Biomedical subjects

S Luhowskyj

Publications and source records attributed to S Luhowskyj.

11 recordsLinked to original sources

PubMed1996

Mutational analysis of the alpha subunit of the human interleukin-3 receptor.

The alpha subunit of the human interleukin-3 receptor (IL-3R alpha) is a 70-kD glycoprotein member of the hematopoietin receptor superfamily. This protein associates with a beta subunit common to the receptors for IL-5 and granulocyte-macrophage colony-stimulating factor (GM-CSF) to form a high-affinity receptor for IL-3. To identify regions of IL-3R alpha critical for ligand binding and receptor function, cDNAs encoding mutant receptors were generated and expressed in COS cells along with the beta subunit. Mutant receptors lacking almost the entire cytoplasmic domain of IL-3R alpha [IL-3R alpha(CD)] or carrying a substitution of trp for leu in the membrane proximal leu-ser-x-trp-ser (LSXWS) box bound 125I-IL-3 with nearly the same affinity as wild-type IL-3R alpha. In contrast, a mutant lacking the entire "LSXWS" motif failed to bind 125I-IL-3 with high affinity despite showing surface expression. In addition, hybrid receptors composed of the first 104 amino acids (aa) of IL-3R alpha joined to aa 118 through 400 of the alpha subunit of the GM-CSF receptor (GM-R alpha) [IL-3R alpha/GM-R alpha] or the first 118 aa of GM-R alpha joined to aa 104 through 378 of IL-3R alpha [GM-R alpha/IL-3R alpha] failed to bind 125I-IL-3 in the presence of the beta subunit. A third hybrid receptor composed of the first 281 residues of IL-3R alpha fused to residues 306 through 379 of GM-R alpha [IL-3R alpha/GM-R alpha-DS] also failed to bind 125I-IL-3 in the presence of the beta subunit but, in contrast to the IL-3R alpha/GM-R alpha hybrid, demonstrated weak surface expression. Mutant receptors lacking the N-terminal 30 aa and the N-terminal 9 aa also did not bind 125I-IL-3 with high affinity, although both were expressed on the cell surface. These data suggest that although the cytoplasmic domain and the leucine residue of the "LSXWS" box are not critical for ligand binding or beta-subunit association, the "LSXWS" motif and amino-terminal sequences are important for these functions.

Amino Acid Sequence↗

PubMed1994

Quantitation of interferon-induced Mx protein in whole blood lysates by an immunochemiluminescent assay: elimination of protease activity of cell lysates in toto.

Due to the rapidly expanding usage of interferons and its costliness of therapy, it is important to evaluate the clinical efficacy of the various interferons. Directly assaying circulating interferon is technically quite difficult. Here, we present an alternate method to evaluate interferon therapy by assaying a unique protein, called Mx protein, which is a 78 kDa cytoplasmic protein selectively induced by type-1 interferon in human leukocytes. The current assay is a two-site chemiluminescent immunoassay, designed to detect Mx protein in whole blood lysates. Since the Mx protein once solubilized, is highly susceptible to proteolysis in whole blood lysates, we have devised a new procedure both to maximize its solubility and virtually eliminate its proteolytic degradation. A mouse monoclonal antibody conjugated to the derivatized-paramagnetic particles and an acridinium ester-labeled antibody serve as the solid phase capture and detector antibodies, respectively. This assay is applicable to both manual and automated modes with a detection limit of Mx protein at 20 ng/ml whole blood. Availability of a reliable assay for Mx protein should facilitate the clinical evaluation of many of the newly constructed type-1 interferons.

Animals↗

PubMed1992

Structure/function studies on vascular cell adhesion molecule-1.

Vascular cell adhesion molecule-1 (VCAM1) is a member of the immunoglobulin (Ig) superfamily which interacts with the integrin very late antigen-4 (VLA4). The VCAM1/VLA4 interaction mediates both adhesion and signal transduction and is thought to play an important role in inflammatory and immune responses in vivo. The major form of human VCAM1 contains seven extracellular Ig-like domains, with domain 1 designated as the most N-terminal. We have examined the relationship between human VCAM1 structure and function using a combination of domain truncation mutants and proteolytic fragmentation of recombinant soluble VCAM1. We have characterized two regions of VCAM1, localized to domains 4 and 5, which are highly sensitive to proteolytic cleavage, localized the epitope of the blocking monoclonal antibody 4B9 to domain 1, and found that domains 1-3 are sufficient for both its adhesive function and its ability to initiate T cell activation.

Amino Acid Sequence↗

PubMed1991

Expression and functional characterization of a soluble form of vascular cell adhesion molecule 1.

