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PubMed1983

Hyaluronic acid content of deep and subcutaneous bursae of man.

To provide a comparison of the contents of subcutaneous and deep bursae we dissected these structures from unfixed cadavers without apparent joint disease. No free fluid was found within any olecranon or prepatellar bursae (examples of subcutaneous bursae), while viscous fluid was invariably present in the (deep) retrocalcaneal bursae. The hyaluronic acid content of the washings of 5 rectrocalcaneal bursae ranged from 142 to 591 nmol hexosamine (mean = 281 nmol hexosamine). In contrast, the hyaluronic acid content of 4 olecranon bursae was much lower (range 35-72 nmol, mean 53 nmol hexosamine), and hyaluronate was not detected in washings from either of 2 prepatellar bursae. The greater hyaluronate content of the retrocalcaneal bursae did not appear to be due to a greater surface area, since on the basis of calculations made from plaster casts the surface areas of the olecranon and prepatellar bursae were approximately 3 times and 2 times, respectively, greater than that of the retrocalcaneal bursae. The data suggest that, although hyaluronic acid may lubricate deep bursae, other factors may be more important in reducing friction within superficial bursae.

Aged↗
PubMed1986

Copper and cobalt alter the cell wall composition of Cunninghamella blakesleeana.

Cunninghamella blakesleeana was highly sensitive to Cu and Co on a medium containing NaNO3 as the sole nitrogen source. The nitrate reductive pathway was altered by Cu and Co, and NO-2 accumulated in the medium. Under conditions of Cu toxicity, the mycelium and the cell walls acquired a blue color, and most of the Cu was located in the cell walls, which differed in several aspects from cell walls derived from Co-containing or control cultures. At half-maximal growth inhibition by Cu (2.5 micrograms/mL or 39.3 microM) or Co (3.5 micrograms/mL or 59.4 microM), the mycelia contained 1.5 micrograms Cu or 1.0 microgram Co/mg dry tissue, respectively, but the isolated cell walls contained 33.5 micrograms Cu or 1.8 micrograms Co/mg dry cell wall. The phosphorous content of mycelia from Co-containing cultures was the same as that from control cultures, whereas that of mycelia from Cu-containing cultures contained 36% less. However, the phosphorous content of the cell walls from mycelia cultured in the presence of Cu or Co was two- and three-fold higher, respectively, than that of cell walls from control cultures. The cell walls of Cu-containing cultures contained significantly less hexosamine than the control cell walls, and chitin and chitosan were present in equal quantities. The cell walls of Co-containing cultures had the same amount of hexosamine as the control cell walls, but 88% of the hexosamine was present as chitosan and bound very little Co. The control cell walls contained approximately 60% chitosan.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Wall↗
PubMed1972

Studies on human platelet gangliosides.

Gangliosides, glycosphingolipids which contain sialic acid, were studied in human platelets. They represented 0.5% of the platelet lipids and accounted for 6% of the total neuraminic acid content of platelets. Three major ganglioside fractions were identified and characterized. Ganglioside I was hematoside (G(6)) and comprised 92% of the platelet gangliosides. It contained glucose, galactose, and sialic acid in molar ratios of 1:1:1 and no hexosamine. The major fatty acid was behenate (22:0). Ganglioside I was also identified in isolated platelet granules and membranes. Ganglioside II (5%) contained glucose, galactose, sialic acid, and hexosamines (molar ratios 1:2:1:1). The hexosamines were glucosamine (72%) and galactosamine (28%). It was therefore designated as ganglioside lacto-N-neotetraose. Ganglioside III (2%) contained disialosyllactosyl ceramide (G(3A)) as well as two other gangliosides which could not be precisely characterized. Gangliosides I, II, and III were susceptible to the action of Clostridium perfringens neuraminidase as evidenced by full recovery of sialic acid in its free form after incubation. Neutral platelet glycolipids were qualitatively examined by thin-layer chromatography. The major component was lactosyl ceramide. Interactions of gangliosides I and III and serotonin-(14)C were examined in an equilibrium dialysis system at 4 degrees C. The gangliosides bound serotonin-(14)C in relatively small quantities, whereas control lipids were negative. The binding was essentially unchanged by reverse dialysis, ultracentrifugation and subsequent thin-layer chromatography. The results are comparable to the previously observed nonmetabolic interactions between whole platelets and serotonin in the cold. It is suggested that the orientation and specific distribution of platelet membrane glycolipids may be important determinants of the unique surface properties of platelets.

