Sulfated proteoglycans as modulators of neuronal migration and axonal decussation in the developing midbrain

Proteoglycans are abundant in the developing brain and there is much circumstantial evidence for their roles in directional neuronal movements such as cell body migration and axonal growth. We have developed an in vitro model of astrocyte cultures of the lateral and medial sectors of the embryonic mouse midbrain, that differ in their ability to support neuritic growth of young midbrain neurons, and we have searched for the role of interactive proteins and proteoglycans in this model. Neurite production in co-cultures reveals that, irrespective of the previous location of neurons in the midbrain, medial astrocytes exert an inhibitory or nonpermissive effect on neuritic growth that is correlated to a higher content of both heparan and chondroitin sulfates (HS and CS). Treatment of astrocytes with chondroitinase ABC revealed a growth-promoting effect of CS on lateral glia but treatment with exogenous CS-4 indicated a U-shaped dose-response curve for CS. In contrast, the growth-inhibitory action of medial astrocytes was reversed by exogenous CS-4. Treatment of astrocytes with heparitinase indicated that the growth-inhibitory action of medial astrocytes may depend heavily on HS by an as yet unknown mechanism. The results are discussed in terms of available knowledge on the binding of HS proteoglycans to interactive proteins, with emphasis on the importance of unraveling the physiological functions of glial glycoconjugates for a better understanding of neuron-glial interactions.

Collagens and proteoglycans of the corneal extracellular matrix

The cornea is a curved and transparent structure that provides the initial focusing of a light image into the eye. It consists of a central stroma that constitutes 90% of the corneal depth, covered anteriorly with epithelium and posteriorly with endothelium. Its transparency is the result of the regular spacing of collagen fibers with remarkably uniform diameter and interfibrillar space. Corneal collagen is composed of heterotypic fibrils consisting of type I and type V collagen molecules. The cornea also contains unusually high amounts of type VI collagen, which form microfibrillar structures, FACIT collagens (XII and XIV), and other nonfibrillar collagens (XIII and XVIII). FACIT collagens and other molecules, such as leucine-rich repeat proteoglycans, play important roles in modifying the structure and function of collagen fibrils.Proteoglycans are macromolecules composed of a protein core with covalently linked glycosaminoglycan side chains. Four leucine-rich repeat proteoglycans are present in the extracellular matrix of corneal stroma: decorin, lumican, mimecan and keratocan. The first is a dermatan sulfate proteoglycan, and the other three are keratan sulfate proteoglycans. Experimental evidence indicates that the keratan sulfate proteoglycans are involved in the regulation of collagen fibril diameter, and dermatan sulfate proteoglycan participates in the control of interfibrillar spacing and in the lamellar adhesion properties of corneal collagens. Heparan sulfate proteoglycans are minor components of the cornea, and are synthesized mainly by epithelial cells. The effect of injuries on proteoglycan synthesis is discussed.

Glycosaminoglycans affect the interaction of human plasma kallikrein with plasminogen, factor XII and inhibitors

Human plasma kallikrein, a serine proteinase, plays a key role in intrinsic blood clotting, in the kallikrein-kinin system, and in fibrinolysis. The proteolytic enzymes involved in these processes are usually controlled by specific inhibitors and may be influenced by several factors including glycosaminoglycans, as recently demonstrated by our group. The aim of the present study was to investigate the effect of glycosaminoglycans (30 to 250 µg/ml) on kallikrein activity on plasminogen and factor XII and on the inhibition of kallikrein by the plasma proteins C1-inhibitor and antithrombin. Almost all available glycosaminoglycans (heparin, heparan sulfate, bovine and tuna dermatan sulfate, chondroitin 4- and 6-sulfates) reduced (1.2 to 3.0 times) the catalytic efficiency of kallikrein (in a nanomolar range) on the hydrolysis of plasminogen (0.3 to 1.8 µM) and increased (1.9 to 7.7 times) the enzyme efficiency in factor XII (0.1 to 10 µM) activation. On the other hand, heparin, heparan sulfate, and bovine and tuna dermatan sulfate improved (1.2 to 3.4 times) kallikrein inhibition by antithrombin (1.4 µM), while chondroitin 4- and 6-sulfates reduced it (1.3 times). Heparin and heparan sulfate increased (1.4 times) the enzyme inhibition by the C1-inhibitor (150 nM).

