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PubMed1979

Articular cartilage preservation and storage. I. Application of tissue culture techniques to the storage of viable articular cartilage.

Articular cartilage slice explants were stored under various conditions, including freezing-thawing at various rates by using dimethyl sulfoxide (DMSO) as a cryoprotective agent, incubating in standard tissue culture medium (MEM Eagle:NCTC 135:15% fetal calf serum) in 5% CO2 and air at 4 degrees, 21 degrees, and 37 degrees C, and incubating in standard tissue culture medium containing 200 micrograms/ml alpha-tocopherol (vitamin E) at 37 degrees C after first ascertaining a dose-response curve of vitamin E. Results indicated that articular cartilage slice explants did not survive freezing or storage at 4 degrees and 21 degrees C as measured by 35S uptake. When stored at 37 degrees C in standard tissue culture in 5% CO2 and air, the slice explants remained viable for up to 60 days. The addition of alpha-tocopherol to the medium resulted in significantly less release of previously incorporated 35Sin stored cartilage slices and significantly less reduction of the amount of hexosamine present in the stored explants. alpha-Tocopherol in the medium also preserved safranin O staining. Thus, the application of tissue culture techniques to the storage of articular cartilage made it possible to preserve cartilage slice explants in a viable, biochemically "normal" state.

Animals↗

PubMed2004

An in vivo study of hydrogels based on physiologically clotted fibrin-gelatin composites as wound-dressing materials.

In this study we have reported the efficacy of three biomaterials: (a) physiologically clotted fibrin-gelatin composite (PFG), (b) PFG graft copolymerized with 2-hydroxyethyl methacrylate (PFG-HEMA), and (c) PFG graft copolymerized with 2-hydroxypropyl methacrylate (PFG-HPMA) as temporary wound-dressing materials using the rat as an animal model. Full-thickness excision wounds were made on the back of female rats weighing about 150 +/- 10 g. The dressings were applied on the wounds and changed periodically at an interval of 4 days with the respective materials. The wounds treated with PFG-HEMA healed completely on 15th day after wound creation, whereas those treated with PFG and PFG-HPMA resulted in complete healing on the 17th day. The concentrations of collagen, hexosamine, and uronic acid in the granulation tissue were determined. The PFG and its graft copolymers acted as hydrogels, thereby absorbing excess exudates, while still maintaining a moist environment at the wound site. The enhanced wound healing in the experimental animals was reflected in the increased rate of wound contraction. The results of the histological and mechanical studies of the experimental groups revealed that reepithelialization and remodeling of the skin have been achieved by providing a moist environment at the wound site by the biomaterials and thereby hastening the migration of keratinocytes.

Animals↗

PubMed1990

Seasonally occurring lectins from the bryozoan Bugula neritina.

Two different lectins (termed BnA-I and BnA-II) with distinct carbohydrate specificities were identified and subsequently isolated from the marine bryozoan Bugula neritina. BnA-I hemagglutinating activity was inhibited by N-acetylated hexosamines, their polymers, and glycoproteins rich in these moieties. BnA-II-induced hemagglutination was not blocked by any simple sugars but could be inhibited by several complex glycoproteins (e.g., thyroglobulin and orosomucoid). Both lectins required the presence of Ca(+)+ for reactivity and were purified by affinity chromatographic procedures. Purified BnA-I was determined to have a native molecular weight of 240 Kd and appeared to be a hexameric homopolymer while BnA-II was shown to be a 65-70 Kd monomer. Both lectins showed seasonality in expression, BnA-I appearing in animal extracts prepared in the spring and fall while BnA-II was expressed only during the summer and winter.

Animals↗

PubMed1984

Sequential changes in the mechanical properties of viable articular cartilage stored in vitro.

