Distribution Of Glycosaminoglycans Research Articles

Stromal accumulation of chondroitin sulphate in mammary tumours of dogs.

To contribute to the investigation of the composition of the extracellular matrix in epithelial tumours, mammary gland tissues of dogs (including tumours, hyperplasias and normal tissue as well as metastatic lesions in lymph nodes and lung) were studied histochemically and immunohistochemically for distribution of sulphated glycosaminoglycans (s-GAGs). The formaline-fixed tissue was stained by alcian blue at pH 5.8, using the ‘critical electrolyte concentration’ to study the degree of sulphation of s-GAGs. s-GAGs were characterized by degradation with enzymes and nitrous acid and by immunohistochemistry with two anti-chondroitin sulphate monoclonal antibodies. The light microscopic investigation of s-GAG deposits revealed a limited number of patterns of their distribution. The main s-GAGs found in the mammary gland tumours of dogs and in metastatic lesions were chondroitin sulphate (CS) and heparin/heparan sulphate (HEP/HS). CS accumulated in diffuse structures between epithelial cells as well as around clusters of tumour cells. The latter pattern, possibly representing a mesenchymal reaction to the tumour, was present in 74% of the tumours, and in 67% of these, highly sulphated CS was present. A diffuse accumulation of CS was present almost exclusively in complex and mixed tumours; because of the expression of the 3B3 epitope for CS in immature cartilage the spindle cells of complex tumours are argued to be the precursors of the cartilage in mixed tumours. HEP/HS was stored mainly in mast cells that were found in increased numbers in hyperplasias and tumours. By pretreatment of microscopic slides with chondroitinase AC or ABC immunostaining of fibronectin could be made possible in areas in which CS was abundantly present, suggesting that CS may mask fibronectin epitopes. It is concluded that CS with different degrees of sulphation is the most important s-GAG in the extracellular matrix of mammary tumours of dogs. CS and other s-GAGs accumulate at different sites and may have a different pathogenetic significance. © 1999 Cancer Research Campaign

Monitoring glycosaminoglycan replenishment in cartilage explants with gadolinium-enhanced magnetic resonance imaging.

We previously devised a magnetic resonance imaging method that allows for the nondestructive and quantitative determination of glycosaminoglycan concentration in excised cartilage. The technique measures the concentration of the charged contrast agent Gd-DTPA2- (gadolinium diethylenetriamine-pentaacetic acid) equilibrated within cartilage, from which the tissue distribution of glycosaminoglycan can be calculated. The goals of our study were to determine the practicality of nondestructively monitoring glycosaminoglycan concentration in cartilage explants over a long-term culture period and to determine if glycosaminoglycan could be restored to glycosaminoglycan-depleted cartilage explants maintained in long-term culture. To meet our objectives, we harvested bovine cartilage explants, treated them initially with trypsin to reduce the glycosaminoglycan concentration, and cultured them for as long as 8 weeks. Images depicting glycosaminoglycan concentration were calculated from magnetic resonance images acquired at selected intervals during the trypsinization process and the subsequent culture period. The results indicate that gadolinium-enhanced magnetic resonance imaging can follow the reduction of glycosaminoglycan concentration over the course of enzymatic digestion and the replenishment of glycosaminoglycan over several weeks of culture and that cultured cartilage explants are capable of restoring glycosaminoglycan to 85% of its initial concentration. Of particular interest, samples cultured for 5 weeks indicated a depth dependence of glycosaminoglycan regeneration to values similar to the initial physiologic distribution. Thus, this magnetic resonance imaging method may be a very powerful means for exploring the spatial and temporal evolution of glycosaminoglycan in cartilage.

