Ets factors play a critical role in oncogenic Ras- and growth
factor-mediated regulation of the proximal rat prolactin (rPRL)
promoter in pituitary cells. The rPRL promoter contains two key
functional Ets binding sites (EBS): a composite EBS/Pit-1
element located at –212 and an EBS that co-localizes with
the basal transcription element (BTE, or A-site) located at –96. Oncogenic
Ras exclusively signals to the –212 site, which we have
named the Ras response element (RRE); whereas the response of multiple
growth factors (FGFs, EGF, IGF, insulin and TRH) maps to both EBSs.
Although Ets-1 and GA binding protein (GABP) have been implicated
in the Ras and insulin responses, respectively, the precise identity
of the pituitary Ets factors that specifically bind to the RRE and
BTE sites remains unknown. In order to identify the Ets factor(s)
present in GH4 and GH3 nuclear extracts (GH4NE and GH3NE) that bind
to the EBSs contained in the RRE and BTE, we used EBS-RRE and BTE
oligonucleotides in electrophoretic mobility shift assays (EMSAs),
antibody supershift assays, western blot analysis of partially purified
fractions and UV-crosslinking studies. EMSAs, using either the BTE
or EBS-RRE probes, identified a specific protein–DNA complex...
The Biomolecular Interaction Network Database (BIND; http://binddb.org)
is a database designed to store full descriptions of interactions,
molecular complexes and pathways. Development of the BIND 2.0 data
model has led to the incorporation of virtually all components of
molecular mechanisms including interactions between any two molecules
composed of proteins, nucleic acids and small molecules. Chemical
reactions, photochemical activation and conformational changes can
also be described. Everything from small molecule biochemistry to signal
transduction is abstracted in such a way that graph theory methods
may be applied for data mining. The database can be used to study
networks of interactions, to map pathways across taxonomic branches
and to generate information for kinetic simulations. BIND anticipates
the coming large influx of interaction information from high-throughput proteomics
efforts including detailed information about post-translational
modifications from mass spectrometry. Version 2.0 of the BIND data
model is discussed as well as implementation, content and the open
nature of the BIND project. The BIND data specification is available
as ASN.1 and XML DTD.
p53 is a tumor-suppressor protein that can activate and repress transcription. Using the yeast two-hybrid system, we identified two previously uncharacterized human proteins, designated 53BP1 and 53BP2, that bind to p53. 53BP1 shows no significant homology to proteins in available databases, whereas 53BP2 contains two adjacent ankyrin repeats and a Src homology 3 domain. In vitro binding analyses indicate that both of these proteins bind to the central domain of p53 (residues 80-320) required for site-specific DNA binding. Consistent with this finding, p53 cannot bind simultaneously to 53BP1 or 53BP2 and to a DNA fragment containing a consensus p53 binding site. Unlike other cellular proteins whose binding to p53 has been characterized, both 53BP1 and 53BP2 bind to the wild-type but not to two mutant p53 proteins identified in human tumors, suggesting that binding is dependent on p53 conformation. The characteristics of these interactions argue that 53BP1 and 53BP2 are involved in some aspect of p53-mediated tumor suppression.
The replication of γ origin, a minimal replicon derived from plasmid R6K, is controlled by the Rep protein π. At low intracellular concentrations, π activates the γ origin, while it inhibits replication at elevated concentrations. Additionally, π acts as a transcription factor (auto)repressing its own synthesis. These varied regulatory functions depend on π binding to reiterated DNA sequences bearing a TGAGNG motif. However, π also binds to a “non-iteron” site (i.e., not TGAGNG) that resides in the A+T-rich region adjacent to the iterons. This positioning places the non-iteron site near the start sites for leading-strand synthesis that also occur in the A+T-rich region of γ origin. We have hypothesized that origin activation (at low π levels) would require the binding of π monomers to iterons, while the binding of π dimers to the non-iteron site (at high π levels) would be required to inhibit priming. Although monomers as well as dimers can bind to an iteron, we demonstrate that only dimers bind to the non-iteron site. Two additional pieces of data support the hypothesis of negative replication control by π binding to the non-iteron site. First, π binds to the non-iteron site about eight times less well than it binds to a single iteron. Second...
