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Sample records for protein response protects

  1. HACE1-dependent protein degradation provides cardiac protection in response to haemodynamic stress

    NASA Astrophysics Data System (ADS)

    Zhang, Liyong; Chen, Xin; Sharma, Parveen; Moon, Mark; Sheftel, Alex D.; Dawood, Fayez; Nghiem, Mai P.; Wu, Jun; Li, Ren-Ke; Gramolini, Anthony O.; Sorensen, Poul H.; Penninger, Josef M.; Brumell, John H.; Liu, Peter P.

    2014-03-01

    The HECT E3 ubiquitin ligase HACE1 is a tumour suppressor known to regulate Rac1 activity under stress conditions. HACE1 is increased in the serum of patients with heart failure. Here we show that HACE1 protects the heart under pressure stress by controlling protein degradation. Hace1 deficiency in mice results in accelerated heart failure and increased mortality under haemodynamic stress. Hearts from Hace1-/- mice display abnormal cardiac hypertrophy, left ventricular dysfunction, accumulation of LC3, p62 and ubiquitinated proteins enriched for cytoskeletal species, indicating impaired autophagy. Our data suggest that HACE1 mediates p62-dependent selective autophagic turnover of ubiquitinated proteins by its ankyrin repeat domain through protein-protein interaction, which is independent of its E3 ligase activity. This would classify HACE1 as a dual-function E3 ligase. Our finding that HACE1 has a protective function in the heart in response to haemodynamic stress suggests that HACE1 may be a potential diagnostic and therapeutic target for heart disease.

  2. Oxidative stress induces mitochondrial dysfunction and a protective unfolded protein response in RPE cells.

    PubMed

    Cano, Marisol; Wang, Lei; Wan, Jun; Barnett, Bradley P; Ebrahimi, Katayoon; Qian, Jiang; Handa, James T

    2014-04-01

    How cells degenerate from oxidative stress in aging-related disease is incompletely understood. This study's intent was to identify key cytoprotective pathways activated by oxidative stress and determine the extent of their protection. Using an unbiased strategy with microarray analysis, we found that retinal pigmented epithelial (RPE) cells treated with cigarette smoke extract (CSE) had overrepresented genes involved in the antioxidant and unfolded protein response (UPR). Differentially expressed antioxidant genes were predominantly located in the cytoplasm, with no induction of genes that neutralize superoxide and H2O2 in the mitochondria, resulting in accumulation of superoxide and decreased ATP production. Simultaneously, CSE induced the UPR sensors IRE1α, p-PERK, and ATP6, including CHOP, which was cytoprotective because CHOP knockdown decreased cell viability. In mice given intravitreal CSE, the RPE had increased IRE1α and decreased ATP and developed epithelial-mesenchymal transition, as suggested by decreased LRAT abundance, altered ZO-1 immunolabeling, and dysmorphic cell shape. Mildly degenerated RPE from early age-related macular degeneration (AMD) samples had prominent IRE1α, but minimal mitochondrial TOM20 immunolabeling. Although oxidative stress is thought to induce an antioxidant response with cooperation between the mitochondria and the ER, herein we show that mitochondria become impaired sufficiently to induce epithelial-mesenchymal transition despite a protective UPR. With similar responses in early AMD samples, these results suggest that mitochondria are vulnerable to oxidative stress despite a protective UPR during the early phases of aging-related disease.

  3. The Mitochondrial Unfolded Protein Response Protects against Anoxia in Caenorhabditis elegans

    PubMed Central

    Peña, Salvador; Sherman, Teresa; Brookes, Paul S.; Nehrke, Keith

    2016-01-01

    The mitochondrial unfolded protein response (UPRmt) is a surveillance pathway that defends proteostasis in the “powerhouse” of the cell. Activation of the UPRmt protects against stresses imposed by reactive oxygen species, respiratory chain deficits, and pathologic bacteria. Consistent with the UPRmt’s role in adaption, we found that either its pharmacological or genetic activation by ethidium bromide (EtBr) or RNAi of the mitochondrial AAA-protease spg-7 was sufficient to reduce death in an anoxia-based Caenorhabditis elegans model of ischemia-reperfusion injury. The UPRmt-specific transcription factor atfs-1 was necessary for protection and atfs-1 gain-of-function (gf) mutants were endogenously protected from both death and dysfunction. Neurons exhibited less axonal degeneration following non-lethal anoxia-reperfusion (A-R) when the UPRmt was pre-activated, and consistent with the concept of mitochondrial stress leading to cell non-autonomous (ie. “remote”) effects, we found that restricted activation of the UPRmt in neurons decreased A-R death. However, expression of the atfs-1(gf) mutant in neurons, which resulted in a robust activation of a neuronal UPRmt, did not upregulate the UPRmt in distal tissues, nor did it protect the worms from A-R toxicity. These findings suggest that remote signaling requires additional component(s) acting downstream of de facto mitochondrial stress. PMID:27459203

  4. Mechanisms of protective immune responses induced by the Plasmodium falciparum circumsporozoite protein-based, self-assembling protein nanoparticle vaccine

    PubMed Central

    2013-01-01

    Background A lack of defined correlates of immunity for malaria, combined with the inability to induce long-lived sterile immune responses in a human host, demonstrate a need for improved understanding of potentially protective immune mechanisms for enhanced vaccine efficacy. Protective sterile immunity (>90%) against the Plasmodium falciparum circumsporozoite protein (CSP) has been achieved using a transgenically modified Plasmodium berghei sporozoite (Tg-Pb/PfCSP) and a self-assembling protein nanoparticle (SAPN) vaccine presenting CSP epitopes (PfCSP-SAPN). Here, several possible mechanisms involved in the independently protective humoral and cellular responses induced following SAPN immunization are described. Methods Inbred mice were vaccinated with PfCSP-SAPN in PBS. Serum antibodies were harvested and effects on P. falciparum sporozoites mobility and integrity were examined using phase contrast microscopy. The functionality of SAPN-induced antibodies on inhibition of sporozoite invasion and growth within primary human hepatocytes was also examined. The internal processing of SAPN by bone marrow-derived dendritic cells (BMDDC), using organelle-specific, fluorescent-tagged antibody or gold-encapsulated SAPN, was observed using confocal or electron microscopy, respectively. Results The results of this work demonstrate that PfCSP-SAPN induces epitope-specific antibody titers, predominantly of the Th2 isotype IgG1, and that serum antibodies from PfCSP-SAPN-immunized mice appear to target P. falciparum sporozoites via the classical pathway of complement. This results in sporozoite death as indicated by cessation of motility and the circumsporozoite precipitation reaction. Moreover, PfCSP-SAPN-induced antibodies are able to inhibit wild-type P. falciparum sporozoite invasion and growth within cultured primary human hepatocytes. In addition, the observation that PfCSP-SAPN are processed (and presented) to the immune system by dendritic cells in a slow and continuous

  5. The unfolded protein response has a protective role in yeast models of classic galactosemia.

    PubMed

    De-Souza, Evandro A; Pimentel, Felipe S A; Machado, Caio M; Martins, Larissa S; da-Silva, Wagner S; Montero-Lomelí, Mónica; Masuda, Claudio A

    2014-01-01

    Classic galactosemia is a human autosomal recessive disorder caused by mutations in the GALT gene (GAL7 in yeast), which encodes the enzyme galactose-1-phosphate uridyltransferase. Here we show that the unfolded protein response pathway is triggered by galactose in two yeast models of galactosemia: lithium-treated cells and the gal7Δ mutant. The synthesis of galactose-1-phosphate is essential to trigger the unfolded protein response under these conditions because the deletion of the galactokinase-encoding gene GAL1 completely abolishes unfolded protein response activation and galactose toxicity. Impairment of the unfolded protein response in both yeast models makes cells even more sensitive to galactose, unmasking its cytotoxic effect. These results indicate that endoplasmic reticulum stress is induced under galactosemic conditions and underscores the importance of the unfolded protein response pathway to cellular adaptation in these models of classic galactosemia.

  6. Induction of a protective response in mice by the dengue virus NS3 protein using DNA vaccines.

    PubMed

    Costa, Simone M; Yorio, Anna Paula; Gonçalves, Antônio J S; Vidale, Mariana M; Costa, Emmerson C B; Mohana-Borges, Ronaldo; Motta, Marcia A; Freire, Marcos S; Alves, Ada M B

    2011-01-01

    The dengue non-structural 3 (NS3) is a multifunctional protein, containing a serino-protease domain, located at the N-terminal portion, and helicase, NTPase and RTPase domains present in the C-terminal region. This protein is considered the main target for CD4+ and CD8+ T cell responses during dengue infection, which may be involved in protection. However, few studies have been undertaken evaluating the use of this protein as a protective antigen against dengue, as well as other flavivirus. In the present work, we investigate the protective efficacy of DNA vaccines based on the NS3 protein from DENV2. Different recombinant plasmids were constructed, encoding either the full-length NS3 protein or only its functional domains (protease and helicase), fused or not to a signal peptide (t-PA). The recombinant proteins were successfully expressed in transfected BHK-21 cells, and only plasmids encoding the t-PA signal sequence mediated protein secretion. Balb/c mice were immunized with the different DNA vaccines and challenged with a lethal dose of DENV2. Most animals immunized with plasmids encoding the full-length NS3 or the helicase domain survived challenge, regardless of the presence of the t-PA. However, some mice presented clinical signs of infection with high morbidity (hind leg paralysis and hunched posture), mainly in animal groups immunized with the DNA vaccines based on the helicase domain. On the other hand, inoculation with plasmids encoding the protease domain did not induce any protection, since mortality and morbidity rates in these mouse groups were similar to those detected in the control animals. The cellular immune response was analyzed by ELISPOT with a specific-CD8+ T cell NS3 peptide. Results revealed that the DNA vaccines based on the full-length protein induced the production of INF-γ, thus suggesting the involvement of this branch of the immune system in the protection.

  7. The mitochondrial unfolded protein response activator ATFS-1 protects cells from inhibition of the mevalonate pathway

    PubMed Central

    Rauthan, Manish; Ranji, Parmida; Aguilera Pradenas, Nataly; Pitot, Christophe; Pilon, Marc

    2013-01-01

    Statins are cholesterol-lowering drugs that inhibit 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase, the rate-limiting enzyme in the synthesis of cholesterol via the mevalonate pathway. This pathway also produces coenzyme Q (a component of the respiratory chain), dolichols (important for protein glycosylation), and isoprenoids (lipid moieties responsible for the membrane association of small GTPases). We previously showed that the nematode Caenorhabditis elegans is useful to study the noncholesterol effects of statins because its mevalonate pathway lacks the sterol synthesis branch but retains all other branches. Here, from a screen of 150,000 mutagenized genomes, we isolated four C. elegans mutants resistant to statins by virtue of gain-of-function mutations within the first six amino acids of the protein ATFS-1, the key regulator of the mitochondrial unfolded protein response that includes activation of the chaperones HSP-6 and HSP-60. The atfs-1 gain-of-function mutants are also resistant to ibandronate, an inhibitor of an enzyme downstream of HMG-CoA reductase, and to gliotoxin, an inhibitor acting on a subbranch of the pathway important for protein prenylation, and showed improved mitochondrial function and protein prenylation in the presence of statins. Additionally, preinduction of the mitochondrial unfolded protein response in wild-type worms using ethidium bromide or paraquat triggered statin resistance, and similar observations were made in Schizosaccharomyces pombe and in a mammalian cell line. We conclude that statin resistance through maintenance of mitochondrial homeostasis is conserved across species, and that the cell-lethal effects of statins are caused primarily through impaired protein prenylation that results in mitochondria dysfunction. PMID:23530189

  8. A Computationally Designed Hemagglutinin Stem-Binding Protein Provides In Vivo Protection from Influenza Independent of a Host Immune Response

    PubMed Central

    Koday, Merika Treants; Nelson, Jorgen; Chevalier, Aaron; Koday, Michael; Kalinoski, Hannah; Stewart, Lance; Carter, Lauren; Nieusma, Travis; Lee, Peter S.; Ward, Andrew B.; Wilson, Ian A.; Dagley, Ashley; Smee, Donald F.; Baker, David; Fuller, Deborah Heydenburg

    2016-01-01

    Broadly neutralizing antibodies targeting a highly conserved region in the hemagglutinin (HA) stem protect against influenza infection. Here, we investigate the protective efficacy of a protein (HB36.6) computationally designed to bind with high affinity to the same region in the HA stem. We show that intranasal delivery of HB36.6 affords protection in mice lethally challenged with diverse strains of influenza independent of Fc-mediated effector functions or a host antiviral immune response. This designed protein prevents infection when given as a single dose of 6.0 mg/kg up to 48 hours before viral challenge and significantly reduces disease when administered as a daily therapeutic after challenge. A single dose of 10.0 mg/kg HB36.6 administered 1-day post-challenge resulted in substantially better protection than 10 doses of oseltamivir administered twice daily for 5 days. Thus, binding of HB36.6 to the influenza HA stem region alone, independent of a host response, is sufficient to reduce viral infection and replication in vivo. These studies demonstrate the potential of computationally designed binding proteins as a new class of antivirals for influenza. PMID:26845438

  9. The Rai (Shc C) adaptor protein regulates the neuronal stress response and protects against cerebral ischemia

    PubMed Central

    Troglio, Flavia; Echart, Cinara; Gobbi, Alberto; Pawson, Tony; Pelicci, Pier Giuseppe; De Simoni, Maria Grazia; Pelicci, Giuliana

    2004-01-01

    Rai (Shc C or N-Shc) is a neuron-specific member of the family of Shc-like adaptor proteins. Rai functions in the cytoplasmic propagation of Ret-dependent survival signals and regulates, in vivo, the number of sympathetic neurons. We report here a function of Rai, i.e., the regulation of the neuronal adaptive response to environmental stresses. We demonstrate that (i) primary cultures of cortical neurons from Rai-/- mice are more sensitive to apoptosis induced by hypoxia or oxidative stress; (ii) in Rai-/- mice, ischemia/reperfusion injury induces severe neurological deficits, increased apoptosis and size of the infarct area, and significantly higher mortality; and (iii) Rai functions as a stress-response gene that increases phosphatidylinositol 3-kinase activation and Akt phosphorylation after hypoxic or oxidation insults. These data suggest that Rai has a functional neuroprotective role in brain injury, with possible implications in the treatment of stroke. PMID:15494442

  10. Protective Antibody and CD8+ T-Cell Responses to the Plasmodium falciparum Circumsporozoite Protein Induced by a Nanoparticle Vaccine

    PubMed Central

    Kaba, Stephen A.; McCoy, Margaret E.; Doll, Tais A. P. F.; Brando, Clara; Guo, Qin; Dasgupta, Debleena; Yang, Yongkun; Mittelholzer, Christian; Spaccapelo, Roberta; Crisanti, Andrea; Burkhard, Peter; Lanar, David E.

    2012-01-01

    Background The worldwide burden of malaria remains a major public health problem due, in part, to the lack of an effective vaccine against the Plasmodium falciparum parasite. An effective vaccine will most likely require the induction of antigen specific CD8+ and CD4+ T-cells as well as long-lasting antibody responses all working in concert to eliminate the infection. We report here the effective modification of a self-assembling protein nanoparticle (SAPN) vaccine previously proven effective in control of a P. berghei infection in a rodent model to now present B- and T-cell epitopes of the human malaria parasite P. falciparum in a platform capable of being used in human subjects. Methodology/Principal Findings To establish the basis for a SAPN-based vaccine, B- and CD8+ T-cell epitopes from the P. falciparum circumsporozoite protein (PfCSP) and the universal CD4 T-helper epitope PADRE were engineered into a versatile small protein (∼125 amino acids) that self-assembles into a spherical nanoparticle repetitively displaying the selected epitopes. P. falciparum epitope specific immune responses were evaluated in mice using a transgenic P. berghei malaria parasite of mice expressing the human malaria full-length P. falciparum circumsporozoite protein (Tg-Pb/PfCSP). We show that SAPN constructs, delivered in saline, can induce high-titer, long-lasting (1 year) protective antibody and poly-functional (IFNγ+, IL-2+) long-lived central memory CD8+ T-cells. Furthermore, we demonstrated that these Ab or CD8+ T–cells can independently provide sterile protection against a lethal challenge of the transgenic parasites. Conclusion The SAPN construct induces long-lasting antibody and cellular immune responses to epitope specific sequences of the P. falciparum circumsporozoite protein (PfCSP) and prevents infection in mice by a transgenic P. berghei parasite displaying the full length PfCSP. PMID:23144750

  11. Immune responses and protective effect in mice vaccinated orally with surface sporozoite protein of Eimeria falciformis in ISCOMs.

    PubMed

    Kazanji, M; Laurent, F; Péry, P

    1994-07-01

    Immunostimulating complexes (ISCOMs) were built after treatment of a purified surface protein from Eimeria falciformis sporozoites with a palmitic acid derivation, leading to a high ratio (33-64%) of P27 incorporation in these cage-like structures. P27 kept its antigenicity after incorporation in ISCOMs, which induced, after iterative intubations by the oral route to groups of mice, a systemic IgG response, a local IgA response, and a local enhanced cellular response as demonstrated by lymphoproliferation of mesenteric lymph node cells upon in vitro stimulation with antigen. This immunization (120 micrograms in six oral doses at 2-day intervals) afforded mice a partial protection (60%) against a subsequent 400 oocyst challenge. The reduction in daily oocyst excretion was corroborated by significantly different weight losses between immunized and control mice on days 9 and 10 postinfection and the subsequent death of these control mice. These observations provide the first application of ISCOMs to parasitic intestinal diseases.

  12. The Respiratory Syncytial Virus G Protein Conserved Domain Induces a Persistent and Protective Antibody Response in Rodents

    PubMed Central

    Nguyen, Thien N.; Power, Ultan F.; Robert, Alain; Haeuw, Jean-François; Helffer, Katia; Perez, Amadeo; Asin, Miguel-Angel; Corvaia, Nathalie; Libon, Christine

    2012-01-01

    Respiratory syncytial virus (RSV) is an important cause of severe upper and lower respiratory disease in infants and in the elderly. There are 2 main RSV subtypes A and B. A recombinant vaccine was designed based on the central domain of the RSV-A attachment G protein which we had previously named G2Na (aa130–230). Here we evaluated immunogenicity, persistence of antibody (Ab) response and protective efficacy induced in rodents by: (i) G2Na fused to DT (Diphtheria toxin) fragments in cotton rats. DT fusion did not potentiate neutralizing Ab responses against RSV-A or cross-reactivity to RSV-B. (ii) G2Nb (aa130–230 of the RSV-B G protein) either fused to, or admixed with G2Na. G2Nb did not induce RSV-B-reactive Ab responses. (iii) G2Na at low doses. Two injections of 3 µg G2Na in Alum were sufficient to induce protective immune responses in mouse lungs, preventing RSV-A and greatly reducing RSV-B infections. In cotton rats, G2Na-induced RSV-reactive Ab and protective immunity against RSV-A challenge that persisted for at least 24 weeks. (iv) injecting RSV primed mice with a single dose of G2Na/Alum or G2Na/PLGA [poly(D,L-lactide-co-glycolide]. Despite the presence of pre-existing RSV-specific Abs, these formulations effectively boosted anti-RSV Ab titres and increased Ab titres persisted for at least 21 weeks. Affinity maturation of these Abs increased from day 28 to day 148. These data indicate that G2Na has potential as a component of an RSV vaccine formulation. PMID:22479601

  13. Screening and Characterization of Drugs That Protect Corneal Endothelial Cells Against Unfolded Protein Response and Oxidative Stress

    PubMed Central

    Kim, Eun Chul; Toyono, Tetsuya; Berlinicke, Cynthia A.; Zack, Donald J.; Jurkunas, Ula; Usui, Tomohiko; Jun, Albert S.

    2017-01-01

    Purpose To screen for and characterize compounds that protect corneal endothelial cells against unfolded protein response (UPR) and oxidative stress. Methods Bovine corneal endothelial cells (BCECs) were treated for 48 hours with 640 compounds from a Food and Drug Administration (FDA)-approved drug library and then challenged with thapsigargin or H2O2 to induce UPR or oxidative stress, respectively. Cell viability was measured using the CellTiter-Glo survival assay. Selected “hits” were subjected to further dose-response testing, and their ability to modulate expression of UPR and oxidative stress markers was assessed by RT-PCR, Western blot, and measurement of protein carbonyl and 8-hydroxydeoxyguanosine (8-OHdG) adducts in immortalized human corneal endothelial cells (iHCECs). Results Forty-one drugs at 20 μM and 55 drugs at 100 μM increased survival of H2O2-challenged cells, and 8 drugs at 20 μM and 2 drugs at 100 μM increased survival of thapsigargin-challenged cells, compared with untreated control cells. Nicergoline, ergothioneine, nimesulide, oxotremorine, and mefenamic acid increased survival of both H2O2- and thapsigargin-challenged cells. Oxotremorine altered DNA damage inducible 3 (CHOP) gene expression, glucose-regulated protein 78 kDa (GRP78) and activating transcription factor 4 (ATF4) protein expression, and protein carbonyl and 8-OHdG levels. Mefenamic acid altered GRP78 protein expression and protein carbonyl and 8-OHdG levels. Conclusions Oxotremorine and mefenamic acid are potential survival factors for corneal endothelial cells under UPR and oxidative stress. The described assay can be further expanded to screen additional drugs for potential therapeutic effect in corneal endothelial diseases such as Fuchs' endothelial corneal dystrophy. PMID:28159976

  14. Connecting two-component regulatory systems by a protein that protects a response regulator from dephosphorylation by its cognate sensor

    PubMed Central

    Kato, Akinori; Groisman, Eduardo A.

    2004-01-01

    A fundamental question in signal transduction is how an organism integrates multiple signals into a cellular response. Here we report the mechanism by which the Salmonella PmrA/PmrB two-component system responds to the signal controlling the PhoP/PhoQ two-component system. We establish that the PhoP-activated PmrD protein binds to the phosphorylated form of the response regulator PmrA, preventing both its intrinsic dephosphorylation and that promoted by its cognate sensor kinase PmrB. This results in PmrA-mediated transcription because phosphorylated PmrA exhibits higher affinity for its target promoters than unphosphorylated PmrA. A PmrD-independent form of the PmrA protein was resistant to PmrB-catalyzed dephosphorylation and promoted transcription of PmrA-activated genes in the absence of inducing signals. This is the first example of a protein that enables a two-component system to respond to the signal governing a different two-component system by protecting the phosphorylated form of a response regulator. PMID:15371344

  15. The responsibility to protect.

    PubMed

    Deutscher, Matt

    2005-01-01

    The decision whether, if ever, to intervene in the affairs of a sovereign state with military force has become a critical issue of the post Cold War era. In 2000 the Canadian government launched the International Commission on Intervention and State Sovereignty (ICISS), which in 2001 published its findings in The Responsibility to Protect. The Commission found broad support for the notion of sovereignty not only as a right, but also a responsibility, the responsibility of a state to provide protection for its people. The primary responsibility for protecting citizens rests with states. But when states are unable or unwilling to provide this protection, or are themselves the perpetrators of atrocities, the Commission argues that the international community has a responsibility temporarily to step in, forcefully if necessary. The Commission resisted the temptation to identify human rights violations falling short of outright killing or ethnic cleansing. This eliminates the possibility of intervening on the basis of systematic oppression of human rights or intervening to remove a military dictatorship. The intention of the report was to provoke debate; to strengthen the role of the United Nations and ensure that such interventions were multilateral and meeting the wider needs of a region and not the interests of major powers. There is an ongoing need to ensure that the Security Council is effective and that resources match the political will. These debates must continue within a UN framework.

  16. Recombinant VP1 protein expressed in Pichia pastoris induces protective immune responses against EV71 in mice.

    PubMed

    Wang, Man; Jiang, Shuai; Wang, Yefu

    2013-01-04

    Human enterovirus 71 (EV71) is one of the major causative agents of hand, foot and mouth disease and is also associated with serious neurological diseases in children. Currently, there are no effective antiviral drugs or vaccines against EV71 infection. VP1, one of the major immunogenic capsid proteins of EV71, is widely considered to be the candidate antigen for an EV71 vaccine. In this study, VP1 of EV71 was expressed as a secretory protein with an N-terminal histidine tag in the methylotrophic yeast Pichia pastoris, and purified by Ni-NTA affinity chromatography. Immunogenicity and vaccine efficacy of the recombinant VP1 were assessed in mouse models. The results showed that the recombinant VP1 could efficiently induce anti-VP1 antibodies in BALB/c mice, which were able to neutralize EV71 viruses in an in vitro neutralization assay. Passive protection of neonatal mice further confirmed the prophylactic efficacy of the antisera from VP1 vaccinated mice. Furthermore, VP1 vaccination induced strong lymphoproliferative and Th1 cytokine responses. Taken together, our study demonstrated that the yeast-expressed VP1 protein retained good immunogenicity and was a potent EV71 vaccine candidate.

  17. The Conserved G-Protein Coupled Receptor FSHR-1 Regulates Protective Host Responses to Infection and Oxidative Stress.

    PubMed

    Miller, Elizabeth V; Grandi, Leah N; Giannini, Jennifer A; Robinson, Joseph D; Powell, Jennifer R

    2015-01-01

    The innate immune system's ability to sense an infection is critical so that it can rapidly respond if pathogenic microorganisms threaten the host, but otherwise maintain a quiescent baseline state to avoid causing damage to the host or to commensal microorganisms. One important mechanism for discriminating between pathogenic and non-pathogenic bacteria is the recognition of cellular damage caused by a pathogen during the course of infection. In Caenorhabditis elegans, the conserved G-protein coupled receptor FSHR-1 is an important constituent of the innate immune response. FSHR-1 activates the expression of antimicrobial infection response genes in infected worms and delays accumulation of the ingested pathogen Pseudomonas aeruginosa. FSHR-1 is central not only to the worm's survival of infection by multiple pathogens, but also to the worm's survival of xenobiotic cadmium and oxidative stresses. Infected worms produce reactive oxygen species to fight off the pathogens; FSHR-1 is required at the site of infection for the expression of detoxifying genes that protect the host from collateral damage caused by this defense response. Finally, the FSHR-1 pathway is important for the ability of worms to discriminate pathogenic from benign bacteria and subsequently initiate an aversive learning program that promotes selective pathogen avoidance.

  18. Protective humoral response against pneumococcal infection in mice elicited by recombinant bacille Calmette-Guerin vaccines expressing pneumococcal surface protein A

    PubMed Central

    1994-01-01

    Pneumococcal surface protein A (PspA), a cell-surface protein present on all strains of pneumococci, has been shown to elicit protective antibody responses in mice in the absence of capsular polysaccharide. Whereas PspA is polymorphic, considerable cross-reactivity and cross- protection have been demonstrated among PspA proteins of pneumococci exhibiting different capsular and PspA serotypes. A gene segment encoding the nonrepetitive variable NH2-terminal portion of PspA has been cloned into three distinct recombinant Bacille Calmette-Guerin (rBCG) vectors, allowing for expression of PspA as a cytoplasmic or secreted protein, or a chimeric exported membrane-associated lipoprotein. All rBCG-PspA strains elicited comparable anti-PspA ELISA titers, ranging from 10(4) to 10(5) (reciprocal titers) in both BALB/c and C3H/HeJ mice. However, protective responses were observed only in animals immunized with the rBCG-PspA vaccines expressing PspA as a secreted protein or chimeric exported lipoprotein. In addition, anti- PspA immune sera elicited by the rBCG vaccines passively protected X- linked immunodeficient mice from lethal challenge with the highly virulent, encapsulated WU2 strain of Streptococcus pneumoniae and two additional virulent strains exhibiting heterologous PspA and capsular serotypes. These studies confirm previous PspA immunization studies showing cross-protection against heterologous serotypes of S. pneumoniae and demonstrate a potential for rBCG-based PspA vaccines to elicit protective humoral responses against pneumococcal disease in humans. PMID:7964500

  19. Evaluation of immune response to recombinant potential protective antigens of Mycoplasma hyopneumoniae delivered as cocktail DNA and/or recombinant protein vaccines in mice.

    PubMed

    Chen, Austen Y; Fry, Scott R; Daggard, Grant E; Mukkur, Trilochan K S

    2008-08-12

    Intramuscular immunization of mice with DNA cocktail vaccines, comprising potential protective antigens P36, P46, NrdF, and P97or P97R1 of Mycoplasma hyopneumoniae, induced strong Th1-polarized immune responses against each antigen, with only P46 eliciting a serum IgG response. Subcutaneous immunization with protein cocktail vaccines, surprisingly, induced both Th1-polarized immune response as well as antibody response whereas mice immunized with DNA cocktail vaccines followed by boosting with protein cocktail vaccines generated strong Th1-polarized and humoral immune responses. P97 was not recognized by serum antibodies from commercial bacterin-immunized mice indicating potential lack of expression of this important antigen in inactivated whole-cell vaccines.

  20. Recombinant gp90 protein expressed in Pichia pastoris induces a protective immune response against reticuloendotheliosis virus in chickens.

    PubMed

    Li, Kai; Gao, Honglei; Gao, Li; Qi, Xiaole; Gao, Yulong; Qin, Liting; Wang, Yongqiang; Wang, Xiaomei

    2012-03-16

    Reticuloendotheliosis virus (REV) causes an oncogenic, immunosuppressive and runting syndrome in multiple avian hosts worldwide. In this study, the gp90 protein of REV was secretory expressed in Pichia pastoris with high production level and good antigenicity. To fully utilize the expression potential of the P. pastoris expression system, a panel of Pichia clones carrying increasing copies of the gp90 expression cassette was created using an in vitro multimerization approach and the effects of gene dosage on gp90 expression were investigated. Results demonstrated that an increase in gp90 copy number can significantly improve the yields of gp90 protein. Following expression and scale-up, the gp90 protein production level could reach up to 400mg/L, and the protein could be detected by gp90-specific monoclonal antibody. Investigations of its vaccine efficacy demonstrated that the recombinant gp90 protein was able to induce sustained high levels of antibodies against REV as being detected by ELISA and virus neutralizing test. Furthermore, immunization of chickens with the recombinant gp90 vaccine fully protected the animals from viremia after REV infection. Overall, the yeast-expressed gp90 protein retains good immunogenicity and could be used as a potential subunit vaccine candidate for REV prevention.

  1. Depletion of the cereblon gene activates the unfolded protein response and protects cells from ER stress-induced cell death.

    PubMed

    Lee, Kwang Min; Yang, Seung-Joo; Park, Sojung; Choi, Yoo Duk; Shin, Hwa Kyoung; Pak, Jhang Ho; Park, Chul-Seung; Kim, Inki

    2015-02-27

    Previous studies showed that cereblon (CRBN) binds to various cellular target proteins, implying that CRBN regulates a wide range of cell responses. In this study, we found that deletion of the Crbn gene desensitized mouse embryonic fibroblast cells to various cell death-promoting stimuli, including endoplasmic reticulum stress inducers. Mechanistically, deletion of Crbn activates pathways involved in the unfolded protein response prior to ER stress induction. Loss of Crbn activated PKR-like ER kinase (PERK) with enhanced phosphorylation of eIF2α. Following ER stress induction, loss of Crbn delayed dephosphorylation of eIF2α, while reconstitution of Crbn reversed enhanced phosphorylation of PERK and eIF2α. Lastly, we found that activation of the PERK/eIF2α pathway following Crbn deletion is caused by activation of AMP-activated protein kinase (AMPK). We propose that CRBN plays a role in cellular stress signaling, including the unfolded protein response, by controlling the activity of AMPK.

  2. A novel recombinant BCG-expressing pro-apoptotic protein BAX enhances Th1 protective immune responses in mice.

    PubMed

    Li, Guanghua; Liu, Guoyuan; Song, Na; Kong, Cong; Huang, Qi; Su, Haibo; Bi, Aixiao; Luo, Liulin; Zhu, Lin; Xu, Ying; Wang, Honghai

    2015-08-01

    One-third of the world's population is infected with Mycobacterium tuberculosis (MTB). The protective efficacy of bacille Calmette Guérin (BCG) vaccine against tuberculosis (TB) in adults is highly controversial even though the BCG vaccine has been available for more than 90 years. Because BCG is effective against infantile tuberculosis meningitis and miliary tuberculosis in young children and provides cost-effective prevention from tuberculosis for developing countries, it would be desirable to modify the existing BCG vaccine to provide more comprehensive protection. In our study, we constructed a novel recombinant BCG strain expressing pro-apoptotic BAX (rBCG::BAX) and demonstrated that it significantly induced the apoptosis of macrophages infected with rBCG::BAX both in vitro and in vivo. In addition, it significantly enhanced Ag85B-specific IFN-γ enzyme-linked immunospot responses, IFN-γ secretion, IL-2 secretion and the ratio of Ag85B-specific IgG2b/IgG1, and it significantly decreased Ag85B-specific IL-4. Furthermore, it presumably facilitated antigen presentation by inducing a significant up-regulation in the expression of MHC-II and B7.1 (CD80) co-stimulatory molecules on macrophages. In conclusion, these results suggest that the rBCG::BAX strain elicited predominantly a Th1 protective immune responses and might be a potential tuberculosis vaccine candidate for further study.

  3. Protective immune response induced by co-immunization with the Trichinella spiralis recombinant Ts87 protein and a Ts87 DNA vaccine.

    PubMed

    Yang, Yaping; Yang, Xiaodi; Gu, Yuan; Wang, Yunyun; Zhao, Xi; Zhu, Xinping

    2013-05-20

    Ts87 is an immunodominant antigen that induces protective immunity against Trichinella spiralis larval challenge in mice. To determine if a combination of recombinant Ts87 protein and its coding DNA induces a stronger immune response in female C57BL/6 mice were immunized with 100 μg of recombinant Ts87 protein plus its coding DNA cloned in vector pVAX1, or the same amount of recombinant protein or DNA only. Mouse subclass IgG responses showed that both co-immunized and single-immunized mice produced a balanced IgG2a/IgG1 (Th1/Th2) response. T-cell proliferation in co-immunized animals was significantly higher than in single-immunized mice. Cytokine profiling in the co-immunization group showed a significant increase in the levels of IL-2, IL-4, IL-6 and IFN-γ in the splenocytes of mice upon stimulation with the recombinant Ts87 protein; however, the expression of IL-17 was down-regulated. Challenge results showed that mice immunized with the recombinant Ts87 protein and its coding DNA produced reduced the muscle larval burden to a greater extent (43.8%) than the groups immunized with only the protein (39.7%) or the DNA (9.7%). A better Th1/Th2 immune response and consequent protection induced by co-immunization with the recombinant Ts87 protein and its coding DNA may result from an adjuvant effect of DNA and a specific persistent expression of Ts87.

  4. Protective and disease-enhancing immune responses induced by recombinant modified vaccinia Ankara (MVA) expressing respiratory syncytial virus proteins.

    PubMed

    Olszewska, Wieslawa; Suezer, Yasemin; Sutter, Gerd; Openshaw, Peter J M

    2004-11-25

    Modified vaccinia Ankara (MVA) recombinants expressing single or multiple RSV surface proteins (F or G) are promising potential vaccines. We studied humoral and cellular responses induced by MVA-F and MVA-G in mice, comparing them to a formalin inactivated RSV preparation (FI-RSV) known to increase disease severity. MVA-F or MVA-G vaccination enhanced weight loss during RSV challenge, but did not show the lung eosinophilia seen after FI-RSV vaccination. FI-RSV induced a stronger total RSV IgG response than the MVA recombinants, but very little IgG2a. MVA recombinants induced cytokine responses biased towards IFNgamma and IL-12, while FI-RSV induced strong IL-4/5 responses in the lungs during RSV challenge. Thus, MVA vaccines induce a favourable immune profile in RSV disease but retain the potential to enhance disease.

  5. A novel fusion protein domain III-capsid from dengue-2, in a highly aggregated form, induces a functional immune response and protection in mice

    SciTech Connect

    Valdes, Iris; Bernardo, Lidice; Pavon, Alekis; Guzman, Maria G.

    2009-11-25

    Based on the immunogenicity of domain III from the Envelope protein of dengue virus as well as the proven protective capacity of the capsid antigen, we have designed a novel domain III-capsid chimeric protein with the goal of obtaining a molecule potentially able to induce both humoral and cell-mediated immunity (CMI). After expression of the recombinant gene in Escherichia coli, the domain III moiety retained its antigenicity as evaluated with anti-dengue sera. In order to explore alternatives for modulating the immunogenicity of the protein, it was mixed with oligodeoxynucleotides in order to obtain particulated aggregates and then immunologically evaluated in mice in comparison with non-aggregated controls. Although the humoral immune response induced by both forms of the protein was equivalent, the aggregated variant resulted in a much stronger CMI as measured by in vitro IFN-gamma secretion and protection experiments, mediated by CD4{sup +} and CD8{sup +} cells. The present work provides additional evidence in support for a crucial role of CMI in protection against dengue virus and describes a novel vaccine candidate against the disease based on a recombinant protein that can stimulate both arms of the acquired immune system.

  6. A novel fusion protein domain III-capsid from dengue-2, in a highly aggregated form, induces a functional immune response and protection in mice.

    PubMed

    Valdés, Iris; Bernardo, Lidice; Gil, Lázaro; Pavón, Alekis; Lazo, Laura; López, Carlos; Romero, Yaremis; Menendez, Ivón; Falcón, Viviana; Betancourt, Lázaro; Martín, Jorge; Chinea, Glay; Silva, Ricardo; Guzmán, María G; Guillén, Gerardo; Hermida, Lisset

    2009-11-25

    Based on the immunogenicity of domain III from the Envelope protein of dengue virus as well as the proven protective capacity of the capsid antigen, we have designed a novel domain III-capsid chimeric protein with the goal of obtaining a molecule potentially able to induce both humoral and cell-mediated immunity (CMI). After expression of the recombinant gene in Escherichia coli, the domain III moiety retained its antigenicity as evaluated with anti-dengue sera. In order to explore alternatives for modulating the immunogenicity of the protein, it was mixed with oligodeoxynucleotides in order to obtain particulated aggregates and then immunologically evaluated in mice in comparison with non-aggregated controls. Although the humoral immune response induced by both forms of the protein was equivalent, the aggregated variant resulted in a much stronger CMI as measured by in vitro IFN-gamma secretion and protection experiments, mediated by CD4(+) and CD8(+) cells. The present work provides additional evidence in support for a crucial role of CMI in protection against dengue virus and describes a novel vaccine candidate against the disease based on a recombinant protein that can stimulate both arms of the acquired immune system.

  7. Changes in the amount of lysine in protective proteins and immune cells after a systemic response to dead Escherichia coli: implications for the nutritional costs of immunity.

    PubMed

    Iseri, V J; Klasing, K C

    2014-11-01

    The nutritional demands of the immune system may result in tradeoffs with competing processes such as growth and reproduction. The magnitude of the nutritional costs of immunity is largely unknown. Thus, we examine the lysine content of the systemic components of the immune system in adult male chickens (Gallus gallus domesticus) in a healthy condition (maintenance) and following a robust Escherichia coli-specific immune response. Lysine was used as a metric, because it is found both in leukocytes and in protective proteins. The dynamics of subsets of leukocytes were monitored in primary and secondary immune tissues (thymus, bone marrow, and spleen) that would be expected to be involved in the response following iv injection of E. coli. The systemic immune system at maintenance has the same lysine content as 332 average-sized feathers, 16% of an egg, or 5.4% of a pectoralis muscle from an adult chicken. During the acute-phase response to E. coli, the additional lysine needed would equal 355 feathers, 17% of an egg, or 5.5% of a pectoralis muscle. The acute-phase proteins accounted for the greatest proportion of lysine in the immune system at maintenance and the proportion increased substantially during an acute-phase response. Hypertrophy of the liver required more lysine than all of the leukocytes and protective proteins that were produced during the acute-phase response. Size of the liver and levels of protein during the acute phase returned to normal during the time when the adaptive response began to utilize significant quantities of lysine. The catabolism would release a surfeit of lysine to provision the anabolic processes of the adaptive response, thus making proliferation of lymphocytes and production of immunoglobulins very cheap.

  8. Immunity to Sand Fly Salivary Protein LJM11 Modulates Host Response to Vector-Transmitted Leishmania Conferring Ulcer-Free Protection

    PubMed Central

    Gomes, Regis; Oliveira, Fabiano; Teixeira, Clarissa; Meneses, Claudio; Gilmore, Dana C; Elnaiem, Dia-Eldin; Kamhawi, Shaden; Valenzuela, Jesus G

    2012-01-01

    Leishmania vaccines that protect against needle challenge fail against the potency of a Leishmania-infected sand fly transmission. Here, we demonstrate that intradermal immunization of mice with 500 ng of the sand fly salivary recombinant protein LJM11 (rLJM11) from Lutzomyia longipalpis, in the absence of adjuvant, induces long-lasting immunity that results in ulcer-free protection against Leishmania major delivered by vector bites. This protection is antibody independent and abrogated by depletion of CD4+ T cells. Two weeks after challenge, early induction of IFN-γ specifically to rLJM11 correlates to diminished parasite replication in protected animals. At this time point, Leishmania-specific induction of IFN-γ in these mice is low in comparison with its high level in non-protected controls. We hypothesize that early control of parasites in a T-cell helper type 1 environment induced by immunity to LJM11 permits the slow development of Leishmania-specific immunity in the absence of open ulcers. Leishmania-specific immunity observed 5 weeks after infection in rLJM11-immunized mice shows a twofold increase over controls in the percentage of IFN-γ-producing CD4+ T cells. We propose LJM11 as an immunomodulator that drives an efficient and controlled protective immune response to a sand fly–transmitted Leishmania somewhat mimicking “leishmanization”-induced protective immunity but without its associated lesions. PMID:22739793

  9. Recombinant Bivalent Fusion Protein rVE Induces CD4+ and CD8+ T-Cell Mediated Memory Immune Response for Protection Against Yersinia enterocolitica Infection

    PubMed Central

    Singh, Amit K.; Kingston, Joseph J.; Gupta, Shishir K.; Batra, Harsh V.

    2015-01-01

    Studies investigating the correlates of immune protection against Yersinia infection have established that both humoral and cell mediated immune responses are required for the comprehensive protection. In our previous study, we established that the bivalent fusion protein (rVE) comprising immunologically active regions of Y. pestis LcrV (100–270 aa) and YopE (50–213 aa) proteins conferred complete passive and active protection against lethal Y. enterocolitica 8081 challenge. In the present study, cohort of BALB/c mice immunized with rVE or its component proteins rV, rE were assessed for cell mediated immune responses and memory immune protection against Y. enterocolitica 8081. rVE immunization resulted in extensive proliferation of both CD4 and CD8 T cell subsets; significantly high antibody titer with balanced IgG1: IgG2a/IgG2b isotypes (1:1 ratio) and up-regulation of both Th1 (TNF-α, IFN-γ, IL-2, and IL-12) and Th2 (IL-4) cytokines. On the other hand, rV immunization resulted in Th2 biased IgG response (11:1 ratio) and proliferation of CD4+ T-cell; rE group of mice exhibited considerably lower serum antibody titer with predominant Th1 response (1:3 ratio) and CD8+ T-cell proliferation. Comprehensive protection with superior survival (100%) was observed among rVE immunized mice when compared to the significantly lower survival rates among rE (37.5%) and rV (25%) groups when IP challenged with Y. enterocolitica 8081 after 120 days of immunization. Findings in this and our earlier studies define the bivalent fusion protein rVE as a potent candidate vaccine molecule with the capability to concurrently stimulate humoral and cell mediated immune responses and a proof of concept for developing efficient subunit vaccines against Gram negative facultative intracellular bacterial pathogens. PMID:26733956

  10. Sestrin2 is induced by glucose starvation via the unfolded protein response and protects cells from non-canonical necroptotic cell death

    PubMed Central

    Ding, Boxiao; Parmigiani, Anita; Divakaruni, Ajit S.; Archer, Kellie; Murphy, Anne N.; Budanov, Andrei V.

    2016-01-01

    Sestrin2 is a member of a family of stress responsive proteins, which controls cell viability via antioxidant activity and regulation of the mammalian target of rapamycin protein kinase (mTOR). Sestrin2 is induced by different stress insults, which diminish ATP production and induce energetic stress in the cells. Glucose is a critical substrate for ATP production utilized via glycolysis and mitochondrial respiration as well as for glycosylation of newly synthesized proteins in the endoplasmic reticulum (ER) and Golgi. Thus, glucose starvation causes both energy deficiency and activation of ER stress followed by the unfolding protein response (UPR). Here, we show that UPR induces Sestrin2 via ATF4 and NRF2 transcription factors and demonstrate that Sestrin2 protects cells from glucose starvation-induced cell death. Sestrin2 inactivation sensitizes cells to necroptotic cell death that is associated with a decline in ATP levels and can be suppressed by Necrostatin 7. We propose that Sestrin2 protects cells from glucose starvation-induced cell death via regulation of mitochondrial homeostasis. PMID:26932729

  11. Protective Effects of Transforming Growth Factor β2 in Intestinal Epithelial Cells by Regulation of Proteins Associated with Stress and Endotoxin Responses

    PubMed Central

    Nguyen, Duc Ninh; Jiang, Pingping; Jacobsen, Susanne; Sangild, Per T.; Bendixen, Emøke; Chatterton, Dereck E. W.

    2015-01-01

    Transforming growth factor (TGF)-β2 is an important anti-inflammatory protein in milk and colostrum. TGF-β2 supplementation appears to reduce gut inflammatory diseases in early life, such as necrotizing enterocolitis (NEC) in young mice. However, the molecular mechanisms by which TGF-β2 protects immature intestinal epithelial cells (IECs) remain to be more clearly elucidated before interventions in infants can be considered. Porcine IECs PsIc1 were treated with TGF-β2 and/or lipopolysaccharide (LPS), and changes in the cellular proteome were subsequently analyzed using two-dimensional gel electrophoresis-MS and LC-MS-based proteomics. TGF-β2 alone induced the differential expression of 13 proteins and the majority of the identified proteins were associated with stress responses, TGF-β and Toll-like receptor 4 signaling cascades. In particular, a series of heat shock proteins had similar differential trends as previously shown in the intestine of NEC-resistant preterm pigs and young mice. Furthermore, LC-MS-based proteomics and Western blot analyses revealed 20 differentially expressed proteins following treatment with TGF-β2 in LPS-challenged IECs. Thirteen of these proteins were associated with stress response pathways, among which five proteins were altered by LPS and restored by TGF-β2, whereas six were differentially expressed only by TGF-β2 in LPS-challenged IECs. Based on previously reported biological functions, these patterns indicate the anti-stress and anti-inflammatory effects of TGF-β2 in IECs. We conclude that TGF-β2 of dietary or endogenous origin may regulate the IEC responses against LPS stimuli, thereby supporting cellular homeostasis and innate immunity in response to bacterial colonization, and the first enteral feeding in early life. PMID:25668313

  12. Alphavirus-based vaccines encoding nonstructural proteins of hepatitis C virus induce robust and protective T-cell responses.

    PubMed

    Ip, Peng Peng; Boerma, Annemarie; Regts, Joke; Meijerhof, Tjarko; Wilschut, Jan; Nijman, Hans W; Daemen, Toos

    2014-04-01

    An absolute prerequisite for a therapeutic vaccine against hepatitis C virus (HCV) infection is the potency to induce HCV-specific vigorous and broad-spectrum T-cell responses. Here, we generated three HCV vaccines based on a recombinant Semliki Forest virus (rSFV) vector expressing all- or a part of the conserved nonstructural proteins (nsPs) of HCV. We demonstrated that an rSFV vector was able to encode a transgene as large as 6.1 kb without affecting its vaccine immunogenicity. Prime-boost immunizations of mice with rSFV expressing all nsPs induced strong and long-lasting NS3-specific CD8(+) T-cell responses. The strength and functional heterogeneity of the T-cell response was similar to that induced with rSFV expressing only NS3/4A. Furthermore this leads to a significant growth delay and negative selection of HCV-expressing EL4 tumors in an in vivo mouse model. In general, as broad-spectrum T-cell responses are only seen in patients with resolved HCV infection, this rSFV-based vector, which expresses all nsPs, inducing robust T-cell activity has a potential for the treatment of HCV infections.

  13. Inter-Clade Protection Offered by Mw-Adjuvanted Recombinant HA, NP Proteins, and M2e Peptide Combination Vaccine in Mice Correlates with Cellular Immune Response

    PubMed Central

    Ingle, Nilesh B.; Virkar, Rashmi G.; Arankalle, Vidya A.

    2017-01-01

    We documented earlier that Mw (heat-killed suspension of Mycobacterium indicus pranii) adjuvant when used with conserved antigens, nucleoprotein (NP), and ectodomain of matrix (M2) protein (M2e) provided complete protection against homologous (clade 2.2) virus challenge in mice. The present study extends these observations to inter-clade challenge (clade 2.3.2.1) H5N1 virus and attempts to understand preliminary immunologic basis for the observed protection. Female BALB/c mice immunized with a single or two doses of vaccine formulations (clade 2.2 antigens) were challenged with 100LD50 homologous or heterologous (clade 2.3.2.1) virus. To understand the preliminary immunologic mechanism, we studied proportions of selected immune cell types, immune response gene expression, and Th1/Th2 cytokines induced by antigen-stimulated splenocytes from immunized mice, at different time points. Complete protection was conferred by Mw-HA, Mw-HA + NP, and Mw-HA + NP + M2e against homologous challenge. The protection correlated with IgG2a antibody titers indicating important role of Th1 response. Despite high inter-cladal antigenic differences, complete protection against the heterologous strain was achieved with Mw-HA + NP + M2e. Of note, a single dose with higher antigen concentrations (50 µg HA + 50 μg NP + 50 μg M2e) led to 80% protection against clade 2.3.2.1 strain. The protection conferred by Mw-HNM correlated with induction of IFN-γ, CD8+ T cytotoxic cells, and CD4+ T helper cells. Mw-adjuvanted HA + NP + M2e combination represents a promising vaccine candidate deserving further evaluation. PMID:28119689

  14. Effect of vaccination with carrier protein on response to meningococcal C conjugate vaccines and value of different immunoassays as predictors of protection.

    PubMed

    Burrage, Moya; Robinson, Andrew; Borrow, Ray; Andrews, Nick; Southern, Joanna; Findlow, Jamie; Martin, Sarah; Thornton, Carol; Goldblatt, David; Corbel, Michael; Sesardic, Dorothea; Cartwight, Keith; Richmond, Peter; Miller, Elizabeth

    2002-09-01

    In order to plan for the wide-scale introduction of meningococcal C conjugate (MCC) vaccine for United Kingdom children up to 18 years old, phase II trials were undertaken to investigate whether there was any interaction between MCC vaccines conjugated to tetanus toxoid (TT) or a derivative of diphtheria toxin (CRM(197)) and diphtheria-tetanus vaccines given for boosting at school entry or leaving. Children (n = 1,766) received a diphtheria-tetanus booster either 1 month before, 1 month after, or concurrently with one of three MCC vaccines conjugated to CRM(197) or TT. All of the MCC vaccines induced high antibody responses to the serogroup C polysaccharide that were indicative of protection. The immune response to the MCC-TT vaccine was reduced as a result of prior immunization with a tetanus-containing vaccine, but antibody levels were still well above the lower threshold for protection. Prior or simultaneous administration of a diphtheria-containing vaccine did not affect the response to MCC-CRM(197) vaccines. The immune responses to the carrier proteins were similar to those induced by a comparable dose of diphtheria or tetanus vaccine. The results also demonstrate that, for these conjugate vaccines in these age groups, both standard enzyme-linked immunosorbent assays and those that measure high-avidity antibodies to meningococcal C polysaccharide correlated equally well with assays that measure serum bactericidal antibodies, the established serological correlate of protection for MCC vaccines.

  15. The bovine viral diarrhea virus E2 protein formulated with a novel adjuvant induces strong, balanced immune responses and provides protection from viral challenge in cattle.

    PubMed

    Snider, Marlene; Garg, Ravendra; Brownlie, Robert; van den Hurk, Jan V; van Drunen Littel-van den Hurk, Sylvia

    2014-11-28

    Bovine viral diarrhea virus (BVDV) is still one of the most serious pathogens in cattle, meriting the development of improved vaccines. Recently, we developed a new adjuvant consisting of poly[di(sodium carboxylatoethylphenoxy)]-phosphazene (PCEP), either CpG ODN or poly(I:C), and an immune defense regulator (IDR) peptide. As this adjuvant has been shown to mediate the induction of robust, balanced immune responses, it was evaluated in an E2 subunit vaccine against BVDV in lambs and calves. The BVDV type 2 E2 protein was produced at high levels in a mammalian expression system and purified. When formulated with either CpG ODN or poly(I:C), together with IDR and PCEP, the E2 protein elicited high antibody titers and production of IFN-γ secreting cells in lambs. As the immune responses were stronger when poly(I:C) was used, the E2 protein with poly(I:C), IDR and PCEP was subsequently tested in cattle. Robust virus neutralizing antibodies as well as cell-mediated immune responses, including CD8(+) cytotoxic T cell (CTL) responses, were induced. The fact that CTL responses were demonstrated in calves vaccinated with an E2 protein subunit vaccine indicates that this adjuvant formulation promotes cross-presentation. Furthermore, upon challenge with a high dose of virulent BVDV-2, the vaccinated calves showed almost no temperature response, weight loss, leukopenia or virus replication, in contrast to the control animals, which had severe clinical disease. These data suggest that this E2 subunit formulation induces significant protection from BVDV-2 challenge, and thus is a promising BVDV vaccine candidate; in addition, the adjuvant platform has applications in bovine vaccines in general.

  16. Th1 stimulatory proteins of Leishmania donovani: comparative cellular and protective responses of rTriose phosphate isomerase, rProtein disulfide isomerase and rElongation factor-2 in combination with rHSP70 against visceral leishmaniasis.

    PubMed

    Jaiswal, Anil Kumar; Khare, Prashant; Joshi, Sumit; Kushawaha, Pramod Kumar; Sundar, Shyam; Dube, Anuradha

    2014-01-01

    In visceral leishmaniasis, the recovery from the disease is always associated with the generation of Th1-type of cellular responses. Based on this, we have previously identified several Th1-stimulatory proteins of Leishmania donovani -triose phosphate isomerase (TPI), protein disulfide isomerase (PDI) and elongation factor-2 (EL-2) etc. including heat shock protein 70 (HSP70) which induced Th1-type of cellular responses in both cured Leishmania patients/hamsters. Since, HSPs, being the logical targets for vaccines aimed at augmenting cellular immunity and can be early targets in the immune response against intracellular pathogens; they could be exploited as vaccine/adjuvant to induce long-term immunity more effectively. Therefore, in this study, we checked whether HSP70 can further enhance the immunogenicity and protective responses of the above said Th1-stimulatory proteins. Since, in most of the studies, immunogenicity of HSP70 of L. donovani was assessed in native condition, herein we generated recombinant HSP70 and tested its potential to stimulate immune responses in lymphocytes of cured Leishmania infected hamsters as well as in the peripheral blood mononuclear cells (PBMCs) of cured patients of VL either individually or in combination with above mentioned recombinant proteins. rLdHSP70 alone elicited strong cellular responses along with remarkable up-regulation of IFN-γ and IL-12 cytokines and extremely lower level of IL-4 and IL-10. Among the various combinations, rLdHSP70 + rLdPDI emerged as superior one augmenting improved cellular responses followed by rLdHSP70 + rLdEL-2. These combinations were further evaluated for its protective potential wherein rLdHSP70 + rLdPDI again conferred utmost protection (∼80%) followed by rLdHSP70 + rLdEL-2 (∼75%) and generated a strong cellular immune response with significant increase in the levels of iNOS transcript as well as IFN-γ and IL-12 cytokines which was further supported by the high level of IgG2 antibody

  17. Characterization of the antibody response elicited by immunization with pneumococcal surface protein A (PspA) as recombinant protein or DNA vaccine and analysis of protection against an intranasal lethal challenge with Streptococcus pneumoniae.

    PubMed

    Vadesilho, Cintia F M; Ferreira, Daniela M; Moreno, Adriana T; Chavez-Olortegui, Carlos; Machado de Avila, Ricardo A; Oliveira, Maria Leonor S; Ho, Paulo L; Miyaji, Eliane N

    2012-01-01

    Pneumococcal surface protein A (PspA) is an important candidate for a vaccine against pneumococcal infections. DNA vaccines expressing PspA were shown to protect mice against intraperitoneal and colonization challenge models in mice. We now show that a DNA vaccine expressing PspA from clade 4 (pSec-pspA4Pro) is also able to elicit protection against an intranasal lethal challenge model at levels similar to the recombinant protein PspA4Pro adjuvanted with alum. PspA4Pro + alum induced an IgG response characterized by a high IgG1/IgG2a ratio, leading to a lack of binding of anti-PspA IgG2a antibodies to intact pneumococci in vitro, which is in contrast to the response elicited by pSec-pspA4Pro. Epitopes recognized by the sera were mapped and antibodies induced by immunization with PspA4Pro + alum showed positive reaction with several synthetic peptides, mostly located in the first half of the protein. On the other hand, antibodies induced by the DNA vaccine showed reactivity with only two peptides. Though both strategies were protective against the intranasal lethal challenge model, the elicited humoral responses differ significantly, with the detection of important differences in the Fc (IgG1/IgG2a ratios) and Fab (recognized epitopes) regions of the induced antibodies.

  18. Immunological responses induced by a DNA vaccine expressing RON4 and by immunogenic recombinant protein RON4 failed to protect mice against chronic toxoplasmosis.

    PubMed

    Rashid, Imran; Hedhli, Dorsaf; Moiré, Nathalie; Pierre, Josette; Debierre-Grockiego, Françoise; Dimier-Poisson, Isabelle; Mévélec, Marie Noëlle

    2011-11-08

    The development of an effective vaccine against Toxoplasma gondii infection is an important issue due to the seriousness of the related public health problems, and the economic importance of this parasitic disease worldwide. Rhoptry neck proteins (RONs) are components of the moving junction macromolecular complex formed during invasion. The aim of this study was to evaluate the vaccine potential of RON4 using two vaccination strategies: DNA vaccination by the intramuscular route, and recombinant protein vaccination by the nasal route. We produced recombinant RON4 protein (RON4S2) using the Schneider insect cells expression system, and validated its antigenicity and immunogenicity. We also constructed optimized plasmids encoding full length RON4 (pRON4), or only the N-terminal (pNRON4), or the C-terminal part (pCRON4) of RON4. CBA/J mice immunized with pRON4, pNRON4 or pCRON4 plus a plasmid encoding the granulocyte-macrophage-colony-stimulating factor showed high IgG titers against rRON4S2. Mice immunized by the nasal route with rRON4S2 plus cholera toxin exhibited low levels of anti-RON4S2 IgG antibodies, and no intestinal IgA antibodies specific to RON4 were detected. Both DNA and protein vaccination generated a mixed Th1/Th2 response polarized towards the IgG1 antibody isotype. Both DNA and protein vaccination primed CD4+ T cells in vivo. In addition to the production of IFN-γ, and IL-2, Il-10 and IL-5 were also produced by the spleen cells of the immunized mice stimulated with RON4S2, suggesting that a mixed Th1/Th2 type immune response occurred in all the immunized groups. No cytokine was detectable in stimulated mesenteric lymph nodes from mice immunized by the nasal route. Immune responses were induced by both DNA and protein vaccination, but failed to protect the mice against a subsequent oral challenge with T. gondii cysts. In conclusion, strategies designed to enhance the immunogenicity and to redirect the cellular response towards a Th1 type response

  19. Protective immune responses against systemic candidiasis mediated by phage-displayed specific epitope of Candida albicans heat shock protein 90 in C57BL/6J mice.

    PubMed

    Wang, Guiyun; Sun, Meiyan; Fang, Jinbo; Yang, Qiong; Tong, Haibin; Wang, Li

    2006-08-28

    Specific epitope (DEPAGE) of Candida albicans heat shock protein 90 (SE-CA-HSP90) was successfully expressed on the surface of filamentous phage fd, fused to the major coat protein pVIII. Protective immune responses mediated by hybrid-phage expressing SE-CA-HSP90 in C57BL/6J mice were evaluated in this paper. The results showed that hybrid-phage particles induced the specific antibody response against SE-CA-HSP90, enhanced delayed-type hypersensitivity (DTH) response, natural killer (NK) cell activity and concanavalin A (ConA)-induced splenocyte proliferation. Furthermore, this study demonstrated that hybrid-phage-immunized mice had fewer colony forming unites (CFU) in the kidneys compared with wild-type phage-immunized mice and TE (1.0mM EDTA, 0.01M Tris-HCl, pH 8.0)-injected mice and showed statistically significant survival advantage over TE-injected group. In conclusion, the results suggest that the hybrid-phage displaying SE-CA-HSP90 may be considered as a potential candidate for producing vaccines against systemic candidiasis.

  20. The immunization-induced antibody response to the Anaplasma marginale major surface protein 2 and its association with protective immunity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Many vector-borne pathogens evade clearance via rapid variation in immunogenic surface expressed proteins. In the case of A. marginale, the generation of major surface protein 2 (Msp2) variants allows for immune escape and long-term pathogen persistence. In the experiments reported here, we pose t...

  1. Antibody Responses to a Novel Plasmodium falciparum Merozoite Surface Protein Vaccine Correlate with Protection against Experimental Malaria Infection in Aotus Monkeys

    PubMed Central

    Cavanagh, David R.; Kocken, Clemens H. M.; White, John H.; Cowan, Graeme J. M.; Samuel, Kay; Dubbeld, Martin A.; der Wel, Annemarie Voorberg-van; Thomas, Alan W.; McBride, Jana S.; Arnot, David E.

    2014-01-01

    The Block 2 region of the merozoite surface protein-1 (MSP-1) of Plasmodium falciparum has been identified as a target of protective immunity by a combination of seroepidemiology and parasite population genetics. Immunogenicity studies in small animals and Aotus monkeys were used to determine the efficacy of recombinant antigens derived from this region of MSP-1 as a potential vaccine antigen. Aotus lemurinus griseimembra monkeys were immunized three times with a recombinant antigen derived from the Block 2 region of MSP-1 of the monkey-adapted challenge strain, FVO of Plasmodium falciparum, using an adjuvant suitable for use in humans. Immunofluorescent antibody assays (IFA) against erythrocytes infected with P. falciparum using sera from the immunized monkeys showed that the MSP-1 Block 2 antigen induced significant antibody responses to whole malaria parasites. MSP-1 Block 2 antigen-specific enzyme-linked immunosorbent assays (ELISA) showed no significant differences in antibody titers between immunized animals. Immunized animals were challenged with the virulent P. falciparum FVO isolate and monitored for 21 days. Two out of four immunized animals were able to control their parasitaemia during the follow-up period, whereas two out of two controls developed fulminating parasitemia. Parasite-specific serum antibody titers measured by IFA were four-fold higher in protected animals than in unprotected animals. In addition, peptide-based epitope mapping of serum antibodies from immunized Aotus showed distinct differences in epitope specificities between protected and unprotected animals. PMID:24421900

  2. Brucella abortus Omp19 recombinant protein subcutaneously co-delivered with an antigen enhances antigen-specific T helper 1 memory responses and induces protection against parasite challenge.

    PubMed

    Coria, Lorena M; Ibañez, Andrés E; Pasquevich, Karina A; Cobiello, Paula L González; Frank, Fernanda M; Giambartolomei, Guillermo H; Cassataro, Juliana

    2016-01-20

    The discovery of effective adjuvants for many vaccines especially those with limited commercial appeal, such as vaccines to poverty-related diseases, is required. In this work, we demonstrated that subcutaneous co-administration of mice with the outer membrane protein U-Omp19 from Brucella spp. plus OVA as antigen (Ag) increases Ag-specific T cell proliferation and T helper (Th) 1 immune responses in vitro and in vivo. U-Omp19 treated dendritic cells promote IFN-γ production by specific CD4(+) T cells and increases T cell proliferation. U-Omp19 co-administration induces the production of Ag specific effector memory T cell populations (CD4(+) CD44(high) CD62L(low) T cells). Finally, subcutaneous co-administration of U-Omp19 with Trypanosoma cruzi Ags confers protection against virulent parasite challenge, reducing parasitemia and weight loss while increasing mice survival. These results indicate that the bacterial protein U-Omp19 when delivered subcutaneously could be a suitable component of vaccine formulations against infectious diseases requiring Th1 immune responses.

  3. Display of enterovirus 71 VP1 on baculovirus as a type II transmembrane protein elicits protective B and T cell responses in immunized mice.

    PubMed

    Kolpe, Annasaheb B; Kiener, Tanja K; Grotenbreg, Gijsbert M; Kwang, Jimmy

    2012-09-01

    Human enterovirus 71 (EV71) has become a major public health threat across Asia Pacific. The virus causes hand, foot, and mouth disease which can lead to neurological complications in young children. There are no specific antivirals or vaccines against EV71 infection. The major neutralizing epitope of EV71 is located in the carboxy-terminal half of the VP1 protein at amino acid positions 215-219 (Lim et al., 2012). To study the immunogenicity of VP1 we have developed a baculovirus vector which displays VP1 as a type II transmembrane protein, providing an accessible C-terminus. Immunization of mice with this recombinant baculovirus elicited neutralizing antibodies against heterologous EV71 in an in vitro microneutralization assay. Passive protection of neonatal mice confirmed the prophylactic efficacy of the antisera. Additionally, EV71 specific T cell responses were stimulated. Taken together, our results demonstrate that the display of VP1 as a type II transmembrane protein efficiently stimulated both humoral and cellular immunities.

  4. Evaluation of Protective Immune Responses Induced by Recombinant TrxLp and ENO2 Proteins against Toxoplasma gondii Infection in BALB/c Mice

    PubMed Central

    Wang, Meng; Yang, Xiao-Yu; Zhang, De-Lin

    2016-01-01

    Toxoplasma gondii is an obligate intracellular parasitic protozoan that can infect almost all species of warm-blooded animals. As any chemical-based drugs could not act against the tissue cyst stage of T. gondii, vaccination may be one of the ideal control strategies. In the present study, two new vaccine candidates, named TgENO2 and TgTrxLp, were purified from Escherichia coli with pET-30a(+) expression system and then were injected into BALB/c mice to evaluate the protective efficacy against acute and chronic toxoplasmosis. The results showed that both the recombinant proteins, either alone or in combination, could elicit strong humoral and cellular immune responses with a higher level of IgG antibodies, IFN-γ, IL-2, CD4+, and CD8+ T cells as compared to those in mice from control groups. After acute challenge with tachyzoites of the GJS strain, mice immunized with rTgTrxLp (8 ± 2.77 d), rTgENO2 (7.4 ± 1.81 d), and rTgTrxLp + rTgENO2 (8.38 ± 4.57 d) proteins showed significantly longer survival time than those that received Freund's adjuvant (6.78 ± 2.08 d) and PBS (6.38 ± 4.65 d) (χ2 = 9.687, df = 4, P = 0.046). The protective immunity of rTgTrxLp, rTgENO2, and rTgTrxLp + rTgENO2 proteins against chronic T. gondii infection showed 69.77%, 58.14%, and 20.93% brain cyst reduction as compared to mice that received PBS. The present study suggested that both TgENO2 and TgTrxLp were potential candidates for the development of multicomponent vaccines against toxoplasmosis. PMID:27803923

  5. Antibiotic resistance free plasmid DNA expressing LACK protein leads towards a protective Th1 response against Leishmania infantum infection.

    PubMed

    Ramos, I; Alonso, A; Peris, A; Marcen, J M; Abengozar, M A; Alcolea, P J; Castillo, J A; Larraga, V

    2009-11-12

    Canine visceral leishmaniasis is a serious public health concern in the Mediterranean basin since dogs are the main Leishmania infantum reservoir. However, there is not a vaccination method in veterinary use in this area, and therefore the development of a vaccine against this parasite is essential for the possible control of the disease. Previous reports have shown the efficacy of heterologous prime-boost vaccination with the pCIneo plasmid and the poxvirus VV (both Western Reserve and MVA strains) expressing L. infantum LACK antigen against canine leishmaniasis. As pCIneo-LACK plasmid contains antibiotic resistance genes, its use as a profilactic method is not recommended. Hence, the antibiotic resistance gene free pORT-LACK plasmid is a more suitable tool for its use as a vaccine. Here we report the protective and immunostimulatory effect of the prime-boost pORT-LACK/MVA-LACK vaccination tested in a canine experimental model. Vaccination induced a reduction in clinical signs and in parasite burden in the liver, an induction of the Leishmania-specific T cell activation, as well as an increase of the expression of Th1 type cytokines in PBMC and target organs.

  6. Aging promotes B-1b cell responses to native, but not protein-conjugated, pneumococcal polysaccharides: implications for vaccine protection in older adults.

    PubMed

    Haas, Karen M; Blevins, Maria W; High, Kevin P; Pang, Bing; Swords, W Edward; Yammani, Rama D

    2014-01-01

    The efficacy of different vaccines in protecting elderly individuals against Streptococcus pneumoniae infections is not clear. In the current study, aged mice (22-25 months old) exhibited significantly increased susceptibility to respiratory infection with serotype 3 S. pneumoniae relative to younger adult mice, regardless of whether mice were naive or immunized with native pneumococcal polysaccharide (PPS; Pneumovax23) or protein-PPS conjugate (Prevnar-13) vaccines. Nonetheless, Pneumovax-immunized aged mice developed limited bacteremia following respiratory challenge and exhibited significantly increased survival following systemic challenge relative to Prevnar-immune aged mice and young mice that had received either vaccine. This was explained by >10-fold increases in PPS-specific immunoglobulin G (IgG) levels in Pneumovax-immunized aged mice relative to other groups. Remarkably, PPS3-specific B-cell expansion, IgG switching, plasmablast differentiation, and spleen and bone marrow antibody-secreting cell frequencies were 10-fold higher in aged mice following Pneumovax immunization relative to young mice, due to significantly increased B-1b cell participation. In summary, this study highlights (1) the need to devise strategies to enhance respiratory immunity in aged populations, (2) the diverse responses young and aged populations generate to Pneumovax vs Prevnar vaccines, and (3) the potential value of exploiting B-1b cell responses in aged individuals for increased vaccine efficacy.

  7. The budding yeast orthologue of Parkinson's disease-associated DJ-1 is a multi-stress response protein protecting cells against toxic glycolytic products.

    PubMed

    Natkańska, Urszula; Skoneczna, Adrianna; Sieńko, Marzena; Skoneczny, Marek

    2016-10-27

    Saccharomyces cerevisiae Hsp31p is a DJ-1/ThiJ/PfpI family protein that was previously shown to be important for survival in the stationary phase of growth and under oxidative stress. Recently, it was identified as a chaperone or as glutathione-independent glyoxalase. To elucidate the role played by this protein in budding yeast cells, we investigated its involvement in the protection against diverse environmental stresses. Our study revealed that HSP31 gene expression is controlled by multiple transcription factors, including Yap1p, Cad1p, Msn2p, Msn4p, Haa1p and Hsf1p. These transcription factors mediate the HSP31 promoter responses to oxidative, osmotic and thermal stresses, to potentially toxic products of glycolysis, such as methylglyoxal and acetic acid, and to the diauxic shift. We also demonstrated that the absence of the HSP31 gene sensitizes cells to these stressors. Overproduction of Hsp31p and its homologue Hsp32p rescued the sensitivity of glo1Δ cells to methylglyoxal. Hsp31p also reversed the increased sensitivity of the ald6Δ strain to acetic acid. Since Hsp31p glyoxalase III coexists in S. cerevisiae cells with thousand-fold more potent glyoxalase I/II system, its biological purpose requires substantiation. We postulate that S. cerevisiae Hsp31p may have broader substrate specificity than previously proposed and is able to eliminate various toxic products of glycolysis. Alternatively, Hsp31p might be effective under high concentration of exogenous methylglyoxal present in some natural environmental niches populated by budding yeast, when glyoxalase I/II system capacity is saturated.

  8. Oral immunization with an attenuated vaccine strain of Salmonella typhimurium expressing the serine-rich Entamoeba histolytica protein induces an antiamebic immune response and protects gerbils from amebic liver abscess.

    PubMed Central

    Zhang, T; Stanley, S L

    1996-01-01

    Attenuated salmonellae represent attractive candidates for the delivery of foreign antigens by oral vaccination. In this report, we describe the high-level expression of a recombinant fusion protein containing the serine-rich Entamoeba histolytica protein (SREHP), a protective antigen derived from virulent amebae, and a bacterially derived maltose-binding protein (MBP) in an attenuated strain of Salmonella typhimurium. Mice and gerbils immunized with S. typhimurium expressing SREHP-MBP produced mucosal immunoglobulin A antiamebic antibodies and serum immunoglobulin G antiamebic antibodies. Gerbils vaccinated with S typhimurium SREHP-MBP were protected against amebic liver abscess, the most common extraintestinal complication of amebiasis. Our findings indicate that the induction of mucosal and immune responses to the amebic SREHP antigen is dependent on the level of SREHP-MBP expression in S. typhimurium and establish that oral vaccination with SREHP can produce protective immunity to invasive amebiasis. PMID:8613356

  9. Foot-and-mouth disease virus-like particles produced by a SUMO fusion protein system in Escherichia coli induce potent protective immune responses in guinea pigs, swine and cattle.

    PubMed

    Guo, Hui-Chen; Sun, Shi-Qi; Jin, Ye; Yang, Shun-Li; Wei, Yan-Quan; Sun, De-Hui; Yin, Shuang-Hui; Ma, Jun-Wu; Liu, Zai-Xin; Guo, Jian-Hong; Luo, Jian-Xun; Yin, Hong; Liu, Xiang-Tao; Liu, Ding Xiang

    2013-07-04

    Foot-and-mouth disease virus (FMDV) causes a highly contagious infection in cloven-hoofed animals. The format of FMD virus-like particles (VLP) as a non-replicating particulate vaccine candidate is a promising alternative to conventional inactivated FMDV vaccines. In this study, we explored a prokaryotic system to express and assemble the FMD VLP and validated the potential of VLP as an FMDV vaccine candidate. VLP composed entirely of FMDV (Asia1/Jiangsu/China/2005) capsid proteins (VP0, VP1 and VP3) were simultaneously produced as SUMO fusion proteins by an improved SUMO fusion protein system in E. coli. Proteolytic removal of the SUMO moiety from the fusion proteins resulted in the assembly of VLP with size and shape resembling the authentic FMDV. Immunization of guinea pigs, swine and cattle with FMD VLP by intramuscular inoculation stimulated the FMDV-specific antibody response, neutralizing antibody response, T-cell proliferation response and secretion of cytokine IFN-γ. In addition, immunization with one dose of the VLP resulted in complete protection of these animals from homologous FMDV challenge. The 50% protection dose (PD50) of FMD VLP in cattle is up to 6.34. These results suggest that FMD VLP expressed in E. coli are an effective vaccine in guinea pigs, swine and cattle and support further development of these VLP as a vaccine candidate for protection against FMDV.

  10. Vaccination with a Fusion Protein That Introduces HIV-1 Gag Antigen into a Multitrimer CD40L Construct Results in Enhanced CD8+ T Cell Responses and Protection from Viral Challenge by Vaccinia-Gag

    PubMed Central

    Gupta, Sachin; Termini, James M.; Raffa, Francesca N.; Williams, Cindi-Ann; Kornbluth, Richard S.

