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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., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

A series of biaxial V-shaped, shape-persistent molecules has been synthesised by stepwise coupling of phenylene ethynylene arms to an oxadiazole bending unit. Studies of their thermotropic nematic phases point to phase biaxiality.

Human skin fibroblasts efficiently internalize the matrikine decorin by receptor-mediated endocytosis, however, very little is known about its intracellular trafficking routes up to lysosomal degradation. In an in vitro system measuring uptake and degradation of [(35)S]sulfate-labeled decorin, endocytosis was blocked by 46% when clathrin assembly/disassembly was inhibited using chlorpromazine. Pharmacological inhibition of EGF receptor signaling caused 34% reduction of decorin uptake, whereas inhibition of the IGF receptor had no effect. Using confocal immunofluorescence microscopy, we determined that only about 5-10% of internalized decorin colocalized with the EGFR. Thus, uptake depends on EGFR signaling rather than trafficking along the same pathway. Decorin passes through early endosomes towards trafficking to lysosomes, since more than 50% of decorin colocalized with EEA1. Moreover, inhibition of endosomal fusion by wortmannin caused a profound inhibition of decorin endocytosis. Overexpression of the clathrin-binding Hrs protein, which has previously been shown to inibit EGFR degradation blocked the degradation of decorin. Cholesterol depletion by filipin inhibited uptake of decorin by 34%, however, nearly no intracellular colocalization was found between decorin and caveolin-1. The combined use of filipin and chlorpromazine had an additive inhibitory effect on decorin endocytosis. Moreover, chlorpromazine diverted decorin from the chlorpromazine-sensitive pathway to an alternative uptake route. The CD44/hyaluronan pathway was excluded as an endocytic route for decorin. Our observations indicate that decorin is taken up by more than one endocytic pathway. Of note, lipid-raft-dependent EGFR signaling modulates decorin uptake, suggesting the presence of a potential feedback regulation mechanism for desensitization of signaling events mediated by decorin.

A combination of negative ion nano-electrospray ionization Fourier-transform ion cyclotron resonance and quadrupole time-of-flight mass spectrometry was applied to analysis of oversulfation in glycosaminoglycan oligosaccharides of the chondroitin sulfate type from bovine aorta. Taking advantage of the high-resolution and high mass accuracy provided by the FT-ICR instrument, a direct compositional assignment of all species present in the mixture can be obtained. An oligosaccharide fraction containing mainly hexasaccharides exhibited different levels of sulfation, indicated by the presence of species with regular sulfation pattern as well as oversulfated oligosaccharides with one additional sulfate group. Oversulfation can be directly identified from the high-resolution/high mass accuracy FT-ICR mass spectra according to their specific isotopic fine structure. Location of sulfate groups was analyzed by Q-TOF MS and low-energy CID MS/MS. Tetrasulfated hexasaccharides were analyzed by use of collision-induced dissociation at variable collision energy for an unambiguous assignment of the attachment site of the sulfate groups by minimizing unspecific neutral losses. Cleavage of glycosidic bonds gave rise to B- and C-type ions and their respective complementary Y- and Z-type fragment ions.

A novel class of bent-core molecules with oligo(siloxane) or carbosilane units at both ends was synthesized and the self-organization of these molecules was investigated by polarizing microscopy, DSC, X-ray scattering, dielectric and electrooptical methods. Depending on the size of the silicon-containing segments, smectic and columnar liquid crystalline phases are formed. Most smectic phases are low birefringent and composed of macroscopic domains of opposite handedness (dark conglomerate phases). The switching process in these smectic phases is surface stabilized ferroelectric and, depending on the conditions, two distinct slow relaxation processes to nonpolar structures were observed. It is proposed that the smectic phases are built up by chiral and polar SmCsPF layer stacks which are separated by anticlinic interfaces. If the size of these layer stacks is sufficiently large a coupling to the substrate surfaces takes place and ferroelectric switching is observed. It is also suggested that the sponge-like layer distortion, occurring in the low birefringent mesophases, is due to an escape from the local polar order within these SmCsPF layer stacks. For compounds with larger silylated units a steric frustration arises, which leads to layer modulation (columnar ribbon phases) and this is associated with a transition from ferroelectric to antiferroelectric switching. All compounds show a switching of the molecules around the long axis which reverses the layer chirality.