Distribution and composition of glycosaminoglycans in the left human coronary arterial branches under myocardial bridge

The distribution and composition of glycosaminoglycans (GAGs) are reported in the anterior interventricular branch under the intermyocardial bridge (MB) and the ventricular branch without bridge, both from the left human coronary artery. Chondroitin sulfate (CS), dermatan sulfate (DS) and heparan sulfate (HS) were purified and quantified by a combination of electrophoretic migration and enzymatic degradation. The absolute amounts of GAGs in the intermyocardial bridge segment (MB) increased by 47%, when compared to the pre (PreMB) and post (PostMB) segments and the ventricular arterial branch (VB). Furthermore, the relative distribution of GAGs in the intermyocardial bridge segment differs when compared to the pre and post segments as well as in the ventricular arterial branch, due to a change in the proportion of DS and CS of 41.9 and 32.4%, compared to 36.4 and 39.7%, respectively. These findings give support to the possible involvement of GAGs in the intermyocardial bridge segment, avoiding local thrombus deposition, reducing atherosclerotic development and moreover giving protection against vessel deformation caused by the systolic pressure.

Biochemical basis of the beta-glucuronidase gene defect causing canine mucopolysaccharidosis VII.

Mucopolysaccharidosis type VII (MPS VII), or Sly syndrome, is an autosomal recessive lysosomal storage disorder resulting from the deficiency in the activity of the enzyme beta-glucuronidase (GUSB). To characterize the biochemical and molecular defect in GUSB-deficient MPS VII dogs, we have measured lysosomal enzyme activities, analyzed distribution of glycosaminoglycans (GAGs), and estimated the size and abundance of the GUSB gene product at the mRNA and protein level in normal, homozygous affected, and heterozygous carrier retinal pigment epithelium (RPE) samples. Compared to normal, only 2-5% and 40-60% of GUSB activity was detected in the affected and the carrier samples, respectively. The decrease in GUSB activity resulted in storage of GAGs predominantly heparan sulfate and chondroitin sulfate. A slight increase in storage of GAGs was also observed in the carrier sample. Northern blot analysis of affected and carrier RPE samples detected a 2.4 kb GUSB transcript similar in size and abundance to that of normal controls. In western blot analysis using anti-human GUSB antibody, three bands of size 78, 56, and 38 kDa were detected in normal samples, which were present at lower intensity in the carrier RPE samples and absent in the MPS VII-affected RPE samples. These results suggest that the mutant GUSB gene causes a posttranscriptional defect and produces an unstable protein.

Glycosaminoglycan and collagen distribution in the developing human vitreous.

We determined the distribution of glycosaminoglycans and collagens in the developing human vitreous. Eighty human eyes from 5 gestational weeks to 2 postnatal years of age were used. Glycosaminoglycan components were determined by enzyme digestion with hyaluronidase or chondroitinase AC and ABC and immunohistochemistry for chondroitin, chondroitin-4-sulfate, chondroitin-6-sulfate, and dermatan sulfate. Collagen distribution was determined by immunohistochemistry for types I, II, and III collagens. Enzyme digestion showed that throughout development hyaluronic acid is the main glycosaminoglycan in the vitreous and in the extraocular space at 5-7 gestational weeks. Both areas were filled with mesenchymal cells. Immunohistochemistry showed chondroitin-6-sulfate in the vitreous between 6 and 40 gestational weeks, and chondroitin-4-sulfate between 12 and 40 gestational weeks. Hyaluronic acid and chondroitin sulfate appeared in the retina and around the hyaloid vessels at 12-40 weeks. Immunohistochemistry showed type III collagen in the vitreous and around the mesenchymal cells at 5-7 weeks that was replaced by type II collagen after 8 weeks. Hyaluronic acid is the major glycosaminoglycan in the vitreous throughout development, except for the transient appearance of chondroitin sulfate at 6-40 gestational weeks. Type III is the main collagen in the early developing vitreous that converts to type II collagen at 8 weeks. The primary and secondary vitreous has the same components as these macromolecules. These vitreous glycosamino-glycans and collagens seem to be produced by mesenchymal cells at an early stage and by the retina and hyaloid vessels during middle and late development.

