Targeting foreign major histocompatibility complex molecules to tumors by tumor cell specific single chain antibody (scFv).

PubMed

Li, Jinhua; Franek, Karl J; Patterson, Andrea L; Holmes, Lillia M; Burgin, Kelly E; Ji, Jianfei; Yu, Xianzhong; Wagner, Thomas E; Wei, Yanzhang

2003-11-01

Down-regulation of the major histocompatibility complex (MHC) is one of the major mechanisms that tumor cells adopted to escape immunosurveillance. Therefore, specifically coating tumor cells with foreign MHC may make tumor cells a better target for immune recognition and surveillance. In this study, we designed and generated a fusion protein, H2Kd/scPSMA, consisting of a single chain antibody against human prostate specific membrane antigen (PSMA) and the extracellular domain of mouse H-2Kd. The expression of this fusion protein in B16F0 mouse melanoma cells was confirmed by RT-PCR and fluorescent activated cell sorting (FACS). Our animal study showed that the expression of H2Kd/scPSMA in B16F0/PSMA5, a B16F0 cell line expressing human PSMA, significantly inhibited tumor growth as demonstrated in the pulmonary metastasis assay and tumor growth study and improved overall survival.

Insect cell transformation vectors that support high level expression and promoter assessment in insect cell culture

USDA-ARS?s Scientific Manuscript database

A somatic transformation vector, pDP9, was constructed that provides a simplified means of producing permanently transformed cultured insect cells that support high levels of protein expression of foreign genes. The pDP9 plasmid vector incorporates DNA sequences from the Junonia coenia densovirus th...

A potyvirus-based gene vector allows producing active human S-COMT and animal GFP, but not human sorcin, in vector-infected plants.

PubMed

Kelloniemi, Jani; Mäkinen, Kristiina; Valkonen, Jari P T

2006-05-01

Potato virus A (PVA), a potyvirus with a (+)ssRNA genome translated to a large polyprotein, was engineered and used as a gene vector for expression of heterologous proteins in plants. Foreign genes including jellyfish GFP (Aequorea victoria) encoding the green fluorescent protein (GFP, 27 kDa) and the genes of human origin (Homo sapiens) encoding a soluble resistance-related calcium-binding protein (sorcin, 22 kDa) and the catechol-O-methyltransferase (S-COMT; 25 kDa) were cloned between the cistrons for the viral replicase and coat protein (CP). The inserts caused no adverse effects on viral infectivity and virulence, and the inserted sequences remained intact in progeny viruses in the systemically infected leaves. The heterologous proteins were released from the viral polyprotein following cleavage by the main viral proteinase, NIa, at engineered proteolytic processing sites flanking the insert. Active GFP, as indicated by green fluorescence, and S-COMT with high levels of enzymatic activity were produced. In contrast, no sorcin was detected despite the expected equimolar amounts of the foreign and viral proteins being expressed as a polyprotein. These data reveal inherent differences between heterologous proteins in their suitability for production in plants.

Rapid and efficient introduction of a foreign gene into bacterial artificial chromosome-cloned varicella vaccine by Tn7-mediated site-specific transposition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Somboonthum, Pranee; Koshizuka, Tetsuo; Okamoto, Shigefumi

2010-06-20

Using a rapid and reliable system based on Tn7-mediated site-specific transposition, we have successfully constructed a recombinant Oka varicella vaccine (vOka) expressing the mumps virus (MuV) fusion protein (F). The backbone of the vector was our previously reported vOka-BAC (bacterial artificial chromosome) genome. We inserted the transposon Tn7 attachment sequence, LacZ{alpha}-mini-attTn7, into the region between ORF12 and ORF13 to generate a vOka-BAC-Tn genome. The MuV-F expressing cassette was transposed into the vOka-BAC genome at the mini-attTn7 transposition site. MuV-F protein was expressed in recombinant virus, rvOka-F infected cells. In addition, the MuV-F protein was cleaved in the rvOka-F infected cellsmore » as in MuV-infected cells. The growth of rvOka-F was similar to that of the original recombinant vOka without the F gene. Thus, we show that Tn7-mediated transposition is an efficient method for introducing a foreign gene expression cassette into the vOka-BAC genome as a live virus vector.« less

Connexin 43 expression of foreign body giant cells after implantation of nanoparticulate hydroxyapatite.

PubMed

Herde, Katja; Hartmann, Sonja; Brehm, Ralph; Kilian, Olaf; Heiss, Christian; Hild, Anne; Alt, Volker; Bergmann, Martin; Schnettler, Reinhard; Wenisch, Sabine

2007-11-01

In bone a role of connexin 43 has been implicated with the fusion of mononuclear precursors of the monocyte/macrophage lineage into multinucleated cells. In order to investigate the putative role of connexin 43 in formation of bone osteoclast-like foreign body giant cells which are formed in response to implantation of biomaterials, nanoparticulate hydroxyapatite had been implanted into defects of minipig femura. After 20 days the defect areas were harvested and connexin 43 expression and synthesis were investigated by using immunohistochemistry, Western Blot, and in situ hybridization within macrophages and osteoclast-like foreign body giant cells. Morphological analysis of gap junctions is performed ultrastructurally. As shown on protein and mRNA level numerous connexin 43 positive macrophages and foreign body giant cells (FBGC) were localized within the granulation tissue and along the surfaces of the implanted hydroxyapatite (HA). Besides, the formation of FBGC by fusion of macrophages could be shown ultrastructurally. Connexin 43 labeling observed on the protein and mRNA level could be attributed to gap junctions identified ultrastructurally between macrophages, between FBGC, and between FBGC and macrophages. Annular gap junctions in the cytoplasm of FBGC pointed to degradation of the channels, and the ubiquination that had occurred in the course of degradation was confirmed by Western blot analysis. All in all, the presently observed pattern of connexin 43 labeling refers to an functional role of gap junctional communication in the formation of osteoclast-like foreign body giant cells formed in response to implantation of the nanoparticulate HA.

Recombinant rabies virus expressing the H protein of canine distemper virus protects dogs from the lethal distemper challenge.

PubMed

Wang, Feng-Xue; Zhang, Shu-Qin; Zhu, Hong-Wei; Yang, Yong; Sun, Na; Tan, Bin; Li, Zhen-Guang; Cheng, Shi-Peng; Fu, Zhen F; Wen, Yong-Jun

2014-12-05

The rabies virus (RV) vector LBNSE expressing foreign antigens have shown considerable promise as vaccines against viral and bacteria diseases, which is effective and safe. We produced a new RV-based vaccine vehicle expressing 1.824 kb hemagglutinin (H) gene of the canine distemper virus (CDV) by reverse genetics technology. The recombinant virus LBNSE-CDV-H retained growth properties similar to those of vector LBNSE both in BSR and mNA cell culture. The H gene of CDV was expressed and detected by immunostaining. To compare the immunogenicity of LBNSE-CDV-H, dogs were immunized with each of these recombinant viruses by intramuscular (i.m.). The dogs were bled at third weeks after the immunization for the measurement of virus neutralizing antibody (VNA) and then challenged with virulent virus (ZJ 7) at fourth weeks. The parent virus (LBNSE) without expression of any foreign molecules was included for comparison. Dogs inoculated with LBNSE-CDV-H showed no any signs of disease and exhibited seroconversion against both RV and CDV H protein. The LBNSE-CDV-H did not cause disease in dogs and conferred protection from challenge with a lethal wild type CDV strain, demonstrating its potential value for wildlife conservation efforts. Together, these studies suggest that recombinant RV expressing H protein from CDV stimulated high levels of adaptive immune responses (VNA), and protected all dogs challenge infection. Copyright © 2014 Elsevier B.V. All rights reserved.

Increasing the yield of middle silk gland expression system through transgenic knock-down of endogenous sericin-1.

PubMed

Ma, Sanyuan; Xia, Xiaojuan; Li, Yufeng; Sun, Le; Liu, Yue; Liu, Yuanyuan; Wang, Xiaogang; Shi, Run; Chang, Jiasong; Zhao, Ping; Xia, Qingyou

2017-08-01

Various genetically modified bioreactor systems have been developed to meet the increasing demands of recombinant proteins. Silk gland of Bombyx mori holds great potential to be a cost-effective bioreactor for commercial-scale production of recombinant proteins. However, the actual yields of proteins obtained from the current silk gland expression systems are too low for the proteins to be dissolved and purified in a large scale. Here, we proposed a strategy that reducing endogenous sericin proteins would increase the expression yield of foreign proteins. Using transgenic RNA interference, we successfully reduced the expression of BmSer1 to 50%. A total 26 transgenic lines expressing Discosoma sp. red fluorescent protein (DsRed) in the middle silk gland (MSG) under the control of BmSer1 promoter were established to analyze the expression of recombinant. qRT-PCR and western blotting showed that in BmSer1 knock-down lines, the expression of DsRed had significantly increased both at mRNA and protein levels. We did an additional analysis of DsRed/BmSer1 distribution in cocoon and effect of DsRed protein accumulation on the silk fiber formation process. This study describes not only a novel method to enhance recombinant protein expression in MSG bioreactor, but also a strategy to optimize other bioreactor systems.

Development of a Newcastle disease virus vector expressing a foreign gene through an internal ribosomal entry site provides direct proof for a sequential transcription mechanism.

PubMed

Zhang, Zhenyu; Zhao, Wei; Li, Deshan; Yang, Jinlong; Zsak, Laszlo; Yu, Qingzhong

2015-08-01

In the present study, we developed a novel approach for foreign gene expression by Newcastle disease virus (NDV) from a second ORF through an internal ribosomal entry site (IRES). Six NDV LaSota strain-based recombinant viruses vectoring the IRES and a red fluorescence protein (RFP) gene behind the nucleocapsid (NP), phosphoprotein (P), matrix (M), fusion (F), haemagglutinin-neuraminidase (HN) or large polymerase (L) gene ORF were generated using reverse genetics technology. The insertion of the second ORF slightly attenuated virus pathogenicity, but did not affect ability of the virus to grow. Quantitative measurements of RFP expression in virus-infected DF-1 cells revealed that the abundance of viral mRNAs and red fluorescence intensity were positively correlated with the gene order of NDV, 3'-NP-P-M-F-HN-L-5', proving the sequential transcription mechanism for NDV. The results herein suggest that the level of foreign gene expression could be regulated by selecting the second ORF insertion site to maximize the efficacy of vaccine and gene therapy.

Requirements for cell rounding and surface protein down-regulation by Ebola virus glycoprotein.

PubMed

Francica, Joseph R; Matukonis, Meghan K; Bates, Paul

2009-01-20

Ebola virus causes an acute hemorrhagic fever that is associated with high morbidity and mortality. The viral glycoprotein is thought to contribute to pathogenesis, though precise mechanisms are unknown. Cellular pathogenesis can be modeled in vitro by expression of the Ebola viral glycoprotein (GP) in cells, which causes dramatic morphological changes, including cell rounding and surface protein down-regulation. These effects are known to be dependent on the presence of a highly glycosylated region of the glycoprotein, the mucin domain. Here we show that the mucin domain from the highly pathogenic Zaire subtype of Ebola virus is sufficient to cause characteristic cytopathology when expressed in the context of a foreign glycoprotein. Similarly to full length Ebola GP, expression of the mucin domain causes rounding, detachment from the extracellular matrix, and the down-regulation of cell surface levels of beta1 integrin and major histocompatibility complex class 1. These effects were not seen when the mucin domain was expressed in the context of a glycophosphatidylinositol-anchored isoform of the foreign glycoprotein. In contrast to earlier analysis of full length Ebola glycoproteins, chimeras carrying the mucin domains from the Zaire and Reston strains appear to cause similar levels of down-modulation and cell detachment. Cytopathology associated with Ebola glycoprotein expression does not occur when GP expression is restricted to the endoplasmic reticulum. In contrast to a previously published report, our results demonstrate that GP-induced surface protein down-regulation is not mediated through a dynamin-dependent pathway. Overall, these results support a model in which the mucin domain of Ebola GP acts at the cell surface to induce protein down modulation and cytopathic effects.

Effect of Plasmid Design and Type of Integration Event on Recombinant Protein Expression in Pichia pastoris.

PubMed

Vogl, Thomas; Gebbie, Leigh; Palfreyman, Robin W; Speight, Robert

2018-03-15

Pichia pastoris (syn. Komagataella phaffii ) is one of the most common eukaryotic expression systems for heterologous protein production. Expression cassettes are typically integrated in the genome to obtain stable expression strains. In contrast to Saccharomyces cerevisiae , where short overhangs are sufficient to target highly specific integration, long overhangs are more efficient in P. pastoris and ectopic integration of foreign DNA can occur. Here, we aimed to elucidate the influence of ectopic integration by high-throughput screening of >700 transformants and whole-genome sequencing of 27 transformants. Different vector designs and linearization approaches were used to mimic the most common integration events targeted in P. pastoris Fluorescence of an enhanced green fluorescent protein (eGFP) reporter protein was highly uniform among transformants when the expression cassettes were correctly integrated in the targeted locus. Surprisingly, most nonspecifically integrated transformants showed highly uniform expression that was comparable to specific integration, suggesting that nonspecific integration does not necessarily influence expression. However, a few clones (<10%) harboring ectopically integrated cassettes showed a greater variation spanning a 25-fold range, surpassing specifically integrated reference strains up to 6-fold. High-expression strains showed a correlation between increased gene copy numbers and high reporter protein fluorescence levels. Our results suggest that for comparing expression levels between strains, the integration locus can be neglected as long as a sufficient numbers of transformed strains are compared. For expression optimization of highly expressible proteins, increasing copy number appears to be the dominant positive influence rather than the integration locus, genomic rearrangements, deletions, or single-nucleotide polymorphisms (SNPs). IMPORTANCE Yeasts are commonly used as biotechnological production hosts for proteins and metabolites. In the yeast Saccharomyces cerevisiae , expression cassettes carrying foreign genes integrate highly specifically at the targeted sites in the genome. In contrast, cassettes often integrate at random genomic positions in nonconventional yeasts, such as Pichia pastoris (syn. Komagataella phaffii ). Hence, cells from the same transformation event often behave differently, with significant clonal variation necessitating the screening of large numbers of strains. The importance of this study is that we systematically investigated the influence of integration events in more than 700 strains. Our findings provide novel insight into clonal variation in P. pastoris and, thus, how to avoid pitfalls and obtain reliable results. The underlying mechanisms may also play a role in other yeasts and hence could be generally relevant for recombinant yeast protein production strains. Copyright © 2018 American Society for Microbiology.

Generate Optimized Genetic Rhythm for Enzyme Expression in Non-native systems

DOE Office of Scientific and Technical Information (OSTI.GOV)

2016-11-03

Most amino acids are represented by more than one codon, resulting in redundancy in the genetic code. Silent codon substitutions that do not alter the amino acid sequence still have an effect on protein expression. We have developed an algorithm, GoGREEN, to enhance the expression of foreign proteins in a host organism. GoGREEN selects codons according to frequency patterns seen in the gene of interest using the codon usage table from the host organism. GoGREEN is also designed to accommodate gaps in the sequence.This software takes for input (1) the aligned protein sequences for genes the user wishes to express,more » (2) the codon usage table for the host organism, (3) and the DNA sequence for the target protein found in the host organism. The program will select codons based on codon usage patterns for the target DNA sequence. The program will also select codons for “gaps” found in the aligned protein sequences using the codon usage table from the host organism.« less

Improvement of foreign-protein production in Aspergillus niger var. awamori by constitutive induction of the unfolded-protein response.

PubMed

Valkonen, Mari; Ward, Michael; Wang, Huaming; Penttilä, Merja; Saloheimo, Markku

2003-12-01

Unfolded-protein response (UPR) denotes the upregulation of endoplasmic reticulum (ER)-resident chaperone and foldase genes and numerous other genes involved in secretory functions during the accumulation of unfolded proteins into the ER. Overexpression of individual foldases and chaperones has been used in attempts to improve protein production in different production systems. We describe here a novel strategy to improve foreign-protein production. We show that the constitutive induction of the UPR pathway in Aspergillus niger var. awamori can be achieved by expressing the activated form of the transcription factor hacA. This induction enhances the production of Trametes versicolor laccase by up to sevenfold and of bovine preprochymosin by up to 2.8-fold in this biotechnically important fungus. The regulatory range of UPR was studied by analyzing the mRNA levels of novel A. niger var. awamori genes involved in different secretory functions. This revealed both similarities and differences to corresponding studies in Saccharomyces cerevisiae.

The potential of transgenic green microalgae; a robust photobioreactor to produce recombinant therapeutic proteins.

PubMed

Akbari, Fariba; Eskandani, Morteza; Khosroushahi, Ahmad Yari

2014-11-01

Microalgae have been used in food, cosmetic, and biofuel industries as a natural source of lipids, vitamins, pigments and antioxidants for a long time. Green microalgae, as potent photobioreactors, can be considered as an economical expression system to produce recombinant therapeutical proteins at large-scale due to low cost of production and scaling-up capitalization owning to the inexpensive medium requirement, fast growth rate, and the ease of manipulation. These microalgae possess all benefit eukaryotic expression systems including the ability of post-translational modifications required for proper folding and stability of active proteins. Among the many items regarded as recombinant protein production, this review compares the different expression systems with green microalgae like Dunaliella by viewing the nuclear/chloroplast transformation challenges/benefits, related selection markers/reporter genes, and crucial factors/strategies affecting the increase of foreign protein expression in microalgae transformants. Some important factors were discussed regarding the increase of protein yielding in microalgae transformants including: transformation-associated genotypic modifications, endogenous regulatory factors, promoters, codon optimization, enhancer elements, and milking of recombinant protein.

Baculovirus-mediated gene transfer in butterfly wings in vivo: an efficient expression system with an anti-gp64 antibody.

PubMed

Dhungel, Bidur; Ohno, Yoshikazu; Matayoshi, Rie; Otaki, Joji M

2013-03-25

Candidate genes for color pattern formation in butterfly wings have been known based on gene expression patterns since the 1990s, but their functions remain elusive due to a lack of a functional assay. Several methods of transferring and expressing a foreign gene in butterfly wings have been reported, but they have suffered from low success rates or low expression levels. Here, we developed a simple, practical method to efficiently deliver and express a foreign gene using baculovirus-mediated gene transfer in butterfly wings in vivo. A recombinant baculovirus containing a gene for green fluorescent protein (GFP) was injected into pupae of the blue pansy butterfly Junonia orithya (Nymphalidae). GFP fluorescence was detected in the pupal wings and other body parts of the injected individuals three to five days post-injection at various degrees of fluorescence. We obtained a high GFP expression rate at relatively high virus titers, but it was associated with pupal death before color pattern formation in wings. To reduce the high mortality rate caused by the baculovirus treatment, we administered an anti-gp64 antibody, which was raised against baculovirus coat protein gp64, to infected pupae after the baculovirus injection. This treatment greatly reduced the mortality rate of the infected pupae. GFP fluorescence was observed in pupal and adult wings and other body parts of the antibody-treated individuals at various degrees of fluorescence. Importantly, we obtained completely developed wings with a normal color pattern, in which fluorescent signals originated directly from scales or the basal membrane after the removal of scales. GFP fluorescence in wing tissues spatially coincided with anti-GFP antibody staining, confirming that the fluorescent signals originated from the expressed GFP molecules. Our baculovirus-mediated gene transfer system with an anti-gp64 antibody is reasonably efficient, and it can be an invaluable tool to transfer, express, and functionally examine foreign genes in butterfly wings and also in other non-model insect systems.

Rescue of infectious rift valley fever virus entirely from cDNA, analysis of virus lacking the NSs gene, and expression of a foreign gene.

PubMed

Ikegami, Tetsuro; Won, Sungyong; Peters, C J; Makino, Shinji

2006-03-01

Rift Valley fever virus (RVFV) (genus Phlebovirus, family Bunyaviridae) has a tripartite negative-strand genome, causes a mosquito-borne disease that is endemic in sub-Saharan African countries and that also causes large epidemics among humans and livestock. Furthermore, it is a bioterrorist threat and poses a risk for introduction to other areas. In spite of its danger, neither veterinary nor human vaccines are available. We established a T7 RNA polymerase-driven reverse genetics system to rescue infectious clones of RVFV MP-12 strain entirely from cDNA, the first for any phlebovirus. Expression of viral structural proteins from the protein expression plasmids was not required for virus rescue, whereas NSs protein expression abolished virus rescue. Mutants of MP-12 partially or completely lacking the NSs open reading frame were viable. These NSs deletion mutants replicated efficiently in Vero and 293 cells, but not in MRC-5 cells. In the latter cell line, accumulation of beta interferon mRNA occurred after infection by these NSs deletion mutants, but not after infection by MP-12. The NSs deletion mutants formed larger plaques than MP-12 did in Vero E6 cells and failed to shut off host protein synthesis in Vero cells. An MP-12 mutant carrying a luciferase gene in place of the NSs gene replicated as efficiently as MP-12 did, produced enzymatically active luciferase during replication, and stably retained the luciferase gene after 10 virus passages, representing the first demonstration of foreign gene expression in any bunyavirus. This reverse genetics system can be used to study the molecular virology of RVFV, assess current vaccine candidates, produce new vaccines, and incorporate marker genes into animal vaccines.

An infectious recombinant equine arteritis virus expressing green fluorescent protein from its replicase gene.

PubMed

van den Born, Erwin; Posthuma, Clara C; Knoops, Kèvin; Snijder, Eric J

2007-04-01

Thus far, systems developed for heterologous gene expression from the genomes of nidoviruses (arteriviruses and coronaviruses) have relied mainly on the translation of foreign genes from subgenomic mRNAs, whose synthesis is a key feature of the nidovirus life cycle. In general, such expression vectors often suffered from relatively low and unpredictable expression levels, as well as genome instability. In an attempt to circumvent these disadvantages, the possibility to express a foreign gene [encoding enhanced green fluorescent protein (eGFP)] from within the nidovirus replicase gene, which encodes two large polyproteins that are processed proteolytically into the non-structural proteins (nsps) required for viral RNA synthesis, has now been explored. A viable recombinant of the arterivirus Equine arteritis virus, EAV-GFP2, was obtained, which contained the eGFP insert at the site specifying the junction between the two most N-proximal replicase-cleavage products, nsp1 and nsp2. EAV-GFP2 replication could be launched by transfection of cells with either in vitro-generated RNA transcripts or a DNA launch plasmid. EAV-GFP2 displayed growth characteristics similar to those of the wild-type virus and was found to maintain the insert stably for at least eight passages. It is proposed that EAV-GFP2 has potential for arterivirus vector development and as a tool in inhibitor screening. It can also be used for fundamental studies into EAV replication, which was illustrated by the fact that the eGFP signal of EAV-GFP2, which largely originated from an eGFP-nsp2 fusion protein, could be used to monitor the formation of the membrane-bound EAV replication complex in real time.

Deep sequencing of Salmonella RNA associated with heterologous Hfq proteins in vivo reveals small RNAs as a major target class and identifies RNA processing phenotypes.

PubMed

Sittka, Alexandra; Sharma, Cynthia M; Rolle, Katarzyna; Vogel, Jörg

2009-01-01

The bacterial Sm-like protein, Hfq, is a key factor for the stability and function of small non-coding RNAs (sRNAs) in Escherichia coli. Homologues of this protein have been predicted in many distantly related organisms yet their functional conservation as sRNA-binding proteins has not entirely been clear. To address this, we expressed in Salmonella the Hfq proteins of two eubacteria (Neisseria meningitides, Aquifex aeolicus) and an archaeon (Methanocaldococcus jannaschii), and analyzed the associated RNA by deep sequencing. This in vivo approach identified endogenous Salmonella sRNAs as a major target of the foreign Hfq proteins. New Salmonella sRNA species were also identified, and some of these accumulated specifically in the presence of a foreign Hfq protein. In addition, we observed specific RNA processing defects, e.g., suppression of precursor processing of SraH sRNA by Methanocaldococcus Hfq, or aberrant accumulation of extracytoplasmic target mRNAs of the Salmonella GcvB, MicA or RybB sRNAs. Taken together, our study provides evidence of a conserved inherent sRNA-binding property of Hfq, which may facilitate the lateral transmission of regulatory sRNAs among distantly related species. It also suggests that the expression of heterologous RNA-binding proteins combined with deep sequencing analysis of RNA ligands can be used as a molecular tool to dissect individual steps of RNA metabolism in vivo.

Development of a reverse genetics system to generate a recombinant Ebola virus Makona expressing a green fluorescent protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Albariño, César G., E-mail: calbarino@cdc.gov; Wiggleton Guerrero, Lisa; Lo, Michael K.

Previous studies have demonstrated the potential application of reverse genetics technology in studying a broad range of aspects of viral biology, including gene regulation, protein function, cell entry, and pathogenesis. Here, we describe a highly efficient reverse genetics system used to generate recombinant Ebola virus (EBOV) based on a recent isolate from a human patient infected during the 2014–2015 outbreak in Western Africa. We also rescued a recombinant EBOV expressing a fluorescent reporter protein from a cleaved VP40 protein fusion. Using this virus and an inexpensive method to quantitate the expression of the foreign gene, we demonstrate its potential usefulnessmore » as a tool for screening antiviral compounds and measuring neutralizing antibodies. - Highlights: • Recombinant Ebola virus (EBOV) derived from Makona variant was rescued. • New protocol for viral rescue allows 100% efficiency. • Modified EBOV expresses a green fluorescent protein from a VP40-fused protein. • Modified EBOV was tested as tool to screen antiviral compounds and measure neutralizing antibodies.« less

Bacteriophage 5' untranslated regions for control of plastid transgene expression.

PubMed

Yang, Huijun; Gray, Benjamin N; Ahner, Beth A; Hanson, Maureen R

2013-02-01

Expression of foreign proteins from transgenes incorporated into plastid genomes requires regulatory sequences that can be recognized by the plastid transcription and translation machinery. Translation signals harbored by the 5' untranslated region (UTR) of plastid transcripts can profoundly affect the level of accumulation of proteins expressed from chimeric transgenes. Both endogenous 5' UTRs and the bacteriophage T7 gene 10 (T7g10) 5' UTR have been found to be effective in combination with particular coding regions to mediate high-level expression of foreign proteins. We investigated whether two other bacteriophage 5' UTRs could be utilized in plastid transgenes by fusing them to the aadA (aminoglycoside-3'-adenyltransferase) coding region that is commonly used as a selectable marker in plastid transformation. Transplastomic plants containing either the T7g1.3 or T4g23 5' UTRs fused to Myc-epitope-tagged aadA were successfully obtained, demonstrating the ability of these 5' UTRs to regulate gene expression in plastids. Placing the Thermobifida fusca cel6A gene under the control of the T7g1.3 or T4g23 5' UTRs, along with a tetC downstream box, resulted in poor expression of the cellulase in contrast with high-level accumulation while using the T7g10 5' UTR. However, transplastomic plants with the bacteriophage 5' UTRs controlling the aadA coding region exhibited fewer undesired recombinant species than plants containing the same marker gene regulated by the Nicotiana tabacum psbA 5' UTR. Furthermore, expression of the T7g1.3 and T4g23 5' UTR::aadA fusions downstream of the cel6A gene provided sufficient spectinomycin resistance to allow selection of homoplasmic transgenic plants and had no effect on Cel6A accumulation.

Outer Membrane Targeting of Passenger Proteins by the Vacuolating Cytotoxin Autotransporter of Helicobacter pylori

PubMed Central

Fischer, Wolfgang; Buhrdorf, Renate; Gerland, Elke; Haas, Rainer

2001-01-01

Helicobacter pylori produces a number of proteins associated with the outer membrane, including adhesins and the vacuolating cytotoxin. These proteins are supposed to integrate into the outer membrane by β-barrel structures, characteristic of the family of autotransporter proteins. By using the SOMPES (shuttle vector-based outer membrane protein expression) system for outer membrane protein production, we were able to functionally express in H. pylori the cholera toxin B subunit genetically fused to the C-terminal VacA domain. We demonstrate that the fusion protein is translocated to the H. pylori outer membrane and that the CtxB domain is exposed on the H. pylori surface. Thus, we provide the first experimental evidence that the C-terminal β-domain of VacA can transport a foreign passenger protein to the H. pylori surface and hence acts as a functional autotransporter. PMID:11598049

A novel regulation mechanism of the T7 RNA polymerase based expression system improves overproduction and folding of membrane proteins.

PubMed

Angius, Federica; Ilioaia, Oana; Amrani, Amira; Suisse, Annabelle; Rosset, Lindsay; Legrand, Amélie; Abou-Hamdan, Abbas; Uzan, Marc; Zito, Francesca; Miroux, Bruno

2018-06-05

Membrane protein (MP) overproduction is one of the major bottlenecks in structural genomics and biotechnology. Despite the emergence of eukaryotic expression systems, bacteria remain a cost effective and powerful tool for protein production. The T7 RNA polymerase (T7RNAP)-based expression system is a successful and efficient expression system, which achieves high-level production of proteins. However some foreign MPs require a fine-tuning of their expression to minimize the toxicity associated with their production. Here we report a novel regulation mechanism for the T7 expression system. We have isolated two bacterial hosts, namely C44(DE3) and C45(DE3), harboring a stop codon in the T7RNAP gene, whose translation is under the control of the basal nonsense suppressive activity of the BL21(DE3) host. Evaluation of hosts with superfolder green fluorescent protein (sfGFP) revealed an unprecedented tighter control of transgene expression with a marked accumulation of the recombinant protein during stationary phase. Analysis of a collection of twenty MP fused to GFP showed an improved production yield and quality of several bacterial MPs and of one human monotopic MP. These mutant hosts are complementary to the other existing T7 hosts and will increase the versatility of the T7 expression system.

Influence of insertion site of the avian influenza virus haemagglutinin (HA) gene within the Newcastle disease virus genome on HA expression.

PubMed

Ramp, Kristina; Skiba, Martin; Karger, Axel; Mettenleiter, Thomas C; Römer-Oberdörfer, Angela

2011-02-01

Members of the order Mononegavirales express their genes in a transcription gradient from 3' to 5'. To assess how this impacts on expression of a foreign transgene, the haemagglutinin (HA) of highly pathogenic avian influenza virus (HPAIV) A/chicken/Vietnam/P41/05 (subtype H5N1) was inserted between the phosphoprotein (P) and matrix protein (M), M and fusion protein (F), or F and haemagglutinin-neuraminidase protein (HN) genes of attenuated Newcastle disease virus (NDV) Clone 30. In addition, the gene encoding the neuraminidase of HPAIV A/duck/Vietnam/TG24-01/05 (subtype H5N1) was inserted into the NDV genome either alone or in combination with the HA gene. All recombinants replicated well in embryonated chicken eggs. The expression levels of HA-specific mRNA and protein were quantified by Northern blot analysis and mass spectrometry, with good correlation. HA expression levels differed only moderately and were highest in the recombinant carrying the HA insertion between the F and HN genes of NDV.

The HSP terminator of Arabidopsis thaliana increases gene expression in plant cells.

PubMed

Nagaya, Shingo; Kawamura, Kazue; Shinmyo, Atsuhiko; Kato, Ko

2010-02-01

To express a foreign gene in plants effectively, a good expression system is required. Here we describe the identification of a transcriptional terminator that supports increased levels of expression. The terminators of several Arabidopsis genes were examined in transfected Arabidopsis T87 protoplasts. The heat shock protein 18.2 (HSP) terminator was the most effective in supporting increased levels of expression. The HSP terminator increases mRNA levels of both transiently and stably expressed transgenes approximately 2-fold more than the NOS (nopaline synthase) terminator. When combined with the HSP terminator, a translational enhancer increased gene expression levels approximately 60- to 100-fold in transgenic plants.

The therapeutic potential of plant-derived vaccines and antibodies.

PubMed

Rodgers, P B; Hamilton, W D; Adair, J R

1999-03-01

The production of recombinant proteins in plants is reviewed with a particular focus on plant-derived vaccines and antibodies for human healthcare. Issues relating to foreign gene expression, such as protein yield, localisation and glycosylation are also considered. Emphasis is placed on reporting progress with preclinical and clinical evaluation of plant-derived vaccines and antibodies. An assessment is made of the likely future direction of research and development in this area.

The xenoestrogens, bisphenol A and para-nonylphenol, decrease the expression of the ABCG2 transporter protein in human term placental explant cultures.

PubMed

Sieppi, E; Vähäkangas, K; Rautio, A; Ietta, F; Paulesu, L; Myllynen, P

2016-07-05

Many endogenous and xenobiotic compounds are substrates and regulators of human placental ABC transporters. ABCG2 is protecting fetus against foreign chemicals. Environmental xenoestrogens, like bisphenol A (BPA) and p-nonylphenol (p-NP), mimic natural estrogens and can affect hormonal systems. Effects of BPA, p-NP, DES (diethylstilbestrol) and estradiol (E2), on ABCG2 expression were studied using human first trimester and term placental explants. Role of estrogen receptors (ER) in the effects of chemicals was studied by ER antagonist. Term placenta expressed less ABCG2 protein. In term placentas BPA (p < 0.05), p-NP (p < 0.01) and E2 (p < 0.05) decreased the ABCG2 protein expression after 48 h exposure while after 24 h exposure, only E2 decreased the expression (p < 0.05). The chemicals did not affect ABCG2 in first trimester placentas. The ER antagonist affected differently the responses of chemicals. In conclusion, environmental xenoestrogens downregulate placental ABCG2 protein expression depending on gestational age. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Gene and process level modulation to overcome the bottlenecks of recombinant proteins expression in Pichia pastoris.

PubMed

Prabhu, Ashish A; Boro, Bibari; Bharali, Biju; Chakraborty, Shuchishloka; Dasu, Veeranki V

2017-01-01

Process development involving system metabolic engineering and bioprocess engineering has become one of the major thrust for the development of therapeutic proteins or enzymes. Pichia pastoris has emerged as a prominent host for the production of therapeutic protein or enzymes. Regardless of producing high protein titers, various cellular and process level bottlenecks restrict the expression of recombinant proteins in P. pastoris. In the present review, we have summarized the recent developments in the expression of foreign proteins in P. pastoris. Further, we have discussed various cellular engineering strategies which include codon optimization, pathway engineering, signal peptide processing, development of protease deficient strain and glyco-engineered strains for the high yield protein secretion of recombinant protein. Bioprocess development of recombinant proteins in large-scale bioreactor including medium optimization, optimum feeding strategy and co-substrate feeding in fed-batch as well as continuous cultivation have been described. The recent advances in system and synthetic biology studies including metabolic flux analysis in understanding the phenotypic characteristics of recombinant Pichia and genome editing with CRISPR-CAS system have also been summarized. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Baculovirus-mediated gene transfer in butterfly wings in vivo: an efficient expression system with an anti-gp64 antibody

PubMed Central

2013-01-01

Background Candidate genes for color pattern formation in butterfly wings have been known based on gene expression patterns since the 1990s, but their functions remain elusive due to a lack of a functional assay. Several methods of transferring and expressing a foreign gene in butterfly wings have been reported, but they have suffered from low success rates or low expression levels. Here, we developed a simple, practical method to efficiently deliver and express a foreign gene using baculovirus-mediated gene transfer in butterfly wings in vivo. Results A recombinant baculovirus containing a gene for green fluorescent protein (GFP) was injected into pupae of the blue pansy butterfly Junonia orithya (Nymphalidae). GFP fluorescence was detected in the pupal wings and other body parts of the injected individuals three to five days post-injection at various degrees of fluorescence. We obtained a high GFP expression rate at relatively high virus titers, but it was associated with pupal death before color pattern formation in wings. To reduce the high mortality rate caused by the baculovirus treatment, we administered an anti-gp64 antibody, which was raised against baculovirus coat protein gp64, to infected pupae after the baculovirus injection. This treatment greatly reduced the mortality rate of the infected pupae. GFP fluorescence was observed in pupal and adult wings and other body parts of the antibody-treated individuals at various degrees of fluorescence. Importantly, we obtained completely developed wings with a normal color pattern, in which fluorescent signals originated directly from scales or the basal membrane after the removal of scales. GFP fluorescence in wing tissues spatially coincided with anti-GFP antibody staining, confirming that the fluorescent signals originated from the expressed GFP molecules. Conclusions Our baculovirus-mediated gene transfer system with an anti-gp64 antibody is reasonably efficient, and it can be an invaluable tool to transfer, express, and functionally examine foreign genes in butterfly wings and also in other non-model insect systems. PMID:23522444

High glucose enhances cAMP level and extracellular signal-regulated kinase phosphorylation in Chinese hamster ovary cell: Usage of Br-cAMP in foreign protein β-galactosidase expression.

PubMed

Lin, Hsiao-Hsien; Lee, Tsung-Yih; Liu, Ting-Wei; Tseng, Ching-Ping

2017-07-01

Glucose is a carbon source for Chinese hamster ovary (CHO) cell growth, while low growth rate is considered to enhance the production of recombinant proteins. The present study reveals that glucose concentrations higher than 1 g/L reduce the growth rate and substantially increase in cAMP (∼300%) at a high glucose concentration (10 g/L). High glucose also enhances the phosphorylation of extracellular signal-regulated kinase (ERK) and p27 kip by Western blot analysis. To determine whether the phosphorylation of ERK is involved in the mechanism, a cyclic-AMP dependent protein kinase A (PKA) inhibitor (H-8) or MEK (MAPKK) inhibitor (PD98059) was added to block ERK phosphorylation. We show that both the high glucose-induced ERK phosphorylation and growth rate return to baseline levels. These results suggest that the cAMP/PKA and MAP signaling pathways are involved in the abovementioned mechanism. Interestingly, the direct addition of 8-bromo-cAMP (Br-cAMP), a membrane-permeable cAMP analog, can mimic the similar effects produced by high glucose. Subsequently Br-cAMP could induce β-galactosidase (β-Gal) recombinant protein expression by 1.6-fold. Furthermore, Br-cAMP can additionally enhance the β-Gal production (from 2.8- to 4.5-fold) when CHO cells were stimulated with glycerol, thymidine, dimethyl sulfoxide, pentanoic acid, or sodium butyrate. Thus, Br-cAMP may be used as an alternative agent in promoting foreign protein expression for CHO cells. Copyright © 2017. Published by Elsevier B.V.

High-level expression of human immunodeficiency virus antigens from the tobacco and tomato plastid genomes.

PubMed

Zhou, Fei; Badillo-Corona, Jesus A; Karcher, Daniel; Gonzalez-Rabade, Nuria; Piepenburg, Katrin; Borchers, A-M Inka; Maloney, Alan P; Kavanagh, Tony A; Gray, John C; Bock, Ralph

2008-12-01

Transgene expression from the plant's plastid genome represents a promising strategy in molecular farming because of the plastid's potential to accumulate foreign proteins to high levels and the increased biosafety provided by the maternal mode of organelle inheritance. In this article, we explore the potential of transplastomic plants to produce human immunodeficiency virus (HIV) antigens as potential components of an acquired immunodeficiency syndrome (AIDS) vaccine. It is shown that the HIV antigens p24 (the major target of T-cell-mediated immune responses in HIV-positive individuals) and Nef can be expressed to high levels in plastids of tobacco, a non-food crop, and tomato, a food crop with an edible fruit. Optimized p24-Nef fusion gene cassettes trigger antigen protein accumulation to up to approximately 40% of the plant's total protein, demonstrating the great potential of transgenic plastids to produce AIDS vaccine components at low cost and high yield.

Extremotolerant tardigrade genome and improved radiotolerance of human cultured cells by tardigrade-unique protein

PubMed Central

Hashimoto, Takuma; Horikawa, Daiki D.; Saito, Yuki; Kuwahara, Hirokazu; Kozuka-Hata, Hiroko; Shin-I, Tadasu; Minakuchi, Yohei; Ohishi, Kazuko; Motoyama, Ayuko; Aizu, Tomoyuki; Enomoto, Atsushi; Kondo, Koyuki; Tanaka, Sae; Hara, Yuichiro; Koshikawa, Shigeyuki; Sagara, Hiroshi; Miura, Toru; Yokobori, Shin-ichi; Miyagawa, Kiyoshi; Suzuki, Yutaka; Kubo, Takeo; Oyama, Masaaki; Kohara, Yuji; Fujiyama, Asao; Arakawa, Kazuharu; Katayama, Toshiaki; Toyoda, Atsushi; Kunieda, Takekazu

2016-01-01

Tardigrades, also known as water bears, are small aquatic animals. Some tardigrade species tolerate almost complete dehydration and exhibit extraordinary tolerance to various physical extremes in the dehydrated state. Here we determine a high-quality genome sequence of Ramazzottius varieornatus, one of the most stress-tolerant tardigrade species. Precise gene repertoire analyses reveal the presence of a small proportion (1.2% or less) of putative foreign genes, loss of gene pathways that promote stress damage, expansion of gene families related to ameliorating damage, and evolution and high expression of novel tardigrade-unique proteins. Minor changes in the gene expression profiles during dehydration and rehydration suggest constitutive expression of tolerance-related genes. Using human cultured cells, we demonstrate that a tardigrade-unique DNA-associating protein suppresses X-ray-induced DNA damage by ∼40% and improves radiotolerance. These findings indicate the relevance of tardigrade-unique proteins to tolerability and tardigrades could be a bountiful source of new protection genes and mechanisms. PMID:27649274

Extremotolerant tardigrade genome and improved radiotolerance of human cultured cells by tardigrade-unique protein.

PubMed

Hashimoto, Takuma; Horikawa, Daiki D; Saito, Yuki; Kuwahara, Hirokazu; Kozuka-Hata, Hiroko; Shin-I, Tadasu; Minakuchi, Yohei; Ohishi, Kazuko; Motoyama, Ayuko; Aizu, Tomoyuki; Enomoto, Atsushi; Kondo, Koyuki; Tanaka, Sae; Hara, Yuichiro; Koshikawa, Shigeyuki; Sagara, Hiroshi; Miura, Toru; Yokobori, Shin-Ichi; Miyagawa, Kiyoshi; Suzuki, Yutaka; Kubo, Takeo; Oyama, Masaaki; Kohara, Yuji; Fujiyama, Asao; Arakawa, Kazuharu; Katayama, Toshiaki; Toyoda, Atsushi; Kunieda, Takekazu

2016-09-20

Tardigrades, also known as water bears, are small aquatic animals. Some tardigrade species tolerate almost complete dehydration and exhibit extraordinary tolerance to various physical extremes in the dehydrated state. Here we determine a high-quality genome sequence of Ramazzottius varieornatus, one of the most stress-tolerant tardigrade species. Precise gene repertoire analyses reveal the presence of a small proportion (1.2% or less) of putative foreign genes, loss of gene pathways that promote stress damage, expansion of gene families related to ameliorating damage, and evolution and high expression of novel tardigrade-unique proteins. Minor changes in the gene expression profiles during dehydration and rehydration suggest constitutive expression of tolerance-related genes. Using human cultured cells, we demonstrate that a tardigrade-unique DNA-associating protein suppresses X-ray-induced DNA damage by ∼40% and improves radiotolerance. These findings indicate the relevance of tardigrade-unique proteins to tolerability and tardigrades could be a bountiful source of new protection genes and mechanisms.

Over-Expression of a Tobacco Nitrate Reductase Gene in Wheat (Triticum aestivum L.) Increases Seed Protein Content and Weight without Augmenting Nitrogen Supplying

PubMed Central

Zhao, Xiao-Qiang; Nie, Xuan-Li; Xiao, Xing-Guo

2013-01-01

Heavy nitrogen (N) application to gain higher yield of wheat (Triticum aestivum L.) resulted in increased production cost and environment pollution. How to diminish the N supply without losing yield and/or quality remains a challenge. To meet the challenge, we integrated and expressed a tobacco nitrate reductase gene (NR) in transgenic wheat. The 35S-NR gene was transferred into two winter cultivars, “Nongda146” and “Jimai6358”, by Agrobacterium-mediation. Over-expression of the transgene remarkably enhanced T1 foliar NR activity and significantly augmented T2 seed protein content and 1000-grain weight in 63.8% and 68.1% of T1 offspring (total 67 individuals analyzed), respectively. Our results suggest that constitutive expression of foreign nitrate reductase gene(s) in wheat might improve nitrogen use efficiency and thus make it possible to increase seed protein content and weight without augmenting N supplying. PMID:24040315

Over-expression of a tobacco nitrate reductase gene in wheat (Triticum aestivum L.) increases seed protein content and weight without augmenting nitrogen supplying.

PubMed

Zhao, Xiao-Qiang; Nie, Xuan-Li; Xiao, Xing-Guo

2013-01-01

Heavy nitrogen (N) application to gain higher yield of wheat (Triticum aestivum L.) resulted in increased production cost and environment pollution. How to diminish the N supply without losing yield and/or quality remains a challenge. To meet the challenge, we integrated and expressed a tobacco nitrate reductase gene (NR) in transgenic wheat. The 35S-NR gene was transferred into two winter cultivars, "Nongda146" and "Jimai6358", by Agrobacterium-mediation. Over-expression of the transgene remarkably enhanced T1 foliar NR activity and significantly augmented T2 seed protein content and 1000-grain weight in 63.8% and 68.1% of T1 offspring (total 67 individuals analyzed), respectively. Our results suggest that constitutive expression of foreign nitrate reductase gene(s) in wheat might improve nitrogen use efficiency and thus make it possible to increase seed protein content and weight without augmenting N supplying.

Loss of monocyte chemoattractant protein-1 alters macrophage polarization and reduces NFκB activation in the foreign body response.

PubMed

Moore, Laura Beth; Sawyer, Andrew J; Charokopos, Antonios; Skokos, Eleni A; Kyriakides, Themis R

2015-01-01

Implantation of biomaterials elicits a foreign body response characterized by fusion of macrophages to form foreign body giant cells and fibrotic encapsulation. Studies of the macrophage polarization involved in this response have suggested that alternative (M2) activation is associated with more favorable outcomes. Here we investigated this process in vivo by implanting mixed cellulose ester filters or polydimethylsiloxane disks in the peritoneal cavity of wild-type (WT) and monocyte chemoattractant protein-1 (MCP-1) knockout mice. We analyzed classical (M1) and alternative (M2) gene expression via quantitative polymerase chain reaction, immunohistochemistry and enzyme-linked immunosorbent assay in both non-adherent cells isolated by lavage and implant-adherent cells. Our results show that macrophages undergo unique activation that displays features of both M1 and M2 polarization including induction of tumor necrosis factor α (TNF), which induces the expression and nuclear translocation of p50 and RelA determined by immunofluorescence and Western blot. Both processes were compromised in fusion-deficient MCP-1 KO macrophages in vitro and in vivo. Furthermore, inclusion of BAY 11-7028, an inhibitor of NFκB activation, reduced nuclear translocation of RelA and fusion in WT macrophages. Our studies suggest that peritoneal implants elicit a unique macrophage polarization phenotype leading to induction of TNF and activation of the NFκB pathway. Published by Elsevier Ltd.

Limits to sulfur accumulation in transgenic lupin seeds expressing a foreign sulfur-rich protein.

PubMed

Tabe, Linda M; Droux, Michel

2002-03-01

The low sulfur amino acid content of legume seeds restricts their nutritive value for animals. We have investigated the limitations to the accumulation of sulfur amino acids in the storage proteins of narrow leaf lupin (Lupinus angustifolius) seeds. Variation in sulfur supply to lupin plants affected the sulfur amino acid accumulation in the mature seed. However, when sulfur was in abundant supply, it accumulated to a large extent in oxidized form, rather than reduced form, in the seeds. At all but severely limiting sulfur supply, addition of a transgenic (Tg) sink for organic sulfur resulted in an increase in seed sulfur amino acid content. We hypothesize that demand, or sink strength for organic sulfur, which is itself responsive to environmental sulfur supply, was the first limit to the methionine (Met) and cysteine (Cys) content of wild-type lupin seed protein under most growing conditions. In Tg, soil-grown seeds expressing a foreign Met- and Cys-rich protein, decreased pools of free Met, free Cys, and glutathione indicated that the rate of synthesis of sulfur amino acids in the cotyledon had become limiting. Homeostatic mechanisms similar to those mediating the responses of plants to environmental sulfur stress resulted in an adjustment of endogenous protein composition in Tg seeds, even when grown at adequate sulfur supply. Uptake of sulfur by lupin cotyledons, as indicated by total seed sulfur at maturity, responded positively to increased sulfur supply, but not to increased demand in the Tg seeds.

Limits to Sulfur Accumulation in Transgenic Lupin Seeds Expressing a Foreign Sulfur-Rich Protein

PubMed Central

Tabe, Linda M.; Droux, Michel

2002-01-01

The low sulfur amino acid content of legume seeds restricts their nutritive value for animals. We have investigated the limitations to the accumulation of sulfur amino acids in the storage proteins of narrow leaf lupin (Lupinus angustifolius) seeds. Variation in sulfur supply to lupin plants affected the sulfur amino acid accumulation in the mature seed. However, when sulfur was in abundant supply, it accumulated to a large extent in oxidized form, rather than reduced form, in the seeds. At all but severely limiting sulfur supply, addition of a transgenic (Tg) sink for organic sulfur resulted in an increase in seed sulfur amino acid content. We hypothesize that demand, or sink strength for organic sulfur, which is itself responsive to environmental sulfur supply, was the first limit to the methionine (Met) and cysteine (Cys) content of wild-type lupin seed protein under most growing conditions. In Tg, soil-grown seeds expressing a foreign Met- and Cys-rich protein, decreased pools of free Met, free Cys, and glutathione indicated that the rate of synthesis of sulfur amino acids in the cotyledon had become limiting. Homeostatic mechanisms similar to those mediating the responses of plants to environmental sulfur stress resulted in an adjustment of endogenous protein composition in Tg seeds, even when grown at adequate sulfur supply. Uptake of sulfur by lupin cotyledons, as indicated by total seed sulfur at maturity, responded positively to increased sulfur supply, but not to increased demand in the Tg seeds. PMID:11891268

A recombinant rabies virus carrying GFP between N and P affects viral transcription in vitro.

PubMed

Luo, Jun; Zhao, Jing; Tian, Qin; Mo, Weiyu; Wang, Yifei; Chen, Hao; Guo, Xiaofeng

2016-06-01

Several studies have demonstrated the rabies virus to be a perfect potential vaccine vector to insert foreign genes into the target genome. For this study, a green fluorescent protein (GFP) gene was cloned into the rabies virus (RABV) genome between the N and P gene. CT dinucleotide was inserted as intergenic region. The recombinant high egg passage Flury strain (HEP-Flury) of RABV, carrying GFP (rHEP-NP-GFP), was generated in BHK-21 cells using reverse genetics. According to the viral growth kinetics assay, the addition of GFP between N and P gene has little effect on the viral growth compared to the parental strain HEP-Flury. Quantitative real-time PCR (qPCR) indicated that rHEP-NP-GFP showed different viral gene transcription, especially for G gene, compared to HEP-Flury. The same is true for one other recombinant RABV carrying GFP between G and L gene in NA cells. In addition, parent HEP-Flury showed more expression of innate immune-related molecules in NA cells. Compared to HEP-Flury, Western blotting (WB) indicated that insertion of a foreign gene following N gene enhanced the expression of M and G proteins. According to the qPCR and WB, GFP expression levels of rHEP-NP-GFP were significantly higher than rHEP-GFP. This study indicates HEP-Flury as valid vector to express exogenous genes between N and P.

Tandem Fusion of Hepatitis B Core Antigen Allows Assembly of Virus-Like Particles in Bacteria and Plants with Enhanced Capacity to Accommodate Foreign Proteins

PubMed Central

Peyret, Hadrien; Gehin, Annick; Thuenemann, Eva C.; Blond, Donatienne; El Turabi, Aadil; Beales, Lucy; Clarke, Dean; Gilbert, Robert J. C.; Fry, Elizabeth E.; Stuart, David I.; Holmes, Kris; Stonehouse, Nicola J.; Whelan, Mike; Rosenberg, William; Lomonossoff, George P.; Rowlands, David J.

2015-01-01

The core protein of the hepatitis B virus, HBcAg, assembles into highly immunogenic virus-like particles (HBc VLPs) when expressed in a variety of heterologous systems. Specifically, the major insertion region (MIR) on the HBcAg protein allows the insertion of foreign sequences, which are then exposed on the tips of surface spike structures on the outside of the assembled particle. Here, we present a novel strategy which aids the display of whole proteins on the surface of HBc particles. This strategy, named tandem core, is based on the production of the HBcAg dimer as a single polypeptide chain by tandem fusion of two HBcAg open reading frames. This allows the insertion of large heterologous sequences in only one of the two MIRs in each spike, without compromising VLP formation. We present the use of tandem core technology in both plant and bacterial expression systems. The results show that tandem core particles can be produced with unmodified MIRs, or with one MIR in each tandem dimer modified to contain the entire sequence of GFP or of a camelid nanobody. Both inserted proteins are correctly folded and the nanobody fused to the surface of the tandem core particle (which we name tandibody) retains the ability to bind to its cognate antigen. This technology paves the way for the display of natively folded proteins on the surface of HBc particles either through direct fusion or through non-covalent attachment via a nanobody. PMID:25830365

Enhanced transgene expression in rice following selection controlled by weak promoters.

PubMed

Zhou, Jie; Yang, Yong; Wang, Xuming; Yu, Feibo; Yu, Chulang; Chen, Juan; Cheng, Ye; Yan, Chenqi; Chen, Jianping

2013-03-27

Techniques that enable high levels of transgene expression in plants are attractive for the commercial production of plant-made recombinant pharmaceutical proteins or other gene transfer related strategies. The conventional way to increase the yield of desired transgenic products is to use strong promoters to control the expression of the transgene. Although many such promoters have been identified and characterized, the increase obtainable from a single promoter is ultimately limited to a certain extent. In this study, we report a method to magnify the effect of a single promoter by using a weak promoter-based selection system in transgenic rice. tCUP1, a fragment derived from the tobacco cryptic promoter (tCUP), was tested for its activity in rice by fusion to both a β-glucuronidase (GUS) reporter and a hygromycin phosphotransferase (HPT) selectable marker. The tCUP1 promoter allowed the recovery of transformed rice plants and conferred tissue specific expression of the GUS reporter, but was much weaker than the CaMV 35S promoter in driving a selectable marker for growth of resistant calli. However, in the resistant calli and regenerated transgenic plants selected by the use of tCUP1, the constitutive expression of green fluorescent protein (GFP) was dramatically increased as a result of the additive effect of multiple T-DNA insertions. The correlation between attenuated selection by a weak promoter and elevation of copy number and foreign gene expression was confirmed by using another relatively weak promoter from nopaline synthase (Nos). The use of weak promoter derived selectable markers leads to a high T-DNA copy number and then greatly increases the expression of the foreign gene. The method described here provides an effective approach to robustly enhance the expression of heterogenous transgenes through copy number manipulation in rice.

Altered Escherichia coli membrane protein assembly machinery allows proper membrane assembly of eukaryotic protein vitamin K epoxide reductase.

PubMed

Hatahet, Feras; Blazyk, Jessica L; Martineau, Eugenie; Mandela, Eric; Zhao, Yongxin; Campbell, Robert E; Beckwith, Jonathan; Boyd, Dana

2015-12-08

Functional overexpression of polytopic membrane proteins, particularly when in a foreign host, is often a challenging task. Factors that negatively affect such processes are poorly understood. Using the mammalian membrane protein vitamin K epoxide reductase (VKORc1) as a reporter, we describe a genetic selection approach allowing the isolation of Escherichia coli mutants capable of functionally expressing this blood-coagulation enzyme. The isolated mutants map to components of membrane protein assembly and quality control proteins YidC and HslV. We show that changes in the VKORc1 sequence and in the YidC hydrophilic groove along with the inactivation of HslV promote VKORc1 activity and dramatically increase its expression level. We hypothesize that such changes correct for mismatches in the membrane topogenic signals between E. coli and eukaryotic cells guiding proper membrane integration. Furthermore, the obtained mutants allow the study of VKORc1 reaction mechanisms, inhibition by warfarin, and the high-throughput screening for potential anticoagulants.

Altered Escherichia coli membrane protein assembly machinery allows proper membrane assembly of eukaryotic protein vitamin K epoxide reductase

PubMed Central

Hatahet, Feras; Blazyk, Jessica L.; Martineau, Eugenie; Mandela, Eric; Zhao, Yongxin; Campbell, Robert E.; Beckwith, Jonathan; Boyd, Dana

2015-01-01

Functional overexpression of polytopic membrane proteins, particularly when in a foreign host, is often a challenging task. Factors that negatively affect such processes are poorly understood. Using the mammalian membrane protein vitamin K epoxide reductase (VKORc1) as a reporter, we describe a genetic selection approach allowing the isolation of Escherichia coli mutants capable of functionally expressing this blood-coagulation enzyme. The isolated mutants map to components of membrane protein assembly and quality control proteins YidC and HslV. We show that changes in the VKORc1 sequence and in the YidC hydrophilic groove along with the inactivation of HslV promote VKORc1 activity and dramatically increase its expression level. We hypothesize that such changes correct for mismatches in the membrane topogenic signals between E. coli and eukaryotic cells guiding proper membrane integration. Furthermore, the obtained mutants allow the study of VKORc1 reaction mechanisms, inhibition by warfarin, and the high-throughput screening for potential anticoagulants. PMID:26598701

[In vitro renaturation of proteins from inclusion bodies].

PubMed

Porowińska, Dorota; Marszałek, Ewelina; Wardęcka, Paulina; Komoszyński, Michał

2012-06-11

Recombinant proteins and enzymes are commonly used in many areas of our life, such as diagnostics, industry and medicine, due to heterologous synthesis in prokaryotic expression systems. However, a high expression level of foreign protein in bacteria cells results in formation of inactive and insoluble aggregates--inclusion bodies. Reactivation of aggregated proteins is a complex and time-consuming process. Every protein requires experimental optimization of the process conditions. The choice of the refolding method depends on the type of recombinant protein and its physical, chemical and biological properties. Recovery of the activity of proteins accumulated in inclusion bodies can be divided into 4 steps: 1) inclusion bodies isolation, 2) solubilization of aggregates, 3) renaturation, 4) purification of catalytically active molecules. Efficiency of the refolding process depends on many physical factors and chemical and biological agents. The above parameters determine the time of the folding and prevent protein aggregation. They also assist the folding and have an influence on the solubility and stability of native molecules. To date, dilution, dialysis and chromatography are the most often used methods for protein refolding.

Identification of Immunogenic Hot Spots within Plum Pox Potyvirus Capsid Protein for Efficient Antigen Presentation

PubMed Central

Fernández-Fernández, M. Rosario; Martínez-Torrecuadrada, Jorge L.; Roncal, Fernando; Domínguez, Elvira; García, Juan Antonio

2002-01-01

PEPSCAN analysis has been used to characterize the immunogenic regions of the capsid protein (CP) in virions of plum pox potyvirus (PPV). In addition to the well-known highly immunogenic N- and C-terminal domains of CP, regions within the core domain of the protein have also shown high immunogenicity. Moreover, the N terminus of CP is not homogeneously immunogenic, alternatively showing regions frequently recognized by antibodies and others that are not recognized at all. These results have helped us to design efficient antigen presentation vectors based on PPV. As predicted by PEPSCAN analysis, a small displacement of the insertion site in a previously constructed vector, PPV-γ, turned the derived chimeras into efficient immunogens. Vectors expressing foreign peptides at different positions within a highly immunogenic region (amino acids 43 to 52) in the N-terminal domain of CP were the most effective at inducing specific antibody responses against the foreign sequence. PMID:12438590

Expression of non-toxic mutant of Escherichia coli heat-labile enterotoxin in tobacco chloroplasts.

PubMed

Kang, Tae-Jin; Han, So-Chon; Kim, Mi-Young; Kim, Young-Sook; Yang, Moon-Sik

2004-11-01

Chloroplast transformation systems offer unique advantages in biotechnology, including high level of foreign gene expression, maternal inheritance, and polycistronic expression. We studied chloroplast expression of LTK63 (change Ser-->Lys at position 63 in the A subunit) which is the mutant of Escherichia coli heat-labile toxin. LTK63 is devoid of any toxic activity, but still retains its mucosal adjuvanticity. The LTK63 was cloned into chloroplast targeting vector and transformed to tobacco chloroplasts by particle bombardment. PCR and Southern blot analyses confirmed stable homologous recombination of the LTK63 gene into the chloroplast genome. The amount of LTK63 protein detected in tobacco chloroplasts was approximately 3.7% of the total soluble protein. The GM1-ganglioside binding assay confirmed that chloroplast-synthesized LTB of LTK63 binds to the intestinal membrane GM1-ganglioside receptor. Thus, the expression of LTK63 in chloroplasts provides a potential route toward the development of a plant-based edible vaccine for high expression system and environmentally friendly approach.

Increased expression of Interleukin-13 and connective tissue growth factor, and their potential roles during foreign body encapsulation of subcutaneous implants.

PubMed

Ward, W Kenneth; Li, Allen G; Siddiqui, Yasmin; Federiuk, Isaac F; Wang, Xiao-Jing

2008-01-01

The purpose of this study was to better understand whether interleukin-13 (IL-13) and connective tissue growth factor (CTGF) are highly expressed during foreign body encapsulation of subcutaneous devices. Mock biosensors were implanted into rats for three lengths of time (7-, 21- and 48-55 days) to address different stages of the foreign body response. Using quantitative real-time PCR and immunofluorescence, the expression of IL13, CTGF, collagen 1, decorin and fibronectin were measured in this tissue. IL-13, a product of Th2 cells, was highly expressed at all time points, with greatest expression at day 21. The IL-13 expression was paralleled by increased presence of T-cells at all time points. CTGF was also found to be more highly expressed in foreign body tissue than in controls. Collagen and decorin were highly expressed at the middle and later stages. Given the increased expression of IL-13 and CTGF in foreign body tissue, and their roles in other fibrotic disorders, these cytokines may well contribute to the formation of the foreign body capsule. Since the peak gene expression of IL-13 occurred later than the previously-reported TGFbeta expression peak, IL-13 is probably not the major stimulus to TGFbeta expression during foreign body encapsulation and may contribute to fibrosis independently.

Sensing Self and Foreign Circular RNAs by Intron Identity.

PubMed

Chen, Y Grace; Kim, Myoungjoo V; Chen, Xingqi; Batista, Pedro J; Aoyama, Saeko; Wilusz, Jeremy E; Iwasaki, Akiko; Chang, Howard Y

2017-07-20

Circular RNAs (circRNAs) are single-stranded RNAs that are joined head to tail with largely unknown functions. Here we show that transfection of purified in vitro generated circRNA into mammalian cells led to potent induction of innate immunity genes and confers protection against viral infection. The nucleic acid sensor RIG-I is necessary to sense foreign circRNA, and RIG-I and foreign circRNA co-aggregate in cytoplasmic foci. CircRNA activation of innate immunity is independent of a 5' triphosphate, double-stranded RNA structure, or the primary sequence of the foreign circRNA. Instead, self-nonself discrimination depends on the intron that programs the circRNA. Use of a human intron to express a foreign circRNA sequence abrogates immune activation, and mature human circRNA is associated with diverse RNA binding proteins reflecting its endogenous splicing and biogenesis. These results reveal innate immune sensing of circRNA and highlight introns-the predominant output of mammalian transcription-as arbiters of self-nonself identity. Copyright © 2017 Elsevier Inc. All rights reserved.

A CGMMV genome-replicon vector with partial sequences of coat protein gene efficiently expresses GFP in Nicotiana benthamiana.

PubMed

Jailani, A Abdul Kader; Solanki, Vikas; Roy, Anirban; Sivasudha, T; Mandal, Bikash

2017-04-02

A highly infectious clone of Cucumber green mottle mosaic virus (CGMMV), a cucurbit-infecting tobamovirus was utilized for designing of gene expression vectors. Two versions of vector were examined for their efficacy in expressing the green fluorescent protein (GFP) in Nicotiana benthamiana. When the GFP gene was inserted at the stop codon of coat protein (CP) gene of the CGMMV genome without any read-through codon, systemic expression of GFP, as well as virion formation and systemic symptoms expression were obtained in N. benthamiana. The qRT-PCR analysis showed 23 fold increase of GFP over actin at 10days post inoculation (dpi), which increased to 45 fold at 14dpi and thereafter the GFP expression was significantly declined. Further, we show that when the most of the CP sequence is deleted retaining only the first 105 nucleotides, the shortened vector containing GFP in frame of original CP open reading frame (ORF) resulted in 234 fold increase of GFP expression over actin at 5dpi in N. benthamiana without the formation of virions and disease symptoms. Our study demonstrated that a simple manipulation of CP gene in the CGMMV genome while preserving the translational frame of CP resulted in developing a virus-free, rapid and efficient foreign protein expression system in the plant. The CGMMV based vectors developed in this study may be potentially useful for the production of edible vaccines in cucurbits. Copyright © 2017 Elsevier B.V. All rights reserved.

