962 resultados para Core-binding-factor
Resumo:
Burkholderia cenocepacia is an opportunistic pathogen that displays a remarkably high resistance to antimicrobial peptides. We hypothesize that high resistance to antimicrobial peptides in these bacteria is because of the barrier properties of the outer membrane. Here we report the identification of genes for the biosynthesis of the core oligosaccharide (OS) moiety of the B. cenocepacia lipopolysaccharide. We constructed a panel of isogenic mutants with truncated core OS that facilitated functional gene assignments and the elucidation of the core OS structure in the prototypic strain K56-2. The core OS structure consists of three heptoses in the inner core region, 3-deoxy-d-manno-octulosonic acid, d-glycero-d-talo-octulosonic acid, and 4-amino-4-deoxy-l-arabinose linked to d-glycero-d-talo-octulosonic acid. Also, glucose is linked to heptose I, whereas heptose II carries a second glucose and a terminal heptose, which is the site of attachment of the O antigen. We established that the level of core truncation in the mutants was proportional to their increased in vitro sensitivity to polymyxin B (PmB). Binding assays using fluorescent 5-dimethylaminonaphthalene-1-sulfonyl-labeled PmB demonstrated a correlation between sensitivity and increased binding of PmB to intact cells. Also, the mutant producing a heptoseless core OS did not survive in macrophages as compared with the parental K56-2 strain. Together, our results demonstrate that a complete core OS is required for full PmB resistance in B. cenocepacia and that resistance is due, at least in part, to the ability of B. cenocepacia to prevent binding of the peptide to the bacterial cell envelope.
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Adrenomedullin (AM) is an important regulatory peptide involved in both physiological and pathological states. We have previously demonstrated the existence of a specific AM-binding protein (AMBP-1) in human plasma. In the present study, we developed a nonradioactive ligand blotting assay, which, together with high pressure liquid chromatography/SDS-polyacrylamide gel electrophoresis purification techniques, allowed us to isolate AMBP-1 to homogeneity. The purified protein was identified as human complement factor H. We show that AM/factor H interaction interferes with the established methodology for quantification of circulating AM. Our data suggest that this routine procedure does not take into account the AM bound to its binding protein. In addition, we show that factor H affects AM in vitro functions. It enhances AM-mediated induction of cAMP in fibroblasts, augments the AM-mediated growth of a cancer cell line, and suppresses the bactericidal capability of AM on Escherichia coli. Reciprocally, AM influences the complement regulatory function of factor H by enhancing the cleavage of C3b via factor I. In summary, we report on a potentially new regulatory mechanism of AM biology, the influence of factor H on radioimmunoassay quantification of AM, and the possible involvement of AM as a regulator of the complement cascade.
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Insulin-like growth factor binding protein (IGFBP)-3 modulates vascular development by regulating endothelial progenitor cell (EPC) behavior, specifically stimulating EPC cell migration. This study was undertaken to investigate the mechanism of IGFBP-3 effects on EPC function and how IGFBP-3 mediates cytoprotection following vascular injury.
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The interactions of epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) with the epidermal growth factor receptor (EGFR) were examined by insertion mutagenesis of the receptor. Seventeen insertions were made throughout a construct containing only the extracellular domain. This truncated receptor (sEGFR) was secreted and had a dissociation constant similar to that of the full-length solubilized receptor. Receptors with insertions within subdomain III were not secreted. Two receptors with insertions at positions 291 and 474, which border subdomain III, have significantly decreased binding to both EGF and TGF alpha relative to wild type. This confirms previous work demonstrating that subdomain III forms the primary binding site for EGF and TGF alpha. Four of the mutants within subdomain II had a decreased binding to TGF alpha relative to wild type, but had wild type binding to EGF. These results suggest that a region within subdomain II may selectively regulate the binding of TGF alpha. Two receptors which contained insertions within subdomains II and IV, approximately equidistant from the center of subdomain III, bound twofold more ligand molecules than wild type receptor, with an affinity similar to that of wild type receptor. These findings suggest that insertion at these positions allows the access of more than one ligand molecule to the binding site.
