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Sample records for protein expression elicited

  1. 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

  2. Comparison of antibody avidity and titre elicited by peptide as a protein conjugate or as expressed in vaccinia.

    PubMed Central

    Lew, A M; Anders, R F; Edwards, S J; Langford, C J

    1988-01-01

    The immunogenicity of a malaria peptide presented in various forms was tested in terms of the quality and quantity of the antibody response in rabbits. Peptide conjugated to a protein carrier, diphtheria toxoid (DT), required strong adjuvants (e.g. muramyl dipeptide, MDP and Freund's adjuvant, FCA) to elicit high levels of antibody with high avidity. Alum was a poor adjuvant, producing the lowest titre and avidity of antibody compared with all the other groups. Peptide expressed in vaccinia (and without carrier) produced intermediate levels of antibody but the avidity of the antibodies produced were comparable to that produced by peptide conjugates given with muramyl dipeptide. PMID:3056855

  3. 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.

  4. 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

  5. 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

  6. 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. PMID:27551358

  7. 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.

  8. Deregulation of Fragile X-related protein 1 by the lipodystrophic lamin A p.R482W mutation elicits a myogenic gene expression program in preadipocytes.

    PubMed

    Oldenburg, Anja R; Delbarre, Erwan; Thiede, Bernd; Vigouroux, Corinne; Collas, Philippe

    2014-03-01

    The nuclear lamina is implicated in the regulation of various nuclear functions. Several laminopathy-causing mutations in the LMNA gene, notably the p.R482W substitution linked to familial partial lipodystrophy type 2 (FPLD2), are clustered in the immunoglobulin fold of lamin A. We report a functional association between lamin A and fragile X-related protein 1 (FXR1P), a protein of the fragile X-related family involved in fragile X syndrome. Searching for proteins differentially interacting with the immunoglobulin fold of wild-type and R482W mutant lamin A, we identify FXR1P as a novel component of the lamin A protein network. The p.R482W mutation abrogates interaction of FXR1P with lamin A. Fibroblasts from FPLD2 patients display elevated levels of FXR1P and delocalized FXR1P. In human adipocyte progenitors, deregulation of lamin A expression leads to FXR1P up-regulation, impairment of adipogenic differentiation and induction of myogenin expression. FXR1P overexpression also stimulates a myogenic gene expression program in these cells. Our results demonstrate a cross-talk between proteins hitherto implicated in two distinct mesodermal pathologies. We propose a model where the FPLD2 lamin A p.R482W mutation elicits, through up-regulation of FXR1P, a remodeling of an adipogenic differentiation program into a myogenic program.

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

    PubMed

    Merchant, Soroush; Huang, Naiyan; Korbelik, Mladen

    2010-12-01

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

  10. 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

  11. Arbuscular mycorrhizal protein mRNA over-expression in bread wheat seedlings by Trichoderma harzianum Raifi (KRL-AG2) elicitation.

    PubMed

    Al-Asbahi, Adnan A S

    2012-02-25

    Association between arbuscular mycorrhizal fungi (AMF) and majority of terrestrial plant species provides many benefits to plants that range from stress alleviation and bioremediation in soils polluted with heavy metals to plant growth promotion and yield quantity. Some non-arbuscular mycorrhizal fungi such as, Trichoderma harzianum, are known to enhance the AMF symbiosis with vascular plants. However, information about their role in AMF symbiosis is still limited. Shoots of (Avocet S) wheat seedlings were sprayed with the fungal culture filtrate and gene expression patterns were analyzed in the treated tissues. An increase in the level of mRNA of arbuscular mycorrhizal protein comparing with control was found. The over-expression of this protein in wheat tissues might contribute in initiation of AMF colonization in wheat tissues. The result of this study can spark future researches to elucidate possible role of this protein in the symbiotic interaction mechanisms between soil AMF and various plant roots.

  12. 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.

  13. The L1 protein of human papilloma virus 16 expressed by a fowlpox virus recombinant can assemble into virus-like particles in mammalian cell lines but elicits a non-neutralising humoral response.

    PubMed

    Bissa, Massimiliano; Zanotto, Carlo; Pacchioni, Sole; Volonté, Luca; Venuti, Aldo; Lembo, David; De Giuli Morghen, Carlo; Radaelli, Antonia

    2015-04-01

    Human papilloma virus (HPV)-16 is the prevalent genotype associated with cervical tumours. Virus-like-particle (VLP)-based vaccines have proven to be effective in limiting new infections of high-risk HPVs, but their high cost has hampered their use, especially in the poor developing countries. Avipox-based recombinants are replication-restricted to avian species and represent efficient and safe vectors also for immunocompromised hosts, as they can elicit a complete immune response. A new fowlpox virus recombinant encoding HPV-L1 (FPL1) was engineered and evaluated side-by-side with a FP recombinant co-expressing L1 and green fluorescent protein (FPL1GFP) for correct expression of L1 in vitro in different cell lines, as confirmed by Western blotting, immunofluorescence, real-time PCR, and electron microscopy. Mice were also immunised to determine its immunogenicity. Here, we demonstrate that the FPL1 recombinant better expresses L1 in the absence of GFP, correctly assembles structured capsomers into VLPs, and elicits an immune response in a preclinical animal model. To our knowledge, this is the first report of HPV VLPs assembled in eukaryotic cells using an avipox recombinant.

  14. 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…

  15. Vaccination with a recombinant protein encoding the tumor-specific antigen NY-ESO-1 elicits an A2/157-165-specific CTL repertoire structurally distinct and of reduced tumor reactivity than that elicited by spontaneous immune responses to NY-ESO-1-expressing Tumors.

    PubMed

    Bioley, Gilles; Guillaume, Philippe; Luescher, Immanuel; Bhardwaj, Nina; Mears, Gregory; Old, Lloyd; Valmori, Danila; Ayyoub, Maha

    2009-01-01

    In a recent vaccination trial assessing the immunogenicity of an NY-ESO-1 (ESO) recombinant protein administered with Montanide and CpG, we have obtained evidence that this vaccine induces specific cytolytic T lymphocytes (CTL) in half of the patients. Most vaccine-induced CTLs were directed against epitopes located in the central part of the protein, between amino acids 81 and 110. This immunodominant region, however, is distinct from another ESO CTL region, 157-165, that is a frequent target of spontaneous CTL responses in A2+ patients bearing ESO tumors. In this study, we have investigated the CTL responses to ESO 157-165 in A2+ patients vaccinated with the recombinant protein. Our data indicate that after vaccination with the protein, CTL responses to ESO 157-165 are induced in some, but not all, A2+ patients. ESO 157-165-specific CTLs induced by vaccination with the ESO protein were functionally heterogeneous in terms of tumor recognition and often displayed decreased tumor reactivity as compared with ESO 157-165-specific CTLs isolated from patients with spontaneous immune responses to ESO. Remarkably, protein-induced CTLs used T-cell receptors similar to those previously isolated from patients vaccinated with synthetic ESO peptides (Vbeta4.1) and distinct from those used by highly tumor-reactive CTLs isolated from patients with spontaneous immune responses (Vbeta1.1, Vbeta8.1, and Vbeta13.1). Together, these results demonstrate that vaccination with the ESO protein elicits a repertoire of ESO 157-165-specific CTLs bearing T-cell receptors that are structurally distinct from those of CTLs found in spontaneous immune responses to the antigen and that are heterogeneous in terms of tumor reactivity, being often poorly tumor reactive.

  16. Surface expression and rapid internalization of macrosialin (mouse CD68) on elicited mouse peritoneal macrophages.

    PubMed

    Kurushima, H; Ramprasad, M; Kondratenko, N; Foster, D M; Quehenberger, O; Steinberg, D

    2000-01-01

    Macrosialin, the mouse homolog of human CD68, is a heavily glycosylated transmembrane protein found almost exclusively in macrophages. Its function remains uncertain. It has a high affinity for oxidized low-density lipoprotein (LDL) in ligand blots and antibodies against the human homolog, CD68, inhibit the binding of oxidized LDL to a human monocyte-derived cell line (THP-1). However, there is still controversy as to whether macrosialin, found predominantly in late endosomes, is expressed at all on the plasma membrane. The present studies, done in thioglycollate-elicited peritoneal macrophages, confirm that macrosialin is predominantly intracellular but show clearly that 10-15% of it is expressed on the cell surface. Exchange with intracellular pools occurs at an extremely high rate. The results are compatible with a surface function, including internalization of bound ligands or adhesion to surfaces.

  17. 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.

  18. BcIEB1, a Botrytis cinerea secreted protein, elicits a defense response in plants.

    PubMed

    Frías, Marcos; González, Mario; González, Celedonio; Brito, Nélida

    2016-09-01

    BcIEB1 is a very abundant protein in the secretome of Botrytis cinerea but it has no known function and no similarity to any characterized protein family. Previous results suggested that this protein is an elicitor of the plant defense system. In this work we have generated loss-of-function B. cinerea mutants lacking BcIEB1 and we have expressed the protein in yeast to assay its activity on plants. Analysis of the Δbcieb1 mutants did not result in any observable phenotype, including no difference in the virulence on a variety of hosts. However, when BcIEB1 was applied to plant tissues it produced necrosis as well as a whole range of symptoms: inhibition of seedling growth in Arabidopsis and tobacco, ion leakage from tobacco leaf disks, a ROS burst, cell death and autofluorescence in onion epidermis, as well as the expression of defense genes in tobacco. Moreover, tobacco plants treated with BcIEB1 showed an increased systemic resistance to B. cinerea. A small 35-amino acids peptide derived from a conserved region of BcIEB1 is almost as active on plants as the whole protein. These results clearly indicate that BcIEB1 elicits plant defenses, probably as a consequence of its recognition as a pathogen associated molecular pattern. PMID:27457989

  19. [Protein expression and purification].

    PubMed

    Růčková, E; Müller, P; Vojtěšek, B

    2014-01-01

    Production of recombinant proteins is essential for many applications in both basic research and also in medicine, where recombinant proteins are used as pharmaceuticals. This review summarizes procedures involved in recombinant protein expression and purification, including molecular cloning of target genes into expression vectors, selection of the appropriate expression system, and protein purification techniques. Recombinant DNA technology allows protein engineering to modify protein stability, activity and function or to facilitate protein purification by affinity tag fusions. A wide range of cloning systems enabling fast and effective design of expression vectors is currently available. A first choice of protein expression system is usually the bacteria Escherichia coli. The main advantages of this prokaryotic expression system are low cost and simplicity; on the other hand this system is often unsuitable for production of complex mammalian proteins. Protein expression mediated by eukaryotic cells (yeast, insect and mammalian cells) usually produces properly folded and posttranslationally modified proteins. How-ever, cultivation of insect and, especially, mammalian cells is time consuming and expensive. Affinity tagged recombinant proteins are purified efficiently using affinity chromatography. An affinity tag is a protein or peptide that mediates specific binding to a chromatography column, unbound proteins are removed during a washing step and pure protein is subsequently eluted. PMID:24945544

  20. Co-expression of four baculovirus proteins, IE1, LEF3, P143, and PP31, elicits a cellular chromatin-containing reticulate structure in the nuclei of uninfected cells

    SciTech Connect

    Nagamine, Toshihiro; Abe, Atsushi; Suzuki, Takehiro; Dohmae, Naoshi; Matsumoto, Shogo

    2011-08-15

    Baculovirus DNA replication, transcription, and nucleocapsid assembly occur within a subnuclear structure called the virogenic stroma (VS) that consists of two subcompartments. Specific components of the VS sub-compartments have not been identified except for PP31, a DNA-binding protein that localizes specifically to the electron-dense region of VS. Here, we investigate the dynamic structure of VS using a GFP-tagged PP31 molecule (GFP-PP31). GFP-PP31 localizes to the VS throughout the course of infection. At later times post-infection, a PP31 reticulum distributed within VS was also apparent, indicating that VS sub-compartments compose a reticulate structure. Transient expression of PP31 with the viral proteins, IE1, LEF3, and P143, in uninfected cells resulted in the formation of a reticulate structure containing cellular chromatin and the spatial arrangements of the four proteins within the induced reticulum were the same as those within VS reticulum, suggesting that the two reticula are formed by a similar mechanism.

  1. 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

  2. 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.

  3. Emotional expressions preferentially elicit implicit evaluations of faces also varying in race or age.

    PubMed

    Craig, Belinda M; Lipp, Ottmar V; Mallan, Kimberley M

    2014-10-01

    Both facial cues of group membership (race, age, and sex) and emotional expressions can elicit implicit evaluations to guide subsequent social behavior. There is, however, little research addressing whether group membership cues or emotional expressions are more influential in the formation of implicit evaluations of faces when both cues are simultaneously present. The current study aimed to determine this. Emotional expressions but not race or age cues elicited implicit evaluations in a series of affective priming tasks with emotional Caucasian and African faces (Experiments 1 and 2) and young and old faces (Experiment 3). Spontaneous evaluations of group membership cues of race and age only occurred when those cues were task relevant, suggesting the preferential influence of emotional expressions in the formation of implicit evaluations of others when cues of race or age are not salient. Implications for implicit prejudice, face perception, and person construal are discussed. PMID:25046242

  4. 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. PMID:26780420

  5. 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.

  6. 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

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

    PubMed

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

    2016-01-01

    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

  8. 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

  9. 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

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

    PubMed

    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.

  11. 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.

  12. Invasion-inhibitory antibodies elicited by immunization with Plasmodium vivax apical membrane antigen-1 expressed in Pichia pastoris yeast.

    PubMed

    Vicentin, Elaine C; Françoso, Kátia S; Rocha, Mariana V; Iourtov, Dmitri; Dos Santos, Fernanda L; Kubrusly, Flávia S; Sakauchi, Maria A; Raw, Isaias; Nosten, Francois; Rénia, Laurent; Rodrigues, Mauricio M; Russell, Bruce; Soares, Irene S

    2014-03-01

    In a recent vaccine trial performed with African children, immunization with a recombinant protein based on Plasmodium falciparum apical membrane antigen 1 (AMA-1) conferred a significant degree of strain-specific resistance against malaria. To contribute to the efforts of generating a vaccine against Plasmodium vivax malaria, we expressed the ectodomain of P. vivax AMA-1 (PvAMA-1) as a secreted soluble protein in the methylotrophic yeast Pichia pastoris. Recognized by a high percentage of sera from individuals infected by P. vivax, this recombinant protein was found to have maintained its antigenicity. The immunogenicity of this protein was evaluated in mice using immunization protocols that included homologous and heterologous prime-boost strategies with plasmid DNA and recombinant protein. We used the following formulations containing different adjuvants: aluminum salts (Alum), Bordetella pertussis monophosphoryl lipid A (MPLA), flagellin FliC from Salmonella enterica serovar Typhimurium, saponin Quil A, or incomplete Freund's adjuvant (IFA). The formulations containing the adjuvants Quil A or IFA elicited the highest IgG antibody titers. Significant antibody titers were also obtained using a formulation developed for human use containing MPLA or Alum plus MPLA. Recombinant PvAMA-1 produced under "conditions of good laboratory practice" provided a good yield, high purity, low endotoxin levels, and no microbial contaminants and reproduced the experimental immunizations. Most relevant for vaccine development was the fact that immunization with PvAMA-1 elicited invasion-inhibitory antibodies against different Asian isolates of P. vivax. Our results show that AMA-1 expressed in P. pastoris is a promising antigen for use in future preclinical and clinical studies.

  13. The critical protein interactions and structures that elicit growth deregulation in cancer and viral replication.

    PubMed

    Ou, Horng D; May, Andrew P; O'Shea, Clodagh C

    2011-01-01

    One of the greatest challenges in biomedicine is to define the critical targets and network interactions that are subverted to elicit growth deregulation in human cells. Understanding and developing rational treatments for cancer requires a definition of the key molecular targets and how they interact to elicit the complex growth deregulation phenotype. Viral proteins provide discerning and powerful probes to understand both how cells work and how they can be manipulated using a minimal number of components. The small DNA viruses have evolved to target inherent weaknesses in cellular protein interaction networks to hijack the cellular DNA and protein replication machinery. In the battle to escape the inevitability of senescence and programmed cell death, cancers have converged on similar mechanisms, through the acquisition and selection of somatic mutations that drive unchecked cellular replication in tumors. Understanding the dynamic mechanisms through which a minimal number of viral proteins promote host cells to undergo unscheduled and pathological replication is a powerful strategy to identify critical targets that are also disrupted in cancer. Viruses can therefore be used as tools to probe the system-wide protein-protein interactions and structures that drive growth deregulation in human cells. Ultimately this can provide a path for developing system context-dependent therapeutics. This review will describe ongoing experimental approaches using viruses to study pathways deregulated in cancer, with a particular focus on viral cellular protein-protein interactions and structures. PMID:21061422

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

    PubMed

    Wood, Charles E; Boue, Stephen M; Collins-Burow, Bridgette M; Rhodes, Lyndsay V; Register, Thomas C; Cline, J Mark; Dewi, Fitriya N; Burow, Matthew E

    2012-01-11

    Glyceollins are stress-induced compounds in soybeans with bioactive properties distinct from parent soy isoflavones. The goals of this study were to evaluate the effects of dietary glyceollin-enriched and standard soy protein isolates and identify candidate target pathways of glyceollins on transcriptional profiles within mammary gland tissue. Thirty female postmenopausal cynomolgus monkeys were randomized to diets containing one of three protein sources for 3 weeks: (1) control casein/lactalbumin (C/L), (2) standard soy protein containing 194 mg/day isoflavones (SOY), and (3) glyceollin-enriched soy protein containing 189 mg/day isoflavones + 134 mg/day glyceollins (GLY). All diets contained a physiologic dose of estradiol (E2) (1 mg/day). All doses are expressed in human equivalents scaled by caloric intake. Relative to the control C/L diet, the GLY diet resulted in greater numbers of differentially regulated genes, which showed minimal overlap with those of SOY. Effects of GLY related primarily to pathways involved in lipid and carbohydrate metabolism, including peroxisome proliferator-activated receptor (PPAR)-γ and AMP-activated protein kinase (AMPK) signaling, adipocytokine expression, triglyceride synthesis, and lipase activity. Notable genes upregulated by the GLY diet included PPAR-γ, adiponectin, leptin, lipin 1, and lipoprotein lipase. The GLY diet also resulted in lower serum total cholesterol, specifically nonhigh-density lipoprotein cholesterol, and increased serum triglycerides as compared to the C/L diet. No effects of GLY or SOY were seen on serum insulin, adipocytokines, or vascular and bone turnover markers. These preliminary findings suggest that glyceollin-enriched soy protein has divergent effects from standard soy with some specificity for adipocyte activity and nutrient metabolism.

  15. A Lentiviral Vector Expressing Japanese Encephalitis Virus-like Particles Elicits Broad Neutralizing Antibody Response in Pigs

    PubMed Central

    Souque, Philippe; Frenkiel, Marie-Pascale; Paulous, Sylvie; Garcìa-Nicolàs, Obdulio; Summerfield, Artur; Charneau, Pierre; Desprès, Philippe

    2015-01-01

    Background Japanese encephalitis virus (JEV) is the major cause of viral encephalitis in Southeast Asia. Vaccination of domestic pigs has been suggested as a “one health” strategy to reduce viral disease transmission to humans. The efficiency of two lentiviral TRIP/JEV vectors expressing the JEV envelope prM and E glycoproteins at eliciting protective humoral response was assessed in a mouse model and piglets. Methodology/Principal Findings A gene encoding the envelope proteins prM and E from a genotype 3 JEV strain was inserted into a lentiviral TRIP vector. Two lentiviral vectors TRIP/JEV were generated, each expressing the prM signal peptide followed by the prM protein and the E glycoprotein, the latter being expressed either in its native form or lacking its two C-terminal transmembrane domains. In vitro transduction of cells with the TRIP/JEV vector expressing the native prM and E resulted in the efficient secretion of virus-like particles of Japanese encephalitis virus. Immunization of BALB/c mice with TRIP/JEV vectors resulted in the production of IgGs against Japanese encephalitis virus, and the injection of a second dose one month after the prime injection greatly boosted antibody titers. The TRIP/JEV vectors elicited neutralizing antibodies against JEV strains belonging to genotypes 1, 3, and 5. Immunization of piglets with two doses of the lentiviral vector expressing JEV virus-like particles led to high titers of anti-JEV antibodies, that had efficient neutralizing activity regardless of the JEV genotype tested. Conclusions/Significance Immunization of pigs with the lentiviral vector expressing JEV virus-like particles is particularly efficient to prime antigen-specific humoral immunity and trigger neutralizing antibody responses against JEV genotypes 1, 3, and 5. The titers of neutralizing antibodies elicited by the TRIP/JEV vector are sufficient to confer protection in domestic pigs against different genotypes of JEV and this could be of a great

  16. 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.

  17. 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

  18. 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.

  19. Distinct transcriptional responses elicited by unfolded nuclear or cytoplasmic protein in mammalian cells

    PubMed Central

    Miyazaki, Yusuke; Chen, Ling-chun; Chu, Bernard W; Swigut, Tomek; Wandless, Thomas J

    2015-01-01

    Eukaryotic cells possess a variety of signaling pathways that prevent accumulation of unfolded and misfolded proteins. Chief among these is the heat shock response (HSR), which is assumed to respond to unfolded proteins in the cytosol and nucleus alike. In this study, we probe this axiom further using engineered proteins called ‘destabilizing domains’, whose folding state we control with a small molecule. The sudden appearance of unfolded protein in mammalian cells elicits a robust transcriptional response, which is distinct from the HSR and other known pathways that respond to unfolded proteins. The cellular response to unfolded protein is strikingly different in the nucleus and the cytosol, although unfolded protein in either compartment engages the p53 network. This response provides cross-protection during subsequent proteotoxic stress, suggesting that it is a central component of protein quality control networks, and like the HSR, is likely to influence the initiation and progression of human pathologies. DOI: http://dx.doi.org/10.7554/eLife.07687.001 PMID:26314864

  20. Protective Humoral Immunity Elicited by a Needle-Free Malaria Vaccine Comprised of a Chimeric Plasmodium falciparum Circumsporozoite Protein and a Toll-Like Receptor 5 Agonist, Flagellin

    PubMed Central

    Carapau, Daniel; Mitchell, Robert; Nacer, Adéla; Shaw, Alan; Othoro, Caroline; Frevert, Ute

    2013-01-01

    Immunization with Plasmodium sporozoites can elicit high levels of sterile immunity, and neutralizing antibodies from protected hosts are known to target the repeat region of the circumsporozoite (CS) protein on the parasite surface. CS-based subunit vaccines have been hampered by suboptimal immunogenicity and the requirement for strong adjuvants to elicit effective humoral immunity. Pathogen-associated molecular patterns (PAMPs) that signal through Toll-like receptors (TLRs) can function as potent adjuvants for innate and adaptive immunity. We examined the immunogenicity of recombinant proteins containing a TLR5 agonist, flagellin, and either full-length or selected epitopes of the Plasmodium falciparum CS protein. Mice immunized with either of the flagellin-modified CS constructs, administered intranasally (i.n.) or subcutaneously (s.c.), developed similar levels of malaria-specific IgG1 antibody and interleukin-5 (IL-5)-producing T cells. Importantly, immunization via the i.n. but not the s.c. route elicited sporozoite neutralizing antibodies capable of inhibiting >90% of sporozoite invasion in vitro and in vivo, as measured using a transgenic rodent parasite expressing P. falciparum CS repeats. These findings demonstrate that functional sporozoite neutralizing antibody can be elicited by i.n. immunization with a flagellin-modified P. falciparum CS protein and raise the potential of a scalable, safe, needle-free vaccine for the 40% of the world's population at risk of malaria. PMID:24042110

  1. 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

  2. Vaccination with major outer membrane protein proteosomes elicits protection in mice against a Chlamydia respiratory challenge.

    PubMed

    Tifrea, Delia F; Pal, Sukumar; Toussi, Deana N; Massari, Paola; de la Maza, Luis M

    2013-11-01

    Vaccines formulated with the Chlamydia muridarum native major outer membrane protein (nMOMP) have so far been shown to elicit the most robust protection against this pathogen. nMOMP is a membrane protein and therefore, detergents are used to keep it in solution. Detergents however, have toxic effects. To address this limitation, we tested a nMOMP proteosome vaccine and compared its ability to elicit protection against nMOMP solubilized in the detergent Z3-14. The two preparations were formulated with or without CpG + Montanide (C/M). As a control antigen we used ovalbumin. Mice vaccinated with nMOMP developed strong humoral and cell mediated Chlamydia-specific immune responses. Based on the IgG2a/IgG1 levels in serum and amounts of IFN-γ in splenocytes supernatants the immune responses were predominantly Th1-biased. The animals were subsequently challenged intranasally with 2 × 10(3)Chlamydia inclusion forming units (IFU) and the course of the infection was followed for 10 days when the mice were euthanized. Based on changes in body weight, weight of the lungs and number of IFU recovered from the lungs, mice immunized with nMOMP-Ps and nMOMP + Z3-14 adjuvanted with C/M showed the most robust protection. In summary, nMOMP-Ps should be considered as Chlamydia vaccine candidates.

  3. Chlamydial disease pathogenesis. Ocular hypersensitivity elicited by a genus-specific 57-kD protein

    PubMed Central

    1989-01-01

    Recurrent or persistent infections with Chlamydia trachomatis are thought to provide the antigenic stimulus for the chronic inflammation associated with blinding trachoma. We used the guinea pig model of inclusion conjunctivitis to identify chlamydial antigens that may be involved in this deleterious immune response. We purified from chlamydial elementary bodies a genus-specific 57-kD protein that elicited an ocular hypersensitivity response when placed topically onto the conjunctiva of ocular immune guinea pigs. This response was characterized by a predominantly mononuclear macrophage and lymphocyte cellular infiltrate of the submucosal epithelium. The clinical and histological findings were consistent with those of a delayed hypersensitivity response. These data demonstrated that the 57-kD chlamydial protein was a potent stimulator of ocular delayed hypersensitivity. Our findings may be critical to understanding the pathogenesis of the debilitating chlamydial diseases associated with chronic inflammation, such as trachoma and many urogenital syndromes. PMID:2926323

  4. Leptospira Protein Expression During Infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  5. 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

  6. Eliciting candidate anatomical routes for protein interactions: a scenario from endocrine physiology

    PubMed Central

    2013-01-01

    Background In this paper, we use: i) formalised anatomical knowledge of connectivity between body structures and ii) a formal theory of physiological transport between fluid compartments in order to define and make explicit the routes followed by proteins to a site of interaction. The underlying processes are the objects of mathematical models of physiology and, therefore, the motivation for the approach can be understood as using knowledge representation and reasoning methods to propose concrete candidate routes corresponding to correlations between variables in mathematical models of physiology. In so doing, the approach projects physiology models onto a representation of the anatomical and physiological reality which underpins them. Results The paper presents a method based on knowledge representation and reasoning for eliciting physiological communication routes. In doing so, the paper presents the core knowledge representation and algorithms using it in the application of the method. These are illustrated through the description of a prototype implementation and the treatment of a simple endocrine scenario whereby a candidate route of communication between ANP and its receptors on the external membrane of smooth muscle cells in renal arterioles is elicited. The potential of further development of the approach is illustrated through the informal discussion of a more complex scenario. Conclusions The work presented in this paper supports research in intercellular communication by enabling knowledge‐based inference on physiologically‐related biomedical data and models. PMID:23590598

  7. 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

  8. Circadian RNA expression elicited by 3'-UTR IRAlu-paraspeckle associated elements.

    PubMed

    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. PMID:27441387

  9. 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

  10. Co-opting the unfolded protein response to elicit olfactory receptor feedback

    PubMed Central

    Dalton, Ryan P.; Lyons, David B.

    2013-01-01

    Summary Olfactory receptor (OR) expression requires the transcriptional activation of one out of thousands of OR alleles and a feedback signal that preserves this transcriptional choice. The mechanism by which olfactory sensory neurons (OSNs) detect ORs to signal to the nucleus remains elusive. Here, we show that OR proteins generate this feedback by activating the unfolded protein response (UPR). OR expression induces Perk-mediated phosphorylation of the translation initiation factor eif2α causing selective translation of Activating Transcription Factor 5 (ATF5). ATF5 induces the transcription of Adenylyl Cyclase 3 (Adcy3), which relieves the UPR. Our data provide a novel role for the UPR in defining neuronal identity and cell fate commitment and support a two-step model for the feedback signal: first OR protein, as a stress stimulus, alters the translational landscape of the OSN and induces Adcy3 expression; then, Adcy3 relieves that stress, restores global translation and makes OR choice permanent. PMID:24120133

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  12. Impact of elicited mood on movement expressivity during a fitness task.

    PubMed

    Giraud, Tom; Focone, Florian; Isableu, Brice; Martin, Jean-Claude; Demulier, Virginie

    2016-10-01

    The purpose of the present study was to evaluate the impact of four mood conditions (control, positive, negative, aroused) on movement expressivity during a fitness task. Motion capture data from twenty individuals were recorded as they performed a predefined motion sequence. Moods were elicited using task-specific scenarii to keep a valid context. Movement qualities inspired by Effort-Shape framework (Laban & Ullmann, 1971) were computed (i.e., Impulsiveness, Energy, Directness, Jerkiness and Expansiveness). A reduced number of computed features from each movement quality was selected via Principal Component Analyses. Analyses of variance and Generalized Linear Mixed Models were used to identify movement characteristics discriminating the four mood conditions. The aroused mood condition was strongly associated with increased mean Energy compared to the three other conditions. The positive and negative mood conditions showed more subtle differences interpreted as a result of their moderate activation level. Positive mood was associated with more impulsive movements and negative mood was associated with more tense movements (i.e., reduced variability and increased Jerkiness). Findings evidence the key role of movement qualities in capturing motion signatures of moods and highlight the importance of task context in their interpretations.

  13. Gatekeepers of Chromatin: Small Metabolites Elicit Big Changes in Gene Expression

    PubMed Central

    Kaochar, Salma; Tu, Benjamin P.

    2012-01-01

    Eukaryotes are constantly fine-tuning their gene expression programs in response to the demands of the environment and the availability of nutrients. Such dynamic regulation of the genome necessitates versatile chromatin architecture. Rapid changes in transcript levels are brought about via a wide range of posttranslational modifications of the histone proteins that control chromatin structure. Many enzymes responsible for these modifications have been identified and they require various metabolic cofactors or substrates for their activity. Herein, we highlight recent developments that have begun to reveal particular cellular metabolites that might in fact be underappreciated regulators of gene expression through their ability to modulate particular histone modifications. PMID:22944281

  14. 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

  15. DOWNREGULATION OF HYPOTHALAMIC INSULIN RECEPTOR EXPRESSION ELICITS DEPRESSIVE-LIKE BEHAVIORS IN RATS

    PubMed Central

    Grillo, Claudia A.; Piroli, Gerardo G.; Kaigler, Kris F.; Wilson, Steven P.; Wilson, Marlene A.; Reagan, Lawrence P.

    2011-01-01

    Ongoing epidemiological studies estimate that greater than 60% of the adult US population may be categorized as either overweight or obese. There is a growing appreciation that the complications of obesity extend to the central nervous system (CNS) and may result in increased risk for neurological co-morbidities like depressive illness. One potential mechanistic mediator linking obesity and depressive illness is the adipocyte derived hormone leptin. We previously demonstrated that lentivirus-mediated downregulation of hypothalamic insulin receptors increases body weight, adiposity and plasma leptin levels, which is consistent with features of the metabolic syndrome. Using this novel model of obesity, we examined performance in the forced swim test (FST), the sucrose preference test and the elevated plus maze (EPM), approaches that are often used as measures of depressive-like and anxiety-like behaviors, in rats that received third ventricular injections of either an insulin receptor antisense lentivirus (hypo-IRAS) or a control lentivirus (hypo-Con). Hypo-IRAS rats exhibited significant increases in immobility time and corresponding decreases in active behaviors in the FST and exhibited anhedonia as measured by decreased sucrose intake compared to hypo-Con rats. Hypo-IRAS rats also exhibited increases in anxiety-like behaviors in the EPM. Plasma, hippocampal and amygdalar brain-derived neurotrophic factor (BDNF) levels were reduced in hypo-IRAS rats, suggesting that the obesity/hyperleptinemic phenotype may elicit this behavioral phenotype through modulation of neurotrophic factor expression. Collectively, these data support the hypothesis for an increased risk for mood disorders in obesity, which may be related to decreased expression of hippocampal and amygdalar BDNF. PMID:21458499

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 ...

  17. 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.

  18. Relating protein adduction to gene expression changes: a systems approach

    PubMed Central

    Zhang, Bing; Shi, Zhiao; Duncan, Dexter T; Prodduturi, Naresh; Marnett, Lawrence J; Liebler, Daniel C

    2013-01-01

    Modification of proteins by reactive electrophiles such as the 4-hydroxy-2-nonenal (HNE) plays a critical role in oxidant-associated human diseases. However, little is known about protein adduction and the mechanism by which protein damage elicits adaptive effects and toxicity. We developed a systems approach for relating protein adduction to gene expression changes through the integration of protein adduction, gene expression, protein-DNA interaction, and protein-protein interaction data. Using a random walk strategy, we expanded a list of responsive transcription factors inferred from gene expression studies to upstream signaling networks, which in turn allowed overlaying protein adduction data on the network for the prediction of stress sensors and their associated regulatory mechanisms. We demonstrated the general applicability of transcription factor-based signaling network inference using 103 known pathways. Applying our workflow on gene expression and protein adduction data from HNE-treatment not only rediscovered known mechanisms of electrophile stress but also generated novel hypotheses regarding protein damage sensors. Although developed for analyzing protein adduction data, the framework can be easily adapted for phosphoproteomics and other types of protein modification data. PMID:21594272

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

    PubMed Central

    Gilbert, Merle L.; Yang, Linghai; Su, Thomas

    2015-01-01

    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. PMID:25587115

  20. 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.

  1. 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

    2015-01-01

    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

  2. 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

  3. 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.

  4. Comparative Analysis of AhR-Mediated TCDD-Elicited Gene Expression in Human Liver Adult Stem Cells

    PubMed Central

    Kim, Suntae; Dere, Edward; Burgoon, Lyle D.; Chang, Chia-Cheng; Zacharewski, Timothy R.

    2009-01-01

    Time course and dose-response studies were conducted in HL1-1 cells, a human liver cell line with stem cell–like characteristics, to assess the differential gene expression elicited by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) compared with other established models. Cells were treated with 0.001, 0.01, 0.1, 1, 10, or 100nM TCDD or dimethyl sulfoxide vehicle control for 12 h for the dose-response study, or with 10nM TCDD or vehicle for 1, 2, 4, 8, 12, 24, or 48 h for the time course study. Elicited changes were monitored using a human cDNA microarray with 6995 represented genes. Empirical Bayes analysis identified 144 genes differentially expressed at one or more time points following treatment. Most genes exhibited dose-dependent responses including CYP1A1, CYP1B1, ALDH1A3, and SLC7A5 genes. Comparative analysis of HL1-1 differential gene expression to human HepG2 data identified 74 genes with comparable temporal expression profiles including 12 putative primary responses. HL1-1–specific changes were related to lipid metabolism and immune responses, consistent with effects elicited in vivo. Furthermore, comparative analysis of HL1-1 cells with mouse Hepa1c1c7 hepatoma cell lines and C57BL/6 hepatic tissue identified 18 and 32 commonly regulated orthologous genes, respectively, with functions associated with signal transduction, transcriptional regulation, metabolism and transport. Although some common pathways are affected, the results suggest that TCDD elicits species- and model-specific gene expression profiles. PMID:19684285

  5. 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.

  6. 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

  7. Fish reovirus GCReV-109 VP33 protein elicits protective immunity in rare minnows.

    PubMed

    Liu, Jia; Pei, Chao; Gao, Xiao-chan; Chen, Zhong-yuan; Zhang, Qi-Ya

    2016-03-01

    Grass carp reovirus strain 109 (GCReV-109) was previously isolated from a grass carp (Ctenopharyngodon idellus) with hemorrhagic disease, and its complete genome has been sequenced. However, the infectivity of GCReV-109 has not been studied, and the viral protein VP33, encoded on genome segment S11, had no detectable sequence homology to other known reovirus proteins. In this study, we characterized GCReV-109 infections in vivo and in vitro, as well as the VP33 protein. Infectivity analysis showed that GCReV-109 caused severe hemorrhagic disease and 100% mortality at dilutions up to 10(-4) in rare minnows (Gobiocypris rarus) by 8 days postinfection, but no visible cytopathic effect was observed in GCReV-109-infected subcultured grass carp muscle (GCM) cells. To confirm that GCReV-109 could be propagated in GCM cells, three virus genome segments were detected by RT-PCR, and large numbers of virus particles were observed by transmission electron microscopy in samples from the infected GCM cells. The suspension of GCReV-109-infected GCM cells was pathogenic to rare minnows. VP33 protein was expressed and purified for generation of an anti-VP33 antiserum. In western blot analysis of purified GCReV-109 particles, the antiserum specifically recognized a protein band (approximately 33 kDa). This revealed that VP33 is a major structural protein of GCReV-109 that might have immunogenic properties. The protective efficacy of the anti-VP33 antiserum against GCReV-109 infection was tested. The death of infected fish was delayed and the mortality fell to 10% when fish were treated with the anti-VP33 antiserum, suggesting that it might be useful for the prevention and control of fish reoviral disease. PMID:26615551

  8. Fowlpox virus recombinants expressing HPV-16 E6 and E7 oncogenes for the therapy of cervical carcinoma elicit humoral and cell-mediated responses in rabbits

    PubMed Central

    2010-01-01

    Background Around half million new cases of cervical cancer arise each year, making the development of an effective therapeutic vaccine against HPV a high priority. As the E6 and E7 oncoproteins are expressed in all HPV-16 tumour cells, vaccines expressing these proteins might clear an already established tumour and support the treatment of HPV-related precancerous lesions. Methods Three different immunisation regimens were tested in a pre-clinical trial in rabbits to evaluate the humoral and cell-mediated responses of a putative HPV-16 vaccine. Fowlpoxvirus (FP) recombinants separately expressing the HPV-16 E6 (FPE6) and E7 (FPE7) transgenes were used for priming, followed by E7 protein boosting. Results All of the protocols were effective in eliciting a high antibody response. This was also confirmed by interleukin-4 production, which increased after simultaneous priming with both FPE6 and FPE7 and after E7 protein boost. A cell-mediated immune response was also detected in most of the animals. Conclusion These results establish a preliminary profile for the therapy with the combined use of avipox recombinants, which may represent safer immunogens than vaccinia-based vectors in immuno-compromised individuals, as they express the transgenes in most mammalian cells in the absence of a productive replication. PMID:20409340

  9. Alveolar Macrophages Infected with Ames or Sterne Strain of Bacillus anthracis Elicit Differential Molecular Expression Patterns

    PubMed Central

    Lane, Douglas; Kenny, Tara; Ojeda, Jenifer F.; Zhong, Yang; Che, Jianwei; Zhou, Yingyao; Ribot, Wilson; Kota, Krishna P.; Bavari, Sina; Panchal, Rekha G.

    2014-01-01

    Alveolar macrophages (AMs) phagocytose Bacillus anthracis following inhalation and induce the production of pro-inflammatory cytokines and chemokines to mediate the activation of innate immunity. Ames, the virulent strain of B. anthracis, contains two plasmids that encode the antiphagocytic poly-γ-d-glutamic acid capsule and the lethal toxin. The attenuated Sterne strain of B. anthracis, which lacks the plasmid encoding capsule, is widely adapted as a vaccine strain. Although differences in the outcome of infection with the two strains may have originated from the presence or absence of an anti-phagocytic capsule, the disease pathogenesis following infection will be manifested via the host responses, which is not well understood. To gain understanding of the host responses at cellular level, a microarray analysis was performed using primary rhesus macaque AMs infected with either Ames or Sterne spores. Notably, 528 human orthologs were identified to be differentially expressed in AMs infected with either strain of the B. anthracis. Meta-analyses revealed genes differentially expressed in response to B. anthracis infection were also induced upon infections with multiple pathogens such as Francisella Novicida or Staphylococcus aureus. This suggests the existence of a common molecular signature in response to pathogen infections. Importantly, the microarray and protein expression data for certain cytokines, chemokines and host factors provide further insights on how cellular processes such as innate immune sensing pathways, anti-apoptosis versus apoptosis may be differentially modulated in response to the virulent or vaccine strain of B. anthracis. The reported differences may account for the marked difference in pathogenicity between these two strains. PMID:24516547

  10. Protective MCMV immunity by vaccination of the salivary gland via Wharton's duct: replication-deficient recombinant adenovirus expressing individual MCMV genes elicits protection similar to that of MCMV.

    PubMed

    Liu, Guangliang; Zhang, Fangfang; Wang, Ruixue; London, Lucille; London, Steven D

    2014-04-01

    Salivary glands, a major component of the mucosal immune system, confer antigen-specific immunity to mucosally acquired pathogens. We investigated whether a physiological route of inoculation and a subunit vaccine approach elicited MCMV-specific and protective immunity. Mice were inoculated by retrograde perfusion of the submandibular salivary glands via Wharton's duct with tcMCMV or MCMV proteins focused to the salivary gland via replication-deficient adenovirus expressing individual MCMV genes (gB, gH, IE1; controls: saline and replication deficient adenovirus without MCMV inserts). Mice were evaluated for MCMV-specific antibodies, T-cell responses, germinal center formation, and protection against a lethal MCMV challenge. Retrograde perfusion with tcMCMV or adenovirus expressed MCMV proteins induced a 2- to 6-fold increase in systemic and mucosal MCMV-specific antibodies, a 3- to 6-fold increase in GC marker expression, and protection against a lethal systemic challenge, as evidenced by up to 80% increased survival, decreased splenic pathology, and decreased viral titers from 10(6) pfu to undetectable levels. Thus, a focused salivary gland immunization via a physiological route with a protein antigen induced systemic and mucosal protective immune responses. Therefore, salivary gland immunization can serve as an alternative mucosal route for administering vaccines, which is directly applicable for use in humans.

  11. 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.

  12. 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. PMID:26370303

  13. Recombinant outer membrane protein C of Aeromonas hydrophila elicits mixed immune response and generates agglutinating antibodies.

    PubMed

    Yadav, Sunita Kumari; Meena, Jitendra Kumar; Sharma, Mahima; Dixit, Aparna

    2016-08-01

    Aeromonas hydrophila is a gram-negative fish pathogenic bacterium, also responsible for causing opportunistic pathological conditions in humans. It causes a number of diseases in fish due to which the fish industry incurs huge economic losses annually. Due to problems of antibiotic resistance, and the rapidity with which the infection spreads among fishes, vaccination remains the most effective strategy to combat this infection in fish populations. Among various virulence factors associated with bacterial virulence, outer membrane proteins have been widely evaluated for their vaccine potential owing to their surface exposure and related role in pathogenicity. In the present study, we have investigated the immunogenic potential of a non-specific porin, outer membrane protein C (OmpC) whose expression is regulated by the two-component regulatory system and plays a major role in the survival of A. hydrophila under different osmolaric conditions. The full-length gene (~1 kb) encoding OmpC of A. hydrophila was cloned, characterized and expressed in E. coli. High yield (~112 mg/L at shake flask level) of the recombinant OmpC (rOmpC) (~40 kDa) of A. hydrophila was obtained upon purification from inclusion bodies using Ni(2+)-NTA affinity chromatography. Immunization with purified rOmpC in murine model generated high endpoint (>1:40,000) titers. IgG isotyping, ELISA and ELISPOT assay indicated mixed immune response with a TH2 bias. Also, the anti-rOmpC antibodies were able to agglutinate A. hydrophila in vitro and exhibited specific cross-reactivity with different Aeromonas strains, which will facilitate easy detection of different Aeromonas isolates in infected samples. Taken together, these data clearly indicate that rOmpC could serve as an effective vaccine against different strains of Aeromonas, a highly heterogenous group of bacteria. PMID:27328672

  14. 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.

  15. Oral administration with attenuated Salmonella encoding a Trichinella cystatin-like protein elicited host immunity.

    PubMed

    Liu, X D; Wang, X L; Bai, X; Liu, X L; Wu, X P; Zhao, Y; Sun, S M; Yu, L; Su, X Z; Wang, Z Q; Wang, F; Liu, M Y

    2014-06-01

    Trichinellosis is a public health problem and is regarded as an emergent/re-emergent disease in various countries. The cDNA encoding a cystatin-like protein (Ts-cystatin) was identified by immunoscreening intestinal muscle larvae cDNA libraries with serum from pigs experimentally infected with 20,000 Trichinella spiralis muscle larvae. To study its impact on host immunity, we chose a eukaryotic expression system based on several comparisons of immunogenicity between the two Salmonella typhimurium administration schemes, which indicated that the eukaryotic expression system was superior. Humoral IgG and mucosal IgA were measured to determine the antibody response. To explore whether Th1 and Th2 responses were responsible for the induced protection, Th1- and Th2-specific cellular transcription factors and the cytokine profile were examined. Changes in the T lymphocyte and macrophage populations were detected by flow cytometry. Lastly, parasitological examination was examined. The results showed that Ts-cystatin induced a Th1/Th2-mixed type of immune response and decreased STAT6 transcription. The intestinal adult recovery increased by 10.9% in the Ts-cystatin group, the Ts-cystatin group fecundity rate was decreased by 91%. Furthermore, the number of muscle larvae did not change compared with the control group. In conclusion, our results suggest that Ts-cystatin plays an important role in Trichinella resistance to rapid expulsion by the host and is worth further study.

  16. 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

  17. 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. PMID:24236215

  18. 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.

  19. 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

  20. 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

  1. 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.

  2. 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

  3. Inducible Expression of a Truncated Form of Tau in Oligodendrocytes Elicits Gait Abnormalities and a Decrease in Myelin: Implications for Selective CNS Degenerative Diseases.

    PubMed

    LoPresti, Patrizia

    2015-11-01

    The cytoskeleton protein Tau present in oligodendrocytes (OLGs) promotes cellular process outgrowth and myelination; whereas abnormally hyperphosphorylated Tau has been shown to be present in the most debilitating form of multiple sclerosis and in selective dementias. This research examined the functional consequences of expressing a truncated form of Tau in OLGs during the second postnatal life. In particular, this truncated form of Tau (∆Tau) retains the Fyn-binding domain but lacks the microtubule-binding domain. Similar to hyperphosphorylated Tau, ∆Tau cannot bind the cytoskeleton and is missorted. The Cre/loxP recombination system was used to generate transgenic (TG) founder lines, which contain a Floxed LacZ-STOP cassette to prevent expression of enhanced green fluorescence protein (EGFP)-∆Tau. The founder lines were then crossed with a Tamoxifen (TM)-inducible proteolipid protein (PLP)-dependent Cre driver line. Myelin PLP is the major myelin protein in the central nervous system (CNS). TM was given at postnatal day (p) 12 for 3 days, and CNS tissues were collected at p22. Only TG mice with both EGFP-∆Tau and Cre manifested an overt phenotype of loss of balance and stumbles starting around p18. CNS tissues obtained from TM-treated EGFP-∆Tau/Cre double transgenic mice had recombined PCR products, GFP, and diminished brain myelin. GFP was expressed in OLGs, but not in neurons or astrocytes. On the contrary, TM-treated TG mice with only one of the two transgenes, i.e., Cre or Tau, did not have recombinant PCR products, GFP, diminished myelin, or abnormal phenotype. Thus, this inducible model shows for the first time that a non-microtubule-associated Tau protein in OLGs elicits both myelin decrease and gait abnormalities, similar to the occurrence in selective demyelinating and neurodegenerative diseases.

  4. 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.

  5. 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.

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

    PubMed

    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.

  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. Anaplasma phagocytophilum and Anaplasma marginale Elicit Different Gene Expression Responses in Cultured Tick Cells

    PubMed Central

    Zivkovic, Zorica; Blouin, Edmour F.; Manzano-Roman, Raúl; Almazán, Consuelo; Naranjo, Victoria; Massung, Robert F.; Jongejan, Frans; Kocan, Katherine M.; de la Fuente, José

    2009-01-01

    The genus Anaplasma (Rickettsiales: Anaplasmataceae) includes obligate tick-transmitted intracellular organisms, Anaplasma phagocytophilum and Anaplasma marginale that multiply in both vertebrate and tick host cells. Recently, we showed that A. marginale affects the expression of tick genes that are involved in tick survival and pathogen infection and multiplication. However, the gene expression profile in A. phagocytophilum-infected tick cells is currently poorly characterized. The objectives of this study were to characterize tick gene expression profile in Ixodes scapularis ticks and cultured ISE6 cells in response to infection with A. phagocypthilum and to compare tick gene expression responses in A. phagocytophilum- and A. marginale-infected tick cells by microarray and real-time RT-PCR analyses. The results of these studies demonstrated modulation of tick gene expression by A. phagocytophilum and provided evidence of different gene expression responses in tick cells infected with A. phagocytophilum and A. marginale. These differences in Anaplasma-tick interactions may reflect differences in pathogen life cycle in the tick cells. PMID:19636428

  9. 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.

  10. 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.

  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. PMID:22438936

  12. Glucopyranosyl lipid A adjuvant significantly enhances HIV specific T and B cell responses elicited by a DNA-MVA-protein vaccine regimen.

    PubMed

    McKay, Paul F; Cope, Alethea V; Mann, Jamie F S; Joseph, Sarah; Esteban, Mariano; Tatoud, Roger; Carter, Darrick; Reed, Steven G; Weber, Jonathan; Shattock, Robin J

    2014-01-01

    Using a unique vaccine antigen matched and single HIV Clade C approach we have assessed the immunogenicity of a DNA-poxvirus-protein strategy in mice and rabbits, administering MVA and protein immunizations either sequentially or simultaneously and in the presence of a novel TLR4 adjuvant, GLA-AF. Mice were vaccinated with combinations of HIV env/gag-pol-nef plasmid DNA followed by MVA-C (HIV env/gag-pol-nef) with HIV CN54gp140 protein (+/-GLA-AF adjuvant) and either co-administered in different muscles of the same animal with MVA-C or given sequentially at 3-week intervals. The DNA prime established a population of B cells that were able to mount a statistically significant anamnestic response to the boost vaccines. The greatest antigen-specific antibody response was observed in animals that received all vaccine components. Moreover, a high proportion of the total mucosal IgG (20 - 50%) present in the vaginal vault of these vaccinated animals was vaccine antigen-specific. The potent elicitation of antigen-specific immune responses to this vaccine modality was also confirmed in rabbits. Importantly, co-administration of MVA-C with the GLA-AF adjuvanted HIV CN54gp140 protein significantly augmented the antigen-specific T cell responses to the Gag antigen, a transgene product expressed by the MVA-C vector in a separate quadriceps muscle. We have demonstrated that co-administration of MVA and GLA-AF adjuvanted HIV CN54gp140 protein was equally effective in the generation of humoral responses as a sequential vaccination modality thus shortening and simplifying the immunization schedule. In addition, a significant further benefit of the condensed vaccination regime was that T cell responses to proteins expressed by the MVA-C were potently enhanced, an effect that was likely due to enhanced immunostimulation in the presence of systemic GLA-AF.

  13. Immunity to malaria elicited by hybrid hepatitis B virus core particles carrying circumsporozoite protein epitopes

    PubMed Central

    1994-01-01

    The hepatitis B virus (HBV) nucleocapsid antigen (HBcAg) was investigated as a carrier moiety for the immunodominant circumsporozoite (CS) protein repeat epitopes of Plasmodium falciparum and the rodent malaria agent P. berghei. For this purpose hybrid genes coding for [NANP]4 (C75CS2) or [DP4NPN]2 (C75CS1) as internal inserts in HBcAg (between amino acids 75 and 81) were constructed and expressed in recombinant Salmonella typhimurium. The resulting hybrid HBcAg-CS polypeptides purified from S. typhimurium were particulate and displayed CS and HBc antigenicity, however, the HBc antigenicity was reduced compared to native recombinant HBcAg. Immunization of several mouse strains with HBcAg-CS1 and HBcAg-CS2 particles resulted in high titer, P.berghei- or P.falciparum-specific anti-CS antibodies representing all murine immunoglobulin G isotypes. The possible influence of carrier-specific immunosuppression was examined, and preexisting immunity to HBcAg did not significantly affect the immunogenicity of the CS epitopes within HBcAg-CS1 particles. Similarly, the choice of adjuvant did not significantly alter the immunogenicity of HBcAg-CS hybrid particles. Immunization in complete or incomplete Freund's adjuvant or alum resulted in equivalent anti-HBc and anti-CS humoral responses. Examination of T cell recognition of HBcAg-CS particles revealed that HBcAg-specific T cells were universally primed and CS-specific T cells were primed if the insert contained a CS-specific T cell recognition site. This indicates that the internal site in HBcAg is permissive for the inclusion of heterologous pathogen-specific T as well as B cell epitopes. Most importantly, 90 and 100% of BALB/c mice immunized with HBcAg-CS1 particles were protected against a P. berghei challenge infection in two independent experiments. Therefore, hybrid HBcAg-CS particles may represent a useful approach for future malaria vaccine development. PMID:7520465

  14. 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

  15. 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.

