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Sample records for dnae2 complement dna

  1. Papillomavirus DNA Complementation in Vivo

    PubMed Central

    Hu, Jiafen; Cladel, Nancy M.; Budgeon, Lynn; Balogh, Karla K.; Christensen, Neil D.

    2009-01-01

    Recent phylogenic studies indicate that DNA recombination could have occurred in ancient papillomaviruses types. However, no experimental data is available to demonstrate this event because of the lack of human papillomavirus infection models. We have used the cottontail rabbit papillomavirus (CRPV)/rabbit model to study pathogenesis and immunogenicity of different mutant genomes in vivo. Although the domestic rabbit is not a natural host for CRPV infection, it is possible to initiate infection with naked CRPV DNA cloned into a plasmid and monitor papilloma outgrowth on these animals. Taking advantage of a large panel of mutants based on a CRPV strain (Hershey CRPV), we tested the hypothesis that two non-viable mutant genomes could induce papillomas by either recombination or complementation. We found that co-infection with a dysfunctional mutant with an E2 transactivation domain mutation and another mutant with an E7 ATG knock out generated papillomas in rabbits. DNA extracted from these papillomas contained genotypes from both parental genomes. Three additional pairs of dysfunctional mutants also showed similar results. Individual wild type genes were also shown to rescue the function of corresponding dysfunctional mutants. Therefore, we suggest that complementation occurred between these two non-viable mutant PV genomes in vivo. PMID:19379784

  2. Complement

    MedlinePlus

    ... the suspected disease are done first. C3 and C4 are the complement components measured most often. A ... normal levels of the complement proteins C3 and C4 . Complement activity varies throughout the body. For example, ...

  3. Detection of specific DNA sequences by fluorescence amplification: a color complementation assay.

    PubMed Central

    Chehab, F F; Kan, Y W

    1989-01-01

    We have developed a color complementation assay that allows rapid screening of specific genomic DNA sequences. It is based on the simultaneous amplification of two or more DNA segments with fluorescent oligonucleotide primers such that the generation of a color, or combination of colors, can be visualized and used for diagnosis. Color complementation assay obviates the need for gel electrophoresis and has been applied to the detection of a large and small gene deletion, a chromosomal translocation, an infectious agent, and a single-base substitution. DNA amplification with fluorescent oligonucleotide primers has also been used to multiplex and discriminate five different amplified DNA loci simultaneously. Each primer set is conjugated to a different dye, and the fluorescence of each dye respective to its amplified DNA locus is scored on a fluorometer. This method is valuable for DNA diagnostics of genetic, acquired, and infectious diseases, as well as in DNA forensics. It also lends itself to complete automation. Images PMID:2594760

  4. Detection and characterization of mammalian DNA polymerase beta mutants by functional complementation in Escherichia coli.

    PubMed Central

    Sweasy, J B; Loeb, L A

    1993-01-01

    We have designed and utilized a bacterial complementation system to identify and characterize mammalian DNA polymerase beta mutants. In this complementation system, wild-type rat DNA polymerase beta replaces both the replicative and repair functions of DNA polymerase I in the Escherichia coli recA718 polA12 double mutant; our 263 DNA polymerase beta mutants replace E. coli polymerase I less efficiently or not at all. Of the 10 mutants that have been shown to contain DNA sequence alterations, 2 exhibit a split phenotype with respect to complementation of the growth defect and methylmethanesulfonate sensitivity of the double mutant; one is a null mutant. The mutants possessing a split phenotype contain amino acid residue alterations within a putative nucleotide binding site of DNA polymerase beta. This approach for the isolation and evaluation of mutants of a mammalian DNA polymerase in E. coli may ultimately lead to a better understanding of the mechanism of action of this enzyme and to precisely defining its role in vertebrate cells. Images Fig. 2 PMID:8506308

  5. Complementation between polymerase- and exonuclease-deficient mitochondrial DNA polymerase mutants in genomically engineered flies

    PubMed Central

    Macao, Bertil; Grönke, Sebastian; Siibak, Triinu; Stewart, James B; Baggio, Francesca; Dols, Jacqueline; Partridge, Linda; Falkenberg, Maria; Wredenberg, Anna; Larsson, Nils-Göran

    2016-01-01

    Replication errors are the main cause of mtDNA mutations and a compelling approach to decrease mutation levels would therefore be to increase the fidelity of the catalytic subunit (POLγA) of the mtDNA polymerase. Here we genomically engineered the tamas locus, encoding fly POLγA, and introduced alleles expressing exonuclease- (exo-) and polymerase-deficient (pol-) POLγA versions. The exo- mutant leads to accumulation of point mutations and linear deletions of mtDNA, whereas pol- mutants cause mtDNA depletion. The mutant tamas alleles are developmentally lethal but can complement each other in trans resulting in viable flies with clonally expanded mtDNA mutations. Reconstitution of human mtDNA replication in vitro confirms that replication is a highly dynamic process where POLγA goes on and off the template to allow complementation during proofreading and elongation. The created fly models are valuable tools to study germ line transmission of mtDNA and the pathophysiology of POLγA mutation disease. PMID:26554610

  6. Mitochondrial fusion provides an 'initial metabolic complementation' controlled by mtDNA.

    PubMed

    Yang, Liang; Long, Qi; Liu, Jinglei; Tang, Haite; Li, Yuxing; Bao, Feixiang; Qin, Dajiang; Pei, Duanqing; Liu, Xingguo

    2015-07-01

    Heteroplasmic cells, harboring both mutant and normal mitochondrial DNAs (mtDNAs), must accumulate mutations to a threshold level before respiratory activity is affected. This phenomenon has led to the hypothesis of mtDNA complementation by inter-mitochondrial content mixing. The precise mechanisms of heteroplasmic complementation are unknown, but it depends both on the mtDNA nucleoid dynamics among mitochondria as well as the mitochondrial dynamics as influenced by mtDNA. We tracked nucleoids among the mitochondria in real time to show that they are shared after complete fusion but not 'kiss-and-run'. Employing a cell hybrid model, we further show that mtDNA-less mitochondria, which have little ATP production and extensive Opa1 proteolytic cleavage, exhibit weak fusion activity among themselves, yet remain competent in fusing with healthy mitochondria in a mitofusin- and OPA1-dependent manner, resulting in restoration of metabolic function. Depletion of mtDNA by overexpression of the matrix-targeted nuclease UL12.5 resulted in heterogeneous mitochondrial membrane potential (ΔΨm) at the organelle level in mitofusin-null cells but not in wild type. In this system, overexpression of mitofusins or application of the fusion-promoting drug M1 could partially rescue the metabolic damage caused by UL12.5. Interestingly, mtDNA transcription/translation is not required for normal mitochondria to restore metabolic function to mtDNA-less mitochondria by fusion. Thus, interplay between mtDNA and fusion capacity governs a novel 'initial metabolic complementation'. PMID:25708700

  7. Complementation of DNA repair in xeroderma pigmentosum group A cell extracts by a protein with affinity for damaged DNA.

    PubMed

    Robins, P; Jones, C J; Biggerstaff, M; Lindahl, T; Wood, R D

    1991-12-01

    Complementation group A of xeroderma pigmentosum (XP) represents one of the most prevalent and serious forms of this cancer-prone disorder. Because of a marked defect in DNA excision repair, cells from individuals with XP-A are hypersensitive to the toxic and mutagenic effects of ultraviolet light and many chemical agents. We report here the isolation of the XP-A DNA repair protein by complementation of cell extracts from a repair-defective human XP-A cell line. XP-A protein purified from calf thymus migrates on denaturing gel electrophoresis as a doublet of 40 and 42 kilodaltons. The XP-A protein binds preferentially to ultraviolet light-irradiated DNA, with a preference for damaged over nondamaged nucleotides of approximately 10(3). This strongly suggests that the XP-A protein plays a direct role in the recognition of and incision at lesions in DNA. We further show that this protein corresponds to the product encoded by a recently isolated gene that can restore excision repair to XP-A cells. Thus, excision repair of plasmid DNA by cell extracts sufficiently resembles genomic repair in cells to reveal accurately the repair defect in an inherited disease. The general approach described here can be extended to the identification and isolation of other human DNA repair proteins. PMID:1935910

  8. Complementation of DNA repair in xeroderma pigmentosum group A cell extracts by a protein with affinity for damaged DNA.

    PubMed Central

    Robins, P; Jones, C J; Biggerstaff, M; Lindahl, T; Wood, R D

    1991-01-01

    Complementation group A of xeroderma pigmentosum (XP) represents one of the most prevalent and serious forms of this cancer-prone disorder. Because of a marked defect in DNA excision repair, cells from individuals with XP-A are hypersensitive to the toxic and mutagenic effects of ultraviolet light and many chemical agents. We report here the isolation of the XP-A DNA repair protein by complementation of cell extracts from a repair-defective human XP-A cell line. XP-A protein purified from calf thymus migrates on denaturing gel electrophoresis as a doublet of 40 and 42 kilodaltons. The XP-A protein binds preferentially to ultraviolet light-irradiated DNA, with a preference for damaged over nondamaged nucleotides of approximately 10(3). This strongly suggests that the XP-A protein plays a direct role in the recognition of and incision at lesions in DNA. We further show that this protein corresponds to the product encoded by a recently isolated gene that can restore excision repair to XP-A cells. Thus, excision repair of plasmid DNA by cell extracts sufficiently resembles genomic repair in cells to reveal accurately the repair defect in an inherited disease. The general approach described here can be extended to the identification and isolation of other human DNA repair proteins. Images PMID:1935910

  9. Interallelic complementation provides functional evidence for cohesin–cohesin interactions on DNA

    PubMed Central

    Eng, Thomas; Guacci, Vincent; Koshland, Douglas

    2015-01-01

    The cohesin complex (Mcd1p, Smc1p, Smc3p, and Scc3p) has multiple roles in chromosome architecture, such as promoting sister chromatid cohesion, chromosome condensation, DNA repair, and transcriptional regulation. The prevailing embrace model for sister chromatid cohesion posits that a single cohesin complex entraps both sister chromatids. We report interallelic complementation between pairs of nonfunctional mcd1 alleles (mcd1-1 and mcd1-Q266) or smc3 alleles (smc3-42 and smc3-K113R). Cells bearing individual mcd1 or smc3 mutant alleles are inviable and defective for both sister chromatid cohesion and condensation. However, cells coexpressing two defective mcd1 or two defective smc3 alleles are viable and have cohesion and condensation. Because cohesin contains only a single copy of Smc3p or Mcd1p, these examples of interallelic complementation must result from interplay or communication between the two defective cohesin complexes, each harboring one of the mutant allele products. Neither mcd1-1p nor smc3-42p is bound to chromosomes when expressed individually at its restrictive temperature. However, their chromosome binding is restored when they are coexpressed with their chromosome-bound interallelic complementing partner. Our results support a mechanism by which multiple cohesin complexes interact on DNA to mediate cohesion and condensation. PMID:26378250

  10. A Human Orthologue of Archaeal DNA Repair Protein Hef is Defective in Fanconi Anemia Complementation Group M

    PubMed Central

    Meetei, Amom Ruhikanta; Medhurst, Annette L.; Ling, Chen; Xue, Yutong; Singh, Thiyam Ramsing; Bier, Patrick; Steltenpool, Jurgen; Stone, Stacie; Dokal, Inderjeet; Mathew, Christopher G.; Hoatlin, Maureen; Joenje, Hans; de Winter, Johan P.; Wang, Weidong

    2005-01-01

    Fanconi anemia (FA) is a genetic disease featuring genomic instability and cancer predisposition1. Nine FA genes have been identified, and their products participate in a DNA damage response network involving BRCA1 and BRCA22,3. We have previously purified a FA core complex containing the FANCL ubiquitin ligase and 6 other FA proteins4–6. Each protein in this complex is essential for monoubiquitination of FANCD2, a key reaction in the FA DNA damage response pathway2,7. Here we show that another component of this complex, FAAP250, is mutated in FA patients of a new complementation group (FA-M). FAAP250, renamed FANCM, has sequence similarity to known DNA repair proteins, including archaeal Hef, yeast Mph1 and human ERCC4/XPF. FANCM can dissociate DNA triplex, possibly due to its ability to translocate on duplex DNA. FANCM is essential for FANCD2 monoubiquitination and becomes hyperphosphorylated in response to DNA damage. Our data suggest an evolutionary link between FA proteins and DNA repair; FANCM may act as an engine that translocates the FA core complex along DNA. PMID:16116422

  11. Molecular cloning of a mouse DNA repair gene that complements the defect of group-A xeroderma pigmentosum

    SciTech Connect

    Tanaka, K.; Satokata, I.; Ogita, Z.; Uchida, T.; Okada, Y.

    1989-07-01

    For isolation of the gene responsible for xeroderma pigmentosum (XP) complementation group A, plasmid pSV2gpt and genomic DNA from a mouse embryo were cotransfected into XP2OSSV cells, a group-A XP cell line. Two primary UV-resistant XP transfectants were isolated from about 1.6 X 10(5) pSV2gpt-transformed XP colonies. pSV2gpt and genomic DNA from the primary transfectants were again cotransfected into XP2OSSV cells and a secondary UV-resistant XP transfectant was obtained by screening about 4.8 X 10(5) pSV2gpt-transformed XP colonies. The secondary transfectant retained fewer mouse repetitive sequences. A mouse gene that complements the defect of XP2OSSV cells was cloned into an EMBL3 vector from the genome of a secondary transfectant. Transfections of the cloned DNA also conferred UV resistance on another group-A XP cell line but not on XP cell lines of group C, D, F, or G. Northern blot analysis of poly(A)+ RNA with a subfragment of cloned mouse DNA repair gene as the probe revealed that an approximately 1.0 kilobase mRNA was transcribed in the donor mouse embryo and secondary transfectant, and approximately 1.0- and approximately 1.3-kilobase mRNAs were transcribed in normal human cells, but none of these mRNAs was detected in three strains of group-A XP cells. These results suggest that the cloned DNA repair gene is specific for group-A XP and may be the mouse homologue of the group-A XP human gene.

  12. Molecular cloning of a mouse DNA repair gene that complements the defect of group-A xeroderma pigmentosum.

    PubMed Central

    Tanaka, K; Satokata, I; Ogita, Z; Uchida, T; Okada, Y

    1989-01-01

    For isolation of the gene responsible for xeroderma pigmentosum (XP) complementation group A, plasmid pSV2gpt and genomic DNA from a mouse embryo were cotransfected into XP2OSSV cells, a group-A XP cell line. Two primary UV-resistant XP transfectants were isolated from about 1.6 X 10(5) pSV2gpt-transformed XP colonies. pSV2gpt and genomic DNA from the primary transfectants were again cotransfected into XP2OSSV cells and a secondary UV-resistant XP transfectant was obtained by screening about 4.8 X 10(5) pSV2gpt-transformed XP colonies. The secondary transfectant retained fewer mouse repetitive sequences. A mouse gene that complements the defect of XP2OSSV cells was cloned into an EMBL3 vector from the genome of a secondary transfectant. Transfections of the cloned DNA also conferred UV resistance on another group-A XP cell line but not on XP cell lines of group C, D, F, or G. Northern blot analysis of poly(A)+ RNA with a subfragment of cloned mouse DNA repair gene as the probe revealed that an approximately 1.0 kilobase mRNA was transcribed in the donor mouse embryo and secondary transfectant, and approximately 1.0- and approximately 1.3-kilobase mRNAs were transcribed in normal human cells, but none of these mRNAs was detected in three strains of group-A XP cells. These results suggest that the cloned DNA repair gene is specific for group-A XP and may be the mouse homologue of the group-A XP human gene. Images PMID:2748601

  13. cDNA sequence coding for the alpha'-chain of the third complement component in the African lungfish.

    PubMed

    Sato, A; Sültmann, H; Mayer, W E; Figueroa, F; Tichy, H; Klein, J

    1999-04-01

    cDNA clones coding for almost the entire C3 alpha-chain of the African lungfish (Protopterus aethiopicus), a representative of the Sarcopterygii (lobe-finned fishes), were sequenced and characterized. From the sequence it is deduced that the lungfish C3 molecule is probably a disulphide-bonded alpha:beta dimer similar to that of the C3 components of other jawed vertebrates. The deduced sequence contains conserved sites presumably recognized by proteolytic enzymes (e.g. factor I) involved in the activation and inactivation of the component. It also contains the conserved thioester region and the putative site for binding properdin. However, the site for the interaction with complement receptor 2 and factor H are poorly conserved. Either complement receptor 2 and factor H are not present in the lungfish or they bind to different residues at the same or a different site than mammalian complement receptor 2 and factor H. The C3 alpha-chain sequences faithfully reflect the phylogenetic relationships among vertebrate classes and can therefore be used to help to resolve the long-standing controversy concerning the origin of the tetrapods. PMID:10219761

  14. Lac Repressor Mediated DNA Looping: Monte Carlo Simulation of Constrained DNA Molecules Complemented with Current Experimental Results

    PubMed Central

    Biton, Yoav Y.; Kumar, Sandip; Dunlap, David; Swigon, David

    2014-01-01

    Tethered particle motion (TPM) experiments can be used to detect time-resolved loop formation in a single DNA molecule by measuring changes in the length of a DNA tether. Interpretation of such experiments is greatly aided by computer simulations of DNA looping which allow one to analyze the structure of the looped DNA and estimate DNA-protein binding constants specific for the loop formation process. We here present a new Monte Carlo scheme for accurate simulation of DNA configurations subject to geometric constraints and apply this method to Lac repressor mediated DNA looping, comparing the simulation results with new experimental data obtained by the TPM technique. Our simulations, taking into account the details of attachment of DNA ends and fluctuations of the looped subsegment of the DNA, reveal the origin of the double-peaked distribution of RMS values observed by TPM experiments by showing that the average RMS value for anti-parallel loop types is smaller than that of parallel loop types. The simulations also reveal that the looping probabilities for the anti-parallel loop types are significantly higher than those of the parallel loop types, even for loops of length 600 and 900 base pairs, and that the correct proportion between the heights of the peaks in the distribution can only be attained when loops with flexible Lac repressor conformation are taken into account. Comparison of the in silico and in vitro results yields estimates for the dissociation constants characterizing the binding affinity between O1 and Oid DNA operators and the dimeric arms of the Lac repressor. PMID:24800809

  15. Morphogenesis and Auxin Sensitivity of Transgenic Tobacco with Different Complements of Ri T-DNA 1

    PubMed Central

    Spanò, Laura; Mariotti, Domenico; Cardarelli, Maura; Branca, Camillo; Costantino, Paolo

    1988-01-01

    Leaf explants of hairy root tobacco (Nicotiana tabacum) regenerants characteristically differentiate roots from the wound margins on hormonefree medium. The same response can be elicited on normal tobacco by culturing the explants in the presence of auxin. We show here that the spontaneous rooting of transformed plants is neither due to the activity of right T-DNA-borne auxin genes nor to a substantially altered balance of endogenous hormones. Rather, an increased sensitivity to auxin is conferred to transformed cells by the left T-DNA (TL-DNA). Analysis of the morphogenetic behavior of transgenic tobacco plants obtained by transferring segments of TL-DNA cloned in a binary vector system allowed us to pinpoint TL-DNA genes responsible for this increased auxin sensitivity of hairy root tissues. Three genes (open reading frames 10, 11, 12) are responsible for the spontaneous rooting of leaf explants and confer to transgenic plants an exaggerated response to auxin. Images Fig. 1 Fig. 2 PMID:16666168

  16. Preferential association of a functional variant in complement receptor 2 with antibodies to double-stranded DNA

    PubMed Central

    Zhao, Jian; Giles, Brendan M; Taylor, Rhonda L; Yette, Gabriel A; Lough, Kara M; Ng, Han Leng; Abraham, Lawrence J; Wu, Hui; Kelly, Jennifer A; Glenn, Stuart B; Adler, Adam J; Williams, Adrienne H; Comeau, Mary E; Ziegler, Julie T; Marion, Miranda; Alarcón-Riquelme, Marta E; Alarcón, Graciela S; Anaya, Juan-Manuel; Bae, Sang-Cheol; Kim, Dam; Lee, Hye-Soon; Criswell, Lindsey A; Freedman, Barry I; Gilkeson, Gary S; Guthridge, Joel M; Jacob, Chaim O; James, Judith A; Kamen, Diane L; Merrill, Joan T; Sivils, Kathy Moser; Niewold, Timothy B; Petri, Michelle A; Ramsey-Goldman, Rosalind; Reveille, John D; Scofield, R Hal; Stevens, Anne M; Vilá, Luis M; Vyse, Timothy J; Kaufman, Kenneth M; Harley, John B; Langefeld, Carl D; Gaffney, Patrick M; Brown, Elizabeth E; Edberg, Jeffrey C; Kimberly, Robert P; Ulgiati, Daniela; Tsao, Betty P; Boackle, Susan A

    2016-01-01

    Objectives Systemic lupus erythematosus (SLE; OMIM 152700) is characterised by the production of antibodies to nuclear antigens. We previously identified variants in complement receptor 2 (CR2/CD21) that were associated with decreased risk of SLE. This study aimed to identify the causal variant for this association. Methods Genotyped and imputed genetic variants spanning CR2 were assessed for association with SLE in 15 750 case-control subjects from four ancestral groups. Allele-specific functional effects of associated variants were determined using quantitative real-time PCR, quantitative flow cytometry, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP)-PCR. Results The strongest association signal was detected at rs1876453 in intron 1 of CR2 (pmeta=4.2×10−4, OR 0.85), specifically when subjects were stratified based on the presence of dsDNA autoantibodies (case-control pmeta=7.6×10−7, OR 0.71; case-only pmeta=1.9×10−4, OR 0.75). Although allele-specific effects on B cell CR2 mRNA or protein levels were not identified, levels of complement receptor 1 (CR1/CD35) mRNA and protein were significantly higher on B cells of subjects harbouring the minor allele (p=0.0248 and p=0.0006, respectively). The minor allele altered the formation of several DNA protein complexes by EMSA, including one containing CCCTC-binding factor (CTCF), an effect that was confirmed by ChIP-PCR. Conclusions These data suggest that rs1876453 in CR2 has long-range effects on gene regulation that decrease susceptibility to lupus. Since the minor allele at rs1876453 is preferentially associated with reduced risk of the highly specific dsDNA autoantibodies that are present in preclinical, active and severe lupus, understanding its mechanisms will have important therapeutic implications. PMID:25180293

  17. DNA sequences of Alu elements indicate a recent replacement of the human autosomal genetic complement

    SciTech Connect

    Knight, A.; Deininger, P.L.; Batzer, M.A.

    1996-04-30

    DNA sequences of neutral nuclear autosomal loci, compared across diverse human populations, provide a previously untapped perspective into the mode and tempo of the emergence of modern humans and a critical comparison with published clonally inherited mitochondrial DNA and Y chromosome measurements of human diversity. We obtained over 55 kilobases of sequence from three autosomal loci encompassing Alu repeats for representatives of diverse human populations as well as orthologous sequences for other hominoid species at one of these loci. Nucleotide diversity was exceedingly low. Most individuals and populations were identical. Only a single nucleotide difference distinguished presumed ancestral alleles from descendants. These results differ from those expected if alleles from divergent archaic populations were maintained through multiregional continuity. The observed virtual lack of sequence polymorphism is the signature of a recent single origin for modern humans, with general replacement of archaic populations. 47 refs., 2 figs., 1 tab.

  18. DNA sequences of Alu elements indicate a recent replacement of the human autosomal genetic complement.

    PubMed Central

    Knight, A; Batzer, M A; Stoneking, M; Tiwari, H K; Scheer, W D; Herrera, R J; Deininger, P L

    1996-01-01

    DNA sequences of neutral nuclear autosomal loci, compared across diverse human populations, provide a previously untapped perspective into the mode and tempo of the emergence of modern humans and a critical comparison with published clonally inherited mitochondrial DNA and Y chromosome measurements of human diversity. We obtained over 55 kilobases of sequence from three autosomal loci encompassing Alu repeats for representatives of diverse human populations as well as orthologous sequences for other hominoid species at one of these loci. Nucleotide diversity was exceedingly low. Most individuals and populations were identical. Only a single nucleotide difference distinguished presumed ancestral alleles from descendants. These results differ from those expected if alleles from divergent archaic populations were maintained through multiregional continuity. The observed virtual lack of sequence polymorphism is the signature of a recent single origin for modern humans, with general replacement of archaic populations. PMID:8633071

  19. A partial cDNA clone of trypomastigote decay-accelerating factor (T-DAF), a developmentally regulated complement inhibitor of Trypanosoma cruzi, has genetic and functional similarities to the human complement inhibitor DAF.

    PubMed Central

    Tambourgi, D V; Kipnis, T L; da Silva, W D; Joiner, K A; Sher, A; Heath, S; Hall, B F; Ogden, G B

    1993-01-01

    Resistance to complement-mediated lysis in Trypanosoma cruzi is due to the expression of complement-regulatory factors by the virulent developmental forms of this protozoan parasite. An 87- to 93-kDa molecule, which we have termed T-DAF (trypomastigote decay-accelerating factor), is present on the surface of the parasite and inhibits complement activation in a manner functionally similar to the mammalian complement regulatory component, decay-accelerating factor. In this report, we characterized monospecific polyclonal and monoclonal antibodies which were obtained from mice and rabbits immunized with fast protein liquid chromatography-purified T-DAF. These polyclonal antibodies were shown to inhibit T-DAF activity and were capable of inducing lysis of the parasites. Both the polyclonal and monoclonal antibodies were used to screen a cDNA expression library prepared from T. cruzi trypomastigote mRNA. From this library, we obtained a partial lambda gt11 cDNA clone which showed genetic and functional similarity to the human C3 convertase inhibitor DAF (A. Nicholson-Weller, J. Burge, D. T. Fearon, P. F. Weller, and K. F. Austen, J. Immunol. 129:184-189, 1982). Images PMID:7689538

  20. In vivo and in vitro complementation study comparing the function of DnaK chaperone systems from halophilic lactic acid bacterium Tetragenococcus halophilus and Escherichia coli.

    PubMed

    Sugimoto, Shinya; Saruwatari, Kozue; Higashi, Chihana; Tsuruno, Keigo; Matsumoto, Shunsuke; Nakayama, Jiro; Sonomoto, Kenji

    2008-03-01

    In this study, we characterized the DnaK chaperone system from Tetragenococcus halophilus, a halophilic lactic acid bacterium. An in vivo complementation test showed that under heat stress conditions, T. halophilus DnaK did not rescue the growth of the Escherichia coli dnaK deletion mutant, whereas T. halophilus DnaJ and GrpE complemented the corresponding mutations of E. coli. Purified T. halophilus DnaK showed intrinsic weak ATPase activity and holding chaperone activity in vitro, but T. halophilus DnaK did not cooperate with the purified DnaJ and GrpE from either T. halophilus or E. coli in ATP hydrolysis or luciferase-refolding reactions under the conditions tested. E. coli DnaK, however, cross-reacted with those from both bacteria. This difference in the cooperation with DnaJ and GrpE appears to result in an inability of T. halophilus DnaK to replace the in vivo function of the DnaK chaperone of E. coli. PMID:18323638

  1. How DNA Barcodes Complement Taxonomy and Explore Species Diversity: The Case Study of a Poorly Understood Marine Fauna

    PubMed Central

    Chen, Jun; Li, Qi; Kong, Lingfeng; Yu, Hong

    2011-01-01

    Background The species boundaries of some venerids are difficult to define based solely on morphological features due to their indistinct intra- and interspecific phenotypic variability. An unprecedented biodiversity crisis caused by human activities has emerged. Thus, to access the biological diversity and further the conservation of this taxonomically muddling bivalve group, a fast and simple approach that can efficiently examine species boundaries and highlight areas of unrecognized diversity is urgently needed. DNA barcoding has proved its effectiveness in high-volume species identification and discovery. In the present study, Chinese fauna was chosen to examine whether this molecular biomarker is sensitive enough for species delimitation, and how it complements taxonomy and explores species diversity. Methodology/Principal Findings A total of 315 specimens from around 60 venerid species were included, qualifying the present study as the first major analysis of DNA barcoding for marine bivalves. Nearly all individuals identified to species level based on morphological traits possessed distinct barcode clusters, except for the specimens of one species pair. Among the 26 individuals that were not assigned binomial names a priori, twelve respectively nested within a species genealogy. The remaining individuals formed five monophyletic clusters that potentially represent species new to science or at least unreported in China. Five putative hidden species were also uncovered in traditional morphospecies. Conclusions/Significance The present study shows that DNA barcoding is effective in species delimitation and can aid taxonomists by indicating useful diagnostic morphological traits, informing needful revision, and flagging unseen species. Moreover, the BOLD system, which deposits barcodes, morphological, geographical and other data, has the potential as a convenient taxonomic platform. PMID:21698181

  2. Human ERCC5 cDNA-cosmid complementation for excision repair and bipartite amino acid domains conserved with RAD proteins of saccharomyces cerevisiae and schizosaccharomyces pombe

    SciTech Connect

    MacInnes, M.A.; Dickson, J.A.; Hernandez, R.R.; Lin, G.Y.; Park, M.S.; Schauer, S.; Reynolds, R.J.; Strniste, G.F. ); Learmonth, D. ); Mudgett, J.S. ); Yu, J.Y. )

    1993-10-01

    Several human genes related to DNA excision repair (ER) have been isolated via ER cross-species complementation (ERCC) of UV-sensitive CHO cells. The authors have now isolated and characterized cDNAs for the human ERCC5 gene that complement CHO UV135 cells. The ERCC5 mRNA size is about 4.6 kb. Their available cDNA clones are partial length, and no single clone was active for UV135 complementation. When cDNAs were mixed pairwise with a cosmid clone containing an overlapping 5[prime]-end segment of the ERCC5 gene, DNA transfer produced UV-resistant colonies with 60 to 95% correction of UV resistance relative to either a genomic ERCC5 DNA transformant or the CHO AA8 progenitor cells. cDNA-cosmid transformants regained intermediate levels (20 to 45%) of ER-dependent reactivation of a UV-damaged pSVCATgpt reporter plasmid. Their evidence strongly implicates an in situ recombination mechanism in cDNA-cosmid complementation for ER. The complete deduced amino acid sequence of ERCC5 was reconstructed for several cDNA clones encoding a predicted protein of 1,186 amino acids. The ERCC5 protein has extensive sequence similarities, in bipartite domains A and B, to products of RAD repair genes of two yeast, Saccharomyces cerevisiae RAD2 and Schizosaccharomyces pombe rad13. Sequence, structural, and functional data taken together indicate that ERCC5 and its relatives are probable functional homologs. A second locus represented by S. cerevisiae YKL510 and S. pombe rad2 genes is structurally distinct from the ERCC5 locus but retains vestigial A and B domain similarities. Their analyses suggest that ERCC5 is a nuclear-localized protein with one or more highly conserved helix-loop-helix segments within domains A and B. 69 refs., 6 figs., 1 tab.

  3. Sundanese Complementation

    ERIC Educational Resources Information Center

    Kurniawan, Eri

    2013-01-01

    The focus of this thesis is the description and analysis of clausal complementation in Sundanese, an Austronesian language spoken in Indonesia. The thesis examined a range of clausal complement types in Sundanese, which consists of (i) "yen/(wi)rehna" "that" complements, (ii) "pikeun" "for" complements,…

  4. Infectious delivery and expression of a 135 kb human FRDA genomic DNA locus complements Friedreich's ataxia deficiency in human cells.

    PubMed

    Gomez-Sebastian, Silvia; Gimenez-Cassina, Alfredo; Diaz-Nido, Javier; Lim, Filip; Wade-Martins, Richard

    2007-02-01

    Friedreich's ataxia (FA) is the most common recessive ataxia, affecting 1-2 in 50,000 Caucasians, and there is currently no effective cure or treatment. FA results from a deficiency of the mitochondrial protein frataxin brought about by a repeat expansion in intron 1 of the FRDA gene. The main areas affected are the central nervous system (particularly the spinocerebellar system) and cardiac tissue. Therapies aimed at alleviating the neurological degeneration have proved unsuccessful to date. Here, we describe the construction and delivery of high capacity herpes simplex virus type 1 (HSV-1) amplicon vectors expressing the entire 80 kb FRDA genomic locus, driven by the endogenous FRDA promoter and including all introns and flanking regulatory sequences within a 135 kb genomic DNA insert. FA patient primary fibroblasts deficient in frataxin protein and exhibiting sensitivity to oxidative stress were transduced at high efficiency by FRDA genomic locus vectors. Following vector transduction, expression of FRDA protein by immunofluorescence was shown. Finally, functional complementation studies demonstrated restoration of the wild-type cellular phenotype in response to oxidative stress in transduced FA patient cells. These results suggest the potential of the infectious bacterial artificial chromosome-FRDA vectors for gene therapy of FA. PMID:17235301

  5. Complementation of defective translesion synthesis and UV light sensitivity in xeroderma pigmentosum variant cells by human and mouse DNA polymerase eta.

    PubMed

    Yamada, A; Masutani, C; Iwai, S; Hanaoka, F

    2000-07-01

    Defects in the human gene XPV result in the variant form of the genetic disease xeroderma pigmentosum (XP-V). XPV encodes DNA polymerase eta, a novel DNA polymerase that belongs to the UmuC/DinB/Rad30 superfamily. This polymerase catalyzes the efficient and accurate translesion synthesis of DNA past cis-syn cyclobutane di-thymine lesions. In this report we present the cDNA sequence and expression profiles of the mouse XPV gene and demonstrate its ability to complement defective DNA synthesis in XP-V cells. The mouse XPV protein shares 80.3% amino acid identity and 86.9% similarity with the human XPV protein. The recombinant mouse XPV protein corrected the inability of XP-V cell extracts to carry out DNA replication, by bypassing thymine dimers on template DNA. Transfection of the mouse or human XPV cDNA into human XP-V cells corrected UV sensitivity. Northern blot analysis revealed that the mouse XPV gene is expressed ubiquitously, but at a higher level in testis, liver, skin and thymus compared to other tissues. Although the mouse XPV gene was not induced by UV irradiation, its expression was elevated approximately 4-fold during cell proliferation. These results suggest that DNA polymerase eta plays a role in DNA replication, though the enzyme is not essential for viability. PMID:10871396

  6. A YAC contig encompassing the XRCC5 (Ku80) DNA repair gene and complementation of defective cells by YAC protoplast fusion

    SciTech Connect

    Blunt, T.; Priestley, A.; Hafezparast, M.; McMillan, T.

    1995-11-20

    The Chinese hamster ovary xrs mutants are sensitive to ionizing radiation, defective in DNA double-strand break rejoining, and unable to carry out V(D)J recombination effectively. Recently, the gene defective in these mutants, XRCC5, has been shown to encode Ku80, a component of the Ku protein and DNA-dependent protein kinase. We present here a YAC contig involving 25 YACs mapping to the region 2q33-q34, which encompasses the XRCC5 gene. Eight new markers for this region of chromosome 2 are identified. YACs encoding the Ku80 gene were transferred to xrs cells by protoplast fusion, and complementation of all the defective phenotypes has been obtained with two YACs. We discuss the advantages and disadvantages of this approach as a strategy for cloning human genes complementing defective rodent cell lines. 44 refs., 2 figs., 4 tabs.

  7. Complete cDNA sequence of human complement C1s and close physical linkage of the homologous genes C1s and C1r

    SciTech Connect

    Tosi, M.; Duponchel, C.; Meo, T.; Julier, C.