Three mesogenic dimers have been synthesized in which a five-ring bent-core moiety is connected with different calamitic units flexible spacers. The mesophase behavior of the dimers have been investigated by polarizing microscopy, differential scanning calorimetry, X-ray diffraction on oriented samples and by dielectric and electro-optical measurements. We found that two dimers exhibit a dimorphism columnar-nematic whereas the third one forms a columnar phase only. On the basis of the X-ray data a possible structure model of one of the columnar phases is proposed. The nematic phase exhibits unusual properties. A smectic-like texture can be induced by applying an electric field, which is unknown for nematic phases formed by rod-like 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.

The influence of the direction of ester linkage groups on the structural and electronic properties of five-ring banana-shaped molecules with a central 1,3-phenylene unit has been investigated including hexyloxy and dodecyloxy terminal chains. DFT studies on the B3LYP/6-31G(d) level were performed on the conformational behaviour of the ten isomers in a systematic way. The one- and two-fold potential energy scans show that the flexibility of the wings significantly depends on the orientation of the carboxyl linkage groups. Moreover, the different directions of the carboxyl groups between the aromatic rings cause remarkable changes on the dipole moment and its components of the molecules. These findings are supported by investigations on the global charge pattern of the molecules calculated from electrostatic potential group charges. The bending angle alpha obtained from a simple model for the five-ring bent-core molecules is a characteristic structural parameter which can be correlated with experimental findings. Calculations on the bent-core molecules in an external electric dipole field related to the direction of their polar axis show remarkable effects with respect to the flexibility and polarity of the isomers. First molecular dynamics simulations on the banana-shaped molecules were carried out within the AMBER 7 package. The trajectories of relevant structural parameters support the findings of the DFT studies. The results concerning the structure and polarity revealed from the DFT and MD calculations of the ten isomers can be correlated with data from dielectric measurements and mesophase properties.

The small leucine-rich proteoglycan (SLRP) decorin is efficiently internalized by a variety of cultured cells. A 51-kDa protein has previously been described as a receptor mediating endocytosis of decorin and of the structurally related SLRP biglycan. Recent findings suggest that endocytosis of SLRPs may also be mediated by additional receptors. The class-A scavenger receptor, the endocytic mannose receptor, the epidermal growth factor receptor, and insulin-like growth factor-I receptor have emerged as candidates. We used a combined approach of immunoprecipitation and photoactivated cross-linking to identify endocytosis receptors for decorin in human skin fibroblasts. Decorin was purified by HPLC-DEAE-ion exchange chromatography from the secretions of human skin fibroblasts under nondenaturing conditions. Confocal immunofluorescence microscopy revealed that both biotinylated decorin and decorin conjugated to the heterobifunctional cross-linker sulfosuccinimidyl 2-(p-azidosalicylamido)ethyl-1-3'-dithiopropionate (SASD) were endocytosed with equal efficiency. SASD-conjugated decorin was added to [35S]-methionine-labeled fibroblasts and cross-linked intracellularly to receptor molecules by photoactivation on endocytic uptake. Cross-linked decorin-receptor complexes were purified from the extracts of trypsin-treated fibroblasts by anion exchange chromatography and immunoprecipitation with a decorin-specific antiserum. Analysis by 2D electrophoresis and autoradiography revealed that decorin was specifically cross-linked to a protein of 110 kDa, which exhibited an isoelectric point of 5.5. In a second approach, unlabeled fibroblasts were subjected to decorin endocytosis and photoactivated cross-linking followed by Western blotting of DEAE-purified cell extracts. A shift of biotinylated decorin immunoreactivity from 165 kDa (decorin-receptor complex) to 54 kDa (SASD-conjugated biotinylated decorin) was noted on reductive cleavage of the cross-linker, representing a difference in molecular weight of approximately 110 kDa. The identification of a 110-kDa protein as a novel endocytosis receptor for decorin provides further support for the emerging concept of a redundancy of receptor molecules in the endocytosis of SLRP.

