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C B Underhill

Publications and source records attributed to C B Underhill.

At least 55 records · Page 3Linked to original sources
PubMed1987

The hyaluronate receptor is identical to a glycoprotein of Mr 85,000 (gp85) as shown by a monoclonal antibody that interferes with binding activity.

The cell surface receptor for hyaluronate is an integral membrane glycoprotein of Mr 85,000 (Underhill, C. B., Thurn, A. L., and Lacy, B. E. (1985) J. Biol. Chem. 260, 8128-8133), which appears to be associated with actin filaments. This protein is similar in many respects to another protein, termed gp85, which was originally identified by Tarone, G., Ferracini, R., Galeto, G., and Comoglio, P. (1984) J. Cell Biol. 99, 512-519), using a monoclonal antibody designated as K-3. The gp85 is also a membrane glycoprotein of Mr 85,000 which is associated with the cytoskeleton. Indeed, immunohistological staining has shown that it is distributed in patches along stress fibers of spread baby hamster kidney (BHK) cells. In the present study, we have used the K-3 monoclonal antibody to determine whether gp85 is identical to the hyaluronate receptor. Initial studies showed that the K-3 antibody reacted with material at Mr 85,000 on immunoblots of a purified preparation of the hyaluronate receptor. In addition, the K-3 antibody specifically blocked the binding of [3H]hyaluronate to detergent extracts of the receptor from both BHK and polyoma virus transformed baby hamster kidney (PY-BHK) cells, as well as to intact PY-BHK cells. These results indicate that the K-3 antibody is directed against the hyaluronate receptor, which therefore must be identical to gp85. The K-3 antibody was then used to determine the relative number of hyaluronate receptors associated with parent (BHK) and transformed (PY-BHK) cells. Using an enzyme-linked assay, we found that parent cells had a substantially greater number of receptors than their transformed counterparts. These results were consistent with those obtained when detergent extracts of cells were directly assayed for [3H]hyaluronate binding activity.

Animals↗
PubMed1987

The hyaluronate receptor is associated with actin filaments.

The cell-surface receptor for hyaluronate is an integral membrane glycoprotein of Mr 85,000 (Underhill, C. B., A. L. Thurn, and B. E. Lacy, 1985, J. Biol. Chem., 260:8128-8133) that is thought to mediate many of the effects that hyaluronate has on cell behavior, such as migration, angiogenesis, and phagocytosis. To determine if the receptor is associated with the underlying cytoskeleton, Swiss 3T3 cells were extracted with a solution of Triton X-100, which solubilized most of the cellular components, but which left behind an insoluble residue containing the cytoskeleton. This detergent-insoluble residue was found to contain the bulk of the hyaluronate-binding activity, suggesting that the receptor might indeed be associated with the cytoskeleton. To further define the cytoskeletal element with which the receptor interacts, 3T3 cells were extracted with Triton X-100 under a variety of different ionic conditions. In each case, the amount of hyaluronate-binding activity in the detergent-insoluble residue was related to the amount of actin present, but not to either tubulin or vimentin. In addition, the recovery of hyaluronate-binding activity was dramatically enhanced (to 100% in most cases) if the cells were extracted in the presence of phalloidin, a drug that stabilizes actin filaments. However, the recovery of binding activity was dramatically decreased when whole cells were treated with cytochalasin B before extraction, and when extracted cells were treated with DNase I, which promotes the depolymerization of actin filaments. In addition, preincubating an extract of SV-40-transformed Swiss 3T3 cell membranes with DNase I caused a change in the elution profile of the receptor as judged by molecular-sieve chromatography. Presumably this decrease in the size of the receptor is due to the loss of associated actin filaments. The results of these experiments strongly suggest that the receptor for hyaluronate is associated either directly or indirectly with cytosolic actin filaments.

Actins↗
PubMed1987

The hyaluronate-binding site from the plasma membrane is distinct from the binding protein present in brain.

