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J B Rothbard

Publications and source records attributed to J B Rothbard.

88 records · Page 5Linked to original sources
PubMed1987

Class I MHC molecules rather than other mouse genes dictate influenza epitope recognition by cytotoxic T cells.

Influenza nucleoprotein (NP) is an important target antigen for influenza A virus cross-reactive cytotoxic T cells (Tc). Here we examine the NP epitope recognized by cloned and polyclonal BALB/c Tc and the genetics of this recognition pattern. We can define NP residues 147-161 as the epitope seen in conjunction with Kd, the only H-2d class I responder allele for NP restriction. H-2d/H-2b F1 mice (C57BL X DBA/2) primed by influenza infection lyse only H-2d target cells treated with peptide 147-161 while H-2b targets are recognized only after treatment with NP residues 365-379 (previously found to be recognized by Db restricted Tc cells). Tc cell recognition of NP peptide 147-161 is entirely dictated by expression of Kd and not by other B10 or C3H background genes of congenic mice. Restriction of a unique NP sequence by each responder class I major histocompatibility complex (MHC) allele suggests that antigen and class I MHC interact for Tc recognition.

Animals↗
PubMed1986

Amino acid residues 56 to 69 of HLA-A2 specify an antigenic determinant shared by HLA-A2 and HLA-B17.

The mouse monoclonal antibody MA2.1 was previously used to define an epitope shared by native HLA-A2 and HLA-B17 molecules and amino acid sequence comparison of nine HLA-A,B,C molecules identified residues 62 to 65 as the region most likely to form this epitope. An unabsorbed rabbit antiserum raised against a peptide corresponding to residues 56 to 69 of HLA-A2 gives highly specific reactions with HLA-A2 and HLA-B17 heavy chains in Western blots. No interactions with native HLA-A2 and B17 molecules were detected in a variety of assays. Although the topographic relationship between the epitopes recognized by the rabbit antiserum and the monoclonal antibody could not be determined, the results show that residues 56 to 69 of HLA-A2 can form epitopes with specificity for HLA-A2 and HLA-B17.

Amino Acid Sequence↗
PubMed1985

Expression of the L11 neuropeptide gene in the Aplysia central nervous system.

Neuron L11 in the abdominal ganglion of Aplysia californica is thought to be both cholinergic and peptidergic. In previous studies, we isolated a cDNA clone encoding the precursor for an L11 secreted protein(s) by differentially screening an abdominal ganglion cDNA library. We now report the isolation of genomic clones encoding the L11 cDNA sequences. Analysis of these clones reveals that the gene is present in a single copy per haploid genome. RNA blotting and cDNA cloning demonstrate that the L11 gene is expressed not only in the abdominal ganglion but in the head ganglia as well. To define the positions of cells expressing this gene and to follow their processes, we raised antibodies to synthetic peptides defined by the cDNA sequence. Histochemistry revealed about 100 neurons containing immunoreactive material. These cells arborize in the neuropil and are distributed throughout the central nervous system, representing about 0.5% of the Aplysia central neurons. In addition, cells in the abdominal ganglion send processes to the mantle floor at the base of the gill via the genital and branchial nerves. Our data suggest that this network of cells expresses the single L11 peptide gene.

Animals↗
PubMed1985

Syngeneic antiidiotypic immune responses to a B cell lymphoma. Comparison between heavy chain hypervariable region peptides and intact Ig as immunogens.

The nucleic acid sequence of the heavy chain variable region (VH) expressed by 38C13, a B cell tumor of C3H origin, was determined by a combination of direct (messenger RNA) mRNA sequencing by primer extension and complementary DNA (cDNA) isolation and sequencing in M13. The VH amino acid sequence was deduced, and hypervariable regions were identified. From an analysis of predicted secondary structure, regions of predicted antigenicity were chosen, and a series of synthetic peptides corresponding to CDR2 and CDR3 (complementarity-determining region) were produced. These peptides were coupled to protein carriers and used to immunize syngeneic C3H mice. All peptides gave rise to a vigorous antibody response. However, only the CDR3 peptides induced antibodies that crossreacted with the isolated H chain protein. Only one CDR3 peptide induced antibody-producing clones, isolated as hybridomas, that reacted with the intact IgM protein. However, the appearance of these clones was a low-frequency event. All antibodies reacting with the H chain or the intact IgM protein were idiotypically specific for 38C13. These monoclonal antiidiotype (anti-Id) antibodies, raised against CDR3 peptides, gave strong reactions in enzyme-linked immunosorbent assays and immunoblots, but they were of low affinity compared to syngeneic anti-Id raised against the intact IgM protein. Moreover, while the intact IgM was capable of inducing tumor immunity, the CDR peptides were not able to do so.

Animals↗
PubMed1985

Antibodies to peptides corresponding to a conserved sequence of gonococcal pilins block bacterial adhesion.