In vivo cells exist in a three-dimensional environment generated and maintained by multiple cell-cell and cell-matrix interactions. Proteoglycans, like decorin, affect these complex interactions. Thus, we sought to investigate the role of decorin in a three-dimensional environment where the matrix was generated over time by decorin-deficient fibroblasts in the presence of L-ascorbic acid 2-phosphate. The cells were viable and proliferated in response to FGF2. Decorin was incorporated in the matrix and caused a approximately 2 nm shift in the average diameter of the collagen fibrils, and the range and distribution of the fibrils became narrower and more uniform. Although there were no appreciable changes in collagen composition, we found that exogenous decorin induced the de novo synthesis of collagen I and V and cross-linked beta(I). In the early phases of the three-dimensional culture, decorin reduced apoptosis. However, following the establishment of a three-dimensional matrix, the cells did not require decorin for their survival.

Linear dielectric relaxation studies performed on two isotropic liquids composed of the molecules of the same moment of inertia and a quite different polarity: C10H21-O-Ph-COO-Ph-CN (the dipole moment of about 5 D) and C10H21-O-Ph-OOC-Ph-CN (2.5 D) showed that, at given temperature, the relaxation times corresponding to the rotation around the short axis of the two kinds of molecules coincide to each other, regardless the polarity of the molecules and their abilities to accomplish dipolar aggregation. The studies allow one to estimate the lifetime of the intermolecular aggregates due to the dipolar interactions in liquids as no longer than 0.1 ns.

The small dermatan sulfate proteoglycan decorin is involved in the regulation of collagen fibrillogenesis, cell adhesion and migration, and growth factor signaling. In a progeroid patient carrying two point mutations in beta4 galactosyltransferase I (beta4GalT-7) only 50% of the decorin core protein molecules are substituted with glycosaminoglycan chains. We expressed decorin, as well as wild-type and mutant alleles of beta4GalT-7 in galactosyltransferase-deficient CHO618 cells. Decorin was less efficiently substituted with glycosaminoglycan chains upon expression of beta4GalT-7(186D) compared to beta4GalT-7-expressing cells. Decorin from beta4GalT-7-expressing cells displayed increased molecular heterogeneity. Decorin glycosaminoglycan chains were completely susceptible to chondroitinase ABC treatment. Cells expressing beta4GalT-7(206P) did not synthesize the proteoglycanform of decorin. Thus, the beta4GalT-7 mutations directly affect the molecular phenotype of decorin observed in a patient with the progeroid form of Ehlers-Danlos syndrome, which may be a major mechanistic cause for the skin and wound healing defects observed in this patient.

Bisdermican (PG760) is a large, heterodimeric, dermatan sulfate proteoglycan found in selected basement membranes, smooth muscle cell layers, and different extracellular matrices. Age-dependent and developmentally regulated alterations in glycosaminoglycan structure and quantity have been shown to be functionally relevant for a number of physiological and pathological processes. Bisdermican was purified from human skin fibroblast cultures of different age and confluency. Following beta-elimination, glycosaminoglycan chains were analyzed by Sephacryl-S-300 chromatography. Glycosaminoglycan chains of Bisdermican from infantile fibroblasts had a molecular weight of 19 kDa, whereas the glycosaminoglycan chain of the large Bisdermican subunit purified from confluent fetal fibroblast secretions was slightly larger (Mr = 24 kDa). Bisdermican derived from subconfluent cultures of fetal fibroblasts displayed the largest glycosaminoglycan chains with a molecular weight of 31.5 kDa for the large subunit, and a molecular weight of 22 kDa for the small subunit. Thus, Bisdermican displays a molecular polymorphism that is related to its chronological age and proliferative state.

Small leucine-rich proteoglycans play important roles in the organization of the extracellular matrix as well as for the regulation of cell behavior; two biological processes that are essential for angiogenesis. We investigated consequences of the targeted ablation of decorin (DCN), biglycan (BGN) and fibromodulin (FMOD) genes on inflammation-induced angiogenesis in the cornea. In wild-type mice, DCN was localized exclusively to the corneal stroma, while FMOD and BGN were more prominently expressed in epithelial cells. Endothelial cells from limbus blood vessels expressed BGN and FMOD, but no DCN. However, after induction of angiogenesis by chemical cauterization, DCN was expressed in the newly formed capillaries, together with BGN and FMOD. Notably, in DCN-deficient mice, the growth of vessels was significantly diminished, whereas it did not significantly change in FMOD- or BGN-deficient animals. Moreover, blood vessels of DCN-deficient mice exhibited a similar expression level of BGN as control mice, while FMOD was increased on day 3 after injury. These results indicate that DCN, in addition to its effects on fibrillogenesis, plays a regulatory role in angiogenesis and that FMOD in endothelial cells may be able to partially substitute for DCN., (Copyright 2004 S. Karger AG, Basel.)

