The Murine Factor H-Related Protein FHR-B Promotes Complement Activation

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Marcell Cserhalmi

2017-09-01

Full Text Available Factor H-related (FHR proteins consist of varying number of complement control protein domains that display various degrees of sequence identity to respective domains of the alternative pathway complement inhibitor factor H (FH. While such FHR proteins are described in several species, only human FHRs were functionally investigated. Their biological role is still poorly understood and in part controversial. Recent studies on some of the human FHRs strongly suggest a role for FHRs in enhancing complement activation via competing with FH for binding to certain ligands and surfaces. The aim of the current study was the functional characterization of a murine FHR, FHR-B. To this end, FHR-B was expressed in recombinant form. Recombinant FHR-B bound to human C3b and was able to compete with human FH for C3b binding. FHR-B supported the assembly of functionally active C3bBb alternative pathway C3 convertase via its interaction with C3b. This activity was confirmed by demonstrating C3 activation in murine serum. In addition, FHR-B bound to murine pentraxin 3 (PTX3, and this interaction resulted in murine C3 fragment deposition due to enhanced complement activation in mouse serum. FHR-B also induced C3 deposition on C-reactive protein, the extracellular matrix (ECM extract Matrigel, and endothelial cell-derived ECM when exposed to mouse serum. Moreover, mouse C3 deposition was strongly enhanced on necrotic Jurkat T cells and the mouse B cell line A20 by FHR-B. FHR-B also induced lysis of sheep erythrocytes when incubated in mouse serum with FHR-B added in excess. Altogether, these data demonstrate that, similar to human FHR-1 and FHR-5, mouse FHR-B modulates complement activity by promoting complement activation via interaction with C3b and via competition with murine FH.

Evasion Mechanisms Used by Pathogens to Escape the Lectin Complement Pathway.

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Rosbjerg, Anne; Genster, Ninette; Pilely, Katrine; Garred, Peter

2017-01-01

The complement system is a crucial defensive network that protects the host against invading pathogens. It is part of the innate immune system and can be initiated via three pathways: the lectin, classical and alternative activation pathway. Overall the network compiles a group of recognition molecules that bind specific patterns on microbial surfaces, a group of associated proteases that initiates the complement cascade, and a group of proteins that interact in proteolytic complexes or the terminal pore-forming complex. In addition, various regulatory proteins are important for controlling the level of activity. The result is a pro-inflammatory response meant to combat foreign microbes. Microbial elimination is, however, not a straight forward procedure; pathogens have adapted to their environment by evolving a collection of evasion mechanisms that circumvent the human complement system. Complement evasion strategies features different ways of exploiting human complement proteins and moreover features different pathogen-derived proteins that interfere with the normal processes. Accumulated, these mechanisms target all three complement activation pathways as well as the final common part of the cascade. This review will cover the currently known lectin pathway evasion mechanisms and give examples of pathogens that operate these to increase their chance of invasion, survival and dissemination.

Complement system proteins which interact with C3b or C4b A superfamily of structurally related proteins

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Reid, K B M; Bentley, D R; Campbell, R D

1986-01-01

Recent cDNA sequencing data has allowed the prediction of the entire amino acid sequences of complement components factor B and C2, the complement control proteins factor H and C4b-binding protein and a partial sequence for the Cab/C4b receptor CR1. These proteins all contain internal repeating u...

Phagocytosis escape by a Staphylococcus aureus protein that connects complement and coagulation proteins at the bacterial surface.

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Ya-Ping Ko

Full Text Available Upon contact with human plasma, bacteria are rapidly recognized by the complement system that labels their surface for uptake and clearance by phagocytic cells. Staphylococcus aureus secretes the 16 kD Extracellular fibrinogen binding protein (Efb that binds two different plasma proteins using separate domains: the Efb N-terminus binds to fibrinogen, while the C-terminus binds complement C3. In this study, we show that Efb blocks phagocytosis of S. aureus by human neutrophils. In vitro, we demonstrate that Efb blocks phagocytosis in plasma and in human whole blood. Using a mouse peritonitis model we show that Efb effectively blocks phagocytosis in vivo, either as a purified protein or when produced endogenously by S. aureus. Mutational analysis revealed that Efb requires both its fibrinogen and complement binding residues for phagocytic escape. Using confocal and transmission electron microscopy we show that Efb attracts fibrinogen to the surface of complement-labeled S. aureus generating a 'capsule'-like shield. This thick layer of fibrinogen shields both surface-bound C3b and antibodies from recognition by phagocytic receptors. This information is critical for future vaccination attempts, since opsonizing antibodies may not function in the presence of Efb. Altogether we discover that Efb from S. aureus uniquely escapes phagocytosis by forming a bridge between a complement and coagulation protein.

Further structural insights into the binding of complement factor H by complement regulator-acquiring surface protein 1 (CspA) of Borrelia burgdorferi

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Caesar, Joseph J. E.; Wallich, Reinhard; Kraiczy, Peter; Zipfel, Peter F.; Lea, Susan M.

2013-01-01

B. burgdorferi binds complement factor H using a dimeric surface protein, CspA (BbCRASP-1). Presented here is a new structure of CspA that suggests that there is a degree of flexibility between subunits which may have implications for complement regulator binding. Borrelia burgdorferi has evolved many mechanisms of evading the different immune systems across its range of reservoir hosts, including the capture and presentation of host complement regulators factor H and factor H-like protein-1 (FHL-1). Acquisition is mediated by a family of complement regulator-acquiring surface proteins (CRASPs), of which the atomic structure of CspA (BbCRASP-1) is known and shows the formation of a homodimeric species which is required for binding. Mutagenesis studies have mapped a putative factor H binding site to a cleft between the two subunits. Presented here is a new atomic structure of CspA which shows a degree of flexibility between the subunits which may be critical for factor H scavenging by increasing access to the binding interface and allows the possibility that the assembly can clamp around the bound complement regulators

Prediction of Protein-Protein Interactions by NanoLuc-Based Protein-Fragment Complementation Assay | Office of Cancer Genomics

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The CTD2 Center at Emory has developed a new NanoLuc®-based protein-fragment complementation assay (NanoPCA) which allows the detection of novel protein-protein interactions (PPI). NanoPCA allows the study of PPI dynamics with reversible interactions.  Read the abstract. Experimental Approaches Read the detailed Experimetnal Approaches. 

Complement factor H family proteins in their non-canonical role as modulators of cellular functions.

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Józsi, Mihály; Schneider, Andrea E; Kárpáti, Éva; Sándor, Noémi

2018-01-04

Complement factor H is a major regulator of the alternative pathway of the complement system. The factor H-related proteins are less characterized, but recent data indicate that they rather promote complement activation. These proteins have some common ligands with factor H and have both overlapping and distinct functions depending on domain composition and the degree of conservation of amino acid sequence. Factor H and some of the factor H-related proteins also appear in a non-canonical function that is beyond their role in the modulation of complement activation. This review covers our current understanding on this emerging role of factor H family proteins in modulating the activation and function of various cells by binding to receptors or receptor ligands. Copyright © 2018 Elsevier Ltd. All rights reserved.

Mesenchymal stromal cells engage complement and complement receptor bearing innate effector cells to modulate immune responses.

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Guido Moll

Full Text Available Infusion of human third-party mesenchymal stromal cells (MSCs appears to be a promising therapy for acute graft-versus-host disease (aGvHD. To date, little is known about how MSCs interact with the body's innate immune system after clinical infusion. This study shows, that exposure of MSCs to blood type ABO-matched human blood activates the complement system, which triggers complement-mediated lymphoid and myeloid effector cell activation in blood. We found deposition of complement component C3-derived fragments iC3b and C3dg on MSCs and fluid-phase generation of the chemotactic anaphylatoxins C3a and C5a. MSCs bound low amounts of immunoglobulins and lacked expression of complement regulatory proteins MCP (CD46 and DAF (CD55, but were protected from complement lysis via expression of protectin (CD59. Cell-surface-opsonization and anaphylatoxin-formation triggered complement receptor 3 (CD11b/CD18-mediated effector cell activation in blood. The complement-activating properties of individual MSCs were furthermore correlated with their potency to inhibit PBMC-proliferation in vitro, and both effector cell activation and the immunosuppressive effect could be blocked either by using complement inhibitor Compstatin or by depletion of CD14/CD11b-high myeloid effector cells from mixed lymphocyte reactions. Our study demonstrates for the first time a major role of the complement system in governing the immunomodulatory activity of MSCs and elucidates how complement activation mediates the interaction with other immune cells.

Complement activation in Ghanaian children with severe Plasmodium falciparum malaria

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Ofori Michael F

2007-12-01

Full Text Available Abstract Background Severe anaemia (SA, intravascular haemolysis (IVH and respiratory distress (RD are severe forms of Plasmodium falciparum malaria, with RD reported to be of prognostic importance in African children with malarial anaemia. Complement factors have been implicated in the mechanism leading to excess anaemia in acute P. falciparum infection. Methods The direct Coombs test (DCT and flow cytometry were used to investigate the mean levels of RBC-bound complement fragments (C3d and C3bαβ and the regulatory proteins [complement receptor 1 (CD35 and decay accelerating factor (CD55] in children with discrete clinical forms of P. falciparum malaria. The relationship between the findings and clinical parameters including coma, haemoglobin (Hb levels and RD were investigated. Results Of the 484 samples tested, 131(27% were positive in DCT, out of which 115/131 (87.8% were positive for C3d alone while 16/131 (12.2% were positive for either IgG alone or both. 67.4% of the study population were below 5 years of age and DCT positivity was more common in this age group relative to children who were 5 years or older (Odds ratio, OR = 3.8; 95%CI, 2.2–6.7, p Conclusion These results suggest that complement activation contributed to anaemia in acute childhood P. falciparum malaria, possibly through induction of erythrophagocytosis and haemolysis. In contrast to other studies, this study did not find association between levels of the complement regulatory proteins, CD35 and CD55 and malarial anaemia. These findings suggest that complement activation could also be involved in the pathogenesis of RD but larger studies are needed to confirm this finding.

The Surface-Exposed Protein SntA Contributes to Complement Evasion in Zoonotic Streptococcus suis.

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Deng, Simin; Xu, Tong; Fang, Qiong; Yu, Lei; Zhu, Jiaqi; Chen, Long; Liu, Jiahui; Zhou, Rui

2018-01-01

Streptococcus suis is an emerging zoonotic pathogen causing streptococcal toxic shock like syndrome (STSLS), meningitis, septicemia, and even sudden death in human and pigs. Serious septicemia indicates this bacterium can evade the host complement surveillance. In our previous study, a functionally unknown protein SntA of S. suis has been identified as a heme-binding protein, and contributes to virulence in pigs. SntA can interact with the host antioxidant protein AOP2 and consequently inhibit its antioxidant activity. In the present study, SntA is identified as a cell wall anchored protein that functions as an important player in S. suis complement evasion. The C3 deposition and membrane attack complex (MAC) formation on the surface of sntA -deleted mutant strain Δ sntA are demonstrated to be significantly higher than the parental strain SC-19 and the complementary strain CΔ sntA . The abilities of anti-phagocytosis, survival in blood, and in vivo colonization of Δ sntA are obviously reduced. SntA can interact with C1q and inhibit hemolytic activity via the classical pathway. Complement activation assays reveal that SntA can also directly activate classical and lectin pathways, resulting in complement consumption. These two complement evasion strategies may be crucial for the pathogenesis of this zoonotic pathogen. Concerning that SntA is a bifunctional 2',3'-cyclic nucleotide 2'-phosphodiesterase/3'-nucleotidase in many species of Gram-positive bacteria, these complement evasion strategies may have common biological significance.

ErpC, a member of the complement regulator-acquiring family of surface proteins from Borrelia burgdorferi, possesses an architecture previously unseen in this protein family

International Nuclear Information System (INIS)

Caesar, Joseph J. E.; Johnson, Steven; Kraiczy, Peter; Lea, Susan M.

2013-01-01

The structure of ErpC, a member of the complement regulator-acquiring surface protein family from B. burgdorferi, has been solved, providing insights into the strategies of complement evasion by this zoonotic bacterium and suggesting a common architecture for other members of this protein family. Borrelia burgdorferi is a spirochete responsible for Lyme disease, the most commonly occurring vector-borne disease in Europe and North America. The bacterium utilizes a set of proteins, termed complement regulator-acquiring surface proteins (CRASPs), to aid evasion of the human complement system by recruiting and presenting complement regulator factor H on its surface in a manner that mimics host cells. Presented here is the atomic resolution structure of a member of this protein family, ErpC. The structure provides new insights into the mechanism of recruitment of factor H and other factor H-related proteins by acting as a molecular mimic of host glycosaminoglycans. It also describes the architecture of other CRASP proteins belonging to the OspE/F-related paralogous protein family and suggests that they have evolved to bind specific complement proteins, aiding survival of the bacterium in different hosts

Surviving mousepox infection requires the complement system.

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Elizabeth A Moulton

2008-12-01

Full Text Available Poxviruses subvert the host immune response by producing immunomodulatory proteins, including a complement regulatory protein. Ectromelia virus provides a mouse model for smallpox where the virus and the host's immune response have co-evolved. Using this model, our study investigated the role of the complement system during a poxvirus infection. By multiple inoculation routes, ectromelia virus caused increased mortality by 7 to 10 days post-infection in C57BL/6 mice that lack C3, the central component of the complement cascade. In C3(-/- mice, ectromelia virus disseminated earlier to target organs and generated higher peak titers compared to the congenic controls. Also, increased hepatic inflammation and necrosis correlated with these higher tissue titers and likely contributed to the morbidity in the C3(-/- mice. In vitro, the complement system in naïve C57BL/6 mouse sera neutralized ectromelia virus, primarily through the recognition of the virion by natural antibody and activation of the classical and alternative pathways. Sera deficient in classical or alternative pathway components or antibody had reduced ability to neutralize viral particles, which likely contributed to increased viral dissemination and disease severity in vivo. The increased mortality of C4(-/- or Factor B(-/- mice also indicates that these two pathways of complement activation are required for survival. In summary, the complement system acts in the first few minutes, hours, and days to control this poxviral infection until the adaptive immune response can react, and loss of this system results in lethal infection.

vProtein: identifying optimal amino acid complements from plant-based foods.

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Peter J Woolf

Full Text Available BACKGROUND: Indispensible amino acids (IAAs are used by the body in different proportions. Most animal-based foods provide these IAAs in roughly the needed proportions, but many plant-based foods provide different proportions of IAAs. To explore how these plant-based foods can be better used in human nutrition, we have created the computational tool vProtein to identify optimal food complements to satisfy human protein needs. METHODS: vProtein uses 1251 plant-based foods listed in the United States Department of Agriculture standard release 22 database to determine the quantity of each food or pair of foods required to satisfy human IAA needs as determined by the 2005 daily recommended intake. The quantity of food in a pair is found using a linear programming approach that minimizes total calories, total excess IAAs, or the total weight of the combination. RESULTS: For single foods, vProtein identifies foods with particularly balanced IAA patterns such as wheat germ, quinoa, and cauliflower. vProtein also identifies foods with particularly unbalanced IAA patterns such as macadamia nuts, degermed corn products, and wakame seaweed. Although less useful alone, some unbalanced foods provide unusually good complements, such as Brazil nuts to legumes. Interestingly, vProtein finds no statistically significant bias toward grain/legume pairings for protein complementation. These analyses suggest that pairings of plant-based foods should be based on the individual foods themselves instead of based on broader food group-food group pairings. Overall, the most efficient pairings include sweet corn/tomatoes, apple/coconut, and sweet corn/cherry. The top pairings also highlight the utility of less common protein sources such as the seaweeds laver and spirulina, pumpkin leaves, and lambsquarters. From a public health perspective, many of the food pairings represent novel, low cost food sources to combat malnutrition. Full analysis results are available online

Induction of complement proteins in a mouse model for cerebral microvascular Aβ deposition

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DeFilippis Kelly

2007-09-01

Full Text Available Abstract The deposition of amyloid β-protein (Aβ in cerebral vasculature, known as cerebral amyloid angiopathy (CAA, is a common pathological feature of Alzheimer's disease and related disorders. In familial forms of CAA single mutations in the Aβ peptide have been linked to the increase of vascular Aβ deposits accompanied by a strong localized activation of glial cells and elevated expression of neuroinflammatory mediators including complement proteins. We have developed human amyloid-β precursor protein transgenic mice harboring two CAA Aβ mutations (Dutch E693Q and Iowa D694N that mimic the prevalent cerebral microvascular Aβ deposition observed in those patients, and the Swedish mutations (K670N/M671L to increase Aβ production. In these Tg-SwDI mice, we have reported predominant fibrillar Aβ along microvessels in the thalamic region and diffuse plaques in cortical region. Concurrently, activated microglia and reactive astrocytes have been detected primarily in association with fibrillar cerebral microvascular Aβ in this model. Here we show that three native complement components in classical and alternative complement pathways, C1q, C3, and C4, are elevated in Tg-SwDI mice in regions rich in fibrillar microvascular Aβ. Immunohistochemical staining of all three proteins was increased in thalamus, hippocampus, and subiculum, but not frontal cortex. Western blot analysis showed significant increases of all three proteins in the thalamic region (with hippocampus as well as the cortical region, except C3 that was below detection level in cortex. Also, in the thalamic region (with hippocampus, C1q and C3 mRNAs were significantly up-regulated. These complement proteins appeared to be expressed largely by activated microglial cells associated with the fibrillar microvascular Aβ deposits. Our findings demonstrate that Tg-SwDI mice exhibit elevated complement protein expression in response to fibrillar vascular Aβ deposition that is

Yersinia enterocolitica serum resistance proteins YadA and ail bind the complement regulator C4b-binding protein.

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Vesa Kirjavainen

Full Text Available Many pathogens are equipped with factors providing resistance against the bactericidal action of complement. Yersinia enterocolitica, a Gram-negative enteric pathogen with invasive properties, efficiently resists the deleterious action of human complement. The major Y. enterocolitica serum resistance determinants include outer membrane proteins YadA and Ail. Lipopolysaccharide (LPS O-antigen (O-ag and outer core (OC do not contribute directly to complement resistance. The aim of this study was to analyze a possible mechanism whereby Y. enterocolitica could inhibit the antibody-mediated classical pathway of complement activation. We show that Y. enterocolitica serotypes O:3, O:8, and O:9 bind C4b-binding protein (C4bp, an inhibitor of both the classical and lectin pathways of complement. To identify the C4bp receptors on Y. enterocolitica serotype O:3 surface, a set of mutants expressing YadA, Ail, O-ag, and OC in different combinations was tested for the ability to bind C4bp. The studies showed that both YadA and Ail acted as C4bp receptors. Ail-mediated C4bp binding, however, was blocked by the O-ag and OC, and could be observed only with mutants lacking these LPS structures. C4bp bound to Y. enterocolitica was functionally active and participated in the factor I-mediated degradation of C4b. These findings show that Y. enterocolitica uses two proteins, YadA and Ail, to bind C4bp. Binding of C4bp could help Y. enterocolitica to evade complement-mediated clearance in the human host.

X-ray repair cross complementing protein 1 in base excision repair

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Hanssen-Bauer, Audun; Solvang-Garten, Karin; Akbari, Mansour

2012-01-01

X-ray Repair Cross Complementing protein 1 (XRCC1) acts as a scaffolding protein in the converging base excision repair (BER) and single strand break repair (SSBR) pathways. XRCC1 also interacts with itself and rapidly accumulates at sites of DNA damage. XRCC1 can thus mediate the assembly of large...

Evaluation of complement proteins as screening markers for colorectal cancer

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Storm, Line; Christensen, Ib J; Jensenius, Jens C

2015-01-01

BACKGROUND: Colorectal cancer (CRC) is a leading cause of cancer death worldwide. Lack of symptoms results in late detection and increased mortality. Inflammation, including complement activation, plays an important role in tumorigenesis. EXPERIMENTAL DESIGN: The concentrations of nine proteins...

Detection and characterisation of Complement protein activity in bovine milk by bactericidal sequestration assay.

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Maye, Susan; Stanton, Catherine; Fitzgerald, Gerald F; Kelly, Philip M

2015-08-01

While the Complement protein system in human milk is well characterised, there is little information on its presence and activity in bovine milk. Complement forms part of the innate immune system, hence the importance of its contribution during milk ingestion to the overall defences of the neonate. A bactericidal sequestration assay, featuring a Complement sensitive strain, Escherichia coli 0111, originally used to characterise Complement activity in human milk was successfully applied to freshly drawn bovine milk samples, thus, providing an opportunity to compare Complement activities in both human and bovine milks. Although not identical in response, the levels of Complement activity in bovine milk were found to be closely comparable with that of human milk. Differential counts of Esch. coli 0111 after 2 h incubation were 6.20 and 6.06 log CFU/ml, for raw bovine and human milks, respectively - the lower value representing a stronger Complement response. Exposing bovine milk to a range of thermal treatments e.g. 42, 45, 65, 72, 85 or 95 °C for 10 min, progressively inhibited Complement activity by increasing temperature, thus confirming the heat labile nature of this immune protein system. Low level Complement activity was found, however, in 65 and 72 °C heat treated samples and in retailed pasteurised milk which highlights the outer limit to which high temperature, short time (HTST) industrial thermal processes should be applied if retention of activity is a priority. Concentration of Complement in the fat phase was evident following cream separation, and this was also reflected in the further loss of activity recorded in low fat variants of retailed pasteurised milk. Laboratory-based churning of the cream during simulated buttermaking generated an aqueous (buttermilk) phase with higher levels of Complement activity than the fat phase, thus pointing to a likely association with the milk fat globule membrane (MFGM) layer.

Lack of evidence from studies of soluble protein fragments that Knops blood group polymorphisms in complement receptor-type 1 are driven by malaria.

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Patience B Tetteh-Quarcoo

Full Text Available Complement receptor-type 1 (CR1, CD35 is the immune-adherence receptor, a complement regulator, and an erythroid receptor for Plasmodium falciparum during merozoite invasion and subsequent rosette formation involving parasitized and non-infected erythrocytes. The non-uniform geographical distribution of Knops blood group CR1 alleles Sl1/2 and McC(a/b may result from selective pressures exerted by differential exposure to infectious hazards. Here, four variant short recombinant versions of CR1 were produced and analyzed, focusing on complement control protein modules (CCPs 15-25 of its ectodomain. These eleven modules encompass a region (CCPs 15-17 key to rosetting, opsonin recognition and complement regulation, as well as the Knops blood group polymorphisms in CCPs 24-25. All four CR1 15-25 variants were monomeric and had similar axial ratios. Modules 21 and 22, despite their double-length inter-modular linker, did not lie side-by-side so as to stabilize a bent-back architecture that would facilitate cooperation between key functional modules and Knops blood group antigens. Indeed, the four CR1 15-25 variants had virtually indistinguishable affinities for immobilized complement fragments C3b (K(D = 0.8-1.1 µM and C4b (K(D = 5.0-5.3 µM. They were all equally good co-factors for factor I-catalysed cleavage of C3b and C4b, and they bound equally within a narrow affinity range, to immobilized C1q. No differences between the variants were observed in assays for inhibition of erythrocyte invasion by P. falciparum or for rosette disruption. Neither differences in complement-regulatory functionality, nor interactions with P. falciparum proteins tested here, appear to have driven the non-uniform geographic distribution of these alleles.

Complement-related proteins control the flavivirus infection of Aedes aegypti by inducing antimicrobial peptides.

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Xiaoping Xiao

2014-04-01

Full Text Available The complement system functions during the early phase of infection and directly mediates pathogen elimination. The recent identification of complement-like factors in arthropods indicates that this system shares common ancestry in vertebrates and invertebrates as an immune defense mechanism. Thioester (TE-containing proteins (TEPs, which show high similarity to mammalian complement C3, are thought to play a key role in innate immunity in arthropods. Herein, we report that a viral recognition cascade composed of two complement-related proteins limits the flaviviral infection of Aedes aegypti. An A. aegypti macroglobulin complement-related factor (AaMCR, belonging to the insect TEP family, is a crucial effector in opposing the flaviviral infection of A. aegypti. However, AaMCR does not directly interact with DENV, and its antiviral effect requires an A. aegypti homologue of scavenger receptor-C (AaSR-C, which interacts with DENV and AaMCR simultaneously in vitro and in vivo. Furthermore, recognition of DENV by the AaSR-C/AaMCR axis regulates the expression of antimicrobial peptides (AMPs, which exerts potent anti-DENV activity. Our results both demonstrate the existence of a viral recognition pathway that controls the flaviviral infection by inducing AMPs and offer insights into a previously unappreciated antiviral function of the complement-like system in arthropods.

Complementation studies with the novel "Bungowannah" virus provide new insights in the compatibility of pestivirus proteins.

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Richter, Maria; Reimann, Ilona; Wegelt, Anne; Kirkland, Peter D; Beer, Martin

2011-09-30

In recent years several atypical pestiviruses have been described. Bungowannah virus is the most divergent virus in this group. Therefore, heterologous complementation was used to clarify the phylogenetic relationship and to analyze the exchangeability of genome regions encoding structural proteins. Using a BVDV type 1 backbone, chimeric constructs with substituted envelope proteins E(rns), E1 and E2, were investigated. While all constructs replicated autonomously, infectious high titer chimeric virus could only be observed after exchanging the complete E1-E2 encoding region. The complementation of E1 and E2 alone resulted only in replicons. Complementation of BVDV-E(rns) was only efficient if Bungowannah virus-E(rns) was expressed from a bicistronic construct. Our data provide new insights in the compatibility of pestivirus proteins and demonstrate that heterologous complementation could be useful to characterize new pestiviruses. Copyright © 2011 Elsevier Inc. All rights reserved.

Plasmin cleaves fibrinogen and the human complement proteins C3b and C5 in the presence of Leptospira interrogans proteins: A new role of LigA and LigB in invasion and complement immune evasion.

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Castiblanco-Valencia, Mónica Marcela; Fraga, Tatiana Rodrigues; Pagotto, Ana Helena; Serrano, Solange Maria de Toledo; Abreu, Patricia Antonia Estima; Barbosa, Angela Silva; Isaac, Lourdes

2016-05-01

Plasminogen is a single-chain glycoprotein found in human plasma as the inactive precursor of plasmin. When converted to proteolytically active plasmin, plasmin(ogen) regulates both complement and coagulation cascades, thus representing an important target for pathogenic microorganisms. Leptospira interrogans binds plasminogen, which is converted to active plasmin. Leptospiral immunoglobulin-like (Lig) proteins are surface exposed molecules that interact with extracellular matrix components and complement regulators, including proteins of the FH family and C4BP. In this work, we demonstrate that these multifunctional molecules also bind plasminogen through both N- and C-terminal domains. These interactions are dependent on lysine residues and are affected by ionic strength. Competition assays suggest that plasminogen does not share binding sites with C4BP or FH on Lig proteins at physiological molar ratios. Plasminogen bound to Lig proteins is converted to proteolytic active plasmin in the presence of urokinase-type plasminogen activator (uPA). Lig-bound plasmin is able to cleave the physiological substrates fibrinogen and the complement proteins C3b and C5. Taken together, our data point to a new role of LigA and LigB in leptospiral invasion and complement immune evasion. Plasmin(ogen) acquisition by these versatile proteins may contribute to Leptospira infection, favoring bacterial survival and dissemination inside the host. Copyright © 2016. Published by Elsevier GmbH.

The paralogous salivary anti-complement proteins IRAC I and IRAC II encoded by Ixodes ricinus ticks have broad and complementary inhibitory activities against the complement of different host species.

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Schroeder, Hélène; Daix, Virginie; Gillet, Laurent; Renauld, Jean-Christophe; Vanderplasschen, Alain

2007-02-01

Several observations suggest that inhibition of the host complement alternative pathway by Ixodes tick saliva is crucial to achieve blood feeding. We recently described two paralogous anti-complement proteins called Ixodes ricinus anti-complement (IRAC) proteins I and II co-expressed in I. ricinus salivary glands. Phylogenetic analyses suggested that these sequences were diversifying by a process of positive Darwinian selection, possibly leading to molecules with different biological properties. In the present study, we tested the hypothesis that each paralogue may have different inhibitory activities against the complement of different natural host species, thereby contributing to broaden the host range of I. ricinus ticks. IRAC I and IRAC II were tested against the complement of eight I. ricinus natural host species (six mammals and two birds). The results demonstrate that IRAC I and IRAC II have broad and complementary inhibition activities against the complement of different host species. This report is the first description of paralogous anti-complement molecules encoded by a pathogen with broad and complementary inhibitory activities against the complement of different host species.

Cyclosporine Induces Endothelial Cell Release of Complement-Activating Microparticles

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Renner, Brandon; Klawitter, Jelena; Goldberg, Ryan; McCullough, James W.; Ferreira, Viviana P.; Cooper, James E.; Christians, Uwe

2013-01-01

Defective control of the alternative pathway of complement is an important risk factor for several renal diseases, including atypical hemolytic uremic syndrome. Infections, drugs, pregnancy, and hemodynamic insults can trigger episodes of atypical hemolytic uremic syndrome in susceptible patients. Although the mechanisms linking these clinical events with disease flares are unknown, recent work has revealed that each of these clinical conditions causes cells to release microparticles. We hypothesized that microparticles released from injured endothelial cells promote intrarenal complement activation. Calcineurin inhibitors cause vascular and renal injury and can trigger hemolytic uremic syndrome. Here, we show that endothelial cells exposed to cyclosporine in vitro and in vivo release microparticles that activate the alternative pathway of complement. Cyclosporine-induced microparticles caused injury to bystander endothelial cells and are associated with complement-mediated injury of the kidneys and vasculature in cyclosporine-treated mice. Cyclosporine-induced microparticles did not bind factor H, an alternative pathway regulatory protein present in plasma, explaining their complement-activating phenotype. Finally, we found that in renal transplant patients, the number of endothelial microparticles in plasma increases 2 weeks after starting tacrolimus, and treatment with tacrolimus associated with increased C3 deposition on endothelial microparticles in the plasma of some patients. These results suggest that injury-associated release of endothelial microparticles is an important mechanism by which systemic insults trigger intravascular complement activation and complement-dependent renal diseases. PMID:24092930

A versatile selection system for folding competent proteins using genetic complementation in a eukaryotic host

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Lyngsø, C.; Kjaerulff, S.; Muller, S.

2010-01-01

in vivo selection system for folded proteins. It is based on genetic complementation of the Schizosaccharomyces pombe growth marker gene invertase fused C-terminally to a protein library. The fusion proteins are directed to the secretion system, utilizing the ability of the eukaryotic protein quality...

Complement factor H protects mice from ischemic acute kidney injury but is not critical for controlling complement activation by glomerular IgM.

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Goetz, Lindsey; Laskowski, Jennifer; Renner, Brandon; Pickering, Matthew C; Kulik, Liudmila; Klawitter, Jelena; Stites, Erik; Christians, Uwe; van der Vlag, Johan; Ravichandran, Kameswaran; Holers, V Michael; Thurman, Joshua M

2018-05-01

Natural IgM binds to glomerular epitopes in several progressive kidney diseases. Previous work has shown that IgM also binds within the glomerulus after ischemia/reperfusion (I/R) but does not fully activate the complement system. Factor H is a circulating complement regulatory protein, and congenital or acquired deficiency of factor H is a strong risk factor for several types of kidney disease. We hypothesized that factor H controls complement activation by IgM in the kidney after I/R, and that heterozygous factor H deficiency would permit IgM-mediated complement activation and injury at this location. We found that mice with targeted heterozygous deletion of the gene for factor H developed more severe kidney injury after I/R than wild-type controls, as expected, but that complement activation within the glomeruli remained well controlled. Furthermore, mice that are unable to generate soluble IgM were not protected from renal I/R, even in the setting of heterozygous factor H deficiency. These results demonstrate that factor H is important for limiting injury in the kidney after I/R, but it is not critical for controlling complement activation by immunoglobulin within the glomerulus in this setting. IgM binds to glomerular epitopes after I/R, but it is not a significant source of injury. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The salivary scavenger and agglutinin (SALSA binds MBL and regulates the lectin pathway of complement in solution and on surfaces

Directory of Open Access Journals (Sweden)

Martin eParnov Reichhardt

2012-07-01

Full Text Available The scavenger receptor cysteine-rich (SRCR protein SALSA, also known as gp340, salivary agglutinin (SAG and deleted in malignant brain tumor 1 (DMBT1, is a 340 kDa glycoprotein expressed on mucosal surfaces and secreted into several body fluids. SALSA binds to a broad variety of microbes and endogenous ligands, such as complement factor C1q, surfactant proteins D and A (SP-D and SP-A and IgA. Our search for novel ligands of SALSA by direct protein-interaction studies led to the identification of mannan binding lectin (MBL as a new binding partner. We observed that surface-associated SALSA activates complement via binding of MBL. On the other hand, soluble SALSA was found to inhibit C. albicans-induced complement activation. Thus, SALSA has a dual complement regulatory function. It activates the lectin pathway when bound to a surface and inhibits it when free in the fluid-phase. These activities are mediated via a direct interaction with MBL.

Coordinated Expression of Borrelia burgdorferi Complement Regulator-Acquiring Surface Proteins during the Lyme Disease Spirochete's Mammal-Tick Infection Cycle▿

OpenAIRE

Bykowski, Tomasz; Woodman, Michael E.; Cooley, Anne E.; Brissette, Catherine A.; Brade, Volker; Wallich, Reinhard; Kraiczy, Peter; Stevenson, Brian

2007-01-01

The Lyme disease spirochete, Borrelia burgdorferi, is largely resistant to being killed by its hosts’ alternative complement activation pathway. One possible resistance mechanism of these bacteria is to coat their surfaces with host complement regulators, such as factor H. Five different B. burgdorferi outer surface proteins having affinities for factor H have been identified: complement regulator-acquiring surface protein 1 (BbCRASP-1), encoded by cspA; BbCRASP-2, encoded by cspZ; and three ...

A functional SNP in the regulatory region of the decay-accelerating factor gene associates with extraocular muscle pareses in myasthenia gravis

KAUST Repository

Heckmann, J M; Uwimpuhwe, H; Ballo, R; Kaur, M; Bajic, Vladimir B.; Prince, S

2009-01-01

Complement activation in myasthenia gravis (MG) may damage muscle endplate and complement regulatory proteins such as decay-accelerating factor (DAF) or CD55 may be protective. We hypothesize that the increased prevalence of severe extraocular

Staphylococcus aureus SdrE captures complement factor H's C-terminus via a novel 'close, dock, lock and latch' mechanism for complement evasion.

Science.gov (United States)

Zhang, Yingjie; Wu, Minhao; Hang, Tianrong; Wang, Chengliang; Yang, Ye; Pan, Weimin; Zang, Jianye; Zhang, Min; Zhang, Xuan

2017-05-04

Complement factor H (CFH) is a soluble complement regulatory protein essential for the down-regulation of the alternative pathway on interaction with specific markers on the host cell surface. It recognizes the complement component 3b (C3b) and 3d (C3d) fragments in addition to self cell markers (i.e. glycosaminoglycans, sialic acid) to distinguish host cells that deserve protection from pathogens that should be eliminated. The Staphylococcus aureus surface protein serine-aspartate repeat protein E (SdrE) was previously reported to bind human CFH as an immune-evasion tactic. However, the molecular mechanism underlying SdrE-CFH-mediated immune evasion remains unknown. In the present study, we identified a novel region at CFH's C-terminus (CFH 1206-1226 ), which binds SdrE N2 and N3 domains (SdrE N2N3 ) with high affinity, and determined the crystal structures of apo-SdrE N2N3 and the SdrE N2N3 -CFH 1206-1226 complex. Comparison of the structure of the CFH-SdrE complex with other CFH structures reveals that CFH's C-terminal tail flips from the main body to insert into the ligand-binding groove of SdrE. In addition, SdrE N2N3 adopts a 'close' state in the absence of CFH, which undergoes a large conformational change on CFH binding, suggesting a novel 'close, dock, lock and latch' (CDLL) mechanism for SdrE to recognize its ligand. Our findings imply that SdrE functions as a 'clamp' to capture CFH's C-terminal tail via a unique CDLL mechanism and sequesters CFH on the surface of S. aureus for complement evasion. © 2017 The Author(s).

Trans-acting translational regulatory RNA binding proteins.

Science.gov (United States)

Harvey, Robert F; Smith, Tom S; Mulroney, Thomas; Queiroz, Rayner M L; Pizzinga, Mariavittoria; Dezi, Veronica; Villenueva, Eneko; Ramakrishna, Manasa; Lilley, Kathryn S; Willis, Anne E

2018-05-01

The canonical molecular machinery required for global mRNA translation and its control has been well defined, with distinct sets of proteins involved in the processes of translation initiation, elongation and termination. Additionally, noncanonical, trans-acting regulatory RNA-binding proteins (RBPs) are necessary to provide mRNA-specific translation, and these interact with 5' and 3' untranslated regions and coding regions of mRNA to regulate ribosome recruitment and transit. Recently it has also been demonstrated that trans-acting ribosomal proteins direct the translation of specific mRNAs. Importantly, it has been shown that subsets of RBPs often work in concert, forming distinct regulatory complexes upon different cellular perturbation, creating an RBP combinatorial code, which through the translation of specific subsets of mRNAs, dictate cell fate. With the development of new methodologies, a plethora of novel RNA binding proteins have recently been identified, although the function of many of these proteins within mRNA translation is unknown. In this review we will discuss these methodologies and their shortcomings when applied to the study of translation, which need to be addressed to enable a better understanding of trans-acting translational regulatory proteins. Moreover, we discuss the protein domains that are responsible for RNA binding as well as the RNA motifs to which they bind, and the role of trans-acting ribosomal proteins in directing the translation of specific mRNAs. This article is categorized under: RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes Translation > Translation Regulation Translation > Translation Mechanisms. © 2018 Medical Research Council and University of Cambridge. WIREs RNA published by Wiley Periodicals, Inc.

Optimizing complement-activating antibody-based cancer immunotherapy: a feasible strategy?

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Maio Michele

2004-06-01

Full Text Available Abstract Passive immunotherapy with monoclonal antibodies (mAb targeted to specific tumor-associated antigens is amongst the most rapidly expanding approaches to biological therapy of cancer. However, until now a limited number of therapeutic mAb has demonstrated clinical efficacy in selected neoplasia. Results emerging from basic research point to a deeper characterization of specific biological features of neoplastic cells as crucial to optimize the clinical potential of therapeutic mAb, and to identify cancer patients who represent the best candidates to antibody-based immunotherapy. Focus on the tissue distribution and on the functional role of membrane complement-regulatory proteins such as Protectin (CD59, which under physiologic conditions protects tissues from Complement (C-damage, might help to optimize the efficacy of immunotherapeutic strategies based on C-activating mAb.

Complement factor H-related proteins in IgA nephropathy-sometimes a gentle nudge does the trick.

Science.gov (United States)

Thurman, Joshua M; Laskowski, Jennifer

2017-10-01

Complement activation probably contributes to glomerular inflammation and damage in IgA nephropathy. In this issue, 2 groups report that levels of factor H-related protein 1 are elevated in patients with IgA nephropathy and correlate with disease progression. These studies provide new evidence that the complement cascade is important to the pathogenesis of this disease. These results also suggest that factor H-related protein 1 levels may be useful for identifying those patients at high risk of disease progression. Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

Staphylococcus aureus SdrE captures complement factor H's C-terminus via a novel ‘close, dock, lock and latch' mechanism for complement evasion

Science.gov (United States)

Zhang, Yingjie; Wu, Minhao; Hang, Tianrong; Wang, Chengliang; Yang, Ye; Pan, Weimin; Zang, Jianye

2017-01-01

Complement factor H (CFH) is a soluble complement regulatory protein essential for the down-regulation of the alternative pathway on interaction with specific markers on the host cell surface. It recognizes the complement component 3b (C3b) and 3d (C3d) fragments in addition to self cell markers (i.e. glycosaminoglycans, sialic acid) to distinguish host cells that deserve protection from pathogens that should be eliminated. The Staphylococcus aureus surface protein serine–aspartate repeat protein E (SdrE) was previously reported to bind human CFH as an immune-evasion tactic. However, the molecular mechanism underlying SdrE–CFH-mediated immune evasion remains unknown. In the present study, we identified a novel region at CFH's C-terminus (CFH1206–1226), which binds SdrE N2 and N3 domains (SdrEN2N3) with high affinity, and determined the crystal structures of apo-SdrEN2N3 and the SdrEN2N3–CFH1206–1226 complex. Comparison of the structure of the CFH–SdrE complex with other CFH structures reveals that CFH's C-terminal tail flips from the main body to insert into the ligand-binding groove of SdrE. In addition, SdrEN2N3 adopts a ‘close’ state in the absence of CFH, which undergoes a large conformational change on CFH binding, suggesting a novel ‘close, dock, lock and latch' (CDLL) mechanism for SdrE to recognize its ligand. Our findings imply that SdrE functions as a ‘clamp' to capture CFH's C-terminal tail via a unique CDLL mechanism and sequesters CFH on the surface of S. aureus for complement evasion. PMID:28258151

CipA of Acinetobacter baumannii Is a Novel Plasminogen Binding and Complement Inhibitory Protein.

Science.gov (United States)

Koenigs, Arno; Stahl, Julia; Averhoff, Beate; Göttig, Stephan; Wichelhaus, Thomas A; Wallich, Reinhard; Zipfel, Peter F; Kraiczy, Peter

2016-05-01

Acinetobacter baumannii is an emerging opportunistic pathogen, responsible for up to 10% of gram-negative, nosocomial infections. The global increase of multidrug-resistant and pan-resistant Acinetobacter isolates presents clinicians with formidable challenges. To establish a persistent infection,A. baumannii must overcome the detrimental effects of complement as the first line of defense against invading microorganisms. However, the immune evasion principles underlying serum resistance inA. baumannii remain elusive. Here, we identified a novel plasminogen-binding protein, termed CipA. Bound plasminogen, upon conversion to active plasmin, degraded fibrinogen and complement C3b and contributed to serum resistance. Furthermore, CipA directly inhibited the alternative pathway of complement in vitro, irrespective of its ability to bind plasminogen. A CipA-deficient mutant was efficiently killed by human serum and showed a defect in the penetration of endothelial monolayers, demonstrating that CipA is a novel multifunctional protein that contributes to the pathogenesis ofA. baumannii. © The Author 2015. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

Inhibition of the complement system by saliva of Anopheles (Nyssorhynchus) aquasalis.

Science.gov (United States)

Mendes-Sousa, Antonio Ferreira; Vale, Vladimir Fazito; Queiroz, Daniel Costa; Pereira-Filho, Adalberto Alves; da Silva, Naylene Carvalho Sales; Koerich, Leonardo Barbosa; Moreira, Luciano Andrade; Pereira, Marcos Horácio; Sant'Anna, Maurício Roberto; Araújo, Ricardo Nascimento; Andersen, John; Valenzuela, Jesus Gilberto; Gontijo, Nelder Figueiredo

2018-01-01

Anopheline mosquitoes are vectors of malaria parasites. Their saliva contains anti-hemostatic and immune-modulator molecules that favor blood feeding and parasite transmission. In this study, we describe the inhibition of the alternative pathway of the complement system (AP) by Anopheles aquasalis salivary gland extracts (SGE). According to our results, the inhibitor present in SGE acts on the initial step of the AP blocking deposition of C3b on the activation surfaces. Properdin, which is a positive regulatory molecule of the AP, binds to SGE. When SGE was treated with an excess of properdin, it was unable to inhibit the AP. Through SDS-PAGE analysis, A. aquasalis presented a salivary protein with the same molecular weight as recombinant complement inhibitors belonging to the SG7 family described in the saliva of other anopheline species. At least some SG7 proteins bind to properdin and are AP inhibitors. Searching for SG7 proteins in the A. aquasalis genome, we retrieved a salivary protein that shared an 85% identity with albicin, which is the salivary alternative pathway inhibitor from A. albimanus. This A. aquasalis sequence was also very similar (81% ID) to the SG7 protein from A. darlingi, which is also an AP inhibitor. Our results suggest that the salivary complement inhibitor from A. aquasalis is an SG7 protein that can inhibit the AP by binding to properdin and abrogating its stabilizing activity. Albicin, which is the SG7 from A. albimanus, can directly inhibit AP convertase. Given the high similarity of SG7 proteins, the SG7 from A. aquasalis may also directly inhibit AP convertase in the absence of properdin. Copyright © 2017 Elsevier Ltd. All rights reserved.

Complement activation and inhibition: a delicate balance

DEFF Research Database (Denmark)

Sjöberg, A P; Trouw, L A; Blom, A M

2009-01-01

proteins, pentraxins, amyloid deposits, prions and DNA, all bind the complement activator C1q, but also interact with complement inhibitors C4b-binding protein and factor H. This contrasts to the interaction between C1q and immune complexes, in which case no inhibitors bind, resulting in full complement...

conformational complexity of complement component C3

NARCIS (Netherlands)

Janssen, B.J.C.

2007-01-01

The complement system is an important part of the immune system and critical for the elimination of pathogens. In mammals the complement system consists of an intricate set of about 35 soluble and cell-surface plasma proteins. Central to complement is component C3, a large protein of 1,641 residues.

Trypanosoma cruzi Evades the Complement System as an Efficient Strategy to Survive in the Mammalian Host: The Specific Roles of Host/Parasite Molecules and Trypanosoma cruzi Calreticulin

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Galia Ramírez-Toloza

2017-09-01

Full Text Available American Trypanosomiasis is an important neglected reemerging tropical parasitism, infecting about 8 million people worldwide. Its agent, Trypanosoma cruzi, exhibits multiple mechanisms to evade the host immune response and infect host cells. An important immune evasion strategy of T. cruzi infective stages is its capacity to inhibit the complement system activation on the parasite surface, avoiding opsonizing, immune stimulating and lytic effects. Epimastigotes, the non-infective form of the parasite, present in triatomine arthropod vectors, are highly susceptible to complement-mediated lysis while trypomastigotes, the infective form, present in host bloodstream, are resistant. Thus T. cruzi susceptibility to complement varies depending on the parasite stage (amastigote, trypomastigotes or epimastigote and on the T. cruzi strain. To avoid complement-mediated lysis, T. cruzi trypomastigotes express on the parasite surface a variety of complement regulatory proteins, such as glycoprotein 58/68 (gp58/68, T. cruzi complement regulatory protein (TcCRP, trypomastigote decay-accelerating factor (T-DAF, C2 receptor inhibitor trispanning (CRIT and T. cruzi calreticulin (TcCRT. Alternatively, or concomitantly, the parasite captures components with complement regulatory activity from the host bloodstream, such as factor H (FH and plasma membrane-derived vesicles (PMVs. All these proteins inhibit different steps of the classical (CP, alternative (AP or lectin pathways (LP. Thus, TcCRP inhibits the CP C3 convertase assembling, gp58/68 inhibits the AP C3 convertase, T-DAF interferes with the CP and AP convertases assembling, TcCRT inhibits the CP and LP, CRIT confers ability to resist the CP and LP, FH is used by trypomastigotes to inhibit the AP convertases and PMVs inhibit the CP and LP C3 convertases. Many of these proteins have similar molecular inhibitory mechanisms. Our laboratory has contributed to elucidate the role of TcCRT in the host

Identification and functional characterisation of Complement Regulator Acquiring Surface Protein-1 of serum resistant Borrelia garinii OspA serotype 4

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Zipfel Peter F

2010-02-01

Full Text Available Abstract Background B. burgdorferi sensu lato (sl is the etiological agent of Lyme borreliosis in humans. Spirochetes have adapted themselves to the human immune system in many distinct ways. One important immune escape mechanism for evading complement activation is the binding of complement regulators Factor H (CFH or Factor H-like protein1 (FHL-1 to Complement Regulator-Acquiring Surface Proteins (CRASPs. Results We demonstrate that B. garinii OspA serotype 4 (ST4 PBi resist complement-mediated killing by binding of FHL-1. To identify the primary ligands of FHL-1 four CspA orthologs from B. garinii ST4 PBi were cloned and tested for binding to human CFH and FHL-1. Orthologs BGA66 and BGA71 were found to be able to bind both complement regulators but with different intensities. In addition, all CspA orthologs were tested for binding to mammalian and avian CFH. Distinct orthologs were able to bind to CFH of different animal origins. Conclusions B. garinii ST4 PBi is able to evade complement killing and it can bind FHL-1 to membrane expressed proteins. Recombinant proteins BGA66 can bind FHL-1 and human CFH, while BGA71 can bind only FHL-1. All recombinant CspA orthologs from B. garinii ST4 PBi can bind CFH from different animal origins. This partly explains the wide variety of animals that can be infected by B. garinii.

Radioassays for quantitation of intact complement proteins C2 and B in human serum

Energy Technology Data Exchange (ETDEWEB)

Oglesby, T J; Ueda, A; Volanakis, J E

1988-05-25

Availability of polyclonal and monoclonal antibodies recognizing determinants on the major cleavage fragments of complement proteins C2 and B enabled development of sensitive radioassays which can be used to quantitate the intact proteins in human sera. Changes in C2 and B concentrations indicative of classical or alternative pathway activation, or both, were seen in normal serum after incubation with complement activators. The authors determined the normal range of C2 concentration to be 11-35 ..mu..g/ml in 32 healthy individuals, and that of protein B to be 74-286 ..mu..g/ml. Sera from patients with systemic lupus erythematosus (SLE), septic shock, infections, and following orthopedic surgery were then assayed. Mean protein B concentration was significantly higher in SLE sera and in the infected and post-operative sera, and the mean C2 concentration in the septic shock group was significantly lower than the mean of healthy individuals. Intact C2 was not detected in known C2-deficient individuals. These assays allow parallel quantitation of the structurally and functionally homologous proteins of the classical (C2) and alternative (B) pathways, which is of interest in patients with genetic and acquired hypocomplementemia. 22 refs.; 3 figs.

A new location to split Cre recombinase for protein fragment complementation.

Science.gov (United States)

Rajaee, Maryam; Ow, David W

2017-11-01

We have previously described a recombinase-mediated gene stacking system in which the Cre recombinase is used to remove lox-site flanked DNA no longer needed after each round of Bxb1 integrase-mediated site-specific integration. The Cre recombinase can be conveniently introduced by hybridization with a cre-expressing plant. However, maintaining an efficient cre-expressing line over many generations can be a problem, as high production of this DNA-binding protein might interfere with normal chromosome activities. To counter this selection against high Cre activity, we considered a split-cre approach, in which Cre activity is reconstituted after separate parts of Cre are brought into the same genome by hybridization. To insure that the recombinase-mediated gene stacking system retains its freedom to operate, we tested for new locations to split Cre into complementing fragments. In this study, we describe testing four new locations for splitting the Cre recombinase for protein fragment complementation and show that the two fragments of Cre split between Lys244 and Asn245 can reconstitute activity that is comparable to that of wild-type Cre. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

complement C3, Complement C4 and C-reactive protein

African Journals Online (AJOL)

ajl yemi

2011-12-19

Dec 19, 2011 ... (IL-6), E-selectin and P-selectin (Perlstein and Lee,. 2006). Studies have ... of cigarette smoke causes complement activation which is in turn ..... are decreased by long term smoking cessation in male smokers. Prevent. Med.

Structure-function mapping of BbCRASP-1, the key complement factor H and FHL-1 binding protein of Borrelia burgdorferi.

Science.gov (United States)

Cordes, Frank S; Kraiczy, Peter; Roversi, Pietro; Simon, Markus M; Brade, Volker; Jahraus, Oliver; Wallis, Russell; Goodstadt, Leo; Ponting, Chris P; Skerka, Christine; Zipfel, Peter F; Wallich, Reinhard; Lea, Susan M

2006-05-01

Borrelia burgdorferi, a spirochaete transmitted to human hosts during feeding of infected Ixodes ticks, is the causative agent of Lyme disease, the most frequent vector-borne disease in Eurasia and North America. Sporadically Lyme disease develops into a chronic, multisystemic disorder. Serum-resistant B. burgdorferi strains bind complement factor H (FH) and FH-like protein 1 (FHL-1) on the spirochaete surface. This binding is dependent on the expression of proteins termed complement-regulator acquiring surface proteins (CRASPs). The atomic structure of BbCRASP-1, the key FHL-1/FH-binding protein of B. burgdorferi, has recently been determined. Our analysis indicates that its protein topology apparently evolved to provide a high affinity interaction site for FH/FHL-1 and leads to an atomic-level hypothesis for the functioning of BbCRASP-1. This work demonstrates that pathogens interact with complement regulators in ways that are distinct from the mechanisms used by the host and are thus obvious targets for drug design.

Feast/famine regulatory proteins (FFRPs): Escherichia coli Lrp, AsnC and related archaeal transcription factors.

Science.gov (United States)

Yokoyama, Katsushi; Ishijima, Sanae A; Clowney, Lester; Koike, Hideaki; Aramaki, Hironori; Tanaka, Chikako; Makino, Kozo; Suzuki, Masashi

2006-01-01

Feast/famine regulatory proteins comprise a diverse family of transcription factors, which have been referred to in various individual identifications, including Escherichia coli leucine-responsive regulatory protein and asparagine synthase C gene product. A full length feast/famine regulatory protein consists of the N-terminal DNA-binding domain and the C-domain, which is involved in dimerization and further assembly, thereby producing, for example, a disc or a chromatin-like cylinder. Various ligands of the size of amino acids bind at the interface between feast/famine regulatory protein dimers, thereby altering their assembly forms. Also, the combination of feast/famine regulatory protein subunits forming the same assembly is altered. In this way, a small number of feast/famine regulatory proteins are able to regulate a large number of genes in response to various environmental changes. Because feast/famine regulatory proteins are shared by archaea and eubacteria, the genome-wide regulation by feast/famine regulatory proteins is traceable back to their common ancestor, being the prototype of highly differentiated transcription regulatory mechanisms found in organisms nowadays.

Molecular basis for genetic deficiency of the second component of human complement

International Nuclear Information System (INIS)

Cole, F.S.; Whitehead, A.S.; Auerbach, H.S.; Lint, T.; Zeitz, H.J.; Kilbridge, P.; Colten, H.R.

1985-01-01

Genetic deficiency of the second component of complement (C2) is the most common complement-deficiency state among Western Europeans and is frequently associated with autoimmune diseases. To examine the molecular basis of this deficiency, the authors established cultures of blood monocytes from four families with C2-deficient members. Using a hemolytic-plaque assay, [ 35 S]methionine metabolic labeling of proteins in tissue culture and immunoprecipitation, RNA extraction and Northern blot analysis, and DNA restriction-enzyme digestion and Southern blot analysis, the authors found that C2 deficiency is not due to a major gene deletion or rearrangement but is the result of a specific and selective pretranslational regulatory defect in C2 gene expression. This leads to a lack of detectable C2 mRNA and a lack of synthesis of C2 protein. The approach used in this study should prove useful in examination of other plasma protein deficiencies, especially those in which the deficient gene is normally expressed in peripheral-blood monocytes or tissue macrophages and in which ethical considerations preclude the use of liver or other tissue for study

Protein kinase A regulatory subunit distribution in medulloblastoma

International Nuclear Information System (INIS)

Mucignat-Caretta, Carla; Denaro, Luca; Redaelli, Marco; D'Avella, Domenico; Caretta, Antonio

2010-01-01

Previous studies showed a differential distribution of the four regulatory subunits of cAMP-dependent protein kinases inside the brain, that changed in rodent gliomas: therefore, the distribution of these proteins inside the brain can give information on the functional state of the cells. Our goal was to examine human brain tumors to provide evidence for a differential distribution of protein kinase A in different tumors. The distribution of detergent insoluble regulatory (R1 and R2) and catalytic subunits of cAMP dependent kinases was examined in pediatric brain tumors by immunohistochemistry and fluorescent cAMP analogues binding. R2 is organized in large single dots in medulloblastomas, while it has a different appearance in other tumors. Fluorescent cAMP labelling was observed only in medulloblastoma. A different distribution of cAMP dependent protein kinases has been observed in medulloblastoma

Controlling the complement system in inflammation.

Science.gov (United States)

Kirschfink, M

1997-12-01

Inappropriate or excessive activation of the complement system can lead to harmful, potentially life-threatening consequences due to severe inflammatory tissue destruction. These consequences are clinically manifested in various disorders, including septic shock, multiple organ failure and hyperacute graft rejection. Genetic complement deficiencies or complement depletion have been proven to be beneficial in reducing tissue injury in a number of animal models of severe complement-dependent inflammation. It is therefore believed that therapeutic inhibition of complement is likely to arrest the process of certain diseases. Attempts to efficiently inhibit complement include the application of endogenous soluble complement inhibitors (C1-inhibitor, recombinant soluble complement receptor 1- rsCR1), the administration of antibodies, either blocking key proteins of the cascade reaction (e.g. C3, C5), neutralizing the action of the complement-derived anaphylatoxin C5a, or interfering with complement receptor 3 (CR3, CD18/11b)-mediated adhesion of inflammatory cells to the vascular endothelium. In addition, incorporation of membrane-bound complement regulators (DAF-CD55, MCP-CD46, CD59) has become possible by transfection of the correspondent cDNA into xenogeneic cells. Thereby, protection against complement-mediated inflammatory tissue damage could be achieved in various animal models of sepsis, myocardial as well as intestinal ischemia/reperfusion injury, adult respiratory distress syndrome, nephritis and graft rejection. Supported by results from first clinical trials, complement inhibition appears to be a suitable therapeutic approach to control inflammation. Current strategies to specifically inhibit complement in inflammation have been discussed at a recent meeting on the 'Immune Consequences of Trauma, Shock and Sepsis', held from March 4-8, 1997, in Munich, Germany. The Congress (chairman: E. Faist, Munich, Germany), which was held in close cooperation with various

Human factor H-related protein 2 (CFHR2 regulates complement activation.

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Hannes U Eberhardt

Full Text Available Mutations and deletions within the human CFHR gene cluster on chromosome 1 are associated with diseases, such as dense deposit disease, CFHR nephropathy or age-related macular degeneration. Resulting mutant CFHR proteins can affect complement regulation. Here we identify human CFHR2 as a novel alternative pathway complement regulator that inhibits the C3 alternative pathway convertase and terminal pathway assembly. CFHR2 is composed of four short consensus repeat domains (SCRs. Two CFHR2 molecules form a dimer through their N-terminal SCRs, and each of the two C-terminal ends can bind C3b. C3b bound CFHR2 still allows C3 convertase formation but the CFHR2 bound convertases do not cleave the substrate C3. Interestingly CFHR2 hardly competes off factor H from C3b. Thus CFHR2 likely acts in concert with factor H, as CFHR2 inhibits convertases while simultaneously allowing factor H assisted degradation by factor I.

Borrelia burgdorferi outer surface protein C (OspC) binds complement component C4b and confers bloodstream survival.

Science.gov (United States)

Caine, Jennifer A; Lin, Yi-Pin; Kessler, Julie R; Sato, Hiromi; Leong, John M; Coburn, Jenifer

2017-12-01

Borrelia burgdorferi (Bb) is the causative agent of Lyme disease in the United States, a disease that can result in carditis, and chronic and debilitating arthritis and/or neurologic symptoms if left untreated. Bb survives in the midgut of the Ixodes scapularis tick, or within tissues of immunocompetent hosts. In the early stages of infection, the bacteria are present in the bloodstream where they must resist clearance by the innate immune system of the host. We have found a novel role for outer surface protein C (OspC) from B. burgdorferi and B. garinii in interactions with the complement component C4b and bloodstream survival in vivo. Our data show that OspC inhibits the classical and lectin complement pathways and competes with complement protein C2 for C4b binding. Resistance to complement is important for maintenance of the lifecycle of Bb, enabling survival of the pathogen within the host as well as in the midgut of a feeding tick when ospC expression is induced. © 2017 John Wiley & Sons Ltd.

Pleiotropy constrains the evolution of protein but not regulatory sequences in a transcription regulatory network influencing complex social behaviours

Directory of Open Access Journals (Sweden)

Daria eMolodtsova

2014-12-01

Full Text Available It is increasingly apparent that genes and networks that influence complex behaviour are evolutionary conserved, which is paradoxical considering that behaviour is labile over evolutionary timescales. How does adaptive change in behaviour arise if behaviour is controlled by conserved, pleiotropic, and likely evolutionary constrained genes? Pleiotropy and connectedness are known to constrain the general rate of protein evolution, prompting some to suggest that the evolution of complex traits, including behaviour, is fuelled by regulatory sequence evolution. However, we seldom have data on the strength of selection on mutations in coding and regulatory sequences, and this hinders our ability to study how pleiotropy influences coding and regulatory sequence evolution. Here we use population genomics to estimate the strength of selection on coding and regulatory mutations for a transcriptional regulatory network that influences complex behaviour of honey bees. We found that replacement mutations in highly connected transcription factors and target genes experience significantly stronger negative selection relative to weakly connected transcription factors and targets. Adaptively evolving proteins were significantly more likely to reside at the periphery of the regulatory network, while proteins with signs of negative selection were near the core of the network. Interestingly, connectedness and network structure had minimal influence on the strength of selection on putative regulatory sequences for both transcription factors and their targets. Our study indicates that adaptive evolution of complex behaviour can arise because of positive selection on protein-coding mutations in peripheral genes, and on regulatory sequence mutations in both transcription factors and their targets throughout the network.

RNA-Binding Proteins in Trichomonas vaginalis: Atypical Multifunctional Proteins Involved in a Posttranscriptional Iron Regulatory Mechanism

Science.gov (United States)

Figueroa-Angulo, Elisa E.; Calla-Choque, Jaeson S.; Mancilla-Olea, Maria Inocente; Arroyo, Rossana

2015-01-01

Iron homeostasis is highly regulated in vertebrates through a regulatory system mediated by RNA-protein interactions between the iron regulatory proteins (IRPs) that interact with an iron responsive element (IRE) located in certain mRNAs, dubbed the IRE-IRP regulatory system. Trichomonas vaginalis, the causal agent of trichomoniasis, presents high iron dependency to regulate its growth, metabolism, and virulence properties. Although T. vaginalis lacks IRPs or proteins with aconitase activity, possesses gene expression mechanisms of iron regulation at the transcriptional and posttranscriptional levels. However, only one gene with iron regulation at the transcriptional level has been described. Recently, our research group described an iron posttranscriptional regulatory mechanism in the T. vaginalis tvcp4 and tvcp12 cysteine proteinase mRNAs. The tvcp4 and tvcp12 mRNAs have a stem-loop structure in the 5'-coding region or in the 3'-UTR, respectively that interacts with T. vaginalis multifunctional proteins HSP70, α-Actinin, and Actin under iron starvation condition, causing translation inhibition or mRNA stabilization similar to the previously characterized IRE-IRP system in eukaryotes. Herein, we summarize recent progress and shed some light on atypical RNA-binding proteins that may participate in the iron posttranscriptional regulation in T. vaginalis. PMID:26703754

Dextrose-mediated aggregation of therapeutic monoclonal antibodies in human plasma: Implication of isoelectric precipitation of complement proteins.