  16. 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.

  17. 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

  18. 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

  19. ATP released by electrical stimuli elicits calcium transients and gene expression in skeletal muscle.

    PubMed

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

    2009-12-11

    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 Ca(2+) concentration, with an EC(50) value of 7.8 +/- 3.1 microm. Exogenous ADP, UTP, and UDP also promoted calcium transients. Both fast and slow calcium signals evoked by tetanic stimulation were inhibited by either 100 mum 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 P2Y(2) receptor and pannexin-1. As reported previously for electrical stimulation, 500 mum 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 Ca(2+) homeostasis and muscle physiology. PMID:19822518

  20. 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.

  1. Ionic responses rapidly elicited by activation of protein kinase C in quiescent Swiss 3T3 cells

    SciTech Connect

    Vara, F.; Schneider, J.A.; Rozengurt, E.

    1985-04-01

    Diacylglycerol and phorbol esters activate protein kinase C in intact cells. The authors report here that addition of the synthetic diacylglycerol 1-oleoyl-2-acetylglycerol (OAG) to quiescent cultures of Swiss 3T3 cells caused a marked increase in the rate of ouabain-sensitive YWRb uptake, a measure of the activity of the Na /K pump. The effect was dose-dependent and could be detected after 1 min of exposure to the diacylglycerol. OAG stimulated Na influx via an amiloride-sensitive pathway and increased intracellular pH by 0.15 pH unit. Phorbol 12,13-dibutyrate (PBt2) also enhanced ouabain sensitive YWRb uptake and amiloride-sensitive SSNa influx. Prolonged treatment (40 hr) of 3T3 cells with PBt2 at a saturating dose, which reduces the number of PBt2 binding sites and protein kinase C activity, abolished the ionic response of the cells to a subsequent addition of either OAG or PBt2. They suggest that activation of protein kinase C elicits, either directly or indirectly, enhanced Na /H antiport activity, which, in turn, leads to Na influx, intracellular pH modulation, and stimulation of the Na /K pump.

  2. 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.

  3. 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

  4. 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.

  5. Endoplasmic reticulum chaperone glucose regulated protein 170-Pokemon complexes elicit a robust antitumor immune response in vivo.

    PubMed

    Yuan, Bangqing; Xian, Ronghua; Wu, Xianqu; Jing, Junjie; Chen, Kangning; Liu, Guojun; Zhou, Zhenhua

    2012-07-01

    Previous evidence suggested that the stress protein grp170 can function as a highly efficient molecular chaperone, binding to large protein substrates and acting as a potent vaccine against specific tumors when purified from the same tumor. In addition, Pokemon can be found in almost all malignant tumor cells and is regarded to be a promising candidate for the treatment of tumors. However, the potential of the grp170-Pokemon chaperone complex has not been well described. In the present study, the natural chaperone complex between grp170 and the Pokemon was formed by heat shock, and its immunogenicity was detected by ELISPOT and (51)Cr-release assays in vitro and by tumor bearing models in vivo. Our results demonstrated that the grp170-Pokemon chaperone complex could elicit T cell responses as determined by ELISPOT and (51)Cr-release assays. In addition, immunized C57BL/6 mice were challenged with subcutaneous (s.c.) injection of Lewis cancer cells to induce primary tumors. Treatment of mice with the grp170-Pokemon chaperone complex also significantly inhibited tumor growth and prolonged the life span of tumor-bearing mice. Our results indicated that the grp170-Pokemon chaperone complex might represent a powerful approach to tumor immunotherapy and have significant potential for clinical application. PMID:22317751

  6. Endoplasmic reticulum chaperone glucose regulated protein 170-Pokemon complexes elicit a robust antitumor immune response in vivo.

    PubMed

    Yuan, Bangqing; Xian, Ronghua; Wu, Xianqu; Jing, Junjie; Chen, Kangning; Liu, Guojun; Zhou, Zhenhua

    2012-07-01

    Previous evidence suggested that the stress protein grp170 can function as a highly efficient molecular chaperone, binding to large protein substrates and acting as a potent vaccine against specific tumors when purified from the same tumor. In addition, Pokemon can be found in almost all malignant tumor cells and is regarded to be a promising candidate for the treatment of tumors. However, the potential of the grp170-Pokemon chaperone complex has not been well described. In the present study, the natural chaperone complex between grp170 and the Pokemon was formed by heat shock, and its immunogenicity was detected by ELISPOT and (51)Cr-release assays in vitro and by tumor bearing models in vivo. Our results demonstrated that the grp170-Pokemon chaperone complex could elicit T cell responses as determined by ELISPOT and (51)Cr-release assays. In addition, immunized C57BL/6 mice were challenged with subcutaneous (s.c.) injection of Lewis cancer cells to induce primary tumors. Treatment of mice with the grp170-Pokemon chaperone complex also significantly inhibited tumor growth and prolonged the life span of tumor-bearing mice. Our results indicated that the grp170-Pokemon chaperone complex might represent a powerful approach to tumor immunotherapy and have significant potential for clinical application.

  7. Withania somnifera Dunal (Indian ginseng) impairs acquisition and expression of ethanol-elicited conditioned place preference and conditioned place aversion.

    PubMed

    Spina, Liliana; Longoni, Rosanna; Rosas, Michela; Collu, Maria; Peana, Alessandra T; Espa, Elena; Kasture, Sanjay; Cotti, Elisabetta; Acquas, Elio

    2015-11-01

    Withania somnifera Dunal (Indian Ginseng) has recently been shown to impair ethanol self-administration. In order to gain further insights on the ability of the Withania somnifera standardised root extract (WSE) to affect the motivational properties of ethanol, this study investigated whether WSE may also affect ethanol (2 g/kg)-elicited conditioned place preference (CPP) and aversion (CPA). To this end male CD-1 mice were conditioned under two distinct schedules: in backward conditioning experiments ethanol was administered before mice were placed in the conditioning apparatus (CPP) while, in forward conditioning experiments, ethanol was administered immediately after removing mice from the apparatus (CPA). Following these schedules, mice developed significant CPP and CPA, respectively. Administration of WSE significantly impaired both the acquisition (50 and 100 mg/kg) and the expression (50 mg/kg) of CPP and CPA without affecting spatial memory (50 mg/kg), as determined by a two-trial memory recognition task. Overall, the study highlights the ability of WSE to interfere with both positive and negative motivational properties of ethanol and suggests that the effects of WSE may target both ethanol's motivational properties and underpinning associative learning mechanisms. In conclusion, these results cast new light on Withania somnifera as an agent potentially useful to counteract distinct aspects of ethanol effects.

  8. Withania somnifera Dunal (Indian ginseng) impairs acquisition and expression of ethanol-elicited conditioned place preference and conditioned place aversion.

    PubMed

    Spina, Liliana; Longoni, Rosanna; Rosas, Michela; Collu, Maria; Peana, Alessandra T; Espa, Elena; Kasture, Sanjay; Cotti, Elisabetta; Acquas, Elio

    2015-11-01

    Withania somnifera Dunal (Indian Ginseng) has recently been shown to impair ethanol self-administration. In order to gain further insights on the ability of the Withania somnifera standardised root extract (WSE) to affect the motivational properties of ethanol, this study investigated whether WSE may also affect ethanol (2 g/kg)-elicited conditioned place preference (CPP) and aversion (CPA). To this end male CD-1 mice were conditioned under two distinct schedules: in backward conditioning experiments ethanol was administered before mice were placed in the conditioning apparatus (CPP) while, in forward conditioning experiments, ethanol was administered immediately after removing mice from the apparatus (CPA). Following these schedules, mice developed significant CPP and CPA, respectively. Administration of WSE significantly impaired both the acquisition (50 and 100 mg/kg) and the expression (50 mg/kg) of CPP and CPA without affecting spatial memory (50 mg/kg), as determined by a two-trial memory recognition task. Overall, the study highlights the ability of WSE to interfere with both positive and negative motivational properties of ethanol and suggests that the effects of WSE may target both ethanol's motivational properties and underpinning associative learning mechanisms. In conclusion, these results cast new light on Withania somnifera as an agent potentially useful to counteract distinct aspects of ethanol effects. PMID:26349555

  9. Vaccine-elicited Human T Cells Recognizing Conserved Protein Regions Inhibit HIV-1

    PubMed Central

    Borthwick, Nicola; Ahmed, Tina; Ondondo, Beatrice; Hayes, Peter; Rose, Annie; Ebrahimsa, Umar; Hayton, Emma-Jo; Black, Antony; Bridgeman, Anne; Rosario, Maximillian; Hill, Adrian VS; Berrie, Eleanor; Moyle, Sarah; Frahm, Nicole; Cox, Josephine; Colloca, Stefano; Nicosia, Alfredo; Gilmour, Jill; McMichael, Andrew J; Dorrell, Lucy; Hanke, Tomáš

    2014-01-01

    Virus diversity and escape from immune responses are the biggest challenges to the development of an effective vaccine against HIV-1. We hypothesized that T-cell vaccines targeting the most conserved regions of the HIV-1 proteome, which are common to most variants and bear fitness costs when mutated, will generate effectors that efficiently recognize and kill virus-infected cells early enough after transmission to potentially impact on HIV-1 replication and will do so more efficiently than whole protein-based T-cell vaccines. Here, we describe the first-ever administration of conserved immunogen vaccines vectored using prime-boost regimens of DNA, simian adenovirus and modified vaccinia virus Ankara to uninfected UK volunteers. The vaccine induced high levels of effector T cells that recognized virus-infected autologous CD4+ cells and inhibited HIV-1 replication by up to 5.79 log10. The virus inhibition was mediated by both Gag- and Pol- specific effector CD8+ T cells targeting epitopes that are typically subdominant in natural infection. These results provide proof of concept for using a vaccine to target T cells at conserved epitopes, showing that these T cells can control HIV-1 replication in vitro. PMID:24166483

  10. Export of the HR eliciting protein, Harpin(Es), of the maize pathogen Erwinia stewartii is species-specific but is independent of the growth temperature.

    PubMed

    Ahmad, Musharaf; Alam, Syed Sartaj; Alam, Shah; Usman, Amjad; Coplin, David L

    2007-01-01

    The extra-cellular export of the HR-eliciting protein, Harpin(Es) of the maize pathogen Erwinia stewartii was studied to find out if the protein needs any species-specific signal for its export and to determine if the export of the protein to the medium is affected in any way by the growth temperature. Based upon the experimental evidence, it was proved that the protein (i.e., Harpin(Es)) does require its own export system (species-specific) to get out of the bacterial cell and can not be exported by the export system of even the very closely related bacterium, Erwinia amylovora. It was also found that the export of Harpin(Es) is, unlike the case of Harpin(Ea) (HR-eliciting protein of Erwinia amylovora), independent of the growth temperature.

  11. Protein expression strategies for identification of novel target proteins.

    PubMed

    Schuster, M; Wasserbauer, E; Einhauer, A; Ortner, C; Jungbauer, A; Hammerschmid, F; Werner, G

    2000-04-01

    Identification of new target proteins is a novel paradigm in drug discovery. A major bottleneck of this strategy is the rapid and simultaneous expression of proteins from differential gene expression to identify eligible candidates. By searching for a generic system enabling high throughput expression analysis and purification of unknown cDNAs, we evaluated the YEpFLAG-1 yeast expression system. We have selected cDNAs encoding model proteins (eukaryotic initiation factor-5A [eIF-5A] and Homo sapiens differentiation-dependent protein-A4) and cDNA encoding an unknown protein (UP-1) for overexpression in Saccharomyces cerevisiae using fusions with a peptide that changes its conformation in the presence of Ca2+ ions, the FLAG tag (Eastman Kodak, Rochester, NY). The cDNAs encoding unknown proteins originating from a directionally cloned cDNA library were expressed in all three possible reading frames. The expressed proteins were detected by an antibody directed against the FLAG tag and/or by antibodies against the model proteins. The alpha-leader sequence, encoding a yeast mating pheromone, upstream of the gene fusion site facilitates secretion into the culture supernatant. EIF-5A could be highly overexpressed and was secreted into the culture supernatant. In contrast, the Homo sapiens differentiation-dependent protein-A4 as well as the protein UP-1, whose cDNA did not match to any known gene, could not be detected in the culture supernatant. The expression product of the correct frame remained in the cells, whereas the FLAG-tagged proteins secreted into the supernatant were short, out-of-frame products. The presence of transmembrane domains or patches of hydrophobic amino acids may preclude secretion of these proteins into the culture supernatant. Subsequently, isolation and purification of the various proteins was accomplished by affinity chromatography or affinity extraction using magnetizable beads coated with the anti-FLAG monoclonal antibody. The purity of

  12. Elicitation of T-cell responses by structural and non-structural proteins of coxsackievirus B4.

    PubMed

    Bengs, Suvi; Marttila, Jane; Susi, Petri; Ilonen, Jorma

    2015-02-01

    Coxsackievirus B4 (CV-B4) belongs to the genus Enterovirus within the family Picornaviridae. To investigate target proteins recognized by T-cells in human enterovirus B infections, virus-encoded structural [VP0 (VP4 and VP2), VP1, VP3] and non-structural (2A, 2B, 2C, 3C and 3D) proteins were expressed and purified in Escherichia coli. Peripheral blood of 19 healthy adult donors was used to create enterovirus-specific T-cell lines by repeated stimulation with CV-B4 cell lysate antigen. T-cell lines responded in individual patterns, and responses to all purified proteins were observed. The most often recognized enteroviral protein was VP0, which is the fusion between the most conserved structural proteins, VP4 and VP2. T-cell responses to VP0 were detected in 15 of the 19 (79 %) donor lines. Non-structural 2C protein was recognized in 11 of the 19 (58 %) lines, and 11 of the 19 (58 %) lines also had a response to 3D protein. Furthermore, responses to other non-structural proteins (2A, 2B and 3C) were also detected. T-cell responses did not correlate clearly to the individual HLA-DR-DQ phenotype or the history of past coxsackie B virus infections of the donors.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  14. A nuclear fraction of turnip crinkle virus capsid protein is important for elicitation of the host resistance response.

    PubMed

    Kang, Sung-Hwan; Qu, Feng; Morris, T Jack

    2015-12-01

    The N-terminal 25 amino acids (AAs) of turnip crinkle virus (TCV) capsid protein (CP) are recognized by the resistance protein HRT to trigger a hypersensitive response (HR) and systemic resistance to TCV infection. This same region of TCV CP also contains a motif that interacts with the transcription factor TIP, as well as a nuclear localization signal (NLS). However, it is not yet known whether nuclear localization of TCV CP is needed for the induction of HRT-mediated HR and resistance. Here we present new evidence suggesting a tight correlation between nuclear inclusions formed by CP and the manifestation of HR. We show that a fraction of TCV CP localized to cell nuclei to form discrete inclusion-like structures, and a mutated CP (R6A) known to abolish HR failed to form nuclear inclusions. Notably, TIP-CP interaction augments the inclusion-forming activity of CP by tethering inclusions to the nuclear membrane. This TIP-mediated augmentation is also critical for HR resistance, as another CP mutant (R8A) known to elicit a less restrictive HR, though still self-associated into nuclear inclusions, failed to direct inclusions to the nuclear membrane due to its inability to interact with TIP. Finally, exclusion of CP from cell nuclei abolished induction of HR. Together, these results uncovered a strong correlation between nuclear localization and nuclear inclusion formation by TCV CP and induction of HR, and suggest that CP nuclear inclusions could be the key trigger of the HRT-dependent, yet TIP-reinforced, resistance to TCV.

  15. A nuclear fraction of turnip crinkle virus capsid protein is important for elicitation of the host resistance response.

    PubMed

    Kang, Sung-Hwan; Qu, Feng; Morris, T Jack

    2015-12-01

    The N-terminal 25 amino acids (AAs) of turnip crinkle virus (TCV) capsid protein (CP) are recognized by the resistance protein HRT to trigger a hypersensitive response (HR) and systemic resistance to TCV infection. This same region of TCV CP also contains a motif that interacts with the transcription factor TIP, as well as a nuclear localization signal (NLS). However, it is not yet known whether nuclear localization of TCV CP is needed for the induction of HRT-mediated HR and resistance. Here we present new evidence suggesting a tight correlation between nuclear inclusions formed by CP and the manifestation of HR. We show that a fraction of TCV CP localized to cell nuclei to form discrete inclusion-like structures, and a mutated CP (R6A) known to abolish HR failed to form nuclear inclusions. Notably, TIP-CP interaction augments the inclusion-forming activity of CP by tethering inclusions to the nuclear membrane. This TIP-mediated augmentation is also critical for HR resistance, as another CP mutant (R8A) known to elicit a less restrictive HR, though still self-associated into nuclear inclusions, failed to direct inclusions to the nuclear membrane due to its inability to interact with TIP. Finally, exclusion of CP from cell nuclei abolished induction of HR. Together, these results uncovered a strong correlation between nuclear localization and nuclear inclusion formation by TCV CP and induction of HR, and suggest that CP nuclear inclusions could be the key trigger of the HRT-dependent, yet TIP-reinforced, resistance to TCV. PMID:26299399

  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. Immune responses elicited in mice with recombinant Lactococcus lactis expressing F4 fimbrial adhesin FaeG by oral immunization.

    PubMed

    Liu, Shujie; Li, Yongming; Xu, Ziwei; Wang, Yicheng

    2010-08-01

    Enterotoxigenic Escherichia coli (ETEC) is a major pathogenic agent causing piglet diarrhea. The major subunit and adhesin FaeG of F4(+) ETEC is an important virulence factor with strong immunogenicity. To determine whether Lactococcus lactis (L. lactis) could effectively deliver FaeG to the mucosal immune system, recombinant L. lactis expressing FaeG was constructed, and immune responses in mice following oral route delivery of recombinant L. lactis were explored. The production of FaeG expressed in L. lactis was up to approximately 10% of soluble whole-cell proteins, and recombinant FaeG (rFaeG) possessed good immunoreactivity by Western blot analysis. Oral immunization with recombinant L. lactis expressing FaeG induced F4-specific mucosal and systemic immune responses in the mice. In addition, high dose recombinant L. lactis or co-administration of high dose recombinant L. lactis with CTB enhanced the immune responses. These results suggested that L. lactis expressing FaeG was a promising candidate vaccine against ETEC. PMID:20532816

  18. MOPED: Model Organism Protein Expression Database.

    PubMed

    Kolker, Eugene; Higdon, Roger; Haynes, Winston; Welch, Dean; Broomall, William; Lancet, Doron; Stanberry, Larissa; Kolker, Natali

    2012-01-01

    Large numbers of mass spectrometry proteomics studies are being conducted to understand all types of biological processes. The size and complexity of proteomics data hinders efforts to easily share, integrate, query and compare the studies. The Model Organism Protein Expression Database (MOPED, htttp://moped.proteinspire.org) is a new and expanding proteomics resource that enables rapid browsing of protein expression information from publicly available studies on humans and model organisms. MOPED is designed to simplify the comparison and sharing of proteomics data for the greater research community. MOPED uniquely provides protein level expression data, meta-analysis capabilities and quantitative data from standardized analysis. Data can be queried for specific proteins, browsed based on organism, tissue, localization and condition and sorted by false discovery rate and expression. MOPED empowers users to visualize their own expression data and compare it with existing studies. Further, MOPED links to various protein and pathway databases, including GeneCards, Entrez, UniProt, KEGG and Reactome. The current version of MOPED contains over 43,000 proteins with at least one spectral match and more than 11 million high certainty spectra.

  19. 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.

  20. 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.

  1. 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. PMID:26614281

  2. 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. PMID:24403082

  3. 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

  4. 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.

  5. Membrane protein expression in Lactococcus lactis.

    PubMed

    King, Martin S; Boes, Christoph; Kunji, Edmund R S

    2015-01-01

    The Gram-positive bacterium Lactococcus lactis has many properties that are ideal for the overproduction of membrane proteins in a functional form. Growth of lactococci is rapid, proceeds to high cell densities, and does not require aeration, which facilitates large-scale fermentation. The available promoter systems are strong and tightly regulated, allowing expression of toxic gene products in a controlled manner. Expressed membrane proteins are targeted exclusively to the cytoplasmic membrane, allowing the use of ionophores, ligands, and inhibitors to study activity of the membrane protein in whole cells. Constructed plasmids are stable and expression levels are highly reproducible. The relatively small genome size of the organism causes little redundancy, which facilitates complementation studies and allows for easier purification. The produced membrane proteins are often stable, as the organism has limited proteolytic capability, and they are readily solubilized from the membrane with mild detergents. Lactococci are multiple amino acid auxotrophs, allowing the incorporation of labels, such as selenomethionine. Among the few disadvantages are the low transformation frequency, AT-rich codon usage, and resistance to lysis by mechanical means, but these problems can be overcome fairly easily. We will describe in detail the protocols used to express membrane proteins in L. lactis, from cloning of the target gene to the isolation of membrane vesicles for the determination of expression levels. PMID:25857778

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 ...

  7. Protein expression in the baculovirus system.

    PubMed

    Bernard, A; Payton, M; Radford, K R

    2001-05-01

    Insect cell-recombinant baculovirus co-cultures offer a protein production system that complements microbial systems by providing recombinant proteins in soluble form and with most post-translational modifications. Moreover, the large size of the viral genome enables cloning of large segments of DNA and consequent expression of complex protein aggregates. This unit describes methods associated with the large-scale production of recombinant proteins in the baculovirus expression system. A method for large-scale production of viral stocks is described and methods for titration of virus are provided (a plaque assay and an end-point assay). Once viral stocks have been prepared and titered, a protocol for testing the virus in small-scale cultures is provided to determine the kinetics of expression, which allows evaluation of various cell culture and infection conditions aimed at developing optimal levels of protein production (e.g., comparisons of different host cell lines, media, and environmental parameters). Support protocols provide instructions for preparing culture samples for protein analysis by SDS-PAGE and discuss analytical methods for monitoring nutrient levels in cell culture fluids. Once optimal process parameters are identified, protocols describe production of the target protein on a large scale in fermentors using either regular batch production in bioreactors or a fed-batch procedure of production in perfusion cultures. Techniques for harvesting cultures from bioreactors are also provided.

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

    PubMed

    Zhu, Shanshan; Zhang, Chunyan; 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

  9. 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

  10. Expression of human milk proteins in plants.

    PubMed

    Lönnerdal, Bo

    2002-06-01

    Human milk proteins are believed to have a multitude of biological activities benefiting the newborn infant. Such functions include antibacterial and antiviral activities, enhancement of the immune system and increased nutrient absorption. To date, only breast-fed infants have been exposed to these proteins. However, by using genetic engineering it is now possible to express these proteins in plants, such as rice, at very high levels. Recombinant human milk proteins can subsequently be added to infant formula and baby foods. Prior to such addition, safety tests and efficacy trials need to be conducted. The safety tests will initially be done in rats and then in humans. The efficacy trials should also evaluate stability against heat treatment (processing), pH (stomach conditions) and proteolytic enzymes (digestion). To date, we have expressed recombinant human lactoferrin, lysozyme and alpha1-antitrypsin in rice at very high expression levels. These recombinant proteins showed a stability and activities similar to those of the native milk proteins, suggesting that they may be able to exert biological activities in infants when added to formula or baby foods.

  11. Enhanced expression of adenovirus transforming proteins.

    PubMed Central

    Gaynor, R B; Tsukamoto, A; Montell, C; Berk, A J

    1982-01-01

    Proteins encoded in regions EIA and EIB of human adenoviruses cause transformation of rodent cells. One protein from EIA also stimulates transcription of other early regions at early times in a productive infection. In the past, direct analysis of these proteins synthesized in vivo has been difficult because of the low levels produced in both transformed cells and productively infected cells. We present a simple method which leads to expression of EIA and EIB mRNAs and proteins at 30-fold greater levels than those observed during the early phase of a standard productive infection. Under these conditions, these proteins are among the most prominent translation products of infected cells. This allowed direct visualization of EIA and EIB proteins on two-dimensional gels of pulse-labeled total cell protein. Experiments with EIA and EIB mutants confirm that the identified proteins are indeed encoded in these regions. Two EIA proteins are observed, one translated from each of the major early EIA mRNAs. Both of these EIA proteins are phosphorylated. Images PMID:7143568

  12. Inflammatory mediators alter the astrocyte transcriptome and calcium signaling elicited by multiple G-protein-coupled receptors.

    PubMed

    Hamby, Mary E; Coppola, Giovanni; Ao, Yan; Geschwind, Daniel H; Khakh, Baljit S; Sofroniew, Michael V

    2012-10-17

    Inflammation features in CNS disorders such as stroke, trauma, neurodegeneration, infection, and autoimmunity in which astrocytes play critical roles. To elucidate how inflammatory mediators alter astrocyte functions, we examined effects of transforming growth factor-β1 (TGF-β1), lipopolysaccharide (LPS), and interferon-gamma (IFNγ), alone and in combination, on purified, mouse primary cortical astrocyte cultures. We used microarrays to conduct whole-genome expression profiling, and measured calcium signaling, which is implicated in mediating dynamic astrocyte functions. Combinatorial exposure to TGF-β1, LPS, and IFNγ significantly modulated astrocyte expression of >6800 gene probes, including >380 synergistic changes not predicted by summing individual treatment effects. Bioinformatic analyses revealed significantly and markedly upregulated molecular networks and pathways associated in particular with immune signaling and regulation of cell injury, death, growth, and proliferation. Highly regulated genes included chemokines, growth factors, enzymes, channels, transporters, and intercellular and intracellular signal transducers. Notably, numerous genes for G-protein-coupled receptors (GPCRs) and G-protein effectors involved in calcium signaling were significantly regulated, mostly down (for example, Cxcr4, Adra2a, Ednra, P2ry1, Gnao1, Gng7), but some up (for example, P2ry14, P2ry6, Ccrl2, Gnb4). We tested selected cases and found that changes in GPCR gene expression were accompanied by significant, parallel changes in astrocyte calcium signaling evoked by corresponding GPCR-specific ligands. These findings identify pronounced changes in the astrocyte transcriptome induced by TGF-β1, LPS, and IFNγ, and show that these inflammatory stimuli upregulate astrocyte molecular networks associated with immune- and injury-related functions and significantly alter astrocyte calcium signaling stimulated by multiple GPCRs.

  13. 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.

  14. Maternal immunization of mice with group B streptococcal type III polysaccharide-beta C protein conjugate elicits protective antibody to multiple serotypes.

    PubMed Central

    Madoff, L C; Paoletti, L C; Tai, J Y; Kasper, D L

    1994-01-01

    Group B streptococcal infection is a major cause of neonatal mortality. Antibody to the capsular polysaccharide protects against invasive neonatal disease, but immunization with capsular polysaccharides fails to elicit protective antibody in many recipients. Conjugation of the polysaccharide to tetanus toxoid has been shown to increase immune response to the polysaccharide. In animal models, C proteins of group B streptococci are also protective determinants. We examined the ability of the beta C protein to serve in the dual role of carrier for the polysaccharide and protective immunogen. Type III polysaccharide was covalently coupled to beta C protein by reductive amination. Immunization of rabbits with the polysaccharide-protein conjugate elicited high titers of antibody to both components, and the serum induced opsonophagocytic killing of type III, Ia/C, and Ib/C strains of group B streptococci. Female mice were immunized with the conjugate vaccine and then bred; 93% of neonatal pups born to these dams vaccinated with conjugate survived type III group B streptococcal challenge and 76% survived type Ia/C challenge, compared with 3% and 8% survival, respectively, in controls (P < 0.001). The beta C protein acted as an effective carrier for the type III polysaccharide while simultaneously induced protective immunity against beta C protein--containing strains of group B streptococci. Images PMID:7518832

  15. The Pore-Forming Protein Cry5B Elicits the Pathogenicity of Bacillus sp. against Caenorhabditis elegans

    PubMed Central

    Kho, Melanie F.; 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. PMID:22216181

  16. Expression of a naturally occurring angiotensin AT1 receptor cleavage fragment elicits caspase-activation and apoptosis

    PubMed Central

    Singh, Akannsha; deHaro, Dawn; Alam, Jawed; Re, Richard N.

    2011-01-01

    Several transmembrane receptors are documented to accumulate in nuclei, some as holoreceptors and others as cleaved receptor products. Our prior studies indicate that a population of the 7-transmembrane angiotensin type-1 receptor (AT1R) is cleaved in a ligand-augmented manner after which the cytoplasmic, carboxy-terminal cleavage fragment (CF) traffics to the nucleus. In the present report, we determine the precise cleavage site within the AT1R by mass spectrometry and Edman sequencing. Cleavage occurs between Leu(305) and Gly(306) at the junction of the seventh transmembrane domain and the intracellular cytoplasmic carboxy-terminal domain. To evaluate the function of the CF distinct from the holoreceptor, we generated a construct encoding the CF as an in-frame yellow fluorescent protein fusion. The CF accumulates in nuclei and induces apoptosis in CHO-K1 cells, rat aortic smooth muscle cells (RASMCs), MCF-7 human breast adenocarcinoma cells, and H9c2 rat cardiomyoblasts. All cell types show nuclear fragmentation and disintegration, as well as evidence for phosphotidylserine displacement in the plasma membrane and activated caspases. RASMCs specifically showed a 5.2-fold increase (P < 0.001) in CF-induced active caspases compared with control and a 7.2-fold increase (P < 0.001) in cleaved caspase-3 (Asp174). Poly(ADP-ribose)polymerase was upregulated 4.8-fold (P < 0.001) in CF expressing cardiomyoblasts and colocalized with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). CF expression also induces DNA laddering, the gold-standard for apoptosis in all cell types studied. CF-induced apoptosis, therefore, appears to be a general phenomenon as it is observed in multiple cell types including smooth muscle cells and cardiomyoblasts. PMID:21813711

  17. Expression of a naturally occurring angiotensin AT(1) receptor cleavage fragment elicits caspase-activation and apoptosis.

    PubMed

    Cook, Julia L; Singh, Akannsha; DeHaro, Dawn; Alam, Jawed; Re, Richard N

    2011-11-01

    Several transmembrane receptors are documented to accumulate in nuclei, some as holoreceptors and others as cleaved receptor products. Our prior studies indicate that a population of the 7-transmembrane angiotensin type-1 receptor (AT(1)R) is cleaved in a ligand-augmented manner after which the cytoplasmic, carboxy-terminal cleavage fragment (CF) traffics to the nucleus. In the present report, we determine the precise cleavage site within the AT(1)R by mass spectrometry and Edman sequencing. Cleavage occurs between Leu(305) and Gly(306) at the junction of the seventh transmembrane domain and the intracellular cytoplasmic carboxy-terminal domain. To evaluate the function of the CF distinct from the holoreceptor, we generated a construct encoding the CF as an in-frame yellow fluorescent protein fusion. The CF accumulates in nuclei and induces apoptosis in CHO-K1 cells, rat aortic smooth muscle cells (RASMCs), MCF-7 human breast adenocarcinoma cells, and H9c2 rat cardiomyoblasts. All cell types show nuclear fragmentation and disintegration, as well as evidence for phosphotidylserine displacement in the plasma membrane and activated caspases. RASMCs specifically showed a 5.2-fold increase (P < 0.001) in CF-induced active caspases compared with control and a 7.2-fold increase (P < 0.001) in cleaved caspase-3 (Asp174). Poly(ADP-ribose)polymerase was upregulated 4.8-fold (P < 0.001) in CF expressing cardiomyoblasts and colocalized with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). CF expression also induces DNA laddering, the gold-standard for apoptosis in all cell types studied. CF-induced apoptosis, therefore, appears to be a general phenomenon as it is observed in multiple cell types including smooth muscle cells and cardiomyoblasts.

  18. Expression, purification, and crystallisationof membrane proteins

    NASA Astrophysics Data System (ADS)

    Byrne, Bernadette

    Approximately, 29,000 protein structures are deposited in the Protein Databank (January 2005), but only about 90 of which are independent membrane protein structures. This represents a significant increase in knowledge compared with a matter of only 5 years ago when a mere handful of membrane protein structures were available. Despite the advances, our understanding of the structure-function relationships and mechanism of action of many membrane proteins is still lacking. This is particularly true of many of the more clinically relevant membrane proteins, such as the G-protein-coupled receptors (GPCRs). The GPCRs regulate cellular responses to a wide range of biologically active molecules including hormones and drugs and are thus important targets for therapeutic intervention in a number of disease states. However, the increasing number of membrane protein structures has provided a critical mass of information which has yielded a more rational approach to the process of obtaining diffraction quality crystals. It is the different stages of this process; expression, solubilisation, purification, and crystallisation that will be covered in this lecture.

  19. Candida-Elicited Murine Th17 Cells Express High CTLA-4 Compared to Th1 Cells and Are Resistant to Costimulation Blockade

    PubMed Central

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

    2014-01-01

    Effector and memory T cells may cross-react with allogeneic antigens to mediate graft rejection. While 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 antigen-specific model in which Th1 and Th17 cells were elicited via Mycobacterium tuberculosis (M.Tb) and Candida albicans (Candida) immunization, respectively. Candida immunization elicited a higher frequency of Th17 cells and conferred resistance to costimulation blockade following transplantation. Compared to the M.Tb group, Candida 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 to 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. PMID:24493820

  20. 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.

  1. Expression Differentiation Is Constrained to Low-Expression Proteins over Ecological Timescales.

    PubMed

    Margres, Mark J; Wray, Kenneth P; Seavy, Margaret; McGivern, James J; Herrera, Nathanael D; Rokyta, Darin R

    2016-01-01

    Protein expression level is one of the strongest predictors of protein sequence evolutionary rate, with high-expression protein sequences evolving at slower rates than low-expression protein sequences largely because of constraints on protein folding and function. Expression evolutionary rates also have been shown to be negatively correlated with expression level across human and mouse orthologs over relatively long divergence times (i.e., ∼100 million years). Long-term evolutionary patterns, however, often cannot be extrapolated to microevolutionary processes (and vice versa), and whether this relationship holds for traits evolving under directional selection within a single species over ecological timescales (i.e., <5000 years) is unknown and not necessarily expected. Expression is a metabolically costly process, and the expression level of a particular protein is predicted to be a tradeoff between the benefit of its function and the costs of its expression. Selection should drive the expression level of all proteins close to values that maximize fitness, particularly for high-expression proteins because of the increased energetic cost of production. Therefore, stabilizing selection may reduce the amount of standing expression variation for high-expression proteins, and in combination with physiological constraints that may place an upper bound on the range of beneficial expression variation, these constraints could severely limit the availability of beneficial expression variants. To determine whether rapid-expression evolution was restricted to low-expression proteins owing to these constraints on highly expressed proteins over ecological timescales, we compared venom protein expression levels across mainland and island populations for three species of pit vipers. We detected significant differentiation in protein expression levels in two of the three species and found that rapid-expression differentiation was restricted to low-expression proteins. Our

  2. 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. PMID:25540276

  3. 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.

  4. 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

  5. Microgravity alters the expression of salivary proteins.

    PubMed

    Mednieks, Maija; Khatri, Aditi; Rubenstein, Renee; Burleson, Joseph A; Hand, Arthur R

    2014-06-01

    Spaceflight provides a unique opportunity to study how physiologic responses are influenced by the external environment. Microgravity has been shown to alter the function of a number of tissues and organ systems. Very little, however, is known about how microgravity affects the oral cavity. The rodent model is useful for study in that their salivary gland morphology and physiology is similar to that of humans. Useful also is the fact that saliva, a product of the salivary glands with a major role in maintaining oral health, can be easily collected in humans whereas the glands can be studied in experimental animals. Our working hypothesis is that expression of secretory proteins in saliva will respond to microgravity and will be indicative of the nature of physiologic reactions to travel in space. This study was designed to determine which components of the salivary proteome are altered in mice flown on the US space shuttle missions and to determine if a subset with predictive value can be identified using microscopy and biochemistry methods. The results showed that the expression of secretory proteins associated with beta-adrenergic hormone regulated responses and mediated via the cyclic AMP pathway was significantly altered, whereas that of a number of unrelated proteins was not. The findings are potentially applicable to designing a biochemical test system whereby specific salivary proteins can be biomarkers for stress associated with travel in space and eventually for monitoring responses to conditions on earth.

  6. Microgravity alters the expression of salivary proteins.

    PubMed

    Mednieks, Maija; Khatri, Aditi; Rubenstein, Renee; Burleson, Joseph A; Hand, Arthur R

    2014-06-01

    Spaceflight provides a unique opportunity to study how physiologic responses are influenced by the external environment. Microgravity has been shown to alter the function of a number of tissues and organ systems. Very little, however, is known about how microgravity affects the oral cavity. The rodent model is useful for study in that their salivary gland morphology and physiology is similar to that of humans. Useful also is the fact that saliva, a product of the salivary glands with a major role in maintaining oral health, can be easily collected in humans whereas the glands can be studied in experimental animals. Our working hypothesis is that expression of secretory proteins in saliva will respond to microgravity and will be indicative of the nature of physiologic reactions to travel in space. This study was designed to determine which components of the salivary proteome are altered in mice flown on the US space shuttle missions and to determine if a subset with predictive value can be identified using microscopy and biochemistry methods. The results showed that the expression of secretory proteins associated with beta-adrenergic hormone regulated responses and mediated via the cyclic AMP pathway was significantly altered, whereas that of a number of unrelated proteins was not. The findings are potentially applicable to designing a biochemical test system whereby specific salivary proteins can be biomarkers for stress associated with travel in space and eventually for monitoring responses to conditions on earth. PMID:24984624

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

    PubMed Central

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

    2014-01-01

    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. PMID:23238561

  8. Reliable protein production in a Pseudomonas fluorescens expression system.

    PubMed

    Retallack, Diane M; Jin, Hongfan; Chew, Lawrence

    2012-02-01

    A bottleneck to product development can be reliable expression of active target protein. A wide array of recombinant proteins in development, including an ever growing number of non-natural proteins, is being expressed in a variety of expression systems. A Pseudomonas fluorescens expression platform has been developed specifically for recombinant protein production. The development of an integrated molecular toolbox of expression elements and host strains, along with automation of strain screening is described. Examples of strain screening and scale-up experiments show rapid development of expression strains producing a wide variety of proteins in a soluble active form.

  9. Regulation of Mutant p53 Protein Expression

    PubMed Central

    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. PMID:26734569

  10. 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.

  11. 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

  12. Novel Staphylococcus aureus Secreted Protein Alters Keratinocyte Proliferation and Elicits a Proinflammatory Response In Vitro and In Vivo.

    PubMed

    Merriman, Joseph A; Klingelhutz, Aloysius J; Diekema, Daniel J; Leung, Donald Y M; Schlievert, Patrick M

    2015-08-11

    Staphylococcus aureus is a leading cause of surgical site infections that results in increased hospital stays due to the development of chronic wounds. Little is known about factors involved in S. aureus' ability to prevent wounds from healing. We discovered a novel secreted protein produced by a surgical site isolate of S. aureus that prevents keratinocyte proliferation. The protein has a molecular weight of 15.7 kDa and an isoelectric point of 8.9. The cloned and purified protein has cytotoxic and proinflammatory properties, as shown in vitro and in vivo. Potent biological effects on keratinocytes and rabbit skin suggest that this protein may play an important role in preventing re-epithelialization. Its lack of homology to known exotoxins suggests that this protein is novel, and this observation is likely to open a new field of research in S. aureus exotoxins. Due to its cytotoxic activities, we call this new protein ε-cytotoxin. PMID:26177220

  13. 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.

  14. Expressed protein ligation-mediated template protein extension.

    PubMed

    Kamei, Ayako; Hauser, Paul S; Beckstead, Jennifer A; Weers, Paul M M; Ryan, Robert O

    2012-06-01

    Expressed protein ligation (EPL) was performed to investigate sequence requirements for a variant human apolipoprotein A-I (apoA-I) to adopt a folded structure. A C-terminal truncated apoA-I, corresponding to residues 1-172, was expressed and isolated from Escherichia coli. Compared to full length apoA-I (243 amino acids), apoA-I(1-172) displayed less α-helix secondary structure and lower stability in solution. To determine if extension of this polypeptide would confer secondary structure content and/or stability, 20 residues were added to the C-terminus of apoA-I(1-172) by EPL, creating apoA-I(Milano)(1-192). The EPL product displayed biophysical properties similar to full-length apoA-I(Milano). The results provide a general protein engineering strategy to modify the length of a recombinant template polypeptide using synthetic peptides as well as a convenient, cost effective way to investigate the structure/function relations in apolipoprotein fragments or domains of different size.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  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. 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. PMID:24813672

  18. HOXA1 drives melanoma tumor growth and metastasis and elicits an invasion gene expression signature that prognosticates clinical outcome

    PubMed Central

    Wardwell-Ozgo, Joanna; Dogruluk, Turgut; Gifford, Armel; Zhang, Yiqun; Heffernan, Timothy P.; van Doorn, Remco; Creighton, Chad J.; Chin, Lynda; Scott, Kenneth L.

    2014-01-01

    Metastatic melanoma is a highly lethal disease notorious for its aggressive clinical course and eventual resistance to existing therapies. Currently we possess a limited understanding of the genetic events driving melanoma progression, and much effort is focused on identifying pro-metastatic aberrations or perturbed signaling networks that constitute new therapeutic targets. In this study, we validate and assess the mechanism by which homeobox transcription factor A1 (HOXA1), a pro-invasion oncogene previously identified in a metastasis screen by our group, contributes to melanoma progression. Transcriptome and pathway profiling analyses of cells expressing HOXA1 reveals up-regulation of factors involved in diverse cytokine pathways that include the TGFβ signaling axis, which we further demonstrate to be required for HOXA1-mediated cell invasion in melanoma cells. Transcriptome profiling also shows HOXA1’s ability to potently down-regulate expression of microphthalmia-associated transcription factor (MITF) and other genes required for melanocyte differentiation, suggesting a mechanism by which HOXA1 expression de-differentiates cells into a pro-invasive cell state concomitant with TGFβ activation. Our analysis of publicly available datasets indicate that the HOXA1-induced gene signature successfully categorizes melanoma specimens based on their metastatic potential and, importantly, is capable of stratifying melanoma patient risk for metastasis based on expression in primary tumors. Together, these validation data and mechanistic insights suggest that patients whose primary tumors express HOXA1 are among a high-risk metastasis subgroup that should be considered for anti-TGFβ therapy in adjuvant settings. Moreover, further analysis of HOXA1 target genes in melanoma may reveal new pathways or targets amenable to therapeutic intervention. PMID:23435427

  19. HOXA1 drives melanoma tumor growth and metastasis and elicits an invasion gene expression signature that prognosticates clinical outcome.

    PubMed

    Wardwell-Ozgo, J; Dogruluk, T; Gifford, A; Zhang, Y; Heffernan, T P; van Doorn, R; Creighton, C J; Chin, L; Scott, K L

    2014-02-20

    Melanoma is a highly lethal malignancy notorious for its aggressive clinical course and eventual resistance to existing therapies. Currently, we possess a limited understanding of the genetic events driving melanoma progression, and much effort is focused on identifying pro-metastatic aberrations or perturbed signaling networks that constitute new therapeutic targets. In this study, we validate and assess the mechanism by which homeobox transcription factor A1 (HOXA1), a pro-invasion oncogene previously identified in a metastasis screen by our group, contributes to melanoma progression. Transcriptome and pathway profiling analyses of cells expressing HOXA1 reveals upregulation of factors involved in diverse cytokine pathways that include the transforming growth factor beta (TGFβ) signaling axis, which we further demonstrate to be required for HOXA1-mediated cell invasion in melanoma cells. Transcriptome profiling also shows HOXA1's ability to potently downregulate expression of microphthalmia-associated transcription factor (MITF) and other genes required for melanocyte differentiation, suggesting a mechanism by which HOXA1 expression de-differentiates cells into a pro-invasive cell state concomitant with TGFβ activation. Our analysis of publicly available data sets indicate that the HOXA1-induced gene signature successfully categorizes melanoma specimens based on their metastatic potential and, importantly, is capable of stratifying melanoma patient risk for metastasis based on expression in primary tumors. Together, these validation data and mechanistic insights suggest that patients whose primary tumors express HOXA1 are among a high-risk metastasis subgroup that should be considered for anti-TGFβ therapy in adjuvant settings. Moreover, further analysis of HOXA1 target genes in melanoma may reveal new pathways or targets amenable to therapeutic intervention. PMID:23435427

  20. 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

  1. 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 Central

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

    2007-01-01

    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. PMID:17900304

  2. Protein binding and its potential for eliciting minimal systemic side effects with a novel inhaled corticosteroid, ciclesonide.

    PubMed

    Rohatagi, Shashank; Luo, Yongyi; Shen, Liduo; Guo, Zuyu; Schemm, Christina; Huang, Yongqing; Chen, Kelly; David, Michael; Nave, Ruediger; King, S Peter

    2005-01-01

    Freely circulating, protein unbound, active inhaled corticosteroid (ICS) can cause systemic adverse effects. Desisobutyryl-ciclesonide (des-CIC) is the active metabolite of ciclesonide, an effective, novel ICS for persistent asthma. This study examines the free fraction of ciclesonide and des-CIC and determines whether the presence of other agents or disease states affects protein binding. Protein binding of des-CIC (0.5, 5.0, 25, 100, and 500 ng/mL) was determined, using both equilibrium dialysis and ultrafiltration, in plasma from humans (healthy and either renally or hepatically impaired) and several animal species and in the presence of either salicylates or warfarin. Dialyzed samples were analyzed by liquid chromatography with tandem mass spectroscopy to determine both free and bound concentrations of des-CIC. After ultrafiltration, spiked plasma plus H-des-CIC was separated into free and bound fractions by centrifugation and quantified by scintillation counting. Additionally, in another study, protein binding of ciclesonide was determined by equilibrium dialysis. For equilibrium dialysis, the mean percentages of des-CIC (0.5-500 ng/mL) plasma protein binding across species were high, approximately 99%, and no apparent saturation of protein binding was observed. Results were similar for ultrafiltration analysis. Protein binding of des-CIC did not change in the presence of warfarin or salicylates or in the plasma of renally or hepatically impaired patients. The protein binding of ciclesonide was 99.4% in human serum. The very low fraction of unbound des-CIC in the systemic circulation suggests minimal systemic exposure of unbound des-CIC, thus suggesting a low potential for systemic adverse effects after administration of inhaled ciclesonide. PMID:15891262

  3. 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

  4. 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

  5. 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.

  6. Oral Immunization with a Salmonella typhimurium Vaccine Vector Expressing Recombinant Enterotoxigenic Escherichia coli K99 Fimbriae Elicits Elevated Antibody Titers for Protective Immunity

    PubMed Central

    Ascón, Miguel A.; Hone, David M.; Walters, Nancy; Pascual, David W.

    1998-01-01

    Bovine enterotoxigenic Escherichia coli (ETEC) continues to cause mortality in piglets and newborn calves. In an effort to develop a safe and effective vaccine for the prevention of F5+ ETEC infections, a balanced lethal asd+ plasmid carrying the complete K99 operon was constructed and designated pMAK99-asd+. Introduction of this plasmid into an attenuated Salmonella typhimurium Δaro Δasd strain, H683, resulted in strain AP112, which stably expresses E. coli K99 fimbriae. A single oral immunization of BALB/c and CD-1 mice with strain AP112 elicited significant mucosal immunoglobulin A (IgA) titers that remained elevated for >11 weeks. IgA and IgG responses in serum specific for K99 fimbriae were also induced, with a prominent IgG1, as well as IgG2a and IgG2b, titer. To assess the derivation of these antibodies, a K99 isotype-specific B-cell ELISPOT analysis was conducted by using mononuclear cells from the lamina propria of the small intestines (LP), Peyer’s patches (PP), and spleens of vaccinated and control BALB/c mice. This analysis revealed elevated numbers of K99 fimbria-specific IgA-producing cells in the LP, PP, and spleen, whereas elevated K99 fimbria-specific IgG-producing cells were detected only in the PP and spleen. These antibodies were important for protective immunity. One-day-old neonates from dams orally immunized with AP112 were provided passive protection against oral challenge with wild-type ETEC, in contrast to challenged neonates from unvaccinated dams or from dams vaccinated with a control Salmonella vector. These results confirm that oral Salmonella vaccine vectors effectively deliver K99 fimbriae to mucosal inductive sites for sustained elevation of IgA and IgG antibodies and for eliciting protective immunity. PMID:9784559

  7. Rabbits immunized with Epstein-Barr virus gH/gL or gB recombinant proteins elicit higher serum virus neutralizing activity than gp350.

    PubMed

    Cui, Xinle; Cao, Zhouhong; Chen, Quanyi; Arjunaraja, Swadhinya; Snow, Andrew L; Snapper, Clifford M

    2016-07-25

    Epstein-Barr virus (EBV) is the primary cause of infectious mononucleosis and has been strongly implicated in the etiology of multiple epithelial and lymphoid cancers, such as nasopharyngeal carcinoma, gastric carcinoma, Hodgkin lymphoma, Burkitt lymphoma, non-Hodgkin lymphoma and post-transplant lymphoproliferative disorder. There is currently no licensed prophylactic vaccine for EBV. Most efforts to develop prophylactic vaccines have focused on EBV gp350, which binds to CD21/CD35 to gain entry into B cells, and is a major target of serum neutralizing antibody in EBV seropositive humans. However, a recombinant monomeric gp350 protein failed to prevent EBV infection in a phase II clinical trial. Thus, alternative or additional target antigens may be necessary for a successful prophylactic vaccine. EBV gH/gL and gB proteins coordinately mediate EBV fusion and entry into B cells and epithelial cells, strongly suggesting that vaccination with these proteins might elicit antibodies that will prevent EBV infection. We produced recombinant trimeric and monomeric EBV gH/gL heterodimeric proteins and a trimeric EBV gB protein, in addition to tetrameric and monomeric gp350(1-470) proteins, in Chinese hamster ovary cells. We demonstrated that vaccination of rabbits with trimeric and monomeric gH/gL, trimeric gB, and tetrameric gp350(1-470) induced serum EBV-neutralizing titers, using cultured human B cells, that were >100-fold, 20-fold, 18-fold, and 4-fold higher, respectively, than monomeric gp350(1-470). These data strongly suggest a role for testing EBV gH/gL and EBV gB in a future prophylactic vaccine to prevent EBV infection of B cells, as well as epithelial cells.

  8. 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

  9. A membrane vesicle/ribosome preparation from Serratia marcescens elicits peritoneal exudate cells expressing both tumoricidal and bactericidal activity.

    PubMed

    McCall, C; Weimer, L; Baldwin, S; Riches, D W; Canono, B; Campbell, P A

    1992-08-01

    A biological response modifier called ImuVert, derived from the bacterium Serratia marcescens, produced long-lasting elevation of peritoneal exudate cell (PEC) numbers after intraperitoneal injection into mice. These cells had enhanced ability to phagocytose both latex beads and opsonized Listeria monocytogenes. PEC harvested 2-14 days after a single injection of ImuVert killed L. monocytogenes, and ImuVert protected mice from infection by L. monocytogenes, measured both by LD50 and bacterial growth in vivo. Cells harvested 7 and 14 days after ImuVert injection also were tumoricidal, measured as killing of P815 mastocytoma cells, and ImuVert induced macrophages to express tumoricidal properties in vitro. These data suggest that ImuVert has a unique ability to induce a chronic inflammatory response, as other agents do not induce such a long-lasting influx of bactericidal inflammatory cells that also show tumoricidal activity. The consequences of this response appear to include protection from infection by certain bacteria.

  10. Artificial antigen-presenting cells engineered by recombinant vaccinia viruses expressing antigen, MHC class II, and costimulatory molecules elicit proliferation of CD4+ lymphocytes in vitro.