Immunolocalization of glycosaminoglycans in ageing, healthy and periodontally diseased human cementum

The distribution of glycosaminoglycans in the extracellular matrix of human cementum was investigated in periodontally involved and periodontal disease-free teeth separated into eight different age groups (from 12 to 90 years), to investigate possible changes in the distribution of glycosaminoglycan species associated with ageing and periodontal disease. A standard indirect immunoperoxidase technique was used, with a panel of monoclonal antibodies, 2B6, 3B3, 5D4, and 7D4, that recognize epitopes in chondroitin-4-sulphate/dermatan sulphate (C-4S/DS), chondroitin-6-sulphate (C-6S), keratan sulphate (KS) and a novel sulphated chondroitin sulphate (CS) epitope, respectively. Intense positive staining for C4-S/DS was observed at the margins and lumina of almost all the lacunae and canaliculi in cellular cementum in all sections. Immunoreactivity to C6-S, KS and novel CS epitopes was limited to a proportion of lacunae and canaliculi in all sections, although C6-S and the novel CS epitopes were more widely distributed than KS. In acellular cementum, there was no demonstrable staining for any of the glycosaminoglycans except where periodontal ligament (Sharpey's) fibres insert; periodontal ligament fibres inserting in cellular cementum also demonstrated positive immunoreactivity. In addition, the cementoblasts on the outer root surface, as well as the pericellular areas around a proportion of these cells, demonstrated positive immunoreactivity. These results indicate that glycosaminoglycan species present in human cementum include C4-S, DS, C6-S, and novel sulphated CS epitopes. KS is also present in cementum but is limited to a more restricted proportion of lacunae and canaliculi. Regional differences in the distribution of glycosaminoglycans exist between the two cementum types, but no qualitative differences in that distribution were observed between the various age groups or between periodontally involved and periodontal disease-free teeth. The immunoreactivity observed in a proportion of lacunae after staining for C6-S, KS, and novel sulphated CS epitopes could suggest the existence of different cementocyte subpopulations.

Changes in distribution of glycosaminoglycans during the progression of cholesterol induced atherosclerosis in Japanese quail

The temporal and spatial distribution and relative concentration of the proteoglycan glycosaminoglycan component were studied during the progression of atherosclerosis in the systemic arteries of Japanese quail selected for cholesterol induced atherosclerosis (CIA). The CIA quail were placed on either control or 0.5% added cholesterol diets at 3 months of age. The major systemic arteries (dorsal aorta, right and left brachiocephalic) were collected at 1- or 2-week intervals over the 10-week period of cholesterol feeding. In the cholesterol fed quail, alcian blue staining of the dorsal aorta showed elevations of glycosaminoglycans in regions of the artery with atherosclerotic plaque, beginning at the 6-week time point. By biochemical analysis, increases in glycosaminoglycan relative concentration was detected at the 10-week time point. In addition to the change in glycosaminoglycan relative concentration and distribution, the cholesterol fed animals also formed foam cells characteristic of atherosclerotic plaques. Therefore, the conclusion reached was that the CIA line of Japanese quail is a valid animal model for the study of alterations in proteoglycan metabolism in atherosclerotic plaques induced by hypercholesterolemia.

Distribution of collagens and glycosaminoglycans in the joint capsule of the proximal interphalangeal joint of the human finger.

The capsule of the proximal interphalangeal joint consists of the central slip of the extensor tendon dorsally, the collateral ligaments at the sides and the palmar ligament ventrally. Fibrocartilaginous menisci have been reported extending into the joint cavity and the central slip has a sesamoid fibrocartilage articulating with the proximal phalanx. This study relates ECM composition in the joint capsule to function. Each part of the capsule from 24 fingers amputated because of trauma, carcinoma, isthaemia, fixed-flexion deformities or Dupuytren's contracture, was dissected out. Sections were prepared for routine histology or immunolabelled with a panel of monoclonal and polyclonal antibodies against collagens and glycosaminoglycans using the avidin/biotin/peroxidase procedure. All parts of the capsule consistently labelled for types I, III and VI collagens and for dermatan and keratan sulphate, though labelling was more pericellular in fibrocartilaginous regions. In contrast, only certain regions of the capsule in some fingers labelled for type II collagen, chondroitin 4 or 6 sulphate. The sesamoid fibrocartilage in the central slip showed the greatest degree of fibrocartilage differentiation, especially in fixed-flexion deformity fingers, and the palmar ligament the least. The immunolabelling patterns suggest that there is an ordered sequence of matrix changes accompanying fibrocartilage differentiation. Chondroitin sulphate-containing proteoglycans accumulate first, and type II collagen appears later. The presence or absence of type II collagen probably relates to different levels of compressive loading. No fibrocartilaginous menisci were found in normal joints and those described previously are regarded as synovial folds.

