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Biomedical subjects

B D Stollar

Publications and source records attributed to B D Stollar.

At least 145 records · Page 8Linked to original sources
PubMed1981

Antigen-binding diversity and idiotypic cross-reactions among hybridoma autoantibodies to DNA.

Hybridoma anti-DNA antibodies produced by clones derived from fusions of spleen cells from unimmunized MRL/l mice with BALB/c plasmacytomas were examined for their binding to nucleic acid antigens and for their reactivities with anti-idiotype antibodies. All the antibodies bound to a variety of nucleic acids and polynucleotides. This finding suggests that epitopes shared by nucleic acid antigens can account for some of the serologic diversity in lupus serum. Each hybridoma autoantibody had a unique pattern of reactivity with nuclei acid antigens, yet cross-reactions among some of them were found by analyses with anti-idiotype antibodies. These cross-reactions were due to similarities in the antigen-binding regions of the autoantibodies because anti-idiotype Ig blocked the binding of the hybridoma antibody DNA; moreover, polynucleotides inhibited the idiotype anti-idiotype reaction. Cross-reactive idiotypes were found not only among hybridoma anti-DNA antibodies produced by clones derived from the same MRL/l mice but also among antibodies produced by clones derived from different MRL/l mice. The latter finding suggests that families of germ line genes may encode some autoantibodies against DNA.

Animals↗
PubMed1980

Conformational changes in the H3 . H4 histone complex. Serological and circular dichroism studies.

The H3.H4 complex has been extracted from calf thymus chromatin in either 0.05 M NaOAc, pH 5.0, or in 2.0 M KCl, 0.1 M KPO4, pH 6.7, and both forms have been shown to consist solely of histones H3 and H4 when examined on sodium dodecyl sulfate-polyacrylamide gels. Serological and circular dichroism analyses indicate the existence of structural differences between the two forms. Dilution of the high salt form of the complex into low salt produces a time-dependent conformational change which is related to reduction in the alpha helix content of the complex and which exposes antigenic sites on the complex. The existence of multiple forms of the H3.H4 complex may be related to the dynamic equilibrium of nucleosome structure in vivo.

Animals↗
PubMed1980

Interferon induction by two 2'-modified double-helical RNAs, poly(2'-fluoro-2'-deoxyinosinic acid) x poly(cytidylic acid) and poly(2'-chloro-2'-deoxyinosinic acid) x poly(cytidylic acid).

In addition to the 2'-azido analogue of (I)n x (C)n, (dIn3)n x (C)n, we have found two other (I)n x (C)n analogues, (dIfl)n x (C)n and (dIcl)n x (C)n, in which the 2'-hydroxyls of the (I)n strand are replaced by either fluorine or chlorine, to be highly effective in inducing interferon. This contrasted with the lack of interferon-inducing activity noted for various other 2'-halogeno analogues of (I)n x (C)n and (A)n x (U)n, i.e. (I)n x (dCcl)n, (dAfl)n x (U)n, (dAcl)n x (U)n, (A)n x (dUfl)n and (A)n x (dUcl)n. In most assay systems, viz. primary rabbit kidney cells, human diploid fibroblasts, HeLa cells, interferon-primed mouse L-929 cells, and intact rabbits, (dIfl)n x (C)n and (dIcl)n x (C)n induced interferon levels that were comparable to those induced by (I)n x (C)n. There was one particular system (L-929 cells treated with DEAE-dextran), however, in which (dIfl)n x (C)n and (dIcl)n x (C)n, unlike (I)n x (C)n, failed to stimulate interferon production. As monitored by both radiochemical and biological means, (dIfl)n x (C)n and, to a lesser extent, (dIcl)n x (C)n were more resistant to degradation by ribonuclease A, T1 and human serum nucleases than was (I)n x (C)n. In their reactivity towards antibodies to double-stranded RNA (dIfl)n x (C)n and (dIcl)n x (C)n conformed more closely to (I)n x (C)n than did other 2'-substituted (e.g. 2'-O-methyl or 2'-O-ethyl) analogues of (I)n x (C)n. The high interferon-inducing potency of (dIfl)n x (C)n and (dIcl)n x (C)n has both theoretical and practical implications. While our findings suggest that (dIfl)n x (C)n and (dIcl)n x (C)n should be further explored for their therapeutic potentials, they also strengthen the notion that the interferon-inducing capacity, and possibly other biological functions of double-stranded RNAs is dependent on the recognition of the overall conformation of the polynucleotide rather than on the binding of specific functional groups such as the 2'-hydroxyl group.

