Two-dimensional NMR spectroscopy of urinary glycosaminoglycans from patients with different mucopolysaccharidoses

Patients with different types of mucopolysaccharidoses (MPS) lack specific lysosomal enzymes, which leads to tissue accumulation and urinary excretion of glycosaminoglycans (GAGs). Since little is known about the molecular composition of the excreted GAG fragments, we used two-dimensional [1H,13C]-correlation nuclear magnetic resonance (NMR) spectroscopy for a detailed analysis of the urinary GAGs of patients with MPS types I, II, IIIA, IVA and VI. The method revealed that the molecular structures of the excreted GAGs, i.e. heparan sulfate (HS), dermatan sulfate (DS), chondroitin sulfate (CS), and keratan sulfate (KS) are clearly distinct for the different MPS types. The chain terminal residues that are the normal substrates for the defective enzymes constitute characteristic sets of signals for each MPS type. The GAG chains show variations in carbohydrate composition and sulfation patterns that can be related to the different MPS types and clinical features. For example, two patients with MPS IIIA (M. Sanfilippo) with signs of CNS degeneration but only mild somatic features excrete a highly sulfated variant of HS, resembling HS in porcine brain, whereas a patient with MPS I (M. Scheie) and two patients with MPS II (M. Hunter), who present primarily with coarse facial features, joint contractures and skeletal deformities excrete a different type of HS with lower sulfation. In another case study, a patient with MPS IVA (M. Morquio), who presented mainly with skeletal dysplasia, excreted not only excessive amounts of KS but also a highly sulfated CS variant, resembling CS in articular cartilage. The high-resolution NMR analysis of urinary GAGs presented here for the first time provides a solid basis for future studies with a larger number of patients to further explore pathogenesis and course of the MPS diseases.     

Molecular recognition and modulation of hepatocyte growth factor activity by heparan and dermatan sulfates

Introduction Hepatocyte growth factor/scatter factor (HGF/SF) is an unusual growth factor in that it binds both heparan sulfate (HS) ( Lyon et al. 1994 ) and dermatan sulfate (DS) ( Lyon et al. 1998 ) glycosaminoglycans (GAGs) with similar high affinities. Both these GAGs act as co‐receptors for HGF/SF in the activation of the Met receptor ( Lyon et al. 2002 ). Our aim was to determine the sequences in HS and DS that specifically interact with and modulate HGF/SF activity. Materials and methods A structurally unique DS, which possesses O‐sulfation at carbon‐6 of the hexosamine residue (and not carbon‐4 as in mammalian DS), was obtained from the sea cucumber, Ascidia nigra. A variety of HS‐ and DS‐like structures were also generated using various chemical modification procedures (specific desulfations and carboxyl reductions). The ability of these various GAG species to compete with cell surface GAGs for HGF/SF binding was tested using radiolabelled HGF/SF and MDCK cells. The modified GAG structures and the A. nigra DS are currently being tested for their ability to act as co‐receptors for the interaction between HGF/SF and Met by studying cell signalling and cellular response assays, using the sulfated GAG‐deficient CHO‐745 cell line. Results Unexpectedly, A. nigra DS was found to bind HGF/SF strongly with a KD of around 1 nm . This interaction is 20‐fold stronger than that of between HGF/SF and mammalian DS, but similar to that of with HS. A. nigra DS also stimulated HGF/SF‐mediated Erk activation and migration in CHO‐745 cells. Studies using the modified GAG species showed that, in the case of HS, 6‐O‐sulfate and N‐sulfate groups are most important for HGF/SF binding. For HGF/SF binding to DS, hexosamine O‐sulfate is most important. HGF/SF was also found to bind 6‐O‐sulfated GAGs more strongly than 4‐O‐sulfated ones. Discussion The data show that there is flexibility in the structures recognized by HGF/SF, and this explains the ability of the growth factor to bind both HS and DS. However, there are still observable preferences in GAG structure, such as 6‐O‐sulfation over 4‐O‐sulfation. Information on HGF/SF‐binding GAG structures is valuable for the design of HGF/SF antagonists that could be useful therapeutically in the treatment of solid tumours where HGF/SF‐Met activity is up‐regulated.     