DNA transformation via local heat shock

NASA Astrophysics Data System (ADS)

Li, Sha; Meadow Anderson, L.; Yang, Jui-Ming; Lin, Liwei; Yang, Haw

2007-07-01

This work describes transformation of foreign DNA into bacterial host cells by local heat shock using a microfluidic system with on-chip, built-in platinum heaters. Plasmid DNA encoding ampicillin resistance and a fluorescent protein can be effectively transformed into the DH5α chemically competent E. coli using this device. Results further demonstrate that only one-thousandth of volume is required to obtain transformation efficiencies as good as or better than conventional practices. As such, this work complements other lab-on-a-chip technologies for potential gene cloning/therapy and protein expression applications.

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. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Geminivirus vectors for high-level expression of foreign proteins in plant cells.

PubMed

Mor, Tsafrir S; Moon, Yong-Sun; Palmer, Kenneth E; Mason, Hugh S

2003-02-20

Bean yellow dwarf virus (BeYDV) is a monopartite geminivirus that can infect dicotyledonous plants. We have developed a high-level expression system that utilizes elements of the replication machinery of this single-stranded DNA virus. The replication initiator protein (Rep) mediates release and replication of a replicon from a DNA construct ("LSL vector") that contains an expression cassette for a gene of interest flanked by cis-acting elements of the virus. We used tobacco NT1 cells and biolistic delivery of plasmid DNA for evaluation of replication and expression of reporter genes contained within an LSL vector. By codelivery of a GUS reporter-LSL vector and a Rep-supplying vector, we obtained up to 40-fold increase in expression levels compared to delivery of the reporter-LSL vectors alone. High-copy replication of the LSL vector was correlated with enhanced expression of GUS. Rep expression using a whole BeYDV clone, a cauliflower mosaic virus 35S promoter driving either genomic rep or an intron-deleted rep gene, or 35S-rep contained in the LSL vector all achieved efficient replication and enhancement of GUS expression. We anticipate that this system can be adapted for use in transgenic plants or plant cell cultures with appropriately regulated expression of Rep, with the potential to greatly increase yield of recombinant proteins. Copyright 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 430-437, 2003.

Baculovirus expression system and method for high throughput expression of genetic material

DOEpatents

Clark, Robin; Davies, Anthony

2001-01-01

The present invention provides novel recombinant baculovirus expression systems for expressing foreign genetic material in a host cell. Such expression systems are readily adapted to an automated method for expression foreign genetic material in a high throughput manner. In other aspects, the present invention features a novel automated method for determining the function of foreign genetic material by transfecting the same into a host by way of the recombinant baculovirus expression systems according to the present invention.

Proteomic Analysis of Fetal Ovary Reveals That Ovarian Developmental Potential Is Greater in Meishan Pigs than in Yorkshire Pigs

PubMed Central

Che, Long; Wang, Dingyue; Yang, Zhenguo; Zhang, Pan; Lin, Yan; Fang, Zhengfeng; Che, Lianqiang; Li, Jian; Chen, Daiwen; Wu, De

2015-01-01

Time-dependent expression of functional proteins in fetal ovaries is important to understand the developmental process of the ovary. This study was carried out to enhance our understanding of the developmental process of porcine fetal ovaries and to better address the differences in fetal ovary development of local and foreign pigs. The objective of the present study is to test the expression of key proteins that regulate the growth and development of fetal ovaries in Meishan and Yorkshire porcine breeds by using proteomics technology. Six Meishan and 6 Yorkshire pregnant gilts were used in this experiment. Fetal ovaries were obtained from Yorkshire and Meishan gilts on days 55 and 90 of the gestation period. Using 2D-DIGE (two dimensional-difference in gel electrophoresis) analysis, the results showed that there are about 1551 and 1400 proteins in gilt fetal ovaries on days 55 and 90, respectively of the gestation. Using MALDI TOF-TOF MS analysis, 27 differentially expressed proteins were identified in the fetal ovaries of the 2 breeds on day 55 of gestation, and a total of 18 proteins were identified on day 90 of gestation. These differentially expressed proteins were involved in the regulation of biological processes (cell death, stress response, cytoskeletal proteins) and molecular functions (enzyme regulator activity). We also found that alpha-1-antitrypsin, actin, vimentin, and PP2A proteins promote the formation of primordial follicles in the ovaries of Yorkshire pigs on day 55 of gestation while low expression heat shock proteins and high expression alpha-fetoproteins (AFP) may promote Meishan fetal ovarian follicular development on day 90 of gestation. These findings provide a deeper understanding of how reduced expression of heat shock proteins and increased expression of AFP can significantly reduce the risk of reproductive disease in obese Meishan sows. Our study also shows how these proteins can increase the ovulation rate and may be responsible for the low reproductive efficiency reported in other obese breeds. The ovarian developmental potential was found to be greater in Meishan pigs than in Yorkshire pigs. PMID:26305539

Nuclear sensing of viral DNA, epigenetic regulation of herpes simplex virus infection, and innate immunity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Knipe, David M., E-mail: david_knipe@hms.harvard.edu

Herpes simplex virus (HSV) undergoes a lytic infection in epithelial cells and a latent infection in neuronal cells, and epigenetic mechanisms play a major role in the differential gene expression under the two conditions. HSV viron DNA is not associated with histones but is rapidly loaded with heterochromatin upon entry into the cell. Viral proteins promote reversal of the epigenetic silencing in epithelial cells while the viral latency-associated transcript promotes additional heterochromatin in neuronal cells. The cellular sensors that initiate the chromatinization of foreign DNA have not been fully defined. IFI16 and cGAS are both essential for innate sensing ofmore » HSV DNA, and new evidence shows how they work together to initiate innate signaling. IFI16 also plays a role in the heterochromatinization of HSV DNA, and this review will examine how IFI16 integrates epigenetic regulation and innate sensing of foreign viral DNA to show how these two responses are related. - Highlights: • HSV lytic and latent gene expression is regulated differentially by epigenetic processes. • The sensors of foreign DNA have not been defined fully. • IFI16 and cGAS cooperate to sense viral DNA in HSV-infected cells. • IFI16 plays a role in both innate sensing of HSV DNA and in restricting its expression.« less

Improved Innate and Adaptive Immunostimulation by Genetically Modified HIV-1 Protein Expressing NYVAC Vectors

PubMed Central

Quakkelaar, Esther D.; Redeker, Anke; Haddad, Elias K.; Harari, Alexandre; McCaughey, Stella Mayo; Duhen, Thomas; Filali-Mouhim, Abdelali; Goulet, Jean-Philippe; Loof, Nikki M.; Ossendorp, Ferry; Perdiguero, Beatriz; Heinen, Paul; Gomez, Carmen E.; Kibler, Karen V.; Koelle, David M.; Sékaly, Rafick P.; Sallusto, Federica; Lanzavecchia, Antonio; Pantaleo, Giuseppe; Esteban, Mariano; Tartaglia, Jim; Jacobs, Bertram L.; Melief, Cornelis J. M.

2011-01-01

Attenuated poxviruses are safe and capable of expressing foreign antigens. Poxviruses are applied in veterinary vaccination and explored as candidate vaccines for humans. However, poxviruses express multiple genes encoding proteins that interfere with components of the innate and adaptive immune response. This manuscript describes two strategies aimed to improve the immunogenicity of the highly attenuated, host-range restricted poxvirus NYVAC: deletion of the viral gene encoding type-I interferon-binding protein and development of attenuated replication-competent NYVAC. We evaluated these newly generated NYVAC mutants, encoding HIV-1 env, gag, pol and nef, for their ability to stimulate HIV-specific CD8 T-cell responses in vitro from blood mononuclear cells of HIV-infected subjects. The new vectors were evaluated and compared to the parental NYVAC vector in dendritic cells (DCs), RNA expression arrays, HIV gag expression and cross-presentation assays in vitro. Deletion of type-I interferon-binding protein enhanced expression of interferon and interferon-induced genes in DCs, and increased maturation of infected DCs. Restoration of replication competence induced activation of pathways involving antigen processing and presentation. Also, replication-competent NYVAC showed increased Gag expression in infected cells, permitting enhanced cross-presentation to HIV-specific CD8 T cells and proliferation of HIV-specific memory CD8 T-cells in vitro. The recombinant NYVAC combining both modifications induced interferon-induced genes and genes involved in antigen processing and presentation, as well as increased Gag expression. This combined replication-competent NYVAC is a promising candidate for the next generation of HIV vaccines. PMID:21347234

The Foreign Body Giant Cell Cannot Resorb Bone, But Dissolves Hydroxyapatite Like Osteoclasts.

PubMed

ten Harkel, Bas; Schoenmaker, Ton; Picavet, Daisy I; Davison, Noel L; de Vries, Teun J; Everts, Vincent

2015-01-01

Foreign body multinucleated giant cells (FBGCs) and osteoclasts share several characteristics, like a common myeloid precursor cell, multinuclearity, expression of tartrate-resistant acid phosphatase (TRAcP) and dendritic cell-specific transmembrane protein (DC-STAMP). However, there is an important difference: osteoclasts form and reside in the vicinity of bone, while FBGCs form only under pathological conditions or at the surface of foreign materials, like medical implants. Despite similarities, an important distinction between these cell types is that osteoclasts can resorb bone, but it is unknown whether FBGCs are capable of such an activity. To investigate this, we differentiated FBGCs and osteoclasts in vitro from their common CD14+ monocyte precursor cells, using different sets of cytokines. Both cell types were cultured on bovine bone slices and analyzed for typical osteoclast features, such as bone resorption, presence of actin rings, formation of a ruffled border, and characteristic gene expression over time. Additionally, both cell types were cultured on a biomimetic hydroxyapatite coating to discriminate between bone resorption and mineral dissolution independent of organic matrix proteolysis. Both cell types differentiated into multinucleated cells on bone, but FBGCs were larger and had a higher number of nuclei compared to osteoclasts. FBGCs were not able to resorb bone, yet they were able to dissolve the mineral fraction of bone at the surface. Remarkably, FBGCs also expressed actin rings, podosome belts and sealing zones--cytoskeletal organization that is considered to be osteoclast-specific. However, they did not form a ruffled border. At the gene expression level, FBGCs and osteoclasts expressed similar levels of mRNAs that are associated with the dissolution of mineral (e.g., anion exchange protein 2 (AE2), carbonic anhydrase 2 (CAII), chloride channel 7 (CIC7), and vacuolar-type H+-ATPase (v-ATPase)), in contrast the matrix degrading enzyme cathepsin K, which was hardly expressed by FBGCs. Functionally, the latter cells were able to dissolve a biomimetic hydroxyapatite coating in vitro, which was blocked by inhibiting v-ATPase enzyme activity. These results show that FBGCs have the capacity to dissolve the mineral phase of bone, similar to osteoclasts. However, they are not able to digest the matrix fraction of bone, likely due to the lack of a ruffled border and cathepsin K.

The Foreign Body Giant Cell Cannot Resorb Bone, But Dissolves Hydroxyapatite Like Osteoclasts

PubMed Central

ten Harkel, Bas; Schoenmaker, Ton; Picavet, Daisy I.; Davison, Noel L.; de Vries, Teun J.; Everts, Vincent

2015-01-01

Foreign body multinucleated giant cells (FBGCs) and osteoclasts share several characteristics, like a common myeloid precursor cell, multinuclearity, expression of tartrate-resistant acid phosphatase (TRAcP) and dendritic cell-specific transmembrane protein (DC-STAMP). However, there is an important difference: osteoclasts form and reside in the vicinity of bone, while FBGCs form only under pathological conditions or at the surface of foreign materials, like medical implants. Despite similarities, an important distinction between these cell types is that osteoclasts can resorb bone, but it is unknown whether FBGCs are capable of such an activity. To investigate this, we differentiated FBGCs and osteoclasts in vitro from their common CD14+ monocyte precursor cells, using different sets of cytokines. Both cell types were cultured on bovine bone slices and analyzed for typical osteoclast features, such as bone resorption, presence of actin rings, formation of a ruffled border, and characteristic gene expression over time. Additionally, both cell types were cultured on a biomimetic hydroxyapatite coating to discriminate between bone resorption and mineral dissolution independent of organic matrix proteolysis. Both cell types differentiated into multinucleated cells on bone, but FBGCs were larger and had a higher number of nuclei compared to osteoclasts. FBGCs were not able to resorb bone, yet they were able to dissolve the mineral fraction of bone at the surface. Remarkably, FBGCs also expressed actin rings, podosome belts and sealing zones—cytoskeletal organization that is considered to be osteoclast-specific. However, they did not form a ruffled border. At the gene expression level, FBGCs and osteoclasts expressed similar levels of mRNAs that are associated with the dissolution of mineral (e.g., anion exchange protein 2 (AE2), carbonic anhydrase 2 (CAII), chloride channel 7 (CIC7), and vacuolar-type H+-ATPase (v-ATPase)), in contrast the matrix degrading enzyme cathepsin K, which was hardly expressed by FBGCs. Functionally, the latter cells were able to dissolve a biomimetic hydroxyapatite coating in vitro, which was blocked by inhibiting v-ATPase enzyme activity. These results show that FBGCs have the capacity to dissolve the mineral phase of bone, similar to osteoclasts. However, they are not able to digest the matrix fraction of bone, likely due to the lack of a ruffled border and cathepsin K. PMID:26426806

The non-essential UL50 gene of avian infectious laryngotracheitis virus encodes a functional dUTPase which is not a virulence factor.

PubMed

Fuchs, W; Ziemann, K; Teifke, J P; Werner, O; Mettenleiter, T C

2000-03-01

The DNA sequence of the infectious laryngotracheitis virus (ILTV) UL50, UL51 and UL52 gene homologues was determined. Although the deduced UL50 protein lacks the first of five conserved domains of the corresponding proteins of mammalian alphaherpesviruses, the ILTV gene product was also shown to possess dUTPase activity. The generation of UL50-negative ILTV mutants was facilitated by recombination plasmids encoding green fluorescent protein (GFP), and expression constructs of predicted transactivator proteins of ILTV (alphaTIF, ICP4) were successfully used to increase the infectivity of viral genomic DNA. A GFP-expressing UL50-deletion mutant of ILTV showed reduced cell-to-cell spread in vitro, and was attenuated in vivo. A similar deletion mutant without the foreign gene, however, propagated like wild-type ILTV in cell culture and was pathogenic in chickens. We conclude that the viral dUTPase is not required for efficient replication of ILTV in the respiratory tract of infected animals. The replication defect of the GFP-expressing ILTV recombinant is most likely caused by toxic effects of the reporter gene product, since spontaneously occurring inactivation mutants exhibited wild-type-like growth.

Activity of foreign proteins targeted within the bacteriophage T4 head and prohead: implications for packaged DNA structure.

PubMed

Mullaney, J M; Black, L W

1998-11-13

The phage-derived expression, packaging, and processing (PEPP) system was used to target foreign proteins into the bacteriophage capsid to probe the intracapsid environment and the structure of packaged DNA. Small proteins with minimal requirements for activity were selected, staphylococcal nuclease (SN) and green fluorescent protein (GFP). These proteins were targeted into the T4 head by means of IPIII (internal protein III) fusions or CTS (capsid targeting sequence) fusions. Additional evidence is provided that foreign proteins are targeted into T4 by the N-terminal ten amino acid residue consensus CTS of IPIII identified in previous work. Fusion proteins were produced within host bacteria by expression from plasmids or by produc tion from recombinant phage carrying the fusion genes. Packaged fusion proteins CTS IPIII SN, CTS IPIII TSN, CTS IPIII GFP, CTS IPIII TGFP, and CTS GFP, where [symbol: see text] indicates a linkage peptide sequence Leu(Ile)-N-Glu cleaved by the T4 head morphogenetic proteinase gp21 during head maturation, are observed to exhibit intracapsid activity. SN activity within the head is demonstrated by loss of phage viability and by digested genomic DNA patterns visualized by gel electrophoresis when viable phage are incubated in Ca2+. Green fluorescent phage result immediately after packaging GFP produced at 30 degreesC and below, and continue to give green fluorescence under 470 nm light after CsCl purification. Non-fluorescent GFP-fusions are produced in bacteria at 37 degreesC, and phage packaged with these proteins achieve a fluorescent state after incubation for several months at 4 degreesC. GFP-packaged phage and proheads analyzed by fluorescence spectroscopy show that the mature head and the DNA-empty prohead package identical numbers of GFP-fusion proteins. Encapsidated GFP and SN can be injected into bacteria and rapidly exhibit intracellular activity. In vivo SN digestion of encapsidated DNA gives an intriguing pattern of DNA fragments by gel analysis, predominantly a repeat pattern of 160 bp multiples, reminiscent of a nucleosome digestion ladder, This quasi-limit DNA digestion pattern, reached >100-fold more slowly than the loss of titer, is invariant over a range

Enhanced heterologous expression of biologically active human granulocyte colony stimulating factor in transgenic tobacco BY-2 cells by localization to endoplasmic reticulum.

PubMed

Nair, Nisha R; Chidambareswaren, M; Manjula, S

2014-09-01

Tobacco Bright Yellow-2 (BY-2) cells, one of the best characterized cell lines is an attractive expression system for heterologous protein expression. However, the expression of foreign proteins is currently hampered by their low yield, which is partially the result of proteolytic degradation. Human granulocyte colony stimulating factor (hG-CSF) is a hematopoietic cytokine. Recombinant hG-CSF is successfully being used for the treatment of chemotherapy-induced neutropenia in cancer patients. Here, we describe a simple strategy for producing biologically active hG-CSF in tobacco BY-2 cells, localized in the apoplast of BY-2 cells, as well as targeted to the endoplasmic reticulum (ER). ER targeting significantly enhanced recombinant production which scaled to 17.89 mg/l from 4.19 mg/l when expressed in the apoplasts. Southern blotting confirmed the stable integration of hG-CSF in the BY-2 nuclear genome, and the expression of hG-CSF was analysed by Western blotting. Total soluble protein containing hG-CSF isolated from positive calli showed proliferative potential when tested on HL-60 cell lines by MTT assay. We also report the potential of a Fluorescence-activated cell sorting approach for an efficient sorting of the hG-CSF-expressing cell lines, which will enable the generation of homogenous high-producing cell lines.

Essential and Dispensable Virus-Encoded Replication Elements Revealed by Efforts To Develop Hypoviruses as Gene Expression Vectors

PubMed Central

Suzuki, Nobuhiro; Geletka, Lynn M.; Nuss, Donald L.

2000-01-01

We have investigated whether hypoviruses, viral agents responsible for virulence attenuation (hypovirulence) of the chestnut blight fungus Cryphonectria parasitica, could serve as gene expression vectors. The infectious cDNA clone of the prototypic hypovirus CHV1-EP713 was modified to generate 20 different vector candidates. Although transient expression was achieved for a subset of vectors that contained the green fluorescent protein gene from Aequorea victoria, long-term expression (past day 8) was not observed for any vector construct. Analysis of viral RNAs recovered from transfected fungal colonies revealed that the foreign genes were readily deleted from the replicating virus, although small portions of foreign sequences were retained by some vectors after months of replication. However, the results of vector viability and progeny characterization provided unexpected new insights into essential and dispensable elements of hypovirus replication. The N-terminal portion (codons 1 to 24) of the 5′-proximal open reading frame (ORF), ORF A, was found to be required for virus replication, while the remaining 598 codons of this ORF were completely dispensable. Substantial alterations were tolerated in the pentanucleotide UAAUG that contains the ORF A termination codon and the overlapping putative initiation codon of the second of the two hypovirus ORFs, ORF B. Replication competence was maintained following either a frameshift mutation that caused a two-codon extension of ORF A or a modification that produced a single-ORF genomic organization. These results are discussed in terms of determinants of hypovirus replication, the potential utility of hypoviruses as gene expression vectors, and possible mechanisms by which hypoviruses recognize and delete foreign sequences. PMID:10906211

Phaseolin expression in tobacco chloroplast reveals an autoregulatory mechanism in heterologous protein translation.

PubMed

De Marchis, Francesca; Bellucci, Michele; Pompa, Andrea

2016-02-01

Plastid DNA engineering is a well-established research area of plant biotechnology, and plastid transgenes often give high expression levels. However, it is still almost impossible to predict the accumulation rate of heterologous protein in transplastomic plants, and there are many cases of unsuccessful transgene expression. Chloroplasts regulate their proteome at the post-transcriptional level, mainly through translation control. One of the mechanisms to modulate the translation has been described in plant chloroplasts for the chloroplast-encoded subunits of multiprotein complexes, and the autoregulation of the translation initiation of these subunits depends on the availability of their assembly partners [control by epistasy of synthesis (CES)]. In Chlamydomonas reinhardtii, autoregulation of endogenous proteins recruited in the assembly of functional complexes has also been reported. In this study, we revealed a self-regulation mechanism triggered by the accumulation of a soluble recombinant protein, phaseolin, in the stroma of chloroplast-transformed tobacco plants. Immunoblotting experiments showed that phaseolin could avoid this self-regulation mechanism when targeted to the thylakoids in transplastomic plants. To inhibit the thylakoid-targeted phaseolin translation as well, this protein was expressed in the presence of a nuclear version of the phaseolin gene with a transit peptide. Pulse-chase and polysome analysis revealed that phaseolin mRNA translation on plastid ribosomes was repressed due to the accumulation in the stroma of the same soluble polypeptide imported from the cytosol. We suggest that translation autoregulation in chloroplast is not limited to heteromeric protein subunits but also involves at least some of the foreign soluble recombinant proteins, leading to the inhibition of plastome-encoded transgene expression in chloroplast. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Purification of phage display-modified bacteriophage T4 by affinity chromatography

PubMed Central

2011-01-01

Background Affinity chromatography is one of the most efficient protein purification strategies. This technique comprises a one-step procedure with a purification level in the order of several thousand-fold, adaptable for various proteins, differentiated in their size, shape, charge, and other properties. The aim of this work was to verify the possibility of applying affinity chromatography in bacteriophage purification, with the perspective of therapeutic purposes. T4 is a large, icosahedral phage that may serve as an efficient display platform for foreign peptides or proteins. Here we propose a new method of T4 phage purification by affinity chromatography after its modification with affinity tags (GST and Histag) by in vivo phage display. As any permanent introduction of extraneous DNA into a phage genome is strongly unfavourable for medical purposes, integration of foreign motifs with the phage genome was not applied. The phage was propagated in bacteria expressing fusions of the phage protein Hoc with affinity tags from bacterial plasmids, independently from the phage expression system. Results Elution profiles of phages modified with the specific affinity motifs (compared to non-specific phages) document their binding to the affinity resins and effective elution with standard competitive agents. Non-specific binding was also observed, but was 102-105 times weaker than the specific one. GST-modified bacteriophages were also effectively released from glutathione Sepharose by proteolytic cleavage. The possibility of proteolytic release was designed at the stage of expression vector construction. Decrease in LPS content in phage preparations was dependent on the washing intensity; intensive washing resulted in preparations of 11-40 EU/ml. Conclusions Affinity tags can be successfully incorporated into the T4 phage capsid by the in vivo phage display technique and they strongly elevate bacteriophage affinity to a specific resin. Affinity chromatography can be considered as a new phage purification method, appropriate for further investigations and development. PMID:21627821

Robust Transgene Expression from Bicistronic mRNA in the Green Alga Chlamydomonas reinhardtii

PubMed Central

Onishi, Masayuki; Pringle, John R.

2016-01-01

The unicellular green alga Chlamydomonas reinhardtii is a model organism that provides an opportunity to understand the evolution and functional biology of the lineage that includes the land plants, as well as aspects of the fundamental core biology conserved throughout the eukaryotic phylogeny. Although many tools are available to facilitate genetic, molecular biological, biochemical, and cell biological studies in Chlamydomonas, expression of unselected transgenes of interest (GOIs) has been challenging. In most methods used previously, the GOI and a selectable marker are expressed from two separate mRNAs, so that their concomitant expression is not guaranteed. In this study, we developed constructs that allow expression of an upstream GOI and downstream selectable marker from a single bicistronic mRNA. Although this approach in other systems has typically required a translation-enhancing element such as an internal ribosome entry site for the downstream marker, we found that a short stretch of unstructured junction sequence was sufficient to obtain adequate expression of the downstream gene, presumably through post-termination reinitiation. With this system, we obtained robust expression of both endogenous and heterologous GOIs, including fluorescent proteins and tagged fusion proteins, in the vast majority of transformants, thus eliminating the need for tedious secondary screening for GOI-expressing transformants. This improved efficiency should greatly facilitate a variety of genetic and cell-biological studies in Chlamydomonas and also enable new applications such as expression-based screens and large-scale production of foreign proteins. PMID:27770025

Efficient renaturation of inclusion body proteins denatured by SDS.

PubMed

He, Chuan; Ohnishi, Kouhei

2017-09-02

Inclusion bodies are often formed when the foreign protein is over expressed in Escherichia coli. Since proteins in inclusion bodies are inactive, denaturing and refolding of inclusion body proteins are necessary to obtain the active form. Instead of the conventional denaturants, urea and guanidine hydrochloride, a strong anionic detergent SDS was used to solubilize C-terminal His-tag form of ulvan lyase in the inclusion bodies. Solution containing SDS-solubilized enzyme were kept on ice to precipitate SDS, followed by SDS-KCl insoluble crystal formation to remove SDS completely. After removing the precipitate by centrifugation, the supernatant was applied to Ni-NTA column to purify His-tagged ulvan lyase. The purified protein showed a dimeric form and ulvan lyase activity, demonstrating that SDS-denatured protein was renatured and recovered enzyme activity. This simple method could be useful for refolding other inclusion body proteins. Copyright © 2017 Elsevier Inc. All rights reserved.

Expression of endogenous and foreign ribulose 1,5-bisphosphate carboxylase-oxygenase (RubisCO) genes in a RubisCO deletion mutant of Rhodobacter sphaeroides.

PubMed Central

Falcone, D L; Tabita, F R

1991-01-01

A Rhodobacter sphaeroides ribulose 1,5-bisphosphate carboxylase-oxygenase (RubisCO) deletion strain was constructed that was complemented by plasmids containing either the form I or form II CO2 fixation gene cluster. This strain was also complemented by genes encoding foreign RubisCO enzymes expressed from a Rhodospirillum rubrum RubisCO promoter. In R. sphaeroides, the R. rubrum promoter was regulated, resulting in variable levels of disparate RubisCO molecules under different growth conditions. Photosynthetic growth of the R. sphaeroides deletion strain complemented with cyanobacterial RubisCO revealed physiological properties reflective of the unique cellular environment of the cyanobacterial enzyme. The R. sphaeroides RubisCO deletion strain and R. rubrum promoter system may be used to assess the properties of mutagenized proteins in vivo, as well as provide a potential means to select for altered RubisCO molecules after random mutagenesis of entire genes or gene regions encoding RubisCO enzymes. Images PMID:1900508

Device-based local delivery of siRNA against mammalian target of rapamycin (mTOR) in a murine subcutaneous implant model to inhibit fibrous encapsulation

PubMed Central

Takahashi, Hironobu; Wang, Yuwei; Grainger, David W.

2010-01-01

Fibrous encapsulation of surgically implant devices is associated with elevated proliferation and activation of fibroblasts in tissues surrounding these implants, frequently causing foreign body complications. Here we test the hypothesis that inhibition of the expression of mammalian target of rapamycin (mTOR) in fibroblasts can mitigate the soft tissue implant foreign body response by suppressing fibrotic responses around implants. In this study, mTOR was knocked down using small interfering RNA conjugated with branched cationic polyethylenimine (bPEI) in fibroblastic lineage cells in serum-based cell culture as shown by both gene and protein analysis. This mTOR knockdown led to an inhibition in fibroblast proliferation by 70% and simultaneous down-regulation in the expression of type I collagen in fibroblasts in vitro. These siRNA/bPEI complexes were released from poly(ethylene glycol) (PEG)-based hydrogel coatings surrounding model polymer implants in a subcutaneous rodent model in vivo. No significant reduction in fibrous capsule thickness and mTOR expression in the foreign body capsules was observed. Observed siRNA inefficacy in this in vivo implant model was attributed to siRNA dosing limitations in the gel delivery system, and lack of targeting ability of the siRNA complex specifically to fibroblasts. While in vitro data supported mTOR knock-down in fibroblast cultures, in vivo siRNA delivery must be further improved to produce clinically relevant effects on fibrotic encapsulation around implants. PMID:20727922

Elevation of transforming growth factor beta (TGFbeta) and its downstream mediators in subcutaneous foreign body capsule tissue.

PubMed

Li, Allen G; Quinn, Matthew J; Siddiqui, Yasmin; Wood, Michael D; Federiuk, Isaac F; Duman, Heather M; Ward, W Kenneth

2007-08-01

Foreign body encapsulation represents a chronic fibrotic response and has been a major obstacle that reduces the useful life of implanted biomedical devices. The precise mechanism underlying such an encapsulation is still unknown. We hypothesized that, considering its central role in many other fibrotic conditions, transforming growth factor beta (TGFbeta) may play an important role during the formation of foreign body capsule (FBC). In the present study, we implanted mock sensors in rats subcutaneously and excised FBC samples at day 7, 21, and 48-55 postimplantation. The most abundant TGFbeta isoform in all tissues was TGFbeta1, which was expressed minimally in control tissue. The expression of both TGFbeta1 RNA and protein was significantly increased in FBC tissues at all time points, with the highest level in day 7 FBC. The number of cells stained for phosphorylated Smad2, an indication of activated TGFbeta signaling, paralleled the expression of TGFbeta. A similar dynamic change was also observed in the numbers of FBC myofibroblasts, which in response to TGFbeta, differentiate from quiescent fibroblasts and synthesize collagen. Type I collagen, the most prominent downstream target of TGFbeta in fibrosis, was found in abundance in the FBC, especially during the latter time periods. We suggest that TGFbeta plays an important role in the FBC formation. Inhibition of TGFbeta signaling could be a promising strategy in the prevention of FBC formation, thereby extending the useful life of subcutaneous implants.

A virus vector based on Canine Herpesvirus for vaccine applications in canids.

PubMed

Strive, T; Hardy, C M; Wright, J; Reubel, G H

2007-01-31

Canine Herpesvirus (CHV) is being developed as a virus vector for the vaccination of European red foxes. However, initial studies using recombinant CHV vaccines in foxes revealed viral attenuation and lack of antibody response to inserted foreign antigens. These findings were attributed both to inactivation of the thymidine kinase (TK) gene and excess foreign genetic material in the recombinant viral genome. In this study, we report an improved CHV-bacterial artificial chromosome (BAC) vector system designed to overcome attenuation in foxes. A non-essential region was identified in the CHV genome as an alternative insertion site for foreign genes. Replacement of a guanine/cytosine (GC)-rich intergenic region between UL21 and UL22 of CHV with a marker gene did not change growth behaviour in vitro, showing that this region is not essential for virus growth in cell culture. We subsequently produced a CHV-BAC vector with an intact TK gene in which the bacterial genes and the antigen expression cassette were inserted into this GC-rich locus. Unlike earlier constructs, the new CHV-BAC allowed self-excision of the bacterial genes via homologous recombination after transfection of BACs into cell culture. The BAC-CHV system was used to produce a recombinant virus that constitutively expressed porcine zona pellucida subunit C protein between the UL21 and UL22 genes of CHV. Complete self-excision of the bacterial genes from CHV was achieved within one round of replication whilst retaining antigen gene expression.

Development of a duplex real-time RT-qPCR assay to monitor genome replication, gene expression and gene insert stability during in vivo replication of a prototype live attenuated canine distemper virus vector encoding SIV gag.

PubMed

Coleman, John W; Wright, Kevin J; Wallace, Olivia L; Sharma, Palka; Arendt, Heather; Martinez, Jennifer; DeStefano, Joanne; Zamb, Timothy P; Zhang, Xinsheng; Parks, Christopher L

2015-03-01

Advancement of new vaccines based on live viral vectors requires sensitive assays to analyze in vivo replication, gene expression and genetic stability. In this study, attenuated canine distemper virus (CDV) was used as a vaccine delivery vector and duplex 2-step quantitative real-time RT-PCR (RT-qPCR) assays specific for genomic RNA (gRNA) or mRNA have been developed that concurrently quantify coding sequences for the CDV nucleocapsid protein (N) and a foreign vaccine antigen (SIV Gag). These amplicons, which had detection limits of about 10 copies per PCR reaction, were used to show that abdominal cavity lymphoid tissues were a primary site of CDV vector replication in infected ferrets, and importantly, CDV gRNA or mRNA was undetectable in brain tissue. In addition, the gRNA duplex assay was adapted for monitoring foreign gene insert genetic stability during in vivo replication by analyzing the ratio of CDV N and SIV gag genomic RNA copies over the course of vector infection. This measurement was found to be a sensitive probe for assessing the in vivo genetic stability of the foreign gene insert. Copyright © 2014 Elsevier B.V. All rights reserved.

Membrane penetrating peptides greatly enhance baculovirus transduction efficiency into mammalian cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Hong-Zhang; Institute of Molecular Biology, Academia Sinica, Taipei 115, Taiwan, ROC; Wu, Carol P.

2011-02-11

Research highlights: {yields} Ligation of CTP with GP64 enhances baculovirus transduction into mammalian cells. {yields} Fusion of PTD with VP39 enhances baculovirus transduction into mammalian cells. {yields} CTP and PTD-carrying viruses improve the transduction of co-transduced baculoviruses. {yields} Virus entry and gene expression can be separate events in different cell types. -- Abstract: The baculovirus group of insect viruses is widely used for foreign gene introduction into mammalian cells for gene expression and protein production; however, the efficiency of baculovirus entry into mammalian cells is in general still low. In this study, two recombinant baculoviruses were engineered and their abilitymore » to improve viral entry was examined: (1) cytoplasmic transduction peptide (CTP) was fused with baculovirus envelope protein, GP64, to produce a cytoplasmic membrane penetrating baculovirus (vE-CTP); and (2) the protein transduction domain (PTD) of HIV TAT protein was fused with the baculovirus capsid protein VP39 to form a nuclear membrane penetrating baculovirus (vE-PTD). Transduction experiments showed that both viruses had better transduction efficiency than vE, a control virus that only expresses EGFP in mammalian cells. Interestingly, vE-CTP and vE-PTD were also able to improve the transduction efficiency of a co-transduced baculovirus, resulting in higher levels of gene expression. Our results have described new routes to further enhance the development of baculovirus as a tool for gene delivery into mammalian cells.« less

Targeted entry of enveloped viruses: measles and herpes simplex virus I.

PubMed

Navaratnarajah, Chanakha K; Miest, Tanner S; Carfi, Andrea; Cattaneo, Roberto

2012-02-01

We compare the receptor-based mechanisms that a small RNA virus and a larger DNA virus have evolved to drive the fusion of viral and cellular membranes. Both systems rely on tight control over triggering the concerted refolding of a trimeric fusion protein. While measles virus entry depends on a receptor-binding protein and a fusion protein only, the herpes simplex virus (HSV) is more complex and requires four viral proteins. Nevertheless, in both viruses a receptor-binding protein is required for triggering the membrane fusion process. Moreover, specificity domains can be appended to these receptor-binding proteins to target virus entry to cells expressing a designated receptor. We discuss how principles established with measles and HSV can be applied to targeting other enveloped viruses, and alternatively how retargeted envelopes can be fitted on foreign capsids. Copyright © 2011 Elsevier B.V. All rights reserved.

A minimal peach type II chlorophyll a/b-binding protein promoter retains tissue-specificity and light regulation in tomato

PubMed Central

Bassett, Carole L; Callahan, Ann M; Artlip, Timothy S; Scorza, Ralph; Srinivasan, Chinnathambi

2007-01-01

Background Promoters with tissue-specificity are desirable to drive expression of transgenes in crops to avoid accumulation of foreign proteins in edible tissues/organs. Several photosynthetic promoters have been shown to be strong regulators of expression of transgenes in light-responsive tissues and would be good candidates for leaf and immature fruit tissue-specificity, if expression in the mature fruit were minimized. Results A minimal peach chlorophyll a/b-binding protein gene (Lhcb2*Pp1) promoter (Cab19) was isolated and fused to an uidA (β-glucuronidase [GUS]) gene containing the PIV2 intron. A control vector carrying an enhanced mas35S CaMV promoter fused to uidA was also constructed. Two different orientations of the Cab19::GUS fusion relative to the left T-DNA border of the binary vector were transformed into tomato. Ten independent regenerants of each construct and an untransformed control line were assessed both qualitatively and quantitatively for GUS expression in leaves, fruit and flowers, and quantitatively in roots. Conclusion The minimal CAB19 promoter conferred GUS activity primarily in leaves and green fruit, as well as in response to light. GUS activity in the leaves of both Cab19 constructs averaged about 2/3 that observed with mas35S::GUS controls. Surprisingly, GUS activity in transgenic green fruit was considerably higher than leaves for all promoter constructs; however, in red, ripe fruit activities were much lower for the Cab19 promoter constructs than the mas35S::GUS. Although GUS activity was readily detectable in flowers and roots of mas35S::GUStransgenic plants, little activity was observed in plants carrying the Cab19 promoter constructs. In addition, the light-inducibility of the Cab19::GUS constructs indicated that all the requisite cis-elements for light responsiveness were contained on the Cab19 fragment. The minimal Cab19 promoter retains both tissue-specificity and light regulation and can be used to drive expression of foreign genes with minimal activity in mature, edible fruit. PMID:17697347

Expression of ORF2 partial gene of hepatitis E virus in tomatoes and immunoactivity of expression products.

PubMed

Ma, Ying; Lin, Shun-Quan; Gao, Yi; Li, Mei; Luo, Wen-Xin; Zhang, Jun; Xia, Ning-Shao

2003-10-01

To transfer hepatitis E virus (HEV) ORF2 partial gene to tomato plants, to investigate its expression in transformants and the immunoactivity of expression products, and to explore the feasibility of developing a new type of plant-derived HEV oral vaccine. Plant binary expression vector p1301E2, carrying a fragment of HEV open reading frame-2 (named HEV-E2), was constructed by linking the fragment to a constitutive CaMV35s promoter and nos terminator, then directly introduced into Agrobacterium tumefaciens EHA105. With leaf-disc method, tomato plants medicated by EHA105 were transformed and hygromycin-resistant plantlets were obtained in selective medium containing hygromycin. The presence and integration of foreign DNA in transgenic tomato genome were confirmed by Gus gene expression, PCR amplification and Southern dot blotting. The immunoactivity of recombinant protein extracted from transformed plants was examined by enzyme-linked immunosorbant assay (ELISA) using a monoclonal antibody specifically against HEV. ELISA was also used to estimate the recombinant protein content in leaves and fruits of the transformants. Seven positive lines of HEV-E2-transgenic tomato plants confirmed by PCR and Southern blotting were obtained and the immunoactivity of recombinant protein could be detected in extracts of transformants. The expression levels of recombinant protein were 61.22 ng/g fresh weight in fruits and 6.37-47.9 ng/g fresh weight in leaves of the transformants. HEV-E2 gene was correctly expressed in transgenic tomatoes and the recombinant antigen derived from them has normal immunoactivity. Transgenic tomatoes may hold a good promise for producing a new type of low-cost oral vaccine for hepatitis E virus.

Higher accumulation of F1-V fusion recombinant protein in plants after induction of protein body formation.

PubMed

Alvarez, M Lucrecia; Topal, Emel; Martin, Federico; Cardineau, Guy A

2010-01-01

Improving foreign protein accumulation is crucial for enhancing the commercial success of plant-based production systems since product yields have a major influence on process economics. Cereal grain evolved to store large amounts of proteins in tightly organized aggregates. In maize, gamma-Zein is the major storage protein synthesized by the rough endoplasmic reticulum (ER) and stored in specialized organelles called protein bodies (PB). Zera (gamma-Zein ER-accumulating domain) is the N-terminal proline-rich domain of gamma-zein that is sufficient to induce the assembly of PB formation. Fusion of the Zera domain to proteins of interest results in assembly of dense PB-like, ER-derived organelles, containing high concentration of recombinant protein. Our main goal was to increase recombinant protein accumulation in plants in order to enhance the efficiency of orally-delivered plant-made vaccines. It is well known that oral vaccination requires substantially higher doses than parental formulations. As a part of a project to develop a plant-made plague vaccine, we expressed our model antigen, the Yersinia pestis F1-V antigen fusion protein, with and without a fused Zera domain. We demonstrated that Zera-F1-V protein accumulation was at least 3x higher than F1-V alone when expressed in three different host plant systems: Ncotiana benthamiana, Medicago sativa (alfalfa) and Nicotiana tabacum NT1 cells. We confirmed the feasibility of using Zera technology to induce protein body formation in non-seed tissues. Zera expression and accumulation did not affect plant development and growth. These results confirmed the potential exploitation of Zera technology to substantially increase the accumulation of value-added proteins in plants.

Rabies virus glycoprotein as a carrier for anthrax protective antigen

DOE Office of Scientific and Technical Information (OSTI.GOV)

Smith, Mary Ellen; Koser, Martin; Xiao Sa

2006-09-30

Live viral vectors expressing foreign antigens have shown great promise as vaccines against viral diseases. However, safety concerns remain a major problem regarding the use of even highly attenuated viral vectors. Using the rabies virus (RV) envelope protein as a carrier molecule, we show here that inactivated RV particles can be utilized to present Bacillus anthracis protective antigen (PA) domain-4 in the viral membrane. In addition to the RV glycoprotein (G) transmembrane and cytoplasmic domains, a portion of the RV G ectodomain was required to express the chimeric RV G anthrax PA on the cell surface. The novel antigen wasmore » also efficiently incorporated into RV virions. Mice immunized with the inactivated recombinant RV virions exhibited seroconversion against both RV G and anthrax PA, and a second inoculation greatly increased these responses. These data demonstrate that a viral envelope protein can carry a bacterial protein and that a viral carrier can display whole polypeptides compared to the limited epitope presentation of previous viral systems.« less

Effects of proteome rebalancing and sulfur nutrition on the accumulation of methionine rich δ-zein in transgenic soybeans

PubMed Central

Kim, Won-Seok; Jez, Joseph M.; Krishnan, Hari B.

2014-01-01

Expression of heterologous methionine-rich proteins to increase the overall sulfur amino acid content of soybean seeds has been only marginally successful, presumably due to low accumulation of transgenes in soybeans or due to gene silencing. Proteome rebalancing of seed proteins has been shown to promote the accumulation of foreign proteins. In this study, we have utilized RNAi technology to suppress the expression of the β-conglycinin, the abundant 7S seed storage proteins of soybean. Western blot and 2D-gel analysis revealed that β-conglycinin knockdown line (SAM) failed to accumulate the α′, α, and β-subunits of β-conglycinin. The proteome rebalanced SAM retained the overall protein and oil content similar to that of wild-type soybean. We also generated transgenic soybean lines expressing methionine-rich 11 kDa δ-zein under the control of either the glycinin or β-conglycinin promoter. The introgression of the 11 kDa δ-zein into β-conglycinin knockdown line did not enhance the accumulation of the 11 kDa δ-zein. However, when the same plants were grown in sulfur-rich medium, we observed 3- to 16-fold increased accumulation of the 11 kDa δ-zein. Transmission electron microscopy observation revealed that seeds grown in sulfur-rich medium contained numerous endoplasmic reticulum derived protein bodies. Our findings suggest that sulfur availability, not proteome rebalancing, is needed for high-level accumulation of heterologous methionine-rich proteins in soybean seeds. PMID:25426134

Efficient and stable transformation of Lactuca sativa L. cv. Cisco (lettuce) plastids.

PubMed

Kanamoto, Hirosuke; Yamashita, Atsushi; Asao, Hiroshi; Okumura, Satoru; Takase, Hisabumi; Hattori, Masahira; Yokota, Akiho; Tomizawa, Ken-Ichi

2006-04-01

Transgenic plastids offer unique advantages in plant biotechnology, including high-level foreign protein expression. However, broad application of plastid genome engineering in biotechnology has been largely hampered by the lack of plastid transformation systems for major crops. Here we describe the development of a plastid transformation system for lettuce, Lactuca sativa L. cv. Cisco. The transforming DNA carries a spectinomycin-resistance gene (aadA) under the control of lettuce chloroplast regulatory expression elements, flanked by two adjacent lettuce plastid genome sequences allowing its targeted insertion between the rbcL and accD genes. On average, we obtained 1 transplastomic lettuce plant per bombardment. We show that lettuce leaf chloroplasts can express transgene-encoded GFP to approximately 36% of the total soluble protein. All transplastomic T0 plants were fertile and the T1 progeny uniformly showed stability of the transgene in the chloroplast genome. This system will open up new possibilities for the efficient production of edible vaccines, pharmaceuticals, and antibodies in plants.

77 FR 22333 - Prospective Grant of Exclusive License: Development of Oncolytic Viral Cancer Therapies

Federal Register 2010, 2011, 2012, 2013, 2014

2012-04-13

..., ``Recombinant Vaccinia Virus Containing a Chimeric Gene Having Foreign DNA Flanked by Vaccinia Regulatory DNA..., ``Compositions Containing Recombinant Poxviruses Having Foreign DNA Expressed under the Control of Poxvirus... entitled, ``Methods of Immunization Using Recombinant Poxviruses Having Foreign DNA Expressed under the...

Display of a maize cDNA library on baculovirus infected insect cells.

PubMed

Meller Harel, Helene Y; Fontaine, Veronique; Chen, Hongying; Jones, Ian M; Millner, Paul A

2008-08-12

Maize is a good model system for cereal crop genetics and development because of its rich genetic heritage and well-characterized morphology. The sequencing of its genome is well advanced, and new technologies for efficient proteomic analysis are needed. Baculovirus expression systems have been used for the last twenty years to express in insect cells a wide variety of eukaryotic proteins that require complex folding or extensive posttranslational modification. More recently, baculovirus display technologies based on the expression of foreign sequences on the surface of Autographa californica (AcMNPV) have been developed. We investigated the potential of a display methodology for a cDNA library of maize young seedlings. We constructed a full-length cDNA library of young maize etiolated seedlings in the transfer vector pAcTMVSVG. The library contained a total of 2.5 x 10(5) independent clones. Expression of two known maize proteins, calreticulin and auxin binding protein (ABP1), was shown by western blot analysis of protein extracts from insect cells infected with the cDNA library. Display of the two proteins in infected insect cells was shown by selective biopanning using magnetic cell sorting and demonstrated proof of concept that the baculovirus maize cDNA display library could be used to identify and isolate proteins. The maize cDNA library constructed in this study relies on the novel technology of baculovirus display and is unique in currently published cDNA libraries. Produced to demonstrate proof of principle, it opens the way for the development of a eukaryotic in vivo display tool which would be ideally suited for rapid screening of the maize proteome for binding partners, such as proteins involved in hormone regulation or defence.

[Application of dhfr gene negative Chinese hamster ovary cell line to express hepatitis B virus surface antigen].

PubMed

Yi, Y; Zhang, M; Liu, C

2001-06-01

To set up an efficient expressing system for recombinant hepatitis B virus surface antigen (HBsAg) in dhfr gene negative CHO cell line. HBsAg gene expressing plasmid pCI-dhfr-S was constructed by integrating HBsAg gene into plasmid pCI which carries dhfr gene. The HBsAg expressing cell line was set up by transfection of plasmid pCI-dhfr-S into dhfr gene negative CHO cell line in the way of lipofectin. Under the selective pressure of MTX, 18 of 28 clonized cell lines expressed HBsAg, 4 of them reached a high titer of 1:32 and protein content 1-3 micrograms/ml. In this study, the high level expression of HBsAg demonstrated that the dhfr negative mammalian cell line when recombined with plasmid harboring the corresponding deleted gene can efficiently express the foreign gene. The further steps toward building optimum conditions of the expressing system and the increase of expressed product are under study.

Effects of Trx2p and Sec23p expression on stable production of hepatitis B surface antigen S domain in recombinant Saccharomyces cerevisiae.

PubMed

Park, Young-Kyoung; Jung, Sang-Min; Lim, Hyung-Kwon; Son, Young-Jin; Park, Yong-Cheol; Seo, Jin-Ho

2012-08-31

The S domain of hepatitis B virus surface antigen (sHBsAg) is the primary component for vaccine development against virus infection. For stable expression of sHBsAg in recombinant Saccharomyces cerevisiae, new accessory genes necessary for foreign protein expression were screened by DNA microarray. Among 600 genes of interest, genes coding for an activating protein of ATPase in Hsp90 (Aha1p), S. cerevisiae DnaJ (Scj1p), thioredoxin 2 (Trx2p) and a GTPase-activator specific for Sar1 (Sec23p) as well as Pdi1p were selected in transcriptome analysis, which are known to facilitate disulfide bond formation or induce protein transport in the secretion pathway. Individual and combinatorial expression of SEC23, TRX2 and PDI1 increased total sHBsAg concentration by 1.9-6.5-fold, relative to the control strain expressing sHBsAg only. Additionally, moderate expression of Kex2p protease able to cut off the signal peptide enhanced the portion of the authentic sHBsAg to total sHBsAg. Fed-batch fermentation of the S. cerevisiae 2805 strain coexpressing the sHBsAg, SEC23, PDI1 and KEX2 genes resulted in 70.6mg/L final sHBsAg concentration which was 5.6 times higher than that of the control. Transmission electron microscopic analysis of the yeast cells elucidated the effects of the accessory gene coexpression on the intracellular localization of sHBsAg. Like PDI1, coexpression of both SEC23 and/or TRX2 newly isolated in this study is expected to improve the target protein expression in S. cerevisiae. Copyright © 2012 Elsevier B.V. All rights reserved.

Device-based local delivery of siRNA against mammalian target of rapamycin (mTOR) in a murine subcutaneous implant model to inhibit fibrous encapsulation.

PubMed

Takahashi, Hironobu; Wang, Yuwei; Grainger, David W

2010-11-01

Fibrous encapsulation of surgically implanted devices is associated with elevated proliferation and activation of fibroblasts in tissues surrounding these implants, frequently causing foreign body complications. Here we test the hypothesis that inhibition of the expression of mammalian target of rapamycin (mTOR) in fibroblasts can mitigate the soft tissue implant foreign body response by suppressing fibrotic responses around implants. In this study, mTOR was knocked down using small interfering RNA (siRNA) conjugated with branched polyethylenimine (bPEI) in fibroblastic lineage cells in serum-based cell culture as shown by both gene and protein analysis. This mTOR knock-down led to an inhibition in fibroblast proliferation by 70% and simultaneous down-regulation in the expression of type I collagen in fibroblasts in vitro. These siRNA/bPEI complexes were released from poly(ethylene glycol) (PEG)-based hydrogel coatings surrounding model polymer implants in a subcutaneous rodent model in vivo. No significant reduction in fibrous capsule thickness and mTOR expression in the foreign body capsules were observed. The siRNA inefficacy in this in vivo implant model was attributed to siRNA dosing limitations in the gel delivery system, and lack of targeting ability of the siRNA complex specifically to fibroblasts. While in vitro data supported mTOR knock-down in fibroblast cultures, in vivo siRNA delivery must be further improved to produce clinically relevant effects on fibrotic encapsulation around implants. Copyright © 2010 Elsevier B.V. All rights reserved.

Single-Vector, Single-Injection Recombinant Vesicular Stomatitis Virus Vaccines Against High-Containment Viruses.

PubMed

Whitt, Michael A; Geisbert, Thomas W; Mire, Chad E

2016-01-01

There are many avenues for making an effective vaccine against viruses. Depending on the virus these can include one of the following: inactivation of whole virions; attenuation of viruses; recombinant viral proteins; non-replication-competent virus particles; or surrogate virus vector systems such as vesicular stomatitis virus (VSV). VSV is a prototypic enveloped animal virus that has been used for over four decades to study virus replication, entry, and assembly due to its ability to replicate to high titers in a wide variety of mammalian and insect cells. The use of reverse genetics to recover infectious and single-cycle replicating VSV from plasmid DNA transfected in cell culture began a revolution in the study of recombinant VSV (rVSV). This platform can be manipulated to study the viral genetic sequences and proteins important in the virus life cycle. Additionally, foreign genes can be inserted between naturally occurring or generated start/stop signals and polyadenylation sites within the VSV genome. VSV has a tolerance for foreign gene expression which has led to numerous rVSVs reported in the literature. Of particular interest are the very effective single-dose rVSV vaccine vectors against high-containment viruses such as filoviruses, henipaviruses, and arenaviruses. Herein we describe the methods for selecting foreign antigenic genes, selecting the location within the VSV genome for insertion, generation of rVSV using reverse genetics, and proper vaccine study designs.

Integrative functional analyses using rainbow trout selected for tolerance to plant diets reveal nutrigenomic signatures for soy utilization without the concurrence of enteritis.

PubMed

Abernathy, Jason; Brezas, Andreas; Snekvik, Kevin R; Hardy, Ronald W; Overturf, Ken

2017-01-01

Finding suitable alternative protein sources for diets of carnivorous fish species remains a major concern for sustainable aquaculture. Through genetic selection, we created a strain of rainbow trout that outperforms parental lines in utilizing an all-plant protein diet and does not develop enteritis in the distal intestine, as is typical with salmonids on long-term plant protein-based feeds. By incorporating this strain into functional analyses, we set out to determine which genes are critical to plant protein utilization in the absence of gut inflammation. After a 12-week feeding trial with our selected strain and a control trout strain fed either a fishmeal-based diet or an all-plant protein diet, high-throughput RNA sequencing was completed on both liver and muscle tissues. Differential gene expression analyses, weighted correlation network analyses and further functional characterization were performed. A strain-by-diet design revealed differential expression ranging from a few dozen to over one thousand genes among the various comparisons and tissues. Major gene ontology groups identified between comparisons included those encompassing central, intermediary and foreign molecule metabolism, associated biosynthetic pathways as well as immunity. A systems approach indicated that genes involved in purine metabolism were highly perturbed. Systems analysis among the tissues tested further suggests the interplay between selection for growth, dietary utilization and protein tolerance may also have implications for nonspecific immunity. By combining data from differential gene expression and co-expression networks using selected trout, along with ontology and pathway analyses, a set of 63 candidate genes for plant diet tolerance was found. Risk loci in human inflammatory bowel diseases were also found in our datasets, indicating rainbow trout selected for plant-diet tolerance may have added utility as a potential biomedical model.

Disabling a Type I-E CRISPR-Cas Nuclease with a Bacteriophage-Encoded Anti-CRISPR Protein.

PubMed

Pawluk, April; Shah, Megha; Mejdani, Marios; Calmettes, Charles; Moraes, Trevor F; Davidson, Alan R; Maxwell, Karen L

2017-12-12

CRISPR (clustered regularly interspaced short palindromic repeat)-Cas adaptive immune systems are prevalent defense mechanisms in bacteria and archaea. They provide sequence-specific detection and neutralization of foreign nucleic acids such as bacteriophages and plasmids. One mechanism by which phages and other mobile genetic elements are able to overcome the CRISPR-Cas system is through the expression of anti-CRISPR proteins. Over 20 different families of anti-CRISPR proteins have been described, each of which inhibits a particular type of CRISPR-Cas system. In this work, we determined the structure of type I-E anti-CRISPR protein AcrE1 by X-ray crystallography. We show that AcrE1 binds to the CRISPR-associated helicase/nuclease Cas3 and that the C-terminal region of the anti-CRISPR protein is important for its inhibitory activity. We further show that AcrE1 can convert the endogenous type I-E CRISPR system into a programmable transcriptional repressor. IMPORTANCE The CRISPR-Cas immune system provides bacteria with resistance to invasion by potentially harmful viruses, plasmids, and other foreign mobile genetic elements. This study presents the first structural and mechanistic insight into a phage-encoded protein that inactivates the type I-E CRISPR-Cas system in Pseudomonas aeruginosa The interaction of this anti-CRISPR protein with the CRISPR-associated helicase/nuclease proteins Cas3 shuts down the CRISPR-Cas system and protects phages carrying this gene from destruction. This interaction also allows the repurposing of the endogenous type I-E CRISPR system into a programmable transcriptional repressor, providing a new biotechnological tool for genetic studies of bacteria encoding this type I-E CRISPR-Cas system. Copyright © 2017 Pawluk et al.

In vivo production of recombinant proteins using occluded recombinant AcMNPV-derived baculovirus vectors.

PubMed

Guijarro-Pardo, Eva; Gómez-Sebastián, Silvia; Escribano, José M

2017-12-01

Trichoplusia ni insect larvae infected with vectors derived from the Autographa californica multiple nucleopolyhedrovirus (AcMNPV), are an excellent alternative to insect cells cultured in conventional bioreactors to produce recombinant proteins because productivity and cost-efficiency reasons. However, there is still a lot of work to do to reduce the manual procedures commonly required in this production platform that limit its scalability. To increase the scalability of this platform technology, a current bottleneck to be circumvented in the future is the need of injection for the inoculation of larvae with polyhedrin negative baculovirus vectors (Polh-) because of the lack of oral infectivity of these viruses, which are commonly used for production in insect cell cultures. In this work we have developed a straightforward alternative to obtain orally infective vectors derived from AcMNPV and expressing recombinant proteins that can be administered to the insect larvae (Trichoplusia ni) by feeding, formulated in the insect diet. The approach developed was based on the use of a recombinant polyhedrin protein expressed by a recombinant vector (Polh+), able to co-occlude any recombinant Polh- baculovirus vector expressing a recombinant protein. A second alternative was developed by the generation of a dual vector co-expressing the recombinant polyhedrin protein and the foreign gene of interest to obtain the occluded viruses. Additionally, by the incorporation of a reporter gene into the helper Polh+ vector, it was possible the follow-up visualization of the co-occluded viruses infection in insect larvae and will help to homogenize infection conditions. By using these methodologies, the production of recombinant proteins in per os infected larvae, without manual infection procedures, was very similar in yield to that obtained by manual injection of recombinant Polh- AcMNPV-based vectors expressing the same proteins. However, further analyses will be required for a detailed comparison of production yields reached by injection vs oral infections for different recombinant proteins. In conclusion, these results open the possibility of future industrial scaling-up production of recombinant proteins in insect larvae by reducing manual operations. Copyright © 2017 Elsevier B.V. All rights reserved.

The CRISPR RNA-guided surveillance complex in Escherichia coli accommodates extended RNA spacers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Luo, Michelle L.; Jackson, Ryan N.; Denny, Steven R.

Bacteria and archaea acquire resistance to foreign genetic elements by integrating fragments of foreign DNA into CRISPR (clustered regularly interspaced short palindromic repeats) loci. In Escherichia coli, CRISPR-derived RNAs (crRNAs) assemble with Cas proteins into a multi-subunit surveillance complex called Cascade (CRISPR-associated complex for antiviral defense). Cascade recognizes DNA targets via protein-mediated recognition of a protospacer adjacent motif and complementary base pairing between the crRNA spacer and the DNA target. Previously determined structures of Cascade showed that the crRNA is stretched along an oligomeric protein assembly, leading us to ask how crRNA length impacts the assembly and function of thismore » complex. We found that extending the spacer portion of the crRNA resulted in larger Cascade complexes with altered stoichiometry and preserved in vitro binding affinity for target DNA. Longer spacers also preserved the in vivo ability of Cascade to repress target gene expression and to recruit the Cas3 endonuclease for target degradation. Lastly, longer spacers exhibited enhanced silencing at particular target locations and were sensitive to mismatches within the extended region. These findings demonstrate the flexibility of the Type I-E CRISPR machinery and suggest that spacer length can be modified to fine-tune Cascade activity.« less

The CRISPR RNA-guided surveillance complex in Escherichia coli accommodates extended RNA spacers

DOE PAGES

Luo, Michelle L.; Jackson, Ryan N.; Denny, Steven R.; ...

2016-05-12

Bacteria and archaea acquire resistance to foreign genetic elements by integrating fragments of foreign DNA into CRISPR (clustered regularly interspaced short palindromic repeats) loci. In Escherichia coli, CRISPR-derived RNAs (crRNAs) assemble with Cas proteins into a multi-subunit surveillance complex called Cascade (CRISPR-associated complex for antiviral defense). Cascade recognizes DNA targets via protein-mediated recognition of a protospacer adjacent motif and complementary base pairing between the crRNA spacer and the DNA target. Previously determined structures of Cascade showed that the crRNA is stretched along an oligomeric protein assembly, leading us to ask how crRNA length impacts the assembly and function of thismore » complex. We found that extending the spacer portion of the crRNA resulted in larger Cascade complexes with altered stoichiometry and preserved in vitro binding affinity for target DNA. Longer spacers also preserved the in vivo ability of Cascade to repress target gene expression and to recruit the Cas3 endonuclease for target degradation. Lastly, longer spacers exhibited enhanced silencing at particular target locations and were sensitive to mismatches within the extended region. These findings demonstrate the flexibility of the Type I-E CRISPR machinery and suggest that spacer length can be modified to fine-tune Cascade activity.« less

Effects of insertion sites in a Newcastle disease virus vector on foreign gene expression through an internal ribosomal entry site

USDA-ARS?s Scientific Manuscript database

Newcastle disease virus (NDV), avian paramyxovirus type 1, has been developed as a vector to express foreign genes for vaccine and gene therapy purposes. The foreign genes are usually inserted into a non-coding region of the NDV genome as an independent transcription unit (ITU), which potentially a...

[Progress of genome engineering technology via clustered regularly interspaced short palindromic repeats--a review].

PubMed

Li, Hao; Qiu, Shaofu; Song, Hongbin

2013-10-04

In survival competition with phage, bacteria and archaea gradually evolved the acquired immune system--Clustered regularly interspaced short palindromic repeats (CRISPR), presenting the trait of transcribing the crRNA and the CRISPR-associated protein (Cas) to silence or cleaving the foreign double-stranded DNA specifically. In recent years, strong interest arises in prokaryotes primitive immune system and many in-depth researches are going on. Recently, researchers successfully repurposed CRISPR as an RNA-guided platform for sequence-specific gene expression, which provides a simple approach for selectively perturbing gene expression on a genome-wide scale. It will undoubtedly bring genome engineering into a more convenient and accurate new era.

Efficient Secretion of Recombinant Proteins from Rice Suspension-Cultured Cells Modulated by the Choice of Signal Peptide.

PubMed

Huang, Li-Fen; Tan, Chia-Chun; Yeh, Ju-Fang; Liu, Hsin-Yi; Liu, Yu-Kuo; Ho, Shin-Lon; Lu, Chung-An

2015-01-01

Plant-based expression systems have emerged as a competitive platform in the large-scale production of recombinant proteins. By adding a signal peptide, αAmy3sp, the desired recombinant proteins can be secreted outside transgenic rice cells, making them easy to harvest. In this work, to improve the secretion efficiency of recombinant proteins in rice expression systems, various signal peptides including αAmy3sp, CIN1sp, and 33KDsp have been fused to the N-terminus of green fluorescent protein (GFP) and introduced into rice cells to explore the efficiency of secretion of foreign proteins. 33KDsp had better efficiency than αAmy3sp and CIN1sp for the secretion of GFP from calli and suspension-cultured cells. 33KDsp was further applied for the secretion of mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF) from transgenic rice suspension-cultured cells; approximately 76%-92% of total rice-derived mGM-CSF (rmGM-CSF) was detected in the culture medium. The rmGM-CSF was bioactive and could stimulate the proliferation of a murine myeloblastic leukemia cell line, NSF-60. The extracellular yield of rmGM-CSF reached 31.7 mg/L. Our study indicates that 33KDsp is better at promoting the secretion of recombinant proteins in rice suspension-cultured cell systems than the commonly used αAmy3sp.

Enhancer activity of Helitron in sericin-1 gene promoter from Bombyx mori.

PubMed

Huang, Ke; Li, Chun-Feng; Wu, Jie; Wei, Jun-Hong; Zou, Yong; Han, Min-Jin; Zhou, Ze-Yang

2016-06-01

Sericin is a kind of water-soluble protein expressed specifically in the middle silk gland of Bombyx mori. When the sericin-1 gene promoter was cloned and a transgenic vector was constructed to express a foreign protein, a specific Helitron, Bmhel-8, was identified in the sericin-1 gene promoter sequence in some genotypes of Bombyx mori and Bombyx mandarina. Given that the Bmhel-8 Helitron transposon was present only in some genotypes, it could be the source of allelic variation in the sericin-1 promoter. The length of the sericin-1 promoter sequence is approximately 1063 or 643 bp. The larger size of the sequence or allele is ascribed to the presence of Bmhel-8. Silkworm genotypes can be homozygous for either the shorter or larger promoter sequence or heterozygous, containing both alleles. Bmhel-8 in the sericin-1 promoter exhibits enhancer activity, as demonstrated by a dual-luciferase reporter system in BmE cell lines. Furthermore, Bmhel-8 displays enhancer activity in a sericin-1 promoter-driven gene expression system but does not regulate the tissue-specific expression of sericin-1. © 2016 Institute of Zoology, Chinese Academy of Sciences.