To determine whether the hyaluronate-binding protein from brain is similar or identical to the hyaluronate-binding site from the cell surface, the two molecules were compared with respect to their physical and binding properties. The hyaluronate-binding protein was purified from mouse brains by lectin-affinity chromatography, and then analyzed by molecular-sieve chromatography and rate-zonal centrifugation, which showed that it has a Stokes radius of 6.3 nm, and a sedimentation coefficient of 4.8 S. These values are remarkably close to those obtained previously for the membrane-associated binding site (a = 6.5 nm, s20,w = 4.8 S), indicating that the two molecules have similar shapes and sizes. Binding studies of the semi-purified proteins showed that the dissociation constant for the brain derived binding protein (Kd = 270 ng/0.5 ml) was similar to that of the cell-surface binding site (Kd = 350 ng/0.5 ml). However, when the two molecules were compared with respect to oligosaccharide inhibition of binding, significant differences were observed. The hexasaccharide significantly inhibited the binding of [3H] hyaluronate to the cell-surface binding site but had only a small effect on the binding to the brain derived protein. Differences were also found between the two molecules with respect to the effects of a monoclonal antibody (K-3). This antibody blocked most of the binding activity of the membrane-associated binding site, but had no effect on the protein from brain. Taken together, these results indicate that although the hyaluronate-binding protein derived from brain and the cell-surface binding site are physically similar, they are distinct proteins.

Animals↗
PubMed1986

A protein binding assay for hyaluronate.

A relatively quick and simple assay for hyaluronate was developed using the specific binding protein, hyaluronectin. The hyaluronection was obtained by homogenizing the brains of Sprague-Dawley rats, and then centrifuging the homogenate. The resulting supernatant was used as a source of crude hyaluronectin. In the binding assay, the hyaluronectin was mixed with [3H]hyaluronate, followed by an equal volume of saturated (NH4)2SO4, which precipitated the hyaluronectin and any [3H]hyaluronate associated with it, but left free [3H]hyaluronate in solution. The mixture was then centrifuged, and the amount of bound [3H]hyaluronate in the precipitate was determined. Using this assay, we found that hyaluronectin specifically bound hyaluronate, since other glycosaminoglycans failed to compete for the binding protein. In addition, the interaction between hyaluronectin and hyaluronate was of relatively high affinity (Kd = 5.7 X 10(-10) M), and the size of the hyaluronate did not appear to substantially alter the amount of binding. To determine the amount of hyaluronate in an unknown sample, we used a competition assay in which the binding of a set amount of [3H]hyaluronate was blocked by the addition of unlabeled hyaluronate. By comparing the degree of competition of the unknown samples with that of known amounts of hyaluronate, it was possible to determine the amount of hyaluronate in the unknowns. We have found that this method is sensitive to 1 microgram or less of hyaluronate, and is unaffected by the presence of proteins.

Animals↗
PubMed1986

Heparin-induced aggregation of lymphoid cells.

The effects of different carbohydrates on cell-to-cell adhesion were examined in an aggregation assay, which consisted of swirling a suspension of cells and monitoring the loss of single cells with a Coulter Counter. Of the carbohydrates tested, only heparin and dextran sulfate induced cell aggregation. This effect occurred in freshly isolated mouse splenocytes and in cultured cells of lymphoid origin (P388, YAA-CI) but not in cell lines of fibroblastic origins (3T3, SV-3T3, BHK, and PY-BHK). Using the YAA-CI cell line for further study, we found that aggregation could be induced by relatively small amounts of heparin (less than 10 micrograms/ml). Binding experiments with 3H-heparin showed that under normal physiological conditions each YAA-CI cell bound approximately 2 X 10(6) molecules of heparin at saturation with a Kd of 3.5 X 10(-7) M. This binding was blocked by both unlabelled heparin and dextran sulfate but not by other carbohydrates. When the pH of the medium was decreased, the heparin-induced aggregation was inhibited, and the Kd of the 3H-heparin binding was increased. In a similar fashion, when the ionic strength of the medium was increased, heparin-induced aggregation was inhibited and the Kd of the interaction was increased. These results suggest that the aggregation is inversely related to the Kd of the interaction and that the binding of heparin to the cell surface is primarily of an ionic nature.

Animals↗
PubMed1985

Characterization and identification of the hyaluronate binding site from membranes of SV-3T3 cells.