    1987-12-29

    Overlapping molecular clones encoding the complement subcomponent C1s were isolated from a human liver cDNA library. The nucleotide sequence reconstructed from these clones spans about 85% of the length of the liver C1s messenger RNAs, which occur in three distinct size classes around 3 kilobases in length. Comparisons with the sequence of C1r, the other enzymatic subcomponent of C1, reveal 40% amino acid identity and conservation of all the cysteine residues. Beside the serine protease domain, the following sequence motifs, previously described in C1r, were also found in C1s: (a) two repeats of the type found in the Ba fragment of complement factor B and in several other complement but also noncomplement proteins, (b) a cysteine-rich segment homologous to the repeats of epidermal growth factor precursor, and (c) a duplicated segment found only in C1r and C1s. Differences in each of these structural motifs provide significant clues for the interpretation of the functional divergence of these interacting serine protease zymogens. Hybridizations of C1r and C1s probes to restriction endonuclease fragments of genomic DNA demonstrate close physical linkage of the corresponding genes. The implications of this finding are discussed with respect to the evolution of C1r and C1s after their origin by tandem gene duplication and to the previously observed combined hereditary deficiencies of Clr and Cls.

  8. Selective enrichment of environmental DNA libraries for genes encoding nonribosomal peptides and polyketides by phosphopantetheine transferase-dependent complementation of siderophore biosynthesis

    PubMed Central

    Charlop-Powers, Zachary; Banik, Jacob J.; Owen, Jeremy G.; Craig, Jeffrey W.; Brady, Sean F.

    2012-01-01

    The cloning of DNA directly from environmental samples provides a means to functionally access biosynthetic gene clusters present in the genomes of the large fraction of bacteria that remains recalcitrant to growth in the laboratory. Herein we demonstrate a method by which complementation of phosphopantetheine transferase deletion mutants can be used to restore siderophore biosynthesis and to therefore selectively enrich eDNA libraries for nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) gene sequences to unprecedented levels. The common use of NRPS/PKS-derived siderophores across bacterial taxa makes this method generalizable and should allow for the facile selective enrichment of NRPS/PKS-containing biosynthetic gene clusters from large environmental DNA libraries using a wide variety of phylogenetically diverse bacterial hosts. PMID:23072412

  9. Complement Test

    MedlinePlus

    ... helpful? Also known as: C1; C1q; C2; C3; C4; CH50; CH100 (among others) Formal name: Complement Activity; ... whether the system is functioning normally. C3 and C4 are the most frequently measured complement proteins. Total ...

  10. Functional complementation of Leishmania (Leishmania) amazonensis AP endonuclease gene (lamap) in Escherichia coli mutant strains challenged with DNA damage agents

    PubMed Central

    Verissimo-Villela, Erika; Kitahara-Oliveira, Milene Yoko; dos Reis, Ana Beatriz de Bragança; Albano, Rodolpho Mattos; Da-Cruz, Alda Maria; Bello, Alexandre Ribeiro

    2016-01-01

    During its life cycle Leishmania spp. face several stress conditions that can cause DNA damages. Base Excision Repair plays an important role in DNA maintenance and it is one of the most conserved mechanisms in all living organisms. DNA repair in trypanosomatids has been reported only for Old World Leishmania species. Here the AP endonuclease from Leishmania (L.) amazonensis was cloned, expressed in Escherichia coli mutants defective on the DNA repair machinery, that were submitted to different stress conditions, showing ability to survive in comparison to the triple null mutant parental strain BW535. Phylogenetic and multiple sequence analyses also confirmed that LAMAP belongs to the AP endonuclease class of proteins. PMID:27223868

  11. Dynamics of interaction between complement-fixing antibody/dsDNA immune complexes and erythrocytes. In vitro studies and potential general applications to clinical immune complex testing

    SciTech Connect

    Taylor, R.P.; Horgan, C.; Hooper, M.; Burge, J.

    1985-01-01

    Soluble antibody//sub 3/H-double-stranded PM2 DNA (dsDNA) immune complexes were briefly opsonized with complement and then allowed to bind to human erythrocytes (via complement receptors). The cells were washed and subsequently a volume of autologous blood in a variety of media was added, and the release of the bound immune complexes from the erythrocytes was studied as a function of temperature and time. After 1-2 h, the majority of the bound immune complexes were not released into the serum during blood clotting at either 37 degrees C or room temperature, but there was a considerably greater release of the immune complexes into the plasma of blood that was anticoagulated with EDTA. Similar results were obtained using various conditions of opsonization and also using complexes that contained lower molecular weight dsDNA. Thus, the kinetics of release of these antibody/dsDNA immune complexes differed substantially from the kinetics of release of antibody/bovine serum albumin complexes that was reported by others. Studies using the solution phase C1q immune complex binding assay confirmed that in approximately half of the SLE samples that were positive for immune complexes, there was a significantly higher level of detectable immune complexes in plasma vs. serum. Freshly drawn erythrocytes from some SLE patients exhibiting this plasma/serum discrepancy had IgG antigen on their surface that was released by incubation in EDTA plasma. Thus, the higher levels of immune complexes observed in EDTA plasma vs. serum using the C1q assay may often reflect the existence of immune complexes circulating in vivo bound to erythrocytes.

  12. Virgin Olive Oil Enriched with Its Own Phenols or Complemented with Thyme Phenols Improves DNA Protection against Oxidation and Antioxidant Enzyme Activity in Hyperlipidemic Subjects.

    PubMed

    Romeu, Marta; Rubió, Laura; Sánchez-Martos, Vanessa; Castañer, Olga; de la Torre, Rafael; Valls, Rosa M; Ras, Rosa; Pedret, Anna; Catalán, Úrsula; López de las Hazas, María del Carmen; Motilva, María J; Fitó, Montserrat; Solà, Rosa; Giralt, Montserrat

    2016-03-01

    The effects of virgin olive oil (VOO) enriched with its own phenolic compounds (PC) and/or thyme PC on the protection against oxidative DNA damage and antioxidant endogenous enzymatic system (AEES) were estimated in 33 hyperlipidemic subjects after the consumption of VOO, VOO enriched with its own PC (FVOO), or VOO complemented with thyme PC (FVOOT). Compared to pre-intervention, 8-hydroxy-2'-deoxyguanosine (a marker for DNA damage) decreased in the FVOO intervention and to a greater extent in the FVOOT with a parallel significant increase in olive and thyme phenolic metabolites. Superoxide dismutase (AEES enzyme) significantly increased in the FVOO intervention and to a greater extent in the FVOOT with a parallel significant increase in thyme phenolic metabolites. When all three oils were compared, FVOOT appeared to have the greatest effect in protecting against oxidative DNA damage and improving AEES. The sustained intake of a FVOOT improves DNA protection against oxidation and AEES probably due to a greater bioavailability of thyme PC in hyperlipidemic subjects. PMID:26889783

  13. Characterization of vanadate-dependent NADH oxidation activity and isolation of yeast DNA which complements a class 1 vanadate resistance mutation

    SciTech Connect

    Minasi, L.E.

    1989-01-01

    A vanadate-dependent NADH oxidation activity has been characterized in plasma membranes from the yeast S cerevisiae. NADH oxidation activity was maximally stimulated at pH 5.0 in phosphate buffer. NADH oxidation was not dependent on the concentration of plasma membranes. The vanadate-dependent NADH oxidation activity was abolished under anaerobic conditions and the concomitant uptake of oxygen occurred during NADH oxidation. The activity was inhibited by superoxide dismutase and stimulated by the presence of paraquat. These results indicate that the vanadate stimulation of NADH oxidation in yeast plasma membranes occurs as a result of the vanadate-dependent oxidation of NADH by superoxide, generated by a plasma membrane NADH oxidase. {sup 51}V-NMR results indicated that a phosphate-vanadate anhydride was the stimulatory species in pH 5.0 and pH 7.0 phosphate buffer. Yeast DNA has been isolated which complements a class 1 vanadate resistance mutation.

  14. Structural basis for the targeting of complement anaphylatoxin C5a using a mixed L-RNA/L-DNA aptamer

    NASA Astrophysics Data System (ADS)

    Yatime, Laure; Maasch, Christian; Hoehlig, Kai; Klussmann, Sven; Andersen, Gregers R.; Vater, Axel

    2015-04-01

    L-Oligonucleotide aptamers (Spiegelmers) consist of non-natural L-configured nucleotides and are of particular therapeutic interest due to their high resistance to plasma nucleases. The anaphylatoxin C5a, a potent inflammatory mediator generated during complement activation that has been implicated with organ damage, can be efficiently targeted by Spiegelmers. Here, we present the first crystallographic structures of an active Spiegelmer, NOX-D20, bound to its physiological targets, mouse C5a and C5a-desArg. The structures reveal a complex 3D architecture for the L-aptamer that wraps around C5a, including an intramolecular G-quadruplex stabilized by a central Ca2+ ion. Functional validation of the observed L-aptamer:C5a binding mode through mutational studies also rationalizes the specificity of NOX-D20 for mouse and human C5a against macaque and rat C5a. Finally, our structural model provides the molecular basis for the Spiegelmer affinity improvement through positional L-ribonucleotide to L-deoxyribonucleotide exchanges and for its inhibition of the C5a:C5aR interaction.

  15. Structural basis for the targeting of complement anaphylatoxin C5a using a mixed L-RNA/L-DNA aptamer

    PubMed Central

    Yatime, Laure; Maasch, Christian; Hoehlig, Kai; Klussmann, Sven; Andersen, Gregers R.; Vater, Axel

    2015-01-01

    L-Oligonucleotide aptamers (Spiegelmers) consist of non-natural L-configured nucleotides and are of particular therapeutic interest due to their high resistance to plasma nucleases. The anaphylatoxin C5a, a potent inflammatory mediator generated during complement activation that has been implicated with organ damage, can be efficiently targeted by Spiegelmers. Here, we present the first crystallographic structures of an active Spiegelmer, NOX-D20, bound to its physiological targets, mouse C5a and C5a-desArg. The structures reveal a complex 3D architecture for the L-aptamer that wraps around C5a, including an intramolecular G-quadruplex stabilized by a central Ca2+ ion. Functional validation of the observed L-aptamer:C5a binding mode through mutational studies also rationalizes the specificity of NOX-D20 for mouse and human C5a against macaque and rat C5a. Finally, our structural model provides the molecular basis for the Spiegelmer affinity improvement through positional L-ribonucleotide to L-deoxyribonucleotide exchanges and for its inhibition of the C5a:C5aR interaction. PMID:25901944

  16. A critical evaluation of how ancient DNA bulk bone metabarcoding complements traditional morphological analysis of fossil assemblages

    NASA Astrophysics Data System (ADS)

    Grealy, Alicia C.; McDowell, Matthew C.; Scofield, Paul; Murray, Dáithí C.; Fusco, Diana A.; Haile, James; Prideaux, Gavin J.; Bunce, Michael

    2015-11-01

    When pooled for extraction as a bulk sample, the DNA within morphologically unidentifiable fossil bones can, using next-generation sequencing, yield valuable taxonomic data. This method has been proposed as a means to rapidly and cost-effectively assess general ancient DNA preservation at a site, and to investigate temporal and spatial changes in biodiversity; however, several caveats have yet to be considered. We critically evaluated the bulk bone metabarcoding (BBM) method in terms of its: (i) repeatability, by quantifying sampling and technical variance through a nested experimental design containing sub-samples and replicates at several stages; (ii) accuracy, by comparing morphological and molecular family-level identifications; and (iii) overall utility, by applying the approach to two independent Holocene fossil deposits, Bat Cave (Kangaroo Island, Australia) and Finsch's Folly (Canterbury, New Zealand). For both sites, bone and bone powder sub-sampling were found to contribute significantly to variance in molecularly identified family assemblage, while the contribution of library preparation and sequencing was almost negligible. Nevertheless, total variance was small. Sampling over 80% fewer bones than was required to morphologically identify the taxonomic assemblages, we found that the families identified molecularly are a subset of the families identified morphologically and, for the most part, represent the most abundant families in the fossil record. In addition, we detected a range of extinct, extant and endangered taxa, including some that are rare in the fossil record. Given the relatively low sampling effort of the BBM approach compared with morphological approaches, these results suggest that BBM is largely consistent, accurate, sensitive, and therefore widely applicable. Furthermore, we assessed the overall benefits and caveats of the method, and suggest a workflow for palaeontologists, archaeologists, and geneticists that will help mitigate these

  17. Decreased complement mediated binding of antibody//sup 3/-dsDNA immune complexes to the red blood cells of patients with systemic lupus erythematosus, rheumatoid arthritis, and hematologic malignancies

    SciTech Connect

    Taylor, R.P.; Horgan, C.; Buschbacher, R.; Brunner, C.M.; Hess, C.E.; O'Brien, W.M.; Wanebo, H.J.

    1983-06-01

    The complement mediated binding of prepared antibody//sup 3/H-dsDNA immune complexes to the red blood cells obtained from a number of patient populations has been investigated. Patients with solid tumors have binding activity similar to that seen in a normal group of individuals. However, a significant fraction of patients with systemic lupus erythematosus, rheumatoid arthritis, and hematologic malignancies have lowered binding activity compared with normal subjects. Quantitative studies indicate the lowered activity probably arises due to a decrease in complement receptors on the respective red blood cells. The potential importance and implications of these findings are briefly discussed.

  18. Phylogenetic and Complementation Analysis of a Single-Stranded DNA Binding Protein Family from Lactococcal Phages Indicates a Non-Bacterial Origin

    PubMed Central

    Mariadassou, Mahendra; Bardowski, Jacek K.; Bidnenko, Elena

    2011-01-01

    Background The single-stranded-nucleic acid binding (SSB) protein superfamily includes proteins encoded by different organisms from Bacteria and their phages to Eukaryotes. SSB proteins share common structural characteristics and have been suggested to descend from an ancestor polypeptide. However, as other proteins involved in DNA replication, bacterial SSB proteins are clearly different from those found in Archaea and Eukaryotes. It was proposed that the corresponding genes in the phage genomes were transferred from the bacterial hosts. Recently new SSB proteins encoded by the virulent lactococcal bacteriophages (Orf14bIL67-like proteins) have been identified and characterized structurally and biochemically. Methodology/Principal Findings This study focused on the determination of phylogenetic relationships between Orf14bIL67-like proteins and other SSBs. We have performed a large scale phylogenetic analysis and pairwise sequence comparisons of SSB proteins from different phyla. The results show that, in remarkable contrast to other phage SSBs, the Orf14bIL67–like proteins form a distinct, self-contained and well supported phylogenetic group connected to the archaeal SSBs. Functional studies demonstrated that, despite the structural and amino acid sequence differences from bacterial SSBs, Orf14bIL67 protein complements the conditional lethal ssb-1 mutation of Escherichia coli. Conclusions/Significance Here we identified for the first time a group of phages encoded SSBs which are clearly distinct from their bacterial counterparts. All methods supported the recognition of these phage proteins as a new family within the SSB superfamily. Our findings suggest that unlike other phages, the virulent lactococcal phages carry ssb genes that were not acquired from their hosts, but transferred from an archaeal genome. This represents a unique example of a horizontal gene transfer between Archaea and bacterial phages. PMID:22073223

  19. Cloning of a human cDNA encoding a CDC2-related kinase by complementation of a budding yeast cdc28 mutation

    SciTech Connect

    Ninomiya-Tsuji, Jun ); Nomoto, Satoshi; Matsumoto, Kunihiro ); Yasuda, Hideyo ); Reed, S.I. )

    1991-10-15

    The authors have cloned two different human cDNAs that can complement cdc28 mutations of budding yeast Saccharomyces cerevisiae. One corresponds to a gene encoding human p34{sup CDC2} kinase, and the other to a gene (CDK2; cell division kinase) that has not been characterized previously. The CDK2 protein is highly homologous to p34{sup CDC2} kinase and more significantly is homologous to Xenopus Eg1 kinase, suggesting that CDK2 is the human homolog of Eg1. The human CDC2 and CDK2 genes were both able to complement the inviability of a null allele of S. cerevisiae CDC28. This result indicates that the CDK2 protein has a biological activity closely related to the CDC28 and p34{sup CDC2} kinases. However, CDK2 was unable to complement cdc2 mutants in fission yeast Schizosaccharomyces pombe under the condition where the human CDC2 gene could complement them. CDK2 mRNA appeared late in G{sub 1} or in early S phase, slightly before CDC2 mRNA, after growth stimulation in normal human fibroblast cells. These results suggest that in human cells, two different CDC2-like kinases may regulate the cell cycle at distinct stages.

  20. Complementation of the embryo-lethal T-DNA insertion mutant of AUXIN-BINDING-PROTEIN 1 (ABP1) with abp1 point mutated versions reveals crosstalk of ABP1 and phytochromes

    PubMed Central

    Effendi, Yunus; Ferro, Noel; Labusch, Corinna; Geisler, Markus; Scherer, Günther F. E.

    2015-01-01

    The function of the extracytoplasmic AUXIN-BINDING-PROTEIN1 (ABP1) is largely enigmatic. We complemented a homozygous T-DNA insertion null mutant of ABP1 in Arabidopsis thaliana Wassilewskia with three mutated and one wild-type (wt) ABP1 cDNA, all tagged C-terminally with a strepII–FLAG tag upstream the KDEL signal. Based on in silico modelling, the abp1 mutants were predicted to have altered geometries of the auxin binding pocket and calculated auxin binding energies lower than the wt. Phenotypes linked to auxin transport were compromised in these three complemented abp1 mutants. Red light effects, such as elongation of hypocotyls in constant red (R) and far-red (FR) light, in white light supplemented by FR light simulating shade, and inhibition of gravitropism by R or FR, were all compromised in the complemented lines. Using auxin- or light-induced expression of marker genes, we showed that auxin-induced expression was delayed already after 10min, and light-induced expression within 60min, even though TIR1/AFB or phyB are thought to act as receptors relevant for gene expression regulation. The expression of marker genes in seedlings responding to both auxin and shade showed that for both stimuli regulation of marker gene expression was altered after 10–20min in the wild type and phyB mutant. The rapidity of expression responses provides a framework for the mechanics of functional interaction of ABP1 and phyB to trigger interwoven signalling pathways. PMID:25392478

  1. PEX12, the Pathogenic Gene of Group III Zellweger Syndrome: cDNA Cloning by Functional Complementation on a CHO Cell Mutant, Patient Analysis, and Characterization of Pex12p

    PubMed Central

    Okumoto, Kanji; Shimozawa, Nobuyuki; Kawai, Atsusi; Tamura, Shigehiko; Tsukamoto, Toshiro; Osumi, Takashi; Moser, Hugo; Wanders, Ronald J. A.; Suzuki, Yasuyuki; Kondo, Naomi; Fujiki, Yukio

    1998-01-01

    Rat PEX12 cDNA was isolated by functional complementation of peroxisome deficiency of a mutant CHO cell line, ZP109 (K. Okumoto, A. Bogaki, K. Tateishi, T. Tsukamoto, T. Osumi, N. Shimozawa, Y. Suzuki, T. Orii, and Y. Fujiki, Exp. Cell Res. 233:11–20, 1997), using a transient transfection assay and an ectopic, readily visible marker, green fluorescent protein. This cDNA encodes a 359-amino-acid membrane protein of peroxisomes with two transmembrane segments and a cysteine-rich zinc finger, the RING motif. A stable transformant of ZP109 with the PEX12 was morphologically and biochemically restored for peroxisome biogenesis. Pex12p was shown by expression of bona fide as well as epitope-tagged Pex12p to expose both N- and C-terminal regions to the cytosol. Fibroblasts derived from patients with the peroxisome deficiency Zellweger syndrome of complementation group III (CG-III) were also complemented for peroxisome biogenesis with PEX12. Two unrelated patients of this group manifesting peroxisome deficiency disorders possessed homozygous, inactivating PEX12 mutations: in one, Arg180Thr by one point mutation, and in the other, deletion of two nucleotides in codons for 291Asn and 292Ser, creating an apparently unchanged codon for Asn and a codon 292 for termination. These results indicate that the gene encoding peroxisome assembly factor Pex12p is a pathogenic gene of CG-III peroxisome deficiency. Moreover, truncation and site mutation studies, including patient PEX12 analysis, demonstrated that the cytoplasmically oriented N- and C-terminal parts of Pex12p are essential for biological function. PMID:9632816

  2. Complementary DNA and derived amino acid sequence of the. beta. subunit of human complement protein C8: identification of a close structural and ancestral relationship to the. cap alpha. subunit and C9

    SciTech Connect

    Howard, O.M.Z.; Rao, A.G.; Sodetz, J.M.

    1987-06-16

    A cDNA clone encoding the ..beta.. subunit (M/sub r/ 64,000) of the eighth component of complement (C8) has been isolated from a human liver cDNA library. This clone has a cDNA insert of 1.95 kilobases (kb) and contains the entire ..beta.. sequence (1608 base pairs (bp)). Analysis of total cellular RNA isolated from the hepatoma cell line HepG2 revealed the mRNA for ..beta.. to be approx. 2.5 kb. This is similar to the message size for the ..cap alpha.. subunit of C8 and confirms the existence of different mRNAs for ..cap alpha.. and ..beta... This finding supports genetic evidence that ..cap alpha.. and ..beta.. are encoded at different loci. Analysis of the derived amino acid sequence revealed several membrane surface seeking segments that may facilitate ..beta.. interaction with target membranes during complement-mediated cytolysis. Determined of the carbohydrate composition indicated 1 or 2 asparagine-linked but no O-linked oligosaccharide chains. Comparison of the ..beta.. sequence to that reported earlier and to that of human C9 revealed a striking homology between all three proteins. For ..beta.. and ..cap alpha.., the overall homology is 33% on the basis of identity and 53% when conserved substitutions are allowed. For ..beta.. and C9, the values are 26% and 47/sup 5/, respectively. All three have a large internal domain that is nearly cysteine free and N- and C-termini that are cysteine-rich and homologous to the low-density lipoprotein receptor repeat and epidermal growth factor type sequences, respectively. The overall homology and similarities in size and structural organization are indicative of a close ancestral relationship. It is concluded that ..cap alpha.., ..beta.. and C9 are members of a family of structurally related proteins that are capable of interacting to produce a hydrophilic to amphiphilic transition and membrane association.

  3. Drugs that inhibit complement.

    PubMed

    Schrezenmeier, Hubert; Höchsmann, Britta

    2012-02-01

    The complement system is an important part of the innate immune system. Complement plays a crucial role in the pathophysiology of many disorders. Despite the pivotal role of the complement system, an approved targeted inhibitor of a complement factor became available only recently. Eculizumab is a humanized monoclonal antibody that inhibits complement factor C5. It is a targeted, disease modifying, treatment of paroxysmal nocturnal hemoglobinuria (PNH). It was approved be the US FDA and the European Commission in 2007. In this review we will update the experience with eculizumab in PNH and discuss potential use of eculizumab in other disorders (e.g. cold agglutinin disease; atypical HUS) and new approaches to complement inhibition with drugs other than eculizumab. PMID:22169380

  4. Complement and Viral Pathogenesis

    PubMed Central

    Stoermer, Kristina A.; Morrison, Thomas E.

    2011-01-01

    The complement system functions as an immune surveillance system that rapidly responds to infection. Activation of the complement system by specific recognition pathways triggers a protease cascade, generating cleavage products that function to eliminate pathogens, regulate inflammatory responses, and shape adaptive immune responses. However, when dysregulated, these powerful functions can become destructive and the complement system has been implicated as a pathogenic effector in numerous diseases, including infectious diseases. This review highlights recent discoveries that have identified critical roles for the complement system in the pathogenesis of viral infection. PMID:21292294

  5. Platelet-mediated transformation of mtDNA-less human cells: Analysis of phenotypic variability among clones from normal individuals-and complementation behavior of the tRNA[sup Lys] mutation causing myoclonic epilepsy and ragged red fibers

    SciTech Connect

    Chomyn, A.; Lai, S.T.; Shakeley, R.; Attardi, G. ); Bresolin, N.; Scarlato, G. )

    1994-06-01

    In the present work, the authors demonstrate the possibility of using human blood platelets as mitochondrial donors for the repopulation of mtDNA-less ([rho][sup o]) cells. The noninvasive nature of platelet isolation, combined with the prolonged viability of platelet mitochondria and the simplicity and efficiency of the mitochondria-transfer procedure, has substantially increased the applicability of the [rho][sup o] cell transformation approach for mitochondrial genetic analysis and for the study of mtDNA-linked diseases. This approach has been applied to platelets from several normal human individuals and one individual affected by the myoclonic-epilepsy-and-ragged-red-fibers (MERRF) encephalomyopathy. A certain variability in respiratory capacity was observed among the platelet-derived [rho][sup o] cell transformants from a given normal subject, and it was shown to be unrelated to their mtDNA content. The results of sequential transfer of mitochondria from selected transformants into a [rho][sup o] cell line different from the first [rho][sup o] acceptor strongly suggest that this variability reflected, at least in part, differences in nuclear gene content and/or activity among the original recipient cells. A much greater variability in respiratory capacity was observed among the transformants derived from the MERRF patient and was found to be related to the presence and amount of the mitochondrial tRNA[sup Lys] mutation associated with the MERRF syndrome. An analysis of the relationship between proportion of mtDNA carrying the MERRF mutation and degree of respiratory activity in various transformations derived from the MERRF patient revealed an unusual complementation behavior of the tRNA[sup Lys] mutation, possibly reflecting the distribution of mutant mtDNA among the platelet mitochondria. 29 refs., 4 figs., 1 tab.

  6. A Two-Locus Global DNA Barcode for Land Plants: The Coding rbcL Gene Complements the Non-Coding trnH-psbA Spacer Region

    PubMed Central

    Kress, W. John; Erickson, David L.

    2007-01-01

    Background A useful DNA barcode requires sufficient sequence variation to distinguish between species and ease of application across a broad range of taxa. Discovery of a DNA barcode for land plants has been limited by intrinsically lower rates of sequence evolution in plant genomes than that observed in animals. This low rate has complicated the trade-off in finding a locus that is universal and readily sequenced and has sufficiently high sequence divergence at the species-level. Methodology/Principal Findings Here, a global plant DNA barcode system is evaluated by comparing universal application and degree of sequence divergence for nine putative barcode loci, including coding and non-coding regions, singly and in pairs across a phylogenetically diverse set of 48 genera (two species per genus). No single locus could discriminate among species in a pair in more than 79% of genera, whereas discrimination increased to nearly 88% when the non-coding trnH-psbA spacer was paired with one of three coding loci, including rbcL. In silico trials were conducted in which DNA sequences from GenBank were used to further evaluate the discriminatory power of a subset of these loci. These trials supported the earlier observation that trnH-psbA coupled with rbcL can correctly identify and discriminate among related species. Conclusions/Significance A combination of the non-coding trnH-psbA spacer region and a portion of the coding rbcL gene is recommended as a two-locus global land plant barcode that provides the necessary universality and species discrimination. PMID:17551588

  7. Targeting complement in therapy.

    PubMed

    Kirschfink, M

    2001-04-01

    With increasing evidence that complement activation significantly contributes to the pathogenesis of a large number of inflammatory diseases, strategies that interfere with its deleterious action have become a major focus in pharmacological research. Endogenous soluble complement inhibitors (C1 inhibitor, recombinant soluble complement receptor 1, antibodies) blocking key proteins of the cascade reaction, 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 have successfully been tested in various animal models over the past years. Promising results consequently led to clinical trials. Furthermore, incorporation of membrane-bound complement regulators (decay-accelerating factor (CD55), membrane co-factor protein (CD46), CD59) in transgenic animals has provided a major step forward in protecting xenografts from hyperacute rejection. At the same time, the poor contribution of complement to the antitumor response, which is caused by multiple resistance mechanisms that hamper the efficacy of antibody-based tumor therapy, is increasingly recognized and requires pharmacologic intervention. First attempts have now been made to interfere with the resistance mechanisms, thereby improving complement-mediated tumor cell destruction. PMID:11414360

  8. Complement in hemolytic anemia.

    PubMed

    Brodsky, Robert A

    2015-11-26

    Complement is increasingly being recognized as an important driver of human disease, including many hemolytic anemias. Paroxysmal nocturnal hemoglobinuria (PNH) cells are susceptible to hemolysis because of a loss of the complement regulatory proteins CD59 and CD55. Patients with atypical hemolytic uremic syndrome (aHUS) develop a thrombotic microangiopathy (TMA) that in most cases is attributable to mutations that lead to activation of the alternative pathway of complement. For optimal therapy, it is critical, but often difficult, to distinguish aHUS from other TMAs, such as thrombotic thrombocytopenic purpura; however, novel bioassays are being developed. In cold agglutinin disease (CAD), immunoglobulin M autoantibodies fix complement on the surface of red cells, resulting in extravascular hemolysis by the reticuloendothelial system. Drugs that inhibit complement activation are increasingly being used to treat these diseases. This article discusses the pathophysiology, diagnosis, and therapy for PNH, aHUS, and CAD. PMID:26582375

  9. Complement in hemolytic anemia.

    PubMed

    Brodsky, Robert A

    2015-01-01

    Complement is increasingly being recognized as an important driver of human disease, including many hemolytic anemias. Paroxysmal nocturnal hemoglobinuria (PNH) cells are susceptible to hemolysis because of a loss of the complement regulatory proteins CD59 and CD55. Patients with atypical hemolytic uremic syndrome (aHUS) develop a thrombotic microangiopathy (TMA) that in most cases is attributable to mutations that lead to activation of the alternative pathway of complement. For optimal therapy, it is critical, but often difficult, to distinguish aHUS from other TMAs, such as thrombotic thrombocytopenic purpura; however, novel bioassays are being developed. In cold agglutinin disease (CAD), immunoglobulin M autoantibodies fix complement on the surface of red cells, resulting in extravascular hemolysis by the reticuloendothelial system. Drugs that inhibit complement activation are increasingly being used to treat these diseases. This article discusses the pathophysiology, diagnosis, and therapy for PNH, aHUS, and CAD. PMID:26637747

  10. Trans-complementation by human apurinic endonuclease (Ape) of hypersensitivity to DNA damage and spontaneous mutator phenotype in apn1-yeast.

    PubMed Central

    Wilson, D M; Bennett, R A; Marquis, J C; Ansari, P; Demple, B

    1995-01-01

    Abasic (AP) sites in DNA are potentially lethal and mutagenic. 'Class II' AP endonucleases initiate the repair of these and other DNA lesions. In yeast, the predominant enzyme of this type is Apn1, and its elimination sensitizes the cells to killing by simple alkylating agents or oxidants, and raises the rate of spontaneous mutation. We investigated the ability of the major human class II AP endonuclease, Ape, which is structurally unrelated to Apn1, to replace the yeast enzyme in vivo. Confocal immunomicroscopy studies indicate that approximately 25% of the Ape expressed in yeast is present in the nucleus. High-level Ape expression corresponding to approximately 7000 molecules per nucleus, equal to the normal Apn1 copy number, restored resistance to methyl methanesulfonate to near wild-type levels in Apn1-deficient (apn1-) yeast. Ape expression in apn1- yeast provided little protection against H2O2 challenges, consistent with the weak 3'-repair diesterase activity of the human enzyme. Ape expression at approximately 2000 molecules per nucleus reduced the spontaneous mutation rate of apn1- yeast to that seen for wild-type cells. Because Ape has a powerful AP endonuclease but weak 3'-diesterase activity, these findings indicate that endogenously generated AP sites can drive spontaneous mutagenesis. Images PMID:8559661

  11. CSF coccidioides complement fixation

    MedlinePlus

    ... eds. Henry's Clinical Diagnosis and Management by Laboratory Methods . 22nd ed. Philadelphia, PA: Elsevier Saunders; 2011:chap 61. Read More Complement Update Date 5/1/2015 Updated by: Jatin M. Vyas, MD, ...

  12. In Silico Screening for Novel Inhibitors of DNA Polymerase III Alpha Subunit of Mycobacterium tuberculosis (MtbDnaE2, H37Rv)

    PubMed Central

    Jadaun, Alka; Sudhakar D, Raja; Subbarao, N.; Dixit, Aparna

    2015-01-01

    Tuberculosis, a pandemic disease is caused by Mycobacterium tuberculosis (Mtb). DNA polymerase III encoded by DnaE2 of Mtb is specifically required for its survival in vivo, and hence can be considered to be a potential drug target. Amino acid sequence analysis of the MtbDnaE2 and its human counterpart does not show any significant similarity. Therefore, a 3D model of the MtbDnaE2 was generated using Modeller 9v10 with the template structure of E. Coli DNA polymerase III alpha subunit (2HNH_A). The generated models were validated using a number of programmes such as RAMPAGE/PROCHECK, VERIFY_3D, and ProSA. MtbDnaE2 has few conserved residues and four conserved domains similar to that present in DNA polymerase III of E. coli. In silico screening was performed with bioactive anti-tuberculosis compounds and 6-AU (a known inhibitor of DNA polymerase III of Bacillus subtilis) and its analogues against the modeled MtbDnaE2 structure. Docking was performed using GOLD v5.2 software which resulted in the identification of top ten compounds with high GOLD fitness scores and binding affinity (X-Score). To further evaluate the efficacy of these compounds, in silico ADMET analysis was performed using MedChem Designer v3. Given their high binding affinity to the targeted MtbDnaE2, which is essential for DNA replication in the Mtb and good ADMET properties, these compounds are promising candidates for further evaluation and development as anti-tubercular agents. PMID:25811866

  13. Complement component 3 (C3)

    MedlinePlus

    C3 and C4 are the most commonly measured complement components. A complement test may be used to monitor people with an ... normal levels of the complement proteins C3 and C4 . Complement activity varies throughout the body. For example, ...

