Interferon-gamma inhibits 35S incorporation in heparan sulfate synthesized by human skin fibroblasts

Glycosaminoglycans synthesized by human skin fibroblasts were simultaneously radiolabelled with D-[1-(3H)]glucosamine and Na2(35)SO4. Considering 3H incorporation, we found that IFNgamma increased the production of glycosaminoglycan synthesis, including hyaluronic acid, heparan and chondroitin/dermatan sulfate. In contrast, the production of heparan and chondroitin/dermatan sulfate was slightly decreased on the basis of the 35S signal. Furthermore, when heparan sulfate was treated with nitrous acid, the release of free 35S was greater in control than in treated cells, although the 3H patterns of depolymerization with this agent were similar. These data demonstrate that IFNgamma inhibits the incorporation of sulfate from extracellular medium into heparan sulfate.     

Synthesis of corneal keratan sulfate proteoglycans by bovine keratocytes in vitro

Keratan sulfate proteoglycans (KSPGs) are the major proteoglycans of the cornea and are secreted by keratocytes in the corneal stroma. Previous studies have been able to show only transient secretion of KSPG in cell culture. In this study, cultures of bovine keratocytes were found to secrete the three previously characterized KSPG proteins into culture medium. Reactivity with monoclonal antibody I22 demonstrated substitution of these proteins with keratan sulfate chains. KSPG constituted 15% of the proteoglycan metabolically labeled with [35S]sulfate in keratocyte culture medium. This labeled KSPG contained keratan sulfate chains of 4700 Da compared to 21,000 Da for bovine corneal keratan sulfate. Labeled keratan sulfate from cultures contained nonsulfated, monosulfated, and disulfated disaccharides that were released by digestion with endo-beta-galactosidase or keratanase II. Nonsulfated disaccharides were relatively more abundant in keratan sulfate from culture than in corneal keratan sulfate. These results show that cultured bovine keratocytes maintain the ability to express all three of the known KSPG proteins, modified with keratan sulfate chains and sulfated on both N-acetylglucosamine and galactose moieties. KSPG made in vitro differs from that found in vivo in the length and sulfation of its keratan sulfate chains. The availability of cell cultures secreting corneal keratan sulfate proteoglycans provides an opportunity to examine biosynthesis and control of this important class of molecules.     

The biosynthesis of glycosaminoglycans in normal rat liver and in response to experimental hepatic injury

The synthesis of glycosaminoglycans in slices from normal and acutely injured rat liver was studied. The rates of incorporation of [14C]-glucosamine into specific types of glycosaminoglycans varied markedly; nearly 90% was incorporated into a fraction containing predominantly heparan sulfate and far less if any heparin; about 9.5% was incorporated into chondroitin 4-and 6-sulfate, and only 0.2% of the radioactivity was found in hyaluronic acid. The rate of synthesis of a fraction having several of the characteristics of keratan sulfate comprised only 0.3% of the synthesis of total glycosaminoglycans. No [14C]hexosamine was incorporated into dermatan sulfate. Following acute hepatic injury, the synthesis of glycosaminoglycans was stimulated by 80 to 100%, and the proportions of various types changed. If calculated on the basis of the specific activity of the precursors of glycosaminoglycans, which was found to be strongly reduced in injured liver, the maximum enhancement of total glycosaminoglycan synthesis was 6.6-fold 5 days after onset of liver injury.     

Purification of chondroitin 6-sulfotransferase secreted from cultured chick embryo chondrocytes

Chondroitin 6-sulfotransferase, which transfers sulfate from 3'-phosphoadenylyl sulfate to position 6 of N-acetylgalactosamine in chondroitin, was purified 1,430-fold to apparent homogeneity with a 22% yield from the serum-free culture medium of chick embryo chondrocytes by affinity chromatography on heparin-Sepharose CL-6B, wheat germ agglutinin-agarose, and 3',5'-ADP-agarose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed a single broad protein band with an apparent molecular weight of 75,000. Since the purified enzyme has an apparent molecular weight of 160,000 as judged by gel chromatography on Superose 12, the active form of chondroitin 6-sulfotransferase may be a dimer. The purified enzyme transferred sulfate to chondroitin, chondroitin sulfate, and corneal keratan sulfate. Chondroitin sulfate E from squid cartilage, dermatan, sulfate, and heparan sulfate hardly served as acceptors of the sulfotransferase. The sulfated product derived from keratan sulfate was degraded by keratanase but not by chondroitinase ABC.     