Science.gov (United States)

Luo, Shen; Zhang, Baolin

2015-01-01

Many therapeutic monoclonal antibodies (mAbs) are clinically administered through intravenous infusion after mixing with a diluent, e.g., saline, 5% dextrose. Such a clinical setting increases the likelihood of interactions among mAb molecules, diluent, and plasma components, which may adversely affect product safety and efficacy. Avastin® (bevacizumab) and Herceptin® (trastuzumab), but not Remicade® (infliximab), were shown to undergo rapid aggregation upon dilution into 5% dextrose when mixed with human plasma in vitro; however, the biochemical pathways leading to the aggregation were not clearly defined. Here, we show that dextrose-mediated aggregation of Avastin or Herceptin in plasma involves isoelectric precipitation of complement proteins. Using mass spectrometry, we found that dextrose-induced insoluble aggregates were composed of mAb itself and multiple abundant plasma proteins, namely complement proteins C3, C4, factor H, fibronectin, and apolipoprotein. These plasma proteins, which are characterized by an isoelectronic point of 5.5-6.7, lost solubility at the resulting pH in the mixture with formulated Avastin (pH 6.2) and Herceptin (pH 6.0). Notably, switching formulation buffers for Avastin (pH 6.2) and Remicade (pH 7.2) reversed their aggregation profiles. Avastin formed little, if any, insoluble aggregates in dextrose-plasma upon raising the buffer pH to 7.2 or above. Furthermore, dextrose induced pH-dependent precipitation of plasma proteins, with massive insoluble aggregates being detected at pH 6.5-6.8. These data show that isoelectric precipitation of complement proteins is a prerequisite of dextrose-induced aggregation of mAb in human plasma. This finding highlights the importance of assessing the compatibility of a therapeutic mAb with diluent and human plasma during product development.

Autocrine Effects of Tumor-Derived Complement

Directory of Open Access Journals (Sweden)

Min Soon Cho

2014-03-01

Full Text Available We describe a role for the complement system in enhancing cancer growth. Cancer cells secrete complement proteins that stimulate tumor growth upon activation. Complement promotes tumor growth via a direct autocrine effect that is partially independent of tumor-infiltrating cytotoxic T cells. Activated C5aR and C3aR signal through the PI3K/AKT pathway in cancer cells, and silencing the PI3K or AKT gene in cancer cells eliminates the progrowth effects of C5aR and C3aR stimulation. In patients with ovarian or lung cancer, higher tumoral C3 or C5aR mRNA levels were associated with decreased overall survival. These data identify a role for tumor-derived complement proteins in promoting tumor growth, and they therefore have substantial clinical and therapeutic implications.

Soya bean Gα proteins with distinct biochemical properties exhibit differential ability to complement Saccharomyces cerevisiae gpa1 mutant.

Science.gov (United States)

Roy Choudhury, Swarup; Wang, Yuqi; Pandey, Sona

2014-07-01

Signalling pathways mediated by heterotrimeric G-proteins are common to all eukaryotes. Plants have a limited number of each of the G-protein subunits, with the most elaborate G-protein network discovered so far in soya bean (Glycine max, also known as soybean) which has four Gα, four Gβ and ten Gγ proteins. Biochemical characterization of Gα proteins from plants suggests significant variation in their properties compared with the well-characterized non-plant proteins. Furthermore, the four soya bean Gα (GmGα) proteins exhibit distinct biochemical activities among themselves, but the extent to which such biochemical differences contribute to their in vivo function is also not known. We used the yeast gpa1 mutant which displays constitutive signalling and growth arrest in the pheromone-response pathway as an in vivo model to evaluate the effect of distinct biochemical activities of GmGα proteins. We showed that specific GmGα proteins can be activated during pheromone-dependent receptor-mediated signalling in yeast and they display different strengths towards complementation of yeast gpa1 phenotypes. We also identified amino acids that are responsible for differential complementation abilities of specific Gα proteins. These data establish that specific plant Gα proteins are functional in the receptor-mediated pheromone-response pathway in yeast and that the subtle biochemical differences in their activity are physiologically relevant.

[Renal risks of dietary complements: a forgotten cause].

Science.gov (United States)

Dori, Olympia; Humbert, Antoine; Burnier, Michel; Teta, Daniel

2014-02-26

The use of dietary complements like vitamins, minerals, trace elements, proteins, aminoacids and plant-derived agents is prevalent in the general population, in order to promote health and treat diseases. Dietary complements are considered as safe natural products and are easily available without prescription. However, these can lead to severe renal toxicity, especially in cases of unknown pre-existing chronic kidney disease (CKD). In particular, Chinese herbs including aristolochic acid, high doses of vitamine C, creatine and protein complements may lead to acute and chronic renal failure, sometimes irreversible. Dietary complement toxicity should be suspected in any case of unexplained renal impairement. In the case of pre-existing CKD, the use of potentially nephrotoxic dietary complements should be screened for.

Pathogens' toolbox to manipulate human complement.

Science.gov (United States)

Fernández, Francisco J; Gómez, Sara; Vega, M Cristina

2017-12-14

The surveillance and pathogen fighting functions of the complement system have evolved to protect mammals from life-threatening infections. In turn, pathogens have developed complex molecular mechanisms to subvert, divert and evade the effector functions of the complement. The study of complement immunoevasion by pathogens sheds light on their infection drivers, knowledge that is essential to implement therapies. At the same time, complement evasion also acts as a discovery ground that reveals important aspects of how complement works under physiological conditions. In recent years, complex interrelationships between infection insults and the onset of autoimmune and complement dysregulation diseases have led to propose that encounters with pathogens can act as triggering factors for disease. The correct management of these diseases involves the recognition of their triggering factors and the development and administration of complement-associated molecular therapies. Even more recently, unsuspected proteins from pathogens have been shown to possess moonlighting functions as virulence factors, raising the possibility that behind the first line of virulence factors there be many more pathogen proteins playing secondary, helping and supporting roles for the pathogen to successfully establish infections. In an era where antibiotics have a progressively reduced effect on the management and control of infectious diseases worldwide, knowledge on the mechanisms of pathogenic invasion and evasion look more necessary and pressing than ever. Copyright © 2017 Elsevier Ltd. All rights reserved.

Anchoring tick salivary anti-complement proteins IRAC I and IRAC II to membrane increases their immunogenicity.

Science.gov (United States)

Gillet, Laurent; Schroeder, Hélène; Mast, Jan; Thirion, Muriel; Renauld, Jean-Christophe; Dewals, Benjamin; Vanderplasschen, Alain

2009-01-01

Tick salivary proteins are promising targets for the development of anti-tick vaccines. Recently, we described two paralogous anti-complement proteins, called Ixodes ricinus anti-complement (IRAC) proteins I and II, that are co-expressed in tick I. ricinus salivary glands. However, our previous attempts to immunize rabbits against IRAC via infection with recombinant Bovine herpesvirus 4 (BoHV-4) vectors invariably failed although both recombinants expressed high levels of functional IRAC proteins in vitro. As IRAC are soluble monovalent antigens, one of the possible explanations is that monovalent ligation of the B-cell receptor induces receptor activation but fails to promote antigen presentation, a phenomenon that is thought to induce a state of B-cell tolerance. In the present study, we tried to increase IRAC immunogenicity by expressing them as oligovalent antigens. To this end, IRAC were fused to membrane anchors and BoHV-4 vectors expressing these recombinant forms were produced. The immunization potentials of recombinant viruses expressing either secreted or transmembrane IRAC proteins were then compared. While the former did not induce a detectable immune response against IRAC, the latter led to high titres of anti-IRAC antibodies that only marginally affected tick blood feeding. All together, the data presented in this study demonstrate that the immunogenicity of a soluble antigen can be greatly improved by anchoring it in membrane.

C-reactive protein and pentraxin-3 binding of factor H-like protein 1 differs from complement factor H: Implications for retinal inflammation

NARCIS (Netherlands)

Swinkels, M. (Maurice); Zhang, J.H. (Justine H.); Tilakaratna, V. (Viranga); Black, G. (Graeme); Perveen, R. (Rahat); McHarg, S. (Selina); Inforzato, A. (Antonio); Day, A.J. (Anthony J.); Clark, S.J. (Simon J.)

2018-01-01

textabstractRetinal inflammation plays a key role in the progression of age-related macular degeneration (AMD), a condition that leads to loss of central vision. The deposition of the acute phase pentraxin C-reactive protein (CRP) in the macula activates the complement system, thereby contributing

Functional genomics of tick thioester-containing proteins reveal the ancient origin of the complement system

Czech Academy of Sciences Publication Activity Database

Burešová, Veronika; Hajdušek, Ondřej; Franta, Zdeněk; Loosová, Gabriela; Grunclová, Lenka; Levashina, E.A.; Kopáček, Petr

2011-01-01

Roč. 3, č. 6 (2011), s. 623-630 ISSN 1662-811X R&D Projects: GA ČR GAP506/10/2136; GA MŠk(CZ) LC06009 Institutional research plan: CEZ:AV0Z60220518 Keywords : tick * thioester-containing proteins * complement Subject RIV: EC - Immunology Impact factor: 4.209, year: 2011

Viral mimicry of the complement system

Indian Academy of Sciences (India)

The complement system is a potent innate immune mechanism consisting of cascades of proteins which are designed to fight against and annul intrusion of all the foreign pathogens. Although viruses are smaller in size and have relatively simple structure, they are not immune to complement attack. Thus, activation of the ...

A functional SNP in the regulatory region of the decay-accelerating factor gene associates with extraocular muscle pareses in myasthenia gravis

KAUST Repository

Heckmann, J M

2009-08-13

Complement activation in myasthenia gravis (MG) may damage muscle endplate and complement regulatory proteins such as decay-accelerating factor (DAF) or CD55 may be protective. We hypothesize that the increased prevalence of severe extraocular muscle (EOM) dysfunction among African MG subjects reported earlier may result from altered DAF expression. To test this hypothesis, we screened the DAF gene sequences relevant to the classical complement pathway and found an association between myasthenics with EOM paresis and the DAF regulatory region c.-198CG SNP (odds ratio8.6; P0.0003). This single nucleotide polymorphism (SNP) results in a twofold activation of a DAF 5?-flanking region luciferase reporter transfected into three different cell lines. Direct matching of the surrounding SNP sequence within the DAF regulatory region with the known transcription factor-binding sites suggests a loss of an Sp1-binding site. This was supported by the observation that the c.-198CG SNP did not show the normal lipopolysaccharide-induced DAF transcriptional upregulation in lymphoblasts from four patients. Our findings suggest that at critical periods during autoimmune MG, this SNP may result in inadequate DAF upregulation with consequent complement-mediated EOM damage. Susceptible individuals may benefit from anti-complement therapy in addition to immunosuppression. © 2010 Macmillan Publishers Limited. All rights reserved.

Early Components of the Complement Classical Activation Pathway in Human Systemic Autoimmune Diseases

Science.gov (United States)

Lintner, Katherine E.; Wu, Yee Ling; Yang, Yan; Spencer, Charles H.; Hauptmann, Georges; Hebert, Lee A.; Atkinson, John P.; Yu, C. Yung

2016-01-01

The complement system consists of effector proteins, regulators, and receptors that participate in host defense against pathogens. Activation of the complement system, via the classical pathway (CP), has long been recognized in immune complex-mediated tissue injury, most notably systemic lupus erythematosus (SLE). Paradoxically, a complete deficiency of an early component of the CP, as evidenced by homozygous genetic deficiencies reported in human, are strongly associated with the risk of developing SLE or a lupus-like disease. Similarly, isotype deficiency attributable to a gene copy-number (GCN) variation and/or the presence of autoantibodies directed against a CP component or a regulatory protein that result in an acquired deficiency are relatively common in SLE patients. Applying accurate assay methodologies with rigorous data validations, low GCNs of total C4, and heterozygous and homozygous deficiencies of C4A have been shown as medium to large effect size risk factors, while high copy numbers of total C4 or C4A as prevalent protective factors, of European and East-Asian SLE. Here, we summarize the current knowledge related to genetic deficiency and insufficiency, and acquired protein deficiencies for C1q, C1r, C1s, C4A/C4B, and C2 in disease pathogenesis and prognosis of SLE, and, briefly, for other systemic autoimmune diseases. As the complement system is increasingly found to be associated with autoimmune diseases and immune-mediated diseases, it has become an attractive therapeutic target. We highlight the recent developments and offer a balanced perspective concerning future investigations and therapeutic applications with a focus on early components of the CP in human systemic autoimmune diseases. PMID:26913032

Complement activating soluble pattern recognition molecules with collagen-like regions, mannan-binding lectin, ficolins and associated proteins

DEFF Research Database (Denmark)

Thiel, Steffen

2007-01-01

Mannan-binding lectin (MBL), L-ficolin, M-ficolin and H-ficolin are all complement activating soluble pattern recognition molecules with recognition domains linked to collagen-like regions. All four may form complexes with four structurally related proteins, the three MBL-associated serine...... proteases (MASPs), MASP-1, MASP-2 and MASP-3, and a smaller MBL-associated protein (MAp19). The four recognition molecules recognize patterns of carbohydrate or acetyl-group containing ligands. After binding to the relevant targets all four are able to activate the complement system. We thus have a system...... where four different and/or overlapping patterns of microbial origin or patterns of altered-self may be recognized, but in all cases the signalling molecules, the MASPs, are shared. MASP-1 and MASP-3 are formed from one gene, MASP1/3, by alternative splicing generating two different mRNAs from a single...

Evaluation of lysozyme, complement C3, and total protein in different developmental stages of Caspian kutum (Rutilus frisii kutum K.

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Abdollahi Razieh

2016-03-01

Full Text Available In this study, non–specific immune parameters in fertilized eggs, eyed embryos, larvae 10, 25, 50, 60, and 70 days post hatch (DPH, and female broodstock of Caspian kutum, Rutilus frisii kutum (Kamensky, were evaluated. The lysozyme activity, complement C3, and total protein levels were measured with the turbidimetric, immunoturbidimetric, and Bradford methods, respectively. The results showed that lysozyme levels decreased from levels noted in the fertilized eggs until the larvae were 10 days old. Subsequently, significant increases in lysozyme levels were observed until 70 DPH. An increasing trend of complement component C3 was noted from the levels in fertilized eggs to 10 DPH, following which it decreased significantly. Total protein levels differed significantly in early developmental stages of Caspian kutum. The higher values of complement component C3 than of lysozyme in the early life stages could be indicative of the former’s more fundamental role.

Dynamic SPR monitoring of yeast nuclear protein binding to a cis-regulatory element

International Nuclear Information System (INIS)

Mao, Grace; Brody, James P.

2007-01-01

Gene expression is controlled by protein complexes binding to short specific sequences of DNA, called cis-regulatory elements. Expression of most eukaryotic genes is controlled by dozens of these elements. Comprehensive identification and monitoring of these elements is a major goal of genomics. In pursuit of this goal, we are developing a surface plasmon resonance (SPR) based assay to identify and monitor cis-regulatory elements. To test whether we could reliably monitor protein binding to a regulatory element, we immobilized a 16 bp region of Saccharomyces cerevisiae chromosome 5 onto a gold surface. This 16 bp region of DNA is known to bind several proteins and thought to control expression of the gene RNR1, which varies through the cell cycle. We synchronized yeast cell cultures, and then sampled these cultures at a regular interval. These samples were processed to purify nuclear lysate, which was then exposed to the sensor. We found that nuclear protein binds this particular element of DNA at a significantly higher rate (as compared to unsynchronized cells) during G1 phase. Other time points show levels of DNA-nuclear protein binding similar to the unsynchronized control. We also measured the apparent association complex of the binding to be 0.014 s -1 . We conclude that (1) SPR-based assays can monitor DNA-nuclear protein binding and that (2) for this particular cis-regulatory element, maximum DNA-nuclear protein binding occurs during G1 phase

Guanine nucleotide-binding regulatory proteins in retinal pigment epithelial cells.

OpenAIRE

Jiang, M; Pandey, S; Tran, V T; Fong, H K

1991-01-01

The expression of GTP-binding regulatory proteins (G proteins) in retinal pigment epithelial (RPE) cells was analyzed by RNA blot hybridization and cDNA amplification. Both adult and fetal human RPE cells contain mRNA for multiple G protein alpha subunits (G alpha) including Gs alpha, Gi-1 alpha, Gi-2 alpha, Gi-3 alpha, and Gz alpha (or Gx alpha), where Gs and Gi are proteins that stimulate or inhibit adenylyl cyclase, respectively, and Gz is a protein that may mediate pertussis toxin-insensi...

The C-type lectin of the aggrecan G3 domain activates complement.

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Camilla Melin Fürst

Full Text Available Excessive complement activation contributes to joint diseases such as rheumatoid arthritis and osteoarthritis during which cartilage proteins are fragmented and released into the synovial fluid. Some of these proteins and fragments activate complement, which may sustain inflammation. The G3 domain of large cartilage proteoglycan aggrecan interacts with other extracellular matrix proteins, fibulins and tenascins, via its C-type lectin domain (CLD and has important functions in matrix organization. Fragments containing G3 domain are released during normal aggrecan turnover, but increasingly so in disease. We now show that the aggrecan CLD part of the G3 domain activates the classical and to a lesser extent the alternative pathway of complement, via binding of C1q and C3, respectively. The complement control protein (CCP domain adjacent to the CLD showed no effect on complement initiation. The binding of C1q to G3 depended on ionic interactions and was decreased in D2267N mutant G3. However, the observed complement activation was attenuated due to binding of complement inhibitor factor H to CLD and CCP domains. This was most apparent at the level of deposition of terminal complement components. Taken together our observations indicate aggrecan CLD as one factor involved in the sustained inflammation of the joint.

The complement inhibitor eculizumab in paroxysmal nocturnal hemoglobinuria.

NARCIS (Netherlands)

Hillmen, P.; Young, N.S.; Schubert, J.; Brodsky, R.A.; Socie, G.; Muus, P.; Roth, A.; Szer, J.; Elebute, M.O.; Nakamura, R.; Browne, P.; Risitano, A.M.; Hill, A.; Schrezenmeier, H.; Fu, C.L.; Maciejewski, J; Rollins, S.A.; Mojcik, C.F.; Rother, R.P.; Luzzatto, L.

2006-01-01

BACKGROUND: We tested the safety and efficacy of eculizumab, a humanized monoclonal antibody against terminal complement protein C5 that inhibits terminal complement activation, in patients with paroxysmal nocturnal hemoglobinuria (PNH). METHODS: We conducted a double-blind, randomized,

Co-suppression of sterol-regulatory element binding protein ...

African Journals Online (AJOL)

Administrator

2011-06-22

Jun 22, 2011 ... In Arabidopsis,. At5g35220 gene being sterol regulatory element-binding protein site 2, protease and metalloendopeptidase activity were required for chloroplast development and play a role in regulation of endodermal plastid size and number that are involved in ethylene-dependent gravitropism of light-.

Hepatitis C virus NS3/4A protease inhibits complement activation by cleaving complement component 4.

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Seiichi Mawatari

Full Text Available BACKGROUND: It has been hypothesized that persistent hepatitis C virus (HCV infection is mediated in part by viral proteins that abrogate the host immune response, including the complement system, but the precise mechanisms are not well understood. We investigated whether HCV proteins are involved in the fragmentation of complement component 4 (C4, composed of subunits C4α, C4β, and C4γ, and the role of HCV proteins in complement activation. METHODS: Human C4 was incubated with HCV nonstructural (NS 3/4A protease, core, or NS5. Samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then subjected to peptide sequencing. The activity of the classical complement pathway was examined using an erythrocyte hemolysis assay. The cleavage pattern of C4 in NS3/4A-expressing and HCV-infected cells, respectively, was also examined. RESULTS: HCV NS3/4A protease cleaved C4γ in a concentration-dependent manner, but viral core and NS5 did not. A specific inhibitor of NS3/4A protease reduced C4γ cleavage. NS3/4A protease-mediated cleavage of C4 inhibited classical pathway activation, which was abrogated by a NS3/4A protease inhibitor. In addition, co-transfection of cells with C4 and wild-type NS3/4A, but not a catalytic-site mutant of NS3/4A, produced cleaved C4γ fragments. Such C4 processing, with a concomitant reduction in levels of full-length C4γ, was also observed in HCV-infected cells expressing C4. CONCLUSIONS: C4 is a novel cellular substrate of the HCV NS3/4A protease. Understanding disturbances in the complement system mediated by NS3/4A protease may provide new insights into the mechanisms underlying persistent HCV infection.

Protein Kinase A Regulatory Subunits in Human Adipose Tissue

Science.gov (United States)

Mantovani, Giovanna; Bondioni, Sara; Alberti, Luisella; Gilardini, Luisa; Invitti, Cecilia; Corbetta, Sabrina; Zappa, Marco A.; Ferrero, Stefano; Lania, Andrea G.; Bosari, Silvano; Beck-Peccoz, Paolo; Spada, Anna

2009-01-01

OBJECTIVE—In human adipocytes, the cAMP-dependent pathway mediates signals originating from β-adrenergic activation, thus playing a key role in the regulation of important metabolic processes, i.e., lipolysis and thermogenesis. Cyclic AMP effects are mainly mediated by protein kinase A (PKA), whose R2B regulatory isoform is the most expressed in mouse adipose tissue, where it protects against diet-induced obesity and fatty liver development. The aim of the study was to investigate possible differences in R2B expression, PKA activity, and lipolysis in adipose tissues from obese and nonobese subjects. RESEARCH DESIGN AND METHODS—The expression of the different PKA regulatory subunits was evaluated by immunohistochemistry, Western blot, and real-time PCR in subcutaneous and visceral adipose tissue samples from 20 nonobese and 67 obese patients. PKA activity and glycerol release were evaluated in total protein extract and adipocytes isolated from fresh tissue samples, respectively. RESULTS—Expression techniques showed that R2B was the most abundant regulatory protein, both at mRNA and protein level. Interestingly, R2B mRNA levels were significantly lower in both subcutaneous and visceral adipose tissues from obese than nonobese patients and negatively correlated with BMI, waist circumference, insulin levels, and homeostasis model assessment of insulin resistance. Moreover, both basal and stimulated PKA activity and glycerol release were significantly lower in visceral adipose tissue from obese patients then nonobese subjects. CONCLUSIONS—Our results first indicate that, in human adipose tissue, there are important BMI-related differences in R2B expression and PKA activation, which might be included among the multiple determinants involved in the different lipolytic response to β-adrenergic activation in obesity. PMID:19095761

Complement-mediated solubilization of immune complexes and their interaction with complement C3 receptors

DEFF Research Database (Denmark)

Petersen, Ivan; Baatrup, Gunnar; Jepsen, H H

1985-01-01

Some of the molecular events in the complement (C)-mediated solubilization of immune complexes (IC) have been clarified in recent years. The solubilization is primarily mediated by alternative C pathway proteins whereas factors in the classical pathway accelerate the process. Components of the me......Some of the molecular events in the complement (C)-mediated solubilization of immune complexes (IC) have been clarified in recent years. The solubilization is primarily mediated by alternative C pathway proteins whereas factors in the classical pathway accelerate the process. Components...... of the cellular localization, expression and structure of the C3 receptors, especially the C3b (CR1) receptor, has been considerably extended in the last few years, whereas our understanding of the physiological role of these receptors is still fragmentary. However, it is becoming increasingly evident...

Plasma complement biomarkers distinguish multiple sclerosis and neuromyelitis optica spectrum disorder.

Science.gov (United States)

Hakobyan, Svetlana; Luppe, Sebastian; Evans, David Rs; Harding, Katharine; Loveless, Samantha; Robertson, Neil P; Morgan, B Paul

2017-06-01

Multiple sclerosis (MS) and neuromyelitis optica spectrum disorder (NMOSD) are autoimmune inflammatory demyelinating diseases of the central nervous system. Although distinguished by clinicoradiological and demographic features, early manifestations can be similar complicating management. Antibodies against aquaporin-4 support the diagnosis of NMOSD but are negative in some patients. Therefore, there is unmet need for biomarkers that enable early diagnosis and disease-specific intervention. We investigated whether plasma complement proteins are altered in MS and NMOSD and provide biomarkers that distinguish these diseases. Plasma from 54 NMOSD, 40 MS and 69 control donors was tested in multiplex assays measuring complement activation products and proteins. Using logistic regression, we tested whether combinations of complement analytes distinguished NMOSD from controls and MS. All activation products were elevated in NMOSD compared to either control or MS. Four complement proteins (C1inh, C1s, C5 and FH) were higher in NMOSD compared to MS or controls. A model comprising C1inh and terminal complement complex (TCC) distinguished NMOSD from MS (area under the curve (AUC): 0.98), while C1inh and C5 distinguished NMOSD from controls (AUC: 0.94). NMOSD is distinguished from MS by plasma complement biomarkers. Selected complement analytes enable differential diagnosis. Findings support trials of anti-complement therapies in NMOSD.

Complement-mediated bactericidal activity of anti-factor H binding protein monoclonal antibodies against the meningococcus relies upon blocking factor H binding.

Science.gov (United States)

Giuntini, Serena; Reason, Donald C; Granoff, Dan M

2011-09-01

Binding of the complement-downregulating protein factor H (fH) to the surface of the meningococcus is important for survival of the organism in human serum. The meningococcal vaccine candidate factor H binding protein (fHbp) is an important ligand for human fH. While some fHbp-specific monoclonal antibodies (MAbs) block binding of fH to fHbp, the stoichiometry of blocking in the presence of high serum concentrations of fH and its effect on complement-mediated bactericidal activity are unknown. To investigate this question, we constructed chimeric antibodies in which the human IgG1 constant region was paired with three murine fHbp-specific binding domains designated JAR 3, JAR 5, and MAb502. By surface plasmon resonance, the association rates for binding of all three MAbs to immobilized fHbp were >50-fold higher than that for binding of fH to fHbp, and the MAb dissociation rates were >500-fold lower than that for fH. While all three MAbs elicited similar C1q-dependent C4b deposition on live bacteria (classical complement pathway), only those antibodies that inhibited binding of fH to fHbp (JAR 3 and JAR 5) had bactericidal activity with human complement. MAb502, which did not inhibit fH binding, had complement-mediated bactericidal activity only when tested with fH-depleted human complement. When an IgG1 anti-fHbp MAb binds to sparsely exposed fHbp on the bacterial surface, there appears to be insufficient complement activation for bacteriolysis unless fH binding also is inhibited. The ability of fHbp vaccines to elicit protective antibodies, therefore, is likely to be enhanced if the antibody repertoire is of high avidity and includes fH-blocking activity.

Solid-phase classical complement activation by C-reactive protein (CRP) is inhibited by fluid-phase CRP-C1q interaction

International Nuclear Information System (INIS)

Sjoewall, Christopher; Wetteroe, Jonas; Bengtsson, Torbjoern; Askendal, Agneta; Almroth, Gunnel; Skogh, Thomas; Tengvall, Pentti

2007-01-01

C-reactive protein (CRP) interacts with phosphorylcholine (PC), Fcγ receptors, complement factor C1q and cell nuclear constituents, yet its biological roles are insufficiently understood. The aim was to characterize CRP-induced complement activation by ellipsometry. PC conjugated with keyhole limpet hemocyanin (PC-KLH) was immobilized to cross-linked fibrinogen. A low-CRP serum with different amounts of added CRP was exposed to the PC-surfaces. The total serum protein deposition was quantified and deposition of IgG, C1q, C3c, C4, factor H, and CRP detected with polyclonal antibodies. The binding of serum CRP to PC-KLH dose-dependently triggered activation of the classical pathway. Unexpectedly, the activation was efficiently down-regulated at CRP levels >150 mg/L. Using radial immunodiffusion, CRP-C1q interaction was observed in serum samples with high CRP concentrations. We propose that the underlying mechanism depends on fluid-phase interaction between C1q and CRP. This might constitute another level of complement regulation, which has implications for systemic lupus erythematosus where CRP is often low despite flare-ups

A mechanism of acquired resistance to complement-mediated lysis by Entamoeba histolytica.

Science.gov (United States)

Gutiérrez-Kobeh, L; Cabrera, N; Pérez-Montfort, R

1997-04-01

Some Entamoeba histolytica strains resist complement-mediated lysis by serum. Susceptible and resistant strains activate the complement system equivalently, but resistant amebas evade killing by membrane attack complexes. Our objective was to determine the mechanism by which trophozoites of E. histolytica resist lysis by human serum. Amebas were made resistant to lysis by incubation with increasing concentrations of normal human serum. The possibility that resistant cells ingest membrane attack complexes was explored by subcellular fractionation of susceptible and resistant trophozoites treated with sublytic concentrations of human serum containing radiolabeled C9. In both cases, most of the label was in the fractions containing plasma membrane. The susceptible strain consistently showed more label associated with these fractions than the resistant strain. Thus, the possibility that the membrane attack complexes were released to the medium was explored. Both resistant and susceptible trophozoites release to the medium similar amounts of material excluded by Sepharose CL-2B in the presence or absence of normal human serum. Labeled C9 elutes together with the main bulk of proteins from the medium: this indicates that it is not in vesicles or high molecular weight aggregates. Coincubation of susceptible amebas with lysates of resistant trophozoites confers resistance to susceptible cells within 30 min. Resistance to lysis by serum can also be acquired by susceptible amebas after coincubation with lysates from human erythrocytes or after feeding them with whole human red blood cells. Resistant but not susceptible trophozoites show intense immunofluorescent staining on their surface with anti-human erythrocytic membrane antibody. These results suggest that amebas acquire resistance to lysis by serum by incorporating into their membranes complement regulatory proteins.

The meningococcal vaccine candidate neisserial surface protein A (NspA binds to factor H and enhances meningococcal resistance to complement.

Directory of Open Access Journals (Sweden)

Lisa A Lewis

2010-07-01

Full Text Available Complement forms an important arm of innate immunity against invasive meningococcal infections. Binding of the alternative complement pathway inhibitor factor H (fH to fH-binding protein (fHbp is one mechanism meningococci employ to limit complement activation on the bacterial surface. fHbp is a leading vaccine candidate against group B Neisseria meningitidis. Novel mechanisms that meningococci employ to bind fH could undermine the efficacy of fHbp-based vaccines. We observed that fHbp deletion mutants of some meningococcal strains showed residual fH binding suggesting the presence of a second receptor for fH. Ligand overlay immunoblotting using membrane fractions from one such strain showed that fH bound to a approximately 17 kD protein, identified by MALDI-TOF analysis as Neisserial surface protein A (NspA, a meningococcal vaccine candidate whose function has not been defined. Deleting nspA, in the background of fHbp deletion mutants, abrogated fH binding and mAbs against NspA blocked fH binding, confirming NspA as a fH binding molecule on intact bacteria. NspA expression levels vary among strains and expression correlated with the level of fH binding; over-expressing NspA enhanced fH binding to bacteria. Progressive truncation of the heptose (Hep I chain of lipooligosaccharide (LOS, or sialylation of lacto-N-neotetraose LOS both increased fH binding to NspA-expressing meningococci, while expression of capsule reduced fH binding to the strains tested. Similar to fHbp, binding of NspA to fH was human-specific and occurred through fH domains 6-7. Consistent with its ability to bind fH, deleting NspA increased C3 deposition and resulted in increased complement-dependent killing. Collectively, these data identify a key complement evasion mechanism with important implications for ongoing efforts to develop meningococcal vaccines that employ fHbp as one of its components.

Interactions among the early Escherichia coli divisome proteins revealed by bimolecular fluorescence complementation.

Science.gov (United States)

Pazos, Manuel; Natale, Paolo; Margolin, William; Vicente, Miguel

2013-12-01

We used bimolecular fluorescence complementation (BiFC) assays to detect protein-protein interactions of all possible pairs of the essential Escherichia coli proto-ring components, FtsZ, FtsA and ZipA, as well as the non-essential FtsZ-associated proteins ZapA and ZapB. We found an unexpected interaction between ZipA and ZapB at potential cell division sites, and when co-overproduced, they induced long narrow constrictions at division sites that were dependent on FtsZ. These assays also uncovered an interaction between ZipA and ZapA that was mediated by FtsZ. BiFC with ZapA and ZapB showed that in addition to their expected interaction at midcell, they also interact at the cell poles. BiFC detected interaction between FtsZ and ZapB at midcell and close to the poles. Results from the remaining pairwise combinations confirmed known interactions between FtsZ and ZipA, and ZapB with itself. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.

Acquisition of complement inhibitor serine protease factor I and its cofactors C4b-binding protein and factor H by Prevotella intermedia.

Science.gov (United States)

Malm, Sven; Jusko, Monika; Eick, Sigrun; Potempa, Jan; Riesbeck, Kristian; Blom, Anna M

2012-01-01

Infection with the Gram-negative pathogen Prevotella intermedia gives rise to periodontitis and a growing number of studies implies an association of P. intermedia with rheumatoid arthritis. The serine protease Factor I (FI) is the central inhibitor of complement degrading complement components C3b and C4b in the presence of cofactors such as C4b-binding protein (C4BP) and Factor H (FH). Yet, the significance of complement inhibitor acquisition in P. intermedia infection and FI binding by Gram-negative pathogens has not been addressed. Here we show that P. intermedia isolates bound purified FI as well as FI directly from heat-inactivated human serum. FI bound to bacteria retained its serine protease activity as shown in degradation experiments with (125)I-labeled C4b. Since FI requires cofactors for its activity we also investigated the binding of purified cofactors C4BP and FH and found acquisition of both proteins, which retained their activity in FI mediated degradation of C3b and C4b. We propose that FI binding by P. intermedia represents a new mechanism contributing to complement evasion by a Gram-negative bacterial pathogen associated with chronic diseases.

Implementing maintenance complement changes and experience with regulatory guide G-323

International Nuclear Information System (INIS)

Bernston, K.; Budau, J.

2009-01-01

G-323, the Canadian Nuclear Safety Commission (CNSC) guidance document on ensuring minimum staff complement, was released as an official document in July 2007. Shortly before the release of this document, Bruce Power was reviewing minimum complement with a particular focus on minimizing the number of maintenance staff on rotating shifts. The goals were both to increase the maintenance being performed on days, where it would be less error likely, and to reduce the number of personnel exposed to the health impacts of shift work. Although G-323 was not in effect at the time, a decision was made to work to the expectations of the document to the extent possible. This paper outlines the experience in performing this work, as well as lessons learned. (author)

Structural evaluation of a nanobody targeting complement receptor Vsig4 and its cross reactivity.

Science.gov (United States)

Wen, Yurong; Ouyang, Zhenlin; Schoonooghe, Steve; Luo, Siyu; De Baetselier, Patrick; Lu, Wuyuan; Muyldermans, Serge; Raes, Geert; Zheng, Fang

2017-06-01

Vsig4 is a recently identified immune regulatory protein related to the B7 family with dual functionality: a negative regulator of T cell activation and a receptor for the complement components C3b and C3c. Here we present a structural evaluation of a nanobody, Nb119, against the extracellular IgV domain protein of both mouse and human recombinant Vsig4, which have a high degree of sequence identity. Although mouse and human Vsig4 bind to Nb119 with a 250 times difference in dissociation constants, the interaction results in a highly identical assembly with a RMSD of 0.4Å. The molecular determinants for Vsig4 recognition and cross reactivity unveiled by the atomic structure of Nb119 in complex with mVsig4 and hVsig4 afford new insights useful for the further optimization of the nanobody for potential use in humans. Additionally, structural analysis of the Vsig4-Nb119 complexes indicates that Nb119 occupies the interface on Vsig4 recognized by the macroglobulin-like domains MG4 and MG5 of C3b. Thus an affinity-improved Nb119 may have the potential to influence the activation of both T cells and complement. Copyright © 2016. Published by Elsevier GmbH.

Construction of a bimolecular fluorescence complementation (BiFC ...

African Journals Online (AJOL)

Protein–protein interactions are essential for signal transduction in cells. Bimolecular fluorescence complementation (BiFC) is a novel technology that utilises green fluorescent proteins to visualize protein–protein interactions and subcellular protein localisation. BiFC based on pSATN vectors are a good system for ...

Systemic complement activation in age-related macular degeneration.

Directory of Open Access Journals (Sweden)

Hendrik P N Scholl

Full Text Available Dysregulation of the alternative pathway (AP of complement cascade has been implicated in the pathogenesis of age-related macular degeneration (AMD, the leading cause of blindness in the elderly. To further test the hypothesis that defective control of complement activation underlies AMD, parameters of complement activation in blood plasma were determined together with disease-associated genetic markers in AMD patients. Plasma concentrations of activation products C3d, Ba, C3a, C5a, SC5b-9, substrate proteins C3, C4, factor B and regulators factor H and factor D were quantified in patients (n = 112 and controls (n = 67. Subjects were analyzed for single nucleotide polymorphisms in factor H (CFH, factor B-C2 (BF-C2 and complement C3 (C3 genes which were previously found to be associated with AMD. All activation products, especially markers of chronic complement activation Ba and C3d (p<0.001, were significantly elevated in AMD patients compared to controls. Similar alterations were observed in factor D, but not in C3, C4 or factor H. Logistic regression analysis revealed better discriminative accuracy of a model that is based only on complement activation markers Ba, C3d and factor D compared to a model based on genetic markers of the complement system within our study population. In both the controls' and AMD patients' group, the protein markers of complement activation were correlated with CFH haplotypes.This study is the first to show systemic complement activation in AMD patients. This suggests that AMD is a systemic disease with local disease manifestation at the ageing macula. Furthermore, the data provide evidence for an association of systemic activation of the alternative complement pathway with genetic variants of CFH that were previously linked to AMD susceptibility.

Comprehensive approach to study complement C4 in systemic lupus erythematosus: Gene polymorphisms, protein levels and functional activity

NARCIS (Netherlands)

Tsang-A-Sjoe, M. W. P.; Bultink, I. E. M.; Korswagen, L. A.; van der Horst, A. [=Anneke; Rensink, I.; de Boer, M.; Hamann, D.; Voskuyl, A. E.; Wouters, D.

2017-01-01

Genetic variation of the genes encoding complement component C4 is strongly associated with systemic lupus erythematosus (SLE), a chronic multi-organ auto-immune disease. This study examined C4 and its isotypes on a genetic, protein, and functional level in 140 SLE patients and 104 healthy controls.

Evasion Mechanisms Used by Pathogens to Escape the Lectin Complement Pathway

DEFF Research Database (Denmark)

Rosbjerg, Anne; Genster, Ninette; Pilely, Katrine

2017-01-01

The complement system is a crucial defensive network that protects the host against invading pathogens. It is part of the innate immune system and can be initiated via three pathways: the lectin, classical and alternative activation pathway. Overall the network compiles a group of recognition...... the level of activity. The result is a pro-inflammatory response meant to combat foreign microbes. Microbial elimination is, however, not a straight forward procedure; pathogens have adapted to their environment by evolving a collection of evasion mechanisms that circumvent the human complement system....... Complement evasion strategies features different ways of exploiting human complement proteins and moreover features different pathogen-derived proteins that interfere with the normal processes. Accumulated, these mechanisms target all three complement activation pathways as well as the final common part...

CONSTRUCTION AND ANALYSIS OF IPBR/XYLS HYBRID REGULATORY PROTEINS

Science.gov (United States)

IpbR and XylS are related regulatory proteins (having 56% identity). IpbR responds to isopropylbenzene as well as to a variety of hydrophobic chemicals to activate expression of the isopropylbenzene catabolic pathway operon of pRE4 from ipbOP. XylS responds to substituted benzoic...

C-Reactive Protein Binds to Cholesterol Crystals and Co-Localizes with the Terminal Complement Complex in Human Atherosclerotic Plaques

DEFF Research Database (Denmark)

Pilely, Katrine; Fumagalli, Stefano; Rosbjerg, Anne

2017-01-01

Inflammation is a part of the initial process leading to atherosclerosis and cholesterol crystals (CC), found in atherosclerotic plaques, which are known to induce complement activation. The pentraxins C-reactive protein (CRP), long pentraxin 3 (PTX3), and serum amyloid P component (SAP) are seru...

Intragenic complementation by the nifJ-coded protein of Klebsiella pneumoniae.

OpenAIRE

Stacey, G; Zhu, J; Shah, V K; Shen, S C; Brill, W J

1982-01-01

A single mutation, nifC1005 (Jin et al. Sci. Sin. 23:108-118, 1980), located between nifH and nifJ in the nif cluster of Klebsiella pneumoniae, genetically complemented mutations in each of the 17 known nif genes. This suggested that the mutation is located in a new nif gene. We showed by complementation analyses that only 3 of 12 nifJ mutations tested were complemented by nifC1005. Nitrogenase activity in cell extracts of the mutant with nifC1005 as well as NifJ- mutants was stimulated by th...

Evasion Mechanisms Used by Pathogens to Escape the Lectin Complement Pathway

DEFF Research Database (Denmark)

Rosbjerg, Anne; Genster, Ninette; Pilely, Katrine

2017-01-01

the level of activity. The result is a pro-inflammatory response meant to combat foreign microbes. Microbial elimination is, however, not a straight forward procedure; pathogens have adapted to their environment by evolving a collection of evasion mechanisms that circumvent the human complement system....... Complement evasion strategies features different ways of exploiting human complement proteins and moreover features different pathogen-derived proteins that interfere with the normal processes. Accumulated, these mechanisms target all three complement activation pathways as well as the final common part...... of the cascade. This review will cover the currently known lectin pathway evasion mechanisms and give examples of pathogens that operate these to increase their chance of invasion, survival and dissemination....

Human SAP is a novel peptidoglycan recognition protein that induces complement- independent phagocytosis of Staphylococcus aureus

Science.gov (United States)

An, Jang-Hyun; Kurokawa, Kenji; Jung, Dong-Jun; Kim, Min-Jung; Kim, Chan-Hee; Fujimoto, Yukari; Fukase, Koichi; Coggeshall, K. Mark; Lee, Bok Luel

2014-01-01

The human pathogen Staphylococcus aureus is responsible for many community-acquired and hospital-associated infections and is associated with high mortality. Concern over the emergence of multidrug-resistant strains has renewed interest in the elucidation of host mechanisms that defend against S. aureus infection. We recently demonstrated that human serum mannose-binding lectin (MBL) binds to S. aureus wall teichoic acid (WTA), a cell wall glycopolymer, a discovery that prompted further screening to identify additional serum proteins that recognize S. aureus cell wall components. In this report, we incubated human serum with 10 different S. aureus mutants and determined that serum amyloid P component (SAP) bound specifically to a WTA-deficient S. aureus ΔtagO mutant, but not to tagO-complemented, WTA-expressing cells. Biochemical characterization revealed that SAP recognizes bacterial peptidoglycan as a ligand and that WTA inhibits this interaction. Although SAP binding to peptidoglycan was not observed to induce complement activation, SAP-bound ΔtagO cells were phagocytosed by human polymorphonuclear leukocytes in an Fcγ receptor-dependent manner. These results indicate that SAP functions as a host defense factor, similar to other peptidoglycan recognition proteins and nucleotide-binding oligomerization domain (NOD)-like receptors. PMID:23966633

Changes in blood levels of proteinase inhibitors, pregnancy zone protein, steroid carriers and complement factors induced by oral contraceptives

DEFF Research Database (Denmark)

Nielsen, C H; Poulsen, H K; Teisner, B

1993-01-01

levels of antithrombin III (AT III), alpha 2-macroglobulin (alpha 2M) alpha 1-antitrypsin (alpha 1at), complement factors (factor B, C3, C4), pregnancy zone protein (PZP), corticosteroid binding globulin (CBG), sex hormone binding globulin (SHBG) and albumin were measured before treatment and during...

Conserved Patterns of Microbial Immune Escape: Pathogenic Microbes of Diverse Origin Target the Human Terminal Complement Inhibitor Vitronectin via a Single Common Motif.

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Teresia Hallström

Full Text Available Pathogenicity of many microbes relies on their capacity to resist innate immunity, and to survive and persist in an immunocompetent human host microbes have developed highly efficient and sophisticated complement evasion strategies. Here we show that different human pathogens including Gram-negative and Gram-positive bacteria, as well as the fungal pathogen Candida albicans, acquire the human terminal complement regulator vitronectin to their surface. By using truncated vitronectin fragments we found that all analyzed microbial pathogens (n = 13 bound human vitronectin via the same C-terminal heparin-binding domain (amino acids 352-374. This specific interaction leaves the terminal complement complex (TCC regulatory region of vitronectin accessible, allowing inhibition of C5b-7 membrane insertion and C9 polymerization. Vitronectin complexed with the various microbes and corresponding proteins was thus functionally active and inhibited complement-mediated C5b-9 deposition. Taken together, diverse microbial pathogens expressing different structurally unrelated vitronectin-binding molecules interact with host vitronectin via the same conserved region to allow versatile control of the host innate immune response.

Structure of the extracellular portion of CD46 provides insights into its interactions with complement proteins and pathogens.

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B David Persson

2010-09-01

Full Text Available The human membrane cofactor protein (MCP, CD46 is a central component of the innate immune system. CD46 protects autologous cells from complement attack by binding to complement proteins C3b and C4b and serving as a cofactor for their cleavage. Recent data show that CD46 also plays a role in mediating acquired immune responses, and in triggering autophagy. In addition to these physiologic functions, a significant number of pathogens, including select adenoviruses, measles virus, human herpes virus 6 (HHV-6, Streptococci, and Neisseria, use CD46 as a cell attachment receptor. We have determined the crystal structure of the extracellular region of CD46 in complex with the human adenovirus type 11 fiber knob. Extracellular CD46 comprises four short consensus repeats (SCR1-SCR4 that form an elongated structure resembling a hockey stick, with a long shaft and a short blade. Domains SCR1, SCR2 and SCR3 are arranged in a nearly linear fashion. Unexpectedly, however, the structure reveals a profound bend between domains SCR3 and SCR4, which has implications for the interactions with ligands as well as the orientation of the protein at the cell surface. This bend can be attributed to an insertion of five hydrophobic residues in a SCR3 surface loop. Residues in this loop have been implicated in interactions with complement, indicating that the bend participates in binding to C3b and C4b. The structure provides an accurate framework for mapping all known ligand binding sites onto the surface of CD46, thereby advancing an understanding of how CD46 acts as a receptor for pathogens and physiologic ligands of the immune system.

Is complement good, bad, or both? New functions of the complement factors associated with inflammation mechanisms in the central nervous system.

Science.gov (United States)

Tahtouh, Muriel; Croq, Françoise; Lefebvre, Christophe; Pestel, Joël

2009-09-01

The complement system is well known as an enzyme cascade that helps to defend against infections. Indeed, this ancestral system bridges innate and adaptive immunity. Its implication in diseases of the central nervous system (CNS), has led to an increased number of studies. Complement activation in the CNS has been generally considered to contribute to tissue damage. However, recent studies suggest that complement may be neuroprotective, and can participate in maintenance and repair of the adult brain. Here, we will review this dual role of complement proteins and some of their functional interactions with part of the chemokine and cytokine network associated with the protection of CNS integrity.

Complement and the control of HIV infection: an evolving story.

Science.gov (United States)

Frank, Michael M; Hester, Christopher; Jiang, Haixiang

2014-05-01

Thirty years ago, investigators isolated and later determined the structure of HIV-1 and its envelope proteins. Using techniques that were effective with other viruses, they prepared vaccines designed to generate antibody or T-cell responses, but they were ineffective in clinical trials. In this article, we consider the role of complement in host defense against enveloped viruses, the role it might play in the antibody response and why complement has not controlled HIV-1 infection. Complement consists of a large group of cell-bound and plasma proteins that are an integral part of the innate immune system. They provide a first line of defense against microbes and also play a role in the immune response. Here we review the studies of complement-mediated HIV destruction and the role of complement in the HIV antibody response. HIV-1 has evolved a complex defense to prevent complement-mediated killing reviewed here. As part of these studies, we have discovered that HIV-1 envelope, on administration into animals, is rapidly broken down into small peptides that may prove to be very inefficient at provident the type of antigenic stimulation that leads to an effective immune response. Improving complement binding and stabilizing envelope may improve the vaccine response.

Pasteurella pneumotropica evades the human complement system by acquisition of the complement regulators factor H and C4BP.

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Alfredo Sahagún-Ruiz

Full Text Available Pasteurella pneumotropica is an opportunist Gram negative bacterium responsible for rodent pasteurellosis that affects upper respiratory, reproductive and digestive tracts of mammals. In animal care facilities the presence of P. pneumotropica causes severe to lethal infection in immunodeficient mice, being also a potential source for human contamination. Indeed, occupational exposure is one of the main causes of human infection by P. pneumotropica. The clinical presentation of the disease includes subcutaneous abscesses, respiratory tract colonization and systemic infections. Given the ability of P. pneumotropica to fully disseminate in the organism, it is quite relevant to study the role of the complement system to control the infection as well as the possible evasion mechanisms involved in bacterial survival. Here, we show for the first time that P. pneumotropica is able to survive the bactericidal activity of the human complement system. We observed that host regulatory complement C4BP and Factor H bind to the surface of P. pneumotropica, controlling the activation pathways regulating the formation and maintenance of C3-convertases. These results show that P. pneumotropica has evolved mechanisms to evade the human complement system that may increase the efficiency by which this pathogen is able to gain access to and colonize inner tissues where it may cause severe infections.

Influence of endurance training on skeletal muscle mitophagy regulatory proteins in type 2 diabetic men.

Science.gov (United States)

Brinkmann, Christian; Przyklenk, Axel; Metten, Alexander; Schiffer, Thorsten; Bloch, Wilhelm; Brixius, Klara; Gehlert, Sebastian

2017-11-01

Mitophagy is a form of autophagy for the elimination of mitochondria. Mitochondrial content and function are reduced in the skeletal muscle of patients with type 2 diabetes mellitus (T2DM). Physical training has been shown to restore mitochondrial capacity in T2DM patients, but the role of mitophagy has not been examined in this context. This study analyzes the impact of a 3-month endurance training on important skeletal muscle mitophagy regulatory proteins and oxidative phosphorylation (OXPHOS) complexes in T2DM patients. Muscle biopsies were obtained from eight overweight/obese T2DM men (61±10 years) at T1 (6 weeks pre-training), T2 (1 week pre-training), and T3 (3 to 4 days post-training). Protein contents were determined by Western blotting. The training increased mitochondrial complex II significantly (T2-T3: +29%, p = 0.037). The protein contents of mitophagy regulatory proteins (phosphorylated form of forkhead box O3A (pFOXO3A), mitochondrial E3 ubiquitin protein ligase-1 (MUL1), Bcl-2/adenovirus E1B 19-kD interacting protein-3 (BNIP3), microtubule-associated protein 1 light chain-3B (the ratio LC3B-II/LC3B-I was determined)) did not differ significantly between T1, T2, and T3. The results imply that training-induced changes in OXPHOS subunits (significant increase in complex II) are not accompanied by changes in mitophagy regulatory proteins in T2DM men. Future studies should elucidate whether acute exercise might affect mitophagic processes in T2DM patients (and whether a transient regulation of mitophagy regulatory proteins is evident) to fully clarify the role of physical activity and mitophagy for mitochondrial health in this particular patient group.

In vitro biosynthesis of complement protein D

International Nuclear Information System (INIS)

Barnum, S.R.

1985-01-01

The aim of this study was twofold: to determine site(s) of complement protein D biosynthesis and to examine D biosynthesis with respect to the kinetics of D secretion, the post-translational modification of D and the tissue-specific differences in D secretion and processing. Antigenic D was detected in the culture supernatants of two cell lines, U937 and HepG2, and adherent blood monocytes by a solid-phase radioimmunoassay. D secreted by U937 cells was hemolytically active with a specific activity comparable to D in serum. De novo synthesis of D by U937 cells was demonstrated with the use of cycloheximide. Biosynthetic labeling using 35 S labeled methionine or cysteine, followed by immunoprecipitation demonstrated a single d band intra- and extra-cellularly in all three cell types as analyzed by SDS-PAGE and auto-radiography. Elevated serum D levels in individuals with IgA nephropathy led to studies on the D levels in serum and urine of individuals with chronic renal failure and an individual with Fanconi's syndrome. The former group had elevated serum D levels, compared to normals, and insignificant levels of D in their urine while the patient with Fanconi's syndrome had normal serum D levels but markedly elevated urinary D levels. These studies demonstrate that the monocyte and hepatocyte are both sites of D synthesis and that there are no apparent differences in the secretion rates and processing of D produced by these cell types. The results also suggest that D is not synthesized or secreted as a precursor molecule. Additionally, these studies suggest that the kidney is a major site of D catabolism

Structural studies of bacterial transcriptional regulatory proteins by multidimensional heteronuclear NMR

Energy Technology Data Exchange (ETDEWEB)

Volkman, Brian Finley [Univ. of California, Berkeley, CA (United States)

1995-02-01

Nuclear magnetic resonance spectroscopy was used to elucidate detailed structural information for peptide and protein molecules. A small peptide was designed and synthesized, and its three-dimensional structure was calculated using distance information derived from two-dimensional NMR measurements. The peptide was used to induce antibodies in mice, and the cross-reactivity of the antibodies with a related protein was analyzed with enzyme-linked immunosorbent assays. Two proteins which are involved in regulation of transcription in bacteria were also studied. The ferric uptake regulation (Fur) protein is a metal-dependent repressor which controls iron uptake in bacteria. Two- and three-dimensional NMR techniques, coupled with uniform and selective isotope labeling allowed the nearly complete assignment of the resonances of the metal-binding domain of the Fur protein. NTRC is a transcriptional enhancer binding protein whose N-terminal domain is a "receiver domain" in the family of "two-component" regulatory systems. Phosphorylation of the N-terminal domain of NTRC activates the initiation of transcription of aeries encoding proteins involved in nitrogen regulation. Three- and four-dimensional NMR spectroscopy methods have been used to complete the resonance assignments and determine the solution structure of the N-terminal receiver domain of the NTRC protein. Comparison of the solution structure of the NTRC receiver domain with the crystal structures of the homologous protein CheY reveals a very similar fold, with the only significant difference being the position of helix 4 relative to the rest of the protein. The determination of the structure of the NTRC receiver domain is the first step toward understanding a mechanism of signal transduction which is common to many bacterial regulatory systems.

Does host complement kill Borrelia burgdorferi within ticks?

Science.gov (United States)

Rathinavelu, Sivaprakash; Broadwater, Anne; de Silva, Aravinda M

2003-02-01

The Lyme disease spirochete, Borrelia burgdorferi, inhabits the gut lumen of the tick vector. At this location the spirochete is exposed to host blood when a tick feeds. We report here on studies that were done with normal and complement-deficient (C3-knockout) mice to determine if the host complement system killed spirochetes within the vector. We found that spirochete numbers within feeding nymphs were not influenced by complement, most likely because host complement was inactivated within the vector. The Lyme disease outer surface protein A (OspA) vaccine is a transmission-blocking vaccine that targets spirochetes in the vector. In experiments with mice hyperimmunized with OspA, complement was not required to kill spirochetes within nymphs and to block transmission from nymphs to the vaccinated host. However, host complement did enhance the ability of OspA antibody to block larvae from acquiring spirochetes. Thus, the effects of OspA antibody on nymphal transmission and larval acquisition appear to be based on different mechanisms.

Inactivation of complement by Loxosceles reclusa spider venom.

Science.gov (United States)

Gebel, H M; Finke, J H; Elgert, K D; Cambell, B J; Barrett, J T

1979-07-01

Zymosan depletion of serum complement in guinea pigs rendered them highly resistant to lesion by Loxosceles reclusa spider venom. Guinea pigs deficient in C4 of the complement system are as sensitive to the venom as normal guinea pigs. The injection of 35 micrograms of whole recluse venom intradermally into guinea pigs lowered their complement level by 35.7%. Brown recluse spider venom in concentrations as slight as 0.02 micrograms protein/ml can totally inactivate one CH50 of guinea pig complement in vitro. Bee, scorpion, and other spider venoms had no influence on the hemolytic titer of complement. Fractionation of recluse spider venom by Sephadex G-200 filtration separated the complement-inactivating property of the venom into three major regions which could be distinguished on the basis of heat stability as well as size. None was neutralized by antivenom. Polyacrylamide gel electrophoresis of venom resolved the complement inactivators into five fractions. Complement inactivated by whole venom or the Sephadex fractions could be restored to hemolytic activity by supplements of fresh serum but not by heat-inactivated serum, pure C3, pure C5, or C3 and C5 in combination.

The Role of Complement Inhibition in Thrombotic Angiopathies and Antiphospholipid Syndrome

Science.gov (United States)

Erkan, Doruk; Salmon, Jane E.

2016-01-01

Antiphospholipid syndrome (APS) is characterized by thrombosis (arterial, venous, small vessel) and/or pregnancy morbidity occurring in patients with persistently positive antiphospholipid antibodies (aPL). Catastrophic APS is the most severe form of the disease, characterized by multiple organ thromboses occurring in a short period and commonly associated with thrombotic microangiopathy (TMA). Similar to patients with complement regulatory gene mutations developing TMA, increased complement activation on endothelial cells plays a role in hypercoagulability in aPL-positive patients. In mouse models of APS, activation of the complement is required and interaction of complement (C) 5a with its receptor C5aR leads to aPL-induced inflammation, placental insufficiency, and thrombosis. Anti-C5 antibody and C5aR antagonist peptides prevent aPL-mediated pregnancy loss and thrombosis in these experimental models. Clinical studies of anti-C5 monoclonal antibody in aPL-positive patients are limited to a small number of case reports. Ongoing and future clinical studies of complement inhibitors will help determine the role of complement inhibition in the management of aPL-positive patients. PMID:27020721

AKT1, LKB1, and YAP1 revealed as MYC interactors with NanoLuc-based protein-fragment complementation assay. | Office of Cancer Genomics

Science.gov (United States)

The c-Myc (MYC) transcription factor is a major cancer driver and a well-validated therapeutic target. However, directly targeting MYC has been challenging. Thus, identifying proteins that interact with and regulate MYC may provide alternative strategies to inhibit its oncogenic activity. Here we report the development of a NanoLuc®-based protein-fragment complementation assay (NanoPCA) and mapping of the MYC protein interaction hub in live mammalian cells.

SALO, a novel classical pathway complement inhibitor from saliva of the sand fly Lutzomyia longipalpis

OpenAIRE

Viviana P. Ferreira; Vladimir Fazito Vale; Michael K. Pangburn; Maha Abdeladhim; Antonio Ferreira Mendes-Sousa; Iliano V. Coutinho-Abreu; Manoochehr Rasouli; Elizabeth A. Brandt; Claudio Meneses; Kolyvan Ferreira Lima; Ricardo Nascimento Araújo; Marcos Horácio Pereira; Michalis Kotsyfakis; Fabiano Oliveira; Shaden Kamhawi

2016-01-01

Blood-feeding insects inject potent salivary components including complement inhibitors into their host's skin to acquire a blood meal. Sand fly saliva was shown to inhibit the classical pathway of complement; however, the molecular identity of the inhibitor remains unknown. Here, we identified SALO as the classical pathway complement inhibitor. SALO, an 11 kDa protein, has no homology to proteins of any other organism apart from New World sand flies. rSALO anti-complement activity has the sa...

Complement modulation of T cell immune responses during homeostasis and disease.

Science.gov (United States)

Clarke, Elizabeth V; Tenner, Andrea J

2014-11-01

The complement system is an ancient and critical effector mechanism of the innate immune system as it senses, kills, and clears infectious and/or dangerous particles and alerts the immune system to the presence of the infection and/or danger. Interestingly, an increasing number of reports have demonstrated a clear role for complement in the adaptive immune system as well. Of note, a number of recent studies have identified previously unknown roles for complement proteins, receptors, and regulators in T cell function. Here, we will review recent data demonstrating the influence of complement proteins C1q, C3b/iC3b, C3a (and C3aR), and C5a (and C5aR) and complement regulators DAF (CD55) and CD46 (MCP) on T cell function during homeostasis and disease. Although new concepts are beginning to emerge in the field of complement regulation of T cell function, future experiments should focus on whether complement is interacting directly with the T cell or is having an indirect effect on T cell function via APCs, the cytokine milieu, or downstream complement activation products. Importantly, the identification of the pivotal molecular pathways in the human systems will be beneficial in the translation of concepts derived from model systems to therapeutic targeting for treatment of human disorders. © 2014 Society for Leukocyte Biology.

EWS and FUS bind a subset of transcribed genes encoding proteins enriched in RNA regulatory functions

DEFF Research Database (Denmark)

Luo, Yonglun; Friis, Jenny Blechingberg; Fernandes, Ana Miguel

2015-01-01

at different levels. Gene Ontology analyses showed that FUS and EWS target genes preferentially encode proteins involved in regulatory processes at the RNA level. Conclusions The presented results yield new insights into gene interactions of EWS and FUS and have identified a set of FUS and EWS target genes...... involved in pathways at the RNA regulatory level with potential to mediate normal and disease-associated functions of the FUS and EWS proteins.......Background FUS (TLS) and EWS (EWSR1) belong to the FET-protein family of RNA and DNA binding proteins. FUS and EWS are structurally and functionally related and participate in transcriptional regulation and RNA processing. FUS and EWS are identified in translocation generated cancer fusion proteins...

Evolution and Function of Thioester-Containing Proteins and the Complement System in the Innate Immune Response

Directory of Open Access Journals (Sweden)

Upasana Shokal

2017-06-01

Full Text Available The innate immune response is evolutionary conserved among organisms. The complement system forms an important and efficient immune defense mechanism. It consists of plasma proteins that participate in microbial detection, which ultimately results in the production of various molecules with antimicrobial activity. Thioester-containing proteins (TEPs are a superfamily of secreted effector proteins. In vertebrates, certain TEPs act in the innate immune response by promoting recruitment of immune cells, phagocytosis, and direct lysis of microbial invaders. Insects are excellent models for dissecting the molecular basis of innate immune recognition and response to a wide range of microbial infections. Impressive progress in recent years has generated crucial information on the role of TEPs in the antibacterial and antiparasite response of the tractable model insect Drosophila melanogaster and the mosquito malaria vector Anopheles gambiae. This knowledge is critical for better understanding the evolution of TEPs and their involvement in the regulation of the host innate immune system.

Enzymatic Mercury Detoxification: The Regulatory Protein MerR

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Ctortecka, B; Walsh, C T; Comess, K M

2002-01-01

Mercury ions and organomercurial reagents are extremely toxic due to their affinity for thiol groups. Many bacteria contain an elaborate detoxification system for a metabolic conversion of toxic Hg$^{2+}$ or organomercurials to less toxic elemental Hg$^0$. The main components of the enzymatic mercury detoxification (see Fig. 1) are the regulatory protein MerR (mercury responsive genetic switch), the organomercurial lyase MerB (cleavage of carbon mercury bonds), and the mercuric ion reductase MerA (reduction of mercuric ions). In these proteins Hg$^{2+}$ is usually coordinated by the thiol groups of cysteines. We utilize the nuclear quadrupole interaction (NQI) of ${\\rm^{199m}}$Hg detected by time differential perturbed angular correlation (TDPAC) to identify the Hg metal site geometries in these proteins in order to elucidate the molecular origin of the ultrasensitivity, selectivity and reaction mechanism of this detoxification system. The short lived TDPAC probe ${\\rm^{199m}}$Hg ($\\tau_{1/2} =$ 43 min) is su...

Assembly and activation of alternative complement components on endothelial cell-anchored ultra-large von Willebrand factor links complement and hemostasis-thrombosis.

