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B D Stollar

Publications and source records attributed to B D Stollar.

At least 73 records · Page 4Linked to original sources
PubMed1989

Amino acid sequence of a platelet-binding human anti-DNA monoclonal autoantibody.

The complete amino acid sequences of the variable regions of the heavy and light chains of a human IgM monoclonal platelet-binding autoantibody have been determined. This antibody, HF2-1/17, produced by a human x human hybridoma prepared from lymphocytes of a patient with systemic lupus erythematosus and thrombocytopenia, is polyreactive with single-stranded DNA, synthetic polynucleotides, sulfated carbohydrates, and acidic glycolipids isolated from platelet membranes. The heavy chain is of the VHIII subgroup, and the light chain is of the VKI subgroup. The heavy chain is the expression product of the VH26 germline gene. The light chain bears significant homology to other immunoglobulins of known primary structure, including WEA, GAL, HAU, HK101, and DEE. These results suggest that HF2-1/17 may be an autoantibody derived with little or no modification from germline genes. A model of the antibody combining site suggests that arginine 24 and arginine 30 in the light chain (CDR1) interact with a surface defined by phosphate or sulfate groups of the antigen.

Amino Acid Sequence↗
PubMed1988

Immunochemical detection of multiple conformations within a 36 base pair oligonucleotide.

A 36 base pair chimeric oligonucleotide containing a central core of DNA duplex flanked by RNA/DNA hybrid at each end was synthesized. These distinct regions of the oligonucleotide adopt different conformations which were detected with antibody probes. Enzyme linked immunosorbent assays (ELISA) and a gel electrophoresis retardation assay were used to demonstrate the binding of antibodies which recognize B-DNA, Z-DNA and RNA/DNA hybrid. The DNA duplex core of this oligonucleotide adopts the B-conformation in 0.14 M NaCl. In high salt solution (4 M NaCl) the DNA core adopts the Z-conformation. The RNA/DNA hybrid at the ends of the oligomer adopt a conformation which is distinct from both B-DNA and A-RNA.

Antibodies, Monoclonal↗
PubMed1988

Nucleic acid-binding specificity and idiotypic expression of canine anti-DNA antibodies.

Serum samples collected from eleven lupus dogs during an active phase of the disease all bound native and denatured DNA, poly(dT), poly(I) and poly(G). Nine bound poly(C); 10 bound poly(U); and 3 bound poly(A). Sera from 22 normal dogs were negative with all of these antigens. The canine sera were also probed for the presence of three idiotypic markers, one related to human lupus anti-DNA antibodies and two related to murine lupus antibodies. One canine lupus serum expressed idiotopes related to murine anti-DNA idiotype (Id) termed H130: (a) the canine serum bound to anti-H130 anti-Id; (b) it inhibited the binding of anti-H130 Id to its homologous Id; and (c) the anti-H130 Id inhibited the binding of the canine serum to DNA. These findings suggest that anti-DNA variable regions exhibit interspecies similarities, probably reflecting the conservation of the encoding gene segments through evolution.

Animals↗
PubMed1988

Disease activity in systemic lupus erythematosus related to a range of antibodies binding DNA and synthetic polynucleotides.

Antibodies to dDNA, nDNA, Z-DNA, poly(dT), poly(I), poly(dG.dC), poly(dA.dT), and total IgG and IgM were measured in five serial bleeds from 39 patients with systemic lupus erythematosus (SLE). The main findings were that those patients with renal disease form a distinct subset whose antibody levels correlate well with disease activity; anti-poly(dT) antibodies showed the best overall correlation with disease activity; and discriminant functional analysis demonstrated a major improvement in correlation of disease activity with combinations of antibodies to dDNA/nDNA/Z-DNA/poly(dT) (generally 50% or more were correctly classified) than with dDNA or nDNA alone (generally less than 25% correct). Serum IgG (but not IgM) correlated significantly (p less than 0.01) with six antibodies, suggesting that polyclonal activation plays a part in the development of these antibodies, though antibody cross reactivity is not excluded.

