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Biomedical subjects

S Muller

Publications and source records attributed to S Muller.

At least 235 records · Page 13Linked to original sources
PubMed1993

The carboxyl-terminal domain of human poly(ADP-ribose) polymerase. Overproduction in Escherichia coli, large scale purification, and characterization.

The cDNA encoding the carboxyl-terminal 40-kDa domain of human poly(ADP-ribose) polymerase was inserted into an expression vector. The recombinant protein was overproduced in Escherichia coli, and purified to homogeneity. The 40-kDa domain had the same affinity (Km) for NAD+ as the full-length enzyme, expressed abortive NAD+ glycohydrolase activity, catalyzed the initiation, elongation, and branching of ADP-ribose polymers, but exhibited no DNA dependence. Its specific activity was approximately 500-fold lower than that of the whole enzyme activated by DNA strand breaks. Surprisingly, the carboxyl-terminal 40-kDa domain exhibited the processive mode of polymer attachment typical of full-length poly(ADP-ribose) polymerase and was able to modify histones H1 and H2B. Finally, the polymer sizes formed by the 40-kDa domain were influenced by histone H1.

Base Sequence↗
PubMed1993

Anti-"dividing cell antigen" autoantibody: a novel antinuclear antibody pattern related to histones in systemic lupus erythematosus.

This study aims to describe a novel antinuclear antibody directed to proteins only accessible during the mitosis: anti-"dividing cell antigen" (DCA) antibody. A total of 709 disease-associated and control sera was tested by indirect immunofluorescence using a variety of cell lines as substrate. Cells were treated with enzymes and antibodies absorbed with nuclear antigens. Antibodies to DNA, histone subfractions, and synthetic peptides were evaluated using enzyme-linked immunosorbent assays. Cell extracts were electrophoresed before and after synchronization and sera tested on the blots. The anti-DCA antibody was demonstrated in 10 of 183 SLE patients but virtually never in other connective tissue diseases. The DCA was sensitive to HCl and proteolytic enzymes and the anti-DCA binding inhibited by histones H2A and H2B. Differences of anti-H2A and anti-H2B were observed between anti-DCA antibody-positive and anti-DCA antibody-negative sera, and antibodies specific for the 1-15 region of H2A, the 1-25 region of H2B and the 1-29 region of H4 were more frequent in the former sera than in the latter. The anti-DCA antibody was shown to react with a 60-kDa protein. Our findings suggest that the anti-DCA antibody is directed to a protein complex containing H2A and H2B.

Adolescent↗
PubMed1993

Induction of immune response against a short synthetic peptide antigen coupled to small neutral liposomes containing monophosphoryl lipid A.

We have investigated the parameters affecting the immunogenicity of a short synthetic hexapeptide associated with liposomes. The model peptide used had the sequence IRGERA which corresponds to the C-terminal hexapeptide region of histone H3. Immunogenicity was measured by the ability of anti-peptide antibodies to cross-react with the parent protein. By itself, the peptide was not able to induce significant antibody production. However, liposomes were shown to be able to render the peptide immunogenic, nevertheless a number of parameters were important: to be immunogenic the peptide had to be surface bound, rather than entrapped within the liposomes, and an adjuvant, monophosphoryl lipid A (MPLA), had to be present in the same population of liposomes. Additionally, the intensity and duration of the immune response were found to be dependent both on the charge of the liposomes; neutral liposomes yielding a longer lasting response than negatively charged liposomes, and on the immunisation schedule where a long time period between immunisation and boosting yielded a better result than a short time period. To account for these phenomena we propose a model in which surface-bound antigen targets liposomal MPLA to B lymphocytes specific for the antigen. These results demonstrate that liposomes containing the non-toxic adjuvant MPLA can act as carriers to induce a long-lasting IgG response against peptides, eliminating the need of protein carriers and conventional adjuvants. Such an approach may be useful for designing synthetic vaccines.

Adjuvants, Immunologic↗
PubMed1993

Structure and binding properties of monoclonal antibodies to core histones from autoimmune mice.

