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Biomedical subjects

B D Stollar

Publications and source records attributed to B D Stollar.

At least 127 records · Page 7Linked to original sources
PubMed1983

Z-DNA-specific antibodies in human systemic lupus erythematosus.

Naturally occurring antibodies to left-handed Z-DNA have been shown to be present in the sera of human patients with systemic lupus erythematosus (SLE). These antibodies are of two types. One type reacts with both denatured DNA and Z-DNA. The other type is specific for Z-DNA and remained in the serum after removal of the cross-reactive antibody by extensive absorption on a denatured DNA affinity column. The antibodies appear to be specific for SLE and do not appear frequently in other rheumatic diseases. The presence of the antibody in SLE is correlated with the clinical manifestations of the disease, in parallel with antibodies to native and denatured DNA.

Antibody Specificity↗
PubMed1982

Monoclonal antibodies recognize different parts of Z-DNA.

Brominated poly(dG-dC) . poly(dG-dC), which forms a stable Z-DNA helix under physiological salt conditions, was used as an antigen to produce monoclonal antibodies from mice. The anti-Z-DNA antibodies were specific for the Z conformation as was shown by direct binding studies and a competitive solid phase assay. Competition could only be seen by brominated poly(dG-dC) . poly(dG-dC) under low and high salt conditions and unmodified poly(dG-dC) . poly(dG-dC) under high salt conditions. No binding could be seen to a number of other polynucleotides, including native and denatured DNA. Among the anti-Z-DNA monoclonal antibodies, examples differed in specificity: binding of one monoclonal antibody was blocked by methylation of cytosine; whereas the other antibody reacted equally well with the unmodified polymer or methyl cytosine-containing polymer in high ionic strength. The two antibodies are specific for different localized features of Z-DNA structure.

Animals↗
PubMed1982

Immunofluorescent characterization of DNA . RNA hybrids on polytene chromosomes of Trichosia pubescens (Diptera, sciaridae).

We have studied the distribution of DNA X RNA hybrids on polytene chromosomes with the aid of a goat antibody against DNA X RNA hybrids using the immunofluorescence technique. Fixed polytene chromosomes of the sciarid Trichosia pubescens (Diptera) show distinct, stage-specific labelling patterns throughout larval development. Controls for the staining procedure - including preincubation with hybrid-specific endoribonuclease H - prove that DNA X RNA hybrids are present on fixed chromosomes. They are revealed only under mild fixation conditions which do not efficiently immobilize all chromosomal proteins, indicating that some proteins have to be removed to make the antigens accessible to antibody. Certain fixation conditions may also cause local denaturation of chromosomal DNA, and some hybrids may possibly form during specimen preparation. After incorporation of radioactive uridine, a combination of phase contrast, fluorescent, and autoradiographic images of one and the same chromosomal preparation demonstrates that hybrid fluorescence is confined to transcriptionally active regions. Two puff classes can be distinguished. The first binds antibody and includes most RNA puffs and all DNA puffs so far studied; the second, comprising some RNA puffs, does not show bright fluorescence in spite of the fact that RNA synthesis is high as revealed by 3H-uridine incorporation. DNA X RNA hybrids are not found at DNA puff sites during the DNA amplification period; these sites contain detectable hybrids only when transcription is taking place. - Combination of the fluorescent technique with its excellent resolution and autoradiography should be helpful in studying detailed topological aspects of transcriptionally active chromosomal regions.

Animals↗
PubMed1982

Negatively supercoiled plasmids contain left-handed Z-DNA segments as detected by specific antibody binding.