    PubMed

    Oertli, D; Marti, W R; Norton, J A; Tsung, K

    1997-10-01

    The current study was designed to test the ability of recombinant Vaccinia virus (rVV) encoding essential components of an artificial antigen-presenting cell to activate antigen-specific T cells in vitro. We have constructed a set of rVV encoding separately or in combination a CD4+ T cell-specific epitope (the 133-145 peptide of chicken conalbumin), the MHC class II molecule I-Ak, and costimulatory molecules (mB7-1 and mB7-2). Cultured cells infected with rVV encoding both the antigen and the presenting MHC, but not either one alone, could activate cloned CD4+ T cells specific for the virus-encoded epitope. Additional co-expression of mB7-1 and mB7-2 resulted in further enhancement of T cell response. Thus, our rVV vector expressing four different foreign gene products elicited the highest proliferation rates of antigen-specific cloned T cells. PMID:9353162

  11. 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

  12. Reduced toxicity and broad spectrum resistance to viral and fungal infection in transgenic plants expressing pokeweed antiviral protein II.

    PubMed

    Wang, P; Zoubenko, O; Tumer, N E

    1998-12-01

    Pokeweed antiviral protein II (PAPII), a 30 kDa protein isolated from leaves of Phytolacca americana, inhibits translation by catalytically removing a specific adenine residue from the large rRNA of the 60S subunit of eukaryotic ribosomes. The protein sequence of PAPII shows only 41% identity to PAP and PAP-S, two other antiviral proteins isolated from pokeweed. We isolated a cDNA corresponding to PAPII and introduced it into tobacco plants. PAPII expressed in transgenic tobacco was correctly processed to the mature form as in pokeweed and accumulated to at least 10-fold higher levels than wild-type PAP. We had previously observed a significant decrease in transformation frequency with PAP and recovered only two transgenic lines expressing 1-2 ng per mg protein. In contrast, eight different transgenic lines expressing up to 250 ng/mg PAPII were recovered, indicating that PAPII is less toxic than PAP. Two symptomless transgenic lines expressing PAPII were resistant to tobacco mosaic virus, potato virus X and the fungal pathogen Rhizoctonia solani. The level of viral and fungal resistance observed correlated well with the amount of PAPII protein accumulated. Pathogenesis-related protein PR1 was constitutively expressed in transgenic lines expressing PAPII. Although PR1 was constitutively expressed, no increase in salicylic acid levels was detected, indicating that PAPII may elicit a salicylic acid-independent signal transduction pathway.

  13. 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.

  14. 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.

  15. Proteins with molecular masses of 25 to 40 kilodaltons elicit optimal protective responses against Plasmodium chabaudi adami infection.

    PubMed

    Kima, P E; Srivastava, I K; Long, C A

    1992-12-01

    The presence of the CD4+ T cell has been shown to be crucial for resolution of acute infection in the Plasmodium chabaudi adami murine malaria model. This model is, therefore, suitable for the isolation of malaria antigens that are capable of activating protective T cells. In light of this, we set out to identify P. chabaudi adami molecules that activate protective responses in this model. Denatured P. chabaudi adami proteins were isolated by continuous-flow electrophoresis on the basis of their apparent molecular masses and then sequentially assessed for the ability to protect mice in immunization experiments. We report here that low-molecular-mass P. chabaudi adami polypeptides in the range from 25 to 40 kDa are most effective at immunizing mice against a challenge infection with viable P. chabaudi adami. The method used to obtain these proteins could also be applied to identify molecules that activate protective cell-mediated responses in other infectious disease models.

  16. Tetravalent dengue DIIIC protein together with alum and ODN elicits a Th1 response and neutralizing antibodies in mice.

    PubMed

    Zuest, Roland; Valdes, Iris; Skibinski, David; Lin, Yufang; Toh, Ying Xiu; Chan, Katherine; Hermida, Lisset; Connolly, John; Guillen, Gerardo; Fink, Katja

    2015-03-17

    Dengue disease is a global challenge for healthcare systems particularly during outbreaks, and millions of dollars are spent every year for vector control. An efficient and safe vaccine that is cost-effective could resolve the burden that dengue virus imposes on affected countries. We describe here the immunogenicity of a tetravalent formulation of a recombinant fusion protein consisting of E domain III and the capsid protein of dengue serotypes 1-4 (Tetra DIIIC). E domain III is an epitope for efficient neutralizing antibodies while the capsid protein contains T cell epitopes. Besides combining B and T cell epitopes, Tetra DIIIC is highly immunogenic due to its aggregate form and a two-component adjuvant. Following previous studies assessing the monovalent DIIIC formulations, we addressed here the quality and breadth of the T cell- and antibody response of Tetra DIIIC in mice. Tetra DIIIC induced a Th1-type response against all four DENV serotypes and dengue-specific antibodies were predominantly IgG1 and IgG2a and neutralizing, while the induction of neutralizing antibodies was dependent on IFN signaling. Importantly, the Th1 and IgG1/IgG2a profile of the DIIIC vaccine approach is similar to an efficient natural anti-dengue response.

  17. 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.

  18. Cloning, Expression, and Purification of Pseudomonas aeruginosa Flagellin, and Characterization of the Elicited Anti-Flagellin Antibody

    PubMed Central

    Behrouz, Bahador; Amirmozafari, Nour; Khoramabadi, Nima; Bahroudi, Mahboobeh; Legaee, Parisa; Mahdavi, Mehdi

    2016-01-01

    Background Pseudomonas aeruginosa is an important opportunistic human pathogen that causes serious infections in immunocompromised hosts. The single polar flagellum is an important factor in both virulence and colonization. Objectives As flagellin is the major component of the flagellar filament, the main aims of the present study are to identify, clone, express, and purify the recombinant type B flagellin (r-B-flagellin) of P. aeruginosa, as well as to evaluate the functional activity of the rabbit polyclonal antibody raised against this r-B-flagellin. Materials and Methods In the current experimental study, the r-B-flagellin gene was isolated from the P. aeruginosa PAO1 strain by PCR. It was cloned into the pET-28a vector and then transformed into the E. coli BL21 strain. Next, r-B-flagellin was overexpressed and affinity purified by Ni-NTA agarose-affinity chromatography, followed by on-column resolubilization. Polyclonal antisera against the recombinant flagellin were raised in rabbits, and the functional activity of the anti-r-B-flagellin antibody was determined by in vitro assays. Results The polyclonal antibodies raised against this r-B-flagellin inhibited the motility of the homologous PAO1 strain of P. aeruginosa, which significantly decreased the invasion of the PAO1 strain into the A549 cells and also enhanced the opsonophagocytosis of this strain. However, our polyclonal antibody showed little effect on the heterologous PAK strain. Conclusions The r-B-flagellin carried antigenic epitopes just like the native flagellin, while the polyclonal antibody raised against it exhibited functional activity. PMID:27621933

  19. Cloning, Expression, and Purification of Pseudomonas aeruginosa Flagellin, and Characterization of the Elicited Anti-Flagellin Antibody

    PubMed Central

    Behrouz, Bahador; Amirmozafari, Nour; Khoramabadi, Nima; Bahroudi, Mahboobeh; Legaee, Parisa; Mahdavi, Mehdi

    2016-01-01

    Background Pseudomonas aeruginosa is an important opportunistic human pathogen that causes serious infections in immunocompromised hosts. The single polar flagellum is an important factor in both virulence and colonization. Objectives As flagellin is the major component of the flagellar filament, the main aims of the present study are to identify, clone, express, and purify the recombinant type B flagellin (r-B-flagellin) of P. aeruginosa, as well as to evaluate the functional activity of the rabbit polyclonal antibody raised against this r-B-flagellin. Materials and Methods In the current experimental study, the r-B-flagellin gene was isolated from the P. aeruginosa PAO1 strain by PCR. It was cloned into the pET-28a vector and then transformed into the E. coli BL21 strain. Next, r-B-flagellin was overexpressed and affinity purified by Ni-NTA agarose-affinity chromatography, followed by on-column resolubilization. Polyclonal antisera against the recombinant flagellin were raised in rabbits, and the functional activity of the anti-r-B-flagellin antibody was determined by in vitro assays. Results The polyclonal antibodies raised against this r-B-flagellin inhibited the motility of the homologous PAO1 strain of P. aeruginosa, which significantly decreased the invasion of the PAO1 strain into the A549 cells and also enhanced the opsonophagocytosis of this strain. However, our polyclonal antibody showed little effect on the heterologous PAK strain. Conclusions The r-B-flagellin carried antigenic epitopes just like the native flagellin, while the polyclonal antibody raised against it exhibited functional activity.

  20. Immune responses elicited against rotavirus middle layer protein VP6 inhibit viral replication in vitro and in vivo.

    PubMed

    Lappalainen, Suvi; Pastor, Ana Ruth; Tamminen, Kirsi; López-Guerrero, Vanessa; Esquivel-Guadarrama, Fernando; Palomares, Laura A; Vesikari, Timo; Blazevic, Vesna

    2014-01-01

    Rotavirus (RV) is a common cause of severe gastroenteritis (GE) in children worldwide. Live oral RV vaccines protect against severe RVGE, but the immune correlates of protection are not yet clearly defined. Inner capsid VP6 protein is a highly conserved, abundant, and immunogenic RV protein, and VP6-specific mucosal antibodies, especially IgA, have been implicated to protect against viral challenge in mice. In the present study systemic and mucosal IgG and IgA responses were induced by immunizing BALB/c mice intranasally with a combination of recombinant RV VP6 protein (subgroup II [SGII]) and norovirus (NoV) virus-like particles (VLPs) used in a candidate vaccine. Following immunization mice were challenged orally with murine RV strain EDIMwt (SG non-I-non-II, G3P10[16]). In order to determine neutralizing activity of fecal samples, sera, and vaginal washes (VW) against human Wa RV (SGII, G1P1A[8]) and rhesus RV (SGI, G3P5B[3]), the RV antigen production was measured with an ELISA-based antigen reduction neutralization assay. Only VWs of immunized mice inhibited replication of both RVs, indicating heterotypic protection of induced antibodies. IgA antibody depletion and blocking experiments using recombinant VP6 confirmed that neutralization was mediated by anti-VP6 IgA antibodies. Most importantly, after the RV challenge significant reduction in viral shedding was observed in feces of immunized mice. These results suggest a significant role for mucosal RV VP6-specific IgA for the inhibition of RV replication in vitro and in vivo. In addition, these results underline the importance of non-serotype-specific immunity induced by the conserved subgroup-specific RV antigen VP6 in clearance of RV infection. PMID:25424814

  1. Immune responses elicited against rotavirus middle layer protein VP6 inhibit viral replication in vitro and in vivo

    PubMed Central

    Lappalainen, Suvi; Pastor, Ana Ruth; Tamminen, Kirsi; López-Guerrero, Vanessa; Esquivel-Guadarrama, Fernando; Palomares, Laura A; Vesikari, Timo; Blazevic, Vesna

    2014-01-01

    Rotavirus (RV) is a common cause of severe gastroenteritis (GE) in children worldwide. Live oral RV vaccines protect against severe RVGE, but the immune correlates of protection are not yet clearly defined. Inner capsid VP6 protein is a highly conserved, abundant, and immunogenic RV protein, and VP6-specific mucosal antibodies, especially IgA, have been implicated to protect against viral challenge in mice. In the present study systemic and mucosal IgG and IgA responses were induced by immunizing BALB/c mice intranasally with a combination of recombinant RV VP6 protein (subgroup II [SGII]) and norovirus (NoV) virus-like particles (VLPs) used in a candidate vaccine. Following immunization mice were challenged orally with murine RV strain EDIMwt (SG non-I-non-II, G3P10[16]). In order to determine neutralizing activity of fecal samples, sera, and vaginal washes (VW) against human Wa RV (SGII, G1P1A[8]) and rhesus RV (SGI, G3P5B[3]), the RV antigen production was measured with an ELISA-based antigen reduction neutralization assay. Only VWs of immunized mice inhibited replication of both RVs, indicating heterotypic protection of induced antibodies. IgA antibody depletion and blocking experiments using recombinant VP6 confirmed that neutralization was mediated by anti-VP6 IgA antibodies. Most importantly, after the RV challenge significant reduction in viral shedding was observed in feces of immunized mice. These results suggest a significant role for mucosal RV VP6-specific IgA for the inhibition of RV replication in vitro and in vivo. In addition, these results underline the importance of non-serotype-specific immunity induced by the conserved subgroup-specific RV antigen VP6 in clearance of RV infection. PMID:25424814

  2. Purify First: rapid expression and purification of proteins from XMRV.

    PubMed

    Gillette, William K; Esposito, Dominic; Taylor, Troy E; Hopkins, Ralph F; Bagni, Rachel K; Hartley, James L

    2011-04-01

    Purifying proteins from recombinant sources is often difficult, time-consuming, and costly. We have recently instituted a series of improvements in our protein purification pipeline that allows much more accurate choice of expression host and conditions and purification protocols. The key elements are parallel cloning, small scale parallel expression and lysate preparation, and small scale parallel protein purification. Compared to analyzing expression data only, results from multiple small scale protein purifications predict success at scale-up with greatly improved reliability. Using these new procedures we purified eight of nine proteins from xenotropic murine leukemia virus-related virus (XMRV) on the first attempt at large scale. PMID:21146612

  3. 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

  4. 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. PMID:27556928

  5. 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.

  6. 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

  7. TCDD and a putative endogenous AhR ligand, ITE, elicit the same immediate changes in gene expression in mouse lung fibroblasts.

    PubMed

    Henry, Ellen C; Welle, Stephen L; Gasiewicz, Thomas A

    2010-03-01

    The aryl hydrocarbon receptor (AhR), a ligand-dependent transcription factor, mediates toxicity of several classes of xenobiotics and also has important physiological roles in differentiation, reproduction, and immunity, although the endogenous ligand(s) mediating these functions is/are as yet unidentified. One candidate endogenous ligand, 2-(1'H-indolo-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE), is a potent AhR agonist in vitro, activates the murine AhR in vivo, but does not induce toxicity. We hypothesized that ITE and the toxic ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), may modify transcription of different sets of genes to account for their different toxicity. To test this hypothesis, primary mouse lung fibroblasts were exposed to 0.5muM ITE, 0.2nM TCDD, or vehicle for 4 h, and total gene expression was evaluated using microarrays. After this short-term and low-dose treatment, several hundred genes were changed significantly, and the response to ITE and TCDD was remarkably similar, both qualitatively and quantitatively. Induced gene sets included the expected battery of AhR-dependent xenobiotic-metabolizing enzymes, as well as several sets that reflect the inflammatory role of lung fibroblasts. Real time quantitative RT-qPCR assay of several selected genes confirmed these microarray data and further suggested that there may be kinetic differences in expression between ligands. These data suggest that ITE and TCDD elicit an analogous change in AhR conformation such that the initial transcription response is the same. Furthermore, if the difference in toxicity between TCDD and ITE is mediated by differences in gene expression, then it is likely that secondary changes enabled by the persistent TCDD, but not by the shorter lived ITE, are responsible.

  8. 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). PMID:27427967

  9. 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.

  10. Melatonin elicits protein kinase C-mediated calcium response in immortalized GT1-7 GnRH neurons.

    PubMed

    Kelestimur, Haluk; Ozcan, Mete; Kacar, Emine; Alcin, Ergul; Yılmaz, Bayram; Ayar, Ahmet

    2012-01-30

    Melatonin is suggested to have effects on hypothalamic-pituitary-gonadal (HPG) axis. The pulsatile pattern of GnRH release, which results in the intermittent release of gonadotropic hormones from the pituitary, has a critical importance for reproductive function but the factors responsible from this release pattern are not known. Calcium is a second messenger involved in hormone release. Therefore, investigation of the effects of melatonin on intracellular free calcium levels ([Ca(2+)](i)) would provide critical information on hormone release in immortalized GnRH neurons. The pattern of melatonin-induced intracellular calcium signaling was investigated by fluorescence calcium imaging using the immortalized GnRH-secreting GT1-7 hypothalamic neurons. Melatonin caused a significant increase in [Ca(2+)](i,) which was greatly blocked by luzindole, a melatonin antagonist, or attenuated by pre-treatment with protein kinase C inhibitor. This study suggests that melatonin seems to have a direct effect on GnRH neurons.

  11. Evaluation of the immune response elicited by vaccination with viral vectors encoding FMDV capsid proteins and boosted with inactivated virus.

    PubMed

    Romanutti, Carina; D'Antuono, Alejandra; Palacios, Carlos; Quattrocchi, Valeria; Zamorano, Patricia; La Torre, Jose; Mattion, Nora

    2013-08-30

    The aim of the present study was to assess the effect of introducing a priming step with replication-defective viral vectors encoding the capsid proteins of FMDV, followed by a boost with killed virus vaccines, using a suitable BALB/c mice model. Additionally, the immune response to other combined vector immunization regimens was studied. For this purpose, we analyzed different prime-boost immunizations with recombinant adenovirus (Ad), herpesvirus amplicons (Hs) and/or killed virus (KV) vaccines. The highest antibody titers were found in the group that received two doses of adjuvanted KV (P<0.002). Antibody titers were higher in those groups receiving a mixed regimen of vectors, compared to immunization with either vector alone (P<0.0001). Priming with any of the viral vectors induced a shift of the cytokine balance toward a Th1 type immune response regardless of the delivery system used for boosting. The highest IgG1 titer was induced by two doses of adjuvanted KV (P=0.0002) and the highest IgG2a titer corresponded to the group primed with Ad and boosted with KV (P=0.01). Re-stimulation of all groups of mice with 0.5 μg of inactivated virus five months later resulted in a fast increase of antibody titers in all the groups tested. After virus stimulation, antibody titers in the groups that received KV alone or Ad prime-KV boost, were indistinguishable (P=0.800). Protection from challenge was similar (75%) in the groups of animals that received Ad prime-Hs boost or Ad prime-KV boost, or two doses of oil-adjuvanted KV. The data presented in this study suggest that sequential immunization with viral vectors-based vaccines combined with protein-based vaccines have the potential to enhance the quality of the immune response against FMDV. PMID:23683999

  12. Evaluation of the immune response elicited by vaccination with viral vectors encoding FMDV capsid proteins and boosted with inactivated virus.

    PubMed

    Romanutti, Carina; D'Antuono, Alejandra; Palacios, Carlos; Quattrocchi, Valeria; Zamorano, Patricia; La Torre, Jose; Mattion, Nora

    2013-08-30

    The aim of the present study was to assess the effect of introducing a priming step with replication-defective viral vectors encoding the capsid proteins of FMDV, followed by a boost with killed virus vaccines, using a suitable BALB/c mice model. Additionally, the immune response to other combined vector immunization regimens was studied. For this purpose, we analyzed different prime-boost immunizations with recombinant adenovirus (Ad), herpesvirus amplicons (Hs) and/or killed virus (KV) vaccines. The highest antibody titers were found in the group that received two doses of adjuvanted KV (P<0.002). Antibody titers were higher in those groups receiving a mixed regimen of vectors, compared to immunization with either vector alone (P<0.0001). Priming with any of the viral vectors induced a shift of the cytokine balance toward a Th1 type immune response regardless of the delivery system used for boosting. The highest IgG1 titer was induced by two doses of adjuvanted KV (P=0.0002) and the highest IgG2a titer corresponded to the group primed with Ad and boosted with KV (P=0.01). Re-stimulation of all groups of mice with 0.5 μg of inactivated virus five months later resulted in a fast increase of antibody titers in all the groups tested. After virus stimulation, antibody titers in the groups that received KV alone or Ad prime-KV boost, were indistinguishable (P=0.800). Protection from challenge was similar (75%) in the groups of animals that received Ad prime-Hs boost or Ad prime-KV boost, or two doses of oil-adjuvanted KV. The data presented in this study suggest that sequential immunization with viral vectors-based vaccines combined with protein-based vaccines have the potential to enhance the quality of the immune response against FMDV.

  13. Post-expression strategies for structural investigations of membrane proteins.

    PubMed

    Columbus, Linda

    2015-06-01

    Currently, membrane proteins only comprise 1.5% of the protein data bank and, thus, still remain a challenge for structural biologists. Expression, stabilization in membrane mimics (e.g. detergent), heterogeneity (conformational and chemical), and crystallization in the presence of a membrane mimic are four major bottlenecks encountered. In response, several post-expression protein modifications have been utilized to facilitate structure determination of membrane proteins. This review highlights four approaches: limited proteolysis, deglycosylation, cysteine alkylation, and lysine methylation. Combined these approaches have facilitated the structure determination of more than 40 membrane proteins and, therefore, are a useful addition to the membrane protein structural biologist's toolkit.

  14. Data presenting a modified bacterial expression vector for expressing and purifying Nus solubility-tagged proteins.

    PubMed

    Gupta, Nidhi; Wu, Heng; Terman, Jonathan R

    2016-09-01

    Bacteria are the predominant source for producing recombinant proteins but while many exogenous proteins are expressed, only a fraction of those are soluble. We have found that a new actin regulatory enzyme Mical is poorly soluble when expressed in bacteria but the use of a Nus fusion protein tag greatly increases its solubility. However, available vectors containing a Nus tag have been engineered in a way that hinders the separation of target proteins from the Nus tag during protein purification. We have now used recombinant DNA approaches to overcome these issues and reengineer a Nus solubility tag-containing bacterial expression vector. The data herein present a modified bacterial expression vector useful for expressing proteins fused to the Nus solubility tag and separating such target proteins from the Nus tag during protein purification. PMID:27547802

  15. Functional Analysis of Avr9/Cf-9 Rapidly Elicited Genes Identifies a Protein Kinase, ACIK1, That Is Essential for Full Cf-9–Dependent Disease Resistance in TomatoW⃞

    PubMed Central

    Rowland, Owen; Ludwig, Andrea A.; Merrick, Catherine J.; Baillieul, Fabienne; Tracy, Frances E.; Durrant, Wendy E.; Fritz-Laylin, Lillian; Nekrasov, Vladimir; Sjölander, Kimmen; Yoshioka, Hirofumi; Jones, Jonathan D.G.

    2005-01-01

    Tomato (Lycopersicon esculentum) Cf genes confer resistance to the fungal pathogen Cladosporium fulvum through recognition of secreted avirulence (Avr) peptides. Plant defense responses, including rapid alterations in gene expression, are immediately activated upon perception of the pathogen. Previously, we identified a collection of Avr9/Cf-9 rapidly (15 to 30 min) elicited (ACRE) genes from tobacco (Nicotiana tabacum). Many of the ACRE genes encode putative signaling components and thus may play pivotal roles in the initial development of the defense response. To assess the requirement of 42 of these genes in the hypersensitive response (HR) induced by Cf-9/Avr9 or by Cf-4/Avr4, we used virus-induced gene silencing (VIGS) in N. benthamiana. Three genes were identified that when silenced compromised the Cf-mediated HR. We further characterized one of these genes, which encodes a Ser/Thr protein kinase called Avr9/Cf-9 induced kinase 1 (ACIK1). ACIK1 mRNA was rapidly upregulated in tobacco and tomato upon elicitation by Avr9 and by wounding. Silencing of ACIK1 in tobacco resulted in a reduced HR that correlated with loss of ACIK1 transcript. Importantly, ACIK1 was found to be required for Cf-9/Avr9- and Cf-4/Avr4-mediated HRs but not for the HR or resistance mediated by other resistance/Avr systems, such as Pto/AvrPto, Rx/Potato virus X, or N/Tobacco mosaic virus. Moreover, VIGS of LeACIK1 in tomato decreased Cf-9–mediated resistance to C. fulvum, showing the importance of ACIK1 in disease resistance. PMID:15598806

  16. 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.

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

    PubMed

    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. 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

  19. 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.

  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. Cloning and expression of special F protein from human liver

    PubMed Central

    Liu, Shu-Ye; Yu, Xin-Da; Song, Chun-Juan; Lu, Wei; Zhang, Jian-Dong; Shi, Xin-Rong; Duan, Ying; Zhang, Ju

    2007-01-01

    AIM: To clone human liver special F protein and to express it in a prokaryotic system. METHODS: Total RNA was isolated from human liver tissue and first-strand cDNA was reverse transcribed using the PCR reverse primer. Following this, cDNA of the F protein was ligated into the clone vector pUCm-T. The segment of F protein’s cDNA was subcloned into the expression vector pET-15b and transformed into E. coli BL21 (DE3) pLyss. Isopropy-β-D-thiogalactoside (IPTG) was then used to induce expression of the target protein. RESULTS: The cDNA clone of human liver special F protein (1134bp) was successfully produced, with the cDNA sequence being published in Gene-bank: DQ188836. We confirmed the expression of F protein by Western blot with a molecular weight of 43 kDa. The expressed protein accounted for 40% of the total protein extracted. CONCLUSION: F protein expresses cDNA clone in a prokaryotic system, which offers a relatively simple way of producing sufficient quantities of F protein and contributes to understanding the principal biological functions of this protein. PMID:17465469

  2. Transient Expression of Viral Proteins in Plants Using Agrobacterium tumefaciens.

    PubMed

    Hitzeroth, Inga I; van Zyl, Albertha R

    2016-01-01

    Transient expression of viral proteins in plants is a novel alternative to other expression platforms. The viral proteins can be used as potential vaccines or in diagnostics. Nicotiana benthamiana leaves or whole plants are infiltrated with recombinant Agrobacterium that harbor the gene of interest. Protein expression in the plants is rapid and results are obtained within 2-7 days. Here we describe how to make electrocompetent Agrobacterium, how to transform Agrobacterium, how to infiltrate leaves or plants with the recombinant Agrobacterium, and lastly how to extract the protein for analysis by gel electrophoresis. PMID:27076324

  3. WRKY proteins: signaling and regulation of expression during abiotic stress responses.

    PubMed

    Banerjee, Aditya; Roychoudhury, Aryadeep

    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

  4. 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

  5. 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. PMID:27029048

  6. Expression strategies for structural studies of eukaryotic membrane proteins.

    PubMed

    Lyons, Joseph A; Shahsavar, Azadeh; Paulsen, Peter Aasted; Pedersen, Bjørn Panyella; Nissen, Poul

    2016-06-01

    Integral membrane proteins in eukaryotes are central to various cellular processes and key targets in structural biology, biotechnology and drug development. However, the number of available structures for eukaryotic membrane protein belies their physiological importance. Recently, the number of available eukaryotic membrane protein structures has been steadily increasing due to the development of novel strategies in construct design, expression and structure determination. Here, we examine the major expression systems exploited for eukaryotic membrane proteins. Additionally we strive to tabulate and describe the recent expression strategies in eukaryotic membrane protein structural biology. We find that a majority of targets have been expressed in advanced host systems and modified from their wild-type form with distinct focus on conformation and thermostabilisation. However, strategies for native protein purification should also be considered where possible, particularly in light of the recent advances in single particle cryo electron microscopy.

  7. Expression strategies for structural studies of eukaryotic membrane proteins.

    PubMed

    Lyons, Joseph A; Shahsavar, Azadeh; Paulsen, Peter Aasted; Pedersen, Bjørn Panyella; Nissen, Poul

    2016-06-01

    Integral membrane proteins in eukaryotes are central to various cellular processes and key targets in structural biology, biotechnology and drug development. However, the number of available structures for eukaryotic membrane protein belies their physiological importance. Recently, the number of available eukaryotic membrane protein structures has been steadily increasing due to the development of novel strategies in construct design, expression and structure determination. Here, we examine the major expression systems exploited for eukaryotic membrane proteins. Additionally we strive to tabulate and describe the recent expression strategies in eukaryotic membrane protein structural biology. We find that a majority of targets have been expressed in advanced host systems and modified from their wild-type form with distinct focus on conformation and thermostabilisation. However, strategies for native protein purification should also be considered where possible, particularly in light of the recent advances in single particle cryo electron microscopy. PMID:27362979

  8. 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.

  9. 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.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  11. 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.

  12. 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

  13. The expression of molecule CD28 and CD38 on CD4⁺/CD8⁺ T lymphocytes in thymus and spleen elicited by Schistosoma japonicum infection in mice model.

    PubMed

    Li, Na; Ji, Peng-yu; Song, Lan-gui; Lei, Jun-xia; Lv, Zhi-yue; Wu, Zhong-dao; Shao, Xiao; Sun, Xi

    2015-08-01

    Schistosomiasis caused by human schistosomes such as Schistosoma japonicum (S. japonicum) is considered as an immune-related disease. It was demonstrated that specific cytokine antibodies' response elicited by S. japonicum infection was gradually downregulated with the progress of the disease, resulting in a Th1/Th2 polarization and suppression of immune response. CD28 (cluster of differentiation 28) is one of the proteins expressed on T cells that provide co-stimulatory signals required for T cell activation and survival, and CD38 is an activating marker of T lymphocyte with high expression in many acute or chronic infections. The immune signature of CD28null T cells in the peripheral circulation associates with chronic inflammation in many diseases, such as HIV and CMV infection. In the thymus, CD28 expression on developing thymocytes appears to play a role for their selection, and it synergizes with CD38 to induce apoptosis of DP (double-positive) thymocytes. Few reports about CD28 and CD38 have been published in schistosomiasis. Here, we investigated the dynamic patterns of the expression of molecules CD28 and CD38 on CD4(+)/CD8(+) T lymphocytes of the thymus and spleen in mice model with S. japonicum infection. Our data indicated that at an early period of infection, the frequency of CD8(+)CD28(-) T cell in the spleen decreased significantly, but higher at chronic infection than that in control. However, it demonstrated an increasing trend in the thymus with the progression of infection. The frequency of CD4(+)CD28(-) T cells increased from acute infection in the thymus, while from chronic infection in the spleen. The expression of CD38 on CD8(+) T cells began to increase at 4 weeks post infection both in the thymus and spleen; its elevated expression on CD4(+) T cells emerged at 6 weeks post infection in the thymus and at 10 weeks post infection in the spleen. Praziquantel (PZQ) treatment could partially restore the frequency of CD28(+) T cell of CD4(+) T

  14. Differential protein expression analysis following olfactory learning in Apis cerana.

    PubMed

    Zhang, Li-Zhen; Yan, Wei-Yu; Wang, Zi-Long; Guo, Ya-Hui; Yi, Yao; Zhang, Shao-Wu; Zeng, Zhi-Jiang

    2015-11-01

    Studies of olfactory learning in honeybees have helped to elucidate the neurobiological basis of learning and memory. In this study, protein expression changes following olfactory learning in Apis cerana were investigated using isobaric tags for relative and absolute quantification (iTRAQ) technology. A total of 2406 proteins were identified from the trained and untrained groups. Among these proteins, 147 were differentially expressed, with 87 up-regulated and 60 down-regulated in the trained group compared with the untrained group. These results suggest that the differentially expressed proteins may be involved in the regulation of olfactory learning and memory in A. cerana. The iTRAQ data can provide information on the global protein expression patterns associated with olfactory learning, which will facilitate our understanding of the molecular mechanisms of learning and memory of honeybees. PMID:26427996

  15. Differential protein expression analysis following olfactory learning in Apis cerana.

    PubMed

    Zhang, Li-Zhen; Yan, Wei-Yu; Wang, Zi-Long; Guo, Ya-Hui; Yi, Yao; Zhang, Shao-Wu; Zeng, Zhi-Jiang

    2015-11-01

    Studies of olfactory learning in honeybees have helped to elucidate the neurobiological basis of learning and memory. In this study, protein expression changes following olfactory learning in Apis cerana were investigated using isobaric tags for relative and absolute quantification (iTRAQ) technology. A total of 2406 proteins were identified from the trained and untrained groups. Among these proteins, 147 were differentially expressed, with 87 up-regulated and 60 down-regulated in the trained group compared with the untrained group. These results suggest that the differentially expressed proteins may be involved in the regulation of olfactory learning and memory in A. cerana. The iTRAQ data can provide information on the global protein expression patterns associated with olfactory learning, which will facilitate our understanding of the molecular mechanisms of learning and memory of honeybees.

  16. 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

  17. Optimizing transient recombinant protein expression in mammalian cells.

    PubMed

    Hopkins, Ralph F; Wall, Vanessa E; Esposito, Dominic

    2012-01-01

    Transient gene expression (TGE) in mammalian cells has become a routine process for expressing recombinant proteins in cell lines such as human embryonic kidney 293 and Chinese hamster ovary cells. The rapidly increasing need for recombinant proteins requires further improvements in TGE technology. While a great deal of focus has been directed toward optimizing the secretion of antibodies and other naturally secreted targets, much less work has been done on ways to improve cytoplasmic expression in mammalian cells. The benefits to protein production in mammalian cells, particularly for eukaryotic proteins, should be very significant - glycosylation and other posttranslational modifications will likely be native or near-native, solubility and protein folding would likely improve overexpression in heterologous hosts, and expression of proteins in their proper intracellular compartments is much more likely to occur. Improvements in this area have been slow, however, due to limited development of the cell culture processes needed for low-cost, higher-throughput expression in mammalian cells, and the relatively low diversity of DNA vectors for protein production in these systems. Here, we describe how the use of recombinational cloning, coupled with improvements in transfection protocols which increase speed and lower cost, can be combined to make mammalian cells much more amenable for routine recombinant protein expression. PMID:21987258

  18. 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.

  19. Insulin influenced expression of myelin proteins in diabetic peripheral neuropathy.

    PubMed

    Rachana, Kuruvanthe S; Manu, Mallahalli S; Advirao, Gopal M

    2016-08-26

    Diabetic peripheral neuropathy (DPN) is one of the downstream complications of diabetes. This complication is caused by the deficiency of insulin action and subsequent hyperglycemia, but the details of their pathogenesis remain unclear. Hence, it is of critical importance to understand how such hormonal variation affects the expression of myelin proteins such as myelin basic protein (MBP) and myelin associated glycoprotein (MAG) in the peripheral nerve. An earlier report from our lab has demonstrated the expression of insulin receptors (IR) in Schwann cells (SCs) of sciatic nerve. To assess the neurotrophic role of insulin in diabetic neuropathy, we studied the expression of these myelin proteins under control, DPN and insulin treated DPN subjects at developmental stages. Further, the expression of these myelin proteins was correlated with the expression of insulin receptor. Expression of myelin proteins was significantly reduced in the diabetic model compared to normal, and upregulated in insulin treated diabetic rats. Similarly, an in vitro study was also carried out in SCs grown at high glucose and insulin treated conditions. The expression pattern of myelin proteins in SCs was comparable to that of in vivo samples. In addition, quantitative study of myelin genes by real time PCR has also showed the significant expression pattern change in the insulin treated and non-treated DPN subjects. Taken together, these results corroborate the critical importance of insulin as a neurotrophic factor in demyelinized neurons in diabetic neuropathy.

  20. 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. PMID:27373596

  1. 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.

  2. 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.

  3. Nasal Immunization with Lactococcus lactis Expressing the Pneumococcal Protective Protein A Induces Protective Immunity in Mice▿

    PubMed Central

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

    2008-01-01

    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. PMID:18390997

  4. A LexA-related protein regulates redox-sensitive expression of the cyanobacterial RNA helicase, crhR

    PubMed Central

    Patterson-Fortin, Laura M.; Colvin, Kimberley R.; Owttrim, George W.

    2006-01-01

    Expression of the cyanobacterial DEAD-box RNA helicase, crhR, is regulated in response to conditions, which elicit reduction of the photosynthetic electron transport chain. A combination of electrophoretic mobility shift assay (EMSA), DNA affinity chromatography and mass spectrometry identified that a LexA-related protein binds specifically to the crhR gene. Transcript analysis indicates that lexA and crhR are divergently expressed, with lexA and crhR transcripts accumulating differentially under conditions, which respectively oxidize and reduce the electron transport chain. In addition, expression of the Synechocystis lexA gene is not DNA damage inducible and its amino acid sequence lacks two of three residues required for activity of prototypical LexA proteins, which repress expression of DNA repair genes in a range of prokaryotes. A direct effect of recombinant LexA protein on crhR expression was confirmed from the observation that LexA reduces crhR expression in a linear manner in an in vitro transcription/translation assay. The results indicate that the Synechocystis LexA-related protein functions as a regulator of redox-responsive crhR gene expression, and not DNA damage repair genes. PMID:16840531

  5. 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…

  6. Characterization and heterologous expression of hydroxycinnamoyl/benzoyl-CoA:anthranilate N-hydroxycinnamoyl/benzoyltransferase from elicited cell cultures of carnation, Dianthus caryophyllus L.

    PubMed

    Yang, Q; Reinhard, K; Schiltz, E; Matern, U

    1997-12-01

    Benzoyl-CoA:anthranilate N-benzoyltransferase catalyzes the first committed reaction of phytoalexin biosynthesis in carnation (Dianthus caryophyllus L.), and the product N-benzoylanthranilate is the precursor of several sets of dianthramides. The transferase activity is constitutively expressed in suspension-cultured carnation cells and can be rapidly induced by the addition of yeast extract. The enzyme was purified to homogeneity from yeast-induced carnation cells and shown to consist of a single polypeptide chain of 53 kDa. Roughly 20% of the sequence was identified by micro-sequencing of tryptic peptides, and some of these sequences differed in a few amino acid residues only suggesting the presence of isoenzymes. A specific 0.8 kb cDNA probe was generated by RT-PCR, employing degenerated oligonucleotide primers complementary to two of the tryptic peptides and using poly(A)+ RNA from elicited carnation cells. Five distinct benzoyltransferase clones were isolated from a cDNA library, and three cDNAs, pchcbt1-3, were sequenced and shown to encode full-size N-benzoyltransferases. The translated peptide sequences revealed more than 95% identity among these three clones. The additional two clones harbored insert sequences mostly homologous with pchcbt 1 but differing in the 3'-flanking regions due to variable usage of poly(A) addition sites. The identity of the clones was confirmed by matching the translated polypeptides with the tryptic enzyme sequences as well as by the activity of the benzoyltransferase expressed in Escherichia coli. Therefore, carnation encodes a small family of anthranilate N-benzoyltransferase genes. In vitro, the benzoyltransferases exhibited narrow substrate specificity for anthranilate but accepted a variety of aromatic acyl-CoAs. Catalytic rates with cinnamoyl- or 4-coumaroyl-CoA exceeded those observed with benzoyl-CoA, although the corresponding dianthramides did not accumulate in vivo. Thus the cDNAs described represent also the first

  7. Effective isotope labeling of proteins in a mammalian expression system.

    PubMed

    Sastry, Mallika; Bewley, Carole A; Kwong, Peter D

    2015-01-01

    Isotope labeling of biologically interesting proteins is a prerequisite for structural and dynamics studies by NMR spectroscopy. Many of these proteins require mammalian cofactors, chaperons, or posttranslational modifications such as myristoylation, glypiation, disulfide bond formation, or N- or O-linked glycosylation; and mammalian cells have the necessary machinery to produce them in their functional forms. Here, we describe recent advances in mammalian expression, including an efficient adenoviral vector-based system, for the production of isotopically labeled proteins. This system enables expression of mammalian proteins and their complexes, including proteins that require posttranslational modifications. We describe a roadmap to produce isotopically labeled (15)N and (13)C posttranslationally modified proteins, such as the outer domain of HIV-1 gp120, which has four disulfide bonds and 15 potential sites of N-linked glycosylation. These methods should allow NMR spectroscopic analysis of the structure and function of posttranslationally modified and secreted, cytoplasmic, or membrane-bound proteins.

  8. Differential Expression of Potato Tuber Protein Genes 1

    PubMed Central

    Hannapel, David J.

    1990-01-01

    Patatin and the 22-kilodalton protein complex make up more than 50% of the soluble protein present in potato (Solanum tuberosum) tubers and these two proteins are coordinately regulated during tuber development. Although genomic sequences related to these tuber genes exist in the genome of potato species that do not bear tubers, they cannot be induced into expression under the tested conditions. These genes are not expressed during substantial starch accumulation in petioles from a model petiole-leaf cutting system in nontuber-bearing plants, indicating that starch accumulation and synthesis of the major tuber proteins occur independently. Tuber protein gene expression also has been examined in hybrid potato plants that contain genomes from both tuberizing and nontuberizing species. One such triploid hybrid produced only stolons, whereas a pentaploid hybrid with an increased number of tuber genomes produced tubers. It was shown, using immunoblotting and Northern blot hybridization, that these two hybrids actively expressed both patatin and the 22-kilodalton tuber protein in induced petioles from the leaf-cutting system. The induced accumulation of patatin transcripts was consistent in all genotypes containing some tuberizing genome. The induced accumulation of the 22-kilodalton protein transcripts, however, was lower in genotypes containing some nontuberizing genome. Sucrose induction of these genes in leaves corroborates the induction patterns in petioles. A correlation exists between 22-kilodalton protein gene expression and a potato plant's ability to produce stolons or tubers. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 6 PMID:16667872

  9. Network Clustering Revealed the Systemic Alterations of Mitochondrial Protein Expression

    PubMed Central

    Koo, Hyun-Jung; Park, Wook-Ha; Yang, Jae-Seong; Yu, Myeong-Hee; Kim, Sanguk; Pak, Youngmi Kim

    2011-01-01

    The mitochondrial protein repertoire varies depending on the cellular state. Protein component modifications caused by mitochondrial DNA (mtDNA) depletion are related to a wide range of human diseases; however, little is known about how nuclear-encoded mitochondrial proteins (mt proteome) changes under such dysfunctional states. In this study, we investigated the systemic alterations of mtDNA-depleted (ρ0) mitochondria by using network analysis of gene expression data. By modularizing the quantified proteomics data into protein functional networks, systemic properties of mitochondrial dysfunction were analyzed. We discovered that up-regulated and down-regulated proteins were organized into two predominant subnetworks that exhibited distinct biological processes. The down-regulated network modules are involved in typical mitochondrial functions, while up-regulated proteins are responsible for mtDNA repair and regulation of mt protein expression and transport. Furthermore, comparisons of proteome and transcriptome data revealed that ρ0 cells attempted to compensate for mtDNA depletion by modulating the coordinated expression/transport of mt proteins. Our results demonstrate that mt protein composition changed to remodel the functional organization of mitochondrial protein networks in response to dysfunctional cellular states. Human mt protein functional networks provide a framework for understanding how cells respond to mitochondrial dysfunctions. PMID:21738461

  10. Attenuated influenza virus construct with enhanced hemagglutinin protein expression.

    PubMed

    Maamary, Jad; Pica, Natalie; Belicha-Villanueva, Alan; Chou, Yi-ying; Krammer, Florian; Gao, Qinshan; García-Sastre, Adolfo; Palese, Peter

    2012-05-01

    Influenza A viruses encoding an altered viral NS1 protein have emerged as promising live attenuated vaccine platforms. A carboxy-terminal truncation in the NS1 protein compromises its interferon antagonism activity, making these viruses attenuated in the host yet still able to induce protection from challenge with wild-type viruses. However, specific viral protein expression by NS1-truncated viruses is known to be decreased in infected cells. In this report, we show that recombinant H5N1 and H1N1 influenza viruses encoding a truncated NS1 protein expressed lower levels of hemagglutinin (HA) protein in infected cells than did wild-type viruses. This reduction in HA protein expression correlated with a reduction in HA mRNA levels in infected cells. NS1 truncation affected the expression of HA protein but not that of the nucleoprotein (NP). This segment specificity was mapped to the terminal sequences of their specific viral RNAs. Since the HA protein is the major immunogenic component in influenza virus vaccines, we sought to restore its expression levels in NS1-truncated viruses in order to improve their vaccine efficacy. For this purpose, we generated an NS1-truncated recombinant influenza A/Puerto Rico/8/34 (rPR8) virus carrying the G3A C8U "superpromoter" mutations in the HA genomic RNA segment. This strategy retained the attenuation properties of the recombinant virus but enhanced the expression level of HA protein in infected cells. Finally, mice immunized with rPR8 viruses encoding a truncated NS1 protein and carrying the G3A C8U mutations in the HA segment demonstrated enhanced protection from wild-type virus challenge over that for mice vaccinated with an rPR8 virus encoding the truncated NS1 protein alone.

  11. 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

  12. 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

  13. Surface protein expression in group B streptococcal invasive isolates.

    PubMed

    Ferrieri, P; Flores, A E

    1997-01-01

    Results from characterization of 211 GBS isolates from early-onset disease indicated that serotypes Ia, III and V accounted for almost 80% of the isolates, and that alpha was the protein most often expressed. Each of the common polysaccharide types had a characteristic predominant protein expression pattern: alpha for Ia, R4 for type III and R1+R4 for type V isolates. Expression of alpha protein was always mutually exclusive of R proteins. The presence of more than one species of R by a given isolate was confirmed by IEP. In addition, PAGE/WB studies verified the multiple MW forms of R1, and the variation from strain to strain in the highest form of R4 that we had previously reported. Our data not only showed the great complexity of the GBS cell surface but also demonstrated the advantage of using both type polysaccharides and surface-localized proteins as markers for characterization of GBS strains.

  14. Development of polyclonal antibodies for detection of aflatoxigenic molds involving culture filtrate and chimeric proteins expressed in Escherichia coli.

    PubMed

    Shapira, R; Paster, N; Menasherov, M; Eyal, O; Mett, A; Meiron, T; Kuttin, E; Salomon, R

    1997-03-01

    Polyclonal antibodies (PAb) were raised against an aflatoxigenic strain of Aspergillus parasiticus by using two different sources for antibody elicitation: (i) filtrate of a culture on which the fungus had been grown (ii) and two chimeric proteins, expressed in Escherichia coli as separate products, of the genes ver-1 and apa-2, which are involved in aflatoxin biosynthesis. The gene products were amplified by PCR, and each was cloned into the E. coli expression vector pGEX2T. Upon induction, the bacteria overexpressed 38- and 33-kDa chimeric proteins corresponding to the N-terminal domains of the genes ver-1 and apa-2, respectively. The chimeric proteins were isolated and affinity purified for use as antigens. The specificity of the raised antibodies was examined by enzyme-linked immunosorbent assay (ELISA). The PAbs raised against the culture filtrate reacted with all the species of Aspergillus and Penicillium tested but not with Fusarium species or corn gain. However, the PAbs elicited against the chimeric proteins were highly specific, showing significantly higher ELISA absorbance values (A405) against A. parasiticus and A. flavus than against the other fungi tested and the corn grain. The approach of utilizing gene products associated with aflatoxin biosynthesis for antibody production therefore appears to be feasible. Such a multiantibody system combined with the PCR technique, could provide a useful tool for the rapid, sensitive, and accurate detection of aflatoxin producers present in grains and foods. PMID:9055416

  15. 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

  16. A DNA Vaccine Coding for the Brucella Outer Membrane Protein 31 Confers Protection against B. melitensis and B. ovis Infection by Eliciting a Specific Cytotoxic Response

    PubMed Central

    Cassataro, Juliana; Velikovsky, Carlos A.; de la Barrera, Silvia; Estein, Silvia M.; Bruno, Laura; Bowden, Raúl; Pasquevich, Karina A.; Fossati, Carlos A.; Giambartolomei, Guillermo H.

    2005-01-01

    The development of an effective subunit vaccine against brucellosis is a research area of intense interest. The outer membrane proteins (Omps) of Brucella spp. have been extensively characterized as potential immunogenic and protective antigens. This study was conducted to evaluate the immunogenicity and protective efficacy of the B. melitensis Omp31 gene cloned in the pCI plasmid (pCIOmp31). Immunization of BALB/c mice with pCIOmp31 conferred protection against B. ovis and B. melitensis infection. Mice vaccinated with pCIOmp31 developed a very weak humoral response, and in vitro stimulation of their splenocytes with recombinant Omp31 did not induced the secretion of gamma interferon. Splenocytes from Omp31-vaccinated animals induced a specific cytotoxic-T-lymphocyte activity, which leads to the in vitro lysis of Brucella-infected macrophages. pCIOmp31 immunization elicited mainly CD8+ T cells, which mediate cytotoxicity via perforins, but also CD4+ T cells, which mediate lysis via the Fas-FasL pathway. In vivo depletion of T-cell subsets showed that the pCIOmp31-induced protection against Brucella infection is mediated predominantly by CD8+ T cells, although CD4+T cells also contribute. Our results demonstrate that the Omp31 DNA vaccine induces cytotoxic responses that have the potential to contribute to protection against Brucella infection. The protective response could be related to the induction of CD8+ T cells that eliminate Brucella-infected cells via the perforin pathway. PMID:16177328

  17. 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.

  18. 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

  19. 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

  20. 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.

  1. Small-scale expression of proteins in E. coli.

    PubMed

    Zerbs, Sarah; Giuliani, Sarah; Collart, Frank

    2014-01-01

    Proteins participate in virtually every cellular activity, and a knowledge of protein function is essential for an understanding of biological systems. However, protein diversity necessitates the application of an array of in vivo and in vitro approaches for characterization of the functional and biochemical properties of proteins. Methods that enable production of proteins for in vitro studies are critical for determination of the molecular, kinetic, and thermodynamic properties of these molecules. Ideally, proteins could be purified from the original source; however, the native host is often unsuitable for a number of reasons. Consequently, systems for heterologous protein production are commonly used to produce large amounts of protein. Heterologous expression hosts are chosen using a number of criteria, including genetic tractability, advantageous production or processing characteristics (secretion or posttranslational modifications), or economy of time and growth requirements. The subcloning process also provides an opportunity to introduce purification tags, epitope tags, fusions, truncations, and mutations into the coding sequence that may be useful in downstream purification or characterization applications. Bacterial systems for heterologous protein expression have advantages in ease of use, cost, short generation times, and scalability. These expression systems have been widely used by high-throughput protein production projects and often represent an initial experiment for any expression target. Escherichia coli has been studied for many years as a model bacterial organism and is one of the most popular hosts for heterologous protein expression (Terpe, 2006). Its protein production capabilities have been intensively studied, and the ease of genetic manipulation in this organism has led to the development of strains engineered exclusively for use in protein expression. These resources are widely available from commercial sources and public repositories

  2. Small-scale expression of proteins in E. coli.

    PubMed

    Zerbs, Sarah; Giuliani, Sarah; Collart, Frank

    2014-01-01

    Proteins participate in virtually every cellular activity, and a knowledge of protein function is essential for an understanding of biological systems. However, protein diversity necessitates the application of an array of in vivo and in vitro approaches for characterization of the functional and biochemical properties of proteins. Methods that enable production of proteins for in vitro studies are critical for determination of the molecular, kinetic, and thermodynamic properties of these molecules. Ideally, proteins could be purified from the original source; however, the native host is often unsuitable for a number of reasons. Consequently, systems for heterologous protein production are commonly used to produce large amounts of protein. Heterologous expression hosts are chosen using a number of criteria, including genetic tractability, advantageous production or processing characteristics (secretion or posttranslational modifications), or economy of time and growth requirements. The subcloning process also provides an opportunity to introduce purification tags, epitope tags, fusions, truncations, and mutations into the coding sequence that may be useful in downstream purification or characterization applications. Bacterial systems for heterologous protein expression have advantages in ease of use, cost, short generation times, and scalability. These expression systems have been widely used by high-throughput protein production projects and often represent an initial experiment for any expression target. Escherichia coli has been studied for many years as a model bacterial organism and is one of the most popular hosts for heterologous protein expression (Terpe, 2006). Its protein production capabilities have been intensively studied, and the ease of genetic manipulation in this organism has led to the development of strains engineered exclusively for use in protein expression. These resources are widely available from commercial sources and public repositories

  3. High-Throughput Baculovirus Expression System for Membrane Protein Production.

    PubMed

    Kalathur, Ravi C; Panganiban, Marinela; Bruni, Renato

    2016-01-01

    The ease of use, robustness, cost-effectiveness, and posttranslational machinery make baculovirus expression system a popular choice for production of eukaryotic membrane proteins. This system can be readily adapted for high-throughput operations. This chapter outlines the techniques and procedures for cloning, transfection, small-scale production, and purification of membrane protein samples in a high-throughput manner. PMID:27485337

  4. The Proteome Response to Amyloid Protein Expression In Vivo

    PubMed Central

    Gomes, Ricardo A.; Franco, Catarina; Da Costa, Gonçalo; Planchon, Sébastien; Renaut, Jenny; Ribeiro, Raquel M.; Pinto, Francisco; Silva, Marta Sousa; Coelho, Ana Varela; Freire, Ana Ponces; Cordeiro, Carlos

    2012-01-01

    Protein misfolding disorders such as Alzheimer, Parkinson and transthyretin amyloidosis are characterized by the formation of protein amyloid deposits. Although the nature and location of the aggregated proteins varies between different diseases, they all share similar molecular pathways of protein unfolding, aggregation and amyloid deposition. Most effects of these proteins are likely to occur at the proteome level, a virtually unexplored reality. To investigate the effects of an amyloid protein expression on the cellular proteome, we created a yeast expression system using human transthyretin (TTR) as a model amyloidogenic protein. We used Saccharomyces cerevisiae, a living test tube, to express native TTR (non-amyloidogenic) and the amyloidogenic TTR variant L55P, the later forming aggregates when expressed in yeast. Differential proteome changes were quantitatively analyzed by 2D-differential in gel electrophoresis (2D-DIGE). We show that the expression of the amyloidogenic TTR-L55P causes a metabolic shift towards energy production, increased superoxide dismutase expression as well as of several molecular chaperones involved in protein refolding. Among these chaperones, members of the HSP70 family and the peptidyl-prolyl-cis-trans isomerase (PPIase) were identified. The latter is highly relevant considering that it was previously found to be a TTR interacting partner in the plasma of ATTR patients but not in healthy or asymptomatic subjects. The small ubiquitin-like modifier (SUMO) expression is also increased. Our findings suggest that refolding and degradation pathways are activated, causing an increased demand of energetic resources, thus the metabolic shift. Additionally, oxidative stress appears to be a consequence of the amyloidogenic process, posing an enhanced threat to cell survival. PMID:23185553

  5. 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

  6. 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

  7. Detecting protein complexes from active protein interaction networks constructed with dynamic gene expression profiles

    PubMed Central

    2013-01-01

    Background Protein interaction networks (PINs) are known to be useful to detect protein complexes. However, most available PINs are static, which cannot reflect the dynamic changes in real networks. At present, some researchers have tried to construct dynamic networks by incorporating time-course (dynamic) gene expression data with PINs. However, the inevitable background noise exists in the gene expression array, which could degrade the quality of dynamic networkds. Therefore, it is needed to filter out contaminated gene expression data before further data integration and analysis. Results Firstly, we adopt a dynamic model-based method to filter noisy data from dynamic expression profiles. Then a new method is proposed for identifying active proteins from dynamic gene expression profiles. An active protein at a time point is defined as the protein the expression level of whose corresponding gene at that time point is higher than a threshold determined by a standard variance involved threshold function. Furthermore, a noise-filtered active protein interaction network (NF-APIN) is constructed. To demonstrate the efficiency of our method, we detect protein complexes from the NF-APIN, compared with those from other dynamic PINs. Conclusion A dynamic model based method can effectively filter out noises in dynamic gene expression data. Our method to compute a threshold for determining the active time points of noise-filtered genes can make the dynamic construction more accuracy and provide a high quality framework for network analysis, such as protein complex prediction. PMID:24565281

  8. Cell-Free Expression of G Protein-Coupled Receptors.

    PubMed

    Segers, Kenneth; Masure, Stefan

    2015-01-01

    The large-scale production of recombinant G protein-coupled receptors (GPCRs) is one of the major bottlenecks that hamper functional and structural studies of this important class of integral membrane proteins. Heterologous overexpression of GPCRs often results in low yields of active protein, usually due to a combination of several factors, such as low expression levels, protein insolubility, host cell toxicity, and the need to use harsh and often denaturing detergents (e.g., SDS, LDAO, OG, and DDM, among others) to extract the recombinant receptor from the host cell membrane. Many of these problematic issues are inherently linked to cell-based expression systems and can therefore be circumvented by the use of cell-free systems. In this unit, we provide a range of protocols for the production of GPCRs in a cell-free expression system. Using this system, we typically obtain GPCR expression levels of ∼1 mg per ml of reaction mixture in the continuous-exchange configuration. Although the protocols in this unit have been optimized for the cell-free expression of GPCRs, they should provide a good starting point for the production of other classes of membrane proteins, such as ion channels, aquaporins, carrier proteins, membrane-bound enzymes, and even large molecular complexes.