Hypereosinophilic syndrome in a child mosaic for a congenital triplication of the short arm of chromosome 8

Fibroblast growth factors are important heparin binding, mitogenic proteins. The binding site in heparin and heparan sulfate for fibroblast growth factor-2 (basic fibroblast growth factor) has been described as rich in glucosamine-2-sulfate 1-->4 linked to iduronic acid-2-sulfate. The glucosamine residue in the heparin binding site is also 6-sulfated. A new glycosaminoglycan, acharan sulfate, has been chemically modified to prepare a polysaccharide, N-sulfoacharan sulfate, consisting of glucosamine-2-sulfate 1-->4 linked to iduronic acid-2-sulfate. Acharan sulfate binds very weakly to fibroblast growth factor-2 while N-sulfoacharan sulfate binds with nearly the same affinity as heparin. Mitogenicity studies were performed using heparan sulfate-free cells stably transfected with fibroblast growth factor receptor-1. Acharan sulfate inhibits heparin's enhancement of fibroblast growth factor-2 mitogenic activity, without affecting cell viability, while N-sulfoacharan sulfate shows heparin-like activity but at a greatly reduced level. These results suggest additional mechanisms not requiring high affinity glycosaminoglycan binding to fibroblast growth factor-2 may be important in its mitogenic activity.     

Foam cell conversion of macrophages alters the biosynthesis of heparan sulfate

Heparan sulfate is thought to regulate the biological activities of several proteins implicated in the pathogenesis of atherosclerosis. While the interactions of heparan sulfate with lipoprotein lipase and various growth factors have been actively studied, little is known of the cellular regulation of heparan sulfate biosynthesis in response to lipid accumulation. We have investigated heparan sulfate biosynthesis during conversion of murine J774 macrophages into lipid-laden foam cells. Such conversion is shown to accelerate the rate of glycosaminoglycan synthesis and the transport of newly synthesized proteoglycans into the medium. Moreover, the structure of heparan sulfate is specifically altered due to an approximately 30% increase in the 6-O-sulfation of glucosamine residues within the N-sulfated heparan sulfate domains, whereas the sulfation of chondroitin sulfate remains unaffected. These results suggest a selective effect of foam cell conversion on the biosynthesis of heparan sulfate.     

Activation of the autophagy, c-FOS and ubiquitin expression, and nucleolar alterations in Schwann cells precede demyelination in tellurium-induced neuropathy

The method of affinity coelectrophoresis was used to study the binding of nine representative glycosaminoglycan (GAG)-binding proteins, all thought to play roles in nervous system development, to GAGs and proteoglycans isolated from developing rat brain. Binding to heparin and non-neural heparan and chondroitin sulfates was also measured. All nine proteins-laminin-1, fibronectin, thrombospondin-1, NCAM, L1, protease nexin-1, urokinase plasminogen activator, thrombin, and fibroblast growth factor-2-bound brain heparan sulfate less strongly than heparin, but the degree of difference in affinity varied considerably. Protease nexin-1 bound brain heparan sulfate only 1.8-fold less tightly than heparin (Kdvalues of 35 vs. 20 nM, respectively), whereas NCAM and L1 bound heparin well (Kd approximately 140 nM) but failed to bind detectably to brain heparan sulfate (Kd>3 microM). Four proteins bound brain chondroitin sulfate, with affinities equal to or a few fold stronger than the same proteins displayed toward cartilage chondroitin sulfate. Overall, the highest affinities were observed with intact heparan sulfate proteoglycans: laminin-1's affinities for the proteoglycans cerebroglycan (glypican-2), glypican-1 and syndecan-3 were 300- to 1800-fold stronger than its affinity for brain heparan sulfate. In contrast, the affinities of fibroblast growth factor-2 for cerebroglycan and for brain heparan sulfate were similar. Interestingly, partial proteolysis of cerebroglycan resulted in a >400-fold loss of laminin affinity. These data support the views that (1) GAG-binding proteins can be differentially sensitive to variations in GAG structure, and (2) core proteins can have dramatic, ligand-specific influences on protein-proteoglycan interactions.     