The Biomolecular Interaction Network Database (BIND: http://bind.ca) archives biomolecular interaction, complex and pathway information. A web-based system is available to query, view and submit records. BIND continues to grow with the addition of individual submissions as well as interaction data from the PDB and a number of large-scale interaction and complex mapping experiments using yeast two hybrid, mass spectrometry, genetic interactions and phage display. We have developed a new graphical analysis tool that provides users with a view of the domain composition of proteins in interaction and complex records to help relate functional domains to protein interactions. An interaction network clustering tool has also been developed to help focus on regions of interest. Continued input from users has helped further mature the BIND data specification, which now includes the ability to store detailed information about genetic interactions. The BIND data specification is available as ASN.1 and XML DTD.
Rhodococcus equi is a facultative intracellular bacterium of macrophages that causes disease in immunocompromised individuals, particularly those with AIDS. In this report, we demonstrate that R. equi binding to mammalian cells requires complement and is mediated primarily by the leukocyte complement receptor, Mac-1. Bacteria bind to macrophages poorly unless exogenous complement is added to the incubation medium. The addition of fresh nonimmune serum, which contains no detectable antibodies to R. equi, greatly enhances bacterial binding to macrophages, whereas heat inactivation of this serum or immunological depletion of C3 from the serum reduces binding to levels only slightly higher than those of binding under serum-free conditions. Human serum depleted of C2 or C4 is fully opsonic, indicating that complement activation and fixation occur by the alternative pathway. The serum-dependent binding of rhodococci to macrophages is mediated primarily by the macrophage complement receptor type 3, Mac-1 (CD11b/CD18). Bacteria do not bind to fibroblastoid or epithelial cells that lack this receptor. Most of the bacterial binding to macrophages is inhibited by a monoclonal antibody to Mac-1 but is unaffected by a monoclonal antibody to complement receptor type 1. Furthermore...
Pneumonia is one of the most common causes of death from infectious disease in the United States. To examine the possible role of carbohydrates as adhesion receptors for infection, several pulmonary pathogenic bacteria were studied for binding to glycosphingolipids. Radiolabeled bacteria were layered on thin-layer chromatograms of separated glycosphingolipids, and bound bacteria were detected by autoradiography. The classic triad of infectious bacteria found in cystic fibrosis, Pseudomonas aeruginosa, Haemophilus influenzae, and Staphylococcus aureus, along with other bacteria commonly implicated in typical pneumonia, such as Streptococcus pneumoniae, Klebsiella pneumoniae, and certain Escherichia coli, bind specifically to fucosylasialo-GM1 (Fuc alpha 1-2Gal beta 1-3GalNAc beta 1-4Gal beta 1-4Cer), asialo-GM1 (Gal beta 1-3GalNAc beta 1-4Gal beta-1-4Galc beta 1-1Cer), and asialo-GM2 (GalNAc beta 1-4Gal beta 1-4Glc beta 1-1Cer). Bacteria maintained in nutrient medium bind better than the same cells suspended in buffer. They do not bind to galactosylceramide, glucosylceramide, lactosylceramide, trihexosylceramide, globoside, paragloboside, Forssman glycosphingolipid, or several other glycosphingolipids tested, including the gangliosides GM1...
