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J C Gorga

Publications and source records attributed to J C Gorga.

33 records · Page 2Linked to original sources
PubMed1989

Comparison of the secondary structures of human class I and class II major histocompatibility complex antigens by Fourier transform infrared and circular dichroism spectroscopy.

We have examined the secondary structures of human class I and class II histocompatibility antigens in solution by Fourier transform infrared spectroscopy and circular dichroism in order to compare the relative amounts of alpha-helix, beta-sheet, and other structures, which are crucial elements in the comparison of the protein structures. Quantitation of infrared spectra of papain-solubilized HLA-A2, HLA-B7, and DR1 in phosphate buffer gave alpha-helix contents of 17%, 8%, and 10% and beta-sheet contents of 41%, 48%, and 53%, respectively. By circular dichroism, papain-solubilized HLA-A2, HLA-B7, and DR1 were also found to have comparable alpha-helix contents (e.g., 8%, 20%, and 17%, respectively). Circular dichroism analysis for beta-sheet gave 29% for papain-solubilized HLA-B7 and 42% for papain-solubilized DR1. The value for papain-solubilized HLA-A2 (74%) was anomalous. It is proposed that Trp-107 of HLA-A2, missing in both HLA-B7 and DR1, may be responsible for much of the anomaly. Due to the uncertainties inherent in quantitation of the amounts of secondary structures by both spectral methods, the differences in the contents of alpha-helix and beta-sheet in the three proteins are not considered significant. However, differences in the nature of the beta-sheet structures are suggested by infrared spectroscopy. These results provide physical evidence for an overall structure of class II antigens modeled on that of class I antigens.

Buffers↗
PubMed1988

Synergistic T cell activation via the physiological ligands for CD2 and the T cell receptor.

T cells may be activated either by the antigen-specific T cell receptor (TCR)-CD3 complex or the cell surface receptor CD2. A natural ligand for CD2 has been found to be lymphocyte function-associated antigen 3 (LFA-3), a widely distributed cell surface glycoprotein. To investigate the interaction of these two pathways, we have expressed the cDNA encoding the human CD2 molecule in a murine T cell hybridoma that produces IL-2 in response to HLA-DR antigens. Expression of the CD2 molecule markedly enhances IL-2 production in response to LFA-3+ antigen-bearing stimulator cells, and this stimulation is inhibited by anti-CD2 and anti-LFA-3 mAb. To further define the role of LFA-3 in antigen-dependent T cell activation, we have studied the ability of the purified ligands of CD2 and the TCR to stimulate the hybridoma. Neither liposomes containing purified HLA-DR antigens nor liposomes containing purified LFA-3 were able to stimulate the parent or the CD2+ hybridoma. However, liposomes containing both purified LFA-3 and HLA-DR, the physiological ligands for CD2 and the TCR, respectively, stimulate IL-2 production by the CD2+ but not the parent hybridoma, suggesting that complementary interactions between the TCR-CD3 complex and the CD2 pathway may regulate lymphocyte activation. To determine whether the CD2/LFA-3 interaction participates in cell-cell adhesion and provides an activation signal, we have constructed a cytoplasmic deletion mutant of CD2, CD2 delta B, in which the COOH-terminal 100 amino acids of CD2 have been replaced with a serine. Hybridomas expressing the CD2 delta B molecule were examined. Deletion of the cytoplasmic domain of CD2 did not alter binding of LFA-3 but eliminated the ability of CD2 to increase the response of the hybridoma to liposomes containing both HLA-DR and LFA-3, demonstrating that adhesion of LFA-3 to CD2 alone was insufficient for activation, and that the cytoplasmic domain was required for LFA-3 stimulation through the CD2 molecule. T cells may be activated by purified LFA-3 binding to CD2 and the TCR interacting with its ligand, and these signals appear to be synergistic for the T cell. These results suggest that the CD2/LFA-3 interaction not only plays a role in cell-cell adhesion but provides a stimulatory signal for T cell activation.

Amino Acid Sequence↗
PubMed1988

Functional analysis of a cytoplasmic domain-deleted mutant of the CD4 molecule.

The CD4 molecule is a receptor found on a subset of T lymphocytes. It has been proposed that, upon binding MHC class II molecules expressed on APC, the CD4 molecule enhances the responsiveness of the T cell by increasing intercellular avidity and/or by transducing an intracellular signal. We have analyzed the effect of removing the cytoplasmic domain of the CD4 molecule on the ability of the CD4 molecule to enhance T cell responsiveness. The cytoplasmic domain-deleted mutant of the CD4 molecule (CD4 delta) was found to be as efficient as the CD4 molecule at enhancing responsiveness to cells bearing the appropriate Ag. If subcellular Ag in the form of purified Ag incorporated into liposomes was used, the CD4 molecule was found to be much more efficient than the CD4 delta molecule at enhancing responsiveness. However, the defect in the ability of the CD4 delta molecule to enhance responsiveness could be compensated for by increasing the level of expression of the CD4 delta molecule.

