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[Quantitative isolation of acid mucopolysaccharides from urine in progressive scleroderma (author's transl)].

After filtration the urine was concentrated to about 1/10 of the original volume. The urine was centrifuged for 15 min at 5000 rev/min. The supernatant was chromatographed on Bio-Gel P-2-with a 10% aqueous solution of ethanol. Then the glycosaminoglycan fraction was separated into seven further fractions by chromatography on Dowex 1 X 2 and elution with increasing concentration of sodium chlorid. The desalted fractions were analysed. From the analytical data presented no definite correlation between excretion of glycosaminoglycans in urine and progressive scleroderma can be observed. These results were discussed with further data presented by Hexosamine determination.

Glycosaminoglycans↗

Breakdown of articular cartilage proteoglycans by lymphokine-activated macrophages.

Lymphokine supernatants (LE) prepared from antigen sensitive lymphocytes caused an inhibition of migration of macrophages from capillary tubes. Control supernatants (LC) had no effect. The lymphokine supernatants, when added to macrophage cultures (the equivalent of 60 x 10(6) lymphocytes added to 40 x 10(6) macrophages), activated the macrophages so that they secreted the enzyme collagenase after 48 h and 72 h of culture. No collagenase was detected before 48 h or from macrophage supernatants to which LC was added. The macrophage supernatants (LE but not LC) also contained factors (probably enzymes) that, when added to a piece of articular cartilage in medium, caused a partial loss of the hexosamine content of the articular cartilage. These changes were seen as early as after 24 h of culture. Activated macrophages therefore release enzymes that can completely destroy cartilage. Both collagenase and a proteoglycan-hydrolyzing enzyme are released which in vivo might be responsible for the cartilage damage that is found in diseases such as rheumatoid arthritis.

Animals↗

Effects of ionizing radiations on human costal cartilage and exploration of the procedures to protect the tissue from radiation damage.

The chemical changes produced following 60Co-gamma-irradiation of human costal cartilage have been monitored using "critical electrolyte concentration" (CEC) measurements utilizing Alcian Blue--magnesium chloride and Toluidine Blue--sodium chloride systems. The decrease in the CEC with radiation can be related to the decrease in hexose and hexosamine contents of the cartilage. Such changes arise as a result of degradation of the glycosaminoglycan component of the tissue. The state of the collagen fibres after irradiation has been examined using the Van Giesson stain and by biochemical assay of the hydroxyproline moeity. Energy transfer methods have been utilized to protect the glycosaminoglycan component of the cartilage from radiation damage.

Aged↗

Histochemical studies of epithelial cell glycoproteins in normal rat colon.

Two general classes of glycoproteins have been identified in the colonic epithelial cells of the Sprague Dawley rat. Glycoproteins belonging to the first of these classes contain sialic acids both with and without side chain o-acyl substituents, abundant o-sulphate ester and 'neutral sugars' (hexose, 6-deoxyhexose or N-acetyl hexosamine residues) with oxidisable vicinal diols and are located in the goblet cells of the descending colon and in goblet cells populating the upper halves of the crypts of the ascending colon. In the descending colon, the sulphosialoglycoproteins in the goblet cells in the base of the crypts contain sialic acids without side chain o-acyl substituents. It appears that as these cells migrate up the crypts, there is o-acylation of the side chains of the sialic acids of the glycoproteins and an increase in the quantity of 'neutral sugars' without a corresponding increase in sialic acid. Glycoproteins with similar properties to those of the goblet cells of the upper halves of the crypts of the descending colon, but containing less sulphate, are found in the goblet cells of the upper half of the crypts of the ascending colon. The second general class of glycoproteins contain sialic acids all, or almost all of which, are substituted at position C8 and only relatively small quantities of sulphate. They are located in the mucous cells of the descending colon, the deep crypt secretory cells of the ascending colon and the columnar absorptive cell brush border.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Further studies on hepatic acidic glycosaminoglycans in the Hurler syndrome.

Acid GAG were isolated from hepatic tissue from 3 patients with Hurler syndrome and 3 normal controls. Gross elevations in the uronic acid and hexosamine contents were found in Hurler livers compared with the normal ones. The total GAG concentration was significantly increased (about 25fold) in Hurler patients.

Child, Preschool↗

Studies in relation to endocrine exophthalmos: the biochemical composition of human retrobulbar connective tissue.

As part of studies on the pathogenesis of exophthalmos of Graves' disease, the biochemical composition of human retrobulbar tissue was investigated. The connective tissue was composed of 72.7% lipid, 23.8% water and 3.5% dried defatted tissue. Total tissue glycosaminoglycan (GAG) amounted to 0.18% of dried defatted tissue. Approximately 27% of the total hexosamine and 19% of the total tissue galactosamine were recovered in the GAG fraction. Cellulose microcolumn fractionation of GAG showed that the hyaluronic acid and dermatan sulfate were the two major GAG species.

