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Biomedical subjects

L E Cannon

Publications and source records attributed to L E Cannon.

At least 19 recordsLinked to original sources
PubMed1989

Mouse antibody response to group A streptococcal carbohydrate.

In an attempt to more fully understand the generation of antibody diversity to carbohydrate (CHO) Ag, we produced and characterized a panel of hybridoma cell lines specific for group A streptococcal CHO from mice injected with the intact bacteria (minus the hyaluronic acid capsule and cell wall protein Ag). We have analyzed the use of H and L chain V region genes in the early (day 7) and late response (hyperimmune) and have sequenced the dominant VH gene used in several of our hybridomas. Our data allowed us to assess the extent to which the recombination of various V, D, and J gene segments and somatic mutation contribute to antibody diversification in this system. In this report we confirm that a minimum of two VH and four VK gene segments are used to encode this response. We extend this analysis to show that multiple D and J gene segments are used and that a significant amount of junctional variability is tolerated in CDR 3. Our results indicate that the level of somatic mutation in the hyperimmune response is generally low in comparison with the response to haptens and protein Ag. These data also suggest that there is a positive selection for mutation in CDR 1 during the hyperimmune response to group A streptococcal CHO.

Amino Acid Sequence↗
PubMed1984

Actin of Naegleria gruberi. Absence of N tau-methylhistidine.

Actin from amebae of Naegleria gruberi has been purified to homogeneity. The purified actin shares many attributes with numerous other actins that have been characterized, including molecular weight, strong binding to DEAE-cellulose, binding to DNase I, reversible polymerization to F-actin, binding of rabbit myosin subfragment 1 to give distinctive arrowheads , formation of Mg paracrystals, and activation of myosin Mg2+-ATPase. In two respects the attributes of Naegleria actin are unusual. Isoelectric focusing resolves three distinct isoforms of the actin, which raises questions about the function of multiple isoforms in a unicellular eukaryote. The amino acid composition closely resembles other actins except that Naegleria actin lacks N tau-methylhistidine. This result indicates that N tau-methylhistidine is not a prerequisite for actin-actin or actin-myosin interactions.

Actins↗
PubMed1983

Modulation of a neural antigen during metamorphosis in Drosophila melanogaster.

Major reorganization in the central nervous system (CNS) takes place during metamorphosis in holometabolan insects (G. Technau and M. Heisenberg, 1982, Nature (London) 295, 405-407. J. W. Truman and S. E. Reiss, 1976, Science 192, 477-479). In this report, two monoclonal antibodies specific for antigens localized in the CNS of Drosophila melanogaster are utilized to examine the behavior of these neuronal antigens during metamorphosis. One of the antigens studied is concentrated in synaptic areas of the CNS in adults and larvae. The other antigen is more ubiquitously distributed, being present in both cortical and synaptic regions. Metamorphosis is accompanied by dramatic change in the concentration of the antigen localized to synaptic areas. In contrast, the ubiquitously present antigen persists throughout.

Animals↗
PubMed1983

Rapid and sensitive procedure for assigning idiotypic determinants to heavy or light chains: application to idiotopes associated with the major cross-reactive idiotype of A/J anti-phenylarsonate antibodies.

A rapid and sensitive procedure is described for assigning idiotypic determinants to heavy or light polypeptide chains. Heavy and light chains are resolved by electrophoresis in the presence of sodium dodecyl sulfate. The electrophoretically resolved polypeptides are then transferred to nitrocellulose filters. Filters containing bound heavy and light chains are incubated with 125I-labelled anti-idiotypic antibody, and idiotype-anti-idiotype reactivity visualized by autoradiography. This procedure is illustrated with three monoclonal anti-idiotopic antibodies which recognize determinants associated with the major cross-reactive idiotype family of A/J anti-phenylarsonate antibodies. All three anti-idiotopic antibodies are shown to react with electrophoretically resolved idiotype heavy chain, but not with idiotype light chain.

Animals↗
PubMed1982

Sulfhydryl groups of native myosin and of the myosin heavy chains from Physarum polycephalum compared to vertebrate skeletal, smooth, and non-muscle myosins.

