Pattern Of Glycosaminoglycans Research Articles

Sulfated glycosaminoglycans of cultured fibroblasts from guinea-pig embryo kidney

The sulfated glycosaminoglycan content of primary cultures of fibroblasts from guinea-pig embryo kidney is reported. A hybrid chondroitin sulfate comprises approx. 90% of these glycosaminoglycans from the cell coat. Changes in the proportion of labelled heparitin sulfate were also observed after successive subcultures. We postulate a possible correlation between the pattern of glycosaminoglycans and processes of cell selection and cell dedifferentiation in these cultures.

Glycosaminoglycan metabolism of cultured cornea cells derived from bovine and human stroma and from bovine epithelium

The glycosaminoglycan metabolism of cultured epithelial and stroma cells from bovine cornea and of human stroma cells was investigated in comparative study. The following results were obtained: 1. (1) All cell types investigated distributed newly synthesized glycosaminoglycans into extracellular, pericellular and intracellular compartments. Epithelial cells, however, produced lower amounts of sulfated glycosaminoglycans and secreted a smaller proportion of them into the culture medium. 2. (2) Epithelial cells exhibited a more rapid turnover of intracellular glycosaminoglycans than the stroma cell lines. All cell types internalized [ 35S]proteoglycans to a similar extent, but epithelial cells degraded them more rapidly. 3. (3) The three cell types studied synthesized hyaluronic acid, undersulfated chondroitin sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, heparan sulfate and small amounts of keratin sulfate. A characteristic distribution pattern of sulfated glycosaminoglycans was observed with respect to the origin of the cells as well as with regard to topographically distinct compartments. Heparan sulfate was found to be the main constituent of pericellular glycosaminoglycans. Bovine stroma cells secreted predominantly chondroitin-4-sulfate, human stroma cells heparan sulfate and epithelial cells dermatan sulfate into the culture medium. Intracellular glycosaminoglycans of bovine stroma cells consisted mainly of chondroitin-6-sulfate, whereas in human cells dermatan sulfate and in epithelial cells dermatan sulfate and chondroitin-6-sulfate were present in high proportions. Human stroma cells produced more hyaluronic acid and undersulfated chondroitin-sulfate than the other cell types.

Developmental changes in glycosaminoglycans during skeletal muscle cell differentiation in culture

Tissue specificity and developmental changes in glycosaminoglycans were studied in chick embryo skeletal muscle cells differentiating in vitro. Myogenic cells appear to be able to synthesize three main kinds of glycosaminoglycans, namely hyaluronic acid, heparan sulfate and chondroitin sulfate. The patterns of glycosaminoglycans synthesized by terminally differentiated multinucleated myotubes are clearly distinguishable from those synthesized by duplicating mononucleated myogenic cells and by sparse or confluent fibroblasts. These multinucleated myotubes, in particular, exhibit a specific increase of heparan sulfate and a specific decrease of hyaluronic acid in their cell layer-associated fraction of the total synthesized glycosaminoglycans.

A method of screening test for excretion pattern of urinary glycosaminoglycans and its application to normal human urine.

To obtain a clue for the metabolic disorder of glycosaminoglycans (GAG) in connective tissue diseases (CTD), a device of screening test for excretion pattern of urinary GAG was achieved. The crude GAG were separated from voluntary 100-20 ml urine by precipitation with cetylpyridinium chloride, followed by digestion with pronase P. The crude GAG were then separated by one-dimensional Separax (cellulose acetate membrane) electrophoresis in 0.2 M aqueous calcium acetate. GAG, in terms of hexosamine, in each GAG-band were quantitated after extraction from the strip with water. All the urines examined gave three regular bands, bands 1, 2 and 3, but urines from most of the pupils at puberty and from several adults gave irregular bands, besides the regular ones. As for the regular bands, a significant difference was observed in percent distribution of bands 1, 2 and 3 between the samples from male and female of the normal youth aged 20-29, and excretion pattern of three regular bands reached to an adult type much younger in female than in male. GAG in the regular bands were identified as follows: band 1, partially desulfated chondroitin sulfates A and C; band 2, dermatan sulfate plus low-sulfated chondroitin sulfates (LSC) in most cases; band 3, heparan sulfate plus LSC in most cases. Usefulness in clinical field of the screening test is indicated.

Metabolism of cell surface-associated sulfated glycosaminoglycans in cultured human smooth muscle cells.

