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Sample records for protein expression elicited

  1. 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.

  2. Distinct patterns of gene and protein expression elicited by organophosphorus pesticides in Caenorhabditis elegans

    PubMed Central

    Lewis, John A; Szilagyi, Maria; Gehman, Elizabeth; Dennis, William E; Jackson, David A

    2009-01-01

    Background The wide use of organophosphorus (OP) pesticides makes them an important public health concern. Persistent effects of exposure and the mechanism of neuronal degeneration are continuing issues in OP toxicology. To elucidate early steps in the mechanisms of OP toxicity, we studied alterations in global gene and protein expression in Caenorhabditis elegans exposed to OPs using microarrays and mass spectrometry. We tested two structurally distinct OPs (dichlorvos and fenamiphos) and employed a mechanistically different third neurotoxicant, mefloquine, as an out-group for analysis. Treatment levels used concentrations of chemical sufficient to prevent the development of 10%, 50% or 90% of mid-vulval L4 larvae into early gravid adults (EGA) at 24 h after exposure in a defined, bacteria-free medium. Results After 8 h of exposure, the expression of 87 genes responded specifically to OP treatment. The abundance of 34 proteins also changed in OP-exposed worms. Many of the genes and proteins affected by the OPs are expressed in neuronal and muscle tissues and are involved in lipid metabolism, cell adhesion, apoptosis/cell death, and detoxification. Twenty-two genes were differentially affected by the two OPs; a large proportion of these genes encode cytochrome P450s, UDP-glucuronosyl/UDP-glucosyltransferases, or P-glycoproteins. The abundance of transcripts and the proteins they encode were well correlated. Conclusion Exposure to OPs elicits a pattern of changes in gene expression in exposed worms distinct from that of the unrelated neurotoxicant, mefloquine. The functional roles and the tissue location of the genes and proteins whose expression is modulated in response to exposure is consistent with the known effects of OPs, including damage to muscle due to persistent hypercontraction, neuronal cell death, and phase I and phase II detoxification. Further, the two different OPs evoked distinguishable changes in gene expression; about half the differences are in

  3. Expression of three topologically distinct membrane proteins elicits unique stress response pathways in the yeast Saccharomyces cerevisiae

    PubMed Central

    Buck, Teresa M.; Jordan, Rick; Lyons-Weiler, James; Adelman, Joshua L.; Needham, Patrick G.; Kleyman, Thomas R.

    2015-01-01

    Misfolded membrane proteins are retained in the endoplasmic reticulum (ER) and are subject to ER-associated degradation, which clears the secretory pathway of potentially toxic species. While the transcriptional response to environmental stressors has been extensively studied, limited data exist describing the cellular response to misfolded membrane proteins. To this end, we expressed and then compared the transcriptional profiles elicited by the synthesis of three ER retained, misfolded ion channels: The α-subunit of the epithelial sodium channel, ENaC, the cystic fibrosis transmembrane conductance regulator, CFTR, and an inwardly rectifying potassium channel, Kir2.1, which vary in their mass, membrane topologies, and quaternary structures. To examine transcriptional profiles in a null background, the proteins were expressed in yeast, which was previously used to examine the degradation requirements for each substrate. Surprisingly, the proteins failed to induce a canonical unfolded protein response or heat shock response, although messages encoding several cytosolic and ER lumenal protein folding factors rose when αENaC or CFTR was expressed. In contrast, the levels of these genes were unaltered by Kir2.1 expression; instead, the yeast iron regulon was activated. Nevertheless, a significant number of genes that respond to various environmental stressors were upregulated by all three substrates, and compared with previous microarray data we deduced the existence of a group of genes that reflect a novel misfolded membrane protein response. These data indicate that aberrant proteins in the ER elicit profound yet unique cellular responses. PMID:25759377

  4. Proteins differentially expressed in elicited cell suspension culture of Podophyllum hexandrum with enhanced podophyllotoxin content

    PubMed Central

    2012-01-01

    Background Podophyllotoxin (PTOX), the precursor for semi-synthesis of cancer therapeutics like etoposide, teniposide and etophos, is primarily obtained from an endangered medicinal herb, Podophyllum hexandrum Royle. PTOX, a lignan is biosynthetically derived from the phenylpropanoid pathway. The aim of this study is to investigate changes in the P. hexandrum cell proteome potentially related to PTOX accumulation in response to methyl jasmonate (MeJA) elicitation. High-resolution two-dimensional gel electrophoresis (2-DE) followed by colloidal Coomassie staining and mass spectrometric analysis was used to detect statistically significant changes in cell’s proteome. Result The HPLC analysis showed approximately 7–8 fold change in accumulation of PTOX, in the 12day old cell suspension culture (i.e. after 9days of elicitation) elicited with 100 μM MeJA as compared to the control. Using 2-DE a total of 233 spots was detected, out of which 105 spots were identified by MALDI TOF-TOF MS/MS. Data were subjected to functional annotation from a biological point of view through KEGG. The phenylpropanoid and monolignol pathway enzymes were identified, amongst these, chalcone synthase, polyphenol oxidase, caffeoyl CoA 3-O-methyltransferase, S-adenosyl-L-methionine-dependent methyltransferases, caffeic acid-O-methyl transferase etc. are noted as important. The relation of other differentially accumulated proteins with varied effects caused by elicitors on P. hexandrum cells namely stress and defense related protein, transcription and DNA replication and signaling are also discussed. Conclusions Elicitor-induced PTOX accumulation in P. hexandrum cell cultures provides a responsive model system to profile modulations in proteins related to phenylpropanoid/monolignol biosynthesis and other defense responses. Present findings form a baseline for future investigation on a non-sequenced medicinal herb P. hexandrum at molecular level. PMID:22621772

  5. Influence of protein expression system on elicitation of IgE antibody responses: experience with lactoferrin.

    PubMed

    Almond, Rachael J; Flanagan, Brian F; Kimber, Ian; Dearman, Rebecca J

    2012-11-15

    With increased interest in genetically modified (GM) crop plants there is an important need to understand the properties that contribute to the ability of such novel proteins to provoke immune and/or allergic responses. One characteristic that may be relevant is glycosylation, particularly as novel expression systems (e.g. bacterial to plant) will impact on the protein glycoprofile. The allergenicity (IgE inducing) and immunogenicity (IgG inducing) properties of wild type native human lactoferrin (NLF) from human milk (hm) and neutrophil granules (n) and a recombinant molecule produced in rice (RLF) have been assessed. These forms of lactoferrin have identical amino acid sequences, but different glycosylation patterns: hmNLF and nNLF have complex glycoprofiles including Lewis (Le)(x) structures, with particularly high levels of Le(x) expressed by nNLF, whereas RLF is simpler and rich in mannose residues. Antibody responses induced in BALB/c strain mice by intraperitoneal exposure to the different forms of lactoferrin were characterised. Immunisation with both forms of NLF stimulated substantial IgG and IgE antibody responses. In contrast, the recombinant molecule was considerably less immunogenic and failed to stimulate detectable IgE, irrespective of endotoxin and iron content. The glycans did not contribute to epitope formation, with equivalent IgE and IgG binding recorded for high titre anti-NLF antisera regardless of whether the immunising NLF or the recombinant molecule were used substrates in the analyses. These data demonstrate that differential glycosylation profiles can have a profound impact on protein allergenicity and immunogenicity, with mannose and Le(x) exhibiting opposing effects. These results have clear relevance for characterising the allergenic hazards of novel proteins in GM crops.

  6. Distinct Patterns of Gene and Protein Expression Elicited by Organophosphorus Pesticides in Caenorhabditis elegans

    DTIC Science & Technology

    2009-01-01

    pro- vide adequate biomass for RNA and protein preparation. The worms were treated with mefloquine ( Ash Stevens, Inc., Detroit, MI), dichlorvos, or...recommendations except that (1) a high pressure liquid chromatography ( HPLC )-purified T24T7 promoter primer (Integrated DNA Technologies, Coralville, IA) was...early gravid adult; EGTA: ethylene glycol tetraacetic acid; FDR: false discovery rate; g: gravity; h: hour; HPLC : high pressure liquid chromatog- raphy

  7. DNA vaccines expressing soluble CD4-envelope proteins fused to C3d elicit cross-reactive neutralizing antibodies to HIV-1

    SciTech Connect

    Bower, Joseph F.; Green, Thomas D.; Ross, Ted M. . E-mail: tmr15@pitt.edu

    2004-10-25

    DNA vaccines expressing the envelope (Env) of the human immunodeficiency virus type 1 (HIV-1) have been relatively ineffective at generating high-titer, long-lasting, neutralizing antibodies in a variety of animal models. In this study, DNA vaccines were constructed to express a fusion protein of the soluble human CD4 (sCD4) and the gp120 subunit of the HIV-1 envelope. To enhance the immunogenicity of the expressed fusion protein, three copies of the murine C3d (mC3d{sub 3}) were added to the carboxyl terminus of the complex. Monoclonal antibodies that recognize CD4-induced epitopes on gp120 efficiently bound to sCD4-gp120 or sCD4-gp120-mC3d{sub 3}. In addition, both sCD4-gp120 and sCD4-gp120-mC3d{sub 3} bound to cells expressing appropriate coreceptors in the absence of cell surface hCD4. Mice (BALB/c) vaccinated with DNA vaccines expressing either gp120-mC3d{sub 3} or sCD4-gp120-mC3d{sub 3} elicited antibodies that neutralized homologous virus infection. However, the use of sCD4-gp120-mC3d{sub 3}-DNA elicited the highest titers of neutralizing antibodies that persisted after depletion of anti-hCD4 antibodies. Interestingly, only mice vaccinated with DNA expressing sCD4-gp120-mC3d{sub 3} had antibodies that elicited cross-protective neutralizing antibodies. The fusion of sCD4 to the HIV-1 envelope exposes neutralizing epitopes that elicit broad protective immunity when the fusion complex is coupled with the molecular adjuvant, C3d.

  8. 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

  9. Recombinant receptor-binding domain of SARS-CoV spike protein expressed in mammalian, insect and E. coli cells elicits potent neutralizing antibody and protective immunity.

    PubMed

    Du, Lanying; Zhao, Guangyu; Chan, Chris C S; Sun, Shihui; Chen, Min; Liu, Zhonghua; Guo, Hongxiang; He, Yuxian; Zhou, Yusen; Zheng, Bo-Jian; Jiang, Shibo

    2009-10-10

    Severe acute respiratory syndrome (SARS) is a newly emerging infectious disease. The potential recurrence of the disease from animal reservoirs highlights the significance of development of safe and efficient vaccines to prevent a future SARS epidemic. In this study, we expressed the recombinant receptor-binding domain (rRBD) in mammalian (293T) cells, insect (Sf9) cells, and E. coli, respectively, and compared their immunogenicity and protection against SARS-CoV infection in an established mouse model. Our results show that all rRBD proteins expressed in the above systems maintained intact conformation, being able to induce highly potent neutralizing antibody responses and complete protective immunity against SARS-CoV challenge in mice, albeit the rRBD expressed in 293T cells elicited stronger humoral immune responses with significantly higher neutralizing activity (P<0.05) than those expressed in Sf9 and E. coli cells. These results suggest that all three rRBDs are effective in eliciting immune responses and protection against SARS-CoV and any of the above expression systems can be used for production of rRBD-based SARS subunit vaccines. Preference will be given to rRBD expressed in mammalian cells for future evaluation of the vaccine efficacy in a non-human primate model of SARS because of its ability to refold into a native conformation more readily and to induce higher level of neutralizing antibody responses than those expressed in E. coli and insect cells.

  10. 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

  11. Stereoscopy Amplifies Emotions Elicited by Facial Expressions

    PubMed Central

    Kätsyri, Jari; Häkkinen, Jukka

    2015-01-01

    Mediated facial expressions do not elicit emotions as strongly as real-life facial expressions, possibly due to the low fidelity of pictorial presentations in typical mediation technologies. In the present study, we investigated the extent to which stereoscopy amplifies emotions elicited by images of neutral, angry, and happy facial expressions. The emotional self-reports of positive and negative valence (which were evaluated separately) and arousal of 40 participants were recorded. The magnitude of perceived depth in the stereoscopic images was manipulated by varying the camera base at 15, 40, 65, 90, and 115 mm. The analyses controlled for participants’ gender, gender match, emotional empathy, and trait alexithymia. The results indicated that stereoscopy significantly amplified the negative valence and arousal elicited by angry expressions at the most natural (65 mm) camera base, whereas stereoscopy amplified the positive valence elicited by happy expressions in both the narrowed and most natural (15–65 mm) base conditions. Overall, the results indicate that stereoscopy amplifies the emotions elicited by mediated emotional facial expressions when the depth geometry is close to natural. The findings highlight the sensitivity of the visual system to depth and its effect on emotions. PMID:27551358

  12. Stereoscopy Amplifies Emotions Elicited by Facial Expressions.

    PubMed

    Hakala, Jussi; Kätsyri, Jari; Häkkinen, Jukka

    2015-12-01

    Mediated facial expressions do not elicit emotions as strongly as real-life facial expressions, possibly due to the low fidelity of pictorial presentations in typical mediation technologies. In the present study, we investigated the extent to which stereoscopy amplifies emotions elicited by images of neutral, angry, and happy facial expressions. The emotional self-reports of positive and negative valence (which were evaluated separately) and arousal of 40 participants were recorded. The magnitude of perceived depth in the stereoscopic images was manipulated by varying the camera base at 15, 40, 65, 90, and 115 mm. The analyses controlled for participants' gender, gender match, emotional empathy, and trait alexithymia. The results indicated that stereoscopy significantly amplified the negative valence and arousal elicited by angry expressions at the most natural (65 mm) camera base, whereas stereoscopy amplified the positive valence elicited by happy expressions in both the narrowed and most natural (15-65 mm) base conditions. Overall, the results indicate that stereoscopy amplifies the emotions elicited by mediated emotional facial expressions when the depth geometry is close to natural. The findings highlight the sensitivity of the visual system to depth and its effect on emotions.

  13. Bacterially Expressed Full-Length Recombinant Plasmodium falciparum RH5 Protein Binds Erythrocytes and Elicits Potent Strain-Transcending Parasite-Neutralizing Antibodies

    PubMed Central

    Reddy, K. Sony; Pandey, Alok K.; Singh, Hina; Sahar, Tajali; Emmanuel, Amlabu; Chitnis, Chetan E.; Chauhan, Virander S.

    2014-01-01

    Plasmodium falciparum reticulocyte binding-like homologous protein 5 (PfRH5) is an essential merozoite ligand that binds with its erythrocyte receptor, basigin. PfRH5 is an attractive malaria vaccine candidate, as it is expressed by a wide number of P. falciparum strains, cannot be genetically disrupted, and exhibits limited sequence polymorphisms. Viral vector-induced PfRH5 antibodies potently inhibited erythrocyte invasion. However, it has been a challenge to generate full-length recombinant PfRH5 in a bacterial-cell-based expression system. In this study, we have produced full-length recombinant PfRH5 in Escherichia coli that exhibits specific erythrocyte binding similar to that of the native PfRH5 parasite protein and also, importantly, elicits potent invasion-inhibitory antibodies against a number of P. falciparum strains. Antibasigin antibodies blocked the erythrocyte binding of both native and recombinant PfRH5, further confirming that they bind with basigin. We have thus successfully produced full-length PfRH5 as a functionally active erythrocyte binding recombinant protein with a conformational integrity that mimics that of the native parasite protein and elicits potent strain-transcending parasite-neutralizing antibodies. P. falciparum has the capability to develop immune escape mechanisms, and thus, blood-stage malaria vaccines that target multiple antigens or pathways may prove to be highly efficacious. In this regard, antibody combinations targeting PfRH5 and other key merozoite antigens produced potent additive inhibition against multiple worldwide P. falciparum strains. PfRH5 was immunogenic when immunized with other antigens, eliciting potent invasion-inhibitory antibody responses with no immune interference. Our results strongly support the development of PfRH5 as a component of a combination blood-stage malaria vaccine. PMID:24126527

  14. Microgravity elicits reproducible alterations in cytoskeletal and metabolic gene and protein expression in space-flown Caenorhabditis elegans

    PubMed Central

    Higashibata, Akira; Hashizume, Toko; Nemoto, Kanako; Higashitani, Nahoko; Etheridge, Timothy; Mori, Chihiro; Harada, Shunsuke; Sugimoto, Tomoko; Szewczyk, Nathaniel J; Baba, Shoji A; Mogami, Yoshihiro; Fukui, Keiji; Higashitani, Atsushi

    2016-01-01

    Although muscle atrophy is a serious problem during spaceflight, little is known about the sequence of molecular events leading to atrophy in response to microgravity. We carried out a spaceflight experiment using Caenorhabditis elegans onboard the Japanese Experiment Module of the International Space Station. Worms were synchronously cultured in liquid media with bacterial food for 4 days under microgravity or on a 1-G centrifuge. Worms were visually observed for health and movement and then frozen. Upon return, we analyzed global gene and protein expression using DNA microarrays and mass spectrometry. Body length and fat accumulation were also analyzed. We found that in worms grown from the L1 larval stage to adulthood under microgravity, both gene and protein expression levels for muscular thick filaments, cytoskeletal elements, and mitochondrial metabolic enzymes decreased relative to parallel cultures on the 1-G centrifuge (95% confidence interval (P⩽0.05)). In addition, altered movement and decreased body length and fat accumulation were observed in the microgravity-cultured worms relative to the 1-G cultured worms. These results suggest protein expression changes that may account for the progressive muscular atrophy observed in astronauts. PMID:28725720

  15. Virus replicon particles expressing porcine reproductive and respiratory syndrome virus proteins elicit immune priming but do not confer protection from viremia in pigs.

    PubMed

    Eck, Melanie; Durán, Margarita García; Ricklin, Meret E; Locher, Samira; Sarraseca, Javier; Rodríguez, María José; McCullough, Kenneth C; Summerfield, Artur; Zimmer, Gert; Ruggli, Nicolas

    2016-02-19

    Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of one of the most devastating and economically significant viral disease of pigs worldwide. The vaccines currently available on the market elicit only limited protection. Recombinant vesicular stomatitis virus (VSV) replicon particles (VRP) have been used successfully to induce protection against influenza A virus (IAV) in chickens and bluetongue virus in sheep. In this study, VSV VRP expressing the PRRSV envelope proteins GP5, M, GP4, GP3, GP2 and the nucleocapsid protein N, individually or in combination, were generated and evaluated as a potential vector vaccine against PRRSV infection. High level expression of the recombinant PRRSV proteins was demonstrated in cell culture. However, none of the PRRSV antigens expressed from VRP, with the exception of the N protein, did induce any detectable antibody response in pigs before challenge infection with PRRSV. After challenge however, the antibody responses against GP5, GP4 and GP3 appeared in average 2 weeks earlier than in pigs vaccinated with the empty control VRP. No reduction of viremia was observed in the vaccinated group compared with the control group. When pigs were co-vaccinated with VRP expressing IAV antigens and VRP expressing PRRSV glycoproteins, only antibody responses to the IAV antigens were detectable. These data show that the VSV replicon vector can induce immune responses to heterologous proteins in pigs, but that the PRRSV envelope proteins expressed from VSV VRP are poorly immunogenic. Nevertheless, they prime the immune system for significantly earlier B-cell responses following PRRSV challenge infection.

  16. In Vivo-Expressed Proteins of Virulent Leptospira interrogans Serovar Autumnalis N2 Elicit Strong IgM Responses of Value in Conclusive Diagnosis

    PubMed Central

    Raja, Veerapandian; Shanmughapriya, Santhanam; Kanagavel, Murugesan; Artiushin, Sergey C.; Velineni, Sridhar; Timoney, John F.

    2015-01-01

    Leptospirosis is a serious zoonosis that is underdiagnosed because of limited access to laboratory facilities in Southeast Asia, Central and South America, and Oceania. Timely diagnosis of locally distributed serovars of high virulence is crucial for successful care and outbreak management. Using pooled patient sera, an expression gene library of a virulent Leptospira interrogans serovar Autumnalis strain N2 isolated in South India was screened. The identified genes were characterized, and the purified recombinant proteins were used as antigens in IgM enzyme-linked immunosorbent assay (ELISA) either singly or in combination. Sera (n = 118) from cases of acute leptospirosis along with sera (n = 58) from healthy subjects were tested for reactivity with the identified proteins in an ELISA designed to detect specific IgM responses. We have identified nine immunoreactive proteins, ArgC, RecA, GlpF, FliD, TrmD, RplS, RnhB, Lp28.6, and Lrr44.9, which were found to be highly conserved among pathogenic leptospires. Apparently, the proteins ArgC, RecA, GlpF, FliD, TrmD, and Lrr44.9 are expressed during natural infection of the host and undetectable in in vitro cultures. Among all the recombinant proteins used as antigens in IgM ELISA, ArgC had the highest sensitivity and specificity, 89.8% and 95.5%, respectively, for the conclusive diagnosis of leptospirosis. The use of ArgC and RecA in combination for IgM ELISA increased the sensitivity and specificity to 95.7% and 94.9%, respectively. ArgC and RecA thus elicited specific IgM responses and were therefore effective in laboratory confirmation of Leptospira infection. PMID:26607308

  17. Recombinant human adenovirus-5 expressing capsid proteins of Indian vaccine strains of foot-and-mouth disease virus elicits effective antibody response in cattle.

    PubMed

    Sreenivasa, B P; Mohapatra, J K; Pauszek, S J; Koster, M; Dhanya, V C; Tamil Selvan, R P; Hosamani, M; Saravanan, P; Basagoudanavar, Suresh H; de Los Santos, T; Venkataramanan, R; Rodriguez, L L; Grubman, M J

    2017-05-01

    Recombinant adenovirus-5 vectored foot-and-mouth disease constructs (Ad5- FMD) were made for three Indian vaccine virus serotypes O, A and Asia 1. Constructs co-expressing foot-and- mouth disease virus (FMDV) capsid and viral 3C protease sequences, were evaluated for their ability to induce a neutralizing antibody response in indigenous cattle (Bos indicus). Purified Ad5-FMD viruses were inoculated in cattle as monovalent (5×10(9) pfu/animal) or trivalent (5×10(9) pfu/animal per serotype) vaccines. Animals vaccinated with monovalent Ad5-FMD vaccines were boosted 63days later with the same dose. After primary immunization, virus neutralization tests (VNT) showed seroconversion in 83, 67 and 33% of animals vaccinated with Ad5-FMD O, A and Asia 1, respectively. Booster immunization elicited seroconversion in all of the animals (100%) in the monovalent groups. When used in a trivalent form, the Ad5-FMD vaccine induced neutralizing antibodies in only 33, 50 and 16% of animals against serotypes O, A and Asia 1, respectively on primo-vaccination, and titers were significantly lower than when the same vectors were used in monovalent form. Neutralizing antibody titers differed by serotype for both Ad5-FMD monovalent and trivalent vaccines, with Asia 1 serotype inducing the lowest titers. Antibody response to Ad5 vector in immunized cattle was also assessed by VNT. It appeared that the vector immunity did not impact the recall responses to expressed FMDV antigens on booster immunization. In summary, the study suggested that the recombinant Ad5-FMD vaccine has a potential use in monovalent form, while its application in multivalent form is not currently encouraging. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Priming with two DNA vaccines expressing hepatitis C virus NS3 protein targeting dendritic cells elicits superior heterologous protective potential in mice.

    PubMed

    Guan, Jie; Deng, Yao; Chen, Hong; Yin, Xiao; Yang, Yang; Tan, Wenjie

    2015-10-01

    Development an effective vaccine may offer an alternative preventive and therapeutic strategy against HCV infection. DNA vaccination has been shown to induce robust humoral and cellular immunity and overcome many problems associated with conventional vaccines. In this study, mice were primed with either conventional pVRC-based or suicidal pSC-based DNA vaccines carrying DEC-205-targeted NS3 antigen (DEC-NS3) and boosted with type 5 adenoviral vectors encoding the partial NS3 and core antigens (C44P). The prime boost regimen induced a marked increase in antigen-specific humoral and T-cell responses in comparison with either rAd5-based vaccines or DEC-205-targeted DNA immunization in isolation. The protective effect against heterogeneous challenge was correlated with high levels of anti-NS3 IgG and T-cell-mediated immunity against NS3 peptides. Moreover, priming with a suicidal DNA vaccine (pSC-DEC-NS3), which elicited increased TNF-α-producing CD4+ and CD8+ T-cells against NS3-2 peptides (aa 1245-1461), after boosting, showed increased heterogeneous protective potential compared with priming with a conventional DNA vaccine (pVRC-DEC-NS3). In conclusion, a suicidal DNA vector (pSC-DEC-NS3) expressing DEC-205-targeted NS3 combined with boosting using an rAd5-based HCV vaccine (rAd5-C44P) is a good candidate for a safe and effective vaccine against HCV infection.

  19. A host-range restricted parainfluenza virus type 3 (PIV3) expressing the human metapneumovirus (hMPV) fusion protein elicits protective immunity in African green monkeys.

    PubMed

    Tang, Roderick S; Mahmood, Kutubuddin; Macphail, Mia; Guzzetta, Jeanne M; Haller, Aurelia A; Liu, Hui; Kaur, Jasmine; Lawlor, Heather A; Stillman, Elizabeth A; Schickli, Jeanne H; Fouchier, Ron A M; Osterhaus, Albert D M E; Spaete, Richard R

    2005-02-25

    Human metapneumovirus (hMPV) infection causes respiratory tract disease similar to that observed during human respiratory syncytial virus infection (hRSV). hMPV infections have been reported across the entire age spectrum although the most severe disease occurs in young children. No vaccines, chemotherapeutics or antibodies are presently available for preventing or treating hMPV infections. In this study, a bovine/human chimeric parainfluenza virus type 3 (b/h PIV3) expressing the human parainfluenza type 3 (hPIV3) fusion (F) and hemagglutinin-neuraminidase (HN) proteins was engineered to express hMPV fusion (F) protein from the second genome position (b/h PIV3/hMPV F2) with the goal of generating a novel hMPV vaccine. b/h PIV3/hMPV F2 was previously shown to protect hamsters from challenge with wt hMPV (Tang RS, Schickli JH, Macphail M, Fernandes F, Bicha L, Spaete J, et al. Effects of human metapneumovirus and respiratory syncytial virus antigen insertion in two 3' proximal genome positions of bovine/human parainfluenza virus type 3 on virus replication and immunogenicity. J Virol 2003;77:10819-28) and is here further evaluated for efficacy and immunogenicity in African green monkeys (AGMs). AGMs immunized intranasally and intratracheally with b/h PIV3/hMPV F2 generated hMPV- and hPIV3-specific humoral and cellular immune responses and were protected from wt hMPV infection. In a separate study, the host-range restriction of b/h PIV3/hMPV F2 replication relative to wt hPIV3 was performed in rhesus monkeys to demonstrate attenuation. These studies showed that b/h PIV3/hMPV F2 was immunogenic, protective and attenuated in non-human primates and warrants further evaluation in humans as a vaccine candidate for prevention of hMPV-associated respiratory tract diseases.

  20. Recombinant human adenovirus-5 expressing capsid proteins of Indian vaccine strains of foot-and-mouth disease virus elicits effective antibody response in cattle

    USDA-ARS?s Scientific Manuscript database

    Recombinant adenovirus-5 vectored foot-and-mouth disease constructs (Ad5- FMD) were made for three Indian vaccine virus serotypes O,A and Asia 1. Constructs co-expressing foot-and- mouth disease virus (FMDV) capsid and viral 3C protease sequences, were evaluated for their ability to induce a neutral...

  1. Recombinant sheep pox virus proteins elicit neutralizing antibodies

    USDA-ARS?s Scientific Manuscript database

    The aim of this study was to evaluate the immunogenicity and neutralizing activity of bacterially-expressed sheep pox virus (SPPV) structural proteins as candidate subunit vaccines to control sheep pox disease. SPPV structural proteins were identified by sequence homology with proteins from vaccinia...

  2. Acinetobacter baumannii Outer Membrane Vesicles Elicit a Potent Innate Immune Response via Membrane Proteins

    PubMed Central

    Jun, So Hyun; Lee, Jung Hwa; Kim, Bo Ra; Kim, Seung Il; Park, Tae In

    2013-01-01

    Acinetobacter baumannii is increasingly becoming a major nosocomial pathogen. This opportunistic pathogen secretes outer membrane vesicles (OMVs) that interact with host cells. The aim of this study was to investigate the ability of A. baumannii OMVs to elicit a pro-inflammatory response in vitro and the immunopathology in response to A. baumannii OMVs in vivo. OMVs derived from A. baumannii ATCC 19606T induced expression of pro-inflammatory cytokine genes, interleukin (IL)-1β and IL-6, and chemokine genes, IL-8, macrophage inflammatory protein-1α, and monocyte chemoattractant protein-1, in epithelial cells in a dose-dependent manner. Disintegration of OMV membrane with ethylenediaminetetraacetic acid resulted in low expression of pro-inflammatory cytokine genes, as compared with the response to intact OMVs. In addition, proteinase K-treated A. baumannii OMVs did not induce significant increase in expression of pro-inflammatory cytokine genes above the basal level, suggesting that the surface-exposed membrane proteins in intact OMVs are responsible for pro-inflammatory response. Early inflammatory processes, such as vacuolization and detachment of epithelial cells and neutrophilic infiltration, were clearly observed in lungs of mice injected with A. baumannii OMVs. Our data demonstrate that OMVs produced by A. baumannii elicit a potent innate immune response, which may contribute to immunopathology of the infected host. PMID:23977136

  3. Naturalistic Observations of Elicited Expressive Communication of Children with Autism: An Analysis of Teacher Instructions

    ERIC Educational Resources Information Center

    Chiang, Hsu-Min

    2009-01-01

    This study observed expressive communication of 17 Australian and 15 Taiwanese children with autism who were mute or had limited spoken language during 2 hour regular school routines and analyzed teacher instructions associated with elicited expressive communication. Results indicated: (a) the frequency of occurrence of elicited expressive…

  4. Protein modification elicited by oxidized low-density lipoprotein (LDL) in endothelial cells: protection by (-)-epicatechin.

    PubMed

    Steffen, Yvonne; Jung, Tobias; Klotz, Lars-Oliver; Schewe, Tankred; Grune, Tilman; Sies, Helmut

    2007-04-01

    The action of oxidatively modified low-density lipoprotein (oxLDL) on vascular endothelial cells has been proposed to be a crucial process leading to endothelial dysfunction and atherogenesis. OxLDL was shown here to elicit oxidative stress in bovine aortic endothelial cells or human umbilical vein endothelial cells, as judged by an increase in 2',7'-dichlorofluorescein fluorescence and elevated levels of carbonylated, nitrated, and 2-hydroxynonenal-coupled proteins. These effects were sensitive to apocynin, indicating involvement of NADPH oxidase. A 170-kDa polypeptide carbonylated upon exposure of cells to oxLDL was identified by immunoprecipitation as EGF receptor. Immunocytochemical visualization by confocal microscopy revealed the highest levels of modified proteins in the perinuclear region. Exposure of endothelial cells to oxLDL led to modulation of the expression levels of *NO synthases; the endothelial isoform (eNOS) was down-regulated via proteasomal degradation, whereas the inducible isoform (iNOS) was up-regulated in an enzymatically active state. eNOS protein was found to be both carbonylated and nitrated upon exposure of cells to oxLDL. iNOS contributed to the generation of modified proteins as judged by the effects of the selective inhibitor L-NIO. These oxLDL-elicited changes in vascular endothelial cells described were suppressed by (-)-epicatechin, a dietary polyphenol, which inhibited NADPH oxidase activity in these cells.

  5. Emotional scenes and facial expressions elicit different psychophysiological responses.

    PubMed

    Alpers, Georg W; Adolph, Dirk; Pauli, Paul

    2011-06-01

    We examined if emotional faces elicit physiological responses similar to pictures of emotional scenes. Forty one students viewed emotional scenes (negative, neutral, and positive) and emotional faces (angry, neutral, and happy). Heart rate, orbicularis oculi and electrodermal activity were measured continuously, and the startle reflex was elicited. Although the patterns of valence and arousal ratings were comparable, physiological response patterns differed. For scenes we replicated the valence-specific modulation of the startle response, heart rate deceleration, and the arousal-related modulation of the electrodermal response. In contrast, for faces we found valence-specific modulation only for the electrodermal response, but the startle and heart rate deceleration were modulated by arousal. Although arousal differences may account for some differences in physiological responding this shows that not all emotional material that is decoded similarly leads to the same psychophysiological output. 2011 Elsevier B.V. All rights reserved.

  6. Elicitation of hypersensitive responses in Nicotiana glutinosa by the suppressor of RNA silencing protein P0 from poleroviruses.

    PubMed

    Wang, Ken-Der; Empleo, Roman; Nguyen, Tan Tri V; Moffett, Peter; Sacco, Melanie Ann

    2015-06-01

    Plant disease resistance (R) proteins that confer resistance to viruses recognize viral gene products with diverse functions, including viral suppressors of RNA silencing (VSRs). The P0 protein from poleroviruses is a VSR that targets the ARGONAUTE1 (AGO1) protein for degradation, thereby disrupting RNA silencing and antiviral defences. Here, we report resistance against poleroviruses in Nicotiana glutinosa directed against Turnip yellows virus (TuYV) and Potato leafroll virus (PLRV). The P0 proteins from TuYV (P0(T) (u) ), PLRV (P0(PL) ) and Cucurbit aphid-borne yellows virus (P0(CA) ) were found to elicit a hypersensitive response (HR) in N. glutinosa accession TW59, whereas other accessions recognized P0(PL) only. Genetic analysis showed that recognition of P0(T) (u) by a resistance gene designated RPO1 (Resistance to POleroviruses 1) is inherited as a dominant allele. Expression of P0 from a Potato virus X (PVX) expression vector transferred recognition to the recombinant virus on plants expressing RPO1, supporting P0 as the unique Polerovirus factor eliciting resistance. The induction of HR required a functional P0 protein, as P0(T) (u) mutants with substitutions in the F-box motif that abolished VSR activity were unable to elicit HR. We surmised that the broad P0 recognition seen in TW59 and the requirement for the F-box protein motif could indicate detection of P0-induced AGO1 degradation and disruption of RNA silencing; however, other viral silencing suppressors, including the PVX P25 that also causes AGO1 degradation, failed to elicit HR in N. glutinosa. Investigation of P0 elicitation of RPO1 could provide insight into P0 activities within the cell that trigger resistance. © 2014 BSPP AND JOHN WILEY & SONS LTD.

  7. Induction of Cd36 expression elicited by fish oil PUFA in spontaneously hypertensive rats.

    PubMed

    Alexander Aguilera, Alfonso; Hernández Díaz, Guillermo; Lara Barcelata, Martín; Angulo Guerrero, Ofelia; Oliart Ros, Rosa M

    2006-11-01

    Cd36 is an integral membrane glycoprotein expressed on the surface of cells active in fatty acid metabolism (adipocytes, muscle cells, platelets, monocytes, heart and intestine cells). This protein plays diverse functions including uptake of long-chain fatty acids and oxidized low-density lipoproteins. A recent report demonstrates that Cd36 deficiency underlies insulin resistance, defective fatty acid metabolism and hypertriglyceridemia in spontaneously hypertensive rats (SHRs). Cd36 is a tightly regulated protein whose expression is modulated through peroxisome proliferator-activated receptor (PPAR) transcription factors, by conditions that alter lipid metabolism such as diabetes mellitus and high-fat feeding. The purpose of this study was to evaluate the effect of dietary fish oil, rich in n-3 polyunsaturated fatty acids (PUFAs), on metabolic parameters and on the expression levels of Cd36 in adipose tissue in the SHR. Spontaneously hypertensive rats showed lower Cd36 mRNA levels when compared to Kyoto-Wistar (KW) rats (control). After 6 weeks of fish oil (FO) administration, this group of SHRs (FO-SHR) presented increased levels of Cd36 mRNA, concomitantly with decreased insulin, free fatty acids (FFAs), triglycerides, cholesterol, LDL, HDL, total lipids and blood pressure, in comparison to control rats that received a corn-canola oil diet. The study confirmed the beneficial effects of fish oil administration on the metabolic syndrome, suggesting that the induction of Cd36 expression could be one of the molecular mechanisms elicited by fish oil PUFAs.

  8. Protein expression-yeast.

    PubMed

    Nielsen, Klaus H

    2014-01-01

    Yeast is an excellent system for the expression of recombinant eukaryotic proteins. Both endogenous and heterologous proteins can be overexpressed in yeast (Phan et al., 2001; Ton and Rao, 2004). Because yeast is easy to manipulate genetically, a strain can be optimized for the expression of a specific protein. Many eukaryotic proteins contain posttranslational modifications that can be performed in yeast but not in bacterial expression systems. In comparison with mammalian cell culture expression systems, growing yeast is both faster and less expensive, and large-scale cultures can be performed using fermentation. While several different yeast expression systems exist, this chapter focuses on the budding yeast Saccharomyces cerevisiae and will briefly describe some options to consider when selecting vectors and tags to be used for protein expression. Throughout this chapter, the expression and purification of yeast eIF3 is shown as an example alongside a general scheme outline.

  9. Hypoxia elicits broad and systematic changes in protein subcellular localization

    PubMed Central

    Henke, Robert Michael; Dastidar, Ranita Ghosh; Shah, Ajit; Cadinu, Daniela; Yao, Xiao; Hooda, Jagmohan

    2011-01-01

    Oxygen provides a crucial energy source in eukaryotic cells. Hence, eukaryotes ranging from yeast to humans have developed sophisticated mechanisms to respond to changes in oxygen levels. Regulation of protein localization, like protein modifications, can be an effective mechanism to control protein function and activity. However, the contribution of protein localization in oxygen signaling has not been examined on a genomewide scale. Here, we examine how hypoxia affects protein distribution on a genomewide scale in the model eukaryote, the yeast Saccharomyces cerevisiae. We demonstrate, by live cell imaging, that hypoxia alters the cellular distribution of 203 proteins in yeast. These hypoxia-redistributed proteins include an array of proteins with important functions in various organelles. Many of them are nuclear and are components of key regulatory complexes, such as transcriptional regulatory and chromatin remodeling complexes. Under hypoxia, these proteins are synthesized and retained in the cytosol. Upon reoxygenation, they relocalize effectively to their normal cellular compartments, such as the nucleus, mitochondria, endoplasmic reticulum, and cell periphery. The resumption of the normal cellular locations of many proteins can occur even when protein synthesis is inhibited. Furthermore, we show that the changes in protein distribution induced by hypoxia follow a slower trajectory than those induced by reoxygenation. These results show that the regulation of protein localization is a common and potentially dominant mechanism underlying oxygen signaling and regulation. These results may have broad implications in understanding oxygen signaling and hypoxia responses in higher eukaryotes such as humans. PMID:21753182

  10. Naturalistic observations of elicited expressive communication of children with autism: an analysis of teacher instructions.

    PubMed

    Chiang, Hsu-Min

    2009-03-01

    This study observed expressive communication of 17 Australian and 15 Taiwanese children with autism who were mute or had limited spoken language during 2 hour regular school routines and analyzed teacher instructions associated with elicited expressive communication. Results indicated: (a) the frequency of occurrence of elicited expressive communication was very low; (b) the incidence of elicited expressive communication was negatively correlated with autism severity; (c) verbal prompt and a combination of verbal prompt and modeling were the most common types of teacher instruction and the use of physical prompt was a rate event; (d) modeling and verbal prompt were positively correlated with speech and unaided augmentative and alternative communication (AAC) and a combination of verbal prompt and modeling was positively associated with aided AAC; and (e) modeling, verbal prompt, and a combination of modeling and verbal prompt were positively correlated with requesting function and commenting function was positively correlated with modeling and verbal prompt.

  11. Recombinant Sheep Pox Virus Proteins Elicit Neutralizing Antibodies

    PubMed Central

    Chervyakova, Olga V.; Zaitsev, Valentin L.; Iskakov, Bulat K.; Tailakova, Elmira T.; Strochkov, Vitaliy M.; Sultankulova, Kulyaisan T.; Sandybayev, Nurlan T.; Stanbekova, Gulshan E.; Beisenov, Daniyar K.; Abduraimov, Yergali O.; Mambetaliyev, Muratbay; Sansyzbay, Abylay R.; Kovalskaya, Natalia Y.; Nemchinov, Lev. G.; Hammond, Rosemarie W.

    2016-01-01

    The aim of this work was to evaluate the immunogenicity and neutralizing activity of sheep pox virus (SPPV; genus Capripoxvirus, family Poxviridae) structural proteins as candidate subunit vaccines to control sheep pox disease. SPPV structural proteins were identified by sequence homology with proteins of vaccinia virus (VACV) strain Copenhagen. Four SPPV proteins (SPPV-ORF 060, SPPV-ORF 095, SPPV-ORF 117, and SPPV-ORF 122), orthologs of immunodominant L1, A4, A27, and A33 VACV proteins, respectively, were produced in Escherichia coli. Western blot analysis revealed the antigenic and immunogenic properties of SPPV-060, SPPV-095, SPPV-117 and SPPV-122 proteins when injected with adjuvant into experimental rabbits. Virus-neutralizing activity against SPPV in lamb kidney cell culture was detected for polyclonal antisera raised to SPPV-060, SPPV-117, and SPPV-122 proteins. To our knowledge, this is the first report demonstrating the virus-neutralizing activities of antisera raised to SPPV-060, SPPV-117, and SPPV-122 proteins. PMID:27338444

  12. An adenoviral vector expressing lipoprotein A, a major antigen of Mycoplasma mycoides subspecies mycoides, elicits robust immune responses in mice.

    PubMed

    Carozza, Marlène; Rodrigues, Valérie; Unterfinger, Yves; Galea, Sandra; Coulpier, Muriel; Klonjkowski, Bernard; Thiaucourt, François; Totté, Philippe; Richardson, Jennifer

    2015-01-01

    Contagious bovine pleuropneumonia (CBPP), caused by Mycoplasma mycoides subsp. mycoides small colony type (MmmSC), is a devastating respiratory disease of cattle. In sub-Saharan Africa, where CBPP is enzootic, live attenuated vaccines are deployed but afford only short-lived protection. In cattle, recovery from experimental MmmSC infection has been associated with the presence of CD4(+) T lymphocytes that secrete interferon gamma in response to MmmSC, and in particular to the lipoprotein A (LppA) antigen. In an effort to develop a better vaccine against CBPP, a viral vector (Ad5-LppA) that expressed LppA was generated from human adenovirus type 5. The LppA-specific immune responses elicited by the Ad5-LppA vector were evaluated in mice, and compared to those elicited by recombinant LppA formulated with a potent adjuvant. Notably, a single administration of Ad5-LppA, but not recombinant protein, sufficed to elicit a robust LppA-specific humoral response. After a booster administration, both vector and recombinant protein elicited strong LppA-specific humoral and cell-mediated responses. Ex vivo stimulation of splenocytes induced extensive proliferation of CD4(+) T cells for mice immunized with vector or protein, and secretion of T helper 1-associated and proinflammatory cytokines for mice immunized with Ad5-LppA. Our study - by demonstrating the potential of a viral-vectored prototypic vaccine to elicit prompt and robust immune responses against a major antigen of MmmSC - represents a first step in developing a recombinant vaccine against CBPP.

  13. Systematic identification of proteins that elicit drug side effects

    PubMed Central

    Kuhn, Michael; Al Banchaabouchi, Mumna; Campillos, Monica; Jensen, Lars Juhl; Gross, Cornelius; Gavin, Anne-Claude; Bork, Peer

    2013-01-01

    Side effect similarities of drugs have recently been employed to predict new drug targets, and networks of side effects and targets have been used to better understand the mechanism of action of drugs. Here, we report a large-scale analysis to systematically predict and characterize proteins that cause drug side effects. We integrated phenotypic data obtained during clinical trials with known drug–target relations to identify overrepresented protein–side effect combinations. Using independent data, we confirm that most of these overrepresentations point to proteins which, when perturbed, cause side effects. Of 1428 side effects studied, 732 were predicted to be predominantly caused by individual proteins, at least 137 of them backed by existing pharmacological or phenotypic data. We prove this concept in vivo by confirming our prediction that activation of the serotonin 7 receptor (HTR7) is responsible for hyperesthesia in mice, which, in turn, can be prevented by a drug that selectively inhibits HTR7. Taken together, we show that a large fraction of complex drug side effects are mediated by individual proteins and create a reference for such relations. PMID:23632385

  14. Differences between Spontaneous and Elicited Expressive Communication in Children with Autism

    ERIC Educational Resources Information Center

    Chiang, Hsu-Min

    2009-01-01

    The purpose of this study was to investigate the differences between spontaneous and elicited expressive communication in Australian and Taiwanese children with autism who were nonverbal or had limited speech. Thirty-four children with autism (17 Australian and 17 Taiwanese children) participated in this study. Each participant was observed for 2…

  15. Cytofluorometric Quantification of Cell Death Elicited by NLR Proteins.

    PubMed

    Sica, Valentina; Manic, Gwenola; Kroemer, Guido; Vitale, Ilio; Galluzzi, Lorenzo

    2016-01-01

    Nucleotide-binding domain and leucine-rich repeat containing (NLR) proteins, also known as NOD-like receptors, are critical components of the molecular machinery that senses intracellular danger signals to initiate an innate immune response against invading pathogens or endogenous sources of hazard. The best characterized effect of NLR signaling is the secretion of various cytokines with immunostimulatory effects, including interleukin (IL)-1β and IL-18. Moreover, at least under specific circumstances, NLRs can promote regulated variants of cell death. Here, we detail two protocols for the cytofluorometric quantification of cell death-associated parameters that can be conveniently employed to assess the lethal activity of specific NLRs or their ligands.

  16. Eliciting the Functional Taxonomy from protein annotations and taxa.

    PubMed

    Falda, Marco; Lavezzo, Enrico; Fontana, Paolo; Bianco, Luca; Berselli, Michele; Formentin, Elide; Toppo, Stefano

    2016-08-18

    The advances of omics technologies have triggered the production of an enormous volume of data coming from thousands of species. Meanwhile, joint international efforts like the Gene Ontology (GO) consortium have worked to provide functional information for a vast amount of proteins. With these data available, we have developed FunTaxIS, a tool that is the first attempt to infer functional taxonomy (i.e. how functions are distributed over taxa) combining functional and taxonomic information. FunTaxIS is able to define a taxon specific functional space by exploiting annotation frequencies in order to establish if a function can or cannot be used to annotate a certain species. The tool generates constraints between GO terms and taxa and then propagates these relations over the taxonomic tree and the GO graph. Since these constraints nearly cover the whole taxonomy, it is possible to obtain the mapping of a function over the taxonomy. FunTaxIS can be used to make functional comparative analyses among taxa, to detect improper associations between taxa and functions, and to discover how functional knowledge is either distributed or missing. A benchmark test set based on six different model species has been devised to get useful insights on the generated taxonomic rules.

  17. Eliciting the Functional Taxonomy from protein annotations and taxa

    PubMed Central

    Falda, Marco; Lavezzo, Enrico; Fontana, Paolo; Bianco, Luca; Berselli, Michele; Formentin, Elide; Toppo, Stefano

    2016-01-01

    The advances of omics technologies have triggered the production of an enormous volume of data coming from thousands of species. Meanwhile, joint international efforts like the Gene Ontology (GO) consortium have worked to provide functional information for a vast amount of proteins. With these data available, we have developed FunTaxIS, a tool that is the first attempt to infer functional taxonomy (i.e. how functions are distributed over taxa) combining functional and taxonomic information. FunTaxIS is able to define a taxon specific functional space by exploiting annotation frequencies in order to establish if a function can or cannot be used to annotate a certain species. The tool generates constraints between GO terms and taxa and then propagates these relations over the taxonomic tree and the GO graph. Since these constraints nearly cover the whole taxonomy, it is possible to obtain the mapping of a function over the taxonomy. FunTaxIS can be used to make functional comparative analyses among taxa, to detect improper associations between taxa and functions, and to discover how functional knowledge is either distributed or missing. A benchmark test set based on six different model species has been devised to get useful insights on the generated taxonomic rules. PMID:27534507

  18. The interplay of elicitation and evaluation of trait-expressive behavior: Evidence in assessment center exercises.

    PubMed

    Lievens, Filip; Schollaert, Eveline; Keen, Gert

    2015-07-01

    In assessment centers (ACs), research on eliciting candidate behavior and evaluating candidate behavior have largely followed independent paths. This study integrates trait activation and trait rating models to posit hypotheses about the effects of behavior elicitation via situational cues on key assessor observation and rating variables. To test the hypotheses, a series of experimental and field studies are conducted. Only when trait-expressive behavior activation and evaluation models work in conjunction, increases in observability are coupled with increases in the interrater reliability, convergent validity, discriminant validity, and accuracy of AC ratings. Implications of these findings for AC theory and practice are formulated.

  19. Magnaporthe oryzae-Secreted Protein MSP1 Induces Cell Death and Elicits Defense Responses in Rice.

    PubMed

    Wang, Yiming; Wu, Jingni; Kim, Sang Gon; Tsuda, Kenichi; Gupta, Ravi; Park, Sook-Young; Kim, Sun Tae; Kang, Kyu Young

    2016-04-01

    The Magnaporthe oryzae snodprot1 homolog (MSP1), secreted by M. oryzae, is a cerato-platanin family protein. msp1-knockout mutants have reduced virulence on barley leaves, indicating that MSP1 is required for the pathogenicity of rice blast fungus. To investigate the functional roles of MSP1 and its downstream signaling in rice, recombinant MSP1 was produced in Escherichia coli and was assayed for its functionality. Application of MSP1 triggered cell death and elicited defense responses in rice. MSP1 also induced H2O2 production and autophagic cell death in both suspension-cultured cells and rice leaves. One or more protein kinases triggered cell death, jasmonic acid and abscisic acid enhanced cell death, while salicylic acid suppressed it. We demonstrated that the secretion of MSP1 into the apoplast is a prerequisite for triggering cell death and activating defense-related gene expression. Furthermore, pretreatment of rice with a sublethal MSP1 concentration potentiated resistance to the pathogen. Taken together, our results showed that MSP1 induces a high degree of cell death in plants, which might be essential for its virulence. Moreover, rice can recognize MSP1, resulting in the induction of pathogen-associated molecular pattern-triggered immunity.

  20. Immune response and protective profile elicited by a multi-epitope chimeric protein derived from Leptospira interrogans.

    PubMed

    Fernandes, Luis G V; Teixeira, Aline F; Filho, Antonio F S; Souza, Gisele O; Vasconcellos, Silvio A; Heinemann, Marcos B; Romero, Eliete C; Nascimento, Ana L T O

    2017-04-01

    Pathogenic Leptospira is the causative agent of leptospirosis, a widely disseminated disease of human and veterinary concern. The development of vaccines that elicit cross-protective immunity through multiple leptospiral serovars has long been pursued. The aim of this study was to develop a novel chimeric multi-epitope fusion antigen, containing sequences of previously studied outer membrane proteins (OMPs) of Leptospira. The chimeric protein was designed based on the amino acid sequences of the LigA, Mce, Lsa45, OmpL1, and LipL41 proteins, cloned into pAE vector, the protein expressed in Escherichia coli, and its immune response evaluated in the hamster infection model. The recombinant chimeric protein (rChi) was recognized by antibodies present in serum samples of confirmed cases of human leptospirosis and experimentally infected hamsters, demonstrating that the rChi protein participates in the immune response activation during infection. However, despite high antibody titers achieved when the rChi protein was administered with either Alhydrogel or Bordetella pertussis monophosphoryl lipid A (MPLA), only 50% of the hamsters were protected against infection. Although a complete characterization of the immune response elicited by rChi/adjuvant in hamsters is required, it is believed that the construction of chimeric genes is an important attempt towards the generation of an effective vaccine against leptospirosis. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

  1. Distinct contractile and cytoskeletal protein patterns in the Antarctic midge are elicited by desiccation and rehydration.

    PubMed

    Li, Aiqing; Benoit, Joshua B; Lopez-Martinez, Giancarlo; Elnitsky, Michael A; Lee, Richard E; Denlinger, David L

    2009-05-01

    Desiccation presents a major challenge for the Antarctic midge, Belgica antarctica. In this study, we use proteomic profiling to evaluate protein changes in the larvae elicited by dehydration and rehydration. Larvae were desiccated at 75% relative humidity (RH) for 12 h to achieve a body water loss of 35%, approximately half of the water that can be lost before the larvae succumb to dehydration. To evaluate the rehydration response, larvae were first desiccated, then rehydrated for 6 h at 100% RH and then in water for 6 h. Controls were held continuously at 100% RH. Protein analysis was performed using 2-DE and nanoscale capillary LC/MS/MS. Twenty-four identified proteins changed in abundance in response to desiccation: 16 were more abundant and 8 were less abundant; 84% of these proteins were contractile or cytoskeletal proteins. Thirteen rehydration-regulated proteins were identified: 8 were more abundant and 5 were less abundant, and 69% of these proteins were also contractile or cytoskeletal proteins. Additional proteins responsive to desiccation and rehydration were involved in functions including stress responses, energy metabolism, protein synthesis, glucogenesis and membrane transport. We conclude that the major protein responses elicited by both desiccation and rehydration are linked to body contraction and cytoskeleton rearrangements.

  2. In Vivo Visualization of Tumor Antigen-containing Microparticles Generated in Fluorescent-protein-elicited Immunity

    PubMed Central

    Yang, Fei; Liu, Shun; Liu, Xiuli; Liu, Lei; Luo, Meijie; Qi, Shuhong; Xu, Guoqiang; Qiao, Sha; Lv, Xiaohua; Li, Xiangning; Fu, Ling; Luo, Qingming; Zhang, Zhihong

    2016-01-01

    In vivo optical spatio-temporal imaging of the tumor microenvironment is useful to explain how tumor immunotherapies work. However, the lack of fluorescent antigens with strong immunogenicity makes it difficult to study the dynamics of how tumors are eliminated by any given immune response. Here, we develop an effective fluorescent model antigen based on the tetrameric far-red fluorescent protein KatushkaS158A (tfRFP), which elicits both humoral and cellular immunity. We use this fluorescent antigen to visualize the dynamic behavior of immunocytes as they attack and selectively eliminate tfRFP-expressing tumors in vivo; swarms of immunocytes rush toward tumors with high motility, clusters of immunocytes form quickly, and numerous antigen-antibody complexes in the form of tfRFP+ microparticles are generated in the tumor areas and ingested by macrophages in the tumor microenvironment. Therefore, tfRFP, as both a model antigen and fluorescent reporter, is a useful tool to visualize specific immune responses in vivo. PMID:27375792

  3. Analysis of Globodera rostochiensis effectors reveals conserved functions of SPRYSEC proteins in suppressing and eliciting plant immune responses

    PubMed Central

    Ali, Shawkat; Magne, Maxime; Chen, Shiyan; Obradovic, Natasa; Jamshaid, Lubna; Wang, Xiaohong; Bélair, Guy; Moffett, Peter

    2015-01-01

    Potato cyst nematodes (PCNs), including Globodera rostochiensis (Woll.), are important pests of potato. Plant parasitic nematodes produce multiple effector proteins, secreted from their stylets, to successfully infect their hosts. These include proteins delivered to the apoplast and to the host cytoplasm. A number of effectors from G. rostochiensis predicted to be delivered to the host cytoplasm have been identified, including several belonging to the secreted SPRY domain (SPRYSEC) family. SPRYSEC proteins are unique to members of the genus Globodera and have been implicated in both the induction and the repression of host defense responses. We have tested the properties of six different G. rostochiensis SPRYSEC proteins by expressing them in Nicotiana benthamiana and N. tabacum. We have found that all SPRYSEC proteins tested are able to suppress defense responses induced by NB-LRR proteins as well as cell death induced by elicitors, suggesting that defense repression is a common characteristic of members of this effector protein family. At the same time, GrSPRYSEC-15 elicited a defense responses in N. tabacum, which was found to be resistant to a virus expressing GrSPRYSEC-15. These results suggest that SPRYSEC proteins may possess characteristics that allow them to be recognized by the plant immune system. PMID:26322064

  4. Induction of sesquiterpenes, phytoesterols and extracellular pathogenesis-related proteins in elicited cell cultures of Capsicum annuum.

    PubMed

    Sabater-Jara, Ana Belén; Almagro, Lorena; Belchí-Navarro, Sarai; Ferrer, María Angeles; Barceló, Alfonso Ros; Pedreño, María Angeles

    2010-10-15

    Capsicum annuum suspension cell cultures were used to evaluate the effect of cyclodextrins and methyl jasmonate as elicitors of defence responses. The induced defence responses included the accumulation of sesquiterpenes and phytosterols and the activation of pathogenesis-related proteins, leading to reinforcement and modification of the cell wall architecture during elicitation and protection cells against biotic stress. The results showed that the addition of both cyclodextrins and methyl jasmonate induced the biosynthesis of two sesquiterpenes, aromadendrene and solavetivone. This response was clearly synergistic since the increase in the levels of these compounds was much greater in the presence of both elicitors than when they were used separately. The biosynthesis of phytosterols was also induced in the combined treatment, as the result of an additive effect. Likewise, the exogenous application of methyl jasmonate induced the accumulation of pathogenesis-related proteins. The analysis of the extracellular proteome showed the presence of amino acid sequences homologous to PR1 and 4, NtPRp27-like proteins and class I chitinases, peroxidases and the hydrolytic enzymes LEXYL1 and 2, arabinosidases, pectinases, nectarin IV and leucin-rich repeat protein, which suggests that methyl jasmonate plays a role in mediating defence-related gene product expression in C. annuum. Apart from these methyl jamonate-induced proteins, other PR proteins were found in both the control and elicited cell cultures of C. annuum. These included class IV chitinases, beta-1,3-glucanases, thaumatin-like proteins and peroxidases, suggesting that their expression is mainly constitutive since they are involved in growth, development and defence processes. Copyright (c) 2010 Elsevier GmbH. All rights reserved.

  5. Nesfatin-1 induces Fos expression and elicits dipsogenic responses in subfornical organ.

    PubMed

    Moreau, Jason M; Ciriello, John

    2013-08-01

    Nesfatin-1 (Nes-1), an 82-amino acid protein cleaved from nucleobindin-2, has been suggested to play a role in ingestive behaviors. Intracerebroventricular (icv) injections of Nes-1 reduce water intake, although the sites of action for this effect are not known. Two series of experiments were done to identify potential sites of action of Nes-1 in drinking behavior. In the first series, icv injections of Nes-1 were made in urethane-anesthetized rats to investigate the distribution of neurons containing Fos-like immunoreactivity (Fos-ir) within the forebrain. Circumventricular organs, including subfornical organ (SFO), were found to contain neurons expressing Fos-ir. Additionally, several hypothalamic, thalamic and limbic nuclei also contained Fos-labeled neurons. As SFO is a pivotal central site in the regulation of water intake, a second series of experiments was done to investigate the role of direct injections of Nes-1 into SFO on water intake in conscious, freely moving rats. Nes-1 (2pmol) injections into SFO induced an increase in water intake compared to vehicle injections. However, when food was made available for ingestion after the Nes-1 injection, the dipsogenic effects of Nes-1 were attenuated. Additionally, the drinking response to Nes-1 was found to be more potent than that observed after injections of ANG II into SFO. Neither simultaneous injections ANG II nor the ANG II type-1 receptor blocker losartan affected the Nes-1 dipsogenic response. Taken together, these results suggest that Nes-1 is a potent dipsogenic agent in SFO, and that Nes-1 may act independently of the SFO angiotensinergic system to elicit the dipsogenic effect. Copyright © 2013 Elsevier B.V. All rights reserved.

  6. Autoinduction of Protein Expression

    PubMed Central

    Fox, Brian G.; Blommel, Paul G.

    2017-01-01

    This unit contains protocols for the use of lactose-derived autoinduction in Escherichia coli. The protocols allow for reproducible expression trials to be undertaken with minimal user intervention. A basic protocol covers production of unlabeled proteins for functional studies. Alternate protocols for selenomethionine labeling for X-ray structural studies, and multi-well plate growth for screening and optimization are also included. PMID:19365792

  7. Protein expression in liposomes.

    PubMed

    Oberholzer, T; Nierhaus, K H; Luisi, P L

    1999-08-02

    Compartmentalization is one of the key steps in the evolution of cellular structures and, so far, only few attempts have been made to model this kind of "compartmentalized chemistry" using liposomes. The present work shows that even such complex reactions as the ribosomal synthesis of polypeptides can be carried out in liposomes. A method is described for incorporating into 1-palmitoyl-2-oleoyl-sn-3-phosphocholine (POPC) liposomes the ribosomal complex together with the other components necessary for protein expression. Synthesis of poly(Phe) in the liposomes is monitored by trichloroacetic acid of the (14)C-labelled products. Control experiments carried out in the absence of one of the ribosomal subunits show by contrast no significant polypeptide expression. This methodology opens up the possibility of using liposomes as minimal cell bioreactors with growing degree of synthetic complexity, which may be relevant for the field of origin of life as well as for biotechnological applications. Copyright 1999 Academic Press.

  8. A novel, broad spectrum therapeutic HPV vaccine targeting the E7 proteins of HPV16, 18, 31, 45 and 52 that elicits potent E7-specific CD8T cell immunity and regression of large, established, E7-expressing TC-1 tumors.

    PubMed

    Wick, Darin A; Webb, John R

    2011-10-13

    Persistent infection by high risk genotypes of human papillomavirus (HPV) is the cause of cervical cancer, which remains one of the most common cancers among women worldwide. In addition, there is a growing appreciation that high risk HPVs are associated with a number of other cancers including anogenital cancers as well as a subset of head and neck cancers. Recently, prophylactic HPV vaccines targeting the two most prevalent high risk HPVs (HPV16 and HPV18) have been deployed in large-scale vaccination campaigns. However, the extent to which these prophylactic vaccines confer protection against other high risk HPV genotypes is largely unknown and prophylactic vaccines have been shown to be ineffective against pre-existing infection. Thus there continues to be an urgent need for effective therapeutic vaccines against HPV. The E7 protein of HPV16 has been widely studied as a target for therapeutic vaccines in HPV-associated cancer settings because HPV16 is the most prevalent of the high risk HPV genotypes. However, HPV16 accounts for only about 50% of cervical cancers and there are at least 15 other high risk HPVs that are known to be oncogenic. We have developed a novel, broad-spectrum, therapeutic vaccine (Pentarix) directed at the E7 proteins from five of the most prevalent high-risk genotypes of HPV worldwide (HPV16, 18, 31, 45 and 52) that together account for more than 80% of all HPV-associated cancers. Pentarix is a recombinant protein-based vaccine that elicits strong, multi-genotype specific CD8 T cell immunity when administered to mice in combination with adjuvants comprised of agonists of the TLR3 or TLR9 family of innate immune receptors. Furthermore, large, established E7-expressing TC-1 tumors undergo rapid and complete regression after therapeutic vaccination of mice with Pentarix. Together, these data suggest that Pentarix may be of clinical value for patients with E7-positive, HPV-associated precancerous lesions or malignant disease.

  9. The effects of anesthetics on cortical spreading depression elicitation and c-fos expression in rats.

    PubMed

    Kitahara, Y; Taga, K; Abe, H; Shimoji, K

    2001-01-01

    The effects of anesthetics on the generation of cortical spreading depression (CSD) were investigated. Volatile anesthetics halothane, isoflurane, sevoflurane (0.5, 1.0, and 2.0 MAC), and the intravenous anesthetic pentobarbital were studied. Cortical spreading depression was induced by 3M-KCl applied to a surface of brain cortex for 30 minutes. Direct current (DC) potential was recorded, and the number, amplitude, and duration of CSDs were observed. With increasing concentrations of each volatile anesthetic, there was a dose-related reduction in CSD frequency but not in CSD amplitude. At 2.0 MAC of sevoflurane the suppression of CSD was less than with the other volatile anesthetics. In addition, the influence of anesthetics on expression of c-fos mRNA was investigated. Additional animals anesthetized by isoflurane or sevoflurane were studied. Five CSDs were elicited by electric stimulation (0.5 mV, 1 second) in each animal. In situ hybridization with 35S-labeled oligonucleotides was used to evaluate the level of c-fos mRNA. The expression of c-fos was observed in the hemisphere in which CSD was elicited, but there was no difference in expression of c-fos among the groups. We conclude that volatile anesthetics can induce suppression of CSD elicitation in a dose dependent manner, but that at high concentrations sevoflurane is significantly less effective than other volatile agents. Pentobarbital has the least effect on KCl-induced CSD. These data suggest that the choice of anesthetics can impact the results of studies examining membrane depolarization and the ionic changes initiated by CSD.

  10. Videos of conspecifics elicit interactive looking patterns and facial expressions in monkeys.

    PubMed

    Mosher, Clayton P; Zimmerman, Prisca E; Gothard, Katalin M

    2011-08-01

    A broader understanding of the neural basis of social behavior in primates requires the use of species-specific stimuli that elicit spontaneous, but reproducible and tractable behaviors. In this context of natural behaviors, individual variation can further inform about the factors that influence social interactions. To approximate natural social interactions similar to those documented by field studies, we used unedited video footage to induce in viewer monkeys spontaneous facial expressions and looking patterns in the laboratory setting. Three adult male monkeys (Macaca mulatta), previously behaviorally and genetically (5-HTTLPR) characterized, were monitored while they watched 10 s video segments depicting unfamiliar monkeys (movie monkeys) displaying affiliative, neutral, and aggressive behaviors. The gaze and head orientation of the movie monkeys alternated between "averted" and "directed" at the viewer. The viewers were not reinforced for watching the movies, thus their looking patterns indicated their interest and social engagement with the stimuli. The behavior of the movie monkey accounted for differences in the looking patterns and facial expressions displayed by the viewers. We also found multiple significant differences in the behavior of the viewers that correlated with their interest in these stimuli. These socially relevant dynamic stimuli elicited spontaneous social behaviors, such as eye-contact induced reciprocation of facial expression, gaze aversion, and gaze following, that were previously not observed in response to static images. This approach opens a unique opportunity to understanding the mechanisms that trigger spontaneous social behaviors in humans and nonhuman primates.

  11. Videos of conspecifics elicit interactive looking patterns and facial expressions in monkeys

    PubMed Central

    Mosher, Clayton P.; Zimmerman, Prisca E.; Gothard, Katalin M.

    2014-01-01

    A broader understanding of the neural basis of social behavior in primates requires the use of species-specific stimuli that elicit spontaneous, but reproducible and tractable behaviors. In this context of natural behaviors, individual variation can further inform about the factors that influence social interactions. To approximate natural social interactions similar to those documented by field studies, we used unedited video footage to induce in viewer monkeys spontaneous facial expressions and looking patterns in the laboratory setting. Three adult male monkeys, previously behaviorally and genetically (5-HTTLPR) characterized (Gibboni et al., 2009), were monitored while they watched 10 s video segments depicting unfamiliar monkeys (movie monkeys) displaying affiliative, neutral, and aggressive behaviors. The gaze and head orientation of the movie monkeys alternated between ‘averted’ and ‘directed’ at the viewer. The viewers were not reinforced for watching the movies, thus their looking patterns indicated their interest and social engagement with the stimuli. The behavior of the movie monkey accounted for differences in the looking patterns and facial expressions displayed by the viewers. We also found multiple significant differences in the behavior of the viewers that correlated with their interest in these stimuli. These socially relevant dynamic stimuli elicited spontaneous social behaviors, such as eye-contact induced reciprocation of facial expression, gaze aversion, and gaze following, that were previously not observed in response to static images. This approach opens a unique opportunity to understanding the mechanisms that trigger spontaneous social behaviors in humans and non-human primates. PMID:21688888

  12. Critical molecular regions for elicitation of the sweetness of the sweet-tasting protein, thaumatin I.

    PubMed

    Ohta, Keisuke; Masuda, Tetsuya; Ide, Nobuyuki; Kitabatake, Naofumi

    2008-07-01

    Thaumatin is an intensely sweet-tasting protein. To identify the critical amino acid residue(s) responsible for elicitation of the sweetness of thaumatin, we prepared mutant thaumatin proteins, using Pichia pastoris, in which alanine residues were substituted for lysine or arginine residues, and the sweetness of each mutant protein was evaluated by sensory analysis in humans. Four lysine residues (K49, K67, K106 and K163) and three arginine residues (R76, R79 and R82) played significant roles in thaumatin sweetness. Of these residues, K67 and R82 were particularly important for eliciting the sweetness. We also prepared two further mutant thaumatin I proteins: one in which an arginine residue was substituted for a lysine residue, R82K, and one in which a lysine residue was substituted for an arginine residue, K67R. The threshold value for sweetness was higher for R82K than for thaumatin I, indicating that not only the positive charge but also the structure of the side chain of the arginine residue at position 82 influences the sweetness of thaumatin, whereas only the positive charge of the K67 side chain affects sweetness.

  13. Arginine Vasopressin gene expression changes within the nucleus accumbens during environment elicited cocaine-conditioned response in rats

    PubMed Central

    Rodríguez-Borrero, E.; Rivera-Escalera, F.; Candelas, F.; Montalvo, J.; Muñoz-Miranda, W.J.; Walker, J.R.; Maldonado-Vlaar, C.S.

    2009-01-01

    It is known that changes in gene expression within the nucleus accumbens (NAc) occur during cocaine dependence development. However, identification of specific genes involved in cocaine conditioning awaits further investigation. We conducted a high throughput gene expression profile analysis of the NAc, during different stages of the environment-elicited cocaine conditioning. Rats were assigned to two different environmental conditions. Cocaine conditioned group received a cocaine injection (10 mg/kg, i.p.) prior to being placed in the activity chambers. Control rats received saline injections before being exposed to their environment. Both groups received a saline injection in their home cage. Conditioning training lasted for 10 days. Animals were then re-exposed to their previously paired environments only on day 12 (test session). We found that the gene for arginine vasopressin (AVP) was differentially expressed on experimental subjects during all stages of environment-elicited cocaine conditioning. To further validate our molecular results, biochemical and immunolocalization experiments were conducted. We found the presence of AVP within accumbal fibers and changes in AVP protein levels following cocaine conditioning. Moreover, we tested the effects of accumbal microinfusions of either AVP receptor V1A agonist [pGlu4, Cyt6, Arg8] AVP 4-9 1.0 ng/0.5μl, or V1A antagonist (CH2) 5[Tyr (Me) 2] AVP, 1.0 ng/0.5μl or vehicle solution (0.9% saline solution) during different stages of the cocaine conditioning. Blockade of V1A receptors within the NAc during acquisition interrupted the expression of the conditioned response, while activation leads to an increase in this response. Our findings propose a new role for AVP in cocaine addiction. PMID:19596360

  14. Toward selective elicitation of TH1-controlled vaccination responses: vaccine applications of bacterial surface layer proteins.

    PubMed

    Jahn-Schmid, B; Messner, P; Unger, F M; Sleytr, U B; Scheiner, O; Kraft, D

    1996-01-26

    Bacterial surface layer proteins have been utilized as combined vaccine carrier/adjuvants and offer a number of advantages in these applications. The crystalline protein arrays contain functional groups in precisely defined orientations for coupling of haptens. Conventional applications of S-layer vaccines do not cause observable trauma or side effects. Depending on the nature of the S-layer preparations, antigenic conjugates will induce immune responses of a predominantly cellular or predominantly humoral nature. Immune responses to S-layer-hapten conjugates are also observed following oral/nasal application. In the present contribution, the status of investigations with S-layer conjugates in three main immunological projects is reviewed. In a project aimed at immunotherapy of cancer, conjugates of S-layer with small, tumor-associated oligosaccharides have been found to elicit hapten-specific DTH responses. An enlarged program of chemical synthesis has now been initiated to prepare a complete set of mucin-derived, tumor-associated oligosaccharides and their chemically modified analogues for elicitation of cell-mediated immune responses to certain tumors in humans. In another application, oligosaccharides derived from capsules of Streptococcus pneumoniae type 8 have been linked to S-layer proteins and have been found to elicit protective antibody responses in animals. Most recently, allergen S-layer conjugates have been prepared with the intention to suppress the TH2-directed, IgE-mediated allergic responses to Bet nu 1, the major allergen of birch pollen. In the former two applications, the S-layer vaccine technology appears to offer the versatility needed to direct vaccination responses toward predominant control by TH1 or TH2 lymphocytes to meet the different therapeutic or prophylactic requirements in each case. In the third application, work has progressed to a preliminary stage only.

  15. Immune Response Elicited by DNA Vaccination Using Lactococcus lactis Is Modified by the Production of Surface Exposed Pathogenic Protein

    PubMed Central

    Pontes, Daniela; Azevedo, Marcela; Innocentin, Silvia; Blugeon, Sébastien; Lefévre, François; Azevedo, Vasco; Miyoshi, Anderson; Courtin, Pascal; Chapot-Chartier, Marie-Pierre; Langella, Philippe; Chatel, Jean-Marc

    2014-01-01

    In this study, we compared immune responses elicited by DNA immunization using Lactococcus lactis or L. lactis expressing the Staphylococcus aureus invasin Fibronectin Binding Protein A (FnBPA) at its surface. Both strains carried pValac:BLG, a plasmid containing the cDNA of Beta-Lactoglobulin (BLG), and were designated LL-BLG and LL-FnBPA+ BLG respectively. A TH2 immune response characterized by the secretion of IL-4 and IL-5 in medium of BLG reactivated splenocytes was detected after either oral or intranasal administration of LL-FnBPA+ BLG. In contrast, intranasal administration of LL-BLG elicited a TH1 immune response. After BLG sensitization, mice previously intranasally administered with LL-BLG showed a significantly lower concentration of BLG-specific IgE than the mice non-administered. Altenatively administration of LL-FnBPA+ BLG didn't modify the BLG-specific IgE concentration obtained after sensitization, thus confirming the TH2 orientation of the immune response. To determine if the TH2-skewed immune response obtained with LL-FnBpA+ BLG was FnBPA-specific or not, mice received another L. lactis strain producing a mutated form of the Listeria monocytogenes invasin Internalin A intranasally, allowing thus the binding to murine E-cadherin, and containing pValac:BLG (LL-mInlA+ BLG). As with LL-FnBPA+ BLG, LL-mInlA+ BLG was not able to elicit a TH1 immune response. Furthermore, we observed that these difference were not due to the peptidoglycan composition of the cell wall as LL-FnBPA+ BLG, LL-mInlA+ BLG and LL-BLG strains shared a similar composition. DNA vaccination using LL-BLG elicited a pro-inflammatory TH1 immune response while using LL-FnBPA+ BLG or LL-mInlA+ BLG elicited an anti-inflammatory TH2 immune response. PMID:24465412

  16. Immune response elicited by DNA vaccination using Lactococcus lactis is modified by the production of surface exposed pathogenic protein.

    PubMed

    Pontes, Daniela; Azevedo, Marcela; Innocentin, Silvia; Blugeon, Sébastien; Lefévre, François; Azevedo, Vasco; Miyoshi, Anderson; Courtin, Pascal; Chapot-Chartier, Marie-Pierre; Langella, Philippe; Chatel, Jean-Marc

    2014-01-01

    In this study, we compared immune responses elicited by DNA immunization using Lactococcus lactis or L. lactis expressing the Staphylococcus aureus invasin Fibronectin Binding Protein A (FnBPA) at its surface. Both strains carried pValac:BLG, a plasmid containing the cDNA of Beta-Lactoglobulin (BLG), and were designated LL-BLG and LL-FnBPA+ BLG respectively. A TH2 immune response characterized by the secretion of IL-4 and IL-5 in medium of BLG reactivated splenocytes was detected after either oral or intranasal administration of LL-FnBPA+ BLG. In contrast, intranasal administration of LL-BLG elicited a TH1 immune response. After BLG sensitization, mice previously intranasally administered with LL-BLG showed a significantly lower concentration of BLG-specific IgE than the mice non-administered. Altenatively administration of LL-FnBPA+ BLG didn't modify the BLG-specific IgE concentration obtained after sensitization, thus confirming the TH2 orientation of the immune response. To determine if the TH2-skewed immune response obtained with LL-FnBpA+ BLG was FnBPA-specific or not, mice received another L. lactis strain producing a mutated form of the Listeria monocytogenes invasin Internalin A intranasally, allowing thus the binding to murine E-cadherin, and containing pValac:BLG (LL-mInlA+ BLG). As with LL-FnBPA+ BLG, LL-mInlA+ BLG was not able to elicit a TH1 immune response. Furthermore, we observed that these difference were not due to the peptidoglycan composition of the cell wall as LL-FnBPA+ BLG, LL-mInlA+ BLG and LL-BLG strains shared a similar composition. DNA vaccination using LL-BLG elicited a pro-inflammatory TH1 immune response while using LL-FnBPA+ BLG or LL-mInlA+ BLG elicited an anti-inflammatory TH2 immune response.