Decorin is one of the proteoglycans expressed in Walker 256 rat mammary carcinoma

Proteoglycan and glycosaminoglycan content was analyzed in a model of rat mammary carcinoma to study the roles of these compounds in tumorigenesis. Hyaluronic acid and proteoglycans bearing chondroitin and/or dermatan sulfate chains were detected in solid tumors obtained after subcutaneous inoculation of Walker 256 rat carcinoma cells. About 10% of sulfated glycosaminoglycan chains corresponded to heparan sulfate. The small leucine-rich proteoglycan, decorin, was identified as one of the proteoglycans, in addition to others of higher molecular weight, by cross-reaction with an antiserum raised against pig laryngeal decorin and by N-terminal amino acid sequencing. Decorin was separated from other proteoglycans by hydrophobic chromatography and its complete structure was determined. It has a molecular weight of about 85 kDa and a dermatan chain of 45 kDa with 4-sulfated disaccharides. After degradation of the glycosaminoglycan chain, three core proteins of different molecular weight (36, 46 and 56 kDa) were identified. The presence of hyaluronic acid and decorin has been reported in a variety of tumors and tumor cells. In the Walker 256 mammary carcinoma model, hyaluronic acid may play an important role in tumor progression, since it provides a more hydrated extracellular matrix. On the other hand, decorin, which is expressed by stromal cells, represents a host defense response to tumor growth.

Heterofucans from Dictyota menstrualis have anticoagulant activity

Fucan is a term used to denote a family of sulfated L-fucose-rich polysaccharides which are present in the extracellular matrix of brown seaweed and in the egg jelly coat of sea urchins. Plant fucans have several biological activities, including anticoagulant and antithrombotic, related to the structural and chemical composition of polysaccharides. We have extracted sulfated polysaccharides from the brown seaweed Dictyota menstrualis by proteolytic digestion, followed by separation into 5 fractions by sequential acetone precipitation. Gel electrophoresis using 0.05 M 1,3-diaminopropane-acetate buffer, pH 9.0, stained with 0.1% toluidine blue, showed the presence of sulfated polysaccharides in all fractions. The chemical analyses demonstrated that all fractions are composed mainly of fucose, xylose, galactose, uronic acid, and sulfate. The anticoagulant activity of these heterofucans was determined by activated partial thromboplastin time (APTT) using citrate normal human plasma. Only the fucans F1.0v and F1.5v showed anticoagulant activity. To prolong the coagulation time to double the baseline value in the APTT, the required concentration of fucan F1.0v (20 µg/ml) was only 4.88-fold higher than that of the low molecular weight heparin Clexane® (4.1 µg/ml), whereas 80 µg/ml fucan 1.5 was needed to obtain the same effect. For both fucans this effect was abolished by desulfation. These polymers are composed of fucose, xylose, uronic acid, galactose, and sulfate at molar ratios of 1.0:0.8:0.7:0.8:0.4 and 1.0:0.3:0.4:1.5:1.3, respectively. This is the fist report indicating the presence of a heterofucan with higher anticoagulant activity from brown seaweed.