Complexing of fibronectin glycosaminoglycans and collagen

Collagen-fibronectin complexes, formed by binding of fibronectin to gelatin or collagen insolubilized on Sepharose, were found to bind 20-40% of radioactivity in [35S]heparin. Fibronectin attached directly to Sepharose also bound [35S]heparin, while gelatin-Sepharose without fibronectin did not. Unlabeled heparin and highly sulfated heparan sulfate efficiently inhibited the binding of [35S]heparin, hyaluronic acid and dermatan sulfate were slightly inhibitory, while chondroitin sulfates and heparan sulfate with a low sulfate content did not inhibit. The interaction of heparin with fibronectin bound to gelatin resulted in complexes which required higher concentrations of urea to dissociate than complexes of fibronectin and gelatin alone. Heparin as well as highly sulfate heparan sulfate and hyaluronic acid brought about agglutination of plastic beads coated with gelatin when fibronectin was present. Neither fibronectin nor glycosaminoglycans alone agglutinated the beads. It is proposed that the multiple interactions of fibronectin, collagen and glycosaminoglycans revealed in these assays could play a role in the deposition of these substances as an insoluble extracellular matrix. Alterations of the quality or quantity of any one of these components could have important effects on cell surface interactions, including the lack of cell surface fibronectin in malignant cells.     

Thermoresponsive release from poly(Glu(OMe))-block-poly(Sar) microcapsules with surface-grafting of poly(N-isopropylacrylamide)

We have designed a synthetic cornea that has a transparent hydrogel optic and a porous skirt. The device has been implanted in rabbit corneas. We have shown that keratocytes migrate into the device and deposit a complex extracellular matrix. The immediate response is detected in the surrounding stroma, and the secondary response is seen after cells have deposited a matrix in the disc. After implantation, a decrease in keratan sulfate accompanied by an increase in dermatan sulfate was detected in the surrounding tissue compared to the unwounded corneal stroma. The glycosaminoglycans in the disc resemble that of an injured stroma. The appearance of heparan sulfate and growth factors, bFGF and TGFbeta, was not detected until 6 weeks after implantation. The growth factors were detected at the interface between the device and the tissue and become more diffuse over time. Methods of controlled release in vivo were used to enhance the rate of fibroplasia and wound repair. While these were successful in the cornea itself, when combined with the synthetic cornea the response was magnified and the initial attempts yielded neovascularization and edema. Currently, efforts are being directed at controlling the release within the porous haptic so that fibroplasia is enhanced while minimizing an inflammatory response.     

A new glycosaminoglycan from the giant African snail Achatina fulica

A new glycosaminoglycan has been isolated from the giant African snail Achatina fulica. This polysaccharide had a molecular weight of 29,000, calculated based on the viscometry, and a uniform repeating disaccharide structure of -->4)-2-acetyl,2-deoxy-alpha-D-glucopyranose (1-->4)-2-sulfo-alpha-L-idopyranosyluronic acid (1-->. This polysaccharide represents a new, previously undescribed glycosaminoglycan. It is related to the heparin and heparan sulfate families of glycosaminoglycans but is distinctly different from all known members of these classes of glycosaminoglycans. The structure of this polysaccharide, with adjacent N-acetylglucosamine and 2-sulfo-iduronic acid residues, also poses interesting questions about how it is made in light of our current understanding of the biosynthesis of heparin and heparan sulfate. This glycosaminoglycan represents 3-5% of the dry weight of this snail's soft body tissues, suggesting important biological roles for the survival of this organism, and may offer new means to control this pest. Snail glycosaminoglycan tightly binds divalent cations, such as copper(II), suggesting a primary role in metal uptake in the snail. Finally, this new polysaccharide might be applied, like the Escherichia coli K5 capsular polysaccharide, to the study of glycosaminoglycan biosynthesis and to the semisynthesis of new glycosaminoglycan analogs having important biological activities.     