Previous research has shown that binding sites for hyaluronate are present on the surfaces of a number of different cell types. To further characterize these binding sites, membranes were prepared from SV-3T3 cells and dissolved in a solution of sodium deoxycholate. Hyaluronate binding activity was detected by mixing the sodium deoxycholate extract with [3H]hyaluronate and then adding an equal volume of saturated (NH4)2SO4, which precipitated the binding protein and any [3H]hyaluronate associated with it, but left free [3H]hyaluronate in solution. Following partial purification by hydroxylapatite chromatography, the binding site was examined by molecular sieve chromatography and by rate-zonal centrifugation, which revealed that it has a Stokes radius of 6.5 nm and a sedimentation coefficient of 4.8 S. From these values, it was possible to calculate that the sodium deoxycholate-solubilized binding site has a frictional coefficient of 1.87 and a molecular weight of 132,000. Since this latter value applies to the complex of both detergent and protein, the binding protein by itself must have a molecular weight lower than 132,000. To determine the molecular weight of the hyaluronate binding site itself, the protein was purified by the sequential application of hydroxylapatite chromatography, molecular sieve chromatography, rate-zonal centrifugation, and finally lectin-affinity chromatography on concanavalin A-agarose. Analysis of the purified material by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed an 85,000 Mr protein which has been identified as the binding site. This protein was also detected on nitrocellulose blots which had been specifically stained for concanavalin A binding material, suggesting that the binding site is a glycoprotein.

Animals↗
PubMed1983

Effects of detergent solubilization on the hyaluronate-binding protein from membranes of simian virus 40-transformed 3T3 cells.

Simian virus 40-transformed 3T3 (SV-3T3) cells have been shown to possess on their surfaces binding sites for hyaluronate which mediate the divalent cation independent aggregation of these cells. To further characterize these binding sites, membranes were prepared from SV-3T3 cells and solubilized with the detergent, sodium deoxycholate. The binding activity present in the detergent solution was measured by the addition of [3H]hyaluronate followed by separation of free and bound ligand by (NH4)2SO4 precipitation. Using this assay, the soluble hyaluronate-binding protein was compared with the membrane-associated protein in situ. In both cases, binding was found to be saturable, linear with protein content, competitively inhibited by unlabeled hyaluronate and dependent on a minimum of 6 sugar residues of hyaluronate for recognition. However, the solubilized binding protein was found to differ from the membrane-associated protein in the following characteristics: a) the affinity of the interaction with hyaluronate was reduced (the Kd was higher), while the amount of ligand bound at saturation (Bmax) was increased; b) in competition experiments with unlabeled preparations of hyaluronate, the effect that the MWv of the hyaluronate had on its inhibitory potency was greatly reduced; and c) the binding was inhibited to a greater extent by chondroitin sulfate and dermatan sulfate. All of these differences can be accounted for by assuming that the detergent solubilization changes the nature of the hyaluronate-binding site interaction from one that is multivalent (i.e. one molecule of hyaluronate is attached to several sites) to one that is monovalent.

Animals↗
PubMed1982

Hyaluronate degradation in 3T3 and simian virus-transformed 3T3 cells.

The cellular control of hyaluronate levels was examined in cultures of simian virus 40-transformed 3T3 (SV3T3) and 3T3 cells which are known to differ in their metabolism of hyaluronate. When [3H]hyaluronate was added to cultures of the two cell lines, four times more ligand was bound per mg of protein by the SV3T3 cells than by the 3T3 cells. Of the bound [3H] hyaluronate, 40% was degraded by the SV3T3 cells to oligosaccharides characteristic of the breakdown of hyaluronate, but only 2% was degraded by 3T3 cells. Hyaluronidase activity was found in the cell layer and medium of the SV3T3 cultures, but was not detectable in 3T3 cells. The SV3T3 enzyme was active only at acidic pH, but at neutral pH the secreted SV3T3 hyaluronidase was thermally more stable then the cell-associated enzyme. In contrast, both cell lines were found to contain similar amounts of beta-glucuronidase and beta-N-acetylglucosaminidase activity. We conclude that the elevated capacity of SV3T3 cells to degrade hyaluronate may be partially responsible for their lack of the hyaluronate-containing pericellular coat which is prominent around 3T3 cells.

Acetylglucosaminidase↗
PubMed1982

Transformation-dependent loss of the hyaluronate-containing coats of cultured cells.

