Sub-cellular damage by copper in the cnidarian Zoanthus robustus.

Sessile organisms may experience chronic exposure to copper that is released into the marine environment from antifoulants and stormwater runoff. We have identified the site of damage caused by copper to the symbiotic cnidarian, Zoanthus robustus (Anthozoa, Hexacorallia). External changes to the zoanthids were apparent when compared with controls. The normally flexible bodies contracted and became rigid. Histological examination of the zoanthid tissue revealed that copper had caused sub-cellular changes to proteins within the extracellular matrix (ECM) of the tubular body. Collagen in the ECM and the internal septa increased in thickness to five and seven times that of controls respectively. The epithelium, which stained for elastin, was also twice as thick and tough to cut, but exposure to copper did not change the total amount of desmosine which is found only in elastin. We conclude that copper stimulated collagen synthesis in the ECM and also caused cross-linking of existing proteins. However, there was no expulsion of the symbiotic algae (Symbiodinium sp.) and no effect on algal pigments or respiration (44, 66 and 110 microg Cu L(-1)). A decrease in net photosynthesis was observed only at the highest copper concentration (156 microg Cu L(-1)). These results show that cnidarians may be more susceptible to damage by copper than their symbiotic algae.

Field-induced phase transitions and reversible field-induced inversion of chirality in tilted smectic phases of bent-core mesogens.

Three homologous achiral five-ring bent-core mesogens are presented where 4-chlororesorcinol is the central core and the aromatic rings are linked by ester groups. These compounds form smectic phases with a tilted arrangement of the molecules (tilt angle approximately 45 degrees). On cooling the isotropic liquid this phase adopts a fan-like texture which shows for two homologues at relatively high electric fields ( 25-35 V microm(-1)) an antiferroelectric electro-optical response based on the collective rotation of the molecules around their long axes. At lower temperature the application of a sufficiently high electric field leads to a continuous transition into a non-birefringent texture which exhibits randomly distributed domains of opposite handedness. These domains can be reversibly switched into a state of opposite chirality by reversal of the field polarity. This switching is bistable and shows a current response typical for a ferroelectric ground state. The possible mechanism of the field-induced phase transition, of the ferroelectric switching and of the field-induced inversion of the chirality is discussed on the base of XRD, 13C- and 1H-NMR investigations, dielectric and electro-optical measurements.

Decorin affects endothelial cells by Akt-dependent and -independent pathways.

Decorin, a small multifunctional proteoglycan, has been shown to be causally involved in the formation of capillary-like structures and a decrease in apoptosis. Here we investigated signal transduction pathways mediating effects of decorin on endothelial cells (ECs). Addition of decorin led to a fourfold increase in phosphorylation of Akt/protein kinase B on Thr307 and a l.4-fold increase on Ser473 after 10 min, but this phosphorylation could not be blocked by preincubation with Ly29400 (10 micro M). Six hours after the addition of decorin, the synthesis of p21 and p27, two inhibitors of cyclin-dependent kinases, started and increased up to 18 h, while synthesis of cyclin A peaked at 12 h and decreased after 24 h below base level. Induction of dominan-negative Akt by a replication-deficient adenovirus blocked p21 and cyclin A synthesis, but had no effect on p27. Dominant-negative Akt also blocked the antiapoptotic effect of decorin on ECs, but induction of dominant-positive Akt could not rescue the cells from apoptosis. Thus, the matrix proteoglycan decorin is a signaling molecule in ECs that affects cell survival by Akt-dependent and -independent pathways.

Four-helix bundle topology re-engineered: monomeric Rop protein variants with different loop arrangements.

We converted the small homodimeric four-helix bundle repressor of primer protein (Rop) into a monomeric four-helix bundle by introduction of connecting loops. Both left- and right-handed four-helix bundles were produced. The left-handed bundles were more stable and were used to introduce biologically interesting peptides in one of the loops.

Proteoglycans of the extracellular matrix and growth control.