Generation of Recombinant Modified Vaccinia Virus Ankara Encoding VP2, NS1, and VP7 Proteins of Bluetongue Virus.

PubMed

Marín-López, Alejandro; Ortego, Javier

2016-01-01

Modified Vaccinia Virus Ankara (MVA) is employed widely as an experimental vaccine vector for its lack of replication in mammalian cells and high expression level of foreign/heterologous genes. Recombinant MVAs (rMVAs) are used as platforms for protein production as well as vectors to generate vaccines against a high number of infectious diseases and other pathologies. The portrait of the virus combines desirable elements such as high-level biological safety, the ability to activate appropriate innate immune mediators upon vaccination, and the capacity to deliver substantial amounts of heterologous antigens. Recombinant MVAs encoding proteins of bluetongue virus (BTV), an Orbivirus that infects domestic and wild ruminants transmitted by biting midges of the Culicoides species, are excellent vaccine candidates against this virus. In this chapter we describe the methods for the generation of rMVAs encoding VP2, NS1, and VP7 proteins of bluetongue virus as a model example for orbiviruses. The protocols included cover the cloning of VP2, NS1, and VP7 BTV-4 genes in a transfer plasmid, the construction of recombinant MVAs, the titration of virus working stocks and the protein expression analysis by immunofluorescence and radiolabeling of rMVA infected cells as well as virus purification.

Development of a Newcastle disease virus vector expressing a foreign gene through an internal ribosomal entry site provides direct proof for a sequential transcription mechanism

USDA-ARS?s Scientific Manuscript database

Objectives: Newcastle disease virus (NDV), a member of the Paramxoviridae family, has been developed as a vector to express foreign genes for vaccine and gene therapy purposes. The foreign genes are usually inserted into a non-coding region of the NDV genome as an independent transcription unit (ITU...

Yeast Genetics and Biotechnological Applications

NASA Astrophysics Data System (ADS)

Mishra, Saroj; Baranwal, Richa

Yeast can be recognized as one of the very important groups of microorganisms on account of its extensive use in the fermentation industry and as a basic eukaryotic model cellular system. The yeast Saccharomyces cerevisiae has been extensively used to elucidate the genetics and regulation of several key functions in the cell such as cell mating, electron transport chain, protein trafficking, cell cycle events and others. Even before the genome sequence of the yeast was out, the structural organization and function of several of its genes was known. With the availability of the origin of replication from the 2 μm plasmid and the development of transformation system, it became the host of choice for expression of a number of important proteins. A large number of episomal and integrative shuttle vectors are available for expression of mammalian proteins. The latest developments in genomics and micro-array technology have allowed investigations of individual gene function by site-specific deletion method. The application of metabolic profiling has also assisted in understanding the cellular network operating in this yeast. This chapter is aimed at reviewing the use of this system as an experimental tool for conducting classical genetics. Various vector systems available, foreign genes expressed and the limitations as a host will be discussed. Finally, the use of various yeast enzymes in biotechnology sector will be reviewed.

A novel transgenic chimaeric mouse system for the rapid functional evaluation of genes encoding secreted proteins

PubMed Central

Kakitani, Makoto; Oshima, Takeshi; Horikoshi, Kaori; Yoshitome, Tetsuo; Ueda, Akiko; Kajikawa, Miwa; Iba, Yumi; Ozone, Yoshinao; Ijima, Yuki; Yoshino, Tohko; Itoh, Mikiko; Seki, Sachiko; Aoki, Ayako; Ishihara, Toshie; Shionoya, Michiyo; Makino, Utako; Kitada, Rina; Ohguma, Atsuko; Ohta, Takami; Yoshida, Yoshimasa; Kudoh, Hiroe; Hanaoka, Kazunori; Sibuya, Kazunori; Ishida, Isao; Kakeda, Minoru; Yagi, Mikio; Yoneya, Takashi; Tomizuka, Kazuma

2005-01-01

A major challenge of the post-genomic era is the functional characterization of anonymous open reading frames (ORFs) identified by the Human Genome Project. In this context, there is a strong requirement for the development of technologies that enhance our ability to analyze gene functions at the level of the whole organism. Here, we describe a rapid and efficient procedure to generate transgenic chimaeric mice that continuously secrete a foreign protein into the systemic circulation. The transgene units were inserted into the genomic site adjacent to the endogenous immunoglobulin (Ig) κ locus by homologous recombination, using a modified mouse embryonic stem (ES) cell line that exhibits a high frequency of homologous recombination at the Igκ region. The resultant ES clones were injected into embryos derived from a B-cell-deficient host strain, thus producing chimaerism-independent, B-cell-specific transgene expression. This feature of the system eliminates the time-consuming breeding typically implemented in standard transgenic strategies and allows for evaluating the effect of ectopic transgene expression directly in the resulting chimaeric mice. To demonstrate the utility of this system we showed high-level protein expression in the sera and severe phenotypes in human EPO (hEPO) and murine thrombopoietin (mTPO) transgenic chimaeras. PMID:15914664

A downstream box fusion allows stable accumulation of a bacterial cellulase in Chlamydomonas reinhardtii chloroplasts.

PubMed

Richter, Lubna V; Yang, Huijun; Yazdani, Mohammad; Hanson, Maureen R; Ahner, Beth A

2018-01-01

We investigated strategies to improve foreign protein accumulation in the chloroplasts of the model algae Chlamydomonas reinhardtii and tested the outcome in both standard culture conditions as well as one pertinent to algal biofuel production. The downstream box (DB) of the TetC or NPTII genes, the first 15 codons following the start codon, was N -terminally fused to the coding region of cel6A , an endoglucanase from Thermobifida fusca . We also employed a chimeric regulatory element, consisting of the 16S rRNA promoter and the atpA 5'UTR, previously reported to enhance protein expression, to regulate the expression of the TetC- cel6A gene. We further investigated the accumulation of TetC-Cel6A under N -deplete growth conditions. Both of the DB fusions improved intracellular accumulation of Cel6A in transplastomic C. reinhardtii strains though the TetC DB was much more effective than the NPTII DB. Furthermore, using the chimeric regulatory element, the TetC-Cel6A protein accumulation displayed a significant increase to 0.3% total soluble protein (TSP), whereas NPTII-Cel6A remained too low to quantify. Comparable levels of TetC- and NPTII- cel6A transcripts were observed, which suggests that factors other than transcript abundance mediate the greater TetC-Cel6A accumulation. The TetC-Cel6A accumulation was stable regardless of the growth stage, and the transplastomic strain growth rate was not altered. When transplastomic cells were suspended in N -deplete medium, cellular levels of TetC-Cel6A increased over time along with TSP, and were greater than those in cells suspended in N -replete medium. The DB fusion holds great value as a tool to enhance foreign protein accumulation in C. reinhardtii chloroplasts and its influence is related to translation or other post-transcriptional processes. Our results also suggest that transplastomic protein production can be compatible with algal biofuel production strategies. Cells displayed a consistent accumulation of recombinant protein throughout the growth phase and nitrogen starvation, a strategy used to induce lipid production in algae, led to higher cellular heterologous protein content. The latter result is contrary to what might have been expected a priori and is an important result for the development of future algal biofuel systems, which will likely require co-products for economic sustainability.

Use of oil bodies and oleosins in recombinant protein production and other biotechnological applications.

PubMed

Bhatla, S C; Kaushik, V; Yadav, M K

2010-01-01

Oil bodies obtained from oilseeds have been exploited for a variety of applications in biotechnology in the recent past. These applications are based on their non-coalescing nature, ease of extraction and presence of unique membrane proteins-oleosins. In suspension, oil bodies exist as separate entities and, hence, they can serve as emulsifying agent for a wide variety of products, ranging from vaccines, food, cosmetics and personal care products. Oil bodies have found significant uses in the production and purification of recombinant proteins with specific applications. The desired protein can be targeted to oil bodies in oilseeds by affinity tag or by fusing it directly to the N or C terminal of oleosins. Upon targeting, the hydrophobic domain of oleosin embeds into the TAG matrix of oil body, whereas the protein fused with N and/or C termini is exposed on the oil body surface, where it acquires correct confirmation spontaneously. Oil bodies with the attached foreign protein can be separated easily from other cellular components. They can be used directly or the protein can be cleaved from the fusion. The desired protein can be a pharmaceutically important polypeptide (e.g. hirudin, insulin and epidermal growth factor), a neutraceutical polypeptide (somatotropin), a commercially important enzyme (e.g. xylanase), a protein important for improvement of crops (e.g. chitinase) or a multimeric protein. These applications can further be widened as oil bodies can also be made artificially and oleosin gene can be expressed in bacterial systems. Thus, a protein fused to oleosin can be expressed in Escherichia coli and after cell lysis it can be incorporated into artificial oil bodies, thereby facilitating the extraction and purification of the desired protein. Artificial oil bodies can also be used for encapsulation of probiotics. The manipulation of oleosin gene for the expression of polyoleosins has further expanded the arena of the applications of oil bodies in biotechnology. (c) 2009 Elsevier Inc. All rights reserved.

Shape of Key Malaria Protein Could Help Improve Vaccine Efficacy

MedlinePlus

... Award Negotiation & Initial Award After Award Foreign Grants Management Getting Your Initial International Award Actions You Can Take as the Project Leader on a Foreign Grant Subawards for Foreign ...

Improving solubility of Shewanella oneidensis MR-1 and Clostridium thermocellum JW-20 proteins expressed into Esherichia coli.

PubMed

Kataeva, Irina; Chang, Jessie; Xu, Hao; Luan, Chi-Hao; Zhou, Jizhong; Uversky, Vladimir N; Lin, Dawei; Horanyi, Peter; Liu, Z J; Ljungdahl, Lars G; Rose, John; Luo, Ming; Wang, Bi-Cheng

2005-01-01

Low solubility of proteins overexpressed in E. coli is a frequent problem in high-throughput structural genomics. To improve solubility of proteins from mesophilic Shewanella oneidensis MR-1 and thermophilic Clostridium thermocellum JW20, an approach was attempted that included a fusion of the target protein to a maltose-binding protein (MBP) and a decrease of induction temperature. The MBP was selected as the most efficient solubilizing carrier when compared to a glutathione S-transferase and a Nus A protein. A tobacco etch virus (TEV) protease recognition site was introduced between fused proteins using a double polymerase-chain reaction and four primers. In this way, 79 S. oneidensis proteins have been expressed in one case with an N-terminal 30-residue tag and in another case as a fusion protein with MBP. A foreign tag might significantly affect the properties of the target polypeptide. At 37 degrees C and 18 degrees C induction temperatures, only 5 and 17 tagged proteins were soluble, respectively. In fusion with MBP 4, 34, and 38 proteins were soluble upon induction at 37 degrees, 28 degrees, and 18 degrees C, respectively. The MBP is assumed to increase stability and solubility of a target protein by changing both the mechanism and the cooperativity of folding/unfolding. The 66 C. thermocellum proteins were expressed as fusion proteins with MBP. Induction at 37 degrees, 28 degrees, and 18 degrees C produced 34, 57, and 60 soluble proteins, respectively. The higher solubility of C. thermocellum proteins in comparison with the S. oneidensis proteins under similar conditions of induction correlates with the thermophilicity of the host. The two-factor Wilkinson-Harrison statistical model was used to identify soluble and insoluble proteins. Theoretical and experimental data showed good agreement for S. oneidensis proteins; however, the model failed to identify soluble/insoluble Clostridium proteins. A suggestion has been made that the Wilkinson-Harrison model is not applicable to C. thermocellum proteins because it did not account for the peculiarities of protein sequences from thermophiles.

An efficient Foxtail mosaic virus vector system with reduced environmental risk

PubMed Central

2010-01-01

Background Plant viral vectors offer high-yield expression of pharmaceutical and commercially important proteins with a minimum of cost and preparation time. The use of Agrobacterium tumefaciens has been introduced to deliver the viral vector as a transgene to each plant cell via a simple, nonsterile infiltration technique called "agroinoculation". With agroinoculation, a full length, systemically moving virus is no longer necessary for excellent protein yield, since the viral transgene is transcribed and replicates in every infiltrated cell. Viral genes may therefore be deleted to decrease the potential for accidental spread and persistence of the viral vector in the environment. Results In this study, both the coat protein (CP) and triple gene block (TGB) genetic segments were eliminated from Foxtail mosaic virus to create the "FECT" vector series, comprising a deletion of 29% of the genome. This viral vector is highly crippled and expresses little or no marker gene within the inoculated leaf. However, when co-agroinoculated with a silencing suppressor (p19 or HcPro), FECT expressed GFP at 40% total soluble protein in the tobacco host, Nicotiana benthamiana. The modified FoMV vector retained the full-length replicase ORF, the TGB1 subgenomic RNA leader sequence and either 0, 22 or 40 bases of TGB1 ORF (in vectors FECT0, FECT22 and FECT40, respectively). As well as N. benthamiana, infection of legumes was demonstrated. Despite many attempts, expression of GFP via syringe agroinoculation of various grass species was very low, reflecting the low Agrobacterium-mediated transformation rate of monocots. Conclusions The FECT/40 vector expresses foreign genes at a very high level, and yet has a greatly reduced biohazard potential. It can form no virions and can effectively replicate only in a plant with suppressed silencing. PMID:21162736

[Transgenic plants as medicine production systems].

PubMed

Okada, Y

1997-10-01

Transgenic plants are emerging as an important system for the expression of many recombinant proteins, especially those intended for therapeutic purpose. The production of foreign proteins in plants has several advantages. In terms of required equipment and cost, mass production in plants is far easier to achieve than techniques involving animal cells. Successful production of several proteins in plants, including human serum albumin, haemoglobin, monoclonal antibodies, viral antigens (vaccines), enkephalin, and trichosanthin, has been reported. Particularly, the demonstration that vaccine antigens can be produced in plants in their native, immunogenic forms opens exciting possibilities for the "bio-farming" of vaccines. If the antigens are orally active, food-based "edible vaccines" could allow economical production. In this review, I will discuss the progress that has been made by several groups in what is now an expanding area of medicine research that utilizes transgenic plants.

Incorporation of chimeric gag protein into retroviral particles.

PubMed Central

Weldon, R A; Erdie, C R; Oliver, M G; Wills, J W

1990-01-01

The product of the Rous sarcoma virus (RSV) gag gene, Pr76gag, is a polyprotein precursor which is cleaved by the viral protease to yield the major structural proteins of the virion during particle assembly in avian host cells. We have recently shown that myristylated forms of the RSV Gag protein can induce particle formation with very high efficiency when expressed in mammalian cells (J. W. Wills, R. C. Craven, and J. A. Achacoso, J. Virol. 63:4331-4343, 1989). We made use of this mammalian system to examine the abilities of foreign antigens to be incorporated into particles when fused directly to the myristylated Gag protein. Our initial experiments showed that removal of various portions of the viral protease located at the carboxy terminus of the RSV Gag protein did not disrupt particle formation. We therefore chose this region for coupling of iso-1-cytochrome c from Saccharomyces cerevisiae to Gag. This was accomplished by constructing an in-frame fusion of the CYC1 and gag coding sequences at a common restriction endonuclease site. Expression of the chimeric gene resulted in synthesis of the Gag-cytochrome fusion protein and its release into the cell culture medium. The chimeric particles were readily purified by simple centrifugation, and transmission electron microscopy of cells that produced them revealed a morphology similar to that of immature type C retrovirions. Images PMID:2166812

Attenuation of Recombinant Yellow Fever 17D Viruses Expressing Foreign Protein Epitopes at the Surface

PubMed Central

Bonaldo, Myrna C.; Garratt, Richard C.; Marchevsky, Renato S.; Coutinho, Evandro S. F.; Jabor, Alfredo V.; Almeida, Luís F. C.; Yamamura, Anna M. Y.; Duarte, Adriana S.; Oliveira, Prisciliana J.; Lizeu, Jackeline O. P.; Camacho, Luiz A. B.; Freire, Marcos S.; Galler, Ricardo

2005-01-01

The yellow fever (YF) 17D vaccine is a live attenuated virus. Three-dimensional (3D) homology modeling of the E protein structure from YF 17D virus and its comparison with that from tick-borne encephalitis virus revealed that it is possible to accommodate inserts of different sizes and amino acid compositions in the flavivirus E protein fg loop. This is consistent with the 3D structures of both the dimeric and trimeric forms in which the fg loop lies exposed to solvents. We demonstrate here that YF 17D viruses bearing foreign humoral (17D/8) and T-cell (17D/13) epitopes, which vary in sequence and length, displayed growth restriction. It is hypothesized that interference with the dimer-trimer transition and with the formation of a ring of such trimers in order to allow fusion compromises the capability of the E protein to induce fusion of viral and endosomal membranes, and a slower rate of fusion may delay the extent of virus production. This would account for the lower levels of replication in cultured cells and of viremia in monkeys, as well as for the more attenuated phenotype of the recombinant viruses in monkeys. Testing of both recombinant viruses (17D/8 and 17D/13) for monkey neurovirulence also suggests that insertion at the 17D E protein fg loop does not compromise the attenuated phenotype of YF 17D virus, further confirming the potential use of this site for the development of new live attenuated 17D virus-based vaccines. PMID:15956601

Disabling a Type I-E CRISPR-Cas Nuclease with a Bacteriophage-Encoded Anti-CRISPR Protein

PubMed Central

Shah, Megha; Mejdani, Marios; Calmettes, Charles; Moraes, Trevor F.

2017-01-01

ABSTRACT CRISPR (clustered regularly interspaced short palindromic repeat)-Cas adaptive immune systems are prevalent defense mechanisms in bacteria and archaea. They provide sequence-specific detection and neutralization of foreign nucleic acids such as bacteriophages and plasmids. One mechanism by which phages and other mobile genetic elements are able to overcome the CRISPR-Cas system is through the expression of anti-CRISPR proteins. Over 20 different families of anti-CRISPR proteins have been described, each of which inhibits a particular type of CRISPR-Cas system. In this work, we determined the structure of type I-E anti-CRISPR protein AcrE1 by X-ray crystallography. We show that AcrE1 binds to the CRISPR-associated helicase/nuclease Cas3 and that the C-terminal region of the anti-CRISPR protein is important for its inhibitory activity. We further show that AcrE1 can convert the endogenous type I-E CRISPR system into a programmable transcriptional repressor. PMID:29233895

Transgenic approach to improve wheat (Triticum aestivum L.) nutritional quality.

PubMed

Tamás, Cecília; Kisgyörgy, Boglárka N; Rakszegi, Mariann; Wilkinson, Mark D; Yang, Moon-Sik; Láng, László; Tamás, László; Bedo, Zoltán

2009-07-01

An amaranth (Amaranthus hypochondriacus) albumin gene, encoding the 35-kDa AmA1 protein of the seed, with a high content of essential amino acids, was used in the biolistic transformation of bread wheat (Triticum aestivum L.) variety Cadenza. The transformation cassette carried the ama1 gene under the control of a powerful wheat endosperm-specific promoter (1Bx17 HMW-GS). Southern-blot analysis of T(1) lines confirmed the integration of the foreign gene, while RT-PCR and Western-blot analyses of the samples confirmed the transcription and translation of the transgene. The effects of the extra albumin protein on the properties of flour, produced from bulked T(2) seeds, were calculated using total protein and essential amino acid content analysis, polymeric/monomeric protein and HMW/LMW glutenin subunit ratio measurements. The results indicated that not only can essential amino acid content be increased, but some parameters associated with functional quality may also be improved because of the expression of the AmA1 protein.

Utility of temporally distinct baculovirus promoters for constitutive and baculovirus-inducible transgene expression in transformed insect cells.

PubMed

Lin, Chi-Hung; Jarvis, Donald L

2013-05-10

Genetically transformed lepidopteran insect cell lines have biotechnological applications as constitutive recombinant protein production platforms and improved hosts for baculovirus-mediated recombinant protein production. Insect cell transformation is often accomplished with a DNA construct(s) encoding a foreign protein(s) under the transcriptional control of a baculovirus immediate early promoter, such as the ie1 promoter. However, the potential utility of increasingly stronger promoters from later baculovirus gene classes, such as delayed early (39K), late (p6.9), and very late (polh), has not been systematically assessed. Hence, we produced DNA constructs encoding secreted alkaline phosphatase (SEAP) under the transcriptional control of each of the four temporally distinct classes of baculovirus promoters, used them to transform insect cells, and compared the levels of SEAP RNA and protein production obtained before and after baculovirus infection. The ie1 construct was the only one that supported SEAP protein production by transformed insect cells prior to baculovirus infection, confirming that only immediate early promoters can be used to isolate transformed insect cells for constitutive recombinant protein production. However, baculovirus infection activated transgene expression by all four classes of baculovirus promoters. After infection, cells transformed with the very late (polh) and late (p6.9) promoter constructs produced the highest levels of SEAP RNA, but only low levels of SEAP protein. Conversely, cells transformed with the immediate early (ie1) and delayed early (39K) promoter constructs produced lower levels of RNA, but equal or higher levels of SEAP protein. Unexpectedly, the 39K promoter construct provided tightly regulated, baculovirus-inducible protein production at higher levels than the later promoter constructs. Thus, this study demonstrated the utility of the 39K promoter for insect cell engineering, particularly when one requires higher levels of effector protein production than obtained with ie1 and/or when constitutive transgene expression adversely impacts host cell fitness and/or genetic stability. Copyright © 2013 Elsevier B.V. All rights reserved.

Effects of Human Metapneumovirus and Respiratory Syncytial Virus Antigen Insertion in Two 3′ Proximal Genome Positions of Bovine/Human Parainfluenza Virus Type 3 on Virus Replication and Immunogenicity

PubMed Central

Tang, Roderick S.; Schickli, Jeanne H.; MacPhail, Mia; Fernandes, Fiona; Bicha, Leenas; Spaete, Joshua; Fouchier, Ron A. M.; Osterhaus, Albert D. M. E.; Spaete, Richard; Haller, Aurelia A.

2003-01-01

A live attenuated bovine parainfluenza virus type 3 (PIV3), harboring the fusion (F) and hemagglutinin-neuraminidase (HN) genes of human PIV3, was used as a virus vector to express surface glycoproteins derived from two human pathogens, human metapneumovirus (hMPV) and respiratory syncytial virus (RSV). RSV and hMPV are both paramyxoviruses that cause respiratory disease in young children, the elderly, and immunocompromised individuals. RSV has been known for decades to cause acute lower respiratory tract infections in young children, which often result in hospitalization, while hMPV has only been recently identified as a novel human respiratory pathogen. In this study, the ability of bovine/human PIV3 to express three different foreign transmembrane surface glycoproteins and to induce a protective immune response was evaluated. The RNA-dependent RNA polymerase of paramyxoviruses binds to a single site at the 3′ end of the viral RNA genome to initiate transcription of viral genes. The genome position of the viral gene determines its level of gene expression. The promoter-proximal gene is transcribed with the highest frequency, and each downstream gene is transcribed less often due to attenuation of transcription at each gene junction. This feature of paramyxoviruses was exploited using the PIV3 vector by inserting the foreign viral genes at the 3′ terminus, at position 1 or 2, of the viral RNA genome. These locations were expected to yield high levels of foreign viral protein expression stimulating a protective immune response. The immunogenicity and protection results obtained with a hamster model showed that bovine/human PIV3 can be employed to generate bivalent PIV3/RSV or PIV3/hMPV vaccine candidates that will be further evaluated for safety and efficacy in primates. PMID:14512532

Differential distribution of proteins expressed in companion cells in the sieve element-companion cell complex of rice plants.

PubMed

Fukuda, Akari; Fujimaki, Syu; Mori, Tomoko; Suzui, Nobuo; Ishiyama, Keiki; Hayakawa, Toshihiko; Yamaya, Tomoyuki; Fujiwara, Toru; Yoneyama, Tadakatsu; Hayashi, Hiroaki

2005-11-01

Sieve tubes are comprised of sieve elements, enucleated cells that are incapable of RNA and protein synthesis. The proteins in sieve elements are supplied from the neighboring companion cells through plasmodesmata. In rice plants, it was unclear whether or not all proteins produced in companion cells had the same distribution pattern in the sieve element-companion cell complex. In this study, the distribution pattern of four proteins, beta-glucuronidase (GUS), green fluorescent protein (GFP), thioredoxin h (TRXh) and glutathione S-transferase (GST) were analyzed. The foreign proteins GUS and GFP were expressed in transgenic rice plants under the control of the TRXh gene promoter (PTRXh), a companion cell-specific promoter. Analysis of leaf cross-sections of PTRXh-GUS and PTRXh-GFP plants indicated high accumulation of GUS and GFP, respectively, in companion cells rather than in sieve elements. GUS and GFP were also detected in phloem sap collected from leaf sheaths of the transgenic rice plants, suggesting these proteins could enter sieve elements. Relative amounts of GFP and endogenous phloem proteins, TRXh and GST, in phloem sap and total leaf extracts were compared. Compared to TRXh and GST, GFP content was higher in total leaf extracts, but lower in phloem sap, suggesting that GFP accumulated mainly in companion cells rather than in sieve elements. On the other hand, TRXh and GST appeared to accumulate in sieve elements rather than in companion cells. These results indicate the evidence for differential distribution of proteins between sieve elements and companion cells in rice plants.

p62/SQSTM1 promotes rapid ubiquitin conjugation to target proteins after endosome rupture during xenophagy.

PubMed

Tsuchiya, Megumi; Ogawa, Hidesato; Koujin, Takako; Mori, Chie; Osakada, Hiroko; Kobayashi, Shouhei; Hiraoka, Yasushi; Haraguchi, Tokuko

2018-03-01

Autophagy is a bulk degradation pathway, and selective autophagy to remove foreign entities is called xenophagy. The conjugation of ubiquitin to target pathogens is an important process in xenophagy but when and where this ubiquitination occurs remains unclear. Here, we analyzed the temporal sequence and subcellular location of ubiquitination during xenophagy using time-lapse observations, with polystyrene beads mimicking invading pathogens. Results revealed accumulation of a ubiquitination marker around the beads within 3 min after endosome rupture. Recruitment of ubiquitin to the beads was significantly delayed in p62-knockout murine embryonic fibroblast cells, and this delay was rescued by ectopic p62 expression. Ectopic expression of a phosphorylation-mimicking p62 mutated at serine residue 405 (equivalent to human serine residue 403) rescued this delay, but its unphosphorylated form did not. These results indicate that ubiquitination mainly occurs after endosome rupture and suggest that p62, specifically the phosphorylated form, promotes ubiquitin conjugation to target proteins in xenophagy.

Engineering T7 bacteriophage as a potential DNA vaccine targeting delivery vector.

PubMed

Xu, Hai; Bao, Xi; Wang, Yiwei; Xu, Yue; Deng, Bihua; Lu, Yu; Hou, Jibo

2018-03-20

DNA delivery with bacteriophage by surface-displayed mammalian cell penetrating peptides has been reported. Although, various phages have been used to facilitate DNA transfer by surface displaying the protein transduction domain of human immunodeficiency virus type 1 Tat protein (Tat peptide), no similar study has been conducted using T7 phage. In this study, we engineeredT7 phage as a DNA targeting delivery vector to facilitate cellular internalization. We constructed recombinant T7 phages that displayed Tat peptide on their surface and carried eukaryotic expression box (EEB) as a part of their genomes (T7-EEB-Tat). We demonstrated that T7 phage harboring foreign gene insertion had packaged into infective progeny phage particles. Moreover, when mammalian cells that were briefly exposed to T7-EEB-Tat, expressed a significant higher level of the marker gene with the control cells infected with the wide type phage without displaying Tat peptides. These data suggested that the potential of T7 phage as an effective delivery vector for DNA vaccine transfer.

Glove-derived foreign proteins induce allergen-specific IgE in a mouse model.

PubMed

Busch, Marion; Schröder, Claudia; Baron, Jens-Malte; Ott, Hagen; Bruckner, Thomas; Diepgen, Thomas L; Mahler, Vera

2008-04-01

Currently, most medical gloves are produced with a low content of natural rubber latex (NRL) protein. However, they may be substituted by proteins of foreign origin to maintain specific properties of the material. The aim of this study was to investigate the allergenicity and immunogenicity of unexpected proteins (i.e., soy and casein) compared with NRL proteins in a murine model in BALB/c mice. All respective allergen sources (extracts from three brands of NRL gloves, soy, and casein) were able to induce significant allergen-specific IgE and IgG(1) responses. On average, the highest IgE induction occurred after immunization with NRL, followed by soy and casein. Certain individuals from each treatment group exhibited levels of specific IgE as high as due to NRL. To analyze further specific IgE responses on a single allergen level, we established a microarray based on recombinant allergens for allergen-specific murine IgE detection. Besides specific IgE against rHev b 3, -6, -7, -8, and -11, specific IgE against kappa-casein could be detected in mice immunized with NRL glove extract, indicating a sensitization potential of the contained foreign protein. The substitution of genuine latex proteins by proteins of foreign origin may lead to a shift and de novo increase in sensitization to the finished products.

Oral immunization with a recombinant bacterial antigen produced in transgenic plants.

PubMed

Haq, T A; Mason, H S; Clements, J D; Arntzen, C J

1995-05-05

The binding subunit of Escherichia coli heat-labile enterotoxin (LT-B) is a highly active oral immunogen. Transgenic tobacco and potato plants were made with the use of genes encoding LT-B or an LT-B fusion protein with a microsomal retention sequence. The plants expressed the foreign peptides, both of which formed oligomers that bound the natural ligand. Mice immunized by gavage produced serum and gut mucosal anti-LT-B immunoglobulins that neutralized the enterotoxin in cell protection assays. Feeding mice fresh transgenic potato tubers also caused oral immunization.

Evaluation of Measles Vaccine Virus as a Vector to Deliver Respiratory Syncytial Virus Fusion Protein or Epstein-Barr Virus Glycoprotein gp350

PubMed Central

Mok, Hoyin; Cheng, Xing; Xu, Qi; Zengel, James R; Parhy, Bandita; Zhao, Jackie; Wang, C. Kathy; Jin, Hong

2012-01-01

Live attenuated recombinant measles vaccine virus (MV) Edmonston-Zagreb (EZ) strain was evaluated as a viral vector to express the ectodomains of fusion protein of respiratory syncytial virus (RSV F) or glycoprotein 350 of Epstein-Barr virus (EBV gp350) as candidate vaccines for prophylaxis of RSV and EBV. The glycoprotein gene was inserted at the 1st or the 3rd position of the measles virus genome and the recombinant viruses were generated. Insertion of the foreign gene at the 3rd position had a minimal impact on viral replication in vitro. RSV F or EBV gp350 protein was secreted from infected cells. In cotton rats, EZ-RSV F and EZ-EBV gp350 induced MV- and insert-specific antibody responses. In addition, both vaccines also induced insert specific interferon gamma (IFN-γ) secreting T cell response. EZ-RSV F protected cotton rats from pulmonary replication of RSV A2 challenge infection. In rhesus macaques, although both EZ-RSV F and EZ-EBV gp350 induced MV specific neutralizing antibody responses, only RSV F specific antibody response was detected. Thus, the immunogenicity of the foreign antigens delivered by measles vaccine virus is dependent on the nature of the insert and the animal models used for vaccine evaluation. PMID:22383906

Bcl11b, a novel GATA3-interacting protein, suppresses Th1 while limiting Th2 cell differentiation.

PubMed

Fang, Difeng; Cui, Kairong; Hu, Gangqing; Gurram, Rama Krishna; Zhong, Chao; Oler, Andrew J; Yagi, Ryoji; Zhao, Ming; Sharma, Suveena; Liu, Pentao; Sun, Bing; Zhao, Keji; Zhu, Jinfang

2018-05-07

GATA-binding protein 3 (GATA3) acts as the master transcription factor for type 2 T helper (Th2) cell differentiation and function. However, it is still elusive how GATA3 function is precisely regulated in Th2 cells. Here, we show that the transcription factor B cell lymphoma 11b (Bcl11b), a previously unknown component of GATA3 transcriptional complex, is involved in GATA3-mediated gene regulation. Bcl11b binds to GATA3 through protein-protein interaction, and they colocalize at many important cis-regulatory elements in Th2 cells. The expression of type 2 cytokines, including IL-4, IL-5, and IL-13, is up-regulated in Bcl11b -deficient Th2 cells both in vitro and in vivo; such up-regulation is completely GATA3 dependent. Genome-wide analyses of Bcl11b- and GATA3-regulated genes (from RNA sequencing), cobinding patterns (from chromatin immunoprecipitation sequencing), and Bcl11b-modulated epigenetic modification and gene accessibility suggest that GATA3/Bcl11b complex is involved in limiting Th2 gene expression, as well as in inhibiting non-Th2 gene expression. Thus, Bcl11b controls both GATA3-mediated gene activation and repression in Th2 cells. This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.

Potential large scale production of meningococcal vaccines by stable overexpression of fHbp in the rice seeds.

PubMed

Ma, Jian; Wang, Yunpeng; Xu, Nuo; Jin, Libo; Liu, Jia; Xing, Shaochen; Li, Xiaokun

2018-06-25

Factor H binding protein (fHbp) is the most promising vaccine candidate against serogroup B of Neisseria meningitidis which is a major cause of morbidity and mortality in children. In order to facilitate large scale production of a commercial vaccine, we previously used transgenic Arabidopsis thaliana, but plant-derived fHbp is still far away from a commercial vaccine due to less biomass production. Herein, we presented an alternative route for the production of recombinant fHbp from the seeds of transgenic rice. The OsrfHbp gene encoding recombinant fHbp fused protein was introduced into the genome of rice via Agrobacterium-mediated transformation. The both stable integration and transcription of the foreign OsrfHbp were confirmed by Southern blotting and RT-PCR analysis respectively. Further, the expression of fHbp protein was measured by immunoblotting analysis and quantified by ELISA. The results indicated that fHbp was successfully expressed and the highest yield of fHbp was 0.52 ± 0.03% of TSP in the transgenic rice seeds. The purified fHbp protein showed good antigenicity and immunogenicity in the animal model. The results of this experiment offer a novel approach for large-scale production of plant-derived commercial vaccine fHbp. Copyright © 2018. Published by Elsevier Inc.

Matricellular homologs in the foreign body response: hevin suppresses inflammation, but hevin and SPARC together diminish angiogenesis.

PubMed

Barker, Thomas H; Framson, Paul; Puolakkainen, Pauli A; Reed, May; Funk, Sarah E; Sage, E Helene

2005-03-01

Implanted foreign materials, used to restore or assist tissue function, elicit an initial acute inflammatory response followed by chronic fibrosis that leads to the entrapment of the biomaterial in a thick, poorly vascularized collagenous capsule. Matricellular proteins, secreted macromolecules that interact with extracellular matrix proteins but do not in themselves serve structural roles, have been identified as important mediators of the foreign body response that includes inflammation, angiogenesis, and collagen synthesis and assembly. In this report we delineate functions of hevin and SPARC, two homologs of the SPARC family of matricellular proteins, in the foreign body response. Despite their sequence similarity, hevin and SPARC mediate different aspects of this fibrotic response. Using mice with targeted gene deletions, we show that hevin is central to the progression of biomaterial-induced inflammation whereas SPARC regulates the formation of the collagenous capsule. Although vascular density within the capsule is unaltered in the absence of either protein, SPARC-hevin double-null capsules show substantially increased numbers of vessels, indicating compensatory functions for these two proteins in the inhibition of angiogenesis. These results provide important information for further development of implant technology.

Comparative studies of lepidopteran baculovirus-specific protein FP25K: development of a novel Bombyx mori nucleopolyhedrovirus-based vector with a modified fp25K gene.

PubMed

Nakanishi, Tadashi; Goto, Chie; Kobayashi, Michihiro; Kang, Wonkyung; Suzuki, Takehiro; Dohmae, Naoshi; Matsumoto, Shogo; Shimada, Toru; Katsuma, Susumu

2010-05-01

Lepidopteran baculovirus-specific protein FP25K performs many roles during the infection cycle, including functions in the production of occlusion bodies (OBs) and budded viruses (BVs), oral infection, and postmortem host degradation. To explore the common and specific functions of FP25K proteins among lepidopteran baculoviruses, we performed comparative analyses of FP25K proteins from group I and group II nucleopolyhedroviruses (NPVs) and granulovirus (GV). Using recombinant Bombyx mori NPVs (BmNPVs), we showed that the FP25Ks from NPVs were able to eliminate all the phenotypic defects observed in an infection with a BmNPV mutant lacking functional fp25K but that FP25K from GV did not show abilities to recover oral infectivity and postmortem host degradation. We also observed that introduction of Autographa californica multiple NPV (AcMNPV) fp25K into the BmNPV genome enhanced OB and BV production. According to these results, we generated a novel BmNPV-based expression vector with AcMNPV fp25K and examined its potential in BmN cells and B. mori larvae. Our results showed that the introduction of AcMNPV fp25K significantly increases the expression of foreign gene products in cultured cells and shortens the time for obtaining the secreted recombinant proteins from larval hemolymph.

Integrative functional analyses using rainbow trout selected for tolerance to plant diets reveal nutrigenomic signatures for soy utilization without the concurrence of enteritis

PubMed Central

Brezas, Andreas; Snekvik, Kevin R.; Hardy, Ronald W.; Overturf, Ken

2017-01-01

Finding suitable alternative protein sources for diets of carnivorous fish species remains a major concern for sustainable aquaculture. Through genetic selection, we created a strain of rainbow trout that outperforms parental lines in utilizing an all-plant protein diet and does not develop enteritis in the distal intestine, as is typical with salmonids on long-term plant protein-based feeds. By incorporating this strain into functional analyses, we set out to determine which genes are critical to plant protein utilization in the absence of gut inflammation. After a 12-week feeding trial with our selected strain and a control trout strain fed either a fishmeal-based diet or an all-plant protein diet, high-throughput RNA sequencing was completed on both liver and muscle tissues. Differential gene expression analyses, weighted correlation network analyses and further functional characterization were performed. A strain-by-diet design revealed differential expression ranging from a few dozen to over one thousand genes among the various comparisons and tissues. Major gene ontology groups identified between comparisons included those encompassing central, intermediary and foreign molecule metabolism, associated biosynthetic pathways as well as immunity. A systems approach indicated that genes involved in purine metabolism were highly perturbed. Systems analysis among the tissues tested further suggests the interplay between selection for growth, dietary utilization and protein tolerance may also have implications for nonspecific immunity. By combining data from differential gene expression and co-expression networks using selected trout, along with ontology and pathway analyses, a set of 63 candidate genes for plant diet tolerance was found. Risk loci in human inflammatory bowel diseases were also found in our datasets, indicating rainbow trout selected for plant-diet tolerance may have added utility as a potential biomedical model. PMID:28723948

Foreign Experience in Mastering Medical Professional Terminology by Foreign Students at Medical Universities

ERIC Educational Resources Information Center

Homonyuk, Olena; Avramenko, Natalia

2017-01-01

Professional broadcasting of future medical foreign workers in the medical sector is a compulsory component of their professional readiness. The diversity of professional broadcasting functions of foreign students, the skillful use of the entire range of speech functionality, its external expressive attributability of speech; these are the most…

A Pathogenic Nematode Targets Recognition Proteins to Avoid Insect Defenses

PubMed Central

Toubarro, Duarte; Avila, Mónica Martinez; Montiel, Rafael; Simões, Nelson

2013-01-01

Steinernema carpocapsae is a nematode pathogenic in a wide variety of insect species. The great pathogenicity of this nematode has been ascribed to its ability to overcome the host immune response; however, little is known about the mechanisms involved in this process. The analysis of an expressed sequence tags (EST) library in the nematode during the infective phase was performed and a highly abundant contig homologous to serine protease inhibitors was identified. In this work, we show that this contig is part of a 641-bp cDNA that encodes a BPTI-Kunitz family inhibitor (Sc-KU-4), which is up-regulated in the parasite during invasion and installation. Recombinant Sc-KU-4 protein was produced in Escherichia coli and shown to inhibit chymotrypsin and elastase activities in a dose-dependent manner by a competitive mechanism with Ki values of 1.8 nM and 2.6 nM, respectively. Sc-KU-4 also inhibited trypsin and thrombin activities to a lesser extent. Studies of the mode of action of Sc-KU-4 and its effects on insect defenses suggest that although Sc-KU-4 did not inhibit the activation of hemocytes or the formation of clotting fibers, it did inhibit hemocyte aggregation and the entrapment of foreign particles by fibers. Moreover, Sc-KU-4 avoided encapsulation and the deposition of clotting materials, which usually occurs in response to foreign particles. We show by protein-protein interaction that Sc-KU-4 targets recognition proteins of insect immune system such as masquerade-like and serine protease-like homologs. The interaction of Sc-KU-4 with these proteins explains the ability of the nematode to overcome host reactions and its large pathogenic spectrum, once these immune proteins are well conserved in insects. The discovery of this inhibitor targeting insect recognition proteins opens new avenues for the development of S . carpocapsae as a biological control agent and provides a new tool to study host-pathogen interactions. PMID:24098715

A stable RNA virus-based vector for citrus trees

DOE Office of Scientific and Technical Information (OSTI.GOV)

Folimonov, Alexey S.; Folimonova, Svetlana Y.; Bar-Joseph, Moshe

Virus-based vectors are important tools in plant molecular biology and plant genomics. A number of vectors based on viruses that infect herbaceous plants are in use for expression or silencing of genes in plants as well as screening unknown sequences for function. Yet there is a need for useful virus-based vectors for woody plants, which demand much greater stability because of the longer time required for systemic infection and analysis. We examined several strategies to develop a Citrus tristeza virus (CTV)-based vector for transient expression of foreign genes in citrus trees using a green fluorescent protein (GFP) as a reporter.more » These strategies included substitution of the p13 open reading frame (ORF) by the ORF of GFP, construction of a self-processing fusion of GFP in-frame with the major coat protein (CP), or expression of the GFP ORF as an extra gene from a subgenomic (sg) mRNA controlled either by a duplicated CTV CP sgRNA controller element (CE) or an introduced heterologous CE of Beet yellows virus. Engineered vector constructs were examined for replication, encapsidation, GFP expression during multiple passages in protoplasts, and for their ability to infect, move, express GFP, and be maintained in citrus plants. The most successful vectors based on the 'add-a-gene' strategy have been unusually stable, continuing to produce GFP fluorescence after more than 4 years in citrus trees.« less

Protein-mRNA interactome capture: cartography of the mRNP landscape

PubMed Central

Ryder, Sean P.

2016-01-01

RNA-binding proteins play a variety of roles in cellular physiology. Some regulate mRNA processing, mRNA abundance, and translation efficiency. Some fight off invader RNA through small RNA-driven silencing pathways. Others sense foreign sequences in the form of double-stranded RNA and activate the innate immune response. Yet others, for example cytoplasmic aconitase, act as bi-functional proteins, processing metabolites in one conformation and regulating metabolic gene expression in another. Not all are involved in gene regulation. Some play structural roles, for example, connecting the translational machinery to the endoplasmic reticulum outer membrane. Despite their pervasive role and relative importance, it has remained difficult to identify new RNA-binding proteins in a systematic, unbiased way. A recent body of literature from several independent labs has defined robust, easily adaptable protocols for mRNA interactome discovery. In this review, I summarize the methods and review some of the intriguing findings from their application to a wide variety of biological systems. PMID:29098073

A full-length cDNA infectious clone of North American type 1 porcine reproductive and respiratory syndrome virus: expression of green fluorescent protein in the Nsp2 region.

PubMed

Fang, Ying; Rowland, Raymond R R; Roof, Michael; Lunney, Joan K; Christopher-Hennings, Jane; Nelson, Eric A

2006-12-01

The recent emergence of a unique group of North American type 1 porcine reproductive and respiratory syndrome virus (PRRSV) in the United States presents new disease control problems for a swine industry that has already been impacted seriously by North American type 2 PRRSV. In this study, a full-length cDNA infectious clone was generated from a low-virulence North American type 1 PRRSV isolate, SD01-08. In vitro studies demonstrated that the cloned virus maintained growth properties similar to those of the parental virus. Virological, pathological, and immunological observations from animals challenged with cloned viruses were similar to those from animals challenged with the parental virus and a modified live virus vaccine. To further explore the potential use as a viral backbone for expressing foreign genes, the green fluorescent protein (GFP) was inserted into a unique deletion site located at amino acid positions 348 and 349 of the predicted Nsp2 region in the virus, and expression of the Nsp2-GFP fusion protein was visualized by fluorescent microscopy. The availability of this North American type 1 infectious clone provides an important research tool for further study of the basic viral biology and pathogenic mechanisms of this group of type 1 PRRSV in the United States.

A Full-Length cDNA Infectious Clone of North American Type 1 Porcine Reproductive and Respiratory Syndrome Virus: Expression of Green Fluorescent Protein in the Nsp2 Region▿

PubMed Central

Fang, Ying; Rowland, Raymond R. R.; Roof, Michael; Lunney, Joan K.; Christopher-Hennings, Jane; Nelson, Eric A.

2006-01-01

The recent emergence of a unique group of North American type 1 porcine reproductive and respiratory syndrome virus (PRRSV) in the United States presents new disease control problems for a swine industry that has already been impacted seriously by North American type 2 PRRSV. In this study, a full-length cDNA infectious clone was generated from a low-virulence North American type 1 PRRSV isolate, SD01-08. In vitro studies demonstrated that the cloned virus maintained growth properties similar to those of the parental virus. Virological, pathological, and immunological observations from animals challenged with cloned viruses were similar to those from animals challenged with the parental virus and a modified live virus vaccine. To further explore the potential use as a viral backbone for expressing foreign genes, the green fluorescent protein (GFP) was inserted into a unique deletion site located at amino acid positions 348 and 349 of the predicted Nsp2 region in the virus, and expression of the Nsp2-GFP fusion protein was visualized by fluorescent microscopy. The availability of this North American type 1 infectious clone provides an important research tool for further study of the basic viral biology and pathogenic mechanisms of this group of type 1 PRRSV in the United States. PMID:16971421

[Construction of Lactobacillus rhamnosus GG particles surface display system].

PubMed

Su, Runyu; Nie, Boyao; Yuan, Shengling; Tao, Haoxia; Liu, Chunjie; Yang, Bailiang; Wang, Yanchun

2017-01-25

To describe a novel particles surface display system which is consisted of gram-positive enhancer matrix (GEM) particles and anchor proteins for bacteria-like particles vaccines, we treated Lactobacillus rhamnosus GG bacteria with 10% heated-TCA for preparing GEM particles, and then identified the harvested GEM particles by electron microscopy, RT-PCR and SDS-PAGE. Meanwhile, Escherichia coli was induced to express hybrid proteins PA3-EGFP and P60-EGFP, and GEM particles were incubated with them. Then binding of anchor proteins were determined by Western blotting, transmission electron microscopy, fluorescence microscopy and spectrofluorometry. GEM particles preserved original size and shape, and proteins and DNA contents of GEM particles were released substantially. The two anchor proteins both had efficiently immobilized on the surface of GEM. GEM particles that were bounded by anchor proteins were brushy. The fluorescence of GEM particles anchoring PA3 was slightly brighter than P60, but the difference was not significant (P>0.05). GEM particles prepared from L. rhamnosus GG have a good binding efficiency with anchor proteins PA3-EGFP and P60-EGFP. Therefore, this novel foreign protein surface display system could be used for bacteria-like particle vaccines.

Avian Paramyxovirus Type-3 as a Vaccine Vector: Identification of a Genome Location for High Level Expression of a Foreign Gene

PubMed Central

Yoshida, Asuka; Samal, Siba K.

2017-01-01

Avian paramyxovirus serotype 3 (APMV-3) causes infection in a wide variety of avian species, but it does not cause apparent diseases in chickens. On the contrary, APMV-1, also known as Newcastle disease virus (NDV), can cause severe disease in chickens. Currently, natural low virulence strains of NDV are used as live-attenuated vaccines throughout the world. NDV is also being evaluated as a vaccine vector against poultry pathogens. However, due to routine vaccination programs, chickens often possess pre-existing antibodies against NDV, which may cause the chickens to be less sensitive to recombinant NDV vaccines expressing antigens of other avian pathogens. Therefore, it may be possible for an APMV-3 vector vaccine to circumvent this issue. In this study, we determined the optimal insertion site in the genome of APMV-3 for high level expression of a foreign gene. We generated recombinant APMV-3 viruses expressing the green fluorescent protein (GFP) by inserting the GFP gene at five different intergenic regions in the genome. The levels of GFP transcription and translation were evaluated. Interestingly, the levels of GFP transcription and translation did not follow the 3′-to-5′ attenuation mechanism of non-segmented, negative-sense RNA viruses. The insertion of GFP gene into the P-M gene junction resulted in higher level of expression of GFP than when the gene was inserted into the upstream N-P gene junction. Unlike NDV, insertion of GFP did not attenuate the growth efficiency of AMPV-3. Thus, APMV-3 could be a more useful vaccine vector for avian pathogens than NDV. PMID:28473820

Avian Paramyxovirus Type-3 as a Vaccine Vector: Identification of a Genome Location for High Level Expression of a Foreign Gene.

PubMed

Yoshida, Asuka; Samal, Siba K

2017-01-01

Avian paramyxovirus serotype 3 (APMV-3) causes infection in a wide variety of avian species, but it does not cause apparent diseases in chickens. On the contrary, APMV-1, also known as Newcastle disease virus (NDV), can cause severe disease in chickens. Currently, natural low virulence strains of NDV are used as live-attenuated vaccines throughout the world. NDV is also being evaluated as a vaccine vector against poultry pathogens. However, due to routine vaccination programs, chickens often possess pre-existing antibodies against NDV, which may cause the chickens to be less sensitive to recombinant NDV vaccines expressing antigens of other avian pathogens. Therefore, it may be possible for an APMV-3 vector vaccine to circumvent this issue. In this study, we determined the optimal insertion site in the genome of APMV-3 for high level expression of a foreign gene. We generated recombinant APMV-3 viruses expressing the green fluorescent protein (GFP) by inserting the GFP gene at five different intergenic regions in the genome. The levels of GFP transcription and translation were evaluated. Interestingly, the levels of GFP transcription and translation did not follow the 3'-to-5' attenuation mechanism of non-segmented, negative-sense RNA viruses. The insertion of GFP gene into the P-M gene junction resulted in higher level of expression of GFP than when the gene was inserted into the upstream N-P gene junction. Unlike NDV, insertion of GFP did not attenuate the growth efficiency of AMPV-3. Thus, APMV-3 could be a more useful vaccine vector for avian pathogens than NDV.

Novel polyvalent live vaccine against varicella-zoster and mumps virus infections.

PubMed

Matsuura, Masaaki; Somboonthum, Pranee; Murakami, Kouki; Ota, Megumi; Shoji, Masaki; Kawabata, Kenji; Mizuguchi, Hiroyuki; Gomi, Yasuyuki; Yamanishi, Koichi; Mori, Yasuko

2013-10-01

The varicella-zoster virus (VZV) Oka vaccine strain (vOka) is a highly immunogenic and safe live vaccine that has long been used worldwide. Because its genome is large, making it suitable for inserting foreign genes, vOka is considered a candidate vector for novel polyvalent vaccines. Previously, a recombinant vOka, rvOka-HN, that expresses mumps virus (MuV) hemagglutinin-neuraminidase (HN) was generated by the present team. rvOka-HN induces production of neutralizing antibodies against MuV in guinea pigs. MuV also expresses fusion (F) protein, which is important for inducing neutralizing antibodies, in its viral envelope. To induce a more robust immune response against MuV than that obtained with rvOka-HN, here an rvOka expressing both HN and F (rvOka-HN-F) was generated. However, co-expression of HN and F caused the infected cells to form syncytia, which reduced virus titers. To reduce the amount of cell fusion, an rvOka expressing HN and a mutant F, F(S195Y) were generated. Almost no syncytia formed among the rvOka-HN-F(S195Y)-infected cells and the growth of rvOka-HN-F(S195Y) was similar to that of the original vOka clone. Moreover, replacement of serine 195 with tyrosine had no effect on the immunogenicity of F in mice and guinea pigs. Although obvious augmentation of neutralizing antibody production was not observed after adding F protein to vOka-HN, the anti-F antibodies did have neutralizing activity. These data suggest that F protein contributes to induction of immune protection against MuV. Therefore this recombinant virus is a promising candidate vaccine for polyvalent protection against both VZV and MuV. © 2013 The Societies and Wiley Publishing Asia Pty Ltd.

[Preparation of polyacrylamide gel electrophoresis for human chorionic gonadotropin chimeric peptide 12 expressed in E. coli].

PubMed

Zou, Yong-Shui; Xu, Wan-Xiang; He, Yuan; Sun, Zhi-Da; Xue, Xiao-Lin

2002-09-01

In recent years, development of chimeric peptide (CP) immunogens is a trend in the vaccinological field. The CPs contain a B cell epitope(s) of target antigen and a promiscuous self - or foreign- T cell epitope(s). However, such constructed CPs were all expressed in prokaryotic or eukaryotic systems at lower levels. To purify the human chorionic gonadotropin (hCG) CP12 expressed in E. coli at the level of about 1% of the total cell proteins, an improved method of preparative gel polyacrylamide gel electrophoresis (PAGE) was developed. The important improvement to routine preparative PAGE involves: (1) running reversed electrophoresis by rearranging the gel- carrying plate when the bromophenol blue band arrived at 1-1.5 centimeter from the bottom of the gel; (2) making a collecting trough between the gel and a dialytic membrane that was used to isolate the upper tank buffer. About 8 fractions were collected at regular intervals of 15 minutes after bromophenol blue running out of gel. And then 0.2 ml was taken from each fraction and the protein was precipitated by sequentially adding trichloroacetic acid and acetone. Each sample was dissolved in 20 microL sample buffer and analyzed and identified by SDS-PAGE and Western blotting. As a result, the hCG CP12 expression product with 95% relative homogeneity was harvested at a 50-100 microgram level after a single-step purification of this preparative PAGE, with respect to the sample which contained 3-4 mg of cell proteins.

Complete plastid genome sequence of Daucus carota: implications for biotechnology and phylogeny of angiosperms.

PubMed

Ruhlman, Tracey; Lee, Seung-Bum; Jansen, Robert K; Hostetler, Jessica B; Tallon, Luke J; Town, Christopher D; Daniell, Henry

2006-08-31

Carrot (Daucus carota) is a major food crop in the US and worldwide. Its capacity for storage and its lifecycle as a biennial make it an attractive species for the introduction of foreign genes, especially for oral delivery of vaccines and other therapeutic proteins. Until recently efforts to express recombinant proteins in carrot have had limited success in terms of protein accumulation in the edible tap roots. Plastid genetic engineering offers the potential to overcome this limitation, as demonstrated by the accumulation of BADH in chromoplasts of carrot taproots to confer exceedingly high levels of salt resistance. The complete plastid genome of carrot provides essential information required for genetic engineering. Additionally, the sequence data add to the rapidly growing database of plastid genomes for assessing phylogenetic relationships among angiosperms. The complete carrot plastid genome is 155,911 bp in length, with 115 unique genes and 21 duplicated genes within the IR. There are four ribosomal RNAs, 30 distinct tRNA genes and 18 intron-containing genes. Repeat analysis reveals 12 direct and 2 inverted repeats > or = 30 bp with a sequence identity > or = 90%. Phylogenetic analysis of nucleotide sequences for 61 protein-coding genes using both maximum parsimony (MP) and maximum likelihood (ML) were performed for 29 angiosperms. Phylogenies from both methods provide strong support for the monophyly of several major angiosperm clades, including monocots, eudicots, rosids, asterids, eurosids II, euasterids I, and euasterids II. The carrot plastid genome contains a number of dispersed direct and inverted repeats scattered throughout coding and non-coding regions. This is the first sequenced plastid genome of the family Apiaceae and only the second published genome sequence of the species-rich euasterid II clade. Both MP and ML trees provide very strong support (100% bootstrap) for the sister relationship of Daucus with Panax in the euasterid II clade. These results provide the best taxon sampling of complete chloroplast genomes and the strongest support yet for the sister relationship of Caryophyllales to the asterids. The availability of the complete plastid genome sequence should facilitate improved transformation efficiency and foreign gene expression in carrot through utilization of endogenous flanking sequences and regulatory elements.

22 CFR 1421.15 - Secret ballot.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 22 Foreign Relations 2 2013-04-01 2009-04-01 true Secret ballot. 1421.15 Section 1421.15 Foreign Relations FOREIGN SERVICE LABOR RELATIONS BOARD; FEDERAL LABOR RELATIONS AUTHORITY; GENERAL COUNSEL OF THE... THIS SUBCHAPTER § 1421.15 Secret ballot. Secret ballot means the expression by ballot, voting machine...

22 CFR 1421.15 - Secret ballot.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 22 Foreign Relations 2 2014-04-01 2014-04-01 false Secret ballot. 1421.15 Section 1421.15 Foreign Relations FOREIGN SERVICE LABOR RELATIONS BOARD; FEDERAL LABOR RELATIONS AUTHORITY; GENERAL COUNSEL OF THE... THIS SUBCHAPTER § 1421.15 Secret ballot. Secret ballot means the expression by ballot, voting machine...

22 CFR 1421.15 - Secret ballot.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 22 Foreign Relations 2 2011-04-01 2009-04-01 true Secret ballot. 1421.15 Section 1421.15 Foreign Relations FOREIGN SERVICE LABOR RELATIONS BOARD; FEDERAL LABOR RELATIONS AUTHORITY; GENERAL COUNSEL OF THE... THIS SUBCHAPTER § 1421.15 Secret ballot. Secret ballot means the expression by ballot, voting machine...

22 CFR 1421.15 - Secret ballot.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 22 Foreign Relations 2 2012-04-01 2009-04-01 true Secret ballot. 1421.15 Section 1421.15 Foreign Relations FOREIGN SERVICE LABOR RELATIONS BOARD; FEDERAL LABOR RELATIONS AUTHORITY; GENERAL COUNSEL OF THE... THIS SUBCHAPTER § 1421.15 Secret ballot. Secret ballot means the expression by ballot, voting machine...

22 CFR 1421.15 - Secret ballot.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 22 Foreign Relations 2 2010-04-01 2010-04-01 true Secret ballot. 1421.15 Section 1421.15 Foreign Relations FOREIGN SERVICE LABOR RELATIONS BOARD; FEDERAL LABOR RELATIONS AUTHORITY; GENERAL COUNSEL OF THE... THIS SUBCHAPTER § 1421.15 Secret ballot. Secret ballot means the expression by ballot, voting machine...

BRACEROS, MEXICO, AND FOREIGN TRADE.

ERIC Educational Resources Information Center

1966

CRITICISM OF GOVERNMENTAL POLICY RESTRICTING USE OF FOREIGN CONTRACT WORKERS ON AMERICAN FARMS IN 1965 EXPRESSES THREE POSSIBILITIES -- (1) A SEVERE DISRUPTION OF DOMESTIC FRUIT AND VEGETABLE PRODUCTION, (2) AN INCREASE OF AMERICAN INVESTMENT IN FOREIGN, PARTICULARLY MEXICAN, AGRICULTURAL PRODUCTION, AND (3) A LOSS OF UNITED STATES EXPORT MARKET…

Knock-in of Enhanced Green Fluorescent Protein or/and Human Fibroblast Growth Factor 2 Gene into β-Casein Gene Locus in the Porcine Fibroblasts to Produce Therapeutic Protein.

PubMed

Lee, Sang Mi; Kim, Ji Woo; Jeong, Young-Hee; Kim, Se Eun; Kim, Yeong Ji; Moon, Seung Ju; Lee, Ji-Hye; Kim, Keun-Jung; Kim, Min-Kyu; Kang, Man-Jong

2014-11-01

Transgenic animals have become important tools for the production of therapeutic proteins in the domestic animal. Production efficiencies of transgenic animals by conventional methods as microinjection and retrovirus vector methods are low, and the foreign gene expression levels are also low because of their random integration in the host genome. In this study, we investigated the homologous recombination on the porcine β-casein gene locus using a knock-in vector for the β-casein gene locus. We developed the knock-in vector on the porcine β-casein gene locus and isolated knock-in fibroblast for nuclear transfer. The knock-in vector consisted of the neomycin resistance gene (neo) as a positive selectable marker gene, diphtheria toxin-A gene as negative selection marker, and 5' arm and 3' arm from the porcine β-casein gene. The secretion of enhanced green fluorescent protein (EGFP) was more easily detected in the cell culture media than it was by western blot analysis of cell extract of the HC11 mouse mammary epithelial cells transfected with EGFP knock-in vector. These results indicated that a knock-in system using β-casein gene induced high expression of transgene by the gene regulatory sequence of endogenous β-casein gene. These fibroblasts may be used to produce transgenic pigs for the production of therapeutic proteins via the mammary glands.

Knock-in of Enhanced Green Fluorescent Protein or/and Human Fibroblast Growth Factor 2 Gene into β-Casein Gene Locus in the Porcine Fibroblasts to Produce Therapeutic Protein

PubMed Central

Lee, Sang Mi; Kim, Ji Woo; Jeong, Young-Hee; Kim, Se Eun; Kim, Yeong Ji; Moon, Seung Ju; Lee, Ji-Hye; Kim, Keun-Jung; Kim, Min-Kyu; Kang, Man-Jong

2014-01-01

Transgenic animals have become important tools for the production of therapeutic proteins in the domestic animal. Production efficiencies of transgenic animals by conventional methods as microinjection and retrovirus vector methods are low, and the foreign gene expression levels are also low because of their random integration in the host genome. In this study, we investigated the homologous recombination on the porcine β-casein gene locus using a knock-in vector for the β-casein gene locus. We developed the knock-in vector on the porcine β-casein gene locus and isolated knock-in fibroblast for nuclear transfer. The knock-in vector consisted of the neomycin resistance gene (neo) as a positive selectable marker gene, diphtheria toxin-A gene as negative selection marker, and 5′ arm and 3′ arm from the porcine β-casein gene. The secretion of enhanced green fluorescent protein (EGFP) was more easily detected in the cell culture media than it was by western blot analysis of cell extract of the HC11 mouse mammary epithelial cells transfected with EGFP knock-in vector. These results indicated that a knock-in system using β-casein gene induced high expression of transgene by the gene regulatory sequence of endogenous β-casein gene. These fibroblasts may be used to produce transgenic pigs for the production of therapeutic proteins via the mammary glands. PMID:25358326

Children's Sociolinguistic Evaluations of Nice Foreigners and Mean Americans

ERIC Educational Resources Information Center

Kinzler, Katherine D.; DeJesus, Jasmine M.

2013-01-01

Three experiments investigated 5- to 6-year-old monolingual English-speaking American children's sociolinguistic evaluations of others based on their accent (native, foreign) and social actions (nice, mean, neutral). In Experiment 1, children expressed social preferences for native-accented English speakers over foreign-accented speakers, and they…

Dividing to unveil protein microheterogeneities: A Traveling Wave Ion Mobility study

PubMed Central

Halgand, F.; Habchi, Johnny; Cravello, Laetitia; Martinho, Marlène; Guigliarelli, Bruno; Longhi, Sonia

2011-01-01

Over-expression of a protein in a foreign host is often the only route toward an exhaustive characterization especially when purification from the natural source(s) is hardly achievable. The key issue in these studies relies on quality control of the purified recombinant protein to precisely determining its identity as well as any undesirable micro-heterogeneities. While standard proteomics approaches preclude unbiased search for modifications, the optional technique of top down MSMS requires the use of highly accurate and highly resolved experiments to reveal subtle sequence modifications. In the present study, the top down MSMS approach combined with Traveling Wave Ion Mobility (TWIM) separation was evaluated for its ability to achieve high sequence coverage and to reveal subtle micro-heterogeneities that were hitherto only accessible with FTICR-MS instruments. The power of this approach is herein illustrated in an in-depth analysis of both wt and K496C variant of the recombinant X domain (XD, aa 459-507) of the measles virus phosphoprotein expressed in E. coli. Using top down MSMS combined to TWIM, we show that XD samples occasionally exhibit a micro-heterogeneity that could not be anticipated from the nucleotide sequence of the encoding constructs and that likely reflects a genetic drift, neutral or not, occurring during expression. In addition, an MTSL nitroxide probe that was grafted on the K496C XD variant was shown to undergo oxidation and/or protonation in the ESI source leading to artifactual mass increases. PMID:21800924

High-level systemic expression of conserved influenza epitope in plants on the surface of rod-shaped chimeric particles.