The alga Bryothamnion seaforthii contains carbohydrates with antinociceptive activity

Bryothamnion seaforthii, a red alga common to the Northeastern coast of Brazil, was used to prepare the protein fraction F0/60 by ammonium sulfate precipitation. The chromatography of F0/60 on DEAE-Sephadel column resulted in two lectin fractions, PI and PII, which have antinociceptive properties in rodents. We determined the antinociceptive activity of the PII fraction and of a carbohydrate-containing fraction (CF) in mice. The CF was prepared from the dried algae, after digestion with 100 mM sodium acetate, pH 6.0, containing 5 mM cysteine, EDTA and 0.4% papain, at 60ºC. A 10% cetylpyridinium chloride was added to the filtrate, and the precipitate was dissolved with 2 M NaCl:ethanol (100:15, v/v) followed by the carbohydrate precipitation with ethanol. The final precipitate, in acetone, was dried at 25ºC. The PII fraction markedly inhibited acetic acid-induced abdominal writhing after ip administration (control: 27.1 ± 2.20; PII 0.1 mg/kg: 5.5 ± 1.85; 1 mg/kg: 1.6 ± 0.72 writhes/20 min) and after oral administration (control: 32.0 ± 3.32; PII 0.1 mg/kg: 13.1 ± 2.50; 1 mg/kg: 9.4 ± 3.96 writhes/20 min). PII was also effective against both phases of pain induced by 1% formalin (control, ip: 48.2 ± 2.40 and 27.7 ± 2.56 s; PII: 1 mg/kg, ip: 34.3 ± 5.13 and 5.6 ± 2.14 s; control, po: 44.5 ± 3.52 and 25.6 ± 2.39 s; PII 5 mg/kg, po: 26.5 ± 4.67 and 15.3 ± 3.54 s for the 1st and 2nd phases, respectively) and in the hot-plate test. The CF (ip) also displayed significant antinociceptive properties in all tests but at higher doses (1 and 5 mg/kg, ip and po). Thus, CF at the dose of 5 mg/kg significantly inhibited writhes (ip: 7.1 ± 2.47 and po: 14.5 ± 2.40 writhes/20 min) as well as the 1st (po: 19.6 ± 1.74 s) and 2nd (po: 7.1 ± 2.24 s) phases of the formalin test compared to controls ip and po. The antinociceptive effects of both the PII and CF in the formalin and hot-plate tests were prevented at least partially by pretreatment with the opioid receptor antagonist naloxone (2 mg/kg, sc). Moreover, both fractions retained antinociceptive activity in the acetic acid-induced writhing test following heating, a procedure which abolished the hemagglutinating activity of the fraction, presumably due to lectins also present. Finally, both fractions also prolonged the barbiturate-induced sleeping time. These results indicate that carbohydrate molecules present in the PII (26.8% carbohydrate) and CF (21% of the alga dried weight) obtained from B. seaforthii display pronounced antinociceptive activity which is resistant to heat denaturation and is mediated by an opioid mechanism, as indicated by naloxone inhibition.

Inotropic effect of Citrus sinensis (L.) Osbeck leaf extracts on the guinea pig atrium

The objective of the present investigation was to determine the contractile effect of crude and acetone leaf extracts of Citrus sinensis (L.) Osb. on mammalian myocardium. Crude leaf extracts have been used in folk medicine to treat neurological disorders. Some flavonoids isolated from this plant presented a positive inotropic effect on myocardium. This motivated us to test the extracts on the atria of guinea pigs of both sexes (300-500 g) and surprisingly we observed inotropic depression instead of an increase in force. The maximum effect of the crude extract was 79.4 ± 8.1% of the control force amplitude (N = 5 hearts, 10 trials, 27 ± 0.1ºC, stimulus: 2 Hz, 400 V, 0.5 ms). The EC50 for crude, ethanol, acetic, aqueous, and acetone extracts was 300, 300, 600, 1000, and 140 µg/ml, respectively, with a Hill constant of 1.8, 2.0, 2.5, 2.0, and 1.4, respectively. Blockade of cholinergic, beta-adrenergic, or opioid membrane receptors with 1.5 µM atropine sulfate, 1 µM propranolol, and 10 µM naloxone, respectively, did not change the effect of the crude extract. The acetone extract abolished the Bowditch positive staircase phenomenon (N = 5 hearts, 10 trials, 27 ± 0.1ºC), suggesting a possible reduction of the calcium inward current, and also promoted the so-called Woodworth phenomenon. The effect was concentration-dependent and indicated the existence of another inhibitory contractile mechanism such as the simultaneous activation of some of the membrane potassium channels reducing the myocardial action potential duration and further decreasing the cellular calcium entry.