Smooth muscle cells grown from human aorta synthesize chondroitin sulfate, dermatan sulfate, and heparan sulfate as sulfated glycosaminoglycans. These polymers are found mainly extracellularly and in association with the cell membrane. Each compartment is characterized by a distinct distribution pattern of sulfated glycosaminoglycans though considerable variability was noted between different cell lines. On incubation of the cells in the presence of [35S]sulfate for up to 72 h no significant change in the distribution pattern of newly synthesized extracellular and membrane-associated glycosaminoglycans was found. Prelabeling experiments revealed that pericellular glycosaminoglycans are metabolically heterogenous. they leave their compartment with half-lives of less than 10 h and 1--3 days respectively. During the initial period of the chase experiment (up to 12 h) about equal proportions of the material disappeared either by shedding into the culture medium or by endocytosis. Thereafter, release of macromolecules into the medium exceeded endocytotic uptake. None of the individual glycosaminoglycans on the cell surface showed a clear preference for its removal either by shedding or by endocytosis. It is concluded that cell surface-associated glycosaminoglycans are neither direct precursors of the extracellular glycosaminoglycans nor do they represent mainly extracellular glycosaminoglycans which are in the process of endocytosis.

Pattern of glycosaminoglycans and glycoproteins associated with nuclei of regenerating liver of rat

1. 1. Hyaluronic acid was detected as the largest glycosaminoglycan component in the glycosaminoglycan fraction from purified nuclei of regenerating livers as in the case of normal livers (Furukawa, K. and Tarayama, H. (1977) Biochim. Biophys. Acta 499, 278–289). However, the nuclear content of glycosaminoglycans tended to decrease after partial hepatectomy, reaching one-third of the normal liver level at 24–30 h after partial hepatectomy. On the other hand, two new polyanionic components were detected in the glycosaminoglycan fraction from regenerating liver nuclei. 2. 2. One of these new components seems to be a sulfated glycopeptide. The 35SO 4 incorporation into this component was stimulated biphasically after partial hepatectomy; the first stimulation occurring immediately after partial hepatectomy and the second stimulation occurring almost in parallel to the DNA synthesis. 3. 3. Another polyanionic component which also increases in the nuclear content after partial hepatectomy lacks hexuronic acid, sialic acid and 35SO 4 and yet it is intensely stained by Alcian Blue. Preliminary investigations revealed the presence of hexose, ribose and phosphate as the major components. 4. 4. In contrast to the primary localization of hyaluronic acid in the chromatin fraction and also in the nonhistone chromosomal protein fraction from it, these new polyanionic components were detected mainly in the karyosol fraction.

The cervix of the sheep and goat during parturition

The change in physical character of the wall of the cervix has been investigated in sheep and goats in relation to the induction of parturition. Two types of observation were made, extension of isolated tissue in vitro and dilation of the cervical canal with balloons in vivo. A decrease in the elastance of the tissue was detectable within 12 hours of first administration of dexamethasone and the decrease was progressive thereafter. This change was unrelated to the plasma concentration of prostaglandins F. There was some relationship in time to changes in circulating concentrations of oestrogen and progesterone but this was not consistent in all animals. Biochemical investigations of cervical wall revealed a marked increase in the liberation of hydroxyproline from collagen and a change in the pattern of extractable glycosaminoglycans, as induction advanced.

Metabolism of sulfated glycosaminoglycans in cultured endothelial cells and smooth muscle cells from bovine aorta

The glycosaminoglycan metabolism of cultured endothelial cells and of cells grown from the intima and from the media layer of bovine aorta thoracica was investigated in a comparative study. The following results were obtained: 1. 1. Endothelial cells have in common with intima and media cells the distribution of newly formed sulfated glycosaminoglycans into extracellular, pericellular and intracellular compartments. Endothelial cells, however, synthesize lower amounts of glycosaminoglycans and distribute them in a different ratio into the three pools. 2. 2. Though all the various cell lines synthesize chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate, heparan sulfate and small amounts of keratan sulfate, endothelial cells exhibit a unique distribution pattern of sulfated glycosaminoglycans in each of the three compartments. Generally, a high proportion of heparan sulfate and chondroitin 6-sulfate and a very low dermatan sulfate content was detected. 3. 3. Heparan sulfate produced by endothelial cells has a higher N-sulfonate content when compared with that from other sources. The cell membrane-associated heparan sulfate, especially, exhibits some heparin-like features as judged by nitrous acid degradation and susceptibility towards heparitinase.