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Nancy A Turner

Full Text Available Vascular endothelial cells (ECs express and release protein components of the complement pathways, as well as secreting and anchoring ultra-large von Willebrand factor (ULVWF multimers in long string-like structures that initiate platelet adhesion during hemostasis and thrombosis. The alternative complement pathway (AP is an important non-antibody-requiring host defense system. Thrombotic microangiopathies can be associated with defective regulation of the AP (atypical hemolytic-uremic syndrome or with inadequate cleavage by ADAMTS-13 of ULVWF multimeric strings secreted by/anchored to ECs (thrombotic thrombocytopenic purpura. Our goal was to determine if EC-anchored ULVWF strings caused the assembly and activation of AP components, thereby linking two essential defense mechanisms.We quantified gene expression of these complement components in cultured human umbilical vein endothelial cells (HUVECs by real-time PCR: C3 and C5; complement factor (CF B, CFD, CFP, CFH and CFI of the AP; and C4 of the classical and lectin (but not alternative complement pathways. We used fluorescent microscopy, monospecific antibodies against complement components, fluorescent secondary antibodies, and the analysis of >150 images to quantify the attachment of HUVEC-released complement proteins to ULVWF strings secreted by, and anchored to, the HUVECs (under conditions of ADAMTS-13 inhibition. We found that HUVEC-released C4 did not attach to ULVWF strings, ruling out activation of the classical and lectin pathways by the strings. In contrast, C3, FB, FD, FP and C5, FH and FI attached to ULVWF strings in quantitative patterns consistent with assembly of the AP components into active complexes. This was verified when non-functional FB blocked the formation of AP C3 convertase complexes (C3bBb on ULVWF strings.AP components are assembled and activated on EC-secreted/anchored ULVWF multimeric strings. Our findings provide one possible molecular mechanism for clinical

A cytoplasmic serine protein kinase binds and may regulate the Fanconi anemia protein FANCA.

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Yagasaki, H; Adachi, D; Oda, T; Garcia-Higuera, I; Tetteh, N; D'Andrea, A D; Futaki, M; Asano, S; Yamashita, T

2001-12-15

Fanconi anemia (FA) is an autosomal recessive disease with congenital anomalies, bone marrow failure, and susceptibility to leukemia. Patient cells show chromosome instability and hypersensitivity to DNA cross-linking agents. At least 8 complementation groups (A-G) have been identified and 6 FA genes (for subtypes A, C, D2, E, F, and G) have been cloned. Increasing evidence indicates that a protein complex assembly of multiple FA proteins, including FANCA and FANCG, plays a crucial role in the FA pathway. Previously, it was reported that FANCA was phosphorylated in lymphoblasts from normal controls, whereas the phosphorylation was defective in those derived from patients with FA of multiple complementation groups. The present study examined phosphorylation of FANCA ectopically expressed in FANCA(-) cells. Several patient-derived mutations abrogated in vivo phosphorylation of FANCA in this system, suggesting that FANCA phosphorylation is associated with its function. In vitro phosphorylation studies indicated that a physiologic protein kinase for FANCA (FANCA-PK) forms a complex with the substrate. Furthermore, at least a part of FANCA-PK as well as phosphorylated FANCA were included in the FANCA/FANCG complex. Thus, FANCA-PK appears to be another component of the FA protein complex and may regulate function of FANCA. FANCA-PK was characterized as a cytoplasmic serine kinase sensitive to wortmannin. Identification of the protein kinase is expected to elucidate regulatory mechanisms that control the FA pathway.

Reference distributions for complement proteins C3 and C4: a practical, simple and clinically relevant approach in a large cohort.

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Ritchie, Robert F; Palomaki, Glenn E; Neveux, Louis M; Navolotskaia, Olga; Ledue, Thomas B; Craig, Wendy Y

2004-01-01

The two serum proteins of the complement cascade in the highest concentrations, C3 and C4, respond to various conditions in much the same manner as do other positive acute-phase proteins. A major difference is that they are relatively sluggish in response to cytokine drive, requiring several days rather than hours to be detectably elevated by serial measurements. As with other acute-phase proteins, there are many processes that up- or down-regulate synthesis, including infection or inflammation, hepatic failure, and immune-complex formation. Clinicians may find it difficult to distinguish among these processes, because they often occur simultaneously. The situation is further complicated by genetic polymorphism, with rare instances of markedly reduced synthesis and circulating levels, and consequent vulnerability to infection. C3 and C4 are measured for clinical purposes to help define certain rheumatic and immunologically mediated renal diseases. Interpreting the measured blood levels of these two components requires one to consider the intensity of the inflammatory drive, the timing of the suspected clinical process, the production of complement-consuming immune complexes, and the possible existence of benign circumstances. In this fifth article in a series, reference ranges for serum levels of two complement proteins (C3 and C4) are examined. The study is based on a cohort of over 55,000 Caucasian individuals from northern New England, who were tested in our laboratory in 1994-1999. Measurements were standardized against certified reference material (CRM) 470/reference preparation for proteins in human serum (RPPHS), and analyzed using a previously described statistical approach. Individuals with unequivocal laboratory evidence of inflammation (C-reactive protein of 10 mg/L or higher) were excluded. Our results show that the levels of C3 and C4 change little during life and between the sexes, except that they increase slightly and then fall after age 20 in males

Regulatory T-Cell-Associated Cytokines in Systemic Lupus Erythematosus

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Akiko Okamoto

2011-01-01

Full Text Available Systemic lupus erythematosus (SLE is an autoimmune disease characterized by autoantibody production, complement activation, and immune complex deposition, resulting in tissue and organ damage. An understanding of the mechanisms responsible for homeostatic control of inflammation, which involve both innate and adoptive immune responses, will enable the development of novel therapies for SLE. Regulatory T cells (Treg play critical roles in the induction of peripheral tolerance to self- and foreign antigens. Naturally occurring CD4+CD25+ Treg, which characteristically express the transcription factor forkhead box protein P3 (Foxp3, have been intensively studied because their deficiency abrogates self-tolerance and causes autoimmune disease. Moreover, regulatory cytokines such as interleukin-10 (IL-10 also play a central role in controlling inflammatory processes. This paper focuses on Tregs and Treg-associated cytokines which might regulate the pathogenesis of SLE and, hence, have clinical applications.

Espins are multifunctional actin cytoskeletal regulatory proteins in the microvilli of chemosensory and mechanosensory cells

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Sekerková, Gabriella; Zheng, Lili; Loomis, Patricia A.; Changyaleket, Benjarat; Whitlon, Donna S.; Mugnaini, Enrico; Bartles, James R.

2010-01-01

Espins are associated with the parallel actin bundles of hair cell stereocilia and are the target of mutations that cause deafness and vestibular dysfunction in mice and humans. Here, we report that espins are also concentrated in the microvilli of a number of other sensory cells: vomeronasal organ sensory neurons, solitary chemoreceptor cells, taste cells and Merkel cells. Moreover, we show that hair cells and these other sensory cells contain novel espin isoforms that arise from a different transcriptional start site and differ significantly from other espin isoforms in their complement of ligand-binding activities and their effects on actin polymerization. The novel espin isoforms of sensory cells bundled actin filaments with high affinity in a Ca2+-resistant fashion, bound actin monomer via a WASP homology 2 domain, bound profilin via a single proline-rich peptide, and caused a dramatic elongation of microvillus-type parallel actin bundles in transfected epithelial cells. In addition, the novel espin isoforms of sensory cells differed from other espin isoforms in that they potently inhibited actin polymerization in vitro, did not bind the Src homology 3 domain of the adapter protein insulin receptor substrate p53 and did not bind the acidic, signaling phospholipid phosphatidylinositol 4,5- bisphosphate. Thus, the espins constitute a family of multifunctional actin cytoskeletal regulatory proteins with the potential to differentially influence the organization, dimensions, dynamics and signaling capabilities of the actin filament-rich, microvillus-type specializations that mediate sensory transduction in a variety of mechanosensory and chemosensory cells. PMID:15190118

Complementation of essential yeast GPI mannosyltransferase mutations suggests a novel specificity for certain Trypanosoma and Plasmodium PigB proteins.

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Leslie K Cortes

Full Text Available The glycosylphosphatidylinositol (GPI anchor is an essential glycolipid that tethers certain eukaryotic proteins to the cell surface. The core structure of the GPI anchor is remarkably well conserved across evolution and consists of NH2-CH2-CH2-PO4-6Manα1,2Manα1,6Manα1,4-GlcNα1,6-myo-inositol-PO4-lipid. The glycan portion of this structure may be modified with various side-branching sugars or other compounds that are heterogeneous and differ from organism to organism. One such modification is an α(1,2-linked fourth mannose (Man-IV that is side-branched to the third mannose (Man-III of the trimannosyl core. In fungi and mammals, addition of Man-III and Man-IV occurs by two distinct Family 22 α(1,2-mannosyltransferases, Gpi10/PigB and Smp3/PigZ, respectively. However, in the five protozoan parasite genomes we examined, no genes encoding Smp3/PigZ proteins were observed, despite reports of tetramannosyl-GPI structures (Man4-GPIs being produced by some parasites. In this study, we tested the hypothesis that the Gpi10/PigB proteins produced by protozoan parasites have the ability to add both Man-III and Man-IV to GPI precursors. We used yeast genetics to test the in vivo specificity of Gpi10/PigB proteins from several Plasmodium and Trypanosoma species by examining their ability to restore viability to Saccharomyces cerevisiae strains harboring lethal defects in Man-III (gpi10Δ or Man-IV (smp3Δ addition to GPI precursor lipids. We demonstrate that genes encoding PigB enzymes from T. cruzi, T. congolense and P. falciparum are each capable of separately complementing essential gpi10Δ and smp3Δ mutations, while PIGB genes from T. vivax and T. brucei only complement gpi10Δ. Additionally, we show the ability of T. cruzi PIGB to robustly complement a gpi10Δ/smp3Δ double mutant. Our data suggest that certain Plasmodium and Trypanosoma PigB mannosyltransferases can transfer more than one mannose to GPI precursors in vivo, and suggest a novel

Evolutionary conservation of regulatory elements in vertebrate HOX gene clusters

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Santini, Simona; Boore, Jeffrey L.; Meyer, Axel

2003-12-31

Due to their high degree of conservation, comparisons of DNA sequences among evolutionarily distantly-related genomes permit to identify functional regions in noncoding DNA. Hox genes are optimal candidate sequences for comparative genome analyses, because they are extremely conserved in vertebrates and occur in clusters. We aligned (Pipmaker) the nucleotide sequences of HoxA clusters of tilapia, pufferfish, striped bass, zebrafish, horn shark, human and mouse (over 500 million years of evolutionary distance). We identified several highly conserved intergenic sequences, likely to be important in gene regulation. Only a few of these putative regulatory elements have been previously described as being involved in the regulation of Hox genes, while several others are new elements that might have regulatory functions. The majority of these newly identified putative regulatory elements contain short fragments that are almost completely conserved and are identical to known binding sites for regulatory proteins (Transfac). The conserved intergenic regions located between the most rostrally expressed genes in the developing embryo are longer and better retained through evolution. We document that presumed regulatory sequences are retained differentially in either A or A clusters resulting from a genome duplication in the fish lineage. This observation supports both the hypothesis that the conserved elements are involved in gene regulation and the Duplication-Deletion-Complementation model.

Exploration of the dynamic properties of protein complexes predicted from spatially constrained protein-protein interaction networks.

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Eric A Yen

2014-05-01

Full Text Available Protein complexes are not static, but rather highly dynamic with subunits that undergo 1-dimensional diffusion with respect to each other. Interactions within protein complexes are modulated through regulatory inputs that alter interactions and introduce new components and deplete existing components through exchange. While it is clear that the structure and function of any given protein complex is coupled to its dynamical properties, it remains a challenge to predict the possible conformations that complexes can adopt. Protein-fragment Complementation Assays detect physical interactions between protein pairs constrained to ≤8 nm from each other in living cells. This method has been used to build networks composed of 1000s of pair-wise interactions. Significantly, these networks contain a wealth of dynamic information, as the assay is fully reversible and the proteins are expressed in their natural context. In this study, we describe a method that extracts this valuable information in the form of predicted conformations, allowing the user to explore the conformational landscape, to search for structures that correlate with an activity state, and estimate the abundance of conformations in the living cell. The generator is based on a Markov Chain Monte Carlo simulation that uses the interaction dataset as input and is constrained by the physical resolution of the assay. We applied this method to an 18-member protein complex composed of the seven core proteins of the budding yeast Arp2/3 complex and 11 associated regulators and effector proteins. We generated 20,480 output structures and identified conformational states using principle component analysis. We interrogated the conformation landscape and found evidence of symmetry breaking, a mixture of likely active and inactive conformational states and dynamic exchange of the core protein Arc15 between core and regulatory components. Our method provides a novel tool for prediction and

Complement evasion by Bordetella pertussis: implications for improving current vaccines.

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Jongerius, Ilse; Schuijt, Tim J; Mooi, Frits R; Pinelli, Elena

2015-04-01

Bordetella pertussis causes whooping cough or pertussis, a highly contagious disease of the respiratory tract. Despite high vaccination coverage, reported cases of pertussis are rising worldwide and it has become clear that the current vaccines must be improved. In addition to the well-known protective role of antibodies and T cells during B. pertussis infection, innate immune responses such as the complement system play an essential role in B. pertussis killing. In order to evade this complement activation and colonize the human host, B. pertussis expresses several molecules that inhibit complement activation. Interestingly, one of the known complement evasion proteins, autotransporter Vag8, is highly expressed in the recently emerged B. pertussis isolates. Here, we describe the current knowledge on how B. pertussis evades complement-mediated killing. In addition, we compare this to complement evasion strategies used by other bacterial species. Finally, we discuss the consequences of complement evasion by B. pertussis on adaptive immunity and how identification of the bacterial molecules and the mechanisms involved in complement evasion might help improve pertussis vaccines.

Effects of sterol regulatory element-binding protein (SREBP in chickens

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Alipour Fahimeh

2012-02-01

Full Text Available Abstract Sterol regulatory element binding protein- 1 and -2 (SREBP-1 and -2 are key transcription factors involved in the biosynthesis of cholesterol and fatty acids. The SREBP have mostly been studied in rodents in which lipogenesis is regulated in both liver and adipose tissue. There is, though, a paucity of information on birds, in which lipogenesis occurs essentially in the liver as in humans. Since a prelude to the investigation of the role of SREBP in lipid metabolism regulation in chicken, we review Size and Tissue expression Pattern of SREBP and role of this protein in chickens.

Correlation of systemic lupus erythematosus disease activity with classical complement (CH50 function and related protein levels

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Salesi M

2008-09-01

Full Text Available "nBackground: The components of the classical complement pathway play an important role in the pathogenesis of systemic lupus erythematosus (SLE and are reportedly useful biomarkers of disease activity. In this study, we evaluate disease activity, complement function (total hemolytic complement, CH50 and complement protein levels (C3, C4, C3d, C4d, SC5b-9, comparing the results of patients with active disease versus those with inactive disease."n"nMethods: This cross-sectional study included 78 hospitalized women with SLE, 24 of whom were in the active group, with SLE disease activity indexes (SLEDAI.2K of >6, and 54 in the inactive group, with SLEDAI.2K of ≤6. Serum CH50 was measured using a red blood cell hemolytic assay. C3 and C4 levels were determined by nephlometry and plasma levels of C3d, C4d, SC5b-9 by ELISA. The data were statistically analyzed using SPSS."n"nResults: The mean (±standard error C4d levels of the inactive group were significantly higher than those of the active group (23.39±1.1µg/ml and 16.9±1.6µg/ml, respectively; p=0.003. There was also a significant correlation between C3 and C4 levels (p=0.807. The mean values of the other proteins (C3, C4, CH50, SC5b-9, and C3d circulating immune complex concentrations were not significantly different between the inactive group vs. the active group: 89.35±6.8 vs. 85.54±7.6mg/dl, 18.33±2.3 vs. 20.45±2.4mg/dl, 149.03±4.3 vs. 157±4.3U, 1414.4±114.94 vs. 1471.1±216.9ng/ml, 9.43±0.96 vs. 13.31±3.16µgEq/ml, respectively (p>0.05."n"nConclusions: According to our results, C4d levels may be used as a biomarker of disease activity. The significant correlation between C3 and C4 may confirm the activity of the classical pathway in SLE patients."n"nKeywords: Systemic lupus erythematosus, CH50, C3, C4, C3d, C4d, SC5b-9, inactive, flare.

Vernonia DGATs can complement the disrupted oil and protein metabolism in epoxygenase-expressing soybean seeds.

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Li, Runzhi; Yu, Keshun; Wu, Yongmei; Tateno, Mizuki; Hatanaka, Tomoko; Hildebrand, David F

2012-01-01

Plant oils can be useful chemical feedstocks such as a source of epoxy fatty acids. High seed-specific expression of a Stokesia laevis epoxygenase (SlEPX) in soybeans only results in 3-7% epoxide levels. SlEPX-transgenic soybean seeds also exhibited other phenotypic alterations, such as altered seed fatty acid profiles, reduced oil accumulation, and variable protein levels. SlEPX-transgenic seeds showed a 2-5% reduction in total oil content and protein levels of 30.9-51.4%. To address these pleiotrophic effects of SlEPX expression on other traits, transgenic soybeans were developed to co-express SlEPX and DGAT (diacylglycerol acyltransferase) genes (VgDGAT1 & 2) isolated from Vernonia galamensis, a high accumulator of epoxy fatty acids. These side effects of SlEPX expression were largely overcome in the DGAT co-expressing soybeans. Total oil and protein contents were restored to the levels in non-transgenic soybeans, indicating that both VgDGAT1 and VgDGAT2 could complement the disrupted phenotypes caused by over-expression of an epoxygenase in soybean seeds. Copyright © 2011 Elsevier Inc. All rights reserved.

Complement factor H deficiency results in decreased neuroretinal expression of Cd59a in aged mice

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Faber, Carsten; Williams, Jennifer; Juel, Helene Bæk

2012-01-01

Purpose. The complement system is closely linked to the pathogenesis of AMD. Several complement genes are expressed in RPE, and complement proteins accumulate in drusen. Further, a common variant of complement factor H (CFH) confers increased risk of developing AMD. Because the mechanisms by which...

Complement Factor H-Related Protein 4A Is the Dominant Circulating Splice Variant of CFHR4

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Richard B. Pouw

2018-04-01

Full Text Available Recent research has elucidated circulating levels of almost all factor H-related (FHR proteins. Some of these proteins are hypothesized to act as antagonists of the important complement regulator factor H (FH, fine-tuning complement regulation on human surfaces. For the CFHR4 splice variants FHR-4A and FHR-4B, the individual circulating levels are unknown, with only total levels being described. Specific reagents for FHR-4A or FHR-4B are lacking due to the fact that the unique domains in FHR-4A show high sequence similarity with FHR-4B, making it challenging to distinguish them. We developed an assay that specifically measures FHR-4A using novel, well-characterized monoclonal antibodies (mAbs that target unique domains in FHR-4A only. Using various FHR-4A/FHR-4B-specific mAbs, no FHR-4B was identified in any of the serum samples tested. The results demonstrate that FHR-4A is the dominant splice variant of CFHR4 in the circulation, while casting doubt on the presence of FHR-4B. FHR-4A levels (avg. 2.55 ± 1.46 µg/mL were within the range of most of the previously reported levels for all other FHRs. FHR-4A was found to be highly variable among the population, suggesting a strong genetic regulation. These results shed light on the physiological relevance of the previously proposed role of FHR-4A and FHR-4B as antagonists of FH in the circulation.

Architecture of the 99 bp DNA-six-protein regulatory complex of the lambda att site.

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Sun, Xingmin; Mierke, Dale F; Biswas, Tapan; Lee, Sang Yeol; Landy, Arthur; Radman-Livaja, Marta

2006-11-17

The highly directional and tightly regulated recombination reaction used to site-specifically excise the bacteriophage lambda chromosome out of its E. coli host chromosome requires the binding of six sequence-specific proteins to a 99 bp segment of the phage att site. To gain structural insights into this recombination pathway, we measured 27 FRET distances between eight points on the 99 bp regulatory DNA bound with all six proteins. Triangulation of these distances using a metric matrix distance-geometry algorithm provided coordinates for these eight points. The resulting path for the protein-bound regulatory DNA, which fits well with the genetics, biochemistry, and X-ray crystal structures describing the individual proteins and their interactions with DNA, provides a new structural perspective into the molecular mechanism and regulation of the recombination reaction and illustrates a design by which different families of higher-order complexes can be assembled from different numbers and combinations of the same few proteins.

Anti-complement activities of human breast-milk.

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Ogundele, M O

1999-08-01

It has long been observed that the human milk possesses significant anti-inflammatory properties, while simultaneously protecting the infant against many intestinal and respiratory pathogens. There is, however, a paucity of information on the degree and extent of this anti-inflammatory activity. In the present study, the inhibitory effects of different fractions of human milk on serum complement activity were analysed. Colostrum and milk samples from healthy voluntary lactating donors at different postpartum ages were obtained and pooled normal human serum was used as source of complement in a modified CH50 assay. Inherent complement activity in human milk was also investigated by measuring the deposition of an activated C3 fragment on a serum-sensitive bacteria, and by haemolytic assays. Most whole- and defatted-milk samples consistently showed a dose-dependent inhibition of the serum complement activity. This inhibition was greater in mature milk compared to transitional milk samples. It was enhanced by inactivation of milk complement, and diminished by centrifugation of milk samples, which partly removed fat and larger protein components including casein micelles. Inherent complement activity in human milk was also demonstrated by haemolysis of sensitised sheep erythrocytes and deposition of C3 fragments on solid-phase bacteria. These activities were highest in the colostrum and gradually decreased as lactation proceeded. Several natural components abundant in the fluid phase of the human breast-milk have been shown to be inhibitors of complement activation in vitro. Their physiological significance probably reside in their ability to prevent inflammatory-induced tissue damage of the delicate immature gastrointestinal tract of the new-born as well as the mammary gland itself, which may arise from ongoing complement activation.

High protein complementation with high fiber substrates for oyster ...

African Journals Online (AJOL)

Agricultural residues have been world widely accepted for oyster mushroom culture. In this study, we used wheat straw, barley straw, maize stem residue, and lawn residue as substrates coupled with wheat bran, rice bran and soybean powder as complements for the growth of Pleurotus florida and Pleurotus ostreatus as ...

Myristoylated α subunits of guanine nucleotide-binding regulatory proteins

International Nuclear Information System (INIS)

Buss, J.E.; Mumby, S.M.; Casey, P.J.; Gilman, A.G.; Sefton, B.M.

1987-01-01

Antisera directed against specific subunits of guanine nucleotide-binding regulatory proteins (G proteins) were used to immunoprecipitate these polypeptides from metabolically labeled cells. This technique detects, in extracts of a human astrocytoma cell line, the α subunits of G/sub s/ (stimulatory) (α 45 and α 52 ), a 41-kDa subunit of G/sub i/ (inhibitory) (α 41 ), a 40-kDa protein (α 40 ), and the 36-kDa β subunit. No protein that comigrated with the α subunit of G 0 (unknown function) (α 39 ) was detected. In cells grown in the presence of [ 3 H]myristic acid, α 41 and α 40 contained 3 H label, while the β subunit did not. Chemical analysis of lipids attached covalently to purified α 41 and α 39 from bovine brain also revealed myristic acid. Similar analysis of brain G protein β and γ subunits and of G/sub t/ (Transducin) subunits (α, β, and γ) failed to reveal fatty acids. The fatty acid associated with α 41 , α 40 , and α 39 was stable to treatment with base, suggesting that the lipid is linked to the polypeptide via an amide bond. These GTP binding proteins are thus identified as members of a select group of proteins that contains myristic acid covalently attached to the peptide backbone. Myristate may play an important role in stabilizing interactions of G proteins with phospholipid or with membrane-bound proteins

EWS and FUS bind a subset of transcribed genes encoding proteins enriched in RNA regulatory functions.

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Luo, Yonglun; Blechingberg, Jenny; Fernandes, Ana Miguel; Li, Shengting; Fryland, Tue; Børglum, Anders D; Bolund, Lars; Nielsen, Anders Lade

2015-11-14

FUS (TLS) and EWS (EWSR1) belong to the FET-protein family of RNA and DNA binding proteins. FUS and EWS are structurally and functionally related and participate in transcriptional regulation and RNA processing. FUS and EWS are identified in translocation generated cancer fusion proteins and involved in the human neurological diseases amyotrophic lateral sclerosis and fronto-temporal lobar degeneration. To determine the gene regulatory functions of FUS and EWS at the level of chromatin, we have performed chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq). Our results show that FUS and EWS bind to a subset of actively transcribed genes, that binding often is downstream the poly(A)-signal, and that binding overlaps with RNA polymerase II. Functional examinations of selected target genes identified that FUS and EWS can regulate gene expression at different levels. Gene Ontology analyses showed that FUS and EWS target genes preferentially encode proteins involved in regulatory processes at the RNA level. The presented results yield new insights into gene interactions of EWS and FUS and have identified a set of FUS and EWS target genes involved in pathways at the RNA regulatory level with potential to mediate normal and disease-associated functions of the FUS and EWS proteins.

The Complement Binding and Inhibitory Protein CbiA of Borrelia miyamotoi Degrades Extracellular Matrix Components by Interacting with Plasmin(ogen

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Ngoc T. T. Nguyen

2018-02-01

Full Text Available The emerging relapsing fever spirochete Borrelia (B. miyamotoi is transmitted by ixodid ticks and causes the so-called hard tick-borne relapsing fever or B. miyamotoi disease (BMD. More recently, we identified a surface-exposed molecule, CbiA exhibiting complement binding and inhibitory capacity and rendering spirochetes resistant to complement-mediated lysis. To gain deeper insight into the molecular principles of B. miyamotoi-host interaction, we examined CbiA as a plasmin(ogen receptor that enables B. miyamotoi to interact with the serine protease plasmin(ogen. Recombinant CbiA was able to bind plasminogen in a dose-dependent fashion. Moreover, lysine residues appear to play a crucial role in the protein-protein interaction as binding of plasminogen was inhibited by the lysine analog tranexamic acid as well as increasing ionic strength. Of relevance, plasminogen bound to CbiA can be converted by urokinase-type plasminogen activator (uPa to active plasmin which cleaved both, the chromogenic substrate S-2251 and its physiologic substrate fibrinogen. Concerning the involvement of specific amino acids in the interaction with plasminogen, lysine residues located at the C-terminus are frequently involved in the binding as reported for various other plasminogen-interacting proteins of Lyme disease spirochetes. Lysine residues located within the C-terminal domain were substituted with alanine to generate single, double, triple, and quadruple point mutants. However, binding of plasminogen to the mutated CbiA proteins was not affected, suggesting that lysine residues distant from the C-terminus might be involved in the interaction.

Complementation of Saccharomyces cerevisiae mutations in genes involved in translation and protein folding (EFB1 and SSB1) with Candida albicans cloned genes.

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Maneu, V; Roig, P; Gozalbo, D

2000-11-01

We have demonstrated that the expression of Candida albicans genes involved in translation and protein folding (EFB1 and SSB1) complements the phenotype of Saccharomyces cerevisiae mutants. The elongation factor 1beta (EF-1beta) is essential for growth and efb1 S. cerevisiae null mutant cells are not viable; however, viable haploid cells, carrying the disrupted chromosomal allele of the S. cerevisiae EFB1 gene and pEFB1, were isolated upon sporulation of a diploid strain which was heterozygous at the EFB1 locus and transformed with pEFB1 (a pEMBLYe23 derivative plasmid containing an 8-kb DNA fragment from the C. albicans genome which contains the EFB1 gene). This indicates that the C. albicans EFB1 gene encodes a functional EF-1beta. Expression of the SSB1 gene from C. albicans, which codes for a member of the 70-kDa heat shock protein family, in S. cerevisiae ssb1 ssb2 double mutant complements the mutant phenotype (poor growth particularly at low temperature, and sensitivity to certain protein synthesis inhibitors, such as paromomycin). This complementation indicates that C. albicans Ssbl may function as a molecular chaperone on the translating ribosomes, as described in S. cerevisiae. Northern blot analysis showed that SSB mRNA levels increased after mild cold shift (28 degrees C to 23 degrees C) and rapidly decreased after mild heat shift (from 28 degrees C to 37 degrees C, and particularly to 42 degrees C), indicating that SSB1 expression is regulated by temperature. Therefore, Ssb1 may be considered as a molecular chaperone whose pattern of expression is similar to that found in ribosomal proteins, according to its common role in translation.

Identifying pathogenicity of human variants via paralog-based yeast complementation.

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Fan Yang

2017-05-01

Full Text Available To better understand the health implications of personal genomes, we now face a largely unmet challenge to identify functional variants within disease-associated genes. Functional variants can be identified by trans-species complementation, e.g., by failure to rescue a yeast strain bearing a mutation in an orthologous human gene. Although orthologous complementation assays are powerful predictors of pathogenic variation, they are available for only a few percent of human disease genes. Here we systematically examine the question of whether complementation assays based on paralogy relationships can expand the number of human disease genes with functional variant detection assays. We tested over 1,000 paralogous human-yeast gene pairs for complementation, yielding 34 complementation relationships, of which 33 (97% were novel. We found that paralog-based assays identified disease variants with success on par with that of orthology-based assays. Combining all homology-based assay results, we found that complementation can often identify pathogenic variants outside the homologous sequence region, presumably because of global effects on protein folding or stability. Within our search space, paralogy-based complementation more than doubled the number of human disease genes with a yeast-based complementation assay for disease variation.

Association between lectin complement pathway initiators, C-reactive protein and left ventricular remodeling in myocardial infarction-a magnetic resonance study

DEFF Research Database (Denmark)

Schoos, Mikkel Malby; Munthe-Fog, Lea; Skjoedt, Mikkel-Ole

2013-01-01

Lectin complement pathway (LP) activation is an important mechanism in myocardial ischemia reperfusion injury (IRI). LP is activated via the recognition molecules mannose-binding lectin (MBL), ficolins-2 and-3 and is regulated by MBL/Ficolin-associated Protein-1 (MAP-1). Also, C-reactive protein...... (CRP) and ficolin-2 interact in vitro, but the role of the ficolins in IRI is unknown.Methods and results In 55 patients with ST segment elevation myocardial infarction, we investigated the association of LP components and CRP in plasma samples with left ventricular (LV) end systolic and diastolic......-activation in IRI and LV remodeling....

Complement 5a Enhances Hepatic Metastases of Colon Cancer via Monocyte Chemoattractant Protein-1-mediated Inflammatory Cell Infiltration*

Science.gov (United States)

Piao, Chunmei; Cai, Lun; Qiu, Shulan; Jia, Lixin; Song, Wenchao; Du, Jie

2015-01-01

Complement 5a (C5a), a potent immune mediator generated by complement activation, promotes tumor growth; however, its role in tumor metastasis remains unclear. We demonstrate that C5a contributes to tumor metastases by modulating tumor inflammation in hepatic metastases of colon cancer. Colon cancer cell lines generate C5a under serum-free conditions, and C5a levels increase over time in a murine syngeneic colon cancer hepatic metastasis model. Furthermore, in the absence of C5a receptor or upon pharmacological inhibition of C5a production with an anti-C5 monoclonal antibody, tumor metastasis is severely impaired. A lack of C5a receptor in colon cancer metastatic foci reduces the infiltration of macrophages, neutrophils, and dendritic cells, and the role for C5a receptor on these cells were further verified by bone marrow transplantation experiments. Moreover, C5a signaling increases the expression of the chemokine monocyte chemoattractant protein-1 and the anti-inflammatory molecules arginase-1, interleukin 10, and transforming growth factor β, but is inversely correlated with the expression of pro-inflammatory molecules, which suggests a mechanism for the role of C5a in the inflammatory microenvironment required for tumor metastasis. Our results indicate a new and potentially promising therapeutic application of complement C5a inhibitor for the treatment of malignant tumors. PMID:25739439

Identification of high-confidence RNA regulatory elements by combinatorial classification of RNA-protein binding sites.

Science.gov (United States)

Li, Yang Eric; Xiao, Mu; Shi, Binbin; Yang, Yu-Cheng T; Wang, Dong; Wang, Fei; Marcia, Marco; Lu, Zhi John

2017-09-08

Crosslinking immunoprecipitation sequencing (CLIP-seq) technologies have enabled researchers to characterize transcriptome-wide binding sites of RNA-binding protein (RBP) with high resolution. We apply a soft-clustering method, RBPgroup, to various CLIP-seq datasets to group together RBPs that specifically bind the same RNA sites. Such combinatorial clustering of RBPs helps interpret CLIP-seq data and suggests functional RNA regulatory elements. Furthermore, we validate two RBP-RBP interactions in cell lines. Our approach links proteins and RNA motifs known to possess similar biochemical and cellular properties and can, when used in conjunction with additional experimental data, identify high-confidence RBP groups and their associated RNA regulatory elements.

Sterol regulatory element binding protein-1 (SREBP1) gene expression is similarly increased in polycystic ovary syndrome and endometrial cancer.

Science.gov (United States)

Shafiee, Mohamad N; Mongan, Nigel; Seedhouse, Claire; Chapman, Caroline; Deen, Suha; Abu, Jafaru; Atiomo, William

2017-05-01

Women with polycystic ovary syndrome have a three-fold higher risk of endometrial cancer. Insulin resistance and hyperlipidemia may be pertinent factors in the pathogenesis of both conditions. The aim of this study was to investigate endometrial sterol regulatory element binding protein-1 gene expression in polycystic ovary syndrome and endometrial cancer endometrium, and to correlate endometrial sterol regulatory element binding protein-1 gene expression with serum lipid profiles. A cross-sectional study was performed at Nottingham University Hospital, UK. A total of 102 women (polycystic ovary syndrome, endometrial cancer and controls; 34 participants in each group) were recruited. Clinical and biochemical assessments were performed before endometrial biopsies were obtained from all participants. Taqman real-time polymerase chain reaction for endometrial sterol regulatory element binding protein-1 gene and its systemic protein expression were analyzed. The body mass indices of women with polycystic ovary syndrome (29.28 ± 2.91 kg/m 2 ) and controls (28.58 ± 2.62 kg/m 2 ) were not significantly different. Women with endometrial cancer had a higher mean body mass index (32.22 ± 5.70 kg/m 2 ). Sterol regulatory element binding protein-1 gene expression was significantly increased in polycystic ovary syndrome and endometrial cancer endometrium compared with controls (p ovary syndrome, but this was not statistically significant. Similarly, statistically insignificant positive correlations were found between endometrial sterol regulatory element binding protein-1 gene expression and body mass index in endometrial cancer (r = 0.643, p = 0.06) and waist-hip ratio (r = 0.096, p = 0.073). Sterol regulatory element binding protein-1 gene expression was significantly positively correlated with triglyceride in both polycystic ovary syndrome and endometrial cancer (p = 0.028 and p = 0.027, respectively). Quantitative serum sterol regulatory element

BIOSENSORS FOR ENVIRONMENTAL MONITORING: A REGULATORY PERSPECTIVE

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Biosensors show the potential to complement laboratory-based analytical methods for environmental applications. Although biosensors for potential environmental-monitoring applications have been reported for a wide range of environmental pollutants, from a regulatory perspective, ...

An Ixodes ricinus Tick Salivary Lectin Pathway Inhibitor Protects Borrelia burgdorferi sensu lato from Human Complement.

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Wagemakers, Alex; Coumou, Jeroen; Schuijt, Tim J; Oei, Anneke; Nijhof, Ard M; van 't Veer, Cornelis; van der Poll, Tom; Bins, Adriaan D; Hovius, Joppe W R

2016-04-01

We previously identified tick salivary lectin pathway inhibitor (TSLPI) in Ixodes scapularis, a vector for Borrelia burgdorferi sensu stricto (s.s.) in North America. TSLPI is a salivary protein facilitating B. burgdorferi s.s. transmission and acquisition by inhibiting the host lectin complement pathway through interference with mannose binding lectin (MBL) activity. Since Ixodes ricinus is the predominant vector for Lyme borreliosis in Europe and transmits several complement sensitive B. burgdorferi sensu lato (s.l.) strains, we aimed to identify, describe, and characterize the I. ricinus ortholog of TSLPI. We performed (q)PCRs on I. ricinus salivary gland cDNA to identify a TSLPI ortholog. Next, we generated recombinant (r)TSLPI in a Drosophila expression system and examined inhibition of the MBL complement pathway and complement-mediated killing of B. burgdorferi s.l. in vitro. We identified a TSLPI ortholog in I. ricinus salivary glands with 93% homology at the RNA and 89% at the protein level compared to I. scapularis TSLPI, which was upregulated during tick feeding. In silico analysis revealed that TSLPI appears to be part of a larger family of Ixodes salivary proteins among which I. persulcatus basic tail salivary proteins and I. scapularis TSLPI and Salp14. I. ricinus rTSLPI inhibited the MBL complement pathway and protected B. burgdorferi s.s. and Borrelia garinii from complement-mediated killing. We have identified a TSLPI ortholog, which protects B. burgdorferi s.l. from complement-mediated killing in I. ricinus, the major vector for tick-borne diseases in Europe.

A selection that reports on protein-protein interactions within a thermophilic bacterium.

Science.gov (United States)

Nguyen, Peter Q; Silberg, Jonathan J

2010-07-01

Many proteins can be split into fragments that exhibit enhanced function upon fusion to interacting proteins. While this strategy has been widely used to create protein-fragment complementation assays (PCAs) for discovering protein-protein interactions within mesophilic organisms, similar assays have not yet been developed for studying natural and engineered protein complexes at the temperatures where thermophilic microbes grow. We describe the development of a selection for protein-protein interactions within Thermus thermophilus that is based upon growth complementation by fragments of Thermotoga neapolitana adenylate kinase (AK(Tn)). Complementation studies with an engineered thermophile (PQN1) that is not viable above 75 degrees C because its adk gene has been replaced by a Geobacillus stearothermophilus ortholog revealed that growth could be restored at 78 degrees C by a vector that coexpresses polypeptides corresponding to residues 1-79 and 80-220 of AK(Tn). In contrast, PQN1 growth was not complemented by AK(Tn) fragments harboring a C156A mutation within the zinc-binding tetracysteine motif unless these fragments were fused to Thermotoga maritima chemotaxis proteins that heterodimerize (CheA and CheY) or homodimerize (CheX). This enhanced complementation is interpreted as arising from chemotaxis protein-protein interactions, since AK(Tn)-C156A fragments having only one polypeptide fused to a chemotaxis protein did not complement PQN1 to the same extent. This selection increases the maximum temperature where a PCA can be used to engineer thermostable protein complexes and to map protein-protein interactions.

Resonance assignment for a particularly challenging protein based on systematic unlabeling of amino acids to complement incomplete NMR data sets

International Nuclear Information System (INIS)

Bellstedt, Peter; Seiboth, Thomas; Häfner, Sabine; Kutscha, Henriette; Ramachandran, Ramadurai; Görlach, Matthias

2013-01-01

NMR-based structure determination of a protein requires the assignment of resonances as indispensable first step. Even though heteronuclear through-bond correlation methods are available for that purpose, challenging situations arise in cases where the protein in question only yields samples of limited concentration and/or stability. Here we present a strategy based upon specific individual unlabeling of all 20 standard amino acids to complement standard NMR experiments and to achieve unambiguous backbone assignments for the fast precipitating 23 kDa catalytic domain of human aprataxin of which only incomplete standard NMR data sets could be obtained. Together with the validation of this approach utilizing the protein GB1 as a model, a comprehensive insight into metabolic interconversion ('scrambling”) of NH and CO groups in a standard Escherichia coli expression host is provided

Guanine nucleotide-binding regulatory proteins in retinal pigment epithelial cells

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Jiang, Meisheng; Tran, V.T.; Fong, H.K.W. (Univ. of Southern California, Los Angeles (United States)); Pandey, S. (Doheny Eye Inst., Los Angeles, CA (United States))

1991-05-01

The expression of GTP-binding regulatory proteins (G proteins) in retinal pigment epithelial (RPE) cells was analyzed by RNA blot hybridization and cDNA amplification. Both adult and fetal human RPE cells contain mRNA for multiple G protein {alpha} subunits (G{alpha}) including G{sub s}{alpha}, G{sub i-1}{alpha}, G{sub i-2}{alpha}, G{sub i-3}{alpha}, and G{sub z}{alpha} (or G{sub x}{alpha}), where G{sub s} and G{sub i} are proteins that stimulate or inhibit adenylyl cyclase, respectively, and G{sub z} is a protein that may mediate pertussis toxin-insensitive events. Other G{alpha}-related mRNA transcripts were detected in fetal RPE cells by low-stringency hybridization to G{sub i-2}{alpha} and G{sub s}{alpha} protein-coding cDNA probes. The diversity of G proteins in RPE cells was further studied by cDNA amplification with reverse transcriptase and the polymerase chain reaction. This approach revealed that, besides the above mentioned members of the G{alpha} gene family, at least two other G{alpha} subunits are expressed in RPE cells. Human retinal cDNA clones that encode one of the additional G{alpha} subunits were isolated and characterized. The results indicate that this G{alpha} subunit belongs to a separate subfamily of G proteins that may be insensitive to inhibition by pertussis toxin.

Shark complement: an assessment.

Science.gov (United States)

Smith, S L

1998-12-01

The classical (CCP) and alternative (ACP) pathways of complement activation have been established for the nurse shark (Ginglymostoma cirratum). The isolation of a cDNA clone encoding a mannan-binding protein-associated serine protease (MASP)-1-like protein from the Japanese dogfish (Triakis scyllia) suggests the presence of a lectin pathway. The CCP consists of six functionally distinct components: C1n, C2n, C3n, C4n, C8n and C9n, and is activated by immune complexes in the presence of Ca++ and Mg++ ions. The ACP is antibody independent, requiring Mg++ ions and a heat-labile 90 kDa factor B-like protein for activity. Proteins considered homologues of C1q, C3 and C4 (C2n) of the mammalian complement system have been isolated from nurse shark serum. Shark C1q is composed of at least two chain types each showing 50% identity to human C1q chains A and B. Partial sequence of the globular domain of one of the chains shows it to be C1q-like rather than like mannan-binding protein. N-terminal amino acid sequences of the alpha and beta chain of shark C3 and C4 molecules show significant identity with corresponding human C3 and C4 chains. A sequence representing shark C4 gamma chain, shows little similarity to human C4 gamma chain. The terminal shark components C8n and C9n are functional analogues of mammalian C8 and C9. Anaphylatoxin activity has been demonstrated in activated shark serum, and porcine C5a desArg induces shark leucocyte chemotaxis. The deduced amino acid sequence of a partial C3 cDNA clone from the nurse shark shows 50%, 30% and 24% homology with the corresponding region of mammalian C3, C4 and alpha 2-macroglobulin. Deduced amino acid sequence data from partial Bf/C2 cDNA clones, two from the nurse shark and one from the Japanese dogfish, suggest that at least one species of elasmobranch has two distinct Bf/C2 genes.

A conserved regulatory mechanism in bifunctional biotin protein ligases.

Science.gov (United States)

Wang, Jingheng; Beckett, Dorothy

2017-08-01

Class II bifunctional biotin protein ligases (BirA), which catalyze post-translational biotinylation and repress transcription initiation, are broadly distributed in eubacteria and archaea. However, it is unclear if these proteins all share the same molecular mechanism of transcription regulation. In Escherichia coli the corepressor biotinoyl-5'-AMP (bio-5'-AMP), which is also the intermediate in biotin transfer, promotes operator binding and resulting transcription repression by enhancing BirA dimerization. Like E. coli BirA (EcBirA), Staphylococcus aureus, and Bacillus subtilis BirA (Sa and BsBirA) repress transcription in vivo in a biotin-dependent manner. In this work, sedimentation equilibrium measurements were performed to investigate the molecular basis of this biotin-responsive transcription regulation. The results reveal that, as observed for EcBirA, Sa, and BsBirA dimerization reactions are significantly enhanced by bio-5'-AMP binding. Thus, the molecular mechanism of the Biotin Regulatory System is conserved in the biotin repressors from these three organisms. © 2017 The Protein Society.

SALO, a novel classical pathway complement inhibitor from saliva of the sand fly Lutzomyia longipalpis.

Science.gov (United States)

Ferreira, Viviana P; Fazito Vale, Vladimir; Pangburn, Michael K; Abdeladhim, Maha; Mendes-Sousa, Antonio Ferreira; Coutinho-Abreu, Iliano V; Rasouli, Manoochehr; Brandt, Elizabeth A; Meneses, Claudio; Lima, Kolyvan Ferreira; Nascimento Araújo, Ricardo; Pereira, Marcos Horácio; Kotsyfakis, Michalis; Oliveira, Fabiano; Kamhawi, Shaden; Ribeiro, Jose M C; Gontijo, Nelder F; Collin, Nicolas; Valenzuela, Jesus G

2016-01-13

Blood-feeding insects inject potent salivary components including complement inhibitors into their host's skin to acquire a blood meal. Sand fly saliva was shown to inhibit the classical pathway of complement; however, the molecular identity of the inhibitor remains unknown. Here, we identified SALO as the classical pathway complement inhibitor. SALO, an 11 kDa protein, has no homology to proteins of any other organism apart from New World sand flies. rSALO anti-complement activity has the same chromatographic properties as the Lu. longipalpis salivary gland homogenate (SGH)counterparts and anti-rSALO antibodies blocked the classical pathway complement activity of rSALO and SGH. Both rSALO and SGH inhibited C4b deposition and cleavage of C4. rSALO, however, did not inhibit the protease activity of C1s nor the enzymatic activity of factor Xa, uPA, thrombin, kallikrein, trypsin and plasmin. Importantly, rSALO did not inhibit the alternative or the lectin pathway of complement. In conclusion our data shows that SALO is a specific classical pathway complement inhibitor present in the saliva of Lu. longipalpis. Importantly, due to its small size and specificity, SALO may offer a therapeutic alternative for complement classical pathway-mediated pathogenic effects in human diseases.

Complement 5a Enhances Hepatic Metastases of Colon Cancer via Monocyte Chemoattractant Protein-1-mediated Inflammatory Cell Infiltration.

Science.gov (United States)

Piao, Chunmei; Cai, Lun; Qiu, Shulan; Jia, Lixin; Song, Wenchao; Du, Jie

2015-04-24

Complement 5a (C5a), a potent immune mediator generated by complement activation, promotes tumor growth; however, its role in tumor metastasis remains unclear. We demonstrate that C5a contributes to tumor metastases by modulating tumor inflammation in hepatic metastases of colon cancer. Colon cancer cell lines generate C5a under serum-free conditions, and C5a levels increase over time in a murine syngeneic colon cancer hepatic metastasis model. Furthermore, in the absence of C5a receptor or upon pharmacological inhibition of C5a production with an anti-C5 monoclonal antibody, tumor metastasis is severely impaired. A lack of C5a receptor in colon cancer metastatic foci reduces the infiltration of macrophages, neutrophils, and dendritic cells, and the role for C5a receptor on these cells were further verified by bone marrow transplantation experiments. Moreover, C5a signaling increases the expression of the chemokine monocyte chemoattractant protein-1 and the anti-inflammatory molecules arginase-1, interleukin 10, and transforming growth factor β, but is inversely correlated with the expression of pro-inflammatory molecules, which suggests a mechanism for the role of C5a in the inflammatory microenvironment required for tumor metastasis. Our results indicate a new and potentially promising therapeutic application of complement C5a inhibitor for the treatment of malignant tumors. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Elevated factor H-related protein 1 and factor H pathogenic variants decrease complement regulation in IgA nephropathy.

Science.gov (United States)

Tortajada, Agustín; Gutiérrez, Eduardo; Goicoechea de Jorge, Elena; Anter, Jaouad; Segarra, Alfons; Espinosa, Mario; Blasco, Miquel; Roman, Elena; Marco, Helena; Quintana, Luis F; Gutiérrez, Josué; Pinto, Sheila; Lopez-Trascasa, Margarita; Praga, Manuel; Rodriguez de Córdoba, Santiago

2017-10-01

IgA nephropathy (IgAN), a frequent cause of chronic kidney disease worldwide, is characterized by mesangial deposition of galactose-deficient IgA1-containing immune complexes. Complement involvement in IgAN pathogenesis is suggested by the glomerular deposition of complement components and the strong protection from IgAN development conferred by the deletion of the CFHR3 and CFHR1 genes (Δ CFHR3-CFHR1 ). Here we searched for correlations between clinical progression and levels of factor H (FH) and FH-related protein 1 (FHR-1) using well-characterized patient cohorts consisting of 112 patients with IgAN, 46 with non-complement-related autosomal dominant polycystic kidney disease (ADPKD), and 76 control individuals. Patients with either IgAN or ADPKD presented normal FH but abnormally elevated FHR-1 levels and FHR-1/FH ratios compared to control individuals. Highest FHR-1 levels and FHR-1/FH ratios are found in patients with IgAN with disease progression and in patients with ADPKD who have reached chronic kidney disease, suggesting that renal function impairment elevates the FHR-1/FH ratio, which may increase FHR-1/FH competition for activated C3 fragments. Interestingly, Δ CFHR3-CFHR1 homozygotes are protected from IgAN, but not from ADPKD, and we found five IgAN patients with low FH carrying CFH or CFI pathogenic variants. These data support a decreased FH activity in IgAN due to increased FHR-1/FH competition or pathogenic CFH variants. They also suggest that alternative pathway complement activation in patients with IgAN, initially triggered by galactose-deficient IgA1-containing immune complexes, may exacerbate in a vicious circle as renal function deterioration increase FHR-1 levels. Thus, a role of FHR-1 in IgAN pathogenesis is to compete with complement regulation by FH. Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

Deciphering complement mechanisms: The contributions of structural biology.

NARCIS (Netherlands)

Arlaud, G.J.; Barlow, P.N.; Gaboriaud, C.; Gros, P.; Narayana, S.V.L.

2007-01-01

Since the resolution of the first three-dimensional structure of a complement component in 1980, considerable efforts have been put into the investigation of this system through structural biology techniques, resulting in about a hundred structures deposited in the Protein Data Bank by the beginning

In vitro and in vivo mapping of the Prunus necrotic ringspot virus coat protein C-terminal dimerization domain by bimolecular fluorescence complementation.

Science.gov (United States)

Aparicio, Frederic; Sánchez-Navarro, Jesús A; Pallás, Vicente

2006-06-01

Interactions between viral proteins are critical for virus viability. Bimolecular fluorescent complementation (BiFC) technique determines protein interactions in real-time under almost normal physiological conditions. The coat protein (CP) of Prunus necrotic ringspot virus is required for multiple functions in its replication cycle. In this study, the region involved in CP dimerization has been mapped by BiFC in both bacteria and plant tissue. Full-length and C-terminal deleted forms of the CP gene were fused in-frame to the N- and C-terminal fragments of the yellow fluorescent protein. The BiFC analysis showed that a domain located between residues 9 and 27 from the C-end plays a critical role in dimerization. The importance of this C-terminal region in dimer formation and the applicability of the BiFC technique to analyse viral protein interactions are discussed.

Identification of Open Stomata1-Interacting Proteins Reveals Interactions with Sucrose Non-fermenting1-Related Protein Kinases2 and with Type 2A Protein Phosphatases That Function in Abscisic Acid Responses1[OPEN

Science.gov (United States)

Waadt, Rainer; Manalansan, Bianca; Rauniyar, Navin; Munemasa, Shintaro; Booker, Matthew A.; Brandt, Benjamin; Waadt, Christian; Nusinow, Dmitri A.; Kay, Steve A.; Kunz, Hans-Henning; Schumacher, Karin; DeLong, Alison; Yates, John R.; Schroeder, Julian I.

2015-01-01

The plant hormone abscisic acid (ABA) controls growth and development and regulates plant water status through an established signaling pathway. In the presence of ABA, pyrabactin resistance/regulatory component of ABA receptor proteins inhibit type 2C protein phosphatases (PP2Cs). This, in turn, enables the activation of Sucrose Nonfermenting1-Related Protein Kinases2 (SnRK2). Open Stomata1 (OST1)/SnRK2.6/SRK2E is a major SnRK2-type protein kinase responsible for mediating ABA responses. Arabidopsis (Arabidopsis thaliana) expressing an epitope-tagged OST1 in the recessive ost1-3 mutant background was used for the copurification and identification of OST1-interacting proteins after osmotic stress and ABA treatments. These analyses, which were confirmed using bimolecular fluorescence complementation and coimmunoprecipitation, unexpectedly revealed homo- and heteromerization of OST1 with SnRK2.2, SnRK2.3, OST1, and SnRK2.8. Furthermore, several OST1-complexed proteins were identified as type 2A protein phosphatase (PP2A) subunits and as proteins involved in lipid and galactolipid metabolism. More detailed analyses suggested an interaction network between ABA-activated SnRK2-type protein kinases and several PP2A-type protein phosphatase regulatory subunits. pp2a double mutants exhibited a reduced sensitivity to ABA during seed germination and stomatal closure and an enhanced ABA sensitivity in root growth regulation. These analyses add PP2A-type protein phosphatases as another class of protein phosphatases to the interaction network of SnRK2-type protein kinases. PMID:26175513

The role of complement in CD4⁺ T cell homeostasis and effector functions.

Science.gov (United States)

Kolev, Martin; Le Friec, Gaëlle; Kemper, Claudia

2013-02-01

The complement system is among the evolutionary oldest 'players' of the immune system. It was discovered in 1896 by Jules Bordet as a heat-labile fraction of the serum responsible for the opsonisation and subsequent killing of bacteria. The decades between the 1920s and 1990s then marked the discovery and biochemical characterization of the proteins comprising the complement system. Today, complement is defined as a complex system consisting of more than 30 membrane-bound and soluble plasma proteins, which are activated in a cascade-like manner, very similarly to the caspase proteases and blood coagulation systems. Complement is engrained in the immunologist's mind as a serum-effective, quintessential part of innate immunity, vitally required for the detection and removal of pathogens or other dangerous entities. Three decades ago, this rather confined definition was challenged and then refined when it was shown that complement participates vitally in the induction and regulation of B cell responses, thus adaptive immunity. Similarly, research work published in more recent years supports an equally important role for the complement system in shaping T cell responses. Today, we are again facing paradigm shifts in the field: complement is actively involved in the negative control of T cell effector immune responses, and thus, by definition in immune homeostasis. Further, while serum complement activity is without doubt fundamental in the defence against invading pathogens, local immune cell-derived production of complement emerges as key mediator of complement's impact on adaptive immune responses. And finally, the impact of complement on metabolic pathways and the crosstalk between complement and other immune effector systems is likely more extensive than previously anticipated and is fertile ground for future discoveries. In this review, we will discuss these emerging new roles of complement, with a focus on Th1 cell biology. Copyright © 2013 Elsevier Ltd. All

Interactions of PLGA nanoparticles with blood components: protein adsorption, coagulation, activation of the complement system and hemolysis studies.

Science.gov (United States)

Fornaguera, Cristina; Calderó, Gabriela; Mitjans, Montserrat; Vinardell, Maria Pilar; Solans, Conxita; Vauthier, Christine

2015-04-14

The intravenous administration of poly(lactic-co-glycolic) acid (PLGA) nanoparticles has been widely reported as a promising alternative for delivery of drugs to specific cells. However, studies on their interaction with diverse blood components using different techniques are still lacking. Therefore, in the present work, the interaction of PLGA nanoparticles with blood components was described using different complementary techniques. The influence of different encapsulated compounds/functionalizing agents on these interactions was also reported. It is worth noting that all these techniques can be simply performed, without the need for highly sophisticated apparatus or skills. Moreover, their transference to industries and application of quality control could be easily performed. Serum albumin was adsorbed onto all types of tested nanoparticles. The saturation concentration was dependent on the nanoparticle size. In contrast, fibrinogen aggregation was dependent on nanoparticle surface charge. The complement activation was also influenced by the nanoparticle functionalization; the presence of a functionalizing agent increased complement activation, while the addition of an encapsulated compound only caused a slight increase. None of the nanoparticles influenced the coagulation cascade at low concentrations. However, at high concentrations, cationized nanoparticles did activate the coagulation cascade. Interactions of nanoparticles with erythrocytes did not reveal any hemolysis. Interactions of PLGA nanoparticles with blood proteins depended both on the nanoparticle properties and the protein studied. Independent of their loading/surface functionalization, PLGA nanoparticles did not influence the coagulation cascade and did not induce hemolysis of erythrocytes; they could be defined as safe concerning induction of embolization and cell lysis.

The interaction between circulating complement proteins and cutaneous microvascular endothelial cells in the development of childhood Henoch-Schonlein Purpura.

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Yao-Hsu Yang

Full Text Available In addition to IgA, the deposition of complement (C3 in dermal vessels is commonly found in Henoch-Schönlein purpura (HSP. The aim of this study is to elucidate the role of circulating complement proteins in the pathogenesis of childhood HSP.Plasma levels of C3a, C4a, C5a, and Bb in 30 HSP patients and 30 healthy controls were detected by enzyme-linked immunosorbent assay (ELISA. The expression of C3a receptor (C3aR, C5a receptor (CD88, E-selectin, intercellular adhesion molecule 1 (ICAM-1, C3, C5, interleukin (IL-8, monocyte chemotactic protein (MCP-1, and RANTES by human dermal microvascular endothelial cells (HMVEC-d was evaluated either by flow cytometry or by ELISA.At the acute stage, HSP patients had higher plasma levels of C3a (359.5 ± 115.3 vs. 183.3 ± 94.1 ng/ml, p < 0.0001, C5a (181.4 ± 86.1 vs. 33.7 ± 26.3 ng/ml, p < 0.0001, and Bb (3.7 ± 2.6 vs. 1.0 ± 0.6 μg/ml, p < 0.0001, but not C4a than healthy controls. Although HSP patient-derived acute phase plasma did not alter the presentation of C3aR and CD88 on HMVEC-d, it enhanced the production of endothelial C3 and C5. Moreover, C5a was shown in vitro to up-regulate the expression of IL-8, MCP-1, E-selectin, and ICAM-1 by HMVEC-d with a dose-dependent manner.In HSP, the activation of the complement system in part through the alternative pathway may have resulted in increased plasma levels of C3a and C5a, which, especially C5a, may play a role in the disease pathogenesis by activating endothelium of cutaneous small vessels.

The proteins of intra-nuclear bodies: a data-driven analysis of sequence, interaction and expression

Directory of Open Access Journals (Sweden)

Bodén Mikael

2010-04-01

Full Text Available Abstract Background Cajal bodies, nucleoli, PML nuclear bodies, and nuclear speckles are morpohologically distinct intra-nuclear structures that dynamically respond to cellular cues. Such nuclear bodies are hypothesized to play important regulatory roles, e.g. by sequestering and releasing transcription factors in a timely manner. While the nucleolus and nuclear speckles have received more attention experimentally, the PML nuclear body and the Cajal body are still incompletely characterized in terms of their roles and protein complement. Results By collating recent experimentally verified data, we find that almost 1000 proteins in the mouse nuclear proteome are known to associate with one or more of the nuclear bodies. Their gene ontology terms highlight their regulatory roles: splicing is confirmed to be a core activity of speckles and PML nuclear bodies house a range of proteins involved in DNA repair. We train support-vector machines to show that nuclear proteins contain discriminative sequence features that can be used to identify their intra-nuclear body associations. Prediction accuracy is highest for nucleoli and nuclear speckles. The trained models are also used to estimate the full protein complement of each nuclear body. Protein interactions are found primarily to link proteins in the nuclear speckles with proteins from other compartments. Cell cycle expression data provide support for increased activity in nucleoli, nuclear speckles and PML nuclear bodies especially during S and G2 phases. Conclusions The large-scale analysis of the mouse nuclear proteome sheds light on the functional organization of physically embodied intra-nuclear compartments. We observe partial support for the hypothesis that the physical organization of the nucleus mirrors functional modularity. However, we are unable to unambiguously identify proteins' intra-nuclear destination, suggesting that critical drivers behind of intra-nuclear translocation are yet to

Rivaroxaban limits complement activation compared with warfarin in antiphospholipid syndrome patients with venous thromboembolism.

Science.gov (United States)

Arachchillage, D R J; Mackie, I J; Efthymiou, M; Chitolie, A; Hunt, B J; Isenberg, D A; Khamashta, M; Machin, S J; Cohen, H

2016-11-01

Essentials Complement activation has a pathogenic role in thrombotic antiphospholipid syndrome (APS). Coagulation proteases such as factor Xa can activate complement proteins. Complement activation markers were elevated in anticoagulated thrombotic APS patients. Complement activation decreased in APS patients switching from warfarin to rivaroxaban. Background Complement activation may play a major role in the pathogenesis of thrombotic antiphospholipid syndrome (APS). Coagulation proteases such as factor Xa can activate complement proteins. Aims To establish whether rivaroxaban, a direct factor Xa inhibitor, limits complement activation compared with warfarin in APS patients with previous venous thromboembolism (VTE). Methods A total of 111 APS patients with previous VTE, on warfarin target INR 2.5, had blood samples taken at baseline and at day 42 after randomization in the RAPS (Rivaroxaban in Antiphospholipid Syndrome) trial. Fifty-six patients remained on warfarin and 55 switched to rivaroxaban. Fifty-five normal controls (NC) were also studied. Markers of complement activation (C3a, C5a, terminal complement complex [SC5b-9] and Bb fragment) were assessed. Results APS patients had significantly higher complement activation markers compared with NC at both time-points irrespective of the anticoagulant. There were no differences between the two patient groups at baseline, or patients remaining on warfarin at day 42. In 55 patients randomized to rivaroxaban, C3a, C5a and SC5b-9 were lower at day 42 (median (ng mL -1 ) [confidence interval] 64 [29-125] vs. 83 [35-147], 9 [2-15] vs. 12 [4-18] and 171 [56-245] vs. 201 [66-350], respectively) but levels of Bb fragment were unchanged. There were no correlations between rivaroxaban levels and complement activation markers. Conclusions APS patients with previous VTE on warfarin exhibit increased complement activation, which is likely to occur via the classical pathway and is decreased by rivaroxaban administration

Virulence of Group A Streptococci Is Enhanced by Human Complement Inhibitors

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Ermert, David; Shaughnessy, Jutamas; Joeris, Thorsten

2015-01-01

Streptococcus pyogenes, also known as Group A Streptococcus (GAS), is an important human bacterial pathogen that can cause invasive infections. Once it colonizes its exclusively human host, GAS needs to surmount numerous innate immune defense mechanisms, including opsonization by complement and c...... in studies of GAS pathogenesis and for developing vaccines and therapeutics that rely on human complement activation for efficacy.......Streptococcus pyogenes, also known as Group A Streptococcus (GAS), is an important human bacterial pathogen that can cause invasive infections. Once it colonizes its exclusively human host, GAS needs to surmount numerous innate immune defense mechanisms, including opsonization by complement...... and consequent phagocytosis. Several strains of GAS bind to human-specific complement inhibitors, C4b-binding protein (C4BP) and/or Factor H (FH), to curtail complement C3 (a critical opsonin) deposition. This results in diminished activation of phagocytes and clearance of GAS that may lead to the host being...

Activation of the human complement system by cholesterol-rich and pegylated liposomes - Modulation of cholesterol-rich liposome-mediated complement activation by elevated serum LDL and HDL levels

DEFF Research Database (Denmark)

Moghimi, S.M.; Hamad, I.; Bunger, R.

2006-01-01

level of S-protein-bound form of the terminal complex (SC5b-9). However, liposome-induced rise of SC5b-9 was significantly suppressed when serum HDL cholesterol levels increased by 30%. Increase of serum LDL to levels similar to that observed in heterozygous familial hypercholesterolemia also suppressed......Intravenously infused liposomes may induce cardiopulmonary distress in some human subjects, which is a manifestation of "complement activation-related pseudoallergy." We have now examined liposome-mediated complement activation in human sera with elevated lipoprotein (LDL and HDL) levels, since...... abnormal or racial differences in serum lipid profiles seem to modulate the extent of complement activation and associated adverse responses. In accordance with our earlier observations, cholesterol-rich (45 mol% cholesterol) liposomes activated human complement, as reflected by a significant rise in serum...