Regulated cell growth results from the biological balance between soluble growth-regulating factors, their receptors and the elicited signal cascade on the one hand side and from extracellular macromolecular components and their interplay with membrane receptors on the other side. Proteoglycans have recently been recognized not only to play a part in providing shape and biomechanical strength of organs and tissues, but also to exhibit direct and indirect cell signalling properties. In this review, we discuss the direct growth-regulating role of proteoglycans with special emphasis on the lectican family and on the family of small proteoglycans with leucine-rich repeats (SLRPs). Indirect actions of proteoglycans by modulation of growth factor activities and growth factor distribution are exemplified by discussing the TGF-beta-binding properties of SLRPs and the interactions of core proteins of matrix proteoglycans with other growth factors. It is emphasized that the modulatory role of proteoglycans on cell proliferation cannot be separated from their participation in tissue organization in general, thereby explaining the diverse and sometimes contradictory reports on the effects of proteoglycans on cell proliferation and differentiation.

Experimental evidence for an achiral orthogonal biaxial smectic phase without in-plane order exhibiting antiferroelectric switching behavior.

We report unambiguous experimental evidence for an achiral orthogonal biaxial smectic-A phase which exhibits antiferroelectric switching behavior. The evidence is based on x-ray-diffraction measurements, texture observation, and the results of dielectric and electro-optical measurements.

The small proteoglycans decorin and biglycan in human articular cartilage of late-stage osteoarthritis.

OBJECTIVE: Disturbances in proteoglycan metabolism of hyaline cartilage play an essential role in the pathology of degenerative joint disease. We investigated the relation between transcript expression, protein synthesis and the ultrastructural localization of the matrix-organizing proteoglycans decorin and biglycan within intra- and extracellular compartments of late-stage osteoarthritic human articular cartilage. METHODS: Human cartilage samples of a macroscopically intact area, the adjoining area and an area of the main defect from knee joints of 10 patients with late stage osteoarthritis were investigated. In situ hybridization and immunogold histochemistry were carried out separately and in combination at the light and electron microscopic level. RESULTS: Ultrastructurally, three main chondrocyte types were identified. The highest levels of mRNA of decorin and biglycan were produced by elongated secretory type 2 cells, already known to synthesize type I collagen. Cells with high levels of mRNA also translated the corresponding proteins to be found in the extracellular compartment. The highest production rate of decorin and biglycan was seen in the tissue area adjoining the main defect. CONCLUSION: The results indicate that at late stages of osteoarthritis the levels of transcription and translation for decorin and biglycan are up-regulated, probably in an effort to compensate for the general proteoglycan loss, characteristic of this disease stage.

Decorin-mediated signal transduction in endothelial cells. Involvement of Akt/protein kinase B in up-regulation of p21(WAF1/CIP1) but not p27(KIP1).

Endothelial cells undergoing angiogenesis express decorin, a small multifunctional proteoglycan. We have shown that decorin is causally involved in the formation of capillary-like structures and a decrease in apoptosis in endothelial cells cultured in a collagen lattice. Here we investigate signal transduction pathways mediating the effects of decorin. Reverse transcription-polymerase chain reaction demonstrated that p21 and p27, two inhibitors of cyclin-dependent kinases, were up-regulated by decorin induction. Decorin also increased protein levels of p21 and caused its translocation into the nucleus. p21 synthesis started 6 h after decorin addition and reached a plateau after 18 h, while cyclin A, which was also induced, peaked at 12 h and declined below basal levels within 24 h. These effects were mediated by the Akt/protein kinase B pathway. Akt phosphorylation at Thr-308 increased 4-fold and at Ser-473 1.4-fold within 10 min after decorin addition. Overexpression of dominant negative Akt inhibited the decorin-mediated induction of p21 and cyclin A, but had no effect on p27. These results show that decorin is a signaling molecule in sprouting endothelial cells where it acts via different pathways, one of them involving Akt/protein kinase B.

Expression of decorin and biglycan in rat gastric tissue: effects of ulceration and basic fibroblast growth factor.

