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Biomedical subjects

L F Boyd

Publications and source records attributed to L F Boyd.

36 records · Page 2Linked to original sources
PubMed1992

Solution binding of an antigenic peptide to a major histocompatibility complex class I molecule and the role of beta 2-microglobulin.

The major histocompatibility complex-encoded class I molecule, a noncovalent dimer of a polymorphic 45-kDa heavy chain and a nonpolymorphic 12-kDa beta 2-microglobulin (beta 2m) light chain, binds peptide antigen prior to its interaction with T-cell antigen receptors. We report here that the binding in aqueous solution at 37 degrees C of a soluble purified murine major histocompatibility complex class I protein, H-2Lds (a soluble analogue of H-2Ld consisting of the alpha 1 and alpha 2 domains of H-2Ld, the alpha 3 domain and the C terminus of Q10b), to an antigenic peptide is controlled by the light-chain subunit beta 2m. Analysis of the equilibrium binding data favors a model in which two classes of peptide binding sites exist, the high-affinity class having an equilibrium constant for dissociation, KH, of 3.7 x 10(-7) M and accounting for 12% of the theoretically available sites. Studies of binding in the presence of excess beta 2m indicate that this increases the concentration of available high-affinity sites. These data are consistent with a ternary model in which high-affinity sites are generated by the interaction of beta 2m with the peptide-binding class I heavy chain.

Amino Acid Sequence↗
PubMed1991

Excess beta 2 microglobulin promoting functional peptide association with purified soluble class I MHC molecules.

T lymphocytes expressing alpha beta receptors recognize antigenic peptide fragments bound to major histocompatibility complex class I or class II molecules present on the surface membranes of other cells. Peptide fragments are present in the two available HLA crystal structures and recent data indicate that peptide is required for the stable folding of the class I heavy chain and maintenance of its association with the class I light chain, beta 2-microglobulin (beta 2m), at physiological temperature. To explain how the exogenous peptide used to create targets for cytotoxic cells bearing CD8 antigen could associate with apparently peptide-filled extracellular class I molecules, we hypothesized that stable binding of exogenous peptide to mature class I molecules reflects either the replacement of previously bound peptide during the well documented beta 2m exchange process or the loading of 'empty' class I heavy chains dependent on the availability of excess beta 2m. In either case, free beta 2m should enhance peptide/class I binding. Using either isolated soluble class I molecules or living cells, we show here that free purified beta 2m markedly augments the generation of antigenic complexes capable of T-cell stimulation.

Cell-Free System↗
PubMed1990

The extracellular domains of MHC class II molecules determine their processing requirements for antigen presentation.

We have evaluated the relative contributions of the extracellular and cytoplasmic domains of MHC class II molecules in determining the Ag-processing requirements for class II-restricted Ag presentation to T cells. Hybrid genes were constructed to encode a heterodimeric I-Ak molecule in which the extracellular portion of the molecule resembled wild type I-Ak but where the connecting stalk, transmembrane and cytoplasmic domains of both the alpha- and beta-chain were derived from the class I molecule H-2Dd. Mutant I-Ak molecules were expressed as heterodimeric membrane glycoproteins reactive with mAb specific for wild type I-Ak. Fibroblast and B lymphoma cells expressing either wild type or mutant I-Ak molecules were able to process and present hen egg lysozyme (HEL) and conalbumin to Ag-specific, I-Ak-restricted, T cell hybridomas or clones. The mutant-expressing cells presented native and peptide Ag less efficiently than the wild type-expressing cells, suggesting that the disparity in presentation efficiency was not due to a difference in Ag processing. CD4 interaction was intact on the mutant I-Ak molecules. Presentation of native Ag by mutant and wild type-I-Ak-expressing cells was abolished by preincubation with chloroquine, or after paraformaldehyde fixation. After transfection of a cDNA encoding the gene for HEL, neither mutant nor wild type-I-Ak-expressing cells presented endogenously synthesized HEL to a specific T hybrid. Newly synthesized mutant I-Ak molecules were associated with invariant chain. These data demonstrate the ability of hybrid class II molecules to associate intracellularly with invariant chain and degraded foreign Ag in a conventional class II-restricted processing pathway indicating that the extracellular domains of class II molecules play a dominant role in controlling these Ag-processing requirements.