Antisera generated against each of seven synthetic peptides corresponding to constant and variable sequences of the pilin from gonococcal strain MS11 were assayed for their ability to crossreact with intact pili from both homologous and heterologous strains. The peptides elicited roughly equal antipeptide responses but varied substantially in their ability to elicit antisera that crossreacted with intact pili. Of the antisera to peptides corresponding to regions of conserved sequence, antisera directed against residues 69-84 were the most efficient in binding pili from all strains tested in both solid-phase assays and immunoblots. Anti-69-84 also efficiently precipitated a tryptic fragment of pilin known to bind human endocervical cells. Sera against the two peptides (121-134 and 135-151) previously shown to contain strain-specific epitopes crossreacted with MS11 pili equally well, but differed in their ability to bind pili from heterologous strains. Anti-121-134 was strain-specific whereas anti-135-151 bound all pilin tested. Each of the sera was examined for its ability to inhibit bacterial adhesion to a human endometrial carcinoma cell line. Sera generated against residues 41-50 and 69-84 successfully inhibited a heterologous gonococcal strain from binding. These peptides could be important components of an effective vaccine for the prevention of gonorrhea.

Adhesiveness↗
PubMed1984

All eight unassigned reading frames of mouse mitochondrial DNA are expressed.

Animal mitochondrial DNA contains genes for 13 potential polypeptides of significant size. Five of these genes have been assigned to distinct proteins and eight remained unassigned reading frames (URFs). Short peptides corresponding to URF protein sequences were synthesized chemically. Antibodies raised to these synthetic peptides were used to establish the existence of all eight URF proteins in mouse tissues and cells by the complementary techniques of immunoperoxidase staining, protein blotting and immunoprecipitation. Immunoperoxidase staining of thin-sectioned, freeze-substituted tissue may prove generally useful for the identification of gene products for which no formal genetic data exist. Furthermore, the ability to determine the cellular and tissue distribution of such proteins may provide the first insight into their function.

Adenosine Triphosphatases↗
PubMed1984

Antigenic analysis of gonococcal pili using monoclonal antibodies.

A bank of mouse monoclonal antibodies has been produced with reactivity to gonococcal pili to investigate epitopes of the pilus structural protein, pilin. Pili of Neisseria gonorrhoeae strains R10 and MS11 were used as immunogens to elicit 19 monoclonal antibodies reactive with the homologous pili type in ELISA. Of the 19 antibodies, 16 demonstrated type-specific reactivity and 3 were cross-reactive with heterologous pili. Reactivity of the antibodies with the carboxyterminal, cyanogen bromide fragment (CB-3) of R10 pilin allowed their classification into three groups. The first group (10 antibodies) were R10 specific and equally reactive with the R10 CB-3 fragment. The second group (6) were also type specific but demonstrated poor reactivity with the CB-3 fragment. This suggested that the epitopes of the first group are linear, and those of the second group, nonlinear. The third group (3), consisting of the cross-reactive antibodies, were not reactive with the CB-3 fragment. Two of the antibodies in group 3 were examined in detail to localize their epitopes. The epitope of one, 9B9/H5, was shown to be a linear determinant. This antibody was reactive with a fragment of MS11 pilin (residues 31-111) and to a synthetic peptide representing residues 69-84 in MS11 pilin. The epitope was more finely mapped, with shorter synthetic peptides conjugated to bovine serum albumin, to an eight amino acid segment (residues 69-76). The epitope of 1E8/G8, a strongly reactive antibody, proved elusive to this type of analysis and probably results from conformational restraints. The significance of species-specific epitopes in the pilin protein is discussed.

Amino Acid Sequence↗
PubMed1984

Strain-specific and common epitopes of gonococcal pili.

The antigenic structure of gonococcal pilin, strain MS11 (Tr), was investigated by assaying the binding of antisera engendered by intact pili from strains MS11 and R10 and their two major cyanogen bromide-generated fragments, CNBr-2 (residues 9-92) and CNBr-2 (residues 93-159), to synthetic peptides corresponding to the amino acid sequence of MS11 pilin. Four peptides were synthesized corresponding to regions of sequence variation between MS11 and R10 gonococcal pilin. Antisera against the homologous pilus filament and against its CNBr-3 fragment bind peptides equivalent to residues 121-134 and 135-151, which comprise the 30 amino acid disulfide loop near the carboxyl terminus of the protein. Heterologous pili antisera did not bind these peptides. Absorption studies proved that each peptide contained an independent, strain-specific epitope. Synthetic peptides corresponding to regions of identical sequence between MS11 and R10 pilin were used in similar binding experiments to localize a weakly immunogenic, common determinant between residues 48 and 60. less than 15% of the antibodies raised against intact pili were directed at this site. Antisera raised against MS11 or R10 CNBr-2 bind a separate peptide, residues 69-80. This region is immunogenic only as a fragment, not in the intact pilus filament.

Amino Acid Sequence↗
PubMed1984

Antibodies to synthetic peptides defined by cDNA cloning reveal a network of peptidergic neurons in Aplysia.

