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PubMed1976

The chemistry of human neonatal femoral epiphyseal cartilage.

The estimation of cartilage constituents such as hydroxyproline and hexosamine may be used to detect gross changes in the total amount of collagen and matrix. This simple approach, which can only be regarded as a "screening" procedure prior to more exhaustive analysis, has been applied to samples of neonatal human cartilage. It is suggested that this method would be useful in analysis cartilage biopsies, especially from patients with bone dysplasias.

Calcium↗
PubMed1976

Three cases of GM1-gangliosidosis.

A biochemical analysis was carried out on three cases of GM1-gangliosidosis which showed different clinical manifestations. These cases were classified in a previous study as Type 1, Type 2 (2B) and Type 2 (2A), an intermediate type between classical Type 1 and Type 2 (2B), by the determination of the chromatographic profile of the liver beta-galactosidase activities. Gangliosides, neutral glycolipids; phospholipids and glycopeptides were analyzed in the brain and the liver of these cases. The concentration of total ganglioside was increased in the brain in all cases. The elevation was due to an increase of GM1-ganglioside, which accounted for 63% or more of the total ganglioside, while in the control brain about 20% of the total ganglioside was GM1-ganglioside. In type 2A, increases of GM1-ganglioside and and asialo-GM1 in the liver were more prominent than those in the liver of Type 2B. The non-dialyzable glycopeptides were analyzed only in Type 2A. In the liver of Type 2A, the hexosamine and hexose contents of the non-dialyzable glycopeptides were about 10 times and 5 times higher than those of the control. These biochemical analyses revealed that Type 2A had intermediate characteristics between two Types. In this classification of the three Types, biochemical data were well correlated with clinical features.

Brain↗
PubMed1979

Identification of keratan sulfate in liver affected by Morquio syndrome.

Glycosaminoglycan content, composition and molecular weight were determined in liver obtained from a patient with Morquio syndrome (Mucopolysaccharidosis IV). There was about a four-fold increase in glycosaminoglycan content (as hexosamine) of the affected liver as compared to the control liver. The major glycosaminoglycan accumulated in the liver was keratan sulfate, which was not found in the control liver. Chondroitin sulfates, especially chondroitin 6-sulfate, were also increased. Heparan sulfate isolated from the liver of a patient with Morquio syndrome was structurally different to that from control liver, and the glycosaminoglycans from Morquio syndrome were of a much lower molecular weight than those from control.

Adolescent↗
PubMed1983

Proteoglycans of rabbit cornea: labelling in organ culture and in vivo.

Rabbit corneas maintained with radioactively-labelled precursors in organ culture for up to 42 hr produced labelled proteoglycans of the same kind as those that exist normally or that are produced by labelling in vivo. Whole corneas, including a narrow strip of sclera, were kept in culture in the presence of [3H]-glucosamine and [35S]-sulfate. The rate of incorporation of sulfate into extractable proteoglycans was linear over the time investigated, as was the rate of incorporation of glucosamine after a short lag. Three labelled proteoglycans were isolated and found to behave in ion-exchange chromatography and gel chromatography in the same way as they did in previous studies by chemical analysis. Their labelled glycosaminoglycans were primarily dermatan sulfate and keratan sulfate, with traces of hyaluronic acid and heparan sulfate. When labelled precursors were injected directly into the anterior chamber of rabbit eyes, the resulting labelled proteoglycans were similar to those obtained in organ culture. Both in vivo and during organ culture, the specific activity of hexosamine in the keratan sulfate proteoglycans was about one-half that in dermatan sulfate, probably because of different synthetic rates or different specific activities of immediate precursors.

Animals↗
PubMed1992

Leishmania donovani: purification and partial characterization of a glycophosphosphingolipid antigen expressed on promastigote surface.