Preeclampsia in autologous and oocyte donation pregnancy: is there a different pathophysiology?

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Lashley, Lisa E E L O; Buurma, Aletta; Swings, Godelieve M J S; Eikmans, Michael; Anholts, Jacqueline D H; Bakker, Jaap A; Claas, Frans H J

2015-06-01

Oocyte donation (OD) is a specific method of artificial reproductive technology that is accompanied by a higher risk of preeclampsia during pregnancy. The pathophysiological mechanism underlying preeclampsia in OD pregnancies is thought to differ from preeclampsia in autologous pregnancies. As preeclampsia in autologous pregnancies is suggested to be associated with complement activation, we studied C4d deposition, circulating complement components and placental complement regulatory proteins in preeclamptic OD pregnancies. Women with uncomplicated and preeclamptic pregnancies after OD or spontaneous conception were selected. We stained the placentas for C4d, marker for complement activation, measured complement factors C1q, C3 and C4 in maternal sera and quantified the placental mRNA expression of complement regulatory proteins CD46, CD55 and CD59. A significantly (p preeclampsia compared with uncomplicated pregnancies, both OD and autologous. The level of complement factors in serum did not differ between the groups. Children born in the autologous preeclampsia group were significantly lower in birth weight (p preeclampsia pregnancies, there is excessive activation of complement in preeclamptic OD pregnancies. However, in contrast to autologous pregnancies this is not associated with counterbalancing upregulation of complement regulatory proteins. Furthermore, C4d deposition in OD pregnancies is not related to the severity of preeclampsia, suggesting another trigger or regulatory mechanism of placental C4d deposition in preeclamptic OD pregnancies. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Involvement of the iron regulatory protein from Eisenia andrei earthworms in the regulation of cellular iron homeostasis.

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Petra Procházková

Full Text Available Iron homeostasis in cells is regulated by iron regulatory proteins (IRPs that exist in different organisms. IRPs are cytosolic proteins that bind to iron-responsive elements (IREs of the 5'- or 3'-untranslated regions (UTR of mRNAs that encode many proteins involved in iron metabolism. In this study, we have cloned and described a new regulatory protein belonging to the family of IRPs from the earthworm Eisenia andrei (EaIRP. The earthworm IRE site in 5'-UTR of ferritin mRNA most likely folds into a secondary structure that differs from the conventional IRE structures of ferritin due to the absence of a typically unpaired cytosine that participates in protein binding. Prepared recombinant EaIRP and proteins from mammalian liver extracts are able to bind both mammalian and Eisenia IRE structures of ferritin mRNA, although the affinity of the rEaIRP/Eisenia IRE structure is rather low. This result suggests the possible contribution of a conventional IRE structure. When IRP is supplemented with a Fe-S cluster, it can function as a cytosolic aconitase. Cellular cytosolic and mitochondrial fractions, as well as recombinant EaIRP, exhibit aconitase activity that can be abolished by the action of oxygen radicals. The highest expression of EaIRP was detected in parts of the digestive tract. We can assume that earthworms may possess an IRE/IRP regulatory network as a potential mechanism for maintaining cellular iron homeostasis, although the aconitase function of EaIRP is most likely more relevant.

Involvement of the Iron Regulatory Protein from Eisenia andrei Earthworms in the Regulation of Cellular Iron Homeostasis

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Procházková, Petra; Škanta, František; Roubalová, Radka; Šilerová, Marcela; Dvořák, Jiří; Bilej, Martin

2014-01-01

Iron homeostasis in cells is regulated by iron regulatory proteins (IRPs) that exist in different organisms. IRPs are cytosolic proteins that bind to iron-responsive elements (IREs) of the 5′- or 3′-untranslated regions (UTR) of mRNAs that encode many proteins involved in iron metabolism. In this study, we have cloned and described a new regulatory protein belonging to the family of IRPs from the earthworm Eisenia andrei (EaIRP). The earthworm IRE site in 5′-UTR of ferritin mRNA most likely folds into a secondary structure that differs from the conventional IRE structures of ferritin due to the absence of a typically unpaired cytosine that participates in protein binding. Prepared recombinant EaIRP and proteins from mammalian liver extracts are able to bind both mammalian and Eisenia IRE structures of ferritin mRNA, although the affinity of the rEaIRP/Eisenia IRE structure is rather low. This result suggests the possible contribution of a conventional IRE structure. When IRP is supplemented with a Fe-S cluster, it can function as a cytosolic aconitase. Cellular cytosolic and mitochondrial fractions, as well as recombinant EaIRP, exhibit aconitase activity that can be abolished by the action of oxygen radicals. The highest expression of EaIRP was detected in parts of the digestive tract. We can assume that earthworms may possess an IRE/IRP regulatory network as a potential mechanism for maintaining cellular iron homeostasis, although the aconitase function of EaIRP is most likely more relevant. PMID:25279857

Overexpression of KH-type splicing regulatory protein regulates proliferation, migration, and implantation ability of osteosarcoma.

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Pruksakorn, Dumnoensun; Teeyakasem, Pimpisa; Klangjorhor, Jeerawan; Chaiyawat, Parunya; Settakorn, Jongkolnee; Diskul-Na-Ayudthaya, Penchatr; Chokchaichamnankit, Daranee; Pothacharoen, Peraphan; Srisomsap, Chantragan

2016-09-01

Osteosarcoma is a common malignant bone tumor in children and adolescents. The current 5-year survival rate is ~60% and that seems to be reaching a plateau. In order to improve treatment outcomes of osteosarcoma, a better understanding of tumorigenesis and underlying molecular mechanisms is required for searching out possible new treatment targets. This study aimed to identify the potential proteins involving the pathogenesis of osteosarcoma using a proteomics approach. Proteins extracted from primary cell culture of osteosarcoma (n=7) and osteoblasts of cancellous bone (n=7) were studied. Using 2-DE based proteomics and LC-MS/MS analysis, we successfully determined seven differentially expressed protein spots. Four upregulated proteins and three downregulated proteins were observed in this study in which KH-type splicing regulatory protein (KSRP) was selected for further exploration. KSRP was significantly upregulated in osteosarcoma cells compared to osteoblasts using western blot assay. In addition, immunohistochemistry demonstrated that KSRP was also highly expressed in osteosarcoma tissue of independent cases from the experimental group. More importantly, KSRP silencing of osteosarcoma cell lines significantly decreased cell proliferation, migration ability, as well as implantation and growth ability in chick chorioallantoic membrane assay. Taken together, these findings demonstrate, that KSRP plays important roles in regulatory controls of osteosarcoma pathogenesis and serves as a potentially therapeutic target of osteosarcoma.

Complement System in the Pathogenesis of Benign Lymphoepithelial Lesions of the Lacrimal Gland.

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Jing Li

Full Text Available We aimed to examine the potential involvement of local complement system gene expression in the pathogenesis of benign lymphoepithelial lesions (BLEL of the lacrimal gland.We collected data from 9 consecutive pathologically confirmed patients with BLEL of the lacrimal gland and 9 cases with orbital cavernous hemangioma as a control group, and adopted whole genome microarray to screen complement system-related differential genes, followed by RT-PCR verification and in-depth enrichment analysis (Gene Ontology analysis of the gene sets.The expression of 14 complement system-related genes in the pathologic tissue, including C2, C3, ITGB2, CR2, C1QB, CR1, ITGAX, CFP, C1QA, C4B|C4A, FANCA, C1QC, C3AR1 and CFHR4, were significantly upregulated while 7 other complement system-related genes, C5, CFI, CFHR1|CFH, CFH, CD55, CR1L and CFD were significantly downregulated in the lacrimal glands of BLEL patients. The microarray results were consistent with RT-PCR analysis results. Immunohistochemistry analysis of C3c and C1q complement component proteins in the resected tissue were positive in BLEL patients, while the control group had negative expression of these proteins. Gene ontology (GO analysis revealed that activation of the genes of complement system-mediated signaling pathways were the most enriched differential gene group in BLEL patients.Local expression of complement components is prominently abnormal in BLEL, and may well play a role in its pathogenesis.

Role of regulatory subunits and protein kinase inhibitor (PKI) in determining nuclear localization and activity of the catalytic subunit of protein kinase A.

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Wiley, J C; Wailes, L A; Idzerda, R L; McKnight, G S

1999-03-05

Regulation of protein kinase A by subcellular localization may be critical to target catalytic subunits to specific substrates. We employed epitope-tagged catalytic subunit to correlate subcellular localization and gene-inducing activity in the presence of regulatory subunit or protein kinase inhibitor (PKI). Transiently expressed catalytic subunit distributed throughout the cell and induced gene expression. Co-expression of regulatory subunit or PKI blocked gene induction and prevented nuclear accumulation. A mutant PKI lacking the nuclear export signal blocked gene induction but not nuclear accumulation, demonstrating that nuclear export is not essential to inhibit gene induction. When the catalytic subunit was targeted to the nucleus with a nuclear localization signal, it was not sequestered in the cytoplasm by regulatory subunit, although its activity was completely inhibited. PKI redistributed the nuclear catalytic subunit to the cytoplasm and blocked gene induction, demonstrating that the nuclear export signal of PKI can override a strong nuclear localization signal. With increasing PKI, the export process appeared to saturate, resulting in the return of catalytic subunit to the nucleus. These results demonstrate that both the regulatory subunit and PKI are able to completely inhibit the gene-inducing activity of the catalytic subunit even when the catalytic subunit is forced to concentrate in the nuclear compartment.

An overview of the gene regulatory network controlling trichome development in the model plant, Arabidopsis

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Sitakanta ePattanaik

2014-06-01

Full Text Available Trichomes are specialized epidermal cells located on aerial parts of plants and are associated with a wide array of biological processes. Trichomes protect plants from adverse conditions including UV light and herbivore attack and are also an important source of a number of phytochemicals. The simple unicellular trichomes of Arabidopsis serve as an excellent model to study molecular mechanism of cell differentiation and pattern formation in plants. The emerging picture suggests that the developmental process is controlled by a transcriptional network involving three major groups of transcription factors: the R2R3 MYB, basic helix-loop-helix (bHLH and WD40 repeat (WDR protein. These regulatory proteins form a trimeric activator complex that positively regulates trichome development. The single repeat R3 MYBs act as negative regulators of trichome development. They compete with the R2R3 MYBs to bind the bHLH factor and form a repressor complex. In addition to activator-repressor mechanism, a depletion mechanism may operate in parallel during trichome development. In this mechanism, the bHLH factor traps the WDR protein which results in depletion of WDR protein in neighboring cells. Consequently, the cells with high levels of bHLH and WDR proteins are developed into trichomes. A group of C2H2 zinc finger TFs has also been implicated in trichome development. Phytohormones, including gibberellins and jasmonic acid, play significant roles in this developmental process. Recently, microRNAs have been shown to be involved in trichome development. Furthermore, it has been demonstrated that the activities of the key regulatory proteins involved in trichome development are controlled by the 26S/ubiquitin proteasome system (UPS, highlighting the complexity of the regulatory network controlling this developmental process. To complement several excellent recent relevant reviews, this review focuses on the transcriptional network and hormonal interplay

Multiple activities of LigB potentiate virulence of Leptospira interrogans: inhibition of alternative and classical pathways of complement.

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Henry A Choy

Full Text Available Microbial pathogens acquire the immediate imperative to avoid or counteract the formidable defense of innate immunity as soon as they overcome the initial physical barriers of the host. Many have adopted the strategy of directly disrupting the complement system through the capture of its components, using proteins on the pathogen's surface. In leptospirosis, pathogenic Leptospira spp. are resistant to complement-mediated killing, in contrast to the highly vulnerable non-pathogenic strains. Pathogenic L. interrogans uses LenA/LfhA and LcpA to respectively sequester and commandeer the function of two regulators, factor H and C4BP, which in turn bind C3b or C4b to interrupt the alternative or classical pathways of complement activation. LigB, another surface-proximal protein originally characterized as an adhesin binding multiple host proteins, has other activities suggesting its importance early in infection, including binding extracellular matrix, plasma, and cutaneous repair proteins and inhibiting hemostasis. In this study, we used a recent model of ectopic expression of LigB in the saprophyte, L. biflexa, to test the hypothesis that LigB also interacts with complement proteins C3b and C4b to promote the virulence of L. interrogans. The surface expression of LigB partially rescued the non-pathogen from killing by 5% normal human serum, showing 1.3- to 48-fold greater survival 4 to 6 d following exposure to complement than cultures of the non-expressing parental strain. Recombinant LigB7'-12 comprising the LigB-specific immunoglobulin repeats binds directly to human complement proteins, C3b and C4b, with respective K(ds of 43±26 nM and 69±18 nM. Repeats 9 to 11, previously shown to contain the binding domain for fibronectin and fibrinogen, are also important in LigB-complement interactions, which interfere with the alternative and classical pathways measured by complement-mediated hemolysis of erythrocytes. Thus, LigB is an adaptable interface

Structural analysis of human complement protein H: homology with C4b binding protein, beta 2-glycoprotein I, and the Ba fragment of B2

DEFF Research Database (Denmark)

Kristensen, Torsten; Wetsel, R A; Tack, B F

1986-01-01

We report here a partial primary structure for human complement protein H. Tryptic peptides comprising 27% of the H molecule were isolated by conventional techniques and were sequenced (333 amino acid residues). Several mixed-sequence oligonucleotide probes were constructed, based on the peptide...... sequence data, and were used to screen a human liver cDNA library. The largest recombinant plasmid (pH1050), which hybridized with two probes, was further characterized. The cDNA insert of this plasmid contained coding sequence (672 bp) for 224 amino acids of H. The 3' end of this clone had...... a polyadenylated tail preceded by a polyadenylation recognition site (ATTAAA) and a 3'-untranslated region (229 bp). Four regions of internal homology, each about 60 amino acids in length, were observed in the derived protein sequence from this cDNA clone, and a further seven from the tryptic peptide sequences...

Activation of the classical pathway of complement by tobacco glycoprotein (TGP).

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Koethe, S M; Nelson, K E; Becker, C G

1995-07-15

Tobacco glycoprotein (TGP), a polyphenol-rich glycoprotein isolated from tobacco leaves, activates the classical complement pathway through a mechanism that appears to involve direct interaction with C1q. A binding site on C1q for TGP can be localized by competitive inhibition with DNA to a region located in the junction between the collagen-like and globular regions of the molecule. A protein with activity similar to TGP has also been isolated from cigarette smoke condensate (TGP-S); it shares a binding site on C1q with TGP and has similar functional activity, with the exception that complement activation does not proceed to formation of a C3 cleaving enzyme. The ability of TGP and TGP-S to activate complement can be partially duplicated using polyphenols associated with tobacco leaf and smoke, i.e., chlorogenic acid and rutin. These polyphenols also compete with TGP for a binding site on immobilized C1q, suggesting that the polyphenol portion of TGP is critical for activation of complement. These results provide an additional mechanism for complement activation by cigarette products that, in vivo, could result in a localized complement depletion, generation of biologically active complement cleavage products, and initiation of an inflammatory response.

Fcγ and Complement Receptors and Complement Proteins in Neutrophil Activation in Rheumatoid Arthritis: Contribution to Pathogenesis and Progression and Modulation by Natural Products

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Adriana Balbina Paoliello-Paschoalato

2015-01-01

Full Text Available Rheumatoid arthritis (RA is a highly disabling disease that affects all structures of the joint and significantly impacts on morbidity and mortality in RA patients. RA is characterized by persistent inflammation of the synovial membrane lining the joint associated with infiltration of immune cells. Eighty to 90% of the leukocytes infiltrating the synovia are neutrophils. The specific role that neutrophils play in the onset of RA is not clear, but recent studies have evidenced that they have an important participation in joint damage and disease progression through the release of proteolytic enzymes, reactive oxygen species (ROS, cytokines, and neutrophil extracellular traps, in particular during frustrated phagocytosis of immune complexes (ICs. In addition, the local and systemic activation of the complement system contributes to the pathogenesis of RA and other IC-mediated diseases. This review discusses (i the participation of Fcγ and complement receptors in mediating the effector functions of neutrophils in RA; (ii the contribution of the complement system and ROS-dependent and ROS-independent mechanisms to joint damage in RA; and (iii the use of plant extracts, dietary compounds, and isolated natural compounds in the treatment of RA, focusing on modulation of the effector functions of neutrophils and the complement system activity and/or activation.

Binding of complement proteins C1q and C4bp to serum amyloid P component (SAP) in solid contra liquid phase

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Sørensen, Inge Juul; Nielsen, EH; Andersen, Ove

1996-01-01

Serum amyloid P component (SAP), a member of the conserved pentraxin family of plasma proteins, binds calcium dependently to its ligands. The authors investigated SAPs interaction with the complement proteins C4b binding protein (C4bp) and C1q by ELISA, immunoelectrophoresis and electron microscopy....... Binding of these proteins to SAP was demonstrated when SAP was immobilized using F(ab')2 anti-SAP, but not when SAP reacted with these proteins in liquid phase; thus the binding to human SAP was markedly phase state dependent. Presaturation of solid phase SAP with heparin, which binds SAP with high...... affinity, did not interfere with the subsequent binding of C4bp or C1q to SAP. In contrast, collagen I and IV showed partial competition with the binding of C1q to SAP. Using fresh serum, immobilized native SAP bound C4bp whereas binding of C1q/C1 could not be demonstrated. Altogether the results indicate...

Dietary β-glucan stimulate complement and C-reactive protein acute phase responses in common carp (Cyprinus carpio) during an Aeromonas salmonicida infection.

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Pionnier, Nicolas; Falco, Alberto; Miest, Joanna; Frost, Patrick; Irnazarow, Ilgiz; Shrive, Annette; Hoole, Dave

2013-03-01

The effect of β-glucans as feed additive on the profile of C-reactive protein (CRP) and complement acute phase responses was studied in common carp Cyprinus carpio after exposition to a bacterial infection with Aeromonas salmonicida. Carp were orally administered with β-glucan (MacroGard®) for 14 days with a daily β-glucan intake of 6 mg per kg body weight. Fish were then intraperitoneally injected with either PBS or 1 × 10⁸ bacteria per fish and sampled at time 0, 6, 12, 24, 48, 72, 96 and 120 h post-injection (p.i.) for serum and head kidney, liver and mid-gut tissues. CRP levels and complement activity were determined in the serum samples whilst the gene expression profiles of CRP and complement related genes (crp1, crp2, c1r/s, bf/c2, c3 and masp2) were analysed in the tissues by quantitative PCR. Results obtained showed that oral administration of β-glucan for 14 days significantly increased serum CRP levels up to 2 fold and serum alternative complement activity (ACP) up to 35 fold. The bacterial infection on its own (i.e. not combined with a β-glucan feeding) did have significant effects on complement response whilst CRP was not detectably induced during the carp acute phase reaction. However, the combination of the infection and the β-glucan feeding did show significant effects on both CRP and complement profiles with higher serum CRP levels and serum ACP activity in the β-glucan fed fish than in the control fed fish. In addition, a distinct organ and time dependent expression profile pattern was detected for all the selected genes: a peak of gene expression first occurred in the head kidney tissue (6 h p.i. or 12 h p.i.), then an up-regulation in the liver several hours later (24 h p.i.) and finally up- or down-regulations in the mid-gut at 24 h p.i. and 72 h p.i. In conclusion, the results of this study suggest that MacroGard® stimulated CRP and complement responses to A. salmonicida infection in common carp. Copyright © 2013 Elsevier Ltd. All

Interactions of the humoral pattern recognition molecule PTX3 with the complement system

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Doni, Andrea; Garlanda, Cecilia; Bottazzi, Barbara

2012-01-01

The innate immune system comprises a cellular and a humoral arm. The long pentraxin PTX3 is a fluid phase pattern recognition molecule, which acts as an essential component of the humoral arm of innate immunity. PTX3 has antibody-like properties including interactions with complement components....... PTX3 interacts with C1q, ficolin-1 and ficolin-2 as well as mannose-binding lectin, recognition molecules in the classical and lectin complement pathways. The formation of these heterocomplexes results in cooperative pathogen recognition and complement activation. Interactions with C4b binding protein...

Zinc deficiency-induced iron accumulation, a consequence of alterations in iron regulatory protein-binding activity, iron transporters, and iron storage proteins.

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Niles, Brad J; Clegg, Michael S; Hanna, Lynn A; Chou, Susan S; Momma, Tony Y; Hong, Heeok; Keen, Carl L

2008-02-22

One consequence of zinc deficiency is an elevation in cell and tissue iron concentrations. To examine the mechanism(s) underlying this phenomenon, Swiss 3T3 cells were cultured in zinc-deficient (D, 0.5 microM zinc), zinc-supplemented (S, 50 microM zinc), or control (C, 4 microM zinc) media. After 24 h of culture, cells in the D group were characterized by a 50% decrease in intracellular zinc and a 35% increase in intracellular iron relative to cells in the S and C groups. The increase in cellular iron was associated with increased transferrin receptor 1 protein and mRNA levels and increased ferritin light chain expression. The divalent metal transporter 1(+)iron-responsive element isoform mRNA was decreased during zinc deficiency-induced iron accumulation. Examination of zinc-deficient cells revealed increased binding of iron regulatory protein 2 (IRP2) and decreased binding of IRP1 to a consensus iron-responsive element. The increased IRP2-binding activity in zinc-deficient cells coincided with an increased level of IRP2 protein. The accumulation of IRP2 protein was independent of zinc deficiency-induced intracellular nitric oxide production but was attenuated by the addition of the antioxidant N-acetylcysteine or ascorbate to the D medium. These data support the concept that zinc deficiency can result in alterations in iron transporter, storage, and regulatory proteins, which facilitate iron accumulation.

The selective phosphorylation of a guanine nucleotide-binding regulatory protein

International Nuclear Information System (INIS)

Carlson, K.E.

1989-01-01

Receptor-activated signal transduction pathways regulate the responsiveness of cells to external stimuli. These transduction pathways themselves are subject to regulation, most commonly by phosphorylation. Guanine nucleotide-binding regulatory proteins (G Proteins), as requisite signal transducing elements for many plasma membrane receptors, are considered likely targets for regulation by phosphorylation. Protein kinase C (PKC) has been shown to phosphorylate the α subunit of G i and other G proteins in solution. However, the occurrence of the phosphorylation of G 1 within intact cells in response to activation of PKC has not been rigorously demonstrated. In this thesis, the extent to which the α subunits of G i undergo phosphorylation within human platelets in response to activation of PKC was examined by means of radiolabeling and immunoprecipitation. Incubation of platelets with phorbol-12-myristate-13-acetate (PMA), a potent activator of PKC, promoted the phosphorylation of several proteins within saponin-permeabilized and intact platelets incubated with [γ 32 P]ATP and [ 32 P]H 3 PO 4 , respectively. None of the phosphoproteins, however, were precipitated by either of two antisera containing antibodies differing in specificities for epitopes within G iα -despite precipitation of a substantial fraction of the subunit itself. In contrast, other antisera, containing antibodies specific for the recently describe G zα , or antibodies for both G zα and G iα , precipitated a 40-kDa phosphoprotein

Early Intra-Articular Complement Activation in Ankle Fractures

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Hagen Schmal

2014-01-01

Full Text Available Cytokine regulation possibly influences long term outcome following ankle fractures, but little is known about synovial fracture biochemistry. Eight patients with an ankle dislocation fracture were included in a prospective case series and matched with patients suffering from grade 2 osteochondritis dissecans (OCD of the ankle. All fractures needed external fixation during which joint effusions were collected. Fluid analysis was done by ELISA measuring aggrecan, bFGF, IL-1β, IGF-1, and the complement components C3a, C5a, and C5b-9. The time periods between occurrence of fracture and collection of effusion were only significantly associated with synovial aggrecan and C5b-9 levels (P<0.001. Furthermore, synovial expressions of both proteins correlated with each other (P<0.001. Although IL-1β expression was relatively low, intra-articular levels correlated with C5a (P<0.01 and serological C-reactive protein concentrations 2 days after surgery (P<0.05. Joint effusions were initially dominated by neutrophils, but the portion of monocytes constantly increased reaching 50% at day 6 after fracture (P<0.02. Whereas aggrecan and IL-1β concentrations were not different in fracture and OCD patients, bFGF, IGF-1, and all complement components were significantly higher concentrated in ankle joints with fractures (P<0.01. Complement activation and inflammatory cell infiltration characterize the joint biology following acute ankle fractures.

Complement Mutations in Diacylglycerol Kinase-ε–Associated Atypical Hemolytic Uremic Syndrome

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Sánchez Chinchilla, Daniel; Pinto, Sheila; Hoppe, Bernd; Adragna, Marta; Lopez, Laura; Justa Roldan, Maria Luisa; Peña, Antonia; Lopez Trascasa, Margarita; Sánchez-Corral, Pilar; Rodríguez de Córdoba, Santiago

2014-01-01

Background and objectives Atypical hemolytic uremic syndrome is characterized by vascular endothelial damage caused by complement dysregulation. Consistently, complement inhibition therapies are highly effective in most patients with atypical hemolytic uremic syndrome. Recently, it was shown that a significant percentage of patients with early-onset atypical hemolytic uremic syndrome carry mutations in diacylglycerol kinase-ε, an intracellular protein with no obvious role in complement. These data support an alternative, complement-independent mechanism leading to thrombotic microangiopathy that has implications for treatment of early-onset atypical hemolytic uremic syndrome. To get additional insights into this new form of atypical hemolytic uremic syndrome, the diacylglycerol kinase-ε gene in a cohort with atypical hemolytic uremic syndrome was analyzed. Design, setting, participants, & measurements Eighty-three patients with early-onset atypical hemolytic uremic syndrome (<2 years) enrolled in the Spanish atypical hemolytic uremic syndrome registry between 1999 and 2013 were screened for mutations in diacylglycerol kinase-ε. These patients were also fully characterized for mutations in the genes encoding factor H, membrane cofactor protein, factor I, C3, factor B, and thrombomodulin CFHRs copy number variations and rearrangements, and antifactor H antibodies. Results Four patients carried mutations in diacylglycerol kinase-ε, one p.H536Qfs*16 homozygote and three compound heterozygotes (p.W322*/p.P498R, two patients; p.Q248H/p.G484Gfs*10, one patient). Three patients also carried heterozygous mutations in thrombomodulin or C3. Extensive plasma infusions controlled atypical hemolytic uremic syndrome recurrences and prevented renal failure in the two patients with diacylglycerol kinase-ε and thrombomodulin mutations. A positive response to plasma infusions and complement inhibition treatment was also observed in the patient with concurrent diacylglycerol

Complement mutations in diacylglycerol kinase-ε-associated atypical hemolytic uremic syndrome.

Science.gov (United States)

Sánchez Chinchilla, Daniel; Pinto, Sheila; Hoppe, Bernd; Adragna, Marta; Lopez, Laura; Justa Roldan, Maria Luisa; Peña, Antonia; Lopez Trascasa, Margarita; Sánchez-Corral, Pilar; Rodríguez de Córdoba, Santiago

2014-09-05

Atypical hemolytic uremic syndrome is characterized by vascular endothelial damage caused by complement dysregulation. Consistently, complement inhibition therapies are highly effective in most patients with atypical hemolytic uremic syndrome. Recently, it was shown that a significant percentage of patients with early-onset atypical hemolytic uremic syndrome carry mutations in diacylglycerol kinase-ε, an intracellular protein with no obvious role in complement. These data support an alternative, complement-independent mechanism leading to thrombotic microangiopathy that has implications for treatment of early-onset atypical hemolytic uremic syndrome. To get additional insights into this new form of atypical hemolytic uremic syndrome, the diacylglycerol kinase-ε gene in a cohort with atypical hemolytic uremic syndrome was analyzed. Eighty-three patients with early-onset atypical hemolytic uremic syndrome (<2 years) enrolled in the Spanish atypical hemolytic uremic syndrome registry between 1999 and 2013 were screened for mutations in diacylglycerol kinase-ε. These patients were also fully characterized for mutations in the genes encoding factor H, membrane cofactor protein, factor I, C3, factor B, and thrombomodulin CFHRs copy number variations and rearrangements, and antifactor H antibodies. Four patients carried mutations in diacylglycerol kinase-ε, one p.H536Qfs*16 homozygote and three compound heterozygotes (p.W322*/p.P498R, two patients; p.Q248H/p.G484Gfs*10, one patient). Three patients also carried heterozygous mutations in thrombomodulin or C3. Extensive plasma infusions controlled atypical hemolytic uremic syndrome recurrences and prevented renal failure in the two patients with diacylglycerol kinase-ε and thrombomodulin mutations. A positive response to plasma infusions and complement inhibition treatment was also observed in the patient with concurrent diacylglycerol kinase-ε and C3 mutations. Data suggest that complement dysregulation influences

Reforms to the European Union Financial Supervisory and Regulatory Architecture and Their Implications for Asia

NARCIS (Netherlands)

Z. Darvas (Zsolt); D. Schoenmaker (Dirk); N. Véron (Nicolas)

2017-01-01

textabstractEuropean Union (EU) countries offer a unique experience of financial regulatory and supervisory integration, complementing various other European integration efforts following the Second World War. Financial regulatory and supervisory integration was a very slow process before 2008,

Activation of the lectin pathway of complement in experimental human keratitis with Pseudomonas aeruginosa.

Science.gov (United States)

Osthoff, Michael; Brown, Karl D; Kong, David C M; Daniell, Mark; Eisen, Damon P

2014-01-01

Pseudomonas aeruginosa (P. aeruginosa) microbial keratitis (MK) is a sight-threatening disease. Previous animal studies have identified an important contribution of the complement system to the clearance of P. aeruginosa infection of the cornea. Mannose-binding lectin (MBL), a pattern recognition receptor of the lectin pathway of complement, has been implicated in the host defense against P. aeruginosa. However, studies addressing the role of the lectin pathway in P. aeruginosa MK are lacking. Hence, we sought to determine the activity of the lectin pathway in human MK caused by P. aeruginosa. Primary human corneal epithelial cells (HCECs) from cadaveric donors were exposed to two different P. aeruginosa strains. Gene expression of interleukin (IL)-6, IL-8, MBL, and other complement proteins was determined by reverse transcription-polymerase chain reaction (RT-PCR) and MBL synthesis by enzyme-linked immunosorbent assay and intracellular flow cytometry. MBL gene expression was not detected in unchallenged HCECs. Exposure of HCECs to P. aeruginosa resulted in rapid induction of the transcriptional expression of MBL, IL-6, and IL-8. In addition, expression of several complement proteins of the classical and lectin pathways, but not the alternative pathway, were upregulated after 5 h of challenge, including MBL-associated serine protease 1. However, MBL protein secretion was not detectable 18 h after challenge with P. aeruginosa. MK due to P. aeruginosa triggers activation of MBL and the lectin pathway of complement. However, the physiologic relevance of this finding is unclear, as corresponding MBL oligomer production was not observed.

Lectin Complement Pathway Proteins in Healthy Individuals

DEFF Research Database (Denmark)

Troldborg, Anne; Hansen, Annette Gudmann; Hansen, Søren W K

2017-01-01

, it is pivotal to know the normal. Our aim was to describe the concentrations of the eleven known proteins of the lectin pathway in serum and plasma and to uncover possible gender differences, age and diurnal variations, which must be taken into account for investigations in different cohorts. We examined...... morning to the middle of night. There were gender differences for most proteins, whereas age did not seem to influence concentration. The present study underlines the necessity of considering which material to use, correct matching and a trial design that takes the nature of the protein into account...

Anopheles Midgut Epithelium Evades Human Complement Activity by Capturing Factor H from the Blood Meal

Science.gov (United States)

Khattab, Ayman; Barroso, Marta; Miettinen, Tiera; Meri, Seppo

2015-01-01

Hematophagous vectors strictly require ingesting blood from their hosts to complete their life cycles. Exposure of the alimentary canal of these vectors to the host immune effectors necessitates efficient counteractive measures by hematophagous vectors. The Anopheles mosquito transmitting the malaria parasite is an example of hematophagous vectors that within seconds can ingest human blood double its weight. The innate immune defense mechanisms, like the complement system, in the human blood should thereby immediately react against foreign cells in the mosquito midgut. A prerequisite for complement activation is that the target cells lack complement regulators on their surfaces. In this work, we analyzed whether human complement is active in the mosquito midgut, and how the mosquito midgut cells protect themselves against complement attack. We found that complement remained active for a considerable time and was able to kill microbes within the mosquito midgut. However, the Anopheles mosquito midgut cells were not injured. These cells were found to protect themselves by capturing factor H, the main soluble inhibitor of the alternative complement pathway. Factor H inhibited complement on the midgut cells by promoting inactivation of C3b to iC3b and preventing the activity of the alternative pathway amplification C3 convertase enzyme. An interference of the FH regulatory activity by monoclonal antibodies, carried to the midgut via blood, resulted in increased mosquito mortality and reduced fecundity. By using a ligand blotting assay, a putative mosquito midgut FH receptor could be detected. Thereby, we have identified a novel mechanism whereby mosquitoes can tolerate human blood. PMID:25679788

Anopheles midgut epithelium evades human complement activity by capturing factor H from the blood meal.

Directory of Open Access Journals (Sweden)

Ayman Khattab

2015-02-01

Full Text Available Hematophagous vectors strictly require ingesting blood from their hosts to complete their life cycles. Exposure of the alimentary canal of these vectors to the host immune effectors necessitates efficient counteractive measures by hematophagous vectors. The Anopheles mosquito transmitting the malaria parasite is an example of hematophagous vectors that within seconds can ingest human blood double its weight. The innate immune defense mechanisms, like the complement system, in the human blood should thereby immediately react against foreign cells in the mosquito midgut. A prerequisite for complement activation is that the target cells lack complement regulators on their surfaces. In this work, we analyzed whether human complement is active in the mosquito midgut, and how the mosquito midgut cells protect themselves against complement attack. We found that complement remained active for a considerable time and was able to kill microbes within the mosquito midgut. However, the Anopheles mosquito midgut cells were not injured. These cells were found to protect themselves by capturing factor H, the main soluble inhibitor of the alternative complement pathway. Factor H inhibited complement on the midgut cells by promoting inactivation of C3b to iC3b and preventing the activity of the alternative pathway amplification C3 convertase enzyme. An interference of the FH regulatory activity by monoclonal antibodies, carried to the midgut via blood, resulted in increased mosquito mortality and reduced fecundity. By using a ligand blotting assay, a putative mosquito midgut FH receptor could be detected. Thereby, we have identified a novel mechanism whereby mosquitoes can tolerate human blood.

Age-related macular degeneration and modification of systemic complement factor H production through liver transplantation.

Science.gov (United States)

Khandhadia, Samir; Hakobyan, Svetlana; Heng, Ling Z; Gibson, Jane; Adams, David H; Alexander, Graeme J; Gibson, Jonathan M; Martin, Keith R; Menon, Geeta; Nash, Kathryn; Sivaprasad, Sobha; Ennis, Sarah; Cree, Angela J; Morgan, B Paul; Lotery, Andrew J

2013-08-01

To investigate whether modification of liver complement factor H (CFH) production, by alteration of liver CFH Y402H genotype through liver transplantation (LT), influences the development of age-related macular degeneration (AMD). Multicenter, cross-sectional study. We recruited 223 Western European patients ≥ 55 years old who had undergone LT ≥ 5 years previously. We determined AMD status using a standard grading system. Recipient CFH Y402H genotype was obtained from DNA extracted from recipient blood samples. Donor CFH Y402H genotype was inferred from recipient plasma CFH Y402H protein allotype, measured using enzyme-linked immunosorbent assays. This approach was verified by genotyping donor tissue from a subgroup of patients. Systemic complement activity was ascertained by measuring levels of plasma complement proteins using an enzyme-linked immunosorbent assay, including substrates (C3, C4), activation products (C3a, C4a, and terminal complement complex), and regulators (total CFH, C1 inhibitor). We evaluated AMD status and recipient and donor CFH Y402H genotype. In LT patients, AMD was associated with recipient CFH Y402H genotype (P = 0.036; odds ratio [OR], 1.6; 95% confidence interval [CI], 1.0-2.4) but not with donor CFH Y402H genotype (P = 0.626), after controlling for age, sex, smoking status, and body mass index. Recipient plasma CFH Y402H protein allotype predicted donor CFH Y402H genotype with 100% accuracy (n = 49). Plasma complement protein or activation product levels were similar in LT patients with and without AMD. Compared with previously reported prevalence figures (Rotterdam Study), LT patients demonstrated a high prevalence of both AMD (64.6% vs 37.1%; OR, 3.09; Pproduction. In addition, AMD is not associated with systemic complement activity in LT patients. These findings suggest that local intraocular complement activity is of greater importance in AMD pathogenesis. The high AMD prevalence observed in LT patients may be associated with

Streptococcus pneumoniae PspC Subgroup Prevalence in Invasive Disease and Differences in Contribution to Complement Evasion.

Science.gov (United States)

van der Maten, Erika; van den Broek, Bryan; de Jonge, Marien I; Rensen, Kim J W; Eleveld, Marc J; Zomer, Aldert L; Cremers, Amelieke J H; Ferwerda, Gerben; de Groot, Ronald; Langereis, Jeroen D; van der Flier, Michiel

2018-04-01

The pneumococcal capsular serotype is an important determinant of complement resistance and invasive disease potential, but other virulence factors have also been found to contribute. Pneumococcal surface protein C (PspC), a highly variable virulence protein that binds complement factor H to evade C3 opsonization, is divided into two subgroups: choline-bound subgroup I and LPxTG-anchored subgroup II. The prevalence of different PspC subgroups in invasive pneumococcal disease (IPD) and functional differences in complement evasion are unknown. The prevalence of PspC subgroups in IPD isolates was determined in a collection of 349 sequenced strains of Streptococcus pneumoniae isolated from adult patients. pspC deletion mutants and isogenic pspC switch mutants were constructed to study differences in factor H binding and complement evasion in relation to capsule thickness. Subgroup I pspC was far more prevalent in IPD isolates than subgroup II pspC The presence of capsule was associated with a greater ability of bound factor H to reduce complement opsonization. Pneumococcal subgroup I PspC bound significantly more factor H and showed more effective complement evasion than subgroup II PspC in isogenic encapsulated pneumococci. We conclude that variation in the PspC subgroups, independent of capsule serotypes, affects pneumococcal factor H binding and its ability to evade complement deposition. Copyright © 2018 American Society for Microbiology.

Human antibodies fix complement to inhibit Plasmodium falciparum invasion of erythrocytes and are associated with protection against malaria.

Science.gov (United States)

Boyle, Michelle J; Reiling, Linda; Feng, Gaoqian; Langer, Christine; Osier, Faith H; Aspeling-Jones, Harvey; Cheng, Yik Sheng; Stubbs, Janine; Tetteh, Kevin K A; Conway, David J; McCarthy, James S; Muller, Ivo; Marsh, Kevin; Anders, Robin F; Beeson, James G

2015-03-17

Antibodies play major roles in immunity to malaria; however, a limited understanding of mechanisms mediating protection is a major barrier to vaccine development. We have demonstrated that acquired human anti-malarial antibodies promote complement deposition on the merozoite to mediate inhibition of erythrocyte invasion through C1q fixation and activation of the classical complement pathway. Antibody-mediated complement-dependent (Ab-C') inhibition was the predominant invasion-inhibitory activity of human antibodies; most antibodies were non-inhibitory without complement. Inhibitory activity was mediated predominately via C1q fixation, and merozoite surface proteins 1 and 2 were identified as major targets. Complement fixation by antibodies was very strongly associated with protection from both clinical malaria and high-density parasitemia in a prospective longitudinal study of children. Ab-C' inhibitory activity could be induced by human immunization with a candidate merozoite surface-protein vaccine. Our findings demonstrate that human anti-malarial antibodies have evolved to function by fixing complement for potent invasion-inhibitory activity and protective immunity. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

The Contribution of Serine 194 Phosphorylation to Steroidogenic Acute Regulatory Protein Function

OpenAIRE

Sasaki, Goro; Zubair, Mohamad; Ishii, Tomohiro; Mitsui, Toshikatsu; Hasegawa, Tomonobu; Auchus, Richard J.

2014-01-01

The steroidogenic acute regulatory protein (StAR) facilitates the delivery of cholesterol to the inner mitochondrial membrane, where the cholesterol side-chain cleavage enzyme catalyzes the initial step of steroid hormone biosynthesis. StAR was initially identified in adrenocortical cells as a phosphoprotein, the expression and phosphorylation of which were stimulated by corticotropin. A number of in vitro studies have implicated cAMP-dependent phosphorylation at serine 194 (S194, S195 in hum...

The Complement System: A Prey of Trypanosoma cruzi

Directory of Open Access Journals (Sweden)

Kárita C. F. Lidani

2017-04-01

Full Text Available Trypanosoma cruzi is a protozoan parasite known to cause Chagas disease (CD, a neglected sickness that affects around 6–8 million people worldwide. Originally, CD was mainly found in Latin America but more recently, it has been spread to countries in North America, Asia, and Europe due the international migration from endemic areas. Thus, at present CD represents an important concern of global public health. Most of individuals that are infected by T. cruzi may remain in asymptomatic form all lifelong, but up to 40% of them will develop cardiomyopathy, digestive mega syndromes, or both. The interaction between the T. cruzi infective forms and host-related immune factors represents a key point for a better understanding of the physiopathology of CD. In this context, the complement, as one of the first line of host defense against infection was shown to play an important role in recognizing T. cruzi metacyclic trypomastigotes and in controlling parasite invasion. The complement consists of at least 35 or more plasma proteins and cell surface receptors/regulators, which can be activated by three pathways: classical (CP, lectin (LP, and alternative (AP. The CP and LP are mainly initiated by immune complexes or pathogen-associated molecular patterns (PAMPs, respectively, whereas AP is spontaneously activated by hydrolysis of C3. Once activated, several relevant complement functions are generated which include opsonization and phagocytosis of particles or microorganisms and cell lysis. An important step during T. cruzi infection is when intracellular trypomastigotes are release to bloodstream where they may be target by complement. Nevertheless, the parasite uses a sequence of events in order to escape from complement-mediated lysis. In fact, several T. cruzi molecules are known to interfere in the initiation of all three pathways and in the assembly of C3 convertase, a key step in the activation of complement. Moreover, T. cruzi promotes secretion

Electroluminescent TCC, C3dg and fB/Bb epitope assays for profiling complement cascade activation in vitro using an activated complement serum calibration standard.

Science.gov (United States)

van Vuuren, B Jansen; Bergseth, G; Mollnes, T E; Shaw, A M

2014-01-15

Electroluminescent assays for epitopes on the complement components C3dg, terminal complement complex (TCC) and factor B/Bb (fB/Bb) have been developed with capture and detection antibodies to produce detection limits C3dg=91±9ng/mL, TCC=3±0.1ng/mL and fB=55.7±0.1ng/mL. The assay performance was assessed against a series of zymosan and heat aggregated IgG (HAIgG) in vitro activations of complement using a calibrated activated complement serum (ACS) as calibration standard. The ACS standard was stable within 20% accuracy over a 6-month period with freeze-thaw cycles as required. Differential activation of the complement cascade was observed for TCC showing a pseudo-first order formation half-life of 3.5h after activation with zymosan. The C3dg activation fragment indicates a 10% total activation for both activation agents. The kinetic-epitope analysis for fB indicates that the capture epitope is on the fB/Bb protein fragment which can then become covered by the formation of C3bBb or C3bBbP complexes during the time course of the cascade. Copyright © 2013 Elsevier B.V. All rights reserved.

Feeding common carp Cyprinus carpio with β-glucan supplemented diet stimulates C-reactive protein and complement immune acute phase responses following PAMPs injection.

Science.gov (United States)

Pionnier, Nicolas; Falco, Alberto; Miest, Joanna J; Shrive, Annette K; Hoole, Dave

2014-08-01

The effect of β-glucan as a feed additive on the serum and gene profile of C-reactive protein (CRP) and complement acute phase responses was ascertained in common carp Cyprinus carpio. In addition effects of subsequent intraperitoneal injections of pathogen-associated molecular patterns (PAMPs), i.e. LPS or poly(I:C), to mimic bacterial or viral infection respectively, were studied. Carp were first orally fed with β-glucan (MacroGard®) with a daily β-glucan intake of 6 mg per kg body weight or with control food for 25 days and then injected with PBS containing either LPS (4 mg/kg) or poly(I:C) (5 mg/kg) or PBS alone. Fish were sampled during the 25 days of the feeding period and up to 7 days post-PAMPs injections for serum and liver, head kidney and mid-gut tissues. Oral administration of β-glucan for 25 days significantly increased serum CRP levels and alternative complement activity (ACP). In addition, the subsequent LPS and poly(I:C) challenges significantly affected CRP and complement related gene expression profiles (crp1, crp2, c1r/s, bf/c2, c3 and masp2), with the greatest effects observed in the β-glucan fed fish. However, in fish fed β-glucan the PAMPs injections had less effects on CRP levels and complement activity in the serum than in control fed fish, suggesting that the 25 days of β-glucan immunostimulation was sufficient enough to reduce the effects of LPS and poly(I:C) injections. Results suggest that MacroGard® stimulated CRP and complement responses to PAMPs immunological challenges in common carp thus highlighting the beneficial β-glucan immunostimulant properties. Copyright © 2014 Elsevier Ltd. All rights reserved.

Protection by meningococcal outer membrane protein PorA-specific antibodies and a serogroup B capsular polysaccharide-specific antibody in complement-sufficient and C6-deficient infant rats.

Science.gov (United States)

Toropainen, Maija; Saarinen, Leena; Vidarsson, Gestur; Käyhty, Helena

2006-05-01

The relative contributions of antibody-induced complement-mediated bacterial lysis and antibody/complement-mediated phagocytosis to host immunity against meningococcal infections are currently unclear. Further, the in vivo effector functions of antibodies may vary depending on their specificity and Fc heavy-chain isotype. In this study, a mouse immunoglobulin G2a (mIgG2a) monoclonal antibody (MN12H2) to meningococcal outer membrane protein PorA (P1.16), its human IgG subclass derivatives (hIgG1 to hIgG4), and an mIgG2a monoclonal antibody (Nmb735) to serogroup B capsular polysaccharide (B-PS) were evaluated for passive protection against meningococcal serogroup B strain 44/76-SL (B:15:P1.7,16) in an infant rat infection model. Complement component C6-deficient (PVG/c-) rats were used to assess the importance of complement-mediated bacterial lysis for protection. The PorA-specific parental mIgG2a and the hIgG1 to hIgG3 derivatives all induced efficient bactericidal activity in vitro in the presence of human or infant rat complement and augmented bacterial clearance in complement-sufficient HsdBrlHan:WIST rats, while the hIgG4 was unable to do so. In C6-deficient PVG/c- rats, lacking complement-mediated bacterial lysis, the augmentation of bacterial clearance by PorA-specific mIgG2a and hIgG1 antibodies was impaired compared to that in the syngeneic complement-sufficient PVG/c+ rat strain. This was in contrast to the case for B-PS-specific mIgG2a, which conferred similar protective activity in both rat strains. These data suggest that while anti-B-PS antibody can provide protection in the infant rats without membrane attack complex formation, the protection afforded by anti-PorA antibody is more dependent on the activation of the whole complement pathway and subsequent bacterial lysis.

Characterization of a Gene Coding for the Complement System Component FB from Loxosceles laeta Spider Venom Glands.

Science.gov (United States)

Myamoto, Daniela Tiemi; Pidde-Queiroz, Giselle; Gonçalves-de-Andrade, Rute Maria; Pedroso, Aurélio; van den Berg, Carmen W; Tambourgi, Denise V

2016-01-01

The human complement system is composed of more than 30 proteins and many of these have conserved domains that allow tracing the phylogenetic evolution. The complement system seems to be initiated with the appearance of C3 and factor B (FB), the only components found in some protostomes and cnidarians, suggesting that the alternative pathway is the most ancient. Here, we present the characterization of an arachnid homologue of the human complement component FB from the spider Loxosceles laeta. This homologue, named Lox-FB, was identified from a total RNA L. laeta spider venom gland library and was amplified using RACE-PCR techniques and specific primers. Analysis of the deduced amino acid sequence and the domain structure showed significant similarity to the vertebrate and invertebrate FB/C2 family proteins. Lox-FB has a classical domain organization composed of a control complement protein domain (CCP), a von Willebrand Factor domain (vWFA), and a serine protease domain (SP). The amino acids involved in Mg2+ metal ion dependent adhesion site (MIDAS) found in the vWFA domain in the vertebrate C2/FB proteins are well conserved; however, the classic catalytic triad present in the serine protease domain is not conserved in Lox-FB. Similarity and phylogenetic analyses indicated that Lox-FB shares a major identity (43%) and has a close evolutionary relationship with the third isoform of FB-like protein (FB-3) from the jumping spider Hasarius adansoni belonging to the Family Salcitidae.

Characterization of a Gene Coding for the Complement System Component FB from Loxosceles laeta Spider Venom Glands.

Directory of Open Access Journals (Sweden)

Daniela Tiemi Myamoto

Full Text Available The human complement system is composed of more than 30 proteins and many of these have conserved domains that allow tracing the phylogenetic evolution. The complement system seems to be initiated with the appearance of C3 and factor B (FB, the only components found in some protostomes and cnidarians, suggesting that the alternative pathway is the most ancient. Here, we present the characterization of an arachnid homologue of the human complement component FB from the spider Loxosceles laeta. This homologue, named Lox-FB, was identified from a total RNA L. laeta spider venom gland library and was amplified using RACE-PCR techniques and specific primers. Analysis of the deduced amino acid sequence and the domain structure showed significant similarity to the vertebrate and invertebrate FB/C2 family proteins. Lox-FB has a classical domain organization composed of a control complement protein domain (CCP, a von Willebrand Factor domain (vWFA, and a serine protease domain (SP. The amino acids involved in Mg2+ metal ion dependent adhesion site (MIDAS found in the vWFA domain in the vertebrate C2/FB proteins are well conserved; however, the classic catalytic triad present in the serine protease domain is not conserved in Lox-FB. Similarity and phylogenetic analyses indicated that Lox-FB shares a major identity (43% and has a close evolutionary relationship with the third isoform of FB-like protein (FB-3 from the jumping spider Hasarius adansoni belonging to the Family Salcitidae.

Expression of complement and pentraxin proteins in acute phase response elicited by tumor photodynamic therapy: the engagement of adrenal hormones.

Science.gov (United States)

Merchant, Soroush; Huang, Naiyan; Korbelik, Mladen

2010-12-01

Treatment of solid tumors by photodynamic therapy (PDT) was recently shown to trigger a strong acute phase response. Using the mouse Lewis lung carcinoma (LLC) model, the present study examined complement and pentraxin proteins as PDT-induced acute phase reactants. The results show a distinct pattern of changes in the expression of genes encoding these proteins in the tumor, as well as host liver and spleen, following PDT mediated by photosensitizer Photofrin™. These changes were influenced by glucocorticoid hormones, as evidenced by transcriptional activation of glucocorticoid receptor and the upregulation of gene encoding this receptor. The expression of gene for glucocorticoid-induced zipper (GILZ) protein, whose activity is particularly susceptible to glucocorticoid regulation, was also changed in PDT-treated tumors. A direct demonstration that tumor PDT induces glucocorticoid hormone upregulation is provided by documenting elevated levels of serum corticosterone in mice bearing PDT-treated LLC tumors. Tumor response to PDT was negatively affected by blocking glucocorticoid receptor activity, which suggests that glucocorticoid hormones have a positive impact on the therapeutic outcome with this therapy. Copyright © 2010 Elsevier B.V. All rights reserved.

Nickel decreases cellular iron level and converts cytosolic aconitase to iron-regulatory protein 1 in A549 cells

International Nuclear Information System (INIS)

Chen Haobin; Davidson, Todd; Singleton, Steven; Garrick, Michael D.; Costa, Max

2005-01-01

Nickel (Ni) compounds are well-established carcinogens and are known to initiate a hypoxic response in cells via the stabilization and transactivation of hypoxia-inducible factor-1 alpha (HIF-1α). This change may be the consequence of nickel's interference with the function of several Fe(II)-dependent enzymes. In this study, the effects of soluble nickel exposure on cellular iron homeostasis were investigated. Nickel treatment decreased both mitochondrial and cytosolic aconitase (c-aconitase) activity in A549 cells. Cytosolic aconitase was converted to iron-regulatory protein 1, a form critical for the regulation of cellular iron homeostasis. The increased activity of iron-regulatory protein 1 after nickel exposure stabilized and increased transferrin receptor (Tfr) mRNA and antagonized the iron-induced ferritin light chain protein synthesis. The decrease of aconitase activity after nickel treatment reflected neither direct interference with aconitase function nor obstruction of [4Fe-4S] cluster reconstitution by nickel. Exposure of A549 cells to soluble nickel decreased total cellular iron by about 40%, a decrease that likely caused the observed decrease in aconitase activity and the increase of iron-regulatory protein 1 activity. Iron treatment reversed the effect of nickel on cytosolic aconitase and iron-regulatory protein 1. To assess the mechanism for the observed effects, human embryonic kidney (HEK) cells over expressing divalent metal transporter-1 (DMT1) were compared to A549 cells expressing only endogenous transporters for inhibition of iron uptake by nickel. The inhibition data suggest that nickel can enter via DMT1 and compete with iron for entry into the cell. This disturbance of cellular iron homeostasis by nickel may have a great impact on the ability of the cell to regulate a variety of cell functions, as well as create a state of hypoxia in cells under normal oxygen tension. These effects may be very important in how nickel exerts phenotypic

Characterization and expression analysis of a complement component gene in sea cucumber ( Apostichopus japonicus)

Science.gov (United States)

Chen, Zhong; Zhou, Zunchun; Yang, Aifu; Dong, Ying; Guan, Xiaoyan; Jiang, Bei; Wang, Bai

2015-12-01

The complement system plays a crucial role in the innate immune system of animals. It can be activated by distinct yet overlapping classical, alternative and lectin pathways. In the alternative pathway, complement factor B (Bf) serves as the catalytic subunit of complement component 3 (C3) convertase, which plays the central role among three activation pathways. In this study, the Bf gene in sea cucumber ( Apostichopus japonicus), termed AjBf, was obtained by rapid amplification of cDNA ends (RACE). The full-length cDNA of AjBf was 3231 bp in length barring the poly (A) tail. It contained an open reading frame (ORF) of 2742 bp encoding 913 amino acids, a 105 bp 5'-UTR (5'-terminal untranslated region) and a 384 bp 3'-UTR. AjBf was a mosaic protein with six CCP (complement control protein) domains, a VWA (von Willebrand factor A) domain, and a serine protease domain. The deduced molecular weight of AjBf protein was 101 kDa. Quantitative real time PCR (qRT-PCR) analysis indicated that the expression level of AjBf in A. japonicus was obviously higher at larval stage than that at embryonic stage. Expression detection in different tissues showed that AjBf expressed higher in coelomocytes than in other four tissues. In addation, AjBf expression in different tissues was induced significantly after LPS or PolyI:C challenge. These results indicated that AjBf plays an important role in immune responses to pathogen infection.

Vaccinia complement control protein: Multi-functional protein and a ...

Indian Academy of Sciences (India)

Unknown

naturally occurring antagonist of the proinflammatory cytokine IL-18. Another strategy used by ... receptors or binding proteins for tumour necrosis factor. (TNF) ... immune regulators, such as the viral IL-10 and vascular endothelial growth factor ...

Protein- protein interaction detection system using fluorescent protein microdomains

Science.gov (United States)

Waldo, Geoffrey S.; Cabantous, Stephanie

2010-02-23

The invention provides a protein labeling and interaction detection system based on engineered fragments of fluorescent and chromophoric proteins that require fused interacting polypeptides to drive the association of the fragments, and further are soluble and stable, and do not change the solubility of polypeptides to which they are fused. In one embodiment, a test protein X is fused to a sixteen amino acid fragment of GFP (.beta.-strand 10, amino acids 198-214), engineered to not perturb fusion protein solubility. A second test protein Y is fused to a sixteen amino acid fragment of GFP (.beta.-strand 11, amino acids 215-230), engineered to not perturb fusion protein solubility. When X and Y interact, they bring the GFP strands into proximity, and are detected by complementation with a third GFP fragment consisting of GFP amino acids 1-198 (strands 1-9). When GFP strands 10 and 11 are held together by interaction of protein X and Y, they spontaneous association with GFP strands 1-9, resulting in structural complementation, folding, and concomitant GFP fluorescence.

Complement is activated in progressive multiple sclerosis cortical grey matter lesions.

Science.gov (United States)

Watkins, Lewis M; Neal, James W; Loveless, Sam; Michailidou, Iliana; Ramaglia, Valeria; Rees, Mark I; Reynolds, Richard; Robertson, Neil P; Morgan, B Paul; Howell, Owain W

2016-06-22

The symptoms of multiple sclerosis (MS) are caused by damage to myelin and nerve cells in the brain and spinal cord. Inflammation is tightly linked with neurodegeneration, and it is the accumulation of neurodegeneration that underlies increasing neurological disability in progressive MS. Determining pathological mechanisms at play in MS grey matter is therefore a key to our understanding of disease progression. We analysed complement expression and activation by immunocytochemistry and in situ hybridisation in frozen or formalin-fixed paraffin-embedded post-mortem tissue blocks from 22 progressive MS cases and made comparisons to inflammatory central nervous system disease and non-neurological disease controls. Expression of the transcript for C1qA was noted in neurons and the activation fragment and opsonin C3b-labelled neurons and glia in the MS cortical and deep grey matter. The density of immunostained cells positive for the classical complement pathway protein C1q and the alternative complement pathway activation fragment Bb was significantly increased in cortical grey matter lesions in comparison to control grey matter. The number of cells immunostained for the membrane attack complex was elevated in cortical lesions, indicating complement activation to completion. The numbers of classical (C1-inhibitor) and alternative (factor H) pathway regulator-positive cells were unchanged between MS and controls, whilst complement anaphylatoxin receptor-bearing microglia in the MS cortex were found closely apposed to cortical neurons. Complement immunopositive neurons displayed an altered nuclear morphology, indicative of cell stress/damage, supporting our finding of significant neurodegeneration in cortical grey matter lesions. Complement is activated in the MS cortical grey matter lesions in areas of elevated numbers of complement receptor-positive microglia and suggests that complement over-activation may contribute to the worsening pathology that underlies the

Evolution of context dependent regulation by expansion of feast/famine regulatory proteins.

Science.gov (United States)

Plaisier, Christopher L; Lo, Fang-Yin; Ashworth, Justin; Brooks, Aaron N; Beer, Karlyn D; Kaur, Amardeep; Pan, Min; Reiss, David J; Facciotti, Marc T; Baliga, Nitin S

2014-11-14

Expansion of transcription factors is believed to have played a crucial role in evolution of all organisms by enabling them to deal with dynamic environments and colonize new environments. We investigated how the expansion of the Feast/Famine Regulatory Protein (FFRP) or Lrp-like proteins into an eight-member family in Halobacterium salinarum NRC-1 has aided in niche-adaptation of this archaeon to a complex and dynamically changing hypersaline environment. We mapped genome-wide binding locations for all eight FFRPs, investigated their preference for binding different effector molecules, and identified the contexts in which they act by analyzing transcriptional responses across 35 growth conditions that mimic different environmental and nutritional conditions this organism is likely to encounter in the wild. Integrative analysis of these data constructed an FFRP regulatory network with conditionally active states that reveal how interrelated variations in DNA-binding domains, effector-molecule preferences, and binding sites in target gene promoters have tuned the functions of each FFRP to the environments in which they act. We demonstrate how conditional regulation of similar genes by two FFRPs, AsnC (an activator) and VNG1237C (a repressor), have striking environment-specific fitness consequences for oxidative stress management and growth, respectively. This study provides a systems perspective into the evolutionary process by which gene duplication within a transcription factor family contributes to environment-specific adaptation of an organism.

[Construction of enterohemorrhagic Escherichia coli O157:H7 strains with espF gene deletion and complementation].

Science.gov (United States)

Hua, Ying; Sun, Qi; Wang, Xiangyu; DU, Yanli; Shao, Na; Zhang, Qiwei; Zhao, Wei; Wan, Chengsong

2015-11-01

To construct enterohemorrhagic Escherichia coli (EHEC) O157:H7 strains with delection espF gene and its nucleotide fragment and with espF gene complementation. A pair of homologous arm primers was designed to amplify the gene fragment of kanamycin resistance, which was transformed into EHEC O157:H7 EDL933w strain via the PKD46 plasmid by electroporation. The replacement of the espF gene by kanamycin resistance gene through the PKD46-mediated red recombination system was confirmed by PCR and sequencing. The entire coding region of espF along with its nucleotide fragment was amplified by PCR and cloned into pBAD33 plasmid, which was transformed into a mutant strain to construct the strain with espF complementation. RT-PCR was used to verify the transcription of espF and its nucleotide fragment in the complemented mutant strain. We established EHEC O157:H7 EDL933w strains with espF gene deletion and with espF gene complementation. Both espF and its nucleotide fragment were transcribed in the complemented mutant strain. The two strains provide a basis for further study of the regulatory mechanism of espF.

Inhibitors Alter the Stochasticity of Regulatory Proteins to Force Cells to Switch to the Other State in the Bistable System.

Science.gov (United States)

Jhang, Wun-Sin; Lo, Shih-Chiang; Yeh, Chen-Chao; Shu, Che-Chi

2017-06-30

The cellular behaviors under the control of genetic circuits are subject to stochastic fluctuations, or noise. The stochasticity in gene regulation, far from a nuisance, has been gradually appreciated for its unusual function in cellular activities. In this work, with Chemical Master Equation (CME), we discovered that the addition of inhibitors altered the stochasticity of regulatory proteins. For a bistable system of a mutually inhibitory network, such a change of noise led to the migration of cells in the bimodal distribution. We proposed that the consumption of regulatory protein caused by the addition of inhibitor is not the only reason for pushing cells to the specific state; the change of the intracellular stochasticity is also the main cause for the redistribution. For the level of the inhibitor capable of driving 99% of cells, if there is no consumption of regulatory protein, 88% of cells were guided to the specific state. It implied that cells were pushed, by the inhibitor, to the specific state due to the change of stochasticity.

Ca2+-regulatory proteins in cardiomyocytes from the right ventricle in children with congenital heart disease

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Wu Yihe

2012-04-01

Full Text Available Abstract Background Hypoxia and hypertrophy are the most frequent pathophysiological consequence of congenital heart disease (CHD which can induce the alteration of Ca2+-regulatory proteins and inhibit cardiac contractility. Few studies have been performed to examine Ca2+-regulatory proteins in human cardiomyocytes from the hypertrophic right ventricle with or without hypoxia. Methods Right ventricle tissues were collected from children with tetralogy of Fallot [n = 25, hypoxia and hypertrophy group (HH group], pulmonary stenosis [n = 25, hypertrophy group (H group], or small isolated ventricular septal defect [n = 25, control group (C group] during open-heart surgery. Paraffin sections of tissues were stained with 3,3′-dioctadecyloxacarbocyanine perchlorate to measure cardiomyocyte size. Expression levels of Ca2+-regulatory proteins [sarcoplasmic reticulum Ca2+-ATPase (SERCA2a, ryanodine receptor (RyR2, sodiumcalcium exchanger (NCX, sarcolipin (SLN and phospholamban (PLN] were analysed by means of real-time PCR, western blot, or immunofluorescence. Additionally, phosphorylation level of RyR and PLN and activity of protein phosphatase (PP1 were evaluated using western blot. Results Mild cardiomyocyte hypertrophy of the right ventricle in H and HH groups was confirmed by comparing cardiomyocyte size. A significant reduction of SERCA2a in mRNA (P16-phosphorylated PLN was down-regulated (PP Conclusions The decreased SERCA2a mRNA may be a biomarker of the pathological process in the early stage of cyanotic CHD with the hypertrophic right ventricle. A combination of hypoxia and hypertrophy can induce the adverse effect of PLN-Ser16 dephosphorylation. Increased PP1 could result in the decreased PLN-Ser16 and inhibition of PP1 is a potential therapeutic target for heart dysfunction in pediatrics.