Histones are frequent targets of self-reactive antibodies during autoimmune syndromes. We report the specificities and V region genes of three IgG anti-histone MAbs obtained from autoimmune mice. Each of the MAbs, named LG2-1, LG2-2 and BWA3, is directed against a different determinant located in the basic amino-terminal domain of core histones. LG2-1 reacts with a peptide from histone H3 (residues 30-45), LG2-2 recognizes the amino-terminus of H2B (residues 1-13) and BWA3 binds an epitope corresponding to a region of high sequence similarity between H2A and H4 (residues 1-20 and 1-29, respectively). The analysis of their V region sequences indicates that the H chain CDRs of these MAbs are remarkable for the presence of negatively charged amino acid residues that may play a role in the binding to cationic histones. The H chain importance in conferring reactivity to histones is corroborated by the observation that each of the VH gene segments of these MAbs is very similar to VH genes of previously described murine anti-histone antibodies.

Amino Acid Sequence↗
PubMed1993

[Immunogenetics of systemic lupus: adequacy between HLA molecules class II and the germinal genes of antigen receptor of T-lymphocytes favours the production of various antibodies].

The study of 30 patients with systemic lupus and of 153 relatives failed to show any differences in HLA and TCR beta haplotype frequencies between patients and relatives. A significant interaction between TCR V beta and HLA-DR/DQ genes in the response to the peptide U1-RNP A 35-58 was demonstrated.

Antibody Formation↗
PubMed1993

Membrane expression of HIV envelope glycoproteins triggers apoptosis in CD4 cells.

The cytopathic effect of HIV-1 and HIV-2 in CD4+ lymphocytes has been shown to be associated with apoptosis or programmed cell death. Using different experimental conditions, we demonstrate here that apoptosis is triggered by cell membrane expression of the mature HIV envelope glycoproteins, gp120-gp41 complex, and their interaction with CD4 receptor molecules. Viral entry alone did not induce apoptosis but virus replication was required in order to produce the gp120-gp41 complex. Indeed, expression of the HIV env gene alone in the CD4+ T cell line (CEM) was sufficient for the induction of apoptosis. In general, syncytium formation and apoptosis induction were closely associated as both events require functional envelope glycoproteins and CD4 molecules. Nevertheless, apoptosis but not syncytium formation was suppressed by a monoclonal antibody against CD4 that does not affect gp120 binding. Furthermore, single-cell killing by apoptosis was observed in infected cell cultures treated with a monoclonal antibody against gp41, which completely abolishes the formation of syncytia. These results indicate that apoptosis is not the consequence of toxic effects induced by the formation of syncytia but is triggered by the HIV envelope glycoproteins. Therefore, cell death during HIV infection in CD4+ lymphocyte cultures is due to a specific event triggered by the gp120-gp41 heterodimer complex programming death in metabolically active cells.

Amino Acid Sequence↗
PubMed1993

Study of antibodies to histones and histone synthetic peptides in pauciarticular juvenile chronic arthritis.

The objective of this study was to determine the prevalence of histone antibodies in early onset pauciarticular juvenile chronic arthritis (JCA), and to assess any association with disease activity in terms of arthritis or uveitis. Forty-four children with early onset pauciarticular JCA have been assessed prospectively using clinical and laboratory parameters. Sera were collected from these patients during clinical follow-up. Some of them had serial measurements during the period of study. Patient sera were analysed for histone antibodies by ELISA. A further study using 30 synthetic peptides of calf thymus core histones was performed by ELISA. Seventy-five per cent of children have IgM antibodies to histone, usually to more than one type. The highest values were anti H3 and H4 antibodies. There was no correlation between the level of anti-histone antibodies and anterior uveitis. There were increased levels of IgG antibodies to histone peptides, in particular the N- and C-terminal peptides of H3 in 70.5% of children. Antibodies to H3 peptides tend to be in patients with chronic anterior uveitis (P = 0.04).

Antibodies↗
PubMed1993

Swallowing disturbances associated with drooling in cerebral-palsied children.

The oral stage of swallowing was studied in two groups of 10 cerebral-palsied (CP) children (one drooled and the other did not), and 10 normal children, aged six to 14 years. Small amounts of liquid (0.5 to 1mL) were placed under the tongue or behind the lower lip and intra-oral pressure was measured during the suction and propulsion stages. The CP children who drooled showed no abnormality in the propulsion of liquid towards the pharynx, but all showed abnormal suction of liquid onto the tongue. The difficulty seems to be associated with three types of disturbance: incomplete lip-closure during swallowing, low suction-pressure and prolonged delay between the suction and propelling stages.

Adolescent↗
PubMed1993

Immunization of rabbits with spermine induces antibodies to self antigens.