BACKGROUND: The small chondroitin/dermatan sulphate proteoglycans decorin and biglycan participate in organizing the network of collagen fibrils and interact with non-collagenous matrix proteins. In addition, via interactions with cytokines they are directly or indirectly involved in signalling, growth and cell differentiation. We aimed to analyse their expression in normal gastric tissue and during gastric ulcer healing. METHODS: Proteoglycan expression was studied by immunohistochemistry and in situ hybridization in acetic acid-induced gastric ulcers in rat during early phases and during chronic ulceration. The effects of treatment with an acid stable mutein of FGF-2 (bFGF) were also studied. RESULTS: In normal gastric tissue, both proteoglycans were most strongly expressed in the submucosal layer. However, some epithelial cells were positive for biglycan and, surprisingly, also for decorin. In the early phase after ulcer induction exclusively decorin became induced in the muscularis mucosae, while biglycan became detectable in this layer only after 2 weeks. There was no up-regulation of either proteoglycan in other layers, nor could an effect of FGF-2 treatment be seen. CONCLUSIONS: The expression of decorin could be observed for the first time in epithelial cells. Decorin, but not biglycan, appears as an early phase reactant in the muscularis mucosae in accordance with its putative role during angiogenesis and the prevention of apoptosis.

Matrix metalloproteinase expression by endothelial cells in collagen lattices changes during co-culture with fibroblasts and upon induction of decorin expression.

EA.hy 926 cells, a derivative of human umbilical vein endothelial cells, in the presence of fibroblasts show the phenomena of angiogenesis, express the proteoglycan decorin and escape apoptosis, when they are maintained in collagen lattices, while fibroblast-free cultures do not show these changes. Virus-mediated decorin expression can substitute for the presence of fibroblasts. Since the expression of matrix metalloproteinases (MMPs) is an essential step in the formation of capillaries, several MMPs and tissue inhibitors of metalloproteinases (TIMPs) were investigated. MMP-1, MMP-2, MMP-9, and the cell-associated MMP-14 were augmented on the protein level in the presence of fibroblasts. No effect was seen with respect to MMP-3, TIMP-1, and TIMP-2. Semiquantitative RT-PCRs of endothelial cells in co-culture revealed a 7-, 19-, and 11-fold increase for mRNAs of MMP-1, MMP-2, and MMP-14 after six days, respectively. Virus-mediated decorin expression also was accompanied by an up-regulation of these MMPs. The expression of MMP-1 mRNAs increased 5-fold after 2 days and gradually declined thereafter. In contrast, MMP-2 and MMP-14 showed a 7-fold and a 14-fold increase on day two which returned to basal levels within 24 h, indicating that the expression of MMP-1 is differentially regulated from MMP-2 and MMP-14. In spite of the upregulation of the proteases, an enhanced degradation of decorin was not observed. These results indicate that the expression of decorin is a sufficient signal in EA.hy 926 cells for a finely tuned induction of selected MMPs which are involved in angiogenesis whereas the up-regulation of MMPs does not lead to the degradation of the responsible proteoglycan.

Small proteoglycans in human diabetic nephropathy: discrepancy between glomerular expression and protein accumulation of decorin, biglycan, lumican, and fibromodulin.

Small leucine-rich proteoglycans (SLRPs), for example, decorin, biglycan, fibromodulin, and lumican, are extracellular matrix organizers and binding partners of TGF-b. Decorin is also involved in growth control and angiogenesis. Hence, these proteoglycans are likely of importance in the pathogenesis of diabetic glomerulosclerosis. In normal kidney, SLRPs were preferentially expressed in the tubulointerstitium. Weak expression occurred in the mesangial matrix. Biglycan was expressed by glomerular endothelial cells and, together with fibromodulin, by distal tubular cells and in collecting ducts. In all stages of diabetic nephropathy, there was a marked up-regulation of the proteoglycans in tubulointerstitium and glomeruli. Decorin and lumican became expressed in tubuli. However, in glomeruli, overexpression was not mirrored by local proteoglycan accumulation except in advanced nephropathy. In severe glomerulosclerosis, increased decorin concentrations were found in plasma and urine, and urinary TGF-b/decorin complexes could be demonstrated indirectly. The failure to detect an increased glomerular proteoglycan quantity during the development of nephropathy could be explained by assuming that they are secreted into the mesangial matrix, but cleared via the vasculature or the urinary tract, in part as complexes with TGF-b. They could thereby counteract the vicious circle being characterized by increased TGF-b production and increased matrix deposition in diabetic nephropathy.

Different usage of the glycosaminoglycan attachment sites of biglycan.