Chemical sulfonation and anticoagulant activity of acharan sulfate

Acharan sulfate is a glycosaminoglycan prepared from the giant African snail, Achatina fulica. This polysaccharide has a repeating disaccharide structure of -->4)-2-deoxy-2-acetamido-alpha-D-glucopyranose (1-->4)-2-sulfo-alpha-L-idopyranosyluronic acid (1-->). Its structure is related to heparin and heparan sulfate but is distinctly different from all known members of these classes of glycosaminoglycans. Because of its structural similarities to heparin, chemically modified acharan sulfate was studied to understand the chemical structure effected its anticoagulant activity. After de-N-acetylation, acharan sulfate was N-sulfonated using either chlorosulfonic acid-pyridine or sulfur trioxide-trimethylamine complex. The sulfate level in these products ranged from 22 to 24%(w/w), significantly less than that of heparin at 36%. The molecular weight of both N-sulfoacharan sulfates were comparable with that of heparin. In vitro anticoagulant activity assays showed that N-sulfoacharan sulfate derivatives were moderately active for the inhibition of thrombin and neither product showed any measurable anti-factor Xa activity. The differences in the activities of N-sulfoacharan sulfates produced by these two methods are probably ascribable to a small level of concomitant O-sulfonation obtained when using chlorosulfonic acid-pyridine.     

Synthesis of glycosaminoglycans by human skin fibroblasts cultured on collagen gels

A comparison has been made of the synthesis of glycosaminoglycans by human skin fibroblasts cultured on plastic or collagen gel substrata. Confluent cultures were incubated with [3H]glucosamine and Na235SO4 for 48h. Radiolabelled glycosaminoglycans were then analysed in the spent media and trypsin extracts from cells on plastic and in the medium, trypsin and collagenase extracts from cells on collagen gels. All enzyme extracts and spent media contained hyaluronic acid, heparan sulphate and dermatan sulphate. Hyaluronic acid was the main 3H-labelled component in media and enzyme extracts from cells on both substrata, although it was distributed mainly to the media fractions. Heparan sulphate was the major [35S]sulphated glycosaminoglycan in trypsin extracts of cells on plastic, and dermatan sulphate was the minor component. In contrast, dermatan sulphate was the principal [35S]sulphated glycosaminoglycan in trypsin and collagenase extracts of cells on collagen gels. The culture substratum also influenced the amounts of [35S]sulphated glycosaminoglycans in media and enzyme extracts. With cells on plastic, the medium contained most of the heparan sulphate (75%) and dermatan sulphate (> 90%), whereas the collagenase extract was the main source of heparan sulphate (60%) and dermatan sulphate (80%) from cells on collagen gels; when cells were grown on collagen, the medium contained only 5-20% of the total [35S]sulphated glycosaminoglycans. Depletion of the medium pool was probably caused by binding of [35S]sulphated glycosaminoglycans to the network of native collagen fibres that formed the insoluble fraction of the collagen gel. Furthermore, cells on collagen showed a 3-fold increase in dermatan sulphate synthesis, which could be due to a positive-feedback mechanism activated by the accumulation of dermatan sulphate in the microenvironment of the cultured cells. For comparative structural analyses of glycosaminoglycans synthesized on different substrata labelling experiments were carried out by incubating cells on plastic with [3H]glucosamine, and cells on collagen gels with [14C]glucosamine. Co-chromatography on DEAE-cellulose of mixed media and enzyme extracts showed that heparan sulphate from cells on collagen gels eluted at a lower salt concentration than did heparan sulphate from cells on plastic, whereas with dermatan sulphate the opposite result was obtained, with dermatan sulphate from cells on collagen eluting at a higher salt concentration than dermatan sulphate from cells on plastic. These differences did not correspond to changes in the molecular size of the glycosaminoglycan chains, but they may be caused by alterations in polymer sulphation.     

