Histone deposition and metabolism in embryonic chick lenses during differentiation.
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Animals↗
Biomedical subjects
S Muller
Publications and source records attributed to S Muller.
Chondromyxoid fibroma of the mandible. Diagnostic image cytometry findings and review of the literature.
A case of chondromyxoid fibroma in the mandible of a 12-year-old boy is reported. A review of the literature is presented with emphasis on flow and image cytometric analysis and evaluation of the radiologic and histologic differences between chondromyxoid fibroma and chondrosarcoma.
Cell Nucleus↗
Double reactivity of monoclonal and polyclonal rheumatoid factors for IgG and histones: mapping of binding sites by means of histone synthetic peptides and anti-Id antibodies.
Polyreactive antibodies able to bind various apparently unrelated structures represent a frequent antibody population in autoimmune diseases. In this work, the structural basis of the double reactivity of such autoantibodies was investigated using as models polyclonal and monoclonal human rheumatoid factors (RF) reacting with histones. Both direct ELISA binding and competitive inhibition experiments were performed. A more precise delineation of the histone regions recognized by the RFs was made by means of 27 synthetic peptides of these proteins. Anti-idiotope (Id) murine antibodies were used to map the binding sites involved on RF in the interaction with IgG and histones. Among the 13 polyclonal and six monoclonal RFs tested, four and two respectively were found to cross-react with IgG and histones. The fragments shown to be the most frequently recognized by RFs were located in residues 1-16 and 204-218 of H1, 1-20 and 65-85 of H2A, and 1-21 of H3. The results obtained by competitive ELISA assays using IgG, histone peptides and anti-Id monoclonal antibodies led us to confirm and characterize more precisely our previous finding suggesting the existence of topographically distinct binding sites for the different targets recognized by RFs.
Animals↗
Endothelial cell injury secondary to surgical preparation of reversed and in situ saphenous vein bypass grafts.
Failure of infra-inguinal vein grafts appears to be due to the development of intrinsic lesions (intimal hyperplasia, fibrous stenoses) within the graft which lead to narrowing of the lumen, poor blood flow and thrombosis. The cause of these lesions remains unknown but recently it has been suggested that endothelial injury might be an aetiological factor. The damage that can occur after preparation of reversed vein grafts includes loss of endothelial cells and functional impairment of those cells that remain, in that the ability to produce prostacyclin and endothelium-derived relaxing factor (EDRF) is reduced. The preparation of in situ grafts is different to that for reversed grafts in that a valvulotome is passed along the lumen of the vein to destroy the valves. However, little is known about the degree of endothelial injury that this technique causes. Vein samples were obtained from patients undergoing infra-inguinal and coronary artery bypass grafting. The veins were mounted in an organ bath system to measure isometric tension and exposed to the endothelium-dependent agents acetylcholine, bradykinin, adenosine, histamine and the endothelium-independent agent sodium nitroprusside. The results indicate that preparation of reversed vein grafts leads to some loss of endothelial cells with functional impairment of the cells that remain. However, preparation of in situ grafts leads to severe or total loss of endothelial cells which resulted in an absence of detectable EDRF release. These findings were confirmed by histological examination of the vein samples.
Aged↗
Diffusion-enhanced energy transfer investigation of histone H5 in chromatin with a fluorescently-labelled antibody fragment Fab'.
The location of chicken erythrocyte H5 histone relative to the axis the 30 nm chromatin fibre axis has been investigated by diffusion-enhanced energy transfer. In this investigation, a neutral lanthanide chelate as donor and a fluorescent probe specific to H5 as acceptor have been used. The acceptor probe consists of H5 antibody Fab' fragment, which has been labeled with 5-iodoacetamidofluorescein (5-IAF). Using H5 fragments we have shown by ELISA that the antibodies recognized the N- and C-terminal ends of this histone. A neutral chelate of terbium (TbHED3A) was chosen as a suitable donor for energy transfer with IAF-labelled Fab' (Fab'-IAF) bound to H5 in various chromatin structures. The ionic strength dependence of the energy transfer from TbHED3A to chromatin-bound Fab'-IAF was used to estimate the accessibility and the location of the Fab' in chromatin. The rate constants for energy transfer, obtained from the lifetimes of the TbHED3A excited state in presence and absence of acceptor, indicated a decrease in transfer efficiency upon increase of salt concentration from 5 to 80 mM NaCl. This can be correlated with the chromatin folding occurring in this ionic strength range and is consistent with the location of at least some of the N and C-termini of H5 within the condensed chromatin structure.
Animals↗
Autoantibodies typical of non-organ-specific autoimmune diseases in HIV-seropositive patients.
