Characterization of heparin-binding site of tissue transglutaminase:its importance in cell surface targeting, matrix deposition, and cell signaling

Tissue transglutaminase (TG2) is a multifunctional Ca2+ activated protein crosslinking enzyme secreted into the extracellular matrix (ECM), where it is involved in wound healing and scarring, tissue fibrosis, celiac disease and metastatic cancer. Extracellular TG2 can also facilitate cell adhesion important in wound healing through a non-transamidating mechanism via its association with fibronectin (FN), heparan sulphates (HS) and integrins. Regulating the mechanism how TG2 is translocated into the ECM therefore provides a strategy for modulating these physiological and pathological functions of the enzyme. Here, through molecular modelling and mutagenesis we have identified the HS binding site of TG2 202KFLKNAGRDCSRRSSPVYVGR222. We demonstrate the requirement of this binding site for translocation of TG2 into the ECM through a mechanism involving cell surface shedding of HS. By synthesizing a peptide NPKFLKNAGRDCSRRSS corresponding to the HS binding site within TG2, we also demonstrate how this mimicking peptide can in isolation compensate the RGD-induced loss of cell adhesion on FN via binding to syndecan-4, leading to activation of PKCa, pFAK-397 and ERK1/2 and the subsequent formation of focal adhesions and actin cytoskeleton organization. A novel regulatory mechanism for TG2 translocation into the extracellular compartment that depends upon TG2 conformation and the binding of HS is proposed.

Liposomal cationic charge and antigen adsorption are important properties for the efficient deposition of antigen at the injection site and ability of the vaccine to induce a CMI response

With respect to liposomes as delivery vehicles and adjuvants for vaccine antigens, the role of vesicle surface charge remains disputed. In the present study we investigate the influence of liposome surface charge and antigen-liposome interaction on the antigen depot effect at the site of injection (SOI). The presence of liposome and antigen in tissue at the SOI as well as the draining lymphatic tissue was quantified to analyse the lymphatic draining of the vaccine components. Furthermore investigations detailing cytokine production and T-cell antigen specificity were undertaken to investigate the relationship between depot effect and the ability of the vaccine to induce an immune response. Our results suggest that cationic charge is an important factor for the retention of the liposomal component at the SOI, and a moderate to high (>50%) level of antigen adsorption to the cationic vesicle surface was required for efficient antigen retention in the same tissue. Furthermore, neutral liposomes expressing poor levels of antigen retention were limited in their ability to mediate long term (14 days) antigen presentation to circulating antigen specific T-cells and to induce the Th1 and Th17 arms of the immune system, as compared to antigen adsorbing cationic liposomes. The neutral liposomes did however induce the production of IL-5 at levels comparable to those induced by cationic liposomes, indicating that neutral liposomes can induce a weak Th2 response.

Increased TG2 expression can result in induction of transforming growth factor β1, causing increased synthesis and deposition of matrix proteins, which can be regulated by nitric oxide

In fibrotic conditions increases in TG2 activity has been linked to an increase in the deposition of extracellular matrix proteins. Using TG2 transfected Swiss 3T3 fibroblasts expressing TG2 under the control of the tetracycline-regulated inducible promoter, we demonstrate that induction of TG2 not only stimulates an increase in collagen and fibronectin deposition but also an increase in the expression of these proteins. Increased TG2 expression in these fibroblasts led to NF-kappaB activation, resulting in the increased expression of transforming growth factor (TGF) beta(1). In addition, cells overexpressing TG2 demonstrated an increase in biologically active TGFbeta(1) in the extracellular environment. A specific site-directed inhibitor of TG abolished the NF-kappaB and TGFbeta1 activation and the subsequent elevation in the synthesis and deposition of extracellular matrix proteins, confirming that this process depends on the induction of transglutaminase activity. Treatment of TG2-induced fibroblasts with nontoxic doses of nitric oxide donor S-nitroso-N-acetylpenicillamine resulted in decreased TG2 activity and apprehension of the inactive enzyme on the cell surface. This was paralleled by a reduction in activation of NF-kappaB and TGFbeta(1) production with a subsequent decrease in collagen expression and deposition. These findings support a role for NO in the regulation of TG2 function in the extracellular environment.