Resumo:
BACKGROUND: Neisseria meningitidis can cause severe infection in humans. Polymorphism of Complement Factor H (CFH) is associated with altered risk of invasive meningococcal disease (IMD). We aimed to find whether polymorphism of other complement genes altered risk and whether variation of N. meningitidis factor H binding protein (fHBP) affected the risk association.
METHODS: We undertook a case-control study with 309 European cases and 5,200 1958 Birth Cohort and National Blood Service cohort controls. We used additive model logistic regression, accepting P<0.05 as significant after correction for multiple testing. The effects of fHBP subfamily on the age at infection and severity of disease was tested using the independent samples median test and Student's T test. The effect of CFH polymorphism on the N. meningitidis fHBP subfamily was investigated by logistic regression and Chi squared test.
RESULTS: Rs12085435 A in C8B was associated with odds ratio (OR) of IMD (0.35 [95% CI 0.19-0.67]; P = 0.03 after correction). A CFH haplotype tagged by rs3753396 G was associated with IMD (OR 0.56 [95% CI 0.42-0.76], P = 1.6x10-4). There was no bacterial load (CtrA cycle threshold) difference associated with carriage of this haplotype. Host CFH haplotype and meningococcal fHBP subfamily were not associated. Individuals infected with meningococci expressing subfamily A fHBP were younger than those with subfamily B fHBP meningococci (median 1 vs 2 years; P = 0.025).
DISCUSSION: The protective CFH haplotype alters odds of IMD without affecting bacterial load for affected heterozygotes. CFH haplotype did not affect the likelihood of infecting meningococci having either fHBP subfamily. The association between C8B rs12085435 and IMD requires independent replication. The CFH association is of interest because it is independent of known functional polymorphisms in CFH. As fHBP-containing vaccines are now in use, relationships between CFH polymorphism and vaccine effectiveness and side-effects may become important.
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Cervical cancer is a multi-stage disease caused by human papillomaviruses (HPV) infection of cervical epithelial cells, but the mechanisms regulating disease progression are not clearly defined. Using 3-dimensional organotypic cultures, we demonstrate that HPV16 E6 and E7 proteins alter the secretome of primary human keratinocytes resulting in local epithelial invasion. Mechanistically, absence of the IGF-binding protein 2 (IGFBP2) caused increases in IGFI/II signalling and through crosstalk with KGF/FGFR2b/AKT, cell invasion. Repression of IGFBP2 is mediated by histone deacetylation at the IGFBP2 promoter and was reversed by treatment with histone deacetylase (HDAC) inhibitors. Our in vitro findings were confirmed in 50 invasive cancers and 79 cervical intra-epithelial neoplastic lesions caused by HPV16 infection, where IGFBP2 levels were reduced with increasing disease severity. In summary, the loss of IGFBP2 is associated with progression of premalignant disease, and sensitises cells to pro-invasive IGF signalling, and together with stromal derived factors promotes epithelial invasion.
