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Biomedical subjects

B F Erlanger

Publications and source records attributed to B F Erlanger.

At least 73 records · Page 4Linked to original sources
PubMed1983

Evidence for extensive methylation of ribosomal RNA genes in a rat XC cell line.

XC cell line was established from a rat fibrosarcoma induced by avian sarcoma virus. Analysis of ribosomal RNA genes in the DNA of this cell line indicated that there was a 3- to 4-fold increase in the number of the rDNA repeating units. [methyl-14C]Methionine labeling experiments as well as Southern blot analysis of the restriction endonuclease fragments showed extensive methylation of the cytosine residues in the rDNA. Further analysis by HpaII, HhaI and MspI suggested that the entire rDNA repeat unit was uniformly methylated in the recognition sequence for these enzymes. Parallel experiments with two other rat cell lines (rat embryo fibroblasts and adenovirus-transformed rat embryo cells) indicated no evidence of methylation in the rDNA.

Animals↗
PubMed1982

The rat XC sarcoma cell line: ribosomal RNA gene amplification and banded karyotype.

Ribosomal RNA gene amplification has been demonstrated in the rat XC sarcoma cell line. Cells of this rat line have a fairly stable karyotype with several unusual features, which have been clarified by in situ hybridization, silver staining, and binding of antibodies to 5-methylcytosine. There are one or two tiny acrocentric chromosomes containing transcriptionally active 18S and 28S ribosomal RNA (rRNA) genes. The short arm of one chromosome No. 12 has been replaced by a GC-rich, C-band negative, differentially staining region (DSR) containing an increased number of rRNA genes; most of these are transcriptionally inactive and located in regions containing highly methylated DNA. The short are of a small chromosome, probably a No. 20, has been replaced by a GC-rich, C-band negative, homogeneously staining region (HSR) that presumably represents amplification of a DNA sequence other than the 18S and 28S rRNA coding sequences. The DNA in this HSR is not enriched in 5-methylcytosine and neither is that in the HSRs of methotrexate-resistant Syrian hamster cells.

Animals↗
PubMed1982

Anti-idiotypic route to anti-acetylcholine receptor antibodies and experimental myasthenia gravis.

trans-3,3'-Bis[alpha-(trimethylammonio)methyl]azobenzene bromide (BisQ) is a potent agonist of the acetylcholine receptor (AcChoR) of Electrophorus electricus. BisQ is highly constrained, suggesting that its structure is complementary to the combining site of the AcChoR when the latter is in its activated state. Antibodies produced in rabbits to a conjugate of bovine serum albumin and a derivative of BisQ mimicked the binding characteristics of the AcChoR with respect to the order of binding of a variety of agonists and to the preferred recognition of decamethonium ion (an agonist) over hexamethonium ion (an antagonist). Immunization of three rabbits with purified anti-BisQ yielded antisera having binding characteristics of anti-AcChoR in that, by complement fixation and enzyme immunoassay, crossreactions with receptor preparations from rat, Torpedo, and eel could be demonstrated in sera of all three rabbits immunized. Two of the three rabbits showed signs of muscle weakness similar to that seen after immunization with the AcChoR. One of the rabbits was injected intramuscularly with neostigmine and showed temporary improvement. Another showed post-tetanic exhaustion of hind-limb muscles after stimulation of the sciatic nerve at 50 Hz. Antibodies reactive with the AcChoR, therefore, were elicited by immunization with an antibody to a potent ligand of the AcChoR without the necessity of isolating the receptor itself. A similar mechanism may play a part in the etiology of at least some autoimmune diseases in which antibodies to various other receptors are involved.

Animals↗
PubMed1982

A photoisomerizable muscarinic antagonist. Studies of binding and of conductance relaxations in frog heart.