Blood Platelets↗
PubMed1988

Serum glycoconjugates in patients with anemia and myeloid leukemia.

Because of carbohydrate alterations in malignant cells, serum glycoproteins have drawn considerable attention. In the current investigation we determined total sialic acid (TSA), lipid bound sialic acid (LSA), protein bound hexoses (galactose + mannose), fucose, hexosamines (galactosamine + glucosamine) and mucoid protein concentrations in the serum of patients with anemia and myeloid leukemia. The results were compared with those obtained in healthy individuals. In the leukemia patients we observed significant increases in glycoconjugates compared with the controls (P less than 0.001), and in TSA and fucose levels compared with the anemia patients (P less than 0.001). LSA and hexosamine levels were significantly lower in anemia patients with respect to the leukemia patients (P less than 0.01 and P less than 0.05 respectively), whereas levels of mucoid proteins and hexoses did not show significant differences. Except for hexosamines, all the markers tested were significantly elevated in the anemia patients compared with the controls. The present study suggests that the glycoconjugates investigated might be useful biochemical markers for differentiating anemic from leukemic conditions.

Anemia↗
PubMed1995

Regulation of glutamine:fructose-6-phosphate amidotransferase gene transcription by epidermal growth factor and glucose.

In preparation for the cellular proliferation stimulated by growth factors, the rate of macromolecular synthesis must be increased to allow for the enlargement of the cell that proceeds mitosis. The increased glycoprotein synthesis that follows growth factor stimulation would consume the hexosamines required for protein modification. Glutamine:fructose-6-phosphate amidotransferase (GFAT) is the rate-limiting enzyme controlling the synthesis of the hexosamines used in these biosynthetic pathways. We tested the idea that growth factors might activate the transcription of the GFAT gene to increase the cellular content of this rate-limiting enzyme in hexosamine synthesis. We employed a human breast cancer cell line, MDA468 cells, which express high numbers of epidermal growth factor (EGF) receptors, to determine whether EGF could stimulate transcription of the GFAT gene. Our experiments showed that EGF stimulated the accumulation of GFAT messenger RNA (mRNA) to a level 4-fold higher than that in unstimulated cells. This accumulation could be largely accounted for by an increase in transcription, as assessed by nuclear run-on experiments. Furthermore, the GFAT mRNA was highly stable and not further stabilized by EGF. This effect of EGF on GFAT gene transcription required stimulation for 12-16 h with EGF. Interestingly, when cells were exposed to 25 mM glucose instead of 5 mM glucose, this effect of EGF was blocked. Glucose had no effect on the stability of the GFAT mRNA, implying that the effect of glucose was to antagonize the transcriptional effect of EGF on the GFAT gene. Glucosamine had an effect opposite that of glucose, in that it stimulated GFAT mRNA accumulation and had an additive effect with EGF on the accumulation of this mRNA. These results demonstrate that the GFAT gene undergoes a late transcriptional response to EGF and that the provision of high glucose concentrations to the cells blocks this EGF activation. This effect of glucose does not appear to result from its metabolism through GFAT to glucosamine.

Animals↗
PubMed2004

Common variants in glutamine:fructose-6-phosphate amidotransferase 2 (GFPT2) gene are associated with type 2 diabetes, diabetic nephropathy, and increased GFPT2 mRNA levels.

