were measured every 1-2 days during one full cycle. RESULTS: Serum levels of leptin and CRP behaved differently during ovulatory cycles, with higher concentrations for leptin only during certain phases. Significant correlations were found in the follicular phase between leptin and PRL and leptin and free testosterone. CONCLUSIONS: Leptin levels change during the menstrual cycle. Leptin levels are more stable on cycle days 1-5 than later in the cycle. For precise cycle-independent measurements, these fluctuations have to be taken into account. There is no similar cyclic pattern for CRP.

Angiopoietin-1 and angiopoietin-2 share the same binding domains in the Tie-2 receptor involving the first Ig-like loop and the epidermal growth factor-like repeats

Angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) have been identified as ligands with different effector functions of the vascular assembly and maturation-mediating receptor tyrosine kinase Tie-2. To understand the molecular interactions of the angiopoietins with their receptor, we have studied the binding of Ang-1 and Ang-2 to the Tie-2 receptor. Enzyme-linked immunosorbent assay-based competition assays and co-immunoprecipitation experiments analyzing the binding of Ang-1 and Ang-2 to truncation mutants of the extracellular domain of Tie-2 showed that the first Ig-like loop of Tie-2 in combination with the epidermal growth factor (EGF)-like repeats (amino acids 1-360) is required for angiopoietin binding. The first Ig-like domain or the EGF-like repeats alone are not capable of binding Ang-1 and Ang-2. Concomitantly, we made the surprising finding that Tie-2 exon-2 knockout mice do express a mutated Tie-2 protein that lacks 104 amino acids of the first Ig-like domain. This mutant Tie-2 receptor is functionally inactive as shown by the lack of ligand binding and receptor phosphorylation. Collectively, the data show that the first 104 amino acids of the Tie-2 receptor are essential but not sufficient for angiopoietin binding. Conversely, the first 360 amino acids (Ig-like domain plus EGF-like repeats) of the Tie-2 receptor are necessary and sufficient to bind both Ang-1 and Ang-2, which suggests that differential receptor binding is not likely to be responsible for the different functions of Ang-1 and Ang-2.

Recurrent rearrangements of chromosome 1q21.1 and variable pediatric phenotypes

BACKGROUND: Duplications and deletions in the human genome can cause disease or predispose persons to disease. Advances in technologies to detect these changes allow for the routine identification of submicroscopic imbalances in large numbers of patients. METHODS: We tested for the presence of microdeletions and microduplications at a specific region of chromosome 1q21.1 in two groups of patients with unexplained mental retardation, autism, or congenital anomalies and in unaffected persons. RESULTS: We identified 25 persons with a recurrent 1.35-Mb deletion within 1q21.1 from screening 5218 patients. The microdeletions had arisen de novo in eight patients, were inherited from a mildly affected parent in three patients, were inherited from an apparently unaffected parent in six patients, and were of unknown inheritance in eight patients. The deletion was absent in a series of 4737 control persons (P=1.1x10(-7)). We found considerable variability in the level of phenotypic expression of the microdeletion; phenotypes included mild-to-moderate mental retardation, microcephaly, cardiac abnormalities, and cataracts. The reciprocal duplication was enriched in nine children with mental retardation or autism spectrum disorder and other variable features (P=0.02). We identified three deletions and three duplications of the 1q21.1 region in an independent sample of 788 patients with mental retardation and congenital anomalies. CONCLUSIONS: We have identified recurrent molecular lesions that elude syndromic classification and whose disease manifestations must be considered in a broader context of development as opposed to being assigned to a specific disease. Clinical diagnosis in patients with these lesions may be most readily achieved on the basis of genotype rather than phenotype.

Differential expression of stx2 variants in Shiga toxin-producing Escherichia coli belonging to seropathotypes A and C

Only a subset of Shiga toxin (Stx)-producing Escherichia coli (STEC) are human pathogens, but the characteristics that account for differences in pathogenicity are not well understood. In this study, we investigated the distribution of the stx variants coding for Stx2 and its variants in highly virulent STEC of seropathotype A and low-pathogenic STEC of seropathotype C. We analysed and compared transcription of the corresponding genes, production of Shiga toxins, and stx-phage release in basal as well as in induced conditions. We found that the stx(2) variant was mainly associated with strains of seropathotype A, whereas most of the strains of seropathotype C possessed the stx(2-vhb) variant, which was frequently associated with stx(2), stx(2-vha) or stx(2c). Levels of stx(2) and stx(2)-related mRNA were higher in strains belonging to seropathotype A and in those strains of seropathotype C that express the stx(2) variant than in the remaining strains of seropathotype C. The stx(2-vhb) genes were the least expressed, in basal as well as in induced conditions, and in many cases did not seem to be carried by an inducible prophage. A clear correlation was observed between stx mRNA levels and stx-phage DNA in the culture supernatants, suggesting that most stx(2)-related genes are expressed only when they are carried by a phage. In conclusion, some relationship between stx(2)-related gene expression in vitro and the seropathotype of the STEC strains was observed. A higher expression of the stx(2) gene and a higher release of its product, in basal as well as in induced conditions, was observed in pathogenic strains of seropathotype A. A subset of strains of seropathotype C shows the same characteristics and could be a high risk to human health.