CFD analyses of fibre transport and fibre deposition at plunging jet conditions

The investigation of insulation debris generation, transport and sedimentation becomes important with regard to reactor safety research for PWR and BWR, when considering the long-term behaviour of emergency core cooling systems during all types of loss of coolant accidents. A joint research project on such questions is being performed in cooperation between the University of Applied Sciences Zittau/Görlitz (HSZG) and the Helmholtz-Zentrum Dresden-Rossendorf (HZDR). The project deals with the experimental investigation of particle transport phenomena in coolant flow and the development of CFD models for its description (see [10-12]). While the experiments are performed at the University at Zittau/Görlitz, the theoretical modelling efforts are concentrated in Rossendorf. In the current paper, the basic concepts for CFD modelling are described and feasibility studies are presented. The model capabilities are demonstrated via complex flow situations, where a plunging jet agitates insulation debris. © Carl Hanser Verlag, München.

β-Amyloid (Aβ) deposition in the medial temporal lobe of patients with dementia with Lewy bodies

The distribution and density of diffuse, primitive and classic β-amyloid (Aβ) deposits in the medial temporal lobe (MTL) was studied in cases of dementia with Lewy bodies (DLB) with and without associated Alzheimer's disease (AD) and 15 cases of sporadic AD. In the 'pure' DLB cases, virtually no Aβ deposits were observed in the CA regions of the hippocampus or dentate gyrus whereas deposits were distributed throughout the MTL in DLB/AD and AD cases. Densities of diffuse and primitive Aβ deposits were similar in AD and DLB/AD cases but density was significantly reduced in the 'pure' DLB cases. The density of the classic deposits was significantly reduced in DLB cases with or without associated AD compared with AD cases. These results suggest that Aβ deposition in the MTL in 'pure' DLB cases is similar to that of elderly non-demented patients while, with the exception of the classic deposits, Aβ deposition in DLB/AD cases is similar to that in cases of AD alone.

Beta-amyloid deposition in the medial temporal lobe in elderly non-demented brains and in Alzheimer's disease

The density of diffuse, primitive, classic and compact β-amyloid (β/A4) deposits was estimated in the medial temporal lobe in elderly non-demented brains and in Alzheimer's disease (AD). In the non-demented cases, β/A4 deposits were absent in the hippocampus but in 8/14 cases they were present in the adjacent cortical regions. Variation in β/A4 deposition in the non-demented cases was large and overlapped with that of the AD cases. The ratio of mature to diffuse β/A4 deposits was greater in the non-demented than in the AD cases. In both the non-demented cases and AD, the β/A4 deposits were clustered with, in many tissues, a regular distribution of clusters along the cortex parallel to the pia. However, the mean cluster size of the deposits in the cortex was greater in AD than in the non-demented cases. These results suggest that the spread of β/A4 pathology between the modular units of the cortex and into the hippocampus could be important factors in the development of AD.

β-Amyloid (Aβ) deposition in elderly non-demented patients and patients with Alzheimer's disease

β-amyloid (Aβ) deposition in the medial temporal lobe (MTL) was studied in elderly non-demented (ND) cases and in patients with Alzheimer's disease (AD). In AD, Aβ deposits were present throughout the MTL although density was less in the hippocampus than the adjacent cortical regions. In the ND cases, no Aβ deposits were recorded in 6 cases and in the remaining 8 cases, Aβ deposits were confined to the cortical regions adjacent to the hippocampus. The mean density of Aβ deposits in the cortical regions examined was greater in AD than in the ND cases but there was a significant overlap between the two groups. The ratio of mature to diffuse Aβ deposits was greater in the ND than the AD cases. In both patient groups, Aβ deposits formed clusters in the cortex and many tissues exhibited a regular distribution of clusters along the cortex parallel to the pia. The mean dimension of the Aβ clusters was greater in AD than in the ND cases. Hence, few aspects of Aβ deposition appeared to consistently separate AD from ND cases. However, the spread of Aβ pathology between modular units of the cortex and into regions of the hippocampus could be factors in the development of AD. © 1994.

