Search PubMed⌕ Search

Biomedical subjects

P Parham

Publications and source records attributed to P Parham.

At least 307 records · Page 17Linked to original sources
PubMed1985

Exon shuffling in vivo can generate novel HLA class I molecules.

The human class I histocompatibility molecule HLA-Aw69 has serological and structural properties which suggested it was a hybrid of the allelic products HLA-A2 and HLA-Aw68. We have now isolated three genes for HLA-Aw69 and one gene for HLA-Aw68. The sequences of exons encoding the entire extracellular portion of the molecule and of intron 2 have been determined. Their comparison with the published sequence of HLA-A2 proves that HLA-Aw69 is a hybrid molecule with complete identity to HLA-Aw68 in the alpha 1 domain and with HLA-A2 in the alpha 2 and alpha 3 domains. This comparison also localised regions involved in the epitopes recognised by monoclonal antibodies. The three HLA-Aw69 genes obtained from unrelated individuals of diverse ethnic backgrounds are identical. All results are consistent with HLA-Aw69 having arisen by a single reciprocal recombination event between the HLA-Aw68 and HLA-A2 genes somewhere in a region of 86 bp about the 3' donor splice site of exon 2. Estimates of the silent mutation rate in HLA genes suggest this event occurred not more than 330 000 years ago. Intra-allelic reciprocal recombination thus represents a further mechanism in addition to gene conversion for the generation of novel class I histocompatibility alleles.

Alleles↗
PubMed1985

In vitro production of a hybrid monoclonal antibody that preferentially binds to cells that express both HLA-A2 and HLA-B7.

A hybrid mouse monoclonal IgGl having one low affinity combining site for HLA-A2 and one low affinity combining site for HLA-B7 was made by the chemical method of Nisonoff and Palmer (Science 143:376,1964). This involved selective reduction of interchain disulphides, a splitting of the IgGl into half molecules at low pH and ionic strength, and reassociation of the half molecules by neutralization. Serologically active hybrids were separated from parental IgGl by an absorbtion procedure and recovered in about 10% yield. The hybrid discriminated between cells that express either HLA-A2 or HLA-B7 from cells that express both A2 and B7. This is because it could bind bivalently to the cell with both A2 and B7 but could only bind with a single combining site to cells expressing A2 or B7. The consequence of these different modes of attachment was to give up to sevenfold greater binding to the cell expressing A2 and B7 in comparison to the cell expressing only A2 or B7.

Animals↗
PubMed1985

Monoclonal antibodies against seven sites on the head and tail of Dictyostelium myosin.

Ten monoclonal antibodies (My1-10) against Dictyostelium discoideum myosin were prepared and characterized. Nine bound to the 210-kD heavy chain and one (My8) bound to the 18-kD light chain. They defined six topographically distinct antigenic sites of the heavy chain. Five binding sites (the My1, My5, My10 site, and the My2, My3, My4, and My9 sites) are located on the rod portion of the myosin molecule. The position of the sixth site (the My6 and My7 site) is less certain, but it appears to be near the junction of the globular heads and the rod. Three of the antibodies (My2, My3, and My6) bound to myosin filaments in solution and could be sedimented in stoichiometric amounts with the filamentous myosin. In contrast, My4, which recognized a site on the rod, inhibited the polymerization of monomeric myosin into filaments. A single antibody (My6) affected the actin-activated ATPase of myosin. The nature of the effect depended on the valency of the antibody and the myosin. Bivalent IgG and F(ab')2 fragments of My6 inhibited the actin-activated ATPase of filamentous myosin by 50% whereas univalent Fab' fragments increased the activity by 50%. The actin-activated ATPase activity of the soluble chymotryptic fragment of myosin was increased 80-90% by both F(ab')2 and Fab' of My6.

Adenosine Triphosphatases↗
PubMed1985

Site-specific inhibition of myosin-mediated motility in vitro by monoclonal antibodies.