  9. Hypoxic-induced stress protein expression in rat cardiac myocytes

    SciTech Connect

    Howard, G.; Geoghegan, T.E.

    1986-05-01

    Mammalian stress proteins can be induced in cells and tissues exposed to a variety of conditions including hyperthermia and diminished O/sub 2/ supply. The authors have previously shown that the expression of three stress proteins (71, 85, and 95 kDa) was induced in cardiac tissue from mice exposed to hypoxic conditions. The expression of mRNAs coding for the 85 and 95 kDa proteins increase with time of exposure to hypoxia, while the mRNA coding for the 71 kDa protein is transiently induced. The authors extended these studies to investigate the expression of stress proteins in isolated rat cardiac myocytes. Freshly prepared myocytes were exposed to control, hypoxic, anoxic, or heat-shock environments for up to 16 h. The proteins were then labeled for 6 hours with (/sup 35/S)methionine. Analysis of the solubilized proteins by SDS-PAGE and autoradiography showed that there was a 6-fold increase in synthesis of the 85 kDa protein upon exposure to hypoxia but not heat-shock conditions. The 71 kDa protein was present at high levels in both control and treated myocyte protein preparations, and presumably had been induced during the isolation procedure. Total RNA isolated from intact rat heart and isolated myocytes was compared by cell-free translation analysis and showed induction of RNAs coding for several stress proteins in the myocyte preparation. The induced proteins at 85 and 95 kDa have molecular weights similar to reported cell stress and/or glucose-regulated proteins.

  10. 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.

  11. Expression of Eukaryotic Membrane Proteins in Pichia pastoris.

    PubMed

    Hartmann, Lucie; Kugler, Valérie; Wagner, Renaud

    2016-01-01

    A key point when it comes to heterologous expression of eukaryotic membrane proteins (EMPs) is the choice of the best-suited expression platform. The yeast Pichia pastoris has proven to be a very versatile system showing promising results in a growing number of cases. Indeed, its particular methylotrophic characteristics combined to the very simple handling of a eukaryotic microorganism that possesses the majority of mammalian-like machineries make it a very competitive expression system for various complex proteins, in amounts compatible with functional and structural studies. This chapter describes a set of robust methodologies routinely used for the successful expression of a variety of EMPs, going from yeast transformation with the recombinant plasmid to the analysis of the quality and quantity of the proteins produced. PMID:27485335

  12. Expression of Eukaryotic Membrane Proteins in Pichia pastoris.

    PubMed

    Hartmann, Lucie; Kugler, Valérie; Wagner, Renaud

    2016-01-01

    A key point when it comes to heterologous expression of eukaryotic membrane proteins (EMPs) is the choice of the best-suited expression platform. The yeast Pichia pastoris has proven to be a very versatile system showing promising results in a growing number of cases. Indeed, its particular methylotrophic characteristics combined to the very simple handling of a eukaryotic microorganism that possesses the majority of mammalian-like machineries make it a very competitive expression system for various complex proteins, in amounts compatible with functional and structural studies. This chapter describes a set of robust methodologies routinely used for the successful expression of a variety of EMPs, going from yeast transformation with the recombinant plasmid to the analysis of the quality and quantity of the proteins produced.

  13. 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

  14. 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

  15. Quorum-sensing Salmonella selectively trigger protein expression within tumors

    PubMed Central

    Swofford, Charles A.; Van Dessel, Nele; Forbes, Neil S.

    2015-01-01

    Salmonella that secrete anticancer proteins have the potential to eliminate tumors, but nonspecific expression causes damage to healthy tissue. We hypothesize that Salmonella, integrated with a density-dependent switch, would only express proteins in tightly packed colonies within tumors. To test this hypothesis, we cloned the lux quorum-sensing (QS) system and a GFP reporter into nonpathogenic Salmonella. Fluorescence and bacterial density were measured in culture and in a tumor-on-a-chip device to determine the critical density necessary to initiate expression. QS Salmonella were injected into 4T1 tumor-bearing mice to quantify GFP expression in vivo using immunofluorescence. At densities below 0.6 × 1010 cfu/g in tumors, less than 3% of QS Salmonella expressed GFP. Above densities of 4.2 × 1010 cfu/g, QS Salmonella had similar expression levels to constitutive controls. GFP expression by QS colonies was dependent upon the distance to neighboring bacteria. No colonies expressed GFP when the average distance to neighbors was greater than 155 µm. Calculations of autoinducer concentrations showed that expression was sigmoidally dependent on density and inversely dependent on average radial distance. Based on bacterial counts from excised tissue, the liver density (0.0079 × 1010 cfu/g) was less than the critical density (0.11 × 1010 cfu/g) necessary to initiate expression. QS Salmonella are a promising tool for cancer treatment that will target drugs to tumors while preventing damage to healthy tissue. PMID:25737556

  16. Expression of pokeweed antiviral protein in mammalian cells activates c-Jun NH2-terminal kinase without causing apoptosis.

    PubMed

    Chan Tung, Kelvin W; Mansouri, Sheila; Hudak, Katalin A

    2008-01-01

    Pokeweed antiviral protein (PAP) is a ribosome inactivating protein isolated from the pokeweed plant (Phytolacca americana L.) that exhibits broad range antiviral activity against several human viruses including HIV and influenza. This characteristic suggests that PAP may have therapeutic applications; however, it is not known whether the protein elicits a ribotoxic stress response that would result in cell death. Therefore, we expressed PAP in 293T cells and showed that the enzyme did not inhibit protein translation even though approximately 15% of the ribosomal RNA (rRNA) was depurinated. PAP expression induced the activation of c-Jun NH2-terminal kinase (JNK), which was specific to rRNA depurination, as the enzymatically inactive mutant PAPx did not affect kinase activity. Moreover, incubation of PAP-expressing cells with translation inhibitors diminished JNK activation, indicating that the signal for induction of the kinase pathway originated from ribosomes. JNK activation did not result in apoptosis as demonstrated by the absence of caspase-3 and poly(ADP-ribose) polymerase cleavage and by the lack of cell staining for morphological changes in membrane permeability. Unlike all ribosome inactivating proteins tested thus far, the stress response triggered by PAP expression did not result in cell death, which supports further investigation of the enzyme in the design of novel antiviral agents.

  17. Expression of genes encoding extracellular matrix proteins: a macroarray study.

    PubMed

    Futyma, Konrad; Miotła, Paweł; Różyńska, Krystyna; Zdunek, Małgorzata; Semczuk, Andrzej; Rechberger, Tomasz; Wojcierowski, Jacek

    2014-12-01

    Endometrial cancer (EC) is one of the most common gynecological malignancies in Poland, with well-established risk factors. Genetic instability and molecular alterations responsible for endometrial carcinogenesis have been systematically investigated. The aim of the present study was to investigate, by means of cDNA macroarrays, the expression profiles of genes encoding extracellular matrix (ECM) proteins in ECs. Tissue specimens were collected during surgical procedures from 40 patients with EC, and control tissue was collected from 9 patients with uterine leiomyomas. RNA was isolated and RT-PCR with radioisotope-labeled cDNA was performed. The levels of ECM protein gene expression in normal endometrial tissues were compared to the expression of these genes in EC specimens. Statistically significant differences in gene expression, stratified by clinical stage of the ECs, were detected for aggrecan, vitronectin, tenascin R, nidogen and two collagen proteins: type VIII chain α1 and type XI chain α2. All of these proteins were overexpressed in stage III endometrial carcinomas compared to levels in stage I and II uterine neoplasms. In conclusion, increased expression of genes encoding ECM proteins may play an important role in facilitating accelerated disease progression of human ECs.

  18. High-throughput insect cell protein expression applications.

    PubMed

    Buchs, Mirjam; Kim, Ernie; Pouliquen, Yann; Sachs, Michael; Geisse, Sabine; Mahnke, Marion; Hunt, Ian

    2009-01-01

    The Baculovirus Expression Vector System (BEVS) is one of the most efficient systems for production of recombinant proteins and consequently its application is wide-spread in industry as well as in academia. Since the early 1970s, when the first stable insect cell lines were established and the infectivity of bacu-lovirus in an in vitro culture system was demonstrated (1, 2), virtually thousands of reports have been published on the successful expression of proteins using this system as well as on method improvement. However, despite its popularity the system is labor intensive and time consuming. Moreover, adaptation of the system to multi-parallel (high-throughput) expression is much more difficult to achieve than with E. coli due to its far more complex nature. However, recent years have seen the development of strategies that have greatly enhanced the stream-lining and speed of baculovirus protein expression for increased throughput via use of automation and miniaturization. This chapter therefore tries to collate these developments in a series of protocols (which are modifications to standard procedure plus several new approaches) that will allow the user to expedite the speed and throughput of baculovirus-mediated protein expression and facilitate true multi-parallel, high-throughput protein expression profiling in insect cells. In addition we also provide a series of optimized protocols for small and large-scale transient insect cell expression that allow for both the rapid analysis of multiple constructs and the concomitant scale-up of those selected for on-going analysis. Since this approach is independent of viral propagation, the timelines for this approach are markedly shorter and offer a significant advantage over standard bacu-lovirus expression approach strategies in the context of HT applications.

  19. 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. PMID:26371433

  20. Subcellular localization of transiently expressed fluorescent fusion proteins.

    PubMed

    Collings, David A

    2013-01-01

    The recent and massive expansion in plant genomics data has generated a large number of gene sequences for which two seemingly simple questions need to be answered: where do the proteins encoded by these genes localize in cells, and what do they do? One widespread approach to answering the localization question has been to use particle bombardment to transiently express unknown proteins tagged with green fluorescent protein (GFP) or its numerous derivatives. Confocal fluorescence microscopy is then used to monitor the localization of the fluorescent protein as it hitches a ride through the cell. The subcellular localization of the fusion protein, if not immediately apparent, can then be determined by comparison to localizations generated by fluorescent protein fusions to known signalling sequences and proteins, or by direct comparison with fluorescent dyes. This review aims to be a tour guide for researchers wanting to travel this hitch-hiker's path, and for reviewers and readers who wish to understand their travel reports. It will describe some of the technology available for visualizing protein localizations, and some of the experimental approaches for optimizing and confirming localizations generated by particle bombardment in onion epidermal cells, the most commonly used experimental system. As the non-conservation of signal sequences in heterologous expression systems such as onion, and consequent mis-targeting of fusion proteins, is always a potential problem, the epidermal cells of the Argenteum mutant of pea are proposed as a model system.

  1. Subcellular localization of transiently expressed fluorescent fusion proteins.

    PubMed

    Collings, David A

    2013-01-01

    The recent and massive expansion in plant genomics data has generated a large number of gene sequences for which two seemingly simple questions need to be answered: where do the proteins encoded by these genes localize in cells, and what do they do? One widespread approach to answering the localization question has been to use particle bombardment to transiently express unknown proteins tagged with green fluorescent protein (GFP) or its numerous derivatives. Confocal fluorescence microscopy is then used to monitor the localization of the fluorescent protein as it hitches a ride through the cell. The subcellular localization of the fusion protein, if not immediately apparent, can then be determined by comparison to localizations generated by fluorescent protein fusions to known signalling sequences and proteins, or by direct comparison with fluorescent dyes. This review aims to be a tour guide for researchers wanting to travel this hitch-hiker's path, and for reviewers and readers who wish to understand their travel reports. It will describe some of the technology available for visualizing protein localizations, and some of the experimental approaches for optimizing and confirming localizations generated by particle bombardment in onion epidermal cells, the most commonly used experimental system. As the non-conservation of signal sequences in heterologous expression systems such as onion, and consequent mis-targeting of fusion proteins, is always a potential problem, the epidermal cells of the Argenteum mutant of pea are proposed as a model system. PMID:23996319

  2. 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.

  3. Agrobacterium induces expression of a host F-box protein required for tumorigenicity

    PubMed Central

    Zaltsman, Adi; Krichevsky, Alexander; Loyter, Abraham; Citovsky, Vitaly

    2010-01-01

    In plant-pathogen interactions, the host defends against the invading pathogen and the pathogen aims to suppress or subvert this defense. Whereas the defense suppression strategy is relatively well understood for many pathogens, the mechanisms by which pathogens can actively utilize the defense machinery of the host remain obscure. We report that Agrobacterium, a microorganism that elicits neoplastic growths on many plant species, induces expression of a plant defense-related F-box protein, VBF, which it incorporates into its own pathway for genetic transformation. Our data suggest that VBF may function to uncoat the bacterial transferred DNA from its associated virulence VirE2 and host VIP1 proteins via the SCFVBF pathway. Suppression of VBF elevates the intracellular content of VIP1, but renders the plant largely resistant to Agrobacterium, indicating that, in the infection pathway, VBF is functionally epistatic to VIP1. When expressed in Agrobacterium and exported into the plant cell, VBF facilitates tumor formation. PMID:20227663

  4. Effects of Chemically Modified Messenger RNA on Protein Expression.

    PubMed

    Li, Bin; Luo, Xiao; Dong, Yizhou

    2016-03-16

    Chemically modified nucleotides play significant roles in the effectiveness of mRNA translation. Here, we describe the synthesis of two sets of chemically modified mRNAs [encoding firefly Luciferase (FLuc) and enhanced green fluorescent protein (eGFP), respectively], evaluation of protein expression, and correlation analysis of expression level under various conditions. The results indicate that chemical modifications of mRNAs are able to significantly improve protein expression, which is dependent on cell types and coding sequences. Moreover, eGFP mRNAs with N1-methylpseudouridine (me(1)ψ), 5-methoxyuridine (5moU), and pseudouridine (ψ) modifications ranked top three in cell lines tested. Interestingly, 5moU-modified eGFP mRNA was more stable than other eGFP mRNAs. Consequently, me(1)ψ, 5moU, and ψ are promising nucleotides for chemical modification of mRNAs. PMID:26906521

  5. 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

    2015-01-01

    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. PMID

  6. Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

    PubMed Central

    Bird, Louise E.; Rada, Heather; Verma, Anil; Gasper, Raphael; Birch, James; Jennions, Matthew; Lӧwe, Jan; Moraes, Isabel; Owens, Raymond J.

    2015-01-01

    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. PMID

  7. 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.

  8. Combining in Vitro Folding with Cell Free Protein Synthesis for Membrane Protein Expression.

    PubMed

    Focke, Paul J; Hein, Christopher; Hoffmann, Beate; Matulef, Kimberly; Bernhard, Frank; Dötsch, Volker; Valiyaveetil, Francis I

    2016-08-01

    Cell free protein synthesis (CFPS) has emerged as a promising methodology for protein expression. While polypeptide production is very reliable and efficient using CFPS, the correct cotranslational folding of membrane proteins during CFPS is still a challenge. In this contribution, we describe a two-step protocol in which the integral membrane protein is initially expressed by CFPS as a precipitate followed by an in vitro folding procedure using lipid vesicles for converting the protein precipitate to the correctly folded protein. We demonstrate the feasibility of using this approach for the K(+) channels KcsA and MVP and the amino acid transporter LeuT. We determine the crystal structure of the KcsA channel obtained by CFPS and in vitro folding to show the structural similarity to the cellular expressed KcsA channel and to establish the feasibility of using this two-step approach for membrane protein production for structural studies. Our studies show that the correct folding of these membrane proteins with complex topologies can take place in vitro without the involvement of the cellular machinery for membrane protein biogenesis. This indicates that the folding instructions for these complex membrane proteins are contained entirely within the protein sequence. PMID:27384110

  9. Expression and export: recombinant protein production systems for Aspergillus.

    PubMed

    Fleissner, André; Dersch, Petra

    2010-07-01

    Several Aspergillus species, in particular Aspergillus niger and Aspergillus oryzae, are widely used as protein production hosts in various biotechnological applications. In order to improve the expression and secretion of recombinant proteins in these filamentous fungi, several novel genetic engineering strategies have been developed in recent years. This review describes state-of-the-art genetic manipulation technologies used for strain improvement, as well as recent advances in designing the most appropriate engineering strategy for a particular protein production process. Furthermore, current developments in identifying bottlenecks in the protein production and secretion pathways are described and novel approaches to overcome these limitations are introduced. An appropriate combination of expression vectors and optimized host strains will provide cell factories customized for each production process and expand the great potential of Aspergilli as biotechnology workhorses to more complex multi-step industrial applications.

  10. Expression, delivery and function of insecticidal proteins expressed by recombinant baculoviruses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  11. [PPR proteins--modular factors regulating expression of organellar genomes].

    PubMed

    Zapisek, Bartosz; Piątkowski, Jakub

    2015-01-01

    PPR proteins make up the most numerous family of RNA-binding proteins identified to date. They localize almost exclusively to plastids and mitochondria of eukaryotic organisms. The most striking feature of this family is the expansion of PPR protein-encoding genes in vascular plants, which likely coincided with plants colonizing land. PPR proteins participate in stabilizing, editing, splicing, degradation and processing of policistronic transcripts, as well as translation activation in mitochondria and plastids. Although the number of PPR proteins in non-plant organisms is significantly smaller than in plants, they still play a crucial role in regulating the expression of mtDNA. Disruptions of PPR protein-encoding genes usually result in severe phenotypic consequences. Plant PPR proteins bind RNA in a sequence-specific manner, where a single PPR motif recognizes an individual nucleotide in a given sequence. This opens up possibilities for engineering de novo synthetic protein sequences that would interact with precisely determined organellar sequences, thus enabling modulation of mtDNA and ctDNA expression.

  12. Beyond protein expression, MOPED goes multi-omics.

    PubMed

    Montague, Elizabeth; Janko, Imre; Stanberry, Larissa; Lee, Elaine; Choiniere, John; Anderson, Nathaniel; Stewart, Elizabeth; Broomall, William; Higdon, Roger; Kolker, Natali; Kolker, Eugene

    2015-01-01

    MOPED (Multi-Omics Profiling Expression Database; http://moped.proteinspire.org) has transitioned from solely a protein expression database to a multi-omics resource for human and model organisms. Through a web-based interface, MOPED presents consistently processed data for gene, protein and pathway expression. To improve data quality, consistency and use, MOPED includes metadata detailing experimental design and analysis methods. The multi-omics data are integrated through direct links between genes and proteins and further connected to pathways and experiments. MOPED now contains over 5 million records, information for approximately 75,000 genes and 50,000 proteins from four organisms (human, mouse, worm, yeast). These records correspond to 670 unique combinations of experiment, condition, localization and tissue. MOPED includes the following new features: pathway expression, Pathway Details pages, experimental metadata checklists, experiment summary statistics and more advanced searching tools. Advanced searching enables querying for genes, proteins, experiments, pathways and keywords of interest. The system is enhanced with visualizations for comparing across different data types. In the future MOPED will expand the number of organisms, increase integration with pathways and provide connections to disease.

  13. 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

  14. Interfacial polymerization for colorimetric labeling of protein expression in cells.

    PubMed

    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.

  15. Genomic and expression analysis of transition proteins in Drosophila.

    PubMed

    Alvi, Zain A; Chu, Tin-Chun; Schawaroch, Valerie; Klaus, Angela V

    2015-01-01

    The current study was aimed at analyzing putative protein sequences of the transition protein-like proteins in 12 Drosophila species based on the reference sequences of transition protein-like protein (Tpl (94D) ) expressed in Drosophila melanogaster sperm nuclei. Transition proteins aid in transforming chromatin from a histone-based nucleosome structure to a protamine-based structure during spermiogenesis - the post-meiotic stage of spermatogenesis. Sequences were obtained from NCBI Ref-Seq database using NCBI ORF-Finder (PSI-BLAST). Sequence alignments and analysis of the amino acid content indicate that orthologs for Tpl (94D) are present in the melanogaster species subgroup (D. simulans, D. sechellia, D. erecta, and D. yakuba), D. ananassae, and D. pseudoobscura, but absent in D. persmilis, D. willistoni, D. mojavensis, D. virilis, and D. grimshawi. Transcriptome next generation sequence (RNA-Seq) data for testes and ovaries was used to conduct differential gene expression analysis for Tpl (94D) in D. melanogaster, D. simulans, D. yakuba, D. ananassae, and D. pseudoobscura. The identified Tpl (94D) orthologs show high expression in the testes as compared to the ovaries. Additionally, 2 isoforms of Tpl (94D) were detected in D. melanogaster with isoform A being much more highly expressed than isoform B. Functional analyses of the conserved region revealed that the same high mobility group (HMG) box/DNA binding region is conserved for both Drosophila Tpl (94D) and Drosophila protamine-like proteins (MST35Ba and MST35Bb). Based on the rigorous bioinformatic approach and the conservation of the HMG box reported in this work, we suggest that the Drosophila Tpl (94D) orthologs should be classified as their own transition protein group.

  16. 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.

  17. Differential expression of receptor protein tyrosine phosphatases accompanies the reorganisation of the retina upon laser lesion.

    PubMed

    Besser, Manuela; Horvat-Bröcker, Andrea; Eysel, Ulf T; Faissner, Andreas

    2009-09-01

    The regulation of protein phosphorylation plays an essential role in virtually all aspects of eukaryotic development. Beginning with the regulation of the cell cycle to cellular proliferation and differentiation, the delicate balance between the phosphorylating activity of kinases and the dephosphorylation by phosphatases controls the outcome of many signal transduction cascades. The generation of cellular diversity occurs in an environment that is structured by the extracellular matrix (ECM) which forms a surrounding niche for stem and progenitor cells. Cell-cell and cell-matrix interactions elicit specific signaling pathways that control cellular behavior. In pathological situations such as neural degenerating diseases, gene expression patterns and finally the composition of the ECM change dramatically. This leads to changes of cell behavior and finally results in the failure of regeneration and functional restoration in the adult central nervous system. In order to study the roles of tyrosine phosphatases and ECM in this context, we analyzed the effects of laser-induced retinal injury on the regulation of the receptor protein tyrosine phosphatases (RPTP) RPTPBr7, Phogrin and RPTPbeta/zeta. The latter occurs in several isoforms, including the soluble released chondroitin sulfate proteoglycan phosphacan that is expressed in the developing retina. The receptor variants RPTPbeta/zeta(long) and RPTPbeta/zeta(short) may serve as receptors of tenascin-proteins and serve as modulators of cell intrinsic signaling in response to the ECM. Using quantitative real-time RT-PCR analysis, we show here a time-dependent pattern of gene expression of these molecules following laser lesions of the retina.

  18. Controlling for gene expression changes in transcription factor protein networks.

    PubMed

    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

    2014-06-01

    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.

  19. Phase variation mediates reductions in expression of surface proteins during persistent meningococcal carriage.

    PubMed

    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; Bayliss, Christopher D

    2014-06-01

    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

  20. 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.

  1. Fos expression in pontomedullary catecholaminergic cells following rapid eye movement sleep-like episodes elicited by pontine carbachol in urethane-anesthetized rats.

    PubMed

    Rukhadze, I; Fenik, V B; Branconi, J L; Kubin, L

    2008-03-01

    Pontine noradrenergic neurons of the locus coeruleus (LC) and sub-coeruleus (SubC) region cease firing during rapid eye movement sleep (REMS). This plays a permissive role in the generation of REMS and may contribute to state-dependent modulation of transmission in the CNS. Whether all pontomedullary catecholaminergic neurons, including those in the A1/C1, A2/C2 and A7 groups, have REMS-related suppression of activity has not been tested. We used Fos protein expression as an indirect marker of the level of neuronal activity and linear regression analysis to determine whether pontomedullary cells identified by tyrosine hydroxylase (TH) immunohistochemistry have reduced Fos expression following REMS-like state induced by pontine microinjections of a cholinergic agonist, carbachol in urethane-anesthetized rats. The percentage of Fos-positive TH cells was negatively correlated with the cumulative duration of REMS-like episodes induced during 140 min prior to brain harvesting in the A7 and rostral A5 groups bilaterally (P < 0.01 for both), and in SubC neurons on the side opposite to carbachol injection (P < 0.05). Dorsal medullary A2/C2 neurons did not exhibit such correlation, but their Fos expression (and that in A7, rostral A5 and SubC neurons) was positively correlated with the duration of the interval between the last REMS-like episode and the time of perfusion (P < 0.05). In contrast, neither of these correlations was significant for A1 /C1 or caudal A5 neurons. These findings suggest that, similar to the prototypic LC neurons, neurons of the A7, rostral A5 and A2/C2 groups have reduced or abolished activity during REMS, whereas A1 /IC1 and caudal A5 neurons do not have this feature. The reduced activity of A2/C2, A5 and A7 neurons during REMS, and the associated decrements in norepinephrine release, may cause state-dependent modulation of.transmission in brain somato- and viscerosensory, somatomotor, and cardiorespiratory pathways.

  2. Production of dengue virus envelope protein domain III-based antigens in tobacco chloroplasts using inducible and constitutive expression systems.

    PubMed

    Gottschamel, Johanna; Lössl, Andreas; Ruf, Stephanie; Wang, Yanliang; Skaugen, Morten; Bock, Ralph; Clarke, Jihong Liu

    2016-07-01

    Dengue fever is a disease in many parts of the tropics and subtropics and about half the world's population is at risk of infection according to the World Health Organization. Dengue is caused by any of the four related dengue virus serotypes DEN-1, -2, -3 and -4, which are transmitted to people by Aedes aegypti mosquitoes. Currently there is only one vaccine (Dengvaxia(®)) available (limited to a few countries) on the market since 2015 after half a century's intensive efforts. Affordable and accessible vaccines against dengue are hence still urgently needed. The dengue envelop protein domain III (EDIII), which is capable of eliciting serotype-specific neutralizing antibodies, has become the focus for subunit vaccine development. To contribute to the development of an accessible and affordable dengue vaccine, in the current study we have used plant-based vaccine production systems to generate a dengue subunit vaccine candidate in tobacco. Chloroplast genome engineering was applied to express serotype-specific recombinant EDIII proteins in tobacco chloroplasts using both constitutive and ethanol-inducible expression systems. Expression of a tetravalent antigen fusion construct combining EDIII polypeptides from all four serotypes was also attempted. Transplastomic EDIII-expressing tobacco lines were obtained and homoplasmy was verified by Southern blot analysis. Northern blot analyses showed expression of EDIII antigen-encoding genes. EDIII protein accumulation levels varied for the different recombinant EDIII proteins and the different expression systems, and reached between 0.8 and 1.6 % of total cellular protein. Our study demonstrates the suitability of the chloroplast compartment as a production site for an EDIII-based vaccine candidate against dengue fever and presents a Gateway(®) plastid transformation vector for inducible transgene expression. PMID:27116001

  3. 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…

  4. Expression of pokeweed antiviral proteins in creeping bentgrass.

    PubMed

    Dai, W D; Bonos, S; Guo, Z; Meyer, W A; Day, P R; Belanger, F C

    2003-01-01

    Fungal diseases of creeping bentgrass, an important amenity grass used extensively on golf courses, are a serious problem in golf course management. Transgenic approaches to improving disease resistance to fungal diseases are being explored in many species, and in some cases ribosome-inactivating proteins have been found to be effective. We have generated transgenic creeping bentgrass plants expressing three forms of ribosome-inactivating proteins from pokeweed, which are termed pokeweed antiviral proteins (PAP). PAP-Y and PAP-C are nontoxic mutants of PAP; PAPII is the native form of another ribosome-inactivating protein from pokeweed. In creeping bentgrass, PAP-C transformants did not accumulate the protein, suggesting that it is unstable, and in a field test these plants were not protected from infection by the fungal pathogen Sclerotinia homoeocarpa, the causal agent of dollar spot disease. PAPII transformants could accumulate stable levels of the protein but had symptoms of toxicity; one low-expressing line exhibited good disease resistance. PAP-Y transformants accumulated stable levels of protein, and under greenhouse conditions they appeared to be phenotypically normal.

  5. The protein expression landscape of the Arabidopsis root

    PubMed Central

    Petricka, Jalean J.; Schauer, Monica A.; Megraw, Molly; Breakfield, Natalie W.; Thompson, J. Will; Georgiev, Stoyan; Soderblom, Erik J.; Ohler, Uwe; Moseley, Martin Arthur; Grossniklaus, Ueli; Benfey, Philip N.

    2012-01-01

    Because proteins are the major functional components of cells, knowledge of their cellular localization is crucial to gaining an understanding of the biology of multicellular organisms. We have generated a protein expression map of the Arabidopsis root providing the identity and cell type-specific localization of nearly 2,000 proteins. Grouping proteins into functional categories revealed unique cellular functions and identified cell type-specific biomarkers. Cellular colocalization provided support for numerous protein–protein interactions. With a binary comparison, we found that RNA and protein expression profiles are weakly correlated. We then performed peak integration at cell type-specific resolution and found an improved correlation with transcriptome data using continuous values. We performed GeLC-MS/MS (in-gel tryptic digestion followed by liquid chromatography-tandem mass spectrometry) proteomic experiments on mutants with ectopic and no root hairs, providing complementary proteomic data. Finally, among our root hair-specific proteins we identified two unique regulators of root hair development. PMID:22447775

  6. 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.

  7. Function of PPR proteins in plastid gene expression.

    PubMed

    Shikanai, Toshiharu; Fujii, Sota

    2013-01-01

    PPR proteins form a huge family in flowering plants and are involved in RNA maturation in plastids and mitochondria. These proteins are sequence-specific RNA-binding proteins that recruit the machinery of RNA processing. We summarize progress in the research on the functional mechanisms of divergent RNA maturation and on the mechanism by which RNA sequences are recognized. We further focus on two topics. RNA editing is an enigmatic process of RNA maturation in organelles, in which members of the PLS subfamily contribute to target site recognition. As the first topic, we speculate on why the PLS subfamily was selected by the RNA editing machinery. Second, we discuss how the regulation of plastid gene expression contributes to efficient photosynthesis. Although the molecular functions of PPR proteins have been studied extensively, information on the physiological significance of regulation by these proteins remains very limited.

  8. Heterologous expression of G-protein-coupled receptors in yeast.

    PubMed

    Bertheleme, Nicolas; Singh, Shweta; Dowell, Simon; Byrne, Bernadette

    2015-01-01

    Heterologous yeast expression systems have been successfully used for the production of G-protein-coupled receptors (GPCRs) for both structural and functional studies. Yeast combine comparatively low cost and short culture times with straightforward generation of expression clones. They also perform some key posttranslational modifications not possible in bacterial systems. There are two major yeast expression systems, Pichia pastoris and Saccharomyces cerevisiae, both of which have been used for the production of GPCRs. P. pastoris has a proven track record for the production of large amounts of GPCR for structural studies. High-resolution crystal structures of both the adenosine A2A and the histamine H1 receptors have been obtained using protein expressed in this system. S. cerevisiae is relatively easy to engineer and this has resulted in the development of sophisticated tools for the functional characterization of GPCRs. In this chapter, we provide protocols for both large-scale receptor expression in P. pastoris for structural studies and small-scale receptor expression in S. cerevisiae for functional characterization. In both cases, the receptor used is the human adenosine A2A receptor. The results that both we and others have obtained using these protocols show the wide utility of the yeast expression systems for the production of GPCRs.

  9. Correlation of protein and gene expression profiles of inflammatory proteins after endotoxin challenge in human subjects.

    PubMed

    Prabhakar, Uma; Conway, Theresa M; Murdock, Paul; Mooney, Jeff L; Clark, Steve; Hedge, Priti; Bond, Brian C; Jazwinska, Elizabeth C; Barnes, Michael R; Tobin, Frank; Damian-Iordachi, Valeriu; Greller, Larry; Hurle, Mark; Stubbs, Andrew P; Li, Zhong; Valoret, Elizabeth I; Erickson-Miller, Connie; Cass, Lisa; Levitt, Blanche; Davis, Hugh M; Jorkasky, Diane K; Williams, William V

    2005-07-01

    Administration of endotoxin (LPS) in humans results in profound physiological responses, including activation of peripheral blood mononuclear cells and the release of inflammatory factors. The time course of the response of selected inflammatory proteins was examined in healthy subjects (n = 6) administered a single intravenous dose of the purified derivative of endotoxin (3.0 ng/kg). Microarray analysis demonstrated changes in the expression of a number of genes, which were confirmed in separate in vitro endotoxin stimulation experiments. Subsequent TaqMan analysis of genes of interest indicated time-dependent changes in the expression of many of these genes. This included pre-B cell enhancing factor, which was identified on microarray analysis as being markedly upregulated following endotoxin stimulation. Protein expression of the genes examined by TaqMan analysis was measured and demonstrated the appearance of tumor necrosis factor (TNF)-alpha and sTNF-R proteins in the plasma beginning within 1 h after dosing, followed by other cytokines/ inflammatory markers (e.g., IL-1ra, G-CSF, IL-6, IL-8, and IL-10) and suppressors of cytokine signaling (SOCS-1 and SOCS-3). In general, cytokine protein expression correlated well with gene expression; however, the temporal profile of expression of some genes did not correlate well with the protein data. For many of these proteins, the lack of correlation was attributable to alternate tissue sources, which were demonstrated on TaqMan analysis. Principal component analysis indicated that cytokines could be grouped according to their temporal pattern of response, with most transcript levels returning to baseline 24 h following endotoxin administration. The combination of cDNA microarray and TaqMan analysis to identify and quantify changes in gene expression, along with the analysis of protein expression, can be useful in investigating inflammatory and other diseases.

  10. 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 I reflect on different social science methods for eliciting sound memories such as the use of sonic prompts, emplaced interviewing, and sound walks. I include examples from my research on medical listening. The article considers the relevance of this work for the conduct of oral histories, arguing that such methods "break the frame," allowing room for collaborative research connections and insights into the otherwise unarticulatable.

  11. 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

  12. Methods and constructs for expression of foreign proteins in photosynthetic organisms

    DOEpatents

    Laible, Philip D.; Hanson, Deborah K.

    2002-01-01

    A method for expressing and purifying foreign proteins in photosynthetic organisms comprising the simultaneous expression of both the heterologous protein and a means for compartmentalizing or sequestering of the protein.

  13. Quorum-sensing Salmonella selectively trigger protein expression within tumors.

    PubMed

    Swofford, Charles A; Van Dessel, Nele; Forbes, Neil S

    2015-03-17

    Salmonella that secrete anticancer proteins have the potential to eliminate tumors, but nonspecific expression causes damage to healthy tissue. We hypothesize that Salmonella, integrated with a density-dependent switch, would only express proteins in tightly packed colonies within tumors. To test this hypothesis, we cloned the lux quorum-sensing (QS) system and a GFP reporter into nonpathogenic Salmonella. Fluorescence and bacterial density were measured in culture and in a tumor-on-a-chip device to determine the critical density necessary to initiate expression. QS Salmonella were injected into 4T1 tumor-bearing mice to quantify GFP expression in vivo using immunofluorescence. At densities below 0.6 × 10(10) cfu/g in tumors, less than 3% of QS Salmonella expressed GFP. Above densities of 4.2 × 10(10) cfu/g, QS Salmonella had similar expression levels to constitutive controls. GFP expression by QS colonies was dependent upon the distance to neighboring bacteria. No colonies expressed GFP when the average distance to neighbors was greater than 155 µm. Calculations of autoinducer concentrations showed that expression was sigmoidally dependent on density and inversely dependent on average radial distance. Based on bacterial counts from excised tissue, the liver density (0.0079 × 10(10) cfu/g) was less than the critical density (0.11 × 10(10) cfu/g) necessary to initiate expression. QS Salmonella are a promising tool for cancer treatment that will target drugs to tumors while preventing damage to healthy tissue.

  14. Raf-1 kinase inhibitory protein expression in thyroid carcinomas.

    PubMed

    Kim, Hyun-Soo; Kim, Gou Young; Lim, Sung-Jig; Kim, Youn Wha

    2010-12-01

    Raf-1 kinase inhibitory protein (RKIP) has been implicated in several fundamental signal transduction pathways that control cellular growth, differentiation, apoptosis and migration. RKIP is reduced in a variety of human carcinomas, but RKIP expression in thyroid carcinomas has not been analyzed at the protein level. In this study, we examined the immunohistochemical expression of RKIP in various subtypes of thyroid carcinoma. Immunostaining for RKIP was performed on 104 cases of primary thyroid carcinoma (40 papillary, 29 follicular, 11 medullary, 11 poorly differentiated, and 13 anaplastic carcinomas) and 26 cases of nodal metastatic tumor (17 papillary, 4 medullary, and 5 anaplastic carcinomas). Normal thyroid tissue and all cases of follicular, papillary, and medullary carcinomas showed uniform, strong cytoplasmic immunoreactivity for RKIP. With the exception of one case, poorly differentiated carcinomas also revealed strong RKIP expression. In contrast, RKIP expression was completely absent in all anaplastic carcinomas. The transition zone from the differentiated carcinoma component (strong RKIP expression) to the anaplastic carcinoma component (no RKIP expression) demonstrated a completely opposite pattern of RKIP immunoreactivity. This reduction of RKIP expression in anaplastic carcinoma was statistically significant (P < 0.0001). Additionally, RKIP expression of nodal metastatic tumors corresponded with that of primary tumors: metastatic papillary and medullary carcinomas showed uniform, strong cytoplasmic RKIP immunoreactivity, in contrast, in metastatic anaplastic carcinomas, RKIP expression was completely absent. RKIP expression is significantly reduced in anaplastic thyroid carcinoma as compared to other subtypes of thyroid carcinoma. Further studies are necessary to elucidate the precise mechanism of RKIP action in anaplastic thyroid carcinoma.

  15. 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.

  16. Heterologous expression of the lipid transfer protein CERT increases therapeutic protein productivity of mammalian cells.

    PubMed

    Florin, Lore; Pegel, Antje; Becker, Eric; Hausser, Angelika; Olayioye, Monilola A; Kaufmann, Hitto

    2009-04-20

    Recent studies have demonstrated that the introduction of transgenes regulating protein transport or affecting post-translational modifications can further improve industrial processes for the production of therapeutic proteins in mammalian cells. Our study on improving therapeutic protein production in CHO cells by heterologous expression of the ceramide transfer protein (CERT) was initiated by the recent discovery that CERT is involved in protein kinase D (PKD)-dependent protein transport from the Golgi to the plasma membrane. We generated a set of CHO DG44 cell lines by stable integration of constructs expressing either CERT wild-type or CERT S132A, a mutant conferring increased lipid transfer activity, or a mock plasmid. CHO cells expressing heterologous CERT demonstrated significantly higher specific productivities of the therapeutic protein HSA when grown in inoculum suspension cultures. This effect translated into significantly increased overall HSA titers in a fed-batch format where cells are grown in chemically defined serum-free media. Furthermore, we could show that CERT also enhanced monoclonal antibody secretion in two IgG production cell lines with different basal productivities. The data demonstrate the potential of CERT engineering to improve mammalian cell culture production processes to yield high amounts of a therapeutic protein product of desired quality. To our knowledge, this is the first study showing a bottle neck in recombinant protein secretion at the Golgi complex in mammalian cells. PMID:19428735

  17. 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.

  18. 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

  19. Easy mammalian expression and crystallography of maltose-binding protein-fused human proteins.

    PubMed

    Bokhove, Marcel; Sadat Al Hosseini, Hamed; Saito, Takako; Dioguardi, Elisa; Gegenschatz-Schmid, Katharina; Nishimura, Kaoru; Raj, Isha; de Sanctis, Daniele; Han, Ling; Jovine, Luca

    2016-04-01

    We present a strategy to obtain milligrams of highly post-translationally modified eukaryotic proteins, transiently expressed in mammalian cells as rigid or cleavable fusions with a mammalianized version of bacterial maltose-binding protein (mMBP). This variant was engineered to combine mutations that enhance MBP solubility and affinity purification, as well as provide crystal-packing interactions for increased crystallizability. Using this cell type-independent approach, we could increase the expression of secreted and intracellular human proteins up to 200-fold. By molecular replacement with MBP, we readily determined five novel high-resolution structures of rigid fusions of targets that otherwise defied crystallization. PMID:26850170

  20. Easy mammalian expression and crystallography of maltose-binding protein-fused human proteins

    PubMed Central

    Bokhove, Marcel; Sadat Al Hosseini, Hamed; Saito, Takako; Dioguardi, Elisa; Gegenschatz-Schmid, Katharina; Nishimura, Kaoru; Raj, Isha; de Sanctis, Daniele; Han, Ling; Jovine, Luca

    2016-01-01

    We present a strategy to obtain milligrams of highly post-translationally modified eukaryotic proteins, transiently expressed in mammalian cells as rigid or cleavable fusions with a mammalianized version of bacterial maltose-binding protein (mMBP). This variant was engineered to combine mutations that enhance MBP solubility and affinity purification, as well as provide crystal-packing interactions for increased crystallizability. Using this cell type-independent approach, we could increase the expression of secreted and intracellular human proteins up to 200-fold. By molecular replacement with MBP, we readily determined five novel high-resolution structures of rigid fusions of targets that otherwise defied crystallization. PMID:26850170

  1. 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

  2. Altered Expression of Bone Morphogenetic Protein Accessory Proteins in Murine and Human Pulmonary Fibrosis.

    PubMed

    Murphy, Noelle; Gaynor, Katherine U; Rowan, Simon C; Walsh, Sinead M; Fabre, Aurelie; Boylan, John; Keane, Michael P; McLoughlin, Paul

    2016-03-01

    Idiopathic pulmonary fibrosis is a chronic, progressive fibrotic disease with a poor prognosis. The balance between transforming growth factor β1 and bone morphogenetic protein (BMP) signaling plays an important role in tissue homeostasis, and alterations can result in pulmonary fibrosis. We hypothesized that multiple BMP accessory proteins may be responsible for maintaining this balance in the lung. Using the bleomycin mouse model for fibrosis, we examined an array of BMP accessory proteins for changes in mRNA expression. We report significant increases in mRNA expression of gremlin 1, noggin, follistatin, and follistatin-like 1 (Fstl1), and significant decreases in mRNA expression of chordin, kielin/chordin-like protein, nephroblastoma overexpressed gene, and BMP and activin membrane-bound inhibitor (BAMBI). Protein expression studies demonstrated increased levels of noggin, BAMBI, and FSTL1 in the lungs of bleomycin-treated mice and in the lungs of idiopathic pulmonary fibrosis patients. Furthermore, we demonstrated that transforming growth factor β stimulation resulted in increased expression of noggin, BAMBI, and FSTL1 in human small airway epithelial cells. These results provide the first evidence that multiple BMP accessory proteins are altered in fibrosis and may play a role in promoting fibrotic injury.

  3. Expression and localization of X11 family proteins in neurons.

    PubMed

    Motodate, Rika; Saito, Yuhki; Hata, Saori; Suzuki, Toshiharu

    2016-09-01

    The X11/Mint family of proteins comprises X11/X11α/Mint1, X11L/X11β/Mint2, and X11L2/X11γ/Mint3. Each of these molecules is an adaptor protein that contains a phosphotyrosine interaction/binding (PI/PTB) and two PDZ domains in its carboxy-terminal region. X11/Mint family members associate with a broad spectrum of membrane proteins, including Alzheimer's β-amyloid precursor protein (APP), alcadeins, and low density lipoprotein receptor proteins, as well as various cytoplasmic proteins including Arf, kalirin-7, and Munc18. In particular, X11 and X11L are thought to play various roles in the regulation of neural functions in brain. Nevertheless, the protein levels and respective localization of individual family members remain controversial. We analyzed the protein levels of X11 and X11L in the corresponding single- and double-knockout mice. X11 and X11L did not exhibit obvious changes of their protein levels when the other was absent, especially in cerebrum in which they were widely co-expressed. In cerebellum, X11 and X11L localized in characteristic patterns in various types of neurons, and X11 protein level increased without an obvious ectopic localization in X11L-knockout mice. Interestingly, only X11L protein existed specifically in brain, whereas, contrary to the accepted view, X11 protein was detected at the highest levels in brain but was also strongly detected in pancreas, testis, and paranephros. Together, our results indicate that both X11 and X11L exert largely in brain neurons, but X11 may also function in peripheral tissues. PMID:27268412

  4. LC–MS Based Detection of Differential Protein Expression

    PubMed Central

    Tuli, Leepika; Ressom, Habtom W.

    2010-01-01

    While several techniques are available in proteomics, LC-MS based analysis of complex protein/peptide mixtures has turned out to be a mainstream analytical technique for quantitative proteomics. Significant technical advances at both sample preparation/separation and mass spectrometry levels have revolutionized comprehensive proteome analysis. Moreover, automation and robotics for sample handling process permit multiple sampling with high throughput. For LC-MS based quantitative proteomics, sample preparation turns out to be critical step, as it can significantly influence sensitivity of downstream analysis. Several sample preparation strategies exist, including depletion of high abundant proteins or enrichment steps that facilitate protein quantification but with a compromise of focusing on a smaller subset of a proteome. While several experimental strategies have emerged, certain limitations such as physiochemical properties of a peptide/protein, protein turnover in a sample, analytical platform used for sample analysis and data processing, still imply challenges to quantitative proteomics. Other aspects that make analysis of a proteome a challenging task include dynamic nature of a proteome, need for efficient and fast analysis of protein due to its constant modifications inside a cell, concentration range of proteins that exceed dynamic range of a single analytical method, and absence of appropriate bioinformatics tools for analysis of large volume and high dimensional data. This paper gives an overview of various LC-MS methods currently used in quantitative proteomics and their potential for detecting differential protein expression. Fundamental steps such as sample preparation, LC separation, mass spectrometry, quantitative assessment and protein identification are discussed. For quantitative assessment of protein expression, both label and label free approaches are evaluated for their set of merits and demerits. While most of these methods edge on providing

  5. Expression, Delivery and Function of Insecticidal Proteins Expressed by Recombinant Baculoviruses

    PubMed Central

    Kroemer, Jeremy A.; Bonning, Bryony C.; Harrison, Robert L.

    2015-01-01

    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 potential by shortening the time required for infection to kill or incapacitate insect pests and reducing the quantity of crop damage as a consequence. A wide variety of neurotoxic peptides, proteins that regulate insect physiology, degradative enzymes, and other potentially insecticidal proteins have been evaluated for their capacity to reduce the survival time of baculovirus-infected lepidopteran host larvae. Researchers have investigated the factors involved in the efficient expression and delivery of baculovirus-encoded insecticidal peptides and proteins, with much effort dedicated to identifying ideal promoters for driving transcription and signal peptides that mediate secretion of the expressed target protein. Other factors, particularly translational efficiency of transcripts derived from recombinant insecticidal genes and post-translational folding and processing of insecticidal proteins, remain relatively unexplored. The discovery of RNA interference as a gene-specific regulation mechanism offers a new approach for improvement of baculovirus biopesticidal efficacy through genetic modification. PMID:25609310

  6. Expression and Targeting of Secreted Proteins from Chlamydia trachomatis

    PubMed Central

    Bauler, Laura D.