Glycosaminoglycan composition and biosynthesis in the endothelium-covered neointima of de-endothelialized rabbit aorta

The biosynthesis and composition of glycosaminoglycans (GAG) in the endothelium-covered neointima, formed in response to de-endothelialization of the rabbit aorta by a balloon catheter, was examined. The [14C]glucosamine incorporation into GAG during an in vitro incubation with intimal-medial tissue was monitored periodically up to 24 hr. The GAG were isolated after an exhaustive proteolytic digestion with pronase and protease followed by ethanolic precipitation at 4 degrees C. Electrophoretic migration on cellulose acetate paper was compared for identification. The distribution of GAG was determined after a selective enzymatic digestion of isolated GAG using specific enzymes. Heparan sulfates were estimated after nitrous acid treatment. The concentration of GAG was measured spectrophotometrically by forming colored complexes with Alcian blue dye. In addition, the specific activity (dpm/microgram GAG) and the rate of GAG synthesis (ng/mg dry defatted tissue/day) were determined. The results indicate that the rate of GAG synthesis by de-endothelialized neointima (DEA) was twice that of intact aorta (control). In the re-endothelialized neointima (REA), the GAG synthetic rate was three times more than in control. However, the release of GAG into medium from REA accounts for only 25% of the GAG synthesized by this tissue type, and the release from DEA accounts for 60% of the synthesized GAG. Similarly, a threefold increase in the GAG concentration in REA compared to control was found. The relative distribution as chondroitin-6-sulfate (C6S), chondroitin-4-sulfate (C4S), dermatan sulfate (DS), heparan sulfate (HS) and hyaluronic acid (HA) was markedly altered in the injured neointima. There was an increase in chondroitin sulfates (CS) and DS concomitant with a decrease in HS. It is concluded that injury to aortic endothelium induces stimulation of GAG synthesis in the arterial wall. Furthermore, the greater release of GAG from DEA, compared to control and REA, suggests that endothelium may function as a "reverse" barrier in the neointima covered by regenerated endothelium.     

Genetic Heterogeneity and Clinical Variability in Musculocontractural Ehlers–Danlos Syndrome Caused by Impaired Dermatan Sulfate Biosynthesis

Bi‐allelic variants in CHST14, encoding dermatan 4‐O‐sulfotransferase‐1 (D4ST1), cause musculocontractural Ehlers–Danlos syndrome (MC‐EDS), a recessive disorder characterized by connective tissue fragility, craniofacial abnormalities, congenital contractures, and developmental anomalies. Recently, the identification of bi‐allelic variants in DSE, encoding dermatan sulfate epimerase‐1 (DS‐epi1), in a child with MC‐EDS features, suggested locus heterogeneity for this condition. DS‐epi1 and D4ST1 are crucial for biosynthesis of dermatan sulfate (DS) moieties in the hybrid chondroitin sulfate (CS)/DS glycosaminoglycans (GAGs). Here, we report four novel families with severe MC‐EDS caused by unique homozygous CHST14 variants and the second family with a homozygous DSE missense variant, presenting a somewhat milder MC‐EDS phenotype. The glycanation of the dermal DS proteoglycan decorin is impaired in fibroblasts from D4ST1‐ as well as DS‐epi1‐deficient patients. However, in D4ST1‐deficiency, the decorin GAG is completely replaced by CS, whereas in DS‐epi1‐deficiency, still some DS moieties are present. The multisystemic abnormalities observed in our patients support a tight spatiotemporal control of the balance between CS and DS, which is crucial for multiple processes including cell differentiation, organ development, cell migration, coagulation, and connective tissue integrity.     