  14. Clinical significance of complement deficiencies.

    PubMed

    Pettigrew, H David; Teuber, Suzanne S; Gershwin, M Eric

    2009-09-01

    The complement system is composed of more than 30 serum and membrane-bound proteins, all of which are needed for normal function of complement in innate and adaptive immunity. Historically, deficiencies within the complement system have been suspected when young children have had recurrent and difficult-to-control infections. As our understanding of the complement system has increased, many other diseases have been attributed to deficiencies within the complement system. Generally, complement deficiencies within the classical pathway lead to increased susceptibility to encapsulated bacterial infections as well as a syndrome resembling systemic lupus erythematosus. Complement deficiencies within the mannose-binding lectin pathway generally lead to increased bacterial infections, and deficiencies within the alternative pathway usually lead to an increased frequency of Neisseria infections. However, factor H deficiency can lead to membranoproliferative glomerulonephritis and hemolytic uremic syndrome. Finally, deficiencies within the terminal complement pathway lead to an increased incidence of Neisseria infections. Two other notable complement-associated deficiencies are complement receptor 3 and 4 deficiency, which result from a deficiency of CD18, a disease known as leukocyte adhesion deficiency type 1, and CD59 deficiency, which causes paroxysmal nocturnal hemoglobinuria. Most inherited deficiencies of the complement system are autosomal recessive, but properidin deficiency is X-linked recessive, deficiency of C1 inhibitor is autosomal dominant, and mannose-binding lectin and factor I deficiencies are autosomal co-dominant. The diversity of clinical manifestations of complement deficiencies reflects the complexity of the complement system. PMID:19758139

  15. Complement, complement activation and anaphylatoxins in human ovarian follicular fluid.

    PubMed Central

    Perricone, R; de Carolis, C; Moretti, C; Santuari, E; de Sanctis, G; Fontana, L

    1990-01-01

    Functionally active complement was sought and detected in human follicular fluids obtained during the pre-ovulatory period. All the functional complement activities tested, including total haemolytic complement, classical pathway activity and alternative pathway activity were present in nine fluids from four different donors with values within the normal serum range. The immunochemical analysis demonstrated the presence of complement factors from C1 to C9, of B and of C1 INH, H, I. Complement anaphylatoxins were found employing RIA techniques in amounts significantly higher than in human plasma, thus demonstrating that follicular fluid complement, at least during the pre-ovulatory period, is partially activated. A possible role for urokinase-like substances in such an activation was indicated by further in vitro experiments. The presence of active complement in follicular fluid can be relevant for the function of the enzymatic multi-factorial mechanism of ovulation. PMID:2242616

  16. Laboratory tests for disorders of complement and complement regulatory proteins.

    PubMed

    Shih, Angela R; Murali, Mandakolathur R

    2015-12-01

    The complement pathway is a cascade of proteases that is involved in immune surveillance and innate immunity, as well as adaptive immunity. Dysfunction of the complement cascade may be mediated by aberrations in the pathways of activation, complement regulatory proteins, or complement deficiencies, and has been linked to a number of hematologic disorders, including paroxysmal noctural hemoglobinuria (PNH), hereditary angioedema (HAE), and atypical hemolytic-uremic syndrome (aHUS). Here, current laboratory tests for disorders of the complement pathway are reviewed, and their utility and limitations in hematologic disorders and systemic diseases are discussed. Current therapeutic advances targeting the complement pathway in treatment of complement-mediated hematologic disorders are also reviewed. PMID:26437749

  17. Complement amplification revisited.

    PubMed

    Lutz, Hans U; Jelezarova, Emiliana

    2006-01-01

    Complement amplification in blood takes place not only on activating surfaces, but in plasma as well, where it is maintained primarily by C3b2-IgG complexes. Regular products of C3 activation in serum, these complexes are inherently very efficient precursors of the alternative pathway C3 convertase. Moreover, they can bind properdin bivalently, thus creating preferred sites for convertase formation. C3b2-IgG complexes have a half-life that is substantially longer than that of free C3b, since both C3b molecules are partially protected from inactivation by factor H and I. These complexes are preferentially generated on certain naturally occurring and induced antibodies that exhibit a paratope-independent affinity for C3/C3b. Such antibodies are known to stimulate alternative complement pathway activation. We have assembled the evidence for the generation and the functional potency of the C3b2-IgG complexes, which have been studied during the last two decades. We illustrate their roles in immune complex solubilization, phagocytosis, immune response, and their ability to initiate devastating effects in ischemia/reperfusion and in aggravating inflammation. PMID:16023211

  18. The extracellular RNA complement of Escherichia coli

    PubMed Central

    Ghosal, Anubrata; Upadhyaya, Bimal Babu; Fritz, Joëlle V; Heintz-Buschart, Anna; Desai, Mahesh S; Yusuf, Dilmurat; Huang, David; Baumuratov, Aidos; Wang, Kai; Galas, David; Wilmes, Paul

    2015-01-01

    The secretion of biomolecules into the extracellular milieu is a common and well-conserved phenomenon in biology. In bacteria, secreted biomolecules are not only involved in intra-species communication but they also play roles in inter-kingdom exchanges and pathogenicity. To date, released products, such as small molecules, DNA, peptides, and proteins, have been well studied in bacteria. However, the bacterial extracellular RNA complement has so far not been comprehensively characterized. Here, we have analyzed, using a combination of physical characterization and high-throughput sequencing, the extracellular RNA complement of both outer membrane vesicle (OMV)-associated and OMV-free RNA of the enteric Gram-negative model bacterium Escherichia coli K-12 substrain MG1655 and have compared it to its intracellular RNA complement. Our results demonstrate that a large part of the extracellular RNA complement is in the size range between 15 and 40 nucleotides and is derived from specific intracellular RNAs. Furthermore, RNA is associated with OMVs and the relative abundances of RNA biotypes in the intracellular, OMV and OMV-free fractions are distinct. Apart from rRNA fragments, a significant portion of the extracellular RNA complement is composed of specific cleavage products of functionally important structural noncoding RNAs, including tRNAs, 4.5S RNA, 6S RNA, and tmRNA. In addition, the extracellular RNA pool includes RNA biotypes from cryptic prophages, intergenic, and coding regions, of which some are so far uncharacterised, for example, transcripts mapping to the fimA-fimL and ves-spy intergenic regions. Our study provides the first detailed characterization of the extracellular RNA complement of the enteric model bacterium E. coli. Analogous to findings in eukaryotes, our results suggest the selective export of specific RNA biotypes by E. coli, which in turn indicates a potential role for extracellular bacterial RNAs in intercellular communication. PMID:25611733

  19. Complementing Gender Analysis Methods.

    PubMed

    Kumar, Anant

    2016-01-01

    The existing gender analysis frameworks start with a premise that men and women are equal and should be treated equally. These frameworks give emphasis on equal distribution of resources between men and women and believe that this will bring equality which is not always true. Despite equal distribution of resources, women tend to suffer and experience discrimination in many areas of their lives such as the power to control resources within social relationships, and the need for emotional security and reproductive rights within interpersonal relationships. These frameworks believe that patriarchy as an institution plays an important role in women's oppression, exploitation, and it is a barrier in their empowerment and rights. Thus, some think that by ensuring equal distribution of resources and empowering women economically, institutions like patriarchy can be challenged. These frameworks are based on proposed equality principle which puts men and women in competing roles. Thus, the real equality will never be achieved. Contrary to the existing gender analysis frameworks, the Complementing Gender Analysis framework proposed by the author provides a new approach toward gender analysis which not only recognizes the role of economic empowerment and equal distribution of resources but suggests to incorporate the concept and role of social capital, equity, and doing gender in gender analysis which is based on perceived equity principle, putting men and women in complementing roles that may lead to equality. In this article the author reviews the mainstream gender theories in development from the viewpoint of the complementary roles of gender. This alternative view is argued based on existing literature and an anecdote of observations made by the author. While criticizing the equality theory, the author offers equity theory in resolving the gender conflict by using the concept of social and psychological capital. PMID:25941756

  20. Examining coagulation-complement crosstalk: complement activation and thrombosis.

    PubMed

    Foley, Jonathan H

    2016-05-01

    The coagulation and complement systems are ancestrally related enzymatic cascades of the blood. Although their primary purposes have diverged over the past few hundred million years, they remain inextricably connected. Both complement and coagulation systems limit infection by pathogens through innate immune mechanisms. Recently, it has been shown that hyperactive complement (in particular, elevated C5a/C5b-9) is involved in the pathogenesis (including thrombosis) of diseases such as paroxysmal nocturnal hemoglobinuria, atypical haemolytic uremic syndrome, antiphospholipid syndrome and bacteremia. Although these diseases together account for many thrombosis cases, there are many more where complement activation is not considered a causative factor leading to thrombosis. To better understand what role complement may play in the pathogenesis of thrombosis a better understanding of the mechanisms that cause over-active complement in thrombotic disease is required. PMID:27207425

  1. DNA.

    ERIC Educational Resources Information Center

    Felsenfeld, Gary

    1985-01-01

    Structural form, bonding scheme, and chromatin structure of and gene-modification experiments with deoxyribonucleic acid (DNA) are described. Indicates that DNA's double helix is variable and also flexible as it interacts with regulatory and other molecules to transfer hereditary messages. (DH)

  2. Complement System in Lung Disease

    PubMed Central

    Pandya, Pankita H.

    2014-01-01

    In addition to its established contribution to innate immunity, recent studies have suggested novel roles for the complement system in the development of various lung diseases. Several studies have demonstrated that complement may serve as a key link between innate and adaptive immunity in a variety of pulmonary conditions. However, the specific contributions of complement to lung diseases based on innate and adaptive immunity are just beginning to emerge. Elucidating the role of complement-mediated immune regulation in these diseases will help to identify new targets for therapeutic interventions. PMID:24901241

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

    PubMed Central

    Khoa, D. V. A.; Wimmers, K.

    2015-01-01

    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. PMID:26194222

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

    PubMed

    Khoa, D V A; Wimmers, K

    2015-09-01

    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. PMID:26194222

  5. Gene for ataxia-telangiectasia complementation group D (ATDC)

    DOEpatents

    Murnane, J.P.; Painter, R.B.; Kapp, L.N.; Yu, L.C.

    1995-03-07

    Disclosed herein is a new gene, an AT gene for complementation group D, the ATDC gene and fragments thereof. Nucleic acid probes for the gene are provided as well as proteins encoded by the gene, cDNA therefrom, preferably a 3 kilobase (kb) cDNA, and recombinant nucleic acid molecules for expression of the proteins. Further disclosed are methods to detect mutations in the gene, preferably methods employing the polymerase chain reaction (PCR). Also disclosed are methods to detect AT genes from other AT complementation groups. 30 figs.

  6. Gene for ataxia-telangiectasia complementation group D (ATDC)

    DOEpatents

    Murnane, John P.; Painter, Robert B.; Kapp, Leon N.; Yu, Loh-Chung

    1995-03-07

    Disclosed herein is a new gene, an AT gene for complementation group D, the ATDC gene and fragments thereof. Nucleic acid probes for said gene are provided as well as proteins encoded by said gene, cDNA therefrom, preferably a 3 kilobase (kb) cDNA, and recombinant nucleic acid molecules for expression of said proteins. Further disclosed are methods to detect mutations in said gene, preferably methods employing the polymerase chain reaction (PCR). Also disclosed are methods to detect AT genes from other AT complementation groups.

  7. Complement fixation by rheumatoid factor.

    PubMed Central

    Tanimoto, K; Cooper, N R; Johnson, J S; Vaughan, J H

    1975-01-01

    The capacity for fixation and activation of hemolytic complement by polyclonal IgM rheumatoid factors (RF) isolated from sera of patients with rheumatoid arthritis and monoclonal IgM-RF isolated from the cryoprecipitates of patients with IgM-IgG mixed cryoglobulinemia was examined. RF mixed with aggregated, reduced, and alkylated human IgG (Agg-R/A-IgG) in the fluid phase failed to significantly reduce the level of total hemolytic complement, CH50, or of individual complement components, C1, C2, C3, and C5. However, sheep erythrocytes (SRC) coated with Agg-R/A-IgG or with reduced and alkylated rabbit IgG anti-SRC antibody were hemolyzed by complement in the presence of polyclonal IgM-RF. Human and guinea pig complement worked equally well. The degree of hemolysis was in direct proportion to the hemagglutination titer of the RF against the same coated cells. Monoclonal IgM-RF, normal human IgM, and purified Waldenström macroglobulins without antiglobulin activity were all inert. Hemolysis of coated SRC by RF and complement was inhibited by prior treatment of the complement source with chelating agents, hydrazine, cobra venom factor, specific antisera to C1q, CR, C5, C6, or C8, or by heating at 56 degrees C for 30 min. Purified radiolabeled C4, C3, and C8 included in the complement source were bound to hemolysed SRC in direct proportion to the degree of hemolysis. These data indicate that polyclonal IgM-RF fix and activate complement via the classic pathway. The system described for assessing complement fixation by isolated RF is readily adaptable to use with whole human serum. PMID:1078825

  8. Bimolecular fluorescence complementation.

    PubMed

    Wong, Katy A; O'Bryan, John P

    2011-01-01

    Defining the subcellular distribution of signaling complexes is imperative to understanding the output from that complex. Conventional methods such as immunoprecipitation do not provide information on the spatial localization of complexes. In contrast, BiFC monitors the interaction and subcellular compartmentalization of protein complexes. In this method, a fluororescent protein is split into amino- and carboxy-terminal non-fluorescent fragments which are then fused to two proteins of interest. Interaction of the proteins results in reconstitution of the fluorophore (Figure 1). A limitation of BiFC is that once the fragmented fluorophore is reconstituted the complex is irreversible. This limitation is advantageous in detecting transient or weak interactions, but precludes a kinetic analysis of complex dynamics. An additional caveat is that the reconstituted flourophore requires 30min to mature and fluoresce, again precluding the observation of real time interactions. BiFC is a specific example of the protein fragment complementation assay (PCA) which employs reporter proteins such as green fluorescent protein variants (BiFC), dihydrofolate reductase, b-lactamase, and luciferase to measure protein:protein interactions. Alternative methods to study protein:protein interactions in cells include fluorescence co-localization and Förster resonance energy transfer (FRET). For co-localization, two proteins are individually tagged either directly with a fluorophore or by indirect immunofluorescence. However, this approach leads to high background of non-interacting proteins making it difficult to interpret co-localization data. In addition, due to the limits of resolution of confocal microscopy, two proteins may appear co-localized without necessarily interacting. With BiFC, fluorescence is only observed when the two proteins of interest interact. FRET is another excellent method for studying protein:protein interactions, but can be technically challenging. FRET

  9. Identification of a partial cDNA clone for the C3d/Epstein-Barr virus receptor of human B lymphocytes: homology with the receptor for fragments C3b and C4b of the third and fourth components of complement.

    PubMed

    Weis, J J; Fearon, D T; Klickstein, L B; Wong, W W; Richards, S A; de Bruyn Kops, A; Smith, J A; Weis, J H

    1986-08-01

    Human complement receptor type 2 (CR2) is the B-lymphocyte receptor both for the C3d fragment of the third component of complement and for the Epstein-Barr virus. Amino acid sequence analysis of tryptic peptides of CR2 revealed a strong degree of homology with the human C3b/C4b receptor, CR1. This homology suggested that CR1 gene sequences could be used to detect the CR2 sequences at conditions of low-stringency hybridization. Upon screening a human tonsillar cDNA library with CR1 cDNA sequences, two clones were identified that hybridized at low, but not at high, stringency. Redundant oligonucleotides specific for CR2 sequences were synthesized and used to establish that the two cDNA clones weakly hybridizing with the CR1 cDNA contained CR2 sequences. One of these CR2 cDNA clones hybridized to oligonucleotides derived from two distinct CR2 tryptic peptides, whereas the other, smaller cDNA clone hybridized to oligonucleotides derived from only one of the CR2 peptides. Nucleotide sequence analysis of the CR2 cDNA confirmed that the site of oligonucleotide hybridization was identical to that predicted from the peptide sequence, including flanking sequences not included within the oligonucleotide probes. The CR2-specific cDNA sequences identified a poly(A)+ RNA species of 5 kilobases in RNA extracted from human B cells but did not hybridize to any RNA obtained from the CR2-negative T-cell line HSB-2, thus confirming the appropriate size and tissue-specific distribution for the CR2 mRNA. The striking peptide sequence homology between CR2 and CR1 and the cross-hybridization of the CR2 cDNA with the CR1-specific sequences allow the placement of CR2 in a recently defined gene family of C3- and C4-binding proteins consisting of CR1, C4-binding protein, factor H, and now, CR2. PMID:3016712

  10. The Complement System in Schizophrenia

    PubMed Central

    Mayilyan, Karine R.; Weinberger, Daniel R.; Sim, Robert B.

    2009-01-01

    summary Several lines of evidence suggest that immunological factors contribute to schizophrenia. Since 1989, the role of complement, a major effector of innate immunity and an adjuvant of adaptive immunity, has been explored in schizophrenia. Increased activity of C1, C3, C4 complement components in schizophrenia has been reported by two or more groups. Two studies on different subject cohorts showed increased MBL-MASP-2 activity in patients versus controls. More then one report indicated a significant high frequency of FB*F allotype and low prevalence of the FS phenotype of complement factor B in schizophrenia. From the data reported, it is likely that the disorder is accompanied by alterations of the complement classical and lectin pathways, which undergo dynamic changes, depending on the illness course and the state of neuro-immune crosstalk. Recent findings, implicating complement in neurogenesis, synapse remodeling and pruning during brain development, suggest a reexamination of the potential role of complement in neurodevelopmental processes contributing to schizophrenia susceptibility. It is plausible that the multicomponent complement system has more than one dimensional association with schizophrenia susceptibility, pathopsychology and illness course, understanding of which will bring a new perspective for possible immunomodulation and immunocorrection of the disease. PMID:18560619

  11. DNA

    ERIC Educational Resources Information Center

    Stent, Gunther S.

    1970-01-01

    This history for molecular genetics and its explanation of DNA begins with an analysis of the Golden Jubilee essay papers, 1955. The paper ends stating that the higher nervous system is the one major frontier of biological inquiry which still offers some romance of research. (Author/VW)

  12. The human papillomavirus type 16 E7 protein complements adenovirus type 5 E1A amino-terminus-dependent transactivation of adenovirus type 5 early genes and increases ATF and Oct-1 DNA binding activity.

    PubMed Central

    Wong, H K; Ziff, E B

    1996-01-01

    We have previously shown that conserved region 1 (CR1) of the adenovirus type 5 (Ad5) E1A protein synergizes with CR3 in the transactivation of Ad5 early genes (H.K. Wong and E. B. Ziff, J. Virol. 68:4910-4920, 1994). CR1 lies within the E1A amino terminus and binds host regulatory proteins such as the RB protein, p107, p130, and p300. Since simian virus 40 (SV40) large T antigen and human papillomavirus type 16 (HPV16) E7 protein also bind host regulatory factors, we investigated whether these viral proteins can complement E1A mutants which are defective in early gene activation. We show that the HPV16 E7 protein but not SV40 T antigen can complement mutations in the Ad5 E1A CR1 in the transactivation of viral early promoters. The inability of SV40 T antigen to complement suggests that RB binding on its own is not sufficient for early promoter transactivation by the E1A amino terminus. Nuclear runoff assays show that complementation by HPV16 E7 restores the ability of the E1A mutants to stimulate early gene expression at the level of transcription. Furthermore, nuclear extracts from the E7-transformed cells show increased binding activity of ATF and Oct-1, factors that can recognize the elements of Ad5 early genes, consistent with gene activation by E1A and E7 at the transcriptional level. PMID:8523545

  13. Luciferase fragment complementation imaging in preclinical cancer studies

    PubMed Central

    Lake, Madryn C.; Aboagye, Eric O.

    2014-01-01

    The luciferase fragment complementation assay (LFCA) enables molecular events to be non-invasively imaged in live cells in vitro and in vivo in a comparatively cheap and safe manner. It is a development of previous enzyme complementation assays in which reporter genes are split into two, individually enzymatically inactive, fragments that are able to complement one another upon interaction. This complementation can be used to externally visualize cellular activities. In recent years, the number of studies which have used LFCAs to probe questions relevant to cancer have increased, and this review summarizes the most significant and interesting of these. In particular, it focuses on work conducted on the epidermal growth factor, nuclear and chemokine receptor families, and intracellular signaling pathways, including IP3, cAMP, Akt, cMyc, NRF2 and Rho GTPases. LFCAs which have been developed to image DNA methylation and detect RNA transcripts are also discussed. PMID:25594026

  14. DNA Sequence Variants in PPARGC1A, a Gene Encoding a Coactivator of the ω-3 LCPUFA Sensing PPAR-RXR Transcription Complex, Are Associated with NV AMD and AMD-Associated Loci in Genes of Complement and VEGF Signaling Pathways

    PubMed Central

    SanGiovanni, John Paul; Chen, Jing; Sapieha, Przemyslaw; Aderman, Christopher M.; Stahl, Andreas; Clemons, Traci E.; Chew, Emily Y.; Smith, Lois E. H.

    2013-01-01

    Background Increased intake of ω-3 long-chain polyunsaturated fatty acids (LCPUFAs) and use of peroxisome proliferator activator receptor (PPAR)-activating drugs are associated with attenuation of pathologic retinal angiogenesis. ω-3 LCPUFAs are endogenous agonists of PPARs. We postulated that DNA sequence variation in PPAR gamma (PPARG) co-activator 1 alpha (PPARGC1A), a gene encoding a co-activator of the LCPUFA-sensing PPARG-retinoid X receptor (RXR) transcription complex, may influence neovascularization (NV) in age-related macular degeneration (AMD). Methods We applied exact testing methods to examine distributions of DNA sequence variants in PPARGC1A for association with NV AMD and interaction of AMD-associated loci in genes of complement, lipid metabolism, and VEGF signaling systems. Our sample contained 1858 people from 3 elderly cohorts of western European ancestry. We concurrently investigated retinal gene expression profiles in 17-day-old neonatal mice on a 2% LCPUFA feeding paradigm to identify LCPUFA-regulated genes both associated with pathologic retinal angiogenesis and known to interact with PPARs or PPARGC1A. Results A DNA coding variant (rs3736265) and a 3'UTR-resident regulatory variant (rs3774923) in PPARGC1A were independently associated with NV AMD (exact P = 0.003, both SNPs). SNP-SNP interactions existed for NV AMD (P<0.005) with rs3736265 and a AMD-associated variant in complement factor B (CFB, rs512559). PPARGC1A influences activation of the AMD-associated complement component 3 (C3) promoter fragment and CFB influences activation and proteolysis of C3. We observed interaction (P≤0.003) of rs3736265 with a variant in vascular endothelial growth factor A (VEGFA, rs3025033), a key molecule in retinal angiogenesis. Another PPARGC1A coding variant (rs8192678) showed statistical interaction with a SNP in the VEGFA receptor fms-related tyrosine kinase 1 (FLT1, rs10507386; P≤0.003). C3 expression was down-regulated 2-fold in retinas of

  15. Cloning of genes that complement yeast hexokinase and glucokinase mutants.

    PubMed Central

    Walsh, R B; Kawasaki, G; Fraenkel, D G

    1983-01-01

    Genes complementing the glucose-negative fructose-negative Saccharomyces cerevisiae triple mutant strain (hxkl hxk2 glk1), which lacks hexokinase PI, hexokinase PII, and glucokinase, were obtained from a pool of yeast DNA in the multicopy plasmid YEp13. Images PMID:6341351

  16. Characterization of group A Streptococcus strains recovered from Mexican children with pharyngitis by automated DNA sequencing of virulence-related genes: unexpectedly large variation in the gene (sic) encoding a complement-inhibiting protein.

    PubMed

    Mejia, L M; Stockbauer, K E; Pan, X; Cravioto, A; Musser, J M

    1997-12-01

    Sequence variation was studied in several target genes in 54 strains of group A Streptococcus (GAS) cultured from children with pharyngitis in Mexico City. Although 16 distinct emm alleles were identified, only 4 had not been previously described. Virtually all bacteria (31 of 33 [94%] with the streptococcal pyrogenic exotoxin gene (speA) had emm1-related, emm3, or emm6 alleles. The gene (sic) encoding an extracellular GAS protein that inhibits complement function was unusually variable among isolates with the emm1 family of alleles, with a total of seven variants identified. The data suggest that many GAS strains infecting Mexican children are genetically similar to organisms commonly encountered in the United States and western Europe. Sequence variation in the sic gene is useful for rapid differentiation among GAS isolates with the emm1 family of alleles. PMID:9399523

  17. Characterization of group A Streptococcus strains recovered from Mexican children with pharyngitis by automated DNA sequencing of virulence-related genes: unexpectedly large variation in the gene (sic) encoding a complement-inhibiting protein.

    PubMed Central

    Mejia, L M; Stockbauer, K E; Pan, X; Cravioto, A; Musser, J M

    1997-01-01

    Sequence variation was studied in several target genes in 54 strains of group A Streptococcus (GAS) cultured from children with pharyngitis in Mexico City. Although 16 distinct emm alleles were identified, only 4 had not been previously described. Virtually all bacteria (31 of 33 [94%] with the streptococcal pyrogenic exotoxin gene (speA) had emm1-related, emm3, or emm6 alleles. The gene (sic) encoding an extracellular GAS protein that inhibits complement function was unusually variable among isolates with the emm1 family of alleles, with a total of seven variants identified. The data suggest that many GAS strains infecting Mexican children are genetically similar to organisms commonly encountered in the United States and western Europe. Sequence variation in the sic gene is useful for rapid differentiation among GAS isolates with the emm1 family of alleles. PMID:9399523

  18. DNA codes

    SciTech Connect

    Torney, D. C.

    2001-01-01

    We have begun to characterize a variety of codes, motivated by potential implementation as (quaternary) DNA n-sequences, with letters denoted A, C The first codes we studied are the most reminiscent of conventional group codes. For these codes, Hamming similarity was generalized so that the score for matched letters takes more than one value, depending upon which letters are matched [2]. These codes consist of n-sequences satisfying an upper bound on the similarities, summed over the letter positions, of distinct codewords. We chose similarity 2 for matches of letters A and T and 3 for matches of the letters C and G, providing a rough approximation to double-strand bond energies in DNA. An inherent novelty of DNA codes is 'reverse complementation'. The latter may be defined, as follows, not only for alphabets of size four, but, more generally, for any even-size alphabet. All that is required is a matching of the letters of the alphabet: a partition into pairs. Then, the reverse complement of a codeword is obtained by reversing the order of its letters and replacing each letter by its match. For DNA, the matching is AT/CG because these are the Watson-Crick bonding pairs. Reversal arises because two DNA sequences form a double strand with opposite relative orientations. Thus, as will be described in detail, because in vitro decoding involves the formation of double-stranded DNA from two codewords, it is reasonable to assume - for universal applicability - that the reverse complement of any codeword is also a codeword. In particular, self-reverse complementary codewords are expressly forbidden in reverse-complement codes. Thus, an appropriate distance between all pairs of codewords must, when large, effectively prohibit binding between the respective codewords: to form a double strand. Only reverse-complement pairs of codewords should be able to bind. For most applications, a DNA code is to be bi-partitioned, such that the reverse-complementary pairs are separated

  19. Improvisation: A Complement to Curriculum

    ERIC Educational Resources Information Center

    Ronald, Green A.

    2006-01-01

    With the growth of standardized assessment benchmarks in both the public and private paradigms, testing performance matters to institutions more than ever. In an attempt to take as many hindering variables out of this process, such as test anxiety, socioeconomic influences, and latency in cognition, Improvisation: A Complement to Curriculum seeks…

  20. Cloning of three human multifunctional de novo purine biosynthetic genes by functional complementation of yeast mutations.

    PubMed Central

    Schild, D; Brake, A J; Kiefer, M C; Young, D; Barr, P J

    1990-01-01

    Functional complementation of mutations in the yeast Saccharomyces cerevisiae has been used to clone three multifunctional human genes involved in de novo purine biosynthesis. A HepG2 cDNA library constructed in a yeast expression vector was used to transform yeast strains with mutations in adenine biosynthetic genes. Clones were isolated that complement mutations in the yeast ADE2, ADE3, and ADE8 genes. The cDNA that complemented the ade8 (phosphoribosylglycinamide formyltransferase, GART) mutation, also complemented the ade5 (phosphoribosylglycinamide synthetase) and ade7 [phosphoribosylaminoimidazole synthetase (AIRS; also known as PAIS)] mutations, indicating that it is the human trifunctional GART gene. Supporting data include homology between the AIRS and GART domains of this gene and the published sequence of these domains from other organisms, and localization of the cloned gene to human chromosome 21, where the GART gene has been shown to map. The cDNA that complemented ade2 (phosphoribosylaminoimidazole carboxylase) also complemented ade1 (phosphoribosylaminoimidazole succinocarboxamide synthetase), supporting earlier data suggesting that in some organisms these functions are part of a bifunctional protein. The cDNA that complemented ade3 (formyltetrahydrofolate synthetase) is different from the recently isolated human cDNA encoding this enzyme and instead appears to encode a related mitochondrial enzyme. Images PMID:2183217

  1. Differential Expression of Complement Markers in Normal and AMD Transmitochondrial Cybrids

    PubMed Central

    Nashine, Sonali; Chwa, Marilyn; Kazemian, Mina; Thaker, Kunal; Lu, Stephanie; Nesburn, Anthony; Kuppermann, Baruch D.; Kenney, M. Cristina

    2016-01-01

    Purpose Variations in mitochondrial DNA (mtDNA) and abnormalities in the complement pathways have been implicated in the pathogenesis of age-related macular degeneration (AMD). This study was designed to determine the effects of mtDNA from AMD subjects on the complement pathway. Methods Transmitochondrial cybrids were prepared by fusing platelets from AMD and age-matched Normal subjects with Rho0 (lacking mtDNA) human ARPE-19 cells. Quantitative PCR and Western blotting were performed to examine gene and protein expression profiles, respectively, of complement markers in these cybrids. Bioenergetic profiles of Normal and AMD cybrids were examined using the Seahorse XF24 flux analyzer. Results Significant decreases in the gene and protein expression of complement inhibitors, along with significantly higher levels of complement activators, were found in AMD cybrids compared to Older-Normal cybrids. Seahorse flux data demonstrated that the bioenergetic profiles for Older-Normal and Older-AMD cybrid samples were similar to each other but were lower compared to Young-Normal cybrid samples. Conclusion In summary, since all cybrids had identical nuclei and differed only in mtDNA content, the observed changes in components of complement pathways can be attributed to mtDNA variations in the AMD subjects, suggesting that mitochondrial genome and retrograde signaling play critical roles in this disease. Furthermore, the similar bioenergetic profiles of AMD and Older-Normal cybrids indicate that the signaling between mitochondria and nuclei are probably not via a respiratory pathway. PMID:27486856

  2. Complement Activation in Placental Malaria

    PubMed Central

    McDonald, Chloe R.; Tran, Vanessa; Kain, Kevin C.

    2015-01-01

    Sixty percent of all pregnancies worldwide occur in malaria endemic regions. Pregnant women are at greater risk of malaria infection than their non-pregnant counterparts and have a higher risk of adverse birth outcomes including low birth weight resulting from intrauterine growth restriction and/or preterm birth. The complement system plays an essential role in placental and fetal development as well as the host innate immune response to malaria infection. Excessive or dysregulated complement activation has been associated with the pathobiology of severe malaria and with poor pregnancy outcomes, dependent and independent of infection. Here we review the role of complement in malaria and pregnancy and discuss its part in mediating altered placental angiogenesis, malaria-induced adverse birth outcomes, and disruptions to the in utero environment with possible consequences on fetal neurodevelopment. A detailed understanding of the mechanisms underlying adverse birth outcomes, and the impact of maternal malaria infection on fetal neurodevelopment, may lead to biomarkers to identify at-risk pregnancies and novel therapeutic interventions to prevent these complications. PMID:26733992

  3. The complement system and adverse pregnancy outcomes.

    PubMed

    Regal, Jean F; Gilbert, Jeffrey S; Burwick, Richard M

    2015-09-01

    Adverse pregnancy outcomes significantly contribute to morbidity and mortality for mother and child, with lifelong health consequences for both. The innate and adaptive immune system must be regulated to insure survival of the fetal allograft, and the complement system is no exception. An intact complement system optimizes placental development and function and is essential to maintain host defense and fetal survival. Complement regulation is apparent at the placental interface from early pregnancy with some degree of complement activation occurring normally throughout gestation. However, a number of pregnancy complications including early pregnancy loss, fetal growth restriction, hypertensive disorders of pregnancy and preterm birth are associated with excessive or misdirected complement activation, and are more frequent in women with inherited or acquired complement system disorders or complement gene mutations. Clinical studies employing complement biomarkers in plasma and urine implicate dysregulated complement activation in components of each of the adverse pregnancy outcomes. In addition, mechanistic studies in rat and mouse models of adverse pregnancy outcomes address the complement pathways or activation products of importance and allow critical analysis of the pathophysiology. Targeted complement therapeutics are already in use to control adverse pregnancy outcomes in select situations. A clearer understanding of the role of the complement system in both normal pregnancy and complicated or failed pregnancy will allow a rational approach to future therapeutic strategies for manipulating complement with the goal of mitigating adverse pregnancy outcomes, preserving host defense, and improving long term outcomes for both mother and child. PMID:25802092

  4. Metformin-mediated downregulation of p38 mitogen-activated protein kinase-dependent excision repair cross-complementing 1 decreases DNA repair capacity and sensitizes human lung cancer cells to paclitaxel.

    PubMed

    Tseng, Sheng-Chieh; Huang, Yu-Ching; Chen, Huang-Jen; Chiu, Hsien-Chun; Huang, Yi-Jhen; Wo, Ting-Yu; Weng, Shao-Hsing; Lin, Yun-Wei

    2013-02-15

    Metformin, an extensively used and well-tolerated drug for treating individuals with type 2 diabetes, has recently gained significant attention as an anticancer drug. On the other hand, paclitaxel (Taxol) is a new antineoplastic drug that has shown promise in the treatment of non-small cell lung cancer (NSCLC). High expression levels of excision repair cross-complementary 1 (ERCC1) in cancers have been positively associated with the DNA repair capacity and a poor prognosis in NSCLC patients treated with platinum-containing chemotherapy. In this current study, paclitaxel was found to increase phosphorylation of mitogen-activated protein kinase (MAPK) kinase 3/6 (MKK3/6)-p38 MAPK as well as protein and mRNA levels of ERCC1 in H1650 and H1703 cells. Moreover, paclitaxel-induced ERCC1 protein and mRNA levels significantly decreased via the downregulation of p38 activity by either a p38 MAPK inhibitor SB202190 or p38 knockdown with specific small interfering RNA (siRNA). Specific inhibition of ERCC1 with siRNA was found to enhance the paclitaxel-induced cytotoxic effect and growth inhibition. Furthermore, metformin was able to not only decrease the paclitaxel-induced p38 MAPK-mediated ERCC1 expression, but also augment the cytotoxic effect induced by paclitaxel. Finally, expression of constitutive activate MKK6 or HA-p38 MAPK vectors in lung cancer cells was able to abrogate ERCC1 downregulation by metformin and paclitaxel as well as cell viability and DNA repair capacity. Overall, our results suggest that inhibition of the p38 MAPK signaling by metformin coupled with paclitaxel therapy in human NSCLC cells may be a clinically useful combination, which however will require further validation. PMID:23228696

  5. Binding of HIV-1 Vpr Protein to the Human Homolog of the Yeast DNA Repair Protein RAD23 (hHR23A) Requires Its Xeroderma Pigmentosum Complementation Group C Binding (XPCB) Domain as Well as the Ubiquitin-associated 2 (UBA2) Domain*

    PubMed Central

    Jung, Jinwon; Byeon, In-Ja L.; DeLucia, Maria; Koharudin, Leonardus M. I.; Ahn, Jinwoo; Gronenborn, Angela M.

    2014-01-01

    The human homolog of the yeast DNA repair protein RAD23, hHR23A, has been found previously to interact with the human immunodeficiency virus, type 1 accessory protein Vpr. hHR23A is a modular protein containing an N-terminal ubiquitin-like (UBL) domain and two ubiquitin-associated domains (UBA1 and UBA2) separated by a xeroderma pigmentosum complementation group C binding (XPCB) domain. All domains are connected by flexible linkers. hHR23A binds ubiquitinated proteins and acts as a shuttling factor to the proteasome. Here, we show that hHR23A utilizes both the UBA2 and XPCB domains to form a stable complex with Vpr, linking Vpr directly to cellular DNA repair pathways and their probable exploitation by the virus. Detailed structural mapping of the Vpr contacts on hHR23A, by NMR, revealed substantial contact surfaces on the UBA2 and XPCB domains. In addition, Vpr binding disrupts an intramolecular UBL-UBA2 interaction. We also show that Lys-48-linked di-ubiquitin, when binding to UBA1, does not release the bound Vpr from the hHR23A-Vpr complex. Instead, a ternary hHR23A·Vpr·di-UbK48 complex is formed, indicating that Vpr does not necessarily abolish hHR23A-mediated shuttling to the proteasome. PMID:24318982

  6. Genetics Home Reference: complement component 8 deficiency

    MedlinePlus

    ... the membranes surrounding the brain and spinal cord (meningitis). Although meningitis can be life-threatening, individuals with complement component ... leaves affected individuals prone to recurrent episodes of meningitis. Learn more about the genes associated with complement ...

  7. Complement fixation test to C. burnetii

    MedlinePlus

    ... ency/article/003520.htm Complement fixation test to C burnetii To use the sharing features on this ... JavaScript. The complement fixation test to Coxiella burnetii ( C burnetti ) is a blood test that checks for ...

  8. Complement component 3 inhibition by an antioxidant is neuroprotective after cerebral ischemia and reperfusion in mice

    PubMed Central

    Yang, Jiwon; Ahn, Hye-na; Chang, Minsun; Narasimhan, Purnima; Chan, Pak H.; Song, Yun Seon

    2012-01-01

    Oxidative stress after stroke is associated with the inflammatory system activation in the brain. The complement cascade, especially the degradation products of complement component 3, is a key inflammatory mediator of cerebral ischemia. We have shown that proinflammatory complement component 3 is increased by oxidative stress after ischemic stroke in mice using DNA array. In this study, we investigated whether up-regulation of complement component 3 is directly related to oxidative stress after transient focal cerebral ischemia in mice and oxygen-glucose deprivation in brain cells. Persistent up-regulation of complement component 3 expression was reduced in copper/zinc-superoxide dismutase transgenic mice, and manganese-superoxide dismutase knockout mice showed highly increased complement component 3 levels after transient focal cerebral ischemia. Antioxidant N-tert-butyl-α-phenylnitrone treatment suppressed complement component 3 expression after transient focal cerebral ischemia. Accumulation of complement component 3 in neurons and microglia was decreased by N-tert-butyl-α-phenylnitrone, which reduced infarct volume and impaired neurological deficiency after cerebral ischemia and reperfusion in mice. Small interfering RNA specific for complement component 3 transfection showed a significant increase in brain cells viability after oxygen-glucose deprivation. Our study suggests that the neuroprotective effect of antioxidants through complement component 3 suppression is a new strategy for potential therapeutic approaches in stroke. PMID:23199288

  9. MOLECULAR CLONING AND EXPRESSION OF CHANNEL CATFISH, ICTALURUS PUNCTATUS, COMPLEMENT MEMBRANE ATTACK COMPLEX INHIBITOR CD59

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The channel catfish, Ictalurus punctatus, complement membrane attack complex inhibitor CD59 gene was cloned and analyzed. Total RNA from tissues was isolated and cDNA libraries were constructed by the rapid amplification cDNA end (RACE) method. The gene-specific primers in conjunction with the RAC...