Animals↗
PubMed1980

Reaction of systemic lupus erythematosus antinative DNA antibodies with native DNA fragments from 20 to 1,200 base pairs.

Double-stranded DNA fragments of varying sizes were isolated and tested for binding to systemic lupus erythematosus (SLE) antinative DNA antibodies. Fragments of 20-25, 40-50, 90-110, and 160-180 base pairs (bp), along with intermediate-size pieces were isolated by preparative gel electrophoresis of a limited micrococcal nuclease digest of calf thymus DNA. Larger helical polynucleotides of 160-200, 380, 600-1,000, and 1,200 bp were isolated by preparative gel electrophoresis of DNA from chicken erythrocyte nucleosomes and oligonucleosomes. The fragments behaved as base-paired structures as tested by thermal denaturation, resistance to S1 nuclease, and serological assays with antibodies to native or denatured DNA. At a concentration of 0.27 muM, fragments of 20-25 bp were able to react with two SLE sera in competition with native DNA. With these and two other sera, DNA of 40-50 bp was a much more effective competitor. One serum required DNA greater than 180 bp for competition in the concentration range tested. Denatured fragments were much less effective than native fragments. The results emphasize the heterogeneity of SLE antinative DNA antibodies, confirm that secondary structure of the antigen is important for specific binding to these antibodies, and support the suggestion that bivalent binding to one molecule may be important for high functional affinity.

Antibodies↗
PubMed1980

Secondary structure in denatured DNA is responsible for its reaction with antinative DNA antibodies of systemic lupus erythematosus sera.

Experiments were designed to determine the basis for the strong competitive reaction of denatured DNA with systemic lupus erythematosus (SLE) antinative DNA antibodies. Secondary structure in denatured DNA was reflected in hyperchromicity upon heating and in multiphase kinetics of its digestion by S1 nuclease. Partial digestion by S1 nuclease completely eliminated the ability of denatured DNA to react with antidenatured DNA antibodies, but not its ability to react with SLE sera. S1 nuclease-resistant cores were isolated from extensively digested denatured DNA. These cores had secondary structure, including some stable fold-back helical regions. The cores, from 20 to several hundred base pairs in size, competed with native DNA for binding by SLE sera. Other experiments measured reactions of denatured DNA under conditions that affected its secondary structure content. Its competitive activity decreased as temperature was increased from 0 degrees to 37 degrees C, whereas the activity of native DNA was not altered in this temperature range. With DNA pieces of 90-110 base pairs, native fragments were much more effective than the denatured fragments, in which stable helical structure is less likely to occur than in high molecular weight denatured DNA. Competitive assays with mononucleotides, oligonucleotides, homopolymers, and RNA-DNA hybrids also indicated that two strands of polydeoxyribonucleotide were required for optimal reactions with these SLE serum antibodies. The antibodies can measure stable helical regions in denatured DNA; they may also stabilize short helical regions that occur in an equilibrium of conformational forms.

Antibody Specificity↗
PubMed1980

Hybridoma autoantibodies to DNA.

MRL/1 mice develop a severe form of systemic lupus erythematosus and spontaneously produce high levels of autoantibodies to DNA. Spleen cells from these animals were used as fusion partners to prepare hybridomas that produce autoantibodies to DNA. Since exogenous immunization was not employed, the hybridoma antibodies were derived from populations of B cells that spontaneously produced autoantibodies. Each hybridoma autoantibody had a distinctive binding specificity with S-DNA, N-DNA, or oligonucleotides.