Urinary glycosaminoglycan patterns in human hepatic angiosarcoma, hepatoma, and in workers at risk for angiosarcoma

Urinary glycosaminoglycan patterns in human hepatic angiosarcoma, hepatoma, and in workers at risk for angiosarcoma

Urinary glycosaminoglycan patterns in human hepatic angiosarcoma, hepatoma, and in workers at risk for angiosarcoma

Urinary glycosaminoglycan patterns in human hepatic angiosarcoma, hepatoma, and in workers at risk for angiosarcoma

Excretion patterns of glycosaminoglycans in the urine of normal children and adults.

The glycosaminoglycans excretion was studied in the urine of normal human subjects from newborns to 68 years of age. The glycosaminoglycans preparation was made by digesting the urine with proteolytic enzymes followed by removal of enzyme and undigested protein and finally freeze-drying of the supernatant. Cellulose acetate electrophoresis was carried out on these preparations. Electrophoresis of urinary concentrates results in three alcian blue positive fractions. The proportion with the mobility of chondroitin sulfates fraction was high in childhood and thereafter decreased gradually with age, and that of heparan sulfate and keratan sulfate fraction was low in childhood. Any significant change in the fraction corresponding with hyaluronic acid could not be observed throughout the life.

Hormonal effect on glycosaminoglycans and glycoproteins in uteri of ovariectomized rats

1. 1. Glycosaminoglycans such as chondroitin sulfate A (or C), chondroitin sulfate B (dermatan sulfate), heparitin sulfate (hepara sulfate) and hyaluronic acd were identified as major glycosaminoglycan components in whole uteri as well as in uterine stroma of rats. Two types of sialoglycoproteins with different electrophoretic mobilities (fast- and slow-migrating) were detected in the glycosaminoglycan fraction from teh luminal epithelia. 2. 2. Treatment of ovariectomized rats with estradiol-17β makedly increased the uterine contents of glycosaminoglycans. Chondroitin sulfate A (or C) was found to increase more than chondroitin sulfate B. Furthermore, it was found that the estrogen treatment specifically increases the fast-migrating sialoglycoprotein level in the luminal epithelia and results in the appearance of it in the uterine fluid. 3. 3. Administration of progesterone to overiectomized rats slightly increased the uterine glycosaminoglycans content without appreciable alteration of the uterine glycosaminoglycan pattern.

Bidimensional electrophoresis in the diagnosis of mucopolysaccharidosis

Using the modified bidimensional electrophoresis method, characteristic patterns of urinary glycosaminoglycans were obtained for nine different syndromes of mucopolysaccharidoses and normal. Each pattern, portrayed on a 55 mm square cellulose acetate membrane, was visualized by staining the electro-phoretically separated GAGs with alcian blue. The method, tested with 38 cases, was precise and sensitive; the first direction of electrophoresis was run for 25 min and the second direction for 60 min. Because the method is qualitative, the usual quantitative measurements and calculations are circumvented. Moreover, any irregular pattern can readily be verified by repeating the bidimensional electrophoresis. Thus, the clarity of the pattern itself offers assurance of its diagnostic reliability; consequently, false negative and false positive results should be minimized.

Surface exposure of glycosaminoglycans in resting, growing and virus transformed 3T3 cells

Glycosaminoglycans (GAG's) were released by trypsin from the surface of cultured mouse cells (3T3) in two different growing states: during log-growth phase and during resting due to serum starvation. Doubly labelled molecules from resting cells were compared with those from growing as well as from trnsformed cells. Reproducible differences in the elution pattern during ion exchange chromatography and in susceptibility to specific hydrolytic enzymes have been demonstrated: the GAGs pattern of growing normal cells is similar to the pattern of the cells transformed by either Polyoma or SV-40 viruses and very different from the pattern of resting cells. Growing and transformed 3T3 show a relatively low amount of trypsin removable heparan sulphate (HS) and a relatively high amount of hyaluronic acid (HA) while resting cells exhibit an opposite ratio between the two GAG'S. The lowering of HS and the increase of HA in the cell coat is therefore suspected to be more dependent upon growth than upon transformation.

The synthesis of glycosaminoglycans by cultures of rabbit corneal endothelial and stromal cells.

Confluent monolayer cultures of rabbit corneal endothelial and stromal cells were incubated independently with [35S]sulphate and [3H]glucosamine for 3 days. AFter incubation, labelled glycosaminoglycans were isolated from the growth medium and from a cellular fraction. These glycosaminoglycans were further characterized by DEAE-cellulose column chromatography and by sequential treatment with various glycosamino-glycan-degrading enzymes. Both endothelial and stromal cultures synthesized hyaluronic acid as the principal product. The cell fraction from the stromal cultures, however, had significantly less hyaluronic acid than that from the endothelial cultures. In addition, both types of cells synthesized a variety of sulphated glycosaminoglycans. The relative amounts of each sulphated glycosaminoglycan in the two cell lines were similar, with chondroitin 4-sulphate, chondroitin 6-sulphate and dermatan sulphate as the major components. Heparan sulphate was present in smaller amounts. Keratan sulphate was also identified, but only in very small amounts (1-3%). The presence of dermatan sulphate and the high content of hyaluronic acid are similar to the pattern of glycosaminoglycans seen in regenerating or developing tissues, including cornea.