  17. Live attenuated rubella vectors expressing SIV and HIV vaccine antigens replicate and elicit durable immune responses in rhesus macaques

    PubMed Central

    2013-01-01

    Background Live attenuated viruses are among our most potent and effective vaccines. For human immunodeficiency virus, however, a live attenuated strain could present substantial safety concerns. We have used the live attenuated rubella vaccine strain RA27/3 as a vector to express SIV and HIV vaccine antigens because its safety and immunogenicity have been demonstrated in millions of children. One dose protects for life against rubella infection. In previous studies, rubella vectors replicated to high titers in cell culture while stably expressing SIV and HIV antigens. Their viability in vivo, however, as well as immunogenicity and antibody persistence, were unknown. Results This paper reports the first successful trial of rubella vectors in rhesus macaques, in combination with DNA vaccines in a prime and boost strategy. The vectors grew robustly in vivo, and the protein inserts were highly immunogenic. Antibody titers elicited by the SIV Gag vector were greater than or equal to those elicited by natural SIV infection. The antibodies were long lasting, and they were boosted by a second dose of replication-competent rubella vectors given six months later, indicating the induction of memory B cells. Conclusions Rubella vectors can serve as a vaccine platform for safe delivery and expression of SIV and HIV antigens. By presenting these antigens in the context of an acute infection, at a high level and for a prolonged duration, these vectors can stimulate a strong and persistent immune response, including maturation of memory B cells. Rhesus macaques will provide an ideal animal model for demonstrating immunogenicity of novel vectors and protection against SIV or SHIV challenge. PMID:24041113

  18. Mitochondrial respiratory dysfunction-elicited oxidative stress and posttranslational protein modification in mitochondrial diseases.

    PubMed

    Wu, Yu-Ting; Wu, Shi-Bei; Lee, Wan-Yu; Wei, Yau-Huei

    2010-07-01

    Pathogenic mutation in mtDNA and mitochondrial dysfunction are associated with mitochondrial diseases. In this review, we discuss the oxidative stress-elicited mitochondrial protein modifications that may contribute to the pathophysiology of mitochondrial diseases. We demonstrated that excess ROS produced by defective mitochondria could increase the acetylation of microtubule proteins through the suppression of Sirt2, which results in perinuclear distribution of mitochondria in skin fibroblasts of patients with CPEO syndrome. Our recent work showed that mitochondrial dysfunction-induced oxidative stress can disrupt protein degradation system by inhibiting the ubiquitin-proteasome pathway and protease activity in human cells harboring mutant mtDNA. This in turn causes accumulation of aberrant proteins in mitochondria and renders the mutant cells more susceptible to apoptosis induced by oxidative stress. Furthermore, oxidative stress can modulate phosphorylation of mitochondrial proteins, which can affect metabolism in a number of diseases. Taken together, we suggest that oxidative stress-triggered protein modifications and defects in protein turnover play an important role in the pathogenesis and progression of mitochondrial diseases.

  19. Differential Gene Expression Analysis in Polygonum minus Leaf upon 24 h of Methyl Jasmonate Elicitation

    PubMed Central

    Rahnamaie-Tajadod, Reyhaneh; Loke, Kok-Keong; Goh, Hoe-Han; Noor, Normah M.

    2017-01-01

    Polygonum minus is an herbal plant that grows in Southeast Asian countries and traditionally used as medicine. This plant produces diverse secondary metabolites such as phenolic compounds and their derivatives, which are known to have roles in plant abiotic and biotic stress responses. Methyl jasmonate (MeJA) is a plant signaling molecule that triggers transcriptional reprogramming in secondary metabolism and activation of defense responses against many biotic and abiotic stresses. However, the effect of MeJA elicitation on the genome-wide expression profile in the leaf tissue of P. minus has not been well-studied due to the limited genetic information. Hence, we performed Illumina paired-end RNA-seq for de novo reconstruction of P. minus leaf transcriptome to identify differentially expressed genes (DEGs) in response to MeJA elicitation. A total of 182,111 unique transcripts (UTs) were obtained by de novo assembly of 191.57 million paired-end clean reads using Trinity analysis pipeline. A total of 2374 UTs were identified to be significantly up-/down-regulated 24 h after MeJA treatment. These UTs comprising many genes related to plant secondary metabolite biosynthesis, defense and stress responses. To validate our sequencing results, we analyzed the expression of 21 selected DEGs by quantitative real-time PCR and found a good correlation between the two analyses. The single time-point analysis in this work not only provides a useful genomic resource for P. minus but also gives insights on molecular mechanisms of stress responses in P. minus. PMID:28220135

  20. 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

  1. Leptospira Protein Expression During Infection

    USDA-ARS?s Scientific Manuscript database

    We are characterizing protein expression in vivo during experimental leptospirosis using immunofluorescence microscopy. Coding regions for several proteins were identified through analysis of Leptospira interrogans serovar Copenhageni and L. borgpetersenii serovar Hardjo genomes. In addition, codi...

  2. Withania somnifera prevents acquisition and expression of morphine-elicited conditioned place preference.

    PubMed

    Ruiu, Stefania; Longoni, Rosanna; Spina, Liliana; Orrù, Alessandro; Cottiglia, Filippo; Collu, Maria; Kasture, Sanjay; Acquas, Elio

    2013-04-01

    Previous studies have reported that some of the central effects of morphine are counteracted by the administration of the methanolic extract of the root of Indian ginseng, Withania somnifera Dunal (WSE). The present study sought to determine whether WSE affects acquisition and expression of morphine-elicited conditioned place preference (CPP) in CD-1 mice. In CPP acquisition experiments, WSE (0, 25, 50, and 100 mg/kg) was administered, during conditioning, 30 min before morphine (10 mg/kg), whereas in expression experiments, WSE (0, 25, 50, and 100 mg/kg) was administered 30 min before the postconditioning test. The results demonstrate (i) that WSE was devoid of motivational properties; (ii) that WSE (100 mg/kg) was devoid of effects on spontaneous and morphine-stimulated motor activity and on spatial memory; and (iii) that WSE (50 and 100 mg/kg) significantly prevented the acquisition and expression of CPP. Further, to characterize the receptor(s) involved in these effects, we studied, by receptor-binding assay, the affinity of WSE for µ-opioid and γ-aminobutyric acid B receptors. These experiments revealed a higher affinity of WSE for γ-aminobutyric acid B than for µ-opioid receptors. Overall, these results point to WSE as an interesting alternative tool, worthy of further investigation, to study opiate addiction.

  3. Maspin expression in prostate tumor elicits host anti-tumor immunity

    PubMed Central

    Dzinic, Sijana H.; Chen, Kang; Thakur, Archana; Kaplun, Alexander; Daniel Bonfil, R.; Li, Xiaohua; Liu, Jason; Margarida Bernardo, M.; Saliganan, Allen; Back, Jessica B.; Yano, Hiroshi; Schalk, Dana L.; Tomaszewski, Elyse N.; Beydoun, Ahmed S.; Dyson, Gregory; Mujagic, Adelina; Krass, David; Dean, Ivory; Mi, Qing-Sheng; Heath, Elisabeth; Sakr, Wael; Lum, Lawrence G.; Sheng, Shijie

    2014-01-01

    The goal of the current study is to examine the biological effects of epithelial-specific tumor suppressor maspin on tumor host immune response. Accumulated evidence demonstrates an anti-tumor effect of maspin on tumor growth, invasion and metastasis. The molecular mechanism underlying these biological functions of maspin is thought to be through histone deacetylase inhibition, key to the maintenance of differentiated epithelial phenotype. Since tumor-driven stromal reactivities co-evolve in tumor progression and metastasis, it is not surprising that maspin expression in tumor cells inhibits extracellular matrix degradation, increases fibrosis and blocks hypoxia-induced angiogenesis. Using the athymic nude mouse model capable of supporting the growth and progression of xenogeneic human prostate cancer cells, we further demonstrate that maspin expression in tumor cells elicits neutrophil- and B cells-dependent host tumor immunogenicity. Specifically, mice bearing maspin-expressing tumors exhibited increased systemic and intratumoral neutrophil maturation, activation and antibody-dependent cytotoxicity, and decreased peritumoral lymphangiogenesis. These results reveal a novel biological function of maspin in directing host immunity towards tumor elimination that helps explain the significant reduction of xenograft tumor incidence in vivo and the clinical correlation of maspin with better prognosis of several types of cancer. Taken together, our data raised the possibility for novel maspin-based cancer immunotherapies. PMID:25373490

  4. Why do fearful facial expressions elicit behavioral approach? Evidence from a combined approach-avoidance implicit association test.

    PubMed

    Hammer, Jennifer L; Marsh, Abigail A

    2015-04-01

    Despite communicating a "negative" emotion, fearful facial expressions predominantly elicit behavioral approach from perceivers. It has been hypothesized that this seemingly paradoxical effect may occur due to fearful expressions' resemblance to vulnerable, infantile faces. However, this hypothesis has not yet been tested. We used a combined approach-avoidance/implicit association test (IAT) to test this hypothesis. Participants completed an approach-avoidance lever task during which they responded to fearful and angry facial expressions as well as neutral infant and adult faces presented in an IAT format. Results demonstrated an implicit association between fearful facial expressions and infant faces and showed that both fearful expressions and infant faces primarily elicit behavioral approach. The dominance of approach responses to both fearful expressions and infant faces decreased as a function of psychopathic personality traits. Results suggest that the prosocial responses to fearful expressions observed in most individuals may stem from their associations with infantile faces. (PsycINFO Database Record

  5. Specific epitopes of the structural and hypothetical proteins elicit variable humoral responses in SARS patients

    PubMed Central

    Chow, S C S; Ho, C Y S; Tam, T T Y; Wu, C; Cheung, T; Chan, P K S; Ng, M H L; Hui, P K; Ng, H K; Au, D M Y; Lo, A W I

    2006-01-01

    Background Severe acute respiratory syndrome (SARS) is an infectious disease which was caused by a novel coronavirus (SARS‐CoV). SARS has caused an outbreak in the world during 2003 and 2004, with 8098 individuals being infected and a death toll of 774 in 28 regions around the world. Specific humoral responses to viral infection remain unclear. Objective To analyse the antigenicity of the SARS‐CoV genome and identify potential antigenic epitopes in the structural proteins. Methods Potential antigenic epitopes were identified in the structural proteins (nucleocapsid, membrane, spike, and small envelope proteins) and hypothetical proteins (SARS3a, 3b, 6, 7a, and 9b) that are specific for SARS‐CoV. A peptide chip platform was created and the profiles of antibodies to these epitopes were investigated in 59 different SARS patients' sera obtained 6–103 days after the onset of the illness. Serial sera from five additional patients were also studied. Results Epitopes at the N‐terminus of the membrane protein and the C‐terminus of nucleocapsid protein elicited strong antibody responses. Epitopes on the spike protein were only moderately immunogenic but the effects were persistent. Antibodies were also detected for some putative proteins, noticeably the C‐termini of SARS3a and SARS6. Conclusions Important epitopes of the SARS‐CoV genome that may serve as potential markers for the viral infection are identified. These specific antigenic sites may also be important for vaccine development against this new fatal infectious disease. PMID:16461566

  6. Retinoic acid-dependent regulation of miR-19 expression elicits vertebrate axis defects

    PubMed Central

    Franzosa, Jill A.; Bugel, Sean M.; Tal, Tamara L.; La Du, Jane K.; Tilton, Susan C.; Waters, Katrina M.; Tanguay, Robert L.

    2013-01-01

    Retinoic acid (RA) is involved in multifarious and complex functions necessary for vertebrate development. RA signaling is reliant on strict enzymatic regulation of RA synthesis and metabolism. Improper spatiotemporal expression of RA during development can result in vertebrate axis defects. microRNAs (miRNAs) are also pivotal in orchestrating developmental processes. While mechanistic links between miRNAs and axial development are established, the role of miRNAs in regulating metabolic enzymes responsible for RA abundance during axis formation has yet to be elucidated. Our results uncovered a role of miR-19 family members in controlling RA metabolism through the regulation of CYP26A1 during vertebrate axis formation. Global miRNA expression profiling showed that developmental RA exposure suppressed the expression of miR-19 family members during zebrafish somitogenesis. A reporter assay confirmed that cyp26a1 is a bona fide target of miR-19 in vivo. Transient knockdown of miR-19 phenocopied axis defects caused by RA exposure. Exogenous miR-19 rescued the axis defects induced by RA exposure. Taken together, these results indicate that the teratogenic effects of RA exposure result, in part, from repression of miR-19 expression and subsequent misregulation of cyp26a1. This highlights a previously unidentified role of miR-19 in facilitating vertebrate axis development via regulation of RA signaling.—Franzosa, J. A., Bugel, S. M., Tal, T. L., La Du, J. K., Tilton, S. C., Waters, K. M., Tanguay, R. L. Retinoic acid-dependent regulation of miR-19 expression elicits vertebrate axis defects. PMID:23975936

  7. A foreign protein incorporated on the Tip of T3 pili in Lactococcus lactis elicits systemic and mucosal immunity.

    PubMed

    Quigley, Bernard R; Hatkoff, Matthew; Thanassi, David G; Ouattara, Mahamoudou; Eichenbaum, Zehava; Scott, June R

    2010-03-01

    The use of Lactococcus lactis to deliver a chosen antigen to the mucosal surface has been shown to elicit an immune response in mice and is a possible method of vaccination in humans. The recent discovery on Gram-positive bacteria of pili that are covalently attached to the bacterial surface and the elucidation of the residues linking the major and minor subunits of such pili suggests that the presentation of an antigen on the tip of pili external to the surface of L. lactis might constitute a successful vaccine strategy. As a proof of principle, we have fused a foreign protein (the Escherichia coli maltose-binding protein) to the C-terminal region of the native tip protein (Cpa) of the T3 pilus derived from Streptococcus pyogenes and expressed this fusion protein (MBP*) in L. lactis. We find that MBP* is incorporated into pili in this foreign host, as shown by Western blot analyses of cell wall proteins and by immunogold electron microscopy. Furthermore, since the MBP* on these pili retains its native biological activity, it appears to retain its native structure. Mucosal immunization of mice with this L. lactis strain expressing pilus-linked MBP* results in production of both a systemic and a mucosal response (IgG and IgA antibodies) against the MBP antigen. We suggest that this type of mucosal vaccine delivery system, which we term UPTOP (for unhindered presentation on tips of pili), may provide an inexpensive and stable alternative to current mechanisms of immunization for many serious human pathogens.

  8. Translation elicits a growth rate-dependent, genome-wide, differential protein production in Bacillus subtilis.

    PubMed

    Borkowski, Olivier; Goelzer, Anne; Schaffer, Marc; Calabre, Magali; Mäder, Ulrike; Aymerich, Stéphane; Jules, Matthieu; Fromion, Vincent

    2016-05-17

    Complex regulatory programs control cell adaptation to environmental changes by setting condition-specific proteomes. In balanced growth, bacterial protein abundances depend on the dilution rate, transcript abundances and transcript-specific translation efficiencies. We revisited the current theory claiming the invariance of bacterial translation efficiency. By integrating genome-wide transcriptome datasets and datasets from a library of synthetic gfp-reporter fusions, we demonstrated that translation efficiencies in Bacillus subtilis decreased up to fourfold from slow to fast growth. The translation initiation regions elicited a growth rate-dependent, differential production of proteins without regulators, hence revealing a unique, hard-coded, growth rate-dependent mode of regulation. We combined model-based data analyses of transcript and protein abundances genome-wide and revealed that this global regulation is extensively used in B. subtilis We eventually developed a knowledge-based, three-step translation initiation model, experimentally challenged the model predictions and proposed that a growth rate-dependent drop in free ribosome abundance accounted for the differential protein production. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

  9. Why Do Fearful Facial Expressions Elicit Behavioral Approach? Evidence From a Combined Approach-Avoidance Implicit Association Test

    PubMed Central

    Hammer, Jennifer L.; Marsh, Abigail A.

    2015-01-01

    Despite communicating a “negative” emotion, fearful facial expressions predominantly elicit behavioral approach from perceivers. It has been hypothesized that this seemingly paradoxical effect may occur due to fearful expressions’ resemblance to vulnerable, infantile faces. However, this hypothesis has not yet been tested. We used a combined approach-avoidance/implicit association test (IAT) to test this hypothesis. Participants completed an approach-avoidance lever task during which they responded to fearful and angry facial expressions as well as neutral infant and adult faces presented in an IAT format. Results demonstrated an implicit association between fearful facial expressions and infant faces and showed that both fearful expressions and infant faces primarily elicit behavioral approach. The dominance of approach responses to both fearful expressions and infant faces decreased as a function of psychopathic personality traits. Results suggest that the prosocial responses to fearful expressions observed in most individuals may stem from their associations with infantile faces. PMID:25603135

  10. Amphetamine-elicited striatal Fos expression is attenuated in neurotensin null mutant mice.

    PubMed

    Fadel, Jim; Dobner, Paul R; Deutch, Ariel Y

    2006-07-10

    Neurotensin (NT) has been suggested to interact with dopamine systems in different forebrain sites to exert both antipsychotic- and psychostimulant-like effects. We previously found that genetic or pharmacological manipulations that disrupt endogenous NT signaling attenuate antipsychotic drug-induced Fos expression in the dorsolateral and central striatum but not other striatal regions. To assess the role of NT in psychostimulant responses, we examined the ability of d-amphetamine (AMP) to induce Fos in wild-type and NT null mutant mice. AMP-elicited Fos expression was significantly attenuated in the medial striatum of NT null mutant mice, but was unaffected in other striatal territories. Similar results were obtained in rats and mice pretreated with the high affinity neurotensin receptor (NTR1) antagonist SR 48692. The effect of the NTR1 antagonist was particularly apparent in the striatal patch (striosome) compartment, as defined by mu-opioid receptor immunoreactivity. These data suggest that NT is required for the full activation by AMP of medial striatal neurons.

  11. Ectopic expression of the striatal-enriched GTPase Rhes elicits cerebellar degeneration and an ataxia phenotype in Huntington's disease.

    PubMed

    Swarnkar, Supriya; Chen, Youjun; Pryor, William M; Shahani, Neelam; Page, Damon T; Subramaniam, Srinivasa

    2015-10-01

    Huntington's disease (HD) is caused by an expansion of glutamine repeats in the huntingtin protein (mHtt) that invokes early and prominent damage of the striatum, a region that controls motor behaviors. Despite its ubiquitous expression, why certain brain regions, such as the cerebellum, are relatively spared from neuronal loss by mHtt remains unclear. Previously, we implicated the striatal-enriched GTPase, Rhes (Ras homolog enriched in the striatum), which binds and SUMOylates mHtt and increases its solubility and cellular cytotoxicity, as the cause for striatal toxicity in HD. Here, we report that Rhes deletion in HD mice (N171-82Q), which express the N-terminal fragment of human Htt with 82 glutamines (Rhes(-/-)/N171-82Q), display markedly reduced HD-related behavioral deficits, and absence of lateral ventricle dilatation (secondary to striatal atrophy), compared to control HD mice (N171-82Q). To further validate the role of GTPase Rhes in HD, we tested whether ectopic Rhes expression would elicit a pathology in a brain region normally less affected in HD. Remarkably, ectopic expression of Rhes in the cerebellum of N171-82Q mice, during the asymptomatic period led to an exacerbation of motor deficits, including loss of balance and motor incoordination with ataxia-like features, not apparent in control-injected N171-82Q mice or Rhes injected wild-type mice. Pathological and biochemical analysis of Rhes-injected N171-82Q mice revealed a cerebellar lesion with marked loss of Purkinje neuron layer parvalbumin-immunoreactivity, induction of caspase 3 activation, and enhanced soluble forms of mHtt. Similarly reintroducing Rhes into the striatum of Rhes deleted Rhes(-/-)Hdh(150Q/150Q) knock-in mice, elicited a progressive HD-associated rotarod deficit. Overall, these studies establish that Rhes plays a pivotal role in vivo for the selective toxicity of mHtt in HD. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. Circadian RNA expression elicited by 3’-UTR IRAlu-paraspeckle associated elements

    PubMed Central

    Torres, Manon; Becquet, Denis; Blanchard, Marie-Pierre; Guillen, Séverine; Boyer, Bénédicte; Moreno, Mathias; Franc, Jean-Louis; François-Bellan, Anne-Marie

    2016-01-01

    Paraspeckles are nuclear bodies form around the long non-coding RNA, Neat1, and RNA-binding proteins. While their role is not fully understood, they are believed to control gene expression at a post-transcriptional level by means of the nuclear retention of mRNA containing in their 3’-UTR inverted repeats of Alu sequences (IRAlu). In this study, we found that, in pituitary cells, all components of paraspeckles including four major proteins and Neat1 displayed a circadian expression pattern. Furthermore the insertion of IRAlu at the 3’-UTR of the EGFP cDNA led to a rhythmic circadian nuclear retention of the egfp mRNA that was lost when paraspeckles were disrupted whereas insertion of a single antisense Alu had only a weak effect. Using real-time video-microscopy, these IRAlu were further shown to drive a circadian expression of EGFP protein. This study shows that paraspeckles, thanks to their circadian expression, control circadian gene expression at a post-transcriptional level. DOI: http://dx.doi.org/10.7554/eLife.14837.001 PMID:27441387

  13. Sprouty2 and Spred1-2 Proteins Inhibit the Activation of the ERK Pathway Elicited by Cyclopentenone Prostanoids

    PubMed Central

    Gragera, Teresa; Pérez-Rodríguez, Andrea; Retana, Diana; León, Gonzalo; Sánchez, Agustín; Oliva, José Luis; Pérez-Sala, Dolores; Rojas, José M.

    2011-01-01

    Sprouty and Spred proteins have been widely implicated in the negative regulation of the fibroblast growth factor receptor-extracellular regulated kinase (ERK) pathway. In considering the functional role of these proteins, we explored their effects on ERK activation induced by cyclopentenone prostanoids, which bind to and activate Ras proteins. We therefore found that ectopic overexpression in HeLa cells of human Sprouty2, or human Spred1 or 2, inhibits ERK1/2 and Elk-1 activation triggered by the cyclopentenone prostanoids PGA1 and 15d-PGJ2. Furthermore, we found that in HT cells that do not express Sprouty2 due to hypermethylation of its gene-promoter, PGA1-provoked ERK activation was more intense and sustained compared to other hematopoietic cell lines with unaltered Sprouty2 expression. Cyclopentenone prostanoids did not induce Sprouty2 tyrosine phosphorylation, in agreement with its incapability to activate tyrosine-kinase receptors. However, Sprouty2 Y55F, which acts as a defective mutant upon tyrosine-kinase receptor stimulation, did not inhibit cyclopentenone prostanoids-elicited ERK pathway activation. In addition, Sprouty2 did not affect the Ras-GTP levels promoted by cyclopentenone prostanoids. These results unveil both common and differential features in the activation of Ras-dependent pathways by cyclopentenone prostanoids and growth factors. Moreover, they provide the first evidence that Sprouty and Spred proteins are negative regulators of the ERK/Elk-1 pathway activation induced not only by growth-factors, but also by reactive lipidic mediators. PMID:21364986

  14. Recombinant protein expression in Nicotiana.

    PubMed

    Matoba, Nobuyuki; Davis, Keith R; Palmer, Kenneth E

    2011-01-01

    Recombinant protein pharmaceuticals are now widely used in treatment of chronic diseases, and several recombinant protein subunit vaccines are approved for human and veterinary use. With growing demand for complex protein pharmaceuticals, such as monoclonal antibodies, manufacturing capacity is becoming limited. There is increasing need for safe, scalable, and economical alternatives to mammalian cell culture-based manufacturing systems, which require substantial capital investment for new manufacturing facilities. Since a seminal paper reporting immunoglobulin expression in transgenic plants was published in 1989, there have been many technological advances in plant expression systems to the present time where production of proteins in leaf tissues of nonfood crops such as Nicotiana species is considered a viable alternative. In particular, transient expression systems derived from recombinant plant viral vectors offer opportunities for rapid expression screening, construct optimization, and expression scale-up. Extraction of recombinant proteins from Nicotiana leaf tissues can be achieved by collection of secreted protein fractions, or from a total protein extract after grinding the leaves with buffer. After separation from solids, the major purification challenge is contamination with elements of the photosynthetic complex, which can be solved by application of a variety of facile and proven strategies. In conclusion, the technologies required for safe, efficient, scalable manufacture of recombinant proteins in Nicotiana leaf tissues have matured to the point where several products have already been tested in phase I clinical trials and will soon be followed by a rich pipeline of recombinant vaccines, microbicides, and therapeutic proteins.

  15. Glyceollin-elicited soy protein consumption induces distinct transcriptional effects as compared to standard soy protein

    USDA-ARS?s Scientific Manuscript database

    Glyceollins are stress-induced compounds in soybeans with bioactive properties distinct from parent soy isoflavones. The goals of this study were to evaluate effects of dietary glyceollin-enriched and standard soy protein isolates and identify candidate target pathways of glyceollins on transcripti...

  16. Biphasic gene expression changes elicited by Phakopsora pachyrhizi in soybean correlates with fungal penetration and haustoria formation

    USDA-ARS?s Scientific Manuscript database

    Inoculation of soybean plants with Phakopsora pachyrhizi, the causal organism of Asian soybean rust, elicits a biphasic response characterized by a burst of differential gene expression in the first 12 h. A quiescent period occurs from 24 to 48 h after inoculation in which P. pachyrhizi continues t...

  17. Correctly folded Pfs48/45 protein of Plasmodium falciparum elicits malaria transmission-blocking immunity in mice

    PubMed Central

    Outchkourov, Nikolay S.; Roeffen, Will; Kaan, Anita; Jansen, Josephine; Luty, Adrian; Schuiffel, Danielle; van Gemert, Geert Jan; van de Vegte-Bolmer, Marga; Sauerwein, Robert W.; Stunnenberg, Hendrik G.

    2008-01-01

    Malaria kills >1 million people each year, in particular in sub-Saharan Africa. Although asexual forms are directly responsible for disease and death, sexual stages account for the transmission of Plasmodium parasites from human to the mosquito vector and therefore the spread of the parasite in the population. Development of a malaria vaccine is urgently needed to reduce morbidity and mortality. Vaccines against sexual stages of Plasmodium falciparum are meant to decrease the force of transmission and consequently reduce malaria burden. Pfs48/45 is specifically expressed in sexual stages and is a well established transmission-blocking (TB) vaccine candidate. However, production of correctly folded recombinant Pfs48/45 protein with display of its TB epitopes has been a major challenge. Here, we show the production of a properly folded Pfs48/45 C-terminal fragment by simultaneous coexpression with four periplasmic folding catalysts in Escherichia coli. This C-terminal fragment fused to maltose binding protein was produced at medium scale with >90% purity and a stability over at least a 9-month period. It induces uniform and high antibody titers in mice and elicits functional TB antibodies in standard membrane feeding assays in 90% of the immunized mice. Our data provide a clear perspective on the clinical development of a Pfs48/45-based TB malaria vaccine. PMID:18332422

  18. Human papillomavirus type 16 virus-like particles expressed in attenuated Salmonella typhimurium elicit mucosal and systemic neutralizing antibodies in mice.

    PubMed Central

    Nardelli-Haefliger, D; Roden, R B; Benyacoub, J; Sahli, R; Kraehenbuhl, J P; Schiller, J T; Lachat, P; Potts, A; De Grandi, P

    1997-01-01

    Attenuated strains of Salmonella are attractive live vaccine candidates for eliciting mucosal as well as systemic immune responses. The ability to induce immune responses in the reproductive tract may be critical for the effectiveness of a prophylactic vaccine against genital human papillomaviruses (HPV), which are important etiologic agents in the development of cervical cancer. To examine the potential of a live Salmonella-based vaccine to prevent genital HPV infection, the L1 major capsid protein from HPV type 16 (HPV16) was constitutively expressed in the PhoPc strain of Salmonella typhimurium. As demonstrated by electron microscopy, the L1 protein expressed in these bacteria assembled into virus-like particles (VLPs) that resemble authentic papillomavirus virions. This is the first demonstration that papillomavirus VLPs can self-assemble in prokaryotes. BALB/c mice were immunized with the HPV16 L1 recombinant PhoPc strain by the oral and nasal routes. Despite a low stability of the L1-expressing plasmid in vivo, a double nasal immunization was effective in inducing L1-specific serum antibodies that recognized mainly native, but not disassembled, VLPs. These antibodies effectively neutralized HPV16 pseudotyped virions in an in vitro infectivity assay. Conformationally dependent anti-VLP immunoglobulin A (IgA) and IgG were also detected in oral and vaginal secretions, indicating that potentially protective antibody responses were elicited at mucosal sites. Recombinant attenuated Salmonella expressing HPV capsids may represent a promising vaccine candidate against genital HPV infection. PMID:9234794

  19. RepA Protein Encoded by Oat dwarf virus Elicits a Temperature-Sensitive Hypersensitive Response-Type Cell Death That Involves Jasmonic Acid-Dependent Signaling.

    PubMed

    Qian, Yajuan; Hou, Huwei; Shen, Qingtang; Cai, Xinzhong; Sunter, Garry; Zhou, Xueping

    2016-01-01

    The hypersensitive response (HR) is a component of disease resistance that is often induced by pathogen infection, but essentially no information is available for members of the destructive mastreviruses. We have investigated an HR-type response elicited in Nicotiana species by Oat dwarf virus (ODV) and have found that expression of the ODV RepA protein but not other ODV-encoded proteins elicits the HR-type cell death associated with a burst of H2O2. Deletion mutagenesis indicates that the first nine amino acids (aa) at the N terminus of RepA and the two regions located between aa residues 173 and 195 and between aa residues 241 and 260 near the C terminus are essential for HR-type cell-death elicitation. Confocal and electron microscopy showed that the RepA protein is localized in the nuclei of plant cells and might contain bipartite nuclear localization signals. The HR-like lesions mediated by RepA were inhibited by temperatures above 30°C and involvement of jasmonic acid (JA) in HR was identified by gain- and loss-of-function experiments. To our knowledge, this is the first report of an elicitor of HR-type cell death from mastreviruses.

  20. Die another day: molecular mechanisms of effector-triggered immunity elicited by type III secreted effector proteins

    USDA-ARS?s Scientific Manuscript database

    Bacterial pathogens inject type III secreted effector (T3SE) proteins into their hosts where they display dual roles depending on the host genotype. T3SEs promote bacterial virulence in susceptible hosts, and elicit immunity in resistant hosts. T3SEs are typically recognized when they modify a host ...

  1. Alcohol odor elicits appetitive facial expressions in human neonates prenatally exposed to the drug.

    PubMed

    Faas, Ana E; March, Samanta M; Moya, Pedro R; Molina, Juan C

    2015-09-01

    OH-Lem-EtOH sequence) and c) when merging both samples of babies, a positive and significant correlation was found between overall maternal absolute alcohol consumption per month and frequency of appetitive facial expressions elicited by alcohol odor. In conjunction with previous preclinical research, the present results indicate that human prenatal exposure to the drug that yields no evident teratological effects is sufficient to modify the hedonic value of alcohol's chemosensory attributes.

  2. Selected prfA* mutations in recombinant attenuated Listeria monocytogenes strains augment expression of foreign immunogens and enhance vaccine-elicited humoral and cellular immune responses.

    PubMed

    Yan, Lin; Qiu, Jin; Chen, Jianbo; Ryan-Payseur, Bridgett; Huang, Dan; Wang, Yunqi; Rong, Lijun; Melton-Witt, Jody A; Freitag, Nancy E; Chen, Zheng W

    2008-08-01

    While recombinant Listeria monocytogenes strains can be explored as vaccine candidates, it is important to develop attenuated but highly immunogenic L. monocytogenes vaccine vectors. Here, prfA* mutations selected on the basis of upregulated expression of L. monocytogenes PrfA-dependent genes and proteins were assessed to determine their abilities to augment expression of foreign immunogens in recombinant L. monocytogenes vectors and therefore enhance vaccine-elicited immune responses (a prfA* mutation is a mutation that results in constitutive overexpression of PrfA and PrfA-dependent virulence genes; the asterisk distinguishes the mutation from inactivation or stop mutations). A total of 63 recombinant L. monocytogenes vaccine vectors expressing seven individual viral or bacterial immunogens each in nine different L. monocytogenes strains carrying wild-type prfA or having prfA* mutations were constructed and investigated. Mutations selected on the basis of increased PrfA activation in recombinant L. monocytogenes prfA* vaccine vectors augmented expression of seven individual protein immunogens remarkably. Consistently, prime and boost vaccination studies with mice indicated that the prfA(G155S) mutation in recombinant L. monocytogenes DeltaactA prfA* strains enhanced vaccine-elicited cellular immune responses. Surprisingly, the prfA(G155S) mutation was found to enhance vaccine-elicited humoral immune responses as well. The highly immunogenic recombinant L. monocytogenes DeltaactA prfA* vaccine strains were as attenuated as the recombinant parent L. monocytogenes DeltaactA vaccine vector. Thus, recombinant attenuated L. monocytogenes DeltaactA prfA* vaccine vectors potentially are better antimicrobial and anticancer vaccines.

  3. Low zone tolerance requires ICAM-1 expression to limit contact hypersensitivity elicitation.

    PubMed

    Komura, Kazuhiro; Iwata, Yohei; Ogawa, Fumihide; Yoshizaki, Ayumi; Yamaoka, Toshifumi; Akiyama, Yuichiro; Hara, Toshihide; Hasegawa, Minoru; Fujimoto, Manabu; Sato, Shinichi

    2009-11-01

    Painting subsensitizing doses of contact sensitizers on skin (low-dose tolerization) induces antigen (Ag)-specific tolerance, known as low zone tolerance (LZT), which has been experimentally demonstrated by the inhibition of contact hypersensitivity (CHS). Although LZT resulted from the inhibition of the sensitization phase, the effects on the effector/elicitation phase remain unknown. L-selectin and ICAM-1 regulate leukocyte influx into inflamed tissues during the elicitation phase of CHS. LZT was investigated in mice lacking either L-selectin or ICAM-1 to evaluate the roles these leukocyte receptors play in LZT during the elicitation phase. Low-dose tolerization effectively suppressed CHS in wild-type and L-selectin-deficient mice, but not in ICAM-1-deficient mice. Low-dose-tolerized ICAM-1-deficient splenocytes effectively suppressed the elicitation phase in naive wild-type recipients. Sensitized ICAM-1-deficient splenocytes showed normal proliferative responses to the sensitizing Ag and generated normal CHS in wild-type recipients. Thus, ICAM-1 deficiency did not affect sensitization. LZT was associated with a lack of ICAM-1 upregulation after elicitation, suggesting a potentially mechanistic role for ICAM-1. The blockade of IL-10, a possible mediator of LZT, produced by hapten-specific suppressor cells, abrogated LZT and restored ICAM-1 upregulation. These results indicate that low-dose tolerization controls CHS by abrogating ICAM-1 upregulation during the elicitation phase.

  4. Biphasic gene expression changes elicited by Phakopsora pachyrhizi in soybean correlate with fungal penetration and haustoria formation.

    PubMed

    Schneider, Katherine T; van de Mortel, Martijn; Bancroft, Timothy J; Braun, Edward; Nettleton, Dan; Nelson, Rex T; Frederick, Reid D; Baum, Thomas J; Graham, Michelle A; Whitham, Steven A

    2011-09-01

    Inoculation of soybean (Glycine max) plants with Phakopsora pachyrhizi, the causal organism of Asian soybean rust, elicits a biphasic response characterized by a burst of differential gene expression in the first 12 h. A quiescent period occurs from 24 to 48 h after inoculation, in which P. pachyrhizi continues to develop but does not elicit strong host responses, followed by a second phase of intense gene expression. To correlate soybean responses with P. pachyrhizi growth and development, we inoculated the soybean cultivar Ankur (accession PI462312), which carries the Rpp3 resistance gene, with avirulent and virulent isolates of P. pachyrhizi. The avirulent isolate Hawaii 94-1 elicits hypersensitive cell death that limits fungal growth on Ankur and results in an incompatible response, while the virulent isolate Taiwan 80-2 grows extensively, sporulates profusely, and produces a compatible reaction. Inoculated leaves were collected over a 288-h time course for microarray analysis of soybean gene expression and microscopic analysis of P. pachyrhizi growth and development. The first burst in gene expression correlated with appressorium formation and penetration of epidermal cells, while the second burst of gene expression changes followed the onset of haustoria formation in both compatible and incompatible interactions. The proliferation of haustoria coincided with the inhibition of P. pachyrhizi growth in the incompatible interaction or the beginning of accelerated growth in the compatible interaction. The temporal relationships between P. pachyrhizi growth and host responses provide an important context in which to view interacting gene networks that mediate the outcomes of their interactions.

  5. Expression of a dominant negative PKA mutation in the kidney elicits a diabetes insipidus phenotype.

    PubMed

    Gilbert, Merle L; Yang, Linghai; Su, Thomas; McKnight, G Stanley

    2015-03-15

    PKA plays a critical role in water excretion through regulation of the production and action of the antidiuretic hormone arginine vasopressin (AVP). The AVP prohormone is produced in the hypothalamus, where its transcription is regulated by cAMP. Once released into the circulation, AVP stimulates antidiuresis through activation of vasopressin 2 receptors in renal principal cells. Vasopressin 2 receptor activation increases cAMP and activates PKA, which, in turn, phosphorylates aquaporin (AQP)2, triggering apical membrane accumulation, increased collecting duct permeability, and water reabsorption. We used single-minded homolog 1 (Sim1)-Cre recombinase-mediated expression of a dominant negative PKA regulatory subunit (RIαB) to disrupt kinase activity in vivo and assess the role of PKA in fluid homeostasis. RIαB expression gave rise to marked polydipsia and polyuria; however, neither hypothalamic Avp mRNA expression nor urinary AVP levels were attenuated, indicating a primary physiological effect on the kidney. RIαB mice displayed a marked deficit in urinary concentrating ability and greatly reduced levels of AQP2 and phospho-AQP2. Dehydration induced Aqp2 mRNA in the kidney of both control and RIαB-expressing mice, but AQP2 protein levels were still reduced in RIαB-expressing mutants, and mice were unable to fully concentrate their urine and conserve water. We conclude that partial PKA inhibition in the kidney leads to posttranslational effects that reduce AQP2 protein levels and interfere with apical membrane localization. These findings demonstrate a distinct physiological role for PKA signaling in both short- and long-term regulation of AQP2 and characterize a novel mouse model of diabetes insipidus.

  6. The 50 distal amino acids of the 2A(HP) homing protein of Grapevine fanleaf virus elicit a hypersensitive reaction on Nicotiana occidentalis.

    PubMed

    Martin, Isabelle R; Vigne, Emmanuelle; Berthold, François; Komar, Véronique; Lemaire, Olivier; Fuchs, Marc; Schmitt-Keichinger, Corinne

    2017-04-07

    Avirulence factors are critical for the arm's race between a virus and its host in determining incompatible reactions. The response of plants to viruses from the genus Nepovirus in the family Secoviridae, including Grapevine fanleaf virus (GFLV), is well characterized, although the nature and characteristics of the viral avirulence factor remain elusive. By using infectious clones of GFLV strains F13 and GHu in a reverse genetics approach with wild-type, assortant and chimeric viruses, the determinant of necrotic lesions caused by GFLV-F13 on inoculated leaves of Nicotiana occidentalis was mapped to the RNA2-encoded protein 2A(HP) , particularly to its 50 C-terminal amino acids. The necrotic response showed hallmark characteristics of a genuine hypersensitive reaction, such as the accumulation of phytoalexins, reactive oxygen species, pathogenesis-related protein 1c and hypersensitivity-related (hsr) 203J transcripts. Transient expression of the GFLV-F13 protein 2A(HP) fused to an enhanced green fluorescent protein (EGFP) tag in N. occidentalis by agroinfiltration was sufficient to elicit a hypersensitive reaction. In addition, the GFLV-F13 avirulence factor, when introduced in GFLV-GHu, which causes a compatible reaction on N. occidentalis, elicited necrosis and partially restricted the virus. This is the first identification of a nepovirus avirulence factor that is responsible for a hypersensitive reaction in both the context of virus infection and transient expression. © 2017 BSPP AND JOHN WILEY & SONS LTD.

  7. Recombinant Eimeria protozoan protein elicits resistance to acute phlebovirus infection in mice but not hamsters.

    PubMed

    Gowen, Brian B; Smee, Donald F; Wong, Min-Hui; Judge, John W; Jung, Kie-Hoon; Bailey, Kevin W; Pace, Anne M; Rosenberg, Barnett; Sidwell, Robert W

    2006-06-01

    A protein antigen from an Eimeria protozoan has recently been reported to induce antitumor activity in mice. This activity most likely results from the strong induction of interkeukin-12 (IL-12) and gamma interferon (IFN-gamma), which are also essential factors in the establishment of protective immunity against viral infection. We evaluated recombinant Eimeria antigen (rEA) as a potential immunotherapeutic agent in mouse and hamster models of acute phleboviral disease. Punta Toro virus (PTV) was highly sensitive to a single dose of nanogram quantities of rEA in the mouse infection model. Intraperitoneal treatment with rEA also reduced virus load and liver damage associated with PTV infection. IL-12 was elicited following exposure of uninfected mice to quantities of rEA of 10 ng or greater, and the levels peaked at between 3 and 8 h postexposure. IFN-gamma release was induced more slowly and required less rEA (1 ng) to produce a significant rise in systemic levels. The induction of IL-12 and IFN-gamma involved in the coordination of innate and adaptive immune responses to microbial pathogens required myeloid differentiation factor 88, a signaling adaptor shared by most members of the Toll-like receptor (TLR) family. Despite encouraging results in the murine system, rEA failed to protect hamsters challenged with PTV. Our findings suggest that hamsters may lack functional TLR11, which has recently been shown to recognize a profilin-like protein homologous to rEA from the protozoan Toxoplasma gondii. Further investigation into the immunostimulatory capacity of rEA in other mammalian systems is necessary.

  8. A 24 kDa Excretory-Secretory Protein of Anisakis simplex Larvae Could Elicit Allergic Airway Inflammation in Mice

    PubMed Central

    Park, Hye-Kyung; Cho, Min Kyoung; Park, Mi Kyung; Kang, Shin Ae; Kim, Yun Seong; Kim, Ki Uk; Lee, Min Ki; Ock, Mee Sun; Cha, Hee Jae

    2011-01-01

    We have reported that a 24 kDa protein (22U homologous; As22U) of Anisakis simplex larvae could elicit several Th2-related chemokine gene expressions in the intestinal epithelial cell line which means that As22U may play a role as an allergen. In order to determine the contribution of As22U to allergic reactions, we treated mice with 6 times intra-nasal application of recombinant As22U (rAs22U). In the group challenged with rAs22U and ovalbumin (OVA), the number of eosinophils in the bronchial alveolar lavage fluid (BALF) was significantly increased, as compared to the group receiving only OVA. In addition, mice treated with rAs22U and OVA showed significantly increased airway hyperresponsiveness. Thus, severe inflammation around the airway and immune cell recruitment was observed in mice treated with rAs22U plus OVA. The levels of IL-4, IL-5, and IL-13 cytokines in the BALF increased significantly after treatment with rAs22U and OVA. Similarly, the levels of anti-OVA specific IgE and IgG1 increased in mice treated with rAs22U and OVA, compared to those treated only with OVA. The Gro-α (CXCL1) gene expression in mouse lung epithelial cells increased instantly after treatment with rAs22U, and allergy-specific chemokines eotaxin (CCL11) and thymus-and-activation-regulated-chemokine (CCL17) gene expressions significantly increased at 6 hr after treatment. In conclusion, rAs22U may induce airway allergic inflammation, as the result of enhanced Th2 and Th17 responses. PMID:22355204

  9. Adaptation to acetaminophen exposure elicits major changes in expression and distribution of the hepatic proteome.

    PubMed

    Eakins, R; Walsh, J; Randle, L; Jenkins, R E; Schuppe-Koistinen, I; Rowe, C; Starkey Lewis, P; Vasieva, O; Prats, N; Brillant, N; Auli, M; Bayliss, M; Webb, S; Rees, J A; Kitteringham, N R; Goldring, C E; Park, B K

    2015-11-26

    Acetaminophen overdose is the leading cause of acute liver failure. One dose of 10-15 g causes severe liver damage in humans, whereas repeated exposure to acetaminophen in humans and animal models results in autoprotection. Insight of this process is limited to select proteins implicated in acetaminophen toxicity and cellular defence. Here we investigate hepatic adaptation to acetaminophen toxicity from a whole proteome perspective, using quantitative mass spectrometry. In a rat model, we show the response to acetaminophen involves the expression of 30% of all proteins detected in the liver. Genetic ablation of a master regulator of cellular defence, NFE2L2, has little effect, suggesting redundancy in the regulation of adaptation. We show that adaptation to acetaminophen has a spatial component, involving a shift in regionalisation of CYP2E1, which may prevent toxicity thresholds being reached. These data reveal unexpected complexity and dynamic behaviour in the biological response to drug-induced liver injury.

  10. Adaptation to acetaminophen exposure elicits major changes in expression and distribution of the hepatic proteome

    PubMed Central

    Eakins, R.; Walsh, J.; Randle, L.; Jenkins, R. E.; Schuppe-Koistinen, I.; Rowe, C.; Starkey Lewis, P.; Vasieva, O.; Prats, N.; Brillant, N.; Auli, M.; Bayliss, M.; Webb, S.; Rees, J. A.; Kitteringham, N. R.; Goldring, C. E.; Park, B. K.

    2015-01-01

    Acetaminophen overdose is the leading cause of acute liver failure. One dose of 10–15 g causes severe liver damage in humans, whereas repeated exposure to acetaminophen in humans and animal models results in autoprotection. Insight of this process is limited to select proteins implicated in acetaminophen toxicity and cellular defence. Here we investigate hepatic adaptation to acetaminophen toxicity from a whole proteome perspective, using quantitative mass spectrometry. In a rat model, we show the response to acetaminophen involves the expression of 30% of all proteins detected in the liver. Genetic ablation of a master regulator of cellular defence, NFE2L2, has little effect, suggesting redundancy in the regulation of adaptation. We show that adaptation to acetaminophen has a spatial component, involving a shift in regionalisation of CYP2E1, which may prevent toxicity thresholds being reached. These data reveal unexpected complexity and dynamic behaviour in the biological response to drug-induced liver injury. PMID:26607827

  11. Multiple antigen peptide dendrimer elicits antibodies for detecting rat and mouse growth hormone binding proteins

    PubMed Central

    Aguilar, Roberto M.; Talamantes, Frank J.; Bustamante, Juan J.; Muñoz, Jesus; Treviño, Lisa R.; Martinez, Andrew O.; Haro, Luis S.

    2009-01-01

    The membrane-bound rat growth hormone receptor (GH-R) and an alternatively spliced isoform, the soluble rat GH binding protein (GH-BP), are comprised of identical N-terminal GH binding domains, however, their C-terminal sequences differ. Immunological reagents are needed to distinguish between the two isoforms in order to understand their respective roles in mediating the actions of GH. Accordingly, a tetravalent multiple antigen peptide (MAP) dendrimer with four identical branches of a C-terminal peptide sequence of the rat GH-BP (GH-BP263-279) was synthesized and used as an immunogen in rabbits. Solid-phase peptide synthesis of four GH-BP263-279 segments onto a tetravalent Lys2-Lys-β-Ala-OH core peptide was carried out using N-(9-fluorenyl)methoxycarbonyl chemistry. The mass of the RP-HPLC purified synthetic product, 8398 Da, determined by ESI-MS, was identical to expected mass. Three anti-rat GH-BP263-279 MAP antisera, BETO-8039, BETO-8040 and BETO-8041, at dilutions of 10-3, recognized both the rat GH-BP263-279 MAP and recombinant mouse GH-BP with ED50s within a range of 5-10 fmol but did not cross-react with BSA in dot blot analyses. BETO-8041 antisera (10-3 dilution) recognized GH-BPs of rat serum and liver having Mrs ranging from 35-130 kDa but did not recognize full-length rat GH-Rs. The antisera also detected recombinant mouse GH-BPs. In summary, the tetravalent rat GH-BP263-279 MAP dendrimer served as an effective immunogenic antigen in eliciting high titer antisera specific for the C-termini of both rat and mouse GH-BPs. The antisera will facilitate studies aimed at improving our understanding of the biology of GH-BPs. PMID:19089805

  12. Modeling Protein Expression and Protein Signaling Pathways

    PubMed Central

    Telesca, Donatello; Müller, Peter; Kornblau, Steven M.; Suchard, Marc A.; Ji, Yuan

    2015-01-01

    High-throughput functional proteomic technologies provide a way to quantify the expression of proteins of interest. Statistical inference centers on identifying the activation state of proteins and their patterns of molecular interaction formalized as dependence structure. Inference on dependence structure is particularly important when proteins are selected because they are part of a common molecular pathway. In that case, inference on dependence structure reveals properties of the underlying pathway. We propose a probability model that represents molecular interactions at the level of hidden binary latent variables that can be interpreted as indicators for active versus inactive states of the proteins. The proposed approach exploits available expert knowledge about the target pathway to define an informative prior on the hidden conditional dependence structure. An important feature of this prior is that it provides an instrument to explicitly anchor the model space to a set of interactions of interest, favoring a local search approach to model determination. We apply our model to reverse-phase protein array data from a study on acute myeloid leukemia. Our inference identifies relevant subpathways in relation to the unfolding of the biological process under study. PMID:26246646

  13. Induction of extracellular defense-related proteins in suspension cultured-cells of Daucus carota elicited with cyclodextrins and methyl jasmonate.

    PubMed

    Sabater-Jara, Ana B; Almagro, Lorena; Pedreño, María A

    2014-04-01

    Suspension cultured-cells (SCC) of Daucus carota were used to evaluate the effect of methyl jasmonate and cyclodextrins, separately or in combination, on the induction of defense responses, particularly the accumulation of pathogenesis-related proteins. A comparative study of the extracellular proteome (secretome) between control and elicited carrot SCC pointed to the presence of amino acid sequences homologous to glycoproteins which have inhibitory activity against the cell-wall-degrading enzymes secreted by pathogens and/or are induced when carrot cells are exposed to a pathogen elicitor. Other amino acid sequences were homologous to Leucine-Rich Repeat domain-containing proteins, which play an essential role in defense against pathogens, as well as in the recognition of microorganisms, making them important players in the innate immunity of this plant. Also, some tryptic peptides were shown to be homologous to a thaumatin-like protein, showing high specificity to abiotic stress and to different reticuline oxidase-like proteins that displayed high levels of antifungal activity, suggesting that methyl jasmonate and cyclodextrins could play a role in mediating defense-related gene product expression in SCC of D. carota. Apart from these elicitor-inducible proteins, we observed the presence of PR-proteins in both control and elicited carrot SCC, suggesting that their expression is mainly constitutive. These PR-proteins are putative class IV chitinases, which also have inhibitory activity against pathogen growth and the class III peroxidases that participate in response to environmental stress (e.g. pathogen attack and oxidative), meaning that they are involved in defense responses triggered by both biotic and abiotic factors. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  14. Yellow fever vaccination elicits broad functional CD4+ T cell responses that recognize structural and nonstructural proteins.

    PubMed

    James, Eddie A; LaFond, Rebecca E; Gates, Theresa J; Mai, Duy T; Malhotra, Uma; Kwok, William W

    2013-12-01

    Yellow fever virus (YFV) can induce acute, life-threatening disease that is a significant health burden in areas where yellow fever is endemic, but it is preventable through vaccination. The live attenuated 17D YFV strain induces responses characterized by neutralizing antibodies and strong T cell responses. This vaccine provides an excellent model for studying human immunity. While several studies have characterized YFV-specific antibody and CD8(+) T cell responses, less is known about YFV-specific CD4(+) T cells. Here we characterize the epitope specificity, functional attributes, and dynamics of YFV-specific T cell responses in vaccinated subjects by investigating peripheral blood mononuclear cells by using HLA-DR tetramers. A total of 112 epitopes restricted by seven common HLA-DRB1 alleles were identified. Epitopes were present within all YFV proteins, but the capsid, envelope, NS2a, and NS3 proteins had the highest epitope density. Antibody blocking demonstrated that the majority of YFV-specific T cells were HLA-DR restricted. Therefore, CD4(+) T cell responses could be effectively characterized with HLA-DR tetramers. Ex vivo tetramer analysis revealed that YFV-specific T cells persisted at frequencies ranging from 0 to 100 cells per million that are detectable years after vaccination. Longitudinal analysis indicated that YFV-specific CD4(+) T cells reached peak frequencies, often exceeding 250 cells per million, approximately 2 weeks after vaccination. As frequencies subsequently declined, YFV-specific cells regained CCR7 expression, indicating a shift from effector to central memory. Cells were typically CXCR3 positive, suggesting Th1 polarization, and produced gamma interferon and other cytokines after reactivation in vitro. Therefore, YFV elicits robust early effector CD4(+) T cell responses that contract, forming a detectable memory population.

  15. 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.

  16. Brain gene expression changes elicited by peripheral vitellogenin knockdown in the honey bee.

    PubMed

    Wheeler, M M; Ament, S A; Rodriguez-Zas, S L; Robinson, G E

    2013-10-01

    Vitellogenin (Vg) is best known as a yolk protein precursor. Vg also functions to regulate behavioural maturation in adult honey bee workers, but the underlying molecular mechanisms by which it exerts this novel effect are largely unknown. We used abdominal vitellogenin (vg) knockdown with RNA interference (RNAi) and brain transcriptomic profiling to gain insights into how Vg influences honey bee behavioural maturation. We found that vg knockdown caused extensive gene expression changes in the bee brain, with much of this transcriptional response involving changes in central biological functions such as energy metabolism. vg knockdown targeted many of the same genes that show natural, maturation-related differences, but the direction of change for the genes in these two contrasts was not correlated. By contrast, vg knockdown targeted many of the same genes that are regulated by juvenile hormone (JH) and there was a significant correlation for the direction of change for the genes in these two contrasts. These results indicate that the tight coregulatory relationship that exists between JH and Vg in the regulation of honey bee behavioural maturation is manifest at the genomic level and suggest that these two physiological factors act through common pathways to regulate brain gene expression and behaviour. © 2013 Royal Entomological Society.

  17. Pediatric measles vaccine expressing a dengue tetravalent antigen elicits neutralizing antibodies against all four dengue viruses.

    PubMed

    Brandler, Samantha; Ruffie, Claude; Najburg, Valérie; Frenkiel, Marie-Pascale; Bedouelle, Hughes; Desprès, Philippe; Tangy, Frédéric

    2010-09-24

    Dengue disease is an increasing global health problem that threatens one-third of the world's population. To control this emerging arbovirus, an efficient preventive vaccine is still needed. Because four serotypes of dengue virus (DV) coexist and antibody-dependent enhanced infection may occur, most strategies developed so far rely on the administration of tetravalent formulations of four live attenuated or chimeric viruses. Here, we evaluated a new strategy based on the expression of a single minimal tetravalent DV antigen by a single replicating viral vector derived from pediatric live-attenuated measles vaccine (MV). We generated a recombinant MV vector expressing a DV construct composed of the four envelope domain III (EDIII) from the four DV serotypes fused with the ectodomain of the membrane protein (ectoM). After two injections in mice susceptible to MV infection, the recombinant vector induced neutralizing antibodies against the four serotypes of dengue virus. When immunized mice were further inoculated with live DV from each serotype, a strong memory neutralizing response was raised against all four serotypes. A combined measles-dengue vaccine might be attractive to immunize infants against both diseases where they co-exist.

  18. Different neurotropic pathogens elicit neurotoxic CCR9- or neurosupportive CXCR3-expressing microglia.

    PubMed

    Li, He; Gang, Zhou; Yuling, He; Luokun, Xie; Jie, Xiong; Hao, Lei; Li, Wei; Chunsong, Hu; Junyan, Liu; Mingshen, Jiang; Youxin, Jin; Feili, Gong; Boquan, Jin; Jinquan, Tan

    2006-09-15

    What mechanism that determines microglia accomplishing destructive or constructive role in CNS remains nebulous. We report here that intracranial priming and rechallenging with Toxoplasma gondii in mice elicit neurotoxic CCR9+ Irg1+ (immunoresponsive gene 1) microglia, which render resistance to apoptosis and produce a high level of TNF-alpha; priming and rechallenging with lymphocytic choriomeningitis virus elicit neurosupportive CXCR3+ Irg1- microglia, which are sensitive to apoptosis and produce a high level of IL-10 and TGF-beta. Administration of CCR9 and/or Irg1 small interfering RNA alters the frequency and functional profiles of neurotoxic CCR9+ Irg1+ and neurosupportive CXCR3+ Irg1- microglia in vivo. Moreover, by using a series of different neurotropic pathogens, including intracellular parasites, chronic virus, bacteria, toxic substances, and CNS injury to intracranially prime and subsequent rechallenge mice, the bi-directional elicitation of microglia has been confirmed as neurotoxic CCR9+ Irg1+ and neurosupportive CXCR3+ Irg1- cells in these mouse models. These data suggest that there exist two different types of microglia, providing with a novel insight into microglial involvement in neurodegenerative and neuroinflammatory pathogenesis such as Alzheimer's disease and AIDS dementia.

  19. Antitumor response elicited by a superantigen-transmembrane sequence fusion protein anchored onto tumor cells.

    PubMed

    Wahlsten, J L; Mills, C D; Ramakrishnan, S

    1998-12-15

    Superantigens stimulate T cells bearing certain TCR beta-chain variable regions when bound to MHC II molecules. We investigated whether the superantigen toxic shock syndrome toxin-1 (TSST1) could induce an antitumor immune response when anchored onto MHC II-negative tumor cells. Our approach was to facilitate association of TSST1 with cell membranes by fusing its coding region to the transmembrane region (TM) sequence of the proto-oncogene c-erb-B-2. TSST1-TM was expressed in bacteria with an N-terminal histidine tag and purified using nickel-agarose affinity chromatography. Purified TSST1-TM added to cultures of several different MHC II-negative tumor cells spontaneously associated with cell membranes, as detected by flow cytometry. Because superantigens can direct cell-mediated cytotoxicity against MHC II-positive cells, a TM fusion protein lacking the TSST1 MHC II binding domain (TSST(88-194)-TM) was also constructed. Tumor cells precoated with TSST1-TM or TSST(88-194)-TM stimulated proliferation of human peripheral blood lymphocytes in vitro whereas uncoated tumor cells did not. Mice preimmunized with TSST1-TM- or TSST(88-194)-TM-coated tumor cells mounted a systemic response that resulted in significant antitumor immunity as measured by regression of a parental tumor challenge. TSST1-TM and TSST(88-194)-TM fusion proteins represent a useful new strategy for attaching superantigens or potentially other proteins onto tumor cell surfaces without genetic manipulation.

  20. Cocaine-elicited imbalances in ventromedial prefrontal cortex Homer1 versus Homer2 expression: Implications for relapse

    PubMed Central

    Gould, Adam T.; Sacramento, Arianne D.; Wroten, Melissa G.; Miller, Bailey W.; von Jonquieres, Georg; Klugmann, Matthias; Ben-Shahar, Osnat; Szumlinski, Karen K.

    2013-01-01

    Withdrawal from a history of extended access to self-administered cocaine produces a time-dependent intensification of drug-seeking, which might relate to a cocaine-induced imbalance in the relative expression of constitutively expressed Homer1 versus Homer2 isoforms within the ventromedial aspect of the prefrontal cortex (vmPFC). Thus, we employed immunoblotting to examine the relation between cue-reinforced lever-pressing at 3 versus 30 days withdrawal from a 10-day history of extended access (6 hrs/day) to intravenous cocaine (0.25 mg/infusion) or saline (Sal6h) and the expression of Homer1b/c and Homer2a/b within the vmPFC versus the more dorsomedial aspect of this structure (dmPFC). Behavioral studies employed adeno-associated viral vectors (AAVs) to reverse cocaine-elicited changes in the relative expression of Homer1 vs. Homer2 isoforms and tested animals for cocaine prime-, and cue-induced responding following extinction training. Cocaine self-administration elevated both Homer1b/c and Homer2a/b levels within the vmPFC at 3 days withdrawal and the rise in Homer2a/b persisted for at least 30 days. dmPFC Homer levels did not change as a function of self-administration history. Reversing the relative increase in Homer2 versus Homer1 expression via Homer1c over-expression or Homer2b knock-down failed to influence cue-reinforced lever-pressing when animals were tested in a drug-free state, but both AAV treatments prevented cocaine-primed reinstatement of lever-pressing behavior. These data suggest that a cocaine-elicited imbalance in the relative expression of constitutively expressed Homer2 versus Homer1 within the vmPFC is necessary for the capacity of cocaine to reinstate drug-seeking behavior, posing drug-induced changes in vmPFC Homer expression as a molecular trigger contributing to drug-elicited relapse. PMID:24118426

  1. 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.

  2. Ultraviolet Radiation-Elicited Enhancement of Isoflavonoid Accumulation, Biosynthetic Gene Expression, and Antioxidant Activity in Astragalus membranaceus Hairy Root Cultures.

    PubMed

    Jiao, Jiao; Gai, Qing-Yan; Wang, Wei; Luo, Meng; Gu, Cheng-Bo; Fu, Yu-Jie; Ma, Wei

    2015-09-23

    In this work, Astragalus membranaceus hairy root cultures (AMHRCs) were exposed to ultraviolet radiation (UV-A, UV-B, and UV-C) for promoting isoflavonoid accumulation. The optimum enhancement for isoflavonoid production was achieved in 34-day-old AMHRCs elicited by 86.4 kJ/m(2) of UV-B. The resulting isoflavonoid yield was 533.54 ± 13.61 μg/g dry weight (DW), which was 2.29-fold higher relative to control (232.93 ± 3.08 μg/g DW). UV-B up-regulated the transcriptional expressions of all investigated genes involved in isoflavonoid biosynthetic pathway. PAL and C4H were found to be two potential key genes that controlled isoflavonoid biosynthesis. Moreover, a significant increase was noted in antioxidant activity of extracts from UV-B-elicited AMHRCs (IC50 values = 0.85 and 1.08 mg/mL) in comparison with control (1.38 and 1.71 mg/mL). Overall, this study offered a feasible elicitation strategy to enhance isoflavonoid accumulation in AMHRCs and also provided a basis for metabolic engineering of isoflavonoid biosynthesis in the future.

  3. Cyclic GMP-dependent protein kinase and soluble guanylyl cyclase disappear in elicited rat neutrophils.

    PubMed

    Ciuman, Małgorzata; Siednienko, Jakub; Czyzyk, Rafał; Witwicka, Hanna; Kołosionek, Ewa; Kobiałka, Marcin; Gorczyca, Wojciech A

    2006-11-01

    The nitric oxide/soluble guanylyl cyclase/cGMP-dependent protein kinase (NO/sGC/PKG) cascade has been shown to affect important functions of circulating neutrophils. We demonstrate that neutrophils isolated from rats treated intraperitoneally with peptone protease cannot use this signaling pathway. Although PKG was detected at both the mRNA and protein levels in peripheral blood neutrophils (PBNs) of control rats, it was expressed neither in PBNs nor in peritoneal exudate neutrophils (PENs) of provoked rats. Also, mRNA of the alpha and beta chains of heterodimeric sGC was present in PBNs, but absent in PENs. Consistently, PBNs responded to activators of sGC with cGMP synthesis, while PENs did not. These results showed that neutrophils recruited by a provoking agent lost PKG and, in the case of PENs, also sGC and thus the capacity to respond to NO with cGMP signaling. We speculate that such downregulation of the sGC/PKG pathway is likely a result of the high activity of inducible NO synthase observed in inflammatory neutrophils.

  4. Adaptation Aftereffects in Vocal Emotion Perception Elicited by Expressive Faces and Voices

    PubMed Central

    Skuk, Verena G.; Schweinberger, Stefan R.

    2013-01-01

    The perception of emotions is often suggested to be multimodal in nature, and bimodal as compared to unimodal (auditory or visual) presentation of emotional stimuli can lead to superior emotion recognition. In previous studies, contrastive aftereffects in emotion perception caused by perceptual adaptation have been shown for faces and for auditory affective vocalization, when adaptors were of the same modality. By contrast, crossmodal aftereffects in the perception of emotional vocalizations have not been demonstrated yet. In three experiments we investigated the influence of emotional voice as well as dynamic facial video adaptors on the perception of emotion-ambiguous voices morphed on an angry-to-happy continuum. Contrastive aftereffects were found for unimodal (voice) adaptation conditions, in that test voices were perceived as happier after adaptation to angry voices, and vice versa. Bimodal (voice + dynamic face) adaptors tended to elicit larger contrastive aftereffects. Importantly, crossmodal (dynamic face) adaptors also elicited substantial aftereffects in male, but not in female participants. Our results (1) support the idea of contrastive processing of emotions (2), show for the first time crossmodal adaptation effects under certain conditions, consistent with the idea that emotion processing is multimodal in nature, and (3) suggest gender differences in the sensory integration of facial and vocal emotional stimuli. PMID:24236215

  5. Adaptation aftereffects in vocal emotion perception elicited by expressive faces and voices.

    PubMed

    Skuk, Verena G; Schweinberger, Stefan R

    2013-01-01

    The perception of emotions is often suggested to be multimodal in nature, and bimodal as compared to unimodal (auditory or visual) presentation of emotional stimuli can lead to superior emotion recognition. In previous studies, contrastive aftereffects in emotion perception caused by perceptual adaptation have been shown for faces and for auditory affective vocalization, when adaptors were of the same modality. By contrast, crossmodal aftereffects in the perception of emotional vocalizations have not been demonstrated yet. In three experiments we investigated the influence of emotional voice as well as dynamic facial video adaptors on the perception of emotion-ambiguous voices morphed on an angry-to-happy continuum. Contrastive aftereffects were found for unimodal (voice) adaptation conditions, in that test voices were perceived as happier after adaptation to angry voices, and vice versa. Bimodal (voice + dynamic face) adaptors tended to elicit larger contrastive aftereffects. Importantly, crossmodal (dynamic face) adaptors also elicited substantial aftereffects in male, but not in female participants. Our results (1) support the idea of contrastive processing of emotions (2), show for the first time crossmodal adaptation effects under certain conditions, consistent with the idea that emotion processing is multimodal in nature, and (3) suggest gender differences in the sensory integration of facial and vocal emotional stimuli.

  6. A region of the N-terminal domain of meningococcal factor H-binding protein that elicits bactericidal antibody across antigenic variant groups.

    PubMed

    Beernink, Peter T; LoPasso, Carla; Angiolillo, Antonella; Felici, Franco; Granoff, Dan

    2009-05-01

    Meningococcal factor H-binding protein (fHbp) is a promising vaccine antigen. Previous studies described three fHbp antigenic variant groups and identified amino acid residues between 100 and 255 as important targets of variant-specific bactericidal antibodies. We investigated residues affecting expression of an epitope recognized by a murine IgG2a anti-fHbp mAb, designated JAR 4, which cross-reacted with fHbps in variant group 1 or 2 (95% of strains), and elicited human complement-mediated, cooperative bactericidal activity with other non-bactericidal anti-fHbp mAbs with epitopes involving residues between 121 and 216. From filamentous bacteriophage libraries containing random peptides that were recognized by JAR 4, we identified a consensus tripeptide, DHK that matched residues 25-27 in the N-terminal domain of fHbp. Since DHK was present in both JAR 4-reactive and non-reactive fHbps, the tripeptide was necessary but not sufficient for reactivity. Based on site-directed mutagenesis studies, the JAR 4 epitope could either be knocked out of a reactive variant 1 fHbp, or introduced into a non-reactive variant 3 protein. Collectively, the data indicated that the JAR 4 epitope was discontinuous and involved DHK residues beginning at position 25; YGN residues beginning at position 57; and a KDN tripeptide that was present in variant 3 proteins beginning at position 67 that negatively affected expression of the epitope. Thus, the region of fHbp encompassing residues 25-59 in the N-terminal domain is important for eliciting antibodies that can cooperate with other anti-fHbp antibodies for cross-reactive bactericidal activity against strains expressing fHbp from different antigenic variant groups.

  7. Induction of trans-resveratrol and extracellular pathogenesis-related proteins in elicited suspension cultured cells of Vitis vinifera cv Monastrell.

    PubMed

    Belchí-Navarro, Sarai; Almagro, Lorena; Sabater-Jara, Ana Belén; Fernández-Pérez, Francisco; Bru, Roque; Pedreño, Maria Angeles

    2013-02-15

    Suspension-cultured cells of Vitis vinifera cv Monastrell were used to investigate the effects of methyljasmonate, ethylene and salicylic acid separately or in combination with cyclodextrins on both trans-resveratrol production and the induction of defense responses. The results showed that the addition of methyljasmonate or ethylene to suspension-cultured cells jointly treated with cyclodextrins and salicylic acid provoked a decrease of trans-resveratrol levels suggesting that salicylic acid has a negative and antagonistic effect with methyljasmonate or ethylene on trans-resveratrol production. Likewise, the exogenous application of these compounds induced the accumulation of pathogenesis-related proteins. Analysis of the extracellular proteome showed the presence of amino acid sequences homologous to an specific β-1,3-glucanase, class III peroxidases and a β-1,4-mannanase, which suggests that these signal molecules could play a role in mediating defense-related gene product expression in V. vinifera cv Monastrell. Apart from these inducible proteins, other proteins were found in both the control and elicited cell cultures of V. vinifera. These included class IV chitinase, polygalacturonase inhibitor protein and reticuline oxidase-like protein, suggesting that their expression is constitutive being involved in the modification of the cell wall architecture during cell culture growth and in the prevention of pathogen attack. Copyright © 2012 Elsevier GmbH. All rights reserved.

  8. Cyclic Lipopeptides from Bacillus subtilis ABS–S14 Elicit Defense-Related Gene Expression in Citrus Fruit

    PubMed Central

    Waewthongrak, Waewruedee; Leelasuphakul, Wichitra; McCollum, Greg

    2014-01-01

    Effects of cyclic lipopeptides (CLPs) obtained from Bacillus subtilis ABS-S14 on eliciting defense-related gene transcription and activity of defense-related enzymes; glucanase (GLU), chitinase (CHI), peroxidase (POX) and lipoxygenase (LOX) in Citrus sinensis cv. Valencia fruit were determined. The maximum level of GLU transcripts induced in fruit treated with fengycin was significantly greatest among treatments at 48 h. Surfactin enhanced the LOX and POX transcripts. In parallel, corresponding enzyme activities were correlated with changes in gene expression observed in fruit inoculated with Penicillium digitatum following treatment with individual CLPs. Synergistic effects of fengycin and iturin A, fengycin and surfactin were shown in gene transcript of GLU and CHI, respectively, and surfactin induced POX and LOX gene expression of citrus flavedo without pathogen infection. These results suggest that fengycin and surfactin act as elicitors of defense-related gene expression in “Valencia” fruit following infection. PMID:25329301

  9. Cyclic LIPopeptides from Bacillus subtilis ABS-S14 elicit defense-related gene expression in citrus fruit.

    PubMed

    Waewthongrak, Waewruedee; Leelasuphakul, Wichitra; McCollum, Greg

    2014-01-01

    Effects of cyclic lipopeptides (CLPs) obtained from Bacillus subtilis ABS-S14 on eliciting defense-related gene transcription and activity of defense-related enzymes; glucanase (GLU), chitinase (CHI), peroxidase (POX) and lipoxygenase (LOX) in Citrus sinensis cv. Valencia fruit were determined. The maximum level of GLU transcripts induced in fruit treated with fengycin was significantly greatest among treatments at 48 h. Surfactin enhanced the LOX and POX transcripts. In parallel, corresponding enzyme activities were correlated with changes in gene expression observed in fruit inoculated with Penicillium digitatum following treatment with individual CLPs. Synergistic effects of fengycin and iturin A, fengycin and surfactin were shown in gene transcript of GLU and CHI, respectively, and surfactin induced POX and LOX gene expression of citrus flavedo without pathogen infection. These results suggest that fengycin and surfactin act as elicitors of defense-related gene expression in "Valencia" fruit following infection.

  10. Linoleic acid and stearic acid elicit opposite effects on AgRP expression and secretion via TLR4-dependent signaling pathways in immortalized hypothalamic N38 cells.

    PubMed

    Wang, Songbo; Xiang, Nana; Yang, Liusong; Zhu, Canjun; Zhu, Xiaotong; Wang, Lina; Gao, Ping; Xi, Qianyun; Zhang, Yongliang; Shu, Gang; Jiang, Qingyan

    2016-03-18

    The regulation of food intake is a promising way to combat obesity. It has been implicated that various fatty acids exert different effects on food intake and body weight. However, the underlying mechanism remains poorly understood. The aim of the present study was to investigate the effects of linoleic acid (LA) and stearic acid (SA) on agouti-related protein (AgRP) expression and secretion in immortalized mouse hypothalamic N38 cells and to explore the likely underlying mechanisms. Our results demonstrated that LA inhibited, while SA stimulated AgRP expression and secretion of N38 cells in a dose-dependent manner. In addition, LA suppressed the protein expression of toll-like receptor 4 (TLR4), phosphorylation levels of JNK and IKKα/β, suggesting the inhibition of TLR4-dependent inflammation pathway. However, the above mentioned inhibitory effects of LA were eliminated by TLR4 agonist lipopolysaccharide (LPS). In contrast, SA promoted TLR4 protein expression and activated TLR4-dependent inflammation pathway, with elevated ratio of p-JNK/JNK. While TLR4 siRNA reversed the stimulatory effects of SA on AgRP expression and TLR4-dependent inflammation. Moreover, we found that TLR4 was also involved in LA-enhanced and SA-impaired leptin/insulin signal pathways in N38 cells. In conclusion, our findings indicated that LA elicited inhibitory while SA exerted stimulatory effects on AgRP expression and secretion via TLR4-dependent inflammation and leptin/insulin pathways in N38 cells. These data provided a better understanding of the mechanism underlying fatty acids-regulated food intake and suggested the potential role of long-chain unsaturated fatty acids such as LA in reducing food intake and treating obesity.

  11. Bactericidal antibody responses elicited by a meningococcal outer membrane vesicle vaccine with overexpressed factor H-binding protein and genetically attenuated endotoxin.

    PubMed

    Koeberling, Oliver; Seubert, Anja; Granoff, Dan M

    2008-07-15

    Outer membrane vesicle (OMV) vaccines from mutant Neisseria meningitidis strains engineered to overexpress factor H-binding protein (fHbp) have elicited broadly protective serum antibody responses in mice. The vaccines investigated were not treated with detergents to avoid extracting fHbp, which is a lipoprotein. Because of their high endotoxin content, the vaccines would not be safe to administer to humans. We prepared a native OMV vaccine from a strain engineered to overexpress fHbp and in which the gene encoding LpxL1 was inactivated, which reportedly decreases endotoxin activity. The OMV vaccine from the mutant had a similar or lower ability to induce the expression of proinflammatory cytokines by human peripheral blood mononuclear cells, compared with a detergent-extracted wild-type OMV, and 1000-10,000-fold lower activity than a native wild-type OMV. In mice, the OMV vaccine from the mutant elicited higher serum bactericidal antibody responses to a panel of heterologous N. meningitidis strains than did a control multicomponent recombinant protein vaccine or a detergent-extracted OMV vaccine that has been demonstrated to confer protection against meningococcal disease in humans. The data illustrate the potential to develop a broadly immunogenic native OMV vaccine that has decreased endotoxin activity and is potentially suitable for testing in humans.