PubMed

Petukhova, Natalia V; Gasanova, Tatiana V; Ivanov, Peter A; Atabekov, Joseph G

2014-04-21

Recombinant viruses based on the cDNA copy of the tobacco mosaic virus (TMV) genome carrying different versions of the conserved M2e epitope from influenza virus A cloned into the coat protein (CP) gene were obtained and partially characterized by our group previously; cysteines in the human consensus M2e sequence were changed to serine residues. This work intends to show some biological properties of these viruses following plant infections. Agroinfiltration experiments on Nicotiana benthamiana confirmed the efficient systemic expression of M2e peptides, and two point amino acid substitutions in recombinant CPs significantly influenced the symptoms and development of viral infections. Joint expression of RNA interference suppressor protein p19 from tomato bushy stunt virus (TBSV) did not affect the accumulation of CP-M2e-ser recombinant protein in non-inoculated leaves. RT-PCR analysis of RNA isolated from either infected leaves or purified TMV-M2e particles proved the genetic stability of TMV‑based viral vectors. Immunoelectron microscopy of crude plant extracts demonstrated that foreign epitopes are located on the surface of chimeric virions. The rod‑shaped geometry of plant-produced M2e epitopes is different from the icosahedral or helical filamentous arrangement of M2e antigens on the carrier virus-like particles (VLP) described earlier. Thereby, we created a simple and efficient system that employs agrobacteria and plant viral vectors in order to produce a candidate broad-spectrum flu vaccine.

Overlap and Uniqueness: Linguistic Componential Traits Contributing to Expressive Skills in English as a Foreign Language

ERIC Educational Resources Information Center

Pae, Hye K.; O'Brien, Beth

2018-01-01

This study identified robust predictors of expressive skills in academic English as a foreign language. The participants were 92 Korean-speaking learners of English. The field test of the Pearson Test of English Academic was used as a secondary data analysis. Four communicative skills (reading, writing, listening, and speaking) and six enabling…

Improved delivery of the OVA-CD4 peptide to T helper cells by polymeric surface display on Salmonella

PubMed Central

2014-01-01

Background Autotransporter proteins represent a treasure trove for molecular engineers who modify Gram-negative bacteria for the export or secretion of foreign proteins across two membrane barriers. A particularly promising direction is the development of autotransporters as antigen display or secretion systems. Immunologists have been using ovalbumin as a reporter antigen for years and have developed sophisticated tools to detect specific T cells that respond to ovalbumin. Although ovalbumin-expressing bacteria are being used to trace T cell responses to colonizing or invading pathogens, current constructs for ovalbumin presentation have not been optimized. Results The activation of T helper cells in response to ovalbumin was improved by displaying the OVA-CD4 reporter epitope as a multimer on the surface of Salmonella and fused to the autotransporter MisL. Expression was optimized by including tandem in vivo promoters and two post-segregational killing systems for plasmid stabilization. Conclusions The use of an autotransporter protein to present relevant epitope repeats on the surface of bacteria, combined with additional techniques favoring stable and efficient in vivo transcription, optimizes antigen presentation to T cells. The technique of multimeric epitope surface display should also benefit the development of new Salmonella or other enterobacterial vaccines. PMID:24898796

Vacuolar deposition of recombinant proteins in plant vegetative organs as a strategy to increase yields.

PubMed

Marin Viegas, Vanesa Soledad; Ocampo, Carolina Gabriela; Petruccelli, Silvana

2017-05-04

Delivery of recombinant proteins to vegetative tissue vacuoles was considered inconvenient since this compartment was expected to be hydrolytic; nevertheless there is growing evidence that certain foreign proteins accumulate at high yields in vacuoles. For example avidin, cellulolytic enzymes, endolysin, and transglutaminases were produced at high yields when were sorted to leaf central vacuole avoiding the detrimental effect of these proteins on plant growth. Also, several secretory mammalian proteins such as collagen, α1-proteinase inhibitor, complement-5a, interleukin-6 and immunoglobulins accumulated at higher yields in leaf vacuoles than in the apoplast or cytosol. To reach this final destination, fusions to sequence specific vacuolar sorting signals (ssVSS) typical of proteases or proteinase inhibitors and/or Ct-VSS representative of storage proteins or plant lectins were used and both types of motifs were capable to increase accumulation. Importantly, the type of VSSs or position, either the N or C-terminus, did not alter protein stability, levels or pos-translational modifications. Vacuolar sorted glycoproteins had different type of oligosaccharides indicating that foreign proteins reached the vacuole by 2 different pathways: direct transport from the ER, bypassing the Golgi (high mannose oligosaccharides decorated proteins) or trafficking through the Golgi (Complex oligosaccharide containing proteins). In addition, some glycoproteins lacked of paucimannosidic oligosaccharides suggesting that vacuolar trimming of glycans did not occur. Enhanced accumulation of foreign proteins fused to VSS occurred in several plant species such as tobacco, Nicotiana benthamiana, sugarcane, tomato and in carrot and the obtained results were influenced by plant physiological state. Ten different foreign proteins fused to vacuolar sorting accumulated at higher levels than their apoplastic or cytosolic counterparts. For proteins with cytotoxic effects vacuolar sorted forms yields were superior than ER retained variants, but for other proteins the results were the opposite an there were also examples of similar levels for ER and vacuolar variants. In conclusion vacuolar sorting in vegetative tissues is a satisfactory strategy to enhance protein yields that can be used in several plant species.

Protein Homeostasis Imposes a Barrier on Functional Integration of Horizontally Transferred Genes in Bacteria.

PubMed

Bershtein, Shimon; Serohijos, Adrian W R; Bhattacharyya, Sanchari; Manhart, Michael; Choi, Jeong-Mo; Mu, Wanmeng; Zhou, Jingwen; Shakhnovich, Eugene I

2015-10-01

Horizontal gene transfer (HGT) plays a central role in bacterial evolution, yet the molecular and cellular constraints on functional integration of the foreign genes are poorly understood. Here we performed inter-species replacement of the chromosomal folA gene, encoding an essential metabolic enzyme dihydrofolate reductase (DHFR), with orthologs from 35 other mesophilic bacteria. The orthologous inter-species replacements caused a marked drop (in the range 10-90%) in bacterial growth rate despite the fact that most orthologous DHFRs are as stable as E.coli DHFR at 37°C and are more catalytically active than E. coli DHFR. Although phylogenetic distance between E. coli and orthologous DHFRs as well as their individual molecular properties correlate poorly with growth rates, the product of the intracellular DHFR abundance and catalytic activity (kcat/KM), correlates strongly with growth rates, indicating that the drop in DHFR abundance constitutes the major fitness barrier to HGT. Serial propagation of the orthologous strains for ~600 generations dramatically improved growth rates by largely alleviating the fitness barriers. Whole genome sequencing and global proteome quantification revealed that the evolved strains with the largest fitness improvements have accumulated mutations that inactivated the ATP-dependent Lon protease, causing an increase in the intracellular DHFR abundance. In one case DHFR abundance increased further due to mutations accumulated in folA promoter, but only after the lon inactivating mutations were fixed in the population. Thus, by apparently distinguishing between self and non-self proteins, protein homeostasis imposes an immediate and global barrier to the functional integration of foreign genes by decreasing the intracellular abundance of their products. Once this barrier is alleviated, more fine-tuned evolution occurs to adjust the function/expression of the transferred proteins to the constraints imposed by the intracellular environment of the host organism.

Minimalist Antibodies and Mimetics: An Update and Recent Applications.

PubMed

Bruce, Virginia J; Ta, Angeline N; McNaughton, Brian R

2016-10-17

The immune system utilizes antibodies to recognize foreign or disease-relevant receptors, initiating an immune response to destroy unwelcomed guests. Because researchers can evolve antibodies to bind virtually any target, it is perhaps unsurprising that these reagents, and their small-molecule conjugates, are used extensively in clinical and basic research environments. However, virtues of antibodies are countered by significant challenges. Foremost among these is the need for expression in mammalian cells (largely due to often necessary post-translational modifications). In response to these challenges, researchers have developed an array of minimalist antibodies and mimetics, which are smaller, more stable, simpler to express in Escherichia coli, and amendable to laboratory evolution and protein engineering. Here we describe these scaffolds and discuss recent applications of minimalist antibodies and mimetics. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Live-cell Imaging of Pol II Promoter Activity to Monitor Gene expression with RNA IMAGEtag reporters

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shin, Ilchung; Ray, Judhajeet; Gupta, Vinayak

2014-04-20

We describe a ribonucleic acid (RNA) reporter system for live-cell imaging of gene expression to detect changes in polymerase II activity on individual promoters in individual cells. The reporters use strings of RNA aptamers that constitute IMAGEtags (Intracellular MultiAptamer GEnetic tags) that can be expressed from a promoter of choice. For imaging, the cells are incubated with their ligands that are separately conjugated with one of the FRET pair, Cy3 and Cy5. The IMAGEtags were expressed in yeast from the GAL1, ADH1 or ACT1 promoters. Transcription from all three promoters was imaged in live cells and transcriptional increases from themore » GAL1 promoter were observed with time after adding galactose. Expression of the IMAGEtags did not affect cell proliferation or endogenous gene expression. Advantages of this method are that no foreign proteins are produced in the cells that could be toxic or otherwise influence the cellular response as they accumulate, the IMAGEtags are short lived and oxygen is not required to generate their signals. The IMAGEtag RNA reporter system provides a means of tracking changes in transcriptional activity in live cells and in real time.« less

17 CFR 232.306 - Foreign language documents and symbols.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 17 Commodity and Securities Exchanges 2 2010-04-01 2010-04-01 false Foreign language documents and symbols. 232.306 Section 232.306 Commodity and Securities Exchanges SECURITIES AND EXCHANGE COMMISSION....14d-102), or Schedule 14D-9F (§ 240.14d-103). (e) Foreign currency denominations must be expressed in...

17 CFR 232.306 - Foreign language documents and symbols.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 17 Commodity and Securities Exchanges 2 2011-04-01 2011-04-01 false Foreign language documents and symbols. 232.306 Section 232.306 Commodity and Securities Exchanges SECURITIES AND EXCHANGE COMMISSION....14d-102), or Schedule 14D-9F (§ 240.14d-103). (e) Foreign currency denominations must be expressed in...

Notable Aspects of Glycan-Protein Interactions

PubMed Central

Cohen, Miriam

2015-01-01

This mini review highlights several interesting aspects of glycan-mediated interactions that are common between cells, bacteria, and viruses. Glycans are ubiquitously found on all living cells, and in the extracellular milieu of multicellular organisms. They are known to mediate initial binding and recognition events of both immune cells and pathogens with their target cells or tissues. The host target tissues are hidden under a layer of secreted glycosylated decoy targets. In addition, pathogens can utilize and display host glycans to prevent identification as foreign by the host’s immune system (molecular mimicry). Both the host and pathogens continually evolve. The host evolves to prevent infection and the pathogens evolve to evade host defenses. Many pathogens express both glycan-binding proteins and glycosidases. Interestingly, these proteins are often located at the tip of elongated protrusions in bacteria, or in the leading edge of the cell. Glycan-protein interactions have low affinity and, as a result, multivalent interactions are often required to achieve biologically relevant binding. These enable dynamic forms of adhesion mechanisms, reviewed here, and include rolling (cells), stick and roll (bacteria) or surfacing (viruses). PMID:26340640

Linear Lepidopteran ambidensovirus 1 sequences drive random integration of a reporter gene in transfected Spodoptera frugiperda cells.

PubMed

Rizk, Francine; Laverdure, Sylvain; d'Alençon, Emmanuelle; Bossin, Hervé; Dupressoir, Thierry

2018-01-01

The Lepidopteran ambidensovirus 1 isolated from Junonia coenia (hereafter JcDV) is an invertebrate parvovirus considered as a viral transduction vector as well as a potential tool for the biological control of insect pests. Previous works showed that JcDV-based circular plasmids experimentally integrate into insect cells genomic DNA. In order to approach the natural conditions of infection and possible integration, we generated linear JcDV- gfp based molecules which were transfected into non permissive Spodoptera frugiperda ( Sf9 ) cultured cells. Cells were monitored for the expression of green fluorescent protein (GFP) and DNA was analyzed for integration of transduced viral sequences. Non-structural protein modulation of the VP-gene cassette promoter activity was additionally assayed. We show that linear JcDV-derived molecules are capable of long term genomic integration and sustained transgene expression in Sf9 cells. As expected, only the deletion of both inverted terminal repeats (ITR) or the polyadenylation signals of NS and VP genes dramatically impairs the global transduction/expression efficiency. However, all the integrated viral sequences we characterized appear "scrambled" whatever the viral content of the transfected vector. Despite a strong GFP expression, we were unable to recover any full sequence of the original constructs and found rearranged viral and non-viral sequences as well. Cellular flanking sequences were identified as non-coding ones. On the other hand, the kinetics of GFP expression over time led us to investigate the apparent down-regulation by non-structural proteins of the VP-gene cassette promoter. Altogether, our results show that JcDV-derived sequences included in linear DNA molecules are able to drive efficiently the integration and expression of a foreign gene into the genome of insect cells, whatever their composition, provided that at least one ITR is present. However, the transfected sequences were extensively rearranged with cellular DNA during or after random integration in the host cell genome. Lastly, the non-structural proteins seem to participate in the regulation of p9 promoter activity rather than to the integration of viral sequences.

Production of Bioactive Recombinant Bovine Chymosin in Tobacco Plants

PubMed Central

Wei, Zheng-Yi; Zhang, Yu-Ying; Wang, Yun-Peng; Fan, Ming-Xia; Zhong, Xiao-Fang; Xu, Nuo; Lin, Feng; Xing, Shao-Chen

2016-01-01

Chymosin (also known as rennin) plays an essential role in the coagulation of milk in the cheese industry. Chymosin is traditionally extracted from the rumen of calves and is of high cost. Here, we present an alternative method to producing bovine chymosin from transgenic tobacco plants. The CYM gene, which encodes a preprochymosin from bovine, was introduced into the tobacco nuclear genome under control of the viral 35S cauliflower mosaic promoter. The integration and transcription of the foreign gene were confirmed with Southern blotting and reverse transcription PCR (RT-PCR) analyses, respectively. Immunoblotting analyses were performed to demonstrate expression of chymosin, and the expression level was quantified by enzyme-linked immunosorbent assay (ELISA). The results indicated recombinant bovine chymosin was successfully expressed at an average level of 83.5 ng/g fresh weight, which is 0.52% of the total soluble protein. The tobacco-derived chymosin exhibited similar native milk coagulation bioactivity as the commercial product extracted from bovine rumen. PMID:27136529

Enhancement of Recombinant Protein Production in Transgenic Nicotiana benthamiana Plant Cell Suspension Cultures with Co-Cultivation of Agrobacterium Containing Silencing Suppressors.

PubMed

Huang, Ting-Kuo; Falk, Bryce W; Dandekar, Abhaya M; McDonald, Karen A

2018-05-24

We have previously demonstrated that the inducible plant viral vector (CMViva) in transgenic plant cell cultures can significantly improve the productivity of extracellular functional recombinant human alpha-1-antiryspin (rAAT) compared with either a common plant constitutive promoter ( Cauliflower mosaic virus (CaMV) 35S) or a chemically inducible promoter (estrogen receptor-based XVE) system. For a transgenic plant host system, however, viral or transgene-induced post-transcriptional gene silencing (PTGS) has been identified as a host response mechanism that may dramatically reduce the expression of a foreign gene. Previous studies have suggested that viral gene silencing suppressors encoded by a virus can block or interfere with the pathways of transgene-induced PTGS in plant cells. In this study, the capability of nine different viral gene silencing suppressors were evaluated for improving the production of rAAT protein in transgenic plant cell cultures (CMViva, XVE or 35S system) using an Agrobacterium -mediated transient expression co-cultivation process in which transgenic plant cells and recombinant Agrobacterium carrying the viral gene silencing suppressor were grown together in suspension cultures. Through the co-cultivation process, the impacts of gene silencing suppressors on the rAAT production were elucidated, and promising gene silencing suppressors were identified. Furthermore, the combinations of gene silencing suppressors were optimized using design of experiments methodology. The results have shown that in transgenic CMViva cell cultures, the functional rAAT as a percentage of total soluble protein is increased 5.7 fold with the expression of P19, and 17.2 fold with the co-expression of CP, P19 and P24.

Evaluation of Trichodysplasia Spinulosa-Associated Polyomavirus Capsid Protein as a New Carrier for Construction of Chimeric Virus-Like Particles Harboring Foreign Epitopes

PubMed Central

Gedvilaite, Alma; Kucinskaite-Kodze, Indre; Lasickiene, Rita; Timinskas, Albertas; Vaitiekaite, Ausra; Ziogiene, Danguole; Zvirbliene, Aurelija

2015-01-01

Recombinant virus-like particles (VLPs) represent a promising tool for protein engineering. Recently, trichodysplasia spinulosa-associated polyomavirus (TSPyV) viral protein 1 (VP1) was efficiently produced in yeast expression system and shown to self-assemble to VLPs. In the current study, TSPyV VP1 protein was exploited as a carrier for construction of chimeric VLPs harboring selected B and T cell-specific epitopes and evaluated in comparison to hamster polyomavirus VP1 protein. Chimeric VLPs with inserted either hepatitis B virus preS1 epitope DPAFR or a universal T cell-specific epitope AKFVAAWTLKAAA were produced in yeast Saccharomyces cerevisiae. Target epitopes were incorporated either at the HI or BC loop of the VP1 protein. The insertion sites were selected based on molecular models of TSPyV VP1 protein. The surface exposure of the insert positions was confirmed using a collection of monoclonal antibodies raised against the intact TSPyV VP1 protein. All generated chimeric proteins were capable to self-assemble to VLPs, which induced a strong immune response in mice. The chimeric VLPs also activated dendritic cells and T cells as demonstrated by analysis of cell surface markers and cytokine production profiles in spleen cell cultures. In conclusion, TSPyV VP1 protein represents a new potential carrier for construction of chimeric VLPs harboring target epitopes. PMID:26230706

A Model of Foreign Language Anxiety in the Saudi EFL Context

ERIC Educational Resources Information Center

Alrabai, Fakieh

2014-01-01

Feelings of anxiety are commonly expressed by Saudi learners in their English as a foreign language (EFL) classes. These feelings typically exert detrimental effects on these learners' foreign language attainment. This paper reports on the findings of a large-scale study for which three data collection iterations were conducted over three years to…

Macrophage Fusion Is Controlled by the Cytoplasmic Protein Tyrosine Phosphatase PTP-PEST/PTPN12

PubMed Central

Rhee, Inmoo; Davidson, Dominique; Souza, Cleiton Martins; Vacher, Jean

2013-01-01

Macrophages can undergo cell-cell fusion, leading to the formation of multinucleated giant cells and osteoclasts. This process is believed to promote the proteolytic activity of macrophages toward pathogens, foreign bodies, and extracellular matrices. Here, we examined the role of PTP-PEST (PTPN12), a cytoplasmic protein tyrosine phosphatase, in macrophage fusion. Using a macrophage-targeted PTP-PEST-deficient mouse, we determined that PTP-PEST was not needed for macrophage differentiation or cytokine production. However, it was necessary for interleukin-4-induced macrophage fusion into multinucleated giant cells in vitro. It was also needed for macrophage fusion following implantation of a foreign body in vivo. Moreover, in the RAW264.7 macrophage cell line, PTP-PEST was required for receptor activator of nuclear factor kappa-B ligand (RANKL)-triggered macrophage fusion into osteoclasts. PTP-PEST had no impact on expression of fusion mediators such as β-integrins, E-cadherin, and CD47, which enable macrophages to become fusion competent. However, it was needed for polarization of macrophages, migration induced by the chemokine CC chemokine ligand 2 (CCL2), and integrin-induced spreading, three key events in the fusion process. PTP-PEST deficiency resulted in specific hyperphosphorylation of the protein tyrosine kinase Pyk2 and the adaptor paxillin. Moreover, a fusion defect was induced upon treatment of normal macrophages with a Pyk2 inhibitor. Together, these data argue that macrophage fusion is critically dependent on PTP-PEST. This function is seemingly due to the ability of PTP-PEST to control phosphorylation of Pyk2 and paxillin, thereby regulating cell polarization, migration, and spreading. PMID:23589331

Macrophage fusion is controlled by the cytoplasmic protein tyrosine phosphatase PTP-PEST/PTPN12.

PubMed

Rhee, Inmoo; Davidson, Dominique; Souza, Cleiton Martins; Vacher, Jean; Veillette, André

2013-06-01

Macrophages can undergo cell-cell fusion, leading to the formation of multinucleated giant cells and osteoclasts. This process is believed to promote the proteolytic activity of macrophages toward pathogens, foreign bodies, and extracellular matrices. Here, we examined the role of PTP-PEST (PTPN12), a cytoplasmic protein tyrosine phosphatase, in macrophage fusion. Using a macrophage-targeted PTP-PEST-deficient mouse, we determined that PTP-PEST was not needed for macrophage differentiation or cytokine production. However, it was necessary for interleukin-4-induced macrophage fusion into multinucleated giant cells in vitro. It was also needed for macrophage fusion following implantation of a foreign body in vivo. Moreover, in the RAW264.7 macrophage cell line, PTP-PEST was required for receptor activator of nuclear factor kappa-B ligand (RANKL)-triggered macrophage fusion into osteoclasts. PTP-PEST had no impact on expression of fusion mediators such as β-integrins, E-cadherin, and CD47, which enable macrophages to become fusion competent. However, it was needed for polarization of macrophages, migration induced by the chemokine CC chemokine ligand 2 (CCL2), and integrin-induced spreading, three key events in the fusion process. PTP-PEST deficiency resulted in specific hyperphosphorylation of the protein tyrosine kinase Pyk2 and the adaptor paxillin. Moreover, a fusion defect was induced upon treatment of normal macrophages with a Pyk2 inhibitor. Together, these data argue that macrophage fusion is critically dependent on PTP-PEST. This function is seemingly due to the ability of PTP-PEST to control phosphorylation of Pyk2 and paxillin, thereby regulating cell polarization, migration, and spreading.

FOREIGN BODY REACTION TO BIOMATERIALS

PubMed Central

Anderson, James M.; Rodriguez, Analiz; Chang, David T.

2008-01-01

The foreign body reaction composed of macrophages and foreign body giant cells is the end-stage response of the inflammatory and wound healing responses following implantation of a medical device, prosthesis, or biomaterial. A brief, focused overview of events leading to the foreign body reaction is presented. The major focus of this review is on factors that modulate the interaction of macrophages and foreign body giant cells on synthetic surfaces where the chemical, physical, and morphological characteristics of the synthetic surface are considered to play a role in modulating cellular events. These events in the foreign body reaction include protein adsorption, monocyte/macrophage adhesion, macrophage fusion to form foreign body giant cells, consequences of the foreign body response on biomaterials, and cross-talk between macrophages/foreign body giant cells and inflammatory/wound healing cells. Biomaterial surface properties play an important role in modulating the foreign body reaction in the first two to four weeks following implantation of a medical device, even though the foreign body reaction at the tissue/material interface is present for the in vivo lifetime of the medical device. An understanding of the foreign body reaction is important as the foreign body reaction may impact the biocompatibility (safety) of the medical device, prosthesis, or implanted biomaterial and may significantly impact short- and long-term tissue responses with tissue-engineered constructs containing proteins, cells, and other biological components for use in tissue engineering and regenerative medicine. Our perspective has been on the inflammatory and wound healing response to implanted materials, devices, and tissue-engineered constructs. The incorporation of biological components of allogeneic or xenogeneic origin as well as stem cells into tissue-engineered or regenerative approaches opens up a myriad of other challenges. An in depth understanding of how the immune system interacts with these cells and how biomaterials or tissue-engineered constructs influences these interactions may prove pivotal to the safety, biocompatibility, and function of the device or system under consideration. PMID:18162407

Premethylation of Foreign DNA Improves Integrative Transformation Efficiency in Synechocystis sp. Strain PCC 6803

PubMed Central

Wang, Bo; Yu, Jianping

2015-01-01

Restriction digestion of foreign DNA is one of the key biological barriers against genetic transformation in microorganisms. To establish a high-efficiency transformation protocol in the model cyanobacterium, Synechocystis sp. strain PCC 6803 (Synechocystis 6803), we investigated the effects of premethylation of foreign DNA on the integrative transformation of this strain. In this study, two type II methyltransferase-encoding genes, i.e., sll0729 (gene M) and slr0214 (gene C), were cloned from the chromosome of Synechocystis 6803 and expressed in Escherichia coli harboring an integration plasmid. After premethylation treatment in E. coli, the integration plasmid was extracted and used for transformation of Synechocystis 6803. The results showed that although expression of methyltransferase M had little impact on the transformation of Synechocystis 6803, expression of methyltransferase C resulted in 11- to 161-fold-higher efficiency in the subsequent integrative transformation of Synechocystis 6803. Effective expression of methyltransferase C, which could be achieved by optimizing the 5′ untranslated region, was critical to efficient premethylation of the donor DNA and thus high transformation efficiency in Synechocystis 6803. Since premethylating foreign DNA prior to transforming Synechocystis avoids changing the host genetic background, the study thus provides an improved method for high-efficiency integrative transformation of Synechocystis 6803. PMID:26452551

Dengue subgenomic RNA binds TRIM25 to inhibit interferon expression for epidemiological fitness

PubMed Central

Manokaran, Gayathri; Finol, Esteban; Wang, Chunling; Gunaratne, Jayantha; Bahl, Justin; Ong, Eugenia Z.; Tan, Hwee Cheng; Sessions, October M.; Ward, Alex M.; Gubler, Duane J.; Harris, Eva; Garcia-Blanco, Mariano A.; Ooi, Eng Eong

2016-01-01

The global spread of dengue virus (DENV) infections has increased viral genetic diversity, some of which appears associated with greater epidemic potential. The mechanisms governing viral fitness in epidemiological settings, however, remain poorly defined. We identified a determinant of fitness in a foreign dominant (PR-2B) DENV serotype 2 (DENV-2) clade, which emerged during the 1994 epidemic in Puerto Rico and replaced an endemic (PR-1) DENV-2 clade. The PR-2B DENV-2 produced increased levels of subgenomic flavivirus RNA (sfRNA) relative to genomic RNA during replication. PR-2B sfRNA showed sequence-dependent binding to and prevention of tripartite motif 25 (TRIM25) deubiquitylation, which is critical for sustained and amplified retinoic acid–inducible gene 1 (RIG-I)–induced type I interferon expression. Our findings demonstrate a distinctive viral RNA–host protein interaction to evade the innate immune response for increased epidemiological fitness. PMID:26138103

African Swine Fever Virus NP868R Capping Enzyme Promotes Reovirus Rescue during Reverse Genetics by Promoting Reovirus Protein Expression, Virion Assembly, and RNA Incorporation into Infectious Virions

PubMed Central

Eaton, Heather E.; Kobayashi, Takeshi; Dermody, Terence S.; Johnston, Randal N.

2017-01-01

ABSTRACT Reoviruses, like many eukaryotic viruses, contain an inverted 7-methylguanosine (m7G) cap linked to the 5′ nucleotide of mRNA. The traditional functions of capping are to promote mRNA stability, protein translation, and concealment from cellular proteins that recognize foreign RNA. To address the role of mRNA capping during reovirus replication, we assessed the benefits of adding the African swine fever virus NP868R capping enzyme during reovirus rescue. C3P3, a fusion protein containing T7 RNA polymerase and NP868R, was found to increase protein expression 5- to 10-fold compared to T7 RNA polymerase alone while enhancing reovirus rescue from the current reverse genetics system by 100-fold. Surprisingly, RNA stability was not increased by C3P3, suggesting a direct effect on protein translation. A time course analysis revealed that C3P3 increased protein synthesis within the first 2 days of a reverse genetics transfection. This analysis also revealed that C3P3 enhanced processing of outer capsid μ1 protein to μ1C, a previously described hallmark of reovirus assembly. Finally, to determine the rate of infectious-RNA incorporation into new virions, we developed a new recombinant reovirus S1 gene that expressed the fluorescent protein UnaG. Following transfection of cells with UnaG and infection with wild-type virus, passage of UnaG through progeny was significantly enhanced by C3P3. These data suggest that capping provides nontraditional functions to reovirus, such as promoting assembly and infectious-RNA incorporation. IMPORTANCE Our findings expand our understanding of how viruses utilize capping, suggesting that capping provides nontraditional functions to reovirus, such as promoting assembly and infectious-RNA incorporation, in addition to enhancing protein translation. Beyond providing mechanistic insight into reovirus replication, our findings also show that reovirus reverse genetics rescue is enhanced 100-fold by the NP868R capping enzyme. Since reovirus shows promise as a cancer therapy, efficient reovirus reverse genetics rescue will accelerate production of recombinant reoviruses as candidates to enhance therapeutic potency. NP868R-assisted reovirus rescue will also expedite production of recombinant reovirus for mechanistic insights into reovirus protein function and structure. PMID:28298603

Tobacco mosaic virus Movement Protein Enhances the Spread of RNA Silencing

PubMed Central

Vogler, Hannes; Kwon, Myoung-Ok; Dang, Vy; Sambade, Adrian; Fasler, Monika; Ashby, Jamie; Heinlein, Manfred

2008-01-01

Eukaryotic cells restrain the activity of foreign genetic elements, including viruses, through RNA silencing. Although viruses encode suppressors of silencing to support their propagation, viruses may also exploit silencing to regulate host gene expression or to control the level of their accumulation and thus to reduce damage to the host. RNA silencing in plants propagates from cell to cell and systemically via a sequence-specific signal. Since the signal spreads between cells through plasmodesmata like the viruses themselves, virus-encoded plasmodesmata-manipulating movement proteins (MP) may have a central role in compatible virus:host interactions by suppressing or enhancing the spread of the signal. Here, we have addressed the propagation of GFP silencing in the presence and absence of MP and MP mutants. We show that the protein enhances the spread of silencing. Small RNA analysis indicates that MP does not enhance the silencing pathway but rather enhances the transport of the signal through plasmodesmata. The ability to enhance the spread of silencing is maintained by certain MP mutants that can move between cells but which have defects in subcellular localization and do not support the spread of viral RNA. Using MP expressing and non-expressing virus mutants with a disabled silencing suppressing function, we provide evidence indicating that viral MP contributes to anti-viral silencing during infection. Our results suggest a role of MP in controlling virus propagation in the infected host by supporting the spread of silencing signal. This activity of MP involves only a subset of its properties implicated in the spread of viral RNA. PMID:18389061

Clustering of adhesion receptors following exposure of insect blood cells to foreign surfaces.

PubMed

Nardi, James B; Zhuang, Shufei; Pilas, Barbara; Bee, Charles Mark; Kanost, Michael R

2005-05-01

Cell-mediated immune responses of insects involve interactions of two main classes of blood cells (hemocytes) known as granular cells and plasmatocytes. In response to a foreign surface, these hemocytes suddenly transform from circulating, non-adherent cells to cells that interact and adhere to each other and the foreign surface. This report presents evidence that during this adhesive transformation the extracellular matrix (ECM) proteins lacunin and a ligand for peanut agglutinin (PNA) lectin are released by granular cells and bind to surfaces of both granular cells and plasmatocytes. ECM protein co-localizes on cell surfaces with the adhesive receptors integrin and neuroglian, a member of the immunoglobulin superfamily. The ECM protein(s) secreted by granular cells are hypothesized to interact with adhesion receptors such as neuroglian and integrin by cross linking and clustering them on hemocyte surfaces. This clustering of receptors is known to enhance the adhesiveness (avidity) of interacting mammalian immune cells. The formation of ring-shaped clusters of these adhesion receptors on surfaces of insect immune cells represents an evolutionary antecedent of the mammalian immunological synapse.

Foreign bodies; or, how did Darwin invent the symptom?

PubMed

Rowlinson, Matthew

2010-01-01

Beginning with a discussion of the sources in Darwin's writing for Freud's theory of the hysterical symptom, this essay proceeds to a symptomatic reading of Darwin himself. With reference to "The Origin of Species," "The Descent of Man," and "The Expression of the Emotions," this essay shows that Darwin's theories of involuntary expressive behavior and of aesthetic preference in sexual selection are linked by their role in his understanding of racial difference and also by their reliance on the idea that learned habits can be inherited as instincts, a view often identified with Lamarck. They are thus at once theories of the foreign body and theories that appear as foreigners within the body of Darwin's work.

A Bivalent Typhoid Live Vector Vaccine Expressing both Chromosome- and Plasmid-Encoded Yersinia pestis Antigens Fully Protects against Murine Lethal Pulmonary Plague Infection

PubMed Central

Wang, Jin Yuan; Carrasco, Jose A.; Lloyd, Scott A.; Mellado-Sanchez, Gabriela; Diaz-McNair, Jovita; Franco, Olga; Buskirk, Amanda D.; Nataro, James P.; Pasetti, Marcela F.

2014-01-01

Live attenuated bacteria hold great promise as multivalent mucosal vaccines against a variety of pathogens. A major challenge of this approach has been the successful delivery of sufficient amounts of vaccine antigens to adequately prime the immune system without overattenuating the live vaccine. Here we used a live attenuated Salmonella enterica serovar Typhi strain to create a bivalent mucosal plague vaccine that produces both the protective F1 capsular antigen of Yersinia pestis and the LcrV protein required for secretion of virulence effector proteins. To reduce the metabolic burden associated with the coexpression of F1 and LcrV within the live vector, we balanced expression of both antigens by combining plasmid-based expression of F1 with chromosomal expression of LcrV from three independent loci. The immunogenicity and protective efficacy of this novel vaccine were assessed in mice by using a heterologous prime-boost immunization strategy and compared to those of a conventional strain in which F1 and LcrV were expressed from a single low-copy-number plasmid. The serum antibody responses to lipopolysaccharide (LPS) induced by the optimized bivalent vaccine were indistinguishable from those elicited by the parent strain, suggesting an adequate immunogenic capacity maintained through preservation of bacterial fitness; in contrast, LPS titers were 10-fold lower in mice immunized with the conventional vaccine strain. Importantly, mice receiving the optimized bivalent vaccine were fully protected against lethal pulmonary challenge. These results demonstrate the feasibility of distributing foreign antigen expression across both chromosomal and plasmid locations within a single vaccine organism for induction of protective immunity. PMID:25332120

[Tobacco--a highly efficient producer of vaccines].

PubMed

Budzianowski, Jaromir

2010-01-01

Along with the depreciation of tobacco as a source of nicotine-containing commercial products, the increase of its appreciation as a potential producer of recombinant therapeutical proteins can be observed. Two species of tobacco--Nicotiana tabacum L. and N. benthamiana are easily grown by well established methods of field or green-house cultivation or cell culture, yield high biomass and soluble protein content, can be easily transformed by several methods and are not food for humans or feed for animals. Expression of foreign proteins, including vaccines, can be achieved in those plants either through stable transformation of nuclear or plastid (chloroplast) genomes or by transient transformation using infection with plant virus or bacteria--Agrobacterium tumefaciens (agroinfiltration). The most advanced mode of agrofiltration termed magnifection, which combines benefits of virus and Agrobacterium and depends on using Agrobacterium with viral pro-vectors, enables high-yield and rapid expression of therapeutical proteins, even in a few days, and can be employed on an industrial scale. Expression of many antigenic proteins, which may serve as antiviral, antibacterial, antiprotozoan and anticancer vaccines, and additionally a few autoantigens designed for the treatment of autoimunogenic diseases, like diabetes, have been achieved in tobacco. To date, a vaccine against Newcastle virus disease in poultry produced by tobacco cell culture has been approved for commercial application and several other vaccines are in advanced stage of development. The possibility of a high-level production of vaccines in tobacco against pandemic influenza or anthrax and plague due to a bioterroristic attack, as well as of individualised anticancer vaccines against non-Hodgkin's lymphoma (NHL) in a much shorter period of time than by traditional methods became realistic and hence caused increased interest in tobacco as a high-efficient producer of vaccines not only of specialistic biotechnology firms but also a big pharmaceutical corporation and a department of defence.

IFIT3 and IFIT2/3 promote IFIT1-mediated translation inhibition by enhancing binding to non-self RNA.

PubMed

Fleith, Renata C; Mears, Harriet V; Leong, Xin Yun; Sanford, Thomas J; Emmott, Edward; Graham, Stephen C; Mansur, Daniel S; Sweeney, Trevor R

2018-06-01

Interferon-induced proteins with tetratricopeptide repeats (IFITs) are highly expressed during the cell-intrinsic immune response to viral infection. IFIT1 inhibits translation by binding directly to the 5' end of foreign RNAs, particularly those with non-self cap structures, precluding the recruitment of the cap-binding eukaryotic translation initiation factor 4F and ribosome recruitment. The presence of IFIT1 imposes a requirement on viruses that replicate in the cytoplasm to maintain mechanisms to avoid its restrictive effects. Interaction of different IFIT family members is well described, but little is known of the molecular basis of IFIT association or its impact on function. Here, we reconstituted different complexes of IFIT1, IFIT2 and IFIT3 in vitro, which enabled us to reveal critical aspects of IFIT complex assembly. IFIT1 and IFIT3 interact via a YxxxL motif present in the C-terminus of each protein. IFIT2 and IFIT3 homodimers dissociate to form a more stable heterodimer that also associates with IFIT1. We show for the first time that IFIT3 stabilizes IFIT1 protein expression, promotes IFIT1 binding to a cap0 Zika virus reporter mRNA and enhances IFIT1 translation inhibition. This work reveals molecular aspects of IFIT interaction and provides an important missing link between IFIT assembly and function.

Complete plastid genome sequence of Daucus carota: Implications for biotechnology and phylogeny of angiosperms

PubMed Central

Ruhlman, Tracey; Lee, Seung-Bum; Jansen, Robert K; Hostetler, Jessica B; Tallon, Luke J; Town, Christopher D; Daniell, Henry

2006-01-01

Background Carrot (Daucus carota) is a major food crop in the US and worldwide. Its capacity for storage and its lifecycle as a biennial make it an attractive species for the introduction of foreign genes, especially for oral delivery of vaccines and other therapeutic proteins. Until recently efforts to express recombinant proteins in carrot have had limited success in terms of protein accumulation in the edible tap roots. Plastid genetic engineering offers the potential to overcome this limitation, as demonstrated by the accumulation of BADH in chromoplasts of carrot taproots to confer exceedingly high levels of salt resistance. The complete plastid genome of carrot provides essential information required for genetic engineering. Additionally, the sequence data add to the rapidly growing database of plastid genomes for assessing phylogenetic relationships among angiosperms. Results The complete carrot plastid genome is 155,911 bp in length, with 115 unique genes and 21 duplicated genes within the IR. There are four ribosomal RNAs, 30 distinct tRNA genes and 18 intron-containing genes. Repeat analysis reveals 12 direct and 2 inverted repeats ≥ 30 bp with a sequence identity ≥ 90%. Phylogenetic analysis of nucleotide sequences for 61 protein-coding genes using both maximum parsimony (MP) and maximum likelihood (ML) were performed for 29 angiosperms. Phylogenies from both methods provide strong support for the monophyly of several major angiosperm clades, including monocots, eudicots, rosids, asterids, eurosids II, euasterids I, and euasterids II. Conclusion The carrot plastid genome contains a number of dispersed direct and inverted repeats scattered throughout coding and non-coding regions. This is the first sequenced plastid genome of the family Apiaceae and only the second published genome sequence of the species-rich euasterid II clade. Both MP and ML trees provide very strong support (100% bootstrap) for the sister relationship of Daucus with Panax in the euasterid II clade. These results provide the best taxon sampling of complete chloroplast genomes and the strongest support yet for the sister relationship of Caryophyllales to the asterids. The availability of the complete plastid genome sequence should facilitate improved transformation efficiency and foreign gene expression in carrot through utilization of endogenous flanking sequences and regulatory elements. PMID:16945140

Recombinant Expression of Tandem-HBc Virus-Like Particles (VLPs).

PubMed

Stephen, Sam L; Beales, Lucy; Peyret, Hadrien; Roe, Amy; Stonehouse, Nicola J; Rowlands, David J

2018-01-01

The hepatitis B virus (HBV) core protein (HBc) has formed the building block for virus-like particle (VLP) production for more than 30 years. The ease of production of the protein, the robust ability of the core monomers to dimerize and assemble into intact core particles, and the strong immune responses they elicit when presenting antigenic epitopes all demonstrate its promise for vaccine development (reviewed in Pumpens and Grens (Intervirology 44: 98-114, 2001)). HBc has been modified in a number of ways in attempts to expand its potential as a novel vaccine platform. The HBc protein is predominantly α-helical in structure and folds to form an L-shaped molecule. The structural subunit of the HBc particle is a dimer of monomeric HBc proteins which together form an inverted T-shaped structure. In the assembled HBc particle the four-helix bundle formed at each dimer interface appears at the surface as a prominent "spike." The tips of the "spikes" are the preferred sites for the insertion of foreign sequences for vaccine purposes as they are the most highly exposed regions of the assembled particles. In the tandem-core modification two copies of the HBc protein are covalently linked by a flexible amino acid sequence which allows the fused dimer to fold correctly and assemble into HBc particles. The advantage of the modified structure is that the assembly of the dimeric subunits is defined and not formed by random association. This facilitates the introduction of single, larger sequences at the tip of each surface "spike," thus overcoming the conformational clashes contingent on insertion of large structures into monomeric HBc proteins.Differences in inserted sequences influence the assembly characteristics of the modified proteins, and it is important to optimize the design of each novel construct to maximize efficiency of assembly into regular VLPs. In addition to optimization of the construct, the expression system used can also influence the ability of recombinant structures to assemble into regular isometric particles. Here, we describe the production of recombinant tandem-core particles in bacterial, yeast and plant expression systems.

Mutant alpha-synuclein overexpression mediates early proinflammatory activity.

PubMed

Su, Xiaomin; Federoff, Howard J; Maguire-Zeiss, Kathleen A

2009-10-01

Microglia provide immune surveillance for the brain through both the removal of cellular debris and protection against infection by microorganisms and "foreign" molecules. Upon activation, microglia display an altered morphology and increased expression of proinflammatory molecules. Increased numbers of activated microglia have been identified in a number of neurodegenerative diseases including Parkinson's disease (PD). What remains to be determined is whether activated microglia result from ongoing cell death or are involved in disease initiation and progression. To address this question we utilized a transgenic mouse model that expresses a mutated form of a key protein involved in Parkinson's disease, alpha-synuclein. Herein, we report an increase in activated microglia and proinflammatory molecules in 1-month-old transgenic mice well before cell death occurs in this model. Frank microglial activation is resolved by 6 months of age while a subset of proinflammatory molecules remain elevated for 12 months. Both tyrosine hydroxylase mRNA expression and alpha-synuclein protein are decreased in the striatum of older animals evidence of dystrophic neuritic projections. To determine whether mutated alpha-synuclein could directly activate microglia primary microglia-enriched cell cultures were treated with exogenous mutated alpha-synuclein. The data reveal an increase in activated microglia and proinflammatory molecules due to direct interaction with mutated alpha-synuclein. Together, these data demonstrate that mutated alpha-synuclein mediates a proinflammatory response in microglia and this activity may participate in PD pathogenesis.

Serial transmission in rodents of neurodegeneration from transgenic mice expressing mutant prion protein.

PubMed Central

Hsiao, K K; Groth, D; Scott, M; Yang, S L; Serban, H; Rapp, D; Foster, D; Torchia, M; Dearmond, S J; Prusiner, S B

1994-01-01

Two lines of transgenic (Tg) mice expressing high (H) levels of the mutant P101L prion protein (PrP) developed a neurologic illness and central nervous system pathology indistinguishable from experimental murine scrapie; these mice were designated Tg(MoPrP-P101L)H. Brain homogenates from Tg(MoPrP-P101L)H mice were inoculated intracerebrally into CD-1 Swiss mice, Syrian hamsters, and Tg196 mice, Tg mice expressing the MoPrP-P101L transgene at low levels. None of the CD-1 mice developed central nervous system dysfunction, whereas approximately 10% of hamsters and approximately 40% of the Tg196 mice manifested neurologic signs between 117 and 639 days after inoculation. Serial transmission of neurodegeneration in Tg196 mice and Syrian hamsters was initiated with brain extracts, producing incubation times of approximately 400 and approximately 75 days, respectively. Although the Tg(MoPrP-P101L)H mice appear to accumulate only low levels of infections prions in their brains, the serial transmission of disease to inoculated recipients argues that prion formation occurs de novo in the brains of these uninoculated animals. These Tg mouse studies, taken together with similar findings in humans dying of inherited prion diseases, provide additional evidence that prions lack a foreign nucleic acid. Images PMID:7916462

Characterizing and modeling protein-surface interactions in lab-on-chip devices

NASA Astrophysics Data System (ADS)

Katira, Parag

Protein adsorption on surfaces determines the response of other biological species present in the surrounding solution. This phenomenon plays a major role in the design of biomedical and biotechnological devices. While specific protein adsorption is essential for device function, non-specific protein adsorption leads to the loss of device function. For example, non-specific protein adsorption on bioimplants triggers foreign body response, in biosensors it leads to reduced signal to noise ratios, and in hybrid bionanodevices it results in the loss of confinement and directionality of molecular shuttles. Novel surface coatings are being developed to reduce or completely prevent the non-specific adsorption of proteins to surfaces. A novel quantification technique for extremely low protein coverage on surfaces has been developed. This technique utilizes measurement of the landing rate of microtubule filaments on kinesin proteins adsorbed on a surface to determine the kinesin density. Ultra-low limits of detection, dynamic range, ease of detection and availability of a ready-made kinesin-microtubule kit makes this technique highly suitable for detecting protein adsorption below the detection limits of standard techniques. Secondly, a random sequential adsorption model is presented for protein adsorption to PEO-coated surfaces. The derived analytical expressions accurately predict the observed experimental results from various research groups, suggesting that PEO chains act as almost perfect steric barriers to protein adsorption. These expressions can be used to predict the performance of a variety of systems towards resisting protein adsorption and can help in the design of better non-fouling surface coatings. Finally, in biosensing systems, target analytes are captured and concentrated on specifically adsorbed proteins for detection. Non-specific adsorption of proteins results in the loss of signal, and an increase in the background. The use of nanoscale transducers as receptors is beneficial from the point of view of signal enhancement, but has a strong mass transport limitation. To overcome this, the use of molecular shuttles has been proposed that can selectively capture analytes and actively transport them to the nanoreceptors. The effect of employing such a two-stage capture process on biosensor sensitivity is theoretically investigated and an optimum design for a kinesin-microtubule-driven hybrid biosensor is proposed.

Biochemical Diversification through Foreign Gene Expression in Bdelloid Rotifers

PubMed Central

Eyres, Isobel; Wang-Koh, Yuan; Lubzens, Esther; Barraclough, Timothy G.; Micklem, Gos; Tunnacliffe, Alan

2012-01-01

Bdelloid rotifers are microinvertebrates with unique characteristics: they have survived tens of millions of years without sexual reproduction; they withstand extreme desiccation by undergoing anhydrobiosis; and they tolerate very high levels of ionizing radiation. Recent evidence suggests that subtelomeric regions of the bdelloid genome contain sequences originating from other organisms by horizontal gene transfer (HGT), of which some are known to be transcribed. However, the extent to which foreign gene expression plays a role in bdelloid physiology is unknown. We address this in the first large scale analysis of the transcriptome of the bdelloid Adineta ricciae: cDNA libraries from hydrated and desiccated bdelloids were subjected to massively parallel sequencing and assembled transcripts compared against the UniProtKB database by blastx to identify their putative products. Of ∼29,000 matched transcripts, ∼10% were inferred from blastx matches to be horizontally acquired, mainly from eubacteria but also from fungi, protists, and algae. After allowing for possible sources of error, the rate of HGT is at least 8%–9%, a level significantly higher than other invertebrates. We verified their foreign nature by phylogenetic analysis and by demonstrating linkage of foreign genes with metazoan genes in the bdelloid genome. Approximately 80% of horizontally acquired genes expressed in bdelloids code for enzymes, and these represent 39% of enzymes in identified pathways. Many enzymes encoded by foreign genes enhance biochemistry in bdelloids compared to other metazoans, for example, by potentiating toxin degradation or generation of antioxidants and key metabolites. They also supplement, and occasionally potentially replace, existing metazoan functions. Bdelloid rotifers therefore express horizontally acquired genes on a scale unprecedented in animals, and foreign genes make a profound contribution to their metabolism. This represents a potential mechanism for ancient asexuals to adapt rapidly to changing environments and thereby persist over long evolutionary time periods in the absence of sex. PMID:23166508

Chimaera and its modern virus-like descendants.

PubMed

Ulrich, R; Gerlich, W H; Krüger, D H

1996-01-01

Chimaera was a monster in ancient Greek mythology combining elements from different animal species in its body. Modern molecular biology enabled the generation of harmless but useful chimaeras consisting of elements from different nonrelated viruses. The objective is that the resulting chimaeras form highly immunogenic virus-like particles (VLPs). Such chimaeric VLPs can be used as highly efficient carriers for sequential and conformational B cell epitopes and T cell epitopes. Most VLPs are readily produced in heterologous hosts and are easy to purify. This article deals with various systems of VLPs described in this topical issue of Intervirology and makes comparisons with chimaeric replication-competent viruses, recombinant virus vectors expressing foreign proteins, and DNA vaccines.

Hypertonic stress induces rapid and widespread protein damage in C. elegans

PubMed Central

Burkewitz, Kris; Choe, Keith

2011-01-01

Proteostasis is defined as the homeostatic mechanisms that maintain the function of all cytoplasmic proteins. We recently demonstrated that the capacity of the proteostasis network is a critical factor that defines the limits of cellular and organismal survival in hypertonic environments. The current studies were performed to determine the extent of protein damage induced by cellular water loss. Using worm strains expressing fluorescently tagged foreign and endogenous proteins and proteins with temperature-sensitive point mutations, we demonstrate that hypertonic stress causes aggregation and misfolding of diverse proteins in multiple cell types. Protein damage is rapid. Aggregation of a polyglutamine yellow fluorescent protein reporter is observable with <1 h of hypertonic stress, and aggregate volume doubles approximately every 10 min. Aggregate formation is irreversible and occurs after as little as 10 min of exposure to hypertonic conditions. To determine whether endogenous proteins are aggregated by hypertonic stress, we quantified the relative amount of total cellular protein present in detergent-insoluble extracts. Exposure for 4 h to 400 mM or 500 mM NaCl induced a 55–120% increase in endogenous protein aggregation. Inhibition of insulin signaling or acclimation to mild hypertonic stress increased survival under extreme hypertonic conditions and prevented aggregation of endogenous proteins. Our results demonstrate that hypertonic stress causes widespread and dramatic protein damage and that cells have a significant capacity to remodel the network of proteins that function to maintain proteostasis. These findings have important implications for understanding how cells cope with hypertonic stress and other protein-damaging stressors. PMID:21613604

Engineering RNA phage MS2 virus-like particles for peptide display

NASA Astrophysics Data System (ADS)

Jordan, Sheldon Keith

Phage display is a powerful and versatile technology that enables the selection of novel binding functions from large populations of randomly generated peptide sequences. Random sequences are genetically fused to a viral structural protein to produce complex peptide libraries. From a sufficiently complex library, phage bearing peptides with practically any desired binding activity can be physically isolated by affinity selection, and, since each particle carries in its genome the genetic information for its own replication, the selectants can be amplified by infection of bacteria. For certain applications however, existing phage display platforms have limitations. One such area is in the field of vaccine development, where the goal is to identify relevant epitopes by affinity-selection against an antibody target, and then to utilize them as immunogens to elicit a desired antibody response. Today, affinity selection is usually conducted using display on filamentous phages like M13. This technology provides an efficient means for epitope identification, but, because filamentous phages do not display peptides in the high-density, multivalent arrays the immune system prefers to recognize, they generally make poor immunogens and are typically useless as vaccines. This makes it necessary to confer immunogenicity by conjugating synthetic versions of the peptides to more immunogenic carriers. Unfortunately, when introduced into these new structural environments, the epitopes often fail to elicit relevant antibody responses. Thus, it would be advantageous to combine the epitope selection and immunogen functions into a single platform where the structural constraints present during affinity selection can be preserved during immunization. This dissertation describes efforts to develop a peptide display system based on the virus-like particles (VLPs) of bacteriophage MS2. Phage display technologies rely on (1) the identification of a site in a viral structural protein that is present on the surface of the virus particle and can accept foreign sequence insertions without disruption of protein folding and viral particle assembly, and (2) on the encapsidation of nucleic acid sequences encoding both the VLP and the peptide it displays. The experiments described here are aimed at satisfying the first of these two requirements by engineering efficient peptide display at two different sites in MS2 coat protein. First, we evaluated the suitability of the N-terminus of MS2 coat for peptide insertions. It was observed that random N-terminal 10-mer fusions generally disrupted protein folding and VLP assembly, but by bracketing the foreign sequences with certain specific dipeptides, these defects could be suppressed. Next, the suitability of a coat protein surface loop for foreign sequence insertion was tested. Specifically, random sequence peptides were inserted into the N-terminal-most AB-loop of a coat protein single-chain dimer. Again we found that efficient display required the presence of appropriate dipeptides bracketing the peptide insertion. Finally, it was shown that an N-terminal fusion that tended to interfere specifically with capsid assembly could be efficiently incorporated into mosaic particles when co-expressed with wild-type coat protein.

Functional complementation of yeast cytosolic pyrophosphatase by bacterial and plant H+-translocating pyrophosphatases.

PubMed

Perez-Castineira, Jose R; Lopez-Marques, Rosa L; Villalba, Jose M; Losada, Manuel; Serrano, Aurelio

2002-12-10

Two types of proteins that hydrolyze inorganic pyrophosphate (PPi), very different in both amino acid sequence and structure, have been characterized to date: soluble and membrane-bound proton-pumping pyrophosphatases (sPPases and H(+)-PPases, respectively). sPPases are ubiquitous proteins that hydrolyze PPi releasing heat, whereas H+-PPases, so far unidentified in animal and fungal cells, couple the energy of PPi hydrolysis to proton movement across biological membranes. The budding yeast Saccharomyces cerevisiae has two sPPases that are located in the cytosol and in the mitochondria. Previous attempts to knock out the gene coding for a cytosolic sPPase (IPP1) have been unsuccessful, thus suggesting that this protein is essential for growth. Here, we describe the generation of a conditional S. cerevisiae mutant (named YPC-1) whose functional IPP1 gene is under the control of a galactose-dependent promoter. Thus, YPC-1 cells become growth arrested in glucose but they regain the ability to grow on this carbon source when transformed with autonomous plasmids bearing diverse foreign H+-PPase genes under the control of a yeast constitutive promoter. The heterologously expressed H+-PPases are distributed among different yeast membranes, including the plasma membrane, functional complementation by these integral membrane proteins being consistently sensitive to external pH. These results demonstrate that hydrolysis of cytosolic PPi is essential for yeast growth and that this function is not substantially affected by the intrinsic characteristics of the PPase protein that accomplishes it. Moreover, this is, to our knowledge, the first direct evidence that H+-PPases can mediate net hydrolysis of PPi in vivo. YPC-1 mutant strain constitutes a convenient expression system to perform studies aimed at the elucidation of the structure-function relationships of this type of proton pumps.

Biological and immunological characterization of recombinant Yellow Fever 17D viruses expressing a Trypanosoma cruzi Amastigote Surface Protein-2 CD8+ T cell epitope at two distinct regions of the genome.

PubMed

Nogueira, Raquel T; Nogueira, Alanderson R; Pereira, Mirian C S; Rodrigues, Maurício M; Galler, Ricardo; Bonaldo, Myrna C

2011-03-18

The attenuated Yellow fever (YF) 17D vaccine virus is one of the safest and most effective viral vaccines administered to humans, in which it elicits a polyvalent immune response. Herein, we used the YF 17D backbone to express a Trypanosoma cruzi CD8+ T cell epitope from the Amastigote Surface Protein 2 (ASP-2) to provide further evidence for the potential of this virus to express foreign epitopes. The TEWETGQI CD8+ T cell epitope was cloned and expressed based on two different genomic insertion sites: in the fg loop of the viral Envelope protein and the protease cleavage site between the NS2B and NS3. We investigated whether the site of expression had any influence on immunogenicity of this model epitope. Recombinant viruses replicated similarly to vaccine virus YF 17D in cell culture and remained genetically stable after several serial passages in Vero cells. Immunogenicity studies revealed that both recombinant viruses elicited neutralizing antibodies to the YF virus as well as generated an antigen-specific gamma interferon mediated T-cell response in immunized mice. The recombinant viruses displayed a more attenuated phenotype than the YF 17DD vaccine counterpart in mice. Vaccination of a mouse lineage highly susceptible to infection by T. cruzi with a homologous prime-boost regimen of recombinant YF viruses elicited TEWETGQI specific CD8+ T cells which might be correlated with a delay in mouse mortality after a challenge with a lethal dose of T. cruzi. We conclude that the YF 17D platform is useful to express T. cruzi (Protozoan) antigens at different functional regions of its genome with minimal reduction of vector fitness. In addition, the model T. cruzi epitope expressed at different regions of the YF 17D genome elicited a similar T cell-based immune response, suggesting that both expression sites are useful. However, the epitope as such is not protective and it remains to be seen whether expression of larger domains of ASP-2, which include the TEWETGQI epitope, will elicit better T-CD8+ responses to the latter. It is likely that additional antigens and recombinant virus formulations will be necessary to generate a protective response.

Development and immunogenicity of recombinant GapA(+) Mycoplasma gallisepticum vaccine strain ts-11 expressing infectious bronchitis virus-S1 glycoprotein and chicken interleukin-6.

PubMed

Shil, Pollob K; Kanci, Anna; Browning, Glenn F; Markham, Philip F

2011-04-12

Mycoplasma gallisepticum (MG) is a major pathogen of poultry that causes chronic respiratory disease in chickens and infectious sinusitis in turkeys. A live attenuated vaccine, ts-11, has been used for the control of MG in several countries. The efficacy of this vaccine is highly dose dependent and the flock antibody response is weak. To improve the functionality of the vaccine and investigate its potential as a delivery vector for foreign antigens and immunomodulatory proteins, we developed a derivative of ts-11 expressing infectious bronchitis virus-S1 glycoprotein (IBV-S1) and releasing chicken interleukin-6 into the extracellular milieu (MG ts-11 C3 (+CS)) using a transposon-based delivery vector. Following administration of MG ts-11 C3 (+CS) to chickens by eye-drop, an antibody response to MG and IBV-S1, as determined by the rapid serum agglutination test (RSA) and Western blotting, respectively, could be detected. Birds inoculated with the recombinant vaccine had significantly enhanced weight gain and were partially protected against damage by pathogenic IBV. These results indicate that the ChIL-6 released by MG ts-11 C3 (+CS) may have had a non-specific effect on growth rate. They also suggest that ts-11 is a promising vaccine vector, capable of delivering heterologous protective antigens, and may also provide non-specific benefits when engineered to express immunomodulatory proteins. With some improvements in the expression system, it could be used to induce a targeted immune response against specific mucosal pathogens, and co-expression of several antigens would allow development of a novel multivalent vaccine. Copyright © 2011 Elsevier Ltd. All rights reserved.

Transgenic APP expression during postnatal development causes persistent locomotor hyperactivity in the adult.

PubMed

Rodgers, Shaefali P; Born, Heather A; Das, Pritam; Jankowsky, Joanna L

2012-06-18

Transgenic mice expressing disease-associated proteins have become standard tools for studying human neurological disorders. Transgenes are often expressed using promoters chosen to drive continuous high-level expression throughout life rather than temporal and spatial fidelity to the endogenous gene. This approach has allowed us to recapitulate diseases of aging within the two-year lifespan of the laboratory mouse, but has the potential for creating aberrant phenotypes by mechanisms unrelated to the human disorder. We show that overexpression of the Alzheimer's-related amyloid precursor protein (APP) during early postnatal development leads to severe locomotor hyperactivity that can be significantly attenuated by delaying transgene onset until adulthood. Our data suggest that exposure to transgenic APP during maturation influences the development of neuronal circuits controlling motor activity. Both when matched for total duration of APP overexpression and when matched for cortical amyloid burden, animals exposed to transgenic APP as juveniles are more active in locomotor assays than animals in which APP overexpression was delayed until adulthood. In contrast to motor activity, the age of APP onset had no effect on thigmotaxis in the open field as a rough measure of anxiety, suggesting that the interaction between APP overexpression and brain development is not unilateral. Our findings indicate that locomotor hyperactivity displayed by the tet-off APP transgenic mice and several other transgenic models of Alzheimer's disease may result from overexpression of mutant APP during postnatal brain development. Our results serve as a reminder of the potential for unexpected interactions between foreign transgenes and brain development to cause long-lasting effects on neuronal function in the adult. The tet-off APP model provides an easy means of avoiding developmental confounds by allowing transgene expression to be delayed until the mice reach adulthood.

The Commercial Course: Defense et Illustration.

ERIC Educational Resources Information Center

Scavillo, Anthony

The development of commercial foreign language courses by colleges and universities would help to satisfy the need for language enrollments and the expressed need of the international business community for foreign language skills in American business employees. Three major problems inhibit commercial language course development: limited…

26 CFR 1.882-0 - Table of contents.

Code of Federal Regulations, 2010 CFR

2010-04-01

... for foreign currency. (5)Coordination with other sections. (6)Special rule for foreign governments. (7... expressed in a single currency. (3)Adjustments. (4) Elective fixed ratio method of determining U.S... error. (iv)Liabilities of insurance companies. (v)Liabilities used to increase artificially interest...

Pluridicta, Numbers 1-27.

ERIC Educational Resources Information Center

Pluridicta, 1993

1993-01-01

The following titles are included: (1) "Computer in Foreign Language Instruction"; (2) "Two Sides of the Same Coin: Prose and Poetry in Translation"; (3) "Notions of Culture in Foreign Language Departments in Different Countries"; (4) "Developing Materials for Teaching Danish to Adult Immigrants"; (5) "Conversational Strategies in Expressive,…

Addressing practical issues related to nursing care for international visitors to Hiroshima.

PubMed

Nishikawa, Mariko; Niiya, Kiyoka; Okayasu, Masako

2014-04-01

When nine million foreigners visited Japan in 2013, the federal government set a goal to attract an additional two and a half million visitors including medical tourists by 2020. This research investigates the attitudes and concerns of Japanese nurses when they are in a situation dealing with foreign patients. The data were collected from March through September 2010, from 114 nurses at three hospitals, in close proximity to popular tourist destinations in Hiroshima. A questionnaire was developed for this research, named Mari Meter, which included a section to write answers to an open question for the nurses to express their opinions. These responses were examined statistically and by word analysis using Text Mining Studio. Japanese nurses expressed greatest concern about payment options, foreign language skills, and issues of informed consent, when dealing with foreigners. The results confirm that, in order to provide a high quality of patient care, extra preparation and a greater knowledge of international workers and visitors are required by nursing professionals in Japan.

27 CFR 28.251 - Railway express receipts.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Railway express receipts... § 28.251 Railway express receipts. Where the exportation is to a contiguous foreign country and the shipment is by railway express, a receipt issued by the railway express agency may be accepted in lieu of...

Advertisements: An Overlooked Resource in the Foreign Language Classroom.

ERIC Educational Resources Information Center

Deutsch, Rena

The use of newspaper and magazine advertisements for teaching foreign language skills in listening, speaking, reading, and writing as well as vocabulary, idiomatic expressions, grammar, pronunciation, and culture is encouraged and discussed. Suggested lessons and classroom activities are presented in four categories: vocabulary, grammatical rules…

Structural and Functional Assessment of APOBEC3G Macromolecular Complexes

PubMed Central

Polevoda, Bogdan; McDougall, William M.; Bennett, Ryan P.; Salter, Jason D.; Smith, Harold C.