Negative superhelical coiling of covalently closed DNA plasmids facilitates the formation of left-handed Z-DNA segments. This was demonstrated by binding of antibodies specific for Z-DNA. Plasmid pBR322 and two derivatives from it, pLP32 and pLP014, carrying inserts of alternating CG sequences of 32 bp and 14 bp, respectively, were used. Longer inserts required less negative superhelical density to induce the B-Z transitions. Antibody binding to supercoiled plasmids was also visualized by electron microscopy. Cross-linking of the antibody to the negatively supercoiled plasmid and restriction of the DNA with restriction endonucleases demonstrated that the antibodies combine with the CG insert in pLP32. For pBR322, however, evidence suggests that the antibody combines with a section of DNA containing 14 bases with alternating purine and pyrimidine residues with one residue out of alternation: CACGGGTGCGCATG. These cross-linking studies provide evidence for the binding specificity of the anti-Z-DNA antibodies. On the basis of experimental findings, we have calculated the changes in free energy associated with B-Z transitions in superhelical plasmids.

Antibodies↗
PubMed1982

Comparison of poly(A).poly(dT) and poly(I).poly(dC) as immunogens for the induction of antibodies to RNA-DNA hybrids.

A goat immunized with poly(A).poly(dT) produced three distinct antibody populations. The major one was specific for RNA-DNA hybrids and was purified from precipitates made with poly(I).poly(dC). It also reacted with hybrids of mixed base composition made with E. coli RNA polymerase. The other populations were purified with poly(A).poly(U) or poly(dT). Three rabbits also produced mainly hybrid-specific antibody in response to poly(A) . poly(dT). A goat immunized with poly(I).poly(dC) formed antibodies reactive with poly(I) and others reactive with poly(dC) but none specific for hybrid structure. Three rabbits did not respond to poly(I).poly(dC). Measurements of reactions with anti-inosine sera, thermal denaturation and sensitivity to S1 nuclease indicated that poly(I).poly(dC) is a less stable helix than poly(A).poly(dT) or poly(I).poly(C). Poly(A).poly(dT) is the more suitable synthetic immunogen for the production of hybrid-specific antibodies.

Animals↗
PubMed1982

Inhibitory monoclonal antibody to calf thymus RNA polymerase II blocks formation of enzyme-DNA complexes.

Monoclonal antibodies were prepared against purified calf thymus RNA polymerase II (RNA nucleotidyltransferase, EC 2.7.7.6). Two distinct antibodies were identified on the basis of their ability to bind RNA polymerase II in a solid-phase assay and to remove enzyme activity from solution in complexes precipitated by anti-immunoglobulin antibodies. One monoclonal antibody directly inhibited enzyme activity in solution. Detailed study of the mechanism of enzyme inhibition by this antibody revealed that it prevented the formation of enzyme-DNA complexes and had no effect on chain elongation. Inhibition of DNA binding and catalytic activity depended on preincubation of antibody with enzyme and was irreversible. Without preincubation, the enzyme readily bound to DNA in the presence of antibody, indicating that the binding of the free enzyme to DNA is faster than that of the antibody to the enzyme. In a competitive immunoassay enzyme-DNA complexes were much less reactive with antibody than was free enzyme, suggesting that DNA either blocks the antigenic site or alters the enzyme's conformation, preventing antibody recognition. These antibodies may serve as useful reagents for analysis of RNA polymerase structure and function.

Animals↗
PubMed1982

Production of autoantibodies by human-human hybridomas.

Peripheral blood lymphocytes and splenocytes of patients with autoimmune disease were used to prepare human-human hybridomas that produce autoantibodies. Because exogenous immunization was not used, the hybridoma antibodies were derived from B cells that spontaneously produced autoantibodies. 108 hybrids grew from 4,254 wells (2.5%). Optimal conditions for obtaining hybridomas with the GM 4672 myeloma line included initial growth in 2-ml wells, the use of 44% polyethylene glycol, a mononuclear cell/GM 4672 cell ratio 5:1, and prior stimulation of the B lymphocytes with pokeweed mitogen. Hybridoma supernatants had activity against ssDNA, platelets, and erythrocytes. The results demonstrate the feasibility of producing human-human hybridomas from lymphocytes of patients with various autoimmune diseases.

Anemia, Hemolytic, Autoimmune↗
PubMed1982

A high frequency idiotypic marker of anti-DNA autoantibodies in MRL-Ipr/Ipr mice.