    2014-01-01

    Chlamydia trachomatis is an obligate intracellular pathogen that replicates in a vacuole termed the inclusion. Many of the interactions of chlamydiae with the host cell are dependent upon bacterial protein synthesis and presumably exposure of these proteins to the cytosol. Because of the dearth of genetic tools for chlamydiae, previous studies examining secreted proteins required the use of heterologous bacterial systems. Recent advances in genetic manipulation of chlamydia now allow for transformation of the bacteria with plasmids. We describe here a shuttle vector system, pBOMB4, that permits expression of recombinant proteins under constitutive or conditional promoter control. We show that the inclusion membrane protein IncD is secreted in a type III-dependent manner from Yersinia pseudotuberculosis and also secreted from C. trachomatis in infected cells where it localizes appropriately to the inclusion membrane. IncD truncated of the first 30 amino acids containing the secretion signal is no longer secreted and is retained by the bacteria. Cytosolic exposure of secreted proteins can be confirmed by using CyaA, GSK, or microinjection assays. A protein predicted to be retained within the bacteria, NrdB is indeed localized to the chlamydia. In addition, we have shown that the chlamydial effector protein, CPAF, which is secreted into the host cell cytosol by a Sec-dependent pathway, also accesses the cytosol when expressed from this system. These assays should prove useful to assess the secretion of other chlamydial proteins that are potentially exposed to the cytosol of the host cell. PMID:24443531

  7. Changes in protein expression during honey bee larval development

    PubMed Central

    Chan, Queenie WT; Foster, Leonard J

    2008-01-01

    Background The honey bee (Apis mellifera), besides its role in pollination and honey production, serves as a model for studying the biochemistry of development, metabolism, and immunity in a social organism. Here we use mass spectrometry-based quantitative proteomics to quantify nearly 800 proteins during the 5- to 6-day larval developmental stage, tracking their expression profiles. Results We report that honey bee larval growth is marked by an age-correlated increase of protein transporters and receptors, as well as protein nutrient stores, while opposite trends in protein translation activity and turnover were observed. Levels of the immunity factors prophenoloxidase and apismin are positively correlated with development, while others surprisingly were not significantly age-regulated, suggesting a molecular explanation for why bees are susceptible to major age-associated bee bacterial infections such as American Foulbrood or fungal diseases such as chalkbrood. Previously unreported findings include the reduction of antioxidant and G proteins in aging larvae. Conclusion These data have allowed us to integrate disparate findings in previous studies to build a model of metabolism and maturity of the immune system during larval development. This publicly accessible resource for protein expression trends will help generate new hypotheses in the increasingly important field of honey bee research. PMID:18959778

  8. G-protein coupled receptor expression patterns delineate medulloblastoma subgroups

    PubMed Central

    2013-01-01

    Background Medulloblastoma is the most common malignant brain tumor in children. Genetic profiling has identified four principle tumor subgroups; each subgroup is characterized by different initiating mutations, genetic and clinical profiles, and prognoses. The two most well-defined subgroups are caused by overactive signaling in the WNT and SHH mitogenic pathways; less is understood about Groups 3 and 4 medulloblastoma. Identification of tumor subgroup using molecular classification is set to become an important component of medulloblastoma diagnosis and staging, and will likely guide therapeutic options. However, thus far, few druggable targets have emerged. G-protein coupled receptors (GPCRs) possess characteristics that make them ideal targets for molecular imaging and therapeutics; drugs targeting GPCRs account for 30-40% of all current pharmaceuticals. While expression patterns of many proteins in human medulloblastoma subgroups have been discerned, the expression pattern of GPCRs in medulloblastoma has not been investigated. We hypothesized that analysis of GPCR expression would identify clear subsets of medulloblastoma and suggest distinct GPCRs that might serve as molecular targets for both imaging and therapy. Results Our study found that medulloblastoma tumors fall into distinct clusters based solely on GPCR expression patterns. Normal cerebellum clustered separately from the tumor samples. Further, two of the tumor clusters correspond with high fidelity to the WNT and SHH subgroups of medulloblastoma. Distinct over-expressed GPCRs emerge; for example, LGR5 and GPR64 are significantly and uniquely over-expressed in the WNT subgroup of tumors, while PTGER4 is over-expressed in the SHH subgroup. Uniquely under-expressed GPCRs were also observed. Our key findings were independently validated using a large international dataset. Conclusions Our results identify GPCRs with potential to act as imaging and therapeutic targets. Elucidating tumorigenic pathways

  9. Tools to cope with difficult-to-express proteins.

    PubMed

    Saccardo, Paolo; Corchero, José Luís; Ferrer-Miralles, Neus

    2016-05-01

    The identification of DNA coding sequences contained in the genome of many organisms coupled to the use of high throughput approaches has fueled the field of recombinant protein production. Apart from basic research interests, the growing relevance of this field is highlighted by the global sales of the top ten biopharmaceuticals on the market, which exceeds the trillion USD in a steady increasing tendency. Therefore, the demand of biological compounds seems to have a long run on the market. One of the most popular expression systems is based on Escherichia coli cells which apart from being cost-effective counts with a large selection of resources. However, a significant percentage of the genes of interest are not efficiently expressed in this system, or the expressed proteins are accumulated within aggregates, degraded or lacking the desired biological activity, being finally discarded. In some instances, expressing the gene in a homologous expression system might alleviate those drawbacks but then the process usually increases in complexity and is not as cost-effective as the prokaryotic systems. An increasing toolbox is available to approach the production and purification of those difficult-to-express proteins, including different expression systems, promoters with different strengths, cultivation media and conditions, solubilization tags and chaperone coexpression, among others. However, in most cases, the process follows a non-integrative trial and error strategy with discrete success. This review is focused on the design of the whole process by using an integrative approach, taken into account the accumulated knowledge of the pivotal factors that affect any of the key processes, in an attempt to rationalize the efforts made in this appealing field. PMID:27079572

  10. Differential Expression of Borrelia burgdorferi Proteins during Growth In Vitro

    PubMed Central

    Ramamoorthy, Ramesh; Philipp, Mario T.

    1998-01-01

    In an earlier paper we described the transcriptionally regulated differential levels of expression of two lipoproteins of Borrelia burgdorferi, P35 and P7.5, during growth of the spirochetes in culture from logarithmic phase to stationary phase (K. J. Indest, R. Ramamoorthy, M. Solé, R. D. Gilmore, B. J. B. Johnson, and M. T. Philipp, Infect. Immun. 65:1165–1171, 1997). Here we further assess this phenomenon by investigating whether the expression of other antigens of B. burgdorferi, including some well-characterized ones, are also regulated in a growth-phase-dependent manner in vitro. These studies revealed 13 additional antigens, including OspC, BmpD, and GroEL, that were upregulated 2- to 66-fold and a 28-kDa protein that was downregulated 2- to 10-fold, during the interval between the logarithmic- and stationary-growth phases. Unlike with these in vitro-regulated proteins, the levels of expression of OspA, OspB, P72, flagellin, and BmpA remained unchanged throughout growth of the spirochetes in culture. Furthermore, ospAB, bmpAB, groEL, and fla all exhibited similar mRNA profiles, which is consistent with the constitutive expression of these genes. By contrast, the mRNA and protein profiles of ospC and bmpD indicated regulated expression of these genes. While bmpD exhibited a spike in mRNA expression in early stationary phase, ospC maintained a relatively higher level of mRNA throughout culture. These findings demonstrate that there are additional genes besides P7.5 and P35 whose regulated expression can be investigated in vitro and which may thus serve as models to facilitate the study of regulatory mechanisms in an organism that cycles between an arthropod and a vertebrate host. PMID:9784512

  11. Expression of bone morphogenetic proteins of human neoplastic epithelial cells.

    PubMed

    Hatakeyama, S; Gao, Y H; Ohara-Nemoto, Y; Kataoka, H; Satoh, M

    1997-07-01

    Bone morphogenetic proteins (BMPs) are crucial factors of osteogenesis. We investigated the expressions of BMP subtypes in human salivary adenocarcinoma cell line (HSG-S8), tongue squamous cell (HSC-4) and gingival squamous cell (Ca9-22) carcinoma cell lines, gastric poorly differentiated adenocarcinoma cell (MNK45) and signet ring cell (KATOIII) carcinoma cell lines, rectal adenocarcinoma (RCM-1, RCM-2, and RCM-3), and thyroid (8505C) and bladder (T24) carcinoma cell lines by reverse transcription-polymerase chain reaction (RT-PCR). RT-PCR disclosed that BMP-1 was expressed in all cell lines examined, and BMP-2 was amplified in almost all cells except MKN45. Two squamous cell carcinomas, HSC-4 and Ca9-22, and KATOIII expressed only BMP-1 and BMP-2. MKN45 did not express BMP-2, but expressed BMP-7 and weakly BMP-4 and BMP-5. In addition to the expression BMP-7, and HSG-S8 expressed BMP-6. These findings indicated that the neoplastic epithelial cells possessed a rather great potency to express BMP mRNAs. On the other hand, among these carcinoma cells, HSG-S8 solely induced bone in nude mouse tumors, and HSC-4 and KATOIII contained many calcified masses in tumors while the rest did not induce either. PMID:9247707

  12. Thermostable tag (TST) protein expression system: engineering thermotolerant recombinant proteins and vaccines.

    PubMed

    Luke, Jeremy M; Carnes, Aaron E; Sun, Ping; Hodgson, Clague P; Waugh, David S; Williams, James A

    2011-02-10

    Methods to increase temperature stability of vaccines and adjuvants are needed to reduce dependence on cold chain storage. We report herein creation and application of pVEX expression vectors to improve vaccine and adjuvant manufacture and thermostability. Defined media fermentation yields of 6g/L thermostable toll-like receptor 5 agonist flagellin were obtained using an IPTG inducible pVEX-flagellin expression vector. Alternative pVEX vectors encoding Pyrococcus furiosus maltodextrin-binding protein (pfMBP) as a fusion partner improved Influenza hemagglutinin antigen vaccine solubility and thermostability. A pfMBP hemagglutinin HA2 domain fusion protein was a potent immunogen. Manufacturing processes that combined up to 5 g/L defined media fermentation yields with rapid, selective, thermostable pfMBP fusion protein purification were developed. The pVEX pfMBP-based thermostable tag (TST) platform is a generic protein engineering approach to enable high yield manufacture of thermostable recombinant protein vaccine components.

  13. Expression of low molecular weight proteins in patients with leukaemia.

    PubMed

    Sheikh, N; Abid, R; Qureshi, A W; Basheer, T

    2012-06-01

    The current study is conducted to observe the differences in the level of low molecular weight proteins in the sera of patients with leukaemia in comparison to healthy subjects (control group). The sera of patients with leukaemia showed 15 peaks in the densitometric curve in comparison to the seven peaks of the controls. The peaks in the experimental samples that coincide with those in the control were of 134.14, 113.15, 76.06, 63.25, 48.07, 22.85 and 16.47 kDa molecular weights, respectively. Most of the new peaks appeared between the proteins of molecular weight 36-29 kDa in the experimental groups. Mean density of the 134.14 kDa protein band showed an increase in the protein in experimental groups I and II only whereas 113.15 and 22.85 kDa protein were increased in all experimental groups of patients with leukaemia. The expression of 76.06 and 63.25 kDa protein fraction was downregulated in the patients with leukaemia. A decline in the level of the protein of 48.07 kDa was observed in patients with leukaemia except in group I. Unlike the other protein fractions, the level of the protein of 16.47 kDa was significantly (p < 0.05) increased with a maximum density in group II. Intergroup experimental) comparison revealed an increasing pattern of 95.44 and 89.21 kDa with maximum level in group III sera. However the protein fractions of 38.07 and 34.94 kDa varied in the serum with maximum density in Group IV Protein fractions of 32.92 and 31.24 kDa were expressed in all age groups of patients with leukaemia with a maximum density in group III whereas the percentage densities of 14.42 and 13.56 kDa protein were quite different. This preliminary study will provide a basis to study the role of different proteins in patients with leukaemia.

  14. CD8+ T-cells expressing interferon gamma or perforin play antagonistic roles in heart injury in experimental Trypanosoma cruzi-elicited cardiomyopathy.

    PubMed

    Silverio, Jaline Coutinho; Pereira, Isabela Resende; Cipitelli, Márcio da Costa; Vinagre, Nathália Ferreira; Rodrigues, Maurício Martins; Gazzinelli, Ricardo Tostes; Lannes-Vieira, Joseli

    2012-01-01

    In Chagas disease, CD8(+) T-cells are critical for the control of Trypanosoma cruzi during acute infection. Conversely, CD8(+) T-cell accumulation in the myocardium during chronic infection may cause tissue injury leading to chronic chagasic cardiomyopathy (CCC). Here we explored the role of CD8(+) T-cells in T. cruzi-elicited heart injury in C57BL/6 mice infected with the Colombian strain. Cardiomyocyte lesion evaluated by creatine kinase-MB isoenzyme activity levels in the serum and electrical abnormalities revealed by electrocardiogram were not associated with the intensity of heart parasitism and myocarditis in the chronic infection. Further, there was no association between heart injury and systemic anti-T. cruzi CD8(+) T-cell capacity to produce interferon-gamma (IFNγ) and to perform specific cytotoxicity. Heart injury, however, paralleled accumulation of anti-T. cruzi cells in the cardiac tissue. In T. cruzi infection, most of the CD8(+) T-cells segregated into IFNγ(+) perforin (Pfn)(neg) or IFNγ(neg)Pfn(+) cell populations. Colonization of the cardiac tissue by anti-T. cruzi CD8(+)Pfn(+) cells paralleled the worsening of CCC. The adoptive cell transfer to T. cruzi-infected cd8(-/-) recipients showed that the CD8(+) cells from infected ifnγ(-/-)pfn(+/+) donors migrate towards the cardiac tissue to a greater extent and caused a more severe cardiomyocyte lesion than CD8(+) cells from ifnγ(+/+)pfn(-/-) donors. Moreover, the reconstitution of naïve cd8(-/-) mice with CD8(+) cells from naïve ifnγ(+/+)pfn(-/-) donors ameliorated T. cruzi-elicited heart injury paralleled IFNγ(+) cells accumulation, whereas reconstitution with CD8(+) cells from naïve ifnγ(-/-)pfn(+/+) donors led to an aggravation of the cardiomyocyte lesion, which was associated with the accumulation of Pfn(+) cells in the cardiac tissue. Our data support a possible antagonist effect of CD8(+)Pfn(+) and CD8(+)IFNγ(+) cells during CCC. CD8(+)IFNγ(+) cells may exert a beneficial role

  15. CD8+ T-Cells Expressing Interferon Gamma or Perforin Play Antagonistic Roles in Heart Injury in Experimental Trypanosoma Cruzi-Elicited Cardiomyopathy

    PubMed Central

    Cipitelli, Márcio da Costa; Vinagre, Nathália Ferreira; Rodrigues, Maurício Martins; Gazzinelli, Ricardo Tostes; Lannes-Vieira, Joseli

    2012-01-01

    In Chagas disease, CD8+ T-cells are critical for the control of Trypanosoma cruzi during acute infection. Conversely, CD8+ T-cell accumulation in the myocardium during chronic infection may cause tissue injury leading to chronic chagasic cardiomyopathy (CCC). Here we explored the role of CD8+ T-cells in T. cruzi-elicited heart injury in C57BL/6 mice infected with the Colombian strain. Cardiomyocyte lesion evaluated by creatine kinase-MB isoenzyme activity levels in the serum and electrical abnormalities revealed by electrocardiogram were not associated with the intensity of heart parasitism and myocarditis in the chronic infection. Further, there was no association between heart injury and systemic anti-T. cruzi CD8+ T-cell capacity to produce interferon-gamma (IFNγ) and to perform specific cytotoxicity. Heart injury, however, paralleled accumulation of anti-T. cruzi cells in the cardiac tissue. In T. cruzi infection, most of the CD8+ T-cells segregated into IFNγ+ perforin (Pfn)neg or IFNγnegPfn+ cell populations. Colonization of the cardiac tissue by anti-T. cruzi CD8+Pfn+ cells paralleled the worsening of CCC. The adoptive cell transfer to T. cruzi-infected cd8−/− recipients showed that the CD8+ cells from infected ifnγ−/−pfn+/+ donors migrate towards the cardiac tissue to a greater extent and caused a more severe cardiomyocyte lesion than CD8+ cells from ifnγ+/+pfn−/− donors. Moreover, the reconstitution of naïve cd8−/− mice with CD8+ cells from naïve ifnγ+/+pfn−/− donors ameliorated T. cruzi-elicited heart injury paralleled IFNγ+ cells accumulation, whereas reconstitution with CD8+ cells from naïve ifnγ−/−pfn+/+ donors led to an aggravation of the cardiomyocyte lesion, which was associated with the accumulation of Pfn+ cells in the cardiac tissue. Our data support a possible antagonist effect of CD8+Pfn+ and CD8+IFNγ+ cells during CCC. CD8+IFNγ+ cells may exert a beneficial role, whereas CD8+Pfn+ may play a detrimental role

  16. Effects of influenza A virus NS1 protein on protein expression: the NS1 protein enhances translation and is not required for shutoff of host protein synthesis.

    PubMed

    Salvatore, Mirella; Basler, Christopher F; Parisien, Jean-Patrick; Horvath, Curt M; Bourmakina, Svetlana; Zheng, Hongyong; Muster, Thomas; Palese, Peter; García-Sastre, Adolfo

    2002-02-01

    The influenza A virus NS1 protein, a virus-encoded alpha/beta interferon (IFN-alpha/beta) antagonist, appears to be a key regulator of protein expression in infected cells. We now show that NS1 protein expression results in enhancement of reporter gene activity from transfected plasmids. This effect appears to be mediated at the translational level, and it is reminiscent of the activity of the adenoviral virus-associated I (VAI) RNA, a known inhibitor of the antiviral, IFN-induced, PKR protein. To study the effects of the NS1 protein on viral and cellular protein synthesis during influenza A virus infection, we used recombinant influenza viruses lacking the NS1 gene (delNS1) or expressing truncated NS1 proteins. Our results demonstrate that the NS1 protein is required for efficient viral protein synthesis in COS-7 cells. This activity maps to the amino-terminal domain of the NS1 protein, since cells infected with wild-type virus or with a mutant virus expressing a truncated NS1 protein-lacking approximately half of its carboxy-terminal end-showed similar kinetics of viral and cellular protein expression. Interestingly, no major differences in host cell protein synthesis shutoff or in viral protein expression were found among NS1 mutant viruses in Vero cells. Thus, another viral component(s) different from the NS1 protein is responsible for the inhibition of host protein synthesis during viral infection. In contrast to the earlier proposal suggesting that the NS1 protein regulates the levels of spliced M2 mRNA, no effects on M2 protein accumulation were seen in Vero cells infected with delNS1 virus.

  17. Survivin and related proteins in canine mammary tumors: immunohistochemical expression.

    PubMed

    Bongiovanni, L; Romanucci, M; Malatesta, D; D'Andrea, A; Ciccarelli, A; Della Salda, L

    2015-03-01

    Survivin is reexpressed in most human breast cancers, where its expression has been associated with tumor aggressiveness, poor prognosis, and poor response to therapy. Survivin expression was evaluated in 41 malignant canine mammary tumors (CMTs) by immunohistochemistry, in relation to histological grade and stage, and correlated with that of some related molecules (β-catenin, caspase 3, heat shock proteins) to understand their possible role in canine mammary tumorigenesis. An increase in nuclear survivin expression, compared with healthy mammary glands, was observed in CMTs, where nuclear immunolabeling was related to the presence of necrosis. No statistically significant relation was found between the expression of the investigated molecules and the histological grade or stage. The present study may suggest an important involvement of survivin in CMT tumorigenesis. Its overexpression in most of the cases evaluated might suggest that targeting survivin in CMTs may be a valid anticancer therapy. PMID:24686389

  18. Expression of Tyrosine Hydroxylase is Negatively Regulated Via Prion Protein.

    PubMed

    da Luz, Marcio Henrique Mello; Glezer, Isaias; Xavier, Andre Machado; da Silva, Marcelo Alberti Paiva; Pino, Jessica Monteiro Volejnik; Zamith, Thiago Panaro; Vieira, Taynara Fernanda; Antonio, Bruno Brito; Antunes, Hanna Karen Moreira; Martins, Vilma Regina; Lee, Kil Sun

    2016-07-01

    Cellular prion protein (PrP(C)) is a glycoprotein of the plasma membrane that plays pleiotropic functions by interacting with multiple signaling complexes at the cell surface. Recently, a number of studies have reported the involvement of PrP(C) in dopamine metabolism and signaling, including its interactions with tyrosine hydroxylase (TH) and dopamine receptors. However, the outcomes reported by independent studies are still debatable. Therefore in this study, we investigated the effects of PrP(C) on the TH expression during the differentiation of N2a cells with dibutyryl-cAMP, a well-known cAMP analog that activates TH transcription. Upon differentiation, TH was induced with concomitant reduction of PrP(C) at protein level, but not at mRNA level. shRNA-mediated PrP(C) reduction increased the basal level of TH at both mRNA and protein levels without dibutyryl-cAMP treatment. This phenotype was reversed by re-expression of PrP(C). PrP(C) knockdown also potentiated the effect of dibutyryl-cAMP on TH expression. Our findings suggest that PrP(C) has suppressive effects on TH expression. As a consequence, altered PrP(C) functions may affect the regulation of dopamine metabolism and related neurological disorders.

  19. Vaccination with recombinant adenoviruses expressing Ebola virus glycoprotein elicits protection in the interferon alpha/beta receptor knock-out mouse.

    PubMed

    O'Brien, Lyn M; Stokes, Margaret G; Lonsdale, Stephen G; Maslowski, David R; Smither, Sophie J; Lever, Mark S; Laws, Thomas R; Perkins, Stuart D

    2014-03-01

    The resistance of adult immunocompetent mice to infection with ebolaviruses has led to the development of alternative small animal models that utilise immunodeficient mice, for example the interferon α/β receptor knock-out mouse (IFNR(-/-)). IFNR(-/-) mice have been shown to be susceptible to infection with ebolaviruses by multiple routes but it is not known if this murine model is suitable for testing therapeutics that rely on the generation of an immune response for efficacy. We have tested recombinant adenovirus vectors for their ability to protect IFNR(-/-) mice from challenge with Ebola virus and have analysed the humoral response generated after immunisation. The recombinant vaccines elicited good levels of protection in the knock-out mouse and the antibody response in IFNR(-/-) mice was similar to that observed in vaccinated wild-type mice. These results indicate that the IFNR(-/-) mouse is a relevant small animal model for studying ebolavirus-specific therapeutics.

  20. Graphene Nanoribbons Elicit Cell Specific Uptake and Delivery Via Activation of Epidermal Growth Factor Receptor Enhanced by Human PapillomaVirus E5 Protein

    PubMed Central

    Chowdhury, Sayan Mullick; Mannepalli, Prady; Sitharaman, Balaji

    2014-01-01

    Ligands such as peptides, antibodies or other epitopes bind and activate specific cell receptors, and are employed for targeted cellular delivery of pharmaceuticals such as drugs, genes and imaging agents. Herein, we show that oxidized graphene nanoribbons, non-covalently functionalized with PEG-DSPE (1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N[amino(polyethyleneglycol)]) (O-GNR-PEG-DSPE) activate epidermal growth factor receptors (EGFRs). This activation generates predominantly dynamin-dependent macropinocytosis-like response, and results in significant O-GNR-PEG-DSPE uptake into cells with high EGFR expression. Cells with an integrated human papillomavirus (HPV) genome also show increased uptake due to the modulation of the activated EFGR by the viral protein E5. We demonstrate that this cell specific uptake of O-GNR-PEG-DSPE can be exploited to achieve significantly enhanced drug efficacies even in drug resistant cells. These results have implications towards the development of active targeting and delivery agents without ligand functionalization for use in the diagnosis and treatment of pathologies that overexpress EGFR or mediated by HPV. PMID:24980059

  1. Determination of protein markers in human serum: Analysis of protein expression in toxic oil syndrome studies.

    PubMed

    Quero, Carmen; Colomé, Nuria; Prieto, Maria Rosario; Carrascal, Montserrat; Posada, Manuel; Gelpí, Emilio; Abian, Joaquin

    2004-02-01

    Toxic oil syndrome (TOS) is a disease that appeared in Spain in 1981. It affected more than 20 000 people and produced over 300 deaths in the first 2 years. In this paper, a prospective study on the differences in gene expression in sera between a control versus a TOS-affected population, both originally exposed to the toxic oil, is presented. Differential protein expression was analyzed by two-dimensional electrophoresis (2-DE). Several problems related with serum analysis by 2-DE were addressed in order to improve protein detection in the gel images. Three new commercial systems for albumin depletion were tested to optimize the detection of minor proteins that can be obscured by the presence of a few families of high abundance proteins (albumin, immunoglobulins). Other factors, such as the use of nonionic reductants or the presence of thiourea in the gels, were also tested. From these optimized images, a group of 329 major gel spots was located, matched and compared in serum samples. Thirty-five of these protein spots were found to be under- or overexpressed in TOS patients (> three-fold increase or decrease). Proteins in the differential spots were identified by matrix-assisted laser desorption/ionization-time of flight peptide map fingerprinting and database search. Several haptoglobin isoforms were found to be differentially expressed, showing expression phenotypes that could be related with TOS affection. Haptoglobin phenotypes have been previously reported to have important biological and clinical consequences and have been described as risk factors for several diseases.

  2. Alternative Eukaryotic Expression Systems for the Production of Proteins and Protein Complexes.

    PubMed

    Gómez, Sara; López-Estepa, Miguel; Fernández, Francisco J; Suárez, Teresa; Vega, M Cristina

    2016-01-01

    Besides the most established expression hosts, several eukaryotic microorganisms and filamentous fungi have also been successfully used as platforms for the production of foreign proteins. Filamentous fungi and Dictyostelium discoideum are two prominent examples. Filamentous fungi, typically Aspergillus and Trichoderma, are usually employed for the industrial production of enzymes and secondary metabolites for food processing, pharmaceutical drugs production, and textile and paper applications, with multiple products already accepted for their commercialization. The low cost of culture medium components, high secretion capability directly to the extracellular medium, and the intrinsic ability to produce post-translational modifications similar to the mammalian type, have promoted this group as successful hosts for the expression of proteins, including examples from phylogenetically distant groups: humans proteins such as IL-2, IL-6 or epithelial growth factor; α-galactosidase from plants; or endoglucanase from Cellulomonas fimi, among others. D. discoideum is a social amoeba that can be used as an expression platform for a variety of proteins, which has been extensively illustrated for cytoskeletal proteins. New vectors for heterologous expression in D. discoideum have been recently developed that might increase the usefulness of this system and expand the range of protein classes that can be tackled. Continuous developments are ongoing to improve strains, promoters, production and downstream processes for filamentous fungi, D. discoideum, and other alternative eukaryotic hosts. Either for the overexpression of individual genes, or in the coexpression of multiples genes, this chapter illustrates the enormous possibilities offered by these groups of eukaryotic organisms. PMID:27165325

  3. Alternative Eukaryotic Expression Systems for the Production of Proteins and Protein Complexes.

    PubMed

    Gómez, Sara; López-Estepa, Miguel; Fernández, Francisco J; Suárez, Teresa; Vega, M Cristina

    2016-01-01

    Besides the most established expression hosts, several eukaryotic microorganisms and filamentous fungi have also been successfully used as platforms for the production of foreign proteins. Filamentous fungi and Dictyostelium discoideum are two prominent examples. Filamentous fungi, typically Aspergillus and Trichoderma, are usually employed for the industrial production of enzymes and secondary metabolites for food processing, pharmaceutical drugs production, and textile and paper applications, with multiple products already accepted for their commercialization. The low cost of culture medium components, high secretion capability directly to the extracellular medium, and the intrinsic ability to produce post-translational modifications similar to the mammalian type, have promoted this group as successful hosts for the expression of proteins, including examples from phylogenetically distant groups: humans proteins such as IL-2, IL-6 or epithelial growth factor; α-galactosidase from plants; or endoglucanase from Cellulomonas fimi, among others. D. discoideum is a social amoeba that can be used as an expression platform for a variety of proteins, which has been extensively illustrated for cytoskeletal proteins. New vectors for heterologous expression in D. discoideum have been recently developed that might increase the usefulness of this system and expand the range of protein classes that can be tackled. Continuous developments are ongoing to improve strains, promoters, production and downstream processes for filamentous fungi, D. discoideum, and other alternative eukaryotic hosts. Either for the overexpression of individual genes, or in the coexpression of multiples genes, this chapter illustrates the enormous possibilities offered by these groups of eukaryotic organisms.

  4. Expression, purification and crystallization of a lyssavirus matrix (M) protein

    SciTech Connect

    Assenberg, René; Delmas, Olivier; Graham, Stephen C.; Verma, Anil; Berrow, Nick; Stuart, David I.; Owens, Raymond J.; Bourhy, Hervé; Grimes, Jonathan M.

    2008-04-01

    The expression, purification and crystallization of the full-length matrix protein from three lyssaviruses is described. The matrix (M) proteins of lyssaviruses (family Rhabdoviridae) are crucial to viral morphogenesis as well as in modulating replication and transcription of the viral genome. To date, no high-resolution structural information has been obtained for full-length rhabdovirus M. Here, the cloning, expression and purification of the matrix proteins from three lyssaviruses, Lagos bat virus (LAG), Mokola virus and Thailand dog virus, are described. Crystals have been obtained for the full-length M protein from Lagos bat virus (LAG M). Successful crystallization depended on a number of factors, in particular the addition of an N-terminal SUMO fusion tag to increase protein solubility. Diffraction data have been recorded from crystals of native and selenomethionine-labelled LAG M to 2.75 and 3.0 Å resolution, respectively. Preliminary analysis indicates that these crystals belong to space group P6{sub 1}22 or P6{sub 5}22, with unit-cell parameters a = b = 56.9–57.2, c = 187.9–188.6 Å, consistent with the presence of one molecule per asymmetric unit, and structure determination is currently in progress.

  5. Heat Shock Protein 90 (Hsp90) Expression and Breast Cancer

    PubMed Central

    Zagouri, Flora; Bournakis, Evangelos; Koutsoukos, Konstantinos; Papadimitriou, Christos A.

    2012-01-01

    Hsp90 is an abundant protein in mammalian cells. It forms several discrete complexes, each containing distinct groups of co-chaperones that assist protein folding and refolding during stress, protein transport and degradation. It interacts with a variety of proteins that play key roles in breast neoplasia including estrogen receptors, tumor suppressor p53 protein, angiogenesis transcription factor HIF-1alpha, antiapoptotic kinase Akt, Raf-1 MAP kinase and a variety of receptor tyrosine kinases of the erbB family. Elevated Hsp90 expression has been documented in breast ductal carcinomas contributing to the proliferative activity of breast cancer cells; whilst a significantly decreased Hsp90 expression has been shown in infiltrative lobular carcinomas and lobular neoplasia. Hsp90 overexpression has been proposed as a component of a mechanism through which breast cancer cells become resistant to various stress stimuli. Therefore, pharmacological inhibition of HSPs can provide therapeutic opportunities in the field of cancer treatment. 17-allylamino,17-demethoxygeldanamycin is the first Hsp90 inhibitor that has clinically been investigated in phase II trial, yielding promising results in patients with HER2-overexpressing metastatic breast cancer, whilst other Hsp90 inhibitors (retaspimycin HCL, NVP-AUY922, NVP-BEP800, CNF2024/BIIB021, SNX-5422, STA-9090, etc.) are currently under evaluation. PMID:24280702

  6. Stepwise optimization of a low-temperature Bacillus subtilis expression system for "difficult to express" proteins.

    PubMed

    Welsch, Norma; Homuth, Georg; Schweder, Thomas

    2015-08-01

    In order to improve the overproduction of "difficult to express" proteins, a low-temperature expression system for Bacillus subtilis based on the cold-inducible promoter of the desaturase-encoding des gene was constructed. Selected regulatory DNA sequence elements from B. subtilis genes known to be cold-inducible were fused to different model genes. It could be demonstrated that these regulatory elements are able to mediate increased heterologous gene expression, either by improved translation efficiency or by higher messenger RNA (mRNA) stability. In case of a cold-adapted β-galactosidase from Pseudoalteromonas haloplanktis TAE79A serving as the model, significantly higher expression was achieved by fusing its coding sequence to the so-called "downstream box" sequence of cspB encoding the major B. subtilis cold-shock protein. The combination of this fusion with a cspB 5'-UTR stem-loop structure resulted in further enhancement of the β-galactosidase expression. In addition, integration of the transcription terminator of the B. subtilis cold-inducible bkd operon downstream of the target genes caused a higher mRNA stability and enabled thus a further significant increase in expression. Finally, the fully optimized expression system was validated by overproducing a B. subtilis xylanase as well as an α-glucosidase from Saccharomyces cerevisiae, the latter known for tending to form inclusion bodies. These analyses verified the applicability of the engineered expression system for extracellular and intracellular protein synthesis in B. subtilis, thereby confirming the suitability of this host organism for the overproduction of critical, poorly soluble proteins. PMID:25851716

  7. tincar encodes a novel transmembrane protein expressed in the Tinman-expressing cardioblasts of Drosophila.

    PubMed

    Hirota, Yuki; Sawamoto, Kazunobu; Okano, Hideyuki

    2002-12-01

    We cloned and characterized the Drosophila gene, tincar (tinc), which encodes a novel protein with eight putative transmembrane domains. The tinc mRNA was expressed specifically in four of the six pairs of cardioblasts in each segment, in a pattern identical to that of tinman (tin), a homeobox gene required for the specification of the dorsal vessel. In the non-Tin-expressing pairs of cardioblasts, tinc transcription seemed to be repressed by Seven-up. PMID:14516698

  8. tincar encodes a novel transmembrane protein expressed in the Tinman-expressing cardioblasts of Drosophila.

    PubMed

    Hirota, Yuki; Sawamoto, Kazunobu; Okano, Hideyuki

    2002-12-01

    We cloned and characterized the Drosophila gene, tincar (tinc), which encodes a novel protein with eight putative transmembrane domains. The tinc mRNA was expressed specifically in four of the six pairs of cardioblasts in each segment, in a pattern identical to that of tinman (tin), a homeobox gene required for the specification of the dorsal vessel. In the non-Tin-expressing pairs of cardioblasts, tinc transcription seemed to be repressed by Seven-up. PMID:12617821

  9. Stepwise optimization of a low-temperature Bacillus subtilis expression system for "difficult to express" proteins.

    PubMed

    Welsch, Norma; Homuth, Georg; Schweder, Thomas

    2015-08-01

    In order to improve the overproduction of "difficult to express" proteins, a low-temperature expression system for Bacillus subtilis based on the cold-inducible promoter of the desaturase-encoding des gene was constructed. Selected regulatory DNA sequence elements from B. subtilis genes known to be cold-inducible were fused to different model genes. It could be demonstrated that these regulatory elements are able to mediate increased heterologous gene expression, either by improved translation efficiency or by higher messenger RNA (mRNA) stability. In case of a cold-adapted β-galactosidase from Pseudoalteromonas haloplanktis TAE79A serving as the model, significantly higher expression was achieved by fusing its coding sequence to the so-called "downstream box" sequence of cspB encoding the major B. subtilis cold-shock protein. The combination of this fusion with a cspB 5'-UTR stem-loop structure resulted in further enhancement of the β-galactosidase expression. In addition, integration of the transcription terminator of the B. subtilis cold-inducible bkd operon downstream of the target genes caused a higher mRNA stability and enabled thus a further significant increase in expression. Finally, the fully optimized expression system was validated by overproducing a B. subtilis xylanase as well as an α-glucosidase from Saccharomyces cerevisiae, the latter known for tending to form inclusion bodies. These analyses verified the applicability of the engineered expression system for extracellular and intracellular protein synthesis in B. subtilis, thereby confirming the suitability of this host organism for the overproduction of critical, poorly soluble proteins.

  10. Protein inhibitor of activated STAT3 inhibits adipogenic gene expression

    SciTech Connect

    Deng Jianbei; Hua Kunjie; Caveney, Erica J.; Takahashi, Nobuyuki; Harp, Joyce B. . E-mail: jharp@unc.edu

    2006-01-20

    Protein inhibitor of activated STAT3 (PIAS3), a cytokine-induced repressor of signal transducer and activator of transcription 3 (STAT3) and a modulator of a broad array of nuclear proteins, is expressed in white adipose tissue, but its role in adipogenesis is not known. Here, we determined that PIAS3 was constitutively expressed in 3T3-L1 cells at all stages of adipogenesis. However, it translocated from the nucleus to the cytoplasm 4 days after induction of differentiation by isobutylmethylxanthine, dexamethasone, and insulin (MDI). In ob/ob mice, PIAS3 expression was increased in white adipose tissue depots compared to lean mice and was found in the cytoplasm of adipocytes. Overexpression of PIAS3 in differentiating preadipocytes, which localized primarily to the nucleus, inhibited mRNA level gene expression of adipogenic transcription factors C/EBP{alpha} and PPAR{gamma}, as well as their downstream target genes aP2 and adiponectin. PIAS3 also inhibited C/EBP{alpha} promoter activation mediated specifically by insulin, but not dexamethasone or isobutylmethylxanthine. Taken together, these data suggest that PIAS3 may play an inhibitory role in adipogenesis by modulating insulin-activated transcriptional activation events. Increased PIAS3 expression in adipose tissue may play a role in the metabolic disturbances of obesity.

  11. Expression of Superficial Zone Protein in Mandibular Condyle Cartilage

    PubMed Central

    Ohno, S; Schmid, T; Tanne, Y; Kamiya, T; Honda, K; Ohno-Nakahara, M; Swentko, N; Desai, T A; Tanne, K; Knudson, CB; Knudson, W

    2011-01-01

    Objective Superficial zone protein (SZP) has been shown to function in the boundary lubrication of articular cartilages of the extremities. However, the expression of SZP has not been clarified in mandibular cartilage which is a tissue that includes a thick fibrous layer on the surface. This study was conducted to clarify the distribution of SZP on the mandibular condyle and the regulatory effects of humoral factors on the expression in both explants and fibroblasts derived from mandibular condyle. Methods The distribution of SZP was determined in bovine mandibular condyle cartilage, and the effects of IL-1β and TGF-β on SZP expression were examined in condyle explants and, fibroblasts derived from the fibrous zone of condyle cartilage. Results SZP was highly distributed in the superficial zone of intact condyle cartilage. The SZP expression was up-regulated by TGF-β in both explants and cultured fibroblasts, whereas the expression was slightly down-regulated by IL-1β. A significant increase in accumulation of SZP protein was also observed in the culture medium of the fibroblasts treated with TGF-β. Conclusions These results suggest that SZP plays an important role in boundary lubrication of mandible condylar cartilage, is synthesized locally within the condyle itself and, exhibits differential regulation by cell mediators relevant to mandibular condyle repairing and pathologies. PMID:16563813

  12. Heterogeneity mapping of protein expression in tumors using quantitative immunofluorescence.

    PubMed

    Faratian, Dana; Christiansen, Jason; Gustavson, Mark; Jones, Christine; Scott, Christopher; Um, InHwa; Harrison, David J

    2011-10-25

    Morphologic heterogeneity within an individual tumor is well-recognized by histopathologists in surgical practice. While this often takes the form of areas of distinct differentiation into recognized histological subtypes, or different pathological grade, often there are more subtle differences in phenotype which defy accurate classification (Figure 1). Ultimately, since morphology is dictated by the underlying molecular phenotype, areas with visible differences are likely to be accompanied by differences in the expression of proteins which orchestrate cellular function and behavior, and therefore, appearance. The significance of visible and invisible (molecular) heterogeneity for prognosis is unknown, but recent evidence suggests that, at least at the genetic level, heterogeneity exists in the primary tumor(1,2), and some of these sub-clones give rise to metastatic (and therefore lethal) disease. Moreover, some proteins are measured as biomarkers because they are the targets of therapy (for instance ER and HER2 for tamoxifen and trastuzumab (Herceptin), respectively). If these proteins show variable expression within a tumor then therapeutic responses may also be variable. The widely used histopathologic scoring schemes for immunohistochemistry either ignore, or numerically homogenize the quantification of protein expression. Similarly, in destructive techniques, where the tumor samples are homogenized (such as gene expression profiling), quantitative information can be elucidated, but spatial information is lost. Genetic heterogeneity mapping approaches in pancreatic cancer have relied either on generation of a single cell suspension(3), or on macrodissection(4). A recent study has used quantum dots in order to map morphologic and molecular heterogeneity in prostate cancer tissue(5), providing proof of principle that morphology and molecular mapping is feasible, but falling short of quantifying the heterogeneity. Since immunohistochemistry is, at best, only semi

  13. Effect of D-aspartate uptake on uncoupling protein-3 and α-tubulin expressions in rat Harderian gland.

    PubMed

    Santillo, Alessandra; Burrone, Lavinia; Senese, Rosalba; Cioffi, Federica; Lanni, Antonia; Chieffi Baccari, Gabriella

    2011-11-01

    Although D-aspartate (D-Asp) has been recognized as having an important physiological role within different organs, high concentrations could elicit detrimental effects on those same organs. In this study, we evaluated the oxidative stress response to D-Asp treatment in rat Harderian gland (HG) by measuring total cellular hydroperoxide levels. Further, we examined the effect of D-Asp uptake on the expression of the mitochondrial uncoupling protein-3 (UCP3), β-actin, and α-tubulin. In rat HG, elevated levels of D-Asp significantly increased hydroperoxide production. This phenomenon was probably due to D-Asp uptake as well as lipid and porphyrin increased levels. Higher UCP3 levels and lower α-tubulin expression were also observed after D-Asp treatment. On the contrary, β-actin expression was unchanged. Given the possible role of UCP3 in lipid handling, the higher expression of mitochondria UCP3 protein in D-Asp-treated HG may reflect a major need to export excessive amounts of hydroperoxides deriving from a greater fatty acid flux across these organelles and higher mitochondrial porphyrin levels. Moreover, abundance of hydroperoxides in D-Asp treated rat HG could determine the decrease of α-tubulin expression. Thus, our findings indicate that a high concentration of D-Asp is critical in initiating a cascade of events determined by oxidative stress.

  14. Expression of mammalian protein kinase C in Schizosaccharomyces pombe: isotype-specific induction of growth arrest, vesicle formation, and endocytosis.

    PubMed Central

    Goode, N T; Hajibagheri, M A; Warren, G; Parker, P J

    1994-01-01

    Mammalian protein kinase C (PKC) isotypes elicit a number of effects on expression in Schizosaccharomyces pombe. A small decrease in growth rate results from PKC-gamma expression, and treatment of these cells with phorbol esters leads to marked growth inhibition and vesicle formation. PKC-delta and -eta expression causes growth inhibition and vesiculation, and the magnitude of both of these effects is increased by phorbol esters. In contrast, PKC-epsilon expression produces growth inhibition but no vesicle accumulation, and this effect is not responsive to phorbol ester. Finally, PKC-zeta has no observable effect. Thus, isotype-specific biological effects are observed. The accumulation of vesicles correlates with phorbol ester-dependent growth inhibition and occurs only with expression of those isotypes that down-regulate in response to phorbol esters in these cells. Antibodies against mammalian clathrin light chain 1a identified clathrin-coated vesicles and up-regulation of clathrin expression in those cells where vesicles accumulate; the increased vesicular traffic includes an element of endocytosis. Thus expression of specific mammalian PKC isotypes up-regulates endocytosis in S. pombe, providing a likely explanation for PKC-mediated receptor internalization in higher eukaryotes. Images PMID:7803858

  15. HIV-1 Tat Protein Enhances Expression and Function of Breast Cancer Resistance Protein.

    PubMed

    Zhou, Yancong; Zhang, Kun; Yin, Xiaojie; Nie, Qichang; Ma, Yonggang

    2016-01-01

    ATP binding cassette (ABC) transporters can transfer a variety of antiviral agents from the cytoplasm to body fluid, which results in a reduced intracellular concentration of the drugs. Proteins of HIV-1, e.g., Tat and gp120, altered some types of ABC transporter expression in brain microvascular endothelial cells and astrocytes. However, the effect of Tat on ABC transporters in T lymphocytes is unclear. In this study the status of breast cancer resistance protein (BCRP) in Tat expressing cell lines was examined with real-time PCR and flow cytometry. It was found that HIV-1 Tat protein upregulated BCRP expression and enhanced efflux mediated by BCRP significantly, which could inhibit antiviral drugs from entering infected cells and interfere with the therapeutic effect of HAART. PMID:26367065

  16. Bacteriophage membrane protein P9 as a fusion partner for the efficient expression of membrane proteins in Escherichia coli.

    PubMed

    Jung, Yuna; Jung, Hyeim; Lim, Dongbin

    2015-12-01

    Despite their important roles and economic values, studies of membrane proteins have been hampered by the difficulties associated with obtaining sufficient amounts of protein. Here, we report a novel membrane protein expression system that uses the major envelope protein (P9) of phage φ6 as an N-terminal fusion partner. Phage membrane protein P9 facilitated the synthesis of target proteins and their integration into the Escherichia coli cell membrane. This system was used to produce various multi-pass transmembrane proteins, including G-protein-coupled receptors, transporters, and ion channels of human origin. Green fluorescent protein fusion was used to confirm the correct folding of the expressed proteins. Of the 14 membrane proteins tested, eight were highly expressed, three were moderately expressed, and three were barely expressed in E. coli. Seven of the eight highly expressed proteins could be purified after extraction with the mild detergent lauryldimethylamine-oxide. Although a few proteins have previously been developed as fusion partners to augment membrane protein production, we believe that the major envelope protein P9 described here is better suited to the efficient expression of eukaryotic transmembrane proteins in E. coli.

  17. Flunitrazepam rapidly reduces GABAA receptor subunit protein expression via a protein kinase C-dependent mechanism

    PubMed Central

    Johnston, Jonathan D; Price, Sally A; Bristow, David R

    1998-01-01

    Acute flunitrazepam (1 μM) exposure for 1 h reduced GABAA receptor α1 (22±4%, mean±s.e.mean) and β2/3 (21±4%) subunit protein levels in cultured rat cerebellar granule cells. This rapid decrease in subunit proteins was completely prevented by bisindolymaleimide 1 (1 μM), an inhibitor of protein kinase C, but not by N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide (H-89, 4.8 μM), an inhibitor of protein kinases A and G. These results suggest the existence of a benzodiazepine-induced mechanism to rapidly alter GABAA receptor protein expression, that appears to be dependent on protein kinase C activity. PMID:9723942

  18. Morphine-conditioned cue alters c-Fos protein expression in the brain of crayfish.

    PubMed

    Dziopa, Leah; Imeh-Nathaniel, Adebobola; Baier, Dana; Kiel, Michael; Sameera, Sayeed; Brager, Adam; Beatriz, Vega; Nathaniel, Thomas I

    2011-07-15

    With a highly organized stereotypic behavior and a simplified neuronal system that is characterized by cellular modularity, crayfish (Orconectes rusticus) represents an excellent model that we used in this study to explore how a drug-conditioned-cue alters c-Fos protein expression in the brain of an invertebrate species. The first set of experiments revealed that a single injection of different doses of morphine (3.0 μg/g, 6.0 μg/g and 12.0 μg/g) into the circulatory system of crayfish significantly increased locomotor activity. Repeated injections of morphine increased locomotion at lower doses (3.0 μg/g and 6.0 μg/g), and decreased locomotion at a higher dose of 12.0 μg/g. The second experiment revealed that a repeated or single injection of morphine serves as reward when paired with a distinct visual environment. In the third experiment, we found that the c-Fos profile of morphine treated crayfish in an unconditioned environment did not show a significant increase from the basal level comparable to saline treated crayfish. The brains of crayfish were more active during exposure to the cue-elicited drug conditioned environment than the unconditioned environment. These results indicate that chronic morphine treatment alone is not sufficient to induce changes in the expression of c-Fos; instead, morphine-environment pairing in a specific context contributes to the expression of alterations in c-Fos regulation. The enhancement of c-Fos expression in the brain of crayfish seems to reflect the sensory or anticipatory facets of conditioning that suggests that potential and even unanticipated hypotheses in drug addiction can emerge from studies of addiction in crayfish.

  19. Expression data on liver metabolic pathway genes and proteins

    PubMed Central

    Raja Gopal Reddy, Mooli; Pavan Kumar, Chodisetti; Mahesh, Malleswarapu; Sravan Kumar, Manchiryala; Jeyakumar, Shanmugam M.

    2016-01-01

    Here, we present the expression data on various metabolic pathways of liver with special emphasize on lipid and carbohydrate metabolism and long chain polyunsaturated fatty acid (PUFA) synthesis, both at gene and protein levels. The data were obtained to understand the effect of vitamin A deficiency on the expression status (both gene and protein levels) of some of the key factors involved in lipogenesis, fatty acid oxidation, triglyceride secretion, long chain PUFA, resolvin D1 synthesis, glucose transport and glycogen synthesis of liver, using modern biology tools, such as quantitative real-time PCR (RT-PCR) and immunoblotting techniques. This data article provides the supporting evidence to the article “Vitamin A deficiency suppresses high fructose-induced triglyceride synthesis and elevates resolvin D1 levels” [1] and therefore, these data may be referred back, for comprehensive understanding and interpretations and for future studies. PMID:26909377

  20. Recombinant Dragline Silk-Like Proteins-Expression and Purification.

    PubMed

    Gaines, William A; Marcotte, William R

    2011-03-01

    Spider dragline silk is a proteinaceous fiber with impressive physical characteristics making it attractive for use in advanced materials. The fiber is composed of two proteins (spidroins MaSp1 and MaSp2), each of which contains a large central repeat array flanked by non-repetitive N- and C-terminal domains. The repeat arrays appear to be largely responsible for the tensile properties of the fiber, suggesting that the N- and C-terminal domains may be involved in self-assembly. We recently isolated the MaSp1 and MaSp2 N-terminal domains from Nephila clavipes and have incorporated these into mini-silk genes for expression in transgenic systems. Current efforts involve the development of expression vectors that will allow purification using a removable affinity tag for scalable protein purification.

  1. Disposable bioreactors for inoculum production and protein expression.

    PubMed

    Eibl, Regine; Löffelholz, Christian; Eibl, Dieter

    2014-01-01

    Disposable bioreactors have been increasingly implemented over the past ten years. This relates to both R & D and commercial manufacture, in particular, in animal cell-based processes. Among the numerous disposable bioreactors which are available today, wave-mixed bag bioreactors and stirred bioreactors are predominant. Whereas wave-mixed bag bioreactors represent the system of choice for inoculum production, stirred systems are often preferred for protein expression. For this reason, the authors present protocols instructing the reader how to use the wave-mixed BIOSTAT CultiBag RM 20 L for inoculum production and the stirred UniVessel SU 2 L for recombinant protein production at benchtop scale. All methods described are based on a Chinese hamster ovary (CHO) suspension cell line expressing the human placental secreted alkaline phosphatase (SEAP).

  2. Ribozymes, riboswitches and beyond: regulation of gene expression without proteins

    PubMed Central

    Serganov, Alexander; Patel, Dinshaw J.

    2015-01-01

    Although various functions of RNA are carried out in conjunction with proteins, some catalytic RNAs, or ribozymes, which contribute to a range of cellular processes, require little or no assistance from proteins. Furthermore, the discovery of metabolite-sensing riboswitches and other types of RNA sensors has revealed RNA-based mechanisms that cells use to regulate gene expression in response to internal and external changes. Structural studies have shown how these RNAs can carry out a range of functions. In addition, the contribution of ribozymes and riboswitches to gene expression is being revealed as far more widespread than was previously appreciated. These findings have implications for understanding how cellular functions might have evolved from RNA-based origins. PMID:17846637

  3. Human ductal adenocarcinomas of the pancreas express extracellular matrix proteins.

    PubMed Central

    Löhr, M.; Trautmann, B.; Göttler, M.; Peters, S.; Zauner, I.; Maillet, B.; Klöppel, G.

    1994-01-01

    Pancreatic ductal adenocarcinomas are characterised by a dense connective tissue reaction. To test the hypothesis that stroma components are synthesised and produced by the tumour cells themselves, eight cell lines as well as six xenografted tumours from human ductal adenocarcinomas of the pancreas were examined for the expression of extracellular matrix proteins (ECM), using cDNA probes and antibodies to collagen types I, III and IV, vitronectin, fibronectin, undulin and laminin. All tumour cell lines (CAPAN-1, CAPAN-2, AsPC-1, BxPC-3, PANC-1, PaCa-2, PaCa-3, PaCa-44) and xenografted human pancreatic tumours expressed at least one of the examined ECM at the RNA (collagen type IV > laminin = fibronectin = vitronectin > collagen type III > undulin > collagen type I) or protein level (collagen type IV = collagen type III > vitronectin > laminin > collagen type I = fibronectin > undulin). In nude mouse tumours expression of laminin and collagen I was most pronounced in well-differentiated carcinomas. In a few tumours, collagen type III, vitronectin and undulin were expressed on the luminal side of the neoplastic glands, suggesting loss of normal polar differentiation. Incubation with fetal calf serum modulated ECM RNA levels to a varying extent in all but one cell line (AsPC-1). The results suggest that human pancreatic ductal adenocarcinomas cells are capable of synthesising and producing extracellular matrix proteins in vitro and in vivo, but that the extent and pattern of ECM expression differs between the various tumours and conditions tested. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:8286197

  4. Differential rates of gene expression monitored by green fluorescent protein.

    PubMed

    Lu, Canghai; Albano, C Renee; Bentley, William E; Rao, Govind

    2002-08-20

    The use of green fluorescent protein (GFP) as a reporter gene has made a broad impact in several areas, especially in studies of protein trafficking, localization, and expression analysis. GFP's many advantages are that it is small, autocatalytic, and does not require fixation, cell disruption, or the addition of cofactors or substrates. Two characteristics of GFP, extreme stability and chromophore cyclization lag time, pose a hindrance to the application of GFP as a real-time gene expression reporter in bioprocess applications. In this report, we present analytical methods that overcome these problems and enable the temporal visualization of discrete gene regulatory events. The approach we present measures the rate of change in GFP fluorescence, which in turn reflects the rate of gene expression. We conducted fermentation and microplate experiments using a protein synthesis inhibitor to illustrate the feasibility of this system. Additional experiments using the classic gene regulation of the araBAD operon show the utility of GFP as a near real-time indicator of gene regulation. With repetitive induction and repression of the arabinose promoter, the differential rate of GFP fluorescence emission shows corresponding cyclical changes during the culture.