    2014-01-01

    CD40 ligand (CD40L, CD154) is a membrane protein that is important for the activation of dendritic cells (DCs) and DC-induced CD8+ T cell responses. To be active, CD40L must cluster CD40 receptors on responding cells. To produce a soluble form of CD40L that clusters CD40 receptors necessitates the use of a multitrimer construct. With this in mind, a tripartite fusion protein was made from surfactant protein D (SPD), HIV-1 Gag as a test antigen, and CD40L, where SPD serves as a scaffold for the multitrimer protein complex. This SPD-Gag-CD40L protein activated CD40-bearing cells and bone marrow-derived DCs in vitro. Compared to a plasmid for Gag antigen alone (pGag), DNA vaccination of mice with pSPD-Gag-CD40L induced an increased number of Gag-specific CD8+ T cells with increased avidity for major histocompatibility complex class I-restricted Gag peptide and improved vaccine-induced protection from challenge by vaccinia-Gag virus. The importance of the multitrimeric nature of the complex was shown using a plasmid lacking the N terminus of SPD that produced a single trimer fusion protein. This plasmid, pTrimer-Gag-CD40L, was only weakly active on CD40-bearing cells and did not elicit strong CD8+ T cell responses or improve protection from vaccinia-Gag challenge. An adenovirus 5 (Ad5) vaccine incorporating SPD-Gag-CD40L was much stronger than Ad5 expressing Gag alone (Ad5-Gag) and induced complete protection (i.e., sterilizing immunity) from vaccinia-Gag challenge. Overall, these results show the potential of a new vaccine design in which antigen is introduced into a construct that expresses a multitrimer soluble form of CD40L, leading to strongly protective CD8+ T cell responses. PMID:24227853

  11. Persistence of the protective immunity and kinetics of the isotype specific antibody response against the viral nucleocapsid protein after experimental Schmallenberg virus infection of sheep.

    PubMed

    Poskin, Antoine; Verite, Stephanie; Comtet, Loic; Van der Stede, Yves; Cay, Brigitte; De Regge, Nick

    2015-10-15

    Schmallenberg virus (SBV) is an Orthobunyavirus that induces abortion, stillbirths and congenital malformations in ruminants. SBV infection induces a long lasting seroconversion under natural conditions. The persistence of the protective immunity and the isotype specific antibody response upon SBV infection of sheep has however not been studied in detail. Five sheep were kept in BSL3 facilities for more than 16 months and subjected to repeated SBV infections. Blood was regularly sampled and organs were collected at euthanasia. The presence of SBV RNA in serum and organs was measured with quantitative real-time PCR. The appearance and persistence of neutralizing and SBV nucleoprotein (N) isotype specific antibodies was determined with virus neutralization tests (VNT) and ELISAs. The primo SBV infection protected ewes against clinical signs, viraemia and virus replication in organs upon challenge infections more than 15 months later. Production of neutralizing SBV specific antibodies was first detected around 6 days post primo-inoculation with VNT and correlated with the appearance of SBV-N specific IgM antibodies. These IgM antibodies remained present for 2 weeks. SBV-N specific IgG antibodies were first detected between 10 and 21 dpi and reached a plateau at 28 dpi. This plateau remained consistently high and no significant decrease in titre was found over a period of more than 1 year. Similar results were found for the neutralising antibody response. In conclusion, the SBV specific IgM response probably eliminates SBV from the blood and the protective immunity induced by SBV infection protects sheep against reinfection for at least 16 months.

  12. Induction of protective immune responses against EV71 in mice by baculovirus encoding a novel expression cassette for capsid protein VP1.

    PubMed

    Premanand, Balraj; Kiener, Tanja K; Meng, Tao; Tan, Yun Rui; Jia, Qiang; Chow, Vincent T K; Kwang, Jimmy

    2012-09-01

    EV71 is a major causative agent of hand, foot and mouth disease (HFMD) and is responsible for large outbreaks in various Asian Pacific countries. In the present study, we generated the recombinant baculovirus (Bac-VP1) encoding VP1 in a novel expression cassette. The transmembrane domain of hemagglutinin of the H3N2 influenza virus was included in the cassette as a minimal membrane anchor for VP1. The protective immunity of Bac-VP1 was investigated in a mouse model. The results showed that mice vaccinated with live Bac-VP1 had strong VP1 specific antibody responses. In an in vitro neutralization assay Bac-VP1 sera exhibited cross-neutralization against homologous and heterologous EV71 strains with a maximum titer of 1:512. Passive immunization studies confirmed that these sera were able to provide 100% protection against 5 MLD(50) of mouse adapted EV71 (B4 strain). This study revealed that baculovirus displaying VP1 with a HA transmembrane domain efficiently induced cross-neutralizing antibody responses in mice.

  13. Heat shock proteins and protection against ischemic injury.

    PubMed Central

    Dillmann, W H

    1999-01-01

    Heat shock proteins present a complex family of proteins exerting chaperone-like activities that are classified according to their molecular weight. We especially explored protective functions of inducible heat shock protein 70, the mitochondrial heat shock protein 60 and 10, and the small heat shock proteins HSP27 and alphaB-crystallin against ischemic, reoxygenation-mediated injury using transgenic animals and hearts under in vivo conditions and in isolated cardiac myocyte-derived cells using adenoviral vectors. We noted with great interest that differential protective effects are exerted by specific hsps. For example, alpha-B-crystallin and constitutive hsp70 markedly protect microtubular structure in cardiac myocytes from ischemia-induced injury. Inducible hsp70, hsp60 and hsp10 when coexpressed, and hsp27 and alphaB-crystallin have an overall protective effect against ischemic injury as determined by the release of enzymes like creatine kinase and LDH. We did not note inflammatory or immune responses elicited by the expression of hsps in transgenic animals and cardiac myocytes. The specific cell types in which hsps are expressed may contribute to the protective effect of hsps versus their inflammatory and immunogenic effects when expressed in other cell types. PMID:10231010

  14. A Single B-repeat of Staphylococcus epidermidis accumulation-associated protein induces protective immune responses in an experimental biomaterial-associated infection mouse model.

    PubMed

    Yan, Lin; Zhang, Lei; Ma, Hongyan; Chiu, David; Bryers, James D

    2014-09-01

    Nosocomial infections are the fourth leading cause of morbidity and mortality in the United States, resulting in 2 million infections and ∼100,000 deaths each year. More than 60% of these infections are associated with some type of biomedical device. Staphylococcus epidermidis is a commensal bacterium of the human skin and is the most common nosocomial pathogen infecting implanted medical devices, especially those in the cardiovasculature. S. epidermidis antibiotic resistance and biofilm formation on inert surfaces make these infections hard to treat. Accumulation-associated protein (Aap), a cell wall-anchored protein of S. epidermidis, is considered one of the most important proteins involved in the formation of S. epidermidis biofilm. A small recombinant protein vaccine comprising a single B-repeat domain (Brpt1.0) of S. epidermidis RP62A Aap was developed, and the vaccine's efficacy was evaluated in vitro with a biofilm inhibition assay and in vivo in a murine model of biomaterial-associated infection. A high IgG antibody response against S. epidermidis RP62A was detected in the sera of the mice after two subcutaneous immunizations with Brpt1.0 coadministered with Freund's adjuvant. Sera from Brpt1.0-immunized mice inhibited in vitro S. epidermidis RP62A biofilm formation in a dose-dependent pattern. After receiving two immunizations, each mouse was surgically implanted with a porous scaffold disk containing 5 × 10(6) CFU of S. epidermidis RP62A. Weight changes, inflammatory markers, and histological assay results after challenge with S. epidermidis indicated that the mice immunized with Brpt1.0 exhibited significantly higher resistance to S. epidermidis RP62A implant infection than the control mice. Day 8 postchallenge, there was a significantly lower number of bacteria in scaffold sections and surrounding tissues and a lower residual inflammatory response to the infected scaffold disks for the Brpt1.0-immunized mice than for of the ovalbumin (Ova

  15. Biomimetic Antigenic Nanoparticles Elicit Controlled Protective Immune Response to Influenza

    PubMed Central

    Patterson, Dustin P.; Rynda-Apple, Agnieszka; Harmsen, Ann L.; Harmsen, Allen G.; Douglas, Trevor

    2013-01-01

    Here we present a biomimetic strategy towards nanoparticle design for controlled immune response through encapsulation of conserved internal influenza proteins on the interior of virus like particles (VLPs) to direct CD8+ cytotoxic T cell protection. Programmed encapsulation and sequestration of the conserved nucleoprotein (NP) from influenza on the interior of a VLP, derived from the bacteriophage P22, results in a vaccine that provides multi-strain protection against 100 times lethal doses of influenza in an NP specific CD8+ T cell-dependent manner. VLP assembly and encapsulation of the immunogenic NP cargo protein is the result of a genetically programmed self-assembly making this strategy amendable to the quick production of vaccines to rapidly emerging pathogens. Addition of adjuvants or targeting molecules were not required for eliciting the protective response. PMID:23540530

  16. Intradermal Immunization of Leishmania donovani Centrin Knock-Out Parasites in Combination with Salivary Protein LJM19 from Sand Fly Vector Induces a Durable Protective Immune Response in Hamsters

    PubMed Central

    Fiuza, Jacqueline Araújo; Dey, Ranadhir; Davenport, Dwann; Abdeladhim, Maha; Meneses, Claudio; Oliveira, Fabiano; Kamhawi, Shaden; Valenzuela, Jesus G.; Gannavaram, Sreenivas; Nakhasi, Hira L.

    2016-01-01

    Background Visceral leishmaniasis (VL) is a neglected tropical disease and is fatal if untreated. There is no vaccine available against leishmaniasis. The majority of patients with cutaneous leishmaniasis (CL) or VL develop a long-term protective immunity after cure from infection, which indicates that development of an effective vaccine against leishmaniasis is possible. Such protection may also be achieved by immunization with live attenuated parasites that do not cause disease. We have previously reported a protective response in mice, hamsters and dogs with Leishmania donovani centrin gene knock-out parasites (LdCen-/-), a live attenuated parasite with a cell division specific centrin1 gene deletion. In this study we have explored the effects of salivary protein LJM19 as an adjuvant and intradermal (ID) route of immunization on the efficacy of LdCen-/- parasites as a vaccine against virulent L. donovani. Methodology/Principal Findings To explore the potential of a combination of LdCen-/- parasites and salivary protein LJM19 as vaccine antigens, LdCen-/- ID immunization followed by ID challenge with virulent L. donovani were performed in hamsters in a 9-month follow up study. We determined parasite burden (serial dilution), antibody production (ELISA) and cytokine expression (qPCR) in these animals. Compared to controls, animals immunized with LdCen-/- + LJM19 induced a strong antibody response, a reduction in spleen and liver parasite burden and a higher expression of pro-inflammatory cytokines after immunization and one month post-challenge. Additionally, a low parasite load in lymph nodes, spleen and liver, and a non-inflamed spleen was observed in immunized animals 9 months after the challenge infection. Conclusions Our results demonstrate that an ID vaccination using LdCen-/-parasites in combination with sand fly salivary protein LJM19 has the capability to confer long lasting protection against visceral leishmaniasis that is comparable to intravenous or

  17. The mitochondrial UPR - protecting organelle protein homeostasis.

    PubMed

    Haynes, Cole M; Ron, David

    2010-11-15

    Mitochondria are required for numerous essential metabolic processes including the regulation of apoptosis; therefore, proper maintenance of the mitochondrial proteome is crucial. The protein-folding environment in mitochondria is challenged by organelle architecture, the presence of reactive oxygen species and the difficulties associated with assembly of the electron transport chain, which consists of components encoded by both the mitochondrial and the nuclear genomes. Mitochondria have dedicated molecular chaperones and proteases that promote proper protein folding, complex assembly and quality control. Work in cultured mammalian cells and Caenorhabditis elegans has yielded clues to the mechanisms linking perturbations in the protein-folding environment in the mitochondrial matrix to the expression of nuclear genes encoding mitochondrial proteins. Here, we review the current knowledge of this mitochondrial unfolded protein response (UPR(mt)), compare it with the better understood UPR of the endoplasmic reticulum and highlight its potential impact on development and disease.

  18. Immunization with the cysteine proteinase Ldccys1 gene from Leishmania (Leishmania) chagasi and the recombinant Ldccys1 protein elicits protective immune responses in a murine model of visceral leishmaniasis.

    PubMed

    Ferreira, Josie Haydée L; Gentil, Luciana Girotto; Dias, Suzana Souza; Fedeli, Carlos Eduardo C; Katz, Simone; Barbiéri, Clara Lúcia

    2008-01-30

    The gene Ldccys1 encoding a cysteine proteinase of 30 kDa from Leishmania (Leishmania) chagasi, as well as the recombinant cysteine proteinase rLdccys1, obtained by cloning and expression of the Ldccys1 gene in the pHIS vector, were used to evaluate their ability to induce immune protective responses in BALB/c mice against L. (L.) chagasi infection. Mice were immunized subcutaneously with rLdccys1 plus Bacille Calmette Guerin (BCG) or Propionibacterium acnes as adjuvants or intramuscularly with a plasmid carrying the Ldccys1 gene (Ldccys1/pcDNA3) and CpG ODN as the adjuvant, followed by a booster with rLdccys1 plus CpG ODN. Two weeks after immunization the animals were challenged with 1 x 10(7) amastigotes of L. (L.) chagasi. Both immunization protocols induced significant protection against L. (L.) chagasi infection as shown by a very low parasite load in the spleen of immunized mice compared to the non-immunized controls. However, DNA immunization was 10-fold more protective than immunization with the recombinant protein. Whereas rLdccys1 induced a significant secretion of IFN-gamma and nitric oxide (NO), animals immunized with the Ldccys1 gene increased the production of IgG2a antibodies, IFN-gamma and NO. These results indicated that protection triggered by the two immunization protocols was correlated to a predominant Th1 response.

  19. Administration of recombinant Reishi immunomodulatory protein (rLZ-8) diet enhances innate immune responses and elicits protection against nervous necrosis virus in grouper Epinephelus coioides.

    PubMed

    Kuan, Yen-Chou; Sheu, Fuu; Lee, Guo-Chi; Tsai, Ming-Wei; Hung, Chih-Liang; Nan, Fan-Hua

    2012-06-01

    Nervous necrosis virus (NNV) infection during larvae and juvenile stage in grouper (Epinephelus coioides) has caused severe economic losses in the aquaculture industry in Asia. The aims of this study were to evaluate the influence of recombinant Reishi protein, rLZ-8, on the innate immune responses and the viral resisting ability in fish. Groupers were fed with rLZ-8 supplemented diet (1.25-37.5 mg (rLZ-8)/kg(diet)), and the cytokine gene expression, innate immune responses, and survival rate after NNV challenge were examined. The fish fed with rLZ-8 diet showed 6- to 11-fold upregulated TNF-α and IL-1β gene expression, along with significant increased respiratory burst and phagocytic activity. Moreover, feeding the fish with 37.5 mg/kg rLZ-8 diet elicited significant improvement in post viral challenge survival rate (85.7%). These discoveries indicated that rLZ-8 could be utilized as an ant-pathogen immunostimulant, and provided a new candidate to fight against NNV infection in fish.

  20. The Phage Shock Protein Response.

    PubMed

    Flores-Kim, Josué; Darwin, Andrew J

    2016-09-08

    The phage shock protein (Psp) system was identified as a response to phage infection in Escherichia coli, but rather than being a specific response to a phage, it detects and mitigates various problems that could increase inner-membrane (IM) permeability. Interest in the Psp system has increased significantly in recent years due to appreciation that Psp-like proteins are found in all three domains of life and because the bacterial Psp response has been linked to virulence and other important phenotypes. In this article, we summarize our current understanding of what the Psp system detects and how it detects it, how four core Psp proteins form a signal transduction cascade between the IM and the cytoplasm, and current ideas that explain how the Psp response keeps bacterial cells alive. Although recent studies have significantly improved our understanding of this system, it is an understanding that is still far from complete.

  1. Development of a DNA vaccine for chicken infectious anemia and its immunogenicity studies using high mobility group box 1 protein as a novel immunoadjuvant indicated induction of promising protective immune responses.

    PubMed

    Sawant, Pradeep Mahadev; Dhama, Kuldeep; Rawool, Deepak Bhiva; Wani, Mohd Yaqoob; Tiwari, Ruchi; Singh, Shambhu Dayal; Singh, Raj Kumar

    2015-01-03

    Chicken infectious anaemia (CIA) is an economically important and emerging poultry disease reported worldwide. Current CIA vaccines have limitations like, the inability of the virus to grow to high titres in embryos/cell cultures, possession of residual pathogenicity and a risk of reversion to virulence. In the present study, a DNA vaccine, encoding chicken infectious anaemia virus (CIAV) VP1 and VP2 genes, was developed and co-administered with truncated chicken high mobility group box 1 (HMGB1ΔC) protein in young chicks for the evaluation of vaccine immune response. CIAV VP1 and VP2 genes were cloned in pTARGET while HMGB1ΔC in PET32b vector. In vitro expression of these gene constructs was evaluated by Western blotting. Further, recombinant HMGB1ΔC was evaluated for its biological activity. The CIAV DNA vaccine administration in specific pathogen free chicks resulted in moderately protective ELISA antibody titres in the range of 4322.87 ± 359.72 to 8288.19 ± 136.38, increased CD8(+) cells, and a higher titre was observed by co-administration of novel adjuvant (HMGB1ΔC) and booster immunizations. The use of vaccine with adjuvant showed achieving antibody titres nearly 8500, titre considered as highly protective, which indicates that co-immunization of HMGB1ΔC may have a strong adjuvant activity on CIAV DNA vaccine induced immune responses. The able potential of HMGB1 protein holding strong adjuvant activity could be exploited further with trials with vaccines for other important pathogens for achieving the required protective immune responses.

  2. Commercial Building Motor Protection Response Report

    SciTech Connect

    James, Daniel P.; Kueck, John

    2015-06-17

    When voltages recover, motors may immediately reenergize and reaccelerate, or delay for a few minutes, or stay stalled. The estimated motor response is given for both the voltage sag magnitude and voltage sag duration. These response estimates are based on experience and available test data. Good data is available for voltage sag response for many components such as relays and contactors, but little data is available for both voltage sag and recovery response. The tables in Appendix A include data from recent voltage sag and recovery tests performed by SCE and BPA on air conditioners and energy management systems. The response of the motor can vary greatly depending on the type of protection and control. The time duration for the voltage sag consists of those times that are of interest for bulk power system modelers.

  3. Thermal response properties of protective clothing fabrics

    SciTech Connect

    Baitinger, W.F.

    1995-12-31

    In the industrial workplace, it becomes increasingly incumbent upon employers to require employees to use suitable protective equipment and to wear protective apparel. When workers may be subjected to accidental radiant, flame, or electric arc heat sources, work clothing should be used that does not become involved in burning. It is axiomatic that work clothing should not become a primary fuel source, adding to the level of heat exposure, since clothing is usually in intimate contact with the skin. Further, clothing should provide sufficient insulation to protect the skin from severe burn injury. If the worker receives such protection from clothing, action then may be taken to escape the confronted thermal hazard. Published laboratory test methods are used to measure flame resistance and thermal responses of flame resistant fabrics in protective clothing. The purpose of this article is to review these test methods, to discuss certain limitations in application, and to suggest how flame resistant cotton fabrics may be used to enhance worker safety.

  4. A prime/boost PfCS14K(M)/MVA-sPfCS(M) vaccination protocol generates robust CD8(+) T cell and antibody responses to Plasmodium falciparum circumsporozoite protein and protects mice against malaria.

    PubMed

    Vijayan, Aneesh; Mejías-Pérez, Ernesto; Espinosa, Diego A; Raman, Suresh C; Sorzano, Carlos Oscar S; Zavala, Fidel; Esteban, Mariano

    2017-03-15

    Vaccines against the pre-erythrocytic stages of malaria are appealing, since the parasite can be eliminated before disease onset and since they offer the unique possibility of targeting the parasite with both antibodies and T cells. Although the role CD8(+) T cells in pre-erythrocytic malaria stages is well documented, a highly effective T cell-inducing vaccine remains to be advanced. Here we report the development of a prime-boost immunization regimen with the Plasmodium falciparum circumsporozoite protein (PfCS) fused to the oligomer-forming vaccinia virus A27 protein and a modified vaccinia virus Ankara (MVA) vector expressing PfCS. This protocol induced polyfunctional CD8(+) T cells with effector memory phenotype and high PfCS antibody levels. These immune responses correlated with inhibition of liver stage parasitemia in 80% and sterile protection in 40% of mice challenged with a transgenic P. berghei parasite line that expressed PfCS. Our findings underscore the potential of T and B cell immunization strategies in improving protective effectiveness against malaria.

  5. Mosaic tetracycline resistance genes encoding ribosomal protection proteins.

    PubMed

    Warburton, Philip J; Amodeo, Nina; Roberts, Adam P

    2016-12-01

    First reported in 2003, mosaic tetracycline resistance genes are a subgroup of the genes encoding ribosomal protection proteins (RPPs). They are formed when two or more RPP-encoding genes recombine resulting in a functional chimera. To date, the majority of mosaic genes are derived from sections of three RPP genes, tet(O), tet(W) and tet(32), with others comprising tet(M) and tet(S). In this first review of mosaic genes, we report on their structure, diversity and prevalence, and suggest that these genes may be responsible for an under-reported contribution to tetracycline resistance in bacteria.

  6. Mosaic tetracycline resistance genes encoding ribosomal protection proteins

    PubMed Central

    Warburton, Philip J.; Amodeo, Nina; Roberts, Adam P.

    2016-01-01

    First reported in 2003, mosaic tetracycline resistance genes are a subgroup of the genes encoding ribosomal protection proteins (RPPs). They are formed when two or more RPP-encoding genes recombine resulting in a functional chimera. To date, the majority of mosaic genes are derived from sections of three RPP genes, tet(O), tet(W) and tet(32), with others comprising tet(M) and tet(S). In this first review of mosaic genes, we report on their structure, diversity and prevalence, and suggest that these genes may be responsible for an under-reported contribution to tetracycline resistance in bacteria. PMID:27494928

  7. Clustered epitopes within the Gag-Pol fusion protein DNA vaccine enhance immune responses and protection against challenge with recombinant vaccinia viruses expressing HIV-1 Gag and Pol antigens.

    PubMed

    Bolesta, Elizabeth; Gzyl, Jaroslaw; Wierzbicki, Andrzej; Kmieciak, Dariusz; Kowalczyk, Aleksandra; Kaneko, Yutaro; Srinivasan, Alagarsamy; Kozbor, Danuta

    2005-02-20

    We have generated a codon-optimized hGagp17p24-Polp51 plasmid DNA expressing the human immunodeficiency virus type 1 (HIV-1) Gag-Pol fusion protein that consists of clusters of highly conserved cytotoxic T lymphocyte (CTL) epitopes presented by multiple MHC class I alleles. In the hGagp17p24-Polp51 construct, the ribosomal frameshift site had been deleted together with the potentially immunosuppressive Gag nucleocapsid (p15) as well as Pol protease (p10) and integrase (p31). Analyses of the magnitude and breadth of cellular responses demonstrated that immunization of HLA-A2/K(b) transgenic mice with the hGagp17p24-Polp51 construct induced 2- to 5-fold higher CD8+ T-cell responses to Gag p17-, p24-, and Pol reverse transcriptase (RT)-specific CTL epitopes than the full-length hGag-PolDeltaFsDeltaPr counterpart. The increases were correlated with higher protection against challenge with recombinant vaccinia viruses (rVVs) expressing gag and pol gene products. Consistent with the profile of Gag- and Pol-specific CD8+ T cell responses, an elevated level of type 1 cytokine production was noted in p24- and RT-stimulated splenocyte cultures established from hGagp17p24-Polp51-immunized mice compared to responses induced with the hGag-PolDeltaFsDeltaPr vaccine. Sera of mice immunized with the hGagp17p24-Polp51 vaccine also exhibited an increased titer of p24- and RT-specific IgG2 antibody responses. The results from our studies provide insights into approaches for boosting the breadth of Gag- and Pol-specific immune responses.

  8. Protective Immunogenicity of Group A Streptococcal M-Related Proteins

    PubMed Central

    Niedermeyer, Shannon E.; Agbaosi, Tina; Hysmith, Nicholas D.; Penfound, Thomas A.; Hohn, Claudia M.; Pullen, Matthew; Bright, Michael I.; Murrell, Daniel S.; Shenep, Lori E.; Courtney, Harry S.

    2015-01-01

    Many previous studies have focused on the surface M proteins of group A streptococci (GAS) as virulence determinants and protective antigens. However, the majority of GAS isolates express M-related protein (Mrp) in addition to M protein, and both have been shown to be required for optimal virulence. In the current study, we evaluated the protective immunogenicity of Mrp to determine its potential as a vaccine component that may broaden the coverage of M protein-based vaccines. Sequence analyses of 33 mrp genes indicated that there are three families of structurally related Mrps (MrpI, MrpII, and MrpIII). N-terminal peptides of Mrps were cloned, expressed, and purified from M type 2 (M2) (MrpI), M4 (MrpII), and M49 (MrpIII) GAS. Rabbit antisera against the Mrps reacted at high titers with the homologous Mrp, as determined by enzyme-linked immunosorbent assay, and promoted bactericidal activity against GAS emm types expressing Mrps within the same family. Mice passively immunized with rabbit antisera against MrpII were protected against challenge infections with M28 GAS. Assays for Mrp antibodies in serum samples from 281 pediatric subjects aged 2 to 16 indicated that the Mrp immune response correlated with increasing age of the subjects. Affinity-purified human Mrp antibodies promoted bactericidal activity against a number of GAS representing different emm types that expressed an Mrp within the same family but showed no activity against emm types expressing an Mrp from a different family. Our results indicate that Mrps have semiconserved N-terminal sequences that contain bactericidal epitopes which are immunogenic in humans. These findings may have direct implications for the development of GAS vaccines. PMID:25630406

  9. Novel influenza virus vectors expressing Brucella L7/L12 or Omp16 proteins in cattle induced a strong T-cell immune response, as well as high protectiveness against B. abortus infection.

    PubMed

    Tabynov, Kaissar; Kydyrbayev, Zhailaubay; Ryskeldinova, Sholpan; Yespembetov, Bolat; Zinina, Nadezhda; Assanzhanova, Nurika; Kozhamkulov, Yerken; Inkarbekov, Dulat; Gotskina, Tatyana; Sansyzbay, Abylai

    2014-04-11

    This paper presents the results of a study of the immunogenicity and protectiveness of new candidate vector vaccine against Brucella abortus - a bivalent vaccine formulation consisting of a mixture of recombinant influenza A subtype H5N1 or H1N1 (viral constructs vaccine formulation) viruses expressing Brucella ribosomal protein L7/L12 and Omp16, in cattle. To increase the effectiveness of the candidate vaccine, adjuvants such as Montanide Gel01 or chitosan were included in its composition. Immunization of cattle (heifers aged 1-1.5 years, 5 animals per group) with the viral constructs vaccine formulation only, or its combination with adjuvants Montanide Gel01 or chitosan, was conducted via the conjunctival method using cross prime (influenza virus subtype H5N1) and booster (influenza virus subtype H1N1) vaccination schedules at an interval of 28 days. Vaccine candidates were evaluated in comparison with the positive (B. abortus S19) and negative (PBS) controls. The viral constructs vaccine formulations, particularly in combination with Montanide Gel01 adjuvant promoted formation of IgG antibodies (with a predominance of antibodies of isotype IgG2a) against Brucella L7/L12 and Omp16 proteins in ELISA. Moreover, these vaccines in cattle induced a strong antigen-specific T-cell immune response, as indicated by a high number of CD4(+) and CD8(+) cells, as well as the concentration of IFN-γ, and most importantly provided a high level of protectiveness comparable to the commercial B. abortus S19 vaccine and superior to the B. abortus S19 vaccine in combination with Montanide Gel01 adjuvant. Based on these findings, we recommended the bivalent vaccine formulation containing the adjuvant Montanide Gel01 for practical use in cattle.

  10. A bicistronic DNA vaccine containing apical membrane antigen 1 and merozoite surface protein 4/5 can prime humoral and cellular immune responses and partially protect mice against virulent Plasmodium chabaudi adami DS malaria.

    PubMed

    Rainczuk, A; Scorza, T; Spithill, T W; Smooker, P M

    2004-10-01

    The ultimate malaria vaccine will require the delivery of multiple antigens from different stages of the complex malaria life cycle. In order to efficiently deliver multiple antigens with use of DNA vaccine technology, new antigen delivery systems must be assessed. This study utilized a bicistronic vector construct, containing an internal ribosome entry site, expressing a combination of malarial candidate antigens: merozoite surface protein 4/5 (MSP4/5) (fused to a monocyte chemotactic protein 3 chemoattractant sequence) and apical membrane antigen 1 (AMA-1) (fused to a tissue plasminogen activator secretion signal). Transfection of COS 7 cells with bicistronic plasmids resulted in production and secretion of both AMA-1 and MSP4/5 in vitro. Vaccination of BALB/c mice via intraepidermal gene gun and intramuscular routes against AMA-1 and MSP4/5 resulted in antibody production and significant in vitro proliferation of splenocytes stimulated by both AMA-1 and MSP4/5. Survival of BALB/c mice vaccinated with bicistronic constructs after lethal Plasmodium chabaudi adami DS erythrocytic-stage challenge was variable, although significant increases in survival and reductions in peak parasitemia were observed in several challenge trials when the vaccine was delivered by the intramuscular route. This study using a murine model demonstrates that the delivery of malarial antigens via bicistronic vectors is feasible. Further experimentation with bicistronic delivery systems is required for the optimization and refinement of DNA vaccines to effectively prime protective immune responses against malaria.

  11. 5-Aminoimidazole-4-carboxamide ribonucleoside-mediated adenosine monophosphate-activated protein kinase activation induces protective innate responses in bacterial endophthalmitis.

    PubMed

    Kumar, Ajay; Giri, Shailendra; Kumar, Ashok

    2016-12-01

    The retina is considered to be the most metabolically active tissue in the body. However, the link between energy metabolism and retinal inflammation, as incited by microbial infection such as endophthalmitis, remains unexplored. In this study, using a mouse model of Staphylococcus aureus (SA) endophthalmitis, we demonstrate that the activity (phosphorylation) of 5' adenosine monophosphate-activated protein kinase alpha (AMPKα), a cellular energy sensor and its endogenous substrate; acetyl-CoA carboxylase is down-regulated in the SA-infected retina. Intravitreal administration of an AMPK activator, 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), restored AMPKα and acetyl-CoA carboxylase phosphorylation. AICAR treatment reduced both the bacterial burden and intraocular inflammation in SA-infected eyes by inhibiting NF-kB and MAP kinases (p38 and JNK) signalling. The anti-inflammatory effects of AICAR were diminished in eyes pretreated with AMPK inhibitor, Compound C. The bioenergetics (Seahorse) analysis of SA-infected microglia and bone marrow-derived macrophages revealed an increase in glycolysis, which was reinstated by AICAR treatment. AICAR also reduced the expression of SA-induced glycolytic genes, including hexokinase 2 and glucose transporter 1 in microglia, bone marrow-derived macrophages and the mouse retina. Interestingly, AICAR treatment enhanced the bacterial phagocytic and intracellular killing activities of cultured microglia, macrophages and neutrophils. Furthermore, AMPKα1 global knockout mice exhibited increased susceptibility towards SA endophthalmitis, as evidenced by increased inflammatory mediators and bacterial burden and reduced retinal function. Together, these findings provide the first evidence that AMPK activation promotes retinal innate defence in endophthalmitis by modulating energy metabolism and that it can be targeted therapeutically to treat ocular infections.

  12. Rabbit immunoglobulin responses to the flagella, somatic, and protective antigens of a highly protective strain of Clostridium chauvoei.

    PubMed

    Chandler, H M

    1975-07-01

    The immunoglobulin response of rabbits to the flagells (H), somatic (O), and protective antigens of a highly protective strain of Clostridium chauvoei was studied using antisera that had been fractionated by Sephadex G-200 chromatography. The H antigen elicited the characteristic agglutinin response to a protein antigen--early production of 19S globulin followed by persistent 7S globulin production. The O antigen stimulated a transient agglutinin response which was detected in both the 19S and 7S serum fractions. Protective antibody was assayed by passive protection tests in mice. Using these tests the protective activity of the rabbit sera was found to be confined exclusively to the 7S serum fractions. Purified immunoglobulin G, prepared by DEAE-cellulose chromatography of the above sera, was also tested and found to confer considerable passive protection on mice. It is considered that either the protective antigen fails to stimulate an immunoglobulin M response or that immunoglobulin M is relatively ineffective in conferring protection against infection in the mouse passive protection tests.

  13. Environmental Protection Agency Award Recipient Responsibilities

    EPA Pesticide Factsheets

    Itemized Award Phase information. Information about the Recipient's Responsibilities Upon Notification of the Award, The EPA Project Officer Responsibilities, and EPA Grant Specialists Responsibilities.

  14. DNA vaccines expressing pneumococcal surface protein A (PspA) elicit protection levels comparable to recombinant protein.

    PubMed

    Ferreira, Daniela M; Miyaji, Eliane N; Oliveira, Maria Leonor S; Darrieux, Michelle; Arêas, Ana Paula M; Ho, Paulo L; Leite, Luciana C C

    2006-04-01

    Pneumococcal surface protein A (PspA) is a promising candidate for the development of cost-effective vaccines against Streptococcus pneumoniae. In the present study, BALB/c mice were immunized with DNA vaccine vectors expressing the N-terminal region of PspA. Animals immunized with a vector expressing secreted PspA developed higher levels of antibody than mice immunized with the vector expressing the antigen in the cytosol. However, both immunogens elicited similar levels of protection against intraperitoneal challenge. Furthermore, immunization with exactly the same fragment in the form of a recombinant protein, with aluminium hydroxide as an adjuvant, elicited even higher antibody levels, but this increased humoral response did not correlate with enhanced protection. These results show that DNA vaccines expressing PspA are able to elicit protection levels comparable to recombinant protein, even though total anti-PspA IgG response is considerably lower.

  15. Identification of polyvalent protective immunogens from outer membrane proteins in Vibrio parahaemolyticus to protect fish against bacterial infection.

    PubMed

    Peng, Bo; Ye, Jin-Zhou; Han, Yi; Zeng, Li; Zhang, Jian-Ying; Li, Hui

    2016-07-01

    Vaccination is one of the most effective and economic way to prevent infectious diseases in aquaculture. The development of effective vaccines, however, is still limited, especially for polyvalent vaccines, which are against multiple species. With this regard, identification of polyvalent protective immunogens, serving as polyvalent vaccines, became a key step in vaccine development. In the current study, 17 outer membrane proteins from Vibrio parahaemolyticus were identified as immunogens. Further, four of the 17 proteins including VP2309, VP0887, VPA0548 and VP1019 were characterized as efficiently protective immunogens against V. parahaemolyticus' infection through passive and active immunizations in zebrafish. Importantly, these four proteins showed cross-protective capability against infections by Aeromonas hydrophila or/and Pseudomonas fluorescens, which shared similar epitopes with V. parahaemolyticus in homology of these proteins. Further investigation showed that the expression level of the four protective immunogens elevated in response to fish plasma in a dose-dependent manner. These results indicate that the four protective immunogens are polyvalent vaccine candidates in aquaculture.

  16. Structure-based design of broadly protective group a streptococcal M protein-based vaccines

    SciTech Connect

    Dale, James B.; Smeesters, Pierre R.; Courtney, Harry S.; Penfound, Thomas A.; Hohn, Claudia M.; Smith, Jeremy C.; Baudry, Jerome Y.

    2016-11-24

    Here, a major obstacle to the development of broadly protective M protein-based group A streptococcal (GAS) vaccines is the variability within the N-terminal epitopes that evoke potent bactericidal antibodies. The concept of M type-specific protective immune responses has recently been challenged based on the observation that multivalent M protein vaccines elicited cross-reactive bactericidal antibodies against a number of non-vaccine M types of GAS. Additionally, a new “cluster-based” typing system of 175 M proteins identified a limited number of clusters containing closely related M proteins. In the current study, we used the emm cluster typing system, in combination with computational structure-based peptide modeling, as a novel approach to the design of potentially broadly protective M protein-based vaccines.

  17. Structure-based design of broadly protective group a streptococcal M protein-based vaccines

    DOE PAGES

    Dale, James B.; Smeesters, Pierre R.; Courtney, Harry S.; ...

    2016-11-24

    Here, a major obstacle to the development of broadly protective M protein-based group A streptococcal (GAS) vaccines is the variability within the N-terminal epitopes that evoke potent bactericidal antibodies. The concept of M type-specific protective immune responses has recently been challenged based on the observation that multivalent M protein vaccines elicited cross-reactive bactericidal antibodies against a number of non-vaccine M types of GAS. Additionally, a new “cluster-based” typing system of 175 M proteins identified a limited number of clusters containing closely related M proteins. In the current study, we used the emm cluster typing system, in combination with computational structure-basedmore » peptide modeling, as a novel approach to the design of potentially broadly protective M protein-based vaccines.« less

  18. UVR8 mediated plant protective responses under low UV-B radiation leading to photosynthetic acclimation.

    PubMed

    Singh, Suruchi; Agrawal, S B; Agrawal, Madhoolika

    2014-08-01

    The UV-B photoreceptor UVR8 regulates the expression of several genes leading to acclimation responses in plants. Direct role of UVR8 in maintaining the photosynthesis is not defined but it is known to increase the expression of some chloroplastic proteins like SIG5 and ELIP. It provides indirect protection to photosynthesis by regulating the synthesis of secondary metabolites and photomorphogenesis. Signaling cascades controlled by UVR8 mediate many protective responses thus promotes plant acclimation against stress and secures its survival.

  19. A Translocated Bacterial Protein Protects Vascular Endothelial Cells from Apoptosis

    PubMed Central

    Schmid, Michael C; Scheidegger, Florine; Dehio, Michaela; Balmelle-Devaux, Nadège; Schulein, Ralf; Guye, Patrick; Chennakesava, Cuddapah S; Biedermann, Barbara; Dehio, Christoph

    2006-01-01

    The modulation of host cell apoptosis by bacterial pathogens is of critical importance for the outcome of the infection process. The capacity of Bartonella henselae and B. quintana to cause vascular tumor formation in immunocompromised patients is linked to the inhibition of vascular endothelial cell (EC) apoptosis. Here, we show that translocation of BepA, a type IV secretion (T4S) substrate, is necessary and sufficient to inhibit EC apoptosis. Ectopic expression in ECs allowed mapping of the anti-apoptotic activity of BepA to the Bep intracellular delivery domain, which, as part of the signal for T4S, is conserved in other T4S substrates. The anti-apoptotic activity appeared to be limited to BepA orthologs of B. henselae and B. quintana and correlated with (i) protein localization to the host cell plasma membrane, (ii) elevated levels of intracellular cyclic adenosine monophosphate (cAMP), and (iii) increased expression of cAMP-responsive genes. The pharmacological elevation of cAMP levels protected ECs from apoptosis, indicating that BepA mediates anti-apoptosis by heightening cAMP levels by a plasma membrane–associated mechanism. Finally, we demonstrate that BepA mediates protection of ECs against apoptosis triggered by cytotoxic T lymphocytes, suggesting a physiological context in which the anti-apoptotic activity of BepA contributes to tumor formation in the chronically infected vascular endothelium. PMID:17121462

  20. Protective immune responses to fungal infections.

    PubMed

    Rivera, A

    2014-09-01

    The incidence of fungal infections has been on the rise over several decades. Fungal infections threaten animals, plants and humans alike and are thus of significant concern to scientists across disciplines. Over the last decade, significant advances on fungal immunology have lead to a better understanding of important mechanisms of host protection against fungi. In this article, I review recent advances of relevant mechanisms of immune-mediated protection to fungal infections.

  1. TpUB05, a Homologue of the Immunodominant Plasmodium falciparum Protein UB05, Is a Marker of Protective Immune Responses in Cattle Experimentally Vaccinated against East Coast Fever

    PubMed Central

    Dinga, Jerome Nyhalah; Wamalwa, Mark; Njimoh, Dieudonné Lemuh; Njahira, Moses N.; Djikeng, Appolinaire; Skilton, Rob; Titanji, Vincent Pryde Kehdingha; Pellé, Roger

    2015-01-01

    Introduction East Coast fever, a devastating disease of cattle, can be controlled partially by vaccination with live T. parva sporozoites. The antigens responsible for conferring immunity are not fully characterized. Recently it was shown that the P. falciparum immunodominant protein UB05 is highly conserved in T. parva, the causative agent of East Coast fever. The aim of the present investigation was to determine the role of the homologue TpUB05 in protective immunity to East Coast fever. Methods The cloning, sequencing and expression of TpUB05 were done according to standard protocols. Bioinformatics analysis of TpUB05 gene was carried out using algorithms found in the public domain. Polyclonal antiserum against recombinant TpUB05 were raised in rabbits and used for further analysis by Western blotting, ELISA, immunolocalization and in vitro infection neutralization assay. The ability of recombinant TpUB05 (r-TpUB05) to stimulate bovine PBMCs ex-vivo to produce IFN-γ or to proliferate was tested using ELISpot and [3H]-thymidine incorporation assays, respectively. Results All the 20 cattle immunised by the infection and treatment method (ITM) developed significantly higher levels of TpUB05 specific antibodies (p<0.0001) compared to the non-vaccinated ones. Similarly, r-TpUB05 highly stimulated bovine PMBCs from 8/12 (67%) of ITM-immunized cattle tested to produce IFN-γ and proliferate (p< 0.029) as compared to the 04 naїve cattle included as controls. Polyclonal TpUB05 antiserum raised against r-TpUB05 also marginally inhibited infection (p < 0.046) of bovine PBMCs by T. parva sporozoites. In further experiments RT-PCR showed that the TpUB05 gene is expressed by the parasite. This was confirmed by immunolocalization studies which revealed TpUB05 expression by schizonts and piroplasms. Bioinformatics analysis also revealed that this antigen possesses two transmembrane domains, a N-glycosylation site and several O-glycosylation sites. Conclusion It was concluded

  2. Antibody response that protects against disseminated candidiasis.

    PubMed Central

    Han, Y; Cutler, J E

    1995-01-01

    We previously showed that surface mannans of Candida albicans function as adhesins during yeast cell attachment to mouse splenic marginal zone macrophages. The mannan adhesin fraction was encapsulated into liposomes and used to vaccinate mice over a 5- to 6-week period. Circulating agglutinins specific for the fraction correlated with increased resistance to disseminated candidiasis. Antiserum from vaccinated animals protected naive BALB/cByJ mice against C. albicans serotype A and B strains and Candida tropicalis. Antiserum also protected SCID mice against disseminated disease. The serum protective ability was stable at 56 degrees C, but this ability was adsorbed by C. albicans cells. The antiserum was divided into three fractions after separation by high-performance liquid chromatography. One fraction contained all of the agglutinin activity and transferred resistance to naive mice. A second fraction also transferred resistance. Two monoclonal antibodies (MAbs) specific for candidal surface determinants were obtained. MAb B6.1 is specific for a mannan epitope in the adhesin fraction, and MAb B6 is specific for a different epitope in the fraction. Both MAbs are immunoglobulin M, and both strongly agglutinate candidal cells, but only MAb B6.1 protected both normal and SCID mice against disseminated candidiasis. In one experiment, 10 normal mice were given MAb B6.1 and challenged with yeast cells. Six mice survived the 67-day observation period; 4 of the survivors were cured as evidenced by the lack of CFU in the kidney and spleen. Our studies show that antibodies against certain cell surface antigens of C. albicans help the host resist disseminated candidiasis. PMID:7790089

  3. How Egg Case Proteins Can Protect Cuttlefish Offspring?

    PubMed

    Cornet, Valérie; Henry, Joël; Goux, Didier; Duval, Emilie; Bernay, Benoit; Le Corguillé, Gildas; Corre, Erwan; Zatylny-Gaudin, Céline

    2015-01-01

    Sepia officinalis egg protection is ensured by a complex capsule produced by the female accessory genital glands and the ink bag. Our study is focused on the proteins constituting the main egg case. De novo transcriptomes from female genital glands provided essential databases for protein identification. A proteomic approach in SDS-PAGE coupled with MS unveiled a new egg case protein family: SepECPs, for Sepia officinalis Egg Case Proteins. N-glycosylation was demonstrated by PAS staining SDS-PAGE gels. These glycoproteins are mainly produced in the main nidamental glands. SepECPs share high sequence homology, especially in the signal peptide and the three cysteine-rich domains. SepECPs have a high number of cysteines, with conserved motifs involved in 3D-structure. SDS-PAGE showed that SepECPs could form dimers; this result was confirmed by TEM observations, which also revealed a protein network. This network is similar to the capsule network, and it associates these structural proteins with polysaccharides, melanin and bacteria to form a tight mesh. Its hardness and elasticity provide physical protection to the embryo. In addition, SepECPs also have bacteriostatic antimicrobial activity on GRAM- bacteria. By observing the SepECP / Vibrio aestuarianus complex in SEM, we demonstrated the ability of these proteins to agglomerate bacteria and thus inhibit their growth. These original proteins identified from the outer egg case ensure the survival of the species by providing physical and chemical protection to the embryos released in the environment without any maternal protection.

  4. How Egg Case Proteins Can Protect Cuttlefish Offspring?

    PubMed Central

    Cornet, Valérie; Henry, Joël; Goux, Didier; Duval, Emilie; Bernay, Benoit; Le Corguillé, Gildas; Corre, Erwan; Zatylny-Gaudin, Céline

    2015-01-01

    Sepia officinalis egg protection is ensured by a complex capsule produced by the female accessory genital glands and the ink bag. Our study is focused on the proteins constituting the main egg case. De novo transcriptomes from female genital glands provided essential databases for protein identification. A proteomic approach in SDS-PAGE coupled with MS unveiled a new egg case protein family: SepECPs, for Sepia officinalis Egg Case Proteins. N-glycosylation was demonstrated by PAS staining SDS-PAGE gels. These glycoproteins are mainly produced in the main nidamental glands. SepECPs share high sequence homology, especially in the signal peptide and the three cysteine-rich domains. SepECPs have a high number of cysteines, with conserved motifs involved in 3D-structure. SDS-PAGE showed that SepECPs could form dimers; this result was confirmed by TEM observations, which also revealed a protein network. This network is similar to the capsule network, and it associates these structural proteins with polysaccharides, melanin and bacteria to form a tight mesh. Its hardness and elasticity provide physical protection to the embryo. In addition, SepECPs also have bacteriostatic antimicrobial activity on GRAM- bacteria. By observing the SepECP / Vibrio aestuarianus complex in SEM, we demonstrated the ability of these proteins to agglomerate bacteria and thus inhibit their growth. These original proteins identified from the outer egg case ensure the survival of the species by providing physical and chemical protection to the embryos released in the environment without any maternal protection. PMID:26168161

  5. ABC-F Proteins Mediate Antibiotic Resistance through Ribosomal Protection

    PubMed Central

    Sharkey, Liam K. R.; Edwards, Thomas A.

    2016-01-01

    ABSTRACT Members of the ABC-F subfamily of ATP-binding cassette proteins mediate resistance to a broad array of clinically important antibiotic classes that target the ribosome of Gram-positive pathogens. The mechanism by which these proteins act has been a subject of long-standing controversy, with two competing hypotheses each having gained considerable support: antibiotic efflux versus ribosomal protection. Here, we report on studies employing a combination of bacteriological and biochemical techniques to unravel the mechanism of resistance of these proteins, and provide several lines of evidence that together offer clear support to the ribosomal protection hypothesis. Of particular note, we show that addition of purified ABC-F proteins to an in vitro translation assay prompts dose-dependent rescue of translation, and demonstrate that such proteins are capable of displacing antibiotic from the ribosome in vitro. To our knowledge, these experiments constitute the first direct evidence that ABC-F proteins mediate antibiotic resistance through ribosomal protection. PMID:27006457

  6. A Federal Response: The President's Critical Infrastructure Protection Board.

    ERIC Educational Resources Information Center

    Schmidt, Howard

    2002-01-01

    Outlines the U.S. Critical Infrastructure Protection Board's purpose, budget, principles, and priorities. Describes the board's role in coordinating all federal activities related to protection of information systems and networks supporting critical infrastructures. Also discusses its responsibility in creating a policy and road map for government…

  7. Attenuated vaccinia virus-circumsporozoite protein recombinants confer protection against rodent malaria.

    PubMed Central

    Lanar, D E; Tine, J A; de Taisne, C; Seguin, M C; Cox, W I; Winslow, J P; Ware, L A; Kauffman, E B; Gordon, D; Ballou, W R; Paoletti, E; Sadoff, J C

    1996-01-01

    NYVAC-based vaccinia virus recombinants expressing the circumsporozoite protein (CSP) were evaluated in the Plasmodium berghei rodent malaria model system. Immunization of mice with a NYVAC-based CSP recombinant elicited a high level of protection (60 to 100%). Protection did not correlate with CS repeat-specific antibody responses and was abrogated by in vivo CD8+ T-cell depletion. Protection was not enhanced by modification of the subcellular localization of CSP. These results suggest the potential of poxvirus-based vectors for the development of vaccine candidates for human malaria. PMID:8613376

  8. ABC-F Proteins Mediate Antibiotic Resistance through Ribosomal Protection.

    PubMed

    Sharkey, Liam K R; Edwards, Thomas A; O'Neill, Alex J

    2016-03-22

    Members of the ABC-F subfamily of ATP-binding cassette proteins mediate resistance to a broad array of clinically important antibiotic classes that target the ribosome of Gram-positive pathogens. The mechanism by which these proteins act has been a subject of long-standing controversy, with two competing hypotheses each having gained considerable support: antibiotic efflux versus ribosomal protection. Here, we report on studies employing a combination of bacteriological and biochemical techniques to unravel the mechanism of resistance of these proteins, and provide several lines of evidence that together offer clear support to the ribosomal protection hypothesis. Of particular note, we show that addition of purified ABC-F proteins to anin vitrotranslation assay prompts dose-dependent rescue of translation, and demonstrate that such proteins are capable of displacing antibiotic from the ribosomein vitro To our knowledge, these experiments constitute the first direct evidence that ABC-F proteins mediate antibiotic resistance through ribosomal protection.IMPORTANCEAntimicrobial resistance ranks among the greatest threats currently facing human health. Elucidation of the mechanisms by which microorganisms resist the effect of antibiotics is central to understanding the biology of this phenomenon and has the potential to inform the development of new drugs capable of blocking or circumventing resistance. Members of the ABC-F family, which includelsa(A),msr(A),optr(A), andvga(A), collectively yield resistance to a broader range of clinically significant antibiotic classes than any other family of resistance determinants, although their mechanism of action has been controversial since their discovery 25 years ago. Here we present the first direct evidence that proteins of the ABC-F family act to protect the bacterial ribosome from antibiotic-mediated inhibition.

  9. Protective host immune responses to Salmonella infection.

    PubMed

    Pham, Oanh H; McSorley, Stephen J

    2015-01-01

    Salmonella enterica serovars Typhi and Paratyphi are the causative agents of human typhoid fever. Current typhoid vaccines are ineffective and are not widely used in endemic areas. Greater understanding of host-pathogen interactions during Salmonella infection should facilitate the development of improved vaccines to combat typhoid and nontyphoidal Salmonellosis. This review will focus on our current understanding of Salmonella pathogenesis and the major host immune components that participate in immunity to Salmonella infection. In addition, recent findings regarding host immune mechanisms in response to Salmonella infection will be also discussed, providing a new perspective on the utility of improved tools to study the immune response to Salmonella infections.

  10. Shigella outer membrane protein PSSP-1 is broadly protective against Shigella infection.

    PubMed

    Kim, Jae-Ouk; Rho, Semi; Kim, Su Hee; Kim, Heejoo; Song, Hyo Jin; Kim, Eun Jin; Kim, Ryang Yeo; Kim, Eun Hye; Sinha, Anuradha; Dey, Ayan; Yang, Jae Seung; Song, Man Ki; Nandy, Ranjan Kumar; Czerkinsky, Cecil; Kim, Dong Wook

    2015-04-01

    In developing countries, Shigella is a primary cause of diarrhea in infants and young children. Although antibiotic therapy is an effective treatment for shigellosis, therapeutic options are narrowing due to the emergence of antibiotic resistance. Thus, preventive vaccination could become the most efficacious approach for controlling shigellosis. We have identified several conserved protein antigens that are shared by multiple Shigella serotypes and species. Among these, one antigen induced cross-protection against experimental shigellosis, and we have named it pan-Shigella surface protein 1 (PSSP-1). PSSP-1-induced protection requires a mucosal administration route and coadministration of an adjuvant. When PSSP-1 was administered intranasally, it induced cross-protection against Shigella flexneri serotypes 2a, 5a, and 6, Shigella boydii, Shigella sonnei, and Shigella dysenteriae serotype 1. Intradermally administered PSSP-1 induced strong serum antibody responses but failed to induce protection in the mouse lung pneumonia model. In contrast, intranasal administration elicited efficient local and systemic antibody responses and production of interleukin 17A and gamma interferon. Interestingly, blood samples from patients with recent-onset shigellosis showed variable but significant mucosal antibody responses to other conserved Shigella protein antigens but not to PSSP-1. We suggest that PSSP-1 is a promising antigen for a broadly protective vaccine against Shigella.

  11. ABC proteins protect the human body and maintain optimal health.

    PubMed

    Ueda, Kazumitsu

    2011-01-01

    Human MDR1, a multi-drug transporter gene, was isolated as the first of the eukaryote ATP Binding Cassette (ABC) proteins from a multidrug-resistant carcinoma cell line in 1986. To date, over 25 years, many ABC proteins have been found to play important physiological roles by transporting hydrophobic compounds. Defects in their functions cause various diseases, indicating that endogenous hydrophobic compounds, as well as water-soluble compounds, are properly transported by transmembrane proteins. MDR1 transports a large number of structurally unrelated drugs and is involved in their pharmacokinetics, and thus is a key factor in drug interaction. ABCA1, an ABC protein, eliminates excess cholesterol in peripheral cells by generating HDL. Because ABCA1 is a key molecule in cholesterol homeostasis, its function and expression are highly regulated. Eukaryote ABC proteins function on the body surface facing the outside and in organ pathways to adapt to the extracellular environment and protect the body to maintain optimal health.

  12. Theoretical tests of the mechanical protection strategy in protein nanomechanics.

    PubMed

    Chwastyk, Mateusz; Galera-Prat, Albert; Sikora, Mateusz; Gómez-Sicilia, Àngel; Carrión-Vázquez, Mariano; Cieplak, Marek

    2014-05-01

    We provide theoretical tests of a novel experimental technique to determine mechanostability of proteins based on stretching a mechanically protected protein by single-molecule force spectroscopy. This technique involves stretching a homogeneous or heterogeneous chain of reference proteins (single-molecule markers) in which one of them acts as host to the guest protein under study. The guest protein is grafted into the host through genetic engineering. It is expected that unraveling of the host precedes the unraveling of the guest removing ambiguities in the reading of the force-extension patterns of the guest protein. We study examples of such systems within a coarse-grained structure-based model. We consider systems with various ratios of mechanostability for the host and guest molecules and compare them to experimental results involving cohesin I as the guest molecule. For a comparison, we also study the force-displacement patterns in proteins that are linked in a serial fashion. We find that the mechanostability of the guest is similar to that of the isolated or serially linked protein. We also demonstrate that the ideal configuration of this strategy would be one in which the host is much more mechanostable than the single-molecule markers. We finally show that it is troublesome to use the highly stable cystine knot proteins as a host to graft a guest in stretching studies because this would involve a cleaving procedure.

  13. [Protective immune response of guinea pigs against challenge with foot and mouth disease virus by immunization with foliar extracts from transgenic tomato plants expressing the FMDV structural protein VP1].

    PubMed

    Pan, Li; Zhang, Yong-Guang; Wang, Yong-Lu; Wang, Bao-Qin; Xie, Qing-Ge

    2006-10-01

    The plant constitutive expression vector pBin438/VP1 for VP1 gene of foot-and-mouth disease virus strain O/ China/99 was constructed. Mediated with Agrobacterium tumefaciens GV3101 harboring pBin438/VP1, VP1 gene was transferred into cotyledons of tomato. After selected by Kanamysin, sixty resistant lines were obtained. The integration and transcription of the VP1 gene in transformed plants was detected by PCR and RT-PCR. After being detected by sandwich-ELISA assays, about 40% transformed plants confirmed to express the recombinant protein. The leave extracts of two positive lines were respectively emulsified in Freund's adjuvant and guinea pigs were intramuscular inoculation at days 0, 15 and 30d. According to the sera antibody levels and the protection of the vaccinated guinea pigs against challenge with 100ID50 FMDV, probed into the immunogenicity of the target protein expressed in transgenic plants. Experimental results showed that the plant expression vector was successfully constructed. PCR and RT-PCR analyses confirmed VP1 gene was transformed into tomato plants and got expression at the transcription levels. The expressed VP1 protein of FMDV, which was identified by ELISA and Western blot, can be specifically recognized by polyclonal antibodies against FMDV. Indirect-ELISA antibody titers reached 1:64 twenty-one days after the third inoculation. In the challenge test, the protection against FMDV challenge in two groups was 80% and 40% respectively. The immunization test in guinea pigs indicated that the expression product of transgenic tomato plants had immunogenicity and could effectively induce the specific antibodies against FMDV.

  14. The Nrf2–Keap1 cellular defense pathway and heat shock protein 70 (Hsp70) response. Role in protection against oxidative stress in early neonatal unilateral ureteral obstruction (UUO)

    PubMed Central

    Rinaldi Tosi, Martin E.; Bocanegra, Victoria; Manucha, Walter; Gil Lorenzo, Andrea

    2010-01-01

    Perturbation of renal tubular antioxidants and overproduction of reactive oxygen species may amplify the proinflammatory state of renal obstruction, culminating in oxidative stress and tubular loss. Here, we analyzed the heat shock protein 70 (Hsp70) response and the function and signal transduction of NF-E2-related protein 2 (Nrf2) transcription factor on oxidative stress modulation in obstruction. Rats were subjected to unilateral ureteral obstruction or sham operation and kidneys harvested at 5, 7, 10, and 14 days after obstruction. Hsp70 expression and Nrf2 activity and its downstream target gene products were assessed. After 10 and 14 days of obstruction, enhanced lipid peroxidation through higher thiobarbituric acid reactive substances levels and increased oxidative stress resulted in reduced total antioxidant activity and enhanced nicotinamide adenine dinucleotide phosphate reduced (NADPH) oxidase activity were demonstrated. This was accompanied by decreased inducible Hsp70 expression and a progressive reduction of nuclear Nrf2 and its target gene products glutathione S-transferase A2 (GSTA2) and NADPH/quinone oxidoreductase 1 (NQO1), whereas the Nrf2 repressor Kelch-like ECH-associated protein-1 (Keap1) was upregulated. By contrast, on early obstruction for 7 days, lack of increased oxidative markers associated with higher inducible Hsp70 protein levels and a rapid nuclear accumulation of Nrf2, Keap1 downregulation, and mRNA induction of the identified Nrf2-dependent genes, NQO1 and GSTA2, were shown. For these results, we suggest that the magnitude of cytoprotection in early obstruction depends on the combined contribution of induced activation of Nrf2 upregulating its downstream gene products and Hsp70 response. Impaired ability to mount the biological response to the prevailing oxidative stress leading to renal injury was shown in prolonged obstruction. PMID:20734248

  15. Identification of a protective protein from stationary-phase exoproteome of Brucella abortus.

    PubMed

    Jain, Shikha; Kumar, Subodh; Dohre, Sudhir; Afley, Prachiti; Sengupta, Nabonita; Alam, Syed I

    2014-02-01

    Brucellosis is a worldwide zoonotic disease. No Brucella vaccine is available for use in humans, and existing animal vaccines have limitations. To search the putative vaccine candidates, we studied the exoproteome of Brucella abortus NCTC 10093 using 2-DE-MS approach. Twenty-six proteins were identified using MALDI-TOF/TOF tandem mass spectrometry. Outer membrane protein 25, d-galactose periplasmic-binding protein, oligopeptide ABC transporter protein and isopropylmalate synthase were found to be the most abundant proteins. Most proteins (6, 23%) were predicted to be involved in amino acid transport and metabolism followed by carbohydrate transport and metabolism (4, 15%). Outer membrane protein 25, Omp2b porin and one hypothetical protein were predicted as outer membrane proteins. In addition, Omp28, Omp31 and one ribosomal protein (L9) were also identified. The ribosomal protein L9 was produced as a recombinant protein and was studied in mouse model for vaccine potential. It was found to be immunogenic in terms of generating serum antibody response and release of IFN-γ from mice spleen cells. Recombinant L9-immunized mice were protected against challenge with virulent B. abortus strain 544, suggesting usefulness of ribosomal protein L9 as a good vaccine candidate against brucellosis.

  16. Transduced Tat-SAG fusion protein protects against oxidative stress and brain ischemic insult.

    PubMed

    Kim, Dae Won; Lee, Sun Hwa; Jeong, Min Seop; Sohn, Eun Jeong; Kim, Mi Jin; Jeong, Hoon Jae; An, Jae Jin; Jang, Sang Ho; Won, Moo Ho; Hwang, In Koo; Cho, Sung-Woo; Kang, Tae-Cheon; Lee, Kil Soo; Park, Jinseu; Yoo, Ki-Yeon; Eum, Won Sik; Choi, Soo Young

    2010-04-01

    Reactive oxygen species (ROS) have been implicated in the pathogenesis of ischemic brain injury. Sensitive to apoptosis gene (SAG) is a RING-finger protein that exhibits antioxidant activity against a variety of redox reagents. However, the protective effect of SAG in brain ischemic injury is unclear. Here, we investigated the protective effects of a Tat-SAG fusion protein against cell death and ischemic insult. When Tat-SAG fusion protein was added to the culture medium of astrocytes, it rapidly entered the cells and protected them against oxidative stress-induced cell death. Immunohistochemical analysis revealed that, when Tat-SAG fusion protein was intraperitoneally injected into gerbils, wild-type Tat-SAG prevented neuronal cell death in the CA1 region of the hippocampus in response to transient forebrain ischemia. In addition, wild-type Tat-SAG fusion protein decreased lipid peroxidation in the brain compared with mutant Tat-SAG- or vehicle-treated animals. Our results demonstrate that Tat-SAG fusion protein is a tool for the treatment of ischemic insult and it can be used in protein therapy for various disorders related to ROS, including stroke.

  17. Immunogenicity and protective efficacy of Clostridium difficile spore proteins.

    PubMed

    Ghose, Chandrabali; Eugenis, Ioannis; Edwards, Adrianne N; Sun, Xingmin; McBride, Shonna M; Ho, David D

    2016-02-01

    Clostridium difficile is a spore-forming, anaerobic, Gram-positive organism that is the leading cause of antibiotic-associated infectious diarrhea, commonly known as C. difficile infection (CDI). C. difficile spores play an important role in the pathogenesis of CDI. Spore proteins, especially those that are surface-bound may play an essential role in the germination, colonization and persistence of C. difficile in the human gut. In our current study, we report the identification of two surface-bound spore proteins, CdeC and CdeM that may be utilized as immunization candidates against C. difficile. These spore proteins are immunogenic in mice and are able to protect mice against challenge with C. difficile UK1, a clinically-relevant 027/B1/NAP1 strain. These spore proteins are also able to afford high levels of protection against challenge with C. difficile 630Δerm in golden Syrian hamsters. This unprecedented study shows the vaccination potential of C. difficile spore exosporium proteins.

  18. Prostate cancer and the unfolded protein response

    PubMed Central

    Arnoldussen, Yke Jildouw; Saatcioglu, Fahri

    2016-01-01

    The endoplasmic reticulum (ER) is an essential organelle that contributes to several key cellular functions, including lipogenesis, gluconeogenesis, calcium storage, and organelle biogenesis. The ER also serves as the major site for protein folding and trafficking, especially in specialized secretory cells. Accumulation of misfolded proteins and failure of ER adaptive capacity activates the unfolded protein response (UPR) which has been implicated in several chronic diseases, including cancer. A number of recent studies have implicated UPR in prostate cancer (PCa) and greatly expanded our understanding of this key stress signaling pathway and its regulation in PCa. Here we summarize these developments and discuss their potential therapeutic implications. PMID:27303918

  19. Hybrid cluster proteins and flavodiiron proteins afford protection to Desulfovibrio vulgaris upon macrophage infection.

    PubMed

    Figueiredo, Mafalda C O; Lobo, Susana A L; Sousa, Sara H; Pereira, Fábio P; Wall, Judy D; Nobre, Lígia S; Saraiva, Lígia M

    2013-06-01

    Desulfovibrio species are Gram-negative anaerobic sulfate-reducing bacteria that colonize the human gut. Recently, Desulfovibrio spp. have been implicated in gastrointestinal diseases and shown to stimulate the epithelial immune response, leading to increased production of inflammatory cytokines by macrophages. Activated macrophages are key cells of the immune system that impose nitrosative stress during phagocytosis. Hence, we have analyzed the in vitro and in vivo responses of Desulfovibrio vulgaris Hildenborough to nitric oxide (NO) and the role of the hybrid cluster proteins (HCP1 and HCP2) and rubredoxin oxygen oxidoreductases (ROO1 and ROO2) in NO protection. Among the four genes, hcp2 was the gene most highly induced by NO, and the hcp2 transposon mutant exhibited the lowest viability under conditions of NO stress. Studies in murine macrophages revealed that D. vulgaris survives incubation with these phagocytes and triggers NO production at levels similar to those stimulated by the cytokine gamma interferon (IFN-γ). Furthermore, D. vulgaris hcp and roo mutants exhibited reduced viability when incubated with macrophages, revealing that these gene products contribute to the survival of D. vulgaris during macrophage infection.

  20. Cellular unfolded protein response against viruses used in gene therapy

    PubMed Central

    Sen, Dwaipayan; Balakrishnan, Balaji; Jayandharan, Giridhara R.

    2014-01-01

    Viruses are excellent vehicles for gene therapy due to their natural ability to infect and deliver the cargo to specific tissues with high efficiency. Although such vectors are usually “gutted” and are replication defective, they are subjected to clearance by the host cells by immune recognition and destruction. Unfolded protein response (UPR) is a naturally evolved cyto-protective signaling pathway which is triggered due to endoplasmic reticulum (ER) stress caused by accumulation of unfolded/misfolded proteins in its lumen. The UPR signaling consists of three signaling pathways, namely PKR-like ER kinase, activating transcription factor 6, and inositol-requiring protein-1. Once activated, UPR triggers the production of ER molecular chaperones and stress response proteins to help reduce the protein load within the ER. This occurs by degradation of the misfolded proteins and ensues in the arrest of protein translation machinery. If the burden of protein load in ER is beyond its processing capacity, UPR can activate pro-apoptotic pathways or autophagy leading to cell death. Viruses are naturally evolved in hijacking the host cellular translation machinery to generate a large amount of proteins. This phenomenon disrupts ER homeostasis and leads to ER stress. Alternatively, in the case of gutted vectors used in gene therapy, the excess load of recombinant vectors administered and encountered by the cell can trigger UPR. Thus, in the context of gene therapy, UPR becomes a major roadblock that can potentially trigger inflammatory responses against the vectors and reduce the efficiency of gene transfer. PMID:24904562

  1. The specificity of protection against cationic antimicrobial peptides by lactoferrin binding protein B.

    PubMed

    Morgenthau, Ari; Partha, Sarathy K; Adamiak, Paul; Schryvers, Anthony B

    2014-10-01

    A variety of Gram-negative pathogens possess host-specific lactoferrin (Lf) receptors that mediate the acquisition of iron from host Lf. The integral membrane protein component of the receptor, lactoferrin binding protein A specifically binds host Lf and is required for acquisition of iron from Lf. In contrast, the role of the bi-lobed surface lipoprotein, lactoferrin binding protein B (LbpB), in Lf binding and iron acquisition is uncertain. A common feature of LbpBs from most species is the presence of clusters of negatively charged amino acids in the protein's C-terminal lobe. Recently it has been shown that the negatively charged regions from the Neisseria meningitidis LbpB are responsible for protecting against an 11 amino acid cationic antimicrobial peptide (CAP), lactoferricin (Lfcin), derived from human Lf. In this study we investigated whether the LbpB confers resistance to other CAPs since N. meningitidis is likely to encounter other CAPs from the host. LbpB provided protection against the cathelicidin derived peptide, cathelicidin related antimicrobial peptide (mCRAMP), but did not confer protection against Tritrp 1 or LL37 under our experimental conditions. When tested against a range of rationally designed synthetic peptides, LbpB was shown to protect against IDR-1002 and IDR-0018 but not against HH-2 or HHC10.

  2. Artemin protects cells and proteins against oxidative and salt stress.

    PubMed

    Takalloo, Zeinab; Sajedi, Reza H; Hosseinkhani, Saman; Moazzenzade, Taghi

    2017-02-01

    Artemin is an abundant thermostable protein in Artemia encysted embryos under environmental stresses. It is confirmed that high regulatory expression of artemin is relevant to stress resistance in this crustacean. Here, the protective role of artemin from Artemia urmiana has been investigated on survival of bacterial cells under salt and oxidative shocks. Also, for continuous monitoring of the effect of artemin in prevention of proteins aggregation/inactivation, co-expression of artemin and luciferase (as an intracellular reporter) in bacterial cells was performed. According to the results, residual activity of luciferase in artemin expressing E. coli cells exposing to different concentrations of H2O2 and NaCl was significantly higher than non-expressing cells. The luciferase activity was rapidly lost in control cells under salt treatments while in co-transformed cells, the activity was considerably retained at higher salt concentrations. Also, analysis from cell viability assays showed that artemin-expressing cells exhibited more resistance to both stress conditions. In the present study, we document for the first time that artemin can protect proteins and bacterial cells against oxidative and salt stress conditions. These results can declare the resistance property of this crustacean against harsh environmental conditions.

  3. Chlamydia trachomatis: Protective Adaptive Responses and Prospects for a Vaccine.

    PubMed

    Poston, Taylor B; Darville, Toni

    2016-04-01

    Chlamydia trachomatis is the most common cause of sexually transmitted bacterial infection globally. These infections translate to a significant public health burden, particularly women's healthcare costs due to serious disease sequelae such as pelvic inflammatory disease (PID), tubal factor infertility, chronic pelvic pain, and ectopic pregnancy. There is no evidence that natural immunity can provide complete, long-term protection necessary to prevent chronic pathology, making human vaccine development critical. Vaccine design will require careful consideration of protective versus pathological host-response mechanisms in concert with elucidation of optimal antigens and adjuvants. Evidence suggests that a Th1 response, facilitated by IFN-γ-producing CD4 T cells, will be instrumental in generating long-term, sterilizing immunity. Although the role of antibodies is not completely understood, they have exhibited a protective effect by enhancing chlamydial clearance. Future work will require investigation of broadly neutralizing antibodies and antibody-augmented cellular immunity to successfully design a vaccine that potently elicits both arms of the immune response. Sterilizing immunity is the ultimate goal. However, vaccine-induced partial immunity that prevents upper genital tract infection and inflammation would be cost-effective compared to current screening and treatment strategies. In this chapter, we examine evidence from animal and human studies demonstrating protective adaptive immune responses to Chlamydia and discuss future challenges and prospects for vaccine development.

  4. Characterization of culture supernatant proteins from Brucella abortus and its protection effects against murine brucellosis.

    PubMed

    Lee, Jin Ju; Lim, Jeong Ju; Kim, Dae Geun; Simborio, Hannah Leah; Kim, Dong Hyeok; Reyes, Alisha Wehdnesday Bernardo; Min, WonGi; Lee, Hu Jang; Kim, Dong Hee; Chang, Hong Hee; Kim, Suk

    2014-09-01

    In this study, we characterized the secreted proteins of Brucella abortus into the enriched media under the bacterial laboratory growth condition and investigated the pathogenic importance of culture supernatant (CS) proteins to B. abortus infection. CS proteins from stationary phase were concentrated and analyzed using 2D electrophoresis. In MALDI TOF/TOF analysis, more than 27 proteins including CuZn SOD, Dps, Tat, OMPs, Adh, LivF, Tuf, SucC, GroEL and DnaK were identified. Cytotoxic effects of CS proteins were found to increase in a dose-dependent manner in RAW 264.7 cells. Upon B. abortus challenge into phagocytes, however, CS proteins pre-treated cells exhibited lower bacterial uptake and intracellular replication compared to untreated cells. Immunization with CS proteins induced a strong humoral and cell mediated immune responses and exhibited significant higher degree of protection against virulence of B. abortus infection compared to mice immunized with Brucella broth protein (BBP). Taken together, these results indicate that B. abortus secreted a number of soluble immunogenic proteins under laboratory culture condition, which can promote antibody production resulted in enhancing host defense against to subsequently bacterial infection. Moreover, further analysis of CS proteins may help to understand the pathogenic mechanism of B. abortus infection and host-pathogen interaction.

  5. Vitamin D binding protein polymorphism protects against development of blastomycosis.

    PubMed

    Sainsbury, J P; Trajtman, A; Stalker, A T; Embil, J M; Keynan, Y

    2014-12-01

    Blastomycosis is an uncommon endemic fungal infection. It is presumed that in the endemic regions, the number of exposed individuals is significantly greater than those in whom clinical manifestations develop. We conducted a case-control study of individuals with clinical blastomycosis and controls with similar exposure but who did not develop disease. A genetic association was observed between the Gc-2 allele of vitamin D binding protein and reduced susceptibility to blastomycosis in a Canadian cohort. The Gc-2 allele can affect increased antimicrobial activity of macrophages. It may be possible to mimic this mechanism of protection by vitamin D supplementation.

  6. A thrombospondin-dependent pathway for a protective ER stress response

    PubMed Central

    Lynch, Jeffrey M.; Maillet, Marjorie; Vanhoutte, Davy; Schloemer, Aryn; Sargent, Michelle A.; Blair, N. Scott; Lynch, Kaari A.; Okada, Tetsuya; Aronow, Bruce J.; Osinska, Hanna; Prywes, Ron; Lorenz, John N.; Mori, Kazutoshi; Lawler, Jack; Robbins, Jeffrey; Molkentin, Jeffery D.

    2012-01-01

    SUMMARY Thrombospondin (Thbs) proteins are induced in sites of tissue damage or active remodeling. The endoplasmic reticulum (ER) stress response is also prominently induced with disease where it regulates protein production and resolution of misfolded proteins. Here we describe a novel function for Thbs’ as ER resident effectors of an adaptive ER stress response. Thbs4 cardiac-specific transgenic mice were protected from myocardial injury while Thbs4−/− mice were sensitized to cardiac maladaptation. Thbs induction produced a unique profile of adaptive ER stress response factors and expansion of the ER and downstream vesicles. The type-3 repeat domain in Thbs’ bind the ER luminal domain of activating transcription factor 6α (Atf6α) to promote its nuclear shuttling. Thbs4−/−mice failed to show activation of Atf6α and other ER stress response factors with injury, and Thbs4-mediated protection was lost when Atf6α was deleted. Hence, Thbs’ can function inside the cell during disease/remodeling to augment ER function and protect through a mechanism involving regulation of Atf6α. PMID:22682248

  7. 75 FR 66385 - Protecting Tenants at Foreclosure Act: Guidance on Notification Responsibilities Under the Act...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-10-28

    ... URBAN DEVELOPMENT Protecting Tenants at Foreclosure Act: Guidance on Notification Responsibilities Under... the notice, entitled ``Protecting Tenants at Foreclosure: Notice of Responsibilities Placed on... existing tenants under the Protecting Tenants at Foreclosure Act of 2009 (PTFA). FHA's existing...

  8. Functional analysis of insect molting fluid proteins on the protection and regulation of ecdysis.

    PubMed

    Zhang, Jie; Lu, Anrui; Kong, Lulu; Zhang, Qiaoli; Ling, Erjun

    2014-12-26

    Molting fluid accumulates between the old and new cuticles during periodical ecdysis in Ecdysozoa. Natural defects in insect ecdysis are frequently associated with melanization (an immunity response) occurring primarily in molting fluids, suggesting that molting fluid may impact immunity as well as affect ecdysis. To address this hypothesis, proteomic analysis of molting fluids from Bombyx mori during three different types of ecdysis was performed. Many proteins were newly identified, including immunity-related proteins, in each molting fluid. Molting fluids inhibited the growth of bacteria in vitro. The entomopathogenic fungi Beauveria bassiana, which can escape immune responses in feeding larvae, is quickly recognized by larvae during ecdysis, followed by melanization in molting fluid and old cuticle. Fungal conidia germination was delayed, and no hyphae were detected in the hemocoels of pharate instar insects. Molting fluids protect the delicate pharate instar insects with extremely thin cuticles against microorganisms. To explore the function of molting fluids in ecdysis regulation, based on protein similarity, 32 genes were selected for analysis in ecdysis regulation through RNAi in Tribolium castaneum, a model commonly used to study integument development because RNAi is difficult to achieve in B. mori. We identified 24 molting proteins that affected ecdysis after knockdown, with different physiological functions, including old cuticle protein recycling, molting fluid pressure balance, detoxification, and signal detection and transfer of molting fluids. We report that insects secrete molting fluid for protection and regulation of ecdysis, which indicates a way to develop new pesticides through interrupting insect ecdysis in the future.