The small leucine-rich proteoglycan decorin can bind via its core protein to different types of collagens such as type I and type VI. To test whether decorin can act as a bridging molecule between these collagens, the binding properties of wild-type decorin, two full-length decorin species with single amino acid substitutions (DCN E180K, DCN E180Q), which previously showed reduced binding to collagen type I fibrils, and a truncated form of decorin (DCN Q153) to the these collagens were investigated. In a solid phase assay dissociation constants for wild-type decorin bound to methylated, therefore monomeric, triple helical type I collagen were in the order of 10(-10) m, while dissociation constants for fibrillar type I collagen were approximately 10(-9) m. The dissociation constant for type VI was approximately 10(-7) m. Using real-time analysis for a more detailed investigation DCN E180Q and DCN E180K exhibited lower association and higher dissociation constants to type I collagen, compared to wild-type decorin, deviating by at least one order of magnitude. In contrast, the affinities of these mutants to type VI collagen were 10 times higher than the affinity of wild-type decorin (K(D) approximately 10(-8) m). Further investigations verified that complexes of type VI collagen and decorin bound type I collagen and that the affinity of collagen type VI to type I was increased by the presence of decorin. These data show that decorin not only can regulate collagen fibril formation but that it also can act as an intermediary between type I and type VI collagen and that these two types of collagen interact via different binding sites.

A novel approach in glycosaminoglycomics, based on sheathless on-line capillary electrophoresis/nanoelectrospray ionization-quadrupole time of flight-mass spectrometry (CE/nanoESI-QTOF-MS) and tandem MS of extended chondroitin sulfate/dermatan (CS/DS) oligosaccharide chains is described. The methodology required the construction of a new sheathless CE/nanoESI-QTOF-MS configuration, its implementation and optimization for the high sensitivity analysis of CS/DS oligosaccharide mixtures from conditioned culture medium of decorin transfected human embryonic kidney (HEK) 293 cells. Under newly established sheathless on-line CE/(-)nanoESI conditions for glycosaminoglycan (GAG) ionization and MS detection, single CS/DS oligosaccharide components of extended chain length and increased sulfation degree were identified. Molecular ions corresponding to species carrying 5 and 6 negative charges could be generated for large GAG oligosaccharide species in the negative ion nanoESI-MS. The optimized on-line conditions enabled the detection of molecular ions assigned to oversulfated tetradeca-, octadeca-, and eicosasaccharide CS/DS molecules, which represent the category of largest sulfated GAG-derived oligosaccharides evidenced by CE/ESI-MS. By on-line CE/ESI tandem MS in data-dependent acquisition mode the oversulfated eicosasaccharide species could be sequenced and the localization of the additional sulfate group along the chain could be determined.

Decorin, a small multifunctional proteoglycan, is expressed by sprouting endothelial cells (ECs) during inflammation-induced angiogenesis in vivo and by human ECs co-cultured with fibroblasts in a collagen lattice. To investigate how decorin is induced, human EA.hy 926 ECs and/or human umbilical vein ECs were treated with interleukin (IL)-10 and IL-6. Both treatments induced decorin mRNA in human ECs. IL-6 and IL-10 led to a dose-dependent mRNA increase with a maximum at 10 and 50 ng/ml, respectively. The combination of both interleukins together had a stronger effect than one alone. Immunostaining demonstrated that both interleukins caused decorin synthesis in ECs and the formation of capillary-like structures in a collagen lattice. However, immunoprecipitations of interleukin-treated ECs cultured on plastic were negative. Only interleukin-stimulated ECs grown on a collagen type I matrix or growth factor-reduced Matrigel were able to synthesize the proteoglycan. Acid-soluble collagen type I did not support decorin protein synthesis. The addition of antibodies to alpha(1) or alpha(2) integrins or the alpha(2) integrin inhibitor rhodocetin led to an inhibition of synthesis. These data show that IL-10 and IL-6 induce decorin mRNA transcription, but additional signals from the extracellular matrix are necessary for its translation.