Molecular size distribution analysis of human gingival proteoglycans and glycosaminoglycans in specific periodontal diseases.

In order to determine the molecular-size distribution of gingival proteoglycans (PGs) and glycosaminoglycans (GAGs) both in periodontal health and disease states, gingival tissues were obtained from patients with early onset periodontitis (EOP) and adult periodontitis (AP) and also from periodontally healthy subjects. Gel filtration chromatography of gingival PGs revealed different profiles for periodontally diseased and healthy gingiva. Healthy gingiva was mainly composed of high-molecular size proteins and PGs, while diseased gingival tissue presented a decrease in high-molecular size PG forms and a shift towards low-molecular size proteins and PGs. This indicates the degradation of PG macromolecules during periodontal disease activity. Furthermore, this shift towards low-molecular size forms was more intense in EOP patients when compared to AP patients. Gel filtration of gingival GAGs also demonstrated depolymerization of GAGs, with low-molecular size GAGs being more intense in periodontally diseased gingiva, while healthy gingival GAGs profile was mainly composed of high-molecular size GAGs. Similar to the profile of gingival PGs, low-molecular size gingival GAGs were more prominent in gingival tissue from patients with EOP. These findings suggest that both PGs and GAGs, essential components of the extracellular matrix (ECM), are depolymerized during periodontal disease activity, which is more prominent in EOP. Since the basic feature of periodontal disease is matrix degradation, ECM components, more specifically PGs and GAGs, are likely to provide valuable information for a better understanding of periodontal disease activity.

Distribution of glycosaminoglycans in rat renal tubular epithelium.

Polyanionic constituents of the glomerular capillary wall have been previously shown to have a primary role in the control of glomerular filtration. In the study presented here, the distribution and biochemical nature of polyanionic constituents in proximal (PT) and distal (DT) tubules have been investigated as possible determinants of tubulointerstitial function. For histochemical localization of sialic acid, paraffin sections were treated with Arachis hypogaea lectin (PNA) before and after neuraminidase treatment. Electron microscopic characterization of glycosaminoglycans (GAG) was performed on thin LR-white sections, using cationic colloidal gold (CCG) as an histochemical probe, and GAG-degrading enzymes. Without neuraminidase, PNA binded to collecting ducts but not to PT or DT. Neuraminidase pretreatment resulted in intense PNA binding to the tubulointerstitial blood vessels but only in mild apical tubular binding, which implies a lack of sialoglycoconjugates in the tubular basolateral membranes. In contrast, all PT and DT showed intense CCG binding to basolateral, but not to apical, membranes. All basement membranes showed CCG labeling, with considerable variations in labeling densities between PT (124 +/- 8.8/micron 2) and DT (52 +/- 1.8/micron 2), as well as between tubules and Bowman's capsule (P < 0.0001). Heparinase III treatment induced an almost complete loss of CCG binding in all basement and basolateral membranes, whereas chondroitinase ABC treatment led to a lesser but significant loss (P < 0.0001). The results indicate that rat tubulointerstitium expresses polyanionic constituents, consisting mainly of heparan and chondroitin sulfate. The role of these anionic sites in tubular function has yet to be clarified.

Injury-induced alterations in newly synthesized sulphated proteoglycans from rabbit arterial neointima covered by regenerated endothelium.