Histones can mediate the binding of DNA and anti-DNA to the glomerular basement membrane (GBM). In ELISA histone/DNA/anti-DNA complexes are able to bind to heparan sulfate (HS), an intrinsic constituent of the GBM. We questioned whether histone containing immune complexes are able to bind to the GBM, and if so, whether the ligand in the GBM is HS. Monoclonal antibodies (mAbs) complexed to nucleosomal antigens and noncomplexed mAbs were isolated from culture supernatants of four IgG anti-nuclear mAbs. All noncomplexed mAbs showed strong anti-nucleosome reactivity in ELISA. One of them showed in addition anti-DNA reactivity in noncomplexed form. The other three mAbs only showed anti-DNA reactivity when they were complexed to nucleosomal antigens. After renal perfusion a fine granular binding of complexed mAbs to the glomerular capillary wall and activation of complement was observed in immunofluorescence, whereas noncomplexed mAbs did not bind. Immuno-electron microscopy showed binding of complexes to the whole width of the GBM. When HS in the GBM was removed by renal heparinase perfusion the binding of complexed mAb decreased, but did not disappear completely. We conclude that anti-nucleosome mAbs, which do not bind DNA, become DNA reactive once complexed to nucleosomal antigens. These complexed mAbs can bind to the GBM. The binding ligand in the GBM is partly...
In vertebrates, densely methylated DNA is associated with inactive transcription. Actors in this process include proteins of the MBD family that can recognize methylated CpGs and repress transcription. Kaiso, a structurally unrelated protein, has also been shown to bind methylated CGCGs through its three Krüppel-like C2H2 zinc fingers. The human genome contains two uncharacterized proteins, ZBTB4 and ZBTB38, that contain Kaiso-like zinc fingers. We report that ZBTB4 and ZBTB38 bind methylated DNA in vitro and in vivo. Unlike Kaiso, they can bind single methylated CpGs. When transfected in mouse cells, the proteins colocalize with foci of heavily methylated satellite DNA and become delocalized upon loss of DNA methylation. Chromatin immunoprecipitation suggests that both of these proteins specifically bind to the methylated allele of the H19/Igf2 differentially methylated region. ZBTB4 and ZBTB38 repress the transcription of methylated templates in transfection assays. The two genes have distinct tissue-specific expression patterns, but both are highly expressed in the brain. Our results reveal the existence of a family of Kaiso-like proteins that bind methylated CpGs. Like proteins of the MBD family, they are able to repress transcription in a methyl-dependent manner...
More than 90% of the major histocompatibility complex (MHC) class II molecules on antigen-presenting cells (APC) have in their binding site a peptide derived from an extracellular protein ingested by the APC or from a protein of the APC itself. These self-peptides can be eluted from affinity-purified MHC class II molecules by acid elution, and have been studied with a variety of techniques. We show here that the self-peptides eluted from the mouse MHC class II molecules Ad, Ed and Ek bind specifically to MHC class II molecules of the allelic type from which they were derived. The pH optimum for binding is around 5.0, i.e. the same optimum at which synthetic peptides representing sequences of foreign antigens bind to MHC class II molecules. This suggests that the physiological compartment where MHC class II molecules bind self-peptides may be very late in the endocytic pathway. The chemical properties of the eluted and labelled MHC class II peptides were studied by isoelectric focusing. This method was able to separate the peptides very efficiently, and enabled a rapid comparison of peptides eluted from different MHC molecules. The 125I-labelled peptides displayed a broad range of isoelectric points with values predominantly below neutral. This suggests that such peptides bind to MHC in a predominantly non-charged state.
Patients with systemic lupus erythematosus (SLE) frequently have anti-lymphocyte autoantibodies, some of which also bind to surfaces of neurons. Since anti-ribosomal P protein autoantibodies (anti-P) from SLE patients also bind to surfaces of neurons, we hypothesized that anti-P are anti-lymphocyte antibodies. A panel of human T lymphocytes was evaluated for anti-P binding by indirect immunofluorescence. Affinity-purified anti-ribosomal antibodies were used as a source of anti-P. These autoantibodies bound to the surfaces of all transformed T cell lines tested. This binding was not mediated by Fc receptors. It was inhibitable by ribosomes. Anti-P bound to circulating T lymphocytes from healthy adults and children. They also bound to thymocytes and cord blood T cells from normal neonates. Circulating T cells from SLE patients with anti-P bound less anti-P than cells from healthy controls. Two patients were studied on multiple occasions. The capacity of their T cells to bind anti-P correlated inversely with titres of anti-ribosomal antibodies. Anti-ribosomal antibodies, other than anti-P, also appear to bind to T cells. The surface of T cells contains a protein with the size and antigenicity of the ribosomal P protein, P0. We conclude that anti-ribosomal antibodies are a subset of anti-lymphocyte autoantibodies. Their possible role in the pathogenesis of lymphopenia or lymphocyte dysfunction in SLE has to be defined in further studies.