  10. Molecular cloning and expression of channel catfish, Ictalurus punctatus, complement membrane attack complex inhibitor CD59

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The channel catfish, Ictalurus punctatus, complement membrane attack complex inhibitor CD59 gene was cloned and analyzed. Total RNA from tissues was isolated and cDNA libraries were constructed by the rapid amplification cDNA end (RACE) method. The gene-specific primers in conjunction with the RAC...

  11. Meningococcal disease and the complement system

    PubMed Central

    Lewis, Lisa A; Ram, Sanjay

    2014-01-01

    Despite considerable advances in the understanding of the pathogenesis of meningococcal disease, this infection remains a major cause of morbidity and mortality globally. The role of the complement system in innate immune defenses against invasive meningococcal disease is well established. Individuals deficient in components of the alternative and terminal complement pathways are highly predisposed to invasive, often recurrent meningococcal infections. Genome-wide analysis studies also point to a central role for complement in disease pathogenesis. Here we review the pathophysiologic events pertinent to the complement system that accompany meningococcal sepsis in humans. Meningococci use several often redundant mechanisms to evade killing by human complement. Capsular polysaccharide and lipooligosaccharide glycan composition play critical roles in complement evasion. Some of the newly described protein vaccine antigens interact with complement components and have sparked considerable research interest. PMID:24104403

  12. Identification and characterization of complement factor H in Branchiostoma belcheri.

    PubMed

    Cai, Lu; Zhu, Jiu; Yin, Denghua; Chen, Liming; Jin, Ping; Ma, Fei

    2014-12-10

    Complement factor H (CFH) is an essential regulator of the complement system and plays very important roles in animal innate immunity. Although the complement system of amphioxus has been extensively studied, the expression in amphioxus and evolution of CFH gene remain unknown. In this study, we identified and characterized an amphioxus (Branchiostoma belcheri) CFH gene (designated as AmphiCFH). Our results showed that the full-length cDNA of AmphiCFH gene consists of 1295 bp nucleotides containing an 855 bp open reading frame (ORF) that was predicted to encode a 284 amino acid protein. The putative AmphiCFH protein possessed the characteristic of the CFH protein family, including typical CCP (complement control protein) domain. Real-time PCR analysis showed that the AmphiCFH was ubiquitously and differentially expressed in five investigated tissues (intestine, gills, notochord, muscles, and hepatic cecum). The expression level of the AmphiCFH gene was induced upon lipopolysaccharide stimulation, indicating that the AmphiCFH gene might be involved in innate immunity. In addition, phylogenetic analysis showed that the AmphiCFH gene was located between that of invertebrates and vertebrates, suggesting that the AmphiCFH gene is a member of the CFH gene family. In conclusion, our findings provided an insight into animal innate immunity and evolution of the CFH gene family. PMID:25281822

  13. The Role of Complement in Tumor Growth

    PubMed Central

    Pio, Ruben; Corrales, Leticia; Lambris, John D.

    2015-01-01

    Complement is a central part of the immune system that has developed as a first defense against non-self cells. Neoplastic transformation is accompanied by an increased capacity of the malignant cells to activate complement. In fact, clinical data demonstrate complement activation in cancer patients. On the basis of the use of protective mechanisms by malignant cells, complement activation has traditionally been considered part of the body's immunosurveillance against cancer. Inhibitory mechanisms of complement activation allow cancer cells to escape from complement-mediated elimination and hamper the clinical efficacy of monoclonal antibody–based cancer immunotherapies. To overcome this limitation, many strategies have been developed with the goal of improving complement-mediated effector mechanisms. However, significant work in recent years has identified new and surprising roles for complement activation within the tumor microenvironment. Recent reports suggest that complement elements can promote tumor growth in the context of chronic inflammation. This chapter reviews the data describing the role of complement activation in cancer immunity, which offers insights that may aid the development of more effective therapeutic approaches to control cancer. PMID:24272362

  14. Complement Activation and Inhibition in Wound Healing

    PubMed Central

    Cazander, Gwendolyn; Jukema, Gerrolt N.; Nibbering, Peter H.

    2012-01-01

    Complement activation is needed to restore tissue injury; however, inappropriate activation of complement, as seen in chronic wounds can cause cell death and enhance inflammation, thus contributing to further injury and impaired wound healing. Therefore, attenuation of complement activation by specific inhibitors is considered as an innovative wound care strategy. Currently, the effects of several complement inhibitors, for example, the C3 inhibitor compstatin and several C1 and C5 inhibitors, are under investigation in patients with complement-mediated diseases. Although (pre)clinical research into the effects of these complement inhibitors on wound healing is limited, available data indicate that reduction of complement activation can improve wound healing. Moreover, medicine may take advantage of safe and effective agents that are produced by various microorganisms, symbionts, for example, medicinal maggots, and plants to attenuate complement activation. To conclude, for the development of new wound care strategies, (pre)clinical studies into the roles of complement and the effects of application of complement inhibitors in wound healing are required. PMID:23346185

  15. Role of Complement in Autoimmune Hemolytic Anemia.

    PubMed

    Berentsen, Sigbjørn

    2015-09-01

    The classification of autoimmune hemolytic anemias and the complement system are reviewed. In autoimmune hemolytic anemia of the warm antibody type, complement-mediated cell lysis is clinically relevant in a proportion of the patients but is hardly essential for hemolysis in most patients. Cold antibody-mediated autoimmune hemolytic anemias (primary cold agglutinin disease, secondary cold agglutinin syndrome and paroxysmal cold hemoglobinuria) are entirely complement-mediated disorders. In cold agglutinin disease, efficient therapies have been developed in order to target the pathogenic B-cell clone, but complement modulation remains promising in some clinical situations. No established therapy exists for secondary cold agglutinin syndrome and paroxysmal cold hemoglobinuria, and the possibility of therapeutic complement inhibition is interesting. Currently, complement modulation is not clinically documented in any autoimmune hemolytic anemia. The most relevant candidate drugs and possible target levels of action are discussed. PMID:26696798

  16. Role of Complement in Autoimmune Hemolytic Anemia

    PubMed Central

    Berentsen, Sigbjørn

    2015-01-01

    Summary The classification of autoimmune hemolytic anemias and the complement system are reviewed. In autoimmune hemolytic anemia of the warm antibody type, complement-mediated cell lysis is clinically relevant in a proportion of the patients but is hardly essential for hemolysis in most patients. Cold antibody-mediated autoimmune hemolytic anemias (primary cold agglutinin disease, secondary cold agglutinin syndrome and paroxysmal cold hemoglobinuria) are entirely complement-mediated disorders. In cold agglutinin disease, efficient therapies have been developed in order to target the pathogenic B-cell clone, but complement modulation remains promising in some clinical situations. No established therapy exists for secondary cold agglutinin syndrome and paroxysmal cold hemoglobinuria, and the possibility of therapeutic complement inhibition is interesting. Currently, complement modulation is not clinically documented in any autoimmune hemolytic anemia. The most relevant candidate drugs and possible target levels of action are discussed. PMID:26696798

  17. Complement (C3), nutrition, and infection*

    PubMed Central

    Kielmann, A. A.; Curcio, L. M.

    1979-01-01

    Complement (C3) was determined and related to various parameters of nutritional status and past infectious disease experience in a group of 53 rural preschool children in North India. Mean complement level was 25% lower than in an age-matched European reference population. Low complement (C3) levels were associated mainly with children who were both stunted and wasted, as well as with those who had experienced frequent purulent skin infections in the past. PMID:311708

  18. Complement and dysbiosis in periodontal disease

    PubMed Central

    Hajishengallis, George; Lambris, John D.

    2012-01-01

    Signaling crosstalk between complement and Toll-like receptors (TLRs) normally serves to coordinate host immunity. However, the periodontal bacterium Porphyromonas gingivalis expresses C5 convertase-like enzymatic activity and adeptly exploits complement-TLR crosstalk to subvert host defenses and escape elimination. Intriguingly, this defective immune surveillance leads to the remodeling of the periodontal microbiota to a dysbiotic state that causes inflammatory periodontitis. Understanding the mechanisms by which P. gingivalis modulates complement function to cause dysbiosis offers new targets for complement therapeutics. PMID:22964237

  19. Role of complement in multiorgan failure.

    PubMed

    Rittirsch, Daniel; Redl, Heinz; Huber-Lang, Markus

    2012-01-01

    Multiorgan failure (MOF) represents the leading cause of death in patients with sepsis and systemic inflammatory response syndrome (SIRS) following severe trauma. The underlying immune response is highly complex and involves activation of the complement system as a crucial entity of innate immunity. Uncontrolled activation of the complement system during sepsis and SIRS with in excessive generation of complement activation products contributes to an ensuing dysfunction of various organ systems. In the present review, mechanisms of the inflammatory response in the development of MOF in sepsis and SIRS with particular focus on the complement system are discussed. PMID:23320021

  20. Human chromosome 15 confers partial complementation of phenotypes to xeroderma pigmentosum group F cells.

    PubMed Central

    Saxon, P J; Schultz, R A; Stanbridge, E J; Friedberg, E C

    1989-01-01

    Microcell-mediated transfer of a single human chromosome from repair-proficient human cells to genetic complementation group F cells from the hereditary disease xeroderma pigmentosum (XP) results in partial complementation of repair-defective phenotypes. The complementing chromosome was identified by cytogenetic and molecular analysis as human chromosome 15. Transfer of this chromosome to XP-F cells restores approximately 20% of the resistance of wild-type cells to killing by UV radiation or by the UV-mimetic chemical 4-nitroquinoline-1-oxide (4NQO), as well as partial repair synthesis of DNA measured as unscheduled DNA synthesis. Additionally, complemented XP-F cells have an enhanced capacity for reactivation of the plasmid-borne E. coli cat gene following its inactivation by UV radiation. Phenotypic complementation of XP cells by chromosome 15 is specific to genetic complementation group F; no effect on the UV sensitivity of XP-A, XP-C, or XP-D cells was detected. The observation that phenotypic complementation is partial is open to several interpretations and does not allow the definitive conclusion that the XP-F locus is carried on chromosome 15. Images Figure 3 Figure 4 PMID:2929593

  1. Complement receptors and the shaping of the natural antibody repertoire.

    PubMed

    Holers, V Michael

    2005-03-01

    Complement and complement receptors have been known for several decades to play important roles in immune effector mechanisms related to pathogen elimination and tissue inflammation. In addition, studies over the last 10 years have clearly demonstrated a key role for the complement C3d activation fragment receptor designated CR2 (complement receptor type 2) in the switched-isotype, high-affinity and memory humoral immune responses to T-dependent foreign antigens. More recent studies have extended those observations to include a key role for CR2 and C3d in the humoral immune response to T-independent foreign antigens. Conversely, as these studies have proceeded, a parallel series of analyses have linked defects in expression or function of complement C4 and other classical pathway activation pathway proteins, as well as CR2 and the closely related CR1, to the loss of self tolerance to nuclear antigens such as double-stranded DNA and chromatin in systemic lupus erythematosus. With regard to the topic of this issue, it is now becoming increasingly clear that CR2 also plays a major role in the development of the natural antibody repertoire. Specifically, in the absence of this receptor natural IgM and IgG develop in the naïve animal that demonstrate clearly altered recognition patterns for specific natural antibody targets. This repertoire change is important physiologically in at least one setting because these CR2-dependent natural antibodies are necessary for the recognition of ischemic self tissues. In addition, it is possible that certain of the phenotypes manifest by CR2-deficient mice may be strongly influenced not only by effects on later stages of B cell activation and maturation, as commonly thought, but also by alterations in the pre-existing pool of natural antibodies that are influenced by this receptor. This review will examine the evidence that has accumulated over the last few years supporting these hypotheses. PMID:15614507

  2. Infectious diseases associated with complement deficiencies.

    PubMed Central

    Figueroa, J E; Densen, P

    1991-01-01

    The complement system consists of both plasma and membrane proteins. The former influence the inflammatory response, immune modulation, and host defense. The latter are complement receptors, which mediate the cellular effects of complement activation, and regulatory proteins, which protect host cells from complement-mediated injury. Complement activation occurs via either the classical or the alternative pathway, which converge at the level of C3 and share a sequence of terminal components. Four aspects of the complement cascade are critical to its function and regulation: (i) activation of the classical pathway, (ii) activation of the alternative pathway, (iii) C3 convertase formation and C3 deposition, and (iv) membrane attack complex assembly and insertion. In general, mechanisms evolved by pathogenic microbes to resist the effects of complement are targeted to these four steps. Because individual complement proteins subserve unique functional activities and are activated in a sequential manner, complement deficiency states are associated with predictable defects in complement-dependent functions. These deficiency states can be grouped by which of the above four mechanisms they disrupt. They are distinguished by unique epidemiologic, clinical, and microbiologic features and are most prevalent in patients with certain rheumatologic and infectious diseases. Ethnic background and the incidence of infection are important cofactors determining this prevalence. Although complement undoubtedly plays a role in host defense against many microbial pathogens, it appears most important in protection against encapsulated bacteria, especially Neisseria meningitidis but also Streptococcus pneumoniae, Haemophilus influenzae, and, to a lesser extent, Neisseria gonorrhoeae. The availability of effective polysaccharide vaccines and antibiotics provides an immunologic and chemotherapeutic rationale for preventing and treating infection in patients with these deficiencies. PMID

  3. Complement resistance mechanisms of Klebsiella pneumoniae.

    PubMed

    Doorduijn, Dennis J; Rooijakkers, Suzan H M; van Schaik, Willem; Bardoel, Bart W

    2016-10-01

    The current emergence of antibiotic-resistant bacteria causes major problems in hospitals worldwide. To survive within the host, bacterial pathogens exploit several escape mechanisms to prevent detection and killing by the immune system. As a major player in immune defense, the complement system recognizes and destroys bacteria via different effector mechanisms. The complement system can label bacteria for phagocytosis or directly kill Gram-negative bacteria via insertion of a pore-forming complex in the bacterial membrane. The multi-drug resistant pathogen Klebsiella pneumoniae exploits several mechanisms to resist complement. In this review, we present an overview of strategies used by K. pneumoniae to prevent recognition and killing by the complement system. Understanding these complement evasion strategies is crucial for the development of innovative strategies to combat K. pneumoniae. PMID:27364766

  4. Serological and Genetic Evidence for Altered Complement System Functionality in Systemic Lupus Erythematosus: Findings of the GAPAID Consortium

    PubMed Central

    Prechl, József; Papp, Krisztián; Hérincs, Zoltán; Péterfy, Hajna; Lóránd, Veronika; Szittner, Zoltán; Estonba, Andone; Rovero, Paolo; Paolini, Ilaria; del Amo, Jokin; Uribarri, Maria; Alcaro, Maria Claudia; Ruiz-Larrañaga, Otsanda; Migliorini, Paola; Czirják, László

    2016-01-01

    Systemic lupus erythematosus is a chronic autoimmune disease with multifactorial ethiopathogenesis. The complement system is involved in both the early and late stages of disease development and organ damage. To better understand autoantibody mediated complement consumption we examined ex vivo immune complex formation on autoantigen arrays. We recruited patients with SLE (n = 211), with other systemic autoimmune diseases (n = 65) and non-autoimmune control subjects (n = 149). Standard clinical and laboratory data were collected and serum complement levels were determined. The genotype of SNP rs1143679 in the ITGAM gene was also determined. Ex vivo formation of immune complexes, with respect to IgM, IgG, complement C4 and C3 binding, was examined using a functional immunoassay on autoantigen microarray comprising nucleic acids, proteins and lipids. Complement consumption of nucleic acids increased upon binding of IgM and IgG even when serum complement levels were decreased due to consumption in SLE patients. A negative correlation between serum complement levels and ex vivo complement deposition on nucleic acid autoantigens is demonstrated. On the contrary, complement deposition on tested protein and lipid autoantigens showed positive correlation with C4 levels. Genetic analysis revealed that the non-synonymous variant rs1143679 in complement receptor type 3 is associated with an increased production of anti-dsDNA IgG antibodies. Notwithstanding, homozygous carriers of the previously reported susceptible allele (AA) had lower levels of dsDNA specific IgM among SLE patients. Both the non-synonymous variant rs1143679 and the high ratio of nucleic acid specific IgG/IgM were associated with multiple organ involvement. In summary, secondary complement deficiency in SLE does not impair opsonization of nucleic-acid-containing autoantigens but does affect other antigens and potentially other complement dependent processes. Dysfunction of the receptor recognizing complement

  5. Serological and Genetic Evidence for Altered Complement System Functionality in Systemic Lupus Erythematosus: Findings of the GAPAID Consortium.

    PubMed

    Prechl, József; Papp, Krisztián; Hérincs, Zoltán; Péterfy, Hajna; Lóránd, Veronika; Szittner, Zoltán; Estonba, Andone; Rovero, Paolo; Paolini, Ilaria; Del Amo, Jokin; Uribarri, Maria; Alcaro, Maria Claudia; Ruiz-Larrañaga, Otsanda; Migliorini, Paola; Czirják, László

    2016-01-01

    Systemic lupus erythematosus is a chronic autoimmune disease with multifactorial ethiopathogenesis. The complement system is involved in both the early and late stages of disease development and organ damage. To better understand autoantibody mediated complement consumption we examined ex vivo immune complex formation on autoantigen arrays. We recruited patients with SLE (n = 211), with other systemic autoimmune diseases (n = 65) and non-autoimmune control subjects (n = 149). Standard clinical and laboratory data were collected and serum complement levels were determined. The genotype of SNP rs1143679 in the ITGAM gene was also determined. Ex vivo formation of immune complexes, with respect to IgM, IgG, complement C4 and C3 binding, was examined using a functional immunoassay on autoantigen microarray comprising nucleic acids, proteins and lipids. Complement consumption of nucleic acids increased upon binding of IgM and IgG even when serum complement levels were decreased due to consumption in SLE patients. A negative correlation between serum complement levels and ex vivo complement deposition on nucleic acid autoantigens is demonstrated. On the contrary, complement deposition on tested protein and lipid autoantigens showed positive correlation with C4 levels. Genetic analysis revealed that the non-synonymous variant rs1143679 in complement receptor type 3 is associated with an increased production of anti-dsDNA IgG antibodies. Notwithstanding, homozygous carriers of the previously reported susceptible allele (AA) had lower levels of dsDNA specific IgM among SLE patients. Both the non-synonymous variant rs1143679 and the high ratio of nucleic acid specific IgG/IgM were associated with multiple organ involvement. In summary, secondary complement deficiency in SLE does not impair opsonization of nucleic-acid-containing autoantigens but does affect other antigens and potentially other complement dependent processes. Dysfunction of the receptor recognizing complement

  6. Complement System Part II: Role in Immunity

    PubMed Central

    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

  7. Molecules Great and Small: The Complement System.

    PubMed

    Mathern, Douglas R; Heeger, Peter S

    2015-09-01

    The complement cascade, traditionally considered an effector arm of innate immunity required for host defense against pathogens, is now recognized as a crucial pathogenic mediator of various kidney diseases. Complement components produced by the liver and circulating in the plasma undergo activation through the classical and/or mannose-binding lectin pathways to mediate anti-HLA antibody-initiated kidney transplant rejection and autoantibody-initiated GN, the latter including membranous glomerulopathy, antiglomerular basement membrane disease, and lupus nephritis. Inherited and/or acquired abnormalities of complement regulators, which requisitely limit restraint on alternative pathway complement activation, contribute to the pathogenesis of the C3 nephropathies and atypical hemolytic uremic syndrome. Increasing evidence links complement produced by endothelial cells and/or tubular cells to the pathogenesis of kidney ischemia-reperfusion injury and progressive kidney fibrosis. Data emerging since the mid-2000s additionally show that immune cells, including T cells and antigen-presenting cells, produce alternative pathway complement components during cognate interactions. The subsequent local complement activation yields production of the anaphylatoxins C3a and C5a, which bind to their respective receptors (C3aR and C5aR) on both partners to augment effector T-cell proliferation and survival, while simultaneously inhibiting regulatory T-cell induction and function. This immune cell-derived complement enhances pathogenic alloreactive T-cell immunity that results in transplant rejection and likely contributes to the pathogenesis of other T cell-mediated kidney diseases. C5a/C5aR ligations on neutrophils have additionally been shown to contribute to vascular inflammation in models of ANCA-mediated renal vasculitis. New translational immunology efforts along with the development of pharmacologic agents that block human complement components and receptors now permit

  8. Complement regulation: physiology and disease relevance

    PubMed Central

    2015-01-01

    The complement system is part of the innate immune response and as such defends against invading pathogens, removes immune complexes and damaged self-cells, aids organ regeneration, confers neuroprotection, and engages with the adaptive immune response via T and B cells. Complement activation can either benefit or harm the host organism; thus, the complement system must maintain a balance between activation on foreign or modified self surfaces and inhibition on intact host cells. Complement regulators are essential for maintaining this balance and are classified as soluble regulators, such as factor H, and membrane-bound regulators. Defective complement regulators can damage the host cell and result in the accumulation of immunological debris. Moreover, defective regulators are associated with several autoimmune diseases such as atypical hemolytic uremic syndrome, dense deposit disease, age-related macular degeneration, and systemic lupus erythematosus. Therefore, understanding the molecular mechanisms by which the complement system is regulated is important for the development of novel therapies for complement-associated diseases. PMID:26300937

  9. Infections Revealing Complement Deficiency in Adults

    PubMed Central

    Audemard-Verger, A.; Descloux, E.; Ponard, D.; Deroux, A.; Fantin, B.; Fieschi, C.; John, M.; Bouldouyre, A.; Karkowsi, L.; Moulis, G.; Auvinet, H.; Valla, F.; Lechiche, C.; Davido, B.; Martinot, M.; Biron, C.; Lucht, F.; Asseray, N.; Froissart, A.; Buzelé, R.; Perlat, A.; Boutboul, D.; Fremeaux-Bacchi, V.; Isnard, S.; Bienvenu, B.

    2016-01-01

    Abstract Complement system is a part of innate immunity, its main function is to protect human from bacterial infection. As genetic disorders, complement deficiencies are often diagnosed in pediatric population. However, complement deficiencies can also be revealed in adults but have been poorly investigated. Herein, we describe a case series of infections revealing complement deficiency in adults to study clinical spectrum and management of complement deficiencies. A nationwide retrospective study was conducted in French university and general hospitals in departments of internal medicine, infectious diseases enrolling patients older than 15 years old who had presented at least one infection leading to a complement deficiency diagnosis. Forty-one patients included between 2002 and 2015 in 19 different departments were enrolled in this study. The male-to-female ratio was 1.3 and the mean age at diagnosis was 28 ± 14 (15–67) years. The main clinical feature was Neisseria meningitidis meningitis 75% (n = 31/41) often involving rare serotype: Y (n = 9) and W 135 (n = 7). The main complement deficiency observed was the common final pathway deficiency 83% (n = 34/41). Half of the cohort displayed severe sepsis or septic shock at diagnosis (n = 22/41) but no patient died. No patient had family history of complement deficiency. The mean follow-up was 1.15 ± 1.95 (0.1–10) years. Half of the patients had already suffered from at least one infection before diagnosis of complement deficiency: meningitis (n = 13), pneumonia (n = 4), fulminans purpura (n = 1), or recurrent otitis (n = 1). Near one-third (n = 10/39) had received prophylactic antibiotics (cotrimoxazole or penicillin) after diagnosis of complement deficiency. The vaccination coverage rate, at the end of the follow-up, for N meningitidis, Streptococcus pneumonia, and Haemophilius influenzae were, respectively, 90% (n = 33/37), 47% (n = 17/36), and 35

  10. Genetics Home Reference: complement factor I deficiency

    MedlinePlus

    ... Page Baracho GV, Nudelman V, Isaac L. Molecular characterization of homozygous hereditary factor I deficiency. Clin Exp ... G, Sánchez-Corral P, López-Trascasa M. Molecular characterization of Complement Factor I deficiency in two Spanish ...

  11. Nomenclature for human complement component C2*

    PubMed Central

    1992-01-01

    This note describes the designations for variants of the human complement component C2, which were approved by the Nomenclature Committee of the International Union of Immunological Societies (IUIS). PMID:1394787

  12. Virus-induced gene complementation in tomato

    PubMed Central

    Kong, Jinhua; Chen, Weiwei; Shen, Jiajia; Qin, Cheng; Lai, Tongfei; Zhang, Pengcheng; Wang, Ying; Wu, Chaoqun; Yang, Xin; Hong, Yiguo

    2013-01-01

    Virus-induced gene complementation (VIGC), a plant virus technology based on Potato virus X for transient overexpression of endogenous genes complemented tomato mutants, resulting in non-ripening fruits to ripen. This efficient “gain-of-function” approach involves no stable transformation, and reveals a fruit-specific transcriptional network that may exist among key transcription factors in modulating tomato ripening. Thus, VIGC represents a novel and feasible strategy for gene functional analysis in plants. PMID:24305652

  13. Complement regulatory proteins are incorporated into lentiviral vectors and protect particles against complement inactivation.

    PubMed

    Schauber-Plewa, C; Simmons, A; Tuerk, M J; Pacheco, C D; Veres, G

    2005-02-01

    Lentiviral vectors pseudotyped with G glycoprotein from vesicular stomatitis virus (VSV-G) and baculovirus gp64 are inactivated by human complement. The extent of vector inactivation in serum from individual donors was examined and results showed wide donor-dependent variation in complement sensitivity for VSV-G-pseudotyped lentivectors. Amphotropic envelope (Ampho)-pseudotyped vectors were generally resistant to serum from all donors, while gp64-pseudotyped vectors were inactivated but showed less donor-to-donor variation than VSV-G. In animal sera, the vectors were mostly resistant to inactivation by rodent complement, whereas canine complement caused a moderate reduction in titer. In a novel advance for the lentiviral vector system, human complement-resistant-pseudotyped lentivector particles were produced through incorporation of complement regulatory proteins (CRPs). Decay accelerating factor (DAF)/CD55 provided the most effective protection using this method, while membrane cofactor protein (MCP)/CD46 showed donor-dependent protection and CD59 provided little or no protection against complement inactivation. Unlike previous approaches using CRPs to produce complement-resistant viral vectors, CRP-containing lentivectors particles were generated for this study without engineering the CRP molecules. Thus, through overexpression of native DAF/CD55 in the viral producer cell, an easy method was developed for generation of lentiviral vectors that are almost completely resistant to inactivation by human complement. Production of complement-resistant lentiviral particles is a critical step toward use of these vectors for in vivo gene therapy applications. PMID:15550926

  14. Polyphosphate suppresses complement via the terminal pathway

    PubMed Central

    Wat, Jovian M.; Foley, Jonathan H.; Krisinger, Michael J.; Ocariza, Linnette Mae; Lei, Victor; Wasney, Gregory A.; Lameignere, Emilie; Strynadka, Natalie C.; Smith, Stephanie A.; Morrissey, James H.

    2014-01-01

    Polyphosphate, synthesized by all cells, is a linear polymer of inorganic phosphate. When released into the circulation, it exerts prothrombotic and proinflammatory activities by modulating steps in the coagulation cascade. We examined the role of polyphosphate in regulating the evolutionarily related proteolytic cascade complement. In erythrocyte lysis assays, polyphosphate comprising more than 1000 phosphate units suppressed total hemolytic activity with a concentration to reduce maximal lysis to 50% that was 10-fold lower than with monophosphate. In the ion- and enzyme-independent terminal pathway complement assay, polyphosphate suppressed complement in a concentration- and size-dependent manner. Phosphatase-treated polyphosphate lost its ability to suppress complement, confirming that polymer integrity is required. Sequential addition of polyphosphate to the terminal pathway assay showed that polyphosphate interferes with complement only when added before formation of the C5b-7 complex. Physicochemical analyses using native gels, gel filtration, and differential scanning fluorimetry revealed that polyphosphate binds to and destabilizes C5b,6, thereby reducing the capacity of the membrane attack complex to bind to and lyse the target cell. In summary, we have added another function to polyphosphate in blood, demonstrating that it dampens the innate immune response by suppressing complement. These findings further establish the complex relationship between coagulation and innate immunity. PMID:24335501

  15. Phylogenetic aspects of the complement system.

    PubMed

    Zarkadis, I K; Mastellos, D; Lambris, J D

    2001-01-01

    During evolution two general systems of immunity have emerged: innate or, natural immunity and adaptive (acquired), or specific immunity. The innate system is phylogenetically older and is found in some form in all multicellular organisms, whereas the adaptive system appeared about 450 million years ago and is found in all vertebrates except jawless fish. The complement system in higher vertebrates plays an important role as an effector of both the innate and the acquired immune response, and also participates in various immunoregulatory processes. In lower vertebrates complement is activated by the alternative and lectin pathways and is primarily involved in the opsonization of foreign material. The Agnatha (the most primitive vertebrate species) possess the alternative and lectin pathways while cartilaginous fish are the first species in which the classical pathway appears following the emergence of immunoglobulins. The rest of the poikilothermic species, ranging from teleosts to reptilians, appear to contain a well-developed complement system resembling that of the homeothermic vertebrates. It seems that most of the complement components have appeared after the duplication of primordial genes encoding C3/C4/C5, fB/C2, C1s/C1r/MASP-1/MASP-2, and C6/C7/C8/C9 molecules, in a process that led to the formation of distinct activation pathways. However, unlike homeotherms, several species of poikilotherms (e.g. trout) have recently been shown to possess multiple forms of complement components (C3, factor B) that are structurally and functionally more diverse than those of higher vertebrates. We hypothesize that this remarkable diversity has allowed these animals to expand their innate capacity for immune recognition and response. Recent studies have also indicated the possible presence of complement receptors in protochordates and lower vertebrates. In conclusion, there is considerable evidence suggesting that the complement system is present in the entire lineage of

  16. Platelets and the complement cascade in atherosclerosis

    PubMed Central

    Patzelt, Johannes; Verschoor, Admar; Langer, Harald F.

    2015-01-01

    Atherosclerosis and its late sequels are still the number one cause of death in western societies. Platelets are a driving force not only during the genesis of atherosclerosis, but especially in its late stages, as evidenced by complications such as arterial thrombosis, myocardial infarction, and ischemic stroke. Atherosclerosis is increasingly recognized as an inflammatory disease, influenced by various immune mechanisms. The complement system is part of our innate immune system, and its diverse roles in atherosclerosis have become evident over the past years. In this review we identify points of intersection between platelets and the complement system and discuss their relevance for atherosclerosis. Specifically, we will focus on roles for platelets in the onset as well as progression of the disease, a possible dual role for complement in the genesis and development of atherosclerosis, and review emerging literature revealing previously unrecognized cross-talk between platelets and the complement system and discuss its possible impact for atherosclerosis. Finally, we identify limitations of current research approaches and discuss perspectives of complement modulation in the control of the disease. PMID:25784879

  17. Complement factor H related proteins (CFHRs).

    PubMed

    Skerka, Christine; Chen, Qian; Fremeaux-Bacchi, Veronique; Roumenina, Lubka T

    2013-12-15

    Factor H related proteins comprise a group of five plasma proteins: CFHR1, CFHR2, CFHR3, CFHR4 and CFHR5, and each member of this group binds to the central complement component C3b. Mutations, genetic deletions, duplications or rearrangements in the individual CFHR genes are associated with a number of diseases including atypical hemolytic uremic syndrome (aHUS), C3 glomerulopathies (C3 glomerulonephritis (C3GN), dense deposit disease (DDD) and CFHR5 nephropathy), IgA nephropathy, age related macular degeneration (AMD) and systemic lupus erythematosus (SLE). Although complement regulatory functions were attributed to most of the members of the CFHR protein family, the precise role of each CFHR protein in complement activation and the exact contribution to disease pathology is still unclear. Recent publications show that CFHR proteins form homo- as well as heterodimers. Genetic abnormalities within the CFHR gene locus can result in hybrid proteins with affected dimerization or recognition domains which cause defective functions. Here we summarize the recent data about CFHR genes and proteins in order to better understand the role of CFHR proteins in complement activation and in complement associated diseases. PMID:23830046

  18. Complement and the severity of pulmonary failure.

    PubMed

    Weigelt, J A; Chenoweth, D E; Borman, K R; Norcross, J F

    1988-07-01

    Complement-induced granulocyte aggregation is suspected as a cause of the adult respiratory distress syndrome. Quantifying the lung damage in these patients is difficult, and complement levels combined with clinical parameters of oxygenation might help define the severity of pulmonary deterioration. Forty-five high-risk patients, selected by arterial blood gas criteria, had their pulmonary insult related to C3a and C5a levels. Patients were stratified by pulmonary shunt, alveolar-arterial oxygen gradient, and radiographic findings into two categories of severity: pulmonary dysfunction, a milder insult, and ARDS, a major aberration in pulmonary function. The clinical assignment of a diagnostic category required at least 96 hours of monitoring. During this 96-hour period, multiple complement levels were obtained. These complement levels were then compared in pulmonary dysfunction and ARDS patients. ARDS patients had significantly higher C3a and C5a values after the patients were selected as high risk. These results suggest that the amount of complement activated in patients with incipient respiratory failure correlates with the severity of eventual pulmonary insult. The use of arterial blood gases and C3a and C5a levels should allow better and earlier definition of patients at risk for ARDS. PMID:3260964

  19. Complement activation in discordant hepatic xenotransplantation.

    PubMed

    Tector, A J; Chen, X; Soderland, C; Tchervenkov, J I

    1998-11-01

    Little is known about hyperacute rejection in hepatic xenotransplantation. Information from clinical xenoperfusions suggests that the liver may be rejected by a mechanism less vigorous than either kidney or heart xenografts. We used the in vitro model of porcine hepatic sinusoidal endothelial cells (PHEC) incubated with either complement replete or deficient human serum to determine the relative roles of the classical and alternate pathways of complement in the immediate response to hepatic xenotransplantation. Our results suggest that either the classical or alternate pathways are capable of independently activating the complement cascade upon exposure to the porcine hepatic sinusoidal endothelium. Our results also imply that either pathway alone is capable of initiating similar degrees of injury as the entire cascade. PMID:9915253

  20. Supramolecular Control over Split-Luciferase Complementation.

    PubMed

    Bosmans, Ralph P G; Briels, Jeroen M; Milroy, Lech-Gustav; de Greef, Tom F A; Merkx, Maarten; Brunsveld, Luc

    2016-07-25

    Supramolecular split-enzyme complementation restores enzymatic activity and allows for on-off switching. Split-luciferase fragment pairs were provided with an N-terminal FGG sequence and screened for complementation through host-guest binding to cucurbit[8]uril (Q8). Split-luciferase heterocomplex formation was induced in a Q8 concentration dependent manner, resulting in a 20-fold upregulation of luciferase activity. Supramolecular split-luciferase complementation was fully reversible, as revealed by using two types of Q8 inhibitors. Competition studies with the weak-binding FGG peptide revealed a 300-fold enhanced stability for the formation of the ternary heterocomplex compared to binding of two of the same fragments to Q8. Stochiometric binding by the potent inhibitor memantine could be used for repeated cycling of luciferase activation and deactivation in conjunction with Q8, providing a versatile module for in vitro supramolecular signaling networks. PMID:27356091

  1. Applying complement therapeutics to rare diseases.

    PubMed

    Reis, Edimara S; Mastellos, Dimitrios C; Yancopoulou, Despina; Risitano, Antonio M; Ricklin, Daniel; Lambris, John D

    2015-12-01

    Around 350 million people worldwide suffer from rare diseases. These may have a genetic, infectious, or autoimmune basis, and several include an inflammatory component. Launching of effective treatments can be very challenging when there is a low disease prevalence and limited scientific insights into the disease mechanisms. As a key trigger of inflammatory processes, complement has been associated with a variety of diseases and has become an attractive therapeutic target for conditions involving inflammation. In view of the clinical experience acquired with drugs licensed for the treatment of rare diseases such as hereditary angioedema and paroxysmal nocturnal hemoglobinuria, growing evidence supports the safety and efficacy of complement therapeutics in restoring immune balance and preventing aggravation of clinical outcomes. This review provides an overview of the candidates currently in the pharmaceutical pipeline with potential to treat orphan diseases and discusses the molecular mechanisms triggered by complement involved with the disease pathogenesis. PMID:26341313

  2. Complement inhibition in C3 glomerulopathy.

    PubMed

    Nester, Carla M; Smith, Richard J H

    2016-06-01

    C3 glomerulopathy (C3G) describes a spectrum of glomerular diseases defined by shared renal biopsy pathology: a predominance of C3 deposition on immunofluorescence with electron microscopy permitting disease sub-classification. Complement dysregulation underlies the observed pathology, a causal relationship that is supported by well described studies of genetic and acquired drivers of disease. In this article, we provide an overview of the features of C3G, including a discussion of disease definition and a review of the causal role of complement. We discuss molecular markers of disease and how biomarkers are informing our evolving understanding of underlying pathology. Research advances are laying the foundation for complement inhibition as a targeted approach to treatment of C3G. PMID:27402056

  3. Xeroderma pigmentosum complementation group G associated with Cockayne syndrome

    SciTech Connect

    Vermeulen, W.; Jaspers, N.G.J.; Bootsma, D.; Hoeijmakers, J.H.J. ); Jaeken, J. )

    1993-07-01

    Xeroderma pigmentosum (XP) and Cockayne syndrome (CS) are two rare inherited disorders with a clinical and cellular hypersensitivity to the UV component of the sunlight spectrum. Although the two traits are generally considered as clinically and genetically distinct entities, on the biochemical level a defect in the nucleotide excision-repair (NER) pathway is involved in both. Classical CS patients are primarily deficient in the preferential repair of DNA damage in actively transcribed genes, whereas in most XP patients the genetic defect affects both [open quotes]preferential[close quotes] and [open quotes]overall[close quotes] NER modalities. Here the authors report a genetic study of two unrelated, severely affected patients with the clinical characteristics of CS but with a biochemical defect typical of XP. By complementation analysis, using somatic cell fusion and nuclear microinjection of cloned repair genes, they assign these two patients to XP complementation group G, which previously was not associated with CS. This observation extends the earlier identification of two patients with a rare combined XP/CS phenotype within XP complementation groups B and D, respectively. It indicates that some mutations in at least three of the seven genes known to be involved in XP also can result in a picture of partial or even full-blown CS. It is concluded that the syndromes XP and CS are biochemically closely related and may be part of a broader clinical disease spectrum. The authors suggest, as a possible molecular mechanism underlying this relation, that the XPGC repair gene has an additional vital function, as shown for some other NER genes. 33 refs., 5 tabs.