Concepts for preparation of novel regioselective modified cellulose derivatives sulfated,aminated, carboxylated and acetylated for hemocompatible ultrathin coatings on biomaterials

Some regioselective cellulose derivatives were synthesized on the basis of the just started longterm concept by variation of O—SO₃, N—SO₃, N—Ac, and N‐carboxymethyl groups solely or in combination for the development of athrombogenic and antithrombogenic nanocoatings on cellulose membranes. Similar regioselectively arranged functional groups are known partially in type 1 glycosaminoglycane (dermatan sulfate DS, and chondroitin sulfate CS) and all the groups are present in type 2 GAG (heparin HE and heparansulfat HS). The goal of this concept was to use instead of the backbone structure β 1–> 4, β 1–>3 (DS, CS) or β 1–> 4, α 1–> 4 (HE, HS or regioselective desulfated HE), the β 1–> 4 backbone structure of cellulose or chitosan, with the same functional groups as in HE. Furthermore different regioselective sulfated cellulose derivatives, such as cellulose‐3‐sulfate, cellulose‐2,6‐disulfate, and cellulose‐2,3‐sulfates with varied sulfation degrees have been synthesized and immobilized ionically on cellulose membranes. They were tested concerning their reduction of platelet adhesion from citrated whole blood in a perfusion system as well for AT III affinity. The lowest platelet adhesion and AT III affinity was observed using cellulose‐2,3‐sulfates and cellulose‐3‐sulfate derivatives, whereas cellulose‐6‐O‐sulfate derivatives show high AT III affinity and high platelet adhesion. This means that a high 6‐O‐sulfate content seems to promote anticoagulant properties of the derivatives with high (AT III) affinity, whereas low concentrations of 2‐O‐ and 3‐O‐sulfate groups show the lowest platelet adhesion. The latter seems to be important for developing athrombogenic coatings for biomaterials. The starting material chitosan with molecular weight of 150 KD was used to synthesize additionally some of 2‐deoxyaminocellulose derivatives containing N—Ac or N—SO₃ or N—CH₂—COOH and O—SO₃ groups. These derivatives are structurally closer to HE and HS and should enable us to work out the influence of each heparin like functional group in cellulose derivative on the athrombogenic and antithrombogenic properties. We synthesized only some of the latter mentioned derivatives with a distribution of the functional groups according to a Bernoulli statistics. Totally sulfated 2‐deoxyaminocellulose‐3,6‐disulfate derivatives will be regioselectively desulfated by reactions known from heparin chemistry.     

Histochemical studies on glycosaminoglycans in Bruch's membrane of postnatal rat eyes

Localization of glycosaminoglycans (GAG) in Bruch's membrane of postnatal rat eyeballs was examined histochemically. Fixed eyeballs from postnatal rats (ages 5 days and 8 weeks) were routinely processed and embedded in paraffin wax or Quetol 651 resin. Paraffin-embedded tissue sections were stained with hematoxylin and eosin or sensitized high iron diamine procedure in combination with selective methods such as GAG-degrading enzyme digestions and/or a chemical modification, and examined by light microscopy. Quetol 651-embedded ultrathin sections were stained with heavy metals and examined by electron microscopy. In rats at postnatal day 5, Bruch's membrane contained mainly chondroitin sulfate (CS) and heparan sulfate (HS). In contrast, at 8 weeks after birth the membrane included a large amount of dermatan sulfate (DS) and HS. According to electron microscopic findings, Bruch's membrane on day 5 consisted of only 3 layers without a central elastic layer. However, at 8 weeks after birth the membrane was constructed of 5 layers. These findings suggested that the difference in GAG molecular species in the membranes at 5 days and at 8 weeks after birth could be correlated with the development and maturation of the collagenous layer in Bruch's membrane. Moreover, maturation of Bruch's membrane may contributes to the architectural stabilization of the outer portions of the photoreceptor cells.     