Rabbits were immunized under different schedules with spermine in the free form or with random noncovalent complexes of spermine or spermidine with ovalbumin. The specificity of the induced antibodies was determined by ELISA and by dot-immunobinding assay. Our results show that in vitro conjugation of spermine and spermidine to a carrier is not an obligatory prerequisite for obtaining corresponding antibodies. Anti-spermine antibodies were found in 9 of 19 animals injected with spermine. Furthermore, all 19 rabbits produced distinct populations of IgM and IgG antibodies which reacted with histones, various synthetic peptides of histones, as well with ubiquitin, a peptide of ubiquitin, dsDNA and two 29-base 5' synthetic oligodeoxynucleotides. Even in antisera with no detectable reactivity with spermine, antibodies to some of the unrelated antigens were found. The pattern of reactivity of the antisera with the various antigens was different for each immunized animal. These findings lend support to the view that polyamines may play a role in the appearance of autoantibodies.

Animals↗
PubMed1992

Application and limitations of the multiple antigen peptide (MAP) system in the production and evaluation of anti-peptide and anti-protein antibodies.

The multiple antigen peptide (MAP) system has been proposed as a novel and valuable approach for eliciting antibodies to peptides and developing synthetic vaccines. The MAP system consists of a small immunogenically inert core matrix of lysine residues with alpha- and epsilon-amino groups for anchoring multiple copies of the same or different synthetic peptides. Several MAP systems, each containing eight copies of 6-15 residue-long peptides derived from the terminal and central regions of various proteins were analyzed in this study. The immunogenicity of MAPs was compared to that of the same peptides linked to carrier protein by means of conventional conjugation procedures. The various peptide antisera were tested in ELISA with homologous peptides conjugated to a carrier protein via their C terminal (as in the MAP system) or their N terminal end, or with their parent proteins. The antigenic properties of MAPs were studied with anti-peptide sera obtained by classical methods and with anti-protein sera. The results showed that the MAP system was an efficient antigen in ELISA except when the peptide corresponded to a C terminal epitope. However, the value of MAPs for raising anti-peptide antibodies cross-reactive with the cognate protein appeared much more limited. In the case of one N terminal peptide, the MAP construction was not immunogenic while the conventionally conjugated peptide induced antibodies that reacted strongly with the corresponding protein. In the case of the two C terminal peptides tested, the antibodies raised against MAP constructs reacted well with homologous MAPs but did not cross-react with the whole protein. Only in the case of a peptide from an internal domain of histone H2A did immunization with a MAP generate antibodies that cross-reacted with the protein.

Amino Acid Sequence↗
PubMed1992

Rearrangements of the nucleosome structure in chromatin by poly(ADP-ribose).

In order to approach and clarify the effect of poly(ADP-ribose) on the nucleosomal structure, polynucleosomes from calf thymus were incubated with long poly(ADP-ribose) chains prepared in vitro and examined by ELISA with antibodies directed against the five individual histones H1, H2A, H2B, H3 and H4 as well as against two synthetic peptides in residues 1-25 of H2B and 130-135 of H3. The results showed that: (i) free ADP-ribose polymers did indeed interact with the nucleosomes; (ii) the accessibility of epitopes recognized by any of the different antibodies was altered, the binding of antibodies being increased or decreased depending on the quantity of poly(ADP-ribose) added thereby suggesting a modulation in nucleosome structure; (iii) for any ADP-ribose polymer concentration, core histones as well as histone H1 were always recognized by their respective antibodies, thus suggesting that poly(ADP-ribose) does not seem to cause complete stripping of histones from nucleosomal DNA.

Adenosine Diphosphate Ribose↗
PubMed1992

Immunoelectron microscopical distribution of histones H2B and H3 and protamines in the course of mouse spermiogenesis.

We have followed the fine structural distribution of two nucleosomal core histones, H2B and H3, and of protamines in the course of mouse spermiogenesis by means of specific antibodies and ultrastructural immunocytochemistry. Our results demonstrate that the nuclear labeling density of histone H2B decreases during steps 6-8 and then increases again in step 9-10 spermatids, while the labeling for histone H3 is constant throughout this period. In step 12 spermatids, the anti-H2B antibody labels mainly the central area of the nucleus. The first signs of protamine labeling are present in step 12 spermatids, where the gold grains can be found over the periphery of the nucleus. Later on, protamine labeling constantly increases and, by the end of spermiogenesis, the whole nucleus is labeled. We suggest that the morphological and structural differences between the central area and the periphery of mouse spermatids are, at least partly, due to a difference in the protein moiety associated with DNA. The central area, which is peculiar to the mouse and has been previously considered as a focus of chromatin condensation, represents, however, the last nuclear region containing histones and consequently the last area where the substitution of histones by protamines takes place.