  5. Differential expression of ribosomal proteins in myelodysplastic syndromes.

    PubMed

    Rinker, Elizabeth B; Dueber, Julie C; Qualtieri, Julianne; Tedesco, Jason; Erdogan, Begum; Bosompem, Amma; Kim, Annette S

    2016-02-01

    Aberrations of ribosomal biogenesis have been implicated in several congenital bone marrow failure syndromes, such as Diamond-Blackfan anaemia, Shwachman-Diamond syndrome and Dyskeratosis Congenita. Recent studies have identified haploinsufficiency of RPS14 in the acquired bone marrow disease isolated 5q minus syndrome, a subtype of myelodysplastic syndromes (MDS). However, the expression of various proteins comprising the ribosomal subunits and other proteins enzymatically involved in the synthesis of the ribosome has not been explored in non-5q minus MDS. Furthermore, differences in the effects of these expression alterations among myeloid, erythroid and megakaryocyte lineages have not been well elucidated. We examined the expression of several proteins related to ribosomal biogenesis in bone marrow biopsy specimens from patients with MDS (5q minus patients excluded) and controls with no known myeloid disease. Specifically, we found that there is overexpression of RPS24, DKC1 and SBDS in MDS. This overexpression is in contrast to the haploinsufficiency identified in the congenital bone marrow failure syndromes and in acquired 5q minus MDS. Potential mechanisms for these differences and aetiology for these findings in MDS are discussed.

  6. Proteasome inhibitors suppress the protein expression of mutant p53.

    PubMed

    Halasi, Marianna; Pandit, Bulbul; Gartel, Andrei L

    2014-01-01

    Tumor suppressor p53 is one of the most frequently mutated genes in cancer, with almost 50% of all types of cancer expressing a mutant form of p53. p53 transactivates the expression of its primary negative regulator, HDM2. HDM2 is a ubiquitin ligase, which initiates the proteasomal degradation of p53 following ubiquitination. Proteasome inhibitors, by targeting the ubiquitin proteasome pathway inhibit the degradation of the majority of cellular proteins including wild-type p53. In contrast, in this study we found that the protein expression of mutant p53 was suppressed following treatment with established or novel proteasome inhibitors. Furthermore, for the first time we demonstrated that Arsenic trioxide, which was previously shown to suppress mutant p53 protein level, exhibits proteasome inhibitory activity. Proteasome inhibitor-mediated suppression of mutant p53 was partially rescued by the knockdown of HDM2, suggesting that the stabilization of HDM2 by proteasome inhibitors might be responsible for mutant p53 suppression to some extent. This study suggests that suppression of mutant p53 is a general property of proteasome inhibitors and it provides additional rationale to use proteasome inhibitors for the treatment of tumors with mutant p53.

  7. Proteasome inhibitors suppress the protein expression of mutant p53

    PubMed Central

    Halasi, Marianna; Pandit, Bulbul; Gartel, Andrei L

    2014-01-01

    Tumor suppressor p53 is one of the most frequently mutated genes in cancer, with almost 50% of all types of cancer expressing a mutant form of p53. p53 transactivates the expression of its primary negative regulator, HDM2. HDM2 is a ubiquitin ligase, which initiates the proteasomal degradation of p53 following ubiquitination. Proteasome inhibitors, by targeting the ubiquitin proteasome pathway inhibit the degradation of the majority of cellular proteins including wild-type p53. In contrast, in this study we found that the protein expression of mutant p53 was suppressed following treatment with established or novel proteasome inhibitors. Furthermore, for the first time we demonstrated that Arsenic trioxide, which was previously shown to suppress mutant p53 protein level, exhibits proteasome inhibitory activity. Proteasome inhibitor-mediated suppression of mutant p53 was partially rescued by the knockdown of HDM2, suggesting that the stabilization of HDM2 by proteasome inhibitors might be responsible for mutant p53 suppression to some extent. This study suggests that suppression of mutant p53 is a general property of proteasome inhibitors and it provides additional rationale to use proteasome inhibitors for the treatment of tumors with mutant p53. PMID:25485499

  8. Axons modulate the expression of proteolipid protein in the CNS.

    PubMed

    Scherer, S S; Vogelbacker, H H; Kamholz, J

    1992-06-01

    We examined the expression of mRNA encoding proteolipid protein (PLP), the major myelin protein in the CNS, in developing rat cerebrum, and in normal and degenerating optic nerves. PLP transcripts were initiated at two clusters of start sites that were separated by about 30 base pairs. During the peak of PLP mRNA expression in developing cerebrum, a higher proportion of PLP transcripts were initiated from the distal start site, furthest from the open reading frame, than in mature cerebrum. We enucleated one eye of immature rats to cause Wallerian degeneration in the optic nerve. In these degenerating optic nerves, the steady state levels of PLP mRNA fell markedly, and the proportion of distally initiated PLP transcripts declined to the same proportion found in normal adult nerves. Changes in myelin gene expression were not limited to PLP mRNA, as the steady-state levels of myelin basic protein (MBP) mRNA paralleled those of PLP mRNA in the developing cerebrum and in degenerating optic nerves. Thus, oligodendrocytes require axons to maintain their normal levels of PLP and MBP transcripts and the high proportion of distally initiated PLP transcripts that characterize early myelination.

  9. Mutational Analysis of the Rift Valley Fever Virus Glycoprotein Precursor Proteins for Gn Protein Expression

    PubMed Central

    Phoenix, Inaia; Lokugamage, Nandadeva; Nishiyama, Shoko; Ikegami, Tetsuro

    2016-01-01

    The Rift Valley fever virus (RVFV) M-segment encodes the 78 kD, NSm, Gn, and Gc proteins. The 1st AUG generates the 78 kD-Gc precursor, the 2nd AUG generates the NSm-Gn-Gc precursor, and the 3rd AUG makes the NSm’-Gn-Gc precursor. To understand biological changes due to abolishment of the precursors, we quantitatively measured Gn secretion using a reporter assay, in which a Gaussia luciferase (gLuc) protein is fused to the RVFV M-segment pre-Gn region. Using the reporter assay, the relative expression of Gn/gLuc fusion proteins was analyzed among various AUG mutants. The reporter assay showed efficient secretion of Gn/gLuc protein from the precursor made from the 2nd AUG, while the removal of the untranslated region upstream of the 2nd AUG (AUG2-M) increased the secretion of the Gn/gLuc protein. Subsequently, recombinant MP-12 strains encoding mutations in the pre-Gn region were rescued, and virological phenotypes were characterized. Recombinant MP-12 encoding the AUG2-M mutation replicated slightly less efficiently than the control, indicating that viral replication is further influenced by the biological processes occurring after Gn expression, rather than the Gn abundance. This study showed that, not only the abolishment of AUG, but also the truncation of viral UTR, affects the expression of Gn protein by the RVFV M-segment. PMID:27231931

  10. Efficient expression and purification of biologically active human cystatin proteins.

    PubMed

    Chauhan, Sakshi; Tomar, Raghuvir S

    2016-02-01

    Cystatins are reversible cysteine protease inhibitor proteins. They are known to play important roles in controlling cathepsins, neurodegenerative disease, and in immune system regulation. Production of recombinant cystatin proteins is important for biochemical and function characterization. In this study, we cloned and expressed human stefin A, stefin B and cystatin C in Escherichia coli. Human stefin A, stefin B and cystatin C were purified from soluble fraction. For cystatin C, we used various chaperone plasmids to make cystatin C soluble, as it is reported to localize in inclusion bodies. Trigger factor, GroES-GroEL, DnaK-DnaJ-GrpE chaperones lead to the presence of cystatin C in the soluble fraction. Immobilized metal affinity chromatography, glutathione sepharose and anion exchange chromatography techniques were employed for efficient purification of these proteins. Their biological activities were tested by inhibition assays against cathepsin L and H3 protease.

  11. The expression and induction of heat shock proteins in molluscs.

    PubMed

    Liu, Dongwu; Chen, Zhiwei

    2013-05-01

    Living cells respond to stress stimuli by triggering rapid changes in the protein profiles, and the induction of heat shock proteins (HSPs) plays an important part in this process. HSPs, mainly acting as molecular chaperones, are constitutively expressed in cells and involved in protein folding, assembly, degradation, and intracellular localization. The overexpression of HSPs represents a ubiquitous molecular mechanism to cope with stress. Compared to vertebrates, molluscs have a biphasic life cycle where pelagic larvae go through settlement and metamorphosis. HSPs may play an important role in the survival strategy of molluscs during the biphasic life stages. Since aquatic environments are highly dynamic, molluscs may be subject to a variety of sources of stress and HSPs might play a more important role in the adaptation of these animals. Moreover, the mechanisms of stress tolerance in molluscs can offer fundamental insights into the adaptation of organisms for a wide range of environmental challenges. The cDNA of HSPs has been cloned from some molluscs, and HSPs can be induced by heat stress, hypoxia, heavy metal contamination, and aestivation, etc. The expression of HSPs was detected in the neuroendocrine system, mollusc development, and reproductive process. Furthermore, the induction of HSPs is related with the phosphorylation of stress-activated p38 mitogen-activated protein kinase (p38 MAPK) and cJun-N-terminal kinases (JNKs) in molluscs.

  12. Grizzly bear corticosteroid binding globulin: Cloning and serum protein expression.

    PubMed

    Chow, Brian A; Hamilton, Jason; Alsop, Derek; Cattet, Marc R L; Stenhouse, Gordon; Vijayan, Mathilakath M

    2010-06-01

    Serum corticosteroid levels are routinely measured as markers of stress in wild animals. However, corticosteroid levels rise rapidly in response to the acute stress of capture and restraint for sampling, limiting its use as an indicator of chronic stress. We hypothesized that serum corticosteroid binding globulin (CBG), the primary transport protein for corticosteroids in circulation, may be a better marker of the stress status prior to capture in grizzly bears (Ursus arctos). To test this, a full-length CBG cDNA was cloned and sequenced from grizzly bear testis and polyclonal antibodies were generated for detection of this protein in bear sera. The deduced nucleotide and protein sequences were 1218 bp and 405 amino acids, respectively. Multiple sequence alignments showed that grizzly bear CBG (gbCBG) was 90% and 83% identical to the dog CBG nucleotide and amino acid sequences, respectively. The affinity purified rabbit gbCBG antiserum detected grizzly bear but not human CBG. There were no sex differences in serum total cortisol concentration, while CBG expression was significantly higher in adult females compared to males. Serum cortisol levels were significantly higher in bears captured by leg-hold snare compared to those captured by remote drug delivery from helicopter. However, serum CBG expression between these two groups did not differ significantly. Overall, serum CBG levels may be a better marker of chronic stress, especially because this protein is not modulated by the stress of capture and restraint in grizzly bears.

  13. Grizzly bear corticosteroid binding globulin: Cloning and serum protein expression.

    PubMed

    Chow, Brian A; Hamilton, Jason; Alsop, Derek; Cattet, Marc R L; Stenhouse, Gordon; Vijayan, Mathilakath M

    2010-06-01

    Serum corticosteroid levels are routinely measured as markers of stress in wild animals. However, corticosteroid levels rise rapidly in response to the acute stress of capture and restraint for sampling, limiting its use as an indicator of chronic stress. We hypothesized that serum corticosteroid binding globulin (CBG), the primary transport protein for corticosteroids in circulation, may be a better marker of the stress status prior to capture in grizzly bears (Ursus arctos). To test this, a full-length CBG cDNA was cloned and sequenced from grizzly bear testis and polyclonal antibodies were generated for detection of this protein in bear sera. The deduced nucleotide and protein sequences were 1218 bp and 405 amino acids, respectively. Multiple sequence alignments showed that grizzly bear CBG (gbCBG) was 90% and 83% identical to the dog CBG nucleotide and amino acid sequences, respectively. The affinity purified rabbit gbCBG antiserum detected grizzly bear but not human CBG. There were no sex differences in serum total cortisol concentration, while CBG expression was significantly higher in adult females compared to males. Serum cortisol levels were significantly higher in bears captured by leg-hold snare compared to those captured by remote drug delivery from helicopter. However, serum CBG expression between these two groups did not differ significantly. Overall, serum CBG levels may be a better marker of chronic stress, especially because this protein is not modulated by the stress of capture and restraint in grizzly bears. PMID:20347821

  14. Expression cloning of genes encoding human peroxisomal proteins

    SciTech Connect

    Spathaky, J.M.; Tate, A.W.; Cox, T.M.

    1994-09-01

    Numerous metabolic disorders associated with diverse peroxisomal defects have been identified but their molecular characterization has been hampered by difficulties associated with the purification of proteins from this fragile organelle. We have utilized antibodies directed against the C-terminal tripeptide peroxisomal targeting signal to detect hitherto unknown peroxisomal proteins in tissue fractions and to isolate genes encoding peroxisonal proteins from human expression libraries. We immunized rabbits with a peptide conjugate encompassing the C-terminal nine amino acids of rat peroxisomal acyl CoA oxidase. Immunoprecipitation assays using radio-labelled peptide showed that the antibody specifically recognizes the terminal SKL motif as well as C-terminal SHL and SRL but not SHL at an internal position. Affinity-purified antibody was used to probe Western blots of crude and peroxisome-enriched monkey liver preparations and detected 8-10 proteins specifically in the peroxisome fractions. 100 positive clones were identified on screening a human liver cDNA expression library in {lambda}-gt11. Sequence analysis has confirmed the identity of cDNA clones for human acyl CoA oxidase and epoxide hydrolase. Four clones show no sequence identity and their putative role in the human peroxisome is being explored.

  15. Phylogeny and expression of carbonic anhydrase-related proteins

    PubMed Central

    2010-01-01

    Background Carbonic anhydrases (CAs) are found in many organisms, in which they contribute to several important biological processes. The vertebrate α-CA family consists of 16 subfamilies, three of which (VIII, X and XI) consist of acatalytic proteins. These are named carbonic anhydrase related proteins (CARPs), and their inactivity is due to absence of one or more Zn-binding histidine residues. In this study, we analyzed and evaluated the distribution of genes encoding CARPs in different organisms using bioinformatic methods, and studied their expression in mouse tissues using immunohistochemistry and real-time quantitative PCR. Results We collected 84 sequences, of which 22 came from novel or improved gene models which we created from genome data. The distribution of CARP VIII covers vertebrates and deuterostomes, and CARP X appears to be universal in the animal kingdom. CA10-like genes have had a separate history of duplications in the tetrapod and fish lineages. Our phylogenetic analysis showed that duplication of CA10 into CA11 has occurred only in tetrapods (found in mammals, frogs, and lizards), whereas an independent duplication of CA10 was found in fishes. We suggest the name CA10b for the second fish isoform. Immunohistochemical analysis showed a high expression level of CARP VIII in the mouse cerebellum, cerebrum, and also moderate expression in the lung, liver, salivary gland, and stomach. These results also demonstrated low expression in the colon, kidney, and Langerhans islets. CARP X was moderately expressed in the cerebral capillaries and the lung and very weakly in the stomach and heart. Positive signals for CARP XI were observed in the cerebellum, cerebrum, liver, stomach, small intestine, colon, kidney, and testis. In addition, the results of real-time quantitative PCR confirmed a wide distribution for the Car8 and Car11 mRNAs, whereas the expression of the Car10 mRNA was restricted to the frontal cortex, parietal cortex, cerebellum, midbrain

  16. Expression of odorant-binding proteins and chemosensory proteins in some Hymenoptera.

    PubMed

    Calvello, M; Brandazza, A; Navarrini, A; Dani, F R; Turillazzi, S; Felicioli, A; Pelosi, P

    2005-04-01

    The expression of chemosensory proteins (CSPs) and odorant-binding proteins (OBPs) in individuals of different castes and ages have been monitored in three species of social hymenopterans, Polistes dominulus (Hymenoptera, Vespidae), Vespa crabro (Hymenoptera, Vespidae) and Apis mellifera (Hymenoptera, Apidae), using PCR with specific primers and polyclonal antibodies. In the paper wasp P. dominulus, OBP is equally expressed in antennae, wings and legs of all castes and ages, while CSP is often specifically present in antennae and in some cases also in legs. In the vespine species V. crabro CSP is antennal specific, while OBP is also expressed in legs and wings. The three CSPs and the five OBPs of A. mellifera show a complex pattern of expression, where both classes of proteins include members specifically expressed in antennae and others present in other parts of the body. These data indicate that at least in some hymenopteran species CSPs are specifically expressed in antennae and could perform roles in chemosensory perception so far assigned only to OBPs. PMID:15763466

  17. Abscisic acid (ABA) regulation of Arabidopsis SR protein gene expression.

    PubMed

    Cruz, Tiago M D; Carvalho, Raquel F; Richardson, Dale N; Duque, Paula

    2014-01-01

    Serine/arginine-rich (SR) proteins are major modulators of alternative splicing, a key generator of proteomic diversity and flexible means of regulating gene expression likely to be crucial in plant environmental responses. Indeed, mounting evidence implicates splicing factors in signal transduction of the abscisic acid (ABA) phytohormone, which plays pivotal roles in the response to various abiotic stresses. Using real-time RT-qPCR, we analyzed total steady-state transcript levels of the 18 SR and two SR-like genes from Arabidopsis thaliana in seedlings treated with ABA and in genetic backgrounds with altered expression of the ABA-biosynthesis ABA2 and the ABA-signaling ABI1 and ABI4 genes. We also searched for ABA-responsive cis elements in the upstream regions of the 20 genes. We found that members of the plant-specific SC35-Like (SCL) Arabidopsis SR protein subfamily are distinctively responsive to exogenous ABA, while the expression of seven SR and SR-related genes is affected by alterations in key components of the ABA pathway. Finally, despite pervasiveness of established ABA-responsive promoter elements in Arabidopsis SR and SR-like genes, their expression is likely governed by additional, yet unidentified cis-acting elements. Overall, this study pinpoints SR34, SR34b, SCL30a, SCL28, SCL33, RS40, SR45 and SR45a as promising candidates for involvement in ABA-mediated stress responses. PMID:25268622

  18. Cullin-3 protein expression levels correlate with breast cancer progression

    PubMed Central

    Haagenson, Kelly K.; Tait, Larry; Wang, Juan; Shekhar, Malathy P.; Polin, Lisa; Chen, Wei; Wu, Gen Sheng

    2012-01-01

    Cullin-3 is a component of the Cullin-Ring ubiquitin ligase (CRL) family that plays an important role in mediating protein degradation. Deregulation of Cullin-3 expression has been observed in human cancers; however, a role for Cullin-3 in tumor progression has not been previously recognized. Using the MCF10DCIS.com human breast cancer xenograft model, we show that Cullin-3 is increasingly expressed during progression from comedo ductal carcinoma in situ (DCIS) to invasive carcinomas. Cullin-3 protein is not detected in early lesions but is noticeably increased in DCIS tumors and significantly overexpressed in invasive cancers. In experimental metastasis assays, high expression of Cullin-3 was observed in the lung site. Importantly, Cullin-3 staining is detected in human breast cancer tissues, not in normal breast tissues and its expression level positively correlates with tumor stage. These data suggest that Cullin-3 may play an important role in tumor progression from DCIS to invasive cancer and may serve as a biomarker for the diagnosis of aggressive breast cancer. PMID:22825334

  19. Regulation of RAG-2 protein expression in avian thymocytes.

    PubMed Central

    Ferguson, S E; Accavitti, M A; Wang, D D; Chen, C L; Thompson, C B

    1994-01-01

    The recombinase-activating genes, RAG-1 and RAG-2, have been shown to be necessary to initiate the process of V(D)J recombination during the ontogeny of lymphocytes. While much is known about the end products of this rearrangement process, little is known about the function or regulation of the components of the recombinase system. To this end, we have generated a monoclonal antibody to the chicken RAG-2 protein. Chicken thymocytes were found to express high levels of RAG-2, part of which is phosphorylated. Within thymocytes, RAG-2 is expressed primarily within the nucleus. RAG-2 protein levels are high in the CD4- CD8- and CD4+ CD8+ immature thymocytes but absent at the single-positive CD4+ CD8- or CD4- CD8+ stage of thymocyte development. Mitogenic stimulation of thymocytes with phorbol myristate acetate and ionomycin results in down-regulation of RAG-2 expression. Consistent with these data, in vivo levels of RAG-2 are markedly lower in proliferating thymocytes than in smaller, G0/G1 cells. Down-regulation of RAG-2 expression appears to occur before cells enter S phase, suggesting that RAG-2 function may be limited to noncycling cells. Images PMID:7935443

  20. Abscisic Acid (ABA) Regulation of Arabidopsis SR Protein Gene Expression

    PubMed Central

    Cruz, Tiago M. D.; Carvalho, Raquel F.; Richardson, Dale N.; Duque, Paula

    2014-01-01

    Serine/arginine-rich (SR) proteins are major modulators of alternative splicing, a key generator of proteomic diversity and flexible means of regulating gene expression likely to be crucial in plant environmental responses. Indeed, mounting evidence implicates splicing factors in signal transduction of the abscisic acid (ABA) phytohormone, which plays pivotal roles in the response to various abiotic stresses. Using real-time RT-qPCR, we analyzed total steady-state transcript levels of the 18 SR and two SR-like genes from Arabidopsis thaliana in seedlings treated with ABA and in genetic backgrounds with altered expression of the ABA-biosynthesis ABA2 and the ABA-signaling ABI1 and ABI4 genes. We also searched for ABA-responsive cis elements in the upstream regions of the 20 genes. We found that members of the plant-specific SC35-Like (SCL) Arabidopsis SR protein subfamily are distinctively responsive to exogenous ABA, while the expression of seven SR and SR-related genes is affected by alterations in key components of the ABA pathway. Finally, despite pervasiveness of established ABA-responsive promoter elements in Arabidopsis SR and SR-like genes, their expression is likely governed by additional, yet unidentified cis-acting elements. Overall, this study pinpoints SR34, SR34b, SCL30a, SCL28, SCL33, RS40, SR45 and SR45a as promising candidates for involvement in ABA-mediated stress responses. PMID:25268622

  1. Purification by reflux electrophoresis of whey proteins and of a recombinant protein expressed in Dictyostelium discoideum.

    PubMed

    Corthals, G L; Collins, B M; Mabbutt, B C; Williams, K L; Gooley, A A

    1997-06-27

    Protein purification that combines the use of molecular mass exclusion membranes with electrophoresis is particularly powerful as it uses properties inherent to both techniques. The use of membranes allows efficient processing and is easily scaled up, while electrophoresis permits high resolution separation under mild conditions. The Gradiflow apparatus combines these two technologies as it uses polyacrylamide membranes to influence electrokinetic separations. The reflux electrophoresis process consists of a series of cycles incorporating a forward phase and a reverse phase. The forward phase involves collection of a target protein that passes through a separation membrane before trailing proteins in the same solution. The forward phase is repeated following clearance of the membrane in the reverse phase by reversing the current. We have devised a strategy to establish optimal reflux separation parameters, where membranes are chosen for a particular operating range and protein transfer is monitored at different pH values. In addition, forward and reverse phase times are determined during this process. Two examples of the reflux method are described. In the first case, we described the purification strategy for proteins from a complex mixture which contains proteins of higher electrophoretic mobility than the target protein. This is a two-step procedure, where first proteins of higher mobility than the target protein are removed from the solution by a series of reflux cycles, so that the target protein remains as the leading fraction. In the second step the target protein is collected, as it has become the leading fraction of the remaining proteins. In the second example we report the development of a reflux strategy which allowed a rapid one-step preparative purification of a recombinant protein, expressed in Dictyostelium discoideum. These strategies demonstrate that the Gradiflow is amenable to a wide range of applications, as the protein of interest is not

  2. Expression and Localization of Lung Surfactant Proteins in Human Testis

    PubMed Central

    Wagner, Walter; Matthies, Cord; Ruf, Christian; Hartmann, Arndt; Garreis, Fabian; Paulsen, Friedrich

    2015-01-01

    Background Surfactant proteins (SPs) have been described in various tissues and fluids including tissues of the nasolacrimal apparatus, airways and digestive tract. Human testis have a glandular function as a part of the reproductive and the endocrine system, but no data are available on SPs in human testis and prostate under healthy and pathologic conditions. Objective The aim of the study was the detection and characterization of the surfactant proteins A, B, C and D (SP-A, SP-B, SP-C, SP-D) in human testis. Additionally tissue samples affected by testicular cancer were investigated. Results Surfactant proteins A, B, C and D were detected using RT-PCR in healthy testis. By means of Western blot analysis, these SPs were detected at the protein level in normal testis, seminoma and seminal fluid, but not in spermatozoa. Expression of SPs was weaker in seminoma compared to normal testicular tissue. SPs were localized in combination with vimentin immunohistochemically in cells of Sertoli and Leydig. Conclusion Surfactant proteins seem to be inherent part of the human testis. By means of physicochemical properties the proteins appear to play a role during immunological and rheological process of the testicular tissue. The presence of SP-B and SP-C in cells of Sertoli correlates with their function of fluid secretion and may support transportation of spermatozoa. In seminoma the expression of all SP's was generally weaker compared to normal germ cells. This could lead to a reduction of immunomodulatory and rheology processes in the germ cell tumor. PMID:26599233

  3. Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production.

    PubMed

    Daly, Rachel; Hearn, Milton T W

    2005-01-01

    The use of the methylotrophic yeast, Pichia pastoris, as a cellular host for the expression of recombinant proteins has become increasing popular in recent times. P. pastoris is easier to genetically manipulate and culture than mammalian cells and can be grown to high cell densities. Equally important, P. pastoris is also a eukaryote, and thereby provides the potential for producing soluble, correctly folded recombinant proteins that have undergone all the post-translational modifications required for functionality. Additionally, linearized foreign DNA can be inserted in high efficiency via homologous recombination procedures to generate stable cell lines whilst expression vectors can be readily prepared that allow multiple copies of the target protein, multimeric proteins with different subunit structures, or alternatively the target protein and its cognate binding partners, to be expressed. A further benefit of the P. pastoris system is that strong promoters are available to drive the expression of a foreign gene(s) of interest, thus enabling production of large amounts of the target protein(s) with relative technical ease and at a lower cost than most other eukaryotic systems. The purpose of this review is to summarize important developments and features of this expression system and, in particular, to examine from an experimental perspective the genetic engineering, protein chemical and molecular design considerations that have to be taken into account for the successful expression of the target recombinant protein. Included in these considerations are the influences of P. pastoris strain selection; the choice of expression vectors and promoters; procedures for the transformation and integration of the vectors into the P. pastoris genome; the consequences of rare codon usage and truncated transcripts; and techniques employed to achieve multi-copy integration numbers. The impact of the alcohol oxidase (AOX) pathways in terms of the mut+ and mut(s) phenotypes

  4. Establishment of Salvia castanea Diels f. tomentosa Stib. hairy root cultures and the promotion of tanshinone accumulation and gene expression with Ag⁺, methyl jasmonate, and yeast extract elicitation.

    PubMed

    Li, Bo; Wang, Bangqing; Li, Hongyan; Peng, Liang; Ru, Mei; Liang, Zongsuo; Yan, Xijun; Zhu, Yonghong

    2016-01-01

    Salvia castanea Diels f. tomentosa Stib. is an endemic medicinal plant distributed in China, and the notably high content of tanshinone IIA in the root is proven effective for the therapy of heart diseases. Hairy root induction of this Salvia species was inoculated with Agrobacterium rhizogenes strain ATCC 15834. Transformed hairy root was cultured in 6,7-V liquid medium for growth kinetics assessment and elicitation. An S curve was present in the hairy root cultures based on the fresh and dry weights with an interval of 3 days. An optimum concentration of the applied elicitors (15 μM Ag(+), 200 μM methyl jasmonate, and 200 mg l(-1) yeast extract elicitor) benefitted both the growth status and tanshinone accumulation in the hairy root cultures. Tanshinone IIA contents were mostly stimulated 1.8-fold and 1.99-fold compared with the control by Ag(+) and methyl jasmonate elicitation, respectively. Yeast extract dramatically enhanced dry mass accumulation, while it promoted cryptotanshinone content of 2.84 ± 0.33 mg g(-1) dry weight at most in the hairy root cultures. Selected elicitors diversely influenced tanshinone accumulation in the time courses of hairy root cultures within 7 days. Furthermore, transcripts of selected genes in the tanshinone biosynthetic pathway were remarkably upregulated with elicitation. Yeast extract elicitor heightened 13.9-fold of isopentenyl diphosphate isomerase expression level at 12 h, while it increased 16.7-fold of geranylgeranyl diphosphate synthase transcript at 24 h compared with that of the control, which was more effective than Ag(+) and methyl jasmonate. This study provided a convenient hairy root culture system of S. castanea Diels f. tomentosa Stib. for tanshinone production for the first time.

  5. Identification of differentially expressed serum proteins in gastric adenocarcinoma☆

    PubMed Central

    Subbannayya, Yashwanth; Mir, Sartaj Ahmad; Renuse, Santosh; Manda, Srikanth S.; Pinto, Sneha M.; Puttamallesh, Vinuth N.; Solanki, Hitendra Singh; Manju, H.C.; Syed, Nazia; Sharma, Rakesh; Christopher, Rita; Vijayakumar, M.; Kumar, K.V. Veerendra; Prasad, T.S. Keshava; Ramaswamy, Girija; Kumar, Rekha V.; Chatterjee, Aditi; Pandey, Akhilesh; Gowda, Harsha

    2015-01-01

    Gastric adenocarcinoma is an aggressive cancer with poor prognosis. Blood based biomarkers of gastric cancer have the potential to improve diagnosis and monitoring of these tumors. Proteins that show altered levels in the circulation of gastric cancer patients could prove useful as putative biomarkers. We used an iTRAQ-based quantitative proteomic approach to identify proteins that show altered levels in the sera of patients with gastric cancer. Our study resulted in identification of 643 proteins, of which 48 proteins showed increased levels and 11 proteins showed decreased levels in serum from gastric cancer patients compared to age and sex matched healthy controls. Proteins that showed increased expression in gastric cancer included inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), Mannose-binding protein C (MBL2), sex hormone-binding globulin (SHBG), insulin-like growth factor-binding protein 2 (IGFBP2), serum amyloid A protein (SAA1), Orosomucoid 1 (ORM1) and extracellular superoxide dismutase [Cu–Zn] (SOD3). We used multiple reaction monitoring assays and validated elevated levels of ITIH4 and SAA1 proteins in serum from gastric cancer patients. Biological significance Gastric cancer is a highly aggressive cancer associated with high mortality. Serum-based biomarkers are of considerable interest in diagnosis and monitoring of various diseases including cancers. Gastric cancer is often diagnosed at advanced stages resulting in poor prognosis and high mortality. Pathological diagnosis using biopsy specimens remains the gold standard for diagnosis of gastric cancer. Serum-based biomarkers are of considerable importance as they are minimally invasive. In this study, we carried out quantitative proteomic profiling of serum from gastric cancer patients to identify proteins that show altered levels in gastric cancer patients. We identified more than 50 proteins that showed altered levels in gastric cancer patient sera. Validation in a large cohort of well

  6. An inducible expression system for high-level expression of recombinant proteins in slow growing mycobacteria.

    PubMed

    Leotta, Lisa; Spratt, Joanne M; Kong, Carlyn U; Triccas, James A

    2015-09-01

    A novel protein expression vector utilising the inducible hspX promoter of Mycobacterium tuberculosis was constructed and evaluated in this study. High-level induction of three mycobacterial antigens, comprising up to 9% of bacterial sonicate, was demonstrated in recombinant Mycobacterium bovis BCG when grown under low-oxygen tension, which serves to enhance hspX promoter activity. Recombinant proteins were efficiently purified from bacterial lysates in a soluble form by virtue of a C-terminal 6-histidine tag. Purification of the immunodominant M. tuberculosis Ag85B antigen using this system resulted in a recombinant protein that stimulated significant IFN-γ release from Ag85B-reactive T cells generated after vaccination of mice with an Ag85B-expressing vaccine. Further, the M. tuberculosis L-alanine dehydrogenase (Ald) protein purified from recombinant BCG displayed strong enzymatic activity in recombinant form. This study demonstrated that high levels of native-like recombinant mycobacterial proteins can be produced in mycobacterial hosts, and this may aid the analysis of mycobacterial protein function and the development of new treatments. PMID:26021569

  7. Protein body formation in stable transgenic tobacco expressing elastin-like polypeptide and hydrophobin fusion proteins

    PubMed Central

    2013-01-01

    Background Plants are recognized as an efficient and inexpensive system to produce valuable recombinant proteins. Two different strategies have been commonly used for the expression of recombinant proteins in plants: transient expression mediated by Agrobacterium; or stable transformation of the plant genome. However, the use of plants as bioreactors still faces two main limitations: low accumulation levels of some recombinant proteins and lack of efficient purification methods. Elastin-like polypeptide (ELP), hydrophobin I (HFBI) and Zera® are three fusion partners found to increase the accumulation levels of recombinant proteins and induce the formation of protein bodies (PBs) in leaves when targeted to the endoplasmic reticulum (ER) in transient expression assays. In this study the effects of ELP and HFBI fusion tags on recombinant protein accumulation levels and PB formation was examined in stable transgenic Nicotiana tabacum. Results The accumulation of recombinant protein and PB formation was evaluated in two cultivars of Nicotiana tabacum transformed with green fluorescent protein (GFP) fused to ELP or HFBI, both targeted and retrieved to the ER. The ELP and HFBI tags increased the accumulation of the recombinant protein and induced the formation of PBs in leaves of stable transgenic plants from both cultivars. Furthermore, these tags induced the formation of PBs in a concentration-dependent manner, where a specific level of recombinant protein accumulation was required for PBs to appear. Moreover, agro-infiltration of plants accumulating low levels of recombinant protein with p19, a suppressor of post-transcriptional gene silencing (PTGS), increased accumulation levels in four independent transgenic lines, suggesting that PTGS might have caused the low accumulation levels in these plants. Conclusion The use of ELP and HFBI tags as fusion partners in stable transgenic plants of tobacco is feasible and promising. In a constitutive environment, these tags

  8. Expression of P53 protein after exposure to ionizing radiation

    NASA Astrophysics Data System (ADS)

    Salazar, A. M.; Salvador, C.; Ruiz-Trejo, C.; Ostrosky, P.; Brandan, M. E.

    2001-10-01

    One of the most important tumor suppressor genes is p53 gene, which is involved in apoptotic cell death, cell differentiation and cell cycle arrest. The expression of p53 gene can be evaluated by determining the presence of P53 protein in cells using Western Blot assay with a chemiluminescent method. This technique has shown variabilities that are due to biological factors. Film developing process can influence the quality of the p53 bands obtained. We irradiated tumor cell lines and human peripheral lymphocytes with 137Cs and 60Co gamma rays to standardize irradiation conditions, to compare ionizing radiation with actinomycin D and to reduce the observed variability of P53 protein induction levels. We found that increasing radiation doses increase P53 protein induction while it decreases viability. We also conclude that ionizing radiation could serve as a positive control for Western Blot analysis of protein P53. In addition, our results show that the developing process may play an important role in the quality of P53 protein bands and data interpretation.

  9. Protein expression and characterization of SEP3 from Arabidopsis thaliana.

    PubMed

    Shi, Q; Zhou, J; Wang, P; Lin, X; Xu, Y

    2015-01-01

    SEPALLATA (SEP) MADS-box genes play crucial roles in the regulation of floral growth and development. They are required for the specification of sepals, petals, stamens, and carpels as well as for floral determinacy. SEPs perform their functions through the formation of homo- or hetero-polymers, which are the molecular basis of floral quartets. In vitro assays indicated that SEP3 forms a tetramer after binding to DNA, but it is unclear whether DNA binding induces the tetramer, because SEP3 is often reported to form a dimer. Here, we analyzed the oligomeric status of SEP3 domains in the absence of the DNA-binding MADS-box domain. The truncated SEP3 was constructed as a fusion protein and expressed in prokaryotic cells. The purified protein fragment displayed as a tetramer in the size exclusion chromatographic column, and a glutaraldehyde cross-linking assay demonstrated that the protein contained a dimer unit. Yeast two-hybrid tests further verified that the fragments form homologous polymers in vivo, and that the K domain is involved in tetramer formation. Current results imply that the SEP3 protein regulates the formation of flower meristems using the tetramer as a unit, and that the DNA-binding MADS-box is dispensable for polymer formation. The C-terminal region does not contribute to homo-tetramer formation, but it may be reserved to glue other proteins. PMID:26505403

  10. Resveratrol upregulates Egr-1 expression and activity involving extracellular signal-regulated protein kinase and ternary complex factors

    SciTech Connect

    Rössler, Oliver G.; Glatzel, Daniel; Thiel, Gerald

    2015-03-01

    Many intracellular functions have been attributed to resveratrol, a polyphenolic phytoalexin found in grapes and in other plants. Here, we show that resveratrol induces the expression of the transcription factor Egr-1 in human embryonic kidney cells. Using a chromosomally embedded Egr-1-responsive reporter gene, we show that the Egr-1 activity was significantly elevated in resveratrol-treated cells, indicating that the newly synthesized Egr-1 protein was biologically active. Stimulus-transcription coupling leading to the resveratrol-induced upregulation of Egr-1 expression and activity requires the protein kinases Raf and extracellular signal-regulated protein kinase ERK, while MAP kinase phosphatase-1 functions as a nuclear shut-off device that interrupts the signaling cascade connecting resveratrol stimulation with enhanced Egr-1 expression. On the transcriptional level, Elk-1, a key transcriptional regulator of serum response element-driven gene transcription, connects the intracellular signaling cascade elicited by resveratrol with transcription of the Egr-1 gene. These data were corroborated by the observation that stimulation of the cells with resveratrol increased the transcriptional activation potential of Elk-1. The SRE as well as the GC-rich DNA binding site of Egr-1 function as resveratrol-responsive elements. Thus, resveratrol regulates gene transcription via activation of the stimulus-regulated protein kinases Raf and ERK and the stimulus-responsive transcription factors TCF and Egr-1. - Highlights: • The plant polyphenol resveratrol upregulates Egr-1 expression and activity. • The stimulation of Egr-1 requires the protein kinases ERK and Raf. • Resveratrol treatment upregulates the transcriptional activation potential of Elk-1. • Resveratrol-induced stimulation of Egr-1 requires ternary complex factors. • Two distinct resveratrol-responsive elements were identified.

  11. Cyclin D1 expression is regulated by the retinoblastoma protein.

    PubMed Central

    Müller, H; Lukas, J; Schneider, A; Warthoe, P; Bartek, J; Eilers, M; Strauss, M

    1994-01-01

    The product of the retinoblastoma susceptibility gene, pRb, acts as a tumor suppressor and loss of its function is involved in the development of various types of cancer. DNA tumor viruses are supposed to disturb the normal regulation of the cell cycle by inactivating pRb. However, a direct function of pRb in regulation of the cell cycle has hitherto not been shown. We demonstrate here that the cell cycle-dependent expression of one of the G1-phase cyclins, cyclin D1, is dependent on the presence of a functional Rb protein. Rb-deficient tumor cell lines as well as cells expressing viral oncoproteins (large tumor antigen of simian virus 40, early region 1A of adenovirus, early region 7 of papillomavirus) have low or barely detectable levels of cyclin D1. Expression of cyclin D1, but not of cyclins A and E, is induced by transfection of the Rb gene into Rb-deficient tumor cells. Cotransfection of a reporter gene under the control of the D1 promoter, together with the Rb gene, into Rb-deficient cell lines demonstrates stimulation of the D1 promoter by Rb, which parallels the stimulation of endogenous cyclin D1 gene expression. Our finding that pRb stimulates expression of a key component of cell cycle control, cyclin D1, suggests the existence of a regulatory loop between pRb and cyclin D1 and extends existing models of tumor suppressor function. Images PMID:8159685

  12. A molecular clock regulates angiopoietin-like protein 2 expression.

    PubMed

    Kadomatsu, Tsuyoshi; Uragami, Shota; Akashi, Makoto; Tsuchiya, Yoshiki; Nakajima, Hiroo; Nakashima, Yukiko; Endo, Motoyoshi; Miyata, Keishi; Terada, Kazutoyo; Todo, Takeshi; Node, Koichi; Oike, Yuichi

    2013-01-01

    Various physiological and behavioral processes exhibit circadian rhythmicity. These rhythms are usually maintained by negative feedback loops of core clock genes, namely, CLOCK, BMAL, PER, and CRY. Recently, dysfunction in the circadian clock has been recognized as an important foundation for the pathophysiology of lifestyle-related diseases, such as obesity, cardiovascular disease, and some cancers. We have reported that angiopoietin-like protein 2 (ANGPTL2) contributes to the pathogenesis of these lifestyle-related diseases by inducing chronic inflammation. However, molecular mechanisms underlying regulation of ANGPTL2 expression are poorly understood. Here, we assess circadian rhythmicity of ANGPTL2 expression in various mouse tissues. We observed that ANGPTL2 rhythmicity was similar to that of the PER2 gene, which is regulated by the CLOCK/BMAL1 complex. Promoter activity of the human ANGPTL2 gene was significantly induced by CLOCK and BMAL1, an induction markedly attenuated by CRY co-expression. We also identified functional E-boxes in the ANGPTL2 promoter and observed occupancy of these sites by endogenous CLOCK in human osteosarcoma cells. Furthermore, Cry-deficient mice exhibited arrhythmic Angptl2 expression. Taken together, these data suggest that periodic expression of ANGPTL2 is regulated by a molecular clock.

  13. Vaccine-Elicited V3 Loop-Specific Antibodies in Rhesus Monkeys and Control of a Simian-Human Immunodeficiency Virus Expressing a Primary Patient Human Immunodeficiency Virus Type 1 Isolate Envelope

    PubMed Central

    Letvin, Norman L.; Robinson, Suzanne; Rohne, Daniela; Axthelm, Michael K.; Fanton, John W.; Bilska, Miroslawa; Palker, Thomas J.; Liao, Hua-Xin; Haynes, Barton F.; Montefiori, David C.

    2001-01-01

    Vaccine-elicited antibodies specific for the third hypervariable domain of the surface gp120 of human immunodeficiency virus type 1 (HIV-1) (V3 loop) were assessed for their contribution to protection against infection in the simian-human immunodeficiency virus (SHIV)/rhesus monkey model. Peptide vaccine-elicited anti-V3 loop antibody responses were examined for their ability to contain replication of SHIV-89.6, a nonpathogenic SHIV expressing a primary patient isolate HIV-1 envelope, as well as SHIV-89.6P, a pathogenic variant of that virus. Low-titer neutralizing antibodies to SHIV-89.6 that provided partial protection against viremia following SHIV-89.6 infection were generated. A similarly low-titer neutralizing antibody response to SHIV-89.6P that did not contain viremia after infection with SHIV-89.6P was generated, but a trend toward protection against CD4+ T-lymphocyte loss was seen in these infected monkeys. These observations suggest that the V3 loop on some primary patient HIV-1 isolates may be a partially effective target for neutralizing antibodies induced by peptide immunogens. PMID:11287566

  14. Expression of recombinant green fluorescent protein in Bacillus methanolicus.

    PubMed

    Nilasari, Dewi; Dover, Nir; Rech, Sabine; Komives, Claire

    2012-01-01

    Microbial biocatalysts are used in a wide range of industries to produce large scale quantities of proteins, amino acids, and commodity chemicals. While the majority of these processes use glucose or other low-cost sugars as the substrate, Bacillus methanolicus is one example of a biocatalyst that has shown sustained growth on methanol as a carbon source at elevated temperature (50-53°C optimum) resulting in reduced feed and utility costs. Specifically, the complete chemical process enabled by this approach takes methane from natural gas, and following a low-cost conversion to methanol, can be used for the production of high value products. In this study, production of recombinant green fluorescent protein (GFPuv) by B. methanolicus is explored. A plasmid was constructed that incorporates the methanol dehydrogenase (mdh) promoter of B. methanolicus MGA3 together with the GFPuv gene. The plasmid, pNW33N, was shown to be effective for expression in other Bacillus strains, although not previously in B. methanolicus. A published electroporation protocol for transformation of B. methanolicus was modified to result in expression of GFP using plasmid pNW33N-mdh-GFPuv (pNmG). Transformation was confirmed by both agarose gel electrophoresis and by observation of green fluorescence under UV light exposure. The mass yield of cells and protein were measured in shake flask experiments. The optimum concentration of methanol for protein production was found to be at 200 mM. Higher concentrations than 200 mM resulted in slightly higher biomass production but lower amounts of recombinant protein. PMID:22275315

  15. Bacteria expressed hepatitis E virus capsid proteins maintain virion-like epitopes.

    PubMed

    Wei, Minxi; Zhang, Xiao; Yu, Hai; Tang, Zi-Min; Wang, Kaihang; Li, Zhongyi; Zheng, Zizheng; Li, Shaowei; Zhang, Jun; Xia, Ningshao; Zhao, Qinjian

    2014-05-19

    The protein encoded by ORF2 in hepatitis E virus (HEV) is the only capsid protein for this single-stranded RNA virus. It was previously shown that 148 aa (aa 459-606) was needed for dimer formation, whereas 239 aa (aa 368-606) was necessary to form virus-like particles (VLPs). The self-assembled VLPs of p239 were characterized with a series of methods including high performance size-exclusion chromatography to demonstrate the particulate nature of purified and properly refolded p239. A neutralizing and protective mouse monoclonal antibody (mAb) 8C11 was previously shown to bind three discontinuous peptide segments in the dimer. In addition to the good binding activity to recombinant dimeric form, E2s or E2, and VLP form p239, we demonstrated that 8C11 was able to capture the authentic HEV virions. The capability of virus capturing was demonstrated with a titration curve from 10(5) to 10(7) HEV genome copies, making binding activity to 8C11 a surrogate marker of virion-like epitopes on recombinant VLPs as well as vaccine efficacy in eliciting protective and neutralizing antibodies. Taken together, it was demonstrated that Escherichia coli expressed pORF2 proteins, p239 in particular, maintain the virion-like epitopes on VLP surface. This is consistent with the fact that p239 was demonstrated to be an effective prophylactic vaccine (recently licensed as Hecolin(®) in China) against HEV-induced hepatitis in a large scale clinical trial.

  16. Blue Light Modulates Murine Microglial Gene Expression in the Absence of Optogenetic Protein Expression

    PubMed Central

    Cheng, Kevin P.; Kiernan, Elizabeth A.; Eliceiri, Kevin W.; Williams, Justin C.; Watters, Jyoti J.

    2016-01-01

    Neural optogenetic applications over the past decade have steadily increased; however the effects of commonly used blue light paradigms on surrounding, non-optogenetic protein-expressing CNS cells are rarely considered, despite their simultaneous exposure. Here we report that blue light (450 nm) repetitively delivered in both long-duration boluses and rapid optogenetic bursts gene-specifically altered basal expression of inflammatory and neurotrophic genes in immortalized and primary murine wild type microglial cultures. In addition, blue light reduced pro-inflammatory gene expression in microglia activated with lipopolysaccharide. These results demonstrate previously unreported, off-target effects of blue light in cells not expressing optogenetic constructs. The unexpected gene modulatory effects of blue light on wild type CNS resident immune cells have novel and important implications for the neuro-optogenetic field. Further studies are needed to elucidate the molecular mechanisms and potential therapeutic utility of blue light modulation of the wild type CNS. PMID:26883795

  17. Heat shock protein 70-hom gene polymorphism and protein expression in multiple sclerosis.

    PubMed

    Boiocchi, C; Monti, M C; Osera, C; Mallucci, G; Pistono, C; Ferraro, O E; Nosari, G; Romani, A; Cuccia, M; Govoni, S; Pascale, A; Montomoli, C; Bergamaschi, R

    2016-09-15

    Immune-mediated and neurodegenerative mechanisms are involved in multiple sclerosis (MS). Growing evidences highlight the role of HSP70 genes in the susceptibility of some neurological diseases. In this explorative study we analyzed a polymorphism (i.e. HSP70-hom rs2227956) of the gene HSPA1L, which encodes for the protein hsp70-hom. We sequenced the polymorphism by polymerase chain reaction (PCR), in 191 MS patients and 365 healthy controls. The hsp70-hom protein expression was quantified by western blotting. We reported a strong association between rs2227956 polymorphism and MS risk, which is independent from the association with HSP70-2 rs1061581, and a significant link between hsp70-hom protein expression and MS severity. PMID:27609295

  18. Simvastatin enhances bone morphogenetic protein receptor type II expression

    SciTech Connect

    Hu Hong; Sung, Arthur; Zhao, Guohua; Shi, Lingfang; Qiu Daoming; Nishimura, Toshihiko; Kao, Peter N. . E-mail: peterkao@stanford.edu

    2006-01-06

    Statins confer therapeutic benefits in systemic and pulmonary vascular diseases. Bone morphogenetic protein (BMP) receptors serve essential signaling functions in cardiovascular development and skeletal morphogenesis. Mutations in BMP receptor type II (BMPR2) are associated with human familial and idiopathic pulmonary arterial hypertension, and pathologic neointimal proliferation of vascular endothelial and smooth muscle cells within small pulmonary arteries. In severe experimental pulmonary hypertension, simvastatin reversed disease and conferred a 100% survival advantage. Here, modulation of BMPR2 gene expression by simvastatin is characterized in human embryonic kidney (HEK) 293T, pulmonary artery smooth muscle, and lung microvascular endothelial cells (HLMVECs). A 1.4 kb BMPR2 promoter containing Egr-1 binding sites confers reporter gene activation in 293T cells which is partially inhibited by simvastatin. Simvastatin enhances steady-state BMPR2 mRNA and protein expression in HLMVEC, through posttranscriptional mRNA stabilization. Simvastatin induction of BMPR2 expression may improve BMP-BMPR2 signaling thereby enhancing endothelial differentiation and function.

  19. Transformation of Escherichia coli and protein expression using lipoplex mimicry.

    PubMed

    Yun, Chul-Ho; Bae, Chun-Sik; Ahn, Taeho

    2016-11-01

    We investigated a "one-step" method for transformation of and protein expression in Escherichia coli (E. coli) using a complex of n-stearylamine, a cationic lipid, and plasmid DNA, which mimics lipoplex-based approaches. When E. coli cells were treated with the cationic lipid-plasmid complex, the transformation efficiencies were in the range of approximately 2-3 × 10(6) colony-forming units. Further increase in the efficiency was obtained by co-treatment with calcium chloride (or rubidium chloride) and the complexes. Moreover, after DNA transfer, E. coli cells successfully expressed plasmid-encoded proteins such as cytochrome P450s and glutathione-S-transferase without overnight incubation of the cells to form colonies, an indispensable step in other bacterial transformation methods. In this study, we provide a simple method for E. coli transformation, which does not require the preparation of competent cells. The present method also shortens the overall procedures for transformation and gene expression in E. coli by omitting the colony-forming step. PMID:27416742

  20. Transformation of Escherichia coli and protein expression using lipoplex mimicry.

    PubMed

    Yun, Chul-Ho; Bae, Chun-Sik; Ahn, Taeho

    2016-11-01

    We investigated a "one-step" method for transformation of and protein expression in Escherichia coli (E. coli) using a complex of n-stearylamine, a cationic lipid, and plasmid DNA, which mimics lipoplex-based approaches. When E. coli cells were treated with the cationic lipid-plasmid complex, the transformation efficiencies were in the range of approximately 2-3 × 10(6) colony-forming units. Further increase in the efficiency was obtained by co-treatment with calcium chloride (or rubidium chloride) and the complexes. Moreover, after DNA transfer, E. coli cells successfully expressed plasmid-encoded proteins such as cytochrome P450s and glutathione-S-transferase without overnight incubation of the cells to form colonies, an indispensable step in other bacterial transformation methods. In this study, we provide a simple method for E. coli transformation, which does not require the preparation of competent cells. The present method also shortens the overall procedures for transformation and gene expression in E. coli by omitting the colony-forming step.

  1. Non-additive hepatic gene expression elicited by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 2,2',4,4',5,5'-hexachlorobiphenyl (PCB153) co-treatment in C57BL/6 mice

    SciTech Connect

    Kopec, Anna K.; D'Souza, Michelle L.; Mets, Bryan D.; Burgoon, Lyle D.; Reese, Sarah E.; Archer, Kellie J.; Potter, Dave; Tashiro, Colleen; Sharratt, Bonnie; Harkema, Jack R.; Zacharewski, Timothy R.