The sizes of hyaluronate-containing coats on the surfaces of parent and virus-transformed cell lines (3T3 vs. SV-3T3; BHK vs. PY-BHK) were compared according to the method of Clarris and Fraser (1968, Exp. Cell Res., 49: 181-193) in which fixed red blood cells were allowed to settle slowly on the surface of culture dishes containing the cells. The coats were seen as areas devoid of red blood cells surrounding each of the cultured cells and could be destroyed by the addition of small amounts of streptomyces hyaluronidase, an enzyme specific for hyaluronate. In the case of the parent cell lines (3T3 and BHK), the coats were clearly visible, whereas for their virus-transformed counterparts (SV-3T3 and PY-BHK), the coats were either greatly reduced or absent. To confirm these observations, the amount of hyaluronate associated with each of the cell lines was measured using a direct chemical assay and shown to be significantly greater for the parent cell lines than for their virus-transformed counterparts. In addition, the parent cell lines secreted greater amounts of hyaluronate into the medium and retained a larger fraction of the total amount of hyaluronate at the cell surface than the virus-transformed cells. Thus the larger amount of hyaluronate on the surfaces of the parent cell types may be the result of both a faster rate of production and a decreased rate of release.

Animals↗
PubMed1982

Interaction of hyaluronate with the surface of simian virus 40-transformed 3T3 cells: aggregation and binding studies.

Previous studies have shown that the divalent-cation independent aggregation of simian virus 40-transformed 3T3 cells (SV-3T3) is mediated by the interaction of endogenous hyaluronate with binding sites on the cell surface. In the present study, the nature of the interaction of hyaluronate with the binding sites was characterized further by examining both the aggregation of SV-3T3 cells and the binding of exogenously added [3H]hyaluronate. The aggregation of SV-3T3 cells was found to be: (1) optimal at pH 7; (2) only slightly affected by variations in temperature between 0 and 40 degrees C; (3) more potently inhibited by the addition of high molecular weight preparations of hyaluronate than by lower molecular weight preparations; and (4) enhanced by increasing the ionic strength of the medium. With the exception of the effects of temperature, these characteristics correspond closely to those previously described for binding of [3H]hyaluronate. The effect of ionic strength on the binding of [3H]hyaluronate by SV-3T3 cells was found to correspond closely to its effect on lowering the viscosity of hyaluronate. Both effects (i.e. binding and reduction in viscosity) had similar dose-response curves for NaCl and MgCl2. This close correspondence between binding and the reduction of viscosity suggested that the effect of ionic strength on binding was due to a change in the structure of hyaluronate. Lowering the temperature (40 to 0 degrees C) also enhanced the binding of [3H]hyaluronate to SV-3T3 cells. However, this effect was blocked if the cells were first fixed with glutaraldehyde, suggesting that temperature mediates its effect through the binding sites. Additional experiments showed that the binding of [3H]hyaluronate by SV-3T3 cells could be prevented by preincubating the cells with several types of lectins.

Animals↗
PubMed1981

Divalent cation-independent aggregation of rat-1 fibroblasts infected with a temperature-sensitive mutant of Rous sarcoma virus.

Rat-1 fibroblasts infected with the temperature-sensitive transformation mutant LA 24 of Rous sarcoma virus have a high rate of divalent cation-independent homotypic cell aggregation when grown at the permissive temperature, 34 degrees. Cells grown at the nonpermissive temperature, 39 degrees, have a low rate of homotypic cell aggregation. Hyaluronic acid is involved in the homotypic aggregation of permissively grown cells since aggregation is blocked by either treatment of the cells with hyaluronidase or the presence of exogenously added hyaluronic acid. Despite their low rate of homotypic aggregation, nonpermissively grown cell are capable of heterotypically adhering to premissively grown cells. The formation of heterotypic aggregates is blocked by treatment of the nonpermissively grown cells with hyaluronidase but not by treatment of permissively grown cells with hyaluronidase. These results provide evidence that the divalent cation-independent aggregation of Rat-1 LA 24 cells is mediated by interactions between hyaluronic acid and other adhesive components on the cell surface.

Animals↗
PubMed1980

Physical characteristics of hyaluronate binding to the surface of simian virus 40-transformed 3T3 cells.