Aged↗

Some observations on the calcium ion binding to the eggshell matrix.

The calcified matrix of the hen eggshell has been demineralized with the EDTA. Aliquots of this material are soluble in water and have been characterized by column chromatography and by chemical analyses. Of particular interest is the high hexosamine and uronic acid content, which confirms the protein-polysaccharide nature of this water-soluble material. The calcium ion binding to the eggshell matrix has been studied by the equilibrium dialysis technique at different pH values, with both free and blocked carboxylic groups. The material with the free carboxylic side chain groups binds more calcium ions with increasing pH value. When the carboxylic groups have been previously blocked with a water-soluble carbodiimide, the calcium ion binding rapidly decreases. The residual capacity to bind calcium ions in the material with the carboxylic functions modified is probably due to the sulfate ions. In agreement with previous observations on other calcified substrates, the calcium ion binding seems to depend on the presence of ionized carboxylic functions of the matrix.

Amino Acids↗

Physical training and connective tissues in young mice-heart.

The effect of physical training on the chemical properties of the heart tissues was studied in male mice of NMRI-strain. The mice to be trained and their controls were about 2 weeks old at the beginning of the training, which took place on 5 degrees inclined treadmill 5 days a week for 3-22 weeks. The duration of daily exercise was progressively increased over the first 3 weeks. The final daily exercise bouts were 50 and 80 min for moderate programs and 180 min for the intensive program at a speed of 30 cm/s. The whole heart or the ventricles were used for the analyses. We found no significant changes related to training in the concentrations of nitrogen, hexosamines, and hydroxyproline both in the normal-sized and the hypertrophied hearts. The hydroxyproline concentration of the ventricles was lower than that of the whole heart tissue, but no difference was observed in the concentration of nitrogen. The hydroxyproline concentration of the heart tissue increased with age both in the trained and untrained mice (39% over 19 weeks). We conclude that collagen and non-collagen proteins in the heart tissue of young mice are stimulated in equal proportions by physical training.

Animals↗

Loss of proteoglycans during decalcification of fresh metaphyses with disodium ethylenediaminetetraacetate (EDTA).

Recent immunofluorescent and histochemical data did not detect changes in the concentration of proteoglycans between noncalcified and calcified cartilage in fetal bovine growth plate or metaphyseal bone. These findings were constant, regardless of prior fixation before demineralization with disodium ethylenediaminetetraacetate (EDTA) or prior demineralization before fixation. Previous experience has shown that EDTA can extract proteoglycans from calcified cartilage. With this in mind, we determined the amount of proteoglycan extracted from calcified cartilage in metaphyseal bone and uncalcified growth plate cartilages during decalcification of unfixed fresh tissues with EDTA. To this end, fresh growth plate cartilages and metaphyses were decalcified at 5 degrees C for 48 hours in a buffered solution of EDTA to which several protease inhibitors were added. Under these conditions 20-25% of the total proteoglycan (measured as uronic acid and hexosamine) was extracted from mineralized cartilage but only about 1% from the uncalcified (growth plate) cartilages. Thus, histochemical and immunohistochemical studies appear to be insensitive measures of proteoglycan concentrations in histological sections of mineralized tissue and may not give quantitative information.

Animals↗

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.

Animals↗

Chemical analysis of the lamella walls of Agaricus bisporus fruit bodies.

Purified lamella wall fragments of Agaricus bisporus fruit bodies were analyzed and shown to consist of neutral sugars (46.5%), hexosamines (31.7%), proteins (9.5%), some lipid material (10.0%), and ash (1.4%). The cell walls were fractionated on the basis of their polysaccharide solubility in water and alkaline solutions. The isolated fractions, using methylation analysis, exhibited striking chemical structural differences compared with the same fractions obtained from the corresponding vegetative cells and fruit bodies (stipe and pileus) walls. The structural differences detected in the wall seem to correspond to the ultimate differentiation of the mycelium inside the fruit body of A. bisporus.

Agaricus↗

A role for N-acetylglucosamine as a nutrient sensor and mediator of insulin resistance.

