Importance of syndecan-4 and syndecan -2 in osteoblast cell adhesion and survival mediated by a tissue transglutaminase-fibronectin complex

Tissue transglutaminase (TG2) has been identified as an important extracellular crosslinking enzyme involved in matrix turnover and in bone differentiation. Here we report a novel cell adhesion/survival mechanism in human osteoblasts (HOB) which requires association of FN bound TG2 with the cell surface heparan sulphates in a transamidase independent manner. This novel pathway not only enhances cell adhesion on FN but also mediates cell adhesion and survival in the presence of integrin competing RGD peptides. We investigate the involvement of cell surface receptors and their intracellular signalling molecules to further explore the pathway mediated by this novel TG-FN heterocomplex. We demonstrate by siRNA silencing the crucial importance of the cell surface heparan sulphate proteoglycans syndecan-2 and syndecan-4 in regulating the compensatory effect of TG-FN on osteoblast cell adhesion and actin cytoskeletal formation in the presence of RGD peptides. By use of immunoprecipitation and inhibitory peptides we show that syndecan-4 interacts with TG2 and demonstrate that syndecan-2 and the a5ß1 integrins, but not a4ß1 function as downstream modulators in this pathway. Using function blocking antibodies, we show activation of a5ß1 occurs by an inside out signalling mechanism involving activation and binding of protein kinase PKCa and phosphorylation of focal adhesion kinase (FAK) at Tyr861 and activation of ERK1/2.

The role of heterodimerization between VEGFR-1 and VEGFR-2 in the regulation of endothelial cell homeostasis

VEGF-A activity is tightly regulated by ligand and receptor availability. Here we investigate the physiological function of heterodimers between VEGF receptor-1 (VEGFR-1; Flt-1) and VEGFR-2 (KDR; Flk-1) (VEGFR(1-2)) in endothelial cells with a synthetic ligand that binds specifically to VEGFR(1-2). The dimeric ligand comprises one VEGFR-2-specific monomer (VEGF-E) and a VEGFR-1-specific monomer (PlGF-1). Here we show that VEGFR(1-2) activation mediates VEGFR phosphorylation, endothelial cell migration, sustained in vitro tube formation and vasorelaxation via the nitric oxide pathway. VEGFR(1-2) activation does not mediate proliferation or elicit endothelial tissue factor production, confirming that these functions are controlled by VEGFR-2 homodimers. We further demonstrate that activation of VEGFR(1-2) inhibits VEGF-A-induced prostacyclin release, phosphorylation of ERK1/2 MAP kinase and mobilization of intracellular calcium from primary endothelial cells. These findings indicate that VEGFR-1 subunits modulate VEGF activity predominantly by forming heterodimer receptors with VEGFR-2 subunits and such heterodimers regulate endothelial cell homeostasis.

Viral and bacterial septicaemic infections modulate the expression of PACAP splicing variants and VIP/PACAP receptors in brown trout immune organs

Copyright © 2015. Published by Elsevier Ltd. Acknowledgments The authors thank Richard Paley, Georgina Rimmer and Tom Hill for their contribution during the brown trout infection challenges carried out in CEFAS-Weymouth biosecurity facilities. Bartolomeo Gorgoglione and Nick G. H. Taylor were supported by a DEFRA contract C3490

Heterodimerisation between VEGFR-1 and VEGFR-2 and not the homodimers of VEGFR-1 inhibit VEGFR-2 activity