Differences in β-amyloid ( β A4) deposition in human patients with Down's syndrome and sporadic Alzheimer's disease

The density of diffuse, primitive, classic and compact β-amyloid ( β A4) deposits was estimated in the hippocampus and adjacent gyri in human patients with Down's syndrome (DS) and sporadic Alzheimer's disease (AD). The objective of the study was to determine whether there were differences in β A4 deposition in DS and sporadic AD and whether these differences could be attributed to overexpression of the amyloid precursor gene (APP) in DS. Total β A4 deposit density was greater in DS than AD in all brain regions studied but the DS/AD density ratios varied between brain regions. In the majority of brain regions, the ratio of primitive to diffuse β A4 deposits was greater in DS but the ratio of classic to diffuse deposits was greater in AD. The data were consistent with the hypothesis that overexpression of the APP gene in DS may lead to increased β A4 deposition. However, local brain factors also appear to be important in β A4 deposition in DS. Overexpression of the APP gene may also be responsible for increased production of paired helical filaments (PHF) and result in enhanced formation of primitive β A4 deposits in DS. In addition, increased formation of classic deposits in AD suggests that factors necessary for the production of a compact amyloid core are enhanced in AD compared with DS. © 1994.

Is beta-amyloid (Abeta) deposition in the frontal cortex of patients with Alzheimer’s disease related to blood vessels?

The density of the diffuse, primitive and classic beta-amyloid (Abeta) deposits and the incidence of large and small diameter blood vessels was studied in the upper laminae of the frontal cortex of 10 patients with sporadic Alzheimer’s disease (AD). The data were analysed using the partial correlation coefficient to determine whether variations in the density of Abeta deposit subtypes along the cortex were related to blood vessels. Significant correlations between the density of the diffuse or primitive Abeta deposits and blood vessels were found in only a small number of patients. However, the classic Abeta deposits were positively correlated with the large blood vessels in all 10 patients, the correlations remaining when the effects of gyral location and mutual correlations between Abeta deposits were removed. These results suggest that the larger blood vessels are involved specifically in the formation of the classic Abeta deposits and are less important in the formation of the diffuse and primitive deposits.

A comparison of βamyloid (aβ deposition in the medial temporal lobe in late-onset sporadic Alzheimer's disease, and down's syndrome

The density of diffuse, primitive, classic and compact βamyloid (Aβ deposits was estimated in regions of the medial temporal lobe (MTL) in 15 cases of late-onset sporadic Alzheimer's disease (AD) and 12 cases of Down's syndrome (DS). A similar pattern of Aβ deposition was observed in the MTL in the AD and DS cases with a reduced density of deposits in the hippocampus compared with the adjacent cortical regions. Total Aβ deposit density was greater in DS than in AD in all brain regions examined. This could be attributable to overexpression of the amyloid precursor protein gene. The ratio of the primitive to the diffuse Aβ deposits was greater in DS than in AD which suggests that the formation of mature amyloid deposits is enhanced in DS. The diffuse deposits exhibited a parabolic and the primitive deposits an inverted parabolic response with age in the DS cases. This suggests either that the diffuse and primitive deposits are sequentially related or that there are alternate pathways of Aβ deposition. © 1995 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted.

