Biomedical subjects

D Gullberg

Publications and source records attributed to D Gullberg.

44 records · Page 3Linked to original sources

PubMed1990

gamma-Glutamyltranspeptidase-positive rat hepatocytes are protected from GSH depletion, oxidative stress and reversible alterations of collagen receptors.

The aim of this study has been to define cytotoxic mechanisms that may cause clonal expansion in the liver of pre-carcinogenic cells. An in vitro model, which has been described previously, was used. Hepatocytes were isolated from carcinogen-treated rats and a high proportion of the cells were gamma-glutamyltranspeptidase (GGT)-positive. The cells were incubated in suspension and exposed to toxic agents in concentrations that induced a moderate increase in cellular leakage within 3 h. Samples were withdrawn and sampled cells were then allowed to attach to collagen-coated plates. Attached cells were stained and the ratio of GGT-positive/GGT-negative cells (GGT-ratio) was determined. The initial GGT-ratio was 10.4 +/- 4.7% and an increased ratio was taken as a sign of toxicity that resulted in a selection of GGT-positive cells. In a first series of experiments it was shown that hydroquinone and menadione increase the GGT-ratio, while diquat, sodium selenite, diethyl maleate or phorone do not. However, diethyl maleate in combination with diquat increased the GGT-ratio. Hydrogen peroxide (5 mM) increased the GGT-ratio as effectively as hydroquinone (0.3 mM). Lower concentrations of H2O2 (0.05 mM) increased the GGT-ratio in GSH-depleted cells. The changes induced by hydroquinone and H2O2 in low concentration were reversible. In another series of experiments, plates coated with antibodies against beta 1-integrin were used. An increase in the GGT-ratio was obtained with anti beta 1-integrin, but not with broad spectrum anti-rat hepatocyte or anti-rat beta 2-microglobulin antibodies as substrata. These data suggested an involvement of the beta 1-integrin in the selection. Taken together, these data indicate that GGT-positive hepatocytes are protected against GSH depletion and oxidative stress that may result in reversible receptor alterations.

Animals↗

PubMed1989

Identification of integrin-like matrix receptors with affinity for interstitial collagens.

Antibodies to a rat liver membrane glycoprotein with an Mr of 115,000 (nonreduced) inhibited the attachment of rat hepatocytes and primary rat heart fibroblasts to both collagen and fibronectin. The Mr 115,000 glycoprotein cross-reacted immunologically with the beta 1-chain of the rat hepatocyte fibronectin receptor (HFNR), and the two proteins showed identical peptide maps after proteolytic cleavage. It was concluded that the Mr 115,000 protein was similar or identical to the beta 1-chain of Arg-Gly-Asp (RGD)-directed matrix receptors. Although collagen type I contains several RGD sequences, the attachment of hepatocytes and fibroblasts to collagen type I was not inhibited by the synthetic peptide GRGDTP in concentrations that blocked adhesion to fibronectin. Furthermore, hepatocytes adhered equally well to collagen fragments, generated by cyanogen bromide cleavage, lacking RGD sequences as to fragments containing this sequence. Antibodies to the Mr 115,000 protein inhibited the adhesion of hepatocytes to both types of collagen fragments. Taken together, these data indicate the presence of collagen receptors that share the beta-subunit with the HFNR but that are not directed to RGD sequences. Tentative alpha-chains of the collagen matrix receptor complex were isolated by immunoprecipitation of surface 125I-labeled fibroblast membrane proteins purified by affinity chromatography on immobilized collagen type I. Data are presented indicating that proteins with Mr around 145,000 and 170,000 (nonreduced) are associated in noncovalently linked complexes with the Mr 115,000 protein. These complexes have affinity for collagen and thus have properties expected for integrin-like collagen receptors.

Amino Acid Sequence↗

PubMed1989

Expression of collagen adhesion proteins and their association with the cytoskeleton in cardiac myocytes.

Previous investigations have shown that specific cell surface glycoproteins on rat hepatocytes (COLL-CAM) are involved in the recognition of interstitial collagens (Rubin et al., Exp. Cell Res., 164:127-138, 1986). Western blot analysis with anti-COLL-CAM antibodies revealed the presence of a variable but restricted number (two) of glycoproteins in detergent-extracted membranes from rat hearts at various developmental stages. Using antibodies against these collagen adhesion proteins, we show an expression of the antigens during different developmental stages of the rat heart and during cardiac hypertrophy. This expression is described morphologically by immunohistochemical staining of cell surfaces of freshly isolated myocytes from neonates, normal adults, and hypertrophied adult hearts. Antibodies made against COLL-CAM were localized on the cell surface of cardiac myocytes and antibodies against talin and vinculin co-localized in a similar position on the inside of the cell. Antibody staining appears to be increased at times when collagen synthesis is high (neonate and cardiac hypertrophy) and low when collagen synthesis is low, as in the normal adult. These results indicate that collagen adhesion proteins may play an important role in linking the extracellular matrix to the cytoskeleton in the heart.

Animals↗

PubMed1988

In vitro studies on adult cardiac myocytes: attachment and biosynthesis of collagen type IV and laminin.