Loss‐of‐function mutations of CHST14 in a new type of Ehlers‐Danlos syndrome

Ehlers‐Danlos syndrome (EDS) is a heterogeneous connective tissue disorder involving skin and joint laxity and tissue fragility. A new type of EDS, similar to kyphoscoliosis type but without lysyl hydroxylase deficiency, has been investigated. We have identified a homozygous CHST14 (carbohydrate sulfotransferase 14) mutation in the two familial cases and compound heterozygous mutations in four sporadic cases. CHST14 encodes dermatan 4‐O‐sulfotransferase 1 (D4ST1), which transfers active sulfate from 3′‐phosphoadenosine 5′‐phosphosulfate to position 4 of the N‐acetyl‐D‐galactosamine (GalNAc) residues of dermatan sulfate (DS). Transfection experiments of mutants and enzyme assays using fibroblast lysates of patients showed the loss of D4ST1 activity. CHST14 mutations altered the glycosaminoglycan (GAG) components in patients' fibroblasts. Interestingly, DS of decorin proteoglycan, a key regulator of collagen fibril assembly, was completely lost and replaced by chondroitin sulfate (CS) in the patients' fibroblasts, leading to decreased flexibility of GAG chains. The loss of the decorin DS proteoglycan due to CHST14 mutations may preclude proper collagen bundle formation or maintenance of collagen bundles while the sizes and shapes of collagen fibrils are unchanged as observed in the patients' dermal tissues. These findings indicate the important role of decorin DS in the extracellular matrix and a novel pathomechanism in EDS. Hum Mutat 31:1–9, 2010. © 2010 Wiley‐Liss, Inc.     

Heparan sulfate proteoglycan in diffuse plaques of hippocampus but not of cerebellum in Alzheimer's disease brain.

Previous studies have shown the basement membrane form of heparan sulfate proteoglycan (HSPG) known as perlecan, co-localized to beta-amyloid protein (A beta)-containing amyloid deposits in brains of patients with Alzheimer's disease (AD) and Down's syndrome. Although HSPG was localized to diffuse A beta plaques in hippocampus, amygdala, and neocortex, it is not known whether they are present in diffuse A beta plaques in cerebellum. In the present study, Alcian blue staining and immunocytochemical techniques were used to determine whether highly sulfated glycosaminoglycans (GAGs) and/or HSPG (perlecan) were also present in diffuse A beta plaques of cerebellum. Tissues from cases of AD were examined for the co-localization of highly sulfated GAGs, HSPGs, and A beta in diffuse plaques in cerebellum in comparison with hippocampus. Consecutive serial sections of AD brain tissue were stained or immunostained with 1) the modified Bielschowsky stain; 2) a polyclonal antibody directed against synthetic A beta (1-40); 3) Congo red; 4) Alcian blue (pH 5.7) with varying concentrations of magnesium chloride for identification of sulfated and highly sulfated GAGs; and 5) polyclonal and monoclonal antibodies recognizing either the core protein or a specific GAG epitope on perlecan. All cases (7 of 7) of AD contained diffuse A beta plaques in the cerebellum as identified by positive Bielschowsky staining and A beta immunoreactivity. None of these cases demonstrated positive Alcian blue staining (at 0.3 and 0.7 mol/L MgCl2), HSPG, or HS GAG immunoreactivity in the same diffuse cerebellar plaques on adjacent serial sections. However, Alcian blue staining, HSPG, and/or HS GAG immunoreactivity were observed in blood vessel walls, choroid plexus, and within Purkinje cells, suggesting that the techniques used were reliable and specific. In cerebellum, all plaques containing amyloid cores that were Congo red-positive were also positive for highly sulfated GAGs (by Alcian blue staining at 0.7 mol/L MgCl2) and HSPG (both core protein and GAG chain) immunoreactivity. Even though HSPG immunoreactivity was not present in cerebellar diffuse plaques, all cases (4 of 4) examined demonstrated HSPG (both core protein and GAG chain) immunoreactivity in diffuse A beta plaques in hippocampus. Therefore, by Alcian blue staining and immunocytochemical methods, highly sulfated GAGs and HSPGs are not present in A beta diffuse plaques in cerebellum. Since previous studies indicate that the cerebellum contains relatively few amyloid-containing plaques in comparison with diffuse plaques, these studies suggest that HSPG may be an essential component needed for amyloid formation and/or persistence in brain as observed in cortical areas.(ABSTRACT TRUNCATED AT 400 WORDS)     