These experiments employ the photoisomerizable compound, 3,3'-bis-[alpha-(trimethylammonium)methyl]azobenzene (Bis-Q), to study the response to muscarinic agents in frog myocardium. In homogenates from the heart, trans-Bis-Q blocks the binding of [3H]-N-methylscopolamine to muscarinic receptors. In voltage-clamped atrial trabeculae, trans-Bis-Q blocks the agonist-induced potassium conductance. The equilibrium dose-response curve for carbachol is shifted to the right, suggesting competitive blockade. Both the biochemical and electrophysiological data yield a dissociation constant of 4-5 microM for trans-Bis-Q; the cis configuration is severalfold less potent as a muscarinic blocker. Voltage-clamped preparations were exposed simultaneously to carbachol and Bis-Q and were subjected to appropriately filtered flashes (less than 1 ms duration) from a xenon flashlamp. Trans leads to cis and cis leads to trans photoisomerizations cause small (less than 20%) increases and decreases, respectively, in the agonist-induced current. The relaxation follows an S-shaped time course, including an initial delay or period of zero slope. The entire waveform is described by [1 - exp(-kt)]n. At 23 degrees C, k is approximately 3 s-1 and n is 2. Neither k nor n is affected when: (a) [Bis-Q] is varied between 5 and 100 microM; (b) [carbachol] is varied between 1 and 50 microM; (c) carbachol is replaced by other agonists (muscarine, acetylcholine, or acetyl-beta-methylcholine); or (d) the voltage is varied between the normal resting potential and a depolarization of 80 mV. However, in the range of 13-30 degrees C, k increases with temperature; the Q10 is between 2 and 2.5. In the same range, n does not change significantly. Like other investigators, we conclude that the activation kinetics of the muscarinic K+ conductance are not determined by ligand-receptor binding, but rather by a subsequent sequence of two (or more) steps with a high activation energy.

Animals↗
PubMed1982

Is DNA methylation responsible for mammalian X chromosome inactivation?

MOHANDAS et al. (1981) have proposed that mammalian X chromosome inactivation involves segmental methylation of cytosine residues in the DNA at multiple sites along the X chromosome. Using antibodies specific for 5-methylcytosine, we have found no detectable difference in the extent of methylation of the DNA in the two X chromosomes of owl monkey or human females. Thus inactivation (facultative heterochromatization) of the X chromosome is not due to, or associated with, intense DNA methylation comparable to that seen in most constitutive heterochromatin.

5-Methylcytosine↗
PubMed1981

Photoregulated ion binding.

A photoregulated chelating agent has been synthesized. It is a photochromic azobenzene compound containing two iminodiacetic acid groups and can exists as cis and trans stereoisomers. The planar trans isomer does not bind zinc ions. On exposure to light of 320 nanometers, the trans isomer is converted to a nonplanar cis isomer, which, because of cooperativity between the two iminodiacetic acid groups, binds zinc ions with the value of the binding constant estimated to be 1.1 x 10(5) +/- 9.2 x 10(5) liters per mole at a ratio of one molecule of chelating agent to one zinc ion. The interconversion of the cis and trans isomers is reversible, suggesting possible application of this class of compounds as photoresponsive ion pumps.

Chelating Agents↗
PubMed1981

Evidence for methylation of inactive human rRNA genes in amplified regions.

In two unrelated families, the short arm of a 14p+ marker chromosome contains an increased number of copies of the 18S + 28S rRNA genes without a comparable increase in the transcriptional activity, as shown by silver staining. The DNA in this region is highly enriched in 5-methylcytosine, as shown by specific antibody binding. In contrast, the owl monkey and cat have a single major nucleolus organizer region (NOR) per haploid genome; these NORs contain about the same number of rRNA genes as the 14p+ chromosome but are not methylated. These findings suggest that most of the amplified human rRNA genes on the 14p+ chromosomes have been inactivated by a process involving DNA methylation.

Animals↗
PubMed1981

Polymorphism of 5-methylcytosine-rich DNA in human acrocentric chromosomes.