OBJECTIVE: To analyse serological aspects of systemic autoimmunity in HIV-1-seropositive patients and in individuals at risk for AIDS. DESIGN AND METHODS: The reactivity of antibodies in the serum of 100 HIV-1-seropositive patients was investigated by enzyme-linked immunosorbent assay (ELISA) using a series of antigens known to be recognized by antibodies from patients with multisystemic autoimmune diseases, such as systemic lupus erythematosus, mixed-connective tissue disease and Sjögren's syndrome. RESULTS: High levels of immunoglobulin G (IgG) antibodies reacting with double-stranded DNA (dsDNA), synthetic peptides of ubiquitinated histone H2A, Sm-D antigen, U1-A RNP antigen and 60 kD SSA/Ro antigen were found in 44-95% of HIV-infected patients. Among histone antibodies, the most frequent reactions were towards the carboxy-terminal region of histone H1 and to histone H2B and its amino-terminal domain 1-25. Eight HIV-1-seropositive patients at different stages of disease according to the Centers for Disease Control classification were also studied. In most cases, no obvious fluctuations were observed over several years. Antibodies were found early, and their specificity and apparent level of activity remained relatively constant. There was no evidence of such an autoimmune response in the serum of high-risk homosexual seronegative men. CONCLUSIONS: Although the aetiology of AIDS is known, in general the aetiology of multisystemic autoimmune diseases remains to be determined, and the sequence of events taking place remains obscure in both cases. It is possible that the large spectrum of antibodies found in HIV-infected patients reflects a specific stimulation of B-cells by nuclear antigens released by apoptosis during an early stage of disease.
Autoantibodies↗
Glomerular immune deposits in murine lupus models may contain histones.
Two types of lupus mice, NZB/NZW F1 female hybrids and mice with graft-versus-host disease (GVHD), were studied. Histones H3 and H2A were detected by immunofluorescence in glomeruli of 22/22 proteinuric GVHD and 8/12 proteinuric NZB/W F1 female mice; in non-proteinuric animals, 3/5 GVHD and 2/27 NZB/W F1 female were positive. Using antibodies to histone peptides it was shown that mainly the N-terminal regions of histones H3 and H2A were exposed in glomerular deposits. Western blot analysis revealed antibodies to histone subfractions in sera of 33/34 lupus mice that developed proteinuria. This study provides evidence that histones are involved in the pathogenesis of lupus nephritis.
Animals↗
IgG antibodies from patients with primary Sjögren's syndrome and systemic lupus erythematosus recognize different epitopes in 60-kD SSA/Ro protein.
Five synthetic peptides corresponding to the N-, the C- and a central domain in 60-kD SSA/Ro protein were prepared and tested with sera from 112 patients with systemic lupus erythematosus (SLE), 55 with primary Sjögren's syndrome (pSS) and 29 with rheumatoid arthritis. Among these five fragments, one representing residues 21-41, was recognized by antibodies in 57% of pSS patients. Interestingly, this peptide was recognized by only a few (less than or equal to 7%) of SLE sera, while 63% of pSS sera and 46% of SLE sera tested in parallel possessed antibodies reacting in ELISA with purified 60-kD SSA protein. The ELISA results were compared with the pattern of reactivity obtained in immunodiffusion and immunoblotting. The results indicate that the sensitivity of ELISA using peptide 21-41 and pSS sera was in the same range as immunoblotting and higher than immunodiffusion. Thus the peptide 21-41 proved useful for the detection of anti-SSA antibodies in the sera of patients with pSS. Furthermore, a positive ELISA using peptide 21-41 could be of potential use to discriminate pSS with systemic features from SLE. The fact that peptide 21-41 is recognized by antibodies in pSS but only by very few SLE sera implies that different mechanisms are involved in the anti-SSA immune response in these two autoimmune diseases.
Amino Acid Sequence↗
Haemolysin-derived synthetic peptides with pore-forming and haemolytic activity.
Escherichia coli haemolysin (Hlya) is a pore-forming protein which belongs to the family of 'Repeat-toxins' (RTX) (Lo et al., 1987; Lally et al., 1989; Kraig et al., 1990). A model for the pore-forming structure of HlyA has been proposed (Ludwig et al., 1991) which consists of eight transmembrane segments all present in this hydrophobic region of HlyA. We report here that two synthetic peptides of 10 and 8 amino acids in length (Pep1 and Pep2, respectively), which are derived from transmembrane segment V, are able to form pores in an artificial lipid bilayer. In addition, Pep1 exhibits strong haemolytic activity when tested on human red blood cells (HRBCs). The haemolytic activity of Pep1 and of E. coli haemolysin is completely inhibited by antibodies raised against Pep1.
Amino Acid Sequence↗
[Ubiquitin and degenerative diseases of the central nervous system].