  9. Conformational Nature of the Borrelia burgdorferi Decorin Binding Protein A Epitopes That Elicit Protective Antibodies

    PubMed Central

    Ulbrandt, Nancy D.; Cassatt, David R.; Patel, Nita K.; Roberts, William C.; Bachy, Christine M.; Fazenbaker, Christine A.; Hanson, Mark S.

    2001-01-01

    Decorin binding protein A (DbpA) has been shown by several laboratories to be a protective antigen for the prevention of experimental Borrelia burgdorferi infection in the mouse model of Lyme borreliosis. However, different recombinant forms of the antigen having either lipidated amino termini, approximating the natural secretion and posttranslational processing, or nonprocessed cytosolic forms have elicited disparate levels of protection in the mouse model. We have now used the unique functional properties of this molecule to investigate the structural requirements needed to elicit a protective immune response. Genetic and physicochemical alterations to DbpA showed that the ability to bind to the ligand decorin is indicative of a potent immunogen but is not conclusive. By mutating the two carboxy-terminal nonconserved cysteines of DbpA from B. burgdorferi strain N40, we have determined that the stability afforded by the putative disulfide bond is essential for the generation of protective antibodies. This mutated protein was more sensitive to thermal denaturation and proteolysis, suggesting that it is in a less ordered state. Immunization with DbpA that was thermally denatured and functionally inactivated stimulated an immune response that was not protective and lacked bactericidal antibodies. Antibodies against conformationally altered forms of DbpA also failed to kill heterologous B. garinii and B. afzelii strains. Additionally, nonsecreted recombinant forms of DbpAN40 were found to be inferior to secreted lipoprotein DbpAN40 in terms of functional activity and antigenic potency. These data suggest that elicitation of a bactericidal and protective immune response to DbpA requires a properly folded conformation for the production of functional antibodies. PMID:11447153

  10. Aggresomes: A Cellular Response to Misfolded Proteins

    PubMed Central

    Johnston, Jennifer A.; Ward, Cristina L.; Kopito, Ron R.

    1998-01-01

    Intracellular deposition of misfolded protein aggregates into ubiquitin-rich cytoplasmic inclusions is linked to the pathogenesis of many diseases. Why these aggregates form despite the existence of cellular machinery to recognize and degrade misfolded protein and how they are delivered to cytoplasmic inclusions are not known. We have investigated the intracellular fate of cystic fibrosis transmembrane conductance regulator (CFTR), an inefficiently folded integral membrane protein which is degraded by the cytoplasmic ubiquitin-proteasome pathway. Overexpression or inhibition of proteasome activity in transfected human embryonic kidney or Chinese hamster ovary cells led to the accumulation of stable, high molecular weight, detergent-insoluble, multiubiquitinated forms of CFTR. Using immunofluorescence and transmission electron microscopy with immunogold labeling, we demonstrate that undegraded CFTR molecules accumulate at a distinct pericentriolar structure which we have termed the aggresome. Aggresome formation is accompanied by redistribution of the intermediate filament protein vimentin to form a cage surrounding a pericentriolar core of aggregated, ubiquitinated protein. Disruption of microtubules blocks the formation of aggresomes. Similarly, inhibition of proteasome function also prevented the degradation of unassembled presenilin-1 molecules leading to their aggregation and deposition in aggresomes. These data lead us to propose that aggresome formation is a general response of cells which occurs when the capacity of the proteasome is exceeded by the production of aggregation-prone misfolded proteins. PMID:9864362

  11. C-reactive protein mediates protection from lipopolysaccharide through interactions with Fc gamma R.

    PubMed

    Mold, Carolyn; Rodriguez, Wilfredo; Rodic-Polic, Bojana; Du Clos, Terry W

    2002-12-15

    C-reactive protein (CRP) is a component of the acute phase response to infection, inflammation, and trauma. A major activity of acute phase proteins is to limit the inflammatory response. It has been demonstrated that CRP protects mice from lethal doses of LPS. In the mouse, CRP binds to the regulatory receptor, FcgammaRIIb, and to the gamma-chain-associated receptor, FcgammaRI. The goal ofthis study was to determine whether FcgammaRs are necessary for the protective effect of CRP. The ability of CRP to protect mice from a lethal dose of LPS was confirmed using injections of 500 and 250 micro g of CRP at 0 and 12 h. CRP treatment of FcgammaRIIb-deficient mice increased mortality after LPS challenge and increased serum levels of TNF and IL-12 in response to LPS. CRP did not protect FcR gamma-chain-deficient mice from LPS-induced mortality. Treatment of normal mice, but not gamma-chain-deficient mice, with CRP increased IL-10 levels following LPS injection. In vitro, in the presence of LPS, CRP enhanced IL-10 synthesis and inhibited IL-12 synthesis by bone marrow macrophages from normal, but not gamma-chain-deficient mice. The protective effect of CRP appears to be mediated by binding to FcgammaRI and FcgammaRII resulting in enhanced secretion of the anti-inflammatory cytokine IL-10 and the down-regulation of IL-12. These results suggest that CRP can alter the cytokine profile of mouse macrophages by acting through FcgammaR leading to a down-regulation of the inflammatory response.

  12. Drinking a hot blood meal elicits a protective heat shock response in mosquitoes.

    PubMed

    Benoit, Joshua B; Lopez-Martinez, Giancarlo; Patrick, Kevin R; Phillips, Zachary P; Krause, Tyler B; Denlinger, David L

    2011-05-10

    The mosquito's body temperature increases dramatically when it takes a blood meal from a warm-blooded, vertebrate host. By using the yellow fever mosquito, Aedes aegypti, we demonstrate that this boost in temperature following a blood meal prompts the synthesis of heat shock protein 70 (Hsp70). This response, elicited by the temperature of the blood meal, is most robust in the mosquito's midgut. When RNA interference is used to suppress expression of hsp70, protein digestion of the blood meal is impaired, leading to production of fewer eggs. We propose that Hsp70 protects the mosquito midgut from the temperature stress incurred by drinking a hot blood meal. Similar increases in hsp70 were documented immediately after blood feeding in two other mosquitoes (Culex pipiens and Anopheles gambiae) and the bed bug, Cimex lectularius, suggesting that this is a common protective response in blood-feeding arthropods.

  13. Trachoma: Protective and Pathogenic Ocular Immune Responses to Chlamydia trachomatis

    PubMed Central

    Hu, Victor H.; Holland, Martin J.; Burton, Matthew J.

    2013-01-01

    Trachoma, caused by Chlamydia trachomatis (Ct), is the leading infectious blinding disease worldwide. Chronic conjunctival inflammation develops in childhood and leads to eyelid scarring and blindness in adulthood. The immune response to Ct provides only partial protection against re-infection, which can be frequent. Moreover, the immune response is central to the development of scarring pathology, leading to loss of vision. Here we review the current literature on both protective and pathological immune responses in trachoma. The resolution of Ct infection in animal models is IFNγ-dependent, involving Th1 cells, but whether this is the case in human ocular infection still needs to be confirmed. An increasing number of studies indicate that innate immune responses arising from the epithelium and other innate immune cells, along with changes in matrix metalloproteinase activity, are important in the development of tissue damage and scarring. Current trachoma control measures, which are centred on repeated mass antibiotic treatment of populations, are logistically challenging and have the potential to drive antimicrobial resistance. A trachoma vaccine would offer significant advantages. However, limited understanding of the mechanisms of both protective immunity and immunopathology to Ct remain barriers to vaccine development. PMID:23457650

  14. Arsenal of elevated defense proteins fails to protect tomato against Verticillium dahliae.

    PubMed

    Robb, Jane; Shittu, Hakeem; Soman, Kizhake V; Kurosky, Alexander; Nazar, Ross N

    2012-08-01

    Although the hypersensitive reaction in foliar plant diseases has been extensively described, little is clear regarding plant defense strategies in vascular wilt diseases affecting numerous economically important crops and trees. We have examined global genetic responses to Verticillium wilt in tomato (Lycopersicon esculentum Mill.) plants differing in Ve1 resistance alleles. Unexpectedly, mRNA analyses in the susceptible plant (Ve1-) based on the microarrays revealed a very heroic but unsuccessful systemic response involving many known plant defense genes. In contrast, the response is surprisingly low in plants expressing the Ve1+ R-gene and successfully resisting the pathogen. Similarly, whole-cell protein analyses, based on 2D gel electrophoresis and mass spectrometry, demonstrate large systemic increases in a variety of known plant defense proteins in the stems of susceptible plants but only modest changes in the resistant plant. Taken together, the results indicate that the large systemic increases in plant defense proteins do not protect the susceptible plant. Indeed, since a number of the highly elevated proteins are known to participate in the plant hypersensitive response as well as natural senescence, the results suggest that some or all of the disease symptoms, including ultimate plant death, actually may be the result of this exaggerated plant response.

  15. Melanoma and the Unfolded Protein Response

    PubMed Central

    Sykes, Erin K.; Mactier, Swetlana; Christopherson, Richard I.

    2016-01-01

    The UPR (unfolded protein response) has been identified as a key factor in the progression and metastasis of cancers, notably melanoma. Several mediators of the UPR are upregulated in cancers, e.g., high levels of GRP78 (glucose-regulator protein 78 kDa) correlate with progression and poor outcome in melanoma patients. The proliferative burden of cancer induces stress and activates several cellular stress responses. The UPR is a tightly orchestrated stress response that is activated upon the accumulation of unfolded proteins within the ER (endoplasmic reticulum). The UPR is designed to mediate two conflicting outcomtes, recovery and apoptosis. As a result, the UPR initiates a widespread signaling cascade to return the cell to homeostasis and failing to achieve cellular recovery, initiates UPR-induced apoptosis. There is evidence that ER stress and subsequently the UPR promote tumourigenesis and metastasis. The complete role of the UPR has yet to be defined. Understanding how the UPR allows for adaption to stress and thereby assists in cancer progression is important in defining an archetype of melanoma pathology. In addition, elucidation of the mechanisms of the UPR may lead to development of effective treatments of metastatic melanoma. PMID:26927180

  16. The responsibility to protect: a new notion of state sovereignty.

    PubMed

    Ashford, Mary-Wynne

    2003-01-01

    The recent policies of the United States Administration threaten the entire structure of international law. Widespread concern in the US and elsewhere has been expressed in a different cluster of principles, in particular in the report of the Canadian-based Commission on Intervention and State Sovereignty. Its report states that the primary responsibility of a state is to protect its citizens; such protection includes prevention, reaction and rebuilding. The implications of this for military intervention are discussed; this must be a last resort after all non-military options have failed in response to the actual or probable large-scale loss of life and the objective to prevent further suffering. The action must be authorized by the United Nations Security Council or, if this fails to respond, the General Assembly. A further commission should examine how the international community should respond to states that refuse to comply with international law regarding weapons of mass destruction.

  17. Protein Degradation and the Stress Response

    PubMed Central

    Flick, Karin; Kaiser, Peter

    2012-01-01

    Environmental stresses are manifold and so are the responses they elicit. This is particularly true for higher eukaryotes where various tissues and cell types are differentially affected by the insult. Type and scope of the stress response can therefore differ greatly among cell types. Given the importance of the Ubiquitin Proteasome System (UPS) for most cellular processes, it comes as no surprise that the UPR plays a pivotal role in counteracting the effects of stressors. Here we outline contributions of the UPS to stress sensing, signaling, and response pathways. We make no claim to comprehensiveness but choose selected examples to illustrate concepts and mechanisms by which protein modification with ubiquitin and proteasomal degradation of key regulators ensures cellular integrity during stress situations. PMID:22414377

  18. Vaccination with Recombinant Microneme Proteins Confers Protection against Experimental Toxoplasmosis in Mice.

    PubMed

    Pinzan, Camila Figueiredo; Sardinha-Silva, Aline; Almeida, Fausto; Lai, Livia; Lopes, Carla Duque; Lourenço, Elaine Vicente; Panunto-Castelo, Ademilson; Matthews, Stephen; Roque-Barreira, Maria Cristina

    2015-01-01

    Toxoplasmosis, a zoonotic disease caused by Toxoplasma gondii, is an important public health problem and veterinary concern. Although there is no vaccine for human toxoplasmosis, many attempts have been made to develop one. Promising vaccine candidates utilize proteins, or their genes, from microneme organelle of T. gondii that are involved in the initial stages of host cell invasion by the parasite. In the present study, we used different recombinant microneme proteins (TgMIC1, TgMIC4, or TgMIC6) or combinations of these proteins (TgMIC1-4 and TgMIC1-4-6) to evaluate the immune response and protection against experimental toxoplasmosis in C57BL/6 mice. Vaccination with recombinant TgMIC1, TgMIC4, or TgMIC6 alone conferred partial protection, as demonstrated by reduced brain cyst burden and mortality rates after challenge. Immunization with TgMIC1-4 or TgMIC1-4-6 vaccines provided the most effective protection, since 70% and 80% of mice, respectively, survived to the acute phase of infection. In addition, these vaccinated mice, in comparison to non-vaccinated ones, showed reduced parasite burden by 59% and 68%, respectively. The protective effect was related to the cellular and humoral immune responses induced by vaccination and included the release of Th1 cytokines IFN-γ and IL-12, antigen-stimulated spleen cell proliferation, and production of antigen-specific serum antibodies. Our results demonstrate that microneme proteins are potential vaccines against T. gondii, since their inoculation prevents or decreases the deleterious effects of the infection.

  19. Redox-responsive self-healing for corrosion protection.

    PubMed

    Vimalanandan, Ashokanand; Lv, Li-Ping; Tran, The Hai; Landfester, Katharina; Crespy, Daniel; Rohwerder, Michael

    2013-12-23

    Raspberry-shaped redox-responsive capsules for storing corrosion inhibitors are introduced, targeted to solve the drawbacks of conducting-polymer-based coating systems for corrosion protection. These capsules synthesized via the miniemulsion technique have a remarkable release property upon reduction (onset of corrosion) and cease release upon reoxidation (passivation of the defect). The self-healing capability is demonstrated by application of these capsules as part of a composite coating on zinc.

  20. Operationalizing the Impossible: The Responsibility to Protect Movement

    DTIC Science & Technology

    2015-05-23

    complex phenomenon of genocide or mass atrocity has carried on in human history to the present. Because it possesses both complexity and a rich yet dark ...Responsibility to Protect Movement, by MAJ Todd L. Poindexter, 57 pages. Genocide is a complex phenomenon that has a long and dark history. The...and a rich yet dark history, it has survived many linear political solutions that seek its demise. The durability of this violent social interaction

  1. Feeding truncated heat shock protein 70s protect Artemia franciscana against virulent Vibrio campbellii challenge.

    PubMed

    Baruah, Kartik; Norouzitallab, Parisa; Shihao, Li; Sorgeloos, Patrick; Bossier, Peter

    2013-01-01

    The 70 kDa heat shock proteins (Hsp70s) are highly conserved in evolution, leading to striking similarities in structure and composition between eukaryotic Hsp70s and their homologs in prokaryotes. The eukaryotic Hsp70 like the DnaK (Escherichia coli equivalent Hsp70) protein, consist of three functionally distinct domains: an N-terminal 44-kDa ATPase portion, an 18-kDa peptide-binding domain and a C-terminal 10-kDa fragment. Previously, the amino acid sequence of eukaryotic (the brine shrimp Artemia franciscana) Hsp70 and DnaK proteins were shown to share a high degree of homology, particularly in the peptide-binding domain (59.6%, the putative innate immunity-activating portion) compared to the N-terminal ATPase (48.8%) and the C-terminal lid domains (19.4%). Next to this remarkable conservation, these proteins have been shown to generate protective immunity in Artemia against pathogenic Vibrio campbellii. This study, aimed to unravel the Vibrio-protective domain of Hsp70s in vivo, demonstrated that gnotobiotically cultured Artemia fed with recombinant C-terminal fragment (containing the conserved peptide binding domain) of Artemia Hsp70 or DnaK protein were well protected against subsequent Vibrio challenge. In addition, the prophenoloxidase (proPO) system, at both mRNA and protein activity levels, was also markedly induced by these truncated proteins, suggesting epitope(s) responsible for priming the proPO system and presumably other immune-related genes, consequently boosting Artemia survival upon challenge with V. campbellii, might be located within this conserved region of the peptide binding domain.

  2. Thermal response of integral multicomponent composite thermal protection systems

    NASA Technical Reports Server (NTRS)

    Stewart, D. A.; Leiser, D. B.; Smith, M.; Kolodziej, P.

    1985-01-01

    Integral-multicomponent thermal-protection materials are discussed in terms of their thermal response to an arc-jet airstream. In-depth temperature measurements are compared with predictions from a one-dimensional, finite-difference code using calculated thermal conductivity values derived from an engineering model. The effect of composition, as well as the optical properties of the bonding material between components, on thermal response is discussed. The performance of these integral-multicomponent composite materials is compared with baseline Space Shuttle insulation.

  3. Protein response to ligation reactions in myoglobin

    SciTech Connect

    Causgrove, T.P.; Dyer, R.B.

    1992-01-01

    The protein response to the photodissociation, escape and subsequent rebinding of carbon monoxide in myoglobin is studied using time-resolved infrared (TRIR) spectroscopy. All phases of these reactions areinvestigated, from ultrafast phenomena (picoseconds) to relatively slow processes (milliseconds). Conformational changes in myoglobin (Mb) are detected by time-resolved absorption changes in the amide I band. On the hundreds of nanoseconds to milliseconds timescale, a real-time'' apparatus is used. This apparatus is based on a tunable diode laser operating in the region of 1650 cm[sup [minus]1] . The time course ofchanges in the amide I band are shown to follow the recombination of CO with photolyzed Mb. On the basis of the rise times of the amide I and FE-CO signals, it is concluded that protein motion is complete within 100 n. A time-resolved difference spectrum in the amide I region is generated from single wavelength transients taken throughout the amide I envelope. A static difference spectrum is also generated by subtracting FTIR spectra of carbonmonoxy and deoxy myoglobin. The two difference spectra are compared and are interpreted in terms of the three-dimensional structures of deoxy and carbonmonoxy Mb. Preliminary picosecond TRIR data are also given for the ultrafast response of the protein immediately following photodissociation of CO.

  4. Protein response to ligation reactions in myoglobin

    SciTech Connect

    Causgrove, T.P.; Dyer, R.B.

    1992-12-31

    The protein response to the photodissociation, escape and subsequent rebinding of carbon monoxide in myoglobin is studied using time-resolved infrared (TRIR) spectroscopy. All phases of these reactions areinvestigated, from ultrafast phenomena (picoseconds) to relatively slow processes (milliseconds). Conformational changes in myoglobin (Mb) are detected by time-resolved absorption changes in the amide I band. On the hundreds of nanoseconds to milliseconds timescale, a ``real-time`` apparatus is used. This apparatus is based on a tunable diode laser operating in the region of 1650 cm{sup {minus}1} . The time course ofchanges in the amide I band are shown to follow the recombination of CO with photolyzed Mb. On the basis of the rise times of the amide I and FE-CO signals, it is concluded that protein motion is complete within 100 n. A time-resolved difference spectrum in the amide I region is generated from single wavelength transients taken throughout the amide I envelope. A static difference spectrum is also generated by subtracting FTIR spectra of carbonmonoxy and deoxy myoglobin. The two difference spectra are compared and are interpreted in terms of the three-dimensional structures of deoxy and carbonmonoxy Mb. Preliminary picosecond TRIR data are also given for the ultrafast response of the protein immediately following photodissociation of CO.

  5. Protein response to ligation reactions in myoglobin

    NASA Astrophysics Data System (ADS)

    Causgrove, Timothy P.; Dyer, R. B.

    1993-05-01

    The protein response to the photodissociation, escape and subsequent rebinding of carbon monoxide in myoglobin is studied using time-resolved infrared (TRIR) spectroscopy. All phases of these reactions are investigated, from ultrafast phenomena (picoseconds) to relatively slow processes (milliseconds). Conformational changes in myoglobin (Mb) are detected by time-resolved infrared absorption changes in the amide I band. On the hundreds of nanoseconds to milliseconds timescale, a 'real-time' apparatus is used. This apparatus is based on a tunable diode laser operating in the region of 1650 cm-1. The time course of changes in the amide I band are shown to follow the recombination of CO with photolyzed Mb. On the basis of the rise times of the amide I and Fe-CO signals, it is concluded that protein motion is complete within 100 ns. A time-resolved difference spectrum in the amide I region is generated from single wavelength transients taken throughout the amide I envelope. A static difference spectrum is also generated by subtracting FTIR spectra of carbonmonoxy and deoxy myoglobin. The two difference spectra are compared and are interpreted in terms of the three-dimensional structures of deoxy and carbonmonoxy Mb. Preliminary picosecond TRIR data are also given for the ultrafast response of the protein immediately following photodissociation of CO.

  6. Determinants of immunogenic response to protein therapeutics.

    PubMed

    Singh, Satish K; Cousens, Leslie P; Alvarez, David; Mahajan, Pramod B

    2012-09-01

    Protein therapeutics occupy a very significant position in the biopharmaceutical market. In addition to the preclinical, clinical and post marketing challenges common to other drugs, unwanted immunogenicity is known to affect efficacy and/or safety of most biotherapeutics. A standard set of immunogenicity risk factors are routinely used to inform monitoring strategies in clinical studies. A number of in-silico, in vivo and in vitro approaches have also been employed to predict immunogenicity of biotherapeutics, but with limited success. Emerging data also indicates the role of immune tolerance mechanisms and impact of several product-related factors on modulating host immune responses. Thus, a comprehensive discussion of the impact of innate and adaptive mechanisms and molecules involved in induction of host immune responses on immunogenicity of protein therapeutics is needed. A detailed understanding of these issues is essential in order to fully exploit the therapeutic potential of this class of drugs. This Roundtable Session was designed to provide a common platform for discussing basic immunobiological and pharmacological issues related to the role of biotherapeutic-associated risk factors, as well as host immune system in immunogenicity against protein therapeutics. The session included overview presentations from three speakers, followed by a panel discussion with audience participation.

  7. DDRprot: a database of DNA damage response-related proteins

    PubMed Central

    Andrés-León, Eduardo; Cases, Ildefonso; Arcas, Aida; Rojas, Ana M.

    2016-01-01

    The DNA Damage Response (DDR) signalling network is an essential system that protects the genome’s integrity. The DDRprot database presented here is a resource that integrates manually curated information on the human DDR network and its sub-pathways. For each particular DDR protein, we present detailed information about its function. If involved in post-translational modifications (PTMs) with each other, we depict the position of the modified residue/s in the three-dimensional structures, when resolved structures are available for the proteins. All this information is linked to the original publication from where it was obtained. Phylogenetic information is also shown, including time of emergence and conservation across 47 selected species, family trees and sequence alignments of homologues. The DDRprot database can be queried by different criteria: pathways, species, evolutionary age or involvement in (PTM). Sequence searches using hidden Markov models can be also used. Database URL: http://ddr.cbbio.es. PMID:27577567

  8. Lack of protective efficacy in buffaloes vaccinated with Fasciola gigantica leucine aminopeptidase and peroxiredoxin recombinant proteins.

    PubMed

    Raina, O K; Nagar, Gaurav; Varghese, Anju; Prajitha, G; Alex, Asha; Maharana, B R; Joshi, P

    2011-06-01

    Gene coding for leucine aminopeptidase (LAP), a metalloprotease, was identified in the tropical liver fluke, Fasciola gigantica; that on sequence analysis showed a close homology (98.6%) with leucine aminopeptidase of the temperate liver fluke, Fasciola hepatica. The recombinant leucine aminopeptidase protein was expressed in Escherichia coli. F. gigantica peroxiredoxin, a hydrogen peroxide scavenger and an immunomodulating protein, was also cloned and expressed in E. coli. A vaccination trial in buffaloes was conducted with these two recombinant proteins, with 150 and 300 μg of leucine aminopeptidase and a cocktail of 150 μg each of recombinant leucine aminopeptidase and peroxiredoxin in three groups, respectively. Both Th1- and Th2-associated humoral immune responses were elicited to immunization with these antigens. A challenge study with 400 metacercariae did not show a significant protection in terms of reduction in the worm burden (8.4%) or anti-fecundity/embryonation effect in the immunized groups, as to the non-immunized control animals. Our observations in this buffalo vaccination trial are contrary to the earlier promise shown by leucine aminopeptidase of F. hepatica as a leading candidate vaccine molecule. Identification of leucine aminopeptidase gene and evaluation of the protein for its protective efficacy in buffaloes is the first scientific report on this protein in F. gigantica.

  9. Interphotoreceptor Retinoid-Binding Protein Protects Retinoids from Photodegradation

    PubMed Central

    Gonzalez-Fernandez, Federico; Betts-Obregon, Brandi; Yust, Brian; Mimun, Joshua; Sung, Dongjin; Sardar, Dhiraj; Tsin, Andrew T.

    2015-01-01

    Retinol degrades rapidly in light into a variety of photoproducts. It is remarkable that visual cycle retinoids can evade photodegradation as they are exchanged between the photoreceptors, retinal pigment epithelium and Müller glia. Within the interphotoreceptor matrix, all-trans retinol, 11-cis retinol and retinal are bound by interphotoreceptor retinoid-binding protein (IRBP). Apart from its role in retinoid trafficking and targeting, could IRBP have a photoprotective function? HPLC was used to evaluate the ability of IRBP to protect all-trans and 11-cis retinols from photodegradation when exposed to incandescent light (0 to 8,842 μW/cm2); time periods of 0 – 60 min, and bIRBP: retinol molar ratios of 1:1 to 1:5. bIRBP afforded a significant prevention of both all-trans and 11-cis retinol to rapid photodegradation. The effect was significant over the entire light intensity range tested, and extended to the bIRBP: retinol ratio 1:5. In view of the continual exposure of the retina to light, and the high oxidative stress in the outer retina, our results suggest IRBP may have an important protective role in the visual cycle by reducing photodegradation of all-trans and 11-cis retinols. This role of IRBP is particularly relevant in the high flux conditions of the cone visual cycle. PMID:25565073

  10. Analysis of Unfolded Protein Response in Arabidopsis

    PubMed Central

    Chen, Yani; Brandizzi, Federica

    2014-01-01

    The unfolded protein response (UPR) is fundamental for development and adaption in eukaryotic cells. Arabidopsis has become one of the best model systems to uncover conserved mechanisms of the UPR in multicellular eukaryotes as well as organism-specific regulation of the UPR in plants. Monitoring the UPR in planta is an elemental approach to identifying regulatory components and to revealing molecular mechanisms of the plant UPR. In this chapter, we provide protocols for the induction and analyses of plant UPR at a molecular level in Arabidopsis. Three kinds of ER stress treatment methods and quantitation of the plant UPR activation are described here. PMID:23913037

  11. Group 3 LEA Protein, ZmLEA3, Is Involved in Protection from Low Temperature Stress

    PubMed Central

    Liu, Yang; Liang, Jianan; Sun, Liping; Yang, Xinghong; Li, Dequan

    2016-01-01

    Late embryogenesis abundant (LEA) proteins are a family of small highly hydrophilic proteins that accumulate at the onset of seed desiccation and in response to adverse conditions such as drought, salinity, low temperature, or water deficit. In previous studies, we demonstrated that ZmLEA3 could enhance the transgenic tobacco tolerance to osmotic and oxidative stresses. Here, we demonstrated that the transcription of ZmLEA3 in the maize stems could be significantly induced by low temperature and osmotic stresses and by treatment with abscisic acid (ABA) and H2O2. Further study indicated that ZmLEA3 is a single copy gene in the maize genome. The ZmLEA3 protein could protect lactate dehydrogenase (LDH) activity at low temperatures. The overexpression of ZmLEA3 conferred tolerance to low-temperature stress to transgenic tobacco, yeast (GS115) and E. coli (BL21). PMID:27471509

  12. Immunofluorescence Imaging of DNA Damage Response Proteins

    PubMed Central

    Bennett, Brian T.; Bewersdorf, Jörg; Knight, Kendall L.

    2013-01-01

    Immunofluorescence imaging has provided captivating visual evidence for numerous cellular events, from vesicular trafficking, organelle maturation and cell division to nuclear processes including the appearance of various proteins and chromatin components in distinct foci in response to DNA damaging agents. With the advent of new super-resolution microscope technologies such as 4Pi microscopy, standard immunofluorescence protocols deserve some reevaluation in order to take full advantage of these new technological accomplishments. Here we describe several methodological considerations that will help overcome some of the limitations that may result from the use of currently applied procedures, with particular attention paid to the analysis of possible colocalization of fluorescent signals. We conclude with an example of how application of optimized methods led to a breakthrough in super-resolution imaging of nuclear events occurring in response to DNA damage. PMID:19245833

  13. The Dose Window for Radiation-Induced Protective Adaptive Responses

    PubMed Central

    Mitchel, Ronald E. J.

    2009-01-01

    Adaptive responses to low doses of low LET radiation occur in all organisms thus far examined, from single cell lower eukaryotes to mammals. These responses reduce the deleterious consequences of DNA damaging events, including radiation-induced or spontaneous cancer and non-cancer diseases in mice. The adaptive response in mammalian cells and mammals operates within a certain window that can be defined by upper and lower dose thresholds, typically between about 1 and 100 mGy for a single low dose rate exposure. However, these thresholds for protection are not a fixed function of total dose, but also vary with dose rate, additional radiation or non-radiation stressors, tissue type and p53 functional status. Exposures above the upper threshold are generally detrimental, while exposures below the lower threshold may or may not increase either cancer or non-cancer disease risk. PMID:20585438

  14. Protein disulphide isomerase protects against protein aggregation and is S-nitrosylated in amyotrophic lateral sclerosis.

    PubMed

    Walker, Adam K; Farg, Manal A; Bye, Chris R; McLean, Catriona A; Horne, Malcolm K; Atkin, Julie D

    2010-01-01

    Amyotrophic lateral sclerosis is a rapidly progressing fatal neurodegenerative disease characterized by the presence of protein inclusions within affected motor neurons. Endoplasmic reticulum stress leading to apoptosis was recently recognized to be an important process in the pathogenesis of sporadic human amyotrophic lateral sclerosis as well as in transgenic models of mutant superoxide dismutase 1-linked familial amyotrophic lateral sclerosis. Endoplasmic reticulum stress occurs early in disease, indicating a critical role in pathogenesis, and involves upregulation of an important endoplasmic reticulum chaperone, protein disulphide isomerase. We aimed to investigate the involvement of protein disulphide isomerase in endoplasmic reticulum stress induction, protein aggregation, inclusion formation and toxicity in amyotrophic lateral sclerosis. Motor neuron-like NSC-34 cell lines were transfected with superoxide dismutase 1 and protein disulphide isomerase encoding vectors and small interfering RNA, and examined by immunocytochemistry and immunoblotting. Expression of mutant superoxide dismutase 1 induced endoplasmic reticulum stress, predominantly in cells bearing mutant superoxide dismutase 1 inclusions but also in a proportion of cells expressing mutant superoxide dismutase 1 without visible inclusions. Over-expression of protein disulphide isomerase decreased mutant superoxide dismutase 1 aggregation, inclusion formation, endoplasmic reticulum stress induction and toxicity, whereas small interfering RNA targeting protein disulphide isomerase increased mutant superoxide dismutase 1 inclusion formation, indicating a protective role for protein disulphide isomerase against superoxide dismutase 1 misfolding. Aberrant modification of protein disulphide isomerase by S-nitrosylation of active site cysteine residues has previously been shown as an important process in neurodegeneration in Parkinson's and Alzheimer's disease brain tissue, but has not been described in

  15. Cellular prion protein and NMDA receptor modulation: protecting against excitotoxicity

    PubMed Central

    Black, Stefanie A. G.; Stys, Peter K.; Zamponi, Gerald W.; Tsutsui, Shigeki

    2014-01-01

    Although it is well established that misfolding of the cellular prion protein (PrPC) into the β-sheet-rich, aggregated scrapie conformation (PrPSc) causes a variety of transmissible spongiform encephalopathies (TSEs), the physiological roles of PrPC are still incompletely understood. There is accumulating evidence describing the roles of PrPC in neurodegeneration and neuroinflammation. Recently, we identified a functional regulation of NMDA receptors by PrPC that involves formation of a physical protein complex between these proteins. Excessive NMDA receptor activity during conditions such as ischemia mediates enhanced Ca2+ entry into cells and contributes to excitotoxic neuronal death. In addition, NMDA receptors and/or PrPC play critical roles in neuroinflammation and glial cell toxicity. Inhibition of NMDA receptor activity protects against PrPSc-induced neuronal death. Moreover, in mice lacking PrPC, infarct size is increased after focal cerebral ischemia, and absence of PrPC increases susceptibility of neurons to NMDA receptor-dependent death. Recently, PrPC was found to be a receptor for oligomeric beta-amyloid (Aβ) peptides, suggesting a role for PrPC in Alzheimer's disease (AD). Our recent findings suggest that Aβ peptides enhance NMDA receptor current by perturbing the normal copper- and PrPC-dependent regulation of these receptors. Here, we review evidence highlighting a role for PrPC in preventing NMDA receptor-mediated excitotoxicity and inflammation. There is a need for more detailed molecular characterization of PrPC-mediated regulation of NMDA receptors, such as determining which NMDA receptor subunits mediate pathogenic effects upon loss of PrPC-mediated regulation and identifying PrPC binding site(s) on the receptor. This knowledge will allow development of novel therapeutic interventions for not only TSEs, but also for AD and other neurodegenerative disorders involving dysfunction of PrPC. PMID:25364752

  16. Arthritis protective regulatory potential of self–heat shock protein cross-reactive T cells

    PubMed Central

    van Eden, Willem; Wendling, Uwe; Paul, Liesbeth; Prakken, Berent; van Kooten, Peter; van der Zee, Ruurd

    2000-01-01

    Immunization with heat shock proteins has protective effects in models of induced arthritis. Analysis has shown a reduced synovial inflammation in such protected animals. Adoptive transfer and immunization with selected T cell epitopes (synthetic peptides) have indicated the protection to be mediated by T cells directed to conserved hsp epitopes. This was shown first for mycobacterial hsp60 and later for mycobacterial hsp70. Fine specificity analysis showed that such T cells were cross-reactive with the homologous self hsp. Therefore protection by microbial hsp reactive T cells can be by cross-recognition of self hsp overexpressed in the inflamed tissue. Preimmunization with hsp leads to a relative expansion of such self hsp cross-responsive T cells. The regulatory nature of such T cells may originate from mucosal tolerance maintained by commensal flora derived hsp or from partial activation through recognition of self hsp as a partial agonist (Altered Peptide Ligand) or in the absence of proper costimulation. Recently, we reported the selective upregulation of B7.2 on microbial hsp60 specific T cells in response to self hsp60. Through a preferred interaction with CTLA-4 on proinflammatory T cells this may constitute an effector mechanism of regulation. Also, regulatory T cells produced IL10. PMID:11189451

  17. TLR2-targeted secreted proteins from Mycobacterium tuberculosis are protective as powdered pulmonary vaccines.

    PubMed

    Tyne, Anneliese S; Chan, John Gar Yan; Shanahan, Erin R; Atmosukarto, Ines; Chan, Hak-Kim; Britton, Warwick J; West, Nicholas P

    2013-09-13

    Despite considerable research efforts towards effective treatments, tuberculosis (TB) remains a staggering burden on global health. Suitably formulated sub-unit vaccines offer potential as safe and effective generators of protective immunity. The Mycobacterium tuberculosis antigens, cutinase-like proteins (Culp) 1 and 6 and MPT83, were conjugated directly to the novel adjuvant Lipokel (Lipotek Pty Ltd), a TLR2 ligand that delivers antigen to immune cells in a self-adjuvanting context. Protein-Lipokel complexes were formulated as dry powders for pulmonary delivery directly to the lungs of mice by intra-tracheal insufflation, leading to recruitment of neutrophils and antigen presenting cell populations to the lungs at 72 h, that persisted at 7 days post immunisation. Significant increases in the frequency of activated dendritic cells were observed in the mediastinal lymph node (MLN) at 1 and 4 weeks after homologous boosting with protein-Lipokel vaccine. This was associated with the increased recruitment of effector CD4(+) and CD8(+) T-lymphocytes to the MLN and systemic antigen-specific, IFN-γ producing T-lymphocyte and IgG responses. Notably, pulmonary immunisation with either Culp1-6-Lipokel or MPT83-Lipokel powder vaccines generated protective responses in the lungs against aerosol M. tuberculosis challenge. The successful combination of TLR2-targeting and dry powder vaccine formulation, together with important practical benefits, offers potential for pulmonary vaccination against M. tuberculosis.

  18. UV protection of euglenoids: computation of the electromagnetic response

    NASA Astrophysics Data System (ADS)

    Dolinko, Andrés.; Valencia, Claudio; Skigin, Diana C.; Inchaussandague, Marina E.; Tolivia, Analía.; Conforti, Visitación

    2015-06-01

    Euglenoids are a group of predominantly free-living unicellular microorganisms that mostly live in freshwater bodies but can also be found in marine and brackish waters. These organisms have a characteristic that distinguishes them form the other protists: they are covered by a surface pellicle formed by S-shaped overlapping bands which resemble a diffraction grating. These microorganisms have developed numerous protection mechanisms intended to avoid or reduce the damage produced by UV radiation, such as the production of pigments and the repair mechanisms in hours of darkness and during daylight. In a recent paper we have investigated the role played by the pellicle of Euglenoids in the protection of the cell against UV radiation, by means of an electromagnetic approach based on the approximation of the pellicle profile by a one-dimensional diffraction grating. This simplified model allowed us to confirm that under certain incidence conditions, the corrugation of the pellicle helps increase the UV reflection, and consequently, diminish the UV radiation that enters the cell. In order to analyze the electromagnetic response of the whole cell, we extend two different approaches to calculate the reflected response: a simulation method especially developed to deal with complex biological structures that permits the introduction of the scattering object via an electron microscopy image, and the integral method, which has been widely used to compute the electromagnetic response of finite structures. Numerical results of near and far fields are shown.

  19. Novel protein-protein interaction family proteins involved in chloroplast movement response.

    PubMed

    Kodama, Yutaka; Suetsugu, Noriyuki; Wada, Masamitsu

    2011-04-01

    To optimize photosynthetic activity, chloroplasts change their intracellular location in response to ambient light conditions; chloroplasts move toward low intensity light to maximize light capture, and away from high intensity light to avoid photodamage. Although several proteins have been reported to be involved in the chloroplast photorelocation movement response, any physical interaction among them was not found so far. We recently found a physical interaction between two plant-specific coiled-coil proteins, WEB1 (Weak Chloroplast Movement under Blue Light 1) and PMI2 (Plastid Movement Impaired 2), that were identified to regulate chloroplast movement velocity. Since the both coiled-coil regions of WEB1 and PMI2 were classified into an uncharacterized protein family having DUF827 (DUF: Domain of Unknown Function) domain, it was the first report that DUF827 proteins could mediate protein-protein interaction. In this mini-review article, we discuss regarding molecular function of WEB1 and PMI2, and also define a novel protein family composed of WEB1, PMI2 and WEB1/PMI2-like proteins for protein-protein interaction in land plants.

  20. From protein denaturant to protectant: Comparative molecular dynamics study of alcohol/protein interactions

    NASA Astrophysics Data System (ADS)

    Shao, Qiang; Fan, Yubo; Yang, Lijiang; Qin Gao, Yi

    2012-03-01

    It is well known that alcohols can have strong effects on protein structures. For example, monohydric methanol and ethanol normally denature, whereas polyhydric glycol and glycerol protect, protein structures. In a recent combined theoretical and NMR experimental study, we showed that molecular dynamics simulations can be effectively used to understand the molecular mechanism of methanol denaturing protein. In this study, we used molecular dynamics simulations to investigate how alcohols with varied hydrophobicity and different numbers of hydrophilic groups (hydroxyl groups) exert effects on the structure of the model polypeptide, BBA5. First, we showed that methanol and trifluoroethanol (TFE) but not glycol or glycerol disrupt hydrophobic interactions. The latter two alcohols instead protect the assembly of the α- and β-domains of the polypeptide. Second, all four alcohols were shown to generally increase the stability of secondary structures, as revealed by the increased number of backbone hydrogen bonds formed in alcohol/water solutions compared to that in pure water, although individual hydrogen bonds can be weakened by certain alcohols, such as TFE. The two monohydric alcohols, methanol and TFE, display apparently different sequence-dependence in affecting the backbone hydrogen bond stability: methanol tends to enhance the stability of backbone hydrogen bonds of which the carbonyl groups are from polar residues, whereas TFE tends to stabilize those involving non-polar residues. These results demonstrated that subtle differences in the solution environment could have distinct consequences on protein structures.

  1. Rapid outer-surface protein C DNA tattoo vaccination protects against Borrelia afzelii infection.

    PubMed

    Wagemakers, A; Mason, L M K; Oei, A; de Wever, B; van der Poll, T; Bins, A D; Hovius, J W R

    2014-12-01

    Borrelia afzelii is the predominant Borrelia species causing Lyme borreliosis in Europe. Currently there is no human vaccine against Lyme borreliosis, and most research focuses on recombinant protein vaccines against Borrelia burgdorferi sensu stricto. DNA tattooing is a novel vaccination method that can be applied in a rapid vaccination schedule. We vaccinated C3H/HeN mice with B. afzelii strain PKo OspC (outer-surface protein C) using a codon-optimized DNA vaccine tattoo and compared this with recombinant protein vaccination in a 0-2-4 week vaccination schedule. We also assessed protection by DNA tattoo in a 0-3-6 day schedule. DNA tattoo and recombinant OspC vaccination induced comparable total IgG responses, with a lower IgG1/IgG2a ratio after DNA tattoo. Two weeks after syringe-challenge with 5 × 10(5) B. afzelii spirochetes most vaccinated mice had negative B. afzelii tissue DNA loads and all were culture negative. Furthermore, DNA tattoo vaccination in a 0-3-6 day regimen also resulted in negative Borrelia loads and cultures after challenge. To conclude, DNA vaccination by tattoo was fully protective against B. afzelii challenge in mice in a rapid vaccination protocol, and induces a favorable humoral immunity compared to recombinant protein vaccination. Rapid DNA tattoo is a promising vaccination strategy against spirochetes.

  2. Extracellular proteins of Clostridium chauvoei are protective in a mouse model.

    PubMed

    Mattar, María A; Cortiñas, Teresa I; Stefanini, Ana M

    2007-06-01

    The anaerobic bacillus Clostridium chauvoei is the causative agent of blackleg, a lethal disease that has an important impact on the sheep and cattle industry worldwide. Immunity to C. chauvoei is considered to be mainly anticellular, and for this reason there is scarce information about the immunogenicity of extracellular proteins. In this work variations in protein profiles, immune response by ELISA and protective capacity of culture supernatants of three C. chauvoei strains, collected at different growth phases, are reported. Sera raised against extracellular antigens also recognised cellular antigens of the same molecular masses. Partially purified cell-free supernatants and those concentrated 10 times by ultrafiltration (C-CFS), obtained at the early stationary phase of growth, induced a strong immunoprotective response, even at low doses, that was more marked for C. chauvoei strain ATCC 10092 (p < or = 0.05). With C-CFS formulations, a clear relationship was observed between IgG titres, protective capacity and concentration of the antigen doses, indicating a specific immune response.

  3. 77 FR 15379 - Protecting Tenants at Foreclosure Act: Additional Guidance on Notification Responsibilities Under...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-03-15

    ... guidance on the notice, entitled ``Protecting Tenants at Foreclosure: Notice of Responsibilities Placed on... URBAN DEVELOPMENT Protecting Tenants at Foreclosure Act: Additional Guidance on Notification Responsibilities Under the Act With Respect to Occupied Conveyance AGENCY: Office of the Assistant Secretary...

  4. Protective immunity induced in Aotus monkeys by a recombinant SERA protein of Plasmodium falciparum: adjuvant effects on induction of protective immunity.

    PubMed Central

    Inselburg, J; Bathurst, I C; Kansopon, J; Barchfeld, G L; Barr, P J; Rossan, R N

    1993-01-01

    We report the results of vaccination trial 2 of Panamanian Aotus monkeys with a recombinant blood-stage antigen, SERA 1, of the malaria parasite Plasmodium falciparum. Monkeys were immunized with SERA 1, a 262-amino-acid fragment (amino acids 24 to 285) of the 989-amino-acid SERA protein produced by the Honduras 1 strain of the parasite. Immunization mixtures contained 100 micrograms of recombinant SERA 1 protein per dose mixed with one of five different adjuvants. The protein mixed with either Freund's adjuvant or MF75.2 adjuvant stimulated protective immunity. When other P. falciparum antigens were included in the SERA 1-Freund's adjuvant mixture, no protective immunity was observed, although high anti-SERA 1 antibody titers were produced. Three other adjuvants mixed with SERA 1 failed to induce a protective immune response. These results, their relationship to those reported previously in the first vaccination trial (trial 1), and their relationships to the quantitative measurement of anti-SERA 1 antibodies in enzyme-linked immunosorbent assays provided insights into the induction of a protective immune response in vaccinated monkeys. PMID:8478092

  5. Electromagnetic response of the protective pellicle of different unicellular microalgae

    NASA Astrophysics Data System (ADS)

    Inchaussandague, Marina E.; Skigin, Diana C.; Tolivia, Analía.; Fuertes Vila, Isabel; Conforti, Visitación

    2014-03-01

    Euglenoids are unicellular aquatic organisms. These microalgae show a typical surface structure that distinguishes them from the other protists. Most cells are naked and bounded by a plasma membrane surrounded by a pellicle formed by overlapping bands. It is well known that all terrestrial and aquatic organisms are exposed to UV-A and UV-B radiation. This radiation is potentially harmful to life and since it can penetrate up to 12 meters in the water, it can reduce survival, growth and production of phytoplankton. However, the organisms have developed numerous protection mechanisms intended to reduce such damage, such as the production of pigments and other repair mechanisms. However, the possible protection that could provide the first barriers before entering into the cell has not been explored yet. In this paper we investigate, from an electromagnetic point of view, the role played by the pellicle of euglenoids in the protection of the cell against UV radiation. To do so, we investigate the electromagnetic response of different species that exhibit different behaviors against the UV radiation. We solve the diffraction problem by using the Chandezon Method and obtain the reflectance of the pellicle for the UV wavelengths. The results show that the corrugated pellicle could contribute to increase the reflectance, thus reducing the penetration of the UV radiation within the cell and therefore, minimizing the damage and increasing the survival of these organisms.

  6. Vaccines displaying mycobacterial proteins on biopolyester beads stimulate cellular immunity and induce protection against tuberculosis.

    PubMed

    Parlane, Natalie A; Grage, Katrin; Mifune, Jun; Basaraba, Randall J; Wedlock, D Neil; Rehm, Bernd H A; Buddle, Bryce M

    2012-01-01

    New improved vaccines are needed for control of both bovine and human tuberculosis. Tuberculosis protein vaccines have advantages with regard to safety and ease of manufacture, but efficacy against tuberculosis has been difficult to achieve. Protective cellular immune responses can be preferentially induced when antigens are displayed on small particles. In this study, Escherichia coli and Lactococcus lactis were engineered to produce spherical polyhydroxybutyrate (PHB) inclusions which displayed a fusion protein of Mycobacterium tuberculosis, antigen 85A (Ag85A)-early secreted antigenic target 6-kDa protein (ESAT-6). L. lactis was chosen as a possible production host due its extensive use in the food industry and reduced risk of lipopolysaccharide contamination. Mice were vaccinated with PHB bead vaccines with or without displaying Ag85A-ESAT-6, recombinant Ag85A-ESAT-6, or M. bovis BCG. Separate groups of mice were used to measure immune responses and assess protection against an aerosol M. bovis challenge. Increased amounts of antigen-specific gamma interferon, interleukin-17A (IL-17A), IL-6, and tumor necrosis factor alpha were produced from splenocytes postvaccination, but no or minimal IL-4, IL-5, or IL-10 was produced, indicating Th1- and Th17-biased T cell responses. Decreased lung bacterial counts and less extensive foci of inflammation were observed in lungs of mice receiving BCG or PHB bead vaccines displaying Ag85A-ESAT-6 produced in either E. coli or L. lactis compared to those observed in the lungs of phosphate-buffered saline-treated control mice. No differences between those receiving wild-type PHB beads and those receiving recombinant Ag85A-ESAT-6 were observed. This versatile particulate vaccine delivery system incorporates a relatively simple production process using safe bacteria, and the results show that it is an effective delivery system for a tuberculosis protein vaccine.

  7. Vaccines Displaying Mycobacterial Proteins on Biopolyester Beads Stimulate Cellular Immunity and Induce Protection against Tuberculosis

    PubMed Central

    Parlane, Natalie A.; Grage, Katrin; Mifune, Jun; Basaraba, Randall J.; Wedlock, D. Neil; Rehm, Bernd H. A.

    2012-01-01

    New improved vaccines are needed for control of both bovine and human tuberculosis. Tuberculosis protein vaccines have advantages with regard to safety and ease of manufacture, but efficacy against tuberculosis has been difficult to achieve. Protective cellular immune responses can be preferentially induced when antigens are displayed on small particles. In this study, Escherichia coli and Lactococcus lactis were engineered to produce spherical polyhydroxybutyrate (PHB) inclusions which displayed a fusion protein of Mycobacterium tuberculosis, antigen 85A (Ag85A)–early secreted antigenic target 6-kDa protein (ESAT-6). L. lactis was chosen as a possible production host due its extensive use in the food industry and reduced risk of lipopolysaccharide contamination. Mice were vaccinated with PHB bead vaccines with or without displaying Ag85A–ESAT-6, recombinant Ag85A–ESAT-6, or M. bovis BCG. Separate groups of mice were used to measure immune responses and assess protection against an aerosol M. bovis challenge. Increased amounts of antigen-specific gamma interferon, interleukin-17A (IL-17A), IL-6, and tumor necrosis factor alpha were produced from splenocytes postvaccination, but no or minimal IL-4, IL-5, or IL-10 was produced, indicating Th1- and Th17-biased T cell responses. Decreased lung bacterial counts and less extensive foci of inflammation were observed in lungs of mice receiving BCG or PHB bead vaccines displaying Ag85A–ESAT-6 produced in either E. coli or L. lactis compared to those observed in the lungs of phosphate-buffered saline-treated control mice. No differences between those receiving wild-type PHB beads and those receiving recombinant Ag85A–ESAT-6 were observed. This versatile particulate vaccine delivery system incorporates a relatively simple production process using safe bacteria, and the results show that it is an effective delivery system for a tuberculosis protein vaccine. PMID:22072720

  8. Moonlight-like proteins of the cell wall protect sessile cells of Candida from oxidative stress.

    PubMed

    Serrano-Fujarte, Isela; López-Romero, Everardo; Cuéllar-Cruz, Mayra

    2016-01-01

    Biofilms of Candida species are associated with high morbidity and hospital mortality. Candida forms biofilms by adhering to human host epithelium through cell wall proteins (CWP) and simultaneously neutralizing the reactive oxygen species (ROS) produced during the respiratory burst by phagocytic cells. The purpose of this paper is to identify the CWP of Candida albicans, Candida glabrata, Candida krusei and Candida parapsilosis expressed after exposure to different concentrations of H2O2 using a proteomic approach. CWP obtained from sessile cells, both treated and untreated with the oxidizing agent, were resolved by one and two-dimensional (2D-PAGE) gels and identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Some of these proteins were identified and found to correspond to moonlighting CWP such as: (i) glycolytic enzymes, (ii) heat shock, (iii) OSR proteins, (iv) general metabolic enzymes and (v) highly conserved proteins, which are up- or down-regulated in the presence or absence of ROS. We also found that the expression of these CWP is different for each Candida species. Moreover, RT-PCR assays allowed us to demonstrate that transcription of the gene coding for Eno1, one of the moonlight-like CWP identified in response to the oxidant agent, is differentially regulated. To our knowledge this is the first demonstration that, in response to oxidative stress, each species of Candida, differentially regulates the expression of moonlighting CWP, which may protect the organism from the ROS generated during phagocytosis. Presumptively, these proteins allow the pathogen to adhere and form a biofilm, and eventually cause invasive candidiasis in the human host. We propose that, in addition to the antioxidant mechanisms present in Candida, the moonlighting CWP also confer protection to these pathogens from oxidative stress.

  9. HDLs protect pancreatic β-cells against ER stress by restoring protein folding and trafficking.

    PubMed

    Pétremand, Jannick; Puyal, Julien; Chatton, Jean-Yves; Duprez, Jessica; Allagnat, Florent; Frias, Miguel; James, Richard W; Waeber, Gérard; Jonas, Jean-Christophe; Widmann, Christian

    2012-05-01

    Endoplasmic reticulum (ER) homeostasis alteration contributes to pancreatic β-cell dysfunction and death and favors the development of diabetes. In this study, we demonstrate that HDLs protect β-cells against ER stress induced by thapsigargin, cyclopiazonic acid, palmitate, insulin overexpression, and high glucose concentrations. ER stress marker induction and ER morphology disruption mediated by these stimuli were inhibited by HDLs. Using a temperature-sensitive viral glycoprotein folding mutant, we show that HDLs correct impaired protein trafficking and folding induced by thapsigargin and palmitate. The ability of HDLs to protect β-cells against ER stress was inhibited by brefeldin A, an ER to Golgi trafficking blocker. These results indicate that HDLs restore ER homeostasis in response to ER stress, which is required for their ability to promote β-cell survival. This study identifies a cellular mechanism mediating the beneficial effect of HDLs on β-cells against ER stress-inducing factors.

  10. HDLs Protect Pancreatic β-Cells Against ER Stress by Restoring Protein Folding and Trafficking

    PubMed Central

    Pétremand, Jannick; Puyal, Julien; Chatton, Jean-Yves; Duprez, Jessica; Allagnat, Florent; Frias, Miguel; James, Richard W.; Waeber, Gérard; Jonas, Jean-Christophe; Widmann, Christian

    2012-01-01

    Endoplasmic reticulum (ER) homeostasis alteration contributes to pancreatic β-cell dysfunction and death and favors the development of diabetes. In this study, we demonstrate that HDLs protect β-cells against ER stress induced by thapsigargin, cyclopiazonic acid, palmitate, insulin overexpression, and high glucose concentrations. ER stress marker induction and ER morphology disruption mediated by these stimuli were inhibited by HDLs. Using a temperature-sensitive viral glycoprotein folding mutant, we show that HDLs correct impaired protein trafficking and folding induced by thapsigargin and palmitate. The ability of HDLs to protect β-cells against ER stress was inhibited by brefeldin A, an ER to Golgi trafficking blocker. These results indicate that HDLs restore ER homeostasis in response to ER stress, which is required for their ability to promote β-cell survival. This study identifies a cellular mechanism mediating the beneficial effect of HDLs on β-cells against ER stress-inducing factors. PMID:22399686

  11. The Unfolded Protein Response and Gastrointestinal Disease

    PubMed Central

    Kaser, Arthur; Adolph, Timon Erik; Blumberg, Richard S

    2013-01-01

    As the inner lining of the gastrointestinal tract, the intestinal epithelium serves an essential role in innate immune function at the interface between the host and microbiota. Given the unique environmental challenges and thus physiologic secretory functions of this surface, it is exquisitely sensitive to perturbations that affect its capacity to resolve endoplasmic reticulum (ER) stress. Genetic deletion of factors involved in the unfolded protein response (UPR), which functions in the resolution of ER stress that arises from misfolded proteins, result in spontaneous intestinal inflammation closely mimicking human inflammatory bowel disease (IBD). This is demonstrated by observations wherein deletion of genes such as Xbp1 and Agr2 profoundly affects the intestinal epithelium and results in spontaneous intestinal inflammation. Moreover, both genes, along with others (e.g. ORDML3) represent genetic risk factors for human IBD, both Crohn’s disease and ulcerative colitis. Here we review the current mechanistic understanding for how unresolved ER stress can lead to intestinal inflammation, and highlight the findings that implicate ER stress as a genetically affected biological pathway in IBD. We further discuss environmental and microbial factors that might impact on the epithelium’s capacity to resolve ER stress, and which may constitute exogenous factors that may precipitate disease in genetically susceptible individuals. PMID:23588234

  12. G protein-coupled estrogen receptor protects from atherosclerosis.

    PubMed

    Meyer, Matthias R; Fredette, Natalie C; Howard, Tamara A; Hu, Chelin; Ramesh, Chinnasamy; Daniel, Christoph; Amann, Kerstin; Arterburn, Jeffrey B; Barton, Matthias; Prossnitz, Eric R

    2014-12-23

    Coronary atherosclerosis and myocardial infarction in postmenopausal women have been linked to inflammation and reduced nitric oxide (NO) formation. Natural estrogen exerts protective effects on both processes, yet also displays uterotrophic activity. Here, we used genetic and pharmacologic approaches to investigate the role of the G protein-coupled estrogen receptor (GPER) in atherosclerosis. In ovary-intact mice, deletion of gper increased atherosclerosis progression, total and LDL cholesterol levels and inflammation while reducing vascular NO bioactivity, effects that were in some cases aggravated by surgical menopause. In human endothelial cells, GPER was expressed on intracellular membranes and mediated eNOS activation and NO formation, partially accounting for estrogen-mediated effects. Chronic treatment with G-1, a synthetic, highly selective small molecule agonist of GPER, reduced postmenopausal atherosclerosis and inflammation without uterotrophic effects. In summary, this study reveals an atheroprotective function of GPER and introduces selective GPER activation as a novel therapeutic approach to inhibit postmenopausal atherosclerosis and inflammation in the absence of uterotrophic activity.

  13. G Protein-coupled Estrogen Receptor Protects from Atherosclerosis

    PubMed Central

    Meyer, Matthias R.; Fredette, Natalie C.; Howard, Tamara A.; Hu, Chelin; Ramesh, Chinnasamy; Daniel, Christoph; Amann, Kerstin; Arterburn, Jeffrey B.; Barton, Matthias; Prossnitz, Eric R.

    2014-01-01

    Coronary atherosclerosis and myocardial infarction in postmenopausal women have been linked to inflammation and reduced nitric oxide (NO) formation. Natural estrogen exerts protective effects on both processes, yet also displays uterotrophic activity. Here, we used genetic and pharmacologic approaches to investigate the role of the G protein-coupled estrogen receptor (GPER) in atherosclerosis. In ovary-intact mice, deletion of gper increased atherosclerosis progression, total and LDL cholesterol levels and inflammation while reducing vascular NO bioactivity, effects that were in some cases aggravated by surgical menopause. In human endothelial cells, GPER was expressed on intracellular membranes and mediated eNOS activation and NO formation, partially accounting for estrogen-mediated effects. Chronic treatment with G-1, a synthetic, highly selective small molecule agonist of GPER, reduced postmenopausal atherosclerosis and inflammation without uterotrophic effects. In summary, this study reveals an atheroprotective function of GPER and introduces selective GPER activation as a novel therapeutic approach to inhibit postmenopausal atherosclerosis and inflammation in the absence of uterotrophic activity. PMID:25532911

  14. HDAC6 controls major cell response pathways to cytotoxic accumulation of protein aggregates

    PubMed Central

    Boyault, Cyril; Zhang, Yu; Fritah, Sabrina; Caron, Cécile; Gilquin, Benoit; Kwon, So Hee; Garrido, Carmen; Yao, Tso-Pang; Vourc’h, Claire; Matthias, Patrick; Khochbin, Saadi

    2007-01-01

    A cellular defense mechanism counteracts the deleterious effects of misfolded protein accumulation by eliciting a stress response. The cytoplasmic deacetylase HDAC6 (histone deacetylase 6) was previously shown to be a key element in this response by coordinating the clearance of protein aggregates through aggresome formation and their autophagic degradation. Here, for the first time, we demonstrate that HDAC6 is involved in another crucial cell response to the accumulation of ubiquitinated protein aggregates, and unravel its molecular basis. Indeed, our data show that HDAC6 senses ubiquitinated cellular aggregates and consequently induces the expression of major cellular chaperones by triggering the dissociation of a repressive HDAC6/HSF1 (heat-shock factor 1)/HSP90 (heat-shock protein 90) complex and a subsequent HSF1 activation. HDAC6 therefore appears as a master regulator of the cell protective response to cytotoxic protein aggregate formation. PMID:17785525

  15. Acidic chitinase primes the protective immune response to gastrointestinal nematodes.

    PubMed

    Vannella, Kevin M; Ramalingam, Thirumalai R; Hart, Kevin M; de Queiroz Prado, Rafael; Sciurba, Joshua; Barron, Luke; Borthwick, Lee A; Smith, Allen D; Mentink-Kane, Margaret; White, Sandra; Thompson, Robert W; Cheever, Allen W; Bock, Kevin; Moore, Ian; Fitz, Lori J; Urban, Joseph F; Wynn, Thomas A

    2016-05-01

    Acidic mammalian chitinase (AMCase) is known to be induced by allergens and helminths, yet its role in immunity is unclear. Using AMCase-deficient mice, we show that AMCase deficiency reduced the number of group 2 innate lymphoid cells during allergen challenge but was not required for establishment of type 2 inflammation in the lung in response to allergens or helminths. In contrast, AMCase-deficient mice showed a profound defect in type 2 immunity following infection with the chitin-containing gastrointestinal nematodes Nippostrongylus brasiliensis and Heligmosomoides polygyrus bakeri. The impaired immunity was associated with reduced mucus production and decreased intestinal expression of the signature type 2 response genes Il13, Chil3, Retnlb, and Clca1. CD103(+) dendritic cells, which regulate T cell homing, were also reduced in mesenteric lymph nodes of infected AMCase-deficient mice. Thus, AMCase functions as a critical initiator of protective type 2 responses to intestinal nematodes but is largely dispensable for allergic responses in the lung.

  16. The Unfolded Protein Response Supports Plant Development and Defense as well as Responses to Abiotic Stress

    PubMed Central

    Bao, Yan; Howell, Stephen H.

    2017-01-01

    The unfolded protein response (UPR) is a stress response conserved in eukaryotic organisms and activated by the accumulation of misfolded proteins in the endoplasmic reticulum (ER). Adverse environmental conditions disrupt protein folding in the ER and trigger the UPR. Recently, it was found that the UPR can be elicited in the course of plant development and defense. During vegetative plant development, the UPR is involved in normal root growth and development, the effect of which can be largely attributed to the influence of the UPR on plant hormone biology. The UPR also functions in plant reproductive development by protecting male gametophyte development from heat stress. In terms of defense, the UPR has been implicated in virus and microbial defense. Viral defense represents a double edge sword in that various virus infections activate the UPR, however, in a number of cases, the UPR actually supports viral infections. The UPR also plays a role in plant immunity to bacterial infections, again through the action of plant hormones in regulating basal immunity responses. PMID:28360918

  17. Normal Cellular Prion Protein Protects against Manganese-induced Oxidative Stress and Apoptotic Cell Death

    PubMed Central

    Choi, Christopher J.; Anantharam, Vellareddy; Saetveit, Nathan J.; Houk, Robert. S.; Kanthasamy, Arthi; Kanthasamy, Anumantha G.

    2012-01-01

    The normal prion protein is abundantly expressed in the CNS, but its biological function remains unclear. The prion protein has octapeptide repeat regions that bind to several divalent metals, suggesting that the prion proteins may alter the toxic effect of environmental neurotoxic metals. In the present study, we systematically examined whether prion protein modifies the neurotoxicity of manganese (Mn) by comparing the effect of Mn on mouse neural cells expressing prion protein (PrPC -cells) and prion-knockout (PrPKO -cells). Exposure to Mn (10 μM-1 mM) for 24 hr produced a dose-dependent cytotoxic response in both PrPC -cells and PrPKO -cells. Interestingly, PrPC -cells (EC50 117.6μM) were more resistant to Mn-induced cytotoxicity, as compared to PrPKO -cells (EC50 59.9μM), suggesting a protective role for PrPC against Mn neurotoxicity. Analysis of intracellular Mn levels showed less Mn accumulation in PrPC -cells as compared to PrPKO -cells. Furthermore, Mn-induced mitochondrial depolarization and ROS generation were significantly attenuated in PrPC -cells as compared to PrPKO -cells. Measurement of antioxidant status revealed similar basal levels of glutathione (GSH) in PrPC -cells and PrPKO -cells; however, Mn treatment caused greater depletion of GSH in PrPKO -cells. Mn-induced mitochondrial depolarization and ROS production were followed by time- and dose-dependent activation of the apoptotic cell death cascade involving caspase-9 and -3. Notably, DNA fragmentation induced by both Mn treatment and oxidative stress-inducer hydrogen peroxide (100μM) was significantly suppressed in PrPC -cells as compared to PrPKO -cells. Together, these results demonstrate that prion protein interferes with divalent metal Mn uptake and protects against Mn-induced oxidative stress and apoptotic cell death. PMID:17483122

  18. Association between Interferon Response and Protective Efficacy of NS1-Truncated Mutants as Influenza Vaccine Candidates in Chickens

    PubMed Central

    Jang, Hyesun; Ngunjiri, John M.; Lee, Chang-Won

    2016-01-01

    Influenza virus mutants that encode C-terminally truncated NS1 proteins (NS1-truncated mutants) are attractive candidates for avian live attenuated influenza vaccine (LAIV) development because they are both attenuated and immunogenic in chickens. We previously showed that a high protective efficacy of NS1-truncated LAIV in chickens corresponds with induction of high levels of type I interferon (IFN) responses in chicken embryonic fibroblast cells. In this study, we investigated the relationship between induction of IFN and IFN-stimulated gene responses in vivo and the immunogenicity and protective efficacy of NS1-truncated LAIV. Our data demonstrates that accelerated antibody induction and protective efficacy of NS1-truncated LAIV correlates well with upregulation of IFN-stimulated genes. Further, through oral administration of recombinant chicken IFN alpha in drinking water, we provide direct evidence that type I IFN can promote rapid induction of adaptive immune responses and protective efficacy of influenza vaccine in chickens. PMID:27257989

  19. Glycaemic Response to Quality Protein Maize Grits

    PubMed Central

    Panlasigui, Leonora N.; Bayaga, Cecile L. T.; Barrios, Erniel B.; Cochon, Kim L.

    2010-01-01

    Background. Carbohydrates have varied rates of digestion and absorption that induces different hormonal and metabolic responses in the body. Given the abundance of carbohydrate sources in the Philippines, the determination of the glycaemic index (GI) of local foods may prove beneficial in promoting health and decreasing the risk of diabetes in the country. Methods. The GI of Quality Protein Maize (QPM) grits, milled rice, and the mixture of these two food items were determined in ten female subjects. Using a randomized crossover design, the control bread and three test foods were given on separate occasions after an overnight fast. Blood samples were collected through finger prick at time intervals of 0, 15, 30, 45, 60, 90, and 120 min and analyzed for glucose concentrations. Results. The computed incremental area under the glucose response curve (IAUC) varies significantly across test foods (P < .0379) with the pure QPM grits yielding the lowest IAUC relative to the control by 46.38. Resulting GI values of the test foods (bootstrapped) were 80.36 (SEM 14.24), 119.78 (SEM 18.81), and 93.17 (SEM 27.27) for pure QPM grits, milled rice, and rice-QPM grits mixture, respectively. Conclusion. Pure QPM corn grits has a lower glycaemic response compared to milled rice and the rice-corn grits mixture, which may be related in part to differences in their dietary fibre composition and physicochemical characteristics. Pure QPM corn grits may be a more health beneficial food for diabetic and hyperlipidemic individuals. PMID:20862364

  20. The Stress Granule RNA-Binding Protein TIAR-1 Protects Female Germ Cells from Heat Shock in Caenorhabditis elegans.

    PubMed

    Huelgas-Morales, Gabriela; Silva-García, Carlos Giovanni; Salinas, Laura S; Greenstein, David; Navarro, Rosa E

    2016-04-07

    In response to stressful conditions, eukaryotic cells launch an arsenal of regulatory programs to protect the proteome. One major protective response involves the arrest of protein translation and the formation of stress granules, cytoplasmic ribonucleoprotein complexes containing the conserved RNA-binding proteins TIA-1 and TIAR. The stress granule response is thought to preserve mRNA for translation when conditions improve. For cells of the germline-the immortal cell lineage required for sexual reproduction-protection from stress is critically important for perpetuation of the species, yet how stress granule regulatory mechanisms are deployed in animal reproduction is incompletely understood. Here, we show that the stress granule protein TIAR-1 protects the Caenorhabditis elegans germline from the adverse effects of heat shock. Animals containing strong loss-of-function mutations in tiar-1 exhibit significantly reduced fertility compared to the wild type following heat shock. Analysis of a heat-shock protein promoter indicates that tiar-1 mutants display an impaired heat-shock response. We observed that TIAR-1 was associated with granules in the gonad core and oocytes during several stressful conditions. Both gonad core and oocyte granules are dynamic structures that depend on translation; protein synthesis inhibitors altered their formation. Nonetheless, tiar-1 was required for the formation of gonad core granules only. Interestingly, the gonad core granules did not seem to be needed for the germ cells to develop viable embryos after heat shock. This suggests that TIAR-1 is able to protect the germline from heat stress independently of these structures.

  1. The Stress Granule RNA-Binding Protein TIAR-1 Protects Female Germ Cells from Heat Shock in Caenorhabditis elegans

    PubMed Central

    Huelgas-Morales, Gabriela; Silva-García, Carlos Giovanni; Salinas, Laura S.; Greenstein, David; Navarro, Rosa E.

    2016-01-01

    In response to stressful conditions, eukaryotic cells launch an arsenal of regulatory programs to protect the proteome. One major protective response involves the arrest of protein translation and the formation of stress granules, cytoplasmic ribonucleoprotein complexes containing the conserved RNA-binding proteins TIA-1 and TIAR. The stress granule response is thought to preserve mRNA for translation when conditions improve. For cells of the germline—the immortal cell lineage required for sexual reproduction—protection from stress is critically important for perpetuation of the species, yet how stress granule regulatory mechanisms are deployed in animal reproduction is incompletely understood. Here, we show that the stress granule protein TIAR-1 protects the Caenorhabditis elegans germline from the adverse effects of heat shock. Animals containing strong loss-of-function mutations in tiar-1 exhibit significantly reduced fertility compared to the wild type following heat shock. Analysis of a heat-shock protein promoter indicates that tiar-1 mutants display an impaired heat-shock response. We observed that TIAR-1 was associated with granules in the gonad core and oocytes during several stressful conditions. Both gonad core and oocyte granules are dynamic structures that depend on translation; protein synthesis inhibitors altered their formation. Nonetheless, tiar-1 was required for the formation of gonad core granules only. Interestingly, the gonad core granules did not seem to be needed for the germ cells to develop viable embryos after heat shock. This suggests that TIAR-1 is able to protect the germline from heat stress independently of these structures. PMID:26865701

  2. Immunization with individual proteins of the Lrp/AsnC family induces protection against Brucella melitensis 16M challenges in mice

    PubMed Central

    Wang, Xinhui; An, Chang; Yang, Mingjuan; Li, Xinran; Ke, Yuehua; Lei, Shuangshuang; Xu, Xiaoyang; Yu, Jiuxuan; Ren, Hang; Du, Xinying; Wang, Zhoujia; Qiu, Yefeng; Liu, Bo; Chen, Zeliang

    2015-01-01

    Brucellosis is one of the most common zoonoses worldwide. Subunit vaccines are promising for the prevention of human brucellosis. In our previous protective antigen screening studies, we identified a new protective antigen, BMEI0357, which belongs to the Lrp/asnC protein family, a conserved transcriptional regulator in bacteria that is absent in eukaryotes. In the present study, the Brucella genome annotation was screened and a total of six proteins were identified as members of the Lrp/AsnC family. Lrp/AsnC proteins have two domains that are conserved among the family members. However, sequence similarities between these proteins ranged from 9 to 50%, indicating high sequence heterogeneity. To test whether proteins of this family have similar characteristics, all six proteins were cloned and expressed in Escherichia coli. The recombinant proteins were purified and their protective efficacy was evaluated in BALB/c mice challenged with Brucella melitensis 16M. The results show that all six Lrp/AsnC proteins could induce a protective immune response against Brucella melitensis 16M. Antibodies against the Lrp/AsnC proteins were detected in the immunized mice. However, levels of antibodies against these proteins were relatively variable in human brucellosis sera. Taken together, our results show that these six proteins of the Lrp/AsnC family in Brucella could induce protective immune responses in mice. PMID:26579099

  3. Evaluation of the protective immunity of the Legionella pneumophila recombinant protein FlaA/MompS/PilE in an A/J mouse model.

    PubMed

    Xu, Ying; Guan, Wang; Xu, Jia-nan; Cao, De-ping; Yang, Bin-bin; Chen, Da-li; Chen, Jian-ping

    2011-05-23

    To investigate the protect effects of the recombinant protein FlaA/MompS/PilE against Legionella pneumophila (L. pneumophila), the coding sequences of the three proteins were optimized by DNA Star software firstly, cloned, expressed by Escherichia coli BL21, and purified. To give an enhanced the immunological response, the proteins were linked together with (Linker) or without a linker insert (NLinker) and were purified from E. coli BL21. The A/J mouse model was used to determine the level of the induction of protective immunity from the purified proteins. Our results showed that the IgG titer, which was measured by ELISA, was increased after the administration of the five proteins. Compared to the administration of the individual proteins, the chimeric Linker and NLinker proteins displayed lasting immunity to a lethal dose of L. pneumophila challenge. The Linker protein protected the A/J mouse against a higher dose of L. pneumonia compared to the other proteins used in this study, as it contained a more effective immunogen. The work presented here demonstrates that the bioinformatics software, DNA Star, is a valid tool to analyse the epitopes of proteins and was useful in the optimization of proteins that could induce the protective immune response to L. pneumophila. The cross-immunity of recombinant proteins, such as the Linker and the NLinker chimera, have higher generates a greater immune than the single proteins.

  4. Immune Responses and Protection of Aotus Monkeys Immunized with Irradiated Plasmodium vivax Sporozoites

    PubMed Central

    Jordán-Villegas, Alejandro; Perdomo, Anilza Bonelo; Epstein, Judith E.; López, Jesús; Castellanos, Alejandro; Manzano, María R.; Hernández, Miguel A.; Soto, Liliana; Méndez, Fabián; Richie, Thomas L.; Hoffman, Stephen L.; Arévalo-Herrera, Myriam; Herrera, Sócrates

    2011-01-01

    A non-human primate model for the induction of protective immunity against the pre-erythrocytic stages of Plasmodium vivax malaria using radiation-attenuated P. vivax sporozoites may help to characterize protective immune mechanisms and identify novel malaria vaccine candidates. Immune responses and protective efficacy induced by vaccination with irradiated P. vivax sporozoites were evaluated in malaria-naive Aotus monkeys. Three groups of six monkeys received two, five, or ten intravenous inoculations, respectively, of 100,000 irradiated P. vivax sporozoites; control groups received either 10 doses of uninfected salivary gland extract or no inoculations. Immunization resulted in the production low levels of antibodies that specifically recognized P. vivax sporozoites and the circumsporozoite protein. Additionally, immunization induced low levels of antigen-specific IFN-γ responses. Intravenous challenge with viable sporozoites resulted in partial protection in a dose-dependent manner. These findings suggest that the Aotus monkey model may be able to play a role in preclinical development of P. vivax pre-erythrocytic stage vaccines. PMID:21292877

  5. Barrier-protective effects of activated protein C in human alveolar epithelial cells.

    PubMed

    Puig, Ferranda; Fuster, Gemma; Adda, Mélanie; Blanch, Lluís; Farre, Ramon; Navajas, Daniel; Artigas, Antonio

    2013-01-01

    Acute lung injury (ALI) is a clinical manifestation of respiratory failure, caused by lung inflammation and the disruption of the alveolar-capillary barrier. Preservation of the physical integrity of the alveolar epithelial monolayer is of critical importance to prevent alveolar edema. Barrier integrity depends largely on the balance between physical forces on cell-cell and cell-matrix contacts, and this balance might be affected by alterations in the coagulation cascade in patients with ALI. We aimed to study the effects of activated protein C (APC) on mechanical tension and barrier integrity in human alveolar epithelial cells (A549) exposed to thrombin. Cells were pretreated for 3 h with APC (50 µg/ml) or vehicle (control). Subsequently, thrombin (50 nM) or medium was added to the cell culture. APC significantly reduced thrombin-induced cell monolayer permeability, cell stiffening, and cell contraction, measured by electrical impedance, optical magnetic twisting cytometry, and traction microscopy, respectively, suggesting a barrier-protective response. The dynamics of the barrier integrity was also assessed by western blotting and immunofluorescence analysis of the tight junction ZO-1. Thrombin resulted in more elongated ZO-1 aggregates at cell-cell interface areas and induced an increase in ZO-1 membrane protein content. APC attenuated the length of these ZO-1 aggregates and reduced the ZO-1 membrane protein levels induced by thrombin. In conclusion, pretreatment with APC reduced the disruption of barrier integrity induced by thrombin, thus contributing to alveolar epithelial barrier protection.