2016-01-01

There are eleven members in the human APOBEC family of proteins that are evolutionarily related through their zinc-dependent cytidine deaminase domains. The human APOBEC gene clusters arose on chromosome 6 and 22 through gene duplication and divergence to where current day APOBEC proteins are functionally diverse and broadly expressed in tissues. APOBEC serve enzymatic and non enzymatic functions in cells. In both cases, formation of higher-order structures driven by APOBEC protein-protein interactions and binding to RNA and/or single stranded DNA are integral to their function. In some circumstances, these interactions are regulatory and modulate APOBEC activities. We are just beginning to understand how macromolecular interactions drive processes such as APOBEC subcellular compartmentalization, formation of holoenzyme complexes, gene targeting, foreign DNA restriction, anti-retroviral activity, formation of ribonucleoprotein particles and APOBEC degradation. Protein-protein and protein-nucleic acid cross-linking methods coupled with mass spectrometry, electrophoretic mobility shift assays, glycerol gradient sedimentation, fluorescence anisotropy and APOBEC deaminase assays are enabling mapping of interacting surfaces that are essential for these functions. The goal of this methods review is through example of our research on APOBEC3G, describe the application of cross-linking methods to characterize and quantify macromolecular interactions and their functional implications. Given the homology in structure and function, it is proposed that these methods will be generally applicable to the discovery process for other APOBEC and RNA and DNA editing and modifying proteins. PMID:26988126

An automated workflow for enhancing microbial bioprocess optimization on a novel microbioreactor platform

PubMed Central

2012-01-01

Background High-throughput methods are widely-used for strain screening effectively resulting in binary information regarding high or low productivity. Nevertheless achieving quantitative and scalable parameters for fast bioprocess development is much more challenging, especially for heterologous protein production. Here, the nature of the foreign protein makes it impossible to predict the, e.g. best expression construct, secretion signal peptide, inductor concentration, induction time, temperature and substrate feed rate in fed-batch operation to name only a few. Therefore, a high number of systematic experiments are necessary to elucidate the best conditions for heterologous expression of each new protein of interest. Results To increase the throughput in bioprocess development, we used a microtiter plate based cultivation system (Biolector) which was fully integrated into a liquid-handling platform enclosed in laminar airflow housing. This automated cultivation platform was used for optimization of the secretory production of a cutinase from Fusarium solani pisi with Corynebacterium glutamicum. The online monitoring of biomass, dissolved oxygen and pH in each of the microtiter plate wells enables to trigger sampling or dosing events with the pipetting robot used for a reliable selection of best performing cutinase producers. In addition to this, further automated methods like media optimization and induction profiling were developed and validated. All biological and bioprocess parameters were exclusively optimized at microtiter plate scale and showed perfect scalable results to 1 L and 20 L stirred tank bioreactor scale. Conclusions The optimization of heterologous protein expression in microbial systems currently requires extensive testing of biological and bioprocess engineering parameters. This can be efficiently boosted by using a microtiter plate cultivation setup embedded into a liquid-handling system, providing more throughput by parallelization and automation. Due to improved statistics by replicate cultivations, automated downstream analysis, and scalable process information, this setup has superior performance compared to standard microtiter plate cultivation. PMID:23113930

A physical/psychological and biological stress combine to enhance endoplasmic reticulum stress

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mondal, Tapan Kumar; Emeny, Rebecca T.; Gao, Donghong

The generation of an immune response against infectious and other foreign agents is substantially modified by allostatic load, which is increased with chemical, physical and/or psychological stressors. The physical/psychological stress from cold-restraint (CR) inhibits host defense against Listeria monocytogenes (LM), due to early effects of the catecholamine norepinephrine (NE) from sympathetic nerves on β1-adrenoceptors (β1AR) of immune cells. Although CR activates innate immunity within 2 h, host defenses against bacterial growth are suppressed 2–3 days after infection (Cao and Lawrence 2002). CR enhances inducible nitric oxide synthase (iNOS) expression and NO production. The early innate activation leads to cellular reduction-oxidationmore » (redox) changes of immune cells. Lymphocytes from CR-treated mice express fewer surface thiols. Splenic and hepatic immune cells also have fewer proteins with free thiols after CR and/or LM, and macrophages have less glutathione after the in vivo CR exposure or exposure to NE in vitro. The early induction of CR-induced oxidative stress elevates endoplasmic reticulum (ER) stress, which could interfere with keeping phagocytized LM within the phagosome or re-encapsuling LM by autophagy once they escape from the phagosome. ER stress-related proteins, such as glucose-regulated protein 78 (GRP78), have elevated expression with CR and LM. The results indicate that CR enhances the unfolded protein response (UPR), which interferes with host defenses against LM. Thus, it is postulated that increased stress, as exists with living conditions at low socioeconomic conditions, can lower host defenses against pathogens because of oxidative and ER stress processes. - Highlights: • Cold-restraint (physical/psychological stress) induces early oxidative stress. • The oxidative stress relates to catecholamine signaling beta-adrenoceptors. • Physical/psychological stress combines infection enhancing inflammation. • Endoplasmic reticulum stress interferes with host defenses and autophagy.« less

Current immunotherapeutic strategies in pancreatic cancer.

PubMed

Plate, Janet M D

2007-10-01

The immune systems of patients with newly diagnosed pancreatic cancers are functional, with T-cell responses capable of responding to tumor antigen presentation. Pancreatic tumors have been demonstrated to express tumor antigens as mutated, altered, underglycosylated and/or inappropriately overexpressed proteins. Considering these two facts, it should be possible for patients' bodies to recognize their tumors as foreign and to reject them. A number of clinical trials have been initiated to exploit this immune activation to eradicate or stabilize tumor growth. Immunotherapeutic trials include the specific testing of a variety of tumor vaccines, of cytokines as adjuvants or directed cytotoxicity, and of monoclonal antibodies to target specific molecules. This article reviews evidence for immune-cell activation and function in patients with pancreatic cancer, and evidence that pancreatic tumor cells express tumor antigens, or mutated (or altered) proteins. Nevertheless, tumors survive immune attacks by producing products that help them to circumvent effector T cells. The article thus examines complications of immune evasion by cancer cells, as well as the challenges of trying to exploit the immune system in solid tumors where tumor cell products can turn off invading immune T cells set to kill them. Finally, the article discusses the choices of a variety of clinical trials using immune modulation for patients with pancreatic cancer.

Tomato ASR1 abrogates the response to abscisic acid and glucose in Arabidopsis by competing with ABI4 for DNA binding.

PubMed

Shkolnik, Doron; Bar-Zvi, Dudy

2008-05-01

The manipulation of transacting factors is commonly used to achieve a wide change in the expression of a large number of genes in transgenic plants as a result of a change in the expression of a single gene product. This is mostly achieved by the overexpression of transactivator or repressor proteins. In this study, it is demonstrated that the overexpression of an exogenous DNA-binding protein can be used to compete with the expression of an endogenous transcription factor sharing the same DNA-binding sequence. Arabidopsis was transformed with cDNA encoding tomato abscisic acid stress ripening 1 (ASR1), a sequence-specific DNA protein that has no orthologues in the Arabidopsis genome. ASR1-overexpressing (ASR1-OE) plants display an abscisic acid-insensitive 4 (abi4) phenotype: seed germination is not sensitive to inhibition by abscisic acid (ABA), glucose, NaCl and paclobutrazol. ASR1 binds coupling element 1 (CE1), a cis-acting element bound by the ABI4 transcription factor, located in the ABI4-regulated promoters, including that of the ABI4 gene. Chromatin immunoprecipitation demonstrates that ASR1 is bound in vivo to the promoter of the ABI4 gene in ASR1-OE plants, but not to promoters of genes known to be regulated by the transcription factors ABI3 or ABI5. Real-time polymerase chain reaction (PCR) analysis confirmed that the expression of ABI4 and ABI4-regulated genes is markedly reduced in ASR1-OE plants. Therefore, it is concluded that the abi4 phenotype of ASR1-OE plants is the result of competition between the foreign ASR1 and the endogenous ABI4 on specific promoter DNA sequences. The biotechnological advantage of using this approach in crop plants from the Brassicaceae family to reduce the transactivation activity of ABI4 is discussed.

22 CFR 303.13 - Fees.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 22 Foreign Relations 2 2014-04-01 2014-04-01 false Fees. 303.13 Section 303.13 Foreign Relations PEACE CORPS PROCEDURES FOR DISCLOSURE OF INFORMATION UNDER THE FREEDOM OF INFORMATION ACT § 303.13 Fees...) Special delivery or express mail: Actual charges as incurred. (f) Fee waivers: Fees will be waived or...

22 CFR 303.13 - Fees.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 22 Foreign Relations 2 2011-04-01 2009-04-01 true Fees. 303.13 Section 303.13 Foreign Relations PEACE CORPS PROCEDURES FOR DISCLOSURE OF INFORMATION UNDER THE FREEDOM OF INFORMATION ACT § 303.13 Fees...) Special delivery or express mail: Actual charges as incurred. (f) Fee waivers: Fees will be waived or...

Selected Economic Translations on Eastern Europe (165th in the series)

DTIC Science & Technology

1960-04-18

requires the;clarification of many other prob- lems (cost computation, the determination of domestic prices,* foreign exchange rates , etc.).as well...expressed in clearing rubles are derived to a considerable extent from the 70 ■p^^^^ capitalist world market prices, because the foreign exchange rates of

GREM1 is expressed in the cancer-associated myofibroblasts of basal cell carcinomas.

PubMed

Kim, Hye Sung; Shin, Myung Soo; Cheon, Min Seok; Kim, Jae Wang; Lee, Cheol; Kim, Woo Ho; Kim, Young Sill; Jang, Bo Gun

2017-01-01

Cancer-associated fibroblasts (CAFs) play important roles in cancer progression through their complex interactions with cancer cells. The secreted bone morphogenetic protein antagonist, gremlin1 (GREM1) is expressed by the CAFs of basal cell carcinomas (BCCs), and promotes the growth of cancer cells. In this study, we investigated the expression of GREM1 mRNAs in various benign and malignant skin tumors, including various BCC subtypes. Analysis by RNA in situ hybridization (ISH) revealed that fibroblasts in the scar tissue expressed GREM1 and α-smooth muscle actin (α-SMA), whereas resident fibroblasts in the dermis of the normal skin did not express GREM1. Real-time polymerase chain reaction analysis showed significantly higher GREM1 expression in skin cancers and pilomatricomas (PMCs) than in other benign skin tumors. Tissue microarrays analyzed by RNA ISH for GREM1 expression also demonstrated that 23% of BCCs, 42% of squamous cell carcinomas, 20% of melanomas, and 90% of PMCs were positive for GREM1 expression, whereas trichoepitheliomas, eccrine poromas, hidradenomas, and spiradenomas were negative for GREM1 expression. Most BCCs that were GREM1 expression positive were of desmoplastic or mixed subtypes, and GREM1 expression was localized to activated myofibroblasts at the tumoral-stromal interface. Interestingly, most PMCs harbored GREM1-expressing fibroblasts, probably because of the inflammatory responses caused by foreign body reactions to keratin. Additionally, in BCCs, stromal GREM1 expression had a strong correlation with CD10 expression. In conclusion, GREM1 is frequently expressed by myofibroblasts in scars or in the stroma of basal cell carcinomas, suggesting that GREM1 expression can be a marker for activated myofibroblasts in the cancer stroma or in scar tissue.

Dengue subgenomic RNA binds TRIM25 to inhibit interferon expression for epidemiological fitness.

PubMed

Manokaran, Gayathri; Finol, Esteban; Wang, Chunling; Gunaratne, Jayantha; Bahl, Justin; Ong, Eugenia Z; Tan, Hwee Cheng; Sessions, October M; Ward, Alex M; Gubler, Duane J; Harris, Eva; Garcia-Blanco, Mariano A; Ooi, Eng Eong

2015-10-09

The global spread of dengue virus (DENV) infections has increased viral genetic diversity, some of which appears associated with greater epidemic potential. The mechanisms governing viral fitness in epidemiological settings, however, remain poorly defined. We identified a determinant of fitness in a foreign dominant (PR-2B) DENV serotype 2 (DENV-2) clade, which emerged during the 1994 epidemic in Puerto Rico and replaced an endemic (PR-1) DENV-2 clade. The PR-2B DENV-2 produced increased levels of subgenomic flavivirus RNA (sfRNA) relative to genomic RNA during replication. PR-2B sfRNA showed sequence-dependent binding to and prevention of tripartite motif 25 (TRIM25) deubiquitylation, which is critical for sustained and amplified retinoic acid-inducible gene 1 (RIG-I)-induced type I interferon expression. Our findings demonstrate a distinctive viral RNA-host protein interaction to evade the innate immune response for increased epidemiological fitness. Copyright © 2015, American Association for the Advancement of Science.

Plant-based immunocontraceptive control of wildlife--"potentials, limitations, and possums".

PubMed

Polkinghorne, Ian; Hamerli, Denes; Cowan, Phil; Duckworth, Janine

2005-03-07

Possums (Trichosurus vulpecula), originally introduced from Australia, are spread over 90% of New Zealand and cause major economic and environmental damage. Immunocontraception has been suggested as a humane means to control them. Marsupial-specific reproductive antigens expressed at high levels in edible transgenic plant tissue might provide a safe, effective, and cheap oral delivery bait for immunocontraceptive control. As proof of concept, female possums vaccinated with immunocontraceptive antigens showed reduced fertility, and possums fed with potato-expressed heat labile toxin-B (LT-B) had mucosal and systemic immune responses to the antigen. This demonstrated that immunocontraception was effective in possums and that oral delivery in edible plant material might be possible. Nuclear transformation with reporter genes showed that transgenic carrot roots accumulate high levels of foreign protein in edible tissues, indicating their potential as a delivery vector. However, prior to attempts at large scale production, more effective immunocontraceptive antigen-adjuvant formulations are probably required before plant-based immunocontraception can become a major tool for immunocontraceptive control of overabundant vertebrate pests.

Attenuated Shigella as a DNA Delivery Vehicle for DNA-Mediated Immunization

NASA Astrophysics Data System (ADS)

Sizemore, Donata R.; Branstrom, Arthur A.; Sadoff, Jerald C.

1995-10-01

Direct inoculation of DNA, in the form of purified bacterial plasmids that are unable to replicate in mammalian cells but are able to direct cell synthesis of foreign proteins, is being explored as an approach to vaccine development. Here, a highly attenuated Shigella vector invaded mammalian cells and delivered such plasmids into the cytoplasm of cells, and subsequent production of functional foreign protein was measured. Because this Shigella vector was designed to deliver DNA to colonic mucosa, the method is a potential basis for oral and other mucosal DNA immunization and gene therapy strategies.

DECREASED PRODUCTION OF SURFACTANT PROTEINS AFTER DIESEL EXHAUST EXPOSURE INCREASES SUSCEPTIBILITY TO INFLUENZA INFECTION

EPA Science Inventory

Pulmonary surfactant proteins A and D (SP-A and SP-D), termed collectins, enhance the opsonization of foreign particles and pathogens by phagocytic cells. Inhaled pollutants such as diesel exhaust (DE) have a possible role in suppressing the production of surfactant proteins whic...

Primer on the Immune System.

PubMed

Spiering, Martin J

2015-01-01

The human body regularly encounters and combats many pathogenic organisms and toxic molecules. Its ensuing responses to these disease-causing agents involve two interrelated systems: innate immunity and adaptive (or acquired) immunity. Innate immunity is active at several levels, both at potential points of entry and inside the body (see figure). For example, the skin represents a physical barrier preventing pathogens from invading internal tissues. Digestive enzymes destroy microbes that enter the stomach with food. Macrophages and lymphocytes, equipped with molecular detectors, such as Toll-like receptors (TLRs), which latch onto foreign structures and activate cellular defenses, patrol the inside of the body. These immune cells sense and devour microbes, damaged cells, and other foreign materials in the body. Certain proteins in the blood (such as proteins of the complement system and those released by natural killer cells, along with antimicrobial host-defense peptides) attach to foreign organisms and toxins to initiate their destruction.

76 FR 68257 - Notice of Applications for Certificates of Public Convenience and Necessity and Foreign Air...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-11-03

... Public Convenience and Necessity and Foreign Air Carrier Permits Filed Under Subpart B (Formerly Subpart Q) During the Week Ending September 10, 2011 The following Applications for Certificates of Public.... Description: Application of Sun Air Express, LLC d/b/a Sun Air International requesting authority to operate...

Fusion tags for protein solubility, purification and immunogenicity in Escherichia coli: the novel Fh8 system

PubMed Central

Costa, Sofia; Almeida, André; Castro, António; Domingues, Lucília

2014-01-01

Proteins are now widely produced in diverse microbial cell factories. The Escherichia coli is still the dominant host for recombinant protein production but, as a bacterial cell, it also has its issues: the aggregation of foreign proteins into insoluble inclusion bodies is perhaps the main limiting factor of the E. coli expression system. Conversely, E. coli benefits of cost, ease of use and scale make it essential to design new approaches directed for improved recombinant protein production in this host cell. With the aid of genetic and protein engineering novel tailored-made strategies can be designed to suit user or process requirements. Gene fusion technology has been widely used for the improvement of soluble protein production and/or purification in E. coli, and for increasing peptide’s immunogenicity as well. New fusion partners are constantly emerging and complementing the traditional solutions, as for instance, the Fh8 fusion tag that has been recently studied and ranked among the best solubility enhancer partners. In this review, we provide an overview of current strategies to improve recombinant protein production in E. coli, including the key factors for successful protein production, highlighting soluble protein production, and a comprehensive summary of the latest available and traditionally used gene fusion technologies. A special emphasis is given to the recently discovered Fh8 fusion system that can be used for soluble protein production, purification, and immunogenicity in E. coli. The number of existing fusion tags will probably increase in the next few years, and efforts should be taken to better understand how fusion tags act in E. coli. This knowledge will undoubtedly drive the development of new tailored-made tools for protein production in this bacterial system. PMID:24600443

Efficient gusA transient expression in Porphyra yezoensis protoplasts mediated by endogenous beta-tubulin flanking sequences

NASA Astrophysics Data System (ADS)

Gong, Qianhong; Yu, Wengong; Dai, Jixun; Liu, Hongquan; Xu, Rifu; Guan, Huashi; Pan, Kehou

2007-01-01

Endogenous tubulin promoter has been widely used for expressing foreign genes in green algae, but the efficiency and feasibility of endogenous tubulin promoter in the economically important Porphyra yezoensis (Rhodophyta) are unknown. In this study, the flanking sequences of beta-tubulin gene from P. yezoensis were amplified and two transient expression vectors were constructed to determine their transcription promoting feasibility for foreign gene gusA. The testing vector pATubGUS was constructed by inserting 5'-and 3'-flanking regions ( Tub5' and Tub3') up-and down-stream of β-glucuronidase (GUS) gene ( gusA), respectively, into pA, a derivative of pCAT®3-enhancer vector. The control construct, pAGUSTub3, contains only gusA and Tub3'. These constructs were electroporated into P. yezoensis protoplasts and the GUS activities were quantitatively analyzed by spectrometry. The results demonstrated that gusA gene was efficiently expressed in P. yezoensis protoplasts under the regulation of 5'-flanking sequence of the beta-tubulin gene. More interestingly, the pATubGUS produced stronger GUS activity in P. yezoensis protoplasts when compared to the result from pBI221, in which the gusA gene was directed by a constitutive CaMV 35S promoter. The data suggest that the integration of P. yezoensis protoplast and its endogenous beta-tubulin flanking sequences is a potential novel system for foreign gene expression.

Where are you from? A validation of the Foreigner Objectification Scale and the psychological correlates of foreigner objectification among Asian Americans and Latinos.

PubMed

Armenta, Brian E; Lee, Richard M; Pituc, Stephanie T; Jung, Kyoung-Rae; Park, Irene J K; Soto, José A; Kim, Su Yeong; Schwartz, Seth J

2013-04-01

Many ethnic minorities in the United States consider themselves to be just as American as their European American counterparts. However, there is a persistent cultural stereotype of ethnic minorities as foreigners (i.e., the perpetual foreigner stereotype) that may be expressed during interpersonal interactions (i.e., foreigner objectification). The goal of the present study was to validate the Foreigner Objectification Scale, a brief self-report measure of perceived foreigner objectification, and to examine the psychological correlates of perceived foreigner objectification. Results indicated that the Foreigner Objectification Scale is structurally (i.e., factor structure) and metrically (i.e., factor loadings) invariant across foreign-born and U.S.-born Asian Americans and Latinos. Scalar (i.e., latent item intercepts) invariance was demonstrated for the two foreign-born groups and the two U.S.-born groups, but not across foreign-born and U.S.-born individuals. Multiple-group structural equation models indicated that, among U.S.-born individuals, perceived foreigner objectification was associated with less life satisfaction and more depressive symptoms, and was indirectly associated with lower self-esteem via identity denial, operationalized as the perception that one is not viewed by others as American. Among foreign-born individuals, perceived foreigner objectification was not significantly associated directly with self-esteem, life satisfaction, or depressive symptoms. However, perceived foreigner objectification was positively associated with identity denial, and identity denial was negatively associated with life satisfaction. This study illustrates the relevance of perceived foreigner objectification to the psychological well-being of U.S.-born Asian Americans and Latinos.

Satellite cell proliferation in adult skeletal muscle

NASA Technical Reports Server (NTRS)

Morrison, Paul R. (Inventor); Thomason, Donald B. (Inventor); Stancel, George M. (Inventor); Booth, Frank W. (Inventor)

1995-01-01

Novel methods of retroviral-mediated gene transfer for the in vivo corporation and stable expression of eukaryotic or prokaryotic foreign genes in tissues of living animals is described. More specifically, methods of incorporating foreign genes into mitotically active cells are disclosed. The constitutive and stable expression of E. coli .beta.-galactosidase gene under the promoter control of the Moloney murine leukemia virus long terminal repeat is employed as a particularly preferred embodiment, by way of example, establishes the model upon which the incorporation of a foreign gene into a mitotically-active living eukaryotic tissue is based. Use of the described methods in therapeutic treatments for genetic diseases, such as those muscular degenerative diseases, is also presented. In muscle tissue, the described processes result in genetically-altered satellite cells which proliferate daughter myoblasts which preferentially fuse to form a single undamaged muscle fiber replacing damaged muscle tissue in a treated animal. The retroviral vector, by way of example, includes a dystrophin gene construct for use in treating muscular dystrophy. The present invention also comprises an experimental model utilizable in the study of the physiological regulation of skeletal muscle gene expression in intact animals.

C-Terminal DxD-Containing Sequences within Paramyxovirus Nucleocapsid Proteins Determine Matrix Protein Compatibility and Can Direct Foreign Proteins into Budding Particles

PubMed Central

Ray, Greeshma; Schmitt, Phuong Tieu

2016-01-01

ABSTRACT Paramyxovirus particles are formed by a budding process coordinated by viral matrix (M) proteins. M proteins coalesce at sites underlying infected cell membranes and induce other viral components, including viral glycoproteins and viral ribonucleoprotein complexes (vRNPs), to assemble at these locations from which particles bud. M proteins interact with the nucleocapsid (NP or N) components of vRNPs, and these interactions enable production of infectious, genome-containing virions. For the paramyxoviruses parainfluenza virus 5 (PIV5) and mumps virus, M-NP interaction also contributes to efficient production of virus-like particles (VLPs) in transfected cells. A DLD sequence near the C-terminal end of PIV5 NP protein was previously found to be necessary for M-NP interaction and efficient VLP production. Here, we demonstrate that 15-residue-long, DLD-containing sequences derived from either the PIV5 or Nipah virus nucleocapsid protein C-terminal ends are sufficient to direct packaging of a foreign protein, Renilla luciferase, into budding VLPs. Mumps virus NP protein harbors DWD in place of the DLD sequence found in PIV5 NP protein, and consequently, PIV5 NP protein is incompatible with mumps virus M protein. A single amino acid change converting DLD to DWD within PIV5 NP protein induced compatibility between these proteins and allowed efficient production of mumps VLPs. Our data suggest a model in which paramyxoviruses share an overall common strategy for directing M-NP interactions but with important variations contained within DLD-like sequences that play key roles in defining M/NP protein compatibilities. IMPORTANCE Paramyxoviruses are responsible for a wide range of diseases that affect both humans and animals. Paramyxovirus pathogens include measles virus, mumps virus, human respiratory syncytial virus, and the zoonotic paramyxoviruses Nipah virus and Hendra virus. Infectivity of paramyxovirus particles depends on matrix-nucleocapsid protein interactions which enable efficient packaging of encapsidated viral RNA genomes into budding virions. In this study, we have defined regions near the C-terminal ends of paramyxovirus nucleocapsid proteins that are important for matrix protein interaction and that are sufficient to direct a foreign protein into budding particles. These results advance our basic understanding of paramyxovirus genome packaging interactions and also have implications for the potential use of virus-like particles as protein delivery tools. PMID:26792745

C-Terminal DxD-Containing Sequences within Paramyxovirus Nucleocapsid Proteins Determine Matrix Protein Compatibility and Can Direct Foreign Proteins into Budding Particles.

PubMed

Ray, Greeshma; Schmitt, Phuong Tieu; Schmitt, Anthony P

2016-01-20

Paramyxovirus particles are formed by a budding process coordinated by viral matrix (M) proteins. M proteins coalesce at sites underlying infected cell membranes and induce other viral components, including viral glycoproteins and viral ribonucleoprotein complexes (vRNPs), to assemble at these locations from which particles bud. M proteins interact with the nucleocapsid (NP or N) components of vRNPs, and these interactions enable production of infectious, genome-containing virions. For the paramyxoviruses parainfluenza virus 5 (PIV5) and mumps virus, M-NP interaction also contributes to efficient production of virus-like particles (VLPs) in transfected cells. A DLD sequence near the C-terminal end of PIV5 NP protein was previously found to be necessary for M-NP interaction and efficient VLP production. Here, we demonstrate that 15-residue-long, DLD-containing sequences derived from either the PIV5 or Nipah virus nucleocapsid protein C-terminal ends are sufficient to direct packaging of a foreign protein, Renilla luciferase, into budding VLPs. Mumps virus NP protein harbors DWD in place of the DLD sequence found in PIV5 NP protein, and consequently, PIV5 NP protein is incompatible with mumps virus M protein. A single amino acid change converting DLD to DWD within PIV5 NP protein induced compatibility between these proteins and allowed efficient production of mumps VLPs. Our data suggest a model in which paramyxoviruses share an overall common strategy for directing M-NP interactions but with important variations contained within DLD-like sequences that play key roles in defining M/NP protein compatibilities. Paramyxoviruses are responsible for a wide range of diseases that affect both humans and animals. Paramyxovirus pathogens include measles virus, mumps virus, human respiratory syncytial virus, and the zoonotic paramyxoviruses Nipah virus and Hendra virus. Infectivity of paramyxovirus particles depends on matrix-nucleocapsid protein interactions which enable efficient packaging of encapsidated viral RNA genomes into budding virions. In this study, we have defined regions near the C-terminal ends of paramyxovirus nucleocapsid proteins that are important for matrix protein interaction and that are sufficient to direct a foreign protein into budding particles. These results advance our basic understanding of paramyxovirus genome packaging interactions and also have implications for the potential use of virus-like particles as protein delivery tools. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Vesicle-associated membrane protein 7 (VAMP-7) is essential for target cell killing in a natural killer cell line

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marcet-Palacios, Marcelo; Odemuyiwa, Solomon O.; Coughlin, Jason J.

2008-02-15

Natural killer cells recognize and induce apoptosis in foreign, transformed or virus-infected cells through the release of perforin and granzymes from secretory lysosomes. Clinically, NK-cell mediated killing is a major limitation to successful allo- and xenotransplantation. The molecular mechanisms that regulate the fusion of granzyme B-containing secretory lysosomes to the plasma membrane in activated NK cells, prior to target cell killing, are not fully understood. Using the NK cell line YT-Indy as a model, we have investigated the expression of SNAP REceptors (SNAREs), both target (t-) and vesicular (v-) SNAREs, and their function in granzyme B-mediated target cell killing. Ourmore » data showed that YT-Indy cells express VAMP-7 and SNAP-23, but not VAMP-2. VAMP-7 was associated with granzyme B-containing lysosomal granules. Using VAMP-7 small interfering RNA (siRNA), we successfully knocked down the expression of VAMP-7 protein in YT-Indy to less than 10% of untreated cells in 24 h. VAMP7-deficient YT-Indy cells activated via co-culture with Jurkat cells released <1 ng/mL of granzyme B, compared to 1.5-2.5 {mu}g/mL from controls. Using Jurkat cells as targets, we showed a 7-fold reduction in NK cell-mediated killing by VAMP-7 deficient YT-Indy cells. Our results show that VAMP-7 is a crucial component of granzyme B release and target cell killing in the NK cell line YT-Indy. Thus, targeting VAMP-7 expression specifically with siRNA, following transplantation, may be a viable strategy for preventing NK cell-mediated transplant rejection, in vivo.« less

Isolation and assessment of gut bacteria from the Formosan subterranean termite, Coptotermes formosanus (Isoptera: Rhinotermitidae), for paratransgenesis research and application.

PubMed

Tikhe, Chinmay V; Sethi, Amit; Delatte, Jennifer; Husseneder, Claudia

2017-02-01

Paratransgenesis targeting the gut protozoa is being developed as an alternative method for the control of the Formosan subterranean termite (FST). This method involves killing the cellulose-digesting gut protozoa using a previously developed antiprotozoal peptide consisting of a target specific ligand coupled to an antimicrobial peptide (Hecate). In the future, we intend to genetically engineer termite gut bacteria as "Trojan Horses" to express and spread ligand-Hecate in the termite colony. The aim of this study was to assess the usefulness of bacteria strains isolated from the gut of FST as "Trojan Horses." We isolated 135 bacteria from the guts of workers from 3 termite colonies. Sequencing of the 16S rRNA gene identified 20 species. We tested 5 bacteria species that were previously described as part of the termite gut community for their tolerance against Hecate and ligand-Hecate. Results showed that the minimum concentration required to inhibit bacteria growth was always higher than the concentration required to kill the gut protozoa. Out of the 5 bacteria tested, we engineered Trabulsiella odontotermitis, a termite specific bacterium, to express green fluorescent protein as a proof of concept that the bacteria can be engineered to express foreign proteins. Engineered T. odontotermitis was fed to FST to study if the bacteria are ingested. This feeding experiment confirmed that engineered T. odontotermitis is ingested by termites and can survive in the gut for at least 48 h. Here we report that T. odontotermitis is a suitable delivery and expression system for paratransgenesis in a termite species. © 2015 Institute of Zoology, Chinese Academy of Sciences.

Characterization of urea transport in Bufo arenarum oocytes.

PubMed

Silberstein, Claudia; Zotta, Elsa; Ripoche, Pierre; Ibarra, Cristina

2003-07-01

Xenopus laevis oocytes have been extensively used for expression cloning, structure/function relationships, and regulation analysis of transporter proteins. Urea transporters have been expressed in Xenopus oocytes and their properties have been described. In order to establish an alternative system in which urea transporters could be efficiently expressed and studied, we determined the urea transport properties of ovarian oocytes from Bufo arenarum, a toad species common in Argentina. Bufo oocytes presented a high urea permeability of 22.3 x 10(-6) cm/s, which was significantly inhibited by the incubation with phloretin. The urea uptake in these oocytes was also inhibited by mercurial reagents, and high-affinity urea analogues. The urea uptake was not sodium dependent. The activation energy was 3.2 Kcal/mol, suggesting that urea movement across membrane oocytes may be through a facilitated urea transporter. In contrast, Bufo oocytes showed a low permeability for mannitol and glycerol. From these results, we propose that one or several specific urea transporters are present in ovarian oocytes from Bufo arenarum. Therefore, these oocytes cannot be used in expression studies of foreign urea transporters. The importance of Bufo urea transporter is not known but could be implicated in osmotic regulation during the laying of eggs in water. Copyright 2003 Wiley-Liss, Inc.

MIRAgel: the immunohistochemical expression of CD3, CD34, and CD68 in the surrounding capsule

PubMed Central

Roldan-Pallares, M; LLanes-Estrada, M; LLanes-Menendez, F

2016-01-01

Purpose To study the immunohistochemical features of the capsule tissue surrounding MIRAgel episcleral buckles. Patients and methods This Institutional interventional clinical cohort study examined a consecutive series of 21 referred patients who required MIRAgel removal from July 2009 to July 2013. All patients with hydrated and fragmented MIRAgel episcleral buckles were included in this study. Capsule biopsies from MIRAgel episcleral buckles were obtained from all patients. Capsule specimens of seven patients with extruded silicone bands were processed as controls. Paraffin-embedded specimens were examined using light microscopy and immunohistochemistry (via the PAP horseradish peroxidase technique) to detect the expression of CD3, CD20, CD34 and CD68, and S-100 protein. Results Inflammation with granuloma, which was primarily related to sutures, was found in all (n=36) of the MIRAgel specimens and foreign body granulomas with multinucleated giant cells, histiocytes, and macrophages (CD68+ cells) surrounded the MIRAgel fragments. Average number of CD68+ cells was higher (P<0.001) for MIRAgel than for silicone rubber. The lymphocytic inflammatory infiltrate related to the MIRAgel fragments was CD3+ and CD20– (delayed T cell-mediated immune response). Moderate neoangiogenesis was indicated by the presence of CD34+ cells. Conclusions The immunohistochemical analysis revealed that the immune system is able to identify the fragments of MIRAgel (after its hydrolytic degradation) as a foreign body during a delayed T cell-mediated immune response. The phagocytosis by macrophages likely triggers and perpetuates local disease. Removal of MIRAgel explants before hydrolysis should be considered. PMID:27341317

Recombinant Listeria monocytogenes as a Live Vaccine Vehicle for the Induction of Protective Anti-Viral Cell-Mediated Immunity

NASA Astrophysics Data System (ADS)

Shen, Hao; Slifka, Mark K.; Matloubian, Mehrdad; Jensen, Eric R.; Ahmed, Rafi; Miller, Jeff F.

1995-04-01

Listeria monocytogenes (LM) is a Gram-positive bacterium that is able to enter host cells, escape from the endocytic vesicle, multiply within the cytoplasm, and spread directly from cell to cell without encountering the extracellular milieu. The ability of LM to gain access to the host cell cytosol allows proteins secreted by the bacterium to efficiently enter the pathway for major histocompatibility complex class I antigen processing and presentation. We have established a genetic system for expression and secretion of foreign antigens by recombinant strains, based on stable site-specific integration of expression cassettes into the LM genome. The ability of LM recombinants to induce protective immunity against a heterologous pathogen was demonstrated with lymphocytic choriomeningitis virus (LCMV). LM strains expressing the entire LCMV nucleoprotein or an H-2L^d-restricted nucleoprotein epitope (aa 118-126) were constructed. Immunization of mice with LM vaccine strains conferred protection against challenge with virulent strains of LCMV that otherwise establish chronic infection in naive adult mice. In vivo depletion of CD8^+ T cells from vaccinated mice abrogated their ability to clear viral infection, showing that protective anti-viral immunity was due to CD8^+ T cells.

Characterization of a small (25-kilodalton) derivative of the Rous sarcoma virus Gag protein competent for particle release.

PubMed Central

Weldon, R A; Wills, J W

1993-01-01

Retroviral Gag proteins have the ability to induce budding and particle release from the plasma membrane when expressed in the absence of all of the other virus-encoded components; however, the locations of the functional domains within the Gag protein that are important for this process are poorly understood. It was shown previously that the protease sequence of the Rous sarcoma virus (RSV) Gag protein can be replaced with a foreign polypeptide, iso-1-cytochrome c from a yeast, without disrupting particle assembly (R. A. Weldon, Jr., C. R. Erdie, M. G. Oliver, and J. W. Wills, J. Virol. 64:4169-4179, 1990). An unexpected product of the chimeric gag gene is a small, Gag-related protein named p25C. This product was of interest because of its high efficiency of packaging into particles. The goal of the experiments described here was to determine the mechanism by which p25C is synthesized and packaged into particles. The results demonstrate that it is not the product of proteolytic processing of the Gag-cytochrome precursor but is derived from an unusual spliced mRNA. cDNA clones of the spliced mRNA were obtained, and each expressed a product of approximately 25 kDa, designated p25M1, which was released into the growth medium in membrane-enclosed particles that were much lighter than authentic retrovirions as measured in sucrose density gradients. DNA sequencing revealed that the clones encode the first 180 of the 701 amino acids of the RSV Gag protein and no residues from iso-1-cytochrome c. This suggested that a domain in the carboxy-terminal half of Gag is important for the packaging of Gag proteins into dense arrays within the particles. In support of this hypothesis, particles of the correct density were obtained when a small segment from the carboxy terminus of the RSV Gag protein (residues 417 to 584) was included on the end of p25. Images PMID:8394460

A Review of the Foreign-body Response to Subcutaneously-implanted Devices: The Role of Macrophages and Cytokines in Biofouling and Fibrosis

PubMed Central

Kenneth Ward, W.

2008-01-01

The biological response to implanted biomaterials in mammals is a complex series of events that involves many biochemical pathways. Shortly after implantation, fibrinogen and other proteins bind to the device surface, a process known as biofouling. Macrophages then bind to receptors on the proteins, join into multinucleated giant cells, and release transforming growth factor β and other inflammatory cytokines. In response to these signals, quiescent fibroblasts are transformed into myofibroblasts, which synthesize procollagen via activation of Smad mediators. The procollagen becomes crosslinked after secretion into the extracellular space. Mature crosslinked collagen and other extracellular matrix proteins gradually contribute to formation of a hypocellular dense fibrous capsule that becomes impermeable or hypopermeable to many compounds. Porous substrates and angiogenic growth factors can stimulate formation of microvessels, which to some extent can maintain analyte delivery to implanted sensors. However, stimulation by vascular endothelial growth factor alone may lead to formation of leaky, thin-walled, immature vessels. Other growth factors are most probably needed to act upon these immature structures to create more robust vessels. During implantation of foreign bodies, the foreign-body response is difficult to overcome, and thousands of biomaterials have been tested. Biomimicry (i.e., creating membranes whose chemical structure mimics natural cellular compounds) may diminish the response, but as of this writing, it has not been possible to create a stealth material that circumvents the ability of the mammalian surveillance systems to distinguish foreign from self. PMID:19885259

Teachers' Perceptions of School Climate as an Indicator of Their Beliefs of Effective Teaching

ERIC Educational Resources Information Center

Oder, Tuuli; Eisenschmidt, Eve

2018-01-01

The ability to express oneself clearly in both a mother tongue and a foreign language is a foundation principle of the new national curriculum in Estonia. Therefore, research was conducted to determine whether there was a possible relationship between English as a foreign language (EFL) teachers' perceptions of effective teaching and the…

Effects of FLES on Reading Comprehension and Vocabulary Achievement: A Multi-Method Longitudinal Study

ERIC Educational Resources Information Center

Taylor, Gregory; Feyten, Carine; Meros, John; Nutta, Joyce

2008-01-01

Historically, school officials, parents, teachers and others have expressed concerns about the implementation of foreign language in the elementary school (FLES) programs believing that precious time was sacrificed from the rest of the curriculum to make room for foreign language study. Some wondered if second language instruction made sense for…

Morality and Foreign Policy. A Symposium on President Carter's Stance.

ERIC Educational Resources Information Center

Lefever, Ernest W., Ed.

This monograph contains a critical examination of President Carter's view on ethics and foreign policy as expressed in his commencement speech at Notre Dame University on May 22, 1977. The book is organized into three parts. Part 1 contains Mr. Carter's speech entitled, "Power for Humane Purposes." Part 2 contains nine responses to the…

A mathematical model for foreign body reactions in 2D.

PubMed

Su, Jianzhong; Gonzales, Humberto Perez; Todorov, Michail; Kojouharov, Hristo; Tang, Liping

2011-02-01

The foreign body reactions are commonly referred to the network of immune and inflammatory reactions of human or animals to foreign objects placed in tissues. They are basic biological processes, and are also highly relevant to bioengineering applications in implants, as fibrotic tissue formations surrounding medical implants have been found to substantially reduce the effectiveness of devices. Despite of intensive research on determining the mechanisms governing such complex responses, few mechanistic mathematical models have been developed to study such foreign body reactions. This study focuses on a kinetics-based predictive tool in order to analyze outcomes of multiple interactive complex reactions of various cells/proteins and biochemical processes and to understand transient behavior during the entire period (up to several months). A computational model in two spatial dimensions is constructed to investigate the time dynamics as well as spatial variation of foreign body reaction kinetics. The simulation results have been consistent with experimental data and the model can facilitate quantitative insights for study of foreign body reaction process in general.

Construction of heat-inducible expression vector of Corynebacterium glutamicum and C. ammoniagenes: fusion of lambda operator with promoters isolated from C. ammoniagenes.

PubMed

Park, Jong-Uk; Jo, Jae-Hyung; Kim, Young-Ji; Chung, So-Sun; Lee, Jin-Ho; Lee, Hyune Hwan

2008-04-01

The heat-inducible expression vectors for Corynebacterium glutamicum and C. ammoniagenes were constructed by using the lambdaOL1 and the cryptic promoters, CJ1 and CJ4 that express genes constitutively in C. ammoniagenes.. Although the promoters were isolated from C. ammoniagenes, CJ1 and CJ4 were also active in C. glutamicum. To construct vectors, the OL1 from the lambdaPL promoter was isolated and fused to the CJ1 and CJ4 promoters by recombinant PCR. The resulting artificial promoters, CJ1O and CJ4O, which have one lambdaOL1, and CJ1OX2, which has two successive lambdaOL1, were fused to the green fluorescent protein (GFP) gene followed by subcloning into pCES208. The expression of GFP in the corynebacteria harboring the vectors was regulated successfully by the temperature sensitive cI857 repressor. Among them, C. ammoniagenes harboring plasmid pCJ1OX2G containing GFP fused to CJ1OX2 showed more GFP than the other ones and the expression was tightly regulated by the repressor. To construct the generally applicable expression vector using the plasmid pCJ1OX2G, the His-tag, enterokinase (EK) moiety, and the MCS were inserted in front of the GFP gene. Using the vector, the expression of pyrR from C. glutamicum was tried by temperature shift-up. The results indicated that the constructed vectors (pCeHEMG) can be successfully used in the expression and regulation of foreign genes in corynebacteria.

The polarized distribution of poly(A+)-mRNA-induced functional ion channels in the Xenopus oocyte plasma membrane is prevented by anticytoskeletal drugs.

PubMed

Peter, A B; Schittny, J C; Niggli, V; Reuter, H; Sigel, E

1991-08-01

Foreign mRNA was expressed in Xenopus laevis oocytes. Newly expressed ion currents localized in defined plasma membrane areas were measured using the two-electrode voltage clamp technique in combination with a specially designed chamber, that exposed only part of the surface on the oocytes to channel agonists or inhibitors. Newly expressed currents were found to be unequally distributed in the surface membrane of the oocyte. This asymmetry was most pronounced during the early phase of expression, when channels could almost exclusively be detected in the animal hemisphere of the oocyte. 4 d after injection of the mRNA, or later, channels could be found at a threefold higher density at the animal than at the vegetal pole area. The pattern of distribution was observed to be similar with various ion channels expressed from crude tissue mRNA and from cRNAs coding for rat GABAA receptor channel subunits. Electron microscopical analysis revealed very similar microvilli patterns at both oocyte pole areas. Thus, the asymmetric current distribution is not due to asymmetric surface structure. Upon incubation during the expression period in either colchicine or cytochalasin D, the current density was found to be equal in both pole areas. The inactive control substance beta-lumicolchicine had no effect on the asymmetry of distribution. Colchicine was without effect on the amplitude of the expressed whole cell current. Our measurements reveal a pathway for plasma membrane protein expression endogenous to the Xenopus oocyte, that may contribute to the formation and maintenance of polarity of this highly organized cell.

Benefits and concerns associated with biotechnology-derived foods: can additional research reduce children health risks?

PubMed

Cantani, A

2009-01-01

The development of techniques devised for the genetic manipulation of foods poses new risks for children with food allergy (FA). The introduction of foreign allergenic proteins from different foods into previously tolerated foods may trigger allergic reactions, often complicating with anaphylactic shock in a subset of allergic babies. Children with FA, even if subjected to preventative diets, always challenge the risk of developing allergic manifestations after unintentional intake of a non tolerated food in restaurant settings, with relatives or schoolmates, etc, where product labelling is necessarily lacking. The introduction of potentially allergenic proteins into foods generally considered safe for allergic children can be done deliberately, by either substantially altering the food ingredients, or by genetic manipulation which change the composition or transfer allergens, or unintentionally by qualitycontrol failures, due to contaminations in the production process, or to genetic mismanipulation. There is a controversy between multinationals often favored by governments and consumer association resistance, thus an equidistant analysis poses some unprecedented impediments. The importance of FA and the potential of transgenic plants to bring food allergens into the food supply should not be disregarded. The expression in soybeans of a Brazil nut protein resulted in a food allergen ex-pressed in widely used infant formulas, so paving the way to an often reported multinational debacle. Genetic engineering poses innovative ethical and social concerns, as well as serious challenges to the environment, human health, animal welfare, and the future of agriculture. In this paper will be emphasized practical concepts more crucial for pediatricians.

Systemic spread of an RNA insect virus in plants expressing plant viral movement protein genes

PubMed Central

Dasgupta, Ranjit; Garcia, Bradley H.; Goodman, Robert M.

2001-01-01

Flock house virus (FHV), a single-stranded RNA insect virus, has previously been reported to cross the kingdom barrier and replicate in barley protoplasts and in inoculated leaves of several plant species [Selling, B. H., Allison, R. F. & Kaesberg, P. (1990) Proc. Natl. Acad. Sci. USA 87, 434–438]. There was no systemic movement of FHV in plants. We tested the ability of movement proteins (MPs) of plant viruses to provide movement functions and cause systemic spread of FHV in plants. We compared the growth of FHV in leaves of nontransgenic and transgenic plants expressing the MP of tobacco mosaic virus or red clover necrotic mosaic virus (RCNMV). Both MPs mobilized cell-to-cell and systemic movement of FHV in Nicotiana benthamiana plants. The yield of FHV was more than 100-fold higher in the inoculated leaves of transgenic plants than in the inoculated leaves of nontransgenic plants. In addition, FHV accumulated in the noninoculated upper leaves of both MP-transgenic plants. RCNMV MP was more efficient in mobilizing FHV to noninoculated upper leaves. We also report here that FHV replicates in inoculated leaves of six additional plant species: alfalfa, Arabidopsis, Brassica, cucumber, maize, and rice. Our results demonstrate that plant viral MPs cause cell-to-cell and long-distance movement of an animal virus in plants and offer approaches to the study of the evolution of viruses and mechanisms governing mRNA trafficking in plants as well as to the development of promising vectors for transient expression of foreign genes in plants. PMID:11296259

Herpes B Virus, Macacine Herpesvirus 1, Breaks Simplex Virus Tradition via Major Histocompatibility Complex Class I Expression in Cells from Human and Macaque Hosts

PubMed Central

Vasireddi, Mugdha

2012-01-01

B virus of the family Herpesviridae is endemic to rhesus macaques but results in 80% fatality in untreated humans who are zoonotically infected. Downregulation of major histocompatibility complex (MHC) class I in order to evade CD8+ T-cell activation is characteristic of most herpesviruses. Here we examined the cell surface presence and total protein expression of MHC class I molecules in B virus-infected human foreskin fibroblast cells and macaque kidney epithelial cells in culture, which are representative of foreign and natural host initial target cells of B virus. Our results show <20% downregulation of surface MHC class I molecules in either type of host cells infected with B virus, which is statistically insignificantly different from that observed in uninfected cells. We also examined the surface expression of MHC class Ib molecules, HLA-E and HLA-G, involved in NK cell inhibition. Our results showed significant upregulation of HLA-E and HLA-G in host cells infected with B virus relative to the amounts observed in other herpesvirus-infected cells. These results suggest that B virus-infected cell surfaces maintain normal levels of MHC class Ia molecules, a finding unique among simplex viruses. This is a unique divergence in immune evasion for B virus, which, unlike human simplex viruses, does not inhibit the transport of peptides for loading onto MHC class Ia molecules because B virus ICP47 lacks a transporter-associated protein binding domain. The fact that MHC class Ib molecules were significantly upregulated has additional implications for host-pathogen interactions. PMID:22973043

The modulation of extracellular superoxide dismutase in the specifically enhanced cellular immune response against secondary challenge of Vibrio splendidus in Pacific oyster (Crassostrea gigas).

PubMed

Liu, Conghui; Zhang, Tao; Wang, Lingling; Wang, Mengqiang; Wang, Weilin; Jia, Zhihao; Jiang, Shuai; Song, Linsheng

2016-10-01

Extracellular superoxide dismutase (EcSOD) is a copper-containing glycoprotein playing an important role in antioxidant defense of living cells exposed to oxidative stress, and also participating in microorganism internalization and cell adhesion in invertebrates. EcSOD from oyster (designated CgEcSOD) had been previously reported to bind lipopolysaccharides (LPS) and act as a bridge molecule in Vibrio splendidus internalization. Its mRNA expression pattern, PAMP binding spectrum and microorganism binding capability were examined in the present study. The mRNA expression of CgEcSOD in hemocytes was significantly up-regulated at the initial phase and decreased sharply at 48 h post V. splendidus stimulation. The recombinant CgEcSOD protein (rCgEcSOD) could bind LPS, PGN and poly (I:C), as well as various microorganisms including Micrococcus luteus, Staphylococcus aureus, Escherichia coli, Vibrio anguillarum, V. splendidus, Pastoris pastoris and Yarrowia lipolytica at the presence of divalent metal ions Cu(2+). After the secondary V. splendidus stimulation, the mRNA and protein of CgEcSOD were both down-regulated significantly. The results collectively indicated that CgEcSOD could not only function in the immune recognition, but also might contribute to the immune priming of oyster by inhibiting the foreign microbe invasion through a specific down-regulation. Copyright © 2016 Elsevier Ltd. All rights reserved.

"Techniques d'expression,""approche communicative," meme combat? ("Expressive Techniques,""Communicative Approach," Same Struggle?)

ERIC Educational Resources Information Center

Vives, Robert

1983-01-01

Based on a literature review and analysis of teaching methods and objectives, it is proposed that the emphasis on communicative competence ascendant in French foreign language instruction is closely related to, and borrows from, expressive techniques taught in French native language instruction in the 1960s. (MSE)

Ofrendas/BorderCrossings: A Studio-Based Model for Cross Cultural Traveling.

ERIC Educational Resources Information Center

Congdon, Kristin G.

While creating an art form which is foreign to an artist's cultural expression may seem like taking a risk in today's world, it may provide a way to successfully negotiate a fulfilling living and working space on this diverse planet. Movement into a foreign space, often referred to as a border crossing, can be understood as a central meeting…

Fimbrial subunit protein FaeG expressed in transgenic tobacco inhibits the binding of F4ac enterotoxigenic Escherichia coli to porcine enterocytes.

PubMed

Joensuu, Jussi J; Kotiaho, Mirkka; Riipi, Tero; Snoeck, Veerle; Palva, E Tapio; Teeri, Teemu H; Lång, Hannu; Cox, Eric; Goddeeris, Bruno M; Niklander-Teeri, Viola

2004-06-01

Plants offer a promising alternative for the production of foreign proteins for pharmaceutical purposes in tissues that are consumed as food and/or feed. Our long-term strategy is to develop edible vaccines against piglet diarrhoea caused by enterotoxigenic Escherichia coli (F4 ETEC) in feed plants. In this work, we isolated a gene, faeG, encoding for a major F4ac fimbrial subunit protein. Our goal was to test whether the FaeG protein, when isolated from its fimbrial background and produced in a plant cell, would retain the key properties of an oral vaccine, that is, stability in gastrointestinal conditions, binding to intestinal receptors and inhibition of the F4 ETEC attachment. For this purpose, tobacco was first transformed with a faeG construct that included a transit peptide encoding sequence to target the FaeG protein to the chloroplast. The best transgenic lines produced FaeG protein in amounts of 1% total soluble protein. The stability of the plant-produced FaeG was tested in fluids simulating piglet gastric (SGF) and intestinal (SIF) conditions. Plant-produced FaeG proved to be stable up to 2 h under these conditions. The binding and inhibition properties were tested with isolated piglet villi. These results showed that the plant-produced FaeG could bind to the receptors on the villi and subsequently inhibit F4 ETEC binding in a dose-dependent manner. Thus, the first two prerequisites for the development of an oral vaccine have been met.

Epigenetics of Peripheral B-Cell Differentiation and the Antibody Response

PubMed Central

Zan, Hong; Casali, Paolo

2015-01-01

Epigenetic modifications, such as histone post-translational modifications, DNA methylation, and alteration of gene expression by non-coding RNAs, including microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), are heritable changes that are independent from the genomic DNA sequence. These regulate gene activities and, therefore, cellular functions. Epigenetic modifications act in concert with transcription factors and play critical roles in B cell development and differentiation, thereby modulating antibody responses to foreign- and self-antigens. Upon antigen encounter by mature B cells in the periphery, alterations of these lymphocytes epigenetic landscape are induced by the same stimuli that drive the antibody response. Such alterations instruct B cells to undergo immunoglobulin (Ig) class switch DNA recombination (CSR) and somatic hypermutation (SHM), as well as differentiation to memory B cells or long-lived plasma cells for the immune memory. Inducible histone modifications, together with DNA methylation and miRNAs modulate the transcriptome, particularly the expression of activation-induced cytidine deaminase, which is essential for CSR and SHM, and factors central to plasma cell differentiation, such as B lymphocyte-induced maturation protein-1. These inducible B cell-intrinsic epigenetic marks guide the maturation of antibody responses. Combinatorial histone modifications also function as histone codes to target CSR and, possibly, SHM machinery to the Ig loci by recruiting specific adaptors that can stabilize CSR/SHM factors. In addition, lncRNAs, such as recently reported lncRNA-CSR and an lncRNA generated through transcription of the S region that form G-quadruplex structures, are also important for CSR targeting. Epigenetic dysregulation in B cells, including the aberrant expression of non-coding RNAs and alterations of histone modifications and DNA methylation, can result in aberrant antibody responses to foreign antigens, such as those on microbial pathogens, and generation of pathogenic autoantibodies, IgE in allergic reactions, as well as B cell neoplasia. Epigenetic marks would be attractive targets for new therapeutics for autoimmune and allergic diseases, and B cell malignancies. PMID:26697022

A Herpes Simplex Virus Type 1 Mutant Expressing a Baculovirus Inhibitor of Apoptosis Gene in Place of Latency-Associated Transcript Has a Wild-Type Reactivation Phenotype in the Mouse

PubMed Central

Jin, Ling; Perng, Guey-Chuen; Mott, Kevin R.; Osorio, Nelson; Naito, Julia; Brick, David J.; Carpenter, Dale; Jones, Clinton; Wechsler, Steven L.

2005-01-01

The latency-associated transcript (LAT) is essential for the wild-type herpes simplex virus type 1 (HSV-1) high-reactivation phenotype since LAT− mutants have a low-reactivation phenotype. We previously reported that LAT can decrease apoptosis and proposed that this activity is involved in LAT's ability to enhance the HSV-1 reactivation phenotype. The first 20% of the primary 8.3-kb LAT transcript is sufficient for enhancing the reactivation phenotype and for decreasing apoptosis, supporting this proposal. For this study, we constructed an HSV-1 LAT− mutant that expresses the baculovirus antiapoptosis gene product cpIAP under control of the LAT promoter and in place of the LAT region mentioned above. Mice were ocularly infected with this mutant, designated dLAT-cpIAP, and the reactivation phenotype was determined using the trigeminal ganglion explant model. dLAT-cpIAP had a reactivation phenotype similar to that of wild-type virus and significantly higher than that of (i) the LAT− mutant dLAT2903; (ii) dLAT1.5, a control virus containing the same LAT deletion as dLAT-cpIAP, but with no insertion of foreign DNA, thereby controlling for potential readthrough transcription past the cpIAP insert; and (iii) dLAT-EGFP, a control virus identical to dLAT-cpIAP except that it contained the enhanced green fluorescent protein open reading frame (ORF) in place of the cpIAP ORF, thereby controlling for expression of a random foreign gene instead of the cpIAP gene. These results show that an antiapoptosis gene with no sequence similarity to LAT can efficiently substitute for the LAT function involved in enhancing the in vitro-induced HSV-1 reactivation phenotype in the mouse. PMID:16160155

STATs and macrophage fusion.

PubMed

Miyamoto, Takeshi

2013-07-01

Macrophages play a pivotal role in host defense against multiple foreign materials such as bacteria, parasites and artificial devices. Some macrophage lineage cells, namely osteoclasts and foreign body giant cells (FBGCs), form multi-nuclear giant cells by the cell-cell fusion of mono-nuclear cells. Osteoclasts are bone-resorbing cells, and are formed in the presence of RANKL on the surface of bones, while FBGCs are formed in the presence of IL-4 or IL-13 on foreign materials such as artificial joints, catheters and parasites. Recently, fusiogenic mechanisms and the molecules required for the cell-cell fusion of these macrophage lineage cells were, at least in part, clarified. Dendritic cell specific transmembrane protein (DC-STAMP) and osteoclast stimulatory transmembrane protein (OC-STAMP), both of which comprise seven transmembrane domains, are required for both osteoclast and FBGC cell-cell fusion. STAT6 was demonstrated to be required for the cell-cell fusion of FBGCs but not osteoclasts. In this review, advances in macrophage cell-cell fusion are discussed.

Diatom centromeres suggest a mechanism for nuclear DNA acquisition

DOE PAGES

Diner, Rachel E.; Noddings, Chari M.; Lian, Nathan C.; ...

2017-07-18

Centromeres are essential for cell division and growth in all eukaryotes, and knowledge of their sequence and structure guides the development of artificial chromosomes for functional cellular biology studies. Centromeric proteins are conserved among eukaryotes; however, centromeric DNA sequences are highly variable. We combined forward and reverse genetic approaches with chromatin immunoprecipitation to identify centromeres of the model diatom Phaeodactylum tricornutum. We observed 25 unique centromere sequences typically occurring once per chromosome, a finding that helps to resolve nuclear genome organization and indicates monocentric regional centromeres. Diatom centromere sequences contain low-GC content regions but lack repeats or other conserved sequencemore » features. Native and foreign sequences with similar GC content to P. tricornutum centromeres can maintain episomes and recruit the diatom centromeric histone protein CENH3, suggesting nonnative sequences can also function as diatom centromeres. Thus, simple sequence requirements may enable DNA from foreign sources to persist in the nucleus as extrachromosomal episomes, revealing a potential mechanism for organellar and foreign DNA acquisition.« less

Diatom centromeres suggest a mechanism for nuclear DNA acquisition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Diner, Rachel E.; Noddings, Chari M.; Lian, Nathan C.

Centromeres are essential for cell division and growth in all eukaryotes, and knowledge of their sequence and structure guides the development of artificial chromosomes for functional cellular biology studies. Centromeric proteins are conserved among eukaryotes; however, centromeric DNA sequences are highly variable. We combined forward and reverse genetic approaches with chromatin immunoprecipitation to identify centromeres of the model diatom Phaeodactylum tricornutum. We observed 25 unique centromere sequences typically occurring once per chromosome, a finding that helps to resolve nuclear genome organization and indicates monocentric regional centromeres. Diatom centromere sequences contain low-GC content regions but lack repeats or other conserved sequencemore » features. Native and foreign sequences with similar GC content to P. tricornutum centromeres can maintain episomes and recruit the diatom centromeric histone protein CENH3, suggesting nonnative sequences can also function as diatom centromeres. Thus, simple sequence requirements may enable DNA from foreign sources to persist in the nucleus as extrachromosomal episomes, revealing a potential mechanism for organellar and foreign DNA acquisition.« less

Genetics Home Reference: Graves disease

MedlinePlus

... risk factors for Graves disease . Some of these genes are part of a family called the human leukocyte antigen (HLA) complex . The HLA complex helps the immune system distinguish the body's own proteins from proteins made by foreign ... Other genes that have been associated with Graves disease help ...

Comparison of sensory perception and verbal expression of Korean Kimchi stews between Chinese consumers and Korean trained panelists.

PubMed

Park, Hye-Won; Kim, Young Choul; Jang, Seong-Ho; Hong, Jae-Hee

2018-05-25

Sensory drivers of liking in foreign food markets are difficult to identify because expression of perceived characteristics varies depending on cultural differences. We aimed to identify differences in perception and expression of a Korean home meal replacement product (Kimchi stew) between 10 Korean trained panelists and 50 eastern Chinese consumers (EC) and 54 northern Chinese consumers (NC) using descriptive analysis and rate-all-that-apply (RATA) tests. Regional differences between EC and NC groups were also investigated. Sensory representations of the six Kimchi stew samples were similar between the Korean trained and Chinese consumer panels. Use of simple sensory RATA terms was similar among the groups. However, EC whose daily diet has mild flavors associated consumer terms with negative connotations, such as odd flavor and sharp, with burning sensation and seasoning, implying the influence of regional food cultures. RATA could elicit foreign consumers' sensory representations of an unfamiliar ethnic food that was comparable to that from descriptive analysis, assisting researchers in understanding target consumers' sensory perceptions in a more cost- and time-effective manner. Inclusion of consumer terms in a RATA list and its correlation with descriptive analysis by a native descriptive panel can help in understanding foreign consumers' verbal expressions This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

A recombinant Bacillus anthracis strain producing the Clostridium perfringens Ib component induces protection against iota toxins.

PubMed

Sirard, J C; Weber, M; Duflot, E; Popoff, M R; Mock, M

1997-06-01

The Bacillus anthracis toxinogenic Sterne strain is currently used as a live veterinary vaccine against anthrax. The capacity of a toxin-deficient derivative strain to produce a heterologous antigen by using the strong inducible promoter of the B. anthracis pag gene was investigated. The expression of the foreign gene ibp, encoding the Ib component of iota toxin from Clostridium perfringens, was analyzed. A pag-ibp fusion was introduced by allelic exchange into a toxin-deficient Sterne strain, thereby replacing the wild-type pag gene. This recombinant strain, called BAIB, was stable and secreted large quantities of Ib protein in induced culture conditions. Mice given injections of live BAIB spores developed an antibody response specific to the Ib protein. The pag-ibp fusion was therefore functional both in vitro and in vivo. Moreover, the immunized animals were protected against a challenge with C. perfringens iota toxin or with the homologous Clostridium spiroforme toxin. The protective immunity was mediated by neutralizing antibodies. In conclusion, B. anthracis is promising for the development of live veterinary vaccines.

A recombinant Bacillus anthracis strain producing the Clostridium perfringens Ib component induces protection against iota toxins.

PubMed Central

Sirard, J C; Weber, M; Duflot, E; Popoff, M R; Mock, M

1997-01-01

The Bacillus anthracis toxinogenic Sterne strain is currently used as a live veterinary vaccine against anthrax. The capacity of a toxin-deficient derivative strain to produce a heterologous antigen by using the strong inducible promoter of the B. anthracis pag gene was investigated. The expression of the foreign gene ibp, encoding the Ib component of iota toxin from Clostridium perfringens, was analyzed. A pag-ibp fusion was introduced by allelic exchange into a toxin-deficient Sterne strain, thereby replacing the wild-type pag gene. This recombinant strain, called BAIB, was stable and secreted large quantities of Ib protein in induced culture conditions. Mice given injections of live BAIB spores developed an antibody response specific to the Ib protein. The pag-ibp fusion was therefore functional both in vitro and in vivo. Moreover, the immunized animals were protected against a challenge with C. perfringens iota toxin or with the homologous Clostridium spiroforme toxin. The protective immunity was mediated by neutralizing antibodies. In conclusion, B. anthracis is promising for the development of live veterinary vaccines. PMID:9169728

Generation of a Recombinant Akabane Virus Expressing Enhanced Green Fluorescent Protein

PubMed Central

Takenaka-Uema, Akiko; Murata, Yousuke; Gen, Fumihiro; Ishihara-Saeki, Yukari; Watanabe, Ken-ichi; Uchida, Kazuyuki; Kato, Kentaro; Murakami, Shin; Haga, Takeshi

2015-01-01

ABSTRACT We generated a recombinant Akabane virus (AKAV) expressing enhanced green fluorescence protein (eGFP-AKAV) by using reverse genetics. We artificially constructed an ambisense AKAV S genome encoding N/NSs on the negative-sense strand, and eGFP on the positive-sense strand with an intergenic region (IGR) derived from the Rift Valley fever virus (RVFV) S genome. The recombinant virus exhibited eGFP fluorescence and had a cytopathic effect in cell cultures, even after several passages. These results indicate that the gene encoding eGFP in the ambisense RNA could be stably maintained. Transcription of N/NSs and eGFP mRNAs of eGFP-AKAV was terminated within the IGR. The mechanism responsible for this appears to be different from that in RVFV, where the termination sites for N and NSs are determined by a defined signal sequence. We inoculated suckling mice intraperitoneally with eGFP-AKAV, which resulted in neurological signs and lethality equivalent to those seen for the parent AKAV. Fluorescence from eGFP in frozen brain slices from the eGFP-AKAV-infected mice was localized to the cerebellum, pons, and medulla oblongata. Our approach to producing a fluorescent virus, using an ambisense genome, helped obtain eGFP-AKAV, a fluorescent bunyavirus whose viral genes are intact and which can be easily visualized. IMPORTANCE AKAV is the etiological agent of arthrogryposis-hydranencephaly syndrome in ruminants, which causes considerable economic loss to the livestock industry. We successfully generated a recombinant enhanced green fluorescent protein-tagged AKAV containing an artificial ambisense S genome. This virus could become a useful tool for analyzing AKAV pathogenesis in host animals. In addition, our approach of using an ambisense genome to generate an orthobunyavirus stably expressing a foreign gene could contribute to establishing alternative vaccine strategies, such as bivalent vaccine virus constructs, for veterinary use against infectious diseases. PMID:26157127

Antigen specific suppression of humoral immunity by anergic Ars/A1 B cells1

PubMed Central

Aviszus, Katja; MacLeod, Megan K.L.; Kirchenbaum, Greg A.; Detanico, Thiago O.; Heiser, Ryan A.; St. Clair, James B.; Guo, Wenzhong; Wysocki, Lawrence J.