A ceramide-anchored glycophosphosphingolipid antigen was isolated from the lipid extract of Leishmania donovani promastigotes. The affinity-purified glycolipid antigen contained galactose, mannose, myo-inositol, phosphate, ceramide, and hexosamine but no sialic acid. The phosphate group was present internally at the core of the structure: inositol (1-O)-phosphorylceramide. The phosphate group became susceptible to alkaline phosphatase only after alkali-catalyzed hydrolysis of the glycolipid.

Animals↗
PubMed1983

Incorporation of radioactive 35SO4(2-) into immunoreactive pituitary lutropin.

The terminal hexosamines of bovine pituitary lutropin are thought to contain a sulfate moiety. In order to test this, a biosynthetic approach was adopted. When rat and buffalo (bovine) pituitaries were incubated with radioactive 35SO4(2-) for 2 h in vitro it was observed that radioactivity test incorporated into trichloroacetic acid-precipitable proteins. When the radioactive proteins were treated with an anti-sheep lutropin serum, radioactivity was found in the immunoprecipitate. The incorporation into rat lutropin like material was very marginal while it was very significant in the case of buffalo lutropin.

Animals↗
PubMed1984

Amino acid sequence of the N-terminal region of human hemopexin.

Cyanogen bromide digestion of hemopexin at its 6 methionine residues results in 7 fragments (CB1-CB7) partially connected by disulfide bridges. By sequence studies of fragments CB1-CB4 and peptides prepared by their enzyme cleavage, a continuous amino acid sequence of the N-terminal region of human hemopexin, comprising 220 amino acid residues, was determined. The presence of intramolecular disulfide bonds, connecting half-cystine residues 126/130 and 165/170, was proved in fragments CB2 and CB3. Fragments CB1-CB4 include 5 sites, where hexosamine oligosaccharides are attached (positions 1,41,164,217 and probably 223). In the sequenced region two sites sensitive to acid hydrolysis--bonds ... Asp--Pro ... in positions 20/21 and 187/188 were found. In spite of the fact that pooled material of many donors was studied, no sequence heterogeneity was discovered.

Amino Acid Sequence↗
PubMed1985

Polydispersity of acidic glycosaminoglycan components in human liver and the changes at different stages in liver cirrhosis.

The aim of this study was to characterize acidic glycosaminoglycan components in normal human liver and in alcoholic cirrhosis, and to determine whether the proportions of individual glycosaminoglycans change with advancing cirrhosis. Acidic glycosaminoglycans are components of extracellular matrices and consist of repeating disaccharides of hexosamine and hexuronic acid with molecular weights ranging from 5 X 10(3) to 5 X 10(4), except for hyaluronic acid, whose molecular weight ranges from 3 X 10(4) to 1.6 X 10(6). The acidic glycosaminoglycan components in normal liver and at different stages of liver cirrhosis were found to be polydisperse as to the molecular weight and the degree of sulfation. The increased content of glycosaminoglycans with advancing liver cirrhosis was related to those of heparan sulfate and dermatan sulfate components. Heparan sulfates were the most prominent components of smaller molecular weight fractions. In the normal state, moderate amounts of dermatan sulfate and the oversulfated isomer were present in intermediate molecular weight fractions, but increasing amounts of the components shifted to higher molecular weight fractions with advancing cirrhosis. A small amount of hyaluronic acid was found in higher molecular weight fractions and the amount increased at the initial stage as a reversible phenomenon. The possible roles of hepatic glycosaminoglycan components in the process of fibrosis are discussed.

Chromatography, Gel↗
PubMed1986

Purification, characterization, and bioassay of teleocalcin, a glycoprotein from salmon corpuscles of Stannius.