TRPC1 is regulated by caveolin-1 and is involved in oxidized LDL-induced apoptosis of vascular smooth muscle cells

Oxidized low-density lipoprotein (oxLDL) induced-apoptosis of vascular cells may participate in plaque instability and rupture. We have previously shown that vascular smooth muscle cells (VSMC) stably expressing caveolin-1 were more susceptible to oxLDL-induced apoptosis than VSMC expressing lower level of caveolin-1, and this was correlated with enhanced Ca(2+) entry and pro-apoptotic events. In this study we aimed to identify the molecular events involved in oxLDL-induced Ca(2+) influx and their regulation by the structural protein caveolin-1. In VSMC, transient receptor potential canonical-1 (TRPC1) silencing by ARN interference, prevents the Ca(2+) influx and reduces the toxicity induced by oxLDL. Moreover, caveolin-1 silencing induces concomitant decrease of TRPC1 expression and reduces oxLDL-induced-apoptosis of VSMC. OxLDL enhanced the cell surface expression of TRPC1, as shown by biotinylation of cell surface proteins, and induced TRPC1 translocation into caveolar compartment, as assessed by subcellular fractionation. OxLDL-induced TRPC1 translocation was dependent on actin cytoskeleton and associated with a dramatic rise of 7-ketocholesterol (a major oxysterol in oxLDL) into caveolar membranes, whereas the caveolar content of cholesterol was unchanged. Altogether, the reported results show that TRPC1 channels play a role in Ca(2+) influx and Ca(2+) homeostasis deregulation that mediate apoptosis induced by oxLDL. These data also shed new light on the role of caveolin-1 and caveolar compartment as important regulators of TRPC1 trafficking to the plasma membrane and apoptotic processes that play a major role in atherosclerosis.

Clinical and intravascular imaging outcomes at 1 and 2 years after implantation of absorb everolimus eluting bioresorbable vascular scaffolds in small vessels. Late lumen enlargement: does bioresorption matter with small vessel size? Insight from the ABSORB cohort B trial

BACKGROUND The long-term results after second generation everolimus eluting bioresorbable vascular scaffold (Absorb BVS) placement in small vessels are unknown. Therefore, we investigated the impact of vessel size on long-term outcomes, after Absorb BVS implantation. METHODS In ABSORB Cohort B Trial, out of the total study population (101 patients), 45 patients were assigned to undergo 6-month and 2-year angiographic follow-up (Cohort B1) and 56 patients to have angiographic follow-up at 1-year (Cohort B2). The pre-reference vessel diameter (RVD) was <2.5 mm (small-vessel group) in 41 patients (41 lesions) and ≥2.5 mm (large-vessel group) in 60 patients (61 lesions). Outcomes were compared according to pre-RVD. RESULTS At 2-year angiographic follow-up no differences in late lumen loss (0.29±0.16 mm vs 0.25±0.22 mm, p=0.4391), and in-segment binary restenosis (5.3% vs 5.3% p=1.0000) were demonstrated between groups. In the small-vessel group, intravascular ultrasound analysis showed a significant increase in vessel area (12.25±3.47 mm(2) vs 13.09±3.38 mm(2) p=0.0015), scaffold area (5.76±0.96 mm(2) vs 6.41±1.30 mm(2) p=0.0008) and lumen area (5.71±0.98 mm(2) vs 6.20±1.27 mm(2) p=0.0155) between 6-months and 2-year follow-up. No differences in plaque composition were reported between groups at either time point. At 2-year clinical follow-up, no differences in ischaemia-driven major adverse cardiac events (7.3% vs 10.2%, p=0.7335), myocardial infarction (4.9% vs 1.7%, p=0.5662) or ischaemia-driven target lesion revascularisation (2.4% vs 8.5%, p=0.3962) were reported between small and large vessels. No deaths or scaffold thrombosis were observed. CONCLUSIONS Similar clinical and angiographic outcomes at 2-year follow-up were reported in small and large vessel groups. A significant late lumen enlargement and positive vessel remodelling were observed in small vessels.

MQN-Mapplet: Visualization of Chemical Space with Interactive Maps of DrugBank, ChEMBL, PubChem, GDB-11, and GDB-13

The MQN-mapplet is a Java application giving access to the structure of small molecules in large databases via color-coded maps of their chemical space. These maps are projections from a 42-dimensional property space defined by 42 integer value descriptors called molecular quantum numbers (MQN), which count different categories of atoms, bonds, polar groups, and topological features and categorize molecules by size, rigidity, and polarity. Despite its simplicity, MQN-space is relevant to biological activities. The MQN-mapplet allows localization of any molecule on the color-coded images, visualization of the molecules, and identification of analogs as neighbors on the MQN-map or in the original 42-dimensional MQN-space. No query molecule is necessary to start the exploration, which may be particularly attractive for nonchemists. To our knowledge, this type of interactive exploration tool is unprecedented for very large databases such as PubChem and GDB-13 (almost one billion molecules). The application is freely available for download at www.gdb.unibe.ch.