Increased flux of glucose through the hexosamine biosynthetic pathway has been implicated in insulin resistance, altered insulin secretion, and diabetic nephropathy. Glutamine:fructose-6-phosphate amidotransferase (GFPT), the rate limiting enzyme in hexosamine biosynthesis, is encoded by the unlinked but highly homologous genes GFPT1 and GFPT2. We tested the hypothesis that GFPT2 sequence variation contributed to the susceptibility to type 2 diabetes mellitus (T2DM) and diabetic nephropathy in Caucasian and African-American individuals. We identified 11 single nucleotide polymorphisms (SNPs), of which seven were common. A single variant in exon 14, I471V, altered the amino acid sequence, is conserved between human and mouse genes, and was associated with T2DM among Caucasians (P = 0.05). A trend to an association was noted with diabetic nephropathy among African-American individuals (P = 0.15). Several variants in the 3' untranslated region (UTR) and exon 18 were also associated with T2DM in Caucasian individuals (P < 0.05), and the SNP in the 3' UTR was associated with diabetic nephropathy in African-American subjects (P = 0.047). GFPT2 mRNA levels in transformed lymphocytes from study subjects were significantly increased among African-American subjects compared with Caucasian individuals, regardless of diagnosis. Furthermore, the associated allele of the 3' UTR SNP was approximately 2-fold overexpressed. We propose that the 3' UTR variant results in increased GFPT2 mRNA levels with resultant increased hexosamine flux. The I471V variant may contribute to altered protein function or may simply be in linkage disequilibrium with the 3' UTR.

3' Untranslated Regions↗
PubMed2001

Short-term glucosamine infusion does not affect insulin sensitivity in humans.

Overactivity of the hexosamine biosynthetic pathway may underlie hyperglycemia-associated insulin resistance, but to date human studies are lacking. Hexosamine pathway activation can be mimicked by glucosamine (GlcN). In the present placebo-controlled study we determined whether GlcN infusion affects insulin resistance in vivo. In 18 healthy subjects, we applied the double forearm balance technique (infused arm vs. control arm) combined with the euglycemic hyperinsulinemic clamp (60 mU/m(2).min insulin) for at least 300 min. During the clamp, subjects received infusions in the brachial artery of 4 micromol/dL.min GlcN from 90-240 min (n = 6) or from 0-300 min (n = 6) or saline (placebo; n = 6). We studied the effects of GlcN on forearm glucose uptake (FGU; infused arm vs. control arm, and vs. placebo experiments) and on whole body glucose uptake. GlcN infusion raised the plasma GlcN concentration in the infusion arms to 0.42 +/- 0.14 and 0.81 +/- 0.46 mmol/L; plasma GlcN remained very low (< 0.07 mmol/L) in the control arms and in the placebo group. GlcN infusion did not change forearm blood flow. During insulin, FGU increased more than 10-fold. At all time points, FGU was similar in the GlcN-infused arm compared with the control arm and was not different from FGU in the placebo experiments. Similar results were obtained for forearm arteriovenous glucose differences or extraction and for whole body glucose uptake. Thus, despite relevant GlcN concentrations for 5 h in the infused forearm, GlcN had no effect on insulin-induced glucose uptake. These results do not support involvement of the hexosamine pathway in the regulation of insulin sensitivity in humans, at least not in the short-term setting.

Adult↗
PubMed1979

Immuno-chemical studies on the alkali-labile carbohydrate chains of human serum glycoproteins.

Human serum glycoproteins can be classified into those containing N-acetyl-D-galactosamine and into those lacking this hexosamine. The N-acetyl-D-galactosamine-containing serum glycoproteins have alkali-labile chains containing this hexosamine linked O-glycosidically to hydroxy amino acids. These alkali-labile chains can be demonstrated in neuraminic acid free serum glycoproteins by gas liquid chromatography and by using precipitating lectins from invertebrates and plants. They are represented by two chains, one containing only N-acetyl-D-galactosamine, the other with D-galactose linked (1--3) beta-glycosidically to this hexosamine forming a disaccharide. Serologically these two chains, which usually occur together on one molecule, can be characterized by their reaction with lectins from Helix pomatia (anti-A like) and from Agaricus bisporus and Arachis hypogaea (anti-TF specificity).

