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Biochemical changes in the articular cartilage of the patella and femoral condyles in the lateral hyperpatellar syndrome.

The biochemical changes in the articular cartilage of the femoral condyles and the patella were studied during the earliest stage of patello-femoral arthrosis, with particular reference to chondromalacia of the patella in the "lateral hypertension syndrome", with the object of comparing them with the results obtained by Boni et al. (1977) in the initial phases of experimental arthrosis induced in rabbits by means of vitamin A. The biochemical determinations were done on samples of cartilage removed at operation from the medial and lateral femoral condyles and the medial and lateral patellar articular facets in ten patients. The biopsy samples were fixed in 80% alcohol and dried in the oven at 50 degrees C for twenty-four hours. The hexosamine and hydroxyproline content was then determined. This investigation demonstrated significant biochemical changes in this syndrome in the four areas examined. The data obtained indicate that only the mucopolysaccharide component of the joint cartilage is involved at this stage. The most interesting finding was marked diminution in the galactosamine/glucosamine ratio, which was markedly diminished - the opposite of what occurs in established arthrosis. These data appear to be identical with those found in the early phases of vitamin A induced arthrosis in experimental rabbits. The diminution of hexosamine content and the diminished galactosamine/glucosamine ratio were more marked in the femoral condyles, which appeared to have few lesions macroscopically. These changes were also present in both articular facets of patella, but were less marked, probably because the degeneration was more advanced in those areas, thus more closely resembling frank arthrosis.

Adolescent↗

Glycohydrolases and lysosomal stability in adjuvant induced arthritis.

Adjuvant induced arthritis in rats was studied by the changes in serum and urinary protein-bound carbohydrate metabolites, changes in serum and tissue lysosomal glycohydrolases and lysosomal fragility. From the second week onwards the urinary excretion of hexosamine and uronic acid is increased. Serum levels of protein bound hexose, hexosamine, sialic acid and fucose are increased significantly in both the acute and chronic phases of the disease. There is no change in the total activity of lysosomal glycohydrolases, viz., beta-glucuronidase, beta-N-acetyl glucosaminidase and cathepsin D in the tissues of liver, kidney and spleen except that of liver enzymes in the chronic phase which are elevated significantly. The free activities of lysosomal glycohydrolases investigated, viz., beta-glucuronidase, beta-N-acetyl glucosaminidase, beta-galactosidase, alpha-mannosidase and cathepsin D are increased in liver and spleen in the acute phase. The free activities of beta-glucuronidase, beta-N-acetyl glucosaminidase and cathepsin D of kidney showed no change whereas those of beta-galactosidase and alpha-mannosidase are increased. In the chronic phase of the disease the free activities of all glycohydrolases are significantly increased in all tissues. Serum glycohydrolases are significantly increased in both acute and chronic phases. Studies on lysosomal preparations showed increased fragility of lysosomes derived from liver and kidney of arthritic rats in both phases of the disease.

Acetylglucosaminidase↗

Cell-wall proteins of Staphylococcus aureus : a kinetic study of release by lysostaphin.

The kinetics of cell-wall hydrolysis of Staphylococcus aureus (strain Cowan I) by lysostaphin was studied by turbidity, quantitative determination of hexosamines, phosphorus, total proteins, protein A and by electron microscopy. In our experimental conditions, the lytic activity of lysostaphin was very fast and no characteristic cell-wall structures could be seen after five minutes. The release of hexosamines, phosphorus and protein A was synchronous and could be recorded even though turbidity had become stable. At the end of the hydrolysis, the totality of these components was solubilized. Total proteins represented about 14% of the cell-wall dry weight. During hydrolysis protein A was the only protein solubilized.

Bacterial Proteins↗

Role of mucus in gastric mucosal injury induced by local ischemia/reperfusion.

