Persistent mercury hot spot in Central Europe and Skalka Dam reservoir as a long-term mercury trap.

This study aimed to evaluate the relevance of the floodplain pollution sinks of the legacy mercury (Hg) hot spot in Kössein-Röslau river system (east Bavaria, Germany) for further mobilisation and fluvial transport of mercury in suspended particulate matter (SPM), as an important transport medium of Hg in aquatic systems. The channel belt fluvial erosion as the secondary pollution pathway was also considered. The hot spot has originated from the production of Hg compounds such as C2H5HgCN and C6H5HgCl in Chemical Factory Marktredwitz, and even more than 30 years after the factory abandonment, the Kössein and the Röslau rivers still export polluted fine grained SPM (median 25-35 µm) with mean annual concentrations of 17.4 mg/kg. SPM sampling was performed by floating samplers, supported by floodplain drill cores and by recent channel sediments manually collected along the polluted rivers further. Based on long-term monitoring data set from state enterprise Povodí Ohre, fish in the Skalka Reservoir have had Hg concentrations in their muscles up to 6 mg/kg for at least the last 14 years, exceeding the European maximal limit of 0.5 mg/Hg/kg. In addition, the Hg inventory in the Kössein-Röslau river stretches was therefore calculated; it produced an estimate of ca. 21 t Hg in a 22-km-long channel belt, prone to fluvial remobilisation during floods. Although a major portion of the fluvially transported Hg has yet been trapped by the Skalka Reservoir, the Hg content in the SPM exported farther downstream still varies between 2 and 10 mg/kg Hg. Due to the considerable Hg inventory in the Kössein-Röslau rivers, an improvement will not occur downstream unless specific measures target the secondary pollution mechanism(s).

Piezo1 Channels in Vascular Development and the Sensing of Shear Stress.

A critical point in mammalian development occurs before mid-embryogenesis when the heart starts to beat, pushing blood into the nascent endothelial lattice. This pushing force is a signal, detected by endothelial cells as a frictional force (shear stress) to trigger cellular changes that underlie the essential processes of vascular remodeling and expansion required for embryonic growth. The processes are complex and multifactorial and Piezo1 became a recognized player only 2years ago, 4years after Piezo1's initial discovery as a functional membrane protein. Piezo1 is now known to be critical in murine embryonic development just at the time when the pushing force is first detected by endothelial cells. Murine Piezo1 gene disruption in endothelial cells is embryonic lethal and mutations in human PIEZO1 associate with severe disease phenotype due to abnormal lymphatic vascular development. Piezo1 proteins coassemble to form calcium-permeable nonselective cationic channels, most likely as trimers. They are large proteins with little if any resemblance to other proteins or ion channel subunits. The channels appear to sense mechanical force directly, including the force imposed on endothelial cells by physiological shear stress. Here, we review current knowledge of Piezo1 in the vascular setting and discuss hypotheses about how it might serve its vascular functions and integrate with other mechanisms. Piezo1 is a new important player for investigators in this field and promises much as a basis for better understanding of vascular physiology and pathophysiology and perhaps also discovery of new therapies.

Epidermal transformation leads to increased perlecan synthesis with heparin-binding-growth-factor affinity.

Perlecan, a proteoglycan of basement membrane and extracellular matrices, has important roles in both normal biological and pathological processes. As a result of its ability to store and protect growth factors, perlecan may have crucial roles in tumour-cell growth and invasion. Since the biological functions of different types of glycosaminoglycan vary with cellular origin and structural modifications, we analysed the expression and biological functions of perlecan produced by a normal epidermal cell line (JB6) and its transformed counterpart (RT101). Expression of perlecan in tumorigenic cells was significantly increased in both mRNA and protein levels. JB6 perlecan was exclusively substituted with heparan sulphate, whereas that of RT101 contained some additional chondroitin sulphate. Detailed structural analysis of the heparan sulphate (HS) chains from perlecan of both cell types revealed that their overall sulphation and chain length were similar (approximately 60 kDa), but the HS chains of tumour-cell-derived perlecan were less sulphated. This resulted from reduced 2-O- and 6-O-sulphation, but not N-sulphation, and an increase in the proportion of unsulphated disaccharides. Despite this, the heparan sulphate of RT101- and JB6-derived perlecan bound fibroblast growth factor-1, -2, -4 and -7 and heparin-binding epidermal growth factor with similar affinity. Therefore abundant tumour-derived perlecan may support the angiogenic responses seen in vivo and be a key player in tumorigenesis.