This report describes the isolation of teleocalcin, a glycoprotein hormone from the corpuscles of Stannius (CS) of sockeye salmon (Oncorhynchus nerka), using affinity, gel exclusion, and ion exchange chromatography. In discontinuous, gradient (10-20%) SDS gels under nonreducing conditions, teleocalcin had an estimated molecular weight of of 39,300 and migrated as a single band. Two bands with estimated molecular weights of 28,000 and 32,000 were obtained upon reduction and carboxymethylation of the molecule. In acid-urea gels, teleocalcin migrated as one band with an Rf of 0.17. Amino acid sequence analysis revealed single residues for the first 19 amino acids, with phenylalanine as the N-terminal residue. Teleocalcin did not resemble parathyroid hormone (PTH) with respect to amino acid composition and exhibited no cross-reactivity in two PTH radioimmunoassays. Gas-liquid chromatographic analysis of teleocalcin demonstrated that mannose was the principal sugar present (1.86%) and glucosamine was the only hexosamine identified (2.49%). Smaller quantities of galactose (0.49%), fucose (0.44%), and sialic acid (1.6%) were also found. In bioassays using juvenile rainbow trout (Salmo gairdneri), teleocalcin significantly reduced the rate of net branchial 45Ca uptake at dosages as low as 0.02 microgram/g body wt. This inhibitory effect was dependent upon the branchial calcium uptake cycle that has been identified in this species. Pronounced inhibition was observed during periods of high uptake whereas little or no effect was obtained during the low uptake phase. The results are discussed in relation to a possible role of teleocalcin in regulating this cycle.

Amino Acid Sequence↗
PubMed1995

Effect of radiation on serum glycoproteins and glycosidases in patients with cervical carcinoma.

OBJECTIVE: The serum glycan moieties of glycoproteins and the activities of glycosidases were analyzed in order to elucidate the metabolism of glycoprotein in uterine cervical cancer. METHODS: Serum was collected from patients at different stages of cervical cancer before and after treatment. The results were analyzed using Student's t-test. RESULTS: The glycan moieties such as sialic acid and fucose were significantly elevated even in the early stages of carcinoma, whereas a significant elevation in hexose and hexosamine levels was observed only in the advanced stages. Among the investigated lysosomal glycohydrolases, beta-D-galactosidase, N-acetyl-beta-D-glucosaminidase and alpha-L-fucosidase activities were elevated significantly at all stages except for stage I. The altered levels of glycoproteins and glycosidases were reduced to near-normal levels after radiotherapy. CONCLUSION: The levels of glycan moieties and the activities of glycosidases can be used as diagnostic markers to assess the stage of cancer and can be used as prognostic markers during therapy.

Adult↗
PubMedDate not supplied

Metabolism of glycosaminoglycans in atheromatous rats. Enzymes concerned with synthesis, degradation and sulphation of glycosaminoglycans.

Some important enzymes concerned with the biosynthesis of the precursors of glycosaminoglycans (gg), degradation of gg and biological sulphation have been studied in rats fed an atherogenic diet. L-Glutamine-D-fructose-6-phosphate amino-transferase and glucosamine-6-phosphate-N-acetylase--2 enzymes concerned with the biosynthesis of hexosamine precursors of gg--decreased in the liver in rats fed the atherogenic diet. UDPG pyrophosphorylase, UDPG dehydrogenase and UDPG glucuronic acid-5'-epimerase, which are concerned with the biosynthesis of the uronic precursors of gg, also decreased in the liver in the diet-fed rats. The activities of some of the enzymes concerned with degradation of gg-hyaluronidase, beta-glucuronidase beta-hexosaminidase, cathepsin and aryl sulphatase--increased both in the liver and aorta. The hepatic concentration of PAPS significantly decreased in the diet-fed rats. The sulphate-activating system, which includes ATP sulphurylase, APS kinase and sulphotransferase, also decreased. Thus the overall picture is one of decreased synthesis of gg and their increased degradation in the atheromatous rats.

Animals↗
PubMedDate not supplied

Lipid transport in the avian species. Part I. Isolation and characterization of apolipoproteins and major lipoprotein density classes of male turkey serum.