  12. The Pseudomonas syringae pv. syringae 61 hrpH product, an envelope protein required for elicitation of the hypersensitive response in plants.

    PubMed Central

    Huang, H C; He, S Y; Bauer, D W; Collmer, A

    1992-01-01

    Pseudomonas syringae pv. syringae 61 contains a 25-kb cluster of hrp genes that are required for elicitation of the hypersensitive response (HR) in tobacco. TnphoA mutagenesis of cosmid pHIR11, which contains the hrp cluster, revealed two genes encoding exported or inner-membrane-spanning proteins (H.-C. Huang, S. W. Hutcheson, and A. Collmer, Mol. Plant-Microbe Interact. 4:469-476, 1991). The gene in complementation group X, designated hrpH, was subcloned on a 3.1-kb SalI fragment into pCPP30, a broad-host-range, mobilizable vector. The subclone restored the ability of hrpH mutant P. syringae pv. syringae 61-2089 to elicit the HR in tobacco. DNA sequence analysis of the 3.1-kb SalI fragment revealed a single open reading frame encoding an 81,956-Da preprotein with a typical amino-terminal signal peptide and no likely inner-membrane-spanning hydrophobic regions. hrpH was expressed in the presence of [35S]methionine by using the T7 RNA polymerase-promoter system and vector pT7-3 in Escherichia coli and was shown to encode a protein with an apparent molecular weight of 83,000 on sodium dodecyl sulfate-polyacrylamide gels. The HrpH protein in E. coli was located in the membrane fraction and was absent from the periplasm and cytoplasm. The HrpH protein possessed similarity with several outer membrane proteins that are known to be involved in protein or phage secretion, including the Klebsiella oxytoca PulD protein, the Yersinia enterocolitica YscC protein, and the pIV protein of filamentous coliphages. All of these proteins possess a possible secretion motif, GG(X)12VP(L/F)LXXIPXIGXL(F/L), near the carboxyl terminus, and they lack a carboxyl-terminal phenylalanine, in contrast to other outer membrane proteins with no known secretion function. These results suggest that the P. syringae pv. syringae HrpH protein is involved in the secretion of a proteinaceous HR elicitor. Images PMID:1400238

  13. Vaccination with a recombinant Saccharomyces cerevisiae expressing a tumor antigen breaks immune tolerance and elicits therapeutic antitumor responses

    PubMed Central

    Wansley, Elizabeth K.; Chakraborty, Mala; Hance, Kenneth W.; Bernstein, Michael B.; Boehm, Amanda L.; Guo, Zhimin; Quick, Deborah; Franzusoff, Alex; Greiner, John W.; Schlom, Jeffrey; Hodge, James W.

    2009-01-01

    Purpose Saccharomyces cerevisiae, a nonpathogenic yeast, has previously been used as a vehicle to elicit immune responses to foreign antigens, and tumor-associated antigens, and has been shown to reduce tumorburden in mice. Studies were designed to determine if vaccination of human carcinoembryonic antigen (CEA)-transgenic mice (where CEA is a self-antigen) with a recombinant S. cerevisiae construct expressing human CEA (yeast-CEA) elicits CEA-specific T-cell responses and antitumor activity. Experimental Design CEA-transgenic mice were vaccinated with yeast-CEA, and CD4+ and CD8+ T-cell responses were assessed after one and multiple administrations or vaccinations at multiple sites per administration. Antitumor activity was determined by tumor growth and overall survival in both pulmonary metastasis and subcutaneous pancreatic tumor models. Results These studies demonstrate that recombinant yeast can break tolerance and that a) yeast-CEA constructs elicit both CEA-specific CD4+ and CD8+ T-cell responses; b) repeated yeast-CEA administration causes increased antigen-specific T-cell responses after each vaccination; c) vaccination with yeast-CEA at multiple sites induces a greater T-cell response than the same dose given at a single site; d) tumor-bearing mice vaccinated with yeast-CEA show a reduction in tumor burden and increased overall survival compared to mock-treated or control yeast-vaccinated mice in both pulmonary metastasis and subcutaneous pancreatic tumor models. Conclusions Vaccination with a heat-killed recombinant yeast expressing the tumor-associated antigen CEA induces CEA-specific immune responses, reduces tumor burden, and extends overall survival in CEA-transgenic mice. These studies thus form the rationale for the incorporation of recombinant yeast-CEA and other recombinant yeast constructs in cancer immunotherapy protocols. PMID:18594015

  14. Schistosoma mansoni Infection of Mice, Rats and Humans Elicits a Strong Antibody Response to a Limited Number of Reduction-Sensitive Epitopes on Five Major Tegumental Membrane Proteins

    PubMed Central

    Tremblay, Jacqueline M.; Oliveira, Sergio C.; Da’dara, Akram A.; Skelly, Patrick J.

    2017-01-01

    Schistosomiasis is a major disease of the developing world for which no vaccine has been successfully commercialized. While numerous Schistosoma mansoni worm antigens have been identified that elicit antibody responses during natural infections, little is known as to the identities of the schistosome antigens that are most prominently recognized by antibodies generated through natural infection. Non-reducing western blots probed with serum from schistosome-infected mice, rats and humans on total extracts of larval or adult schistosomes revealed that a small number of antigen bands predominate in all cases. Recognition of each of these major bands was lost when the blots were run under reducing condition. We expressed a rationally selected group of schistosome tegumental membrane antigens in insect host cells, and used the membrane extracts of these cells to unambiguously identify the major antigens recognized by S. mansoni infected mouse, rat and human serum. These results revealed that a limited number of dominant, reduction-sensitive conformational epitopes on five major tegumental surface membrane proteins: SmTsp2, Sm23, Sm29, SmLy6B and SmLy6F, are primary targets of mouse, rat and human S. mansoni infection sera antibodies. We conclude that, Schistosoma mansoni infection of both permissive (mouse) and non-permissive (rat) rodent models, as well as humans, elicit a dominant antibody response recognizing a limited number of conformational epitopes on the same five tegumental membrane proteins. Thus it appears that neither infecting schistosomula nor mature adult schistosomes are substantively impacted by the robust circulating anti-tegumental antibody response they elicit to these antigens. Importantly, our data suggest a need to re-evaluate host immune responses to many schistosome antigens and has important implications regarding schistosome immune evasion mechanisms and schistosomiasis vaccine development. PMID:28095417

  15. Wounding tomato fruit elicits ripening-stage specific changes in gene expression and production of volatile compounds

    PubMed Central

    Baldassarre, Valentina; Cabassi, Giovanni; Spadafora, Natasha D.; Aprile, Alessio; Müller, Carsten T.; Rogers, Hilary J.; Ferrante, Antonio

    2015-01-01

    Fleshy fruits develop from an unripe organ that needs to be protected from damage to a ripe organ that attracts frugivores for seed dispersal through production of volatile organic compounds (VOCs). Thus, different responses to wounding damage are predicted. The aim of this study was to discover whether wound-induced changes in the transcriptome and VOC production alter as tomato transitions from unripe to ripe. Transcript changes were analysed 3h post-wounding using microarray analysis in two commercial salad-tomato (Solanum lycopersicum L.) cultivars: Luna Rossa and AVG, chosen for their high aroma production. This was followed by quantitative PCR on Luna Rossa genes involved in VOC biosynthesis and defence responses. VOCs elicited by wounding at different ripening stages were analysed by solid phase micro extraction and gas chromatography–mass spectrometry. Approximately 4000 differentially expressed genes were identified in the cultivar AVG and 2500 in Luna Rossa. In both cultivars the majority of genes were up-regulated and the most affected pathways were metabolism of terpenes, carotenoids, and lipids. Defence-related genes were mostly up-regulated in immature stages of development, whereas expression of genes related to VOCs changed at riper stages. More than 40 VOCs were detected and profiles changed with ripening stage. Thus, both transcriptome and VOC profiles elicited by wounding depend on stage of ripening, indicating a shift from defence to attraction. PMID:25614658

  16. Wounding tomato fruit elicits ripening-stage specific changes in gene expression and production of volatile compounds.

    PubMed

    Baldassarre, Valentina; Cabassi, Giovanni; Spadafora, Natasha D; Aprile, Alessio; Müller, Carsten T; Rogers, Hilary J; Ferrante, Antonio

    2015-03-01

    Fleshy fruits develop from an unripe organ that needs to be protected from damage to a ripe organ that attracts frugivores for seed dispersal through production of volatile organic compounds (VOCs). Thus, different responses to wounding damage are predicted. The aim of this study was to discover whether wound-induced changes in the transcriptome and VOC production alter as tomato transitions from unripe to ripe. Transcript changes were analysed 3h post-wounding using microarray analysis in two commercial salad-tomato (Solanum lycopersicum L.) cultivars: Luna Rossa and AVG, chosen for their high aroma production. This was followed by quantitative PCR on Luna Rossa genes involved in VOC biosynthesis and defence responses. VOCs elicited by wounding at different ripening stages were analysed by solid phase micro extraction and gas chromatography-mass spectrometry. Approximately 4000 differentially expressed genes were identified in the cultivar AVG and 2500 in Luna Rossa. In both cultivars the majority of genes were up-regulated and the most affected pathways were metabolism of terpenes, carotenoids, and lipids. Defence-related genes were mostly up-regulated in immature stages of development, whereas expression of genes related to VOCs changed at riper stages. More than 40 VOCs were detected and profiles changed with ripening stage. Thus, both transcriptome and VOC profiles elicited by wounding depend on stage of ripening, indicating a shift from defence to attraction.

  17. Post-translational intracellular trafficking determines the type of immune response elicited by DNA vaccines expressing Gag antigen of Human Immunodeficiency Virus Type 1 (HIV-1).

    PubMed

    Wallace, Aaron; West, Kim; Rothman, Alan L; Ennis, Francis A; Lu, Shan; Wang, Shixia

    2013-10-01

    In the current study, immune responses induced by Gag DNA vaccines with different designs were evaluated in Balb/C mice. The results demonstrated that the DNA vaccine with the full length wild type gag gene (Wt-Gag) mainly produced Gag antigens intracellularly and induced a higher level of cell-mediated immune (CMI) responses, as measured by IFN-gamma ELISPOT, intracellular cytokine staining (ICS), and cytotoxic T lymphocytes (CTL) assays against a dominant CD8(+) T cell epitope (AMQMLKETI). In contrast, the addition of a tissue plasminogen activator (tPA) leader sequence significantly improved overall Gag protein expression/secretion and Gag-specific antibody responses; however, Gag-specific CMI responses were decreased. The mutation of zinc-finger motif changed Gag protein expression patterns and reduced the ability to generate both CMI and antibody responses against Gag. These findings indicate that the structure and post-translational processing of antigens expressed by DNA vaccines play a critical role in eliciting optimal antibody or CMI responses.

  18. Post-translational intracellular trafficking determines the type of immune response elicited by DNA vaccines expressing Gag antigen of Human Immunodeficiency Virus Type 1 (HIV-1)

    PubMed Central

    Wallace, Aaron; West, Kim; Rothman, Alan L; Ennis, Francis A; Lu, Shan; Wang, Shixia

    2013-01-01

    In the current study, immune responses induced by Gag DNA vaccines with different designs were evaluated in Balb/C mice. The results demonstrated that the DNA vaccine with the full length wild type gag gene (Wt-Gag) mainly produced Gag antigens intracellularly and induced a higher level of cell-mediated immune (CMI) responses, as measured by IFN-gamma ELISPOT, intracellular cytokine staining (ICS), and cytotoxic T lymphocytes (CTL) assays against a dominant CD8+ T cell epitope (AMQMLKETI). In contrast, the addition of a tissue plasminogen activator (tPA) leader sequence significantly improved overall Gag protein expression/secretion and Gag-specific antibody responses; however, Gag-specific CMI responses were decreased. The mutation of zinc-finger motif changed Gag protein expression patterns and reduced the ability to generate both CMI and antibody responses against Gag. These findings indicate that the structure and post-translational processing of antigens expressed by DNA vaccines play a critical role in eliciting optimal antibody or CMI responses. PMID:23941868

  19. Overproduction of a Model Sec- and Tat-Dependent Secretory Protein Elicits Different Cellular Responses in Streptomyces lividans.

    PubMed

    Gullón, Sonia; Marín, Silvia; Mellado, Rafael P

    2015-01-01

    Streptomyces lividans is considered an efficient host for the secretory production of homologous and heterologous proteins. To identify possible bottlenecks in the protein production process, a comparative transcriptomic approach was adopted to study cellular responses during the overproduction of a Sec-dependent model protein (alpha-amylase) and a Tat-dependent model protein (agarase) in Streptomyces lividans. The overproduction of the model secretory proteins via the Sec or the Tat route in S. lividans does elicit a different major cell response in the bacterium. The stringent response is a bacterial response to nutrients' depletion, which naturally occurs at late times of the bacterial cell growth. While the induction of the stringent response at the exponential phase of growth may limit overall productivity in the case of the Tat route, the induction of that response does not take place in the case of the Sec route, which comparatively is an advantage in secretory protein production processes. Hence, this study identifies a potential major drawback in the secretory protein production process depending on the secretory route, and provides clues to improving S. lividans as a protein production host.

  20. Overproduction of a Model Sec- and Tat-Dependent Secretory Protein Elicits Different Cellular Responses in Streptomyces lividans

    PubMed Central

    Gullón, Sonia; Marín, Silvia; Mellado, Rafael P.

    2015-01-01

    Streptomyces lividans is considered an efficient host for the secretory production of homologous and heterologous proteins. To identify possible bottlenecks in the protein production process, a comparative transcriptomic approach was adopted to study cellular responses during the overproduction of a Sec-dependent model protein (alpha-amylase) and a Tat-dependent model protein (agarase) in Streptomyces lividans. The overproduction of the model secretory proteins via the Sec or the Tat route in S. lividans does elicit a different major cell response in the bacterium. The stringent response is a bacterial response to nutrients’ depletion, which naturally occurs at late times of the bacterial cell growth. While the induction of the stringent response at the exponential phase of growth may limit overall productivity in the case of the Tat route, the induction of that response does not take place in the case of the Sec route, which comparatively is an advantage in secretory protein production processes. Hence, this study identifies a potential major drawback in the secretory protein production process depending on the secretory route, and provides clues to improving S. lividans as a protein production host. PMID:26200356

  1. Protein synthesis rate is the predominant regulator of protein expression during differentiation

    PubMed Central

    Kristensen, Anders R; Gsponer, Joerg; Foster, Leonard J

    2013-01-01

    External perturbations, by forcing cells to adapt to a new environment, often elicit large-scale changes in gene expression resulting in an altered proteome that improves the cell's fitness in the new conditions. Steady-state levels of a proteome depend on transcription, the levels of transcripts, translation and protein degradation but system-level contribution that each of these processes make to the final protein expression change has yet to be explored. We therefore applied a systems biology approach to characterize the regulation of protein expression during cellular differentiation using quantitative proteomics. As a general rule, it seems that protein expression during cellular differentiation is largely controlled by changes in the relative synthesis rate, whereas the relative degradation rate of the majority of proteins stays constant. In these data, we also observe that the proteins in defined sub-structures of larger protein complexes tend to have highly correlated synthesis and degradation rates but that this does not necessarily extend to the holo-complex. Finally, we provide strong evidence that the generally poor correlation observed between transcript and protein levels can fully be explained once the protein synthesis and degradation rates are taken into account. PMID:24045637

  2. Children's Prototypic Facial Expressions During Emotion-Eliciting Conversations With Their Mothers.

    PubMed

    Castro, Vanessa L; Camras, Linda A; Halberstadt, Amy G; Shuster, Michael

    2017-07-17

    Despite theoretical claims that emotions are primarily communicated through prototypic facial expressions, empirical evidence is surprisingly scarce. This study aimed to (a) test whether children produced more components of a prototypic emotional facial expression during situations judged or self-reported to involve the corresponding emotion than situations involving other emotions (termed "intersituational specificity"), (b) test whether children produced more components of the prototypic expression corresponding to a situation's judged or self-reported emotion than components of other emotional expressions (termed "intrasituational specificity"), and (c) examine coherence between children's self-reported emotional experience and observers' judgments of children's emotions. One hundred and 20 children (ages 7-9) were video-recorded during a discussion with their mothers. Emotion ratings were obtained for children in 441 episodes. Children's nonverbal behaviors were judged by observers and coded by FACS-trained researchers. Children's self-reported emotion corresponded significantly to observers' judgments of joy, anger, fear, and sadness but not surprise. Multilevel modeling results revealed that children produced joy facial expressions more in joy episodes than nonjoy episodes (supporting intersituational specificity for joy) and more joy and surprise expressions than other emotional expressions in joy and surprise episodes (supporting intrasituational specificity for joy and surprise). However, children produced anger, fear, and sadness expressions more in noncorresponding episodes and produced these expressions less than other expressions in corresponding episodes. Findings suggest that communication of negative emotion during social interactions-as indexed by agreement between self-report and observer judgments-may rely less on prototypic facial expressions than is often theoretically assumed. (PsycINFO Database Record (c) 2017 APA, all rights reserved).

  3. Data Mining for Expressivity of Recombinant Protein Expression

    NASA Astrophysics Data System (ADS)

    Kira, Satoshi; Isoai, Atsushi; Yamamura, Masayuki

    We analyzed the expressivity of recombinant proteins by using data mining methods. The expression technique of recombinant protein is a key step towards elucidating the functions of genes discovered through genomic sequence projects. We have studied the productive efficiency of recombinant proteins in fission yeast, Schizosaccharomyces pombe (S.pombe), by mining the expression results. We gathered 57 proteins whose expression levels were known roughly in the host. Correlation analysis, principal component analysis and decision tree analysis were applied to these expression data. Analysis featuring codon usage and amino acid composition clarified that the amino acid composition affected to the expression levels of a recombinant protein strongly than the effect of codon usage. Furthermore, analysis of amino acid composition showed that protein solubility and the metabolism cost of amino acids correlated with a protein expressivity. Codon usage was often interesting in the field of recombinant expressions. However, our analysis found the weak correlation codon features with expressivities. These results indicated that ready-made indices of codon bias were irrelevant ones for modeling the expressivities of recombinant proteins. Our data driven approach was an easy and powerful method to improve recombinant protein expression, and this approach should be concentrated attention with the huge amount of expression data accumulating through the post-genome era.

  4. Protein status elicits compensatory changes in food intake and food preferences.

    PubMed

    Griffioen-Roose, Sanne; Mars, Monica; Siebelink, Els; Finlayson, Graham; Tomé, Daniel; de Graaf, Cees

    2012-01-01

    Protein is an indispensable component within the human diet. It is unclear, however, whether behavioral strategies exist to avoid shortages. The objective was to investigate the effect of a low protein status compared with a high protein status on food intake and food preferences. We used a randomized crossover design that consisted of a 14-d fully controlled dietary intervention involving 37 subjects [mean ± SD age: 21 ± 2 y; BMI (in kg/m(2)): 21.9 ± 1.5] who consumed individualized, isoenergetic diets that were either low in protein [0.5 g protein · kg body weight (BW)(-1) · d(-1)] or high in protein (2.0 g protein · kg BW(-1) · d(-1)). The diets were followed by an ad libitum phase of 2.5 d, during which a large array of food items was available, and protein and energy intakes were measured. We showed that in the ad libitum phase protein intake was 13% higher after the low-protein diet than after the high-protein diet (253 ± 70 compared with 225 ± 63 g, P < 0.001), whereas total energy intake was not different. The higher intake of protein was evident throughout the ad libitum phase of 2.5 d. In addition, after the low-protein diet, food preferences for savory high-protein foods were enhanced. After a protein deficit, food intake and food preferences show adaptive changes that suggest that compensatory mechanisms are induced to restore adequate protein status. This indicates that there are human behavioral strategies present to avoid protein shortage and that these involve selection of savory high-protein foods. This trial was registered with the Dutch Trial register at http://www.trialregister.nl as NTR2491.

  5. Protein status elicits compensatory changes in food intake and food preferences123

    PubMed Central

    Mars, Monica; Siebelink, Els; Finlayson, Graham; Tomé, Daniel; de Graaf, Cees

    2012-01-01

    Background: Protein is an indispensable component within the human diet. It is unclear, however, whether behavioral strategies exist to avoid shortages. Objective: The objective was to investigate the effect of a low protein status compared with a high protein status on food intake and food preferences. Design: We used a randomized crossover design that consisted of a 14-d fully controlled dietary intervention involving 37 subjects [mean ± SD age: 21 ± 2 y; BMI (in kg/m2): 21.9 ± 1.5] who consumed individualized, isoenergetic diets that were either low in protein [0.5 g protein · kg body weight (BW)−1 · d−1] or high in protein (2.0 g protein · kg BW−1 · d−1). The diets were followed by an ad libitum phase of 2.5 d, during which a large array of food items was available, and protein and energy intakes were measured. Results: We showed that in the ad libitum phase protein intake was 13% higher after the low-protein diet than after the high-protein diet (253 ± 70 compared with 225 ± 63 g, P < 0.001), whereas total energy intake was not different. The higher intake of protein was evident throughout the ad libitum phase of 2.5 d. In addition, after the low-protein diet, food preferences for savory high-protein foods were enhanced. Conclusions: After a protein deficit, food intake and food preferences show adaptive changes that suggest that compensatory mechanisms are induced to restore adequate protein status. This indicates that there are human behavioral strategies present to avoid protein shortage and that these involve selection of savory high-protein foods. This trial was registered with the Dutch Trial register at http://www.trialregister.nl as NTR2491. PMID:22158729

  6. Elevated myocardial Na+/H+ exchanger isoform 1 activity elicits gene expression that leads to cardiac hypertrophy

    PubMed Central

    Xue, Jin; Mraiche, Fatima; Zhou, Dan; Karmazyn, Morris; Oka, Tatsujiro; Fliegel, Larry

    2010-01-01

    In myocardial disease, elevated expression and activity of Na+/H+ exchanger isoform 1 (NHE1) are detrimental. To better understand the involvement of NHE1, transgenic mice with elevated heart-specific NHE1 expression were studied. N-line mice expressed wild-type NHE1, and K-line mice expressed activated NHE1. Cardiac morphology, interstitial fibrosis, and cardiac function were examined by histological staining and echocardiography. Differences in gene expression between the N-line or K-line and nontransgenic littermates were probed with genechip analysis. We found that NHE1 K-line (but not N-line) hearts developed hypertrophy, including elevated heart weight-to-body weight ratio and increased cross-sectional area of the cardiomyocytes, interstitial fibrosis, as well as depressed cardiac function. N-line hearts had modest changes in gene expression (50 upregulations and 99 downregulations, P < 0.05), whereas K-line hearts had a very strong transcriptional response (640 upregulations and 677 downregulations, P < 0.05). In addition, the magnitude of expression alterations was much higher in K-line than N-line mice. The most significant changes in gene expression were involved in cardiac hypertrophy, cardiac necrosis/cell death, and cardiac infarction. Secreted phosphoprotein 1 and its signaling pathways were upregulated while peroxisome proliferator-activated receptor γ signaling was downregulated in K-line mice. Our study shows that expression of activated NHE1 elicits specific pathways of gene activation in the myocardium that lead to cardiac hypertrophy, cell death, and infarction. PMID:20460605

  7. Expressive suppression and enhancement during music-elicited emotions in younger and older adults

    PubMed Central

    Vieillard, Sandrine; Harm, Jonathan; Bigand, Emmanuel

    2015-01-01

    When presented with emotional visual scenes, older adults have been found to be equally capable to regulate emotion expression as younger adults, corroborating the view that emotion regulation skills are maintained or even improved in later adulthood. However, the possibility that gaze direction might help achieve an emotion control goal has not been taken into account, raising the question whether the effortful processing of expressive regulation is really spared from the general age-related decline. Since it does not allow perceptual attention to be redirected away from the emotional source, music provides a useful way to address this question. In the present study, affective, behavioral, and physiological consequences of free expression of emotion, expressive suppression and expressive enhancement were measured in 31 younger and 30 older adults while they listened to positive and negative musical excerpts. The main results indicated that compared to younger adults, older adults reported experiencing less emotional intensity in response to negative music during the free expression of emotion condition. No age difference was found in the ability to amplify or reduce emotional expressions. However, an age-related decline in the ability to reduce the intensity of emotional state and an age-related increase in physiological reactivity were found when participants were instructed to suppress negative expression. Taken together, the current data support previous findings suggesting an age-related change in response to music. They also corroborate the observation that older adults are as efficient as younger adults at controlling behavioral expression. But most importantly, they suggest that when faced with auditory sources of negative emotion, older age does not always confer a better ability to regulate emotions. PMID:25741278

  8. Rotavirus VP7 epitope chimeric proteins elicit cross-immunoreactivity in guinea pigs.

    PubMed

    Zhao, Bingxin; Pan, Xiaoxia; Teng, Yumei; Xia, Wenyue; Wang, Jing; Wen, Yuling; Chen, Yuanding

    2015-10-01

    VP7 of group A rotavirus (RVA) contains major neutralizing epitopes. Using the antigenic protein VP6 as the vector, chimeric proteins carrying foreign epitopes have been shown to possess good immunoreactivity and immunogenicity. In the present study, using modified VP6 as the vector, three chimeric proteins carrying epitopes derived from VP7 of RVA were constructed. The results showed that the chimeric proteins reacted with anti-VP6 and with SA11 and Wa virus strains. Antibodies from guinea pigs inoculated with the chimeric proteins recognized VP6 and VP7 of RVA and protected mammalian cells from SA11 and Wa infection in vitro. The neutralizing activities of the antibodies against the chimeric proteins were significantly higher than those against the vector protein VP6F. Thus, development of chimeric vaccines carrying VP7 epitopes using VP6 as a vector could be a promising alternative to enhance immunization against RVAs.

  9. Microarray analysis of differential gene expression elicited in Trametes versicolor during interspecific mycelial interactions.

    PubMed

    Eyre, Catherine; Muftah, Wafa; Hiscox, Jennifer; Hunt, Julie; Kille, Peter; Boddy, Lynne; Rogers, Hilary J

    2010-08-01

    Trametes versicolor is an important white rot fungus of both industrial and ecological interest. Saprotrophic basidiomycetes are the major decomposition agents in woodland ecosystems, and rarely form monospecific populations, therefore interspecific mycelial interactions continually occur. Interactions have different outcomes including replacement of one species by the other or deadlock. We have made subtractive cDNA libraries to enrich for genes that are expressed when T. versicolor interacts with another saprotrophic basidiomycete, Stereum gausapatum, an interaction that results in the replacement of the latter. Expressed sequence tags (ESTs) (1920) were used for microarray analysis, and their expression compared during interaction with three different fungi: S. gausapatum (replaced by T. versicolor), Bjerkandera adusta (deadlock) and Hypholoma fasciculare (replaced T. versicolor). Expression of significantly more probes changed in the interaction between T. versicolor and S. gausapatum or B. adusta compared to H. fasciculare, suggesting a relationship between interaction outcome and changes in gene expression. Copyright © 2010 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

  10. Satellite panicum mosaic virus capsid protein elicits symptoms on a nonhost plant and interferes with a suppressor of virus-induced gene silencing.

    PubMed

    Qiu, Wenping; Scholthof, Karen-Beth G

    2004-03-01

    The capsid protein (CP) of satellite panicum mosaic virus (SPMV) has been implicated as a pathogenicity factor, inducing severe chlorosis on millet plants co-infected with SPMV and its helper virus, Panicum mosaic virus (PMV). In this study, we tested the effects of SPMV CP on Nicotiana benthamiana, a plant that does not support PMV+SPMV infections. SPMV CP expressed from a Potato virus X (PVX) gene vector elicited necrotic lesions on N. benthamiana. Pathogenicity factors often have the additional feature of acting as suppressors of gene silencing; therefore, several assays were developed to test if SPMV CP could act in such a capacity. The results showed that SPMV CP failed to act as a suppressor of posttranscriptional gene silencing when such tests were performed with transgenic N. benthamiana plants silenced for green fluorescent protein (GFP) expression by agroinfiltration or plant virus vectors. However SPMV CP expressed from the PVX gene vector did interfere with suppressor activity associated with PVX p25. This included a rebounded level of GFP silencing along the vascular tissues, including the veins on upper noninoculated leaves. Therefore, the roles of the SPMV CP now include encapsidation of the SPMV RNA, activity as a pathogenicity factor in both host and nonhost plants, and the enigmatic feature of interfering with suppression of gene silencing.

  11. A selective emotional decision-making bias elicited by facial expressions.

    PubMed

    Furl, Nicholas; Gallagher, Shannon; Averbeck, Bruno B

    2012-01-01

    Emotional and social information can sway otherwise rational decisions. For example, when participants decide between two faces that are probabilistically rewarded, they make biased choices that favor smiling relative to angry faces. This bias may arise because facial expressions evoke positive and negative emotional responses, which in turn may motivate social approach and avoidance. We tested a wide range of pictures that evoke emotions or convey social information, including animals, words, foods, a variety of scenes, and faces differing in trustworthiness or attractiveness, but we found only facial expressions biased decisions. Our results extend brain imaging and pharmacological findings, which suggest that a brain mechanism supporting social interaction may be involved. Facial expressions appear to exert special influence over this social interaction mechanism, one capable of biasing otherwise rational choices. These results illustrate that only specific types of emotional experiences can best sway our choices.

  12. Roles for soluble guanylate cyclase and a thiol oxidation-elicited subunit dimerization of protein kinase G in pulmonary artery relaxation to hydrogen peroxide

    PubMed Central

    Neo, Boon Hwa; Kandhi, Sharath

    2010-01-01

    We have previously provided evidence that hydrogen peroxide (H2O2) stimulates soluble guanylate cyclase (sGC) under conditions where it relaxes isolated endothelium-removed bovine pulmonary arteries (BPAs). Since it was recently reported that H2O2 induces coronary vasorelaxation associated with a nitric oxide/cGMP-independent thiol oxidation/subunit dimerization-elicited activation of protein kinase G (PKG), we investigated whether this mechanism participates in the relaxation of BPAs to H2O2. BPAs precontracted with serotonin (incubated under hypoxia to lower endogenous H2O2) were exposed to increasing concentrations of H2O2. It was observed that 0.1–1 mM H2O2 caused increased PKG dimerization and relaxation. These responses were associated with increased phosphorylation of vasodilator-stimulated phosphoprotein (VASP) at the serine-239 site known to be mediated by PKG. Treatment of BPAs with 1 mM DTT attenuated PKG dimerization, VASP phosphorylation, and relaxation to H2O2. An organoid culture of BPAs for 48 h with 10 μM 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a heme oxidant inhibitor of sGC activation, depleted sGC expression by 85%, associated with a 67% attenuation of VASP phosphorylation and 48% inhibition of relaxation elicited by 100 μM H2O2. Thus both a sGC activation/cGMP-dependent and a thiol oxidation subunit dimerization/cGMP-independent activation of PKG appear to contribute to the relaxation of BPAs elicited by H2O2. PMID:20709865

  13. TNFα-Induced Mucin 4 Expression Elicits Trastuzumab Resistance in HER2-Positive Breast Cancer.

    PubMed

    Mercogliano, María F; De Martino, Mara; Venturutti, Leandro; Rivas, Martín A; Proietti, Cecilia J; Inurrigarro, Gloria; Frahm, Isabel; Allemand, Daniel H; Deza, Ernesto Gil; Ares, Sandra; Gercovich, Felipe G; Guzmán, Pablo; Roa, Juan C; Elizalde, Patricia V; Schillaci, Roxana

    2017-02-01

    Although trastuzumab administration improved the outcome of HER2-positive breast cancer patients, resistance events hamper its clinical benefits. We demonstrated that TNFα stimulation in vitro induces trastuzumab resistance in HER2-positive breast cancer cell lines. Here, we explored the mechanism of TNFα-induced trastuzumab resistance and the therapeutic strategies to overcome it. Trastuzumab-sensitive breast cancer cells, genetically engineered to stably overexpress TNFα, and de novo trastuzumab-resistant tumors, were used to evaluate trastuzumab response and TNFα-blocking antibodies effectiveness respectively. Immunohistochemistry and antibody-dependent cell cytotoxicity (ADCC), together with siRNA strategy, were used to explore TNFα influence on the expression and function of its downstream target, mucin 4 (MUC4). The clinical relevance of MUC4 expression was studied in a cohort of 78 HER2-positive breast cancer patients treated with adjuvant trastuzumab. TNFα overexpression turned trastuzumab-sensitive cells and tumors into resistant ones. Histopathologic findings revealed mucin foci in TNFα-producing tumors. TNFα induced upregulation of MUC4 that reduced trastuzumab binding to its epitope and impaired ADCC. Silencing MUC4 enhanced trastuzumab binding, increased ADCC, and overcame trastuzumab and trastuzumab-emtansine antiproliferative effects in TNFα-overexpressing cells. Accordingly, administration of TNFα-blocking antibodies downregulated MUC4 and sensitized de novo trastuzumab-resistant breast cancer cells and tumors to trastuzumab. In HER2-positive breast cancer samples, MUC4 expression was found to be an independent predictor of poor disease-free survival (P = 0.008). We identified TNFα-induced MUC4 expression as a novel trastuzumab resistance mechanism. We propose MUC4 expression as a predictive biomarker of trastuzumab efficacy and a guide to combination therapy of TNFα-blocking antibodies with trastuzumab. Clin Cancer Res; 23(3); 636-48.

  14. Characterization and differential expression of microRNAs elicited by sulfur deprivation in Chlamydomonas reinhardtii

    PubMed Central

    2012-01-01

    Background microRNAs (miRNAs) have been found to play an essential role in the modulation of numerous biological processes in eukaryotes. Chlamydomonas reinhardtii is an ideal model organism for the study of many metabolic processes including responses to sulfur-deprivation. We used a deep sequencing platform to extensively profile and identify changes in the miRNAs expression that occurred under sulfur-replete and sulfur-deprived conditions. The aim of our research was to characterize the differential expression of Chlamydomonas miRNAs under sulfur-deprived conditions, and subsequently, the target genes of miRNA involved in sulfur-deprivation were further predicted and analyzed. Results By using high-throughput sequencing, we characterized the microRNA transcriptomes under sulphur-replete and sulfur-deprived conditions in Chlamydomonas reinhardtii. We predicted a total of 310 miRNAs which included 85 known miRNAs and 225 novel miRNAs. 13 miRNAs were the specific to the sulfur-deprived conditions. 47 miRNAs showed significantly differential expressions responding to sulfur-deprivation, and most were up-regulated in the small RNA libraries with sulfur-deprivation. Using a web-based integrated system (Web MicroRNAs Designer 3) and combing the former information from a transcriptome of Chlamydomonas reinhardtii, 22 miRNAs and their targets involved in metabolism regulation with sulfur-deprivation were verified. Conclusions Our results indicate that sulfur-deprivation may have a significant influence on small RNA expression patterns, and the differential expressions of miRNAs and interactions between miRNA and its targets might further reveal the molecular mechanism responding to sulfur-deprivation in Chlamydomonas reinhardtii. PMID:22439676

  15. Identification of Group B Streptococcal Sip Protein, Which Elicits Cross-Protective Immunity

    PubMed Central

    Brodeur, Bernard R.; Boyer, Martine; Charlebois, Isabelle; Hamel, Josée; Couture, France; Rioux, Clément R.; Martin, Denis

    2000-01-01

    A protein of group B streptococci (GBS), named Sip for surface immunogenic protein, which is distinct from previously described surface proteins, was identified after immunological screening of a genomic library. Immunoblots using a Sip-specific monoclonal antibody indicated that a protein band with an approximate molecular mass of 53 kDa which did not vary in size was present in every GBS strain tested. Representatives of all nine GBS serotypes were included in the panel of strains. Cloning and sequencing of the sip gene revealed an open reading frame of 1,305 nucleotides coding for a polypeptide of 434 amino acid residues, with a calculated pI of 6.84 and molecular mass of 45.5 kDa. Comparison of the nucleotide sequences from six different strains confirmed with 98% identity that the sip gene is highly conserved among GBS isolates. N-terminal amino acid sequencing also indicated the presence of a 25-amino-acid signal peptide which is cleaved in the mature protein. More importantly, immunization with the recombinant Sip protein efficiently protected CD-1 mice against deadly challenges with six GBS strains of serotypes Ia/c, Ib, II/R, III, V, and VI. The data presented in this study suggest that this highly conserved protein induces cross-protective immunity against GBS infections and emphasize its potential as a universal vaccine candidate. PMID:10992461

  16. Tamoxifen-elicited uterotrophy: cross-species and cross-ligand analysis of the gene expression program

    PubMed Central

    Kwekel, Joshua C; Forgacs, Agnes L; Burgoon, Lyle D; Williams, Kurt J; Zacharewski, Timothy R

    2009-01-01

    Background Tamoxifen (TAM) is a well characterized breast cancer drug and selective estrogen receptor modulator (SERM) which also has been associated with a small increase in risk for uterine cancers. TAM's partial agonist activation of estrogen receptor has been characterized for specific gene promoters but not at the genomic level in vivo.Furthermore, reducing uncertainties associated with cross-species extrapolations of pharmaco- and toxicogenomic data remains a formidable challenge. Results A comparative ligand and species analysis approach was conducted to systematically assess the physiological, morphological and uterine gene expression alterations elicited across time by TAM and ethynylestradiol (EE) in immature ovariectomized Sprague-Dawley rats and C57BL/6 mice. Differential gene expression was evaluated using custom cDNA microarrays, and the data was compared to identify conserved and divergent responses. 902 genes were differentially regulated in all four studies, 398 of which exhibit identical temporal expression patterns. Conclusion Comparative analysis of EE and TAM differentially expressed gene lists suggest TAM regulates no unique uterine genes that are conserved in the rat and mouse. This demonstrates that the partial agonist activities of TAM extend to molecular targets in regulating only a subset of EE-responsive genes. Ligand-conserved, species-divergent expression of carbonic anhydrase 2 was observed in the microarray data and confirmed by real time PCR. The identification of comparable temporal phenotypic responses linked to related gene expression profiles demonstrates that systematic comparative genomic assessments can elucidate important conserved and divergent mechanisms in rodent estrogen signalling during uterine proliferation. PMID:19400957

  17. Dehydroepiandrosterone promotes pulmonary artery relaxation by NADPH oxidation-elicited subunit dimerization of protein kinase G 1α

    PubMed Central

    Patel, Dhara; Kandhi, Sharath; Kelly, Melissa; Neo, Boon Hwa

    2013-01-01

    The activity of glucose-6-phosphate dehydrogenase (G6PD) controls a vascular smooth muscle relaxing mechanism promoted by the oxidation of cytosolic NADPH, which has been associated with activation of the 1α form of protein kinase G (PKG-1α) by a thiol oxidation-elicited subunit dimerization. This PKG-1α-activation mechanism appears to contribute to responses of isolated endothelium-removed bovine pulmonary arteries (BPA) elicited by peroxide, cytosolic NADPH oxidation resulting from G6PD inhibition, and hypoxia. Dehydroepiandrosterone (DHEA) is a steroid hormone with pulmonary vasodilator activity, which has beneficial effects in treating pulmonary hypertension. Because multiple mechanisms have been suggested for the vascular effects of DHEA and one of the known actions of DHEA is inhibiting G6PD, we investigated whether it promoted relaxation associated with NADPH oxidation, PKG-1α dimerization, and PKG activation detected by increased vasodilator-stimulated phosphoprotein (VASP) phosphorylation. Relaxation of BPA to DHEA under aerobic or hypoxic conditions was associated with NADPH oxidation, PKG-1α dimerization, and increased VASP phosphorylation. The vasodilator activity of DHEA was markedly attenuated in pulmonary arteries and aorta from a PKG knockin mouse containing a serine in place of a cysteine involved in PKG dimerization. DHEA promoted increased PKG dimerization in lungs from wild-type mice, which was not detected in the PKG knockin mouse model. Thus PKG-1α dimerization is a major contributing factor to the vasodilator actions of DHEA and perhaps its beneficial effects in treating pulmonary hypertension. PMID:24375799

  18. Expression of multiple proteins in transgenic plants

    DOEpatents

    Vierstra, Richard D.; Walker, Joseph M.

    2002-01-01

    A method is disclosed for the production of multiple proteins in transgenic plants. A DNA construct for introduction into plants includes a provision to express a fusion protein of two proteins of interest joined by a linking domain including plant ubiquitin. When the fusion protein is produced in the cells of a transgenic plant transformed with the DNA construction, native enzymes present in plant cells cleave the fusion protein to release both proteins of interest into the cells of the transgenic plant. Since the proteins are produced from the same fusion protein, the initial quantities of the proteins in the cells of the plant are approximately equal.

  19. Nerve Agent Exposure Elicits Site-Specific Changes in Protein Phosphorylation in Mouse Brain

    PubMed Central

    Zhu, Hongwen; O’Brien, Jennifer J.; O’Callaghan, James P.; Miller, Diane B.; Zhang, Qiang; Rana, Minal; Tsui, Tiffany; Peng, Youyi; Tomesch, John; Hendrick, Joseph P.; Wennogle, Lawrence P; Snyder, Gretchen L.

    2010-01-01

    Organophosphorus (OP) compounds cause toxic symptoms, including convulsions, coma, and death, as the result of irreversible inhibition of acetylcholinesterase (AChE). The development of effective treatments to block these effects and attenuate long-term cognitive and motor disabilities that result from OP intoxication is hampered by a limited understanding of the CNS pathways responsible for these actions. We employed a candidate method (called CNSProfile™) to identify changes in the phosphorylation state of key neuronal phosphoproteins evoked by the OP compound, diisopropyl fluorophosphate (DFP). Focused microwave fixation was used to preserve the phosphorylation state of phosphoproteins in brains of DFP-treated mice; hippocampus and striatum were analyzed by immunoblotting with a panel of phospho-specific antibodies. DFP exposure elicited comparable effects on phosphorylation of brain phosphoproteins in both C57BL/6 and FVB mice. DFP treatment significantly altered phosphorylation at regulatory residues on glutamate receptors, including Serine897 (S897) of the NR1 NMDA receptor. NR1 phosphorylation was bi-directionally regulated after DFP in striatum versus hippocampus. NR1 phosphorylation was reduced in striatum, but elevated in hippocampus, compared with controls. DARPP-32 phosphorylation in striatum was selectively increased at the Cdk5 kinase substrate, Threonine75 (T75). Phencynonate hydrochloride, a muscarinic cholinergic antagonist, prevented seizure-like behaviors and the observed changes in phosphorylation induced by DFP. The data reveal region-specific effects of nerve agent exposure on intracellular signaling pathways that correlate with seizure-like behavior and which are reversed by the muscarinic receptor blockade. This approach identifies specific targets for nerve agents, including substrates for Cdk5 kinase, which may be the basis for new anti-convulsant therapies. PMID:20423708

  20. Alternative Splicing of a Multi-Drug Transporter from Pseudoperonospora cubensis Generates an RXLR Effector Protein That Elicits a Rapid Cell Death

    PubMed Central

    Savory, Elizabeth A.; Zou, Cheng; Adhikari, Bishwo N.; Hamilton, John P.; Buell, C. Robin; Shiu, Shin-Han; Day, Brad

    2012-01-01

    Pseudoperonospora cubensis, an obligate oomycete pathogen, is the causal agent of cucurbit downy mildew, a foliar disease of global economic importance. Similar to other oomycete plant pathogens, Ps. cubensis has a suite of RXLR and RXLR-like effector proteins, which likely function as virulence or avirulence determinants during the course of host infection. Using in silico analyses, we identified 271 candidate effector proteins within the Ps. cubensis genome with variable RXLR motifs. In extending this analysis, we present the functional characterization of one Ps. cubensis effector protein, RXLR protein 1 (PscRXLR1), and its closest Phytophthora infestans ortholog, PITG_17484, a member of the Drug/Metabolite Transporter (DMT) superfamily. To assess if such effector-non-effector pairs are common among oomycete plant pathogens, we examined the relationship(s) among putative ortholog pairs in Ps. cubensis and P. infestans. Of 271 predicted Ps. cubensis effector proteins, only 109 (41%) had a putative ortholog in P. infestans and evolutionary rate analysis of these orthologs shows that they are evolving significantly faster than most other genes. We found that PscRXLR1 was up-regulated during the early stages of infection of plants, and, moreover, that heterologous expression of PscRXLR1 in Nicotiana benthamiana elicits a rapid necrosis. More interestingly, we also demonstrate that PscRXLR1 arises as a product of alternative splicing, making this the first example of an alternative splicing event in plant pathogenic oomycetes transforming a non-effector gene to a functional effector protein. Taken together, these data suggest a role for PscRXLR1 in pathogenicity, and, in total, our data provide a basis for comparative analysis of candidate effector proteins and their non-effector orthologs as a means of understanding function and evolutionary history of pathogen effectors. PMID:22496844

  1. Cocoa butter and safflower oil elicit different effects on hepatic gene expression and lipid metabolism in rats.

    PubMed

    Gustavsson, Carolina; Parini, Paolo; Ostojic, Jovanca; Cheung, Louisa; Hu, Jin; Zadjali, Fahad; Tahir, Faheem; Brismar, Kerstin; Norstedt, Gunnar; Tollet-Egnell, Petra

    2009-11-01

    The aim of this study was to compare the effects of cocoa butter and safflower oil on hepatic transcript profiles, lipid metabolism and insulin sensitivity in healthy rats. Cocoa butter-based high-fat feeding for 3 days did not affect plasma total triglyceride (TG) levels or TG-rich VLDL particles or hepatic insulin sensitivity, but changes in hepatic gene expression were induced that might lead to increased lipid synthesis, lipotoxicity, inflammation and insulin resistance if maintained. Safflower oil increased hepatic beta-oxidation, was beneficial in terms of circulating TG-rich VLDL particles, but led to reduced hepatic insulin sensitivity. The effects of safflower oil on hepatic gene expression were partly overlapping with those exerted by cocoa butter, but fewer transcripts from anabolic pathways were altered. Increased hepatic cholesterol levels and increased expression of hepatic CYP7A1 and ABCG5 mRNA, important gene products in bile acid production and cholesterol excretion, were specific effects elicited by safflower oil only. Common effects on gene expression included increased levels of p8, DIG-1 IGFBP-1 and FGF21, and reduced levels of SCD-1 and SCD-2. This indicates that a lipid-induced program for hepatic lipid disposal and cell survival was induced by 3 days of high-fat feeding, independent on the lipid source. Based on the results, we speculate that hepatic TG infiltration leads to reduced expression of SCD-1, which might mediate either neutral, beneficial or unfavorable effects on hepatic metabolism upon high-fat feeding, depending on which fatty acids were provided by the diet.

  2. Responsibility modulates pain-matrix activation elicited by the expressions of others in pain.

    PubMed

    Cui, Fang; Abdelgabar, Abdel-Rahman; Keysers, Christian; Gazzola, Valeria

    2015-07-01

    Here we examine whether brain responses to dynamic facial expressions of pain are influenced by our responsibility for the observed pain. Participants played a flanker task with a confederate. Whenever either erred, the confederate was seen to receive a noxious shock. Using functional magnetic resonance imaging, we found that regions of the functionally localized pain-matrix of the participants (the anterior insula in particular) were activated most strongly when seeing the confederate receive a noxious shock when only the participant had erred (and hence had full responsibility). When both or only the confederate had erred (i.e. participant's shared or no responsibility), significantly weaker vicarious pain-matrix activations were measured. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  3. Responsibility modulates pain-matrix activation elicited by the expressions of others in pain

    PubMed Central

    Cui, Fang; Abdelgabar, Abdel-Rahman; Keysers, Christian; Gazzola, Valeria

    2015-01-01

    Here we examine whether brain responses to dynamic facial expressions of pain are influenced by our responsibility for the observed pain. Participants played a flanker task with a confederate. Whenever either erred, the confederate was seen to receive a noxious shock. Using functional magnetic resonance imaging, we found that regions of the functionally localized pain-matrix of the participants (the anterior insula in particular) were activated most strongly when seeing the confederate receive a noxious shock when only the participant had erred (and hence had full responsibility). When both or only the confederate had erred (i.e. participant's shared or no responsibility), significantly weaker vicarious pain-matrix activations were measured. PMID:25800210

  4. ATP Released by Electrical Stimuli Elicits Calcium Transients and Gene Expression in Skeletal Muscle*

    PubMed Central

    Buvinic, Sonja; Almarza, Gonzalo; Bustamante, Mario; Casas, Mariana; López, Javiera; Riquelme, Manuel; Sáez, Juan Carlos; Huidobro-Toro, Juan Pablo; Jaimovich, Enrique

    2009-01-01

    ATP released from cells is known to activate plasma membrane P2X (ionotropic) or P2Y (metabotropic) receptors. In skeletal muscle cells, depolarizing stimuli induce both a fast calcium signal associated with contraction and a slow signal that regulates gene expression. Here we show that nucleotides released to the extracellular medium by electrical stimulation are partly involved in the fast component and are largely responsible for the slow signals. In rat skeletal myotubes, a tetanic stimulus (45 Hz, 400 1-ms pulses) rapidly increased extracellular levels of ATP, ADP, and AMP after 15 s to 3 min. Exogenous ATP induced an increase in intracellular free Ca2+ concentration, with an EC50 value of 7.8 ± 3.1 μm. Exogenous ADP, UTP, and UDP also promoted calcium transients. Both fast and slow calcium signals evoked by tetanic stimulation were inhibited by either 100 μm suramin or 2 units/ml apyrase. Apyrase also reduced fast and slow calcium signals evoked by tetanus (45 Hz, 400 0.3-ms pulses) in isolated mouse adult skeletal fibers. A likely candidate for the ATP release pathway is the pannexin-1 hemichannel; its blockers inhibited both calcium transients and ATP release. The dihydropyridine receptor co-precipitated with both the P2Y2 receptor and pannexin-1. As reported previously for electrical stimulation, 500 μm ATP significantly increased mRNA expression for both c-fos and interleukin 6. Our results suggest that nucleotides released during skeletal muscle activity through pannexin-1 hemichannels act through P2X and P2Y receptors to modulate both Ca2+ homeostasis and muscle physiology. PMID:19822518

  5. Cloning of a Mycobacterium tuberculosis gene encoding a purifed protein derivative protein that elicits strong tuberculosis-specific delayed-type hypersensitivity.

    PubMed

    Coler, R N; Skeiky, Y A; Ovendale, P J; Vedvick, T S; Gervassi, L; Guderian, J; Jen, S; Reed, S G; Campos-Neto, A

    2000-07-01

    The purified protein derivative (PPD) skin test has been used for the diagnosis of tuberculosis for more than 75 years. However, the test lacks specificity because all mycobacteria share antigens present in PPD. Therefore, sensitization with nontuberculous pathogenic or with environmental nonpathogenic mycobacteria can lead to positive skin tests. This communication describes a novel PPD protein present only in tuberculous complex mycobacteria. A recombinant protein was obtained and named DPPD on the basis of the first 4 amino acids of its N-terminus sequence. DPPD elicited delayed-type hypersensitivity (DTH) in 100% of Mycobacterium tuberculosis-infected guinea pigs but in no animals sensitized with several organisms representative of all members of the Mycobacterium genus. Preliminary results indicate that DPPD induces strong and specific DTH in humans. This work points to the definition of a single recombinant M. tuberculosis protein that may be an alternative to the PPD test.

  6. Eliciting an antibody response against a recombinant TSH containing fusion protein.

    PubMed

    Mard-Soltani, Maysam; Rasaee, Mohamad Javad; Sheikhi, AbdolKarim; Hedayati, Mehdi

    2016-10-27

    Designing novel antigens to rise specific antibodies for Thyroid Stimulating Hormone (TSH) detection is of great significance. A novel fusion protein consisting of the C termini sequence of TSH beta subunit and a fusion sequence was designed and produced for rabbit immunization. Thereafter, the produced antibodies were purified and characterized for TSH detection. Our results indicate that the produced antibody is capable of sensitive and specific detection of TSH with low cross reactivity. This study underscores the applicability of designed fusion protein for specific and sensitive polyclonal antibody production and the importance of selecting an amenable region of the TSH for immunization.

  7. Herpesvirus saimiri STP-A oncoprotein utilizes Src family protein tyrosine kinase and tumor necrosis factor receptor-associated factors to elicit cellular signal transduction.

    PubMed

    Garcia, Maria I; Kaserman, Joseph; Chung, Young-Hwa; Jung, Jae U; Lee, Sun-Hwa

    2007-03-01

    The saimiri transforming protein oncogene, called STP-A, of herpesvirus saimiri (HVS) subgroup A is not required for viral replication but is required for lymphoid cell immortalization in culture and lymphoma induction in primates. Here we report that STP-A interacts with cellular tumor necrosis factor receptor-associated factors (TRAF2 and TRAF6) and Src family protein tyrosine kinases (SF-PTKs) in a genetically and functionally separable manner and that each interaction constitutively elicits independent cellular signal transduction. The amino-terminal and central proline-rich motifs of STP-A were responsible for TRAF6 and TRAF2 interactions, respectively, and STP-A and TRAF6 interaction contributed to the majority of NF-kappaB activation, whereas STP-A and TRAF2 interaction played a minor role in NF-kappaB activation. On the other hand, interaction of STP-A with SF-PTKs through its SH2 binding motif effectively elicited AP-1 and NF-AT transcription factor activity. One cellular gene targeted by STP-A is intercellular adhesion molecule 1 (ICAM-1), which participates in a wide range of inflammatory and immune responses. Both TRAF and SF-PTK signal transductions induced by STP-A were required for the marked increase of ICAM-1 expression. These results demonstrate that the viral oncogene STP-A independently targets two vital cellular signaling molecules and that these activities likely contribute to HVS-mediated lymphoid cell immortalization in culture and lymphoma induction in primates.

  8. Caffeine elicits c-Fos expression in horizontal diagonal band cholinergic neurons.

    PubMed

    Reznikov, Leah R; Pasumarthi, Ravi K; Fadel, Jim R

    2009-12-09

    Caffeine is a widely self-administered psychostimulant with purported neuroprotective and procognitive effects in rodent models of aging. The cholinergic basal forebrain is important for arousal and attention and is implicated in age-related cognitive decline. Accordingly, we determined the effects of caffeine on cholinergic neuron activation in the rat basal forebrain. Young adult (age 2 months) male rats were treated with caffeine (0, 10, or 50 mg/kg) and killed 2 h later. Caffeine significantly increased c-Fos expression in cholinergic neurons of the horizontal limb of the diagonal band of Broca but not other basal forebrain regions such as the medial septum or substantia innominata. The horizontal limb of the diagonal band of Broca provides cholinergic innervation to the olfactory bulb, suggesting that deficits in this structure may contribute to diminished olfactory function observed in Alzheimer's disease patients. These results suggest that part of the cognitive-enhancing effects of caffeine may be mediated through activation of this part of the cholinergic basal forebrain.

  9. Enhancement of phytosterols, taraxasterol and induction of extracellular pathogenesis-related proteins in cell cultures of Solanum lycopersicum cv Micro-Tom elicited with cyclodextrins and methyl jasmonate.

    PubMed

    Briceño, Zuleika; Almagro, Lorena; Sabater-Jara, Ana B; Calderón, Antonio A; Pedreño, María Angeles; Ferrer, María Angeles

    2012-07-15

    Suspension-cultured cells of Solanum lycopersicum cv Micro-Tom were used to evaluate the effect of methyl jasmonate and cyclodextrins, separately or in combination, on the induction of defense responses. An extracellular accumulation of two sterols (isofucosterol and β-sitosterol) and taraxasterol, a common tomato fruit cuticular triterpene, were observed. Their levels were higher in Micro-Tom tomato suspension cultured cells elicited with cyclodextrins than in control and methyl jasmonate-treated cells. Also, their accumulation profiles during the cell growth phase were markedly different. The most striking feature in response to cyclodextrin treatments was the observed enhancement of taraxasterol accumulation. Likewise, the exogenous application of methyl jasmonate and cyclodextrins induced the accumulation of pathogenesis-related proteins. Analysis of the extracellular proteome showed the presence of amino acid sequences homologous to pathogenesis-related 1 and 5 proteins, a cationic peroxidase and a biotic cell death-associated protein, which suggests that methyl jasmonate and cyclodextrins could play a role in mediating defense-related gene product expression in S. lycopersicum cv Micro-Tom. Copyright © 2012 Elsevier GmbH. All rights reserved.

  10. Protein structure protection commits gene expression patterns.

    PubMed

    Chen, Jianping; Liang, Han; Fernández, Ariel

    2008-01-01

    Gene co-expressions often determine module-defining spatial and temporal concurrences of proteins. Yet, little effort has been devoted to tracing coordinating signals for expression correlations to the three-dimensional structures of gene products. We performed a global structure-based analysis of the yeast and human proteomes and contrasted this information against their respective transcriptome organizations obtained from comprehensive microarray data. We show that protein vulnerability quantifies dosage sensitivity for metabolic adaptation phases and tissue-specific patterns of mRNA expression, determining the extent of co-expression similarity of binding partners. The role of protein intrinsic disorder in transcriptome organization is also delineated by interrelating vulnerability, disorder propensity and co-expression patterns. Extremely vulnerable human proteins are shown to be subject to severe post-transcriptional regulation of their expression through significant micro-RNA targeting, making mRNA levels poor surrogates for protein-expression levels. By contrast, in yeast the expression of extremely under-wrapped proteins is likely regulated through protein aggregation. Thus, the 85 most vulnerable proteins in yeast include the five confirmed prions, while in human, the genes encoding extremely vulnerable proteins are predicted to be targeted by microRNAs. Hence, in both vastly different organisms protein vulnerability emerges as a structure-encoded signal for post-transcriptional regulation. Vulnerability of protein structure and the concurrent need to maintain structural integrity are shown to quantify dosage sensitivity, compelling gene expression patterns across tissue types and temporal adaptation phases in a quantifiable manner. Extremely vulnerable proteins impose additional constraints on gene expression: They are subject to high levels of regulation at the post-transcriptional level.

  11. Heat exposure in female rats elicits abnormal fear expression and cellular changes in prefrontal cortex and hippocampus.

    PubMed

    Gruene, Tina M; Lipps, Jennifer; Rey, Colin D; Bouck, Anna; Shansky, Rebecca M

    2014-11-01

    Despite a twofold higher prevalence of fear-related disorders in women, the neurobiological factors that modulate and drive fear expression are rarely studied in female animals. Fear conditioning and extinction are useful tools for dissecting these mechanisms, and here we tested the effects of environmental manipulations - four days of exposure to 31°C temperatures in the animal housing facility - on fear learning and memory exclusively in female rats. We found that heat exposure disrupted freezing to tone during fear conditioning, and elicited enhanced freezing during extinction and extinction retrieval. We also performed immunohistochemistry for c-fos expression in the infralimbic (IL) and prelimbic (PL) regions of the prefrontal cortex during extinction retrieval, and found that heat exposure induced a switch from IL-dominated activity to PL-dominated activity. Finally, morphological analysis of spines in hippocampal CA3 neurons revealed an increase in spine head diameter in heat-exposed animals, which may partly underlie the persistent freezing observed in these animals. Together, our data show that heat exposure can induce changes at behavioral, physiological, and structural levels, and add to a woefully lacking body of literature on fear processes in female animals. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. Silencing Nuclear Pore Protein Tpr Elicits a Senescent-Like Phenotype in Cancer Cells

    PubMed Central

    David-Watine, Brigitte

    2011-01-01

    Background Tpr is a large coiled-coil protein located in the nuclear basket of the nuclear pore complex for which many different functions were proposed from yeast to human. Methodology/Principal Findings Here we show that depletion of Tpr by RNA interference triggers G0–G1 arrest and ultimately induces a senescent-like phenotype dependent on the presence of p53. We also found that Tpr depletion impairs the NES [nuclear export sequence]-dependent nuclear export of proteins and causes partial co-depletion of Nup153. In addition Tpr depletion impacts on level and function of the SUMO-protease SENP2 thus affecting SUMOylation regulation at the nuclear pore and overall SUMOylation in the cell. Conclusions Our data for the first time provide evidence that a nuclear pore component plays a role in controlling cellular senescence. Our findings also point to new roles for Tpr in the regulation of SUMO-1 conjugation at the nuclear pore and directly confirm Tpr involvement in the nuclear export of NES-proteins. PMID:21811608

  13. A tumor vessel-targeting fusion protein elicits a chemotherapeutic bystander effect in pancreatic ductal adenocarcinoma

    PubMed Central

    Chen, Chun-Te; Chen, Yi-Chun; Du, Yi; Han, Zhenbo; Ying, Haoqiang; Bouchard, Richard R; Hsu, Jennifer L; Hsu, Jung-Mao; Mitcham, Trevor M; Chen, Mei-Kuang; Sun, Hui-Lung; Chang, Shih-Shin; Li, Donghui; Chang, Ping; DePinho, Ronald A; Hung, Mien-Chie

    2017-01-01

    Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease characterized by a prominent desmoplastic stroma that may constrain tumor progression but also limit the access of therapeutic drugs. In this study, we explored a tumor-targeting strategy that enlists an engineered anti-angiogenic protein consisting of endostatin and cytosine deaminase linked to uracil phosphoribosyltransferase (EndoCD). This protein selectively binds to tumor vessels to compromise tumor angiogenesis and converts the non-toxic 5-fluorocytosine (5-FC) to the cytotoxic 5-fluorouracil to produce a chemotherapeutic bystander effect at the pancreatic tumor site. We found that resveratrol increased the protein stability of EndoCD through suppression of chymotrypsin-like proteinase activity and synergistically enhances EndoCD-mediated 5-FC-induced cell killing. In various PDAC mouse models, the EndoCD/5-FC/resveratrol regimen decreased intratumoral vascular density and stroma formation and enhances apoptosis in tumors cells as well as in surrounding endothelial, pancreatic stellate, and immune cells, leading to reduced tumor growth and extended survival. Thus, the EndoCD/5-FC/resveratrol combination may be an effective treatment option for PDAC. PMID:28401019

  14. Oral administration of bovine whey proteins to mice elicits opposing immunoregulatory responses and is adjuvant dependent

    PubMed Central

    AFUWAPE, A O; TURNER, M W; STROBEL, S

    2004-01-01

    Most studies investigating the induction of oral tolerance (OT) use purified proteins such as ovalbumin (OVA), bovine serum albumin (BSA) and beta-lactoglobulin (β-LG). Little information is available regarding the induction of OT to a protein mixture, e.g. cow's milk. In this study we compared the regulatory mechanisms induced after the oral administration of a whey protein concentrate (WP) derived from cow's milk following immunization with two different adjuvants, complete Freund's adjuvant (CFA) and alum. OVA was used as a control antigen. Animals were given a single feed of these proteins at an equivalent dose of 1 mg/g body weight before they were immunized seven days later with the antigen in Freund's adjuvant or alum. Delayed type hypersensitivity (DTH) responses were suppressed by both a feed of WP and OVA after immunization with CFA. However, only OVA feeding suppressed antigen specific IgG responses. In an attempt to investigate whether WP would tolerize the more susceptible IgE responses, alum immunization replaced CFA as the adjuvant used for systemic immunizations. WP, after a single feed, significantly primed for DTH and IgE responses indicating oral sensitization to WP. In contrast, OVA suppressed DTH, IgE and IgG responses. Antigen specific proliferation of mononuclear cells was suppressed in mice fed OVA, but primed in those fed with WP. In addition cells taken from sensitized mice fed WP up-regulated levels of specific interleukin (IL) -4, -10 and -12 in vitro whereas these cytokines were suppressed in cultures from tolerant WP fed mice. Global suppression was obtained in cultures from tolerant OVA fed mice. TGF-β was not detected in draining PLN cell cultures of either tolerant or sensitized mice. These data suggest that a whey protein mixture induces divergent responses following immunization with either CFA or alum despite being fed at an identical dose. We suggest that that the choice of the adjuvant may determine the immunoregulatory

  15. A recombinant adenovirus expressing CFP10, ESAT6, Ag85A and Ag85B of Mycobacterium tuberculosis elicits strong antigen-specific immune responses in mice.

    PubMed

    Li, Wu; Deng, Guangcun; Li, Min; Zeng, Jin; Zhao, Liping; Liu, Xiaoming; Wang, Yujiong

    2014-11-01

    Tuberculosis (TB) is caused by an infection of Mycobacterium tuberculosis (Mtb) and remains an enormous and increasing health burden worldwide. To date, Mycobacterium bovis Bacillus Calmette Guerin (BCG) is the only licensed anti-TB vaccine worldwide, which provides an important but limited protection from the Mtb infection. The development of alternative anti-TB vaccines is therefore urgently needed. Here we report, the generation of Ad5-CEAB, a recombinant adenovirus expressing Mtb antigens of CFP10, ESAT6, Ag85A and Ag85B proteins in a form of mixture. In order to evaluate the immunogenicity of Ad5-CEAB, mice were immunized with Ad5-CEAB by intranasal instillation three times with 2-week intervals. The results demonstrated that Ad5-CEAB elicited a strong antigen-specific immune response, particularly of the Th1 immune responses that were characterized by an increased ratio of IgG2a/IgG1 and secretions of Th1 type cytokines, IFN-γ, TNF-α, IL-2 and IL-12. In addition, the Ad5-CEAB also showed an ability to enhance humoral responses with a dramatically augmented antigen-specific serum IgG. Furthermore, an elevated sIgA were also found in the bronchoalveolar lavage fluid of the immunized mice, suggesting the elicitation of mucosal immune responses. These data indicate that Ad5-CEAB can induce a broad range of antigen-specific immune responses in vivo, which provides a promising and novel route for developing anti-TB vaccines and warrants further investigation.

  16. Coevolution of gene expression among interacting proteins

    SciTech Connect

    Fraser, Hunter B.; Hirsh, Aaron E.; Wall, Dennis P.; Eisen,Michael B.

    2004-03-01

    Physically interacting proteins or parts of proteins are expected to evolve in a coordinated manner that preserves proper interactions. Such coevolution at the amino acid-sequence level is well documented and has been used to predict interacting proteins, domains, and amino acids. Interacting proteins are also often precisely coexpressed with one another, presumably to maintain proper stoichiometry among interacting components. Here, we show that the expression levels of physically interacting proteins coevolve. We estimate average expression levels of genes from four closely related fungi of the genus Saccharomyces using the codon adaptation index and show that expression levels of interacting proteins exhibit coordinated changes in these different species. We find that this coevolution of expression is a more powerful predictor of physical interaction than is coevolution of amino acid sequence. These results demonstrate previously uncharacterized coevolution of gene expression, adding a different dimension to the study of the coevolution of interacting proteins and underscoring the importance of maintaining coexpression of interacting proteins over evolutionary time. Our results also suggest that expression coevolution can be used for computational prediction of protein protein interactions.

  17. Glutamine Amide Flip Elicits Long Distance Allosteric Responses in the LOV Protein Vivid.

    PubMed

    Ganguly, Abir; Thiel, Walter; Crane, Brian R

    2017-03-01

    Light-oxygen-voltage (LOV) domains sense blue light through the photochemical formation of a cysteinyl-flavin covalent adduct. Concurrent protonation at the flavin N5 position alters the hydrogen bonding interactions of an invariant Gln residue that has been proposed to flip its amide side chain as a critical step in the propagation of conformational change. Traditional molecular dynamics (MD) and replica-exchange MD (REMD) simulations of the well-characterized LOV protein Vivid (VVD) demonstrate that the Gln182 amide indeed reorients by ∼180° in response to either adduct formation or reduction of the isoalloxazine ring to the neutral semiquinone, both of which involve N5 protonation. Free energy simulations reveal that the relative free energies of the flipped Gln conformation and the flipping barrier are significantly lower in the light-adapted state. The Gln182 flip stabilizes an important hinge-bβ region between the PAS β-sheet and the N-terminal cap helix that in turn destabilizes an N-terminal latch region against the PAS core. Release of the latch, observed both experimentally and in the simulations, is known to mediate light-induced VVD dimerization. This computational study of a LOV protein, unprecedented in its agreement with experiment, provides an atomistic view of long-range allosteric coupling in a photoreceptor.

  18. Recombinant Mycoplasma mycoides proteins elicit protective immune responses against contagious bovine pleuropneumonia.

    PubMed

    Nkando, Isabel; Perez-Casal, Jose; Mwirigi, Martin; Prysliak, Tracy; Townsend, Hugh; Berberov, Emil; Kuria, Joseph; Mugambi, John; Soi, Reuben; Liljander, Anne; Jores, Joerg; Gerdts, Volker; Potter, Andrew; Naessens, Jan; Wesonga, Hezron

    2016-03-01

    Mycoplasma mycoides subsp. mycoides (Mmm) is the causative agent of contagious bovine pleuropneumonia (CBPP), a devastating respiratory disease mainly affecting cattle in sub-Saharan Africa. The current vaccines are based on live-attenuated Mmm strains and present problems with temperature stability, duration of immunity and adverse reactions, thus new vaccines are needed to overcome these issues. We used a reverse vaccinology approach to identify 66 Mmm potential vaccine candidates. The selection and grouping of the antigens was based on the presence of specific antibodies in sera from CBPP-positive animals. The antigens were used to immunize male Boran cattle (Bos indicus) followed by a challenge with the Mmm strain Afadé. Two of the groups immunized with five proteins each showed protection after the Mmm challenge (Groups A and C; P<0.05) and in one group (Group C) Mmm could not be cultured from lung specimens. A third group (Group N) showed a reduced number of animals with lesions and the cultures for Mmm were also negative. While immunization with some of the antigens conferred protection, others may have increased immune-related pathology. This is the first report that Mmm recombinant proteins have been successfully used to formulate a prototype vaccine and these results pave the way for the development of a novel commercial vaccine. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. MetQ of Neisseria gonorrhoeae Is a Surface-Expressed Antigen That Elicits Bactericidal and Functional Blocking Antibodies

    PubMed Central

    Semchenko, Evgeny A.; Day, Christopher J.

    2016-01-01

    ABSTRACT Neisseria gonorrhoeae, the causative agent of the sexually transmitted infection (STI) gonorrhea, is a growing public health threat for which a vaccine is urgently needed. We characterized the functional role of the gonococcal MetQ protein, which is the methionine binding component of an ABC transporter system, and assessed its potential as a candidate antigen for inclusion in a gonococcal vaccine. MetQ has been found to be highly conserved in all strains investigated to date, it is localized on the bacterial surface, and it binds l-methionine with a high affinity. MetQ is also involved in gonococcal adherence to cervical epithelial cells. Mutants lacking MetQ have impaired survival in human monocytes, macrophages, and serum. Furthermore, antibodies raised against MetQ are bactericidal and are able to block gonococcal adherence to epithelial cells. These data suggest that MetQ elicits both bactericidal and functional blocking antibodies and is a valid candidate antigen for additional investigation and possible inclusion in a vaccine for prevention of gonorrhea. PMID:27895130

  20. Sensitizing and Eliciting Capacity of Egg White Proteins in BALB/c Mice As Affected by Processing.

    PubMed

    Pablos-Tanarro, Alba; Lozano-Ojalvo, Daniel; Martínez-Blanco, Mónica; López-Fandiño, Rosina; Molina, Elena

    2017-06-07

    This study assesses to what extent technological processes that lead to different degrees of denaturation of egg white proteins affect their allergenicity. We focused on heat (80 °C, 10 min) and high-pressure (400 MPa and 37 °C, 10 min) treatments and used a BALB/c mouse model of food allergy. Oral sensitization to egg white using cholera toxin as adjuvant induced the production of IgE and IgG1 isotypes and led to severe clinical signs following challenge with the allergen. Extensive protein denaturation caused by heat treatment increased its ability to induce Th1 responses and reduced both its sensitizing and eliciting capacity. Heated egg white stimulated the production of IgE over IgG1 antibodies directed, at least in part, toward new epitopes exposed as a result of heat treatment. Conversely, partial denaturation caused by high-pressure treatment increased the ability of egg white to stimulate Th2 responses and its allergenic potential.

  1. Bacterial Infection Elicits Heat Shock Protein 72 Release from Pleural Mesothelial Cells

    PubMed Central

    Varano della Vergiliana, Julius F.; Lansley, Sally M.; Porcel, Jose M.; Bielsa, Silvia; Brown, Jeremy S.; Creaney, Jenette; Temple, Suzanna E. L.; Waterer, Grant W.; Lee, Y. C. Gary

    2013-01-01

    Heat shock protein 70 (HSP70) has been implicated in infection-related processes and has been found in body fluids during infection. This study aimed to determine whether pleural mesothelial cells release HSP70 in response to bacterial infection in vitro and in mouse models of serosal infection. In addition, the in vitro cytokine effects of the HSP70 isoform, Hsp72, on mesothelial cells were examined. Further, Hsp72 was measured in human pleural effusions and levels compared between non-infectious and infectious patients to determine the diagnostic accuracy of pleural fluid Hsp72 compared to traditional pleural fluid parameters. We showed that mesothelial release of Hsp72 was significantly raised when cells were treated with live and heat-killed Streptococcus pneumoniae. In mice, intraperitoneal injection of S. pneumoniae stimulated a 2-fold increase in Hsp72 levels in peritoneal lavage (p<0.01). Extracellular Hsp72 did not induce or inhibit mediator release from cultured mesothelial cells. Hsp72 levels were significantly higher in effusions of infectious origin compared to non-infectious effusions (p<0.05). The data establish that pleural mesothelial cells can release Hsp72 in response to bacterial infection and levels are raised in infectious pleural effusions. The biological role of HSP70 in pleural infection warrants exploration. PMID:23704948

  2. Human Cytomegalovirus IE1 Protein Elicits a Type II Interferon-Like Host Cell Response That Depends on Activated STAT1 but Not Interferon-γ

    PubMed Central

    Knoblach, Theresa; Grandel, Benedikt; Seiler, Jana

    2011-01-01

    Human cytomegalovirus (hCMV) is a highly prevalent pathogen that, upon primary infection, establishes life-long persistence in all infected individuals. Acute hCMV infections cause a variety of diseases in humans with developmental or acquired immune deficits. In addition, persistent hCMV infection may contribute to various chronic disease conditions even in immunologically normal people. The pathogenesis of hCMV disease has been frequently linked to inflammatory host immune responses triggered by virus-infected cells. Moreover, hCMV infection activates numerous host genes many of which encode pro-inflammatory proteins. However, little is known about the relative contributions of individual viral gene products to these changes in cellular transcription. We systematically analyzed the effects of the hCMV 72-kDa immediate-early 1 (IE1) protein, a major transcriptional activator and antagonist of type I interferon (IFN) signaling, on the human transcriptome. Following expression under conditions closely mimicking the situation during productive infection, IE1 elicits a global type II IFN-like host cell response. This response is dominated by the selective up-regulation of immune stimulatory genes normally controlled by IFN-γ and includes the synthesis and secretion of pro-inflammatory chemokines. IE1-mediated induction of IFN-stimulated genes strictly depends on tyrosine-phosphorylated signal transducer and activator of transcription 1 (STAT1) and correlates with the nuclear accumulation and sequence-specific binding of STAT1 to IFN-γ-responsive promoters. However, neither synthesis nor secretion of IFN-γ or other IFNs seems to be required for the IE1-dependent effects on cellular gene expression. Our results demonstrate that a single hCMV protein can trigger a pro-inflammatory host transcriptional response via an unexpected STAT1-dependent but IFN-independent mechanism and identify IE1 as a candidate determinant of hCMV pathogenicity. PMID:21533215

  3. 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.

