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PubMed1992

The extracellular matrix of rat spinal cord: a comparative study on the localization of hyaluronic acid, glial hyaluronate-binding protein, and chondroitin sulfate proteoglycan.

The localization of hyaluronic acid (HA), glial hyaluronate-binding protein (GHAP), and chondroitin sulfate (CS) proteoglycan was compared in cryostat sections of rat spinal cord. HA, GHAP, and CS proteoglycan were similarly distributed in white matter where they surrounded myelinated axons. In gray matter, large motoneurons were surrounded by a rim of reaction product in sections stained for HA and CS proteoglycan. GHAP immunoreactivity as well as HA had disappeared in hyaluronidase-digested sections, while CS proteoglycan immunoreactivity was not abolished under these conditions.

Animals↗
PubMed2000

Human mast cells express the hyaluronic-acid-binding isoform of CD44 and adhere to hyaluronic acid.

CD44 is expressed in various isoforms on multiple cell lineages including those of hematopoietic origin and is believed in part to mediate cell adhesion to hyaluronic acid. Elevated levels of soluble CD44 (sCD44) have been identified in the serum of some patients with specific neoplasms. We thus sought to determine whether human mast cells express functional CD44 and whether sCD44 might be associated with systemic mast cell disease. Using a standard assay, CD34(+)-derived cultured human mast cells were first demonstrated to adhere to hyaluronic-acid-coated surfaces. Human mast cells were then found by flow cytometry to express CD44S, but not the v5, v6, v7, and v8 isoforms, and to shed CD44S following activation induced by PMA or aggregation of FcvarepsilonRI. However, CD44S was not found to be consistently elevated in serum obtained from patients with mastocytosis or individuals experiencing anaphylaxis. Thus, human cultured mast cells express and shed CD44S, which appears to mediate the attachment of these cells to hyaluronic acid.

Anaphylaxis↗
PubMedDate not supplied

Controlled degradation and mechanical behavior of photopolymerized hyaluronic acid networks.

Hyaluronic acid is a natural polysaccharide found abundantly throughout the body with many desirable properties for application as a biomaterial, including scaffolding for tissue engineering. In this work, hyaluronic acid with molecular weights ranging from 50 to 1100 kDa was modified with methacrylic anhydride and photopolymerized into networks with a wide range of physical properties. With macromer concentrations from 2 to 20 wt %, networks exhibited volumetric swelling ratios ranging from approximately 42 to 8, compressive moduli ranging from approximately 2 to over 100 kPa, and degradation times ranging from less than 1 day up to almost 38 days in the presence of 100 U/mL of hyaluronidase. When 3T3-fibroblasts were photoencapsulated in the hydrogels, cells remained viable with low macromer concentrations but decreased sequentially as the macromer concentration increased. Finally, auricular swine chondrocytes produced neocartilage when photoencapsulated in the hyaluronic acid networks. This work presents a next step toward the development of advanced in vivo curable biomaterials.

3T3 Cells↗
PubMed1985

The hyaluronic acid binding region as a specific probe for the localization of hyaluronic acid in tissue sections. Application to chick embryo and rat brain.

The hyaluronic acid binding region was prepared by clostripain digestion of chondroitin sulfate proteoglycan isolated from the Swarm rat chondrosarcoma, and biotinylated in the presence of associated hyaluronic acid and link protein. After removal of hyaluronic acid by gel filtration in 4 M guanidine HCl, the biotinylated binding region-link protein complex was used as a specific histochemical probe in conjunction with avidin-peroxidase. Its utility was initially evaluated by comparison with Alcian blue staining of the axial region of 2 to 5 day chick embryos, where staining was seen in the dorsolateral area between the neural tube and the ectoderm, in the perichordal mesenchyme, and in developing limb buds. Light and electron microscopic studies of early postnatal rat cerebellum indicate that hyaluronic acid is primarily localized in the extracellular space of immature brain. Staining specificity was demonstrated by the ability of hyaluronic acid oligosaccharides of appropriate size to block the staining reaction, and by the absence of staining after treatment of tissue sections with protease-free Streptomyces hyaluronidase, which degrades only this glycosaminoglycan.

Animals↗
PubMed1975

Studies on hyaluronic acid. V. Relationship between the protein content and viscosity of rooster comb dermis hyaluronic acid.