Distribution of small proteoglycans and glycosaminoglycans in humerus-related articular cartilage of chickens

The expression of components present in the cartilaginous extracellular matrix is related to development, gender, and genotype, as well as to the biomechanical properties of each type of cartilage. In the present study, we analyzed small proteoglycans and glycosaminoglycans present in different cartilages of the chicken wing after extraction with guanidine hydrochloride or papain. Quantitative analysis of glycosaminoglycans showed a larger amount in humeral cartilage (around 200 mg/g tissue) than in articular cartilage of the radius and ulna, with 138 and 80 mg/g tissue, respectively. Non-collagenous proteins isolated were predominantly from cartilage in the proximal regions of the humerus and radius. D4 fractions obtained by ultracentrifugation were separated by DEAE-Sephacel and Octyl-Sepharose chromatography and analyzed by SDS-PAGE. Two bands of 57 and 70-90 kDa were observed for all samples treated with ß-mercaptoethanol. Immunoblotting of these proteins was positive for the small proteoglycans fibromodulin and decorin, respectively. Apparently, the 57-kDa protein is present in macromolecular complexes of 160 and 200 kDa. Chondroitin sulfate was detected in all regions. HPLC analysis of the products formed by chondroitinase AC and ABC digestion mainly revealed ß-D-glucuronic acid and N-acetyl ß-D-galactosamine residues. The 4-sulfation/6-sulfation ratio was close to 3, except for the proximal cartilage of the radius (2.5). These results suggest functional differences between the scapula-humerus, humerus-ulna, and humerus-radius joints of the chicken wing. This study contributes to the understanding of the physiology of cartilage and joints of birds under different types of mechanical stress.

Partial characterization and anticoagulant activity of a heterofucan from the brown seaweed Padina gymnospora

The brown algae Padina gymnospora contain different fucans. Powdered algae were submitted to proteolysis with the proteolytic enzyme maxataze. The first extract of the algae was constituted of polysaccharides contaminated with lipids, phenols, etc. Fractionation of the fucans with increasing concentrations of acetone produced fractions with different proportions of fucose, xylose, uronic acid, galactose, and sulfate. One of the fractions, precipitated with 50% acetone (v/v), contained an 18-kDa heterofucan (PF1), which was further purified by gel-permeation chromatography on Sephadex G-75 using 0.2 M acetic acid as eluent and characterized by agarose gel electrophoresis in 0.05 M 1,3 diaminopropane/acetate buffer at pH 9.0, methylation and nuclear magnetic resonance spectroscopy. Structural analysis indicates that this fucan has a central core consisting mainly of 3-ß-D-glucuronic acid 1-> or 4-ß-D-glucuronic acid 1 ->, substituted at C-2 with alpha-L-fucose or ß-D-xylose. Sulfate groups were only detected at C-3 of 4-alpha-L-fucose 1-> units. The anticoagulant activity of the PF1 (only 2.5-fold lesser than low molecular weight heparin) estimated by activated partial thromboplastin time was completely abolished upon desulfation by solvolysis in dimethyl sulfoxide, indicating that 3-O-sulfation at C-3 of 4-alpha-L-fucose 1-> units is responsible for the anticoagulant activity of the polymer.

