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Chemicals/CAS: Blood Coagulation Factors; Tissue Plasminogen Activator, EC 3.4.21.68

We have previously shown that the melanotrope population of the pituitary intermediate lobe of Rana ridibunda is composed of two subpopulations, of low (LD) and high density (HD), that show distinct ultrastructural features and display different synthetic and secretory rates. To investigate whether LD and HD melanotrope cells also differ in proopiomelanocortin (POMC) processing, we have analyzed the POMC-end products in single cells from both subpopulations by means of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The mass spectra revealed the presence of 8 POMC-derived peptides in HD and LD melanotrope cells, indicating a similar processing of the precursor in both subpopulations. However, the relative abundance of three POMC-end products (i.e. lys-γ1- MSH, acetyl-α-MSH, and CLIP fragment) was higher in the HD subset. Moreover, two peptides with molecular weights of 1030 and 1818 Da, respectively, were detected that could not be assigned to any product deduced from the frog POMC sequence. The relative amount of the 1030 Da peptide was higher in LD melanotrope cells. Taken together, our results suggest that POMC processing is differentially regulated in the two melanotrope cell subsets.

Dietary fiber intake is associated with lower incidence and mortality from disease, but the underlying mechanisms of these protective effects are unclear.We hypothesized that β2→1-fructan dietary fibers confer protection on intestinal epithelial cell barrier function via Toll-like receptor 2 (TLR2), and we studied whether β2→1-fructan chain-length differences affect this process. T84 human intestinal epithelial cell monolayers were incubated with 4 β2→1-fructan formulations of different chain-length compositions and were stimulated with the proinflammatory phorbol 12-myristate 13-acetate (PMA). Transepithelial electrical resistance (TEER) was analyzed by electric cell substrate impedance sensing (ECIS) as a measure for tight junction-mediated barrier function. To confirm TLR2 involvement in barrier modulation by β2→1-fructans, ECIS experiments were repeated using TLR2 blocking antibody. After preincubation of T84 cells with short-chain β2→1-fructans, the decrease in TEER as induced by PMA (62.3 ± 5.2%, P < 0.001) was strongly attenuated (15.2 ± 8.8%, P < 0.01). However, when PMA was applied first, no effect on recovery was observed during addition of the fructans. By blocking TLR2 on the T84 cells, the protective effect of short-chain β2→1-fructans was substantially inhibited. Stimulation of human embryonic kidney human TLR2 reporter cells with β2→1-fructans induced activation of nuclear factor kappa-light-chain-enhancer of activated B cells, confirming that β2→1-fructans are specific ligands for TLR2. To conclude, β2→1-fructans exert time-dependent and chain length-dependent protective effects on the T84 intestinal epithelial cell barrier mediated via TLR2. These results suggest that TLR2 located on intestinal epithelial cells could be a target of β2→1-fructan-mediated health effects. © 2014 American Society for Nutrition.Chemicals/CAS: inulinase, 9025-67-6; phorbol 13 acetate 12 myristate, 16561-29-8; toll like receptor 2, 203811-81-8

The value of the multivariate data analysis tools principal component analysis (PCA) and principal component discriminant analysis (PCDA) for prioritizing leads generated by microarrays was evaluated. To this end, Pseudomonas putida S12 was grown in independent triplicate fermentations on four different carbon sources, i.e. fructose, glucose, gluconate and succinate. RNA isolated from these samples was analysed in duplicate on an anonymous clone-based array to avoid bias during data analysis. The relevant transcripts were identified by analysing the loadings of the principal components (PC) and discriminants (D) in PCA and PCDA, respectively. Even more specifically, the relevant transcripts for a specific phenotype could also be ranked from the loadings under an angle (biplot) obtained after PCDA analysis. The leads identified in this way were compared with those identified using the commonly applied fold-difference and hierarchical clustering approaches. The different data analysis methods gave different results. The methods used were complementary and together resulted in a comprehensive picture of the processes important for the different carbon sources studied. For the more subtle, regulatory processes in a cell, the PCDA approach seemed to be the most effective. Except for glucose and gluconate dehydrogenase, all genes involved in the degradation of glucose, gluconate and fructose were identified. Moreover, the transcriptomics approach resulted in potential new insights into the physiology of the degradation of these carbon sources. Indications of iron limitation were observed with cells grown on glucose, gluconate or succinate but not with fructose-grown cells. Moreover, several cytochrome- or quinone-associated genes seemed to be specifically up- or downregulated, indicating that the composition of the electron-transport chain in P. putida S12 might change significantly in fructose-grown cells compared to glucose-, gluconate- or succinate-grown cells. © 2006 SGM.