Although a previously reported analysis of Physarum myosin detected no cysteine residues in the molecule, the myosin ATPase activity was inhibited by p-chloromercuribenzoate. We have re-examined this apparently contradictory finding. We found highly purified plasmodial myosin to be very sensitive to N-ethylmaleimide inhibition of the K+, Ca2+ -activated ATPase. An estimate of the number of reactive sulfhydryls of the native myosin using Ellman's reagent showed only 1.5 mol 11 min-reactive sulfhydryl/mol as compared to 4.5 for chicken breast myosin in 5 min. 3H- and 14C-labelled N-ethylmaleimide was used to estimate the total sulfhydryls of the SDS-denatured heavy chains. Plasmodial myosin heavy chains bound 10-13% of the N-ethylmaleimide bound by chicken breast myosin heavy chains. Smooth muscle myosin heavy chains as well as heavy chains of embryonic chicken presumptive myoblasts had 65-70% of the reactive groups of chicken myotube myosin heavy chains. Amino acid analyses of purified Physarum myosin showed that some preparations contained cysteic acid residues even before performic oxidation. After the performic oxidation a mean value of 3 mol cysteic acid per 10(5) g Physarum myosin was found, or less than half that reported for striated muscle myosin. Our results show that in the sulfhydryl-poor plasmodial myosin each heavy chain contains at least two sulfhydryls, and probably more, but that there is variable oxidation of the total sulfhydryls. It has been reported that plasmodial myosin lacks rapidly reacting sulfhydryls groups when tested with an ATP analogue which reacts with light chains of vertebrate muscle myosins. Therefore, the 1-2 sulfhydryls of plasmodial myosin which react rapidly with Ellman's reagent appear to be on the heavy chain. Our results also suggest that during development of myotubes changes occur in the myosin heavy chains.

Amino Acids↗
PubMed1982

Presecretory and cytoplasmic invertase polypeptides encoded by distinct mRNAs derived from the same structural gene differ by a signal sequence.

Saccharomyces cerevisiae strain FH4C carries a single invertase structural gene, SUC2, but produces distinct invertase mRNAs and polypeptides for the secreted and cytoplasmic forms of the enzyme. The two major invertase cell-free translation products are polypeptides of 60,000 daltons (p60) and 62,000 daltons (p62) and correspond to the nonglycosylated cytoplasmic form of invertase and the precursor of glycosylated secreted invertase, respectively. This paper describes amino acid sequence and peptide map analyses of invertase polypeptides. The peptide maps demonstrate that p62, p60, and the in vivo secreted polypeptide have significant structural homology. Sequence analysis, however, revealed differences between p62 and p60 at their amino termini. p62 contains an amino-terminal signal sequence of 19 amino acid residues that is specifically cleaved during secretion in a cell-free system to generate the secreted 87,000-dalton invertase glycopeptide gp87. This signal sequence is not present in p60. p60 synthesis begins with a methionine which can be aligned with a methionine at residue 21 in p 62. During translation, the p60 initiator methionine is removed and the newly generated amino terminus is acetylated. Based on peptide map similarities, partial amino-terminal sequence data, and common genetic origin, it is suggested that p60 and p62 have identical amino acid sequences carboxy-terminal to the p60 initiator methionine (residue 21 of p62). The reciprocal correlations of signal sequence with secretion and absence of signal sequence with cytoplasmic localization provide proof of the signal hypothesis for secreted proteins. Two mechanisms are proposed for the derivation of p60 and p62 from a single structural gene: alternative promoter sites, and differential processing of a single primary transcript.

Amino Acid Sequence↗
PubMed1981

Unique V kappa group associated with two mouse L chain genetic markers.