Ubiquitin is an ubiquitous 76 aminoacids protein that is present in all cellular compartments. It intervenes in numerous functions of cell metabolism, and in particular in non-lysosomal (but also lysosomal) lysis of altered or short-lived proteins. Immunohistochemical studies have shown that it is present in many inclusions characteristic of neurodegenerative diseases, notably in Lewy bodies, neurofibrillary tangles of Alzheimer's disease, Pick bodies and also in inclusions characteristic of certain motor neuron diseases. The presence of ubiquitin in these inclusions raises 2 questions: (1) the nature of the target proteins, probably altered proteins of the cytoskeleton; (2) the significance of ubiquination: it might reflect the degeneration process or participate in the protection of cells against degeneration or be an active factor in programmed cell death.
Brain Chemistry↗
Structural and functional smooth muscle injury after surgical preparation of reversed and non-reversed (in situ) saphenous vein bypass grafts.
Intimal hyperplasia and isolated stenoses are major factors in determining the long-term patency of reversed and in situ vein grafts. Surgical handling causes functional endothelial damage in reversed vein grafts but the effects of valvulotome use are unknown. Saphenous vein samples were taken after minimal dissection (control), distension with saline, or after the passage of a valvulotome. Rectangular strips of vein were mounted in an organ bath and cumulative noradrenaline (10(-8)-10(-5) M) dose contraction curves were performed. Compared with controls there was a significant reduction in the contractile response of both the distended and valvulotome-prepared veins (P less than 0.05). Histological studies revealed partial endothelial cell loss in the distended group and total endothelial cell loss with patchy necrosis of the smooth muscle in the valvulotome group. These results suggest that the current use of a valvulotome not only renders the valves incompetent but also seriously damages the function of the smooth muscle layer.
Aged↗
Parameters affecting the immunogenicity of a liposome-associated synthetic hexapeptide antigen.
The effect of liposome association on the immunogenicity of the hexapeptide IRGERA was investigated. When administered in the absence of a carrier and adjuvant this peptide, which corresponds to a linear epitope located at the C-terminus of histone H3, was not immunogenic. When mice were immunized with the peptide covalently linked to the surface of small unilamellar vesicles containing monophosphoryl lipid A as adjuvant, a relatively long-lasting response with memory cell induction was observed. The anti-peptide antibodies raised in this way reacted with the cognate sequence in the native histone. In contrast, coupling of the peptide to the surface of large vesicles yielded both an IgM and IgG response of short duration whereas encapsulation of the free peptide in large vesicles was ineffective. These results indicate that with short synthetic peptides, liposomes provide a substitute for a carrier protein. However, an adjuvant has to be incorporated in the vesicles in order to obtain an efficient immune response. Such an approach may be useful for designing synthetic vaccines.
Adjuvants, Immunologic↗
Detection of poly(ADP ribose) polymerase in crude extracts by activity-blot.
We have recently devised an activity-blot procedure permitting the detection, on the same nitrocellulose sheet, of the functional poly(ADP ribose) polymerase (PARP) activity as well as the immunostained active peptide(s) after renaturation of the transferred protein(s). Using this technique we have analyzed the PARP activity in higher and lower eukaryotes directly on crude extracts from cell cultures. This procedure has been extended also to in situ screening of bacterial colonies expressing the PARP enzymatic activity.
Cells, Cultured↗
The cytopathic effect of HIV is associated with apoptosis.
Large amounts of histones, H1, H2A, H2B, H3, and H4, were observed in total extracts of T4 lymphocytes and derived cell lines infected with the human immunodeficiency virus (HIV) type 1 or type 2. These histones were simply detectable by analysis of crude cellular extracts by polyacrylamide gel electrophoresis in SDS and staining the proteins with Coomassie blue or by immunoblot assays using specific polyclonal antibodies. The histones were found to be localized in the nucleoplasm, bound to low molecular weight (LMW) DNA in the form of nucleosomes. The mechanism responsible for the accumulation of nucleosomes during HIV infection was found to be due to fragmentation of cellular DNA, a mechanism referred to as apoptosis or programmed cell death in which a nuclear endonuclease becomes activated and cleaves DNA at internucleosomal regions. Accordingly, the LMW DNA accumulated in the course of infection was found to have a characteristic pattern of nucleosomal ladder and its accumulation was reduced in the presence of zinc, a known inhibitor of the endonuclease. Routinely in acute HIV infections, the accumulation of nucleosomes was observed at least 24 hr before lysis of infected cells. In a particular HIV-1 infection, in which the first signals of the cytopathic effect (vacuolization of cells and appearance of syncytia) was observed at Days 6-7 whereas maximal virus production occurred at Days 10-17, the accumulation of nucleosomes was at its maximal level already on Day 6 postinfection. In the nucleoplasm of chronically infected cells producing virus but not manifesting a cytopathic effect, no LMW DNA or histones were detectable. These observations indicate that the cytopathic effect of HIV infection is associated with apoptosis. The detection of histones and oligonucleosomal DNA fragments in the nucleoplasm can be used as a convenient marker for chromatin fragmentation during this process.