UV irradiation analysis of complementation between, and replication of, RNA-negative temperature-sensitivie mutants of Newcastle disease virus

International Nuclear Information System (INIS)

Peeples, M.E.; Bratt, M.A.

1982-01-01

Random uv irradiation-induced lesions destroy the infectivity of Newcastle disease virus (NDV) by blocking downstream transcription from the single viral promoter. The nucleocapsid-associated polypeptides most likely to be involved in RNA synthesis are located at the extreme ends of the genome: NP and P are promoter proximal genes, and L is the most distal gene. We attempted to order the two temperature-sensitive (ts) RNA-negative (RNA-) mutant groups of NDV by determining the uv target sizes for the complementing abilities of mutants A1 and E1. After uv irradiation, E1 was unable to complement A1, a result compatible with the A mutation lying in the L gene. In contrast, after uv irradiation A1 was able to complement E1 for both virus production and viral protein synthesis, with a target size most consistent with the E mutation lying in the P gene. UV-irradiated virus was unable to replicate as indicated by its absence in the yields of multiply infected cells, either as infectious virus or as particles with complementing activity. After irradiation, ts mutant B1ΔP, with a non-ts mutation affecting the electrophoretic mobility of the P protein, complemented E1 in a manner similar to A1, but it did not amplify the expression of ΔP in infected cells. This too is consistent with irradiated virus being unable to replicate despite the presence of the components needed for replication of E1. At high uv doses, A1 was able to complement E1 in a different, uv-resistant manner, probably by direct donation of input polypeptides. Multiplicity reactivation has previously been observed at high-multiplicity infection by uv-irradiated paramyxoviruses. In this case, virions which are noninfectious because they lack a protein component may be activated by a protein from irradiated virions

Pro-protein convertases control the maturation and processing of the iron-regulatory protein, RGMc/hemojuvelin

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Rotwein Peter

2008-04-01

Full Text Available Abstract Background Repulsive guidance molecule c (RGMc or hemojuvelin, a glycosylphosphatidylinositol-linked glycoprotein expressed in liver and striated muscle, plays a central role in systemic iron balance. Inactivating mutations in the RGMc gene cause juvenile hemochromatosis (JH, a rapidly progressing iron storage disorder with severe systemic manifestations. RGMc undergoes complex biosynthetic steps leading to membrane-bound and soluble forms of the protein, including both 50 and 40 kDa single-chain species. Results We now show that pro-protein convertases (PC are responsible for conversion of 50 kDa RGMc to a 40 kDa protein with a truncated COOH-terminus. Unlike related molecules RGMa and RGMb, RGMc encodes a conserved PC recognition and cleavage site, and JH-associated RGMc frame-shift mutants undergo COOH-terminal cleavage only if this site is present. A cell-impermeable peptide PC inhibitor blocks the appearance of 40 kDa RGMc in extra-cellular fluid, as does an engineered mutation in the conserved PC recognition sequence, while the PC furin cleaves 50 kDa RGMc in vitro into a 40 kDa molecule with an intact NH2-terminus. Iron loading reduces release of RGMc from the cell membrane, and diminishes accumulation of the 40 kDa species in cell culture medium. Conclusion Our results define a role for PCs in the maturation of RGMc that may have implications for the physiological actions of this critical iron-regulatory protein.

Increased complement C1q level marks active disease in human tuberculosis.

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Yi Cai

Full Text Available BACKGROUND: Complement functions as an important host defense system and complement C5 and C7 have been implicated in immunopathology of tuberculosis. However, little is known about the role of other complement components in tuberculosis. METHODS: Complement gene expression in peripheral blood mononuclear cells of tuberculosis patients and controls were determined using whole genome transcriptional microarray assays. The mRNA and protein levels of three C1q components, C1qA, C1qB, and C1qC, were further validated by qRT-PCR and enzyme-linked immunosorbent assay, respectively. The percentages of C1q expression in CD14 positive cells were determined by flow cytometry. Finally, C1qC protein level was quantified in the pleural fluid of tuberculosis and non-tuberculosis pleurisy. RESULTS: C1q expression increases significantly in the peripheral blood of patients with active tuberculosis compared to healthy controls and individuals with latent TB infection. The percentage of C1q-expressing CD14 positive cells is significantly increased in active TB patients. C1q expression in the peripheral blood correlates with sputum smear positivity in tuberculosis patients and is reduced after anti-tuberculosis chemotherapy. Notably, receiver operating characteristic analysis showed that C1qC mRNA levels in peripheral blood efficiently discriminate active from latent tuberculosis infection and healthy controls. Additionally, C1qC protein level in pleural effusion shows improved power in discriminating tuberculosis from non-tuberculosis pleurisy when compared to other inflammatory markers, such as IL-6 and TNF-α. CONCLUSIONS: C1q expression correlates with active disease in human tuberculosis. C1q could be a potential diagnostic marker to discriminate active tuberculosis from latent tuberculosis infection as well as tuberculosis pleurisy from non-tuberculosis pleurisy.

Complementing the sugar code: role of GAGs and sialic acid in complement regulation

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Alex eLangford-Smith

2015-02-01

Full Text Available Sugar molecules play a vital role on both microbial and mammalian cells, where they are involved in cellular communication, govern microbial virulence and modulate host immunity and inflammatory responses. The complement cascade, as part of a host’s innate immune system, is a potent weapon against invading bacteria but has to be tightly regulated to prevent inappropriate attack and damage to host tissues. A number of complement regulators, such as factor H and properdin, interact with sugar molecules, such as glycosaminoglycans and sialic acid, on host and pathogen membranes and direct the appropriate complement response by either promoting the binding of complement activators or inhibitors. The binding of these complement regulators to sugar molecules can vary from location to location, due to their different specificities and because distinct structural and functional subpopulations of sugars are found in different human organs, such as the brain, kidney and eye. This review will cover recent studies that have provided important new insights into the role of glycosaminoglycans and sialic acid in complement regulation and how sugar recognition may be compromised in disease

Functional analysis of the putative peroxidase domain of FANCA, the Fanconi anemia complementation group A protein.

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Ren, J; Youssoufian, H

2001-01-01

Fanconi anemia (FA) is an autosomal recessive disorder manifested by chromosomal breakage, birth defects, and susceptibility to bone marrow failure and cancer. At least seven complementation groups have been identified, and the genes defective in four groups have been cloned. The most common subtype is complementation group A. Although the normal functions of the gene products defective in FA cells are not completely understood, a clue to the function of the FA group A gene product (FANCA) was provided by the detection of limited homology in the amino terminal region to a class of heme peroxidases. We evaluated this hypothesis by mutagenesis and functional complementation studies. We substituted alanine residues for the most conserved FANCA residues in the putative peroxidase domain and tested their effects on known biochemical and cellular functions of FANCA. While the substitution mutants were comparable to wild-type FANCA with regard to their stability, subcellular localization, and interaction with FANCG, only the Trp(183)-to-Ala substitution (W183A) abolished the ability of FANCA to complement the sensitivity of FA group A cells to mitomycin C. By contrast, TUNEL assays for apoptosis after exposure to H2O2 showed no differences between parental FA group A cells, cells complemented with wild-type FANCA, and cells complemented with the W183A of FANCA. Moreover, semiquantitative RT-PCR analysis for the expression of the peroxide-sensitive heme oxygenase gene showed appropriate induction after H2O2 exposure. Thus, W183A appears to be essential for the in vivo activity of FANCA in a manner independent of its interaction with FANCG. Moreover, neither wild-type FANCA nor the W183A mutation appears to alter the peroxide-induced apoptosisor peroxide-sensing ability of FA group A cells. Copyright 2001 Academic Press.

Phosphorylation of protein kinase A (PKA) regulatory subunit RIα by protein kinase G (PKG) primes PKA for catalytic activity in cells.

Science.gov (United States)

Haushalter, Kristofer J; Casteel, Darren E; Raffeiner, Andrea; Stefan, Eduard; Patel, Hemal H; Taylor, Susan S

2018-03-23

cAMP-dependent protein kinase (PKAc) is a pivotal signaling protein in eukaryotic cells. PKAc has two well-characterized regulatory subunit proteins, RI and RII (each having α and β isoforms), which keep the PKAc catalytic subunit in a catalytically inactive state until activation by cAMP. Previous reports showed that the RIα regulatory subunit is phosphorylated by cGMP-dependent protein kinase (PKG) in vitro , whereupon phosphorylated RIα no longer inhibits PKAc at normal (1:1) stoichiometric ratios. However, the significance of this phosphorylation as a mechanism for activating type I PKA holoenzymes has not been fully explored, especially in cellular systems. In this study, we further examined the potential of RIα phosphorylation to regulate physiologically relevant "desensitization" of PKAc activity. First, the serine 101 site of RIα was validated as a target of PKGIα phosphorylation both in vitro and in cells. Analysis of a phosphomimetic substitution in RIα (S101E) showed that modification of this site increases PKAc activity in vitro and in cells, even without cAMP stimulation. Numerous techniques were used to show that although Ser 101 variants of RIα can bind PKAc, the modified linker region of the S101E mutant has a significantly reduced affinity for the PKAc active site. These findings suggest that RIα phosphorylation may be a novel mechanism to circumvent the requirement of cAMP stimulus to activate type I PKA in cells. We have thus proposed a model to explain how PKG phosphorylation of RIα creates a "sensitized intermediate" state that is in effect primed to trigger PKAc activity.

Complement Activation in Inflammatory Skin Diseases

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Jenny Giang

2018-04-01

Full Text Available The complement system is a fundamental part of the innate immune system, playing a crucial role in host defense against various pathogens, such as bacteria, viruses, and fungi. Activation of complement results in production of several molecules mediating chemotaxis, opsonization, and mast cell degranulation, which can contribute to the elimination of pathogenic organisms and inflammation. Furthermore, the complement system also has regulating properties in inflammatory and immune responses. Complement activity in diseases is rather complex and may involve both aberrant expression of complement and genetic deficiencies of complement components or regulators. The skin represents an active immune organ with complex interactions between cellular components and various mediators. Complement involvement has been associated with several skin diseases, such as psoriasis, lupus erythematosus, cutaneous vasculitis, urticaria, and bullous dermatoses. Several triggers including auto-antibodies and micro-organisms can activate complement, while on the other hand complement deficiencies can contribute to impaired immune complex clearance, leading to disease. This review provides an overview of the role of complement in inflammatory skin diseases and discusses complement factors as potential new targets for therapeutic intervention.

Acquisition of negative complement regulators by the saprophyte Leptospira biflexa expressing LigA or LigB confers enhanced survival in human serum.

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Castiblanco-Valencia, Mónica M; Fraga, Tatiana R; Breda, Leandro C D; Vasconcellos, Sílvio A; Figueira, Cláudio P; Picardeau, Mathieu; Wunder, Elsio; Ko, Albert I; Barbosa, Angela S; Isaac, Lourdes

2016-05-01

Leptospiral immunoglobulin-like (Lig) proteins are surface exposed molecules present in pathogenic but not in saprophytic Leptospira species. We have previously shown that Lig proteins interact with the soluble complement regulators Factor H (FH), FH like-1 (FHL-1), FH related-1 (FHR-1) and C4b Binding Protein (C4BP). In this study, we used the saprophyte L. biflexa serovar Patoc as a surrogate host to address the specific role of LigA and LigB proteins in leptospiral complement evasion. L. biflexa expressing LigA or LigB was able to acquire FH and C4BP. Bound complement regulators retained their cofactor activities of FI in the proteolytic cleavage of C3b and C4b. Moreover, heterologous expression of ligA and ligB genes in the saprophyte L. biflexa enhanced bacterial survival in human serum. Complement deposition on lig-transformed L. biflexa was assessed by flow cytometry analysis. With regard to MAC deposition, L. biflexa expressing LigA or LigB presented an intermediate profile: MAC deposition levels were greater than those found in the pathogenic L. interrogans, but lower than those observed for L. biflexa wildtype. In conclusion, Lig proteins contribute to in vitro control of complement activation on the leptospiral surface, promoting an increased bacterial survival in human serum. Copyright © 2016 European Federation of Immunological Societies. All rights reserved.

Cefditoren and ceftriaxone enhance complement-mediated immunity in the presence of specific antibodies against antibiotic-resistant pneumococcal strains.

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Elisa Ramos-Sevillano

Full Text Available BACKGROUND: Specific antibodies mediate humoral and cellular protection against invading pathogens such as Streptococcus pneumoniae by activating complement mediated immunity, promoting phagocytosis and stimulating bacterial clearance. The emergence of pneumococcal strains with high levels of antibiotic resistance is of great concern worldwide and a serious threat for public health. METHODOLOGY/PRINCIPAL FINDINGS: Flow cytometry was used to determine whether complement-mediated immunity against three antibiotic-resistant S. pneumoniae clinical isolates is enhanced in the presence of sub-inhibitory concentrations of cefditoren and ceftriaxone. The binding of acute phase proteins such as C-reactive protein and serum amyloid P component, and of complement component C1q, to pneumococci was enhanced in the presence of serum plus either of these antibiotics. Both antibiotics therefore trigger the activation of the classical complement pathway against S. pneumoniae. C3b deposition was also increased in the presence of specific anti-pneumococcal antibodies and sub-inhibitory concentrations of cefditoren and ceftriaxone confirming that the presence of these antibiotics enhances complement-mediated immunity to S. pneumoniae. CONCLUSIONS/SIGNIFICANCE: Using cefditoren and ceftriaxone to promote the binding of acute phase proteins and C1q to pneumococci, and to increase C3b deposition, when anti-pneumococcal antibodies are present, might help reduce the impact of antibiotic resistance in S. pneumoniae infections.

Crystallization and quaternary structure analysis of an Lrp-like regulatory protein from the hyperthermophile Pyrococcus furiosus

NARCIS (Netherlands)

Sedelnikova, S.E.; Smits, S.H.J.; Leonard, P.M.; Brinkman, A.B.; Oost, van der J.; Rafferty, J.B.

2001-01-01

The LrpA transcriptional regulator from Pyrococcus furiosus, a member of the leucine-responsive regulatory protein (Lrp) family, has been crystallized by the hanging-drop method of vapour diffusion using ammonium sulfate as the precipitant. The crystals belong to the tetragonal system and are in

A tick mannose-binding lectin inhibits the vertebrate complement cascade to enhance transmission of the Lyme disease agent

OpenAIRE

Schuijt, Tim J.; Coumou, Jeroen; Narasimhan, Sukanya; Dai, Jianfeng; DePonte, Kathleen; Wouters, Diana; Brouwer, Mieke; Oei, Anneke; Roelofs, Joris J.T.H.; van Dam, Alje P.; van der Poll, Tom; van ’t Veer, Cornelis; Hovius, Joppe W.; Fikrig, Erol

2011-01-01

The Lyme disease agent, Borrelia burgdorferi, is primarily transmitted to vertebrates by Ixodes ticks. The classical and alternative complement pathways are important in Borrelia eradication by the vertebrate host. We recently identified a tick salivary protein, designated P8 that reduced complement-mediated killing of Borrelia. We now discover that P8 interferes with the human lectin complement cascade resulting in impaired neutrophil phagocytosis and chemotaxis, and diminished Borrelia lysi...

Serum immunoglobulin and complement levels in prematures with parenteral feeding--preliminary results.

Science.gov (United States)

Tamaro, G; Morena, C; Uxa, F; Candusso, M; Trappan, A; de Vonderweid, U

1993-01-01

Immunoglobulins IgA, IgG and IgM and complement factors C3 and C4 have been measured in a population of premature infants to evaluate their degree of immunological maturity. All the infants were receiving complete parenteral nutrition. In parallel, the same parameters were measured in twenty two full term, healthy neonates. To explore maturation and liver function, the authors used other proteins as nutritional markers. Differences in the immunoglobulins, but not in the complement fractions were seen between the two groups. Two applications are suggested: incidence of infections and post partum maturation.

Human Complement Receptor Type 1/CD35 Is an Epstein-Barr Virus Receptor

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Javier G. Ogembo

2013-02-01

Full Text Available Epstein-Barr virus (EBV attachment to primary B cells initiates virus entry. Although CD21 is the only known receptor for EBVgp350/220, a recent report documents EBV-infected B cells from a patient genetically deficient in CD21. On normal resting B cells, CD21 forms two membrane complexes: one with CD19 and another with CD35. Whereas the CD21/CD19 complex is widely retained on immortalized and B cell tumor lines, the related complement-regulatory protein CD35 is lost. To determine the role(s of CD35 in initial infection, we transduced a CD21-negative pre-B cell and myeloid leukemia line with CD35, CD21, or both. Cells expressing CD35 alone bound gp350/220 and became latently infected when the fusion receptor HLA II was coexpressed. Temporal, biophysical, and structural characteristics of CD35-mediated infection were distinct from CD21. Identification of CD35 as an EBV receptor uncovers a salient role in primary infection, addresses unsettled questions of virus tropism, and underscores the importance of EBVgp350/220 for vaccine development.

Tuning complement activation and pathway through controlled molecular architecture of dextran chains in nanoparticle corona.

Science.gov (United States)

Coty, Jean-Baptiste; Eleamen Oliveira, Elquio; Vauthier, Christine

2017-11-05

The understanding of complement activation by nanomaterials is a key to a rational design of safe and efficient nanomedicines. This work proposed a systematic study investigating how molecular design of nanoparticle coronas made of dextran impacts on mechanisms that trigger complement activation. The nanoparticles used for this work consisted of dextran-coated poly(isobutylcyanoacrylate) (PIBCA) nanoparticles have already been thoroughly characterized. Their different capacity to trigger complement activation established on the cleavage of the protein C3 was also already described making these nanoparticles good models to investigate the relation between the molecular feature of their corona and the mechanism by which they triggered complement activation. Results of this new study show that complement activation pathways can be selected by distinct architectures formed by dextran chains composing the nanoparticle corona. Assumptions that explain the relation between complement activation mechanisms triggered by the nanoparticles and the nanoparticle corona molecular feature were proposed. These results are of interest to better understand how the design of dextran-coated nanomaterials will impact interactions with the complement system. It can open perspectives with regard to the selection of a preferential complement activation pathway or prevent the nanoparticles to activate the complement system, based on a rational choice of the corona configuration. Copyright © 2017 Elsevier B.V. All rights reserved.

The stability of complement-mediated bactericidal activity in human serum against Salmonella.

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Colette M O'Shaughnessy

Full Text Available The complement cascade includes heat-labile proteins and care is required when handling serum in order to preserve its functional integrity. We have previously used a whole human serum bactericidal assay to show that antibody and an intact complement system are required in blood for killing of invasive isolates of Salmonella. The aim of the present study was to evaluate the conditions under which human serum can be stored and manipulated while maintaining complement integrity. Serum bactericidal activity against Salmonella was maintained for a minimum of 35 days when stored at 4°C, eight days at 22°C and 54 hours at 37°C. Up to three freeze-thaw cycles had no effect on the persistence of bactericidal activity and hemolytic complement assays confirmed no effect on complement function. Delay in the separation of serum for up to four days from clotted blood stored at 22°C did not affect bactericidal activity. Dilution of serum resulted in an increased rate of loss of bactericidal activity and so serum should be stored undiluted. These findings indicate that the current guidelines concerning manipulation and storage of human serum to preserve complement integrity and function leave a large margin for safety with regards to bactericidal activity against Salmonella. The study provides a scheme for determining the requirements for serum handling in relation to functional activity of complement in other systems.

Regulation of Cre recombinase by ligand-induced complementation of inactive fragments.

Science.gov (United States)

Jullien, Nicolas; Sampieri, François; Enjalbert, Alain; Herman, Jean-Paul

2003-11-01

Cre recombinase is extensively used to engineer the genome of experimental animals. However, its usefulness is still limited by the lack of an efficient temporal control over its activity. To overcome this, we have developed DiCre, a regulatable fragment complementation system for Cre. The enzyme was split into two moieties that were fused to FKBP12 (FK506-binding protein) and FRB (binding domain of the FKBP12-rapamycin-associated protein), respectively. These can be efficiently heterodimerized by rapamycin. Several variants, based on splitting Cre at different sites and using different linker peptides, were tested in an indicator cell line. The fusion proteins, taken separately, had no recombinase activity. Stable transformants, co-expressing complementing fragments based on splitting Cre between Asn59 and Asn60, displayed low background activity affecting 0.05-0.4% of the cells. Rapamycin induced a rapid recombination, reaching 100% by 48-72 h, with an EC50 of 0.02 nM. Thus, ligand-induced dimerization can efficiently regulate Cre, and should be useful to achieve a tight temporal control of its activity, such as in the case of the creation of conditional knock-out animals.

Complement activation cascade triggered by PEG-PL engineered nanomedicines and carbon nanotubes: The challenges ahead

DEFF Research Database (Denmark)

Moghimi, S.M.; Andersen, Alina Joukainen; Hashemi, S.H.

2010-01-01

reactions to certain PEG-PL engineered nanomedicines in both experimental animals and man. These reactions are classified as pseudoallergy and may be associated with cardiopulmonary disturbance and other related symptoms of anaphylaxis. Recent studies suggest that complement activation may be a contributing......, but not a rate limiting factor, in eliciting hypersensitivity reactions to such nanomedicines in sensitive individuals. This is rather surprising since PEGylated structures are generally assumed to suppress protein adsorption and blood opsonization events including complement. Here, we examine the molecular...... basis of complement activation by PEG-PL engineered nanomedicines and carbon nanotubes and discuss the challenges ahead....

Hepatic expression of the GH/JAK/STAT/IGF pathway, acute-phase response signalling and complement system are affected in mouse offspring by prenatal and early postnatal exposure to maternal high-protein diet.

Science.gov (United States)

Vanselow, Jens; Kucia, Marzena; Langhammer, Martina; Koczan, Dirk; Rehfeldt, Charlotte; Metges, Cornelia C

2011-12-01

Effects of pre- and early postnatal exposure to maternal high-protein diets are not well understood. Transcription profiling was performed in male mouse offspring exposed to maternal high-protein diet during pregnancy and/or lactation to identify affected hepatic molecular pathways. Dams were fed isoenergetic diets with control (20% w/w) or high protein levels (40%). The hepatic expression profiles were evaluated by differential microarray analysis 3 days (d3) and 3 weeks (d21) after birth. Offspring from three different high-protein dietary groups, HP (d3, high-protein diet during pregnancy), HPHP (d21, high-protein diet during pregnancy and lactation) and CHP (d21, control diet during pregnancy and high-protein diet during lactation), were compared with age-matched offspring from dams fed control diet. Offspring body and liver mass of all high-protein groups were decreased. Prenatal high-protein diet affected hepatic expression of genes mapping to the acute response/complement system and the GH/JAK/STAT/IGF signalling pathways. Maternal exposure to high-protein diet during lactation affected hepatic gene expression of the same pathways but additionally affected genes mapping to protein, fatty acid, hexose and pyruvate metabolism. (1) Genes of the acute response/complement system and GH/JAK/STAT/IGF pathways were down-regulated in offspring of dams exposed to high-protein diets during pregnancy and/or lactation. (2) Genes related to nutrient and energy metabolism, however, were only affected when high-protein diet was administered during lactation. (3) Modulation of the GH/JAK/STAT/IGF pathway might be responsible for reduced body and liver masses by maternal high-protein diet.

Changes in cis-regulatory elements of a key floral regulator are associated with divergence of inflorescence architectures.

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Kusters, Elske; Della Pina, Serena; Castel, Rob; Souer, Erik; Koes, Ronald

2015-08-15

Higher plant species diverged extensively with regard to the moment (flowering time) and position (inflorescence architecture) at which flowers are formed. This seems largely caused by variation in the expression patterns of conserved genes that specify floral meristem identity (FMI), rather than changes in the encoded proteins. Here, we report a functional comparison of the promoters of homologous FMI genes from Arabidopsis, petunia, tomato and Antirrhinum. Analysis of promoter-reporter constructs in petunia and Arabidopsis, as well as complementation experiments, showed that the divergent expression of leafy (LFY) and the petunia homolog aberrant leaf and flower (ALF) results from alterations in the upstream regulatory network rather than cis-regulatory changes. The divergent expression of unusual floral organs (UFO) from Arabidopsis, and the petunia homolog double top (DOT), however, is caused by the loss or gain of cis-regulatory promoter elements, which respond to trans-acting factors that are expressed in similar patterns in both species. Introduction of pUFO:UFO causes no obvious defects in Arabidopsis, but in petunia it causes the precocious and ectopic formation of flowers. This provides an example of how a change in a cis-regulatory region can account for a change in the plant body plan. © 2015. Published by The Company of Biologists Ltd.

COMK ENCODES THE COMPETENCE TRANSCRIPTION FACTOR, THE KEY REGULATORY PROTEIN FOR COMPETENCE DEVELOPMENT IN BACILLUS-SUBTILIS

NARCIS (Netherlands)

VANSINDEREN, D; LUTTINGER, A; KONG, LY; DUBNAU, D; VENEMA, G; HAMOEN, L

comK is a positive autoregulatory gene occupying a central position in the com petence-signal-transduction network. All regulatory routes identified in this network converge at the level of comK expression. The ComK protein is required for the transcriptional induction of comK and the late

Characterization of noncoding regulatory DNA in the human genome.

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Elkon, Ran; Agami, Reuven

2017-08-08

Genetic variants associated with common diseases are usually located in noncoding parts of the human genome. Delineation of the full repertoire of functional noncoding elements, together with efficient methods for probing their biological roles, is therefore of crucial importance. Over the past decade, DNA accessibility and various epigenetic modifications have been associated with regulatory functions. Mapping these features across the genome has enabled researchers to begin to document the full complement of putative regulatory elements. High-throughput reporter assays to probe the functions of regulatory regions have also been developed but these methods separate putative regulatory elements from the chromosome so that any effects of chromatin context and long-range regulatory interactions are lost. Definitive assignment of function(s) to putative cis-regulatory elements requires perturbation of these elements. Genome-editing technologies are now transforming our ability to perturb regulatory elements across entire genomes. Interpretation of high-throughput genetic screens that incorporate genome editors might enable the construction of an unbiased map of functional noncoding elements in the human genome.

Crystal structures of the Erp protein family members ErpP and ErpC from Borrelia burgdorferi reveal the reason for different affinities for complement regulator factor H.

Science.gov (United States)

Brangulis, Kalvis; Petrovskis, Ivars; Kazaks, Andris; Akopjana, Inara; Tars, Kaspars

2015-05-01

Borrelia burgdorferi is the causative agent of Lyme disease, which can be acquired after the bite of an infected Ixodes tick. As a strategy to resist the innate immunity and to successfully spread and proliferate, B. burgdorferi expresses a set of outer membrane proteins that are capable of binding complement regulator factor H (CFH), factor H-like protein 1 (CFHL-1) and factor H-related proteins (CFHR) to avoid complement-mediated killing. B. burgdorferi B31 contains three proteins that belong to the Erp (OspE/F-related) protein family and are capable of binding CFH and some CFHRs, namely ErpA, ErpC and ErpP. We have determined the crystal structure of ErpP at 2.53Å resolution and the crystal structure of ErpC at 2.15Å resolution. Recently, the crystal structure of the Erp family member OspE from B. burgdorferi N40 was determined in complex with CFH domains 19-20, revealing the residues involved in the complex formation. Despite the high sequence conservation between ErpA, ErpC, ErpP and the homologous protein OspE (78-80%), the affinity for CFH and CFHRs differs markedly among the Erp family members, suggesting that ErpC may bind only CFHRs but not CFH. A comparison of the binding site in OspE with those of ErpC and ErpP revealed that the extended loop region, which is only observed in the potential binding site of ErpC, plays an important role by preventing the binding of CFH. These results can explain the inability of ErpC to bind CFH, whereas ErpP and ErpA still possess the ability to bind CFH. Copyright © 2015 Elsevier B.V. All rights reserved.

Fanconi anemia complementation group A (FANCA) protein has intrinsic affinity for nucleic acids with preference for single-stranded forms.

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Yuan, Fenghua; Qian, Liangyue; Zhao, Xinliang; Liu, Jesse Y; Song, Limin; D'Urso, Gennaro; Jain, Chaitanya; Zhang, Yanbin

2012-02-10

The Fanconi anemia complementation group A (FANCA) gene is one of 15 disease-causing genes and has been found to be mutated in ∼60% of Fanconi anemia patients. Using purified protein, we report that human FANCA has intrinsic affinity for nucleic acids. FANCA binds to both single-stranded (ssDNA) and double-stranded (dsDNA) DNAs; however, its affinity for ssDNA is significantly higher than for dsDNA in an electrophoretic mobility shift assay. FANCA also binds to RNA with an intriguingly higher affinity than its DNA counterpart. FANCA requires a certain length of nucleic acids for optimal binding. Using DNA and RNA ladders, we determined that the minimum number of nucleotides required for FANCA recognition is ∼30 for both DNA and RNA. By testing the affinity between FANCA and a variety of DNA structures, we found that a 5'-flap or 5'-tail on DNA facilitates its interaction with FANCA. A patient-derived FANCA truncation mutant (Q772X) has diminished affinity for both DNA and RNA. In contrast, the complementing C-terminal fragment of Q772X, C772-1455, retains the differentiated nucleic acid-binding activity (RNA > ssDNA > dsDNA), indicating that the nucleic acid-binding domain of FANCA is located primarily at its C terminus, where most disease-causing mutations are found.

Fanconi Anemia Complementation Group A (FANCA) Protein Has Intrinsic Affinity for Nucleic Acids with Preference for Single-stranded Forms*

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Yuan, Fenghua; Qian, Liangyue; Zhao, Xinliang; Liu, Jesse Y.; Song, Limin; D'Urso, Gennaro; Jain, Chaitanya; Zhang, Yanbin

2012-01-01

The Fanconi anemia complementation group A (FANCA) gene is one of 15 disease-causing genes and has been found to be mutated in ∼60% of Fanconi anemia patients. Using purified protein, we report that human FANCA has intrinsic affinity for nucleic acids. FANCA binds to both single-stranded (ssDNA) and double-stranded (dsDNA) DNAs; however, its affinity for ssDNA is significantly higher than for dsDNA in an electrophoretic mobility shift assay. FANCA also binds to RNA with an intriguingly higher affinity than its DNA counterpart. FANCA requires a certain length of nucleic acids for optimal binding. Using DNA and RNA ladders, we determined that the minimum number of nucleotides required for FANCA recognition is ∼30 for both DNA and RNA. By testing the affinity between FANCA and a variety of DNA structures, we found that a 5′-flap or 5′-tail on DNA facilitates its interaction with FANCA. A patient-derived FANCA truncation mutant (Q772X) has diminished affinity for both DNA and RNA. In contrast, the complementing C-terminal fragment of Q772X, C772–1455, retains the differentiated nucleic acid-binding activity (RNA > ssDNA > dsDNA), indicating that the nucleic acid-binding domain of FANCA is located primarily at its C terminus, where most disease-causing mutations are found. PMID:22194614

Contribution of a fuzzy expert system to regulatory impact analysis

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Marco Antônio da Cunha

2015-09-01

Full Text Available Regulatory Impact Analysis (RIA has been consolidating in Brazilian regulatory agencies throughout the last decades. The RIA methodology aims to examine the regulatory process, measure the costs and benefits generated, as well as other effects of social, political or economic nature caused by a new or an existing regulation. By analysing each regulatory option, the expert or regulator faces a myriad of variables, usually of qualitative nature, that are difficult to measure and with a high degree of uncertainty. This research complements the existing literature, given the scarcity of decision support models in RIA that – regardless of the problem treated – incorporate the tacit knowledge of the regulation expert. This paper proposes an exploratory approach using a Fuzzy Expert System, which therefore helps to enrich the decision process in the final stage of comparison of the regulatory options.

Nasal immunization of mice with Lactobacillus casei expressing the Pneumococcal Surface Protein A: induction of antibodies, complement deposition and partial protection against Streptococcus pneumoniae challenge.

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Campos, Ivana B; Darrieux, Michelle; Ferreira, Daniela M; Miyaji, Eliane N; Silva, Débora A; Arêas, Ana Paula M; Aires, Karina A; Leite, Luciana C C; Ho, Paulo L; Oliveira, Maria Leonor S

2008-04-01

Strategies for the development of new vaccines against Streptococcus pneumoniae infections try to overcome problems such as serotype coverage and high costs, present in currently available vaccines. Formulations based on protein candidates that can induce protection in animal models have been pointed as good alternatives. Among them, the Pneumococcal Surface Protein A (PspA) plays an important role during systemic infection at least in part through the inhibition of complement deposition on the pneumococcal surface, a mechanism of evasion from the immune system. Antigen delivery systems based on live recombinant lactic acid bacteria (LAB) represents a promising strategy for mucosal vaccination, since they are generally regarded as safe bacteria able to elicit both systemic and mucosal immune responses. In this work, the N-terminal region of clade 1 PspA was constitutively expressed in Lactobacillus casei and the recombinant bacteria was tested as a mucosal vaccine in mice. Nasal immunization with L. casei-PspA 1 induced anti-PspA antibodies that were able to bind to pneumococcal strains carrying both clade 1 and clade 2 PspAs and to induce complement deposition on the surface of the bacteria. In addition, an increase in survival of immunized mice after a systemic challenge with a virulent pneumococcal strain was observed.

Finding trans-regulatory genes and protein complexes modulating meiotic recombination hotspots of human, mouse and yeast.

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Wu, Min; Kwoh, Chee-Keong; Li, Xiaoli; Zheng, Jie

2014-09-11

The regulatory mechanism of recombination is one of the most fundamental problems in genomics, with wide applications in genome wide association studies (GWAS), birth-defect diseases, molecular evolution, cancer research, etc. Recombination events cluster into short genomic regions called "recombination hotspots". Recently, a zinc finger protein PRDM9 was reported to regulate recombination hotspots in human and mouse genomes. In addition, a 13-mer motif contained in the binding sites of PRDM9 is found to be enriched in human hotspots. However, this 13-mer motif only covers a fraction of hotspots, indicating that PRDM9 is not the only regulator of recombination hotspots. Therefore, the challenge of discovering other regulators of recombination hotspots becomes significant. Furthermore, recombination is a complex process. Hence, multiple proteins acting as machinery, rather than individual proteins, are more likely to carry out this process in a precise and stable manner. Therefore, the extension of the prediction of individual trans-regulators to protein complexes is also highly desired. In this paper, we introduce a pipeline to identify genes and protein complexes associated with recombination hotspots. First, we prioritize proteins associated with hotspots based on their preference of binding to hotspots and coldspots. Second, using the above identified genes as seeds, we apply the Random Walk with Restart algorithm (RWR) to propagate their influences to other proteins in protein-protein interaction (PPI) networks. Hence, many proteins without DNA-binding information will also be assigned a score to implicate their roles in recombination hotspots. Third, we construct sub-PPI networks induced by top genes ranked by RWR for various species (e.g., yeast, human and mouse) and detect protein complexes in those sub-PPI networks. The GO term analysis show that our prioritizing methods and the RWR algorithm are capable of identifying novel genes associated with

SACE_5599, a putative regulatory protein, is involved in morphological differentiation and erythromycin production in Saccharopolyspora erythraea.

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Kirm, Benjamin; Magdevska, Vasilka; Tome, Miha; Horvat, Marinka; Karničar, Katarina; Petek, Marko; Vidmar, Robert; Baebler, Spela; Jamnik, Polona; Fujs, Štefan; Horvat, Jaka; Fonovič, Marko; Turk, Boris; Gruden, Kristina; Petković, Hrvoje; Kosec, Gregor

2013-12-17

Erythromycin is a medically important antibiotic, biosynthesized by the actinomycete Saccharopolyspora erythraea. Genes encoding erythromycin biosynthesis are organized in a gene cluster, spanning over 60 kbp of DNA. Most often, gene clusters encoding biosynthesis of secondary metabolites contain regulatory genes. In contrast, the erythromycin gene cluster does not contain regulatory genes and regulation of its biosynthesis has therefore remained poorly understood, which has for a long time limited genetic engineering approaches for erythromycin yield improvement. We used a comparative proteomic approach to screen for potential regulatory proteins involved in erythromycin biosynthesis. We have identified a putative regulatory protein SACE_5599 which shows significantly higher levels of expression in an erythromycin high-producing strain, compared to the wild type S. erythraea strain. SACE_5599 is a member of an uncharacterized family of putative regulatory genes, located in several actinomycete biosynthetic gene clusters. Importantly, increased expression of SACE_5599 was observed in the complex fermentation medium and at controlled bioprocess conditions, simulating a high-yield industrial fermentation process in the bioreactor. Inactivation of SACE_5599 in the high-producing strain significantly reduced erythromycin yield, in addition to drastically decreasing sporulation intensity of the SACE_5599-inactivated strains when cultivated on ABSM4 agar medium. In contrast, constitutive overexpression of SACE_5599 in the wild type NRRL23338 strain resulted in an increase of erythromycin yield by 32%. Similar yield increase was also observed when we overexpressed the bldD gene, a previously identified regulator of erythromycin biosynthesis, thereby for the first time revealing its potential for improving erythromycin biosynthesis. SACE_5599 is the second putative regulatory gene to be identified in S. erythraea which has positive influence on erythromycin yield. Like bld

CSF coccidioides complement fixation

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... this page: //medlineplus.gov/ency/article/003526.htm CSF coccidioides complement fixation test To use the sharing features on this page, please enable JavaScript. CSF coccidioides complement fixation is a test that checks ...

Live cell imaging of interactions between replicase and capsid protein of Brome mosaic virus using Bimolecular Fluorescence Complementation: implications for replication and genome packaging.

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Chaturvedi, Sonali; Rao, A L N

2014-09-01

In Brome mosaic virus, it was hypothesized that a physical interaction between viral replicase and capsid protein (CP) is obligatory to confer genome packaging specificity. Here we tested this hypothesis by employing Bimolecular Fluorescent Complementation (BiFC) as a tool for evaluating protein-protein interactions in living cells. The efficacy of BiFC was validated by a known interaction between replicase protein 1a (p1a) and protein 2a (p2a) at the endoplasmic reticulum (ER) site of viral replication. Additionally, co-expression in planta of a bona fide pair of interacting protein partners of p1a and p2a had resulted in the assembly of a functional replicase. Subsequent BiFC assays in conjunction with mCherry labeled ER as a fluorescent cellular marker revealed that CP physically interacts with p2a, but not p1a, and this CP:p2a interaction occurs at the cytoplasmic phase of the ER. The significance of the CP:p2a interaction in BMV replication and genome packaging is discussed. Copyright © 2014 Elsevier Inc. All rights reserved.

Mutational robustness of gene regulatory networks.

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Aalt D J van Dijk

Full Text Available Mutational robustness of gene regulatory networks refers to their ability to generate constant biological output upon mutations that change network structure. Such networks contain regulatory interactions (transcription factor-target gene interactions but often also protein-protein interactions between transcription factors. Using computational modeling, we study factors that influence robustness and we infer several network properties governing it. These include the type of mutation, i.e. whether a regulatory interaction or a protein-protein interaction is mutated, and in the case of mutation of a regulatory interaction, the sign of the interaction (activating vs. repressive. In addition, we analyze the effect of combinations of mutations and we compare networks containing monomeric with those containing dimeric transcription factors. Our results are consistent with available data on biological networks, for example based on evolutionary conservation of network features. As a novel and remarkable property, we predict that networks are more robust against mutations in monomer than in dimer transcription factors, a prediction for which analysis of conservation of DNA binding residues in monomeric vs. dimeric transcription factors provides indirect evidence.

Relationship of Circulating C5a and Complement Factor H Levels With Disease Control in Pregnant Women With Asthma.

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Bohács, Anikó; Bikov, András; Ivancsó, István; Czaller, Ibolya; Böcskei, Renáta; Müller, Veronika; Rigó, János; Losonczy, György; Tamási, Lilla

2016-04-01

Asthma often complicates pregnancy and represents a risk of serious pregnancy complications. The complement system contributes to asthma pathogenesis and is up-regulated in healthy gestation as well. The anaphylatoxin C5a has a major pro-inflammatory role, and the complement factor H is a main soluble regulator protein both in asthma and during pregnancy; however, peripheral levels of these complement factors and their relationship to disease control have not yet been evaluated in pregnant subjects with asthma. The present study aimed to investigate circulating C5a and complement factor H levels in asthma (non-pregnant subjects with asthma; n = 19) and in pregnancy with asthma (pregnant subjects with asthma; n = 22), compared with healthy non-pregnant (n = 21) and healthy pregnant women (n = 13) and to test their relationship to clinical parameters of asthma (lung function, airway inflammation, and symptoms). Circulating C5a levels were higher in the pregnant asthma subject group compared with the healthy non-pregnant, healthy pregnant, and non-pregnant asthma groups: median 2.629 (interquartile range [IQR] 2.257-3.052) ng/mL versus 1.84 (IQR 1.576-2.563), 1.783 (IQR 0.6064-2.786), and 2.024 (IQR 1.232-2.615) ng/mL, respectively (P = .02 in all cases). C5a correlated negatively with FEV1 (r = -0.44, P = .039) and FVC values (r = -0.64, P = .001) in the pregnant asthma group and positively with fraction of exhaled nitric oxide levels in the non-pregnant asthma group (n = 12, r = 0.78, P = .004). Complement factor H levels were elevated in both the healthy pregnant and pregnant asthma subject groups compared with the healthy non-pregnant group (median 1,082 [IQR 734.9-1,224] and 910.7 [IQR 614.5-1076] μg/mL vs. 559.7 [IQR 388.7-783.1] μg/mL, P = .002 and P = .004, respectively) but not in the pregnant asthma group compared with the non-pregnant asthma group (median 687.4 [IQR 441.6-947.6] μg/mL, P = .10). Asthma during pregnancy increases the circulating level of

Solubilization and reconstitution of the formylmethionylleucylphenylalanine receptor coupled to guanine nucleotide regulatory protein

International Nuclear Information System (INIS)

Williamson, K.; Dickey, B.F.; Pyun, H.Y.; Navarro, J.

1988-01-01

The authors describe the solubilization, resolution, and reconstitution of the formylmethionylleucylphenylalanine (fMet-Leu-Phe) receptor and guanine nucleotide regulatory proteins (G-proteins). The receptor was solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Guanine nucleotides decreased the number of high-affinity binding sites and accelerated the rate of dissociation of the receptor-ligand complex, suggesting that the solubilized receptor remained coupled to endogenous G-proteins. The solubilized receptor was resolved from endogenous G-proteins by fractionation on a wheat germ agglutinin (WGA)-Sepharose 4B column. High-affinity [ 3 H]fMet-Leu-Phe binding to the WGA-purified receptor was diminished and exhibited reduced guanine nucleotide sensitivity. High-affinity [ 3 H]fMET-Leu-Phe binding and guanine nucleotide sensitivity were reconstituted upon the addition of purified brain G-proteins. Similar results were obtained when the receptor was reconstituted with brain G-proteins into phospholipid vesicles by gel filtration chromatography. In addition, they also demonstrated fMET-Leu-Phe-dependent GTP hydrolysis in the reconstituted vesicles. The results of this work indicate that coupling of the fMet-Leu-Phe receptor to G-proteins converts the receptor to a high-affinity binding state and that agonist produces activation of G-proteins. The resolution and functional reconstitution of this receptor should provide an important step toward the elucidation of the molecular mechanism of the fMet-Leu-Phe transduction system in neutrophils

Nanomedicine and the complement paradigm.

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Moghimi, S Moein; Farhangrazi, Z Shadi

2013-05-01

The role of complement in idiosyncratic reactions to nanopharmaceutical infusion is receiving increasing attention. We discuss this in relation to nanopharmaceutical development and the possible use of complement inhibitors to prevent related adverse reactions. We further call on initiation of genetic association studies to unravel the genetic basis of nanomedicine infusion-related adverse responses, since most of the polymorphic genes in the genome belong to the immune system. In this paper, idiosyncratic reactions based on complement activation are discussed in the context of newly available complement inhibitors. Copyright © 2013 Elsevier Inc. All rights reserved.

Study of the plant COPII vesicle coat subunits by functional complementation of yeast Saccharomyces cerevisiae mutants.

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Johan-Owen De Craene

Full Text Available The formation and budding of endoplasmic reticulum ER-derived vesicles depends on the COPII coat protein complex that was first identified in yeast Saccharomyces cerevisiae. The ER-associated Sec12 and the Sar1 GTPase initiate the COPII coat formation by recruiting the Sec23-Sec24 heterodimer following the subsequent recruitment of the Sec13-Sec31 heterotetramer. In yeast, there is usually one gene encoding each COPII protein and these proteins are essential for yeast viability, whereas the plant genome encodes multiple isoforms of all COPII subunits. Here, we used a systematic yeast complementation assay to assess the functionality of Arabidopsis thaliana COPII proteins. In this study, the different plant COPII subunits were expressed in their corresponding temperature-sensitive yeast mutant strain to complement their thermosensitivity and secretion phenotypes. Secretion was assessed using two different yeast cargos: the soluble α-factor pheromone and the membranous v-SNARE (vesicle-soluble NSF (N-ethylmaleimide-sensitive factor attachment protein receptor Snc1 involved in the fusion of the secretory vesicles with the plasma membrane. This complementation study allowed the identification of functional A. thaliana COPII proteins for the Sec12, Sar1, Sec24 and Sec13 subunits that could represent an active COPII complex in plant cells. Moreover, we found that AtSec12 and AtSec23 were co-immunoprecipitated with AtSar1 in total cell extract of 15 day-old seedlings of A. thaliana. This demonstrates that AtSar1, AtSec12 and AtSec23 can form a protein complex that might represent an active COPII complex in plant cells.

Host-pathogen interactions in Lyme disease and their application in diagnostics

NARCIS (Netherlands)

Burgel, Nathalie Daniëlle van

2013-01-01

B. burgdorferi has a wide variety of strategies to hide from the host immune system. Complement regulatory binding proteins have been described for almost all complement resistant B. burgdorferi sl, except for the complement resistant B. bavariensis, one of the species that is known to frequently

Thyroid status influence on adiponectin, acylation stimulating protein (ASP and complement C3 in hyperthyroid and hypothyroid subjects

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Zhang Jianhua

2006-02-01

Full Text Available Abstract Background Thyroid abnormalities (hyperthyroid and hypothyroid are accompanied by changes in intermediary metabolism including alterations in body weight, insulin resistance and lipid profile. The aims of this study were to examine plasma ASP, its precursor C3 and adiponectin in hyperthyroid and hypothyroid subjects as compared to controls. Methods A total of 99 subjects were recruited from endocrinology/out-patient clinics: 46 hyperthyroid subjects, 23 hypothyroid subjects and 30 control subjects. Subjects were evaluated for FT4, FT3, TSH, glucose, insulin, complete lipid profile and the adipokines: adiponectin, acylation stimulating protein (ASP and complement C3. Results Hyperthyroidism was associated with a 95% increase in adiponectin (p = 0.0002, a 47% decrease in C3 (p Conclusion These changes suggest that thyroid disease may be accompanied by changes in adipokines, which may contribute to the phenotype expressed.

Nickel and low CO2-controlled motility in Chlamydomonas through complementation of a paralyzed flagella mutant with chemically regulated promoters

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Rosenbaum Joel L

2011-01-01

Full Text Available Abstract Background Chlamydomonas reinhardtii is a model system for the biology of unicellular green algae. Chemically regulated promoters, such as the nickel-inducible CYC6 or the low CO2-inducible CAH1 promoter, may prove useful for expressing, at precise times during its cell cycle, proteins with relevant biological functions, or complementing mutants in genes encoding such proteins. To this date, this has not been reported for the above promoters. Results We fused the CYC6 and CAH1 promoters to an HA-tagged RSP3 gene, encoding a protein of the flagellar radial spoke complex. The constructs were used for chemically regulated complementation of the pf14 mutant, carrying an ochre mutation in the RSP3 gene. 7 to 8% of the transformants showed cells with restored motility after induction with nickel or transfer to low CO2 conditions, but not in non-inducing conditions. Maximum complementation (5% motile cells was reached with very different kinetics (5-6 hours for CAH1, 48 hours for CYC6. The two inducible promoters drive much lower levels of RSP3 protein expression than the constitutive PSAD promoter, which shows almost complete rescue of motility. Conclusions To our knowledge, this is the first example of the use of the CYC6 or CAH1 promoters to perform a chemically regulated complementation of a Chlamydomonas mutant. Based on our data, the CYC6 and CAH1 promoters should be capable of fully complementing mutants in genes whose products exert their biological activity at low concentrations.

Omics-Based Approach Reveals Complement-Mediated Inflammation in Chronic Lymphocytic Inflammation With Pontine Perivascular Enhancement Responsive to Steroids (CLIPPERS

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Morten Blaabjerg

2018-04-01

Full Text Available ObjectiveChronic lymphocytic inflammation with pontine perivascular enhancement responsive to steroids (CLIPPERS is a rare syndrome with relapsing brainstem/cerebellar symptoms. To examine the pathogenic processes and investigate potential biomarkers, we analyzed combined materials of brain and cerebrospinal fluid (CSF by comprehensive methodologies.Materials and methodsTo identify major pathways of perivascular inflammation in CLIPPERS, we first compared the CSF proteome (n = 5 to a neurodegenerative condition, Alzheimer’s disease (AD, n = 5. Activation of complement was confirmed by immunohistochemistry (IHC on CLIPPERS brain samples (n = 3 and by ELISA in the CSF. For potential biomarkers, we used biomarker arrays, and compared inflammatory and vessel-associated proteins in the CSF of CLIPPERS (n = 5 with another inflammatory relapsing CNS disease, multiple sclerosis (RMS, n = 9 and healthy subjects (HS, n = 7.ResultsTwo hundred and seven proteins in the CSF discriminated CLIPPERS from AD. The complement cascade, immunoglobulins, and matrix proteins were among the most frequently represented pathways. Pathway analysis of upstream regulators suggested the importance of vascular cell adhesion protein 1 (VCAM1, IFN-γ, interleukin (IL-1, and IL-10. Differential regulation of more than 10 complement proteins of the 3 complement pathways in the CSF pointed to the role of complement activation. IHC on brain samples confirmed the perivascular complement activation, i.e., deposition of C3bc, C3d, and the terminal C5b-9 complement complex that partially overlapped with accumulation of IgG in the vessel wall. Besides endothelial cell damage, reactivity to smooth muscle actin was lost in the walls of inflamed vessels, but the glia limitans was preserved. The semi-quantitative array indicated that increased level of IL-8/CXCL8 (p < 0.05, eotaxin/CCL11 (p < 0.01, and granulocyte colony-stimulating factor (p < 0.05 in

Down-regulation of complement receptors on the surface of host monocyte even as in vitro complement pathway blocking interferes in dengue infection.

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Cintia Ferreira Marinho

Full Text Available In dengue virus (DENV infection, complement system (CS activation appears to have protective and pathogenic effects. In severe dengue fever (DF, the levels of DENV non-structural-1 protein and of the products of complement activation, including C3a, C5a and SC5b-9, are higher before vascular leakage occurs, supporting the hypothesis that complement activation contributes to unfavourable outcomes. The clinical manifestations of DF range from asymptomatic to severe and even fatal. Here, we aimed to characterise CS by their receptors or activation product, in vivo in DF patients and in vitro by DENV-2 stimulation on monocytes. In comparison with healthy controls, DF patients showed lower expression of CR3 (CD11b, CR4 (CD11c and, CD59 on monocytes. The DF patients who were high producers of SC5b-9 were also those that showed more pronounced bleeding or vascular leakage. Those findings encouraged us to investigate the role of CS in vitro, using monocytes isolated from healthy subjects. Prior blocking with CR3 alone (CD11b or CR3 (CD11b/CD18 reduced viral infection, as quantified by the levels of intracellular viral antigen expression and soluble DENV non-structural viral protein. However, we found that CR3 alone (CD11b or CR3 (CD11b/CD18 blocking did not influence major histocompatibility complex presentation neither active caspase-1 on monocytes, thus probably ruling out inflammasome-related mechanisms. Although it did impair the secretion of tumour necrosis factor alpha and interferon alpha. Our data provide strategies of blocking CR3 (CD11b pathways could have implications for the treatment of viral infection by antiviral-related mechanisms.

Regulatory network controlling extracellular proteins in Erwinia carotovora subsp. carotovora: FlhDC, the master regulator of flagellar genes, activates rsmB regulatory RNA production by affecting gacA and hexA (lrhA) expression.

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Cui, Yaya; Chatterjee, Asita; Yang, Hailian; Chatterjee, Arun K

2008-07-01

Erwinia carotovora subsp. carotovora produces an array of extracellular proteins (i.e., exoproteins), including plant cell wall-degrading enzymes and Harpin, an effector responsible for eliciting hypersensitive reaction. Exoprotein genes are coregulated by the quorum-sensing signal, N-acyl homoserine lactone, plant signals, an assortment of transcriptional factors/regulators (GacS/A, ExpR1, ExpR2, KdgR, RpoS, HexA, and RsmC) and posttranscriptional regulators (RsmA, rsmB RNA). rsmB RNA production is positively regulated by GacS/A, a two-component system, and negatively regulated by HexA (PecT in Erwinia chrysanthemi; LrhA [LysR homolog A] in Escherichia coli) and RsmC, a putative transcriptional adaptor. While free RsmA, an RNA-binding protein, promotes decay of mRNAs of exoprotein genes, binding of RsmA with rsmB RNA neutralizes the RsmA effect. In the course of studies of GacA regulation, we discovered that a locus bearing strong homology to the flhDC operon of E. coli also controls extracellular enzyme production. A transposon insertion FlhDC(-) mutant produces very low levels of pectate lyase, polygalacturonase, cellulase, protease, and E. carotovora subsp. carotovora Harpin (Harpin(Ecc)) and is severely attenuated in its plant virulence. The production of these exoproteins is restored in the mutant carrying an FlhDC(+) plasmid. Sequence analysis and transcript assays disclosed that the flhD operon of E. carotovora subsp. carotovora, like those of other enterobacteria, consists of flhD and flhC. Complementation analysis revealed that the regulatory effect requires functions of both flhD and flhC products. The data presented here show that FlhDC positively regulates gacA, rsmC, and fliA and negatively regulates hexA (lrhA). Evidence shows that FlhDC controls extracellular protein production through cumulative effects on hexA and gacA. Reduced levels of GacA and elevated levels of HexA in the FlhDC(-) mutant are responsible for the inhibition of rsmB RNA

Regulatory Network Controlling Extracellular Proteins in Erwinia carotovora subsp. carotovora: FlhDC, the Master Regulator of Flagellar Genes, Activates rsmB Regulatory RNA Production by Affecting gacA and hexA (lrhA) Expression▿

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Cui, Yaya; Chatterjee, Asita; Yang, Hailian; Chatterjee, Arun K.

2008-01-01

Erwinia carotovora subsp. carotovora produces an array of extracellular proteins (i.e., exoproteins), including plant cell wall-degrading enzymes and Harpin, an effector responsible for eliciting hypersensitive reaction. Exoprotein genes are coregulated by the quorum-sensing signal, N-acyl homoserine lactone, plant signals, an assortment of transcriptional factors/regulators (GacS/A, ExpR1, ExpR2, KdgR, RpoS, HexA, and RsmC) and posttranscriptional regulators (RsmA, rsmB RNA). rsmB RNA production is positively regulated by GacS/A, a two-component system, and negatively regulated by HexA (PecT in Erwinia chrysanthemi; LrhA [LysR homolog A] in Escherichia coli) and RsmC, a putative transcriptional adaptor. While free RsmA, an RNA-binding protein, promotes decay of mRNAs of exoprotein genes, binding of RsmA with rsmB RNA neutralizes the RsmA effect. In the course of studies of GacA regulation, we discovered that a locus bearing strong homology to the flhDC operon of E. coli also controls extracellular enzyme production. A transposon insertion FlhDC− mutant produces very low levels of pectate lyase, polygalacturonase, cellulase, protease, and E. carotovora subsp. carotovora Harpin (HarpinEcc) and is severely attenuated in its plant virulence. The production of these exoproteins is restored in the mutant carrying an FlhDC+ plasmid. Sequence analysis and transcript assays disclosed that the flhD operon of E. carotovora subsp. carotovora, like those of other enterobacteria, consists of flhD and flhC. Complementation analysis revealed that the regulatory effect requires functions of both flhD and flhC products. The data presented here show that FlhDC positively regulates gacA, rsmC, and fliA and negatively regulates hexA (lrhA). Evidence shows that FlhDC controls extracellular protein production through cumulative effects on hexA and gacA. Reduced levels of GacA and elevated levels of HexA in the FlhDC− mutant are responsible for the inhibition of rsmB RNA production

Neuron-derived IgG protects neurons from complement-dependent cytotoxicity.

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Zhang, Jie; Niu, Na; Li, Bingjie; McNutt, Michael A

2013-12-01

Passive immunity of the nervous system has traditionally been thought to be predominantly due to the blood-brain barrier. This concept must now be revisited based on the existence of neuron-derived IgG. The conventional concept is that IgG is produced solely by mature B lymphocytes, but it has now been found to be synthesized by murine and human neurons. However, the function of this endogenous IgG is poorly understood. In this study, we confirm IgG production by rat cortical neurons at the protein and mRNA levels, with 69.0 ± 5.8% of cortical neurons IgG-positive. Injury to primary-culture neurons was induced by complement leading to increases in IgG production. Blockage of neuron-derived IgG resulted in more neuronal death and early apoptosis in the presence of complement. In addition, FcγRI was found in microglia and astrocytes. Expression of FcγR I in microglia was increased by exposure to neuron-derived IgG. Release of NO from microglia triggered by complement was attenuated by neuron-derived IgG, and this attenuation could be reversed by IgG neutralization. These data demonstrate that neuron-derived IgG is protective of neurons against injury induced by complement and microglial activation. IgG appears to play an important role in maintaining the stability of the nervous system.

Complement fixation test to C burnetii

Science.gov (United States)

... complement fixation test; Coxiella burnetii - complement fixation test; C burnetii - complement fixation test ... a specific foreign substance ( antigen ), in this case, C burnetii . Antibodies defend the body against bacteria, viruses, ...

Subversion of complement by hematophagous parasites.

Science.gov (United States)

Schroeder, Hélène; Skelly, Patrick J; Zipfel, Peter F; Losson, Bertrand; Vanderplasschen, Alain

2009-01-01

The complement system is a crucial part of innate and adaptive immunity which exerts a significant evolutionary pressure on pathogens. It has selected for those pathogens, mainly microorganisms but also parasites, that have evolved countermeasures. The characterization of how pathogens evade complement attack is a rapidly developing field of current research. In recent years, multiple complement evasion strategies have been characterized. In this review, we focus on complement escape mechanisms expressed by hematophagous parasites, a heterogeneous group of metazoan parasites that share the property of ingesting the whole blood of their host. Complement inhibition is crucial for parasite survival within the host tissue or to facilitate blood feeding. Finally, complement inhibition by hematophagous parasites may also contribute to their success as pathogen vectors.

The complement system: a gateway to gene-environment interactions in schizophrenia pathogenesis.

Science.gov (United States)

Nimgaonkar, V L; Prasad, K M; Chowdari, K V; Severance, E G; Yolken, R H

2017-11-01

The pathogenesis of schizophrenia is considered to be multi-factorial, with likely gene-environment interactions (GEI). Genetic and environmental risk factors are being identified with increasing frequency, yet their very number vastly increases the scope of possible GEI, making it difficult to identify them with certainty. Accumulating evidence suggests a dysregulated complement pathway among the pathogenic processes of schizophrenia. The complement pathway mediates innate and acquired immunity, and its activation drives the removal of damaged cells, autoantigens and environmentally derived antigens. Abnormalities in complement functions occur in many infectious and autoimmune disorders that have been linked to schizophrenia. Many older reports indicate altered serum complement activity in schizophrenia, though the data are inconclusive. Compellingly, recent genome-wide association studies suggest repeat polymorphisms incorporating the complement 4A (C4A) and 4B (C4B) genes as risk factors for schizophrenia. The C4A/C4B genetic associations have re-ignited interest not only in inflammation-related models for schizophrenia pathogenesis, but also in neurodevelopmental theories, because rodent models indicate a role for complement proteins in synaptic pruning and neurodevelopment. Thus, the complement system could be used as one of the 'staging posts' for a variety of focused studies of schizophrenia pathogenesis. They include GEI studies of the C4A/C4B repeat polymorphisms in relation to inflammation-related or infectious processes, animal model studies and tests of hypotheses linked to autoimmune diseases that can co-segregate with schizophrenia. If they can be replicated, such studies would vastly improve our understanding of pathogenic processes in schizophrenia through GEI analyses and open new avenues for therapy.

Noun complement clauses as referential modifiers

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Carlos de Cuba

2017-01-01

Full Text Available A number of recent analyses propose that so-called noun complement clauses should be analyzed as a type of relative clause. In this paper, I present a number of complications for any analysis that equates noun complement clauses to relative clauses, and conclude that this type of analysis is on the wrong track. I present cross-linguistic evidence showing that the syntactic behavior of noun complement clauses does not pattern with relative clauses. Patterns of complementizer choice and complementizer drop as well as patterns involving main clause phenomena and extraction differ in the two constructions, which I argue is unexpected under a relative clause analysis that involves operator movement. Instead I present an alternative analysis in which I propose that the referentiality of a noun complement clause is linked to its syntactic behavior. Following recent work, I claim that referential clauses have a syntactically truncated left-periphery, and this truncation can account for the lack of main clause phenomena in noun complement clauses. I argue that the truncation analysis is also able to accommodate complementizer data patterns more easily than relative clause analyses that appeal to operator movement.

CCAAT/enhancer-binding proteins regulate expression of the human steroidogenic acute regulatory protein (StAR) gene.

Science.gov (United States)

Christenson, L K; Johnson, P F; McAllister, J M; Strauss, J F

1999-09-10

Two putative CCAAT/enhancer-binding protein (C/EBP) response elements were identified in the proximal promoter of the human steroidogenic acute regulatory protein (StAR) gene, which encodes a key protein-regulating steroid hormone synthesis. Expression of C/EBPalpha and -beta increased StAR promoter activity in COS-1 and HepG2 cells. Cotransfection of C/EBPalpha or -beta and steroidogenic factor 1, a transcription factor required for cAMP regulation of StAR expression, into COS-1 augmented 8-bromoadenosine 3':5'-cyclic monophosphate (8-Br-cAMP)-stimulated promoter activity. When the putative C/EBP response elements were mutated, individually or together, a pronounced decline in basal StAR promoter activity in human granulosa-lutein cells resulted, but the fold stimulation of promoter activity by 8-Br-cAMP was unaffected. Recombinant C/EBPalpha and -beta bound to the two identified sequences but not the mutated elements. Human granulosa-lutein cell nuclear extracts also bound these elements but not the mutated sequences. An antibody to C/EBPbeta, but not C/EBPalpha, supershifted the nuclear protein complex associated with the more distal element. The complex formed by nuclear extracts with the proximal element was not supershifted by either antibody. Western blot analysis revealed the presence of C/EBPalpha and C/EBPbeta in human granulosa-lutein cell nuclear extracts. C/EBPbeta levels were up-regulated 3-fold by 8-Br-cAMP treatment. Our studies demonstrate a role for C/EBPbeta as well as yet to be identified proteins, which can bind to C/EBP response elements, in the regulation of StAR gene expression and suggest a mechanism by which C/EBPbeta participates in the cAMP regulation of StAR gene transcription.