  6. Multiple antioxidant proteins protect Chlorobaculum tepidum against oxygen and reactive oxygen species.

    PubMed

    Li, Hui; Jubelirer, Sara; Garcia Costas, Amaya M; Frigaard, Niels-Ulrik; Bryant, Donald A

    2009-11-01

    The genome of the green sulfur bacterium Chlorobaculum (Cba.) tepidum, a strictly anaerobic photolithoautotroph, is predicted to encode more than ten genes whose products are potentially involved in protection from reactive oxygen species and an oxidative stress response. The encoded proteins include cytochrome bd quinol oxidase, NADH oxidase, rubredoxin oxygen oxidoreductase, several thiol peroxidases, alkyl hydroperoxide reductase, superoxide dismutase, methionine sulfoxide reductase, and rubrerythrin. To test the physiological functions of some of these proteins, ten genes were insertionally inactivated. Wild-type Cba. tepidum cells were very sensitive to oxygen in the light but were remarkably resistant to oxygen in the dark. When wild-type and mutant cells were subjected to air for various times under dark or light condition, significant decreases in viability were detected in most of the mutants relative to wild type. Treatments with hydrogen peroxide (H(2)O(2)), tert-butyl hydroperoxide (t-BOOH) and methyl viologen resulted in more severe effects in most of the mutants than in the wild type. The results demonstrated that these putative antioxidant proteins combine to form an effective defense against oxygen and reactive oxygen species. Reverse-transcriptase polymerase chain reaction studies showed that the genes with functions in oxidative stress protection were constitutively transcribed under anoxic growth conditions.

  7. Platelets protect lung from injury induced by systemic inflammatory response

    PubMed Central

    Luo, Shuhua; Wang, Yabo; An, Qi; Chen, Hao; Zhao, Junfei; Zhang, Jie; Meng, Wentong; Du, Lei

    2017-01-01

    Systemic inflammatory responses can severely injure lungs, prompting efforts to explore how to attenuate such injury. Here we explored whether platelets can help attenuate lung injury in mice resulting from extracorporeal circulation (ECC)-induced systemic inflammatory responses. Mice were subjected to ECC for 30 min, then treated with phosphate-buffered saline, platelets, the GPIIb/IIIa inhibitor Tirofiban, or the combination of platelets and Tirofiban. Blood and lung tissues were harvested 60 min later, and lung injury and inflammatory status were assessed. As expected, ECC caused systemic inflammation and pulmonary dysfunction, and platelet transfusion resulted in significantly milder lung injury and higher lung function. It also led to greater numbers of circulating platelet-leukocyte aggregates and greater platelet accumulation in the lung. Platelet transfusion was associated with higher production of transforming growth factor-β and as well as lower levels of tumour necrosis factor-α and neutrophil elastase in plasma and lung. None of these platelet effects was observed in the presence of Tirofiban. Our results suggest that, at least under certain conditions, platelets can protect lung from injury induced by systemic inflammatory responses. PMID:28155889

  8. Ebolavirus Glycoprotein Fc Fusion Protein Protects Guinea Pigs against Lethal Challenge

    PubMed Central

    Konduru, Krishnamurthy; Shurtleff, Amy C.; Bradfute, Steven B.; Nakamura, Siham; Bavari, Sina; Kaplan, Gerardo

    2016-01-01

    Ebola virus (EBOV), a member of the Filoviridae that can cause severe hemorrhagic fever in humans and nonhuman primates, poses a significant threat to the public health. Currently, there are no licensed vaccines or therapeutics to prevent and treat EBOV infection. Several vaccines based on the EBOV glycoprotein (GP) are under development, including vectored, virus-like particles, and protein-based subunit vaccines. We previously demonstrated that a subunit vaccine containing the extracellular domain of the Ebola ebolavirus (EBOV) GP fused to the Fc fragment of human IgG1 (EBOVgp-Fc) protected mice against EBOV lethal challenge. Here, we show that the EBOVgp-Fc vaccine formulated with QS-21, alum, or polyinosinic-polycytidylic acid-poly-L-lysine carboxymethylcellulose (poly-ICLC) adjuvants induced strong humoral immune responses in guinea pigs. The vaccinated animals developed anti-GP total antibody titers of approximately 105−106 and neutralizing antibody titers of approximately 103 as assessed by a BSL-2 neutralization assay based on vesicular stomatitis virus (VSV) pseudotypes. The poly-ICLC formulated EBOVgp-Fc vaccine protected all the guinea pigs against EBOV lethal challenge performed under BSL-4 conditions whereas the same vaccine formulated with QS-21 or alum only induced partial protection. Vaccination with a mucin-deleted EBOVgp-Fc construct formulated with QS-21 adjuvant did not have a significant effect in anti-GP antibody levels and protection against EBOV lethal challenge compared to the full-length GP construct. The bulk of the humoral response induced by the EBOVgp-Fc vaccine was directed against epitopes outside the EBOV mucin region. Our findings indicate that different adjuvants can eliciting varying levels of protection against lethal EBOV challenge in guinea pigs vaccinated with EBOVgp-Fc, and suggest that levels of total anti-GP antibodies elicit by protein-based GP subunit vaccines do not correlate with protection. Our data further support

  9. Immunization with different formulations of Mycobacterium tuberculosis antigen 85A induces immune responses with different specificity and protective efficacy.

    PubMed

    Tchilian, Elma; Ahuja, Diksha; Hey, Ariann; Jiang, Shisong; Beverley, Peter

    2013-09-23

    To test the relative efficacy of CD4 and CD8T cells in mediating protective immunity to Mycobacterium tuberculosis (Mtb), we compared three immunization regimes designed to induce preferentially each subset. BALB/c mice were immunized intranasally (i.n.) or parenterally with antigen 85A either in a recombinant Adenoviral vector (Ad85A), as recombinant protein (r85A) or as a set of overlapping 15mer peptides (p85A). For the first time we show that i.n. immunization with overlapping 85A synthetic peptides as well as Ad85A or r85A can provide protection against Mtb challenge. For all forms of the antigen, i.n. induces greater protection against Mtb challenge than parenteral immunization. Ad85A induces a predominantly CD8T cell response against the 85A(70-78) epitope, r85A a CD4 response to 85A(99-118) and p85A a balanced CD4/CD8 response to the CD4 85A(99-118 )and CD8 85A(145-152) epitopes. Immune responses to CD4 85A(99-118) and CD8 85A(70-78) but not CD8 85A(145-152) are protective. Although Ad85A induces a strong response to the protective CD8 85A(70-78) epitope, we could not induce any response to this epitope by peptide immunization. These results show that although peptide immunization can induce protective immunity to Mtb challenge, it can also induce a response to a non-protective epitope in antigen 85A, indicating that the specificity of an immune response may be more important for protection against Mtb than its magnitude. These findings have important implications for the application of such vaccines in humans.

  10. Dichotomy of protective cellular immune responses to human visceral leishmaniasis.

    PubMed

    Khalil, E A G; Ayed, N B; Musa, A M; Ibrahim, M E; Mukhtar, M M; Zijlstra, E E; Elhassan, I M; Smith, P G; Kieny, P M; Ghalib, H W; Zicker, F; Modabber, F; Elhassan, A M

    2005-05-01

    Healing/protective responses in human visceral leishmaniasis (VL) are associated with stimulation/production of Th1 cytokines, such as interferon IFN-gamma, and conversion in the leishmanin skin test (LST). Such responses were studied for 90 days in 44 adult healthy volunteers from VL non-endemic areas, with no past history of VL/cutaneous leishmaniasis (CL) and LST non-reactivity following injection with one of four doses of Alum-precipitated autoclaved Leishmania major (Alum/ALM) +/- bacille Calmette-Guerin (BCG), a VL candidate vaccine. The vaccine was well tolerated with minimal localized side-effects and without an increase in antileishmanial antibodies or interleukin (IL)-5. Five volunteers (5/44; 11.4%) had significant IFN-gamma production by peripheral blood mononuclear cells (PBMCs) in response to Leishmania antigens in their prevaccination samples (P = 0.001) but were LST non-reactive. On day 45, more than half the volunteers (26/44; 59.0%) had significantly high LST indurations (mean 9.2 +/- 2.7 mm) and high IFN-gamma levels (mean 1008 +/- 395; median 1247 pg/ml). Five volunteers had significant L. donovani antigen-induced IFN-gamma production (mean 873 +/- 290; median 902; P = 0.001), but were non-reactive in LST. An additional five volunteers (5/44; 11.4%) had low IFN-gamma levels (mean 110 +/- 124 pg/ml; median 80) and were non-reactive in LST (induration = 00 mm). The remaining eight volunteers had low IFN-gamma levels, but significant LST induration (mean 10 +/- 2.9 mm; median 11). By day 90 the majority of volunteers (27/44; 61.4%) had significant LST induration (mean 10.8 +/- 9.9 mm; P < 0.001), but low levels of L. donovani antigen-induced IFN-gamma (mean 66.0 +/- 62 pg/ml; P > 0.05). Eleven volunteers (11/44; 25%) had significantly high levels of IFN-gamma and LST induration, while five volunteers had low levels of IFN-gamma (<100 pg/ml) and no LST reactivity (00 mm). One volunteer was lost to follow-up. In conclusion, it is hypothesized that

  11. Protective Immunity and Reduced Renal Colonization Induced by Vaccines Containing Recombinant Leptospira interrogans Outer Membrane Proteins and Flagellin Adjuvant

    PubMed Central

    Monaris, D.; Sbrogio-Almeida, M. E.; Dib, C. C.; Canhamero, T. A.; Souza, G. O.; Vasconcellos, S. A.; Ferreira, L. C. S.

    2015-01-01

    Leptospirosis is a global zoonotic disease caused by different Leptospira species, such as Leptospira interrogans, that colonize the renal tubules of wild and domestic animals. Thus far, attempts to develop effective leptospirosis vaccines, both for humans and animals, have failed to induce immune responses capable of conferring protection and simultaneously preventing renal colonization. In this study, we evaluated the protective immunity induced by subunit vaccines containing seven different recombinant Leptospira interrogans outer membrane proteins, including the carboxy-terminal portion of the immunoglobulinlike protein A (LigAC) and six novel antigens, combined with aluminum hydroxide (alum) or Salmonella flagellin (FliC) as adjuvants. Hamsters vaccinated with the different formulations elicited high antigen-specific antibody titers. Immunization with LigAC, either with alum or flagellin, conferred protective immunity but did not prevent renal colonization. Similarly, animals immunized with LigAC or LigAC coadministered with six leptospiral proteins with alum adjuvant conferred protection but did not reduce renal colonization. In contrast, immunizing animals with the pool of seven antigens in combination with flagellin conferred protection and significantly reduced renal colonization by the pathogen. The present study emphasizes the relevance of antigen composition and added adjuvant in the efficacy of antileptospirosis subunit vaccines and shows the complex relationship between immune responses and renal colonization by the pathogen. PMID:26108285

  12. Protective Immunity and Reduced Renal Colonization Induced by Vaccines Containing Recombinant Leptospira interrogans Outer Membrane Proteins and Flagellin Adjuvant.

    PubMed

    Monaris, D; Sbrogio-Almeida, M E; Dib, C C; Canhamero, T A; Souza, G O; Vasconcellos, S A; Ferreira, L C S; Abreu, P A E

    2015-08-01

    Leptospirosis is a global zoonotic disease caused by different Leptospira species, such as Leptospira interrogans, that colonize the renal tubules of wild and domestic animals. Thus far, attempts to develop effective leptospirosis vaccines, both for humans and animals, have failed to induce immune responses capable of conferring protection and simultaneously preventing renal colonization. In this study, we evaluated the protective immunity induced by subunit vaccines containing seven different recombinant Leptospira interrogans outer membrane proteins, including the carboxy-terminal portion of the immunoglobulinlike protein A (LigA(C)) and six novel antigens, combined with aluminum hydroxide (alum) or Salmonella flagellin (FliC) as adjuvants. Hamsters vaccinated with the different formulations elicited high antigen-specific antibody titers. Immunization with LigA(C), either with alum or flagellin, conferred protective immunity but did not prevent renal colonization. Similarly, animals immunized with LigA(C) or LigA(C) coadministered with six leptospiral proteins with alum adjuvant conferred protection but did not reduce renal colonization. In contrast, immunizing animals with the pool of seven antigens in combination with flagellin conferred protection and significantly reduced renal colonization by the pathogen. The present study emphasizes the relevance of antigen composition and added adjuvant in the efficacy of antileptospirosis subunit vaccines and shows the complex relationship between immune responses and renal colonization by the pathogen.

  13. Cellular prion protein protects from inflammatory and neuropathic pain.

    PubMed

    Gadotti, Vinicius M; Zamponi, Gerald W

    2011-08-16

    Cellular prion protein (PrPC) inhibits N-Methyl-D-Aspartate (NMDA) receptors. Since NMDA receptors play an important role in the transmission of pain signals in the dorsal horn of spinal cord, we thus wanted to determine if PrPC null mice show a reduced threshold for various pain behaviours.We compared nociceptive thresholds between wild type and PrPC null mice in models of inflammatory and neuropathic pain, in the presence and the absence of a NMDA receptor antagonist. 2-3 months old male PrPC null mice exhibited an MK-801 sensitive decrease in the paw withdrawal threshold in response both mechanical and thermal stimuli. PrPC null mice also exhibited significantly longer licking/biting time during both the first and second phases of formalin-induced inflammation of the paw, which was again prevented by treatment of the mice with MK-801, and responded more strongly to glutamate injection into the paw. Compared to wild type animals, PrPC null mice also exhibited a significantly greater nociceptive response (licking/biting) after intrathecal injection of NMDA. Sciatic nerve ligation resulted in MK-801 sensitive neuropathic pain in wild-type mice, but did not further augment the basal increase in pain behaviour observed in the null mice, suggesting that mice lacking PrPC may already be in a state of tonic central sensitization. Altogether, our data indicate that PrPC exerts a critical role in modulating nociceptive transmission at the spinal cord level, and fit with the concept of NMDA receptor hyperfunction in the absence of PrPC.

  14. Protective Capacity of the Human Anamnestic Antibody Response during Acute Dengue Virus Infection

    PubMed Central

    Xu, Meihui; Züst, Roland; Toh, Ying Xiu; Pfaff, Jennifer M.; Kahle, Kristen M.; Davidson, Edgar; Doranz, Benjamin J.; Velumani, Sumathy; Tukijan, Farhana; Wang, Cheng-I

    2016-01-01

    ABSTRACT Half of the world's population is exposed to the risk of dengue virus infection. Although a vaccine for dengue virus is now available in a few countries, its reported overall efficacy of about 60% is not ideal. Protective immune correlates following natural dengue virus infection remain undefined, which makes it difficult to predict the efficacy of new vaccines. In this study, we address the protective capacity of dengue virus-specific antibodies that are produced by plasmablasts a few days after natural secondary infection. Among a panel of 18 dengue virus-reactive human monoclonal antibodies, four groups of antibodies were identified based on their binding properties. While antibodies targeting the fusion loop of the glycoprotein of dengue virus dominated the antibody response, two smaller groups of antibodies bound to previously undescribed epitopes in domain II of the E protein. The latter, largely serotype-cross-reactive antibodies, demonstrated increased stability of binding at pH 5. These antibodies possessed weak to moderate neutralization capacity in vitro but were the most efficacious in promoting the survival of infected mice. Our data suggest that the cross-reactive anamnestic antibody response has a protective capacity despite moderate neutralization in vitro and a moderate decrease of viremia in vivo. IMPORTANCE Antibodies can protect from symptomatic dengue virus infection. However, it is not easy to assess which classes of antibodies provide protection because in vitro assays are not always predictive of in vivo protection. During a repeat infection, dengue virus-specific immune memory cells are reactivated and large amounts of antibodies are produced. By studying antibodies cloned from patients with heterologous secondary infection, we tested the protective value of the serotype-cross-reactive “recall” or “anamnestic” response. We found that results from in vitro neutralization assays did not always correlate with the ability of

  15. Iron regulatory protein-1 protects against mitoferrin-1-deficient porphyria.

    PubMed

    Chung, Jacky; Anderson, Sheila A; Gwynn, Babette; Deck, Kathryn M; Chen, Michael J; Langer, Nathaniel B; Shaw, George C; Huston, Nicholas C; Boyer, Leah F; Datta, Sumon; Paradkar, Prasad N; Li, Liangtao; Wei, Zong; Lambert, Amy J; Sahr, Kenneth; Wittig, Johannes G; Chen, Wen; Lu, Wange; Galy, Bruno; Schlaeger, Thorsten M; Hentze, Matthias W; Ward, Diane M; Kaplan, Jerry; Eisenstein, Richard S; Peters, Luanne L; Paw, Barry H

    2014-03-14

    Mitochondrial iron is essential for the biosynthesis of heme and iron-sulfur ([Fe-S]) clusters in mammalian cells. In developing erythrocytes, iron is imported into the mitochondria by MFRN1 (mitoferrin-1, SLC25A37). Although loss of MFRN1 in zebrafish and mice leads to profound anemia, mutant animals showed no overt signs of porphyria, suggesting that mitochondrial iron deficiency does not result in an accumulation of protoporphyrins. Here, we developed a gene trap model to provide in vitro and in vivo evidence that iron regulatory protein-1 (IRP1) inhibits protoporphyrin accumulation. Mfrn1(+/gt);Irp1(-/-) erythroid cells exhibit a significant increase in protoporphyrin levels. IRP1 attenuates protoporphyrin biosynthesis by binding to the 5'-iron response element (IRE) of alas2 mRNA, inhibiting its translation. Ectopic expression of alas2 harboring a mutant IRE, preventing IRP1 binding, in Mfrn1(gt/gt) cells mimics Irp1 deficiency. Together, our data support a model whereby impaired mitochondrial [Fe-S] cluster biogenesis in Mfrn1(gt/gt) cells results in elevated IRP1 RNA-binding that attenuates ALAS2 mRNA translation and protoporphyrin accumulation.

  16. Conformational Epitopes Recognized by Protective Anti-Neisserial Surface Protein A Antibodies

    PubMed Central

    Hou, Victor C.; Moe, Gregory R.; Raad, Zyde; Wuorimaa, Tomi; Granoff, Dan M.

    2003-01-01

    NspA is a conserved membrane protein that elicits protective antibody responses in mice against Neisseria meningitidis. A recent crystallographic study showed that NspA adopts an eight-stranded β-barrel structure when reconstituted in detergent. In order to define the segments of NspA-containing epitopes recognized by protective murine anti-NspA antibodies, we studied the binding of two bactericidal and protective anti-NspA monoclonal antibodies (MAbs), AL12 and 14C7. Neither MAb binds to overlapping synthetic peptides (10-mers, 12-mers, and cyclic 12-mers) corresponding to the entire mature sequence of NspA, or to denatured recombinant NspA (rNspA), although binding to the protein can be restored by refolding in liposomes. Based on the ability of the two MAbs to bind to Escherichia coli microvesicles prepared from a set of rNspA variants created by site-specific mutagenesis, the most important contacts between the MAbs and NspA appear to be located within the LGG segment of loop 3. The conformation of loop 2 also appears to be an important determinant, as particular combinations of residues in this segment resulted in loss of antibody binding. Thus, the two anti-NspA MAbs recognize discontinuous conformational epitopes that result from the close proximity of loops 2 and 3 in the three-dimensional structure of NspA. The data suggest that optimally immunogenic vaccines using rNspA will require formulations that permit proper folding of the protein. PMID:14638771

  17. Maresin 1 Mitigates Inflammatory Response and Protects Mice from Sepsis

    PubMed Central

    Li, Ruidong; Wang, Yaxin; Ma, Zhijun; Ma, Muyuan; Wang, Di; Xie, Gengchen; Yin, Yuping

    2016-01-01

    Sepsis, frequently caused by infection of bacteria, is considered as an uncontrollable systematic inflammation response syndrome (SIRS). Maresin 1 (Mar1) is a new proresolving mediator with potent anti-inflammatory effect in several animal models. However, its effect in sepsis is still not investigated. To address this question, we developed sepsis model in BALB/c mice by cecal ligation and puncture (CLP) with or without Mar1 treatment. Our data showed that Mar1 markedly improved survival rate and decreased the levels of proinflammatory cytokines in CLP mice such as interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β). Furthermore, Mar1 reduced serum level of lipopolysaccharide (LPS) and enhanced the bacteria clearance in mice sepsis model. Moreover, Mar1 attenuated lung injury and decreased level of alanine transaminase (ALT), aspartate transaminase (AST), creatinine (Cre), and blood urea nitrogen (BUN) in serum in mice after CLP surgery. Treatment with Mar1 inhibited activation of nuclear factor kappa B (NF-κb) pathway. In conclusion, Mar1 exhibited protective effect in sepsis by reducing LPS, bacteria burden in serum, inhibiting inflammation response, and improving vital organ function. The possible mechanism is partly involved in inhibition of NF-κb activation. PMID:28042205

  18. Recent findings about the Yersinia enterocolitica phage shock protein response.

    PubMed

    Yamaguchi, Saori; Darwin, Andrew J

    2012-02-01

    The phage shock protein (Psp) system is a conserved extracytoplasmic stress response in bacteria that is essential for virulence of the human pathogen Yersinia enterocolitica. This article summarizes some recent findings about Y. enterocolitica Psp system function. Increased psp gene expression requires the transcription factor PspF, but under non-inducing conditions PspF is inhibited by an interaction with another protein, PspA, in the cytoplasm. A Psp-inducing stimulus causes PspA to relocate to the cytoplasmic membrane, freeing PspF to induce psp gene expression. This PspA relocation requires the integral cytoplasmic membrane proteins, PspB and PspC, which might sense an inducing trigger and sequester PspA by direct interaction. The subsequent induction of psp gene expression increases the PspA concentration, which also allows it to contact the membrane directly, perhaps for its physiological function. Mutational analysis of the PspB and PspC proteins has revealed that they both positively and negatively regulate psp gene expression and has also identified PspC domains associated with each function. We also compare the contrasting physiological roles of the Psp system in the virulence of Y. enterocolitica and Salmonella enterica sv. Typhimurium (S. Typhimurium). In S. Typhimurium, PspA maintains the proton motive force, which provides the energy needed to drive ion importers required for survival within macrophages. In contrast, in the extracellular pathogen Y. enterocolitica, PspB and PspC, but not PspA, are the Psp components needed for virulence. PspBC protect Y. enterocolitica from damage caused by the secretin component of its type 3 secretion system, an essential virulence factor.

  19. Protective responses induced by herbicide safeners in wheat.

    PubMed

    Taylor, Victoria L; Cummins, Ian; Brazier-Hicks, Melissa; Edwards, Robert

    2013-04-01

    Safeners are agrochemicals which enhance tolerance to herbicides in cereals including wheat (Triticum aestivum L.) by elevating the expression of xenobiotic detoxifying enzymes, such as glutathione transferases (GSTs). When wheat plants were spray-treated with three safener chemistries, namely cloquintocet mexyl, mefenpyr diethyl and fenchlorazole ethyl, an apparently identical subset of GSTs derived from the tau, phi and lambda classes accumulated in the foliage. Treatment with the closely related mefenpyr diethyl and fenchlorazole ethyl enhanced seedling shoot growth, but this effect was not determined with the chemically unrelated cloquintocet mexyl. Focussing on cloquintocet mexyl, treatments were found to only give a transient induction of GSTs, with the period of elevation being dose dependent. Examining the role of safener metabolism in controlling these responses, it was determined that cloquintocet mexyl was rapidly hydrolysed to the respective carboxylic acid. Studies with cloquintocet showed that the acid was equally effective at inducing GSTs as the ester and appeared to be the active safener. Studies on the tissue induction of GSTs showed that whilst phi and tau class enzymes were induced in all tissues, the induction of the lambda enzymes was restricted to the meristems. To test the potential protective effects of cloquintocet mexyl in wheat on chemicals other than herbicides, seeds were pre-soaked in safeners prior to sowing on soil containing oil and a range of heavy metals. Whilst untreated seeds were unable to germinate on the contaminated soil, safener treatments resulted in seedlings briefly growing before succumbing to the pollutants. Our results show that safeners exert a range of protective and growth promoting activities in wheat that extend beyond enhancing tolerance to herbicides.

  20. Protective responses induced by herbicide safeners in wheat

    PubMed Central

    Taylor, Victoria L.; Cummins, Ian; Brazier-Hicks, Melissa; Edwards, Robert

    2013-01-01

    Safeners are agrochemicals which enhance tolerance to herbicides in cereals including wheat (Triticum aestivum L.) by elevating the expression of xenobiotic detoxifying enzymes, such as glutathione transferases (GSTs). When wheat plants were spray-treated with three safener chemistries, namely cloquintocet mexyl, mefenpyr diethyl and fenchlorazole ethyl, an apparently identical subset of GSTs derived from the tau, phi and lambda classes accumulated in the foliage. Treatment with the closely related mefenpyr diethyl and fenchlorazole ethyl enhanced seedling shoot growth, but this effect was not determined with the chemically unrelated cloquintocet mexyl. Focussing on cloquintocet mexyl, treatments were found to only give a transient induction of GSTs, with the period of elevation being dose dependent. Examining the role of safener metabolism in controlling these responses, it was determined that cloquintocet mexyl was rapidly hydrolysed to the respective carboxylic acid. Studies with cloquintocet showed that the acid was equally effective at inducing GSTs as the ester and appeared to be the active safener. Studies on the tissue induction of GSTs showed that whilst phi and tau class enzymes were induced in all tissues, the induction of the lambda enzymes was restricted to the meristems. To test the potential protective effects of cloquintocet mexyl in wheat on chemicals other than herbicides, seeds were pre-soaked in safeners prior to sowing on soil containing oil and a range of heavy metals. Whilst untreated seeds were unable to germinate on the contaminated soil, safener treatments resulted in seedlings briefly growing before succumbing to the pollutants. Our results show that safeners exert a range of protective and growth promoting activities in wheat that extend beyond enhancing tolerance to herbicides. PMID:23564986

  1. S-Bacillithiolation Protects Conserved and Essential Proteins Against Hypochlorite Stress in Firmicutes Bacteria

    PubMed Central

    Chi, Bui Khanh; Roberts, Alexandra A.; Huyen, Tran Thi Thanh; Bäsell, Katrin; Becher, Dörte; Albrecht, Dirk; Hamilton, Chris J.

    2013-01-01

    Abstract Aims: Protein S-bacillithiolations are mixed disulfides between protein thiols and the bacillithiol (BSH) redox buffer that occur in response to NaOCl in Bacillus subtilis. We used BSH-specific immunoblots, shotgun liquid chromatography (LC)–tandem mass spectrometry (MS/MS) analysis and redox proteomics to characterize the S-bacillithiolomes of B. subtilis, B. megaterium, B. pumilus, B. amyloliquefaciens, and Staphylococcus carnosus and also measured the BSH/oxidized bacillithiol disulfide (BSSB) redox ratio after NaOCl stress. Results: In total, 54 proteins with characteristic S-bacillithiolation (SSB) sites were identified, including 29 unique proteins and eight proteins conserved in two or more of these bacteria. The methionine synthase MetE is the most abundant S-bacillithiolated protein in Bacillus species after NaOCl exposure. Further, S-bacillithiolated proteins include the translation elongation factor EF-Tu and aminoacyl-tRNA synthetases (ThrS), the DnaK and GrpE chaperones, the two-Cys peroxiredoxin YkuU, the ferredoxin–NADP+ oxidoreductase YumC, the inorganic pyrophosphatase PpaC, the inosine-5′-monophosphate dehydrogenase GuaB, proteins involved in thiamine biosynthesis (ThiG and ThiM), queuosine biosynthesis (QueF), biosynthesis of aromatic amino acids (AroA and AroE), serine (SerA), branched-chain amino acids (YwaA), and homocysteine (LuxS and MetI). The thioredoxin-like proteins, YphP and YtxJ, are S-bacillithiolated at their active sites, suggesting a function in the de-bacillithiolation process. S-bacillithiolation is accompanied by a two-fold increase in the BSSB level and a decrease in the BSH/BSSB redox ratio in B. subtilis. Innovation: Many essential and conserved proteins, including the dominant MetE, were identified in the S-bacillithiolome of different Bacillus species and S. carnosus using shotgun-LC-MS/MS analyses. Conclusion: S-bacillithiolation is a widespread redox control mechanism among Firmicutes bacteria that protects

  2. SALIVARY ANTIMICROBIAL PROTEIN RESPONSE TO PROLONGED RUNNING

    PubMed Central

    Kuennen, M.; Gourley, C.; Schneider, S.; Dokladny, K.; Moseley, P.

    2013-01-01

    Introduction Prolonged exercise may compromise immunity through a reduction of salivary antimicrobial proteins (AMPs). Salivary IgA (IgA) has been extensively studied, but little is known about the effect of acute, prolonged exercise on AMPs including lysozyme (Lys) and lactoferrin (Lac). Objective To determine the effect of a 50-km trail race on salivary cortisol (Cort), IgA, Lys, and Lac. Methods 14 subjects: (6 females, 8 males) completed a 50km ultramarathon. Saliva was collected pre, immediately after (post) and 1.5 hrs post race (+1.5). Results Lac concentration was higher at +1.5 hrs post race compared to post exercise (p < 0.05). Lys was unaffected by the race (p > 0.05). IgA concentration, secretion rate, and IgA/Osm were lower +1.5 hrs post compared to pre race (p < 0.05). Cort concentration was higher at post compared to +1.5 (p < 0.05), but was unaltered from pre race levels. Subjects finished in 7.81±1.2 hrs. Saliva flow rate did not differ between time points. Saliva Osm increased at post (p < 0.05) compared to pre race. Conclusions The intensity could have been too low to alter Lys and Lac secretion rates and thus, may not be as sensitive as IgA to changes in response to prolonged running. Results expand our understanding of the mucosal immune system and may have implications for predicting illness after prolonged running. PMID:24744458

  3. Stress proteins and glial cell functions during chronic aluminium exposures: protective role of curcumin.

    PubMed

    Sood, Pooja Khanna; Nahar, Uma; Nehru, Bimla

    2012-03-01

    Involved in the ongoing debate is the speculation that aluminium is somehow toxic for neurons. Glial cells cope up to protect neurons from this toxic insult by maintaining the glutathione homeostasis. Of late newer and newer roles of glial cells have been depicted. The present work looks into the other regulatory mechanisms that show the glial cells response to pro-oxidant effects of aluminium exposure. In the present investigation we have evaluated the inflammatory responses of the glial cells as well as HSP70-induction during aluminium exposure. Further, the protective role of curcumin is also evaluated. Aluminium was administered by oral gavage at a dose level of 100 mg/kg b.wt/day for a period of 8 weeks. Curcumin was administered i.p. at a dose of 50 mg/kg b.wt./day on alternate days. Enhanced gene and protein expression of HSP70 in the glial fractions of the aluminium exposed animals as compared to the corresponding neuronal population. Aluminium exposure resulted in a significant increase in the NF-κB and TNF-α expression suggesting inflammatory responses. In the conjunctive treatment group of aluminium and curcumin exposure marked reduction in the gene and protein expression of NF-κB and TNF-α was observed. This was further reflected in histopathological studies showing no evidence of inflammation in conjunctive group as compared to aluminium treatment. From the present study, it can be concluded that curcumin has a potential anti-inflammatory action and can be exploited in other toxicological conditions also.

  4. The Immune Response to Sand Fly Salivary Proteins and Its Influence on Leishmania Immunity

    PubMed Central

    Gomes, Regis; Oliveira, Fabiano

    2012-01-01

    Leishmaniasis is a vector-borne disease transmitted by bites of phlebotomine sand flies. During Leishmania transmission, sand fly saliva is co-inoculated with parasites into the skin of the mammalian host. Sand fly saliva consists of roughly thirty different salivary proteins, many with known roles linked to blood feeding facilitation. Apart from the anti-hemostatic capacity of saliva, several sand fly salivary proteins have been shown to be immunogenic. Immunization with a single salivary protein or exposure to uninfected bites was shown to result in a protective immune response against leishmaniasis. Antibodies to saliva were not required for this protection. A strong body of evidence points to the role for saliva-specific T cells producing IFN-γ in the form of a delayed-type hypersensitivity reaction at the bite site as the main protective response. Herein, we review the immunity to sand fly salivary proteins in the context of its vector–parasite–host combinations and their vaccine potential, as well as some recent advances to shed light on the mechanism of how an immune response to sand fly saliva protects against leishmaniasis. PMID:22593758

  5. Efficacy of heterologous and homologous heat shock protein 70s as protective agents to Artemia franciscana challenged with Vibrio campbellii.

    PubMed

    Baruah, Kartik; Ranjan, Jayant; Sorgeloos, Patrick; Bossier, Peter

    2010-11-01

    The Hsp70 class of heat shock proteins (Hsps) has been implicated at multiple points in the immune response of both vertebrates and invertebrates. This class of chaperones is highly conserved in both sequence and structure, from prokaryotes to higher eukaryotes. In view of their high degree of homology, it was assumed that these Hsp70 proteins derived either from the prokaryotes or eukaryotes would have similar functions, especially in relation to their protective ability in a challenge assay. To verify this, we compared two evolutionary diverse Hsp70s, Artemia Hsp70 and Escherichia coli Hsp70 equivalent DnaK (each overproduced in E.coli), for their ability to protect Artemia against Vibrio challenge. Results showed that Artemia fed with E. coli producing Artemia Hsp70 or DnaK proteins, as assessed by immune-probing in western blots, survived better in a Vibrio challenge assay. The observed effects could be due to enhancement of the Artemia immune system as phenoloxidase activity was found to be increased by these proteins. These two Hsp70 proteins exhibit a high degree of homology, particularly in the peptide-binding domain (the putative innate immunity-activating portion) with 59.6% identity, indicating that the observed protective capacity of homologous or heterologous Hsp70 proteins might reside within this peptide-binding domain.

  6. Protein A Suppresses Immune Responses during Staphylococcus aureus Bloodstream Infection in Guinea Pigs

    PubMed Central

    Kim, Hwan Keun; Falugi, Fabiana; Thomer, Lena; Missiakas, Dominique M.

    2015-01-01

    ABSTRACT   Staphylococcus aureus infection is not associated with the development of protective immunity, and disease relapses occur frequently. We hypothesize that protein A, a factor that binds immunoglobulin Fcγ and cross-links VH3 clan B cell receptors (IgM), is the staphylococcal determinant for host immune suppression. To test this, vertebrate IgM was examined for protein A cross-linking. High VH3 binding activity occurred with human and guinea immunoglobulin, whereas mouse and rabbit immunoglobulins displayed little and no binding, respectively. Establishing a guinea pig model of S. aureus bloodstream infection, we show that protein A functions as a virulence determinant and suppresses host B cell responses. Immunization with SpAKKAA, which cannot bind immunoglobulin, elicits neutralizing antibodies that enable guinea pigs to develop protective immunity. Importance  Staphylococcus aureus is the leading cause of soft tissue and bloodstream infections; however, a vaccine with clinical efficacy is not available. Using mice to model staphylococcal infection, earlier work identified protective antigens; however, corresponding human clinical trials did not reach their endpoints. We show that B cell receptor (IgM) cross-linking by protein A is an important immune evasion strategy of S. aureus that can be monitored in a guinea pig model of bloodstream infection. Further, immunization with nontoxigenic protein A enables infected guinea pigs to elicit antibody responses that are protective against S. aureus. Thus, the guinea pig model may support preclinical development of staphylococcal vaccines. PMID:25564466

  7. Balancing Immune Protection and Immune Pathology by CD8+ T-Cell Responses to Influenza Infection

    PubMed Central

    Duan, Susu; Thomas, Paul G.

    2016-01-01

    Influenza A virus (IAV) is a significant human pathogen causing annual epidemics and periodic pandemics. CD8+ cytotoxic T lymphocyte (CTL)-mediated immunity contributes to the clearance of virus-infected cells, and CTL immunity targeting the conserved internal proteins of IAVs is a key protection mechanism when neutralizing antibodies are absent during heterosubtypic IAV infection. However, CTL infiltration into the airways, its cytotoxicity, and the effects of produced proinflammatory cytokines can cause severe lung tissue injury, thereby contributing to immunopathology. Studies have discovered complicated and exquisite stimulatory and inhibitory mechanisms that regulate CTL magnitude and effector activities during IAV infection. Here, we review the state of knowledge on the roles of IAV-specific CTLs in immune protection and immunopathology during IAV infection in animal models, highlighting the key findings of various requirements and constraints regulating the balance of immune protection and pathology involved in CTL immunity. We also discuss the evidence of cross-reactive CTL immunity as a positive correlate of cross-subtype protection during secondary IAV infection in both animal and human studies. We argue that the effects of CTL immunity on protection and immunopathology depend on multiple layers of host and viral factors, including complex host mechanisms to regulate CTL magnitude and effector activity, the pathogenic nature of the IAV, the innate response milieu, and the host historical immune context of influenza infection. Future efforts are needed to further understand these key host and viral factors, especially to differentiate those that constrain optimally effective CTL antiviral immunity from those necessary to restrain CTL-mediated non-specific immunopathology in the various contexts of IAV infection, in order to develop better vaccination and therapeutic strategies for modifying protective CTL immunity. PMID:26904022

  8. Spliced X-box Binding Protein 1 Couples the Unfolded Protein Response to Hexosamine Biosynthetic Pathway

    PubMed Central

    Wang, Zhao V.; Deng, Yingfeng; Gao, Ningguo; Pedrozo, Zully; Li, Dan L.; Morales, Cyndi R.; Criollo, Alfredo; Luo, Xiang; Tan, Wei; Jiang, Nan; Lehrman, Mark A.; Rothermel, Beverly A.; Lee, Ann-Hwee; Lavandero, Sergio; Mammen, Pradeep P.A.; Ferdous, Anwarul; Gillette, Thomas G.; Scherer, Philipp E.; Hill, Joseph A.

    2014-01-01

    SUMMARY The hexosamine biosynthetic pathway (HBP) generates UDP-GlcNAc (uridine diphosphate N-acetylglucosamine) for glycan synthesis and O-linked GlcNAc (O-GlcNAc) protein modifications. Despite the established role of the HBP in metabolism and multiple diseases, regulation of the HBP remains largely undefined. Here, we show that spliced X-box binding protein 1 (Xbp1s), the most conserved signal transducer of the unfolded protein response (UPR), is a direct transcriptional activator of the HBP. We demonstrate that the UPR triggers HBP activation via Xbp1s-dependent transcription of genes coding for key, rate-limiting enzymes. We further establish that this previously unrecognized UPR-HBP axis is triggered in a variety of stress conditions. Finally, we demonstrate a physiologic role for the UPR-HBP axis, by showing that acute stimulation of Xbp1s in heart by ischemia/reperfusion confers robust cardioprotection in part through induction of the HBP. Collectively, these studies reveal that Xbp1s couples the UPR to the HBP to protect cells under stress. PMID:24630721

  9. Spliced X-box binding protein 1 couples the unfolded protein response to hexosamine biosynthetic pathway.

    PubMed

    Wang, Zhao V; Deng, Yingfeng; Gao, Ningguo; Pedrozo, Zully; Li, Dan L; Morales, Cyndi R; Criollo, Alfredo; Luo, Xiang; Tan, Wei; Jiang, Nan; Lehrman, Mark A; Rothermel, Beverly A; Lee, Ann-Hwee; Lavandero, Sergio; Mammen, Pradeep P A; Ferdous, Anwarul; Gillette, Thomas G; Scherer, Philipp E; Hill, Joseph A

    2014-03-13

    The hexosamine biosynthetic pathway (HBP) generates uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) for glycan synthesis and O-linked GlcNAc (O-GlcNAc) protein modifications. Despite the established role of the HBP in metabolism and multiple diseases, regulation of the HBP remains largely undefined. Here, we show that spliced X-box binding protein 1 (Xbp1s), the most conserved signal transducer of the unfolded protein response (UPR), is a direct transcriptional activator of the HBP. We demonstrate that the UPR triggers HBP activation via Xbp1s-dependent transcription of genes coding for key, rate-limiting enzymes. We further establish that this previously unrecognized UPR-HBP axis is triggered in a variety of stress conditions. Finally, we demonstrate a physiologic role for the UPR-HBP axis by showing that acute stimulation of Xbp1s in heart by ischemia/reperfusion confers robust cardioprotection in part through induction of the HBP. Collectively, these studies reveal that Xbp1s couples the UPR to the HBP to protect cells under stress.

  10. Potential protective immunogenicity of tetanus toxoid, diphtheria toxoid and Cross Reacting Material 197 (CRM197) when used as carrier proteins in glycoconjugates.

    PubMed

    Bröker, Michael

    2016-03-03

    When tetanus toxoid (TT), diphtheria toxoid (DT) or Cross Reacting Material 197 (CRM197), a non-toxic diphtheria toxin mutant protein, are used as carrier proteins in glycoconjugate vaccines, these carriers induce a protein specific antibody response as measured by in vitro assays. Here, it was evaluated whether or not glycoconjugates based on TT, DT or CRM197 can induce a protective immune response as measured by potency tests according to the European Pharmacopoeia. It could be shown, that the conjugate carriers TT and DT can induce a protective immune response against a lethal challenge by toxins in animals, while glycoconjugates based on CRM197 failed to induce a protective immune response. Opportunities for new applications of glycoconjugates are discussed.

  11. Protective activity of the CnaBE3 domain conserved among Staphylococcus aureus Sdr proteins.

    PubMed

    Becherelli, Marco; Prachi, Prachi; Viciani, Elisa; Biagini, Massimiliano; Fiaschi, Luigi; Chiarot, Emiliano; Nosari, Sarah; Brettoni, Cecilia; Marchi, Sara; Biancucci, Marco; Fontana, Maria Rita; Montagnani, Francesca; Bagnoli, Fabio; Barocchi, Michèle A; Manetti, Andrea G O

    2013-01-01

    Staphylococcus aureus is an opportunistic pathogen, commensal of the human skin and nares, but also responsible for invasive nosocomial as well as community acquired infections. Staphylococcus aureus adheres to the host tissues by means of surface adhesins, such as SdrC, SdrD, and SdrE proteins. The Sdr family of proteins together with a functional A domain, contain respectively two, three or five repeated sequences called B motifs which comprise the CnaB domains. SdrD and SdrE proteins were reported to be protective in animal models against invasive diseases or lethal challenge with human clinical S. aureus isolates. In this study we identified a 126 amino acid sequence containing a CnaB domain, conserved among the three Sdr proteins. The three fragments defined here as CnaBC2, D5 and E3 domains even though belonging to phylogenetically distinct strains, displayed high sequence similarity. Based on the sequence conservation data, we selected the CnaBE3 domain for further analysis and characterization. Polyclonal antibodies raised against the recombinant CnaBE3 domain recognized SdrE, SdrC and SdrD proteins of different S. aureus lineages. Moreover, we demonstrated that the CnaBE3 domain was expressed in vivo during S. aureus infections, and that immunization of this domain alone significantly reduces the bacterial load in mice challenged with S. aureus. Furthermore, we show that the reduction of bacteria by CnaBE3 vaccination is due to functional antibodies. Finally, we demonstrated that the region of the SdrE protein containing the CnaBE3 domain was resistant to trypsin digestion, a characteristic often associated with the presence of an isopeptide bond.

  12. Immunization with Pneumococcal Surface Protein K of Nonencapsulated Streptococcus pneumoniae Provides Protection in a Mouse Model of Colonization.

    PubMed

    Keller, Lance E; Luo, Xiao; Thornton, Justin A; Seo, Keun-Seok; Moon, Bo Youn; Robinson, D Ashley; McDaniel, Larry S

    2015-11-01

    Current vaccinations are effective against encapsulated strains of Streptococcus pneumoniae, but they do not protect against nonencapsulated Streptococcus pneumoniae (NESp), which is increasing in colonization and incidence of pneumococcal disease. Vaccination with pneumococcal proteins has been assessed for its ability to protect against pneumococcal disease, but several of these proteins are not expressed by NESp. Pneumococcal surface protein K (PspK), an NESp virulence factor, has not been assessed for immunogenic potential or host modulatory effects. Mammalian cytokine expression was determined in an in vivo mouse model and in an in vitro cell culture system. Systemic and mucosal mouse immunization studies were performed to determine the immunogenic potential of PspK. Murine serum and saliva were collected to quantitate specific antibody isotype responses and the ability of antibody and various proteins to inhibit epithelial cell adhesion. Host cytokine response was not reduced by PspK. NESp was able to colonize the mouse nasopharynx as effectively as encapsulated pneumococci. Systemic and mucosal immunization provided protection from colonization by PspK-positive (PspK(+)) NESp. Anti-PspK antibodies were recovered from immunized mice and significantly reduced the ability of NESp to adhere to human epithelial cells. A protein-based pneumococcal vaccine is needed to provide broad protection against encapsulated and nonencapsulated pneumococci in an era of increasing antibiotic resistance and vaccine escape mutants. We demonstrate that PspK may serve as an NESp target for next-generation pneumococcal vaccines. Immunization with PspK protected against pneumococcal colonization, which is requisite for pneumococcal disease.

  13. Recombinant Phage Elicits Protective Immune Response against Systemic S. globosa Infection in Mouse Model

    PubMed Central

    Chen, Feng; Jiang, Rihua; Wang, Yicun; Zhu, Mingji; Zhang, Xu; Dong, Shuai; Shi, Hongxi; Wang, Li

    2017-01-01

    Sporothrix globosa is a type of fungus that typically infects immunocompromised patients. Its prevention continues to pose a challenge. A 70-KDa glycoprotein (Gp70) of Sporothrix has been previously reported to protect host against infection from this fungus. Here, we displayed an epitope peptide (kpvqhalltplgldr) of Gp70 on the major coat protein (pIII), and investigated its efficiency as a vaccine for preventing S. globosa infection. The recombinant phage and the heat-killed S. globosa were used to immunize mice separately. In this study, we evaluated the humoral and cellular immune responses in the mice and demonstrated that recombinant phage could induce mice to produce a stronger immune response and generate antibodies to inhibit S. globosa infection. Furthermore, immunization with recombinant phage could increase the survival rate of S. globosa infection in mice. All these results together indicated that recombinant phages displaying kpvqhalltplgldr are a potential vaccine candidate against S. globosa infection. PMID:28165018

  14. Single strand DNA binding proteins 1 and 2 protect newly replicated telomeres

    PubMed Central

    Gu, Peili; Deng, Wei; Lei, Ming; Chang, Sandy

    2013-01-01

    Human single-strand (ss) DNA binding proteins 1 and 2 (hSSB1 and 2) are components of the hSSB1/2-INTS3-C9orf80 heterotrimeric protein complex shown to participate in DNA damage response and maintenance of genome stability. However, their roles at telomeres remain unknown. Here, we generated murine SSB1 conditional knockout mice and cells and found that mSSB1 plays a critical role in telomere end protection. Both mSSB1 and mSSB2 localize to a subset of telomeres and are required to repair TRF2-deficient telomeres. Deletion of mSSB1 resulted in increased chromatid-type fusions involving both leading- and lagging-strand telomeric DNA, suggesting that it is required for the protection of G-overhangs. mSSB1's interaction with INTS3 is required for its localization to damaged DNA. mSSB1 interacts with Pot1a, but not Pot1b, and its association with telomeric ssDNA requires Pot1a. mSSB1Δ/Δ mice die at birth with developmental abnormalities, while mice with the hypomorphic mSSB1F/F allele are born alive and display increased sensitivity to ionizing radiation (IR). Our results suggest that mSSB1 is required to maintain genome stability, and document a previously unrecognized role for mSSB1/2 in the protection of newly replicated leading- and lagging-strand telomeres. PMID:23459151

  15. α/β-hydrolase domain containing protein 15 (ABHD15)--an adipogenic protein protecting from apoptosis.

    PubMed

    Walenta, Evelyn; Pessentheiner, Ariane R; Pelzmann, Helmut J; Deutsch, Alexander; Goeritzer, Madeleine; Kratky, Dagmar; Hackl, Hubert; Oh, Da Young; Prokesch, Andreas; Bogner-Strauss, Juliane G

    2013-01-01

    Our knowledge about adipocyte metabolism and development is steadily growing, yet many players are still undefined. Here, we show that α/β-hydrolase domain containing protein 15 (Abhd15) is a direct and functional target gene of peroxisome proliferator-activated receptor gamma (PPARγ), the master regulator of adipogenesis. In line, Abhd15 is mainly expressed in brown and white adipose tissue and strongly upregulated during adipogenesis in various murine and human cell lines. Stable knockdown of Abhd15 in 3T3-L1 cells evokes a striking differentiation defect, as evidenced by low lipid accumulation and decreased expression of adipocyte marker genes. In preconfluent cells, knockdown of Abhd15 leads to impaired proliferation, which is caused by apoptosis, as we see an increased SubG1 peak, caspase 3/7 activity, and BAX protein expression as well as a reduction in anti-apoptotic BCL-2 protein. Furthermore, apoptosis-inducing amounts of palmitic acid evoke a massive increase of Abhd15 expression, proposing an apoptosis-protecting role for ABHD15. On the other hand, in mature adipocytes physiological (i.e. non-apoptotic) concentrations of palmitic acid down-regulate Abhd15 expression. Accordingly, we found that the expression of Abhd15 in adipose tissue is reduced in physiological situations with high free fatty acid levels, like high-fat diet, fasting, and aging as well as in genetically obese mice. Collectively, our results position ABHD15 as an essential component in the development of adipocytes as well as in apoptosis, thereby connecting two substantial factors in the regulation of adipocyte number and size. Together with its intricate regulation by free fatty acids, ABHD15 might be an intriguing new target in obesity and diabetes research.

  16. Engineering of chaperone systems and of the unfolded protein response

    PubMed Central

    Khan, Saeed U.

    2008-01-01

    Production of recombinant proteins in mammalian cells is a successful technology that delivers protein pharmaceuticals for therapies and for diagnosis of human disorders. Cost effective production of protein biopharmaceuticals requires extensive optimization through cell and fermentation process engineering at the upstream and chemical engineering of purification processes at the downstream side of the production process. The majority of protein pharmaceuticals are secreted proteins. Accumulating evidence suggests that the folding and processing of these proteins in the endoplasmic reticulum (ER) is a general rate- and yield limiting step for their production. We will summarize our knowledge of protein folding in the ER and of signal transduction pathways activated by accumulation of unfolded proteins in the ER, collectively called the unfolded protein response (UPR). On the basis of this knowledge we will evaluate engineering approaches to increase cell specific productivities through engineering of the ER-resident protein folding machinery and of the UPR. PMID:19003179

  17. Topical CpG Adjuvantation of a Protein-Based Vaccine Induces Protective Immunity to Listeria monocytogenes

    PubMed Central

    Cheng, Wing Ki; Wee, Kathleen; Kollmann, Tobias R.

    2014-01-01

    Robust CD8+ T cell responses are essential for immune protection against intracellular pathogens. Using parenteral administration of ovalbumin (OVA) protein as a model antigen, the effect of the Toll-like receptor 9 (TLR9) agonist, CpG oligodeoxynucleotide (ODN) 1826, as an adjuvant delivered either topically, subcutaneously, or intramuscularly on antigen-specific CD8+ T cell responses in a mouse model was evaluated. Topical CpG adjuvant increased the frequency of OVA-specific CD8+ T cells in the peripheral blood and in the spleen. The more effective strategy to administer topical CpG adjuvant to enhance CD8+ T cell responses was single-dose administration at the time of antigen injection with a prime-boost regimen. Topical CpG adjuvant conferred both rapid and long-lasting protection against systemic challenge with recombinant Listeria monocytogenes expressing the cytotoxic T lymphocyte (CTL) epitope of OVA257–264 (strain Lm-OVA) in a TLR9-dependent manner. Topical CpG adjuvant induced a higher proportion of CD8+ effector memory T cells than parenteral administration of the adjuvant. Although traditional vaccination strategies involve coformulation of antigen and adjuvant, split administration using topical adjuvant is effective and has advantages of safety and flexibility. Split administration of topical CpG ODN 1826 with parenteral protein antigen is superior to other administration strategies in enhancing both acute and memory protective CD8+ T cell immune responses to subcutaneous protein vaccines. This vaccination strategy induces rapid and persistent protective immune responses against the intracellular organism L. monocytogenes. PMID:24391136

  18. A protein-truncating R179X variant in RNF186 confers protection against ulcerative colitis

    PubMed Central

    Rivas, Manuel A.; Graham, Daniel; Sulem, Patrick; Stevens, Christine; Desch, A. Nicole; Goyette, Philippe; Gudbjartsson, Daniel; Jonsdottir, Ingileif; Thorsteinsdottir, Unnur; Degenhardt, Frauke; Mucha, Sören; Kurki, Mitja I.; Li, Dalin; D'Amato, Mauro; Annese, Vito; Vermeire, Severine; Weersma, Rinse K.; Halfvarson, Jonas; Paavola-Sakki, Paulina; Lappalainen, Maarit; Lek, Monkol; Cummings, Beryl; Tukiainen, Taru; Haritunians, Talin; Halme, Leena; Koskinen, Lotta L. E.; Ananthakrishnan, Ashwin N.; Luo, Yang; Heap, Graham A.; Visschedijk, Marijn C.; Barrett, J; de Lange, K; Edwards, C; Hart, A; Hawkey, C; Jostins, L; Kennedy, N; Lamb, C; Lee, J; Lees, C; Mansfield, J; Mathew, C; Mowatt, C; Newman, W; Nimmo, E; Parkes, M; Pollard, M; Prescott, N; Randall, J; Rice, D; Satsangi, J; Simmons, A; Tremelling, M; Uhlig, H; Wilson, D; Abraham, C; Achkar, J.P; Bitton, A; Boucher, G; Croitoru, K; Fleshner, P; Glas, J; Kugathasan, S; Limbergen, J.V; Milgrom, R; Proctor, D; Regueiro, M; Schumm, P.L; Sharma, Y; Stempak, J.M; Targan, S.R; Wang, M.H; MacArthur, Daniel G.; Neale, Benjamin M.; Ahmad, Tariq; Anderson, Carl A.; Brant, Steven R.; Duerr, Richard H.; Silverberg, Mark S.; Cho, Judy H; Palotie, Aarno; Saavalainen, Päivi; Kontula, Kimmo; Färkkilä, Martti; McGovern, Dermot P. B.; Franke, Andre; Stefansson, Kari; Rioux, John D.; Xavier, Ramnik J.; Daly, Mark J.; Barrett, J.; de Lane, K.; Edwards, C.; Hart, A.; Hawkey, C.; Jostins, L.; Kennedy, N.; Lamb, C.; Lee, J.; Lees, C.; Mansfield, J.; Mathew, C.; Mowatt, C.; Newman, B.; Nimmo, E.; Parkes, M.; Pollard, M.; Prescott, N.; Randall, J.; Rice, D.; Satsangi, J.; Simmons, A.; Tremelling, M.; Uhlig, H.; Wilson, D.; Abraham, C.; Achkar, J. P.; Bitton, A.; Boucher, G.; Croitoru, K.; Fleshner, P.; Glas, J.; Kugathasan, S.; Limbergen, J. V.; Milgrom, R.; Proctor, D.; Regueiro, M.; Schumm, P. L.; Sharma, Y.; Stempak, J. M.; Targan, S. R.; Wang, M. H.

    2016-01-01

    Protein-truncating variants protective against human disease provide in vivo validation of therapeutic targets. Here we used targeted sequencing to conduct a search for protein-truncating variants conferring protection against inflammatory bowel disease exploiting knowledge of common variants associated with the same disease. Through replication genotyping and imputation we found that a predicted protein-truncating variant (rs36095412, p.R179X, genotyped in 11,148 ulcerative colitis patients and 295,446 controls, MAF=up to 0.78%) in RNF186, a single-exon ring finger E3 ligase with strong colonic expression, protects against ulcerative colitis (overall P=6.89 × 10−7, odds ratio=0.30). We further demonstrate that the truncated protein exhibits reduced expression and altered subcellular localization, suggesting the protective mechanism may reside in the loss of an interaction or function via mislocalization and/or loss of an essential transmembrane domain. PMID:27503255

  19. HSF transcription factor family, heat shock response, and protein intrinsic disorder.

    PubMed

    Westerheide, Sandy D; Raynes, Rachel; Powell, Chase; Xue, Bin; Uversky, Vladimir N

    2012-02-01

    Intrinsically disordered proteins are highly abundant in all kingdoms of life, and several protein functional classes, such as transcription factors, transcriptional regulators, hub and scaffold proteins, signaling proteins, and chaperones are especially enriched in intrinsic disorder. One of the unique cellular reactions to protein damaging stress is the so-called heat shock response that results in the upregulation of heat shock proteins including molecular chaperones. This molecular protective mechanism is conserved from prokaryotes to eukaryotes and allows an organism to respond to various proteotoxic stressors, such as heat shock, oxidative stress, exposure to heavy metals, and drugs. The heat shock response- related proteins can be expressed during normal conditions (e.g., during the cell growth and development) or can be induced by various pathological conditions, such as infection, inflammation, and protein conformation diseases. The initiation of the heat shock response is manifested by the activation of the heat shock transcription factors HSF 1, part of a family of related HSF transcription factors. This review analyzes the abundance and functional roles of intrinsic disorder in various heat shock transcription factors and clearly shows that the heat shock response requires HSF flexibility to be more efficient.

  20. Protective contributions against invasive Streptococcus pneumoniae pneumonia of antibody and Th17-cell responses to nasopharyngeal colonisation.

    PubMed

    Cohen, Jonathan M; Khandavilli, Suneeta; Camberlein, Emilie; Hyams, Catherine; Baxendale, Helen E; Brown, Jeremy S

    2011-01-01

    The nasopharyngeal commensal bacteria Streptococcus pneumoniae is also a frequent cause of serious infections. Nasopharyngeal colonisation with S. pneumoniae inhibits subsequent re-colonisation by inducing Th17-cell adaptive responses, whereas vaccination prevents invasive infections by inducing antibodies to S. pneumoniae capsular polysaccharides. In contrast, protection against invasive infection after nasopharyngeal colonisation with mutant S. pneumoniae strains was associated with antibody responses to protein antigens. The role of colonisation-induced Th17-cell responses during subsequent invasive infections is unknown. Using mouse models, we show that previous colonisation with S. pneumoniae protects against subsequent lethal pneumonia mainly by preventing bacteraemia with a more modest effect on local control of infection within the lung. Previous colonisation resulted in CD4-dependent increased levels of Th17-cell cytokines during subsequent infectious challenge. However, mice depleted of CD4 cells prior to challenge remained protected against bacteraemia, whereas no protection was seen in antibody deficient mice and similar protection could be achieved through passive transfer of serum. Serum from colonised mice but not antibody deficient mice promoted phagocytosis of S. pneumoniae, and previously colonised mice were able to rapidly clear S. pneumoniae from the blood after intravenous inoculation. Thus, despite priming for a Th17-cell response during subsequent infection, the protective effects of prior colonisation in this model was not dependent on CD4 cells but on rapid clearance of bacteria from the blood by antibody-mediated phagocytosis. These data suggest that whilst nasopharyngeal colonisation induces a range of immune responses, the effective protective responses depend upon the site of subsequent infection.

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

    PubMed Central

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

    2016-01-01

    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

  2. Pediatric Response to a Large-Scale Child Protection Intervention

    ERIC Educational Resources Information Center

    Lukefahr, James L.; Kellogg, Nancy D.; Anderst, James D.; Gavril, Amy R.; Wehner, Karl K.

    2011-01-01

    Objective: In a rural area of the US state of Texas, in April 2008, the Texas Department of Family and Protective Services (DFPS) responded to evidence of widespread child abuse in an isolated religious compound by removing 463 individuals into state custody. This mass child protection intervention is the largest such action that has ever occurred…

  3. Heat shock (stress response) proteins and renal ischemia/reperfusion injury.

    PubMed

    Kelly, Katherine J

    2005-01-01

    Acute renal failure occurs frequently, may be increasing, carries an unacceptably high mortality, yet there is no specific treatment. The induction of stress response (heat shock) proteins (HSPs) is a highly conserved response that protects many cell types from diverse physiological and environmental stressors. HSP families of different sizes function as molecular chaperones that facilitate the folding of enzymes and other proteins into functional conformations. After injury, HSPs are believed to facilitate the restoration of normal function by assisting in the refolding of denatured proteins and degradation of irreparably damaged proteins and toxic metabolites, limitation of aggregation of damaged peptides and aiding appropriate folding of newly synthesized essential polypeptides. HSPs may also regulate apoptosis and immune functions. We have demonstrated protection from the functional deficits and histological evidence of experimental ischemic renal injury with heat stress 6 but not 48 h prior to ischemia. Limitation of the induction of HSPs (either with a short period of hyperthermia or pharmacologically) attenuated the protection observed. Other investigators have demonstrated a correlation between the levels of HSP25 and renal ischemic preconditioning in the mouse. Several pharmacological agents have been shown to increase HSP expression. Enhancement of these endogenous protective mechanisms has potential benefit in human disease.

  4. Genetic and antigenic diversity of the surface protective antigen proteins of Erysipelothrix rhusiopathiae.

    PubMed

    To, Ho; Nagai, Shinya

    2007-07-01

    The surface protective antigen (Spa) protein of Erysipelothrix rhusiopathiae has been shown to be highly immunogenic and is a potential candidate for a new vaccine against erysipelas. In this study, we cloned and sequenced spa genes from all E. rhusiopathiae serovar reference strains as well as from a serovar 18 strain which was not classified as any species in the genus Erysipelothrix. Sequence analysis revealed that the Spa proteins could be classified into three molecular species, including SpaA, which was previously found in serovars 1a and 2, and the newly designated SpaB and SpaC proteins. The SpaA protein is produced by E. rhusiopathiae serovars 1a, 1b, 2, 5, 8, 9, 12, 15, 16, 17, and N, the SpaB protein is produced by E. rhusiopathiae serovars 4, 6, 11, 19, and 21, and the SpaC protein is produced only by serovar 18. The amino acid sequence similarity was high among members of each Spa type (96 to 99%) but low between different Spa types ( approximately 60%). The greatest diversity in Spa proteins was found in the N-terminal half of the molecule (50 to 57% similarity), which was shown to be involved in immunoprotection. Coinciding with this, immunoblot analysis revealed that rabbit antisera specific to each Spa reacted strongly with the homologous Spa protein but weakly with heterologous Spa proteins. A mouse cross-protection study showed that the three recombinant Spa (rSpa) proteins elicited complete protection against challenge with homologous strains but that the level of protection against challenge with heterologous strains varied depending on the rSpa protein used for immunization. Our study is the first to demonstrate sequence and antigenic diversity in Spa proteins and to indicate that rSpaC may be the most promising antigen for use as a vaccine component because of its broad cross-protectiveness.

  5. Electroporation of a multivalent DNA vaccine cocktail elicits a protective immune response against anthrax and plague.

    PubMed

    Albrecht, Mark T; Livingston, Brian D; Pesce, John T; Bell, Matt G; Hannaman, Drew; Keane-Myers, Andrea M

    2012-07-06

    Electroporation of DNA vaccines represents a platform technology well positioned for the development of multivalent biodefense vaccines. To evaluate this hypothesis, three vaccine constructs were produced using codon-optimized genes encoding Bacillus anthracis Protective Antigen (PA), and the Yersinia pestis genes LcrV and F1, cloned into pVAX1. A/J mice were immunized on a prime-boost schedule with these constructs using the electroporation-based TriGrid Delivery System. Immunization with the individual pDNA vaccines elicited higher levels of antigen-specific IgG than when used in combination. DNA vaccine effectiveness was proven, the pVAX-PA titers were toxin neutralizing and fully protective against a lethal B. anthracis spore challenge when administered alone or co-formulated with the plague pDNA vaccines. LcrV and F1 pVAX vaccines against plague were synergistic, resulting in 100% survival, but less protective individually and when co-formulated with pVAX-PA. These DNA vaccine responses were Th1/Th2 balanced with high levels of IFN-γ and IL-4 in splenocyte recall assays, contrary to complimentary protein Alum vaccinations displaying a Th2 bias with increased IL-4 and low levels of IFN-γ. These results demonstrate the feasibility of electroporation to deliver and maintain the overall efficacy of an anthrax-plague DNA vaccine cocktail whose individual components have qualitative immunological differences when combined.

  6. Novel protein targets of the humoral immune response to Listeria monocytogenes infection in rabbits.

    PubMed

    Yu, Wei Ling; Dan, Hanhong; Lin, Min

    2007-07-01

    The role of the humoral immune response in protective immunity against listerial infection has been overlooked and is essentially unknown. This study aimed to discover the protein targets of Listeria monocytogenes that elicit an antibody response following infection in a rabbit model. A genomic expression library for L. monocytogenes was constructed and differentially screened to identify genes encoding proteins that reacted with antiserum from rabbits infected with live L. monocytogenes serotype 4b (RalphaL), but not with that from animals immunized with heat-killed bacteria (RalphaK). Thirty-one clones expressing proteins that reacted exclusively with RalphaL were identified and sequenced. Sequence analysis, together with Western blot analysis of the proteins expressed from positive clones, led to the identification of eight L. monocytogenes proteins as targets of humoral immune responses during listerial infection: three internalin members (InlA, InlD and InlC2) and five novel proteins of unknown function (designated IspA, IspB, IspC, IspD and IspE, respectively). Exhibition of humoral immune responses to these proteins in actively infected rabbits but not in animals receiving heat-killed L. monocytogenes suggested that they were induced or significantly upregulated in vivo during infection and thus are important in Listeria pathogenesis. With the exception of antibodies to InlA, this is the first demonstration of antibodies to the other seven proteins in infected hosts. These immunogenic proteins may be useful candidates for elucidation of the role of antibodies in protective immunity in the context of listerial infection, as well as potential targets for serodiagnostic reagents and vaccine and drug development.

  7. 20 CFR 670.220 - Are we responsible for the protection and maintenance of center facilities?

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... maintenance of center facilities? 670.220 Section 670.220 Employees' Benefits EMPLOYMENT AND TRAINING... and Protection and Maintenance of Facilities § 670.220 Are we responsible for the protection and maintenance of center facilities? (a) Yes, the Secretary establishes procedures for the protection...

  8. [Immunogenic and protective characteristics of recombinant Lassa virus NP protein].

    PubMed

    Ignat'ev, G M

    2002-01-01

    Recombinant fragment of Lassa virus (strain Josiah) nucleocapsid protein (corresponding to amino acid residues 141 + 569) constructed by Dr. Jan ter Meulen (Tropenmedizine, Hamburg) was used for immunizing CBA/calac mice. The preparation was injected intraperitoneally twice with 2-week interval in a dose of 10 micrograms. The parameters of the resultant specific humoral and cell-mediated immunity were comparable to those in reference animals immunized with inactivated Lassa virus. Challenge with Lassa virus (10,000 PFU) resulted in 100% death of the reference animals, while of 15 animals immunized with the recombinant NP protein 8 survived.

  9. The phage-shock-protein response.

    PubMed

    Darwin, Andrew J

    2005-08-01

    The phage-shock-protein (Psp) system responds to extracytoplasmic stress that may reduce the energy status of the cell. It is conserved in many different bacteria and has been linked to several important phenotypes. Escherichia coli psp mutants have defects in maintenance of the proton-motive force, protein export by the sec and tat pathways, survival in stationary phase at alkaline pH, and biofilm formation. Yersinia enterocolitica psp mutants cannot grow when the secretin component of a type III secretion system is mislocalized, and have a severe virulence defect in animals. A Salmonella enterica psp mutation exacerbates some phenotypes of an rpoE null mutant and the psp genes of S. enterica and Shigella flexneri are highly induced during macrophage infection. PspA, the most abundant of the Psp proteins, is required for most of the phenotypes associated with the Psp system. Therefore, PspA is probably an effector that may play a role in maintaining cytoplasmic membrane integrity and/or the proton-motive force. However, PspA is not required for the ability to tolerate secretin mislocalization, which suggests an important physiological role for other Psp proteins. This article summarizes our current understanding of the Psp system: inducing signals, the underlying signal transduction mechanisms, the physiological roles it may play, and a genomic analysis of its conservation.

  10. Invasive Escherichia coli vaccines expressing Brucella melitensis outer membrane proteins 31 or 16 or periplasmic protein BP26 confer protection in mice challenged with B. melitensis.

    PubMed

    Gupta, V K; Radhakrishnan, G; Harms, J; Splitter, G

    2012-06-08

    Because of the serious economic and medical consequences of brucellosis, efforts are to prevent infection of domestic animals through vaccines. Many disadvantages are associated with the current Brucella melitensis Rev.1 vaccine prompting development of alternative vaccines and delivery. Escherichia coli (DH5α) was engineered to express a plasmid containing the inv gene from Yersinia pseudotuberculosis and the hly gene from Listeria monocytogenes. These recombinant invasive E. coli expressing B. melitensis outer membrane proteins (Omp31 or 16) or the periplasmic protein BP26 were evaluated for protection of mice against virulent B. melitensis. Importantly, these invasive E. coli vaccines induced significant protection against B. melitensis challenged mice. Invasive E. coli may be an ideal vaccine platform with natural adjuvant properties for application against B. melitensis since the E. coli delivery system is non-pathogenic and can deliver antigens to antigen-presenting cells promoting cellular immune responses.

  11. Influenza virus-like particles engineered by protein transfer with tumor-associated antigens induces protective antitumor immunity.

    PubMed

    Patel, Jaina M; Vartabedian, Vincent F; Kim, Min-Chul; He, Sara; Kang, Sang-Moo; Selvaraj, Periasamy

    2015-06-01

    Delivery of antigen in particulate form using either synthetic or natural particles induces stronger immunity than soluble forms of the antigen. Among naturally occurring particles, virus-like particles (VLPs) have been genetically engineered to express tumor-associated antigens (TAAs) and have shown to induce strong TAA-specific immune responses due to their nano-particulate size and ability to bind and activate antigen-presenting cells. In this report, we demonstrate that influenza VLPs can be modified by a protein transfer technology to express TAAs for induction of effective antitumor immune responses. We converted the breast cancer HER-2 antigen to a glycosylphosphatidylinositol (GPI)-anchored form and incorporated GPI-HER-2 onto VLPs by a rapid protein transfer process. Expression levels on VLPs depended on the GPI-HER-2 concentration added during protein transfer. Vaccination of mice with protein transferred GPI-HER-2-VLPs induced a strong Th1 and Th2-type anti-HER-2 antibody response and protected mice against a HER-2-expressing tumor challenge. The Soluble form of GPI-HER-2 induced only a weak Th2 response under similar conditions. These results suggest that influenza VLPs can be enriched with TAAs by protein transfer to develop effective VLP-based subunit vaccines against cancer without chemical or genetic modifications and thus preserve the immune stimulating properties of VLPs for easier production of antigen-specific therapeutic cancer vaccines.

  12. A novel bacterial Water Hypersensitivity-like protein shows in vivo protection against cold and freeze damage.

    PubMed

    Anderson, Dominique; Ferreras, Eloy; Trindade, Marla; Cowan, Don

    2015-08-01

    Metagenomic library screening, by functional or sequence analysis, has become an established method for the identification of novel genes and gene products, including genetic elements implicated in microbial stress response and adaptation. We have identified, using a sequence-based approach, a fosmid clone from an Antarctic desert soil metagenome library containing a novel gene which codes for a protein homologous to a Water Hypersensitivity domain (WHy). The WHy domain is typically found as a component of specific LEA (Late Embryogenesis Abundant) proteins, particularly the LEA-14 (LEA-8) variants, which occur widely in plants, nematodes, bacteria and archaea and which are typically induced by exposure to stress conditions. The novel WHy-like protein (165 amino acid, 18.6 kDa) exhibits a largely invariant NPN motif at the N-terminus and has high sequence identity to genes identified in Pseudomonas genomes. Expression of this protein in Escherichia coli significantly protected the recombinant host against cold and freeze stress.

  13. Proteomic analysis of membrane proteins of vero cells: exploration of potential proteins responsible for virus entry.

    PubMed

    Guo, Donghua; Zhu, Qinghe; Zhang, Hong; Sun, Dongbo

    2014-01-01

    Vero cells are highly susceptible to many viruses in humans and animals, and its membrane proteins (MPs) are responsible for virus entry. In our study, the MP proteome of the Vero cells was investigated using a shotgun LC-MS/MS approach. Six hundred twenty-seven proteins, including a total of 1839 peptides, were identified in MP samples of the Vero cells. In 627 proteins, 307 proteins (48.96%) were annotated in terms of biological process of gene ontology (GO) categories; 356 proteins (56.78%) were annotated in terms of molecular function of GO categories; 414 proteins (66.03%) were annotated in terms of cellular components of GO categories. Of 627 identified proteins, seventeen proteins had been revealed to be virus receptor proteins. The resulting protein lists and highlighted proteins may provide valuable information to increase understanding of virus infection of Vero cells.

  14. Protective effect of taraxasterol against rheumatoid arthritis by the modulation of inflammatory responses in mice.

    PubMed

    Jiang, Shu-Hua; Ping, Li-Feng; Sun, Feng-Yan; Wang, Xiao-Lei; Sun, Zhi-Juan

    2016-12-01

    Taraxasterol is an effective component of dandelion that has anti-inflammatory effects in vivo and in vitro. The present study was performed to explore whether taraxasterol exhibits a protective effect against rheumatoid arthritis through the modulation of inflammatory responses in mice. Eight-week-old CCR9-deficient mice were injected with a collagen II monoclonal antibody cocktail to create a rheumatoid arthritis model. In the experimental group, arthritic model mice were treated with 10 mg/kg taraxasterol once per day for 5 days. Treatment with taraxasterol significantly increased the pain thresholds and reduced the clinical arthritic scores of the mice in the experimental group compared with those of the model group. Furthermore, treatment with taraxasterol significantly suppressed tumor necrosis factor-α, interleukin (IL)-1β, IL-6 and nuclear factor-κB protein expression levels compared with those in the rheumatoid arthritis model mice. Taraxasterol treatment also significantly reduced nitric oxide, prostaglandin E2 and cyclooxygenase-2 levels compared with those in the rheumatoid arthritis model group. These observations indicate that the protective effect of taraxasterol against rheumatoid arthritis is mediated via the modulation of inflammatory responses in mice.

