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Previously, spatially resolved analysis of glycosaminoglycans (GAGs), by type and sulfation state, was unobtainable. Here, we describe a mass spectrometry imaging (MSI) approach which enables the detection, identification, localization, and profiling of GAG oligosaccharides directly from retinal tissue. Through in situ treatment of tissues with relevant chondroitinase enzymes, we liberate and spatially resolve chondroitin, dermatan, and hyaluronan from disaccharides through to hexasaccharides, directly from tissue sections. We demonstrate the separation of isomeric GAG oligosaccharide ions at different histologically relevant regions using trapped ion mobility spectrometry (TIMS). This paper describes the first spatially resolved analysis of multiple GAGs and their oligosaccharide sulfation state(s) directly from tissues.

Chondroitin, the precursor of chondroitin sulfate, which is an important polysaccharide, has drawn significant attention due to its applications in many fields. In the present study, a heterologous biosynthesis pathway of chondroitin was designed in a GRAS (generally recognized as safe) strain C. glutamicum . CgkfoC and CgkfoA genes with host codon preference were synthesized and driven by promoter P tac , which was confirmed as a strong promoter via GFPuv reporter assessment. In a lactate dehydrogenase ( ldh ) deficient host, intracellular chondroitin titer increased from 0.25 to 0.88 g/l compared with that in a wild-type host. Moreover, precursor enhancement via overexpressing precursor synthesizing gene ugdA further improved chondroitin titers to 1.09 g/l. Chondroitin production reached 1.91 g/l with the engineered strain C. glutamicum ΔL-CgCAU in a 5-L fed-batch fermentation with a single distribution M w of 186 kDa. This work provides an alternative, safe and novel means of producing chondroitin for industrial applications.

Two samples of fucosylated chondroitin sulfate (FCS), AJ and AM, were isolated from holothurian species Apostichopus japonicus and Actinopyga mauritiana, respectively. Purification of FCS was performed by ion exchange chromatography followed by gel filtration. Structure of the biopolymers was elucidated using chemical and NMR spectroscopic methods. Both polysaccharides were shown to contain a typical chondroitin core built up of repeating disaccharide units →3)-β-d-GalNAc-(1→4)-β-d-GlcA-(1→ and decorated by sulfate groups and α-l-Fuc branches. Two polysaccharides were different in pattern of sulfation of GalNAc and fucosyl branches connected to O-3 of GlcA. The ratio of GalNAc4S6S:GalNAc4S for AJ was about 2:1, whereas for AM this value was approximately 1:1. AJ contained Fucp2S4S and Fucp3S4S residues linked to O-3 of GlcA in a ratio of 3:1, while for AM this ratio was 1:4. Small portions of Fucp4S units attached to O-3 of GlcA were also found in both polysaccharides. Moreover, in a structure of AM the presence of Fucp3S residues linked to O-6 of GalNAc were determined using the data of NMR spectra., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Multiple glycosyltransferases (GTases) that produce glycosaminoglycans (GAGs) have been identified; -several distinct putative architectures and catalytic abilities have been noted from microbes and vertebrates. Here the preparation and use of a class of enzymes (Class II) from the gram-negative bacterium, Pasteurella multocida, with utility in chemoenzymatic synthesis of GAGs is described. The analyses of sugar products include thin layer chromatography (TLC), matrix-assisted laser desorption ionization mass spectroscopy (MALDI-ToF MS), and polyacrylamide gel electrophoresis (PAGE). The related Chapter 18 is focused on larger molecular weight GAGs analyses as well as the Class I membrane streptococcal and mammalian HA synthases.

Reflectance near-IR (RNIR) spectroscopy was used for the simultaneous determination of chondroitin (CH), glucosamine (GO), and methyl sulfonyl methane (MSM) in tablets. Simple sample preparation was done by grinding, sieving, and compression of the tablets for improving RNIR spectra. Principal component regression and partial least squares (PLS-1 and PLS-2) were successfully applied to quantify the three components in the studied mixture using information included in RNIR spectra in the range of 4350-9100 cm(-1). The calibration model was developed with drug concentration ranges of 14.5-44.2% (w/w) for CH, 18.4-55.3% (w/w) for GO, and 6-18.6% (w/w) for MSM with addition of tablet excipients to the calibration set in the same ratio as in the tested tablets. The calibration models were evaluated by internal validation, cross-validation, and external validation using synthetic and pharmaceutical preparations. The proposed method was applied for analysis of six batches of the pharmaceutical product. The results of the proposed method were compared with the results of the pharmacopoeial method for the same batch of the pharmaceutical product. No significant differences between the results were found. The RNIR method is accurate and precise, and can be used for QC of pharmaceutical products.

Recently, the possibility of producing fructosylated chondroitin from the capsular polysaccharide of Escherichia coli O5:K4:H4, in fed-batch and microfiltration experiments was assessed on a 2 L bioreactor. In this work, a first scale-up step was set on a 22 L membrane reactor with modified baffles to insert ad hoc designed microfiltration modules permanently inside the bioreactor vessel. Moreover, the downstream polysaccharide purification process, recently established on the A¨︁KTA cross-flow instrument, was translated to a UNIFLUX-10, a tangential flow filtration system suitable for prepilot scale. In particular, the microfiltered permeates obtained throughout the fermentation, and the supernatant recovered from the centrifuged broth at the end of the process, were treated as two separate samples in the following ultrafiltration procedure, and the differences in the two streams and how these affected the ultrafiltration/diafiltration process performance were analysed. The total amount of K4 capsular polysaccharide was about 85% in the broth and 15% in the microfiltered permeates. However, the downstream treatment was more efficient when applied to the latter. The major contaminant, the lipopolysaccharide, could easily be separated by a mild hydrolysis that also results in the elimination of the unwanted fructosyl residue, which is linked to the C-3 of glucuronic acid residues. The tangential ultrafiltration/diafiltration protocols developed in a previous work were effectively scaled-up, and therefore in this research proof of principle was established for the biotechnological production of chondroitin from the wild-type strain E. coli O5:K4:H4. The complete downstream procedure yielded about 80% chondroitin with 90% purity., (Copyright © 2012 American Institute of Chemical Engineers (AIChE).)