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One of the various functions of proteins in biological systems is the transport of small molecules, for this purpose proteins have naturally evolved special mechanisms to allow both ligand binding and its subsequent release to a target site; a process fundamental to many biological processes. Transport of Vitamin E (a-tocopherol), a lipid soluble antioxidant, to membranes helps in the protection of polyunsaturated fatty acids against peroxidative damage. In this research, the ligand binding characteristics of several members of the CRALTRIO family of lipid binding proteins was examined; the recombinant human a-Tocopherol Transfer Protein (a-TIP), Supernatant Protein Factor (SPF)ffocopherol Associated Protein (TAP), Cellular Retinaldehyde Binding Protein (CRALBP) and the phosphatidylinositol transfer protein from S. cerevisiae Sec 14p. Recombinant Sec 14p was expressed and purified from E. coli for comparison of tocopherol binding to the two other recombinant proteins postulated to traffic a-tocopherol. Competitive binding assays using [3H]-a-tocopherol and Lipidex-l000 resin allowed determination of the dissociation constants ~) of the CRAL-TRIO proteins for a-tocopherol and - 20 hydrophobic ligands for evaluation of the possible biological relevance of the binding interactions observed. The KIs (nM) for RRR-a-tocopherol are: a-TIP: 25.0, Sec 14p: 373, CRALBP: 528 and SPFffAP: 615. This indicates that all proteins recognize tocopherol but not with the same affinity. Sec 14p bound its native ligand PI with a KI of381 whereas SPFffAP bound PI (216) and y-tocopherol (268) similarly in contrast to the preferential binding ofRRR-a-tocopherol by a-TIP. Efforts to adequately represent biologically active SPFff AP involved investigation of tocopherol binding for several different recombinant proteins derived from different constructs and in the presence of different potential modulators (Ca+2, Mg+2, GTP and GDP); none of these conditions enhanced or inhibited a-tocopherol binding to SPF. This work suggests that only aTTP serves as the physiological mediator of a-tocopherol, yet structural homology between proteins allows common recognition of similar ligand features. In addition, several photo-affmity analogs of a-tocopherol were evaluated for their potential utility in further elucidation of a-TTP function or identification of novel tocopherol binding proteins.
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The Vpu accessory protein promotes HIV-1 release by counteracting Tetherin/BST-2, an interferon-regulated restriction factor, which retains virions at the cell-surface. Recent reports proposed beta-TrCP-dependent proteasomal and/or endo-lysosomal degradation of Tetherin as potential mechanisms by which Vpu could down-regulate Tetherin cell-surface expression and antagonize this restriction. In all of these studies, Tetherin degradation did not, however, entirely account for Vpu anti-Tetherin activity. Here, we show that Vpu can promote HIV-1 release without detectably affecting Tetherin steady-state levels or turnover, suggesting that Tetherin degradation may not be necessary and/or sufficient for Vpu anti-Tetherin activity. Even though Vpu did not enhance Tetherin internalization from the plasma membrane (PM), it did significantly slow-down the overall transport of the protein towards the cell-surface. Accordingly, Vpu expression caused a specific removal of cell-surface Tetherin and a re-localization of the residual pool of Tetherin in a perinuclear compartment that co-stained with the TGN marker TGN46 and Vpu itself. This re-localization of Tetherin was also observed with a Vpu mutant unable to recruit beta-TrCP, suggesting that this activity is taking place independently from beta-TrCP-mediated trafficking and/or degradation processes. We also show that Vpu co-immunoprecipitates with Tetherin and that this interaction involves the transmembrane domains of the two proteins. Importantly, this association was found to be critical for reducing cell-surface Tetherin expression, re-localizing the restriction factor in the TGN and promoting HIV-1 release. Overall, our results suggest that association of Vpu to Tetherin affects the outward trafficking and/or recycling of the restriction factor from the TGN and as a result promotes its sequestration away from the PM where productive HIV-1 assembly takes place. This mechanism of antagonism that results in TGN trapping is likely to be augmented by beta-TrCP-dependent degradation, underlining the need for complementary and perhaps synergistic strategies to effectively counteract the powerful restrictive effects of human Tetherin.