Viable articular cartilage from the medial femoral condyles of rabbits was stored in vitro in tissue culture medium with various additives and the same site of each specimen was mechanically tested sequentially throughout a 12-day storage period. Indentation testing was performed with instantaneous and sustained loads. Preservation of sustained-load carrying capacity was observed in the condyles stored with additives, indicating maintenance of an intact cartilage matrix. However, initial testing with small sustained loads (preload) showed changes not observed at higher load levels. The changes noted at small sustained initial loads may reflect alterations in cartilage surface structure and may be an early indicator of its mechanical integrity. Chondrocyte viability and proteoglycan content, as measured by 35S incorporation and hexosamine concentration, were unchanged in comparison to fresh articular cartilage.

Animals↗

PubMed1988

Metabolic and biochemical abnormalities of articular cartilage induced by implantation of a sterile sheet of polyethylene in the rabbit patellofemoral joint.

A polyethylene sheet was implanted in the patellofemoral joint of the right knee of the rabbit and the biochemical and metabolic changes in the articular cartilage from femoral trochleas (in contact with the implant) and femoral condyles (free of direct contact) were compared with those in their sham-operated counterparts 7, 15, and 30 days after joint implantation. The results showed that there was an increase in the water content; the extraction yields of uronic acid--, 35SO4-, and [3H]glycine-containing compounds; and the incorporation of [3H]thymidine, [3H]glycine, and 35SO4. Concomitantly, the contents of uronic acid--, hexosamine-, neutral sugars-, and hydroxyproline-containing substances decreased in the femoral trochlear cartilage and, to a much lesser extent, in the femoral condylar cartilage from implanted joints. The increased capacity of viable chondrocytes to incorporate metabolic radiolabeled precursors into newly synthesized macromolecules may represent a reparative cell response to the tissue injury induced by the implant. This is therefore a useful model for studying the response of chondrocytes to mechanical injury and tissue tolerance to intraarticularly implanted prosthetic materials.

Animals↗

PubMed1992

Metabolic and biochemical status of articular cartilage following cryopreservation and transplantation: a rabbit model.

To determine the fate of transplanted cryopreserved articular cartilage, an animal model employing the proximal humerus in the rabbit has been developed. Previous studies have been hindered by problems of postoperative joint instability, secondary injury due to immobilization, and paucity of cartilage for analysis. This experiment demonstrates the survival and function of transplanted cartilage by quantitative assessment of metabolic and biochemical parameters. Forty-five New Zealand white rabbits underwent transplantation of the right proximal humerus. In 29 animals, the proximal half of the humerus was resected and replaced by a cryopreserved osteoarticular allograft. Autograft procedures were carried out in the remaining animals. Following sacrifice at 3, 6, 9, and 12 months postoperatively, articular cartilage was analyzed for gross appearance, collagen synthesis, proteoglycan synthesis, and water, hydroxyproline, hexosamine, and hexuronic acid contents. The results indicate that the cryopreserved osteoarticular allografts retained their metabolic and biochemical integrity and behaved as viable and biologically functional units 1 year postoperatively.

Animals↗

PubMed1976

Mechanism of action of retinyl compounds on wound healing IV: effect of desmethylretinoic acid and its vinylogs on granuloma formation.

The effect of side-chain length of desmethylretinoic acid on granuloma formation induced by implantation of cotton pellets was studied. As in the homologous series of retinoic acid studies, compounds with side chains shorter than retinoic acid were not active while compounds with side chains as long or longer than retinoic acid were active. The compound with a chain two carbons longer than retinoic acid was the most active. The hydroxyproline and hexosamine contents of the granuloma were analyzed. The mechanisms of action of these active compounds on wound healing are discussed.

Animals↗

PubMed1980

Further observations in lincomycin-induced cholelithiasis in guinea-pigs.

Further experiments are reported on Lincomycin-induced cholelithiasis in guinea-pigs. The biochemical events in the bile and blood, and the chemical composition of gallstones, have been studied. The gallstones resemble human pigment stones in chemical composition. The clear hepatic bile and the normality of the bile salt--phospholipid--cholesterol equilibrium, the rise in beta-glucuronidase and hexosamine levels with the gallbladder, have reaffirmed that epithelial injury is most probably the primary lithogenic factor. Ligation of the cystic duct and the construction of a common hepatic duct-duodenum bypass did not prevent the development of acute cholecystitis, suggesting the lithogenic factor was mediated through the blood circulation and not through the enterohepatic circulation of bile.