Objective: Previous studies have shown that decorin and biglycan account for over 70% of the proteoglycans (PGs) synthesized by human peritoneal mesothelial cells (HPMCs). Since these PGs are involved in the control of cell growth, cell differentiation, and matrix assembly, we investigated their turnover in cultured HPMCs., Methods: Confluent HPMCs were metabolically labeled with [35S]-sulfate and the labeled products isolated from the cell medium and the cell layer characterized by sensitivity to bacterial eliminases. Experiments were undertaken with exogenous labeled decorin, and its metabolic state was studied., Results: In a 24-hour labeling period, 75% of the newly synthesized chondroitin sulfate/dermatan sulfate (CS/DS) PGs appeared in the culture medium, the majority of which (90%) was decorin. In the cell layer, protein-free glycosaminoglycan (GAG) chains accounted for 21% of the total CS/DS at 24 hours and exhibited constant specific activity at 12-16 hours. The latter material was turned over with a half-life of approximately 2.5 hours. Exogenous decorin underwent receptor-mediated endocytosis and subsequent intracellular degradation. Uptake but not degradation could be inhibited by heparin., Conclusions: HPMCs are distinguished by a rapid turnover of decorin. A characteristic metabolic feature is the existence of a large intracellular pool of protein-free DS-GAGs. Understanding the control of decorin turnover in HPMCs might lead to delineation of its potential role in both the physiology and pathophysiology of the membrane in PD patients.

Mesogenic compounds belonging to the two well-known -cyanophenyl, and -isothiocyanatophenyl homologous series, which distinctly differ in the molecular polarity (-C identical with N, 5D; -N=C=S, 2.5D), show an essential difference in the pretransitional dielectric behavior in the vicinity of the isotropic to nematic (I-N) phase transition. Taking into account the results presented in Phys. Rev. E 67, 041705 (2003), the features of the I-N transition observed for the less polar mesogens are characteristic for the first order phase transition, whereas in the case of the strongly polar ones, the I-N transition is undoubtedly close to the second order.

The small leucine-rich proteoglycan biglycan (BGN) is abundantly expressed in mesenchymal tissues. Its expression level is related to the phenotypic differentiation of cells. A dysregulation in BGN expression occurs under several pathological conditions, including glomerulonephritis, mesothelioma, pancreatic cancer and a mouse model of osteoporosis. Since the extracellular concentration of BGN is regulated both by secretion and endocytosis, we performed mechanistic studies on BGN endocytosis in human skin fibroblasts in vitro, using inhibitors of different endocytic routes. Chlorpromazine, an inhibitor of the clathrin-coated pit-pathway reduced endocytosis of BGN in human skin fibroblasts by 40%, and decreased degradation of BGN by 66% Filipin, an inhibitor of the caveolae pathway, and Tyrphostin AG 1478, a specific inhibitor of EGF-receptor phosphorylation that partially inhibits endocytosis of the structurally related proteoglycan decorin, had no influence on BGN internalization and degradation. Our data indicates that the classical clathrin-mediated endocytic pathway is a major route for the internalization of BGN. Based on the differential susceptibility to pharmacological inhibition, it appears that BGN endocytosis seems to be at least in part mechanistically different from decorin uptake.

Objective: Endothelin-1 concentrations are increased in patients with increased mean pulmonary arterial pressure, pulmonary blood flow, and pulmonary vascular resistance. However, endothelin-1 concentrations have not been well characterized in patients with congenital heart disease and normal pulmonary vascular resistance. In particular, it is unclear whether pressure or flow is the key regulator of endothelin- 1 in this setting. We tested the hypothesis that pulmonary blood pressure and not flow is associated with net endothelin-1 production in patients with congenital heart disease and normal pulmonary vascular resistance., Methods: With a commercially available immunoassay, we measured endothelin-1 concentrations in pulmonary arterial and pulmonary venous plasma of 56 consecutive patients with congenital heart disease and pulmonary vascular resistance less than 2 U. m(2) undergoing cardiac catheterization. We used multiple linear regression to analyze the effect of demographic and hemodynamic variables on pulmonary arterial and venous endothelin-1 concentrations and on the change of endothelin-1 concentration over the pulmonary vascular bed., Results: Multiple linear regression revealed that of all the hemodynamic variables tested, mean pulmonary arterial pressure had the greatest effect on increasing the change of endothelin-1 concentration over the pulmonary vascular bed (P <.0001). Pulmonary blood flow did not have any effect on endothelin-1 concentrations or on the change of endothelin-1 concentration over the pulmonary vascular bed., Conclusions: This study shows that pulmonary blood pressure and not flow is associated with net endothelin-1 production in patients with congenital heart disease and normal pulmonary vascular resistance.