Anti-idiotypic antibodies were used to analyze relatedness of anti-DNA autoantibodies of MRL mice. The antiidiotypic antibodies, produced by immunization of rabbits with MRL-Ipr-derived hybridoma anti-DNA antibodies, were defined by their ability to inhibit specifically the binding of hybridoma anti-DNA antibody to DNA. One monoclonal anti-DNA antibody, H130, cross-reacted with two thirds of other hybridoma anti-DNA antibodies. The H130/anti-H130 system detected high amounts (up to 5 mg/ml) of immunoreactivity in sera of MRL-Ipr mice, whereas levels in MRL-+ sera were relatively low. The H130 family increased 10-fold in MRL-Ipr mice over the course of 5 mo, but its levels did not correlate chronologically with those of anti-DNA antibodies, cardiolipin-binding antibodies, or total serum immunoglobulins. H130 cross-reactivity was found, by quantitative immunoadsorption, in 40 to 60% of serum anti-DNA antibodies; however, a large excess of H130-related serum antibodies did not bind to DNA. The H130 family and anti-DNA antibodies therefore seem to constitute overlapping populations.

Aging↗
PubMed1981

Antibodies to left-handed Z-DNA bind to interband regions of Drosophila polytene chromosomes.

Antibodies which are specific to the Z-DNA conformation have been purified and characterized on the basis of their binding to three different DNA polymers which can form this left-handed helix. These antibodies bind specifically to polytene chromosomes of Drosophila melanogaster as visualized by fluorescent staining. The staining is found in the interband regions and its intensity varies among different interbands in a reproducible manner. This is the first identification of the Z-DNA conformation in material of biological origin.

Animals↗
PubMed1981

Serological detection of homologies of H1o with H5 and H1 histones.

Immunospecifically purified anti-chicken H5 antibodies caused bright staining of mouse liver nuclei in an indirect immunofluorescence assay. Analysis with the fluorescence-activated cell sorter showed that 95% of the nuclei were in the brightest category. Histones extracted from mouse liver nuclei, analyzed by sodium dodecyl sulfate-gel electrophoresis, presented an extra band with the same mobility as chick erythrocyte H5. Electrophoretic blots on nitrocellulose paper were treated with anti-H5 or anti-H1 antibodies and iodinated protein A. The affinity purified anti-H5 bound to chicken H5 and mouse H1o fractions only. Anti-calf H1 bound to calf, chicken, and mouse H1 proteins and to mouse H1o as well. Antibodies to purified subfractions of rat thymus H1 showed binding to both H1 and H1o. The results support the suggestions that H1o bears significant structural homology with H5 and H1.

Animals↗
PubMed1981

Immunological and organizational heterogeneity of histone H2a variants within chromatin of cells at different stages of Friend leukemia.

We have used antibodies directed against two histone H2a variants, H2a.1 and H2a.2, to probe chromatin structure in Friend erythroleukemia cells. Each molecule has at least one unique antigenic determinant, as well as determinants shared by both variants. Some Friend leukemia cell types contained H2a molecules which showed altered immunologic reactivity with the two antisera. The accessibility of the H2a variants in chromatin to anti-H2a antibody was different as measured by the use of whole chromatin as an immunoabsorbent and by binding of antibody to nucleosomes in a solid phase radioimmunoassay. While anti-H2a.1 IgG bound to chromatin, anti-H2a.2 IgG did not. Moreover, anti-H2a.1 IgG binding to chromatin from different Friend cell types reflected, in general, the relative amounts of H2a.1 in total chromatin. The different reactivity of the two antisera with chromatin was also observed with isolated nucleosomes: anti-H2a.1 IgG bound but anti-H2a.2 did not. Furthermore, the binding of anti-H2a.1 Ig with subfractions of nucleosomes varied; H1-depleted, high mobility group-enriched nucleosomes reacted better than H1-containing, high mobility group-depleted nucleosomes. These findings demonstrated a heterogeneity in the organization of H2a variants in chromatin within nucleosomal subfractions of chromatin and among chromatin of different Friend leukemia cell types. Moreover, most of the antigenic determinants common to both H2a variants were shown to be buried within the nucleosome core; only H2a.1-unique determinants were accessible to an anti-H2a.1 IgG molecule.