A reflectance near-infrared (RNIR) spectroscopy method was developed for simultaneous determination of chondroitin (CH), glucosamine (GO), and ascorbic acid (AS) in capsule powder. A simple preparation of the sample was done by grinding, sieving, and compression of the powder sample for improving RNIR spectra. Partial least squares (PLS-1 and PLS-2) was successfully applied to quantify the three components in the studied mixture using information included in RNIR spectra in the 4240-9780 cm(-1) range. The calibration model was developed with the three drug concentrations ranging from 50 to 150% of the labeled amount. The calibration models using pure standards were evaluated by internal validation, cross-validation, and external validation using synthetic and pharmaceutical preparations. The proposed method was applied for analysis of two pharmaceutical products. Both pharmaceutical products had the same active principle and similar excipients, but with different nominal concentration values. The results of the proposed method were compared with the results of a pharmacopoeial method for the same pharmaceutical products. No significant differences between the results were found. The standard error of prediction was 0.004 for CH, 0.003 for GO, and 0.005 for AS. The correlation coefficient was 0.9998 for CH, 0.9999 for GO, and 0.9997 for AS. The highly accurate and precise RNIR method can be used for QC of pharmaceutical products.

Sulfated glycans play critical roles during the development, differentiation and growth of various organisms. The most well-studied sulfated molecules are sulfated glycosaminoglycans (GAGs). Recent incidents of heparin drug contamination convey the importance of having a convenient and sensitive method for detecting different GAGs. Here, we describe a molecular method to detect GAGs in biological and biomedical samples. Because the sulfation of GAGs is generally not saturated in vivo, it is possible to introduce the radioisotope (35)S in vitro using recombinant sulfotransferases, thereby allowing detection of minute quantities of these molecules. This strategy was also successfully applied in the detection of other glycans. As examples, we detected contaminant GAGs in commercial heparin, heparan sulfate and chondroitin samples. The identities of the contaminant GAGs were further confirmed by lyase digestion. Oversulfated chondroitin sulfate was detectable only following a simple desulfation step. Additionally, in vitro sulfation by sulfotransferases allowed us to map glycan epitopes in biological samples. This was illustrated using mouse embryo and rat organ tissue sections labeled with the following carbohydrate sulfotransferases: CHST3, CHST15, HS3ST1, CHST4 and CHST10.

Purpose: To describe and identify unknown opaque material between the optic of an AR40 intraocular lens (IOL) injected with the Emerald Series implantation system (both AMO, Inc.) and the posterior capsule at the conclusion of routine phacoemulsification to prevent an outbreak of toxic anterior segment syndrome (TASS)., Setting: Ambulatory care center operating room, University of North Carolina Hospitals and Department of Ophthalmology, University of North Carolina School of Medicine at Chapel Hill, Chapel Hill, North Carolina, USA., Methods: After coaxial phacoemulsification in multiple patients, opaque material was present between the optic of a posterior chamber IOL and the posterior capsule. Although there was no TASS, the material was removed from 2 eyes and analyzed with scanning electron microscopy (SEM) and x-ray microanalysis (XRM). Similarly, crystalline lens, Klenzyme (Steris Corp.), Viscoat (sodium hyaluronate 3.0%-chondroitin sulfate 4.0%), and Provisc (sodium hyaluronate 1.0%) were analyzed., Results: On SEM, the material had an irregular undulating surface similar to that of Provisc. Viscoat and the crystalline lens had smoother surfaces. On XRM, the material contained sodium, chlorine, and calcium, like Viscoat and Provisc, and phosphorous and sulfur, like Viscoat. The material also contained silicone, magnesium, aluminum, titanium, iron, and zinc. Klenzyme had smaller peaks of sodium, chlorine, and calcium and a higher carbon background than the unknown material., Conclusions: The material was likely ophthalmic viscosurgical device that was chemically and structurally altered by the cleaning and sterilization process. The silicone and metallic elements were probably from the Emerald Series implantation system as the disposable cartridge is coated with silicone and the reusable injector is metal.

The hydrozoan is the simplest organism whose movements are governed by the neuromuscular system, and its de novo morphogenesis can be easily induced by the removal of body parts. These features make the hydrozoan an excellent model for studying the regeneration of tissues in vivo, especially in the nervous system. Although glycosaminoglycans (GAGs) and proteoglycans (PGs) have been implicated in the signaling functions of various growth factors and play critical roles in the development of the central nervous system, the isolation and characterization of GAGs from hydrozoans have never been reported. Here, we characterized GAGs of Hydra magnipapillata. Immunostaining using anti-GAG antibodies showed chondroitin or chondroitin sulfate (CS) in the developing nematocyst, which is a sting organelle specific to cnidarians. The CS-PGs might furnish an environment for assembling nematocyst components, and might themselves be components of nematocysts. Therefore, GAGs were isolated from Hydra and their structural features were examined. A considerable amount of CS, three orders of magnitude less heparan sulfate (HS), but no hyaluronan were found, as in Caenorhabditis elegans. Analysis of the disaccharide composition of HS revealed glucosamine 2-N-sulfation, glucosamine 6-O-sulfation, and uronate 2-O-sulfation. CS contains not only nonsulfated and 4-O-sulfated N-acetylgalactosamine (GalNAc) but also 6-O-sulfated GalNAc. The average molecular size of CS and HS was 110 and 10 kDa, respectively. It has also been established here that CS chains are synthesized on the core protein through the ubiquitous linkage region tetrasaccharide, suggesting that indispensable functions of the linkage region in the synthesis of GAGs have been conserved during evolution.