  4. High throughput gene complementation screening permits identification of a mammalian mitochondrial protein synthesis (ρ(-)) mutant.

    PubMed

    Potluri, Prasanth; Procaccio, Vincent; Scheffler, Immo E; Wallace, Douglas C

    2016-08-01

    To identify nuclear DNA (nDNA) oxidative phosphorylation (OXPHOS) gene mutations using cultured cells, we have developed a complementation system based on retroviral transduction with a full length cDNA expression library and selection for OXHOS function by growth in galactose. We have used this system to transduce the Chinese hamster V79-G7 OXPHOS mutant cell line with a defect in mitochondrial protein synthesis. The complemented cells were found to have acquired the cDNA for the bS6m polypeptide of the small subunit of the mitochondrial ribosome. bS6m is a 14 kDa polypeptide located on the outside of the mitochondrial 28S ribosomal subunit and interacts with the rRNA. The V79-G7 mutant protein was found to harbor a methionine to threonine missense mutation at codon 13. The hamster bS6m null mutant could also be complemented by its orthologs from either mouse or human. bS6m protein tagged at its C-terminus by HA, His or GFP localized to the mitochondrion and was fully functional. Through site-directed mutagenesis we identified the probable RNA interacting residues of the bS6m peptide and tested the functional significance of mammalian specific C-terminal region. The N-terminus of the bS6m polypeptide functionally corresponds to that of the prokaryotic small ribosomal subunit, but deletion of C-terminal residues along with the zinc ion coordinating cysteine had no functional effect. Since mitochondrial diseases can result from hundreds to thousands of different nDNA gene mutations, this one step viral complementation cloning may facilitate the molecular diagnosis of a range of nDNA mitochondrial disease mutations. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi. PMID:26946086

  5. Primer Extension Reactions for the PCR- based α- complementation Assay

    PubMed Central

    Achuthan, Vasudevan; DeStefano, Jeffrey J.

    2016-01-01

    The PCR- based- α- complementation assay is an effective technique to measure the fidelity of polymerases, especially RNA-dependent RNA polymerases (RDRP) and Reverse Transcriptases (RT). It has been successfully employed to determine the fidelity of the poliovirus polymerase 3D-pol (DeStefano, 2010) as well as the human immunodeficiency virus Reverse Transcriptase (HIV RT) (Achuthan et al., 2014). A major advantage of the assay is that since the PCR step is involved, even the low yield of products obtained after two rounds of low yield of RNA synthesis (for RDRP) or reverse transcription (for RT) can be measured using the assay. The assay also mimics the reverse transcription process, since both RNA- and DNA- directed RT synthesis steps are performed. We recently used this assay to show that the HIV RT, at physiologically relevant magnesium concentration, has accuracy in the same range as other reverse transcriptases (Achuthan et al., 2014). Here, we describe in detail how to prepare the inserts using the primer extension reactions. The prepared inserts are then processed further in the PCR- based- α- complementation assay.

  6. Genetics Home Reference: complement component 2 deficiency

    MedlinePlus

    ... Page Jönsson G, Sjöholm AG, Truedsson L, Bengtsson AA, Braconier JH, Sturfelt G. Rheumatological manifestations, organ damage ... 31. Review. Citation on PubMed Truedsson L, Bengtsson AA, Sturfelt G. Complement deficiencies and systemic lupus erythematosus. ...

  7. Pneumococcal psoas pyomyositis associated with complement deficiency.

    PubMed

    Tuerlinckx, David; Bodart, Eddy; de Bilderling, Georges; Nisolle, Jean-François

    2004-04-01

    A 4.5-year-old boy with complement deficiency developed infection of the psoas caused by Streptococcus pneumoniae. Pyomyositis of the psoas muscle is uncommon but should be included in the differential diagnosis of fever and lameness. The most useful diagnostic test is computed tomography guided needle aspiration, and underlying conditions should be sought. PMID:15071302

  8. 21 CFR 866.4100 - Complement reagent.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Complement reagent. 866.4100 Section 866.4100 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunology Laboratory Equipment and Reagents §...

  9. 21 CFR 866.4100 - Complement reagent.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Complement reagent. 866.4100 Section 866.4100 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunology Laboratory Equipment and Reagents §...

  10. 21 CFR 866.4100 - Complement reagent.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Complement reagent. 866.4100 Section 866.4100 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunology Laboratory Equipment and Reagents §...

  11. 21 CFR 866.4100 - Complement reagent.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Complement reagent. 866.4100 Section 866.4100 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunology Laboratory Equipment and Reagents §...

  12. 21 CFR 866.4100 - Complement reagent.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Complement reagent. 866.4100 Section 866.4100 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunology Laboratory Equipment and Reagents §...

  13. How Preschool Children Understand Missing Complement Subjects.

    ERIC Educational Resources Information Center

    Maratsos, Michael P.

    Two studies investigated preschool children's comprehension of the missing subject of infinitival complement clauses. In the first study, use of a Surface Structure Minimal Distance principle of the type outlined by C. Chomsky was distinguished from use of a Semantic Role Principle. Preschoolers acted out sentences in which the use of the two…

  14. Complement Constructions in English: Fairly Difficult for EFL Language Learners

    ERIC Educational Resources Information Center

    Fazeli, Fatemeh; Shokrpour, Nasrin

    2012-01-01

    Complement constructions vary significantly in English and Persian. There are more complementation structures in English than in Persian and a complement structure in Persian might have more than one equivalent in English. Producing complement structures (CSs) in English is very difficult for native speakers of Persian, especially in an EFL…

  15. Engineering of human complement component C3 for catalytic inhibition of complement.

    PubMed

    Kölln, Johanna; Bredehorst, Reinhard; Spillner, Edzard

    2005-04-15

    As a novel therapeutic approach in complement-mediated pathologies, we recently developed a human C3 derivative capable of obliterating functional complement by a catalytic, non-inhibitory mechanism. In this derivative, the C-terminal region of hC3 was substituted by a 275 amino acid sequence derived from the corresponding sequence of cobra venom factor (CVF), a complement-activating C3b homologue from snake venom. In this study, we replaced shorter C-terminal sequences of hC3 by corresponding CVF sequences to further reduce potential immunogenicity and to identify domains essential for the formation of functionally stable C3 convertases. In one of these derivatives that is still capable of obliterating functional complement in vitro, the non-human portion could be reduced to a small domain located in the C-terminus of different complement proteins. This conserved NTR/C345C motif is known to be involved in assembly of different convertases of the complement system. These results suggest a major role of the C345C domain in the regulation of the half-life of the C3 convertase. Moreover, its overall identity of 96% to human C3 renders this derivative a promising candidate for therapeutic intervention in complement-mediated pathologies. PMID:15790508

  16. Utilizing complement evasion strategies to design complement-based antibacterial immunotherapeutics: Lessons from the pathogenic Neisseriae.

    PubMed

    Ram, Sanjay; Shaughnessy, Jutamas; DeOliveira, Rosane B; Lewis, Lisa A; Gulati, Sunita; Rice, Peter A

    2016-10-01

    Novel therapies are urgently needed to combat the global threat of multidrug-resistant pathogens. Complement forms an important arm of innate defenses against infections. In physiological conditions, complement activation is tightly controlled by soluble and membrane-associated complement inhibitors, but must be selectively activated on invading pathogens to facilitate microbial clearance. Many pathogens, including Neisseria gonorrhoeae and N. meningitidis, express glycans, including N-acetylneuraminic acid (Neu5Ac), that mimic host structures to evade host immunity. Neu5Ac is a negatively charged 9-cabon sugar that inhibits complement, in part by enhancing binding of the complement inhibitor factor H (FH) through C-terminal domains (19 and 20) on FH. Other microbes also bind FH, in most instances through FH domains 6 and 7 or 18-20. Here we describe two strategies to target complement activation on Neisseriae. First, microbial binding domains of FH were fused to IgG Fc to create FH18-20/Fc (binds gonococci) and FH6,7/Fc (binds meningococci). A point mutation in FH domain 19 eliminated hemolysis caused by unmodified FH18-20, but retained binding to gonococci. FH18-20/Fc and FH6,7/Fc mediated complement-dependent killing in vitro and showed efficacy in animal models of gonorrhea and meningococcal bacteremia, respectively. The second strategy utilized CMP-nonulosonate (CMP-NulO) analogs of sialic acid that were incorporated into LOS and prevented complement inhibition by physiologic CMP-Neu5Ac and resulted in attenuated gonococcal infection in mice. While studies to establish the safety of these agents are needed, enhancing complement activation on microbes may represent a promising strategy to treat antimicrobial resistant organisms. PMID:27297292

  17. Susceptibility to advanced age-related macular degeneration and alleles of complement factor H, complement factor B, complement component 2, complement component 3, and age-related maculopathy susceptibility 2 genes in a Mexican population

    PubMed Central

    Buentello-Volante, Beatriz; Rodriguez-Ruiz, Gabriela; Miranda-Duarte, Antonio; Pompa-Mera, Ericka N.; Graue-Wiechers, Federico; Bekker-Méndez, Carolina; Ayala-Ramirez, Raul; Quezada, Carlos; Rodríguez-Loaiza, Jose L.

    2012-01-01

    Purpose To investigate the association of age-related macular degeneration (AMD)–high risk alleles of the complement factor H (CFH), complement factor B (CFB), complement component 2 (C2), complement component 3 (C3), and age-related maculopathy susceptibility 2 (ARMS2) genes in a Mexican population for the first time. Methods Genotyping was performed for the Y402H variant of CFH, for the L9H, R32Q, and K565E variants of CFB, the E318D variant of C2, the A69S variant of ARMS2, and the R102G variant of C3 in 159 Mexican mestizo patients at advanced stages of AMD, i.e., CARMS (Clinical Age-Related Maculopathy Staging System) grade 4 or 5. The frequency of these variants was also investigated in a group of 152 control subjects without AMD. Genomic DNA was extracted from blood leukocytes, and genotyping was performed using PCR followed by direct sequencing. Allele-specific restriction enzyme digestion was used to detect the R102G polymorphism in C3. Results There were significant differences in the allelic distribution between the two groups for CFH Y402H (p=1×10−5), ARMS A69S (p=4×10−7), and CFB R32Q (p=0.01). The odds ratios (95% confidence interval) obtained for the risk alleles of these three variants were 3.8 (2.4–5.9), 3.04 (2.2–4.3), and 2.5 (1.1–5.7), respectively. Haplotype analysis including the two most significantly associated alleles (CFH Y402H and ARMS A69S) indicated that the C-T combination conferred an odds ratio (95% confidence interval) of 6.9 (3.2–14.8). The exposed attributable risk for this particular haplotype was 85.5%. Conclusions This is the first case-control investigation of AMD–high risk alleles in a Latino population. Our results support that CFH, ARMS2, and CFB AMD-risk alleles are consistently associated with the disease, even in ethnic groups with a complex admixture of ancestral populations such as Mexican mestizos. PMID:23112567

  18. Chronic Low Level Complement Activation within the Eye Is Controlled by Intraocular Complement Regulatory Proteins

    PubMed Central

    Sohn, Jeong-Hyeon; Kaplan, Henry J.; Suk, Hye-Jung; Bora, Puran S.; Bora, Nalini S.

    2007-01-01

    Purpose To explore the role of the complement system and complement regulatory proteins in an immune-privileged organ, the eye. Methods Eyes of normal Lewis rats were analyzed for the expression of complement regulatory proteins, membrane cofactor protein (MCP), decay-acceleration factor (DAF), membrane inhibitor of reactive lysis (MIRL, CD59), and cell surface regulator of complement (Crry), using immunohistochemistry, Western blot analysis, and reverse transcription–polymerase chain reaction (RT-PCR). Zymosan, a known activator of the alternative pathway of complement system was injected into the anterior chamber of the eye of Lewis rats. Animals were also injected intracamerally with 5 μl (25 μg) of neutralizing monoclonal antibody (mAb) against rat Crry (5I2) or CD59 (6D1) in an attempt to develop antibody induced anterior uveitis; control animals received 5 μl of sterile phosphate-buffered saline (PBS), OX-18 (25 μg), G-16-510E3 (25 μg), or MOPC-21 (25 μg). The role of complement system in antibody-induced uveitis was explored by intraperitoneal injection of 35 U cobra venom factor (CVF), 24 hours before antibody injection. Immunohistochemical staining and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) with Western blot analysis were used to detect the presence of membrane attack complex (MAC) and C3 activation products, respectively, in normal and antibody-injected rat eyes. Results Complement activation product MAC was present in the normal rat eye, and intraocular injection of zymosan induced severe anterior uveitis. The complement regulatory proteins, MCP, DAF, CD59, and Crry, were identified in the normal rat eye. Soluble forms of Crry and CD59 were also detected in normal rat aqueous humor. Severe anterior uveitis developed in Lewis rats injected with a neutralizing mAb against Crry, with increased formation of C3 split products. Systemic complement depletion by CVF prevented the induction of anterior uveitis by anti

  19. Molecular nature of the complement lesion.

    PubMed Central

    Bhakdi, S; Tranum-Jensen, J

    1978-01-01

    The principle molecular event leading to membrane perturbation by complement is the assembly of the terminal five serum complement components (C5b-C9) into a macromolecular C5b-9 complex on the target membrane [Müller-Eberhard, H.-J. (1975) Ann. Rev. Biochem. 44, 697--723]. The present communication reports on the ability of purified C5b-9 complexes isolated from target membranes to become reincorporated into artificial lipid vesicles. The data indicate that the complex is a vertically oriented, hollow, cylindrical macromolecule possessing lipid-binding regions that enable one terminus to penetrate into the lipid bilayer. A transmembrane pore appears to be created at the attachment site of the C5b-9 complex. Images PMID:281714

  20. Trichinella spiralis Paramyosin Binds Human Complement C1q and Inhibits Classical Complement Activation

    PubMed Central

    Sun, Ran; Zhao, Xi; Wang, Zixia; Yang, Jing; Zhao, Limei; Zhan, Bin; Zhu, Xinping

    2015-01-01

    Background Trichinella spiralis expresses paramyosin (Ts-Pmy) as a defense mechanism. Ts-Pmy is a functional protein with binding activity to human complement C8 and C9 and thus plays a role in evading the attack of the host’s immune system. In the present study, the binding activity of Ts-Pmy to human complement C1q and its ability to inhibit classical complement activation were investigated. Methods and Findings The binding of recombinant and natural Ts-Pmy to human C1q were determined by ELISA, Far Western blotting and immunoprecipitation, respectively. Binding of recombinant Ts-Pmy (rTs-Pmy) to C1q inhibited C1q binding to IgM and consequently inhibited C3 deposition. The lysis of antibody-sensitized erythrocytes (EAs) elicited by the classical complement pathway was also inhibited in the presence of rTs-Pmy. In addition to inhibiting classical complement activation, rTs-Pmy also suppressed C1q binding to THP-1-derived macrophages, thereby reducing C1q-induced macrophages migration. Conclusion Our results suggest that T. spiralis paramyosin plays an important role in immune evasion by interfering with complement activation through binding to C1q in addition to C8 and C9. PMID:26720603

  1. Reference instrument complement for IPNS Upgrade

    SciTech Connect

    Crawford, R.K.

    1993-07-01

    A feasibility study for a new 1 MW pulsed neutron source has recently been completed at Argonne. As part of this feasibility study, an instrument package to instrument 24 of the 36 beam ports has been considered. This complement of instruments is outlined, and details of some of the instruments are discussed. Developments required before some of these instruments can be built are also indicated.

  2. Bacteria under stress by complement and coagulation.

    PubMed

    Berends, Evelien T M; Kuipers, Annemarie; Ravesloot, Marietta M; Urbanus, Rolf T; Rooijakkers, Suzan H M

    2014-11-01

    The complement and coagulation systems are two related protein cascades in plasma that serve important roles in host defense and hemostasis, respectively. Complement activation on bacteria supports cellular immune responses and leads to direct killing of bacteria via assembly of the Membrane Attack Complex (MAC). Recent studies have indicated that the coagulation system also contributes to mammalian innate defense since coagulation factors can entrap bacteria inside clots and generate small antibacterial peptides. In this review, we will provide detailed insights into the molecular interplay between these protein cascades and bacteria. We take a closer look at how these pathways are activated on bacterial surfaces and discuss the mechanisms by which they directly cause stress to bacterial cells. The poorly understood mechanism for bacterial killing by the MAC will be reevaluated in light of recent structural insights. Finally, we highlight the strategies used by pathogenic bacteria to modulate these protein networks. Overall, these insights will contribute to a better understanding of the host defense roles of complement and coagulation against bacteria. PMID:25065463

  3. Age-related macular degeneration: Complement in action.

    PubMed

    van Lookeren Campagne, Menno; Strauss, Erich C; Yaspan, Brian L

    2016-06-01

    The complement system plays a key role in host-defense against common pathogens but must be tightly controlled to avoid inflammation and tissue damage. Polymorphisms in genes encoding two important negative regulators of the alternative complement pathway, complement factor H (CFH) and complement factor I (CFI), are associated with the risk for Age-Related Macular Degeneration (AMD), a leading cause of vision impairment in the ageing population. In this review, we will discuss the genetic basis of AMD and the potential impact of complement de-regulation on disease pathogenesis. Finally, we will highlight recent therapeutic approaches aimed at controlling complement activation in patients with AMD. PMID:26742632

  4. Complement research in the 18th-21st centuries: Progress comes with new technology.

    PubMed

    Sim, R B; Schwaeble, W; Fujita, T

    2016-10-01

    The complement system has been studied for about 120 years. Progress in defining this large and complex system has been dependent on the research technologies available, but since the introduction of protein chromatography, electrophoresis, and antibody-based assay methods in the 1950s and 60s, and sequencing of proteins and DNA in the 70s and 80s, there has been very rapid accumulation of data. With more recent improvements in 3D structure determination (nmr and X-ray crystallography), the structures of most of the complement proteins have now been solved. Complement research since 1990 has been greatly stimulated by the discoveries of the multiple proteins in the lectin pathway, the strong association of Factor H, C3, Factor B allelic variants with adult macular degeneration and atypical haemolytic uremic syndrome, and the introduction of the anti-C5 monoclonal antibody as a therapy for paroxysmal nocturnal hemoglobinuria and atypical haemolytic uremic syndrome. Potential new roles for complement in tissue development and the search for novel therapeutics suggest a very active future for complement research. PMID:27371361

  5. Human PEX1 cloned by functional complementation on a CHO cell mutant is responsible for peroxisome-deficient Zellweger syndrome of complementation group I.

    PubMed

    Tamura, S; Okumoto, K; Toyama, R; Shimozawa, N; Tsukamoto, T; Suzuki, Y; Osumi, T; Kondo, N; Fujiki, Y

    1998-04-14

    The peroxisome biogenesis disorders (PBDs), including Zellweger syndrome (ZS) and neonatal adrenoleukodystrophy (NALD), are autosomal recessive diseases caused by defects in peroxisome assembly, for which at least 10 complementation groups have been reported. We have isolated a human PEX1 cDNA (HsPEX1) by functional complementation of peroxisome deficiency of a mutant Chinese hamster ovary (CHO) cell line, ZP107, transformed with peroxisome targeting signal type 1-tagged "enhanced" green fluorescent protein. This cDNA encodes a hydrophilic protein (Pex1p) comprising 1,283 amino acids, with high homology to the AAA-type ATPase family. A stable transformant of ZP107 with HsPEX1 was morphologically and biochemically restored for peroxisome biogenesis. HsPEX1 expression restored peroxisomal protein import in fibroblasts from three patients with ZS and NALD of complementation group I (CG-I), which is the highest-incidence PBD. A CG-I ZS patient (PBDE-04) possessed compound heterozygous, inactivating mutations: a missense point mutation resulting in Leu-664 --> Pro and a deletion of the sequence from Gly-634 to His-690 presumably caused by missplicing (splice site mutation). Both PBDE-04 PEX1 cDNAs were defective in peroxisome-restoring activity when expressed in the patient fibroblasts as well as in ZP107 cells. These results demonstrate that PEX1 is the causative gene for CG-I peroxisomal disorders. PMID:9539740

  6. Complementation cloning and sequence analysis of the Chlamydomonas reinhardtii hemL gene encoding glutamate-1-semialdehyde aminotransferase

    SciTech Connect

    Matters, G.L.; Beale, S.I. )

    1993-05-01

    Glutamate-1-semialdehyde amino-transferase (GSAT) catalyzes formation of the tetrapyrrole precursor, [delta]-aminolevulinic acid. GSAT is encoded by the hemL gene. A Chlamydomonas reinhardtii hemL cDNA was selected from a vegetative stage expression library by complementation of Escherichia coli hemL mutant GE 1377. In vitro GSAT activity was ten-fold higher in an extract of the complemented hemL cells than in an extract of uncomplemented mutant cells. The complementing cDNA is 2010 bp long and includes 591 bp of 3' noncoding DNA and an estimated 27 bp of 5' noncoding DNA. The coding region includes the sequence for a putative 30-amino acid chloroplast transit peptide and a 433-amino acid mature protein. The mature protein deduced from the Chlamydomonas cDNA sequence has a molecular weight of 45,880, compared to the value of 43,000 reported for purified Chlamydomonas GSAT (d. Jahn et al., 1991, J. Biol. Chem. 266:161-167). The deduced peptide is 74% identical to Synechococcus GSAT, 70% identical to barley GSAT and 66% identical to tobacco GSAT. The putative pyridoxal binding region has the sequence TTMGKVIGG, which differs somewhat from those reported for other aminotransferases. The deduced putative chloroplast transit peptide has recognizable similarity to barley GSAT transit peptide. Southern analysis of genomic DNA from Chlamydomonas strain CC124, using the cDNA as a probe, indicates that GSAT is probably encoded by a single gene.

  7. The role of the complement system in innate immunity.

    PubMed

    Rus, Horea; Cudrici, Cornelia; Niculescu, Florin

    2005-01-01

    Complement is a major component of innate immune system involved in defending against all the foreign pathogens through complement fragments that participate in opsonization, chemotaxis, and activation of leukocytes and through cytolysis by C5b-9 membrane attack complex. Bacterias and viruses have adapted in various ways to escape the complement activation, and they take advantage of the complement system by using the host complement receptors to infect various cells. Complement activation also participates in clearance of apoptotic cells and immune complexes. Moreover, at sublytic dose, C5b-9 was shown to promote cell survival. Recently it was also recognized that complement plays a key role in adaptive immunity by modulating and modifying the T cell responses. All these data suggest that complement activation constitutes a critical link between the innate and acquired immune responses. PMID:16234578

  8. Functional evaluation of paraplegin mutations by a yeast complementation assay.

    PubMed

    Bonn, Florian; Pantakani, Krishna; Shoukier, Moneef; Langer, Thomas; Mannan, Ashraf U

    2010-05-01

    An autosomal recessive form of hereditary spastic paraplegia (AR-HSP) is primarily caused by mutations in the SPG7 gene, which codes for paraplegin, a subunit of the hetero-oligomeric m-AAA protease in mitochondria. In the current study, sequencing of the SPG7 gene in the genomic DNA of 25 unrelated HSP individuals/families led to the identification of two HSP patients with compound heterozygous mutations (p.G349S/p.W583C and p.A510V/p.N739KfsX741) in the coding sequence of the SPG7 gene. We used a yeast complementation assay to evaluate the functional consequence of novel SPG7 sequence variants detected in the HSP patients. We assessed the proteolytic activity of hetero-oligomeric m-AAA proteases composed of paraplegin variant(s) and proteolytically inactive forms of AFG3L2 (AFG3L2(E575Q) or AFG3L2(K354A)) upon expression in m-AAA protease-deficient yeast cells. We demonstrate that the newly identified paraplegin variants perturb the proteolytic function of hetero-oligomeric m-AAA protease. Moreover, commonly occurring silent polymorphisms such as p.T503A and p.R688Q could be distinguished from mutations (p.G349S, p.W583C, p.A510V, and p.N739KfsX741) in our HSP cohort. The yeast complementation assay thus can serve as a reliable system to distinguish a pathogenic mutation from a silent polymorphism for any novel SPG7 sequence variant, which will facilitate the interpretation of genetic data for SPG7. PMID:20186691

  9. Functional anatomy of complement factor H.

    PubMed

    Makou, Elisavet; Herbert, Andrew P; Barlow, Paul N

    2013-06-11

    Factor H (FH) is a soluble regulator of the proteolytic cascade at the core of the evolutionarily ancient vertebrate complement system. Although FH consists of a single chain of similar protein modules, it has a demanding job description. Its chief role is to prevent complement-mediated injury to healthy host cells and tissues. This entails recognition of molecular patterns on host surfaces combined with control of one of nature's most dangerous examples of a positive-feedback loop. In this way, FH modulates, where and when needed, an amplification process that otherwise exponentially escalates the production of the pro-inflammatory, pro-phagocytic, and pro-cytolytic cleavage products of complement proteins C3 and C5. Mutations and single-nucleotide polymorphisms in the FH gene and autoantibodies against FH predispose individuals to diseases, including age-related macular degeneration, dense-deposit disease, and atypical hemolytic uremic syndrome. Moreover, deletions or variations of genes for FH-related proteins also influence the risk of disease. Numerous pathogens hijack FH and use it for self-defense. As reviewed herein, a molecular understanding of FH function is emerging. While its functional oligomeric status remains uncertain, progress has been achieved in characterizing its three-dimensional architecture and, to a lesser extent, its intermodular flexibility. Models are proposed, based on the reconciliation of older data with a wealth of recent evidence, in which a latent circulating form of FH is activated by its principal target, C3b tethered to a self-surface. Such models suggest hypotheses linking sequence variations to pathophysiology, but improved, more quantitative, functional assays and rigorous data analysis are required to test these ideas. PMID:23701234

  10. VISUALIZATION OF MOLECULAR INTERACTIONS BY FLUORESCENCE COMPLEMENTATION

    PubMed Central

    Kerppola, Tom K.

    2008-01-01

    The visualization of protein complexes in living cells enables validation of protein interactions in their normal environment and determination of their subcellular localization. The bimolecular fluorescence complementation (BiFC) assay has been used to visualize interactions among multiple proteins in many cell types and organisms. This assay is based on the association between two fluorescent-protein fragments when they are brought together by an interaction between proteins fused to the fragments. Modified forms of this assay have been used to visualize the competition between alternative interaction partners and the covalent modification of proteins by ubiquitin family peptides. PMID:16625152

  11. Complement sensitivity of Entamoeba histolytica and various nonpathogenic amoeba species.

    PubMed

    Förster, B; Ebert, F; Horstmann, R D

    1994-12-01

    Culture forms of the potentially pathogenic Entamoeba histolytica were compared to those of the nonpathogenic species of E. dispar, E. hartmanni, E. coli, Endolimax nana, and E. moshkovskii regarding the sensitivity to lysis by human complement activated through the alternative pathway. E. dispar was found unique in its complement resistance; all other nonpathogenic isolates resembled E. histolytica in that they were complement sensitive. Thus, a state of complement sensitivity is not a particular property of potentially pathogenic amoebae. PMID:7716404

  12. The Production of Complement Clauses in Children with Language Impairment

    ERIC Educational Resources Information Center

    Steel, Gillian; Rose, Miranda; Eadie, Patricia

    2016-01-01

    Purpose: The purpose of this research was to provide a comprehensive description of complement-clause production in children with language impairment. Complement clauses were examined with respect to types of complement structure produced, verb use, and both semantic and syntactic accuracy. Method: A group of 17 children with language impairment…

  13. False Belief, Complementation Language, and Contextual Bias in Preschoolers

    ERIC Educational Resources Information Center

    Ng, Lisa; Cheung, Him; Xiao, Wen

    2010-01-01

    In the present study, we address two questions concerning the relation between children's false belief and their understanding of complex object complements. The first question is whether the previously demonstrated association between tensed complements and false belief generalizes to infinitival complements (de Villiers & Pyers, 2002). The…

  14. Complement activation induced by rabbit rheumatoid factor.

    PubMed Central

    Meyer, R R; Brown, J C

    1980-01-01

    Rabbit rheumatoid factor produced in animals by hyperimmunized with group C streptococcal vaccine activated guinea pig complement. Anti-streptococcal serum was fractionated by Sephacryl S-200 chromatography into excluded (19S) and included (7S) material and examined for hemolytic activity in a sensitive homologous hemolytic assay system. In the presence of complement, both 19S and 7S antistreptococcal serum fractions induced lysis of bovine (ox) erythrocytes coated with mildly reduced and carboxymethylated rabbit anti-erythrocyte immunoglobulin G. That rabbit rheumatoid factor was responsible for the observed hemolytic activity was substantiated by hemolytic inhibition assays. Significant inhibition of hemolysis was effected when antistreptococcal serum fractions were incubated in the presence of human immunoglobulin G, rabbit immunoglobulin G, and Fc, whereas, no inhibition was detected when the same fractions were tested in the presence of rabbit Fab or F(ab')2 fragments. Deaggregation of inhibitor preparations revealed a preferential reactivity of rheumatoid factor for rabbit immunoglobulin G. In addition to the rheumatoid factor-dependent hemolytic activity observed in humoral preparations, immunoglobulin G-specific antibody-forming cells in spleen and peripheral blood lymphocyte isolates were enumerated by plaque-forming cell assay. PMID:7399707

  15. Complement analysis 2016: Clinical indications, laboratory diagnostics and quality control.

    PubMed

    Prohászka, Zoltán; Nilsson, Bo; Frazer-Abel, Ashley; Kirschfink, Michael

    2016-11-01

    In recent years, complement analysis of body fluids and biopsies, going far beyond C3 and C4, has significantly enhanced our understanding of the disease process. Such expanded complement analysis allows for a more precise differential diagnosis and for critical monitoring of complement-targeted therapy. These changes are a result of the growing understanding of the involvement of complement in a diverse set of disorders. To appreciate the importance of proper complement analysis, it is important to understand the role it plays in disease. Historically, it was the absence of complement as manifested in severe infection that was noted. Since then complement has been connected to a variety of inflammatory disorders, such as autoimmune diseases and hereditary angioedema. While the role of complement in the rejection of renal grafts has been known longer, the significant impact of complement. In certain nephropathies has now led to the reclassification of some rare kidney diseases and an increased role for complement analysis in diagnosis. Even more unexpected is that complement has also been implicated in neural, ophtalmological and dermatological disorders. With this level of involvement in some varied and impactful health issues proper complement testing is clearly important; however, analysis of the complement system varies widely among laboratories. Except for a few proteins, such as C3 and C4, there are neither well-characterized standard preparations nor calibrated assays available. This is especially true for the inter-laboratory variation of tests which assess classical, alternative, or lectin pathway function. In addition, there is a need for the standardization of the measurement of complement activation products that are so critical in determining whether clinically relevant complement activation has occurred in vivo. Finally, autoantibodies to complement proteins (e.g. anti-C1q), C3 and C4 convertases (C3 and C4 nephritic factor) or to regulatory proteins

  16. Infliximab treatment reduces complement activation in patients with rheumatoid arthritis

    PubMed Central

    Familian, A; Voskuyl, A; van Mierlo, G J; Heijst, H; Twisk, J; Dijkmans, B; Hack, C

    2005-01-01

    Background: Tumour necrosis factor (TNF) blocking agents decrease C reactive protein (CRP) levels in rheumatoid arthritis (RA). It has been shown that CRP may contribute to complement activation in RA. Objective: To assess the effect of intravenous infliximab treatment on complement activation, especially that mediated by CRP, in RA. Methods: 35 patients with active RA (28 joint count Disease Activity Score (DAS28) >4.4) were treated with intravenous injections of infliximab (3 mg/kg, at weeks 0, 2, 6, 14, and 22). Clinical response and plasma levels of complement activation products, of CRP and of CRP-complement complexes, which are specific markers for CRP mediated complement activation, were assessed at the indicated time points up to 22 weeks. The relationship between CRP and CRP-complement complexes was analysed by paired t test between two time points and by generalised estimated equation, to test differences of variables over time. Results: At 2 weeks after the first dose, infliximab significantly reduced overall C3 and C4 activation and plasma levels of CRP and CRP-complement complexes were also significantly reduced at this time point. The effects of infliximab on CRP and complement continued throughout the observation period and were more pronounced in patients with a good response to infliximab treatment. Conclusion: Treatment with infliximab decreases plasma levels of CRP and CRP dependent complement activation products and concomitantly may reduce complement activation in RA. Complement activation may be among the effector mechanisms of TNF in RA. PMID:15958758

  17. DNA ELECTROPHORESIS AT SURFACES

    SciTech Connect

    RAFAILOVICH, MIRIAM; SOKOLOV, JONATHAN; GERSAPPE, DILIP

    2003-09-01

    During this year we performed two major projects: I. We developed a detailed theoretical model which complements our experiments on surface DNA electrophoresis. We found that it was possible to enhance the separation of DNA chains by imposing a chemical nanoscale pattern on the surface. This approach utilized the surface interaction effect of the DNA chains with the substrate and is a refinement to our previous method in which DNA chains were separated on homogeneous flat surfaces. By introducing the nano-patterns on the surface, the conformational changes of DNA chains of different lengths can be amplified, which results in the different friction strengths with the substrate surface. Our results also show that, when compared to the DNA electrophoresis performed on homogeneous flat surfaces, nanopatterned surfaces offer a larger window in choosing different surface interactions to achieve separation. II. In collaboration with a large international manufacturer of skin care products we also embarked on a project involving photo toxicity of titanium dioxide nanoparticles, which are a key ingredient in sunscreen and cosmetic lotions. The results clearly implicated the nanoparticles in catalyzing damage to chromosomal DNA. We then used this knowledge to develop a polymer/anti-oxidant coating which prevented the photocatalytic reaction on DNA while still retaining the UV absorptive properties of the nanoparticles. The standard gel electrophoresis was not sufficient in determining the extent of the DNA damage. The conclusions of this study were based predominantly on analysis obtained with the surface electrophoresis method.