Proteoglycan (PG) synthesis is a highly regulated, dynamic process that is known to be altered during atherogenesis. Endothelial injury, which may be the primary event in atherosclerosis, has been reported to stimulate PG synthesis and accumulation in the arterial extracellular matrix. The objective of this investigation was to study injury-induced alterations in PG synthesis and accumulation in the neointima, developed in response to a selective balloon catheter de-endothelialization of aortas of normocholesterolaemic rabbits. One group of rabbit aortas was incubated with 35S-Na2SO4 for 8 h, to study in vitro the de novo synthesis of sulphated PG. Another group of rabbit aortas was used to study PG accumulated in aortic neointima vs. PG present in intima-media of normal aortas. Newly synthesized sulphated PG was characterized by light microscopic radioautography and size exclusion chromatography. Purified intimal-medial PG extracts from unlabelled aortas were analysed for protein and glycosaminoglycan (GAG) content and GAG distribution pattern. Results from this study revealed that the neointima of injured aortas synthesized sulphated PG at a significantly higher concentration than the intima of normal aortas. Size exclusion chromatography revealed that neointima synthesized higher molecular weight PG, and in a higher proportion, than its counterpart PG from normal aortas. PG accumulated in neointima of injured aortas showed a significantly altered GAG distribution pattern. These data confirm that neointima developed in response to injury synthesizes and accumulates PG which is altered compared to PG present in the intima-media of normal aorta.

Characterization of ß-glucuronidase in the retinal pigment epithelium

PURPOSE. To study the biochemical and molecular characteristics of the lysosomal enzyme ß-glucuronidase (GUSB) in the retinal pigment epithelium (RPE) and other tissues of different species. METHODS. Freshly isolated and cultured cells were harvested, and GUSB activity was measured fluorimetrically in cell homogenates or tissue culture media using the synthetic substrate 4-methyl-umbelliferyl ß-D-glucuronide (4-MUG). The temperature and pH optima, and thermal stability of GUSB in the RPE and fibroblasts were established. Distribution of glycosaminoglycans (GAGs), the natural substrates of GUSB, in the RPE and fibroblast cell layer and media was examined by cellulose acetate electrophoresis following 72 h of metabolic labeling with Na 2 35 SO 4. Total or poly A + -RNA isolated from cells or tissues of different species were examined in Northern blots to identify GUSB mRNA transcripts. RESULTS. Among all the species, the activity of GUSB and its mRNA level was found to be consistently high in RPE cells. In RPE cultures, the activity was detected in the cell layer and the media, and the activity decreased in both compartments with serial passage. While the temperature and pH optima for the enzyme activity was similar across the species, the thermal stability was remarkably different. The GAG profiles in RPE cells were different from fibroblasts. Supplementation of the cultured cells with selected GAGs moderately increased the GUSB activity. A GUSB transcript was detected in all the tissues examined. In man, mouse, dog, and cat the size of the transcript was 2.4 kb, while the rat GUSB transcript was 2.7 kb. CONCLUSIONS. The ubiquitous distribution of GUSB was evident from the biochemical and molecular studies. Presence of a high level of GUSB activity in the RPE makes it an ideal model for studies of this enzyme both in normal as well as in diseases resulting in GUSB deficiency.

Age-related changes in the localization of glycosaminoglycans in condylar cartilage of the mandible in rats.