Biglycan is a member of the small leucine-rich proteoglycan family. Its core protein comprises two chondroitin/dermatan sulfate attachment sites on serine 42 and serine 47, respectively, which are the fifth and tenth amino acid residues, respectively, after removal of the prepro peptide. Because the regulation of glycosaminoglycan chain assembly is not fully understood and because of the in vivo existence of monoglycanated biglycan, mutant core proteins were stably expressed in human 293 and Chinese hamster ovary cells in which i) either one or both serine residues were converted into alanine or threonine residues, ii) the number of acidic amino acids N-terminal of the respective serine residues was altered, and iii) a hexapeptide was inserted between the mutated site 1 and the unaltered site 2. Labeling experiments with [(35)S]sulfate and [(35)S]methionine indicated that serine 42 was almost fully used as the glycosaminoglycan attachment site regardless of whether site 2 was available or not for chain assembly. In contrast, substitution of site 2 was greatly influenced by the presence or absence of serine 42, although additional mutations demonstrated a direct influence of the amino acid sequence between the two sites. When site 2 was not substituted with a glycosaminoglycan chain, there was also no assembly of the linkage region. These results indicate that xylosyltransferase is the rate-limiting enzyme in glycosaminoglycan chain assembly and implicate a cooperative effect on the xylosyl transfer to site 2 by xylosylation of site 1, which probably becomes manifest before the removal of the propeptide. It is shown additionally that biglycan expressed in 293 cells may still contain the propeptide sequence and may carry heparan sulfate chains as well as sulfated N-linked oligosaccharides.

Antisense inhibition of decorin expression in myoblasts decreases cell responsiveness to transforming growth factor beta and accelerates skeletal muscle differentiation.

Decorin is a member of the family of the small leucine-rich proteoglycans. In addition to its function as an extracellular matrix organizer, it has the ability to activate the epidermal growth factor receptor, and it forms complexes with various isoforms of transforming growth factor beta (TGF-beta). Decorin is expressed during skeletal muscle differentiation and is up-regulated in dystrophic muscle. In this study we investigated the role of decorin in TGF-beta-dependent inhibition of myogenesis. To probe the function of decorin during myogenesis, C(2)C(12) myoblasts were stably transfected with a plasmid expressing antisense decorin mRNA. The resulting inhibition of decorin expression led to the expression of myogenin, a master transcription factor for muscle differentiation, under growth conditions and accelerated skeletal muscle differentiation as determined by the expression of creatine kinase. In contrast myogenin expression was inhibited by adenovirally induced decorin expression or by adding exogenous decorin. Reduced synthesis of decorin resulted in a 7-fold decreased sensitivity to TGF-beta-mediated inhibition of myogenin expression. In contrast, adenovirally induced decorin expression in wild type cells resulted in a 5-fold increased sensitivity to TGF-beta-mediated inhibition of myogenin expression. Transfection studies with the TGF-beta-dependent promoter of the plasminogen activator inhibitor-1 coupled with luciferase revealed that the transducing receptors for TGF-beta1 and TGF-beta2 were involved in the different responses of wild type and antisense decorin myoblasts. These results demonstrate that a reduction of decorin expression or of decorin availability results in a decreased responsiveness to TGF-beta. These findings strongly suggest a new role for decorin during skeletal muscle terminal differentiation by activating TGF-beta-dependent signaling pathways.

Influence of decorin expression on transforming growth factor-beta-mediated collagen gel retraction and biglycan induction.

Complex formation of transforming growth factor-beta (TGF-beta) with the small proteoglycan decorin has been considered to inactivate the cytokine. However, neither the TGF-beta-mediated stimulation of the retraction of collagen lattices in culture nor the enhanced transcription of biglycan were influenced by an excess of native decorin in the culture medium. In contrast, when MG-63 osteosarcoma cells were transfected with sense- or antisense-decorin-cDNA, which led to an over- or under-expression of the proteoglycan, they responded to TGF-beta differently. An inverse correlation between decorin expression and the TGF-beta-mediated stimulation of collagen gel retraction and biglycan induction, respectively, was found. These results are best explained by assuming that decorin is not inactivating but sequestering TGF-beta in the extracellular matrix.

Lipoprotein lipase-mediated interactions of small proteoglycans and low-density lipoproteins.