Ocular compatibility of hydrogel contact lenses:deposition and clinical performance

Currently over 50 million people worldwide wear contact lenses, of which over 75% wear hydrogel lenses. Significant deposition occurs in approximately 80% of hydrogel lenses and many contact lens wearers cease wearing lenses due to problems associated with deposition. The contact lens field is not alone in encountering complications associated with interactions between the body and artificial devices. The widespread use of man-made materials to replace structures in the body has emphasised the importance of studies that examine the interactions between implantation materials and body tissues.This project used carefully controlled, randomized clinical studies to study the interactive effects of contact lens materials, care systems, replacement periods and patient differences. Of principal interest was the influence of these factors on material deposition and their subsequent impact on subjective performance. A range of novel and established analytical techniques were used to examine hydrogel lenses following carefully controlled clinical studies in which clinical performance was meticulously monitored. These studies established the inter-relationship between clinical performance and deposition to be evaluated. This project showed that significant differences exist between individuals in their ability to deposit hydrogel lenses, with approximately 20% of subjects displaying significant deposition irrespective of the lens material. Additionally, materials traditionally categorised together show markedly different spoilation characteristics, which are wholly attributable to their detailed chemical structure. For the first time the in vivo deposition kinetics of both protein and lipid in charged and uncharged polymers was demonstrated. In addition the importance of care systems in the deposition process was shown, clearly demonstrating the significance of the quality rather than the quantity of deposition in influencing subjective performance.

Development of a non-destructive fluorescence technique for analysis of contact lens deposition levels

The primary objective of this research has been to determine the potential of fluorescence spectroscopy as a method for analysis of surface deposition on contact lenses. In order to achieve this it was first necessary to ascertain whether fluorescence analysis would be able to detect and distinguish between protein and lipid deposited on a lens surface. In conjunction with this it was important to determine the specific excitation wavelengths at which these deposited species were detected with the greatest sensitivity. Experimental observations showed that an excitation wavelength of 360nm would detect lipid deposited on a lens surface, and an excitation wavelength of 280nm would detect and distinguish between protein and lipid deposited on a contact lens. It was also very important to determine whether clean unspoilt lenses showed significant levels of fluorescence themselves. Fluorescence spectra recorded from a variety of unworn contact lenses at excitation wavelengths of 360nm and 280nm indicated that most contact lens materials do not fluoresce themselves to any great extent. Following these initial experiments various clinically and laboratory based studies were performed using fluorescence spectroscopy as a method of analysing contact lens deposition levels. The clinically based studies enabled analysis of contact lenses with known wear backgrounds to be rapidly and individually analysed following discontinuation of wear. Deposition levels in the early stages of lens wear were determined for various lens materials. The effect of surfactant cleaning on deposition levels was also investigated. The laboratory based studies involved comparing some of the in vivo results with those of identical lenses that had been spoilt using an in vitro method. Finally, an examination of lysosyme migration into and out of stored ionic high water contact lenses was made.

Analysis of β-amyloid (Aβ) deposition in the temporal lobe in Alzheimer's disease using Fourier (spectral) analysis

To determine the spatial pattern of ß-amyloid (Aß) deposition throughout the temporal lobe in Alzheimer's disease (AD). Methods: Sections of the complete temporal lobe from six cases of sporadic AD were immunolabelled with antibody against Aß. Fourier (spectral) analysis was used to identify sinusoidal patterns in the fluctuation of Aß deposition in a direction parallel to the pia mater or alveus. Results: Significant sinusoidal fluctuations in density were evident in 81/99 (82%) analyses. In 64% of analyses, two frequency components were present with density peaks of Aß deposits repeating every 500–1000 µm and at distances greater than 1000 µm. In 25% of analyses, three or more frequency components were present. The estimated period or wavelength (number of sample units to complete one full cycle) of the first and second frequency components did not vary significantly between gyri of the temporal lobe, but there was evidence that the fluctuations of the classic deposits had longer periods than the diffuse and primitive deposits. Conclusions: (i) Aß deposits exhibit complex sinusoidal fluctuations in density in the temporal lobe in AD; (ii) fluctuations in Aß deposition may reflect the formation of Aß deposits in relation to the modular and vascular structure of the cortex; and (iii) Fourier analysis may be a useful statistical method for studying the patterns of Aß deposition both in AD and in transgenic models of disease.