Antibodies↗
PubMed1987

The carboxyl-terminal domain of murine H1(0). Immunochemical and partial amino acid sequence comparisons with other H1(0)/H1/H5 histones.

The carboxyl-terminal domain of murine H1(0) histone was compared with that of human H1(0), bovine H1(0) and other H1 and H5 histones. Two sets of antibodies were induced by murine H1(0). One set reacted with only the carboxyl-terminal domain of murine H1(0) and preferred the murine over the bovine and human proteins. The second set of antibodies reacted with the globular domain of murine H1(0) and did not distinguish among murine, bovine and human H1(0) species. There were five positions in the first 60 residues of the carboxyl-terminal domain in which the murine H1(0) differed from the human H1(0). In this region, the murine H1(0) had no more than 49% overall homology with other H1 and H5 histones; however, short sequences in the domain were very similar to short sequences that occur in rabbit H1.3, trout H1 and goose or chicken H5. In comparisons based on these and other published data, the carboxyl-terminal domain of H1(0) is found to be more variable among species than is the globular domain; the first two-thirds of the H1(0) carboxyl-terminal domain is largely unique and does not show great overall homology with H1 or H5, whereas the last third is again more conserved. As the first two-thirds of the domain is the only portion where the homology with H5 is less than 50%, it may be responsible for functional differences between H1(0) and H5.

Amino Acid Sequence↗
PubMed1987

Relationship of human variable region heavy chain germ-line genes to genes encoding anti-DNA autoantibodies.

In order to identify the V region genes encoding systemic lupus erythematosus (SLE)-derived anti-DNA autoantibodies, we have determined the nucleotide sequence of heavy chain mRNA from several DNA-binding immunoglobulins secreted by human hybridomas. We used the technique of cDNA primer extension for determining sequences of the VH, D, and JH gene segments of anti-DNA autoantibodies from three different primary hybridoma growths from an SLE patient and one hybridoma from a leprosy patient. Immunoglobulins from two of the SLE hybridomas expressed the same idiotype, Id-16/6, which is also expressed on immunoglobulins in sera of patients with active SLE. Their mRNA sequences showed complete homology to each other in the V, D, and J genes and more than 99% homology to the VH26 germ-line gene sequence, a member of the human VHIII gene family. The VH mRNA sequence of the third SLE hybridoma, 21/28, which was idiotypically unrelated to the other two, was 93% homologous to a different VH germ-line gene sequence, HA2, a member of the human VHI gene family. The fourth anti-DNA-producing hybridoma, 8E10, was derived from a leprosy patient of different ethnic origin than the SLE patient. It was idiotypically related to 21/28 and expressed a VH segment gene identical to that of 21/28. Hybridomas 21/28 and 8E10 shared sequence homology with the VH26 anti-DNA antibodies in the first complementarity-determining region. In addition, 21/28 shared sequence homology with the Id-16/6+ group in the region encoded by the D and J gene segments. Our findings indicate that some SLE autoantibodies are encoded by unmodified or scarcely modified VH germ-line genes that are conserved in the human population and identify two distinct VH germ-line genes that can encode segments of anti-DNA immunoglobulins.

Antibodies, Antinuclear↗
PubMed1987

Identification of human anti-DNA, anti-RNP, anti-SM, and anti-SS-A serum antibodies bearing the cross-reactive 16/6 idiotype.

The human idiotype 16/6, characteristic of lupus-derived anti-DNA autoantibodies, was detected in sera of Japanese patients with autoimmune diseases. The 16/6-positive sera were from patients with active SLE and, in one case, a scleroderma-polymyositis overlap syndrome; all contained anti-ssDNA antibody. Affinity chromatography was used to isolate 16/6-bearing Ig from sera of seven patients having anti-DNA and other autoantibodies. In a preliminary step, rheumatoid factors and nonspecific binding were removed by four passes of the serum through a column of immobilized normal rabbit gamma-globulin. The absorbed sera were then applied to a column of immobilized specific rabbit anti-16/6 globulin. This combined procedure depleted anti-ssDNA activity selectively. From 44 to 60% of total Ig but only 6 to 25% of anti-ssDNA activity was recovered in the material that passed through both columns. Approximately 40% of the anti-RNP/Sm and anti-SS-A/SS-B activities were recovered, reflecting a lesser degree of selective depletion of these antibodies. The specific eluate from the anti-idiotype column contained more IgG than IgM; anti-DNA activity; anti-RNP, anti-Sm, or anti-SS-A activity. Soluble DNA inhibited the binding of one eluate to RNP/Sm but not of another eluate to SS-A/SS-B. The results confirm that the genes encoding the 16/6 idiotype are widely represented in ethnically different human populations, and indicate that 16/6-bearing Ig may bind to other autoantigens as well as to DNA.