The role of gastric mucus was evaluated in a rat model of gastric epithelial damage induced by local ischemia/reperfusion (I/R) stress. In this model, blood-to-lumen chromium 51-labeled ethylenediaminetetraacetic acid (51Cr-EDTA) clearance served as an index of injury. Tetraprenyl acetone (TPA; 100 mg, 200 mg/kg IP) was used to stimulate mucus production. Administration of TPA increased both the hexosamine content in gastric tissue and the amount of alcian blue-periodic acid Schiff (AB-PAS) stained mucus in the mucosa in a dose-dependent manner. Increases in 51Cr-EDTA clearance induced by I/R were significantly attenuated by TPA in a dose-dependent manner. N-acetyl-L-cysteine (NAC; 0.6%, 0.8%) was perfused into the gastric lumen to assess the effect of reduction in mucus on the injury induced by I/R. Although mean values of hexosamine content were increased by perfusion with NAC, AB-PAS-stained mucus in the mucosa was significantly decreased in a dose-dependent manner. Perfusion of NAC did not change basal 51Cr-EDTA clearance but significantly exacerbated the increase in clearance induced by I/R in a dose-dependent manner. These results indicate that gastric mucus protects the gastric mucosa against I/R stress in vivo.

Acetylcysteine↗

Deacetylase activity of human tumor cells producing immunosuppressive aminosugars: its possible role in resistance to cell-mediated cytotoxicity.

In the present study, we examined the presence of deacetylases capable of producing free hexosamines, which we have shown earlier to be immunosuppressive against human natural killer (NK) cell-mediated cytotoxicity, from N-acetylhexosamines in human tumor cells. When human NK-resistant colon cancer cells (Colo-320DM) were incubated with acetyl-D-[1,6-3H(N)]glucosamine, a significant conversion to [3H]glucosamine occurred. Deacetylation was demonstrated as a change of the substrate radioactivity into free glucosamine trapped by a cation exchange resin, and this was subsequently confirmed by paper chromatography. This deacetylase activity was detected in other NK-resistant tumor cell lines, especially in freshly isolated human renal and breast cancer cells and testicular seminoma cells. However, no deacetylase activity was detected in NK-sensitive target cells such as K562, MOLT-4, or HL-60 cells. The ability to produce free hexosamines from N-acetylated aminosugars may provide a new mechanism for the escape of tumor cells from the attack of immune effector cells such as NK cells.

Acetylglucosamine↗

Some characteristics of the residue obtained after pronase treatment of sheep erythrocyte membranes. II. Carbohydrate patterns.

The composition of single carbohydrate classes of intact and pronase treated sheep erythrocyte membranes has been studied. In comparison with the data obtained from untreated stromata after proteolytic digestion the amount of each class of sugars is decreased. A high disappearance of sialic acids and hexosamines can be observed. Nevertheless if the total sugar content is referred to the residual protein content (16% of the native proteins, an enrichment in carbohydrates, specially neutral hexoses, can be observed. The results indicate that most of the carbohydrate fractions solubilized by pronase treatment are sialic acids and hexosamines. Different molar ratios obtained for the single carbohydrate classes in comparison with NANA before and after pronase treatment suggest a microheterogeneity of the glycoprotein structure of sheep erythrocyte membranes. By the use of several analytical methods the residue obtained after pronase treatment shows two major fractions containing proteins, lipids and carbohydrates. The data are discussed in view of the possible arrangement of lipo-glycoproteins in sheep erythrocyte membrane.

Animals↗

Serum leptin levels in female patients with NIDDM.

OBJECTIVE: To compare serum leptin levels of diabetic and non-diabetic female subjects and also assess the relationship of hyperglycemia with serum insulin, C-peptide and leptin levels. DESIGN: It is a case control study. PLACE AND DURATION OF STUDY: The study was conducted at Medicare Hospital, Family Care Clinic and Baqai Institute of Diabetes and Endocrinology between December 1997 to September 1999. SUBJECT AND METHODS: One hundred and forty female subjects with different body mass indices and fasting blood sugar levels were selected from three different diabetic centers. A venous sample was drawn after an overnight fast (12 hours) for determination of blood parameters in all groups. Glycosylated hemoglobin, hexosamine, fructosamine, insulin and C-peptide were determined only in diabetic patients. Blood glucose, triacylglycerol (TAG), total cholesterol, HDL cholesterol, HbA1C, hexosamine and fructosamine were determined enzymatically. Serum leptin, C-peptide and insulin were measured using enzyme-linked immunoassay. RESULTS: Serum leptin levels of obese diabetic and non-diabetic subjects were significantly higher as compared with lean diabetic patients and non-diabetic subjects (P< 0.05). Leptin levels were positively correlated with serum insulin and C-peptide levels. Serum leptin increased with increase in body mass index and waist hip ratio was strongly related with insulin resistance in NIDDM. CONCLUSION: Leptin levels are increased in obesity and may play a role in development of insulin resistance and NIDDM.

