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Biomedical subjects

B F Erlanger

Publications and source records attributed to B F Erlanger.

At least 55 records · Page 3Linked to original sources
PubMed1987

Characterization of a bioeffective monoclonal antibody against thyrotropin beta-subunit.

To describe a region of the TSH molecule participating in binding to receptor, a monoclonal antibody specific for a TSH epitope shared by beta-subunits of bovine (b), ovine (o), and human (h) TSH was obtained by immunization with mixtures of purified bTSH and hTSH. RIAs showed that the antibody also bound the beta-subunits of bLH, oLH, hLH, and hCG, but not the beta-subunits of porcine LH and TSH. Preincubation of [125I]iodo-bTSH with the antibody completely inhibited binding of the hormone to the TSH receptor of bovine thyroid membrane preparations at pH 7.4 in 50 mM NaCl (ED50 = 10 nM). The antibody also inhibited TSH-induced mitogenesis of FRTL-5 cells (ED50 = 50 nM). We conclude that the antibody binds to a site on the bTSH molecule that participates in high affinity binding of hormone to physiological TSH receptor. The target epitope includes a conserved structural determinant in beta-subunits of the glycoprotein hormones as well as a feature that allows discrimination of porcine hormones from those of bovine, ovine, and human origin.

Animals↗
PubMed1987

Immunocytochemical evidence for methylation of the inactive X chromosome in human fetal oogonia.

The state of DNA methylation of the X chromosomes of human interphase oogonia from a 46,XX and a 46,XX/47,XXX fetus at 17 weeks of gestation was tested immunocytochemically with an antibody to 5-methylcytosine (5MeC). Of 1637 oogonial nuclei from the 46,XX fetal ovary, 313 (19.1%) contained Barr bodies, of which 93.6% were positive for 5MeC. Of 1780 oogonia from the 46,XX/47,XXX fetus 327 (18.4%) contained Barr bodies; 175 oogonia had one Barr body and 152 had two. Of the single Barr bodies 145 (82.8%) had positive 5MeC reaction product. Of the 152 oogonia from the XXX line, 97 (63.8%) had positive 5MeC on both Barr bodies, 35 (23%) had one positive and one negative, and 20 (13.1%) had no product on either Barr body. This immunocytochemical evidence supports the hypothesis that the DNA of the inactive X-chromosome of the human 17-week gestation oogonium is methylated.

DNA↗
PubMed1986

Generation of an auto-anti-idiotypic antibody that binds to glucocorticoid receptor.

A monoclonal antibody (8G11-C6) that is directed to a region near the ligand-binding site of the glucocorticoid receptor was obtained by an auto-anti-idiotypic route, using a derivative of triamcinolone coupled to thyroglobulin to immunize a mouse. The resulting hybridomas were screened for anti-idiotypic antibody (anti-antisteroid) with Fab fragments of affinity-purified polyclonal rabbit anti-triamcinolone antibody. The anti-idiotypes were then screened for binding to rat cytosol glucocorticoid receptor by a depletion procedure, yielding a clone, 8G11-C6, whose specificity for receptor was verified by sucrose density and Western blot analyses. Depletion was not significantly reduced by prelabeling the cytosol with [3H]triamcinolone acetonide. The anti-idiotype (8G11-C6) bound to Fab fragments of antisteroid and to partially purified receptor in a concentration-dependent manner. Both binding reactions were inhibited only by rabbit serum albumin conjugates of steroids known to bind to the glucocorticoid receptors. Triamcinolone derivatives of lysine and of oligopeptides containing up to six amino acids inhibited the binding of the anti-idiotype to the Fab fragments but not to the receptor, implying that the target epitope of the antisteroid antibody may be closer to its glucocorticoid-binding site than the cross-reacting epitope of the receptor. Our findings demonstrate further the versatility of the auto-anti-idiotypic route for the preparation of anti-receptor antibodies.

Animals↗
PubMed1986

New amiloride analogue as hapten to raise anti-amiloride antibodies.