    2011-10-15

    Interactions between environmental contaminants can lead to non-additive effects that may affect the toxicity and risk assessment of a mixture. Comprehensive time course and dose-response studies with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), non-dioxin-like 2,2',4,4',5,5'-hexachlorobiphenyl (PCB153) and their mixture were performed in immature, ovariectomized C57BL/6 mice. Mice were gavaged once with 30 {mu}g/kg TCDD, 300 mg/kg PCB153, a mixture of 30 {mu}g/kg TCDD with 300 mg/kg PCB153 (MIX) or sesame oil vehicle for 4,12, 24,72 or 168 h. In the 24 h dose-response study, animals were gavaged with TCDD (0.3,1, 3, 6, 10, 15, 30, 45 {mu}g/kg), PCB153 (3,10, 30, 60, 100, 150, 300, 450 mg/kg), MIX (0.3 + 3, 1 + 10, 3 + 30, 6 + 60, 10 + 100, 15 + 150, 30 + 300, 45 {mu}g/kg TCDD + 450 mg/kg PCB153, respectively) or vehicle. All three treatments significantly increased relative liver weights (RLW), with MIX eliciting significantly greater increases compared to TCDD and PCB153 alone. Histologically, MIX induced hepatocellular hypertrophy, vacuolization, inflammation, hyperplasia and necrosis, a combination of TCDD and PCB153 responses. Complementary lipid analyses identified significant increases in hepatic triglycerides in MIX and TCDD samples, while PCB153 had no effect on lipids. Hepatic PCB153 levels were also significantly increased with TCDD co-treatment. Microarray analysis identified 167 TCDD, 185 PCB153 and 388 MIX unique differentially expressed genes. Statistical modeling of quantitative real-time PCR analysis of Pla2g12a, Serpinb6a, Nqo1, Srxn1, and Dysf verified non-additive expression following MIX treatment compared to TCDD and PCB153 alone. In summary, TCDD and PCB153 co-treatment elicited specific non-additive gene expression effects that are consistent with RLW increases, histopathology, and hepatic lipid accumulation. - Graphical abstract: Display Omitted Highlights: > MIX (TCDD:PCB153 at 1:10,000 ratio) exposure leads to non-additive gene expression

  2. A single bolus meal elicits different regulation of protein synthesis and mTOR signaling in visceral tissues and skeletal muscle of newborn pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Neonates, a highly anabolic population, use dietary amino acids efficiently for growth because they can increase protein synthesis in response to feeding to a greater extent than mature animals. The enhancement of protein synthesis after a meal in neonatal pigs is well known, but the time course of ...

  3. Streptococcus pneumoniae fructose-1,6-bisphosphate aldolase, a protein vaccine candidate, elicits Th1/Th2/Th17-type cytokine responses in mice.

    PubMed

    Elhaik Goldman, Shirin; Dotan, Shahar; Talias, Amir; Lilo, Amit; Azriel, Shalhevet; Malka, Itay; Portnoi, Maxim; Ohayon, Ariel; Kafka, Daniel; Ellis, Ronald; Elkabets, Moshe; Porgador, Angel; Levin, Ditza; Azhari, Rosa; Swiatlo, Edwin; Ling, Eduard; Feldman, Galia; Tal, Michael; Dagan, Ron; Mizrachi Nebenzahl, Yaffa

    2016-04-01

    Streptococcus pneumoniae (S. pneumoniae) is a major pathogen worldwide. The currently available polysaccharide-based vaccines significantly reduce morbidity and mortality. However, the inherent disadvantages of the currently available polysaccharide-based vaccines have motivated the search for other bacterial immunogens capable of eliciting a protective immune response against S. pneumoniae. Fructose-1,6-bisphosphate aldolase (FBA) is a glycolytic enzyme, which was found to localize to the bacterial surface, where it functions as an adhesin. Previously, immunizing mice with recombinant FBA (rFBA) in the presence of alum elicited a protective immune response against a lethal challenge with S. pneumoniae. Thus, the aim of the present study was to determine the cytokine responses that are indicative of protective immunity following immunization with rFBA. The protective effects against pneumococcal challenge in mice immunized with rFBA with complete Freund's adjuvant (CFA) in the initial immunization and with incomplete Freund's adjuvant (IFA) in booster immunizations surpassed the protective effects observed following immunization with either rFBA + alum or pVACfba. CD4+ T-cells obtained from the rFBA/CFA/IFA/IFA-immunized mice co-cultured with rFBA-pulsed antigen-presenting cells (APCs), exhibited a significantly greater proliferative ability than CD4+ T-cells obtained from the adjuvant-immunized mice co-cultured with rFBA‑pulsed APCs. The levels of the Th1-type cytokines, interferon (IFN)-γ, interleukin (IL)-2, tumor necrosis factor (TNF)-α and IL-12, the Th2-type cytokines, IL-4, IL-5 and IL-10, and the Th17-type cytokine, IL-17A, significantly increased within 72 h of the initiation of co-culture with CD4+ T-cells obtained from the rFBA‑immunized mice, in comparison with the co-cultures with CD4+ T-cells obtained from the adjuvant-immunized mice. Immunizing mice with rFBA resulted in an IgG1/IgG2 ratio of 41, indicating a Th2 response with substantial Th1

  4. Streptococcus pneumoniae fructose-1,6-bisphosphate aldolase, a protein vaccine candidate, elicits Th1/Th2/Th17-type cytokine responses in mice.

    PubMed

    Elhaik Goldman, Shirin; Dotan, Shahar; Talias, Amir; Lilo, Amit; Azriel, Shalhevet; Malka, Itay; Portnoi, Maxim; Ohayon, Ariel; Kafka, Daniel; Ellis, Ronald; Elkabets, Moshe; Porgador, Angel; Levin, Ditza; Azhari, Rosa; Swiatlo, Edwin; Ling, Eduard; Feldman, Galia; Tal, Michael; Dagan, Ron; Mizrachi Nebenzahl, Yaffa

    2016-04-01

    Streptococcus pneumoniae (S. pneumoniae) is a major pathogen worldwide. The currently available polysaccharide-based vaccines significantly reduce morbidity and mortality. However, the inherent disadvantages of the currently available polysaccharide-based vaccines have motivated the search for other bacterial immunogens capable of eliciting a protective immune response against S. pneumoniae. Fructose-1,6-bisphosphate aldolase (FBA) is a glycolytic enzyme, which was found to localize to the bacterial surface, where it functions as an adhesin. Previously, immunizing mice with recombinant FBA (rFBA) in the presence of alum elicited a protective immune response against a lethal challenge with S. pneumoniae. Thus, the aim of the present study was to determine the cytokine responses that are indicative of protective immunity following immunization with rFBA. The protective effects against pneumococcal challenge in mice immunized with rFBA with complete Freund's adjuvant (CFA) in the initial immunization and with incomplete Freund's adjuvant (IFA) in booster immunizations surpassed the protective effects observed following immunization with either rFBA + alum or pVACfba. CD4+ T-cells obtained from the rFBA/CFA/IFA/IFA-immunized mice co-cultured with rFBA-pulsed antigen-presenting cells (APCs), exhibited a significantly greater proliferative ability than CD4+ T-cells obtained from the adjuvant-immunized mice co-cultured with rFBA‑pulsed APCs. The levels of the Th1-type cytokines, interferon (IFN)-γ, interleukin (IL)-2, tumor necrosis factor (TNF)-α and IL-12, the Th2-type cytokines, IL-4, IL-5 and IL-10, and the Th17-type cytokine, IL-17A, significantly increased within 72 h of the initiation of co-culture with CD4+ T-cells obtained from the rFBA‑immunized mice, in comparison with the co-cultures with CD4+ T-cells obtained from the adjuvant-immunized mice. Immunizing mice with rFBA resulted in an IgG1/IgG2 ratio of 41, indicating a Th2 response with substantial Th1

  5. The Co-Operonic PE25/PPE41 Protein Complex of Mycobacterium tuberculosis Elicits Increased Humoral and Cell Mediated Immune Response

    PubMed Central

    Tundup, Smanla; Pathak, Niteen; Ramanadham, M.; Mukhopadhyay, Sangita; Murthy, K. J. R.; Ehtesham, Nasreen Z.; Hasnain, Seyed E.

    2008-01-01

    Background Many of the PE/PPE proteins are either surface localized or secreted outside and are thought to be a source of antigenic variation in the host. The exact role of these proteins are still elusive. We previously reported that the PPE41 protein induces high B cell response in TB patients. The PE/PPE genes are not randomly distributed in the genome but are organized as operons and the operon containing PE25 and PPE41 genes co-transcribe and their products interact with each other. Methodology/Principal Finding We now describe the antigenic properties of the PE25, PPE41 and PE25/PPE41 protein complex coded by a single operon. The PPE41 and PE25/PPE41 protein complex induces significant (p<0.0001) B cell response in sera derived from TB patients and in mouse model as compared to the PE25 protein. Further, mice immunized with the PE25/PPE41 complex and PPE41 proteins showed significant (p<0.00001) proliferation of splenocyte as compared to the mice immunized with the PE25 protein and saline. Flow cytometric analysis showed 15–22% enhancement of CD8+ and CD4+ T cell populations when immunized with the PPE41 or PE25/PPE41 complex as compared to a marginal increase (8–10%) in the mice immunized with the PE25 protein. The PPE41 and PE25/PPE41 complex can also induce higher levels of IFN-γ, TNF-α and IL-2 cytokines. Conclusion While this study documents the differential immunological response to the complex of PE and PPE vis-à-vis the individual proteins, it also highlights their potential as a candidate vaccine against tuberculosis. PMID:18974870

  6. Structure and expression of a novel compact myelin protein – Small VCP-interacting protein (SVIP)

    SciTech Connect

    Wu, Jiawen; Peng, Dungeng; Voehler, Markus; Sanders, Charles R.; Li, Jun

    2013-10-11

    Highlights: •SVIP (small p97/VCP-interacting protein) co-localizes with myelin basic protein (MBP) in compact myelin. •We determined that SVIP is an intrinsically disordered protein (IDP). •The helical content of SVIP increases dramatically during its interaction with negatively charged lipid membrane. •This study provides structural insight into interactions between SVIP and myelin membranes. -- Abstract: SVIP (small p97/VCP-interacting protein) was initially identified as one of many cofactors regulating the valosin containing protein (VCP), an AAA+ ATPase involved in endoplasmic-reticulum-associated protein degradation (ERAD). Our previous study showed that SVIP is expressed exclusively in the nervous system. In the present study, SVIP and VCP were seen to be co-localized in neuronal cell bodies. Interestingly, we also observed that SVIP co-localizes with myelin basic protein (MBP) in compact myelin, where VCP was absent. Furthermore, using nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopic measurements, we determined that SVIP is an intrinsically disordered protein (IDP). However, upon binding to the surface of membranes containing a net negative charge, the helical content of SVIP increases dramatically. These findings provide structural insight into interactions between SVIP and myelin membranes.

  7. Modular Broad-Host-Range Expression Vectors for Single-Protein and Protein Complex Purification

    PubMed Central

    Fodor, Barna D.; Kovács, Ákos T.; Csáki, Róbert; Hunyadi-Gulyás, Éva; Klement, Éva; Maróti, Gergely; Mészáros, Lívia S.; Medzihradszky, Katalin F.; Rákhely, Gábor; Kovács, Kornél L.

    2004-01-01

    A set of modular broad-host-range expression vectors with various affinity tags (six-His-tag, FLAG-tag, Strep-tag II, T7-tag) was created. The complete nucleotide sequences of the vectors are known, and these small vectors can be mobilized by conjugation. They are useful in the purification of proteins and protein complexes from gram-negative bacterial species. The plasmids were easily customized for Thiocapsa roseopersicina, Rhodobacter capsulatus, and Methylococcus capsulatus by inserting an appropriate promoter. These examples demonstrate the versatility and flexibility of the vectors. The constructs harbor the T7 promoter for easy overproduction of the desired protein in an appropriate Escherichia coli host. The vectors were useful in purifying different proteins from T. roseopersicina. The FLAG-tag-Strep-tag II combination was utilized for isolation of the HynL-HypC2 protein complex involved in hydrogenase maturation. These tools should be useful for protein purification and for studying protein-protein interactions in a range of bacterial species. PMID:14766546

  8. Detecting Protein Complexes in Protein Interaction Networks Modeled as Gene Expression Biclusters

    PubMed Central

    Hanna, Eileen Marie; Zaki, Nazar; Amin, Amr

    2015-01-01

    Developing suitable methods for the detection of protein complexes in protein interaction networks continues to be an intriguing area of research. The importance of this objective originates from the fact that protein complexes are key players in most cellular processes. The more complexes we identify, the better we can understand normal as well as abnormal molecular events. Up till now, various computational methods were designed for this purpose. However, despite their notable performance, questions arise regarding potential ways to improve them, in addition to ameliorative guidelines to introduce novel approaches. A close interpretation leads to the assent that the way in which protein interaction networks are initially viewed should be adjusted. These networks are dynamic in reality and it is necessary to consider this fact to enhance the detection of protein complexes. In this paper, we present “DyCluster”, a framework to model the dynamic aspect of protein interaction networks by incorporating gene expression data, through biclustering techniques, prior to applying complex-detection algorithms. The experimental results show that DyCluster leads to higher numbers of correctly-detected complexes with better evaluation scores. The high accuracy achieved by DyCluster in detecting protein complexes is a valid argument in favor of the proposed method. DyCluster is also able to detect biologically meaningful protein groups. The code and datasets used in the study are downloadable from https://github.com/emhanna/DyCluster. PMID:26641660

  9. Expression of goose parvovirus whole VP3 protein and its epitopes in Escherichia coli cells.

    PubMed

    Tarasiuk, K; Woźniakowski, G; Holec-Gąsior, L

    2015-01-01

    The aim of this study was the expression of goose parvovirus capsid protein (VP3) and its epitopes in Escherichia coli cells. Expression of the whole VP3 protein provided an insufficient amount of protein. In contrast, the expression of two VP3 epitopes (VP3ep4, VP3ep6) in E. coli, resulted in very high expression levels. This may suggest that smaller parts of the GPV antigenic determinants are more efficiently expressed than the complete VP3 gene.

  10. Immunogenicity of a neutralizing epitope from porcine epidemic diarrhea virus: M cell targeting ligand fusion protein expressed in transgenic rice calli.

    PubMed

    Huy, Nguyen-Xuan; Kim, Sae-Hae; Yang, Moon-Sik; Kim, Tae-Geum

    2012-10-01

    To increase immune responses of plant-based vaccines in intestine mucosal immune systems, a synthetic neutralizing epitope (sCOE) gene of porcine epidemic diarrhea virus (PEDV) was fused with M cell-targeting ligand (Co1) and introduced into a plant expression vector under the control of rice amylase 3D promoter. The sCOE-Co1 fusion gene was introduced into rice calli via the particle bombardment-mediated transformation method. The stable integration and transcriptional expression of the sCOE-Co1 fusion gene was confirmed by genomic DNA PCR amplification and Northern blot analysis, respectively. The expression of the COE-Co1 fusion protein was confirmed by immunoblot analysis. The highest expression level of the COE-Co1 fusion protein reached 0.083 % of the total soluble protein according to quantitative densitometry of Western blot analysis. Mice immunized with transgenic rice calli protein extracts induced significant serum IgG and fecal IgA antibody levels against purified bacterial COE. The systemic and mucosal immune responses were confirmed by measuring COE-specific IgG and IgA antibody-secreting cells in the lymphocytes extracted from the spleen and COE-specific IgA antibody-secreting cells in the Peyer's patches from immunized mice. These results indicated that oral immunization of plant-produced COE-Co1 fusion protein could elicit efficient systemic and mucosal immune responses against the COE antigen. Key message Neutralizing epitope from porcine epidemic diarrhea virus-M cell targeting ligand fusion protein was produced in transgenic rice calli and elicited systemic and mucosal immune responses by oral administration in mice. PMID:22736145

  11. Design of riboregulators for control of cyanobacterial (Synechocystis) protein expression.

    PubMed

    Abe, Koichi; Sakai, Yuta; Nakashima, Saki; Araki, Masataka; Yoshida, Wataru; Sode, Koji; Ikebukuro, Kazunori

    2014-02-01

    Cyanobacteria are attractive host bacteria for biofuel production because they can covert CO2 to biofuel lipids using only sunlight, water, and inorganic ions. For genetically engineering an ideal cyanobacterium, a synthetic biological approach is promising but few genetic components have been characterized in cyanobacteria. Here for controlling cyanobacterial protein expression, we constructed riboregulators, that one of the post-transcriptional regulators composed of RNAs. Riboregulators harboring a ribosome-binding site suitable for Synechocystis sp. were designed by trial and error using Escherichia coli as host bacteria. The designed riboregulators were effective in Synechocystis sp. as well as E. coli with slight interference on growth only observed in E. coli. They will therefore be useful tools for controlling target gene expression. PMID:24068508

  12. Altered gravity downregulates aquaporin-1 protein expression in choroid plexus.

    PubMed

    Masseguin, C; Corcoran, M; Carcenac, C; Daunton, N G; Güell, A; Verkman, A S; Gabrion, J

    2000-03-01

    Aquaporin-1 (AQP1) is a water channel expressed abundantly at the apical pole of choroidal epithelial cells. The protein expression was quantified by immunocytochemistry and confocal microscopy in adult rats adapted to altered gravity. AQP1 expression was decreased by 64% at the apical pole of choroidal cells in rats dissected 5.5-8 h after a 14-day spaceflight. AQP1 was significantly overexpressed in rats readapted for 2 days to Earth's gravity after an 11-day flight (48% overshoot, when compared with the value measured in control rats). In a ground-based model that simulates some effects of weightlessness and alters choroidal structures and functions, apical AQP1 expression was reduced by 44% in choroid plexus from rats suspended head down for 14 days and by 69% in rats suspended for 28 days. Apical AQP1 was rapidly enhanced in choroid plexus of rats dissected 6 h after a 14-day suspension (57% overshoot, in comparison with control rats) and restored to the control level when rats were dissected 2 days after the end of a 14-day suspension. Decreases in the apical expression of choroidal AQP1 were also noted in rats adapted to hypergravity in the NASA 24-ft centrifuge: AQP1 expression was reduced by 47% and 85% in rats adapted for 14 days to 2 G and 3 G, respectively. AQP1 is downregulated in the apical membrane of choroidal cells in response to altered gravity and is rapidly restored after readaptation to normal gravity. This suggests that water transport, which is partly involved in the choroidal production of cerebrospinal fluid, might be decreased during spaceflight and after chronic hypergravity.

  13. Photoregulated gene expression may involve ubiquitous DNA binding proteins.

    PubMed Central

    Schindler, U; Cashmore, A R

    1990-01-01

    Several promoter elements have previously been shown to influence the expression of the cab-E gene in Nicotiana plumbaginifolia. Here we demonstrate, by electrophoretic mobility shift and methylation interference assays, that a complex pattern of protein-DNA interactions characterizes this promoter. Among the multiple proteins identified, we focused on five different factors which either occupied important regulatory elements and/or were present in relatively large amounts in nuclear extracts. All of these proteins were distinguished on the basis of their recognition sequence and other biochemical parameters. One, GBF, interacted with a single sequence within the cab-E promoter homologous to the G-box found in many photoregulated and other plant promoters. A second factor, GA-1, bound to the GATA element which is located between the CAAT and TATA boxes of the cab-E and all other LHCII Type I CAB promoters. GA-1 also interacted in vitro with the I-boxes of the Arabidopsis rbcS-1A promoter and the as-2 site of the CaMV 35S promoter. Two other factors, GC-1 and AT-1, bound to multiple recognition sites localized within the GC-rich and AT-rich elements, respectively. GT-1, a protein which interacts with promoters of other light-regulated genes, bound to seven distinct sites distributed throughout the cab-E promoter. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig.5 Fig.6 Fig.7 PMID:2209551

  14. Expressed protein ligation for metalloprotein design and engineering.

    PubMed

    Clark, Kevin M; van der Donk, Wilfred A; Lu, Yi

    2009-01-01

    Metalloproteins contain highly specialized metal-binding sites that are designed to accept specific metal ions to maintain correct function. Although many of the sites have been modified with success, the relative paucity of functional group availability within proteinogenic amino acids can sometimes leave open questions about specific functions of the metal binding ligands. Attaining a more thorough analysis of individual amino acid function within metalloproteins has been realized using expressed protein ligation (EPL). Here we describe our recent efforts using EPL to incorporate nonproteinogenic cysteine and methionine analogues into the type 1 copper site found in Pseudomonas aeruginosa azurin.

  15. RNA viruses as vectors for the expression of heterologous proteins.

    PubMed

    Schlesinger, S

    1995-04-01

    RNA viruses comprise a wide variety of infectious agents, some of which are the cause of disease in humans, animals, and plants. Recombinant DNA technology is now making it feasible to modify these genomes and engineer them to express heterologous proteins. Several different schemes are being employed that depend on the genome organization of the virus and on the strategy of replication of the particular virus. Several different examples are illustrated and potential uses as well as possible problems are discussed. In the future reverse genetics may convert some of these viruses from agents of disease to agents of cure. PMID:7620976

  16. Expressed Protein Ligation: A Resourceful Tool to Study Protein Structure and Function

    PubMed Central

    Berrade, Luis; Camarero, Julio A.

    2013-01-01

    This review outlines the use of expressed protein ligation (EPL) to study protein structure, function and stability. EPL is a chemoselective ligation method that allows the selective ligation of unprotected polypeptides from synthetic and recombinant origin for the production of semi-synthetic protein samples of well-defined and homogeneous chemical composition. This method has been extensively used for the site-specific introduction of biophysical probes, unnatural amino acids, and increasingly complex post-translational modifications. Since it was introduced 10 years ago, EPL applications have grown increasingly more sophisticated in order to address even more complex biological questions. In this review we highlight how this powerful technology combined with standard biochemical analysis techniques has been used to improve our ability to understand protein structure and function. PMID:19685006

  17. Differential regulation of dentin matrix protein 1 expression during odontogenesis.

    PubMed

    Lu, Yongbo; Zhang, Shubin; Xie, Yixia; Pi, Yuli; Feng, Jian Q

    2005-01-01

    Dentin matrix protein 1 (DMP1) is highly expressed in mineralized tooth and bone. Both in vitro and in vivo data show that DMP1 is critical for mineralization and tooth morphogenesis (growth and development). In this study, we studied Dmp1 gene regulation. The in vitro transient transfection assay identified two important DNA fragments, the 2.4- and 9.6-kb promoter regions. We next generated and analyzed transgenic mice bearing the beta-galactosidase (lacZ) reporter gene driven by the 2.4- or 9.6-kb promoter with the complete 4-kb intron 1. The 9.6-kb Dmp1-lacZ mice conferred a DMP1 expression pattern in odontoblasts identical to that in the endogenous Dmp1 gene. This is reflected by lacZ expression in Dmp1-lacZ knock-in mice during all stages of odontogenesis. In contrast, the 2.4-kb Dmp1-lacZ mice display activity in odontoblast cells only at the early stage of odontogenesis. Thus, we propose that different transcription factors regulate early or later cis-regulatory domains of the Dmp1 promoter, which gives rise to the unique spatial and temporal expression pattern of Dmp1 gene at different stages of tooth development.

  18. Elicited Emotions and Cognitive Functioning in Preschool Children

    ERIC Educational Resources Information Center

    Blau, Rivka; Klein, Pnina S.

    2010-01-01

    In this study, the effects of eliciting positive and negative emotions on various cognitive functions of four- to five-year-old preschool children were examined. Emotions were elicited through presentations of "happy" and "sad" video clips, before the children performed the cognitive tasks. Behavioural (facial expressions) and physiological (heart…

  19. AR-v7 protein expression is regulated by protein kinase and phosphatase

    PubMed Central

    Li, Yinan; Xie, Ning; Gleave, Martin E.; Rennie, Paul S.; Dong, Xuesen

    2015-01-01

    Failure of androgen-targeted therapy and progression of castration-resistant prostate cancer (CRPC) are often attributed to sustained expression of the androgen receptor (AR) and its major splice variant, AR-v7. Although the new generation of anti-androgens such as enzalutamide effectively inhibits AR activity, accumulating pre-clinical and clinical evidence indicates that AR-v7 remains constitutively active in driving CRPC progression. However, molecular mechanisms which control AR-v7 protein expression remain unclear. We apply multiple prostate cancer cell models to demonstrate that enzalutamide induces differential activation of protein phosphatase-1 (PP-1) and Akt kinase depending on the gene context of cancer cells. The balance between PP-1 and Akt activation governs AR phosphorylation status and activation of the Mdm2 ubiquitin ligase. Mdm2 recognizes phosphorylated serine 213 of AR-v7, and induces AR-v7 ubiquitination and protein degradation. These findings highlight the decisive roles of PP-1 and Akt for AR-v7 protein expression and activities when AR is functionally blocked. PMID:26378044

  20. AR-v7 protein expression is regulated by protein kinase and phosphatase.

    PubMed

    Li, Yinan; Xie, Ning; Gleave, Martin E; Rennie, Paul S; Dong, Xuesen

    2015-10-20

    Failure of androgen-targeted therapy and progression of castration-resistant prostate cancer (CRPC) are often attributed to sustained expression of the androgen receptor (AR) and its major splice variant, AR-v7. Although the new generation of anti-androgens such as enzalutamide effectively inhibits AR activity, accumulating pre-clinical and clinical evidence indicates that AR-v7 remains constitutively active in driving CRPC progression. However, molecular mechanisms which control AR-v7 protein expression remain unclear. We apply multiple prostate cancer cell models to demonstrate that enzalutamide induces differential activation of protein phosphatase-1 (PP-1) and Akt kinase depending on the gene context of cancer cells. The balance between PP-1 and Akt activation governs AR phosphorylation status and activation of the Mdm2 ubiquitin ligase. Mdm2 recognizes phosphorylated serine 213 of AR-v7, and induces AR-v7 ubiquitination and protein degradation. These findings highlight the decisive roles of PP-1 and Akt for AR-v7 protein expression and activities when AR is functionally blocked.

  1. The fusion of Toxoplasma gondii SAG1 vaccine candidate to Leishmania infantum heat shock protein 83-kDa improves expression levels in tobacco chloroplasts.

    PubMed

    Albarracín, Romina M; Becher, Melina Laguía; Farran, Inmaculada; Sander, Valeria A; Corigliano, Mariana G; Yácono, María L; Pariani, Sebastián; López, Edwin Sánchez; Veramendi, Jon; Clemente, Marina

    2015-05-01

    Chloroplast transformation technology has emerged as an alternative platform offering many advantages over nuclear transformation. SAG1 is the main surface antigen of the intracellular parasite Toxoplasma gondii and a promising candidate to produce an anti-T. gondii vaccine. The aim of this study was to investigate the expression of SAG1 using chloroplast transformation technology in tobacco plants. In order to improve expression in transplastomic plants, we also expressed the 90-kDa heat shock protein of Leishmania infantum (LiHsp83) as a carrier for the SAG1 antigen. SAG1 protein accumulation in transplastomic plants was approximately 0.1-0.2 μg per gram of fresh weight (FW). Fusion of SAG1 to LiHsp83 significantly increased the level of SAG1 accumulation in tobacco chloroplasts (by up to 500-fold). We also evaluated the functionality of the chLiHsp83-SAG1. Three human seropositive samples reacted with SAG1 expressed in transplastomic chLiHsp83-SAG1 plants. Oral immunization with chLiHsp83-SAG1 elicited a significant reduction of the cyst burden that correlated with an increase of SAG1-specific antibodies. We propose the fusion of foreign proteins to LiHsp83 as a novel strategy to increase the expression level of the recombinant proteins using chloroplast transformation technology, thus addressing one of the current challenges for this approach in antigen protein production.

  2. The fusion of Toxoplasma gondii SAG1 vaccine candidate to Leishmania infantum heat shock protein 83-kDa improves expression levels in tobacco chloroplasts.

    PubMed

    Albarracín, Romina M; Becher, Melina Laguía; Farran, Inmaculada; Sander, Valeria A; Corigliano, Mariana G; Yácono, María L; Pariani, Sebastián; López, Edwin Sánchez; Veramendi, Jon; Clemente, Marina

    2015-05-01

    Chloroplast transformation technology has emerged as an alternative platform offering many advantages over nuclear transformation. SAG1 is the main surface antigen of the intracellular parasite Toxoplasma gondii and a promising candidate to produce an anti-T. gondii vaccine. The aim of this study was to investigate the expression of SAG1 using chloroplast transformation technology in tobacco plants. In order to improve expression in transplastomic plants, we also expressed the 90-kDa heat shock protein of Leishmania infantum (LiHsp83) as a carrier for the SAG1 antigen. SAG1 protein accumulation in transplastomic plants was approximately 0.1-0.2 μg per gram of fresh weight (FW). Fusion of SAG1 to LiHsp83 significantly increased the level of SAG1 accumulation in tobacco chloroplasts (by up to 500-fold). We also evaluated the functionality of the chLiHsp83-SAG1. Three human seropositive samples reacted with SAG1 expressed in transplastomic chLiHsp83-SAG1 plants. Oral immunization with chLiHsp83-SAG1 elicited a significant reduction of the cyst burden that correlated with an increase of SAG1-specific antibodies. We propose the fusion of foreign proteins to LiHsp83 as a novel strategy to increase the expression level of the recombinant proteins using chloroplast transformation technology, thus addressing one of the current challenges for this approach in antigen protein production. PMID:25823559

  3. Exercise prevents downregulation of hippocampal presynaptic proteins following olanzapine-elicited metabolic dysregulation in rats: Distinct roles of inhibitory and excitatory terminals.

    PubMed

    Ramos-Miguel, A; Honer, W G; Boyda, H N; Sawada, K; Beasley, C L; Procyshyn, R M; Barr, A M

    2015-08-20

    Schizophrenia patients treated with olanzapine, or other second-generation antipsychotics, frequently develop metabolic side-effects, such as glucose intolerance and increased adiposity. We previously observed that modeling these adverse effects in rodents also resulted in hippocampal shrinkage. Here, we investigated the impact of olanzapine treatment, and the beneficial influence of routine exercise, on the neurosecretion machinery of the hippocampus. Immunodensities and interactions of three soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins (syntaxin-1, synaptosome-associated protein of 25kDa (SNAP-25) and vesicle-associated membrane protein (VAMP)), synaptotagmin and complexins-1/2 were quantified in the hippocampus of sedentary and exercising rats exposed over 9weeks to vehicle (n=28) or olanzapine (10mg/kg/day, n=28). In addition, brain sections from subgroups of sedentary animals (n=8) were co-immunolabeled with antibodies against vesicular GABA (VGAT) and glutamate (VGLUT1) transporters, along with syntaxin-1, and examined by confocal microscopy to detect selective olanzapine effects within inhibitory or excitatory terminals. Following olanzapine treatment, sedentary, but not exercising rats showed downregulated (33-50%) hippocampal densities of SNARE proteins and synaptotagmin, without altering complexin levels. Strikingly, these effects had no consequences on the amount of SNARE protein-protein interactions. Lower immunodensity of presynaptic proteins was associated with reduced CA1 volume and glucose intolerance. Syntaxin-1 depletion appeared more prominent in VGAT-positive terminals within the dentate gyrus, and in non-VGAT/VGLUT1-overlapping areas of CA3. The present findings suggest that chronic exposure to olanzapine may alter hippocampal connectivity, especially in inhibitory terminals within the dentate gyrus, and along the mossy fibers of CA3. Together with previous studies, we propose that exercise

  4. Exercise prevents downregulation of hippocampal presynaptic proteins following olanzapine-elicited metabolic dysregulation in rats: Distinct roles of inhibitory and excitatory terminals.

    PubMed

    Ramos-Miguel, A; Honer, W G; Boyda, H N; Sawada, K; Beasley, C L; Procyshyn, R M; Barr, A M

    2015-08-20

    Schizophrenia patients treated with olanzapine, or other second-generation antipsychotics, frequently develop metabolic side-effects, such as glucose intolerance and increased adiposity. We previously observed that modeling these adverse effects in rodents also resulted in hippocampal shrinkage. Here, we investigated the impact of olanzapine treatment, and the beneficial influence of routine exercise, on the neurosecretion machinery of the hippocampus. Immunodensities and interactions of three soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins (syntaxin-1, synaptosome-associated protein of 25kDa (SNAP-25) and vesicle-associated membrane protein (VAMP)), synaptotagmin and complexins-1/2 were quantified in the hippocampus of sedentary and exercising rats exposed over 9weeks to vehicle (n=28) or olanzapine (10mg/kg/day, n=28). In addition, brain sections from subgroups of sedentary animals (n=8) were co-immunolabeled with antibodies against vesicular GABA (VGAT) and glutamate (VGLUT1) transporters, along with syntaxin-1, and examined by confocal microscopy to detect selective olanzapine effects within inhibitory or excitatory terminals. Following olanzapine treatment, sedentary, but not exercising rats showed downregulated (33-50%) hippocampal densities of SNARE proteins and synaptotagmin, without altering complexin levels. Strikingly, these effects had no consequences on the amount of SNARE protein-protein interactions. Lower immunodensity of presynaptic proteins was associated with reduced CA1 volume and glucose intolerance. Syntaxin-1 depletion appeared more prominent in VGAT-positive terminals within the dentate gyrus, and in non-VGAT/VGLUT1-overlapping areas of CA3. The present findings suggest that chronic exposure to olanzapine may alter hippocampal connectivity, especially in inhibitory terminals within the dentate gyrus, and along the mossy fibers of CA3. Together with previous studies, we propose that exercise

  5. Trichohyalin-like 1 protein, a member of fused S100 proteins, is expressed in normal and pathologic human skin

    SciTech Connect

    Yamakoshi, Takako; Makino, Teruhiko; Ur Rehman, Mati; Yoshihisa, Yoko; Sugimori, Michiya; Shimizu, Tadamichi

    2013-03-01

    Highlights: ► Trichohyalin-like 1 protein is a member of the fused-type S100 protein gene family. ► Specific antibodies against the C-terminus of the TCHHL1 protein were generated. ► TCHHL1 proteins were expressed in the basal layer of the normal epidermis. ► TCHHL1 proteins were strongly expressed in tumor nests of BCC and SCC. ► The expression of TCHHL1 proteins increased in epidermis of psoriasis vulgaris. - Abstract: Trichohyalin-like 1 (TCHHL1) protein is a novel member of the fused-type S100 protein gene family. The deduced amino acid sequence of TCHHL1 contains an EF-hand domain in the N-terminus, one trans-membrane domain and a nuclear localization signal. We generated specific antibodies against the C-terminus of the TCHHL1 protein and examined the expression of TCHHL1 proteins in normal and pathological human skin. An immunohistochemical study showed that TCHHL1 proteins were expressed in the basal layer of the normal epidermis. In addition, signals of TCHHL1 proteins were observed around the nuclei of cultured growing keratinocytes. Accordingly, TCHHL1 mRNA has been detected in normal skin and cultured growing keratinocytes. Furthermore, TCHHL1 proteins were strongly expressed in the peripheral areas of tumor nests in basal cell carcinomas and squamous cell carcinomas. A dramatic increase in the number of Ki67 positive cells was observed in TCHHL1-expressing areas. The expression of TCHHL1 proteins also increased in non-cancerous hyperproliferative epidermal tissues such as those of psoriasis vulgaris and lichen planus. These findings highlight the possibility that TCHHL1 proteins are expressed in growing keratinocytes of the epidermis and might be associated with the proliferation of keratinocytes.

  6. Vaccines expressing the innate immune modulator EAT-2 elicit potent effector memory T lymphocyte responses despite pre-existing vaccine immunity.

    PubMed

    Aldhamen, Yasser Ali; Seregin, Sergey S; Schuldt, Nathaniel J; Rastall, David P W; Liu, Chyong-Jy J; Godbehere, Sarah; Amalfitano, Andrea

    2012-08-01

    The mixed results from recent vaccine clinical trials targeting HIV-1 justify the need to enhance the potency of HIV-1 vaccine platforms in general. Use of first-generation recombinant adenovirus serotype 5 (rAd5) platforms failed to protect vaccinees from HIV-1 infection. One hypothesis is that the rAd5-based vaccine failed due to the presence of pre-existing Ad5 immunity in many vaccines. We recently confirmed that EAT-2-expressing rAd5 vectors uniquely activate the innate immune system and improve cellular immune responses against rAd5-expressed Ags, inclusive of HIV/Gag. In this study, we report that use of the rAd5-EAT-2 vaccine can also induce potent cellular immune responses to HIV-1 Ags despite the presence of Ad5-specific immunity. Compared to controls expressing a mutant SH2 domain form of EAT-2, Ad5 immune mice vaccinated with an rAd5-wild-type EAT-2 HIV/Gag-specific vaccine formulation significantly facilitated the induction of several arms of the innate immune system. These responses positively correlated with an improved ability of the vaccine to induce stronger effector memory T cell-biased, cellular immune responses to a coexpressed Ag despite pre-existing anti-Ad5 immunity. Moreover, inclusion of EAT-2 in the vaccine mixture improves the generation of polyfunctional cytolytic CD8(+) T cell responses as characterized by enhanced production of IFN-γ, TNF-α, cytotoxic degranulation, and increased in vivo cytolytic activity. These data suggest a new approach whereby inclusion of EAT-2 expression in stringent human vaccination applications can provide a more effective vaccine against HIV-1 specifically in Ad5 immune subjects.

  7. Vaccination with the Recombinant Brucella Outer Membrane Protein 31 or a Derived 27-Amino-Acid Synthetic Peptide Elicits a CD4+ T Helper 1 Response That Protects against Brucella melitensis Infection

    PubMed Central

    Cassataro, Juliana; Estein, Silvia M.; Pasquevich, Karina A.; Velikovsky, Carlos A.; de la Barrera, Silvia; Bowden, Raúl; Fossati, Carlos A.; Giambartolomei, Guillermo H.

    2005-01-01

    The immunogenicity and protective efficacy of the recombinant 31-kDa outer membrane protein from Brucella melitensis (rOmp31), administered with incomplete Freund's adjuvant, were evaluated in mice. Immunization of BALB/c mice with rOmp31 conferred protection against B. ovis and B. melitensis infection. rOmp31 induced a vigorous immunoglobulin G (IgG) response, with higher IgG1 than IgG2 titers. In addition, spleen cells from rOmp31-immunized mice produced interleukin 2 (IL-2) and gamma interferon, but not IL-10 or IL-4, after in vitro stimulation with rOmp31, suggesting the induction of a T helper 1 (Th1) response. Splenocytes from rOmp31-vaccinated animals also induced a specific cytotoxic-T-lymphocyte activity, which led to the in vitro lysis of Brucella-infected macrophages. In vitro T-cell subset depletion indicated that rOmp31 immunization elicited specific CD4+ T cells that secrete IL-2 and gamma interferon, while CD8+ T cells induced cytotoxic-T-lymphocyte activity. In vivo depletion of T-cell subsets showed that the rOmp31-elicited protection against B. melitensis infection is mediated by CD4+ T cells while the contribution of CD8+ T cells may be limited. We then evaluated the immunogenicity and protective efficacy of a known exposed region from Omp31 on the Brucella membrane, a peptide that contains amino acids 48 to 74 of Omp31. Immunization with the synthetic peptide in adjuvant did not elicit a specific humoral response but elicited a Th1 response mediated by CD4+ T cells. The peptide in adjuvant induced levels of protection similar to those induced by rOmp31 against B. melitensis but less protection than was induced by rOmp31 against B. ovis. Our results indicate that rOmp31 could be a useful candidate for the development of subunit vaccines against B. melitensis and B. ovis. PMID:16299302

  8. Recombinant West Nile virus envelope protein E and domain III expressed in insect larvae protects mice against West Nile disease.

    PubMed

    Alonso-Padilla, Julio; de Oya, Nereida Jiménez; Blázquez, Ana-Belén; Escribano-Romero, Estela; Escribano, José M; Saiz, Juan-Carlos

    2011-02-17

    In this study, West Nile virus (WNV) envelope (rE) protein and its domain III (rDIII) were efficiently expressed in a cost-effective system based on insect larvae as non-fermentative living biofactories. Mice immunized with the partially purified rE or rDIII elicited high antibodies titers that neutralized viral infectivity in cell culture and in suckling mice. All vaccinated animals were fully protected when challenged with neurovirulent WNV NY99. Passive transfer of protective antibodies from immunized mothers to their offspring occurred both by transplacental and lactation routes. These results indicate that the insect-derived antigens tested may constitute potential vaccine candidates to be further evaluated.

  9. 3-Methylcholanthrene elicits DNA adduct formation in the CYP1A1 promoter region and attenuates reporter gene expression in rat H4IIE cells

    SciTech Connect

    Moorthy, Bhagavatula . E-mail: bmoorthy@bcm.tmc.edu; Muthiah, Kathirvel; Fazili, Inayat S.; Kondraganti, Sudha R.; Wang Lihua; Couroucli, Xanthi I.; Jiang Weiwu

    2007-03-23

    Cytochrome CYP1A (CYP1A) enzymes catalyze bioactivation of 3-methylcholanthrene (MC) to genotoxic metabolites. Here, we tested the hypothesis that CYP1A2 catalyzes formation of MC-DNA adducts that are preferentially formed in the promoter region of CYP1A1, resulting in modulation of CYP1A1 gene expression. MC bound covalently to plasmid DNA (50 {mu}g) containing human CYP1A1 promoter (pGL3-1A1), when incubated with wild-type (WT) liver microsomes (2 mg) and NAPPH 37 {sup o}C for 2 h, giving rise to 9 adducts, as determined by {sup 32}P-postlabeling. Eighty percent of adducts was located in the promoter region. Transient transfection of the adducted plasmids into rat hepatoma (H4IIE) cells for 16 h, followed by MC (1 {mu}M) treatment for 24 h inhibited reporter (luciferase) gene expression by 75%, compared to unadducted controls. Our results suggest that CYP1A2 plays a key role in sequence-specific MC-DNA adduct formation in the CYP1A1 promoter region, leading to attenuation of CYP1A1 gene expression.

  10. Induction of anti-tumor immunity elicited by tumor cells expressing a murine LFA-3 analog via a recombinant vaccinia virus.

    PubMed

    Lorenz, M G; Kantor, J A; Schlom, J; Hodge, J W

    1999-03-01

    T cell activation requires binding of the T cell receptor to the major histocompatibility molecule-peptide complex in the presence of adhesion and/or costimulatory molecules such as B7-1 (CD80), B7-2 (CD86), ICAM-1 (CD54), and LFA-3 [corrected]. The major ligand of CD2 is CD48, the murine analog of human leukocyte function-associated antigen 3 (LFA-3). To determine the effect of LFA-3 expression on the immunogenicity of tumor cells, we constructed a recombinant vaccinia virus containing the murine LFA-3 gene (designated rV-LFA-3). rV-LFA-3 was shown to be functional in vitro in terms of expression of LFA-3, T cell proliferation, adhesion, and cytotoxicity. Subcutaneous inoculation of rV-LFA-3-infected murine colon adenocarcinoma tumor cells (MC38) into immunocompetent syngeneic C57BL/6 mice resulted in complete lack of tumor growth. Inoculation of MC38 cells infected with equal doses of control wild-type vaccinia virus resulted in tumor growth in all animals. In addition, partial immunological protection was demonstrated against subsequent challenge with uninfected parental tumor cells up to 56 days after vaccination with rV-LFA-3-infected cells. Anti-tumor memory was also demonstrated by using gamma-irradiated MC38 cells and cells from another carcinoma model (CT26). These studies demonstrate that expression of LFA-3 via a poxvirus vector can be used to induce anti-tumor immunity.

  11. Antigenic assessment of a recombinant human CD90 protein expressed in prokaryotic expression system.

    PubMed

    Yousefi-Rad, Narges; Shokrgozar, Mohammad Ali; Behdani, Mahdi; Moradi-Kalbolandi, Shima; Motamedi-Rad, Mahdieh; Habibi-Anbouhi, Mahdi

    2015-12-01

    Cluster of Differentiation 90 (CD90, Thy-1) has been proposed as one of the most important biomarkers in several cancer cells including cancer stem cells (CSCs). CD90 is considered as a potential normal stem cell and CSCs biomarker and also has been identified in lung cancer stem cells, hepatocellular carcinoma cells and high-grade gliomas. Using eukaryotic host systems involves complex procedures and frequently results in low protein yields. The expression of recombinant proteins in Escherichia coli is comparatively easier than eukaryotic host cells. The potential of large scale production of recombinant protein has made this system an economic production platform. In this study we expressed the extra-membrane domain of human CD90 (exCD90) antigen (Gln15-Cys130) in E. coli expression host cells. The epitope integrity of purified recombinant antigen was confirmed by antibody-antigen interaction using 5E10 anti-CD90 monoclonal antibody and binding study through ELISA and florescent staining of CD90(+) cells in a flow cytometry experiment. PMID:26297626

  12. Antigenic assessment of a recombinant human CD90 protein expressed in prokaryotic expression system.

    PubMed

    Yousefi-Rad, Narges; Shokrgozar, Mohammad Ali; Behdani, Mahdi; Moradi-Kalbolandi, Shima; Motamedi-Rad, Mahdieh; Habibi-Anbouhi, Mahdi

    2015-12-01

    Cluster of Differentiation 90 (CD90, Thy-1) has been proposed as one of the most important biomarkers in several cancer cells including cancer stem cells (CSCs). CD90 is considered as a potential normal stem cell and CSCs biomarker and also has been identified in lung cancer stem cells, hepatocellular carcinoma cells and high-grade gliomas. Using eukaryotic host systems involves complex procedures and frequently results in low protein yields. The expression of recombinant proteins in Escherichia coli is comparatively easier than eukaryotic host cells. The potential of large scale production of recombinant protein has made this system an economic production platform. In this study we expressed the extra-membrane domain of human CD90 (exCD90) antigen (Gln15-Cys130) in E. coli expression host cells. The epitope integrity of purified recombinant antigen was confirmed by antibody-antigen interaction using 5E10 anti-CD90 monoclonal antibody and binding study through ELISA and florescent staining of CD90(+) cells in a flow cytometry experiment.

  13. Expression and purification of recombinant polyomavirus VP2 protein and its interactions with polyomavirus proteins

    NASA Technical Reports Server (NTRS)

    Cai, X.; Chang, D.; Rottinghaus, S.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1994-01-01

    Recombinant polyomavirus VP2 protein was expressed in Escherichia coli (RK1448), using the recombinant expression system pFPYV2. Recombinant VP2 was purified to near homogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electroelution, and Extracti-Gel chromatography. Polyclonal serum to this protein which reacted specifically with recombinant VP2 as well as polyomavirus virion VP2 and VP3 on Western blots (immunoblots) was produced. Purified VP2 was used to establish an in vitro protein-protein interaction assay with polyomavirus structural proteins and purified recombinant VP1. Recombinant VP2 interacted with recombinant VP1, virion VP1, and the four virion histones. Recombinant VP1 coimmunoprecipitated with recombinant VP2 or truncated VP2 (delta C12VP2), which lacked the carboxy-terminal 12 amino acids. These experiments confirmed the interaction between VP1 and VP2 and revealed that the carboxyterminal 12 amino acids of VP2 and VP3 were not necessary for formation of this interaction. In vivo VP1-VP2 interaction study accomplished by cotransfection of COS-7 cells with VP2 and truncated VP1 (delta N11VP1) lacking the nuclear localization signal demonstrated that VP2 was capable of translocating delta N11VP1 into the nucleus. These studies suggest that complexes of VP1 and VP2 may be formed in the cytoplasm and cotransported to the nucleus for virion assembly to occur.

  14. Modified recombinant adenoviruses increase porcine circovirus 2 capsid protein expression and induce enhanced immune responses in mice.

    PubMed

    Li, D L; Huang, Y; Chang, L L; DU, Q; Chen, Y; Wang, T T; Luo, X M; Zhao, X M; Tong, D W

    2016-01-01

    Porcine circovirus type 2 (PCV2) is the primary viral pathogen of porcine circovirus associated disease (PCVAD) and vaccination is an important method to prevent and control the disease. The expression of PCV2 capsid protein (Cap) in adenovirus vector system has been investigated, but the poor immune responses limit its application. In this study, transcriptional enhancer element largest intron of the human cytomegalovirus (Intron A) and woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) were applied to increase the immunogenicity of PCV2 Cap adenovirus-based vaccine. Western blot and indirect immunofluorescence assay (IFA) analysis showed that modified adenoviruses with Intron A and WPRE alone or both could significantly increase the expression of Cap compared to the unmodified adenoviruses. Furthermore, the humoral and cellular immune responses of the constructed recombinant adenoviruses were evaluated in mice. Indirect ELISA, virus neutralizing test and western blot showed that modified adenoviruses elicited higher humoral immune responses than unmodified adenovirus, and Intron A-WPRE-modified virus immunized group had better immune response than the others. Besides, the results of lymphocyte proliferation response and cytokines release assay showed that enhanced cellular immune responses were induced by modified adenoviruses. These results demonstrated that Intron A and WPRE significantly improved the expression of the Cap protein in adenovirus vector system and enhanced the immune responses in mice, making the adenovirus vector system more applicable against PCV2. PMID:27640437

  15. Immunogenicity of orally administrated recombinant Lactobacillus casei Zhang expressing Cryptosporidium parvum surface adhesion protein P23 in mice.