  18. The Complement System and Antibody-Mediated Transplant Rejection.

    PubMed

    Stites, Erik; Le Quintrec, Moglie; Thurman, Joshua M

    2015-12-15

    Complement activation is an important cause of tissue injury in patients with Ab-mediated rejection (AMR) of transplanted organs. Complement activation triggers a strong inflammatory response, and it also generates tissue-bound and soluble fragments that are clinically useful markers of inflammation. The detection of complement proteins deposited within transplanted tissues has become an indispensible biomarker of AMR, and several assays have recently been developed to measure complement activation by Abs reactive to specific donor HLA expressed within the transplant. Complement inhibitors have entered clinical use and have shown efficacy for the treatment of AMR. New methods of detecting complement activation within transplanted organs will improve our ability to diagnose and monitor AMR, and they will also help guide the use of complement inhibitory drugs. PMID:26637661

  19. The minimal gene complement of mycoplasma genitalium

    SciTech Connect

    Fraser, C.M.; Gocayne, J.D.; White, O.

    1995-10-20

    The complete nucleotide sequence (580,070 base pairs) of the Mycoplasma genitalium genome, the smallest known genome of any free-living organism, has been determined by whole-genome random sequencing and assembly. A total of only 470 predicted coding regions were identified that include genes required for DNA replication, transcription and translation, DNA repair, cellular transport, and energy metabolism. Comparison of this genome to that of Haemophilus influenzae suggests that differences in genome content are reflected as profound differences in physiology and metabolic capacity between these two organisms. 43 refs., 1 fig., 2 tabs.

  20. Complement-activating ability of leucocytes from patients with complement factor I deficiency.

    PubMed Central

    Marquart, H V; Rasmussen, J M; Leslie, R G

    1997-01-01

    Previous studies from this laboratory have shown that normal peripheral blood B cells are capable of activating complement via the alternative pathway (AP), that the activation is associated with complement receptor type 2 (CR2) expression, and that erythrocytes at normal blood levels partially inhibit the activation. The purpose of the present study was to investigate whether factor I (FI) deficiency, which leads to continued formation of the AP convertase (C3bBb) resulting in the consumption of factor B and C3 and large scale generation of C3b fragments, affects the phenotype and/or function of the patients' B cells. Using flow cytometry, peripheral blood leucocytes (PBL) from two FI-deficient patients were investigated for expression of complement receptors and complement regulatory proteins, in vivo-deposited C3 fragments and in vitro complement-activating ability. CR1 levels on B cells were significantly lower in FI-deficient patients than in normal individuals, whereas CR2 levels were found to be reduced, although not to a significant extent. CR1 levels on monocytes and polymorphonuclear leucocytes (PMN) were found to be normal or slightly raised. All leucocyte subpopulations were found to be covered in vivo with C3b fragments. AP activation on B cells from FI-deficient patients in homologous serum was significantly reduced compared with that for normal individuals, whereas no in vitro activation was seen in autologous serum. In addition, the in vivo-bound C3b fragments were degraded to C3d,g when the patients' PBL were incubated in homologous serum containing EDTA. Finally, the patients, erythrocytes failed to exert any inhibition on AP activation in homologous serum. PMID:9301541

  1. Mitochondria and the Lectin Pathway of Complement*

    PubMed Central

    Brinkmann, Christel R.; Jensen, Lisbeth; Dagnæs-Hansen, Frederik; Holm, Ida E.; Endo, Yuichi; Fujita, Teizo; Thiel, Steffen; Jensenius, Jens C.; Degn, Søren E.

    2013-01-01

    Mitochondria, the powerhouses of our cells, are remnants of a eubacterial endosymbiont. Notwithstanding the evolutionary time that has passed since the initial endosymbiotic event, mitochondria have retained many hallmarks of their eubacterial origin. Recent studies have indicated that during perturbations of normal homeostasis, such as following acute trauma leading to massive necrosis and release of mitochondria, the immune system might mistake symbiont for enemy and initiate an inappropriate immune response. The innate immune system is the first line of defense against invading microbial pathogens, and as such is the primary suspect in the recognition of mitochondria-derived danger-associated molecular patterns and initiation of an aberrant response. Conversely, innate immune mechanisms are also central to noninflammatory clearance of innocuous agents. Here we investigated the role of a central humoral component of innate immunity, the lectin pathway of complement, in recognition of mitochondria in vitro and in vivo. We found that the soluble pattern recognition molecules, mannan-binding lectin (MBL), L-ficolin, and M-ficolin, were able to recognize mitochondria. Furthermore, MBL in complex with MBL-associated serine protease 2 (MASP-2) was able to activate the lectin pathway and deposit C4 onto mitochondria, suggesting that these molecules are involved either in homeostatic clearance of mitochondria or in induction of untoward inflammatory reactions. We found that following mitochondrial challenge, C3 was consumed in vivo in the absence of overt inflammation, indicating a potential role of complement in noninflammatory clearance of mitochondria. Thus, we report here the first indication of involvement of the lectin pathway in mitochondrial immune handling. PMID:23378531

  2. Molecular basis of complement C3 deficiency in guinea pigs.

    PubMed Central

    Auerbach, H S; Burger, R; Dodds, A; Colten, H R

    1990-01-01

    In experiments to ascertain the biochemical basis of a genetically determined deficiency of the third component of complement (C3) in guinea pigs, we found that C3-deficient liver and peritoneal macrophages contain C3 messenger RNA of normal size (approximately 5 kb) and amounts, that this mRNA programs synthesis of pro-C3 in oocytes primed with liver RNA and in primary macrophage cultures. In each instance, heterodimeric native C3 protein was secreted with normal kinetics but the C3 protein product of the deficient cells failed to undergo autolytic cleavage and was unusually susceptible to proteolysis. These data and a selective failure of C3 in plasma of deficient animals to incorporate [14C]methylamine suggested either a mutation in primary structure of the C3 protein or a selective defect in co- or postsynthetic processing affecting the thiolester bridge, a structure important for C3 function. A mutation in the primary structure of C3 was ruled out by comparison of direct sequence analysis of C3 cDNA generated from two C3 deficient and two C3 sufficient guinea pig liver libraries. Three base pair differences, none resulting in derived amino acid sequence differences were identified. Finally, restriction fragment length polymorphisms were identified in the C3 gene that are independent of the deficiency phenotype. This marker of the C3 gene permits testing of these hypotheses using molecular biological and classical genetic methods. Images PMID:1973176

  3. Human PEX1 is mutated in complementation group 1 of the peroxisome biogenesis disorders.

    PubMed

    Portsteffen, H; Beyer, A; Becker, E; Epplen, C; Pawlak, A; Kunau, W H; Dodt, G

    1997-12-01

    Human peroxisome biogenesis disorders (PBDs) are a group of genetically heterogeneous autosomal-recessive disease caused by mutations in PEX genes that encode peroxins, proteins required for peroxisome biogenesis. These lethal diseases include Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD) and infantile Refsum's disease (IRD), three phenotypes now thought to represent a continuum of clinical features that are most severe in ZS, milder in NALD and least severe in IRD2. At least eleven PBD complementation groups have been identified by somatic-cell hybridization analysis compared to the eighteen PEX complementation groups that have been found in yeast. We have cloned the human PEX1 gene encoding a 147-kD member of the AAA protein family (ATPases associated with diverse cellular activities), which is the putative orthologue of Saccharomyces cerevisiae Pex1p (ScPex1p). Human PEX1 has been identified by computer-based 'homology probing' using the ScPex1p sequence to screen databases of expressed sequence tags (dbEST) for human cDNA clones. Expression of PEX1 rescued the cells from the biogenesis defect in human fibroblasts of complementation group 1 (CG1), the largest PBD complementation group. We show that PEX1 is mutated in CG1 patients. PMID:9398848

  4. Bovine viral diarrhea virus structural protein E2 as a complement regulatory protein.

    PubMed

    Ostachuk, Agustín

    2016-07-01

    Bovine viral diarrhea virus (BVDV) is a member of the genus Pestivirus, family Flaviviridae, and is one of the most widely distributed viruses in cattle worldwide. Approximately 60 % of cattle in endemic areas without control measures are infected with BVDV during their lifetime. This wide prevalence of BVDV in cattle populations results in significant economic losses. BVDV is capable of establishing persistent infections in its host due to its ability to infect fetuses, causing immune tolerance. However, this cannot explain how the virus evades the innate immune system. The objective of the present work was to test the potential activity of E2 as a complement regulatory protein. E2 glycoprotein, produced both in soluble and transmembrane forms in stable CHO-K1 cell lines, was able to reduce complement-mediated cell lysis up to 40 % and complement-mediated DNA fragmentation by 50 %, in comparison with cell lines not expressing the glycoprotein. This work provides the first evidence of E2 as a complement regulatory protein and, thus, the finding of a mechanism of immune evasion by BVDV. Furthermore, it is postulated that E2 acts as a self-associated molecular pattern (SAMP), enabling the virus to avoid being targeted by the immune system and to be recognized as self. PMID:27038454

  5. Complement inhibition: a promising concept for cancer treatment

    PubMed Central

    Pio, Ruben; Ajona, Daniel; Lambris, John D.

    2013-01-01

    For decades, complement has been recognized as an effector arm of the immune system that contributes to the destruction of tumor cells. In fact, many therapeutic strategies have been proposed that are based on the intensification of complement-mediated responses against tumors. However, recent studies have challenged this paradigm by demonstrating a tumor-promoting role for complement. Cancer cells seem to be able to establish a convenient balance between complement activation and inhibition, taking advantage of complement initiation without suffering its deleterious effects. Complement activation may support chronic inflammation, promote an immunosuppressive microenvironment, induce angiogenesis, and activate cancer-related signaling pathways. In this context, inhibition of complement activation would be a therapeutic option for treating cancer. This concept is relatively novel and deserves closer attention. In this paper, we will summarize the mechanisms of complement activation on cancer cells, the cancer-promoting effect of complement initiation, and the rationale behind the use of complement inhibition as a therapeutic strategy against cancer. PMID:23706991

  6. A vital role for complement in heart disease.

    PubMed

    Lappegård, Knut T; Garred, Peter; Jonasson, Lena; Espevik, Terje; Aukrust, Pål; Yndestad, Arne; Mollnes, Tom E; Hovland, Anders

    2014-10-01

    Heart diseases are common and significant contributors to worldwide mortality and morbidity. During recent years complement mediated inflammation has been shown to be an important player in a variety of heart diseases. Despite some negative results from clinical trials using complement inhibitors, emerging evidence points to an association between the complement system and heart diseases. Thus, complement seems to be important in coronary heart disease as well as in heart failure, where several studies underscore the prognostic importance of complement activation. Furthermore, patients with atrial fibrillation often share risk factors both with coronary heart disease and heart failure, and there is some evidence implicating complement activation in atrial fibrillation. Moreover, Chagas heart disease, a protozoal infection, is an important cause of heart failure in Latin America, and the complement system is crucial for the protozoa-host interaction. Thus, complement activation appears to be involved in the pathophysiology of a diverse range of cardiac conditions. Determination of the exact role of complement in the various heart diseases will hopefully help to identify patients that might benefit from therapeutic complement intervention. PMID:25037633

  7. The Expression Profile of Complement Components in Podocytes.

    PubMed

    Li, Xuejuan; Ding, Fangrui; Zhang, Xiaoyan; Li, Baihong; Ding, Jie

    2016-01-01

    Podocytes are critical for maintaining the glomerular filtration barrier and are injured in many renal diseases, especially proteinuric kidney diseases. Recently, reports suggested that podocytes are among the renal cells that synthesize complement components that mediate glomerular diseases. Nevertheless, the profile and extent of complement component expression in podocytes remain unclear. This study examined the expression profile of complement in podocytes under physiological conditions and in abnormal podocytes induced by multiple stimuli. In total, 23/32 complement component components were detected in podocyte by conventional RT-PCR. Both primary cultured podocytes and immortalized podocytes expressed the complement factors C1q, C1r, C2, C3, C7, MASP, CFI, DAF, CD59, C4bp, CD46, Protein S, CR2, C1qR, C3aR, C5aR, and Crry (17/32), whereas C4, CFB, CFD, C5, C6, C8, C9, MBL1, and MBL2 (9/32) complement factors were not expressed. C3, Crry, and C1q-binding protein were detected by tandem mass spectrometry. Podocyte complement gene expression was affected by several factors (puromycin aminonucleoside (PAN), angiotensin II (Ang II), interleukin-6 (IL-6), and transforming growth factor-β (TGF-β)). Representative complement components were detected using fluorescence confocal microscopy. In conclusion, primary podocytes express various complement components at the mRNA and protein levels. The complement gene expressions were affected by several podocyte injury factors. PMID:27043537

  8. The Expression Profile of Complement Components in Podocytes

    PubMed Central

    Li, Xuejuan; Ding, Fangrui; Zhang, Xiaoyan; Li, Baihong; Ding, Jie

    2016-01-01

    Podocytes are critical for maintaining the glomerular filtration barrier and are injured in many renal diseases, especially proteinuric kidney diseases. Recently, reports suggested that podocytes are among the renal cells that synthesize complement components that mediate glomerular diseases. Nevertheless, the profile and extent of complement component expression in podocytes remain unclear. This study examined the expression profile of complement in podocytes under physiological conditions and in abnormal podocytes induced by multiple stimuli. In total, 23/32 complement component components were detected in podocyte by conventional RT-PCR. Both primary cultured podocytes and immortalized podocytes expressed the complement factors C1q, C1r, C2, C3, C7, MASP, CFI, DAF, CD59, C4bp, CD46, Protein S, CR2, C1qR, C3aR, C5aR, and Crry (17/32), whereas C4, CFB, CFD, C5, C6, C8, C9, MBL1, and MBL2 (9/32) complement factors were not expressed. C3, Crry, and C1q-binding protein were detected by tandem mass spectrometry. Podocyte complement gene expression was affected by several factors (puromycin aminonucleoside (PAN), angiotensin II (Ang II), interleukin-6 (IL-6), and transforming growth factor-β (TGF-β)). Representative complement components were detected using fluorescence confocal microscopy. In conclusion, primary podocytes express various complement components at the mRNA and protein levels. The complement gene expressions were affected by several podocyte injury factors. PMID:27043537

  9. Genetics and complementation of Haemophilus influenzae mutants deficient in adenosine 5'-triphosphate-dependent nuclease.

    PubMed Central

    Kooistra, J; Small, G D; Setlow, J K; Shapanka, R

    1976-01-01

    Eight different mutations in Haemophilus influenzae leading to deficiency in adenosine 5'-triphosphate (ATP)-dependent nuclease have been investigated in strains in which the mutations of the originally mutagenized strains have been transferred into the wild type. Sensitivity to mitomycin C and deoxycholate and complementation between extracts and deoxyribonucleic acid (DNA)-dependent ATPase activity have been measured. Genetic crosses have provided information on the relative position of the mutations on the genome. There are three complementation groups, corresponding to three genetic groups. The strains most sensitive to mitomycin and deoxycholate, derived from mutants originally selected on the basis of sensitivity to mitomycin C or methyl methanesulfonate, are in one group. Apparently all these sensitive strains lack DNA-dependent ATPase activity, as does a strain intermediate in sensitivity to deoxycholate, which is the sole representative of another group. There are four strains that are relatively resistant to deoxycholate and mitomycin C, and all of these contain the ATPase activity. Three of these are in the same genetic and complementation group, whereas the other incongruously belongs in the same group as the sensitive strains. It is postulated that there are three cistrons in H. influenzae that code for the three known subunits of the ATP-dependent nuclease. PMID:177397

  10. Isolation and complementation of mutants of Anabaena sp. strain PCC 7120 unable to grow aerobically on dinitrogen

    SciTech Connect

    Wolk, C.P.; Cai, Y.; Cardemil, L.; Flores, E.; Hohn, B.; Murry, M.; Schmetterer, G.; Schrautemeier, B.; Wilson, R.

    1988-03-01

    Mutants of Anabaena sp. strain PCC 7120 unable to grow aerobically on dinitrogen were isolated by mutagenesis with UV irradiation, followed by a period of incubation in yellow light and then by penicillin enrichment. A cosmid vector, pRL25C, containing replicons functional in Escherichia coli and in Anabaena species was constructed. DNA from wild-type Anabaena sp. strain PCC 7120 was partially digested with Sau3AI, and size-fractionated fragments about 40 kilobases (kb) in length were ligated into the phosphatase-treated unique BamHI site of pRL25C. A library of 1054 cosmid clones was generated in E. coli DH1 bearing helper plasmid pDS4101. A derivative of conjugative plasmid RP-4 was transferred to this library by conjugation, and the library was replicated to lawns of mutant Anabaena strains with defects in the polysaccharide layer of the envelopes of the heterocysts. Mutant EF116 was complemented by five cosmids, three of which were subjected to detailed restriction mapping; a 2.8-kb fragment of DNA derived from one of the cosmids was found to complement EF116. Mutant EF113 was complemented by a single cosmid, which was also restriction mapped, and was shown to be complemented by a 4.8-kb fragment of DNA derived from this cosmid.

  11. The role of complement in inflammation and adaptive immunity.

    PubMed

    Barrington, R; Zhang, M; Fischer, M; Carroll, M C

    2001-04-01

    Major advances in our understanding of the immunobiology of complement were made within the past 5 years primarily due to the development of gene-targeting technology. New strains of mice bearing specific deficiencies in serum complement proteins or their receptors were developed using this approach. Characterization of these mice has provided new and exciting insights into the biology of the complement system. In this review, we discuss recent results on two important aspects of the complement system, i) host protection and inflammation, and ii) regulation of B lymphocytes of adaptive immunity. While these two roles appear distinct, they are linked. We discuss how natural antibody and classical pathway complement work together in host protection against bacterial infection on the one hand but, on the other, they co-operate to induce inflammation as observed in reperfusion injury. Significantly, the lymphocytes that produce natural antibody, the B-1 lymphocytes, are regulated in part by the complement system. PMID:11414363

  12. Complement, a target for therapy in inflammatory and degenerative diseases.

    PubMed

    Morgan, B Paul; Harris, Claire L

    2015-12-01

    The complement system is a key innate immune defence against infection and an important driver of inflammation; however, these very properties can also cause harm. Inappropriate or uncontrolled activation of complement can cause local and/or systemic inflammation, tissue damage and disease. Complement provides numerous options for drug development as it is a proteolytic cascade that involves nine specific proteases, unique multimolecular activation and lytic complexes, an arsenal of natural inhibitors, and numerous receptors that bind to activation fragments. Drug design is facilitated by the increasingly detailed structural understanding of the molecules involved in the complement system. Only two anti-complement drugs are currently on the market, but many more are being developed for diseases that include infectious, inflammatory, degenerative, traumatic and neoplastic disorders. In this Review, we describe the history, current landscape and future directions for anti-complement therapies. PMID:26493766

  13. Humoral response to herpes simplex virus is complement-dependent

    PubMed Central

    Da Costa, Xavier J.; Brockman, Mark A.; Alicot, Elisabeth; Ma, Minghe; Fischer, Michael B.; Zhou, Xioaning; Knipe, David M.; Carroll, Michael C.

    1999-01-01

    The complement system represents a cascade of serum proteins, which provide a major effector function in innate immunity. Recent studies have revealed that complement links innate and adaptive immunity via complement receptors CD21/CD35 in that it enhances the B cell memory response to noninfectious protein antigens introduced i.v. To examine the importance of complement for immune responses to virus infection in a peripheral tissue, we compared the B cell memory response of mice deficient in complement C3, C4, or CD21/CD35 with wild-type controls. We found that the deficient mice failed to generate a normal memory response, which is characterized by a reduction in IgG antibody and germinal centers. Thus, complement is important not only in the effector function of innate immunity but also in the stimulation of memory B cell responses to viral-infected cell antigens in both blood and peripheral tissues. PMID:10535987

  14. Complement-mediated antiinflammatory effect of bisbenzylisoquinoline alkaloid fangchinoline.

    PubMed

    Hristova, M; Istatkova, R

    1999-11-01

    Complement-mediated mode of action of bisbenzylisoquinoline alkaloid fangchinoline was investigated in vivo and in vitro. The application of fangchinoline intraperitoneally (i.p.) to complement normal mice, strain ICR, inhibited the complement activity in serum and peritoneal exudate. The substance activated serum complement of C5-deficient DBA/2 mice. Fangchinoline was able to provoke local inflammatory reaction in both strains after subcutaneous (s.c.) injection. The alkaloid suppressed paw swelling induced by live Candida albicans in ICR and DBA/2 mice. Its effect depended on the dose and time of injection prior to inflammatory reaction. The in vitro experiments proved the interference of fangchinoline action with post-C5 reactions. The substance augmented C5-convertase formation and functional activity. These results are in correspondence with our previous investigations, proving the complement-mediated action of fangchinoline. The antiinflammatory effect could be a consequence of the caused complement exhaustion. PMID:11962544

  15. Complement-Mediated Regulation of Metabolism and Basic Cellular Processes.

    PubMed

    Hess, Christoph; Kemper, Claudia

    2016-08-16

    Complement is well appreciated as a critical arm of innate immunity. It is required for the removal of invading pathogens and works by directly destroying them through the activation of innate and adaptive immune cells. However, complement activation and function is not confined to the extracellular space but also occurs within cells. Recent work indicates that complement activation regulates key metabolic pathways and thus can impact fundamental cellular processes, such as survival, proliferation, and autophagy. Newly identified functions of complement include a key role in shaping metabolic reprogramming, which underlies T cell effector differentiation, and a role as a nexus for interactions with other effector systems, in particular the inflammasome and Notch transcription-factor networks. This review focuses on the contributions of complement to basic processes of the cell, in particular the integration of complement with cellular metabolism and the potential implications in infection and other disease settings. PMID:27533012

  16. Complement factor H–related hybrid protein deregulates complement in dense deposit disease

    PubMed Central

    Chen, Qian; Wiesener, Michael; Eberhardt, Hannes U.; Hartmann, Andrea; Uzonyi, Barbara; Kirschfink, Michael; Amann, Kerstin; Buettner, Maike; Goodship, Tim; Hugo, Christian; Skerka, Christine; Zipfel, Peter F.

    2013-01-01

    The renal disorder C3 glomerulopathy with dense deposit disease (C3G-DDD) pattern results from complement dysfunction and primarily affects children and young adults. There is no effective treatment, and patients often progress to end-stage renal failure. A small fraction of C3G-DDD cases linked to factor H or C3 gene mutations as well as autoantibodies have been reported. Here, we examined an index family with 2 patients with C3G-DDD and identified a chromosomal deletion in the complement factor H–related (CFHR) gene cluster. This deletion resulted in expression of a hybrid CFHR2-CFHR5 plasma protein. The recombinant hybrid protein stabilized the C3 convertase and reduced factor H–mediated convertase decay. One patient was refractory to plasma replacement and exchange therapy, as evidenced by the hybrid protein quickly returning to pretreatment plasma levels. Subsequently, complement inhibitors were tested on serum from the patient for their ability to block activity of CFHR2-CFHR5. Soluble CR1 restored defective C3 convertase regulation; however, neither eculizumab nor tagged compstatin had any effect. Our findings provide insight into the importance of CFHR proteins for C3 convertase regulation and identify a genetic variation in the CFHR gene cluster that promotes C3G-DDD. Monitoring copy number and sequence variations in the CFHR gene cluster in C3G-DDD and kidney patients with C3G-DDD variations will help guide treatment strategies. PMID:24334459

  17. Expression of complement 3 and complement 5 in newt limb and lens regeneration.

    PubMed

    Kimura, Yuko; Madhavan, Mayur; Call, Mindy K; Santiago, William; Tsonis, Panagiotis A; Lambris, John D; Del Rio-Tsonis, Katia

    2003-03-01

    Some urodele amphibians possess the capacity to regenerate their body parts, including the limbs and the lens of the eye. The molecular pathway(s) involved in urodele regeneration are largely unknown. We have previously suggested that complement may participate in limb regeneration in axolotls. To further define its role in the regenerative process, we have examined the pattern of distribution and spatiotemporal expression of two key components, C3 and C5, during limb and lens regeneration in the newt Notophthalmus viridescens. First, we have cloned newt cDNAs encoding C3 and C5 and have generated Abs specifically recognizing these molecules. Using these newt-specific probes, we have found by in situ hybridization and immunohistochemical analysis that these molecules are expressed during both limb and lens regeneration, but not in the normal limb and lens. The C3 and C5 proteins were expressed in a complementary fashion during limb regeneration, with C3 being expressed mainly in the blastema and C5 exclusively in the wound epithelium. Similarly, during the process of lens regeneration, C3 was detected in the iris and cornea, while C5 was present in the regenerating lens vesicle as well as the cornea. The distinct expression profile of complement proteins in regenerative tissues of the urodele lens and limb supports a nonimmunologic function of complement in tissue regeneration and constitutes the first systematic effort to dissect its involvement in regenerative processes of lower vertebrate species. PMID:12594255

  18. Vitronectin-binding staphylococci enhance surface-associated complement activation.

    PubMed Central

    Lundberg, F; Lea, T; Ljungh, A

    1997-01-01

    Coagulase-negative staphylococci are well recognized in medical device-associated infections. Complement activation is known to occur at the biomaterial surface, resulting in unspecific inflammation around the biomaterial. The human serum protein vitronectin (Vn), a potent inhibitor of complement activation by formation of an inactive terminal complement complex, adsorbs to biomaterial surfaces in contact with blood. In this report, we discuss the possibility that surface-immobilized Vn inhibits complement activation and the effect of Vn-binding staphylococci on complement activation on surfaces precoated with Vn. The extent of complement activation was measured with a rabbit anti-human C3c antibody and a mouse anti-human C9 antibody, raised against the neoepitope of C9. Our data show that Vn immobilized on a biomaterial surface retains its ability to inhibit complement activation. The additive complement activation-inhibitory effect of Vn on a heparinized surface is very small. In the presence of Vn-binding strain, Staphylococcus hemolyticus SM131, complement activation on a surface precoated with Vn occurred as it did in the absence of Vn precoating. For S. epidermidis 3380, which does not express binding of Vn, complement activation on a Vn-precoated surface was significantly decreased. The results could be repeated on heparinized surfaces. These data suggest that Vn adsorbed to a biomaterial surface may serve to protect against surface-associated complement activation. Furthermore, Vn-binding staphylococcal cells may enhance surface-associated complement activation by blocking the inhibitory effect of preadsorbed Vn. PMID:9038294

  19. Initiation and Regulation of Complement during Hemolytic Transfusion Reactions

    PubMed Central

    Stowell, Sean R.; Winkler, Anne M.; Maier, Cheryl L.; Arthur, C. Maridith; Smith, Nicole H.; Girard-Pierce, Kathryn R.; Cummings, Richard D.; Zimring, James C.; Hendrickson, Jeanne E.

    2012-01-01

    Hemolytic transfusion reactions represent one of the most common causes of transfusion-related mortality. Although many factors influence hemolytic transfusion reactions, complement activation represents one of the most common features associated with fatality. In this paper we will focus on the role of complement in initiating and regulating hemolytic transfusion reactions and will discuss potential strategies aimed at mitigating or favorably modulating complement during incompatible red blood cell transfusions. PMID:23118779

  20. Molecular genetics of the human MHC complement gene cluster.

    PubMed

    Yu, C Y

    1998-01-01

    The human major histocompatibility complex (MHC) complement gene cluster (MCGC) is a highly variable region that is characterized by polymorphisms, variations in gene size and gene number, and associations with diseases. Deficiencies in complement C2 are either due to abolition of C2 protein synthesis by mini-deletions that caused frameshift mutations, or blocked secretion of the C2 protein by single amino acid substitutions. One, two or three C4 genes may be present in a human MCGC haplotype and these genes may code for C4A, C4B, or both. Deficiencies of C4A or C4B proteins are attributed to the expression of identical C4 isotypes or allotypes from the C4 loci, the absence or deletion of a C4 gene, 2-bp insertion at exon 29 or 1-bp deletion at exon 20 that caused frameshift mutations. The C4 genes are either 21 or 14.6 kb in size due to the presence of endogenous retrovirus HERV-K(C4) in the intron 9 of long C4 genes. A deletion or duplication of a C4 gene is always accompanied by its neighboring genes, RP at the 5' region, and CYP21 and TNX at the 3' region. These four genes form a genetic unit termed the RCCX module. In an RCCX bimodular structure, the pseudogene CYP21A, and partially duplicated gene segments TNXA and RP2 are present between the two C4 loci. The RCCX modular variations in gene number and gene size contributed to unequal crossovers and exchanges of polymorphic sequences/mutations, resulting in the homogenization of C4 polymorphisms and acquisitions of deleterious mutations in RP1, C4A, C4B, CYP21B and TNXB genes. RD, SKI2W, DOM3Z and RP1 are the four novel genes found between Bf and C4. RD and Ski2w proteins may be related to RNA splicing, RNA turnover and regulation of translation. The functions of Dom3z and RP1 are being investigated. The complete genomic DNA sequence between C2 and TNX is now available. This should facilitate a complete documentation of polymorphisms, mutations and disease associations for the MCGC. PMID:10072631

  1. Complement binding to Leishmania donovani promastigotes (LD)

    SciTech Connect

    Puentes, S.M.; Bates, P.A.; Dwyer, D.M.; Joiner, K.A.

    1986-03-01

    To study the binding and processing of C3 on LD, parasites in various phases of growth were incubated in human serum deficient in complement component 8 containing /sup 125/I-C3. Uptake of /sup 125/I-C3 is rapid, peaking at 1.7-2.1 x 10/sup 6/ C3 molecules bound per parasite at 15 minutes for all growth phases, and decreases thereafter with continued incubation. One half of total C3 bound is spontaneously released by 90 minutes of incubation with all LD phases and occurs at a similar rate for LD washed free of serum and incubated at 37/sup 0/ C in buffer. As assessed by SDS-PAGE autoradiography, C3 on the surface of LD is present as C3b (36 to 50%) and iC3b (50 to 65%), linked covalently via a bond resistant to hydroxylamine treatment, presumably an amide linkage. Immunoblot analysis of purified membranes from serum-incubated LD, using rabbit antibody to C3 and LD surface constituents, strongly suggests that a major C3 acceptor is the LD acid phosphatase (AP). These results, in conjunction with recent studies, suggest a previously unrecognized role of AP as a C3 acceptor and, thus, as a molecule potentially involved in parasite binding and uptake.

  2. Altmetrics - a complement to conventional metrics.

    PubMed

    Melero, Remedios

    2015-01-01

    Emerging metrics based on article-level does not exclude traditional metrics based on citations to the journal, but complements them. Both can be employed in conjunction to offer a richer picture of an article use from immediate to long terms. Article-level metrics (ALM) is the result of the aggregation of different data sources and the collection of content from multiple social network services. Sources used for the aggregation can be broken down into five categories: usage, captures, mentions, social media and citations. Data sources depend on the tool, but they include classic metrics indicators based on citations, academic social networks (Mendeley, CiteULike, Delicious) and social media (Facebook, Twitter, blogs, or Youtube, among others). Altmetrics is not synonymous with alternative metrics. Altmetrics are normally early available and allow to assess the social impact of scholarly outputs, almost at the real time. This paper overviews briefly the meaning of altmetrics and describes some of the existing tools used to apply this new metrics: Public Library of Science--Article-Level Metrics, Altmetric, Impactstory and Plum. PMID:26110028

  3. Altmetrics – a complement to conventional metrics

    PubMed Central

    Melero, Remedios

    2015-01-01

    Emerging metrics based on article-level does not exclude traditional metrics based on citations to the journal, but complements them. Both can be employed in conjunction to offer a richer picture of an article use from immediate to long terms. Article-level metrics (ALM) is the result of the aggregation of different data sources and the collection of content from multiple social network services. Sources used for the aggregation can be broken down into five categories: usage, captures, mentions, social media and citations. Data sources depend on the tool, but they include classic metrics indicators based on citations, academic social networks (Mendeley, CiteULike, Delicious) and social media (Facebook, Twitter, blogs, or Youtube, among others). Altmetrics is not synonymous with alternative metrics. Altmetrics are normally early available and allow to assess the social impact of scholarly outputs, almost at the real time. This paper overviews briefly the meaning of altmetrics and describes some of the existing tools used to apply this new metrics: Public Library of Science - Article-Level Metrics, Altmetric, Impactstory and Plum. PMID:26110028

  4. 21 CFR 866.5240 - Complement components immunological test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... tissues. Complement is a group of serum proteins which destroy infectious agents. Measurements of these proteins aids in the diagnosis of immunologic disorders, especially those associated with deficiencies...

  5. A dual role for complement in allergic asthma.

    PubMed

    Köhl, Jörg; Wills-Karp, Marsha

    2007-06-01

    Complement is an ancient danger-sensor system of innate immunity, providing first-line defence against pathogens. Concordant with its pro-inflammatory properties, complement contributes to airway inflammation, hyperresponsiveness and mucus production during the effector phase of allergic asthma. In contrast to these pro-allergic properties, complement can also protect from the development of the maladaptive Th2-biased immune response that drives airway inflammation and hyperreactivity in allergic asthma. As such, selective targeting of pro-allergic complement pathways appears an attractive therapeutic option in allergic asthma. PMID:17475559

  6. Complement system in dermatological diseases - fire under the skin.

    PubMed

    Panelius, Jaana; Meri, Seppo

    2015-01-01

    The complement system plays a key role in several dermatological diseases. Overactivation, deficiency, or abnormality of the control proteins are often related to a skin disease. Autoimmune mechanisms with autoantibodies and a cytotoxic effect of the complement membrane attack complex on epidermal or vascular cells can cause direct tissue damage and inflammation, e.g., in systemic lupus erythematosus (SLE), phospholipid antibody syndrome, and bullous skin diseases like pemphigoid. By evading complement attack, some microbes like Borrelia spirochetes and staphylococci can persist in the skin and cause prolonged symptoms. In this review, we present the most important skin diseases connected to abnormalities in the function of the complement system. Drugs having an effect on the complement system are also briefly described. On one hand, drugs with free hydroxyl on amino groups (e.g., hydralazine, procainamide) could interact with C4A, C4B, or C3 and cause an SLE-like disease. On the other hand, progress in studies on complement has led to novel anti-complement drugs (recombinant C1-inhibitor and anti-C5 antibody, eculizumab) that could alleviate symptoms in diseases associated with excessive complement activation. The main theme of the manuscript is to show how relevant the complement system is as an immune effector system in contributing to tissue injury and inflammation in a broad range of skin disorders. PMID:25688346

  7. Complement System in Dermatological Diseases – Fire Under the Skin

    PubMed Central

    Panelius, Jaana; Meri, Seppo

    2015-01-01

    The complement system plays a key role in several dermatological diseases. Overactivation, deficiency, or abnormality of the control proteins are often related to a skin disease. Autoimmune mechanisms with autoantibodies and a cytotoxic effect of the complement membrane attack complex on epidermal or vascular cells can cause direct tissue damage and inflammation, e.g., in systemic lupus erythematosus (SLE), phospholipid antibody syndrome, and bullous skin diseases like pemphigoid. By evading complement attack, some microbes like Borrelia spirochetes and staphylococci can persist in the skin and cause prolonged symptoms. In this review, we present the most important skin diseases connected to abnormalities in the function of the complement system. Drugs having an effect on the complement system are also briefly described. On one hand, drugs with free hydroxyl on amino groups (e.g., hydralazine, procainamide) could interact with C4A, C4B, or C3 and cause an SLE-like disease. On the other hand, progress in studies on complement has led to novel anti-complement drugs (recombinant C1-inhibitor and anti-C5 antibody, eculizumab) that could alleviate symptoms in diseases associated with excessive complement activation. The main theme of the manuscript is to show how relevant the complement system is as an immune effector system in contributing to tissue injury and inflammation in a broad range of skin disorders. PMID:25688346

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

    NASA Astrophysics Data System (ADS)

    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.