  15. Protective effect of taraxasterol against rheumatoid arthritis by the modulation of inflammatory responses in mice

    PubMed Central

    Jiang, Shu-Hua; Ping, Li-Feng; Sun, Feng-Yan; Wang, Xiao-Lei; Sun, Zhi-Juan

    2016-01-01

    Taraxasterol is an effective component of dandelion that has anti-inflammatory effects in vivo and in vitro. The present study was performed to explore whether taraxasterol exhibits a protective effect against rheumatoid arthritis through the modulation of inflammatory responses in mice. Eight-week-old CCR9-deficient mice were injected with a collagen II monoclonal antibody cocktail to create a rheumatoid arthritis model. In the experimental group, arthritic model mice were treated with 10 mg/kg taraxasterol once per day for 5 days. Treatment with taraxasterol significantly increased the pain thresholds and reduced the clinical arthritic scores of the mice in the experimental group compared with those of the model group. Furthermore, treatment with taraxasterol significantly suppressed tumor necrosis factor-α, interleukin (IL)-1β, IL-6 and nuclear factor-κB protein expression levels compared with those in the rheumatoid arthritis model mice. Taraxasterol treatment also significantly reduced nitric oxide, prostaglandin E2 and cyclooxygenase-2 levels compared with those in the rheumatoid arthritis model group. These observations indicate that the protective effect of taraxasterol against rheumatoid arthritis is mediated via the modulation of inflammatory responses in mice. PMID:28101182

  16. Protein-surface interactions on stimuli-responsive polymeric biomaterials.

    PubMed

    Cross, Michael C; Toomey, Ryan G; Gallant, Nathan D

    2016-03-04

    Responsive surfaces: a review of the dependence of protein adsorption on the reversible volume phase transition in stimuli-responsive polymers. Specifically addressed are a widely studied subset: thermoresponsive polymers. Findings are also generalizable to other materials which undergo a similarly reversible volume phase transition. As of 2015, over 100,000 articles have been published on stimuli-responsive polymers and many more on protein-biomaterial interactions. Significantly, fewer than 100 of these have focused specifically on protein interactions with stimuli-responsive polymers. These report a clear trend of increased protein adsorption in the collapsed state compared to the swollen state. This control over protein interactions makes stimuli-responsive polymers highly useful in biomedical applications such as wound repair scaffolds, on-demand drug delivery, and antifouling surfaces. Outstanding questions are whether the protein adsorption is reversible with the volume phase transition and whether there is a time-dependence. A clear understanding of protein interactions with stimuli-responsive polymers will advance theoretical models, experimental results, and biomedical applications.

  17. Antibodies are not required to a protective immune response against dengue virus elicited in a mouse encephalitis model.

    PubMed

    Amorim, Jaime Henrique; dos Santos Alves, Rúbens Prince; Bizerra, Raíza; Araújo Pereira, Sara; Ramos Pereira, Lennon; Nascimento Fabris, Denicar Lina; Santos, Robert Andreata; Romano, Camila Malta; de Souza Ferreira, Luís Carlos

    2016-01-01

    Generating neutralizing antibodies have been considered a prerequisite to control dengue virus (DENV) infection. However, T lymphocytes have also been shown to be important in a protective immune state. In order to investigate the contribution of both humoral and cellular immune responses in DENV immunity, we used an experimental model in which a non-lethal DENV2 strain (ACS46) is used to intracranially prime Balb/C mice which develop protective immunity against a lethal DENV2 strain (JHA1). Primed mice generated envelope-specific antibodies and CD8(+) T cell responses targeting mainly non-structural proteins. Immune sera from protected mice did not confer passive protection to naïve mice challenged with the JHA1 strain. In contrast, depletion of CD4(+) and CD8(+) T lymphocytes significantly reduced survival of ACS46-primed mice challenged with the JHA1 strain. Collectively, results presented in this study show that a cellular immune response targeting non-structural proteins are a promising way in vaccine development against dengue.

  18. Subdominant outer membrane antigens in anaplasma marginale: conservation, antigenicity, and protective capacity using recombinant protein

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Anaplasma marginale is a tick-borne rickettsial pathogen of cattle with a worldwide distribution. Currently a safe and efficacious vaccine is unavailable. Outer membrane protein (OMP) extracts or a well- defined surface protein complex reproducibly induce protective immunity. However, there are seve...

  19. Protective effect of black tea on integral membrane proteins in rat liver.

    PubMed

    Szachowicz-Petelska, Barbara; Skrzydlewska, Elżbieta; Figaszewski, Zbigniew

    2013-01-01

    Ethanol intoxication is accompanied by oxidative stress formation. Consequently, it leads to disturbances in cellular metabolism that can alter the structure and function of cell membrane components. Black tea displays antioxidant properties, protects membrane phospholipids and may protect integral membrane proteins. In the present study, we examined whether black tea induces changes in the liver integral membrane proteins of 12-months old rats chronically intoxicated with ethanol. To estimate qualitatively and quantitatively the levels of the liver integral membrane proteins, the proteins were selectively hydrolyzed by trypsin, the obtained peptides were resolved by HPLC and the levels of specific amino acids within the individual peptides were determined. All of the obtained peptides contained phenylalanine (Phe), cysteine (Cys) and lysine (Lys). Compared to the control group, rats in the ethanol intoxication group showed decreased liver levels of integral membrane proteins as well as fewer trypsin-hydrolyzed peptides and amino acids in the hydrolyzed peptides. Administration of black tea to ethanol-intoxicated rats partially protected proteins against the structural changes caused by ethanol. Black tea prevented decreases in the levels of cysteine (in about 90% of cases), lysine (in about 60% of cases), phenylalanine (in about 70% of cases) and examined peptides (in about 60% of cases). The liver protein level was higher (by about 18%) in rats who received black tea and ethanol than in those who received ethanol alone. In conclusion, black tea partially protects the composition and level of rat liver cell integral membrane proteins against changes caused by ethanol intoxication.

  20. Rabbit hemorrhagic disease virus capsid, a versatile platform for foreign B-cell epitope display inducing protective humoral immune responses

    PubMed Central

    Moreno, Noelia; Mena, Ignacio; Angulo, Iván; Gómez, Yolanda; Crisci, Elisa; Montoya, María; Castón, José R.; Blanco, Esther; Bárcena, Juan

    2016-01-01

    Virus-like particles (VLPs), comprised of viral structural proteins devoid of genetic material, are tunable nanoparticles that can be chemically or genetically engineered, to be used as platforms for multimeric display of foreign antigens. Here, we report the engineering of chimeric VLPs, derived from rabbit hemorrhagic disease virus (RHDV) for presentation of foreign B-cell antigens to the immune system. The RHDV capsid comprises 180 copies of a single capsid subunit (VP60). To evaluate the ability of chimeric RHDV VLPs to elicit protective humoral responses against foreign antigens, we tested two B-cell epitopes: a novel neutralizing B-cell epitope, derived from feline calicivirus capsid protein, and a well characterized B-cell epitope from the extracellular domain of influenza A virus M2 protein (M2e). We generated sets of chimeric RHDV VLPs by insertion of the foreign B-cell epitopes at three different locations within VP60 protein (which involved different levels of surface accessibility) and in different copy numbers per site. The immunogenic potential of the chimeric VLPs was analyzed in the mouse model. The results presented here indicated that chimeric RHDV VLPs elicit potent protective humoral responses against displayed foreign B-cell epitopes, demonstrated by both, in vitro neutralization and in vivo protection against a lethal challenge. PMID:27549017

  1. Intein-mediated backbone cyclization of VP1 protein enhanced protection of CVB3-induced viral myocarditis

    PubMed Central

    Qi, Xingmei; Xiong, Sidong

    2017-01-01

    CVB3 is a common human pathogen to be highly lethal to newborns and causes viral myocarditis and pancreatitis in adults. However, there is no vaccine available for clinical use. CVB3 capsid protein VP1 is an immunodominant structural protein, containing several B- and T-cell epitopes. However, immunization of mice with VP1 protein is ineffective. Cyclization of peptide is commonly used to improve their in vivo stability and biological activity. Here, we designed and synthesizd cyclic VP1 protein by using engineered split Rma DnaB intein and the cyclization efficiency was 100% in E. coli. As a result, the cyclic VP1 was significantly more stable against irreversible aggregation upon heating and against carboxypeptidase in vitro and the degradation rate was more slowly in vivo. Compared with linear VP1, immunization mice with circular VP1 significantly increased CVB3-specific serum IgG level and augmented CVB3-specific cellular immune responses, consequently afforded better protection against CVB3-induced viral myocarditis. The cyclic VP1 may be a novel candidate protein vaccine for preventing CVB3 infection and similar approaches could be employed to a variety of protein vaccines to enhance their protection effect. PMID:28148910

  2. Kinetic response of a photoperturbed allosteric protein.

    PubMed

    Buchli, Brigitte; Waldauer, Steven A; Walser, Reto; Donten, Mateusz L; Pfister, Rolf; Blöchliger, Nicolas; Steiner, Sandra; Caflisch, Amedeo; Zerbe, Oliver; Hamm, Peter

    2013-07-16

    By covalently linking an azobenzene photoswitch across the binding groove of a PDZ domain, a conformational transition, similar to the one occurring upon ligand binding to the unmodified domain, can be initiated on a picosecond timescale by a laser pulse. The protein structures have been characterized in the two photoswitch states through NMR spectroscopy and the transition between them through ultrafast IR spectroscopy and molecular dynamics simulations. The binding groove opens on a 100-ns timescale in a highly nonexponential manner, and the molecular dynamics simulations suggest that the process is governed by the rearrangement of the water network on the protein surface. We propose this rearrangement of the water network to be another possible mechanism of allostery.

  3. 10 CFR 35.24 - Authority and responsibilities for the radiation protection program.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 10 Energy 1 2010-01-01 2010-01-01 false Authority and responsibilities for the radiation... MATERIAL General Administrative Requirements § 35.24 Authority and responsibilities for the radiation protection program. (a) In addition to the radiation protection program requirements of § 20.1101 of...

  4. 10 CFR 35.24 - Authority and responsibilities for the radiation protection program.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 10 Energy 1 2013-01-01 2013-01-01 false Authority and responsibilities for the radiation... MATERIAL General Administrative Requirements § 35.24 Authority and responsibilities for the radiation protection program. (a) In addition to the radiation protection program requirements of § 20.1101 of...

  5. 10 CFR 35.24 - Authority and responsibilities for the radiation protection program.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 10 Energy 1 2012-01-01 2012-01-01 false Authority and responsibilities for the radiation... MATERIAL General Administrative Requirements § 35.24 Authority and responsibilities for the radiation protection program. (a) In addition to the radiation protection program requirements of § 20.1101 of...

  6. 10 CFR 35.24 - Authority and responsibilities for the radiation protection program.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 10 Energy 1 2011-01-01 2011-01-01 false Authority and responsibilities for the radiation... MATERIAL General Administrative Requirements § 35.24 Authority and responsibilities for the radiation protection program. (a) In addition to the radiation protection program requirements of § 20.1101 of...

  7. 10 CFR 35.24 - Authority and responsibilities for the radiation protection program.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 10 Energy 1 2014-01-01 2014-01-01 false Authority and responsibilities for the radiation... MATERIAL General Administrative Requirements § 35.24 Authority and responsibilities for the radiation protection program. (a) In addition to the radiation protection program requirements of § 20.1101 of...

  8. From Humanitarian Intervention to the Responsibility to Protect: From Kosovo to Libya and Beyond

    DTIC Science & Technology

    2011-12-01

    10 Noam Chomsky (statement, United Nations General Assembly Thematic Dialogue on the Responsibility to Protect, New York, NY, 23 July 2009). 11... Chomsky , Noam . Statement. United Nations General Assembly Thematic Dialogue on the Responsibility to Protect. New York, NY, 23 July 2009. Cody

  9. Extremophilic Enzymatic Response for Protection against UV-Radiation Damage

    DTIC Science & Technology

    2012-09-17

    ammonium sulphate . 11 Figure 12. SDS–PAGE (12%) analysis of heat treatment of recombinant SOD. Lane 1, markers; Lane 2, TC; Lane 3, CFE; Lane 4...16 After several trials we could obtain the catalase sequence. The catalase gene from I1P has 1437 nucleotides. The protein contains 478 amino acid ...protein preservation. The protein was maintained active in 3.2 ammonium sulphate . Figure 22. SDS-PAGE (12 %) analysis of IMAC purification fractions

  10. Expression of heat shock protein genes in insect stress responses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The heat shock proteins (HSPs) that are abundantly expressed in insects are important modulators of insect survival. Expression of HSP genes in insects is not only developmentally regulated, but also induced by various stressors in order to confer protection against such stressors. The expression o...

  11. Oxidative stress response induced by atrazine in Palaemonetes argentinus: the protective effect of vitamin E.

    PubMed

    Griboff, Julieta; Morales, David; Bertrand, Lidwina; Bonansea, Rocío Inés; Monferrán, Magdalena Victoria; Asis, Ramón; Wunderlin, Daniel Alberto; Amé, María Valeria

    2014-10-01

    The widespread contamination and persistence of the herbicide atrazine residues in the environment resulted in the exposure of non-target organisms. The present study was undertaken to investigate the effect of atrazine in the response of oxidative stress biomarkers in the freshwater shrimp Palaemonetes argentinus and the protective effect of vitamin-E against atrazine-induced toxicity. Therefore, two batches of P. argentinus were fed for 21 days with a commercial food enriched in proteins (D1) or with D2, composed of D1 enriched with vitamin-E (6.8 and 16.0mg% of vitamin-E, respectively). Subsequently, half of the individuals of each group were exposed to atrazine (0.4mgL(-1)) for 24h and the others remained as controls. Atrazine promoted oxidative stress response in P. argentinus fed with D1 as indicated by enhanced H2O2 content and induction of superoxide dismutase, glutathione-S-transferases and glutathione reductase. This antioxidant activity would prevent the increment of thiobarbituric acid reactive substances in the shrimp tissues. P. argentinus fed with D2 reversed the response of the biomarkers measured. However, the activation of antioxidants response had an energetic cost, which was revealed by a decrease in lipids storage in shrimps. These results show the modulatory effect of vit-E on oxidative stress and its potential use as an effective antioxidant to be applied in chemoprotection strategies during aquaculture.

  12. Taurine protects cisplatin induced cardiotoxicity by modulating inflammatory and endoplasmic reticulum stress responses.

    PubMed

    Chowdhury, Sayantani; Sinha, Krishnendu; Banerjee, Sharmistha; Sil, Parames C

    2016-11-12

    Oxidative stress, ER stress, inflammation, and apoptosis results in the pathogenesis of cisplatin-induced cardiotoxicity. The present study was designed to investigate the signaling mechanisms involved in the ameliorating effect of taurine, a conditionally essential amino acid, against cisplatin-mediated cardiac ER stress dependent apoptotic death and inflammation. Mice were simultaneously treated with taurine (150 mg kg(-1) body wt, i.p.) and cisplatin (10 mg kg(-1) body wt, i.p.) for a week. Cisplatin exposure significantly altered serum creatine kinase and troponin T levels. In addition, histological studies revealed disintegration in the normal radiation pattern of cardiac muscle fibers. However, taurine administration could abate such adverse effects of cisplatin. Taurine administration significantly mitigated the reactive oxygen species production, alleviated the overexpression of nuclear factor-κB (NF-κB), and inhibited the elevation of proinflammatoy cytokines, adhesion molecules, and chemokines. Cisplatin exposure resulted in the unfolded protein response (UPR)-regulated CCAAT/enhancer binding protein (CHOP) up-regulation, induction of GRP78: a marker of ER stress and eIF2α signaling. Increase in calpain-1 expression level, activation of caspase-12 and caspase-3, cleavage of the PARP protein as well as the inhibition of antiapoptotic protein Bcl-2 were reflected on cisplatin-triggered apoptosis. Taurine could, however, combat against such cisplatin induced cardiac-abnormalities. The above mentioned findings suggest that taurine plays a beneficial role in providing protection against cisplatin-induced cardiac damage by modulating inflammatory responses and ER stress. © 2016 BioFactors, 42(6):647-664, 2016.

  13. Coherence Dynamics in Photosynthesis: Protein Protection of Excitonic Coherence

    NASA Astrophysics Data System (ADS)

    Lee, Hohjai; Cheng, Yuan-Chung; Fleming, Graham R.

    2007-06-01

    The role of quantum coherence in promoting the efficiency of the initial stages of photosynthesis is an open and intriguing question. We performed a two-color photon echo experiment on a bacterial reaction center that enabled direct visualization of the coherence dynamics in the reaction center. The data revealed long-lasting coherence between two electronic states that are formed by mixing of the bacteriopheophytin and accessory bacteriochlorophyll excited states. This coherence can only be explained by strong correlation between the protein-induced fluctuations in the transition energy of neighboring chromophores. Our results suggest that correlated protein environments preserve electronic coherence in photosynthetic complexes and allow the excitation to move coherently in space, enabling highly efficient energy harvesting and trapping in photosynthesis.

  14. Coherence dynamics in photosynthesis: protein protection of excitonic coherence.

    PubMed

    Lee, Hohjai; Cheng, Yuan-Chung; Fleming, Graham R

    2007-06-08

    The role of quantum coherence in promoting the efficiency of the initial stages of photosynthesis is an open and intriguing question. We performed a two-color photon echo experiment on a bacterial reaction center that enabled direct visualization of the coherence dynamics in the reaction center. The data revealed long-lasting coherence between two electronic states that are formed by mixing of the bacteriopheophytin and accessory bacteriochlorophyll excited states. This coherence can only be explained by strong correlation between the protein-induced fluctuations in the transition energy of neighboring chromophores. Our results suggest that correlated protein environments preserve electronic coherence in photosynthetic complexes and allow the excitation to move coherently in space, enabling highly efficient energy harvesting and trapping in photosynthesis.

  15. The protective immune response against infectious bronchitis virus induced by multi-epitope based peptide vaccines.

    PubMed

    Yang, Tai; Wang, Hong-Ning; Wang, Xue; Tang, Jun-Ni; Lu, Dan; Zhang, Yun-Fei; Guo, Zi-Cheng; Li, Yu-Ling; Gao, Rong; Kang, Run-Min

    2009-07-01

    Peptide vaccine was found to be an effective and powerful approach to a variety of pathogens. To explore multi-epitope based peptide vaccines against infectious bronchitis virus (IBV), the immunogenic peptides were fused to the 3' terminal of glutathione S transferase gene (GST) and expressed in Escherichia coli. ELISA and Western blot analysis showed that the purified fusion proteins had excellent immune activity with chicken anti-IBV serum. During the vaccination course, the candidate peptide vaccines induced strong humoral and cellular response, and provided up to 80.0% immune protection, while all non-immunized chickens in the negative control group manifested obvious typical symptoms and died after virus challenge. Our finding provides a new way to develop multi-epitope based peptide vaccine against IBV.

  16. Protein stability in stored decellularized heart valve scaffolds and diffusion kinetics of protective molecules.

    PubMed

    Wang, Shangping; Oldenhof, Harriëtte; Dai, Xiaolei; Haverich, Axel; Hilfiker, Andres; Harder, Michael; Wolkers, Willem F

    2014-02-01

    Decellularized tissues can be used as matrix implants. The aims of this study were to investigate protein stability and solvent accessibility in decellularized pulmonary heart valve tissues. Protein denaturation profiles of tissues were studied by differential scanning calorimetry. Protein solvent accessibility of tissue exposed to D2O, and diffusion kinetics of various protective molecules were studied by Fourier transform infrared spectroscopy. Little changes were observed in the protein denaturation temperature during storage, at either 5 or 40°C. Glycerol was found to stabilize proteins; it increased the protein denaturation temperature. The stabilizing effect of glycerol disappeared after washing the sample with saline solution. Hydrogen-to-deuterium exchange rates of protein amide groups were fastest in leaflet tissue, followed by artery and muscle tissue. Diffusion of glycerol was found to be fastest in muscle tissue, followed by artery and leaflet tissue. Diffusion coefficients were derived and used to estimate the time needed to reach saturation. Fixation of tissue with glutaraldehyde had little effects on exchange and diffusion rates. Diffusion rates decreased with increasing molecular size. Proteins in decellularized heart valve tissue are stable during storage. Glycerol increases protein stability in a reversible manner. Solvent accessibility studies of protein amide groups provide an additional tool to study proteins in tissues. Diffusion coefficients can be derived to simulate diffusion kinetics of protective molecules in tissues. This study provides novel tools to evaluate protein stability and solvent accessibility in tissues, which can be used to develop biopreservation strategies.

  17. Immunization of N terminus of enterovirus 71 VP4 elicits cross-protective antibody responses

    PubMed Central

    2013-01-01

    Background Enterovirus 71 (EV71) is major cause of hand, foot and mouth disease. Large epidemics of EV71 infection have been recently reported in the Asian-Pacific region. Currently, no vaccine is available to prevent EV71 infection. Results The peptide (VP4N20) consisting of the first 20 amino acids at the N-terminal of VP4 of EV71 genotype C4 were fused to hepatitis B core (HBcAg) protein. Expression of fusion proteins in E. coli resulted in the formation of chimeric virus-like particles (VLPs). Mice immunized with the chimeric VLPs elicited anti-VP4N20 antibody response. In vitro microneutralization experiments showed that anti-chimeric VLPs sera were able to neutralize not only EV71 of genotype C4 but also EV71 of genotype A. Neonatal mice model confirmed the neutralizing ability of anti-chimeric VLPs sera. Eiptope mapping led to the identification of a “core sequence” responsible for antibody recognition within the peptide. Conclusions Immunization of chimeric VLPs is able to elicit antibodies displaying a broad neutralizing activity against different genotypes of EV71 in vitro. The “core sequence” of EV71-VP4 is highly conserved across EV71 genotypes. The chimeric VLPs have a great potential to be a novel vaccine candidate with a broad cross-protection against different EV71 genotypes. PMID:24320792

  18. Protective benefits of AMP-activated protein kinase in hepatic ischemia-reperfusion injury

    PubMed Central

    Zhang, Min; Yang, Dan; Gong, Xianqiong; Ge, Pu; Dai, Jie; Lin, Ling; Zhang, Li

    2017-01-01

    Hepatic ischemia-reperfusion injury (HIRI) is a major cause of hepatic failure and death after liver trauma, haemorrhagic shock, resection surgery and liver transplantation. AMP-activated protein kinase (AMPK) is an energy sensitive kinase that plays crucial roles in the regulation of metabolic homeostasis. In HIRI, ischemia induces the decline of ATP and the increased ratio of AMP/ATP, which promotes the phosphorylation and activation of AMPK. Three AMPK kinases, liver kinase B1 (LKB1), Ca2+/calmodulin-depedent protein kinase kinase β (CaMKKβ) and TGF-β-activated kinase-1 (TAK1), are main upstream kinases for the phosphorylation of AMPK. In addition to the changed AMP/ATP ratio, the activated CaMKKβ by increased intracelluar Ca2+ and the overproduction of reactive oxygen species (ROS) are also involved in the activation of AMPK during HIRI. The activated AMPK might provide protective benefits in HIRI via prevention of energy decline, inhibition of inflammatory response, suppression of hepatocyte apoptosis and attenuation of oxidative stress. Thus, AMPK might become a novel target for the pharmacological intervention of HIRI. PMID:28386315

  19. ULTRAVIOLET PROTECTIVE COMPOUNDS AS A RESPONSE TO ULTRAVIOLET RADIATION EXPOSURE

    EPA Science Inventory

    Life on Earth has evolved adaptations to many environmental stresses over the epochs. One consistent stress has been exposure to ultraviolet radiation. In response to UVR organisms have adapted myriad responses; behavioral, morphological and physiological. Behaviorally, some orga...

  20. FK506 protects against various immune responses and secondary degeneration following cerebral ischemia.

    PubMed

    Brecht, Stephan; Waetzig, Vicki; Hidding, Ute; Hanisch, Uwe-Karsten; Walther, Michael; Herdegen, Thomas; Neiss, Wolfram F

    2009-12-01

    The immunosuppressant FK506 (1 mg/kg, i.p.) reduces the infarct size following 90 min occlusion of the middle cerebral artery (MCAo) in adult rat brain. Here we have investigated the effect of FK506 on cerebral immune cells that are considered to contribute to neurodegeneration. FK506 substantially attenuated the response of resident and peripheral immune cells following transient ischemia. Between 24 hr and 5 days after MCAo, FK506 reduced the T-cell infiltration in the infarct area as well as the presence of activated and/or phagocytic OX-18, OX-42, GSA-IB4, Iba1, and ED1 positive microglia/macrophages. FK506 also lowered the protein levels of TNFalpha and IL-2 in ischemic brain areas. Repetitive application of FK506 over 20 days attenuated the activation of microglia in the substantia nigra (SN), an area of secondary degeneration. Importantly, FK506 conferred also lasting protection of the neurons of SN; these neurons degenerate by withdrawal of neurotrophic factors from the striatum that undergoes necrotic death as part of the ischemic core. To understand the molecular basis of FK506 effects in cerebral immune cells, we determined in primary postnatal day 0/1 (P0/P1) microglia (i) the expression of the FK506 binding proteins FKBP12, FKBP52, and FKPB65 and (ii) that FK506 (1-100 ng/mL) lowered the number of resting or lipopolysaccharide stimulated microglia as well as we induced the lipopolysaccharide release of TNFalpha in a dose-dependent manner. In summary, FK506 confers rescue of brain tissue following cerebral ischemia not only by neuronal protection, but also by suppression of microglial activation and peripheral immune responses.

  1. Protein A suppresses immune responses during Staphylococcus aureus bloodstream infection in guinea pigs

    DOE PAGES

    Kim, Hwan Keun; Falugi, Fabiana; Thomer, Lena; ...

    2015-01-06

    Staphylococcus aureus infection is not associated with the development of protective immunity, and disease relapses occur frequently. We hypothesize that protein A, a factor that binds immunoglobulin Fcγ and cross-links VH3 clan B cell receptors (IgM), is the staphylococcal determinant for host immune suppression. To test this, vertebrate IgM was examined for protein A cross-linking. High VH3 binding activity occurred with human and guinea immunoglobulin, whereas mouse and rabbit immunoglobulins displayed little and no binding, respectively. Establishing a guinea pig model of S. aureus bloodstream infection, we show that protein A functions as a virulence determinant and suppresses host Bmore » cell responses. Immunization with SpAKKAA, which cannot bind immunoglobulin, elicits neutralizing antibodies that enable guinea pigs to develop protective immunity.« less

  2. Hypothesis: NDL proteins function in stress responses by regulating microtubule organization

    PubMed Central

    Khatri, Nisha; Mudgil, Yashwanti

    2015-01-01

    N-MYC DOWNREGULATED-LIKE proteins (NDL), members of the alpha/beta hydrolase superfamily were recently rediscovered as interactors of G-protein signaling in Arabidopsis thaliana. Although the precise molecular function of NDL proteins is still elusive, in animals these proteins play protective role in hypoxia and expression is induced by hypoxia and nickel, indicating role in stress. Homology of NDL1 with animal counterpart N-MYC DOWNREGULATED GENE (NDRG) suggests similar functions in animals and plants. It is well established that stress responses leads to the microtubule depolymerization and reorganization which is crucial for stress tolerance. NDRG is a microtubule-associated protein which mediates the microtubule organization in animals by causing acetylation and increases the stability of α-tubulin. As NDL1 is highly homologous to NDRG, involvement of NDL1 in the microtubule organization during plant stress can also be expected. Discovery of interaction of NDL with protein kinesin light chain- related 1, enodomembrane family protein 70, syntaxin-23, tubulin alpha-2 chain, as a part of G protein interactome initiative encourages us to postulate microtubule stabilizing functions for NDL family in plants. Our search for NDL interactors in G protein interactome also predicts the role of NDL proteins in abiotic stress tolerance management. Based on published report in animals and predicted interacting partners for NDL in G protein interactome lead us to hypothesize involvement of NDL in the microtubule organization during abiotic stress management in plants. PMID:26583023

  3. Immunization of Mice with Recombinant Brucella abortus Organic Hydroperoxide Resistance (Ohr) Protein Protects Against a Virulent Brucella abortus 544 Infection.

    PubMed

    Hop, Huynh Tan; Reyes, Alisha Wehdnesday Bernardo; Simborio, Hannah Leah Tadeja; Arayan, Lauren Togonon; Min, Won Gi; Lee, Hu Jang; Lee, Jin Ju; Chang, Hong Hee; Kim, Suk

    2016-01-01

    In this study, the Brucella abortus ohr gene coding for an organic hydroperoxide resistance protein (Ohr) was cloned into a maltose fusion protein expression system (pMAL), inserted into Escherichia coli, and purified, and its immunogenicity was evaluated by western blot analysis using Brucella-positive mouse sera. The purified recombinant Ohr (rOhr) was treated with adjuvant and injected intraperitoneally into BALB/c mice. A protective immune response analysis revealed that rOhr induced a significant increase in both the IgG1 and IgG2a titers, and IgG2a reached a higher level than IgG1 after the second and third immunizations. Additionally, immunization with rOhr induced high production of IFN-γ as well as proinflammatory cytokines such as TNF, MCP-1, IL-12p70, and IL-6, but a lesser amount of IL-10, suggesting that rOhr predominantly elicited a cell-mediated immune response. In addition, immunization with rOhr caused a significantly higher degree of protection against a virulent B. abortus infection compared with a positive control group consisting of mice immunized with maltose-binding protein. These findings showed that B. abortus rOhr was able to induce both humoral and cell-mediated immunity in mice, which suggested that this recombinant protein could be a potential vaccine candidate for animal brucellosis.

  4. Protective immunity provided by HLA-A2 epitopes for fusion and hemagglutinin proteins of measles virus

    SciTech Connect

    Oh, Sang Kon . E-mail: sangkono@baylorhealth.edu; Stegman, Brian; Pendleton, C. David; Ota, Martin O.; Pan, C.-H.; Griffin, Diane E.; Burke, Donald S.; Berzofsky, Jay A. . E-mail: berzofsk@helix.nih.gov

    2006-09-01

    Natural infection and vaccination with a live-attenuated measles virus (MV) induce CD8{sup +} T-cell-mediated immune responses that may play a central role in controlling MV infection. In this study, we show that newly identified human HLA-A2 epitopes from MV hemagglutinin (H) and fusion (F) proteins induced protective immunity in HLA-A2 transgenic mice challenged with recombinant vaccinia viruses expressing F or H protein. HLA-A2 epitopes were predicted and synthesized. Five and four peptides from H and F, respectively, bound to HLA-A2 molecules in a T2-binding assay, and four from H and two from F could induce peptide-specific CD8{sup +} T cell responses in HLA-A2 transgenic mice. Further experiments proved that three peptides from H (H9-567, H10-250, and H10-516) and one from F protein (F9-57) were endogenously processed and presented on HLA-A2 molecules. All peptides tested in this study are common to 5 different strains of MV including Edmonston. In both A2K{sup b} and HHD-2 mice, the identified peptide epitopes induced protective immunity against recombinant vaccinia viruses expressing H or F. Because F and H proteins induce neutralizing antibodies, they are major components of new vaccine strategies, and therefore data from this study will contribute to the development of new vaccines against MV infection.

  5. Protective Efficacy in Sheep of Adenovirus-Vectored Vaccines against Bluetongue Virus Is Associated with Specific T Cell Responses

    PubMed Central

    Martín, Verónica; Pascual, Elena; Avia, Miguel; Peña, Lourdes; Valcárcel, Félix; Sevilla, Noemí

    2015-01-01

    Bluetongue virus (BTV) is an economically important Orbivirus of the Reoviridae family that causes a hemorrhagic disease in ruminants. Its control has been achieved by inactivated-vaccines that have proven to protect against homologous BTV challenge although unable to induce long-term immunity. Therefore, a more efficient control strategy needs to be developed. Recombinant adenovirus vectors are lead vaccine candidates for protection of several diseases, mainly because of their potency to induce potent T cell immunity. Here we report the induction of humoral and T-cell mediated responses able to protect animals against BTV challenge by recombinant replication-defective human adenovirus serotype 5 (Ad5) expressing either VP7, VP2 or NS3 BTV proteins. First we used the IFNAR(-/-) mouse model system to establish a proof of principle, and afterwards we assayed the protective efficacy in sheep, the natural host of BTV. Mice were completely protected against BTV challenge, developing humoral and BTV-specific CD8+- and CD4+-T cell responses by vaccination with the different rAd5. Sheep vaccinated with Ad5-BTV-VP2 and Ad5-BTV-VP7 or only with Ad5-BTV-VP7 and challenged with BTV showed mild disease symptoms and reduced viremia. This partial protection was achieved in the absence of neutralizing antibodies but strong BTV-specific CD8+ T cell responses in those sheep vaccinated with Ad5-BTV-VP7. These data indicate that rAd5 is a suitable vaccine vector to induce T cell immunity during BTV vaccination and provide new data regarding the relevance of T cell responses in protection during BTV infection. PMID:26619062

  6. Recombinant BCG prime and PPE protein boost provides potent protection against acute Mycobacterium tuberculosis infection in mice.

    PubMed

    Yang, Enzhuo; Gu, Jin; Wang, Feifei; Wang, Honghai; Shen, Hongbo; Chen, Zheng W

    2016-04-01

    Since BCG, the only vaccine widely used against tuberculosis (TB) in the world, provides varied protective efficacy and may not be effective for inducing long-term cellular immunity, it is in an urgent need to develop more effective vaccines and more potent immune strategies against TB. Prime-boost is proven to be a good strategy by inducing long-term protection. In this study, we tested the protective effect against Mycobacterium tuberculosis (Mtb) challenge of prime-boost strategy by recombinant BCG (rBCG) expressing PPE protein Rv3425 fused with Ag85B and Rv3425. Results showed that the prime-boost strategy could significantly increase the protective efficiency against Mtb infection, characterized by reduction of bacterial load in lung and spleen, attenuation of tuberculosis lesions in lung tissues. Importantly, we found that Rv3425 boost, superior to Ag85B boost, provided better protection against Mtb infection. Further research proved that rBCG prime-Rv3425 boost could obviously increase the expansion of lymphocytes, significantly induce IL-2 production by lymphocytes upon PPD stimulation, and inhibit IL-6 production at an early stage. It implied that rBCG prime-Rv3425 boost opted to induce Th1 immune response and provided a long-term protection against TB. These results implicated that rBCG prime-Rv3425 boost is a potent and promising strategy to prevent acute Mtb infection.

  7. Companion Protease Inhibitors for the In Situ Protection of Recombinant Proteins in Plants.

    PubMed

    Robert, Stéphanie; Jutras, Philippe V; Khalf, Moustafa; D'Aoust, Marc-André; Goulet, Marie-Claire; Sainsbury, Frank; Michaud, Dominique

    2016-01-01

    We previously described a procedure for the use of plant protease inhibitors as "companion" accessory proteins to prevent unwanted proteolysis of clinically useful recombinant proteins in leaf crude protein extracts (Benchabane et al. Methods Mol Biol 483:265-273, 2009). Here we describe the use of these inhibitors for the protection of recombinant proteins in planta, before their extraction from leaf tissues. A procedure is first described involving inhibitors co-expressed along-and co-migrating-with the protein of interest in host plant cells. An alternative, single transgene scheme is then described involving translational fusions of the recombinant protein and companion inhibitor. These approaches may allow for a significant improvement of protein steady-state levels in leaves, comparable to yield improvements observed with protease-deficient strains of less complex protein expression hosts such as E. coli or yeasts.

  8. Vaccination with a single CD4 T cell peptide epitope from a Salmonella type III-secreted effector protein provides protection against lethal infection.

    PubMed

    Kurtz, Jonathan R; Petersen, Hailey E; Frederick, Daniel R; Morici, Lisa A; McLachlan, James B

    2014-06-01

    Salmonella infections affect millions worldwide and remain a significant cause of morbidity and mortality. It is known from mouse studies that CD4 T cells are essential mediators of immunity against Salmonella infection, yet it is not clear whether targeting CD4 T cell responses directly with peptide vaccines against Salmonella can be effective in combating infection. Additionally, it is not known whether T cell responses elicited against Salmonella secreted effector proteins can provide protective immunity against infection. In this study, we investigated both of these possibilities using prime-boost immunization of susceptible mice with a single CD4 T cell peptide epitope from Salmonella secreted effector protein I (SseI), a component of the Salmonella type III secretion system. This immunization conferred significant protection against lethal oral infection, equivalent to that conferred by whole heat-killed Salmonella bacteria. Surprisingly, a well-characterized T cell epitope from the flagellar protein FliC afforded no protection compared to immunization with an irrelevant control peptide. The protective response appeared to be most associated with polyfunctional CD4 T cells raised against the SseI peptide, since no antibodies were produced against any of the peptides and very little CD8 T cell response was observed. Overall, this study demonstrates that eliciting CD4 T cell responses against components of the Salmonella type III secretion system can contribute to protection against infection and should be considered in the design of future Salmonella subunit vaccines.

  9. Stimuli-Responsive Nanomaterials for Therapeutic Protein Delivery

    PubMed Central

    Lu, Yue; Sun, Wujin; Gu, Zhen

    2014-01-01

    Protein therapeutics have emerged as a significant role in treatment of a broad spectrum of diseases, including cancer, metabolic disorders and autoimmune diseases. The efficacy of protein therapeutics, however, is limited by their instability, immunogenicity and short half-life. In order to overcome these barriers, tremendous efforts have recently been made in developing controlled protein delivery systems. Stimuli-triggered release is an appealing and promising approach for protein delivery and has made protein delivery with both spatiotemporal- and dosage-controlled manners possible. This review surveys recent advances in controlled protein delivery of proteins or peptides using stimuli-responsive nanomaterials. Strategies utilizing both physiological and external stimuli are introduced and discussed. PMID:25151983

  10. Prion proteins with pathogenic and protective mutations show similar structure and dynamics

    PubMed Central

    Bae, Sung-Hun; Legname, Giuseppe; Serban, Ana; Prusiner, Stanley B.; Wright, Peter E.; Dyson, H. Jane

    2009-01-01

    Conformational change in the prion protein (PrP) is thought to be responsible for a group of rare but fatal neurodegenerative diseases of humans and other animals, including Creutzfeldt-Jakob disease and bovine spongiform encephalopathy. However, little is known about the mechanism by which normal cellular PrPs initiate and propagate the conformational change. Here, we studied backbone dynamics of the inherited pathogenic mutants (P101L and H186R), protective mutants (Q167R and Q218K), and wild type mouse PrP(89−230) at pH 5.5 and 3.5. Mutations result in minor chemical shift changes around the mutation sites except that H186R induces large chemical shift changes at distal regions. At lowered pH values, the C-terminal half of the second helix is significantly disordered for the wild type and all mutant proteins, while other parts of the protein are essentially unaffected. This destabilization is accompanied by protonation of the partially exposed histidine H186 in the second helix of the wild type protein. This region in the mutant protein H186R is disordered even at pH 5.5. The wild type and mutant proteins have similar μs conformational exchange near the two β-strands and have similar ns internal motions in several regions including the C-terminal half of the second helix, but only wild type and P101L have extensive ns internal motions throughout the helices. These motions mostly disappear at lower pH. Our findings raise the possibility that the pathogenic or dominant negative mutations exert their effects on some non-native intermediate form such as PrP* after conversion of cellular PrP (PrPC) into the pathogenic isoform PrPSc has been initiated; additionally, formation of PrPSc might begin within the C-terminal folded region rather than in the disordered N-terminal region. PMID:19618915

  11. Gut microbiota elicits a protective immune response against malaria transmission.

    PubMed

    Yilmaz, Bahtiyar; Portugal, Silvia; Tran, Tuan M; Gozzelino, Raffaella; Ramos, Susana; Gomes, Joana; Regalado, Ana; Cowan, Peter J; d'Apice, Anthony J F; Chong, Anita S; Doumbo, Ogobara K; Traore, Boubacar; Crompton, Peter D; Silveira, Henrique; Soares, Miguel P

    2014-12-04

    Glycosylation processes are under high natural selection pressure, presumably because these can modulate resistance to infection. Here, we asked whether inactivation of the UDP-galactose:β-galactoside-α1-3-galactosyltransferase (α1,3GT) gene, which ablated the expression of the Galα1-3Galβ1-4GlcNAc-R (α-gal) glycan and allowed for the production of anti-α-gal antibodies (Abs) in humans, confers protection against Plasmodium spp. infection, the causative agent of malaria and a major driving force in human evolution. We demonstrate that both Plasmodium spp. and the human gut pathobiont E. coli O86:B7 express α-gal and that anti-α-gal Abs are associated with protection against malaria transmission in humans as well as in α1,3GT-deficient mice, which produce protective anti-α-gal Abs when colonized by E. coli O86:B7. Anti-α-gal Abs target Plasmodium sporozoites for complement-mediated cytotoxicity in the skin, immediately after inoculation by Anopheles mosquitoes. Vaccination against α-gal confers sterile protection against malaria in mice, suggesting that a similar approach may reduce malaria transmission in humans.

  12. Vaccination with recombinant flagellar proteins FlgJ and FliN induce protection against Brucella abortus 544 infection in BALB/c mice.

    PubMed

    Li, Xianbo; Xu, Jie; Xie, Yongfei; Qiu, Yefeng; Fu, Simei; Yuan, Xitong; Ke, Yuehua; Yu, Shuang; Du, Xinying; Cui, Mingquan; Chen, Yanfen; Wang, Tongkun; Wang, Zhoujia; Yu, Yaqing; Huang, Kehe; Huang, Liuyu; Peng, Guangneng; Chen, Zeliang; Wang, Yufei

    2012-12-28

    Brucella has been considered as a non-motile, facultative intracellular pathogenic bacterium. However, the genome sequences of different Brucella species reveal the presence of the flagellar genes needed for the construction of a functional flagellum. Due to its roles in the interaction between pathogen and host, we hypothesized that some of the flagellar proteins might induce protective immune responses and these proteins will be good subunit vaccine candidates. This study was conducted to screening of protective antigens among these flagellar proteins. Firstly, according to the putative functional roles, a total of 30 flagellar genes of Brucella abortus were selected for in vitro expression. 15 of these flagellar genes were successfully expressed as his-tagged recombinant proteins in Escherichia coli ER2566. Then, these proteins were purified and used to analyze their T cell immunity induction activity by an in vitro gamma interferon (IFN-γ) assay. Five of the flagellar proteins could stimulate significantly higher levels of IFN-γ secretion in splenocytes from S19 immunized mice, indicating their T cell induction activity. Finally, immunogenicity and protection activity of these 5 flagellar proteins were evaluated in BALB/c mice. Results showed that immunization with FlgJ (BAB1_0260) or FliN (BAB2_0122) plus adjuvant could provide protection against B. abortus 544 infection. Furthermore, mice immunized with FlgJ and FliN developed a vigorous immunoglobulin G response, and in vitro stimulation of their splenocytes with immunizing proteins induced the secretion of IFN-γ. Altogether, these data suggest that flagellar proteins FlgJ and FliN are protective antigens that could produce humoral and cell-mediated responses in mice and candidates for use in future studies of vaccination against brucellosis.

  13. Defining the humoral immune response to infectious agents using high-density protein microarrays

    PubMed Central

    Vigil, Adam; Davies, D Huw; Felgner, Philip L

    2010-01-01

    A major component of the adaptive immune response to infection is the generation of protective and long-lasting humoral immunity. Traditional approaches to understanding the host’s humoral immune response are unable to provide an integrated understanding of the antibody repertoire generated in response to infection. By studying multiple antigenic responses in parallel, we can learn more about the breadth and dynamics of the antibody response to infection. Measurement of antibody production following vaccination is also a gauge for efficacy, as generation of antibodies can protect from future infections and limit disease. Protein microarrays are well suited to identify, quantify and compare individual antigenic responses following exposure to infectious agents. This technology can be applied to the development of improved serodiagnostic tests, discovery of subunit vaccine antigen candidates, epidemiologic research and vaccine development, as well as providing novel insights into infectious disease and the immune system. In this review, we will discuss the use of protein microarrays as a powerful tool to define the humoral immune response to bacteria and viruses. PMID:20143947

  14. Preclinical evaluation of bacterially produced RSV-G protein vaccine: Strong protection against RSV challenge in cotton rat model

    PubMed Central

    Fuentes, Sandra; Klenow, Laura; Golding, Hana; Khurana, Surender

    2017-01-01

    In current study, we evaluated the safety and protective efficacy of recombinant unglycosylated RSV G protein ectodomain produced in E. coli (in presence and absence of oil-in-water adjuvant) in a preclinical RSV susceptible cotton rat challenge model compared to formaldehyde inactivated RSV (FI-RSV) and live RSV experimental infection. The adjuvanted G protein vaccine induced robust neutralization antibody responses comparable to those generated by live RSV infection. Importantly, adjuvanted G protein significantly reduced viral loads in both the lungs and nose at early time points following viral challenge. Antibody kinetics determined by Surface Plasmon Resonance showed that adjuvanted G generated 10-fold higher G-binding antibodies compared to non-adjvuanted G vaccine and live RSV infection, which correlated strongly with both neutralization titers and viral load titers in the nose and lungs post-viral challenge. Antibody diversity analysis revealed immunodominant antigenic sites in the N- and C-termini of the RSV-G protein, that were boosted >10-fold by adjuvant and inversely correlated with viral load titers. Enhanced lung pathology was observed only in animals vaccinated with FI-RSV, but not in animals vaccinated with unadjuvanted or adjuvanted RSV-G vaccine after viral challenge. The bacterially produced unglycosylated G protein could be developed as a protective vaccine against RSV disease. PMID:28186208

  15. The Immune Response in Measles: Virus Control, Clearance and Protective Immunity.

    PubMed

    Griffin, Diane E

    2016-10-12

    Measles is an acute systemic viral infection with immune system interactions that play essential roles in multiple stages of infection and disease. Measles virus (MeV) infection does not induce type 1 interferons, but leads to production of cytokines and chemokines associated with nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) signaling and activation of the NACHT, LRR and PYD domains-containing protein (NLRP3) inflammasome. This restricted response allows extensive virus replication and spread during a clinically silent latent period of 10-14 days. The first appearance of the disease is a 2-3 day prodrome of fever, runny nose, cough, and conjunctivitis that is followed by a characteristic maculopapular rash that spreads from the face and trunk to the extremities. The rash is a manifestation of the MeV-specific type 1 CD4⁺ and CD8⁺ T cell adaptive immune response with lymphocyte infiltration into tissue sites of MeV replication and coincides with clearance of infectious virus. However, clearance of viral RNA from blood and tissues occurs over weeks to months after resolution of the rash and is associated with a period of immunosuppression. However, during viral RNA clearance, MeV-specific antibody also matures in type and avidity and T cell functions evolve from type 1 to type 2 and 17 responses that promote B cell development. Recovery is associated with sustained levels of neutralizing antibody and life-long protective immunity.

  16. The Immune Response in Measles: Virus Control, Clearance and Protective Immunity

    PubMed Central

    Griffin, Diane E.

    2016-01-01

    Measles is an acute systemic viral infection with immune system interactions that play essential roles in multiple stages of infection and disease. Measles virus (MeV) infection does not induce type 1 interferons, but leads to production of cytokines and chemokines associated with nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) signaling and activation of the NACHT, LRR and PYD domains-containing protein (NLRP3) inflammasome. This restricted response allows extensive virus replication and spread during a clinically silent latent period of 10–14 days. The first appearance of the disease is a 2–3 day prodrome of fever, runny nose, cough, and conjunctivitis that is followed by a characteristic maculopapular rash that spreads from the face and trunk to the extremities. The rash is a manifestation of the MeV-specific type 1 CD4+ and CD8+ T cell adaptive immune response with lymphocyte infiltration into tissue sites of MeV replication and coincides with clearance of infectious virus. However, clearance of viral RNA from blood and tissues occurs over weeks to months after resolution of the rash and is associated with a period of immunosuppression. However, during viral RNA clearance, MeV-specific antibody also matures in type and avidity and T cell functions evolve from type 1 to type 2 and 17 responses that promote B cell development. Recovery is associated with sustained levels of neutralizing antibody and life-long protective immunity. PMID:27754341

  17. Nanoparticles modulate surfactant protein A and D mediated protection against influenza A infection in vitro

    PubMed Central

    McKenzie, Zofi; Kendall, Michaela; Mackay, Rose-Marie; Tetley, Teresa D.; Morgan, Cliff; Griffiths, Mark; Clark, Howard W.; Madsen, Jens

    2015-01-01

    Numerous epidemiological and toxicological studies have indicated that respiratory infections are exacerbated following enhanced exposure to airborne particulates. Surfactant protein A (SP-A) and SP-D form an important part of the innate immune response in the lung and can interact with nanoparticles to modulate the cellular uptake of these particles. We hypothesize that this interaction will also affect the ability of these proteins to combat infections. TT1, A549 and differentiated THP-1 cells, representing the predominant cell types found in the alveolus namely alveolar type I (ATI) epithelial cells, ATII cells and macrophages, were used to examine the effect of two model nanoparticles, 100 nm amine modified (A-PS) and unmodified polystyrene (U-PS), on the ability of SP-A and SP-D to neutralize influenza A infections in vitro. Pre-incubation of low concentrations of U-PS with SP-A resulted in a reduction of SP-A anti-influenza activity in A549 cells, whereas at higher concentrations there was an increase in SP-A antiviral activity. This differential pattern of U-PS concentration on surfactant protein mediated protection against IAV was also shown with SP-D in TT1 cells. On the other hand, low concentrations of A-PS particles resulted in a reduction of SP-A activity in TT1 cells and a reduction in SP-D activity in A549 cells. These results indicate that nanoparticles can modulate the ability of SP-A and SP-D to combat viral challenges. Furthermore, the nanoparticle concentration, surface chemistry and cell type under investigation are important factors in determining the extent of these modulations. PMID:25533100

  18. Pathogenesis-related protein 1b1 (PR1b1) is a major tomato fruit protein responsive to chilling temperature and upregulated in high polyamine transgenic genotypes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plants execute an array of mechanisms in response to stress which include upregulation of defense-related proteins and changes in specific metabolites. A group of commonly found metabolites implicated in protection against stresses such as chilling stress constitute ubiquitous biogenic amines calle...

  19. Transcription Factor Functional Protein-Protein Interactions in Plant Defense Responses

    PubMed Central

    Alves, Murilo S.; Dadalto, Silvana P.; Gonçalves, Amanda B.; de Souza, Gilza B.; Barros, Vanessa A.; Fietto, Luciano G.

    2014-01-01

    Responses to biotic stress in plants lead to dramatic reprogramming of gene expression, favoring stress responses at the expense of normal cellular functions. Transcription factors are master regulators of gene expression at the transcriptional level, and controlling the activity of these factors alters the transcriptome of the plant, leading to metabolic and phenotypic changes in response to stress. The functional analysis of interactions between transcription factors and other proteins is very important for elucidating the role of these transcriptional regulators in different signaling cascades. In this review, we present an overview of protein-protein interactions for the six major families of transcription factors involved in plant defense: basic leucine zipper containing domain proteins (bZIP), amino-acid sequence WRKYGQK (WRKY), myelocytomatosis related proteins (MYC), myeloblastosis related proteins (MYB), APETALA2/ ETHYLENE-RESPONSIVE ELEMENT BINDING FACTORS (AP2/EREBP) and no apical meristem (NAM), Arabidopsis transcription activation factor (ATAF), and cup-shaped cotyledon (CUC) (NAC). We describe the interaction partners of these transcription factors as molecular responses during pathogen attack and the key components of signal transduction pathways that take place during plant defense responses. These interactions determine the activation or repression of response pathways and are crucial to understanding the regulatory networks that modulate plant defense responses. PMID:28250372

  20. Emerging functions of the unfolded protein response in immunity

    PubMed Central

    Janssens, Sophie; Pulendran, Bali; Lambrecht, Bart N.

    2015-01-01

    The unfolded protein response (UPR) has traditionally been viewed as an adaptive response triggered upon accumulation of unfolded proteins in the endoplasmic reticulum (ER), aimed at restoring ER function. The UPR can also be an anticipatory response that is activated well before the disruption of protein homeostasis. UPR signaling intersects at many levels with the innate and adaptive immune response. In some immune cell types like dendritic cells and B cells, particular UPR sensors appear constitutively active in the absence of traditional UPR gene program induction, necessary for antigen presentation and immunoglobulin synthesis. The UPR also influences Toll-like receptor signaling and NF-κB activation, and some pathogens subvert the UPR. This review summarizes these emerging non-canonical functions of the UPR in immunity. PMID:25232821

  1. Biofilm Matrix Exoproteins Induce a Protective Immune Response against Staphylococcus aureus Biofilm Infection

    PubMed Central

    Gil, Carmen; Solano, Cristina; Burgui, Saioa; Latasa, Cristina; García, Begoña; Toledo-Arana, Alejandro

    2014-01-01

    The Staphylococcus aureus biofilm mode of growth is associated with several chronic infections that are very difficult to treat due to the recalcitrant nature of biofilms to clearance by antimicrobials. Accordingly, there is an increasing interest in preventing the formation of S. aureus biofilms and developing efficient antibiofilm vaccines. Given the fact that during a biofilm-associated infection, the first primary interface between the host and the bacteria is the self-produced extracellular matrix, in this study we analyzed the potential of extracellular proteins found in the biofilm matrix to induce a protective immune response against S. aureus infections. By using proteomic approaches, we characterized the exoproteomes of exopolysaccharide-based and protein-based biofilm matrices produced by two clinical S. aureus strains. Remarkably, results showed that independently of the nature of the biofilm matrix, a common core of secreted proteins is contained in both types of exoproteomes. Intradermal administration of an exoproteome extract of an exopolysaccharide-dependent biofilm induced a humoral immune response and elicited the production of interleukin 10 (IL-10) and IL-17 in mice. Antibodies against such an extract promoted opsonophagocytosis and killing of S. aureus. Immunization with the biofilm matrix exoproteome significantly reduced the number of bacterial cells inside a biofilm and on the surrounding tissue, using an in vivo model of mesh-associated biofilm infection. Furthermore, immunized mice also showed limited organ colonization by bacteria released from the matrix at the dispersive stage of the biofilm cycle. Altogether, these data illustrate the potential of biofilm matrix exoproteins as a promising candidate multivalent vaccine against S. aureus biofilm-associated infections. PMID:24343648

  2. Vaccination with TAT-antigen fusion protein induces protective, CD8(+) T cell-mediated immunity against Leishmania major.

    PubMed

    Kronenberg, Katharina; Brosch, Sven; Butsch, Florian; Tada, Yayoi; Shibagaki, Naotaka; Udey, Mark C; von Stebut, Esther

    2010-11-01

    In murine leishmaniasis, healing is mediated by IFN-γ-producing CD4(+) and CD8(+) T cells. Thus, an efficacious vaccine should induce Th1 and Tc1 cells. Dendritic cells (DCs) pulsed with exogenous proteins primarily induce strong CD4-dependent immunity; induction of CD8 responses has proven to be difficult. We evaluated the immunogenicity of fusion proteins comprising the protein transduction domain of HIV-1 TAT and the Leishmania antigen LACK (Leishmania homolog of receptors for activated C kinase), as TAT-fusion proteins facilitate major histocompatibility complex class I-dependent antigen presentation. In vitro, TAT-LACK-pulsed DCs induced stronger proliferation of Leishmania-specific CD8(+) T cells compared with DCs incubated with LACK alone. Vaccination with TAT-LACK-pulsed DCs or fusion proteins plus adjuvant in vivo significantly improved disease outcome in Leishmania major-infected mice and was superior to vaccination with DCs treated with LACK alone. Vaccination with DC+TAT-LACK resulted in stronger proliferation of CD8(+) T cells when compared with immunization with DC+LACK. Upon depletion of CD4(+) or CD8(+) T cells, TAT-LACK-mediated protection was lost. TAT-LACK-pulsed IL-12p40-deficient DCs did not promote protection in vivo. In summary, these data show that TAT-fusion proteins are superior in activating Leishmania-specific Tc1 cells when compared with antigen alone and suggest that IL-12-dependent preferential induction of antigen-specific CD8(+) cells promotes significant protection against this important human pathogen.

  3. Extracellular proteins of Cryptococcus neoformans and host antibody response.

    PubMed Central

    Chen, L C; Pirofski, L A; Casadevall, A

    1997-01-01

    Proteins secreted by the fungal pathogen Cryptococcus neoformans may be involved in invasion and could be useful in vaccine design. Despite the medical importance of this fungus, little is known about its extracellular proteins or the immune response to these antigens. To study C. neoformans extracellular proteins, 12 strains were metabolically radiolabeled and protein supernatants were analyzed. Both strain- and growth condition-dependent differences were observed. Enzymatic assays of filtered culture supernatants revealed butyrate esterase and caprylate esterase lipase activity for 11 of 12 strains, as well as acid phosphatase, naphthol-AS-BI-phosphohydrolase, and beta-glucosidase activities in some strains. Serum from infected rodents immunoprecipitated several secreted proteins, consistent with in vivo expression and development of an antibody response. For strain 24067, two immunodominant species, of approximately 75 and 30 kDa, were recognized. The relative intensity of the autoradiographic bands depended on the route of infection for both rats and mice. In summary, our results indicate that (i) there are multiple proteins in C. neoformans culture supernatants, (ii) there are strain differences in supernatant protein profiles, (iii) there are differences in supernatant protein profile depending on the growth conditions, (iv) there are several new extracellular and/or cell-associated enzymatic activities, and (v) antibodies to several supernatant proteins are made in the course of infection. PMID:9199426

  4. Response of surge protection devices to fast rising pulses

    NASA Technical Reports Server (NTRS)

    Mindel, I. N.

    1980-01-01

    Two types of lightning protection modules incorporating leadless (pill type) Zener like devices were evaluated with regard to their ability to suppress EMP induced transients. Two series of tests were performed to evaluate the ability of these modules to react to fast rate of rise ( 1Kv/ns) transients, and the attenuation introduced and the ability to limit damped sinusoid pulses which may be induced due to an EMP resulting from a nuclear detonation.

  5. Tomato QM-like protein protects Saccharomyces cerevisiae cells against oxidative stress by regulating intracellular proline levels.

    PubMed

    Chen, Changbin; Wanduragala, Srimevan; Becker, Donald F; Dickman, Martin B

    2006-06-01

    Exogenous proline can protect cells of Saccharomyces cerevisiae from oxidative stress. We altered intracellular proline levels by overexpressing the proline dehydrogenase gene (PUT1) of S. cerevisiae. Put1p performs the first enzymatic step of proline degradation in S. cerevisiae. Overexpression of Put1p results in low proline levels and hypersensitivity to oxidants, such as hydrogen peroxide and paraquat. A put1-disrupted yeast mutant deficient in Put1p activity has increased protection from oxidative stress and increased proline levels. Following a conditional life/death screen in yeast, we identified a tomato (Lycopersicon esculentum) gene encoding a QM-like protein (tQM) and found that stable expression of tQM in the Put1p-overexpressing strain conferred protection against oxidative damage from H2O2, paraquat, and heat. This protection was correlated with reactive oxygen species (ROS) reduction and increased proline accumulation. A yeast two-hybrid system assay was used to show that tQM physically interacts with Put1p in yeast, suggesting that tQM is directly involved in modulating proline levels. tQM also can rescue yeast from the lethality mediated by the mammalian proapoptotic protein Bax, through the inhibition of ROS generation. Our results suggest that tQM is a component of various stress response pathways and may function in proline-mediated stress tolerance in plants.

  6. Immediate induction of heat shock proteins is not protective against cryopreservation in normal human fibroblasts.

    PubMed

    Park, S J; Choi, H R; Nam, K M; Na, J I; Huh, C H; Park, K C

    2013-01-01

    Heat shock proteins (HSPs) were first identified as proteins whose synthesis was enhanced by stresses, such as increased temperature. HSPs can protect cells from various cytotoxic factors by stabilizing proteins. Thus, it could be hypothesized that heat induced HSPs can provide protective effects against cryopreservation-induced cell death. The aim of this study was to determine whether induction of HSPs can increase the cell viability of normal human fibroblasts after cryopreservation. Cytotoxic effects of heat treatment were tested and the induction of HSPs was assessed by examining time-dependent HSP expression. A cell counting method using fluorescence microscopy was used to determine the viability of cells. In addition, the effects of geranylgeranylacetone were evaluated in terms of HSP expression and cytoskeleton changes. The results of this study showed that immediate induction of HSPs does not protect normal human fibroblasts against cryopreservation-induced cell death possibly by inducing cytoskeleton changes.

  7. Enhanced protective antibody to a mutant meningococcal factor H-binding protein with low-factor H binding

    PubMed Central

    Granoff, Dan M.; Giuntini, Serena; Gowans, Flor A.; Lujan, Eduardo; Sharkey, Kelsey; Beernink, Peter T.

    2016-01-01

    Meningococcal factor H-binding protein (FHbp) is an antigen in 2 serogroup B meningococcal vaccines. FHbp specifically binds human and some nonhuman primate complement FH. To investigate the effect of binding of FH to FHbp on protective antibody responses, we immunized infant rhesus macaques with either a control recombinant FHbp antigen that bound macaque FH or a mutant antigen with 2 amino acid substitutions and >250-fold lower affinity for FH. The mutant antigen elicited 3-fold higher serum IgG anti-FHbp titers and up to 15-fold higher serum bactericidal titers than the control FHbp vaccine. When comparing sera with similar IgG anti-FHbp titers, the antibodies elicited by the mutant antigen gave greater deposition of complement component C4b on live meningococci (classical complement pathway) and inhibited binding of FH, while the anti-FHbp antibodies elicited by the control vaccine enhanced FH binding. Thus, the mutant FHbp vaccine elicited an anti-FHbp antibody repertoire directed at FHbp epitopes within the FH binding site, which resulted in greater protective activity than the antibodies elicited by the control vaccine, which targeted FHbp epitopes outside of the FH combining site. Binding of a host protein to a vaccine antigen impairs protective antibody responses, which can be overcome with low-binding mutant antigens. PMID:27668287

  8. Aging Is Accompanied by a Blunted Muscle Protein Synthetic Response to Protein Ingestion

    PubMed Central

    Wall, Benjamin Toby; Gorissen, Stefan H.; Pennings, Bart; Koopman, René; Groen, Bart B. L.; Verdijk, Lex B.; van Loon, Luc J. C.

    2015-01-01

    Purpose Progressive loss of skeletal muscle mass with aging (sarcopenia) forms a global health concern. It has been suggested that an impaired capacity to increase muscle protein synthesis rates in response to protein intake is a key contributor to sarcopenia. We assessed whether differences in post-absorptive and/or post-prandial muscle protein synthesis rates exist between large cohorts of healthy young and older men. Procedures We performed a cross-sectional, retrospective study comparing in vivo post-absorptive muscle protein synthesis rates determined with stable isotope methodologies between 34 healthy young (22±1 y) and 72 older (75±1 y) men, and post-prandial muscle protein synthesis rates between 35 healthy young (22±1 y) and 40 older (74±1 y) men. Findings Post-absorptive muscle protein synthesis rates did not differ significantly between the young and older group. Post-prandial muscle protein synthesis rates were 16% lower in the older subjects when compared with the young. Muscle protein synthesis rates were >3 fold more responsive to dietary protein ingestion in the young. Irrespective of age, there was a strong negative correlation between post-absorptive muscle protein synthesis rates and the increase in muscle protein synthesis rate following protein ingestion. Conclusions Aging is associated with the development of muscle anabolic inflexibility which represents a key physiological mechanism underpinning sarcopenia. PMID:26536130

  9. Protective effects of leucine against lipopolysaccharide-induced inflammatory response in Labeo rohita fingerlings.

    PubMed

    Giri, Sib Sankar; Sen, Shib Sankar; Jun, Jin Woo; Sukumaran, Venkatachalam; Park, Se Chang

    2016-05-01

    The present study investigated the protective effects of leucine against lipopolysaccharide (LPS)-induced inflammatory responses in Labeo rohita (rohu) in vivo and in vitro. Primary hepatocytes, isolated from the hepatopancreas, were exposed to different concentrations of LPS for 24 h to induce an inflammatory response, and the protective effects of leucine against LPS-induced inflammation were studied. Finally, we investigated the efficiency of dietary leucine supplementation in attenuating an immune challenge induced by LPS in vivo. Exposure of cells to 10-25 μg mL(-1) of LPS for 24 h resulted in a significant production of nitric oxide and release of lactate dehydrogenase to the medium, whereas cell viability and protein content were reduced (p < 0.05). LPS exposure (10 μg mL(-1)) increased mRNA levels of the pro-inflammatory cytokines TNF-α, IL-1β and IL-8 in vitro (p < 0.05). However, pretreatment with leucine prevented the LPS-induced upregulation of TNF-α, IL-1β and IL-8 mRNAs by downregulating TLR4, MyD88, NF-κBp65, and MAPKp38 mRNA expression. Interestingly, mRNA expression of the anti-inflammatory cytokine, IL-10, which was increased by LPS treatment, was further enhanced (p < 0.05) by leucine pretreatment. The enhanced expression of IL-10 might inhibit the production of other pro-inflammatory cytokines. It was found that leucine pretreatment attenuated the excessive activation of LPS-induced TLR4-MyD88 signaling as manifested by lower level of TLR4, MyD88, MAPKp38, NF-κBp65 and increased level of IκB-α protein in leucine pre-treatment group. In vivo experiments demonstrated that leucine pre-supplementation could protect fish against LPS-induced inflammation through an attenuation of TLR4-MyD88 signaling pathway. Taken together, we propose that leucine pre-supplementation decreases LPS-induced immune damage in rohu by enhancing the expression of IL-10 and by regulating the TLR4-MyD88 signaling pathways.

  10. Immunogenicity and protective role of antigenic regions from five outer membrane proteins of Flavobacterium columnare in grass carp Ctenopharyngodon idella

    NASA Astrophysics Data System (ADS)

    Luo, Zhang; Liu, Zhixin; Fu, Jianping; Zhang, Qiusheng; Huang, Bei; Nie, Pin

    2016-11-01

    Flavobacterium columnare causes columnaris disease in freshwater fish. In the present study, the antigenic regions of five outer membrane proteins (OMPs), including zinc metalloprotease, prolyl oligopeptidase, thermolysin, collagenase and chondroitin AC lyase, were bioinformatically analyzed, fused together, and then expressed as a recombinant fusion protein in Escherichia coli. The expressed protein of 95.6 kDa, as estimated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was consistent with the molecular weight deduced from the amino acid sequence. The purified recombinant protein was used to vaccinate the grass carp, Ctenopharyngodon idella. Following vaccination of the fish their IgM antibody levels were examined, as was the expression of IgM, IgD and IgZ immunoglobulin genes and other genes such as MHC Iα and MHC IIβ, which are also involved in adaptive immunity. Interleukin genes ( IL), including IL-1β, IL-8 and IL-10, and type I and type II interferon ( IFN) genes were also examined. At 3 and 4 weeks post-vaccination (wpv), significant increases in IgM antibody levels were observed in the fish vaccinated with the recombinant fusion protein, and an increase in the expression levels of IgM, IgD and IgZ genes was also detected following the vaccinations, thus indicating that an adaptive immune response was induced by the vaccinations. Early increases in the expression levels of IL and IFN genes were also observed in the vaccinated fish. At four wpv, the fish were challenged with F. columnare, and the vaccinated fish showed a good level of protection against this pathogen, with 39% relative percent survival (RPS) compared with the control group. It can be concluded, therefore, that the five OMPs, in the form of a recombinant fusion protein vaccine, induced an immune response in fish and protection against F. columnare.

  11. Knockdown of Ice-Binding Proteins in Brachypodium distachyon Demonstrates Their Role in Freeze Protection

    PubMed Central

    Bredow, Melissa; Vanderbeld, Barbara; Walker, Virginia K.

    2016-01-01

    Sub-zero temperatures pose a major threat to the survival of cold-climate perennials. Some of these freeze-tolerant plants produce ice-binding proteins (IBPs) that offer frost protection by restricting ice crystal growth and preventing expansion-induced lysis of the plasma membranes. Despite the extensive in vitro characterization of such proteins, the importance of IBPs in the freezing stress response has not been investigated. Using the freeze-tolerant grass and model crop, Brachypodium distachyon, we characterized putative IBPs (BdIRIs) and generated the first ‘IBP-knockdowns’. Seven IBP sequences were identified and expressed in Escherichia coli, with all of the recombinant proteins demonstrating moderate to high levels of ice-recrystallization inhibition (IRI) activity, low levels of thermal hysteresis (TH) activity (0.03−0.09°C at 1 mg/mL) and apparent adsorption to ice primary prism planes. Following plant cold acclimation, IBPs purified from wild-type B. distachyon cell lysates similarly showed high levels of IRI activity, hexagonal ice-shaping, and low levels of TH activity (0.15°C at 0.5 mg/mL total protein). The transfer of a microRNA construct to wild-type plants resulted in the attenuation of IBP activity. The resulting knockdown mutant plants had reduced ability to restrict ice-crystal growth and a 63% reduction in TH activity. Additionally, all transgenic lines were significantly more vulnerable to electrolyte leakage after freezing to −10°C, showing a 13−22% increase in released ions compared to wild-type. IBP-knockdown lines also demonstrated a significant decrease in viability following freezing to −8°C, with some lines showing only two-thirds the survival seen in control lines. These results underscore the vital role IBPs play in the development of a freeze-tolerant phenotype and suggests that expression of these proteins in frost-susceptible plants could be valuable for the production of more winter-hardy crops. PMID:27959937

  12. The Unfolded Protein Response in Homeostasis and Modulation of Mammalian Immune Cells.

    PubMed

    Martins, Ana Sofia; Alves, Inês; Helguero, Luisa; Domingues, Maria Rosário; Neves, Bruno Miguel

    2016-11-01

    The endoplasmic reticulum (ER) plays important roles in eukaryotic protein folding and lipid biosynthesis. Several exogenous and endogenous cellular sources of stress can perturb ER homeostasis leading to the accumulation of unfolded proteins in the lumen. Unfolded protein accumulation triggers a signal-transduction cascade known as the unfolded protein response (UPR), an adaptive mechanism which aims to protect cells from protein aggregates and to restore ER functions. Further to this protective mechanism, in immune cells, UPR molecular effectors have been shown to participate in a wide range of biological processes such as cell differentiation, survival and immunoglobulin and cytokine production. Recent findings also highlight the involvement of the UPR machinery in the maturational program and antigen presentation capacities of dendritic cells. UPR is therefore a key element in immune system homeostasis with direct implications on both adaptive and innate immune responses. The present review summarizes the knowledge on the emerging roles of UPR signaling cascades in mammalian immune cells as well as the consequences of their dysregulation in relation to the pathogenesis of several diseases.

  13. Bioinformatics and variability in drug response: a protein structural perspective

    PubMed Central

    Lahti, Jennifer L.; Tang, Grace W.; Capriotti, Emidio; Liu, Tianyun; Altman, Russ B.

    2012-01-01

    Marketed drugs frequently perform worse in clinical practice than in the clinical trials on which their approval is based. Many therapeutic compounds are ineffective for a large subpopulation of patients to whom they are prescribed; worse, a significant fraction of patients experience adverse effects more severe than anticipated. The unacceptable risk–benefit profile for many drugs mandates a paradigm shift towards personalized medicine. However, prior to adoption of patient-specific approaches, it is useful to understand the molecular details underlying variable drug response among diverse patient populations. Over the past decade, progress in structural genomics led to an explosion of available three-dimensional structures of drug target proteins while efforts in pharmacogenetics offered insights into polymorphisms correlated with differential therapeutic outcomes. Together these advances provide the opportunity to examine how altered protein structures arising from genetic differences affect protein–drug interactions and, ultimately, drug response. In this review, we first summarize structural characteristics of protein targets and common mechanisms of drug interactions. Next, we describe the impact of coding mutations on protein structures and drug response. Finally, we highlight tools for analysing protein structures and protein–drug interactions and discuss their application for understanding altered drug responses associated with protein structural variants. PMID:22552919

  14. Meeting Report: Structural Determination of Environmentally Responsive Proteins

    PubMed Central

    Reinlib, Leslie

    2005-01-01

    The three-dimensional structure of gene products continues to be a missing lynchpin between linear genome sequences and our understanding of the normal and abnormal function of proteins and pathways. Enhanced activity in this area is likely to lead to better understanding of how discrete changes in molecular patterns and conformation underlie functional changes in protein complexes and, with it, sensitivity of an individual to an exposure. The National Institute of Environmental Health Sciences convened a workshop of experts in structural determination and environmental health to solicit advice for future research in structural resolution relative to environmentally responsive proteins and pathways. The highest priorities recommended by the workshop were to support studies of structure, analysis, control, and design of conformational and functional states at molecular resolution for environmentally responsive molecules and complexes; promote understanding of dynamics, kinetics, and ligand responses; investigate the mechanisms and steps in posttranslational modifications, protein partnering, impact of genetic polymorphisms on structure/function, and ligand interactions; and encourage integrated experimental and computational approaches. The workshop participants also saw value in improving the throughput and purity of protein samples and macromolecular assemblies; developing optimal processes for design, production, and assembly of macromolecular complexes; encouraging studies on protein–protein and macromolecular interactions; and examining assemblies of individual proteins and their functions in pathways of interest for environmental health. PMID:16263521

  15. Responsive block copolymer photonics triggered by protein-polyelectrolyte coacervation.

    PubMed

    Fan, Yin; Tang, Shengchang; Thomas, Edwin L; Olsen, Bradley D

    2014-11-25

    Ionic interactions between proteins and polyelectrolytes are demonstrated as a method to trigger responsive transitions in block copolymer (BCP) photonic gels containing one neutral hydrophobic block and one cationic hydrophilic block. Poly(2-vinylpyridine) (P2VP) blocks in lamellar poly(styrene-b-2-vinylpyridine) block copolymer thin films are quaternized with primary bromides to yield swollen gels that show strong reflectivity peaks in the visible range; exposure to aqueous solutions of various proteins alters the swelling ratios of the quaternized P2VP (QP2VP) gel layers in the PS-QP2VP materials due to the ionic interactions between proteins and the polyelectrolyte. Parameters such as charge density, hydrophobicity, and cross-link density of the QP2VP gel layers as well as the charge and size of the proteins play significant roles on the photonic responses of the BCP gels. Differences in the size and pH-dependent charge of proteins provide a basis for fingerprinting proteins based on their temporal and equilibrium photonic response. The results demonstrate that the BCP gels and their photonic effect provide a robust and visually interpretable method to differentiate different proteins.

  16. A recombinant fusion protein displaying murine and human MHC class I- and II-specific epitopes protects against Leishmania amazonensis infection.

    PubMed

    Martins, Vívian T; Lage, Daniela P; Duarte, Mariana C; Carvalho, Ana Maria R S; Costa, Lourena E; Mendes, Tiago A O; Vale, Danniele L; Menezes-Souza, Daniel; Roatt, Bruno M; Tavares, Carlos A P; Soto, Manuel; Coelho, Eduardo A F

    2017-03-01

    Tegumentary leishmaniasis (TL) constitutes a major public health problem with significant morbidity worldwide. Synthetic peptide-based vaccines are attractive candidates to protect against leishmaniasis, since T cell-specific epitopes can be delivery to antigen-presenting cells, leading to the generation of a Th1 cell-mediated immunity. In this context, the present study aims to evaluate the immunogenicity and protective efficacy of a vaccine composed of major histocompatibility complex class I and II-restricted epitopes derived from four Leishmania infantum proteins to protect mice against Leishmania amazonensis infection. This recombinant fusion protein was administered in BALB/c mice alone or with saponin. As controls, animals received saline or saponin. In the results, the administration of the recombinant protein plus saponin induced a specific IFN-γ, IL-12 and GM-CSF production, as well as high IgG2a isotype antibody levels, which protected mice against a challenge using L. amazonensis promastigotes. Lower parasite burden was found in the infected footpads, liver, spleen and draining lymph node of vaccinated mice, when compared to those from the control groups. In addition, protection was associated with a lower IL-4 and IL-10 response, which was accompanied by the antileishmanial nitrite production by spleen cells of the animals. Interestingly, the recombinant protein administered alone induced a partial protection against challenge. In conclusion, this study shows a new vaccine candidate based on T cell-specific epitopes that was able to induce protection against L. amazonensis infection.