Hybrid chondroitin/dermatan sulfate (CS/DS) glycosaminoglycan chains, derived from decorin secreted by human skin fibroblasts, were shown to interact with FGF-2, as did oligosaccharides derived therefrom by chondroitin B lyase digestion. In a first attempt to identify the biologically active sequence, a novel protocol for structural analysis of enzyme-resistant oligosaccharides larger than standard trisulfated hexasaccharides was developed. The method bases on capillary electrophoresis (CE) for separating oversulfated species in offline combination with nanoelectrospray ionization quadrupole time-of-flight tandem mass spectrometry (nanoESI-QTOF-MS/MS) in the negative ion mode. Under optimized CE and ESI-MS conditions, up to 12-mer oligosaccharides with different degrees of sulfation were identified. A novel tandem MS protocol (CID-VE) was applied to elucidate the structure of a previously undescribed pentasulfated CS/DS hexasaccharide, Delta-4,5-IdoAGalNAc[GlcAGalNAc]2(5S). In this molecular species, detected as a triply charged ion at m/z 511.38, three sulfates are found in the IdoAGalNAcGlcA moiety offering two structural variants: one containing sulfated IdoA together with a disulfated GalNAc moiety and in the other one both uronic acids, that is, GlcA and IdoA and the amino sugar each carry a sulfate ester group.

During glomerular inflammation mesangial cells are the major source and target of nitric oxide that pro-foundly influences proliferation, adhesion, and death of mesangial cells. The effect of nitric oxide on the mRNA expression pattern of cultured rat mesangial cells was therefore investigated by RNA-arbitrarily-primed polymerase chain reaction. Employing this approach, biglycan expression turned out to be down-regulated time- and dose-dependently either by interleukin-1beta-stimulated endogenous nitric oxide production or by direct application of the exogenous nitric oxide donor, diethylenetriamine nitric oxide. There was a corresponding decline in the rate of biglycan biosynthesis and in the steady state level of this proteoglycan. In vivo, in a model of mesangioproliferative glomerulonephritis up-regulation of inducible nitric-oxide synthase mRNA was associated with reduced expression of biglycan in isolated glomeruli. Biglycan expression could be normalized, both in vitro and in vivo, by using a specific inhibitor of the inducible nitric-oxide synthase, l-N6-(l-iminoethyl)-l-lysine dihydrochloride. Further studies showed that biglycan inhibited cell adhesion on type I collagen and fibronectin because of its binding to these substrates. More importantly, biglycan protected mesangial cells from apoptosis by decreasing caspase-3 activity, and it counteracted the proliferative effects of platelet-derived growth factor-BB. These findings indicate a signaling role of biglycan and describe a novel pathomechanism by which nitric oxide modulates the course of renal glomerular disease through regulation of biglycan expression.

Decorin is a well-known, ubiquitous proteoglycan that is a normal component of the ECM. Upon transgenic expression of decorin, tumor cells with diverse histogenetic background overexpress p21WAF1, a potent inhibitor of cyclin-dependent kinase activity, become arrested in G1, and fail to generate tumors in immunocompromised animals. Because decorin is a secreted protein, it has been recently suggested that decorin could act as an autocrine and paracrine regulator of tumor growth. Here, we demonstrate that adenovirus (Ad)-mediated transfer and expression of human decorin cDNA induced in vivo apoptosis of xenograft tumor cells in nude mice. This oncolytic activity was observed when the Ad vector encoding the decorin cDNA was injected intratumorally (i.t.) or i.v. Importantly, i.t. injection of the decorin Ad vector led to growth inhibition of the injected tumor associated with similar growth inhibition of a distant contralateral tumor, demonstrating a distant decorin antitumoral effect. Immunochemistry against human decorin and decorin quantitation in tumors confirmed that decorin migrated to the tumor distant site. Furthermore, decorin effect was specific to tumor cells, because neither apoptosis nor growth inhibition were observed in nontumoral human cells such as hepatocytes, endothelial cells, and fibroblasts, despite p21 overexpression.