The ability to regulate energy balance at both the cellular and whole body level is an essential process of life. As western society has shifted to a higher caloric diet and more sedentary lifestyle, the incidence of type 2 diabetes (non-insulin-dependent diabetes mellitus) has increased to epidemic proportions. Thus, type 2 diabetes has been described as a disease of 'chronic overnutrition'. There are abundant data to support the relationship between nutrient availability and insulin action. However, there have been multiple hypotheses and debates as to the mechanism by which nutrient availability modulates insulin signaling and how excess nutrients lead to insulin resistance. One well-established pathway for nutrient sensing is the hexosamine biosynthetic pathway (HSP), which produces the acetylated aminosugar nucleotide uridine 5'-diphospho-N-acetylglucosamine (UDP-Glc-NAc) as its end product. Since UDP-GlcNAc is the donor substrate for modification of nucleocytoplasmic proteins at serine and threonine residues with N-acetylglucosamine (O-GlcNAc), the possibility of this posttranslational modification serving as the nutrient sensor has been proposed. We have recently directly tested this model in adipocytes by examining the effect of elevated levels of O-GlcNAc on insulin-stimulated glucose uptake. In this review, we summarize the existing work that implicates the HSP and O-GlcNAc modification as nutrient sensors and regulators of insulin signaling.

Acetylglucosamine↗

The urinary excretion of glycosaminoglycans and heparan sulphate in lupus nephritis.

Renal involvement in systemic lupus erythematosus (SLE) affects the disease outcome. In order to advance the diagnosis and the initiation of therapy, non-invasive diagnostic techniques are required. In this study, urinary glycosaminoglycans (GAG) and heparan sulphate (HS) were measured in 26 patients with biopsy-proven lupus nephritis and compared to 16 healthy controls. Uronic acid as a representative of GAGs in urine was determined spectrophotometrically with the meta-hydroxydiphenyl, following acid treatment. HS was determined as hexosamine by the method of Smith and Gilkerson. The median values of GAG (3.99 mg/g crea./day) and HS (2.41 mg/g crea./day) in patients were significantly ( P = 0.001) higher than in the control group (1.98 and 0.87, respectively). There was a positive correlation between GAG and HS values ( P = 0.000, r = 0.924) in SLE patients. There were no differences in HS excretion, microalbuminuria and SLE-DAI scores between different classes of lupus nephritis. However, GAG values in class 3 nephritis were significantly ( P = 0.033) higher than from both class 2 and class 4 lupus nephritis. There were no differences in all the measured parameters between normoalbuminuric, microalbuminuric and macroproteinuric patients. Furthermore, there were no correlations between GAG, HS excretions and SLE-DAI scores or microalbuminuria. These results suggest that urinary GAG and HS may serve as useful, independent and non-invasive markers of lupus nephritis.

Adolescent↗

Introduction of hyperglycemia and dyslipidemia in the pathogenesis of diabetic vascular complications.

Hyperglycemia and dyslipidemia are significant and independent risk factors for the vascular complications in patients with diabetes. They have been suggested to cause cardiovascular pathologic changes in diabetic states through the following molecular mechanisms: formation and accumulation of advanced glycation end products; increased oxidative stress; activation of protein kinase C pathway; increased activity of hexosamine pathway; and vascular inflammation and the impairment of insulin action in the vascular tissues.

Diabetic Angiopathies↗

Myocardial infarction in young vs old male rats: pathophysiologic changes.

Young (90 days) and old (15 months) male, Sprague-Dawley rats were subjected to an acute and massive myocardial infarct by giving them two injections of a large dose of isoproterenol. The animals were autopsied at sequential time intervals to ascertain the similarities or dissimilarities in the pathophysiologic events which attend acute myocardial infarction and repair in young vs old rats. Although the signs and severity of hypotensive shock appeared to be equal, mortality was higher in the old rats, especially during the acute necrosis phase. The older rats also manifested more severe and persistent congestive heart failure, i.e., hydrothorax. Serum enzymes (CPK, SGOT, SGPT, and LDH), lipids (triglycerides, free fatty acids, and cholesterol), glucose, and BUN levels manifested a dynamic rise and fall concomitant with the induced myocardial necrosis and repair phases with distinct differences in these metabolic changes between young and old rats. Despite initially higher circulating levels of corticosterone in the old vs young rats, the older animals manifested little or no increase in circulating corticosterone levels during the acute stress of myocardial infarction. This apparent lack of adrenocortical responsiveness was accentuated by the concomitant finding of greatly hypertrophied, hemorrhagic, and lipid-depleted adrenal glands in the old rats vs a dynamic increase in circulating corticosterone levels and alterations in the weight of adrenal and thymus glands of the young rats. During the myocardial repair phase, the young rats manifested extensive endocardial fibrosis whereas the old rats displayed little or no endocardial fibrosis but copous and persistent myocardial edema and ground substance in keeping with their higher concentration of cardiac hexosamine. The pathophysiologic course of events which attends myocardial necrosis and repair is quite different in young vs old rats and may be related to the degree of responsiveness of the pituitary-adrenal axis which changes with age.