Despite their wide occurrence, proteoglycans (PGs) have never been isolated from the saliva of higher animals. We found that the Collocalia glycoproteins isolated from edible birds'-nests (the dried forms of regurgitated saliva of male Collocalia swiftlets) were rich in a PG containing nonsulfated chondroitin glycosaminoglycans (GAGs). We have devised a method to isolate a PG from the water extract of the white nest built by Aerodramus fuciphagus (white nest swiftlets) with a yield of 2-mg PG per gram nest. This PG contained 83% of carbohydrates, of which 79% were GalNAc and GlcUA (D-glucuronic acid) in an equimolar ratio. By using chondroitin AC lyase, the structure of GAGs in this PG was established to be chondroitin ( --> 4GlcUAbeta1 --> 3GalNAcbeta1 --> )(n) chains. The average molecular mass of the chondroitin chain was estimated to be 49 kDa by gel filtration. We have isolated a linkage region hexasaccharide, DeltaHexUAalpha1 --> 3GalNAcbeta1 --> 4GlcUAbeta1 --> 3Galbeta1 --> 3Galbeta1 --> 4Xyl, from this PG by chondroitinase ABC digestion to show that the GAGs in this PG are also linked to the core protein through the common tetrasaccharide linker, GlcUAbeta1 --> 3Galbeta1 --> 3Galbeta1 --> 4Xyl, found in various PGs. As water was not effective in extracting uronic acid-containing glycoconjugates from the black nest built by black nest swiftlets (A. maximus), we used 4 M guanidium chloride and anion-exchange chromatography in the presence of urea to extract and isolate about 30 mg of a chondroitin PG preparation from 10 g of the desialylated black nest. As the biological significance of chondroitin is still not well understood, bird's nest should become a convenient source for preparing this unique GAG to study its biological functions.

Background: We are not aware of any in vitro study comparing the biomechanical, biochemical, and cellular properties of commercial extracellular matrix materials marketed for rotator cuff tendon repair. In this study, the properties of GraftJacket, TissueMend, Restore, and CuffPatch were quantified and compared with each other. The elastic moduli were also compared with that of normal canine infraspinatus tendon., Methods: Samples were tested from different manufacturing lots of four materials: GraftJacket (ten lots), TissueMend (six), Restore (ten), and CuffPatch (six). The Kruskal-Wallis test was used to compare thickness, stiffness, and modulus as well as hydroxyproline, chondroitin/dermatan sulfate glycosaminoglycan, hyaluronan, and DNA contents among these matrices. The moduli of the extracellular matrices were also compared with those of normal canine infraspinatus tendon., Results: All four extracellular matrices required 10% to 30% stretch before they began to carry substantial load. Their maximum moduli were realized in their linear region at 30% to 80% strain. The elastic moduli of all four commercial matrices were an order of magnitude lower than that of canine infraspinatus tendon. TissueMend had significantly higher DNA content than the other three matrices (p<0.0001), although both Restore and GraftJacket also had measurable amounts of DNA., Conclusions: Our data demonstrate chemical and mechanical differences among the four commercial extracellular matrices that we evaluated. Probably, the source (dermis or small intestine submucosa), species (human, porcine, or bovine), age of the donor (fetal or adult), and processing of these matrices all contribute to the unique biophysical properties of the delivered product. The biochemical composition of commercial extracellular matrices is similar to that of tendon. However, the elastic moduli of these materials are an order of magnitude lower than that of tendon, suggesting a limited mechanical role in augmentation of tendon repair.

Purpose: The present study was performed to evaluate the corneal endothelium protection and anterior chamber stagnation abilities of three different types of viscoelastic substances (Healon, Viscoat, HealonV)., Methods: Viscoelastic substances were selected at random for 120 eyes with cataracts, and the postoperative reduction rates of the corneal endothelium cells were compared. The residual viscoelastic substances after filling of the anterior chamber of pig eyes and aspiration with a handpiece were measured by an anterior eye segment image analysis system. The same procedures were performed in rabbit eyes and the residual levels of viscoelastic substances on the corneal endothelium were photographed histologically., Results: The reduction rate of endothelium corneal cells tended to decrease with Viscoat three months after surgery. The results obtained with the anterior eye segment image analysis system showed that the residual level in the anterior chamber was higher with Healon. Histological analyses demonstrated residual Viscoat at the center of the corneal endothelium after perfusion., Conclusion: HealonV was superior in terms of spatial retention and Viscoat had corneal endothelium protection potential.

Objective: To select a high-quality chondroitin dosage form and/or an appropriate source of sodium chondroitin for the National Institutes of Health's Glucosamine/Chondroitin Arthritis Intervention Trial (GAIT)., Design: Controlled experimental trials., Setting: Laboratory., Patients or Participants: Not applicable., Interventions: Commercially available chondroitin products were reviewed, and purified sodium chondroitin from two suppliers was evaluated through tests (infrared and near-infrared identification, moisture content, pH, optical rotation, color and clarity of aqueous solutions prepared from the powders, protein contamination, total residue following ignition and nitrogen content, determination of sodium chondroitin molecular weight, disaccharide analysis, and measurement of chondroitin, sodium, and total glycosaminoglycan content) and an onsite supplier audit., Main Outcome Measures: Purity, potency, and quality of sodium chondroitin powders., Results: No commercially available chondroitin product was deemed appropriate for use in GAIT. Samples of sodium chondroitin powder from two suppliers exhibited similar disaccharide and glycosaminoglycan content. Each contained approximately 2% hyaluronic acid and 8%-9% unsulfated disaccharide. Potency was inconsistent across groups, which might have resulted from different analytical methods and choice of reference standard. Mean potency obtained by five separate methods ranged from 82.2% to 95.5% for one supplier, 92.5% to 110.1% for another, and 95.1% to 112.5% for a commercially obtained reference standard. Critical issues raised by the results include choice of reference standard, selection of assay method, and the consistent appearance of an unidentifiable contaminant present in all three lots from one supplier., Conclusion: This blinded study determined methods to identify acceptable agents and provided results, which, in addition to regulatory compliance supplier audits, formed the basis for chondroitin product selection in GAIT.