Monoclonal antibodies directed against seven different sites on Dictyostelium myosin (Peltz, G., J. A. Spudich, and P. Parham, 1985, J. Cell Biol., 100: 1016-1023) were tested for their ability to inhibit movement of myosin in vitro, using the Nitella-based myosin-mediated bead movement assay (Sheetz, M. P., R. Chasan, and J. A. Spudich, 1984, J. Cell Biol., 99: 1867-1871). To complement this functional assay, we located the binding sites of these antibodies by electron microscopy, using the rotary shadowing technique. One antibody bound to the 18,000-dalton light chain and inhibited movement completely. All of the remaining antibodies bound to various positions along the rod portion of the myosin molecule, which is approximately 1,800 A long. Antibodies that bound to the rod about 470, 680, and 1400 A from the head-tail junction did not alter myosin movement. One antibody appeared to bind very close to the head-tail junction and to inhibit movement 50%. Surprisingly, three antibodies that bound about 1,200 A from the head-tail junction inhibited movement completely. This inhibition did not depend on using intact IgG, since Fab' fragments had the same effect.

Antibodies, Monoclonal↗
PubMed1984

The binding of monoclonal antibodies to cell surface molecules. Quantitative analysis of the reactions and cross-reactions of an antibody (MB40.3) with four HLA-B molecules.

The MB40.3 monoclonal antibody binds to four distinct HLA-B molecules; B7, B40, B40*, and B27. With Fab' fragments only the interaction with B7 and B40 was detected and the affinity for both was the same (1-2 X 10(8) M-1) suggesting the epitope is shared by the two molecules. Unlike many antibodies for which low affinity is due to a high-dissociation constant, that of MB40.3 results from a very low-association rate constant, coupled with a low-dissociation constant. In consequence, the affinity and avidity of Fab', F(ab')2, and IgG for B7 and B40 were found to be of a similar magnitude, soluble B7 was a more efficient competitor for antibody than cell surface B7 and in practice antibody bivalency was of little importance. The forward rate constant could be increased by removing Fc from the antibody or by removing sialic acid from the cells by treatment with neuraminidase. The neuraminidase treatment also produced an increase in the number of detectable cell surface HLA-A,B molecules. The affinity of MB40.3 for B40* and B27 was estimated to be less than 4 X 10(6) as no binding with Fab' was detected due to a high-dissociation rate. For these two HLA-B molecules bivalent attachment was critical, and it increased the strength of interaction with cell surface B40* and B27 to a point where the avidities were comparable to those obtained with B7 and B40, with B40* interacting more strongly than B27. The epitopes recognized by MB40.3 on B40* and B27 were thus shown to be structurally different from each other and from those on B7 and B40. The properties of this antibody contrast with those of other anti-HLA-A,B we have studied (Ways, J.P., and Parham, P. (1983) Biochem. J. 216, 423-432).

Antibodies, Monoclonal↗
PubMed1984

Comparison of the primary structures of clathrin light chains from bovine brain and adrenal gland by peptide mapping.

The structures of the polymorphic forms of clathrin light chains were analyzed by two peptide mapping procedures. Comparison of the products of partial digestion by V8 protease showed no common peptides between LCA and LCB from bovine brain. No similarities between clathrin light chains and tropomyosin chains from bovine brain and skeletal muscle were detected with this technique. The peptides produced by complete tryptic digestion of LCA and LCB from bovine brain and bovine adrenal gland were analyzed by reverse phase h.p.l.c. For both LCA and LCB the polypeptides from different tissues showed considerable homology. LCA from brain and adrenal gland shared 10 out of a total of 15 peptides. LCB from brain and adrenal gland shared 10 out of 14 peptides. In contrast, when LCA was compared with the LCB chain from the same tissue very few peptides were shared; 4/23 for brain and 3/21 for adrenal gland. These results strongly indicate that, within a tissue, LCB is not related to LCA by post-translational processing and that each chain is encoded by a separate gene. The data also demonstrate the close homology of the different forms of LCA and LCB expressed in different tissues within the same organism. Thus the polymorphic differences of clathrin light chains within a tissue are greater than those between tissues.