There is little information available regarding the morphological and biomolecular characteristics of mandibular condylar cartilage. The purpose of this study was to determine the age-related changes in the morphology and immunolocalization of glycosaminoglycans (GAGs) in mandibular condyles. The mandibular condylar cartilages from 4-, 8-, 16-, 32-, and 64-week-old Wistar male rats were examined to verify the localization of chondroitin-4-sulfate (Ch-4S), chondroitin-6-sulfate (Ch-6S) and keratan sulfate (KS) using an indirect immunofluorescent technique with three monoclonal antibodies for glycosaminoglycans, 2-B-6, 3-B-3 and 5-D-4, respectively. Morphologically, the condylar cartilage was a growth cartilage during growing periods, began to differentiate into articular cartilage from the central area of 16-week-old condyles, and became mature articular cartilage at 32 weeks of age. A regional difference was found in the morphological features and distribution of GAGs between the anterior, central, postero-superior and posterior areas of the condyles at each age. The immunohistochemical localizations of these three glycosaminoglycans showed age-related, morphology-dependent changes, from growth cartilage to articular cartilage-like cartilage. Immunoreactions for all of the antibodies decreased progressively with age in the interterritorial matrix, while the pericellular and territorial matrix in the condylar cartilage of the mandible maintained relatively higher immunoreactivity. In conclusion, age-related and regional differences in the localization of glycosaminoglycans Ch-4S, Ch-6S, and KS were found in the mandibular condyles in rats, and these changes are believed to be related to functional and developmental requirements.

An immunohistochemical study of enthesis development in the medial collateral ligament of the rat knee joint.

The changing distributions of collagens and glycosaminoglycans have been studied at the attachments of the medial collateral ligament during postnatal development. The ligament is of particular interest because it has a fibrocartilaginous attachment to the femoral epiphysis, but a fibrous one to the tibial metaphysis. Ligaments were examined in rats killed at birth and at 2, 4, 6, 8, 10, 20, 30, 45, 60, 90 and 120 days after birth. Cryosections were immunolabelled with monoclonal and polyclonal antibodies against types I and II collagen, chondroitin 4 and 6 sulfate, dermatan and keratan sulfate. Although the ligament is attached at both ends to bones that develop from cartilage, there was a striking difference in collagen labelling. Type II collagen was only found in spicules of calcified cartilage in bone beneath the tibial enthesis after ossification had commenced, but there was a continuous band of labelling at all stages of development at the femoral enthesis. Initially, the cartilage at the femoral attachment lacked type I collagen, but by 45 days labelling was continuous from ligament to bone. Continuity of labelling was seen much earlier at the tibial enthesis, as soon as bone had formed. There were also marked changes in glycosaminoglycan distribution. Keratan sulfate was present at both entheses up to 45 days, but only at the femoral enthesis thereafter. Both attachments labelled throughout life for dermatan sulfate, but chondroitin 4 and 6 sulfate were only found at the femoral end. The results suggest that enthesial cartilage at the femoral attachment was initially derived from the cartilaginous bone rudiment but was quickly eroded on its deep surface by endochondral ossification as bone formed at the attachment site. It was replaced by fibrocartilage developing in the ligament. This mechanism allows enthesis cartilage/fibrocartilage to contribute to the growth of a bone at a secondary centre of ossification in addition to dissipating stress at the ligament-bone junction.

Compartmental distribution of sulfated glycosaminoglycans in lateral and medial midbrain astroglial cultures.

Sulfated glycosaminoglycans (S-GAGs) were isolated from the pericellular (P), intracellular (I), and extracellular (E) compartments of astrocytes cultures from lateral (L) and medial (M) sectors of embryonic mouse midbrain; these sectors differ in their ability to support neurite growth (L, permissive, M, non-permissive for growth) and laminin deposition patterns (L, fibrillar; M, punctate pattern). The total amount of S-GAGs in M cultures was twice that in L cultures and was particularly high in the P compartment of M glia. Both glial cultures showed heparan sulfate (HS) in the three cellular compartments but chondroitin sulfate (CS) GAGs were vestigial in I and P compartments of L glia. Our results suggest that M and L astrocytes are heterogeneous concerning the ability to synthesize GAGs and distribute them among the different cellular compartments. Together with other data (Garcia-Abreu et al: J Neurosci Res 40:471, 1995; Garcia-Abreu et al: Neuroreport 6:761, 1995), the present results suggest that this heterogeneous features might be at least partially responsible for the differential effects of L and M glial cultures on the growth of midbrain neurons and may also be involved in complex ways in the guidance of axons at the brain midline.

Chronic sun exposure alters both the content and distribution of dermal glycosaminoglycans.