(1) Lipoproteins from the serum of male turkeys maintained on a normal diet were separated by sequential preparative ultracentrifugation into VLDL (d less than 1.006 g/ml), LDL (d = 1.006-1.063 g/ml), HDL (d = 1.063-1.21 g/ml) and VHDL (d greater than 1.21 g/ml). Lipoprotein density classes were characterized by analytical ultracentrifugation, agarose electrophoresis, immunodiffusion and immunoelectrophoresis, and by quantitative determination of protein, lipids and individual phosphatides. (2) HDL were the major density class representing 75% of the total lipoprotein content, LDL accounted for approximately 20% and VLDL for only 3-5% of the total lipoproteins. (3) VLDL were characterized by a relatively low content of glyceride (34%). Cholesterol esters were the major lipid (38%) of LDL, and the phospholipids (26%) of HDL. Glycerides of all major density classes consisted of equal amounts of triglycerides and diglycerides. (4) Phosphatidylcholine was the major phosphatide in all density classes. The composition of phosphatides was very similar in the VLDL and LDL, but it was different in the HDL. The ratio of phosphatidylcholine/sphingomyelin was higher in HDL than in VLDL and LD. (5) Immunological and electrophoretic studies showed that all three major density classes consisted of two lipoprotein families designated, in analogy to the human serum lipoprotein system [1], as LP-A and LP-B. The exception was HDL3 (d = 1.125-1.21 g/ml) which contained only LP-A. (6) ApoB was insoluble in aqueous buffers but could be solubilized after reduction and carboxymethylation. No C- or N-terminal amino acids were released by the usual chemical methods. The carbohydrate moiety of ApoB contained mannose, galactose and galactosamine. (7) ApoA consisted of a non-identical polypeptides designated in analogy to the human polypeptides as A-I and A-II. A-I was the major ApoA polypeptide and had a molecular weight of about 27,000. This polypeptide contained no half cystine, and the aspartic acid as the N-terminal and alanine as the C-terminal amino acids. A-II had a molecular weight of about 10,000, contained no half cystine and had alanine as the C-terminal amino acid. A-II showed no N-terminal amino acid by either dansylation, dinitrophenylation or Edman's procedure. Neither A-I nor A-II contained neutral sugars or hexosamines. (8) Concentrations of polypetides analogous to human ApoC, ApoD and "arginine-rich" polypeptide, if present, were too low for their unequivocal chemical characterization.

Animals↗
PubMed1977

Elastin sub-fraction as binding site for lipids.

Elastin preparations were isolated from human thoracic aorta, from atherosclerotic and from grossly normal regions. A relatively mild procedure was used to avoid hot alkaline extraction and autoclaving. The elastase digest of the aortic elastin was chromatographed on a Sephadex G-100 column and separated into two fractions: A (larger molecular weight) and B (smaller molecular weight). The ratio of fraction A to total aortic elastin increased with age and the development of the atherosclerosis. Amino acid and sugar analyses showed that fraction A consistently contained more polar amino acids, hexose, hexosamine and L-fucose, and less sialic acid, in comparison with fraction B. Part of the elastin preparation was incubated with human low-density lipoprotein; a considerable amount of lipid, especially cholesterol, was transferred from the lipoprotein to the elastin. Estimation of protein and cholesterol in fractions A and B of the elastase hydrolyzate of incubated elastin showed that most of the cholesterol taken up by elastin had been in fraction A. The increased proportion of fraction A in aortic elastin derived from plaque areas appeared responsible for the marked lipid-binding capacity of plaque elastin.

Age Factors↗
PubMed1988

Low density lipoprotein binding affinity of arterial wall isomeric chondroitin sulfate proteoglycans.