Calf articular cartilage in organ culture in a chemically defined medium. 2. Concentrations of glycosaminoglycans and [35S]-sulfate incorporation at different oxygen tensions.

Articular cartilage from 6-month-old calves was maintained in organ culture in Eagle's minimum essential medium at different oxygen tensions between 20 and 50%. When well standardized cartilage pieces of about 1 mm thickness were used the results showed a high degree of reproducibility. The glycosaminoglycans were studied using the cetylpyridinium chloride cellulose and epichlorohydrin triethanolamine (ECTEOLA) column techniques on a microscale. In some experiments the cartilage was labelled with [35S]sulfate. Small alterations of the concentrations and distribution of different glycosaminoglycans were found and were not affected by various oxygen tensions for up to 4 weeks. A small increase was found in the fractions containing low molecular weight chondroitin sulfate. Devitalized cartilage, maintained under identical conditions, remained largely unaltered during the 4 weeks. The [35S]sulfate activity calculated per mole of hexosamine showed a general decrease during culture maintenance. High specific activities in the fractions containing low molecular weight chondroitin sulfate and keratan sulfate were found between 1 and 3 weeks of maintenance in 20% oxygen. Maintenance in 50% oxygen resulted in severely disturbed synthesis of glycoaminoglycans. The results are interpreted as showing that the synthesis pattern of glycosaminoglycans in articular cartilage can be altered by other factors and without the prior loss of glycosaminoglycans.

Urinary excretion of acidic glycosaminoglycans in the aged.

The excretion of acidic glycosaminoglycans in urines of 30 aged subjects was investigated in relation to age and disease. The pattern of glycosaminoglycans excreted in urines of the aged subjects was compared with each other and with those of healthy young adults (26–31 yrs., male), using two dimensional electrophoresis on cellulose acetate strips. 1. The average amount of acidic glycosaminoglycans excreted in urine of the healthy aged was 1.74±0.51mg/day as uronic acid, indicating that the aged shows a tendency of less excretion of urinary acidic glycosaminoglycans, compared with healthy young adults. 2. Among the aged with individual diseases examined, only patients with diabetes mellitus showed a higher excretion of acidic glycosaminoglycans in their urines, compared with the healthy aged. 3. The analysis of urinary acidic glycosaminoglycans from the aged by two dimensional electrophoresis demonstrated that among 30 aged examined 8 cases excreted low sulfated chondroitin sulfate as a main component and other 7 less but higher than that of young adults. In the remaining 15 cases, rather young adult type of patterns were obtained. 4. Physiological meaning of the excretion of low sulfated chondroitin sulfate in urine is discussed in relation to renal function and aging process.

Studies on corneal polysaccharides: VIII. Changes in the glycosaminoglycans in some human corneal disorders

The glycosaminoglycan pattern in human corneas afflicted with diseases was measured. In maculae corneae, keratitis and lattice degeneration a decrease in the polysaccharide content was observed. In the two latter conditions the presence of dermatan sulfate was demonstrated. The glycosaminoglycan pattern in keratoconus was normal.

Studies on corneal polysaccharides: VII. Changes in the glycosaminoglycans in penetrating corneal grafts

The glycosaminoglycan pattern in penetrating corneal grafts in successful and unsuccessful keratoplasties was investigated in the present study. During the early period of healing a decrease in the polysaccharide content was noticed in both conditions. After 3 months of healing transparent grafts showed a normalization of their polysaccharide pattern, whereas in the nontransparent grafts a further decrease, especially of the keratan sulfate, was observed. The nontransparent grafts also contained dermatan sulfate.

Studies on corneal polysaccharides: V. Changes in corneal glycosaminoglycans in transient stromal edema

The glycosaminoglycan content of corneal stroma during transient edema produced by removal of the endothelium was measured. It was observed that this process is associated with a considerable loss of polysaccharides. Both the keratan sulfate and the chondroitin 4-sulfate content were decreased to approximately 50% of their normal values. After healing the tissue assumed a normal appearance and the polysaccharide content returned to its original level. During this process no qualitative changes in the glycosaminoglycan pattern were observed, nor did any other polysaccharide species appear.