Differential binding of fibroblast growth factor-2 and -7 to basement membrane heparan sulfate: comparison of normal and abnormal human tissues

Fibroblast growth factors (FGFs) play multiple roles during development and in adult tissues as paracrine regulators of growth and differentiation. FGFs signal through transmembrane receptor tyrosine kinases, but heparan sulfate is also required for signaling by members of the FGF family. In addition, heparan sulfate may be involved in determining tissue distribution of FGFs. Using biotinylated FGF-2 and FGF-7 (KGF) as probes, we have identified specific interactions between FGFs and heparan sulfates in human tissues. Both FGF species bind to tissue mast cells and to epithelial cell membranes. Binding to basement membrane heparan sulfate is tissue source dependent and specific. Although FGF-2 strongly binds to basement membrane heparan sulfate in skin and most other tissue sites examined, FGF-7 fails to bind to basement membrane heparan sulfate in most locations. However, in subendothelial matrix in blood vessels and in the basement membrane of a papillary renal cell carcinoma, strong FGF-7 binding is seen. In summary, distinct and specific affinities of heparan sulfates for different FGFs were identified that may affect growth factor activation and local distribution. Heparan sulfate may have a gatekeeper function to either restrict or permit diffusion of heparin-binding growth factors across the basement membrane.     

Nonreducing end structures of chondroitin sulfate chains on aggrecan isolated from Swarm rat chondrosarcoma cultures

Chondrocyte cultures derived from the Swarm rat chondrosarcoma were metabolically labeled with [35S]sulfate or [6-3H]GlcN. Radiolabeled aggrecan was purified from the cell layer and exhaustively digested with chondroitin ABC lyase. Digestion products were resolved into disaccharide and monosaccharide residues using Toyopearl HW40S chromatography. The separated saccharide pools were reduced with NaBH4 and applied onto a CarboPac PA1 column to resolve all of the internal disaccharide alditols (unsaturated) from the nonreducing end disaccharide (saturated) and monosaccharide alditols. Mercuric acetate treatment was used prior to carbohydrate analysis to identify unambiguously the saturated from the unsaturated disaccharides. The chondroitin sulfate (CS) chains from these aggrecan preparations contained: (a) an internal disaccharide composition of unsulfated (3-4 per chain), 4-sulfated (approximately 32 per chain), 6-sulfated (approximately 1 per 14 chains), and 4,6-sulfated disaccharides (approximately 1 per 6 chains) and (b) a nonreducing terminal composition of 4-sulfated GalNAc (approximately 4 out of every 7 chains), 4,6-disulfated GalNAc (approximately 2 out of every 7 chains), and GlcUA adjacent to a 4-sulfated GalNAc residue (approximately 1 out of every 7 chains). Thus, the vast majority of these CS chains terminated with a sulfated GalNAc residue. The presence of 4,6-disulfated GalNAc at nonreducing termini is 60-fold more abundant than 4,6-disulfated GalNAc in interior disaccharides. This observation is consistent with the suggestion that disulfation of terminal GalNAc residues is involved in chain termination.     