Chronic sun exposure leads to structural and functional alterations in exposed skin. Photoageing is a process distinct from the changes taking place due to chronological ageing. Unique alterations in the dermal extracellular matrix occur as a result of photoageing and are responsible for many of these physiological changes taking place in sun-damaged skin. Accompanying the deposition of abnormal elastic tissue, or solar elastosis, are significant alterations in dermal glycosaminoglycans (GAGs). Accumulation of GAGs as a result of photoageing, as demonstrated in both humans and animal models of photoageing, seems almost paradoxical in view of the large amounts of GAGs present in the skin of newborns, making their skin well hydrated and supple, in sharp contrast to the weathered appearance of photoaged skin. We investigate the relative GAG content of photoaged skin using immunoperoxidase stains specific for hyaluronic acid and chondroitin sulphate, and determine the location of these GAGs using confocal laser scanning microscopy. Our results demonstrate significant increases in GAG staining in sun-damaged vs. sun-protected skin from the same individuals, as measured by computer-based image analysis. Furthermore, confocal laser scanning microscopy reveals that the increased dermal GAGs in sun-damaged skin are deposited on the elastotic material of the superficial dermis of photodamaged skin, and not between collagen and elastic fibres as in normal skin. The abnormal location of GAGs on these fibres may explain the apparent paradoxical weathered appearance of photodamaged skin despite increased GAGs.

Developmental distribution of glycosaminoglycans in embryonic rat brain: relationship to axonal tract formation.

Glycosaminoglycans, the sugar moieties of proteoglycans, modulate axonal growth in vitro. However, their anatomical distribution in relation to developing axonal tracts in the rat brain has not been studied. Here, we examined the immunohistochemical distribution of chondroitin-6-sulfate and chondroitin-4-sulfate, two related glycosaminoglycan epitopes, which are present in three types of glycosaminoglycans: chondroitin sulfate C, chondroitin sulfate A, and chondroitin sulfate B. Further, we compared their distribution pattern to that of axonal tract development. Both glycosaminoglycan epitopes showed a heterogeneous spatiotemporal distribution within the developing rat brain. However, the expression of chondroitin-4-sulfate was more restricted than that of chondroitin-6-sulfate, although both epitopes were detected from embryonic day 13 until the day of birth, overlapping in many regions of the central nervous system including cortex, hippocampus, thalamus, and hindbrain. After birth, the levels of expression of both glycosaminoglycan epitopes progressively decreased and were practically undetectable after the first postnatal week. The expression of chondroitin-6-sulfate and, to a lesser extent, that of chondroitin-4-sulfate, was preferentially associated to the extracellular matrix surrounding specific axon bundles. However, the converse association was not true, and several apparently similar types of axon developed on a substrate devoid of both types of glycosaminoglycan epitopes. These results provide an anatomical background for the idea that different types of glycosaminoglycans may contribute to establish the complex set of guidance cues necessary for the specific development of defined axon tracts in the central nervous system.

Congenital enterocyte heparan sulphate deficiency with massive albumin loss, secretory diarrhoea, and malnutrition

Summary Background The molecular basis of protein-losing enteropathy is unknown. However it has been shown that sulphated glycosaminoglycans may be important in regulating vascular and renal albumin loss. Methods We describe three baby boys who presented within the first weeks of life with massive enteric protein loss, secretory diarrhoea, and intolerance of enteral feeds. All required total parenteral nutrition and repeated albumin infusions. No cause could be found in any case despite extensive investigations, including small intestinal biopsy sampling, which were repeatedly normal. Findings By specific histochemistry, we detected gross abnormality in the distribution of small intestinal glycosaminoglycans in all three infants, with complete absence of enterocyte heparan sulphate. The distribution of vascular and lamina propria glycosaminoglycans was, however, normal. Interpretation The presentation of these infants suggests that enterocyte heparan sulphate is important in normal small intestinal function.

Insulin-dependent diabetes mellitus and glycosaminoglycans.