Therapeutic inhibition of the complement system. Y2K update.

Science.gov (United States)

Asghar, S S; Pasch, M C

2000-09-01

Activation of complement is an essential part of the mechanism of pathogenesis of a large number of human diseases; its inhibition by pharmacological means is likely to suppress disease processes in complement mediated diseases. From this point of view low molecular weight synthetic inhibitors of complement are being developed and high molecular weight natural inhibitors of human origin present in plasma or embedded in cell membrane are being purified or produced in their recombinant forms. This review is concerned with high molecular weight inhibitors, some of which are already in clinical use but may be efficacious in many other diseases in which they have not yet been tried. C1-esterase inhibitor (C1-INH) concentrate prepared from human plasma is being successfully used for the treatment of hereditary angioneurotic edema. Recently, C1-INH has been found to be consumed in severe inflammation and has been shown to exert beneficial effects in several inflammatory conditions such as human sepsis, post-operative myocardial dysfunction due to reperfusion injury, severe capillary leakage syndrome after bone marrow transplantation, reperfusion injury after lung transplantation, burn, and cytotoxicity caused by IL-2 therapy in cancer. Factor I has been used for the treatment of factor I deficiency. Recombinant soluble forms of membrane cofactor protein (MCP), and decay accelerating factor (DAF) have not yet been tried in humans but have been shown to be effective in immune complex mediate inflammation in animals. Organs of pigs transgenic for one or more of human membrane regulators of complement namely membrane cofactor protein (MCP), decay accelerating factor (DAF) or CD59, are being produced for transplantation into humans. They have been shown to be resistant to hyperacute rejection in non-human primates; acute vascular rejection is still a problem in their clinical use. It is hoped that these observations together with future developments will make xeno

The Epidermal Growth Factor Receptor Is a Regulator of Epidermal Complement Component Expression and Complement Activation

DEFF Research Database (Denmark)

Abu-Humaidan, Anas H A; Ananthoju, Nageshwar; Mohanty, Tirthankar

2014-01-01

The complement system is activated in response to tissue injury. During wound healing, complement activation seems beneficial in acute wounds but may be detrimental in chronic wounds. We found that the epidermal expression of many complement components was only increased to a minor extent in skin...

Epitopes of human immunodeficiency virus regulatory proteins tat, nef, and rev are expressed in normal human tissue

NARCIS (Netherlands)

Parmentier, H. K.; van Wichen, D. F.; Meyling, F. H.; Goudsmit, J.; Schuurman, H. J.

1992-01-01

The expression of regulatory proteins tat, rev, and nef of human immunodeficiency virus type-1 (HIV-1) and tat of HIV-2 was studied in frozen sections of lymph nodes from HIV-1-infected individuals, and various tissues from uninfected persons. In HIV-1-positive lymph nodes, monoclonal antibodies to

Bacteria-Phagocyte Interactions: Emerging Tactics in an Ancient Rivalry

Science.gov (United States)

1990-01-01

regulated by comple- cyte function (Table 1). Some organisms, like ment regulatory proteins . leptospira , are able to invade host tissue and dis- Many...similarly anchored protein . pili are no longer expressed . Opsonin-mediated C8-binding protein . are complement regulatory phagocytosis may have...diphthamide] and botulinu, bacteria expressing the smooth LPS phenotype (𔃽 [(TP-binding protein ] and C2 [G actin] ISO], were considered to be better

A tick mannose-binding lectin inhibitor interferes with the vertebrate complement cascade to enhance transmission of the lyme disease agent.

Science.gov (United States)

Schuijt, Tim J; Coumou, Jeroen; Narasimhan, Sukanya; Dai, Jianfeng; Deponte, Kathleen; Wouters, Diana; Brouwer, Mieke; Oei, Anneke; Roelofs, Joris J T H; van Dam, Alje P; van der Poll, Tom; Van't Veer, Cornelis; Hovius, Joppe W; Fikrig, Erol

2011-08-18

The Lyme disease agent Borrelia burgdorferi is primarily transmitted to vertebrates by Ixodes ticks. The classical and alternative complement pathways are important in Borrelia eradication by the vertebrate host. We recently identified a tick salivary protein, designated P8, which reduced complement-mediated killing of Borrelia. We now discover that P8 interferes with the human lectin complement cascade, resulting in impaired neutrophil phagocytosis and chemotaxis and diminished Borrelia lysis. Therefore, P8 was renamed the tick salivary lectin pathway inhibitor (TSLPI). TSLPI-silenced ticks, or ticks exposed to TSLPI-immune mice, were hampered in Borrelia transmission. Moreover, Borrelia acquisition and persistence in tick midguts was impaired in ticks feeding on TSLPI-immunized, B. burgdorferi-infected mice. Together, our findings suggest an essential role for the lectin complement cascade in Borrelia eradication and demonstrate how a vector-borne pathogen co-opts a vector protein to facilitate early mammalian infection and vector colonization. Copyright © 2011 Elsevier Inc. All rights reserved.

Subversion of complement by hematophagous parasites

OpenAIRE

Schroeder, Hélène; Skelly, Patrick; Zipfel, Peter F.; Losson, Bertrand; Vanderplasschen, Alain

2009-01-01

The complement system is a crucial part of innate and adaptive immunity which exerts a significant evolutionary pressure on pathogens. It has selected for those pathogens, mainly micro-organisms but also parasites, that have evolved countermeasures. The characterization of how pathogens evade complement attack is a rapidly developing field of current research. In recent years, multiple complement evasion strategies have been characterized. In this review, we focus on complement escape mechani...

Complementation contributes to transcriptome complexity in maize (Zea mays L.) hybrids relative to their inbred parents

Science.gov (United States)

Paschold, Anja; Jia, Yi; Marcon, Caroline; Lund, Steve; Larson, Nick B.; Yeh, Cheng-Ting; Ossowski, Stephan; Lanz, Christa; Nettleton, Dan; Schnable, Patrick S.; Hochholdinger, Frank

2012-01-01

Typically, F1-hybrids are more vigorous than their homozygous, genetically distinct parents, a phenomenon known as heterosis. In the present study, the transcriptomes of the reciprocal maize (Zea mays L.) hybrids B73×Mo17 and Mo17×B73 and their parental inbred lines B73 and Mo17 were surveyed in primary roots, early in the developmental manifestation of heterotic root traits. The application of statistical methods and a suitable experimental design established that 34,233 (i.e., 86%) of all high-confidence maize genes were expressed in at least one genotype. Nearly 70% of all expressed genes were differentially expressed between the two parents and 42%–55% of expressed genes were differentially expressed between one of the parents and one of the hybrids. In both hybrids, ∼10% of expressed genes exhibited nonadditive gene expression. Consistent with the dominance model (i.e., complementation) for heterosis, 1124 genes that were expressed in the hybrids were expressed in only one of the two parents. For 65 genes, it could be shown that this was a consequence of complementation of genomic presence/absence variation. For dozens of other genes, alleles from the inactive inbred were activated in the hybrid, presumably via interactions with regulatory factors from the active inbred. As a consequence of these types of complementation, both hybrids expressed more genes than did either parental inbred. Finally, in hybrids, ∼14% of expressed genes exhibited allele-specific expression (ASE) levels that differed significantly from the parental-inbred expression ratios, providing further evidence for interactions of regulatory factors from one parental genome with target genes from the other parental genome. PMID:23086286

HOXA1 and TALE proteins display cross-regulatory interactions and form a combinatorial binding code on HOXA1 targets.

Science.gov (United States)

De Kumar, Bony; Parker, Hugo J; Paulson, Ariel; Parrish, Mark E; Pushel, Irina; Singh, Narendra Pratap; Zhang, Ying; Slaughter, Brian D; Unruh, Jay R; Florens, Laurence; Zeitlinger, Julia; Krumlauf, Robb

2017-09-01

Hoxa1 has diverse functional roles in differentiation and development. We identify and characterize properties of regions bound by HOXA1 on a genome-wide basis in differentiating mouse ES cells. HOXA1-bound regions are enriched for clusters of consensus binding motifs for HOX, PBX, and MEIS, and many display co-occupancy of PBX and MEIS. PBX and MEIS are members of the TALE family and genome-wide analysis of multiple TALE members (PBX, MEIS, TGIF, PREP1, and PREP2) shows that nearly all HOXA1 targets display occupancy of one or more TALE members. The combinatorial binding patterns of TALE proteins define distinct classes of HOXA1 targets, which may create functional diversity. Transgenic reporter assays in zebrafish confirm enhancer activities for many HOXA1-bound regions and the importance of HOX-PBX and TGIF motifs for their regulation. Proteomic analyses show that HOXA1 physically interacts on chromatin with PBX, MEIS, and PREP family members, but not with TGIF, suggesting that TGIF may have an independent input into HOXA1-bound regions. Therefore, TALE proteins appear to represent a wide repertoire of HOX cofactors, which may coregulate enhancers through distinct mechanisms. We also discover extensive auto- and cross-regulatory interactions among the Hoxa1 and TALE genes, indicating that the specificity of HOXA1 during development may be regulated though a complex cross-regulatory network of HOXA1 and TALE proteins. This study provides new insight into a regulatory network involving combinatorial interactions between HOXA1 and TALE proteins. © 2017 De Kumar et al.; Published by Cold Spring Harbor Laboratory Press.

Delay-independent stability of genetic regulatory networks.

Science.gov (United States)

Wu, Fang-Xiang

2011-11-01

Genetic regulatory networks can be described by nonlinear differential equations with time delays. In this paper, we study both locally and globally delay-independent stability of genetic regulatory networks, taking messenger ribonucleic acid alternative splicing into consideration. Based on nonnegative matrix theory, we first develop necessary and sufficient conditions for locally delay-independent stability of genetic regulatory networks with multiple time delays. Compared to the previous results, these conditions are easy to verify. Then we develop sufficient conditions for global delay-independent stability for genetic regulatory networks. Compared to the previous results, this sufficient condition is less conservative. To illustrate theorems developed in this paper, we analyze delay-independent stability of two genetic regulatory networks: a real-life repressilatory network with three genes and three proteins, and a synthetic gene regulatory network with five genes and seven proteins. The simulation results show that the theorems developed in this paper can effectively determine the delay-independent stability of genetic regulatory networks.

Complement factor H binds malondialdehyde epitopes and protects from oxidative stress

DEFF Research Database (Denmark)

Weismann, David; Hartvigsen, Karsten; Lauer, Nadine

2011-01-01

peroxidation product that accumulates in many pathophysiological processes, including AMD. Here we identify complement factor H (CFH) as a major MDA-binding protein that can block both the uptake of MDA-modified proteins by macrophages and MDA-induced proinflammatory effects in vivo in mice. The CFH...... polymorphism H402, which is strongly associated with AMD, markedly reduces the ability of CFH to bind MDA, indicating a causal link to disease aetiology. Our findings provide important mechanistic insights into innate immune responses to oxidative stress, which may be exploited in the prevention of and therapy...

Complement inhibitors for age-related macular degeneration.

Science.gov (United States)

Williams, Michael A; McKay, Gareth J; Chakravarthy, Usha

2014-01-15

Given the relatively high prevalence of age-related macular degeneration (AMD) and the increased incidence of AMD as populations age, the results of trials of novel treatments are awaited with much anticipation. The complement cascade describes a series of proteolytic reactions occurring throughout the body that generate proteins with a variety of roles including the initiation and promotion of immune reactions against foreign materials or micro-organisms. The complement cascade is normally tightly regulated, but much evidence implicates complement overactivity in AMD and so it is a logical therapeutic target in the treatment of AMD. To assess the effects and safety of complement inhibitors in the prevention or treatment of advanced AMD. We searched CENTRAL (which contains the Cochrane Eyes and Vision Group Trials Register) (The Cochrane Library 2013, Issue 11), Ovid MEDLINE, Ovid MEDLINE In-Process and Other Non-Indexed Citations, Ovid MEDLINE Daily, Ovid OLDMEDLINE (January 1946 to November 2013), EMBASE (January 1980 to November 2013), Allied and Complementary Medicine Database (AMED) (January 1985 to November 2013), Latin American and Caribbean Literature on Health Sciences (LILACS) (January 1982 to November 2013), OpenGrey (System for Information on Grey Literature in Europe) (www.opengrey.eu/), Web of Science Conference Proceedings Citation Index - Science (CPCI-S) (January 1990 to November 2013), the metaRegister of Controlled Trials (mRCT) (www.controlled-trials.com), ClinicalTrials.gov (www.clinicaltrials.gov) and the WHO International Clinical Trials Registry Platform (ICTRP) (www.who.int/ictrp/search/en). We did not use any date or language restrictions in the electronic searches for trials. We last searched the electronic databases on 21 November 2013. We also performed handsearching of proceedings, from 2012 onwards, of meetings and conferences of specific professional organisations. We planned to include randomised controlled trials (RCTs) with

Lack of a Y-Chromosomal Complement in the Majority of Gestational Trophoblastic Neoplasms

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Kai Lee Yap

2010-01-01

Full Text Available Gestational trophoblastic neoplasms (GTNs are a rare group of neoplastic diseases composed of choriocarcinomas, placental site trophoblastic tumors (PSTTs and epithelioid trophoblastic tumors (ETTs. Since these tumors are derivatives of fetal trophoblastic tissue, approximately 50% of GTN cases are expected to originate from a male conceptus and carry a Y-chromosomal complement according to a balanced sex ratio. To investigate this hypothesis, we carried out a comprehensive analysis by genotyping a relatively large sample size of 51 GTN cases using three independent sex chromosome genetic markers; Amelogenin, Protein Kinase and Zinc Finger have X and Y homologues that are distinguishable by their PCR product size. We found that all cases contained the X-chromosomal complement while only five (10% of 51 tumors harbored the Y-chromosomal complement. Specifically, Y-chromosomal signals were detected in one (5% of 19 choriocarcinomas, one (7% of 15 PSTTs and three (18% of 17 ETTs. The histopathological features of those with a Y-chromosome were similar to those without. Our results demonstrate the presence of a Y-chromosomal complement in GTNs, albeit a low 10% of cases. This shortfall of Y-chromosomal complements in GTNs may reinforce the notion that the majority of GTNs are derived from previous molar gestations.

Crystallization and preliminary X-ray diffraction analysis of iron regulatory protein 1 in complex with ferritin IRE RNA

International Nuclear Information System (INIS)

Selezneva, Anna I.; Cavigiolio, Giorgio; Theil, Elizabeth C.; Walden, William E.; Volz, Karl

2006-01-01

The iron regulatory protein IRP1 has been crystallized in a complex with ferritin IRE RNA and a complete data set has been collected to 2.8 Å resolution. Iron regulatory protein 1 (IRP1) is a bifunctional protein with activity as an RNA-binding protein or as a cytoplasmic aconitase. Interconversion of IRP1 between these mutually exclusive states is central to cellular iron regulation and is accomplished through iron-responsive assembly and disassembly of a [4Fe–4S] cluster. When in its apo form, IRP1 binds to iron responsive elements (IREs) found in mRNAs encoding proteins of iron storage and transport and either prevents translation or degradation of the bound mRNA. Excess cellular iron stimulates the assembly of a [4Fe–4S] cluster in IRP1, inhibiting its IRE-binding ability and converting it to an aconitase. The three-dimensional structure of IRP1 in its different active forms will provide details of the interconversion process and clarify the selective recognition of mRNA, Fe–S sites and catalytic activity. To this end, the apo form of IRP1 bound to a ferritin IRE was crystallized. Crystals belong to the monoclinic space group P2 1 , with unit-cell parameters a = 109.6, b = 80.9, c = 142.9 Å, β = 92.0°. Native data sets have been collected from several crystals with resolution extending to 2.8 Å and the structure has been solved by molecular replacement

Genetic Association of the Porcine C9 Complement Component with Hemolytic Complement Activity

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D. V. A. Khoa

2015-09-01

Full Text Available The complement system is a part of the natural immune regulation mechanism against invading pathogens. Complement activation from three different pathways (classical, lectin, and alternative leads to the formation of C5-convertase, an enzyme for cleavage of C5 into C5a and C5b, followed by C6, C7, C8, and C9 in membrane attack complex. The C9 is the last complement component of the terminal lytic pathway, which plays an important role in lysis of the target cells depending on its self-polymerization to form transmembrane channels. To address the association of C9 with traits related to disease resistance, the complete porcine C9 cDNA was comparatively sequenced to detect single nucleotide polymorphisms (SNPs in pigs of the breeds Hampshire (HS, Duroc (DU, Berlin miniature pig (BMP, German Landrace (LR, Pietrain (PIE, and Muong Khuong (Vietnamese potbelly pig. Genotyping was performed in 417 F2 animals of a resource population (DUMI: DU×BMP that were vaccinated with Mycoplasma hyopneumoniae, Aujeszky diseases virus and porcine respiratory and reproductive syndrome virus at 6, 14 and 16 weeks of age, respectively. Two SNPs were detected within the third exon. One of them has an amino acid substitution. The European porcine breeds (LR and PIE show higher allele frequency of these SNPs than Vietnamese porcine breed (MK. Association of the substitution SNP with hemolytic complement activity indicated statistically significant differences between genotypes in the classical pathway but not in the alternative pathway. The interactions between eight time points of measurement of complement activity before and after vaccinations and genotypes were significantly different. The difference in hemolytic complement activity in the both pathways depends on genotype, kind of vaccine, age and the interaction to the other complement components. These results promote the porcine C9 (pC9 as a candidate gene to improve general animal health in the future.

Multi-faceted proteomic characterization of host protein complement of Rift Valley fever virus virions and identification of specific heat shock proteins, including HSP90, as important viral host factors.

Science.gov (United States)

Nuss, Jonathan E; Kehn-Hall, Kylene; Benedict, Ashwini; Costantino, Julie; Ward, Michael; Peyser, Brian D; Retterer, Cary J; Tressler, Lyal E; Wanner, Laura M; McGovern, Hugh F; Zaidi, Anum; Anthony, Scott M; Kota, Krishna P; Bavari, Sina; Hakami, Ramin M

2014-01-01

Rift Valley fever is a potentially fatal disease of humans and domestic animals caused by Rift Valley fever virus (RVFV). Infection with RVFV in ruminants can cause near 100% abortion rates and recent outbreaks in naïve human populations have suggested case fatality rates of greater than thirty percent. To elucidate the roles that host proteins play during RVFV infection, proteomic analysis of RVFV virions was conducted using complementary analytical approaches, followed by functional validation studies of select identified host factors. Coupling the more traditional Gel LC/MS/MS approach (SDS PAGE followed by liquid chromatography tandem mass spectrometry) with an alternative technique that preserves protein complexes allowed the protein complement of these viral particles to be thoroughly examined. In addition to viral proteins present within the virions and virion-associated host proteins, multiple macromolecular complexes were identified. Bioinformatic analysis showed that host chaperones were among over-represented protein families associated with virions, and functional experiments using siRNA gene silencing and small molecule inhibitors identified several of these heat shock proteins, including heat shock protein 90 (HSP90), as important viral host factors. Further analysis indicated that HSP inhibition effects occur during the replication/transcription phase of the virus life cycle, leading to significant lowering of viral titers without compromising the functional capacity of released virions. Overall, these studies provide much needed further insight into interactions between RVFV and host cells, increasing our understanding of the infection process and suggesting novel strategies for anti-viral development. In particular, considering that several HSP90 inhibitors have been advancing through clinical trials for cancer treatment, these results also highlight the exciting potential of repurposing HSP90 inhibitors to treat RVF.

Multi-faceted proteomic characterization of host protein complement of Rift Valley fever virus virions and identification of specific heat shock proteins, including HSP90, as important viral host factors.

Directory of Open Access Journals (Sweden)

Jonathan E Nuss

Full Text Available Rift Valley fever is a potentially fatal disease of humans and domestic animals caused by Rift Valley fever virus (RVFV. Infection with RVFV in ruminants can cause near 100% abortion rates and recent outbreaks in naïve human populations have suggested case fatality rates of greater than thirty percent. To elucidate the roles that host proteins play during RVFV infection, proteomic analysis of RVFV virions was conducted using complementary analytical approaches, followed by functional validation studies of select identified host factors. Coupling the more traditional Gel LC/MS/MS approach (SDS PAGE followed by liquid chromatography tandem mass spectrometry with an alternative technique that preserves protein complexes allowed the protein complement of these viral particles to be thoroughly examined. In addition to viral proteins present within the virions and virion-associated host proteins, multiple macromolecular complexes were identified. Bioinformatic analysis showed that host chaperones were among over-represented protein families associated with virions, and functional experiments using siRNA gene silencing and small molecule inhibitors identified several of these heat shock proteins, including heat shock protein 90 (HSP90, as important viral host factors. Further analysis indicated that HSP inhibition effects occur during the replication/transcription phase of the virus life cycle, leading to significant lowering of viral titers without compromising the functional capacity of released virions. Overall, these studies provide much needed further insight into interactions between RVFV and host cells, increasing our understanding of the infection process and suggesting novel strategies for anti-viral development. In particular, considering that several HSP90 inhibitors have been advancing through clinical trials for cancer treatment, these results also highlight the exciting potential of repurposing HSP90 inhibitors to treat RVF.

Complement-coagulation cross-talk: a potential mediator of the physiological activation of complement by low pH

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Hany Ibrahim Kenawy

2015-05-01

Full Text Available The complement system is a major constituent of the innate immune system. It not only bridges innate and adaptive arms of the immune system but also links the immune system with the coagulation system. Current understanding of the role of complement has extended far beyond fighting of infections, and now encompasses maintenance of homeostasis, tissue regeneration and pathophysiology of multiple diseases. It has been known for many years that complement activation is strongly pH sensitive, but only relatively recently has the physiological significance of this been appreciated. Most complement assays are carried out at the physiological pH 7.4. However, pH in some extracellular compartments, for example renal tubular fluid in parts of the tubule, and extracellular fluid at inflammation loci, is sufficiently acidic to activate complement. The exact molecular mechanism of this activation is still unclear, but possible cross talk between the contact system and complement may exist at low pH with subsequent complement activation. The current article reviews the published data on the effect of pH on the contact system and complement activity, the nature of the pH sensor molecules, and the clinical implications of these effects. Of particular interest is chronic kidney disease (CKD accompanied by metabolic acidosis, in which therapeutic alkalinisation of urine has been shown significantly to reduce tubular complement activation products, an effect which may have important implications for slowing progression of CKD.

Recurrent rewiring and emergence of RNA regulatory networks.

Science.gov (United States)

Wilinski, Daniel; Buter, Natascha; Klocko, Andrew D; Lapointe, Christopher P; Selker, Eric U; Gasch, Audrey P; Wickens, Marvin

2017-04-04

Alterations in regulatory networks contribute to evolutionary change. Transcriptional networks are reconfigured by changes in the binding specificity of transcription factors and their cognate sites. The evolution of RNA-protein regulatory networks is far less understood. The PUF (Pumilio and FBF) family of RNA regulatory proteins controls the translation, stability, and movements of hundreds of mRNAs in a single species. We probe the evolution of PUF-RNA networks by direct identification of the mRNAs bound to PUF proteins in budding and filamentous fungi and by computational analyses of orthologous RNAs from 62 fungal species. Our findings reveal that PUF proteins gain and lose mRNAs with related and emergent biological functions during evolution. We demonstrate at least two independent rewiring events for PUF3 orthologs, independent but convergent evolution of PUF4/5 binding specificity and the rewiring of the PUF4/5 regulons in different fungal lineages. These findings demonstrate plasticity in RNA regulatory networks and suggest ways in which their rewiring occurs.

Incident microalbuminuria and complement factor mannan-binding lectin-associated protein 19 in people with newly diagnosed type 1 diabetes

DEFF Research Database (Denmark)

Ostergaard, J A; Thiel, S; Hoffmann-Petersen, I T

2017-01-01

BACKGROUND: Evidence links the lectin pathway of complement activation to diabetic kidney disease. Upon carbohydrate-recognition by pattern-recognition molecules, e.g., mannan-binding lectin (MBL), the MBL-associated serine protease (MASP-2) is activated and initiates the complement cascade. The ...

GLUCOCORTICOSTEROIDS' EFFECT UPON THE COMPLEMENT LEVEL

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Voja Pavlovic

2001-03-01

Full Text Available The effect of high doses of cortisol upon the level of the overall complements'hemolytic activity and particular complements' components is studies. The experimentsinvolved guinea pigs of male sex of the body mass from 300 to 400 g, namelythose that have not been treated by anything so far. The doses of hydrocortisone(Hemofarm DD were also used for the experiment. The overall complements'activity was determined by testing the capabilities of a series of various solutions ofthe guinea pigs' serum to separate sheep erythrocytes that were made sensitive byrabbit anti-erythrocyte antibodies. The determination of the C1, C2, C3 and C4complements' components was done by the method of the quantitative diffusion ofthe radial type by using the Partigen blocks Behringwerke AG. The series comprised25 guinea pigs of male sex. The low cortisol level rapidly increase the overallhemolytic activity of the complements of the C1 est erase concentration. Along withthe cortisol dose increase the overall hemolytic complements' activity is dropping aswell as that of the C1, C2, C3 and C4 complements' components.

An Anti-C1s Monoclonal, TNT003, Inhibits Complement Activation Induced by Antibodies Against HLA.

Science.gov (United States)

Thomas, K A; Valenzuela, N M; Gjertson, D; Mulder, A; Fishbein, M C; Parry, G C; Panicker, S; Reed, E F

2015-08-01

Antibody-mediated rejection (AMR) of solid organ transplants (SOT) is characterized by damage triggered by donor-specific antibodies (DSA) binding donor Class I and II HLA (HLA-I and HLA-II) expressed on endothelial cells. While F(ab')2 portions of DSA cause cellular activation and proliferation, Fc regions activate the classical complement cascade, resulting in complement deposition and leukocyte recruitment, both hallmark features of AMR. We characterized the ability of an anti-C1s monoclonal antibody, TNT003, to inhibit HLA antibody (HLA-Ab)-induced complement activation. Complement deposition induced by HLA-Ab was evaluated using novel cell- and bead-based assays. Human aortic endothelial cells (HAEC) were cultured with HLA-Ab and human complement; production of activated complement proteins was measured by flow cytometry. Additionally, C3d deposition was measured on single antigen beads (SAB) mixed with HLA-Ab and human complement. TNT003 inhibited HLA-Ab mediated complement deposition on HAEC in a concentration-dependent manner; C3a, C4a and C5a anaphylatoxin production was also diminished by TNT003. Finally, TNT003 blocked C3d deposition induced by Class I (HLAI-Ab)- and Class II (HLAII-Ab)-specific antibodies on SAB. These data suggest TNT003 may be useful for modulating the effects of DSA, as TNT003 inhibits complement deposition and split product formation generated by HLA-I/II-Ab in vitro. © 2015 The Authors. American Journal of Transplantation Published by Wiley Periodicals, Inc.

Expression, purification, cocrystallization and preliminary crystallographic analysis of sucrose octasulfate/human complement regulator factor H SCRs 6–8

Energy Technology Data Exchange (ETDEWEB)

Prosser, Beverly E.; Johnson, Steven; Roversi, Pietro [The Sir William Dunn School of Pathology, The University of Oxford, South Parks Road, Oxford OX1 3RE (United Kingdom); Clark, Simon J. [Faculty of Life Sciences, Manchester University, Michael Smith Building, Oxford Road, Manchester M13 9PT (United Kingdom); Tarelli, Edward [Medical Biomics Centre, St George’s, University of London, Cranmer Terrace, London SW17 0RE (United Kingdom); Sim, Robert B. [The MRC Immunochemistry Unit, The University of Oxford, South Parks Road, Oxford OX1 3RE (United Kingdom); Day, Antony J. [Faculty of Life Sciences, Manchester University, Michael Smith Building, Oxford Road, Manchester M13 9PT (United Kingdom); Lea, Susan M., E-mail: susan.lea@bnc.ox.ac.uk [The Sir William Dunn School of Pathology, The University of Oxford, South Parks Road, Oxford OX1 3RE (United Kingdom)

2007-06-01

The crystallization of human complement regulator FH-678{sub 402H} with a glycosaminoglycan analogue is described. Human plasma protein complement factor H (FH) is an inhibitor of the spontaneously activated alternative complement pathway. An allotypic variant of FH, 402His, has been associated with age-related macular degeneration, the leading cause of blindness in the elderly. Crystals of FH domains 6–8 (FH678) containing 402His have been grown in the presence of a polyanionic sucrose octasulfate ligand (an analogue of the natural glycosaminoglycan ligands of FH) using both native and selenomethionine-derivatized protein. Native data sets diffracting to 2.3 Å and SeMet data sets of up to 2.8 Å resolution have been collected. An anomalous difference Patterson map reveals self- and cross-peaks from two incorporated Se atoms. The corresponding selenium substructure has been solved.

Methodology for the Systematic Assessment of the Regulatory Competence Needs (SARCoN) for Regulatory Bodies of Nuclear Installations

International Nuclear Information System (INIS)

2015-03-01

A regulatory body’s competence is dependent, among other things, on the competence of its staff. A necessary, but not sufficient, condition for a regulatory body to be competent is that its staff can perform the tasks related to the functions of the regulatory body. In 2001, the IAEA published TECDOC 1254, Training the Staff of the Regulatory Body for Nuclear Facilities: A Competency Framework, which examines the manner in which the recognized regulatory functions of a nuclear regulatory body results in competence needs. Using the internationally recognized systematic approach to training, TECDOC 1254 provides a framework for regulatory bodies for managing training and developing, and maintaining the competence of its staff. It has been successfully used by many regulatory bodies all over the world, including States embarking on a nuclear power programme. The IAEA has also introduced a methodology and an assessment tool — Guidelines for Systematic Assessment of Regulatory Competence Needs (SARCoN) — which provides practical guidance on analysing the training and development needs of a regulatory body and, through a gap analysis, guidance on establishing competence needs and how to meet them. In 2013, the IAEA published Safety Reports Series No. 79, Managing Regulatory Body Competence, which provides generic guidance based on IAEA safety requirements in the development of a competence management system within a regulatory body’s integrated management system. An appendix in the Safety Report deals with the special case of building up the competence of regulatory bodies as part of the overall process of establishing an embarking State’s regulatory system. This publication provides guidance for the analysis of required and existing competences to identify those required by the regulatory body to perform its functions and therefore associated needs for acquiring competences. Hence, it is equally applicable to the needs of States embarking on nuclear power

DNA-binding site of major regulatory protein alpha 4 specifically associated with promoter-regulatory domains of alpha genes of herpes simplex virus type 1.

OpenAIRE

Kristie, T M; Roizman, B

1986-01-01

Herpes simplex virus type 1 genes form at least five groups (alpha, beta 1, beta 2, gamma 1, and gamma 2) whose expression is coordinately regulated and sequentially ordered in a cascade fashion. Previous studies have shown that functional alpha 4 gene product is essential for the transition from alpha to beta protein synthesis and have suggested that alpha 4 gene expression is autoregulatory. We have previously reported that labeled DNA fragments containing promoter-regulatory domains of thr...

Feeding common carp Cyprinus carpio with b-glucan supplemented \\ud diet stimulates C-reactive protein and complement immune acute\\ud phase responses following PAMPs injection

OpenAIRE

Pionnier, Nicolas; Falco, Alberto; Miest, Joanna J.; Shrive, Annette K.; Hoole, Dave

2014-01-01

The effect of β-glucan as a feed additive on the serum and gene profile of C-reactive protein (CRP) and complement acute phase responses was ascertained in common carp Cyprinus carpio. In addition effects of subsequent intraperitoneal injections of pathogen-associated molecular patterns (PAMPs), i.e. LPS or poly(I:C), to mimic bacterial or viral infection respectively, were studied. Carp were first orally fed with β-glucan (MacroGard®) with a daily β-glucan intake of 6 mg per kg body weight o...

77 FR 61033 - Self-Regulatory Organizations; National Securities Clearing Corporation; Order Approving Proposed...

Science.gov (United States)

2012-10-05

...) NSCC's automated ACATS-Fund/SERV interface, for eligible mutual fund assets; (iii) NSCC's ACATS-IPS... customer account asset data. \\4\\ ACATS complements Financial Industry Regulatory Authority (``FINRA'') Rule... positions and available securities are allocated by book entry. This allocation of securities is...

The regulatory beta-subunit of protein kinase CK2 regulates cell-cycle progression at the onset of mitosis

DEFF Research Database (Denmark)

Yde, C W; Olsen, B B; Meek, D

2008-01-01

25 dual-specificity phosphatase family members. In somatic cells, Wee1 is downregulated by phosphorylation and ubiquitin-mediated degradation to ensure rapid activation of CDK1 at the beginning of M phase. Here, we show that downregulation of the regulatory beta-subunit of protein kinase CK2 by RNA...

Nuclear Magnetic Resonance structural studies of peptides and proteins from the vaso-regulatory System

International Nuclear Information System (INIS)

Sizun, Philippe

1991-01-01

The aim of the present work is to show how Nuclear Magnetic Resonance (NMR) allows to determine the 3D structure of peptides and proteins in solution. A comparative study of peptides involved in the vaso-regulatory System (form small hormonal peptide to the 65 amido-acid protein hirudin) has allowed to design most efficient NMR 1D and 2D strategies. It rapidly appeared that the size of the peptide plays a key role in the structuration of the molecule, smallest peptides being weakly structured owing to the lack of cooperative effects. As the molecular size increases or if conformational locks are present (disulfide bridges) the probability of stable secondary structure increases. For the protein hirudin, a combination of ail available NMR parameters deduced form dedicated experiments (chemical shifts, coupling constants, overhauser effects, accessibility of amide protons) and molecular modelling under constraints allows a clear 3D structure to be proposed for this protein in solution. Finally, a comparative study of the experimental structures and of those deduced form prediction rules has shed light on the concept of structural predisposition, the latter being of high value for a better understanding of structure-activity relationships. (author) [fr

Identification of the Fanconi Anemia Complementation Group I Gene, FANCI

Directory of Open Access Journals (Sweden)

Josephine C. Dorsman

2007-01-01

Full Text Available To identify the gene underlying Fanconi anemia (FA complementation group I we studied informative FA-I families by a genome-wide linkage analysis, which resulted in 4 candidate regions together encompassing 351 genes. Candidates were selected via bioinformatics and data mining on the basis of their resemblance to other FA genes/proteins acting in the FA pathway, such as: degree of evolutionary conservation, presence of nuclear localization signals and pattern of tissue-dependent expression. We found a candidate, KIAA1794 on chromosome 15q25-26, to be mutated in 8 affected individuals previously assigned to complementation group I. Western blots of endogenous FANCI indicated that functionally active KIAA1794 protein is lacking in FA-I individuals. Knock-down of KIAA1794 expression by siRNA in HeLa cells caused excessive chromosomal breakage induced by mitomycin C, a hallmark of FA cells. Furthermore, phenotypic reversion of a patient-derived cell line was associated with a secondary genetic alteration at the KIAA1794 locus. These data add up to two conclusions. First, KIAA1794 is a FA gene. Second, this gene is identical to FANCI, since the patient cell lines found mutated in this study included the reference cell line for group I, EUFA592.

Complement activation by Pseudomonas aeruginosa biofilms

DEFF Research Database (Denmark)

Jensen, E T; Kharazmi, A; Garred, P

1993-01-01

In chronic infections, such as the bronchopulmonary Pseudomonas aeruginosa infection in cystic fibrosis (CF) patients, bacteria persist despite an intact host immune defense and frequent antibiotic treatment. An important reason for the persistence of the bacteria is their capacity for the biofilm...... mode of growth. In this study we investigated the role of biofilms in activation of complement, a major contributor to the inflammatory process. Complement activation by P. aeruginosa was examined in a complement consumption assay, production of C3 and factor B conversion products assessed by crossed...... immuno-electrophoresis, C5a generation tested by a PMN chemotactic assay, and terminal complement complex formation measured by ELISA. Two of the four assays showed that P. aeruginosa grown in biofilm activated complement less than planktonic bacteria, and all assays showed that activation by intact...

Material properties in complement activation

DEFF Research Database (Denmark)

Moghimi, S. Moein; Andersen, Alina Joukainen; Ahmadvand, Davoud

2011-01-01

activation differently and through different sensing molecules and initiation pathways. The importance of material properties in triggering complement is considered and mechanistic aspects discussed. Mechanistic understanding of complement events could provide rational approaches for improved material design...

Co-evolution of transcriptional silencing proteins and the DNA elements specifying their assembly.

Directory of Open Access Journals (Sweden)

Oliver A Zill

Full Text Available Co-evolution of transcriptional regulatory proteins and their sites of action has been often hypothesized but rarely demonstrated. Here we provide experimental evidence of such co-evolution in yeast silent chromatin, a finding that emerged from studies of hybrids formed between two closely related Saccharomyces species. A unidirectional silencing incompatibility between S. cerevisiae and S. bayanus led to a key discovery: asymmetrical complementation of divergent orthologs of the silent chromatin component Sir4. In S. cerevisiae/S. bayanus interspecies hybrids, ChIP-Seq analysis revealed a restriction against S. cerevisiae Sir4 associating with most S. bayanus silenced regions; in contrast, S. bayanus Sir4 associated with S. cerevisiae silenced loci to an even greater degree than did S. cerevisiae's own Sir4. Functional changes in silencer sequences paralleled changes in Sir4 sequence and a reduction in Sir1 family members in S. cerevisiae. Critically, species-specific silencing of the S. bayanus HMR locus could be reconstituted in S. cerevisiae by co-transfer of the S. bayanus Sir4 and Kos3 (the ancestral relative of Sir1 proteins. As Sir1/Kos3 and Sir4 bind conserved silencer-binding proteins, but not specific DNA sequences, these rapidly evolving proteins served to interpret differences in the two species' silencers presumably involving emergent features created by the regulatory proteins that bind sequences within silencers. The results presented here, and in particular the high resolution ChIP-Seq localization of the Sir4 protein, provided unanticipated insights into the mechanism of silent chromatin assembly in yeast.

Partitioning of genetic variation between regulatory and coding gene segments: the predominance of software variation in genes encoding introvert proteins.

Science.gov (United States)

Mitchison, A

1997-01-01

In considering genetic variation in eukaryotes, a fundamental distinction can be made between variation in regulatory (software) and coding (hardware) gene segments. For quantitative traits the bulk of variation, particularly that near the population mean, appears to reside in regulatory segments. The main exceptions to this rule concern proteins which handle extrinsic substances, here termed extrovert proteins. The immune system includes an unusually large proportion of this exceptional category, but even so its chief source of variation may well be polymorphism in regulatory gene segments. The main evidence for this view emerges from genome scanning for quantitative trait loci (QTL), which in the case of the immune system points to a major contribution of pro-inflammatory cytokine genes. Further support comes from sequencing of major histocompatibility complex (Mhc) class II promoters, where a high level of polymorphism has been detected. These Mhc promoters appear to act, in part at least, by gating the back-signal from T cells into antigen-presenting cells. Both these forms of polymorphism are likely to be sustained by the need for flexibility in the immune response. Future work on promoter polymorphism is likely to benefit from the input from genome informatics.

Cell Cycle and Apoptosis Regulatory Protein (CARP)-1 is Expressed inOsteoblasts and Regulated by PTH

International Nuclear Information System (INIS)

Sharma, Sonali; Mahalingam, Chandrika D.; Das, Varsha; Jamal, Shazia; Levi, Edi; Rishi, Arun K.; Datta, Nabanita S.

2013-01-01

Highlights: •CARP-1 is identified for the first time in bone cells. •PTH downregulates CARP-1 expression in differentiated osteoblasts. •PTH displaces CARP-1 from nucleus to the cytoplasm in differentiated osteoblasts. •Downregulation of CARP-1 by PTH involves PKA, PKC and P-p38 MAPK pathways. -- Abstract: Bone mass is dependent on osteoblast proliferation, differentiation and life-span of osteoblasts. Parathyroid hormone (PTH) controls osteoblast cell cycle regulatory proteins and suppresses mature osteoblasts apoptosis. Intermittent administration of PTH increases bone mass but the mechanism of action are complex and incompletely understood. Cell Cycle and Apoptosis Regulatory Protein (CARP)-1 (aka CCAR1) is a novel transducer of signaling by diverse agents including cell growth and differentiation factors. To gain further insight into the molecular mechanism, we investigated involvement of CARP-1 in PTH signaling in osteoblasts. Immunostaining studies revealed presence of CARP-1 in osteoblasts and osteocytes, while a minimal to absent levels were noted in the chondrocytes of femora from 10 to 12-week old mice. Treatment of 7-day differentiated MC3T3-E1 clone-4 (MC-4) mouse osteoblastic cells and primary calvarial osteoblasts with PTH for 30 min to 5 h followed by Western blot analysis showed 2- to 3-fold down-regulation of CARP-1 protein expression in a dose- and time-dependent manner compared to the respective vehicle treated control cells. H-89, a Protein Kinase A (PKA) inhibitor, suppressed PTH action on CARP-1 protein expression indicating PKA-dependent mechanism. PMA, a Protein Kinase C (PKC) agonist, mimicked PTH action, and the PKC inhibitor, GF109203X, partially blocked PTH-dependent downregulation of CARP-1, implying involvement of PKC. U0126, a Mitogen-Activated Protein Kinase (MAPK) Kinase (MEK) inhibitor, failed to interfere with CARP-1 suppression by PTH. In contrast, SB203580, p38 inhibitor, attenuated PTH down-regulation of CARP-1

Cell Cycle and Apoptosis Regulatory Protein (CARP)-1 is Expressed inOsteoblasts and Regulated by PTH

Energy Technology Data Exchange (ETDEWEB)

Sharma, Sonali; Mahalingam, Chandrika D.; Das, Varsha [Department of Internal Medicine/Endocrinology, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Jamal, Shazia [Department of Oncology, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Levi, Edi [Department of Oncology, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Department of Pathology, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Rishi, Arun K. [Department of Oncology, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, MI 48201 (United States); VA Medical Center, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Datta, Nabanita S., E-mail: ndatta@med.wayne.edu [Department of Internal Medicine/Endocrinology, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Cardiovascular Research Institute, Wayne State University School of Medicine, Detroit, MI 48201 (United States)

2013-07-12

Highlights: •CARP-1 is identified for the first time in bone cells. •PTH downregulates CARP-1 expression in differentiated osteoblasts. •PTH displaces CARP-1 from nucleus to the cytoplasm in differentiated osteoblasts. •Downregulation of CARP-1 by PTH involves PKA, PKC and P-p38 MAPK pathways. -- Abstract: Bone mass is dependent on osteoblast proliferation, differentiation and life-span of osteoblasts. Parathyroid hormone (PTH) controls osteoblast cell cycle regulatory proteins and suppresses mature osteoblasts apoptosis. Intermittent administration of PTH increases bone mass but the mechanism of action are complex and incompletely understood. Cell Cycle and Apoptosis Regulatory Protein (CARP)-1 (aka CCAR1) is a novel transducer of signaling by diverse agents including cell growth and differentiation factors. To gain further insight into the molecular mechanism, we investigated involvement of CARP-1 in PTH signaling in osteoblasts. Immunostaining studies revealed presence of CARP-1 in osteoblasts and osteocytes, while a minimal to absent levels were noted in the chondrocytes of femora from 10 to 12-week old mice. Treatment of 7-day differentiated MC3T3-E1 clone-4 (MC-4) mouse osteoblastic cells and primary calvarial osteoblasts with PTH for 30 min to 5 h followed by Western blot analysis showed 2- to 3-fold down-regulation of CARP-1 protein expression in a dose- and time-dependent manner compared to the respective vehicle treated control cells. H-89, a Protein Kinase A (PKA) inhibitor, suppressed PTH action on CARP-1 protein expression indicating PKA-dependent mechanism. PMA, a Protein Kinase C (PKC) agonist, mimicked PTH action, and the PKC inhibitor, GF109203X, partially blocked PTH-dependent downregulation of CARP-1, implying involvement of PKC. U0126, a Mitogen-Activated Protein Kinase (MAPK) Kinase (MEK) inhibitor, failed to interfere with CARP-1 suppression by PTH. In contrast, SB203580, p38 inhibitor, attenuated PTH down-regulation of CARP-1

iTRAQ-Based Quantitative Proteomics Identifies Potential Regulatory Proteins Involved in Chicken Eggshell Brownness.

Directory of Open Access Journals (Sweden)

Guangqi Li

Full Text Available Brown eggs are popular in many countries and consumers regard eggshell brownness as an important indicator of egg quality. However, the potential regulatory proteins and detailed molecular mechanisms regulating eggshell brownness have yet to be clearly defined. In the present study, we performed quantitative proteomics analysis with iTRAQ technology in the shell gland epithelium of hens laying dark and light brown eggs to investigate the candidate proteins and molecular mechanisms underlying variation in chicken eggshell brownness. The results indicated 147 differentially expressed proteins between these two groups, among which 65 and 82 proteins were significantly up-regulated in the light and dark groups, respectively. Functional analysis indicated that in the light group, the down-regulated iron-sulfur cluster assembly protein (Iba57 would decrease the synthesis of protoporphyrin IX; furthermore, the up-regulated protein solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator, member 5 (SLC25A5 and down-regulated translocator protein (TSPO would lead to increased amounts of protoporphyrin IX transported into the mitochondria matrix to form heme with iron, which is supplied by ovotransferrin protein (TF. In other words, chickens from the light group produce less protoporphyrin IX, which is mainly used for heme synthesis. Therefore, the exported protoporphyrin IX available for eggshell deposition and brownness is reduced in the light group. The current study provides valuable information to elucidate variation of chicken eggshell brownness, and demonstrates the feasibility and sensitivity of iTRAQ-based quantitative proteomics analysis in providing useful insights into the molecular mechanisms underlying brown eggshell pigmentation.

Live cell imaging of interactions between replicase and capsid protein of Brome mosaic virus using Bimolecular Fluorescence Complementation: Implications for replication and genome packaging

International Nuclear Information System (INIS)

Chaturvedi, Sonali; Rao, A.L.N.

2014-01-01

In Brome mosaic virus, it was hypothesized that a physical interaction between viral replicase and capsid protein (CP) is obligatory to confer genome packaging specificity. Here we tested this hypothesis by employing Bimolecular Fluorescent Complementation (BiFC) as a tool for evaluating protein–protein interactions in living cells. The efficacy of BiFC was validated by a known interaction between replicase protein 1a (p1a) and protein 2a (p2a) at the endoplasmic reticulum (ER) site of viral replication. Additionally, co-expression in planta of a bona fide pair of interacting protein partners of p1a and p2a had resulted in the assembly of a functional replicase. Subsequent BiFC assays in conjunction with mCherry labeled ER as a fluorescent cellular marker revealed that CP physically interacts with p2a, but not p1a, and this CP:p2a interaction occurs at the cytoplasmic phase of the ER. The significance of the CP:p2a interaction in BMV replication and genome packaging is discussed. - Highlights: • YFP fusion proteins of BMV p1a and p2a are biologically active. • Self-interaction was observed for p1a, p2a and CP. • CP interacts with p2a but not p1a. • Majority of reconstituted YFP resulting from bona fide fusion protein partners localized on ER

Live cell imaging of interactions between replicase and capsid protein of Brome mosaic virus using Bimolecular Fluorescence Complementation: Implications for replication and genome packaging

Energy Technology Data Exchange (ETDEWEB)

Chaturvedi, Sonali; Rao, A.L.N., E-mail: arao@ucr.edu

2014-09-15

In Brome mosaic virus, it was hypothesized that a physical interaction between viral replicase and capsid protein (CP) is obligatory to confer genome packaging specificity. Here we tested this hypothesis by employing Bimolecular Fluorescent Complementation (BiFC) as a tool for evaluating protein–protein interactions in living cells. The efficacy of BiFC was validated by a known interaction between replicase protein 1a (p1a) and protein 2a (p2a) at the endoplasmic reticulum (ER) site of viral replication. Additionally, co-expression in planta of a bona fide pair of interacting protein partners of p1a and p2a had resulted in the assembly of a functional replicase. Subsequent BiFC assays in conjunction with mCherry labeled ER as a fluorescent cellular marker revealed that CP physically interacts with p2a, but not p1a, and this CP:p2a interaction occurs at the cytoplasmic phase of the ER. The significance of the CP:p2a interaction in BMV replication and genome packaging is discussed. - Highlights: • YFP fusion proteins of BMV p1a and p2a are biologically active. • Self-interaction was observed for p1a, p2a and CP. • CP interacts with p2a but not p1a. • Majority of reconstituted YFP resulting from bona fide fusion protein partners localized on ER.

Heat shock protein 70 inhibits shrinkage-induced programmed cell death via mechanisms independent of effects on cell volume-regulatory membrane transport proteins

DEFF Research Database (Denmark)

Nylandsted, J; Jäättelä, M; Hoffmann, E K

2004-01-01

Cell shrinkage is a ubiquitous feature of programmed cell death (PCD), but whether it is an obligatory signalling event in PCD is unclear. Heat shock protein 70 (Hsp70) potently counteracts PCD in many cells, by mechanisms that are incompletely understood. In the present investigation, we found...... that severe hypertonic stress greatly diminished the viability of murine fibrosarcoma cells (WEHI-902) and immortalized murine embryonic fibroblasts (iMEFs). This effect was attenuated markedly by Hsp70 over-expression. To determine whether the protective effect of Hsp70 was mediated via an effect on volume...... regulatory ion transport, we compared regulatory volume decrease (RVD) and increase (RVI) in control WEHI-902 cells and after increasing Hsp70 levels by heat shock or over-expression (WEHI-912). Hsp70 levels affected neither RVD, RVI nor the relative contributions of the Na(+)/H(+)-exchanger (NHE1) and Na...

Peptide Inhibitor of Complement C1 (PIC1 Rapidly Inhibits Complement Activation after Intravascular Injection in Rats.

Directory of Open Access Journals (Sweden)

Julia A Sharp

Full Text Available The complement system has been increasingly recognized to play a pivotal role in a variety of inflammatory and autoimmune diseases. Consequently, therapeutic modulators of the classical, lectin and alternative pathways of the complement system are currently in pre-clinical and clinical development. Our laboratory has identified a peptide that specifically inhibits the classical and lectin pathways of complement and is referred to as Peptide Inhibitor of Complement C1 (PIC1. In this study, we determined that the lead PIC1 variant demonstrates a salt-dependent binding to C1q, the initiator molecule of the classical pathway. Additionally, this peptide bound to the lectin pathway initiator molecule MBL as well as the ficolins H, M and L, suggesting a common mechanism of PIC1 inhibitory activity occurs via binding to the collagen-like tails of these collectin molecules. We further analyzed the effect of arginine and glutamic acid residue substitution on the complement inhibitory activity of our lead derivative in a hemolytic assay and found that the original sequence demonstrated superior inhibitory activity. To improve upon the solubility of the lead derivative, a pegylated, water soluble variant was developed, structurally characterized and demonstrated to inhibit complement activation in mouse plasma, as well as rat, non-human primate and human serum in vitro. After intravenous injection in rats, the pegylated derivative inhibited complement activation in the blood by 90% after 30 seconds, demonstrating extremely rapid function. Additionally, no adverse toxicological effects were observed in limited testing. Together these results show that PIC1 rapidly inhibits classical complement activation in vitro and in vivo and is functional for a variety of animal species, suggesting its utility in animal models of classical complement-mediated diseases.

Anti-regulatory T cells

DEFF Research Database (Denmark)

Andersen, Mads Hald

2017-01-01

responses to tumours or inhibiting autoimmunity development. However, recent studies report the discovery of self-reactive pro-inflammatory T cells—termed anti-regulatory T cells (anti-Tregs)—that target immune-suppressive cells. Thus, regulatory cells can now be defined as both cells that suppress immune...... reactions as well as effector cells that counteract the effects of suppressor cells and support immune reactions. Self-reactive anti-Tregs have been described that specifically recognize human leukocyte antigen-restricted epitopes derived from proteins that are normally expressed by regulatory immune cells......Our initial understanding of immune-regulatory cells was based on the discovery of suppressor cells that assure peripheral T-cell tolerance and promote immune homeostasis. Research has particularly focused on the importance of regulatory T cells (Tregs) for immune modulation, e.g. directing host...

Regulatory CD4 T cells inhibit HIV-1 expression of other CD4 T cell subsets via interactions with cell surface regulatory proteins.

Science.gov (United States)

Zhang, Mingce; Robinson, Tanya O; Duverger, Alexandra; Kutsch, Olaf; Heath, Sonya L; Cron, Randy Q

2018-03-01

During chronic HIV-1 infection, regulatory CD4 T cells (Tregs) frequently represent the largest subpopulation of CD4 T cell subsets, implying relative resistant to HIV-1. When HIV-1 infection of CD4 T cells was explored in vitro and ex vivo from patient samples, Tregs possessed lower levels of HIV-1 DNA and RNA in comparison with conventional effector and memory CD4 T cells. Moreover, Tregs suppressed HIV-1 expression in other CD4 T cells in an in vitro co-culture system. This suppression was mediated in part via multiple inhibitory surface proteins expressed on Tregs. Antibody blockade of CTLA-4, PD-1, and GARP on Tregs resulted in increased HIV-1 DNA integration and mRNA expression in neighboring CD4 T cells. Moreover, antibody blockade of Tregs inhibitory proteins resulted in increased HIV-1 LTR transcription in co-cultured CD4 T cells. Thus, Tregs inhibit HIV-1 infection of other CD4 T cell subsets via interactions with inhibitory cell surface proteins. Copyright © 2018 Elsevier Inc. All rights reserved.

The Evolution of the Secreted Regulatory Protein Progranulin.

Science.gov (United States)

Palfree, Roger G E; Bennett, Hugh P J; Bateman, Andrew

2015-01-01

Progranulin is a secreted growth factor that is active in tumorigenesis, wound repair, and inflammation. Haploinsufficiency of the human progranulin gene, GRN, causes frontotemporal dementia. Progranulins are composed of chains of cysteine-rich granulin modules. Modules may be released from progranulin by proteolysis as 6kDa granulin polypeptides. Both intact progranulin and some of the granulin polypeptides are biologically active. The granulin module occurs in certain plant proteases and progranulins are present in early diverging metazoan clades such as the sponges, indicating their ancient evolutionary origin. There is only one Grn gene in mammalian genomes. More gene-rich Grn families occur in teleost fish with between 3 and 6 members per species including short-form Grns that have no tetrapod counterparts. Our goals are to elucidate progranulin and granulin module evolution by investigating (i): the origins of metazoan progranulins (ii): the evolutionary relationships between the single Grn of tetrapods and the multiple Grn genes of fish (iii): the evolution of granulin module architectures of vertebrate progranulins (iv): the conservation of mammalian granulin polypeptide sequences and how the conserved granulin amino acid sequences map to the known three dimensional structures of granulin modules. We report that progranulin-like proteins are present in unicellular eukaryotes that are closely related to metazoa suggesting that progranulin is among the earliest extracellular regulatory proteins still employed by multicellular animals. From the genomes of the elephant shark and coelacanth we identified contemporary representatives of a precursor for short-from Grn genes of ray-finned fish that is lost in tetrapods. In vertebrate Grns pathways of exon duplication resulted in a conserved module architecture at the amino-terminus that is frequently accompanied by an unusual pattern of tandem nearly identical module repeats near the carboxyl-terminus. Polypeptide

Critical protein GAPDH and its regulatory mechanisms in cancer cells

International Nuclear Information System (INIS)

Zhang, Jin-Ying; Zhang, Fan; Hong, Chao-Qun; Giuliano, Armando E.; Cui, Xiao-Jiang; Zhou, Guang-Ji; Zhang, Guo-Jun; Cui, Yu-Kun

2015-01-01

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), initially identified as a glycolytic enzyme and considered as a housekeeping gene, is widely used as an internal control in experiments on proteins, mRNA, and DNA. However, emerging evidence indicates that GAPDH is implicated in diverse functions independent of its role in energy metabolism; the expression status of GAPDH is also deregulated in various cancer cells. One of the most common effects of GAPDH is its inconsistent role in the determination of cancer cell fate. Furthermore, studies have described GAPDH as a regulator of cell death; other studies have suggested that GAPDH participates in tumor progression and serves as a new therapeutic target. However, related regulatory mechanisms of its numerous cellular functions and deregulated expression levels remain unclear. GAPDH is tightly regulated at transcriptional and posttranscriptional levels, which are involved in the regulation of diverse GAPDH functions. Several cancer-related factors, such as insulin, hypoxia inducible factor-1 (HIF-1), p53, nitric oxide (NO), and acetylated histone, not only modulate GAPDH gene expression but also affect protein functions via common pathways. Moreover, posttranslational modifications (PTMs) occurring in GAPDH in cancer cells result in new activities unrelated to the original glycolytic function of GAPDH. In this review, recent findings related to GAPDH transcriptional regulation and PTMs are summarized. Mechanisms and pathways involved in GAPDH regulation and its different roles in cancer cells are also described

Novel fusion protein approach for efficient high-throughput screening of small molecule-mediating protein-protein interactions in cells and living animals.

Science.gov (United States)

Paulmurugan, Ramasamy; Gambhir, Sanjiv S

2005-08-15

Networks of protein interactions execute many different intracellular pathways. Small molecules either synthesized within the cell or obtained from the external environment mediate many of these protein-protein interactions. The study of these small molecule-mediated protein-protein interactions is important in understanding abnormal signal transduction pathways in a variety of disorders, as well as in optimizing the process of drug development and validation. In this study, we evaluated the rapamycin-mediated interaction of the human proteins FK506-binding protein (FKBP12) rapamycin-binding domain (FRB) and FKBP12 by constructing a fusion of these proteins with a split-Renilla luciferase or a split enhanced green fluorescent protein (split-EGFP) such that complementation of the reporter fragments occurs in the presence of rapamycin. Different linker peptides in the fusion protein were evaluated for the efficient maintenance of complemented reporter activity. This system was studied in both cell culture and xenografts in living animals. We found that peptide linkers with two or four EAAAR repeat showed higher protein-protein interaction-mediated signal with lower background signal compared with having no linker or linkers with amino acid sequences GGGGSGGGGS, ACGSLSCGSF, and ACGSLSCGSFACGSLSCGSF. A 9 +/- 2-fold increase in signal intensity both in cell culture and in living mice was seen compared with a system that expresses both reporter fragments and the interacting proteins separately. In this fusion system, rapamycin induced heterodimerization of the FRB and FKBP12 moieties occurred rapidly even at very lower concentrations (0.00001 nmol/L) of rapamycin. For a similar fusion system employing split-EGFP, flow cytometry analysis showed significant level of rapamycin-induced complementation.

Cross-species complementation of bacterial- and eukaryotic-type cardiolipin synthases

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Petra Gottier

2017-11-01

Full Text Available The glycerophospholipid cardiolipin is a unique constituent of bacterial and mitochondrial membranes. It is involved in forming and stabilizing high molecular mass membrane protein complexes and in maintaining membrane architecture. Absence of cardiolipin leads to reduced efficiency of the electron transport chain, decreased membrane potential, and, ultimately, impaired respiratory metabolism. For the protozoan parasite Trypanosoma brucei cardiolipin synthesis is essential for survival, indicating that the enzymes involved in cardiolipin production represent potential drug targets. T. brucei cardiolipin synthase (TbCLS is unique as it belongs to the family of phospholipases D (PLD, harboring a prokaryotic-type cardiolipin synthase (CLS active site domain. In contrast, most other eukaryotic CLS, including the yeast ortholog ScCrd1, are members of the CDP-alcohol phosphatidyl­ transferase family. To study if these mechanistically distinct CLS enzymes are able to catalyze cardiolipin production in a cell that normally expresses a different type of CLS, we expressed TbCLS and ScCrd1 in CLS-deficient yeast and trypanosome strains, respectively. Our results show that TbCLS complemented cardiolipin production in CRD1 knockout yeast and partly restored wild-type colony forming capability under stress conditions. Remarkably, CL remodeling appeared to be impaired in the transgenic construct, suggesting that CL production and remodeling are tightly coupled processes that may require a clustering of the involved proteins into specific CL-synthesizing domains. In contrast, no complementation was observed by heterologous expression of ScCrd1 in conditional TbCLS knockout trypanosomes, despite proper mitochondrial targeting of the protein.

Complement System Part II: Role in Immunity

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Merle, Nicolas S.; Noe, Remi; Halbwachs-Mecarelli, Lise; Fremeaux-Bacchi, Veronique; Roumenina, Lubka T.

2015-01-01

The complement system has been considered for a long time as a simple lytic cascade, aimed to kill bacteria infecting the host organism. Nowadays, this vision has changed and it is well accepted that complement is a complex innate immune surveillance system, playing a key role in host homeostasis, inflammation, and in the defense against pathogens. This review discusses recent advances in the understanding of the role of complement in physiology and pathology. It starts with a description of complement contribution to the normal physiology (homeostasis) of a healthy organism, including the silent clearance of apoptotic cells and maintenance of cell survival. In pathology, complement can be a friend or a foe. It acts as a friend in the defense against pathogens, by inducing opsonization and a direct killing by C5b–9 membrane attack complex and by triggering inflammatory responses with the anaphylatoxins C3a and C5a. Opsonization plays also a major role in the mounting of an adaptive immune response, involving antigen presenting cells, T-, and B-lymphocytes. Nevertheless, it can be also an enemy, when pathogens hijack complement regulators to protect themselves from the immune system. Inadequate complement activation becomes a disease cause, as in atypical hemolytic uremic syndrome, C3 glomerulopathies, and systemic lupus erythematosus. Age-related macular degeneration and cancer will be described as examples showing that complement contributes to a large variety of conditions, far exceeding the classical examples of diseases associated with complement deficiencies. Finally, we discuss complement as a therapeutic target. PMID:26074922

Regulatory Roles for Long ncRNA and mRNA

International Nuclear Information System (INIS)

Karapetyan, Armen R.; Buiting, Coen; Kuiper, Renske A.; Coolen, Marcel W.

2013-01-01

Recent advances in high-throughput sequencing technology have identified the transcription of a much larger portion of the genome than previously anticipated. Especially in the context of cancer it has become clear that aberrant transcription of both protein-coding and long non-coding RNAs (lncRNAs) are frequent events. The current dogma of RNA function describes mRNA to be responsible for the synthesis of proteins, whereas non-coding RNA can have regulatory or epigenetic functions. However, this distinction between protein coding and regulatory ability of transcripts may not be that strict. Here, we review the increasing body of evidence for the existence of multifunctional RNAs that have both protein-coding and trans-regulatory roles. Moreover, we demonstrate that coding transcripts bind to components of the Polycomb Repressor Complex 2 (PRC2) with similar affinities as non-coding transcripts, revealing potential epigenetic regulation by mRNAs. We hypothesize that studies on the regulatory ability of disease-associated mRNAs will form an important new field of research

Regulatory Roles for Long ncRNA and mRNA

Energy Technology Data Exchange (ETDEWEB)

Karapetyan, Armen R.; Buiting, Coen; Kuiper, Renske A.; Coolen, Marcel W., E-mail: M.Coolen@gen.umcn.nl [Department of Human Genetics, Nijmegen Centre for Molecular Life Sciences (NCMLS), Radboud University Nijmegen Medical Centre, P.O. Box 9101, Nijmegen 6500 HB (Netherlands)

2013-04-26

Recent advances in high-throughput sequencing technology have identified the transcription of a much larger portion of the genome than previously anticipated. Especially in the context of cancer it has become clear that aberrant transcription of both protein-coding and long non-coding RNAs (lncRNAs) are frequent events. The current dogma of RNA function describes mRNA to be responsible for the synthesis of proteins, whereas non-coding RNA can have regulatory or epigenetic functions. However, this distinction between protein coding and regulatory ability of transcripts may not be that strict. Here, we review the increasing body of evidence for the existence of multifunctional RNAs that have both protein-coding and trans-regulatory roles. Moreover, we demonstrate that coding transcripts bind to components of the Polycomb Repressor Complex 2 (PRC2) with similar affinities as non-coding transcripts, revealing potential epigenetic regulation by mRNAs. We hypothesize that studies on the regulatory ability of disease-associated mRNAs will form an important new field of research.