Although the selective interaction of low density lipoproteins (LDL) with arterial proteoglycans is known, information is lacking on LDL-binding affinity of different subspecies occurring within a proteoglycan family. Isomeric chondroitin sulfate proteoglycan preparations sedimenting at densities of 1.54 g/ml (D1), 1.50 g/ml (D2) and 1.46 g/ml (D3) were isolated from bovine aorta intima-media under dissociative conditions and subjected to equilibrium binding to LDL-agarose gel. D1, D2 and D3 contained 36%, 37% and 11% dermatan sulfate, respectively. Sulfate to hexosamine ratio was low (0.73) in D1 when compared to D2 and D3 (0.94 and 1.04). Of the total proteoglycans contained in D1, D2 and D3, 41%, 52% and 66% interacted with LDL, respectively. LDL-bound proteoglycans dissociated over a wide range of ionic strengths (0.15-1.0); in comparison, LDL-bound heparin dissociated within a narrow range (0.5-0.75). Unlike other preparations, 30% of bound D3 dissociated at an ionic strength of 1.0. In D1 and D2 the proportion of dermatan sulfate increased in proteoglycan fractions that were bound firmly to LDL, whereas a high affinity fraction in D3 contained no dermatan sulfate. Thus, isomeric chondroitin sulfate proteoglycans display considerable divergence with respect to LDL binding. This may depend not only on the degree of sulfation but on other characteristics of the chondroitin sulfate isomers as well.

Animals↗
PubMed1992

Effects of homodimeric isoforms of platelet-derived growth factor (PDGF-AA and PDGF-BB) on wound healing in rat.

Platelet-derived growth factor (PDGF) has been suggested to have a significant role in wound healing. The present work was aimed at studying the effects of PDGF-AA and PDGF-BB homodimers on developing granulation tissue in rats. Subcutaneously implanted hollow cylindrical cellulose sponges were used as an inductive matrix for the ingrowth of granulation tissue. Fifty microliters of solutions containing 0, 5, 50, or 500 ng of PDGF-AA or PDGF-BB homodimers was injected daily into the sponges; 7 days after implantation the granulation tissue in the sponge cylinders was analyzed. Five hundred nanograms of PDGF-BB stimulated significantly the accumulation of collagen, indicated by the elevated hydroxyproline content of the sponge (+34%, P < 0.001). Similarly, the amounts of RNA-ribose, nitrogen, hexosamines, and uronic acids were significantly higher, reflecting a PDGF-BB-induced increase in the accumulation of RNA, protein, and glycosaminoglycans. Analyses of wound fluid showed no essential changes in the composition of different cell types after PDGF-BB-treatment. The PDGF-AA-treatment increased significantly the mean amount of RNA but there were no significant changes in other parameters. In vitro both PDGF-AA and PDGF-BB increased significantly the number of rat granulation tissue derived fibroblasts in culture at concentrations of 10 and 30 ng/ml. This proliferative effect resulted in a lowered level of protein synthesis per cell. To conclude, PDGF-BB accelerates granulation tissue formation both in vitro and in vivo, whereas PDGF-AA is effective in vitro but it is clearly less effective in vivo.

Animals↗
PubMed1986

Influence of serum lipoproteins and carbohydrate metabolism on erythrocyte membrane composition in children: Bogalusa Heart Study.

The influence of serum lipoprotein profile and measures of carbohydrate metabolism on erythrocyte membrane composition were examined in four groups of children (n = 356, ages 6 to 18 years) from a total biracial population whose earlier very low density (VLDL-C) or low density lipoprotein cholesterol (LDL-C) levels (or both) were in the extreme quintiles or quartiles. Erythrocyte membranes of white children contained significantly increased levels of protein (+2.0%) and phospholipid (+2.7%) and decreased levels of neutral sugars (-4.6%) when compared to black children. Membrane neutral sugars were markedly higher (+4.7%) in girls than in boys. Membrane protein was lowest in children characterized by high LDL-C and low VLDL-C. The phospholipid/protein ratio was consistently higher (+1.9%) in the low VLDL-C strata. The relationships between membrane constituents and plasma/serum variables were explored after controlling for race, sex, and age effects and after combining the four selection groups. Less membrane cholesterol was present in the lowest (v highest) quintile of serum LDL-C/high density lipoprotein cholesterol (HDL-C) ratio (P less than 0.01). Membrane phospholipid levels were lower in the highest (v lowest) quintiles of the apoB/apoA-I ratio (P less than 0.01). With respect to measures of carbohydrate tolerance, membrane hexosamine showed low values in the highest (v lowest) quintile of hemoglobin A1c, while the trend was opposite for plasma insulin response (P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1992

Salivary hypofunction in patients with familial amyloidotic polyneuropathy.