Autoantibodies↗
PubMed1987

Detection of antibodies to total histones and their subfractions in systemic lupus erythematosus patients and their asymptomatic relatives.

Sera drawn from 75 patients with systemic lupus erythematosus, 141 healthy relatives (from the families of 51 patients), and 115 healthy control subjects were examined, by enzyme-linked immunosorbent assay, for IgG and IgM antibodies to total histones and their subfractions. Compared with the controls, statistically significant numbers of patients and their relatives had antihistone antibodies of both isotypes. Among the relatives, the sera from females, notably sisters of the patients, contained the highest levels of anti-total histone antibody. Anti-H2A/H2B and H3 antibodies were most prevalent among the lupus patients, but many of the relatives had IgM anti-H4 antibodies. These findings indicate that antihistone antibodies can serve as a genetic marker in patients with systemic lupus erythematosus.

Autoantibodies↗
PubMed1987

Immunochemical approaches to gene probe assays.

Antibodies specific for helical nucleic acids can be applied to assays for hybridized DNA and/or RNA. The assays can use either radioactive or nonradioactive detection systems. Antibodies specific for RNA-DNA hybrids are applicable to assays for measuring hybrid helices that are immobilized on plastic or nitrocellulose, whether the helices are preformed in solution or are formed on the solid-phase support. Alternatively, anti-RNA-DNA hybrid antibodies can be immobilized and used to capture hybrids formed in solution, resulting in an assay with a high signal-to-noise ratio. It has been applied to a test for the presence of ribosomal RNA of Campylobacter jejuni in biological samples.

Campylobacter↗
PubMed1987

Monoclonal antibodies to DNA modified with cis- or trans-diamminedichloroplatinum(II).

Murine monoclonal antibodies that bind selectively to adducts formed on DNA by the antitumor drug cis-diamminedichloroplatinum(II), cis-DDP, or to the chemotherapeutically inactive trans isomer trans-DDP were elicited by immunization with calf thymus DNA modified with either cis- or trans-DDP at ratios of bound platinum per nucleotide, (D/N)b, of 0.06-0.08. The binding of two monoclonal antibodies to cis-DDP-modified DNA was competitively inhibited (50% control) in an enzyme-linked immunosorbent assay (ELISA) by 4-6 nM concentrations (600-900 fmol) of cis-DDP bound to DNA, (D/N)b = 0.031. Similar concentrations (4-6 nM) of cis-DDP-modified poly(dG).poly(dC) also inhibited antibody binding, whereas higher concentrations (17-36 nM) of cis-DDP-modified poly[d(AG)].poly[d(TC)] were required for inhibition. Adducts formed by cis-DDP on other synthetic DNA polymers did not inhibit antibody binding to cis-DDP-DNA. The biologically active compounds [Pt(en)Cl2], [Pt(dach)Cl2], and [Pt(NH3)2(cbdca)] (carboplatin) (where en is ethylenediamine, dach is 1,2-diaminocyclohexane, and cbdca is cyclobutane-1,1-dicarboxylate) all formed antibody-detectable adducts on DNA, whereas the inactive platinum complexes trans-DDP and [Pt(dien)Cl]Cl (dien, diethylenetriamine) did not. The monoclonal antibodies therefore recognize a bifunctional Pt-DNA adduct with cis stereochemistry in which platinum is coordinated by two adjacent guanines or, to a lesser degree, by adjacent adenine and guanine. A monoclonal antibody raised against trans-DDP-DNA was competitively inhibited in an ELISA by 40 nM trans-DDP bound to DNA, (D/N)b = 0.022. This antibody crossreacted with unmodified, denatured DNA. Its binding to trans-DDP-DNA was selectively inhibited by trans-DDP-modified poly[d(GT)].poly[d(CA)] (50% inhibition at 1 nM bound trans-DDP). The recognition of cis- or trans-DDP-modified DNAs by monoclonal antibodies thus parallels the known modes of DNA binding of these compounds and may correlate with their biological activities.