2012-01-01

Autoreactive anergic B lymphocytes are considered to be dangerous because of their potential for activation and recruitment into autoimmune responses. Yet they persist for days and constitute ~5% of the B cell pool. We assessed their functional potential in the Ars/A1 transgene model, where anergic B cells express a dual-reactive antigen receptor that binds, in addition to a self-antigen, the hapten p-azophenylarsonate (Ars). When Ars/A1 B cells were transferred into adoptive recipients that were immunized with foreign proteins covalently conjugated with Ars, endogenous IgG immune responses to both were selectively and severely diminished, and the development of T helper cells was impaired. Approximately 95% inhibition of the anti-Ars response was attained with ~4000 transferred Ars/A1 B cells through redundant mechanisms, one of which depended upon their expression of MHC II but not upon secretion of IL-10 or IgM. This antigen-specific suppressive activity implicates the autoreactive anergic B cell as an enforcer of immunological tolerance to self-antigens. PMID:23008448

Antigen-specific suppression of humoral immunity by anergic Ars/A1 B cells.

PubMed

Aviszus, Katja; Macleod, Megan K L; Kirchenbaum, Greg A; Detanico, Thiago O; Heiser, Ryan A; St Clair, James B; Guo, Wenzhong; Wysocki, Lawrence J

2012-11-01

Autoreactive anergic B lymphocytes are considered to be dangerous because of their potential for activation and recruitment into autoimmune responses. However, they persist for days and constitute ∼5% of the B cell pool. We assessed their functional potential in the Ars/A1 transgene model, where anergic B cells express a dual-reactive Ag receptor that binds, in addition to a self-Ag, the hapten p-azophenylarsonate (Ars). When Ars/A1 B cells were transferred into adoptive recipients that were immunized with foreign proteins covalently conjugated with Ars, endogenous IgG immune responses to both were selectively and severely diminished, and the development of T helper cells was impaired. Approximately 95% inhibition of the anti-Ars response was attained with ∼4000 transferred Ars/A1 B cells through redundant mechanisms, one of which depended on their expression of MHC class II but not upon secretion of IL-10 or IgM. This Ag-specific suppressive activity implicates the autoreactive anergic B cell as an enforcer of immunological tolerance to self-Ags.

Function of the CRISPR-Cas System of the Human Pathogen Clostridium difficile

PubMed Central

Boudry, Pierre; Semenova, Ekaterina; Monot, Marc; Datsenko, Kirill A.; Lopatina, Anna; Sekulovic, Ognjen; Ospina-Bedoya, Maicol; Fortier, Louis-Charles; Severinov, Konstantin; Dupuy, Bruno

2015-01-01

ABSTRACT Clostridium difficile is the cause of most frequently occurring nosocomial diarrhea worldwide. As an enteropathogen, C. difficile must be exposed to multiple exogenous genetic elements in bacteriophage-rich gut communities. CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems allow bacteria to adapt to foreign genetic invaders. Our recent data revealed active expression and processing of CRISPR RNAs from multiple type I-B CRISPR arrays in C. difficile reference strain 630. Here, we demonstrate active expression of CRISPR arrays in strain R20291, an epidemic C. difficile strain. Through genome sequencing and host range analysis of several new C. difficile phages and plasmid conjugation experiments, we provide evidence of defensive function of the CRISPR-Cas system in both C. difficile strains. We further demonstrate that C. difficile Cas proteins are capable of interference in a heterologous host, Escherichia coli. These data set the stage for mechanistic and physiological analyses of CRISPR-Cas-mediated interactions of important global human pathogen with its genetic parasites. PMID:26330515

Xenobiotic metabolizing enzyme (XME) expression in aging humans.

EPA Science Inventory

In the presence of foreign compounds, metabolic homeostasis of the organism is maintained by the liver’s ability to detoxify and eliminate these xenobiotics. This is accomplished, in part, by the expression of XMEs, which metabolize xenobiotics and determine whether exposure will...

Genetics Home Reference: celiac disease

MedlinePlus

... HLA-DQB1 genes belong to a family of genes called the human leukocyte antigen (HLA) complex . The HLA complex helps the immune system distinguish the body's own proteins from proteins made by foreign invaders such as ... and HLA-DQB1 genes attach (bind) to each other to form a ...

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

PubMed Central

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 results suggest that various constraints on high-expression proteins reduce the availability of beneficial expression variants relative to low-expression proteins, enabling low-expression proteins to evolve and potentially lead to more rapid adaptation. PMID:26546003

Lentiviral Protein Transfer Vectors Are an Efficient Vaccine Platform and Induce a Strong Antigen-Specific Cytotoxic T Cell Response

PubMed Central

Uhlig, Katharina M.; Schülke, Stefan; Scheuplein, Vivian A. M.; Malczyk, Anna H.; Reusch, Johannes; Kugelmann, Stefanie; Muth, Anke; Koch, Vivian; Hutzler, Stefan; Bodmer, Bianca S.; Schambach, Axel; Buchholz, Christian J.; Waibler, Zoe; Scheurer, Stephan

2015-01-01

ABSTRACT To induce and trigger innate and adaptive immune responses, antigen-presenting cells (APCs) take up and process antigens. Retroviral particles are capable of transferring not only genetic information but also foreign cargo proteins when they are genetically fused to viral structural proteins. Here, we demonstrate the capacity of lentiviral protein transfer vectors (PTVs) for targeted antigen transfer directly into APCs and thereby induction of cytotoxic T cell responses. Targeting of lentiviral PTVs to APCs can be achieved analogously to gene transfer vectors by pseudotyping the particles with truncated wild-type measles virus (MV) glycoproteins (GPs), which use human SLAM (signaling lymphocyte activation molecule) as a main entry receptor. SLAM is expressed on stimulated lymphocytes and APCs, including dendritic cells. SLAM-targeted PTVs transferred the reporter protein green fluorescent protein (GFP) or Cre recombinase with strict receptor specificity into SLAM-expressing CHO and B cell lines, in contrast to broadly transducing vesicular stomatitis virus G protein (VSV-G) pseudotyped PTVs. Primary myeloid dendritic cells (mDCs) incubated with targeted or nontargeted ovalbumin (Ova)-transferring PTVs stimulated Ova-specific T lymphocytes, especially CD8+ T cells. Administration of Ova-PTVs into SLAM-transgenic and control mice confirmed the observed predominant induction of antigen-specific CD8+ T cells and demonstrated the capacity of protein transfer vectors as suitable vaccines for the induction of antigen-specific immune responses. IMPORTANCE This study demonstrates the specificity and efficacy of antigen transfer by SLAM-targeted and nontargeted lentiviral protein transfer vectors into antigen-presenting cells to trigger antigen-specific immune responses in vitro and in vivo. The observed predominant activation of antigen-specific CD8+ T cells indicates the suitability of SLAM-targeted and also nontargeted PTVs as a vaccine for the induction of cytotoxic immune responses. Since cytotoxic CD8+ T lymphocytes are a mainstay of antitumoral immune responses, PTVs could be engineered for the transfer of specific tumor antigens provoking tailored antitumoral immunity. Therefore, PTVs can be used as safe and efficient alternatives to gene transfer vectors or live attenuated replicating vector platforms, avoiding genotoxicity or general toxicity in highly immunocompromised patients, respectively. Thereby, the potential for easy envelope exchange allows the circumventing of neutralizing antibodies, e.g., during repeated boost immunizations. PMID:26085166

[Immunoreactivity of chimeric proteins carrying poliovirus epitopes on the VP6 of rotavirus as a vector].

PubMed

Pan, X-X; Zhao, B-X; Teng, Y-M; Xia, W-Y; Wang, J; Li, X-F; Liao, G-Y; Yang, С; Chen, Y-D

2016-01-01

Rotavirus and poliovirus continue to present significant risks and burden of disease to children in developing countries. Developing a combined vaccine may effectively prevent both illnesses and may be advantageous in terms of maximizing compliance and vaccine coverage at the same visit. Recently, we sought to generate a vaccine vector by incorporating multiple epitopes into the rotavirus group antigenic protein, VP6. In the present study, a foreign epitope presenting a system using VP6 as a vector was created with six sites on the outer surface of the vector that could be used for insertion of foreign epitopes, and three VP6-based PV1 epitope chimeric proteins were constructed. The chimeric proteins were confirmed by immunoblot, immunofluorescence assay, and injected into guinea pigs to analyze the epitope-specific humoral response. Results showed that these chimeric proteins reacted with anti-VP6F and -PV1 antibodies, and elicited antibodies against both proteins in guinea pigs. Antibodies against the chimeric proteins carrying PV1 epitopes neutralized rotavirus Wa and PV1 infection in vitro. Our study contributes to a better understanding of the use of VP6-based vectors as multiple-epitope delivery vehicles and the epitopes displayed in this form could be considered for development of epitope-based vaccines against rotavirus and poliovirus.

Protein deficiency lowers resistance of Mormon crickets to the pathogenic fungus Beauveria bassiana

USDA-ARS?s Scientific Manuscript database

Little is known about the effects of dietary macronutrients on the capacity of insects to ward off a fungal pathogen. Here we tested the hypothesis that Mormon crickets fed restricted protein diets have lower enzymatic assays of generalized immunity, slower rates of encapsulation of foreign bodies,...

Transgenic cotton over-producing spinach sucrose phosphate synthase showed enhanced leaf sucrose synthesis and improved fiber quality under controlled environmental conditions.

PubMed

Haigler, Candace H; Singh, Bir; Zhang, Deshui; Hwang, Sangjoon; Wu, Chunfa; Cai, Wendy X; Hozain, Mohamed; Kang, Wonhee; Kiedaisch, Brett; Strauss, Richard E; Hequet, Eric F; Wyatt, Bobby G; Jividen, Gay M; Holaday, A Scott

2007-04-01

Prior data indicated that enhanced availability of sucrose, a major product of photosynthesis in source leaves and the carbon source for secondary wall cellulose synthesis in fiber sinks, might improve fiber quality under abiotic stress conditions. To test this hypothesis, a family of transgenic cotton plants (Gossypium hirsutum cv. Coker 312 elite) was produced that over-expressed spinach sucrose-phosphate synthase (SPS) because of its role in regulation of sucrose synthesis in photosynthetic and heterotrophic tissues. A family of 12 independent transgenic lines was characterized in terms of foreign gene insertion, expression of spinach SPS, production of spinach SPS protein, and development of enhanced extractable V (max) SPS activity in leaf and fiber. Lines with the highest V (max) SPS activity were further characterized in terms of carbon partitioning and fiber quality compared to wild-type and transgenic null controls. Leaves of transgenic SPS over-expressing lines showed higher sucrose:starch ratio and partitioning of (14)C to sucrose in preference to starch. In two growth chamber experiments with cool nights, ambient CO(2) concentration, and limited light below the canopy, the transgenic line with the highest SPS activity in leaf and fiber had higher fiber micronaire and maturity ratio associated with greater thickness of the cellulosic secondary wall.

Design strategies to address the effect of hydrophobic epitope on stability and in vitro assembly of modular virus-like particle.

PubMed

Tekewe, Alemu; Connors, Natalie K; Middelberg, Anton P J; Lua, Linda H L

2016-08-01

Virus-like particles (VLPs) and capsomere subunits have shown promising potential as safe and effective vaccine candidates. They can serve as platforms for the display of foreign epitopes on their surfaces in a modular architecture. Depending on the physicochemical properties of the antigenic modules, modularization may affect the expression, solubility and stability of capsomeres, and VLP assembly. In this study, three module designs of a rotavirus hydrophobic peptide (RV10) were synthesized using synthetic biology. Among the three synthetic modules, modularization of the murine polyomavirus VP1 with a single copy of RV10 flanked by long linkers and charged residues resulted in the expression of stable modular capsomeres. Further employing the approach of module titration of RV10 modules on each capsomere via Escherichia coli co-expression of unmodified VP1 and modular VP1-RV10 successfully translated purified modular capomeres into modular VLPs when assembled in vitro. Our results demonstrate that tailoring the physicochemical properties of modules to enhance modular capsomeres stability is achievable through synthetic biology designs. Combined with module titration strategy to avoid steric hindrance to intercapsomere interactions, this allows bioprocessing of bacterially produced in vitro assembled modular VLPs. © 2016 The Protein Society.

A novel helper phage enabling construction of genome-scale ORF-enriched phage display libraries.

PubMed

Gupta, Amita; Shrivastava, Nimisha; Grover, Payal; Singh, Ajay; Mathur, Kapil; Verma, Vaishali; Kaur, Charanpreet; Chaudhary, Vijay K

2013-01-01

Phagemid-based expression of cloned genes fused to the gIIIP coding sequence and rescue using helper phages, such as VCSM13, has been used extensively for constructing large antibody phage display libraries. However, for randomly primed cDNA and gene fragment libraries, this system encounters reading frame problems wherein only one of 18 phages display the translated foreign peptide/protein fused to phagemid-encoded gIIIP. The elimination of phages carrying out-of-frame inserts is vital in order to improve the quality of phage display libraries. In this study, we designed a novel helper phage, AGM13, which carries trypsin-sensitive sites within the linker regions of gIIIP. This renders the phage highly sensitive to trypsin digestion, which abolishes its infectivity. For open reading frame (ORF) selection, the phagemid-borne phages are rescued using AGM13, so that clones with in-frame inserts express fusion proteins with phagemid-encoded trypsin-resistant gIIIP, which becomes incorporated into the phages along with a few copies of AGM13-encoded trypsin-sensitive gIIIP. In contrast, clones with out-of-frame inserts produce phages carrying only AGM13-encoded trypsin-sensitive gIIIP. Trypsin treatment of the phage population renders the phages with out-of-frame inserts non-infectious, whereas phages carrying in-frame inserts remain fully infectious and can hence be enriched by infection. This strategy was applied efficiently at a genome scale to generate an ORF-enriched whole genome fragment library from Mycobacterium tuberculosis, in which nearly 100% of the clones carried in-frame inserts after selection. The ORF-enriched libraries were successfully used for identification of linear and conformational epitopes for monoclonal antibodies specific to mycobacterial proteins.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Su, Qiudong; Yi, Yao; Guo, Minzhuo

Highlights: •The conformational HBV neutralization antigen domain was successfully displayed on the surface of truncated HBc particles. •Appropriate dialysis procedures to support the renaturing environment for the protein refolding. •Efficient purification procedures to obtain high purity and icosahedral particles of mosaic HBV antigen. •Strong immune responses not only including neutralization antibody response but also Th1 cell response were induced in mice. -- Abstract: Hepatitis B capsid protein expressed in Escherichia coli can reassemble into icosahedral particles, which could strongly enhance the immunogenicity of foreign epitopes, especially those inserted into its major immunodominant region. Herein, we inserted the entire ‘α’ antigenicmore » determinant amino acids (aa) 119–152 of HBsAg into the truncated HBc (aa 1–144), between Asp{sup 78} and Pro{sup 79}. Prokaryotic expression showed that the mosaic HBc was mainly in the form of inclusion bodies. After denaturation with urea, it was dialyzed progressively for protein renaturation. We observed that before and after renaturation, mosaic HBc was antigenic as determined by HBsAg ELISA and a lot of viruslike particles were observed after renaturation. Thus, we further purified the mosaic viruslike particles by (NH{sub 4}){sub 2}SO{sub 4} precipitation, DEAE chromatography, and Sepharose 4FF chromatography. Negative staining electron microscopy demonstrated the morphology of the viruslike particles. Immunization of Balb/c mice with mosaic particles induced the production of anti-HBs antibody and Th1 cell immune response supported by ELISPOT and CD4/CD8 proportions assay. In conclusion, we constructed mosaic hepatitis core particles displaying the entire ‘α’ antigenic determinant on the surface and laid a foundation for researching therapeutic hepatits B vaccines.« less

Chimeric classical swine fever (CSF)-Japanese encephalitis (JE) viral replicon as a non-transmissible vaccine candidate against CSF and JE infections.

PubMed

Yang, Zhenhua; Wu, Rui; Li, Robert W; Li, Ling; Xiong, Zhongliang; Zhao, Haizhong; Guo, Deyin; Pan, Zishu

2012-04-01

A trans-complemented chimeric CSF-JE virus replicon was constructed using an infectious cDNA clone of the CSF virus (CSFV) Alfort/187 strain. The CSFV E2 gene was deleted, and a fragment containing the region encoding a truncated envelope protein (tE, amino acid 292-402, domain III) of JE virus (JEV) was inserted into the resultant plasmid, pA187delE2, to generate the recombinant cDNA clone pA187delE2/JEV-tE. Porcine kidney 15 (PK15) cells that constitutively express the CSFV E2p7 proteins were then transfected with in vitro-transcribed RNA from pA187delE2/JEV-tE. As a result, the chimeric CSF-JE virus replicon particle (VRP), rv187delE2/JEV-tE, was rescued. In a mouse model, immunization with the chimeric CSF-JE VRP induced strong production of JEV-specific antibody and conferred protection against a lethal JEV challenge. Pigs immunized with CSF-JE VRP displayed strong anti-CSFV and anti-JEV antibody responses and protection against CSFV and JEV challenge infections. Our evidence suggests that E2-complemented CSF-JE VRP not only has potential as a live-attenuated non-transmissible vaccine candidate against CSF and JE but also serves as a potential DIVA (Differentiating Infected from Vaccinated Animals) vaccine for CSF in pigs. Together, our data suggest that the non-transmissible chimeric VRP expressing foreign antigenic proteins may represent a promising strategy for bivalent DIVA vaccine design. Copyright © 2012 Elsevier B.V. All rights reserved.

A modified acetylcholine receptor δ-subunit enables a null mutant to survive beyond sexual maturation

PubMed Central

Epley, Kimberly E.; Urban, Jason M.; Ikenaga, Takanori; Ono, Fumihito

2008-01-01

The contraction of skeletal muscle is dependent upon synaptic transmission through acetylcholine receptors (AChRs) at the neuromuscular junction (NMJ). The lack of an AChR subunit causes a fetal akinesia in humans, leading to death in the first trimester and characteristic features of Fetal Akinesia Deformation Sequences (FADS). A corresponding null mutation of the δ-subunit in zebrafish (sofa potato; sop−/−) leads to the death of embryos around 5 days post-fertilization (dpf). In sop−/− mutants, we expressed modified δ-subunits, with one (δ1YFP) or two yellow fluorescent protein (δ2YFP) molecules fused at the intracellular loop, under the control of an α-actin promoter. AChRs containing these fusion proteins are fluorescent, assemble on the plasma membrane, make clusters under motor neuron endings, and generate synaptic current. We screened for germ-line transmission of the transgene and established a line of sop−/− fish stably expressing the δ2YFP. These δ2YFP/sop−/− embryos can mount escape behavior close to that of their wild type siblings. Synaptic currents in these embryos had a smaller amplitude, slower rise time, and slower decay when compared to wild type fish. Remarkably, these embryos grow to adulthood and display complex behaviors such as feeding and breeding. To the best of our knowledge, this is the first case of a mutant animal corresponding to first trimester lethality in human that has been rescued by a transgene and survived to adulthood. In the rescued fish, a foreign promoter drove the transgene expression and the NMJ had altered synaptic strength. The survival of the transgenic animal delineates requirements for gene therapies of NMJ. PMID:19052214

Hormone-induced modifications of the chromatin structure surrounding upstream regulatory regions conserved between the mouse and rabbit whey acidic protein genes.

PubMed Central

Millot, Benjamin; Montoliu, Lluís; Fontaine, Marie-Louise; Mata, Teresa; Devinoy, Eve

2003-01-01

The upstream regulatory regions of the mouse and rabbit whey acidic protein (WAP) genes have been used extensively to target the efficient expression of foreign genes into the mammary gland of transgenic animals. Therefore both regions have been studied to elucidate fully the mechanisms controlling WAP gene expression. Three DNase I-hypersensitive sites (HSS0, HSS1 and HSS2) have been described upstream of the rabbit WAP gene in the lactating mammary gland and correspond to important regulatory regions. These sites are surrounded by variable chromatin structures during mammary-gland development. In the present study, we describe the upstream sequence of the mouse WAP gene. Analysis of genomic sequences shows that the mouse WAP gene is situated between two widely expressed genes (Cpr2 and Ramp3). We show that the hypersensitive sites found upstream of the rabbit WAP gene are also detected in the mouse WAP gene. Further, they encompass functional signal transducer and activator of transcription 5-binding sites, as has been observed in the rabbit. A new hypersensitive site (HSS3), not specific to the mammary gland, was mapped 8 kb upstream of the rabbit WAP gene. Unlike the three HSSs described above, HSS3 is also detected in the liver, but similar to HSS1, it does not depend on lactogenic hormone treatments during cell culture. The region surrounding HSS3 encompasses a potential matrix attachment region, which is also conserved upstream of the mouse WAP gene and contains a functional transcription factor Ets-1 (E26 transformation-specific-1)-binding site. Finally, we demonstrate for the first time that variations in the chromatin structure are dependent on prolactin alone. PMID:12580766

Production of red-flowered plants by genetic engineering of multiple flavonoid biosynthetic genes.

PubMed

Nakatsuka, Takashi; Abe, Yoshiko; Kakizaki, Yuko; Yamamura, Saburo; Nishihara, Masahiro

2007-11-01

Orange- to red-colored flowers are difficult to produce by conventional breeding techniques in some floricultural plants. This is due to the deficiency in the formation of pelargonidin, which confers orange to red colors, in their flowers. Previous researchers have reported that brick-red colored flowers can be produced by introducing a foreign dihydroflavonol 4-reductase (DFR) with different substrate specificity in Petunia hybrida, which does not accumulate pelargonidin pigments naturally. However, because these experiments used dihydrokaempferol (DHK)-accumulated mutants as transformation hosts, this strategy cannot be applied directly to other floricultural plants. Thus in this study, we attempted to produce red-flowered plants by suppressing two endogenous genes and expressing one foreign gene using tobacco as a model plant. We used a chimeric RNAi construct for suppression of two genes (flavonol synthase [FLS] and flavonoid 3'-hydroxylase [F3'H]) and expression of the gerbera DFR gene in order to accumulate pelargonidin pigments in tobacco flowers. We successfully produced red-flowered tobacco plants containing high amounts of additional pelargonidin as confirmed by HPLC analysis. The flavonol content was reduced in the transgenic plants as expected, although complete inhibition was not achieved. Expression analysis also showed that reduction of the two-targeted genes and expression of the foreign gene occurred simultaneously. These results demonstrate that flower color modification can be achieved by multiple gene regulation without use of mutants if the vector constructs are designed resourcefully.

Can foreign proteins imported into yeast mitochondria interfere with PIM1p protease and/or chaperone function?

PubMed

Saveliev, A S; Kovaleva, I E; Novikova, L A; Isaeva, L V; Luzikov, V N

1999-03-15

When studying the fate of mammalian apocytochrome P450scc (apo-P450scc) imported in small amounts into isolated yeast mitochondria, we found that it undergoes degradation, this process being retarded if recipient mitochondria are preloaded in vivo (to about 0.2% of total organelle protein) with a fusion protein composed of mammalian adrenodoxin reductase and adrenodoxin (AdR-Ad); in parallel we observed aggregation of apo-P450scc. These effects suggest some overload of Pim1p protease and/or mtHsp70 system by AdR-Ad, as both of them are involved in the degradation of apo-P450scc (see Savel'ev et al. J. Biol. Chem. 273, 20596-20602, 1998). However, under the same conditions AdR-Ad was not able to impede the import of proteins into mitochondria and the development of the mitochondrial respiratory machinery in yeast, the processes requiring the mtHsp70 system and Pim1p, respectively. These data imply that chaperones and Pim1p protease prefer their natural targets in mitochondria to imported foreign proteins. Copyright 1999 Academic Press.

Teaching Vocabulary with Graphic Novels

ERIC Educational Resources Information Center

Basal, Ahmet; Aytan, Talat; Demir, Ibrahim

2016-01-01

Mastery of idiomatic expressions by foreign language learners is often equated with the fluency of native speakers of that language. However, learning these idiomatic expressions is one of the significant problems experienced by learners. The present quasi-experimental study conducted over four weeks in the ELT department of a Turkish university…

T cell receptor cross-reactivity between similar foreign and self peptides influences naïve cell population size and autoimmunity

PubMed Central

Nelson, Ryan W.; Beisang, Daniel; Tubo, Noah J.; Dileepan, Thamotharampillai; Wiesner, Darin L.; Nielsen, Kirsten; Wüthrich, Marcel; Klein, Bruce S.; Kotov, Dmitri I.; Spanier, Justin A.; Fife, Brian T.; Moon, James J.; Jenkins, Marc K.

2014-01-01

SUMMARY T cell receptor (TCR) cross-reactivity between major histocompatibility complex II (MHCII)-binding self and foreign peptides could influence the naïve CD4+ T cell repertoire and autoimmunity. We found that nonamer peptides that bind to the same MHCII molecule only need to share five amino acids to cross-react on the same TCR. This property was biologically relevant since systemic expression of a self peptide reduced the size of a naïve cell population specific for a related foreign peptide by deletion of cells with cross-reactive TCRs. Reciprocally, an incompletely deleted naïve T cell population specific for a tissue-restricted self peptide could be triggered by related microbial peptides to cause autoimmunity. Thus, TCR cross-reactivity between similar self and foreign peptides can reduce the size of certain foreign peptide-specific T cell populations, and may allow T cell populations specific for tissue-restricted self peptides to cause autoimmunity after infection. PMID:25601203

An optimized transit peptide for effective targeting of diverse foreign proteins into chloroplasts in rice.

PubMed

Shen, Bo-Ran; Zhu, Cheng-Hua; Yao, Zhen; Cui, Li-Li; Zhang, Jian-Jun; Yang, Cheng-Wei; He, Zheng-Hui; Peng, Xin-Xiang

2017-04-11

Various chloroplast transit peptides (CTP) have been used to successfully target some foreign proteins into chloroplasts, but for other proteins these same CTPs have reduced localization efficiencies or fail completely. The underlying cause of the failures remains an open question, and more effective CTPs are needed. In this study, we initially observed that two E.coli enzymes, EcTSR and EcGCL, failed to be targeted into rice chloroplasts by the commonly-used rice rbcS transit peptide (rCTP) and were subsequently degraded. Further analyses revealed that the N-terminal unfolded region of cargo proteins is critical for their localization capability, and that a length of about 20 amino acids is required to attain the maximum localization efficiency. We considered that the unfolded region may alleviate the steric hindrance produced by the cargo protein, by functioning as a spacer to which cytosolic translocators can bind. Based on this inference, an optimized CTP, named RC2, was constructed. Analyses showed that RC2 can more effectively target diverse proteins, including EcTSR and EcGCL, into rice chloroplasts. Collectively, our results provide further insight into the mechanism of CTP-mediated chloroplastic localization, and more importantly, RC2 can be widely applied in future chloroplastic metabolic engineering, particularly for crop plants.

Trafficking and processing of bacterial proteins by mammalian cells: Insights from chondroitinase ABC.

PubMed

Muir, Elizabeth; Raza, Mansoor; Ellis, Clare; Burnside, Emily; Love, Fiona; Heller, Simon; Elliot, Matthew; Daniell, Esther; Dasgupta, Debayan; Alves, Nuno; Day, Priscilla; Fawcett, James; Keynes, Roger

2017-01-01

There is very little reported in the literature about the relationship between modifications of bacterial proteins and their secretion by mammalian cells that synthesize them. We previously reported that the secretion of the bacterial enzyme Chondroitinase ABC by mammalian cells requires the strategic removal of at least three N-glycosylation sites. The aim of this study was to determine if it is possible to enhance the efficacy of the enzyme as a treatment for spinal cord injury by increasing the quantity of enzyme secreted or by altering its cellular location. To determine if the efficiency of enzyme secretion could be further increased, cells were transfected with constructs encoding the gene for chondroitinase ABC modified for expression by mammalian cells; these contained additional modifications of strategic N-glycosylation sites or alternative signal sequences to direct secretion of the enzyme from the cells. We show that while removal of certain specific N-glycosylation sites enhances enzyme secretion, N-glycosylation of at least two other sites, N-856 and N-773, is essential for both production and secretion of active enzyme. Furthermore, we find that the signal sequence directing secretion also influences the quantity of enzyme secreted, and that this varies widely amongst the cell types tested. Last, we find that replacing the 3'UTR on the cDNA encoding Chondroitinase ABC with that of β-actin is sufficient to target the enzyme to the neuronal growth cone when transfected into neurons. This also enhances neurite outgrowth on an inhibitory substrate. Some intracellular trafficking pathways are adversely affected by cryptic signals present in the bacterial gene sequence, whilst unexpectedly others are required for efficient secretion of the enzyme. Furthermore, targeting chondroitinase to the neuronal growth cone promotes its ability to increase neurite outgrowth on an inhibitory substrate. These findings are timely in view of the renewed prospects for gene therapy, and of direct relevance to strategies aimed at expressing foreign proteins in mammalian cells, in particular bacterial proteins.

Physics of phagocytosis of foreign versus self-tolerance

NASA Astrophysics Data System (ADS)

Tsai, Richard; Rodriguez, Pia; Discher, Dennis

2009-03-01

The first cells to `attack' an implanted or injected foreign material or microbe are phagocytic cells of the innate immune system. These cells actively and rapidly phagocytose foreign cells, surfaces, or particles, but the process that is inefficient when faced with ``self'' cells. We have examined the biochemistry and some of the physics of this decision to eat or not eat. One particular protein on all animal cell membranes, called CD47, seems to engage phagocytic cell couter-receptors, and deactivate the force-generating myosin machinery that otherwise makes phagocytosis efficient. We will map the phagocytic synapse between phagocytes and particles or cells and describe the physicochemical dynamics that mediate this key decision in compatability.

Internationalization of the Space Station

NASA Technical Reports Server (NTRS)

Lottmann, R. V.

1985-01-01

Attention is given to the NASA Space Station system elements whose production is under consideration by potential foreign partners. The ESA's Columbus Program declaration encompasses studies of pressurized modules, unmanned payload carriers, and ground support facilities. Canada has expressed interest in construction and servicing facilities, solar arrays, and remote sensing facilities. Japanese studies concern a multipurpose experimental module concept. Each of these foreign investments would expand Space Station capabilities and lay the groundwork for long term partnerships.

Behavior of bubbles in glassmelts. II - Dissolution of a stationary bubble containing a diffusing and a nondiffusing gas

NASA Technical Reports Server (NTRS)

Weinberg, M. C.; Onorato, P. I. K.; Uhlmann, D. R.

1980-01-01

The effect of a foreign nondiffusing gas on the rate of shrinkage of an oxygen bubble in a soda-lime-silica melt was studied. The rate of change of bubble radius with time was computed using the quasi-stationary approximation. The effects of melt undersaturation and initial fraction of foreign gas in the bubble are considered and compared with those calculated using previously derived expressions.

Introducing Translation-Based Activities in Teaching English as a Foreign Language: A Step towards the Improvement of Learners' Accurate Use of Words and Expressions in Writing

ERIC Educational Resources Information Center

Mbeudeu, Clovis Delor

2017-01-01

The teaching of English as a Foreign Language (EFL) in the world in general and in Cameroon in particular has witnessed, over the last three decades, heated debates on which methodologies to adopt in the classroom and which learning strategies to apply for effective teaching and learning so that learners do not only acquire a linguistic competence…

High Polyhydroxybutyrate Production in Pseudomonas extremaustralis Is Associated with Differential Expression of Horizontally Acquired and Core Genome Polyhydroxyalkanoate Synthase Genes

PubMed Central

Catone, Mariela V.; Ruiz, Jimena A.; Castellanos, Mildred; Segura, Daniel; Espin, Guadalupe; López, Nancy I.

2014-01-01

Pseudomonas extremaustralis produces mainly polyhydroxybutyrate (PHB), a short chain length polyhydroxyalkanoate (sclPHA) infrequently found in Pseudomonas species. Previous studies with this strain demonstrated that PHB genes are located in a genomic island. In this work, the analysis of the genome of P. extremaustralis revealed the presence of another PHB cluster phbFPX, with high similarity to genes belonging to Burkholderiales, and also a cluster, phaC1ZC2D, coding for medium chain length PHA production (mclPHA). All mclPHA genes showed high similarity to genes from Pseudomonas species and interestingly, this cluster also showed a natural insertion of seven ORFs not related to mclPHA metabolism. Besides PHB, P. extremaustralis is able to produce mclPHA although in minor amounts. Complementation analysis demonstrated that both mclPHA synthases, PhaC1 and PhaC2, were functional. RT-qPCR analysis showed different levels of expression for the PHB synthase, phbC, and the mclPHA synthases. The expression level of phbC, was significantly higher than the obtained for phaC1 and phaC2, in late exponential phase cultures. The analysis of the proteins bound to the PHA granules showed the presence of PhbC and PhaC1, whilst PhaC2 could not be detected. In addition, two phasin like proteins (PhbP and PhaI) associated with the production of scl and mcl PHAs, respectively, were detected. The results of this work show the high efficiency of a foreign gene (phbC) in comparison with the mclPHA core genome genes (phaC1 and phaC2) indicating that the ability of P. extremaustralis to produce high amounts of PHB could be explained by the different expression levels of the genes encoding the scl and mcl PHA synthases. PMID:24887088

Transgenesis in fish.

PubMed

Houdebine, L M; Chourrout, D

1991-09-15

Gene transfer into fish embryo is being performed in several species (trout, salmon, carps, tilapia, medaka, goldfish, zebrafish, loach, catfish, etc.). In most cases, pronuclei are not visible and microinjection must be done into the cytoplasm of early embryos. Several million copies of the gene are generally injected. In medaka, transgenesis was attempted by injection of the foreign gene into the nucleus of oocyte. Several reports indicate that the injected DNA was rapidly replicated in the early phase of embryo development, regardless of the origin and the sequence of the foreign DNA. The survival of the injected embryos was reasonably good and a large number reached maturity. The proportion of transgenic animals ranged from 1 to 50% or more, according to species and to experimentators. The reasons for this discrepancy have not been elucidated. In all species, the transgenic animals were mosaic. The copy number of the foreign DNA was different in the various tissues of an animal and a proportion lower than 50% of F1 offsprings received the gene from their parents. This suggests that the foreign DNA was integrated into the fish genome at the two cells stage or later. An examination of the integrated DNA in different cell types of an animal revealed that integration occurred mainly during early development. The transgene was found essentially unrearranged in the fish genome of the founders and offsprings. The transgenes were therefore stably transmitted to progeny in a Mendelian fashion. Southern blot analysis revealed the presence of possible junction fragments and also of minor bands which may result from a rearrangement of the injected DNA. In all species, the integrated DNA appeared mainly as random end-to-end concatemers. In adult trout blood cells, a small proportion of the foreign DNA was maintained in the form of non-integrated concatemers, as judged by the existence of end fragments. The transgenes were generally only poorly expressed. The majority of the injected gene constructs contained essentially mammalian or higher vertebrates sequences. The comparison of the expression efficiency of these constructs in transfected fish and mammalian cells indicates that some of the mammalian DNA sequences are most efficiently understood by the fish cell machinery. Chloramphenicol acetyl transferase gene under the control of promoters from Rous sarcoma virus, and human cytomegalovirus, was expressed in several tissues of transgenic fish. Chicken delta-crystallin gene was expressed in several tissues of transgenic fish.(ABSTRACT TRUNCATED AT 400 WORDS)

Neuroglian-positive plasmatocytes of Manduca sexta and the initiation of hemocyte attachment to foreign surfaces.

PubMed

Nardi, James B; Pilas, Barbara; Bee, Charles Mark; Zhuang, Shufei; Garsha, Karl; Kanost, Michael R

2006-01-01

Observations of hemocyte aggregation on abiotic surfaces suggested that certain plasmatocytes from larvae of Manduca sexta act as foci for hemocyte aggregation. To establish how these particular plasmatocytes form initial attachments to foreign surfaces, they were cultured separately from other selected populations of hemocytes. While all circulating plasmatocytes immunolabel with anti-beta-integrin monoclonal antibody (MAb), only these larger plasmatocytes immunolabel with a MAb to the adhesion protein neuroglian. Neuroglian-negative plasmatocytes and granular cells that have been magnetically segregated from the majority of granular cells adhere to each other but fail to adhere to foreign substrata; by contrast, neuroglian-positive plasmatocytes that segregate with most granular cells adhere firmly to a substratum. Hemocytes form stable aggregates around the large, neuroglian-positive plasmatocytes. However, if neuroglian-positive plasmatocytes are separated from most granular cells, attachment of these plasmatocytes to foreign surfaces is suppressed.

Are Women More Emotionally Skilled When It Comes to Expression of Emotions in the Foreign Language? Gender, Emotional Intelligence and Personality Traits in Relation to Emotional Expression in the L2

ERIC Educational Resources Information Center

Ozanska-Ponikwia, Katarzyna

2017-01-01

The present study investigates the link between gender, emotional intelligence (EI), personality traits and self-reported emotional expression in the second language (L2). Data analysis suggests that gender might not influence self-perceived emotional expression in the L2, as the results of the t-test show that both males and females declare…

Two Patients with Dry Eye Disease Followed Up Using an Expression Assay of Ocular Surface Mucin.

PubMed

Machida, Yumiko; Shoji, Jun; Harada, Natsuko; Inada, Noriko

2016-01-01

We report 2 patients with dry eye disease followed up using the expression levels of ocular surface mucin. Patient 1: a 57-year-old woman with Sjögren's syndrome-associated dry eyes experienced severe dryness and foreign body sensation in both her eyes, and instilled sodium hyaluronate ophthalmic solution 0.3% about 10-15 times daily. We measured the expression levels of MUC5AC mRNA (MUC5AC) and MUC16 mRNA (MUC16) by using real-time reversed transcription polymerase chain reaction for the specimens of modified impression cytology. Expression levels of MUC5AC and MUC16 on her ocular surface were very low. Subjective symptoms and expression levels of ocular surface mucin improved after combined treatment of rebamipide (4 times daily) and fluorometholone (once daily) ophthalmic suspension. Patient 2: a 62-year-old man with chronic graft-versus-host disease-associated dry eye experienced severe foreign body sensation and developed superficial punctate keratopathy with mucous thread and filamentary keratitis. Expression level of MUC5AC was very high at baseline. Subjective symptoms and expression levels of ocular surface mucin improved by combined treatment of rebamipide (4 times daily) and fluorometholone (once daily) ophthalmic suspension. Clinical test for MUC gene expression on the ocular surface was found to be useful in the follow-up of dry eye treatment.

Production of therapeutic proteins in algae, analysis of expression of seven human proteins in the chloroplast of Chlamydomonas reinhardtii

PubMed Central

Rasala, Beth A; Muto, Machiko; Lee, Philip A; Jager, Michal; Cardoso, Rosa MF; Behnke, Craig A; Kirk, Peter; Hokanson, Craig A; Crea, Roberto; Mendez, Michael; Mayfield, Stephen P

2010-01-01

Summary Recombinant proteins are widely used today in many industries, including the biopharmaceutical industry, and can be expressed in bacteria, yeasts, mammalian and insect cell cultures, or in transgenic plants and animals. In addition, transgenic algae have also been shown to support recombinant protein expression, both from the nuclear and chloroplast genomes. However, to date, there are only a few reports on recombinant proteins expressed in the algal chloroplast. It is unclear if this is due to few attempts or to limitations of the system that preclude expression of many proteins. Thus, we sought to assess the versatility of transgenic algae as a recombinant protein production platform. To do this, we tested whether the algal chloroplast could support the expression of a diverse set of current or potential human therapeutic proteins. Of the seven proteins chosen, greater than 50% expressed at levels sufficient for commercial production. Three expressed at 2% to 3% of total soluble protein, while a forth protein accumulated to similar levels when translationally fused to a well-expressed serum amyloid protein. All of the algal chloroplast-expressed proteins are soluble and showed biological activity comparable to that of the same proteins expressed using traditional production platforms. Thus, the success rate, expression levels, and bioactivty achieved demonstrate the utility of C. reinhardtii as a robust platform for human therapeutic protein production. PMID:20230484

Visualizing High-Efficiency HIV Transfer | Center for Cancer Research

Cancer.gov

The Human Immunodeficiency Virus (HIV), the causative agent of Acquired Immunodeficiency Syndrome (AIDS), infects and eventually kills CD4 receptor-expressing T cells, which are critical for proper immune system function. The gp120 protein on the surface of HIV particles is known to bind CD4 and a co-receptor, either CCR5 or CXCR4, leading to fusion of the virus and T cell membranes and infection of the cell. The most efficient means of viral infection occurs when an uninfected T cell interacts with a dendritic cell (DC) that has previously come in contact with HIV. Antigen presenting cells, such as DCs, normally circulate throughout the body binding or engulfing foreign material and presenting it to T cells to initiate an immune response. HIV takes advantage of this close cell-cell association to propagate, so knowing the cells’ spatial arrangement during viral transmission could elucidate novel modes of treatment.

Reverse Genetics for Newcastle Disease Virus as a Vaccine Vector.

PubMed

Kim, Shin-Hee; Samal, Siba K

2018-02-22

Newcastle disease virus (NDV) is an economically important pathogen in the poultry industry worldwide. Recovery of infectious NDV from cDNA using reverse genetics has made it possible to manipulate the genome of NDV. This has greatly contributed to our understanding of the molecular biology and pathogenesis of NDV. Furthermore, NDV has modular genome and accommodates insertion of a foreign gene as a transcriptional unit, thus enabling NDV as a vaccine vector against diseases of humans and animals. Avirulent NDV strains (e.g., LaSota and B1) have been commonly used as vaccine vectors. In this protocol, we have described reverse genetics of NDV to be used as a vaccine vector by exemplifying the recovery of NDV vectored avian influenza virus vaccine. Specifically, cloning and recovery of NDV expressing the hemagglutinin protein of highly pathogenic influenza virus were explained. © 2018 by John Wiley & Sons, Inc. Copyright © 2018 John Wiley & Sons, Inc.

First characterization of fish CD22: An inhibitory role in the activation of peripheral blood leukocytes.

PubMed

Li, Yi-Qun; Zhang, Jian; Li, Jun; Sun, Li

2017-08-01

In mammals, CD22 is a member of the Ig superfamily that serves as an inhibitor during B cell responses to foreign antigens. In this study, we characterized for the first time a fish CD22 from tongue sole Cynoglossus semilaevis (CsCD22). CsCD22 possesses the conserved structural features of CD22 and shares 35%-54% sequence identities with other fish CD22. mRNA expression of CsCD22 was most abundant in head kidney and heart. CsCD22 protein was detected on the surface of peripheral blood leukocytes (PBL). In the presence of rCsCD22 antibody, the proliferation, phagocytosis, and antibacterial activity of PBL were significantly increased. These results indicate for the first time that fish CD22 plays an inhibitory role in PBL activation. Copyright © 2017 Elsevier B.V. All rights reserved.

Protein deficiency lowers resistance of Mormon crickets to the pathogenic fungus Beauveria bassiana.

PubMed

Srygley, R B; Jaronski, S T

Little is known about the effects of dietary macronutrients on the capacity of insects to ward off a fungal pathogen. Here we tested the hypothesis that Mormon crickets fed restricted protein diets have lower enzymatic assays of generalized immunity, slower rates of encapsulation of foreign bodies, and greater mortality from infection by Beauveria bassiana, a fungal pathogen. Beginning in the last nymphal instar, Mormon crickets were fed a high, intermediate, or low protein diet with correspondingly low, intermediate, or high carbohydrate proportions. After they eclosed to adult, we drew hemolymph, topically applied B. bassiana, maintained them on diet treatments, and measured mortality for 21 days. Mormon crickets fed high protein diets had higher prophenoloxidase titers, greater encapsulation response, and higher survivorship to Beauveria fungal infection than those on low protein diets. We replicated the study adding very high and very low protein diets to the treatments. A high protein diet increased phenoloxidase titers, and those fed the very high protein diet had more circulating prophenoloxidase. Mormon crickets fed the very low protein diet were the most susceptible to B. bassiana infection, but the more concentrated phenoloxidase and prophenoloxidase associated with the highest protein diets did not confer the greatest protection from the fungal pathogen as in the first replicate. We conclude that protein-restricted diets caused Mormon crickets to have lower phenoloxidase titers, slower encapsulation of foreign bodies, and greater mortality from B. bassiana infection than those fed high protein diets. These results support the nutrition-based dichotomy of migrating Mormon crickets, protein-deficient ones are more susceptible to pathogenic fungi whereas carbohydrate-deficient ones are more vulnerable to bacterial challenge. Published by Elsevier Ltd.

Exploiting translational coupling for the selection of cells producing toxic recombinant proteins from expression vectors.

PubMed

Tagliavia, Marcello; Cuttitta, Angela

2016-01-01

High rates of plasmid instability are associated with the use of some expression vectors in Escherichia coli, resulting in the loss of recombinant protein expression. This is due to sequence alterations in vector promoter elements caused by the background expression of the cloned gene, which leads to the selection of fast-growing, plasmid-containing cells that do not express the target protein. This phenomenon, which is worsened when expressing toxic proteins, results in preparations containing very little or no recombinant protein, or even in clone loss; however, no methods to prevent loss of recombinant protein expression are currently available. We have exploited the phenomenon of translational coupling, a mechanism of prokaryotic gene expression regulation, in order to select cells containing plasmids still able to express recombinant proteins. Here we designed an expression vector in which the cloned gene and selection marker are co-expressed. Our approach allowed for the selection of the recombinant protein-expressing cells and proved effective even for clones encoding toxic proteins.

LGP2 Synergy with MDA5 in RLR-Mediated RNA Recognition and Antiviral Signaling

PubMed Central

Bruns, Annie M.; Horvath, Curt M.

2015-01-01

Mammalian cells have the ability to recognize virus infection and mount a powerful antiviral response. Pattern recognition receptor proteins detect molecular signatures of virus infection and activate antiviral signaling. The RIG-I-like receptor (RLR) proteins are expressed in the cytoplasm of nearly all cells and specifically recognize virus-derived RNA species as a molecular feature discriminating the pathogen from the host. The RLR family is composed of three homologous proteins, RIG-I, MDA5, and LGP2. All RLRs have the ability to detect virus-derived dsRNA and hydrolyze ATP, but display individual differences in enzymatic activity, intrinsic ability to recognize RNA, and mechanisms of activation. Emerging evidence suggests that MDA5 and RIG-I utilize distinct mechanisms to form oligomeric complexes along dsRNA. Aligning of their signaling domains creates a platform capable of propagating and amplifying antiviral signaling responses. LGP2 with intact ATP hydrolysis is critical for the MDA5-mediated antiviral response, but LGP2 lacks the domains essential for activation of antiviral signaling, leaving the role of LGP2 in antiviral signaling unclear. Recent studies revealed a mechanistic basis of synergy between LGP2 and MDA5 leading to enhanced antiviral signaling. This review briefly summarizes the RLR system, and focuses on the relationship between LGP2 and MDA5, describing in detail how these two proteins work together to detect foreign RNA and generate a fully functional antiviral response. PMID:25794939

Cultivation to improve in vivo solubility of overexpressed arginine deiminases in Escherichia coli and the enzyme characteristics.

PubMed

Wang, Ying; Li, Yue-Zhong

2014-06-07

Overexpression of foreign genes in Escherichia coli cells is an efficient means to obtain recombinant proteins. The technique is, however, often hampered by misfolding, degradation, aggregation and formation in inclusion bodies of products. In this study, we reported that in vivo solubility of overexpressed arginine deiminases (ADI) improved by changing the cultivation conditions. ADI is enzymes that convert L-arginine to L-citrulline. After codon optimization, we synthesized the ADI gene of Pseudomonas putida and constructed it for overexpression in E. coli cells. The rADI products were mainly in inclusion body forms. We performed a series of optimization to enhance solubility of the protein. Co-expression with the GroES-GroEL chaperone team increased approximately 5-fold of the rADI activity. In addition the combination of L-arginine and D-glucose in the Luria-Bertani (LB) growth medium further increased the total activity to about 15 times. Separate L-arginine and D-glucose or the addition of other saccharides or amino acids had no such effects. The solubilization effects of the combination of L-arginine and D-glucose were further confirmed in the overexpression of another ADI from Listeria welshimeri. The enzymatic and conversion characteristics of the rADI products were further determined. Combined addition of L-arginine and D-glucose in the LB medium significantly improved in vivo solubility of rADI proteins. The present study suggested a new strategy to increase the solubilization of overexpressed recombinant proteins in E. coli cells.

A genetically adjuvanted influenza B virus vector increases immunogenicity and protective efficacy in mice.

PubMed

Kittel, Christian; Wressnigg, Nina; Shurygina, Anna Polina; Wolschek, Markus; Stukova, Marina; Romanovskaya-Romanko, Ekatherina; Romanova, Julia; Kiselev, Oleg; Muster, Thomas; Egorov, Andrej

2015-10-01

The existence of multiple antigenically distinct types and subtypes of influenza viruses allows the construction of a multivalent vector system for the mucosal delivery of foreign sequences. Influenza A viruses have been exploited successfully for the expression of extraneous antigens as well as immunostimulatory molecules. In this study, we describe the development of an influenza B virus vector whose functional part of the interferon antagonist NS1 was replaced by human interleukin 2 (IL2) as a genetic adjuvant. We demonstrate that IL2 expressed by this viral vector displays immune adjuvant activity in immunized mice. Animals vaccinated with the IL2 viral vector showed an increased hemagglutination inhibition antibody response and higher protective efficacy after challenge with a wild-type influenza B virus when compared to mice vaccinated with a control virus. Our results demonstrate that it is feasible to construct influenza B vaccine strains expressing immune-potentiating foreign sequences from the NS genomic segment. Based on these data, it is now hypothetically possible to create a trivalent (or quadrivalent) live attenuated influenza vaccine in which each component expresses a selected genetic adjuvant with tailored expression levels.

Complementation between avirulent Newcastle disease virus and a fusion protein gene expressed from a retrovirus vector: requirements for membrane fusion.

PubMed Central

Morrison, T; McQuain, C; McGinnes, L

1991-01-01

The cDNA derived from the fusion gene of the virulent AV strain of Newcastle disease virus (NDV) was expressed in chicken embryo cells by using a retrovirus vector. The fusion protein expressed in this system was transported to the cell surface and was efficiently cleaved into the disulfide-linked F1-F2 form found in infectious virions. The cells expressing the fusion gene grew normally and could be passaged many times. Monolayers of these cells would plaque, in the absence of trypsin, avirulent NDV strains (strains which encode a fusion protein which is not cleaved in tissue culture). Fusion protein-expressing cells would not fuse if mixed with uninfected cells or uninfected cells expressing the hemagglutinin-neuraminidase (HN) protein. However, the fusion protein-expressing cells, if infected with avirulent strains of NDV, would fuse with uninfected cells, suggesting that fusion requires both the fusion protein and another viral protein expressed in the same cell. Fusion was also seen after transfection of the HN protein gene into fusion protein-expressing cells. Thus, the expressed fusion protein gene is capable of complementing the virus infection, providing an active cleaved fusion protein required for the spread of infection. However, the fusion protein does not mediate cell fusion unless the cell also expresses the HN protein. Fusion protein-expressing cells would not plaque influenza virus in the absence of trypsin, nor would influenza virus-infected fusion protein-expressing cells fuse with uninfected cells. Thus, the influenza virus HA protein will not substitute for the NDV HN protein in cell-to-cell fusion. Images PMID:1987376

Use of adenoviral vectors as veterinary vaccines.

PubMed

Ferreira, T B; Alves, P M; Aunins, J G; Carrondo, M J T

2005-10-01

Vaccines are the most effective and inexpensive prophylactic tool in veterinary medicine. Ideally, vaccines should induce a lifelong protective immunity against the target pathogen while not causing clinical or pathological signs of diseases in the vaccinated animals. However, such ideal vaccines are rare in the veterinary field. Many vaccines are either of limited effectiveness or have harmful side effects. In addition, there are still severe diseases with no effective vaccines. A very important criterion for an ideal vaccine in veterinary medicine is low cost; this is especially important in developing countries and even more so for poultry vaccination, where vaccines must sell for a few cents a dose. Traditional approaches include inactivated vaccines, attenuated live vaccines and subunit vaccines. Recently, genetic engineering has been applied to design new, improved vaccines. Adenovirus vectors are highly efficient for gene transfer in a broad spectrum of cell types and species. Moreover, adenoviruses often induce humoral, mucosal and cellular immune responses to antigens encoded by the inserted foreign genes. Thus, adenoviruses have become a vector of choice for delivery and expression of foreign proteins for vaccination. Consequently, the market requirements for adenovirus vaccines are increasing, creating a need for production methodologies of concentrated vectors with warranted purity and efficacy. This review summarizes recent developments and approaches of adenovirus production and purification as the application of these vectors, including successes and failures in clinical applications to date.

The dsRNA binding protein RDE-4 interacts with RDE-1, DCR-1, and a DExH-box helicase to direct RNAi in C. elegans.

PubMed

Tabara, Hiroaki; Yigit, Erbay; Siomi, Haruhiko; Mello, Craig C

2002-06-28

Double-stranded (ds) RNA induces potent gene silencing, termed RNA interference (RNAi). At an early step in RNAi, an RNaseIII-related enzyme, Dicer (DCR-1), processes long-trigger dsRNA into small interfering RNAs (siRNAs). DCR-1 is also required for processing endogenous regulatory RNAs called miRNAs, but how DCR-1 recognizes its endogenous and foreign substrates is not yet understood. Here we show that the C. elegans RNAi pathway gene, rde-4, encodes a dsRNA binding protein that interacts during RNAi with RNA identical to the trigger dsRNA. RDE-4 protein also interacts in vivo with DCR-1, RDE-1, and a conserved DExH-box helicase. Our findings suggest a model in which RDE-4 and RDE-1 function together to detect and retain foreign dsRNA and to present this dsRNA to DCR-1 for processing.

GENE EXPRESSION PROFILING OF XENOBIOTIC METABOLIZING ENZYMES (XMES) THROUGH THE LIFE STAGES OF THE MALE C57BL/6 MOUSE

EPA Science Inventory

In the presence of foreign compounds, metabolic homeostasis of the organism is maintained by the liver's ability to detoxify and eliminate these xenobiotics. This is accomplished, in part, by the expression of XMEs, which metabolize xenobiotics and determine whether exposure will...

The C. elegans CSR-1 Argonaute pathway counteracts epigenetic silencing to promote germline gene expression

PubMed Central

Seth, Meetu; Shirayama, Masaki; Gu, Weifeng; Ishidate, Takao; Conte, Darryl; Mello, Craig C.

2014-01-01

SUMMARY Organisms can develop adaptive sequence-specific immunity by re-expressing pathogen-specific small RNAs that guide gene silencing. For example, the C. elegans PIWI-Argonaute/piRNA pathway recruits RNA-dependent RNA polymerase RdRP to foreign sequences to amplify a trans-generational small RNA-induced epigenetic silencing signal (termed RNAe). Here we provide evidence that in addition to an adaptive memory of silenced sequences, C. elegans can also develop an opposing adaptive memory of expressed/self mRNAs. We refer to this mechanism, which can prevent or reverse RNAe as RNA-induced epigenetic gene activation (RNAa). We show that CSR-1, which engages RdRP-amplified small RNAs complementary to germline-expressed mRNAs, is required for RNAa. We show that a transgene with RNAa activity also exhibits accumulation of cognate CSR-1 small RNAs. Our findings suggest that C. elegans adaptively acquires and maintains a trans-generational CSR-1 memory that recognizes and protects self mRNAs, allowing piRNAs to recognize foreign sequences innately, without need for prior exposure. PMID:24360782

A non-targeted UHPLC-UV methid with classical and multi-variate data analysis to detect adulteration of skim milk powder with foreign proteins

USDA-ARS?s Scientific Manuscript database

Ultra-High performance liquid chromatography (UHPLC) with single wavelength (215 nm) detection was used to obtain chromatographic profiles of authentic skim milk powder (SMP) and synthetic mixtures of SMP with variable amounts of soy (SPI), pea (PPI), brown rice (BRP), and hydrolyzed wheat protein (...

Expressing the sense of the House of Representatives that judicial determinations regarding the meaning of the Constitution of the United States should not be based on judgments, laws, or pronouncements of foreign institutions unless such foreign judgments, laws, or pronouncements inform an understanding of the original meaning of the Constitution of the United States.

THOMAS, 111th Congress

Rep. Goodlatte, Bob [R-VA-6

2009-05-21

House - 08/19/2009 Referred to the Subcommittee on the Constitution, Civil Rights, and Civil Liberties. (All Actions) Tracker: This bill has the status IntroducedHere are the steps for Status of Legislation:

[Use of a novel baculovirus vector to express nucleoprotein gene of Crimean-Congo hemorrhagic fever virus in both insect and mammalian cells].

PubMed

Ma, Benjiang; Hang, Changshou; Zhao, Yun; Wang, Shiwen; Xie, Yanxiang

2002-09-01

To construct a novel baculovirus vector which is capable of promoting the high-yield expression of foreign gene in mammalian cells and to express by this vector the nucleoprotein (NP) gene of Crimean-Congo hemorrhagic fever virus (CCHFV) Chinese isolate (Xinjiang hemorrhagic fever virus, XHFV) BA88166 in insect and Vero cells. Human cytomegalovirus (CMV) immediate early (IE) promoter was ligated to the baculovirus vector pFastBac1 downstream of the polyhedrin promoter to give rise to the novel vector pCB1. XHFV NP gene was cloned to this vector and was well expressed in COS-7 cells and Vero cells by means of recombinant plasmid transfection and baculovirus infection. The XHFV NP gene in vector pCB1 could be well expressed in mammalian cells. Vero cells infected with recombinant baculovirus harboring NP gene could be employed as antigens to detect XHF serum specimens whose results were in good correlation with those of ELISA and in parallel with clinical diagnoses. This novel baculovirus vector is able to express the foreign gene efficiently in both insect and mammalian cells, which provides not only the convenient diagnostic antigens but also the potential for developing recombinant virus vaccines and gene therapies.

Expression of proteins in Escherichia coli as fusions with maltose-binding protein to rescue non-expressed targets in a high-throughput protein-expression and purification pipeline

PubMed Central

Hewitt, Stephen N.; Choi, Ryan; Kelley, Angela; Crowther, Gregory J.; Napuli, Alberto J.; Van Voorhis, Wesley C.

2011-01-01

Despite recent advances, the expression of heterologous proteins in Escherichia coli for crystallization remains a nontrivial challenge. The present study investigates the efficacy of maltose-binding protein (MBP) fusion as a general strategy for rescuing the expression of target proteins. From a group of sequence-verified clones with undetectable levels of protein expression in an E. coli T7 expression system, 95 clones representing 16 phylogenetically diverse organisms were selected for recloning into a chimeric expression vector with an N-terminal histidine-tagged MBP. PCR-amplified inserts were annealed into an identical ligation-independent cloning region in an MBP-fusion vector and were analyzed for expression and solubility by high-throughput nickel-affinity binding. This approach yielded detectable expression of 72% of the clones; soluble expression was visible in 62%. However, the solubility of most proteins was marginal to poor upon cleavage of the MBP tag. This study offers large-scale evidence that MBP can improve the soluble expression of previously non-expressing proteins from a variety of eukaryotic and prokaryotic organisms. While the behavior of the cleaved proteins was disappointing, further refinements in MBP tagging may permit the more widespread use of MBP-fusion proteins in crystallographic studies. PMID:21904041

Tomato bushy stunt virus (TBSV), a versatile platform for polyvalent display of antigenic epitopes and vaccine design.

PubMed

Kumar, Shantanu; Ochoa, Wendy; Singh, Pratik; Hsu, Catherine; Schneemann, Anette; Manchester, Marianne; Olson, Mark; Reddy, Vijay

2009-05-25

Viruses-like particles (VLPs) are frequently being used as platforms for polyvalent display of foreign epitopes of interest on their capsid surface to improve their presentation enhancing the antigenicity and host immune response. In the present study, we used the VLPs of Tomato bushy stunt virus (TBSV), an icosahedral plant virus, as a platform to display 180 copies of 16 amino acid epitopes of ricin toxin fused to the C-terminal end of a modified TBSV capsid protein (NDelta52). Expression of the chimeric recombinant protein in insect cells resulted in spontaneous assembly of VLPs displaying the ricin epitope. Cryo-electron microscopy and image reconstruction of the chimeric VLPs at 22 A resolution revealed the locations and orientation of the ricin epitope exposed on the TBSV capsid surface. Furthermore, injection of chimeric VLPs into mice generated antisera that detected the native ricin toxin. The ease of fusing of short peptides of 15-20 residues and their ability to form two kinds (T=1, T=3) of bio-nanoparticles that result in the display of 60 or 180 copies of less constrained and highly exposed antigenic epitopes makes TBSV an attractive and versatile display platform for vaccine design.

Improving membrane protein expression and function using genomic edits

DOE PAGES

Jensen, Heather M.; Eng, Thomas; Chubukov, Victor; ...

2017-10-12

Expression of membrane proteins often leads to growth inhibition and perturbs central metabolism and this burden varies with the protein being overexpressed. There are also known strain backgrounds that allow greater expression of membrane proteins but that differ in efficacy across proteins. Here, we hypothesized that for any membrane protein, it may be possible to identify a modified strain background where its expression can be accommodated with less burden. To directly test this hypothesis, we used a bar-coded transposon insertion library in tandem with cell sorting to assess genome-wide impact of gene deletions on membrane protein expression. The expression ofmore » five membrane proteins (CyoB, CydB, MdlB, YidC, and LepI) and one soluble protein (GST), each fused to GFP, was examined. We identified Escherichia coli mutants that demonstrated increased membrane protein expression relative to that in wild type. For two of the proteins (CyoB and CydB), we conducted functional assays to confirm that the increase in protein expression also led to phenotypic improvement in function. This study represents a systematic approach to broadly identify genetic loci that can be used to improve membrane protein expression, and our method can be used to improve expression of any protein that poses a cellular burden.« less

Improving membrane protein expression and function using genomic edits

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jensen, Heather M.; Eng, Thomas; Chubukov, Victor

Expression of membrane proteins often leads to growth inhibition and perturbs central metabolism and this burden varies with the protein being overexpressed. There are also known strain backgrounds that allow greater expression of membrane proteins but that differ in efficacy across proteins. Here, we hypothesized that for any membrane protein, it may be possible to identify a modified strain background where its expression can be accommodated with less burden. To directly test this hypothesis, we used a bar-coded transposon insertion library in tandem with cell sorting to assess genome-wide impact of gene deletions on membrane protein expression. The expression ofmore » five membrane proteins (CyoB, CydB, MdlB, YidC, and LepI) and one soluble protein (GST), each fused to GFP, was examined. We identified Escherichia coli mutants that demonstrated increased membrane protein expression relative to that in wild type. For two of the proteins (CyoB and CydB), we conducted functional assays to confirm that the increase in protein expression also led to phenotypic improvement in function. This study represents a systematic approach to broadly identify genetic loci that can be used to improve membrane protein expression, and our method can be used to improve expression of any protein that poses a cellular burden.« less

The use of transgenic parasites in malaria vaccine research.