The interactions between adult rat cardiac myocytes and the basement membrane components collagen type IV and laminin were investigated in attachment experiments and biosynthesis studies and by immunofluorescence staining. Adult myocytes attached equally well to native collagen type IV and laminin but did not attach to collagen type IV solubilized with pepsin (P-CIV) or to collagen type I. However, when laminin was used to coat P-CIV, attachment was enhanced. Affinity-purified antibodies against laminin inhibited the attachment of myocytes to dishes coated with native collagen type IV, indicating that cell surface-bound laminin mediated attachment of the cells to this substrate. Immunofluorescence staining of freshly isolated myocytes, using antibodies against laminin or collagen type IV, revealed the presence of laminin but not of collagen type IV on the surface of freshly isolated cells, indicating that during the isolation procedure collagen IV was removed from the cell surface. Metabolic labeling followed by immunoprecipitation demonstrated synthesis of both laminin and collagen type IV in cardiac myocytes as they progressed into culture over a 14-day period. This synthesis was accompanied by the deposition of the collagen type IV and laminin into distinctly different patterns as revealed by immunofluorescence staining. As the cells progressed into culture, newly synthesized laminin formed a network radiating from the center of the reorganizing cell into the pseudopods. The laminin was redistributed and remodeled with time in culture to form a dense layer beneath the cell. Collagen type IV was also synthesized with time in culture, but the pattern was a much finer network as opposed to the denser pattern of laminin staining. These studies demonstrate that adult cardiac myocytes synthesize and remodel the basement membrane as they adapt to the culture environment.

Adult↗

PubMed1988

Membrane glycoproteins involved in hepatocyte adhesion to collagen type I.

Liver membrane glycoproteins with affinity for immobilized collagen type I were subjected to preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by electroelution of the separated proteins. Electroeluted glycoproteins with ability to neutralize the inhibitory effect of anti-CollCAM antibodies on hepatocyte adhesion to collagen were collected from several consecutive runs and used to raise a high titer antiserum, denoted anti-CollCAM II. IgG from this antiserum inhibited the attachment of hepatocytes to dishes coated with collagen type I, but not to fibronectin- or collagen type IV-coated dishes. When the antibodies were immobilized to Sepharose CL-4B they bound three sets of glycoproteins with apparent Mr's of 105,000, 115,000, and 130,000 as analyzed by SDS-PAGE under nonreducing (NR) conditions. Upon reduction (R) the glycoproteins migrated with apparent Mr's of 115,000, 130,000, and 160,000, respectively. The Mr 105,000-115,000 (NR) glycoproteins effectively neutralized the inhibitory effect exerted by both anti-CollCAM and anti-CollCAM II antibodies, on hepatocyte spreading and attachment to collagen type I substrates. Peptide mapping suggested the Mr 160,000 (R) species to be different from the Mr 115,000 (R).

Animals↗

PubMed1986

Hepatocyte adhesion to collagen. Isolation of membrane glycoproteins involved in adhesion to collagen.

Adhesion of hepatocytes to collagenous substrates and their spreading have been shown to involve a specific recognition event, possibly mediated by membrane proteins with affinity for collagen. In the present communication, we describe the isolation of membrane components that are involved in the adhesion of rat hepatocytes to collagen. These components could be solubilized from liver microsomal membranes by treatment with detergents or papain--but not by treatment with EDTA, urea or high salt. The purification of detergent-solubilized components was monitored by an assay determining the ability of membrane components to neutralize antibody-mediated inhibition of hepatocyte adhesion to collagen. By affinity chromatography on lentil lectin-Sepharose it was found that the neutralizing activity resided within the glycoprotein fraction. These glycoproteins were purified further by affinity-chromatography on collagen type I linked to Sepharose. Antibodies raised against the glycoproteins with affinity for immobilized collagen, effectively inhibited hepatocyte adhesion to collagen. The bulk of the neutralizing activity migrated with an apparent molecular weight of 120 000-140 000 in preparative SDS-PAGE.

Animals↗

PubMed1985

Characterization of the hydrophobic properties of the receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin.

The hydrophobic character of the receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been estimated by its tendency to adsorb to hydrophobically interacting matrices. The receptor is adsorbed to uncharged pentyl-Sepharose but not to butyl-Sepharose at 1 M NaCl. It is also adsorbed to phenyl-Sepharose or Cibacron blue-Sepharose at lower ionic strengths (0-0.15 M NaCl). Elution of adsorbed receptor could not be achieved under mild conditions (decreasing salt concentration, increasing glycerol concentration). A concentration of 0.2% (w/v) of the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) was required to desorb the receptor from pentyl-Sepharose with an approximate yield of 11-14% of the specific [3H]TCDD-binding activity. The CHAPS-treated receptor exhibited the same physicochemical characteristics as that in crude cytosol (4-5 S, Stokes radius approximately 60 A). Furthermore, the effects of detergents other than CHAPS on hydrodynamic parameters and on [3H]TCDD binding to the receptor were studied. In conclusion the TCDD receptor showed more pronounced hydrophobic properties than those reported for steroid hormone receptors.

Animals↗

PubMed1985

Incidence of arthritis and autoreactivity of anti-collagen antibodies after immunization of DBA/1 mice with heterologous and autologous collagen II.

Native type II collagens of rat, chick, bovine, human and murine origin have been used for immunization of male and female DBA/1 mice of different ages. The four heterologous collagens were able to induce arthritis in both male and female mice although the incidence of arthritis was higher in males. The onset of arthritis was sudden with severe lesions starting to appear 5- to 6-weeks after immunization. Mouse collagen II, on the other hand, caused arthritis exclusively in males. The onset of arthritis was in the latter case less dramatic and usually delayed until 12- to 14-weeks post immunization. Antibody production against both the immunogen and other type II collagens, including mouse collagen was seen in both arthritic and non-arthritic animals, and antibody titres against both the collagen used for immunization and mouse collagen were higher in the sera from arthritic than in identically immunized non-arthritic animals. Total amounts of auto-anticollagen II antibodies were, however, more dependent on which collagen preparation was used for immunization than on whether the immunized mice developed arthritis or not. These results indicate that arthritis induction in mice is not dependent solely on the levels of autoreactive anti-collagen II antibodies.

Animals↗