A spatial pattern analysis of beta-amyloid (Abeta) deposition in the temporal lobe in Alzheimer's disease

To determine the factors influencing the distribution of -amyloid (Abeta) deposits in Alzheimer's disease (AD), the spatial patterns of the diffuse, primitive, and classic A deposits were studied from the superior temporal gyrus (STG) to sector CA4 of the hippocampus in six sporadic cases of the disease. In cortical gyri and in the CA sectors of the hippocampus, the Abeta deposits were distributed either in clusters 200-6400 microm in diameter that were regularly distributed parallel to the tissue boundary or in larger clusters greater than 6400 microm in diameter. In some regions, smaller clusters of Abeta deposits were aggregated into larger 'superclusters'. In many cortical gyri, the density of Abeta deposits was positively correlated with distance below the gyral crest. In the majority of regions, clusters of the diffuse, primitive, and classic deposits were not spatially correlated with each other. In two cases, double immunolabelled to reveal the Abeta deposits and blood vessels, the classic Abeta deposits were clustered around the larger diameter vessels. These results suggest a complex pattern of Abeta deposition in the temporal lobe in sporadic AD. A regular distribution of Abeta deposit clusters may reflect the degeneration of specific cortico-cortical and cortico-hippocampal pathways and the influence of the cerebral blood vessels. Large-scale clustering may reflect the aggregation of deposits in the depths of the sulci and the coalescence of smaller clusters.

The importance of the tissue transglutaminase-fibronectin heterocomplex in the RGD-independent cell adhesion and fibronectin matrix deposition

Tissue transglutaminase (TG2) has been reported as a wound response protein. Once over-expressed by cells under stress such as during wound healing or following tissue damage, TG2 can be secreted and deposited into extracellular matrix, where it forms a heterocomplex (TG-FN) with the abundant matrix protein fibronectin (FN). A further cellular response elicited after tissue damage is that of matrix remodelling leading to the release of the Arg-Gly-Asp (RGD) containing matrix fragments by matrix matelloproteinases (MMPs). These peptides are able to block the interaction between integrin cell surface receptors and ECM proteins, leading to the loss of cell adhesion and ultimately Anoikis. This study provides a mechanism for TG2, as a stress-induced matrix protein, in protecting the cells from the RGD-dependent loss of cell adhesion and rescuing the cells from Anoikis. Mouse fibroblasts were used as a major model for this study, including different types of cell surface receptor knockout mouse embryonic fibroblasts (MEFs) (such as syndecan-4, a5, ß1 or ß3 integrins). In addition specific syndecan-2 targetting siRNAs, ß1 integrin and a4ß1 integrin functional blocking antibodies, and a specific targeting peptide against a5ß1 integrin A5-1 were used to investigate the involvement of these receptors in the RGD-independent cell adhesion on TG-FN. Crucial for TG-FN to compensate the RGD-independent cell adhesion and actin cytoskeleton formation is the direct interaction between the heparan sulfate chains of syndecan-4 and TG2, which elicits the inside-out signalling of a5ß1 integrin and the intracellular activation of syndecan-2 by protein kinase C a (PKCa). By using specific inhibitors, a cell-permeable inhibiting peptide and the detection of the phosphorylation sites for protein kinases and/or the translocation of PKCa via Western blotting, the activation of PKCa, focal adhesion kinase (FAK), ERK1/2 and Rho kinase (ROCK) were confirmed as downstream signalling molecules. Importantly, this study also investigated the influence of TG-FN on matrix turnover and demonstrated that TG-FN can restore the RGD-independent FN deposition process via an a5ß1 integrin and syndecan-4/2 co-signalling pathway linked by PKCa in a transamidating-independent manner. These data provide a novel function for TG2 in wound healing and matrix turnover which is a key event in a number of both physiological and pathological processes.