  4. Viral-mimicking protein nanoparticle vaccine for eliciting anti-tumor responses

    PubMed Central

    Molino, Nicholas M.; Neek, Medea; Tucker, Jo Anne; Nelson, Edward L.; Wang, Szu-Wen

    2016-01-01

    The immune system is a powerful resource for the eradication of cancer, but to overcome the low immunogenicity of tumor cells, a sufficiently strong CD8+ T cell-mediated adaptive immune response is required. Nanoparticulate biomaterials represent a potentially effective delivery system for cancer vaccines, as they can be designed to mimic viruses, which are potent inducers of cellular immunity. We have been exploring the non-viral pyruvate dehydrogenase E2 protein nanoparticle as a biomimetic platform for cancer vaccine delivery. Simultaneous conjugation of a melanoma-associated gp100 epitope and CpG to the E2 nanoparticle (CpG-gp-E2) yielded an antigen-specific increase in the CD8+ T cell proliferation index and IFN-γ secretion by 1.5-fold and 5-fold, respectively, compared to an unbound peptide and CpG formulation. Remarkably, a single nanoparticle immunization resulted in a 120-fold increase in the frequency of melanoma epitope-specific CD8+ T cells in draining lymph nodes and a 30-fold increase in the spleen, relative to free peptide with free CpG. Furthermore, in the very aggressive B16 melanoma murine tumor model, prophylactic immunization with CpG-gp-E2 delayed the onset of tumor growth by approximately 5.5 days and increased animal survival time by approximately 40%, compared to PBS-treated animals. These results show that by combining optimal particle size and simultaneous co-delivery of molecular vaccine components, antigen-specific anti-tumor immune responses can be significantly increased. PMID:26894870

  5. Heterologous and cell free protein expression systems.

    PubMed

    Farrokhi, Naser; Hrmova, Maria; Burton, Rachel A; Fincher, Geoffrey B

    2009-01-01

    In recognition of the fact that a relatively small percentage of 'named' genes in databases have any experimental proof for their annotation, attention is shifting towards the more accurate assignment of functions to individual genes in a genome. The central objective will be to reduce our reliance on nucleotide or amino acid sequence similarities as a means to define the functions of genes and to annotate genome sequences. There are many unsolved technical difficulties associated with the purification of specific proteins from extracts of biological material, especially where the protein is present in low abundance, has multiple isoforms or is found in multiple post-translationally modified forms. The relative ease with which cDNAs can be cloned has led to the development of methods through which cDNAs from essentially any source can be expressed in a limited range of suitable host organisms, so that sufficient levels of the encoded proteins can be generated for functional analysis. Recently, these heterologous expression systems have been supplemented by more robust prokaryotic and eukaryotic cell-free protein synthesis systems. In this chapter, common host systems for heterologous expression are reviewed and the current status of cell-free expression systems will be presented. New approaches to overcoming the special problems encountered during the expression of membrane-associated proteins will also be addressed. Methodological considerations, including the characteristics of codon usage in the expressed DNA, peptide tags that facilitate subsequent purification of the expressed proteins and the role of post-translational modifications, are examined.

  6. Intranasal administration of RSV antigen-expressing MCMV elicits robust tissue-resident effector and effector memory CD8+ T cells in the lung

    PubMed Central

    Morabito, Kaitlyn M.; Ruckwardt, Tracy R.; Redwood, Alec J.; Moin, Syed M.; Price, David A.; Graham, Barney S.

    2016-01-01

    Cytomegalovirus vectors are promising delivery vehicles for vaccine strategies that aim to elicit effector CD8+ T cells. To determine how the route of immunization affects CD8+ T cell responses in the lungs of mice vaccinated with a murine cytomegalovirus vector expressing the respiratory syncytial virus matrix (M) protein, we infected CB6F1 mice via the intranasal or intraperitoneal route and evaluated the M-specific CD8+ T cell response at early and late time points. We found that intranasal vaccination generated robust and durable tissue-resident effector and effector memory CD8+ T cell populations that were undetectable after intraperitoneal vaccination. The generation of these antigen-experienced cells by intranasal vaccination resulted in earlier T cell responses, interferon gamma secretion, and viral clearance after respiratory syncytial virus challenge. Collectively, these findings validate a novel approach to vaccination that emphasizes the route of delivery as a key determinant of immune priming at the site of vulnerability. PMID:27220815

  7. Intranasal administration of RSV antigen-expressing MCMV elicits robust tissue-resident effector and effector memory CD8+ T cells in the lung.

    PubMed

    Morabito, K M; Ruckwardt, T R; Redwood, A J; Moin, S M; Price, D A; Graham, B S

    2017-03-01

    Cytomegalovirus vectors are promising delivery vehicles for vaccine strategies that aim to elicit effector CD8+ T cells. To determine how the route of immunization affects CD8+ T-cell responses in the lungs of mice vaccinated with a murine cytomegalovirus vector expressing the respiratory syncytial virus matrix (M) protein, we infected CB6F1 mice via the intranasal or intraperitoneal route and evaluated the M-specific CD8+ T-cell response at early and late time points. We found that intranasal vaccination generated robust and durable tissue-resident effector and effector memory CD8+ T-cell populations that were undetectable after intraperitoneal vaccination. The generation of these antigen-experienced cells by intranasal vaccination resulted in earlier T-cell responses, interferon gamma secretion, and viral clearance after respiratory syncytial virus challenge. Collectively, these findings validate a novel approach to vaccination that emphasizes the route of delivery as a key determinant of immune priming at the site of vulnerability.

  8. Phenylbutyrate improves nitrogen disposal via alternative pathway without eliciting an increase in protein breakdown and catabolism in control and ornithine transcarbamylace-deficient patients

    USDA-ARS?s Scientific Manuscript database

    Phenylbutyrate (PB) is a drug used in urea cycle disorder patients to elicit alternative pathways for nitrogen disposal. However, PB decreases plasma branched chain amino acid (BCAA) concentrations and prior research suggests that PB may increase leucine oxidation, indicating increased protein degra...

  9. Alterations in metal toxicity and metal-induced metallothionein gene expression elicited by growth medium calcium concentration.

    PubMed

    Singh, Rajendra K; Albrecht, Amy L; Somji, Seema; Sens, Mary Ann; Sens, Donald A; Garrett, Scott H

    2008-06-01

    The calcium content of the growth medium has been shown to influence the growth and differentiation of primary epithelial cells in culture. The goal of the present study was to determine if growth medium calcium concentration could influence the susceptibility to metal toxicity and metallothionein gene expression of an immortalized human prostate-derived epithelial cell line (RWPE-1). The RWPE-1 cell line was grown in medium containing either 0.1 or 1.4 mM calcium. Confluent cells were exposed to either Zn(+2) (50, 100, or 150 microM) or Cd(+2) (3, 6, or 12 microM) for 13 days, and cell toxicity and MT gene expression were determined along the time course of exposure. It was demonstrated that the calcium content of the growth medium had a marked influence on Zn(+2) toxicity and a lesser but significant effect on Cd(+2) toxicity to the RWPE-1 cells. Calcium concentration of the growth medium was also shown to alter the accumulation of MT-1/2 protein and MT-1E, MT-1X, and MT-2A mRNAs. It was shown that MT-1/2 protein was markedly increased for metal-exposed cells grown in medium containing 0.1 mM calcium; however, the increased expression did not cause an increase in the resistance of the cells to Zn(+2) or Cd(+2) exposure. These observations show that growth medium calcium concentration can influence metal toxicity and the pattern of expression of the MT mRNAs and protein for RWPE-1 cells. The results suggest that caution should be exercised when comparing toxicological responses between cell lines that may be grown in growth formulations differing in calcium concentration.

  10. Immunization with a 22-kDa outer membrane protein elicits protective immunity to multidrug-resistant Acinetobacter baumannii

    PubMed Central

    Huang, Weiwei; Yao, Yufeng; Wang, Shijie; Xia, Ye; Yang, Xu; Long, Qiong; Sun, Wenjia; Liu, Cunbao; Li, Yang; Chu, Xiaojie; Bai, Hongmei; Yao, Yueting; Ma, Yanbing

    2016-01-01

    A. baumannii infections are becoming more and more serious health issues with rapid emerging of multidrug and extremely drug resistant strains, and therefore, there is an urgent need for the development of nonantibiotic-based intervention strategies. This study aimed at identifying whether an outer membrane protein with molecular weight of about 22 kDa (Omp22) holds the potentials to be an efficient vaccine candidate and combat A. baumannii infection. Omp22 which has a molecule length of 217 amino acids kept more than 95% conservation in totally 851 reported A. baumannii strains. Recombinant Omp22 efficiently elicited high titers of specific IgG in mice. Both active and passive immunizations of Omp22 increased the survival rates of mice, suppressed the bacterial burdens in the organs and peripheral blood, and reduced the levels of serum inflammatory cytokines and chemokines. Opsonophagocytosis assays showed in vitro that Omp22 antiserum had highly efficient bactericidal activities on clonally distinct clinical A. baumannii isolates, which were partly complements-dependent and opsonophagocytic killing effects. Additionally, administration with as high as 500 μg of Omp22 didn’t cause obvious pathological changes in mice. In conclusion, Omp22 is a novel conserved and probably safe antigen for developing effective vaccines or antisera to control A. baumannii infections. PMID:26853590

  11. Immunization with a 22-kDa outer membrane protein elicits protective immunity to multidrug-resistant Acinetobacter baumannii.

    PubMed

    Huang, Weiwei; Yao, Yufeng; Wang, Shijie; Xia, Ye; Yang, Xu; Long, Qiong; Sun, Wenjia; Liu, Cunbao; Li, Yang; Chu, Xiaojie; Bai, Hongmei; Yao, Yueting; Ma, Yanbing

    2016-02-08

    A. baumannii infections are becoming more and more serious health issues with rapid emerging of multidrug and extremely drug resistant strains, and therefore, there is an urgent need for the development of nonantibiotic-based intervention strategies. This study aimed at identifying whether an outer membrane protein with molecular weight of about 22 kDa (Omp22) holds the potentials to be an efficient vaccine candidate and combat A. baumannii infection. Omp22 which has a molecule length of 217 amino acids kept more than 95% conservation in totally 851 reported A. baumannii strains. Recombinant Omp22 efficiently elicited high titers of specific IgG in mice. Both active and passive immunizations of Omp22 increased the survival rates of mice, suppressed the bacterial burdens in the organs and peripheral blood, and reduced the levels of serum inflammatory cytokines and chemokines. Opsonophagocytosis assays showed in vitro that Omp22 antiserum had highly efficient bactericidal activities on clonally distinct clinical A. baumannii isolates, which were partly complements-dependent and opsonophagocytic killing effects. Additionally, administration with as high as 500 μg of Omp22 didn't cause obvious pathological changes in mice. In conclusion, Omp22 is a novel conserved and probably safe antigen for developing effective vaccines or antisera to control A. baumannii infections.

  12. Activation of dimeric ABA receptors elicits guard cell closure, ABA-regulated gene expression, and drought tolerance.

    PubMed

    Okamoto, Masanori; Peterson, Francis C; Defries, Andrew; Park, Sang-Youl; Endo, Akira; Nambara, Eiji; Volkman, Brian F; Cutler, Sean R

    2013-07-16

    Abscisic acid (ABA) is an essential molecule in plant abiotic stress responses. It binds to soluble pyrabactin resistance1/PYR1-like/regulatory component of ABA receptor receptors and stabilizes them in a conformation that inhibits clade A type II C protein phosphatases; this leads to downstream SnRK2 kinase activation and numerous cellular outputs. We previously described the synthetic naphthalene sulfonamide ABA agonist pyrabactin, which activates seed ABA responses but fails to trigger substantial responses in vegetative tissues in Arabidopsis thaliana. Here we describe quinabactin, a sulfonamide ABA agonist that preferentially activates dimeric ABA receptors and possesses ABA-like potency in vivo. In Arabidopsis, the transcriptional responses induced by quinabactin are highly correlated with those induced by ABA treatments. Quinabactin treatments elicit guard cell closure, suppress water loss, and promote drought tolerance in adult Arabidopsis and soybean plants. The effects of quinabactin are sufficiently similar to those of ABA that it is able to rescue multiple phenotypes observed in the ABA-deficient mutant aba2. Genetic analyses show that quinabactin's effects in vegetative tissues are primarily mediated by dimeric ABA receptors. A PYL2-quinabactin-HAB1 X-ray crystal structure solved at 1.98-Å resolution shows that quinabactin forms a hydrogen bond with the receptor/PP2C "lock" hydrogen bond network, a structural feature absent in pyrabactin-receptor/PP2C complexes. Our results demonstrate that ABA receptors can be chemically controlled to enable plant protection against water stress and define the dimeric receptors as key targets for chemical modulation of vegetative ABA responses.

  13. Activation of dimeric ABA receptors elicits guard cell closure, ABA-regulated gene expression, and drought tolerance

    PubMed Central

    Okamoto, Masanori; Peterson, Francis C.; Defries, Andrew; Park, Sang-Youl; Endo, Akira; Nambara, Eiji; Volkman, Brian F.; Cutler, Sean R.

    2013-01-01

    Abscisic acid (ABA) is an essential molecule in plant abiotic stress responses. It binds to soluble pyrabactin resistance1/PYR1-like/regulatory component of ABA receptor receptors and stabilizes them in a conformation that inhibits clade A type II C protein phosphatases; this leads to downstream SnRK2 kinase activation and numerous cellular outputs. We previously described the synthetic naphthalene sulfonamide ABA agonist pyrabactin, which activates seed ABA responses but fails to trigger substantial responses in vegetative tissues in Arabidopsis thaliana. Here we describe quinabactin, a sulfonamide ABA agonist that preferentially activates dimeric ABA receptors and possesses ABA-like potency in vivo. In Arabidopsis, the transcriptional responses induced by quinabactin are highly correlated with those induced by ABA treatments. Quinabactin treatments elicit guard cell closure, suppress water loss, and promote drought tolerance in adult Arabidopsis and soybean plants. The effects of quinabactin are sufficiently similar to those of ABA that it is able to rescue multiple phenotypes observed in the ABA-deficient mutant aba2. Genetic analyses show that quinabactin’s effects in vegetative tissues are primarily mediated by dimeric ABA receptors. A PYL2-quinabactin-HAB1 X-ray crystal structure solved at 1.98-Å resolution shows that quinabactin forms a hydrogen bond with the receptor/PP2C “lock” hydrogen bond network, a structural feature absent in pyrabactin-receptor/PP2C complexes. Our results demonstrate that ABA receptors can be chemically controlled to enable plant protection against water stress and define the dimeric receptors as key targets for chemical modulation of vegetative ABA responses. PMID:23818638

  14. MyD88 expression by CNS-resident cells is pivotal for eliciting protective immunity in brain abscesses.

    PubMed

    Garg, Sarita; Nichols, Jessica R; Esen, Nilufer; Liu, Shuliang; Phulwani, Nirmal K; Syed, Mohsin Md; Wood, William H; Zhang, Yongqing; Becker, Kevin G; Aldrich, Amy; Kielian, Tammy

    2009-05-05

    MyD88 KO (knockout) mice are exquisitely sensitive to CNS (central nervous system) infection with Staphylococcus aureus, a common aetiological agent of brain abscess, exhibiting global defects in innate immunity and exacerbated tissue damage. However, since brain abscesses are typified by the involvement of both activated CNS-resident and infiltrating immune cells, in our previous studies it has been impossible to determine the relative contribution of MyD88-dependent signalling in the CNS compared with the peripheral immune cell compartments. In the present study we addressed this by examining the course of S. aureus infection in MyD88 bone marrow chimaera mice. Interestingly, chimaeras where MyD88 was present in the CNS, but not bone marrow-derived cells, mounted pro-inflammatory mediator expression profiles and neutrophil recruitment equivalent to or exceeding that detected in WT (wild-type) mice. These results implicate CNS MyD88 as essential in eliciting the initial wave of inflammation during the acute response to parenchymal infection. Microarray analysis of infected MyD88 KO compared with WT mice revealed a preponderance of differentially regulated genes involved in apoptotic pathways, suggesting that the extensive tissue damage characteristic of brain abscesses from MyD88 KO mice could result from dysregulated apoptosis. Collectively, the findings of the present study highlight a novel mechanism for CNS-resident cells in initiating a protective innate immune response in the infected brain and, in the absence of MyD88 in this compartment, immunity is compromised.

  15. The nucleocapsid protein of Rift Valley fever virus is a potent human CD8+ T cell antigen and elicits memory responses.

    PubMed

    Xu, Weidong; Watts, Douglas M; Costanzo, Margaret C; Tang, Xiaolei; Venegas, Leon A; Jiao, Feng; Sette, Alessandro; Sidney, John; Sewell, Andrew K; Wooldridge, Linda; Makino, Shinji; Morrill, John C; Peters, Clarence J; Kan-Mitchell, June

    2013-01-01

    There is no licensed human vaccine currently available for Rift Valley Fever Virus (RVFV), a Category A high priority pathogen and a serious zoonotic threat. While neutralizing antibodies targeting the viral glycoproteins are protective, they appear late in the course of infection, and may not be induced in time to prevent a natural or bioterrorism-induced outbreak. Here we examined the immunogenicity of RVFV nucleocapsid (N) protein as a CD8(+) T cell antigen with the potential for inducing rapid protection after vaccination. HLA-A*0201 (A2)-restricted epitopic determinants were identified with N-specific CD8(+) T cells from eight healthy donors that were primed with dendritic cells transduced to express N, and subsequently expanded in vitro by weekly re-stimulations with monocytes pulsed with 59 15mer overlapping peptides (OLPs) across N. Two immunodominant epitopes, VT9 (VLSEWLPVT, N(121-129)) and IL9 (ILDAHSLYL, N165-173), were defined. VT9- and IL9-specific CD8(+) T cells identified by tetramer staining were cytotoxic and polyfunctional, characteristics deemed important for viral control in vivo. These peptides induced specific CD8(+) T cell responses in A2-transgenic mice, and more importantly, potent N-specific CD8(+) T cell reactivities, including VT9- and IL9-specific ones, were mounted by mice after a booster vaccination with the live attenuated RVF MP-12. Our data suggest that the RVFV N protein is a potent human T cell immunogen capable of eliciting broad, immunodominant CD8(+) T cell responses that are potentially protective. Understanding the immune responses to the nucleocapsid is central to the design of an effective RVFV vaccine irrespective of whether this viral protein is effective as a stand-alone immunogen or only in combination with other RVFV antigens.

  16. The Nucleocapsid Protein of Rift Valley Fever Virus Is a Potent Human CD8+ T Cell Antigen and Elicits Memory Responses

    PubMed Central

    Xu, Weidong; Watts, Douglas M.; Costanzo, Margaret C.; Tang, Xiaolei; Venegas, Leon A.; Jiao, Feng; Sette, Alessandro; Sidney, John; Sewell, Andrew K.; Wooldridge, Linda; Makino, Shinji; Morrill, John C.; Peters, Clarence J.; Kan-Mitchell, June

    2013-01-01

    There is no licensed human vaccine currently available for Rift Valley Fever Virus (RVFV), a Category A high priority pathogen and a serious zoonotic threat. While neutralizing antibodies targeting the viral glycoproteins are protective, they appear late in the course of infection, and may not be induced in time to prevent a natural or bioterrorism-induced outbreak. Here we examined the immunogenicity of RVFV nucleocapsid (N) protein as a CD8+ T cell antigen with the potential for inducing rapid protection after vaccination. HLA-A*0201 (A2)-restricted epitopic determinants were identified with N-specific CD8+ T cells from eight healthy donors that were primed with dendritic cells transduced to express N, and subsequently expanded in vitro by weekly re-stimulations with monocytes pulsed with 59 15mer overlapping peptides (OLPs) across N. Two immunodominant epitopes, VT9 (VLSEWLPVT, N121–129) and IL9 (ILDAHSLYL, N165–173), were defined. VT9- and IL9-specific CD8+ T cells identified by tetramer staining were cytotoxic and polyfunctional, characteristics deemed important for viral control in vivo. These peptides induced specific CD8+ T cell responses in A2-transgenic mice, and more importantly, potent N-specific CD8+ T cell reactivities, including VT9- and IL9-specific ones, were mounted by mice after a booster vaccination with the live attenuated RVF MP-12. Our data suggest that the RVFV N protein is a potent human T cell immunogen capable of eliciting broad, immunodominant CD8+ T cell responses that are potentially protective. Understanding the immune responses to the nucleocapsid is central to the design of an effective RVFV vaccine irrespective of whether this viral protein is effective as a stand-alone immunogen or only in combination with other RVFV antigens. PMID:23527138

  17. Sporozoite neutralizing antibodies elicited in mice and rhesus macaques immunized with a Plasmodium falciparum repeat peptide conjugated to meningococcal outer membrane protein complex

    PubMed Central

    Przysiecki, Craig; Lucas, Bob; Mitchell, Robert; Carapau, Daniel; Wen, Zhiyun; Xu, Hui; Wang, Xin-Min; Nahas, Debbie; Wu, Chengwei; Hepler, Robert; Ottinger, Elizabeth; ter Meulen, Jan; Kaslow, David; Shiver, John; Nardin, Elizabeth

    2012-01-01

    Antibodies that neutralize infectivity of malaria sporozoites target the central repeat region of the circumsporozoite (CS) protein, which in Plasmodium falciparum is comprised primarily of 30–40 tandem NANP tetramer repeats. We evaluated immunogenicity of an alum-adsorbed (NANP)6 peptide conjugated to an outer membrane protein complex (OMPC) derived from Neisseria meningitidis, a carrier protein used in a licensed Haemophilus influenzae pediatric vaccine. Mice immunized with (NANP)6-OMPC adsorbed to Merck's alum adjuvant (MAA), with or without Iscomatrix® as co-adjuvant, developed high levels of anti-repeat peptide antibody that inhibited in vitro invasion of human hepatoma cells by transgenic P. berghei sporozoites that express P. falciparum CS repeats (PfPb). Inhibition of sporozoite invasion in vitro correlated with in vivo resistance to challenge by the bites of PfPb-infected mosquitoes. Challenged mice had >90% reduction of hepatic stage parasites as measured by real-time PCR, and either sterile immunity, i.e., no detectable blood stage parasites, or delayed prepatent periods which indicate neutralization of a majority, but not all, sporozoites. Rhesus macaques immunized with two doses of (NANP)6-OMPC/MAA formulated with Iscomatrix® developed anti-repeat antibodies that persisted for ~2 years. A third dose of (NANP)6-OMPC/MAA+ Iscomatrix® at that time elicited strong anamnestic antibody responses. Rhesus macaque immune sera obtained post second and third dose of vaccine displayed high levels of sporozoite neutralizing activity in vitro that correlated with presence of high anti-repeat antibody titers. These preclinical studies in mice of different MHC haplotypes and a non-human primate support use of CS peptide-OMPC conjugates as a highly immunogenic platform to evaluate CS protective epitopes. Potential pre-erythrocytic vaccines can be combined with sexual blood stage vaccines as a multi-antigen malaria vaccine to block invasion and transmission of

  18. Role of amphotericin B upon enhancement of protective immunity elicited by oral administration with liposome-encapsulated-Japanese encephalitis virus nonstructural protein 1 (NS1) in mice.

    PubMed

    Lin, Tsung-Shun; Chuang, Chuan-Chang; Hsu, Hui-Ling; Liu, Yu-Tien; Lin, Wen-Po; Liang, Chung-Chih; Liu, Wen-Tssann

    2010-09-01

    Amphotericin B (AmB) is an antifungal antibiotic the activity of which has been associated with modulation of pro-inflammatory cytokines expression in cultured cells. Herein we reveal that co-administration with AmB enhances the immunogenicity of oral Lip-JENS1 vaccine which derived from liposomes functionalized with DSPC (distearoylphosphatidylcholine) and cholesterol (2:1, molar ratio)-bearing JE virus NS1 protein (600 microg ml(-1)). Oral single dose of Lip-JENS1 elicited a detectable serum NS1-specific IgG antibody response from a mouse model. Remarkably, the addition of AmB (125 microg per mouse), particularly, 2 h prior to, but not simultaneously with, the administration of Lip-JENS1 significantly enhanced the systemic antigen-specific antibody response, providing superior protection against lethal JEV challenges. Further, we observed AmB-induced the transcription of cytokine expression and translocation of transcriptional factor NF-kappaB from the cytoplasm to the nucleus for the murine macrophage J774A.1. Moreover, Peyer's-patch lymphocytes (PPL) from AmB-treated mice produced high levels of IL-1beta, IL-6 and TNF-alpha expression compared to the corresponding control of cells from non-treated mice. Taken together, the results suggest that AmB exerts a profound influence upon mucosal vaccination with Lip-JENS1, possibly playing an adjuvant-augmented role to "fine-tune" humoral as well as cellular immune response, thus conferring enhanced protective immunity for immunising individuals against JE infection.

  19. PVY(NTN) elicits a diverse gene expression response in different potato genotypes in the first 12 h after inoculation.

    PubMed

    Baebler, Spela; Krecic-Stres, Hana; Rotter, Ana; Kogovsek, Polona; Cankar, Katarina; Kok, Esther J; Gruden, Kristina; Kovac, Maja; Zel, Jana; Pompe-Novak, Marusa; Ravnikar, Maja

    2009-03-01

    Host gene expression changes in the early response to potato virus Y(NTN) interaction were compared in two differently sensitive potato cultivars: the resistant cultivar Santé and the sensitive cultivar Igor. Hybridization of potato TIGR cDNA microarrays allowed us to monitor the expression of approximately 10,000 genes simultaneously at 0.5 and 12 h post-inoculation (hpi). Microarray data, analysed by statistics and data mining, were complemented by subtraction library construction and sequence analysis to validate the findings. The expression profiles of the two cultivars were similar and faint at 0.5 hpi, but they differed substantially at 12 hpi. Although, at 0.5 hpi, cv. Santé responded by the differential expression of a greater number of genes, at 12 hpi the number was higher in cv. Igor. The majority of genes in this cultivar were down-regulated at 12 hpi, indicating a host gene shut-off. Suites of genes that exhibited altered transcript abundance in response to the virus were identified, and included genes involved in the processes of photosynthesis, perception, signalling and defence responses. The expression of the considerable number of genes associated with photosynthesis was surprisingly up-regulated as early as 0.5 hpi and down-regulated at 12 hpi in both cultivars. The expression of genes involved in perception and signalling was increased in the sensitive cultivar at 12 hpi. By contrast, a simultaneous strong defence response at the transcriptional level was evident in the resistant cultivar, as shown by the up-regulation of genes involved in brassinosteroid, polyamine and secondary metabolite biosynthesis, and of genes coding for pathogenesis-related proteins.

  20. Yin Yang 1 Intronic Binding Sequences and Splicing Elicit Intron-Mediated Enhancement of Ubiquitin C Gene Expression

    PubMed Central

    Bianchi, Marzia; Crinelli, Rita; Giacomini, Elisa; Carloni, Elisa; Radici, Lucia; Magnani, Mauro

    2013-01-01

    In a number of organisms, introns affect expression of the gene in which they are contained. Our previous studies revealed that the 5′-UTR intron of human ubiquitin C (UbC) gene is responsible for the boost of reporter gene expression and is able to bind, in vitro, Yin Yang 1 (YY1) trans-acting factor. In this work, we demonstrate that intact YY1 binding sequences are required for maximal promoter activity and YY1 silencing causes downregulation of luciferase mRNA levels. However, YY1 motifs fail to enhance gene expression when the intron is moved upstream of the proximal promoter, excluding the typical enhancer hypothesis and supporting a context-dependent action, like intron-mediated enhancement (IME). Yet, almost no expression is seen in the construct containing an unspliceable version of UbC intron, indicating that splicing is essential for promoter activity. Moreover, mutagenesis of YY1 binding sites and YY1 knockdown negatively affect UbC intron removal from both endogenous and reporter transcripts. Modulation of splicing efficiency by YY1 cis-elements and protein factor may thus be part of the mechanism(s) by which YY1 controls UbC promoter activity. Our data highlight the first evidence of the involvement of a sequence-specific DNA binding factor in IME. PMID:23776572

  1. Heterologous Prime-Boost Regimens with a Recombinant Chimpanzee Adenoviral Vector and Adjuvanted F4 Protein Elicit Polyfunctional HIV-1-Specific T-Cell Responses in Macaques

    PubMed Central

    Lorin, Clarisse; Vanloubbeeck, Yannick; Baudart, Sébastien; Ska, Michaël; Bayat, Babak; Brauers, Geoffroy; Clarinval, Géraldine; Donner, Marie-Noëlle; Marchand, Martine; Koutsoukos, Marguerite; Mettens, Pascal; Cohen, Joe; Voss, Gerald

    2015-01-01

    HIV-1-specific CD4+ and CD8+ T lymphocytes are important for HIV-1 replication control. F4/AS01 consists of F4 recombinant fusion protein (containing clade B Gag/p24, Pol/RT, Nef and Gag/p17) formulated in AS01 Adjuvant System, and was shown to induce F4-specific polyfunctional CD4+ T-cell responses in humans. While replication-incompetent recombinant HIV-1/SIV antigen-expressing human adenoviral vectors can elicit high-frequency antigen-specific CD8+ T-cell responses, their use is hampered by widespread pre-existing immunity to human serotypes. Non-human adenovirus serotypes associated with lower prevalence may offer an alternative strategy. We evaluated the immunogenicity of AdC7-GRN (‘A’), a recombinant chimpanzee adenovirus type 7 vector expressing clade B Gag, RT and Nef, and F4/AS01 (‘P’), when delivered intramuscularly in homologous (PP or AA) and heterologous (AAPP or PPAA) prime-boost regimens, in macaques and mice. Vaccine-induced HIV-1-antigen-specific T cells in peripheral blood (macaques), liver, spleen, and intestinal and genital mucosa (mice) were characterized by intracellular cytokine staining. Vaccine-specific IgG antibodies (macaques) were detected using ELISA. In macaques, only the heterologous prime-boost regimens induced polyfunctional, persistent and balanced CD4+ and CD8+ T-cell responses specific to each HIV-1 vaccine antigen. AdC7-GRN priming increased the polyfunctionality of F4/AS01-induced CD4+ T cells. Approximately 50% of AdC7-GRN-induced memory CD8+ T cells exhibited an effector-memory phenotype. HIV-1-specific antibodies were detected with each regimen. In mice, antigen-specific CD4+ and CD8+ T-cell responses were detected in the mucosal and systemic anatomical compartments assessed. When administered in heterologous prime-boost regimens, AdC7-GRN and F4/AS01 candidate vaccines acted complementarily in inducing potent and persistent peripheral blood HIV-1-specific CD4+ and CD8+ T-cell responses and antibodies in macaques

  2. Expression of clock proteins in developing tooth.

    PubMed

    Zheng, Li; Papagerakis, Silvana; Schnell, Santiago D; Hoogerwerf, Willemijntje A; Papagerakis, Petros

    2011-01-01

    Morphological and functional changes during ameloblast and odontoblast differentiation suggest that enamel and dentin formation is under circadian control. Circadian rhythms are endogenous self-sustained oscillations with periods of 24h that control diverse physiological and metabolic processes. Mammalian clock genes play a key role in synchronizing circadian functions in many organs. However, close to nothing is known on clock genes expression during tooth development. In this work, we investigated the expression of four clock genes during tooth development. Our results showed that circadian clock genes Bmal1, clock, per1, and per2 mRNAs were detected in teeth by RT-PCR. Immunohistochemistry showed that clock protein expression was first detected in teeth at the bell stage (E17), being expressed in EOE and dental papilla cells. At post-natal day four (PN4), all four clock proteins continued to be expressed in teeth but with different intensities, being strongly expressed within the nucleus of ameloblasts and odontoblasts and down-regulated in dental pulp cells. Interestingly, at PN21 incisor, expression of clock proteins was down-regulated in odontoblasts of the crown-analogue side but expression was persisting in root-analogue side odontoblasts. In contrast, both crown and root odontoblasts were strongly stained for all four clock proteins in first molars at PN21. Within the periodontal ligament (PDL) space, epithelial rests of Malassez (ERM) showed the strongest expression among other PDL cells. Our data suggests that clock genes might be involved in the regulation of ameloblast and odontoblast functions, such as enamel and dentin protein secretion and matrix mineralization. Copyright © 2010 Elsevier B.V. All rights reserved.

  3. Transient Protein Expression by Agroinfiltration in Lettuce.

    PubMed

    Chen, Qiang; Dent, Matthew; Hurtado, Jonathan; Stahnke, Jake; McNulty, Alyssa; Leuzinger, Kahlin; Lai, Huafang

    2016-01-01

    Current systems of recombinant protein production include bacterial, insect, and mammalian cell culture. However, these platforms are expensive to build and operate at commercial scales and/or have limited abilities to produce complex proteins. In recent years, plant-based expression systems have become top candidates for the production of recombinant proteins as they are highly scalable, robust, safe, and can produce complex proteins due to having a eukaryotic endomembrane system. Newly developed "deconstructed" viral vectors delivered via Agrobacterium tumefaciens (agroinfiltration) have enabled robust plant-based production of proteins with a wide range of applications. The leafy Lactuca sativa (lettuce) plant with its strong foundation in agriculture is an excellent host for pharmaceutical protein production. Here, we describe a method for agroinfiltration of lettuce that can rapidly produce high levels of recombinant proteins in a matter of days and has the potential to be scaled up to an agricultural level.

  4. Integral Membrane Protein Expression in Saccharomyces cerevisiae.

    PubMed

    Boswell-Casteel, Rebba C; Johnson, Jennifer M; Stroud, Robert M; Hays, Franklin A

    2016-01-01

    Eukaryotic integral membrane proteins are challenging targets for crystallography or functional characterization in a purified state. Since expression is often a limiting factor when studying this difficult class of biological macromolecules, the intent of this chapter is to focus on the expression of eukaryotic integral membrane proteins (IMPs) using the model organism Saccharomyces cerevisiae. S. cerevisiae is a prime candidate for the expression of eukaryotic IMPs because it offers the convenience of using episomal expression plasmids, selection of positive transformants, posttranslational modifications, and it can properly fold and target IMPs. Here we present a generalized protocol and insights based on our collective knowledge as an aid to overcoming the challenges faced when expressing eukaryotic IMPs in S. cerevisiae.

  5. hnRNP G elicits tumor-suppressive activity in part by upregulating the expression of Txnip

    SciTech Connect

    Shin, Ki-Hyuk Kim, Reuben H.; Kim, Roy H.; Kang, Mo K.; Park, No-Hee

    2008-08-08

    Heterogeneous nuclear ribonuclearproteins (hnRNPs) are nucleic acid-binding proteins and have critical roles in DNA repair, telomere regulation, and transcriptional gene regulation. Previously, we showed that hnRNP G has tumor-suppressive activity in human oral squamous cell carcinoma cells. Therefore, the identification of hnRNP G target genes is important for understanding the function of hnRNP G and its tumor-suppressive activity. In this study, we identify a known tumor suppressor gene, thioredoxin-interacting protein (Txnip) gene as a novel target of hnRNP G. Expression of Txnip is upregulated by wild-type (wt) hnRNP G but not by a suppression-defective mutant hnRNP G (K22R) in human squamous cell carcinoma. Wt hnRNP G binds and transactivates the Txnip promoter in vivo, whereas the K22R mutant does not. Furthermore, overexpression of Txnip alone in cancer cells leads to the inhibition of anchorage-independent growth and in vivo tumorigenicity in immunocompromised mice, suggesting a reversion of the transformation phenotype. These studies indicate that hnRNP G promotes the expression of Txnip and mediates its tumor-suppressive effect.

  6. Biotechnology Protein Expression and Purification Facility

    NASA Technical Reports Server (NTRS)

    2003-01-01

    The purpose of the Project Scientist Core Facility is to provide purified proteins, both recombinant and natural, to the Biotechnology Science Team Project Scientists and the NRA-Structural Biology Test Investigators. Having a core facility for this purpose obviates the need for each scientist to develop the necessary expertise and equipment for molecular biology, protein expression, and protein purification. Because of this, they are able to focus their energies as well as their funding on the crystallization and structure determination of their target proteins.

  7. Biotechnology Protein Expression and Purification Facility

    NASA Technical Reports Server (NTRS)

    2003-01-01

    The purpose of the Project Scientist Core Facility is to provide purified proteins, both recombinant and natural, to the Biotechnology Science Team Project Scientists and the NRA-Structural Biology Test Investigators. Having a core facility for this purpose obviates the need for each scientist to develop the necessary expertise and equipment for molecular biology, protein expression, and protein purification. Because of this, they are able to focus their energies as well as their funding on the crystallization and structure determination of their target proteins.

  8. Eliciting the mitochondrial unfolded protein response by nicotinamide adenine dinucleotide repletion reverses fatty liver disease in mice

    PubMed Central

    Gariani, Karim; Menzies, Keir J.; Ryu, Dongryeol; Wegner, Casey J.; Wang, Xu; Ropelle, Eduardo R.; Moullan, Norman; Zhang, Hongbo; Perino, Alessia; Lemos, Vera; Kim, Bohkyung; Park, Young‐Ki; Piersigilli, Alessandra; Pham, Tho X.; Yang, Yue; Ku, Chai Siah; Koo, Sung I.; Fomitchova, Anna; Cantó, Carlos; Schoonjans, Kristina; Sauve, Anthony A.

    2015-01-01

    With no approved pharmacological treatment, nonalcoholic fatty liver disease (NAFLD) is now the most common cause of chronic liver disease in Western countries and its worldwide prevalence continues to increase along with the growing obesity epidemic. Here, we show that a high‐fat high‐sucrose (HFHS) diet, eliciting chronic hepatosteatosis resembling human fatty liver, lowers hepatic nicotinamide adenine dinucleotide (NAD+) levels driving reductions in hepatic mitochondrial content, function, and adenosine triphosphate (ATP) levels, in conjunction with robust increases in hepatic weight, lipid content, and peroxidation in C57BL/6J mice. To assess the effect of NAD+ repletion on the development of steatosis in mice, nicotinamide riboside, a precursor of NAD+ biosynthesis, was added to the HFHS diet, either as a preventive strategy or as a therapeutic intervention. We demonstrate that NR prevents and reverts NAFLD by inducing a sirtuin (SIRT)1‐ and SIRT3‐dependent mitochondrial unfolded protein response, triggering an adaptive mitohormetic pathway to increase hepatic β‐oxidation and mitochondrial complex content and activity. The cell‐autonomous beneficial component of NR treatment was revealed in liver‐specific Sirt1 knockout mice (Sirt1hep−/−), whereas apolipoprotein E‐deficient mice (Apoe −/−) challenged with a high‐fat high‐cholesterol diet affirmed the use of NR in other independent models of NAFLD. Conclusion: Our data warrant the future evaluation of NAD+ boosting strategies to manage the development or progression of NAFLD. (Hepatology 2016;63:1190–1204) PMID:26404765

  9. Chironex fleckeri (box jellyfish) venom proteins: expansion of a cnidarian toxin family that elicits variable cytolytic and cardiovascular effects.

    PubMed

    Brinkman, Diane L; Konstantakopoulos, Nicki; McInerney, Bernie V; Mulvenna, Jason; Seymour, Jamie E; Isbister, Geoffrey K; Hodgson, Wayne C

    2014-02-21

    The box jellyfish Chironex fleckeri produces extremely potent and rapid-acting venom that is harmful to humans and lethal to prey. Here, we describe the characterization of two C. fleckeri venom proteins, CfTX-A (∼40 kDa) and CfTX-B (∼42 kDa), which were isolated from C. fleckeri venom using size exclusion chromatography and cation exchange chromatography. Full-length cDNA sequences encoding CfTX-A and -B and a third putative toxin, CfTX-Bt, were subsequently retrieved from a C. fleckeri tentacle cDNA library. Bioinformatic analyses revealed that the new toxins belong to a small family of potent cnidarian pore-forming toxins that includes two other C. fleckeri toxins, CfTX-1 and CfTX-2. Phylogenetic inferences from amino acid sequences of the toxin family grouped CfTX-A, -B, and -Bt in a separate clade from CfTX-1 and -2, suggesting that the C. fleckeri toxins have diversified structurally and functionally during evolution. Comparative bioactivity assays revealed that CfTX-1/2 (25 μg kg(-1)) caused profound effects on the cardiovascular system of anesthetized rats, whereas CfTX-A/B elicited only minor effects at the same dose. Conversely, the hemolytic activity of CfTX-A/B (HU50 = 5 ng ml(-1)) was at least 30 times greater than that of CfTX-1/2. Structural homology between the cubozoan toxins and insecticidal three-domain Cry toxins (δ-endotoxins) suggests that the toxins have a similar pore-forming mechanism of action involving α-helices of the N-terminal domain, whereas structural diversification among toxin members may modulate target specificity. Expansion of the cnidarian toxin family therefore provides new insights into the evolutionary diversification of box jellyfish toxins from a structural and functional perspective.

  10. Centrally administered lipopolysaccharide elicits sympathetic excitation via NAD(P)H oxidase-dependent mitogen-activated protein kinase signaling

    PubMed Central

    Zhang, Zhi-Hua; Yu, Yang; Wei, Shun-Guang; Felder, Robert B.

    2010-01-01

    Objective The mechanisms by which inflammation activates sympathetic drive in heart failure and hypertension remain ill-defined. In this study, an intracerebroventricular (ICV) injection of lipopolysaccharide (LPS) was used to induce the expression of cytokines and other inflammatory mediators in the brain, in the absence of other excitatory mediators, and the downstream signaling pathways leading to sympathetic activation were examined using ICV injections of blocking or inhibiting agents. Methods and Results In anesthetized rats, ICV injection of LPS (5 µg) increased (p<0.05) renal sympathetic nerve activity, blood pressure and heart rate. LPS increased (p<0.05) hypothalamic mRNA for NAD(P)H oxidase subunits p47 phox and gp91phox, NAD(P)H-oxidase-dependent superoxide generation, hypothalamic mRNA for tumor necrosis factor (TNF)-α, cyclooxygenase-2 (COX-2), and cerebrospinal fluid (CSF) levels of TNF-α and prostaglandin E2 (PGE2). In the paraventricular nucleus of hypothalamus, dihydroethidium staining for superoxide expression and c-Fos activity (indicating neuronal excitation) increased. The superoxide scavenger tempol significantly (p<0.05) diminished the expression of inflammatory mediators, as well as superoxide expression and neuronal excitation in paraventricular nucleus. SB203580 (p38 mitogen-activated protein kinase inhibitor) also reduced the expression of inflammatory mediators in hypothalamus and CSF. Tempol, apocynin (NAD(P)H oxidase inhibitor), SB203580 and NS398 (COX-2 inhibitor) all reduced CSF PGE2 and the sympatho-excitatory response to LPS. LPS also increased angiotensin II type 1 receptor mRNA, a response blocked by apocynin and tempol but not by SB203580. Conclusion These findings suggest that central inflammation in pathophysiological conditions activates the sympathetic nervous system via NAD(P)H-oxidase and p38 mitogen-activated protein kinase dependent synthesis of PGE2. PMID:20027123

  11. Neuroprotection elicited by P2Y13 receptors against genotoxic stress by inducing DUSP2 expression and MAPK signaling recovery.

    PubMed

    Morente, Verónica; Pérez-Sen, Raquel; Ortega, Felipe; Huerta-Cepas, Jaime; Delicado, Esmerilda G; Miras-Portugal, M Teresa

    2014-09-01

    Nucleotides activating P2Y13 receptors display neuroprotective actions against different apoptotic stimuli in cerebellar granule neurons. In the present study, P2Y13 neuroprotection was analyzed in conditions of genotoxic stress. Exposure to cisplatin and UV radiation induced caspase-3-dependent apoptotic cell death, and p38 MAPK signaling de-regulation. Pre-treatment with P2Y13 nucleotide agonist, 2methyl-thio-ADP (2MeSADP), restored granule neuron survival and prevented p38 long-lasting activation induced by cytotoxic treatments. Microarray gene expression analysis in 2MeSADP-stimulated cells revealed over-representation of genes related to protein phosphatase activity. Among them, dual-specificity phosphatase-2, DUSP2, was validated as a transcriptional target for P2Y13 receptors by QPCR. This effect could explain 2MeSADP ability to dephosphorylate a DUSP2 substrate, p38, reestablishing the inactive form. In addition, cisplatin-induced p38 sustained activation correlated perfectly with progressive reduction in DUSP2 expression. In conclusion, P2Y13 receptors regulate DUSP2 expression and contribute to p38 signaling homeostasis and survival in granule neurons. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. Candida-elicited murine Th17 cells express high Ctla-4 compared with Th1 cells and are resistant to costimulation blockade.

    PubMed

    Krummey, Scott M; Floyd, Tamara L; Liu, Danya; Wagener, Maylene E; Song, Mingqing; Ford, Mandy L

    2014-03-01

    Effector and memory T cells may cross-react with allogeneic Ags to mediate graft rejection. Whereas the costimulation properties of Th1 cells are well studied, relatively little is known about the costimulation requirements of microbe-elicited Th17 cells. The costimulation blocker CTLA-4 Ig has been ineffective in the treatment of several Th17-driven autoimmune diseases and is associated with severe acute rejection following renal transplantation, leading us to investigate whether Th17 cells play a role in CD28/CTLA-4 blockade-resistant alloreactivity. We established an Ag-specific model in which Th1 and Th17 cells were elicited via Mycobacterium tuberculosis and Candida albicans immunization, respectively. C. albicans immunization elicited a higher frequency of Th17 cells and conferred resistance to costimulation blockade following transplantation. Compared with the M. tuberculosis group, C. albicans-elicited Th17 cells contained a higher frequency of IL-17(+)IFN-γ(+) producers and a lower frequency of IL-10(+) and IL-10(+)IL-17(+) cells. Importantly, Th17 cells differentially regulated the CD28/CTLA-4 pathway, expressing similarly high CD28 but significantly greater amounts of CTLA-4 compared with Th1 cells. Ex vivo blockade experiments demonstrated that Th17 cells are more sensitive to CTLA-4 coinhibition and therefore less susceptible to CTLA-4 Ig. These novel insights into the differential regulation of CTLA-4 coinhibition on CD4(+) T cells have implications for the immunomodulation of pathologic T cell responses during transplantation and autoimmunity.

  13. CD8+ T cell response in HLA-A*0201 transgenic mice is elicited by epitopes from SARS-CoV S protein.

    PubMed

    Zhao, Kai; Yang, Binyan; Xu, Yanquan; Wu, Changyou

    2010-09-24

    Cytotoxic CD8(+) T lymphocytes (CTLs) play an important role in antiviral immunity. Several human HLA-A*0201 restricted CTL epitopes of severe acute respiratory syndrome (SARS) spike (S) protein have been identified in HLA-A*0201 transgenic (Tg) mice, but the mechanisms and properties of immune responses are still not well understood. In this study, HLA-A*0201 Tg mice were primed intramuscularly with SARS S DNA and boosted subcutaneously with HLA-A*0201 restricted peptides. The lymphocytes from draining lymph nodes, spleens and lungs were stimulated with the cognate peptides. Three different methods (ELISA, ELISPOT and FACS) were used to evaluate the immune responses during short and long periods of time after immunization. Results showed that peptide-specific CD8(+) T cells secreted IFN-γ, TNF-α and IL-2 and expressed CD107a/b on cell surface. IFN-γ(+)CD8(+) T cells and CD107a/b(+)CD8(+) T cells distributed throughout the lymphoid and non-lymphoid tissues, but the frequency of peptide-specific CD8(+) T cells was higher in lungs than in spleens and lymph nodes. The phenotype of the CD8(+) T cells was characterized based on the expression of IFN-γ. Most of the HLA-A*0201 restricted peptide-specific CD8(+) T cells represented a memory subset with CD45RB(high) and CD62L(low). Taken together, these data demonstrate that immunization with SARS S DNA and HLA-A*0201 restricted peptides can elicit antigen-specific CD8(+) T cell immune responses which may have a significant implication in the long-term protection. We provide novel information in cellular immune responses of SARS S antigen-specific CD8(+) T cells, which are important in the development of vaccine against SARS-CoV infection. Copyright © 2010 Elsevier Ltd. All rights reserved.

  14. Cyclic lipopeptides from Bacillus subtilis ABS-S14 elicit defense-related gene expression in citrus fruit

    USDA-ARS?s Scientific Manuscript database

    Effects of cyclic lipopeptides obtained from B. subtilis ABS-S14 on eliciting defense-related gene transcription and activity of defense-related enzymes glucanase (GLU), chitinase (CHI), peroxidase (POX) and lipoxygenase (LOX) in Citrus sinensis cv. Valencia fruit were determined. The maximum level ...

  15. Chronic mild stress and imipramine treatment elicit opposite changes in behavior and in gene expression in the mouse prefrontal cortex.

    PubMed

    Erburu, M; Cajaleon, L; Guruceaga, E; Venzala, E; Muñoz-Cobo, I; Beltrán, E; Puerta, E; Tordera, R M

    2015-08-01

    Many studies suggest that the prefrontal cortex (PFC) is a target limbic region for stress response because a dysfunction here is linked to anhedonia, a decrease in reactivity to rewards, and to anxiety. It is suggested that stress-induced persistent molecular changes in this brain region could bring some light on the mechanisms perpetuating depressive episodes. In order to address this issue, here we have studied the long-term PFC gene expression pattern and behavioral effects induced by a chronic mild stress (CMS) model and antidepressant treatment in mice. CMS was applied to mice for six weeks and imipramine (10mg/kg, i.p.) or saline treatment was administered for five weeks starting from the third week of CMS. Mice were sacrificed one month after CMS and following two weeks after the discontinuation of drug treatment and the PFC was dissected and prepared for gene (mRNA) and protein expression studies. Using the same experimental design, a separate group of mice was tested for anhedonia, recognition memory, social interaction and anxiety. CMS induced a long-term altered gene expression profile in the PFC that was partially reverted by imipramine. Specifically, the circadian rhythm signaling pathway and functions such as gene expression, cell proliferation, survival and apoptosis as well as neurological and psychiatric disorders were affected. Of these, some changes of the circadian rhythm pathway (Hdac5, Per1, and Per2) were validated by RT-PCR and western-blot. Moreover, CMS induced long-lasting anhedonia that was reverted by imipramine treatment. Impaired memory, decreased social interaction and anxiety behavior were also induced by chronic stress. We have identified in the PFC molecular targets oppositely regulated by CMS and imipramine that could be relevant for chronic depression and antidepressant action. Among these, a possible candidate for further investigation could be the circadian rhythm pathway.

  16. Cavernous nerve injury elicits GAP-43 mRNA expression but not regeneration of injured pelvic ganglion neurons.

    PubMed

    Kato, Ryuichi; Kiryu-Seo, Sumiko; Sato, Yoshikazu; Hisasue, Shinichi; Tsukamoto, Taiji; Kiyama, Hiroshi

    2003-10-03

    Recovery of erectile dysfunction after cavernous nerve injury takes a long period. To elucidate this mechanism, unilateral cavernous nerve of male rat was cut, and the expression level of a nerve regeneration marker, the growth associated protein-43 (GAP-43) mRNA was evaluated by in situ hybridization and RT-PCR. While GAP-43 mRNA expression was transiently increased in the injured neurons of the major pelvic ganglion (MPG) at 7 days after nerve injury, continuous increase of GAP-43 mRNA was observed in the contralateral MPG from 7 days to 6 months after the nerve injury. Histochemical double-labeling studies for either neuronal NOS (nNOS) or tyrosine hydroxylase (TH) and the GAP-43 mRNA expression demonstrated that in injured MPG the transient up-regulation of GAP-43 mRNA was mainly seen in nNOS negative and/or TH positive neurons, suggesting non-parasympathetic post-ganglionic neurons, and also demonstrated that in contralateral MPG GAP-43 mRNA positive neurons were gradually increased in nNOS positive but TH negative neurons, suggesting parasympathetic post-ganglionic neurons. When a retrograde tracer Fluorogold (FG) was injected into the penile crus 7 days before histological experiments, FG-positive neurons were, if any, hardly seen in nNOS-positive neurons of the injured MPG for at least 6 months, whereas numerous FG-positive cells were seen in nNOS-positive neurons of the contralateral MPG. These results suggest that post-ganglionic projecting neurons of the intact side, which express increased GAP-43 mRNA, would be most likely to contribute to the recovery of the erectile function after unilateral cavernous nerve injury possibly by a plastic change such as nerve sprouting.

  17. Immunity Elicited by an Experimental Vaccine Based on Recombinant Flagellin-Porcine Circovirus Type 2 Cap Fusion Protein in Piglets

    PubMed Central

    Wang, Jing; Wei, Li; Quan, Rong; Yang, Jiayu; Yan, Xu; Li, Zixuan; She, Ruiping; Hu, Fengjiao; Liu, Jue

    2016-01-01

    In a recent study, we reported that a recombinant protein from fusion expression of flagellin to porcine circovirus type 2 (PCV2) Cap induced robust humoral and cell-mediated immunity that afforded full protection for PCV2 infection using BALB/c mice. Here, we further evaluated the immunogenicity and protection of the recombinant protein using specific pathogen free (SPF) pigs. Twenty-five 3-week-old piglets without passively acquired immunity were divided into 5 groups. All piglets except negative controls were challenged with a virulent PCV2 at 21 days after booster vaccination and necropsied at 21 days post-challenge. Vaccination of piglets with the recombinant protein without adjuvant induced strong humoral and cellular immune responses as observed by high levels of PCV2-specific IgG antibodies and neutralizing antibodies, as well as frequencies of PCV2-specific IFN-γ-secreting cells that conferred good protection against PCV2 challenge, with significant reduced PCV2 viremia, mild lesions, low PCV2 antigen-positive cells, as well as improved body weight gain, comparable to piglets vaccinated with a commercial PCV2 subunit vaccine. These results further demonstrated that the recombinant flagellin-Cap fusion protein is capable of inducing solid protective humoral and cellular immunity when administered to pigs, thereby becoming an effective PCV2 vaccine candidate for control of PCV2 infection. PMID:26848967

  18. Targeting Sentinel Proteins and Extrasynaptic Glutamate Receptors: a Therapeutic Strategy for Preventing the Effects Elicited by Perinatal Asphyxia?

    PubMed

    Herrera-Marschitz, Mario; Perez-Lobos, Ronald; Lespay-Rebolledo, Carolyne; Tapia-Bustos, Andrea; Casanova-Ortiz, Emmanuel; Morales, Paola; Valdes, Jose-Luis; Bustamante, Diego; Cassels, Bruce K

    2017-08-26

    Perinatal asphyxia (PA) is a relevant cause of death at the time of labour, and when survival is stabilised, associated with short- and long-term developmental disabilities, requiring inordinate care by health systems and families. Its prevalence is high (1 to 10/1000 live births) worldwide. At present, there are few therapeutic options, apart from hypothermia, that regrettably provides only limited protection if applied shortly after the insult.PA implies a primary and a secondary insult. The primary insult relates to the lack of oxygen, and the secondary one to the oxidative stress triggered by re-oxygenation, formation of reactive oxygen (ROS) and reactive nitrogen (RNS) species, and overactivation of glutamate receptors and mitochondrial deficiencies. PA induces overactivation of a number of sentinel proteins, including hypoxia-induced factor-1α (HIF-1α) and the genome-protecting poly(ADP-ribose) polymerase-1 (PARP-1). Upon activation, PARP-1 consumes high amounts of ATP at a time when this metabolite is scarce, worsening in turn the energy crisis elicited by asphyxia. The energy crisis also impairs ATP-dependent transport, including glutamate re-uptake by astroglia. Nicotinamide, a PARP-1 inhibitor, protects against the metabolic cascade elicited by the primary stage, avoiding NAD(+) exhaustion and the energetic crisis. Upon re-oxygenation, however, oxidative stress leads to nuclear translocation of the NF-κB subunit p65, overexpression of the pro-inflammatory cytokines IL-1β and TNF-α, and glutamate-excitotoxicity, due to impairment of glial-glutamate transport, extracellular glutamate overflow, and overactivation of NMDA receptors, mainly of the extrasynaptic type. This leads to calcium influx, mitochondrial impairment, and inactivation of antioxidant enzymes, increasing further the activity of pro-oxidant enzymes, thereby making the surviving neonate vulnerable to recurrent metabolic insults whenever oxidative stress is involved. Here, we discuss

  19. Immunization of Mice by BCG Formulated HCV Core Protein Elicited Higher Th1-Oriented Responses Compared to Pluronic-F127 Copolymer

    PubMed Central

    Yazdanian, Maryam; Memarnejadian, Arash; Mahdavi, Mehdi; Sadat, Seyed Mehdi; Motevali, Fatemeh; Vahabpour, Rouhollah; Khanahmad, Hossein; Siadat, Seyed Davar; Aghasadeghi, Mohammad Reza; Roohvand, Farzin

    2013-01-01

    Background A supreme vaccine for Hepatitis C virus (HCV) infection should elicit strong Th1-oriented cellular responses. In the absence of a Th1-specific adjuvant, immunizations by protein antigens generally induce Th2-type and weak cellular responses. Objectives To evaluate the adjuvant effect of BCG in comparison with nonionic copolymer-Pluronic F127 (F127) as a classic adjuvant in the formulation of HCV core protein (HCVcp) as a candidate vaccine for induction of Th1 immune responses. Materials and Methods Expression of N-terminally His-Tagged HCVcp (1-122) by pIVEX2.4a-core vector harboring the corresponding gene under the control of arabinose-inducible (araBAD) promoter was achieved in BL21-AI strain of E.coli and purified through application of nitrilotriacetic acid (Ni-NTA) chromatography. Mice were immunized subcutaneously (s.c.) in base of the tail with 100 μl of immunogen (F127+HCVcp or BCG+HCVcp; 5 μgHCVcp/mouse/dose) or control formulations (PBS, BCG, F127) at weeks 0, 3, 6. Total and subtypes of IgG, as well as cellular immune responses (Proliferation, In vivo CTL and IFN-γ/IL-4 ELISpot assays against a strong and dominant H2-d restricted, CD8+-epitopic peptide, core 39-48; RRGPRLGVRA of HCVcp) were compared in each group of immunized animals. Results Expression and purification of core protein around the expected size (21 kDa) was confirmed by Western blotting. The HCVcp + BCG vaccinated mice showed significantly higher lymphocyte proliferation and IFN-γ production but lower levels of cell lysis (45% versus 62% in CTL assay) than the HCVcp+F127 immunized animals. “Besides, total anti-core IgG and IgG1 levels were significantly higher in HCVcp + F127 immunized mice as compared to HCVcp + BCG vaccinated animals, indicating relatively higher efficacy of F127 for the stimulation of humoral and Th2-oriented immune responses”. Conclusions Results showed that HCVcp + BCG induced a moderate CTL and mixed Th1/Th2 immune responses with higher levels of

  20. Genetic elicitation by inducible expression of β-cryptogein stimulates secretion of phenolics from Coleus blumei hairy roots.

    PubMed

    Vuković, Rosemary; Bauer, Nataša; Curković-Perica, Mirna

    2013-02-01

    The accumulation of phenolic compounds in plants is often part of the defense response against stress and pathogen attack, which can be triggered and activated by elicitors. Oomycetal proteinaceous elicitor, β-cryptogein, induces hypersensitive response and systemic acquired resistance against some pathogens. In order to test the effect of endogenously synthesized cryptogein protein on phenolic compounds accumulation in tissue, and secretion into the culture medium, Coleus blumei hairy roots were generated. Agrobacterium rhizogenes was employed to insert synthetic crypt gene, encoding β-cryptogein, under the control of alcohol-inducible promoter. The expression of β-cryptogein, in C. blumei hairy roots, was controlled by application of 1% and 2% ethanol, during 21 days induction period. Ethanol-induced expression of β-cryptogein caused significant decrease of soluble phenolics and rosmarinic acid (RA) in hairy root lines and increase of phenolics, RA and caffeic acid in culture medium. These data suggest that β-cryptogein might be a potential regulatory factor for phenolics secretion from the roots.

  1. Vaccine-Elicited 10-Kilodalton Culture Filtrate Protein-Specific CD8+ T Cells Are Sufficient To Mediate Protection against Mycobacterium tuberculosis Infection▿

    PubMed Central

    Wu, Ying; Woodworth, Joshua S.; Shin, Daniel S.; Morris, Sheldon; Behar, Samuel M.

    2008-01-01

    The 10-kDa culture filtrate protein (CFP-10) and 6-kDa early secretory antigen of T cells (ESAT-6) are secreted in abundance by Mycobacterium tuberculosis and are frequently recognized by T cells from infected people. The genes encoding these proteins have been deleted from the genome of the vaccine strain Mycobacterium bovis bacillus Calmette-Guérin (BCG), and it is hypothesized that these proteins are important targets of protective immunity. Indeed, vaccination with ESAT-6 elicits protective CD4+ T cells in C57BL/6 mice. We have previously shown that M. tuberculosis infection of C3H mice elicits CFP-10-specific CD8+ and CD4+ T cells. Here we demonstrate that immunization with a CFP-10 DNA vaccine stimulates a specific T-cell response only to the H-2Kk-restricted epitope CFP-1032-39. These CFP-1032-39-specific CD8+ cells undergo a rapid expansion and accumulate in the lung following challenge of immunized mice with aerosolized M. tuberculosis. Protective immunity is induced by CFP-10 DNA vaccination as measured by a CFU reduction in the lung and spleen 4 and 8 weeks after challenge with M. tuberculosis. These data demonstrate that CFP-10 is a protective antigen and that CFP-1032-39-specific CD8+ T cells elicited by vaccination are sufficient to mediate protection against tuberculosis. PMID:18332205

  2. Biphasic Gene Expression Changes Elicited by Phakopsora pachyrhizi in Soybean Correlate with Fungal Penetration and Haustoria Formation1[W][OA

    PubMed Central

    Schneider, Katherine T.; van de Mortel, Martijn; Bancroft, Timothy J.; Braun, Edward; Nettleton, Dan; Nelson, Rex T.; Frederick, Reid D.; Baum, Thomas J.; Graham, Michelle A.; Whitham, Steven A.

    2011-01-01

    Inoculation of soybean (Glycine max) plants with Phakopsora pachyrhizi, the causal organism of Asian soybean rust, elicits a biphasic response characterized by a burst of differential gene expression in the first 12 h. A quiescent period occurs from 24 to 48 h after inoculation, in which P. pachyrhizi continues to develop but does not elicit strong host responses, followed by a second phase of intense gene expression. To correlate soybean responses with P. pachyrhizi growth and development, we inoculated the soybean cultivar Ankur (accession PI462312), which carries the Rpp3 resistance gene, with avirulent and virulent isolates of P. pachyrhizi. The avirulent isolate Hawaii 94-1 elicits hypersensitive cell death that limits fungal growth on Ankur and results in an incompatible response, while the virulent isolate Taiwan 80-2 grows extensively, sporulates profusely, and produces a compatible reaction. Inoculated leaves were collected over a 288-h time course for microarray analysis of soybean gene expression and microscopic analysis of P. pachyrhizi growth and development. The first burst in gene expression correlated with appressorium formation and penetration of epidermal cells, while the second burst of gene expression changes followed the onset of haustoria formation in both compatible and incompatible interactions. The proliferation of haustoria coincided with the inhibition of P. pachyrhizi growth in the incompatible interaction or the beginning of accelerated growth in the compatible interaction. The temporal relationships between P. pachyrhizi growth and host responses provide an important context in which to view interacting gene networks that mediate the outcomes of their interactions. PMID:21791600

  3. ESPRESSO: a system for estimating protein expression and solubility in protein expression systems.

    PubMed

    Hirose, Shuichi; Noguchi, Tamotsu

    2013-05-01

    Recombinant protein technology is essential for conducting protein science and using proteins as materials in pharmaceutical or industrial applications. Although obtaining soluble proteins is still a major experimental obstacle, knowledge about protein expression/solubility under standard conditions may increase the efficiency and reduce the cost of proteomics studies. In this study, we present a computational approach to estimate the probability of protein expression and solubility for two different protein expression systems: in vivo Escherichia coli and wheat germ cell-free, from only the sequence information. It implements two kinds of methods: a sequence/predicted structural property-based method that uses both the sequence and predicted structural features, and a sequence pattern-based method that utilizes the occurrence frequencies of sequence patterns. In the benchmark test, the proposed methods obtained F-scores of around 70%, and outperformed publicly available servers. Applying the proposed methods to genomic data revealed that proteins associated with translation or transcription have a strong tendency to be expressed as soluble proteins by the in vivo E. coli expression system. The sequence pattern-based method also has the potential to indicate a candidate region for modification, to increase protein solubility. All methods are available for free at the ESPRESSO server (http://mbs.cbrc.jp/ESPRESSO). © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Recombinant heat shock protein 70 functional peptide and alpha-fetoprotein epitope peptide vaccine elicits specific anti-tumor immunity

    PubMed Central

    Wang, Xiao-Ping; Wang, Qiao-Xia; Lin, Huan-Ping; Xu, Bing; Zhao, Qian; Chen, Kun

    2016-01-01

    Alpha-fetoprotein (AFP) is a marker of hepatocellular carcinoma (HCC) and serves as a target for immunotherapy. However, current treatments targeting AFP are not reproducible and do not provide complete protection against cancer. This issue may be solved by developing novel therapeutic vaccines with enhanced immunogenicity that could effectively target AFP-expressing tumors. In this study, we report construction of a therapeutic peptide vaccine by linking heat shock protein 70 (HSP70) functional peptide to the AFP epitope to obtain HSP70-P/AFP-P. This novel peptide was administered into BALB/c mice to observe the effects. Quantification of AFP-specific CD8 + T cells that secrete IFN-γ in these mice via ELISPOT revealed the synergistic effects of HSP70-P/AFP-P with increased numbers of AFP-specific CD8 + T cells. Similarly, ELISA analysis showed increased granzyme B and perforin released by natural killer cells. Moreover, in vitro cytotoxic T-lymphocyte assays and in vivo tumor preventive experiments clearly showed the higher antitumor effects of HSP70-P/AFP-P against AFP-expressing tumors. These results show that treatment of BALB/c mice with HSP70-P/AFP-P induced stronger T-cells responses and improved protective immunity. Our data suggest that HSP70-P/AFP-P may be used as a therapeutic approach in the treatment of AFP-expressing cancers. PMID:27713135

  5. Soybean Meal and Soy Protein Concentrate in Early Diet Elicit Different Nutritional Programming Effects on Juvenile Zebrafish.

    PubMed

    Perera, Erick; Yúfera, Manuel

    2016-02-01

    There is now strong evidence that early nutrition plays an important role in shaping later physiology. We assessed here whether soy protein concentrate (SPC) or soybean meal (SBM) in early diet would modify zebrafish responses to these products in later life. We fed zebrafish larvae with SPC-, SBM-, or a control-diet for the first 3 days of feeding and then grew all larvae on the control diet up to juveniles. Finally, we assessed the expression in juveniles of genes involved in inflammation/immunity, the breakdown of extracellular matrix, luminal digestion, and intestinal nutrient absorption/trafficking. First feeding SBM had wider, stronger, and more persistent effects on gene expression with respect to SPC. Juveniles fed with SPC at first feeding were more prone to inflammation after refeeding with SPC than fish that never experienced SPC before. Conversely, zebrafish that faced SBM at first feeding were later less responsive to refeeding with SBM through inflammation and had higher expression of markers of peptide absorption and fatty acid transport. Results indicate that some features of inflammation/remodeling, presumably at the intestine, and nutrient absorption/transport in fish can be programmed by early nutrition. These findings sustain the rationale of using zebrafish for depicting molecular mechanisms involved in nutritional programming.

  6. The pore-forming protein Cry5B elicits the pathogenicity of Bacillus sp. against Caenorhabditis elegans.

    PubMed

    Kho, Melanie F; Bellier, Audrey; Balasubramani, Venkatasamy; Hu, Yan; Hsu, Wayne; Nielsen-LeRoux, Christina; McGillivray, Shauna M; Nizet, Victor; Aroian, Raffi V

    2011-01-01

    The soil bacterium Bacillus thuringiensis is a pathogen of insects and nematodes and is very closely related to, if not the same species as, Bacillus cereus and Bacillus anthracis. The defining characteristic of B. thuringiensis that sets it apart from B. cereus and B. anthracis is the production of crystal (Cry) proteins, which are pore-forming toxins or pore-forming proteins (PFPs). Although it is known that PFPs are important virulence factors since their elimination results in reduced virulence of many pathogenic bacteria, the functions by which PFPs promote virulence are incompletely understood. Here we study the effect of Cry proteins in B. thuringiensis pathogenesis of the nematode Caenorhabditis elegans. We find that whereas B. thuringiensis on its own is not able to infect C. elegans, the addition of the PFP Cry protein, Cry5B, results in a robust lethal infection that consumes the nematode host in 1-2 days, leading to a "Bob" or bag-of-bacteria phenotype. Unlike other infections of C. elegans characterized to date, the infection by B. thuringiensis shows dose-dependency based on bacterial inoculum size and based on PFP concentration. Although the infection process takes 1-2 days, the PFP-instigated infection process is irreversibly established within 15 minutes of initial exposure. Remarkably, treatment of C. elegans with Cry5B PFP is able to instigate many other Bacillus species, including B. anthracis and even "non-pathogenic" Bacillus subtilis, to become lethal and infectious agents to C. elegans. Co-culturing of Cry5B-expressing B. thuringiensis with B. anthracis can result in lethal infection of C. elegans by B. anthracis. Our data demonstrate that one potential property of PFPs is to sensitize the host to bacterial infection and further that C. elegans and probably other roundworms can be common hosts for B. cereus-group bacteria, findings with important ecological and research implications. © 2011 Kho et al.

  7. Streamlined expressed protein ligation using split inteins.

    PubMed

    Vila-Perelló, Miquel; Liu, Zhihua; Shah, Neel H; Willis, John A; Idoyaga, Juliana; Muir, Tom W

    2013-01-09

    Chemically modified proteins are invaluable tools for studying the molecular details of biological processes, and they also hold great potential as new therapeutic agents. Several methods have been developed for the site-specific modification of proteins, one of the most widely used being expressed protein ligation (EPL) in which a recombinant α-thioester is ligated to an N-terminal Cys-containing peptide. Despite the widespread use of EPL, the generation and isolation of the required recombinant protein α-thioesters remain challenging. We describe here a new method for the preparation and purification of recombinant protein α-thioesters using engineered versions of naturally split DnaE inteins. This family of autoprocessing enzymes is closely related to the inteins currently used for protein α-thioester generation, but they feature faster kinetics and are split into two inactive polypeptides that need to associate to become active. Taking advantage of the strong affinity between the two split intein fragments, we devised a streamlined procedure for the purification and generation of protein α-thioesters from cell lysates and applied this strategy for the semisynthesis of a variety of proteins including an acetylated histone and a site-specifically modified monoclonal antibody.

  8. Targeting inhibitor of apoptosis proteins by Smac mimetic elicits cell death in poor prognostic subgroups of chronic lymphocytic leukemia.

    PubMed

    Opel, Daniela; Schnaiter, Andrea; Dodier, Dagmar; Jovanovic, Marjana; Gerhardinger, Andreas; Idler, Irina; Mertens, Daniel; Bullinger, Lars; Stilgenbauer, Stephan; Fulda, Simone

    2015-12-15

    Inhibitor of apoptosis (IAP) proteins are highly expressed in chronic lymphocytic leukemia (CLL) cells and contribute to evasion of cell death and poor therapeutic response. Here, we report that Smac mimetic BV6 dose-dependently induces cell death in 28 of 51 (54%) investigated CLL samples, while B-cells from healthy donors are largely unaffected. Importantly, BV6 is significantly more effective in prognostic unfavorable cases with, e.g., non-mutated VH status and TP53 mutation than samples with unknown or favorable prognosis. The majority of cases with 17p deletion (10/12) and Fludarabine refractory cases respond to BV6, indicating that BV6 acts independently of p53. BV6 also triggers cell death under survival conditions mimicking the microenvironment, e.g., by adding CD40 ligand or conditioned medium. Gene expression profiling identifies cell death, NF-κB and redox signaling among the top pathways regulated by BV6 not only in CLL but also in core-binding factor (CBF) acute myeloid leukemia (AML). Consistently, BV6 stimulates production of reactive oxygen species (ROS), which are contributing to BV6-induced cell death, since antioxidants reduce cell death. While BV6 causes degradation of cellular inhibitor of apoptosis (cIAP)1 and cIAP2 and nuclear factor-kappaB (NF-κB) pathway activation in primary CLL samples, BV6 induces cell death independently of caspase activity, receptor-interacting protein (RIP)1 activity or tumor necrosis factor (TNF)α, as zVAD.fmk, necrostatin-1 or TNFα-blocking antibody Enbrel fail to inhibit cell death. Together, these novel insights into BV6-regulated cell death in CLL have important implications for developing new therapeutic strategies to overcome cell death resistance especially in poor prognostic CLL subgroups. © 2015 UICC.

  9. Wheat streak mosaic virus Coat Protein Deletion Mutants Elicit More Severe Symptoms than Wild-Type Virus in Multiple Cereal Hosts.

    PubMed

    Tatineni, Satyanarayana; ELowsky, Christian; Graybosch, Robert

    2017-08-25

    Previously, we reported that coat protein (CP) of Wheat streak mosaic virus (WSMV; genus Tritimovirus, family Potyviridae) tolerates deletion of amino acids 36 to 84 for efficient systemic infection of wheat. In this study, we demonstrated that WSMV mutants with deletion of CP amino acids 58 to 84, but not 36 to 57, induced severe chlorotic streaks and spots, followed by acute chlorosis in wheat, maize, barley, and rye compared to mild to moderate chlorotic streaks and mosaic symptoms by wild-type virus. Deletion of CP amino acids 58 to 84 from WSMV genome accelerated cell-to-cell movement with increased accumulation of genomic RNAs and CP compared to wild-type virus. Microscopic examination of wheat tissues infected by GFP-tagged mutants revealed that infection by mutants lacking CP amino acids 58 to 84 caused degradation of chloroplasts, resulting in acute macroscopic chlorosis. The profile of CP-specific proteins was altered in wheat infected by mutants causing acute chlorosis compared to mutants eliciting wild-type symptoms. All deletion mutants accumulated CP-specific major protein similar to wild-type virus; however, mutants that elicit acute chlorosis failed to accumulate a 31-kDa minor protein compared to wild-type virus or mutants lacking amino acids 36 to 57. Taken together, these data suggest that deletion of CP amino acids 58 to 84 from WSMV genome enhanced accumulation of CP and genomic RNA, altered CP-specific protein profiles, and caused severe symptom phenotypes in multiple cereal hosts.

  10. Expression and purification of membrane proteins.

    PubMed

    Kubicek, Jan; Block, Helena; Maertens, Barbara; Spriestersbach, Anne; Labahn, Jörg

    2014-01-01

    Approximately 30% of a genome encodes for membrane proteins. They are one of the most important classes of proteins in that they can receive, differentiate, and transmit intra- and intercellular signals. Some examples of classes of membrane proteins include cell-adhesion molecules, translocases, and receptors in signaling pathways. Defects in membrane proteins may be involved in a number of serious disorders such as neurodegenerative diseases (e.g., Alzheimer's) and diabetes. Furthermore, membrane proteins provide natural entry and anchoring points for the molecular agents of infectious diseases. Thus, membrane proteins constitute ~50% of known and novel drug targets. Progress in this area is slowed by the requirement to develop methods and procedures for expression and isolation that are tailored to characteristic properties of membrane proteins. A set of standard protocols for the isolation of the targets in quantities that allow for the characterization of their individual properties for further optimization is required. The standard protocols given below represent a workable starting point. If optimization of yields is desired, a variation of conditions as outlined in the theory section is recommended.

  11. A novel chimeric Hepatitis B virus S/preS1 antigen produced in mammalian and plant cells elicits stronger humoral and cellular immune response than the standard vaccine-constituent, S protein.