We previously isolated and characterized a cDNA clone specifically expressed in neurons R3 to R8 and R14 of the Aplysia abdominal ganglion (Nambu, J.R., R. Taussig, A.C. Mahon, and R.H. Scheller (1983) Cell 35: 47-56). The cDNA nucleotide sequence and the inferred protein amino acid sequence suggest that this gene encodes the precursor for neuroactive peptides used by these cells. Peptides corresponding to three regions of the precursor were synthesized, coupled to a protein carrier, and used to generate antibodies. These antibodies stain a set of cell bodies, R3 to R14, and their processes in the abdominal ganglion; no other cells in the nervous system or the periphery are immunoreactive. R3 to R14 send numerous fine immunoreactive processes into the vascularized sheath that surrounds the ganglion. Each of these cells also has a large axon which exits the ganglion via the branchial nerve and terminates on the heart. In addition, R14 is anatomically distinct from R3 to R13 in that it sends additional immunoreactive processes to the vasculature near the ganglion. Immunoreactive processes and varicosities were observed on the efferent vein of the gill, the abdominal ganglion artery, and the anterior aorta. These data are consistent with previous studies suggesting that one or more neuropeptides released from R3 to R14 function as modulators of cardiovascular physiology.

Abdomen↗
PubMed1980

Structure of the heavy chain of the H-2Kk histocompatibility antigen.

We have used radiochemical techniques to characterize the heavy chain (Mr 46,000) of the murine H-2Kk histocompatibility antigen in terms of six fragments (I-VI) obtained after cleavage of the polypeptide chain with CNBr. The tentative order of the fragments, which account for more than 90% of the heavy chain, was assigned by radiochemical sequence analysis of the intact heavy chain and of each purified CNBr fragment and by analysis of the CNBr fragments obtained from the large papain fragment of the heavy chain. Treatment of cells with tunicamycin yielded H-2 molecules with heavy chains of molecular weight 40,000, suggesting that the carbohydrate moieties have a combined molecular weight of approximately 6000. CNBr cleavage of H-2Kk heavy chains labeled with [3H]fucose indicated that the carbohydrate moieties are located on fragments II and IV.. Incubation of cells with 32PO4 gave H-2 molecules with radioactive phosphoserine in the carboxyl-terminal CNBr fragment (VI) of the heavy chain and in the fraction containing beta 2-microglobulin. Sequence analysis of each CNBr fragment intrinsically labeled with 3H- and 35S-labeled amino acids identified a total of 87 residues in the H-2Kk heavy chain. The sequence closely resembles that of the H-2Kb molecule, and the 11 differences are scattered throughout the polypeptide chain. Comparison with HLA sequences indicates that the two allelic H-2 sequences are more closely related to each other (88% identity) than either is to the HLA-B7 or A2 antigens (approximately 70%). Similarly, the nonallelic HLA antigens are more closely related to each other (83%) than either is to the H-2Kk or H-2Kb molecules.

Amino Acid Sequence↗
PubMedDate not supplied

T-cell epitope of the autoantigen myelin basic protein that induces encephalomyelitis.

Chronic relapsing paralysis and demyelination within the central nervous system (CNS), features associated with the human disease multiple sclerosis (MS), develop in mice after injection of murine T-cell clones specific for the autoantigen myelin basic protein (MBP). We examined the fine specificity of three independently derived encephalitogenic T-cell clones using synthetic polypeptides derived from portions of the N-terminal sequence of MBP. These clones appear functionally identical; they all respond to an epitope in the N-terminal nine amino acid residues in association with the same class II (I-A) molecules of the major histocompatibility complex (MHC). Both the N-terminal acetyl moiety and the first residue (Ala) are necessary for recognition. Only N-terminal MBP peptides recognized by these clones were found to cause encephalomyelitis (EAE) in vivo. These results show that the N-terminal MBP-specific T lymphocytes that mediate autoimmune encephalomyelitis are a small population with a limited repertoire; they all recognise the same combination of MHC and target.

Animals↗
PubMedDate not supplied

Identification of mycobacterial antigens recognized by T lymphocytes.

The development of vaccines capable of inducing protective immunity against mycobacterial infection depends in part on the identification of antigenic determinants that activate T cells with antimycobacterial effector function. Various approaches designed to analyze the recognition of mycobacterial antigens by T cells are reviewed. In addition to the established approach of using serologically defined antigens, alternative methods independent of antibody preselection, such as polyacrylamide gel electrophoresis-fractionated immunoblots of mycobacteria, can be used to probe the specificity of the T cell repertoire. Furthermore, the application of recombinant DNA expression combined with that of synthetic peptides whose sequences are predicted to constitute T cell determinants allow the localization of T cell epitopes within a protein. The use of these techniques in defining potentially "pathogenic and protective" T cell epitopes in mycobacteria is discussed.

Antigens, Bacterial↗