Amino Acid Sequence↗
PubMed1987

Purification and characterization of class II histocompatibility antigens from a homozygous human B cell line.

Human class II histocompatibility antigens were purified from the Epstein-Barr virus-transformed human B lymphoblastoid cell line LG-2 by immunoaffinity chromatography. This is the first time all three subsets have been prepared as nonradioactive materials on a milligram scale. The yields of DR, DQ, and DP from 10 g of cells were approximately 12, 2, and 0.2 mg, respectively. Cross-contamination of the subsets was found to be less than 2% when assayed by measuring the binding of antigen-specific monoclonal antibodies to antigen immobilized on fixed erythrocytes. The three purified subsets were extensively characterized. They contained no detectable invariant chain. The three proteins were distinguished by their migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing. The denatured antigens were susceptible to partial removal of carbohydrate by endoglycosidase H and apparently complete removal of carbohydrate by endoglycosidase F. The isolated, denatured chains differed in their affinities for radiolabeled lectins, suggesting differences in carbohydrate structures. A water-soluble form of each antigen was prepared by a controlled papain digestion of the native antigen. Both native and denatured antigens were analyzed for their reactivities with a panel of class II antigen-specific monoclonal antibodies, allowing a precise definition of the specificities of the antibodies.

Alkylation↗
PubMed1986

The role of T cell accessory molecules in the generation of class II-specific xenogeneic cytolytic T cells.

In the generation of allogeneic, hapten-modified and virus-specific cytotoxic T cell (CTL) responses there is usually a requirement for T-T interaction between the T helper cell (TH) and the precursor CTL (CTL). We have investigated the role of a TH signal in the induction of a xenogeneic mouse antihuman CTL response by using membranes and liposomes bearing the xenogeneic antigen to stimulate primed responders. The TH signal can be achieved by either an Ia-restricted, L3T4+ DR-specific T cell or by the addition of nonspecific T helper factors(s). This signal is delivered to an Lyt-2+, L3T4-DR-specific CTL to generate active xenogeneic (xeno-) CTL. The roles of the T cell accessory molecules L3T4, Lyt-2, and LFA-1 in the generation of and target cell lysis by xeno-CTL are investigated.

Animals↗
PubMed1986

Immunochemically purified DR antigens in liposomes stimulate xenogeneic cytolytic T cells in secondary in vitro cultures.

A monoclonal antibody directed against the human class II major histocompatibility antigen DR was generated. Use of this antibody, LB3.1, allowed isolation of large amounts of highly purified DR by immunoaffinity chromatography. The DR was reconstituted into liposomes and shown to stimulate secondary xenogeneic cytolytic T lymphocytes (CTL) specific for targets expressing DR antigens. DR digested with neuraminidase was equally as effective as native DR at stimulating CTL, while denatured DR and other purified membrane proteins were much less effective. The DR liposome-induced CTL lysed only target cells expressing class II antigens. Cytolysis of targets bearing class II antigens was blocked by DR-specific antisera.

Animals↗
PubMed1986

Characterization of seven new monoclonal antibodies against human DR, DR + DP and DQ1 + DQ3 antigens.

Seven murine monoclonal antibodies were prepared that react with human class II antigens. Four of them (HL-39, MEM-12, MEM-24G, and MEM-32B: react with a monomorphic determinant dependent on association of heavy and light chains of DR antigens, two others (HL-38 and HL-40) recognize a monomorphic determinant localized on the light chains of DR and DP antigens. The antibody HL-37 is directed against a determinant present on DQ1 and DQ3, but not DQ2 molecules; at least in the case of DQ1, the epitope recognized is located on the light chain.

Animals↗
PubMed1985

Determination of anesthetic molecule environments by infrared spectroscopy. I. Effects of solvating molecule structure on nitrous oxide spectra.

The effects of the medium on the infrared spectrum of nitrous oxide (N2O) were determined in the antisymmetric stretch region near 2200 cm-1 for solutions of N2O in 38 different solvents at 25 degrees C. The solvents were chosen to reflect the variety of environments potentially available in sites occupied by N2O in nerve and other tissue. Band parameters of overlapping fundamental and hot bands were obtained with deconvolution techniques. Differences in solvent molecule structure had marked effects on both the frequency and the shape of the infrared bands. The fundamental band frequency (v3) ranged from 2215 cm-1 for carbon disulfide to 2230 cm-1 for water. Among the alcohols, v3 increased nearly linearly with increasing dielectric constant. However, solvent parameters that reflect bulk properties of the solvent did not correlate well with v3 over the entire range of solvents studied. Rather short-range specific solute-solvent molecular interactions appear particularly important. In general, v3 increases with the strength and number of dipoles in adjacent solvent molecules interacting with the vibrating dipole of N2O. Half-bandwidths (delta v1/2) ranged from 7.4 cm-1 for carbon tetrachloride to 14.4 cm-1 for hexane. Variations in bandwidth did not correlate in any direct way with solvent polarity, but delta v1/2 did increase with an increase in the conformational flexibility of the solvent molecule, which results in a greater diversity in the immediate environment about the N2O molecules. The observed sensitivity of the N2O infrared band parameters to changes in solvation environment and the appearance of the antisymmetric stretch band at a frequency within a window of relatively low-energy absorption by water makes infrared spectroscopy potentially useful for the characterization of the sites occupied by the anesthetic molecules within lipid, protein, and aqueous components of intact tissue.