Behavior of hyaluronic acid from gingival epithelium and connective tissue on the analytical ultracentrifuge

The molecular weights of hyaluronic acid (HA) isolated from separated specimens of human gingival epithelium and connective tissue as well as standard hyaluronic acid preparations have been estimated. The values were determined following substitution of sedimentation values into a previously determined empirical relationship between the reciprocal of the sedimentation coefficient at zero concentration (S-1)0 and molecular weights estimated by sedimentation-diffusion (MsD). The values of (S-1)0 for connective tissue and standard low-molecular weight HA preparations obtained by linear regression of all points indicated molecular weights (MsD) of 340,000 and 205,000 respectively. However, epithelial and standard high-molecular weight HA behaved differently during ultracentrifugation generating a curvilinear relationship between s-1 and concentration. Nevertheless linear extrapolation of a line of best fit of the very lowest concentrations (those which approached zero concentration) provided molecular weight estimates of 860,000 and 2,500,000 respectively. Moreover, similar treatment of s-1 values derived from the previously published data of Laurent, Ryan and Pietruszkiewicz has validated the use of linear regression of s-1 at the lower concentrations alone, to calculate high-molecular weight HA. The curvilinear relationship for s-1 throughout the whole concentration range (0.15-2.3 mg/ml) has been regarded only as a qualitative indication that the HA samples are of relatively high-molecular weight, while a straight line through such data points implies a qualitatively lower molecular weight for HA.     

Identification of glycosaminoglycans in human airway secretions

Glycosaminoglycans (GAGs), known to be present in airway mucus, are macromolecules with a variety of structural and biological functions. In the present work, we used fluorophore-assisted carbohydrate electrophoresis (FACE) to identify and relatively quantify GAGs in human tracheal aspirates (HTA) obtained from healthy volunteers. Primary cultures of normal human bronchial epithelial (NHBE) and submucosal gland (SMG) cells were used to assess their differential contribution to GAGs in mucus. Distribution was further assessed by immunofluorescence in human trachea tissue sections and in cell cultures. HTA samples contained keratan sulfate (KS), chondroitin/dermatan sulfate (CS/DS), and hyaluronan (HA), whereas heparan sulfate (HS) was not detected. SMG cultures secreted CS/DS and HA, CS/DS being the most abundant GAGs in these cultures. NHBE cells synthesized KS, HA, and CS/DS. Confocal microscopy showed that KS was exclusively found at the apical border of NHBE cells and on the apical surface of ciliated epithelial cells in tracheal tissues. CS/DS and HA were present in both NHBE and SMG cells. HS was only found in the extracellular matrix in trachea tissue sections. In summary, HTA samples contain KS, CS/DS, and HA, mirroring a mixture of secretions originated in surface epithelial cells and SMGs. We conclude that surface epithelium is responsible for most HA and all KS present in secretions, whereas glands secrete most of CS/DS. These data suggest that, in diseases where the contribution to secretions of glands versus epithelial cells is altered, the relative concentration of individual GAGs, and therefore their biological activities, will also be affected.     

Differences in the synthesis and secretion of sulfated glycosaminoglycans by aorta explant monolayers cultured from atherosclerosis-susceptible and -resistant pigeons.