Animals↗
PubMed1988

T cell activation by purified, soluble, class I MHC molecules. Requirement for polyvalency.

To examine the nature of the interaction of the TCR with the MHC class I Ag, we have studied the stimulation requirements of an H-2Dd-reactive T cell hybridoma, using a homogeneous, purified preparation of a molecularly engineered soluble counterpart of the class I Ag, H-2Dd/Q10b. We demonstrate that this monovalent, soluble MHC Ag is incapable of stimulating the release of IL-2 from this T cell hybridoma. However, the same preparation of the purified protein can elicit a dose-dependent response when made multivalent either by covalent coupling to soluble, high m.w. dextran or to agarose beads, or by adsorption to polystyrene tissue culture plates.

Animals↗
PubMed1986

Alternative processing of H-2Dd pre-mRNAs results in membrane expression of differentially phosphorylated protein products.

Two distinct mRNA species encoding the mouse major histocompatibility antigen H-2Dd have been identified in BALB/c spleen cells as well as in cultured cell lines expressing this cell surface glycoprotein. The alternate transcripts of H-2Dd arise from either removal or inclusion of exon VII (encoding I2) during pre-mRNA processing. The relative levels of each kind of H-2Dd transcript varied considerably between different cell types, and in all cells examined both forms of alloantigen were expressed on the cell membrane. Antigen derived from both types of transcript reacted with H-2Dd-specific monoclonal antibodies, whereas only protein lacking the 13 amino acids of I2 reacted with a specific antiserum raised against a predicted exon VI/VIII fusion peptide. Those H-2Dd proteins translated from full length, but not smaller, transcripts were phosphorylated in resting and phorbol myristate acetate-stimulated BALB/c spleen cells, suggesting that the major site of in vivo phosphorylation is within the highly conserved sequence encoded by exon VII. Thus alternative splicing of pre-mRNA transcripts is a mechanism which leads to membrane expression of two forms of H-2Dd, one of which lacks a major site of phosphorylation.

Animals↗
PubMed1986

Genetic engineering of an H-2Dd/Q10b chimeric histocompatibility antigen: purification of soluble protein from transformant cell supernatants.

We have constructed a recombinant class I gene in which 5' sequences of H-2Dd are linked to the 3' half of a Qa subregion gene, Q10b. This hybrid gene would be expected to direct the synthesis of a protein containing the N and C1 domains of H-2Dd covalently linked to the C2 domain of the secreted, nonpolymorphic, Q10b antigen. Following DNA-mediated gene transfer into mouse L cells, transformants were analyzed by radiolabeling and immunoprecipitation. These cells secreted a molecule reactive with anti-H-2Dd monoclonal antibodies that identify epitopes on the N and C1 domains as well as with an anti-Q10 carboxyl-terminal peptide antiserum. The H-2Dd-derived antigen is associated with beta 2-microglobulin and is readily purified in milligram amounts from culture supernatants by immunoaffinity chromatography.

Animals↗
PubMed1985

Isolation of tryptic peptides of myelin basic protein by reversed-phase high-performance liquid chromatography.

A reversed-phase high-performance liquid chromatography (HPLC) system was developed to obtain individual tryptic peptides of myelin basic protein (BP). Because of the similar charge and hydrophobicity of some of the tryptic peptides of the whole protein, several of these were not clearly separated by a single HPLC system. Therefore, the BP was first cleaved specifically between residues 97 and 98 with thrombin, and the two resulting fragments were separated by ion-exchange chromatography. When the thrombic fragments were digested with trypsin separately and subjected to HPLC, all of the peptides were satisfactorily separated. Elution times of all of the tryptic peptides of human BP were established. Differences among homologous peptides, derived from different mammalian BPs, were readily detected from their elution patterns inasmuch as a change in a single amino acid residue was usually sufficient to cause a shift in the retention time of the peptide. An amino acid difference detected by a peak shift could be confirmed by amino acid analysis. The technique has been used to isolate short peptides of rabbit, monkey, porcine, bovine, and human BP for sequence analysis.