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Context: Pregnant tissues express corticotropin-releasing factor (CRF), a peptide modulating fetal and placental ACTH and cortisol secretion. These actions are modulated by the locally expressed CRF-binding protein (CRF-BP). Objective: The objective of the study was to determine whether CRF, CRF-BP, ACTH, and cortisol concentrations change in amniotic fluid and umbilical cord plasma in the presence of intraamniotic infection/inflammation (IAI) in women with spontaneous labor at term. Design: This was a cross-sectional study. Setting: The study was conducted at a tertiary referral center for obstetric care. Patients: Patients included women in active labor at term with (n = 39) and without (controls; n = 78) IAI. Main Outcome Measures: Amniotic fluid and umbilical cord plasma concentrations of CRF, CRF-BP, ACTH, and cortisol measured by RIA and immunoradiometric assays were measured. Results: In patients with IAI, amniotic fluid CRF (0.97 +/- 0.18 ng/ml) and CRF-BP (33.06 +/- 5.54 nmol/liter) concentrations were significantly (P < 0.001) higher than in controls (CRF: 0.32 +/- 0.04 ng/ml; CRF-BP: 14.69 +/- 2.79 ml). The umbilical cord plasma CRF and CRF-BP concentrations were significantly (P < 0.001 for all) higher in women with IAI than in controls (CRF: 2.96 +/- 0.35 ng/ml vs. 0.38 +/- 0.18 ng/ml; CRF-BP: 152.12 +/- 5.94 nmol/liter vs. 106.9 +/- 5.97 nmol/liter). In contrast, amniotic fluid and umbilical cord plasma ACTH and cortisol concentrations did not differ between groups. Conclusions: Amniotic fluid and umbilical cord plasma CRF and CRF-BP concentrations are increased in women with spontaneous labor at term and IAI. CRF-BP may modulate CRF actions on ACTH and cortisol secretion, playing a pivotal role in limiting the inflammatory process and thus avoiding an overactivation of the fetal/placental hypothalamus-pituitary-adrenal axis at birth.
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In the present study we measured maternal plasma concentrations of two placental neurohormones, corticotropin-releasing factor (CRF) and CRF-binding protein (CRF-BP), in 58 at-risk pregnant women consecutively enrolled between 28 and 29 wk of pregnancy to evaluate whether their evaluation may predict third trimester-onset preeclampsia ( PE). The statistical significance was assessed by t test. The cut-off points for defining altered CRF and CRF-BP levels for prediction of PE were chosen by receiving operator characteristics curve analysis, and the probability of developing PE was calculated for several combinations of hormone testing results. CRF and CRF-BP levels were significantly ( both P < 0.0001) higher and lower, respectively, in the patients (n = 20) who later developed PE than in those who did not present PE at follow-up. CRF at the cut-off 425.95 pmol/liter achieved a sensitivity of 94.8% and a specificity of 96.9%, whereas CRF-BP at the cut-off 125.8 nmol/liter combined a sensitivity of 92.5% and a specificity of 82.5% as single markers for prediction of PE. The probability of PE was 34.5% in the whole study population, 93.75% when both CRF and CRF-BP levels were changed, and 0% if both hormone markers were unaltered. The measurement of CRF and CRF-BP levels may add significant prognostic information for predicting PE in at-risk pregnant women.
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A cellular receptor for the haemagglutinating enteroviruses (HEV), and the protein that mediates haemagglutination, is the membrane complement regulatory protein decay accelerating factor (DAF; CD55). Although primate DAF is highly conserved, significant differences exist to enable cell lines derived from primates to be utilized for the characterization of the DAF binding phenotype of human enteroviruses. Thus, several distinct DAF-binding phenotypes of a selection of HEVs (viz. coxsackievirus A21 and echoviruses 6, 7, 11-13, 29) were identified from binding and infection assays using a panel of primate cells derived from human, orang-utan, African Green monkey and baboon tissues. These studies complement our recent determination of the crystal structure of SCR(34) of human DAF [Williams, P., Chaudhry, Y., Goodfellow, I. G., Billington, J., Powell, R., Spiller, O. B., Evans, D. J. & Lea, S. (2003). J Biol Chem 278, 10691-10696] and have enabled us to better map the regions of DAF with which enteroviruses interact and, in certain cases, predict specific virus-receptor contacts.