The acrocentric chromosomes of 18 unrelated individuals were analyzed by sequential staining by the chromomycin A3/methyl green R-banding technique to identify the chromosomes, followed by an indirect immunoperoxidase technique to detect 5-methylcytosine (5MeC)-rich DNA. The short arms of both chromosomes 15 usually (92% of the chromosomes) had a large collection of 5MeC-rich DNA, which was always rich in AT base pairs. Much less commonly (11% of the possible occasions), a collection of 5MeC-rich DNA was seen on the short arm of a chromosome 13, 14, 21 or 22, and this DNA was always rich in GC base pairs. Sequential distamycin A/DAPI (DA/DAPI) and R-banding studies were carried out in 13 of these 18 individuals. There was bright DA/DAPI fluorescence of the 5MeC-rich region on the short arm of chromosome 15 but not on that of any other acrocentric chromosome. One implication of these findings is that bisatellited or other abnormal chromosomes that are DA/DAPI negative and 5MeC positive cannot be derived from number 15. In the case of a de novo chromosome of this type, the specific origin from any other acrocentric chromosome could be demonstrated by examining 5MeC-binding of the parental chromosomes.

5-Methylcytosine↗
PubMed1981

Agents related to a potent activator of the acetylcholine receptor of Electrophorus electricus.

The synthesis of a number of compounds related to trans-3,3'-bis[alpha-(trimethylammonium)methyl]azobenzene dibromide (trans-3,3'-BisQ) (1) is described. Among the compounds are: [14C]-trans-3,3'-BisQ (1) diiodide, cis-3,3'-BisQ (2) dibromide, the trans-2,2' (7) and 4,4' (11) isomers of BisQ, 2,2', (12), 3,3' (13) and 4,4' (14) isomers of bis-benzyldimethylammonium analogues, and related compounds in which the azo bridge between the two aromatic rings is replaced by diketo and amide bridges. Of them all trans-3,3'-BisQ (1) was the most active cholinergic compound in the electroplax system of Electrophorus electricus; the pure cis isomer (2) was without activity. Intermediate activities were found for some of the other compounds and others were inhibitors. The relationship of the structure of these agents to a proposed conformation and topography of the binding site of the acetylcholine receptor (AChR) is discussed.

Animals↗
PubMed1981

Amplified ribosomal RNA genes in a rat hepatoma cell line are enriched in 5-methylcytosine.

In a rat hepatoma cell line, H4-IIE-C3, a 10-fold excess of 18S and 28S rRNA genes has been found in amplified chromosome regions. Antibodies to 5-methylcytidine bound extensively to the DNA of these regions, indicating a high level of DNA methylation. Most of the amplified rRNA genes were transcriptionally inactive, as shown by their failure to stain with silver. DNAs from the tumor cells and control rat hepatocytes grown with L-[methyl-14C]methionine were digested with restriction endonuclease EcoRI; the DNA fragments were separated by agarose gel electrophoresis, denatured, transferred to nitrocellulose filters, and hybridized to 32P-labeled rRNA or cDNA. Fragments containing the 18S or 28S rRNA coding sequences occurred in three major size classes; all three were rich in 5-methylcytosine. Analysis of EcoRI fragments of DNA from the tumor and control cells after digestion with Hpa II or Msp I endonuclease indicated that the 5'-C-C-G-G-3' sequences in most of the amplified rRNA genes were methylated. Analysis of the fragments produced by digestion with Hha I endonuclease indicated a high degree of methylation within its recognition sequence in the amplified rRNA genes as well. The association of hypermethylation with restricted transcriptional activity suggests that DNA methylation may regulate the activity of the rRNA genes.

5-Methylcytosine↗
PubMed1981

Detection of T cells that secrete molecules which share determinants with antigen-specific T-cell factors.