Animals↗
PubMed1986

A single germline VH gene segment of normal A/J mice encodes autoantibodies characteristic of systemic lupus erythematosus.

These experiments tested the hypothesis that unmutated germline genes from normal mice can encode autoantibodies. We found that the unmutated VHIdCR gene segment, which encodes a large proportion of antiarsonate antibodies in A/J mice, also encodes antibodies with the ability to bind to DNA and cytoskeletal proteins. After Ars immunization, at a time when the VHIdCR gene segment mutates and antibody affinity for the hapten increases, reactivity with the autoantigens was lost. Six antibodies obtained after immunization with Ars bound both the Ars and DNA. Results of competitive inhibition assays suggested that the same variable region site in the antibodies bound to both Ars and DNA. The properties of the individual germline-encoded antibodies, which include reactivity to both DNA and cytoskeletal proteins, suggest that autoantibodies characteristic of SLE might be a subset of antibodies encoded by unmutated germline V genes.

Animals↗
PubMed1986

A rapid ELISA for measurement of antibodies to nucleic acid antigens using UV-treated polystyrene microplates.

Pretreatment of polystyrene microplate wells with certain doses of UV light enhances their capacity for binding to single-stranded DNA, double stranded DNA and various synthetic polynucleotides. The use of UV-irradiated plates to immobilize nucleic acid antigens provides a simple, rapid, and specific ELISA for measuring anti-nucleic acid antibodies. The assay is at least as sensitive as the more complex method of precoating plates with poly(L-lysine). It is useful for detection of anti-DNA antibodies in sera of systemic lupus erythematous patients, as well as in culture fluids of murine and human anti-DNA-secreting hybridomas.

Animals↗
PubMed1986

The effect of anti-Z-DNA antibodies on the B-DNA-Z-DNA equilibrium.

Four different preparations of rabbit and goat anti-Z-DNA antibodies were examined to determine the effects of antibody binding on the B-DNA-Z-DNA equilibrium. One of the four antibodies, a goat IgG, caused a marked lowering in the ionic strength required for the B-DNA to Z-DNA transition in poly(dG-dC) X poly(dG-dC), shifting the midpoint from 2.25 to 2.0 M NaCl. This IgG had a more prominent high affinity antibody population than did the other goat IgG, which caused little change in the midpoint of this transition. The presence of anti-Z-DNA antibodies also reduced the degree of negative supercoiling required for the formation of Z-DNA in (dG-dC)n sequences inserted into closed circular plasmid DNA. The goat IgG with the more marked effect on the salt-induced transition also had a greater effect in favoring Z-DNA formation in negatively supercoiled plasmids. A shift toward Z-DNA formation was observed in circular dichroism measurements upon antibody binding to poly(dG-dC) X poly(dG-dC) in very low ionic strength solution as well. We propose that the stabilization of Z-DNA by antibody binding in poly(dG-dC) X poly(dG-dC) occurs cooperatively, several antibody molecules binding to a single polymer molecule and stabilizing the entire molecule in Z-DNA through their combined binding energies. The stabilization of Z-DNA by antibody binding in a supercoiled plasmid can be significant, and failure to consider this effect and to choose appropriate conditions for measurement can lead to errors in estimating when Z-DNA will form in response to negative supercoiling.

Animals↗
PubMed1986

Binding of cis- and trans-diamminedichloroplatinum(II) to deoxyribonucleic acid exposes nucleosides as measured immunochemically with anti-nucleoside antibodies.