Protein accounted for an average of 8.7% w/w of the hyaluronic acid obtained from rooster comb dermis extracts and three types of peptide constituents appeared to be present. A few collagen-like fibers were closely associated with the hyaluronic acid when samples were examined in the electron microscope and collagenase treatment decreased the intrinsic viscosity from 7000-5000 ml/g to 3900-2700 ml/g. The quantities of collagen present, however, were too small to detect chemically with the methods employed. The major peptide consituent was readily separated from the hyaluronic acid by fractionation in a cesium chloride gradient or by treatment with pronase. The viscosity was decreased by the density gradient procedure but not by the pronase digestion. Repeated fractionation in a cesium chloride gradient decreased the intrinsic viscosity still further and a small peptide constituent with a high glycine and serine content remained associated with a hyaluronic acid. The data suggest that an interaction or entanglement with collagen fibers is responsible for the high viscosity of hyaluronic acid in this tissue extract and that the viscosity of purified hyaluronic acid preparations is dependent upon interactions between adjacent polysaccharide chains. Interactions between the major peptide constituent and polysaccharide chains or the small residual peptide component remaining with hyaluronic acid after extensive purification procedures, however, appear to be involved in some organized structure because the presence of the major peptide constituent minimized the decrease in viscosity that occurred when hyaluronic acid samples were lyophilized.

Amino Acids↗
PubMed1975

[Report on the study of hyaluronic acid in the diagnosis of pleural mesotheliomas: study of 100 hyaluronic acid rich effusions].

Hyaluronic acid was measured in 1580 pleural effusions. We report 100 cases of hyaluronate-rich pleural fluids. Correlations betueen clinical, radiological and histological diagnosis of mesothelioma and the characterization of hyaluronate are discussed. These data suggest the possibility of a biochemical approach for the diagnosis of pleural malignant tumours.

Asbestosis↗
PubMed1999

Oocytectomy does not influence synthesis of hyaluronic acid by pig cumulus cells: retention of hyaluronic acid after insulin-like growth factor-I treatment in serum-free medium.

Mouse oocytes secrete a factor that enables cumulus cells to undergo expansion in response to FSH (1 microg/ml), whereas expansion of the porcine cumulus oophorus has been shown to be independent of the oocyte. The aim of this study was to assess FSH-induced synthesis of hyaluronic acid (HA) by porcine cumulus cells before and after oocytectomy. In addition, we studied the effect of insulin-like growth factor-I (IGF-I) on the ability of cumulus cells to synthesize and retain HA in response to FSH in serum-free medium. Porcine oocyte-cumulus complexes and complexes from which the oocytes had been removed by oocytectomy were cultured for 24 h in the presence of 2.5 microCi of D-[6-(3)H]glucosamine hydrochloride, fetal calf serum (FCS, 5%), and FSH. After 24 h, incorporation of [(3)H]glucosamine into HA was measured either in complexes alone (retained HA) or in medium plus complexes (total HA). Specificity of incorporation of radioactivity into HA was confirmed by the sensitivity to highly specific Streptomyces hyaluronidase. Our results suggest that 1) the synthesis of HA by pig cumulus cells in vitro is stimulated by FSH and that oocytectomy does not change this synthesis; 2) oocytes do not influence retention of HA within the complex; 3) FSH-induced synthesis of HA by cumulus cells is decreased in medium with polyvinylpyrrolidone (PVP)-supplemented (total and retained HA) compared to FCS-supplemented medium; 4) IGF-I enabled cumulus cells to synthesize HA in response to FSH in PVP-supplemented medium in a manner similar to that observed when serum is present in the medium.

Animals↗
PubMed1985

Interaction of hyaluronectin with hyaluronic acid oligosaccharides.

Hyaluronic acid was digested by bovine testicular hyaluronidase, and oligomers were fractionated by gel permeation using AcA 202 Ultrogel, an acrylamide-agarose matrix. Oligosaccharides composed of from two to six disaccharide repeating units were isolated. Two nonasaccharides were prepared by enzymatic or chemical modification of the decasaccharide. Oligosaccharides were compared by a competitive inhibition in the enzyme-linked immunosorbent assay for their ability to inhibit the interaction of hyaluronectin (a hyaluronic acid-binding brain glycoprotein) with hyaluronic acid. Among these oligosaccharides, decasaccharides were the smallest fragments that strongly inhibited the interaction. Octasaccharides inhibited with 700-fold lower affinity than decasaccharides. Dodecasaccharides had the same effect as decasaccharides. Nonasaccharides obtained by beta-glucuronidase splitting of decasaccharides inhibited the interaction more than nonasaccharides prepared by an alkaline treatment.