The extensor tendons of the fingers and toes form part of the capsule of the interphalangeal joint and press against the proximal phalanx during flexion. Previous work on the fingers has shown that there is a "sesamoid" fibrocartilage on the deep surface of each tendon that labels immunohistochemically for a variety of glycosaminoglycans and collagens. However, we know little about the molecular composition of the tendon in the toes. This question is of special interest, because the mechanics of the interphalangeal joints differ in the upper and lower limbs-the toes balance the forefoot, distribute load during the gait cycle, and transmit the pull of larger muscles. This means that their extensor tendons are more often under higher tension than those in the fingers. Here, we report the presence of an equivalent fibrocartilage and compare its immunolabelling characteristics in all the toes. Six forefeet were removed from elderly cadavers, and the interphalangeal (IP) joints were fixed in 90% methanol. The extensor tendon and its enthesis were dissected out from the IP joint of the big toe and from the proximal interphalangeal (PIP) joint of all lesser toes, decalcified, cryosectioned, and immunolabelled with a panel of monoclonal and polyclonal antibodies for type I, II, III, and VI collagens; chondroitin 4 and 6 sulphates; and dermatan and keratan sulphate. Antibody binding was detected with the Vectastain ABC Elite avidin-biotin-peroxidase kit (Vector Laboratories, Burlingame, CA). The extensor tendon in all the toes had a metachromatic, sesamoid fibrocartilage on its deep surface that immunolabelled for all glycosaminoglycans and for type I, III, and VI collagens. Labelling for type II collagen was seen in the sesamoid fibrocartilage of all toes but was particularly characteristic of the 2nd through 5th toes. The immunolabelling patterns of the enthesis fibrocartilage were similar in all toes and to results reported previously for fingers. The normal occurrence of type II collagen in the sesamoid fibrocartilage of the 2nd through 5th toes is in contrast to our published data on the fingers. The finding can be related to the more constant loading of the tendon in the toes. The greater prominence of type II collagen in the sesamoid fibrocartilage of the 2nd through 5th toes could be related to a difference in joint position during walking between the 1st toe and the 2nd through 5th toes--the PIP joints of the latter are usually more flexed than the IP joint of the former.

The type and distribution of sulphated proteoglycans (PGs) in the midshaft subperiosteal bone of 15-18-day embryonic chick femurs were studied immunocytochemically and biochemically, using four monoclonal antibodies (MAb 2B6, 3B3, 1B5, and 5D4). These MAb specifically recognize epitopes in chondroitin 4-sulphate (C4-S) and dermatan sulphate (DS); chondroitin 6-sulphate (C6-S) and unsulphated chondroitin (C0-S); C0-S; and keratan sulphate (KS) respectively. Immunohistochemistry showed that staining of C4-S, DS, and KS, but not of C6-S and C0-S, was limited to osteoid, the cell surface of osteocytes, and to the walls of osteocytic lacunae and bone canaliculi in 15-18-day embryonic specimens. However, no significant difference in the distribution and intensity of immunostaining was observed in these specimens. Bone proteins were extracted from fresh 18-day embryonic specimens with a three extraction procedure, 4 M guanidine HCl (GdnCl, G-1 extract), 0.4 M EDTA (E-extract), followed by GdnCl (G-2 extract), to characterize mineral binding and collagenous matrix associated PGs in E- and G2-extracts respectively. Western blot analysis of E- and G2-extracts demonstrated that chondroitinase ABC-digested PGs with a molecular weight (Mr) approximately of 45,000 containing GAGs predominantly corresponding to C4-S and/or DS, with no detectable C6-S or C0-S present in the mineral and matrix phase, whereas KSPGs having an Mr of approximately 72,000 are only present in the mineral phase. These results indicate that embryonic chick bone contains small PGs having C4-S, DS, and KS chains with preferential localization to osteoid, the cell surface of osteocytes, and to the walls of osteocytic lacunae and bone canaliculi.

Purpose: Synchrotron x-ray diffraction experiments indicate that corneas with macular corneal dystrophy (MCD) contain unusual 4.6-A periodic repeats thought to reside in proteoglycans or glycosaminoglycans. Recently the 4.6-A x-ray reflection was found to be significantly diminished after incubation of MCD specimens in buffer containing chondroitinase ABC or N-glycanase. We examined the sulfated proteoglycans in these glycosidase-digested MCD corneas., Methods: Transmission electron microscopy was used in conjunction with cuprolinic blue-staining for sulfated proteoglycans., Results: Incubation of an MCD specimen in enzyme buffer left both small and large proteoglycan filaments in the stromal matrix, whereas incubation in the presence of chondroitinase ABC removed these molecules from the tissue. Incubation in buffer containing N-glycanase, on the other hand, removed the large proteoglycan filaments from the MCD stroma but left unaffected the small collagen-associated proteoglycans., Conclusion: These results are consistent with the interpretation that 4.6-A periodic repeats in MCD corneas reside in large sulfated proteoglycan filaments (or aggregates thereof) that may contain chondroitin/dermatan sulfate and keratan sulfate or keratan components.

This study investigated the synovial fluid concentrations of glycosaminoglycan (GAG), keratan sulphate (KS) epitope 5D4 and chondroitin sulphate (CS) sulphation patterns in healthy volunteers and patients with osteoarthritis (OA) and rheumatoid arthritis (RA). Synovial fluids were collected from knee joints of healthy volunteers (n = 24), and patients with OA (n = 28) and RA (n = 29). Concentrations of GAG and the keratan sulphate epitope 5D4 were measured in 15 of the healthy volunteers, and all of the OA and RA synovial fluids. Total GAG was measured using a dye-binding method and 5D4 by an ELISA. The unsaturated CS disaccharides delta C4 and delta C6 were measured by capillary electrophoresis in all synovial fluids. The concentrations of GAG, 5D4 and delta C6 in the normal synovial fluid were higher but that of delta C4 lower than those of the disease groups. The delta C6:delta C4 ratios correlated with age (r = -0.437, P < 0.001) and the mean value was lower in females than males (2.92 compared with 5.22, P < 0.001). After allowing for age and sex, the delta C6:delta C4 ratio in the control group was significantly elevated (P < 0.001) compared to both OA and RA. The ratio was also related to proteoglycan markers (r = 0.383 for 5D4 and r = 0.357 for GAG). The finding that 5D4 and delta C6:delta C4 ratios are higher in synovial fluid from healthy volunteers compared to OA and RA suggests that they may be markers of the susceptibility of articular cartilage to early damage in arthritis.