Animals↗
PubMed1979

Detection of antibody-forming cells and humoral antibody to horseradish peroxidase.

Two new simple methods for detecting antibody-forming cells by hemolytic plaque assay and hemagglutinating antibody to horseradish peroxidase have been developed in mice. Both techniques utilize as target, sheep erythrocytes coupled directly with horseradish peroxidase. These assays are sensitive, antigen-specific and are useful to quantitate both direct and indirect antibody-forming cells and humoral antibodies.

Animals↗
PubMed1978

Immunofluorescent detection of nuclear double-stranded RNA in situ in Vero and mosquito cells.

Double-stranded RNA (dsRNA) was detected in situ by indirect immunofluorescence with antibodies to dsRNA. It was seen in nuclei of Vero and Aedes albopictus cells, but not in BHK cells, KB cells, chick embryo fibroblasts, or HeLa cells. Reactive dsRNA was present in the nucleoplasm, but not in nucleoli or cytoplasm. Extracted RNA from the whole cell contained from 0.08 percent (BHK) to 0.46 percent (HeLa) dsRNA, as estimated by serological methods. This dsRNA, found in molecules having the size distribution of heterogeneous nuclear RNA, did not renature rapidly after denaturation. The quantity of dsRNA in total extracted RNA did not correlate with the presence or absence of nuclear staining in situ.

Cell Line↗
PubMed1978

Antigenic structure of double-stranded RNA analogues having varying activity in interferon induction.

Antibodies were induced by immunization of rabbits with methylated bovine serum albumin complexes of: poly(I).poly(BC), an effective interferon inducer; poly(c7A).poly(rT), a noninducer that can block induction by active poly(A).poly(rT); and poly(A).poly(Um), which has neither inducing nor blocking activity. Similar complexes of f2 phage RNA or tRNA did not induce anti-nucleic acid antibodies. Each anti-polynucleotide serum contained some antibodies specific for double-stranded structure. Antibodies were immunospecifically purified from precipitates made with each serum and homologous or cross-reacting double-stranded polynucleotides. The purified antibodies distinguished among varying helices bearing base or ribose modifications. Antipoly(I).poly(BC) specificity paralleled that of the interferon induction system. Anti-poly(A).poly(Um) specificity favored the 2'-modified polymers. Anti-poly(c7A).poly(rT) antibodies were the least discriminating. Cross-reaction results indicated that some antibodies reacted with determinants that included both sugar-phosphate backbones. In far antibody excess, antigen:antibody ratios in precipitating complexes reached a minimum of 7 to 12 base pairs per bivalent IgG molecule. Single antigenic determinants may span about 4 base pairs, with primary contact sites including the phosphate groups and the furanose.

Complement Fixation Tests↗
PubMed1978

Immunochemical measurement of conformational heterogeneity of poly(inosinic acid).

Several pure poly(I) preparations differed in: (a) their complement fixation reactivity with anti-poly(I) antiserum; (b) their ability to bind to a solid-phase anti-poly(I) antibody-Sepharose column; (c) their ability to inactivate serum complement; and (d) their reactivity with purified antibodies to double-stranded RNA. In particular, poly(I) samples that could induce interferon production differed from non-inducer poly(I)s; the inducers reacted weakly with anti-poly(I) antiserum and were the only ones that reacted with antibodies to double-stranded RNA. One inducer poly(I) did not inactivate complement, and differed from non-inducer poly(I) in quantitative aspects of poly(I) . poly(C) formation with varying amounts of poly(C). An additional type of poly(I) preparation reacted poorly with anti-poly(I) antiserum, did not react with anti-double-stranded-RNA antibodies and failed to induce interferon production. The varying forms of poly(I) were not interconvertible by boiling and rapid chilling. These results indicate that several different stable structural forms of poly(I) may result from a standardized synthetic procedure.

Antibodies↗