Animals↗
PubMed1994

Evidence for a regional distribution of hyaluronic acid in the rat brain using a highly specific hyaluronic acid recognizing protein.

By means of a highly specific hyaluronic acid-recognizing protein the localization and regional distribution of hyaluronic acid was demonstrated in the tel- and diencephalon and in the midbrain of the adult rat nervous system. Histochemistry revealed labeling associated with the plasma membrane in highly discrete nerve cell bodies of the frontoparietal cortex, the red nucleus, the zona reticulata of the substantia nigra, the oculomotor nucleus and the reticular thalamic nucleus. A strong labeling without association with perikarya was demonstrated in the subgranular zone of the dentate gyrus of the hippocampal formation. The present results open up the possibility that the hyaluronic acid found in high concentrations associated with some perikarya may have a special role in plasticity responses in these discrete nerve cell populations.

Animals↗
PubMed2006

Synthesis and characterization of a novel hyaluronic acid hydrogel.

Hyaluronic acid (hyaluronan, HA) has many medical applications as a biomaterial. To enhance its biostability, a novel hydrogel of cross-linked hyaluronic acid was prepared using a double cross-linking process, which involves building cross-linkages between hydroxyl group pairs and carboxyl group pairs. The present study explored a number of cross-linking processes in order to obtain different degrees of cross-linking, which were evaluated by the measurement of water absorption capacity as an index of the gel network density. To gain a better understanding of the stability of the gel, the chemical structure and particularly the rheological behaviour of the cross-linked HA, which included the influences of factors, such as degree of cross-linking, HA concentration and gel particle size, were investigated. The in vitro biostability against hyaluronidase and free radical degradation was tested to show that the cross-linked hydrogel had improved resistance to in vitro hyaluronidase and free radical degradation.

Biocompatible Materials↗
PubMed1999

Characterization of a two-component system in Streptococcus pyogenes which is involved in regulation of hyaluronic acid production.

Hyaluronic acid production by group A streptococci is regulated by transcriptional control. In this study, transposon mutagenesis of an unencapsulated strain yielded an encapsulated mutant. Two genes homologous to sensors and response regulators of bacterial two-component systems were identified downstream of the transposon insertion. Inactivation of the putative sensor gene, csrS, in three different unencapsulated strains yielded encapsulated mutant strains. Electrophoretic mobility shift assays determined factor(s) in a cytoplasmic extract of an unencapsulated group A streptococcal strain was binding to a double-stranded DNA fragment derived from the has operon promoter. In contrast, similarly prepared cytoplasmic extracts from a csrS deletion mutant did not shift the fragment. The putative response regulator, CsrR, was partially purified and was shown to bind the has operon promoter fragment. The affinity and specificity of CsrR for the fragment were increased significantly after incubation with acetyl phosphate. DNase I footprinting determined that the acetyl phosphate-treated CsrR was binding to key sequences in the promoter and the coding region of hasA. Therefore, a two-component system is repressing the production of hyaluronic acid in group A streptococci using a phosphorylation-dependent binding interaction between the response regulator CsrR and the promoter region of the has operon.

Alleles↗
PubMed1992

Cytokine regulation of human lung fibroblast hyaluronan (hyaluronic acid) production. Evidence for cytokine-regulated hyaluronan (hyaluronic acid) degradation and human lung fibroblast-derived hyaluronidase.

We characterized the mechanisms by which recombinant (r) tumor necrosis factor (TNF), IFN-gamma, and IL-1, alone and in combination, regulate human lung fibroblast hyaluronic acid (HA) production. Each cytokine stimulated fibroblast HA production. The combination of rTNF and rIFN-gamma resulted in a synergistic increase in the production of high molecular weight HA. This was due to a synergistic increase in hyaluronate synthetase activity and a simultaneous decrease in HA degradation. In contrast, when rTNF and rIL-1 were combined, an additive increase in low molecular weight HA was noted. This was due to a synergistic increase in hyaluronate synthetase activity and a simultaneous increase in HA degradation. Human lung fibroblasts contained a hyaluronidase that, at pH 3.7, depolymerized high molecular weight HA to 10-40 kD end products of digestion. However, hyaluronidase activity did not correlate with fibroblast HA degradation. Instead, HA degradation correlated with fibroblast-HA binding, which was increased by rIL-1 plus rTNF and decreased by rIFN-gamma plus rTNF. Recombinant IL-1 and rTNF weakly stimulated and rIL-1 and rTNF in combination further augmented the levels of CD44 mRNA in lung fibroblasts. In contrast, rIFN-gamma did not significantly alter the levels of CD44 mRNA in unstimulated or rTNF stimulated cells. These studies demonstrate that rIL-1, rTNF, and rIFN-gamma have complex effects on biosynthesis and degradation which alter the quantity and molecular weight of the HA produced by lung fibroblasts. They also show that fibroblast HA degradation is mediated by a previously unrecognized lysosomal-type hyaluronidase whose function may be regulated by altering fibroblast-HA binding. Lastly, they suggest that the CD44 HA receptor may be involved in this process.