    PubMed

    Dobrica, Mihaela-Olivia; Lazar, Catalin; Paruch, Lisa; Skomedal, Hanne; Steen, Hege; Haugslien, Sissel; Tucureanu, Catalin; Caras, Iuliana; Onu, Adrian; Ciulean, Sonya; Branzan, Alexandru; Clarke, Jihong Liu; Stavaru, Crina; Branza-Nichita, Norica

    2017-08-01

    Chronic Hepatitis B Virus (HBV) infection leads to severe liver pathogenesis associated with significant morbidity and mortality. As no curable medication is yet available, vaccination remains the most cost-effective approach to limit HBV spreading and control the infection. Although safe and efficient, the standard vaccine based on production of the small (S) envelope protein in yeast fails to elicit an effective immune response in about 10% of vaccinated individuals, which are at risk of infection. One strategy to address this issue is the development of more immunogenic antigens. Here we describe a novel HBV antigen obtained by combining relevant immunogenic determinants of S and large (L) envelope proteins. Our approach was based on the insertion of residues 21-47 of the preS1 domain of the L protein (nomenclature according to genotype D), involved in virus attachment to hepatocytes, within the external antigenic loop of S. The resulting S/preS1(21-47) chimera was successfully produced in HEK293T and Nicotiana benthamiana plants, as a more economical recombinant protein production platform. Comparative biochemical, functional and electron microscopy analysis indicated assembly of the novel antigen into subviral particles in mammalian and plant cells. Importantly, these particles preserve both S- and preS1-specific epitopes and elicit significantly stronger humoral and cellular immune responses than the S protein, in both expression systems used. Our data promote this antigen as a promising vaccine candidate to overcome poor responsiveness to the conventional, S protein-based, HBV vaccine. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Engineering Genes for Predictable Protein Expression

    PubMed Central

    Gustafsson, Claes; Minshull, Jeremy; Govindarajan, Sridhar; Ness, Jon; Villalobos, Alan; Welch, Mark

    2013-01-01

    The DNA sequence used to encode a polypeptide can have dramatic effects on its expression. Lack of readily available tools has until recently inhibited meaningful experimental investigation of this phenomenon. Advances in synthetic biology and the application of modern engineering approaches now provide the tools for systematic analysis of the sequence variables affecting heterologous expression of recombinant proteins. We here discuss how these new tools are being applied and how they circumvent the constraints of previous approaches, highlighting some of the surprising and promising results emerging from the developing field of gene engineering. PMID:22425659

  13. Engineering genes for predictable protein expression.

    PubMed

    Gustafsson, Claes; Minshull, Jeremy; Govindarajan, Sridhar; Ness, Jon; Villalobos, Alan; Welch, Mark

    2012-05-01

    The DNA sequence used to encode a polypeptide can have dramatic effects on its expression. Lack of readily available tools has until recently inhibited meaningful experimental investigation of this phenomenon. Advances in synthetic biology and the application of modern engineering approaches now provide the tools for systematic analysis of the sequence variables affecting heterologous expression of recombinant proteins. We here discuss how these new tools are being applied and how they circumvent the constraints of previous approaches, highlighting some of the surprising and promising results emerging from the developing field of gene engineering.

  14. Human cytomegalovirus latency-associated protein LUNA is expressed during HCMV infections in vivo.

    PubMed

    Bego, Mariana G; Keyes, Lisa R; Maciejewski, Jarek; St Jeor, Stephen C

    2011-10-01

    Human cytomegalovirus (HCMV) latency is poorly understood. We previously described a novel HCMV latency-associated transcript, UL81-82ast, coding for a protein designated LUNA (latency unique natural antigen). The aim of this study was to confirm the presence of LUNA in HCMV-seropositive donors. Standard co-immunoprecipitation and ELISA assays were used to detect antibodies against the LUNA protein in the sera of HCMV-seropositive donors. Specific antibodies against LUNA were detected in all HCMV-seropositive donors but in none of the seronegative donors. These data confirm that LUNA is expressed during in vivo infections and is capable of eliciting an immune response.

  15. PARP-1 protein expression in glioblastoma multiforme

    PubMed Central

    Galia, A.; Calogero, A.E.; Condorelli, R.A.; Fraggetta, F.; La Corte, C.; Ridolfo, F.; Bosco, P.; Castiglione, R.; Salemi, M.

    2012-01-01

    One of the most common type of primary brain tumors in adults is the glioblastoma multiforme (GBM) (World Health Organization grade IV astrocytoma). It is the most common malignant and aggressive form of glioma and it is among the most lethal ones. Poly (ADP-ribose) polymerase 1 (PARP-1) gene, located to 1q42, plays an important role for the efficient maintenance of genome integrity. PARP-1 protein is required for the apoptosis-inducing factor (AIF) translocation from the mitochondria to the nucleus. PARP-1 is proteolytically cleaved at the onset of apoptosis by caspase-3. Microarray analysis of PARP-1 gene expression in more than 8000 samples revealed that PARP-1 is more highly expressed in several types of cancer compared with the equivalent normal tissues. Overall, the most differences in PARP-1 gene expression have been observed in breast, ovarian, endometrial, lung, and skin cancers, and non-Hodgkin's lymphoma. We evaluated the expression of PARP-1 protein in normal brain tissues and primary GBM by immunohistochemistry. Positive nuclear PARP-1 staining was found in all samples with GBM, but not in normal neurons from controls (n=4) and GBM patients (n=27). No cytoplasmic staining was observed in any sample. In conclusion, PARP-1 gene is expressed in GBM. This finding may be envisioned as an attempt to trigger apoptosis in this tumor, as well as in many other malignancies. The presence of the protein exclusively at the nucleus further support the function played by this gene in genome integrity maintenance and apoptosis. Finally, PARP-1 staining may be used as GBM cell marker. PMID:22472897

  16. Geranylgeranylacetone, an inducer of the 70-kDa heat shock protein (HSP70), elicits unfolded protein response and coordinates cellular fate independently of HSP70.

    PubMed

    Endo, Satoshi; Hiramatsu, Nobuhiko; Hayakawa, Kunihiro; Okamura, Maro; Kasai, Ayumi; Tagawa, Yasuhiro; Sawada, Norifumi; Yao, Jian; Kitamura, Masanori

    2007-11-01

    Geranylgeranylacetone (GGA), an antiulcer agent, has the ability to induce 70-kDa heat shock protein (HSP70) in various cell types and to protect cells from apoptogenic insults. However, little is known about effects of GGA on other HSP families of molecules. We found that, at concentrations >/=100 microM, GGA caused selective expression of 78-kDa glucose-regulated protein (GRP78), an HSP70 family member inducible by endoplasmic reticulum (ER) stress, without affecting the level of HSP70 in various cell types. Induction of ER stress by GGA was also evidenced by expression of another endogenous marker, CCAAT/enhancer-binding protein-homologous protein (CHOP); decreased activity of ER stress-responsive alkaline phosphatase; and unfolded protein response (UPR), including activation of the activating transcription factor 6 (ATF6) pathway and the inositol-requiring ER-to-nucleus signal kinase 1-X-box-binding protein 1 (IRE1-XBP1) pathway. Incubation of mesangial cells with GGA caused significant apoptosis, which was attenuated by transfection with inhibitors of caspase-12 (i.e., a dominant-negative mutant of caspase-12 and MAGE-3). Dominant-negative suppression of IRE1 or XBP1 significantly attenuated apoptosis without affecting the levels of CHOP and GRP78. Inhibition of c-Jun NH(2)-terminal kinase, the molecule downstream of IRE1, by 1,9-pyrazoloanthrone (SP600125) did not improve cell survival. Blockade of ATF6 by 4-(2-aminoethyl) benzenesulfonyl fluoride enhanced apoptosis by GGA, and it was correlated with attenuated induction of both GRP78 and CHOP. Overexpression of GRP78 or dominant-negative inhibition of CHOP significantly attenuated GGA-induced apoptosis. These results suggested that GGA triggers both proapoptotic (IRE1-XBP1, ATF6-CHOP) and antiapoptotic (ATF6-GRP78) UPR and thereby coordinates cellular fate even without induction of HSP70.

  17. A Two-Component DNA-Prime/Protein-Boost Vaccination Strategy for Eliciting Long-Term, Protective T Cell Immunity against Trypanosoma cruzi

    PubMed Central

    Gupta, Shivali; Garg, Nisha J.

    2015-01-01

    In this study, we evaluated the long-term efficacy of a two-component subunit vaccine against Trypanosoma cruzi infection. C57BL/6 mice were immunized with TcG2/TcG4 vaccine delivered by a DNA-prime/Protein-boost (D/P) approach and challenged with T. cruzi at 120 or 180 days post-vaccination (dpv). We examined whether vaccine-primed T cell immunity was capable of rapid expansion and intercepting the infecting T. cruzi. Our data showed that D/P vaccine elicited CD4+ (30-38%) and CD8+ (22-42%) T cells maintained an effector phenotype up to 180 dpv, and were capable of responding to antigenic stimulus or challenge infection by a rapid expansion (CD8>CD4) with type 1 cytokine (IFNγ+ and TFNα+) production and cytolytic T lymphocyte (CTL) activity. Subsequently, challenge infection at 120 or 180 dpv, resulted in 2-3-fold lower parasite burden in vaccinated mice than was noted in unvaccinated/infected mice. Co-delivery of IL-12- and GMCSF-encoding expression plasmids provided no significant benefits in enhancing the anti-parasite efficacy of the vaccine-induced T cell immunity. Booster immunization (bi) with recombinant TcG2/TcG4 proteins 3-months after primary vaccine enhanced the protective efficacy, evidenced by an enhanced expansion (1.2-2.8-fold increase) of parasite-specific, type 1 CD4+ and CD8+ T cells and a potent CTL response capable of providing significantly improved (3-4.5-fold) control of infecting T. cruzi. Further, CD8+T cells in vaccinated/bi mice were predominantly of central memory phenotype, and capable of responding to challenge infection 4-6-months post bi by a rapid expansion to a poly-functional effector phenotype, and providing a 1.5-2.3-fold reduction in tissue parasite replication. We conclude that the TcG2/TcG4 D/P vaccine provided long-term anti-T. cruzi T cell immunity, and bi would be an effective strategy to maintain or enhance the vaccine-induced protective immunity against T. cruzi infection and Chagas disease. PMID:25951312

  18. Immunization with Brucella VirB proteins reduces organ colonization in mice through a Th1-type immune response and elicits a similar immune response in dogs.

    PubMed

    Pollak, Cora N; Wanke, María Magdalena; Estein, Silvia M; Delpino, M Victoria; Monachesi, Norma E; Comercio, Elida A; Fossati, Carlos A; Baldi, Pablo C

    2015-03-01

    VirB proteins from Brucella spp. constitute the type IV secretion system, a key virulence factor mediating the intracellular survival of these bacteria. Here, we assessed whether a Th1-type immune response against VirB proteins may protect mice from Brucella infection and whether this response can be induced in the dog, a natural host for Brucella. Splenocytes from mice immunized with VirB7 or VirB9 responded to their respective antigens with significant and specific production of gamma interferon (IFN-γ), whereas interleukin-4 (IL-4) was not detected. Thirty days after an intraperitoneal challenge with live Brucella abortus, the spleen load of bacteria was almost 1 log lower in mice immunized with VirB proteins than in unvaccinated animals. As colonization reduction seemed to correlate with a Th1-type immune response against VirB proteins, we decided to assess whether such a response could be elicited in the dog. Peripheral blood mononuclear cells (PBMCs) from dogs immunized with VirB proteins (three subcutaneous doses in QuilA adjuvant) produced significantly higher levels of IFN-γ than cells from control animals upon in vitro stimulation with VirB proteins. A skin test to assess specific delayed-type hypersensitivity was positive in 4 out of 5 dogs immunized with either VirB7 or VirB9. As both proteins are predicted to locate in the outer membrane of Brucella organisms, the ability of anti-VirB antibodies to mediate complement-dependent bacteriolysis of B. canis was assessed in vitro. Sera from dogs immunized with either VirB7 or VirB9, but not from those receiving phosphate-buffered saline (PBS), produced significant bacteriolysis. These results suggest that VirB-specific responses that reduce organ colonization by Brucella in mice can be also elicited in dogs.

  19. Immunization with Brucella VirB Proteins Reduces Organ Colonization in Mice through a Th1-Type Immune Response and Elicits a Similar Immune Response in Dogs

    PubMed Central

    Pollak, Cora N.; Wanke, María Magdalena; Estein, Silvia M.; Delpino, M. Victoria; Monachesi, Norma E.; Comercio, Elida A.; Fossati, Carlos A.

    2014-01-01

    VirB proteins from Brucella spp. constitute the type IV secretion system, a key virulence factor mediating the intracellular survival of these bacteria. Here, we assessed whether a Th1-type immune response against VirB proteins may protect mice from Brucella infection and whether this response can be induced in the dog, a natural host for Brucella. Splenocytes from mice immunized with VirB7 or VirB9 responded to their respective antigens with significant and specific production of gamma interferon (IFN-γ), whereas interleukin-4 (IL-4) was not detected. Thirty days after an intraperitoneal challenge with live Brucella abortus, the spleen load of bacteria was almost 1 log lower in mice immunized with VirB proteins than in unvaccinated animals. As colonization reduction seemed to correlate with a Th1-type immune response against VirB proteins, we decided to assess whether such a response could be elicited in the dog. Peripheral blood mononuclear cells (PBMCs) from dogs immunized with VirB proteins (three subcutaneous doses in QuilA adjuvant) produced significantly higher levels of IFN-γ than cells from control animals upon in vitro stimulation with VirB proteins. A skin test to assess specific delayed-type hypersensitivity was positive in 4 out of 5 dogs immunized with either VirB7 or VirB9. As both proteins are predicted to locate in the outer membrane of Brucella organisms, the ability of anti-VirB antibodies to mediate complement-dependent bacteriolysis of B. canis was assessed in vitro. Sera from dogs immunized with either VirB7 or VirB9, but not from those receiving phosphate-buffered saline (PBS), produced significant bacteriolysis. These results suggest that VirB-specific responses that reduce organ colonization by Brucella in mice can be also elicited in dogs. PMID:25540276

  20. β2-Microglobulin elicits itch-related responses in mice through the direct activation of primary afferent neurons expressing transient receptor potential vanilloid 1.

    PubMed

    Andoh, Tsugunobu; Maki, Takahito; Li, Sikai; Uta, Daisuke

    2017-09-05

    Uremic pruritus is an unpleasant symptom in patients undergoing hemodialysis, and the underlying mechanisms remain unclear. β2-Microglobulin (β2-MG) is well-known as an MHC class I molecule and its level is increased in the plasma of patients undergoing hemodialysis. In this study, we investigated whether β2-MG was a pruritogen in mice. Intradermal injections of β2-MG into the rostral back induced scratching in a dose-dependent manner. Intradermal injection of β2-MG into the cheek also elicited scratching, but not wiping. β2-MG-induced scratching was inhibited by the μ-opioid receptor antagonist naltrexone hydrochloride. β2-MG-induced scratching was not inhibited by antagonists of itch-related receptors (e.g., H1 histamine receptor (terfenadine), TP thromboxane receptor (DCHCH), BLT1 leukotriene B4 receptor (CMHVA), and proteinase-activated receptor 2 (FSLLRY-NH2)). However, β2-MG-induced scratching was attenuated in mice desensitized by repeated application of capsaicin and also by a selective transient receptor potential vanilloid 1 (TRPV1) antagonist (BCTC). In addition, β2-MG induced phosphorylation of extracellular signal-regulated kinase (a marker of activated neurons) in primary culture of dorsal root ganglion neurons that expressed TRPV1. These results suggest that β2-MG is a pruritogen and elicits itch-related responses, at least in part, through TRPV1-expressing primary sensory neurons. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Prenylation of Rho G-proteins: a novel mechanism regulating gene expression and protein stability in human trabecular meshwork cells.

    PubMed

    Stubbs, Evan B; Von Zee, Cynthia L

    2012-08-01

    Endogenous prenylation with sesquiterpene or diterpene isoprenoids facilitates membrane localization and functional activation of small monomeric GTP-binding proteins. A direct effect of isoprenoids on regulation of gene expression and protein stability has also been proposed. In this study, we determined the role of sesquiterpene or diterpene isoprenoids on the regulation of Rho G-protein expression, activation, and stability in human trabecular meshwork (TM) cells. In both primary and transformed human TM cells, limiting endogenous isoprenoid synthesis with lovastatin, a potent HMG-CoA reductase inhibitor, elicited marked increases in RhoA and RhoB mRNA and protein content. The effect of lovastatin was dose-dependent with newly synthesized inactive protein accumulating in the cytosol. Supplementation with geranylgeranyl pyrophosphate (GGPP) prevented, while inhibition of geranylgeranyl transferase-I mimicked, the effects of lovastatin on RhoA and RhoB protein content. Similarly, lovastatin-dependent increases in RhoA and RhoB mRNA expression were mimicked by geranylgeranyl transferase-I inhibition. Interestingly, GGPP supplementation selectively promoted the degradation of newly synthesized Rho proteins which was mediated, in part, through the 20S proteasome. Functionally, GGPP supplementation prevented lovastatin-dependent decreases in actin stress fiber organization while selectively facilitating the subcellular redistribution of accumulated Rho proteins from the cytosol to the membrane and increasing RhoA activation. Post-translational prenylation with geranylgeranyl diterpenes selectively facilitates the expression, membrane translocation, functional activation, and turnover of newly synthesized Rho proteins. Geranylgeranyl prenylation represents a novel mechanism by which active Rho proteins are targeted to the 20S proteasome for degradation in human TM cells.

  2. Regulation of Mutant p53 Protein Expression.

    PubMed

    Vijayakumaran, Reshma; Tan, Kah Hin; Miranda, Panimaya Jeffreena; Haupt, Sue; Haupt, Ygal

    2015-01-01

    For several decades, p53 has been detected in cancer biopsies by virtue of its high protein expression level which is considered indicative of mutation. Surprisingly, however, mouse genetic studies revealed that mutant p53 is inherently labile, similar to its wild type (wt) counterpart. Consistently, in response to stress conditions, both wt and mutant p53 accumulate in cells. While wt p53 returns to basal level following recovery from stress, mutant p53 remains stable. In part, this can be explained in mutant p53-expressing cells by the lack of an auto-regulatory loop with Mdm2 and other negative regulators, which are pivotal for wt p53 regulation. Further, additional protective mechanisms are acquired by mutant p53, largely mediated by the co-chaperones and their paralogs, the stress-induced heat shock proteins. Consequently, mutant p53 is accumulated in cancer cells in response to chronic stress and this accumulation is critical for its oncogenic gain of functions (GOF). Building on the extensive knowledge regarding wt p53, the regulation of mutant p53 is unraveling. In this review, we describe the current understanding on the major levels at which mutant p53 is regulated. These include the regulation of p53 protein levels by microRNA and by enzymes controlling p53 proteasomal degradation.

  3. Expression of Contractile Protein Isoforms in Microgravity

    NASA Technical Reports Server (NTRS)

    Anderson, Page A. W.

    1996-01-01

    The general objective of this experiment is to determine the effect of space flight parameters, including microgravity, on ontogenesis and embryogenesis of Japanese quail. Nine U.S. and two Russian investigators are cooperating in this study. Specific objectives of the participating scientists include assessing the gross and microscopic morphological and histological development of the embryo, as well as the temporal and spacial development of specific cells, tissues, and organs. Temporally regulated production of specific proteins is also being investigated. Our objective is to determine the effects of microgravity on developmentally programmed expression of Troponin T and I isoforms known to regulate cardiac and skeletal muscle contraction.

  4. A Comparative Study of the Expression of Cytotoxic Proteins in Allergic Contact Dermatitis and Psoriasis

    PubMed Central

    Yawalkar, Nikhil; Hunger, Robert E.; Buri, Caroline; Schmid, Simone; Egli, Fabienne; Brand, Christoph U.; Mueller, Christoph; Pichler, Werner J.; Braathen, Lasse R.

    2001-01-01

    Recent reports indicate that cytotoxic T cells are critically involved in contact hypersensitivity reactions in animals. In this study we sought to investigate the in vivo expression of cytotoxic granule proteins in the elicitation phase of allergic contact dermatitis in humans. Skin biopsy specimens were obtained from patients with allergic contact dermatitis (n = 8) and psoriasis (n = 6) and from controls with normal skin (n = 6). Expression of perforin and granzyme B was investigated by in situ hybridization and immunohistochemistry. In contrast to normal skin and psoriasis, a significant enhancement of perforin and granzyme B gene expression and immunoreactivity was observed in the mononuclear cell infiltrate of allergic contact dermatitis. Immunoreactivity for perforin and granzyme B was mainly found in the cytoplasm of lymphocytic cells, which were located in the dense perivascular infiltrate as well as at sites of marked spongiosis in the epidermis. Double immunostaining revealed that both CD4+ and CD8+ T cells are capable of expressing perforin and granzyme B. In conclusion, our data suggest that T-cell-mediated mechanisms involving cytotoxic granule proteins may elicit epidermal cell injury in vivo and thereby strongly contribute to the development of allergic contact dermatitis in humans. PMID:11238028

  5. Trypanosoma cruzi expresses diverse repetitive protein antigens.

    PubMed Central

    Hoft, D F; Kim, K S; Otsu, K; Moser, D R; Yost, W J; Blumin, J H; Donelson, J E; Kirchhoff, L V

    1989-01-01

    We screened a Trypanosoma cruzi cDNA expression library with human and rabbit anti-T. cruzi sera and identified cDNA clones that encode polypeptides containing tandemly arranged repeats which are 6 to 34 amino acids in length. The peptide repeats encoded by these cDNAs varied markedly in sequence, copy number, and location relative to the polyadenylation site of the mRNAs from which they were derived. The repeats were specific for T. cruzi, but in each case the sizes of the corresponding mRNAs and the total number of repeat copies encoded varied considerably among different isolates of the parasite. Expression of the peptide repeats was not stage specific. One of the peptide repeats occurred in a protein with an Mr of greater than 200,000 and one was in a protein of Mr 75,000 to 105,000. The frequent occurrence and diversity of these peptide repeats suggested that they may play a role in the ability of the parasite to evade immune destruction in its invertebrate and mammalian hosts, but the primary roles of these macromolecules may be unrelated to the host-parasite relationship. Images PMID:2659529

  6. Characterization and Implementation of a Diverse Simian Immunodeficiency Virus SIVsm Envelope Panel in the Assessment of Neutralizing Antibody Breadth Elicited in Rhesus Macaques by Multimodal Vaccines Expressing the SIVmac239 Envelope

    PubMed Central

    Kilgore, Katie M.; Murphy, Megan K.; Burton, Samantha L.; Wetzel, Katherine S.; Smith, S. Abigail; Xiao, Peng; Reddy, Sharmila; Francella, Nicholas; Sodora, Donald L.; Silvestri, Guido; Cole, Kelly S.; Villinger, Francois; Robinson, James E.; Pulendran, Bali; Hunter, Eric; Collman, Ronald G.; Amara, Rama R.

    2015-01-01

    ABSTRACT Antibodies that can neutralize diverse viral strains are likely to be an important component of a protective human immunodeficiency virus type 1 (HIV-1) vaccine. To this end, preclinical simian immunodeficiency virus (SIV)-based nonhuman primate immunization regimens have been designed to evaluate and enhance antibody-mediated protection. However, these trials often rely on a limited selection of SIV strains with extreme neutralization phenotypes to assess vaccine-elicited antibody activity. To mirror the viral panels used to assess HIV-1 antibody breadth, we created and characterized a novel panel of 14 genetically and phenotypically diverse SIVsm envelope (Env) glycoproteins. To assess the utility of this panel, we characterized the neutralizing activity elicited by four SIVmac239 envelope-expressing DNA/modified vaccinia virus Ankara vector- and protein-based vaccination regimens that included the immunomodulatory adjuvants granulocyte-macrophage colony-stimulating factor, Toll-like receptor (TLR) ligands, and CD40 ligand. The SIVsm Env panel exhibited a spectrum of neutralization sensitivity to SIV-infected plasma pools and monoclonal antibodies, allowing categorization into three tiers. Pooled sera from 91 rhesus macaques immunized in the four trials consistently neutralized only the highly sensitive tier 1a SIVsm Envs, regardless of the immunization regimen. The inability of vaccine-mediated antibodies to neutralize the moderately resistant tier 1b and tier 2 SIVsm Envs defined here suggests that those antibodies were directed toward epitopes that are not accessible on most SIVsm Envs. To achieve a broader and more effective neutralization profile in preclinical vaccine studies that is relevant to known features of HIV-1 neutralization, more emphasis should be placed on optimizing the Env immunogen, as the neutralization profile achieved by the addition of adjuvants does not appear to supersede the neutralizing antibody profile determined by the

  7. TLR2 Elicits IL-17-Mediated RANKL Expression, IL-17, and OPG Production in Neutrophils from Arthritic Mice

    PubMed Central

    Milanova, Viktoriya; Ivanovska, Nina

    2014-01-01

    We investigated the ability of neutrophils to express receptor activator of nuclear factor kappa-B ligand (RANKL), to secrete osteoprotegerin (OPG), and to produce IL-17. Arthritis was induced by intra-articular injection of zymosan, a ligand for Toll-like receptor 2 (TLR2). Frequencies of neutrophils in bone marrow (BM), blood and synovial fluid (SF), receptor expression, and cytokine production were evaluated by flow cytometry. 1A8 antibody (1A8 Ab) was used to deplete neutrophils in zymosan-injected SCID mice. IL-17, RANKL, and OPG amounts in SF, serum, or cell cultures were determined by ELISA. The development of arthritis was associated with increased secretion of IL-17, RANKL, and OPG in serum and SF, elevated frequencies of Ly6G+CD11b+ cells in BM, blood, and SF and upregulated RANKL expression. Both IL-17 and OPG were absent in serum and SF after neutrophil depletion; therefore we assume that they were released by neutrophils. In vitro blood Ly6G+CD11b+ cells from arthritic mice produced spontaneously IL-17, IFN-γ, and OPG and expressed RANKL. This phenotype was sustained by IL-17. TLR2 engagement increased IL-17 and IFN-γ production, potentiated IL-17-mediated RANKL expression, and inhibited OPG secretion. We conclude that TLR2 regulates the destructive potential of neutrophils and its targeting might limit joint alterations in arthritis. PMID:24757287

  8. H2S exposure elicits differential expression of candidate genes in fish adapted to sulfidic and non-sulfidic environments.

    PubMed

    Tobler, Michael; Henpita, Chathurika; Bassett, Brandon; Kelley, Joanna L; Shaw, Jennifer H

    2014-09-01

    Disentangling the effects of plasticity, genetic variation, and their interactions on organismal responses to environmental stressors is a key objective in ecological physiology. We quantified the expression of five candidate genes in response to hydrogen sulfide (H2S) exposure in fish (Poecilia mexicana, Poeciliidae) from a naturally sulfide-rich environment as well as an ancestral, non-sulfidic population to test for constitutive and environmentally dependent population differences in gene expression patterns. Common garden raised individuals that had never encountered environmental H2S during their lifetime were subjected to short or long term H2S exposure treatments or respective non-sulfidic controls. The expression of genes involved in responses to H2S toxicity (cytochrome c oxidase, vascular endothelial growth factor, and cytochrome P450-2J6), H2S detoxification (sulfide:quinone oxidoreductase), and endogenous H2S production (cystathionine γ lyase) was determined in both gill and liver tissues by real time PCR. The results indicated complex changes in expression patterns that--depending on the gene--not only differed between organs and populations, but also on the type of H2S exposure. Populations differences, both constitutive and H2S exposure dependent (i.e., plastic), in gene expression were particularly evident for sulfide:quinone oxidoreductase, vascular endothelial growth factor, and to a lesser degree for cytochrome P450-2J6. Our study uncovered putatively adaptive modifications in gene regulation that parallel previously documented adaptive changes in phenotypic traits.

  9. Wheat germ systems for cell-free protein expression.

    PubMed

    Harbers, Matthias

    2014-08-25

    Cell-free protein expression plays an important role in biochemical research. However, only recent developments led to new methods to rapidly synthesize preparative amounts of protein that make cell-free protein expression an attractive alternative to cell-based methods. In particular the wheat germ system provides the highest translation efficiency among eukaryotic cell-free protein expression approaches and has a very high success rate for the expression of soluble proteins of good quality. As an open in vitro method, the wheat germ system is a preferable choice for many applications in protein research including options for protein labeling and the expression of difficult-to-express proteins like membrane proteins and multiple protein complexes. Here I describe wheat germ cell-free protein expression systems and give examples how they have been used in genome-wide expression studies, preparation of labeled proteins for structural genomics and protein mass spectroscopy, automated protein synthesis, and screening of enzymatic activities. Future directions for the use of cell-free expression methods are discussed.

  10. The Type-Specific Neutralizing Antibody Response Elicited by a Dengue Vaccine Candidate Is Focused on Two Amino Acids of the Envelope Protein

    PubMed Central

    VanBlargan, Laura A.; Mukherjee, Swati; Dowd, Kimberly A.; Durbin, Anna P.; Whitehead, Stephen S.; Pierson, Theodore C.

    2013-01-01

    Dengue viruses are mosquito-borne flaviviruses that circulate in nature as four distinct serotypes (DENV1-4). These emerging pathogens are responsible for more than 100 million human infections annually. Severe clinical manifestations of disease are predominantly associated with a secondary infection by a heterotypic DENV serotype. The increased risk of severe disease in DENV-sensitized populations significantly complicates vaccine development, as a vaccine must simultaneously confer protection against all four DENV serotypes. Eliciting a protective tetravalent neutralizing antibody response is a major goal of ongoing vaccine development efforts. However, a recent large clinical trial of a candidate live-attenuated DENV vaccine revealed low protective efficacy despite eliciting a neutralizing antibody response, highlighting the need for a better understanding of the humoral immune response against dengue infection. In this study, we sought to identify epitopes recognized by serotype-specific neutralizing antibodies elicited by monovalent DENV1 vaccination. We constructed a panel of over 50 DENV1 structural gene variants containing substitutions at surface-accessible residues of the envelope (E) protein to match the corresponding DENV2 sequence. Amino acids that contribute to recognition by serotype-specific neutralizing antibodies were identified as DENV mutants with reduced sensitivity to neutralization by DENV1 immune sera, but not cross-reactive neutralizing antibodies elicited by DENV2 vaccination. We identified two mutations (E126K and E157K) that contribute significantly to type-specific recognition by polyclonal DENV1 immune sera. Longitudinal and cross-sectional analysis of sera from 24 participants of a phase I clinical study revealed a markedly reduced capacity to neutralize a E126K/E157K DENV1 variant. Sera from 77% of subjects recognized the E126K/E157K DENV1 variant and DENV2 equivalently (<3-fold difference). These data indicate the type

  11. GB Virus Type C Envelope Protein E2 Elicits Antibodies That React with a Cellular Antigen on HIV-1 Particles and Neutralize Diverse HIV-1 Isolates

    PubMed Central

    Mohr, Emma L.; Xiang, Jinhua; McLinden, James H.; Kaufman, Thomas M.; Chang, Qing; Montefiori, David C.; Klinzman, Donna; Stapleton, Jack T.

    2012-01-01

    Broadly neutralizing Abs to HIV-1 are well described; however, identification of Ags that elicit these Abs has proven difficult. Persistent infection with GB virus type C (GBV-C) is associated with prolonged survival in HIV-1–infected individuals, and among those without HIV-1 viremia, the presence of Ab to GBV-C glycoprotein E2 is also associated with survival. GBV-C E2 protein inhibits HIV-1 entry, and an antigenic peptide within E2 interferes with gp41-induced membrane perturbations in vitro, suggesting the possibility of structural mimicry between GBV-C E2 protein and HIV-1 particles. Naturally occurring human and experimentally induced GBV-C E2 Abs were examined for their ability to neutralize infectious HIV-1 particles and HIV-1–enveloped pseudovirus particles. All GBV-C E2 Abs neutralized diverse isolates of HIV-1 with the exception of rabbit anti-peptide Abs raised against a synthetic GBV-C E2 peptide. Rabbit anti–GBV-C E2 Abs neutralized HIV-1–pseudotyped retrovirus particles but not HIV-1–pseudotyped vesicular stomatitis virus particles, and E2 Abs immune-precipitated HIV-1 gag particles containing the vesicular stomatitis virus type G envelope, HIV-1 envelope, GBV-C envelope, or no viral envelope. The Abs did not neutralize or immune-precipitate mumps or yellow fever viruses. Rabbit GBV-C E2 Abs inhibited HIV attachment to cells but did not inhibit entry following attachment. Taken together, these data indicate that the GBV-C E2 protein has a structural motif that elicits Abs that cross-react with a cellular Ag present on retrovirus particles, independent of HIV-1 envelope glycoproteins. The data provide evidence that a heterologous viral protein can induce HIV-1–neutralizing Abs. PMID:20826757

  12. Transforming growth factor-beta expression by host cells is elicited locally by the filarial nematode Onchocerca volvulus in hyporeactive patients independently from Wolbachia.

    PubMed

    Korten, Simone; Kaifi, Jussuf T; Büttner, Dietrich W; Hoerauf, Achim

    2010-07-01

    Transforming growth factor-beta (TGF-beta) is a key cytokine in immune regulation, cell differentiation, development, wound healing, and tissue remodelling. It mediates immunosuppression in filarial infections facilitating parasite persistence, while attenuating immunopathology, which is induced by migrating microfilariae. Immunosuppression rises with parasite burden, but it remains unknown whether filariae elicit local release of immunosuppressive cytokines. Therefore, using immunohistology, we investigated the expression of stable, released latent TGF-beta1 in subcutaneous nodules from highly infected, hyporeactive onchocerciasis patients, harbouring adult Onchocerca volvulus. Since many cell types produce TGF-beta, we elucidated the cellular source, distribution and dependency on the worms' sex, productivity and vitality. We found TGF-beta1 to be abundantly expressed by T cells, plasma/B cells, macrophages, mast cells, fibrocytes, and vascular endothelial cells, particularly in onchocercomas with productive or previously productive females, damaged, dead and resorbed adult worms or microfilariae. We conclude TGF-beta to be antigen induced by the filariae since expression was scarce around subcutaneous arthropods or cholesterol crystals in onchocercomas. Enhanced expression after ivermectin or endobacteria-depleting doxycycline treatment indicates induction to depend on filariae and not on Wolbachia endobacteria. TGF-beta(+) cells were reduced in HIV co-infection. This finding of local and sustained TGF-beta induction by vital and dead filariae, untreated and after treatment, adds new aspects to immunomodulation by helminths.

  13. Cutin monomer induces expression of the rice OsLTP5 lipid transfer protein gene.

    PubMed

    Kim, Tae Hyun; Park, Jong Ho; Kim, Moon Chul; Cho, Sung Ho

    2008-01-01

    Treatment with the cutin monomer 16-hydroxypalmitic acid (HPA), a major component of cutin, elicited the synthesis of hydrogen peroxide (H2O2) in rice leaves and induced the expression of the lipid transfer protein gene OsLTP5. Treatment with HPA also induced expression of OsLTP1, OsLTP2, and the pathogen-related PR-10 genes to a lesser extent. The OsLTP5 transcript was expressed prominently in stems and flowers, but was barely detectable in leaves. Expression of OsLTP5 was induced in shoots in response to ABA and salicylic acid. It is proposed that HPA is perceived by rice as a signal, inducing defense reactions.

  14. Analysis of Globodera rostochiensis effectors reveals conserved functions of SPRYSEC proteins in suppressing and eliciting plant immune responses

    USDA-ARS?s Scientific Manuscript database

    Potato cyst nematodes (PCNs), including Globodera rostochiensis (Woll.), are important pests of potato. Plant parasitic nematodes produce multiple effector proteins, secreted from their stylets, to successfully infect their hosts. These include proteins that are delivered to the apoplast, as well as...

  15. Retinal Photoreceptor Expresses Toll-Like Receptors (TLRs) and Elicits Innate Responses Following TLR Ligand and Bacterial Challenge

    PubMed Central

    Singh, Pawan Kumar; Kumar, Ashok

    2015-01-01

    Toll-like receptors (TLRs) play an important role in host defense against microbial pathogens. Our previous studies have shown that TLRs are expressed on various retinal cells (Microglia and Müller glia) and orchestrate retinal innate responses in bacterial endophthalmitis. In this study, we used a well-characterized mouse cone photoreceptor cell line (661W); and demonstrated that these cells express all known TLRs. Although the stimulation of 661W cells with TLR ligands (Pam3Cys, PolyI:C, LPS, Flagellin, Poly DT, and ODN) did not alter TLR expression, downstream TLR-signaling pathways (NF-κB, p38, and ERK) are activated. Moreover, TLR-activated 661W cells secreted significant amounts of inflammatory mediators (IL-6, IL-1β, MIP-2, and KC) in their culture supernatant, as assessed by ELISA. A similar trend was observed in 661W cells challenged with live bacteria (Staphylococcus aureus). Interestingly, the neutralization of TLR2, a major receptor for S. aureus recognition, did not significantly attenuate bacterial-induced inflammatory mediators, suggesting the existence of TLR2-independent mechanisms in photoreceptor cells. Together, these results indicate that photoreceptors constitutively express functional TLRs and possess the ability to initiate innate responses following pathogen challenge, implicating their role in retinal innate immunity. PMID:25767877

  16. Construction and immunogenicity of recombinant Mycobacterium bovis BCG expressing GP5 and M protein of porcine reproductive respiratory syndrome virus.

    PubMed

    Bastos, Reginaldo G; Dellagostin, Odir A; Barletta, Raúl G; Doster, Allan R; Nelson, Eric; Osorio, Fernando A

    2002-11-22

    Mycobacterium bovis BCG was used to express a truncated form of GP5 (lacking the first 30 NH(2)-terminal residues) and M protein of porcine reproductive and respiratory syndrome virus (PRRSV). The PRRSV proteins were expressed in BCG under control of the mycobacterial hsp60 gene promoter either in the mycobacterial cytoplasm (BCGGP5cyt and BCGMcyt) or as MT19-fusion proteins on the mycobacterial surface (BCGGP5surf and BCGMsurf). Mice inoculated with BCGGP5surf and BCGMsurf developed antibodies against the viral proteins at 30 days post-inoculation (dpi) as detected by ELISA and Western blot. By 60 dpi, the animals developed titer of neutralizing antibodies of 8. A PRRSV-specific gamma interferon response was also detected in splenocytes of recombinant BCG-inoculated mice at 60 and 90 dpi. These results indicate that BCG was able to express antigens of PRRSV and elicit an immune response against the viral proteins in mice.

  17. Comparison of TCDD-elicited genome-wide hepatic gene expression in Sprague–Dawley rats and C57BL/6 mice

    SciTech Connect

    Nault, Rance; Kim, Suntae; Zacharewski, Timothy R.

    2013-03-01

    Although the structure and function of the AhR are conserved, emerging evidence suggests that downstream effects are species-specific. In this study, rat hepatic gene expression data from the DrugMatrix database (National Toxicology Program) were compared to mouse hepatic whole-genome gene expression data following treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). For the DrugMatrix study, male Sprague–Dawley rats were gavaged daily with 20 μg/kg TCDD for 1, 3 and 5 days, while female C57BL/6 ovariectomized mice were examined 1, 3 and 7 days after a single oral gavage of 30 μg/kg TCDD. A total of 649 rat and 1386 mouse genes (|fold change| ≥ 1.5, P1(t) ≥ 0.99) were differentially expressed following treatment. HomoloGene identified 11,708 orthologs represented across the rat Affymetrix 230 2.0 GeneChip (12,310 total orthologs), and the mouse 4 × 44K v.1 Agilent oligonucleotide array (17,578 total orthologs). Comparative analysis found 563 and 922 orthologs differentially expressed in response to TCDD in the rat and mouse, respectively, with 70 responses associated with immune function and lipid metabolism in common to both. Moreover, QRTPCR analysis of Ceacam1, showed divergent expression (induced in rat; repressed in mouse) functionally consistent with TCDD-elicited hepatic steatosis in the mouse but not the rat. Functional analysis identified orthologs involved in nucleotide binding and acetyltransferase activity in rat, while mouse-specific responses were associated with steroid, phospholipid, fatty acid, and carbohydrate metabolism. These results provide further evidence that TCDD elicits species-specific regulation of distinct gene networks, and outlines considerations for future comparisons of publicly available microarray datasets. - Highlights: ► We performed a whole-genome comparison of TCDD-regulated genes in mice and rats. ► Previous species comparisons were extended using data from the DrugMatrix database. ► Less than 15% of TCDD

  18. Perforin and gamma interferon expression are required for CD4+ and CD8+ T-cell-dependent protective immunity against a human parasite, Trypanosoma cruzi, elicited by heterologous plasmid DNA prime-recombinant adenovirus 5 boost vaccination.

    PubMed

    de Alencar, Bruna C G; Persechini, Pedro M; Haolla, Filipe A; de Oliveira, Gabriel; Silverio, Jaline C; Lannes-Vieira, Joseli; Machado, Alexandre V; Gazzinelli, Ricardo T; Bruna-Romero, Oscar; Rodrigues, Mauricio M

    2009-10-01

    A heterologous prime-boost strategy using plasmid DNA, followed by replication-defective recombinant adenovirus 5, is being proposed as a powerful way to elicit CD4(+) and CD8(+) T-cell-mediated protective immunity against intracellular pathogens. We confirmed this concept and furthered existing research by providing evidence that the heterologous prime-boost regimen using the gene encoding amastigote surface protein 2 elicited CD4(+) and CD8(+) T-cell-mediated protective immunity (reduction of acute parasitemia and prolonged survival) against experimental infection with Trypanosoma cruzi. Protective immunity correlated with the presence of in vivo antigen-specific cytotoxic activity prior to challenge. Based on this, our second goal was to determine the outcome of infection after heterologous prime-boost immunization of perforin-deficient mice. These mice were highly susceptible to infection. A detailed analysis of the cell-mediated immune responses in immunized perforin-deficient mice showed an impaired gamma interferon (IFN-gamma) secretion by immune spleen cells upon restimulation in vitro with soluble recombinant antigen. In spite of a normal numeric expansion, specific CD8(+) T cells presented several functional defects detected in vivo (cytotoxicity) and in vitro (simultaneous expression of CD107a/IFN-gamma or IFN-gamma/tumor necrosis factor alpha) paralleled by a decreased expression of CD44 and KLRG-1. Our final goal was to determine the importance of IFN-gamma in the presence of highly cytotoxic T cells. Vaccinated IFN-gamma-deficient mice developed highly cytotoxic cells but failed to develop any protective immunity. Our study thus demonstrated a role for perforin and IFN-gamma in a number of T-cell-mediated effector functions and in the antiparasitic immunity generated by a heterologous plasmid DNA prime-adenovirus boost vaccination strategy.

  19. Perforin and Gamma Interferon Expression Are Required for CD4+ and CD8+ T-Cell-Dependent Protective Immunity against a Human Parasite, Trypanosoma cruzi, Elicited by Heterologous Plasmid DNA Prime-Recombinant Adenovirus 5 Boost Vaccination▿

    PubMed Central

    de Alencar, Bruna C. G.; Persechini, Pedro M.; Haolla, Filipe A.; de Oliveira, Gabriel; Silverio, Jaline C.; Lannes-Vieira, Joseli; Machado, Alexandre V.; Gazzinelli, Ricardo T.; Bruna-Romero, Oscar; Rodrigues, Mauricio M.

    2009-01-01

    A heterologous prime-boost strategy using plasmid DNA, followed by replication-defective recombinant adenovirus 5, is being proposed as a powerful way to elicit CD4+ and CD8+ T-cell-mediated protective immunity against intracellular pathogens. We confirmed this concept and furthered existing research by providing evidence that the heterologous prime-boost regimen using the gene encoding amastigote surface protein 2 elicited CD4+ and CD8+ T-cell-mediated protective immunity (reduction of acute parasitemia and prolonged survival) against experimental infection with Trypanosoma cruzi. Protective immunity correlated with the presence of in vivo antigen-specific cytotoxic activity prior to challenge. Based on this, our second goal was to determine the outcome of infection after heterologous prime-boost immunization of perforin-deficient mice. These mice were highly susceptible to infection. A detailed analysis of the cell-mediated immune responses in immunized perforin-deficient mice showed an impaired gamma interferon (IFN-γ) secretion by immune spleen cells upon restimulation in vitro with soluble recombinant antigen. In spite of a normal numeric expansion, specific CD8+ T cells presented several functional defects detected in vivo (cytotoxicity) and in vitro (simultaneous expression of CD107a/IFN-γ or IFN-γ/tumor necrosis factor alpha) paralleled by a decreased expression of CD44 and KLRG-1. Our final goal was to determine the importance of IFN-γ in the presence of highly cytotoxic T cells. Vaccinated IFN-γ-deficient mice developed highly cytotoxic cells but failed to develop any protective immunity. Our study thus demonstrated a role for perforin and IFN-γ in a number of T-cell-mediated effector functions and in the antiparasitic immunity generated by a heterologous plasmid DNA prime-adenovirus boost vaccination strategy. PMID:19651871

  20. Immunization against Clostridium perfringens cells elicits protection against Clostridium tetani in mouse model: identification of cross-reactive proteins using proteomic methodologies.

    PubMed

    Alam, Syed Imteyaz; Bansod, Sunita; Singh, Lokendra

    2008-11-11

    Clostridium tetani and Clostridium perfringens are among the medically important clostridial pathogens causing diseases in man and animals. Several homologous open reading frames (ORFs) have been identified in the genomes of the two pathogens by comparative genomic analysis. We tested a likelihood of extensive sharing of common epitopes between homologous proteins of these two medically important pathogens and the possibility of cross-protection using active immunization. Eight predominant cross-reactive spots were identified by mass spectrometry and had hits in the C. tetani E88 proteome with significant MOWSE scores. Most of the cross-reactive proteins of C. tetani shared 65-78% sequence similarity with their closest homologues in C. perfringens ATCC13124. Electron transfer flavoprotein beta-subunit (CT3) was the most abundant protein (43.3%), followed by methylaspartate ammonia-lyase (36.8%) and 2-phosphoglycerate dehydratase (35.6%). All the proteins were predicted to be cytoplasmic by PSORT protein localization algorithm. Active immunization with C. perfringens whole cells elicited cross-protective immunity against C. tetani infection in a mouse model. Most of the dominant cross-reactive proteins of C. tetani belonged to the cluster of orthologous group (COG) functional category, either of posttranslational modification, protein turnover, and chaperones (O) or energy production and conversion (C). The homologs of the identified proteins have been shown to play role in pathogenesis in other Gram-positive pathogenic bacteria. Our findings provide basis for the search of potential vaccine candidates with broader coverage, encompassing more than one pathogenic clostridial species.

  1. Immunization against Clostridium perfringens cells elicits protection against Clostridium tetani in mouse model: identification of cross-reactive proteins using proteomic methodologies

    PubMed Central

    Alam, Syed Imteyaz; Bansod, Sunita; Singh, Lokendra

    2008-01-01

    Background Clostridium tetani and Clostridium perfringens are among the medically important clostridial pathogens causing diseases in man and animals. Several homologous open reading frames (ORFs) have been identified in the genomes of the two pathogens by comparative genomic analysis. We tested a likelihood of extensive sharing of common epitopes between homologous proteins of these two medically important pathogens and the possibility of cross-protection using active immunization. Results Eight predominant cross-reactive spots were identified by mass spectrometry and had hits in the C. tetani E88 proteome with significant MOWSE scores. Most of the cross-reactive proteins of C. tetani shared 65–78% sequence similarity with their closest homologues in C. perfringens ATCC13124. Electron transfer flavoprotein beta-subunit (CT3) was the most abundant protein (43.3%), followed by methylaspartate ammonia-lyase (36.8%) and 2-phosphoglycerate dehydratase (35.6%). All the proteins were predicted to be cytoplasmic by PSORT protein localization algorithm. Active immunization with C. perfringens whole cells elicited cross-protective immunity against C. tetani infection in a mouse model. Conclusion Most of the dominant cross-reactive proteins of C. tetani belonged to the cluster of orthologous group (COG) functional category, either of posttranslational modification, protein turnover, and chaperones (O) or energy production and conversion (C). The homologs of the identified proteins have been shown to play role in pathogenesis in other Gram-positive pathogenic bacteria. Our findings provide basis for the search of potential vaccine candidates with broader coverage, encompassing more than one pathogenic clostridial species. PMID:19000325

  2. Identification of two immunogenic domains of the prion protein--PrP--which activate class II-restricted T cells and elicit antibody responses against the native molecule.

    PubMed

    Gregoire, Sylvie; Logre, Caroline; Metharom, Pat; Loing, Estelle; Chomilier, Jacques; Rosset, Martine Bruley; Aucouturier, Pierre; Carnaud, Claude

    2004-07-01

    Recent reports suggest that immunity against the prion protein (PrP) retards transmissible spongiform encephalopathies progression in infected mice. A major obstacle to the development of vaccines comes from the fact that PrP is poorly immunogenic, as it is seen as self by the host immune system. Additional questions concern the immune mechanisms involved in protection and the risk of eliciting adverse reactions in the central nervous system of treated patients. Peptide-based vaccines offer an attractive strategy to overcome these difficulties. We have undertaken the identification of the immunogenic regions of PrP, which trigger helper T cells (Th) associated with antibody production. Our results identify two main regions, one between the structured and flexible portion of PrP (98-127) and a second between alpha 1 and alpha 2 helix (143-187). Peptides (30-mer) corresponding to these regions elicit class II-restricted Th cells and antibody production against native PrP and could therefore be of potential interest for a peptide-based vaccination.

  3. Gustatory sensory cells express a receptor responsive to protein breakdown products (GPR92).

    PubMed

    Haid, Désirée; Widmayer, Patricia; Voigt, Anja; Chaudhari, Nirupa; Boehm, Ulrich; Breer, Heinz

    2013-08-01

    The ingestion of dietary protein is of vital importance for the maintenance of fundamental physiological processes. The taste modality umami, with its prototype stimulus, glutamate, is considered to signal the protein content of food. Umami was thought to be mediated by the heterodimeric amino acid receptor, T1R1 + T1R3. Based on knockout studies, additional umami receptors are likely to exist. In addition to amino acids, certain peptides can also elicit and enhance umami taste suggesting that protein breakdown products may contribute to umami taste. The recently deorphanized peptone receptor, GPR92 (also named GPR93; LPAR5), is expressed in gastric enteroendocrine cells where it responds to protein hydrolysates. Therefore, it was of immediate interest to investigate if the receptor GPR92 is expressed in gustatory sensory cells. Using immunohistochemical approaches we found that a large population of cells in murine taste buds was labeled with an GPR92 antibody. A molecular phenotyping of GPR92 cells revealed that the vast majority of GPR92-immunoreactive cells express PLCβ2 and can therefore be classified as type II cells. More detailed analyses have shown that GPR92 is expressed in the majority of T1R1-positive taste cells. These results indicate that umami cells may respond not only to amino acids but also to peptides in protein hydrolysates.

  4. The immunodominant T helper 2 (Th2) response elicited in BALB/c mice by the Leishmania LiP2a and LiP2b acidic ribosomal proteins cannot be reverted by strong Th1 inducers.

    PubMed

    Iborra, S; Abánades, D R; Parody, N; Carrión, J; Risueño, R M; Pineda, M A; Bonay, P; Alonso, C; Soto, M

    2007-11-01

    The search for disease-associated T helper 2 (Th2) Leishmania antigens and the induction of a Th1 immune response to them using defined vaccination protocols is a potential strategy to induce protection against Leishmania infection. Leishmania infantum LiP2a and LiP2b acidic ribosomal protein (P proteins) have been described as prominent antigens during human and canine visceral leishmaniasis. In this study we demonstrate that BALB/c mice infected with Leishmania major develop a Th2-like humoral response against Leishmania LiP2a and LiP2b proteins and that the same response is induced in BALB/c mice when the parasite P proteins are immunized as recombinant molecules without adjuvant. The genetic immunization of BALB/c mice with eukaryotic expression plasmids coding for these proteins was unable to redirect the Th2-like response induced by these antigens, and only the co-administration of the recombinant P proteins with CpG oligodeoxynucleotides (CpG ODN) promoted a mixed Th1/Th2 immune response. According to the preponderance of a Th2 or mixed Th1/Th2 responses elicited by the different regimens of immunization tested, no evidence of protection was observed in mice after challenge with L. major. Although alterations of the clinical outcome were not detected in mice presensitized with the P proteins, the enhanced IgG1 and interleukin (IL)-4 response against total Leishmania antigens in these mice may indicate an exacerbation of the disease.

  5. Baculovirus expression of the glycoprotein gene of Lassa virus and characterization of the recombinant protein.

    PubMed

    Hummel, K B; Martin, M L; Auperin, D D

    1992-09-01

    A recombinant baculovirus was constructed that expresses the glycoprotein gene of Lassa virus (Josiah strain) under the transcriptional control of the polyhedrin promoter. The expressed protein (B-LSGPC) comigrated with the authentic viral glycoprotein as observed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), was reactive with monoclonal antibodies (MAbs) in Western blots, and was glycosylated. Although the recombinant protein was not processed into the mature glycoproteins, G1 and G2, it demonstrated reactivity with all known epitopes as measured by indirect immunofluorescence (IFA), and it was immunogenic, eliciting antisera in rabbits that recognized whole virus in IFAs. Regarding future applications to diagnostic assays, the recombinant glycoprotein proved to be an effective substitute for Lassa virus-infected mammalian cells in IFAs and it was able to distinguish sera from several human cases of Lassa fever, against a panel of known negative sera of African origin, in an enzyme immunoassay (EIA).

  6. Expression of brain-derived neurotrophic factor, IGF-1 and cortisol elicited by regular aerobic exercise in adolescents

    PubMed Central

    Jeon, Yong Kyun; Ha, Chang Ho

    2015-01-01

    [Purpose] This study was conducted on adolescent subjects whose brains are still developing with the purpose of identifying the effect of 8 weeks duration of aerobic exercises on the expression of BDNF, IGF-1 and cortisol, to identify effect of aerobic exercise on the expression of cortisol, BDNF and IGF-1 related to nerve cell growth. [Subjects and Methods] The subjects were 20 junior-high school students with no history of physical illness. The students were divided into an exercise group and a control group. The exercise group performed 3 treadmill exercise times per week for 8 weeks. The exercise time for the consumption of 200 kcal was calculated and the exercises were performed by each individual for 8 weeks. [Results] The exercise group showed statistically significant in increases serum BDNF and IGF-1 after 8 weeks, but cortisol showed no significant change. There were statistically significant differences between the groups in serum BDNF and IGF-1 after 8 weeks, but the difference in cortisol levels was not significant. [Conclusion] We found that long-term regular aerobic exercises has a positive effect on the enhancement of serum BDNF levels at rest and IGF-1 of adolescents who are still undergoing through brain developments. PMID:25931720

  7. Expression of brain-derived neurotrophic factor, IGF-1 and cortisol elicited by regular aerobic exercise in adolescents.

    PubMed

    Jeon, Yong Kyun; Ha, Chang Ho

    2015-03-01

    [Purpose] This study was conducted on adolescent subjects whose brains are still developing with the purpose of identifying the effect of 8 weeks duration of aerobic exercises on the expression of BDNF, IGF-1 and cortisol, to identify effect of aerobic exercise on the expression of cortisol, BDNF and IGF-1 related to nerve cell growth. [Subjects and Methods] The subjects were 20 junior-high school students with no history of physical illness. The students were divided into an exercise group and a control group. The exercise group performed 3 treadmill exercise times per week for 8 weeks. The exercise time for the consumption of 200 kcal was calculated and the exercises were performed by each individual for 8 weeks. [Results] The exercise group showed statistically significant in increases serum BDNF and IGF-1 after 8 weeks, but cortisol showed no significant change. There were statistically significant differences between the groups in serum BDNF and IGF-1 after 8 weeks, but the difference in cortisol levels was not significant. [Conclusion] We found that long-term regular aerobic exercises has a positive effect on the enhancement of serum BDNF levels at rest and IGF-1 of adolescents who are still undergoing through brain developments.

  8. Green Fluorescent Protein as a Marker for Gene Expression

    NASA Astrophysics Data System (ADS)

    Chalfie, Martin; Tu, Yuan; Euskirchen, Ghia; Ward, William W.; Prasher, Douglas C.

    1994-02-01

    A complementary DNA for the Aequorea victoria green fluorescent protein (GFP) produces a fluorescent product when expressed in prokaryotic (Escherichia coli) or eukaryotic (Caenorhabditis elegans) cells. Because exogenous substrates and cofactors are not required for this fluorescence, GFP expression can be used to monitor gene expression and protein localization in living organisms.

  9. Phosphodiesterases reduce spontaneous sinoatrial beating but not the 'fight or flight' tachycardia elicited by agonists through Gs-protein-coupled receptors.

    PubMed

    Kaumann, Alberto J

    2011-07-01

    Cyclic AMP (cAMP) steers the generation of basal heart beat in the sinoatrial node. It also induces sinoatrial tachycardia and increased cardiac force, elicited through activation of Gs-protein-coupled receptors (GsPCRs). Phosphodiesterases (PDEs) hydrolyse cAMP. In the heart mainly PDE3 and PDE4 would be expected to limit those functions, and the PDE isoenzymes do indeed reduce basal sinoatrial beating rate and blunt the positive inotropic effects of agonists, mediated by GsPCRs. By contrast, recent evidence shows that GsPCR-mediated sinoatrial tachycardia is not controlled by PDE1-5. A PDE-resistant cAMP pool in sinoatrial cells, generated through activation of GsPCRs, including β(1)- and β(2)-adrenoceptors, appears to guarantee unrestrained tachycardia during fight or flight stress.

  10. Expression of the Tick-Associated Vtp Protein of Borrelia hermsii in a Murine Model of Relapsing Fever

    PubMed Central

    Marcsisin, Renee A.; Lewis, Eric R. G.; Barbour, Alan G.

    2016-01-01

    Borrelia hermsii, a spirochete and cause of relapsing fever, is notable for its immune evasion by multiphasic antigenic variation within its vertebrate host. This is based on a diverse repertoire of surface antigen genes, only one of which is expressed at a time. Another major surface protein, the Variable Tick Protein (Vtp), is expressed in the tick vector and is invariable at its genetic locus. Given the limited immune systems of ticks, the finding of considerable diversity among the Vtp proteins of different strains of B. hermsii was unexpected. We investigated one explanation for this diversity of Vtp proteins, namely expression of the protein in mammals and a consequent elicitation of a specific immune response. Mice were infected with B. hermsii of either the HS1 or CC1 strain, which have antigenically distinctive Vtp proteins but otherwise have similar repertoires of the variable surface antigens. Subsequently collected sera were examined for antibody reactivities against Vtp and other antigens using Western blot analysis, dot blot, and protein microarray. Week-6 sera of infected mice contained antibodies that were largely specific for the Vtp of the infecting strain and were not attributable to antibody cross-reactivities. The antibody responses of the mice infected with different strains were otherwise similar. Further evidence of in vivo expression of the vtp gene was from enumeration of cDNA sequence reads that mapped to a set of selected B. hermsii genes. This measure of transcription of the infecting strain’s vtp gene was ~10% of that for the abundantly-expressed, serotype-defining variable antigen gene but similar to that of genes known for in vivo expression. The findings of Vtp expression in a vertebrate host and elicitation of a specific anti-Vtp antibody response support the view that balancing selection by host adaptive immunity accounts in part for the observed diversity of Vtp proteins. PMID:26918760

  11. HIV-1 trans activator of transcription protein elicits mitochondrial hyperpolarization and respiratory deficit, with dysregulation of complex IV and nicotinamide adenine dinucleotide homeostasis in cortical neurons.

    PubMed

    Norman, John P; Perry, Seth W; Kasischke, Karl A; Volsky, David J; Gelbard, Harris A

    2007-01-15

    HIV-1 causes a common, progressive neurological disorder known as HIV-associated dementia (HAD). The prevalence of this disorder has increased despite the use of highly active antiretroviral therapy, and its underlying pathogenesis remains poorly understood. However, evidence suggests that some aspects of HAD may be reversible. To model the reversible aspects of HAD, we have used the HIV-1 neurotoxin trans activator of transcription protein (Tat) to investigate nonlethal changes in cultured neurons. Exposure of rodent cortical neurons to sublethal concentrations of Tat elicits mitochondrial hyperpolarization. In this study, we used the cationic lipophilic dye rhodamine 123 to confirm this observation, and then performed follow-up studies to examine the mechanism involved. In intact neurons, we found Tat elicited a rapid drop in internal mitochondrial pH, and addition of Tat to purified mitochondrial extracts inhibited complex IV of the electron transport chain. To correlate enzyme activity in mitochondrial extracts with results in intact cells, we measured neuronal respiration following Tat exposure. Cortical neurons demonstrated decreased respiration upon Tat treatment, consistent with inhibition of complex IV. We examined mitochondrial Ca(2+) homeostasis using a mitochondrial targeted enhanced yellow fluorescent protein-calmodulin construct. We detected a decrease in mitochondrial calcium concentration following exposure to Tat. Finally, we measured the energy intermediate NAD(P)H after Tat treatment, and found a 20% decrease in the autofluorescence. Based on these findings, we suggest that decreased NADPH and calcium concentration contribute to subsequent respiratory decline after exposure to Tat, with detrimental effects on neuronal signaling.

  12. Effects of immunosuppressive treatment on protein expression in rat kidney

    PubMed Central

    Kędzierska, Karolina; Sporniak-Tutak, Katarzyna; Sindrewicz, Krzysztof; Bober, Joanna; Domański, Leszek; Parafiniuk, Mirosław; Urasińska, Elżbieta; Ciechanowicz, Andrzej; Domański, Maciej; Smektała, Tomasz; Masiuk, Marek; Skrzypczak, Wiesław; Ożgo, Małgorzata; Kabat-Koperska, Joanna; Ciechanowski, Kazimierz

    2014-01-01

    The structural proteins of renal tubular epithelial cells may become a target for the toxic metabolites of immunosuppressants. These metabolites can modify the properties of the proteins, thereby affecting cell function, which is a possible explanation for the mechanism of immunosuppressive agents’ toxicity. In our study, we evaluated the effect of two immunosuppressive strategies on protein expression in the kidneys of Wistar rats. Fragments of the rat kidneys were homogenized after cooling in liquid nitrogen and then dissolved in lysis buffer. The protein concentration in the samples was determined using a protein assay kit, and the proteins were separated by two-dimensional electrophoresis. The obtained gels were then stained with Coomassie Brilliant Blue, and their images were analyzed to evaluate differences in protein expression. Identification of selected proteins was then performed using mass spectrometry. We found that the immunosuppressive drugs used in popular regimens induce a series of changes in protein expression in target organs. The expression of proteins involved in drug, glucose, amino acid, and lipid metabolism was pronounced. However, to a lesser extent, we also observed changes in nuclear, structural, and transport proteins’ synthesis. Very slight differences were observed between the group receiving cyclosporine, mycophenolate mofetil, and glucocorticoids (CMG) and the control group. In contrast, compared to the control group, animals receiving tacrolimus, mycophenolate mofetil, and glucocorticoids (TMG) exhibited higher expression of proteins responsible for renal drug metabolism and lower expression levels of cytoplasmic actin and the major urinary protein. In the TMG group, we observed higher expression of proteins responsible for drug metabolism and a decrease in the expression of respiratory chain enzymes (thioredoxin-2) and markers of distal renal tubular damage (heart fatty acid-binding protein) compared to expression in the CMG

  13. Over-expression of secreted proteins from mammalian cell lines

    PubMed Central

    Dalton, Annamarie C; Barton, William A

    2014-01-01

    Secreted mammalian proteins require the development of robust protein over-expression systems for crystallographic and biophysical studies of protein function. Due to complex disulfide bonds and distinct glycosylation patterns preventing folding and expression in prokaryotic expression hosts, many secreted proteins necessitate production in more complex eukaryotic expression systems. Here, we elaborate on the methods used to obtain high yields of purified secreted proteins from transiently or stably transfected mammalian cell lines. Among the issues discussed are the selection of appropriate expression vectors, choice of signal sequences for protein secretion, availability of fusion tags for enhancing protein stability and purification, choice of cell line, and the large-scale growth of cells in a variety of formats. PMID:24510886

  14. Robust expression of a bioactive mammalian protein in Chlamydomonas chloroplast

    DOEpatents

    Mayfield, Stephen P

    2015-01-13

    Methods and compositions are disclosed to engineer chloroplast comprising heterologous mammalian genes via a direct replacement of chloroplast Photosystem II (PSII) reaction center protein coding regions to achieve expression of recombinant protein above 5% of total protein. When algae is used, algal expressed protein is produced predominantly as a soluble protein where the functional activity of the peptide is intact. As the host algae is edible, production of biologics in this organism for oral delivery of proteins/peptides, especially gut active proteins, without purification is disclosed.

  15. Robust expression of a bioactive mammalian protein in chlamydomonas chloroplast

    DOEpatents

    Mayfield, Stephen P.

    2010-03-16

    Methods and compositions are disclosed to engineer chloroplast comprising heterologous mammalian genes via a direct replacement of chloroplast Photosystem II (PSII) reaction center protein coding regions to achieve expression of recombinant protein above 5% of total protein. When algae is used, algal expressed protein is produced predominantly as a soluble protein where the functional activity of the peptide is intact. As the host algae is edible, production of biologics in this organism for oral delivery or proteins/peptides, especially gut active proteins, without purification is disclosed.

  16. Strain engineering for improved expression of recombinant proteins in bacteria

    PubMed Central

    2011-01-01

    Protein expression in Escherichia coli represents the most facile approach for the preparation of non-glycosylated proteins for analytical and preparative purposes. So far, the optimization of recombinant expression has largely remained a matter of trial and error and has relied upon varying parameters, such as expression vector, media composition, growth temperature and chaperone co-expression. Recently several new approaches for the genome-scale engineering of E. coli to enhance recombinant protein expression have been developed. These methodologies now enable the generation of optimized E. coli expression strains in a manner analogous to metabolic engineering for the synthesis of low-molecular-weight compounds. In this review, we provide an overview of strain engineering approaches useful for enhancing the expression of hard-to-produce proteins, including heterologous membrane proteins. PMID:21569582

  17. Sperm-derived WW domain-binding protein, PAWP, elicits calcium oscillations and oocyte activation in humans and mice.

    PubMed

    Aarabi, Mahmoud; Balakier, Hanna; Bashar, Siamak; Moskovtsev, Sergey I; Sutovsky, Peter; Librach, Clifford L; Oko, Richard

    2014-10-01

    Mammalian zygotic development is initiated by sperm-mediated intracellular calcium oscillations, followed by activation of metaphase II-arrested oocytes. Sperm postacrosomal WW binding protein (PAWP) fulfils the criteria set for an oocyte-activating factor by inducing oocyte activation and being stored in the perinuclear theca, the sperm compartment whose content is first released into oocyte cytoplasm during fertilization. However, proof that PAWP initiates mammalian zygotic development relies on demonstration that it acts upstream of oocyte calcium oscillations. Here, we show that PAWP triggers calcium oscillations and pronuclear formation in human and mouse oocytes similar to what is observed during intracytoplasmic sperm injection (ICSI). Most important, sperm-induced calcium oscillations are blocked by coinjection of a competitive inhibitor, derived from the WWI domain-binding motif of PAWP, implying the requirement of sperm PAWP and an oocyte-derived WWI domain protein substrate of PAWP for successful fertilization. Sperm-delivered PAWP is, therefore, a unique protein with a nonredundant role during human and mouse fertilization, required to trigger zygotic development. Presented data confirm our previous findings in nonmammalian models and suggest potential applications of PAWP in the diagnosis and treatment of infertility.- © FASEB.

  18. Nalfurafine prevents 5'-guanidinonaltrindole- and compound 48/80-induced spinal c-fos expression and attenuates 5'-guanidinonaltrindole-elicited scratching behavior in mice.

    PubMed

    Inan, S; Dun, N J; Cowan, A

    2009-09-29

    The aims of the present study were to establish if nalfurafine, a kappa opioid agonist, inhibits compulsive scratching in mice elicited by the s.c. administration (behind the neck) of 5'-guanidinonaltrindole (GNTI), a kappa opioid antagonist; to assess if nalfurafine prevents c-fos expression provoked by GNTI or compound 48/80, two chemically diverse pruritogens; and to distinguish on the basis of neuroanatomy, those neurons in the brainstem activated by either GNTI-induced itch or formalin-induced pain (both compounds given s.c. to the right cheek). Pretreatment of mice with nalfurafine (0.001-0.03 mg/kg s.c.) attenuated GNTI (0.3 mg/kg)-evoked scratching dose-dependently. A standard antiscratch dose of nalfurafine (0.02 mg/kg) had no marked effect on the spontaneous locomotion of mice. Tolerance did not develop to the antiscratch activity of nalfurafine. Both GNTI and compound 48/80 provoked c-fos expression on the lateral side of the superficial layer of the dorsal horn of the cervical spinal cord and pretreating mice with nalfurafine inhibited c-fos expression induced by both pruritogens. In contrast to formalin, GNTI did not induce c-fos expression in the trigeminal nucleus suggesting that pain and itch sensations are projected differently along the sensory trigeminal pathway. Our data indicate that the kappa opioid system is involved, at least in part, in the pathogenesis of itch; and that nalfurafine attenuates excessive scratching and prevents scratch-induced neuronal activity at the spinal level. On the basis of our results, nalfurafine holds promise as a potentially useful antipruritic in human conditions involving itch.

  19. 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.

  20. Platelet proteins, including platelet-derived growth factor, specifically depress a subset of the multiple components of the response elicited by glutathione in Hydra

    PubMed Central

    1987-01-01

    Human serum more strongly depressed the feeding response of Hydra (ball formation) elicited by S-methylglutathione than plasma. On the basis of the effect of several proteins released by platelets, at least five apparent components of the response (R1-R5) were suggested. Each of the platelet proteins examined specifically depressed a subset of these components. Among the platelet proteins examined, platelet-derived growth factor (PDGF) specifically depressed the R2 response (the concentration at which the depressing effect was 50% of the maximum [ED50] was 0.17 pM), and basic fibroblast growth factor depressed the R3 and R5 responses (ED50 0.50 aM) and the R2 response (ED50 0.55 pM). With respect to the depression of the R2 response by PDGF, addition of an anti-PDGF IgG or chemical reduction of PDGF, both of which prevent PDGF from binding to its cell surface receptor on responsive cells, eliminated the depressing effect of PDGF on the hydra response. The implications of these observations are discussed. PMID:3584244

  1. Nifedipine, a calcium channel blocker, inhibits advanced glycation end product (AGE)-elicited mesangial cell damage by suppressing AGE receptor (RAGE) expression via peroxisome proliferator-activated receptor-gamma activation

    SciTech Connect

    Matsui, Takanori; Yamagishi, Sho-ichi; Takeuchi, Masayoshi; Ueda, Seiji; Fukami, Kei; Okuda, Seiya

    2009-07-24

    The interaction between advanced glycation end products (AGE) and their receptor RAGE mediates the progressive alteration in renal architecture and loss of renal function in diabetic nephropathy. Oxidative stress generation and inflammation also play a central role in diabetic nephropathy. This study investigated whether and how nifedipine, a calcium channel blocker (CCB), blocked the AGE-elicited mesangial cell damage in vitro. Nifedipine, but not amlodipine, a control CCB, down-regulated RAGE mRNA levels and subsequently reduced reactive oxygen species (ROS) generation in AGE-exposed mesangial cells. AGE increased mRNA levels of vascular cell adhesion molecule-1 (VCAM-1) and induced monocyte chemoattractant protein-1 (MCP-1) production in mesangial cells, both of which were prevented by the treatment with nifedipine, but not amlodipine. The beneficial effects of nifedipine on AGE-exposed mesangial cells were blocked by the simultaneous treatment of GW9662, an inhibitor of peroxisome proliferator-activated receptor-{gamma} (PPAR-{gamma}). Although nifedipine did not affect expression levels of PPAR-{gamma}, it increased the PPAR-{gamma} transcriptional activity in mesangial cells. Our present study provides a unique beneficial aspect of nifedipine on diabetic nephropathy; it could work as an anti-inflammatory agent against AGE by suppressing RAGE expression in cultured mesangial cells via PPAR-{gamma} activation.

  2. Oral immunization of mice using Bifidobacterium longum expressing VP1 protein from enterovirus 71.

    PubMed

    Yu, Zhijian; Huang, Zhen; Sao, Chongwen; Huang, Yuanjian; Zhang, Fan; Ma, Guihong; Chen, Zhong; Zeng, Zhongming; Qiwen, Deng; Zeng, Weiseng

    2013-05-01

    Bifidobacterium longum is an attractive candidate for delivering biologically active proteins by the mucosal route due to its non-pathogenic and colonizing properties. Enterovirus 71 (EV71) has aroused widespread attention recently due to several epidemics, and great attention should be paid to the fact that there are currently no effective antiviral drugs or vaccines against EV71 infection. In this report, we described a recombinant B. longum that could be used to develop an oral vaccine against EV71 infection. A VP1 expression vector (pBBADs-VP1) was constructed by amplifying the EV71 VP1 gene and inserting it into the E. coli-Bifidobacterium shuttle expression vector pBBAD/Xs. Then, the expression of VP1 protein in pBBADs-VP1-transformed bacteria was demonstrated by western blot. In vivo studies indicated that oral immunization of BALB/c mice with pBBADs-VP1-transformed bacteria induced potent immune responses against EV71 infection, including virus-neutralising titers, anti-EV71-VP1 antibody and the induction of Th1 immune responses in the spleen and Peyer's patches. Importantly, immunization of mother mice with this recombinant VP1-expressing B. longum conferred protection to neonatal mice. These results demonstrate that the novel oral vaccine utilizing B. longum expressing the VP1 protein might successfully elicit a specific immune response against EV71 infection.

  3. Calreticulin: Roles in Cell-Surface Protein Expression

    PubMed Central

    Jiang, Yue; Dey, Sandeepa; Matsunami, Hiroaki

    2014-01-01

    In order to perform their designated functions, proteins require precise subcellular localizations. For cell-surface proteins, such as receptors and channels, they are able to transduce signals only when properly targeted to the cell membrane. Calreticulin is a multi-functional chaperone protein involved in protein folding, maturation, and trafficking. However, evidence has been accumulating that calreticulin can also negatively regulate the surface expression of certain receptors and channels. In these instances, depletion of calreticulin enhances cell-surface expression and function. In this review, we discuss the role of calreticulin with a focus on its negative effects on the expression of cell-surface proteins. PMID:25230046

  4. Avian and Human Seasonal Influenza Hemagglutinin Proteins Elicit CD4 T Cell Responses That Are Comparable in Epitope Abundance and Diversity.

    PubMed

    DiPiazza, Anthony; Richards, Katherine; Poulton, Nicholas; Sant, Andrea J

    2017-03-01

    Avian influenza viruses remain a significant concern due to their pandemic potential. Vaccine trials have suggested that humans respond poorly to avian influenza vaccines relative to seasonal vaccines. It is important to understand, first, if there is a general deficiency in the ability of avian hemagglutinin (HA) proteins to generate immune responses and, if so, what underlies this defect. This question is of particular interest because it has been suggested that in humans, the poor immunogenicity of H7 vaccines may be due to a paucity of CD4 T cell epitopes. Because of the generally high levels of cross-reactive CD4 T cells in humans, it is not possible to compare the inherent immunogenicities of avian and seasonal HA proteins in an unbiased manner. Here, we empirically examine the epitope diversity and abundance of CD4 T cells elicited by seasonal and avian HA proteins. HLA-DR1 and HLA-DR4 transgenic mice were vaccinated with purified HA proteins, and CD4 T cells to specific epitopes were identified and quantified. These studies revealed that the diversity and abundance of CD4 T cells specific for HA do not segregate on the basis of whether the HA was derived from human seasonal or avian influenza viruses. Therefore, we conclude that failure in responses to avian vaccines in humans is likely due to a lack of cross-reactive CD4 T cell memory perhaps coupled with competition with or suppression of naive, HA-specific CD4 T cells by memory CD4 T cells specific for more highly conserved proteins.