  17. Overexpressed heat shock protein 70 protects cells against DNA damage caused by ultraviolet C in a dose-dependent manner

    PubMed Central

    Niu, Piye; Liu, Lin; Gong, Zhiyong; Tan, Hao; Wang, Feng; Yuan, Jing; Feng, Youmei; Wei, Qingyi; Tanguay, Robert M; Wu, Tangchun

    2006-01-01

    Heat shock protein 70 (Hsp70) comprises proteins that have been reported to protect cells, tissues, and organisms against damage from a wide variety of stressful stimuli; however, little is known about whether Hsp70 protects against DNA damage. In this study, we investigated the relationship between Hsp70 expression and the levels of ultraviolet C (UVC)–induced DNA damage in A549 cells with normal, inhibited, and overexpressed Hsp70 levels. Hsp70 expression was inhibited by treatment with quercetin or overexpressed by transfection of plasmids harboring the hsp70 gene. The level of DNA damage was assessed by the comet assay. The results showed that the levels of DNA damage (shown as the percentage of comet cells) in A549 cells increased in all cells after exposure to an incident dose of 0, 10, 20, 40, and 80 J/m2 whether Hsp70 was inhibited or overexpressed. This response was dose dependent: a protection against UVC-induced DNA damage in cells with overexpressed Hsp70 was observed at UVC dose 20 J/ m2 with a maximum at 40 J/m2 when compared with cells with normal Hsp70 levels and in quercetin-treated cells. This differential protection disappeared at 80 J/m2. These results suggest that overexpressed Hsp70 might play a role in protecting A549 cells from DNA damage caused by UVC irradiation, with a threshold of protection from at UVC irradiation-induced DNA damage by Hsp70. The detailed mechanism how Hsp70 is involved in DNA damage and possible DNA repair warrants further investigation. PMID:16817322

  18. A DNA vaccine encoding the E protein of West Nile virus is protective and can be boosted by recombinant domain DIII.

    PubMed

    Schneeweiss, Anne; Chabierski, Stefan; Salomo, Mathias; Delaroque, Nicolas; Al-Robaiy, Samiya; Grunwald, Thomas; Bürki, Kurt; Liebert, Uwe G; Ulbert, Sebastian

    2011-08-26

    West Nile Virus (WNV) is an emerging pathogenic flavivirus with increasing distribution worldwide. Birds are the natural host of the virus, but also mammals, including humans, can be infected. In some cases, a WNV infection can be associated with severe neurological symptoms. All currently available WNV vaccines are in the veterinary sector, and there is a need to develop safe and effective immunization technologies, which can also be used in humans. An alternative to current vaccination methods is DNA immunization. Most current DNA vaccine candidates against flaviviruses simultaneously express the viral envelope (E) and membrane (prM) proteins, which leads to the formation of virus-like particles. Here we generated a DNA plasmid, which expresses only the E-protein ectodomain. Vaccination of mice stimulated anti-WNV T-cell responses and neutralizing antibodies that were higher than those obtained after immunizing with a recombinant protein previously shown to be a protective WNV vaccine. A single dose of the plasmid was sufficient to protect animals from a lethal challenge with the virus. Moreover, immunogenicity could be boosted when DNA injection was followed by immunization with recombinant domain DIII of the E-protein. This resulted in significantly enhanced neutralizing antibody titers and a more prominent cellular immune response. The results suggest that the WNV E-protein is sufficient as a protective antigen in DNA vaccines and that protection can be significantly improved by adding a recombinant protein boost to the DNA prime.

  19. The responsibility to protect and the root causes of conflicts.

    PubMed

    von Horn, Helge

    2005-01-01

    The increase in humanitarian crises due to states failing to observe the human rights of their citizens led to the establishment of the International Commission on Intervention and State Sovereignty. Whilst all agree with the need for concerted action to protect innocent civilians from human rights abuses the focus must be on prevention; the use of military force creates further problems. An analysis of failed states, such as Rwanda, shows how the actions of the international community, especially the most powerful states, contribute to conditions creating the abuse and how appropriate actions by the external players could have prevented these abuses.

  20. Serum Response Factor Protects Retinal Ganglion Cells Against High-Glucose Damage.

    PubMed

    Cao, Yan; Wang, Liang; Zhao, Junhong; Zhang, Hongbing; Tian, Ying; Liang, Houcheng; Ma, Qiang

    2016-06-01

    Serum response factor (SRF), which encodes the MADS-box family of related proteins, is a common transcription factor related to the expression of genes associated with cell survival. However, SRF's role in retinal ganglion cells (RGCs) after high-glucose injury remains unclear. In this study, we investigate the protective role of SRF after high-glucose injury and its underlying mechanism. The in vitro RGC model subjected to high glucose was established by employing a 50 mmol/L glucose culture environment. As detected by real-time quantitative PCR and Western blot, SRF was significantly upregulated in RGCs treated with high glucose. Overexpression of SRF significantly promoted survival among RGCs exposed to high glucose and inhibited RGC apoptosis. Knockdown of SRF exerted an inverse effect. Moreover, SRF upregulation enhanced expression of an antioxidant protein, nuclear factor erythroid 2-related factor (Nrf2), via control of the Fos-related antigen 1 (Fra-1). SRF upregulation also affected RGC survival after high-glucose treatment. Our findings showed that overexpression of SRF promoted survival of RGCs after high-glucose injury by regulating Fra-1 and Nrf2.

  1. Protein-protected luminescent noble metal quantum clusters: an emerging trend in atomic cluster nanoscience.

    PubMed

    Xavier, Paulrajpillai Lourdu; Chaudhari, Kamalesh; Baksi, Ananya; Pradeep, Thalappil

    2012-01-01

    Noble metal quantum clusters (NMQCs) are the missing link between isolated noble metal atoms and nanoparticles. NMQCs are sub-nanometer core sized clusters composed of a group of atoms, most often luminescent in the visible region, and possess intriguing photo-physical and chemical properties. A trend is observed in the use of ligands, ranging from phosphines to functional proteins, for the synthesis of NMQCs in the liquid phase. In this review, we briefly overview recent advancements in the synthesis of protein protected NMQCs with special emphasis on their structural and photo-physical properties. In view of the protein protection, coupled with direct synthesis and easy functionalization, this hybrid QC-protein system is expected to have numerous optical and bioimaging applications in the future, pointers in this direction are visible in the literature.

  2. Induction of Multifunctional Broadly Reactive T Cell Responses by a Plasmodium vivax Circumsporozoite Protein Recombinant Chimera.

    PubMed

    Cabrera-Mora, Monica; Fonseca, Jairo Andres; Singh, Balwan; Oliveira-Ferreira, Joseli; Lima-Junior, Josué da Costa; Calvo-Calle, J Mauricio; Moreno, Alberto

    2015-09-01

    Plasmodium vivax is the most widespread species of Plasmodium, causing up to 50% of the malaria cases occurring outside sub-Saharan Africa. An effective vaccine is essential for successful control and potential eradication. A well-characterized vaccine candidate is the circumsporozoite protein (CSP). Preclinical and clinical trials have shown that both antibodies and cellular immune responses have been correlated with protection induced by immunization with CSP. On the basis of our reported approach of developing chimeric Plasmodium yoelii proteins to enhance protective efficacy, we designed PvRMC-CSP, a recombinant chimeric protein based on the P. vivax CSP (PvCSP). In this engineered protein, regions of the PvCSP predicted to contain human T cell epitopes were genetically fused to an immunodominant B cell epitope derived from the N-terminal region I and to repeat sequences representing the two types of PvCSP repeats. The chimeric protein was expressed in soluble form with high yield. As the immune response to PvCSP has been reported to be genetically restricted in the murine model, we tested the immunogenicity of PvRMC-CSP in groups of six inbred strains of mice. PvRMC-CSP was able to induce robust antibody responses in all the mouse strains tested. Synthetic peptides representing the allelic forms of the P. vivax CSP were also recognized to a similar extent regardless of the mouse strain. Furthermore, the immunization regimen induced high frequencies of multifunctional CD4(+) and CD8(+) PvRMC-CSP-specific T cells. The depth and breadth of the immune responses elicited suggest that immunization with PvRMC-CSP can circumvent the genetic restriction of the immune response to P. vivax CSP. Interestingly, PvRMC-CSP was also recognized by naturally acquired antibodies from individuals living in areas where malaria is endemic. These features make PvRMC-CSP a promising vaccine candidate for further development.

  3. Functional Mapping of Protective Domains and Epitopes in the Rotavirus VP6 Protein

    PubMed Central

    Choi, Anthony H. C.; Basu, Mitali; McNeal, Monica M.; Flint, Jason; VanCott, John L.; Clements, John D.; Ward, Richard L.

    2000-01-01

    The purpose of this study was to determine which regions of the VP6 protein of the murine rotavirus strain EDIM are able to elicit protection against rotavirus shedding in the adult mouse model following intranasal (i.n.) immunization with fragments of VP6 and a subsequent oral EDIM challenge. In the initial experiment, the first (fragment AB), middle (BC), or last (CD) part of VP6 that was genetically fused to maltose-binding protein (MBP) and expressed in Escherichia coli was examined. Mice (BALB/c) immunized with two 9-μg doses of each of the chimeras and 10 μg of the mucosal adjuvant LT(R192G) were found to be protected against EDIM shedding (80, 92, and nearly 100% reduction, respectively; P ≤ 0.01) following challenge. Because CD produced almost complete protection, we prepared four E. coli-expressed, MBP-fused chimeras containing overlapping fragments of the CD region (i.e., CD1, CD2, CD3, and CD4) whose lengths ranged from 61 to 67 amino acid residues. Following i.n. immunization, CD1, CD2, and CD4 induced significant (P ≤ 0.004) protection (88, 84, and 92% reduction, respectively). In addition, 11 peptides (18 to 30 residues) of the CD region with between 0 and 13 overlapping amino acids were synthesized. Two 50-μg doses of each peptide with LT(R192G) were administered i.n. to BALB/c mice. Five peptides were found to elicit significant (P ≤ 0.02) protection. Moreover, a 14-amino-acid region within peptide 6 containing a putative CD4+ T-cell epitope was found to confer nearly complete protection, suggesting a protective role for CD4+ T cells. Mice that were protected by fragments BC and CD1 and four of the five protective synthetic peptides did not develop measurable rotavirus antibodies in serum or stool, implying that protection induced by these domains was not dependent on antibody. Together, these observations suggest that multiple regions of VP6 can stimulate protection, a region of VP6 as small as 14 amino acids containing a CD4+ T

  4. C/EBP homologous protein-induced macrophage apoptosis protects mice from steatohepatitis.

    PubMed

    Malhi, Harmeet; Kropp, Erin M; Clavo, Vinna F; Kobrossi, Christina R; Han, JaeSeok; Mauer, Amy S; Yong, Jing; Kaufman, Randal J

    2013-06-28

    Nonalcoholic fatty liver disease is a heterogeneous disorder characterized by liver steatosis; inflammation and fibrosis are features of the progressive form nonalcoholic steatohepatitis. The endoplasmic reticulum stress response is postulated to play a role in the pathogenesis of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis. In particular, C/EBP homologous protein (CHOP) is undetectable under normal conditions but is induced by cellular stress, including endoplasmic reticulum stress. Chop wild type (Chop(+/+)) and knock-out (Chop(-/-)) mice were used in these studies to elucidate the role of CHOP in the pathogenesis of fatty liver disease. Paradoxically, Chop(-/-) mice developed greater liver injury, inflammation, and fibrosis than Chop(+/+) mice, with greater macrophage activation. Primary, bone marrow-derived, and peritoneal macrophages from Chop(+/+) and Chop(-/-) were challenged with palmitic acid, an abundant saturated free fatty acid in plasma and liver lipids. Where palmitic acid treatment activated Chop(+/+) and Chop(-/-) macrophages, Chop(-/-) macrophages were resistant to its lipotoxicity. Chop(-/-) mice were sensitized to liver injury in a second model of dietary steatohepatitis using the methionine-choline-deficient diet. Analysis of bone marrow chimeras between Chop(-/-) and Chop(+/+) mice demonstrated that Chop in macrophages protects from liver injury and inflammation when fed the methionine-choline-deficient diet. We conclude that Chop deletion has a proinflammatory effect in fatty liver injury apparently due to decreased cell death of activated macrophages, resulting in their net accumulation in the liver. Thus, macrophage CHOP plays a key role in protecting the liver from steatohepatitis likely by limiting macrophage survival during lipotoxicity.

  5. C/EBP Homologous Protein-induced Macrophage Apoptosis Protects Mice from Steatohepatitis*

    PubMed Central

    Malhi, Harmeet; Kropp, Erin M.; Clavo, Vinna F.; Kobrossi, Christina R.; Han, JaeSeok; Mauer, Amy S.; Yong, Jing; Kaufman, Randal J.

    2013-01-01

    Nonalcoholic fatty liver disease is a heterogeneous disorder characterized by liver steatosis; inflammation and fibrosis are features of the progressive form nonalcoholic steatohepatitis. The endoplasmic reticulum stress response is postulated to play a role in the pathogenesis of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis. In particular, C/EBP homologous protein (CHOP) is undetectable under normal conditions but is induced by cellular stress, including endoplasmic reticulum stress. Chop wild type (Chop+/+) and knock-out (Chop−/−) mice were used in these studies to elucidate the role of CHOP in the pathogenesis of fatty liver disease. Paradoxically, Chop−/− mice developed greater liver injury, inflammation, and fibrosis than Chop+/+ mice, with greater macrophage activation. Primary, bone marrow-derived, and peritoneal macrophages from Chop+/+ and Chop−/− were challenged with palmitic acid, an abundant saturated free fatty acid in plasma and liver lipids. Where palmitic acid treatment activated Chop+/+ and Chop−/− macrophages, Chop−/− macrophages were resistant to its lipotoxicity. Chop−/− mice were sensitized to liver injury in a second model of dietary steatohepatitis using the methionine-choline-deficient diet. Analysis of bone marrow chimeras between Chop−/− and Chop+/+ mice demonstrated that Chop in macrophages protects from liver injury and inflammation when fed the methionine-choline-deficient diet. We conclude that Chop deletion has a proinflammatory effect in fatty liver injury apparently due to decreased cell death of activated macrophages, resulting in their net accumulation in the liver. Thus, macrophage CHOP plays a key role in protecting the liver from steatohepatitis likely by limiting macrophage survival during lipotoxicity. PMID:23720735

  6. The stress response system of proteins: Implications for bioreactor scaleup

    NASA Technical Reports Server (NTRS)

    Goochee, Charles F.

    1988-01-01

    Animal cells face a variety of environmental stresses in large scale bioreactors, including periodic variations in shear stress and dissolved oxygen concentration. Diagnostic techniques were developed for identifying the particular sources of environmental stresses for animal cells in a given bioreactor configuration. The mechanisms by which cells cope with such stresses was examined. The individual concentrations and synthesis rates of hundreds of intracellular proteins are affected by the extracellular environment (medium composition, dissolved oxygen concentration, ph, and level of surface shear stress). Techniques are currently being developed for quantifying the synthesis rates and concentrations of the intracellular proteins which are most sensitive to environmental stress. Previous research has demonstrated that a particular set of stress response proteins are synthesized by mammalian cells in response to temperature fluctuations, dissolved oxygen deprivation, and glucose deprivation. Recently, it was demonstrated that exposure of human kidney cells to high shear stress results in expression of a completely distinct set of intracellular proteins.

  7. Characterization of the Bat proteins in the oxidative stress response of Leptospira biflexa

    PubMed Central

    2012-01-01

    Background Leptospires lack many of the homologs for oxidative defense present in other bacteria, but do encode homologs of the Bacteriodes aerotolerance (Bat) proteins, which have been proposed to fulfill this function. Bat homologs have been identified in all families of the phylum Spirochaetes, yet a specific function for these proteins has not been experimentally demonstrated. Results We investigated the contribution of the Bat proteins in the model organism Leptospira biflexa for their potential contributions to growth rate, morphology and protection against oxidative challenges. A genetically engineered mutant strain in which all bat ORFs were deleted did not exhibit altered growth rate or morphology, relative to the wild-type strain. Nor could we demonstrate a protective role for the Bat proteins in coping with various oxidative stresses. Further, pre-exposing L. biflexa to sublethal levels of reactive oxygen species did not appear to induce a general oxidative stress response, in contrast to what has been shown in other bacterial species. Differential proteomic analysis of the wild-type and mutant strains detected changes in the abundance of a single protein only – HtpG, which is encoded by the gene immediately downstream of the bat loci. Conclusion The data presented here do not support a protective role for the Leptospira Bat proteins in directly coping with oxidative stress as previously proposed. L. biflexa is relatively sensitive to reactive oxygen species such as superoxide and H2O2, suggesting that this spirochete lacks a strong, protective defense against oxidative damage despite being a strict aerobe. PMID:23234440

  8. AMP-18 protects barrier function of colonic epithelial cells: role of tight junction proteins

    PubMed Central

    Walsh-Reitz, Margaret M.; Huang, Erick F.; Musch, Mark W.; Chang, Eugene B.; Martin, Terence E.; Kartha, Sreedharan; Toback, F. Gary

    2005-01-01

    AMP-18, a novel gastric antrum mucosal protein, and a synthetic peptide of amino acids 77-97, have mitogenic and motogenic properties for epithelial cells. The possibility that AMP-18 is also protective was evaluated in the colonic mucosa of mice and monolayer cultures of human colonic epithelial Caco2/bbe (C2) cells. Administration of AMP peptide to mice with dextran sulfate sodium (DSS)-induced colonic injury delayed the onset of bloody diarrhea, and reduced weight loss. Treatment of C2 cells with AMP peptide protected monolayers against decreases in transepithelial electrical resistance (TER) induced by the oxidant monochloramine, indomethacin, or DSS. A molecular mechanism for these barrier-protective effects was sought by asking if AMP peptide acted on specific tight junction (TJ) proteins. Immunoblots of detergent-insoluble fractions of C2 cells treated with AMP peptide exhibited increased accumulation of specific TJ proteins. Occludin immunoreactivity was also increased in detergent-insoluble fractions obtained from colonic mucosal cells of mice injected with AMP peptide. Laser scanning confocal microscopy (CF) supported the capacity of AMP peptide to enhance accumulation of occludin and ZO-1 in TJ domains of C2 cell monolayers, and together with immunoblot analysis showed that the peptide protected against loss of these TJ proteins following oxidant injury. AMP peptide also protected against a fall in TER during disruption of actin filaments by cytochalasin D, and stabilized perijunctional actin during oxidant injury when assessed by CF. These findings suggest that AMP-18 could protect the intestinal mucosal barrier by acting on specific TJ proteins and stabilizing perijunctional actin. PMID:15961882

  9. Human immune system mice immunized with Plasmodium falciparum circumsporozoite protein induce protective human humoral immunity against malaria.

    PubMed

    Huang, Jing; Li, Xiangming; Coelho-dos-Reis, Jordana G A; Zhang, Min; Mitchell, Robert; Nogueira, Raquel Tayar; Tsao, Tiffany; Noe, Amy R; Ayala, Ramses; Sahi, Vincent; Gutierrez, Gabriel M; Nussenzweig, Victor; Wilson, James M; Nardin, Elizabeth H; Nussenzweig, Ruth S; Tsuji, Moriya

    2015-12-01

    In this study, we developed human immune system (HIS) mice that possess functional human CD4+ T cells and B cells, named HIS-CD4/B mice. HIS-CD4/B mice were generated by first introducing HLA class II genes, including DR1 and DR4, along with genes encoding various human cytokines and human B cell activation factor (BAFF) to NSG mice by adeno-associated virus serotype 9 (AAV9) vectors, followed by engrafting human hematopoietic stem cells (HSCs). HIS-CD4/B mice, in which the reconstitution of human CD4+ T and B cells resembles to that of humans, produced a significant level of human IgG against Plasmodium falciparum circumsporozoite (PfCS) protein upon immunization. CD4+ T cells in HIS-CD4/B mice, which possess central and effector memory phenotypes like those in humans, are functional, since PfCS protein-specific human CD4+ T cells secreting IFN-γ and IL-2 were detected in immunized HIS-CD4/B mice. Lastly, PfCS protein-immunized HIS-CD4/B mice were protected from in vivo challenge with transgenic P. berghei sporozoites expressing the PfCS protein. The immune sera collected from protected HIS-CD4/B mice reacted against transgenic P. berghei sporozoites expressing the PfCS protein and also inhibited the parasite invasion into hepatocytes in vitro. Taken together, these studies show that our HIS-CD4/B mice could mount protective human anti-malaria immunity, consisting of human IgG and human CD4+ T cell responses both specific for a human malaria antigen.

  10. Proteomic response of wheat embryos to fosthiazate stress in a protected vegetable soil.

    PubMed

    Yin, Chunyan; Teng, Ying; Luo, Yongming; Christie, Peter

    2012-01-01

    A proteomic analysis of wheat defense response induced by the widely used organophosphorus nematicide fosthiazate is reported. Seed germination and two-dimensional gel electrophoresis (2-DE) experiments were performed using a Chinese wheat cultivar, Zhenmai No. 5. Root and shoot elongation decreased but thiobarbituric acid reactive substances (TBARS) content in embryos increased with increasing pesticide concentration. More than 1000 protein spots were reproducibly detected in each silver-stained gel. Thirty-seven protein spots with at least 2-fold changes were identified using MALDI-TOF MS/MS analysis. Of these, 24 spots were up-regulated and 13 were down-regulated. Proteins identified included some well-known classical stress responsive proteins under abiotic or biotic stresses as well as some unusual responsive proteins. Ten responsive proteins were reported for the first time at the proteomic level, including fatty acyl CoA reductase, dihydrodipicolinate synthase, DEAD-box ATPase-RNA-helicase, fimbriata-like protein, waxy B1, rust resistance kinase Lr10, putative In2.1 protein, retinoblastoma-related protein 1, pollen allergen-like protein and S-adenosyl-L-methionine:phosphoethanolamine N-methyltransferase. The proteins identified were involved in several processes such as metabolism, defense/detoxification, cell structure/cell growth, signal transduction/transcription, photosynthesis and energy. Seven candidate proteins were further analyzed at the mRNA level by RT-PCR to compare transcript and protein accumulation patterns, revealing that not all the genes were correlated well with the protein level. Identification of these responsive proteins may provide new insight into the molecular basis of the fosthiazate-stress response in the early developmental stages of plants and may be useful in stress monitoring or stress-tolerant crop breeding for environmentally friendly agricultural production.

  11. Immune Responses and Protection of Aotus Monkeys Immunized with Irradiated Plasmodium vivax Sporozoites

    DTIC Science & Technology

    2011-01-01

    responses and protective efficacy induced by vacci- nation with irradiated P vivax sporozoites were evaluated in malaria-naive Aotus monkeys. Three groups...received either 10 doses of uninfected salivary gland extract or no inoculations. Immunization resulted in the production low levels of antibodies that...responses. Intravenous challenge with viable sporozoites resulted in partial protection in a dose-dependent manner.l11ese findings suggest that the

  12. Zinc finger antiviral protein inhibits coxsackievirus B3 virus replication and protects against viral myocarditis.

    PubMed

    Li, Min; Yan, Kepeng; Wei, Lin; Yang, Jie; Lu, Chenyu; Xiong, Fei; Zheng, Chunfu; Xu, Wei

    2015-11-01

    The host Zinc finger antiviral protein (ZAP) has been reported exhibiting antiviral activity against positive-stranded RNA viruses (Togaviridae), negative-stranded RNA viruses (Filoviridae) and retroviruses (Retroviridae). However, whether ZAP restricts the infection of enterovirus and the development of enterovirus mediated disease remains unknown. Here, we reported the antiviral properties of ZAP against coxsackievirus B3 (CVB3), a single-stranded RNA virus of the Enterovirus genus within the Picornaviridae as a major causative agent of viral myocarditis (VMC). We found that the expression of ZAP was significantly induced after CVB3 infection in heart tissues of VMC mice. ZAP potently inhibited CVB3 replication in cells after infection, while overexpression of ZAP in mice significantly increased the resistance to CVB3 replication and viral myocarditis by significantly reducing cardiac inflammatory cytokine production. The ZAP-responsive elements (ZREs) were mapped to the 3'UTR and 5'UTR of viral RNA. Taken together, ZAP confers resistance to CVB3 infection via directly targeting viral RNA and protects mice from acute myocarditis by suppressing viral replication and cardiac inflammatory cytokine production. Our finding further expands ZAP's range of viral targets, and suggests ZAP as a potential therapeutic target for viral myocarditis caused by CVB3.

  13. Eimeria tenella heat shock protein 70 enhances protection of recombinant microneme protein MIC2 subunit antigen vaccination against E. tenella challenge.

    PubMed

    Zhang, Lei; Ma, Liping; Liu, Renqiang; Zhang, Yunfei; Zhang, Shouping; Hu, Chunmei; Song, Meng; Cai, Jianping; Wang, Ming

    2012-09-10

    Heat shock proteins have been reported to stimulate the immune system via innate receptors. Our study found that the novel immunopotentiator, Eimeria tenella (E. tenella) heat shock protein 70 (HSP70), enhanced protective immunity elicited by E. tenella antigen microneme protein 2 (EtMIC2) against avian coccidiosis. It demonstrated that the expression of TLR2 and TLR4 were strongly upregulated in EtHSP70 and EtMIC2 plus EtHSP70 stimulated chicken embryo fibroblasts (CEF) compared with untreated controls and EtMIC2 alone. In addition, the same treatment induced high levels of interleukin (IL)-12 and interferon (IFN)-γ that are critical cytokines of innate immunity. In vivo experiments involved using broiler chickens subcutaneously immunized with EtMIC2 alone or EtMIC2 plus EtHSP70 at 7 and 14 days post-hatch, which were then orally challenged with live E. tenella at 7 days following secondary immunization. Body weight gains, cecal lesion scores, fecal oocyst shedding, serum antibody responses against MIC2, and intestinal cytokine transcript levels were assessed as measures of protective immunity. Chickens immunized with EtMIC2 plus EtHSP70 showed increased body weight gains, decreased oocyst shedding, increased serum antibody responses, and high levels of IL-12, IFN-γ, and IL-17 compared with the EtMIC2 only or control groups. Moreover, chickens immunized with EtHSP70 alone showed significantly protective effect against E. tenella infection. In summary, this study provides the first evidence of the immunoenhancing activities of EtHSP70 in poultry.

  14. Targeting unfolded protein response in cancer and diabetes.

    PubMed

    Mkrtchian, Souren

    2015-06-01

    The maturation of secretory and membrane proteins in the endoplasmic reticulum (ER) is tightly regulated by the unfolded protein response (UPR), a signal transduction pathway maintaining ER protein folding homeostasis. However, certain ER states are incompatible with cell survival and therefore the UPR may choose to eliminate severely disrupted cells by apoptosis. This is accomplished primarily through the activation of the transcription factor CCAAT-enhancer-binding protein homologous protein (CHOP). In the April 2015 issue of Endocrine-Related Cancer, researchers from the universities of South Carolina and Athens (Greece) suggested a novel mechanism of CHOP-mediated apoptosis connected with the suppression of a prominent cell cycle regulator with anti-apoptotic activity, p21. These findings and suggested clinical applications, such as potentiation of cancer chemotherapy and a novel therapeutic approach for type 2 diabetes, are discussed in the context of UPR.

  15. The unfolded protein response and translation attenuation: a modelling approach.

    PubMed

    Trusina, A; Tang, C

    2010-10-01

    Unfolded protein response (UPR) is a stress response to increased levels of unfolded proteins in the endoplasmic reticulum (ER). To deal with this stress, all eukaryotic cells share a well-conserved strategy--the upregulation of chaperons and proteases to facilitate protein folding and to degrade the misfolded proteins. For metazoans, however, an additional and seemingly redundant strategy has been evolved--translation attenuation (TA) of proteins targeted to the ER via the protein kinase PERK pathway. PERK is essential in secretory cells, such as the pancreatic β-cells, but not in non-secretory cell types. We have recently developed a mathematical model of UPR, focusing on the interplay and synergy between the TA arm and the conserved Ire1 arm of the UPR. The model showed that the TA mechanism is beneficial in highly fluctuating environment, for example, in the case where the ER stress changes frequently. Under highly variable levels of ER stress, tight regulation of the ER load by TA avoids excess amount of chaperons and proteases being produced. The model also showed that TA is of greater importance when there is a large flux of proteins through the ER. In this study, we further expand our model to investigate different types of ER stress and different temporal profiles of the stress. We found that TA is more desirable in dealing with the translation stress, for example, prolonged stimulation of proinsulin biosynthesis, than the chemical stress.

  16. Identification of iron-responsive proteins expressed by Chlamydia trachomatis reticulate bodies during intracellular growth.

    PubMed

    Dill, Brian D; Dessus-Babus, Sophie; Raulston, Jane E

    2009-01-01

    The obligate intracellular bacterium Chlamydia trachomatis serovar E is the most prevalent cause of bacterial sexually transmitted disease. With an established requirement for iron, the developmental cycle arrests at the intracellular reticulate body stage during iron restriction, resulting in a phenomenon termed persistence. Persistence has implications in natural infections for altered expression of virulence factors and antigens, in addition to a potential role in producing chronic infection. In this study, chlamydial proteins in iron-restricted, infected HEC-1B cells were radiolabelled during mid-developmental cycle growth, harvested, and separated using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Of approximately 250 radiolabelled protein species visualized, densitometric analysis revealed 25 proteins that increased in expression under iron restriction compared to iron-sufficient control samples; ten protein species identified by mass spectrometry are involved in the oxidative damage response (alkyl hydroperoxide reductase, 6-phosphogluconolactonase and acyl carrier protein synthase), transcription (RNA polymerase subunit alpha and transcription anti-termination factors NusA and NusG), protein modification (peptide deformylase and trigger factor), and virulence (Chlamydia protein associating with death domains, CADD). Transcript-level expression patterns of ahpC, devB, cadd, fabF and ct538 were measured by quantitative RT-PCR throughout the developmental cycle, and each gene examined demonstrated a significant but small mid-cycle increase in transcript level in iron-restricted cultures compared to iron-replete controls. Taken together, these data suggest that the primary response of chlamydiae to reduced iron availability is to increase expression of proteins involved in protection against oxidative damage via iron-catalysed generation of reactive oxygen species and adaptation to stress by increasing expression of transcriptional machinery

  17. LKTA and PlpE small fragments fusion protein protect against Mannheimia haemolytica challenge.

    PubMed

    Guzmán-Brambila, Carolina; Quintero-Fabián, Saray; González-Castillo, Celia; de Obeso-Fernández del Valle, Álvaro; Flores-Samaniego, Beatriz; de la Mora, Germán; Rojas-Mayorquín, Argelia E; Ortuño-Sahagún, Daniel

    2012-12-01

    Bovine respiratory disease (BRD) complex is a major cause of economic losses for the cattle backgrounding and feedlot industries. Mannheimia haemolytica is considered the most important pathogen associated with this disease. Vaccines against M. haemolytica have been prepared and used for many decades, but traditional bacterins have failed to demonstrate effective protection and their use has often exacerbated disease in vaccinated animals. Thus, the BRD complex continues to exert a strong adverse effect on the health and wellbeing of stocker and feeder cattle. Therefore, generation of recombinant proteins has been helpful in formulating enhanced vaccines against M. haemolytica, which could confer better protection against BRD. In the present study, we formulated a vaccine preparation enriched with recombinant small fragments of leukotoxin A (LKTA) and outer-membrane lipoprotein (PlpE) proteins, and demonstrated its ability to generate high antibody titers in rabbits and sheep, which protected against M. haemolytica bacterial challenge in mice.

  18. Protection from heat-induced protein migration and DNA repair inhibition by cycloheximide.

    PubMed

    Armour, E P; Lee, Y J; Corry, P M; Borrelli, M J

    1988-12-15

    The mechanism by which Cycloheximide (CHM) protects cells from heat induced killing has been investigated. Cycloheximide (10 micrograms/ml) added for 2 hr before and during a 3 hour heating at 43 degrees C prevented a 40% increase of heat-induced protein accumulation in the nucleus and protected cells (0.0001 vs. 0.15 surviving fraction) from heat-induced killing. Heat-induced DNA repair inhibition was also suppressed when cells were treated with CHM in the above manner. This combination of results suggests that protein accumulation in the nucleus and inhibition of DNA repair are related and these events are associated with CHM protection from heat induced cell killing.

  19. The responsibility of healthcare institutions to protect global health security.

    PubMed

    Wilson, Kumanan; McDougall, Christopher; Forster, Alan

    2009-01-01

    New public threats that can rapidly cross borders are continuing to challenge global health securityand will require unprecedented levels of co-operation. At the international level, the response to this challenge led to the approval of revised International Health Regulations (IHR). This unanimously approved document outlines how countries are to prepare for and respond to public health emergencies of international concern in a manner that does not unnecessarily impact on travel and trade.

  20. Current transformer model with hysteresis for improving the protection response in electrical transmission systems

    NASA Astrophysics Data System (ADS)

    Matussek, Robert; Dzienis, Cezary; Blumschein, Jörg; Schulte, Horst

    2014-12-01

    In this paper, a generic enhanced protection current transformer (CT) model with saturation effects and transient behavior is presented. The model is used for the purpose of analysis and design of power system protection algorithms. Three major classes of protection CT have been modeled which all take into account the nonlinear inductance with remanence effects. The transient short-circuit currents in power systems are simulated under CT saturation condition. The response of a common power system protection algorithm with respect to robustness to nominal parameter variations and sensitivity against maloperation is demonstrated by simulation studies.

  1. Sigma 1 receptor stimulation protects against oxidative damage through suppression of the ER stress responses in the human lens.

    PubMed

    Wang, Lixin; Eldred, Julie A; Sidaway, Peter; Sanderson, Julie; Smith, Andrew J O; Bowater, Richard P; Reddan, John R; Wormstone, I Michael

    2012-01-01

    Stimulation of sigma-1 receptors is reported to protect against oxidative stress. The present study uses cells and tissue from the human lens to elucidate the relationship between the sigma 1 receptor, ER stress and oxidative stress-induced damage. Exposure of the human lens cell line FHL124 to increasing concentrations of H(2)O(2) led to reduced cell viability and increased apoptosis. In response to 30 μM H(2)O(2), levels of the ER stress proteins BiP, ATF6 and pEIF2α were significantly increased within 4h of exposure. Expression of the sigma 1 receptor was markedly increased in response to H(2)O(2). Application of 10 and 30 μM (+)-pentazocine, a sigma 1 receptor agonist, significantly inhibited the H(2)O(2) induced cell death. (+)-Pentazocine also suppressed the oxidative stress induced reduction of pro-caspase 12 and suppressed the induction of the ER stress proteins BiP and EIF2α. When applied to cultured human lenses, (+)-pentazocine protected against apoptotic cell death, LDH release and against H(2)O(2) induced opacification. These data demonstrate that stimulation of the sigma 1 receptor provides significant protection from oxidative damage and is, therefore, a putative therapeutic approach to delay the onset of diseases that may be triggered by oxidative damage, including cataract formation.

  2. Characterization of the immune response of domestic fowl following immunization with proteins extracted from Dermanyssus gallinae.

    PubMed

    Harrington, David; Din, Hatem Mohi El; Guy, Jonathan; Robinson, Karen; Sparagano, Olivier

    2009-03-23

    Dermanyssus gallinae is the most significant ectoparasite of European poultry egg laying production systems due to high costs of control and associated production losses as well as adverse effects on bird welfare. In this study, soluble proteins were extracted from unfed D. gallinae (DGE) using a urea-based detergent and ultra-filtration, passed through a 0.22 microm filter and blended aseptically with adjuvant. One group of laying hens was immunized with DGE and adjuvant (Montanide ISA 50 V) whilst another group (Control) received physiological saline and adjuvant. All birds were immunized on two occasions, 21 days apart. Antibody response to immunization was determined by ELISA and western blotting using immunoglobulins (Igs) extracted from egg yolk. DGE immunization of hens resulted in a significant (P<0.05) IgY response compared to controls, although there was no significant difference in IgM response between treatments. A number of proteins were identified by western blotting using IgY antibodies from DGE immunized birds, most prominently at 40 and 230kDa. Analysis of proteins from approximately corresponding bands on SDS-PAGE confirmed the identity of tropomyosin, whilst other proteins showed high sequence homology with myosin and actin from other arachnid and insect species. Immunization of hens with DGE resulted in a 50.6% increase in mite mortality (P<0.001) 17h after feeding when tested by an in vitro mite feeding model. Data in this study demonstrate that somatic antigens from D. gallinae can be used to stimulate a protective immune response in laying hens. Further work is needed to identify other proteins of interest that could confer higher protection against D. gallinae, as well as optimization of the vaccination and in vitro testing protocol.

  3. Identification of Differentially Expressed Proteins and Phosphorylated Proteins in Rice Seedlings in Response to Strigolactone Treatment

    PubMed Central

    Chen, Fangyu; Jiang, Liangrong; Zheng, Jingsheng; Huang, Rongyu; Wang, Houcong; Hong, Zonglie; Huang, Yumin

    2014-01-01

    Strigolactones (SLs) are recently identified plant hormones that inhibit shoot branching and control various aspects of plant growth, development and interaction with parasites. Previous studies have shown that plant D10 protein is a carotenoid cleavage dioxygenase that functions in SL biosynthesis. In this work, we used an allelic SL-deficient d10 mutant XJC of rice (Oryza sativa L. spp. indica) to investigate proteins that were responsive to SL treatment. When grown in darkness, d10 mutant seedlings exhibited elongated mesocotyl that could be rescued by exogenous application of SLs. Soluble protein extracts were prepared from d10 mutant seedlings grown in darkness in the presence of GR24, a synthetic SL analog. Soluble proteins were separated on two-dimensional gels and subjected to proteomic analysis. Proteins that were expressed differentially and phosphoproteins whose phosphorylation status changed in response to GR24 treatment were identified. Eight proteins were found to be induced or down-regulated by GR24, and a different set of 8 phosphoproteins were shown to change their phosphorylation intensities in the dark-grown d10 seedlings in response to GR24 treatment. Analysis of these proteins revealed that they are important enzymes of the carbohydrate and amino acid metabolic pathways and key components of the cellular energy generation machinery. These proteins may represent potential targets of the SL signaling pathway. This study provides new insight into the complex and negative regulatory mechanism by which SLs control shoot branching and plant development. PMID:24699514

  4. Mineral proximity influences mechanical response of proteins in biological mineral-protein hybrid systems.

    PubMed

    Ghosh, Pijush; Katti, Dinesh R; Katti, Kalpana S

    2007-03-01

    The organic phase of nacre, which is composed primarily of proteins, has an extremely high elastic modulus as compared to that of bulk proteins, and also undergoes large deformation before failure. One reason for this unusually high modulus could be the mineral-organic interactions. In this work, we elucidate the specific role of mineral proximity on the structural response of proteins in biological structural composites such as nacre through molecular modeling. The "glycine-serine" domain of a nacre protein Lustrin A has been used as a model system. It is found that the amount of work needed to unfold is significantly higher when the GS domain is pulled in the proximity of aragonite. These results indicate that the proximity of aragonite has a significant effect on the unfolding mechanisms of proteins when pulled. These results will provide very useful information in designing synthetic biocomposites, as well as further our understanding of mechanical response in structural composites in nature.

  5. The Acetic Acid Tolerance Response induces cross-protection to salt stress in Salmonella typhimurium.

    PubMed

    Greenacre, E J; Brocklehurst, T F

    2006-10-15

    Salmonella typhimurium induces an Acid Tolerance Response (ATR) upon exposure to mildly acidic conditions in order to protect itself against severe acid shock. This response can also induce cross-protection to other stresses such as heat and salt. We investigated whether both the acetic acid induced and lactic acid induced ATR in S. typhimurium provided cross-protection to a salt stress at 20 degrees C. Acid-adapted cells were challenged with both a sodium chloride (NaCl) and potassium chloride (KCl) shock and their ability to survive ascertained. Acetic acid adaptation provided cells with protection against both NaCl and KCl stress. However, lactic acid adaptation did not protect against either osmotic stressor and rendered cells hypersensitive to NaCl. These results have implications for the food industry where hurdle technology means multiple sub-lethal stresses such as mild pH and low salt are commonly used in the preservation of products.

  6. Protein S-thiolation targets glycolysis and protein synthesis in response to oxidative stress in the yeast Saccharomyces cerevisiae.

    PubMed Central

    Shenton, Daniel; Grant, Chris M

    2003-01-01

    The irreversible oxidation of cysteine residues can be prevented by protein S-thiolation, a process by which protein SH groups form mixed disulphides with low-molecular-mass thiols such as glutathione. We report here the target proteins which are modified in yeast cells in response to H(2)O(2). In particular, a range of glycolytic and related enzymes (Tdh3, Eno2, Adh1, Tpi1, Ald6 and Fba1), as well as translation factors (Tef2, Tef5, Nip1 and Rps5) are identified. The oxidative stress conditions used to induce S-thiolation are shown to inhibit GAPDH (glyceraldehyde-3-phosphate dehydrogenase), enolase and alcohol dehydrogenase activities, whereas they have no effect on aldolase, triose phosphate isomerase or aldehyde dehydrogenase activities. The inhibition of GAPDH, enolase and alcohol dehydrogenase is readily reversible once the oxidant is removed. In addition, we show that peroxide stress has little or no effect on glucose-6-phosphate dehydrogenase or 6-phosphogluconate dehydrogenase, the enzymes that catalyse NADPH production via the pentose phosphate pathway. Thus the inhibition of glycolytic flux is proposed to result in glucose equivalents entering the pentose phosphate pathway for the generation of NADPH. Radiolabelling is used to confirm that peroxide stress results in a rapid and reversible inhibition of protein synthesis. Furthermore, we show that glycolytic enzyme activities and protein synthesis are irreversibly inhibited in a mutant that lacks glutathione, and hence cannot modify proteins by S-thiolation. In summary, protein S-thiolation appears to serve an adaptive function during exposure to an oxidative stress by reprogramming metabolism and protecting protein synthesis against irreversible oxidation. PMID:12755685

  7. Loss of RAD-23 Protects Against Models of Motor Neuron Disease by Enhancing Mutant Protein Clearance

    PubMed Central

    Jablonski, Angela M.; Lamitina, Todd; Liachko, Nicole F.; Sabatella, Mariangela; Lu, Jiayin; Zhang, Lei; Ostrow, Lyle W.; Gupta, Preetika; Wu, Chia-Yen; Doshi, Shachee; Mojsilovic-Petrovic, Jelena; Lans, Hannes; Wang, Jiou; Kraemer, Brian

    2015-01-01

    Misfolded proteins accumulate and aggregate in neurodegenerative disease. The existence of these deposits reflects a derangement in the protein homeostasis machinery. Using a candidate gene screen, we report that loss of RAD-23 protects against the toxicity of proteins known to aggregate in amyotrophic lateral sclerosis. Loss of RAD-23 suppresses the locomotor deficit of Caenorhabditis elegans engineered to express mutTDP-43 or mutSOD1 and also protects against aging and proteotoxic insults. Knockdown of RAD-23 is further neuroprotective against the toxicity of SOD1 and TDP-43 expression in mammalian neurons. Biochemical investigation indicates that RAD-23 modifies mutTDP-43 and mutSOD1 abundance, solubility, and turnover in association with altering the ubiquitination status of these substrates. In human amyotrophic lateral sclerosis spinal cord, we find that RAD-23 abundance is increased and RAD-23 is mislocalized within motor neurons. We propose a novel pathophysiological function for RAD-23 in the stabilization of mutated proteins that cause neurodegeneration. SIGNIFICANCE STATEMENT In this work, we identify RAD-23, a component of the protein homeostasis network and nucleotide excision repair pathway, as a modifier of the toxicity of two disease-causing, misfolding-prone proteins, SOD1 and TDP-43. Reducing the abundance of RAD-23 accelerates the degradation of mutant SOD1 and TDP-43 and reduces the cellular content of the toxic species. The existence of endogenous proteins that act as “anti-chaperones” uncovers new and general targets for therapeutic intervention. PMID:26490867

  8. Cognitive Functioning and Heat Strain: Performance Responses and Protective Strategies.

    PubMed

    Schmit, Cyril; Hausswirth, Christophe; Le Meur, Yann; Duffield, Rob

    2016-12-17

    Despite the predominance of research on physical performance in the heat, many activities require high cognitive functioning for optimal performance (i.e. decision making) and/or health purposes (i.e. injury risk). Prolonged periods of demanding cognitive activity or exercise-induced fatigue will incur altered cognitive functioning. The addition of hot environmental conditions will exacerbate poor cognitive functioning and negatively affect performance outcomes. The present paper attempts to extract consistent themes from the heat-cognition literature to explore cognitive performance as a function of the level of heat stress encountered. More specifically, experimental studies investigating cognitive performance in conditions of hyperthermia, often via the completion of computerised tasks (i.e. cognitive tests), are used to better understand the relationship between endogenous thermal load and cognitive performance. The existence of an inverted U-shaped relationship between hyperthermia development and cognitive performance is suggested, and highlights core temperatures of ~38.5 °C as the potential 'threshold' for hyperthermia-induced negative cognitive performance. From this perspective, interventions to slow or blunt thermal loads and protect both task- and hyperthermia-related changes in task performances (e.g. cooling strategies) could be used to great benefit and potentially preserve cognitive performance during heat strain.

  9. Nicotinamide mononucleotide adenylyltransferase promotes hypoxic survival by activating the mitochondrial unfolded protein response

    PubMed Central

    Mao, X R; Kaufman, D M; Crowder, C M

    2016-01-01

    Gain-of-function mutations in the mouse nicotinamide mononucleotide adenylyltransferase type 1 (Nmnat1) produce two remarkable phenotypes: protection against traumatic axonal degeneration and reduced hypoxic brain injury. Despite intensive efforts, the mechanism of Nmnat1 cytoprotection remains elusive. To develop a new model to define this mechanism, we heterologously expressed a mouse Nmnat1 non-nuclear-localized gain-of-function mutant gene (m-nonN-Nmnat1) in the nematode Caenorhabditis elegans and show that it provides protection from both hypoxia-induced animal death and taxol-induced axonal pathology. Additionally, we find that m-nonN-Nmnat1 significantly lengthens C. elegans lifespan. Using the hypoxia-protective phenotype in C. elegans, we performed a candidate screen for genetic suppressors of m-nonN-Nmnat1 cytoprotection. Loss of function in two genes, haf-1 and dve-1, encoding mitochondrial unfolded protein response (mitoUPR) factors were identified as suppressors. M-nonN-Nmnat1 induced a transcriptional reporter of the mitoUPR gene hsp-6 and provided protection from the mitochondrial proteostasis toxin ethidium bromide. M-nonN-Nmnat1 was also protective against axonal degeneration in C. elegans induced by the chemotherapy drug taxol. Taxol markedly reduced basal expression of a mitoUPR reporter; the expression was restored by m-nonN-Nmnat1. Taken together, these data implicate the mitoUPR as a mechanism whereby Nmnat1 protects from hypoxic and axonal injury. PMID:26913604

  10. Zea mays L. protein changes in response to potassium dichromate treatments.

    PubMed

    Labra, M; Gianazza, E; Waitt, R; Eberini, I; Sozzi, A; Regondi, S; Grassi, F; Agradi, E

    2006-03-01

    The plant metabolic response to heavy metal stress is largely unknown. The present investigation was undertaken to examine the influence of different concentrations of potassium dichromate on the Zea mays L. plantlets. A clear effect of chromium on maize plantlets growth and seed germination was observed strating from 100-300 ppm up to 1500 ppm. In this concentration range, chromium uptake was dependent on the concentration in the medium. Metallothioneins, involved in heavy metal binding, were measured by capillary electrophoresis (CE), and showed a dose-response induction. Protein profile analyzed by two-dimensional gel electrophoresis showed differential expression of several proteins. Identification of spots of upregulated proteins was performed by MALDI mass spectrometry. Results showed that proteins induced by heavy metal exposure are principally involved in oxidative stress tolerance or in other stress pathways. Induction of proteins implicated in sugar metabolism was also observed. Identification of factors involved in plant response may lead to a better understanding of the mechanisms involved in cell protection and tolerance. This information could be used to improve agricultural production and environmental quality.

  11. Protecting Skeletal Muscle with Protein and Amino Acid during Periods of Disuse

    PubMed Central

    Galvan, Elfego; Arentson-Lantz, Emily; Lamon, Séverine; Paddon-Jones, Douglas

    2016-01-01

    Habitual sedentary behavior increases risk of chronic disease, hospitalization and poor quality of life. Short-term bed rest or disuse accelerates the loss of muscle mass, function, and glucose tolerance. Optimizing nutritional practices and protein intake may reduce the consequences of disuse by preserving metabolic homeostasis and muscle mass and function. Most modes of physical inactivity have the potential to negatively impact the health of older adults more than their younger counterparts. Mechanistically, mammalian target of rapamycin complex 1 (mTORC1) signaling and muscle protein synthesis are negatively affected by disuse. This contributes to reduced muscle quality and is accompanied by impaired glucose regulation. Simply encouraging increased protein and/or energy consumption is a well-intentioned, but often impractical strategy to protect muscle health. Emerging evidence suggests that leucine supplemented meals may partially and temporarily protect skeletal muscle during disuse by preserving anabolism and mitigating reductions in mass, function and metabolic homeostasis. PMID:27376322

  12. Molecular basis for protection of ribosomal protein L4 from cellular degradation

    PubMed Central

    Huber, Ferdinand M.; Hoelz, André

    2017-01-01

    Eukaryotic ribosome biogenesis requires the nuclear import of ∼80 nascent ribosomal proteins and the elimination of excess amounts by the cellular degradation machinery. Assembly chaperones recognize nascent unassembled ribosomal proteins and transport them together with karyopherins to their nuclear destination. We report the crystal structure of ribosomal protein L4 (RpL4) bound to its dedicated assembly chaperone of L4 (Acl4), revealing extensive interactions sequestering 70 exposed residues of the extended RpL4 loop. The observed molecular recognition fundamentally differs from canonical promiscuous chaperone–substrate interactions. We demonstrate that the eukaryote-specific RpL4 extension harbours overlapping binding sites for Acl4 and the nuclear transport factor Kap104, facilitating its continuous protection from the cellular degradation machinery. Thus, Acl4 serves a dual function to facilitate nuclear import and simultaneously protect unassembled RpL4 from the cellular degradation machinery. PMID:28148929

  13. Plasma amino acid response to graded levels of escape protein.

    PubMed

    Gibb, D J; Klopfenstein, T J; Britton, R A; Lewis, A J

    1992-09-01

    A trial was conducted to examine the potential of using plasma amino acid responses to graded levels of escape protein to determine limiting amino acids in cattle. Growing calves (n = 120; mean BW = 220 +/- 21 kg) were fed a basal diet of corncob:sorghum silage (61:39) and were individually supplemented with distillers' dried grains (DDG), heat-damaged DDG (H-DDG), feather meal (FTH), or urea. The urea supplement was mixed with DDG and H-DDG to allow 0, 20, 35, 50, 65, or 80% of the supplemental CP to come from distillers' protein and maintain an 11.5% CP diet. Urea supplement was mixed with FTH to allow 0, 22, 39, 56, 73, or 90% of the supplemental CP to come from FTH. Dietary CP ranged from 11.5% at the 0% level to 17.3% at the 90% level. Plasma concentration of most essential plasma amino acids responded (P less than .10) linearly and(or) quadratically to increased escape protein. The broken-line response of plasma methionine at low DDG intake suggested that methionine was limiting at low levels of escape protein. An initial decrease followed by a plateau fit by a broken line indicated that histidine became limiting in FTH diets, and lysine eventually became limiting for DDG, H-DDG, and FTH diets before maximum BW gain was reached. Results indicate that plasma amino acid responses may identify amino acids that become limiting with increasing escape protein.

  14. Liposomes with diverse compositions are protected during desiccation by LEA proteins from Artemia franciscana and trehalose.

    PubMed

    Moore, Daniel S; Hansen, Richard; Hand, Steven C

    2016-01-01

    Intracellular accumulation of Late Embryogenesis Abundant (LEA) proteins and the disaccharide trehalose is associated with cellular desiccation tolerance in a number of animal species. Two LEA proteins from anhydrobiotic embryos of the brine shrimp Artemia franciscana were tested for the ability to protect liposomes of various compositions against desiccation-induced damage in the presence and absence of trehalose. Damage was assessed by carboxyfluorescein leakage after drying and rehydration. Further, using a cytoplasmic-localized (AfrLEA2) and a mitochondrial-targeted (AfrLEA3m) LEA protein allowed us to evaluate whether each may preferentially stabilize membranes of a particular lipid composition based on the protein's subcellular location. Both LEA proteins were able to offset damage during drying of liposomes that mimicked the lipid compositions of the inner mitochondrial membrane (with cardiolipin), outer mitochondrial membrane, and the inner leaflet of the plasma membrane. Thus liposome stabilization by AfrLEA3m or AfrLEA2 was not dependent on lipid composition, provided physiological amounts of bilayer and non-bilayer-forming lipids were present (liposomes with a non-biological composition of 100% phosphatidylcholine were not protected by either protein). Additive protection by LEA proteins plus trehalose was dependent on the lipid composition of the target membrane. Minimal additional damage occurred to liposomes stored at room temperature in the dried state for one week compared to liposomes rehydrated after 24h. Consistent with the ability to stabilize lipid bilayers, molecular modeling of the secondary structures for AfrLEA2 and AfrLEA3m revealed bands of charged amino acids similar to other amphipathic proteins that interact directly with membranes.

  15. Digoxin-Mediated Upregulation of RGS2 Protein Protects against Cardiac Injury.

    PubMed

    Sjögren, Benita; Parra, Sergio; Atkins, Kevin B; Karaj, Behirda; Neubig, Richard R

    2016-05-01

    Regulator of G protein signaling (RGS) proteins have emerged as novel drug targets since their discovery almost two decades ago. RGS2 has received particular interest in cardiovascular research due to its role in regulating Gqsignaling in the heart and vascular smooth muscle. RGS2(-/-)mice are hypertensive, prone to heart failure, and display accelerated kidney fibrosis. RGS2 is rapidly degraded through the proteasome, and human mutations leading to accelerated RGS2 protein degradation correlate with hypertension. Hence, stabilizing RGS2 protein expression could be a novel route in treating cardiovascular disease. We previously identified cardiotonic steroids, including digoxin, as selective stabilizers of RGS2 protein in vitro. In the current study we investigated the functional effects of digoxin-mediated RGS2 protein stabilization in vivo. Using freshly isolated myocytes from wild-type and RGS2(-/-)mice treated with vehicle or low-dose digoxin (2µg/kg/day for 7 days) we demonstrated that agonist-induced cAMP levels and cardiomyocyte contractility was inhibited by digoxin in wild-type but not in RGS2(-/-)mice. This inhibition was accompanied by an increase in RGS2 protein levels in cardiomyocytes as well as in whole heart tissue. Furthermore, digoxin had protective effects in a model of cardiac injury in wild-type mice and this protection was lost in RGS2(-/-)mice. Digoxin is the oldest known therapy for heart failure; however, beyond its activity at the Na(+)/K(+)-ATPase, the exact mechanism of action is not known. The current study adds a novel mechanism, whereby through stabilizing RGS2 protein levels digoxin could exert its protective effects in the failing heart.

  16. The influence of macrophage inflammatory protein-1α on protective immunity mediated by antiviral cytotoxic T cells

    PubMed Central

    Jones, Emma; Price, David A; Dahm-Vicker, Michaela; Cerundolo, Vincenzo; Klenerman, Paul; Gallimore, Awen

    2003-01-01

    Macrophage inflammatory protein 1α (MIP-1α), a member of the CC-chemokine subfamily, is known to induce chemotaxis of a variety of cell types in vivo. Although the role of MIP-1α in inflammatory responses generated following primary infection of mice with many different pathogens has been characterized, the influence of this chemokine on the generation of antigen-specific T-cell responses in vivo is less well understood. This is important, as virus-specific CD8+ T lymphocytes (CTL) play a crucial role in defence against viral infections, both acutely and in the long term. In this study, we compared the ability of wild-type and MIP-1α-deficient (MIP-1α−/−) mice to mount CTL responses specific for the immunodominant epitope derived from influenza nucleoprotein (NP366–374). Influenza-specific CTL responses were compared with respect to frequency, cytotoxic activity and ability to clear subsequent infections with recombinant vaccinia viruses expressing the influenza NP. The results indicate that antiviral CTL generated in MIP-1α−/− mice are slightly impaired in their ability to protect against a subsequent infection. However, impaired in vivo CTL-mediated antiviral protection was found to be associated with reduced cytotoxicity rather than with a failure of the CTL to migrate to peripheral sites of infection. PMID:12709019

  17. A thermo-responsive protein treatment for dry eyes

    PubMed Central

    Wang, Wan; Jashnani, Aarti; Aluri, Suhaas R.; Gustafson, Joshua A.; Hsueh, Pang-Yu; Yarber, Frances; McKown, Robert L.; Laurie, Gordon W.; Hamm-Alvarez, Sarah F.; MacKay, J. Andrew

    2015-01-01

    Millions of Americans suffer from dry eye disease, and there are few effective therapies capable of treating these patients. A decade ago, an abundant protein component of human tears was discovered and named lacritin(Lacrt). Lacrt has prosecretory activity in the lacrimal gland and mitogenic activity at the corneal epithelium. Similar to other proteins placed on the ocular surface, the durability of its effect is limited by rapid tear turnover. Motivated by the rationale that a thermo-responsive coacervate containing Lacrt would have better retention upon administration, we have constructed and tested the activity of a thermo-responsive Lacrt fused to an Elastin-like polypeptide (ELP). Inspired from the human tropoelastin protein, ELP protein polymers reversibly phase separate into viscous coacervates above a tunable transition temperature. This fusion construct exhibited the prosecretory function of native Lacrt as illustrated by its ability to stimulate β-hexosaminidase secretion from primary rabbit lacrimal gland acinar cells. It also increased tear secretion from non-obese diabetic (NOD) mice, a model of autoimmune dacryoadenitis, when administered via intra-lacrimal injection. Lacrt ELP fusion proteins undergo temperature-mediated assembly to form a depot inside the lacrimal gland. We propose that these Lacrt ELP fusion proteins represent a potential therapy for dry eye disease and the strategy of ELP-mediated phase separation may have applicability to other diseases of the ocular surface. PMID:25481446

  18. Protective antibody responses against Clostridium difficile elicited by a DNA vaccine expressing the enzymatic domain of toxin B.

    PubMed

    Jin, Ke; Wang, Shixia; Zhang, Chunhua; Xiao, Yanling; Lu, Shan; Huang, Zuhu

    2013-01-01

    A DNA vaccination approach was used in the current study to screen for the immunogenicity of different fragments of toxin A and toxin B from Clostridium difficile. With this approach, protein antigens do not need to be produced in vitro and the immunogenicity of candidate C. difficile antigens can be identified directly in animals. Codon optimized toxin gene fragments were individually cloned into the DNA vaccine vector and tested in mice and rabbits for their ability to elicit C. difficile toxin-specific antibody responses. Only a subset of the C. difficile toxin fragments, including the C-terminal receptor binding domain of toxin A and a novel N-terminal enzymatic domain of toxin B, were able to elicit protective antibody responses as determined by protection of target cells in a cytotoxicity assay or by preventing death of mice in a passive antibody protection study. Significantly, antibodies elicited by the novel N-terminus of the toxin B DNA vaccine were able to increase the level of protection when used in combination with anti-toxin A antibodies in a toxin challenge model in mice.

  19. Enzyme-Responsive Delivery of Multiple Proteins with Spatiotemporal Control

    PubMed Central

    Zhu, Suwei; Nih, Lina; Carmichael, S. Thomas; Lu, Yunfeng; Segura, Tatiana

    2015-01-01

    The growth of tissues and organs is regulated by orchestrated signals from biomolecules such as enzymes and growth factors. The ability to deliver signal molecules in response to particular biological events (e.g., enzyme expression and activation) holds great promise towards tissue healing and regeneration. The current delivery vehicles mainly rely on hydrolysable scaffolds and thin films of protein-containing polymers, which cannot be programmed to respond to biological signals. We report herein an injectable delivery platform based on enantiomeric protein nanocapsules, which can deliver multiple proteins with spatiotemporal control in response to the tissue proteases secreted during wound healing. Exemplified by stroke and diabetic wound healing in mice, sequential delivery of vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) greatly enhances tissue revascularization and vessel maturation, providing effective delivery vehicles for tissue engineering and reparative medicine. PMID:25962336

  20. The unfolded protein response in immunity and inflammation

    PubMed Central

    Grootjans, Joep; Kaser, Arthur; Kaufman, Randal J.; Blumberg, Richard S.

    2017-01-01

    The unfolded protein response (UPR) is a highly conserved pathway that allows the cell to manage endoplasmic reticulum (ER) stress that is imposed by the secretory demands associated with environmental forces. In this role, the UPR has increasingly been shown to have crucial functions in immunity and inflammation. In this Review, we discuss the importance of the UPR in the development, differentiation, function and survival of immune cells in meeting the needs of an immune response. In addition, we review current insights into how the UPR is involved in complex chronic inflammatory diseases and, through its role in immune regulation, antitumour responses. PMID:27346803

  1. Co-Activation of Th17 and Antibody Responses Provides Efficient Protection against Mucosal Infection by Group A Streptococcus

    PubMed Central

    Chen, Xianyang; Li, Ning; Bi, Shuai; Wang, Xiaoguang; Wang, Beinan

    2016-01-01

    Conserved protein antigens among serotypes of group A Streptococcus pyogenes (GAS) have been focused for vaccine development because of the diversity of GAS serotypes and risks of autoimmunity post-GAS infection. Precise delineation of protective immune response to each of GAS antigens is critical for vaccine efficacy and safety. We recently reported that immunization with SrtA of GAS provides Th17-dependent clearance of heterologous serotypes of GAS in NALT. SCPA is a surface virulence molecule of GAS and known to induce antibody-mediated protection against GAS. We hypothesized that co-immunization with SrtA and SCPA would provide more efficient protection by eliciting combined Th17 and antibody responses. The present study showed that mice that were intranasally co-immunized with SrtA/SCPA cleared GAS more efficiently than the mice that were immunized with either SrtA or SCPA individually, and as efficient as the mice that experienced repeated GAS infections. The co-immunization induced Th17 and robust SCPA antibody responses, accompanied by a rapid influx of neutrophils and high myeloperoxidase activity in NALT, suggesting that simultaneous induction of mucosal Th17 and neutralizing antibody responses offers more effective GAS elimination through rapid infiltration and activation of neutrophils. Moreover, Th17 response was strongly induced in mice that experienced repeated GAS-infection and maintained at a high level even after the bacteria were cleared; whereas, it was moderately induced and promptly returned to baseline following bacterial elimination in SrtA/SCPA co-immunized mice. Additional results showed that the survival rate of systemic challenge was significantly higher in infection experienced than in co-immunized mice, indicating that more immune elements are required for protection against systemic than mucosal GAS infection. PMID:28030629

  2. Genetic conjugation of components in two pneumococcal fusion protein vaccines enhances paediatric mucosal immune responses.

    PubMed

    Pope, Caroline; Oliver, Elizabeth H; Ma, Jiangtao; Langton Hewer, Claire; Mitchell, Tim J; Finn, Adam

    2015-03-30

    Streptococcus pneumoniae colonises the upper respiratory tract and can cause pneumonia, meningitis and otitis media. Existing pneumococcal conjugate vaccines are expensive to produce and only protect against 13 of the 90+ pneumococcal serotypes; hence there is an urgent need for the development of new vaccines. We have shown previously in mice that pneumolysin (Ply) and a non-toxic variant (Δ6Ply) enhance antibody responses when genetically fused to pneumococcal surface adhesin A (PsaA), a potentially valuable effect for future vaccines. We investigated this adjuvanticity in human paediatric mucosal primary immune cell cultures. Adenoidal mononuclear cells (AMNC) from children aged 0-15 years (n=46) were stimulated with conjugated, admixed or individual proteins, cell viability and CD4+ T-cell proliferative responses were assessed using flow cytometry and cytokine secretion was measured using multiplex technology. Proliferation of CD4+ T-cells in response to PsaAPly, was significantly higher than responses to individual or admixed proteins (p=0.002). In contrast, an enhanced response to PsaAΔ6Ply compared to individual or admixed proteins only occurred at higher concentrations (p<0.01). Evaluation of cytotoxicity suggested that responses occurred when Ply-induced cytolysis was inhibited, either by fusion or mutation, but importantly an additional toxicity independent immune enhancing effect was also apparent as a result of fusion. Responses were MHC class II dependent and had a Th1/Th17 profile. Genetic fusion of Δ6Ply to PsaA significantly modulates and enhances pro-inflammatory CD4+ T-cell responses without the cytolytic effects of some other pneumolysoids. Membrane binding activity of such proteins may confer valuable adjuvant properties as fusion may assist Δ6Ply to deliver PsaA to the APC surface effectively, contributing to the initiation of anti-pneumococcal CD4+ T-cell immunity.

  3. Radioadaptive response for protection against radiation-induced teratogenesis.

    PubMed

    Okazaki, Ryuji; Ootsuyama, Akira; Norimura, Toshiyuki

    2005-03-01

    To clarify the characteristics of the radioadaptive response in mice, we compared the incidence of radiation-induced malformations in ICR mice. Pregnant ICR mice were exposed to a priming dose of 2 cGy (667 muGy/min) on day 9.5 of gestation and to a challenging dose of 2 Gy (1.04 Gy/min) 4 h later and were killed on day 18.5 of gestation. The incidence of malformations and prenatal death and fetal body weights were studied. The incidence of external malformations was significantly lower (by approximately 10%) in the primed (2 cGy + 2 Gy) mice compared to the unprimed (2 Gy alone) mice. However, there were no differences in the incidence of prenatal death or the skeletal malformations or the body weights between primed and unprimed mice. These results suggest that primary conditioning with low doses of radiation suppresses radiation-induced teratogenesis.

  4. 10 CFR 35.2024 - Records of authority and responsibilities for radiation protection programs.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 10 Energy 1 2010-01-01 2010-01-01 false Records of authority and responsibilities for radiation... MATERIAL Records § 35.2024 Records of authority and responsibilities for radiation protection programs. (a... of the Radiation Safety Officer as required by § 35.24(e), and a signed copy of each Radiation...

  5. 10 CFR 35.2024 - Records of authority and responsibilities for radiation protection programs.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 10 Energy 1 2014-01-01 2014-01-01 false Records of authority and responsibilities for radiation... MATERIAL Records § 35.2024 Records of authority and responsibilities for radiation protection programs. (a... of the Radiation Safety Officer as required by § 35.24(e), and a signed copy of each Radiation...

  6. 10 CFR 35.2024 - Records of authority and responsibilities for radiation protection programs.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 10 Energy 1 2012-01-01 2012-01-01 false Records of authority and responsibilities for radiation... MATERIAL Records § 35.2024 Records of authority and responsibilities for radiation protection programs. (a... of the Radiation Safety Officer as required by § 35.24(e), and a signed copy of each Radiation...

  7. 10 CFR 35.2024 - Records of authority and responsibilities for radiation protection programs.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 10 Energy 1 2013-01-01 2013-01-01 false Records of authority and responsibilities for radiation... MATERIAL Records § 35.2024 Records of authority and responsibilities for radiation protection programs. (a... of the Radiation Safety Officer as required by § 35.24(e), and a signed copy of each Radiation...

  8. 10 CFR 35.2024 - Records of authority and responsibilities for radiation protection programs.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 10 Energy 1 2011-01-01 2011-01-01 false Records of authority and responsibilities for radiation... MATERIAL Records § 35.2024 Records of authority and responsibilities for radiation protection programs. (a... of the Radiation Safety Officer as required by § 35.24(e), and a signed copy of each Radiation...

  9. On the HEMP (high-altitude electromagnetic pulse) response of protective relays

    SciTech Connect

    Thomas, D.E.; Wiggins, C.M.; Barnes, P.R.; Oak Ridge National Lab., TN )

    1990-01-01

    An assessment of the susceptibility of protective relays to the transients produced by high-altitude electromagnetic pulse (HEMP) events is presented in this paper. Several mechanisms responsible for coupling of HEMP to relay terminals are examined. The predicted relay responses to HEMP events are compared to measured data on a solid state based relay's impulse. 11 refs., 16 figs.

  10. Immunogenicity and protective efficacy of Semliki forest virus replicon-based DNA vaccines encoding goatpox virus structural proteins

    SciTech Connect

    Zheng Min; Jin Ningyi; Liu Qi; Huo Xiaowei; Li Yang; Hu Bo; Ma Haili; Zhu Zhanbo; Cong Yanzhao; Li Xiao; Jin Minglan; Zhu Guangze

    2009-08-15

    Goatpox, caused by goatpox virus (GTPV), is an acute feverish and contagious disease in goats often associated with high morbidity and high mortality. To resolve potential safety risks and vaccination side effects of existing live attenuated goatpox vaccine (AV41), two Semliki forest virus (SFV) replicon-based bicistronic expression DNA vaccines (pCSm-AAL and pCSm-BAA) which encode GTPV structural proteins corresponding to the Vaccinia virus proteins A27, L1, A33, and B5, respectively, were constructed. Then, theirs ability to induce humoral and cellular response in mice and goats, and protect goats against virulent virus challenge were evaluated. The results showed that, vaccination with pCSm-AAL and pCSm-BAA in combination could elicit strong humoral and cellular responses in mice and goats, provide partial protection against viral challenge in goats, and reduce disease symptoms. Additionally, priming vaccination with the above-mentioned DNA vaccines could significantly reduce the goats' side reactions from boosting vaccinations with current live vaccine (AV41), which include skin lesions at the inoculation site and fevers. Data obtained in this study could not only facilitate improvement of the current goatpox vaccination strategy, but also provide valuable guidance to suitable candidates for evaluation and development of orthopoxvirus vaccines.

  11. Immunogenicity and protective efficacy of Semliki forest virus replicon-based DNA vaccines encoding goatpox virus structural proteins.

    PubMed

    Zheng, Min; Jin, Ningyi; Liu, Qi; Huo, Xiaowei; Li, Yang; Hu, Bo; Ma, Haili; Zhu, Zhanbo; Cong, Yanzhao; Li, Xiao; Jin, Minglan; Zhu, Guangze

    2009-08-15

    Goatpox, caused by goatpox virus (GTPV), is an acute feverish and contagious disease in goats often associated with high morbidity and high mortality. To resolve potential safety risks and vaccination side effects of existing live attenuated goatpox vaccine (AV41), two Semliki forest virus (SFV) replicon-based bicistronic expression DNA vaccines (pCSm-AAL and pCSm-BAA) which encode GTPV structural proteins corresponding to the Vaccinia virus proteins A27, L1, A33, and B5, respectively, were constructed. Then, theirs ability to induce humoral and cellular response in mice and goats, and protect goats against virulent virus challenge were evaluated. The results showed that, vaccination with pCSm-AAL and pCSm-BAA in combination could elicit strong humoral and cellular responses in mice and goats, provide partial protection against viral challenge in goats, and reduce disease symptoms. Additionally, priming vaccination with the above-mentioned DNA vaccines could significantly reduce the goats' side reactions from boosting vaccinations with current live vaccine (AV41), which include skin lesions at the inoculation site and fevers. Data obtained in this study could not only facilitate improvement of the current goatpox vaccination strategy, but also provide valuable guidance to suitable candidates for evaluation and development of orthopoxvirus vaccines.

  12. Oral Immunization with Recombinant Vaccinia Virus Prime and Intramuscular Protein Boost Provides Protection against Intrarectal Simian-Human Immunodeficiency Virus Challenge in Macaques.

    PubMed

    Thippeshappa, Rajesh; Tian, Baoping; Cleveland, Brad; Guo, Wenjin; Polacino, Patricia; Hu, Shiu-Lok

    2015-12-30

    Human immunodeficiency virus type 1 (HIV-1) acquisition occurs predominantly through mucosal transmission. We hypothesized that greater mucosal immune responses and protective efficacy against mucosal HIV-1 infection may be achieved by prime-boost immunization at mucosal sites. We used a macaque model to determine the safety, immunogenicity, and protective efficacy of orally delivered, replication-competent but attenuated recombinant vaccinia viruses expressing full-length HIV-1 SF162 envelope (Env) or simian immunodeficiency virus (SIV) Gag-Pol proteins. We examined the dose and route that are suitable for oral immunization with recombinant vaccinia viruses. We showed that sublingual inoculation of two vaccinia virus-naive pigtailed macaques with 5 × 10(8) PFU of recombinant vaccinia viruses was safe. However, sublingual inoculation with a higher dose or tonsillar inoculation resulted in secondary oral lesions, indicating the need to optimize the dose and route for oral immunization with replication-competent vaccinia virus vectors. Oral priming alone elicited antibody responses to vaccinia virus and to the SF162 Env protein. Intramuscular immunization with the SF162 gp120 protein at either 20 or 21 weeks postpriming resulted in a significant boost in antibody responses in both systemic and mucosal compartments. Furthermore, we showed that immune responses induced by recombinant vaccinia virus priming and intramuscular protein boosting provided protection against intrarectal challenge with the simian-human immunodeficiency virus SHIV-SF162-P4.

  13. Small heat shock proteins mediate cell-autonomous and -nonautonomous protection in a Drosophila model for environmental-stress-induced degeneration.

    PubMed

    Kawasaki, Fumiko; Koonce, Noelle L; Guo, Linda; Fatima, Shahroz; Qiu, Catherine; Moon, Mackenzie T; Zheng, Yunzhen; Ordway, Richard W

    2016-09-01

    Cell and tissue degeneration, and the development of degenerative diseases, are influenced by genetic and environmental factors that affect protein misfolding and proteotoxicity. To better understand the role of the environment in degeneration, we developed a genetic model for heat shock (HS)-stress-induced degeneration in Drosophila This model exhibits a unique combination of features that enhance genetic analysis of degeneration and protection mechanisms involving environmental stress. These include cell-type-specific failure of proteostasis and degeneration in response to global stress, cell-nonautonomous interactions within a simple and accessible network of susceptible cell types, and precise temporal control over the induction of degeneration. In wild-type flies, HS stress causes selective loss of the flight ability and degeneration of three susceptible cell types comprising the flight motor: muscle, motor neurons and associated glia. Other motor behaviors persist and, accordingly, the corresponding cell types controlling leg motor function are resistant to degeneration. Flight motor degeneration was preceded by a failure of muscle proteostasis characterized by diffuse ubiquitinated protein aggregates. Moreover, muscle-specific overexpression of a small heat shock protein (HSP), HSP23, promoted proteostasis and protected muscle from HS stress. Notably, neurons and glia were protected as well, indicating that a small HSP can mediate cell-nonautonomous protection. Cell-autonomous protection of muscle was characterized by a distinct distribution of ubiquitinated proteins, including perinuclear localization and clearance of protein aggregates associated with the perinuclear microtubule network. This network was severely disrupted in wild-type preparations prior to degeneration, suggesting that it serves an important role in muscle proteostasis and protection. Finally, studies of resistant leg muscles revealed that they sustain proteostasis and the microtubule

  14. Small heat shock proteins mediate cell-autonomous and -nonautonomous protection in a Drosophila model for environmental-stress-induced degeneration

    PubMed Central

    Kawasaki, Fumiko; Koonce, Noelle L.; Guo, Linda; Fatima, Shahroz; Qiu, Catherine; Moon, Mackenzie T.; Zheng, Yunzhen

    2016-01-01

    ABSTRACT Cell and tissue degeneration, and the development of degenerative diseases, are influenced by genetic and environmental factors that affect protein misfolding and proteotoxicity. To better understand the role of the environment in degeneration, we developed a genetic model for heat shock (HS)-stress-induced degeneration in Drosophila. This model exhibits a unique combination of features that enhance genetic analysis of degeneration and protection mechanisms involving environmental stress. These include cell-type-specific failure of proteostasis and degeneration in response to global stress, cell-nonautonomous interactions within a simple and accessible network of susceptible cell types, and precise temporal control over the induction of degeneration. In wild-type flies, HS stress causes selective loss of the flight ability and degeneration of three susceptible cell types comprising the flight motor: muscle, motor neurons and associated glia. Other motor behaviors persist and, accordingly, the corresponding cell types controlling leg motor function are resistant to degeneration. Flight motor degeneration was preceded by a failure of muscle proteostasis characterized by diffuse ubiquitinated protein aggregates. Moreover, muscle-specific overexpression of a small heat shock protein (HSP), HSP23, promoted proteostasis and protected muscle from HS stress. Notably, neurons and glia were protected as well, indicating that a small HSP can mediate cell-nonautonomous protection. Cell-autonomous protection of muscle was characterized by a distinct distribution of ubiquitinated proteins, including perinuclear localization and clearance of protein aggregates associated with the perinuclear microtubule network. This network was severely disrupted in wild-type preparations prior to degeneration, suggesting that it serves an important role in muscle proteostasis and protection. Finally, studies of resistant leg muscles revealed that they sustain proteostasis and the

  15. In vivo protein transduction: delivery of PEP-1-SOD1 fusion protein into myocardium efficiently protects against ischemic insult.