The influence of diabetic nephropathy on urinary glycosaminoglycan distribution was assessed in 96 patients with insulin-dependent diabetes mellitus (IDDM, 49 female, age: 16 - 64 yrs, median 35; duration of IDDM: 0 - 43 yrs, median 13 yrs) in comparison to 103 healthy controls (57 female, 17 - 82 yrs, median 40 yrs). Glycosaminoglycan concentration of 24 h urine samples was determined by means of precipitation with cethylpyridinium chloride and potassium acetate in ethanol followed by a colorimetric test with carbazole. A marked difference (p = 0.0008) in urinary glycosaminoglycan excretion between patients (19.0, 12.4, 35.6 mg/24 h, median, 25th, 75th percentile) and controls (15.8, 10.4, 21.6 mg/24h) could be detected. In patients with insulin-dependent diabetes mellitus of longer duration, glycosaminoglycan excretion was increased (<10 years: 17.3, 10.8, 30.5 mg/24 h; > 10 yrs: 23.3, 16.0, 39.1 mg/24 h; p = 0.03). Furthermore, diabetic patients with manifest nephropathy and retinopathy exhibited a significantly higher glycosaminoglycan excretion compared to patients without nephropathy (40.8, 23.3, 42.2 versus 18.2, 11.4, 31.0 mg/24 h, p = 0.005) or retinopathy (31.9, 16.6, 41.6 versus 17.6, 11.2, 26.7 mg/24 h, p = 0.009). Thus, the results of this study demonstrate a close relationship between diabetic nephropathy and the renal glycosaminoglycan excretion.

Characterization of glycoconjugate expression during development of Meckel's cartilage in the rat

The staining patterns of 24 biotinylated lectins were analyzed in serial sections of the mandible of 13- to 21-day-old rat embryos by means of the avidin-biotin-peroxidase method. A ubiquitous distribution of binding sites was demonstrated after incubation with Con A (Canavalia ensiformis), DSL (Datura stramonium; except bone matrix), and WGA (Triticum vulgare). ECL (Erythrina cristagalli), GSL I (Griffonia simplicifolia), SJA (Saphora japonica), VVL (Vicia villosa), DBA (Dolichus biflorus), UEA I (Ulex europeus), and LTA (Lotus tetragonobolus) were constantly negative. In early stages of development, GSL II (Griffonia simplicifolia II) was a selective marker of prechondral blastema. In contrast, PNA (Arachis hypogaea) did not stain condensing mesenchyme. During chondrogenesis of Meckels's cartilage a general decrease of lectin binding was observed. Mature cartilage matrix was constantly negative. Chondrocytes were marked by the lectins PSA (Pisum sativum), WGA, PHA-E, and PHA-L (Phaseolus vulgaris E and L). A strong GSL II binding was restricted to the mesial-superior region of the perichondrium. In later stages, several lectins revealed significant differences between preskeletal ("central") areas and the remaining ("peripheral") mesenchyme. A clear binding reaction was noted in central regions by applying LEA (Lycopersicon esculentum) and STL (Solanum tuberosum), while the peripheral tissue was only faintly stained. Developing bone was specifically marked by succinylated WGA (sWGA). The lectins LCA (Lens culinaris) and RCA (Ricinus communis) bound to fibers and extracellular matrix of the connective tissue. Jacalin (Artocarpus integrifolia) and SBA (Glycine max) binding sites were found in macrophages. Affinity of VAA (Viscum album) increased parallel with maturation of endothelial cells. Specific lectin-binding patterns revealed no correlation with the distribution of glycosaminoglycans. The results demonstrate a general reduction of oligosaccharide structures during development of Meckel's cartilage. From our observations we conclude that intralaminar glucose and/or mannose sequences as well as terminal sialic acid molecules are ubiquitously distributed, while terminal alpha-fucose was constantly negative. Lectin-binding patterns of macrophages may reflect the presence of specifically linked terminal galactose. Our findings indicate that oligosaccharides terminating in N-acetylglucosamine are bone-specific. The significance of the restricted staining of the perichondrium by GSL II remains to be elucidated.