The synthesis and secretion of sulfated glycosaminoglycans (GAGs) by aorta explant monolayers cultured from atherosclerosis-susceptible White Carneau (WC) and atherosclerosis resistant Show Racer SR pigeons have been compared. Primary cultures of WC pigeon aorta incorporation three to four times as much 35-S-sulfate into trichloroacetic acid (TCA) soluble, nondialyzable material that is over 90% sensitive to chondroitinase ABC digestion when compared with parallel cultures of SR pigeon aorta. Qualitatively, the GAGs produced by WC and SR aorta explants were similar in that their electrophoretic profiles were characterized by a prominent slow migrating band that did not coelectrophorese with known GAG standards, a discrete hyaluronic acid and heparan sulfate band and a broad band containing dermatan sulfate and chondroitin 4- and 6-sulfate. Enzyme digestion of the labeled material revealed that cultures of each breed synthesized and secreted predominantly chondroitin sulfate (approximately 60%) with moderate amounts of dermatan sulfate (approximately 35%) and little heparan sulfate (< 5%). This pattern of GAG distribution resembled that of GAGs present in pigeon aortas in vivo. Although differences in the relative percentages of each type of GAG produced by aorta explant cultures from each breed were not evident, densitometric tracings and radioisotopic activity of the electrophoretically separated GAGs indicate more sulfated GAG of each type present in WC as compared with SR cultures. Glycosaminoglycan-containing proteoglycans were also demonstrated morphologically in the aorta explant monolayers of each breed and resembled aortic proteoglycans in vivo. Proteoglycans in vitro existed as discrete 200-500-A polygonal granules, exhibited a marked affinity for ruthenium red, were intimately associated with each other through filamentous projections as well as with other components of the intercellular matrix (collagen and elastic fiber), and were completely sensitive to chondroitinase ABC digestion. This culture system is offered as a useful model for future investigations concerned with relating GAG metabolism to susceptibility to atherosclerosis.     

High-molecular-weight proteins of nontypeable Haemophilus influenzae mediate bacterial adhesion to cellular proteoglycans.

A family of high-molecular-weight (HMW) surface-exposed proteins of nontypeable Haemophilus influenzae (NT H. influenzae) mediated adherence of these organisms to human epithelium. To better understand the molecular basis for this adherence, the role of glycosaminoglycans (GAGs), substances commonly expressed on cell surfaces, was examined. Bacterial adherence to cells with specific deficiencies in GAG biosynthesis was measured. HMW protein-dependent bacterial adherence to normal cells was significantly greater than adherence to cells deficient in sulfated GAGs or to cells deficient in heparan sulfate but overexpressing chondroitin sulfate. Cells expressing undersulfated heparan sulfate exhibited intermediate levels of bacterial adherence. The addition of exogenous dextran sulfate or heparin inhibited over 70% of the adherence of NT H. influenzae to normal cells, whereas hyaluronic acid and chondroitin sulfate tested at the same concentration (100 micrograms/ml) inhibited bacterial adherence by less than 11%. Treatment of cells with heparinase significantly reduced bacterial adherence. Following electrophoretic separation, HMW proteins were shown to bind directly to radiolabeled heparin. These results indicate that HMW protein-dependent adherence of NT H. influenzae is mediated by cellular sulfated GAGs and that heparan sulfate may be the predominant GAG involved in this process. However, the decreased adherence of bacteria to cells expressing undersulfated heparan sulfate and the inhibition of bacterial adherence by the addition of exogenous dextran sulfate suggest that bacterial adhesion to mammalian cells is likely to be influenced by a variety of factors, including the degree of sulfation and the specificity of the carbohydrate moieties contained in the cellular proteoglycans.     