Vascular cell adhesion molecule 1 (VCAM1) is a leukocyte adhesion molecule induced on human endothelium in vitro and in vivo by inflammatory stimuli. A truncated cDNA for VCAM1 was constructed, stably expressed in Chinese Hamster Ovary (CHO) cells, and the secreted recombinant soluble form of VCAM1 (rsVCAM1) purified to homogeneity by immunoaffinity chromatography. Immobilized rsVCAM1 is a functional adhesion protein, and selectively binds only VLA4-expressing cells, including human B and T lymphocytes, NK cells, and certain lymphoblastoid cell lines. T cell subset analyses indicate preferential binding of CD8+ memory cells. rsVCAM1 should prove valuable for the further study of the role of VCAM1 during inflammatory and immune responses in vivo.

Amino Acid Sequence↗

PubMed1991

Expression and functional characterization of a soluble form of endothelial-leukocyte adhesion molecule 1.

Endothelial leukocyte adhesion molecule 1 (ELAM1) is a leukocyte adhesion molecule induced on human venular endothelium in vitro and in vivo by inflammatory stimuli. A truncated cDNA for ELAM1 has been constructed, stably expressed in Chinese hamster ovary cells, and the secreted recombinant soluble form of ELAM1 (rsELAM1) purified to homogeneity by immunoaffinity chromatography. rsELAM1, when immobilized on plastic, is fully functional as an adhesion protein, and selectively binds only cells known to bind cell-surface ELAM1 expressed on human endothelial cells, including the myelomonocytic cell line HL60 and the colon carcinoma cell line HT29. Immobilized rsELAM1 also binds human PMN, monocytes, NK cells, and T cells. T cell subset analyses indicate preferential binding of CD4+ T memory cells. However, rsELAM1 is only a weak inhibitor of ELAM1-mediated adhesion. rsELAM1 should prove valuable for the further study of the role of ELAM1 expressed on the vascular wall during the inflammatory response.

Amino Acid Sequence↗

PubMed1991

Cloning of an alternate form of vascular cell adhesion molecule-1 (VCAM1).

Vascular cell adhesion molecule-1 (VCAM1) of the Ig superfamily is induced by the inflammatory cytokines interleukin-1 and tumor necrosis factor on human umbilical vein endothelial cells (HUVECs). It binds to mononuclear leukocytes via the integrin VLA-4. We have cloned and expressed a cDNA encoding a new form of human VCAM1 containing an additional Ig homologous domain inserted between the third and fourth domains of the original six-domain protein. Characterization of mRNA from HUVECs from three individuals at various time points after induction by tumor necrosis factor indicates that both the long and short VCAM1 mRNAs are made by all three individuals, with the long form predominating quantitatively. Immunoprecipitation of VCAM1 protein from cos7 cells transfected with each cDNA and from cultured endothelial cells followed by deglycosylation suggests that the long form is the major form found on endothelium. The two forms may result from alternate splicing of a precursor mRNA. Both forms support adhesion of VLA-4-expressing cell lines.

Amino Acid Sequence↗

PubMed1990

A blocking monoclonal antibody to endothelial-leukocyte adhesion molecule-1 (ELAM1).

ELAM1 is a leukocyte adhesion molecule induced on human umbilical vein endothelial cells (HUVECs) by inflammatory cytokines. Balb/C mice were immunized with COS cells transiently expressing cell-surface ELAM1 after transfection with ELAM1 cDNA. After fusion, ELAM1-specific monoclonal antibodies (Mabs) were identified by selective adhesion to ELAM1-expressing, but not control, CHO cells, and to cytokine-treated but not untreated HUVECs. One Mab, designated BB11, binds to and immunoprecipitates ELAM1 expressed on HUVECs, COS and CHO cells. BB11 blocks the interaction of ELAM1 with human PMN, the human myelomonocytic cell line HL60, and the human colon carcinoma line HT29.

Antibodies, Monoclonal↗

PubMed1990

VCAM-1 on activated endothelium interacts with the leukocyte integrin VLA-4 at a site distinct from the VLA-4/fibronectin binding site.