Adult↗

Simultaneous, quantitative analysis of UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine, UDP-glucose and UDP-galactose in human peripheral blood cells, muscle biopsies and cultured mesangial cells by capillary zone electrophoresis.

The aim of this study was to develop and evaluate a capillary zone electrophoretic (CE) procedure for the accurate quantification of the UDP-hexosamines as well as for the corresponding UDP-hexoses in samples from various biological origins. Testing different buffer conditions, voltages, capillary dimensions and temperatures, optimal results were achieved with a 90 mM borate buffer, pH 9.0, at 18 degrees C and 15.5 kV in an uncoated fused-silica capillary of 50 cm x 50 microm and a detection wavelength of lambda = 262 nm. The total procedure, i.e., including variations of the sample preparation, showed coefficients of variation for the peak areas between 4. 1% and 10.4% in mesangial cells (n = 7) and between 7.8 and 10.3% (n = 6) in leukocytes for the components of interest. To improve precision, an internal standard was used for calibration. The limit of detection for all compounds is an absolute amount of 180 fmol, sufficient for the precise analysis of UDP-sugars in a limited amount of biological samples, such as human leukocytes (obtained from a 10 mL blood sample), muscle biopsies (< or = 100 mg), and mesangial kidney cells (ca. 2.5 x 10(5) cells). This reproducible, quantitative analysis of all four UDP-sugars from various biomedically relevant origins by CZE is a definite improvement over the generally used high performance liquid chromatography (HPLC) procedures. The CZE method allows the study of the flux through the hexosamine pathway in diabetes mellitus and other diseases in a simple, quantitative and accurate way.

Blood Cells↗

Preferential incorporation of glucosamine into the galactosamine moieties of chondroitin sulfates in articular cartilage explants.

OBJECTIVE: To determine the metabolic fate of glucosamine (GlcN) in intact articular cartilage tissue. METHODS: Intact articular cartilage explants were cultured for up to 13 days in Dulbecco's modified Eagle's medium supplemented with 1) 1-13C-labeled GlcN, 2) 1-13C-labeled glucose (Glc), or 3) no labeling. Every 3-4 days, samples were removed and frozen in liquid nitrogen for carbon-13 magnetic resonance spectroscopic (MRS) analysis. The metabolic products of the labeled precursors were determined from the MRS data based on resonance positions and comparison with known standards and published values. RESULTS: GlcN was taken up by the chondrocytes and incorporated selectively into the hexosamine, but not the hexuronic acid, components of the glycosaminoglycan chains of articular cartilage proteoglycan. The data also demonstrated that GlcN is the substrate of choice for the galactosamine moieties of the chondroitin sulfates, incorporating at levels 300% higher than with an equivalent amount of labeled Glc. CONCLUSION: The results indicate that GlcN facilitates the production of proteoglycan components that are synthesized through the hexosamine biochemical pathway.

Animals↗

Effect of monensin on the sulfation of heparan sulfate proteoglycan from endothelial cells.

Monensin is a monovalent metal ionophore that affects the intracellular translocation of secretory proteins at the level of trans-Golgi cisternae. Exposure of endothelial cells to monensin results in the synthesis of heparan sulfate and chondroitin sulfate with a lower degree of sulfation. The inhibition is dose dependent and affects the ratio [35S]-sulfate/[3H]-hexosamine of heparan sulfate from both cells and medium, with no changes in their molecular weight. By the use of several degradative enzymes (heparitinases, glycuronidase, and sulfatases) the fine structure of the heparan sulfate synthesized by control and monensin-treated cells was investigated. The results have shown that among the six heparan sulfate disaccharides there is a specific decrease of the ones bearing a sulfate ester at the 6-position of the glucosamine moiety. All other biosynthetic steps were not affected by monensin. The results are indicative that monensin affects the hexosamine C-6 sulfation, and that this sterification is the last step of the heparan sulfate biosynthesis and should occur at the trans-Golgi compartment.

