Biomedical subjects

G Chi-Rosso

Publications and source records attributed to G Chi-Rosso.

26 records · Page 2Linked to original sources

PubMed1990

Endothelial leukocyte adhesion molecule 1: direct expression cloning and functional interactions.

A cDNA for endothelial leukocyte adhesion molecule 1 (ELAM-1) was isolated by transient expression in COS-7 cells of a subtracted cDNA library from cytokine-treated human umbilical vein endothelial cells (HUVECs), with selection of ELAM-1-expressing clones by adhesion of transfected cells to the human promyelocytic cell line HL-60. This cloning method requires neither antibody nor purified ligand. ELAM-1-expressing COS cells bind the promyelocytic cell line HL-60 by a Ca2(+)-dependent but temperature-independent mechanism. Although ELAM-1 is homologous to mammalian lectins, its interaction with HL-60 cells is not inhibited by simple carbohydrate structures. ELAM-1-expressing COS cells also bind human neutrophils and the human colon carcinoma cell line HT-29, but not the B-cell line Ramos. However, Ramos cells adhere to cytokine-treated HUVECs but not control HUVECs, confirming the existence of other inducible adhesion molecules. In addition, the binding of HL-60 cells or neutrophils to ELAM-1-expressing COS cells is not inhibited by a monoclonal antibody (60.3) directed to an inhibitory epitope on CD18, indicating that the ELAM-1 ligand, although uncharacterized, is not a member of the CD11/CD18 family.

Amino Acid Sequence↗

PubMed1990

Hyaluronate-binding proteins of murine brain.

The distribution of hyaluronate-binding activity was determined in the soluble and membrane fractions derived from adult mouse brain by sonication in low-ionic-strength buffer. Approximately 60% of the total activity was recovered in the soluble fraction and 33% in membrane fractions. In both cases, the hyaluronate-binding activities were found to be of high affinity (KD = 10(-9) M), specific for hyaluronate, and glycoprotein in nature. Most of the hyaluronate-binding activity from the soluble fraction chromatographed in the void volume of Sepharose CL-4B and CL-6B. Approximately 50% of this activity was highly negatively charged, eluting from diethylaminoethyl (DEAE)-cellulose in 0.5 M NaCl, and contained chondroitin sulfate chains. This latter material also reacted with antibodies raised against cartilage link protein and the core protein of cartilage proteoglycan. Thus, the binding and physical characteristics of this hyaluronate-binding activity are consistent with those of a chondroitin sulfate proteoglycan aggregate similar to that found in cartilage. A 500-fold purification of this proteoglycan-like, hyaluronate-binding material was achieved by wheat germ agglutinin affinity chromatography, molecular sieve chromatography on Sepharose CL-6B, and ion exchange chromatography on DEAE-cellulose. Another class of hyaluronate-binding material (25-50% of that recovered) eluted from DEAE with 0.24 M NaCl; this material had the properties of a complex glycoprotein, did not contain chondroitin sulfate, and did not react with the antibodies against cartilage link protein and proteoglycan. Thus, adult mouse brain contains at least three different forms of hyaluronate-binding macromolecules. Two of these have properties similar to the link protein and proteoglycan of cartilage proteoglycan aggregates; the third is distinguishable from these entities.

Animals↗

PubMed1989

Direct expression cloning of vascular cell adhesion molecule 1, a cytokine-induced endothelial protein that binds to lymphocytes.

We have cloned a previously undescribed adhesion molecule, VCAM-1, which is induced by cytokines on human endothelial cells and binds lymphocytes. Using a novel method requiring neither monoclonal antibodies nor purified protein, VCAM-1-expressing clones were selected by adhesion to human lymphoid cell lines. VCAM-1 mRNA is present in endothelial cells at 2 hr after treatment with IL-1 or TNF-alpha and is maintained for at least 72 hr; leukocyte binding activity parallels mRNA induction. Cells transfected with VCAM-1 bind the human leukemia lines Jurkat, Ramos, Raji, HL60, and THP1, but not peripheral blood neutrophils. VCAM-1, which belongs to the immunoglobulin gene super-family, may be central to recruitment of mononuclear leukocytes into inflammatory sites in vivo.