    PubMed

    Geriletu; Xu, Rihua; Jia, Honglin; Terkawi, Mohamad Alaa; Xuan, Xuenan; Zhang, Heping

    2011-05-01

    Cryptosporidium parvum, an intestinal apicomplexan parasite, is a significant cause of diarrheal diseases in both humans and animals. What is more, there is no promising strategy for controlling cryptosporidiosis. In this study, the P23 immunodominant surface protein of C. parvum sporozoites was stably expressed in the Lactobacillus casei Zhang strain and its immunogenicity was evaluated in a mouse model. The molecular weight (23 kDa) and immunogenicity of p23 gene expressed by L. casei Zhang were similar to that of the native P23 protein. Oral immunization with control L. casei Zhang and recombinant L. casei Zhang-p23 activated the mucosal immune system to elicit serum immunoglobulin G (IgG) and mucosal IgA in mice. Furthermore, the expression of cytokines such as IL-4, IL-6, and IFN-γ in splenocytes of mice was detected by real-time PCR after oral immunization. P23-specific immunocyte activation was also verified. These findings indicate that the live L. casei Zhang vector may be a new tool for the production of mucosal vaccines against cryptosporidiosis in animals. PMID:21336991

  16. Characterization of protein expression levels with label-free detected reverse phase protein arrays.

    PubMed

    Guo, Xuexue; Deng, Yihong; Zhu, Chenggang; Cai, Junlong; Zhu, Xiangdong; Landry, James P; Zheng, Fengyun; Cheng, Xunjia; Fei, Yiyan

    2016-09-15

    In reverse-phase protein arrays (RPPA), one immobilizes complex samples (e.g., cellular lysate, tissue lysate or serum etc.) on solid supports and performs parallel reactions of antibodies with immobilized protein targets from the complex samples. In this work, we describe a label-free detection of RPPA that enables quantification of RPPA data and thus facilitates comparison of studies performed on different samples and on different solid supports. We applied this detection platform to characterization of phosphoserine aminotransferase (PSAT) expression levels in Acanthamoeba lysates treated with artemether and the results were confirmed by Western blot studies. PMID:27372609

  17. Preferential expression and immunogenicity of HIV-1 Tat fusion protein expressed in tomato plant.

    PubMed

    Cueno, Marni E; Hibi, Yurina; Karamatsu, Katsuo; Yasutomi, Yasuhiro; Imai, Kenichi; Laurena, Antonio C; Okamoto, Takashi

    2010-10-01

    HIV-1 Tat plays a major role in viral replication and is essential for AIDS development making it an ideal vaccine target providing that both humoral and cellular immune responses are induced. Plant-based antigen production, due to its cheaper cost, appears ideal for vaccine production. In this study, we created a plant-optimized tat and mutant (Cys30Ala/Lys41Ala) tat (mtat) gene and ligated each into a pBI121 expression vector with a stop codon and a gusA gene positioned immediately downstream. The vector construct was bombarded into tomato leaf calli and allowed to develop. We thus generated recombinant tomato plants preferentially expressing a Tat-GUS fusion protein over a Tat-only protein. In addition, plants bombarded with either tat or mtat genes showed no phenotypic difference and produced 2-4 microg Tat-GUS fusion protein per milligram soluble plant protein. Furthermore, tomato extracts intradermally inoculated into mice were found to induce a humoral and, most importantly, cellular immunity. PMID:20072815

  18. Cell-Free Protein Expression under Macromolecular Crowding Conditions

    PubMed Central

    Ge, Xumeng; Luo, Dan; Xu, Jianfeng

    2011-01-01

    Background Cell-free protein expression (CFPE) comprised of in vitro transcription and translation is currently manipulated in relatively dilute solutions, in which the macromolecular crowding effects present in living cells are largely ignored. This may not only affect the efficiency of protein synthesis in vitro, but also limit our understanding of the functions and interactions of biomolecules involved in this fundamental biological process. Methodology/Principal Findings Using cell-free synthesis of Renilla luciferase in wheat germ extract as a model system, we investigated the CFPE under macromolecular crowding environments emulated with three different crowding agents: PEG-8000, Ficoll-70 and Ficoll-400, which vary in chemical properties and molecular size. We found that transcription was substantially enhanced in the macromolecular crowding solutions; up to 4-fold increase in the mRNA production was detected in the presence of 20% (w/v) of Ficoll-70. In contrast, translation was generally inhibited by the addition of each of the three crowding agents. This might be due to PEG-induced protein precipitation and non-specific binding of translation factors to Ficoll molecules. We further explored a two-stage CFPE in which transcription and translation was carried out under high then low macromolecular crowding conditions, respectively. It produced 2.2-fold higher protein yield than the coupled CFPE control. The macromolecular crowding effects on CFPE were subsequently confirmed by cell-free synthesis of an approximately two-fold larger protein, Firefly luciferase, under macromolecular crowding environments. Conclusions/Significance Three macromolecular crowding agents used in this research had opposite effects on transcription and translation. The results of this study should aid researchers in their choice of macromolecular crowding agents and shows that two-stage CFPE is more efficient than coupled CFPE. PMID:22174874

  19. Neurotoxocarosis alters myelin protein gene transcription and expression.

    PubMed

    Heuer, Lea; Beyerbach, Martin; Lühder, Fred; Beineke, Andreas; Strube, Christina

    2015-06-01

    Neurotoxocarosis is an infection of the central nervous system caused by migrating larvae of the common dog and cat roundworms (Toxocara canis and Toxocara cati), which are zoonotic agents. As these parasites are prevalent worldwide and neuropathological and molecular investigations on neurotoxocarosis are scare, this study aims to characterise nerve fibre demyelination associated with neurotoxocarosis on a molecular level. Transcription of eight myelin-associated genes (Cnp, Mag, Mbp, Mog, Mrf-1, Nogo-A, Plp1, Olig2) was determined in the mouse model during six time points of the chronic phase of infection using qRT-PCR. Expression of selected proteins was analysed by Western blotting or immunohistochemistry. Additionally, demyelination and neuronal damage were investigated histologically. Significant differences (p ≤ 0.05) between transcription rates of T. canis-infected and uninfected control mice were detected for all analysed genes while T. cati affected five of eight investigated genes. Interestingly, 2', 3 ´-cyclic nucleotide 3'-phosphodiesterase (Cnp) and myelin oligodendrocyte glycoprotein (Mog) were upregulated in both T. canis- and T. cati-infected mice preceding demyelination. Later, CNPase expression was additionally enhanced. As expected, myelin basic protein (Mbp) was downregulated in cerebra and cerebella of T. canis-infected mice when severe demyelination was present 120 days post infectionem (dpi). The transcriptional pattern observed in the present study appears to reflect direct traumatic and hypoxic effects of larval migration as well as secondary processes including host immune reactions, demyelination and attempts to remyelinate damaged areas.

  20. PPAR-β/δ activation promotes phospholipid transfer protein expression.

    PubMed

    Chehaibi, Khouloud; Cedó, Lídia; Metso, Jari; Palomer, Xavier; Santos, David; Quesada, Helena; Naceur Slimane, Mohamed; Wahli, Walter; Julve, Josep; Vázquez-Carrera, Manuel; Jauhiainen, Matti; Blanco-Vaca, Francisco; Escolà-Gil, Joan Carles

    2015-03-15

    The peroxisome proliferator-activated receptor (PPAR)-β/δ has emerged as a promising therapeutic target for treating dyslipidemia, including beneficial effects on HDL cholesterol (HDL-C). In the current study, we determined the effects of the PPAR-β/δ agonist GW0742 on HDL composition and the expression of liver HDL-related genes in mice and cultured human cells. The experiments were carried out in C57BL/6 wild-type, LDL receptor (LDLR)-deficient mice and PPAR-β/δ-deficient mice treated with GW0742 (10mg/kg/day) or a vehicle solution for 14 days. GW0742 upregulated liver phospholipid transfer protein (Pltp) gene expression and increased serum PLTP activity in mice. When given to wild-type mice, GW0742 significantly increased serum HDL-C and HDL phospholipids; GW0742 also raised serum potential to generate preβ-HDL formation. The GW0742-mediated effects on liver Pltp expression and serum enzyme activity were completely abolished in PPAR-β/δ-deficient mice. GW0742 also stimulated PLTP mRNA expression in mouse J774 macrophages, differentiated human THP-1 macrophages and human hepatoma Huh7. Collectively, our findings demonstrate a common transcriptional upregulation by GW0742-activated PPAR-β/δ of Pltp expression in cultured cells and in mouse liver resulting in enhanced serum PLTP activity. Our results also indicate that PPAR-β/δ activation may modulate PLTP-mediated preβ-HDL formation and macrophage cholesterol efflux.

  1. Exposure to Paper Mill Effluent at a Site in North Central Florida Elicits Molecular-Level Changes in Gene Expression Indicative of Progesterone and Androgen Exposure

    PubMed Central

    Brockmeier, Erica K.; Jayasinghe, B. Sumith; Pine, William E.; Wilkinson, Krystan A.; Denslow, Nancy D.

    2014-01-01

    Endocrine disrupting compounds (EDCs) are chemicals that negatively impact endocrine system function, with effluent from paper mills one example of this class of chemicals. In Florida, female Eastern mosquitofish (Gambusia holbrooki) have been observed with male secondary sexual characteristics at three paper mill-impacted sites, indicative of EDC exposure, and are still found at one site on the Fenholloway River. The potential impacts that paper mill effluent exposure has on the G. holbrooki endocrine system and the stream ecosystem are unknown. The objective of this study was to use gene expression analysis to determine if exposure to an androgen receptor agonist was occurring and to couple this analysis with in vitro assays to evaluate the presence of androgen and progesterone receptor active chemicals in the Fenholloway River. Focused gene expression analyses of masculinized G. holbrooki from downstream of the Fenholloway River paper mill were indicative of androgen exposure, while genes related to reproduction indicated potential progesterone exposure. Hepatic microarray analysis revealed an increase in the expression of metabolic genes in Fenholloway River fish, with similarities in genes and biological processes compared to G. holbrooki exposed to androgens. Water samples collected downstream of the paper mill and at a reference site indicated that progesterone and androgen receptor active chemicals were present at both sites, which corroborates previous chemical analyses. Results indicate that G. holbrooki downstream of the Fenholloway River paper mill are impacted by a mixture of both androgens and progesterones. This research provides data on the mechanisms of how paper mill effluents in Florida are acting as endocrine disruptors. PMID:25198161

  2. Exposure to paper mill effluent at a site in North Central Florida elicits molecular-level changes in gene expression indicative of progesterone and androgen exposure.

    PubMed

    Brockmeier, Erica K; Jayasinghe, B Sumith; Pine, William E; Wilkinson, Krystan A; Denslow, Nancy D

    2014-01-01

    Endocrine disrupting compounds (EDCs) are chemicals that negatively impact endocrine system function, with effluent from paper mills one example of this class of chemicals. In Florida, female Eastern mosquitofish (Gambusia holbrooki) have been observed with male secondary sexual characteristics at three paper mill-impacted sites, indicative of EDC exposure, and are still found at one site on the Fenholloway River. The potential impacts that paper mill effluent exposure has on the G. holbrooki endocrine system and the stream ecosystem are unknown. The objective of this study was to use gene expression analysis to determine if exposure to an androgen receptor agonist was occurring and to couple this analysis with in vitro assays to evaluate the presence of androgen and progesterone receptor active chemicals in the Fenholloway River. Focused gene expression analyses of masculinized G. holbrooki from downstream of the Fenholloway River paper mill were indicative of androgen exposure, while genes related to reproduction indicated potential progesterone exposure. Hepatic microarray analysis revealed an increase in the expression of metabolic genes in Fenholloway River fish, with similarities in genes and biological processes compared to G. holbrooki exposed to androgens. Water samples collected downstream of the paper mill and at a reference site indicated that progesterone and androgen receptor active chemicals were present at both sites, which corroborates previous chemical analyses. Results indicate that G. holbrooki downstream of the Fenholloway River paper mill are impacted by a mixture of both androgens and progesterones. This research provides data on the mechanisms of how paper mill effluents in Florida are acting as endocrine disruptors. PMID:25198161

  3. Ras protein expression as a marker for breast cancer

    PubMed Central

    CALAF, GLORIA M.; ABARCA-QUINONES, JORGE

    2016-01-01

    Breast cancer, the most common neoplasm in women of all ages, is the leading cause of cancer-related mortality in women worldwide. Markers to help to predict the risk of progression and ultimately provide non-surgical treatment options would be of great benefit. At present, there are no available molecular markers to predict the risk of carcinoma in situ progression to invasive cancer; therefore, all women diagnosed with this type of malignancy must undergo surgery. Breast cancer is a heterogeneous complex disease, and different patients respond differently to different treatments. In breast cancer, analysis using immunohistochemical markers remains an essential component of routine pathological examinations, and plays an import role in the management of the disease by providing diagnostic and prognostic strategies. The aim of the present study was to identify a marker that can be used as a prognostic tool for breast cancer. For this purpose, we firstly used an established breast cancer model. MCF-10F, a spontaneously immortalized breast epithelial cell line was transformed by exposure to estrogen and radiation. MCF-10F cells were exposed to low doses of high linear energy transfer (LET) α particles (150 keV/μm) of radiation, and subsequently cultured in the presence of 17β-estradiol. Three cell lines were used: i) MCF-10F cells as a control; ii) Alpha5 cells, a malignant and tumorigenic cell line; and iii) Tumor2 cells derived from Alpha5 cells injected into nude mice. Secondly, we also used normal, benign and malignant breast specimens obtained from biopsies. The results revealed that the MCF-10F cells were negative for c-Ha-Ras protein expression; however, the Alpha5 and Tumor2 cell lines were positive for c-Ha-Ras protein expression. The malignant breast samples were also strongly positive for c-Ha-Ras expression. The findings of our study indicate that c-Ha-Ras protein expression may be used as a marker to predict the progression of breast cancer; this

  4. mRNA expression and protein localization of dentin matrix protein 1 during dental root formation.

    PubMed

    Toyosawa, S; Okabayashi, K; Komori, T; Ijuhin, N

    2004-01-01

    Dentin matrix protein 1 (DMP1) is an acidic phosphoprotein. DMP1 was initially detected in dentin and later in other mineralized tissues including cementum and bone, but the DMP1 expression pattern in tooth is still controversial. To determine the precise localization of DMP1 messenger RNA (mRNA) and the protein in the tooth, we performed in situ hybridization and immunohistochemical analyses using rat molars and incisors during various stages of root formation. During root dentin formation of molars, DMP1 mRNA was detected in root odontoblasts in parallel with mineralization of the dentin. However, the level of DMP1 mRNA expression in root odontoblasts decreased near the coronal part and was absent in coronal odontoblasts. DMP1 protein was localized along dentinal tubules and their branches in mineralized root dentin, and the distribution of DMP1 shifted from the end of dentinal tubules to the base of the tubules as dentin formation progressed. During the formation of the acellular cementum, DMP1 mRNA was detected in cementoblasts lining the acellular cementum where its protein was localized. During the formation of the cellular cementum, DMP1 mRNA was detected in cementocytes embedded in the cellular cementum but not in cementoblasts, and its protein was localized in the pericellular cementum of cementocytes including their processes. During dentin formation of incisors, DMP1 mRNA was detected in odontoblasts on the cementum-related dentin, where its protein was localized along dentinal tubules near the mineralization front. The localization of DMP1 mRNA and protein in dentin and cementum was related to their mineralization, suggesting that one of the functions of DMP1 may be involved in the mineralization of dentin and cementum during root formation. PMID:14751569

  5. Immunogenicity of Escherichia coli expressed envelope 2 protein of Chikungunya virus.

    PubMed

    Tripathi, Nagesh K; Priya, Raj; Shrivastava, Ambuj

    2014-01-01

    Chikungunya fever, a re-emerging infection, is an arthropod-borne viral disease prevalent in different parts of the world, particularly Africa and South East Asia. Chikungunya virus envelope 2 protein is involved in binding to host receptors and it contains specific epitopes that elicit virus neutralizing antibodies. A highly immunogenic, recombinant Chikungunya virus envelope 2 protein was produced by bioreactor in Escherichia coli for development of a suitable diagnostic and vaccine candidate. This protein was refolded and further purified to achieve biologically active protein. The biological function of refolded and purified recombinant envelope 2 protein of Chikungunya virus was confirmed by its ability to generate envelope 2 specific antibodies with high titers in animal models. These findings suggest that recombinant envelope 2 protein of Chikungunya virus in combination with compatible adjuvant is highly immunogenic. Thus, recombinant envelope 2 protein can be a potential diagnostic reagent and vaccine candidate against Chikungunya virus infection.

  6. Real-time quantification of protein expression at the single-cell level via dynamic protein synthesis translocation reporters.

    PubMed

    Aymoz, Delphine; Wosika, Victoria; Durandau, Eric; Pelet, Serge

    2016-01-01

    Protein expression is a dynamic process, which can be rapidly induced by extracellular signals. It is widely appreciated that single cells can display large variations in the level of gene induction. However, the variability in the dynamics of this process in individual cells is difficult to quantify using standard fluorescent protein (FP) expression assays, due to the slow maturation of their fluorophore. Here we have developed expression reporters that accurately measure both the levels and dynamics of protein synthesis in live single cells with a temporal resolution under a minute. Our system relies on the quantification of the translocation of a constitutively expressed FP into the nucleus. As a proof of concept, we used these reporters to measure the transient protein synthesis arising from two promoters responding to the yeast hyper osmolarity glycerol mitogen-activated protein kinase pathway (pSTL1 and pGPD1). They display distinct expression dynamics giving rise to strikingly different instantaneous expression noise. PMID:27098003

  7. Real-time quantification of protein expression at the single-cell level via dynamic protein synthesis translocation reporters

    PubMed Central

    Aymoz, Delphine; Wosika, Victoria; Durandau, Eric; Pelet, Serge

    2016-01-01

    Protein expression is a dynamic process, which can be rapidly induced by extracellular signals. It is widely appreciated that single cells can display large variations in the level of gene induction. However, the variability in the dynamics of this process in individual cells is difficult to quantify using standard fluorescent protein (FP) expression assays, due to the slow maturation of their fluorophore. Here we have developed expression reporters that accurately measure both the levels and dynamics of protein synthesis in live single cells with a temporal resolution under a minute. Our system relies on the quantification of the translocation of a constitutively expressed FP into the nucleus. As a proof of concept, we used these reporters to measure the transient protein synthesis arising from two promoters responding to the yeast hyper osmolarity glycerol mitogen-activated protein kinase pathway (pSTL1 and pGPD1). They display distinct expression dynamics giving rise to strikingly different instantaneous expression noise. PMID:27098003

  8. Neurodevelopmental alcohol exposure elicits long-term changes to gene expression that alter distinct molecular pathways dependent on timing of exposure

    PubMed Central

    2013-01-01

    Background Maternal alcohol consumption is known to adversely affect fetal neurodevelopment. While it is known that alcohol dose and timing play a role in the cognitive and behavioral changes associated with prenatal alcohol exposure, it is unclear what developmental processes are disrupted that may lead to these phenotypes. Methods Mice (n=6 per treatment per developmental time) were exposed to two acute doses of alcohol (5 g/kg) at neurodevelopmental times representing the human first, second, or third trimester equivalent. Mice were reared to adulthood and changes to their adult brain transcriptome were assessed using expression arrays. These were then categorized based on Gene Ontology annotations, canonical pathway associations, and relationships to interacting molecules. Results The results suggest that ethanol disrupts biological processes that are actively occurring at the time of exposure. These include cell proliferation during trimester one, cell migration and differentiation during trimester two, and cellular communication and neurotransmission during trimester three. Further, although ethanol altered a distinct set of genes depending on developmental timing, many of these show interrelatedness and can be associated with one another via ‘hub’ molecules and pathways such as those related to huntingtin and brain-derived neurotrophic factor. Conclusions These changes to brain gene expression represent a ‘molecular footprint’ of neurodevelopmental alcohol exposure that is long-lasting and correlates with active processes disrupted at the time of exposure. This study provides further support that there is no neurodevelopmental time when alcohol cannot adversely affect the developing brain. PMID:23497526

  9. Expression Analysis of Phenylalanine Ammonia Lyase Gene and Rosmarinic Acid Production in Salvia officinalis and Salvia virgata Shoots Under Salicylic Acid Elicitation.

    PubMed

    Ejtahed, Roghayeh Sadat; Radjabian, Tayebeh; Hoseini Tafreshi, Sayed Ali

    2015-08-01

    Partial fragments of phenylalanine ammonia lyase (PAL) genes were cloned and characterized from Salvia officinalis (SoPAL) and Salvia virgata (SvPAL). Different concentrations (250 and 500 μM) of exogenous salicylic acid (SA) were used when correlation between PAL expression and rosmarinic acid (RA) accumulation was compared. The results showed that the deduced cDNA sequences of the partial genes had high similarities with those of known PAL gene from other plant species. Semi-quantitative reverse transcription PCR (RT-PCR) analysis revealed that exogenous application of SA led to up-regulating of the PAL expression. Further analysis showed that in S. virgata, at higher concentration of SA, higher accumulation of RA was achieved, while in S. officinalis, the higher RA accumulation was observed at lower concentration of SA. It was concluded that there was no positive correlation between the intensity of PAL transcription and the RA accumulation in the studied species. Therefore, despite of the increase in transcription rate of the PAL at the higher concentration of SA, the lower amounts of RA were accumulated in the case of S. officinalis. Consequently, the hypothesis that PAL is the rate-determining step in RA biosynthesis is not always valid and probably some other unknown factors participate in the synthesis of phenolics.

  10. Centrifugal forces within usually-used magnitude elicited a transitory and reversible change in proliferation and gene expression of osteoblastic cells UMR-106.

    PubMed

    Li, Juan; Jiang, Lingyong; Liao, Ga; Chen, Guoping; Liu, Ying; Wang, Jun; Zheng, Yi; Luo, Songjiao; Zhao, Zhihe

    2009-02-01

    Centrifugation is an important step in biochemical and molecular biological researches. But the effects of centrifugal stress on cells are still unclear. In this study, osteoblastic cells UMR-106 were subjected to a moderate centrifugal stress at 209 x g for 10 min. Then the cell proliferation and gene transcription after centrifugation were analyzed with flow cytometry and Real-time RT-PCR techniques, respectively. The result showed that the cell proliferation and mRNA expression of Runx2/Cbfa1, Collagen I and osteocalcin changed shortly after centrifugal loading, but recovered to pre-load levels within 24 h. A dose-response study of exposure cells to centrifugal force at 209, 253 and 301 x g showed that the centrifugal forces within usually-used range can rapidly influenced the mRNA expression of the osteoblast-specific genes, but no statistical differences were found among the three centrifugal magnitudes. And the fast regulation in the investigated genes was proved to be related to increased c-fos mRNA levels and subsequent activation of RTK and integrity of cytoskeleton construction. The result showed that the osteoblastic cells displayed a fast auto-regulation to usually-used centrifugal stress through multiple signal pathways.

  11. Quinacrine induces apoptosis in human leukemia K562 cells via p38 MAPK-elicited BCL2 down-regulation and suppression of ERK/c-Jun-mediated BCL2L1 expression

    SciTech Connect

    Changchien, Jung-Jung; Chen, Ying-Jung; Huang, Chia-Hui; Cheng, Tian-Lu; Lin, Shinne-Ren; Chang, Long-Sen

    2015-04-01

    Although previous studies have revealed the anti-cancer activity of quinacrine, its effect on leukemia is not clearly resolved. We sought to explore the cytotoxic effect and mechanism of quinacrine action in human leukemia K562 cells. Quinacrine induced K562 cell apoptosis accompanied with ROS generation, mitochondrial depolarization, and down-regulation of BCL2L1 and BCL2. Upon exposure to quinacrine, ROS-mediated p38 MAPK activation and ERK inactivation were observed in K562 cells. Quinacrine-induced cell death and mitochondrial depolarization were suppressed by the p38MAPK inhibitor SB202190 and constitutively active MEK1 over-expression. Activation of p38 MAPK was shown to promote BCL2 degradation. Further, ERK inactivation suppressed c-Jun-mediated transcriptional expression of BCL2L1. Over-expression of BCL2L1 and BCL2 attenuated quinacrine-evoked mitochondrial depolarization and rescued the viability of quinacrine-treated cells. Taken together, our data indicate that quinacrine-induced K562 cell apoptosis is mediated through mitochondrial alterations triggered by p38 MAPK-mediated BCL2 down-regulation and suppression of ERK/c-Jun-mediated BCL2L1 expression. - Highlights: • Quinacrine induces K562 cell apoptosis via down-regulation of BCL2 and BCL2L1. • Quinacrine induces p38 MAPK activation and ERK inactivation in K562 cells. • Quinacrine elicits p38 MAPK-mediated BCL2 down-regulation. • Quinacrine suppresses ERK/c-Jun-mediated BCL2L1 expression.

  12. Protein expression patterns of the yeast mating response.

    PubMed

    Yuan, Haiyu; Zhang, Rongfei; Shao, Bin; Wang, Xuan; Ouyang, Qi; Hao, Nan; Luo, Chunxiong

    2016-06-13

    Microfluidics, in combination with time-lapse microscopy, is a transformative technology that significantly enhances our ability to monitor and probe biological processes in living cells. However, high-throughput microfluidic devices mostly require sophisticated preparatory and setup work and are thus hard to adopt by non-experts. In this work, we designed an easy-to-use microfluidic chip, which enables tracking of 48 GFP-tagged yeast strains, with each strain under two different stimulus conditions, in a single experiment. We used this technology to investigate the dynamic pattern of protein expression during the yeast mating differentiation response. High doses of pheromone induce cell cycle arrest and the shmoo morphology, whereas low doses of pheromone lead to elongation and chemotrophic growth. By systematically analyzing the protein dynamics of 156 pheromone-regulated genes, we identified groups of genes that are preferentially induced in response to low-dose pheromone (elongation during growth) or high-dose pheromone (shmoo formation and cell cycle arrest). The protein dynamics of these genes may provide insights into the mechanisms underlying the differentiation switch induced by different doses of pheromone. PMID:27177258

  13. Expression of Water Channel Proteins in Mesembryanthemum crystallinum1

    PubMed Central

    Kirch, Hans-Hubert; Vera-Estrella, Rosario; Golldack, Dortje; Quigley, Francoise; Michalowski, Christine B.; Barkla, Bronwyn J.; Bohnert, Hans J.

    2000-01-01

    We have characterized transcripts for nine major intrinsic proteins (MIPs), some of which function as water channels (aquaporins), from the ice plant Mesembryanthemum crystallinum. To determine the cellular distribution and expression of these MIPs, oligopeptide-based antibodies were generated against MIP-A, MIP-B, MIP-C, or MIP-F, which, according to sequence and functional characteristics, are located in the plasma membrane (PM) and tonoplast, respectively. MIPs were most abundant in cells involved in bulk water flow and solute flux. The tonoplast MIP-F was found in all cells, while signature cell types identified different PM-MIPs: MIP-A predominantly in phloem-associated cells, MIP-B in xylem parenchyma, and MIP-C in the epidermis and endodermis of immature roots. Membrane protein analysis confirmed MIP-F as tonoplast located. MIP-A and MIP-B were found in tonoplast fractions and also in fractions distinct from either the tonoplast or PM. MIP-C was most abundant but not exclusive to PM fractions, where it is expected based on its sequence signature. We suggest that within the cell, MIPs are mobile, which is similar to aquaporins cycling through animal endosomes. MIP cycling and the differential regulation of these proteins observed under conditions of salt stress may be fundamental for the control of tissue water flux. PMID:10806230

  14. Nonnative proteins induce expression of the Bacillus subtilis CIRCE regulon.

    PubMed

    Mogk, A; Völker, A; Engelmann, S; Hecker, M; Schumann, W; Völker, U

    1998-06-01

    The chaperone-encoding groESL and dnaK operons constitute the CIRCE regulon of Bacillus subtilis. Both operons are under negative control of the repressor protein HrcA, which interacts with the CIRCE operator and whose activity is modulated by the GroESL chaperone machine. In this report, we demonstrate that induction of the CIRCE regulon can also be accomplished by ethanol stress and puromycin. Introduction of the hrcA gene and a transcriptional fusion under the control of the CIRCE operator into Escherichia coli allowed induction of this fusion by heat shock, ethanol stress, and overproduction of GroESL substrates. The expression level of this hrcA-bgaB fusion inversely correlated with the amount of GroE machinery present in the cells. Therefore, all inducing conditions seem to lead to induction via titration of the GroE chaperonins by the increased level of nonnative proteins formed. Puromycin treatment failed to induce the sigmaB-dependent general stress regulon, indicating that nonnative proteins in general do not trigger this response. Reconstitution of HrcA-dependent heat shock regulation of B. subtilis in E. coli and complementation of E. coli groESL mutants by B. subtilis groESL indicate that the GroE chaperonin systems of the two bacterial species are functionally exchangeable. PMID:9603878

  15. Discrete phosphorylated Retinoblastoma protein isoform expression in mouse tooth development

    PubMed Central

    Zhang, Weibo; Vazquez, Betsy; Andreeva, Viktoria; Spear, Daisy; Kong, Elizabeth; Hinds, Philip W.; Yelick, Pamela C.

    2015-01-01

    It is widely accepted that Retinoblastoma protein (pRb) phosphorylation plays a central role in mediating cell cycle G1/S stage transition, together with E2 promoter-binding factors (E2F). The binding of pRb to E2F is controlled by the sequential and cumulative phosphorylation of pRb at various amino acids. In addition to the well characterized roles for pRb as a tumor suppressor, pRb has more recently been implicated in osteoprogenitor and other types of stem cell maintenance, proliferation and differentiation, thereby influencing the morphogenesis of developing organs. In this study, we present data characterizing the expression of three phosphorylated pRb (ppRb) isoforms - ppRbS780, ppRbS795, and ppRbS807/811- in developing mouse molar and incisor tooth buds. Also, we analyzed the co-localization of pRb isoforms and histone H3 expression in incisor tooth buds. Our results reveal distinct developmental expression patterns for individual ppRb isoforms in differentiating dental epithelial and dental mesenchymal cells, suggesting discrete functions for each in tooth development. PMID:22476877

  16. Improved protein quality in transgenic soybean expressing a de novo synthetic protein, MB-16.

    PubMed

    Zhang, Yunfang; Schernthaner, Johann; Labbé, Natalie; Hefford, Mary A; Zhao, Jiping; Simmonds, Daina H

    2014-06-01

    To improve soybean [Glycine max (L.) Merrill] seed nutritional quality, a synthetic gene, MB-16 was introduced into the soybean genome to boost seed methionine content. MB-16, an 11 kDa de novo protein enriched in the essential amino acids (EAAs) methionine, threonine, lysine and leucine, was originally developed for expression in rumen bacteria. For efficient seed expression, constructs were designed using the soybean codon bias, with and without the KDEL ER retention sequence, and β-conglycinin or cruciferin seed specific protein storage promoters. Homozygous lines, with single locus integrations, were identified for several transgenic events. Transgene transmission and MB-16 protein expression were confirmed to the T5 and T7 generations, respectively. Quantitative RT-PCR analysis of developing seed showed that the transcript peaked in growing seed, 5-6 mm long, remained at this peak level to the full-sized green seed and then was significantly reduced in maturing yellow seed. Transformed events carrying constructs with the rumen bacteria codon preference showed the same transcription pattern as those with the soybean codon preference, but the transcript levels were lower at each developmental stage. MB-16 protein levels, as determined by immunoblots, were highest in full-sized green seed but the protein virtually disappeared in mature seed. However, amino acid analysis of mature seed, in the best transgenic line, showed a significant increase of 16.2 and 65.9 % in methionine and cysteine, respectively, as compared to the parent. This indicates that MB-16 elevated the sulfur amino acids, improved the EAA seed profile and confirms that a de novo synthetic gene can enhance the nutritional quality of soybean.

  17. Influence of FcγRIIa-Expressing Cells on the Assessment of Neutralizing and Enhancing Serum Antibodies Elicited by a Live-Attenuated Tetravalent Dengue Vaccine

    PubMed Central

    Byers, Anthony M.; Broder, Ryan; Haupfear, Kelly; Timiryasova, Tatyana M.; Hu, Branda T.; Boaz, Mark; Warren, William L.; Jackson, Nicholas; Moser, Janice M.; Guy, Bruno

    2015-01-01

    Background. Recent trials of recombinant, live-attenuated chimeric yellow fever-dengue tetravalent dengue vaccine (CYD-TDV) demonstrated efficacy against symptomatic, virologically confirmed dengue disease with higher point estimates of efficacy toward dengue virus (DENV)3 and DENV4 and moderate levels toward DENV1 and DENV2. It is interesting to note that serotype-specific efficacy did not correlate with absolute neutralizing antibody (nAb) geometric mean titer (GMT) values measured in a Vero-based plaque reduction neutralization test assay. The absence of Fcγ receptors on Vero cells may explain this observation. Methods. We performed parallel seroneutralization assays in Vero cells and CV-1 cells that express FcγRIIa (CV-1-Fc) to determine the neutralizing and enhancing capacity of serotype-specific DENV Abs present in CYD-TDV clinical trial sera. Results. Enhancement of DENV infection was observed in CV-1-Fc cells in naturally exposed nonvaccine sera, mostly for DENV3 and DENV4, at high dilutions. The CYD-TDV-vaccinated sera showed similar enhancement patterns. The CV-1-Fc nAb GMT values were 2- to 9-fold lower than Vero for all serotypes in both naturally infected individuals and CYD-TDV-vaccinated subjects with and without previous dengue immunity. The relative (CV-1-Fc/Vero) GMT decrease for anti-DENV1 and anti-DENV2 responses was not greater than for the other serotypes. Conclusions. In vitro neutralization assays utilizing FcγRIIa-expressing cells provide evidence that serotype-specific Ab enhancement may not be a primary factor in the serotype-specific efficacy differences exhibited in the CYD-TDV trials. PMID:26719844

  18. Regulation of RE1 protein silencing transcription factor (REST) expression by HIP1 protein interactor (HIPPI).

    PubMed

    Datta, Moumita; Bhattacharyya, Nitai P

    2011-09-30

    Earlier we have shown that the proapoptotic protein HIPPI (huntingtin interacting protein 1 (HIP1) protein interactor) along with its molecular partner HIP1 could regulate transcription of the caspase-1 gene. Here we report that RE1-silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) is a new transcriptional target of HIPPI. HIPPI could bind to the promoter of REST and increased its expression in neuronal as well as non-neuronal cells. Such activation of REST down-regulated expression of REST target genes, such as brain-derived neurotrophic factor (BDNF) or proenkephalin (PENK). The ability of HIPPI to activate REST gene transcription was dependent on HIP1, the nuclear transporter of HIPPI. Using a Huntington disease cell model, we have demonstrated that feeble interaction of HIP1 with mutant huntingtin protein resulted in increased nuclear accumulation of HIPPI and HIP1, leading to higher occupancy of HIPPI at the REST promoter, triggering its transcriptional activation and consequent repression of REST target genes. This novel transcription regulatory mechanism of REST by HIPPI may contribute to the deregulation of transcription observed in the cell model of Huntington disease. PMID:21832040

  19. Differentially expressed protein markers in human submandibular and sublingual secretions.

    PubMed

    Hu, Shen; Denny, Patricia; Denny, Paul; Xie, Yongming; Loo, Joseph A; Wolinsky, Lawrence E; Li, Yang; McBride, Jim; Ogorzalek Loo, Rachel R; Navazesh, Mavash; Wong, David T

    2004-11-01

    Proteome analysis of secretions from individual salivary glands is important for understanding the health of the oral cavity and pathogenesis of certain diseases. However, cross-contamination of submandibular (SM) and sublingual (SL) glandular secretions can occur. The close anatomic relationship of the SM and SL ductal orifices can lead to such contamination. Additionally, these glands may share common ducts. To insure the purity of SM/SL secretions for proteomic analysis, it is important to develop unique biomarkers which could be used to verify the integrity of the individual glandular saliva. In this study, a proteomics approach based on mass spectrometry and gel electrophoresis techniques was utilized to identify and verify a set of proteins (cystatin C, calgranulin B and MUC5B mucin), which are differentially expressed in SM/SL secretions. SM/SL fluids were obtained from nine healthy subjects. Cystatin C was found to be an SM-selective protein as it was found in all SM fluids but not detected in two SL fluids. MUC5B mucin and calgranulin B, on the other hand, were found to be SL-selective proteins. All SL samples contained MUC5B mucin, whereas MUC5B mucin was not detected in four SM samples. Eight of the SL samples contained calgranulin B; however, calgranulin B was absent in eight SM samples. This set of protein markers, especially calgranulin B, can be used to determine the purity of SM/SL samples, and therefore identify potential individuals who do not exhibit cross-contaminated SM/SL secretions, an important requirement for subsequent proteome analysis of pure SM and SL secretions.

  20. Production of soluble mammalian proteins in Escherichia coli: identification of protein features that correlate with successful expression

    PubMed Central

    Dyson, Michael R; Shadbolt, S Paul; Vincent, Karen J; Perera, Rajika L; McCafferty, John

    2004-01-01

    Background In the search for generic expression strategies for mammalian protein families several bacterial expression vectors were examined for their ability to promote high yields of soluble protein. Proteins studied included cell surface receptors (Ephrins and Eph receptors, CD44), kinases (EGFR-cytoplasmic domain, CDK2 and 4), proteases (MMP1, CASP2), signal transduction proteins (GRB2, RAF1, HRAS) and transcription factors (GATA2, Fli1, Trp53, Mdm2, JUN, FOS, MAD, MAX). Over 400 experiments were performed where expression of 30 full-length proteins and protein domains were evaluated with 6 different N-terminal and 8 C-terminal fusion partners. Expression of an additional set of 95 mammalian proteins was also performed to test the conclusions of this study. Results Several protein features correlated with soluble protein expression yield including molecular weight and the number of contiguous hydrophobic residues and low complexity regions. There was no relationship between successful expression and protein pI, grand average of hydropathicity (GRAVY), or sub-cellular location. Only small globular cytoplasmic proteins with an average molecular weight of 23 kDa did not require a solubility enhancing tag for high level soluble expression. Thioredoxin (Trx) and maltose binding protein (MBP) were the best N-terminal protein fusions to promote soluble expression, but MBP was most effective as a C-terminal fusion. 63 of 95 mammalian proteins expressed at soluble levels of greater than 1 mg/l as N-terminal H10-MBP fusions and those that failed possessed, on average, a higher molecular weight and greater number of contiguous hydrophobic amino acids and low complexity regions. Conclusions By analysis of the protein features identified here, this study will help predict which mammalian proteins and domains can be successfully expressed in E. coli as soluble product and also which are best targeted for a eukaryotic expression system. In some cases proteins may be truncated

  1. Escherichia coli K88ac fimbriae expressing heat-labile and heat-stable (STa) toxin epitopes elicit antibodies that neutralize cholera toxin and STa toxin and inhibit adherence of K88ac fimbrial E. coli.

    PubMed

    Zhang, Chengxian; Zhang, Weiping

    2010-12-01

    Enterotoxigenic Escherichia coli (ETEC) strains are a major cause of diarrheal disease in humans and animals. Bacterial adhesins and heat-labile (LT) and heat-stable (ST) enterotoxins are the virulence determinants in ETEC diarrhea. It is believed that vaccines inducing anti-adhesin immunity to inhibit bacterial adherence and anti-toxin immunity to eliminate toxin activity would provide broad-spectrum protection against ETEC. In this study, an ETEC fimbrial adhesin was used as a platform to express LT and STa for adhesin-toxin fusion antigens to induce anti-toxin and anti-adhesin immunity. An epitope from the B subunit of LT toxin (LTP1, (8)LCSEYRNTQIYTIN(21)) and an STa toxoid epitope ((5)CCELCCNPQCAGCY(18)) were embedded in the FaeG major subunit of E. coli K88ac fimbriae. Constructed K88ac-toxin chimeric fimbriae were harvested and used for rabbit immunization. Immunized rabbits developed anti-K88ac, anti-LT, and anti-STa antibodies. Moreover, induced antibodies not only inhibited adherence of K88ac fimbrial E. coli to porcine small intestinal enterocytes but also neutralized cholera toxin and STa toxin. Data from this study demonstrated that K88ac fimbriae expressing LT and STa epitope antigens elicited neutralizing anti-toxin antibodies and anti-adhesin antibodies and suggested that E. coli fimbriae could serve as a platform for the development of broad-spectrum vaccines against ETEC. PMID:20980482

  2. Unconsciously elicited perceptual prior

    PubMed Central

    Chang, Raymond; Baria, Alexis T.; Flounders, Matthew W.; He, Biyu J.

    2016-01-01

    Increasing evidence over the past decade suggests that vision is not simply a passive, feed-forward process in which cortical areas relay progressively more abstract information to those higher up in the visual hierarchy, but rather an inferential process with top-down processes actively guiding and shaping perception. However, one major question that persists is whether such processes can be influenced by unconsciously perceived stimuli. Recent psychophysics and neuroimaging studies have revealed that while consciously perceived stimuli elicit stronger responses in higher visual and frontoparietal areas than those that fail to reach conscious awareness, the latter can still drive high-level brain and behavioral responses. We investigated whether unconscious processing of a masked natural image could facilitate subsequent conscious recognition of its degraded counterpart (a black-and-white “Mooney” image) presented many seconds later. We found that this is indeed the case, suggesting that conscious vision may be influenced by priors established by unconscious processing of a fleeting image. PMID:27595010

  3. [Eukaryotic Expression and Immunogenic Research of Recombination Ebola Virus Membrane Protein Gp-Fc].

    PubMed

    Zhang, Xiaoguang; Yang, Ren; Wang, Jiao; Wang, Xuan; Hou, Mieling; An, Lina; Zhu, Ying; Cao, Yuxi; Zeng, Yi

    2016-01-01

    We used 293 cells to express the recombinant membrane protein of the Ebola virus. Then, the immunogenicity of the recombinant protein was studied by immunized BALB/c mice. According to the codon use frequency of humans, the gene encoding the extracellular domain of the Ebola virus membrane protein was optimized, synthesized, and inserted into the eukaryotic expression plasmid pXG-Fc to construct the human IgG Fc and Ebola GP fusion protein expression plasmid pXG-modGP-Fc. To achieve expression, the fusion protein expression vector was transfected into high-density 293 cells using transient transfection technology. The recombinant protein was purified by protein A affinity chromatography. BALB/c mice were immunized with the purified fusion protein, and serum antibody titers evaluated by an indirect enzyme-linked immunosorbent assay (ELISA). Purification and analyses of the protein revealed that the eukaryotic expression vector could express the recombinant protein GP-Fc effectively, and that the recombinant protein in the supernatant of the cell culture was present as a dimer. After immunization with the purified recombinant protein, a high titer of antigen-specific IgG could be detected in the serum of immunized mice by indirect ELISA, showing that the recombinant protein had good immunogenicity. These data suggest that we obtained a recombinant protein with good immunogenicity. Our study is the basis for development of a vaccine against the Ebola virus and for screening of monoclonal antibodies.

  4. Lytic Promoters Express Protein during Herpes Simplex Virus Latency

    PubMed Central

    Russell, Tiffany A.; Tscharke, David C.

    2016-01-01

    Herpes simplex virus (HSV) has provided the prototype for viral latency with previously well-defined acute or lytic and latent phases. More recently, the deep quiescence of HSV latency has been questioned with evidence that lytic genes can be transcribed in this state. However, to date the only evidence that these transcripts might be translated has come from immunological studies that show activated T cells persist in the nervous system during latency. Here we use a highly sensitive Cre-marking model to show that lytic and latent phases are less clearly defined in two significant ways. First, around half of the HSV spread leading to latently infected sites occurred beyond the initial acute infection and second, we show direct evidence that lytic promoters can drive protein expression during latency. PMID:27348812

  5. Andrographis paniculata elicits anti-invasion activities by suppressing TM4SF3 gene expression and by anoikis-sensitization in esophageal cancer cells

    PubMed Central

    Yue, Grace Gar-Lee; Lee, Julia Kin-Ming; Li, Lin; Chan, Kar-Man; Wong, Eric Chun-Wai; Chan, Judy Yuet-Wah; Fung, Kwok-Pui; Lui, Vivian Wai Yan; Chiu, Philip Wai-Yan; Lau, Clara Bik-San

    2015-01-01

    Esophageal cancer is the sixth most common cancer in male causing death worldwide. It is usually diagnosed at advanced stage with high postoperative recurrence and systemic metastasis, which leads to poor prognosis. The potential inhibitory effect of herbal medicines on metastasis of esophageal cancer has drawn researchers’ great attention. In the present study, the anti-invasion activities of Andrographis paniculata (AP) have been evaluated in two esophageal cancer cell lines, EC-109 and KYSE-520, as well as human microvascular endothelial cells (HMEC-1). The anti-tumor and anti-metastatic activities of AP were also evaluated in human esophageal xenograft-bearing mouse models. Our results demonstrated for the first time that aqueous extract of AP inhibited the motility and invasion of esophageal cancer cells, which is the initial step of metastasis, without cytotoxicity. Anoikis resistance has also been reversed in AP-treated cancer cells. Besides, the expression of metastasis-related gene TM4SF3 in EC-109 cells was significantly decreased in AP extract-treated cells in a concentration-dependent manner. Furthermore, the anti-tumor and anti-metastatic efficacies in subcutaneous and intraperitoneal esophageal xenograft-bearing mice were demonstrated after oral administration of AP aqueous extract for 3 weeks. Last but not least, the active component, isoandrographolide, responsible for the anti-migratory activity was firstly revealed here. In conclusion, the AP aqueous extract exerted inhibitory activities on the migration and anoikis resistance of esophageal cancer cells EC-109 and KYSE-520, as well as suppressed the proliferation and motility of endothelial cells. Combining the mentioned effects may account for the anti-tumor and anti-metastasis effects of AP aqueous extract in xenograft-bearing mice. The findings in the present study further enhance the understanding of the therapeutic mechanisms of the herb AP, which may lead to clinical applications. PMID

  6. CXCL10/XCL1 fusokine elicits in vitro and in vivo chemotaxis.

    PubMed

    Sanchez-Lugo, Yessica E; Perez-Trujillo, Jose J; Gutierrez-Puente, Yolanda; Garcia-Garcia, Aracely; Rodriguez-Rocha, Humberto; Barboza-Quintana, Oralia; Muñoz-Maldonado, Gerardo E; Saucedo-Cardenas, Odila; de Oca-Luna, Roberto Montes; Loera-Arias, Maria J

    2015-04-01

    Fusokines are proteins formed by the fusion of two cytokines. They have greater bioavailability and therapeutic potential than individual cytokines or a combination of different cytokines. Interferon-gamma-inducible protein 10 (CXCL10) and lymphotactin (XCL1) are members of the chemotactic family of cytokines, which induce tumor regression by eliciting immune-system cell chemotaxis. We engineered a replication-deficient adenoviral system expressing CXCL10/XCL1 fusokine (Ad FIL) and assessed its chemotactic response in vitro and in vivo. The CXCL10/XCL1 fusokine elicited a greater chemotactic effect in IL-2 stimulated lymphocytes than individual or combined cytokines in vitro. CXCL10/XCL1 fusokine biological activity was demonstrated in vivo by intratumoral chemoattraction of CXCR3+ cells. Thus, this novel CXCL10/XCL1 fusokine may represent a potential tool for gene therapy treatment of cancer and other illnesses that require triggering immune-system cell recruitment.

  7. Phytomonas: A non-pathogenic trypanosomatid model for functional expression of proteins.

    PubMed

    Miranda, Mariana R; Sayé, Melisa; Reigada, Chantal; Carrillo, Carolina; Pereira, Claudio A

    2015-10-01

    Phytomonas are protozoan parasites from the Trypanosomatidae family which infect a wide variety of plants. Herein, Phytomonas Jma was tested as a model for functional expression of heterologous proteins. Green fluorescent protein expression was evaluated in Phytomonas and compared with Trypanosoma cruzi, the etiological agent of Chagas' disease. Phytomonas was able to express GFP at levels similar to T. cruzi although the transgenic selection time was higher. It was possible to establish an efficient transfection and selection protocol for protein expression. These results demonstrate that Phytomonas can be a good model for functional expression of proteins from other trypanosomatids, presenting the advantage of being completely safe for humans. PMID:26142019

  8. Phytomonas: A non-pathogenic trypanosomatid model for functional expression of proteins.

    PubMed

    Miranda, Mariana R; Sayé, Melisa; Reigada, Chantal; Carrillo, Carolina; Pereira, Claudio A

    2015-10-01

    Phytomonas are protozoan parasites from the Trypanosomatidae family which infect a wide variety of plants. Herein, Phytomonas Jma was tested as a model for functional expression of heterologous proteins. Green fluorescent protein expression was evaluated in Phytomonas and compared with Trypanosoma cruzi, the etiological agent of Chagas' disease. Phytomonas was able to express GFP at levels similar to T. cruzi although the transgenic selection time was higher. It was possible to establish an efficient transfection and selection protocol for protein expression. These results demonstrate that Phytomonas can be a good model for functional expression of proteins from other trypanosomatids, presenting the advantage of being completely safe for humans.

  9. Neurotoxocarosis alters myelin protein gene transcription and expression.

    PubMed

    Heuer, Lea; Beyerbach, Martin; Lühder, Fred; Beineke, Andreas; Strube, Christina

    2015-06-01

    Neurotoxocarosis is an infection of the central nervous system caused by migrating larvae of the common dog and cat roundworms (Toxocara canis and Toxocara cati), which are zoonotic agents. As these parasites are prevalent worldwide and neuropathological and molecular investigations on neurotoxocarosis are scare, this study aims to characterise nerve fibre demyelination associated with neurotoxocarosis on a molecular level. Transcription of eight myelin-associated genes (Cnp, Mag, Mbp, Mog, Mrf-1, Nogo-A, Plp1, Olig2) was determined in the mouse model during six time points of the chronic phase of infection using qRT-PCR. Expression of selected proteins was analysed by Western blotting or immunohistochemistry. Additionally, demyelination and neuronal damage were investigated histologically. Significant differences (p ≤ 0.05) between transcription rates of T. canis-infected and uninfected control mice were detected for all analysed genes while T. cati affected five of eight investigated genes. Interestingly, 2', 3 ´-cyclic nucleotide 3'-phosphodiesterase (Cnp) and myelin oligodendrocyte glycoprotein (Mog) were upregulated in both T. canis- and T. cati-infected mice preceding demyelination. Later, CNPase expression was additionally enhanced. As expected, myelin basic protein (Mbp) was downregulated in cerebra and cerebella of T. canis-infected mice when severe demyelination was present 120 days post infectionem (dpi). The transcriptional pattern observed in the present study appears to reflect direct traumatic and hypoxic effects of larval migration as well as secondary processes including host immune reactions, demyelination and attempts to remyelinate damaged areas. PMID:25773181

  10. Despite sequence homologies to gluten, salivary proline-rich proteins do not elicit immune responses central to the pathogenesis of celiac disease.

    PubMed

    Tian, Na; Leffler, Daniel A; Kelly, Ciaran P; Hansen, Joshua; Marietta, Eric V; Murray, Joseph A; Schuppan, Detlef; Helmerhorst, Eva J

    2015-12-01

    Celiac disease (CD) is an inflammatory disorder triggered by ingested gluten, causing immune-mediated damage to the small-intestinal mucosa. Gluten proteins are strikingly similar in amino acid composition and sequence to proline-rich proteins (PRPs) in human saliva. On the basis of this feature and their shared destination in the gastrointestinal tract, we hypothesized that salivary PRPs may modulate gluten-mediated immune responses in CD. Parotid salivary secretions were collected from CD patients, refractory CD patients, non-CD patients with functional gastrointestinal complaints, and healthy controls. Structural similarities of PRPs with gluten were probed with anti-gliadin antibodies. Immune responses to PRPs were investigated toward CD patient-derived peripheral blood mononuclear cells and in a humanized transgenic HLA-DQ2/DQ8 mouse model for CD. Anti-gliadin antibodies weakly cross-reacted with the abundant salivary amylase but not with PRPs. Likewise, the R5 antibody, recognizing potential antigenic gluten epitopes, showed negligible reactivity to salivary proteins from all groups. Inflammatory responses in peripheral blood mononuclear cells were provoked by gliadins whereas responses to PRPs were similar to control levels, and PRPs did not compete with gliadins in immune stimulation. In vivo, PRP peptides were well tolerated and nonimmunogenic in the transgenic HLA-DQ2/DQ8 mouse model. Collectively, although structurally similar to dietary gluten, salivary PRPs were nonimmunogenic in CD patients and in a transgenic HLA-DQ2/DQ8 mouse model for CD. It is possible that salivary PRPs play a role in tolerance induction to gluten early in life. Deciphering the structural basis for the lack of immunogenicity of salivary PRPs may further our understanding of the toxicity of gluten.

  11. Both cell substratum regulation and hormonal regulation of milk protein gene expression are exerted primarily at the posttranscriptional level

    SciTech Connect

    Eisenstein, R.S.; Rosen, J.M.

    1988-08-01

    The mechanism by which individual peptide and steroid hormones and cell-substratum interactions regulate milk protein gene expression has been studied in the COMMA-D mammary epithelial cell line. In the presence of insulin, hydrocortisone, and prolactin, growth of COMMA-D cells on floating collagen gels in comparison with that on a plastic substratum resulted in a 2.5- to 3-fold increase in the relative rate of ..beta..-casein gene transcription but a 37-fold increase in ..beta..-casein mRNA accumulation. In contrast, whey acidic protein gene transcription was constitutive in COMMA-D cells grown on either substratum, but its mRNA was unstable and little intact mature mRNA was detected. Culturing COMMA-D cells on collagen also promoted increased expression of other genes expressed in differentiated mammary epithelial cells, including those encoding ..cap alpha..- and ..gamma..-casein, transferrin, malic enzyme, and phosphoenolpyruvate carboxykinase but decreased the expression of actin and histone genes. Using COMMA-D cells, the authors defined further the role of individual hormones in influencing ..beta..-casein gene transcription. With insulin alone, a basal level of ..beta..-casein gene transcription was detected in COMMA-D cells grown on floating collagen gels. Addition of prolactin but not hydrocortisone resulted in a 2.5- to 3.0-fold increase in ..beta..-casein gene transcription, but both hormones were required to elicit the maximal 73-fold induction in mRNA accumulation. The posttranscriptional effect of hormones on casein mRNA accummulation preceded any detectable changes in the relative rate of transcription. Thus, regulation by both hormones and cell substratum of casein gene expression is exerted primarily at the post transcriptional level.

  12. Integrating clinical, gene expression, protein expression and preanalytical data for in silico cancer research.

    PubMed

    Rossille, Delphine; Burgun, Anita; Pangault-Lorho, Céline; Fest, Thierry

    2008-01-01

    We present the phase I development of an integrative platform for the analysis of clinical, gene expression, protein expression and pre-analytical data. The platform is aimed at providing transparent access and analysis tools to researchers investigating new biomarkers and prognosis factors in the particular field of lymphoma diseases. In this article, we report on the data integration phase. The platform's principal advantage is its completeness as it integrates in a single environment clinical, genomic and proteomic data, allowing for their combined analysis. The architecture consists in a data warehouse including data on patients, clinical trials and array platforms and a DeMilitarized Zone for data exchange. A secure web-based platform allows any collaborative team to request the data warehouse and access basic statistics on integrated data. The presented system is currently in use.

  13. F