Change in Decorin during Aging of Rat Placenta

By immunohistochemistry, with or without chondroitinases, decorin was found to be distributed in the extracellular matrix of chorionic villi and amnia. The strength of staining intensified with increasing gestational age. Decorin was isolated from the placenta of 13- to 20-day-old pregnant rats and identified by Western blotting, using an antidecorin core protein antibody. The molecular weight of decorin is ～100 kDa, whereas the respective figures for the core protein treated with chondroitinase (chase) ABC and with chase B are ～40 kDa and 43 kDa. The difference in the molecular weight between the core protein with chase ABC and B suggests that the glycosaminoglycan (GAG)- base structure on the core protein was chondroitin sulfate (CS) without dermatan sulfate (DS). The decorin content and the proportion of CS to DS in GAG increased with age. We concluded that the age-related changes in the GAG chain may be related to specific functional properties and may have a crucial role in placental tissue organization.     

Facile surface functionalization with glycosaminoglycans by direct coating with mussel adhesive protein

The use of mussel adhesive proteins (MAPs) as a surface coating for cell adhesion has been suggested due to their unique properties of biocompatibility and effective adhesion on diverse inorganic and organic surfaces. The surface functionalization of scaffolds or implants using extracellular matrix (ECM) molecules is important for the enhancement of target cell behaviors such as proliferation and differentiation. In the present work, we suggest a new, simple surface functionalization platform based on the charge interactions between the positively charged MAP linker and negatively charged ECM molecules, such as glycosaminoglycans (GAGs). MAP was efficiently coated onto a titanium model surface using its adhesion ability. Then, several GAG molecules, including hyaluronic acid (HA), heparin sulfate (HS), chondroitin sulfate (CS), and dermatan sulfate (DS), were effectively immobilized on the MAP-coated surfaces by charge interactions. Using HA as a model GAG molecule, we found that the proliferation, spreading, and differentiation behaviors of mouse preosteoblast cells were all significantly improved on MAP/HA-layered titanium. In addition, we successfully constructed a multilayer film on a titanium surface with oppositely charged layer-by-layer coatings of MAP and HA. Collectively, our simple MAP-based surface functionalization strategy can be successfully used for the efficient surface immobilization of negatively charged ECM molecules in various tissue engineering and medical implantation applications.     

Ultrastructural and biochemical aspects of the Sanfilippo syndrome,--type III genetic mucopolysaccharidosis

The Sanfilippo Syndrome (SS) is a recessively inherited connective tissue disorder expressed in early life. It is classified as a genetic mucopolysaccharidosis (MPS) because the underlying defect involves the catabolism of heparan sulfate (HS), one of the glycosaminoglycans (GAG). Four variant forms, i.e., type A, B, C, and D, each associated with a different enzymatic defect, have been recognized in affected children. Biochemical studies show that characteristically HS accounts for most of the increased amounts of GAG excreted in the urine and those stored in viscera and brain. Gangliosides GM2, GM3 and GD2 are elevated considerably in the brain. Morphologically, the very water-soluble substances accumulating in the viscera are metachromatic, and consist ultrastructurally of finely granulo-floccular (or filamentous) material which is bound in cytoplasmic vacuoles. These substances are considered to represent the GAG. In the central nervous system (CNS) the stored substances are not soluble in water or alcohol and xylol, give a PAS-positive reaction, and stain for lipids and with luxol fast blue. Ultrastructurally, they consist of membranous arrays which often are of the "zebra body" variety. The CNS-inclusions are considered to represent the stored gangliosides; they were found also in small numbers in viscera of older children with SS. The search for a common denominator in the pathogenetic mechanism(s) culminating in both types of inclusions, continues.     