The C.C58 and C.AKR congeneic strains of mice differ from BALB/c at loci on chromosome 6 which govern kappa light chain variable region (V kappa) polymorphisms and the Lyt-2 and Lyt-3 alloantigens. Amino acid sequence analysis of light chains of myelomas induced in these strains revealed one light chain, C.C58 M75, that had an NH2-terminal serine and differed sufficiently from published V kappa sequences to define a new V kappa group, V kappa (Ser), apparently not expressed by BALB/c mice. Peptide map analysis indicated that the M75 light chain contained the IB-peptide marker, a V kappa polymorphism expressed by C.C58 but not BALB/c mice, which is determined by the IgK-Trpa allele present on chromosome 6. This same light chain was found by isoelectric focussing to correspond to IgK-Ef1a, another V kappa genetic marker of C.C58 and C.AKR. Isoelectric focussing of approximately 200 C.C58 and C.AKR myeloma light chains revealed three additional C.C58 and four C.AKR light chains that corresponded to IgK-Ef1a-specific light chains. All three additional C.C58 light chains belonged to the V kappa (Ser) group and contained the IB-peptide marker. Thus, the differences in V kappa repertoires represented by the IB-peptide and IgK-Ef1a markers and controlled by genes on chromosome 6 appear to reflect expression (or failure of expression) of a distinct group of V kappa regions.

Amino Acid Sequence↗
PubMed1980

In vitro synthesis of repressible yeast acid phosphatase: identification of multiple mRNAs and products.

Antibodies to repressible nonspecific acid phosphatase [APase; orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2] purified from Saccharomyces cerevisiae were used to detect the in vitro products of APase mRNA. Immunoprecipitation of cell-free synthesized protein and of in vivo enzyme from cell extracts has shown that derepression of enzyme synthesis in situ is the result of de novo appearance of functional mRNA followed by de novo protein synthesis. At least three unique APase polypeptides are synthesized in vitro from separate mRNAs and appear to be glycosylated in vivo to form secreted enzyme.

Acid Phosphatase↗
PubMed1979

Cell-free translation of the mRNAs for the heavy and light chains of HLA-A and HLA-B antigens.

Cell-free translation of poly(A)-containing RNA from the JY lymphoblastoid cell line followed by immunoprecipitation has indicated the presence of mRNAs for both the heavy and light chains of HLA-A and HLA-B antigens in these preparations. Both chains are synthesized with an NH2-terminal extension, approximately 20 residues in length for the light chain, and 20 or 24 residues for the heavy chains. The precursors can be processed by dog pancreatic microsomes to products similar to those obtained in vivo. Immunoprecipitation of the cell-free products has been employed as an assay for partial purification of the mRNAs. Investigation of the Daudi cell line, which cannot synthesize the small subunit, beta 2-microglobin, has indicated that the heavy chains of HLA-A and HLA-B antigens are synthesized intracellularly in vivo and can also be translated from their cognate RNAs in vitro. The implications of these findings for biosynthesis of membrane proteins in general and multimeric membrane proteins in particular, as well as the role of beta 2-microglobulin in expression of HLA-A and HLA-B antigens, are discussed.

Amino Acid Sequence↗
PubMed1978

Light chain variable region sequence of rabbit antipneumococcal type III polysaccharide antibody 3368.

The amino acid sequence of the amino-terminal 111 residues (variable region) for the light chain of the homogeneous rabbit antipneumococcal type III polysaccharide antibody 3368 was determined. This sequence was obtained principally through automated Edmann degradations of the intact light chain and of peptides generated by tryptic digestion of the citraconylated light chain. With these methods only 2 mumol of purified light chain was required to determine the reported sequence. When compared with the light chains of four other antipneumococcal type III polysaccharide antibodies, the 3368 light chain exhibits a unique sequence in those segments of the variable region that contribute to formation of the antigen binding site (complementarity-determining regions) (10 or 11 residue differences in 12 positions). The 3368 light chain also demonstrates an insertion of three residues relative to the other four light chains in the complementarity-determining region at positions 89 to 98. These five light chains have greater than 80% sequence homology for the portion of the variable region which is not involved in antigen binding (framework).

Amino Acid Sequence↗
PubMed1977

The structural basis of rabbit VH allotypes: serologic studies on a1 H chains with defined amino acid sequence.