A new amiloride analogue, "amiloride-caproic acid," was synthesized, coupled to albumin, and used as a hapten to raise anti-amiloride antibodies in rabbits. The antibodies were affinity purified with an amiloride affinity column and characterized. Binding studies using [3H]benzamil showed a dissociation constant of 0.8 nM. Amiloride and amiloride-caproate inhibited [3H]benzamil binding; epsilon-guanidinocaproic acid showed no inhibition. Anti-amiloride antibodies reversed the inhibition by amiloride of sodium transport across toad urinary bladder. Anti-amiloride antibodies and an amiloride affinity column should provide useful tools for the characterization of the epithelial sodium channel.

Albumins↗
PubMed1985

Rates and equilibria for a photoisomerizable antagonist at the acetylcholine receptor of Electrophorus electroplaques.

Voltage-jump and light-flash experiments have been performed on isolated Electrophorus electroplaques exposed simultaneously to nicotinic agonists and to the photoisomerizable compound 2,2'-bis-[alpha-(trimethylammonium)methyl]-azobenzene (2BQ). Dose-response curves are shifted to the right in a nearly parallel fashion by 2BQ, which suggests competitive antagonism; dose-ratio analyses show apparent dissociation constants of 0.3 and 1 microM for the cis and trans isomers, respectively. Flash-induced trans----cis concentration jumps produce the expected decrease in agonist-induced conductance; the time constant is several tens of milliseconds. From the concentration dependence of these rates, we conclude that the association and dissociation rate constants for the cis-2BQ-receptor binding are approximately 10(8) M-1 s-1 and 60 s-1 at 20 degrees C; the Q10 is 3. Flash-induced cis----trans photoisomerizations produce molecular rearrangements of the ligand-receptor complex, but the resulting relaxations probably reflect the kinetics of buffered diffusion rather than of the interaction between trans-2BQ and the receptor. Antagonists seem to bind about an order of magnitude more slowly than agonists at nicotinic receptors.

Animals↗
PubMed1984

Intervening sequences in human fetal globin genes adopt left-handed Z helices.

The large intervening sequences ( IVS2 ) of three human fetal globin genes contain tracts of alternating purine-pyrimidine sequences approximately 40-60 base pairs in length which adopt left-handed Z DNA helices under the influence of negative supercoiling. The amount of negative supercoiling (approximately 0.045) required for the right- to left-handed transitions is within the physiological range. The structural aberrations between the right- and left-handed helices were mapped by sequencing the S1 nuclease cleavage sites. Two-dimensional gel electrophoretic analyses of the supercoil-induced relaxation served to characterize the type and length of left-handed structure. Furthermore, binding studies with several types of antibodies confirmed the presence of left-handed helices. Since these simple sequences appear to be hotspots for recombination and gene conversion, unusual DNA conformations may participate in genetic expression.

Base Sequence↗
PubMed1984

An immunochemical examination of acetylaminofluorene-modified poly(dG-dC) X poly(dG-dC) in the Z-conformation.

Immunization of rabbits with a complex of methylated bovine serum albumin and N-2-acetylaminofluorene (AAF)-modified poly(dG-dC) X poly(dG-dC), a polynucleotide that can assume the Z-DNA conformation, yielded several populations of antibodies specific for Z-DNA determinants. The Z-DNA determinants were analyzed by examination of the antisera and of antibody preparations purified on immunoadsorbents. The following was found: AAF-poly(dG-dC) X poly(dG-dC) shared Z-DNA determinants in common with poly(dG-dC) X poly(dG-dC) in 3.0 M NaCl, poly(dG-m5dC) X poly(dG-m5dC) in 1.5 M NaCl, and brominated poly(dG-dC) X poly(dG-dC) in 0.2, 1.5, and 3.0 M NaCl. Included among the antibodies induced by these determinants was a subpopulation whose reaction with brominated poly(dG-dC) X poly(dG-dC) was sensitive to increased ionic strength. Another distinct population of antibodies recognized determinants present on AAF-poly(dG-dC) X poly(dG-dC) but not on the other Z-DNAs. Only a small portion of this population was specific for the AAF moiety; the greater part appeared to recognize Z-DNA-associated conformational characteristics that were unique to AAF-poly(dG-dC) X poly(dG-dC). These findings are consistent with the existence of a continuum of Z-DNA determinants, which might be capable of functioning as recognition signals for regulatory DNA-binding proteins.