Background: During wound repair, fibrin acts both as a barrier to prevent blood loss and as a temporary matrix for the invasion and ingrowth of endothelial and tissue cells. A well-controlled angiogenesis process in the fibrinous exudate matrix is crucial for optimal wound healing. The composition and structure of the fibrin matrix are important determinants of the invasion of endothelial cells and capillary-tube formation into the matrix. Objective: Fibrinogen circulates in a high and low molecular weight form (HMW and LMW, respectively) and the purpose of this study was to investigate how fibrin matrices from these naturally occurring fibrinogen variants influence angiogenesis. Angiogenesis was studied using an in vitro model in which human microvascular endothelial cells (hMVEC) were cultured on three-dimensional fibrin matrices from different fibrinogen forms, and using two in vivo mouse models. Results: The in vitro angiogenesis in an HMW-fibrin matrix shows increased cell and tubular structure ingrowth compared with unfractionated fibrin matrix (median increase 58%, range 46-234%). The ingrowth of tubular structures in an LMW-fibrin matrices is decreased when compared with unfractionated fibrin (median decrease 70%, range 67-100%). Similar results were observed for in vivo angiogenesis. Conclusions: The naturally occurring fibrinogen variants HMW- and LMW-fibrin modulate the angiogenic capacity of endothelial cells in fibrin matrices. The different effects of the molecular weight fibrinogen variants provide further insight in the matrix characteristics in angiogenesis and could possibly be applied in the context of tissue engineering and wound healing. © 2006 International Society on Thrombosis and Haemostasis.

Although food allergy has emerged as a major health problem, the mechanisms that are decisive in the development of sensitization to dietary Ag remain largely unknown. CTLA-4 signaling negatively regulates immune activation, and may play a crucial role in preventing induction and/or progression of sensitization to food Ag. To elucidate the role of CTLA-4 signaling in responses to food allergens, a murine model of peanut allergy was used. During oral exposure to peanut protein extract (PPE) together with the mucosal adjuvant cholera toxin (CT), which induces peanut allergy, CTLA-4 ligation was prevented using a CTLA-4 mAb. Additionally, the effect of inhibition of the CTLA-4 pathway on oral exposure to PPE in the absence of CT, which leads to unresponsiveness to peanut Ag, was explored. During sensitization, anti-CTLA-4 treatment considerably enhanced IgE responses to PPE and the peanut allergens, Ara h 1, Ara h 3, and Ara h 6, resulting in elevated mast cell degranulation upon an oral challenge. Remarkably, antagonizing CTLA-4 during exposure to PPE in the absence of CT resulted in significant induction of Th2 cytokines and an elevation in total serum IgE levels, but failed to induce allergen-specific IgE responses and mast cell degranulation upon a PPE challenge. These results indicate that CTLA-4 signaling is not the crucial factor in preventing sensitization to food allergens, but plays a pivotal role in regulating the intensity of a food allergic sensitization response. Furthermore, these data indicate that a profoundly Th2-biased cytokine environment is insufficient to induce allergic responses against dietary Ag.

Background-Ginseng is a commonly used nutraceutical. Intriguingly, existing literature reports both wound-healing and antitumor effects of ginseng extract through opposing activities on the vascular system. To elucidate this perplexity, we merged a chemical fingerprinting approach with a deconstructional study of the effects of pure molecules from ginseng extract on angiogenesis. Methods and Results-A mass spectrometric compositional analysis of American, Chinese and Korean, and Sanqi ginseng revealed distinct "sterol ginsenoside" fingerprints, especially in the ratio between a triol, Rg1, and a diol, Rb1, the 2 most prevalent constituents. Using a Matrigel implant model and reconstituting the extracts using distinct ratios of the 2 ginsenosides, we demonstrate that the dominance of Rg1 leads to angiogenesis, whereas Rb1 exerts an opposing effect. Rg1 also promoted functional neovascularization into a polymer scaffold in vivo and the proliferation of, chemoinvasion of, and tubulogenesis by endothelial cells in vitro, an effect mediated through the expression of nitric oxide synthase and the phosphatidylinositol-3 kinase→Akt pathway. In contrast, Rb1 inhibited the earliest step in angiogenesis, the chemoinvasion of endothelial cells. Conclusions-The present study explains, for the first time, the ambiguity about the effects of ginseng in vascular pathophysiology based on the existence of opposing active principles in the extract. We also unraveled a speciogeographic variation impinging on the compositional fingerprint that may modulate the final phenotype. This emphasizes the need for regulations standardizing herbal therapy, currently under the Dietary Supplement and Health Education Act. Furthermore, we propose that Rg1 could be a prototype for a novel group of nonpeptide molecules that can induce therapeutic angiogenesis, such as in wound healing. Chemicals / CAS: nitric oxide synthase, 125978-95-2; phosphatidylinositol 3 kinase, 115926-52-8; protein kinase B, 148640-14-6; 1-Phosphatidylinositol 3-Kinase, EC 2.7.1.137; Angiogenesis Inducing Agents; Angiogenesis Inhibitors; Drug Implants; Enzyme Inhibitors; ginsenoside Rb1; ginsenoside Rg1, 22427-39-0; Ginsenosides; NG-Nitroarginine Methyl Ester, 50903-99-6