The spacing of S-domains on HS glycosaminoglycans determines whether the chain is a substrate for intracellular heparanases.

Heparanases are mammalian endoglucuronidases that degrade heparan sulfate (HS) glycosaminoglycans to short 5-6 kDa pieces. In the Golgi, HS glycosaminoglycans are modified by a series of interdependent reactions which result in chains that have regions rich in N- and O-sulfate groups and iduronate residues (S-domains), separated by regions that are nearly devoid of sulfate. Structural analysis of the short HS chains produced by Chinese hamster ovary (CHO) cell heparanases indicate that the enzymes recognize differences in sulfate content between S-domains and unmodified sequences, and cleave the chain at junctions between these regions. To look more closely at whether the spacing of S-domains on the gly- cosaminoglycan influences its ability to be cleaved by heparanases, we examined the susceptibility of the HS chains synthesized by the proteoglycan synthesis mutant, pgsE-606. PGS:E-606 cells are deficient in the modification enzyme N-deacetylase/N-sulfotransferase I, and synthesize HS chains that have fewer N- and O-sulfate groups and iduronate residues compared to wild-type (Bame et al., (1991), J. Biol. Chem., 266, 10287). HS glycosaminoglycans were isolated from wild-type and pgsE-606 cells and separated into populations based on sulfate content. Compared to wild-type HS, which has 14 S-domains, pgsE-606 cells synthesize three HS species, 606-1, 606-2, and 606-3, with 1, 4, and 8 S-domains, respectively. The spacing of the S-domains on the pgsE-606 HS chains is similar to the spacing the modified sequences on wild-type HS, indicating that each mutant glycosaminoglycan is composed of wild-type-like sequences and sequences devoid of S-domains. When incubated with partially purified CHO heparanases, only the portion of the mutant HS chains that had S-domains were degraded. Structural analysis of the heparanase-products confirmed that both the number and the arrangement of S-domains on the HS glycosaminoglycan are important for heparanase susceptibility. The structure of the different pgsE-606 HS chains also suggests mechanisms for the placement of S-domains when the gly- cosaminoglycan is synthesized.

Heparan sulfate chains from glypican and syndecans bind the Hep II domain of fibronectin similarly despite minor structural differences.

Numerous functions of heparan sulfate proteoglycans are mediated through interactions between their heparan sulfate glycosaminoglycan chains and extracellular ligands. Ligand binding specificity for some molecules, including many growth factors, is determined by complex heparan sulfate fine structure, where highly sulfated, iduronate-rich domains alternate with N-acetylated domains. Syndecan-4, a cell surface heparan sulfate proteoglycan, has a distinct role in cell adhesion, suggesting its chains may differ from those of other cell surface proteoglycans. To determine whether the specific role of syndecan-4 correlates with a distinct heparan sulfate structure, we have analyzed heparan sulfate chains from the different surface proteoglycans of a single fibroblast strain and compared their ability to bind the Hep II domain of fibronectin, a ligand known to promote focal adhesion formation through syndecan-4. Despite distinct molecular masses of glypican and syndecan glycosaminoglycans and minor differences in disaccharide composition and sulfation pattern, the overall proportion and distribution of sulfated regions and the affinity for the Hep II domain were similar. Therefore, adhesion regulation requires core protein determinants of syndecan-4.

Heparan sulfate proteoglycans on the cell surface: versatile coordinators of cellular functions.