Animals↗

Effect of Withania somnifera on B16F-10 melanoma induced metastasis in mice.

Withania somnifera, a plant with known immunopotentiating activity and its bioactive fraction-Withanolide D were studied for their anti-metastatic activity using B16F-10 melanoma cells in C57BL/6 mice. Simultaneous administration of Withania extract (122 +/- 10 tumour nodules) and Withanolide (126 +/- 9 lung tumour nodules) could significantly (p < 0.001) inhibit the metastatic colony formation of the melanoma in lungs. 72.58% by extract and 69.84% by Withanolide treated, as compared to the untreated control animals also increased the survival days. Lung collagen hydroxyproline content was highly elevated in the control animals (23.5 +/- 0.9 micro g/mg protein), which was reduced by the simultaneous administration of both the extract (16.3 +/- 2.0 micro g/mg protein) and Withanolide (15.3 +/- 1.8 micro g/mg protein). The level of lung hexosamines (4.85 +/- 0.20 mg/100 mg tissue) and uronic acids (330.1 +/- 23.7 micro g/100 mg tissue) content was also elevated in the control animals. The elevated level of hexosamine was significantly reduced by the treatment with extract (1.92 +/- 0.05) and Withanolide (1.85 +/- 0.05). Similarly, the uronic acid content was also been reduced by the simultaneous administration of both Withania extract (194.2 +/- 17.4) and Withanolide (183.2 +/- 8.8). The control animals had 35.3 +/- 3.8 U/L gamma-glutamyl transpeptidase (gamma-GT), which was reduced by 50% by the treatment of extract and Withanolide to 17.5 +/- 4.0 U/L and 16.3 +/- 4.4 U/L respectively. There was a significant reduction in the levels of sialic acid in the serum of Withania extract (60.7 +/- 7.7) and Withanolide (67.16 +/- 5.8) treated animals compared to the higher level (102.2 +/- 8.7) in the control animals. Histopathological analysis of the lung tissues also correlated with these findings. Prophylactic administrations of both extract as well as Withanolide were ineffective in inhibiting the metastasis of B16F-10 melanoma cells.

Adjuvants, Immunologic↗

Evaluation of phytoconstituents of Terminalia arjuna for wound healing activity in rats.

The effect of topical application of phytoconstituents (fraction I, II and III) fractionated from a hydroalcohol extract of the bark of the plant, Terminalia arjuna, was assessed on the healing of rat dermal wounds using in vivo models. The results indicated a statistically significant increase in the tensile strength of the incision wounds and the percent epithelialization of excision wounds compared with control (p < 0.05). However, topical treatment with fraction I, consisting mainly of tannins, was found to demonstrate a maximum increase in the tensile strength of incision wounds. Even with respect to excision wounds, the fastest rate of epithelialization was seen with fraction I. Hexosamine estimation of granulation tissue obtained from excision wounds revealed an increase in the hexosamine content with fraction I compared with the control. In addition, fraction I from the hydroalcohol extract of Arjuna bark possessed antimicrobial activity against tested microorganisms such as Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Streptococcus pyogenes but not Candida albicans. These results strongly document the beneficial effects of fraction I, consisting mainly of tannins, of Terminalia arjuna in the acceleration of the healing process. Thus, the present study validates the claim made with respect to the plant as well as corroborating the astringent effect of tannins by drawing the tissues closer together.

Animals↗

The distinction of underivatized monosaccharides using electrospray ionization ion trap mass spectrometry.

A convenient method for distinguishing underivatized isomeric monosaccharides has been established using electrospray ionization ion trap mass spectrometry (ESI-ITMS). Mass spectra of hexoses (glucose, galactose, and mannose), N-acetylhexosamines (N-acetylglucosamine, N-acetylgalactosamine, and N-acetylmannosamine) and hexosamines (glucosamine, galactosamine, and mannosamine) dissolved in solvent containing 1 mM ammonium acetate were obtained in the positive ion mode. Glucose was distinguished from galactose and mannose in the MS(2) spectrum of the [M+NH(4)](+) ion at m/z 198. The MS(3) spectra generated from [M+NH(4)-H(2)O-NH(3)](+) at m/z 163 showed that galactose and mannose could be distinguished by the ratio of peak intensities at m/z 145 and 127, while the three N-acetylhexosamine and hexosamine stereochemical isomers could be identified by the relative abundance ratios of product ions observed in MS(3) spectra. The investigation of MS and MS(2) spectra from complexes of these monosaccharides with Na(+) and Pb(2+) failed to distinguish these monosaccharide isomers. Therefore, multiple stage mass analysis by ESI-ITMS using either [M+NH(4)](+) or [M+H](+) was useful to distinguish between the isomers of monosaccharides.