Subsets of neurons ensheathed by perineuronal nets containing chondroitin unsulfated proteoglycan have been immunohistochemically mapped throughout the rat central nervous system from the olfactory bulb to the spinal cord. A variable proportion of neurons were outlined by immunoreactivity for the monoclonal antibody (Mab 1B5), but only after chondroitinase ABC digestion. In forebrain cortical structures the only immunoreactive nets were around interneurons; in contrast, throughout the brainstem and spinal cord a large proportion of projection neurons were surrounded by intense immunoreactivity. Immunoreactivity was ordinarily found in the neuropil between neurons surrounded by an immunopositive net. By contrast, within the pyriform cortex the neuropil of the plexiform layer was intensely immunoreactive even though no perineuronal net could be found. The presence of perineuronal nets could not be correlated with any single class of neurons; however a few functionally related groups (e.g., motor and motor-related structures: motor neurons both in the spinal cord and in the efferent somatic nuclei of the brainstem, deep cerebellar nuclei, vestibular nuclei; red nucleus, reticular formation; central auditory pathway: ventral cochlear nucleus, trapezoid body, superior olive, nucleus of the lateral lemniscus, inferior colliculus, medial geniculate body) were the main components of the neuronal subpopulation displaying chondroitin unsulfated proteoglycans in the surrounding extracellular matrix. The immunodecorated neurons found in the present study and those shown by different monoclonal antibodies or by lectin cytochemistry, revealed consistent overlapping of their distribution patterns.

To determine the correlation between the types of glycosaminoglycans (GAGs) and the features of rhegmatogenous retinal detachment, we analyzed the types of GAGs in the subretinal fluid of 20 eyes with rhegmatogenous retinal detachment. The GAGs were analyzed by cellulose acetate two-dimensional electrophoresis. Hyaluronic acid alone (HA type) was found in 10 of the 20 eyes. A combination of chondroitin sulfate (chSA) and HA was present (chSA type) in 3 of the 20 eyes. A combination of dermatan sulfate (DS) and HA (DS type) was present in 7 of the 20 eyes. No correlation was found between the type of GAGs and the extent, the duration of detachment, location, size or type of break in the 20 eyes. Some correlation was found between the type of GAGs and the grade of vitreous haze, proliferative vitreoretinopathy (PVR) and the number of surgeries. Retinal detachment with a demarcation line resulted in subretinal strand formation in the DS type eyes, while no such formation was seen in the subretinal space of the eyes of the chSA type. Vitreous haze of grade ( ) was seen in one eye of the DS type, but not seen in the other types. All 3 eyes with PVR of grade C were the DS type. The 2 eyes with reoperated surgeries were the DS type. The presence of DS may indicate an advanced condition of retinal detachment.

A comparative study on compositional analysis of two sets of eight unsaturated disaccharide standards derived from heparin/heparan sulfate and chondroitin/dermatan sulfate was carried out using capillary electrophoresis performed in both normal and reverse polarity modes. While these heparin/heparan sulfate disaccharides (S. A. Ampofo, H. M. Wang, and R. J. Linhardt (1991) Anal. Biochem. 199, 249-255) and chondroitin/dermatan sulfate disaccharides (A. Al-Hakim and R. J. Linhardt (1991) Anal. Biochem. 195, 68-73) have previously been fractionated using normal polarity capillary electrophoresis, multiple buffer systems and conditions were required to separate certain disaccharide isomers and these separations often resulted in poor peak symmetry and significant tailing. This paper demonstrates that reverse polarity capillary electrophoresis completely resolves disaccharide mixtures into all components using a single buffer, 20 mM phosphoric acid-sodium phosphate at pH 3.48. This improved resolution is due primarily to an increase in the sharpness of peaks and improved peak symmetry. Separation of heparin-derived oligosaccharides, ranging from disaccharide to hexasaccharide, had also previously been reported using normal polarity capillary electrophoresis (U.R. Desai, H.M. Wang, S.A. Ampofo, and R.J. Linhardt (1993) Anal. Biochem. 213, 120-127). This paper now demonstrates the separation of 13 heparin-derived oligosaccharides of sizes ranging from disaccharide to tetradecasaccharide using both reverse and normal polarities. An enzymatic digestion of bovine lung heparin containing many of these larger oligosaccharides was also compared in both normal and reverse polarity modes. Mixtures containing oligosaccharides primarily differing in size (number of saccharide units) were better resolved using normal polarity.

Objectives: Chondroitin sulphate is the major sulphated glycosaminoglycan present in the extracellular matrix of soft connective tissues and the aim of this study was to investigate the distribution of chondroitin sulphate species in normal and diseased synovium., Methods: Distribution of chondroitin-4-sulphate/dermatan sulphate (Ch4S/DS) and chondroitin-6-sulphate in normal (n = 6), osteoarthritic (n = 4) and rheumatoid (n = 10) synovium was determined using an immunoperoxidase technique and specific monoclonal antibodies to chondroitinase ABC-digested preparations., Results: Ch4S/DS was expressed throughout the interstitium of all tissues and was also present on blood vessels in rheumatoid samples only. Ch6S was expressed in the lining layer of normal synovium but was absent from this site in osteoarthritic and rheumatoid tissues. Ch6S was also present on all blood vessels in all tissues., Conclusions: The distinct zonal distributions of Ch4S/DS and Ch6S and their alteration in disease suggest these molecules have different and specific functions in normal and diseased synovium.

We examined proteoglycans (PGs) in amphibian epidermal cells by immunofluorescence microscopy. Immunoelectron microscopy with pre- and post-embedding methods, combined with HRP- or gold-conjugated secondary antibody, revealed ultrastructural localization of glycosaminoglycans (GAGs). Embryonic epidermis secretion granules in Xenopus laevis contained chondroitin 6-sulfate and unsulfated chondroitin. Immature secretion granules were also labeled with anti-chondroitin 6-sulfate and anti-chondroitin. A step-wise digestion experiment on chondroitinase ABC revealed that fine filaments in the secretion granules were chondroitin sulfate chains. Inhibition experiments with brefeldin A revealed that the life of a secretion granule was 5-10 hr, suggesting that GAG secretion from embryonic epidermis is routed through a regulated pathway.