Adrenal Glands↗
PubMed1984

Molecular characterization of HLA-A28*, a novel HLA product, and its relationship to HLA-A28 and HLA-A2.

The HLA-A28* molecule expressed by the B-cell line IDF is serologically distinct and intermediate between HLA-A28 and HLA-A2. Comparative tryptic peptide mapping of biosynthetically labeled HLA-A28*, A28, and A2 molecules showed that HLA-A28* is also chemically distinct. Reverse-phase high pressure liquid chromatographic analysis of tryptic peptides labeled with 3H-arginine and 3H-lysine revealed that A28*, A28, and A2 share approximately 65% of their tryptic peptides. Multiple differences were observed between A28* and both A28 and A2. No peptides unique to A28* were detected and 25 peptides were shared with both A28 and A2. These results show that A28* is a novel HLA product that is closely related to A28 and A2. Tryptic peptide map comparisons of these molecules labeled separately with 11 amino acids confirm these results. The data suggest that HLA-A28* may have arisen from a genetic exchange event involving HLA-A28 and -A2. These data are consistent with the hypothesis that A28* is identical with A28 in the first extracellular domain (alpha 1) and identical with A2 in the second domain (alpha 2).

Antibodies, Monoclonal↗
PubMed1984

Monoclonal antibodies prepared against Dictyostelium actin: characterization and interactions with actin.

Three mouse monoclonal antibodies, Act I, Act II, and Act IV, against actin from the cellular slime mold Dictyostelium discoideum, have been made and characterized. All three antibodies are IgG1 and share the following properties: They form stable complexes with monomeric Dictyostelium actin, which prevents polymerization of the actin into filaments. On addition to preformed actin filaments, they cause a reduction in filament size and in the viscosity of the actin solution. They cross-react strongly with actins from the lower eucaryotes Physarum and Acanthamoeba, but not with alpha-actins from rabbit and human muscle or beta- and gamma-actins from human erythrocytes and a human B lymphoid cell line. Act II and Act IV recognize a similar antigenic determinant that is topographically distinct from that identified by Act I. In protein immunoblotting, only Act I bound strongly to Dictyostelium actin. Analysis of actin fragments with this technique showed that amino acids 13 to about 50 are required for Act I binding to actin. A comparison of the amino acid sequences of actins from lower eucaryotes and higher vertebrates implicates threonine 41 as a critical residue in the Act I antigenic site. The properties of Act II and Act IV suggest that they recognize antigenic sites involving the NH2-terminal six residues.

Actins↗
PubMed1984

Changes in conformation with loss of alloantigenic determinants of a histocompatibility antigen (HLA-B7) induced by monoclonal antibodies.

Two conformations of HLA-B7 have been characterized. In one conformation B7 has epitopes that are: specific to B7, shared with B27, shared with B40, and common to many HLA-B locus products. In the second conformation the B7-specific and B7/B27 cross-reactive epitopes are effectively lost and an additional epitope that is shared with B40 has been acquired. The conformation of papain-solubilized and cell surface B7 can be reversibly changed by monoclonal antibodies against the appropriate epitopes. Antibody bivalency is not an important factor in these changes. In the second conformation B7 is antigenically indistinguishable from B40. It is suggested that conformational changes of this magnitude could contribute to the adaptability of histocompatibility antigens in forming functional complexes with many pairs of T cell receptors and foreign antigens.

Animals↗
PubMed1983

The binding of monoclonal antibodies to cell-surface molecules. A quantitative analysis with immunoglobulin G against two alloantigenic determinants of the human transplantation antigen HLA-A2.