Heparan sulfate proteoglycans are complex molecules composed of a core protein with covalently attached glycosaminoglycan chains. While the protein part determines localization of the proteoglycan on the cell surfaces or in the extracellular matrix, the glycosaminoglycan component, heparan sulfate, mediates interactions with a variety of extracellular ligands such as growth factors and adhesion molecules. Through these interactions, heparan sulfate proteoglycans participate in many events during cell adhesion, migration, proliferation and differentiation. We are determining the multitude of proteoglycan functions, as their intricate roles in many pathways are revealed. They act as coreceptors for growth factors, participate in signalling during cell adhesion, modulate the activity of a broad range of molecules, and partake in many developmental and pathological processes, including tumorigenesis and wound repair. This review concentrates on biological roles of cell surface heparan sulfate proteoglycans, namely syndecans and glypicans, and outlines the progress achieved during the last decade in unraveling the molecular interactions behind proteoglycan functions.

Syndecan-4 binding to the high affinity heparin-binding domain of fibronectin drives focal adhesion formation in fibroblasts.

Cell adhesion to extracellular matrix involves signaling mechanisms which control attachment, spreading and the formation of focal adhesions and stress fibers. Fibronectin can provide sufficient signals for all three processes, even when protein synthesis is prevented by cycloheximide. Primary fibroblasts attach and spread following integrin ligation, but do not form focal adhesions unless treated with a heparin-binding fragment of fibronectin (HepII), a peptide from this domain, or phorbol esters to activate protein kinase C. Syndecan-4 heparan sulfate proteoglycan is a transmembrane component present together with integrins in focal adhesions. Syndecan-4 binds and activates protein kinase Calpha, whose activity is needed for focal adhesion formation. We now report that the glycosaminoglycan chains of syndecan-4 bind recombinant HepII and it is incorporated into forming focal adhesions.

Basic fibroblast growth factor does not prevent heparan sulphate proteoglycan catabolism in intact cells, but it alters the distribution of the glycosaminoglycan degradation products.

Heparan sulphate proteoglycans on cell surfaces have been shown to mediate the degradation or recycling of several ligands. Since the interaction with ligand may affect proteoglycan catabolism once the complex is internalized, this could alter the cellular pool of heparan sulphate chains, with possible consequences for heparan sulphate-mediated cellular processes. We have recently demonstrated that the specific binding of basic fibroblast growth factor (bFGF) to heparan sulphate chains prevents the glycosaminoglycan from being degraded by partially purified heparanases from Chinese hamster ovary (CHO) cells [Tumova and Bame (1997) J. Biol. Chem. 272, 9078-9085]. The present study examines the effect of bFGF on heparan sulphate catabolism in intact cells. The distribution and size of the heparan sulphate degradation products in CHO cells was analysed in the presence and absence of bFGF using pulse-chase protocols. Although heparan sulphate molecules and bFGF are internalized through the same pathway, even relatively high concentrations of the growth factor do not have any inhibitory effects on glycosaminoglycan degradation. However, the interaction with the growth factor alters the distribution of heparan sulphate-degradation products, presumably by preventing secretion of the short heparanase-derived species. Our findings show that most of the free and bFGF-bound heparan sulphate chains are destined for lysosomes, which would be consistent with a recent hypothesis that the primary role of proteoglycan-mediated internalization of the growth factor is to remove bFGF from its site of action at the cell surface. However, in the presence of bFGF, a fraction of intracellular, heparanase-degraded heparan sulphate chains is delivered to the nucleus, suggesting that the glycosaminoglycan accompanies the growth factor to the organelle. It may be important for bFGF activity that the growth factor is protected from proteolytic degradation by its interaction with heparan sulphate. This work demonstrates that the internalization of a ligand along with the proteoglycan can affect the sorting of heparan sulphate-degradation products in endosomes, and the ultimate destination of the short glycosaminoglycan. It also provides evidence that formation of heparan sulphate-ligand complexes may regulate the recycling and degradation of both ligands and heparan sulphate chains and, consequently, affect their biological activities.