Proteoglycans stain weakly in polyacrylamide gels by traditional protein stains such as coomassie brilliant blue or silver. In the present work preparations of large aggregating proteoglycan from human articular cartilage were used to evaluate a convenient staining method based on successive staining with alcian blue and neutral silver. The method is developed for sodium dodecyl sulfate-polyacrylamide gel electrophoresis; with ultrathin minigels in a semiautomated electrophoresis system, takes 1 1/2 h, and uses stable reagents. Preparations, electrophoresis, and staining of up to 24 samples can be completed within 2 1/2 h. The method has a detection limit of 0.04-1 ng proteoglycan and less than 0.5 ng of glycosaminoglycan. In addition the method can be adjusted for selective staining of proteoglycans and glycosaminoglycans.

The release rates of specific components of the proteoglycan aggregates (G1 domain, the chondroitin sulfate and keratan sulfate containing portion of the protein core, and link protein) of the articular cartilage of mature beagles were studied at early stages of canine experimental osteoarthritis (OA), generated by transection of the anterior cruciate ligament. Analysis of cartilage explants and synovial fluids indicates that at early stages of experimental OA, there is increased release of the proteoglycan aggregates of the articular cartilage. This involves a release from the tissue of the components of the proteoglycan that are specifically involved with aggregation together with the glycosaminoglycans of the proteoglycan. These components were detected at elevated levels in the media of explants of cartilage from the operated joint, and in the synovial fluids of the operated joints.

The involvement of proteoglycans in hair growth has been recognized through the observation of increased hair growth in diseases such as the mucopolysaccharidoses and pre-tibial myxedema, which involve an increase in skin proteoglycan content. In an attempt to understand this, we have examined the distribution of chondroitin 6 sulphate (C6S), unsulphated chondroitin (COS), dermatan sulphate (DS), and heparan sulphate proteoglycans (HSPG) in frozen tissue sections of normal scalp by immunostaining. Results show that during anagen, the thick connective tissue sheath around the follicle strains strongly for C6S, COS, and DS. COS is uniquely associated with this region and is not found beneath the epidermis or infundibular epithelium. HSPG is, however, localized in the basement membrane zone adjacent to the outer root sheath. In addition, all of these proteoglycans are localized in the dermal papilla. In mid-catagen, we observed significant loss of C6S and COS staining from both the dermal papilla and the connective tissue sheath, but no decrease in staining for HSPG. In late catagen, very little staining of C6S and COS was observed. In early anagen, we observed that C6S was again present in the connective tissue sheath and dermal papilla; however, COS staining appeared to be weaker and less closely associated with the follicle. HSPG staining was observed in early anagen in a pattern very similar to that found for other basement membrane components. Results for DS were not obtained for catagen or early anagen. These results provide further evidence that hair growth is associated with the presence of chondroitin proteoglycans in the follicle environment and that the cessation of growth is associated with their removal. Further studies are underway to characterize the relationship between hair growth and proteoglycans.

We examined biochemically and immunocytochemically the type and distribution of mineral binding proteoglycans (PGs) in rat mid-shaft subperiosteal bone using three monoclonal antibodies (MAb 1-B-5, 9-A-2, and 3-B-3) which specifically recognize unsulfated chondroitin, chondroitin 4-sulfate (C4-S) and dermatan sulfate (DS), and chondroitin 6-sulfate. Bone proteins were extracted from fresh specimens with a three-step technique: 4 M guanidine HCl (GdnCl), aqueous EDTA without GdnCl (E-extract), followed by GdnCl. Western blot analysis of SDS-polyacrylamide gel electrophoresis revealed that E-extract after chondroitinase ABC digestion reacted strongly with MAb 9-A-2 but not with MAb 1-B-5 or 3-B-3. After adehyde fixation, ethanolic trimethylammonium EDTA was used as a demineralizing agent for light and electron immunocytochemistry. This provided good retention of water-soluble PGs in the specimens. After chondroitinase ABC pre-treatment of tissue sections, MAb 9-A-2 specifically stained C4-S and/or DS in the walls of osteocyte lacunae and bone canaliculi in the mineralized matrix as well as in the unmineralized matrix such as pre-bone, vascular canals, and pericellular matrix surrounding osteocytes; the remainder of the mineralized matrix lacked staining. These results indicate that mineral binding PGs contain C4-S and/or DS and are exclusively localized in the walls of the bone lacuna and canaliculus.

The skin of the white mutant axolotl larva is pigmented differently from that of the normal dark due to a local inability of the extracellular matrix (ECM) to support subepidermal migration of neural crest-derived pigment cell precursors. In the present study, we have compared the ECM of neural crest migratory pathways of normal dark and white mutant embryos ultrastructurally, immunohistochemically and biochemically to disclose differences in their structure/composition that could be responsible for the restriction of subepidermal neural crest cell migration in the white mutant axolotl. When examined by electron microscopy, in conjunction with computerized image analysis, the structural assembly of interstitial and basement membrane ECMs of the two embryos was found to be largely comparable. At stages of initial neural crest cell migration, however, fixation of the subepidermal ECM in situ with either Karnovsky-ruthenium red or with periodate-lysine-paraformaldehyde followed by ruthenium red-containing fixatives, revealed that fibrils of the dark matrix were significantly more abundant in associated electron-dense granules. This ultrastructural discrepancy of the white axolotl ECM was specific for the subepidermal region and suggested an abnormal proteoglycan distribution. Dark and white matrices of the medioventral migratory route of neural crest cells had a comparable appearance but differed from the corresponding subepidermal ECMs. Immunohistochemistry revealed only minor differences in the distribution of fibronectin, laminin, collagen types I, and IV, whereas collagen type III appeared differentially distributed in the two embryos. Chondroitin- and chondroitin-6-sulfate-rich proteoglycans were more prevalent in the white mutant embryo than in the dark, especially in the subepidermal space. Membrane microcarriers were utilized to explant site-specifically native ECM for biochemical analysis. Two-dimensional gel electrophoresis of these regional matrices revealed a number of differences in their protein content, principally in constituents of apparent molecular masses of 30-90,000. Taken together our observations suggest that local divergences in the concentration/assembly of low and high molecular mass proteins and proteoglycans of the ECM encountered by the moving neural crest cells account for their disparate migratory behavior in the white mutant axolotl.