2-Acetylaminofluorene↗
PubMed1984

Antisera specific for the oligoribonucleotide sequences AAU and A2U2 and their recognition of oligonucleotide conformation.

Antisera specific for two synthetic oligoribonucleotide sequences, AAU and A2U2, were elicited in rabbits. The oligonucleotides were synthesized using polynucleotide phosphorylase under high salt conditions. Each oligomer was isolated by ion exchange chromatography, and was conjugated to bovine serum albumin, and injected into rabbits as an emulsion with complete Freund's adjuvant. The specificities of the resulting sera were analyzed using a modified Farr-type radioimmunoassay employing homologous oligonucleotide-protein conjugates radiolabeled with [3H]acetic anhydride and unlabeled free oligonucleotides as inhibitors. The antiserum elicited by AAU-BSA reacted well with AAU-RSA but a major fraction of the antibodies was directed to determinants of the conjugate that were not present on the free hapten. With respect to the haptenic determinants, AAU was a better inhibitor than any of the constituent mono- or dinucleotides, implying that features of the entire trinucleotide were being recognized. The other members of the A2Un family reacted to about the same extent as AAU, while other trinucleotides required an up to 21-fold higher concn in order to achieve similar inhibition. The most striking aspect of this antiserum was its failure to bind free ApA, although it could bind the ApA-containing oligonucleotides A3, AAG, AAC and A2Un. It seems likely that the ApA sequence in solution does not contain a significant proportion of a conformation present to a great extent in the ApA-containing oligomers. The antiserum elicited by A2U2-BSA was like anti-AAU-BSA in that some of the antibodies were directed against determinants not present on the free hapten. The most striking result of the inhibition experiments was the specificity of the antiserum for members of the A2Un series. When the A2Un series was compared with AA, AMP or any member of the Un series, approximately four orders of magnitude separated the inhibition curves. The poor binding of component mono- and dinucleotides implies that the conformation recognized by the antibody is present only to a significant extent in the trimeric sequence; the equality of binding of AAU with A2U2, A2U3 and A2U4 suggests that this conformation of the triplet is preserved in the longer sequences. These studies demonstrate the utility of immunochemical procedures for the study of oligonucleotide conformation in solution.

Animals↗
PubMed1984

Hypothesis: the MHC-restricted T-cell receptor as a structure with two multistate allosteric combining sites.

This paper presents a dual-recognition model of the T-cell receptor that has been constructed to account for the phenomenon of MHC restriction as well as the paradoxical ability of T-cells to be both multispecific and precisely specific at the same time. In our model the combining sites for antigen and MHC are not independent as in classical dual-recognition models, but interact with each other by an allosteric mechanism. We envision a flexible receptor with combining sites for antigen and MHC that are capable of existing in a multitude of distinct complementarity states. MHC and antigen molecules act as allosteric effectors such that one ligand perturbs the conformation and therefore the specificity of the site for the other ligand. An essential feature of the model is that different MHC determinants induce different conformations at the anti-antigen site. In this way the receptor acquires multiple specificities. Within a particular complementarity state, precise recognition results from the requirement that antigen and MHC exhibit positive cooperativity in their binding to the T-cell receptor. Positive cooperativity is also the basis for MHC restriction. Reaction mechanisms are presented which describe the requirement that antigen and MHC both induce conformational changes in order to generate high-affinity binding to either ligand. As a precedent for the multistate allosteric receptor model, we discuss the properties of allosteric enzymes, especially ribonucleotide reductase, whose properties are analogous to those we have postulated for the T-cell receptor. Also discussed is the possibility that molecules such as Ly2, L3T4 and the Mls antigen, which have been found to play a role in antigen recognition, function as affinity-enhancing allosteric effectors that interact with the constant portion of the T-cell receptor.

Allosteric Site↗
PubMed1984

Electronmicroscopy and circular dichroism of the dynamics of the formation and dissolution of supramolecular forms of Z-DNA.