Vascular endothelial growth factor (VEGF) signaling is tightly regulated by specific VEGF receptors (VEGF-R). Recently, we identified heterodimerisation between VEGFR-1 and VEGFR-2 (VEGFR1–2) to regulate VEGFR-2 function. However, both the mechanism of action and the relationship with VEGFR-1 homodimers remain unknown. The current study shows that activation of VEGFR1–2, but not VEGFR-1 homodimers, inhibits VEGFR-2 receptor phosphorylation under VEGF stimulation in human endothelial cells. Furthermore, inhibition of phosphatidylinositol 3-kinase (PI3K) increases VEGFR-2 phosphorylation under VEGF stimulation. More importantly, inhibition of PI3K pathway abolishes the VEGFR1–2 mediated inhibition of VEGFR-2 phosphorylation. We further demonstrate that inhibition of PI3K pathway promotes capillary tube formation. Finally, the inhibition of PI3K abrogates the inhibition of in vitro angiogenesis mediated by VEGFR1–2 heterodimers. These findings demonstrate that VEGFR1–2 heterodimers and not VEGFR-1 homodimers inhibit VEGF-VEGFR-2 signaling by suppressing VEGFR-2 phosphorylation via PI3K pathway.

Fluorescence spectrometric study on the interactions of isoprocarb and sodium 2-isopropylphenate with bovine serum albumin

The binding interaction of the pesticide Isoprocarb and its degradation product, sodium 2-isopropylphenate, with bovine serum albumin (BSA) was studied by spectrofluorimetry under simulated physiological conditions. Both Isoprocarb and sodium 2-isopropylphenate quenched the intrinsic fluorescence of BSA. This quenching proceeded via a static mechanism. The thermodynamic parameters (ΔH°, ΔS° and ΔG°) obtained from the fluorescence data measured at two different temperatures showed that the binding of Isoprocarb to BSA involved hydrogen bonds and that of sodium 2-isopropylphenate to BSA involved hydrophobic and electrostatic interactions. Synchronous fluorescence spectroscopy of the interaction of BSA with either Isoprocarb or sodium 2-isopropylphenate showed that the molecular structure of the BSA was changed significantly, which is consistent with the known toxicity of the pesticide, i.e., the protein is denatured. The sodium 2-isopropylphenate, was estimated to be about 4–5 times more toxic than its parent, Isoprocarb. Synchronous fluorescence spectroscopy and the resolution of the three-way excitation–emission fluorescence spectra by the PARAFAC method extracted the relative concentration profiles of BSA, Isoprocab and sodium 2-isopropylphenate as a function of the added sodium 2-isopropylphenate. These profiles showed that the degradation product, sodium 2-isopropylphenate, displaced the pesticide in a competitive reaction with the BSA protein.

Wireless channel modelling for indoor populated environments : statistical characterization of narrowband channels at 2.45 Ghz and 5.2 Ghz

The recent development of indoor wireless local area network (WLAN) standards at 2.45 GHz and 5 GHz has led to increased interest in propagation studies at these frequency bands. Within the indoor environment, human body effects can strongly reduce the quality of wireless communication systems. Human body effects can cause temporal variations and shadowing due to pedestrian movement and antenna- body interaction with portable terminals. This book presents a statistical characterisation, based on measurements, of human body effects on indoor narrowband channels at 2.45 GHz and at 5.2 GHz. A novel cumulative distribution function (CDF) that models the 5 GHz narrowband channel in populated indoor environments is proposed. This novel CDF describes the received envelope in terms of pedestrian traffic. In addition, a novel channel model for the populated indoor environment is proposed for the Multiple-Input Multiple-Output (MIMO) narrowband channel in presence of pedestrians at 2.45 GHz. Results suggest that practical MIMO systems must be sufficiently adaptive if they are to benefit from the capacity enhancement caused by pedestrian movement.

Low-dimensional hydrogen-bonded structures in the 1:1 and 1:2 proton-transfer compounds of 4,5-dichlorophthalic acid with the aliphatic Lewis bases triethylamine, diethylamine, n-butylamine and piperidine