Interactions between the epithelial and lymphocytic components of the thymus are required for T cell maturation, yet the molecular bases for these interactions remain elusive. In the development and function of other endodermally derived organs, glycosaminoglycan-containing proteins are known to play a critical role. In contrast, virtually nothing is known about the macromolecules that are major constituents of thymic interstitial spaces. For these reasons, we undertook metabolic labeling studies in vitro with D-(6-3H)glucosamine and 35SO4(-2) to begin to characterize systematically the relative amounts and types of glycosaminoglycans made by enriched subpopulations of cells within the thymus. Hydrocortisone, which depletes the thymus of 90% of its lymphocytes, was used both to enrich for epithelium-derived glycoconjugates and to determine if significant alterations in glycoconjugate metabolism accompany drug-induced premature thymic involution. Results indicate: 1) glycosaminoglycans account for a substantial proportion of the total glycoconjugates synthesized by both thymocytes and epithelium; 2) Glycosaminoglycans show a tissue-specific distribution. Hyaluronic acid is the major glycosaminoglycan synthesized by thymic epithelium, whereas it accounts for less than 15% of the total glycosaminoglycans made by thymocytes; 3) Similar proportions of sulfated glycosaminoglycans are made by thymic epithelium and thymocytes. Chondroitin sulfates predominate (75 to 90%) over heparan sulfates (10 to 25%). Chondroitin sulfates from both nonstimulated thymocytes and epithelium are nearly exclusively sulfated at the 4-position of their N-acetylgalactosamine residues; 4) The major high m.w. glycoconjugate of thymocytes, however, is nonsulfated and is resistant to pronase, hyaluronidase, chondroitinase ABC, nitrous acid, keratanase, and neuraminidase; 5) Although hydrocortisone treatment causes a dramatic inhibitory effect on the incorporation of radioactivity into smaller oligosaccharide side-chains by "cortisone-resistant" thymocytes, the drug exerts negligible effects on the metabolism of glycoconjugates by epithelium. These data, which quantify and categorize the complex arrays of glycoconjugates synthesized by the major cell types of the thymus, establish the necessary foundations for further investigations into the functional roles of these glycoconjugates in thymic epithelium-induced maturation of T lymphocytes.

1. A rapid and sensitive method for the accurate estimation of the molecular-weight distribution of keratan sulphate and chondroitin sulphate isolated from adult bovine nasal septum and intervertebral disc is described. The method utilizes gel chromatography of reductively labelled glycosaminoglycan and end-group estimation of number-average molecular weight for each fraction across the peak of eluted glycosaminoglycan. 2. Chain-length distribution data obtained by this procedure are used to evaluate mechanisms of chondroitin sulphate biosynthesis.

1. When cultured in medium 199 in an atmosphere of O2+CO2 (95:5) rabbit tracheal explants retained their viability for up to 21 days. 2. The explants secreted into the culture medium three electrophoretically separable components which were eluted in the non-retarded fraction from Sephadex G-200. 3. Digestion with papain and fractionation with a LiCl gradient on DEAE-cellulose resulted in the separation of these components, which were identified as a sialic acid-rich glycoprotein of epithelial origin, and chondroitin sulphate and hyaluronic acid from sub-epithelial cartilage and connective tissue. 4. Incorporation of radioactive precursors ( D-[U-14-C]glucose, D-[1-14-C]glucosamine, D-[1-14-C]galactose and Na2-35SO4) into these secreted macromolecules was indicative of their active biosynthesis by the tracheal tissue.

The fraction of crude glycosaminoglycans was prepared from an emphysematous lung by means of proteolytic digestion, precipitation with ethanol and fractionation with CPC (cetylpyridinium chloride). The above fraction of crude glycosaminoglycans was then subjected to chromatography with a column of Dowex-1. Individual glycosaminoglycan species was identified based on the results of electrophoresis and on those of incubation with specific mucopolysaccharide-lyases. As a result, hyaluronic acid, chondroitin sulfate A (C), dermatan sulfate and heparan sulfate were detected. Quantitation of individual glycosaminoglycan species revealed that the ratio to total glycosaminoglycan of hyaluronic acid was smaller in the emphysematous than in the normal lung. The significance which can be attributed to the change in quantity of glycosaminoglycan of the lung was discussed in relation to pathogenesis of pulmonary emphysema.

Posterior lumbar two-level spinal fusion was undertaken in 10 mature beagles. The animals were sacrificed 6 and 12 months later. Two months before sacrifice control and experimental animals received intravenously Na2(35)SO4 (1 mCi/kg). Discs encompassed by the fusion and those adjacent to it were dissected into the nucleus pulposus and annulus fibrosus (AF), which were analysed separately. Proteoglycans (PGs) were extracted with 4.0 M guanidine HCl and purified by CsCl density gradient ultracentrifugation. The hydrodynamic size and ability of the PG subunits to aggregate in the presence of hyaluronic acid were investigated by Sepharose CL-2B chromatography. The PG subunits were analysed for their galactosamine (galN), glucosamine (glcN), hexuronic acid, and protein content or were subjected to digestion with papain or chondroitin-ABC-lyase to establish the size of the chondroitin (CS) and keratan (KS) sulphate chains and the KS-PG core protein complex. Decreased ability to aggregate of PGs isolated from discs 6 and 12 months after surgery was demonstrated. While their hydrodynamic size after 6 months was generally the same or smaller than those in control tissues, the PG population present after 12 months was larger, particularly in the AF. Analysis of PG subunits from fusion discs afforded galN/glcN, galN/protein, and hexuronic acid/protein ratios that were compatible with the presence in these tissues of PGs in which the proportion of CS attached to core protein was greater than in control tissues. These studies provide the first experimental evidence that a metabolic response of discs in a fused segment may be accompanied by the biosynthesis of a new PG population whose structure is similar to that present in immature tissues.

A simple chemical method for the determination of individual mucopolysaccharides in mixtures of dermatan sulfate and chondroitin sulfates by means of a single reagent was established, utilizing the difference in reaction rates of these polysaccharides with orcinol. To each 1 ml of a sample mixture of standard dermatan sulfate and standard chondroitin sulfate (either 4- or 6-sulfate) was added 3 ml of orcinol reagent and the resulting solution was heated in a boiling-water bath. After 20 and 60 min reaction, absorbances at 660 nm were measured and the concentrations of individual mucopolysaccharides were calculated. High reproducibility was observed for the determination of dermatan sulfate in the presence of chondroitin sulfates. In addition, orcinol reaction for 90 min employing D-glucuronolactone as a standard appeared to be of practical value in the estimation of the uronic acid content of these mucopolysaccharides.

