Immunofluorescence for the detection of photochemical lesions in intracellular DNA.
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Biomedical subjects
Publications and source records attributed to B F Erlanger.
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Highly specific antibodies were raised to histone 1 (H 1) and the histone complexes H32-H42 AND H2A-H2B, isolated by salt extraction. Antibody to H1 could detect irreversible conformational changes in acid- or urea-treated H1. The antibodies showed different reactivities with chromosomes as compared to antibodies in acid-extracted histones and should be useful in studies of native chromatin and chromosome structure.
The centromeric regions of cattle, goat and sheep chromosomes bind anti-5-MeC as revealed by immunofluorescence technique, indicating concentration of 5-MeC at these heterochromatic regions. The centromere of the submetacentric X of cattle remains nearly unstained and so do the centromeres of the acrocentric X chromosomes in goat and sheep. The short arm of the cattle Y exhibits strong anti-5-MeC binding whereas the tiny Y chromosomes of goat and sheep contain no brightly fluorescent material.
A variety of azobenzene compounds having bis-quaternary nitrogens have been shown to accelerate the hydrolysis by chymotrypsin of certain specific substrates by an allosteric mechanism. One of the most potent, 2,2'-bis[alpha-(benzyldimethylammonium)methyl]azobenzene dibromide (2,2'-QBzl) accelerated the hydrolysis of glutaryl-L-phenylalanine p-nitroanilide 40-fold at saturating concentration. Acceleration was by increasing kcat without altering Km. The hydrolysis of acetyl-L-tyrosine p-nitroanilide and acetyl-L-tyrosine anilide was also accelerated by Q-Bzl (25-fold and 1.8-fold respectively) while the hydrolysis of hemoglobin, azocoll and a number of esters was not affected. The inactivation of chymotrypsin by diphenylcarbamyl chloride and diphenylcarbamyl fluoride was accelerated by 2,2'-Q-Bzl. Reac;ivation in the presence of NH2OH was also accelerated, but in the absence of added nucleophile (i.e. of NH20H) no increase in rate was detectable. An allosteric effector was covalently attached to chymotrypsinogen A by reaction with 2,2'-bis[alpha-(o-bromomethylbenzyldimethylammonium)methyl]azobenezene dibromide. The product, when converted to active enzyme, was about 4 times more active than chymotrypsin as a result of an increase in kcat of hydrolysis; Km was unaffected. The mechanism of the allosteric acceleration process is not known but, because for all of the substrates affected acylation of the enzyme is rate-limitimg, it is tentatively suggested that the effectors facilitate proton transfer to the leaving group by an inductive effect on the 'charge relay system'. Spectral studies indicate that the allosteric site is a portion of the enzyme with a polarity near that of water, possibly on the outside surface of the enzyme molecule.
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Human metaphase chromosomes, fixed in methanol-acetic acid, were ultraviolet irradiated to produce single-stranded regions of chromosomal DNA and treated with anti-5-methylcytidine. Using an immunoperoxidase procedure, regions of antibody binding were readily visualized by light microscopy in the centromeric heterochromatin regions of chromosomes 1, 9, 16, the short arm of chromosome 15, and in the distal portion of the Y. Electron microscopic visualization of the same whole mount chromosome preparations transferred to formvarcoated grids revealed additional details of the distribution and arrangement of 5-methylcytosine. A helical arrangement of 5-methylcytosine residues was seen below the centromere of chromosome 1. The Y chromosome showed a concentration of 5-methylcytosine residues on the distal long arm, and in areas just below and slightly above the centromere. In all the above chromosomes, especially chromosome 15, additional 5-methylcytosine residues were detected as isolated foci along the arms. Our findings support the concept that clusters of similar purine or pyrimidine residues exist along the arms of condensed metaphase chromosomes, with the possibility that concentrations of 5-methylcytosine residues might have been enhanced at the surface of the chromosomes during the condensation process.
A broad view is given of photoregulated processes as they occur in algae, fungi, halophilic bacteria, higher plants, invertebrates, and higher animals. Emphasis is on the following: the organs, tissues, and organelles that participate; the nature of the photoreceptor pigments; the light-induced structural changes that occur in the photopigments; and the way in which the photochemical events are believed to be translated into the physiological response. An attempt is made to show that there exist common biochemical attributes in all systems. In particular, they depend upon the ability of a low-molecular-weight to regulate a biologically active macromolecule, which may or may not be incorporated into a membrane. This is a common type of biochemical regulation and is, for example, the basis of allosterism. The additional refinement in photosensitive systems is the ability of light to alter the stereochemistry of the low-molecular-weight effector molecule and thus to modify its effect on the macromolecule. Model photosensitive systems are examined that incorporate control mechanisms that function in natural systems. For example, there are systems in which enzymes, normally insensitive to light, are made subject to photoregulation. In others, membrane permeability is rendered photoresponsive. A comparison of the model systems was processes found in nature permits the formulation of an hypothesis to explain how naturally occurring photoresponsive systems might have evolved.