Animals↗

PubMed2002

Influence of Terminalia chebula on dermal wound healing in rats.

The effects of topical administration of an alcohol extract of the leaves of an evergreen plant, Terminalia chebula, on the healing of rat dermal wounds, in vivo, was assessed. T. chebula treated wounds healed much faster as indicated by improved rates of contraction and a decreased period of epithelialization. Biochemical studies revealed a significant increase in total protein, DNA and collagen contents in the granulation tissues of treated wounds. The levels of hexosamine and uronic acid in these tissues, also increased upto day 8 post-wounding. Reduced lipid peroxide levels in treated wounds, as well as ESR measurement of antioxidant activity by DPPH radical quenching, suggested that T. chebula possessed antioxidant activities. The tensile strength of tissues from extract-treated incision wounds increased by about 40%. In addition, T. chebula possessed antimicrobial activity and was active largely against Staphylococcus aureus and Klebsiella. These results strongly document the beneficial effects of T. chebula in the acceleration of the healing process.

Administration, Topical↗

PubMed1994

Isolation and characterization of a novel 20-kDa sulfated polysaccharide from the extracellular slime layer of Staphylococcus epidermidis.

Slime-producing coagulase-negative staphylococci have emerged as important pathogens especially in immunocompromised hosts and patients with implanted devices. Although the extracellular slime layer is considered an important virulence factor, the chemical composition of the slime polysaccharide(s) remains unknown. The crude slime product derived from two reference Staphylococcus epidermidis strains (ATCC 35983 and 35984) and two clinical isolates was found to contain protein (11-20.5%), hexosamines (8-19%), neutral sugars (12.2-14%), phosphates (4-9.5%), uronic acids (1-13%), and small amounts of sulfates (0.5-3%). Preparative anion-exchange chromatography separated a main carbohydrate component which was isolated by combined chromatographies on DEAE-Sephacel and Sepharose CL-6B. HPLC and electrophoreses on polyacrylamide gel and cellulose acetate membrane revealed the presence of one species of low-sulfated polysaccharide with a relative molecular mass of 20-kDa. Chemical analyses of the polysaccharide showed that it is rich in glucosamine (46%) and neutral sugars (30-34%) with small amounts of sulfates (5.7-6.5%) and glucuronic acid (2.9-3.4%). Ten percent of the glucosamine is sulfated at the amino group. The neutral monosaccharides present are glucose, fucose, and xylose with glucose as the predominant one. It is estimated that the polysaccharide consists of 61-65 molecules of glucosamine (6-7 of which are N-sulfated), 30-35 neutral monosaccharides, 3-4 molecules of glucuronic acid, and 1-3 of fucose and xylose. Isolation and characterization of such a polysaccharide from the extracellular slime layer of S. epidermidis has not been previously reported. Its role to pathogenicity remains to be elucidated.

Bacterial Proteins↗

PubMed1999

Chondrocyte-mediated catabolism of aggrecan: evidence for a glycosylphosphatidylinositol-linked protein in the aggrecanase response to interleukin-1 or retinoic acid.

The control of chondrocyte-mediated degradation of aggrecan has been studied in rat chondrosarcoma cells and bovine cartilage explants treated with either IL-1 or retinoic acid. The capacity of glucosamine to inhibit the aggrecanase-mediated response (J. D. Sandy, D. Gamett, V. Thompson, and C. Verscharen (1998) Biochem. J. 335, 59-66) has been extended to an investigation of the effect of other hexosamines. Mannosamine inhibits the aggrecanase response to both IL-1 and RA at about one-tenth the concentration of glucosamine in both rat cell and bovine explant systems. This effect of mannosamine appears to be due to its capacity to inhibit the synthesis of glycosylphosphatidylinositol (GPI)-linked proteins by chondrocytes since the GPI synthesis inhibitor 2-deoxyfluoroglucose (2-DFG) also inhibited the aggrecanase response to IL-1b and RA in rat cells. Moreover, phosphatidylinositol-specific phospholipase C (PIPLC) treatment of rat cells markedly inhibited the aggrecanase response to IL-1b and RA. These inhibitory effects of mannosamine, 2-DFG, and PIPLC in rat cells did not appear to be due to an interference with general biosynthetic activity of the cells as measured by [3H]proline incorporation into secreted proteins. We suggest that the aggrecanase response by chondrocytes to IL-1 and RA is dependent on the activity of a GPI-anchored protein on the chondrocyte cell surface.