    PubMed

    Zhang, You-En; Wang, Jia-Ning; Tang, Jun-Ming; Guo, Ling-Yun; Yang, Jian-Ye; Huang, Yong-Zhang; Tan, Yan; Fu, Shou-Zhi; Kong, Xia; Zheng, Fei

    2009-02-28

    Myocardial ischemia-reperfusion injury is a medical problem occurring as damage to the myocardium following blood flow restoration after a critical period of coronary occlusion. Oxygen free radicals (OFR) are implicated in reperfusion injury after myocardial ischemia. The antioxidant enzyme, Cu, Zn-superoxide dismutase (Cu, Zn-SOD, also called SOD1) is one of the major means by which cells counteract the deleterious effects of OFR after ischemia. Recently, we reported that a PEP-1-SOD1 fusion protein was efficiently delivered into cultured cells and isolated rat hearts with ischemia-reperfusion injury. In the present study, we investigated the protective effects of the PEP-1-SOD1 fusion protein after ischemic insult. Immunofluorescecnce analysis revealed that the expressed and purified PEP-1-SOD1 fusion protein injected into rat tail veins was efficiently transduced into the myocardium with its native protein structure intact. When injected into Sprague-Dawley rat tail veins, the PEP-1- SOD1 fusion protein significantly attenuated myocardial ischemia-reperfusion damage; characterized by improving cardiac function of the left ventricle, decreasing infarct size, reducing the level of malondialdehyde (MDA), decreasing the release of creatine kinase (CK) and lactate dehydrogenase (LDH), and relieving cardiomyocyte apoptosis. These results suggest that the biologically active intact forms of PEP-1-SOD1 fusion protein will provide an efficient strategy for therapeutic delivery in various diseases related to SOD1 or to OFR.

  16. Parenteral Vaccination Can Be an Effective Means of Inducing Protective Mucosal Responses

    PubMed Central

    Freytag, Lucy C.

    2016-01-01

    The current paradigm in vaccine development is that nonreplicating vaccines delivered parenterally fail to induce immune responses in mucosal tissues. However, both clinical and experimental data have challenged this concept, and numerous studies have shown that induction of mucosal immune responses after parenteral vaccination is not a rare occurrence and might, in fact, significantly contribute to the protection against mucosal infections afforded by parenteral vaccines. While the mechanisms underlying this phenomenon are not well understood, the realization that parenteral vaccination can be an effective means of inducing protective mucosal responses is paradigm-shifting and has potential to transform the way vaccines are designed and delivered. PMID:27122485

  17. Mycobacterial Membrane Vesicles Administered Systemically in Mice Induce a Protective Immune Response to Surface Compartments of Mycobacterium tuberculosis

    PubMed Central

    Carreño, Leandro J.; Batista-Gonzalez, Ana; Baena, Andres; Venkataswamy, Manjunatha M.; Xu, Jiayong; Yu, Xiaobo; Wallstrom, Garrick; Magee, D. Mitchell; LaBaer, Joshua; Achkar, Jacqueline M.; Jacobs, William R.; Chan, John; Porcelli, Steven A.; Casadevall, Arturo

    2014-01-01

    ABSTRACT Pathogenic and nonpathogenic species of bacteria and fungi release membrane vesicles (MV), containing proteins, polysaccharides, and lipids, into the extracellular milieu. Previously, we demonstrated that several mycobacterial species, including bacillus Calmette-Guerin (BCG) and Mycobacterium tuberculosis, release MV containing lipids and proteins that subvert host immune response in a Toll-like receptor 2 (TLR2)-dependent manner (R. Prados-Rosales et al., J. Clin. Invest. 121:1471–1483, 2011, doi:10.1172/JCI44261). In this work, we analyzed the vaccine potential of MV in a mouse model and compared the effects of immunization with MV to those of standard BCG vaccination. Immunization with MV from BCG or M. tuberculosis elicited a mixed humoral and cellular response directed to both membrane and cell wall components, such as lipoproteins. However, only vaccination with M. tuberculosis MV was able to protect as well as live BCG immunization. M. tuberculosis MV boosted BCG vaccine efficacy. In summary, MV are highly immunogenic without adjuvants and elicit immune responses comparable to those achieved with BCG in protection against M. tuberculosis. PMID:25271291

  18. Physiological Response to Membrane Protein Overexpression in E. coli*

    PubMed Central

    Gubellini, Francesca; Verdon, Grégory; Karpowich, Nathan K.; Luff, Jon D.; Boël, Grégory; Gauthier, Nils; Handelman, Samuel K.; Ades, Sarah E.; Hunt, John F.

    2011-01-01

    Overexpression represents a principal bottleneck in structural and functional studies of integral membrane proteins (IMPs). Although E. coli remains the leading organism for convenient and economical protein overexpression, many IMPs exhibit toxicity on induction in this host and give low yields of properly folded protein. Different mechanisms related to membrane biogenesis and IMP folding have been proposed to contribute to these problems, but there is limited understanding of the physical and physiological constraints on IMP overexpression and folding in vivo. Therefore, we used a variety of genetic, genomic, and microscopy techniques to characterize the physiological responses of Escherichia coli MG1655 cells to overexpression of a set of soluble proteins and IMPs, including constructs exhibiting different levels of toxicity and producing different levels of properly folded versus misfolded product on induction. Genetic marker studies coupled with transcriptomic results indicate only minor perturbations in many of the physiological systems implicated in previous studies of IMP biogenesis. Overexpression of either IMPs or soluble proteins tends to block execution of the standard stationary-phase transcriptional program, although these effects are consistently stronger for the IMPs included in our study. However, these perturbations are not an impediment to successful protein overexpression. We present evidence that, at least for the target proteins included in our study, there is no inherent obstacle to IMP overexpression in E. coli at moderate levels suitable for structural studies and that the biochemical and conformational properties of the proteins themselves are the major obstacles to success. Toxicity associated with target protein activity produces selective pressure leading to preferential growth of cells harboring expression-reducing and inactivating mutations, which can produce chemical heterogeneity in the target protein population, potentially

  19. Unfolded protein response in filamentous fungi-implications in biotechnology.

    PubMed

    Heimel, Kai

    2015-01-01

    The unfolded protein response (UPR) represents a mechanism to preserve endoplasmic reticulum (ER) homeostasis that is conserved in eukaryotes. ER stress caused by the accumulation of potentially toxic un- or misfolded proteins in the ER triggers UPR activation and the induction of genes important for protein folding in the ER, ER expansion, and transport from and to the ER. Along with this adaptation, the overall capacity for protein secretion is markedly increased by the UPR. In filamentous fungi, various approaches to employ the UPR for improved production of homologous and heterologous proteins have been investigated. As the effects on protein production were strongly dependent on the expressed protein, generally applicable strategies have to be developed. A combination of transcriptomic approaches monitoring secretion stress and basic research on the UPR mechanism provided novel and important insight into the complex regulatory cross-connections between UPR signalling, cellular physiology, and developmental processes. It will be discussed how this increasing knowledge on the UPR might stimulate the development of novel strategies for using the UPR as a tool in biotechnology.

  20. The effect of the unfolded protein response on the production of recombinant proteins in plants.

    PubMed

    Thomas, David Rhys; Walmsley, Amanda Maree

    2015-02-01

    Recombinant proteins are currently produced through a wide variety of host systems, including yeast, E. coli, insect and mammalian cells. One of the most recent systems developed uses plant cells. While considerable advances have been made in the yields and fidelity of plant-made recombinant proteins, many of these gains have arisen from the development of recombinant factors. This includes elements such as highly effective promoters and untranslated regions, deconstructed viral vectors, silencing inhibitors, and improved DNA delivery techniques. However, unlike other host systems, much of the work on recombinant protein production in plants uses wild-type hosts that have not been modified to facilitate recombinant protein expression. As such, there are still endogenous mechanisms functioning to maintain the health of the cell. The result is that these pathways, such as the unfolded protein response, can actively work to reduce recombinant protein production to maintain the integrity of the cell. This review examines how issues arising from the unfolded protein response have been addressed in other systems, and how these methods may be transferable to plant systems. We further identify several areas of host plant biology that present attractive targets for modification to facilitate recombinant protein production.

  1. Interactions involved in pH protection of the alphavirus fusion protein

    SciTech Connect

    Fields, Whitney; Kielian, Margaret

    2015-12-15

    The alphavirus membrane protein E1 mediates low pH-triggered fusion of the viral and endosome membranes during virus entry. During virus biogenesis E1 associates as a heterodimer with the transmembrane protein p62. Late in the secretory pathway, cellular furin cleaves p62 to the mature E2 protein and a peripheral protein E3. E3 remains bound to E2 at low pH, stabilizing the heterodimer and thus protecting E1 from the acidic pH of the secretory pathway. Release of E3 at neutral pH then primes the virus for fusion during entry. Here we used site-directed mutagenesis and revertant analysis to define residues important for the interactions at the E3–E2 interface. Our data identified a key residue, E2 W235, which was required for E1 pH protection and alphavirus production. Our data also suggest additional residues on E3 and E2 that affect their interacting surfaces and thus influence the pH protection of E1 during alphavirus exit.

  2. Novel Soluble Dietary Fiber-Tannin Self-Assembled Film: A Promising Protein Protective Material.

    PubMed

    Song, Guo-Bin; Xu, Juan; Zheng, Hua; Feng, Ying; Zhang, Wen-Wen; Li, Kun; Ge, Shuang-shuang; Li, Kai; Zhang, Hong

    2015-06-24

    In this experiment, a natural promising protein protective film was fabricated through soluble dietary fiber (SDF)-tannin nanocluster self-assembly. FT-IR, XRD, and DSC tests were employed to investigate the interaction between the SDF and tannins before and after cross-linking induced by calcium ion. On the other hand, referring to the SEM and TEM results, the self-assembly process of the protein protective film could be indicated as follows: first, calcium ion, with its cross-ability, served as the "nucleus"; SDF and tannins were combined to prepare the nanoscale SDF-tannin clusters; then, the clusters were homogeneously deposited on the surface of protein to form a protective film by self-assembling hydrogen bond between tannin component of clusters as "adhesive" and protein in aqueous solutions under very mild conditions. Film thickness could also be controlled by tannin of different concentrations ranging from 114 to 1384 μm. Antibacterial test and in vitro cytotoxicity test proved that the film had a broad spectrum of antimicrobial properties and excellent cell biocompatibility, respectively, which might open up new applications in the food preservation and biomedical fields.

  3. A recombinant capsid protein from Dengue-2 induces protection in mice against homologous virus.

    PubMed

    Lazo, Laura; Hermida, Lisset; Zulueta, Aída; Sánchez, Jorge; López, Carlos; Silva, Ricardo; Guillén, Gerardo; Guzmán, María G

    2007-01-22

    In the present work, we study the immunogenicity and protective capacity of a recombinant capsid protein from Dengue-2 virus. The capsid gene was cloned under the T5 phage promoter and expressed in Escherichia coli. The recombinant protein was obtained mainly associated to the soluble fraction upon cellular disruption and exhibited a pattern of high aggregation, determined by gel filtration chromatography. The semipurified preparation was inoculated in mice and after three doses, no antiviral antibodies were induced. On the other hand, mice intracranially challenged with homologous lethal virus, exhibited statistically significant protection with respect to the control group. These results describe, for the first time, the protective capacity of the capsid protein of Dengue virus indicating the existence of a protector mechanism, which is totally independent of the antibodies. This lack of induction of antiviral antibodies makes the capsid protein an attractive vaccine candidate against dengue since eliminates the potential risk of the induction of antibody dependent enhancement associated to the current vaccines under study.

  4. Priming the prophenoloxidase system of Artemia franciscana by heat shock proteins protects against Vibrio campbellii challenge.

    PubMed

    Baruah, Kartik; Ranjan, Jayant; Sorgeloos, Patrick; Macrae, Thomas H; Bossier, Peter

    2011-07-01

    Like other invertebrates, the brine shrimp Artemia franciscana relies solely on innate immunity, which by definition lacks adaptive characteristics, to combat against invading pathogens. One of the innate mechanisms is melanisation of bacteria mediated by the activation of the prophenoloxidase (proPO) system. The 70 kDa heat shock proteins (Hsp70) derived from either prokaryote (Escherichia coli) or eukaryote (Artemia), well conserved and immune-dominant molecules, protect Artemia against Vibrio campbellii. However, the molecular mechanisms by which these proteins protect Artemia against Vibrio campbellii infection are unknown. Here we demonstrated that feeding gnotobiotically grown Artemia with either Artemia Hsp70 or the E. coli Hsp70 equivalent DnaK, each overproduced in E. coli, followed by V. campbellii challenge enhanced the proPO system, at both mRNA and protein activity levels. Additionally, the Artemia fed with these proteins survived well in a Vibrio challenge assay. These results indicated that Hsp70s derived from either prokaryotic or eukaryotic sources generate protective immunity in the crustacean Artemia against V. campbellii infection by priming the proPO system. This is apparently the first in vivo report on priming activity of Hsp70 in an invertebrate.

  5. Parental transfer of the antimicrobial protein LBP/BPI protects Biomphalaria glabrata eggs against oomycete infections.

    PubMed

    Baron, Olga Lucia; van West, Pieter; Industri, Benoit; Ponchet, Michel; Dubreuil, Géraldine; Gourbal, Benjamin; Reichhart, Jean-Marc; Coustau, Christine

    2013-01-01

    Vertebrate females transfer antibodies via the placenta, colostrum and milk or via the egg yolk to protect their immunologically immature offspring against pathogens. This evolutionarily important transfer of immunity is poorly documented in invertebrates and basic questions remain regarding the nature and extent of parental protection of offspring. In this study, we show that a lipopolysaccharide binding protein/bactericidal permeability increasing protein family member from the invertebrate Biomphalaria glabrata (BgLBP/BPI1) is massively loaded into the eggs of this freshwater snail. Native and recombinant proteins displayed conserved LPS-binding, antibacterial and membrane permeabilizing activities. A broad screening of various pathogens revealed a previously unknown biocidal activity of the protein against pathogenic water molds (oomycetes), which is conserved in human BPI. RNAi-dependent silencing of LBP/BPI in the parent snails resulted in a significant reduction of reproductive success and extensive death of eggs through oomycete infections. This work provides the first functional evidence that a LBP/BPI is involved in the parental immune protection of invertebrate offspring and reveals a novel and conserved biocidal activity for LBP/BPI family members.

  6. Parental Transfer of the Antimicrobial Protein LBP/BPI Protects Biomphalaria glabrata Eggs against Oomycete Infections

    PubMed Central

    Baron, Olga Lucia; van West, Pieter; Industri, Benoit; Ponchet, Michel; Dubreuil, Géraldine; Gourbal, Benjamin; Reichhart, Jean-Marc; Coustau, Christine

    2013-01-01

    Vertebrate females transfer antibodies via the placenta, colostrum and milk or via the egg yolk to protect their immunologically immature offspring against pathogens. This evolutionarily important transfer of immunity is poorly documented in invertebrates and basic questions remain regarding the nature and extent of parental protection of offspring. In this study, we show that a lipopolysaccharide binding protein/bactericidal permeability increasing protein family member from the invertebrate Biomphalaria glabrata (BgLBP/BPI1) is massively loaded into the eggs of this freshwater snail. Native and recombinant proteins displayed conserved LPS-binding, antibacterial and membrane permeabilizing activities. A broad screening of various pathogens revealed a previously unknown biocidal activity of the protein against pathogenic water molds (oomycetes), which is conserved in human BPI. RNAi-dependent silencing of LBP/BPI in the parent snails resulted in a significant reduction of reproductive success and extensive death of eggs through oomycete infections. This work provides the first functional evidence that a LBP/BPI is involved in the parental immune protection of invertebrate offspring and reveals a novel and conserved biocidal activity for LBP/BPI family members. PMID:24367257

  7. A Low-Dose Electron Diffraction Assay for Protection of Protein Structure against Damage from Drying

    NASA Astrophysics Data System (ADS)

    Massover, William H.

    2004-04-01

    A new assay using low-dose electron diffraction to measure the protection of protein structure against damage from drying is described. When thin single crystals of catalase are dried within water alone, low-dose electron diffraction yields no Bragg spots. Drying within an experimental aqueous solution that permits detection of diffraction spots thereby indicates a positive result, and the extent of these Bragg reflections into the high angle range gives a quantitative measure of the degree of protection. Bragg spots out to 3.7 3.9 [Angstrom capital A, ring] are recorded for drying within 100 mM solutions of the known structure-preserving sugars, sucrose, tannin, and trehalose. The ability of trehalose to maintain native protein structure during drying starts between 10 and 25 mM, and changes only slightly at concentrations above this threshold; with drying in 150-mM trehalose, catalase crystals yield diffraction spots out to 3.7 [Angstrom capital A, ring]. Drying within the organic nonsugar polymer polyvinylpyrrolidone gives Bragg spots to 4.0 [Angstrom capital A, ring]. This new assay should be useful to measure the unexamined structure-preserving capabilities of modified sugars, other nonsugars, and mixtures to identify which protective matrix maintains native protein structure to the greatest extent during drying; electron crystallography using that optimal matrix should yield protein structure at improved levels of high resolution.

  8. Creating community responsibility for child protection: possibilities and challenges.

    PubMed

    Daro, Deborah; Dodge, Kenneth A

    2009-01-01

    Deborah Daro and Kenneth Dodge observe that efforts to prevent child abuse have historically focused on directly improving the skills of parents who are at risk for or engaged in maltreatment. But, as experts increasingly recognize that negative forces within a community can overwhelm even well-intentioned parents, attention is shifting toward creating environments that facilitate a parent's ability to do the right thing. The most sophisticated and widely used community prevention programs, say Daro and Dodge, emphasize the reciprocal interplay between individual-family behavior and broader neighborhood, community, and cultural contexts. The authors examine five different community prevention efforts, summarizing for each both the theory of change and the empirical evidence concerning its efficacy. Each program aims to enhance community capacity by expanding formal and informal resources and establishing a normative cultural context capable of fostering collective responsibility for positive child development. Over the past ten years, researchers have explored how neighborhoods influence child development and support parenting. Scholars are still searching for agreement on the most salient contextual factors and on how to manipulate these factors to increase the likelihood parents will seek out, find, and effectively use necessary and appropriate support. The current evidence base for community child abuse prevention, observe Daro and Dodge, offers both encouragement and reason for caution. Although theory and empirical research suggest that intervention at the neighborhood level is likely to prevent child maltreatment, designing and implementing a high-quality, multifaceted community prevention initiative is expensive. Policy makers must consider the trade-offs in investing in strategies to alter community context and those that expand services for known high-risk individuals. The authors conclude that if the concept of community prevention is to move beyond the

  9. Creating Community Responsibility for Child Protection: Possibilities and Challenges

    PubMed Central

    Daro, Deborah; Dodge, Kenneth A.

    2013-01-01

    Summary Deborah Daro and Kenneth Dodge observe that efforts to prevent child abuse have historically focused on directly improving the skills of parents who are at risk for or engaged in maltreatment. But, as experts increasingly recognize that negative forces within a community can overwhelm even well-intentioned parents, attention is shifting toward creating environments that facilitate a parent’s ability to do the right thing. The most sophisticated and widely used community prevention programs, say Daro and Dodge, emphasize the reciprocal interplay between individual-family behavior and broader neighborhood, community, and cultural contexts. The authors examine five different community prevention efforts, summarizing for each both the theory of change and the empirical evidence concerning its efficacy. Each program aims to enhance community capacity by expanding formal and informal resources and establishing a normative cultural context capable of fostering collective responsibility for positive child development. Over the past ten years, researchers have explored how neighborhoods influence child development and support parenting. Scholars are still searching for agreement on the most salient contextual factors and on how to manipulate these factors to increase the likelihood parents will seek out, find, and effectively use necessary and appropriate support. The current evidence base for community child abuse prevention, observe Daro and Dodge, offers both encouragement and reason for caution. Although theory and empirical research suggest that intervention at the neighborhood level is likely to prevent child maltreatment, designing and implementing a high-quality, multifaceted community prevention initiative is expensive. Policy makers must consider the trade-offs in investing in strategies to alter community context and those that expand services for known high-risk individuals. The authors conclude that if the concept of community prevention is to move

  10. Protein phosphorylation and regulation of adaptive responses in bacteria.

    PubMed Central

    Stock, J B; Ninfa, A J; Stock, A M

    1989-01-01

    Bacteria continuously adapt to changes in their environment. Responses are largely controlled by signal transduction systems that contain two central enzymatic components, a protein kinase that uses adenosine triphosphate to phosphorylate itself at a histidine residue and a response regulator that accepts phosphoryl groups from the kinase. This conserved phosphotransfer chemistry is found in a wide range of bacterial species and operates in diverse systems to provide different regulatory outputs. The histidine kinases are frequently membrane receptor proteins that respond to environmental signals and phosphorylate response regulators that control transcription. Four specific regulatory systems are discussed in detail: chemotaxis in response to attractant and repellent stimuli (Che), regulation of gene expression in response to nitrogen deprivation (Ntr), control of the expression of enzymes and transport systems that assimilate phosphorus (Pho), and regulation of outer membrane porin expression in response to osmolarity and other culture conditions (Omp). Several additional systems are also examined, including systems that control complex developmental processes such as sporulation and fruiting-body formation, systems required for virulent infections of plant or animal host tissues, and systems that regulate transport and metabolism. Finally, an attempt is made to understand how cross-talk between parallel phosphotransfer pathways can provide a global regulatory curcuitry. PMID:2556636

  11. Protective immunity elicited by recombinant bacille Calmette-Guerin (BCG) expressing outer surface protein A (OspA) lipoprotein: a candidate Lyme disease vaccine

    PubMed Central

    1993-01-01

    The current vaccine against tuberculosis, Mycobacterium bovis strain bacille Calmette-Guerin (BCG), offers potential advantages as a live, innately immunogenic vaccine vehicle for the expression and delivery of protective recombinant antigens (Stover, C.K., V.F. de la Cruz, T.R. Fuerst, J.E. Burlein, L.A. Benson, L.T. Bennett, G.P. Bansal, J.F. Young, M.H. Lee, G.F. Hatfull et al. 1991. Nature [Lond]. 351:456; Jacobs, W.R., Jr., S.B. Snapper, L. Lugosi and B.R. Bloom. 1990. Curr. Top. Microbiol. Immunol. 155:153; Jacobs, W.R., M. Tuckman, and B.R. Bloom. 1987. Nature [Lond.]. 327:532); but as an attenuated intracellular bacterium residing in macrophages, BCG would seem to be best suited for eliciting cellular responses and not humoral responses. Since bacterial lipoproteins are often among the most immunogenic of bacterial antigens, we tested whether BCG expression of a target antigen as a membrane-associated lipoprotein could enhance the potential for a recombinant BCG vaccine to elicit high-titered protective antibody responses to target antigens. Immunization of mice with recombinant BCG vaccines expressing the outer surface protein A (OspA) antigen of Borrelia burgdorferi as a membrane-associated lipoprotein resulted in protective antibody responses that were 100- 1,000-fold higher than responses elicited by immunization with recombinant BCG expressing OspA cytoplasmically or as a secreted fusion protein. Furthermore, these improved antibody responses were observed in heterogeneous mouse strains that vary in their immune responsiveness to OspA and sensitivity to BCG growth. Thus, expression of protective antigens as chimeric membrane-associated lipoproteins on recombinant BCG may result in the generation of new candidate vaccines against Lyme borreliosis and other human or veterinary diseases where humoral immunity is the protective response. PMID:8315378

  12. Comparative proteomic and physiological analyses reveal the protective effect of exogenous calcium on the germinating soybean response to salt stress.

    PubMed

    Yin, Yongqi; Yang, Runqiang; Han, Yongbin; Gu, Zhenxin

    2015-01-15

    suppressed under salt stress condition. According to previous studies, exogenous calcium counters the harmful effect of salt stress and increases the biomass and GABA content of germinating soybeans. Nevertheless, the precise molecular mechanism underlying the role of calcium in resistance to salt stress is still unknown. This paper is the first study employing comparative proteomic and physiological analyses to reveal the protective effect of exogenous calcium in the germinating soybean response to salt stress. Our study links the biological events with proteomic information and provides detailed peptide information on all identified proteins. The functions of those significantly changed proteins are also analyzed. The physiological and comparative proteomic analyses revealed the putative molecular mechanism of exogenous calcium treatment induced salt stress responses. The findings from this paper are beneficial to high GABA-rich germinating soybean biomass. Additionally, these findings also might be applicable to the genetic engineering of soybean plants to improve stress tolerance.

  13. Cryptosporidium Priming Is More Effective than Vaccine for Protection against Cryptosporidiosis in a Murine Protein Malnutrition Model

    PubMed Central

    Bartelt, Luther A.; Bolick, David T.; Kolling, Glynis L.; Zaenker, Edna I.; Lara, Ana M.; Noronha, Francisco Jose; Cowardin, Carrie A.; Moore, John H.; Turner, Jerrold R.; Warren, Cirle A.; Buck, Gregory A.; Guerrant, Richard L.

    2016-01-01

    Cryptosporidium is a major cause of severe diarrhea, especially in malnourished children. Using a murine model of C. parvum oocyst challenge that recapitulates clinical features of severe cryptosporidiosis during malnutrition, we interrogated the effect of protein malnutrition (PM) on primary and secondary responses to C. parvum challenge, and tested the differential ability of mucosal priming strategies to overcome the PM-induced susceptibility. We determined that while PM fundamentally alters systemic and mucosal primary immune responses to Cryptosporidium, priming with C. parvum (106 oocysts) provides robust protective immunity against re-challenge despite ongoing PM. C. parvum priming restores mucosal Th1-type effectors (CD3+CD8+CD103+ T-cells) and cytokines (IFNγ, and IL12p40) that otherwise decrease with ongoing PM. Vaccination strategies with Cryptosporidium antigens expressed in the S. Typhi vector 908htr, however, do not enhance Th1-type responses to C. parvum challenge during PM, even though vaccination strongly boosts immunity in challenged fully nourished hosts. Remote non-specific exposures to the attenuated S. Typhi vector alone or the TLR9 agonist CpG ODN-1668 can partially attenuate C. parvum severity during PM, but neither as effectively as viable C. parvum priming. We conclude that although PM interferes with basal and vaccine-boosted immune responses to C. parvum, sustained reductions in disease severity are possible through mucosal activators of host defenses, and specifically C. parvum priming can elicit impressively robust Th1-type protective immunity despite ongoing protein malnutrition. These findings add insight into potential correlates of Cryptosporidium immunity and future vaccine strategies in malnourished children. PMID:27467505

  14. Cutting Edge: Innate Immune Augmenting Vesicular Stomatitis Virus Expressing Zika Virus Proteins Confers Protective Immunity.

    PubMed

    Betancourt, Dillon; de Queiroz, Nina M G P; Xia, Tianli; Ahn, Jeonghyun; Barber, Glen N

    2017-04-15

    Zika virus (ZIKV) has become a serious public health concern because of its link to brain damage in developing human fetuses. Recombinant vesicular stomatitis virus (rVSV) was shown to be a highly effective and safe vector for the delivery of foreign immunogens for vaccine purposes. In this study, we generated rVSVs (wild-type and attenuated VSV with mutated matrix protein [VSVm] versions) that express either the full length ZIKV envelope protein (ZENV) alone or include the ZENV precursor to the membrane protein upstream of the envelope protein, and our rVSV-ZIKV constructs showed efficient immunogenicity in murine models. We also demonstrated maternal protective immunity in challenged newborn mice born to female mice vaccinated with VSVm-ZENV containing the transmembrane domain. Our data indicate that rVSVm may be a suitable strategy for the design of effective vaccines against ZIKV.

  15. IMPIPS: The Immune Protection-Inducing Protein Structure Concept in the Search for Steric-Electron and Topochemical Principles for Complete Fully-Protective Chemically Synthesised Vaccine Development

    PubMed Central

    Patarroyo, Manuel Elkin; Bermúdez, Adriana; Alba, Martha Patricia; Vanegas, Magnolia; Moreno-Vranich, Armando; Poloche, Luis Antonio; Patarroyo, Manuel Alfonso

    2015-01-01

    Determining immune protection-inducing protein structures (IMPIPS) involves defining the stereo-electron and topochemical characteristics which are essential in MHC-p-TCR complex formation. Modified high activity binding peptides (mHABP) were thus synthesised to produce a large panel of IMPIPS measuring 26.5 ±3.5Å between the farthest atoms fitting into Pockets 1 to 9 of HLA-DRβ1* structures. They displayed a polyproline II-like (PPIIL) structure with their backbone O and N atoms orientated to establish H-bonds with specific residues from HLA-DRβ1*-peptide binding regions (PBR). Residues having specific charge and gauche+ orientation regarding p3χ1, p5χ2, and p7χ1 angles determined appropriate rotamer orientation for perfectly fitting into the TCR to induce an appropriate immune response. Immunological assays in Aotus monkeys involving IMPIPS mixtures led to promising results; taken together with the aforementioned physicochemical principles, non-interfering, long-lasting, protection-inducing, multi-epitope, multistage, minimal subunit-based chemically-synthesised peptides can be designed against diseases scourging humankind. PMID:25879751

  16. Vaccination of Mice Using the West Nile Virus E-Protein in a DNA Prime-Protein Boost Strategy Stimulates Cell-Mediated Immunity and Protects Mice against a Lethal Challenge

    PubMed Central

    De Filette, Marina; Soehle, Silke; Ulbert, Sebastian; Richner, Justin; Diamond, Michael S.; Sinigaglia, Alessandro; Barzon, Luisa; Roels, Stefan; Lisziewicz, Julianna; Lorincz, Orsolya; Sanders, Niek N.

    2014-01-01

    West Nile virus (WNV) is a mosquito-borne flavivirus that is endemic in Africa, the Middle East, Europe and the United States. There is currently no antiviral treatment or human vaccine available to treat or prevent WNV infection. DNA plasmid-based vaccines represent a new approach for controlling infectious diseases. In rodents, DNA vaccines have been shown to induce B cell and cytotoxic T cell responses and protect against a wide range of infections. In this study, we formulated a plasmid DNA vector expressing the ectodomain of the E-protein of WNV into nanoparticles by using linear polyethyleneimine (lPEI) covalently bound to mannose and examined the potential of this vaccine to protect against lethal WNV infection in mice. Mice were immunized twice (prime – boost regime) with the WNV DNA vaccine formulated with lPEI-mannose using different administration routes (intramuscular, intradermal and topical). In parallel a heterologous boost with purified recombinant WNV envelope (E) protein was evaluated. While no significant E-protein specific humoral response was generated after DNA immunization, protein boosting of DNA-primed mice resulted in a marked increase in total neutralizing antibody titer. In addition, E-specific IL-4 T-cell immune responses were detected by ELISPOT after protein boost and CD8+ specific IFN-γ expression was observed by flow cytometry. Challenge experiments using the heterologous immunization regime revealed protective immunity to homologous and virulent WNV infection. PMID:24503579

  17. Item Randomized-Response Models for Measuring Noncompliance: Risk-Return Perceptions, Social Influences, and Self-Protective Responses

    ERIC Educational Resources Information Center

    Bockenholt, Ulf; Van Der Heijden, Peter G. M.

    2007-01-01

    Randomized response (RR) is a well-known method for measuring sensitive behavior. Yet this method is not often applied because: (i) of its lower efficiency and the resulting need for larger sample sizes which make applications of RR costly; (ii) despite its privacy-protection mechanism the RR design may not be followed by every respondent; and…

  18. A molecular assembly system that renders antigens of choice highly repetitive for induction of protective B cell responses.

    PubMed

    Jegerlehner, Andrea; Tissot, Alain; Lechner, Franziska; Sebbel, Peter; Erdmann, Iris; Kündig, Thomas; Bächi, Thomas; Storni, Tazio; Jennings, Gary; Pumpens, Paul; Renner, Wolfgang A; Bachmann, Martin F

    2002-08-19

    Virus like particles (VLPs) are known to induce potent B cell responses in the absence of adjuvants. Moreover, epitope-specific antibody responses may be induced by VLPs that contain peptides inserted in their immunodominant regions. However, due to steric problems, the size of the peptides capable of being incorporated into VLPs while still permitting capsid assembly, is rather limited. While peptides genetically fused to either the N- or C-terminus of VLPs present fewer assembly problems, the immune responses obtained against such epitopes are often limited, most likely because the epitopes are not optimally exposed. In addition, such particles may be less stable in vivo. Here, we show that peptides and proteins engineered to contain a free cys can be chemically coupled to VLPs formed from the hepatitis B core antigen (HBcAg) containing a lys in the immuno-dominant region. By using this approach steric hindrance of capsid assembly is abrogated. Peptides or protein coupled to VLPs in an oriented fashion are shown to induce strong and protective B cell responses even against self-epitopes in the absence of adjuvants. This molecular assembly system may be used to induce strong B cell responses against most antigens.

  19. Intranuclear localization and UV response of ERCC5/XPG protein

    SciTech Connect

    Park, M.S.; Marrone, B.L.; MacInnes, M.A.

    1995-11-01

    The human ERCC5/XPG protein is defective in the hereditary genetic disorder xeroderma pigmentosum, group-G. The XPG gene encodes a single-strand DNA endonuclease which is essential for the incision step of nucleotide excision repair for a wide variety of DNA damages. We have shown previously by indirect immunofluorescence and biochemical fractionation that the XPG protein is localized in the nucleus, in discrete foci, and probably associated with the nuclear matrix. However, the intranuclear localization of XPG is markedly altered for a short time after UV irradiation. Here, we report the identification of XPG protein regions involved in the UV response, and its putative nuclear localization signals (NLS) using a B-galactosidase (B-gal) reporter gene system. Control and fusion reporter genes were expressed in Hela S3 cells after CaPO{sub 4} transfection. B-gal protein was detected by indirect immuno-fluorescence using an anti B-gal monoclonal antibody and FITC-labeled goat anti-mouse antiserum. Two NLS peptides of the XPG carboxy-terminal region (AA 1029-1069 and 1146-1186 term) were shown to independently localize B-gal fusion proteins to the nucleus (>90%). The C-terminus peptide was observed to further localize B-gal into nuclear foci and the perinucleolar regions. When B-gal was fused with two copies of the C-terminal NLS, in tandem, B-gal was extensively sublocalized to the perinucleolar regions. Shortly after cell UV irradiation (5 J/m{sup 2}) this B-gal fusion protein became dissociated from the perinucleolar regions whereupon it was distributed throughout the nucleus. Within 6 hours post-irradiation, the fusion protein reassociated again with the perinucleolar regions. These observations confirm and extend a similar UV response of endogenous XPG protein in UV-irradiation human cells. The involvement of XPG protein and its UV responses will be discussed in context of models nuclear matrix and preferential DNA repair in actively transcribed genes.

  20. The unfolded protein response triggers site-specific regulatory ubiquitylation of 40S ribosomal proteins

    PubMed Central

    Rising, Lisa; Mak, Raymond; Webb, Kristofor; Kaiser, Stephen E.; Zuzow, Nathan; Riviere, Paul; Yang, Bing; Fenech, Emma; Tang, Xin; Lindsay, Scott A.; Christianson, John C.; Hampton, Randolph Y.; Wasserman, Steven A.; Bennett, Eric J.

    2015-01-01

    Summary Insults to endoplasmic reticulum (ER) homeostasis activate the unfolded protein response (UPR), which elevates protein folding and degradation capacity and attenuates protein synthesis. While a role for ubiquitin in regulating the degradation of misfolded ER-resident proteins is well described, ubiquitin-dependent regulation of translational reprogramming during the UPR remains uncharacterized. Using global quantitative ubiquitin proteomics, we identify evolutionarily conserved, site-specific regulatory ubiquitylation of 40S ribosomal proteins. We demonstrate that these events occur on assembled cytoplasmic ribosomes and are stimulated by both UPR activation and translation inhibition. We further show that ER stress-stimulated regulatory 40S ribosomal ubiquitylation occurs on a timescale similar to eIF2α phosphorylation, is dependent upon PERK signaling, and is required for optimal cell survival during chronic UPR activation. In total, these results reveal regulatory 40S ribosomal ubiquitylation as a previously uncharacterized and important facet of eukaryotic translational control. PMID:26051182

  1. Coadministration of the Three Antigenic Leishmania infantum Poly (A) Binding Proteins as a DNA Vaccine Induces Protection against Leishmania major Infection in BALB/c Mice

    PubMed Central

    Corvo, Laura; Garde, Esther; Ramírez, Laura; Iniesta, Virginia; Bonay, Pedro; Gómez-Nieto, Carlos; González, Víctor M.; Martín, M. Elena; Alonso, Carlos; Coelho, Eduardo A. F.; Barral, Aldina; Barral-Netto, Manoel

    2015-01-01

    Background Highly conserved intracellular proteins from Leishmania have been described as antigens in natural and experimental infected mammals. The present study aimed to evaluate the antigenicity and prophylactic properties of the Leishmania infantum Poly (A) binding proteins (LiPABPs). Methodology/Principal Findings Three different members of the LiPABP family have been described. Recombinant tools based on these proteins were constructed: recombinant proteins and DNA vaccines. The three recombinant proteins were employed for coating ELISA plates. Sera from human and canine patients of visceral leishmaniasis and human patients of mucosal leishmaniasis recognized the three LiPABPs. In addition, the protective efficacy of a DNA vaccine based on the combination of the three Leishmania PABPs has been tested in a model of progressive murine leishmaniasis: BALB/c mice infected with Leishmania major. The induction of a Th1-like response against the LiPABP family by genetic vaccination was able to down-regulate the IL-10 predominant responses elicited by parasite LiPABPs after infection in this murine model. This modulation resulted in a partial protection against L. major infection. LiPABP vaccinated mice showed a reduction on the pathology that was accompanied by a decrease in parasite burdens, in antibody titers against Leishmania antigens and in the IL-4 and IL-10 parasite-specific mediated responses in comparison to control mice groups immunized with saline or with the non-recombinant plasmid. Conclusion/Significance The results presented here demonstrate for the first time the prophylactic properties of a new family of Leishmania antigenic intracellular proteins, the LiPABPs. The redirection of the immune response elicited against the LiPABP family (from IL-10 towards IFN-γ mediated responses) by genetic vaccination was able to induce a partial protection against the development of the disease in a highly susceptible murine model of leishmaniasis. PMID:25955652

  2. The role of lactoferrin binding protein B in mediating protection against human lactoferricin.

    PubMed

    Morgenthau, Ari; Livingstone, Margaret; Adamiak, Paul; Schryvers, Anthony B

    2012-06-01

    Bacteria that inhabit the mucosal surfaces of the respiratory and genitourinary tracts of mammals encounter an iron-deficient environment because of iron sequestration by the host iron-binding proteins transferrin and lactoferrin. Lactoferrin is also present in high concentrations at sites of inflammation where the cationic, antimicrobial peptide lactoferricin is produced by proteolysis of lactoferrin. Several Gram-negative pathogens express a lactoferrin receptor that enables the bacteria to use lactoferrin as an iron source. The receptor is composed of an integral membrane protein, lactoferrin binding protein A (LbpA), and a membrane-bound lipoprotein, lactoferrin binding protein B (LbpB). LbpA is essential for growth with lactoferrin as the sole iron source, whereas the role of LbpB in iron acquisition is not yet known. In this study, we demonstrate that LbpB from 2 different species is capable of providing protection against the killing activity of a human lactoferrin-derived peptide. We investigated the prevalence of lactoferrin receptors in bacteria and examined their sequence diversity. We propose that the protection against the cationic antimicrobial human lactoferrin-derived peptide is associated with clusters of negatively charged amino acids in the C-terminal lobe of LbpB that is a common feature of this protein.

  3. Protective effects of dimethyl sulfoxide on labile protein interactions during electrospray ionization.

    PubMed

    Landreh, Michael; Alvelius, Gunvor; Johansson, Jan; Jörnvall, Hans

    2014-05-06

    Electrospray ionization mass spectrometry is a valuable tool to probe noncovalent interactions. However, the integrity of the interactions in the gas-phase is heavily influenced by the ionization process. Investigating oligomerization and ligand binding of transthyretin (TTR) and the chaperone domain from prosurfactant protein C, we found that dimethyl sulfoxide (DMSO) can improve the stability of the noncovalent interactions during the electrospray process, both regarding ligand binding and the protein quaternary structure. Low amounts of DMSO can reduce in-source dissociation of native protein oligomers and their interactions with hydrophobic ligands, even under destabilizing conditions. We interpret the effects of DMSO as being derived from its enrichment in the electrospray droplets during evaporation. Protection of labile interactions can arise from the decrease in ion charges to reduce the contributions from Coulomb repulsions, as well as from the cooling effect of adduct dissociation. The protective effects of DMSO on labile protein interactions are an important property given its widespread use in protein analysis by electrospray ionization mass spectrometry (ESI-MS).

  4. Influence of Respiratory Syncytial Virus F Glycoprotein Conformation on Induction of Protective Immune Responses

    PubMed Central

    Palomo, Concepción; Mas, Vicente; Thom, Michelle; Vázquez, Mónica; Cano, Olga; Terrón, María C.; Luque, Daniel

    2016-01-01

    ABSTRACT Human respiratory syncytial virus (hRSV) vaccine development has received new impetus from structure-based studies of its main protective antigen, the fusion (F) glycoprotein. Three soluble forms of F have been described: monomeric, trimeric prefusion, and trimeric postfusion. Most human neutralizing antibodies recognize epitopes found exclusively in prefusion F. Although prefusion F induces higher levels of neutralizing antibodies than does postfusion F, postfusion F can also induce protection against virus challenge in animals. However, the immunogenicity and protective efficacy of the three forms of F have not hitherto been directly compared. Hence, BALB/c mice were immunized with a single dose of the three proteins adjuvanted with CpG and challenged 4 weeks later with virus. Serum antibodies, lung virus titers, weight loss, and pulmonary pathology were evaluated after challenge. Whereas small amounts of postfusion F were sufficient to protect mice, larger amounts of monomeric and prefusion F proteins were required for protection. However, postfusion and monomeric F proteins were associated with more pathology after challenge than was prefusion F. Antibodies induced by all doses of prefusion F, in contrast to other F protein forms, reacted predominantly with the prefusion F conformation. At high doses, prefusion F also induced the highest titers of neutralizing antibodies, and all mice were protected, yet at low doses of the immunogen, these antibodies neutralized virus poorly, and mice were not protected. These findings should be considered when developing new hRSV vaccine candidates. IMPORTANCE Protection against hRSV infection is afforded mainly by neutralizing antibodies, which recognize mostly epitopes found exclusively in the viral fusion (F) glycoprotein trimer, folded in its prefusion conformation, i.e., before activation for membrane fusion. Although prefusion F is able to induce high levels of neutralizing antibodies, highly stable postfusion

  5. Stress responses during ageing: molecular pathways regulating protein homeostasis.

    PubMed

    Kyriakakis, Emmanouil; Princz, Andrea; Tavernarakis, Nektarios

    2015-01-01

    The ageing process is characterized by deterioration of physiological function accompanied by frailty and ageing-associated diseases. The most broadly and well-studied pathways influencing ageing are the insulin/insulin-like growth factor 1 signaling pathway and the dietary restriction pathway. Recent studies in diverse organisms have also delineated emerging pathways, which collectively or independently contribute to ageing. Among them the proteostatic-stress-response networks, inextricably affect normal ageing by maintaining or restoring protein homeostasis to preserve proper cellular and organismal function. In this chapter, we survey the involvement of heat stress and endoplasmic reticulum stress responses in the regulation of longevity, placing emphasis on the cross talk between different response mechanisms and their systemic effects. We further discuss novel insights relevant to the molecular pathways mediating these stress responses that may facilitate the development of innovative interventions targeting age-related pathologies such as diabetes, cancer, cardiovascular and neurodegenerative diseases.

  6. Protective effects of zinc on oxidative stress enzymes in liver of protein-deficient rats.

    PubMed

    Sidhu, Pardeep; Garg, M L; Dhawan, D K

    2005-01-01

    Persons afflicted with protein malnutrition are generally deficient in a variety of essential micronutrients like zinc, copper, iron, and selenium, which in turn affects number of metabolic processes in the body. To evaluate the protective effects of zinc on the enzymes involved in oxidative stress induced in liver of protein-deficient rats, the current study was designed. Zinc sulfate at a dose level of 227 mg/L zinc in drinking water was administered to female Sprague-Dawley normal control as well as protein-deficient rats for a total duration of 8 weeks. The effects of zinc treatment in conditions of protein deficiency were studied on rat liver antioxidant enzymes, which included catalase, glutathione peroxidase (GPx), glutathione reductase (GR), superoxide dismutase (SOD), glutathione reduced (GSH), and glutathione-S-transferase (GST). Protein deficiency in normal rats resulted in a significant increase in hepatic activities of catalase, glutathione peroxidase, glutathione reductase, and glutathione-S-transferase and the levels of lipid peroxidation. A significant inhibition in the levels of reduced glutathione and the enzyme activity of superoxide dismutase has been observed after protein deficiency in normal rats. Interestingly, Zn treatment to protein-deficient animals lowered already raised activity catalase, glutathione peroxidase, and glutathione-S-transferase and levels of lipid peroxidation to significant levels when compared to protein-deficient animals. Also, Zn treatment to the protein-deficient animals resulted in a significant elevation in the levels of GSH and SOD activity as compared to their respective controls, thereby indicating its effectiveness in regulating their levels in adverse conditions. It has also been observed that concentrations of zinc, copper, iron, and selenium were found to be decreased significantly in protein-deficient animals. However, the levels of these elements came back to within normal limits when zinc was administrated

  7. Protein O-GlcNAcylation: A critical regulator of the cellular response to stress

    PubMed Central

    Chatham, John C.; Marchase, Richard B.

    2012-01-01

    The post-translational modification of serine and threonine residues of nuclear and cytoplasmic proteins by the O-linked attachment of the monosaccharide ß-N-acetyl-glucosamine (O-GlcNAc) is a highly dynamic and ubiquitous protein modification that plays a critical role in regulating numerous biological processes. Much of our understanding of the mechanisms underlying the role of O-GlcNAc on cellular function has been in the context of chronic disease processes. However, there is increasing evidence that O-GlcNAc levels are increased in response to stress and that acute augmentation of this response is cytoprotective, at least in the short term. Conversely, a reduction in O-GlcNAc levels appears to be associated with decreased cell survival in response to an acute stress. Here we summarize our current understanding of protein O-GlcNAcylation on the cellular response to stress and in mediating cellular protective mechanisms focusing primarily on the cardiovascular system as an example. We consider the potential link between O-GlcNAcylation and cardiomyocyte calcium homeostasis and explore the parallels between O-GlcNAc signaling and redox signaling. We also discuss the apparent paradox between the reported adverse effects of increased O-GlcNAcylation with its recently reported role in mediating cell survival mechanisms. PMID:22308107

  8. Role for the Unfolded Protein Response in Heart Disease and Cardiac Arrhythmias.

    PubMed

    Liu, Man; Dudley, Samuel C

    2015-12-31

    The unfolded protein response (UPR) has been extensively investigated in neurological diseases and diabetes, while its function in heart disease is less well understood. Activated UPR participates in multiple cardiac conditions and can either protect or impair heart function. Recently, the UPR has been found to play a role in arrhythmogenesis during human heart failure by affecting cardiac ion channels expression, and blocking UPR has an antiarrhythmic effect. This review will discuss the rationale for and challenges to targeting UPR in heart disease for treatment of arrhythmias.

  9. Immunogenicity and protective efficacy of recombinant major envelope protein (rH3L) of buffalopox virus in animal models.

    PubMed

    Kumar, Amit; Yogisharadhya, Revanaiah; Venkatesan, Gnanavel; Bhanuprakash, Veerakyathappa; Shivachandra, Sathish Bhadravati

    2016-02-01

    Buffalopox virus, a zoonotic Indian vaccinia-like virus, is responsible for contagious disease affecting mainly buffaloes, cattle and humans. H3L gene, encoding for an immunodominant major envelope protein of intracellular mature virion of orthopoxviruses, is highly conserved and found to elicit neutralizing antibodies. Therefore in the present study, the immunogenicity and protective efficacy of the recombinant H3L protein of buffalopox virus in laboratory animal models has been evaluated. A partial H3L gene encoding for the C-terminal truncated ectodomain of H3L protein (1M to I280) of BPXV-Vij/96 strain was cloned, over-expressed and purified as histidine-tagged fusion protein (50 kDa) from Escherichia coli using Ni-NTA affinity chromatography. The purified rH3L protein was further used for active immunization of guinea pig (250 μg/dose) and adult mice (10 μg and 50 μg/dose) with or without adjuvants (alum, Freund's Complete Adjuvant and CpG). Subsequently, a gradual increase in antigen specific serum IgG as well as neutralizing antibody titres measured by using indirect-ELISA and serum neutralization test respectively, was noted in both guinea pigs and mouse models. Suckling mice immunized passively with anti-H3L serum showed 80% pre-exposure prophylaxis upon challenge with virulent buffalopox virus strain. An indirect-ELISA based on rH3L protein showed no cross-reactivity with hyperimmune sera against sheeppox virus (SPPV), goatpox virus (GTPV), orf virus (ORFV), foot- and- mouth disease virus (FMDV), peste des petits ruminants virus (PPRV) and bluetongue virus (BTV) during the course of study. The study highlights the potential utility of rH3L protein as a safer prophylactic and diagnostic reagent for buffalopox.

  10. Unfolded protein response in cancer: the Physician's perspective

    PubMed Central

    2011-01-01

    The unfolded protein response (UPR) is a cascade of intracellular stress signaling events in response to an accumulation of unfolded or misfolded proteins in the lumen of the endoplasmic reticulum (ER). Cancer cells are often exposed to hypoxia, nutrient starvation, oxidative stress and other metabolic dysregulation that cause ER stress and activation of the UPR. Depending on the duration and degree of ER stress, the UPR can provide either survival signals by activating adaptive and antiapoptotic pathways, or death signals by inducing cell death programs. Sustained induction or repression of UPR pharmacologically may thus have beneficial and therapeutic effects against cancer. In this review, we discuss the basic mechanisms of UPR and highlight the importance of UPR in cancer biology. We also update the UPR-targeted cancer therapeutics currently in clinical trials. PMID:21345215

  11. Unfolded protein response in hepatitis C virus infection

    PubMed Central

    Chan, Shiu-Wan

    2014-01-01

    Hepatitis C virus (HCV) is a single-stranded, positive-sense RNA virus of clinical importance. The virus establishes a chronic infection and can progress from chronic hepatitis, steatosis to fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). The mechanisms of viral persistence and pathogenesis are poorly understood. Recently the unfolded protein response (UPR), a cellular homeostatic response to endoplasmic reticulum (ER) stress, has emerged to be a major contributing factor in many human diseases. It is also evident that viruses interact with the host UPR in many different ways and the outcome could be pro-viral, anti-viral or pathogenic, depending on the particular type of infection. Here we present evidence for the elicitation of chronic ER stress in HCV infection. We analyze the UPR signaling pathways involved in HCV infection, the various levels of UPR regulation by different viral proteins and finally, we propose several mechanisms by which the virus provokes the UPR. PMID:24904547

  12. Integration of the Unfolded Protein and Oxidative Stress Responses through SKN-1/Nrf

    PubMed Central

    Glover-Cutter, Kira M.; Lin, Stephanie; Blackwell, T. Keith

    2013-01-01

    The Unfolded Protein Response (UPR) maintains homeostasis in the endoplasmic reticulum (ER) and defends against ER stress, an underlying factor in various human diseases. During the UPR, numerous genes are activated that sustain and protect the ER. These responses are known to involve the canonical UPR transcription factors XBP1, ATF4, and ATF6. Here, we show in C. elegans that the conserved stress defense factor SKN-1/Nrf plays a central and essential role in the transcriptional UPR. While SKN-1/Nrf has a well-established function in protection against oxidative and xenobiotic stress, we find that it also mobilizes an overlapping but distinct response to ER stress. SKN-1/Nrf is regulated by the UPR, directly controls UPR signaling and transcription factor genes, binds to common downstream targets with XBP-1 and ATF-6, and is present at the ER. SKN-1/Nrf is also essential for resistance to ER stress, including reductive stress. Remarkably, SKN-1/Nrf-mediated responses to oxidative stress depend upon signaling from the ER. We conclude that SKN-1/Nrf plays a critical role in the UPR, but orchestrates a distinct oxidative stress response that is licensed by ER signaling. Regulatory integration through SKN-1/Nrf may coordinate ER and cytoplasmic homeostasis. PMID:24068940

  13. The unfolded protein response is required for dendrite morphogenesis.

    PubMed

    Wei, Xing; Howell, Audrey S; Dong, Xintong; Taylor, Caitlin A; Cooper, Roshni C; Zhang, Jianqi; Zou, Wei; Sherwood, David R; Shen, Kang

    2015-06-08

    Precise patterning of dendritic fields is essential for the formation and function of neuronal circuits. During development, dendrites acquire their morphology by exuberant branching. How neurons cope with the increased load of protein production required for this rapid growth is poorly understood. Here we show that the physiological unfolded protein response (UPR) is induced in the highly branched Caenorhabditis elegans sensory neuron PVD during dendrite morphogenesis. Perturbation of the IRE1 arm of the UPR pathway causes loss of dendritic branches, a phenotype that can be rescued by overexpression of the ER chaperone HSP-4 (a homolog of mammalian BiP/grp78). Surprisingly, a single transmembrane leucine-rich repeat protein, DMA-1, plays a major role in the induction of the UPR and the dendritic phenotype in the UPR mutants. These findings reveal a significant role for the physiological UPR in the maintenance of ER homeostasis during morphogenesis of large dendritic arbors.

  14. Preliminary protective capacity study of a Dicrocoelium dendriticum antigenic protein in hamsters.

    PubMed

    González-Lanza, C; Manga-González, M Y; Revilla-Nuín, B

    2006-11-01

    The purpose of this study was to evaluate the protective capacity of 130 kDa Dicrocoelium dendriticum protein in hamsters experimentally infected with this parasite. Forty hamsters divided into four groups of ten animals each were used: G1 (control), G2 (infected), G3 (immunized with Freund's adjuvant and infected), G4 (130 kDa protein vaccinated + adjuvant and infected). Infection with 40 metacercariae/hamster was carried out 4 weeks after the last immunization. Parasitological studies [number of eggs per gram (epg) and worm burden] and biochemical parameters (total proteins, albumin, and total bilirubin), hepatic enzymes [aspartate aminotransferase (AST) and alanine aminotransferase (ALT)], and total IgG levels were determined. A reduction in epg in G3 and G4 was observed 16 weeks postinfection with the higher reduction percentage in the latter (25.2%). No statistically significant differences were detected in the number of recovered worms among groups, although the mean was slightly less in G4 (12.2 +/- 2.08, mean +/- SE) than in G2 (15.4 +/- 2.90). In G4, global protection was 20.9% and an increase in AST and ALT levels was observed. Total IgG levels were similar in the three infected groups. The protection obtained was inadequate, so the antigen dose, immunization-infection period, adjuvants, and immunization route must be optimized.

  15. Generation of protective immune response against anthrax by oral immunization with protective antigen plant-based vaccine.

    PubMed

    Gorantala, Jyotsna; Grover, Sonam; Rahi, Amit; Chaudhary, Prerna; Rajwanshi, Ravi; Sarin, Neera Bhalla; Bhatnagar, Rakesh

    2014-04-20

    In concern with frequent recurrence of anthrax in endemic areas and inadvertent use of its spores as biological weapon, the development of an effective anthrax vaccine suitable for both human and veterinary needs is highly desirable. A simple oral delivery through expression in plant system could offer promising alternative to the current methods that rely on injectable vaccines extracted from bacterial sources. In the present study, we have expressed protective antigen (PA) gene in Indian mustard by Agrobacterium-mediated transformation and in tobacco by plastid transformation. Putative transgenic lines were verified for the presence of transgene and its expression by molecular analysis. PA expressed in transgenic lines was biologically active as evidenced by macrophage lysis assay. Intraperitoneal (i.p.) and oral immunization with plant PA in murine model indicated high serum PA specific IgG and IgA antibody titers. PA specific mucosal immune response was noted in orally immunized groups. Further, antibodies indicated lethal toxin neutralizing potential in-vitro and conferred protection against in-vivo toxin challenge. Oral immunization experiments demonstrated generation of immunoprotective response in mice. Thus, our study examines the feasibility of oral PA vaccine expressed in an edible plant system against anthrax.

  16. A prime-boost immunization with Tc52 N-terminal domain DNA and the recombinant protein expressed in Pichia pastoris protects against Trypanosoma cruzi infection.

    PubMed

    Matos, Marina N; Sánchez Alberti, Andrés; Morales, Celina; Cazorla, Silvia I; Malchiodi, Emilio L

    2016-06-14

    We have previously reported that the N-terminal domain of the antigen Tc52 (NTc52) is the section of the protein that confers the strongest protection against Trypanosoma cruzi infection. To improve vaccine efficacy, we conducted here a prime-boost strategy (NTc52PB) by inoculating two doses of pcDNA3.1 encoding the NTc52 DNA carried by attenuated Salmonella (SNTc52), followed by two doses of recombinant NTc52 expressed in Picchia pastoris plus ODN-CpG as adjuvant. This strategy was comparatively analyzed with the following protocols: (1) two doses of NTc52+ODN-CpG by intranasal route followed by two doses of NTc52+ODN-CpG by intradermal route (NTc52CpG); (2) four doses of SNTc52; and (3) a control group with four doses of Salmonella carrying the empty plasmid. All immunized groups developed a predominant Th1 cellular immune response but with important differences in antibody development and protection against infection. Thus, immunization with just SNTc52 induces a strong specific cellular response, a specific systemic antibody response that is weak yet functional (considering lysis of trypomastigotes and inhibition of cell invasion), and IgA mucosal immunity, protecting in both the acute and chronic stages of infection. The group that received only recombinant protein (NTc52CpG) developed a strong antibody immune response but weaker cellular immunity than the other groups, and the protection against infection was clear in the acute phase of infection but not in chronicity. The prime-boost strategy, which combines DNA and protein vaccine and both mucosal and systemic immunizations routes, was the best assayed protocol, inducing strong cellular and humoral responses as well as specific mucosal IgA, thus conferring better protection in the acute and chronic stages of infection.

  17. The Stress-responsive Gene ATF3 Mediates Dichotomous UV Responses by Regulating the Tip60 and p53 Proteins.

    PubMed

    Cui, Hongmei; Li, Xingyao; Han, Chunhua; Wang, Qi-En; Wang, Hongbo; Ding, Han-Fei; Zhang, Junran; Yan, Chunhong

    2016-05-13

    The response to UV irradiation is important for a cell to maintain its genetic integrity when challenged by environmental genotoxins. An immediate early response to UV irradiation is the rapid induction of activating transcription factor 3 (ATF3) expression. Although emerging evidence has linked ATF3 to stress pathways regulated by the tumor suppressor p53 and the histone acetyltransferase Tip60, the role of ATF3 in the UV response remains largely unclear. Here, we report that ATF3 mediated dichotomous UV responses. Although UV irradiation enhanced the binding of ATF3 to Tip60, knockdown of ATF3 expression decreased Tip60 stability, thereby impairing Tip60 induction by UV irradiation. In line with the role of Tip60 in mediating UV-induced apoptosis, ATF3 promoted the death of p53-defective cells in response to UV irradiation. However, ATF3 could also activate p53 and promote p53-mediated DNA repair, mainly through altering histone modifications that could facilitate recruitment of DNA repair proteins (such as DDB2) to damaged DNA sites. As a result, ATF3 rather protected the p53 wild-type cells from UV-induced apoptosis. Our results thus indicate that ATF3 regulates cell fates upon UV irradiation in a p53-dependent manner.

  18. An update on polygalacturonase-inhibiting protein (PGIP), a leucine-rich repeat protein that protects crop plants against pathogens

    PubMed Central

    Kalunke, Raviraj M.; Tundo, Silvio; Benedetti, Manuel; Cervone, Felice; De Lorenzo, Giulia; D'Ovidio, Renato

    2015-01-01

    Polygalacturonase inhibiting proteins (PGIPs) are cell wall proteins that inhibit the pectin-depolymerizing activity of polygalacturonases secreted by microbial pathogens and insects. These ubiquitous inhibitors have a leucine-rich repeat structure that is strongly conserved in monocot and dicot plants. Previous reviews have summarized the importance of PGIP in plant defense and the structural basis of PG-PGIP interaction; here we update the current knowledge about PGIPs with the recent findings on the composition and evolution of pgip gene families, with a special emphasis on legume and cereal crops. We also update the information about the inhibition properties of single pgip gene products against microbial PGs and the results, including field tests, showing the capacity of PGIP to protect crop plants against fungal, oomycetes and bacterial pathogens. PMID:25852708

  19. Casitas B-cell lymphoma (Cbl) proteins protect mammary epithelial cells from proteotoxicity of active c-Src accumulation

    PubMed Central

    Mukhopadhyay, Chandrani; Triplett, Aleata; Bargar, Tom; Heckman, Carol; Wagner, Kay-Uwe

    2016-01-01

    Casitas B-cell lymphoma (Cbl) family ubiquitin ligases negatively regulate tyrosine kinase-dependent signal transduction by promoting degradation of active kinases. We and others previously reported that loss of Cbl functions caused hyperproliferation in lymphoid and hematopoietic systems. Unexpectedly, Cbl deletion in Cbl-b–null, Cbl-c–null primary mouse mammary epithelial cells (MECs) (Cbl triple-deficiency) induced rapid cell death despite enhanced MAP kinase and AKT activation. Acute Cbl triple-deficiency elicited distinct transcriptional and biochemical responses with partial overlap with previously described cellular reactions to unfolded proteins and oxidative stress. Although the levels of reactive oxygen species were comparable, detergent-insoluble protein aggregates containing phosphorylated c-Src accumulated in Cbl triple-deficient MECs. Treatment with a broad-spectrum kinase inhibitor dasatinib blocked protein aggregate accumulation and restored in vitro organoid formation. This effect is most likely mediated through c-Src because Cbl triple-deficient MECs were able to form organoids upon shRNA-mediated c-Src knockdown. Taking these data together, the present study demonstrates that Cbl family proteins are required to protect MECs from proteotoxic stress-induced cell death by promoting turnover of active c-Src. PMID:27930322

  20. Monoclonal antibodies of three different immunoglobulin G isotypes produced by immunization with a synthetic peptide or native protein protect mice against challenge with Plasmodium yoelii sporozoites.

    PubMed

    Ak, M; Bower, J H; Hoffman, S L; Sedegah, M; Lees, A; Carter, M; Beaudoin, R L; Charoenvit, Y

    1993-06-01

    Passive transfer of monoclonal antibodies (MAbs) against malaria circumsporozoite (CS) proteins protects animals against malaria. Active immunization with synthetic or recombinant peptides induces a level of polyclonal antibodies to sporozoites comparable to those found after passive immunization but does not provide comparable protection. In the Plasmodium yoelii system, synthetic or recombinant peptide-induced antibodies have never been shown to protect. The current studies were designed to determine whether immunogen structure (native protein versus synthetic peptide) or immunoglobulin G (IgG) subclass of antibodies was responsible for the absolute differences between protective, passively transferred MAbs and nonprotective, actively induced polyclonal antibodies. In this study we produced two MAbs, QGP-S1 (IgG1) and QGP-S2 (IgG2b), by immunization with a synthetic peptide based on the P. yoelii CS major repeat, (QGPGAP)4, conjugated to keyhole limpet hemocyanin. These MAbs were compared tp NYS1 (IgG3), an anti-CS protein MAb previously produced by immunization with irradiated P. yoelii sporozoites, which recognizes (QGP GAP)2. QGP-S1 and QGP-S2 passively transferred protection. However, when compared with NYS1, there was a hierarchy of protection, NYS1 > QGP-S1 > QGP-S2. There was no correlation between antibody level at challenge as determined by immunofluorescent antibody test against sporozoites or enzyme-linked immunosorbent assay against (QGPGAP)2 or apparent antibody avidity for (QGPGAP)2 by sodium thiocyanate elution assay. The data demonstrate that a synthetic peptide can induce protective antibodies and that a specific antibody subclass is not required for protection. Work to determine whether antibody affinity or fine specificity can explain the hierarchy of protection among the MAbs is under way.

  1. A mimotope from a solid-phase peptide library induces a measles virus-neutralizing and protective antibody response.

    PubMed

    Steward, M W; Stanley, C M; Obeid, O E

    1995-12-01

    A solid-phase 8-mer random combinatorial peptide library was used to generate a panel of mimotopes of an epitope recognized by a monoclonal antibody to the F protein of measles virus (MV). An inhibition immunoassay was used to show that these peptides were bound by the monoclonal antibody with different affinities. BALB/c mice were coimmunized with the individual mimotopes and a T-helper epitope peptide (from MV fusion protein), and the reactivity of the induced anti-mimotope antibodies with the corresponding peptides and with MV was determined. The affinities of the antibodies with the homologous peptides ranged from 8.9 x 10(5) to 4.4 x 10(7) liters/mol. However, only one of the anti-mimotope antibodies cross-reacted with MV in an enzyme-linked immunosorbent assay and inhibited MV plaque formation. Coimmunization of mice with this mimotope and the T-helper epitope peptide induced an antibody response which conferred protection against fatal encephalitis induced following challenge with MV and with the structurally related canine distemper virus. These results indicate that peptide libraries can be used to identify mimotopes of conformational epitopes and that appropriate immunization with these mimotopes can induce protective antibody responses.

  2. Acute Responses of Microorganisms from Membrane Bioreactors in the Presence of NaOCl: Protective Mechanisms of Extracellular Polymeric Substances.

    PubMed

    Han, Xiaomeng; Wang, Zhiwei; Chen, Mei; Zhang, Xingran; Tang, Chuyang Y; Wu, Zhichao

    2017-03-21

    Extracellular polymeric substances (EPS) are key foulants in membrane bioreactors (MBRs). However, their positive functions of protecting microorganisms from environmental stresses, e.g., during in situ hypochlorite chemical cleaning of membranes, have not been adequately elucidated. In this work, we investigated the response of microorganisms in an MBR to various dosages of NaOCl, with a particular emphasis on the mechanistic roles of EPS. Results showed that functional groups in EPS such as the hydroxyl and amino groups were attacked by NaOCl, causing the oxidation of polysaccharides, denaturation of amino acids, damage to protein secondary structure, and transformation of tryptophan protein-like substances to condensed aromatic ring substances. The presence of EPS alleviated the negative impacts on catalase and superoxide dismutase, which in turn reduced the concentration of reactive oxygen species (ROS) in microbial cells. The direct extracellular reaction and the mitigated intracellular oxidative responses facilitated the maintenance of microbial metabolism, as indicated by the quantity of adenosine triphosphate and the activity of dehydrogenase. The reaction with NaOCl also led to the changes of cell integrity and adhesion properties of EPS, which promoted the release of organic matter into bulk solution. Our results systematically demonstrate the protective roles of EPS and the underlying mechanisms in resisting the environmental stress caused by NaOCl, which provides important implications for in situ chemical cleaning in MBRs.

  3. Cell-mediated immune response to unrelated proteins and unspecific inflammation blocked by orally tolerated proteins.

    PubMed

    Ramos, Gustavo C; Rodrigues, Claudiney M; Azevedo, Geraldo M; Pinho, Vanessa; Carvalho, Cláudia R; Vaz, Nelson M

    2009-03-01

    Oral tolerance promotes a generalized decrease in specific immune responsiveness to proteins previously encountered via the oral route. In addition, parenteral immunization with a tolerated protein also triggers a significant reduction in the primary responsiveness to a second unrelated antigen. This is generally explained by 'innocent bystander suppression', suggesting that the transient and episodic effects of inhibitory cytokines released by contact with the tolerated antigen would block responses to the second antigen. In disagreement with this view, we have previously shown that: (i) these inhibitory effects do not require concomitance or contiguity of the injections of the two proteins; (ii) that intravenous or intragastric exposures to the tolerated antigen are not inhibitory; and (iii) that the inhibitory effect, once triggered, persists in the absence of further contact with the tolerated protein, possibly by inhibition of secondary responsiveness (immunological memory). The present work confirms that immunological memory of the second unrelated antigen is hindered by exposure to the tolerated antigen and, in addition, shows that this exposure: (i) inhibits the inflammation triggered by an unrelated antigen through the double effect of inhibiting production of leucocytes in the bone marrow and blocking their migration to inflammed sites; and (ii) significantly blocks footpaw swelling triggered by carrageenan. Taken together, these results conclusively demonstrate that inhibitory effects of parenteral injection of tolerated antigens are much more general than suggested by the 'innocent bystander suppression' hypothesis.

  4. Auditing protein therapeutics management by professional APCs: toward prevention of immune responses against therapeutic proteins.

    PubMed

    Dasgupta, Suryasarathi; Bayry, Jagadeesh; André, Sebastien; Dimitrov, Jordan D; Kaveri, Srinivas V; Lacroix-Desmazes, Sebastien

    2008-08-01

    Alloimmunization is a crippling concern in the management of patients undergoing administration of protein therapeutics as evidenced in replacement therapy and other treatment procedures. Several issues in the genesis and modulation of such deleterious immune responses have been studied. While authors have focused on the downstream events of the specific immune response and suggested modification of protein therapeutics to eliminate epitopes that interact with B cell receptors, T cell receptors, or MHCII molecules, the mechanisms underlying Ag interaction with APCs, a step upstream of immune effectors, have been grossly neglected. We wish to emphasize that the recent knowledge in understanding the capacities of an APC to handle an Ag and the importance of the surrounding microenvironment in this process are crucial for designing novel protein therapeutics with reduced immunogenicity.

  5. Protein Phosphatase Methyl-Esterase PME-1 Protects Protein Phosphatase 2A from Ubiquitin/Proteasome Degradation.

    PubMed

    Yabe, Ryotaro; Miura, Akane; Usui, Tatsuya; Mudrak, Ingrid; Ogris, Egon; Ohama, Takashi; Sato, Koichi

    2015-01-01

    Protein phosphatase 2A (PP2A) is a conserved essential enzyme that is implicated as a tumor suppressor based on its central role in phosphorylation-dependent signaling pathways. Protein phosphatase methyl esterase (PME-1) catalyzes specifically the demethylation of the C-terminal Leu309 residue of PP2A catalytic subunit (PP2Ac). It has been shown that PME-1 affects the activity of PP2A by demethylating PP2Ac, but also by directly binding to the phosphatase active site, suggesting loss of PME-1 in cells would enhance PP2A activity. However, here we show that PME-1 knockout mouse embryonic fibroblasts (MEFs) exhibit lower PP2A activity than wild type MEFs. Loss of PME-1 enhanced poly-ubiquitination of PP2Ac and shortened the half-life of PP2Ac protein resulting in reduced PP2Ac levels. Chemical inhibition of PME-1 and rescue experiments with wild type and mutated PME-1 revealed methyl-esterase activity was necessary to maintain PP2Ac protein levels. Our data demonstrate that PME-1 methyl-esterase activity protects PP2Ac from ubiquitin/proteasome degradation.

  6. Recombinant rabies virus expressing the H protein of canine distemper virus protects dogs from the lethal distemper challenge.

    PubMed

    Wang, Feng-Xue; Zhang, Shu-Qin; Zhu, Hong-Wei; Yang, Yong; Sun, Na; Tan, Bin; Li, Zhen-Guang; Cheng, Shi-Peng; Fu, Zhen F; Wen, Yong-Jun

    2014-12-05

    The rabies virus (RV) vector LBNSE expressing foreign antigens have shown considerable promise as vaccines against viral and bacteria diseases, which is effective and safe. We produced a new RV-based vaccine vehicle expressing 1.824 kb hemagglutinin (H) gene of the canine distemper virus (CDV) by reverse genetics technology. The recombinant virus LBNSE-CDV-H retained growth properties similar to those of vector LBNSE both in BSR and mNA cell culture. The H gene of CDV was expressed and detected by immunostaining. To compare the immunogenicity of LBNSE-CDV-H, dogs were immunized with each of these recombinant viruses by intramuscular (i.m.). The dogs were bled at third weeks after the immunization for the measurement of virus neutralizing antibody (VNA) and then challenged with virulent virus (ZJ 7) at fourth weeks. The parent virus (LBNSE) without expression of any foreign molecules was included for comparison. Dogs inoculated with LBNSE-CDV-H showed no any signs of disease and exhibited seroconversion against both RV and CDV H protein. The LBNSE-CDV-H did not cause disease in dogs and conferred protection from challenge with a lethal wild type CDV strain, demonstrating its potential value for wildlife conservation efforts. Together, these studies suggest that recombinant RV expressing H protein from CDV stimulated high levels of adaptive immune responses (VNA), and protected all dogs challenge infection.

  7. Bactericidal/Permeability-Increasing Protein Fold–Containing Family Member A1 in Airway Host Protection and Respiratory Disease

    PubMed Central

    Britto, Clemente J.