We report the use of anti-nucleoside antibodies to probe for local denaturation of calf thymus DNA upon binding of the antitumor drug cis-diamminedichloroplatinum(II), cis-DDP, and the biologically inactive analogues trans-diamminedichloroplatinum(II), trans-DDP, and chloro(diethylenetriamine)platinum(II) chloride, [Pt(dien)Cl]Cl. These antibodies specifically recognize each of the four DNA nucleosides. They bind well to denatured DNA, but not to native DNA in which the bases are less accessible owing to Watson-Crick duplex structure. At relatively high levels of modification (D/N approximately 0.1), cis-DDP causes significant disruption of DNA base pairing as reflected by the increased binding of anti-cytidine, anti-adenosine, and anti-thymidine antibodies. At lower levels of platinum adduct formation, however, all four anti-nucleoside antibodies bind more to DNA modified with trans-DDP. This result indicates that adducts formed by trans-DDP disrupt the DNA structure to a greater extent than those formed by cis-DDP at low D/N ratios. Modification of DNA by the monofunctional complex [Pt(dien)Cl]Cl does not affect its recognition by anti-nucleoside antibodies, demonstrating that base pair disruption is a consequence of bifunctional binding. The relative anti-nucleoside antibody recognition of cis-DDP-modified DNA is anti-cytosine greater than anti-adenosine approximately anti-thymidine much greater than anti-guanosine, consistent with the major adduct being an intrastrand d(GpG) cross-link. These results reveal that base pair disruption in a naturally occurring DNA modified by either cis-DDP or trans-DDP is sufficient to be detected by protein (antibody) binding. The relevance of these findings to current ideas about the molecular mechanism of action of cis-DDP is discussed.

Animals↗
PubMed1986

Analysis of Z-DNA in fixed polytene chromosomes with monoclonal antibodies that show base sequence-dependent selectivity in reactions with supercoiled plasmids and polynucleotides.

Five monoclonal anti-Z-DNA antibodies were characterized with respect to their binding of synthetic nucleic acid polymers and of supercoiled circular plasmid DNA. All of the antibodies reacted only with DNA in the Z-conformation; however, they fell into two classes on the basis of sequence specificity. One class, with broad specificity, reacted well with all sequences in the Z-form, including poly(dG-dC), poly(dG-dm5C), and poly (dG-dBr5C) in linear polymers and poly(dG-dC)n and poly[(dC-dA)n.(dT-dG)n] sequences in supercoiled plasmids. The other class bound only Z-DNA formed by poly(dG-dC). Binding of the monoclonal antibodies specifically to inserts of Z-DNA-forming sequences in plasmids was mapped directly by cross-linking of antibody to the DNA, digestion with restriction nuclease, and electrophoretic analysis of both the unbound fragments and the bound fragments recovered from immune complexes. The monoclonal antibodies were used for indirect immunofluorescence staining of Drosophila polytene chromosomes fixed by two procedures. One procedure yielded chromosomes with Z-specific antibody binding in many interbands, a few specific bands, and parts of some puffs. On chromosomes fixed by the second procedure, antibody staining appeared to follow the DNA concentration, staining all bands brightly. For each fixation procedure, chromosomes showed the same staining pattern with each of the broad specificity monoclonal antibodies that had been seen with polyclonal antibodies. The antibodies that reacted only with poly(dG-dC) and poly (dG-dC)n plasmid inserts did not stain chromosomes fixed by either protocol. We conclude that stretches of poly(dG-dC)n sequences do not contribute significantly to the presence of Z-DNA in fixed polytene chromosomes of Drosophila melanogaster.

Animals↗
PubMed1986

A recognition site on synthetic helical oligonucleotides for monoclonal anti-native DNA autoantibody.

The binding site in native DNA for a murine monoclonal anti-DNA autoantibody was investigated by measurements of competitive binding of a series of synthetic helical oligonucleotides. The antibody bound to a (dG-dC)3 or (dG-dC)4 core in the center of a base-paired octadecanucleotide. Reactions of analogues containing modifications or substitutions at specific sites indicated that the antibody bound to portions of cytosine and guanine in the major groove, a limited region of the backbone, and the 2-amino group of one guanine in the minor groove. For these interactions to occur, the antibody combining site would straddle the backbone of one of the helical strands of DNA.

Animals↗