Purification and characterization of heparan sulfate 2-sulfotransferase from cultured Chinese hamster ovary cells

Heparan sulfate 2-sulfotransferase, which catalyzes the transfer of sulfate from adenosine 3'-phosphate 5'-phosphosulfate to position 2 of L-iduronic acid residue in heparan sulfate, was purified 51,700-fold to apparent homogeneity with a 6% yield from cultured Chinese hamster ovary cells. The isolation procedure included a combination of affinity chromatography on heparin-Sepharose CL-6B and 3',5'-ADP-agarose, which was repeated twice for each, and finally gel chromatography on Superose 12 . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed two protein bands with molecular masses of 47 and 44 kDa. Both proteins appeared to be glycoproteins, because their molecular masses decreased after N-glycanase digestion. When completely desulfated and N-resulfated heparin and mouse Engelbreth-Holm-Swarm tumor heparan sulfate were used as acceptors, the purified enzyme transferred sulfate to position 2 of L-iduronic acid residue but did not transfer sulfate to the amino group of glucosamine residue or to position 6 of N-sulfoglucosamine residue. Heparan sulfates from pig aorta and bovine liver, however, were poor acceptors. The enzyme showed no activities toward chondroitin, chondroitin sulfate, dermatan sulfate, and keratan sulfate. The optimal pH for the enzyme activity was around 5.5. The enzyme activity was minimally affected by dithiothreitol and was stimulated strongly by protamine. The Km value for adenosine 3'-phosphate 5'-phosphosulfate was 0.20 microM.     

Identification of a signaling pathway activated specifically in the somatodendritic compartment by a heparan sulfate that regulates dendrite growth

In two earlier reports we demonstrated that natural heparan sulfate, but not dermatan or chondroitin sulfate glycosaminoglycans, stimulate axonal elongation and inhibit dendrite growth in vitro (Lafont et al., 1992). The latter specific effect on dendrite elongation was reproduced by chemically synthesized heparan sulfates and by SR 80037A, a purified sulfated and hexanoylated heparin fragment (Lafont et al., 1994). Adding radioactive SR 80037A to purified neurons demonstrated the existence, at the neuronal surface, of heparan sulfate-specific and saturable binding sites, suggesting that SR 80037A activates specific signal transduction pathways. In the present study, using rat or mouse neurons from the embryonic cortex, we show that SR 80037A signaling involves one or several G-coupled receptor or receptors, small GTPases rhoA and/or rhoC, and one or several PKCs. We also demonstrate that the rapid soma rounding elicited by SR 80037A does not require protein synthesis but that the long-term effect on dendrite initiation requires protein synthesis in a short period after the addition of the heparan sulfate. Finally, by preparing membranes from the somatodendritic or axonal compartments we demonstrate that the identified signaling pathway is activated by SR 80037A primarily in the somatodendritic compartment and is not sensitive to the addition of a dermatan sulfate glycosaminoglycan that does not induce the axonal phenotype by impairing dendrite initiation and elongation.     

Analysis of domain motions by approximate normal mode calculations

The use of specific enzymes (heparinase and heparitinases from Flavobacterium heparinum, endoglucuronidase, alphaN-acetylglucosaminidase and beta-glucuronidase from the mollusc Anomalocardia brasiliana) and chemical methods (nitrous acid degradation, hydrazine N-deacetylation and borohydride reduction), led to the proposal of the total sequence of a heparan sulfate derived from bovine pancreas and partial sequences of heparan sulfates from different origins (bovine: lung, liver, brain; hog: liver, brain; rabbit liver; dog liver). It was shown that all the heparan sulfates contain common structural features such as: a N-acetylated and a N-sulfated domain made of glucuronic acid-containing disaccharides and a more sulfated region made of iduronic acid-containing disaccharides. Separating the two domains a peculiar tetrasaccharide made of GlcNAc-(alpha1-4)-IdoUA-(alpha1-4)-GlcNS-(alpha1-4)-IdoUA was identified in all the heparan sulfates analyzed. It was also shown that the non-reducing ends of the heparan sulfates contain the monosaccharides glucosamine N-sulfate and/or glucosamine 2,6 disulfate.     