The extracellular matrix provides directional cues for neuronal migration during cerebellar development

Normal central nervous system development relies on accurate intrinsic cellular programs as well as on extrinsic informative cues provided by extracellular molecules. Migration of neuronal progenitors from defined proliferative zones to their final location is a key event during embryonic and postnatal development. Extracellular matrix components play important roles in these processes, and interactions between neurons and extracellular matrix are fundamental for the normal development of the central nervous system. Guidance cues are provided by extracellular factors that orient neuronal migration. During cerebellar development, the extracellular matrix molecules laminin and fibronectin give support to neuronal precursor migration, while other molecules such as reelin, tenascin, and netrin orient their migration. Reelin and tenascin are extracellular matrix components that attract or repel neuronal precursors and axons during development through interaction with membrane receptors, and netrin associates with laminin and heparan sulfate proteoglycans, and binds to the extracellular matrix receptor integrins present on the neuronal surface. Altogether, the dynamic changes in the composition and distribution of extracellular matrix components provide external cues that direct neurons leaving their birthplaces to reach their correct final location. Understanding the molecular mechanisms that orient neurons to reach precisely their final location during development is fundamental to understand how neuronal misplacement leads to neurological diseases and eventually to find ways to treat them.

Transient outward potassium current and Ca2+ homeostasis in the heart: beyond the action potential

Normal central nervous system development relies on accurate intrinsic cellular programs as well as on extrinsic informative cues provided by extracellular molecules. Migration of neuronal progenitors from defined proliferative zones to their final location is a key event during embryonic and postnatal development. Extracellular matrix components play important roles in these processes, and interactions between neurons and extracellular matrix are fundamental for the normal development of the central nervous system. Guidance cues are provided by extracellular factors that orient neuronal migration. During cerebellar development, the extracellular matrix molecules laminin and fibronectin give support to neuronal precursor migration, while other molecules such as reelin, tenascin, and netrin orient their migration. Reelin and tenascin are extracellular matrix components that attract or repel neuronal precursors and axons during development through interaction with membrane receptors, and netrin associates with laminin and heparan sulfate proteoglycans, and binds to the extracellular matrix receptor integrins present on the neuronal surface. Altogether, the dynamic changes in the composition and distribution of extracellular matrix components provide external cues that direct neurons leaving their birthplaces to reach their correct final location. Understanding the molecular mechanisms that orient neurons to reach precisely their final location during development is fundamental to understand how neuronal misplacement leads to neurological diseases and eventually to find ways to treat them.

Noncrystalline uric acid inhibits proteoglycan and glycosaminoglycan synthesis in distal tubular epithelial cells (MDCK)

Hyperuricemia is associated with renal stones, not only consisting of uric acid (UrAc) but also of calcium oxalate (CaOx). Glycosaminoglycans (GAGs) are well-known inhibitors of growth and aggregation of CaOx crystals. We analyzed the effect of noncrystalline UrAc on GAG synthesis in tubular distal cells. MDCK (Madin-Darby canine kidney) cells were exposed to noncrystalline UrAc (80 µg/mL) for 24 h. GAGs were labeled metabolically and characterized by agarose gel electrophoresis. The expression of proteoglycans and cyclooxygenase 2 (COX-2) was assessed by real-time PCR. Necrosis, apoptosis and prostaglandin E2 (PGE2) were determined by acridine orange, HOESCHT 33346, and ELISA, respectively. CaOx crystal endocytosis was evaluated by flow cytometry. Noncrystalline UrAc significantly decreased the synthesis and secretion of heparan sulfate into the culture medium (UrAc: 2127 ± 377; control: 4447 ± 730 cpm) and decreased the expression of perlecan core protein (UrAc: 0.61 ± 0.13; control: 1.07 ± 0.16 arbitrary units), but not versican. Noncrystalline UrAc did not induce necrosis or apoptosis, but significantly increased COX-2 and PGE2 production. The effects of noncrystalline UrAc on GAG synthesis could not be attributed to inflammatory actions because lipopolysaccharide, as the positive control, did not have the same effect. CaOx was significantly endocytosed by MDCK cells, but this endocytosis was inhibited by exposure to noncrystalline UrAc (control: 674.6 ± 4.6, CaOx: 724.2 ± 4.2, and UrAc + CaOx: 688.6 ± 5.4 geometric mean), perhaps allowing interaction with CaOx crystals. Our results indicate that UrAc decreases GAG synthesis in MDCK cells and this effect could be related to the formation of UrAc and CaOx stones.