PubMed

Othman, Ahmad Syibli; Marin-Mogollon, Catherin; Salman, Ahmed M; Franke-Fayard, Blandine M; Janse, Chris J; Khan, Shahid M

2017-07-01

Transgenic malaria parasites expressing foreign genes, for example fluorescent and luminescent proteins, are used extensively to interrogate parasite biology and host-parasite interactions associated with malaria pathology. Increasingly transgenic parasites are also exploited to advance malaria vaccine development. Areas covered: We review how transgenic malaria parasites are used, in vitro and in vivo, to determine protective efficacy of different antigens and vaccination strategies and to determine immunological correlates of protection. We describe how chimeric rodent parasites expressing P. falciparum or P. vivax antigens are being used to directly evaluate and rank order human malaria vaccines before their advancement to clinical testing. In addition, we describe how transgenic human and rodent parasites are used to develop and evaluate live (genetically) attenuated vaccines. Expert commentary: Transgenic rodent and human malaria parasites are being used to both identify vaccine candidate antigens and to evaluate both sub-unit and whole organism vaccines before they are advanced into clinical testing. Transgenic parasites combined with in vivo pre-clinical testing models (e.g. mice) are used to evaluate vaccine safety, potency and the durability of protection as well as to uncover critical protective immune responses and to refine vaccination strategies.

Retrotransposed genes such as Frat3 in the mouse Chromosome 7C Prader-Willi syndrome region acquire the imprinted status of their insertion site.

PubMed

Chai, J H; Locke, D P; Ohta, T; Greally, J M; Nicholls, R D

2001-11-01

Prader-Willi syndrome (PWS) results from loss of function of a 1.0- to 1.5-Mb domain of imprinted, paternally expressed genes in human Chromosome (Chr) 15q11-q13. The loss of imprinted gene expression in the homologous region in mouse Chr 7C leads to a similar neonatal PWS phenotype. Several protein-coding genes in the human PWS region are intronless, possibly arising by retrotransposition. Here we present evidence for continued acquisition of genes by the mouse PWS region during evolution. Bioinformatic analyses identified a BAC containing four genes, Mkrn3, Magel2, Ndn, Frat3, and the Atp5l-ps1 pseudogene, the latter two genes derived from recent L1-mediated retrotransposition. Analyses of eight overlapping BACs indicate that these genes are clustered within 120 kb in two inbred strains, in the order tel-Atp5l-ps1-Frat3-Mkrn3-Magel2-Ndn-cen. Imprinting analyses show that Frat3 is differentially methylated and expressed solely from the paternal allele in a transgenic mouse model of Angelman syndrome, with no expression from the maternal allele in a mouse model of PWS. Loss of Frat3 expression may, therefore, contribute to the phenotype of mouse models of PWS. The identification of five intronless genes in a small genomic interval suggests that this region is prone to retroposition in germ cells or their zygotic and embryonic cell precursors, and that it allows the subsequent functional expression of these foreign sequences. The recent evolutionary acquisition of genes that adopt the same imprint as older, flanking genes indicates that the newly acquired genes become 'innocent bystanders' of a primary epigenetic signal causing imprinting in the PWS domain.

Association of birthplace and self-reported hypertension by racial/ethnic groups among US adults--National Health Interview Survey, 2006-2010.

PubMed

Fang, Jing; Ayala, Carma; Loustalot, Fleetwood

2012-12-01

Over the past few decades, the proportion of US adults who were foreign-born has been increasing, as has the overall prevalence of hypertension. Here, we compared the prevalence of self-reported hypertension among native-born adults with that among foreign-born adults, classified by racial/ethnic group. Using 2006-2010 data from the National Health Interview Survey (NHIS), we compared the age-adjusted prevalence of hypertension among native-born adults to foreign-born adults, specified by continent of birthplace and race/ethnicity. Results are expressed as unadjusted odds ratios (ORs) and three sets of adjusted odds ratios (AORs) adjusted for selected sociodemographic, behavioral and health-related characteristics. All results accounted for NHIS sampling design variables. The analytic sample was 124,260 with 16.3% foreign-born adults. Among the foreign-born adults, 56% were from Central or South America, 22% from Asia, 13% from Europe, and 4% from Africa. Overall and after adjustment, hypertension prevalence was significantly higher among US-born adults than among foreign-born adults (AOR: 1.28, 95% CI: 1.21-1.36). By race/ethnicity, hypertension prevalence was higher among US-born non-Hispanic blacks than either foreign-born non-Hispanic blacks (AOR: 1.24, 95%CI: 1.02-1.50) or all Africa-born immigrants of any race/ethnicity [AOR: 1.45, 95% confidence interval (CI): 1.07-1.97]. Among foreign-born adults, duration of US residence was positively associated with the likelihood of hypertension. Hypertension prevalence was higher among US-born adults than among foreign-born adults and higher among US-born non-Hispanic blacks than in any other group. Among foreign-born adults, hypertension risk increased with the number of years they had lived in the United States.

Insights into plant biomass conversion from the genome of the anaerobic thermophilic bacterium Caldicellulosiruptor bescii DSM 6725

PubMed Central

Dam, Phuongan; Kataeva, Irina; Yang, Sung-Jae; Zhou, Fengfeng; Yin, Yanbin; Chou, Wenchi; Poole, Farris L.; Westpheling, Janet; Hettich, Robert; Giannone, Richard; Lewis, Derrick L.; Kelly, Robert; Gilbert, Harry J.; Henrissat, Bernard; Xu, Ying; Adams, Michael W. W.

2011-01-01

Caldicellulosiruptor bescii DSM 6725 utilizes various polysaccharides and grows efficiently on untreated high-lignin grasses and hardwood at an optimum temperature of ∼80°C. It is a promising anaerobic bacterium for studying high-temperature biomass conversion. Its genome contains 2666 protein-coding sequences organized into 1209 operons. Expression of 2196 genes (83%) was confirmed experimentally. At least 322 genes appear to have been obtained by lateral gene transfer (LGT). Putative functions were assigned to 364 conserved/hypothetical protein (C/HP) genes. The genome contains 171 and 88 genes related to carbohydrate transport and utilization, respectively. Growth on cellulose led to the up-regulation of 32 carbohydrate-active (CAZy), 61 sugar transport, 25 transcription factor and 234 C/HP genes. Some C/HPs were overproduced on cellulose or xylan, suggesting their involvement in polysaccharide conversion. A unique feature of the genome is enrichment with genes encoding multi-modular, multi-functional CAZy proteins organized into one large cluster, the products of which are proposed to act synergistically on different components of plant cell walls and to aid the ability of C. bescii to convert plant biomass. The high duplication of CAZy domains coupled with the ability to acquire foreign genes by LGT may have allowed the bacterium to rapidly adapt to changing plant biomass-rich environments. PMID:21227922

Producing recombinant human milk proteins in the milk of livestock species.

PubMed

Bösze, Zsuzsanna; Baranyi, Mária; Whitelaw, C Bruce A

2008-01-01

Recombinant human proteins produced by the mammary glands of genetically modified transgenic livestock mammals represent a special aspect of milk bioactive components. For therapeutic applications, the often complex posttranslational modifications of human proteins should be recapitulated in the recombinant products. Compared to alternative production methods, mammary gland production is a viable option, underlined by a number of transgenic livestock animal models producing abundant biologically active foreign proteins in their milk. Recombinant proteins isolated from milk have reached different phases of clinical trials, with the first marketing approval for human therapeutic applications from the EMEA achieved in 2006.

Expression of foreign genes in filamentous cyanobacteria

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kuritz, T.; Wolk, C.P.

1993-06-01

Several advantages make cyanobacteria attractive hosts for biodegradative genes and possibly for other exogenous genes that have practical uses. The authors have obtained expression in Anabaena sp. strain PCC 7120 and Nostoc ellipsosporum of a dechlorination operon, fcbAB, from Arthrobacter globiformis, and have also developed a simple method for qualitative assessment of dechlorination by microorganisms, such as cyanobacteria, whose metabolism is dependent on the presence of chloride in the medium. Transcription of fcbAB under the control of a variety of promoters was monitored by placing luxAB (encoding luciferase) downstream from fcbAB, and by measuring light emission from luciferase. They believemore » that the system that they have described has value as a means to screen for factors influencing transcription of foreign genes in cyanobacteria.« less

Transduction of a Foreign Histocompatibility Gene into the Arterial Wall Induces Vasculitis

NASA Astrophysics Data System (ADS)

Nabel, Elizabeth G.; Plautz, Gregory; Nabel, Gary J.

1992-06-01

Autoimmune vasculitis represents a disease characterized by focal inflammation within arteries at multiple sites in the vasculature. Therapeutic interventions in this disease are empirical and often unsuccessful, and the mechanisms of immune injury are not well-defined. The direct transfer of recombinant genes and their expression in the arterial wall provides an opportunity to explore the pathogenesis and treatment of vascular disease. In this report, an animal model for vasculitis has been developed. Inflammation has been elicited by direct gene transfer of a foreign class I major histocompatibility complex gene, HLA-B7, to specific sites in porcine arteries. Transfer and expression of this recombinant gene was confirmed by a polymerase chain reaction and immunohistochemistry, and cytolytic T cells specific for HLA-B7 were detected. These findings demonstrate that expression of a recombinant gene in the vessel wall can induce a focal immune response and suggest that vessel damage induced by cell-mediated immune injury can initiate vasculitis.

[Prokaryotic expression, purification and antigenicity identification of recombinant human survivin protein].

PubMed

Yin, Xiaotao; Wang, Wei; Tian, Renli; Xu, Yuanji; Yan, Jinqi; Zhang, Wei; Gao, Jiangping; Yu, Jiyun

2013-08-01

To construct a prokaryotic expression plasmid pET28a-survivin, optimize the recombinant protein expression conditions in E.coli, and purify the survivin recombinant protein and identify its antigenicity. Survivin cDNA segment was amplified by PCR and cloned into prokaryotic expression vector pET28a(+) to construct the recombinant expression vector pET28a-survivin. The expression vector was transformed into BL21 (DE3) and the fusion protein survivin/His was induced by IPTG. The fusion protein was purified through Ni affinity chromatography. The antigenicity of the purified survivin protein was identified by Western blotting and ELISA. The recombinant expression vector was verified successfully by BamHI and HindIII. The fusion protein induced by IPTG was obtained with Mr; about 24 000. The purity of the purified protein reached 90% by SDS-PAGE analysis. And the antigenicity of the survivin protein was validated by Western blotting and ELISA. The prokaryotic expression plasmid pET28a-survivin was successfully constructed and the survivin protein was expressed and purified in E.coli. The antigenicity of the purified survivin protein was demonstrated desirable.

[Comparison of the English titles of articles in Chinese and foreign academic journals: influences of difference between China and the foreign in thinking and psychology].

PubMed

Wang, Jie-hua

2006-11-01

By analyzing the East-West difference in psychology and thinking patterns, the author compared the frequency of conventional words applied and the usage of subtitle, as well as the selection of sentence structure in title of articles, and thus advocated that the differences of attention psychology and thinking patterns, linear in the West and spiral in China, were the primary causes for different expressions of articles' title.

The Learning of Chinese Idiomatic Expressions as a Foreign Language

ERIC Educational Resources Information Center

Liu, Li; Yao, Jiayi

2017-01-01

Chinese idioms are mostly four-character phrases and are called Quadra-syllabic Idiomatic Expressions (QIEs). It has long been reported that learning of Chinese QIEs poses a great challenge for both young L1 speakers and adult L2 learners as the condensed form is often associated with complicated figurative meanings. The present study explored the…

L'Enquete linguistique et son application a la correction de l'expression orale (Linguistic Inquiry and Its Application to the Correction of Oral Expression)

ERIC Educational Resources Information Center

Niedzielski, Henri

1970-01-01

This article suggests applying the methods of linguistic investigation which dialectologists use in field work to the systematically programmed training of foreign language teachers in hearing and correcting the phonemic deviations in their students' pronunciation of the target language. (Author/FB)

Novel insights in genetic transformation of the probiotic yeast Saccharomyces boulardii.

PubMed

Douradinha, Bruno; Reis, Viviane C B; Rogers, Matthew B; Torres, Fernando A G; Evans, Jared D; Marques, Ernesto T A

2014-01-01

Saccharomyces boulardii (S. boulardii) is a probiotic yeast related to Saccharomyces cerevisiae (S. cerevisiae) but with distinct genetic, taxonomic and metabolic properties. S. cerevisiae has been used extensively in biotechnological applications. Currently, many strains are available, and multiple genetic tools have been developed, which allow the expression of several exogenous proteins of interest with applications in the fields of medicine, biofuels, the food industry, and scientific research, among others. Although S. boulardii has been widely studied due to its probiotic properties against several gastrointestinal tract disorders, very few studies addressed the use of this yeast as a vector for expression of foreign genes of interest with biotechnological applications. Here we show that, despite the similarity of the two yeasts, not all genetic tools used in S. cerevisiae can be applied in S. boulardii. While transformation of the latter could be obtained using a commercial kit developed for the former, consequent screening of successful transformants had to be optimized. We also show that several genes frequently used in genetic manipulation of S. cerevisiae (e.g., promoters and resistance markers) are present in S. boulardii. Sequencing revealed a high rate of homology (> 96%) between the orthologs of the two yeasts. However, we also observed some of them are not eligible to be targeted for transformation of S. boulardii. This work has important applications toward the potential of this probiotic yeast as an expression system for genes of interest.

7 CFR 319.56-49 - Eggplant from Israel.

Code of Federal Regulations, 2011 CFR

2011-01-01

... SERVICE, DEPARTMENT OF AGRICULTURE FOREIGN QUARANTINE NOTICES Fruits and Vegetables § 319.56-49 Eggplant...-exclusionary structures. APHIS-approved fruit fly traps with an approved protein bait must be placed inside the...

High-Throughput Protein Expression Using a Combination of Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein (IFP) Detection

PubMed Central

Dortay, Hakan; Akula, Usha Madhuri; Westphal, Christin; Sittig, Marie; Mueller-Roeber, Bernd

2011-01-01

Protein expression in heterologous hosts for functional studies is a cumbersome effort. Here, we report a superior platform for parallel protein expression in vivo and in vitro. The platform combines highly efficient ligation-independent cloning (LIC) with instantaneous detection of expressed proteins through N- or C-terminal fusions to infrared fluorescent protein (IFP). For each open reading frame, only two PCR fragments are generated (with three PCR primers) and inserted by LIC into ten expression vectors suitable for protein expression in microbial hosts, including Escherichia coli, Kluyveromyces lactis, Pichia pastoris, the protozoon Leishmania tarentolae, and an in vitro transcription/translation system. Accumulation of IFP-fusion proteins is detected by infrared imaging of living cells or crude protein extracts directly after SDS-PAGE without additional processing. We successfully employed the LIC-IFP platform for in vivo and in vitro expression of ten plant and fungal proteins, including transcription factors and enzymes. Using the IFP reporter, we additionally established facile methods for the visualisation of protein-protein interactions and the detection of DNA-transcription factor interactions in microtiter and gel-free format. We conclude that IFP represents an excellent reporter for high-throughput protein expression and analysis, which can be easily extended to numerous other expression hosts using the setup reported here. PMID:21541323

On the fate of orally ingested foreign DNA in mice: chromosomal association and placental transmission to the fetus.

PubMed

Schubbert, R; Hohlweg, U; Renz, D; Doerfler, W

1998-10-01

We have previously shown that, when administered orally to mice, bacteriophage M13 DNA, as a paradigm foreign DNA without homology to the mouse genome, can persist in fragmented form in the gastrointestinal tract, penetrate the intestinal wall, and reach the nuclei of leukocytes, spleen and liver cells. Similar results were obtained when a plasmid containing the gene for the green fluorescent protein (pEGFP-C1) was fed to mice. In spleen, the foreign DNA was detected in covalent linkage to DNA with a high degree of homology to mouse genes, perhaps pseudogenes, or to authentic E. coli DNA. We have now extended these studies to the offspring of mice that were fed regularly during pregnancy with a daily dose of 50 microg of M13 or pEGFP-C1 DNA. Using the polymerase chain reaction (PCR) or the fluorescent in situ hybridization (FISH) method, foreign DNA, orally ingested by pregnant mice, can be discovered in various organs of fetuses and of newborn animals. The M13 DNA fragments have a length of about 830 bp. In various organs of the mouse fetus, clusters of cells contain foreign DNA as revealed by FISH. The foreign DNA is invariably located in the nuclei. We have never found all cells of the fetus to be transgenic for the foreign DNA. This distribution pattern argues for a transplacental pathway rather than for germline transmission which might be expected only after long-time feeding regimens. In rare cells of three different fetuses, whose mothers have been fed with M 13 DNA during gestation, the foreign DNA was detected by FISH in association with both chromatids. Is maternally ingested foreign DNA a potential mutagen for the developing fetus?

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 group. The consequences of the reported changes in protein expression require further study. PMID:25328384

Exploitation of stable nanostructures based on the mouse polyomavirus for development of a recombinant vaccine against porcine circovirus 2

PubMed Central

Fraiberk, Martin; Hájková, Michaela; Krulová, Magdaléna; Kojzarová, Martina; Drda Morávková, Alena; Pšikal, Ivan

2017-01-01

The aim of this study was to develop a suitable vaccine antigen against porcine circovirus 2 (PCV2), the causative agent of post-weaning multi-systemic wasting syndrome, which causes significant economic losses in swine breeding. Chimeric antigens containing PCV2b Cap protein sequences based on the mouse polyomavirus (MPyV) nanostructures were developed. First, universal vectors for baculovirus-directed production of chimeric MPyV VLPs or pentamers of the major capsid protein, VP1, were designed for their exploitation as vaccines against other pathogens. Various strategies were employed based on: A) exposure of selected immunogenic epitopes on the surface of MPyV VLPs by insertion into a surface loop of the VP1 protein, B) insertion of foreign protein molecules inside the VLPs, or C) fusion of a foreign protein or its part with the C-terminus of VP1 protein, to form giant pentamers of a chimeric protein. We evaluated these strategies by developing a recombinant vaccine against porcine circovirus 2. All candidate vaccines induced the production of antibodies against the capsid protein of porcine circovirus after immunization of mice. The candidate vaccine, Var C, based on fusion of mouse polyomavirus and porcine circovirus capsid proteins, could induce the production of antibodies with the highest PCV2 neutralizing capacity. Its ability to induce the production of neutralization antibodies was verified after immunization of pigs. The advantage of this vaccine, apart from its efficient production in insect cells and easy purification, is that it represents a DIVA (differentiating infected from vaccinated animals) vaccine, which also induces an immune response against the mouse polyoma VP1 protein and is thus able to distinguish between vaccinated and naturally infected animals. PMID:28922413

Assessing the utility of gene co-expression stability in combination with correlation in the analysis of protein-protein interaction networks

PubMed Central

2011-01-01

Background Gene co-expression, in the form of a correlation coefficient, has been valuable in the analysis, classification and prediction of protein-protein interactions. However, it is susceptible to bias from a few samples having a large effect on the correlation coefficient. Gene co-expression stability is a means of quantifying this bias, with high stability indicating robust, unbiased co-expression correlation coefficients. We assess the utility of gene co-expression stability as an additional measure to support the co-expression correlation in the analysis of protein-protein interaction networks. Results We studied the patterns of co-expression correlation and stability in interacting proteins with respect to their interaction promiscuity, levels of intrinsic disorder, and essentiality or disease-relatedness. Co-expression stability, along with co-expression correlation, acts as a better classifier of hub proteins in interaction networks, than co-expression correlation alone, enabling the identification of a class of hubs that are functionally distinct from the widely accepted transient (date) and obligate (party) hubs. Proteins with high levels of intrinsic disorder have low co-expression correlation and high stability with their interaction partners suggesting their involvement in transient interactions, except for a small group that have high co-expression correlation and are typically subunits of stable complexes. Similar behavior was seen for disease-related and essential genes. Interacting proteins that are both disordered have higher co-expression stability than ordered protein pairs. Using co-expression correlation and stability, we found that transient interactions are more likely to occur between an ordered and a disordered protein while obligate interactions primarily occur between proteins that are either both ordered, or disordered. Conclusions We observe that co-expression stability shows distinct patterns in structurally and functionally different groups of proteins and interactions. We conclude that it is a useful and important measure to be used in concert with gene co-expression correlation for further insights into the characteristics of proteins in the context of their interaction network. PMID:22369639

Sleeping Beauty transposon-based system for rapid generation of HBV-replicating stable cell lines.

PubMed

Wu, Yong; Zhang, Tian-Ying; Fang, Lin-Lin; Chen, Zi-Xuan; Song, Liu-Wei; Cao, Jia-Li; Yang, Lin; Yuan, Quan; Xia, Ning-Shao

2016-08-01

The stable HBV-replicating cell lines, which carry replication-competent HBV genome stably integrated into the genome of host cell, are widely used to evaluate the effects of antiviral agents. However, current methods to generate HBV-replicating cell lines, which are mostly dependent on random integration of foreign DNA via plasmid transfection, are less-efficient and time-consuming. To address this issue, we constructed an all-in-one Sleeping Beauty transposon system (denoted pTSMP-HBV vector) for robust generation of stable cell lines carrying replication-competent HBV genome of different genotype. This vector contains a Sleeping Beauty transposon containing HBV 1.3-copy genome with an expression cassette of the SV40 promoter driving red fluorescent protein (mCherry) and self-cleaving P2A peptide linked puromycin resistance gene (PuroR). In addition, a PGK promoter-driven SB100X hyperactive transposase cassette is placed in the outside of the transposon in the same plasmid.The HBV-replicating stable cells could be obtained from pTSMP-HBV transfected HepG2 cells by red fluorescence-activated cell sorting and puromycin resistant cell selection within 4-week. Using this system, we successfully constructed four cell lines carrying replication-competent HBV genome of genotypes A-D. The replication and viral protein expression profiles of these cells were systematically characterized. In conclusion, our study provides a high-efficiency strategy to generate HBV-replicating stable cell lines, which may facilitate HBV-related virological study. Copyright © 2016. Published by Elsevier B.V.

The prognosis was poorer in colorectal cancers that expressed both VEGF and PROK1 (No correlation coefficient between VEGF and PROK1).

PubMed

Goi, Takanori; Nakazawa, Toshiyuki; Hirono, Yasuo; Yamaguchi, Akio

2015-10-06

The angiogenic proteins vascular endothelial growth factor (VEGF) and prokineticin1 (PROK1) proteins are considered important in colorectal cancer, the relationship between their simultaneous expression and prognosis was investigated in the present study. VEGF and PROK1 expression in 620 primary human colorectal cancer lesions was confirmed via immunohistochemical staining with anti-VEGF and anti-PROK1 antibodies, and the correlation between the expression of these 2 proteins and recurrence/prognosis were investigated. VEGF protein was expressed in 329 (53.1%) and PROK1 protein was expressed in 223 (36.0%). PROK1 and VEGF were simultaneously expressed in 116 (18.7%) of the 620 cases. The correlation coefficient between VEGF expression and PROK1 expression was r = 0.11, and therefore correlation was not observed. Clinical pathology revealed that substantially lymphnode matastasis, hematogenous metastasis, or TMN advanced-stage IV was significantly more prevalent in cases that expressed both VEGF and PROK1 than in the cases negative for both proteins or those positive for only 1 of the proteins. Also the cases positive for both proteins exhibited the worst recurrence and prognosis. In the Cox proportional hazards model, VEGF and PROK1 expression was an independent prognostic factor. The prognosis was poorer in colorectal cancers that expressed both PROK1 and VEGF relative to the cases that expressed only 1 protein, and the expression of both proteins was found to be an independent prognostic factor.

Recombinant Expression Screening of P. aeruginosa Bacterial Inner Membrane Proteins

PubMed Central

2010-01-01

Background Transmembrane proteins (TM proteins) make up 25% of all proteins and play key roles in many diseases and normal physiological processes. However, much less is known about their structures and molecular mechanisms than for soluble proteins. Problems in expression, solubilization, purification, and crystallization cause bottlenecks in the characterization of TM proteins. This project addressed the need for improved methods for obtaining sufficient amounts of TM proteins for determining their structures and molecular mechanisms. Results Plasmid clones were obtained that encode eighty-seven transmembrane proteins with varying physical characteristics, for example, the number of predicted transmembrane helices, molecular weight, and grand average hydrophobicity (GRAVY). All the target proteins were from P. aeruginosa, a gram negative bacterial opportunistic pathogen that causes serious lung infections in people with cystic fibrosis. The relative expression levels of the transmembrane proteins were measured under several culture growth conditions. The use of E. coli strains, a T7 promoter, and a 6-histidine C-terminal affinity tag resulted in the expression of 61 out of 87 test proteins (70%). In this study, proteins with a higher grand average hydrophobicity and more transmembrane helices were expressed less well than less hydrophobic proteins with fewer transmembrane helices. Conclusions In this study, factors related to overall hydrophobicity and the number of predicted transmembrane helices correlated with the relative expression levels of the target proteins. Identifying physical characteristics that correlate with protein expression might aid in selecting the "low hanging fruit", or proteins that can be expressed to sufficient levels using an E. coli expression system. The use of other expression strategies or host species might be needed for sufficient levels of expression of transmembrane proteins with other physical characteristics. Surveys like this one could aid in overcoming the technical bottlenecks in working with TM proteins and could potentially aid in increasing the rate of structure determination. PMID:21114855

Random heteropolymers preserve protein function in foreign environments

NASA Astrophysics Data System (ADS)

Panganiban, Brian; Qiao, Baofu; Jiang, Tao; DelRe, Christopher; Obadia, Mona M.; Nguyen, Trung Dac; Smith, Anton A. A.; Hall, Aaron; Sit, Izaac; Crosby, Marquise G.; Dennis, Patrick B.; Drockenmuller, Eric; Olvera de la Cruz, Monica; Xu, Ting

2018-03-01

The successful incorporation of active proteins into synthetic polymers could lead to a new class of materials with functions found only in living systems. However, proteins rarely function under the conditions suitable for polymer processing. On the basis of an analysis of trends in protein sequences and characteristic chemical patterns on protein surfaces, we designed four-monomer random heteropolymers to mimic intrinsically disordered proteins for protein solubilization and stabilization in non-native environments. The heteropolymers, with optimized composition and statistical monomer distribution, enable cell-free synthesis of membrane proteins with proper protein folding for transport and enzyme-containing plastics for toxin bioremediation. Controlling the statistical monomer distribution in a heteropolymer, rather than the specific monomer sequence, affords a new strategy to interface with biological systems for protein-based biomaterials.

Receipt of surgical treatment in US women with early stage breast cancer: does place of birth matter?

PubMed

Chavan, Saurabh; Goodman, Michael; Jemal, Ahmedin; Fedewa, Stacey A

2014-01-01

While effects of age, race, place of residence, and marital status on receipt of treatment among female breast cancer patients have been well documented, place of birth is a relatively less studied factor. The purpose of our study was to assess the relationship between birth place and type of surgery performed for early-stage breast cancer among US women of different racial and ethnic backgrounds. Eligible cases (n=119,560) were selected from the SEER registries for the period 2004-2009. US-born and foreign-born patients of different racial/ethnic groups were compared to US-born non-Hispanic Whites (NHW) with respect to receipt of breast conserving surgery (BCS) or mastectomy. Results of multivariable logistic regression analyses were expressed as adjusted odds ratios (OR) and the corresponding 95% confidence intervals (CI). The proportion of BCS was highest in foreign-born Whites (62.5%) and lowest in foreign-born Asians (50.3%). Relative to US-born NHW, BCS was more common in foreign-born Whites (OR=1.21. 95% CI: 1.15-1.28) and foreign-born Blacks (OR=1.21. 95% CI: 1.15-1.28). In contrast, foreign-born Asians received less BCS compared to both US-born NHW (OR=.76, 95% CI: .72-0.80) and US-born Asians (OR=.74, 95% CI: .64-.86). Foreign-born Asian breast cancer patients are less likely to receive BSC compared to US-born Whites or Asian-Americans, whereas foreign-born Whites and foreign-born Blacks are more likely to receive BCS than US-born Whites. Further studies are needed to understand cultural and or health systems factors that may explain these observations.

Construction and characterization of 3A-epitope-tagged foot-and-mouth disease virus.

PubMed

Ma, Xueqing; Li, Pinghua; Sun, Pu; Bai, Xingwen; Bao, Huifang; Lu, Zengjun; Fu, Yuanfang; Cao, Yimei; Li, Dong; Chen, Yingli; Qiao, Zilin; Liu, Zaixin

2015-04-01

Nonstructural protein 3A of foot-and-mouth disease virus (FMDV) is a partially conserved protein of 153 amino acids (aa) in most FMDVs examined to date. Specific deletion in the FMDV 3A protein has been associated with the inability of FMDV to grow in primary bovine cells and cause disease in cattle. However, the aa residues playing key roles in these processes are poorly understood. In this study, we constructed epitope-tagged FMDVs containing an 8 aa FLAG epitope, a 9 aa haemagglutinin (HA) epitope, and a 10 aa c-Myc epitope to substitute residues 94-101, 93-101, and 93-102 of 3A protein, respectively, using a recently developed O/SEA/Mya-98 FMDV infectious cDNA clone. Immunofluorescence assay (IFA), Western blot and sequence analysis showed that the epitope-tagged viruses stably maintained and expressed the foreign epitopes even after 10 serial passages in BHK-21 cells. The epitope-tagged viruses displayed growth properties and plaque phenotypes similar to those of the parental virus in BHK-21 cells. However, the epitope-tagged viruses exhibited lower growth rates and smaller plaque size phenotypes than those of the parental virus in primary fetal bovine kidney (FBK) cells, but similar growth properties and plaque phenotypes to those of the recombinant viruses harboring 93-102 deletion in 3A. These results demonstrate that the decreased ability of FMDV to replicate in primary bovine cells was not associated with the length of 3A, and the genetic determinant thought to play key role in decreased ability to replicate in primary bovine cells could be reduced from 93-102 residues to 8 aa residues at positions 94-101 in 3A protein. Copyright © 2015 Elsevier B.V. All rights reserved.

Redirecting the Cyanobacterial Bicarbonate Transporters BicA and SbtA to the Chloroplast Envelope: Soluble and Membrane Cargos Need Different Chloroplast Targeting Signals in Plants

PubMed Central

Rolland, Vivien; Badger, Murray R.; Price, G. Dean

2016-01-01

Most major crops used for human consumption are C3 plants, which yields are limited by photosynthetic inefficiency. To circumvent this, it has been proposed to implement the cyanobacterial CO2-concentrating mechanism (CCM), principally consisting of bicarbonate transporters and carboxysomes, into plant chloroplasts. As it is currently not possible to recover homoplasmic transplastomic monocots, foreign genes must be introduced in these plants via nuclear transformation. Consequently, it is paramount to ensure that resulting proteins reach the appropriate sub-cellular compartment, which for cyanobacterial transporters BicA and SbtA, is the chloroplast inner-envelope membrane (IEM). At present, targeting signals to redirect large transmembrane proteins from non-chloroplastic organisms to plant chloroplast envelopes are unknown. The goal of this study was to identify such signals, using agrobacteria-mediated transient expression and confocal microscopy to determine the sub-cellular localization of ∼37 GFP-tagged chimeras. Initially, fragments of chloroplast proteins known to target soluble cargos to the stroma were tested for their ability to redirect BicA, but they proved ineffective. Next, different N-terminal regions from Arabidopsis IEM transporters were tested. We demonstrated that the N-terminus of AtHP59, AtPLGG1 or AtNTT1 (92–115 amino acids), containing a cleavable chloroplast transit peptide (cTP) and a membrane protein leader (MPL), was sufficient to redirect BicA or SbtA to the chloroplast envelope. This constitutes the first evidence that nuclear-encoded transmembrane proteins from non-chloroplastic organisms can be targeted to the envelope of plant chloroplasts; a finding which represents an important advance in chloroplast engineering by opening up the door to further manipulation of the chloroplastic envelope. PMID:26973659

The Effects of L2 Proficiency on Pragmatics Instruction: A Web-Based Approach to Teaching Chinese Expressions of Gratitude

ERIC Educational Resources Information Center

Yang, Li

2017-01-01

This study investigated whether the effects of pragmatics instruction delivered via a self-access website in a Chinese as a foreign language learning environment vary according to learners' language proficiency. The website provided learners with explicit instruction in how to express gratitude appropriately in Chinese and offered them pragmatic…

Was the appearance of surfactants in air breathing vertebrates ultimately the cause of decompression sickness and autoimmune disease?

PubMed

Arieli, Ran

2015-01-15

All air breathing vertebrates are endowed with pulmonary surfactants, surface-active lipoprotein complexes formed by type II alveolar cells. Surfactants are deposited in clearly defined areas on the luminal aspect of blood vessels, producing hydrophobic spots. Gas nanobubbles measuring 5-100nm form spontaneously on the smooth hydrophobic spot from dissolved gas. Bubbles nucleate and grow at these spots after decompression from high pressure. Proteins with hydrophobic regions circulating in the blood will adhere to the gas phase-plasma interface. Deformation of their secondary and tertiary configuration will present them as foreign molecules or autoantigens. Components of the intact protein which are also present in a deformed protein may be recognized as foreign too. This process is proposed as the trigger for autoimmune diseases. The presence of autoimmune disease in air breathing vertebrates, increased autoimmunity and the elevated risk of decompression sickness with age, as well as variable sensitivity to both diseases, can be matched with the appearance of surfactant spots. Eliminating these spots may provide protection against both diseases. Copyright © 2014 Elsevier B.V. All rights reserved.

Thymidylate synthase (TS) protein expression as a prognostic factor in advanced colorectal cancer: a comparison with TS mRNA expression.

PubMed

Nakagawa, Tateo; Shimada, Mitsuo; Kurita, Nobuhiro; Iwata, Takashi; Nishioka, Masanori; Yoshikawa, Kozo; Higashijima, Jun; Utsunomiya, Tohru

2012-06-01

The role of intratumoral thymidylate synthase (TS) mRNA or protein expression is still controversial and little has been reported regarding relation of them in colorectal cancer. Forty-six patients with advanced colorectal cancer who underwent surgical resection were included. TS mRNA expression was determined by the Danenberg tumor profile method based on laser-captured micro-dissection of the tumor cells. TS protein expression was evaluated using immunohistochemical staining. TS mRNA expression tended to relate TS protein expression. Statistical significance was not found in overall survival between the TS mRNA high group and low group regardless of performing adjuvant chemotherapy. The overall survival in the TS protein negative group was significantly higher than that in positive group in all and the patients without adjuvant chemotherapy. Multivariate analysis showed TS protein expression was as an independent prognostic factor. TS protein expression tends to be related TS mRNA expression and is an independent prognostic factor in advanced colorectal cancer.

Needs, Acceptability, and Value of Humanitarian Medical Assistance in Remote Peruvian Amazon Riverine Communities

PubMed Central

Sanchez, Juan F.; Halsey, Eric S.; Bayer, Angela M.; Beltran, Martin; Razuri, Hugo R.; Velasquez, Daniel E.; Cama, Vitaliano A.; Graf, Paul C. F.; Quispe, Antonio M.; Maves, Ryan C.; Montgomery, Joel M.; Sanders, John W.; Lescano, Andres G.

2015-01-01

Much debate exists regarding the need, acceptability, and value of humanitarian medical assistance. We conducted a cross-sectional study on 457 children under 5 years from four remote riverine communities in the Peruvian Amazon and collected anthropometric measures, blood samples (1–4 years), and stool samples. Focus groups and key informant interviews assessed perspectives regarding medical aid delivered by foreigners. The prevalence of stunting, anemia, and intestinal parasites was 20%, 37%, and 62%, respectively. Infection with multiple parasites, usually geohelminths, was detected in 41% of children. The prevalence of intestinal parasites both individual and polyparasitism increased with age. Participants from smaller communities less exposed to foreigners expressed lack of trust and fear of them. However, participants from all communities were positive about foreigners visiting to provide health support. Prevalent health needs such as parasitic infections and anemia may be addressed by short-term medical interventions. There is a perceived openness to and acceptability of medical assistance delivered by foreign personnel. PMID:25846293

Construction and characterization of recombinant flaviviruses bearing insertions between E and NS1 genes

PubMed Central

Bonaldo, Myrna C; Mello, Samanta M; Trindade, Gisela F; Rangel, Aymara A; Duarte, Adriana S; Oliveira, Prisciliana J; Freire, Marcos S; Kubelka, Claire F; Galler, Ricardo

2007-01-01

Background The yellow fever virus, a member of the genus Flavivirus, is an arthropod-borne pathogen causing severe disease in humans. The attenuated yellow fever 17D virus strain has been used for human vaccination for 70 years and has several characteristics that are desirable for the development of new, live attenuated vaccines. We described here a methodology to construct a viable, and immunogenic recombinant yellow fever 17D virus expressing a green fluorescent protein variant (EGFP). This approach took into account the presence of functional motifs and amino acid sequence conservation flanking the E and NS1 intergenic region to duplicate and fuse them to the exogenous gene and thereby allow the correct processing of the viral polyprotein precursor. Results YF 17D EGFP recombinant virus was grew in Vero cells and reached a peak titer of approximately 6.45 ± 0.4 log10 PFU/mL at 96 hours post-infection. Immunoprecipitation and confocal laser scanning microscopy demonstrated the expression of the EGFP, which was retained in the endoplasmic reticulum and not secreted from infected cells. The association with the ER compartment did not interfere with YF assembly, since the recombinant virus was fully competent to replicate and exit the cell. This virus was genetically stable up to the tenth serial passage in Vero cells. The recombinant virus was capable to elicit a neutralizing antibody response to YF and antibodies to EGFP as evidenced by an ELISA test. The applicability of this cloning strategy to clone gene foreign sequences in other flavivirus genomes was demonstrated by the construction of a chimeric recombinant YF 17D/DEN4 virus. Conclusion This system is likely to be useful for a broader live attenuated YF 17D virus-based vaccine development for human diseases. Moreover, insertion of foreign genes into the flavivirus genome may also allow in vivo studies on flavivirus cell and tissue tropism as well as cellular processes related to flavivirus infection. PMID:17971212

Expression of the oncoprotein gankyrin and phosphorylated retinoblastoma protein in human testis and testicular germ cell tumor.

PubMed

Ando, Satoshi; Matsuoka, Taeko; Kawai, Koji; Sugita, Shintaro; Joraku, Akira; Kojima, Takahiro; Suetomi, Takahiro; Miyazaki, Jun; Fujita, Jun; Nishiyama, Hiroyuki

2014-10-01

The oncoprotein, gankyrin, is known to facilitate cell proliferation through phosphorylation and degradation of retinoblastoma protein. In the present study, we evaluated the expression of gankyrin and phosphorylated retinoblastoma protein in human testis and testicular germ cell tumors. The effects of suppression of gankyrin by locked nucleic acid on phosphorylation status of retinoblastoma and cell proliferation were analyzed using western blot analysis and testicular tumor cell line NEC8. The expressions of gankyrin, retinoblastoma and retinoblastoma protein were analyzed in 93 testicular germ cell tumor samples and five normal human testis by immunohistochemistry. The retinoblastoma protein expression was determined using an antibody to retinoblastoma protein, Ser795. Gankyrin was expressed in NEC8 cells as well as a normal human testis and testicular tumors. Suppression of gankyrin by locked nucleic acid led to suppression of retinoblastoma protein and cell proliferation in NEC8 cells. Immunohistochemistry of normal testis showed that gankyrin is expressed dominantly in spermatocytes. In testicular germ cell tumors, high expressions of gankyrin and phosphorylated-retinoblastoma protein were observed in seminoma and embryonal carcinoma, whereas the expressions of both proteins were weak in histological subtypes of non-seminoma. Growing teratoma and testicular malignant transformation tissues expressed phosphorylated-retinoblastoma protein strongly, but gankyrin faintly. Gankyrin is dominantly expressed in normal spermatocytes and seminoma/embryonal carcinoma, and its expression correlates well with retinoblastoma protein expression except in the growing teratoma and testicular malignant transformation cases. These data provide new insights into the molecular mechanisms of normal spermatogenesis and pathogenesis of testicular germ cell tumors. © 2014 The Japanese Urological Association.

Colloquium paper: uniquely human evolution of sialic acid genetics and biology.

PubMed

Varki, Ajit

2010-05-11

Darwinian evolution of humans from our common ancestors with nonhuman primates involved many gene-environment interactions at the population level, and the resulting human-specific genetic changes must contribute to the "Human Condition." Recent data indicate that the biology of sialic acids (which directly involves less than 60 genes) shows more than 10 uniquely human genetic changes in comparison with our closest evolutionary relatives. Known outcomes are tissue-specific changes in abundant cell-surface glycans, changes in specificity and/or expression of multiple proteins that recognize these glycans, and novel pathogen regimes. Specific events include Alu-mediated inactivation of the CMAH gene, resulting in loss of synthesis of the Sia N-glycolylneuraminic acid (Neu5Gc) and increase in expression of the precursor N-acetylneuraminic acid (Neu5Ac); increased expression of alpha2-6-linked Sias (likely because of changed expression of ST6GALI); and multiple changes in SIGLEC genes encoding Sia-recognizing Ig-like lectins (Siglecs). The last includes binding specificity changes (in Siglecs -5, -7, -9, -11, and -12); expression pattern changes (in Siglecs -1, -5, -6, and -11); gene conversion (SIGLEC11); and deletion or pseudogenization (SIGLEC13, SIGLEC14, and SIGLEC16). A nongenetic outcome of the CMAH mutation is human metabolic incorporation of foreign dietary Neu5Gc, in the face of circulating anti-Neu5Gc antibodies, generating a novel "xeno-auto-antigen" situation. Taken together, these data suggest that both the genes associated with Sia biology and the related impacts of the environment comprise a relative "hot spot" of genetic and physiological changes in human evolution, with implications for uniquely human features both in health and disease.

Metabolic engineering of Chinese hamster ovary cells: Towards a bioengineered heparin

PubMed Central

Baik, Jong Youn; Gasimli, Leyla; Yang, Bo; Datta, Payel; Zhang, Fuming; Glass, Charles A.; Esko, Jeffrey D.; Linhardt, Robert J.; Sharfstein, Susan T.

2012-01-01

Heparin is the most widely used pharmaceutical to control blood coagulation in modern medicine. A health crisis that took place in 2008 led to a demand for production of heparin from non-animal sources. Chinese hamster ovary (CHO) cells, commonly used mammalian host cells for production of foreign pharmaceutical proteins in the biopharmaceutical industry, are capable of producing heparan sulfate (HS), a related polysaccharide naturally. Since heparin and HS share the same biosynthetic pathway, we hypothesized that heparin could be produced in CHO cells by metabolic engineering. Based on the expression of endogenous enzymes in the HS / heparin pathways of CHO-S cells, human N-deacetylase/N-sulfotransferase (NDST2) and mouse heparan sulfate 3-O-sulfotransferase 1 (Hs3st1) genes were transfected sequentially into CHO host cells growing in suspension culture. Transfectants were screened using quantitative RT-PCR and Western blotting. Out of 120 clones expressing NDST2 and Hs3st1, 2 clones, Dual-3 and Dual-29, were selected for further analysis. An antithrombin III (ATIII) binding assay using flow cytometry, designed to recognize a key sugar structure characteristic of heparin, indicated that Hs3st1 transfection was capable of increasing ATIII binding. An anti-factor Xa assay, which affords a measure of anticoagulant activity, showed a significant increase in activity in the dual-expressing cell lines. Disaccharide analysis of the engineered HS showed a substantial increase in N-sulfo groups, but did not show a pattern consistent with pharmacological heparin, suggesting that further balancing the expression of transgenes with the expression levels of endogenous enzymes involved in HS / heparin biosynthesis might be necessary. PMID:22326251

Metabolic engineering of Chinese hamster ovary cells: towards a bioengineered heparin.

PubMed

Baik, Jong Youn; Gasimli, Leyla; Yang, Bo; Datta, Payel; Zhang, Fuming; Glass, Charles A; Esko, Jeffrey D; Linhardt, Robert J; Sharfstein, Susan T

2012-03-01

Heparin is the most widely used pharmaceutical to control blood coagulation in modern medicine. A health crisis that took place in 2008 led to a demand for production of heparin from non-animal sources. Chinese hamster ovary (CHO) cells, commonly used mammalian host cells for production of foreign pharmaceutical proteins in the biopharmaceutical industry, are capable of producing heparan sulfate (HS), a related polysaccharide naturally. Since heparin and HS share the same biosynthetic pathway, we hypothesized that heparin could be produced in CHO cells by metabolic engineering. Based on the expression of endogenous enzymes in the HS/heparin pathways of CHO-S cells, human N-deacetylase/N-sulfotransferase (NDST2) and mouse heparan sulfate 3-O-sulfotransferase 1 (Hs3st1) genes were transfected sequentially into CHO host cells growing in suspension culture. Transfectants were screened using quantitative RT-PCR and Western blotting. Out of 120 clones expressing NDST2 and Hs3st1, 2 clones, Dual-3 and Dual-29, were selected for further analysis. An antithrombin III (ATIII) binding assay using flow cytometry, designed to recognize a key sugar structure characteristic of heparin, indicated that Hs3st1 transfection was capable of increasing ATIII binding. An anti-factor Xa assay, which affords a measure of anticoagulant activity, showed a significant increase in activity in the dual-expressing cell lines. Disaccharide analysis of the engineered HS showed a substantial increase in N-sulfo groups, but did not show a pattern consistent with pharmacological heparin, suggesting that further balancing the expression of transgenes with the expression levels of endogenous enzymes involved in HS/heparin biosynthesis might be necessary. Copyright © 2012 Elsevier Inc. All rights reserved.

Differential Protein Expressions in Virus-Infected and Uninfected Trichomonas vaginalis.

PubMed

He, Ding; Pengtao, Gong; Ju, Yang; Jianhua, Li; He, Li; Guocai, Zhang; Xichen, Zhang

2017-04-01

Protozoan viruses may influence the function and pathogenicity of the protozoa. Trichomonas vaginalis is a parasitic protozoan that could contain a double stranded RNA (dsRNA) virus, T. vaginalis virus (TVV). However, there are few reports on the properties of the virus. To further determine variations in protein expression of T. vaginalis , we detected 2 strains of T. vaginalis ; the virus-infected (V + ) and uninfected (V - ) isolates to examine differentially expressed proteins upon TVV infection. Using a stable isotope N-terminal labeling strategy (iTRAQ) on soluble fractions to analyze proteomes, we identified 293 proteins, of which 50 were altered in V + compared with V - isolates. The results showed that the expression of 29 proteins was increased, and 21 proteins decreased in V + isolates. These differentially expressed proteins can be classified into 4 categories: ribosomal proteins, metabolic enzymes, heat shock proteins, and putative uncharacterized proteins. Quantitative PCR was used to detect 4 metabolic processes proteins: glycogen phosphorylase, malate dehydrogenase, triosephosphate isomerase, and glucose-6-phosphate isomerase, which were differentially expressed in V + and V - isolates. Our findings suggest that mRNA levels of these genes were consistent with protein expression levels. This study was the first which analyzed protein expression variations upon TVV infection. These observations will provide a basis for future studies concerning the possible roles of these proteins in host-parasite interactions.

SPLICEFINDER – A Fast and Easy Screening Method for Active Protein Trans-Splicing Positions

PubMed Central

Eppmann, Simone; Busche, Alena; Dikovskaya, Dina; Dötsch, Volker; Mootz, Henning D.

2013-01-01

Split intein enabled protein trans-splicing (PTS) is a powerful method for the ligation of two protein fragments, thereby paving the way for various protein modification or protein function control applications. PTS activity is strongly influenced by the amino acids directly flanking the splice junctions. However, to date no reliable prediction can be made whether or not a split intein is active in a particular foreign extein context. Here we describe SPLICEFINDER, a PCR-based method, allowing fast and easy screening for active split intein insertions in any target protein. Furthermore we demonstrate the applicability of SPLICEFINDER for segmental isotopic labeling as well as for the generation of multi-domain and enzymatically active proteins. PMID:24023792

Advances in recombinant protein expression for use in pharmaceutical research.

PubMed

Assenberg, Rene; Wan, Paul T; Geisse, Sabine; Mayr, Lorenz M

2013-06-01

Protein production for structural and biophysical studies, functional assays, biomarkers, mechanistic studies in vitro and in vivo, but also for therapeutic applications in pharma, biotech and academia has evolved into a mature discipline in recent years. Due to the increased emphasis on biopharmaceuticals, the growing demand for proteins used for structural and biophysical studies, the impact of genomics technologies on the analysis of large sets of structurally diverse proteins, and the increasing complexity of disease targets, the interest in innovative approaches for the expression, purification and characterisation of recombinant proteins has steadily increased over the years. In this review, we summarise recent developments in the field of recombinant protein expression for research use in pharma, biotech and academia. We focus mostly on the latest developments for protein expression in the most widely used expression systems: Escherichia coli (E. coli), insect cell expression using the Baculovirus Expression Vector System (BEVS) and, finally, transient and stable expression of recombinant proteins in mammalian cells. Copyright © 2013. Published by Elsevier Ltd.

High-yield, in vitro protein expression using a continuous-exchange, coupled transcription/ translation system.

PubMed

Martin, G A; Kawaguchi, R; Lam, Y; DeGiovanni, A; Fukushima, M; Mutter, W

2001-10-01

The Rapid Translation System (RTS 500) (Roche Molecular Biochemicals) is a high-yield protein expression system that utilizes an enhanced E. coli lysate for an in vitro transcription/translation reaction. In contrast to conventional transcription/translation, this system allows protein expression to continue for more than 24 h. We demonstrated the utility of the RTS 500 by expressing different soluble and active proteins that generally pose problems in cell-based expression systems. We first expressed GFP-lunasin, a fusion protein that, because of its toxicity, has been impossible to produce in whole cells. The second protein we expressed, human interleukin-2 (IL-2), is generally difficult to produce, either as the native molecule or as a GSTfusion protein, in a soluble form in bacteria. Finally, we demonstrated the capacity of the RTS 500 to co-express proteins, by the simultaneous production of GFP and CAT in a single reaction. This new technology appears to be particularly usefulfor the convenient production of preparative amounts (100-900 microg) of proteins that are toxic or insoluble in cell-based systems.

[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.

The growing impact of lyophilized cell-free protein expression systems

PubMed Central

Hunt, J. Porter; Yang, Seung Ook; Wilding, Kristen M.; Bundy, Bradley C.

2017-01-01

ABSTRACT Recently reported shelf-stable, on-demand protein synthesis platforms are enabling new possibilities in biotherapeutics, biosensing, biocatalysis, and high throughput protein expression. Lyophilized cell-free protein expression systems not only overcome cold-storage limitations, but also enable stockpiling for on-demand synthesis and completely sterilize the protein synthesis platform. Recently reported high-yield synthesis of cytotoxic protein Onconase from lyophilized E. coli extract preparations demonstrates the utility of lyophilized cell-free protein expression and its potential for creating on-demand biotherapeutics, vaccines, biosensors, biocatalysts, and high throughput protein synthesis. PMID:27791452

RRE-dependent HIV-1 Env RNA effects on Gag protein expression, assembly and release

DOE Office of Scientific and Technical Information (OSTI.GOV)

López, Claudia S., E-mail: lopezcl@ohsu.edu; Sloan, Rachel; Cylinder, Isabel

The HIV-1 Gag proteins are translated from the full-length HIV-1 viral RNA (vRNA), whereas the envelope (Env) protein is translated from incompletely spliced Env mRNAs. Nuclear export of vRNAs and Env mRNAs is mediated by the Rev accessory protein which binds to the rev-responsive element (RRE) present on these RNAs. Evidence has shown there is a direct or indirect interaction between the Gag protein, and the cytoplasmic tail (CT) of the Env protein. Our current work shows that env gene expression impacts HIV-1 Gag expression and function in two ways. At the protein level, full-length Env expression altered Gag proteinmore » expression, while Env CT-deletion proteins did not. At the RNA level, RRE-containing Env mRNA expression reduced Gag expression, processing, and virus particle release from cells. Our results support models in which Gag is influenced by the Env CT, and Env mRNAs compete with vRNAs for nuclear export. - Highlights: • At the protein level, full-length HIV-1 Env alters Gag protein expression. • HIV-1 Env RNA expression reduces Gag levels and virus release. • Env RNA effects on Gag are dependent on the RRE. • RRE-containing Env RNAs compete with vRNAs for nuclear export.« less

Similar protein expression profiles of ovarian and endometrial high-grade serous carcinomas.

PubMed

Hiramatsu, Kosuke; Yoshino, Kiyoshi; Serada, Satoshi; Yoshihara, Kosuke; Hori, Yumiko; Fujimoto, Minoru; Matsuzaki, Shinya; Egawa-Takata, Tomomi; Kobayashi, Eiji; Ueda, Yutaka; Morii, Eiichi; Enomoto, Takayuki; Naka, Tetsuji; Kimura, Tadashi

2016-03-01

Ovarian and endometrial high-grade serous carcinomas (HGSCs) have similar clinical and pathological characteristics; however, exhaustive protein expression profiling of these cancers has yet to be reported. We performed protein expression profiling on 14 cases of HGSCs (7 ovarian and 7 endometrial) and 18 endometrioid carcinomas (9 ovarian and 9 endometrial) using iTRAQ-based exhaustive and quantitative protein analysis. We identified 828 tumour-expressed proteins and evaluated the statistical similarity of protein expression profiles between ovarian and endometrial HGSCs using unsupervised hierarchical cluster analysis (P<0.01). Using 45 statistically highly expressed proteins in HGSCs, protein ontology analysis detected two enriched terms and proteins composing each term: IMP2 and MCM2. Immunohistochemical analyses confirmed the higher expression of IMP2 and MCM2 in ovarian and endometrial HGSCs as well as in tubal and peritoneal HGSCs than in endometrioid carcinomas (P<0.01). The knockdown of either IMP2 or MCM2 by siRNA interference significantly decreased the proliferation rate of ovarian HGSC cell line (P<0.01). We demonstrated the statistical similarity of the protein expression profiles of ovarian and endometrial HGSC beyond the organs. We suggest that increased IMP2 and MCM2 expression may underlie some of the rapid HGSC growth observed clinically.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yamakoshi, Takako; Makino, Teruhiko, E-mail: tmakino@med.u-toyama.ac.jp; Ur Rehman, Mati

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 andmore » 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.« less

The transcriptional response of Escherichia coli to recombinant protein insolubility.

PubMed

Smith, Harold E

2007-03-01

Bacterial production of recombinant proteins offers several advantages over alternative expression methods and remains the system of choice for many structural genomics projects. However, a large percentage of targets accumulate as insoluble inclusion bodies rather than soluble protein, creating a significant bottleneck in the protein production pipeline. Numerous strategies have been reported that can improve in vivo protein solubility, but most do not scale easily for high-throughput expression screening. To understand better the host cell response to the accumulation of insoluble protein, we determined genome-wide changes in bacterial gene expression upon induction of either soluble or insoluble target proteins. By comparing transcriptional profiles for multiple examples from the soluble or insoluble class, we identified a pattern of gene expression that correlates strongly with protein solubility. Direct targets of the sigma32 heat shock sigma factor, which includes genes involved in protein folding and degradation, were highly expressed in response to induction of insoluble protein. This same group of genes was also upregulated by insoluble protein accumulation under a different growth regime, indicating that sigma32-mediated gene expression is a general response to protein insolubility. This knowledge provides a starting point for the rational design of growth parameters and host strains with improved protein solubility characteristics. Summary Problems with protein solubility are frequently encountered when recombinant proteins are expressed in E. coli. The bacterial host responds to this problem by increasing expression of the protein folding machinery via the heat shock sigma factor sigma32. Manipulation of the sigma32 regulon might provide a general mechanism for improving recombinant protein solubility.

Effective non-denaturing purification method for improving the solubility of recombinant actin-binding proteins produced by bacterial expression.

PubMed

Chung, Jeong Min; Lee, Sangmin; Jung, Hyun Suk

2017-05-01

Bacterial expression is commonly used to produce recombinant and truncated mutant eukaryotic proteins. However, heterologous protein expression may render synthesized proteins insoluble. The conventional method used to express a poorly soluble protein, which involves denaturation and refolding, is time-consuming and inefficient. There are several non-denaturing approaches that can increase the solubility of recombinant proteins that include using different bacterial cell strains, altering the time of induction, lowering the incubation temperature, and employing different detergents for purification. In this study, we compared several non-denaturing protocols to express and purify two insoluble 34 kDa actin-bundling protein mutants. The solubility of the mutant proteins was not affected by any of the approaches except for treatment with the detergent sarkosyl. These results indicate that sarkosyl can effectively improve the solubility of insoluble proteins during bacterial expression. Copyright © 2016. Published by Elsevier Inc.

Purification of proteins from baculovirus-infected insect cells.

PubMed

O'Shaughnessy, Luke; Doyle, Sean

2011-01-01

Expression of recombinant proteins in the baculovirus/insect cell expression system is employed because it enables post-translational protein modification and high yields of recombinant protein. The system is capable of facilitating the functional expression of many proteins - either secreted or intracellularly located within infected insect cells. Strategies for the isolation and extraction of soluble proteins are presented in this chapter and involve selective cell lysis, precipitation and chromatography. Protein insolubility, following recombinant expression in insect cells, can occur. However, using the methods described herein, it is possible to extract and purify insoluble protein using affinity, ion-exchange and gel filtration chromatography. Indeed, protein insolubility often aids protein purification.

On the nature of in vivo requirements for rde-4 in RNAi and developmental pathways in C. elegans

PubMed Central

Lopez-Molina, Javier; Gent, Jonathan; Saynuk, Jamie Fleenor

2011-01-01

C. elegans RDE-4 is a double-stranded RNA binding protein that has been shown to play a key role in response to foreign double-stranded RNA (dsRNA). We have used diverse tools for analysis of gene function to characterize the domain and organismal foci of RDE-4 action in C. elegans. First, we examined the focus of activity within the RDE-4 protein, by testing a series of RDE-4 deletion constructs for their ability to support dsRNA-triggered gene silencing. These assays indicated a molecular requirement for a linker region and the second dsRNA-binding domain of RDE-4, with ancillary contributions to function from the C and N terminal domains. Second, we used mosaic analysis to explore the cellular focus of action of RDE-4. These experiments indicated an ability of RDE-4 to function non-autonomously in foreign RNA responses. Third, we used growth under stressful conditions to search for evidence of an organismal focus of action for RDE-4 distinct from its role in response to foreign dsRNA. Propagation at high temperatures exposed a conditional requirement for RDE-4 for optimal growth and fertility, indicating at least under these conditions that RDE-4 can serve an essential role in C. elegans. PMID:21519199

Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells.

PubMed

Fabrick, Jeffrey A; Hull, J Joe

2017-04-20

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and the determination of the biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for protein production in numerous biotechnological, pharmaceutical, and industrial applications, nonlytic systems that do not involve viral infection have clear benefits but are often overlooked and underutilized. Here, we describe a method for generating nonlytic expression vectors and transient recombinant protein expression. This protocol allows for the efficient cellular localization of recombinant proteins and can be used to rapidly discern protein trafficking within the cell. We show the expression of four recombinant proteins in a commercially available insect cell line, including two aquaporin proteins from the insect Bemisia tabaci, as well as subcellular marker proteins specific for the cell plasma membrane and for intracellular lysosomes. All recombinant proteins were produced as chimeras with fluorescent protein markers at their carboxyl termini, which allows for the direct detection of the recombinant proteins. The double transfection of cells with plasmids harboring constructs for the genes of interest and a known subcellular marker allows for live cell imaging and improved validation of cellular protein localization.

Heterologous expression of proteins from Plasmodium falciparum: results from 1000 genes.

PubMed

Mehlin, Christopher; Boni, Erica; Buckner, Frederick S; Engel, Linnea; Feist, Tiffany; Gelb, Michael H; Haji, Lutfiyah; Kim, David; Liu, Colleen; Mueller, Natascha; Myler, Peter J; Reddy, J T; Sampson, Joshua N; Subramanian, E; Van Voorhis, Wesley C; Worthey, Elizabeth; Zucker, Frank; Hol, Wim G J

2006-08-01

As part of a structural genomics initiative, 1000 open reading frames from Plasmodium falciparum, the causative agent of the most deadly form of malaria, were tested in an E. coli protein expression system. Three hundred and thirty-seven of these targets were observed to express, although typically the protein was insoluble. Sixty-three of the targets provided soluble protein in yields ranging from 0.9 to 406.6 mg from one liter of rich media. Higher molecular weight, greater protein disorder (segmental analysis, SEG), more basic isoelectric point (pI), and a lack of homology to E. coli proteins were all highly and independently correlated with difficulties in expression. Surprisingly, codon usage and the percentage of adenosines and thymidines (%AT) did not appear to play a significant role. Of those proteins which expressed, high pI and a hypothetical annotation were both strongly and independently correlated with insolubility. The overwhelmingly important role of pI in both expression and solubility appears to be a surprising and fundamental issue in the heterologous expression of P. falciparum proteins in E. coli. Twelve targets which did not express in E. coli from the native gene sequence were codon-optimized through whole gene synthesis, resulting in the (insoluble) expression of three of these proteins. Seventeen targets which were expressed insolubly in E. coli were moved into a baculovirus/Sf-21 system, resulting in the soluble expression of one protein at a high level and six others at a low level. A variety of factors conspire to make the heterologous expression of P. falciparum proteins challenging, and these observations lay the groundwork for a rational approach to prioritizing and, ultimately, eliminating these impediments.

Vectors for co-expression of an unrestricted number of proteins

PubMed Central

Scheich, Christoph; Kümmel, Daniel; Soumailakakis, Dimitri; Heinemann, Udo; Büssow, Konrad

2007-01-01

A vector system is presented that allows generation of E. coli co-expression clones by a standardized, robust cloning procedure. The number of co-expressed proteins is not limited. Five ‘pQLink’ vectors for expression of His-tag and GST-tag fusion proteins as well as untagged proteins and for cloning by restriction enzymes or Gateway cloning were generated. The vectors allow proteins to be expressed individually; to achieve co-expression, two pQLink plasmids are combined by ligation-independent cloning. pQLink co-expression plasmids can accept an unrestricted number of genes. As an example, the co-expression of a heterotetrameric human transport protein particle (TRAPP) complex from a single plasmid, its isolation and analysis of its stoichiometry are shown. pQLink clones can be used directly for pull-down experiments if the proteins are expressed with different tags. We demonstrate pull-down experiments of human valosin-containing protein (VCP) with fragments of the autocrine motility factor receptor (AMFR). The cloning method avoids PCR or gel isolation of restriction fragments, and a single resistance marker and origin of replication are used, allowing over-expression of rare tRNAs from a second plasmid. It is expected that applications are not restricted to bacteria, but could include co-expression in other hosts such as Bacluovirus/insect cells. PMID:17311810

The C. elegans CSR-1 argonaute pathway counteracts epigenetic silencing to promote germline gene expression.

PubMed

Seth, Meetu; Shirayama, Masaki; Gu, Weifeng; Ishidate, Takao; Conte, Darryl; Mello, Craig C

2013-12-23

Organisms can develop adaptive sequence-specific immunity by reexpressing pathogen-specific small RNAs that guide gene silencing. For example, the C. elegans PIWI-Argonaute/piwi-interacting RNA (piRNA) pathway recruits RNA-dependent RNA polymerase (RdRP) to foreign sequences to amplify a transgenerational small-RNA-induced epigenetic silencing signal (termed RNAe). Here, we provide evidence that, in addition to an adaptive memory of silenced sequences, C. elegans can also develop an opposing adaptive memory of expressed/self-mRNAs. We refer to this mechanism, which can prevent or reverse RNAe, as RNA-induced epigenetic gene activation (RNAa). We show that CSR-1, which engages RdRP-amplified small RNAs complementary to germline-expressed mRNAs, is required for RNAa. We show that a transgene with RNAa activity also exhibits accumulation of cognate CSR-1 small RNAs. Our findings suggest that C. elegans adaptively acquires and maintains a transgenerational CSR-1 memory that recognizes and protects self-mRNAs, allowing piRNAs to recognize foreign sequences innately, without the need for prior exposure

Topographical modulation of macrophage phenotype by shrink-film multi-scale wrinkles.