Adrenal Glands↗

Effects of the beta-adrenoceptor antagonists atenolol and propranolol on human whole saliva flow rate and composition.

The effects of beta-adrenoceptor antagonists on salivary secretion have been extensively studied in animals but not in man. The aim here was to compare salivary flow rate and composition in man during 1 week of treatment with a non-selective (propranolol 80 mg b.i.d.) and a beta 1-selective (atenolol 50 mg o.d.) antagonist with that of placebo. The randomized, double-blind, cross-over ("Latin square") design was used and 42 healthy male volunteers were recruited to the study. The treatment periods were separated by a wash-out period of 2 weeks. Whole saliva was sampled on day 0 (before) and on day 7 during each treatment. The plasma concentration of propranolol and atenolol was determined from blood samples obtained on day 7. Resting saliva was assessed for flow rate, amylase activity and concentration of total protein, hexosamine and sialic acid. Stimulated saliva was assessed for flow rate, pH, buffer pH, amylase activity and concentration of total protein, Ca2+, Mg2+, Na+, K+, Cl- and PO4(2-). In resting as well as stimulated whole saliva both the total protein concentration and the amylase activity were significantly decreased during the active treatment periods (p less than 0.05-p less than 0.001). At lunchtime during atenolol treatment the hexosamine/total protein and the sialic acid/total protein ratios were significantly increased (p less than 0.05-p less than 0.01), suggesting a possible effect on protein synthesis. In addition, the concentrations of Ca2+, PO4(2-), Cl- and Mg2+ were significantly altered during the active treatment periods (p less than 0.05-p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

The effects of long-term treatment with salmeterol and salbutamol on the flow rate and composition of whole saliva in the rat.

The effect of long-acting beta 2-adrenoceptor agonists on salivary glands and saliva secretion has not been studied before. Sprague-Dawley rats were given either the long-acting beta 2-agonist salmeterol, 1 mg/kg body wt per day or the short-acting agonist salbutamol, 5 mg/kg per day. Saline solution was used as control. After 18 days pilocarpine-stimulated saliva was collected, and after 21 days saliva was collected after stimulation with isoproterol and pilocarpine in combination. The saliva was analysed for total protein, amylase, hexosamine, sialic acid, sodium, potassium and calcium. At day 25 the salivary glands were extirpated and weighed. The weight of the parotid glands increased significantly after both salmeterol and salbutamol treatment, approx. 40%; the submandibular gland weights were not affected by either beta 2-agonist treatment. Pilocarpine-stimulated salivary flow rate was increased in the salbutamol, but not in the salmeterol, group. In the salmeterol group the concentration of sialic acid was increased and that of calcium was decreased. In saliva stimulated with pilocarpine and isoproterenol in combination, the concentrations of total protein, amylase and calcium were decreased after salmeterol. In the salbutamol group, total protein and potassium were decreased. The ratio sialic acid: total protein was increased at both saliva collections in both beta 2-agonist groups. It is concluded that rats treated chronically with the long-acting beta 2-adrenoceptor agonist salmeterol have an impaired secretion of salivary proteins and calcium and that the effect resembles that of salbutamol.

Adrenergic beta-Agonists↗

Sialoglycopeptides from bovine milk fat globule membrane.

Milk fat globule membrane was shown to contain sialic acid, all of which could be released without disruption of the fat globule. Sialoglycopeptides were cleaved from the surface of intact fat globules by Pronase and fractionated on Sephadex G-50. Further fractionation of the major sialoglycopeptide peak on DEAE-Sephadex gave two groups of sialoglycopeptides eluted with 0.1 M NaCl (Group A) and 0.5 M NaCl (Group B), respectively. Refractionation gave a major sialoglycopeptide from each of the two groups together with a total of three minor sialoglycopeptides. All five sialoglycopeptides eluted as single peaks using shallow salt gradients on DEAE-Sephadex and contained a hydrophilic peptide chain together with galactose, mannose, N-acetylgalactosamine, N-acetylglucosamine, and sialic acid. Glycopeptides of Group A but not Group B contained fucose. The major sialoglycopeptide of Group B released 35% of its hexose and hexosamine on treatment with alkaline borohydride leaving a sialoglycopeptide which had reduced serine and threonine and elevated alanine levels and in addition contained 2-aminobutyric acid. An oligosaccharide fraction containing N-acetylgalactosaminitol, galactose and sialic acid in a molar ratio of 1:1:2 was partially characterised from the clevage mixture. The major sialoglycopeptide of Group A had a more complex carbohydrate structure and showed no released carbohydrate on treatment with alkaline borohydride. The sialoglycopeptides of milk fat globule membrane show many similarities with those of erythrocyte membrane and have a potential use in comparative and structural studies.

Amino Acids↗