Isomerism↗

Stimulation by eicosapentaenoic acids of leptin mRNA expression and its secretion in mouse 3T3-L1 adipocytes in vitro.

Recent evidence indicates that both leptin and eicosapentaenoic acids (EPA) improve insulin sensitivity. In the present study, we examined the effect of EPA on endogenous leptin expression in 3T3-L1 adipocytes to clarify whether the EPA's effect is exerted through leptin expression. EPA caused a time- and dose-dependent increase of leptin mRNA levels in 3T3-L1 adipocytes. Leptin mRNA expression was significantly increased up to 309.4 +/- 17.0% of the control by 24 h (P < 0.01; n = 6). Leptin secretion was also significantly increased up to 193.3 +/- 12.1% of the control by 24 h (P < 0.01; n = 6). EPA is a ligand for peroxisome proliferator-activated receptors (PPARs) with the highest affinity to PPARalpha. We examined the effect on leptin expression of clofibrate, a ligand for PPARalpha, bezafibrate, for PPARbeta, or troglitazone, for PPARgamma, to clarify whether these ligands for PPARs could mimic EPA-induced stimulation of leptin expression. Neither clofibrate nor bezafibrate affected leptin mRNA expression, whereas troglitazone significantly suppressed leptin mRNA expression. On the other hand, inhibition by 6-diazo-5-oxo-l-norleucine of the rate-limiting enzyme in hexosamine biosynthesis blunted EPA-induced stimulation of leptin mRNA expression and its secretion. These data suggest that EPA up-regulates leptin gene expression and its secretion probably through a hexosamine biosynthetic pathway.

3T3 Cells↗

Development and comparison of two 3T3-L1 adipocyte models of insulin resistance: increased glucose flux vs glucosamine treatment.

Insulin resistance can be induced in vivo by intravenous infusion of glucosamine or in cells by incubation with glucosamine. However, a publication (Hresko, R. C., et al. (1998) J. Biol. Chem. 273, 20658-20668) suggests a trivial explanation of glucosamine-induced insulin resistance whereby intracellular ATP pools are depleted presumably due to the phosphorylation of glucosamine to glucosamine 6-phosphate, a hexosamine pathway intermediate. The reduced ATP level impaired insulin receptor (IR) autophosphorylation and tyrosine kinase activity toward substrates. The present work describes the development and comparison of two methods for inducing insulin resistance, by treating 3T3-L1 adipocytes overnight using either 25 mM glucose/5 nM insulin or 2 mM glucosamine. Under these conditions basal glucose transport rates were comparable with controls. Insulin-stimulated 2-deoxyglucose uptake, however, was reduced by approximately 45% in response to both high glucose/insulin and glucosamine treatment, relative to control cells. The total relative amounts of the insulin-responsive glucose transporter, Glut4, remained constant under both treatment conditions. The relative phosphotyrosine (Tyr(P)) contents of the insulin receptor and its substrate 1 (IRS-1) were assessed in whole cell homogenates. With both methods to induce insulin resistance, IR/IRS-1 Tyr(P) levels were virtually indistinguishable from those in control cells. Insulin-stimulated phosphorylation of Akt on Ser(473) was not impaired in insulin-resistant cells. Furthermore, the relative Tyr(P) content of the PDGF receptor was comparable in high glucose/insulin- or glucosamine-treated 3T3-L1 adipocytes upon subsequent challenge with PDGF. Finally, the relative amounts of glutamine:fructose-6-phosphate amidotransferase and O-linked N-acetylglucosamine transferase, two important hexosamine pathway enzymes, were similar in both treatments when compared with controls. Thus, 3T3-L1 adipocytes can be used as a model system for studying insulin resistance induced by increased influx of glucose. Under appropriate experimental conditions, glucosamine treatment can mimic the effects of increased glucose flux without impairment of tyrosine phosphorylation-based signaling.