According to numerous studies low-density lipoproteins (LDL) are supposed to interact with the glycosaminoglycan chain(s) of proteoglycans, e.g. with decorin and biglycan, which themselves are subject to receptor-mediated endocytosis. We tested, therefore, whether complexes of LDL and small proteoglycans can be endocytosed by either the LDL- or the small proteoglycan uptake mechanism. However, neither was the endocytosis of LDL significantly influenced by proteoglycans nor that of proteoglycans by LDL. This negative result could be explained by the observation that in vitro complex formation takes place only in buffers of low ionic strength. Under physiological conditions additional molecules may be necessary for complex stabilization. Lipoprotein lipase (LpL) which binds LDL was also able to interact with high affinity with decorin and its glycosaminoglycan-free core protein, both interactions being heparin-sensitive. Regardless of the presence or absence of LDL, LpL stimulated the endocytosis of decorin 1.5-fold, whereas LpL mediated a 4-fold stimulation of LDL uptake in the absence of decorin. No significant additional effect was seen in the presence of small concentrations of proteoglycans whereas in the presence of 1 microM decorin the endocytosis of [125I]LDL was reduced in normal as well as in LDL receptor-deficient fibroblasts. These observations could best be explained by assuming that LpL/LDL complexes are internalized upon binding to membrane-associated heparan sulphate and that small proteoglycans interfere with this process. It could not be ruled out, however, that a small proportion of the complexes is also taken up by the small proteoglycan receptor.

Small proteoglycans of normal adult human kidney: distinct expression patterns of decorin, biglycan, fibromodulin, and lumican.

BACKGROUND: Among the members of the small leucine-rich proteoglycan family, decorin, biglycan, and fibromodulin have been proposed to be potent modulators of transforming growth factor-beta (TGF-beta) activity, thereby playing an important role in the pathogenesis of fibrotic kidney diseases. Furthermore, decorin expression influences the expression of p21WAF1/CIP1, which has been related to kidney hypertrophy and hyperplasia. However, none of the members of this proteoglycan family have been investigated in normal adult human kidney cortex, thus making it impossible to correlate disease-mediated alterations of their expression with the normal situation in vivo. METHODS: The chondroitin/dermatan sulfate proteoglycans, decorin and biglycan, and the keratan sulfate proteoglycans, fibromodulin and lumican, were investigated in normal human adult renal cortex by immunohistochemistry on the light and electron microscopic level and by in situ hybridization. Northern blot and reverse transcription-polymerase chain reaction (RT-PCR) methods were used to get an estimate of their expression in isolated glomeruli. Decorin excretion with the urine was measured by Western blotting. RESULTS: Two bands of decorin and a single band of biglycan mRNA were identified in Northern blots of isolated glomeruli. Amplification by RT-PCR was required to detect the signals for fibromodulin and lumican. All four proteoglycans were preferentially expressed in the renal interstitium with accumulations around tubules. Weak expression was found in the mesangial matrix. Biglycan was expressed by glomerular endothelial cells and, together with fibromodulin, was synthesized and deposited in distal tubular cells and collecting ducts. Immunogold labeling indicated the presence of the proteoglycans in the glomerular basement membrane, which was interpreted as a result of glomerular filtration. Indirect evidence suggested tubular reuptake of decorin after glomerular filtration. CONCLUSION: The data indicate that the different cells of the adult human kidney are characterized by a distinct expression pattern of the four small proteoglycans. It is suggested that these proteoglycans may have distinct pathophysiological roles depending upon whether they are expressed by mesangial cells, endothelial cells, epithelial cells, or cells of the tubulointerstitium.

Alternative exon usage of rat septins.

Septins represent a family of phylogenetically conserved proteins required for cytokinesis. Their presence in pre- and postsynaptic neuronal membranes suggests a general function as scaffolds for membrane reorganization. The transcriptional regulation of all septins examined so far is complex, resulting in alternatively spliced variants. We focus here on the rat homologue of the gene for the human septin MSF, a truncated form of which, designated eseptin, had been described previously. It will be shown here that there is an alternative usage of the first exon by two forms, named exon r1a and r1b, respectively. Exon r1a, but not exon r1b, contains a part of the coding sequence while the start of translation for the remaining coding sequence resides in the second exon. The complete genomic organization was resolved and data on the temporal and spatial expression of this septins are presented.

Synthesis of proteoglycans is augmented in dystrophic mdx mouse skeletal muscle.