Amino Acid Sequence↗

PubMed1988

Subcellular localization of leukotriene D4 receptors in sheep tracheal smooth muscle.

The distribution of [3H]leukotriene D4 [( 3H]LTD4) receptors in subcellular membrane fractions obtained from sheep tracheal smooth muscle was studied. Using differential centrifugation and discontinuous sucrose density gradient centrifugation, the subcellular membranes were separated into six fractions. The [3H]LTD4 receptor distribution profile in these fractions correlated with markers for the plasma membrane (5'-nucleotidase and alkaline phosphodiesterase) and did not correlate with markers for the mitochondria (cytochrome c oxidase and succinate-dependent cytochrome c reductase). The dissociation constant (Kd) and maximum number of binding sites (Bmax) for [3H]LTD4 binding to the receptors in the crude mixture of membranes (PII) were 0.38 +/- 0.2 nM and 77 +/- 14 fmol/mg of protein, respectively. The Kd and Bmax of [3H]LTD4 binding to the receptors in the plasma membrane-enriched fraction (FII) were 0.40 +/- 0.2 nM and 268 +/- 46 fmol/mg of protein, respectively. The specificity profile of the [3H]LTD4 receptors in the plasma membrane-enriched fraction was equivalent to that observed in the crude membrane and correlated with the agonist myotonic activities in the smooth muscle contraction assay system. Furthermore, the binding of [3H]LTD4 to the plasma membrane receptors was modulated by guanine nucleotides in a manner analogous to that observed in crude membranes, suggesting that agonist interaction with the receptors was regulated by guanine nucleotide binding protein. These results suggest that, in sheep tracheal smooth muscle, the plasma membrane is the primary location of specific LTD4 receptors.

Animals↗

PubMed1987

Hyaluronate-binding protein of simian virus 40-transformed 3T3 cells: membrane distribution and reconstitution into lipid vesicles.

Hyaluronate-binding protein (HABP) has been extracted in detergent from the membranes of simian virus 40-transformed 3T3 (SV-3T3) cells (Underhill et al, J Biol Chem 258:8086-8091, 1983). When SV-3T3 cells were treated with trypsin prior to isolation and dissolution of the membranes, no hyaluronate-binding activity could be detected. This indicates that all of the detectable HABP of SV-3T3 cells is located on the external surface of the plasma membrane rather than on internal membranes, which would be inaccessible to the trypsin. The detergent-extracted HABP from SV-3T3 membranes was reconstituted into the membrane of lipid vesicles, which were formed by addition of exogenous phosphatidylcholine and cholic acid to the extracts followed by removal of detergent by dialysis against 0.02 M Tris pH 8.0 in the presence of protease inhibitors. Reconstitution was assessed by sedimentation in a discontinuous sucrose gradient and by gel filtration on Sepharose 4B in the presence and absence of detergent. The characteristics of binding of hyaluronate to the reconstituted HABP were then compared with those studied previously for the original membrane-bound HABP and the detergent-extracted HABP (Underhill et al, J Biol Chem 258:8086-8091, 1983). It was observed previously that binding of hyaluronate to HABP in the cell membranes was of higher affinity and specificity than to HABP in the detergent extracts of these membranes. It was found here that reconstitution of the extracted HABP into the membranes of lipid vesicles led to restoration of affinity of binding to the level observed in the original cell membranes. However, whereas chondroitin sulfate does not compete significantly for binding of hyaluronate to cell membrane-bound HABP, partial competition was observed for the reconstituted HABP as well as for detergent-extracted HABP. Thus, it is concluded that the high affinity of binding of hyaluronate to the plasma membrane of SV-3T3 cells is in part dependent on insertion of the HABP in the membrane, but that other interactions, not duplicated in our reconstitution experiments, must be necessary for the specificity of the HABP.