This study evaluates the cell surface expression of urokinase-type plasminogen activator (u-PA) and the capacity to bind exogenous urokinase as possible parameters for the distinction of various types of human lung tumours. Twelve different tumour cell lines including four small cell carcinoma, two large cell carcinoma, three squamous cell carcinoma, one adenocarcinoma and two mesothelioma cell lines of lung origin were investigated. Surface expression of endogenous u-PA was determined in a cellular radioimmunoassay (CRIA) using the u-PA-specific monoclonal antibody 98/6. To estimate additional u-PA binding capacity, exogenous two-chain, 54 kDa u-PA was employed in the CRIA. The influence of phorbol ester (PMA) treatment on expression and binding of these molecules was studied. Three different groups of lung tumour cell lines could be distinguished according to their expression of u-PA and u-PA-binding ability: (i) non small cell lung carcinoma (NSCLC) cell lines of squamous cell carcinoma/adenocarcinoma origin expressed small amounts of u-PA and bound little u-PA. Large cell carcinoma cell lines expressed high amounts of u-PA and bound large amounts of u-PA. In general, expression of u-PA and u-PA binding was enhanced after PMA treatment. (ii) Mesothelioma cell lines did not express u-PA...
Electron microscopy of myosin-II molecules and filaments reacted with monoclonal antibodies demonstrates directly where the antibodies bind and shows that certain antibodies can inhibit the polymerization of myosin-II into filaments. The binding sites of seven of 23 different monoclonal antibodies were localized by platinum shadowing of myosin monomer-antibody complexes. The antibodies bind to a variety of sites on the myosin-II molecule, including the heads, the proximal end of the tail near the junction of the heads and tail, and the tip of the tail. The binding sites of eight of the 23 antibodies were also localized on myosin filaments by negative staining. Antibodies that bind to either the myosin heads or to the proximal end of the tail decorate the ends of the bipolar filaments. Some of the antibodies that bind to the tip of the myosin-II tail decorate the bare zone of the myosin-II thin filament with 14-nm periodicity. By combining the data from these electron microscope studies and the peptide mapping and competitive binding studies we have established the binding sites of 16 of 23 monoclonal antibodies. Two of the 23 antibodies block the formation of myosin-II filaments and given sufficient time, disassemble preformed myosin-II filaments. Both antibodies bind near one another at the tip of the myosin-II tail and are those that decorate the bare zone of preformed bipolar filaments with 14-nm periodicity. None of the other antibodies affect myosin filament formation...
Polyspecificity is a well-known property of the anti-DNA antibodies produced by autoimmune animals. In our search for antigen targets of anti-DNA antibodies within tissue extracts, we identified a 32-kD polypeptide that was recognized by a large panel of anti-DNA antibodies. Direct sequencing of this protein disclosed its identity with DNase I. 22 monoclonal anti-DNA antibodies bound to DNase I in direct and competitive immunoassays; out of 15 autoantibodies that did not bind DNA, none had the ability to bind DNase I. The ability of anti- DNA antibodies to interfere with DNase I enzymatic activity was evaluated in an assay based on the enzyme digestion of phage double strand DNA. Six monoclonal anti-single strand DNA antibodies that did not bind double strand DNA were tested in this assay. Three out of six inhibited DNase I-mediated digestion of phage DNA. The interaction of anti-DNA antibodies with DNase I was further investigated by testing their ability to bind a synthetic peptide that corresponds to the catalytic site of the molecule. 4 out of 22 anti-DNA antibodies bound the active site peptide; two of these had been shown to inhibit DNase I enzymatic activity. This report show that anti-DNA antibodies recognize both DNA and its natural ligand DNase I. Some anti-DNA antibodies inhibit DNase I enzymatic activity...