Aggrecans↗

PubMed2000

Lectin histochemical examination of rabbit bladder glycoproteins and characterization of a mucin isolated from the bladder mucosa.

The glycocalyx of the mucosal surface of urinary bladder acts as an effective barrier against invasion by pathogenic microorganisms and injury from toxic substances in the urine. Defects in these bladder mucosal components could thus be important factors in the development of diseases such as interstitial cystitis and lower urinary tract infections. However, information on the nature of glycoconjugates of mammalian bladder mucosa is very limited. In this study, the glycoconjugates of rabbit bladder were examined histochemically using biotinylated lectins with specificities for a variety of carbohydrate moieties. Three [Artocarpus integrifolia (Jacalin), Datura stramonium (DSL), and Maackia amurensis II (MAL-II)] of the lectins bound predominantly to the luminal cell layer, with decreased binding to the basal layers of the epithelium. In contrast, Ricinus communis I and Sambucus nigra lectins did not bind to the cells in the epithelium but strongly interacted with the subepithelial layers, especially the lamina propria. The intensity of the staining by Jacalin and MAL-II was significantly reduced by prior treatment of the bladder sections with O-sialoglycoprotein endopeptidase, indicating that the ligands of these lectins are primarily mucin glycoproteins. In parallel biochemical studies, a high-molecular-weight glycoprotein with characteristics typical of epithelial mucins was purified from the mucosa of rabbit bladder explant cultures metabolically labeled with [(3)H]glucosamine. Quantitative analysis of the sialic acid, uronic acid, and hexosamine contents of delipidated rabbit bladder mucosa revealed a larger proportion of sialoglycoproteins compared with glycosaminoglycans. Taken together, the results of histochemical and biochemical analyses indicate that glycoproteins rather than glycosaminoglycans are the major components of the bladder epithelium, and that the former include a mucin.

Animals↗

PubMed1994

Quantitative determination of monosaccharides in glycoproteins by high-performance liquid chromatography with highly sensitive fluorescence detection.

For specific determination of monosaccharides with high sensitivity, glycoprotein acid hydrolysates were derivatized in a simple step with excess anthranilic acid (2-aminobenzoic acid) in the presence of sodium cyanoborohydride to give highly fluorescent stable derivatives. The monosaccharide derivatives were completely separated from the excess reagent and from each other by HPLC on a C-18 reversed-phase column using a 1-butylamine-phosphoric acid-tetrahydrofuran mobile phase. Reductive amination of the monosaccharides in the methanol-acetate-borate medium was complete within 20 min at 80 degrees C. Derivatization of glucosamine with the anthranilic acid was accompanied by epimerization to mannosamine (> 15%) in methanol-acetic acid reaction medium, but it was reduced to < 3% in methanol-acetate-borate reaction medium. Fluorescence intensity of the hexosamines was greater than twice the intensity of the neutral monosaccharides. The fluorescent derivatives had excitation maxima at 230, 245, and 360 nm and an emission maximum at 425 nm. Fluorescence intensity at 230 nm excitation was about 10 times greater than that obtained with excitation at 360 nm for all the monosaccharides. Release and concomitant destruction of the monosaccharides during hydrolysis in 20% TFA at 100 degrees C for 7-8 h resulted in 83-85% recovery of all the monosaccharides from glycoproteins. The monosaccharide compositions determined by this method were in excellent agreement with the expected values for a recombinant immunoglobulin and fetuin and were highly reproducible. Relative standard deviation for the composition determinations and precision was less than 3%.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1999

Preventive effect of teprenone on stress-induced gastric mucosal lesions and its relation to gastric mucosal constitutive nitric oxide synthase activity.