The glycosaminoglycans of Dupuytren's disease

The total and individual glycosaminoglycan (GAG) content at various stages of the Dupuytren disease process and in samples of normal palmar connective tissue (palmar dermis, palmar fascia and digital flexor tendon) from the hands of uninvolved age-matched controls have been assayed and compared. Morphological comparisons between the different tissues were made by histological examination of sections stained to demonstrate collagen fiber patterns and glycosaminoglycan distribution. Significant differences in the type and amount of GAG were found between the various manifestations of the disease process, i.e., nodules, cellular and fibrous bands, and between these and the normal palmar connective tissues. In the most actively proliferating cellular regions chondroitin sulfate levels were 11 times greater than those of the normal palmar connective tissues, whereas dermatan sulfate tissue levels showed a fourfold increase. On the other hand, tissue concentrations of hyaluronate were similar to those of normal palmar connective tissue. The relationship of these differences in GAG levels to the development and maturation of the normal palmar connective tissues and the Dupuytren's process is discussed.     

Change in decorin during aging of rat placenta

By immunohistochemistry, with or without chondroitinases, decorin was found to be distributed in the extracellular matrix of chorionic villi and amnia. The strength of staining intensified with increasing gestational age. Decorin was isolated from the placenta of 13- to 20-day-old pregnant rats and identified by Western blotting, using an antidecorin core protein antibody. The molecular weight of decorin is approximately 100 kDa, whereas the respective figures for the core protein treated with chondroitinase (chase) ABC and with chase B are approximately 40 kDa and 43 kDa. The difference in the molecular weight between the core protein with chase ABC and B suggests that the glycosaminoglycan (GAG)- base structure on the core protein was chondroitin sulfate (CS) without dermatan sulfate (DS). The decorin content and the proportion of CS to DS in GAG increased with age. We concluded that the age-related changes in the GAG chain may be related to specific functional properties and may have a crucial role in placental tissue organization.     

The Glycosaminoglycans of Dupuytren's Disease

The total and individual glycosaminoglycan (GAG) content at various stages of the Dupuytren disease process and in samples of normal palmar connective tissue (palmar dermis, palmar fascia and digital flexor tendon) from the hands of uninvolved age-matched controls have been assayed and compared. Morphological comparisons between the different tissues were made by histological examination of sections stained to demonstrate collagen fiber patterns and glycosaminoglycan distribution.

Significant differences in the type and amount of GAG were found between the various manifestations of the disease process, i.e., nodules, cellular and fibrous bands, and between these and the normal palmar connective tissues.

In the most actively proliferating cellular regions chondroitin sulfate levels were 11 times greater than those of the normal palmar connective tissues, whereas dermatan sulfate tissue levels showed a fourfold increase. On the other hand, tissue concentrations of hyaluronate were similar to those of normal palmar connective tissue.

The relationship of these differences in GAG levels to the development and maturation of the normal palmar connective tissues and the Dupuytren's process is discussed.     

Rational development of GAG-augmented chitosan membranes by fractional factorial design methodology

To develop a novel biomaterial for chondrocyte culture, 8 glycosaminoglycan (GAG)/chitosan membranes (groups N1-N8) were prepared, with the aid of a 2-level 2(4-1) fractional factorial design, by co-immobilizing chondroitin-4-sulfate (CSA), chondroitin-6-sulfate (CSC), dermatan sulfate (DS), and heparin to chitosan membranes. The fractional factorial design allowed us to partly interpret the effects of individual GAGs and two-way interactions between GAGs. Within the level range of -1 and +1, low CSA level (2.6 mg) is favorable for collagen synthesis but not for cell proliferation. High CSC level (1.3 mg) is favorable for GAG production but not for cell proliferation. Conversely, high heparin (0.33 mg) and DS (0.13 mg) levels are desired for cell proliferation but not for the production of collagen and GAG. Moreover, the two-way interactions between GAGs influence the cell behavior. Among the 8 GAG/chitosan membranes, N1 and N4 (containing low CSA and heparin levels) lead to the maintenance of proper chondrocyte phenotype, as judged by the chondrocyte-like morphology, modest cell expansion, higher GAG and collagen production and proper cartilage marker gene expression. In conclusion, this approach provides a means of rationally predicting and evaluating the proper formulation of GAG/chitosan membranes and may facilitate the rational design of other tissue engineering scaffolds.