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Treatment of murine Swiss 3T3 fibroblasts and XB/2 keratinocytes with UV-B light (302 nm) resulted in a dose-dependent inhibition of [125I] epidermal growth factor (EGF) binding. The light dose required to achieve 50% inhibition of binding in both cell types was 80–85 J/m2 Decreased [125I] platelet-derived growth factor binding was not evoked even by light doses of up to 280 J/m2 UV-B irradiation did not stimultate phosphorylation of the 80 kd protein substrate for protein kinase C. Furthermore, its effect on [125I]EGF binding was not altered as a consequence of protein kinase C down-regulation following prolonged exposure of cells to phorbol esters. These results indicate that UV-B-induced transmodulation of the epidermal growth factor receptor is a specific event mediated through a protein kinase C-indepen dent pathway. Transfer of culture medium from irradiated cells to untreated control cells showed this effect was not induced as a result of transforming growth factor α release and subsequent binding to the EGF receptor in these cells.
Resumo:
In view of the reported inflammatory effects of corticotrophin-releasing factor (CRF) and the associated regulatory elements in the gene of its binding protein (BP), we postulate that both BP as well as novel BP-ligands other than CRF may be involved in inflammatory disease. We have investigated BP in the blood of patients with arthritis and septicaemia and have attempted to identify CRF and other BP-ligands in synovial fluid. The BP was found to be significantly elevated in the blood of patients with rheumatoid arthritis and septicaemia. There was less BP-ligand and CRF in synovial fluid from patients with rheumatoid arthritis that from those with osteo- or psoriatic arthritis. There was at least 10-fold more BP-ligand than CRF in the fluid of all three groups of patients. A small amount of immunoreactive human (h)CRF, eluting in the expected position of CRF-41, was detected after high-pressure liquid chromatography of arthritic synovial fluid; however, the bulk of material with BP-ligand binding activity eluted earlier, suggesting that synovial fluid contained novel peptides that interacted with the BP. These results would suggest that the BP and its ligands could play an endocrine immunomodulatory role in inflammatory disease.
Resumo:
We have suggested recently that the fall in plasma CRF-binding protein (BP) during the last few weeks of pregnancy is a direct effect of association with its ligand because of the rapid decrease in plasma BP concentration seen in normal males reaching a nadir some 15 min after a bolus injection of synthetic CRF. In the present study, we have investigated the physicochemical properties of both natural and recombinant BP by gel filtration under physiological conditions and have shown that association of human CRF to this BP results in an increase in molecular weight consistent with the formation of a dimer form of the BP ligand complex. The dimer is more stable when the interaction occurs in the presence of serum or if a peptide with a higher affinity for the BP is substituted as ligand. Experimental evidence would also suggest that the dimer BP has a higher affinity for ligand than the monomeric form. We suggest that this dimerization occurs in vivo when CRF is released into the bloodstream and provides the trigger that causes the uptake of the complex at specific receptor sites.
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TNF alpha is an important mediator of catabolism in cachexia. Most of its effects have been characterized in peripheral tissues, such as skeletal muscle and fat. However, by acting directly in the hypothalamus, TNF alpha can activate thermogenesis and modulate food intake. Here we show that high concentration TNF alpha in the hypothalamus leads to increased O(2) consumption/CO(2) production, increased body temperature, and reduced caloric intake, resulting in loss of body mass. Most of the thermogenic response is produced by beta 3-adrenergic signaling to the brown adipose tissue (BAT), leading to increased BAT relative mass, reduction in BAT lipid quantity, and increased BAT mitochondria density. The expression of proteins involved in BAT thermogenesis, such as beta 3-adrenergic receptor, peroxisomal proliferator-activated receptor-gamma coactivator-1 alpha, and uncoupling protein-1, are increased. In the hypothalamus, TNF alpha produces reductions in neuropeptide Y, agouti gene-related peptide, proopiomelanocortin, and melanin-concentrating hormone, and increases CRH and TRH. The activity of the AMP-activated protein kinase signaling pathway is also decreased in the hypothalamus of TNF alpha-treated rats. Upon intracerebroventricular infliximab treatment, tumor-bearing and septic rats present a significantly increased survival. In addition, the systemic inhibition of beta 3-adrenergic signaling results in a reduced body mass loss and increased survival in septic rats. These data suggest hypothalamic TNF alpha action to be important mediator of the wastage syndrome in cachexia. (Endocrinology 151: 683-694, 2010)