A single-cell secretion assay was used to detect cells that secrete products which react with an antiserum that binds T cell antigen-binding polypeptides. The antiserum (R11), which was produced by immunization of rabbits with a murine trinitrophenyl-specific suppressor factor, reacts with T cells and their products and with a suppressor T-cell clone but not with B cells or their products. The secretory cells this antiserum detected were found to be unevenly distributed among various organs (spleen, lymph node, and thymus) and, to different degrees, in spleens of various strains of mice. In unimmunized CBA/J mice, approximately 1-3% of spleen cells secreted macromolecules precipitable by R11. The majority of the secretory cells could be removed by panning with a mixture of Ly 1 and Ly 2 antibodies but not with either antibody alone. This is consistent with the cells having low surface antigen densities as a result of being either "pre-T" cells or mature secretory cells analogous to B-lineage plasma cells. In agreement with the latter possibility was our finding that the secretory activity of cells detected with antisuppressor factor was comparable to that of Ig-secretory cells as detected with an anti-Ig antiserum. However, higher numbers of R11+ secretory cells were seen in the immunoglobulin-negative fraction of spleen cells from nude mice, which could be interpreted to favor the first possibility. In either case this study shows that the single-cell assay technique is well suited for the detection and characterization of molecules released by immunoregulatory T cells.

Animals↗
PubMed1980

Fine structure mapping of an avian tumor virus RNA by immunoelectron microscopy.

The RNA of a deleted strain (lacking Src gene) of an avian sarcoma virus (ASV) was examined by a newly developed immunoelectron microscopic procedure which uses anti-nucleotide antibodies as probes. After denaturation of the RNA and reaction with a high affinity, highly specific anti-7-methylguanosine-5'-phosphate (anti-pm 7G), 81% of 106 molecules examined were found to have antibody at one terminus, in agreement with the presence of a pm 7G cap in ASV-RNA. Hapten inhibition by pm 7G could be demonstrated. Experiments with anti-A and with anti-poly A gave results consistent with the known structure of ASV-RNA, in particular the presence of a 3' poly A tail. These studies illustrate the feasibility of using anti-nucleotide antibodies in a combined immunochemical and electron microscopic study of the fine structure of nucleic acids.

Alpharetrovirus↗
PubMed1980

The organization of DNA in the mitotic and polytene chromosomes of Sciara corprophila.

The organization of DNA in the mitotic metaphase and polytene chromosomes of the fungus gnat, Sciara coprophila, has been studied using base-specific DNA ligands, including anti-nucleoside antibodies. The DNA of metaphase and polytene chromosomes reacts with AT-specific probes (quinacrine, DAPI, Hoechst 33258 and anti-adenosine) and to a somewhat lesser extent with GC-specific probes (mithramycin, chromomycin A3 and anti-cytidine). In virtually every band of the polytene chromosomes chromomycin A3 fluorescence is almost totally quenched by counterstaining with the AT-specific ligand methyl green. This indicates that GC base pairs in most bands are closely interspersed with AT base pairs. The only exceptions are band IV-8A3 and the nucleolus organizer on the X. In contrast, quinacrine and DAPI fluorescence in every band is only slightly quenched by counterstaining with the GC-specific ligand actinomycin D. Thus, each band contains a moderate proportion of AT-rich DNA sequences with few interspersed GC base pairs. - The C-bands in mitotic and polytene chromosomes can be visualized by Giemsa staining after differential extraction of DNA and those in polytene chromosomes by the use of base-specific fluorochromes or antibodies without prior extraction of DNA. C-bands are located in the centromeric region of every chromosome, and the telomeric region of some. The C-bands in the polytene chromosomes contain AT-rich DNA sequences without closely interspered GC base pairs and lack relatively GC-rich sequences. However, one C-band in the centromeric region of chromosome IV contains relatively GC-rich sequences with closely interspersed AT base pairs. - C-bands make up less than 1% of polytene chromosomes compared to nearly 20% of mitotic metaphase chromosomes. The C-bands in polytene chromosomes are detectable with AT-specific or GC-specific probes while those in metaphase chromosomes are not. Thus, during polytenization there is selective replication of highly At-rich and relatively GC-rich sequences and underreplication of the remainder of the DNA sequences in the constitutive heterochromatin.

Animals↗