Monoclonal IgG1 (immunoglobulin G1) PA2.1 and MA2.1 antibodies recognize polymorphic sites of the human transplantation antigen HLA-A2. They are distinguishable because MA2.1 binds HLA-A2 and HLA-B17, whereas PA2.1 binds HLA-A2 and HLA-A28. The affinities of PA2.1-Fab for HLA-A2, three HLA-A2 variants and HLA-A28 are similar and relatively low (1.9 X 10(7) M-1). The affinities of MA2.1-Fab for HLA-A2, three HLA-A2 variants and HLA-B17 are similar and high (1.2 X 10(9) M-1). The difference in affinity is due to the rates of dissociation, which give half-times of dissociation of 290 min for MA2.1-Fab and 4 min for PA2.1-Fab. For both Fab, equilibrium measurements and kinetic determinations gave consistent estimates for affinity. When PA2.1-F(ab)2 or IgG is incubated with cells it reaches equilibrium within 3 h, with most molecules bound bivalently to the cell. Under similar conditions, MA2.1-F(ab)2 does not reach equilibrium and a significant proportion of molecules bound with one and two sites are found. For the lower-affinity antibody (PA2.1), estimates of the binding constants for one- and two-site interactions could be made. By simple Scatchard analysis the avidity of F(ab)2 or IgG is 1.3 X 10(9) M-1, giving an enhancement factor of 68 between bivalent and univalent binding. This is a measure of the equilibrium constant for the interchange between bivalent and univalent binding. Analysis of the results with more realistic models indicates that the actual value is larger (10(3)-10(4) M-1) than 68 M-1. The avidities of F(ab)2 and IgG for HLA-A2 are identical, showing the Fc does not interfere with bivalent binding to cells.

Antibodies, Monoclonal↗
PubMed1983

Polymorphism in clathrin light chains from different tissues.

Two types of light chains were observed in clathrins from different bovine tissues. Clathrin was purified from seven different bovine tissues using immunoaffinity columns prepared from a monoclonal antibody specific for the heavier light chain (LCa) of bovine brain clathrin. The LCa equivalent in clathrin from non-brain tissue was identified by monoclonal antibody binding to electrophoretic blots of these clathrins. The LCa and LCb light chains of bovine brain clathrin are, respectively, 4 X 10(3) daltons and 3 X 10(3) daltons heavier than their corresponding light chains in other tissues. The light chains from tissues other than brain are similar in molecular weight.

Animals↗
PubMed1983

Arginine 45 is a major part of the antigenic determinant of human beta 2-microglobulin recognized by mouse monoclonal antibody BBM.1.

Monoclonal antibody BBM.1 (Brodsky, F.M., Bodmer, W. F., and Parham, P. (1979) Eur. J. Immunol. 9, 536-545) identifies an antigenic determinant of human beta 2-microglobulin (beta 2-M). The antibody binds free and HLA-A,B-associated beta 2-M with similar affinity, showing that the BBM.1 antigenic determinant does not involve residues of beta 2-M that interact with HLA-A,B heavy chains. Peptides (SWH.1-5) synthesized from residues 35-50 of the beta 2-M sequence specifically inhibit the binding of BBM.1 to cell surfaces. Their inhibitory activity is destroyed by trypsin treatment. The observations (i) that BBM.1 does not bind to beta 2-M of species other than man, gorilla, and chimpanzee and (ii) that arginine 45 is the only human-specific residue between positions 35 and 50 suggested that this residue might be part of the BBM.1 antigenic determinant. This hypothesis was confirmed by reversible modification of arginine residues with cyclohexanedione. Modification of arginines in native beta 2-M and of the single arginine, corresponding to position 45, in the peptide SWH.5 resulted in up to 95% loss of BBM.1 inhibitory activity. Reversal of the modification by treatment with hydroxylamine resulted in complete recovery of activity. Rabbit antibodies elicited by immunization of SWH.5 conjugated to bovine serum albumin showed no detectable reaction with native beta 2-M but did specifically react with human beta 2-M after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoresis onto nitrocellulose. These results thus identify a region around residue 45 of the beta 2-M polypeptide which is exposed to the environment and not involved in binding HLA-A,B heavy chain. Analysis of the beta 2-M sequence by calculating local hydrophilicity indices (Hopp, T. P., and Woods, K. R. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 3824-3828) agree with this region being a major antigenic determinant. Models of beta 2-M structure as an immunoglobulin domain show this region of polypeptide to be part of a loop between the two layers of beta-pleated sheet, also consistent with it being a major antigenic determinant. The position of the loop favors a model in which beta 2-M interacts with HLA across the four-stranded beta-pleated sheet like an immunoglobulin constant region domain.