Animals↗
PubMed1992

A lipoyl synthetic octadecapeptide of dihydrolipoamide acetyltransferase specifically recognized by anti-M2 autoantibodies in primary biliary cirrhosis.

Close to 95% of patients with established clinical, biochemical and histologic features of primary biliary cirrhosis (PBC) possess antimitochondrial M2 antibodies reacting with the E2 component, dihydrolipoamide acetyltransferase, of the pyruvate dehydrogenase complex. We examined the ability of synthetic peptides of E2 to be recognized in ELISA by sera from patients with PBC and autoimmune-related disorders. Sera from 14 PBC M2+ patients, 1 PBC M2- patient, 5 non-PBC M2+ patients, and 6 patients with chronic active hepatitis were studied. Among the seven E2 synthetic peptides tested (namely peptides 87-119, 167-184, 169-202, 267-302, 456-477, 498-513 and 530-543), only peptide 167-184 used as OVA conjugate and prepared with lipoic acid (LA) located on lysine 173 (natural inner lipoyl-binding site) was recognized in direct ELISA by PBC M2+ sera. The conjugated peptide 167-184 LA was not recognized in direct ELISA by non-PBC M2+ sera or by sera from patients with chronic active hepatitis. The free peptide 167-184 LA inhibited the ELISA reaction of PBC antibodies to PDH and totally abolished the typical immunofluorescence reaction of PBC sera on rat kidney, stomach and liver, or human HEp-2 cell substrates. No inhibition of ELISA or immunofluorescence reaction was found with the other E2 fragments including peptide 167-184 without LA. Our results show that the lipoyl moiety forms an integral part of a dominant conformational epitope recognized by PBC sera. Inasmuch as the peptide 167-184 LA was not recognized by non-PBC sera in direct ELISA, it could be used as a valuable probe for PBC diagnosis.

Acetyltransferases↗
PubMed1992

Multiple autoepitope presentation for specific detection of antibodies in primary biliary cirrhosis.

Antimitochondrial autoantibodies are present in sera from close to 95% of patients with primary biliary cirrhosis. The so-called primary biliary cirrhosis-specific antigen, named M2, was found to be associated with an enzyme complex of the inner mitochondrial membrane and, more precisely, with the E2 component, dihydrolipoamide acetyltransferase, of the pyruvate dehydrogenase complex. We recently established that an immunodominant epitope recognized in direct enzyme-linked immunosorbent assay by primary biliary cirrhosis M2+ sera, but not by non-primary biliary cirrhosis M2+ sera, could be mimicked by a synthetic peptide encompassing residues 167-184 of the E2 component and associated with lipoic acid. This fragment is present in the natural inner lipoyl-binding site of the human enzyme, and the presence of lipoic acid located on lysine 173 was found to be essential to allow IgG antibody binding. In this study we have improved the enzyme-linked immunosorbent assay test based on the synthetic peptide-lipoic acid conjugate by using a multiple antigen peptide system containing eight copies of the peptide as antigen. This approach avoids the use of a peptide conjugated to a carrier protein and was found to be particularly efficient because 23 of 27 primary biliary cirrhosis M2+ sera (85%) could be identified. A multiple antigen peptide without lipoic acid was not recognized by primary biliary cirrhosis antibodies. The peptide used in the multiple antigen peptide construction was a short 13-mer peptide encompassing a highly conserved sequence present in both the outer (residues 40-52) and the inner (residues 167-179) lipoyl-binding sites of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetyltransferases↗
PubMed1992

Use of fluorescence anisotropy determinations for indicating the physiological status of hybridoma cell cultures.

The evolution of lipid compartment fluidity during culture of hybridoma cells was studied by fluorescence polarization measurements. The probe partition between the plasma membrane and intracytoplasmic compartments was determined by a quenching fluorescence method. A progressive decrease of the plasma membrane fluidity was observed during the growth phase with an increase during stationary and degeneration phases of the culture. These data suggest that fluidity parameters could be used to follow the behaviour of hybridoma cell cultures.

Cell Membrane↗