  9. Coordinate expression of Escherichia coli dnaA and dnaN genes.

    PubMed

    Sako, T; Sakakibara, Y

    1980-01-01

    The defects of temperature-sensitive dnaA and dnaN mutants of Escherichia coli are complemented by a recombinant lambda phage, which carries the bacterial DNA segment composed of two EcoRI segments of 1.0 and 3.3 kilobases. Derivatives of the phage, which have an insertion segment of Tn3 in the dnaA gene, are much less active in expressing the dnaN gene function than the parent phage. The dnaN gene activity was determined as the efficiency of superinfecting phage to suppress loss of the viability of lambda lysogenic dnaN59 cells at the non-permissive temperature. Deletions that include the end of the dnaA gene distal to the dnaN gene also reduce the expression of the dnaN gene function. Deletion and insertion in the dnaN gene do not affect the expression of the dnaA gene function. The expression of the dnaN gene function by the dnaA- dnaN+ phages remains weak upon simultaneous infection with dnaA+ dnaN- phages. Thus the insertion and deletion of the dnaA gene influence in cis the expresion of the dnaN gene. We propose that the dnaA and dnaN genes constitute an operon, where the former is upstream to the latter. PMID:6449652

  10. Minor Role of Plasminogen in Complement Activation on Cell Surfaces

    PubMed Central

    Hyvärinen, Satu; Jokiranta, T. Sakari

    2015-01-01

    Atypical hemolytic uremic syndrome (aHUS) is a rare, but severe thrombotic microangiopathy. In roughly two thirds of the patients, mutations in complement genes lead to uncontrolled activation of the complement system against self cells. Recently, aHUS patients were described with deficiency of the fibrinolytic protein plasminogen. This zymogen and its protease form plasmin have both been shown to interact with complement proteins in the fluid phase. In this work we studied the potential of plasminogen to restrict complement propagation. In hemolytic assays, plasminogen inhibited complement activation, but only when it had been exogenously activated to plasmin and when it was used at disproportionately high concentrations compared to serum. Addition of only the zymogen plasminogen into serum did not hinder complement-mediated lysis of erythrocytes. Plasminogen could not restrict deposition of complement activation products on endothelial cells either, as was shown with flow cytometry. With platelets, a very weak inhibitory effect on deposition of C3 fragments was observed, but it was considered too weak to be significant for disease pathogenesis. Thus it was concluded that plasminogen is not an important regulator of complement on self cells. Instead, addition of plasminogen was shown to clearly hinder platelet aggregation in serum. This was attributed to plasmin causing disintegration of formed platelet aggregates. We propose that reduced proteolytic activity of plasmin on structures of growing thrombi, rather than on complement activation fragments, explains the association of plasminogen deficiency with aHUS. This adds to the emerging view that factors unrelated to the complement system can also be central to aHUS pathogenesis and suggests that future research on the mechanism of the disease should expand beyond complement dysregulation. PMID:26637181

  11. Complement-Coagulation Cross-Talk: A Potential Mediator of the Physiological Activation of Complement by Low pH

    PubMed Central

    Kenawy, Hany Ibrahim; Boral, Ismet; Bevington, Alan

    2015-01-01

    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 (intrinsic pathway) 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 alkalinization of urine has been shown significantly to reduce tubular complement activation products, an effect, which may have important implications for slowing progression of CKD. PMID:25999953

  12. Degradation of Complement 3 by Streptococcal Pyrogenic Exotoxin B Inhibits Complement Activation and Neutrophil Opsonophagocytosis▿

    PubMed Central

    Kuo, Chih-Feng; Lin, Yee-Shin; Chuang, Woei-Jer; Wu, Jiunn-Jong; Tsao, Nina

    2008-01-01

    Streptococcal pyrogenic exotoxin B (SPE B), a cysteine protease, is an important virulence factor in group A streptococcus (GAS) infection. The inhibition of phagocytic activity by SPE B may help prevent bacteria from being ingested. In this study, we examined the mechanism SPE B uses to enable bacteria to resist opsonophagocytosis. Using an enzyme-linked immunosorbent assay, we found that SPE B-treated serum impaired the activation of the classical, the lectin, and the alternative complement pathways. In contrast, C192S, a SPE B mutant lacking protease activity, had no effect on complement activation. Further study showed that cleavage of serum C3 by SPE B, but not C192S, blocked zymosan-induced production of reactive oxygen species in neutrophils as a result of decreased deposition of C3 fragments on the zymosan surface. Reconstitution of C3 into SPE B-treated serum unblocked zymosan-mediated neutrophil activation dose dependently. SPE B-treated, but not C192S-treated, serum also impaired opsonization of C3 fragments on the surface of GAS strain A20. Moreover, the amount of C3 fragments on the A20 cell surface, a SPE B-producing strain, was less than that on its isogenic mutant strain, SW507, after opsonization with normal serum. A20 opsonized with SPE B-treated serum was more resistant to neutrophil killing than A20 opsonized with normal serum, and SPE B-mediated resistance was C3 dependent. These results suggest a novel SPE B mechanism, one which degrades serum C3 and enables GAS to resist complement damage and opsonophagocytosis. PMID:18174338

  13. Complement activation in leprosy: a retrospective study shows elevated circulating terminal complement complex in reactional leprosy.

    PubMed

    Bahia El Idrissi, N; Hakobyan, S; Ramaglia, V; Geluk, A; Morgan, B Paul; Das, P Kumar; Baas, F

    2016-06-01

    Mycobacterium leprae infection gives rise to the immunologically and histopathologically classified spectrum of leprosy. At present, several tools for the stratification of patients are based on acquired immunity markers. However, the role of innate immunity, particularly the complement system, is largely unexplored. The present retrospective study was undertaken to explore whether the systemic levels of complement activation components and regulators can stratify leprosy patients, particularly in reference to the reactional state of the disease. Serum samples from two cohorts were analysed. The cohort from Bangladesh included multi-bacillary (MB) patients with (n = 12) or without (n = 46) reaction (R) at intake and endemic controls (n = 20). The cohort from Ethiopia included pauci-bacillary (PB) (n = 7) and MB (n = 23) patients without reaction and MB (n = 15) patients with reaction. The results showed that the activation products terminal complement complex (TCC) (P ≤ 0·01), C4d (P ≤ 0·05) and iC3b (P ≤ 0·05) were specifically elevated in Bangladeshi patients with reaction at intake compared to endemic controls. In addition, levels of the regulator clusterin (P ≤ 0·001 without R; P < 0·05 with R) were also elevated in MB patients, irrespective of a reaction. Similar analysis of the Ethiopian cohort confirmed that, irrespective of a reaction, serum TCC levels were increased significantly in patients with reactions compared to patients without reactions (P ≤ 0·05). Our findings suggests that serum TCC levels may prove to be a valuable tool in diagnosing patients at risk of developing reactions. PMID:26749503

  14. Complement inhibitors to treat IgM-mediated autoimmune hemolysis.

    PubMed

    Wouters, Diana; Zeerleder, Sacha

    2015-11-01

    Complement activation in autoimmune hemolytic anemia may exacerbate extravascular hemolysis and may occasionally result in intravascular hemolysis. IgM autoantibodies as characteristically found in cold autoantibody autoimmune hemolytic anemia, in cold agglutinin disease but also in a considerable percentage of patients with warm autoantibodies are very likely to activate complement in vivo. Therapy of IgM-mediated autoimmune hemolytic anemia mainly aims to decrease autoantibody production. However, most of these treatments require time to become effective and will not stop immediate ongoing complement-mediated hemolysis nor prevent hemolysis of transfused red blood cells. Therefore pharmacological inhibition of the complement system might be a suitable approach to halt or at least attenuate ongoing hemolysis and improve the recovery of red blood cell transfusion in autoimmune hemolytic anemia. In recent years, several complement inhibitors have become available in the clinic, some of them with proven efficacy in autoimmune hemolytic anemia. In the present review, we give a short introduction on the pathogenesis of autoimmune hemolytic anemia, followed by an overview on the complement system with a special focus on its regulation. Finally, we will discuss complement inhibitors with regard to their potential efficacy to halt or attenuate hemolysis in complement-mediated autoimmune hemolytic anemia. PMID:26521297

  15. Maximality and Idealized Cognitive Models: The Complementation of Spanish "Tener."

    ERIC Educational Resources Information Center

    Hilferty, Joseph; Valenzuela, Javier

    2001-01-01

    Discusses the bare-noun phrase (NP) complementation pattern of the Spanish verb "tener" (have). Shows that the maximality of the complement NP is dependent upon three factors: (1) idiosyncratic valence requirements; (2) encyclopedic knowledge related to possession; and (3) contextualized semantic construal. (Author/VWL)

  16. Complement inhibitors to treat IgM-mediated autoimmune hemolysis

    PubMed Central

    Wouters, Diana; Zeerleder, Sacha

    2015-01-01

    Complement activation in autoimmune hemolytic anemia may exacerbate extravascular hemolysis and may occasionally result in intravascular hemolysis. IgM autoantibodies as characteristically found in cold autoantibody autoimmune hemolytic anemia, in cold agglutinin disease but also in a considerable percentage of patients with warm autoantibodies are very likely to activate complement in vivo. Therapy of IgM-mediated autoimmune hemolytic anemia mainly aims to decrease autoantibody production. However, most of these treatments require time to become effective and will not stop immediate ongoing complement-mediated hemolysis nor prevent hemolysis of transfused red blood cells. Therefore pharmacological inhibition of the complement system might be a suitable approach to halt or at least attenuate ongoing hemolysis and improve the recovery of red blood cell transfusion in autoimmune hemolytic anemia. In recent years, several complement inhibitors have become available in the clinic, some of them with proven efficacy in autoimmune hemolytic anemia. In the present review, we give a short introduction on the pathogenesis of autoimmune hemolytic anemia, followed by an overview on the complement system with a special focus on its regulation. Finally, we will discuss complement inhibitors with regard to their potential efficacy to halt or attenuate hemolysis in complement-mediated autoimmune hemolytic anemia. PMID:26521297

  17. How antibodies use complement to regulate antibody responses.

    PubMed

    Sörman, Anna; Zhang, Lu; Ding, Zhoujie; Heyman, Birgitta

    2014-10-01

    Antibodies, forming immune complexes with their specific antigen, can cause complete suppression or several 100-fold enhancement of the antibody response. Immune complexes containing IgG and IgM may activate complement and in such situations also complement components will be part of the immune complex. Here, we review experimental data on how antibodies via the complement system upregulate specific antibody responses. Current data suggest that murine IgG1, IgG2a, and IgG2b upregulate antibody responses primarily via Fc-receptors and not via complement. In contrast, IgM and IgG3 act via complement and require the presence of complement receptors 1 and 2 (CR1/2) expressed on both B cells and follicular dendritic cells. Complement plays a crucial role for antibody responses not only to antigen complexed to antibodies, but also to antigen administered alone. Lack of C1q, but not of Factor B or MBL, severely impairs antibody responses suggesting involvement of the classical pathway. In spite of this, normal antibody responses are found in mice lacking several activators of the classical pathway (complement activating natural IgM, serum amyloid P component (SAP), specific intracellular adhesion molecule-grabbing non-integrin R1 (SIGN-R1) or C-reactive protein. Possible explanations to these observations will be discussed. PMID:25001046

  18. Complementation of a Fanconi anemia group A cell line by UbA{sup 52}

    SciTech Connect

    Moses, R.E.; Heina, J.A.; Jakobs, P.M.

    1994-09-01

    Cells from patients with Fanconi anemia (FA) display chromosomal instability and increased sensitivity to mitomycin C (MMC) and diepoxybutane (DEB) relative to normal cells. Several genes act in this pathway of DNA damage processing based upon four known complementation groups in FA. We have made a cDNA expression library in a vector with a G418 selectable marker to identify FA genes other than the FA-C group. Approximately 1 x 10{sup 6} independent cDNA clones were isolated with an average cDNA size of 1.5 kb. Five cell lines resistant to MMC and DEB were isolated from 6 x 10{sup 6} G418-resistant transfectants from 65 individual transfections of the FA-A fibroblast line GM6914. The isolated cell lines also showed normal chromosome stability. The same cDNA (600 bp) was recovered from three independent cell lines by PCR using flanking sequence primers. The gene has sequence identity with a known gene, the ubiquitin fusion gene, UbA{sub 52}. Interestingly, each of the cDNAs were inserted in antisense orientation relative to the cytomegalovirus (CMV) promoter as determined by sequencing and PCR using UbA{sub 52}-specific internal primers. Southern blot analysis indicated the cell lines had distinct chromosomal insertion sites. Mutation analysis by chemical cleavage showed no reading frame mutations, indicating that UbA{sub 52} is not the FA-A gene. Re-transfection with the UbA{sub 52} gene in antisense gave complementation for MMC, DEB and chromosome stability to varying degrees. Re-transfection of the antisense construct with the CMV promotor removed or with a sense construct did not alter the MMC sensitivity. We conclude that the antisense UbA{sub 52} gene has a non-specific effect, perhaps acting by altering the cell cycle or susceptibility to apoptosis.

  19. Protease-dependent mechanisms of complement evasion by bacterial pathogens

    PubMed Central

    Potempa, Michal; Potempa, Jan

    2012-01-01

    The human immune system has evolved a variety of mechanisms for the primary task of neutralizing and eliminating microbial intruders. As the first line of defense, the complement system is responsible for rapid recognition and opsonisation of bacteria, presentation to phagocytes and bacterial cell killing by direct lysis. All successful human pathogens have mechanisms of circumventing the antibacterial activity of the complement system and escaping this stage of the immune response. One of the ways in which pathogens achieve this is the deployment of proteases. Based on the increasing number of recent publications in this area, it appears that proteolytic inactivation of the antibacterial activities of the complement system is a common strategy of avoiding targeting by this arm of host innate immune defense. In this review, we focus on those bacteria that deploy proteases capable of degrading complement system components into non-functional fragments, thus impairing complement-dependent antibacterial activity and facilitating pathogen survival inside the host. PMID:22944688

  20. The alternative pathway of complement and the thrombotic microangiopathies.

    PubMed

    Teoh, Chia Wei; Riedl, Magdalena; Licht, Christoph

    2016-04-01

    Thrombotic microangiopathies (TMA) are disorders defined by microangiopathic hemolytic anemia, non-immune thrombocytopenia and have multi-organ involvement including the kidneys, brain, gastrointestinal, respiratory tract and skin. Emerging evidence points to the central role of complement dysregulation in leading to microvascular endothelial injury which is crucial for the development of TMAs. This key insight has led to the development of complement-targeted therapy. Eculizumab is an anti-C5 monoclonal antibody, which has revolutionized the treatment of atypical hemolytic uremic syndrome. Several other anti-complement therapeutic agents are currently in development, offering a potential armamentarium of therapies available to treat complement-mediated TMAs. The development of sensitive, reliable and easy to perform assays to monitor complement activity and therapeutic efficacy will be key to devising an individualized treatment regime with the potential of safely weaning or discontinuing treatment in the appropriate clinical setting. PMID:27160864

  1. Physicochemical signatures of nanoparticle-dependent complement activation

    NASA Astrophysics Data System (ADS)

    Thomas, Dennis G.; Chikkagoudar, Satish; Heredia-Langner, Alejandro; Tardiff, Mark F.; Xu, Zhixiang; Hourcade, Dennis E.; Pham, Christine T. N.; Lanza, Gregory M.; Weinberger, Kilian Q.; Baker, Nathan A.

    2014-01-01

    Nanoparticles are potentially powerful therapeutic tools that have the capacity to target drug payloads and imaging agents. However, some nanoparticles can activate complement, a branch of the innate immune system, and cause adverse side-effects. Recently, we employed an in vitro hemolysis assay to measure the serum complement activity of perfluorocarbon nanoparticles that differed by size, surface charge, and surface chemistry, quantifying the nanoparticle-dependent complement activity using a metric called Residual Hemolytic Activity (RHA). In the present work, we have used a decision tree learning algorithm to derive the rules for estimating nanoparticle-dependent complement response based on the data generated from the hemolytic assay studies. Our results indicate that physicochemical properties of nanoparticles, namely, size, polydispersity index, zeta potential, and mole percentage of the active surface ligand of a nanoparticle, can serve as good descriptors for prediction of nanoparticle-dependent complement activation in the decision tree modeling framework.

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

    PubMed

    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. PMID:24665660

  3. Novel Evasion Mechanisms of the Classical Complement Pathway.

    PubMed

    Garcia, Brandon L; Zwarthoff, Seline A; Rooijakkers, Suzan H M; Geisbrecht, Brian V

    2016-09-15

    Complement is a network of soluble and cell surface-associated proteins that gives rise to a self-amplifying, yet tightly regulated system with fundamental roles in immune surveillance and clearance. Complement becomes activated on the surface of nonself cells by one of three initiating mechanisms known as the classical, lectin, and alternative pathways. Evasion of complement function is a hallmark of invasive pathogens and hematophagous organisms. Although many complement-inhibition strategies hinge on hijacking activities of endogenous complement regulatory proteins, an increasing number of uniquely evolved evasion molecules have been discovered over the past decade. In this review, we focus on several recent investigations that revealed mechanistically distinct inhibitors of the classical pathway. Because the classical pathway is an important and specific mediator of various autoimmune and inflammatory disorders, in-depth knowledge of novel evasion mechanisms could direct future development of therapeutic anti-inflammatory molecules. PMID:27591336

  4. Recent Developments in Low Molecular Weight Complement Inhibitors

    PubMed Central

    Qu, Hongchang; Ricklin, Daniel; Lambris, John D.

    2009-01-01

    As a key part of the innate immune system, complement plays an important role not only in defending invading pathogens but also in many other biological processes. Inappropriate or excessive activation of complement has been linked to many autoimmune, inflammatory, and neurodegenerative diseases, as well as ischemia-reperfusion injury and cancer. A wide array of low molecular weight complement inhibitors has been developed to target various components of the complement cascade. Their efficacy has been demonstrated in numerous in vitro and in vivo experiments. Though none of these inhibitors has reached the market so far, some of them have entered clinical trials and displayed promising results. This review provides a brief overview of the currently developed low molecular weight complement inhibitors, including short peptides and synthetic small molecules, with an emphasis on those targeting components C1 and C3, and the anaphylatoxin receptors. PMID:19800693

  5. Identification of Rad51 regulation by BRCA2 using Caenorhabditis elegans BRCA2 and bimolecular fluorescence complementation analysis

    SciTech Connect

    Min, Jaewon; Park, Pil-gu; Ko, Eunkyong; Choi, Eunhee; Lee, Hyunsook

    2007-11-03

    BRCA2 is involved in double-stranded DNA break repair by binding and regulating Rad51-mediated homologous recombination. Insights as to how BRCA2 regulates Rad51-mediated DNA repair arose from in vitro biochemical studies on fragments of BRCA2. However, the large 400-kDa BRCA2 protein has hampered our ability to understand the entire process by which full-length BRCA2 regulates Rad51. Here, we show that CeBRC-2, which is only one tenth the size of mammalian BRCA2, complemented BRCA2-deficiency in Rad51 regulation. CeBRC-2 was able to bind to mammalian Rad51 (mRad51) and form distinct nuclear foci when they interacted. In our bimolecular fluorescence complementation analysis (BiFC), we show that the strength of the interaction between CeBRC-2 and mRad51 increased markedly after DNA damage. The BRC motif of CeBRC-2 was responsible for binding mRad51, but without the OB fold, the complex was unable to target damaged DNA. When CeBRC-2 was introduced into BRCA2-deficient cells, it restored Rad51 foci after DNA damage. Our study suggests a mode of action for BRCA2 with regard to DNA repair.

  6. The cytolytic C5b-9 complement complex: feedback inhibition of complement activation.

    PubMed Central

    Bhakdi, S; Maillet, F; Muhly, M; Kazatchkine, M D

    1988-01-01

    We describe a regulatory function of the terminal cytolytic C5b-9 complex [C5b-9(m)] of human complement. Purified C5b-9(m) complexes isolated from target membranes, whether in solution or bound to liposomes, inhibited lysis of sensitized sheep erythrocytes by whole human serum in a dose-dependent manner. C9 was not required for the inhibitory function since C5b-7 and C5b-8 complexes isolated from membranes were also effective. No effect was found with the cytolytically inactive, fluid-phase SC5b-9 complex. However, tryptic modification of SC5b-9 conferred an inhibitory capacity to the complex, due probably to partial removal of the S protein. Experiments using purified components demonstrated that C5b-9(m) exerts a regulatory effect on the formation of the classical- and alternative-pathway C3 convertases and on the utilization of C5 by cell-bound C5 convertases. C5b-9(m) complexes were unable to inhibit the lysis of cells bearing C5b-7(m) by C8 and C9. Addition of C5b-9(m) to whole human serum abolished its bactericidal effect on the serum-sensitive Escherichia coli K-12 strain W 3110 and suppressed its hemolytic function on antibody-sensitized, autologous erythrocytes. Feedback inhibition by C5b-9(m) represents a biologically relevant mechanism through which complement may autoregulate its effector functions. Images PMID:3162317

  7. MEMBRANE ATTACK BY COMPLEMENT: THE ASSEMBLY AND BIOLOGY OF TERMINAL COMPLEMENT COMPLEXES

    PubMed Central

    Tegla, Cosmin A.; Cudrici, Cornelia; Patel, Snehal; Trippe, Richard; Rus, Violeta; Niculescu, Florin; Rus, Horea

    2013-01-01

    Complement system activation plays an important role in both innate and acquired immunity. Activation of complement and the subsequent formation of C5b-9 channels (the membrane attack complex) on cell membranes lead to cell death. However, when the number of channels assembled on the surface of nucleated cells is limited, sublytic C5b-9 can induce cell cycle progression by activating signal transduction pathways and transcription factors and inhibiting apoptosis. This induction by C5b-9 is dependent upon the activation of the phosphatidylinositol 3-kinase/Akt/FOXO1 and ERK1 pathways in a Gi protein-dependent manner. C5b-9 induces sequential activation of CDK4 and CDK2, enabling the G1/S-phase transition and cellular proliferation. In addition, it induces RGC-32, a novel gene that plays a role in cell cycle activation by interacting with Akt and the cyclin B1-CDC2 complex. C5b-9 also inhibits apoptosis by inducing the phosphorylation of Bad and blocking the activation of FLIP, caspase-8, and Bid cleavage. Thus, sublytic C5b-9 plays an important role in cell activation, proliferation, and differentiation, thereby contributing to the maintenance of cell and tissue homeostasis. PMID:21850539

  8. Complement in disease: a defence system turning offensive.

    PubMed

    Ricklin, Daniel; Reis, Edimara S; Lambris, John D

    2016-07-01

    Although the complement system is primarily perceived as a host defence system, a more versatile, yet potentially more harmful side of this innate immune pathway as an inflammatory mediator also exists. The activities that define the ability of the complement system to control microbial threats and eliminate cellular debris - such as sensing molecular danger patterns, generating immediate effectors, and extensively coordinating with other defence pathways - can quickly turn complement from a defence system to an aggressor that drives immune and inflammatory diseases. These host-offensive actions become more pronounced with age and are exacerbated by a variety of genetic factors and autoimmune responses. Complement can also be activated inappropriately, for example in response to biomaterials or transplants. A wealth of research over the past two decades has led to an increasingly finely tuned understanding of complement activation, identified tipping points between physiological and pathological behaviour, and revealed avenues for therapeutic intervention. This Review summarizes our current view of the key activating, regulatory, and effector mechanisms of the complement system, highlighting important crosstalk connections, and, with an emphasis on kidney disease and transplantation, discusses the involvement of complement in clinical conditions and promising therapeutic approaches. PMID:27211870

  9. Complement activation promotes muscle inflammation during modified muscle use

    NASA Technical Reports Server (NTRS)

    Frenette, J.; Cai, B.; Tidball, J. G.

    2000-01-01

    Modified muscle use can result in muscle inflammation that is triggered by unidentified events. In the present investigation, we tested whether the activation of the complement system is a component of muscle inflammation that results from changes in muscle loading. Modified rat hindlimb muscle loading was achieved by removing weight-bearing from the hindlimbs for 10 days followed by reloading through normal ambulation. Experimental animals were injected with the recombinant, soluble complement receptor sCR1 to inhibit complement activation. Assays for complement C4 or factor B in sera showed that sCR1 produced large reductions in the capacity for activation of the complement system through both the classical and alternative pathways. Analysis of complement C4 concentration in serum in untreated animals showed that the classical pathway was activated during the first 2 hours of reloading. Analysis of factor B concentration in untreated animals showed activation of the alternative pathway at 6 hours of reloading. Administration of sCR1 significantly attenuated the invasion of neutrophils (-49%) and ED1(+) macrophages (-52%) that occurred in nontreated animals after 6 hours of reloading. The presence of sCR1 also reduced significantly the degree of edema by 22% as compared to untreated animals. Together, these data show that increased muscle loading activated the complement system which then briefly contributes to the early recruitment of inflammatory cells during modified muscle loading.

  10. Role of Complement Activation in Obliterative Bronchiolitis Post Lung Transplantation

    PubMed Central

    Suzuki, Hidemi; Lasbury, Mark E.; Fan, Lin; Vittal, Ragini; Mickler, Elizabeth A.; Benson, Heather L.; Shilling, Rebecca; Wu, Qiang; Weber, Daniel J.; Wagner, Sarah R.; Lasaro, Melissa; Devore, Denise; Wang, Yi; Sandusky, George E.; Lipking, Kelsey; Pandya, Pankita; Reynolds, John; Love, Robert; Wozniak, Thomas; Gu, Hongmei; Brown, Krista M.; Wilkes, David S.

    2013-01-01

    Obliterative bronchiolitis (OB) post lung transplantation involves IL-17 regulated autoimmunity to type V collagen and alloimmunity, which could be enhanced by complement activation. However, the specific role of complement activation in lung allograft pathology, IL-17 production, and OB are unknown. The current study examines the role of complement activation in OB. Complement regulatory protein (CRP) (CD55, CD46, Crry/CD46) expression was down regulated in human and murine OB; and C3a, a marker of complement activation, was up regulated locally. IL-17 differentially suppressed Crry expression in airway epithelial cells in vitro. Neutralizing IL-17 recovered CRP expression in murine lung allografts and decreased local C3a production. Exogenous C3a enhanced IL-17 production from alloantigen or autoantigen (type V collagen) reactive lymphocytes. Systemically neutralizing C5 abrogated the development of OB, reduced acute rejection severity, lowered systemic and local levels of C3a and C5a, recovered CRP expression, and diminished systemic IL-17 and IL-6 levels. These data indicated that OB induction is in part complement dependent due to IL-17 mediated down regulation of CRPs on airway epithelium. C3a and IL-17 are part of a feed forward loop that may enhance CRP down regulation, suggesting that complement blockade could be a therapeutic strategy for OB. PMID:24043901

  11. Genomic view of the evolution of the complement system

    PubMed Central

    Kimura, Ayuko

    2006-01-01

    The recent accumulation of genomic information of many representative animals has made it possible to trace the evolution of the complement system based on the presence or absence of each complement gene in the analyzed genomes. Genome information from a few mammals, chicken, clawed frog, a few bony fish, sea squirt, fruit fly, nematoda and sea anemone indicate that bony fish and higher vertebrates share practically the same set of complement genes. This suggests that most of the gene duplications that played an essential role in establishing the mammalian complement system had occurred by the time of the teleost/mammalian divergence around 500 million years ago (MYA). Members of most complement gene families are also present in ascidians, although they do not show a one-to-one correspondence to their counterparts in higher vertebrates, indicating that the gene duplications of each gene family occurred independently in vertebrates and ascidians. The C3 and factor B genes, but probably not the other complement genes, are present in the genome of the cnidaria and some protostomes, indicating that the origin of the central part of the complement system was established more than 1,000 MYA. PMID:16896831

  12. Involvement of Antilipoarabinomannan Antibodies in Classical Complement Activation in Tuberculosis

    PubMed Central

    Hetland, Geir; Wiker, Harald G.; Høgåsen, Kolbjørn; Hamasur, Beston; Svenson, Stefan B.; Harboe, Morten

    1998-01-01

    We examined alternative and classical complement activation induced by whole bacilli of Mycobacterium bovis BCG and Mycobacterium tuberculosis products. After exposure to BCG, there were higher levels of the terminal complement complex in sera from Indian tuberculosis patients than in sera from healthy controls. The addition of BCG with or without EGTA to these sera indicated that approximately 70 to 85% of the total levels of the terminal complement complex was formed by classical activation. Sera from Indian tuberculosis patients contained more antibody to lipoarabinomannan (LAM) than sera from healthy Indians. Levels of anti-LAM immunoglobulin G2 (IgG2), but not anti-LAM IgM, correlated positively with classical activation induced by BCG in the sera. By flow cytometry, deposition of C3 and terminal complement complex on bacilli incubated with normal human serum was demonstrated. The anticomplement staining was significantly reduced in the presence of EGTA and EDTA. Flow cytometry also revealed the binding of complement to BCG incubated with rabbit anti-LAM and then with factor B-depleted serum. This indicates that classical activation plays a major role in complement activation induced by mycobacteria and that anti-LAM IgG on the bacilli can mediate this response. Classical complement activation may be important for the extent of phagocytosis of M. tuberculosis by mononuclear phagocytes, which may influence the course after infection. PMID:9521145

  13. [Atypical HUS caused by complement-related abnormalities].

    PubMed

    Yoshida, Yoko; Matsumoto, Masanori

    2015-02-01

    Atypical hemolytic uremic syndrome (aHUS) is a rare disease characterized by the triad of microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure. The term aHUS was historically used to distinguish this disorder from Shiga-toxin producing Escherichia coli (STEC)-HUS. Many aHUS cases (approximately 70%) are reportedly caused by uncontrolled complement activation due to genetic mutations in the alternative pathway, including complement factor H (CFH), complement factor I (CFI), membrane cofactor protein (MCP), thrombomodulin (THBD), complement component C3 (C3), and complement factor B (CFB). Mutations in the coagulation pathway, such as diacylglycerol kinase ε (DGKE) and plasminogen, are also reported to be causes of aHUS. In this review, we have focused on aHUS due to complement dysfunction. aHUS is suspected based on plasma ADAMTS13 activity of 10% or more, and being negative for STEC-HUS, in addition to the aforementioned triad. Complement genetic studies provide a more specific diagnosis of aHUS. Plasma therapy is the first-line treatment for patients with aHUS and should be initiated as soon as the diagnosis is suspected. Recently, eculizumab, a humanized monoclonal antibody against C5, was shown to be an effective treatment for aHUS. Therefore, early diagnosis and identification of the underlying pathogenic mechanism is important for improving the outcome of aHUS. PMID:25765799

  14. Antifungal activity of the local complement system in cerebral aspergillosis.

    PubMed

    Rambach, Günter; Hagleitner, Magdalena; Mohsenipour, Iradj; Lass-Flörl, Cornelia; Maier, Hans; Würzner, Reinhard; Dierich, Manfred P; Speth, Cornelia

    2005-10-01

    Dissemination of aspergillosis into the central nervous system is associated with nearly 100% mortality. To study the reasons for the antifungal immune failure we analyzed the efficacy of cerebral complement to combat the fungus Aspergillus. Incubation of Aspergillus in non-inflammatory cerebrospinal fluid (CSF) revealed that complement levels were sufficient to obtain a deposition on the surface, but opsonization was much weaker than in serum. Consequently complement deposition from normal CSF on fungal surface stimulated a very low phagocytic activity of microglia, granulocytes, monocytes and macrophages compared to stimulation by conidia opsonized in serum. Similarly, opsonization of Aspergillus by CSF was not sufficient to induce an oxidative burst in infiltrating granulocytes, whereas conidia opsonized in serum induced a clear respiratory signal. Thus, granulocytes were capable of considerably reducing the viability of serum-opsonized Aspergillus conidia, but not of conidia opsonized in CSF. The limited efficacy of antifungal attack by cerebral complement can be partly compensated by enhanced synthesis, leading to elevated complement concentrations in CSF derived from a patient with cerebral aspergillosis. This inflammatory CSF was able to induce (i) a higher complement deposition on the Aspergillus surface than non-inflammatory CSF, (ii) an accumulation of complement activation products and (iii) an increase in phagocytic and killing activity of infiltrating granulocytes. However, levels and efficacy of the serum-derived complement were not reached. These data indicate that low local complement synthesis and activation may represent a central reason for the insufficient antifungal defense in the brain and the high mortality rate of cerebral aspergillosis. PMID:16027023

  15. Complement Activation in Trauma Patients Alters Platelet Function.

    PubMed

    Atefi, Gelareh; Aisiku, Omozuanvbo; Shapiro, Nathan; Hauser, Carl; Dalle Lucca, Jurandir; Flaumenhaft, Robert; Tsokos, George C

    2016-09-01

    Trauma remains the main cause of death for both civilians and those in uniform. Trauma-associated coagulopathy is a complex process involving inflammation, coagulation, and platelet dysfunction. It is unknown whether activation of complement, which occurs invariably in trauma patients, is involved in the expression of trauma-associated coagulopathy. We designed a prospective study in which we enrolled 40 trauma patients and 30 healthy donors upon arrival to the emergency department of BIDMC. Platelets from healthy individuals were incubated with sera from trauma patients and their responsiveness to a thrombin receptor-activating peptide was measured using aggregometry. Complement deposition on platelets from trauma patients was measured by flow cytometry. Normal platelets displayed hypoactivity after incubation with trauma sera even though exposure to trauma sera resulted in increased agonist-induced calcium flux. Depletion of complement from sera further blocked activation of hypoactive platelets. Conversely, complement activation increased aggregation of platelets. Platelets from trauma patients were found to have significantly higher amounts of C3a and C4d on their surface compared with platelets from controls. Depletion of complement (C4d, C3a) reversed the ability of trauma sera to augment agonist-induced calcium flux in donor platelets. Our data indicate that complement enhances platelet aggregation. Despite its complement content, trauma sera render platelets hypoactive and complement depletion further blocks activation of hypoactive platelets. The defect in platelet activation induced by trauma sera is distal to receptor activation since agonist-induced Ca2+ flux is elevated in the presence of trauma sera owing to complement deposition. PMID:27355402

  16. Role of the lectin complement pathway in kidney transplantation.

    PubMed

    Farrar, Conrad A; Zhou, Wuding; Sacks, Steven H

    2016-10-01

    In the last 15 years two major advances in the role of complement in the kidney transplant have come about. The first is that ischaemia reperfusion injury and its profound effect on transplant outcome is dependent on the terminal product of complement activation, C5b-9. The second key observation relates to the function of the small biologically active fragments C3a and C5a released by complement activation in increasing antigen presentation and priming the T cell response that results in transplant rejection. In both cases local synthesis of C3 principally by the renal tubule cells plays an essential role that overshadows the role of the circulating pool of C3 generated largely by hepatocyte synthesis. More recent efforts have investigated the molecules expressed by renal tissue that can trigger complement activation. These have revealed a prominent effect of collectin-11 (CL-11), a soluble C-type lectin that is expressed in renal tissue and aligns with its major ligand L-fucose at sites of complement activation following ischaemic stress. Biochemical studies have shown that interaction between CL-11 and L-fucose results in complement activation by the lectin complement pathway, precisely targeting the innate immune response to the ischaemic tubule surface. Therapeutic approaches to reduce inflammatory and immune stimulation in ischaemic kidney have so far targeted C3 or its activation products and several are in clinical trials. The finding that lectin-fucose interaction is an important trigger of lectin pathway complement activation within the donor organ opens up further therapeutic targets where intervention could protect the donor kidney against complement. PMID:27286717

  17. Cloning of Phanerochaete chrysosporium leu2 by complementation of bacterial auxotrophs and transformation of fungal auxotrophs

    SciTech Connect

    Zapanta, L.S.; Hattori, Takefumi; Rzetskaya, M.; Tien, M.