The binding of labeled hyaluronate to the surface of Simian virus 40-transformed 3T3 cells was studied as a function of 1) pH, 2) ionic strength, 3) temperature, and 4) molecular weight of the hyaluronate. Binding occurred over a wide range of pH values with optima at pH 7 and at less than pH 4. Binding at low pH was eliminated at high ionic strength whereas that at physiological pH was enhanced, with a maximum at 0.5 M NaCl. The enhancement of binding at pH 7 was reversible and independent of the particular salt used. Scatchard plot analysis showed that increasing the ionic strength resulted in both a decrease in the dissociation constant (Kd) and an increase in the amount bound at saturation (Bmax). Temperature also influenced the binding of hyaluronate to the cell surface. The amount bound at low temperatures (0 degrees C) was 3 to 5 times that bound at high temperatures (40 degrees C) with a sharp transition occurring at 18 degrees C, the temperature of phase transition of the plasma membrane. The temperature effect was primarily a change in the Bmax and was reversible. Finally the molecular weight of the ligand influenced the binding. High molecular weight preparations of hyaluronate had a higher binding affinity (lower Kd) and a lower Bmax than did smaller molecular weight preparations.

Animals↗
PubMed1979

Binding of hyaluronate to the surface of cultured cells.

The binding of hyaluronate to SV-3T3 cells was measured by incubating a suspension of cells (released from the substratum with EDTA) with 3H-labeled hyaluronate and then applying the suspension to glass fiber filters which retained the cells and the bound hyaluronate. The extent of binding was a function of both the concentration of labeled hyaluronate and the cell number. Most of the binding took place within the first 2 min of the incubation and was not influenced by the presence or absence of divalent cations. The binding of labeled hyaluronate to SV-3T3 cells could be prevented by the addition of an excess of unlabeled hyaluronate. High molecular weight preparations of hyaluronate were more effective in preventing binding than low molecular weight preparations. The binding of [3H]hyaluronate was inhibited by high concentrations of oligosaccharide fragments of hyaluronate consisting of six sugars or more, and by chondroitin. The sulfated glycosaminoglycans (chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, heparin, and heparan sulfate) had little or no effect on the binding. The labeled hyaluronate bound to the cells could be totally removed by incubating the cells with testicular hyaluronidase, streptomyces hyaluronidase, or trypsin, indicating that the hyaluronate-binding sites are located on the cell surface.

Animals↗
PubMed1977

Heparan sulfates of mouse cells. Analysis of parent and transformed 3T3 cell lines.

Heparan sulfate from the surface of a variety of mouse cells at different cell densities was examined by ion-exchange chromatography. The results of this analysis show that: (1) The heparan sulfate from new isolates of Swiss 3T3 cells transformed by SV40 virus (a DNA tumor virus) elutes from DEAE-cellulose at a lower ionic strength than that from the parent cell type. This finding confirms our earlier observation with an established SV40-transformed cell line (Underhill and Keller, '75) and eliminates the possibility that this change is caused by extended passage in culture. (2) For both parent and transformed 3T3 cells, the heparan sulfates from low and high density cultures were the same as judged by chromatography on DEAE-cellulose. This result demonstrates that the transformation-dependent change which we have observed is independent of cell density. (3) The heparan sulfate from Balb/c 3T3 cells transformed with Kirsten murine sarcoma virus (an RNA tumor virus) elutes from DEAE-cellulose prior to that from parent Balb/c 3T3 cells. This result extends the transformation dependent change in heparan sulfate to the Balb/c 3T3 cell line and to cells transformed with an RNA virus.

Cell Count↗
PubMed1976

Density-dependent changes in the amount of sulfated glycosaminoglycans associated with mouse 3T3 cells.

The relative amount of sulfated glycosaminoglycans associated with the cell layer of parent and SV40-transformed Swiss mouse 3T3 cells was determined from the incorporation of labeled sulfate (35SO4) into macromolecular material. In cultures of SV40-transformed cells, the glycosaminoglycan content per cell was constant over a wide range of densities. In cultures of parent 3T3 cells, the glycosaminoglycan content per cell increased directly with density, the highest values being found in contact-inhibited cultures. At high cell densities, the glycosaminoglycan content of 3T3 cells was several-fold higher than that for SV40-transformed cells. Most of the density-dependent increase in glycosaminoglycans of 3T3 cells was accounted for by chondroitin sulfate (dermatan sulfate) which was over 6-fold higher in confluent cultures than in low density cultures.

Cell Count↗