Recently, we demonstrated that teprenone, an anti-ulcer agent, exerts protective and preventive actions against water immersion restraint (WIR) stress-induced gastric mucosal lesions in rats both by inhibiting neutrophil infiltration into the gastric mucosal tissue and by preserving gastric mucus synthesis and secretion. In rats with WIR stress we have also found a decrease in gastric mucosal constitutive nitric oxide synthase (cNOS) activity and a drastic increase in gastric mucosal inducible nitric oxide synthase (iNOS) activity. The decrease in gastric mucosal cNOS activity is closely related to an increase in neutrophil infiltration into the gastric mucosa and a decrease in the level of gastric mucus. In this study of WIR-stressed rats, therefore, we examined whether the inhibitory actions of teprenone on neutrophil infiltration and decreases in mucus synthesis and secretion in the gastric mucosa of rats are related to the change in gastric mucosal cNOS activity during the development of gastric mucosal lesions. Pre-administration of teprenone (200 mg kg-1) prevented the decrease in gastric mucosal cNOS activity with attenuations of neutrophil infiltration into gastric mucosal tissues and decreased levels of gastric mucosal hexosamine, an index of gastric mucin, and adherent mucus in rats with 3 or 6 h of WIR stress. These preventive effects of teprenone on the gastric mucosal neutrophil infiltration and the decrease in gastric mucus levels in rats with WIR stress were completely reversed with inhibition of gastric mucosal cNOS activity by co-administration of NG-monomethyl L-arginine (L-NMMA), a non-selective NOS inhibitor. These results suggest that the inhibitory actions of teprenone on neutrophil infiltration and decreases in mucus synthesis and secretion in the gastric mucosa of rats with WIR stress are closely related to the maintenance of cNOS activity in the gastric mucosal tissue.

Animals↗

PubMed1996

Characterisation of a developmentally related polypeptide with glutelin solubility characteristics from Lupinus albus L.

Proteins from Lupinus albus L. cv. Rio Maior seeds were fractionated according to solubility criteria. Patterns of concanavalin A (ConA)-binding polypeptides from the different classes, albumins, globulins, glutelins and prolamins, were established by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two bands of apparent molecular masses of 29 and 23.5 kDa with glutelin solubility characteristics bound the lectin. The 23.5-kDa band was separated by two-dimensional electrophoresis into two components: one glycosylated and heterogeneous with an isoelectric point of approx. 10 (designated as G23) and another, not detected with ConA, precipitating in the first dimension. The amino acid and hexosamine analysis of G23 showed that it is particularly rich in Gly (11.2%), Glx (10.0%), Ser (9.0%), Leu (8.2%), Asx (7.5%), and Pro (6.7%) and that it has a considerable content of the sulphur-containing amino acids Met (2.0%) and Cys (5.8%) and contains glucosamine. The determined N-terminal amino acid sequence of G23 was: 1KG(R)V5KGTGD10(T)PXXV15XLY(N)R20T, and this had no significant similarity to any of the amino acid sequences contained in the data bank SWISS-PROT 26. The glycoprotein G23 was completely deglycosylated with peptide-N-glycosidase F, yielding a homogeneous 21-kDa polypeptide composed of approximately 191 amino acids. The structures of the major N-linked neutral oligosaccharides of G23, determined by exoglycosidase sequencing, were as follows: Man alpha 2Man alpha 6(Man alpha 3) Man alpha 6(Man alpha 2Man alpha 2Man alpha 3)Man beta 4GlcNAc beta 4 GlcNAc (13%); +/- Man alpha 2Man alpha 6(Man alpha 3)Man alpha 6(+/- Man alpha 2 Man alpha 2 Man alpha 3)Man beta 4GlcNAc beta 4GlcNAc (29%); Man alpha 6(Man alpha 3) Man alpha 6(Man alpha 2Man alpha 3)Man beta 4GlcNAc beta 4GlcNAc (13%); Man alpha 6(Man alpha 3)Man alpha 6(Man alpha 3)Man beta 4GlcNAc beta 4GlcNAc (16%); Man alpha 6(Man alpha 3)(Xyl beta 2)Man beta 4GlcNAc beta 4GlcNAc (28%). Changes in G23 abundance during seed development, germination and seedling growth were monitored with a specific antibody. The glycoprotein G23 started to accumulate appreciably during seed formation between the 40th and the 50th days after anthesis and was detected following seed imbibition, until the 9th day in cotyledons, the 2nd day in roots and the 4th day in hypocotyls and leaves.