Mdx mice uniquely recover from degenerative dystrophic lesions through an intense myoproliferative response. The onset and progression of this process are controlled by a complex set of interactions between myoblasts and their environment. The presence of the extracellular matrix is essential for normal myogenesis. Proteoglycans are abundant components of the extracellular matrix. The synthesis of proteoglycans in mdx mice during skeletal muscle regeneration was evaluated. Incorporation of radioactive sulfate demonstrated a significant increase in the synthesis of several types of proteoglycans in mdx animals compared to age-matched controls. The size and charge of proteoglycans synthesized by the mdx mice remained unchanged. In particular, one of the up-regulated proteoglycans, the small chondroitin/dermatan sulfate proteoglycan decorin which is known to bind and to sequester transforming growth factor-beta, was investigated. Immunocytolocalization and in situ hybridization studies showed that decorin mainly accumulated in the endomysium, i.e. around individual skeletal muscle fibers from M. tibialis anterior and diaphragm.

Interaction of thrombospondin-1 and heparan sulfate from endothelial cells. Structural requirements of heparan sulfate.

Cell surface-associated heparan sulfate proteoglycans, predominantly perlecan, are involved in the process of binding and endocytosis of thrombospondin-1 (TSP-1) by vascular endothelial cells. To investigate the structural properties of heparan sulfate (HS) side chains that mediate this interaction, the proteoglycans were isolated from porcine endothelial cells and HS chains obtained thereof by beta-elimination. To characterize the structural composition of the HS chains and to identify the TSP-1-binding sequences, HS was disintegrated by specific chemical and enzymatic treatments. Cell layer-derived HS chains revealed the typical structural heterogeneity with domains of non-contiguously arranged highly sulfated disaccharides separated by extended sequences containing predominantly N-acetylated sequences of low sulfation. Affinity chromatography on immobilized TSP-1 demonstrated that nearly all intact HS chains possessed binding affinity, whereas after heparinase III treatment only a small proportion of oligosaccharides were bound with similar affinity to the column. Size fractioning of the bound and unbound oligosaccharides revealed that only a specific portion of deca- to tetradecasaccharides possessed TSP-1-binding affinity. The binding fraction contained over 40% di- and trisulfated disaccharide units and was enriched in the content of the trisulfated 2-O-sulfated L-iduronic acid-N-sulfated-6-O-sulfated glucosamine disaccharide unit. Comparison with the disaccharide composition of the intact HS chains and competition experiments with modified heparin species indicated the specific importance of N- and 6-O-sulfated glucosamine residues for binding. Further depolymerization of the binding oligosaccharides revealed that the glucosamine residues within the TSP-1-binding sequences are not continuously N-sulfated. The present findings implicate specific structural properties for the HS domain involved in TSP-1 binding and indicate that they are distinct from the binding sequence described for basic fibroblast growth factor, another HS ligand and a potential antagonist of TSP-1.

Decorin endocytosis: structural features of heparin and heparan sulphate oligosaccharides interfering with receptor binding and endocytosis.

Receptor-mediated endocytosis of decorin depends on its core-protein-mediated interaction with a 51 kDa membrane protein, which, in addition to its core-protein-binding site, carries a binding site for glycosaminoglycan chains. Membrane-associated heparan sulphate as well as heparin are known to have an inhibitory effect on decorin endocytosis by cultured skin fibroblasts. In this study, structural features of both glycosaminoglycans required for binding to the 51 kDa protein and for inhibiting decorin endocytosis, were investigated. Upon digestion of [(3)H]glucosamine-labelled heparan sulphate with heparinase III, dodeca- and higher saccharides were able to interact with the receptor protein. In comparison with unbound fragments of the same size, bound fragments were enriched in N-sulphated disaccharides carrying one or two sulphate ester groups. Using heparinase III-generated fragments from [(35)S]sulphate-labelled heparan sulphate chains, binding of fragments as small as octasaccharides could be detected. Competition experiments between dermatan sulphate and chemically modified heparin revealed that N- and 6-O-sulphation of glucosamine residues are important structural elements for binding to the receptor, whereas iduronate-2-O-sulphate groups contribute to binding only to a limited extent. However, with respect to the inhibition of decorin endocytosis, 2-O-desulphation had a quantitatively similar effect to 6-O-desulphation. Furthermore, for maximal inhibition of decorin endocytosis, longer fragments were required than for binding to the receptor. Thus, it appears that heparin/heparan sulphate has to interact with additional component(s) for effective inhibition of decorin uptake.