Previous electronmicroscopic studies had shown that N-acetylaminofluorene (AFF)-substituted poly(dG-dC)-poly(dG-dC) in the Z conformation, in 10mM Mg++, condensed into periodically banded, branched structures. We now show that similar structures are seen when poly(dG-dC)-poly(dG-dC) is converted to the Z conformation by heating to 60 degrees C in 1mM Mn++ or to 65 degrees in the presence of 0.5mM Mn++. We demonstrate that these banded structures form in solution, i.e. they are not artifacts of the preparative procedures used for electronmicroscopy, by crosslinking the Z conformers in solution with DL-diepoxybutane (DEB), and then restoring the solution to conditions that favor return to the B conformation. Circular dichroism (CD) and immunochemical studies showed that the Z conformation was maintained and the banded supramolecular structures were still seen by electronmicroscopy. Electronmicroscopy and CID were also used to follow the dissolution of the supramolecular structures by controlled scission of the crosslinks with the eventual return to the short double stranded molecules typical of the B conformers. During this process, supercoiled structures, both toroidal and interwound, were observed. The relationship of the toroids to the banded structure is discussed in the context of two possible structures for the condensed polynucleotide. We conclude that DNA, whether in the B or Z conformation, is extremely flexible in the presence of appropriate counter ions, and we present evidence that earlier estimates of their persistence lengths are too high. The inherent tendency to form condensed, highly organized structures is a property of DNA that could play an important role in its "packaging," and in its functions, and might have been critical for the evolution and replication of early life forms.

2-Acetylaminofluorene↗
PubMed1984

Properties of antibodies to thymine glycol, a product of the radiolysis of DNA.

Anti-thymine glycol antibodies were elicited by immunizing rabbits with thymine glycol monophosphate (TMP-glycol) conjugated by carbodiimide to BSA. The antibodies produced are specific for thymine glycol as measured by immunoprecipitation of TMP-glycol-RSA conjugates and hapten inhibition of reactivity with OsO4-treated DNA in an enzyme immunoassay. Using the enzyme immunoassay, the antibody is capable of detecting femtomole and picomole levels of thymine glycol in direct and competitive assays, respectively. This immunochemical assay is potentially suitable for measuring the production and repair of thymine glycol damage in cellular DNA.

Animals↗
PubMed1983

Stabilization of (dG-dC)n.(dG-dC)n in the Z conformation by a crosslinking reaction.

(dG-dC)n.(dG-dC) was converted to the Z conformer by heating in the presence of Mn++n. Reaction of this preparation with the crosslinking reagent, DL-diepoxybutane (DEB), stabilized this conformer so that it retained its structure even when returned to conditions that favored reversion to the B conformation. Treatment of the crosslinked Z conformer with periodate caused scission of the crosslink, allowing reversion to the B conformer. Reaction of (dG-dC)n.(dG-dC)n in the B conformation with DEB did not prevent conversion to the Z conformer in 4M NaC1; dialysis of the high salt solution against low ionic strength buffer allowed return to the B conformer. The Z in equilibrium B transitions were followed by circular dichroism studies and immunochemical procedures. The results suggest the feasibility of stabilizing Z sequences of DNA in chromatin by crosslinking, so that they could then be identified after DNA isolation.

Circular Dichroism↗
PubMed1983

Conformations of oligonucleotides in solution as determined by sequence-specific antibodies.

The conformations of some related oligoribonucleotides in solution were investigated immunochemically with antisera specific for two synthetic oligonucleotide sequences, A-A-U and A-A-U-U. Radioimmunoassay showed differences of as much as three or more orders of magnitude in binding among oligonucleotides with commonly shared sequences. These large differences, which reflect the loss of many points of contact with antibody because of changes in overall conformation, allow the following conclusions: (1) A-A-U and A-A-U-U have conformations distinct from any present in the Un family of oligomers. (2) Conformations of A-A-U and A-A-U-U differ markedly from those of oligomers of A. The dinucleotide A-A, in particular, bears little resemblance in conformation to the A-A sequence in A-A-U and A-A-U-U. (3) The recognizable conformational unit appears to be the triplet A-A-U, which binds as well as A-A-U-U and far better than its component dimers. Interactions between non-adjacent bases may be a factor here, as well as in codon recognition. The immunological data support the conclusion that, in oligonucleotides, as in polypeptides, primary sequence can determine conformation in solution.

Animals↗