Group B streptococci (GBS) are the most common cause of neonatal sepsis and meningitis. Most infants who are colonized with GBS at birth do not develop invasive disease, although many of these uninfected infants lack protective levels of capsular polysaccharide (CPS)-specific antibody. The lectin pathway of complement is a potential mechanism for initiating opsonization of GBS with CPS-specific antibody-deficient serum. In this study, we determined whether mannose-binding lectin (MBL)/MBL-associated serine protease (MASP) complexes and L-ficolin/MASP complexes bind to different strains of GBS to activate the lectin pathway, and we identified the molecules recognized by lectins on the GBS surface. We found that MBL did not bind to any GBS examined, whereas L-ficolin bound to GBS cells of many serotypes. L-ficolin binding to GBS cells correlated with the CPS content in serotypes Ib, III (restriction digestion pattern types III-2 and III-3), and V but not with the group B-specific polysaccharide (GBPS) content or with the lipoteichoic acid (LTA) content. L-ficolin bound to purified CPS and GBPS in a concentration-dependent manner but not to purified LTA. All strains to which L-ficolin/MASP complexes bound consumed C4. When N-acetylneuraminic acid (NeuNAc) was selectively removed from GBS cells by treatment with neuraminidase...
DEC205/CD205, an endocytic receptor of C-type multilectin, is expressed highly in dendritic cells (DCs). DEC205 was shown to efficiently deliver vaccine antigens in surrogate ligands to the antigen processing and presentation machinery of DCs, which resulted in the development of DC-targeted vaccines employing anti-DC monoclonal antibodies (mAbs). During our studies to characterize a variety of anti-DC mAbs including anti-DEC205 by flow cytometric analysis, we discovered that a secondary anti-immunoglobulin antibody conjugated with PE-Cy5.5 bound strongly to the cells expressing mouse DEC205 (mDEC205) without incubation of a primary anti-mDEC205 mAb. In the present study we demonstrate that various antibodies and streptavidin conjugated with PE-Cy5.5 bind to the mDEC205-expressing cells including CHO, KIT6, and HEK293 cells. The interaction between the PE-Cy5.5 conjugates and the cells expressing mDEC205 appears distinctive, since none of PE-Cy5.5 conjugates bind to the cells that express human DEC205 on surface. Besides, only PE-Cy5.5 conjugates bind strongly to mDEC205-expressing cells; PerCP-Cy5.5, APC-Cy5.5, and Cy5.5 conjugates bind weakly; PE, PE-Cy5, Cy5, FITC, or Alexa488 conjugates do not bind. Therefore the use of PE-Cy5.5 conjugates...
p300 is a transcriptional coactivator that participates in many important processes in the cell, including proliferation, differentiation and apoptosis. The viral oncoproteins, adenovirus (Ad) E1A and human papillomavirus (HPV) E7, have been implicated in binding to p300. The Ad-E1A/p300 interaction has been shown to result in an induction of cellular proliferation, epigenetic reprogramming, as well as cellular transformation and cancer. The HPV-E7/p300 interaction, on the other hand, is not well understood. p300 contains three zinc-binding domains, CH1-CH3, and studies have shown that Ad-E1A can bind to the p300 CH1 and CH3 domains, whereas E7 can bind to the CH1 domain and to a lesser extent to the CH2 and CH3 domains. Here we address how high risk HPV16-E7 and Ad5-E1A, which have different structures, can both bind the p300 CH1 domain. Using pull down, gel filtration, and analytical ultracentrifugation studies, we show that the N-terminus and CR1 domains of Ad5-E1A and that the CR1-CR2 domains of HPV16-E7 bind to the p300 CH1 domain competitively and with mid- nanomolar and low micromolar dissociation constants, respectively. We also show that Ad5-E1A can form a ternary complex with the p300 CH1 domain and the retinoblastoma pRb transcriptional repressor...