Acetylgalactosamine↗
PubMed2000

Effects of glucosamine infusion on insulin secretion and insulin action in humans.

Glucose toxicity (i.e., glucose-induced reduction in insulin secretion and action) may be mediated by an increased flux through the hexosamine-phosphate pathway. Glucosamine (GlcN) is widely used to accelerate the hexosamine pathway flux, independently of glucose. We tested the hypothesis that GlcN can affect insulin secretion and/or action in humans. In 10 healthy subjects, we sequentially performed an intravenous glucose (plus [2-3H]glucose) tolerance test (IVGTT) and a euglycemic insulin clamp during either a saline infusion or a low (1.6 micromol x min(-1) x kg(-1)) or high (5 micromol x min(-1) x kg(-1) [n = 5]) GlcN infusion. Beta-cell secretion, insulin (SI*-IVGTT), and glucose (SG*) action on glucose utilization during the IVGTT were measured according to minimal models of insulin secretion and action. Infusion of GlcN did not affect readily releasable insulin levels, glucose-stimulated insulin secretion (GSIS), or the time constant of secretion, but it increased both the glucose threshold of GSIS (delta approximately 0.5-0.8 mmol/l, P < 0.03-0.01) and plasma fasting glucose levels (delta approximately 0.3-0.5 mmol/l, P < 0.05-0.02). GlcN did not change glucose utilization or intracellular metabolism (glucose oxidation and glucose storage were measured by indirect calorimetry) during the clamp. However, high levels of GlcN caused a decrease in SI*-IVGTT (delta approximately 30%, P < 0.02) and in SG* (delta approximately 40%, P < 0.05). Thus, in humans, acute GlcN infusion recapitulates some metabolic features of human diabetes. It remains to be determined whether acceleration of the hexosamine pathway can cause insulin resistance at euglycemia in humans.

Adult↗
PubMed1999

Biochemical & histochemical changes relating to fibrosis following infection with Mycobacterium tuberculosis in the guinea pig.

Guinea pigs infected with M. tuberculosis were studied for parameters relating to fibrosis following infection. The infected animals were followed up to a period of 44 wk and the changes that occurred in the lung, liver and spleen were studied. Corresponding tissues from animals injected with bleomycin, an anti-mitotic drug which has the ability to produce pulmonary fibrosis, served as positive controls. Tissue collagen, elastin and hexosamines were estimated biochemically. The presence of granuloma and stainable collagen in paraffin sections of these tissues was also studied. Establishment of the infection was assessed bacteriologically by culturing the viable organisms from the spleen. It was observed that a self-limiting infection was established in the guinea pigs and none of the animals died of the infection. In the infected animals, collagen, elastin and hexosamines showed an initial decrease followed by an increase. While the elastin and the hexosamine levels returned to the basal levels in all the three organs, collagen levels increased in the lung and were comparable to those of the bleomycin control. Collagen stainable by Van Gieson's method was found to be increased in the lung from the 4th wk onwards. The present report indicates the potential of adopting this system for studying mechanisms of fibrogenesis in tuberculous infection.

Animals↗
PubMed2001

Effects of ovine LH, GH and prolactin, and testosterone on serum testosterone and estradiol-17 beta levels, and seminal vesicle and testicular activity in the catfish Clarias batrachus (L.).

Intraperitoneal administrations of testosterone (0.5 microgram/g body wt), and ovine LH (1.0 microgram/g body wt), GH (5 micrograms/g body wt) and prolactin (10 micrograms/g body wt) daily for 7 days during early prespawning phase (May) in C. batrachus produced varied effects on seminal vesicle (SVSI) and testicular (GSI) weights and biochemical correlates. Testosterone and LH treatments significantly increased serum testosterone level and concentrations of total proteins, fructose, hexosamines and sialic acid in both seminal vesicles and testis. Serum E2 levels increased significantly only after testosterone treatment. GH treatment increased significantly serum testosterone level and only the concentrations of SV hexosamines and testicular protein. Prolactin, however, significantly lowered serum testosterone level and concentrations of total protein, hexosamines in both SV and testis, and testicular fructose and sialic acid levels. The results show that the stimulating effect of LH and GH on SV and testicular activity is mediated through the increased secretion of testosterone and the inhibitory effect of prolactin by decreased testosterone secretion.