Triatoma infestans Calreticulin: Gene Cloning and Expression of a Main Domain That Interacts with the Host Complement System.

Science.gov (United States)

Weinberger, Katherine; Collazo, Norberto; Aguillón, Juan Carlos; Molina, María Carmen; Rosas, Carlos; Peña, Jaime; Pizarro, Javier; Maldonado, Ismael; Cattan, Pedro E; Apt, Werner; Ferreira, Arturo

2017-02-08

Triatoma infestans is an important hematophagous vector of Chagas disease, a neglected chronic illness affecting approximately 6 million people in Latin America. Hematophagous insects possess several molecules in their saliva that counteract host defensive responses. Calreticulin (CRT), a multifunctional protein secreted in saliva, contributes to the feeding process in some insects. Human CRT (HuCRT) and Trypanosoma cruzi CRT (TcCRT) inhibit the classical pathway of complement activation, mainly by interacting through their central S domain with complement component C1. In previous studies, we have detected CRT in salivary gland extracts from T. infestans We have called this molecule TiCRT. Given that the S domain is responsible for C1 binding, we have tested its role in the classical pathway of complement activation in vertebrate blood. We have cloned and characterized the complete nucleotide sequence of CRT from T. infestans , and expressed its S domain. As expected, this S domain binds to human C1 and, as a consequence, it inhibits the classical pathway of complement, at its earliest stage of activation, namely the generation of C4b. Possibly, the presence of TiCRT in the salivary gland represents an evolutionary adaptation in hematophagous insects to control a potential activation of complement proteins, present in the massive blood meal that they ingest, with deleterious consequences at least on the anterior digestive tract of these insects. © The American Society of Tropical Medicine and Hygiene.

Core shell methyl methacrylate chitosan nanoparticles: In vitro mucoadhesion and complement activation

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F Atyabi

2011-10-01

Full Text Available Background and the purpose of the study: Studies show that chitosan nanoparticles increase mucoadhesivity and penetration of large molecules across mucosal surface. The aim of the present study was to investigate the use of thiolated chitosan in the development of polysaccharide-coated nanoparticles in order to confer specific functionality to the system. Methods: Methyl methacrylate nanoparticles were coated with thiolated chitosan using a radical polymerization method. Thiolation was carried out using glutathione (GSH to improve mucoadhesivity and permeation enhancing properties of chitosan. Mucoadhesion studies were carried out by calculating the amount of mucin adsorbed on nanoparticles in a specific period of time. Complement consumption was assessed in human serum (HS by measurement of the hemolytic capacity of the complement system after contact with nanoparticles.   Results:   The FT-IR and 1HNMR spectra both confirmed the synthesis and showed the conjugation of thiolated chitosan to methyl methacrylate (MMA homopolymer. Nanoparticles were spherical having a mean diameter within the range of about 334-650 nm and their positive zeta potential values indicated the presence of the cationic polysaccharide at the nanoparticle surface. Increasing the amount of thiolated chitosan led to mucoadhesivity and complement activation. However there was not dose dependent correlation between these phenomenons and the absence of thiolated chitosan led to particles with larger size, and without ability to activate complement process. Major conclusion: It can be concluded that nanoparticles could be used for the mucosal delivery of peptides and proteins. Results show that the thiolated chitosan had higher mucoadhesion and complement activation than unmodified chitosan.

Complement-mediated solubilization of immune complexes. Solubilization inhibition and complement factor levels in SLE patients

DEFF Research Database (Denmark)

Baatrup, Gunnar; Petersen, Ivan; Kappelgaard, E

1984-01-01

Thirty-two of 36 serum samples from 19 SLE patients showed reduced capacity to mediate complement-dependent solubilization of immune complexes (IC). SLE patients with nephritis exerted the lowest complement-mediated solubilization capacity (CMSC) whereas sera from patients with inactive disease g...

Complement anaphylatoxins as immune regulators in cancer.

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Sayegh, Eli T; Bloch, Orin; Parsa, Andrew T

2014-08-01

The role of the complement system in innate immunity is well characterized. However, a recent body of research implicates the complement anaphylatoxins C3a and C5a as insidious propagators of tumor growth and progression. It is now recognized that certain tumors elaborate C3a and C5a and that complement, as a mediator of chronic inflammation and regulator of immune function, may in fact foster rather than defend against tumor growth. A putative mechanism for this function is complement-mediated suppression of immune effector cells responsible for immunosurveillance within the tumor microenvironment. This paradigm accords with models of immune dysregulation, such as autoimmunity and infectious disease, which have defined a pathophysiological role for abnormal complement signaling. Several types of immune cells express the cognate receptors for the complement anaphylatoxins, C3aR and C5aR, and demonstrate functional modulation in response to complement stimulation. In turn, impairment of antitumor immunity has been intimately tied to tumor progression in animal models of cancer. In this article, the literature was systematically reviewed to identify studies that have characterized the effects of the complement anaphylatoxins on the composition and function of immune cells within the tumor microenvironment. The search identified six studies based upon models of lymphoma and ovarian, cervical, lung, breast, and mammary cancer, which collectively support the paradigm of complement as an immune regulator in the tumor microenvironment. © 2014 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Platelet cytosolic 44-kDa protein is a substrate of cholera toxin-induced ADP-ribosylation and is not recognized by antisera against the α subunit of the stimulatory guanine nucleotide-binding regulatory protein

International Nuclear Information System (INIS)

Molina Y Vedia, L.M.; Reep, B.R.; Lapetina, E.G.

1988-01-01

ADP-ribosylation induced by cholera toxin and pertussis toxin was studied in particulate and cytosolic fractions of human platelets. Platelets were disrupted by a cycle of freezing and thawing in the presence of a hyposmotic buffer containing protease inhibitors. In both fractions, the A subunit of cholera toxin ADP-ribosylates two proteins with molecular masses of 42 and 44 kDa, whereas pertussis toxin ADP-ribosylates a 41-kDa polypeptide. Two antisera against the α subunit of the stimulatory guanine nucleotide-binding regulatory protein recognize only the 42-kDa polypeptide. Cholera toxin-induced ADP-ribosylation of the 42- and 44-kDa proteins is reduced by pretreatment of platelets with iloprost, a prostacyclin analog. The 44-kDa protein, which is substrate of cholera toxin, could be extracted completely from the membrane and recovered in the cytosolic fraction when the cells were disrupted by Dounce homogenization and the pellet was extensively washed. A 44-kDa protein can also be labeled with 8-azidoguanosine 5'-[α- 32 P]triphosphate in the cytosol and membranes. These finding indicate that cholera and pertussis toxins produced covalent modifications of proteins present in particulate and cytosolic platelet fractions. Moreover, the 44-kDa protein might be an α subunit of a guanine nucleotide-binding regulatory protein that is not recognized by available antisera

Complement Test

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... Salicylates Semen Analysis Serotonin Serum Free Light Chains Sex Hormone Binding Globulin (SHBG) Shiga toxin-producing Escherichia ... and forming complexes that respond to infections, non-self tissues (transplants), dead cells ... KJ. Complement determinations in human disease. Ann Allergy Asthma Immunol . 2004; ...

Combined roles of human IgG subclass, alternative complement pathway activation, and epitope density in the bactericidal activity of antibodies to meningococcal factor h binding protein.

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Giuntini, Serena; Reason, Donald C; Granoff, Dan M

2012-01-01

Meningococcal vaccines containing factor H binding protein (fHbp) are in clinical development. fHbp binds human fH, which enables the meningococcus to resist complement-mediated bacteriolysis. Previously, we found that chimeric human IgG1 mouse anti-fHbp monoclonal antibodies (MAbs) had human complement-mediated bactericidal activity only if the MAb inhibited fH binding. Since IgG subclasses differ in their ability to activate complement, we investigated the role of human IgG subclasses on antibody functional activity. We constructed chimeric MAbs in which three different murine fHbp-specific binding domains were each paired with human IgG1, IgG2, or IgG3. Against a wild-type group B isolate, all three IgG3 MAbs, irrespective of their ability to inhibit fH binding, had bactericidal activity that was >5-fold higher than the respective IgG1 MAbs, while the IgG2 MAbs had the least activity. Against a mutant with increased fHbp expression, the anti-fHbp MAbs elicited greater C4b deposition (classical pathway) and greater bactericidal activity than against the wild-type strain, and the IgG1 MAbs had similar or greater activity than the respective IgG3 MAbs. The bactericidal activity against both wild-type and mutant strains also was dependent, in part, on activation of the alternative complement pathway. Thus, at lower epitope density in the wild-type strain, the IgG3 anti-fHbp MAbs had the greatest bactericidal activity. At a higher epitope density in the mutant, the IgG1 MAbs had similar or greater bactericidal activity than the IgG3 MAbs, and the activity was less dependent on the inhibition of fH binding than at a lower epitope density.

Susceptibility to invasive meningococcal disease: polymorphism of complement system genes and Neisseria meningitidis factor H binding protein.

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Declan T Bradley

Full Text Available 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.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.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.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

Network perturbation by recurrent regulatory variants in cancer.

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Kiwon Jang

2017-03-01

Full Text Available Cancer driving genes have been identified as recurrently affected by variants that alter protein-coding sequences. However, a majority of cancer variants arise in noncoding regions, and some of them are thought to play a critical role through transcriptional perturbation. Here we identified putative transcriptional driver genes based on combinatorial variant recurrence in cis-regulatory regions. The identified genes showed high connectivity in the cancer type-specific transcription regulatory network, with high outdegree and many downstream genes, highlighting their causative role during tumorigenesis. In the protein interactome, the identified transcriptional drivers were not as highly connected as coding driver genes but appeared to form a network module centered on the coding drivers. The coding and regulatory variants associated via these interactions between the coding and transcriptional drivers showed exclusive and complementary occurrence patterns across tumor samples. Transcriptional cancer drivers may act through an extensive perturbation of the regulatory network and by altering protein network modules through interactions with coding driver genes.

Identification and functional characterisation of Complement Regulator Acquiring Surface Protein-1 of serum resistant Borrelia garinii OspA serotype 4

NARCIS (Netherlands)

van Burgel, Nathalie D.; Kraiczy, Peter; Schuijt, Tim J.; Zipfel, Peter F.; van Dam, Alje P.

2010-01-01

B. burgdorferi sensu lato (sl) is the etiological agent of Lyme borreliosis in humans. Spirochetes have adapted themselves to the human immune system in many distinct ways. One important immune escape mechanism for evading complement activation is the binding of complement regulators Factor H (CFH)

A targeted complement-dependent strategy to improve the outcome of mAb therapy, and characterization in a murine model of metastatic cancer

Science.gov (United States)

Elvington, Michelle; Huang, Yuxiang; Morgan, B. Paul; Qiao, Fei; van Rooijen, Nico; Atkinson, Carl

2012-01-01

Complement inhibitors expressed on tumor cells provide an evasion mechanism against mAb therapy and may modulate the development of an acquired antitumor immune response. Here we investigate a strategy to amplify mAb-targeted complement activation on a tumor cell, independent of a requirement to target and block complement inhibitor expression or function, which is difficult to achieve in vivo. We constructed a murine fusion protein, CR2Fc, and demonstrated that the protein targets to C3 activation products deposited on a tumor cell by a specific mAb, and amplifies mAb-dependent complement activation and tumor cell lysis in vitro. In syngeneic models of metastatic lymphoma (EL4) and melanoma (B16), CR2Fc significantly enhanced the outcome of mAb therapy. Subsequent studies using the EL4 model with various genetically modified mice and macrophage-depleted mice revealed that CR2Fc enhanced the therapeutic effect of mAb therapy via both macrophage-dependent FcγR-mediated antibody-dependent cellular cytotoxicity, and by direct complement-mediated lysis. Complement activation products can also modulate adaptive immunity, but we found no evidence that either mAb or CR2Fc treatment had any effect on an antitumor humoral or cellular immune response. CR2Fc represents a potential adjuvant treatment to increase the effectiveness of mAb therapy of cancer. PMID:22442351

Genome-wide analysis of regulatory proteases sequences identified through bioinformatics data mining in Taenia solium.

Science.gov (United States)

Yan, Hong-Bin; Lou, Zhong-Zi; Li, Li; Brindley, Paul J; Zheng, Yadong; Luo, Xuenong; Hou, Junling; Guo, Aijiang; Jia, Wan-Zhong; Cai, Xuepeng

2014-06-04

Cysticercosis remains a major neglected tropical disease of humanity in many regions, especially in sub-Saharan Africa, Central America and elsewhere. Owing to the emerging drug resistance and the inability of current drugs to prevent re-infection, identification of novel vaccines and chemotherapeutic agents against Taenia solium and related helminth pathogens is a public health priority. The T. solium genome and the predicted proteome were reported recently, providing a wealth of information from which new interventional targets might be identified. In order to characterize and classify the entire repertoire of protease-encoding genes of T. solium, which act fundamental biological roles in all life processes, we analyzed the predicted proteins of this cestode through a combination of bioinformatics tools. Functional annotation was performed to yield insights into the signaling processes relevant to the complex developmental cycle of this tapeworm and to highlight a suite of the proteases as potential intervention targets. Within the genome of this helminth parasite, we identified 200 open reading frames encoding proteases from five clans, which correspond to 1.68% of the 11,902 protein-encoding genes predicted to be present in its genome. These proteases include calpains, cytosolic, mitochondrial signal peptidases, ubiquitylation related proteins, and others. Many not only show significant similarity to proteases in the Conserved Domain Database but have conserved active sites and catalytic domains. KEGG Automatic Annotation Server (KAAS) analysis indicated that ~60% of these proteases share strong sequence identities with proteins of the KEGG database, which are involved in human disease, metabolic pathways, genetic information processes, cellular processes, environmental information processes and organismal systems. Also, we identified signal peptides and transmembrane helices through comparative analysis with classes of important regulatory proteases

Endogenous Natural Complement Inhibitor Regulates Cardiac Development

DEFF Research Database (Denmark)

Mortensen, Simon A; Skov, Louise L; Kjaer-Sorensen, Kasper

2017-01-01

mechanisms during fetal development and adult homeostasis. In this article, we describe the function of an endogenous complement inhibitor, mannan-binding lectin (MBL)-associated protein (MAp)44, in regulating the composition of a serine protease-pattern recognition receptor complex, MBL-associated serine...... of MAp44 caused impaired cardiogenesis, lowered heart rate, and decreased cardiac output. These defects were associated with aberrant neural crest cell behavior. We found that MAp44 competed with MASP-3 for pattern recognition molecule interaction, and knockdown of endogenous MAp44 expression could...... be rescued by overexpression of wild-type MAp44. Our observations provide evidence that immune molecules are centrally involved in the orchestration of cardiac tissue development....

Novel activation domain derived from Che-1 cofactor coupled with the artificial protein Jazz drives utrophin upregulation.

Science.gov (United States)

Desantis, Agata; Onori, Annalisa; Di Certo, Maria Grazia; Mattei, Elisabetta; Fanciulli, Maurizio; Passananti, Claudio; Corbi, Nicoletta

2009-02-01

Our aim is to upregulate the expression level of the dystrophin related gene utrophin in Duchenne muscular dystrophy, thus complementing the lack of dystrophin functions. To this end, we have engineered synthetic zinc finger based transcription factors. We have previously shown that the artificial three-zinc finger protein named Jazz fused with the Vp16 activation domain, is able to bind utrophin promoter A and to increase the endogenous level of utrophin in transgenic mice. Here, we report on an innovative artificial protein, named CJ7, that consists of Jazz DNA binding domain fused to a novel activation domain derived from the regulatory multivalent adaptor protein Che-1/AATF. This transcriptional activation domain is 100 amino acids in size and it is very powerful as compared to the Vp16 activation domain. We show that CJ7 protein efficiently promotes transcription and accumulation of the acetylated form of histone H3 on the genomic utrophin promoter locus.

Complementation Studies of Bacteriophage λ O Amber Mutants by Allelic Forms of O Expressed from Plasmid, and O-P Interaction Phenotypes.

Science.gov (United States)

Hayes, Sidney; Rajamanickam, Karthic; Hayes, Connie

2018-04-05

λ genes O and P are required for replication initiation from the bacteriophage λ origin site, ori λ, located within gene O . Questions have persisted for years about whether O-defects can indeed be complemented in trans . We show the effect of original null mutations in O and the influence of four origin mutations (three are in-frame deletions and one is a point mutation) on complementation. This is the first demonstration that O proteins with internal deletions can complement for O activity, and that expression of the N-terminal portion of gene P can completely prevent O complementation. We show that O-P co-expression can limit the lethal effect of P on cell growth. We explore the influence of the contiguous small RNA OOP on O complementation and P-lethality.

Nitrosylation of Nitric-Oxide-Sensing Regulatory Proteins Containing [4Fe-4S] Clusters Gives Rise to Multiple Iron-Nitrosyl Complexes

Energy Technology Data Exchange (ETDEWEB)

Serrano, Pauline N. [Department of Chemistry, University of California, Davis CA 95616 USA; Wang, Hongxin [Department of Chemistry, University of California, Davis CA 95616 USA; Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley CA 94720 USA; Crack, Jason C. [Centre for Molecular and Structural Biochemistry, School of Chemistry, University of East Anglia, Norwich Research Park Norwich NR4 7TJ UK; Prior, Christopher [Centre for Molecular and Structural Biochemistry, School of Chemistry, University of East Anglia, Norwich Research Park Norwich NR4 7TJ UK; Hutchings, Matthew I. [School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ UK; Thomson, Andrew J. [Centre for Molecular and Structural Biochemistry, School of Chemistry, University of East Anglia, Norwich Research Park Norwich NR4 7TJ UK; Kamali, Saeed [University of Tennessee Space Institute, Tullahome TN 37388-9700 USA; Yoda, Yoshitaka [Research and Utilization Division, SPring-8/JASRI, 1-1-1 Kouto, Sayo Hyogo 679-5198 Japan; Zhao, Jiyong [Advanced Photon Source, Argonne National Laboratory, Argonne IL 60439 USA; Hu, Michael Y. [Advanced Photon Source, Argonne National Laboratory, Argonne IL 60439 USA; Alp, Ercan E. [Advanced Photon Source, Argonne National Laboratory, Argonne IL 60439 USA; Oganesyan, Vasily S. [Centre for Molecular and Structural Biochemistry, School of Chemistry, University of East Anglia, Norwich Research Park Norwich NR4 7TJ UK; Le Brun, Nick E. [Centre for Molecular and Structural Biochemistry, School of Chemistry, University of East Anglia, Norwich Research Park Norwich NR4 7TJ UK; Cramer, Stephen P. [Department of Chemistry, University of California, Davis CA 95616 USA; Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley CA 94720 USA

2016-10-25

The reaction of protein-bound iron–sulfur (Fe-S) clusters with nitric oxide (NO) plays key roles in NO-mediated toxicity and signaling. Elucidation of the mechanism of the reaction of NO with DNA regulatory proteins that contain Fe-S clusters has been hampered by a lack of information about the nature of the iron-nitrosyl products formed. Herein, we report nuclear resonance vibrational spectroscopy (NRVS) and density functional theory (DFT) calculations that identify NO reaction products in WhiD and NsrR, regulatory proteins that use a [4Fe-4S] cluster to sense NO. This work reveals that nitrosylation yields multiple products structurally related to Roussin's Red Ester (RRE, [Fe₂(NO)₄(Cys)₂]) and Roussin's Black Salt (RBS, [Fe₄(NO)₇S₃]. In the latter case, the absence of ³²S/³⁴S shifts in the Fe-S region of the NRVS spectra suggest that a new species, Roussin's Black Ester (RBE), may be formed, in which one or more of the sulfide ligands is replaced by Cys thiolates.

Human conglutinin-like protein

DEFF Research Database (Denmark)

Jensenius, J C; Thiel, S; Baatrup, G

1985-01-01

The presence in human plasma of a molecule homologous to bovine conglutinin is indicated by the results of biological and immunochemical analysis. The human conglutinin-like protein shows calcium-dependent binding to complement-treated solid phase IgG and immunological cross-reaction with chicken...... anti-bovine conglutinin. The binding of the human protein to complement-treated IgG was inhibited by N-acetyl-D-glucosamine but not by other sugars. Analysis by SDS-PAGE and Western blotting showed reaction of anti-conglutinin with molecules of similar mobility to the monomer and hexamer of bovine...

A Novel, In-solution Separation of Endogenous Cardiac Sarcomeric Proteins and Identification of Distinct Charged Variants of Regulatory Light Chain*

Science.gov (United States)

Scruggs, Sarah B.; Reisdorph, Rick; Armstrong, Mike L.; Warren, Chad M.; Reisdorph, Nichole; Solaro, R. John; Buttrick, Peter M.

2010-01-01

The molecular conformation of the cardiac myosin motor is modulated by intermolecular interactions among the heavy chain, the light chains, myosin binding protein-C, and titin and is governed by post-translational modifications (PTMs). In-gel digestion followed by LC/MS/MS has classically been applied to identify cardiac sarcomeric PTMs; however, this approach is limited by protein size, pI, and difficulties in peptide extraction. We report a solution-based work flow for global separation of endogenous cardiac sarcomeric proteins with a focus on the regulatory light chain (RLC) in which specific sites of phosphorylation have been unclear. Subcellular fractionation followed by OFFGEL electrophoresis resulted in isolation of endogenous charge variants of sarcomeric proteins, including regulatory and essential light chains, myosin heavy chain, and myosin-binding protein-C of the thick filament. Further purification of RLC using reverse-phase HPLC separation and UV detection enriched for RLC PTMs at the intact protein level and provided a stoichiometric and quantitative assessment of endogenous RLC charge variants. Digestion and subsequent LC/MS/MS unequivocally identified that the endogenous charge variants of cardiac RLC focused in unique OFFGEL electrophoresis fractions were unphosphorylated (78.8%), singly phosphorylated (18.1%), and doubly phosphorylated (3.1%) RLC. The novel aspects of this study are that 1) milligram amounts of endogenous cardiac sarcomeric subproteome were focused with resolution comparable with two-dimensional electrophoresis, 2) separation and quantification of post-translationally modified variants were achieved at the intact protein level, 3) separation of intact high molecular weight thick filament proteins was achieved in solution, and 4) endogenous charge variants of RLC were separated; a novel doubly phosphorylated form was identified in mouse, and singly phosphorylated, singly deamidated, and deamidated/phosphorylated forms were

Dengue-Immune Humans Have Higher Levels of Complement-Independent Enhancing Antibody than Complement-Dependent Neutralizing Antibody.

Science.gov (United States)

Yamanaka, Atsushi; Konishi, Eiji

2017-09-25

Dengue is the most important arboviral disease worldwide. We previously reported that most inhabitants of dengue-endemic countries who are naturally immune to the disease have infection-enhancing antibodies whose in vitro activity does not decrease in the presence of complement (complement-independent enhancing antibodies, or CiEAb). Here, we compared levels of CiEAb and complement-dependent neutralizing antibodies (CdNAb) in dengue-immune humans. A typical antibody dose-response pattern obtained in our assay system to measure the balance between neutralizing and enhancing antibodies showed both neutralizing and enhancing activities depending on serum dilution factor. The addition of complement to the assay system increased the activity of neutralizing antibodies at lower dilutions, indicating the presence of CdNAb. In contrast, similar dose-response curves were obtained with and without complement at higher dilutions, indicating higher levels of CiEAb than CdNAb. For experimental support for the higher CiEAb levels, a cocktail of mouse monoclonal antibodies against dengue virus type 1 was prepared. The antibody dose-response curves obtained in this assay, with or without complement, were similar to those obtained with human serum samples when a high proportion of D1-V-3H12 (an antibody exhibiting only enhancing activity and thus a model for CiEAb) was used in the cocktail. This study revealed higher-level induction of CiEAb than CdNAb in humans naturally infected with dengue viruses.

Complement anaphylatoxins as immune regulators in cancer

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Sayegh, Eli T; Bloch, Orin; Parsa, Andrew T

2014-01-01

The role of the complement system in innate immunity is well characterized. However, a recent body of research implicates the complement anaphylatoxins C3a and C5a as insidious propagators of tumor growth and progression. It is now recognized that certain tumors elaborate C3a and C5a and that complement, as a mediator of chronic inflammation and regulator of immune function, may in fact foster rather than defend against tumor growth. A putative mechanism for this function is complement-mediat...

Regulatory Roles for Long ncRNA and mRNA

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Marcel W. Coolen

2013-04-01

Full Text Available Recent advances in high-throughput sequencing technology have identified the transcription of a much larger portion of the genome than previously anticipated. Especially in the context of cancer it has become clear that aberrant transcription of both protein-coding and long non-coding RNAs (lncRNAs are frequent events. The current dogma of RNA function describes mRNA to be responsible for the synthesis of proteins, whereas non-coding RNA can have regulatory or epigenetic functions. However, this distinction between protein coding and regulatory ability of transcripts may not be that strict. Here, we review the increasing body of evidence for the existence of multifunctional RNAs that have both protein-coding and trans-regulatory roles. Moreover, we demonstrate that coding transcripts bind to components of the Polycomb Repressor Complex 2 (PRC2 with similar affinities as non-coding transcripts, revealing potential epigenetic regulation by mRNAs. We hypothesize that studies on the regulatory ability of disease-associated mRNAs will form an important new field of research.

Decorin core protein (decoron) shape complements collagen fibril surface structure and mediates its binding.

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Orgel, Joseph P R O; Eid, Aya; Antipova, Olga; Bella, Jordi; Scott, John E

2009-09-15

Decorin is the archetypal small leucine rich repeat proteoglycan of the vertebrate extracellular matrix (ECM). With its glycosaminoglycuronan chain, it is responsible for stabilizing inter-fibrillar organization. Type I collagen is the predominant member of the fibrillar collagen family, fulfilling both organizational and structural roles in animal ECMs. In this study, interactions between decoron (the decorin core protein) and binding sites in the d and e(1) bands of the type I collagen fibril were investigated through molecular modeling of their respective X-ray diffraction structures. Previously, it was proposed that a model-based, highly curved concave decoron interacts with a single collagen molecule, which would form extensive van der Waals contacts and give rise to strong non-specific binding. However, the large well-ordered aggregate that is the collagen fibril places significant restraints on modes of ligand binding and necessitates multi-collagen molecular contacts. We present here a relatively high-resolution model of the decoron-fibril collagen complex. We find that the respective crystal structures complement each other well, although it is the monomeric form of decoron that shows the most appropriate shape complementarity with the fibril surface and favorable calculated energies of interaction. One molecule of decoron interacts with four to six collagen molecules, and the binding specificity relies on a large number of hydrogen bonds and electrostatic interactions, primarily with the collagen motifs KXGDRGE and AKGDRGE (d and e(1) bands). This work helps us to understand collagen-decorin interactions and the molecular architecture of the fibrillar ECM in health and disease.

Decorin core protein (decoron shape complements collagen fibril surface structure and mediates its binding.

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Joseph P R O Orgel

2009-09-01

Full Text Available Decorin is the archetypal small leucine rich repeat proteoglycan of the vertebrate extracellular matrix (ECM. With its glycosaminoglycuronan chain, it is responsible for stabilizing inter-fibrillar organization. Type I collagen is the predominant member of the fibrillar collagen family, fulfilling both organizational and structural roles in animal ECMs. In this study, interactions between decoron (the decorin core protein and binding sites in the d and e(1 bands of the type I collagen fibril were investigated through molecular modeling of their respective X-ray diffraction structures. Previously, it was proposed that a model-based, highly curved concave decoron interacts with a single collagen molecule, which would form extensive van der Waals contacts and give rise to strong non-specific binding. However, the large well-ordered aggregate that is the collagen fibril places significant restraints on modes of ligand binding and necessitates multi-collagen molecular contacts. We present here a relatively high-resolution model of the decoron-fibril collagen complex. We find that the respective crystal structures complement each other well, although it is the monomeric form of decoron that shows the most appropriate shape complementarity with the fibril surface and favorable calculated energies of interaction. One molecule of decoron interacts with four to six collagen molecules, and the binding specificity relies on a large number of hydrogen bonds and electrostatic interactions, primarily with the collagen motifs KXGDRGE and AKGDRGE (d and e(1 bands. This work helps us to understand collagen-decorin interactions and the molecular architecture of the fibrillar ECM in health and disease.

The small serine-threonine protein SIP2 interacts with STE12 and is involved in ascospore germination in Sordaria macrospora.

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Elleuche, Skander; Bernhards, Yasmine; Schäfers, Christian; Varghese, Jans Manjali; Nolting, Nicole; Pöggeler, Stefanie

2010-12-01

In fungi, the homoeodomain protein STE12 controls diverse developmental processes, and derives its regulatory specificity from different protein interactions. We recently showed that in the homothallic ascomycete Sordaria macrospora, STE12 is essential for ascospore development, and is able to interact with the alpha-domain mating-type protein SMTA-1 and the MADS box protein MCM1. To further evaluate the functional roles of STE12, we used the yeast two-hybrid approach to identify new STE12-interacting partners. Using STE12 as bait, a small, serine-threonine-rich protein (designated STE12-interacting protein 2, SIP2) was identified. SIP2 is conserved among members of the fungal class Sordariomycetes. In vivo localization studies revealed that SIP2 was targeted to the nucleus and cytoplasm. The STE12/SIP2 interaction was further confirmed in vivo by bimolecular fluorescence complementation. Nuclear localization of SIP2 was apparently mediated by STE12. Unlike deletion of ste12, deletion of sip2 in S. macrospora led to only a slight decrease in ascospore germination, and no other obvious morphological phenotype. In comparison to the Δste12 single knockout strain, ascospore germination was significantly increased in a Δsip2/ste12 double knockout strain. Our data provide evidence for a regulatory role of the novel fungal protein SIP2 in ascospore germination. Copyright © 2010 Elsevier GmbH. All rights reserved.

A method for functional trans-complementation of intracellular Francisella tularensis.

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Shaun Steele

trans-complementation using bead-bound bacteria co-infections is a method to rapidly identify mutants that fail to modify a host response. Francisella tularensis is a facultative intracellular bacterial pathogen and is the causative agent of the disease tularemia. F. tularensis enters host cells through phagocytosis, escapes the phagosome, and replicates in the host cell cytosol while suppressing cytokine secretion [1]-[4]. Although substantial progress has been made in understanding the intracellular life cycle of F. tularensis, the F. tularensis proteins responsible for manipulating many host cell pathways are unknown. Identifying novel host-pathogen effector proteins is difficult because there is no rapid method to reliably distinguish between bacterial proteins that modify host processes and proteins that are involved in bacterial processes that are required for the bacteria to survive or replicate in the intracellular environment. The ability to identify mutants that are deficient for host-pathogen interactions is important because it can aid in prioritizing the investigation of genes of interest and in downstream experimental design. Moreover, certain mutant phenotypes, such as decreased phagosomal escape, hinder investigation of other potential phenotypes. A method to specifically complement these phenotypes would allow for further characterizations of certain F. tularensis mutants. Thus we sought to develop a method to easily identify and functionally complement mutants that are deficient for interactions with the host.

Systematic comparison of the response properties of protein and RNA mediated gene regulatory motifs.

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Iyengar, Bharat Ravi; Pillai, Beena; Venkatesh, K V; Gadgil, Chetan J

2017-05-30

We present a framework enabling the dissection of the effects of motif structure (feedback or feedforward), the nature of the controller (RNA or protein), and the regulation mode (transcriptional, post-transcriptional or translational) on the response to a step change in the input. We have used a common model framework for gene expression where both motif structures have an activating input and repressing regulator, with the same set of parameters, to enable a comparison of the responses. We studied the global sensitivity of the system properties, such as steady-state gain, overshoot, peak time, and peak duration, to parameters. We find that, in all motifs, overshoot correlated negatively whereas peak duration varied concavely with peak time. Differences in the other system properties were found to be mainly dependent on the nature of the controller rather than the motif structure. Protein mediated motifs showed a higher degree of adaptation i.e. a tendency to return to baseline levels; in particular, feedforward motifs exhibited perfect adaptation. RNA mediated motifs had a mild regulatory effect; they also exhibited a lower peaking tendency and mean overshoot. Protein mediated feedforward motifs showed higher overshoot and lower peak time compared to the corresponding feedback motifs.

75 FR 51866 - Self-Regulatory Organizations; The Depository Trust Company; Order Approving Proposed Rule Change...

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2010-08-23

... Transfer Service of National Securities Clearing Corporation August 16, 2010. I. Introduction On June 4... complements a Financial Industry Regulatory Authority (``FINRA'') rule requiring FINRA members to use... specified time frames. \\5\\ CNS is an ongoing accounting system which nets today's Settling Trades with...

Molluskan Hemocyanins Activate the Classical Pathway of the Human Complement System through Natural Antibodies.

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Pizarro-Bauerle, Javier; Maldonado, Ismael; Sosoniuk-Roche, Eduardo; Vallejos, Gerardo; López, Mercedes N; Salazar-Onfray, Flavio; Aguilar-Guzmán, Lorena; Valck, Carolina; Ferreira, Arturo; Becker, María Inés

2017-01-01

Molluskan hemocyanins are enormous oxygen-carrier glycoproteins that show remarkable immunostimulatory properties when inoculated in mammals, such as the generation of high levels of antibodies, a strong cellular reaction, and generation of non-specific antitumor immune responses in some types of cancer, particularly for superficial bladder cancer. These proteins have the ability to bias the immune response toward a T h 1 phenotype. However, despite all their current uses with beneficial clinical outcomes, a clear mechanism explaining these properties is not available. Taking into account reports of natural antibodies against the hemocyanin of the gastropod Megathura crenulata [keyhole limpet hemocyanin (KLH)] in humans as well as other vertebrate species, we report here for the first time, the presence, in sera from unimmunized healthy donors, of antibodies recognizing, in addition to KLH, two other hemocyanins from gastropods with documented immunomodulatory capacities: Fisurella latimarginata hemocyanin (FLH) and Concholepas concholepas hemocyanin (CCH). Through an ELISA screening, we found IgM and IgG antibodies reactive with these hemocyanins. When the capacity of these antibodies to bind deglycosylated hemocyanins was studied, no decreased interaction was detected. Moreover, in the case of FLH, deglycosylation increased antibody binding. We evaluated through an in vitro complement deposition assay whether these antibodies activated the classical pathway of the human complement system. The results showed that all three hemocyanins and their deglycosylated counterparts elicited this activation, mediated by C1 binding to immunoglobulins. Thus, this work contributes to the understanding on how the complement system could participate in the immunostimulatory properties of hemocyanins, through natural, complement-activating antibodies reacting with these proteins. Although a role for carbohydrates cannot be completely ruled out, in our experimental setting

The human complement inhibitor Sushi Domain-Containing Protein 4 (SUSD4) expression in tumor cells and infiltrating T cells is associated with better prognosis of breast cancer patients

OpenAIRE

Englund, Emelie; Reitsma, Bart; King, Ben C.; Escudero-Esparza, Astrid; Owen, Sioned; Orimo, Akira; Okroj, Marcin; Anagnostaki, Lola; Jiang, Wen G.; Jirström, Karin; Blom, Anna M.

2015-01-01

Background: The human Sushi Domain-Containing Protein 4 (SUSD4) was recently shown to function as a novel inhibitor of the complement system, but its role in tumor progression is unknown. \\ud \\ud Methods: Using immunohistochemistry and quantitative PCR, we investigated SUSD4 expression in breast cancer tissue samples from two cohorts. The effect of SUSD4 expression on cell migration and invasion was studied in vitro using two human breast cancer cell lines overexpressing SUSD4. \\ud \\ud Result...

Complementation pattern of lexB and recA mutations in Escherichia coli K12; mapping of tif-1, lexB and recA mutations

International Nuclear Information System (INIS)

Morand, P.; Goze, A.; Devoret, R.

1977-01-01

Three lexB mutations, whose phenotypes have been previously characterized, are studied here in relation to a few recA mutations as to their complementation pattern and relative location. The restoration of resistance to UV-light and to X-rays in the hetero-allelic diploid bacteria was used as a test for dominance and complementation. The wild type allele was always dominant over the mutant allele. Only partial complementation was found between lexB and two rexA alleles. There was no complementation between the recA alleles. All the data taken together strongly suggest that the complementations found are intragenic: lexB and recA mutations are in one gene. Mapping of lexB, recA and tif-1 mutations in relation to srl-1 and cysC by phage P1 transduction shows that lexB and the tif-1 mutations form a cluster proximal to srl-1 whereas recA mutations are located at the other extremity of the gene. Variability with temperature of cotransduction frequencies as well as their extended range of values prevent a meaningful calculation of the length of the recA gene. Our hypothesis is that the recA protein has two functional regions called A and B respectively defined at the genetical level by recA and lexB mutations and that it is, in vivo, an oligomeric protein forming a complex with the lexA protein. This complex is postulated to be multifunctional: recombination and control of exonuclease V are effected by the A region while the B region and lexA protein effect induced DNA repair and lysogenic induction. (orig.) [de

Specific alterations in complement protein activity of little brown myotis (Myotis lucifugus hibernating in white-nose syndrome affected sites.

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Marianne S Moore

Full Text Available White-nose syndrome (WNS is the most devastating condition ever reported for hibernating bats, causing widespread mortality in the northeastern United States. The syndrome is characterized by cutaneous lesions caused by a recently identified psychrophilic and keratinophylic fungus (Geomyces destructans, depleted fat reserves, atypical behavior, and damage to wings; however, the proximate cause of mortality is still uncertain. To assess relative levels of immunocompetence in bats hibernating in WNS-affected sites compared with levels in unaffected bats, we describe blood plasma complement protein activity in hibernating little brown myotis (Myotis lucifugus based on microbicidal competence assays using Escherichia coli, Staphylococcus aureus and Candida albicans. Blood plasma from bats collected during mid-hibernation at WNS-affected sites had higher bactericidal ability against E. coli and S. aureus, but lower fungicidal ability against C. albicans when compared with blood plasma from bats collected at unaffected sites. Within affected sites during mid-hibernation, we observed no difference in microbicidal ability between bats displaying obvious fungal infections compared to those without. Bactericidal ability against E. coli decreased significantly as hibernation progressed in bats collected from an affected site. Bactericidal ability against E. coli and fungicidal ability against C. albicans were positively correlated with body mass index (BMI during late hibernation. We also compared complement activity against the three microbes within individuals and found that the ability of blood plasma from hibernating M. lucifugus to lyse microbial cells differed as follows: E. coli>S. aureus>C. albicans. Overall, bats affected by WNS experience both relatively elevated and reduced innate immune responses depending on the microbe tested, although the cause of observed immunological changes remains unknown. Additionally, considerable trade-offs may exist

Specific alterations in complement protein activity of little brown myotis (Myotis lucifugus) hibernating in white-nose syndrome affected sites.

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Moore, Marianne S; Reichard, Jonathan D; Murtha, Timothy D; Zahedi, Bita; Fallier, Renee M; Kunz, Thomas H

2011-01-01

White-nose syndrome (WNS) is the most devastating condition ever reported for hibernating bats, causing widespread mortality in the northeastern United States. The syndrome is characterized by cutaneous lesions caused by a recently identified psychrophilic and keratinophylic fungus (Geomyces destructans), depleted fat reserves, atypical behavior, and damage to wings; however, the proximate cause of mortality is still uncertain. To assess relative levels of immunocompetence in bats hibernating in WNS-affected sites compared with levels in unaffected bats, we describe blood plasma complement protein activity in hibernating little brown myotis (Myotis lucifugus) based on microbicidal competence assays using Escherichia coli, Staphylococcus aureus and Candida albicans. Blood plasma from bats collected during mid-hibernation at WNS-affected sites had higher bactericidal ability against E. coli and S. aureus, but lower fungicidal ability against C. albicans when compared with blood plasma from bats collected at unaffected sites. Within affected sites during mid-hibernation, we observed no difference in microbicidal ability between bats displaying obvious fungal infections compared to those without. Bactericidal ability against E. coli decreased significantly as hibernation progressed in bats collected from an affected site. Bactericidal ability against E. coli and fungicidal ability against C. albicans were positively correlated with body mass index (BMI) during late hibernation. We also compared complement activity against the three microbes within individuals and found that the ability of blood plasma from hibernating M. lucifugus to lyse microbial cells differed as follows: E. coli>S. aureus>C. albicans. Overall, bats affected by WNS experience both relatively elevated and reduced innate immune responses depending on the microbe tested, although the cause of observed immunological changes remains unknown. Additionally, considerable trade-offs may exist between energy

Plasma complement and vascular complement deposition in patients with coronary artery disease with and without inflammatory rheumatic diseases

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2017-01-01

Purpose Inflammatory rheumatic diseases (IRD) are associated with accelerated coronary artery disease (CAD), which may result from both systemic and vascular wall inflammation. There are indications that complement may be involved in the pathogenesis of CAD in Systemic Lupus Erythematosus (SLE) and Rheumatoid Arthritis (RA). This study aimed to evaluate the associations between circulating complement and complement activation products with mononuclear cell infiltrates (MCI, surrogate marker of vascular inflammation) in the aortic media and adventitia in IRDCAD and non-IRDCAD patients undergoing coronary artery bypass grafting (CABG). Furthermore, we compared complement activation product deposition patterns in rare aorta adventitial and medial biopsies from SLE, RA and non-IRD patients. Methods We examined plasma C3 (p-C3) and terminal complement complexes (p-TCC) in 28 IRDCAD (SLE = 3; RA = 25), 52 non-IRDCAD patients, and 32 IRDNo CAD (RA = 32) from the Feiring Heart Biopsy Study. Aortic biopsies taken from the CAD only patients during CABG were previously evaluated for adventitial MCIs. The rare aortic biopsies from 3 SLE, 3 RA and 3 non-IRDCAD were assessed for the presence of C3 and C3d using immunohistochemistry. Results IRDCAD patients had higher p-TCC than non-IRDCAD or IRDNo CAD patients (prheumatic disease, and, in particular, SLE with the complement system. Exaggerated systemic and vascular complement activation may accelerate CVD, serve as a CVD biomarker, and represent a target for new therapies. PMID:28362874

Zonulin as prehaptoglobin2 regulates lung permeability and activates the complement system.

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Rittirsch, Daniel; Flierl, Michael A; Nadeau, Brian A; Day, Danielle E; Huber-Lang, Markus S; Grailer, Jamison J; Zetoune, Firas S; Andjelkovic, Anuska V; Fasano, Alessio; Ward, Peter A

2013-06-15

Zonulin is a protein involved in the regulation of tight junctions (TJ) in epithelial or endothelial cells. Zonulin is known to affect TJ in gut epithelial cells, but little is known about its influences in other organs. Prehaptoglobin2 has been identified as zonulin and is related to serine proteases (MASPs, C1qrs) that activate the complement system. The current study focused on the role of zonulin in development of acute lung injury (ALI) in C57BL/6 male mice following intrapulmonary deposition of IgG immune complexes. A zonulin antagonist (AT-1001) and a related peptide with permeability agonist activities (AT-1002) were employed and given intratracheally or intravenously. Also, zonulin was blocked in lung with a neutralizing antibody. In a dose-dependent manner, AT-1001 or zonulin neutralizing antibody attenuated the intensity of ALI (as quantitated by albumin leak, neutrophil accumulation, and proinflammatory cytokines). A similar pattern was found using the bacterial lipopolysaccharide model of ALI. Using confocal microscopy on sections of injured lungs, staining patterns for TJ proteins were discontinuous, reduced, and fragmented. As expected, the leak of blood products into the alveolar space confirmed the passage of 3 and 20 kDa dextran, and albumin. In contrast to AT-1001, application of the zonulin agonist AT-1002 intensified ALI. Zonulin both in vitro and in vivo induced generation of complement C3a and C5a. Collectively, these data suggest that zonulin facilitates development of ALI both by enhancing albumin leak and complement activation as well as increased buildup of neutrophils and cytokines during development of ALI.

An Ixodes ricinus Tick Salivary Lectin Pathway Inhibitor Protects Borrelia burgdorferi sensu lato from Human Complement

NARCIS (Netherlands)

Wagemakers, Alex; Coumou, Jeroen; Schuijt, Tim J.; Oei, Anneke; Nijhof, Ard M.; van 't Veer, Cornelis; van der Poll, Tom; Bins, Adriaan D.; Hovius, Joppe W. R.

2016-01-01

We previously identified tick salivary lectin pathway inhibitor (TSLPI) in Ixodes scapularis, a vector for Borrelia burgdorferi sensu stricto (s.s.) in North America. TSLPI is a salivary protein facilitating B. burgdorferi s.s. transmission and acquisition by inhibiting the host lectin complement

RNA-Binding Proteins in Trichomonas vaginalis: Atypical Multifunctional Proteins

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Elisa E. Figueroa-Angulo

2015-11-01

Full Text Available Iron homeostasis is highly regulated in vertebrates through a regulatory system mediated by RNA-protein interactions between the iron regulatory proteins (IRPs that interact with an iron responsive element (IRE located in certain mRNAs, dubbed the IRE-IRP regulatory system. Trichomonas vaginalis, the causal agent of trichomoniasis, presents high iron dependency to regulate its growth, metabolism, and virulence properties. Although T. vaginalis lacks IRPs or proteins with aconitase activity, possesses gene expression mechanisms of iron regulation at the transcriptional and posttranscriptional levels. However, only one gene with iron regulation at the transcriptional level has been described. Recently, our research group described an iron posttranscriptional regulatory mechanism in the T. vaginalis tvcp4 and tvcp12 cysteine proteinase mRNAs. The tvcp4 and tvcp12 mRNAs have a stem-loop structure in the 5'-coding region or in the 3'-UTR, respectively that interacts with T. vaginalis multifunctional proteins HSP70, α-Actinin, and Actin under iron starvation condition, causing translation inhibition or mRNA stabilization similar to the previously characterized IRE-IRP system in eukaryotes. Herein, we summarize recent progress and shed some light on atypical RNA-binding proteins that may participate in the iron posttranscriptional regulation in T. vaginalis.

RNA-Binding Proteins in Trichomonas vaginalis: Atypical Multifunctional Proteins.

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Figueroa-Angulo, Elisa E; Calla-Choque, Jaeson S; Mancilla-Olea, Maria Inocente; Arroyo, Rossana

2015-11-26

Iron homeostasis is highly regulated in vertebrates through a regulatory system mediated by RNA-protein interactions between the iron regulatory proteins (IRPs) that interact with an iron responsive element (IRE) located in certain mRNAs, dubbed the IRE-IRP regulatory system. Trichomonas vaginalis, the causal agent of trichomoniasis, presents high iron dependency to regulate its growth, metabolism, and virulence properties. Although T. vaginalis lacks IRPs or proteins with aconitase activity, possesses gene expression mechanisms of iron regulation at the transcriptional and posttranscriptional levels. However, only one gene with iron regulation at the transcriptional level has been described. Recently, our research group described an iron posttranscriptional regulatory mechanism in the T. vaginalis tvcp4 and tvcp12 cysteine proteinase mRNAs. The tvcp4 and tvcp12 mRNAs have a stem-loop structure in the 5'-coding region or in the 3'-UTR, respectively that interacts with T. vaginalis multifunctional proteins HSP70, α-Actinin, and Actin under iron starvation condition, causing translation inhibition or mRNA stabilization similar to the previously characterized IRE-IRP system in eukaryotes. Herein, we summarize recent progress and shed some light on atypical RNA-binding proteins that may participate in the iron posttranscriptional regulation in T. vaginalis.

The Evolution of the Secreted Regulatory Protein Progranulin.

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Roger G E Palfree

Full Text Available Progranulin is a secreted growth factor that is active in tumorigenesis, wound repair, and inflammation. Haploinsufficiency of the human progranulin gene, GRN, causes frontotemporal dementia. Progranulins are composed of chains of cysteine-rich granulin modules. Modules may be released from progranulin by proteolysis as 6kDa granulin polypeptides. Both intact progranulin and some of the granulin polypeptides are biologically active. The granulin module occurs in certain plant proteases and progranulins are present in early diverging metazoan clades such as the sponges, indicating their ancient evolutionary origin. There is only one Grn gene in mammalian genomes. More gene-rich Grn families occur in teleost fish with between 3 and 6 members per species including short-form Grns that have no tetrapod counterparts. Our goals are to elucidate progranulin and granulin module evolution by investigating (i: the origins of metazoan progranulins (ii: the evolutionary relationships between the single Grn of tetrapods and the multiple Grn genes of fish (iii: the evolution of granulin module architectures of vertebrate progranulins (iv: the conservation of mammalian granulin polypeptide sequences and how the conserved granulin amino acid sequences map to the known three dimensional structures of granulin modules. We report that progranulin-like proteins are present in unicellular eukaryotes that are closely related to metazoa suggesting that progranulin is among the earliest extracellular regulatory proteins still employed by multicellular animals. From the genomes of the elephant shark and coelacanth we identified contemporary representatives of a precursor for short-from Grn genes of ray-finned fish that is lost in tetrapods. In vertebrate Grns pathways of exon duplication resulted in a conserved module architecture at the amino-terminus that is frequently accompanied by an unusual pattern of tandem nearly identical module repeats near the carboxyl

Evolutionary Conservation and Emerging Functional Diversity of the Cytosolic Hsp70:J Protein Chaperone Network of Arabidopsis thaliana.

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Verma, Amit K; Diwan, Danish; Raut, Sandeep; Dobriyal, Neha; Brown, Rebecca E; Gowda, Vinita; Hines, Justin K; Sahi, Chandan

2017-06-07

Heat shock proteins of 70 kDa (Hsp70s) partner with structurally diverse Hsp40s (J proteins), generating distinct chaperone networks in various cellular compartments that perform myriad housekeeping and stress-associated functions in all organisms. Plants, being sessile, need to constantly maintain their cellular proteostasis in response to external environmental cues. In these situations, the Hsp70:J protein machines may play an important role in fine-tuning cellular protein quality control. Although ubiquitous, the functional specificity and complexity of the plant Hsp70:J protein network has not been studied. Here, we analyzed the J protein network in the cytosol of Arabidopsis thaliana and, using yeast genetics, show that the functional specificities of most plant J proteins in fundamental chaperone functions are conserved across long evolutionary timescales. Detailed phylogenetic and functional analysis revealed that increased number, regulatory differences, and neofunctionalization in J proteins together contribute to the emerging functional diversity and complexity in the Hsp70:J protein network in higher plants. Based on the data presented, we propose that higher plants have orchestrated their "chaperome," especially their J protein complement, according to their specialized cellular and physiological stipulations. Copyright © 2017 Verma et al.

Francisella tularensis Confronts the Complement System

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Susan R. Brock

2017-12-01

Full Text Available Francisella tularensis has developed a number of effective evasion strategies to counteract host immune defenses, not the least of which is its ability to interact with the complement system to its own advantage. Following exposure of the bacterium to fresh human serum, complement is activated and C3b and iC3b can be found covalently attached to the bacterial surface. However, the lipopolysaccharide and capsule of the F. tularensis cell wall prevent complement-mediated lysis and endow the bacterium with serum resistance. Opsonization of F. tularensis with C3 greatly increases its uptake by human neutrophils, dendritic cells and macrophages. Uptake occurs by an unusual looping morphology in human macrophages. Complement receptor 3 is thought to play an important role in opsonophagocytosis by human macrophages, and signaling through this receptor can antagonize Toll-like receptor 2-initiated macrophage activation. Complement C3 also determines the survival of infected human macrophages and perhaps other cell types. C3-opsonization of F. tularensis subsp. tularensis strain SCHU S4 results in greatly increased death of infected human macrophages, which requires more than complement receptor engagement and is independent of the intracellular replication by the pathogen. Given its entry into the cytosol of host cells, F. tularensis has the potential for a number of other complement-mediated interactions. Studies on the uptake C3-opsonized adenovirus have suggested the existence of a C3 sensing system that initiates cellular responses to cytosolic C3b present on invading microbes. Here we propose that C3 peptides enter the cytosol of human macrophages following phagosome escape of F. tularensis and are recognized as intruding molecular patterns that signal host cell death. With the discovery of new roles for intracellular C3, a better understanding of tularemia pathogenesis is likely to emerge.

Regulatory protein OmpR influences the serum resistance of Yersinia enterocolitica O:9 by modifying the structure of the outer membrane.

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Karolina Skorek

Full Text Available The EnvZ/OmpR two-component system constitutes a regulatory pathway involved in bacterial adaptive responses to environmental cues. Our previous findings indicated that the OmpR regulator in Yersinia enterocolitica O:9 positively regulates the expression of FlhDC, the master flagellar activator, which influences adhesion/invasion properties and biofilm formation. Here we show that a strain lacking OmpR grown at 37°C exhibits extremely high resistance to the bactericidal activity of normal human serum (NHS compared with the wild-type strain. Analysis of OMP expression in the ompR mutant revealed that OmpR reciprocally regulates Ail and OmpX, two homologous OMPs of Y. enterocolitica, without causing significant changes in the level of YadA, the major serum resistance factor. Analysis of mutants in individual genes belonging to the OmpR regulon (ail, ompX, ompC and flhDC and strains lacking plasmid pYV, expressing YadA, demonstrated the contribution of the respective proteins to serum resistance. We show that Ail and OmpC act in an opposite way to the OmpX protein to confer serum resistance to the wild-type strain, but are not responsible for the high resistance of the ompR mutant. The serum resistance phenotype of ompR seems to be multifactorial and mainly attributable to alterations that potentiate the function of YadA. Our results indicate that a decreased level of FlhDC in the ompR mutant cells is partly responsible for the serum resistance and this effect can be suppressed by overexpression of flhDC in trans. The observation that the loss of FlhDC enhances the survival of wild-type cells in NHS supports the involvement of FlhDC regulator in this phenotype. In addition, the ompR mutant exhibited a lower level of LPS, but this was not correlated with changes in the level of FlhDC. We propose that OmpR might alter the susceptibility of Y. enterocolitica O:9 to complement-mediated killing through remodeling of the outer membrane.

Crosstalk between complement and Toll-like receptor activation in relation to donor brain death and renal ischemia-reperfusion injury.

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Damman, Jeffrey; Daha, Mohamed R; van Son, Willem J; Leuvenink, Henri G; Ploeg, Rutger J; Seelen, Marc A

2011-04-01

Two central pathways of innate immunity, complement and Toll-like receptors (TLRs), play an important role in the pathogenesis of renal injury inherent to kidney transplantation. Recent findings indicate close crosstalk between complement and TLR signaling pathways. It is suggested that mitogen activated protein kinases (MAPKs) might be the key molecules linking both the complement and TLR pathways together. Complement and TLRs are important mediators of renal ischemia-reperfusion injury (IRI). Besides IRI, complement C3 can also be upregulated and activated in the kidney before transplantation as a direct result of brain death (BD) in the donor. This local upregulation and activation of complement in the donor kidney has been proven to be detrimental for renal allograft outcome. Also TLR4 and several of its major ligands are upregulated by donor BD compared to living donors. Important and in line with the observations above, kidney transplant recipients have a benefit when receiving a kidney from a TLR4 Asp299Gly/Thr399Ile genotypic donor. The role of complement and TLRs and crosstalk between these two innate immune systems in relation to renal injury during donor BD and ischemia-reperfusion are focus of this review. Future strategies to target complement and TLR activation in kidney transplantation are considered. ©2011 The Authors Journal compilation©2011 The American Society of Transplantation and the American Society of Transplant Surgeons.

Carnivorous Nutrition in Pitcher Plants (Nepenthes spp.) via an Unusual Complement of Endogenous Enzymes.

Science.gov (United States)

Lee, Linda; Zhang, Ye; Ozar, Brittany; Sensen, Christoph W; Schriemer, David C

2016-09-02

Plants belonging to the genus Nepenthes are carnivorous, using specialized pitfall traps called "pitchers" that attract, capture, and digest insects as a primary source of nutrients. We have used RNA sequencing to generate a cDNA library from the Nepenthes pitchers and applied it to mass spectrometry-based identification of the enzymes secreted into the pitcher fluid using a nonspecific digestion strategy superior to trypsin in this application. This first complete catalog of the pitcher fluid subproteome includes enzymes across a variety of functional classes. The most abundant proteins present in the secreted fluid are proteases, nucleases, peroxidases, chitinases, a phosphatase, and a glucanase. Nitrogen recovery involves a particularly rich complement of proteases. In addition to the two expected aspartic proteases, we discovered three novel nepenthensins, two prolyl endopeptidases that we name neprosins, and a putative serine carboxypeptidase. Additional proteins identified are relevant to pathogen-defense and secretion mechanisms. The full complement of acid-stable enzymes discovered in this study suggests that carnivory in the genus Nepenthes can be sustained by plant-based mechanisms alone and does not absolutely require bacterial symbiosis.

Synergistic inhibition of the intrinsic factor X activation by protein S and C4b-binding protein

NARCIS (Netherlands)

Koppelman, S.J.

1995-01-01

The complement protein C4b-binding protein plays an important role in the regulation of the protein C anticoagulant pathway. C4b-binding protein can bind to protein S, thereby inhibiting the cofactor activity of protein S for activated protein C. In this report, we describe a new role for

Complement component C5a Promotes Expression of IL-22 and IL-17 from Human T cells and its Implication in Age-related Macular Degeneration

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Klein Michael L

2011-07-01

Full Text Available Abstract Background Age related macular degeneration (AMD is the leading cause of irreversible blindness in elderly populations worldwide. Inflammation, among many factors, has been suggested to play an important role in AMD pathogenesis. Recent studies have demonstrated a strong genetic association between AMD and complement factor H (CFH, the down-regulatory factor of complement activation. Elevated levels of complement activating molecules including complement component 5a (C5a have been found in the serum of AMD patients. Our aim is to study whether C5a can impact human T cells and its implication in AMD. Methods Human peripheral blood mononuclear cells (PBMCs were isolated from the blood of exudative form of AMD patients using a Ficoll gradient centrifugation protocol. Intracellular staining and enzyme-linked immunosorbent assays were used to measure protein expression. Apoptotic cells were detected by staining of cells with the annexin-V and TUNEL technology and analyzed by a FACS Caliber flow cytometer. SNP genotyping was analyzed by TaqMan genotyping assay using the Real-time PCR system 7500. Results We show that C5a promotes interleukin (IL-22 and IL-17 expression by human CD4+ T cells. This effect is dependent on B7, IL-1β and IL-6 expression from monocytes. We have also found that C5a could protect human CD4+ cells from undergoing apoptosis. Importantly, consistent with a role of C5a in promoting IL-22 and IL-17 expression, significant elevation in IL-22 and IL-17 levels was found in AMD patients as compared to non-AMD controls. Conclusions Our results support the notion that C5a may be one of the factors contributing to the elevated serum IL-22 and IL-17 levels in AMD patients. The possible involvement of IL-22 and IL-17 in the inflammation that contributes to AMD may herald a new approach to treat AMD.

Complement Attack against Aspergillus and Corresponding Evasion Mechanisms

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Cornelia Speth

2012-01-01

Full Text Available Invasive aspergillosis shows a high mortality rate particularly in immunocompromised patients. Perpetually increasing numbers of affected patients highlight the importance of a clearer understanding of interactions between innate immunity and fungi. Innate immunity is considered to be the most significant host defence against invasive fungal infections. Complement represents a crucial part of this first line defence and comprises direct effects against invading pathogens as well as bridging functions to other parts of the immune network. However, despite the potency of complement to attack foreign pathogens, the prevalence of invasive fungal infections is increasing. Two possible reasons may explain that phenomenon: First, complement activation might be insufficient for an effective antifungal defence in risk patients (due to, e.g., low complement levels, poor recognition of fungal surface, or missing interplay with other immune elements in immunocompromised patients. On the other hand, fungi may have developed evasion strategies to avoid recognition and/or eradication by complement. In this review, we summarize the most important interactions between Aspergillus and the complement system. We describe the various ways of complement activation by Aspergillus and the antifungal effects of the system, and also show proven and probable mechanisms of Aspergillus for complement evasion.

Alkhedaide et al., Afr J Tradit Complement Altern Med., (2017) 14 (5 ...

African Journals Online (AJOL)

It regulates the body through helper and suppressor cells and soluble products. ..... It is basically consisted of lymphoid tissue, bone ... Most of cytokines are regulatory proteins, which in turn may cause dysregulation of innate and/or adaptive.

Bioactive lysophospholipids generated by hepatic lipase degradation of lipoproteins lead to complement activation via the classical pathway.