During atherogenesis vascular smooth muscle cells are converted from a contractile into a synthetic phenotype characterized by enhanced matrix production. The transcription factors Gax and GATA-6 are considered negative, and Oct-1 positive regulators of the synthetic phenotype. Since the phenotype transition can be induced by culturing the cells with serum, we followed the expression of Gax, GATA-6 and Oct-1, integrins and matrix genes in quiescent porcine vascular smooth muscle cells after serum application. Comparisons were made between enzymatically released primary smooth muscle cells and cells grown out from explants of the medial layer of porcine aorta. The serum-mediated down-regulation of Gax was more intense than that of GATA-6, and stronger in explant-derived than in primary cells. Serum was without influence on the expression of Oct-1. Changes in the expression of the transcription factors preceded the induction of integrin alpha2 and the down-regulation of decorin, while mRNAs for laminin beta1 and osteopontin rose immediately after serum stimulation. Primary cells reacted more rapidly than explant cells with respect to changes in laminin isoforms. Studies with a Gax-expressing adenovirus indicated that among all the gene products tested only the expression of integrin alpha2 responded to Gax induction. Thus, our data show that i) Gax should be considered a transcription factor being directly responsible for only few aspects of the phenotypic conversion of smooth muscle cells and that ii) explant cells may represent a subpopulation of smooth muscle cells, which differ from the total population of smooth muscle cells, as obtained in primary culture, in their response to serum stimuli.

The first carbosilane dendrimer with peripheral bent-core mesogenic units is reported. This material forms a liquid crystalline phase which is stable over a wide temperature range and forms an LC glass on cooling. Polarizing microscopy, X-ray diffraction, and dielectric and electrooptic investigations reveal the presence of a novel liquid crystalline phase, in which the molecules are tilted and adapt a polar order within the layers, but without long-range correlation between the layers. By applying external electric fields, switching into a ferroelectric organization can be achieved. Once formed the ferroelectric states are stable and can be switched between the different polarization states.

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.

Chondroitin sulfate and dermatan sulfate proteoglycans are distinguished by differences in their proportion of d-glucuronosyl and l-iduronosyl residues, the latter being formed by chondroitin-glucuronate 5-epimerase during or after glycosaminoglycan chain polymerization. To investigate the influence of the core protein on the extent of epimerization, we expressed chimeric proteins in 293 HEK cells constructed from intact or modified Met(1)-Gln(153) of decorin (DCN), which normally has a single dermatan sulfate chain at Ser(34), in combination with intact or modified Leu(241)-Ser(353) of CSF-1, which has a chondroitin sulfate attachment site at Ser(309). Transfected DCN(M1-Q153), like full-length DCN, contained approximately 20% l-iduronate. Conversely, transfected CSF-1(L241-S353), attached C-terminally on the DCN prepropeptide, contained almost exclusively d-glucuronate. Transfected intact chimeric DCN(M1-Q153)-CSF-1(L241-S353), with two glycosaminoglycan chains, also contained almost exclusively d-glucuronate in chains at both sites, as did chimeras in which alanine was substituted for serine at either of the glycosaminoglycan attachment sites. Nevertheless, undersulfated intact chimeric proteoglycan was an effective substrate for epimerization of glucuronate to iduronate residues when incubated with microsomal proteins and 3'-phosphoadenylylphosphosulfate. C-terminal truncation constructs were prepared from the full-length chimera with an alanine substitution at the CSF-1 glycosaminoglycan attachment site. Transfected truncations retaining the alanine-blocked site contained chains with essentially only glucuronate, whereas those further truncated by 49 or more amino acids and missing the modified attachment site contained chains with approximately 15% iduronate. This 49-amino acid region contains a 7-amino acid motif that appears to be conserved in several chondroitin sulfate proteoglycans. The results are consistent with a model in which the core protein, possibly via this motif, is responsible for routing to subcellular compartments with or without sufficient access to chondroitin-glucuronate 5-epimerase for the addition of chains with or without iduronate residues, respectively.