Chloroform↗
PubMed1985

Determination of anesthetic molecule environments by infrared spectroscopy. II. Multiple sites for nitrous oxide in proteins, lipids, and brain tissue.

The presence of molecules of the general anesthetic nitrous oxide (N2O) in oils, esters, proteins, red cells, cream, lipid vesicles, and brain tissue upon exposure to the gas was observed by infrared spectroscopy. Analysis of the N-N-O antisymmetric stretch band reveals a distribution of N2O molecules among several sites of differing polarity in these solutions and tissues. The sensitivity of the band intensity and frequency to the number and strength of the dipoles in the solvating molecules is demonstrated by the resolution of N2O-ester and N2O-alkane interactions in acetic acid ethyl ester and oleic acid methyl ester. In all aqueous solutions and in all tissues a population of N2O molecules in water is observed. At least two sites of N2O-protein interaction are observed in purified hemoglobin A and packed red cells; multiple N2O sites may also be present in bovine serum albumin. Two sites of N2O-lipid interaction are observed in whipping cream and in an aqueous suspension of phosphatidylcholine vesicles. The sites providing the least polar immediate environment to N2O in hemoglobin, cream, and vesicles give similar band frequencies to those found in pure alkane solvents. Infrared spectra of bovine brain tissue, upon exposure to N2O, show N2O molecules present in water and in two less-polar environments. Analysis of spectra of N2O in cerebellum tissue removed from a dog under halothane-N2O anesthesia reveals, in addition to N2O in water, a single population of N2O molecules in an alkane-like environment. Infrared spectroscopy provides a unique means of probing the structure of the environment of N2O and should prove useful in correlating anesthetic potency with anesthetic environment under physiological conditions.

Animals↗
PubMed1984

One transporter per vesicle: determination of the basis of the insulin effect on glucose transport.

The basis for insulin stimulation of glucose transport in rat adipocytes has been investigated by determining the relative number of functional glucose transporters in the plasma and microsomal membranes from basal and insulin-treated cells. Each fraction was solubilized with cholate and then reconstituted into vesicles of about 500 A in diameter through removal of the cholate by dialysis. This procedure distributed the glucose transporters into the vesicles at a density of either one or none per vesicle. Consequently the fraction of the intravesicular volume that rapidly equilibrated with D-glucose provided an estimate of the relative number of functional transporters. By means of this one-transporter-per-vesicle method, it was found that insulin increased the number of transporters in the plasma membrane by a factor of 2.4 and decreased the number in the microsomes to 68% of the original value. These results provide independent evidence for the hypothesis that insulin causes the translocation of functional transporters from an intracellular location to the plasma membrane.

Adipose Tissue↗
PubMed1981

The monosaccharide transporter of the human erythrocyte. Transport activity upon reconstitution.

The transport function of the purified monosaccharide transporter from human erythrocytes has been investigated. By a cycle of cholate solubilization and removal, the purified transporter was incorporated into phospholipid vesicles of about 300 A diameter, at a density of about one per vesicle. This distribution permitted an all-or-none assay for transport activity, in which the fraction of the intravesicular volume that rapidly equilibrated with D-glucose in the medium yielded an estimate of the moles of protein functional in transport. It was found that almost every transporter molecule capable of binding cytochalasin B was also capable of transport. The rate of transporter-catalyzed exchange of D-glucose between the medium and the vesicles at equilibrium was also measured. In this assay, the transport activity of the purified protein, expressed per mol of cytochalasin B binding of site, was about 5% of that of the transporter in the intact erythrocyte under similar conditions. These results show that the reduced transport activity of the purified transporter found in various rate assays is due to most of the molecules functioning at a lower rate, rather than a few molecules functioning at the in vivo rate.

Biological Transport, Active↗
PubMedDate not supplied

Crystallization of HLA-DR antigens.

The class II major histocompatibility antigens HLA-DR1, DR2, DR3, DR4, DR7 and DR8 were purified by immunoaffinity chromatography from homozygous human B lymphoblastoid cell lines. The purified, detergent-soluble molecules were cleaved with the protease papain to remove the hydrophobic transmembrane regions and cytoplasmic tails. Crystals were obtained for each of the papain-solubilized fragments. DR1 crystallized under a variety of different conditions, resulting in two different orthorhombic crystal forms, one of which diffracts as far as 3.5A. Crystals of DR2, DR3, DR4 and DR8 have the same unit cell dimensions as the DR1 crystals, and crystals of DR3 and DR4 have the same diffracting power as the DR1 crystals. The best DR7 crystals obtained thus far are hexagonal and diffract to only about 8A. Crystals of similar hexagonal form have also been observed for most of the other DR subsets.

Alleles↗