Opportunistic fungal pneumonia

Epithelial cells are important components of the thymus microenvironment and are involved in thymocyte differentiation. The production and secretion of sulfated glycosaminoglycans by these cells grown in culture were investigated using labeling with radioactive 35S-Na2SO4 and 3H-glucosamine. The major glycosaminoglycans synthesized by these cells are heparan sulfate and hyaluronic acid. The structure of the heparan sulfate was investigated by the pattern of degradation products formed by deaminative cleavage with nitrous acid. The ratio 35S-sulfate/ H-glucosamine is high in the segments of the heparan sulfate released during the deaminative cleavage with nitrous acid but low in the resistant portion of the molecule. Thus, the heparan sulfate synthesized by the thymic epithelial cells contains a highly sulfated region. Digestion with heparitinase reveals that this highly sulfated region is a heparin-like segment of the molecule. The heparan sulfate is rapidly incorporated into the cell surface but its secretion to the extracellular medium requires a longer incubation period. Finally, heparin was used to mimic the possible effect of this heparan sulfate with a highly sulfated region, as ascertained by its ability to modulate thymocyte adhesion to thymic epithelial cells. Since heparin actually enhanced thymocyte adhesion, it is suggested that the heparan sulfate described herein, secreted by the thymic epithelium, may play a role upon intrathymic heterotypic cellular interactions.     

Genetic evidence that heparin-like glycosaminoglycans are involved in wingless signaling

We have identified the Drosophila UDP-glucose dehydrogenase gene as being involved in wingless signaling. Mutations in this gene, called kiwi, generate a phenotype identical to that of wingless. UDP-glucose dehydrogenase is required for the biosynthesis of UDP-glucuronate, which in turn is utilized in the biosynthesis of glycosaminoglycans. By rescuing the kiwi phenotype with both UDP-glucuronate and the glycosaminoglycan heparan sulfate, we show that kiwi function in the embryo is crucial for the production of heparan sulfate in the extracellular matrix. Further, injection of heparin degrading enzyme, heparinase (and not chondroitin, dermatan or hyaluronic acid degrading enzyme) into wild-type embryos leads to the degradation of heparin-like glycosaminoglycans and a 'wingless-like' cuticular phenotype. Our study thus provides the first genetic evidence for the involvement of heparin-like glycosaminoglycans in signal transduction.     

Effect of irradiation with a low-intensity diode laser on the metabolism of equine articular cartilage in vitro

OBJECTIVE: To determine whether irradiation with a low-intensity diode laser, which produces radiation at a wavelength of 810 nm, will induce nonthermal enhancement of chondrocyte metabolism. SAMPLE POPULATION: 144 grossly normal articular cartilage explants aseptically harvested from the femoral condyles of 6 adult horses. PROCEDURE: Treated cartilage explants were irradiated with a diode laser at 1 of 7 fluence levels that ranged from 8 to 1,600 J/cm2. Explants were incubated for 24 or 72 hours, labeled for 24 hours with [35S]Na2SO4, and assayed for newly synthesized sulfated glycosaminoglycan (GAG; measured incorporation of 35SO4) and endogenous GAG, chondroitin 6-sulfate (CS), and keratan sulfate (KS) content, using a dimethylmethylene blue assay. Laser-induced temperature changes were measured during irradiation with a diode laser and a neodymium:yttrium aluminum garnet (Nd:YAG) laser, which produces radiation at a wavelength of 1,064 nm, using conditions that were reported in previous studies to increase explant metabolism. RESULTS: After incubation for 24 or 72 hours, rate of 33SO4 uptake or endogenous GAG, CS, or KS content in irradiated explants was not significantly different than in nonirradiated explants. Cartilage temperature increased < 4.75 C during diode laser application. Cartilage temperature increased 5 to 12 C during Nd:YAG laser application. CONCLUSIONS: Minimal thermal increases in cartilage explants with use of a low-intensity diode laser resulted in no change in proteoglycan metabolism of chondrocytes. An increase in tissue temperature over a narrow range with use of a Nd:YAG laser may have contributed to the metabolic alteration of chondrocytes reported in previous studies.