Purpose: To elucidate differences, at the macromolecular level, in corneal tissue subjected to repeated argon fluoride excimer treatment., Methods: A light microscopic, electron microscopic, and immunohistochemical study was performed on a scarred human cornea., Results: Keratocytes were enlarged with an expanded endoplasmic reticulum and exhibited a fibroblastic appearance. Amorphous material was observed extracellularly. Collagen fibrils exhibited a disordered arrangement while banding patterns and diameter were normal. Immunohistochemical investigation of several collagen types, of collagen-associated proteoglycans, and of basement membrane components demonstrated an enhanced immunoreactivity of all of them in the scarred area. Type V collagen was found as a normal component of the epithelial basement membrane whereas types I and III collagen were present beneath Bowman's layer. Excimer-laser-treated sections revealed considerably stronger subepithelial staining for collagen types I, III, IV, and V. Laminin-1, a typical component of basement membranes, was detectable throughout the scarred tissue. The small proteoglycans decorin and fibromodulin accumulated in a patch-like manner in the scarred tissue below the epithelium, whereas biglycan was expressed by the epithelium and throughout the stroma. Lumican was expressed most strongly by the epithelium and rather equally distributed in the excimer-laser-treated and in the normal stroma., Conclusion: Effects of argon laser treatment of the cornea must be regarded as a process acting over many months. Intra- and extracellular structures and components are involved and influence the unpredictable shape of the corneal architecture.

Decorin, a small dermatan-sulfate proteoglycan, participates in extracellular matrix assembly and influences directly and indirectly cell behavior via interactions with signaling membrane receptors and transforming growth factor (TGF)-beta. We have therefore compared the development of tubulointerstitial kidney fibrosis in wild-type (WT) and decorin-/- mice in the model of unilateral ureteral obstruction. Without obstruction, kidneys from decorin-/- mice did not differ in any aspect from their WT counterparts. However, already 12 hours after obstruction decorin-/- animals showed lower levels of p27(KIP1) and soon thereafter a more pronounced up-regulation and activation of initiator and effector caspases followed by enhanced apoptosis of tubular epithelial cells. Later, a higher increase of TGF-beta1 became apparent. After 7 days, there was an up to 15-fold transient up-regulation of the related proteoglycan biglycan, which was mainly caused by the appearance of biglycan-expressing mononuclear cells. Other small proteoglycans showed no similar response. Because of enhanced degradation of type I collagen, end-stage kidneys from decorin-/- animals were more atrophic than WT kidneys. These data suggest that decorin exerts beneficial effects on tubulointerstitial fibrosis, primarily by influencing the expression of a key cyclin-dependent kinase inhibitor and by limiting the degree of apoptosis, mononuclear cell infiltration, tubular atrophy, and expression of TGF-beta1.

An analytical approach based on high-performance capillary electrophoresis (CE) in conjunction with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (ESI-QTOF-MS/MS) has been developed for providing the basis to obtain new insights into the domain structure of the glycosaminoglycan (GAG) moiety of proteoglycans. The feasibility and performance of the off-line CE/ESI-QTOF-MS approach in GAG oligosaccharide analysis were assessed by screening a chondroitin/dermatan sulfate (DS) oligosaccharide mixture obtained from bovine aorta by enzymatic depolymerization by chondroitin B lyase. The CS/DS mixture was analyzed by CE using 50 mM ammonium acetate, pH 12.0, dissolved in aqueous methanol (2:3; v/v), as a CE carrier. Structural identification of the GAG components was achieved using off-line CE/nanoESI-QTOF-MS and-MS/MS experiments. ESI-QTOF instrumental parameters were found to play an important role in the MS screening of the CE-separated GAG species. By optimizing the ESI conditions, oligosaccharides differing in chain length and degree of sulfation could be detected. The building block composition, the size of the carbohydrate chain, as well as structural features of the repeating HexA-GalNAc, HexA-GalNAc(S) units, have been determined using MS/MS by applying collision-induced dissociation at low energies. Cleavage of GAG chains by chondroitin B lyase occurs with formation of structural markers useful for identification of IdoA-containing domains., (Copyright 2002 John Wiley & Sons, Ltd.)

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., (Copyright 2001 Wiley-Liss, Inc.)

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.

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.

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.

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., (Copyright 2001 OsteoArthritis Research Society International.)

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.

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.

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., (Copyright 2001 Wiley-Liss, Inc.)

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.

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.

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.

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.

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.

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., (Copyright 2000 Academic Press.)