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An enzyme immunoassay procedure has been developed for the visualization and estimation of lymphocyte surface receptors. beta-Galactosidase (beta-gal'ase) was covalently linked to sheep anti-rabbit immunoglobulin (SARIg), to ovalbumin (OA), and to adenosine (A). Exposure of rabbit peripheral lymphocytes to SARIg-beta-gal'ase and subsequent incubation with the fluorogenic substrate fluorescein-beta-digalactopyranoside allowed visualization of B cells by fluorescence microscopy. Treatment with each of the above beta-gal'ase conjugates and incubation with another fluorogenic substrate, 4-methyl-beta-D-umbelliferylgalactopyranoside, made possible the estimation of the various receptors by spectro fluorimetry. Procedures used for enrichment of T and B cell populations could be monitored. Among the findings was that immunoglobulin-bearing cells (presumably B cells) formed rosettes with sheep erythrocytes. The quantitative procedure was used to study lymphocyte population changes during immunization with an A-O A conjugate. An increase in the binding of A-beta-gal'ase, SARIg-beta-gal'ase, and OA-beta-gal'ase by peripheral lymphocytes occurred repeatedly 6 to 8 days after immunization but lasted only a few days despite a continued high circulating antibody titer. These results are consistent with the possibility that immunization induces migration of lymphocytes into the circulation from bone marrow and/or thymus.
Antibodies specific for the trinucleotide codons AAA, AUG, and AAC were examined for their reactions with nucleic acids. Anti-AUG and anti-AAC precipitated denatured DNA. Anti-AAA did not, and moreover, the binding of a tritiated AAA derivative to anti-AAA was not inhibited by denatured DNA. Radioligand-binding studies showed that anti-AAA was highly specific for the triplet sequence, some cross-reactions occurring with di-A and tetra-A but little with A and poly(A). The anti-codons did not precipitate whole yeast RNA, but binding could be demonstrated with Escherichia coli 3H-rRNA. Anti-AAC and anti-AUG (but not anti-AAA) bound E. coli 14C-tRNA. Anti-AUG was found to inhibit protein synthesis in an in vitro system in which rabbit reticulocyte mRNA was being translated. Inhibition was abolished by absorption of the antibody with AUG-RSA. The results of these and previous experiments (reference 1) are interpreted to indicate that antibodies can recognize trinucleotide sequences but not with absolute specificity, and it is suggested that antibodies to longer nucleotide sequences might show specificity comparable to that shown by antibodies to polysaccharide and peptide sequences.
Quinacrine (Q-band) and centromeric heterochromatin (C-band) patterns of metaphase chromosomes of two subspecies of Mus musculus were compared. M.m. musculus (the laboratory mouse) and M.m. molossinus (a subspecies from Southeast Asia) had similar Q-band patterns along the length of the chromosomes, but differences were observed in the centromeric region of some chromosomes. The two subspecies had very different distributions of C-band material. Antibodies to 5-methyleytosine were bound to regions of the chromosome corresponding to the C-bands in each animal. These findings support the idea that satellite DNA, which is concentrated in the C-band region, changes more quickly than bulk DNA. The interfertility of these two subspecies permits the development of a musculus strain carrying normal marker chromosomes for genetic studies.
Fixed metaphase chromosomes of gorilla and chimpanzee were UV-irradiated to produce regions of single-stranded DNA and then treated with antibodies specific for the minor DNA base 5-methylcytosine (5 MeC). An indirect immunofluorescence technique was used to visualize sites of antibody binding. In the gorilla six pairs of autosomes contained major fluorescent regions, indicating localized regions of highly methylated DNA. These corresponded, with the exception of chromosome 19, to the major regions of constitutive heterochromatin as seen by C-banding. The Y chromosome also contained a highly fluorescent region which was located just proximal to the intense Q-band region. In the chimpanzee no comparable concentrations of highly methylated DNA were seen. Smaller regions of intense 5 MeC binding were present on perhaps six chimpanzee chromosomes, including the Y. Five of these corresponded to chromosomes which were highly methylated in the gorilla.--There is diversity among the human, gorilla and chimpanzee in both the size and location of concentrations of 5 MeC, supporting the idea that satellite DNA evolves more rapidly than DNA in the remainder of the chromosome.
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Interstitial immune complex nephritis in patients with systemic lupus erythematosus (SLE). Renal tissues from 45 patients with SLE nephritis, 34 patients with idiopathic membranous nephropathy (IMN) and 77 patients with minimal glomerular disease (MGD) were studied by light, immunofluorescence and electron microscopy. Interstitial nephritis characterized by focal or diffuse infiltration of inflammatory cells, tubular damage and interstitial fibrosis was observed in 66% of SLE patients. Fluorescein-conjugated antibodies to immunoglobulins or complement or both were bound to peritubular capillaries, interstitium and tubular basement membranes (TBM) in 53% of patients with a granular pattern corresponding to opaque deposits seen by light or electron microscopy or both. Antibodies reactive with thymidine or cytosine or both were bound to interstitial structures in 19% of patients tested and showed the same granular distribution. Interstitial cellular infiltration was rare and deposits of immunoglobulins and complement were rare or absent in IMN and MGD, whereas deposits of DNA products were never observed. The findings are consistent with the interpretation that in patients with SLE nephritis immune deposits, presumably containing DNA-anti-DNA complexes, localize in peritublular capillaries, TBM and interstitum, thereby producing an inflammatory reaction which contributes to development and evolution of renal diseases.
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