An Orcein staining method has been developed which stains mature and immature leukocytes in blood films and bone-marrow smears. Two different patterns of staining are obtained depending upon whether staining is or is not preceded by oxidation. In the latter case, all granulocytes and some monocytes show granular reddish-brown cytoplasmic staining. When prior oxidation is used, the staining is in the form of fine grey or black cytoplasmic granules. All lymphocytes, by both techniques, are negative. It is suggested that Orcein stains sulphated mucosubstances, possibly chondroitin sulphate, which in granulocytes is concentrated in their primary granules.

1. Complex carbohydrate fractions were extracted successively with 40% aqueous EDTA (pH 7.4) and 6M urea (PH 7.8) FROM ACETONE-DRIED bone powder of rabbit femur. 2. The carbohydrate fraction extracted with EDTA (E=Fr) was separated into five fractions,D1approximatelyD5 by DEAE-Dephadex A-50 column chromatography. Chemical and infrared spectral analyses, and enzymatic digestion indicate that D2 contained lessacidic glycoprotein, D3 contained sialoglycoprotein, D4 contained a low sulfated proteokeratan sulfate-like substance, and d5 contained glycoprotein-bound chondroitin sulfate A plus protein-free chondroitin sulfate A. 3. Two fractions, HU-D1 and HU-D2, were isolated from the carbohydrate fraction extracted with urea (HU-Fr) by successive digestion with collagenase [EC 3.4.99.5] and pronase, followed by gel-filtration on Sephadex G-100 and then DEAE-Sephadex A-50 column chromatography. HU-D1 and HU-D2 contained a low sulfated keratan sulfate-like substance linked to peptide and glycopeptide-bound chondroitin sulfated keratan sulfate-like substance linked to peptide and glycopeptide-bound chondroitin sulfate A, respectively. 4. The present findings indicate that rabbit femur contains low sulfated proteokeratan sulfate-like substances with varying sulfate contents and glycoprotein-bound chondroitin sulfate A as the principal glycosaminoglycans. The macromolecules bound more tightly to the tissue contain much more sulfate than the corresponding loosely bound ones.

The composition of acidic glycosaminoglycans (AGAG) in normal and atherosclerotic human cerebral arteries was studied by enzymatic methods. The main cerebral trunk contained more AGAG than the distal branches. The content and relative proportion of heparan sulfates were greater in normal areas than in those affected, but the reverse was found for dermatan sulfate and chondroitin 6-sulfate. Quantitative changes in AGAG with severity of the disease were compared with those in water and lipid content.

Lipoprotein-acid mucopolysaccharide complexes occurring in different types of human atherosclerotic lesions were isolated and partially characterized. Complexes from fatty streaks and fibrous plaques were extracted from pooled tissues with 0.15 M NaCl, purified by gel filtration, and fractionated by ultracentrifugation. Both low and very low density lipoproteins were present; low density lipoprotein was predominant. The complexes were analyzed for uronic acid, cholesterol, phospholipid, and Ca-2+ contents; there was no significant difference in the relative molar ratios between complexes from fatty streaks and fibrous plaques. Acid mucopolysaccharides were isolated from the complexes and identified by electrophoresis and enzymatic studies. Chondroitin sulfate C and hyaluronic acid were found in complexes from fatty streaks and fibrous plaques. Heparin was detected only in fibrous plaques.

Extracellular matrix molecules including glycosaminoglycans have been implicated in several differentiative and morphogenetic processes including cell aggregation and migration. Previous reports have shown that plating of stage 24 limb mesenchyme cells onto hyaluronic acid (HA) bonded to the culture substrate causes an increase in the number of cells exhibiting chondrogenesis. This increased chondrogenesis is now shown to be dependent upon the source of the HA. When limb mesenchymal cells are plated onto HA from bovine vitreous humor, human umbilical cord, or large molecular weight HA (Healon), increased chondrogenesis is observed only on the bovine vitreous humor HA. Unsulfated chondroitin, which has a structure and charge density similar to those of HA, is capable of enhancing chondrogenesis, while cells plated onto sulfated glycosaminoglycan substrates are indistinguishable from controls. The evidence in this report suggests that the differentiation response is related to the molecular size of the HA bound to the culture substrate. Healon and human umbilical cord HA are ineffective because their molecular weight is too large, while smaller HA derived from these larger molecules or normally present in bovine vitreous humor preparations stimulates the chondrogenic differentiation of stage 24 limb mesenchymal cells in culture. The most active size class of HA elutes from a Sepharose CL-2B column with a Kav between 0.6 and 0.7 and, thus, has a molecular weight of approximately 200,000-400,000. These observations reinforce the hypothesis that local cues have an informational effect on the differentiation of chick limb mesenchymal cells.

Heparin (Hep), hyaluronic acid, chondroitins (sulfate) A, B, and C, and heparins (sulfate) A, B, C, and D were subjected to microelectrophoresis in barbital-agarose gel, fixed with cetylpyridinium chloride and stained with toluidine blue. The optical densities of the resulting bands were compared with optical densities obtained upon reaction with azure A in aqueous solution and with the carbazole reagent. A linear relation was obtained between optical density and concentration of purified sulfated mucopolysaccharide (SMP). Less than 1 microgram of Hep and 2 microgram of other SMPs are required for measurement by electrophoresis, while about 30 microgram of each is required with the carbazole reagent. The optical density of a mixture of SMPs was equal to the sum of the densities for the individual SMPs upon microelectrophoresis. It was demonstrated that the individual SMPs in mixtures were distinguishabed by reaction with specific enzymes and by changes in migration in agarose with barbital, phthalate, ethylenediamine, or propanediamine buffers, permitting ready demonstration and quantitation of various SMP species. Examples are shown of the application of the procedure to measure the total SMPs and individual SMPs in tissue extracts. The method is sensitive, reproducible, flexible, and measures quantities 1/30th of those measured colorimetrically, yet is relatively unaffected by protein, carbohydrate, or inorganic electrolytes.