Cytokine-activated human endothelial cells express vascular cell adhesion molecule-1 (VCAM-1), which binds lymphocytes. We now identify the integrin VLA-4 as a receptor for VCAM-1 because VLA-4 surface expression on K-562 cells (following transfection of the VLA alpha 4 subunit cDNA) resulted in specific cell adhesion to VCAM-1, and anti-VLA-4 antibodies completely inhibited VCAM-1-dependent cell-cell attachment. In addition, VLA-4 expression allowed K-562 cells to attach to the heparin II binding region (FN-40) of fibronectin. However, VLA-4/VCAM-1 and VLA-4/FN-40 interactions are readily distinguishable: only the former was inhibited by the anti-VLA-4 monoclonal antibody HP1/3, and only the latter was inhibited by soluble FN-40. The VCAM-1/VLA-4 ligand-receptor pair may play a major role in the recruitment of mononuclear leukocytes to inflammatory sites in vivo.

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↗

PubMed1989

The insulin-like effect of vanadate on lipolysis in rat adipocytes is not accompanied by an insulin-like effect on tyrosine phosphorylation.

Tyrosine phosphorylation of the insulin receptor and other intracellular proteins in rat adipocytes was examined using an immunoblot technique with antiphosphotyrosine antibody. Insulin at 10(-7) M increased the tyrosine phosphorylation of the 95K subunit of the insulin receptor (15-fold) and proteins of 180K (7-fold) and 60K (23-fold). Increases in insulin-dependent phosphorylation of the three proteins were detectable at 10(-10) M insulin and attained steady state within 30 sec of insulin (10(-7) M) addition. Small effects of insulin (less than 30% increases) were observed on proteins of 120K and 53K. In contrast to insulin, the effects of vanadate on tyrosine phosphorylation were small and nonspecific. Vanadate increased tyrosine phosphorylation of the 95K insulin receptor beta-subunit and the 120K and 60K proteins similarly, with increases of 1.5- to 3-fold at 1 mM and 2-fold or less at 200 and 50 microM. Vanadate-dependent tyrosine phosphorylation of the 180K protein increased to a maximum of only 30% at 200 microM. Tyrosine phosphorylation of the 53K protein was somewhat larger, approaching 4-fold at 1 mM vanadate. The concentration of insulin and vanadate that inhibited isoproterenol-dependent lipolysis were not comparable to those that increased tyrosine phosphorylation. Vanadate at 1 mM was more potent as an antilipolytic agent than 10(-9) M insulin (93% vs. 81%), yet increased tyrosine phosphorylation of the 95K insulin receptor beta-subunit only as effectively as 10(-10) M insulin (which inhibited lipolysis only 42%). The dissimilar responses were even more pronounced when antilipolysis was compared to tyrosine phosphorylation of the 180K and 60K proteins. For example, insulin at 10(-9) M increased tyrosine phosphorylation of the 180K protein 2.9-fold, while 1 mM vanadate had a negligible effect (10% increase). Thus, vanadate exerts an insulin-like effect on lipolysis, yet its effects on tyrosine phosphorylation differ from those of insulin.

Adipose Tissue↗

PubMedDate not supplied

Flavopiridol induces apoptosis and caspase-3 activation of a newly characterized Burkitt's lymphoma cell line containing mutant p53 genes.

Burkitt's lymphoma cell lines have been important in vitro models for studying the pathogenesis of Burkitt's lymphoma (BL) and for exploring new treatment strategies. A new EBV(-) Burkitt's lymphoma cell line (GA-10) was established from a patient with a clinically aggressive, chemorefractory BL and characterized. Although functional p-glycoprotein could not be demonstrated by dye-efflux assays, both p53 genes were mutated in the GA-10 cells, perhaps contributing to the resistant phenotype of the original neoplasm. Two properties of BL cells which may be useful targets for novel cytotoxic therapeutics are their surface expression of CD77, the receptor for Shiga toxin (Stx), and their high rate of proliferation. Expression of CD77 on the GA-10 cells was heterogeneous in that certain subclones expressed high levels of CD77 and correspondingly exhibited strong growth inhibition by Stx while others showed low levels of CD77 expression and weak Stx-induced growth inhibition. Flavopiridol, a potent inhibitor of cell cycle progression through G1 and G2, induced cytotoxicity of the GA-10 cells with an LC(50) of approximately 40 nM vs 70 nM for HL-60 cells (P < 0.05). The concentrations of flavopiridol at which only 10% of the cells were viable (LC(10)) were approximately 280 nM for the GA-10 cells and 520 nM for the HL-60 cells (P < 0.05). Dose-related induction of apoptosis in response to flavopiridol was demonstrated in the GA-10 cells by morphology, TUNEL assay, and activation of caspase-3. Flavopiridol was also cytotoxic to seven other BL cell lines tested. These data suggest that flavopiridol may have therapeutic value in the treatment of Burkitt's lymphoma.

ATP Binding Cassette Transporter, Subfamily B, Mem↗