Animals↗
PubMedDate not supplied

Biochemical analysis of rabbit articular cartilage using an amino acid analyzer.

The automated amino acid analyzer has been used to provide a simple, rapid and accurate means of assaying the glycosaminoglycan (GAG) and collagen content, and collagen type of connective tissues. The analysis of rabbit articular cartilage using this method is reported. Cartilage slices taken from the tibial plateaus of rabbits were dried and weighed. A comparison study showed that oven-drying (110 degrees C for 24 hours) was more effective in dehydrating cartilage than vacuum dessication for 96 hours. This drying temperature could be maintained for 24 hours without destroying hexosamine. The dried samples were hydrolyzed for 4 hours in 4 N HCl at 100 degrees C; these conditions were shown to give virtually 100% release of galactosamine from purified chondroitin-6-sulphate with minimal degradation of the hexosamine. The 4-hour hydrolysates were assayed for galactosamine and glucosamine using the amino acid analyzer. From these values the amount of chondroitin sulphate and keratan sulphate were calculated. An aliquot of the 4-hour hydrolysate was hydrolyzed further in 6 N HCl for 24 hours. This destroyed hexosamine but permitted the quantitation of hydroxyproline and hydroxylysine using the amino acid analyzer. Collagen content was calculated from the hydroxyproline value and the hydroxylysine to hydroxyproline ratio was used to determine the collagen type.

Amino Acids↗
PubMed1980

[Variations in the P. aeruginosa polysaccharide synthesis conditioned by aminosugars (author's transl)].

The production of capsular and extracellular polysaccharides by P. aeruginosa species was conditioned by the presence of hexosamines in the growth media. Glucosamine and most remarkably galactosamine, strongly reduced the production of extracellular polysaccharides. Studies on the dynamics of the polysaccharide production show that these products are used by the cells after the carbon source (glucose of glucosamine) of the growth medium has been exhausted. These results suggest the presence of adaptative hexosaminidases. The concentration of total hexosamines in the polysaccharides changed according to the carbon source (glucose, glucosamine or galactosamine) and so did the molecular composition as demonstrate by IR spectrometry. IR spectra show variations in the intensity of some characteristic bands, as those located at 1,650-1,560 cm-1 (N-acetyl-hexosamines), 1,420 and 1,320 cm-1 (aminosugars) and at 1,380 cm-1. This last band disappears from polysaccharides after prolonged incubation. Bands between 1,300 and 700 cm-1 varied and even disappeared according to the aminosugar employed and the length of the incubation period.

Galactosamine↗
PubMedDate not supplied

Connective tissue response to immobility. Correlative study of biomechanical and biochemical measurements of normal and immobilized rabbit knees.

The biomechanical changes in rabbit knee contractures were measured after 9 weeks of immobilization. These results were correlated with the biochemical composition of periarticular connective knee tissue of the same knees. The loss of total hexosamine correlates significantly with joint stiffness on an animal-to-animal basis. Total hexosamine also correlates with the energy required cyclicly to flex and to extend the experimental joints. Of the individual glycosaminoglycan fractions, only hyaluronic acid showed significant correlation with the biomechanical data. The possible mechanism of contracture formation was postulated based on these results.

Animals↗
PubMed1983

Glycosaminoglycan content in skin of the tight-skin mouse.

The tight-skin (TSK) mouse has cutaneous changes similar to those found in the skin of patients with progressive systemic sclerosis (PSS). Previous studies have shown that both have common abnormalities in skin thickness, dry weight, and hydroxyproline content. In this study, glycosaminoglycans (GAGs), major components of the ground substance, were quantitated in skins from TSK mice and compared with age-matched normal mice. Biochemical studies included determinations of hexosamines, uronic acids, and total GAGs by cetylpyridinium chloride precipitation. Dry weights and water-fat content of skin biopsy specimens from TSK mice were also compared with those of normal mice. Hexosamine, uronic acid, total GAGs, and dry weight were increased in TSK mouse skin when compared with normal mouse skin. The water-fat content did not differ significantly. These findings were similar to those known to occur in PSS skin, further suggesting that the TSK mouse might serve as an animal model for the skin changes found in PSS patients.