The amino acid sequences for the VH regions of three homogeneous antibodies elicited by type III pneumococcal vaccine were determined. All three antibodies had the group a allotype a1. Two of the antibody H chains (3372, 3381) had identical amino acid sequences in all framework positions that are considered correlates of the VH allotype, whereas the third H chain (3T72) differed from these at positions 15 and 16. The a1 allotypic specificities of the three homogeneous antibodies were compared by quantitative radiobinding and inhibition assays by using both insolubilized anti-a1 antisera and allotypic antiserum fractions rendered specific for the homogeneous antibody 3374. It was found that antibodies 3374 and 3381 are allotypically indistinguishable and have in common an a1 allotypic specificity that predominates in pooled a1 IgG. The allotypic specificity of the 3T72 antibody, on the other hand, was markedly deficient to those of 3374, 3381, and the a1 IgG pool. This correlation of allotypic difference with amino acid sequence variation at position 15 and 16 of the H chain indicates the involvement of these two residues in a major a1 allotypic determinant.

Amino Acid Sequence↗
PubMed1976

Diversity among rabbit antibody light chain amino terminal sequences.

The amino terminal (positions 0 to 20) amino acid sequences of 22 rabbit antibody light chains are reported and compared with 44 amino terminal sequences previously described in the literature. Rabbit k light chains demonstrate three different amino terminal chain lengths and considerable variability at the amino terminal end. In relating amino terminal sequence to b locus allotype, allotype-specific residues (amino acid residues unique to all light chains having a given allotype) were not identified, although certain residue alternatives were unique to some of the b5 and b9 light chains. There was no correlation of antibody specificity with amino terminal sequence. In the most striking example, identical amino terminal sequences were associated with two different carbohydrate specificities (type VIII pneumococcal polysaccharide and streptococcal group A carbohydrate) and an aminophenyltrimethylammonium specificity. An obligatory association of amino terminal sequence with complete variable region framework sequences was not observed. Thus, it is not always possible to predict the framework homology among a given pair of light chains by examination of their amino terminal sequences. The limited diversity seen among the variable regions of murine myelomas that have the same antigen-binding properties is not found in the light chains of specifically elicited rabbit antibodies.

Amino Acid Sequence↗
PubMed1976

Purification, specificity, and hypervariable region sequence of anti-pneumococcal polysaccharide antibodies elicited in a single rabbit.

Four homogeneous antibodies to type VIII pneumococcal polysaccharide (S8) were isolated from the serum of a single rabbit (3322) by affinity chromatography on an S8 immunoadsoebent by utilizing gradient elution with cellobiose and NaCl. The binding properties of these antibodies were determined by a radioimmunoassay with 125I-bovine gamma-globulin-S8. Cellobiose (a disaccharide unit of S8) was the immunodominant group of each of the four antibodies, but each antibody bound to this disaccharide with different relative affinities. The amino acid sequences (positions 0-40) of three of the four antibody light chains were each different both in framework and first hypervariable region sequences. The fourth antibody light chain has a blocked amino terminus. These findings indicate that antibodies elicited by a relatively simple antigen and examined at one time during the course of immunization in a single rabbit may exhibit common specificities for an oligosaccharide determinant, yet have different binding affinities for that determinant as well as different primary structures in the complementarity (hypervariable) regions and framework regions.

Amino Acid Sequence↗
PubMed1975

Diversity of light chain variable region sequences among rabbit antibodies elicited by the same antigens.

We report the complete variable region sequences of three homogeneous rabbit antibody light chains and the partial sequences of five others. Wehn these are compared to other published rabbit light chain sequences, two regions of markedly increased variability are revealed, which are homologous in position to the first and third hypervariable regions of murine and human myeloma light chains. In addition, there is increased variability among the first three residues at the aminoterminal end. A hypervariable region homologous to that identified at positions 50 to 56 in myeloma light chains is not present in these rabbit antibody light chains. The available three-dimensional models of Fab fragments based on x-ray crystallography indicate that neither the amino-terminal portion of the light chain nor the region homologous to positions 50 to 56 forms a part of the combining site. Comparison of the hypervariable regions among six light chains from antibodies to Type III pneumococcal polysaccharide and among four from antibodies to Type VIII pneumococcal polysaccharide suggests that a large number of different sequences may be found in antibodies specific for these relatively simple antigens. Certain residues outside of the hypervariable regions are invariant in the rabbit light chains and correspond to residues that are required for proper chain folding in human and murine myeloma light chains, indicating that the general conformation of myeloma light chains is the same as that of light chains of elicited antibodies.

Amino Acid Sequence↗