Patients who suffer from familial amyloidotic polyneuropathy frequently complain of mouth dryness and an increased need for dental treatment. The aim of the present investigation was to study saliva secretion rate and composition and other factors related to the risk of dental caries in patients with familial amyloidotic polyneuropathy. Thirty patients with familial amyloidotic polyneuropathy volunteered for the study and were compared with a matched control group. Samples of unstimulated and stimulated whole saliva were collected in a standardized manner. The secretion rates were calculated, and the concentrations of electrolytes, glycoprotein markers, and proteins with antibacterial properties were analyzed. Dental caries and variables related to the risk of dental caries were also scored. The results show that familial amyloidotic polyneuropathy patients frequently have a decreased rate of saliva secretion and that the degree of salivary hypofunction is positively correlated to the progress of familial amyloidotic polyneuropathy. Forty-three percent of the familial amyloidotic polyneuropathy patients in this study had no detectable secretion of unstimulated saliva. A low secretion rate of stimulated saliva (< 0.7 ml/min) was found in 33% of the patients. The concentrations of salivary protein, amylase, lysozyme, salivary peroxidase, secretory IgA, hexosamines, sialic acid, fucose, phosphate, potassium, and the degree of protein glycosylation were higher in the familial amyloidotic polyneuropathy patients than in the control patients. We conclude that patients with familial amyloidotic polyneuropathy have a reduced saliva secretion and are subsequently at risk for increased development of dental caries.

Adult↗
PubMed1989

Isolation and characterization of a fibrinogen-clotting enzyme from venom of the snake, Lachesis muta muta (Peruvian bushmaster).

A fibrinogen-clotting enzyme from the venom of the Peruvian bushmaster snake was purified to homogeneity by gel filtration on Sephadex G-100 followed by DEAE-cellulose ion-exchange chromatography using a linear ionic strength gradient with NaCl. The specific activity of the enzyme was 866 NIH U/mg, representing a 55-fold purification, with a recovery of 45%. The amino acid composition was Asx30, Thr14, Ser15, Glx33, Pro23, Gly22, Ala15, Val22, Cys18, Met3, Ile18, Leu23, Tyr2, Phe13, His8, Lys11, Arg11. The total carbohydrate content was 13.4%, comprised of 3.4% hexose, 8.7% hexosamine and 1.3% sialic acid. The enzyme was active against the synthetic amide substrate alpha-N-benzoyl-DL-arginine-p-nitroanilide (BAPNA) and against the ester substrates alpha-N-benzoyl-L-arginine ethyl ester (BAEE) and tosyl-L-arginine methyl ester (TAME). Kinetic parameters for TAME esterolysis were: Vmax, 135 mumoles/min/mg and Km, 2.5 x 10(-4) M. The pH optimum was 8.0. Vmax for BAPNA amidolysis was 0.363 mumoles/min/mg and Km, 7.5 x 10(-5) M. Enzyme activity was reduced by diethylpyrocarbonate and by photo-oxidation, suggesting that the enzyme is a serine protease with a histidine residue involved in the active site. The enzyme released fibrinopeptide A rapidly from purified human fibrinogen and fibrinopeptide B more slowly. Factor XIII was not activated and the clotting activity was not inhibited by heparin. A dose of 50 micrograms/kg brought about defibrinogenation in anaesthetized rats but rabbits were unaffected. A dose of 80 micrograms/kg defibrinogenated conscious rats after 5 hr. There were no hypotensive or haemorrhagic effects.

Amino Acids↗