Adult↗
PubMed1983

A new chromatographic method for the fractionation of hyaluronic acid.

Sodium hyaluronate from rooster comb, umbilical cord, bovine vitreous and a special commercial hyaluronic acid preparation, "HealonR" have been fractionated at 4 degrees C on DEAE-Sephacel columns using salt gradients from 0 leads to 0.4M NaCl. All samples examined displayed a high degree of polydispersity. The number of well separated fractions observed in these chromatograms ranges from 10-30 depending on the source of the material. A molecular weight distribution effect does not seem to be the main factor controlling this fractionation as no differences could be observed when the limiting viscosity numbers (eta) for numerous fractions isolated from the same sample were compared with each other. As no galactosamine, sulfate or phosphate is found directly linked to these fractions, it is suggested that disaggregation of an hyaluronate molecular aggregate could be the basis for this fractionation.

Animals↗
PubMed2004

Microbial hyaluronic acid production.

Hyaluronic acid (HA) is a commercially valuable medical biopolymer increasingly produced through microbial fermentation. Viscosity limits product yield and the focus of research and development has been on improving the key quality parameters, purity and molecular weight. Traditional strain and process optimisation has yielded significant improvements, but appears to have reached a limit. Metabolic engineering is providing new opportunities and HA produced in a heterologous host is about to enter the market. In order to realise the full potential of metabolic engineering, however, greater understanding of the mechanisms underlying chain termination is required.

Animals↗
PubMed1996

Enzymological characterization of the Pasteurella multocida hyaluronic acid synthase.

Hyaluronic acid (HA), a linear polysaccharide composed of alternating glucuronic acid and N-acetylglucosamine residues, is an essential molecule of higher vertebrates. The fowl cholera pathogen Pasteurella multocida Carter Type A also produces HA in the form of an extracellular capsule in order to evade host defenses. HA synthase activity could be obtained from cell-free membrane preparations of P. multocida. The enzyme utilized UDP-sugar precursors of HA in the presence of Mg2+ or Mn2+ at neutral pH. Mn2+ at 1 mM stimulated approximately 2-fold more incorporation than Mg2+ at 10 mM. On the other hand, the analogous enzyme from group A Streptococcus, HasA, is stimulated more by Mg2+ than Mn2+. The apparent Michaelis constants, K(M), of the P. multocida HA synthase for UDP-N-acetylglucosamine and UDP-glucuronic acid were estimated to be approximately 75 and approximately 20 microM, respectively, in the presence of Mg2+, which suggests that the substrates are bound with 2-3-fold higher affinity than by the HasA enzyme. The rate enhancement observed with Mn2+ is apparently not due to better binding of the sugar nucleotide precursors complexed to Mn ion because the K(M) value, a measure of substrate affinity, increases by 25-50% in comparison to Mg2+. In summary, the HA synthase from P. multocida, a Gram-negative bacterium, has kinetic optima distinct from those of HasA, the analog from the Gram-positive group A Streptococcus.

Cell Membrane↗
PubMedDate not supplied

The influence of molecular weight on the biological activity of heparin like sulphated hyaluronic acids.

Sulphated hyaluronic acids having a sulphation degree of 3.5 per disaccharide unit, HyalS3.5, were prepared with different molecular weights corresponding to 21 x 10(3), 320 x 10(3) and 3500 x 10(3). The thrombin inhibition in plasma and in the presence of purified molecules, i.e. fibrinogen, antithrombin III (AT III) and heparin cofactor II (HC II), were studied for the three different MW compounds at different concentrations. The thrombin time in plasma depended on the length of the chain, and the two lower MW HyalS3.5 inhibited thrombin both by direct aspecific interaction and via HC II, whereas the activity of the highest MW compound was mainly related to the electrostatic interaction with HC II. The inactivation of FXa serine protease was only attributed to HyalS3.5-AT III complex.

Antithrombin III↗