Amino Acid Sequence↗

PubMed1975

The composition and protein metabolism in the immature rabbit intervertebral disc.

The nucleus pulposus (NP) and annulus fibrosus (AF) of immature rabbit intervertebral discs (IVD) have been subjected to the dissociative extraction procedure of Sajdera and Hascall (1969). The soluble, insoluble and unextracted fractions so obtained were analysed for total nitrogen, collagen, tyrosine, uronic acid, hexosamine and sialic acid content. A high proportion of non-collagenous protein, hexose and sialic acid in the NP insoluble fraction suggests the presence of glycopeptides associated with collagen and/or proteoglycans. The levels of proteoglycan in the soluble NP and AF fraction are similar. Immature (soluble) collagen, however, resides largely in the AF region. The metabolism of rabbit IVD protein components was also investigated both chemically and by autoradiography. L-Tyrosine-3,5-H3 was administered intraperitoneally (3 mc/kg) to 4 week-old rabbits. Animals were sacrificed at various time intervals and the harvested tissues extracted as before and lumbar discs collected. The levels of L-Tyrosine-3,5-H3 in the NP and AF insoluble and soluble fractions were determined using a tritium scintillation counting procedure and localisation by autoradiography. Pronounced extracellular activity of proteoglycan and glycoprotein is not evident before 24 hours. Soluble collagen, however, is synthesized and dispersed within 4 hours of isotope administration.

Animals↗

PubMed1977

Bovine serum transferrin phenotypes AA, D1D1, D2D2, EE: their carbohydrate compositions and electrophoretic multiplicity.

Samples of homozygous bovine serum transferrins have been prepared and their purity has been ascertained by immunological techniques and electrophoretic analysis in SDS. Measurements of carbohydrate composition show that no significant differences exist among the phenotype variants AA, D1D1, D2D2, and EE. Chromatography of transferrin AA on DEAE-cellulose separated four subfractions, each of which corresponded well with one band obtained by polyacrylamide gel electrophoresis. Carbohydrate analyses of the individual subfractions did not show significant differences in sialic acid, hexose, or hexosamine contents. After desialylation with neuraminidase, each subfraction was converted to a major band and a minor band on gel electrophoresis. From the relative band positions of the desialylated transferrins, it was concluded that possession of sialyl residued by bovine transferrin is not the primary cause of electrophoretic multiplicity. Rather, sialic acid masks an underlying heterogeneity which most likely resides within the polypeptide chain. Further characterization of this heterogeneity will best be undertaken with the isolated asialotransferrin subfractions.

Animals↗

PubMed1976

Polymorphism of transferrin in carp (Cyprinus carpio L.): genetic determination, isolation, and partial characterization.

Seven transferrin variants (A,B,C,D,E,F, and G) have been found in carp sera (Cyprinus carpio L.). Genetic analysis involves five variants and agrees with the hypothesis of simple codominant autosomal inheritance at one transferrin (Tf) locus in spite of the fact that the carp is a tetraploid in relation to other species of the same family. Carp populations from three regions were studied which differed in gene frequencies. Individual populations were in Hardy-Weinberg equilibrium. The polymorphism of carp transferrins can be used for the identification of offspring of single parent pairs, stocked in one pond. Transferrins have been isolated and characterized. Homozygous phenotypes comprised four iron-binding components differing in electrophoretic mobility. This heterogeneity is not caused by sialic acid, which is absent. Amino acid composition, content of hexoses (1 mole/mole of protein) and hexosamines (1 mole/mole of protein), molecualr weight (70,000), and the isoelectric point (5.0) have been determined. No N-terminal amino acid could be detected.

Amino Acids↗