LPL activity plays an important role in preceding the VLDL remnant clearance via the three major apolipoprotein E (apoE)-recognizing receptors: the LDL receptor (LDLr), LDL receptor-related protein (LRP), and VLDL receptor (VLDLr). The aim of this study was to determine whether LPL activity is also important for VLDL remnant clearance irrespective of these receptors and to determine the mechanisms involved in the hepatic remnant uptake. Administration of an adenovirus expressing LPL (AdLPL) into lrp-ldlr-/-vldlr-/- mice reduced both VLDL-triglyceride (TG) and VLDL-total cholesterol (TC) levels. Conversely, inhibition of LPL by AdAPOC1 increased plasma VLDL-TG and VLDL-TC levels. Metabolic studies with radiolabeled VLDL-like emulsion particles showed that the clearance and hepatic association of their remnants positively correlated with LPL activity. This hepatic association was independent of the bridging function of LPL and HL, since heparin did not reduce the liver association. In vitro studies demonstrated that VLDL-like emulsion particles avidly bound to the cell surface of primary hepatocytes from lrp-ldlr-/-vldlr-/- mice, followed by slow internalization, and involved heparin-releaseable cell surface proteins as well as scavenger receptor class B type I (SR-BI). Collectively, we conclude that hepatic VLDL remnant uptake in the absence of the three classical apoE-recognizing receptors is regulated by LPL activity and involves heparan sulfate proteoglycans and SR-BI. Copyright © 2008 by the American Society for Biochemistry and Molecular Biology, Inc.

Thrombin mediates changes in endothelial barrier function and increases endothelial permeability. A feature of thrombin-enhanced endothelial hyperpermeability is contraction of endothelial cells (ECs), accompanied by formation of focal adhesions (FAs). Recently, a G protein-coupled receptor kinase-interacting protein, GIT1, was shown to regulate FA disassembly. We hypothesized that GIT1 modulates thrombin-induced changes in FAs. In human umbilical vein ECs (HUVECs), thrombin recruited GIT1 to FAs, where GIT1 colocalized with FAK and vinculin. Recruitment of GIT1 to FAs was dependent on activation of the small GTPase RhoA, and Rho kinase, as demonstrated by adenoviral transfection of dominant-negative RhoA and treatment with Y-27632. Thrombin stimulated GIT1 tyrosine phosphorylation with a time course similar to FAK phosphorylation in a Rho kinase- and Src-dependent manner. Depletion of GIT1 with antisense GIT1 oligonucleotides had no effect on basal cell morphology, but increased cell rounding and contraction of HUVECs, increased FA formation, and increased FAK tyrosine phosphorylation in response to thrombin, concomitant with increased endothelial hyperpermeability. These data identify GIT1 as a novel mediator in agonist-dependent signaling in ECs, demonstrate that GIT1 is involved in cell shape changes, and suggest a role for GIT1 as a negative feedback regulator that augments recovery of cell contraction. Chemicals / CAS: 4 (1 aminoethyl) n (4 pyridyl)cyclohexanecarboxamide, 146986-50-7; guanosine triphosphatase, 9059-32-9; protein tyrosine kinase, 80449-02-1; thrombin, 9002-04-4; Adaptor Proteins, Signal Transducing; Amides; Cell Cycle Proteins; Enzyme Inhibitors; Focal Adhesion Kinase 1, EC 2.7.1.112; Focal Adhesion Protein-Tyrosine Kinases, EC 2.7.1.112; GIT1 protein, human; GTPase-Activating Proteins; Oligodeoxyribonucleotides, Antisense; Phosphoproteins; Protein-Tyrosine Kinases, EC 2.7.1.112; Proto-Oncogene Proteins pp60(c-src), EC 2.7.1.112; PTK2 protein, human, EC 2.7.1.112; Pyridines; rac GTP-Binding Proteins, EC 3.6.5.2; rhoA GTP-Binding Protein, EC 3.6.5.2; RNA, Small Interfering; Thrombin, EC 3.4.21.5; Vinculin, 125361-02-6; Y 27632, 138381-45-0