Amino Acids↗
PubMed1984

Enzymatic and nonenzymatic degradation of myelin basic protein.

A procedure for large scale isolation of myelin basic protein (BP) has been modified to insure BP preparations free of neutral proteinase activity. Fractions were monitored by electrophoretic analysis of BP solutions incubated under various conditions of temperature and pH. Maximum degradation of human BP prepared by the old batch procedure occurs at pH 7, approximately 47 degrees C. BP preparations obtained by the new procedure, as well as BP preparations purified by CM-cellulose chromatography, are stable under these conditions. The latter, however, do undergo significant breakdown at pH 9, 100 degrees C. The results suggest that the degradation observed under these conditions is non-enzymatic in nature.

Humans↗
PubMed1975

Temporal changes in tectal cell surface specificity induced by nerve growth factor.

The change in cell surface adhesive specificity previously shown to occur between day 7 and 8 of development in the chick optic tectum ]Gottlieb et al. (1974), Proc. Nat. Acad. Sci. USA 71, 1800-1802] can be induced in rotating cultures of tectal cells by the addition of 10(-7) M mouse submaxillary gland nerve growth factor. Insulin, proinsulin, dexamethasone, and performic-acid-oxidized nerve growth factor are individually inactive in this system, but nerve growth factor in which the three tryptophan residues/subunit have been oxidized with N-bromosuccinimide is active. Thus, the specificity of this system for nerve growth factor is different than that observed with embryonic dorsal root or sympathetic ganglia, where the oxidized tryptophan derivative is inactive in stimulating neurite production. It is possible that in this system nerve growth factor serves as an analog of another specific trophic factor, presumably structurally related to nerve growth factor, may be active at much lower concentrations.

Age Factors↗
PubMed1973

Interaction of nerve growth factor with surface membranes: biological competence of insolubilized nerve growth factor.

Nerve growth factor was insolubilized by covalent attachment to Sepharose beads. Nerve growth factor-Sepharose was biologically active in both the neurite outgrowth assay for nerve growth factor and in preserving responsive neurons in vitro. Modification of the bioassay to detect solubilized activity of nerve growth factor and histological examination of ganglia treated with nerve growth factor-Sepharose revealed that nerve growth factor-Sepharose prepared by reaction in 6 M guanidine hydrochloride released negligible amounts of solubilized nerve growth factor activity. These observations extend the previously noted correlations on the structure and function of nerve growth factor and insulin to include the primary action of these two proteins. Thus nerve growth factor, like insulin, appears to express its biological activity by first binding to a receptor on the surface membrane of responsive cells.

Animals↗
PubMed1973

Mechanism of action of nerve growth factor and cyclic AMP on neurite outgrowth in embryonic chick sensory ganglia: demonstration of independent pathways of stimulation.

The suggested role of adenosine 3':5'-cyclic monophosphate as the "second messenger" in the neurite outgrowth from chick embryonic sensory ganglia mediated by nerve growth factor was examined. Although N(6),O(2)-dibutyryl adenosine 3':5'-cyclic monophosphate induces fiber outgrowth at concentrations of 1-5 mM, this response is morphologically distinct from that produced by nerve growth factor, is pH dependent, is mimicked by sodium butyrate, and does not occur in sympathetic ganglia. In addition, nerve growth factor does not alter the amounts of intracellular cyclic AMP during incubations up to 24 hr and does not stimulate adenylate cyclase in broken-cell preparations. Addition of theophylline, an inhibitor of phosphodiesterase, causes increases in intracellular levels of cyclic AMP but does not affect fiber outgrowth. These observations indicate that the nerve growth factor response is not mediated through cyclic AMp and that stimulation of sensory ganglia by exogenous cyclic AMP derivatives is probably of limited physiological significance. These findings are also compatible with the developing hypothesis, based on structural similarities, that nerve growth factor and insulin exert their effects on their respective responsive tissues by related mechanisms.

Adenylyl Cyclases↗