Science.gov (United States)

Ma, Wanchao; Paik, David C; Barile, Gaetano R

2014-09-09

We determined bioactivity of lysophospholipids generated by degradation of the low-density (LDL), very low-density (VLDL), and high-density (HDL) lipoproteins with hepatic lipase (HL), cholesterol esterase (CE), and lipoprotein-associated phospholipase A2 (Lp-PLA2). The LDL, VLDL, and HDL were treated with HL, CE, and Lp-PLA2 after immobilization on plates, and complement activation studies were performed with diluted human serum. Complement component 3 (C3) fixation, a marker for complement activation, was determined with a monoclonal anti-human C3d antibody. Enzymatic properties of HL and CE were assayed with triglyceride and phosphatidylcholine substrates for triglyceride hydrolase and phospholipase A activities. The ARPE-19 cells were used for viability studies. The HL degradation of human lipoproteins LDL, VLDL, or HDL results in the formation of modified lipoproteins that can activate the complement pathway. Complement activation is dose- and time-dependent upon HL and occurs via the classical pathway. Enzymatic studies suggest that the phospholipase A1 activity of HL generates complement-activating lysophospholipids. C-reactive protein (CRP), known to simultaneously interact with complement C1 and complement factor H (CFH), further enhances HL-induced complement activation. The lysophospholipids, 1-Palmitoyl-sn-glycero-3-phosphocholine and 1-Oleoyl-sn-glycero-3-phosphocholine, can be directly cytotoxic to ARPE-19 cells. The HL degradation of lipoproteins, known to accumulate in the outer retina and in drusen, can lead to the formation of bioactive lysophospholipids that can trigger complement activation and induce RPE cellular dysfunction. Given the known risk associations for age-related macular degeneration (AMD) with HL, CRP, and CFH, this study elucidates a possible damage pathway for age-related macular degeneration (AMD) in genetically predisposed individuals, that HL activity may lead to accumulation of lysophospholipids to initiate complement

Human Properdin Opsonizes Nanoparticles and Triggers a Potent Pro-inflammatory Response by Macrophages without Involving Complement Activation

Science.gov (United States)

Kouser, Lubna; Paudyal, Basudev; Kaur, Anuvinder; Stenbeck, Gudrun; Jones, Lucy A.; Abozaid, Suhair M.; Stover, Cordula M.; Flahaut, Emmanuel; Sim, Robert B.; Kishore, Uday

2018-01-01

Development of nanoparticles as tissue-specific drug delivery platforms can be considerably influenced by the complement system because of their inherent pro-inflammatory and tumorigenic consequences. The complement activation pathways, and its recognition subcomponents, can modulate clearance of the nanoparticles and subsequent inflammatory response and thus alter the intended translational applications. Here, we report, for the first time, that human properdin, an upregulator of the complement alternative pathway, can opsonize functionalized carbon nanotubes (CNTs) via its thrombospondin type I repeat (TSR) 4 and 5. Binding of properdin and TSR4+5 is likely to involve charge pattern/polarity recognition of the CNT surface since both carboxymethyl cellulose-coated carbon nanotubes (CMC-CNT) and oxidized (Ox-CNT) bound these proteins well. Properdin enhanced the uptake of CMC-CNTs by a macrophage cell line, THP-1, mounting a robust pro-inflammatory immune response, as revealed by qRT-PCR, multiplex cytokine array, and NF-κB nuclear translocation analyses. Properdin can be locally synthesized by immune cells in an inflammatory microenvironment, and thus, its interaction with nanoparticles is of considerable importance. In addition, recombinant TSR4+5 coated on the CMC-CNTs inhibited complement consumption by CMC-CNTs, suggesting that nanoparticle decoration with TSR4+5, can be potentially used as a complement inhibitor in a number of pathological contexts arising due to exaggerated complement activation. PMID:29483907

PipY, a Member of the Conserved COG0325 Family of PLP-Binding Proteins, Expands the Cyanobacterial Nitrogen Regulatory Network

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José I. Labella

2017-07-01

Full Text Available Synechococcus elongatus PCC 7942 is a paradigmatic model organism for nitrogen regulation in cyanobacteria. Expression of genes involved in nitrogen assimilation is positively regulated by the 2-oxoglutarate receptor and global transcriptional regulator NtcA. Maximal activation requires the subsequent binding of the co-activator PipX. PII, a protein found in all three domains of life as an integrator of signals of the nitrogen and carbon balance, binds to PipX to counteract NtcA activity at low 2-oxoglutarate levels. PII-PipX complexes can also bind to the transcriptional regulator PlmA, whose regulon remains unknown. Here we expand the nitrogen regulatory network to PipY, encoded by the bicistronic operon pipXY in S. elongatus. Work with PipY, the cyanobacterial member of the widespread family of COG0325 proteins, confirms the conserved roles in vitamin B6 and amino/keto acid homeostasis and reveals new PLP-related phenotypes, including sensitivity to antibiotics targeting essential PLP-holoenzymes or synthetic lethality with cysK. In addition, the related phenotypes of pipY and pipX mutants are consistent with genetic interactions in the contexts of survival to PLP-targeting antibiotics and transcriptional regulation. We also showed that PipY overexpression increased the length of S. elongatus cells. Taken together, our results support a universal regulatory role for COG0325 proteins, paving the way to a better understanding of these proteins and of their connections with other biological processes.

Specific interactions between DNA and regulatory protein controlled by ligand-binding: Ab initio molecular simulation

Energy Technology Data Exchange (ETDEWEB)

Matsushita, Y., E-mail: kurita@cs.tut.ac.jp; Murakawa, T., E-mail: kurita@cs.tut.ac.jp; Shimamura, K., E-mail: kurita@cs.tut.ac.jp; Oishi, M., E-mail: kurita@cs.tut.ac.jp; Ohyama, T., E-mail: kurita@cs.tut.ac.jp; Kurita, N., E-mail: kurita@cs.tut.ac.jp [Department of Computer Science and Engineering, Toyohashi University of Technology, Tempaku-cho, Toyohashi, Aichi, 441-8580 (Japan)

2015-02-27

The catabolite activator protein (CAP) is one of the regulatory proteins controlling the transcription mechanism of gene. Biochemical experiments elucidated that the complex of CAP with cyclic AMP (cAMP) is indispensable for controlling the mechanism, while previous molecular simulations for the monomer of CAP+cAMP complex revealed the specific interactions between CAP and cAMP. However, the effect of cAMP-binding to CAP on the specific interactions between CAP and DNA is not elucidated at atomic and electronic levels. We here considered the ternary complex of CAP, cAMP and DNA in solvating water molecules and investigated the specific interactions between them at atomic and electronic levels using ab initio molecular simulations based on classical molecular dynamics and ab initio fragment molecular orbital methods. The results highlight the important amino acid residues of CAP for the interactions between CAP and cAMP and between CAP and DNA.

Specific interactions between DNA and regulatory protein controlled by ligand-binding: Ab initio molecular simulation

International Nuclear Information System (INIS)

Matsushita, Y.; Murakawa, T.; Shimamura, K.; Oishi, M.; Ohyama, T.; Kurita, N.

2015-01-01

The catabolite activator protein (CAP) is one of the regulatory proteins controlling the transcription mechanism of gene. Biochemical experiments elucidated that the complex of CAP with cyclic AMP (cAMP) is indispensable for controlling the mechanism, while previous molecular simulations for the monomer of CAP+cAMP complex revealed the specific interactions between CAP and cAMP. However, the effect of cAMP-binding to CAP on the specific interactions between CAP and DNA is not elucidated at atomic and electronic levels. We here considered the ternary complex of CAP, cAMP and DNA in solvating water molecules and investigated the specific interactions between them at atomic and electronic levels using ab initio molecular simulations based on classical molecular dynamics and ab initio fragment molecular orbital methods. The results highlight the important amino acid residues of CAP for the interactions between CAP and cAMP and between CAP and DNA

Does Host Complement Kill Borrelia burgdorferi within Ticks?

OpenAIRE

Rathinavelu, Sivaprakash; Broadwater, Anne; de Silva, Aravinda M.

2003-01-01

The Lyme disease spirochete, Borrelia burgdorferi, inhabits the gut lumen of the tick vector. At this location the spirochete is exposed to host blood when a tick feeds. We report here on studies that were done with normal and complement-deficient (C3-knockout) mice to determine if the host complement system killed spirochetes within the vector. We found that spirochete numbers within feeding nymphs were not influenced by complement, most likely because host complement was inactivated within ...

The Protein Model Portal

OpenAIRE

Arnold, Konstantin; Kiefer, Florian; Kopp, J?rgen; Battey, James N. D.; Podvinec, Michael; Westbrook, John D.; Berman, Helen M.; Bordoli, Lorenza; Schwede, Torsten

2008-01-01

Structural Genomics has been successful in determining the structures of many unique proteins in a high throughput manner. Still, the number of known protein sequences is much larger than the number of experimentally solved protein structures. Homology (or comparative) modeling methods make use of experimental protein structures to build models for evolutionary related proteins. Thereby, experimental structure determination efforts and homology modeling complement each other in the exploratio...

Monitoring β-arrestin recruitment via β-lactamase enzyme fragment complementation: purification of peptide E as a low-affinity ligand for mammalian bombesin receptors.

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Yuichi Ikeda

Full Text Available Identification of cognate ligands for G protein-coupled receptors (GPCRs provides a starting point for understanding novel regulatory mechanisms. Although GPCR ligands have typically been evaluated through the activation of heterotrimeric G proteins, recent studies have shown that GPCRs signal not only through G proteins but also through β-arrestins. As such, monitoring β-arrestin signaling instead of G protein signaling will increase the likelihood of identifying currently unknown ligands, including β-arrestin-biased agonists. Here, we developed a cell-based assay for monitoring ligand-dependent GPCR-β-arrestin interaction via β-lactamase enzyme fragment complementation. Inter alia, β-lactamase is a superior reporter enzyme because of its cell-permeable fluorescent substrate. This substrate makes the assay non-destructive and compatible with fluorescence-activated cell sorting (FACS. In a reporter cell, complementary fragments of β-lactamase (α and ω were fused to β-arrestin 2 and GPCR, respectively. Ligand stimulation initiated the interaction of these chimeric proteins (β-arrestin-α and GPCR-ω, and this inducible interaction was measured through reconstituted β-lactamase activity. Utilizing this system, we screened various mammalian tissue extracts for agonistic activities on human bombesin receptor subtype 3 (hBRS3. We purified peptide E as a low-affinity ligand for hBRS3, which was also found to be an agonist for the other two mammalian bombesin receptors such as gastrin-releasing peptide receptor (GRPR and neuromedin B receptor (NMBR. Successful purification of peptide E has validated the robustness of this assay. We conclude that our newly developed system will facilitate the discovery of GPCR ligands.

Targeted organ generation using Mixl1-inducible mouse pluripotent stem cells in blastocyst complementation.

Science.gov (United States)

Kobayashi, Toshihiro; Kato-Itoh, Megumi; Nakauchi, Hiromitsu

2015-01-15

Generation of functional organs from patients' own cells is one of the ultimate goals of regenerative medicine. As a novel approach to creation of organs from pluripotent stem cells (PSCs), we employed blastocyst complementation in organogenesis-disabled animals and successfully generated PSC-derived pancreas and kidneys. Blastocyst complementation, which exploits the capacity of PSCs to participate in forming chimeras, does not, however, exclude contribution of PSCs to the development of tissues-including neural cells or germ cells-other than those specifically targeted by disabling of organogenesis. This fact provokes ethical controversy if human PSCs are to be used. In this study, we demonstrated that forced expression of Mix-like protein 1 (encoded by Mixl1) can be used to guide contribution of mouse embryonic stem cells to endodermal organs after blastocyst injection. We then succeeded in applying this method to generate functional pancreas in pancreatogenesis-disabled Pdx1 knockout mice using a newly developed tetraploid-based organ-complementation method. These findings hold promise for targeted organ generation from patients' own PSCs in livestock animals.

Complement and thrombosis in the antiphospholipid syndrome.

Science.gov (United States)

Oku, Kenji; Nakamura, Hiroyuki; Kono, Michihiro; Ohmura, Kazumasa; Kato, Masaru; Bohgaki, Toshiyuki; Horita, Tetsuya; Yasuda, Shinsuke; Amengual, Olga; Atsumi, Tatsuya

2016-10-01

The involvement of complement activation in the pathophysiology of antiphospholipid syndrome (APS) was first reported in murine models of antiphospholipid antibody (aPL)-related pregnancy morbidities. We previously reported that complement activation is prevalent and may function as a source of procoagulant cell activation in the sera of APS patients. Recently, autoantibodies against C1q, a component of complement 1, were reported to be correlated with complement activation in systemic lupus erythematosus. These antibodies target neoepitopes of deformed C1q bound to various molecules (i.e., anionic phospholipids) and induce accelerated complement activation. We found that anti-C1q antibodies are more frequently detected in primary APS patients than in control patients and in refractory APS patients with repeated thrombotic events. The titer of anti-C1q antibodies was significantly higher in refractory APS patients than in APS patients without flare. The binding of C1q to anionic phospholipids may be associated with the surge in complement activation in patients with anti-C1q antibodies when triggered by 'second-hit' biological stressors such as infection. Such stressors will induce overexpression of anionic phospholipids, with subsequent increases in deformed C1q that is targeted by anti-C1q antibodies. Copyright © 2016. Published by Elsevier B.V.

Integrated analysis of RNA-binding protein complexes using in vitro selection and high-throughput sequencing and sequence specificity landscapes (SEQRS).

Science.gov (United States)

Lou, Tzu-Fang; Weidmann, Chase A; Killingsworth, Jordan; Tanaka Hall, Traci M; Goldstrohm, Aaron C; Campbell, Zachary T

2017-04-15

RNA-binding proteins (RBPs) collaborate to control virtually every aspect of RNA function. Tremendous progress has been made in the area of global assessment of RBP specificity using next-generation sequencing approaches both in vivo and in vitro. Understanding how protein-protein interactions enable precise combinatorial regulation of RNA remains a significant problem. Addressing this challenge requires tools that can quantitatively determine the specificities of both individual proteins and multimeric complexes in an unbiased and comprehensive way. One approach utilizes in vitro selection, high-throughput sequencing, and sequence-specificity landscapes (SEQRS). We outline a SEQRS experiment focused on obtaining the specificity of a multi-protein complex between Drosophila RBPs Pumilio (Pum) and Nanos (Nos). We discuss the necessary controls in this type of experiment and examine how the resulting data can be complemented with structural and cell-based reporter assays. Additionally, SEQRS data can be integrated with functional genomics data to uncover biological function. Finally, we propose extensions of the technique that will enhance our understanding of multi-protein regulatory complexes assembled onto RNA. Copyright © 2016 Elsevier Inc. All rights reserved.

Structure and function of complement protein C1q and its role in the development of autoimmune diseases

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Katarzyna Smykał-Jankowiak

2009-09-01

Full Text Available Complement plays an important role in the immune system. Three different pathways of complement activation are known: the classical, alternative, and lectin dependent. They involve more than 30 serum peptides. C1q is the first subcomponent of the classical pathway of complement activation. It is composed of three types of chains, A, B, and C, which form a molecule containing 18 peptides. Each of the chains has a short amino-terminal region followed by a collagen-like region (playing a role in the activation of C1r2C1s2 and a carboxy-terminal head, which binds to immune complexes. Recent studies have shown a great number of ligands for C1q, including aggregated IgG, IgM, human T-cell lymphotropic virus-I (HTLV-I, gp21 peptide, human immunodeficiency virus-1 (HIV-1 gp21 peptide, β-amyloid, fragments of bacterial walls, apoptotic cells, and many others. However, the role of C1q is not only associated with complement activation. It also helps in the removal of immune complexes and necrotic cells, stimulates the production of some cytokines, and modulates the function of lymphocytes. Complete C1q deficiency is a rare genetic disorder. The C1q gene is located on the short arm of chromosome 1. So far, only a few mutations in C1q gene have been reported. The presence of these mutations is strongly associated with recurrent bacterial infections and the development of systemic lupus erythematosus (SLE. Recent clinical studies point to the significance of anti-C1q antibodies in the diagnosis and assessment of lupus nephritis activity.

Hepatic macrophage complement receptor clearance function following injury.

Science.gov (United States)

Cuddy, B G; Loegering, D J; Blumenstock, F A; Shah, D M

1986-03-01

Previous work has demonstrated that in vivo hepatic macrophage complement receptor clearance function is depressed following thermal injury. The present study was carried out to determine if complement receptor function depression is associated with other states of depressed host defense. Hepatic complement receptor clearance function was determined from the hepatic uptake of rat erythrocytes coated with antierythrocyte IgM (EIgM) in rats. Receptor function was determined following cannulation of a carotid artery, laparotomy plus enterotomy, hemorrhagic shock, trauma, thermal injury, acute bacteremia, acute endotoxemia, and injection of erythrocyte stroma, gelatinized lipid emulsion, or colloidal carbon. Hepatic uptake of EIgM was depressed following each of these experimental interventions except arterial cannulation. This effect was shown not to be due to a decrease in hepatic blood flow or depletion of complement and was therefore due to a depression in hepatic macrophage complement receptor clearance function. Thus, impairment of hepatic macrophage complement receptor function is associated with several states of depressed host defense.

The EbpA-RpoN Regulatory Pathway of the Pathogen Leptospira interrogans Is Essential for Survival in the Environment

Science.gov (United States)

Hu, Wei-Lin; Pappas, Christopher J.; Zhang, Jun-Jie; Yang, You-Yun; Yan, Jie

2016-01-01

ABSTRACT Leptospira interrogans is the agent of leptospirosis, a reemerging zoonotic disease. It is transmitted to humans through environmental surface waters contaminated by the urine of mammals chronically infected by pathogenic strains able to survive in water for long periods. Little is known about the regulatory pathways underlying environmental sensing and host adaptation of L. interrogans during its enzootic cycle. This study identifies the EbpA-RpoN regulatory pathway in L. interrogans. In this pathway, EbpA, a σ54 activator and putative prokaryotic enhancer-binding protein (EBP), and the alternative sigma factor RpoN (σ54) control expression of at least three genes, encoding AmtB (an ammonium transport protein) and two proteins of unknown function. Electrophoresis mobility shift assay demonstrated that recombinant RpoN and EbpA bind to the promoter region and upstream of these three identified genes, respectively. Genetic disruption of ebpA in L. interrogans serovar Manilae virtually abolished expression of the three genes, including amtB in two independent ebpA mutants. Complementation of the ebpA mutant restored expression of these genes. Intraperitoneal inoculation of gerbils with the ebpA mutant did not affect mortality. However, the ebpA mutant had decreased cell length in vitro and had a significantly lowered cell density at stationary phase when grown with l-alanine as the sole nitrogen source. Furthermore, the ebpA mutant has dramatically reduced long-term survival ability in water. Together, these studies identify a regulatory pathway, the EbpA-RpoN pathway, that plays an important role in the zoonotic cycle of L. interrogans. IMPORTANCE Leptospirosis is a reemerging disease with global importance. However, our understanding of gene regulation of the spirochetal pathogen Leptospira interrogans is still in its infancy, largely due to the lack of robust tools for genetic manipulation of this spirochete. Little is known about how the pathogen

The EbpA-RpoN Regulatory Pathway of the Pathogen Leptospira interrogans Is Essential for Survival in the Environment.

Science.gov (United States)

Hu, Wei-Lin; Pappas, Christopher J; Zhang, Jun-Jie; Yang, You-Yun; Yan, Jie; Picardeau, Mathieu; Yang, X Frank

2017-02-01

Leptospira interrogans is the agent of leptospirosis, a reemerging zoonotic disease. It is transmitted to humans through environmental surface waters contaminated by the urine of mammals chronically infected by pathogenic strains able to survive in water for long periods. Little is known about the regulatory pathways underlying environmental sensing and host adaptation of L. interrogans during its enzootic cycle. This study identifies the EbpA-RpoN regulatory pathway in L. interrogans In this pathway, EbpA, a σ 54 activator and putative prokaryotic enhancer-binding protein (EBP), and the alternative sigma factor RpoN (σ 54 ) control expression of at least three genes, encoding AmtB (an ammonium transport protein) and two proteins of unknown function. Electrophoresis mobility shift assay demonstrated that recombinant RpoN and EbpA bind to the promoter region and upstream of these three identified genes, respectively. Genetic disruption of ebpA in L. interrogans serovar Manilae virtually abolished expression of the three genes, including amtB in two independent ebpA mutants. Complementation of the ebpA mutant restored expression of these genes. Intraperitoneal inoculation of gerbils with the ebpA mutant did not affect mortality. However, the ebpA mutant had decreased cell length in vitro and had a significantly lowered cell density at stationary phase when grown with l-alanine as the sole nitrogen source. Furthermore, the ebpA mutant has dramatically reduced long-term survival ability in water. Together, these studies identify a regulatory pathway, the EbpA-RpoN pathway, that plays an important role in the zoonotic cycle of L. interrogans IMPORTANCE: Leptospirosis is a reemerging disease with global importance. However, our understanding of gene regulation of the spirochetal pathogen Leptospira interrogans is still in its infancy, largely due to the lack of robust tools for genetic manipulation of this spirochete. Little is known about how the pathogen achieves its

High concentrations of protein test substances may have non-toxic effects on Daphnia magna: implications for regulatory study designs and ecological risk assessments for GM crops.

Science.gov (United States)

Raybould, Alan; Burns, Andrea; Hamer, Mick

2014-01-01

Laboratory testing for possible adverse effects of insecticidal proteins on non-target organisms (NTOs) is an important part of many ecological risk assessments for regulatory decision-making about the cultivation of insect-resistant genetically modified (IRGM) crops. To increase confidence in the risk assessments, regulatory guidelines for effects testing specify that representative surrogate species for NTOs are exposed to concentrations of insecticidal proteins that are in excess of worst-case predicted exposures in the field. High concentrations in effects tests are achieved by using protein test substances produced in microbes, such as Escherichia coli. In a study that exposed Daphnia magna to a single high concentration of a microbial test substance containing Vip3Aa20, the insecticidal protein in MIR162 maize, small reductions in growth were observed. These effects were surprising as many other studies strongly suggest that the activity of Vip3Aa20 is limited to Lepidoptera. A plausible explanation for the effect on growth is that high concentrations of test substance have a non-toxic effect on Daphnia, perhaps by reducing its feeding rate. A follow-up study tested that hypothesis by exposing D. magna to several concentrations of Vip3Aa20, and a high concentration of a non-toxic protein, bovine serum albumin (BSA). Vip3Aa20 and BSA had sporadic effects on the reproduction and growth of D. magna. The pattern of the effects suggests that they result from non-toxic effects of high concentrations of protein, and not from toxicity. The implications of these results for regulatory NTO effects testing and ERA of IRGM crops are discussed.

A novel processing system of sterol regulatory element-binding protein-1c regulated by polyunsaturated fatty acid.

Science.gov (United States)

Nakakuki, Masanori; Kawano, Hiroyuki; Notsu, Tatsuto; Imada, Kazunori; Mizuguchi, Kiyoshi; Shimano, Hitoshi

2014-05-01

The proteolytic cascade is the key step in transactivation of sterol regulatory element-binding proteins (SREBPs), a transcriptional factor of lipid synthesis. Proteolysis of SREBP-2 is strictly regulated by sterols, but that of SREBP-1c was not strongly sterol-regulated, but inhibited by polyunsaturated fatty acids (PUFAs). In this study, the proteolytic processing of SREBP-1 and -2 was examined by transfection studies of cDNA-encoding mutants in which all the known cleavage sites were disrupted. In cultured cells, sterol-regulated SREBP-2 processing was completely eliminated by mutation of cleavage sites. In contrast, the corresponding SREBP-1c mutants as well as wild type exhibited large amounts of cleaved products in the nuclear extracts from culture cells and murine liver in vivo. The nuclear form of the mutant SREBP-1c was induced by delipidated condition and suppressed by eicosapentaenoic acid, an n-3 PUFA, but not by sterols. This novel processing mechanism was affected by neither SREBP cleavage-activating protein (SCAP) nor insulin-induced gene (Insig)-1, unlike SREBP-2, but abolished by a serine protease inhibitor. Through analysis of deletion mutant, a site-2 protease recognition sequence (DRSR) was identified to be involved in this novel processing. These findings suggest that SREBP-1c cleavage could be subjected to a novel PUFA-regulated cleavage system in addition to the sterol-regulatory SCAP/Insig system.

Protein-protein interactions in the regulation of WRKY transcription factors.

Science.gov (United States)

Chi, Yingjun; Yang, Yan; Zhou, Yuan; Zhou, Jie; Fan, Baofang; Yu, Jing-Quan; Chen, Zhixiang

2013-03-01

It has been almost 20 years since the first report of a WRKY transcription factor, SPF1, from sweet potato. Great progress has been made since then in establishing the diverse biological roles of WRKY transcription factors in plant growth, development, and responses to biotic and abiotic stress. Despite the functional diversity, almost all analyzed WRKY proteins recognize the TTGACC/T W-box sequences and, therefore, mechanisms other than mere recognition of the core W-box promoter elements are necessary to achieve the regulatory specificity of WRKY transcription factors. Research over the past several years has revealed that WRKY transcription factors physically interact with a wide range of proteins with roles in signaling, transcription, and chromatin remodeling. Studies of WRKY-interacting proteins have provided important insights into the regulation and mode of action of members of the important family of transcription factors. It has also emerged that the slightly varied WRKY domains and other protein motifs conserved within each of the seven WRKY subfamilies participate in protein-protein interactions and mediate complex functional interactions between WRKY proteins and between WRKY and other regulatory proteins in the modulation of important biological processes. In this review, we summarize studies of protein-protein interactions for WRKY transcription factors and discuss how the interacting partners contribute, at different levels, to the establishment of the complex regulatory and functional network of WRKY transcription factors.

The alternative complement pathway control protein H binds to immune complexes and serves their detection

International Nuclear Information System (INIS)

Nydegger, U.E.; Corvetta, A.; Spaeth, P.J.; Spycher, M.

1983-01-01

During solubilization of immune complexes C3b becomes fixed to the immunoglobulin part and serves as a receptor for the alternative complement pathway control protein H. The H-C3b immune complex interaction can be made detectable using 4% polyethyleneglycol to separate free from bound 125 I-H. Tetanus toxoid (Te)/anti-Te complexes kept soluble with fresh serum and containing 125 IU of specific antibody bound 18% of 125 I-H; when fresh serum was chelated with 10 mM EDTA, 125 I-H binding was only 5%. On sucrose density gradients, the H-binding material sedimented in the range of 12 to 30 S. In 36 serum samples from rheumatoid arthritis (RA) patients and in 12 serum samples from patients with systemic lupus erythematosus (SLE), 125 I-H binding was significantly elevated to 9.5 +/- 4.7% (mean +/- 1 SD) and 13.3 +/- 5.6%, respectively, while 125 I-H binding by 36 normal human sera was 4 +/- 2%. RA samples (17/36, 47%) and SLE samples (9/12, 75%) had H-binding values increased by more than 2 SD above the normal mean. The serum samples were also assessed for conglutinin- and C1q-binding activities; a significant correlation between H and C1q binding was observed (P less than 0.001); there was no correlation between H and conglutinin binding. Although binding to immune complexes through its interaction with C3b, H clearly detects a population of complexes other than conglutinin, thus expanding the possibilities of further characterizing pathological complexes

Uncovering packaging features of co-regulated modules based on human protein interaction and transcriptional regulatory networks

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He Weiming

2010-07-01

Full Text Available Abstract Background Network co-regulated modules are believed to have the functionality of packaging multiple biological entities, and can thus be assumed to coordinate many biological functions in their network neighbouring regions. Results Here, we weighted edges of a human protein interaction network and a transcriptional regulatory network to construct an integrated network, and introduce a probabilistic model and a bipartite graph framework to exploit human co-regulated modules and uncover their specific features in packaging different biological entities (genes, protein complexes or metabolic pathways. Finally, we identified 96 human co-regulated modules based on this method, and evaluate its effectiveness by comparing it with four other methods. Conclusions Dysfunctions in co-regulated interactions often occur in the development of cancer. Therefore, we focussed on an example co-regulated module and found that it could integrate a number of cancer-related genes. This was extended to causal dysfunctions of some complexes maintained by several physically interacting proteins, thus coordinating several metabolic pathways that directly underlie cancer.

α -Actinin TvACTN3 of Trichomonas vaginalis is an RNA-binding protein that could participate in its posttranscriptional iron regulatory mechanism.

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Calla-Choque, Jaeson Santos; Figueroa-Angulo, Elisa Elvira; Ávila-González, Leticia; Arroyo, Rossana

2014-01-01

Trichomonas vaginalis is a sexually transmitted flagellated protist parasite responsible for trichomoniasis. This parasite is dependent on high levels of iron, favoring its growth and multiplication. Iron also differentially regulates some trichomonad virulence properties by unknown mechanisms. However, there is evidence to support the existence of gene regulatory mechanisms at the transcriptional and posttranscriptional levels that are mediated by iron concentration in T. vaginalis. Thus, the goal of this study was to identify an RNA-binding protein in T. vaginalis that interacts with the tvcp4 RNA stem-loop structure, which may participate in a posttranscriptional iron regulatory mechanism mediated by RNA-protein interactions. We performed RNA electrophoretic mobility shift assay (REMSA) and supershift, UV cross-linking, Northwestern blot, and western blot (WB) assays using cytoplasmic protein extracts from T. vaginalis with the tvcp4 RNA hairpin structure as a probe. We identified a 135-kDa protein isolated by the UV cross-linking assays as α-actinin 3 (TvACTN3) by MALDI-TOF-MS that was confirmed by LS-MS/MS and de novo sequencing. TvACTN3 is a cytoplasmic protein that specifically binds to hairpin RNA structures from trichomonads and humans when the parasites are grown under iron-depleted conditions. Thus, TvACTN3 could participate in the regulation of gene expression by iron in T. vaginalis through a parallel posttranscriptional mechanism similar to that of the IRE/IRP system.

Sterol regulatory element binding protein 2 overexpression is associated with reduced adipogenesis and ectopic fat accumulation in transgenic spontaneously hypertensive rats

Czech Academy of Sciences Publication Activity Database

Landa, Vladimír; Zídek, Václav; Mlejnek, Petr; Šimáková, Miroslava; Šilhavý, Jan; Trnovská, J.; Kazdová, L.; Pravenec, Michal

2014-01-01

Roč. 63, č. 5 (2014), s. 587-590 ISSN 0862-8408 R&D Projects: GA MŠk(CZ) LH12061 Institutional support: RVO:67985823 Keywords : sterol regulatory element binding protein 2 * transgenic * spontaneously hypertensive rat * lipid metabolism Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.293, year: 2014

Evolution of Cis-Regulatory Elements and Regulatory Networks in Duplicated Genes of Arabidopsis.

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Arsovski, Andrej A; Pradinuk, Julian; Guo, Xu Qiu; Wang, Sishuo; Adams, Keith L

2015-12-01

Plant genomes contain large numbers of duplicated genes that contribute to the evolution of new functions. Following duplication, genes can exhibit divergence in their coding sequence and their expression patterns. Changes in the cis-regulatory element landscape can result in changes in gene expression patterns. High-throughput methods developed recently can identify potential cis-regulatory elements on a genome-wide scale. Here, we use a recent comprehensive data set of DNase I sequencing-identified cis-regulatory binding sites (footprints) at single-base-pair resolution to compare binding sites and network connectivity in duplicated gene pairs in Arabidopsis (Arabidopsis thaliana). We found that duplicated gene pairs vary greatly in their cis-regulatory element architecture, resulting in changes in regulatory network connectivity. Whole-genome duplicates (WGDs) have approximately twice as many footprints in their promoters left by potential regulatory proteins than do tandem duplicates (TDs). The WGDs have a greater average number of footprint differences between paralogs than TDs. The footprints, in turn, result in more regulatory network connections between WGDs and other genes, forming denser, more complex regulatory networks than shown by TDs. When comparing regulatory connections between duplicates, WGDs had more pairs in which the two genes are either partially or fully diverged in their network connections, but fewer genes with no network connections than the TDs. There is evidence of younger TDs and WGDs having fewer unique connections compared with older duplicates. This study provides insights into cis-regulatory element evolution and network divergence in duplicated genes. © 2015 American Society of Plant Biologists. All Rights Reserved.

Peroxisome biogenesis disorders: identification of a new complementation group distinct from peroxisome-deficient CHO mutants and not complemented by human PEX 13

NARCIS (Netherlands)

Shimozawa, N.; Suzuki, Y.; Zhang, Z.; Imamura, A.; Tsukamoto, T.; Osumi, T.; Tateishi, K.; Okumoto, K.; Fujiki, Y.; Orii, T.; Barth, P. G.; Wanders, R. J.; Kondo, N.

1998-01-01

Ten complementation groups of generalized peroxisome biogenesis disorders (PBD), (excluding rhizomelic chondrodysplasia punctata) have been identified using complementation analysis. Four of the genes involved have been identified using two different methods of (1) genetic functional complementation

Force Dynamics of Verb Complementation

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Jacek Woźny

2015-12-01

Full Text Available Force Dynamics of Verb Complementation The concepts of motion and force are both extensively discussed in cognitive linguistics literature. But they are discussed separately. The first usually in the context of ‘motion situations’ (Talmy, Slobin, Zlatev, the other as part of the Force Dynamics framework, which was developed by Talmy. The aim of this paper is twofold: first, to argue that the concepts of force and motion should not be isolated but considered as two inseparable parts of force-motion events. The second goal is to prove that the modified Force Dynamics (force-motion framework can be used for precise characterization of the verb complementation patterns. To this end, a random sample of 50 sentences containing the verb ‘went’ is analyzed, demonstrating the differences between the categories of intensive and intransitive complementation with respect to the linguistically coded parameters of force and motion.

Storage of the complement components C4, C3, and C 3-activator in the human liver as PAS-negative globular hyaline bodies.

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Storch, W; Riedel, H; Trautmann, B; Justus, J; Hiemann, D

1982-01-01

Liver biopsies of a 58-year-old clinically healthy patient with a hepatomegaly and intracisternal PAS-negative globular hyaline bodies were immunofluorescent-optically examined for the content of the complement components C 1 q, C 4, C 9, C 1-inactivator, C 3-activator. Further examinations were performed for fibrinogen, IgG, IgA, IgM, IgD, IgE, L-chain (type chi and lambda), alpha 1-antitrypsin, alpha 1-fetoprotein, alpha 1- and alpha 2-glycoprotein, cholinesterase, ceruloplasmin, myoglobin, hemopexin, HBsAg and HBsAg. Th inclusion bodies reacted with antisera against the complement components C 4, C 3 and C 3-activator, as also identified by double immunofluorescence. Probably this is a disturbance of the protein metabolism of the liver cell with abnormal complement storage in the presence of normal total complement and normal complement components in the serum.

The Emerging Role of Complement Lectin Pathway in Trypanosomatids: Molecular Bases in Activation, Genetic Deficiencies, Susceptibility to Infection, and Complement System-Based Therapeutics

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Ingrid Evans-Osses

2013-01-01

Full Text Available The innate immune system is evolutionary and ancient and is the pivotal line of the host defense system to protect against invading pathogens and abnormal self-derived components. Cellular and molecular components are involved in recognition and effector mechanisms for a successful innate immune response. The complement lectin pathway (CLP was discovered in 1990. These new components at the complement world are very efficient. Mannan-binding lectin (MBL and ficolin not only recognize many molecular patterns of pathogens rapidly to activate complement but also display several strategies to evade innate immunity. Many studies have shown a relation between the deficit of complement factors and susceptibility to infection. The recently discovered CLP was shown to be important in host defense against protozoan microbes. Although the recognition of pathogen-associated molecular patterns by MBL and Ficolins reveal efficient complement activations, an increase in deficiency of complement factors and diversity of parasite strategies of immune evasion demonstrate the unsuccessful effort to control the infection. In the present paper, we will discuss basic aspects of complement activation, the structure of the lectin pathway components, genetic deficiency of complement factors, and new therapeutic opportunities to target the complement system to control infection.

Biology of the Heat Shock Response and Protein Chaperones: Budding Yeast (Saccharomyces cerevisiae) as a Model System

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Verghese, Jacob; Abrams, Jennifer; Wang, Yanyu

2012-01-01

Summary: The eukaryotic heat shock response is an ancient and highly conserved transcriptional program that results in the immediate synthesis of a battery of cytoprotective genes in the presence of thermal and other environmental stresses. Many of these genes encode molecular chaperones, powerful protein remodelers with the capacity to shield, fold, or unfold substrates in a context-dependent manner. The budding yeast Saccharomyces cerevisiae continues to be an invaluable model for driving the discovery of regulatory features of this fundamental stress response. In addition, budding yeast has been an outstanding model system to elucidate the cell biology of protein chaperones and their organization into functional networks. In this review, we evaluate our understanding of the multifaceted response to heat shock. In addition, the chaperone complement of the cytosol is compared to those of mitochondria and the endoplasmic reticulum, organelles with their own unique protein homeostasis milieus. Finally, we examine recent advances in the understanding of the roles of protein chaperones and the heat shock response in pathogenic fungi, which is being accelerated by the wealth of information gained for budding yeast. PMID:22688810

Bovine proteins containing poly-glutamine repeats are often polymorphic and enriched for components of transcriptional regulatory complexes

LENUS (Irish Health Repository)

Whan, Vicki

2010-11-23

Abstract Background About forty human diseases are caused by repeat instability mutations. A distinct subset of these diseases is the result of extreme expansions of polymorphic trinucleotide repeats; typically CAG repeats encoding poly-glutamine (poly-Q) tracts in proteins. Polymorphic repeat length variation is also apparent in human poly-Q encoding genes from normal individuals. As these coding sequence repeats are subject to selection in mammals, it has been suggested that normal variations in some of these typically highly conserved genes are implicated in morphological differences between species and phenotypic variations within species. At present, poly-Q encoding genes in non-human mammalian species are poorly documented, as are their functions and propensities for polymorphic variation. Results The current investigation identified 178 bovine poly-Q encoding genes (Q ≥ 5) and within this group, 26 genes with orthologs in both human and mouse that did not contain poly-Q repeats. The bovine poly-Q encoding genes typically had ubiquitous expression patterns although there was bias towards expression in epithelia, brain and testes. They were also characterised by unusually large sizes. Analysis of gene ontology terms revealed that the encoded proteins were strongly enriched for functions associated with transcriptional regulation and many contributed to physical interaction networks in the nucleus where they presumably act cooperatively in transcriptional regulatory complexes. In addition, the coding sequence CAG repeats in some bovine genes impacted mRNA splicing thereby generating unusual transcriptional diversity, which in at least one instance was tissue-specific. The poly-Q encoding genes were prioritised using multiple criteria for their likelihood of being polymorphic and then the highest ranking group was experimentally tested for polymorphic variation within a cattle diversity panel. Extensive and meiotically stable variation was identified

Complement: Alive and Kicking Nanomedicines

DEFF Research Database (Denmark)

Andersen, Alina Joukainen; Hashemi, S.H.; Andresen, Thomas Lars

2009-01-01

Administration of liposome- and polymer-based clinical nanomedicines, as well as many other proposed multifunctional nanoparticles, often triggers hypersensitivity reactions without the involvement of IgE. These anaphylactic reactions are believed to be secondary to activation of the complement...... their procoagulant activity, and has the capacity to elicit non-lytic stimulatory responses from vascular endothelial cells. Here we discuss the molecular basis of complement activation by liposomes, including poly(ethylene glycol) coated vesicles, and other related lipid-based and phospholipid-poly(ethylene glycol...

Cis-regulatory somatic mutations and gene-expression alteration in B-cell lymphomas.

Science.gov (United States)

Mathelier, Anthony; Lefebvre, Calvin; Zhang, Allen W; Arenillas, David J; Ding, Jiarui; Wasserman, Wyeth W; Shah, Sohrab P

2015-04-23

With the rapid increase of whole-genome sequencing of human cancers, an important opportunity to analyze and characterize somatic mutations lying within cis-regulatory regions has emerged. A focus on protein-coding regions to identify nonsense or missense mutations disruptive to protein structure and/or function has led to important insights; however, the impact on gene expression of mutations lying within cis-regulatory regions remains under-explored. We analyzed somatic mutations from 84 matched tumor-normal whole genomes from B-cell lymphomas with accompanying gene expression measurements to elucidate the extent to which these cancers are disrupted by cis-regulatory mutations. We characterize mutations overlapping a high quality set of well-annotated transcription factor binding sites (TFBSs), covering a similar portion of the genome as protein-coding exons. Our results indicate that cis-regulatory mutations overlapping predicted TFBSs are enriched in promoter regions of genes involved in apoptosis or growth/proliferation. By integrating gene expression data with mutation data, our computational approach culminates with identification of cis-regulatory mutations most likely to participate in dysregulation of the gene expression program. The impact can be measured along with protein-coding mutations to highlight key mutations disrupting gene expression and pathways in cancer. Our study yields specific genes with disrupted expression triggered by genomic mutations in either the coding or the regulatory space. It implies that mutated regulatory components of the genome contribute substantially to cancer pathways. Our analyses demonstrate that identifying genomically altered cis-regulatory elements coupled with analysis of gene expression data will augment biological interpretation of mutational landscapes of cancers.

Complement elevation in spinal cord injury.

Science.gov (United States)

Rebhun, J; Botvin, J

1980-05-01

Laboratory studies revealed an elevated complement in 66% of patients with spinal cord injury. It is postulated that the activated complement may be a component of self-feeding immunological mechanism responsible for the failure of regeneration of a mature mammalian spinal cord. There was no evidence that such an injury had any effect on pre-existing atopy.

RNA-ID, a Powerful Tool for Identifying and Characterizing Regulatory Sequences.

Science.gov (United States)

Brule, C E; Dean, K M; Grayhack, E J

2016-01-01

The identification and analysis of sequences that regulate gene expression is critical because regulated gene expression underlies biology. RNA-ID is an efficient and sensitive method to discover and investigate regulatory sequences in the yeast Saccharomyces cerevisiae, using fluorescence-based assays to detect green fluorescent protein (GFP) relative to a red fluorescent protein (RFP) control in individual cells. Putative regulatory sequences can be inserted either in-frame or upstream of a superfolder GFP fusion protein whose expression, like that of RFP, is driven by the bidirectional GAL1,10 promoter. In this chapter, we describe the methodology to identify and study cis-regulatory sequences in the RNA-ID system, explaining features and variations of the RNA-ID reporter, as well as some applications of this system. We describe in detail the methods to analyze a single regulatory sequence, from construction of a single GFP variant to assay of variants by flow cytometry, as well as modifications required to screen libraries of different strains simultaneously. We also describe subsequent analyses of regulatory sequences. © 2016 Elsevier Inc. All rights reserved.

Complement lysis activity in autologous plasma is associated with lower viral loads during the acute phase of HIV-1 infection.

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Michael Huber

2006-11-01

Full Text Available BACKGROUND: To explore the possibility that antibody-mediated complement lysis contributes to viremia control in HIV-1 infection, we measured the activity of patient plasma in mediating complement lysis of autologous primary virus. METHODS AND FINDINGS: Sera from two groups of patients-25 with acute HIV-1 infection and 31 with chronic infection-were used in this study. We developed a novel real-time PCR-based assay strategy that allows reliable and sensitive quantification of virus lysis by complement. Plasma derived at the time of virus isolation induced complement lysis of the autologous virus isolate in the majority of patients. Overall lysis activity against the autologous virus and the heterologous primary virus strain JR-FL was higher at chronic disease stages than during the acute phase. Most strikingly, we found that plasma virus load levels during the acute but not the chronic infection phase correlated inversely with the autologous complement lysis activity. Antibody reactivity to the envelope (Env proteins gp120 and gp41 were positively correlated with the lysis activity against JR-FL, indicating that anti-Env responses mediated complement lysis. Neutralization and complement lysis activity against autologous viruses were not associated, suggesting that complement lysis is predominantly caused by non-neutralizing antibodies. CONCLUSIONS: Collectively our data provide evidence that antibody-mediated complement virion lysis develops rapidly and is effective early in the course of infection; thus it should be considered a parameter that, in concert with other immune functions, steers viremia control in vivo.

Domain structure of human complement C4b extends with increasing NaCl concentration: implications for its regulatory mechanism.

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Fung, Ka Wai; Wright, David W; Gor, Jayesh; Swann, Marcus J; Perkins, Stephen J

2016-12-01

During the activation of complement C4 to C4b, the exposure of its thioester domain (TED) is crucial for the attachment of C4b to activator surfaces. In the C4b crystal structure, TED forms an Arg 104 -Glu 1032 salt bridge to tether its neighbouring macroglobulin (MG1) domain. Here, we examined the C4b domain structure to test whether this salt bridge affects its conformation. Dual polarisation interferometry of C4b immobilised at a sensor surface showed that the maximum thickness of C4b increased by 0.46 nm with an increase in NaCl concentration from 50 to 175 mM NaCl. Analytical ultracentrifugation showed that the sedimentation coefficient s 20,w of monomeric C4b of 8.41 S in 50 mM NaCl buffer decreased to 7.98 S in 137 mM NaCl buffer, indicating that C4b became more extended. Small angle X-ray scattering reported similar R G values of 4.89-4.90 nm for C4b in 137-250 mM NaCl. Atomistic scattering modelling of the C4b conformation showed that TED and the MG1 domain were separated by 4.7 nm in 137-250 mM NaCl and this is greater than that of 4.0 nm in the C4b crystal structure. Our data reveal that in low NaCl concentrations, both at surfaces and in solution, C4b forms compact TED-MG1 structures. In solution, physiologically relevant NaCl concentrations lead to the separation of the TED and MG1 domain, making C4b less capable of binding to its complement regulators. These conformational changes are similar to those seen previously for complement C3b, confirming the importance of this salt bridge for regulating both C4b and C3b. © 2016 The Author(s).

Relative Contribution of Cellular Complement Inhibitors CD59, CD46, and CD55 to Parainfluenza Virus 5 Inhibition of Complement-Mediated Neutralization

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Yujia Li

2018-04-01

Full Text Available The complement system is a part of the innate immune system that viruses need to face during infections. Many viruses incorporate cellular regulators of complement activation (RCA to block complement pathways and our prior work has shown that Parainfluenza virus 5 (PIV5 incorporates CD55 and CD46 to delay complement-mediated neutralization. In this paper, we tested the role of a third individual RCA inhibitor CD59 in PIV5 interactions with complement pathways. Using a cell line engineered to express CD59, we show that small levels of functional CD59 are associated with progeny PIV5, which is capable of blocking assembly of the C5b-C9 membrane attack complex (MAC. PIV5 containing CD59 (PIV5-CD59 showed increased resistance to complement-mediated neutralization in vitro comparing to PIV5 lacking regulators. Infection of A549 cells with PIV5 and RSV upregulated CD59 expression. TGF-beta treatment of PIV5-infected cells also increased cell surface CD59 expression and progeny virions were more resistant to complement-mediated neutralization. A comparison of individual viruses containing only CD55, CD46, or CD59 showed a potency of inhibiting complement-mediated neutralization, which followed a pattern of CD55 > CD46 > CD59.

Tumorigenic Properties of Iron Regulatory Protein 2 (IRP2) Mediated by Its Specific 73-Amino Acids Insert

OpenAIRE

Maffettone, Carmen; Chen, Guohua; Drozdov, Ignat; Ouzounis, Christos; Pantopoulos, Kostas

2010-01-01

Iron regulatory proteins, IRP1 and IRP2, bind to mRNAs harboring iron responsive elements and control their expression. IRPs may also perform additional functions. Thus, IRP1 exhibited apparent tumor suppressor properties in a tumor xenograft model. Here we examined the effects of IRP2 in a similar setting. Human H1299 lung cancer cells or clones engineered for tetracycline-inducible expression of wild type IRP2, or the deletion mutant IRP2(Delta73) (lacking a specific insert of 73 amino acid...

In situ detection of a heat-shock regulatory element binding protein using a soluble short synthetic enhancer sequence

Energy Technology Data Exchange (ETDEWEB)

Harel-Bellan, A; Brini, A T; Farrar, W L [National Cancer Institute, Frederick, MD (USA); Ferris, D K [Program Resources, Inc., Frederick, MD (USA); Robin, P [Institut Gustave Roussy, Villejuif (France)

1989-06-12

In various studies, enhancer binding proteins have been successfully absorbed out by competing sequences inserted into plasmids, resulting in the inhibition of the plasmid expression. Theoretically, such a result could be achieved using synthetic enhancer sequences not inserted into plasmids. In this study, a double stranded DNA sequence corresponding to the human heat shock regulatory element was chemically synthesized. By in vitro retardation assays, the synthetic sequence was shown to bind specifically a protein in extracts from the human T cell line Jurkat. When the synthetic enhancer was electroporated into Jurkat cells, not only the enhancer was shown to remain undegraded into the cells for up to 2 days, but also its was shown to bind intracellularly a protein. The binding was specific and was modulated upon heat shock. Furthermore, the binding protein was shown to be of the expected molecular weight by UV crosslinking. However, when the synthetic enhancer element was co-electroporated with an HSP 70-CAT reporter construct, the expression of the reporter plasmid was consistently enhanced in the presence of the exogenous synthetic enhancer.

Trichinella spiralis Calreticulin Binds Human Complement C1q As an Immune Evasion Strategy.

Science.gov (United States)

Zhao, Limei; Shao, Shuai; Chen, Yi; Sun, Ximeng; Sun, Ran; Huang, Jingjing; Zhan, Bin; Zhu, Xinping

2017-01-01

As a multicellular parasitic nematode, Trichinella spiralis regulates host immune responses by producing a variety of immunomodulatory molecules to escape from host immune attack, but the mechanisms underlying the immune evasion are not well understood. Here, we identified that T. spiralis calreticulin ( Ts -CRT), a Ca 2+ -binding protein, facilitated T. spiralis immune evasion by interacting with the first component of human classical complement pathway, C1q. In the present study, Ts -CRT was found to be expressed on the surface of different developmental stages of T. spiralis as well as in the secreted products of adult and muscle larval worms. Functional analysis identified that Ts -CRT was able to bind to human C1q, resulting in the inhibition of C1q-initiated complement classical activation pathway reflected by reduced C4/C3 generation and C1q-dependent lysis of antibody-sensitized sheep erythrocytes. Moreover, recombinant Ts -CRT (r Ts -CRT) binding to C1q suppressed C1q-induced THP-1-derived macrophages chemotaxis and reduced monocyte-macrophages release of reactive oxygen intermediates (ROIs). Blocking Ts -CRT on the surface of newborn larvae (NBL) of T. spiralis with anti- Ts -CRT antibody increased the C1q-mediated adherence of monocyte-macrophages to larvae and impaired larval infectivity. All of these results suggest that T. spiralis -expressed Ts -CRT plays crucial roles in T. spiralis immune evasion and survival in host mostly by directly binding to host complement C1q, which not only reduces C1q-mediated activation of classical complement pathway but also inhibits the C1q-induced non-complement activation of macrophages.

Kupffer cell complement receptor clearance function and host defense.

Science.gov (United States)

Loegering, D J

1986-01-01

Kupffer cells are well known to be important for normal host defense function. The development of methods to evaluate the in vivo function of specific receptors on Kupffer cells has made it possible to assess the role of these receptors in host defense. The rationale for studying complement receptors is based on the proposed important role of these receptors in host defense and on the observation that the hereditary deficiency of a complement receptor is associated with recurrent severe bacterial infections. The studies reviewed here demonstrate that forms of injury that are associated with depressed host defense including thermal injury, hemorrhagic shock, trauma, and surgery also cause a decrease in complement receptor clearance function. This decrease in Kupffer cell receptor clearance function was shown not to be the result of depressed hepatic blood flow or depletion of complement components. Complement receptor function was also depressed following the phagocytosis of particulates that are known to depress Kupffer cell host defense function. Endotoxemia and bacteremia also were associated with a depression of complement receptor function. Complement receptor function was experimentally depressed in uninjured animals by the phagocytosis of IgG-coated erythrocytes. There was a close association between the depression of complement receptor clearance function and increased susceptibility to the lethal effects of endotoxin and bacterial infection. These studies support the hypotheses that complement receptors on Kupffer cells are important for normal host defense and that depression of the function of these receptors impairs host defense.

Expression of Genes Involved in Bacteriocin Production and Self-Resistance in Lactobacillus brevis 174A Is Mediated by Two Regulatory Proteins.

Science.gov (United States)

Noda, Masafumi; Miyauchi, Rumi; Danshiitsoodol, Narandalai; Matoba, Yasuyuki; Kumagai, Takanori; Sugiyama, Masanori

2018-04-01

We have previously shown that the lactic acid bacterium Lactobacillus brevis 174A, isolated from Citrus iyo fruit, produces a bacteriocin designated brevicin 174A, which is comprised of two antibacterial polypeptides (designated brevicins 174A-β and 174A-γ). We have also found a gene cluster, composed of eight open reading frames (ORFs), that contains genes for the biosynthesis of brevicin 174A, self-resistance to its own bacteriocin, and two transcriptional regulatory proteins. Some lactic acid bacterial strains have a system to start the production of bacteriocin at an adequate stage of growth. Generally, the system consists of a membrane-bound histidine protein kinase (HPK) that senses a specific environmental stimulus and a corresponding response regulator (RR) that mediates the cellular response. We have previously shown that although the HPK- and RR-encoding genes are not found on the brevicin 174A biosynthetic gene cluster in the 174A strain, two putative regulatory genes, designated breD and breG , are in the gene cluster. In the present study, we demonstrate that the expression of brevicin 174A production and self-resistance is positively controlled by two transcriptional regulatory proteins, designated BreD and BreG. BreD is expressed together with BreE as the self-resistance determinant of L. brevis 174A. DNase I footprinting analysis and a promoter assay demonstrated that BreD binds to the breED promoter as a positive autoregulator. The present study also demonstrates that BreG, carrying a transmembrane domain, binds to the common promoter of breB and breC , encoding brevicins 174A-β and 174A-γ, respectively, for positive regulation. IMPORTANCE The problem of the appearance of bacteria that are resistant to practical antibiotics and the increasing demand for safe foods have increased interest in replacing conventional antibiotics with bacteriocin produced by the lactic acid bacteria. This antibacterial substance can inhibit the growth of pathogenic

Complement activation on the surface of cell-derived microparticles during cardiac surgery with cardiopulmonary bypass - is retransfusion of pericardial blood harmful?

Science.gov (United States)

Biró, E; van den Goor, J M; de Mol, B A; Schaap, M C; Ko, L-Y; Sturk, A; Hack, C E; Nieuwland, R

2011-01-01

To investigate whether cell-derived microparticles play a role in complement activation in pericardial blood of patients undergoing cardiac surgery with cardiopulmonary bypass (CPB) and whether microparticles in pericardial blood contribute to systemic complement activation upon retransfusion. Pericardial blood of 13 patients was retransfused in 9 and discarded in 4 cases. Microparticles were isolated from systemic blood collected before anesthesia (T1) and at the end of CPB (T2), and from pericardial blood. The microparticles were analyzed by flow cytometry for bound complement components C1q, C4 and C3, and bound complement activator molecules C-reactive protein (CRP), serum amyloid P-component (SAP), immunoglobulin (Ig)M and IgG. Fluid-phase complement activation products (C4b/c, C3b/c) and activator molecules were determined by ELISA. Compared with systemic T1 blood, pericardial blood contained increased C4b/c and C3b/c, and increased levels of microparticles with bound complement components. In systemic T1 samples, microparticle-bound CRP, whereas in pericardial blood, microparticle-bound SAP and IgM were associated with complement activation. At the end of CPB, increased C3b/c (but not C4b/c) was present in systemic T2 blood compared with T1, while concentrations of microparticles binding complement components and of those binding complement activator molecules were similar. Concentrations of fluid-phase complement activation products and microparticles were similar in patients whether or not retransfused with pericardial blood. In pericardial blood of patients undergoing cardiac surgery with CPB, microparticles contribute to activation of the complement system via bound SAP and IgM. Retransfusion of pericardial blood, however, does not contribute to systemic complement activation.

Activation and binding of opsonic fragments of C3 on encapsulated Cryptococcus neoformans by using an alternative complement pathway reconstituted from six isolated proteins.

Science.gov (United States)

Kozel, T R; Wilson, M A; Pfrommer, G S; Schlageter, A M

1989-07-01

Encapsulated Cryptococcus neoformans yeast cells are potent activators of the complement system. We examined the interaction of the yeast cells with an alternative complement pathway reconstituted from isolated factor D, factor B, factor H, factor I, C3, and properdin. Incubation of encapsulated cryptococci with the reconstituted pathway led to activation and binding of C3 fragments to the yeast cells that was quantitatively and qualitatively identical to that observed with normal human serum. Incubation with either normal serum or a mixture of isolated proteins led to binding of 4 x 10(7) to 5 x 10(7) C3 molecules to the yeast cells. The kinetics for activation and binding of C3 were identical, with maximum binding observed after a 20-min incubation. Immunoglobulin G was not needed for optimal activation kinetics. C3 fragments eluted from the yeast cells by treatment with hydroxylamine and subsequent analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated the presence primarily of iC3b on yeast cells incubated with either normal serum or the reconstituted pathway. Ultrastructural examination of the opsonized yeast cells showed that the cryptococcal capsule was the site for binding of C3 activated from normal serum or the reconstituted pathway, with a dense accumulation of C3 at the periphery of the capsule. Thus, incubation of encapsulated cryptococci in the reconstituted pathway led to deposition of opsonic complement fragments at a site that was appropriate for interaction with phagocyte receptors. Cryptococci opsonized with the reconstituted pathway showed a markedly enhanced interaction with cultured human monocytes compared with unopsonized yeast cells, indicating that the alternative pathway alone is opsonic for yeast cells. However, the results indicate that additional serum factors are needed for optimal opsonization of yeast cells because a 35% reduction in the number of cryptococci bound to macrophages was observed with

The multifaceted activity of the VirF regulatory protein in the Shigella lifestyle

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Maria Letizia Di Martino

2016-09-01

Full Text Available Shigella is a highly adapted human pathogen, mainly found in the developing world and causing a severe enteric syndrome. The highly sophisticated infectious strategy of Shigella banks on the capacity to invade the intestinal epithelial barrier and cause its inflammatory destruction. The cellular pathogenesis and clinical presentation of shigellosis are the sum of the complex action of a large number of bacterial virulence factors mainly located on a large virulence plasmid (pINV. The expression of pINV genes is controlled by multiple environmental stimuli through a regulatory cascade involving proteins and sRNAs encoded by both the pINV and the chromosome. The primary regulator of the virulence phenotype is VirF, a DNA-binding protein belonging to the AraC family of transcriptional regulators. The virF gene, located on the pINV, is expressed only within the host, mainly in response to the temperature transition occurring when the bacterium transits from the outer environment to the intestinal milieu. VirF then acts as anti-H-NS protein and directly activates the icsA and virB genes, triggering the full expression of the invasion program of Shigella. In this review we will focus on the structure of VirF, on its sophisticated regulation, and on its role as major player in the path leading from the non invasive to the invasive phenotype of Shigella. We will address also the involvement of VirF in mechanisms aimed at withstanding adverse conditions inside the host, indicating that this protein is emerging as a global regulator whose action is not limited to virulence systems. Finally, we will discuss recent observations conferring VirF the potential of a novel antibacterial target for shigellosis.

Activation of an immune-regulatory macrophage response and inhibition of lung inflammation in a mouse model of COPD using heat-shock protein alpha B-crystallin-loaded PLGA microparticles

NARCIS (Netherlands)

van Noort, J.M.; Bsibsi, M.; Nacken, P.J.; Gerritsen, W.H.; Amor, S.; Holtman, I.R.; Boddeke, E.; van Ark, I.; Leusink-Muis, T.; Folkerts, G.; Hennink, W.E.; Amidi, M.

2013-01-01

As an extracellular protein, the small heat-shock protein alpha B-crystallin (HSPB5) has anti-inflammatory effects in several mouse models of inflammation. Here, we show that these effects are associated with the ability of HSPB5 to activate an immune-regulatory response in macrophages via

Activation of an immune-regulatory macrophage response and inhibition of lung inflammation in a mouse model of COPD using heat-shock protein alpha B-crystallin-loaded PLGA microparticles

NARCIS (Netherlands)

van Noort, Johannes M.; Bsibsi, Malika; Nacken, Peter J.; Gerritsen, Wouter H.; Amor, Sandra; Holtman, Inge R.; Boddeke, Erik; van Ark, Ingrid; Leusink-Muis, Thea; Folkerts, Gert; Hennink, Wim E.; Amidi, Maryam

As an extracellular protein, the small heat-shock protein alpha B-crystallin (HSPB5) has anti-inflammatory effects in several mouse models of inflammation. Here, we show that these effects are associated with the ability of HSPB5 to activate an immune-regulatory response in macrophages via

Complement pathways and meningococcal disease : diagnostic aspects

DEFF Research Database (Denmark)

Sjöholm, A G; Truedsson, L; Jensenius, Jens Christian

2001-01-01

Complement is an immunological effector system that bridges innate and acquired immunity in several ways. There is a striking association between susceptibility to meningococcal disease and various forms of complement deficiency (1,2). In defense against bacterial infection, the most important fu...

The FasX Small Regulatory RNA Negatively Regulates the Expression of Two Fibronectin-Binding Proteins in Group A Streptococcus.

Science.gov (United States)

Danger, Jessica L; Makthal, Nishanth; Kumaraswami, Muthiah; Sumby, Paul

2015-12-01

The group A Streptococcus (GAS; Streptococcus pyogenes) causes more than 700 million human infections each year. The success of this pathogen can be traced in part to the extensive arsenal of virulence factors that are available for expression in temporally and spatially specific manners. To modify the expression of these virulence factors, GAS use both protein- and RNA-based regulators, with the best-characterized RNA-based regulator being the small regulatory RNA (sRNA) FasX. FasX is a 205-nucleotide sRNA that contributes to GAS virulence by enhancing the expression of the thrombolytic secreted virulence factor streptokinase and by repressing the expression of the collagen-binding cell surface pili. Here, we have expanded the FasX regulon, showing that this sRNA also negatively regulates the expression of the adhesion- and internalization-promoting, fibronectin-binding proteins PrtF1 and PrtF2. FasX posttranscriptionally regulates the expression of PrtF1/2 through a mechanism that involves base pairing to the prtF1 and prtF2 mRNAs within their 5' untranslated regions, overlapping the mRNA ribosome-binding sites. Thus, duplex formation between FasX and the prtF1 and prtF2 mRNAs blocks ribosome access, leading to an inhibition of mRNA translation. Given that FasX positively regulates the expression of the spreading factor streptokinase and negatively regulates the expression of the collagen-binding pili and of the fibronectin-binding PrtF1/2, our data are consistent with FasX functioning as a molecular switch that governs the transition of GAS between the colonization and dissemination stages of infection. More than half a million deaths each year are a consequence of infections caused by GAS. Insights into how this pathogen regulates the production of proteins during infection may facilitate the development of novel therapeutic or preventative regimens aimed at inhibiting this activity. Here, we have expanded insight into the regulatory activity of the GAS small

Altered expression of the cell cycle regulatory protein cyclin D1 in the rat dentate gyrus after adrenalectomy-induced granular cell lass

NARCIS (Netherlands)

Postigo, JA; Van der Werf, YD; Korf, J; Krugers, HJ

1998-01-01

The loss of dentate gyrus (DG) granular cells after removal of the rat adrenal glands (ADX) is mediated by a process that is apoptotic in nature. The present study was initiated to compare changes in the immunocytochemical distribution of the cell-cycle regulatory protein cyclin D1, which has been

Comprehensive approach to study complement C4 in systemic lupus erythematosus: Gene polymorphisms, protein levels and functional activity.

Science.gov (United States)

Tsang-A-Sjoe, M W P; Bultink, I E M; Korswagen, L A; van der Horst, A; Rensink, I; de Boer, M; Hamann, D; Voskuyl, A E; Wouters, D

2017-12-01

Genetic variation of the genes encoding complement component C4 is strongly associated with systemic lupus erythematosus (SLE), a chronic multi-organ auto-immune disease. This study examined C4 and its isotypes on a genetic, protein, and functional level in 140 SLE patients and 104 healthy controls. Gene copy number (GCN) variation, silencing CT-insertion, and the retroviral HERV-K(C4) insertion) were analyzed with multiplex ligation-dependent probe amplification. Increased susceptibility to SLE was found for low GCN (≪2) of C4A. Serositis was the only clinical manifestation associated with low C4A GCN. One additional novel silencing mutation in the C4A gene was found by Sanger sequencing. This mutation causes a premature stop codon in exon 11. Protein concentrations of C4 isoforms C4A and C4B were determined with ELISA and were significantly lower in SLE patients compared to healthy controls. To study C4 isotypes on a functional level, a new C4 assay was developed, which distinguishes C4A from C4B by its binding capacity to amino or hydroxyl groups, respectively. This assay showed high correlation with ELISA and detected crossing over of Rodgers and Chido antigens in 3.2% (8/244) of individuals. The binding capacity of available C4 to its substrates was unaffected in SLE. Our study provides, for the first time, a complete overview of C4 in SLE from genetic variation to binding capacity using a novel test. As this test detects crossing over of Rodgers and Chido antigens, it will allow for more accurate measurement of C4 in future studies. Copyright © 2017 Elsevier Ltd. All rights reserved.

Regulatory elements of the floral homeotic gene AGAMOUS identified by phylogenetic footprinting and shadowing.

Energy Technology Data Exchange (ETDEWEB)

Hong, R. L., Hamaguchi, L., Busch, M. A., and Weigel, D.