Antibodies, Monoclonal↗
PubMed1983

Enhancement of monoclonal antibodies against HLA-A2 is due to antibody bivalency.

Enhancement between two monoclonal antibodies directed against the same antigen is when binding of one antibody, the enhancing antibody, increases the measured binding of the other antibody. This phenomenon has been observed for monoclonal antibodies against a variety of molecules including histocompatibility antigens. The mechanism of enhancement for monoclonal antibodies against HLA-A2 has been studied with purified preparations of IgG, F(ab')2 and Fab. Enhancement was only observed between antibodies against different antigenic sites. It requires bivalency of both antibody species and involves formation of stable, cyclic, tetramolecular complexes consisting of two antigen molecules and one each of the two antibody molecules. Conformational changes of either antigen or antibody do not appear to be important in this system, nor is the formation of combinatorial determinants between antigen and enhancing antibody. The enhancing properties of the antibodies studied are adequately explained in terms of their affinities and association and dissociation constants.

Antibodies, Monoclonal↗
PubMed1983

Antigenic determinants of the HLA-B7 molecule; Bw6- and B7-specific determinants are spatially separate.

Monoclonal antibodies that bind HLA-B7 were used to show that the B7-specific determinant is at a topologically different site from that of the broad polymorphic, Bw6 determinant. The relationship to other antigenic determinants defined by monoclonal antibodies was also assessed. These results were independently obtained in four ways: (1) by cellular blocking assays, in which there was no inhibition of 125I-B7 antibody binding in the presence of Bw6 antibody and no inhibition of 125I-Bw6 antibody binding in the presence of B7 antibody; (2) cellular binding assays under conditions of antibody saturation showed the binding of B7-specific and Bw6 antibodies were additive; (3) solid-phase radioimmune assays demonstrated enhancement between B7-specific and Bw6 antibodies; (4) analysis of antigen antibody complexes by size-exclusion high pressure liquid chromatography showed Bw6 and B7 antibodies could form tetramolecular complexes with papain-solubilized HLA-B7. Limitations were encountered in using cellular blocking assays to map antigenic determinants of HLA-B7. These assays can produce blocking in cases where two antibodies are not competing for an antigenic determinant. Mapping antigenic determinants with assays using purified HLA-B7 as the antigenic target, in addition to cell-based assays, provided a more accurate picture.

Antibodies, Monoclonal↗
PubMed1983

Isolation of heavy chain class switch variants of a monoclonal anti-DC1 hybridoma cell line: effective conversion of noncytotoxic IgG1 antibodies to cytotoxic IgG2 antibodies.

Spontaneously arising class switch variants of the Genox 3.53 hybridoma cell line were isolated. They secrete IgG2a or IgG2b monoclonal antibodies of anti-DC1 specificity identical to that of the IgG1 secreting parental cell. In contrast to the parental monoclonal antibody, those secreted by the variants are cytotoxic to peripheral blood B lymphocytes of DC1 positive individuals and are thus compatible with existing HLA typing techniques. This provides a general method for converting noncytotoxic anti-HLA antibodies into cytotoxic typing reagents.

Animals↗