Abeta(1-40) prevents heparanase-catalyzed degradation of heparan sulfate glycosaminoglycans and proteoglycans in vitro. A role for heparan sulfate proteoglycan turnover in Alzheimer's disease.

Alzheimer's disease is characterized by senile plaques composed of polymeric fibrils of beta amyloid (Abeta), a 39-42-amino acid peptide formed after proteolytic processing of the amyloid precursor protein (betaAPP). Heparan sulfate proteoglycans have been shown to colocalize with Abeta in Alzheimer's disease brain, and experimental evidence indicates that the interactions between the proteoglycan and the peptide are important for the promotion, deposition, and/or persistence of the senile plaques. Studies in rat brain indicated that both the core protein and the heparan sulfate glycosaminoglycan chains are required for amyloid fiber formation and deposition in vivo (Snow, A. D., Sekiguchi, R., Nochlin, D., Fraser, P., Kimata, K. , Mizutani, A., Arai, M., Schreier, W. A., and Morgan, D. G. (1994) Neuron 12, 219-234), suggesting that one mechanism to prevent the formation of Abeta-heparan sulfate proteoglycan complexes that lead to deposition of amyloid would be to degrade the proteoglycan. Normally, heparan sulfate proteoglycans are internalized and degraded to short glycosaminoglycans by intracellular heparanases. These reactions occur in the endosomal-lysosomal pathway, which is the same intracellular location where betaAPP is processed to Abeta. Using partially purified heparanase activities from Chinese hamster ovary cells we examined whether Abeta(1-40) affects the catabolism of Chinese hamster ovary heparan sulfate glycosaminoglycans and proteoglycans in vitro. Abeta(1-40) binds to both the long heparan sulfate glycosaminoglycans attached to core proteins and the short, heparanase-derived chains in a concentration-dependent and pH-dependent manner. When Abeta(1-40) is added to heparanase assays, it prevents the partially purified activities from releasing heparan sulfate chains from core proteins and degrading them to short glycosaminoglycans; however, a large molar excess of the peptide to heparan sulfate is required to see the effect. Our results suggest that normally the levels of Abeta in the endosomal pathway are not sufficient to interfere with heparanase activity in vivo. However, once the level of Abeta-peptides are elevated, as they are in Alzheimer's disease, they could interact with heparan sulfate proteoglycans and prevent their catabolism. This could promote the formation and deposition of amyloid, since the binding of Abeta to the proteoglycan species will predominate.

The interaction between basic fibroblast growth factor and heparan sulfate can prevent the in vitro degradation of the glycosaminoglycan by Chinese hamster ovary cell heparanases.

Heparan sulfate proteoglycans on Chinese hamster ovary (CHO) cell surfaces can bind and internalize basic fibroblast growth factor (bFGF). We have investigated whether this interaction affects heparan sulfate catabolism in vitro by measuring the ability of partially purified CHO heparanase activities to degrade 35S-labeled heparan sulfate glycosaminoglycans in the absence or presence of bFGF. Our studies show that the presence of the growth factor prevents partially purified heparanases from degrading the nascent 81-kDa chains to short 6-kDa products, whether the glycosaminoglycan is free in solution or covalently bound to core proteins. A 30-60 molar excess of the growth factor is required to inhibit completely chain degradation by heparanases, implying that multiple bFGF molecules must be bound to the glycosaminoglycan to prevent heparanase-catalyzed catabolism. This hypothesis is supported by protection studies indicating that nascent CHO heparan sulfate glycosaminoglycans have at least four to eight bFGF binding sites/chain. It does not appear, however, that the growth factor inhibits heparanase-catalyzed degradation of the glycosaminoglycan by binding to the sequence cleaved by the enzyme. Both the nascent and short chains bind bFGF with similar affinity (Kd values of 27.0 +/- 3.5 and 38.9 +/- 5.1 nM, respectively), indicating that heparanase activities do not destroy the bFGF binding sites. Rather, our results suggest that the growth factor interferes sterically with heparanase action by binding the heparan sulfate chain at a sequence next to the cleavage site or at a secondary site recognized by the enzyme.