CD4-Positive T-Lymphocytes↗
Immunogenicity of free histones and of histones complexed with RNA.
Histone antibodies have been obtained by immunizing rabbits with histones H1, H2A, H2B, H3, H4 and triacetylated H4, uncomplexed to RNA. The reactivity of these antibodies was investigated by ELISA using as antigen isolated histones and chromatin as well as thirty-five different synthetic peptides covering the entire sequence of the four core histones, two peptides of H1 and two acetylated peptides of H4. The binding of these antibodies to histones was also measured in immunoblotting and in microcomplement fixation (MCF) tests. In parallel experiments using the same assays the various antigens were tested with antisera raised against histones complexed with RNA. Antibodies induced in the absence of RNA did not react with histones in MCF tests nor with chromatin in ELISA but reacted with the histones in ELISA, although the antibody titers were somewhat lower than in the case of antisera to histone-RNA complexes. Antibodies to RNA-histone complexes reacted with histones in both ELISA and MCF tests. When they were tested with peptide-coated microtiter plates in a direct binding ELISA format, antibodies induced with uncomplexed histones recognized very few fragments which were mainly located in the N- and C-terminal ends of the histones.
Acetylation↗
Plasma membrane-associated proteins with the ability to partially inhibit perforin-mediated lysis.
Cytolytic lymphocytes have previously been reported to be resistant to the lytic effects of perforin. In this work, plasma membrane proteins from a CTL cell line were fractionated by HPLC, and the eluted fractions were collected based on their ability to inhibit perforin-mediated hemolysis. Three proteins with inhibitory activity were thus purified, the serine esterase MCSP-3/granzyme F and the histones H2B and H3. A commercial source of H2B was able to potently inhibit perforin-mediated lysis, and it was confirmed by FACS analysis that H2B is in fact present on the surface of cytolytic cells. However, H2B was also found on the surface of perforin-susceptible tumor cell lines, indicating that the histones may partially inhibit perforin-mediated lysis in vitro, but that they do not represent the factor conferring specific resistance on cytolytic lymphocytes. The origin of the surface histones and the possible role of the surface MCSP-3/granzyme F are discussed.
Animals↗
Mapping of epitopes on U1 snRNP polypeptide A with synthetic peptides and autoimmune sera.
The ability of synthetic peptides encompassing almost the entire sequence of snRNP U1A polypeptide to be recognized in ELISA by sera of autoimmune patients was investigated. Sera from 18 patients with mixed connective tissue disease (MCTD), 145 with systemic lupus erythematosus (SLE) and 120 with other rheumatic autoimmune diseases were tested with 13 overlapping peptides. Among them, peptide 257-282 and, to a lower extent, peptide 1-11 were recognized by MCTD, SLE and Sjögren's syndrome sera. In contrast, peptide 35-58 was recognized by 94% of MCTD and only 19% of SLE sera. It did not react with any of the other patient sera. The ELISA results were compared with the pattern of reactivity observed in immunoblotting. The results indicate that peptide 35-58 probably contains a major epitope recognized by MCTD autoantibodies. It is noteworthy that in snRNP particles, this region of U1A interacts with RNA and presents only limited homology with the corresponding sequence 32-50 of U2B''.
Amino Acid Sequence↗
New Zealand white rabbits immunized with RNA-complexed total histones develop an autoimmune-like response.
The antibody response of rabbits immunized with a total histone mixture containing randomly coiled H1/H5, H2A, H2B, H3 and H4 devoid of DNA was investigated in direct and competitive ELISA. The antisera were tested with isolated histones and chromatin and with a series of overlapping synthetic peptides covering the entire sequences of the four core histones and two peptides of H1. It was found that the New Zealand (NZ) white rabbits immunized with the total histone (TH) mixture complexed with RNA produced IgG antibodies reacting with histones and with a number of histone peptides but not with chromatin. The antisera also contained IgG antibodies which bound components that correspond to common target antigens in autoimmune diseases such as native dsDNA, peptides of Sm-D antigen, ubiquitin, branched peptides of ubiquitinated H2A and poly(ADP-ribose). By competition experiments, it was shown that these antibodies corresponded to non-crossreacting antibody populations. New Zealand rabbits immunized with TH in the absence of RNA or random outbred rabbits immunized with the RNA-complexed histone fraction produced antibodies reacting with histone, chromatin and very few histone peptides, while no activity with non-related antigens was observed. The pattern of reactivity of antisera raised in NZ rabbits with RNA-complexed TH was found to be very similar to that observed in sera of patients with systemic lupus erythematosus while, in contrast, the antibody response was very different in NZ or outbred rabbits immunized with various native nuclear particles and with individual histones. Altered nucleosome particles rather than native nucleosomes may represent the antigenic stimulus giving rise to autoantibodies.
Animals↗