Animals↗
PubMed1981

Polyspecific monoclonal lupus autoantibodies reactive with both polynucleotides and phospholipids.

Hybridomas the produce anti-DNA autoantibodies were prepared from spleen cells of unimmunized MRL/1 mice, a strain that spontaneously develops severe systemic lupus erythematous (SLE). Reactivities of these monoclonal antibodies with a wide range of polynucleotides prompted tests of their reactions with phospholipids which, like polynucleotides, contain diester-linked phosphate groups in their backbones. In competitive radioimmunoassays, cardiolipin, phosphatidic acid, and phosphatidyl glycerol blocked the binding of these hybridoma antibodies to denatured DNA. These phospholipids also specifically inhibited the reaction between a hybridoma antibody and a site-specific anti-idiotypic antibody. The antinuclear reaction of one of these antibodies was specifically inhibited by cardiolipin. This same antibody prolonged the activated partial thromboplastin time in a manner characteristic of a lupus anticoagulant, presumably by binding to phospholipid in the test system. The polyspecific reactivity of a single molecular species of lupus autoantibody suggests that some of the diverse serological abnormalities of SLE may be a result of the binding of certain autoantibodies to a phosphodiester-containing epitope that is present in diverse biological molecules.

Animals↗
PubMed1981

Antibodies specific for left-handed Z-DNA.

We prepared a brominated poly(dG-dC).poly(dG-dC) which forms a stable Z-DNA helix under physiological salt conditions. Rabbits and mice were immunized with brominated and unbrominated poly(dG-dC).poly(dG-dC) complexed with methylated bovine serum albumin. Antibodies specific for Z-DNA were produced. These antibodies were found not only in the sera of animals immunized with the low-salt stabilized Z-DNA [Br-poly(dG-dC).poly(dG-dC)] but also in sera from animals immunized with the unbrominated B-DNA form of the polymer. From this it is inferred that the unbrominated poly(dG-dC).poly(dG-dC) was partially converted to Z-DNA by its combination with the basic protein methylated bovine serum albumin. In addition to specific anti-Z-DNA antibody populations, two other interesting types of antibody populations were found. One of these reacted with both the Z and B forms of poly(dG-dC).poly(dG-dC). This antibody may be converting the polymer from the B-DNA to the Z-DNA form. The other type of antibody was specific for a B form of poly(dG-dC).poly(dG-dC) and did not react at all with the Z form. The antibodies raised to Z-DNA were shown to be highly specific for Z-DNA and did not react with B-DNA, RNA, DNA.RNA hybrids, or a number of other polynucleotides. This specificity for Z-DNA will make possible their use as reagents for determining the presence of Z-DNA in biological systems. Sera of autoimmune lupus mice were also shown to have a considerable amount of naturally occurring anti-Z-DNA antibody activity.

Animals↗
PubMed1981

Differing accessibility in chromatin of the antigenic sites of regions 1-58 and 63-125 of histone H2B.

Experiments with antibodies induced by separated fragments 1-58 and 63-125 of H2B histone indicated that the 1-58 portion of the molecule is much more accessible in chromatin than is the 63-125 region. In immunoabsorption and immunoelectron microscopic assays with bovine and chicken chromatins, anti-1-58 antibodies reacted with sheared or unsheared chromatin both at low ionic strength (1 mM Tris-HCl) and in 0.14 M NaCl. Anti-63-125 antibodies were bound only weakly by chromatin at low ionic strength and not at all in 0.14 M NaCl. Antibodies to whole H2B showed intermediate reactivity with chromatin in both assays. In tests of immunofluorescence with unfixed calf liver nuclei in suspension, anti-1-58 caused nucleolar as well as nucleoplasmic fluorescence, whereas anti-63-125 did not lead to detectable fluorescence; anti-H2B showed intermediate staining intensity. In control experiments, anti-H1 antibody was bound by chromatin at low ionic strength but not in 0.14 M NaCl; anti-H3 antibody was bound poorly under either condition.

Amino Acid Sequence↗