Fonte: Universidade Nacional da AustráliaPublicador: Universidade Nacional da Austrália
Tipo: Thesis (Masters); Master of Philosophy (MPhil)
Relevância na Pesquisa
The present thesis reports on the results of an experiment designed to measure certain aspects of the double bind hypothesis, which was developed by the Bateson group in 1956 to account for the aetiology of schizophrenia. Two ingredients of the total double bind situation were studied, firstly, conflict between levels of communication and the importance of a command keeping the victim of the double bind in the field. These two conditions were seen as important distinguishing points between double bind frustration and the ordinary type of frustration. The experimenter tested 80 normal subjects in a problem solving situation similar to one used by Maier (1949) in his experiments with rats. In the experiment, subjects were given a number of problems to solve and some were frustrated in the middle of the experiment. The general hypothesis was that double bind subjects would show greater performance deterioration after frustration than subjects who experienced the ordinary frustration situation (i.e. contradiction subjects). Performance deterioration was measured in terms of response latencies, number of correct answer, and the number of “fixated”- responses. The result offered limited support for the hypothesis. In general, double bind subjects and contradiction subjects performed equally badly on the problem...
A desconstrução proposta por Derrida nos permite ver a tradução como um acontecimento lingüistico singular. Esta dimensão enseja a reflexão sobre a diferença entre a chamada língua materna e a língua estrangeira. O pressuposto é que, com base nessa diferença, a língua do tradutor adquire um novo papel, como meio de transformação e produção de significado na língua para a qual se traduz. Esse acontecimento lingüístico cria um certo tipo de manifestação da tradução. Meu objetivo principal neste artigo é refletir sobre a relação existente entre as línguas envolvidas no processo de tradução através do double bind. O artigo analisa os prefácios, posfácios e notas de tradução de versões inglesas de alguns textos de Derrida a fim de verificar como os tradutores utilizam a desconstrução para explicar e justificar suas traduções. A hipótese desta reflexão é que entre a língua de Jacques Derrida — o francês — e a língua do tradutor — o inglês — há tradução recíproca.; The manifestation of translation and the ‘double bind’: the writing of Jacques Derrida and his translations. — The deconstruction proposed by Jacques Derrida allows us to see translation as a distinct language event. This dimension calls for a reflection on the distinction between the so-called mother tongue and the foreign language; we assume that...
FUJIWARA, GISLENE MARI; Centro de Pesquisa e Processamento de Alimentos CEPPA; TALAMINI, ANELISE; Centro de Pesquisa e Processamento de Alimentos CEPPA; BEUX, MARCIA REGINA; Centro de Pesquisa e Processamento de Alimentos CEPPA
Este trabalho teve por objetivo avaliar dois métodos para a pesquisa de Salmonella sp em alimentos. O projeto foi dividido em duas etapas: a primeira, realizada com cepas padrão, visou a verificação da especificidade dos métodos, enquanto a segunda comparou os resultados obtidos. Os métodos utilizados foram o cultural clássico, indicado pela Association of Official Analytical Chemists (AOAC), pela Food and Drug Administration (FDA) e pela legislação brasileira e o BIND test rápido, ainda não citado como método usual, mas já aceito pela AOAC. Os resultados demonstraram que não há diferença entre os métodos, ficando a escolha de utilização a critério dos laboratórios.; This work is aimed to evaluate two survey methods for Salmonella in food, by means of simple comparison tests between the methods applied. The project was divided in two stages: the first, accomplished with standard strains, was aimed to verify the specificity of the methods, whereas the second part made the confrontation of the results obtained. The methods used were the Classic Cultural Method, indicated by AOAC, FDA, and by Brazilian Legislation as the chosen method for the research of Salmonella...