    1998-07-01

    A Phanerochaete chrysosporium cDNA library was constructed in an expression vector that allows expression in both Escherichia coli and Saccharomyces cerevisiae. This expression vector, {lambda}YES, contains the lacZ promoter for expression in E. coli and the GAL1 promoter for expression in yeast. A number of genes were cloned by complementation of bacterial amino acid auxotrophs. The cDNA encoding the {beta}-isoproopylmalate dehydrogenase from P. chrysosporium was characterized further. The genomic (gleu2) was subsequently isolated and was used successfully as a selectable marker to transform P. chrysosporium auxotrophs for LEU2. Protoplasts for transportation were prepared with readily obtained conidiospores rather than with basidiospores, which were used in previous P. chrysoporium transformation procedures. The method described here allows other genes to be isolated from P. chrysosportium for use as selectable markers.

  18. Cloning of a human galactokinase gene (GK2) on chromosome 15 by complementation in yeast.

    PubMed Central

    Lee, R T; Peterson, C L; Calman, A F; Herskowitz, I; O'Donnell, J J

    1992-01-01

    A human cDNA encoding a galactokinase (EC 2.7.1.6) was isolated by complementation of a galactokinase-deficient (gal1-) strain of Saccharomyces cerevisiae. This cDNA encodes a predicted protein of 458 amino acids with 29% identity to galactokinase of Saccharomyces carlsbergensis. Previous studies have mapped a human galactokinase gene (GK1) to chromosome 17q23-25, closely linked to thymidine kinase. The galactokinase gene that we have isolated (GK2) is located on chromosome 15. The relationship between the disease locus for galactokinase deficiency galactosemia, which is responsible for cataracts in newborns and possibly presenile cataracts in adults, and the two galactokinase loci is unknown. Images PMID:1438294

  19. Bioplastique as a complement in conventional plastic surgery.

    PubMed

    Nácul, A M; Nácul, A P; Greca de Born, A

    1998-01-01

    This research presents reports of cases where a biocompatible and alloplastic biomaterial-Bioplastique-was used, associated with conventional plastic surgery or as a complement to it, with the aim of achieving a better final aesthetic result. Four cases are presented where Bioplastique was used in association with rhytidoplasty, rhinoplasty, and other surgical techniques. This material has shown itself to be appropriate to complement surgery; achieving a final result which would not be possible without any resort to a complement or any other hard procedure by the surgeon and is not more traumatic for the patient. PMID:9852179

  20. Studies on phenotypic complementation of ataxia-telangiectasia cells by chromosome transfer.

    PubMed Central

    Jongmans, W; Verhaegh, G W; Jaspers, N G; Oshimura, M; Stanbridge, E J; Lohman, P H; Zdzienicka, M Z

    1995-01-01

    Cells derived from patients with the cancer-prone inherited disorder ataxia-telangiectasia (A-T) show an abnormal response to ionizing radiation-induced DNA damage, such as an increased cell killing and a diminished inhibition of DNA synthesis. The enhanced killing of A-T (group D) cells by X-rays can be corrected by multiple cDNAs, mapping to different chromosomes (6, 11, 17, and 18). In order to examine whether genes located on these chromosomes complement AT-D cells, normal neo-tagged chromosomes 6, 11, 17, and 18 were introduced into AT-D cells by microcell-mediated chromosome transfer. However, correction of the enhanced killing of AT-D cells by X-rays could only be achieved by chromosome 11 and by none of the other chromosomes tested. The enhanced killing of A-T (complementation group C) cells was also corrected by chromosome 11. Usually, but not in all microcell hybrid clones, chromosome 11 also corrected the radioresistant DNA synthesis (RDS) phenotype of AT-D and AT-C cells. These results (i) confirm findings by others suggesting assignment of the ATD and ATC genes to chromosome 11, (ii) demonstrate that several genes can modify the cellular radiation response when they are taken out of their normal genomic context and/or control, and (iii) indicate that the RDS phenotype and the enhanced cell killing in A-T are independent pleiotropic features resulting from the primary mutations in A-T. Also, our findings underscore that, in establishing cDNAs as candidate genes for A-T, microcell-mediated chromosome transfer studies are needed to exclude nonspecific correcting effects of these candidate cDNA genes. PMID:7847380

  1. Molecular cloning and characterization of a complement-depleting factor from king cobra, Ophiophagus hannah.

    PubMed

    Zeng, Lin; Sun, Qian-Yun; Jin, Yang; Zhang, Yong; Lee, Wen-Hui; Zhang, Yun

    2012-09-01

    Cobra venom factor (CVF) is an anti-complement factor existing in cobra venom. CVF proteins have been purified from the venoms of Naja haje, Naja siamensis, Naja atra, Naja kaouthia, Naja naja, Naja melanoleuca and Austrelaps superbus, but only three full-length cDNA sequences of CVF are available. In the present work, a cobra venom factor termed OVF was purified from the crude venom of Ophiophagus hannah by successive gel filtration, ion-exchange and heparin affinity chromatography steps. The purified OVF was homogenous on the SDS-PAGE gel with an apparent molecular weight of 140 kDa under non-reducing conditions. Under reducing conditions, OVF was divided into three bands with apparent molecular weight of 72 kDa (α chain), 45 kDa (β chain) and 32 kDa (γ chain), respectively. OVF consumed complement components with anti-complement activity of 154 units per mg. By using Reverse transcription-PCR and 5'-RACE assay, the open reading frame of OVF was obtained. MALDI-TOF and protein sequencing assays confirmed the cloned cDNA coding for OVF protein. The cDNA sequence of OVF is conservative when aligned with that of other CVFs. Phylogenetic analysis revealed OVF is closer to CVF from N. kaouthia than to AVF-1 and AVF-2 from A. superbus. Our results demonstrated that OVF has its unique features as following: 1) The N-terminal amino acid sequence of OVF γ chain is different from that of other known CVFs, suggesting that the OVF γ chain might be further processed; 2) Unlike N. kaouthia CVF and A. superbus AVF-1, which have potential N-linked glycosylation sites located in both α and β chain, OVF only has N-linked glycosylation site in its α chain as revealed by Schiff's reagent staining and protein sequence analysis; 3) In addition to the 27 well conserved cysteine residues in all known CVFs, OVF have an additional cysteine residue in its γ chain. Understanding the importance of above mentioned specific characteristics might provide useful information on structure

  2. Complement factor I from flatfish half-smooth tongue (Cynoglossus semilaevis) exhibited anti-microbial activities.

    PubMed

    Xiang, Jinsong; Li, Xihong; Chen, Yadong; Lu, Yang; Yu, Mengjun; Chen, Xuejie; Zhang, Wenting; Zeng, Yan; Sun, Luming; Chen, Songlin; Sha, Zhenxia

    2015-11-01

    Complement factor I (Cfi) is a soluble serine protease which plays a crucial role in the modulation of complement cascades. In the presence of substrate modulating cofactors (such as complement factor H, C4bp, CR1, etc), Cfi cleaves and inactivates C3b and C4b, thereby controlling the complement-mediated processes. In this study, we sequenced and characterized Cfi gene from Cynoglossus Semilaevis (designated as CsCfi) for the first time. The full-length cDNA of CsCfi was 2230 bp in length, including a 98 bp 5'-untranslated region (UTR), a 164 bp 3'-UTR and a 1968 bp open reading frame (ORF). It encoded a polypeptide of 656 amino acids, with a molecular mass of 72.28 kDa and an isoelectric point of 7.71. A signal peptide was defined at N-terminus, resulting in a 626-residue mature protein. Multiple sequence alignment revealed that Cfi proteins were well conserved with the typical modular architecture and identical active sites throughout the vertebrates, which suggested the conserved function of Cfi. Phylogenetic analysis indicated that CsCfi and the homologous Cfi sequences from teleosts clustered into a clade, separating from another clade from the cartilaginous fish and other vertebrates. Tissue expression profile analysis by quantitative real-time PCR (qRT-PCR) showed that CsCfi mRNA constitutively expressed in all tested tissues, with the predominant expression in liver and the lowest in stomach. Temporal expression levels of CsCfi after challenging with Vibrio anguillarum showed different expression patterns in intestine, spleen, skin, blood, head kidney and liver. The recombinant CsCfi (rCsCfi) protein showed broad-spectrum antimicrobial activities against the Gram-positive bacteria Staphylococcus aureus and the Gram-negative bacteria Escherichia coli, Pseudomonas aeruginosa and Shewanella putrefaciens. The research revealed that CsCfi plays an important role in C. Semilaevis immunity. PMID:26148855

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

    PubMed

    Sharp, Julia A; Hair, Pamela S; Pallera, Haree K; Kumar, Parvathi S; Mauriello, Clifford T; Nyalwidhe, Julius O; Phelps, Cody A; Park, Dalnam; Thielens, Nicole M; Pascal, Stephen M; Chen, Waldon; Duffy, Diane M; Lattanzio, Frank A; Cunnion, Kenji M; Krishna, Neel K

    2015-01-01

    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. PMID:26196285

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

    PubMed Central

    Sharp, Julia A.; Hair, Pamela S.; Pallera, Haree K.; Kumar, Parvathi S.; Mauriello, Clifford T.; Nyalwidhe, Julius O.; Phelps, Cody A.; Park, Dalnam; Thielens, Nicole M.; Pascal, Stephen M.; Chen, Waldon; Duffy, Diane M.; Lattanzio, Frank A.; Cunnion, Kenji M.; Krishna, Neel K.

    2015-01-01

    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. PMID:26196285

  5. Complement - a key system for immune surveillance and homeostasis

    PubMed Central

    Ricklin, Daniel; Hajishengallis, George; Yang, Kun; Lambris, John D.

    2010-01-01

    Nearly a century after the significance of the human complement system was recognized we have come to realize that its versatile functions extend far beyond the elimination of microbes. Indeed, complement acts as a rapid and efficient immune surveillance system that has distinct effects on healthy and altered host cells and foreign intruders. By eliminating cellular debris and infectious microbes, orchestrating immune responses, and sending `danger' signals, complement contributes substantially to homeostasis, but it may also take action against healthy cells if not properly controlled. This review describes our updated view of the function, structure, and dynamics of the complement network, highlights its interconnection with immunity at large and with other endogenous pathways, and illustrates its dual role in homeostasis and disease. PMID:20720586

  6. Complement-coagulation crosstalk on cellular and artificial surfaces.

    PubMed

    Wiegner, Rebecca; Chakraborty, Shinjini; Huber-Lang, Markus

    2016-10-01

    The humoral serine proteases of the complement system and the coagulation system play central roles during the events of an inflammatory response. While the complement system confers immunoprotective and -regulatory functions, the coagulation cascade is responsible to ensure hemostatic maintenance. Although these two systems individually unfold during inflammation, several studies have reported on the "crosstalk" between components of the complement and the coagulation system in the fluid phase. However, both cascades are usually initiated on or in close proximity to foreign or activated surfaces, and there is increasing evidence for interacting complement and coagulation proteins on various superficial areas on endothelium, circulating entities like platelets, leukocytes, microparticles and pathogens, and even on artificial surfaces. This review aims at summarizing these interactions to complete the picture. PMID:27371975

  7. Complementing Gaia from the ground. The DANCe survey

    NASA Astrophysics Data System (ADS)

    Bouy, H.; Bertin, E.; Sarro, L. M.; Moraux, E.; Barrado, D.; Cuillandre, J. C.; Bouvier, J.; Berihuete, A.; Wright, N.; Beletsky, Y.; Brandner, W.; Olivares, J.

    The DANCe survey aims at complementing Gaia by providing proper motion measurements with a comparable accuracy 4 magnitudes fainter. These measurements are used to identify sub-stellar members of young nearby clusters and associations down the planetary mass regime.

  8. Complement involvement in periodontitis: molecular mechanisms and rational therapeutic approaches

    PubMed Central

    Hajishengallis, George; Maekawa, Tomoki; Abe, Toshiharu; Hajishengallis, Evlambia; Lambris, John D.

    2015-01-01

    The complement system is a network of interacting fluid-phase and cell surface-associated molecules that trigger, amplify, and regulate immune and inflammatory signaling pathways. Dysregulation of this finely balanced network can destabilize host-microbe homeostasis and cause inflammatory tissue damage. Evidence from clinical and animal model-based studies suggests that complement is implicated in the pathogenesis of periodontitis, a polymicrobial community-induced chronic inflammatory disease that destroys the tooth-supporting tissues. This review discusses molecular mechanisms of complement involvement in the dysbiotic transformation of the periodontal microbiome and the resulting destructive inflammation, culminating in loss of periodontal bone support. These mechanistic studies have additionally identified potential therapeutic targets. In this regard, interventional studies in preclinical models have provided proof-of-concept for using complement inhibitors for the treatment of human periodontitis. PMID:26306443

  9. Complementing T-cell Function: An Inhibitory Role of the Complement System in T-cell-Mediated Antitumor Immunity.

    PubMed

    Peng, Weiyi; McKenzie, Jodi A; Hwu, Patrick

    2016-09-01

    New data from Wang and colleagues show that complement C3 suppresses the function of CD8(+) tumor-infiltrating T cells by inhibiting IL10 production, and targeting the complement receptors C3aR and C5aR enhances the antitumor activity of immune checkpoint blockade. Their results not only define a new role of complement receptors as T-cell coinhibitory receptors, but also are useful in the development of novel strategies to improve the effectiveness of cancer immunotherapy. Cancer Discov; 6(9); 953-5. ©2016 AACR.See related article by Wang et al., p. 1022. PMID:27587467

  10. Protective responses to sublytic complement in the retinal pigment epithelium.

    PubMed

    Tan, Li Xuan; Toops, Kimberly A; Lakkaraju, Aparna

    2016-08-01

    The retinal pigment epithelium (RPE) is a key site of injury in inherited and age-related macular degenerations. Abnormal activation of the complement system is a feature of these blinding diseases, yet how the RPE combats complement attack is poorly understood. The complement cascade terminates in the cell-surface assembly of membrane attack complexes (MACs), which promote inflammation by causing aberrant signal transduction. Here, we investigated mechanisms crucial for limiting MAC assembly and preserving cellular integrity in the RPE and asked how these are compromised in models of macular degeneration. Using polarized primary RPE and the pigmented Abca4(-/-) Stargardt disease mouse model, we provide evidence for two protective responses occurring within minutes of complement attack, which are essential for maintaining mitochondrial health in the RPE. First, accelerated recycling of the membrane-bound complement regulator CD59 to the RPE cell surface inhibits MAC formation. Second, fusion of lysosomes with the RPE plasma membrane immediately after complement attack limits sustained elevations in intracellular calcium and prevents mitochondrial injury. Cholesterol accumulation in the RPE, induced by vitamin A dimers or oxidized LDL, inhibits these defense mechanisms by activating acid sphingomyelinase (ASMase), which increases tubulin acetylation and derails organelle traffic. Defective CD59 recycling and lysosome exocytosis after complement attack lead to mitochondrial fragmentation and oxidative stress in the RPE. Drugs that stimulate cholesterol efflux or inhibit ASMase restore both these critical safeguards in the RPE and avert complement-induced mitochondrial injury in vitro and in Abca4(-/-) mice, indicating that they could be effective therapeutic approaches for macular degenerations. PMID:27432952

  11. 21 CFR 866.5240 - Complement components immunological test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Complement components immunological test system. 866.5240 Section 866.5240 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... complement components C1q, C1r, C1s, C2, C3, C4, C5, C6, C7, C8, and C9, in serum, other body fluids,...

  12. 21 CFR 866.5240 - Complement components immunological test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Complement components immunological test system. 866.5240 Section 866.5240 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... complement components C1q, C1r, C1s, C2, C3, C4, C5, C6, C7, C8, and C9, in serum, other body fluids,...

  13. 21 CFR 866.5240 - Complement components immunological test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Complement components immunological test system. 866.5240 Section 866.5240 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... complement components C1q, C1r, C1s, C2, C3, C4, C5, C6, C7, C8, and C9, in serum, other body fluids,...

  14. 21 CFR 866.5240 - Complement components immunological test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Complement components immunological test system. 866.5240 Section 866.5240 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... complement components C1q, C1r, C1s, C2, C3, C4, C5, C6, C7, C8, and C9, in serum, other body fluids,...

  15. Alteration of human erythrocyte membrane properties by complement fixation.

    PubMed Central

    Durocher, J R; Gockerman, J P; Conrad, M E

    1975-01-01

    Erythrocyte survival studies of complement-coated radiolabeled erythrocytes have shown rapid removal of these cells from the peripheral blood with a return of these cells into the circulation within a few hours. We studied complement-coated human erythrocytes and measured surface charge and deformability, two parameters believed to be important in erythrocyte survival. Erythrocytes were coated with complement by two in vitro techniques: the addition of (a) low ionic strength sucrose, and (b) IgM cold agglutinins. Erythrocytes obtained from three patients with cold agglutinin disease were used as a source of in vivo complement-coated cells. No difference was found in surface charge as measured by electrophoretic mobility between erythrocytes from normal subjects and complement-coated erythrocytes from any of the three sources. When deformability was measured by filtration through 3-mum polycarbonate sieves, marked decreases in deformability were found in complement-coated erythrocytes. The filtration returned toward control levels by incubating the complement-coated erythrocytes in serum for 1 h and correlated with decreases in immune adherence. Using screen filtration pressure as a measure of deformability, a positive correlation between number of C3 molecules per erythrocyte and decreased deformability was found. C3b appeared responsible for the decreased deformability of the erythrocytes, since conversion of C3b to C3d resulted in a return of deformability toward normal. The data suggested that the sequestration of complement-coated human erythrocytes in the microvasculature can be explained in part by decreased deformability and changes in immune adherence. PMID:1120777

  16. Von Willebrand factor regulates complement on endothelial cells.

    PubMed

    Noone, Damien G; Riedl, Magdalena; Pluthero, Fred G; Bowman, Mackenzie L; Liszewski, M Kathryn; Lu, Lily; Quan, Yi; Balgobin, Steve; Schneppenheim, Reinhard; Schneppenheim, Sonja; Budde, Ulrich; James, Paula; Atkinson, John P; Palaniyar, Nades; Kahr, Walter H A; Licht, Christoph

    2016-07-01

    Atypical hemolytic uremic syndrome and thrombotic thrombocytopenic purpura have traditionally been considered separate entities. Defects in the regulation of the complement alternative pathway occur in atypical hemolytic uremic syndrome, and defects in the cleavage of von Willebrand factor (VWF)-multimers arise in thrombotic thrombocytopenic purpura. However, recent studies suggest that both entities are related as defects in the disease-causing pathways overlap or show functional interactions. Here we investigate the possible functional link of VWF-multimers and the complement system on endothelial cells. Blood outgrowth endothelial cells (BOECs) were obtained from 3 healthy individuals and 2 patients with Type 3 von Willebrand disease lacking VWF. Cells were exposed to a standardized complement challenge via the combination of classical and alternative pathway activation and 50% normal human serum resulting in complement fixation to the endothelial surface. Under these conditions we found the expected release of VWF-multimers causing platelet adhesion onto BOECs from healthy individuals. Importantly, in BOECs derived from patients with von Willebrand disease complement C3c deposition and cytotoxicity were more pronounced than on BOECs derived from normal individuals. This is of particular importance as primary glomerular endothelial cells display a heterogeneous expression pattern of VWF with overall reduced VWF abundance. Thus, our results support a mechanistic link between VWF-multimers and the complement system. However, our findings also identify VWF as a new complement regulator on vascular endothelial cells and suggest that VWF has a protective effect on endothelial cells and complement-mediated injury. PMID:27236750

  17. Complement and cytokine response in acute Thrombotic Thrombocytopenic Purpura

    PubMed Central

    Westwood, John-Paul; Langley, Kathryn; Heelas, Edward; Machin, Samuel J; Scully, Marie

    2014-01-01

    Complement dysregulation is key in the pathogenesis of atypical Haemolytic Uraemic Syndrome (aHUS), but no clear role for complement has been identified in Thrombotic Thrombocytopenic Purpura (TTP). We aimed to assess complement activation and cytokine response in acute antibody-mediated TTP. Complement C3a and C5a and cytokines (interleukin (IL)-2, IL-4, IL-6, IL-10, tumour necrosis factor, interferon-γ and IL-17a) were measured in 20 acute TTP patients and 49 remission cases. Anti-ADAMTS13 immunoglobulin G (IgG) subtypes were measured in acute patients in order to study the association with complement activation. In acute TTP, median C3a and C5a were significantly elevated compared to remission, C3a 63·9 ng/ml vs. 38·2 ng/ml (P < 0·001) and C5a 16·4 ng/ml vs. 9·29 ng/ml (P < 0·001), respectively. Median IL-6 and IL-10 levels were significantly higher in the acute vs. remission groups, IL-6: 8 pg/ml vs. 2 pg/ml (P = 0·003), IL-10: 6 pg/ml vs. 2 pg/ml (P < 0·001). C3a levels correlated with both anti-ADAMTS13 IgG (rs = 0·604, P = 0·017) and IL-10 (rs = 0·692, P = 0·006). No anti-ADAMTS13 IgG subtype was associated with higher complement activation, but patients with the highest C3a levels had 3 or 4 IgG subtypes present. These results suggest complement anaphylatoxin levels are higher in acute TTP cases than in remission, and the complement response seen acutely may relate to anti-ADAMTS13 IgG antibody and IL-10 levels. PMID:24372446

  18. Legionella pneumophila lipopolysaccharide activates the classical complement pathway.

    PubMed Central

    Mintz, C S; Schultz, D R; Arnold, P I; Johnson, W

    1992-01-01

    Legionella pneumophila is a gram-negative bacterium capable of entering and growing in alveolar macrophages and monocytes. Complement and complement receptors are important in the uptake of L. pneumophila by human mononuclear phagocytes. The surface molecules of L. pneumophila that activate the complement system are unknown. To identify these factors, we investigated the effects of L. pneumophila lipopolysaccharide (LPS) on the classical and alternative complement pathways of normal human serum by functional hemolytic assays. Although incubation of LPS in normal human serum at 37 degrees C resulted in the activation of both pathways, complement activation proceeded primarily through the classical pathway. Activation of the classical pathway by LPS was dependent on natural antibodies of the immunoglobulin M class that were present in various quantities in sera from different normal individuals but were absent in an immunoglobulin-deficient serum obtained from an agammaglobulinemic patient. Additional studies using sheep erythrocytes coated with LPS suggested that the antibodies recognized antigenic sites in the carbohydrate portion of LPS. The ability of LPS to interact with the complement system suggests a role for LPS in the uptake of L. pneumophila by mononuclear phagocytes. PMID:1612744

  19. Complement involvement in kidney diseases: From physiopathology to therapeutical targeting

    PubMed Central

    Salvadori, Maurizio; Rosso, Giuseppina; Bertoni, Elisabetta

    2015-01-01

    Complement cascade is involved in several renal diseases and in renal transplantation. The different components of the complement cascade might represent an optimal target for innovative therapies. In the first section of the paper the authors review the physiopathology of complement involvement in renal diseases and transplantation. In some cases this led to a reclassification of renal diseases moving from a histopathological to a physiopathological classification. The principal issues afforded are: renal diseases with complement over activation, renal diseases with complement dysregulation, progression of renal diseases and renal transplantation. In the second section the authors discuss the several complement components that could represent a therapeutic target. Even if only the anti C5 monoclonal antibody is on the market, many targets as C1, C3, C5a and C5aR are the object of national or international trials. In addition, many molecules proved to be effective in vitro or in preclinical trials and are waiting to move to human trials in the future. PMID:25949931

  20. Cleaving DNA with DNA

    NASA Astrophysics Data System (ADS)

    Carmi, Nir; Balkhi, Shameelah R.; Breaker, Ronald R.

    1998-03-01

    A DNA structure is described that can cleave single-stranded DNA oligonucleotides in the presence of ionic copper. This ``deoxyribozyme'' can self-cleave or can operate as a bimolecular complex that simultaneously makes use of duplex and triplex interactions to bind and cleave separate DNA substrates. Bimolecular deoxyribozyme-mediated strand scission proceeds with a kobs of 0.2 min-1, whereas the corresponding uncatalyzed reaction could not be detected. The duplex and triplex recognition domains can be altered, making possible the targeted cleavage of single-stranded DNAs with different nucleotide sequences. Several small synthetic DNAs were made to function as simple ``restriction enzymes'' for the site-specific cleavage of single-stranded DNA.

  1. Antibodies That Efficiently Form Hexamers upon Antigen Binding Can Induce Complement-Dependent Cytotoxicity under Complement-Limiting Conditions.

    PubMed

    Cook, Erika M; Lindorfer, Margaret A; van der Horst, Hilma; Oostindie, Simone; Beurskens, Frank J; Schuurman, Janine; Zent, Clive S; Burack, Richard; Parren, Paul W H I; Taylor, Ronald P

    2016-09-01

    Recently, we demonstrated that IgG Abs can organize into ordered hexamers after binding their cognate Ags expressed on cell surfaces. This process is dependent on Fc:Fc interactions, which promote C1q binding, the first step in classical pathway complement activation. We went on to engineer point mutations that stimulated IgG hexamer formation and complement-dependent cytotoxicity (CDC). The hexamer formation-enhanced (HexaBody) CD20 and CD38 mAbs support faster, more robust CDC than their wild-type counterparts. To further investigate the CDC potential of these mAbs, we used flow cytometry, high-resolution digital imaging, and four-color confocal microscopy to examine their activity against B cell lines and primary chronic lymphocytic leukemia cells in sera depleted of single complement components. We also examined the CDC activity of alemtuzumab (anti-CD52) and mAb W6/32 (anti-HLA), which bind at high density to cells and promote substantial complement activation. Although we observed little CDC for mAb-opsonized cells reacted with sera depleted of early complement components, we were surprised to discover that the Hexabody mAbs, as well as ALM and W6/32, were all quite effective at promoting CDC in sera depleted of individual complement components C6 to C9. However, neutralization studies conducted with an anti-C9 mAb verified that C9 is required for CDC activity against cell lines. These highly effective complement-activating mAbs efficiently focus activated complement components on the cell, including C3b and C9, and promote CDC with a very low threshold of MAC binding, thus providing additional insight into their enhanced efficacy in promoting CDC. PMID:27474078

  2. Antibodies That Efficiently Form Hexamers upon Antigen Binding Can Induce Complement-Dependent Cytotoxicity under Complement-Limiting Conditions

    PubMed Central

    Cook, Erika M.; Lindorfer, Margaret A.; van der Horst, Hilma; Oostindie, Simone; Beurskens, Frank J.; Schuurman, Janine; Zent, Clive S.; Burack, Richard; Parren, Paul W. H. I.

    2016-01-01

    Recently, we demonstrated that IgG Abs can organize into ordered hexamers after binding their cognate Ags expressed on cell surfaces. This process is dependent on Fc:Fc interactions, which promote C1q binding, the first step in classical pathway complement activation. We went on to engineer point mutations that stimulated IgG hexamer formation and complement-dependent cytotoxicity (CDC). The hexamer formation–enhanced (HexaBody) CD20 and CD38 mAbs support faster, more robust CDC than their wild-type counterparts. To further investigate the CDC potential of these mAbs, we used flow cytometry, high-resolution digital imaging, and four-color confocal microscopy to examine their activity against B cell lines and primary chronic lymphocytic leukemia cells in sera depleted of single complement components. We also examined the CDC activity of alemtuzumab (anti-CD52) and mAb W6/32 (anti-HLA), which bind at high density to cells and promote substantial complement activation. Although we observed little CDC for mAb-opsonized cells reacted with sera depleted of early complement components, we were surprised to discover that the Hexabody mAbs, as well as ALM and W6/32, were all quite effective at promoting CDC in sera depleted of individual complement components C6 to C9. However, neutralization studies conducted with an anti-C9 mAb verified that C9 is required for CDC activity against cell lines. These highly effective complement-activating mAbs efficiently focus activated complement components on the cell, including C3b and C9, and promote CDC with a very low threshold of MAC binding, thus providing additional insight into their enhanced efficacy in promoting CDC. PMID:27474078

  3. Bisretinoid-mediated Complement Activation on Retinal Pigment Epithelial Cells Is Dependent on Complement Factor H Haplotype*

    PubMed Central

    Radu, Roxana A.; Hu, Jane; Jiang, Zhichun; Bok, Dean

    2014-01-01

    Age-related macular degeneration (AMD) is a common central blinding disease of the elderly. Homozygosity for a sequence variant causing Y402H and I62V substitutions in the gene for complement factor H (CFH) is strongly associated with risk of AMD. CFH, secreted by many cell types, including those of the retinal pigment epithelium (RPE), is a regulatory protein that inhibits complement activation. Recessive Stargardt maculopathy is another central blinding disease caused by mutations in the gene for ABCA4, a transporter in photoreceptor outer segments (OS) that clears retinaldehyde and prevents formation of toxic bisretinoids. Photoreceptors daily shed their distal OS, which are phagocytosed by the RPE cells. Here, we investigated the relationship between the CFH haplotype of human RPE (hRPE) cells, exposure to OS containing bisretinoids, and complement activation. We show that hRPE cells of the AMD-predisposing CFH haplotype (HH402/VV62) are attacked by complement following exposure to bisretinoid-containing Abca4−/− OS. This activation was dependent on factor B, indicating involvement of the alternative pathway. In contrast, hRPE cells of the AMD-protective CFH haplotype (YY402/II62) showed no complement activation following exposure to either Abca4−/− or wild-type OS. The AMD-protective YY402/II62 hRPE cells were more resistant to the membrane attack complex, whereas HH402/VV62 hRPE cells showed significant membrane attack complex deposition following ingestion of Abca4−/− OS. These results suggest that bisretinoid accumulation in hRPE cells stimulates activation and dysregulation of complement. Cells with an intact complement negative regulatory system are protected from complement attack, whereas cells with reduced CFH synthesis because of the Y402H and I62V substitutions are vulnerable to disease. PMID:24550392

  4. Complement Activation in Patients with Focal Segmental Glomerulosclerosis

    PubMed Central

    Thurman, Joshua M.; Wong, Maria; Renner, Brandon; Frazer-Abel, Ashley; Giclas, Patricia C.; Joy, Melanie S.; Jalal, Diana; Radeva, Milena K.; Gassman, Jennifer; Gipson, Debbie S.; Kaskel, Frederick; Friedman, Aaron; Trachtman, Howard

    2015-01-01

    Background Recent pre-clinical studies have shown that complement activation contributes to glomerular and tubular injury in experimental FSGS. Although complement proteins are detected in the glomeruli of some patients with FSGS, it is not known whether this is due to complement activation or whether the proteins are simply trapped in sclerotic glomeruli. We measured complement activation fragments in the plasma and urine of patients with primary FSGS to determine whether complement activation is part of the disease process. Study Design Plasma and urine samples from patients with biopsy-proven FSGS who participated in the FSGS Clinical Trial were analyzed. Setting and Participants We identified 19 patients for whom samples were available from weeks 0, 26, 52 and 78. The results for these FSGS patients were compared to results in samples from 10 healthy controls, 10 patients with chronic kidney disease (CKD), 20 patients with vasculitis, and 23 patients with lupus nephritis. Outcomes Longitudinal control of proteinuria and estimated glomerular filtration rate (eGFR). Measurements Levels of the complement fragments Ba, Bb, C4a, and sC5b-9 in plasma and urine. Results Plasma and urine Ba, C4a, sC5b-9 were significantly higher in FSGS patients at the time of diagnosis than in the control groups. Plasma Ba levels inversely correlated with the eGFR at the time of diagnosis and at the end of the study. Plasma and urine Ba levels at the end of the study positively correlated with the level of proteinuria, the primary outcome of the study. Limitations Limited number of patients with samples from all time-points. Conclusions The complement system is activated in patients with primary FSGS, and elevated levels of plasma Ba correlate with more severe disease. Measurement of complement fragments may identify a subset of patients in whom the complement system is activated. Further investigations are needed to confirm our findings and to determine the prognostic significance of

  5. Transcriptome Analysis of the Innate Immunity-Related Complement System in Spleen Tissue of Ctenopharyngodon idella Infected with Aeromonas hydrophila

    PubMed Central

    Dang, Yunfei; Xu, Xiaoyan; Shen, Yubang; Hu, Moyan; Zhang, Meng; Li, Lisen; Lv, Liqun; Li, Jiale

    2016-01-01

    The grass carp (Ctenopharyngodon idella) is an important commercial farmed herbivorous fish species in China, but is susceptible to Aeromonas hydrophila infections. In the present study, we performed de novo RNA-Seq sequencing of spleen tissue from specimens of a disease-resistant family, which were given intra-peritoneal injections containing PBS with or without a dose of A. hydrophila. The fish were sampled from the control group at 0 h, and from the experimental group at 4, 8, 12, 24, 48 and 72 h. 122.18 million clean reads were obtained from the normalized cDNA libraries; these were assembled into 425,260 contigs and then 191,795 transcripts. Of those, 52,668 transcripts were annotated with the NCBI Nr database, and 41,347 of the annotated transcripts were assigned into 90 functional groups. 20,569 unigenes were classified into six main categories, including 38 secondary KEGG pathways. 2,992 unigenes were used in the analysis of differentially expressed genes (DEGs). 89 of the putative DEGs were related to the immune system and 41 of them were involved in the complement and coagulation cascades pathway. This study provides insights into the complement and complement-related pathways involved in innate immunity, through expression profile analysis of the genomic resources in C. idella. We conclude that complement and complement-related genes play important roles during defense against A. hydrophila infection. The immune response is activated at 4 h after the bacterial injections, indicating that the complement pathways are activated at the early stage of bacterial infection. The study has improved our understanding of the immune response mechanisms in C. idella to bacterial pathogens. PMID:27383749

  6. 21 CFR 866.5260 - Complement C3b inactivator immunological test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... immunochemical techniques the complement C3b inactivator (a plasma protein) in serum. Complement is a group of serum proteins that destroy infectious agents. Measurement of complement C3b inactivator aids in...

  7. 21 CFR 866.5260 - Complement C3b inactivator immunological test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... immunochemical techniques the complement C3b inactivator (a plasma protein) in serum. Complement is a group of serum proteins that destroy infectious agents. Measurement of complement C3b inactivator aids in...

  8. 21 CFR 866.5260 - Complement C3b inactivator immunological test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... immunochemical techniques the complement C3b inactivator (a plasma protein) in serum. Complement is a group of serum proteins that destroy infectious agents. Measurement of complement C3b inactivator aids in...

  9. 21 CFR 866.5260 - Complement C3b inactivator immunological test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... immunochemical techniques the complement C3b inactivator (a plasma protein) in serum. Complement is a group of serum proteins that destroy infectious agents. Measurement of complement C3b inactivator aids in...

  10. 21 CFR 866.5260 - Complement C3b inactivator immunological test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... immunochemical techniques the complement C3b inactivator (a plasma protein) in serum. Complement is a group of serum proteins that destroy infectious agents. Measurement of complement C3b inactivator aids in...