  5. Phenylbutyrate improves nitrogen disposal via an alternative pathway without eliciting an increase in protein breakdown and catabolism in control and ornithine transcarbamylase–deficient patients123

    PubMed Central

    Marini, Juan C; Lanpher, Brendan C; Scaglia, Fernando; O'Brien, William E; Sun, Qin; Garlick, Peter J; Jahoor, Farook

    2011-01-01

    Background: Phenylbutyrate is a drug used in patients with urea cycle disorder to elicit alternative pathways for nitrogen disposal. However, phenylbutyrate administration decreases plasma branched-chain amino acid (BCAA) concentrations, and previous research suggests that phenylbutyrate administration may increase leucine oxidation, which would indicate increased protein degradation and net protein loss. Objective: We investigated the effects of phenylbutyrate administration on whole-body protein metabolism, glutamine, leucine, and urea kinetics in healthy and ornithine transcarbamylase–deficient (OTCD) subjects and the possible benefits of BCAA supplementation during phenylbutyrate therapy. Design: Seven healthy control and 7 partial-OTCD subjects received either phenylbutyrate or no treatment in a crossover design. In addition, the partial-OTCD and 3 null-OTCD subjects received phenylbutyrate and phenylbutyrate plus BCAA supplementation. A multitracer protocol was used to determine the whole-body fluxes of urea and amino acids of interest. Results: Phenylbutyrate administration reduced ureagenesis by ≈15% without affecting the fluxes of leucine, tyrosine, phenylalanine, or glutamine and the oxidation of leucine or phenylalanine. The transfer of 15N from glutamine to urea was reduced by 35%. However, a reduction in plasma concentrations of BCAAs due to phenylbutyrate treatment was observed. BCAA supplementation did not alter the respective baseline fluxes. Conclusions: Prolonged phenylbutyrate administration reduced ureagenesis and the transfer of 15N from glutamine to urea without parallel reductions in glutamine flux and concentration. There were no changes in total-body protein breakdown and amino acid catabolism, which suggests that phenylbutyrate can be used to dispose of nitrogen effectively without adverse effects on body protein economy. PMID:21490144

  6. Avian and Human Seasonal Influenza Hemagglutinin Proteins Elicit CD4 T Cell Responses That Are Comparable in Epitope Abundance and Diversity

    PubMed Central

    DiPiazza, Anthony; Richards, Katherine; Poulton, Nicholas

    2017-01-01

    ABSTRACT Avian influenza viruses remain a significant concern due to their pandemic potential. Vaccine trials have suggested that humans respond poorly to avian influenza vaccines relative to seasonal vaccines. It is important to understand, first, if there is a general deficiency in the ability of avian hemagglutinin (HA) proteins to generate immune responses and, if so, what underlies this defect. This question is of particular interest because it has been suggested that in humans, the poor immunogenicity of H7 vaccines may be due to a paucity of CD4 T cell epitopes. Because of the generally high levels of cross-reactive CD4 T cells in humans, it is not possible to compare the inherent immunogenicities of avian and seasonal HA proteins in an unbiased manner. Here, we empirically examine the epitope diversity and abundance of CD4 T cells elicited by seasonal and avian HA proteins. HLA-DR1 and HLA-DR4 transgenic mice were vaccinated with purified HA proteins, and CD4 T cells to specific epitopes were identified and quantified. These studies revealed that the diversity and abundance of CD4 T cells specific for HA do not segregate on the basis of whether the HA was derived from human seasonal or avian influenza viruses. Therefore, we conclude that failure in responses to avian vaccines in humans is likely due to a lack of cross-reactive CD4 T cell memory perhaps coupled with competition with or suppression of naive, HA-specific CD4 T cells by memory CD4 T cells specific for more highly conserved proteins. PMID:28100497

  7. Cold Shock Proteins Are Expressed in the Retina Following Exposure to Low Temperatures

    PubMed Central

    Contartese, Daniela S.; Rolón, Federico; Sarotto, Anibal; Dorfman, Veronica B.; Loidl, Cesar F.; Martínez, Alfredo

    2016-01-01

    Hypothermia has been proposed as a therapeutic intervention for some retinal conditions, including ischemic insults. Cold exposure elevates expression of cold-shock proteins (CSP), including RNA-binding motif protein 3 (RBM3) and cold inducible RNA-binding protein (CIRP), but their presence in mammalian retina is so far unknown. Here we show the effects of hypothermia on the expression of these CSPs in retina-derived cell lines and in the retina of newborn and adult rats. Two cell lines of retinal origin, R28 and mRPE, were exposed to 32°C for different time periods and CSP expression was measured by qRT-PCR and Western blotting. Neonatal and adult Sprague-Dawley rats were exposed to a cold environment (8°C) and expression of CSPs in their retinas was studied by Western blotting, multiple inmunofluorescence, and confocal microscopy. RBM3 expression was upregulated by cold in both R28 and mRPE cells in a time-dependent fashion. On the other hand, CIRP was upregulated in R28 cells but not in mRPE. In vivo, expression of CSPs was negligible in the retina of newborn and adult rats kept at room temperature (24°C). Exposure to a cold environment elicited a strong expression of both proteins, especially in retinal pigment epithelium cells, photoreceptors, bipolar, amacrine and horizontal cells, Müller cells, and ganglion cells. In conclusion, CSP expression rapidly rises in the mammalian retina following exposure to hypothermia in a cell type-specific pattern. This observation may be at the basis of the molecular mechanism by which hypothermia exerts its therapeutic effects in the retina. PMID:27556928

  8. Cold Shock Proteins Are Expressed in the Retina Following Exposure to Low Temperatures.

    PubMed

    Larrayoz, Ignacio M; Rey-Funes, Manuel; Contartese, Daniela S; Rolón, Federico; Sarotto, Anibal; Dorfman, Veronica B; Loidl, Cesar F; Martínez, Alfredo

    2016-01-01

    Hypothermia has been proposed as a therapeutic intervention for some retinal conditions, including ischemic insults. Cold exposure elevates expression of cold-shock proteins (CSP), including RNA-binding motif protein 3 (RBM3) and cold inducible RNA-binding protein (CIRP), but their presence in mammalian retina is so far unknown. Here we show the effects of hypothermia on the expression of these CSPs in retina-derived cell lines and in the retina of newborn and adult rats. Two cell lines of retinal origin, R28 and mRPE, were exposed to 32°C for different time periods and CSP expression was measured by qRT-PCR and Western blotting. Neonatal and adult Sprague-Dawley rats were exposed to a cold environment (8°C) and expression of CSPs in their retinas was studied by Western blotting, multiple inmunofluorescence, and confocal microscopy. RBM3 expression was upregulated by cold in both R28 and mRPE cells in a time-dependent fashion. On the other hand, CIRP was upregulated in R28 cells but not in mRPE. In vivo, expression of CSPs was negligible in the retina of newborn and adult rats kept at room temperature (24°C). Exposure to a cold environment elicited a strong expression of both proteins, especially in retinal pigment epithelium cells, photoreceptors, bipolar, amacrine and horizontal cells, Müller cells, and ganglion cells. In conclusion, CSP expression rapidly rises in the mammalian retina following exposure to hypothermia in a cell type-specific pattern. This observation may be at the basis of the molecular mechanism by which hypothermia exerts its therapeutic effects in the retina.

  9. Vaccination of ducks with a whole-cell vaccine expressing duck hepatitis B virus core antigen elicits antiviral immune responses that enable rapid resolution of de novo infection.

    PubMed

    Miller, Darren S; Halpern, Michael; Kotlarski, Ieva; Jilbert, Allison R

    2006-05-10

    As a first step in developing immuno-therapeutic vaccines for patients with chronic hepatitis B virus infection, we examined the ability of a whole-cell vaccine, expressing the duck hepatitis B virus (DHBV) core antigen (DHBcAg), to target infected cells leading to the resolution of de novo DHBV infections. Three separate experiments were performed. In each experiment, ducks were vaccinated at 7 and 14 days of age with primary duck embryonic fibroblasts (PDEF) that had been transfected 48 h earlier with plasmid DNA expressing DHBcAg with and without the addition of anti-DHBcAg (anti-DHBc) antibodies. Control ducks were injected with either 0.7% NaCl or non-transfected PDEF. The ducks were then challenged at 18 days of age by intravenous inoculation with DHBV (5 x 10(8) viral genome equivalents). Liver biopsies obtained on day 4 post-challenge demonstrated that vaccination did not prevent infection of the liver as similar numbers of infected hepatocytes were detected in all vaccinated and control ducks. However, analysis of liver tissue obtained 9 or more days post-challenge revealed that 9 out of 11 of the PDEF-DHBcAg vaccinated ducks and 8 out of 11 ducks vaccinated with PDEF-DHBcAg plus anti-DHBc antibodies had rapidly resolved the DHBV infection with clearance of infected cells. In contrast, 10 out of 11 of the control unvaccinated ducks developed chronic DHBV infection. In conclusion, vaccination of ducks with a whole-cell PDEF vaccine expressing DHBcAg elicited immune responses that induced a rapid resolution of DHBV infection. The results establish that chronic infection can be prevented via the vaccine-mediated induction of a core-antigen-specific immune response.

  10. HIV-1 Subtype C Mosaic Gag Expressed by BCG and MVA Elicits Persistent Effector T Cell Responses in a Prime-Boost Regimen in Mice.

    PubMed

    Jongwe, Tsungai Ivai; Chapman, Ros; Douglass, Nicola; Chetty, Shivan; Chege, Gerald; Williamson, Anna-Lise

    2016-01-01

    Over 90% of HIV/AIDS positive individuals in sub-Saharan Africa are infected with highly heterogeneous HIV-1 subtype C (HIV-1C) viruses. One of the best ways to reduce the burden of this disease is the development of an affordable and effective prophylactic vaccine. Mosaic immunogens are computationally designed to overcome the hurdle of HIV diversity by maximizing the expression of potential T cell epitopes. Mycobacterium bovis BCG ΔpanCD auxotroph and modified vaccinia Ankara (MVA) vaccines expressing HIV-1C mosaic Gag (GagM) were tested in a prime-boost regimen to demonstrate immunogenicity in a mouse study. The BCG-GagM vaccine was stable and persisted 11.5 weeks post vaccination in BALB/c mice. Priming with BCG-GagM and boosting with MVA-GagM elicited higher Gag-specific IFN-γ ELISPOT responses than the BCG-GagM only and MVA-GagM only homologous vaccination regimens. The heterologous vaccination also generated a more balanced and persistent CD4+ and CD8+ T cell Gag-specific IFN-γ ELISPOT response with a predominant effector memory phenotype. A Th1 bias was induced by the vaccines as determined by the predominant secretion of IFN-γ, TNF-α, and IL-2. This study shows that a low dose of MVA (104 pfu) can effectively boost a BCG prime expressing the same mosaic immunogen, generating strong, cellular immune responses against Gag in mice. Our data warrants further evaluation in non-human primates. A low dose vaccine would be an advantage in the resource limited countries of sub-Saharan Africa and India (where the predominating virus is HIV-1 subtype C).

  11. HIV-1 Subtype C Mosaic Gag Expressed by BCG and MVA Elicits Persistent Effector T Cell Responses in a Prime-Boost Regimen in Mice

    PubMed Central

    Jongwe, Tsungai Ivai; Chapman, Ros; Douglass, Nicola; Chetty, Shivan; Chege, Gerald; Williamson, Anna-Lise

    2016-01-01

    Over 90% of HIV/AIDS positive individuals in sub-Saharan Africa are infected with highly heterogeneous HIV-1 subtype C (HIV-1C) viruses. One of the best ways to reduce the burden of this disease is the development of an affordable and effective prophylactic vaccine. Mosaic immunogens are computationally designed to overcome the hurdle of HIV diversity by maximizing the expression of potential T cell epitopes. Mycobacterium bovis BCG ΔpanCD auxotroph and modified vaccinia Ankara (MVA) vaccines expressing HIV-1C mosaic Gag (GagM) were tested in a prime-boost regimen to demonstrate immunogenicity in a mouse study. The BCG-GagM vaccine was stable and persisted 11.5 weeks post vaccination in BALB/c mice. Priming with BCG-GagM and boosting with MVA-GagM elicited higher Gag-specific IFN-γ ELISPOT responses than the BCG-GagM only and MVA-GagM only homologous vaccination regimens. The heterologous vaccination also generated a more balanced and persistent CD4+ and CD8+ T cell Gag-specific IFN-γ ELISPOT response with a predominant effector memory phenotype. A Th1 bias was induced by the vaccines as determined by the predominant secretion of IFN-γ, TNF-α, and IL-2. This study shows that a low dose of MVA (104 pfu) can effectively boost a BCG prime expressing the same mosaic immunogen, generating strong, cellular immune responses against Gag in mice. Our data warrants further evaluation in non-human primates. A low dose vaccine would be an advantage in the resource limited countries of sub-Saharan Africa and India (where the predominating virus is HIV-1 subtype C). PMID:27427967

  12. Efficient protein production method for NMR using soluble protein tags with cold shock expression vector.

    PubMed

    Hayashi, Kokoro; Kojima, Chojiro

    2010-11-01

    The E. coli protein expression system is one of the most useful methods employed for NMR sample preparation. However, the production of some recombinant proteins in E. coli is often hampered by difficulties such as low expression level and low solubility. To address these problems, a modified cold-shock expression system containing a glutathione S-transferase (GST) tag, the pCold-GST system, was investigated. The pCold-GST system successfully expressed 9 out of 10 proteins that otherwise could not be expressed using a conventional E. coli expression system. Here, we applied the pCold-GST system to 84 proteins and 78 proteins were successfully expressed in the soluble fraction. Three other cold-shock expression systems containing a maltose binding protein tag (pCold-MBP), protein G B1 domain tag (pCold-GB1) or thioredoxin tag (pCold-Trx) were also developed to improve the yield. Additionally, we show that a C-terminal proline tag, which is invisible in ¹H-¹⁵N HSQC spectra, inhibits protein degradation and increases the final yield of unstable proteins. The purified proteins were amenable to NMR analyses. These data suggest that pCold expression systems combined with soluble protein tags can be utilized to improve the expression and purification of various proteins for NMR analysis.

  13. Determination of ¹⁵N-incorporation into plant proteins and their absolute quantitation: a new tool to study nitrogen flux dynamics and protein pool sizes elicited by plant-herbivore interactions.

    PubMed

    Ullmann-Zeunert, Lynn; Muck, Alexander; Wielsch, Natalie; Hufsky, Franziska; Stanton, Mariana A; Bartram, Stefan; Böcker, Sebastian; Baldwin, Ian T; Groten, Karin; Svatoš, Aleš

    2012-10-05

    Herbivory leads to changes in the allocation of nitrogen among different pools and tissues; however, a detailed quantitative analysis of these changes has been lacking. Here, we demonstrate that a mass spectrometric data-independent acquisition approach known as LC-MS(E), combined with a novel algorithm to quantify heavy atom enrichment in peptides, is able to quantify elicited changes in protein amounts and (15)N flux in a high throughput manner. The reliable identification/quantitation of rabbit phosphorylase b protein spiked into leaf protein extract was achieved. The linear dynamic range, reproducibility of technical and biological replicates, and differences between measured and expected (15)N-incorporation into the small (SSU) and large (LSU) subunits of ribulose-1,5-bisphosphate-carboxylase/oxygenase (RuBisCO) and RuBisCO activase 2 (RCA2) of Nicotiana attenuata plants grown in hydroponic culture at different known concentrations of (15)N-labeled nitrate were used to further evaluate the procedure. The utility of the method for whole-plant studies in ecologically realistic contexts was demonstrated by using (15)N-pulse protocols on plants growing in soil under unknown (15)N-incorporation levels. Additionally, we quantified the amounts of lipoxygenase 2 (LOX2) protein, an enzyme important in antiherbivore defense responses, demonstrating that the approach allows for in-depth quantitative proteomics and (15)N flux analyses of the metabolic dynamics elicited during plant-herbivore interactions.

  14. Colitis elicits differential changes in the expression levels of receptor tyrosine kinase TrkA and TrkB in colonic afferent neurons: A possible involvement of axonal transport

    PubMed Central

    Qiao, Li-Ya; Grider, John R

    2010-01-01

    The role of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) in colitis-induced hypersensitivity has been suggested. NGF and BDNF facilitate cellular physiology through binding to receptor tyrosine kinase TrkA and TrkB respectively. The present study by examining the mRNA and/or protein levels of TrkA and TrkB in the distal colon and in colonic primary afferent neurons in the dorsal root ganglia (DRG) during colitis demonstrated that colitis elicited location-specific changes in the mRNA and protein levels of TrkA and TrkB in colonic primary sensory pathways. In colitis both the TrkA and TrkB protein levels were increased in the L1 and S1 DRGs in a time-dependent manner; however, the level of TrkB mRNA but not TrkA mRNA was increased in these DRGs. Further experiments showed that colitis facilitated a retrograde transport of TrkA protein toward and an anterograde transport of TrkA mRNA away from the DRG, which may contribute to the increased TrkA mRNA level in the distal colon during colitis. Colitis also increased the level of NGF mRNA but not BDNF mRNA in the distal colon. Double staining showed that the expression of TrkA but not TrkB was increased in the specifically labeled colonic afferent neurons in the L1 and S1 DRGs during colitis; this increase in TrkA level was attenuated by pretreatment with resiniferatoxin. These results suggested that colitis-induced primary afferent activation involved retrograde transport of TrkA but not TrkB from the distal colon to primary afferent neurons in DRG. PMID:20638179

  15. Expression analysis of G Protein-Coupled Receptors in mouse macrophages

    PubMed Central

    Lattin, Jane E; Schroder, Kate; Su, Andrew I; Walker, John R; Zhang, Jie; Wiltshire, Tim; Saijo, Kaoru; Glass, Christopher K; Hume, David A; Kellie, Stuart; Sweet, Matthew J

    2008-01-01

    Background Monocytes and macrophages express an extensive repertoire of G Protein-Coupled Receptors (GPCRs) that regulate inflammation and immunity. In this study we performed a systematic micro-array analysis of GPCR expression in primary mouse macrophages to identify family members that are either enriched in macrophages compared to a panel of other cell types, or are regulated by an inflammatory stimulus, the bacterial product lipopolysaccharide (LPS). Results Several members of the P2RY family had striking expression patterns in macrophages; P2ry6 mRNA was essentially expressed in a macrophage-specific fashion, whilst P2ry1 and P2ry5 mRNA levels were strongly down-regulated by LPS. Expression of several other GPCRs was either restricted to macrophages (e.g. Gpr84) or to both macrophages and neural tissues (e.g. P2ry12, Gpr85). The GPCR repertoire expressed by bone marrow-derived macrophages and thioglycollate-elicited peritoneal macrophages had some commonality, but there were also several GPCRs preferentially expressed by either cell population. Conclusion The constitutive or regulated expression in macrophages of several GPCRs identified in this study has not previously been described. Future studies on such GPCRs and their agonists are likely to provide important insights into macrophage biology, as well as novel inflammatory pathways that could be future targets for drug discovery. PMID:18442421

  16. Ethanol and Cancer Induce Similar Changes on Protein Expression Pattern of Human Fibroblast Cell

    PubMed Central

    Zamanian–Azodi, Mona; Rezaei-Tavirani, Mostafa; Rahmati-Rad, Sara; Rezaei Tavirani, Majid

    2016-01-01

    Ethanol has a vast consumption around the world. Many researches confirmed some adverse effect of this component on human health. In addition, recent studies showed significant alteration in both cellular population, and protein profile of human foreskin fibroblast cell line (HFFF2) in the specific dosage of ethanol. Here, the role and interaction of some proteins (characterized by significant alteration in expression due to ethanol effect) analyzed by proteomics and evaluated by considering cancerous case. 2D-electrophoresis findings of comparison of normal fibroblast cells and treated fibroblast with 270 mM dosage of ethanol analyzed by using SameSpots software, R software, and Cytoscape for protein-protein interaction (PPI) investigation. Six proteins with significantly altered expression associated with fundamental properties in a cell identified in ethanol-treated sample. These include AnnexinA5, Heterogeneous nuclear ribonucleoprotein A1, Rho-GDP dissociation inhibitor, Cathepsin L, Cu/Zn-SOD, Rho-GDP dissociation inhibitor, and Serpin peptidase inhibitor. Surprisingly, all these proteins were down-regulated and this pattern is similar to nasopharyngeal carcinoma-associated stromal fibroblast sample. Additionally, protein-protein interaction (PPI) indicates that HNRNPA1, SERPINE1 are hub proteins. Once their expression alters, it can impose vast changes on other molecular function. Based on this approach, ethanol may target same pathways that are related to cancer onset. In addition, some epidemiologic studies proved that ethanol consumption is related to increment of cancer risk. Therefore, more investigation is required in this regard to elicit the feasible relationship. PMID:28228815

  17. The Saccharomyces cerevisiae poly(A) binding protein Pab1 as a target for eliciting stress tolerant phenotypes

    PubMed Central

    Martani, Francesca; Marano, Francesca; Bertacchi, Stefano; Porro, Danilo; Branduardi, Paola

    2015-01-01

    When exploited as cell factories, Saccharomyces cerevisiae cells are exposed to harsh environmental stresses impairing titer, yield and productivity of the fermentative processes. The development of robust strains therefore represents a pivotal challenge for the implementation of cost-effective bioprocesses. Altering master regulators of general cellular rewiring represents a possible strategy to evoke shaded potential that may accomplish the desirable features. The poly(A) binding protein Pab1, as stress granules component, was here selected as the target for obtaining widespread alterations in mRNA metabolism, resulting in stress tolerant phenotypes. Firstly, we demonstrated that the modulation of Pab1 levels improves robustness against different stressors. Secondly, the mutagenesis of PAB1 and the application of a specific screening protocol on acetic acid enriched medium allowed the isolation of the further ameliorated mutant pab1 A60-9. These findings pave the way for a novel approach to unlock industrially promising phenotypes through the modulation of a post-transcriptional regulatory element. PMID:26658950

  18. Expression, Solubilization, and Purification of Bacterial Membrane Proteins.

    PubMed

    Jeffery, Constance J

    2016-02-02

    Bacterial integral membrane proteins play many important roles, including sensing changes in the environment, transporting molecules into and out of the cell, and in the case of commensal or pathogenic bacteria, interacting with the host organism. Working with membrane proteins in the lab can be more challenging than working with soluble proteins because of difficulties in their recombinant expression and purification. This protocol describes a standard method to express, solubilize, and purify bacterial integral membrane proteins. The recombinant protein of interest with a 6His affinity tag is expressed in E. coli. After harvesting the cultures and isolating cellular membranes, mild detergents are used to solubilize the membrane proteins. Protein-detergent complexes are then purified using IMAC column chromatography. Support protocols are included to help select a detergent for protein solubilization and for use of gel filtration chromatography for further purification.

  19. Intranasal immunization with a replication-deficient adenoviral vector expressing the fusion glycoprotein of respiratory syncytial virus elicits protective immunity in BALB/c mice

    SciTech Connect

    Fu, Yuanhui; He, Jinsheng; Zheng, Xianxian; Wu, Qiang; Zhang, Mei; Wang, Xiaobo; Wang, Yan; Xie, Can; Tang, Qian; Wei, Wei; Wang, Min; Song, Jingdong; Qu, Jianguo; Zhang, Ying; Wang, Xin; Hong, Tao

    2009-04-17

    Human respiratory syncytial virus (RSV) is a serious pediatric pathogen of the lower respiratory tract worldwide. There is currently no clinically approved vaccine against RSV infection. Recently, it has been shown that a replication-deficient first generation adenoviral vector (FGAd), which encodes modified RSV attachment glycoprotein (G), elicits long-term protective immunity against RSV infection in mice. The major problem in developing such a vaccine is that G protein lacks MHC-I-restricted epitopes. However, RSV fusion glycoprotein (F) is a major cytotoxic T-lymphocyte epitope in humans and mice, therefore, an FGAd-encoding F (FGAd-F) was constructed and evaluated for its potential as an RSV vaccine in a murine model. Intranasal (i.n.) immunization with FGAd-F generated serum IgG, bronchoalveolar lavage secretory IgA, and RSV-specific CD8+ T-cell responses in BALB/c mice, with characteristic balanced or mixed Th1/Th2 CD4+ T-cell responses. Serum IgG was significantly elevated after boosting with i.n. FGAd-F. Upon challenge, i.n. immunization with FGAd-F displayed an effective protective role against RSV infection. These results demonstrate FGAd-F is able to induce effective protective immunity and is a promising vaccine regimen against RSV infection.

  20. Integration of Genome-Wide Computation DRE Search, AhR ChIP-chip and Gene Expression Analyses of TCDD-Elicited Responses in the Mouse Liver

    PubMed Central

    2011-01-01

    Background The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor (TF) that mediates responses to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Integration of TCDD-induced genome-wide AhR enrichment, differential gene expression and computational dioxin response element (DRE) analyses further elucidate the hepatic AhR regulatory network. Results Global ChIP-chip and gene expression analyses were performed on hepatic tissue from immature ovariectomized mice orally gavaged with 30 μg/kg TCDD. ChIP-chip analysis identified 14,446 and 974 AhR enriched regions (1% false discovery rate) at 2 and 24 hrs, respectively. Enrichment density was greatest in the proximal promoter, and more specifically, within ± 1.5 kb of a transcriptional start site (TSS). AhR enrichment also occurred distal to a TSS (e.g. intergenic DNA and 3' UTR), extending the potential gene expression regulatory roles of the AhR. Although TF binding site analyses identified over-represented DRE sequences within enriched regions, approximately 50% of all AhR enriched regions lacked a DRE core (5'-GCGTG-3'). Microarray analysis identified 1,896 number of TCDD-responsive genes (|fold change| ≥ 1.5, P1(t) > 0.999). Integrating this gene expression data with our ChIP-chip and DRE analyses only identified 625 differentially expressed genes that involved an AhR interaction at a DRE. Functional annotation analysis of differentially regulated genes associated with AhR enrichment identified overrepresented processes related to fatty acid and lipid metabolism and transport, and xenobiotic metabolism, which are consistent with TCDD-elicited steatosis in the mouse liver. Conclusions Details of the AhR regulatory network have been expanded to include AhR-DNA interactions within intragenic and intergenic genomic regions. Moreover, the AhR can interact with DNA independent of a DRE core suggesting there are alternative mechanisms of AhR-mediated gene regulation. PMID:21762485

  1. Transforming Lepidopteran Insect Cells for Improved Protein Processing and Expression

    USDA-ARS?s Scientific Manuscript database

    The lepidopteran insect cells used with the baculovirus expression vector system (BEVS) are capable of synthesizing and accurately processing foreign proteins. However, proteins expressed in baculovirus-infected cells often fail to be completely processed, or are not processed in a manner that meet...

  2. Changes in protein expression of pacific oyster Crassostrea gigas exposed in situ to urban sewage.

    PubMed

    Flores-Nunes, Fabrício; Gomes, Tânia; Company, Rui; Moraes, Roberta R M; Sasaki, Silvio T; Taniguchi, Satie; Bicego, Márcia C; Melo, Cláudio M R; Bainy, Afonso C D; Bebianno, Maria J

    2015-11-01

    The composition and concentration of substances in urban effluents are complex and difficult to measure. These contaminants elicit biological responses in the exposed organisms. Proteomic analysis is a powerful tool in environmental toxicology by evidencing alterations in protein expression due to exposure to contaminants and by providing a useful framework for the development of new potential biomarkers. The aim of this study was to determine changes in protein expression signatures (PES) in the digestive gland of oysters Crassostrea gigas transplanted to two farming areas (LIS and RIB) and to one area contaminated by sanitary sewage (BUC) after 14 days of exposure. This species is one of the most cultivated molluscs in the world. The identified proteins are related to the cytoskeleton (CKAP5 and ACT2), ubiquitination pathway conjugation (UBE3C), G protein-coupled receptor and signal transduction (SVEP1), and cell cycle/division (CCNB3). CKAP5 showed higher expression in oysters kept at BUC in comparison with those kept at the farming areas, while ACT2, UBE3C, SVEP1, and CCNB3 were suppressed. The results suggest that these changes might lead to DNA damage, apoptosis, and interference with the immune system in oyster C. gigas exposed to sewage and give initial information on PES of C. gigas exposed to sanitary sewage, which can subsequently be useful in the development of more sensitive tools for biomonitoring coastal areas, particularly those devoted mainly to oyster farming activities.

  3. Characterization of NoV P particle-based chimeric protein vaccines developed from two different expression systems.

    PubMed

    Fu, Lu; Jin, Hao; Yu, Yongjiao; Yu, Bin; Zhang, Haihong; Wu, Jiaxin; Yin, Yuhe; Yu, Xianghui; Wu, Hui; Kong, Wei

    2017-02-01

    The Norovirus (NoV) P domain, with three surface loops for foreign antigen insertion, has been demonstrated as an excellent platform for antigen presentation and novel vaccine development. The P domain alone can self-assemble into a P dimer, 12-mer small particle or 24-mer P particle, and vaccines based on those particles may elicit different levels of immunogenicity. Currently, P particles are generally produced in soluble expression systems in Escherichia coli, mainly in the 24-mer form. However, the low yield of the soluble protein has hindered further clinical applications of P particle-based protein vaccines. In this study, we inserted the Alzheimer's disease (AD) immunogen Aβ1-6 into the three loops of the P particle to generate an AD protein vaccine. To increase the yield of this chimeric protein, we tested the generation of proteins in a soluble expression system and an inclusion body expression system separately in E. coli. The result showed that the inclusion body expression system could greatly enhance the product yield of the chimeric protein compared with the soluble expression system. The refolded protein from the inclusion bodies was mainly in the 12-mer form, while the protein generated from the soluble supernatant was mainly in the 24-mer form. Moreover, the immunogenicity of soluble proteins was significantly stronger than that of the refolded proteins. Thus, comparisons between the two expression methods suggested that the soluble expression system generated chimeric P particles with better immunogenicity, while inclusion body expression system yielded more P particle proteins.

  4. Cell-free protein synthesis as a promising expression system for recombinant proteins.

    PubMed

    Ge, Xumeng; Xu, Jianfeng

    2012-01-01

    Cell-free protein synthesis (CFPS) has major advantages over traditional cell-based methods in the capability of high-throughput protein synthesis and special protein production. During recent decades, CFPS has become an alternative protein production platform for both fundamental and applied purposes. Using Renilla luciferase as model protein, we describe a typical process of CFPS in wheat germ extract system, including wheat germ extract preparation, expression vector construction, in vitro protein synthesis (transcription/translation), and target protein assay.

  5. Cell-free expression of G-protein-coupled receptors.

    PubMed

    Orbán, Erika; Proverbio, Davide; Haberstock, Stefan; Dötsch, Volker; Bernhard, Frank

    2015-01-01

    Cell-free expression has emerged as a new standard for the production of membrane proteins. The reduction of expression complexity in cell-free systems eliminates central bottlenecks and allows the reliable and efficient synthesis of many different types of membrane proteins. Furthermore, the open accessibility of cell-free reactions enables the co-translational solubilization of cell-free expressed membrane proteins in a large variety of supplied additives. Hydrophobic environments can therefore be adjusted according to the requirements of individual membrane protein targets. We present different approaches for the preparative scale cell-free production of G-protein-coupled receptors using the extracts of Escherichia coli cells. We exemplify expression conditions implementing detergents, nanodiscs, or liposomes. The generated protein samples could be directly used for further functional characterization.

  6. Maltose-Binding Protein (MBP), a Secretion-Enhancing Tag for Mammalian Protein Expression Systems.

    PubMed

    Reuten, Raphael; Nikodemus, Denise; Oliveira, Maria B; Patel, Trushar R; Brachvogel, Bent; Breloy, Isabelle; Stetefeld, Jörg; Koch, Manuel

    2016-01-01

    Recombinant proteins are commonly expressed in eukaryotic expression systems to ensure the formation of disulfide bridges and proper glycosylation. Although many proteins can be expressed easily, some proteins, sub-domains, and mutant protein versions can cause problems. Here, we investigated expression levels of recombinant extracellular, intracellular as well as transmembrane proteins tethered to different polypeptides in mammalian cell lines. Strikingly, fusion of proteins to the prokaryotic maltose-binding protein (MBP) generally enhanced protein production. MBP fusion proteins consistently exhibited the most robust increase in protein production in comparison to commonly used tags, e.g., the Fc, Glutathione S-transferase (GST), SlyD, and serum albumin (ser alb) tag. Moreover, proteins tethered to MBP revealed reduced numbers of dying cells upon transient transfection. In contrast to the Fc tag, MBP is a stable monomer and does not promote protein aggregation. Therefore, the MBP tag does not induce artificial dimerization of tethered proteins and provides a beneficial fusion tag for binding as well as cell adhesion studies. Using MBP we were able to secret a disease causing laminin β2 mutant protein (congenital nephrotic syndrome), which is normally retained in the endoplasmic reticulum. In summary, this study establishes MBP as a versatile expression tag for protein production in eukaryotic expression systems.

  7. WRKY Proteins: Signaling and Regulation of Expression during Abiotic Stress Responses

    PubMed Central

    Banerjee, Aditya

    2015-01-01

    WRKY proteins are emerging players in plant signaling and have been thoroughly reported to play important roles in plants under biotic stress like pathogen attack. However, recent advances in this field do reveal the enormous significance of these proteins in eliciting responses induced by abiotic stresses. WRKY proteins act as major transcription factors, either as positive or negative regulators. Specific WRKY factors which help in the expression of a cluster of stress-responsive genes are being targeted and genetically modified to induce improved abiotic stress tolerance in plants. The knowledge regarding the signaling cascade leading to the activation of the WRKY proteins, their interaction with other proteins of the signaling pathway, and the downstream genes activated by them are altogether vital for justified targeting of the WRKY genes. WRKY proteins have also been considered to generate tolerance against multiple abiotic stresses with possible roles in mediating a cross talk between abiotic and biotic stress responses. In this review, we have reckoned the diverse signaling pattern and biological functions of WRKY proteins throughout the plant kingdom along with the growing prospects in this field of research. PMID:25879071

  8. A toolkit for graded expression of green fluorescent protein fusion proteins in mammalian cells.

    PubMed

    Nalaskowski, Marcus M; Ehm, Patrick; Giehler, Susanne; Mayr, Georg W

    2012-09-01

    Green fluorescent protein (GFP) and GFP-like proteins of different colors are important tools in cell biology. In many studies, the intracellular targeting of proteins has been determined by transiently expressing GFP fusion proteins and analyzing their intracellular localization by fluorescence microscopy. In most vectors, expression of GFP is driven by the enhancer/promoter cassette of the immediate early gene of human cytomegalovirus (hCMV). This cassette generates high levels of protein expression in most mammalian cell lines. Unfortunately, these nonphysiologically high protein levels have been repeatedly reported to artificially alter the intracellular targeting of proteins fused to GFP. To cope with this problem, we generated a multitude of attenuated GFP expression vectors by modifying the hCMV enhancer/promoter cassette. These modified vectors were transiently expressed, and the expression levels of enhanced green fluorescent protein (EGFP) alone and enhanced yellow fluorescent protein (EYFP) fused to another protein were determined by fluorescence microscopy and/or Western blotting. As shown in this study, we were able to (i) clearly reduce the expression of EGFP alone and (ii) reduce expression of an EYFP fusion protein down to the level of the endogenous protein, both in a graded manner.

  9. Nucleic Acid Programmable Protein Array: A Just-In-Time Multiplexed Protein Expression and Purification Platform

    PubMed Central

    Qiu, Ji; LaBaer, Joshua

    2012-01-01

    Systematic study of proteins requires the availability of thousands of proteins in functional format. However, traditional recombinant protein expression and purification methods have many drawbacks for such study at the proteome level. We have developed an innovative in situ protein expression and capture system, namely NAPPA (nucleic acid programmable protein array), where C-terminal tagged proteins are expressed using an in vitro expression system and efficiently captured/purified by antitag antibodies coprinted at each spot. The NAPPA technology presented in this chapter enable researchers to produce and display fresh proteins just in time in a multiplexed high-throughput fashion and utilize them for various downstream biochemical researches of interest. This platform could revolutionize the field of functional proteomics with it ability to produce thousands of spatially separated proteins in high density with narrow dynamic rand of protein concentrations, reproducibly and functionally. PMID:21943897

  10. Nucleic acid programmable protein array a just-in-time multiplexed protein expression and purification platform.

    PubMed

    Qiu, Ji; LaBaer, Joshua

    2011-01-01

    Systematic study of proteins requires the availability of thousands of proteins in functional format. However, traditional recombinant protein expression and purification methods have many drawbacks for such study at the proteome level. We have developed an innovative in situ protein expression and capture system, namely NAPPA (nucleic acid programmable protein array), where C-terminal tagged proteins are expressed using an in vitro expression system and efficiently captured/purified by antitag antibodies coprinted at each spot. The NAPPA technology presented in this chapter enable researchers to produce and display fresh proteins just in time in a multiplexed high-throughput fashion and utilize them for various downstream biochemical researches of interest. This platform could revolutionize the field of functional proteomics with it ability to produce thousands of spatially separated proteins in high density with narrow dynamic rand of protein concentrations, reproducibly and functionally.

  11. Protein expression in Arabidopsis thaliana after chronic clinorotation

    NASA Technical Reports Server (NTRS)

    Piastuch, William C.; Brown, Christopher S.

    1994-01-01

    Soluble protein expression in Arabidopsis thaliana L. (Heynh.) leaf and stem tissue was examined after chronic clinorotation. Seeds of Arabidopsis were germinated and plants grown to maturity on horizontal or vertical slow-rotating clinostats (1 rpm) or in stationary vertical control units. Total soluble proteins and in vivo-labeled soluble proteins isolated from these plants were analyzed by two-dimensional sodium doedocyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and subsequent fluorography. Visual and computer analysis of the resulting protein patterns showed no significant differences in either total protein expression or in active protein synthesis between horizontal clinorotation and vertical controls in the Arabidopsis leaf and stem tissue. These results show chronic clinorotation does not cause gross changes in protein expression in Arabidopsis.

  12. Protein expression in Arabidopsis thaliana after chronic clinorotation

    NASA Technical Reports Server (NTRS)

    Piastuch, W. C.; Brown, C. S.

    1995-01-01

    Soluble protein expression in Arabidopsis thaliana L. (Heynh.) leaf and stem tissue was examined after chronic clinorotation. Seeds of Arabidopsis were germinated and plants grown to maturity on horizontal or vertical slow-rotating clinostats (1 rpm) or in stationary vertical control units. Total soluble proteins and in vivo-labeled soluble proteins isolated from these plants were analyzed by two-dimensional SDS PAGE and subsequent fluorography. Visual and computer analysis of the resulting protein patterns showed no significant differences in either total protein expression or in active protein synthesis between horizontal clinorotation and vertical controls in the Arabidopsis leaf and stem tissue. These results show chronic clinorotation does not cause gross changes in protein expression in Arabidopsis.

  13. Protein expression in Arabidopsis thaliana after chronic clinorotation

    NASA Technical Reports Server (NTRS)

    Piastuch, W. C.; Brown, C. S.

    1995-01-01

    Soluble protein expression in Arabidopsis thaliana L. (Heynh.) leaf and stem tissue was examined after chronic clinorotation. Seeds of Arabidopsis were germinated and plants grown to maturity on horizontal or vertical slow-rotating clinostats (1 rpm) or in stationary vertical control units. Total soluble proteins and in vivo-labeled soluble proteins isolated from these plants were analyzed by two-dimensional SDS PAGE and subsequent fluorography. Visual and computer analysis of the resulting protein patterns showed no significant differences in either total protein expression or in active protein synthesis between horizontal clinorotation and vertical controls in the Arabidopsis leaf and stem tissue. These results show chronic clinorotation does not cause gross changes in protein expression in Arabidopsis.

  14. Engineering Cells to Improve Protein Expression

    PubMed Central

    Xiao, Su; Shiloach, Joseph; Betenbaugh, Michael J.

    2014-01-01

    Cellular engineering of bacteria, fungi, insect cells and mammalian cells is a promising methodology to improve recombinant protein production for structural, biochemical, and commercial applications. Increased understanding of the host organism biology has suggested engineering strategies targeting bottlenecks in transcription, translation, protein processing and secretory pathways, as well as cell growth and survival. A combination of metabolic engineering and synthetic biology has been used to improve the properties of cells for protein production, which has resulted in enhanced yields of multiple protein classes. PMID:24704806

  15. Engineering cells to improve protein expression.

    PubMed

    Xiao, Su; Shiloach, Joseph; Betenbaugh, Michael J

    2014-06-01

    Cellular engineering of bacteria, fungi, insect cells and mammalian cells is a promising methodology to improve recombinant protein production for structural, biochemical, and commercial applications. Increased understanding of the host organism biology has suggested engineering strategies targeting bottlenecks in transcription, translation, protein processing and secretory pathways, as well as cell growth and survival. A combination of metabolic engineering and synthetic biology has been used to improve the properties of cells for protein production, which has resulted in enhanced yields of multiple protein classes. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  17. Emerging technology of in situ cell free expression protein microarrays.

    PubMed

    Nand, Amita; Gautam, Anju; Pérez, Javier Batista; Merino, Alejandro; Zhu, Jinsong

    2012-02-01

    Recently, in situ protein microarrays have been developed for large scale analysis and high throughput studies of proteins. In situ protein microarrays produce proteins directly on the solid surface from pre-arrayed DNA or RNA. The advances in in situ protein microarrays are exemplified by the ease of cDNA cloning and cell free protein expression. These technologies can evaluate, validate and monitor protein in a cost effective manner and address the issue of a high quality protein supply to use in the array. Here we review the importance of recently employed methods: PISA (protein in situ array), DAPA (DNA array to protein array), NAPPA (nucleic acid programmable protein array) and TUSTER microarrays and the role of these methods in proteomics.

  18. Evolution, diversification, and expression of KNOX proteins in plants

    PubMed Central

    Gao, Jie; Yang, Xue; Zhao, Wei; Lang, Tiange; Samuelsson, Tore

    2015-01-01

    The KNOX (KNOTTED1-like homeobox) transcription factors play a pivotal role in leaf and meristem development. The majority of these proteins are characterized by the KNOX1, KNOX2, ELK, and homeobox domains whereas the proteins of the KNATM family contain only the KNOX domains. We carried out an extensive inventory of these proteins and here report on a total of 394 KNOX proteins from 48 species. The land plant proteins fall into two classes (I and II) as previously shown where the class I family seems to be most closely related to the green algae homologs. The KNATM proteins are restricted to Eudicots and some species have multiple paralogs of this protein. Certain plants are characterized by a significant increase in the number of KNOX paralogs; one example is Glycine max. Through the analysis of public gene expression data we show that the class II proteins of this plant have a relatively broad expression specificity as compared to class I proteins, consistent with previous studies of other plants. In G. max, class I protein are mainly distributed in axis tissues and KNATM paralogs are overall poorly expressed; highest expression is in the early plumular axis. Overall, analysis of gene expression in G. max demonstrates clearly that the expansion in gene number is associated with functional diversification. PMID:26557129

  19. Differential expression of stress-inducible proteins in chronic hepatic iron overload

    SciTech Connect

    Brown, Kyle E. Broadhurst, Kimberly A.; Mathahs, M. Meleah; Weydert, Jamie

    2007-09-01

    Introduction:: Oxidative stress can trigger a cellular stress response characterized by induction of antioxidants, acute phase reactants (APRs) and heat shock proteins (HSPs), which are presumed to play a role in limiting tissue damage. In rodents, hepatic iron overload causes oxidative stress that results in upregulation of antioxidant defenses with minimal progressive liver injury. The aim of this study was to determine whether iron overload modulates expression of other stress-responsive proteins such as APRs and HSPs that may confer protection against iron-induced damage in rodent liver. Methods:: Male rats received repeated injections of iron dextran or dextran alone over a 6-month period. Hepatic transcript levels for a panel of APRs and HSPs were quantitated by real-time PCR and protein expression was evaluated by Western blot and immunohistochemistry. Results:: Hepatic iron concentrations were increased > 50-fold in the iron-loaded rats compared to controls. Iron loading resulted in striking increases in mRNAs for Hsp32 (heme oxygenase-1; 12-fold increase vs. controls) and metallothionein-1 and -2 (both increased {approx} 6-fold). Transcripts for {alpha}1-acid glycoprotein, the major rat APR, were increased {approx} 3-fold, while expression of other classical APRs was unaltered. Surprisingly, although mRNA levels for the HSPs were not altered by iron, the abundance of Hsp25, Hsp70 and Hsp90 proteins was uniformly reduced in the iron-loaded livers, as were levels of NAD(P)H:quinone oxidoreductase 1, an Hsp70 client protein. Conclusions:: Chronic iron administration elicits a unique pattern of stress protein expression. These alterations may modulate hepatic responses to iron overload, as well as other injury processes.

  20. Major cancer protein amplifies global gene expression

    Cancer.gov

    Scientists may have discovered why a protein called MYC can provoke a variety of cancers. Like many proteins associated with cancer, MYC helps regulate cell growth. A new study carried out by researchers at the National Institutes of Health and colleagues

  1. Tools for Co-expressing Multiple Proteins in Mammalian Cells

    PubMed Central

    Assur, Zahra; Hendrickson, Wayne A.; Mancia, Filippo

    2013-01-01

    Summary Structural and functional studies of many mammalian systems are critically dependent on abundant supplies of recombinant multi-protein complexes. Mammalian cells are often the most ideal, if not the only suitable host for such experiments. This is due to their intrinsic capability to generate functional mammalian proteins. This advantage is frequently countered by problems with yields in expression, time required to generate over-expressing lines, and elevated costs. Co-expression of multiple proteins adds another level of complexity to these experiments, as cells need to be screened and selected for expression of suitable levels of each component. Here we present an efficient fluorescence marking procedure for establishing stable cell lines that over-express two proteins in co-ordination, and we validate the method in the production of recombinant monoclonal antibody Fab fragments. This procedure may readily be expanded to systems of greater complexity, comprising more then two components. PMID:21987254

  2. Comparative Protein Profiling of Intraphagosomal Expressed Proteins of Mycobacterium bovis BCG.

    PubMed

    Singhal, Neelja; Kumar, Manish; Sharma, Divakar; Bisht, Deepa

    2016-01-01

    BCG, the only available vaccine against tuberculosis affords a variable protection which wanes with time. In this study we have analyzed and compared the proteins which are expressed differentially during broth-culture and intraphagosomal growth of M.bovis BCG. Eight proteins which showed increased expression during the intraphagosomal growth were identified by MALDI-TOF/MS. These were - a precursor of alanine and proline-rich secreted protein apa, isoforms of malate dehydrogenase, large subunit alpha (Alpha-ETF) of electron transfer flavoprotein, immunogenic protein MPB64 precursor, UPF0036 protein, and two proteins with unknown function. Based on these findings we speculate that higher expression of these proteins has a probable role in intracellular survival, adaptation and/or immunoprotective effect of BCG. Further, these proteins might also be used as gene expression markers for endosome trafficking events of BCG.

  3. Transient protein expression in three Pisum sativum (green pea) varieties.

    PubMed

    Green, Brian J; Fujiki, Masaaki; Mett, Valentina; Kaczmarczyk, Jon; Shamloul, Moneim; Musiychuk, Konstantin; Underkoffler, Susan; Yusibov, Vidadi; Mett, Vadim

    2009-02-01

    The expression of proteins in plants both transiently and via permanently transformed lines has been demonstrated by a number of groups. Transient plant expression systems, due to high expression levels and speed of production, show greater promise for the manufacturing of biopharmaceuticals when compared to permanent transformants. Expression vectors based on a tobacco mosaic virus (TMV) are the most commonly utilized and the primary plant used, Nicotiana benthamiana, has demonstrated the ability to express a wide range of proteins at levels amenable to purification. N. benthamiana has two limitations for its use; one is its relatively slow growth, and the other is its low biomass. To address these limitations we screened a number of legumes for transient protein expression. Using the alfalfa mosaic virus (AMV) and the cucumber mosaic virus (CMV) vectors, delivered via Agrobacterium, we were able to identify three Pisum sativum varieties that demonstrated protein expression transiently. Expression levels of 420 +/- 26.24 mg GFP/kgFW in the green pea variety speckled pea were achieved. We were also able to express three therapeutic proteins indicating promise for this system in the production of biopharmaceuticals.

  4. Vesicular stomatitis virus-based vaccines expressing EV71 virus-like particles elicit strong immune responses and protect newborn mice from lethal challenges.

    PubMed

    Yan, Qin; Wu, Linjuan; Chen, Longyun; Qin, Yali; Pan, Zishu; Chen, Mingzhou

    2016-07-29

    Enterovirus 71 (EV71) belonging to the Picornaviridae family is considered the most frequently detected causative agent in hand-foot-and-mouth disease (HFMD) and is a serious threat to public health in the Asia-Pacific region. There are currently no approved vaccines or effective drugs for EV71. In this study, using recombinant vesicular stomatitis virus (rVSV) expressing viral VP1 protein (mVP1) of EV71 as a control, we generated two types of rVSVs that can form EV71 virus-like particles (VLPs). First, we co-infected two rVSVs singly expressing P1 (mP1) and 3CD (m3CD) of EV71. Second, we inserted P1 and 3CD into one VSV backbone to generate an rVSV expressing P1 and 3CD together (mP1-3CD). When P1 and 3CD were expressed in the cells either co-infected with mP1 and m3CD (mP1/m3CD) or infected with mP1-3CD, P1 was cleaved by 3CD and produced VP1, VP3, and VP0 to form VLPs. Furthermore, mice immunized with mP1/m3CD or mP1-3CD showed higher humoral and cellular immunity responses than mice immunized with mVP1. Finally, the rVSVs expressing the EV71 proteins were evaluated in mice to determine their potential to protect against a lethal EV71 virus challenge, and among all the rVSVs, the mP1-3CD was shown to be the most promising vaccine candidate for EV71 protection.

  5. Mobile phone radiation might alter protein expression in human skin

    PubMed Central

    Karinen, Anu; Heinävaara, Sirpa; Nylund, Reetta; Leszczynski, Dariusz

    2008-01-01

    Background Earlier we have shown that the mobile phone radiation (radiofrequency modulated electromagnetic fields; RF-EMF) alters protein expression in human endothelial cell line. This does not mean that similar response will take place in human body exposed to this radiation. Therefore, in this pilot human volunteer study, using proteomics approach, we have examined whether a local exposure of human skin to RF-EMF will cause changes in protein expression in living people. Results Small area of forearm's skin in 10 female volunteers was exposed to RF-EMF (specific absorption rate SAR = 1.3 W/kg) and punch biopsies were collected from exposed and non-exposed areas of skin. Proteins extracted from biopsies were separated using 2-DE and protein expression changes were analyzed using PDQuest software. Analysis has identified 8 proteins that were statistically significantly affected (Anova and Wilcoxon tests). Two of the proteins were present in all 10 volunteers. This suggests that protein expression in human skin might be affected by the exposure to RF-EMF. The number of affected proteins was similar to the number of affected proteins observed in our earlier in vitro studies. Conclusion This is the first study showing that molecular level changes might take place in human volunteers in response to exposure to RF-EMF. Our study confirms that proteomics screening approach can identify protein targets of RF-EMF in human volunteers. PMID:18267023

  6. Tissue-selective expression of a conditionally-active ROCK2-estrogen receptor fusion protein.

    PubMed

    Samuel, Michael S; Rath, Nicola; Masre, Siti F; Boyle, Sarah T; Greenhalgh, David A; Kochetkova, Marina; Bryson, Sheila; Stevenson, David; Olson, Michael F

    2016-12-01

    The serine/threonine kinases ROCK1 and ROCK2 are central mediators of actomyosin contractile force generation that act downstream of the RhoA small GTP-binding protein. As a result, they have key roles in regulating cell morphology and proliferation, and have been implicated in numerous pathological conditions and diseases including hypertension and cancer. Here we describe the generation of a gene-targeted mouse line that enables CRE-inducible expression of a conditionally-active fusion between the ROCK2 kinase domain and the hormone-binding domain of a mutated estrogen receptor (ROCK2:ER). This two-stage system of regulation allows for tissue-selective expression of the ROCK2:ER fusion protein, which then requires administration of estrogen analogues such as tamoxifen or 4-hydroxytamoxifen to elicit kinase activity. This conditional gain-of-function system was validated in multiple tissues by crossing with mice expressing CRE recombinase under the transcriptional control of cytokeratin14 (K14), murine mammary tumor virus (MMTV) or cytochrome P450 Cyp1A1 (Ah) promoters, driving appropriate expression in the epidermis, mammary or intestinal epithelia respectively. Given the interest in ROCK signaling in normal physiology and disease, this mouse line will facilitate research into the consequences of ROCK activation that could be used to complement conditional knockout models. Birth Defects Research (Part A) 106:636-646, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  7. Nasal immunization with Lactococcus lactis expressing the pneumococcal protective protein A induces protective immunity in mice.

    PubMed

    Medina, Marcela; Villena, Julio; Vintiñi, Elisa; Hebert, Elvira María; Raya, Raúl; Alvarez, Susana

    2008-06-01

    Nisin-controlled gene expression was used to develop a recombinant strain of Lactococcus lactis that is able to express the pneumococcal protective protein A (PppA) on its surface. Immunodetection assays confirmed that after the induction with nisin, the PppA antigen was predictably and efficiently displayed on the cell surface of the recombinant strain, which was termed L. lactis PppA. The production of mucosal and systemically specific antibodies in adult and young mice was evaluated after mice were nasally immunized with L. lactis PppA. Immunoglobulin M (IgM), IgG, and IgA anti-PppA antibodies were detected in the serum and bronchoalveolar lavage fluid of adult and young mice, which showed that PppA expressed in L. lactis was able to induce a strong mucosal and systemic immune response. Challenge survival experiments demonstrated that immunization with L. lactis PppA was able to increase resistance to systemic and respiratory infection with different pneumococcal serotypes, and passive immunization assays of naïve young mice demonstrated a direct correlation between anti-PppA antibodies and protection. The results presented in this study demonstrate three major characteristics of the effectiveness of nasal immunization with PppA expressed as a protein anchored to the cell wall of L. lactis: it elicited cross-protective immunity against different pneumococcal serotypes, it afforded protection against both systemic and respiratory challenges, and it induced protective immunity in mice of different ages.

  8. Impact of Chronic Alcohol Ingestion on Cardiac Muscle Protein Expression

    PubMed Central

    Fogle, Rachel L.; Lynch, Christopher J.; Palopoli, Mary; Deiter, Gina; Stanley, Bruce A.; Vary, Thomas C.

    2014-01-01

    Background Chronic alcohol abuse contributes not only to an increased risk of health-related complications, but also to a premature mortality in adults. Myocardial dysfunction, including the development of a syndrome referred to as alcoholic cardiomyopathy, appears to be a major contributing factor. One mechanism to account for the pathogenesis of alcoholic cardiomyopathy involves alterations in protein expression secondary to an inhibition of protein synthesis. However, the full extent to which myocardial proteins are affected by chronic alcohol consumption remains unresolved. Methods The purpose of this study was to examine the effect of chronic alcohol consumption on the expression of cardiac proteins. Male rats were maintained for 16 weeks on a 40% ethanol-containing diet in which alcohol was provided both in drinking water and agar blocks. Control animals were pair-fed to consume the same caloric intake. Heart homogenates from control- and ethanol-fed rats were labeled with the cleavable isotope coded affinity tags (ICAT™). Following the reaction with the ICAT™ reagent, we applied one-dimensional gel electrophoresis with in-gel trypsin digestion of proteins and subsequent MALDI-TOF-TOF mass spectrometric techniques for identification of peptides. Differences in the expression of cardiac proteins from control- and ethanol-fed rats were determined by mass spectrometry approaches. Results Initial proteomic analysis identified and quantified hundreds of cardiac proteins. Major decreases in the expression of specific myocardial proteins were observed. Proteins were grouped depending on their contribution to multiple activities of cardiac function and metabolism, including mitochondrial-, glycolytic-, myofibrillar-, membrane-associated, and plasma proteins. Another group contained identified proteins that could not be properly categorized under the aforementioned classification system. Conclusions Based on the changes in proteins, we speculate modulation of

  9. Exocyst Complex Protein Expression in the Human Placenta

    PubMed Central

    Gonzalez, I.M.; Ackerman, W.E.; Vandre, D.D.; Robinson, J.M.

    2014-01-01

    Introduction Protein production and secretion are essential to syncytiotrophoblast function and are associated with cytotrophoblast cell fusion and differentiation. Syncytiotrophoblast hormone secretion is a crucial determinant of maternal-fetal health, and can be misregulated in pathological pregnancies. Although, polarized secretion is a key component of placental function, the mechanisms underlying this process are poorly understood. Objective While the octameric exocyst complex is classically regarded as a master regulator of secretion in various mammalian systems, its expression in the placenta remained unexplored. We hypothesized that the syncytiotrophoblast would express all exocyst complex components and effector proteins requisite for vesicle-mediated secretion more abundantly than cytotrophoblasts in tissue specimens. Methods A two-tiered immunobiological approach was utilized to characterize exocyst and ancillary proteins in normal, term human placentas. Exocyst protein expression and localization was documented in tissue homogenates via immunoblotting and immunofluorescence labeling of placental sections. Results The eight exocyst proteins, EXOC1, 2, 3, 4, 5, 6, 7, and 8, were found in the human placenta. In addition, RAB11, an important exocyst complex modulator, was also expressed. Exocyst and Rab protein expression appeared to be regulated during trophoblast differentiation, as the syncytiotrophoblast expressed these proteins with little, if any, expression in cytotrophoblast cells. Additionally, exocyst proteins were localized at or near the syncytiotrophoblast apical membrane, the major site of placental secretion Discussion/Conclusion Our findings highlight exocyst protein expression as novel indicators of trophoblast differentiation. The exocyst’s regulated localization within the syncytiotrophoblast in conjunction with its well known functions suggests a possible role in placental polarized secretion PMID:24856041

  10. Exocyst complex protein expression in the human placenta.

    PubMed

    Gonzalez, I M; Ackerman, W E; Vandre, D D; Robinson, J M

    2014-07-01

    Protein production and secretion are essential to syncytiotrophoblast function and are associated with cytotrophoblast cell fusion and differentiation. Syncytiotrophoblast hormone secretion is a crucial determinant of maternal-fetal health, and can be misregulated in pathological pregnancies. Although, polarized secretion is a key component of placental function, the mechanisms underlying this process are poorly understood. While the octameric exocyst complex is classically regarded as a master regulator of secretion in various mammalian systems, its expression in the placenta remained unexplored. We hypothesized that the syncytiotrophoblast would express all exocyst complex components and effector proteins requisite for vesicle-mediated secretion more abundantly than cytotrophoblasts in tissue specimens. A two-tiered immunobiological approach was utilized to characterize exocyst and ancillary proteins in normal, term human placentas. Exocyst protein expression and localization was documented in tissue homogenates via immunoblotting and immunofluorescence labeling of placental sections. The eight exocyst proteins, EXOC1, 2, 3, 4, 5, 6, 7, and 8, were found in the human placenta. In addition, RAB11, an important exocyst complex modulator, was also expressed. Exocyst and Rab protein expression appeared to be regulated during trophoblast differentiation, as the syncytiotrophoblast expressed these proteins with little, if any, expression in cytotrophoblast cells. Additionally, exocyst proteins were localized at or near the syncytiotrophoblast apical membrane, the major site of placental secretion. Our findings highlight exocyst protein expression as novel indicators of trophoblast differentiation. The exocyst's regulated localization within the syncytiotrophoblast in conjunction with its well known functions suggests a possible role in placental polarized secretion. Copyright © 2014 Elsevier Ltd. All rights reserved.

  11. Systemic and mucosal immunity in mice elicited by a single immunization with human adenovirus type 5 or 41 vector-based vaccines carrying the spike protein of Middle East respiratory syndrome coronavirus

    PubMed Central

    Guo, Xiaojuan; Deng, Yao; Chen, Hong; Lan, Jiaming; Wang, Wen; Zou, Xiaohui; Hung, Tao; Lu, Zhuozhuang; Tan, Wenjie

    2015-01-01

    An ideal vaccine against mucosal pathogens such as Middle East respiratory syndrome coronavirus (MERS-CoV) should confer sustained, protective immunity at both systemic and mucosal levels. Here, we evaluated the in vivo systemic and mucosal antigen-specific immune responses induced by a single intramuscular or intragastric administration of recombinant adenoviral type 5 (Ad5) or type 41 (Ad41) -based vaccines expressing the MERS-CoV spike (S) protein. Intragastric administration of either Ad5-S or Ad41-S induced antigen-specific IgG and neutralizing antibody in serum; however, antigen-specific T-cell responses were not detected. In contrast, after a single intramuscular dose of Ad5-S or Ad41-S, functional antigen-specific T-cell responses were elicited in the spleen and pulmonary lymphocytes of the mice, which persisted for several months. Both rAd-based vaccines administered intramuscularly induced systemic humoral immune responses (neutralizing IgG antibodies). Our results show that a single dose of Ad5-S- or Ad41-S-based vaccines represents an appealing strategy for the control of MERS-CoV infection and transmission. PMID:25762305

  12. The Treponema denticola FhbB Protein Is a Dominant Early Antigen That Elicits FhbB Variant-Specific Antibodies That Block Factor H Binding and Cleavage by Dentilisin.

    PubMed

    Miller, Daniel P; Oliver, Lee D; Tegels, Brittney K; Reed, Lucas A; O'Bier, Nathaniel S; Kurniyati, Kurni; Faust, Lindsay A; Lawson, Christine K; Allard, Anna M; Caimano, Melissa J; Marconi, Richard T

    2016-07-01

    The Treponema denticola FhbB protein contributes to immune evasion by binding factor H (FH). Cleavage of FH by the T. denticola protease, dentilisin, may contribute to the local immune dysregulation that is characteristic of periodontal disease (PD). Although three FhbB phyletic types have been defined (FhbB1, FhbB2, and FhbB3), the in vivo expression patterns and antigenic heterogeneity of FhbB have not been assessed. Here, we demonstrate that FhbB is a dominant early antigen that elicits FhbB type-specific antibody (Ab) responses. Using the murine skin abscess model, we demonstrate that the presence or absence of FhbB or dentilisin significantly influences Ab responses to infection and skin abscess formation. Competitive binding analyses revealed that α-FhbB Ab can compete with FH for binding to T. denticola and block dentilisin-mediated FH cleavage. Lastly, we demonstrate that dentilisin cleavage sites reside within critical functional domains of FH, including the complement regulatory domain formed by CCPs 1 to 4. Analysis of the FH cleavage products revealed that they lack cofactor activity. The data presented here provide insight into the in vivo significance of dentilisin, FhbB and its antigenic diversity, and the potential impact of FH cleavage on the regulation of complement activation. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  13. The Treponema denticola FhbB Protein Is a Dominant Early Antigen That Elicits FhbB Variant-Specific Antibodies That Block Factor H Binding and Cleavage by Dentilisin

    PubMed Central

    Miller, Daniel P.; Oliver, Lee D.; Tegels, Brittney K.; Reed, Lucas A.; O'Bier, Nathaniel S.; Kurniyati, Kurni; Faust, Lindsay A.; Lawson, Christine K.; Allard, Anna M.; Caimano, Melissa J.

    2016-01-01

    The Treponema denticola FhbB protein contributes to immune evasion by binding factor H (FH). Cleavage of FH by the T. denticola protease, dentilisin, may contribute to the local immune dysregulation that is characteristic of periodontal disease (PD). Although three FhbB phyletic types have been defined (FhbB1, FhbB2, and FhbB3), the in vivo expression patterns and antigenic heterogeneity of FhbB have not been assessed. Here, we demonstrate that FhbB is a dominant early antigen that elicits FhbB type-specific antibody (Ab) responses. Using the murine skin abscess model, we demonstrate that the presence or absence of FhbB or dentilisin significantly influences Ab responses to infection and skin abscess formation. Competitive binding analyses revealed that α-FhbB Ab can compete with FH for binding to T. denticola and block dentilisin-mediated FH cleavage. Lastly, we demonstrate that dentilisin cleavage sites reside within critical functional domains of FH, including the complement regulatory domain formed by CCPs 1 to 4. Analysis of the FH cleavage products revealed that they lack cofactor activity. The data presented here provide insight into the in vivo significance of dentilisin, FhbB and its antigenic diversity, and the potential impact of FH cleavage on the regulation of complement activation. PMID:27113359

  14. Human SUMO fusion systems enhance protein expression and solubility.

    PubMed

    Wang, Zhongyuan; Li, Haolong; Guan, Wei; Ling, Haili; Wang, Zhiyong; Mu, Tianyang; Shuler, Franklin D; Fang, Xuexun

    2010-10-01

    A major challenge associated with recombinant protein production in Escherichia coli is generation of large quantities of soluble, functional protein. Yeast SUMO (small ubiquitin-related modifier), has been shown to enhance heterologous protein expression and solubility as fusion tag, however, the effects of human SUMOs on protein expression have not been investigated. Here we describe the use of human SUMO1 and SUMO2 as a useful gene fusion technology. Human SUMO1 and SUMO2 fusion expression vectors were constructed and tested in His-tag and ubiquitin fusion expression systems. Two difficult-to-express model proteins, matrix metalloprotease-13 (MMP13) and enhanced green fluorescence protein (eGFP) were fused to the C-terminus of the human SUMO1 and SUMO2 expression vectors. These constructs were expressed in E. coli and evaluation of MMP13 and eGFP expression and solubility was conducted. We found that both SUMO1 and SUMO2 had the ability to enhance the solubility of MMP13 and eGFP, with the SUMO2 tag having a more significant effect. Since fusion tags produce varying quantities of soluble proteins, we assessed the effect of SUMO2 coupled with ubiquitin (Ub). SUMO2-ubiquitin and ubiquitin-SUMO2 fusion expression plasmids were constructed with eGFP as a passenger protein. Following expression in E. coli, both plasmids could improve eGFP expression and solubility similar to the SUMO2 fusion and better than the ubiquitin fusion. The sequential order of SUMO2 and ubiquitin had little effect on expression and solubility of eGFP. Purification of eGFP from the gene fusion product, SUMO2-ubiquitin-eGFP, involved cleavage by a deubiquitinase (Usp2-cc) and Ni-Sepharose column chromatography. The eGFP protein was purified to high homogeneity. In summary, human SUMO1 and SUMO2 are useful gene fusion technologies enhancing the expression, solubility and purification of model heterologous proteins. Copyright 2010 Elsevier Inc. All rights reserved.

  15. Improving membrane protein expression by optimizing integration efficiency.

    PubMed

    Niesen, Michiel J M; Marshall, Stephen S; Miller, Thomas F; Clemons, William M

    2017-09-16

    The heterologous overexpression of integral membrane proteins in Escherichia coli often yields insufficient quantities of purifiable protein for applications of interest. The current study leverages a recently demonstrated link between co-translational membrane integration efficiency and protein expression levels to predict protein sequence modifications that improve expression. Membrane integration efficiencies, obtained using a coarse-grained simulation approach, robustly predicted effects on expression of the integral membrane protein TatC for a set of 140 sequence modifications, including loop-swap chimeras and single-residue mutations distributed throughout the protein sequence. Mutations that improve simulated integration efficiency were four-fold enriched with respect to improved experimentally observed expression levels. Furthermore, the effect of double mutations, on both simulated integration efficiency and experimentally observed expression levels were cumulative and largely independent, suggesting that multiple mutations can be introduced to yield higher levels of purifiable protein. This work provides a foundation for a general method for the rational overexpression of integral membrane proteins based on computationally simulated membrane integration efficiencies. Copyright © 2017, The American Society for Biochemistry and Molecular Biology.

  16. Ludic Elicitation: Using Games for Knowledge Elicitation

    ERIC Educational Resources Information Center

    Cao, Yan

    2014-01-01

    Knowledge elicitation from human beings is important for many fields, such as decision support systems, risk communication, and customer preference studying. Traditional approaches include observations, questionnaires, structured and semi-structured interviews, and group discussions. Many publications have been studying different techniques for a…

  17. Ludic Elicitation: Using Games for Knowledge Elicitation

    ERIC Educational Resources Information Center

    Cao, Yan

    2014-01-01

    Knowledge elicitation from human beings is important for many fields, such as decision support systems, risk communication, and customer preference studying. Traditional approaches include observations, questionnaires, structured and semi-structured interviews, and group discussions. Many publications have been studying different techniques for a…

  18. Protein Production for Structural Genomics Using E. coli Expression

    PubMed Central

    Makowska-Grzyska, Magdalena; Kim, Youngchang; Maltseva, Natalia; Li, Hui; Zhou, Min; Joachimiak, Grazyna; Babnigg, Gyorgy; Joachimiak, Andrzej

    2014-01-01

    The goal of structural biology is to reveal details of the molecular structure of proteins in order to understand their function and mechanism. X-ray crystallography and NMR are the two best methods for atomic level structure determination. However, these methods require milligram quantities of proteins. In this chapter a reproducible methodology for large-scale protein production applicable to a diverse set of proteins is described. The approach is based on protein expression in E. coli as a fusion with a cleavable affinity tag that was tested on over 20,000 proteins. Specifically, a protocol for fermentation of large quantities of native proteins in disposable culture vessels is presented. A modified protocol that allows for the production of selenium-labeled proteins in defined media is also offered. Finally, a method for the purification of His6-tagged proteins on immobilized metal affinity chromatography columns that generates high-purity material is described in detail. PMID:24590711

  19. An intact signal peptide on dengue virus E protein enhances immunogenicity for CD8(+) T cells and antibody when expressed from modified vaccinia Ankara.

    PubMed

    Quinan, Bárbara R; Flesch, Inge E A; Pinho, Tânia M G; Coelho, Fabiana M; Tscharke, David C; da Fonseca, Flávio G

    2014-05-23

    Dengue is a global public health concern and this is aggravated by a lack of vaccines or antiviral therapies. Despite the well-known role of CD8(+) T cells in the immunopathogenesis of Dengue virus (DENV), only recent studies have highlighted the importance of this arm of the immune response in protection against the disease. Thus, the majority of DENV vaccine candidates are designed to achieve protective titers of neutralizing antibodies, with less regard for cellular responses. Here, we used a mouse model to investigate CD8(+) T cell and humoral responses to a set of potential DENV vaccines based on recombinant modified vaccinia virus Ankara (rMVA). To enable this study, we identified two CD8(+) T cell epitopes in the DENV-3 E protein in C57BL/6 mice. Using these we found that all the rMVA vaccines elicited DENV-specific CD8(+) T cells that were cytotoxic in vivo and polyfunctional in vitro. Moreover, vaccines expressing the E protein with an intact signal peptide sequence elicited more DENV-specific CD8(+) T cells than those expressing E proteins in the cytoplasm. Significantly, it was these same ER-targeted E protein vaccines that elicited antibody responses. Our results support the further development of rMVA vaccines expressing DENV E proteins and add to the tools available for dengue vaccine development.

  20. Protein Expression Dynamics During Postnatal Mouse Brain Development

    PubMed Central

    Laeremans, Annelies; Van de Plas, Babs; Clerens, Stefan; Van den Bergh, Gert; Arckens, Lutgarde; Hu, Tjing-Tjing

    2013-01-01

    We explored differential protein expression profiles in the mouse forebrain at different stages of postnatal development, including 10-day (P10), 30-day (P30), and adult (Ad) mice, by large-scale screening of proteome maps using two-dimensional difference gel electrophoresis. Mass spectrometry analysis resulted in the identification of 251 differentially expressed proteins. Most molecular changes were observed between P10 compared to both P30 and Ad. Computational ingenuity pathway analysis (IPA) confirmed these proteins as crucial molecules in the biological function of nervous system development. Moreover, IPA revealed Semaphorin signaling in neurons and the protein ubiquitination pathway as essential canonical pathways in the mouse forebrain during postnatal development. For these main biological pathways, the transcriptional regulation of the age-dependent expression of selected proteins was validated by means of in situ hybridization. In conclusion, we suggest that proteolysis and neurite outgrowth guidance are key biological processes, particularly during early brain maturation. PMID:25157209

  1. Analysis of incomplete gene expression dataset through protein-protein interaction information.

    PubMed

    Massanet-Vila, Raimon; Padró, Teresa; Cardús, Anna; Badimon, Lina; Caminal, Pere; Perera, Alexandre

    2011-01-01

    This paper shows a graph based method to analyze proteomic expression data. The method allows the prediction of the expression of genes not measured by the gene expression technology based on the local connectivity properties of the measured differentially expressed gene set. The prediction of the expression jointly with the stability of this prediction as a function of the variation of the initial expressed set is computed. The method is able to correctly predict one third of the proteins with independence of variations on the selection of the initial set. The algorithm is validated through a Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometer (MALDI-TOF) protein expression experiment aiming the study of the protein expression patterns and post-translational modifications in human endothelial vascular cells exposed to atherosclerotic levels of Low Density Lipoproteins (LDL).

  2. A bivalent pneumococcal histidine triad protein D-choline-binding protein A vaccine elicits functional antibodies that passively protect mice from Streptococcus pneumoniae challenge

    PubMed Central

    Ochs, Martina M.; Williams, Kimberley; Sheung, Anthony; Lheritier, Philippe; Visan, Lucian; Rouleau, Nicolas; Proust, Emilie; de Montfort, Aymeric; Tang, Mei; Mari, Karine; Hopfer, Robert; Gallichan, Scott; Brookes, Roger H.

    2016-01-01

    ABSTRACT Vaccines based on conserved pneumococcal proteins are being investigated because serotype coverage by pneumococcal polysaccharide and polysaccharide conjugate vaccines is incomplete and may eventually decrease due to serotype replacement. Here, we examined the functionality of human antibodies induced by a candidate bivalent choline-binding protein A- pneumococcal histidine triad protein D (PcpA-PhtD) vaccine. Pre- and post-immune sera from subjects who had been vaccinated with the PcpA-PhtD candidate vaccine were tested in an established passive protection model in which mice were challenged by intravenous injection with Streptococcus pneumoniae serotype 3 strain A66.1. Serum antibody concentrations were determined by enzyme-linked immunosorbent assay (ELISA). Bacterial surface binding by serum antibodies was determined by a flow cytometry-based assay. Sera from 20 subjects were selected based on low activity of pre-immune samples in the passive protection model. Bacterial surface binding correlated more strongly with anti-PcpA (0.87; p < 0.0001) than with anti-PhtD (0.71; p < 0.0001). The odds ratio for predicting survival in the passive protection assay was higher for the anti-PcpA concentration (470 [95% confidence interval (CI), 46.8 to >999.9]) than for the anti-PhtD concentration (3.4 [95% CI, 1.9 to 5.6]) or bacterial surface binding (9.4 [95% CI, 3.6 to 24.3]). Pooled post-immune serum also protected mice against a challenge with S. pneumoniae serotype 3 strain WU2. Both anti-PcpA and anti-PhtD antibodies induced by the bivalent candidate vaccine mediate protection against S. pneumoniae. The results also showed that the ELISA titer might be useful as a surrogate for estimating the functional activity of antibodies induced by pneumococcal protein vaccines. PMID:27392182

  3. A bivalent pneumococcal histidine triad protein D-choline-binding protein A vaccine elicits functional antibodies that passively protect mice from Streptococcus pneumoniae challenge.