Animals↗
PubMed1987

Site-related variations in glycosaminoglycan content and swelling properties of bovine flexor tendon.

The presence of morphologically distinct tissues within the adult bovine deep flexor tendon presented a model which we examined for correlations between proteoglycan content and tissue swelling properties. The proximal portion which experiences only tensile forces contained low levels of glycosaminoglycan (0.2% glycosaminoglycan hexosamine as percentage of dry weight) that were evenly distributed throughout its length and thickness. Collagen accounted for 80% of tissue mass, and collagen fibers formed parallel arrays running longitudinal to the direction of tensile force. In equilibrium bulk swelling tests, the properties of proximal tissue were uniform throughout the tissue and typical of collagenous tissues in which the response to equilibration in low ionic strength buffers or acid pH is dominated by the collagen network. The anterior aspect of the distal deep flexor tendon articulates with the stiff paratendinous sheath and sesamoid bones and is subjected to compressive and frictional forces in addition to longitudinal tensile forces. Along this anterior surface and extending partially into the tendon is tissue that resembles fibrocartilage with collagen fibers organized as a random network. Greatest glycosaminoglycan hexosamine contents (2-3% of dry weight) were found in the distal surface layer, and large proteoglycans were concentrated at the site receiving direct compressive and frictional loads. Equilibrium bulk swelling tests on distal tissue showed that the articulating surface layer possessed unique material properties. Distal surface layers swelled when counter ions were washed from the tissue, whereas lowered pH had little effect on distal tissue volume. Swelling properties of distal tissue correlated directly with proteoglycan content and were similar to swelling properties of articular cartilage. These results suggest that the articulating layer of distal flexor tendon is adapted for a unique set of mechanical requirements and that an elevated proteoglycan content at the site of compressive and frictional forces contributes to meeting these functional needs.

Animals↗
PubMed1999

Enhanced expression of osteopontin by high glucose in cultured rat aortic smooth muscle cells.

Atherosclerotic vascular disease is a major complication of diabetic patients, and osteopontin has recently been implicated in the development of atherosclerosis. In the present study, we have investigated the effects of high glucose on expression of osteopontin in cultured rat aortic smooth muscle cells. High concentrations of glucose increased osteopontin secretion from the cells, and the increased secretion was completely inhibited by an inhibitor of protein kinase C, GF109203X. Northern blot analysis confirmed the enhanced effect of glucose on expression of osteopontin mRNA. Promoter activity of osteopontin, measured using the osteopontin promoter/luciferase expression vector system, was increased by high glucose, and the enhanced effect was completely inhibited by GF109203X. Glucosamine also increased the promoter activity of osteopontin, and azaserine, an inhibitor of glutamine:fructose-6-phosphate amidotransferase (the key enzyme of the hexosamine pathway), profoundly inhibited high glucose-mediated increase in the promoter activity. Taken together, these data indicate that high glucose enhances the expression of osteopontin at the transcriptional level possibly through the activation of protein kinase C as well as the hexosamine pathway. Our results suggest that osteopontin could play a role in the development of diabetic vascular complications.

Animals↗
PubMed1976

[Sephadex G 100- Separation of the mucicrinic and the mucimitiginic factors from an extract of duodenal mucosa (author's transl)].

The mucicrin stimulating the hexosamine gastrointestinal secretion and extracted form porcine duodenum, was concentrated in the zone 7 using sephadex G 100. The molecular weight of 4000-5000 corresponds to this zone. An opposite activity was detected in zone 3. The factor from digestive mucous origin being able to diminish the hexosamine gastric secretion and not influencing the arterial pressure was called mucimitigin. The molecular weight of about 35000 corresponds to this factor.

Animals↗