Effect of activation of canonical Wnt signaling by the Wnt-3a protein on the susceptibility of PC12 cells to oxidative and apoptotic insults

Wnt proteins are involved in tissue development and their signaling pathways play an important role during embryogenesis. Wnt signaling can promote cell survival, which is beneficial for neurons, but could also lead to tumor development in different tissues. The present study investigated the effects of a Wnt protein on the susceptibility of a neural tumor cell line (PC12 cells) to the cytotoxic compounds ferrous sulfate (10 mM), staurosporine (100 and 500 nM), 3-nitropropionic acid (5 mM), and amyloid β-peptide (Aβ25-35; 50 µM). Cells (1 x 10(6) cells/mL) were treated with the Wnt-3a recombinant peptide (200 ng/mL) for 24 h before exposure to toxic insults. The Wnt-3a protein partially protected PC12 cells, with a 6-15% increase in cell viability in the presence of toxic agents, similar to the effect measured using the MTT and lactate dehydrogenase cell viability assays. The Wnt-3a protein increased protein expression of β-catenin by 52% compared to control. These findings suggest that Wnt signaling can protect neural cells against apoptosis induced by toxic agents, which are relevant to the pathogenesis of Alzheimer’s and Huntington’s diseases.

Heparan sulphate, its derivatives and analogues share structural characteristics that can be exploited, particularly in inhibiting microbial attachment

Heparan sulphate (HS) and the related polysaccharide, heparin, exhibit conformational and charge arrangement properties, which provide a degree of redundancy allowing several seemingly distinct sequences to exhibit the same activity. This can also be mimicked by other sulphated polysaccharides, both in overall effect and in the details of interactions and structural consequences of interactions with proteins. Together, these provide a source of active compounds suitable for further development as potential drugs. These polysaccharides also possess considerable size, which bestows upon them an additional useful property: the capability of disrupting processes comprising many individual interactions, such as those characterising the attachment of microbial pathogens to host cells. The range of involvement of HS in microbial attachment is reviewed and examples, which include viral, bacterial and parasitic infections and which, in many cases, are now being investigated as potential targets for intervention, are identified.

Effect of castration on renal glycosaminoglycans and their urinary excretion in male and female rats with chronic renal failure

Glycosaminoglycans (GAGs) participate in a variety of processes in the kidney, and evidence suggests that gender-related hormones participate in renal function. The aim of this study was to analyze the relationship of GAGs, gender, and proteinuria in male and female rats with chronic renal failure (CRF). GAGs were analyzed in total kidney tissue and 24-h urine of castrated (c), male (M), and female (F) Wistar control (C) rats (CM, CMc, CF, CFc) and after 30 days of CRF induced by 5/6 nephrectomy (CRFM, CRFMc, CRFF, CRFFc). Total GAG quantification and composition were determined using agarose and polyacrylamide gel electrophoresis, respectively. Renal GAGs were higher in CF compared to CM. CRFM presented an increase in renal GAGs, heparan sulfate (HS), and proteinuria, while castration reduced these parameters. However, CRFF and CRFFc groups showed a decrease in renal GAGs concomitant with an increase in proteinuria. Our results suggest that, in CRFM, sex hormones quantitatively alter GAGs, mainly HS, and possibly the glomerular filtration barrier, leading to proteinuria. The lack of this response in CRFMc, where HS did not increase, corroborates this theory. This pattern was not observed in females. Further studies of CRF are needed to clarify gender-dependent differences in HS synthesis.