    PubMed

    Ochs, Martina M; Williams, Kimberley; Sheung, Anthony; Lheritier, Philippe; Visan, Lucian; Rouleau, Nicolas; Proust, Emilie; de Montfort, Aymeric; Tang, Mei; Mari, Karine; Hopfer, Robert; Gallichan, Scott; Brookes, Roger H

    2016-11-01

    Vaccines based on conserved pneumococcal proteins are being investigated because serotype coverage by pneumococcal polysaccharide and polysaccharide conjugate vaccines is incomplete and may eventually decrease due to serotype replacement. Here, we examined the functionality of human antibodies induced by a candidate bivalent choline-binding protein A- pneumococcal histidine triad protein D (PcpA-PhtD) vaccine. Pre- and post-immune sera from subjects who had been vaccinated with the PcpA-PhtD candidate vaccine were tested in an established passive protection model in which mice were challenged by intravenous injection with Streptococcus pneumoniae serotype 3 strain A66.1. Serum antibody concentrations were determined by enzyme-linked immunosorbent assay (ELISA). Bacterial surface binding by serum antibodies was determined by a flow cytometry-based assay. Sera from 20 subjects were selected based on low activity of pre-immune samples in the passive protection model. Bacterial surface binding correlated more strongly with anti-PcpA (0.87; p < 0.0001) than with anti-PhtD (0.71; p < 0.0001). The odds ratio for predicting survival in the passive protection assay was higher for the anti-PcpA concentration (470 [95% confidence interval (CI), 46.8 to >999.9]) than for the anti-PhtD concentration (3.4 [95% CI, 1.9 to 5.6]) or bacterial surface binding (9.4 [95% CI, 3.6 to 24.3]). Pooled post-immune serum also protected mice against a challenge with S. pneumoniae serotype 3 strain WU2. Both anti-PcpA and anti-PhtD antibodies induced by the bivalent candidate vaccine mediate protection against S. pneumoniae. The results also showed that the ELISA titer might be useful as a surrogate for estimating the functional activity of antibodies induced by pneumococcal protein vaccines.

  4. Proteins and an Inflammatory Network Expressed in Colon Tumors

    PubMed Central

    Zhu, Wenhong; Fang, Changming; Gramatikoff, Kosi; Niemeyer, Christina C.; Smith, Jeffrey W.

    2011-01-01

    The adenomatous polyposis coli (APC) protein is crucial to homeostasis of normal intestinal epithelia because it suppresses the β-catenin/TCF pathway. Consequently, loss or mutation of the APC gene causes colorectal tumors in humans and mice. Here, we describe our use of Multidimensional Protein Identification Technology (MudPIT) to compare protein expression in colon tumors to that of adjacent healthy colon tissue from ApcMin/+ mice. Twenty-seven proteins were found to be up-regulated in colon tumors and twenty-five down-regulated. As an extension of the proteomic analysis, the differentially expressed proteins were used as “seeds” to search for co-expressed genes. This approach revealed a co-expression network of 45 genes that is up-regulated in colon tumors. Members of the network include the antibacterial peptide cathelicidin (CAMP), Toll-like receptors (TLRs), IL-8, and triggering receptor expressed on myeloid cells 1 (TREM1). The co-expression network is associated with innate immunity and inflammation, and there is significant concordance between its connectivity in humans versus mice (Friedman: p value = 0.0056). This study provides new insights into the proteins and networks that are likely to drive the onset and progression of colon cancer. PMID:21366352

  5. Different Immunity Elicited by Recombinant H5N1 Hemagglutinin Proteins Containing Pauci-Mannose, High-Mannose, or Complex Type N-Glycans

    PubMed Central

    Lin, Shih-Chang; Jan, Jia-Tsrong; Dionne, Ben; Butler, Michael; Huang, Ming-Hsi; Wu, Chung-Yi; Wong, Chi-Huey; Wu, Suh-Chin

    2013-01-01

    Highly pathogenic avian influenza H5N1 viruses can result in poultry and occasionally in human mortality. A safe and effective H5N1 vaccine is urgently needed to reduce the pandemic potential. Hemagglutinin (HA), a major envelope protein accounting for approximately 80% of spikes in influenza virus, is often used as a major antigen for subunit vaccine development. In this study, we conducted a systematic study of the immune response against influenza virus infection following immunization with recombinant HA proteins expressed in insect (Sf9) cells, insect cells that contain exogenous genes for elaborating N-linked glycans (Mimic) and mammalian cells (CHO). While the antibody titers are higher with the insect cell derived HA proteins, the neutralization and HA inhibition titers are much higher with the mammalian cell produced HA proteins. Recombinant HA proteins containing tri- or tetra-antennary complex, terminally sialylated and asialyated-galactose type N-glycans induced better protective immunity in mice to lethal challenge. The results are highly relevant to issues that should be considered in the production of fragment vaccines. PMID:23799128

  6. Lipopolysaccharide-binding protein and leptin are associated with stress-induced interleukin-6 cytokine expression ex vivo in obesity.

    PubMed

    Huang, Chun-Jung; Stewart, Jennifer K; Shibata, Yoshimi; Slusher, Aaron L; Acevedo, Edmund O

    2015-05-01

    Obesity is associated with enhanced inflammation and mental stress, but limited information has addressed the potential additive effect of psychological stress on obesity-associated inflammation. This study examined whether obese subjects would elicit a greater host immune response (IL-6 mRNA and cytokine) to lipopolysaccharide (LPS) in response to mental stress. Blood samples for LPS-stimulated IL-6 mRNA and cytokine were collected prior to and following mental stress. Results showed that obese subjects elicited a greater LPS-induced IL-6 along with its mRNA expression following mental stress compared to normal-weight subjects. Stress-induced IL-6 cytokine response to LPS was correlated with the baseline levels of plasma LPS binding protein (LBP) and leptin. These findings are consistent with the idea that endogenous inflammatory agents (e.g., LBP and leptin), often elevated with obesity, enhance inflammatory responses to psychological stress. © 2014 Society for Psychophysiological Research.

  7. Expression of Yes-associated protein modulates Survivin expression in primary liver malignancies.

    PubMed

    Bai, Haibo; Gayyed, Mariana F; Lam-Himlin, Dora M; Klein, Alison P; Nayar, Suresh K; Xu, Yang; Khan, Mehtab; Argani, Pedram; Pan, Duojia; Anders, Robert A

    2012-09-01

    Hepatocellular carcinoma and intrahepatic cholangiocarcinoma account for 95% of primary liver cancer. For each of these malignancies, the outcome is dismal; incidence is rapidly increasing, and mechanistic understanding is limited. We observed abnormal proliferation of both biliary epithelium and hepatocytes in mice after genetic manipulation of Yes-associated protein, a transcription coactivator. Here, we comprehensively documented Yes-associated protein expression in the human liver and primary liver cancers. We showed that nuclear Yes-associated protein expression is significantly increased in human intrahepatic cholangiocarcinoma and hepatocellular carcinoma. We found that increased Yes-associated protein levels in hepatocellular carcinoma are due to multiple mechanisms including gene amplification and transcriptional and posttranscriptional regulation. Survivin, a member of the inhibitors-of-apoptosis protein family, has been reported as an independent prognostic factor for poor survival in both hepatocellular carcinoma and intrahepatic cholangiocarcinoma. We found that nuclear Yes-associated protein expression correlates significantly with nuclear Survivin expression for both intrahepatic cholangiocarcinoma and hepatocellular carcinoma. Furthermore, using mice engineered to conditionally overexpress Yes-associated protein in the liver, we found that Survivin messenger RNA expression depends upon Yes-associated protein levels. Our findings suggested that Yes-associated protein contributes to primary liver tumorigenesis and likely mediates its oncogenic effects through modulating Survivin expression. Copyright © 2012 Elsevier Inc. All rights reserved.

  8. Roles for cytosolic NADPH redox in regulating pulmonary artery relaxation by thiol oxidation-elicited subunit dimerization of protein kinase G1α

    PubMed Central

    Neo, Boon Hwa; Patel, Dhara; Kandhi, Sharath

    2013-01-01

    The activity of glucose-6-phosphate dehydrogenase (G6PD) appears to control a vascular smooth muscle relaxing mechanism regulated through cytosolic NADPH oxidation. Since our recent studies suggest that thiol oxidation-elicited dimerization of the 1α form of protein kinase G (PKG1α) contributes to the relaxation of isolated endothelium-removed bovine pulmonary arteries (BPA) to peroxide and responses to hypoxia, we investigated whether cytosolic NADPH oxidation promoted relaxation by PKG1α dimerization. Relaxation of BPA to G6PD inhibitors 6-aminonicotinamide (6-AN) and epiandrosterone (studied under hypoxia to minimize basal levels of NADPH oxidation and PKG1α dimerization) was associated with increased PKG1α dimerization and PKG-mediated vasodilator-stimulated phosphoprotein (VASP) phosphorylation. Depletion of PKG1α by small inhibitory RNA (siRNA) inhibited relaxation of BPA to 6-AN and attenuated the increase in VASP phosphorylation. Relaxation to 6-AN did not appear to be altered by depletion of soluble guanylate cyclase (sGC). Depletion of G6PD, thioredoxin-1 (Trx-1), and Trx reductase-1 (TrxR-1) in BPA with siRNA increased PKG1α dimerization and VASP phosphorylation and inhibited force generation under aerobic and hypoxic conditions. Depletion of TrxR-1 with siRNA inhibited the effects of 6-AN and enhanced similar responses to peroxide. Peroxiredoxin-1 depletion by siRNA inhibited PKG dimerization to peroxide, but it did not alter PKG dimerization under hypoxia or the stimulation of dimerization by 6-AN. Thus regulation of cytosolic NADPH redox by G6PD appears to control PKG1α dimerization in BPA through its influence on Trx-1 redox regulation by the NADPH dependence of TrxR-1. NADPH regulation of PKG dimerization may contribute to vascular responses to hypoxia that are associated with changes in NADPH redox. PMID:23709600

  9. Performance benchmarking of four cell-free protein expression systems.

    PubMed

    Gagoski, Dejan; Polinkovsky, Mark E; Mureev, Sergey; Kunert, Anne; Johnston, Wayne; Gambin, Yann; Alexandrov, Kirill

    2016-02-01

    Over the last half century, a range of cell-free protein expression systems based on pro- and eukaryotic organisms have been developed and have found a range of applications, from structural biology to directed protein evolution. While it is generally accepted that significant differences in performance among systems exist, there is a paucity of systematic experimental studies supporting this notion. Here, we took advantage of the species-independent translation initiation sequence to express and characterize 87 N-terminally GFP-tagged human cytosolic proteins of different sizes in E. coli, wheat germ (WGE), HeLa, and Leishmania-based (LTE) cell-free systems. Using a combination of single-molecule fluorescence spectroscopy, SDS-PAGE, and Western blot analysis, we assessed the expression yields, the fraction of full-length translation product, and aggregation propensity for each of these systems. Our results demonstrate that the E. coli system has the highest expression yields. However, we observe that high expression levels are accompanied by production of truncated species-particularly pronounced in the case of proteins larger than 70 kDa. Furthermore, proteins produced in the E. coli system display high aggregation propensity, with only 10% of tested proteins being produced in predominantly monodispersed form. The WGE system was the most productive among eukaryotic systems tested. Finally, HeLa and LTE show comparable protein yields that are considerably lower than the ones achieved in the E. coli and WGE systems. The protein products produced in the HeLa system display slightly higher integrity, whereas the LTE-produced proteins have the lowest aggregation propensity among the systems analyzed. The high quality of HeLa- and LTE-produced proteins enable their analysis without purification and make them suitable for analysis of multi-domain eukaryotic proteins.

  10. Disruption of Visc-2, a Brain-Expressed Conserved Long Noncoding RNA, Does Not Elicit an Overt Anatomical or Behavioral Phenotype

    PubMed Central

    Oliver, Peter L.; Chodroff, Rebecca A.; Gosal, Amrit; Edwards, Benjamin; Cheung, Amanda F.P.; Gomez-Rodriguez, Julio; Elliot, Gene; Garrett, Lisa J.; Lickiss, Tom; Szele, Francis; Green, Eric D.; Molnár, Zoltán; Ponting, Chris P.

    2015-01-01

    Although long noncoding RNAs (lncRNAs) are proposed to play essential roles in mammalian neurodevelopment, we know little of their functions from their disruption in vivo. Combining evidence for evolutionary constraint and conserved expression data, we previously identified candidate lncRNAs that might play important and conserved roles in brain function. Here, we demonstrate that the sequence and neuronal transcription of lncRNAs transcribed from the previously uncharacterized Visc locus are conserved across diverse mammals. Consequently, one of these lncRNAs, Visc-2, was selected for targeted deletion in the mouse, and knockout animals were subjected to an extremely detailed anatomical and behavioral characterization. Despite a neurodevelopmental expression pattern of Visc-2 that is highly localized to the cortex and sites of neurogenesis, anomalies in neither cytoarchitecture nor neuroproliferation were identified in knockout mice. In addition, no abnormal motor, sensory, anxiety, or cognitive behavioral phenotypes were observed. These results are important because they contribute to a growing body of evidence that lncRNA loci contribute on average far less to brain and biological functions than protein-coding loci. A high-throughput knockout program focussing on lncRNAs, similar to that currently underway for protein-coding genes, will be required to establish the distribution of their organismal functions. PMID:25209608

  11. GTP cyclohydrolase I expression, protein, and activity determine intracellular tetrahydrobiopterin levels, independent of GTP cyclohydrolase feedback regulatory protein expression.

    PubMed

    Tatham, Amy L; Crabtree, Mark J; Warrick, Nicholas; Cai, Shijie; Alp, Nicholas J; Channon, Keith M

    2009-05-15

    GTP cyclohydrolase I (GTPCH) is a key enzyme in the synthesis of tetrahydrobiopterin (BH4), a required cofactor for nitricoxide synthases and aromatic amino acid hydroxylases. Alterations of GTPCH activity and BH4 availability play an important role in human disease. GTPCH expression is regulated by inflammatory stimuli, in association with reduced expression of GTP cyclohydrolase feedback regulatory protein (GFRP). However, the relative importance of GTPCH expression versus GTPCH activity and the role of GFRP in relation to BH4 bioavailability remain uncertain. We investigated these relationships in a cell line with tet-regulated GTPCH expression and in the hph-1 mouse model of GTPCH deficiency. Doxycycline exposure resulted in a dose-dependent decrease in GTPCH protein and activity, with a strong correlation between GTPCH expression and BH4 levels (r(2) = 0.85, p < 0.0001). These changes in GTPCH and BH4 had no effect on GFRP expression or protein levels. GFRP overexpression and knockdown in tet-GCH cells did not alter GTPCH activity or BH4 levels, and GTPCH-specific knockdown in sEnd.1 endothelial cells had no effect on GFRP protein. In mouse liver we observed a graded reduction of GTPCH expression, protein, and activity, from wild type, heterozygote, to homozygote littermates, with a striking linear correlation between GTPCH expression and BH4 levels (r(2) = 0.82, p < 0.0001). Neither GFRP expression nor protein differed between wild type, heterozygote, nor homozygote mice, despite the substantial differences in BH4. We suggest that GTPCH expression is the primary regulator of BH4 levels, and changes in GTPCH or BH4 are not necessarily accompanied by changes in GFRP expression.

  12. NOD1 expression elicited by iE-DAP in first trimester human trophoblast cells and its potential role in infection-associated inflammation.

    PubMed

    Zhou, S; Yu, P; Guan, L; Xing, A; Liu, S; Xiong, Q; Peng, B

    2013-10-01

    The underlying mechanisms of protective immunity of placental trophoblast cells against bacterial infection remain largely unknown. NOD1 are intracellular pattern recognition receptors that are activated by bacterial peptides and mediate innate immunity. This study aimed to investigate the expression and function of NOD1 in first trimester trophoblast cells, and evaluate the potential role of trophoblast cells in infection-associated inflammation. Human extravillous trophoblast cell line HTR8 cells were stimulated with various concentrations of iE-DAP for various periods of time. NOD1 expression was detected by immunofluorescence, and the changes in NOD1 and RICK mRNA and protein in H8 cells were determined by real-time polymerase chain reaction and Western blot analysis. The concentrations of interleukin (IL)-8 and IL-6 secreted by H8 cells were examined by enzyme-linked immunosorbent assay. NF-κB transcription activity and P65 expression were detected by electrophoretic mobility shift assay and Western blot analysis. H8 cells expressed NOD1, and the effects of iE-DAP on NOD1 were dose- and time-dependent. The concentration of IL-8 increased gradually with increasing concentration of iE-DAP, and the levels of IL-8 and IL-6 were associated with the duration of exposure to iE-DAP. The dose of iE-DAP was significantly associated with expression of RICK and P65, and stimulation of H8 cells by iE-DAP altered NF-κB transcription activity. NOD1 may have a role in mediating infection-associated inflammation. Once iE-DAP is recognized by NOD1, the inflammatory response may be induced via NOD1-RICK-NF-κB-mediated pathways. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  13. Recombinant Brucella abortus gene expressing immunogenic protein

    SciTech Connect

    Mayfield, J.E.; Tabatabai, L.B.

    1991-06-11

    This patent describes a synthetic recombinant DNA molecule containing a DNA sequence. It comprises a gene of Brucella abortus encoding an immunogenic protein having a molecular weight of approximately 31,000 daltons as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis under denaturing conditions, the protein having an isoelectric point around 4.9, and containing a twenty-five amino acid sequence from its amino terminal end consisting of Gln-Ala-Pro-Thr-Phe-Phe-Arg-Ile-Gly-Thr-Gly-Gly-Thr-Ala-Gly-Thr-Tyr-Tyr-Pro-Ile-Gly-Gly-Leu-Ile-Ala, wherein Gln, Ala, Pro, Thr, Phe, Arg, Ile, Gly, Tyr, and Leu, respectively, represent glutamine, alanine, proline, threonine, phenylalanine, arginine, isolecuine, glycine, tyrosine, and leucine.

  14. Patterns of fluorescent protein expression in Scleractinian corals.

    PubMed

    Gruber, David F; Kao, Hung-Teh; Janoschka, Stephen; Tsai, Julia; Pieribone, Vincent A

    2008-10-01

    Biofluorescence exists in only a few classes of organisms, with Anthozoa possessing the majority of species known to express fluorescent proteins. Most species within the Anthozoan subgroup Scleractinia (reef-building corals) not only express green fluorescent proteins, they also localize the proteins in distinct anatomical patterns.We examined the distribution of biofluorescence in 33 coral species, representing 8 families, from study sites on Australia's Great Barrier Reef. For 28 of these species, we report the presence of biofluorescence for the first time. The dominant fluorescent emissions observed were green (480-520 nm) and red (580-600 nm). Fluorescent proteins were expressed in three distinct patterns (highlighted, uniform, and complementary) among specific anatomical structures of corals across a variety of families. We report no significant overlap between the distribution of fluorescent proteins and the distribution of zooxanthellae. Analysis of the patterns of fluorescent protein distribution provides evidence that the scheme in which fluorescent proteins are distributed among the anatomical structures of corals is nonrandom. This targeted expression of fluorescent proteins in corals produces contrast and may function as a signaling mechanism to organisms with sensitivity to specific wavelengths of light.

  15. A Statistical Study on Oscillatory Protein Expression

    NASA Astrophysics Data System (ADS)

    Yan, Shiwei

    Motivated by the experiments on the dynamics of a common network motif, p53 and Mdm2 feedback loop, by Lahav et al. [Nat. Genet 36, 147(2004)] in individual cells and Lev Bar-or et al. [Proc. Natl. Acad. Sci. USA 97, 11250(2000)] at the population of cells, we propose a statistical signal-response model with aiming to describe the different oscillatory behaviors for the activities of p53 and Mdm2 proteins both in individual and in population of cells in a unified way. At the cellular level, the activities of p53 and Mdm2 proteins are described by a group of nonlinear dynamical equations where the damage-derived signal is assumed to have the form with abrupt transition (”on” leftrightarrow ”off”) as soon as signal strength passes forth and back across a threshold. Each cell responses to the damage with different time duration within which the oscillations persist. For the case of population of cells, the activities of p53 and Mdm2 proteins will be the population average of the individual cells, which results damped oscillations, due to the averaging over the cell population with the different response time.

  16. Fibronectin-Binding Protein of Borrelia hermsii Expressed in the Blood of Mice with Relapsing Fever

    PubMed Central

    Lewis, Eric R. G.; Marcsisin, Renee A.; Campeau Miller, Shelley A.; Hue, Fong; Phillips, April; AuCoin, David P.

    2014-01-01

    To identify and characterize surface proteins expressed by the relapsing fever (RF) agent Borrelia hermsii in the blood of infected mice, we used a cell-free filtrate of their blood to immunize congenic naive mice. The resultant antiserum was used for Western blotting of cell lysates, and gel slices corresponding to reactive bands were subjected to liquid chromatography-tandem mass spectrometry, followed by a search of the proteome database with the peptides. One of the immunogens was identified as the BHA007 protein, which is encoded by a 174-kb linear plasmid. BHA007 had sequence features of lipoproteins, was surface exposed by the criteria of in situ protease susceptibility and agglutination of Vtp− cells by anti-BHA007 antibodies, and was not essential for in vitro growth. BHA007 elicited antibodies during experimental infection of mice, but immunization with recombinant protein did not confer protection against needle-delivered infection. Open reading frames (ORFs) orthologous to BHA007 were found on large plasmids of other RF species, including the coding sequences for the CihC proteins of Borrelia duttonii and B. recurrentis, but not in Lyme disease Borrelia species. Recombinant BHA007 bound both human and bovine fibronectin with Kd (dissociation constant) values of 22 and 33 nM, respectively, and bound to C4-binding protein with less affinity. The distant homology of BHA007 and its orthologs to BBK32 proteins of Lyme disease species, as well as to previously described BBK32-like proteins in relapsing fever species, indicates that BHA007 is a member of a large family of multifunctional proteins in Borrelia species that bind to fibronectin as well as other host proteins. PMID:24686059

  17. Fibronectin-binding protein of Borrelia hermsii expressed in the blood of mice with relapsing fever.

    PubMed

    Lewis, Eric R G; Marcsisin, Renee A; Campeau Miller, Shelley A; Hue, Fong; Phillips, April; Aucoin, David P; Barbour, Alan G

    2014-06-01

    To identify and characterize surface proteins expressed by the relapsing fever (RF) agent Borrelia hermsii in the blood of infected mice, we used a cell-free filtrate of their blood to immunize congenic naive mice. The resultant antiserum was used for Western blotting of cell lysates, and gel slices corresponding to reactive bands were subjected to liquid chromatography-tandem mass spectrometry, followed by a search of the proteome database with the peptides. One of the immunogens was identified as the BHA007 protein, which is encoded by a 174-kb linear plasmid. BHA007 had sequence features of lipoproteins, was surface exposed by the criteria of in situ protease susceptibility and agglutination of Vtp(-) cells by anti-BHA007 antibodies, and was not essential for in vitro growth. BHA007 elicited antibodies during experimental infection of mice, but immunization with recombinant protein did not confer protection against needle-delivered infection. Open reading frames (ORFs) orthologous to BHA007 were found on large plasmids of other RF species, including the coding sequences for the CihC proteins of Borrelia duttonii and B. recurrentis, but not in Lyme disease Borrelia species. Recombinant BHA007 bound both human and bovine fibronectin with Kd (dissociation constant) values of 22 and 33 nM, respectively, and bound to C4-binding protein with less affinity. The distant homology of BHA007 and its orthologs to BBK32 proteins of Lyme disease species, as well as to previously described BBK32-like proteins in relapsing fever species, indicates that BHA007 is a member of a large family of multifunctional proteins in Borrelia species that bind to fibronectin as well as other host proteins.

  18. Expression and Purification of Mini G Proteins from Escherichia coli.

    PubMed

    Carpenter, Byron; Tate, Christopher G

    2017-04-20

    Heterotrimeric G proteins modulate intracellular signalling by transducing information from cell surface G protein-coupled receptors (GPCRs) to cytoplasmic effector proteins. Structural and functional characterisation of GPCR-G protein complexes is important to fully decipher the mechanism of signal transduction. However, native G proteins are unstable and conformationally dynamic when coupled to a receptor. We therefore developed an engineered minimal G protein, mini-Gs, which formed a stable complex with GPCRs, and facilitated the crystallisation and structure determination of the human adenosine A2A receptor (A2AR) in its active conformation. Mini G proteins are potentially useful tools in a variety of applications, including characterising GPCR pharmacology, binding affinity and kinetic experiments, agonist drug discovery, and structure determination of GPCR-G protein complexes. Here, we describe a detailed protocol for the expression and purification of mini-Gs.

  19. Clostridium perfringens enterotoxin elicits rapid and specific cytolysis of breast carcinoma cells mediated through tight junction proteins claudin 3 and 4.

    PubMed

    Kominsky, Scott L; Vali, Mustafa; Korz, Dorian; Gabig, Theodore G; Weitzman, Sigmund A; Argani, Pedram; Sukumar, Saraswati

    2004-05-01

    Clostridium perfringens enterotoxin (CPE) induces cytolysis very rapidly through binding to its receptors, the tight junction proteins CLDN 3 and 4. In this study, we investigated CLDN 3 and 4 expression in breast cancer and tested the potential of CPE-mediated therapy. CLDN 3 and 4 proteins were detected in all primary breast carcinomas tested (n = 21) and, compared to normal mammary epithelium, were overexpressed in approximately 62% and 26%, respectively. Treatment of breast cancer cell lines in culture with CPE resulted in rapid and dose-dependent cytolysis exclusively in cells that expressed CLDN 3 and 4. Intratumoral CPE treatment of xenografts of T47D breast cancer cells in immunodeficient mice resulted in a significant reduction in tumor volume (P = 0.007), with accompanying necrosis. Necrotic reactions were also seen in three freshly resected primary breast carcinoma samples treated with CPE for 12 hours, while isolated primary breast carcinoma cells underwent rapid and complete cytolysis within 1 hour. Thus, expression of CLDN 3 and 4 sensitizes primary breast carcinomas to CPE-mediated cytolysis and emphasizes the potential of CPE in breast cancer therapy.

  20. Clostridium perfringens Enterotoxin Elicits Rapid and Specific Cytolysis of Breast Carcinoma Cells Mediated through Tight Junction Proteins Claudin 3 and 4

    PubMed Central

    Kominsky, Scott L.; Vali, Mustafa; Korz, Dorian; Gabig, Theodore G.; Weitzman, Sigmund A.; Argani, Pedram; Sukumar, Saraswati

    2004-01-01

    Clostridium perfringens enterotoxin (CPE) induces cytolysis very rapidly through binding to its receptors, the tight junction proteins CLDN 3 and 4. In this study, we investigated CLDN 3 and 4 expression in breast cancer and tested the potential of CPE-mediated therapy. CLDN 3 and 4 proteins were detected in all primary breast carcinomas tested (n = 21) and, compared to normal mammary epithelium, were overexpressed in approximately 62% and 26%, respectively. Treatment of breast cancer cell lines in culture with CPE resulted in rapid and dose-dependent cytolysis exclusively in cells that expressed CLDN 3 and 4. Intratumoral CPE treatment of xenografts of T47D breast cancer cells in immunodeficient mice resulted in a significant reduction in tumor volume (P = 0.007), with accompanying necrosis. Necrotic reactions were also seen in three freshly resected primary breast carcinoma samples treated with CPE for 12 hours, while isolated primary breast carcinoma cells underwent rapid and complete cytolysis within 1 hour. Thus, expression of CLDN 3 and 4 sensitizes primary breast carcinomas to CPE-mediated cytolysis and emphasizes the potential of CPE in breast cancer therapy. PMID:15111309

  1. Enhanced membrane protein expression by engineering increased intracellular membrane production

    PubMed Central

    2013-01-01

    Background Membrane protein research is frequently hampered by the low natural abundance of these proteins in cells and typically relies on recombinant gene expression. Different expression systems, like mammalian cells, insect cells, bacteria and yeast are being used, but very few research efforts have been directed towards specific host cell customization for enhanced expression of membrane proteins. Here we show that by increasing the intracellular membrane production by interfering with a key enzymatic step of lipid synthesis, enhanced expression of membrane proteins in yeast is achieved. Results We engineered the oleotrophic yeast, Yarrowia lipolytica, by deleting the phosphatidic acid phosphatase, PAH1, which led to massive proliferation of endoplasmic reticulum (ER) membranes. For all eight tested representatives of different integral membrane protein families, we obtained enhanced protein accumulation levels and in some cases enhanced proteolytic integrity in the ∆pah1 strain. We analysed the adenosine A2AR G-protein coupled receptor case in more detail and found that concomitant induction of the unfolded protein response in the ∆pah1 strain enhanced the specific ligand binding activity of the receptor. These data indicate an improved quality control mechanism for membrane proteins accumulating in yeast cells with proliferated ER. Conclusions We conclude that redirecting the metabolic flux of fatty acids away from triacylglycerol- and sterylester-storage towards membrane phospholipid synthesis by PAH1 gene inactivation, provides a valuable approach to enhance eukaryotic membrane protein production. Complementary to this improvement in membrane protein quantity, UPR co-induction further enhances the quality of the membrane protein in terms of its proper folding and biological activity. Importantly, since these pathways are conserved in all eukaryotes, it will be of interest to investigate similar engineering approaches in other cell types of

  2. Climbazole increases expression of cornified envelope proteins in primary keratinocytes.

    PubMed

    Pople, J E; Moore, A E; Talbot, D C S; Barrett, K E; Jones, D A; Lim, F L

    2014-10-01

    Dandruff is a troubling consumer problem characterized by flaking and pruritus of the scalp and is considered a multifactorial condition with sebum, individual susceptibility and the fungus Malassezia all thought to play a part. The condition is commonly treated with shampoo products containing antifungal ingredients such as zinc pyrithione and climbazole. It is hypothesized that these ingredients may be delivering additional scalp skin benefits besides their antifungal activity helping to relieve dandruff effectively. The objective of this study was to evaluate the anti-dandruff ingredient climbazole for potential skin benefits using genomics and in vitro assays. Microarray analysis was performed to profile gene expression changes in climbazole-treated primary human keratinocyte cells. Results were independently validated using qPCR and analysis of protein expression using ELISA and immunocytochemistry. Microarray analysis of climbazole-treated keratinocytes showed statistically significant expression changes in genes associated with the gene ontology groups encompassing epidermal differentiation, keratinization, cholesterol biosynthesis and immune response. Upregulated genes included a number encoding cornified envelope proteins such as group 3 late-cornified envelope proteins, LCE3 and group 2 small-proline-rich proteins, SPRR2. Protein analysis studies of climbazole-treated primary keratinocytes using ELISA and immunocytochemistry were able to demonstrate that the increase in gene transcripts translated into increased protein expression of these cornified envelope markers. Climbazole treatment of primary keratinocytes results in an upregulation in expression of a number of genes including those encoding proteins involved in cornified envelope formation with further studies demonstrating this did translate into increased protein expression. A climbazole-driven increase in cornified envelope proteins may improve the scalp skin barrier, which is known to be weaker

  3. Expression patterns and binding properties of three pheromone binding proteins in the diamondback moth, Plutella xyllotella.

    PubMed

    Sun, Mengjing; Liu, Yang; Wang, Guirong

    2013-01-01

    Pheromone binding proteins (PBPs) play a key role in transporting hydrophobic sex pheromone components emitted by con-specific female across aqueous sensillar lymph to the surface of olfactory receptor neurons. A number of PBPs have been cloned, however, details of their function are still largely unknown. Here three pheromone binding protein genes in the diamondback moth, Plutella xyllotella were cloned. The three PxylPBP genes are not only expressed in chemosensory tissues but also expressed in female reproductive organs and male legs. To better understand the functions of PxylPBPs in the initial steps of pheromone recognition, three PxylPBPs were expressed in Escherichia coli and the ligand-binding specificities of purified recombinant PBPs were investigated. Fluorescence binding assays indicate that three PxylPBPs not only robustly bound all four sex pheromone components but also significantly bound pheromone analogs with at least one double bond, while weakly bound tested plant volatiles. Although pheromone analogs bound PBPs, they could not elicit the moth's electrophysiological response. These experiments provide evidence that PxylPBPs have limited selectivity of pheromone components and analogs and some downstream components such as odor receptors might be involved in selectivity and specificity of pheromone perception in P. xyllotella.

  4. Expression of the major outer membrane protein of Chlamydia trachomatis in Escherichia coli.

    PubMed Central

    Manning, D S; Stewart, S J

    1993-01-01

    The major outer membrane protein (MOMP) of Chlamydia trachomatis was expressed in Escherichia coli. To assess whether it assembled into a conformationally correct structure at the cell surface, we characterized the recombinant MOMP (rMOMP) by Western immunoblot analysis, indirect immunofluorescence, and immunoprecipitation with monoclonal antibodies (MAbs) that recognize contiguous and conformational MOMP epitopes. Western blot analysis showed that most of the rMOMP comigrated with authentic monomer MOMP, indicating that its signal peptide was recognized and cleaved by E. coli. The rMOMP could not be detected on the cell surface of viable or formalin-killed E. coli organisms by indirect immunofluorescence staining with a MAb specific for a MOMP contiguous epitope. In contrast, the same MAb readily stained rMOMP-expressing E. coli cells that had been permeabilized by methanol fixation. A MAb that recognizes a conformational MOMP epitope and reacted strongly with formalin- or methanol-fixed elementary bodies failed to stain formalin- or methanol-fixed E. coli expressing rMOMP. Moreover, this MAb did not immunoprecipitate rMOMP from expressing E. coli cells even though it precipitated the authentic protein from lysates of C. trachomatis elementary bodies. Therefore we concluded that rMOMP was not localized to the E. coli cell surface and was not recognizable by a conformation-dependent antibody. These results indicate that rMOMP expressed by E. coli is unlikely to serve as an accurate model of MOMP structure and function. They also question the utility of rMOMP as a source of immunogen for eliciting neutralizing antibodies against conformational antigenic sites of the protein. Images PMID:8406797

  5. Mucosal vaccination with a codon-optimized hemagglutinin gene expressed by attenuated Salmonella elicits a protective immune response in chickens against highly pathogenic avian influenza.

    PubMed

    Liljebjelke, Karen A; Petkov, Daniel I; Kapczynski, Darrell R

    2010-06-17

    The purpose of this study was to evaluate clinical protection from challenge conferred by two attenuated Salmonella enteria serovar typhimurium vaccine strains expressing the hemagglutinin (HA1) gene from a highly pathogenic avian influenza (HPAI) H5N1 (A/whooper swan/Mongolia/3/2005), under control of the anaerobically inducible nir15 promoter. Two-week-old White Leghorn chickens were immunized by oral gavage with one milliliter doses of >109 Salmonella colony-forming units once weekly for 4 weeks prior to challenge. Expression of recombinant protein was confirmed via Western blot. Serum and mucosal gavage samples were collected prior to, and following immunization and antibodies against avian influenza HA were confirmed by Western blot and hemagglutination-inhibition (HI) assay. Chickens were challenged with homologous (A/whooper swan/Mongolia/3/2005), or heterologous (A/Chicken/Queretaro/14588-19/95) HPAI virus strains. Chickens immunized with attenuated Salmonella strains containing plasmid expression vector (pTETnir15HA) demonstrated a statistically significant increase in survival compared to control groups. Results provide evidence of effectiveness of attenuated Salmonella strains for delivery of recombinant avian influenza HA antigens and induction of mucosal and systemic immune responses protective against lethal challenge with HPAI.

  6. Proteomic analysis of Clostridium thermocellum core metabolism: relative protein expression profiles and growth phase-dependent changes in protein expression

    PubMed Central

    2012-01-01

    Background Clostridium thermocellum produces H2 and ethanol, as well as CO2, acetate, formate, and lactate, directly from cellulosic biomass. It is therefore an attractive model for biofuel production via consolidated bioprocessing. Optimization of end-product yields and titres is crucial for making biofuel production economically feasible. Relative protein expression profiles may provide targets for metabolic engineering, while understanding changes in protein expression and metabolism in response to carbon limitation, pH, and growth phase may aid in reactor optimization. We performed shotgun 2D-HPLC-MS/MS on closed-batch cellobiose-grown exponential phase C. thermocellum cell-free extracts to determine relative protein expression profiles of core metabolic proteins involved carbohydrate utilization, energy conservation, and end-product synthesis. iTRAQ (isobaric tag for relative and absolute quantitation) based protein quantitation was used to determine changes in core metabolic proteins in response to growth phase. Results Relative abundance profiles revealed differential levels of putative enzymes capable of catalyzing parallel pathways. The majority of proteins involved in pyruvate catabolism and end-product synthesis were detected with high abundance, with the exception of aldehyde dehydrogenase, ferredoxin-dependent Ech-type [NiFe]-hydrogenase, and RNF-type NADH:ferredoxin oxidoreductase. Using 4-plex 2D-HPLC-MS/MS, 24% of the 144 core metabolism proteins detected demonstrated moderate changes in expression during transition from exponential to stationary phase. Notably, proteins involved in pyruvate synthesis decreased in stationary phase, whereas proteins involved in glycogen metabolism, pyruvate catabolism, and end-product synthesis increased in stationary phase. Several proteins that may directly dictate end-product synthesis patterns, including pyruvate:ferredoxin oxidoreductases, alcohol dehydrogenases, and a putative bifurcating hydrogenase

  7. Recombinant protein expression in Escherichia coli: advances and challenges

    PubMed Central

    Rosano, Germán L.; Ceccarelli, Eduardo A.

    2014-01-01

    Escherichia coli is one of the organisms of choice for the production of recombinant proteins. Its use as a cell factory is well-established and it has become the most popular expression platform. For this reason, there are many molecular tools and protocols at hand for the high-level production of heterologous proteins, such as a vast catalog of expression plasmids, a great number of engineered strains and many cultivation strategies. We review the different approaches for the synthesis of recombinant proteins in E. coli and discuss recent progress in this ever-growing field. PMID:24860555

  8. Expression of rabies virus G protein in carrots (Daucus carota).

    PubMed

    Rojas-Anaya, Edith; Loza-Rubio, Elizabeth; Olivera-Flores, Maria Teresa; Gomez-Lim, Miguel

    2009-12-01

    Antigens derived from various pathogens can readily be synthesized at high levels in plants in their authentic forms. Such antigens administered orally can induce an immune response and, in some cases, result in protection against a subsequent challenge. We here report the expression of rabies virus G protein into carrots. The G gene was subcloned into the pUCpSSrabG vector and then used to transform carrot embryogenic cells by particle bombardment. The carrot cells were selected in liquid medium, a method previously unreported. The presence of the transgene was verified by PCR, and by RT-PCR. By western blot, G protein transgene was identified in 93.3% of adult carrot roots. The G protein was quantified by densitometric analysis (range 0.4-1.2%). The expressed protein was antigenic in mice. This confirms that the carrot is an adequate system for antigen expression.

  9. Vectors for the expression of tagged proteins in Drosophila.

    PubMed

    Parker, L; Gross, S; Alphey, L

    2001-12-01

    Regulated expression systems have been extremely useful in developmental studies, allowing the expression of specific proteins in defined spatial and temporal patterns. If these proteins are fused to an appropriate molecular tag, then they can be purified or visualized without the need to raise specific antibodies. If the tag is inherently fluorescent, then the proteins can even be visualized directly, in living tissue. We have constructed a series of P element-based transformation vectors for the most widely used expression system in Drosophila, GAL4/UAS. These vectors provide a series of useful tags for antibody detection, protein purification, and/or direct visualization, together with a convenient multiple cloning site into which the cDNA of interest can be inserted.

  10. Microfluidic chips for protein differential expression profiling.

    PubMed

    Armenta, Jenny M; Dawoud, Abdulilah A; Lazar, Iulia M

    2009-04-01

    Biomarker discovery and screening using novel proteomic technologies is an area that is attracting increased attention in the biomedical community. Early detection of abnormal physiological conditions will be highly beneficial for diagnosing various diseases and increasing survivability rates. Clearly, progress in this area will depend on the development of fast, reliable, and highly sensitive and specific sample bioanalysis methods. Microfluidics has emerged as a technology that could become essential in proteomics research as it enables the integration of all sample preparation, separation, and detection steps, with the added benefit of enhanced sample throughput. The combination of these advantages with the sensitivity and capability of MS detection to deliver precise structural information makes microfluidics-MS a very competitive technology for biomarker discovery. The integration of LC microchip devices with MS detection, and specifically their applicability to biomarker screening applications in MCF-7 breast cancer cellular extracts is reported in this manuscript. Loading approximately 0.1-1 microg of crude protein extract tryptic digest on the chip has typically resulted in the reliable identification of approximately 40-100 proteins. The potential of an LC-ESI-MS chip for comparative proteomic analysis of isotopically labeled MCF-7 breast cancer cell extracts is explored for the first time.

  11. Variation in Protein Intake Induces Variation in Spider Silk Expression

    PubMed Central

    Blamires, Sean J.; Wu, Chun-Lin; Tso, I-Min

    2012-01-01

    Background It is energetically expensive to synthesize certain amino acids. The proteins (spidroins) of spider major ampullate (MA) silk, MaSp1 and MaSp2, differ in amino acid composition. Glutamine and proline are prevalent in MaSp2 and are expensive to synthesize. Since most orb web spiders express high proline silk they might preferentially attain the amino acids needed for silk from food and shift toward expressing more MaSp1 in their MA silk when starved. Methodology/Principal Findings We fed three spiders; Argiope aetherea, Cyrtophora moluccensis and Leucauge blanda, high protein, low protein or no protein solutions. A. aetherea and L. blanda MA silks are high in proline, while C. moluccesnsis MA silks are low in proline. After 10 days of feeding we determined the amino acid compositions and mechanical properties of each species' MA silk and compared them between species and treatments with pre-treatment samples, accounting for ancestry. We found that the proline and glutamine of A. aetherea and L. blanda silks were affected by protein intake; significantly decreasing under the low and no protein intake treatments. Glutmaine composition in C. moluccensis silk was likewise affected by protein intake. However, the composition of proline in their MA silk was not significantly affected by protein intake. Conclusions Our results suggest that protein limitation induces a shift toward different silk proteins with lower glutamine and/or proline content. Contradictions to the MaSp model lie in the findings that C. moluccensis MA silks did not experience a significant reduction in proline and A. aetherea did not experience a significant reduction in serine on low/no protein. The mechanical properties of the silks could not be explained by a MaSp1 expressional shift. Factors other than MaSp expression, such as the expression of spidroin-like orthologues, may impact on silk amino acid composition and spinning and glandular processes may impact mechanics. PMID:22363691

  12. Low activity of LSD1 elicits a pro-inflammatory gene expression profile in riboflavin-deficient human T Lymphoma Jurkat cells.

    PubMed

    Liu, Dandan; Zempleni, Janos

    2014-09-01

    Mono- and dimethylation of lysine (K)-4 in histone H3 (H3K4me1, H3K4me2) create epigenetic gene activation marks that are enriched near the transcription start site of genes. Lysine-specific demethylase 1 (LSD1) is a flavin adenine dinucleotide (FAD)-dependent demethylase that catalyzes the demethylation of H3K4me1 and H3K4me2, thereby mediating gene repression. This study tested the hypothesis that LSD1 activity depends on the concentrations of the FAD precursor, riboflavin, in cell culture media, and that riboflavin deficiency causes derepression of pro-inflammatory cytokines. Human T lymphoma Jurkat cells were cultured in riboflavin-defined media, representing plasma levels of riboflavin in moderately deficient, sufficient, and supplemented humans. The expression of LSD1 mRNA and protein followed the pattern riboflavin-deficient > riboflavin-sufficient > riboflavin-supplemented cells. However, the increase in LSD1 expression was insufficient to compensate for FAD depletion, and LSD activities were more than 30 % higher in riboflavin-supplemented cells compared with the other treatment groups. The enrichment of H3K4me2 marks was 11-137 % greater in riboflavin-deficient cells compared with sufficient cells in exon 1 of genes coding for the pro-inflammatory cytokines interleukin (IL)-1α, IL-1β, IL-6, and tumor necrosis factor-α. Consistent with the enrichment of gene activation marks, the expression of mRNA coding for pro-inflammatory cytokines was 62-487 % higher in riboflavin-deficient cells compared with sufficient cells. These findings support the hypothesis that riboflavin deficiency contributes toward a pro-inflammatory gene expression pattern through a loss of LSD1 activity.

  13. Differential Protein Expression in Congenital and Acquired Cholesteatomas

    PubMed Central

    Kim, Sung Huhn; Choi, Jae Young

    2015-01-01

    Congenital cholesteatomas are epithelial lesions that present as an epithelial pearl behind an intact eardrum. Congenital and acquired cholesteatomas progress quite differently from each other and progress patterns can provide clues about the unique origin and pathogenesis of the abnormality. However, the exact pathogenic mechanisms by which cholesteatomas develop remain unknown. In this study, key proteins that directly affect cholesteatoma pathogenesis are investigated with proteomics and immunohistochemistry. Congenital cholesteatoma matrices and retroauricular skin were harvested during surgery in 4 patients diagnosed with a congenital cholesteatoma. Tissue was also harvested from the retraction pocket in an additional 2 patients during middle ear surgery. We performed 2-dimensional (2D) electrophoresis to detect and analyze spots that are expressed only in congenital cholesteatoma and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/MS) to separate proteins by molecular weight. Protein expression was confirmed by immunohistochemical staining. The image analysis of 2D electrophoresis showed that 4 congenital cholesteatoma samples had very similar protein expression patterns and that 127 spots were exclusively expressed in congenital cholesteatomas. Of these 127 spots, 10 major spots revealed the presence of titin, forkhead transcription activator homolog (FKH 5–3), plectin 1, keratin 10, and leucine zipper protein 5 by MALDI-TOF/MS analysis. Immunohistochemical staining showed that FKH 5–3 and titin were expressed in congenital cholesteatoma matrices, but not in acquired cholesteatomas. Our study shows that protein expression patterns are completely different in congenital cholesteatomas, acquired cholesteatomas, and skin. Moreover, non-epithelial proteins, including FKH 5–3 and titin, were unexpectedly expressed in congenital cholesteatoma tissue. Our data indicates that congenital cholesteatoma origins may differ

  14. Borrelia burgdorferi elicited-IL-10 suppresses the production of inflammatory mediators, phagocytosis, and expression of co-stimulatory receptors by murine macrophages and/or dendritic cells.

    PubMed

    Chung, Yutein; Zhang, Nan; Wooten, R Mark

    2013-01-01

    Borrelia burgdorferi (Bb) is a tick-borne spirochete that is the causative agent for Lyme disease. Our previous studies indicate that virulent Bb can potently enhance IL-10 production by macrophages (MØs) and that blocking IL-10 production significantly enhances bacterial clearance. We hypothesize that skin-associated APC types, such as MØs and dendritic cells (DCs) are potent producers of IL-10 in response to Bb, which may act in autocrine fashion to suppress APC responses critical for efficient Bb clearance. Our goal is to delineate which APC immune functions are dysregulated by Bb-elicited IL-10 using a murine model of Lyme disease. Our in vitro studies indicated that both APCs rapidly produce IL-10 upon exposure to Bb, that these levels inversely correlate with the production of many Lyme-relevant proinflammatory cytokines and chemokines, and that APCs derived from IL-10(-/-) mice produced greater amounts of these proinflammatory mediators than wild-type APCs. Phagocytosis assays determined that Bb-elicited IL-10 levels can diminish Bb uptake and trafficking by MØs, suppresses ROS production, but does not affect NO production; Bb-elicited IL-10 had little effect on phagocytosis, ROS, and NO production by DCs. In general, Bb exposure caused little-to-no upregulation of several critical surface co-stimulatory markers by MØs and DCs, however eliminating Bb-elicited IL-10 allowed a significant upregulation in many of these co-stimulatory receptors. These data indicate that IL-10 elicited from Bb-stimulated MØs and DCs results in decreased production of proinflammatory mediators and co-stimulatory molecules, and suppress phagocytosis-associated events that are important for mediating both innate and adaptive immune responses by APCs.

  15. Putative bacterial volatile-mediated growth in soybean (Glycine max L. Merrill) and expression of induced proteins under salt stress.

    PubMed

    Vaishnav, A; Kumari, S; Jain, S; Varma, A; Choudhary, D K

    2015-08-01

    Plant root-associated rhizobacteria elicit plant immunity referred to as induced systemic tolerance (IST) against multiple abiotic stresses. Among multibacterial determinants involved in IST, the induction of IST and promotion of growth by putative bacterial volatile compounds (VOCs) is reported in the present study. To characterize plant proteins induced by putative bacterial VOCs, proteomic analysis was performed by MALDI-MS/MS after exposure of soybean seedlings to a new strain of plant growth promoting rhizobacteria (PGPR) Pseudomonas simiae strain AU. Furthermore, expression analysis by Western blotting confirmed that the vegetative storage protein (VSP), gamma-glutamyl hydrolase (GGH) and RuBisCo large chain proteins were significantly up-regulated by the exposure to AU strain and played a major role in IST. VSP has preponderant roles in N accumulation and mobilization, acid phosphatase activity and Na(+) homeostasis to sustain plant growth under stress condition. More interestingly, plant exposure to the bacterial strain significantly reduced Na(+) and enhanced K(+) and P content in root of soybean seedlings under salt stress. In addition, high accumulation of proline and chlorophyll content also provided evidence of protection against osmotic stress during the elicitation of IST by bacterial exposure. The present study reported for the first time that Ps. simiae produces a putative volatile blend that can enhance soybean seedling growth and elicit IST against 100 mmol l(-1) NaCl stress condition. The identification of such differentially expressed proteins provide new targets for future studies that will allow assessment of their physiological roles and significance in the response of glycophytes to stresses. Further work should uncover more about the chemical side of VOC compounds and a detailed study about their molecular mechanism responsible for plant growth. © 2015 The Society for Applied Microbiology.

  16. Cloning, expression and characterization of recombinant sweet-protein thaumatin II using the methylotrophic yeast Pichia pastoris.

    PubMed

    Masuda, Tetsuya; Tamaki, Shinobu; Kaneko, Ryosuke; Wada, Ritsuko; Fujita, Yuki; Mehta, Alka; Kitabatake, Naofumi

    2004-03-30

    Thaumatin, an intensely sweet-tasting protein, was secreted by the methylotrophic yeast Pichia pastoris. The mature thaumatin II gene was directly cloned from Taq polymerase-amplified PCR products by using TA cloning methods and fused the pPIC9K expression vector that contains Saccharomyces cerevisiae prepro alpha-mating factor secretion signal. Several additional amino acid residues were introduced at both the N- and C-terminal ends by genetic modification to investigate the role of the terminal end region for elicitation of sweetness in the thaumatin molecule. The secondary and tertiary structures of purified recombinant thaumatin were almost identical to those of the plant thaumatin molecule. Recombinant thaumatin II elicited a sweet taste as native plant thaumatin II; its threshold value of sweetness to humans was around 50 nM, which is the same as that of plant thaumatin II. These results demonstrate that the functional expression of thaumatin II was attained by Pichia pastoris systems and that the N- and C-terminal regions of the thaumatin II molecule do not -play an important role in eliciting the sweet taste of thaumatin. Copyright 2004 Wiley Periodicals, Inc.

  17. Differential protein expression in Phalaenopsis under low temperature.

    PubMed

    Yuan, Xiu-Yun; Liang, Fang; Jiang, Su-Hua; Wan, Mo-Fei; Ma, Jie; Zhang, Xian-Yun; Cui, Bo

    2015-01-01

    A comparative proteomic analysis was carried out to explore the molecular mechanisms of responses to cold stress in Phalaenopsis after treated by low temperature (13/8 °C day/night) for 15 days. Differentially expressed proteins were examined using two-dimensional electrophoresis (2-DE) and matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-TOF/MS). Among 85 differentially expressed proteins, 73 distinct proteins were identified. Comparative analysis revealed that the identified proteins mainly participate in photosynthesis, protein synthesis, folding and degradation, respiration, defense response, amino acid metabolism, energy pathway, cytoskeleton, transcription regulation, signal transduction, and seed storage protein, while the functional classification of the remaining four proteins was not determined. These data suggested that the proteins might work cooperatively to establish a new homeostasis under cold stress; 37 % of the identified cold-responsive proteins were associated with various aspects of chloroplast physiology, and 56 % of them were predicted to be located in the chloroplasts, implying that the cold stress tolerance of Phalaenopsis was achieved, at least partly, by regulation of chloroplast function. Moreover, the protein destination control, which was mediated by chaperones and proteases, plays an important role in tolerance to cold stress.

  18. Global Analysis of Protein Expression of Inner Ear Hair Cells.

    PubMed

    Hickox, Ann E; Wong, Ann C Y; Pak, Kwang; Strojny, Chelsee; Ramirez, Miguel; Yates, John R; Ryan, Allen F; Savas, Jeffrey N

    2017-02-01

    The mammalian inner ear (IE) subserves auditory and vestibular sensations via highly specialized cells and proteins. Sensory receptor hair cells (HCs) are necessary for transducing mechanical inputs and stimulating sensory neurons by using a host of known and as yet unknown protein machinery. To understand the protein composition of these unique postmitotic cells, in which irreversible protein degradation or damage can lead to impaired hearing and balance, we analyzed IE samples by tandem mass spectrometry to generate an unbiased, shotgun-proteomics view of protein identities and abundances. By using Pou4f3/eGFP-transgenic mice in which HCs express GFP driven by Pou4f3, we FACS purified a population of HCs to analyze and compare the HC proteome with other IE subproteomes from sensory epithelia and whole IE. We show that the mammalian HC proteome comprises hundreds of uniquely or highly expressed proteins. Our global proteomic analysis of purified HCs extends the existing HC transcriptome, revealing previously undetected gene products and isoform-specific protein expression. Comparison of our proteomic data with mouse and human databases of genetic auditory/vestibular impairments confirms the critical role of the HC proteome for normal IE function, providing a cell-specific pool of candidates for novel, important HC genes. Several proteins identified exclusively in HCs by proteomics and verified by immunohistochemistry map to human genetic deafness loci, potentially representing new deafness genes.

  19. Prolonged morphine administration alters protein expression in the rat myocardium

    PubMed Central

    2011-01-01

    Background Morphine is used in clinical practice as a highly effective painkiller as well as the drug of choice for treatment of certain heart diseases. However, there is lack of information about its effect on protein expression in the heart. Therefore, here we aimed to identify the presumed alterations in rat myocardial protein levels after prolonged morphine treatment. Methods Morphine was administered to adult male Wistar rats in high doses (10 mg/kg per day) for 10 days. Proteins from the plasma membrane- and mitochondria-enriched fractions or cytosolic proteins isolated from left ventricles were run on 2D gel electrophoresis, scanned and quantified with specific software to reveal differentially expressed proteins. Results Nine proteins were found to show markedly altered expression levels in samples from morphine-treaded rats and these proteins were identified by mass spectrometric analysis. They belong to different cell pathways including signaling, cytoprotective, and structural elements. Conclusions The present identification of several important myocardial proteins altered by prolonged morphine treatment points to global effects of this drug on heart tissue. These findings represent an initial step toward a more complex view on the action of morphine on the heart. PMID:22129148

  20. Opinion Elicitation in Second Life

    NASA Astrophysics Data System (ADS)

    van Vliet, Marijn; Neviarouskaya, Alena; Prendinger, Helmut

    The paper describes a novel method for opinion elicitation, which is based on the popular 3D online world of “Second Life”. Here people, as avatars, are put into a somewhat realistic context related to the topic for which opinions are sought. We hypothesize that this kind of concrete, interactive context supports the evocation of opinions better than non-context methods, e.g. only showing related images. To confirm our hypothesis, we conducted a small pilot study, which compares the influence of static and interactive context methods on the opinions expressed by subjects. The opinion elicitation scenario in Second Life is supported by the automatic retrieval of opinions from the web. The results of a study indicate that subjects show more reasoned opinions in the interactive condition. A demo illustrating the content of this paper is available.

  1. Analysis of protein composition and protein expression in the tear fluid of patients with congenital aniridia.

    PubMed

    Ihnatko, Robert; Edén, Ulla; Lagali, Neil; Dellby, Anette; Fagerholm, Per

    2013-12-06

    Aniridia is a rare congenital genetic disorder caused by haploinsuffiency of the PAX6 gene, the master gene for development of the eye. The expression of tear proteins in aniridia is unknown. To screen for proteins involved in the aniridia pathophysiology, the tear fluid of patients with diagnosed congenital aniridia was examined using two-dimensional electrophoresis (2-DE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Two-dimensional map of tear proteins in aniridia has been established and 7 proteins were differentially expressed with P<0.01 between aniridia patients and control subjects. Five of them were more abundant in healthy subjects, particularly α-enolase, peroxiredoxin 6, cystatin S, gelsolin, apolipoprotein A-1 and two other proteins, zinc-α2-glycoprotein and lactoferrin were more expressed in the tears of aniridia patients. Moreover, immunoblot analysis revealed elevated levels of vascular endothelial growth factor (VEGF) in aniridia tears which is in concordance with clinical finding of pathological blood and lymph vessels in the central and peripheral cornea of aniridia patients. The proteins with different expression in patients' tears may be new candidate molecules involved in the pathophysiology of aniridia and thus may be helpful for development of novel treatment strategies for the symptomatic therapy of this vision threatening condition. This study is first to demonstrate protein composition and protein expression in aniridic tears and identifies proteins with different abundance in tear fluid from patients with congenital aniridia vs. healthy tears. © 2013 Elsevier B.V. All rights reserved.

  2. Enteral delivery of proteins enhances the expression of proteins involved in the cytoskeleton and protein biosynthesis in human duodenal mucosa.

    PubMed

    Goichon, Alexis; Bertrand, Julien; Chan, Philippe; Lecleire, Stéphane; Coquard, Aude; Cailleux, Anne-Françoise; Vaudry, David; Déchelotte, Pierre; Coëffier, Moïse

    2015-08-01

    Amino acids are well known to be key effectors of gut protein turnover. We recently reported that enteral delivery of proteins markedly stimulated global duodenal protein synthesis in carbohydrate-fed healthy humans, but specifically affected proteins remain unknown. We aimed to assess the influence of an enteral protein supply on the duodenal mucosal proteome in carbohydrate-fed humans. Six healthy volunteers received for 5 h, on 2 occasions and in random order, either an enteral infusion of maltodextrins alone (0.25 g · kg⁻¹ · h⁻¹) mimicking the fed state or maltodextrins with a protein powder (0.14 g proteins · kg⁻¹ · h⁻¹). Endoscopic duodenal biopsy specimens were then collected and frozen until analysis. A 2-dimensional polyacrylamide gel electrophoresis-based comparative proteomics analysis was then performed, and differentially expressed proteins (at least ±1.5-fold change; Student's t test, P < 0.05) were identified by mass spectrometry. Protein expression changes were confirmed by Western blot analysis. Thirty-two protein spots were differentially expressed after protein delivery compared with maltodextrins alone: 28 and 4 spots were up- or downregulated, respectively. Among the 22 identified proteins, 11 upregulated proteins were involved either in the cytoskeleton (ezrin, moesin, plastin 1, lamin B1, vimentin, and β-actin) or in protein biosynthesis (glutamyl-prolyl-transfer RNA synthetase, glutaminyl-transfer RNA synthetase, elongation factor 2, elongation factor 1δ, and eukaryotic translation and initiation factor 3 subunit f). Enteral delivery of proteins altered the duodenal mucosal proteome and mainly stimulated the expression of proteins involved in cytoskeleton and protein biosynthesis. These results suggest that protein supply may affect intestinal morphology by stimulating actin cytoskeleton remodeling. © 2015 American Society for Nutrition.

  3. Enhancement of protein expression by alphavirus replicons by designing self-replicating subgenomic RNAs.

    PubMed

    Kim, Dal Young; Atasheva, Svetlana; McAuley, Alexander J; Plante, Jessica A; Frolova, Elena I; Beasley, David W C; Frolov, Ilya

    2014-07-22

    Since the development of infectious cDNA clones of viral RNA genomes and the means of delivery of the in vitro-synthesized RNA into cells, alphaviruses have become an attractive system for expression of heterologous genetic information. Alphaviruses replicate exclusively in the cytoplasm, and their genetic material cannot recombine with cellular DNA. Alphavirus genome-based, self-replicating RNAs (replicons) are widely used vectors for expression of heterologous proteins. Their current design relies on replacement of structural genes, encoded by subgenomic RNAs (SG RNA), with heterologous sequences of interest. The SG RNA is transcribed from a promoter located in the alphavirus-specific RNA replication intermediate and is not further amplified. In this study, we have applied the accumulated knowledge of the mechanism of alphavirus replication and promoter structures, in particular, to increase the expression level of heterologous proteins from Venezuelan equine encephalitis virus (VEEV)-based replicons. During VEEV infection, replication enzymes are produced in excess to RNA replication intermediates, and a large fraction of them are not involved in RNA synthesis. The newly designed constructs encode SG RNAs, which are not only transcribed from the SG promoter, but are additionally amplified by the previously underused VEEV replication enzymes. These replicons produce SG RNAs and encoded proteins of interest 10- to 50-fold more efficiently than those using a traditional design. A modified replicon encoding West Nile virus (WNV) premembrane and envelope proteins efficiently produced subviral particles and, after a single immunization, elicited high titers of neutralizing antibodies, which protected mice from lethal challenge with WNV.

  4. Recent patents on alphavirus protein expression and vector production.

    PubMed

    Aranda, Alejandro; Ruiz-Guillen, Marta; Quetglas, Jose I; Bezunartea, Jaione; Casales, Erkuden; Smerdou, Cristian

    2011-12-01

    Alphaviruses contain a single-strand RNA genome that can be modified to express heterologous genes at high levels. Alphavirus vectors can be packaged within viral particles (VPs) or used as DNA/RNA layered systems. The broad tropism and high expression levels of alphavirus vectors have made them very attractive for applications like recombinant protein expression, vaccination or gene therapy. Expression mediated by alphavirus vectors is generally transient due to induction of apoptosis. However, during the last years several non-cytopathic mutations have been identified within the replicase sequence of different alphaviruses, allowing prolonged protein expression in culture cells. Some of these mutants, which have been patented, have allowed the generation of stable cell lines able to express recombinant proteins for extended periods of time in a constitutive or inducible manner. Production of alphavirus VPs usually requires cotransfection of cells with vector and helper RNAs providing viral structural proteins in trans. During this process full-length wild type (wt) genomes can be generated through recombination between different RNAs. Several new strategies to reduce wt virus generation during packaging, optimize VP production, increase packaging capacity, and provide VPs with specific targeting have been recently patented. Finally, hybrid vectors between alphavirus and other types of viruses have led to a number of patents with applications in vaccination, cancer therapy or retrovirus production.

  5. Mammary differentiation induces expression of Tristetraprolin, a tumor suppressor AU-rich mRNA-binding protein.

    PubMed

    Goddio, M Victoria; Gattelli, Albana; Slomiansky, Victoria; Lacunza, Ezequiel; Gingerich, Timothy; Tocci, Johanna M; Facchinetti, María M; Curino, Alejandro C; LaMarre, Jonathan; Abba, Martín C; Kordon, Edith C

    2012-10-01

    Tristetraprolin (TTP) is a RNA-binding protein that inhibits the expression of pro-inflammatory cytokines and invasiveness-associated genes. TTP levels are decreased in many different cancer types and it has been proposed that this protein could be used as a prognostic factor in breast cancer. Here, using publicly available DNA microarray datasets, "serial analysis of gene expression" libraries and qRT-PCR analysis, we determined that TTP mRNA is present in normal breast cells and its levels are significantly decreased in all breast cancer subtypes. In addition, by immunostaining, we found that TTP expression is higher in normal breast tissue and benign lesions than in infiltrating carcinomas. Among these, lower grade tumors showed increased TTP expression compared to higher grade cancers. Therefore, these data indicate that TTP protein levels would provide a better negative correlation with breast cancer invasiveness than TTP transcript levels. In mice, we found that TTP mRNA and protein expression is also diminished in mammary tumors. Interestingly, a strong positive association of TTP expression and mammary differentiation was identified in normal and tumor cells. In fact, TTP expression is highly increased during lactation, showing good correlation with various mammary differentiation factors. TTP expression was also induced in mammary HC11 cells treated with lactogenic hormones, mainly by prolactin, through Stat5A activation. The effect of this hormone was highly dependent on mammary differentiation status, as prolactin was unable to elicit a similar response in proliferating or neoplastic mammary cells. In summary, these studies show that TTP expression is strongly linked to the mammary differentiation program in human and mice, suggesting that this protein might play specific and relevant roles in the normal physiology of the gland.

  6. Expression and purification of GST-FHL2 fusion protein.

    PubMed

    Yu, H; Ma, Q; Lin, J; Sun, Y F; Zheng, F

    2013-12-06

    Escherichia coli is the most widely used host for the production of recombinant proteins. However, most eukaryotic proteins are typically obtained as insoluble, misfolded inclusion bodies that need solubilization and refolding. The interactions between human FHL2 protein and many types of proteins, including structural proteins, kinases, and several classes of transcription factor, have been found to have important roles in a variety of fundamental processes, including arrhythmia, hypertrophy, atherosclerosis, and angiogenesis. To achieve high-level expression of soluble recombinant human FHL2 protein in E. coli, we have constructed a recombinant expression plasmid, pGEX-4T-1-FHL2, in which we merged FHL2 cDNA with the glutathione S-transferase (GST) coding sequence downstream of the tac inducible promoter. Using this plasmid, we have achieved high expression of soluble FHL2 as a GST fusion protein in E. coli BL21. We have used the engineered plasmid (pGEX-4T-1-FHL2) and the modified E. coli strain to overcome the problem of removing the GST moiety while expressing soluble FHL2. Our results show that: 1) the recombinant plasmid was successfully constructed. Sequencing results showed that FHL2 and GST are in the same reading frame; 2) at 23°C, soluble GST-FHL2 fusion protein was highly expressed after induction with 0.1 mM IPTG; and 3) GST-FHL2 can be detected by Western blotting using mouse monoclonal anti-GST antibody. Our data are the first to show that high yields of soluble FHL2 tagged with GST can be achieved in E.coli.

  7. Enhanced Expression of Hedgehog Pathway Proteins in Oral Epithelial Dysplasia.

    PubMed

    Dias, Rosane Borges; Valverde, Ludmila de Faro; Sales, Caroline Brandi Schlaepfer; Guimarães, Vanessa Sousa Nazaré; Cabral, Márcia Grillo; de Aquino Xavier, Flávia Caló; Dos Santos, Jean Nunes; Ramos, Eduardo Antônio Gonçalves; Gurgel Rocha, Clarissa Araújo

    2016-09-01

    The aim of this study was to characterize the profile of the proteins involved in the Hedgehog signaling pathway to aid in the understanding of the pathogenesis of oral epithelial dysplasia (OED). The proteins SHH, PTCH1, HHIP, SUFU, GLI1, and cyclin D1 were evaluated by immunohistochemistry in 25 cases of OED, 4 of non-neoplasic oral mucosa, 8 of inflammatory fibrous hyperplasia and 5 of hyperkeratosis. SHH proteins were predominant in OED cases. Although PTCH1 protein was observed in all cases, this molecule was more highly expressed in OED. The inhibitor protein SUFU was present in OED and HHIP protein was overexpressed in OED. GLI1 proteins were predominantly found in the nuclei of epithelial cells in OED. Basal and suprabasal cells in the epithelial lining were positive for cyclin D1 only in OED. In conclusion, comparative analysis of the proteins involved in the Hedgehog pathway suggests that enhanced expression of these proteins can play an important role in the biological behavior of OED.

  8. The cationic liposomal adjuvants CAF01 and CAF09 formulated with the major outer membrane protein elicit robust protection in mice against a Chlamydia muridarum respiratory challenge.

    PubMed

    Pal, Sukumar; Tifrea, Delia F; Follmann, Frank; Andersen, Peter; de la Maza, Luis M

    2017-03-23

    Two cationic liposomal adjuvants CAF01 and CAF09 were formulated with the native or the recombinant Chlamydia muridarum major outer membrane protein (nMOMP and rMOMP). BALB/c mice were immunized with the four vaccine formulations using the subcutaneous followed by the intranasal (i.n.) routes. As positive controls mice were inoculated i.n. with live C. muridarum and negative controls received i.n. minimal essential medium (MEM). Four weeks after the last immunization mice were challenged i.n. with 10(4) inclusion forming units (IFU) of C. muridarum. Following the challenge the mice were weighed daily. At 10days post-challenge the mice were euthanized, their lungs weighed and the number of C. muridarum IFU determined. Serum collected the day before the challenge showed that all four groups of mice immunized with CAF01, or CAF09 and MOMP had significant C. muridarum-specific antibody titers. As determined by a T-cell lymphoproliferative assay, these four groups of mice also mounted robust cell mediated immune responses with high production of IFN-γ and IL17 and low levels of IL-4. Following the challenge the four groups of mice lost significantly less body weight than the MEM-immunized group. Lungs of mice vaccinated with CAF01, or CAF09, and nMOMP were significantly lighter than those from mice immunized using rMOMP. The number of IFU recovered from the lungs of mice vaccinated with CAF01, or CAF09, and nMOMP was similar to the number of IFU recovered from mice immunized with live EB. Mice that received rMOMP had significantly higher numbers of IFU than other groups. In conclusion, CAF01 and CAF09 elicited very robust protective humoral and cellular immune responses and were equally effective at adjuntavizing the C. muridarum MOMP. Mice vaccinated with nMOMP were significantly better protected than those immunized with rMOMP, indicative of the importance of the structural conformation of this antigen in protection.

  9. Cell Cycle Programs of Gene Expression Control Morphogenetic Protein Localization

    PubMed Central

    Lord, Matthew; Yang, Melody C.; Mischke, Michelle; Chant, John

    2000-01-01

    Genomic studies in yeast have revealed that one eighth of genes are cell cycle regulated in their expression. Almost without exception, the significance of cell cycle periodic gene expression has not been tested. Given that many such genes are critical to cellular morphogenesis, we wanted to examine the importance of periodic gene expression to this process. The expression profiles of two genes required for the axial pattern of cell division, BUD3 and BUD10/AXL2/SRO4, are strongly cell cycle regulated. BUD3 is expressed close to the onset of mitosis. BUD10 is expressed in late G1. Through promotor-swap experiments, the expression profile of each gene was altered and the consequences examined. We found that an S/G2 pulse of BUD3 expression controls the timing of Bud3p localization, but that this timing is not critical to Bud3p function. In contrast, a G1 pulse of BUD10 expression plays a direct role in Bud10p localization and function. Bud10p, a membrane protein, relies on the polarized secretory machinery specific to G1 to be delivered to its proper location. Such a secretion-based targeting mechanism for membrane proteins provides cells with flexibility in remodeling their architecture or evolving new forms. PMID:11134078

  10. Protein Co-Expression Network Analysis (ProCoNA)

    SciTech Connect

    Gibbs, David L.; Baratt, Arie; Baric, Ralph; Kawaoka, Yoshihiro; Smith, Richard D.; Orwoll, Eric S.; Katze, Michael G.; Mcweeney, Shannon K.

    2013-06-01

    Biological networks are important for elucidating disease etiology due to their ability to model complex high dimensional data and biological systems. Proteomics provides a critical data source for such models, but currently lacks robust de novo methods for network construction, which could bring important insights in systems biology. We have evaluated the construction of network models using methods derived from weighted gene co-expression network analysis (WGCNA). We show that approximately scale-free peptide networks, composed of statistically significant modules, are feasible and biologically meaningful using two mouse lung experiments and one human plasma experiment. Within each network, peptides derived from the same protein are shown to have a statistically higher topological overlap and concordance in abundance, which is potentially important for inferring protein abundance. The module representatives, called eigenpeptides, correlate significantly with biological phenotypes. Furthermore, within modules, we find significant enrichment for biological function and known interactions (gene ontology and protein-protein interactions). Biological networks are important tools in the analysis of complex systems. In this paper we evaluate the application of weighted co-expression network analysis to quantitative proteomics data. Protein co-expression networks allow novel approaches for biological interpretation, quality control, inference of protein abundance, a framework for potentially resolving degenerate peptide-protein mappings, and a biomarker signature discovery.

  11. Thyroid-Related Protein Expression in the Human Thymus

    PubMed Central

    Park, Do Joon; Jung, Kyeong Cheon

    2017-01-01

    Radioiodine whole body scan (WBS), related to sodium iodide symporter (NIS) function, is widely used to detect recurrence/metastasis in postoperative patients with thyroid cancer. However, the normal thymic uptake of radioiodine has occasionally been observed in young patients. We evaluated the expression of thyroid-related genes and proteins in the human thymus. Thymic tissues were obtained from 22 patients with thyroid cancer patients of all ages. The expression of NIS, thyroid-stimulating hormone receptor (TSHR), thyroperoxidase (TPO), and thyroglobulin (Tg) was investigated using immunohistochemistry and quantitative RT-PCR. NIS and TSHR were expressed in 18 (81.8%) and 19 samples (86.4%), respectively, whereas TPO was expressed in five samples (22.7%). Three thyroid-related proteins were localized to Hassall's corpuscles and thymocytes. In contrast, Tg was detected in a single patient (4.5%) localized to vascular endothelial cells. The expression of thyroid-related proteins was not increased in young thymic tissues compared to that in old thymic tissues. In conclusion, the expression of NIS and TSHR was detected in the majority of normal thymus samples, whereas that of TPO was detected less frequently, and that of Tg was detected rarely. The increased thymic uptake of radioiodine in young patients is not due to the increased expression of NIS. PMID:28386277

  12. Protein-directed ribosomal frameshifting temporally regulates gene expression

    PubMed Central

    Napthine, Sawsan; Ling, Roger; Finch, Leanne K.; Jones, Joshua D.; Bell, Susanne; Brierley, Ian; Firth, Andrew E.

    2017-01-01

    Programmed −1 ribosomal frameshifting is a mechanism of gene expression, whereby specific signals within messenger RNAs direct a proportion of translating ribosomes to shift −1 nt and continue translating in the new reading frame. Such frameshifting normally occurs at a set ratio and is utilized in the expression of many viral genes and a number of cellular genes. An open question is whether proteins might function as trans-acting switches to turn frameshifting on or off in response to cellular conditions. Here we show that frameshifting in a model RNA virus, encephalomyocarditis virus, is trans-activated by viral protein 2A. As a result, the frameshifting efficiency increases from 0 to 70% (one of the highest known in a mammalian system) over the course of infection, temporally regulating the expression levels of the viral structural and enzymatic proteins. PMID:28593994

  13. Rhizobacterial exopolysaccharides elicit induced resistance on cucumber.

    PubMed

    Park, Kyungseok; Kloepper, Joseph W; Ryu, Choong-Min

    2008-06-01

    The role of exopolysaccharides (EPSs) from a plant growth-promoting rhizobacterium, Burkholderia gladioli IN26, on elicitation of induced systemic resistance was investigated. A purified EPS induced expression of PR- 1a::GUS on tobacco and elicited induced resistance against Colletotrichum orbiculare on cucumber. The maximum level of disease protection was noted when seeds were soaked in 200 ppm of the EPS. Our results indicate that EPS from specific rhizobacteria can elicit induced resistance and suggest that bacterial EPS might be a useful elicitor of resistance under field conditions.

  14. Using ion exchange chromatography to purify a recombinantly expressed protein.

    PubMed

    Duong-Ly, Krisna C; Gabelli, Sandra B

    2014-01-01

    Ion exchange chromatography (IEX) separates molecules by their surface charge, a property that can vary vastly between different proteins. There are two types of IEX, cation exhange and anion exchange chromatography. The protocol that follows was designed by the authors for anion exchange chromatography of a recombinantly expressed protein having a pI of 4.9 and containing two cysteine residues and one tryptophan residue, using an FPLC system. Prior to anion exchange, the protein had been salted out using ammonium sulfate precipitation and partially purified via hydrophobic interaction chromatography (see Salting out of proteins using ammonium sulfate precipitation and Use and Application of Hydrophobic Interaction Chromatography for Protein Purification). Slight modifications to this protocol may be made to accommodate both the protein of interest and the availability of equipment.