Animals↗

PubMed1986

Leukotriene B4 induces formation of inositol phosphates in rat peritoneal polymorphonuclear leukocytes.

Leukotriene B4 (LTB4) induced rapid breakdown of prelabeled inositol phospholipids in rat peritoneal polymorphonuclear leukocytes (PMNs). Formation of [3H]inositol triphosphate ([3H]IP3) was rapid, with a peak of 250-300% of the control level, after 5-15 sec of exposure to LTB4. Accumulation of [3H]inositol bisphosphate was rapid, peaking after 30 sec of treatment. Accumulation of [3H]inositol monophosphate was also rapid in the presence of LiCl. The kinetics of [3H]IP3, [3H]inositol bisphosphate, and [3H]inositol monophosphate accumulation suggest that LTB4 may interact with receptors in PMNs and activate phospholipase C which in turn induces hydrolysis of inositol-phospholipids. The agonist activities of several LTB4 analogs were employed to investigate the structure-activity relationships of LTB4 receptor-mediated activation of phosphatidylinositol hydrolysis. Increases in [3H]IP3 formation were dependent upon the concentration of LTB4 and the agonist analogs. The rank order potency of these analogs was equivalent to that of the pharmacological activity of LTB4 agonists in the PMN chemotaxis assay. Furthermore, the islet activation protein isolated from Bordetella pertussis inhibited LTB4-induced [3H]IP3 formation. The tumor-promoting phorbol myristate acetate also inhibited LTB4-induced [3H]IP3 formation. The LTB4 receptors on a partially purified PMN membrane were characterized. LTB4 binding to the receptors was stereoselective and specific. The binding affinity (Kd) of [3H] LTB4 to the receptors was 1.3 +/- 0.2 nM. The maximum density of binding was 5.5 +/- 1.8 pmol/mg of protein. The rank order potency of binding affinities of several LTB4 analogs was equivalent to that of the induction of IP3 response induced by LTB4 and analogs. These results suggest that LTB4 may interact with receptors in rat PMNs, activate G protein-regulated phospholipase C, and induce [3H]IP3 formation.

Animals↗

PubMed1984

Endogenous hyaluronate-cell surface interactions in 3T3 and simian virus-transformed 3T3 cells.

3T3 cells have a large, pericellular coat which contains 30 times more hyaluronate than the amount of cell surface hyaluronate associated with simian virus 40-transformed 3T3 (SV-3T3) cells. On the other hand, SV-3T3 cells have high affinity binding sites for exogenously added hyaluronate, whereas 3T3 cells have much lower affinity sites. Removal of cell surface hyaluronate from SV-3T3 cells by treatment with hyaluronidase caused a reproducible increase in their maximum binding capacity for exogenous hyaluronate but no significant change in binding affinity or specificity. For 3T3 cells, however, the maximum amount of binding decreased and the affinity of binding increased after hyaluronidase treatment. When endogenous cell surface hyaluronate was labeled metabolically and then the cells incubated in the presence of exogenous unlabeled hyaluronate, the labeled cell surface hyaluronate was quantitatively displaced from the SV-3T3 cells but was not displaced from the 3T3 cells. Chondroitin sulfate and heparin did not displace cell surface hyaluronate from either cell type. Membranes isolated from SV-3T3 cells bound hyaluronate specifically and with high affinity, whereas membranes from 3T3 cells did not consistently bind a significant amount of hyaluronate. We conclude from these studies that the retention of endogenous hyaluronate on the surface of SV-3T3 cells is mediated by binding sites similar to those detected by the addition of exogenous hyaluronate, and the mechanism of retention of endogenous hyaluronate on the surface of 3T3 cells differs from SV-3T3 cells.

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↗