    2015-01-01

    Bactericidal/permeability-increasing protein fold–containing family member A1 (BPIFA1), formerly known as SPLUNC1, is one of the most abundant proteins in respiratory secretions and has been identified with increasing frequency in studies of pulmonary disease. Its expression is largely restricted to the respiratory tract, being highly concentrated in the upper airways and proximal trachea. BPIFA1 is highly responsive to airborne pathogens, allergens, and irritants. BPIFA1 actively participates in host protection through antimicrobial, surfactant, airway surface liquid regulation, and immunomodulatory properties. Its expression is modulated in multiple lung diseases, including cystic fibrosis, chronic obstructive pulmonary disease, respiratory malignancies, and idiopathic pulmonary fibrosis. However, the role of BPIFA1 in pulmonary pathogenesis remains to be elucidated. This review highlights the versatile properties of BPIFA1 in antimicrobial protection and its roles as a sensor of environmental exposure and regulator of immune cell function. A greater understanding of the contribution of BPIFA1 to disease pathogenesis and activity may clarify if BPIFA1 is a biomarker and potential drug target in pulmonary disease. PMID:25265466

  8. A protective protein antigen of Rickettsia rickettsii has tandemly repeated, near-identical sequences.

    PubMed Central

    Anderson, B E; McDonald, G A; Jones, D C; Regnery, R L

    1990-01-01

    The nucleotide sequence of a Rickettsia rickettsii gene that encodes a high-molecular-mass surface antigen (190 kilodaltons), which elicits protective immunity, was determined. The 6,747-nucleotide gene coded for a 2,249-amino-acid protein with a calculated molecular weight of 224,321. A 3.8-kilobase PstI fragment proximal to the 5' end of the gene was found to consist of 13 highly related tandem repeats which constituted over 40% of the coding region. The repeated sequences could be divided into either a 225-nucleotide, 75-amino-acid unit (type I) or a 216-nucleotide, 72-amino-acid unit (type II), with extensive homology between the two types of repeating units. The deduced amino acid sequence for these repeat units, overall, was slightly hydrophobic with short hydrophilic domains. The carboxy-terminal (nonrepetitive) portion of the deduced protein sequence was hydrophilic, with potential surface-exposed epitopes. The full-length reading frame was reconstructed in Escherichia coli, and transient expression of the 190-kilodalton antigen was demonstrated; however, the protein appeared to be severely degraded by proteases and was apparently toxic to E. coli. The conservation of this unique repetitive gene structure, coupled with results from previous reports showing the protective properties of the 190-kilodalton antigen, suggests that this protein plays an important role in the pathogenesis of and immunity to Rocky Mountain spotted fever. Images PMID:2117568

  9. The core protein of a pestivirus protects the incoming virus against IFN-induced effectors

    PubMed Central

    Riedel, Christiane; Lamp, Benjamin; Hagen, Benedikt; Indik, Stanislav; Rümenapf, Till

    2017-01-01

    A multitude of viral factors - either inhibiting the induction of the IFN-system or its effectors – have been described to date. However, little is known about the role of structural components of the incoming virus particle in protecting against IFN-induced antiviral factors during or immediately after entry. In this study, we take advantage of the previously reported property of Classical swine fever virus (family Flaviviridae, genus Pestivirus) to tolerate a deletion of the core protein if a compensatory mutation is present in the NS3-helicase-domain (Vp447∆c). In contrast to the parental virus (Vp447), which causes a hemorrhagic-fever-like disease in pigs, Vp447∆c is avirulent in vivo. In comparison to Vp447, growth of Vp447∆c in primary porcine cells and IFN-treated porcine cell lines was reduced >20-fold. Also, primary porcine endothelial cells and IFN-pretreated porcine cell lines were 8–24 times less susceptible to Vp447∆c. This reduction of susceptibility could be partially reversed by loading Vp447∆c particles with different levels of core protein. In contrast, expression of core protein in the recipient cell did not have any beneficial effect. Therefore, a protective effect of core protein in the incoming virus particle against the products of IFN-stimulated genes could be demonstrated. PMID:28290554

  10. DNA vaccination with a gene encoding Toxoplasma gondii Rhoptry Protein 17 induces partial protective immunity against lethal challenge in mice

    PubMed Central

    Wang, Hai-Long; Wang, Yu-Jing; Pei, Yan-Jiang; Bai, Ji-Zhong; Yin, Li-Tian; Guo, Rui; Yin, Guo-Rong

    2016-01-01

    Toxoplasma gondii is an obligate intracellular apicomplexan parasite that affects humans and various vertebrate livestock and causes serious economic losses. To develop an effective vaccine against T. gondii infection, we constructed a DNA vaccine encoding the T. gondii rhoptry protein 17 (TgROP17) and evaluated its immune protective efficacy against acute T. gondii infection in mice. The DNA vaccine (p3×Flag-CMV-14-ROP17) was intramuscularly injected to BALB/c mice and the immune responses of the vaccinated mice were determined. Compared to control mice treated with empty vector or PBS, mice immunized with the ROP17 vaccine showed a relatively high level of specific anti-T. gondii antibodies, and a mixed IgG1/IgG2a response with predominance of IgG2a production. The immunized mice also displayed a specific lymphocyte proliferative response, a Th1-type cellular immune response with production of IFN-γ and interleukin-2, and increased number of CD8+ T cells. Immunization with the ROP17 DNA significantly prolonged the survival time (15.6 ± 5.4 days, P < 0.05) of mice after challenge infection with the virulent T. gondii RH strain (Type I), compared with the control groups which died within 8 days. Therefore, our data suggest that DNA vaccination with TgROP17 triggers significant humoral and cellular responses and induces effective protection in mice against acute T. gondii infection, indicating that TgROP17 is a promising vaccine candidate against acute toxoplasmosis. PMID:26842927

  11. The ectromelia virus SPI-2 protein causes lethal mousepox by preventing NK cell responses.

    PubMed

    Melo-Silva, Carolina R; Tscharke, David C; Lobigs, Mario; Koskinen, Aulikki; Wong, Yik Chun; Buller, R Mark; Müllbacher, Arno; Regner, Matthias

    2011-11-01

    Ectromelia virus (ECTV) is a natural pathogen of mice that causes mousepox, and many of its genes have been implicated in the modulation of host immune responses. Serine protease inhibitor 2 (SPI-2) is one of these putative ECTV host response modifier proteins. SPI-2 is conserved across orthopoxviruses, but results defining its mechanism of action and in vivo function are lacking or contradictory. We studied the role of SPI-2 in mousepox by deleting the SPI-2 gene or its serine protease inhibitor reactive site. We found that SPI-2 does not affect viral replication or cell-intrinsic apoptosis pathways, since mutant viruses replicate in vitro as efficiently as wild-type virus. However, in the absence of SPI-2 protein, ECTV is attenuated in mousepox-susceptible mice, resulting in lower viral loads in the liver, decreased spleen pathology, and substantially improved host survival. This attenuation correlates with more effective immune responses in the absence of SPI-2, including an earlier serum gamma interferon (IFN-γ) response, raised serum interleukin 18 (IL-18), increased numbers of granzyme B(+) CD8(+) T cells, and, most notably, increased numbers and activation of NK cells. Both virus attenuation and the improved immune responses associated with SPI-2 deletion from ECTV are lost when mice are depleted of NK cells. Consequently, SPI-2 renders mousepox lethal in susceptible strains by preventing protective NK cell defenses.

  12. Outer membrane vesicles derived from Salmonella Typhimurium mutants with truncated LPS induce cross-protective immune responses against infection of Salmonella enterica serovars in the mouse model.

    PubMed

    Liu, Qiong; Liu, Qing; Yi, Jie; Liang, Kang; Liu, Tian; Roland, Kenneth L; Jiang, Yanlong; Kong, Qingke

    2016-12-01

    Salmonella enterica cause diarrheal and systemic diseases and are of considerable concern worldwide. Vaccines that are cross-protective against multiple serovars could provide effective control of Salmonella-mediated diseases. Bacteria-derived outer membrane vesicles (OMVs) are highly immunogenic and are capable of eliciting protective immune responses. Alterations in lipopolysaccharide (LPS) length can result in outer membrane remodeling and composition of outer membrane proteins (OMPs) changing. In this study, we investigated the impact of truncated LPS on both the production and immunogenicity of Salmonella OMVs, including the ability of OMVs to elicit cross-protection against challenge by heterologous Salmonella strains. We found that mutations in waaJ and rfbP enhanced vesiculation, while mutations in waaC, waaF and waaG inhibited this process. Animal experiments indicated that OMVs from waaC, rfaH and rfbP mutants induced stronger serum immune responses compared to OMVs from the parent strain, while all elicited protective responses against the wild-type S. Typhimurium challenge. Furthermore, intranasal or intraperitoneal immunization with OMVs derived from the waaC and rfbP mutants elicited significantly higher cross-reactive IgG responses and provided enhanced cross-protection against S. Choleraesuis and S. Enteritidis challenge than the wild-type OMVs. These results indicate that truncated-LPS OMVs are capable of conferring cross protection against multiple serotypes of Salmonella infection.

  13. Signal transduction induced by activated protein C: no role in protection against sepsis?

    PubMed

    Slofstra, Sjoukje H; ten Cate, Hugo; Spek, C Arnold

    2006-08-01

    The anticoagulant activated protein C (APC) is historically known as a risk factor for venous thrombosis. However, after the positive results of the protein C worldwide evaluation in severe sepsis (PROWESS) trial, which showed that APC was the first drug that considerably reduced sepsis-related mortality, APC is considered a pleiotropic protein with both anticoagulant and anti-inflammatory properties. In addition, in vitro studies have suggested that APC-induced intracellular signal transduction is a potential mechanism by which APC might be protective against sepsis. Recently, however, the efficacy of APC in sepsis has been argued, and also the extent to which the signal transduction capacity of APC contributes to its pro-survival effects is debated. Here, we review the role of APC in the body natural defense against sepsis and discuss the mechanism by which APC might act at a cellular level.

  14. Dynamics of DNA damage response proteins at DNA breaks: a focus on protein modifications

    PubMed Central

    Polo, Sophie E.; Jackson, Stephen P.

    2011-01-01

    Genome integrity is constantly monitored by sophisticated cellular networks, collectively termed the DNA damage response (DDR). A common feature of DDR proteins is their mobilization in response to genotoxic stress. Here, we outline how the development of various complementary methodologies has provided valuable insights into the spatiotemporal dynamics of DDR protein assembly/disassembly at sites of DNA strand breaks in eukaryotic cells. Considerable advances have also been made in understanding the underlying molecular mechanisms for these events, with post-translational modifications of DDR factors being shown to play prominent roles in controlling the formation of foci in response to DNA-damaging agents. We review these regulatory mechanisms and discuss their biological significance to the DDR. PMID:21363960

  15. A novel neural response algorithm for protein function prediction

    PubMed Central

    2012-01-01

    Background Large amounts of data are being generated by high-throughput genome sequencing methods. But the rate of the experimental functional characterization falls far behind. To fill the gap between the number of sequences and their annotations, fast and accurate automated annotation methods are required. Many methods, such as GOblet, GOFigure, and Gotcha, are designed based on the BLAST search. Unfortunately, the sequence coverage of these methods is low as they cannot detect the remote homologues. Adding to this, the lack of annotation specificity advocates the need to improve automated protein function prediction. Results We designed a novel automated protein functional assignment method based on the neural response algorithm, which simulates the neuronal behavior of the visual cortex in the human brain. Firstly, we predict the most similar target protein for a given query protein and thereby assign its GO term to the query sequence. When assessed on test set, our method ranked the actual leaf GO term among the top 5 probable GO terms with accuracy of 86.93%. Conclusions The proposed algorithm is the first instance of neural response algorithm being used in the biological domain. The use of HMM profiles along with the secondary structure information to define the neural response gives our method an edge over other available methods on annotation accuracy. Results of the 5-fold cross validation and the comparison with PFP and FFPred servers indicate the prominent performance by our method. The program, the dataset, and help files are available at http://www.jjwanglab.org/NRProF/. PMID:23046521

  16. Inclusion bodies enriched for p62 and polyubiquitinated proteins in macrophages protect against atherosclerosis.

    PubMed

    Sergin, Ismail; Bhattacharya, Somashubhra; Emanuel, Roy; Esen, Emel; Stokes, Carl J; Evans, Trent D; Arif, Batool; Curci, John A; Razani, Babak

    2016-01-05

    Autophagy is a catabolic cellular mechanism that degrades dysfunctional proteins and organelles. Atherosclerotic plaque formation is enhanced in mice with macrophages deficient for the critical autophagy protein ATG5. We showed that exposure of macrophages to lipids that promote atherosclerosis increased the abundance of the autophagy chaperone p62 and that p62 colocalized with polyubiquitinated proteins in cytoplasmic inclusions, which are characterized by insoluble protein aggregates. ATG5-null macrophages developed further p62 accumulation at the sites of large cytoplasmic ubiquitin-positive inclusion bodies. Aortas from atherosclerotic mice and plaques from human endarterectomy samples showed increased abundance of p62 and polyubiquitinated proteins that colocalized with plaque macrophages, suggesting that p62-enriched protein aggregates were characteristic of atherosclerosis. The formation of the cytoplasmic inclusions depended on p62 because lipid-loaded p62-null macrophages accumulated polyubiquitinated proteins in a diffuse cytoplasmic pattern. Lipid-loaded p62-null macrophages also exhibited increased secretion of interleukin-1β (IL-1β) and had an increased tendency to undergo apoptosis, which depended on the p62 ubiquitin-binding domain and at least partly involved p62-mediated clearance of NLRP3 inflammasomes. Consistent with our in vitro observations, p62-deficient mice formed greater numbers of more complex atherosclerotic plaques, and p62 deficiency further increased atherosclerotic plaque burden in mice with a macrophage-specific ablation of ATG5. Together, these data suggested that sequestration of cytotoxic ubiquitinated proteins by p62 protects against atherogenesis, a condition in which the clearance of protein aggregates is disrupted.

  17. Plasmodium falciparum synthetic LbL microparticle vaccine elicits protective neutralizing antibody and parasite-specific cellular immune responses.

    PubMed

    Powell, Thomas J; Tang, Jie; Derome, Mary E; Mitchell, Robert A; Jacobs, Andrea; Deng, Yanhong; Palath, Naveen; Cardenas, Edwin; Boyd, James G; Nardin, Elizabeth

    2013-04-08

    Epitopes of the circumsporozoite (CS) protein of Plasmodium falciparum, the most pathogenic species of the malaria parasite, have been shown to elicit protective immunity in experimental animals and human volunteers. The mechanisms of immunity include parasite-neutralizing antibodies that can inhibit parasite motility in the skin at the site of infection and in the bloodstream during transit to the hepatocyte host cell and also block interaction with host cell receptors on hepatocytes. In addition, specific CD4+ and CD8+ cellular mechanisms target the intracellular hepatic forms, thus preventing release of erythrocytic stage parasites from the infected hepatocyte and the ensuing blood stage cycle responsible for clinical disease. An innovative method for producing particle vaccines, layer-by-layer (LbL) fabrication of polypeptide films on solid CaCO3 cores, was used to produce synthetic malaria vaccines containing a tri-epitope CS peptide T1BT comprising the antibody epitope of the CS repeat region (B) and two T-cell epitopes, the highly conserved T1 epitope and the universal epitope T. Mice immunized with microparticles loaded with T1BT peptide developed parasite-neutralizing antibodies and malaria-specific T-cell responses including cytotoxic effector T-cells. Protection from liver stage infection following challenge with live sporozoites from infected mosquitoes correlated with neutralizing antibody levels. Although some immunized mice with low or undetectable neutralizing antibodies were also protected, depletion of T-cells prior to challenge resulted in the majority of mice remaining resistant to challenge. In addition, mice immunized with microparticles bearing only T-cell epitopes were not protected, demonstrating that cellular immunity alone was not sufficient for protective immunity. Although the microparticles without adjuvant were immunogenic and protective, a simple modification with the lipopeptide TLR2 agonist Pam3Cys increased the potency and

  18. Control of Protein Particle Formation During Ultrafiltration/Diafiltration Through Interfacial Protection

    PubMed Central

    Callahan, Daniel J; Stanley, Bradford; Li, Yuling

    2014-01-01

    This study investigates the mechanism of protein particle formation during ultrafiltration/diafiltration (UF/DF), finding that agitation drives particle formation by promoting protein-interface adsorption and desorption. Low conductivity and the presence of surfactant reduced the level of particle formation in small-scale stirring studies, and the same trends were observed in pumping and UF/DF. Polysorbate 80 (PS80) and hydroxypropyl-β-cyclodextrin (HPβCD) reduced particle formation in UF/DF by factors of 15 and 4, respectively. Measurements of conformational stability, colloidal stability, and surface tension demonstrated that PS80 protects against particle formation by preventing protein-interface adsorption, low conductivity improves the colloidal stability of the protein, and the mechanism of action of HPβCD remains unclear. This work demonstrates that interfacial adsorption–desorption of the protein during UF/DF is the principal cause of particle formation, that the level of surfactant-free particle formation depends on the colloidal stability of the protein, and that the inclusion of surfactant greatly reduces in-process particle formation during UF/DF. PMID:24449131

  19. Protein and Essential Amino Acids to Protect Musculoskeletal Health during Spaceflight: Evidence of a Paradox?

    PubMed Central

    Hackney, Kyle J.; English, Kirk L.

    2014-01-01

    Long-duration spaceflight results in muscle atrophy and a loss of bone mineral density. In skeletal muscle tissue, acute exercise and protein (e.g., essential amino acids) stimulate anabolic pathways (e.g., muscle protein synthesis) both independently and synergistically to maintain neutral or positive net muscle protein balance. Protein intake in space is recommended to be 12%–15% of total energy intake (≤1.4 g∙kg−1∙day−1) and spaceflight is associated with reduced energy intake (~20%), which enhances muscle catabolism. Increasing protein intake to 1.5–2.0 g∙kg−1∙day−1 may be beneficial for skeletal muscle tissue and could be accomplished with essential amino acid supplementation. However, increased consumption of sulfur-containing amino acids is associated with increased bone resorption, which creates a dilemma for musculoskeletal countermeasures, whereby optimizing skeletal muscle parameters via essential amino acid supplementation may worsen bone outcomes. To protect both muscle and bone health, future unloading studies should evaluate increased protein intake via non-sulfur containing essential amino acids or leucine in combination with exercise countermeasures and the concomitant influence of reduced energy intake. PMID:25370374

  20. Monotreme lactation protein is highly expressed in monotreme milk and provides antimicrobial protection.

    PubMed

    Enjapoori, Ashwantha Kumar; Grant, Tom R; Nicol, Stewart C; Lefèvre, Christophe M; Nicholas, Kevin R; Sharp, Julie A

    2014-09-22

    Monotremes (platypus and echidna) are the descendants of the oldest ancestor of all extant mammals distinguished from other mammals by mode of reproduction. Monotremes lay eggs following a short gestation period and after an even briefer incubation period, altricial hatchlings are nourished over a long lactation period with milk secreted by nipple-less mammary patches located on the female's abdomen. Milk is the sole source of nutrition and immune protection for the developing young until weaning. Using transcriptome and mass spectrometry analysis of milk cells and milk proteins, respectively, a novel Monotreme Lactation Protein (MLP) was identified as a major secreted protein in milk. We show that platypus and short-beaked echidna MLP genes show significant homology and are unique to monotremes. The MLP transcript was shown to be expressed in a variety of tissues; however, highest expression was observed in milk cells and was expressed constitutively from early to late lactation. Analysis of recombinant MLP showed that it is an N-linked glycosylated protein and biophysical studies predicted that MLP is an amphipathic, α-helical protein, a typical feature of antimicrobial proteins. Functional analysis revealed MLP antibacterial activity against both opportunistic pathogenic Staphylococcus aureus and commensal Enterococcus faecalis bacteria but showed no effect on Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis, and Salmonella enterica. Our data suggest that MLP is an evolutionarily ancient component of milk-mediated innate immunity absent in other mammals. We propose that MLP evolved specifically in the monotreme lineage supporting the evolution of lactation in these species to provide bacterial protection, at a time when mammals lacked nipples.

  1. Monotreme Lactation Protein Is Highly Expressed in Monotreme Milk and Provides Antimicrobial Protection

    PubMed Central

    Enjapoori, Ashwantha Kumar; Grant, Tom R.; Nicol, Stewart C.; Lefèvre, Christophe M.; Nicholas, Kevin R.; Sharp, Julie A.

    2014-01-01

    Monotremes (platypus and echidna) are the descendants of the oldest ancestor of all extant mammals distinguished from other mammals by mode of reproduction. Monotremes lay eggs following a short gestation period and after an even briefer incubation period, altricial hatchlings are nourished over a long lactation period with milk secreted by nipple-less mammary patches located on the female’s abdomen. Milk is the sole source of nutrition and immune protection for the developing young until weaning. Using transcriptome and mass spectrometry analysis of milk cells and milk proteins, respectively, a novel Monotreme Lactation Protein (MLP) was identified as a major secreted protein in milk. We show that platypus and short-beaked echidna MLP genes show significant homology and are unique to monotremes. The MLP transcript was shown to be expressed in a variety of tissues; however, highest expression was observed in milk cells and was expressed constitutively from early to late lactation. Analysis of recombinant MLP showed that it is an N-linked glycosylated protein and biophysical studies predicted that MLP is an amphipathic, α-helical protein, a typical feature of antimicrobial proteins. Functional analysis revealed MLP antibacterial activity against both opportunistic pathogenic Staphylococcus aureus and commensal Enterococcus faecalis bacteria but showed no effect on Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis, and Salmonella enterica. Our data suggest that MLP is an evolutionarily ancient component of milk-mediated innate immunity absent in other mammals. We propose that MLP evolved specifically in the monotreme lineage supporting the evolution of lactation in these species to provide bacterial protection, at a time when mammals lacked nipples. PMID:25245409

  2. Novel Immunity Proteins Associated with Colicin M-like Bacteriocins Exhibit Promiscuous Protection in Pseudomonas

    PubMed Central

    Ghequire, Maarten G. K.; Kemland, Lieselore; De Mot, René

    2017-01-01

    Bacteriocins related to colicin M, acting via cleavage of the cell wall precursor lipid II, have been characterized in γ- and β-proteobacteria. Depending on the species, immunity is provided by either an inner membrane-anchored periplasmic protein or by an integral membrane protein. In Pseudomonas however, the immunity partner of colicin M-like bacteriocins remains unknown. Based on an in silico analysis in pseudomonad genomes, we here identify a gene encoding a putative immunity partner that represents a novel type of integral membrane protein (PmiA, Pseudomonas colicin M-like immunity type A). By heterologous expression of pmiA genes in susceptible strains, we show that immunity to colicin M-like bacteriocins is indeed provided by the cognate PmiA. Sequence homology among PmiA proteins is essentially absent, except for a short motif with a conserved periplasm-exposed aspartate residue. However, PmiA's protective function is not abolished by changing this acidic residue to the uncharged alanine. Immunity by PmiAs appears promiscuous to the extent that PmiA homologs from a clade sharing <40% pairwise amino acid identity, equally provide protection against the bacteriocin linked to the original PmiA. This study shows that multiple immunity factors have evolved independently to silence lipid II-targeting enzymatic bacteriocins. Their relaxed bacteriocin immunization capacity contrasts to the strict specificity of immunity proteins shielding the enzymatic domain of nuclease bacteriocins. The nature of associated immune functions needs consideration when using such natural protein antibiotics or designing novel variants. PMID:28194143

  3. Peroxiredoxin IV regulates pro-inflammatory responses in large yellow croaker (Pseudosciaena crocea) and protects against bacterial challenge.

    PubMed

    Yu, Suhong; Mu, Yinnan; Ao, Jingqun; Chen, Xinhua

    2010-03-05

    In this study, we applied a comparative proteomic approach to the analysis of differentially expressed proteins in the spleens of large yellow croaker following treatment with an inactivated trivalent bacterial vaccine. Twenty-four altered proteins were identified by MALDI-TOF or MALDI-TOF-TOF, including immune-related proteins, antioxidant proteins, signal transducers, protein biosynthesis and catabolism modulators, and carbonic anhydrases. Three Prx family members, namely, Prx I, Prx II, and Prx IV, were upregulated after treatment with the vaccine, indicating potentially important roles for these antioxidant proteins in the antibacterial immune response. Large yellow croaker Prx IV (LycPrxIV), which has thiol-dependent peroxidase activity, was constitutively expressed in all tissues examined. Immunoelectron microscopy showed that LycPrxIV was primarily localized to the rER or peroxisome in spleen cells of healthy fish, and its synthesis on the rER increased following treatment with bacterial vaccine. Suppression of LycPrxIV by siRNA resulted in an increase in NF-kappaB activity in spleen tissues, while in vivo administration of recombinant LycPrxIV (rLycPrxIV) caused a decrease in NF-kappaB activity, indicating that LycPrxIV negatively regulates NF-kappaB activation. Likewise, siRNA-mediated knockdown of LycPrxIV increased the expression of TNF-alpha and CC chemokine, and downregulated the expression of IL-10. However, injection of fish with rLycPrxIV induced the opposite expression pattern of these cytokines, suggesting a role for LycPrxIV in regulating pro-inflammatory responses. Bacterial challenge experiments showed that suppression of LycPrxIV expression by siRNA significantly increased fish mortality as compared to controls, whereas rLycPrxIV provided a protective effect. Together, our data suggest that LycPrxIV may regulate pro-inflammatory responses to protect large yellow croaker from bacterial challenge, revealing a novel antibacterial mechanism in

  4. Neurotoxicity induced by arsenic in Gallus Gallus: Regulation of oxidative stress and heat shock protein response.

    PubMed

    Zhao, Panpan; Guo, Ying; Zhang, Wen; Chai, Hongliang; Xing, Houjuan; Xing, Mingwei

    2017-01-01

    Arsenic, a naturally occurring heavy metal pollutant, is one of the functioning risk factors for neurological toxicity in humans. However, little is known about the effects of arsenic on the nervous system of Gallus Gallus. To investigate whether arsenic induce neurotoxicity and influence the oxidative stress and heat shock proteins (Hsps) response in chickens, seventy-two 1-day-old male Hy-line chickens were treated with different doses of arsenic trioxide (As2O3). The histological changes, antioxidant enzyme activity, and the expressions of Hsps were detected. Results showed slightly histology changes were obvious in the brain tissues exposure to arsenic. The activities of Glutathione peroxidase (GSH-Px) and catalase (CAT) were decreased compared to the control, whereas the malondialdehyde (MDA) content was increased gradually along with increase in diet-arsenic. The mRNA levels of Hsps and protein expressions of Hsp60 and Hsp70 were up-regulated. These results suggested that sub-chronic exposure to arsenic induced neurotoxicity in chickens. Arsenic exposure disturbed the balance of oxidants and antioxidants. Increased heat shock response tried to protect chicken brain tissues from tissues damage caused by oxidative stress. The mechanisms of neurotoxicity induced by arsenic include oxidative stress and heat shock protein response in chicken brain tissues.

  5. Immunogenicity and protective efficacy of heparan sulphate binding proteins of Entamoeba histolytica in a guinea pig model of intestinal amoebiasis.

    PubMed

    Kaur, Upninder; Khurana, Sumeeta; Saikia, Uma Nahar; Dubey, M L

    2013-11-01

    Entamoeba histolytica infection is associated with considerable morbidity and mortality in the form of intestinal and extraintestinal amoebiasis. No vaccine is yet available for amoebiasis. Heparan Sulphate Binding Proteins (HSBPs) from E. histolytica were evaluated for immunogenicity and protective efficacy in a Guinea pig model. Animals were immunized subcutaneously with 30μg of HSBP by three weekly inoculations. The immunogenicity of HSBP was determined by antibody response (IgG, IgM and IgA), splenocyte proliferation assay and in vitro direct amoebicidal assay with splenic lymphocytes and monocytes from vaccinated and control animals. The efficacy of the vaccine was evaluated by challenge infection to vaccinated and control animals by intra-caecal inoculation of E. histolytica trophozoites and comparing gross and histopathological findings in caeca of these animals. HSBP was found to induce specific anti-amoebic response as seen by specific antibody production and direct amoebicidal activity of splenocytes. The vaccine also showed partial protection against challenge infection in vaccinated animals as shown by mild/absent lesions and histopathological findings.

  6. Tofacitinib suppresses antibody responses to protein therapeutics in murine hosts.

    PubMed

    Onda, Masanori; Ghoreschi, Kamran; Steward-Tharp, Scott; Thomas, Craig; O'Shea, John J; Pastan, Ira H; FitzGerald, David J

    2014-07-01

    Immunogenicity remains the "Achilles' heel" of protein-based therapeutics. Anti-drug Abs produced in response to protein therapeutics can severely limit both the safety and efficacy of this expanding class of agent. In this article, we report that monotherapy of mice with tofacitinib (the JAK inhibitor) quells Ab responses to an immunotoxin derived from the bacterial protein Pseudomonas exotoxin A, as well as to the model Ag keyhole limpet hemocyanin. Thousand-fold reductions in IgG1 titers to both Ags were observed 21 d post immunization. In fact, suppression was evident for all IgG isotypes and IgM. A reduction in IgG3 production was also noted with a thymus-independent type II Ag. Mechanistic investigations revealed that tofacitinib treatment led to reduced numbers of CD127+ pro-B cells. Furthermore, we observed fewer germinal center B cells and the impaired formation of germinal centers of mice treated with tofacitinib. Because normal Ig levels were still present during tofacitinib treatment, this agent specifically reduced anti-drug Abs, thus preserving the potential efficacy of biological therapeutics, including those used as cancer therapeutics.

  7. Legionella suppresses the host unfolded protein response via multiple mechanisms

    PubMed Central

    Treacy-Abarca, Sean; Mukherjee, Shaeri

    2015-01-01

    The intracellular pathogen, Legionella pneumophila, secretes ∼300 effector proteins to modulate the host environment. Given the intimate interaction between L. pneumophila and the endoplasmic reticulum, we investigated the role of the host unfolded protein response (UPR) during L. pneumophila infection. Interestingly, we show that the host identifies L. pneumophila infection as a form of endoplasmic reticulum stress and the sensor pATF6 is processed to generate pATF6(N), a transcriptional activator of downstream UPR genes. However, L. pneumophila is able to suppress the UPR and block the translation of prototypical UPR genes, BiP and CHOP. Furthermore, biochemical studies reveal that L. pneumophila uses two effectors (Lgt1 and Lgt2) to inhibit the splicing of XBP1u mRNA to spliced XBP1 (XBP1s), an UPR response regulator. Thus, we demonstrate that L. pneumophila is able to inhibit the UPR by multiple mechanisms including blocking XBP1u splicing and causing translational repression. This observation highlights the utility of L. pneumophila as a powerful tool for studying a critical protein homeostasis regulator. PMID:26219498

  8. Inhibition of the Unfolded Protein Response Mechanism Prevents Cardiac Fibrosis

    PubMed Central

    Jung, Joanna; Dyck, Jason R. B.; Lopaschuk, Gary D.; Agellon, Luis B.; Michalak, Marek

    2016-01-01

    Background Cardiac fibrosis attributed to excessive deposition of extracellular matrix proteins is a major cause of heart failure and death. Cardiac fibrosis is extremely difficult and challenging to treat in a clinical setting due to lack of understanding of molecular mechanisms leading to cardiac fibrosis and effective anti-fibrotic therapies. The objective in this study was to examine whether unfolded protein response (UPR) pathway mediates cardiac fibrosis and whether a pharmacological intervention to modulate UPR can prevent cardiac fibrosis and preserve heart function. Methodology/Principal Findings We demonstrate here that the mechanism leading to development of fibrosis in a mouse with increased expression of calreticulin, a model of heart failure, stems from impairment of endoplasmic reticulum (ER) homeostasis, transient activation of the unfolded protein response (UPR) pathway and stimulation of the TGFβ1/Smad2/3 signaling pathway. Remarkably, sustained pharmacologic inhibition of the UPR pathway by tauroursodeoxycholic acid (TUDCA) is sufficient to prevent cardiac fibrosis, and improved exercise tolerance. Conclusions We show that the mechanism leading to development of fibrosis in a mouse model of heart failure stems from transient activation of UPR pathway leading to persistent remodelling of cardiac tissue. Blocking the activation of the transiently activated UPR pathway by TUDCA prevented cardiac fibrosis, and improved prognosis. These findings offer a window for additional interventions that can preserve heart function. PMID:27441395

  9. Timely and Needed Perspectives on Differential Response in Child Protective Services

    ERIC Educational Resources Information Center

    Ellett, Alberta J.

    2013-01-01

    This article is an invited commentary and analysis of the authors' completed systematic evaluation of Child Protective Services (CPS) differential response (DR) models. I write this commentary based on 25 years of public child welfare experience followed by 13 years as a social work professor and researcher. In their review of DR, the…

  10. USE OF THE RIBONUCLEASE PROTECTION ASSAY FOR IDENTIFYING CHEMICALS WHICH ELLICIT HYPERSENSITIVITY RESPONSES

    EPA Science Inventory

    Use of the Ribonuclease Protection Assay (RPA) for Identifying Chemicals that Elicit Hypersensitivity Responses. L.M. Plitnick, 1, D.M. Sailstad, 2, and R.J. Smialowicz, 2 1UNC, Curriculum in Toxicology, Chapel Hill, NC and 2USEPA, NHEERL, RTP, NC.

    The incidence of aller...

  11. Does infection with environmental mycobacteria suppress the protective response to subsequent vaccination with BCG?

    PubMed

    Smith, D; Reeser, P; Musa, S

    1985-03-01

    Using a guinea pig model of experimental airborne tuberculosis, we were unable to find evidence to support the hypothesis that infection with environmental mycobacteria (M. simiae or M. avium-intracellulare) interferes with the induction of a protective response in animals subsequently vaccinated with BCG.

  12. THE MILLENNIUM CHALLENGE: THE U.S. ENVIRONMENTAL PROTECTION AGENCY'S RESPONSE TO INVASIVE SPECIES

    EPA Science Inventory

    The U.S. Environmental Protection Agency (EPA) is responding to the scientific and regulatory challenges of invasive species in a variety of ways. One response has been to use existing programs and regulations, as appropriate, to address invasive species. A recent example is th...

  13. Rhetorical Dissent as an Adaptive Response to Classroom Problems: A Test of Protection Motivation Theory

    ERIC Educational Resources Information Center

    Bolkan, San; Goodboy, Alan K.

    2016-01-01

    Protection motivation theory (PMT) explains people's adaptive behavior in response to personal threats. In this study, PMT was used to predict rhetorical dissent episodes related to 210 student reports of perceived classroom problems. In line with theoretical predictions, a moderated moderation analysis revealed that students were likely to voice…

  14. A chemical screen to identify inducers of the mitochondrial unfolded protein response in C. elegans

    PubMed Central

    Rauthan, Manish; Pilon, Marc

    2015-01-01

    We previously showed that inhibition of the mevalonate pathway in C. elegans causes inhibition of protein prenylation, developmental arrest and lethality. We also showed that constitutive activation of the mitochondrial unfolded protein response, UPRmt, is an effective way for C. elegans to become resistant to the negative effects of mevalonate pathway inhibition. This was an important finding since statins, a drug class prescribed to lower cholesterol levels in patients, act by inhibiting the mevalonate pathway, and it is therefore possible that some of their undesirable side effects could be alleviated by activating the UPRmt. Here, we screened a chemical library and identified 4 compounds that specifically activated the UPRmt. One of these compounds, methacycline hydrochloride (a tetracycline antibiotic) also protected C. elegans and mammalian cells from statin toxicity. Methacycline hydrochloride and ethidium bromide, a known UPRmt activator, were also tested in mice: only ethidium bromide significantly activate the UPRmt in skeletal muscles. PMID:27123370

  15. Effects of rare earth elements and REE-binding proteins on physiological responses in plants.

    PubMed

    Liu, Dongwu; Wang, Xue; Chen, Zhiwei

    2012-02-01

    Rare earth elements (REEs), which include 17 elements in the periodic table, share chemical properties related to a similar external electronic configuration. REEs enriched fertilizers have been used in China since the 1980s. REEs could enter the cell and cell organelles, influence plant growth, and mainly be bound with the biological macromolecules. REE-binding proteins have been found in some plants. In addition, the chlorophyll activities and photosynthetic rate can be regulated by REEs. REEs could promote the protective function of cell membrane and enhance the plant resistance capability to stress produced by environmental factors, and affect the plant physiological mechanism by regulating the Ca²⁺ level in the plant cells. The focus of present review is to describe how REEs and REE-binding proteins participate in the physiological responses in plants.

  16. Immunogenicity and protective efficacy of recombinant Clostridium difficile flagellar protein FliC.

    PubMed

    Ghose, Chandrabali; Eugenis, Ioannis; Sun, Xingmin; Edwards, Adrianne N; McBride, Shonna M; Pride, David T; Kelly, Ciarán P; Ho, David D

    2016-02-03

    Clostridium difficile is a Gram-positive bacillus and is the leading cause of toxin-mediated nosocomial diarrhea following antibiotic use. C. difficile flagella play a role in colonization, adherence, biofilm formation, and toxin production, which might contribute to the overall virulence of certain strains. Human and animal studies indicate that anti-flagella immune responses may play a role in protection against colonization by C. difficile and subsequent disease outcome. Here we report that recombinant C. difficile flagellin (FliC) is immunogenic and protective in a murine model of C. difficile infection (CDI) against a clinical C. difficile strain, UK1. Passive protection experiments using anti-FliC polyclonal serum in mice suggest this protection to be antibody-mediated. FliC immunization also was able to afford partial protection against CDI and death in hamsters following challenge with C. difficile 630Δerm. Additionally, immunization against FliC does not have an adverse effect on the normal gut flora of vaccinated hamsters as evidenced by comparing the fecal microbiome of vaccinated and control hamsters. Therefore, the use of FliC as a vaccine candidate against CDI warrants further testing.

  17. Adaptor protein cerebral cavernous malformation 3 (CCM3) mediates phosphorylation of the cytoskeletal proteins ezrin/radixin/moesin by mammalian Ste20-4 to protect cells from oxidative stress.

    PubMed

    Fidalgo, Miguel; Guerrero, Ana; Fraile, María; Iglesias, Cristina; Pombo, Celia M; Zalvide, Juan

    2012-03-30

    While studying the functions of CCM3/PDCD10, a gene encoding an adaptor protein whose mutation results in vascular malformations, we have found that it is involved in a novel response to oxidative stress that results in phosphorylation and activation of the ezrin/radixin/moesin (ERM) family of proteins. This phosphorylation protects cells from accidental cell death induced by oxidative stress. We also present evidence that ERM phosphorylation is performed by the GCKIII kinase Mst4, which is activated and relocated to the cell periphery after oxidative stress. The cellular levels of Mst4 and its activation after oxidative stress depend on the presence of CCM3, as absence of the latter impairs the phosphorylation of ERM proteins and enhances death of cells exposed to reactive oxygen species. These findings shed new light on the response of cells to oxidative stress and identify an important pathophysiological situation in which ERM proteins and their phosphorylation play a significant role.

  18. The Unfolded Protein Response Regulates Uterine Myocyte Antioxidant Responsiveness During Pregnancy.

    PubMed

    Ramnarayanan, Saiprasad; Kyathanahalli, Chandrashekara; Ingles, Judith; Park-York, MieJung; Jeyasuria, Pancharatnam; Condon, Jennifer C

    2016-12-01

    There is considerable evidence that implicates oxidative stress in the pathophysiology of human pregnancy complications. However, the role and the mechanism of maintaining an antioxidant prosurvival uterine environment during normal pregnancy is largely unresolved. Herein we report that the highly active uterine unfolded protein response plays a key role in promoting antioxidant activity in the uterine myocyte across gestation. The unfolded protein response (UPR) senses the accumulation of misfolded proteins in the endoplasmic reticulum (ER) and activates a signaling network that consists of the transmembrane protein kinase eukaryotic translation initiation factor 2 alpha kinase 3/PKR-like-ER kinase (EIF2AK3), which acts to decrease protein translation levels, allowing for a lowered need for protein folding during periods of ER stress. However, independent of its translational regulatory capacity, EIF2AK3-dependent signals elicit the activation of the transcription factor, nuclear factor erythroid 2-like 2 (NFE2L2) in response to oxidative stress. NFE2L2 binds to antioxidant response elements in the promoters of a variety of antioxidant genes that minimize the opportunities for generation of reactive oxygen intermediates. Our analysis demonstrates that in the absence of EIF2AK3, the uterine myocyte experiences increased levels of reactive oxygen species due to decreased NFE2L2 activation. Elevated levels of intracellular reactive oxygen species were observed in the EIF2AK3 null cells, and this was associated with the onset of apoptotic cell death. These findings confirm the prosurvival and antioxidant role of UPR-mediated EIF2AK3 activation in the context of the human uterine myocyte.

  19. Protein-responsive ribozyme switches in eukaryotic cells

    PubMed Central

    Kennedy, Andrew B.; Vowles, James V.; d'Espaux, Leo; Smolke, Christina D.

    2014-01-01

    Genetic devices that directly detect and respond to intracellular concentrations of proteins are important synthetic biology tools, supporting the design of biological systems that target, respond to or alter specific cellular states. Here, we develop ribozyme-based devices that respond to protein ligands in two eukaryotic hosts, yeast and mammalian cells, to regulate the expression of a gene of interest. Our devices allow for both gene-ON and gene-OFF response upon sensing the protein ligand. As part of our design process, we describe an in vitro characterization pipeline for prescreening device designs to identify promising candidates for in vivo testing. The in vivo gene-regulatory activities in the two types of eukaryotic cells correlate with in vitro cleavage activities determined at different physiologically relevant magnesium concentrations. Finally, localization studies with the ligand demonstrate that ribozyme switches respond to ligands present in the nucleus and/or cytoplasm, providing new insight into their mechanism of action. By extending the sensing capabilities of this important class of gene-regulatory device, our work supports the implementation of ribozyme-based devices in applications requiring the detection of protein biomarkers. PMID:25274734

  20. Targeting Plant Ethylene Responses by Controlling Essential Protein-Protein Interactions in the Ethylene Pathway.

    PubMed

    Bisson, Melanie M A; Groth, Georg

    2015-08-01

    The gaseous plant hormone ethylene regulates many processes of high agronomic relevance throughout the life span of plants. A central element in ethylene signaling is the endoplasmic reticulum (ER)-localized membrane protein ethylene insensitive2 (EIN2). Recent studies indicate that in response to ethylene, the extra-membranous C-terminal end of EIN2 is proteolytically processed and translocated from the ER to the nucleus. Here, we report that the conserved nuclear localization signal (NLS) mediating nuclear import of the EIN2 C-terminus provides an important domain for complex formation with ethylene receptor ethylene response1 (ETR1). EIN2 lacking the NLS domain shows strongly reduced affinity for the receptor. Interaction of EIN2 and ETR1 is also blocked by a synthetic peptide of the NLS motif. The corresponding peptide substantially reduces ethylene responses in planta. Our results uncover a novel mechanism and type of inhibitor interfering with ethylene signal transduction and ethylene responses in plants. Disruption of essential protein-protein interactions in the ethylene signaling pathway as shown in our study for the EIN2-ETR1 complex has the potential to guide the development of innovative ethylene antagonists for modern agriculture and horticulture.

  1. Killing Me Softly: Connotations to Unfolded Protein Response and Oxidative Stress in Alzheimer's Disease

    PubMed Central

    Pająk, Beata; Kania, Elżbieta; Orzechowski, Arkadiusz

    2016-01-01

    This review is focused on the possible causes of mitochondrial dysfunction in AD, underlying molecular mechanisms of this malfunction, possible causes and known consequences of APP, Aβ, and hyperphosphorylated tau presence in mitochondria, and the contribution of altered lipid metabolism (nonsterol isoprenoids) to pathological processes leading to increased formation and accumulation of the aforementioned hallmarks of AD. Abnormal protein folding and unfolded protein response seem to be the outcomes of impaired glycosylation due to metabolic disturbances in geranylgeraniol intermediary metabolism. The origin and consecutive fate of APP, Aβ, and tau are emphasized on intracellular trafficking apparently influenced by inaccurate posttranslational modifications. We hypothesize that incorrect intracellular processing of APP determines protein translocation to mitochondria in AD. Similarly, without obvious reasons, the passage of Aβ and tau to mitochondria is observed. APP targeted to mitochondria blocks the activity of protein translocase complex resulting in poor import of proteins central to oxidative phosphorylation. Besides, APP, Aβ, and neurofibrillary tangles of tau directly or indirectly impair mitochondrial biochemistry and bioenergetics, with concomitant generation of oxidative/nitrosative stress. Limited protective mechanisms are inadequate to prevent the free radical-mediated lesions. Finally, neuronal loss is observed in AD-affected brains typically by pathologic apoptosis. PMID:26881014

  2. Killing Me Softly: Connotations to Unfolded Protein Response and Oxidative Stress in Alzheimer's Disease.

    PubMed

    Pająk, Beata; Kania, Elżbieta; Orzechowski, Arkadiusz

    2016-01-01

    This review is focused on the possible causes of mitochondrial dysfunction in AD, underlying molecular mechanisms of this malfunction, possible causes and known consequences of APP, Aβ, and hyperphosphorylated tau presence in mitochondria, and the contribution of altered lipid metabolism (nonsterol isoprenoids) to pathological processes leading to increased formation and accumulation of the aforementioned hallmarks of AD. Abnormal protein folding and unfolded protein response seem to be the outcomes of impaired glycosylation due to metabolic disturbances in geranylgeraniol intermediary metabolism. The origin and consecutive fate of APP, Aβ, and tau are emphasized on intracellular trafficking apparently influenced by inaccurate posttranslational modifications. We hypothesize that incorrect intracellular processing of APP determines protein translocation to mitochondria in AD. Similarly, without obvious reasons, the passage of Aβ and tau to mitochondria is observed. APP targeted to mitochondria blocks the activity of protein translocase complex resulting in poor import of proteins central to oxidative phosphorylation. Besides, APP, Aβ, and neurofibrillary tangles of tau directly or indirectly impair mitochondrial biochemistry and bioenergetics, with concomitant generation of oxidative/nitrosative stress. Limited protective mechanisms are inadequate to prevent the free radical-mediated lesions. Finally, neuronal loss is observed in AD-affected brains typically by pathologic apoptosis.

  3. Glycoarray Technologies: Deciphering Interactions from Proteins to Live Cell Responses

    PubMed Central

    Puvirajesinghe, Tania M.; Turnbull, Jeremy. E.

    2016-01-01

    Microarray technologies inspired the development of carbohydrate arrays. Initially, carbohydrate array technology was hindered by the complex structures of glycans and their structural variability. The first designs of glycoarrays focused on the HTP (high throughput) study of protein–glycan binding events, and subsequently more in-depth kinetic analysis of carbohydrate–protein interactions. However, the applications have rapidly expanded and now achieve successful discrimination of selective interactions between carbohydrates and, not only proteins, but also viruses, bacteria and eukaryotic cells, and most recently even live cell responses to immobilized glycans. Combining array technology with other HTP technologies such as mass spectrometry is expected to allow even more accurate and sensitive analysis. This review provides a broad overview of established glycoarray technologies (with a special focus on glycosaminoglycan applications) and their emerging applications to the study of complex interactions between glycans and whole living cells. PMID:27600069

  4. Development of a simple assay system for protein-stabilizing efficiency based on hemoglobin protection against denaturation and measurement of the cooperative effect of mixing protein stabilizers.

    PubMed

    Chen, Siyu; Manabe, Yoshiyuki; Minamoto, Naoya; Saiki, Naoka; Fukase, Koichi

    2016-10-01

    We have elucidated the cooperative stabilization of proteins by sugars, amino acids, and other protein-stabilizing agents using a new and simple assay system. Our system determines the protein-stabilizing ability of various compounds by measuring their ability to protect hemoglobin from denaturation. Hemoglobin denaturation was readily measured by quantitative changes in its ultraviolet-visible absorption spectrum. The efficiency of our assay was confirmed using various sugars such as trehalose and sucrose that are known to be good protein stabilizers. We have also found that mixtures of two different types of protein stabilizers resulted in a cooperative stabilizing effect on protein.

  5. Mobile detection assessment and response systems (MDARS): a force protection physical security operational success

    NASA Astrophysics Data System (ADS)

    Shoop, Brian; Johnston, Michael; Goehring, Richard; Moneyhun, Jon; Skibba, Brian

    2006-05-01

    MDARS is a Semi-autonomous unmanned ground vehicle with intrusion detection & assessment, product & barrier assessment payloads. Its functions include surveillance, security, early warning, incident first response and product and barrier status primarily focused on a depot/munitions security mission at structured/semi-structured facilities. MDARS is in Systems Development and Demonstration (SDD) under the Product Manager for Force Protection Systems (PM-FPS). MDARS capabilities include semi-autonomous navigation, obstacle avoidance, motion detection, day and night imagers, radio frequency tag inventory/barrier assessment and audio challenge and response. Four SDD MDARS Patrol Vehicles have been undergoing operational evaluation at Hawthorne Army Depot, NV (HWAD) since October 2004. Hawthorne personnel were trained to administer, operate and maintain the system in accordance with the US Army Military Police School (USAMPS) Concept of Employment and the PM-FPS MDARS Integrated Logistic Support Plan. The system was subjected to intensive periods of evaluation under the guidance and control of the Army Test and Evaluation Center (ATEC) and PM-FPS. Significantly, in terms of User acceptance, the system has been under the "operational control" of the installation performing security and force protection missions in support of daily operations. This evaluation is intended to assess MDARS operational effectiveness in an operational environment. Initial observations show that MDARS provides enhanced force protection, can potentially reduce manpower requirements by conducting routine tasks within its design capabilities and reduces Soldier exposure in the initial response to emerging incidents and situations. Success of the MDARS program has been instrumental in the design and development of two additional robotic force protection programs. The first was the USAF Force Protection Battle Lab sponsored Remote Detection Challenge & Response (REDCAR) concept demonstration

  6. Protective activity of the purified protein antigen of Erysipelothrix rhusiopathiae in pigs.

    PubMed

    Yamazaki, Y; Sato, H; Sakakura, H; Shigeto, K; Nakano, K; Saito, H; Maehara, N

    1999-02-01

    We purified the protein antigen (P64), which contains 66 and 64 kDa proteins, from the alkaline extract (AE) of whole cells of Erysipelothrix rhusiopathiae strain Agata (serovar 5) to determine the protective activity of the antigen against E. rhusiopathiae infection in pigs. The serum titre of antibody against P64 rapidly increased in pigs immunized with 500 and 100 micrograms of P64 and reached maximum values at 3 weeks after the first immunization (1 week after the second immunization). However, the serum antibody titres were not increased in pigs immunized with 20 micrograms of P64 and in nonimmunized pigs. In the pigs immunized with live cell vaccine (acriflavin-fast attenuated strain Koganei 65-0.15), the serum titres of antibody against P64 also increased at 1-2 weeks after immunization. In a pig challenge test performed on immunized and nonimmunized pigs, all nonimmunized pigs showed typical clinical signs of swine erysipelas (fever, erysipeloid, arthritis), while all pigs immunized with 500 and 100 micrograms of P64 and live cell vaccine showed no clinical signs of this disease. In Western blot analysis, sera from pigs immunized with P64 and live cell vaccine strongly reacted with the 64 kDa protein. In contrast, the serum from nonimmunized pigs did not react with any proteins. From these results, it was suggested that a specific antibody against the 64 kDa protein could be increased in pigs immunized with P64 or live cell vaccine and that this anti-P64 antibody has a strong protective effect against E. rhusiopathiae infection in pigs.

  7. Development of benzimidazole derivatives to inhibit HIV-1 replication through protecting APOBEC3G protein.

    PubMed

    Pan, Ting; He, Xin; Chen, Bing; Chen, Hui; Geng, Guannan; Luo, Haihua; Zhang, Hui; Bai, Chuan

    2015-05-05

    Human APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G, A3G) is a potent restriction factor against human immunodeficiency virus type 1 (HIV-1) by inducing hypermutation of G to A in viral genome after its incorporation into virions. HIV-1 Vif (Virion Infectivity Factor) counteracts A3G by inducing ubiquitination and proteasomal degradation of A3G protein. Vif-A3G axis therefore is a promising therapeutic target of HIV-1. Here we report the screening, synthesis and SAR studies of benzimidazole derivatives as potent inhibitors against HIV-1 replication via protecting A3G protein. Based on the steep SAR of the benzimidazole scaffold, we identified compound 14 and 26 which provided the best potency, with IC50 values of 3.45 nM and 58.03 nM respectively in the anti-HIV-1 replication assay in H9 cells. Compound 14 and 26 also afforded protective effects on A3G protein level. Both compounds have been proved to be safe in acute toxicological studies. Taken together, we suggest that these two benzimidazole derivatives can be further developed as a new category of anti-HIV-1 leads.

  8. Differential protein expression in metallothionein protection from depleted uranium-induced nephrotoxicity

    PubMed Central

    Hao, Yuhui; Huang, Jiawei; Liu, Cong; Li, Hong; Liu, Jing; Zeng, Yiping; Yang, Zhangyou; Li, Rong

    2016-01-01

    The purpose of this study was to investigate the underlying mechanism of metallothionein (MT) protection from depleted uranium (DU) using a proteomics approach to search for a DU toxicity-differential protein. MT−/− and MT+/+ mice were administrated with a single dose of DU (10 mg/kg, i.p.) or equal volume of saline. After 4 days, protein changes in kidney tissues were evaluated using a proteomics approach. A total of 13 differentially expressed proteins were identified using two-dimensional electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The validating results showed that the expression of aminoacylase-3 (ACY-3) and the mitochondrial ethylmalonic encephalopathy 1 (ETHE1) decreased significantly after DU exposure; in addition, the reduction in MT−/− mice was more significant than that in MT+/+ mice. The results also showed that exogenous ETHE1 or ACY-3 could increase the survival rate of human embryonic kidney 293 (HEK293) cells after DU exposure. A specific siRNA of ETHE1 significantly increased cell apoptosis rates after DU exposure, whereas exogenous ETHE1 significantly decreased cell apoptosis rates. In summary, ACY-3 and ETHE1 might involve in protection roles of MT. ETHE1 could be a new sensitive molecular target of DU-induced cell apoptosis. PMID:27966587

  9. Identification of a 68-kilodalton protective protein antigen from Bordetella bronchiseptica.

    PubMed Central

    Montaraz, J A; Novotny, P; Ivanyi, J

    1985-01-01

    A 68-kilodalton (kd) outer membrane protein antigen of Bordetella bronchiseptica has been identified by using monoclonal antibodies that recognized two nonoverlapping determinants. Antibody BB05 also reacted with homologous proteins from Bordetella pertussis and Bordetella parapertussis but not with another 12 organisms from various bacterial genera. Passive injection of BB05 antibody protected mice from aerosol infection with B. bronchiseptica as shown by reduced mortality and reduced pathology of turbinate bones. The 68-kd B. bronchiseptica antigen was purified by BB05-based affinity chromatography and evaluated for its potency to immunize mice actively against either intraperitoneal or aerosol challenge with B. bronchiseptica. Immunization with the 68-kd antigen in incomplete Freund adjuvant significantly reduced the levels of mortality in intraperitoneally challenged mice. In the aerosol infection model, injection of the 68-kd antigen with complete or incomplete Freund adjuvant or saponin reduced the bacterial counts in the lungs of infected mice. These results suggest that the 68-kd protein may represent a potential "protective" antigen of B. bronchiseptica. Images PMID:3972452

  10. Saccharomyces cerevisiae Tti2 Regulates PIKK Proteins and Stress Response

    PubMed Central

    Hoffman, Kyle S.; Duennwald, Martin L.; Karagiannis, Jim; Genereaux, Julie; McCarton, Alexander S.; Brandl, Christopher J.

    2016-01-01

    The TTT complex is composed of the three essential proteins Tel2, Tti1, and Tti2. The complex is required to maintain steady state levels of phosphatidylinositol 3-kinase-related kinase (PIKK) proteins, including mTOR, ATM/Tel1, ATR/Mec1, and TRRAP/Tra1, all of which serve as regulators of critical cell signaling pathways. Due to their association with heat shock proteins, and with newly synthesized PIKK peptides, components of the TTT complex may act as cochaperones. Here, we analyze the consequences of depleting the cellular level of Tti2 in Saccharomyces cerevisiae. We show that yeast expressing low levels of Tti2 are viable under optimal growth conditions, but the cells are sensitive to a number of stress conditions that involve PIKK pathways. In agreement with this, depleting Tti2 levels decreased expression of Tra1, Mec1, and Tor1, affected their localization and inhibited the stress responses in which these molecules are involved. Tti2 expression was not increased during heat shock, implying that it does not play a general role in the heat shock response. However, steady state levels of Hsp42 increase when Tti2 is depleted, and tti2L187P has a synthetic interaction with exon 1 of the human Huntingtin gene containing a 103 residue polyQ sequence, suggesting a general role in protein quality control. We also find that overexpressing Hsp90 or its cochaperones is synthetic lethal when Tti2 is depleted, an effect possibly due to imbalanced stoichiometry of a complex required for PIKK assembly. These results indicate that Tti2 does not act as a general chaperone, but may have a specialized function in PIKK folding and/or complex assembly. PMID:27172216

  11. Caffeine Induces the Stress Response and Up-Regulates Heat Shock Proteins in Caenorhabditis elegans.

    PubMed

    Al-Amin, Mohammad; Kawasaki, Ichiro; Gong, Joomi; Shim, Yhong-Hee

    2016-02-01

    Caffeine has both positive and negative effects on physiological functions in a dose-dependent manner. C. elegans has been used as an animal model to investigate the effects of caffeine on development. Caffeine treatment at a high dose (30 mM) showed detrimental effects and caused early larval arrest. We performed a comparative proteomic analysis to investigate the mode of action of high-dose caffeine treatment in C. elegans and found that the stress response proteins, heat shock protein (HSP)-4 (endoplasmic reticulum [ER] chaperone), HSP-6 (mitochondrial chaperone), and HSP-16 (cytosolic chaperone), were induced and their expression was regulated at the transcriptional level. These findings suggest that high-dose caffeine intake causes a strong stress response and activates all three stress-response pathways in the worms, including the ER-, mitochondrial-, and cytosolic pathways. RNA interference of each hsp gene or in triple combination retarded growth. In addition, caffeine treatment stimulated a food-avoidance behavior (aversion phenotype), which was enhanced by RNAi depletion of the hsp-4 gene. Therefore, up-regulation of hsp genes after caffeine treatment appeared to be the major responses to alleviate stress and protect against developmental arrest.

  12. Semi-permeable coatings fabricated from comb-polymers efficiently protect proteins in vivo

    NASA Astrophysics Data System (ADS)

    Liu, Mi; Johansen, Pål; Zabel, Franziska; Leroux, Jean-Christophe; Gauthier, Marc A.

    2014-11-01

    In comparison to neutral linear polymers, functional and architecturally complex (that is, non-linear) polymers offer distinct opportunities for enhancing the properties and performance of therapeutic proteins. However, understanding how to harness these parameters is challenging, and studies that capitalize on them in vivo are scarce. Here we present an in vivo demonstration that modification of a protein with a polymer of appropriate architecture can impart low immunogenicity, with a commensurably low loss of therapeutic activity. These combined properties are inaccessible by conventional strategies using linear polymers. For the model protein L-asparaginase, a comb-polymer bio-conjugate significantly outperformed the linear polymer control in terms of lower immune response and more sustained bioactivity. The semi-permeability characteristics of the coatings are consistent with the phase diagram of the polymer, which will facilitate the application of this strategy to other proteins and with other therapeutic models.

  13. How gender and task difficulty affect a sport-protective response in young adults

    PubMed Central

    Lipps, David B.; Eckner, James T.; Richardson, James K.; Ashton-Miller, James A.

    2013-01-01

    We tested the hypotheses that gender and task difficulty affect the reaction, movement, and total response times associated with performing a head protective response. Twenty-four healthy young adults (13 females) performed a protective response of raising their hands from waist level to block a foam ball fired at their head from an air cannon. Participants initially stood 8.25 m away from the cannon (‘low difficulty’), and were moved successively closer in 60 cm increments until they failed to block at least 5 of 8 balls (‘high difficulty’). Limb motion was quantified using optoelectronic markers on the participants’ left wrist. Males had significantly faster total response times (p = 0.042), a trend towards faster movement times (p = 0.054), and faster peak wrist velocity (p < .001) and acceleration (p = 0.032) than females. Reaction time, movement time, and total response time were significantly faster under high difficulty conditions for both genders (p < .001). This study suggests that baseball and softball pitchers and fielders should have sufficient time to protect their head from a batted ball under optimal conditions if they are adequately prepared for the task. PMID:23234296

  14. Correlates of Protection for M Protein-Based Vaccines against Group A Streptococcus

    PubMed Central

    Smeesters, Pierre R.; Frost, Hannah R. C.; Steer, Andrew C.

    2015-01-01

    Group A streptococcus (GAS) is known to cause a broad spectrum of illness, from pharyngitis and impetigo, to autoimmune sequelae such as rheumatic heart disease, and invasive diseases. It is a significant cause of infectious disease morbidity and mortality worldwide, but no efficacious vaccine is currently available. Progress in GAS vaccine development has been hindered by a number of obstacles, including a lack of standardization in immunoassays and the need to define human correlates of protection. In this review, we have examined the current immunoassays used in both GAS and other organisms, and explored the various challenges in their implementation in order to propose potential future directions to identify a correlate of protection and facilitate the development of M protein-based vaccines, which are currently the main GAS vaccine candidates. PMID:26101780

  15. Immunisation With Immunodominant Linear B Cell Epitopes Vaccine of Manganese Transport Protein C Confers Protection against Staphylococcus aureus Infection

    PubMed Central

    Yang, Hui-Jie; Zhang, Jin-Yong; Wei, Chao; Yang, Liu-Yang; Zuo, Qian-Fei; Zhuang, Yuan; Feng, You-Jun; Srinivas, Swaminath; Zeng, Hao; Zou, Quan-Ming

    2016-01-01

    Vaccination strategies for Staphylococcus aureus, particularly methicillin-resistant S. aureus (MRSA) infections have attracted much research attention. Recent efforts have been made to select manganese transport protein C, or manganese binding surface lipoprotein C (MntC), which is a metal ion associated with pathogen nutrition uptake, as potential candidates for an S. aureus vaccine. Although protective humoral immune responses to MntC are well-characterised, much less is known about detailed MntC-specific B cell epitope mapping and particularly epitope vaccines, which are less-time consuming and more convenient. In this study, we generated a recombinant protein rMntC which induced strong antibody response when used for immunisation with CFA/IFA adjuvant. On the basis of the results, linear B cell epitopes within MntC were finely mapped using a series of overlapping synthetic peptides. Further studies indicate that MntC113-136, MntC209-232, and MntC263-286 might be the original linear B-cell immune dominant epitope of MntC, furthermore, three-dimensional (3-d) crystal structure results indicate that the three immunodominant epitopes were displayed on the surface of the MntC antigen. On the basis of immunodominant MntC113-136, MntC209-232, and MntC263-286 peptides, the epitope vaccine for S. aureus induces a high antibody level which is biased to TH2 and provides effective immune protection and strong opsonophagocytic killing activity in vitro against MRSA infection. In summary, the study provides strong proof of the optimisation of MRSA B cell epitope vaccine designs and their use, which was based on the MntC antigen in the development of an MRSA vaccine. PMID:26895191

  16. Immunogenicity and Protective Efficacy of Brugia malayi Heavy Chain Myosin as Homologous DNA, Protein and Heterologous DNA/Protein Prime Boost Vaccine in Rodent Model

    PubMed Central

    Gupta, Jyoti; Pathak, Manisha; Misra, Sweta; Misra-Bhattacharya, Shailja

    2015-01-01

    We earlier demonstrated the immunoprophylactic efficacy of recombinant heavy chain myosin (Bm-Myo) of Brugia malayi (B. malayi) in rodent models. In the current study, further attempts have been made to improve this efficacy by employing alternate approaches such as homologous DNA (pcD-Myo) and heterologous DNA/protein prime boost (pcD-Myo+Bm-Myo) in BALB/c mouse model. The gene bm-myo was cloned in a mammalian expression vector pcDNA 3.1(+) and protein expression was confirmed in mammalian Vero cell line. A significant degree of protection (79.2%±2.32) against L3 challenge in pcD-Myo+Bm-Myo immunized group was observed which was much higher than that exerted by Bm-Myo (66.6%±2.23) and pcD-Myo (41.6%±2.45). In the heterologous immunized group, the percentage of peritoneal leukocytes such as macrophages, neutrophils, B cells and T cells marginally increased and their population augmented further significantly following L3 challenge. pcD-Myo+Bm-Myo immunization elicited robust cellular and humoral immune responses as compared to pcD-Myo and Bm-Myo groups as evidenced by an increased accumulation of CD4+, CD8+ T cells and CD19+ B cells in the mouse spleen and activation of peritoneal macrophages. Though immunized animals produced antigen-specific IgG antibodies and isotypes, sera of mice receiving pcD-Myo+Bm-Myo or Bm-Myo developed much higher antibody levels than other groups and there was profound antibody-dependent cellular adhesion and cytotoxicity (ADCC) to B. malayi infective larvae (L3). pcD-Myo+Bm-Myo as well as Bm-Myo mice generated a mixed T helper cell phenotype as evidenced by the production of both pro-inflammatory (IL-2, IFN-γ) and anti-inflammatory (IL-4, IL-10) cytokines. Mice receiving pcD-Myo on contrary displayed a polarized pro-inflammatory immune response. The findings suggest that the priming of animals with DNA followed by protein booster generates heightened and mixed pro- and anti-inflammatory immune responses that are capable of providing

  17. Children's experiences of domestic violence: developing an integrated response from police and child protection services.

    PubMed

    Stanley, Nicky; Miller, Pam; Foster, Helen Richardson; Thomson, Gill

    2011-08-01

    Police notifications of incidents of domestic violence to child protection services constitute an acknowledgement of the harm that domestic violence inflicts on children. However, these notifications represent a substantial demand on child welfare services and the outcomes for children and victims of domestic violence have been questioned. This paper presents findings from the first UK study to examine these notifications in depth and examines the interface between the police and child protection services in responding to domestic violence incidents. The research reports on police interventions in 251 incidents of domestic violence involving children; the communication of information to child protection services and the subsequent filtering and service response. Social workers found that notifications conveyed little information on children's experiences of domestic violence. Forty per cent of families notified had had no previous contact with child protection services in that area, but those cases most likely to receive social work assessment or intervention were those where the case was already open. Notifications triggered a new social work intervention in only 5% of cases. The study also identified a range of innovative approaches for improving the co-ordination of police and child protective services in relation to children's exposure to domestic violence. Arrangements that maximized opportunities for police and social workers to share agency information appeared to offer the best option for achieving informed decisions about the appropriate level of service response to children and families experiencing domestic violence.

  18. Molecular characterization, functional expression, tissue localization and protective potential of a Taenia solium fatty acid-binding protein.

    PubMed

    Illescas, Oscar; Carrero, Julio C; Bobes, Raúl J; Flisser, Ana; Rosas, Gabriela; Laclette, Juan P

    2012-12-01

    The fatty acid-binding proteins (FABPs) comprise a family of proteins that are widely expressed in animal cells and perform a variety of vital functions. Here, we report the identification, characterization, recombinant expression, tissue localization and protective potential of a Taenia solium FABP (TsFABP1). The TsFABP1 primary structure showed all the conserved residues characteristic of the subfamily iv of the intracellular Lipid-Binding Proteins (iLBPs), including those involved in the binding stabilization of the fatty acid molecule. Through a competitive binding assay we found that TsFABP1 is able to bind at least six different fatty acids with preference toward palmitic and stearic acid, suggesting that TsFABP1 is a member of the iLBP subfamily iv. Immunolocalization assays carried out on larval and adult tissues of four species of taeniids using anti-TsFABP1 hyperimmune sera produced in mice and rabbit, showed intense labeling in the tegument of the spiral canal and in subtegumental cytons of the larvae. These findings suggest that the spiral canal might be a major place for FA uptake in the developing scolex. In contrast, only subtegumental cytons in the adult worms stained positive. We propose that TsFABP1 is involved in the mechanism to mobilize fatty acids between compartments in the extensive syncytial tissue of taeniids. Protection assays carried out in a murine model of cysticercosis showed that subcutaneous immunization with TsFABP1 resulted in about 45% reduction of parasite load against an intraperitoneal challenge with Taenia crassiceps cysts. This reduction in parasite load correlated with the level of cellular and humoral immune responses against TsFABP1, as determined in spleen lymphocyte proliferation and ELISA testing.

  19. Role of T3SS-1 SipD Protein in Protecting Mice against Non-typhoidal Salmonella Typhimurium

    PubMed Central

    Jneid, Bakhos; Moreau, Karine; Plaisance, Marc; Rouaix, Audrey; Dano, Julie

    2016-01-01

    Background Salmonella enterica species are enteric pathogens that cause severe diseases ranging from self-limiting gastroenteritis to enteric fever and sepsis in humans. These infectious diseases are still the major cause of morbidity and mortality in low-income countries, especially in children younger than 5 years and immunocompromised adults. Vaccines targeting typhoidal diseases are already marketed, but none protect against non-typhoidal Salmonella. The existence of multiple non-typhoidal Salmonella serotypes as well as emerging antibiotic resistance highlight the need for development of a broad-spectrum protective vaccine. All Salmonella spp. utilize two type III Secretion Systems (T3SS 1 and 2) to initiate infection, allow replication in phagocytic cells and induce systemic disease. T3SS-1, which is essential to invade epithelial cells and cross the barrier, forms an extracellular needle and syringe necessary to inject effector proteins into the host cell. PrgI and SipD form, respectively, the T3SS-1 needle and the tip complex at the top of the needle. Because they are common and highly conserved in all virulent Salmonella spp., they might be ideal candidate antigens for a subunit-based, broad-spectrum vaccine. Principal Findings We investigated the immunogenicity and protective efficacy of PrgI and SipD administered by subcutaneous, intranasal and oral routes, alone or combined, in a mouse model of Salmonella intestinal challenge. Robust IgG (in all immunization routes) and IgA (in intranasal and oral immunization routes) antibody responses were induced against both proteins, particularly SipD. Mice orally immunized with SipD alone or SipD combined with PrgI were protected against lethal intestinal challenge with Salmonella Typhimurium (100 Lethal Dose 50%) depending on antigen, route and adjuvant. Conclusions and Significance Salmonella T3SS SipD is a promising antigen for the development of a protective Salmonella vaccine, and could be developed for

  20. Recombinant Adenovirus Delivery of Calreticulin–ESAT-6 Produces an Antigen-Specific Immune Response but no Protection Against a Mycobacterium Tuberculosis Challenge

    PubMed Central

    Esparza-González, S. C.; Troy, A.; Troudt, J.; Loera-Arias, M. J.; Villatoro-Hernández, J.; Torres-López, E.; Ancer-Rodríguez, J.; Gutiérrez-Puente, Y.; Muñoz-Maldonado, G.; Saucedo-Cárdenas, O.; Montes-de-Oca-Luna, R.; Izzo, A.

    2015-01-01

    Bacillus Calmette–Guerin (BCG) has failed to efficaciously control the worldwide spread of the disease. New vaccine development targets virulence antigens of Mycobacterium tuberculosis that are deleted in Mycobacterium bovis BCG. Immunization with ESAT-6 and CFP10 provides protection against M. tuberculosis in a murine infection model. Further, previous studies have shown that calreticulin increases the cell-mediated immune responses to antigens. Therefore, to test whether calreticulin enhances the immune response against M. tuberculosis antigens, we fused ESAT-6 to calreticulin and constructed a recombinant replication-deficient adenovirus to express the resulting fusion protein (AdCRT–ESAT-6). The adjuvant effect of calreticulin was assayed by measuring cytokine responses specific to ESAT-6. Recombinant adenovirus expressing the fusion protein produced higher levels of interferon-γ and tumour necrosis factor-α in response to ESAT-6. This immune response was not improved by the addition of CFP-10 to the CRT-ESAT-6 fusion protein (AdCRT–ESAT-6–CFP10). Mice immunized with these recombinant adenoviruses did not decrease the mycobacterial burden after low-dose aerosol infection with M. tuberculosis. We conclude that calreticulin can be used as an adjuvant to enhance the immune response against mycobacterial antigens, but it is not enough to protect against tuberculosis. PMID:22010821

  1. Recombinant anthrax toxin receptor-Fc fusion proteins produced in plants protect rabbits against inhalational anthrax.

    PubMed

    Wycoff, Keith L; Belle, Archana; Deppe, Dorothée; Schaefer, Leah; Maclean, James M; Haase, Simone; Trilling, Anke K; Liu, Shihui; Leppla, Stephen H; Geren, Isin N; Pawlik, Jennifer; Peterson, Johnny W

    2011-01-01

    Inhalational anthrax, a zoonotic disease caused by the inhalation of Bacillus anthracis spores, has a ∼50% fatality rate even when treated with antibiotics. Pathogenesis is dependent on the activity of two toxic noncovalent complexes: edema toxin (EdTx) and lethal toxin (LeTx). Protective antigen (PA), an essential component of both complexes, binds with high affinity to the major receptor mediating the lethality of anthrax toxin in vivo, capillary morphogenesis protein 2 (CMG2). Certain antibodies against PA have been shown to protect against anthrax in vivo. As an alternative to anti-PA antibodies, we produced a fusion of the extracellular domain of human CMG2 and human IgG Fc, using both transient and stable tobacco plant expression systems. Optimized expression led to the CMG2-Fc fusion protein being produced at high levels: 730 mg/kg fresh leaf weight in Nicotiana benthamiana and 65 mg/kg in N. tabacum. CMG2-Fc, purified from tobacco plants, fully protected rabbits against a lethal challenge with B. anthracis spores at a dose of 2 mg/kg body weight administered at the time of challenge. Treatment with CMG2-Fc did not interfere with the development of the animals' own immunity to anthrax, as treated animals that survived an initial challenge also survived a rechallenge 30 days later. The glycosylation of the Fc (or lack thereof) had no significant effect on the protective potency of CMG2-Fc in rabbits or on its serum half-life, which was about 5 days. Significantly, CMG2-Fc effectively neutralized, in vitro, LeTx-containing mutant forms of PA that were not neutralized by anti-PA monoclonal antibodies.

  2. Type IV fimbrial subunit protein ApfA contributes to protection against porcine pleuropneumonia

    PubMed Central

    2012-01-01

    Porcine pleuropneumonia caused by Actinobacillus pleuropneumoniae accounts for serious economic losses in the pig farming industry worldwide. We examined here the immunogenicity and protective efficacy of the recombinant type IV fimbrial subunit protein ApfA as a single antigen vaccine against pleuropneumonia, or as a component of a multi-antigen preparation comprising five other recombinant antigens derived from key virulence factors of A. pleuropneumoniae (ApxIA, ApxIIA, ApxIIIA, ApxIVA and TbpB). Immunization of pigs with recombinant ApfA alone induced high levels of specific serum antibodies and provided partial protection against challenge with the heterologous A. pleuropneumoniae serotype 9 strain. This protection was higher than that engendered by vaccination with rApxIVA or rTbpB alone and similar to that observed after immunization with the tri-antigen combination of rApxIA, rApxIIA and rApxIIIA. In addition, rApfA improved the vaccination potential of the penta-antigen mixture of rApxIA, rApxIIA, rApxIIIA, rApxIVA and rTbpB proteins, where the hexa-antigen vaccine containing rApfA conferred a high level of protection on pigs against the disease. Moreover, when rApfA was used for vaccination alone or in combination with other antigens, such immunization reduced the number of pigs colonized with the challenge strain. These results indicate that ApfA could be a valuable component of an efficient subunit vaccine for the prevention of porcine pleuropneumonia. PMID:22240397

  3. Neuropeptide signals cell non-autonomous mitochondrial unfolded protein response

    PubMed Central

    Shao, Li-Wa; Niu, Rong; Liu, Ying

    2016-01-01

    Neurons have a central role in the systemic coordination of mitochondrial unfolded protein response (UPRmt) and the cell non-autonomous modulation of longevity. However, the mechanism by which the nervous system senses mitochondrial stress and communicates to the distal tissues to induce UPRmt remains unclear. Here we employ the tissue-specific CRISPR-Cas9 approach to disrupt mitochondrial function only in the nervous system of Caenorhabditis elegans, and reveal a cell non-autonomous induction of UPRmt in peripheral cells. We further show that a neural sub-circuit composed of three types of sensory neurons, and one interneuron is required for sensing and transducing neuronal mitochondrial stress. In addition, neuropeptide FLP-2 functions in this neural sub-circuit to signal the non-autonomous UPRmt. Taken together, our results suggest a neuropeptide coordination of mitochondrial stress response in the nervous system. PMID:27767096

  4. Compartmentalization and regulation of iron metabolism proteins protect male germ cells from iron overload.

    PubMed

    Leichtmann-Bardoogo, Yael; Cohen, Lyora A; Weiss, Avital; Marohn, Britta; Schubert, Stephanie; Meinhardt, Andreas; Meyron-Holtz, Esther G

    2012-06-15

    The universal importance of iron, its high toxicity, and complex chemistry present a challenge to biological systems in general and to protected compartments in particular. The high mitotic rate and avid mitochondriogenesis of developing male germ cells imply high iron requirements. Yet access to germ cells is tightly regulated by the blood-testis barrier that protects the meiotic and postmeiotic germ cells. To elucidate how iron is supplied to developing male germ cells, we analyzed iron deposition and iron transport proteins in testes of mice with iron overload and with genetic ablation of the iron regulators Hfe and iron regulatory protein 2. Iron accumulated mainly around seminiferous tubules, and only small amounts localized within the seminiferous tubules. The localization and regulation of proteins involved in iron import, storage, and export such as transferrin, transferrin receptor, the divalent metal transporter-1, cytosolic ferritin, and ferroportin strongly support a model of a largely autonomous iron cycle within seminiferous tubules. We show evidence that ferritin secretion from Sertoli cells may play an important role in iron acquisition of primary spermatocytes. During spermatogenic development iron is carried along from primary spermatocytes to spermatids, and from spermatids iron is recycled to the apical compartment of Sertoli cells, which traffic it back to a new generation of spermatocytes. Losses are replenished by the peripheral circulation. Such an internal iron cycle essentially de