3T3 Cells↗

Crystal structure of chondroitin AC lyase, a representative of a family of glycosaminoglycan degrading enzymes.

Glycosaminoglycans (GAGs), highly sulfated polymers built of hexosamine-uronic acid disaccharide units, are major components of the extracellular matrix, mostly in the form of proteoglycans. They interact with a large array of proteins, in particular of the blood coagulation cascade. Degradation of GAGs in mammalian systems occurs by the action of GAG hydrolases. Bacteria express a large number of GAG-degrading lyases that break the hexosamine-uronic acid bond to create an unsaturated sugar ring. Flavobacterium heparinum produces at least five GAG lyases of different specificity. Chondroitin AC lyase (chondroitinase AC, 75 kDa) is highly active toward chondroitin 4-sulfate and chondroitin-6 sulfate. Its crystal structure has been determined to 1.9 A resolution. The enzyme is composed of two domains. The N-terminal domain of approximately 300 residues contains mostly alpha-helices which form a doubly-layered horseshoe (a subset of the (alpha/alpha)6 toroidal topology). The approximately 370 residues long C-terminal domain is made of beta-strands arranged in a four layered beta-sheet sandwich, with the first two sheets having nine strands each. This fold is novel and has no counterpart in full among known structures. The sequence of chondroitinase AC shows low level of homology to several hyaluronate lyases, which likely share its fold. The shape of the molecule, distribution of electrostatic potential, the pattern of conservation of the amino acids and the results of mutagenesis of hyaluronate lyases, indicate that the enzymatic activity resides primarily within the N-terminal domain. The most likely candidate for the catalytic base is His225. Other residues involved in catalysis and/or substrate binding are Arg288, Arg292, Lys298 and Lys299.

Amino Acid Sequence↗

Ultrastructural characterization by ruthenium red of the surface of the fat globule membrane of human and rat milk with data on carbohydrates of fractions of rat milk.

The fat globules of the cream fractions of human and rat milk were stained with ruthenium red. Under the electron microscope, discrete granules and an amorphous coat of lesser density are seen at the surface of the milk fat globules. Since ruthenium red binds anionic groups selectively, it is probable that the granules contain the greatest concentration of these groups. The cream fraction of rat milk contains hexoses, hexosamines, methylpentoses and sialic acid. Methylpentoses and hexosamines are significantly enriched in the cream fraction. It is concluded that the finding of a surface coat in milk fat globules is in keeping with the Bargmann-Knoop model and suggests a distinct mechanism for carrying certain complex carbohydrates in milk. The role of the negative charges at the outer surface of the membrane coat is maintaining fat globules in suspension and in binding certain cations such as calcium is suggested.

Animals↗

Modification of the glomerular basement membrane in sucrose-fed and streptozotocin-diabetic rats.

Rats were fed on diets containing either sucrose or starch as the carbohydrate component (55%) for eight months. Diabetes was induced in animals of both groups by injecting streptozotocin (50 mg/kg body weight). Diabetic rats failed to gain weight, had enlarged kidneys, polyuria and elevated blood glucose levels. Starch and sucrose fed rats gained weight normally and had normal blood glucose levels. Sucrose fed rats had enlarged kidneys. Regional thickening of the glomerular basement membrane was present in sucrose-fed and diabetic rats but not in starch-fed controls. Glomerular basement membrane isolated from pooled kidney cortices from rats in the different experimental groups were analysed for amino acid, disaccharide and hexosamine content. Hydroxylysine (9 to 20%), hydroxyproline (21 to 24%), disaccharide (27%) and hexosamine (26%) were increased in membranes insolated from the three experimental groups, compared with starch-fed non-diabetic controls. An increase in low molecular weight components of the glomerular basement membrane of sucrose-fed and diabetic rats was observed using electrophoresis in sodium dodecyl sulphate. Significantly higher (p < 0.001) glucosyltransferase activity was present in kidney supernatants prepared from sucrose-fed (1050 +/- 60 nmol/2h/kidney) compared to starch-fed rats (510 +/- 40 nmol/2h/kidney). Sucrose feeding induces changes similar to those found in diabetes and the induction of diabetes made little difference over the feeding of sucrose alone.

Amino Acids↗