The structures of proton-transfer compounds of 4,5-dichlorophthalic acid (DCPA) with the aliphatic Lewis bases triethylamine, diethylamine, n-butylamine and piperidine, namely triethylaminium 2-carboxy-4,5-dichlorobenzoate C~6~H~16~N^+^ C~8~H~3~Cl~2~O~4~^-^ (I), diethylaminium 2-carboxy-4,5-dichlorobenzoate C~4~H~12~N^+^ C~8~H~3~Cl~2~O~4~^-^ (II), bis(n-butylaminium) 4,5-dichlorophthalate monohydrate 2(C~4~H~12~N^+^) C~8~H~2~Cl~2~O~4~^2-^ . H~2~O (III) and bis(piperidinium) 4,5-dichlorophthalate monohydrate 2(C~5~H~12~N^+^) C~8~H~2~Cl~2~O~4~^2-^ . H~2~O (IV)have been determined at 200 K. All compounds have hydrogen-bonding associations giving in (I) discrete cation-anion units, linear chains in (II) while (III) and (IV) both have two-dimensional structures. In (I) a discrete cation-anion unit is formed through an asymmetric R2/1(4) N+-H...O,O' hydrogen-bonding association whereas in (II), one-dimensional chains are formed through linear N-H...O associations by both aminium H donors. In compounds (III) and (IV) the primary N-H...O linked cation-anion units are extended into a two-dimensional sheet structure via amide N-H...O(carboxyl) and ...O(carbonyl) interactions. In the 1:1 salts [(I) and (II)], the hydrogen 4,5-dichlorophthalate anions are essentially planar with short intramolecular carboxylic acid O-H...O(carboxyl) hydrogen bonds [O...O, 2.4223(14) and 2.388(2)A respectively]. This work provides a further example of the uncommon zero-dimensional hydrogen-bonded DCPA-Lewis base salt and the one-dimensional chain structure type, while even with the hydrate structures of the 1:2 salts with the primary and secondary amines, the low dimensionality generally associated with 1:1 DCPA salts is also found.

Aggravation of ADAMTS and MMP production and ERK1/2 pathway in the interaction of osteoarthritic subchondral bone osteoblasts and articular cartilage chondrocytes : a possible pathogenic role in osteoarthritis

Introduction: Degradative enzymes, such as A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) and matrix metalloproteinases (MMPs), play key roles in osteoarthritis (OA) development. The aim of the present study was to investigate if cross-talk between subchondral bone osteoblasts (SBOs) and articular cartilage chondrocytes (ACCs) in OA alters the expression and regulation of ADAMTS5, ADAMTS4, MMP-1, MMP-2, MMP-3, MMP-8, MMP-9 and MMP-13, and also to test the possible involvement of mitogen activated protein kinase (MAPK) signaling pathway during this process. Methods: ACCs and SBOs were isolated from normal and OA patients. An in vitro co-culture model was developed to study the regulation of ADAMTS and MMPs under normal and OA joint cross-talk conditions. MAPK-ERK inhibitor, PD98059 was applied to delineate the involvement of specific pathway during this interaction process. Results: Indirect co-culture of OA SBOs with normal ACCs resulted in significantly increased expression of ADAMTS5, ADAMTS4, MMP-2, MMP-3 and MMP-9 in ACCs, whereas co-culture of OA ACCs led to increased MMP-1 and MMP-2 expression in normal SBOs. The upregulation of ADAMTS and MMPs under these conditions was correlated with activation of the MAPK-ERK1/2 signaling pathway and the addition of the MAPK-ERK inhibitor, PD98059, reversed the overexpression of ADAMTS and MMPs in co-cultures. Conclusion: In summary, we believe, these results add to the evidence that in human OA, altered bi-directional signals transmitted between SBOs and ACCs significantly impacts the critical features of both cartilage and bone by producing abnormal levels of ADAMTS and MMPs. Furthermore, we have demonstrated for the first time that this altered cross-talk was mediated by the phosphorylation of MAPK-ERK1/2 signaling pathway.