  15. GILT expression in B cells diminishes cathepsin S steady-state protein expression and activity

    PubMed Central

    Phipps-Yonas, Hannah; Semik, Vikki; Hastings, Karen Taraszka

    2013-01-01

    MHC class II-restricted Ag processing requires protein degradation in the endocytic pathway for the activation of CD4+ T cells. Gamma-interferon-inducible lysosomal thiol reductase (GILT) facilitates Ag processing by reducing protein disulfide bonds in this compartment. Lysosomal cysteine protease cathepsin S (CatS) contains disulfide bonds and mediates essential steps in MHC class II-restricted processing, including proteolysis of large polypeptides and cleavage of the invariant chain. We sought to determine whether GILT’s reductase activity regulates CatS expression and function. Confocal microscopy confirmed that GILT and CatS colocalized within lysosomes of B cells. GILT expression posttranscriptionally decreased the steady-state protein expression of CatS in primary B cells and B-cell lines. GILT did not substantially alter the expression of other lysosomal proteins, including H2-M, H2-O, or CatL. GILT’s reductase active site was necessary for diminished CatS protein levels, and GILT expression decreased the half-life of CatS, suggesting that GILT-mediated reduction of protein disulfide bonds enhances CatS degradation. GILT expression decreased the proteolysis of a CatS selective substrate. This study illustrates a physiologic mechanism that regulates CatS and has implications for fine tuning MHC class II-restricted Ag processing and for the development of CatS inhibitors, which are under investigation for the treatment of autoimmune disease. PMID:23012103

  16. Green fluorescent protein-based expression screening of membrane proteins in Escherichia coli.

    PubMed

    Bird, Louise E; Rada, Heather; Verma, Anil; Gasper, Raphael; Birch, James; Jennions, Matthew; Lӧwe, Jan; Moraes, Isabel; Owens, Raymond J

    2015-01-06

    The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

  17. Increased functional protein expression using nucleotide sequence features enriched in highly expressed genes in zebrafish.

    PubMed

    Horstick, Eric J; Jordan, Diana C; Bergeron, Sadie A; Tabor, Kathryn M; Serpe, Mihaela; Feldman, Benjamin; Burgess, Harold A

    2015-04-20

    Many genetic manipulations are limited by difficulty in obtaining adequate levels of protein expression. Bioinformatic and experimental studies have identified nucleotide sequence features that may increase expression, however it is difficult to assess the relative influence of these features. Zebrafish embryos are rapidly injected with calibrated doses of mRNA, enabling the effects of multiple sequence changes to be compared in vivo. Using RNAseq and microarray data, we identified a set of genes that are highly expressed in zebrafish embryos and systematically analyzed for enrichment of sequence features correlated with levels of protein expression. We then tested enriched features by embryo microinjection and functional tests of multiple protein reporters. Codon selection, releasing factor recognition sequence and specific introns and 3' untranslated regions each increased protein expression between 1.5- and 3-fold. These results suggested principles for increasing protein yield in zebrafish through biomolecular engineering. We implemented these principles for rational gene design in software for codon selection (CodonZ) and plasmid vectors incorporating the most active non-coding elements. Rational gene design thus significantly boosts expression in zebrafish, and a similar approach will likely elevate expression in other animal models.

  18. Protein Poly(ADP-ribosyl)ation Regulates Arabidopsis Immune Gene Expression and Defense Responses

    PubMed Central

    Feng, Baomin; Liu, Chenglong; de Oliveira, Marcos V. V.; Intorne, Aline C.; Li, Bo; Babilonia, Kevin; de Souza Filho, Gonçalo A.; Shan, Libo; He, Ping

    2015-01-01

    Perception of microbe-associated molecular patterns (MAMPs) elicits transcriptional reprogramming in hosts and activates defense to pathogen attacks. The molecular mechanisms underlying plant pattern-triggered immunity remain elusive. A genetic screen identified Arabidopsis poly(ADP-ribose) glycohydrolase 1 (atparg1) mutant with elevated immune gene expression upon multiple MAMP and pathogen treatments. Poly(ADP-ribose) glycohydrolase (PARG) is predicted to remove poly(ADP-ribose) polymers on acceptor proteins modified by poly(ADP-ribose) polymerases (PARPs) with three PARPs and two PARGs in Arabidopsis genome. AtPARP1 and AtPARP2 possess poly(ADP-ribose) polymerase activity, and the activity of AtPARP2 was enhanced by MAMP treatment. AtPARG1, but not AtPARG2, carries glycohydrolase activity in vivo and in vitro. Importantly, mutation (G450R) in atparg1 blocks its activity and the corresponding residue is highly conserved and essential for human HsPARG activity. Consistently, mutant atparp1atparp2 plants exhibited compromised immune gene activation and enhanced susceptibility to pathogen infections. Our study indicates that protein poly(ADP-ribosyl)ation plays critical roles in plant immune gene expression and defense to pathogen attacks. PMID:25569773

  19. Protein poly(ADP-ribosyl)ation regulates arabidopsis immune gene expression and defense responses.

    PubMed

    Feng, Baomin; Liu, Chenglong; de Oliveira, Marcos V V; Intorne, Aline C; Li, Bo; Babilonia, Kevin; de Souza Filho, Gonçalo A; Shan, Libo; He, Ping

    2015-01-01

    Perception of microbe-associated molecular patterns (MAMPs) elicits transcriptional reprogramming in hosts and activates defense to pathogen attacks. The molecular mechanisms underlying plant pattern-triggered immunity remain elusive. A genetic screen identified Arabidopsis poly(ADP-ribose) glycohydrolase 1 (atparg1) mutant with elevated immune gene expression upon multiple MAMP and pathogen treatments. Poly(ADP-ribose) glycohydrolase (PARG) is predicted to remove poly(ADP-ribose) polymers on acceptor proteins modified by poly(ADP-ribose) polymerases (PARPs) with three PARPs and two PARGs in Arabidopsis genome. AtPARP1 and AtPARP2 possess poly(ADP-ribose) polymerase activity, and the activity of AtPARP2 was enhanced by MAMP treatment. AtPARG1, but not AtPARG2, carries glycohydrolase activity in vivo and in vitro. Importantly, mutation (G450R) in atparg1 blocks its activity and the corresponding residue is highly conserved and essential for human HsPARG activity. Consistently, mutant atparp1atparp2 plants exhibited compromised immune gene activation and enhanced susceptibility to pathogen infections. Our study indicates that protein poly(ADP-ribosyl)ation plays critical roles in plant immune gene expression and defense to pathogen attacks.

  20. Global Analysis of Protein Expression of Inner Ear Hair Cells

    PubMed Central

    Wong, Ann C.Y.; Pak, Kwang; Strojny, Chelsee; Ramirez, Miguel

    2017-01-01

    The mammalian inner ear (IE) subserves auditory and vestibular sensations via highly specialized cells and proteins. Sensory receptor hair cells (HCs) are necessary for transducing mechanical inputs and stimulating sensory neurons by using a host of known and as yet unknown protein machinery. To understand the protein composition of these unique postmitotic cells, in which irreversible protein degradation or damage can lead to impaired hearing and balance, we analyzed IE samples by tandem mass spectrometry to generate an unbiased, shotgun-proteomics view of protein identities and abundances. By using Pou4f3/eGFP-transgenic mice in which HCs express GFP driven by Pou4f3, we FACS purified a population of HCs to analyze and compare the HC proteome with other IE subproteomes from sensory epithelia and whole IE. We show that the mammalian HC proteome comprises hundreds of uniquely or highly expressed proteins. Our global proteomic analysis of purified HCs extends the existing HC transcriptome, revealing previously undetected gene products and isoform-specific protein expression. Comparison of our proteomic data with mouse and human databases of genetic auditory/vestibular impairments confirms the critical role of the HC proteome for normal IE function, providing a cell-specific pool of candidates for novel, important HC genes. Several proteins identified exclusively in HCs by proteomics and verified by immunohistochemistry map to human genetic deafness loci, potentially representing new deafness genes. SIGNIFICANCE STATEMENT Hearing and balance rely on specialized sensory hair cells (HCs) in the inner ear (IE) to convey information about sound, acceleration, and orientation to the brain. Genetically and environmentally induced perturbations to HC proteins can result in deafness and severe imbalance. We used transgenic mice with GFP-expressing HCs, coupled with FACS sorting and tandem mass spectrometry, to define the most complete HC and IE proteome to date. We show that

  1. SPINK 1 Protein Expression and Prostate Cancer Progression

    PubMed Central

    Flavin, Richard; Pettersson, Andreas; Hendrickson, Whitney K.; Fiorentino, Michelangelo; Finn, Stephen; Kunz, Lauren; Judson, Gregory L.; Lis, Rosina; Bailey, Dyane; Fiore, Christopher; Nuttall, Elizabeth; Martin, Neil E.; Stack, Edward; Penney, Kathryn L.; Rider, Jennifer R.; Sinnott, Jennifer; Sweeney, Christopher; Sesso, Howard D.; Fall, Katja; Giovannucci, Edward; Kantoff, Philip; Stampfer, Meir; Loda, Massimo; Mucci, Lorelei A.

    2014-01-01

    Purpose SPINK1 over-expression has been described in prostate cancer and is linked with poor prognosis in many cancers. The objective of this study was to characterize the association between SPINK1 over-expression and prostate cancer specific survival. Experimental Design The study included 879 participants in the US Physicians’ Health Study and Health Professionals Follow–Up Study, diagnosed with prostate cancer (1983 – 2004) and treated by radical prostatectomy. Protein tumor expression of SPINK1 was evaluated by immunohistochemistry on tumor tissue microarrays. Results 74/879 (8%) prostate cancer tumors were SPINK1 positive. Immunohistochemical data was available for PTEN, p-Akt, pS6, stathmin, androgen receptor (AR) and ERG (as a measure of the TMPRSS2:ERG translocation). Compared to SPINK1 negative tumors, SPINK1 positive tumors showed higher PTEN and stathmin expression, and lower expression of AR (p<0.01). SPINK1 over-expression was seen in 47 of 427 (11%) ERG negative samples and in 19 of 427 (4%) ERG positive cases (p=0.0003). We found no significant associations between SPINK1 status and Gleason grade or tumor stage. There was no association between SPINK1 expression and biochemical recurrence (p=0.56). Moreover, there was no association between SPINK1 expression and prostate cancer mortality (there were 75 lethal cases of prostate cancer during a mean of 13.5 years follow-up [HR 0.71 (95% confidence interval 0.29–1.76)]). Conclusions Our results suggest that SPINK1 protein expression may not be a predictor of recurrence or lethal prostate cancer amongst men treated by radical prostatectomy. SPINK1 and ERG protein expression do not appear to be entirely mutually exclusive, as some previous studies have suggested. PMID:24687926

  2. Protein Expression of Proteasome Subunits in Elderly Patients with Schizophrenia

    PubMed Central

    Scott, Madeline R; Rubio, Maria D; Haroutunian, Vahram; Meador-Woodruff, James H

    2016-01-01

    The ubiquitin proteasome system (UPS) is a major regulator of protein processing, trafficking, and degradation. While protein ubiquitination is utilized for many cellular processes, one major function of this system is to target proteins to the proteasome for degradation. In schizophrenia, studies have found UPS transcript abnormalities in both blood and brain, and we have previously reported decreased protein expression of ubiquitin-associated proteins in brain. To test whether the proteasome is similarly dysregulated, we measured the protein expression of proteasome catalytic subunits as well as essential subunits from proteasome regulatory complexes in 14 pair-matched schizophrenia and comparison subjects in superior temporal cortex. We found decreased expression of Rpt1, Rpt3, and Rpt6, subunits of the 19S regulatory particle essential for ubiquitin-dependent degradation by the proteasome. Additionally, the α subunit of the 11S αβ regulatory particle, which enhances proteasomal degradation of small peptides and unfolded proteins, was also decreased. Haloperidol-treated rats did not have altered expression of these subunits, suggesting the changes we observed in schizophrenia are likely not due to chronic antipsychotic treatment. Interestingly, expression of the catalytic subunits of both the standard and immunoproteasome were unchanged, suggesting the abnormalities we observed may be specific to the complexed state of the proteasome. Aging has significant effects on the proteasome, and several subunits (20S β2, Rpn10, Rpn13, 11Sβ, and 11Sγ) were significantly correlated with subject age. These data provide further evidence of dysfunction of the ubiquitin-proteasome system in schizophrenia, and suggest that altered proteasome activity may be associated with the pathophysiology of this illness. PMID:26202105

  3. Enhancement of G Protein-Coupled Receptor Surface Expression

    PubMed Central

    Dunham, Jill H.; Hall, Randy A.

    2009-01-01

    G protein-coupled receptors (GPCRs) mediate physiological responses to a diverse array of stimuli and are the molecular targets for numerous therapeutic drugs. GPCRs primarily signal from the plasma membrane, but when expressed in heterologous cells many GPCRs exhibit poor trafficking to the cell surface. Multiple approaches have been taken to enhance GPCR surface expression in heterologous cells, including addition/deletion of receptor sequences, co-expression with interacting proteins, and treatment with pharmacological chaperones. In addition to allowing for enhanced surface expression of certain GPCRs in heterologous cells, these approaches have also shed light on the control of GPCR trafficking in vivo and in some cases have led to new therapeutic approaches for treating human diseases that result from defects in GPCR trafficking. PMID:19679364

  4. Expression, delivery and function of insecticidal proteins expressed by recombinant baculoviruses

    USDA-ARS?s Scientific Manuscript database

    Since the development of methods for inserting and expressing genes in baculoviruses, a line of research has focused on developing recombinant baculoviruses that express insecticidal peptides and proteins. These recombinant viruses have been engineered with the goal of improving their pesticidal po...

  5. p53 and MDM2 protein expression in actinic cheilitis.

    PubMed

    de Freitas, Maria da Conceição Andrade; Ramalho, Luciana Maria Pedreira; Xavier, Flávia Caló Aquino; Moreira, André Luis Gomes; Reis, Sílvia Regina Almeida

    2008-01-01

    Actinic cheilitis is a potentially malignant lip lesion caused by excessive and prolonged exposure to ultraviolet radiation, which can lead to histomorphological alterations indicative of abnormal cell differentiation. In this pathology, varying degrees of epithelial dysplasia may be found. There are few published studies regarding the p53 and MDM2 proteins in actinic cheilitis. Fifty-eight cases diagnosed with actinic cheilitis were histologically evaluated using Banóczy and Csiba (1976) parameters, and were subjected to immunohistochemical analysis using the streptavidin-biotin method in order to assess p53 and MDM2 protein expression. All studied cases expressed p53 proteins in basal and suprabasal layers. In the basal layer, the nuclei testing positive for p53 were stained intensely, while in the suprabasal layer, cells with slightly stained nuclei were predominant. All cases also tested positive for the MDM2 protein, but with varying degrees of nuclear expression and a predominance of slightly stained cells. A statistically significant correlation between the percentage of p53 and MDM2-positive cells was established, regardless of the degree of epithelial dysplasia. The expression of p53 and MDM2 proteins in actinic cheilitis can be an important indicator in lip carcinogenesis, regardless of the degree of epithelial dysplasia.

  6. Argonaute Family Protein Expression in Normal Tissue and Cancer Entities

    PubMed Central

    Bruckmann, Astrid; Hauptmann, Judith; Deutzmann, Rainer; Meister, Gunter; Bosserhoff, Anja Katrin

    2016-01-01

    The members of the Argonaute (AGO) protein family are key players in miRNA-guided gene silencing. They enable the interaction between small RNAs and their respective target mRNA(s) and support the catalytic destruction of the gene transcript or recruit additional proteins for downstream gene silencing. The human AGO family consists of four AGO proteins (AGO1-AGO4), but only AGO2 harbors nuclease activity. In this study, we characterized the expression of the four AGO proteins in cancer cell lines and normal tissues with a new mass spectrometry approach called AGO-APP (AGO Affinity Purification by Peptides). In all analyzed normal tissues, AGO1 and AGO2 were most prominent, but marked tissue-specific differences were identified. Furthermore, considerable changes during development were observed by comparing fetal and adult tissues. We also identified decreased overall AGO expression in melanoma derived cell lines compared to other tumor cell lines and normal tissues, with the largest differences in AGO2 expression. The experiments described in this study suggest that reduced amounts of AGO proteins, as key players in miRNA processing, have impact on several cellular processes. Deregulated miRNA expression has been attributed to chromosomal aberrations, promoter regulation and it is known to have a major impact on tumor development and progression. Our findings will further increase our basic understanding of the molecular basis of miRNA processing and its relevance for disease. PMID:27518285

  7. Expression analysis of BRUCE protein in esophageal squamous cell carcinoma.

    PubMed

    Salehi, Somayeh; Jafarian, Amir Hossein; Forghanifard, Mohammad Mahdi

    2016-10-01

    Apoptosis is a form of cell death in response to diverse stressful physiological or pathological stimuli. One of the most important gene families involved in apoptosis is inhibitors of apoptosis. As a member of inhibitors of apoptosis, BRUCE can suppress apoptosis and promote cell division. Because esophageal squamous cell carcinoma (ESCC) cells, as well as other cancer cells, are immortal, our aim in this study was to analyze BRUCE protein expression in ESCC and evaluate its correlation with tumoral clinicopathologic features. Fifty ESCC specimens were examined for BRUCE protein expression using immunohistochemistry. A defined scoring method was applied. BRUCE protein was detected in 82% of tumors. Tumor progression stage and invasion depth correlated significantly with BRUCE protein expression (P=.019 and .005, respectively). Furthermore, association of BRUCE expression with tumor location was near significant (P=.058). The correlation of BRUCE overexpression in ESCC and disease aggressiveness may confirm the importance of BRUCE in ESCC progression and invasiveness. Therefore, BRUCE protein may be a molecular marker for aggressive ESCC and, thus, a potential therapeutic target to inhibit tumor cell progression and invasion. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Interfacial Polymerization for Colorimetric Labeling of Protein Expression in Cells

    PubMed Central

    Lilly, Jacob L.; Sheldon, Phillip R.; Hoversten, Liv J.; Romero, Gabriela; Balasubramaniam, Vivek; Berron, Brad J.

    2014-01-01

    Determining the location of rare proteins in cells typically requires the use of on-sample amplification. Antibody based recognition and enzymatic amplification is used to produce large amounts of visible label at the site of protein expression, but these techniques suffer from the presence of nonspecific reactivity in the biological sample and from poor spatial control over the label. Polymerization based amplification is a recently developed alternative means of creating an on-sample amplification for fluorescence applications, while not suffering from endogenous labels or loss of signal localization. This manuscript builds upon polymerization based amplification by developing a stable, archivable, and colorimetric mode of amplification termed Polymer Dye Labeling. The basic concept involves an interfacial polymer grown at the site of protein expression and subsequent staining of this polymer with an appropriate dye. The dyes Evans Blue and eosin were initially investigated for colorimetric response in a microarray setting, where both specifically stained polymer films on glass. The process was translated to the staining of protein expression in human dermal fibroblast cells, and Polymer Dye Labeling was specific to regions consistent with desired protein expression. The labeling is stable for over 200 days in ambient conditions and is also compatible with modern mounting medium. PMID:25536421

  9. Patterns of soybean proline-rich protein gene expression.

    PubMed Central

    Wyatt, R E; Nagao, R T; Key, J L

    1992-01-01

    The expression patterns of three members of a gene family that encodes proline-rich proteins in soybean (SbPRPs) were examined using in situ hybridization experiments. In most instances, the expression of SbPRP genes was intense in a limited number of cell types of a particular organ. SbPRP1 RNA was localized in several cell types of soybean hypocotyls, including cells within the phloem and xylem. SbPRP1 expression increased within epidermal cells in the elongating and mature regions of the hypocotyl; expression was detected also in lignified cells surrounding the hilum of mature seeds. SbPRP2 RNA was present in cortical cells and in the vascular tissue of the hypocotyl, especially cells of the phloem. This gene was expressed also in the inner integuments of the mature seed coat. SbPRP3 RNA was localized specifically to the endodermoid layer of cells surrounding the stele in the elongating region of the hypocotyl, as well as in the epidermal cells of leaves and cotyledons. These data show that members of this gene family exhibit cell-specific expression. The members of the SbPRP gene family are expressed in different types of cells and in some cell types that also express the glycine-rich protein or hydroxyproline-rich glycoprotein classes of genes. PMID:1525563

  10. AB223. Expression of tight junction proteins in rat vagina

    PubMed Central

    Oh, Kyung Jin; Lee, Hyun-Suk; Chung, Ho Suck; Ahn, Kyu Youn; Park, Kwangsung

    2014-01-01

    Aim Tight junction plays a role in apical cell-to-cell adhesion and epithelial polarity. In this study, we investigated the expression of tight junction proteins, such as Claudin-1, zonula occludens (ZO)-1, junction adhesion molecule (JAM)-A, and occludin in rat vagina. Methods Female Sprague-dawley rats (230-240 g, n=20) were divided into two groups: control (n=10) and bilateral ovariectomy (n=10). The expression and cellular localization of claudin-1, ZO-1, JAM-A, and occludin were determined in each group by immunohistochemistry and Western blot. Results Immunolabeling of ZO-1 was mainly expressed in the capillaries and venules of the vagina. Claudin-1, JAM-A, and occludin were expressed in the epithelium of the vagina. The immunoreactivity and protein expression of claudin-1 was significantly decreased in the ovariectomy group compared with the control group. Conclusions Our results suggest that tight junction proteins may have an important role in the vagina. Further studies are needed to clarify the role of each tight junction protein on vaginal lubrication.

  11. Functional interaction between co-expressed MAGE-A proteins

    PubMed Central

    Laiseca, Julieta E.; Ladelfa, María F.; Cotignola, Javier; Peche, Leticia Y.; Pascucci, Franco A.; Castaño, Bryan A.; Galigniana, Mario D.; Schneider, Claudio

    2017-01-01

    MAGE-A (Melanoma Antigen Genes-A) are tumor-associated proteins with expression in a broad spectrum of human tumors and normal germ cells. MAGE-A gene expression and function are being increasingly investigated to better understand the mechanisms by which MAGE proteins collaborate in tumorigenesis and whether their detection could be useful for disease prognosis purposes. Alterations in epigenetic mechanisms involved in MAGE gene silencing cause their frequent co-expression in tumor cells. Here, we have analyzed the effect of MAGE-A gene co-expression and our results suggest that MageA6 can potentiate the androgen receptor (AR) co-activation function of MageA11. Database search confirmed that MageA11 and MageA6 are co-expressed in human prostate cancer samples. We demonstrate that MageA6 and MageA11 form a protein complex resulting in the stabilization of MageA11 and consequently the enhancement of AR activity. The mechanism involves association of the Mage A6-MHD domain to MageA11, prevention of MageA11 ubiquitinylation on lysines 240 and 245 and decreased proteasome-dependent degradation. We experimentally demonstrate here for the first time that two MAGE-A proteins can act together in a non-redundant way to potentiate a specific oncogenic function. Overall, our results highlight the complexity of the MAGE gene networking in regulating cancer cell behavior. PMID:28542476

  12. Functional interaction between co-expressed MAGE-A proteins.

    PubMed

    Laiseca, Julieta E; Ladelfa, María F; Cotignola, Javier; Peche, Leticia Y; Pascucci, Franco A; Castaño, Bryan A; Galigniana, Mario D; Schneider, Claudio; Monte, Martin

    2017-01-01

    MAGE-A (Melanoma Antigen Genes-A) are tumor-associated proteins with expression in a broad spectrum of human tumors and normal germ cells. MAGE-A gene expression and function are being increasingly investigated to better understand the mechanisms by which MAGE proteins collaborate in tumorigenesis and whether their detection could be useful for disease prognosis purposes. Alterations in epigenetic mechanisms involved in MAGE gene silencing cause their frequent co-expression in tumor cells. Here, we have analyzed the effect of MAGE-A gene co-expression and our results suggest that MageA6 can potentiate the androgen receptor (AR) co-activation function of MageA11. Database search confirmed that MageA11 and MageA6 are co-expressed in human prostate cancer samples. We demonstrate that MageA6 and MageA11 form a protein complex resulting in the stabilization of MageA11 and consequently the enhancement of AR activity. The mechanism involves association of the Mage A6-MHD domain to MageA11, prevention of MageA11 ubiquitinylation on lysines 240 and 245 and decreased proteasome-dependent degradation. We experimentally demonstrate here for the first time that two MAGE-A proteins can act together in a non-redundant way to potentiate a specific oncogenic function. Overall, our results highlight the complexity of the MAGE gene networking in regulating cancer cell behavior.

  13. ceRNA crosstalk stabilizes protein expression and affects the correlation pattern of interacting proteins

    PubMed Central

    Martirosyan, Araks; De Martino, Andrea; Pagnani, Andrea; Marinari, Enzo

    2017-01-01

    Gene expression is a noisy process and several mechanisms, both transcriptional and post-transcriptional, can stabilize protein levels in cells. Much work has focused on the role of miRNAs, showing in particular that miRNA-mediated regulation can buffer expression noise for lowly expressed genes. Here, using in silico simulations and mathematical modeling, we demonstrate that miRNAs can exert a much broader influence on protein levels by orchestrating competition-induced crosstalk between mRNAs. Most notably, we find that miRNA-mediated cross-talk (i) can stabilize protein levels across the full range of gene expression rates, and (ii) modifies the correlation pattern of co-regulated interacting proteins, changing the sign of correlations from negative to positive. The latter feature may constitute a potentially robust signature of the existence of RNA crosstalk induced by endogenous competition for miRNAs in standard cellular conditions. PMID:28266541

  14. ceRNA crosstalk stabilizes protein expression and affects the correlation pattern of interacting proteins.

    PubMed

    Martirosyan, Araks; De Martino, Andrea; Pagnani, Andrea; Marinari, Enzo

    2017-03-07

    Gene expression is a noisy process and several mechanisms, both transcriptional and post-transcriptional, can stabilize protein levels in cells. Much work has focused on the role of miRNAs, showing in particular that miRNA-mediated regulation can buffer expression noise for lowly expressed genes. Here, using in silico simulations and mathematical modeling, we demonstrate that miRNAs can exert a much broader influence on protein levels by orchestrating competition-induced crosstalk between mRNAs. Most notably, we find that miRNA-mediated cross-talk (i) can stabilize protein levels across the full range of gene expression rates, and (ii) modifies the correlation pattern of co-regulated interacting proteins, changing the sign of correlations from negative to positive. The latter feature may constitute a potentially robust signature of the existence of RNA crosstalk induced by endogenous competition for miRNAs in standard cellular conditions.

  15. Spatiotemporal expression profiling of proteins in rat sciatic nerve regeneration using reverse phase protein arrays

    PubMed Central

    2012-01-01

    Background Protein expression profiles throughout 28 days of peripheral nerve regeneration were characterized using an established rat sciatic nerve transection injury model. Reverse phase protein microarrays were used to identify the spatial and temporal expression profile of multiple proteins implicated in peripheral nerve regeneration including growth factors, extracellular matrix proteins, and proteins involved in adhesion and migration. This high-throughput approach enabled the simultaneous analysis of 3,360 samples on a nitrocellulose-coated slide. Results The extracellular matrix proteins collagen I and III, laminin gamma-1, fibronectin, nidogen and versican displayed an early increase in protein levels in the guide and proximal sections of the regenerating nerve with levels at or above the baseline expression of intact nerve by the end of the 28 day experimental course. The 28 day protein levels were also at or above baseline in the distal segment however an early increase was only noted for laminin, nidogen, and fibronectin. While the level of epidermal growth factor, ciliary neurotrophic factor and fibroblast growth factor-1 and -2 increased throughout the experimental course in the proximal and distal segments, nerve growth factor only increased in the distal segment and fibroblast growth factor-1 and -2 and nerve growth factor were the only proteins in that group to show an early increase in the guide contents. As expected, several proteins involved in cell adhesion and motility; namely focal adhesion kinase, N-cadherin and β-catenin increased earlier in the proximal and distal segments than in the guide contents reflecting the relatively acellular matrix of the early regenerate. Conclusions In this study we identified changes in expression of multiple proteins over time linked to regeneration of the rat sciatic nerve both demonstrating the utility of reverse phase protein arrays in nerve regeneration research and revealing a detailed, composite

  16. Generation and immunogenicity of transgenic potato expressing the GP5 protein of porcine reproductive and respiratory syndrome virus.

    PubMed

    Chen, Xia; Liu, Jinlin

    2011-04-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) is an important pathogen that has caused huge economic losses in the global swine industry. The advent of molecular farming has provided a cost-effective strategy for the development of transgenic plants as bioreactors to produce recombinant proteins. In this study, transgenic potato expressing GP5 protein of PRRSV was produced by Agrobacterium-mediated transformation, and confirmed using Southern blot and RT-PCR analyses. Recombinant GP5 protein was detected by ELISA and Western blot analyses. Mice immunized with transgenic potato extracts generated both serum and gut mucosal-specific antibodies, although low levels of neutralizing antibodies were elicited. This study provides a new approach for the production of vaccines against PRRSV. Copyright © 2011 Elsevier B.V. All rights reserved.

  17. Human gut flora-fermented nondigestible fraction from cooked bean ( Phaseolus vulgaris L.) modifies protein expression associated with apoptosis, cell cycle arrest, and proliferation in human adenocarcinoma colon cancer cells.

    PubMed

    Campos-Vega, Rocio; García-Gasca, Teresa; Guevara-Gonzalez, Ramón; Ramos-Gomez, Minerva; Oomah, B Dave; Loarca-Piña, Guadalupe

    2012-12-26

    Metabolism of the nondigested fraction (NDF) from common bean ( Phaseolus vulgaris L.) by the human gut flora (hgf) produces short-chain fatty acids (SCFAs) that may benefit cancer by reducing colorectal tumor risks. This paper reports the effect of fermentation products (FP) by hgf (FP-hgf) from NDF of cooked beans on survival and protein expression associated with apoptosis, cell cycle arrest, and proliferation in human adenocarcinoma colon cancer cells. FP-hgf was the only inoculum eliciting butyrate production after 24 h of NDF fermentation using different bacterial sources. FP-hgf inhibited HT-29 cell growth and modulated protein expression associated with apoptosis, cell cycle arrest, and proliferation, as well as morphological changes linked to apoptosis evaluated by TUNEL and hematoxylin and eosin stains, confirming previous results on gene expression. The current results suggest that fermentation of NDF from common beans can elicit beneficial chemoprotective effects in colon cancer by modulating protein expression in HT-29 cells.

  18. Human Cementum Protein 1 induces expression of bone and cementum proteins by human gingival fibroblasts.

    PubMed

    Carmona-Rodríguez, Bruno; Alvarez-Pérez, Marco Antonio; Narayanan, A Sampath; Zeichner-David, Margarita; Reyes-Gasga, José; Molina-Guarneros, Juan; García-Hernández, Ana Lilia; Suárez-Franco, José Luis; Chavarría, Ivet Gil; Villarreal-Ramírez, Eduardo; Arzate, Higinio

    2007-07-06

    We recently presented evidence showing that a human cementoblastoma-derived protein, named Cementum Protein 1 (CEMP1) may play a role as a local regulator of cementoblast differentiation and cementum-matrix mineralization. This protein was shown to be expressed by cementoblasts and progenitor cells localized in the periodontal ligament. In this study we demonstrate that transfection of CEMP1 into human gingival fibroblasts (HGF) induces mineralization and expression of bone and cementum-matrix proteins. The transfected HGF cells had higher alkaline phosphatase activity and proliferation rate and they expressed genes for alkaline phosphatase, bone sialoprotein, osteocalcin, osteopontin, the transcription factor Runx2/Cbfa1, and cementum attachment protein (CAP). They also produced biological-type hydroxyapatite. These findings indicate that the CEMP1 might participate in differentiation and mineralization of nonosteogenic cells, and that it might have a potential function in cementum and bone formation.

  19. Human Cementum Protein 1 induces expression of bone and cementum proteins by human gingival fibroblasts

    SciTech Connect

    Carmona-Rodriguez, Bruno; Alvarez-Perez, Marco Antonio; Narayanan, A. Sampath; Zeichner-David, Margarita; Reyes-Gasga, Jose; Molina-Guarneros, Juan; Garcia-Hernandez, Ana Lilia; Suarez-Franco, Jose Luis; Chavarria, Ivet Gil; Villarreal-Ramirez, Eduardo; Arzate, Higinio . E-mail: harzate@servidor.unam.mx

    2007-07-06

    We recently presented evidence showing that a human cementoblastoma-derived protein, named Cementum Protein 1 (CEMP1) may play a role as a local regulator of cementoblast differentiation and cementum-matrix mineralization. This protein was shown to be expressed by cementoblasts and progenitor cells localized in the periodontal ligament. In this study we demonstrate that transfection of CEMP1 into human gingival fibroblasts (HGF) induces mineralization and expression of bone and cementum-matrix proteins. The transfected HGF cells had higher alkaline phosphatase activity and proliferation rate and they expressed genes for alkaline phosphatase, bone sialoprotein, osteocalcin, osteopontin, the transcription factor Runx2/Cbfa1, and cementum attachment protein (CAP). They also produced biological-type hydroxyapatite. These findings indicate that the CEMP1 might participate in differentiation and mineralization of nonosteogenic cells, and that it might have a potential function in cementum and bone formation.

  20. Sublingual vaccination with fusion protein consisting of the functional domain of hemagglutinin A of Porphyromonas gingivalis and Escherichia coli maltose-binding protein elicits protective immunity in the oral cavity.

    PubMed

    Yuzawa, Satoshi; Kurita-Ochiai, Tomoko; Hashizume, Tomomi; Kobayashi, Ryoki; Abiko, Yoshimitsu; Yamamoto, Masafumi

    2012-03-01

    This study demonstrated that sublingual immunization with a fusion protein, 25k-hagA-MBP, which consists of a 25-kDa antigenic region of hemagglutinin A purified from Porphyromonas gingivalis fused to maltose-binding protein (MBP) originating from Escherichia coli as an adjuvant, elicited protective immune responses. Immunization with 25k-hagA-MBP induced high levels of antigen-specific serum IgG and IgA, as well as salivary IgA. High level titers of serum IgG and IgA were also induced for almost 1 year. In an IgG subclass analysis, sublingual immunization with 25k-hagA-MBP induced both IgG1 and IgG2b antibody responses. Additionally, numerous antigen-specific IgA antibody-forming cells were detected from the salivary gland 7 days after the final immunization. Mononuclear cells isolated from submandibular lymph nodes (SMLs) showed significant levels of proliferation upon restimulation with 25k-hagA-MBP. An analysis of cytokine responses showed that antigen-specific mononuclear cells isolated from SMLs produced significantly high levels of IL-4, IFN-γ, and TGF-β. These results indicate that sublingual immunization with 25k-hagA-MBP induces efficient protective immunity against P. gingivalis infection in the oral cavity via Th1-type and Th2-type cytokine production.

  1. Fe-S Proteins that Regulate Gene Expression

    PubMed Central

    Mettert, Erin L.; Kiley, Patricia J.

    2014-01-01

    Iron-sulfur (Fe-S) cluster containing proteins that regulate gene expression are present in most organisms. The innate chemistry of their Fe-S cofactors makes these regulatory proteins ideal for sensing environmental signals, such as gases (e.g. O2 and NO), levels of Fe and Fe-S clusters, reactive oxygen species, and redox cycling compounds, to subsequently mediate an adaptive response. Here we review the recent findings that have provided invaluable insight into the mechanism and function of these highly significant Fe-S regulatory proteins. PMID:25450978

  2. Expression of Aequorea green fluorescent protein in plant cells.

    PubMed

    Hu, W; Cheng, C L

    1995-08-07

    The coding region of the green fluorescent protein (GFP) from Aequorea victoria has been fused to the cauliflower mosaic virus 35S promoter and introduced into maize leaf protoplasts. Transient expression of GFP was observed. In addition, the coding region of GFP was fused to an Arabidopsis heat shock promoter and co-transformed with another construct in which GFP has been replaced with chloramphenicol acetyltransferase (CAT). The heat-induced expression of GFP in maize protoplasts parallels that of CAT. While GFP was expressed in both dark-grown and green maize leaf protoplasts, no green fluorescence was observed in similarly transformed Arabidopsis protoplasts.

  3. Identifying subcellular protein localization with fluorescent protein fusions after transient expression in onion epidermal cells.

    PubMed

    Nebenführ, Andreas

    2014-01-01

    Most biochemical functions of plant cells are carried out by proteins which act at very specific places within these cells, for example, within different organelles. Identifying the subcellular localization of proteins is therefore a useful tool to narrow down the possible functions that a novel or unknown protein may carry out. The discovery of genetically encoded fluorescent markers has made it possible to tag specific proteins and visualize them in vivo under a variety of conditions. This chapter describes a simple method to use transient expression of such fluorescently tagged proteins in onion epidermal cells to determine their subcellular localization relative to known markers.

  4. Fragile X mental retardation protein (FMRP) interacting proteins exhibit different expression patterns during development.

    PubMed

    Bonaccorso, C M; Spatuzza, M; Di Marco, B; Gloria, A; Barrancotto, G; Cupo, A; Musumeci, S A; D'Antoni, S; Bardoni, B; Catania, M V

    2015-05-01

    Fragile X syndrome is caused by the lack of expression of fragile X mental retardation protein (FMRP), an RNA-binding protein involved in mRNA transport and translation. FMRP is a component of mRNA ribonucleoprotein complexes and it can interact with a range of proteins either directly or indirectly, as demonstrated by two-hybrid selection and co-immunoprecipitation, respectively. Most of FMRP-interacting proteins are RNA-binding proteins such as FXR1P, FXR2P and 82-FIP. Interestingly, FMRP can also interact directly with the cytoplasmic proteins CYFIP1 and CYFIP2, which do not bind RNA and link FMRP to the RhoGTPase pathway. The interaction with these different proteins may modulate the functions of FMRP by influencing its affinity to RNA and by affecting the FMRP ability of cytoskeleton remodeling through Rho/Rac GTPases. To better define the relationship of FMRP with its interacting proteins during brain development, we have analyzed the expression pattern of FMRP and its interacting proteins in the cortex, striatum, hippocampus and cerebellum at different ages in wild type (WT) mice. FMRP and FXR2P were strongly expressed during the first week and gradually decreased thereafter, more rapidly in the cerebellum than in the cortex. FXR1P was also expressed early and showed a reduction at later stages of development with a similar developmental pattern in these two regions. CYFIP1 was expressed at all ages and peaked in the third post-natal week. In contrast, CYFIP2 and 82-FIP (only in forebrain regions) were moderately expressed at P3 and gradually increased after P7. In general, the expression pattern of each protein was similar in the regions examined, except for 82-FIP, which exhibited a strong expression at P3 and low levels at later developmental stages in the cerebellum. Our data indicate that FMRP and its interacting proteins have distinct developmental patterns of expression and suggest that FMRP may be preferentially associated to certain proteins in

  5. Rice black-streaked dwarf virus P10 induces membranous structures at the ER and elicits the unfolded protein response in Nicotiana benthamiana.

    PubMed

    Sun, Zongtao; Yang, Di; Xie, Li; Sun, Liying; Zhang, Shanglin; Zhu, Qisong; Li, Junmin; Wang, Xu; Chen, Jianping

    2013-12-01

    Endoplasmic reticular (ER) membrane modifications play an important role in viral RNA replication and virion assembly but little is known about the involvement of ER-membrane remodeling in the infection cycle of fijiviruses in plant cells. The subcellular localization of Rice black-streaked dwarf virus outer capsid P10 was therefore examined using live-cell imaging. P10 fused to eGFP formed vesicular structures associated with ER membranes in Nicotiana benthamiana epidermal cells and in rice protoplasts. Subcellular fractionation experiments confirmed that P10 is an integral membrane protein. Three predicted transmembrane domains and two less-well-defined domains were each able to target eGFP to the ER. Disruption of the actin cytoskeleton with LatB, indicated that the maintenance of P10-induced membrane structures required the intact actin cytoskeleton. P10 induced the expression of ER stress marker genes, including ER stress-related chaperones and transcription factor, indicating that RBSDV P10 triggers ER stress and the unfolded protein response. © 2013 Published by Elsevier Inc.

  6. Phase Variation Mediates Reductions in Expression of Surface Proteins during Persistent Meningococcal Carriage

    PubMed Central

    Alamro, Mohamed; Bidmos, Fadil A.; Chan, Hannah; Oldfield, Neil J.; Newton, Emma; Bai, Xilian; Aidley, Jack; Care, Rory; Mattick, Claire; Turner, David P. J.; Neal, Keith R.; Ala'Aldeen, Dlawer A. A.; Feavers, Ian; Borrow, Ray

    2014-01-01

    Asymptomatic and persistent colonization of the upper respiratory tract by Neisseria meningitidis occurs despite elicitation of adaptive immune responses against surface antigens. A putative mechanism for facilitating host persistence of this bacterial commensal and pathogen is alterations in expression of surface antigens by simple sequence repeat (SSR)-mediated phase variation. We investigated how often phase variation occurs during persistent carriage by analyzing the SSRs of eight loci in multiple isolates from 21 carriers representative of 1 to 6 months carriage. Alterations in repeat number were detected by a GeneScan analysis and occurred at 0.06 mutations/gene/month of carriage. The expression states were determined by Western blotting and two genes, fetA and nadA, exhibited trends toward low expression states. A critical finding from our unique examination of combinatorial expression states, “phasotypes,” was for significant reductions in expression of multiple phase-variable surface proteins during persistent carriage of some strains. The immune responses in these carriers were examined by measuring variant-specific PorA IgG antibodies, capsular group Y IgG antibodies and serum bactericidal activity in concomitant serum samples. Persistent carriage was associated with high levels of specific IgG antibodies and serum bactericidal activity while recent strain acquisition correlated with a significant induction of antibodies. We conclude that phase-variable genes are driven into lower expression states during long-term persistent meningococcal carriage, in part due to continuous exposure to antibody-mediated selection, suggesting localized hypermutation has evolved to facilitate host persistence. PMID:24686058

  7. Using Green and Red Fluorescent Proteins to Teach Protein Expression, Purification, and Crystallization

    ERIC Educational Resources Information Center

    Wu, Yifeng; Zhou, Yangbin; Song, Jiaping; Hu, Xiaojian; Ding, Yu; Zhang, Zhihong

    2008-01-01

    We have designed a laboratory curriculum using the green and red fluorescent proteins (GFP and RFP) to visualize the cloning, expression, chromatography purification, crystallization, and protease-cleavage experiments of protein science. The EGFP and DsRed monomer (mDsRed)-coding sequences were amplified by PCR and cloned into pMAL (MBP-EGFP) or…

  8. Using Green and Red Fluorescent Proteins to Teach Protein Expression, Purification, and Crystallization

    ERIC Educational Resources Information Center

    Wu, Yifeng; Zhou, Yangbin; Song, Jiaping; Hu, Xiaojian; Ding, Yu; Zhang, Zhihong

    2008-01-01

    We have designed a laboratory curriculum using the green and red fluorescent proteins (GFP and RFP) to visualize the cloning, expression, chromatography purification, crystallization, and protease-cleavage experiments of protein science. The EGFP and DsRed monomer (mDsRed)-coding sequences were amplified by PCR and cloned into pMAL (MBP-EGFP) or…

  9. Salicylic acid enhances Staphylococcus aureus extracellular adhesin protein expression.

    PubMed

    Alvarez, Lucía P; Barbagelata, María S; Cheung, Ambrose L; Sordelli, Daniel O; Buzzola, Fernanda R

    2011-11-01

    One of the virulence factors required by Staphylococcus aureus at the early stages of infection is Eap, a secreted adhesin that binds many host proteins and is upregulated by the two-component regulatory system saeRS. The S. aureus Newman strain harbors a mutation in saeS that is thought to be responsible for the high level of Eap expression in this strain. This study was designed to ascertain whether salicylic acid (SAL) affects the expression of Eap and the internalization of S. aureus into epithelial cells. The strain Newman treated with SAL exhibited increased levels of eap transcription and protein expression. Furthermore, SAL treatment increased the eap promoter activity. SAL treatment enhanced Eap expression in the Newman and in other S. aureus strains that do not carry the mutation in saeS. Internalization of S. aureus eap and sae mutants into the MAC-T epithelial cells was significantly decreased compared with the wild-type counterparts. In conclusion, we demonstrated that a low concentration of SAL increased S. aureus Eap expression possibly due to enhancement of sae. SAL may create the conditions for S. aureus persistence in the host, not only by decreasing the capsular polysaccharide expression as shown before, but also by enhancing Eap expression.

  10. Wolbachia endosymbiont of Brugia malayi elicits a T helper type 17-mediated pro-inflammatory immune response through Wolbachia surface protein.

    PubMed

    Pathak, Manisha; Verma, Meenakshi; Srivastava, Mrigank; Misra-Bhattacharya, Shailja

    2015-02-01

    Wolbachia is an endosymbiotic bacterium of the filarial nematode Brugia malayi. The symbiotic relationship between Wolbachia and its filarial host is dependent on interactions between the proteins of both organisms. However, little is known about Wolbachia proteins that are involved in the inflammatory pathology of the host during lymphatic filariasis. In the present study, we cloned, expressed and purified Wolbachia surface protein (r-wsp) from Wolbachia and administered it to mice, either alone or in combination with infective larvae of B. malayi (Bm-L3) and monitored the developing immune response in infected animals. Our results show that spleens and mesenteric lymph nodes of mice immunized with either r-wsp or infected with Bm-L3 show increased percentages of CD4(+) T helper type 17 (Th17) cells and Th1 cytokines like interferon-γ and interleukin-2 (IL-2) along with decreased percentages of regulatory T cells, Th2 cytokines like IL-4 and IL-10 and transforming growth factor β (TGF-β) levels in culture supernatants of splenocytes. These observations were stronger in mice immunized with r-wsp alone. Interestingly, when mice were first immunized with r-wsp and subsequently infected with Bm-L3, percentages of CD4(+) Th17 cells and Th1 cytokines increased even further while that of regulatory T cells, Th2 cytokines and TGF-β levels decreased. These results for the first time show that r-wsp acts synergistically with Bm-L3 in promoting a pro-inflammatory response by increasing Th17 cells and at the same time diminishes host immunological tolerance by decreasing regulatory T cells and TGF-β secretion. © 2014 John Wiley & Sons Ltd.

  11. Cementum attachment protein/protein-tyrosine phosphotase-like member A is not expressed in teeth.

    PubMed

    Schild, Christof; Beyeler, Michael; Lang, Niklaus P; Trueb, Beat

    2009-02-01

    Cementum is a highly specialized connective tissue that covers tooth roots. The only cementum-specific protein described to date is the cementum attachment protein (CAP). A putative sequence for CAP was established from a cDNA clone isolated from a human cementifying fibroma cDNA library. This sequence overlaps with a phosphatase-like protein in muscle termed the protein-tyrosine phosphatase-like member A (PTPLA). To clarify the nature of CAP/PTPLA, we cloned the homologous rat protein and determined its sequence. The rat protein shared 94% sequence identity with the human protein. On Northern blots containing RNA from various rat tissues of different developmental stages, the cDNA hybridized to an mRNA expressed in heart and skeletal muscle but not in teeth. These results were confirmed by real-time PCR. Thus, the sequence deposited in public databanks under the name 'cementum attachment protein' does not represent genuine CAP.

  12. Controlling for Gene Expression Changes in Transcription Factor Protein Networks*

    PubMed Central

    Banks, Charles A. S.; Lee, Zachary T.; Boanca, Gina; Lakshminarasimhan, Mahadevan; Groppe, Brad D.; Wen, Zhihui; Hattem, Gaye L.; Seidel, Chris W.; Florens, Laurence; Washburn, Michael P.

    2014-01-01

    The development of affinity purification technologies combined with mass spectrometric analysis of purified protein mixtures has been used both to identify new protein–protein interactions and to define the subunit composition of protein complexes. Transcription factor protein interactions, however, have not been systematically analyzed using these approaches. Here, we investigated whether ectopic expression of an affinity tagged transcription factor as bait in affinity purification mass spectrometry experiments perturbs gene expression in cells, resulting in the false positive identification of bait-associated proteins when typical experimental controls are used. Using quantitative proteomics and RNA sequencing, we determined that the increase in the abundance of a set of proteins caused by overexpression of the transcription factor RelA is not sufficient for these proteins to then co-purify non-specifically and be misidentified as bait-associated proteins. Therefore, typical controls should be sufficient, and a number of different baits can be compared with a common set of controls. This is of practical interest when identifying bait interactors from a large number of different baits. As expected, we found several known RelA interactors enriched in our RelA purifications (NFκB1, NFκB2, Rel, RelB, IκBα, IκBβ, and IκBε). We also found several proteins not previously described in association with RelA, including the small mitochondrial chaperone Tim13. Using a variety of biochemical approaches, we further investigated the nature of the association between Tim13 and NFκB family transcription factors. This work therefore provides a conceptual and experimental framework for analyzing transcription factor protein interactions. PMID:24722732

  13. Secreted Proteins Defy the Expression Level-Evolutionary Rate Anticorrelation.

    PubMed

    Feyertag, Felix; Berninsone, Patricia M; Alvarez-Ponce, David

    2017-03-01

    The rates of evolution of the proteins of any organism vary across orders of magnitude. A primary factor influencing rates of protein evolution is expression. A strong negative correlation between expression levels and evolutionary rates (the so-called E-R anticorrelation) has been observed in virtually all studied organisms. This effect is currently attributed to the abundance-dependent fitness costs of misfolding and unspecific protein-protein interactions, among other factors. Secreted proteins are folded in the endoplasmic reticulum, a compartment where chaperones, folding catalysts, and stringent quality control mechanisms promote their correct folding and may reduce the fitness costs of misfolding. In addition, confinement of secreted proteins to the extracellular space may reduce misinteractions and their deleterious effects. We hypothesize that each of these factors (the secretory pathway quality control and extracellular location) may reduce the strength of the E-R anticorrelation. Indeed, here we show that among human proteins that are secreted to the extracellular space, rates of evolution do not correlate with protein abundances. This trend is robust to controlling for several potentially confounding factors and is also observed when analyzing protein abundance data for 6 human tissues. In addition, analysis of mRNA abundance data for 32 human tissues shows that the E-R correlation is always less negative, and sometimes nonsignificant, in secreted proteins. Similar observations were made in Caenorhabditis elegans and in Escherichia coli, and to a lesser extent in Drosophila melanogaster, Saccharomyces cerevisiae and Arabidopsis thaliana. Our observations contribute to understand the causes of the E-R anticorrelation. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  14. Selection of soluble protein expression constructs: the experimental determination of protein domain boundaries.

    PubMed

    Dyson, Michael R

    2010-08-01

    Proteins can contain multiple domains each of which is capable of possessing a separate independent function and three-dimensional structure. It is often useful to clone and express individual protein domains to study their biochemical properties and for structure determination. However, the annotated domain boundaries in databases such as Pfam or SMART are not always accurate. The present review summarizes various strategies for the experimental determination of protein domain boundaries.

  15. Expression Trend of Selected Ribosomal Protein Genes in Nasopharyngeal Carcinoma

    PubMed Central

    Ma, Xiang-Ru; Sim, Edmund Ui-Hang; Ling, Teck-Yee; Tiong, Thung-Sing; Subramaniam, Selva Kumar; Khoo, Alan Soo-Beng

    2012-01-01

    Background: Ribosomal proteins are traditionally associated with protein biosynthesis until recent studies that implicated their extraribosomal functions in human diseases and cancers. Our previous studies using GeneFishing™ DEG method and microarray revealed underexpression of three ribosomal protein genes, RPS26, RPS27, and RPL32 in cancer of the nasopharynx. Herein, we investigated the expression pattern and nucleotide sequence integrity of these genes in nasopharyngeal carcinoma to further delineate their involvement in tumourigenesis. The relationship of expression level with clinicopathologic factors was also statistically studied. Methods: Quantitative Polymerase Chain Reaction was performed on nasopharyngeal carcinoma and their paired normal tissues. Expression and sequence of these three genes were analysed. Results: All three ribosomal protein genes showed no significant difference in transcript expressions and no association could be established with clinicopathologic factors studied. No nucleotide aberrancy was detected in the coding regions of these genes. Conclusion: There is no early evidence to substantiate possible involvement of RPS26, RPS27, and RPL32 genes in NPC tumourigenesis. PMID:23613646

  16. Expression of Prokaryotic Integral Membrane Proteins in E. coli.

    PubMed

    Love, James D

    2017-01-01

    Production of prokaryotic membrane proteins for structural and functional studies in E. coli can be parallelized and miniaturized. All stages from cloning, expression, purification to detergent selection can be investigated using high-throughput techniques to rapidly and economically find tractable targets.

  17. Computational codon optimization of synthetic gene for protein expression

    PubMed Central

    2012-01-01

    Background The construction of customized nucleic acid sequences allows us to have greater flexibility in gene design for recombinant protein expression. Among the various parameters considered for such DNA sequence design, individual codon usage (ICU) has been implicated as one of the most crucial factors affecting mRNA translational efficiency. However, previous works have also reported the significant influence of codon pair usage, also known as codon context (CC), on the level of protein expression. Results In this study, we have developed novel computational procedures for evaluating the relative importance of optimizing ICU and CC for enhancing protein expression. By formulating appropriate mathematical expressions to quantify the ICU and CC fitness of a coding sequence, optimization procedures based on genetic algorithm were employed to maximize its ICU and/or CC fitness. Surprisingly, the in silico validation of the resultant optimized DNA sequences for Escherichia coli, Lactococcus lactis, Pichia pastoris and Saccharomyces cerevisiae suggests that CC is a more relevant design criterion than the commonly considered ICU. Conclusions The proposed CC optimization framework can complement and enhance the capabilities of current gene design tools, with potential applications to heterologous protein production and even vaccine development in synthetic biotechnology. PMID:23083100

  18. Expression and detection of LINE-1 ORF-encoded proteins.

    PubMed

    Dai, Lixin; LaCava, John; Taylor, Martin S; Boeke, Jef D

    2014-01-01

    LINE-1 (L1) elements are endogenous retrotransposons active in mammalian genomes. The L1 RNA is bicistronic, encoding two non-overlapping open reading frames, ORF1 and ORF2, whose protein products (ORF1p and ORF2p) bind the L1 RNA to form a ribonucleoprotein (RNP) complex that is presumed to be a critical retrotransposition intermediate. However, ORF2p is expressed at a significantly lower level than ORF1p; these differences are thought to be controlled at the level of translation, due to a low frequency ribosome reinitiation mechanism controlling ORF2 expression. As a result, while ORF1p is readily detectable, ORF2p has previously been very challenging to detect in vitro and in vivo. To address this, we recently tested several epitope tags fused to the N- or C-termini of the ORF proteins in an effort to enable robust detection and affinity purification from native (L1RP) and synthetic (ORFeus-Hs) L1 constructs. An analysis of tagged RNPs from both L1RP and ORFeus-Hs showed similar host-cell-derived protein interactors. Our observations also revealed that the tag sequences affected the retrotransposition competency of native and synthetic L1s differently although they encode identical ORF proteins. Unexpectedly, we observed apparently stochastic expression of ORF2p within seemingly homogenous L1-expressing cell populations.

  19. Escherichia coli Protein Expression System for Acetylcholine Binding Proteins (AChBPs)

    PubMed Central

    Abraham, Nikita; Paul, Blessy; Ragnarsson, Lotten; Lewis, Richard J.

    2016-01-01

    Nicotinic acetylcholine receptors (nAChR) are ligand gated ion channels, identified as therapeutic targets for a range of human diseases. Drug design for nAChR related disorders is increasingly using structure-based approaches. Many of these structural insights for therapeutic lead development have been obtained from co-crystal structures of nAChR agonists and antagonists with the acetylcholine binding protein (AChBP). AChBP is a water soluble, structural and functional homolog of the extracellular, ligand-binding domain of nAChRs. Currently, AChBPs are recombinantly expressed in eukaryotic expression systems for structural and biophysical studies. Here, we report the establishment of an Escherichia coli (E. coli) expression system that significantly reduces the cost and time of production compared to the existing expression systems. E. coli can efficiently express unglycosylated AChBP for crystallography and makes the expression of isotopically labelled forms feasible for NMR. We used a pHUE vector containing an N-terminal His-tagged ubiquitin fusion protein to facilitate AChBP expression in the soluble fractions, and thus avoid the need to recover protein from inclusion bodies. The purified protein yield obtained from the E. coli expression system is comparable to that obtained from existing AChBP expression systems. E. coli expressed AChBP bound nAChR agonists and antagonists with affinities matching those previously reported. Thus, the E. coli expression system significantly simplifies the expression and purification of functional AChBP for structural and biophysical studies. PMID:27304486

  20. The expression of metabolism-related proteins in phyllodes tumors.

    PubMed

    Kwon, Ji Eun; Jung, Woo-Hee; Koo, Ja Seung

    2013-02-01

    The purpose of this study was to investigate the association between the expression of hypoxia-inducible factor (HIF)-1α, insulin-like growth factor (IGF)-1, glucose transporter 1 (Glut-1), carbonic anhydrase IX (CAIX), and monocarboxylate transporter (MCT)4, which are metabolism-related proteins in phyllodes tumors (PTs), and clinicopathologic factors and its implication. We used tissue microarrays to analyze 207 PTs and performed immunohistochemical staining against the glycolysis-related molecules HIF-1α, IGF-1, Glut-1, CAIX, and MCT4. We then compared the immunohistochemical results and clinicopathologic parameters. The expressions of HIF-1α, Glut-1, CAIX, and MCT4 in the stromal component of PTs increased (P = 0.019, P < 0.001, P = 0.045, and P < 0.001, respectively) with increasing tumor grade. According to univariate analysis, factors associated with shorter disease-free survival were Glut-1 expression (P = 0.001) and MCT4 expression (P < 0.001) in the stromal component, and the factors associated with shorter overall survival were IGF-1 expression (P = 0.012), Glut-1 expression (P < 0.001), CAIX expression (P = 0.039), and MCT4 expression (P < 0.001) in the stromal component. Our investigation of stromal expression of the metabolism-related proteins HIF-1α, IGF-1, Glut-1, CAIX, and MCT4 revealed that, as the PT grade increased, the stromal expression of HIF-1α, Glut-1, CAIX, and MCT4 significantly increased. This result suggested that increasing PT grade is associated with increased glycolysis in the stromal component.

  1. Heterologous Expression of Membrane Proteins: Choosing the Appropriate Host

    PubMed Central

    Pochon, Nathalie; Dementin, Sébastien; Hivin, Patrick; Boutigny, Sylvain; Rioux, Jean-Baptiste; Salvi, Daniel; Seigneurin-Berny, Daphné; Richaud, Pierre; Joyard, Jacques; Pignol, David; Sabaty, Monique; Desnos, Thierry; Pebay-Peyroula, Eva; Darrouzet, Elisabeth; Vernet, Thierry; Rolland, Norbert

    2011-01-01

    Background Membrane proteins are the targets of 50% of drugs, although they only represent 1% of total cellular proteins. The first major bottleneck on the route to their functional and structural characterisation is their overexpression; and simply choosing the right system can involve many months of trial and error. This work is intended as a guide to where to start when faced with heterologous expression of a membrane protein. Methodology/Principal Findings The expression of 20 membrane proteins, both peripheral and integral, in three prokaryotic (E. coli, L. lactis, R. sphaeroides) and three eukaryotic (A. thaliana, N. benthamiana, Sf9 insect cells) hosts was tested. The proteins tested were of various origins (bacteria, plants and mammals), functions (transporters, receptors, enzymes) and topologies (between 0 and 13 transmembrane segments). The Gateway system was used to clone all 20 genes into appropriate vectors for the hosts to be tested. Culture conditions were optimised for each host, and specific strategies were tested, such as the use of Mistic fusions in E. coli. 17 of the 20 proteins were produced at adequate yields for functional and, in some cases, structural studies. We have formulated general recommendations to assist with choosing an appropriate system based on our observations of protein behaviour in the different hosts. Conclusions/Significance Most of the methods presented here can be quite easily implemented in other laboratories. The results highlight certain factors that should be considered when selecting an expression host. The decision aide provided should help both newcomers and old-hands to select the best system for their favourite membrane protein. PMID:22216205

  2. Heterologous expression of membrane proteins: choosing the appropriate host.

    PubMed

    Bernaudat, Florent; Frelet-Barrand, Annie; Pochon, Nathalie; Dementin, Sébastien; Hivin, Patrick; Boutigny, Sylvain; Rioux, Jean-Baptiste; Salvi, Daniel; Seigneurin-Berny, Daphné; Richaud, Pierre; Joyard, Jacques; Pignol, David; Sabaty, Monique; Desnos, Thierry; Pebay-Peyroula, Eva; Darrouzet, Elisabeth; Vernet, Thierry; Rolland, Norbert

    2011-01-01

    Membrane proteins are the targets of 50% of drugs, although they only represent 1% of total cellular proteins. The first major bottleneck on the route to their functional and structural characterisation is their overexpression; and simply choosing the right system can involve many months of trial and error. This work is intended as a guide to where to start when faced with heterologous expression of a membrane protein. The expression of 20 membrane proteins, both peripheral and integral, in three prokaryotic (E. coli, L. lactis, R. sphaeroides) and three eukaryotic (A. thaliana, N. benthamiana, Sf9 insect cells) hosts was tested. The proteins tested were of various origins (bacteria, plants and mammals), functions (transporters, receptors, enzymes) and topologies (between 0 and 13 transmembrane segments). The Gateway system was used to clone all 20 genes into appropriate vectors for the hosts to be tested. Culture conditions were optimised for each host, and specific strategies were tested, such as the use of Mistic fusions in E. coli. 17 of the 20 proteins were produced at adequate yields for functional and, in some cases, structural studies. We have formulated general recommendations to assist with choosing an appropriate system based on our observations of protein behaviour in the different hosts. Most of the methods presented here can be quite easily implemented in other laboratories. The results highlight certain factors that should be considered when selecting an expression host. The decision aide provided should help both newcomers and old-hands to select the best system for their favourite membrane protein. © 2011 Bernaudat et al.

  3. Canine Parvovirus VP2 Protein Expressed in Silkworm Pupae Self-Assembles into Virus-Like Particles with High Immunogenicity

    PubMed Central

    Wang, Hua-lei; Liang, Meng; Liang, Hongru; Guo, He; Zhao, Pingsen; Yang, Yu-jiao; Zheng, Xue-xing; Zhang, Zhi-fang; Zhao, Yong-kun; Gao, Yu-wei; Yang, Song-tao; Xia, Xian-zhu

    2014-01-01

    The VP2 structural protein of parvovirus can produce virus-like particles (VLPs) by a self-assembly process in vitro, making VLPs attractive vaccine candidates. In this study, the VP2 protein of canine parvovirus (CPV) was expressed using a baculovirus expression system and assembled into parvovirus-like particles in insect cells and pupae. Electron micrographs of VLPs showed that they were very similar in size and morphology when compared to the wild-type parvovirus. The immunogenicity of the VLPs was investigated in mice and dogs. Mice immunized intramuscularly with purified VLPs, in the absence of an adjuvant, elicited CD4+ and CD8+ T cell responses and were able to elicit a neutralizing antibody response against CPV, while the oral administration of raw homogenates containing VLPs to the dogs resulted in a systemic immune response and long-lasting immunity. These results demonstrate that the CPV-VLPs stimulate both cellular and humoral immune responses, and so CPV-VLPs may be a promising candidate vaccine for the prevention of CPV-associated disease. PMID:24465364

  4. Canine parvovirus VP2 protein expressed in silkworm pupae self-assembles into virus-like particles with high immunogenicity.

    PubMed

    Feng, Hao; Hu, Gui-qiu; Wang, Hua-lei; Liang, Meng; Liang, Hongru; Guo, He; Zhao, Pingsen; Yang, Yu-jiao; Zheng, Xue-xing; Zhang, Zhi-fang; Zhao, Yong-kun; Gao, Yu-wei; Yang, Song-tao; Xia, Xian-zhu

    2014-01-01

    The VP2 structural protein of parvovirus can produce virus-like particles (VLPs) by a self-assembly process in vitro, making VLPs attractive vaccine candidates. In this study, the VP2 protein of canine parvovirus (CPV) was expressed using a baculovirus expression system and assembled into parvovirus-like particles in insect cells and pupae. Electron micrographs of VLPs showed that they were very similar in size and morphology when compared to the wild-type parvovirus. The immunogenicity of the VLPs was investigated in mice and dogs. Mice immunized intramuscularly with purified VLPs, in the absence of an adjuvant, elicited CD4(+) and CD8(+) T cell responses and were able to elicit a neutralizing antibody response against CPV, while the oral administration of raw homogenates containing VLPs to the dogs resulted in a systemic immune response and long-lasting immunity. These results demonstrate that the CPV-VLPs stimulate both cellular and humoral immune responses, and so CPV-VLPs may be a promising candidate vaccine for the prevention of CPV-associated disease.

  5. Optimization of translation profiles enhances protein expression and solubility.

    PubMed

    Hess, Anne-Katrin; Saffert, Paul; Liebeton, Klaus; Ignatova, Zoya

    2015-01-01

    mRNA is translated with a non-uniform speed that actively coordinates co-translational folding of protein domains. Using structure-based homology we identified the structural domains in epoxide hydrolases (EHs) and introduced slow-translating codons to delineate the translation of single domains. These changes in translation speed dramatically improved the solubility of two EHs of metagenomic origin in Escherichia coli. Conversely, the importance of transient attenuation for the folding, and consequently solubility, of EH was evidenced with a member of the EH family from Agrobacterium radiobacter, which partitions in the soluble fraction when expressed in E. coli. Synonymous substitutions of codons shaping the slow-transiting regions to fast-translating codons render this protein insoluble. Furthermore, we show that low protein yield can be enhanced by decreasing the free folding energy of the initial 5'-coding region, which can disrupt mRNA secondary structure and enhance ribosomal loading. This study provides direct experimental evidence that mRNA is not a mere messenger for translation of codons into amino acids but bears an additional layer of information for folding, solubility and expression level of the encoded protein. Furthermore, it provides a general frame on how to modulate and fine-tune gene expression of a target protein.

  6. A characterization of structural proteins expressed by Bombyx mori bidensovirus.

    PubMed

    Lü, Peng; Xing, Yali; Hu, Zhaoyang; Yang, Yanhua; Pan, Ye; Chen, Kangmin; Zhu, Feifei; Zhou, Yajing; Chen, Keping; Yao, Qin

    2017-03-01

    Bombyx mori bidensiovirus (BmBDV) is a species of Bidensovirus that has been was placed into a new genus within the new family Bidnaviridae by the International Committee on Taxonomy of Viruses. BmBDV causes fatal flacherie disease in silkworms, which causes large losses to the sericulture industry. BmBDV contains two sets of complementary linear single-stranded DNAs of approximately 6.5kb (viral DNA 1, VD1) and 6.0kb (viral DNA 2, VD2). VD1 and VD2 are encapsidated in separate icosahedral non-enveloped capsids, which are similar in size and shape. However, the strategies used to express BmBDV structural proteins remains unclear. In this work, a total of six structural proteins were separated by two-dimensional electrophoresis and shown to be encoded by the BmBDV VP gene via mass spectrometry. The transmission electron microscopy results showed that co-expression of the BmBDV VP and SP structural proteins in Spodoptera frugiperda sf9 cells resulted in the formation of 22-24nm virus-like particles. Furthermore, a mutation of the major structural protein-encoding VP gene, in which the second in-frame ATG codon was mutated to GCG, abrogated the production of several structural proteins, indicating that this strategy of expressing BmBDV VP is dependent on a leaky scanning translation mechanism.

  7. Optimization of Translation Profiles Enhances Protein Expression and Solubility

    PubMed Central

    Hess, Anne-Katrin; Saffert, Paul; Liebeton, Klaus; Ignatova, Zoya

    2015-01-01

    mRNA is translated with a non-uniform speed that actively coordinates co-translational folding of protein domains. Using structure-based homology we identified the structural domains in epoxide hydrolases (EHs) and introduced slow-translating codons to delineate the translation of single domains. These changes in translation speed dramatically improved the solubility of two EHs of metagenomic origin in Escherichia coli. Conversely, the importance of transient attenuation for the folding, and consequently solubility, of EH was evidenced with a member of the EH family from Agrobacterium radiobacter, which partitions in the soluble fraction when expressed in E. coli. Synonymous substitutions of codons shaping the slow-transiting regions to fast-translating codons render this protein insoluble. Furthermore, we show that low protein yield can be enhanced by decreasing the free folding energy of the initial 5’-coding region, which can disrupt mRNA secondary structure and enhance ribosomal loading. This study provides direct experimental evidence that mRNA is not a mere messenger for translation of codons into amino acids but bears an additional layer of information for folding, solubility and expression level of the encoded protein. Furthermore, it provides a general frame on how to modulate and fine-tune gene expression of a target protein. PMID:25965266

  8. Raman microscopy of bladder cancer cells expressing green fluorescent protein

    NASA Astrophysics Data System (ADS)

    Mandair, Gurjit S.; Han, Amy L.; Keller, Evan T.; Morris, Michael D.

    2016-11-01

    Gene engineering is a commonly used tool in cellular biology to determine changes in function or expression of downstream targets. However, the impact of genetic modulation on biochemical effects is less frequently evaluated. The aim of this study is to use Raman microscopy to assess the biochemical effects of gene silencing on T24 and UMUC-13 bladder cancer cell lines. Cellular biochemical information related to nucleic acid and lipogenic components was obtained from deconvolved Raman spectra. We show that the green fluorescence protein (GFP), the chromophore that served as a fluorescent reporter for gene silencing, could also be detected by Raman microscopy. Only the gene-silenced UMUC-13 cell lines exhibited low-to-moderate GFP fluorescence as determined by fluorescence imaging and Raman spectroscopic studies. Moreover, we show that gene silencing and cell phenotype had a greater effect on nucleic acid and lipogenic components with minimal interference from GFP expression. Gene silencing was also found to perturb cellular protein secondary structure in which the amount of disorderd protein increased at the expense of more ordered protein. Overall, our study identified the spectral signature for cellular GFP expression and elucidated the effects of gene silencing on cancer cell biochemistry and protein secondary structure.

  9. Developmental expression of Drosophila Wiskott-Aldrich Syndrome family proteins

    PubMed Central

    Rodriguez-Mesa, Evelyn; Abreu-Blanco, Maria Teresa; Rosales-Nieves, Alicia E.; Parkhurst, Susan M.

    2012-01-01

    Background Wiskott-Aldrich Syndrome (WASP) family proteins participate in many cellular processes involving rearrangements of the actin cytoskeleton. To the date, four WASP subfamily members have been described in Drosophila: Wash, WASp, SCAR, and Whamy. Wash, WASp, and SCAR are essential during early Drosophila development where they function in orchestrating cytoplasmic events including membrane-cytoskeleton interactions. A mutant for Whamy has not yet been reported. Results We generated monoclonal antibodies that are specific to Drosophila Wash, WASp, SCAR, and Whamy, and use these to describe their spatial and temporal localization patterns. Consistent with the importance of WASP family proteins in flies, we find that Wash, WASp, SCAR, and Whamy are dynamically expressed throughout oogenesis and embryogenesis. For example, we find that Wash accumulates at the oocyte cortex. WASp is highly expressed in the PNS, while SCAR is the most abundantly expressed in the CNS. Whamy exhibits an asymmetric subcellular localization that overlaps with mitochondria and is highly expressed in muscle. Conclusion All four WASP family members show specific expression patterns, some of which reflect their previously known roles and others revealing new potential functions. The monoclonal antibodies developed offer valuable new tools to investigate how WASP family proteins regulate actin cytoskeleton dynamics. PMID:22275148

  10. Expression of glutamine metabolism-related proteins in thyroid cancer

    PubMed Central

    Kim, Hye Min; Lee, Yu Kyung; Koo, Ja Seung

    2016-01-01

    Purpose This study aimed to investigate the expression of glutamine metabolism-related protein in tumor and stromal compartments among the histologic subtypes of thyroid cancer. Results GLS1 and GDH expression in tumor and stromal compartments were the highest in AC than in other subtypes. Tumoral ASCT2 expression was higher in MC but lower in FC (p < 0.001). In PTC, tumoral GLS1 and tumoral GDH expression was higher in the conventional type than in the follicular variant (p = 0.043 and 0.001, respectively), and in PTC with BRAF V600E mutation than in PTC without BRAF V600E mutation (p<0.001). Stromal GDH positivity was the independent factor associated with short overall survival (hazard ratio: 21.48, 95% confidence interval: 2.178-211.8, p = 0.009). Methods We performed tissue microarrays with 557 thyroid cancer cases (papillary thyroid carcinoma [PTC]: 344, follicular carcinoma [FC]: 112, medullary carcinoma [MC]: 70, poorly differentiated carcinoma [PDC]: 23, and anaplastic carcinoma [AC]: 8) and 152 follicular adenoma (FA) cases. We performed immunohistochemical staining of glutaminolysis-related proteins (glutaminase 1 [GLS1], glutamate dehydrogenase [GDH], and amino acid transporter-2 [ASCT-2]). Conclusion Glutamine metabolism-related protein expression differed among the histologic subtypes of thyroid cancer. PMID:27447554

  11. Combined protein construct and synthetic gene engineering for heterologous protein expression and crystallization using Gene Composer

    SciTech Connect

    Raymond, Amy; Lovell, Scott; Lorimer, Don; Walchli, John; Mixon, Mark; Wallace, Ellen; Thompkins, Kaitlin; Archer, Kimberly; Burgin, Alex; Stewart, Lance

    2009-12-01

    With the goal of improving yield and success rates of heterologous protein production for structural studies we have developed the database and algorithm software package Gene Composer. This freely available electronic tool facilitates the information-rich design of protein constructs and their engineered synthetic gene sequences, as detailed in the accompanying manuscript. In this report, we compare heterologous protein expression levels from native sequences to that of codon engineered synthetic gene constructs designed by Gene Composer. A test set of proteins including a human kinase (P38{alpha}), viral polymerase (HCV NS5B), and bacterial structural protein (FtsZ) were expressed in both E. coli and a cell-free wheat germ translation system. We also compare the protein expression levels in E. coli for a set of 11 different proteins with greatly varied G:C content and codon bias. The results consistently demonstrate that protein yields from codon engineered Gene Composer designs are as good as or better than those achieved from the synonymous native genes. Moreover, structure guided N- and C-terminal deletion constructs designed with the aid of Gene Composer can lead to greater success in gene to structure work as exemplified by the X-ray crystallographic structure determination of FtsZ from Bacillus subtilis. These results validate the Gene Composer algorithms, and suggest that using a combination of synthetic gene and protein construct engineering tools can improve the economics of gene to structure research.

  12. The E4 protein; structure, function and patterns of expression

    SciTech Connect

    Doorbar, John

    2013-10-15

    The papillomavirus E4 open reading frame (ORF) is contained within the E2 ORF, with the primary E4 gene-product (E1{sup ∧}E4) being translated from a spliced mRNA that includes the E1 initiation codon and adjacent sequences. E4 is located centrally within the E2 gene, in a region that encodes the E2 protein′s flexible hinge domain. Although a number of minor E4 transcripts have been reported, it is the product of the abundant E1{sup ∧}E4 mRNA that has been most extensively analysed. During the papillomavirus life cycle, the E1{sup ∧}E4 gene products generally become detectable at the onset of vegetative viral genome amplification as the late stages of infection begin. E4 contributes to genome amplification success and virus synthesis, with its high level of expression suggesting additional roles in virus release and/or transmission. In general, E4 is easily visualised in biopsy material by immunostaining, and can be detected in lesions caused by diverse papillomavirus types, including those of dogs, rabbits and cattle as well as humans. The E4 protein can serve as a biomarker of active virus infection, and in the case of high-risk human types also disease severity. In some cutaneous lesions, E4 can be expressed at higher levels than the virion coat proteins, and can account for as much as 30% of total lesional protein content. The E4 proteins of the Beta, Gamma and Mu HPV types assemble into distinctive cytoplasmic, and sometimes nuclear, inclusion granules. In general, the E4 proteins are expressed before L2 and L1, with their structure and function being modified, first by kinases as the infected cell progresses through the S and G2 cell cycle phases, but also by proteases as the cell exits the cell cycle and undergoes true terminal differentiation. The kinases that regulate E4 also affect other viral proteins simultaneously, and include protein kinase A, Cyclin-dependent kinase, members of the MAP Kinase family and protein kinase C. For HPV16 E1{sup

  13. Eliciting Sound Memories.

    PubMed

    Harris, Anna

    2015-11-01

    Sensory experiences are often considered triggers of memory, most famously a little French cake dipped in lime blossom tea. Sense memory can also be evoked in public history research through techniques of elicitation. In this article