Influence of internal fixator stiffness on murine fracture healing : two types of fracture healing lead to two distinct cellular events and FGF-2 expressions

This study aimed to clarify the relationship between the mechanical environment at the fracture site and endogenous fibroblast growth factor-2 (FGF-2). We compared two types of fracture healing with different callus formations and cellular events using MouseFix(TM) plate fixation systems for murine fracture models. Left femoral fractures were induced in 72 ten-week-old mice and then fixed with a flexible (Group F) or rigid (Group R) Mouse Fix(TM) plate. Mice were sacrificed on days 3, 5, 7, 10, 14, and 21. The callus volumes were measured by 3D micro-CT and tissues were histologically stained with hematoxylin & eosin or safranin-O. Sections from days 3, 5, and 7 were immunostained for FGF-2 and Proliferating Cell Nuclear Antigen (PCNA). The callus in Group F was significantly larger than that in Group R. The rigid plate allowed bone union without a marked external callus or chondrogenesis. The flexible plate formed a large external callus as a result of endochondral ossification. Fibroblastic cells in the granulation tissue on days 5 and 7 in Group F showed marked FGF-2 expression compared with Group R. Fibroblastic cells showed ongoing proliferation in granulation tissue in group F, as indicated by PCNA expression, which explained the relative granulation tissue increase in group F. There were major differences in early phase endogenous FGF-2 expression between these two fracture healing processes, due to different mechanical environments.

Vapour phase assembly of a halogen bonded complex of an isoindoline nitroxide and 1,2-diiodotetrafluorobenzene

Vapour phase assembly has been used for the first time to prepare co-crystals in which the primary intermolecular interaction is halogen bonding. Co-crystals of the nitroxide 1,1,3,3-tetramethylisoindolin-2-yloxyl (TMIO) and 1,2-diiodotetrafluorobenzene (1,2-DITFB) are readily formed under standard sublimation conditions. Single crystal X-ray diffraction confirmed the structure of a 2:2 cyclic tetramer, (TMIO)2·(1,2-DITFB)2, which exhibits a new halogen bonding motif, with each nitroxide oxygen atom accepting two halogen bonds. Powder X-ray diffraction confirmed the homogeneity of the bulk sample. The crystalline complex was further characterized in the solid state using thermal analysis and vibrational spectroscopy (infrared and Raman). Density functional theory calculations were also used to evaluate the enthalpy of formation, electrostatic potential and unpaired electron density of the complex. These findings illustrate the preparation of co-crystals where solution state methodology is problematic and the potential of this approach for the formation of novel organic spin systems.

The effect of a high fat meal on postprandial arterial stiffness in men with obesity and type 2 diabetes

Context: Postprandial dysmetabolism is emerging as an important cardiovascular risk factor. Augmentation index (AIx) is a measure of systemic arterial stiffness and independently predicts cardiovascular outcome. Objective: The objective of this study was to assess the effect of a standardized high-fat meal on metabolic parameters and AIx in 1) lean, 2) obese nondiabetic, and 3) subjects with type 2 diabetes mellitus (T2DM). Design and Setting: Male subjects (lean, n = 8; obese, n = 10; and T2DM, n = 10) were studied for 6 h after a high-fat meal and water control. Glucose, insulin, triglycerides, and AIx (radial applanation tonometry) were measured serially to determine the incremental area under the curve (iAUC). Results: AIx decreased in all three groups after a high-fat meal. A greater overall postprandial reduction in AIx was seen in lean and T2DM compared with obese subjects (iAUC, 2251 +/- 1204, 2764 +/- 1102, and 1187 +/- 429% . min, respectively; P < 0.05). The time to return to baseline AIx was significantly delayed in subjects with T2DM (297 +/- 68 min) compared with lean subjects (161 +/- 88 min; P < 0.05). There was a significant correlation between iAUC AIx and iAUC triglycerides (r = 0.50; P < 0.05). Conclusions: Obesity is associated with an attenuated overall postprandial decrease in AIx. Subjects with T2DM have a preserved, but significantly prolonged, reduction in AIx after a high-fat meal. The correlation between AIx and triglycerides suggests that postprandial dysmetabolism may impact on vascular dynamics. The markedly different response observed in the obese subjects compared with those with T2DM was unexpected and warrants additional evaluation.