PubMed

Wang, Tingting; Luu, Thuy U; Chen, Aaron; Khine, Michelle; Liu, Wendy F

2016-06-24

The host immune response to foreign materials is a major hurdle for implanted medical devices. To control this response, modulation of macrophage behavior has emerged as a promising strategy, given their prominent role in inflammation and wound healing. Towards this goal, we explore the effect of biomimetic multi-scale wrinkles on macrophage adhesion and expression of phenotype markers. We find that macrophages elongate along the direction of the uniaxial wrinkles made from shape memory polymers, and express more arginase-1 and IL-10, and less TNF-α, suggesting polarization towards an alternatively activated, anti-inflammatory phenotype. Materials were further implanted in the subcutaneous space of mice and tissue surrounding the material evaluated by histology and immunohistochemistry. We found that material surface topography altered the distribution of collagen deposition in the adjacent tissue, with denser collagen tissue observed near flat materials when compared to wrinkled materials. Furthermore, cells surrounding wrinkled materials exhibited higher arginase-1 expression. Together these data suggest that wrinkled material surfaces promote macrophage alternative activation, and may influence the foreign body response to implants.

[Plasma proteomic analysis in children with infectious mononucleosis].

PubMed

Ran, Zhi-Ling; Xiao, Bin; Liu, Hong-Rui; Liu, You-Ping; Sheng, Qiao-Ni

2015-03-01

To explore the abnormal expression of plasma proteins by analysis of proteomic expression profile in children with infectious mononucleosis (IM). Two dimensional gel electrophoresis (2-DE) followed by the mass spectrometry was used to examine important protein spots with different expression levels between children with IM and normal controls. Seven differential proteins were obtained: hemopexin, vitamin D binding protein, fetuin A, C-reactive protein, apolipoprotein A, haptoglobin and transthyretin. Compared with the control group, haptoglobin showed a higher expression level in children with IM, and the expression levels of the other proteins were obviously down-regulated. The expression changes of differential proteins identified in this study are all related with the liver acute injury, suggesting that children with IM are associated with acute liver injury. Further studies on the characteristics of above proteins will contribute to the diagnosis and treatment of pediatric IM.

Expression of taste receptors in Solitary Chemosensory Cells of rodent airways

PubMed Central

2011-01-01

Background Chemical irritation of airway mucosa elicits a variety of reflex responses such as coughing, apnea, and laryngeal closure. Inhaled irritants can activate either chemosensitive free nerve endings, laryngeal taste buds or solitary chemosensory cells (SCCs). The SCC population lies in the nasal respiratory epithelium, vomeronasal organ, and larynx, as well as deeper in the airway. The objective of this study is to map the distribution of SCCs within the airways and to determine the elements of the chemosensory transduction cascade expressed in these SCCs. Methods We utilized a combination of immunohistochemistry and molecular techniques (rtPCR and in situ hybridization) on rats and transgenic mice where the Tas1R3 or TRPM5 promoter drives expression of green fluorescent protein (GFP). Results Epithelial SCCs specialized for chemoreception are distributed throughout much of the respiratory tree of rodents. These cells express elements of the taste transduction cascade, including Tas1R and Tas2R receptor molecules, α-gustducin, PLCβ2 and TrpM5. The Tas2R bitter taste receptors are present throughout the entire respiratory tract. In contrast, the Tas1R sweet/umami taste receptors are expressed by numerous SCCs in the nasal cavity, but decrease in prevalence in the trachea, and are absent in the lower airways. Conclusions Elements of the taste transduction cascade including taste receptors are expressed by SCCs distributed throughout the airways. In the nasal cavity, SCCs, expressing Tas1R and Tas2R taste receptors, mediate detection of irritants and foreign substances which trigger trigeminally-mediated protective airway reflexes. Lower in the respiratory tract, similar chemosensory cells are not related to the trigeminal nerve but may still trigger local epithelial responses to irritants. In total, SCCs should be considered chemoreceptor cells that help in preventing damage to the respiratory tract caused by inhaled irritants and pathogens. PMID:21232137

Expression of taste receptors in solitary chemosensory cells of rodent airways.

PubMed

Tizzano, Marco; Cristofoletti, Mirko; Sbarbati, Andrea; Finger, Thomas E

2011-01-13

Chemical irritation of airway mucosa elicits a variety of reflex responses such as coughing, apnea, and laryngeal closure. Inhaled irritants can activate either chemosensitive free nerve endings, laryngeal taste buds or solitary chemosensory cells (SCCs). The SCC population lies in the nasal respiratory epithelium, vomeronasal organ, and larynx, as well as deeper in the airway. The objective of this study is to map the distribution of SCCs within the airways and to determine the elements of the chemosensory transduction cascade expressed in these SCCs. We utilized a combination of immunohistochemistry and molecular techniques (rtPCR and in situ hybridization) on rats and transgenic mice where the Tas1R3 or TRPM5 promoter drives expression of green fluorescent protein (GFP). Epithelial SCCs specialized for chemoreception are distributed throughout much of the respiratory tree of rodents. These cells express elements of the taste transduction cascade, including Tas1R and Tas2R receptor molecules, α-gustducin, PLCβ2 and TrpM5. The Tas2R bitter taste receptors are present throughout the entire respiratory tract. In contrast, the Tas1R sweet/umami taste receptors are expressed by numerous SCCs in the nasal cavity, but decrease in prevalence in the trachea, and are absent in the lower airways. Elements of the taste transduction cascade including taste receptors are expressed by SCCs distributed throughout the airways. In the nasal cavity, SCCs, expressing Tas1R and Tas2R taste receptors, mediate detection of irritants and foreign substances which trigger trigeminally-mediated protective airway reflexes. Lower in the respiratory tract, similar chemosensory cells are not related to the trigeminal nerve but may still trigger local epithelial responses to irritants. In total, SCCs should be considered chemoreceptor cells that help in preventing damage to the respiratory tract caused by inhaled irritants and pathogens.

[Expression of Dengue virus type 2 nonstructural protein 3 and isolation of host proteins interacting with it].

PubMed

Weng, Daihui; Lei, Yingfeng; Dong, Yangchao; Han, Peijun; Ye, Chuantao; Yang, Jing; Wang, Yuan; Yin, Wen

2015-12-01

To construct the plasmid expressing the fusion protein of Dengue virus type 2 (DENV2) nonstructural protein 3 (NS3) with affinity tag, and isolate the cellular proteins interacting with NS3 protein using tandem affinity purification (TAP) assay. Primers for amplifying NS3 gene were designed according to the sequence of DENV2 genome and chemically synthesized. The NS3 fragments, after amplified by PCR with DENV2 cDNA as template, were digested and cloned into the mammalian eukaryotic expression vector pCI-SF with the tandem affinity tag (FLAG-StrepII). The recombinant pCI-NS3-SF was transiently transformed by Lipofectamine(TM) 2000 into HEK293T cells, and the expression of the fusion protein was confirmed by Western blotting. Cellular proteins that interacted with NS3 were isolated and purified by TAP assay. The eukaryotic expression vector expressing NS3 protein was successfully constructed. The host proteins interacting with NS3 protein were isolated by TAP system. TAP is an efficient method to isolate the cellular proteins interacting with DENV2 NS3.

General introduction: recombinant protein production and purification of insoluble proteins.

PubMed

Ferrer-Miralles, Neus; Saccardo, Paolo; Corchero, José Luis; Xu, Zhikun; García-Fruitós, Elena

2015-01-01

Proteins are synthesized in heterologous systems because of the impossibility to obtain satisfactory yields from natural sources. The production of soluble and functional recombinant proteins is among the main goals in the biotechnological field. In this context, it is important to point out that under stress conditions, protein folding machinery is saturated and this promotes protein misfolding and, consequently, protein aggregation. Thus, the selection of the optimal expression organism and the most appropriate growth conditions to minimize the formation of insoluble proteins should be done according to the protein characteristics and downstream requirements. Escherichia coli is the most popular recombinant protein expression system despite the great development achieved so far by eukaryotic expression systems. Besides, other prokaryotic expression systems, such as lactic acid bacteria and psychrophilic bacteria, are gaining interest in this field. However, it is worth mentioning that prokaryotic expression system poses, in many cases, severe restrictions for a successful heterologous protein production. Thus, eukaryotic systems such as mammalian cells, insect cells, yeast, filamentous fungus, and microalgae are an interesting alternative for the production of these difficult-to-express proteins.

Expression of bovine non-classical major histocompatibility complex class I proteins in mouse P815 and human K562 cells.

PubMed

Parasar, Parveen; Wilhelm, Amanda; Rutigliano, Heloisa M; Thomas, Aaron J; Teng, Lihong; Shi, Bi; Davis, William C; Suarez, Carlos E; New, Daniel D; White, Kenneth L; Davies, Christopher J

2016-08-01

Major histocompatibility complex class I (MHC-I) proteins can be expressed as cell surface or secreted proteins. To investigate whether bovine non-classical MHC-I proteins are expressed as cell surface or secreted proteins, and to assess the reactivity pattern of monoclonal antibodies with non-classical MHC-I isoforms, we expressed the MHC proteins in murine P815 and human K562 (MHC-I deficient) cells. Following antibiotic selection, stably transfected cell lines were stained with H1A or W6/32 antibodies to detect expression of the MHC-I proteins by flow cytometry. Two non-classical proteins (BoLA-NC1*00501 and BoLA-NC3*00101) were expressed on the cell surface in both cell lines. Surprisingly, the BoLA-NC4*00201 protein was expressed on the cell membrane of human K562 but not mouse P815 cells. Two non-classical proteins (BoLA-NC1*00401, which lacks a transmembrane domain, and BoLA-NC2*00102) did not exhibit cell surface expression. Nevertheless, Western blot analyses demonstrated expression of the MHC-I heavy chain in all transfected cell lines. Ammonium-sulfate precipitation of proteins from culture supernatants showed that BoLA-NC1*00401 was secreted and that all surface expressed proteins where shed from the cell membrane by the transfected cells. Interestingly, the surface expressed MHC-I proteins were present in culture supernatants at a much higher concentration than BoLA-NC1*00401. This comprehensive study shows that bovine non-classical MHC-I proteins BoLA-NC1*00501, BoLA-NC3*00101, and BoLA-NC4*00201 are expressed as surface isoforms with the latter reaching the cell membrane only in K562 cells. Furthermore, it demonstrated that BoLA-NC1*00401 is a secreted isoform and that significant quantities of membrane associated MHC-I proteins can be shed from the cell membrane. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Genetic Modification of Oncolytic Newcastle Disease Virus for Cancer Therapy.

PubMed

Cheng, Xing; Wang, Weijia; Xu, Qi; Harper, James; Carroll, Danielle; Galinski, Mark S; Suzich, JoAnn; Jin, Hong

2016-06-01

Clinical development of a mesogenic strain of Newcastle disease virus (NDV) as an oncolytic agent for cancer therapy has been hampered by its select agent status due to its pathogenicity in avian species. Using reverse genetics, we have generated a lead candidate oncolytic NDV based on the mesogenic NDV-73T strain that is no longer classified as a select agent for clinical development. This recombinant NDV has a modification at the fusion protein (F) cleavage site to reduce the efficiency of F protein cleavage and an insertion of a 198-nucleotide sequence into the HN-L intergenic region, resulting in reduced viral gene expression and replication in avian cells but not in mammalian cells. In mammalian cells, except for viral polymerase (L) gene expression, viral gene expression is not negatively impacted or increased by the HN-L intergenic insertion. Furthermore, the virus can be engineered to express a foreign gene while still retaining the ability to grow to high titers in cell culture. The recombinant NDV selectively replicates in and kills tumor cells and is able to drive potent tumor growth inhibition following intratumoral or intravenous administration in a mouse tumor model. The candidate is well positioned for clinical development as an oncolytic virus. Avian paramyxovirus type 1, NDV, has been an attractive oncolytic agent for cancer virotherapy. However, this virus can cause epidemic disease in poultry, and concerns about the potential environmental and economic impact of an NDV outbreak have precluded its clinical development. Here we describe generation and characterization of a highly potent oncolytic NDV variant that is unlikely to cause Newcastle disease in its avian host, representing an essential step toward moving NDV forward as an oncolytic agent. Several attenuation mechanisms have been genetically engineered into the recombinant NDV that reduce chicken pathogenicity to a level that is acceptable worldwide without impacting viral production in cell culture. The selective tumor replication of this recombinant NDV, both in vitro and in vivo, along with efficient tumor cell killing makes it an attractive oncolytic virus candidate that may provide clinical benefit to patients. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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.

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

PubMed

Qiu, Ji; LaBaer, Joshua

2011-01-01

Systematic study of proteins requires the availability of thousands of proteins in functional format. However, traditional recombinant protein expression and purification methods have many drawbacks for such study at the proteome level. We have developed an innovative in situ protein expression and capture system, namely NAPPA (nucleic acid programmable protein array), where C-terminal tagged proteins are expressed using an in vitro expression system and efficiently captured/purified by antitag antibodies coprinted at each spot. The NAPPA technology presented in this chapter enable researchers to produce and display fresh proteins just in time in a multiplexed high-throughput fashion and utilize them for various downstream biochemical researches of interest. This platform could revolutionize the field of functional proteomics with it ability to produce thousands of spatially separated proteins in high density with narrow dynamic rand of protein concentrations, reproducibly and functionally. Copyright © 2011 Elsevier Inc. All rights reserved.

Endocrine-immune interactions in human endometrium.

PubMed

Kayisli, U A; Guzeloglu-Kayisli, O; Arici, A

2004-12-01

The immune system is a complex entity designed to eliminate foreign intruding antigens and is influenced by and, in turn, influences the function of the reproductive system. Despite the widespread associations between immunology and reproductive medicine, the study of system interactions remains in its infancy. Many diverse facts are accumulating, and pieces of the puzzle are becoming available to provide a clearer picture. In this review article, we focus on the interactions between endocrine and immune systems in the human endometrium. Understanding the molecular pathways in endocrine-immune interactions in the human endometrium is crucial to understand events such as menstrual bleeding, tissue repair and regeneration, inflammation, angiogenesis, blastocyst implantation, and progression of pregnancy. These events require a balanced regulation of endometrial differentiation, proliferation, cell survival, leukocyte recruitment, apoptosis, and angiogenesis by sex steroids. In this review, we first outline the role of survival factors such as phosphoinositol 3-kinase/protein kinase B, PTEN, NFkappaB, and apoptotic molecules (Fas-FasL, Bcl-2). We then discuss their regulation by estrogen and progesterone in the endometrium. We present evidence for direct and/or indirect roles of steroid hormones on the expression of chemotactic cytokines (interleukin-8 and monocyte chemotactic protein-1) and on the survival versus apoptosis of resident endometrial cells (stromal, epithelial, and endothelial cells) and nonresident cells (leukocytes).

Tomato bushy stunt virus (TBSV), a versatile platform for polyvalent display of antigenic epitopes and vaccine design

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kumar, Shantanu; Ochoa, Wendy; Singh, Pratik

2009-05-25

Viruses-like particles (VLPs) are frequently being used as platforms for polyvalent display of foreign epitopes of interest on their capsid surface to improve their presentation enhancing the antigenicity and host immune response. In the present study, we used the VLPs of Tomato bushy stunt virus (TBSV), an icosahedral plant virus, as a platform to display 180 copies of 16 amino acid epitopes of ricin toxin fused to the C-terminal end of a modified TBSV capsid protein (NDELTA52). Expression of the chimeric recombinant protein in insect cells resulted in spontaneous assembly of VLPs displaying the ricin epitope. Cryo-electron microscopy and imagemore » reconstruction of the chimeric VLPs at 22 A resolution revealed the locations and orientation of the ricin epitope exposed on the TBSV capsid surface. Furthermore, injection of chimeric VLPs into mice generated antisera that detected the native ricin toxin. The ease of fusing of short peptides of 15-20 residues and their ability to form two kinds (T = 1, T = 3) of bio-nanoparticles that result in the display of 60 or 180 copies of less constrained and highly exposed antigenic epitopes makes TBSV an attractive and versatile display platform for vaccine design.« less

Ndfip1 mediates peripheral tolerance to self and exogenous antigen by inducing cell cycle exit in responding CD4+ T cells

PubMed Central

Altin, John A.; Daley, Stephen R.; Howitt, Jason; Rickards, Helen J.; Batkin, Alison K.; Horikawa, Keisuke; Prasad, Simon J.; Nelms, Keats A.; Kumar, Sharad; Wu, Lawren C.; Tan, Seong-Seng; Cook, Matthew C.; Goodnow, Christopher C.

2014-01-01

The NDFIP1 (neural precursor cell expressed, developmentally down-regulated protein 4 family-interacting protein 1) adapter for the ubiquitin ligase ITCH is genetically linked to human allergic and autoimmune disease, but the cellular mechanism by which these proteins enable foreign and self-antigens to be tolerated is unresolved. Here, we use two unique mouse strains—an Ndfip1-YFP reporter and an Ndfip1-deficient strain—to show that Ndfip1 is progressively induced during T-cell differentiation and activation in vivo and that its deficiency causes a cell-autonomous, Forkhead box P3-independent failure of peripheral CD4+ T-cell tolerance to self and exogenous antigen. In small cohorts of antigen-specific CD4+ cells responding in vivo, Ndfip1 was necessary for tolerogen-reactive T cells to exit cell cycle after one to five divisions and to abort Th2 effector differentiation, defining a step in peripheral tolerance that provides insights into the phenomenon of T-cell anergy in vivo and is distinct from the better understood process of Bcl2-interacting mediator of cell death-mediated apoptosis. Ndfip1 deficiency precipitated autoimmune pancreatic destruction and diabetes; however, this depended on a further accumulation of nontolerant anti-self T cells from strong stimulation by exogenous tolerogen. These findings illuminate a peripheral tolerance checkpoint that aborts T-cell clonal expansion against allergens and autoantigens and demonstrate how hypersensitive responses to environmental antigens may trigger autoimmunity. PMID:24520172

In situ synthesis of protein arrays.

PubMed

He, Mingyue; Stoevesandt, Oda; Taussig, Michael J

2008-02-01

In situ or on-chip protein array methods use cell free expression systems to produce proteins directly onto an immobilising surface from co-distributed or pre-arrayed DNA or RNA, enabling protein arrays to be created on demand. These methods address three issues in protein array technology: (i) efficient protein expression and availability, (ii) functional protein immobilisation and purification in a single step and (iii) protein on-chip stability over time. By simultaneously expressing and immobilising many proteins in parallel on the chip surface, the laborious and often costly processes of DNA cloning, expression and separate protein purification are avoided. Recently employed methods reviewed are PISA (protein in situ array) and NAPPA (nucleic acid programmable protein array) from DNA and puromycin-mediated immobilisation from mRNA.

Antigenic characterization of bovine ephemeral fever rhabdovirus G and GNS glycoproteins expressed from recombinant baculoviruses.

PubMed

Johal, Jasjit; Gresty, Karryn; Kongsuwan, Kritaya; Walker, Peter J

2008-01-01

Recombinant baculoviruses expressing the BEFV envelope glycoprotein G and non-structural glycoprotein G(NS) were constructed. The G protein expressed in insect cells was located on the cell surface and induced spontaneous cell fusion at mildly acidic pH. The expressed G protein reacted with MAbs to continuous and conformational neutralization sites (G1, G2, G3b and G4), but not to conformational site G3a. The expressed G(NS) protein was also located on the cell surface but did not exhibit fusogenic activity. The G(NS) protein reacted with polyclonal antiserum produced from vaccinia-virus-expressed recombinant G(NS) but did not react with G protein antibodies. A His(6)-tagged, soluble form of the G protein was expressed and purified by Ni(2+)-NTA chromatography. The purified G protein reacted with BEFV-neutralizing MAbs to all continuous and conformational antigenic sites. The highly protective characteristics of the native BEFV G protein suggest that the secreted, baculovirus-expressed product may be a useful vaccine antigen.

Recombinant protein expression for structural biology in HEK 293F suspension cells: a novel and accessible approach.

PubMed

Portolano, Nicola; Watson, Peter J; Fairall, Louise; Millard, Christopher J; Milano, Charles P; Song, Yun; Cowley, Shaun M; Schwabe, John W R

2014-10-16

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two decades, prokaryotic expression systems such as E. coli have dominated the scientific literature over costly and less efficient eukaryotic cell lines. Despite the clear advantage in terms of yields and costs of expressing recombinant proteins in bacteria, the absence of specific co-factors, chaperones and post-translational modifications may cause loss of function, mis-folding and can disrupt protein-protein interactions of certain eukaryotic multi-subunit complexes, surface receptors and secreted proteins. The use of mammalian cell expression systems can address these drawbacks since they provide a eukaryotic expression environment. However, low protein yields and high costs of such methods have until recently limited their use for structural biology. Here we describe a simple and accessible method for expressing and purifying milligram quantities of protein by performing transient transfections of suspension grown HEK (Human Embryonic Kidney) 293 F cells.

Using Peptide-Level Proteomics Data for Detecting Differentially Expressed Proteins.

PubMed

Suomi, Tomi; Corthals, Garry L; Nevalainen, Olli S; Elo, Laura L

2015-11-06

The expression of proteins can be quantified in high-throughput means using different types of mass spectrometers. In recent years, there have emerged label-free methods for determining protein abundance. Although the expression is initially measured at the peptide level, a common approach is to combine the peptide-level measurements into protein-level values before differential expression analysis. However, this simple combination is prone to inconsistencies between peptides and may lose valuable information. To this end, we introduce here a method for detecting differentially expressed proteins by combining peptide-level expression-change statistics. Using controlled spike-in experiments, we show that the approach of averaging peptide-level expression changes yields more accurate lists of differentially expressed proteins than does the conventional protein-level approach. This is particularly true when there are only few replicate samples or the differences between the sample groups are small. The proposed technique is implemented in the Bioconductor package PECA, and it can be downloaded from http://www.bioconductor.org.

SHuffle, a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm

PubMed Central

2012-01-01

Background Production of correctly disulfide bonded proteins to high yields remains a challenge. Recombinant protein expression in Escherichia coli is the popular choice, especially within the research community. While there is an ever growing demand for new expression strains, few strains are dedicated to post-translational modifications, such as disulfide bond formation. Thus, new protein expression strains must be engineered and the parameters involved in producing disulfide bonded proteins must be understood. Results We have engineered a new E. coli protein expression strain named SHuffle, dedicated to producing correctly disulfide bonded active proteins to high yields within its cytoplasm. This strain is based on the trxB gor suppressor strain SMG96 where its cytoplasmic reductive pathways have been diminished, allowing for the formation of disulfide bonds in the cytoplasm. We have further engineered a major improvement by integrating into its chromosome a signal sequenceless disulfide bond isomerase, DsbC. We probed the redox state of DsbC in the oxidizing cytoplasm and evaluated its role in assisting the formation of correctly folded multi-disulfide bonded proteins. We optimized protein expression conditions, varying temperature, induction conditions, strain background and the co-expression of various helper proteins. We found that temperature has the biggest impact on improving yields and that the E. coli B strain background of this strain was superior to the K12 version. We also discovered that auto-expression of substrate target proteins using this strain resulted in higher yields of active pure protein. Finally, we found that co-expression of mutant thioredoxins and PDI homologs improved yields of various substrate proteins. Conclusions This work is the first extensive characterization of the trxB gor suppressor strain. The results presented should help researchers design the appropriate protein expression conditions using SHuffle strains. PMID:22569138

High-throughput Cloning and Expression of Integral Membrane Proteins in Escherichia coli

PubMed Central

Bruni, Renato

2014-01-01

Recently, several structural genomics centers have been established and a remarkable number of three-dimensional structures of soluble proteins have been solved. For membrane proteins, the number of structures solved has been significantly trailing those for their soluble counterparts, not least because over-expression and purification of membrane proteins is a much more arduous process. By using high throughput technologies, a large number of membrane protein targets can be screened simultaneously and a greater number of expression and purification conditions can be employed, leading to a higher probability of successfully determining the structure of membrane proteins. This unit describes the cloning, expression and screening of membrane proteins using high throughput methodologies developed in our laboratory. Basic Protocol 1 deals with the cloning of inserts into expression vectors by ligation-independent cloning. Basic Protocol 2 describes the expression and purification of the target proteins on a miniscale. Lastly, for the targets that express at the miniscale, basic protocols 3 and 4 outline the methods employed for the expression and purification of targets at the midi-scale, as well as a procedure for detergent screening and identification of detergent(s) in which the target protein is stable. PMID:24510647

Content of intrinsic disorder influences the outcome of cell-free protein synthesis.

PubMed

Tokmakov, Alexander A; Kurotani, Atsushi; Ikeda, Mariko; Terazawa, Yumiko; Shirouzu, Mikako; Stefanov, Vasily; Sakurai, Tetsuya; Yokoyama, Shigeyuki

2015-09-11

Cell-free protein synthesis is used to produce proteins with various structural traits. Recent bioinformatics analyses indicate that more than half of eukaryotic proteins possess long intrinsically disordered regions. However, no systematic study concerning the connection between intrinsic disorder and expression success of cell-free protein synthesis has been presented until now. To address this issue, we examined correlations of the experimentally observed cell-free protein expression yields with the contents of intrinsic disorder bioinformatically predicted in the expressed sequences. This analysis revealed strong relationships between intrinsic disorder and protein amenability to heterologous cell-free expression. On the one hand, elevated disorder content was associated with the increased ratio of soluble expression. On the other hand, overall propensity for detectable protein expression decreased with disorder content. We further demonstrated that these tendencies are rooted in some distinct features of intrinsically disordered regions, such as low hydrophobicity, elevated surface accessibility and high abundance of sequence motifs for proteolytic degradation, including sites of ubiquitination and PEST sequences. Our findings suggest that identification of intrinsically disordered regions in the expressed amino acid sequences can be of practical use for predicting expression success and optimizing cell-free protein synthesis.

A review of protein adsorption on bioceramics

PubMed Central

Wang, Kefeng; Zhou, Changchun; Hong, Youliang; Zhang, Xingdong

2012-01-01

Bioceramics, because of its excellent biocompatible and mechanical properties, has always been considered as the most promising materials for hard tissue repair. It is well know that an appropriate cellular response to bioceramics surfaces is essential for tissue regeneration and integration. As the in vivo implants, the implanted bioceramics are immediately coated with proteins from blood and body fluids, and it is through this coated layer that cells sense and respond to foreign implants. Hence, the adsorption of proteins is critical within the sequence of biological activities. However, the biological mechanisms of the interactions of bioceramics and proteins are still not well understood. In this review, we will recapitulate the recent studies on the bioceramic–protein interactions. PMID:23741605

Gene doping.

PubMed

Azzazy, Hassan M E

2010-01-01

Gene doping abuses the legitimate approach of gene therapy. While gene therapy aims to correct genetic disorders by introducing a foreign gene to replace an existing faulty one or by manipulating existing gene(s) to achieve a therapeutic benefit, gene doping employs the same concepts to bestow performance advantages on athletes over their competitors. Recent developments in genetic engineering have contributed significantly to the progress of gene therapy research and currently numerous clinical trials are underway. Some athletes and their staff are probably watching this progress closely. Any gene that plays a role in muscle development, oxygen delivery to tissues, neuromuscular coordination, or even pain control is considered a candidate for gene dopers. Unfortunately, detecting gene doping is technically very difficult because the transgenic proteins expressed by the introduced genes are similar to their endogenous counterparts. Researchers today are racing the clock because assuring the continued integrity of sports competition depends on their ability to develop effective detection strategies in preparation for the 2012 Olympics, which may mark the appearance of genetically modified athletes.

Recent Advances in Preclinical Developments Using Adenovirus Hybrid Vectors.

PubMed

Ehrke-Schulz, Eric; Zhang, Wenli; Gao, Jian; Ehrhardt, Anja

2017-10-01

Adenovirus (Ad)-based vectors are efficient gene-transfer vehicles to deliver foreign DNA into living organisms, offering large cargo capacity and low immunogenicity and genotoxicity. As Ad shows low integration rates of their genomes into host chromosomes, vector-derived gene expression decreases due to continuous cell cycling in regenerating tissues and dividing cell populations. To overcome this hurdle, adenoviral delivery can be combined with mechanisms leading to maintenance of therapeutic DNA and long-term effects of the desired treatment. Several hybrid Ad vectors (AdV) exploiting various strategies for long-term treatment have been developed and characterized. This review summarizes recent developments of preclinical approaches using hybrid AdVs utilizing either the Sleeping Beauty transposase system for somatic integration into host chromosomes or designer nucleases, including transcription activator-like effector nucleases and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein-9 nuclease for permanent gene editing. Further options on how to optimize these vectors further are discussed, which may lead to future clinical applications of these versatile gene-therapy tools.

Cellular and Molecular Biology of Airway Mucins

PubMed Central

Lillehoj, Erik P.; Kato, Kosuke; Lu, Wenju; Kim, Kwang C.

2017-01-01

Airway mucus constitutes a thin layer of airway surface liquid with component macromolecules that covers the luminal surface of the respiratory tract. The major function of mucus is to protect the lungs through mucociliary clearance of inhaled foreign particles and noxious chemicals. Mucus is comprised of water, ions, mucin glycoproteins, and a variety of other macromolecules, some of which possess anti-microbial, anti-protease, and anti-oxidant activities. Mucins comprise the major protein component of mucus and exist as secreted and cell-associated glycoproteins. Secreted, gel-forming mucins are mainly responsible for the viscoelastic property of mucus, which is crucial for effective mucociliary clearance. Cell-associated mucins shield the epithelial surface from pathogens through their extracellular domains and regulate intracellular signaling through their cytoplasmic regions. However, neither the exact structures of mucin glycoproteins, nor the manner through which their expression is regulated, are completely understood. This chapter reviews what is currently known about the cellular and molecular properties of airway mucins. PMID:23445810

Co-stimulatory function in primary germinal center responses: CD40 and B7 are required on distinct antigen-presenting cells.

PubMed

Watanabe, Masashi; Fujihara, Chiharu; Radtke, Andrea J; Chiang, Y Jeffrey; Bhatia, Sumeena; Germain, Ronald N; Hodes, Richard J

2017-09-04

T cell-dependent germinal center (GC) responses require coordinated interactions of T cells with two antigen-presenting cell (APC) populations, B cells and dendritic cells (DCs), in the presence of B7- and CD40-dependent co-stimulatory pathways. Contrary to the prevailing paradigm, we found unique cellular requirements for B7 and CD40 expression in primary GC responses to vaccine immunization with protein antigen and adjuvant: B7 was required on DCs but was not required on B cells, whereas CD40 was required on B cells but not on DCs in the generation of antigen-specific follicular helper T cells, antigen-specific GC B cells, and high-affinity class-switched antibody production. There was, in fact, no requirement for coexpression of B7 and CD40 on the same cell in these responses. Our findings support a substantially revised model for co-stimulatory function in the primary GC response, with crucial and distinct contributions of B7- and CD40-dependent pathways expressed by different APC populations and with important implications for understanding how to optimize vaccine responses or limit autoimmunity. This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.

Evolving phage vectors for cell targeted gene delivery.

PubMed

Larocca, David; Burg, Michael A; Jensen-Pergakes, Kristen; Ravey, Edward Prenn; Gonzalez, Ana Maria; Baird, Andrew

2002-03-01

We adapted filamentous phage vectors for targeted gene delivery to mammalian cells by inserting a mammalian reporter gene expression cassette (GFP) into the vector backbone and fusing the pIII coat protein to a cell targeting ligand (i.e. FGF2, EGF). Like transfection with animal viral vectors, targeted phage gene delivery is concentration, time, and ligand dependent. Importantly, targeted phage particles are specific for the appropriate target cell surface receptor. Phage have distinct advantages over existing gene therapy vectors because they are simple, economical to produce at high titer, have no intrinsic tropism for mammalian cells, and are relatively simple to genetically modify and evolve. Initially transduction by targeted phage particles was low resulting in foreign gene expression in 1-2% of transfected cells. We increased transduction efficiency by modifying both the transfection protocol and vector design. For example, we stabilized the display of the targeting ligand to create multivalent phagemid-based vectors with transduction efficiencies of up to 45% in certain cell lines when combined with genotoxic treatment. Taken together, these studies establish that the efficiency of phage-mediated gene transfer can be significantly improved through genetic modification. We are currently evolving phage vectors with enhanced cell targeting, increased stability, reduced immunogenicity and other properties suitable for gene therapy.

Ethnobotany and Medicinal Plant Biotechnology: From Tradition to Modern Aspects of Drug Development.

PubMed

Kayser, Oliver

2018-05-24

Secondary natural products from plants are important drug leads for the development of new drug candidates for rational clinical therapy and exhibit a variety of biological activities in experimental pharmacology and serve as structural template in medicinal chemistry. The exploration of plants and discovery of natural compounds based on ethnopharmacology in combination with high sophisticated analytics is still today an important drug discovery to characterize and validate potential leads. Due to structural complexity, low abundance in biological material, and high costs in chemical synthesis, alternative ways in production like plant cell cultures, heterologous biosynthesis, and synthetic biotechnology are applied. The basis for any biotechnological process is deep knowledge in genetic regulation of pathways and protein expression with regard to todays "omics" technologies. The high number genetic techniques allowed the implementation of combinatorial biosynthesis and wide genome sequencing. Consequently, genetics allowed functional expression of biosynthetic cascades from plants and to reconstitute low-performing pathways in more productive heterologous microorganisms. Thus, de novo biosynthesis in heterologous hosts requires fundamental understanding of pathway reconstruction and multitude of genes in a foreign organism. Here, actual concepts and strategies are discussed for pathway reconstruction and genome sequencing techniques cloning tools to bridge the gap between ethnopharmaceutical drug discovery to industrial biotechnology. Georg Thieme Verlag KG Stuttgart · New York.

In Vivo Visualization of Bacterial Colonization, Antigen Expression, and Specific T-Cell Induction following Oral Administration of Live Recombinant Salmonella enterica Serovar Typhimurium

PubMed Central

Bumann, Dirk

2001-01-01

Live attenuated Salmonella strains that express a foreign antigen are promising oral vaccine candidates. Numerous genetic modifications have been empirically tested, but their effects on immunogenicity are difficult to interpret since important in vivo properties of recombinant Salmonella strains such as antigen expression and localization are incompletely characterized and the crucial early inductive events of an immune response to the foreign antigen are not fully understood. Here, methods were developed to directly localize and quantitate the in situ expression of an ovalbumin model antigen in recombinant Salmonella enterica serovar Typhimurium using two-color flow cytometry and confocal microscopy. In parallel, the in vivo activation, blast formation, and division of ovalbumin-specific CD4+ T cells were followed using a well-characterized transgenic T-cell receptor mouse model. This combined approach revealed a biphasic induction of ovalbumin-specific T cells in the Peyer's patches that followed the local ovalbumin expression of orally administered recombinant Salmonella cells in a dose- and time-dependent manner. Interestingly, intact Salmonella cells and cognate T cells seemed to remain in separate tissue compartments throughout induction, suggesting a transport of killed Salmonella cells from the colonized subepithelial dome area to the interfollicular inductive sites. The findings of this study will help to rationally optimize recombinant Salmonella strains as efficacious live antigen carriers for oral vaccination. PMID:11402006

Protein disorder is positively correlated with gene expression in E. coli

PubMed Central

Paliy, Oleg; Gargac, Shawn M.; Cheng, Yugong; Uversky, Vladimir N.; Dunker, A. Keith

2009-01-01

We considered on a global scale the relationship between the predicted fraction of protein disorder and RNA and protein expression in E. coli. Fraction of protein disorder correlated positively with both measured RNA expression levels of E. coli genes in three different growth media and with predicted abundance levels of E. coli proteins. Though weak, the correlation was highly significant. Correlation of protein disorder with RNA expression did not depend on the growth rate of E. coli cultures and was not caused by a small subset of genes showing exceptionally high concordance in their disorder and expression levels. Global analysis was complemented by detailed consideration of several groups of proteins. PMID:18465893

Autoimmune Regulator (AIRE) Is Expressed in Spermatogenic Cells, and It Altered the Expression of Several Nucleic-Acid-Binding and Cytoskeletal Proteins in Germ Cell 1 Spermatogonial (GC1-spg) Cells.

PubMed

Radhakrishnan, Karthika; Bhagya, Kongattu P; Kumar, Anil Tr; Devi, Anandavalli N; Sengottaiyan, Jeeva; Kumar, Pradeep G

2016-08-01

Autoimmune regulator (AIRE) is a gene associated with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED). AIRE is expressed heavily in the thymic epithelial cells and is involved in maintaining self-tolerance through regulating the expression of tissue-specific antigens. The testes are the most predominant extrathymic location where a heavy expression of AIRE is reported. Homozygous Aire-deficient male mice were infertile, possibly due to impaired spermatogenesis, deregulated germ cell apoptosis, or autoimmunity. We report that AIRE is expressed in the testes of neonatal, adolescent, and adult mice. AIRE expression was detected in glial cell derived neurotrophic factor receptor alpha (GFRα)(+) (spermatogonia), GFRα(-)/synaptonemal complex protein (SCP3)(+) (meiotic), and GFRα(-)/Phosphoglycerate kinase 2 (PGK2)(+) (postmeiotic) germ cells in mouse testes. GC1-spg, a germ-cell-derived cell line, did not express AIRE. Retinoic acid induced AIRE expression in GC1-spg cells. Ectopic expression of AIRE in GC1-spg cells using label-free LC-MS/MS identified a total of 371 proteins that were differentially expressed. 100 proteins were up-regulated, and 271 proteins were down-regulated. Data are available via ProteomeXchange with identifier PXD002511. Functional analysis of the differentially expressed proteins showed increased levels of various nucleic-acid-binding proteins and transcription factors and a decreased level of various cytoskeletal and structural proteins in the AIRE overexpressing cells as compared with the empty vector-transfected controls. The transcripts of a select set of the up-regulated proteins were also elevated. However, there was no corresponding decrease in the mRNA levels of the down-regulated set of proteins. Molecular function network analysis indicated that AIRE influenced gene expression in GC1-spg cells by acting at multiple levels, including transcription, translation, RNA processing, protein transport, protein localization, and protein degradation, thus setting the foundation in understanding the functional role of AIRE in germ cell biology. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

High-level expression of soluble recombinant proteins in Escherichia coli using an HE-maltotriose-binding protein fusion tag.

PubMed

Han, Yingqian; Guo, Wanying; Su, Bingqian; Guo, Yujie; Wang, Jiang; Chu, Beibei; Yang, Guoyu

2018-02-01

Recombinant proteins are commonly expressed in prokaryotic expression systems for large-scale production. The use of genetically engineered affinity and solubility enhancing fusion proteins has increased greatly in recent years, and there now exists a considerable repertoire of these that can be used to enhance the expression, stability, solubility, folding, and purification of their fusion partner. Here, a modified histidine tag (HE) used as an affinity tag was employed together with a truncated maltotriose-binding protein (MBP; consisting of residues 59-433) from Pyrococcus furiosus as a solubility enhancing tag accompanying a tobacco etch virus protease-recognition site for protein expression and purification in Escherichia coli. Various proteins tagged at the N-terminus with HE-MBP(Pyr) were expressed in E. coli BL21(DE3) cells to determine expression and solubility relative to those tagged with His6-MBP or His6-MBP(Pyr). Furthermore, four HE-MBP(Pyr)-fused proteins were purified by immobilized metal affinity chromatography to assess the affinity of HE with immobilized Ni 2+ . Our results showed that HE-MBP(Pyr) represents an attractive fusion protein allowing high levels of soluble expression and purification of recombinant protein in E. coli. Copyright © 2017 Elsevier Inc. All rights reserved.

N-terminal SKIK peptide tag markedly improves expression of difficult-to-express proteins in Escherichia coli and Saccharomyces cerevisiae.

PubMed

Ojima-Kato, Teruyo; Nagai, Satomi; Nakano, Hideo

2017-05-01

Despite advances in microbial protein expression systems, low production of proteins remains a great concern for some genes. Here we report that the insertion of a short peptide tag, consisting of Ser-Lys-Ile-Lys (SKIK), adjacent to the start codon of genes encoding difficult-to-express proteins can increase protein expression in Escherichia coli and Saccharomyces cerevisiae. Protein expression levels of a mouse monoclonal antibody (mAb), rabbit mAbs obtained from clonal B cells, and an artificially designed peptide were significantly increased simply by the addition of the SKIK tag in E. coli systems. In particular, a ∼30-fold increase in protein production was observed for the mouse mAb, and the artificially designed peptide band became detectable in sodium dodecyl sulfate-poly acrylamide gel electrophoresis after coomassie brilliant blue staining or western blotting on adding the SKIK tag. The tag also increased the expression of tagged proteins in S. cerevisiae and an E. coli cell-free protein synthesis system. Although the mechanism of high protein expression on addition of the tag is unclear, our findings offer great benefits to biotechnology research and industry. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Development of a baculovirus vector carrying a small hairpin RNA for suppression of sf-caspase-1 expression and improvement of recombinant protein production.

PubMed

Zhang, Xiaoyue; Xu, Keyan; Ou, Yanmei; Xu, Xiaodong; Chen, Hongying

2018-05-02

The Baculovirus expression vector system (BEVS) is a transient expression platform for recombinant protein production in insect cells. Baculovirus infection of insect cells will shutoff host translation and induce apoptosis and lead to the termination of protein expression. Previous reports have demonstrated the enhancement of protein yield in BEVS using stable insect cell lines expressing interference RNA to suppress the expression of caspase-1. In this study, short-hairpin RNA (shRNA) expression cassettes targeting Spodoptera frugiperda caspase-1 (Sf-caspase-1) were constructed and inserted into an Autographa californica multiple nucleopolyhedrovirus (AcMNPV) vector. Using the recombinant baculovirus vectors, we detected the suppression of Sf-caspase-1 expression and cell apoptosis. Green fluorescent protein (GFP), Discosoma sp. Red (DsRed) and firefly luciferase were then expressed as reporter proteins. The results showed that suppression of apoptosis enhanced the accumulation of exogenous proteins at 2 and 3 days post infection. After 4 days post infection, the activity of the reporter proteins remained higher in BEVS using the baculovirus carrying shRNA in comparison with the control without shRNA, but the accumulated protein levels showed no obvious difference between them, suggesting that apoptosis suppression resulted in improved protein folding rather than translation efficiency at the very late stage of baculovirus infection. The baculovirus vector developed in this study would be a useful tool for the production of active proteins suitable for structural and functional studies or pharmaceutical applications in Sf9 cells, and it also has the potential to be adapted for the improvement of protein expression in different insect cell lines that can be infected by AcMNPV.

Novel insights in genetic transformation of the probiotic yeast Saccharomyces boulardii

PubMed Central

Douradinha, Bruno; Reis, Viviane CB; Rogers, Matthew B; Torres, Fernando AG; Evans, Jared D; Marques Jr, Ernesto TA

2014-01-01

Saccharomyces boulardii (S. boulardii) is a probiotic yeast related to Saccharomyces cerevisiae (S. cerevisiae) but with distinct genetic, taxonomic and metabolic properties. S. cerevisiae has been used extensively in biotechnological applications. Currently, many strains are available, and multiple genetic tools have been developed, which allow the expression of several exogenous proteins of interest with applications in the fields of medicine, biofuels, the food industry, and scientific research, among others. Although S. boulardii has been widely studied due to its probiotic properties against several gastrointestinal tract disorders, very few studies addressed the use of this yeast as a vector for expression of foreign genes of interest with biotechnological applications. Here we show that, despite the similarity of the two yeasts, not all genetic tools used in S. cerevisiae can be applied in S. boulardii. While transformation of the latter could be obtained using a commercial kit developed for the former, consequent screening of successful transformants had to be optimized. We also show that several genes frequently used in genetic manipulation of S. cerevisiae (e.g., promoters and resistance markers) are present in S. boulardii. Sequencing revealed a high rate of homology (>96%) between the orthologs of the two yeasts. However, we also observed some of them are not eligible to be targeted for transformation of S. boulardii. This work has important applications toward the potential of this probiotic yeast as an expression system for genes of interest. PMID:24013355

Affective journeys: the emotional structuring of medical tourism in India.

PubMed

Solomon, Harris

2011-04-01

This paper examines the grid of sentiment that structures medical travel to India. In contrast to studies that render emotion as ancillary, the paper argues that affect is fundamental to medical travel's ability to ease the linked somatic, emotional, financial, and political injuries of being ill 'back home'. The ethnographic approach follows the scenes of medical travel within the Indian corporate hospital room, based on observations and interviews among foreign patients, caregivers, and hospital staff in Mumbai, New Delhi, Chennai, and Bangalore. Foreign patients conveyed diverse sentiments about their journey to India ranging from betrayal to gratitude, and their expressions of risk, healthcare costs, and cultural difference help sustain India's popularity as a medical travel destination. However, although the affective dimensions of medical travel promise a remedy for foreign patients, they also reveal the fault lines of market medicine in India.

[Identification of differential proteins of serum in the patients suffering from coal-burning arsenism].

PubMed

Han, Bing; Yang, Qin; Luo, Xin-hua; He, Xiao-fei; Wu, Jun; Cheng, Ming-liang

2009-06-02

To compare and analyze the differential expression of proteins between coal-burning arsenism serum and normal human serum and identify the proteins related with arseniasis caused by coal-burning. Serum samples were collected from 6 normal subjects and 6 patients suffering from coal-burning arsenism. 2-DE was performed to separate serum proteins. After silver staining, the differential expression of proteins was analyzed and then identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). There were an average of 779 +/- 35 spots and 865 +/- 30 spots on 2-DE matching of two groups and the matching rate was 90.1% between two groups. From these two groups, 60 different protein spots were identified. Up-regulated expression was observed in 25 proteins and down-regulated expression in 35 proteins in the patient serum group. Among which 35 with differential expression above three times were singled out and MALDI-TOF-MS analysis was carried out on them. Thirteen proteins were identified, including keratin 10, apolipoprotein A-V, transferrin, alpha-1-antitrypsin, human zinc-alpha-2-glycoprotein, mitogen-activated protein kinase 3, vacuolar protein sorting 33A, O-linked GlcNAc transferase and etc. Up-regulated expression was observed in 5 proteins and down-regulated expression in 8 proteins in the patient serum group. The well-resolved and reproducible 2-DE serum patterns of patients suffering from coal-burning arsenism were established and some differentially expressed proteins characterized. These data will be used to screen the biomarker and to further study arseniasis caused by coal-burning.

Improving membrane protein expression by optimizing integration efficiency

PubMed Central

2017-01-01

The heterologous overexpression of integral membrane proteins in Escherichia coli often yields insufficient quantities of purifiable protein for applications of interest. The current study leverages a recently demonstrated link between co-translational membrane integration efficiency and protein expression levels to predict protein sequence modifications that improve expression. Membrane integration efficiencies, obtained using a coarse-grained simulation approach, robustly predicted effects on expression of the integral membrane protein TatC for a set of 140 sequence modifications, including loop-swap chimeras and single-residue mutations distributed throughout the protein sequence. Mutations that improve simulated integration efficiency were 4-fold enriched with respect to improved experimentally observed expression levels. Furthermore, the effects of double mutations on both simulated integration efficiency and experimentally observed expression levels were cumulative and largely independent, suggesting that multiple mutations can be introduced to yield higher levels of purifiable protein. This work provides a foundation for a general method for the rational overexpression of integral membrane proteins based on computationally simulated membrane integration efficiencies. PMID:28918393

Expression and activity of multidrug resistance proteins in mature endothelial cells and their precursors: A challenging correlation.

PubMed

Krawczenko, Agnieszka; Bielawska-Pohl, Aleksandra; Wojtowicz, Karolina; Jura, Roksana; Paprocka, Maria; Wojdat, Elżbieta; Kozłowska, Urszula; Klimczak, Aleksandra; Grillon, Catherine; Kieda, Claudine; Duś, Danuta

2017-01-01

Active cellular transporters of harmful agents-multidrug resistance (mdr) proteins-are present in tumor, stem and endothelial cells, among others. While mdr proteins are broadly studied in tumor cells, their role in non-tumor cells and the significance of their action not connected with removal of harmful xenobiotics is less extensively documented. Proper assessment of mdr proteins expression is difficult. Mdr mRNA presence is most often evaluated but that does not necessarily correlate with the protein level. The protein expression itself is difficult to determine; usually cells with mdr overexpression are studied, not cells under physiological conditions, in which a low expression level of mdr protein is often insufficient for detection in vitro. Various methods are used to identify mdr mRNA and protein expression, together with functional tests demonstrating their biological drug transporting activities. Data comparing different methods of investigating expression of mdr mRNAs and their corresponding proteins are still scarce. In this article we present the results of a study concerning mdr mRNA and protein expression. Our goal was to search for the best method to investigate the expression level and functional activity of five selected mdr proteins-MDR1, BCRP, MRP1, MRP4 and MRP5-in established in vitro cell lines of human endothelial cells (ECs) and their progenitors. Endothelial cells demonstrated mdr presence at the mRNA level, which was not always confirmed at the protein level or in functional tests. Therefore, several different assays had to be applied for evaluation of mdr proteins expression and functions in endothelial cells. Among them functional tests seemed to be the most conclusive, although not very specific.

Evaluation of detergents for the soluble expression of alpha-helical and beta-barrel-type integral membrane proteins by a preparative scale individual cell-free expression system.

PubMed

Klammt, Christian; Schwarz, Daniel; Fendler, Klaus; Haase, Winfried; Dötsch, Volker; Bernhard, Frank

2005-12-01

Cell-free expression has become a highly promising tool for the fast and efficient production of integral membrane proteins. The proteins can be produced as precipitates that solubilize in mild detergents usually without any prior denaturation steps. Alternatively, membrane proteins can be synthesized in a soluble form by adding detergents to the cell-free system. However, the effects of a representative variety of detergents on the production, solubility and activity of a wider range of membrane proteins upon cell-free expression are currently unknown. We therefore analyzed the cell-free expression of three structurally very different membrane proteins, namely the bacterial alpha-helical multidrug transporter, EmrE, the beta-barrel nucleoside transporter, Tsx, and the porcine vasopressin receptor of the eukaryotic superfamily of G-protein coupled receptors. All three membrane proteins could be produced in amounts of several mg per one ml of reaction mixture. In general, the detergent 1-myristoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] was found to be most effective for the resolubilization of membrane protein precipitates, while long chain polyoxyethylene-alkyl-ethers proved to be most suitable for the soluble expression of all three types of membrane proteins. The yield of soluble expressed membrane protein remained relatively stable above a certain threshold concentration of the detergents. We report, for the first time, the high-level cell-free expression of a beta-barrel type membrane protein in a functional form. Structural and functional variations of the analyzed membrane proteins are evident that correspond with the mode of expression and that depend on the supplied detergent.

GroEL-GroES assisted folding of multiple recombinant proteins simultaneously over-expressed in Escherichia coli.

PubMed

Goyal, Megha; Chaudhuri, Tapan K

2015-07-01

Folding of aggregation prone recombinant proteins through co-expression of chaperonin GroEL and GroES has been a popular practice in the effort to optimize preparation of functional protein in Escherichia coli. Considering the demand for functional recombinant protein products, it is desirable to apply the chaperone assisted protein folding strategy for enhancing the yield of properly folded protein. Toward the same direction, it is also worth attempting folding of multiple recombinant proteins simultaneously over-expressed in E. coli through the assistance of co-expressed GroEL-ES. The genesis of this thinking was originated from the fact that cellular GroEL and GroES assist in the folding of several endogenous proteins expressed in the bacterial cell. Here we present the experimental findings from our study on co-expressed GroEL-GroES assisted folding of simultaneously over-expressed proteins maltodextrin glucosidase (MalZ) and yeast mitochondrial aconitase (mAco). Both proteins mentioned here are relatively larger and aggregation prone, mostly form inclusion bodies, and undergo GroEL-ES assisted folding in E. coli cells during over-expression. It has been reported that the relative yield of properly folded functional forms of MalZ and mAco with the exogenous GroEL-ES assistance were comparable with the results when these proteins were overexpressed alone. This observation is quite promising and highlights the fact that GroEL and GroES can assist in the folding of multiple substrate proteins simultaneously when over-expressed in E. coli. This method might be a potential tool for enhanced production of multiple functional recombinant proteins simultaneously in E. coli. Copyright © 2015 Elsevier Ltd. All rights reserved.

Recombinant expression and purification of a tumor-targeted toxin in Bacillus anthracis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bachran, Christopher; Abdelazim, Suzanne; Fattah, Rasem J.

Highlights: Black-Right-Pointing-Pointer Non-infectious and protease-deficient Bacillus anthracis protein expression system. Black-Right-Pointing-Pointer Successful expression and purification of a tumor-targeted fusion protein drug. Black-Right-Pointing-Pointer Very low endotoxin contamination of purified protein. Black-Right-Pointing-Pointer Efficient protein secretion simplifies purification. Black-Right-Pointing-Pointer Functional anti-tumor fusion protein purified. -- Abstract: Many recombinant therapeutic proteins are purified from Escherichia coli. While expression in E. coli is easily achieved, some disadvantages such as protein aggregation, formation of inclusion bodies, and contamination of purified proteins with the lipopolysaccharides arise. Lipopolysaccharides have to be removed to prevent inflammatory responses in patients. Use of the Gram-positive Bacillus anthracis as an expression hostmore » offers a solution to circumvent these problems. Using the multiple protease-deficient strain BH460, we expressed a fusion of the N-terminal 254 amino acids of anthrax lethal factor (LFn), the N-terminal 389 amino acids of diphtheria toxin (DT389) and human transforming growth factor alpha (TGF{alpha}). The resulting fusion protein was constitutively expressed and successfully secreted by B. anthracis into the culture supernatant. Purification was achieved by anion exchange chromatography and proteolytic cleavage removed LFn from the desired fusion protein (DT389 fused to TGF{alpha}). The fusion protein showed the intended specific cytotoxicity to epidermal growth factor receptor-expressing human head and neck cancer cells. Final analyses showed low levels of lipopolysaccharides, originating most likely from contamination during the purification process. Thus, the fusion to LFn for protein secretion and expression in B. anthracis BH460 provides an elegant tool to obtain high levels of lipopolysaccharide-free recombinant protein.« less

Proteomic analysis of acute responses to copper sulfate stress in larvae of the brine shrimp, Artemia sinica

NASA Astrophysics Data System (ADS)

Zhou, Qian; Wu, Changgong; Dong, Bo; Li, Fuhua; Liu, Fengqi; Xiang, Jianhai

2010-03-01

Proteomics was used to reveal the differential protein expression profiles of acute responses to copper sulfate exposure in larvae of Artemia sinica. Fourteen differentially displayed protein spots were detected and seven of them were identified. Three spots were up-expressed and identified: actin, heat shock protein 70, and chaperone subunit 1; three down-regulated proteins were identified: arginine kinase, elongation factor-2, and glycine-rich protein; and a newly expressed protein was identified as peroxiredoxin. The study indicates the involvement of all the differentially expressed proteins in the early responses of protein expression, and in the survival of A. sinica in the presence of copper and other heavy metals; the findings improve understanding of the organism’s adaptive responses and resistance.

Health care satisfaction among foreign residents in Taiwan - An assessment and improvement.

PubMed

Kumar, Ajit; Maskara, Sanjeev; Chiang, I-Jen

2014-01-21

In 2012, almost half-million foreigners (2.3% of the total population) from 160 countries were estimated to live in Taiwan. In 2010, approximately eighty-seven percent of the population expressed high satisfaction in the national health care system of Taiwan. However, satisfaction level among foreign residents towards the Taiwanese health care system has not been clearly documented in the literature.OBJECTIVE: In this study, we assessed satisfaction level among foreign residents for receiving healthcare facility in Taiwanese hospital. In addition, the study came out with some potential solutions to improve health care received by them. Human-Centered Design (HCD) approach was deployed, which included three phases: observation, pattern recognition, and ideation and delivery. Each phase involved various steps. Techniques, such as ethnography, interview, discussion, and survey were used in accomplishing various steps within each phase. Four participants and two facilitators from Taiwan took part in the study. The study revealed various stories, insights, and themes about the health care received by the foreigners in Taiwan. In addition, some potential solutions and immediate available opportunities were recommended to improve their health care. The results provided a deeper understanding into the satisfaction level among foreign residents in Taiwan. For instance, this study pointed out the need to improve English proficiency of hospital's administrative staffs because a lack of English language proficiencies was found to be a major barrier in delivering quality health care to foreign residents. Therefore, adoption of various training modalities, such as English training using mobile device based games, role-play, and hospital's workflow depiction using English posters were recommended.

Health care satisfaction among foreign residents in Taiwan--an assessment and improvement.

PubMed

Kumar, Ajit; Maskara, Sanjeev; Chiang, I-Jen

2014-01-01

In 2012, almost half-million foreigners (2.3% of the total population) from 160 countries were estimated to live in Taiwan. In 2010, approximately eighty-seven percent of the population expressed high satisfaction in the national health care system of Taiwan. However, satisfaction level among foreign residents towards the Taiwanese health care system has not been clearly documented in the literature. In this study, we assessed satisfaction level among foreign residents for receiving healthcare facility in Taiwanese hospital. In addition, the study came out with some potential solutions to improve health care received by them. Human-Centered Design (HCD) approach was deployed, which included three phases: observation, pattern recognition, and ideation and delivery. Each phase involved various steps. Techniques, such as ethnography, interview, discussion, and survey were used in accomplishing various steps within each phase. Four participants and two facilitators from Taiwan took part in the study. The study revealed various stories, insights, and themes about the health care received by the foreigners in Taiwan. In addition, some potential solutions and immediate available opportunities were recommended to improve their health care. The results provided a deeper understanding into the satisfaction level among foreign residents in Taiwan. For instance, this study pointed out the need to improve English proficiency of hospital's administrative staffs because a lack of English language proficiencies was found to be a major barrier in delivering quality health care to foreign residents. Therefore, adoption of various training modalities, such as English training using mobile device based games, role-play, and hospital's workflow depiction using English posters were recommended.

Human B cells fail to secrete type I interferons upon cytoplasmic DNA exposure.

PubMed

Gram, Anna M; Sun, Chenglong; Landman, Sanne L; Oosenbrug, Timo; Koppejan, Hester J; Kwakkenbos, Mark J; Hoeben, Rob C; Paludan, Søren R; Ressing, Maaike E

2017-11-01

Most cells are believed to be capable of producing type I interferons (IFN I) as part of an innate immune response against, for instance, viral infections. In macrophages, IFN I is potently induced upon cytoplasmic exposure to foreign nucleic acids. Infection of these cells with herpesviruses leads to triggering of the DNA sensors interferon-inducible protein 16 (IFI16) and cyclic GMP-AMP (cGAMP) synthase (cGAS). Thereby, the stimulator of interferon genes (STING) and the downstream molecules TANK-binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3) are sequentially activated culminating in IFN I secretion. Human gamma-herpesviruses, such as Epstein-Barr virus (EBV), exploit B cells as a reservoir for persistent infection. In this study, we investigated whether human B cells, similar to macrophages, engage the cytoplasmic DNA sensing pathway to induce an innate immune response. We found that the B cells fail to secrete IFN I upon cytoplasmic DNA exposure, although they express the DNA sensors cGAS and IFI16 and the signaling components TBK1 and IRF3. In primary human B lymphocytes and EBV-negative B cell lines, this deficiency is explained by a lack of detectable levels of the central adaptor protein STING. In contrast, EBV-transformed B cell lines did express STING, yet both these lines as well as STING-reconstituted EBV-negative B cells did not produce IFN I upon dsDNA or cGAMP stimulation. Our combined data show that the cytoplasmic DNA sensing pathway is dysfunctional in human B cells. This exemplifies that certain cell types cannot induce IFN I in response to cytoplasmic DNA exposure providing a potential niche for viral persistence. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Isolation of a novel plasmid from Couchioplanes caeruleus and construction of two plasmid vectors for gene expression in Actinoplanes missouriensis.

PubMed

Jang, Moon-Sun; Fujita, Azusa; Ikawa, Satomi; Hanawa, Keitaro; Yamamura, Hideki; Tamura, Tomohiko; Hayakawa, Masayuki; Tezuka, Takeaki; Ohnishi, Yasuo

2015-01-01

To date, no plasmid vector has been developed for the rare actinomycete Actinoplanes missouriensis. Moreover, no small circular plasmid has been reported to exist in the genus Actinoplanes. Here, a novel plasmid, designated pCAZ1, was isolated from Couchioplanes caeruleus subsp. azureus via screening for small circular plasmids in Actinoplanes (57 strains) and Couchioplanes (2 strains). Nucleotide sequencing revealed that pCAZ1 is a 5845-bp circular molecule with a G + C content of 67.5%. The pCAZ1 copy number was estimated at 30 per chromosome. pCAZ1 contains seven putative open reading frames, one of which encodes a protein containing three motifs conserved among plasmid-encoded replication proteins that are involved in the rolling-circle mechanism of replication. Detection of single-stranded DNA intermediates in C. caeruleus confirmed that pCAZ1 replicates by this mechanism. The ColE1 origin from pBluescript SK(+) and the oriT sequence with the apramycin resistance gene aac(3)IV from pIJ773 were inserted together into pCAZ1, to construct the Escherichia coli-A. missouriensis shuttle vectors, pCAM1 and pCAM2, in which the foreign DNA fragment was inserted into pCAZ1 in opposite directions. pCAM1 and pCAM2 were successfully transferred to A. missouriensis through the E. coli-mediated conjugative transfer system. The copy numbers of pCAM1 and pCAM2 in A. missouriensis were estimated to be one and four per chromosome, respectively. Thus, these vectors can be used as effective genetic tools for homologous and heterologous gene expression studies in A. missouriensis. Copyright © 2014 Elsevier Inc. All rights reserved.

A cytokine-like factor astakine accelerates the hemocyte production in Pacific oyster Crassostrea gigas.

PubMed

Li, Yiqun; Jiang, Shuai; Li, Meijia; Xin, Lusheng; Wang, Lingling; Wang, Hao; Qiu, Limei; Song, Linsheng

2016-02-01

Astakine has been reported to be a hematopoietic growth factor of prokineticin homolog firstly found in arthropods freshwater crayfish Pacifastacus leniusculus. In the present study, an astakine homologous gene was identified from Pacific oyster Crassostrea gigas (designated CgAstakine). The full length cDNA of CgAstakine encoded a polypeptide of 103 amino acids containing a prokineticin (PK) domain homologous to that in astakine from freshwater crayfish P. leniusculus. The deduced amino acid sequence of CgAstakine shared higher similarity with those of other invertebrate astakines than prokineticins from vertebrates. The mRNA of CgAstakine was highly expressed in hepatopancreas and adductor muscle of oyster, while the CgAstakine protein was mainly distributed in hepatopancreas, gill and hemocytes. The mRNA expression of CgAstakine in hemocytes was significantly increased (p < 0.01) and maintained at a high level from 3 h to 9 h after Vibrio anguillarum challenge. After the oyster hemocytes were incubated with 5 μg/mL recombinant CgAstakine protein (rCgAstakine) for 24 h in vitro, the proliferation of hemocytes was significantly increased to 1.89 fold of that in control group (p < 0.05). Moreover, the total count of oyster hemocytes was significantly upregulated (2.45 fold of that in control group, p < 0.05) at 12 h after the oysters were received an injection of rCgAstakine (0.5 μg/g). These results collectively indicated that CgAstakine could modulate the hemocytes proliferation both in vitro and in vivo, and probably involved in the hematopoietic process fighting against the invasion of foreign pathogens. Copyright © 2015 Elsevier Ltd. All rights reserved.

Transcriptional elements from the human SP-C gene direct expression in the primordial respiratory epithelium of transgenic mice.

PubMed

Wert, S E; Glasser, S W; Korfhagen, T R; Whitsett, J A

1993-04-01

Transgenic animals bearing a chimeric gene containing 5'-flanking regions of the human surfactant protein C (SP-C) gene ligated to the bacterial chloramphenicol acetyltransferase (CAT) gene were analyzed by in situ hybridization histochemistry to determine the temporal and spatial distribution of transgene expression during organogenesis of the murine lung. Ontogenic expression of the SP-C-CAT gene was compared to that of the endogenous SP-C gene and to the Clara cell CC10 gene. High levels of SP-C-CAT expression were observed as early as Day 10 of gestation in epithelial cells of the primordial lung buds. Low levels of endogenous SP-C mRNA were detected a day later, but only in the more distal epithelial cells of the newly formed, primitive, lobar bronchi. On Gestational Days 13 through 16, transcripts for both the endogenous and chimeric gene were restricted to distal epithelial elements of the branching bronchial tubules and were no longer detected in the more proximal regions of the bronchial tree. Although high levels of SP-C-CAT expression were maintained throughout organogenesis, endogenous SP-C expression increased dramatically on Gestational Day 15, coincident with acinar tubule differentiation at the lung periphery. Low levels of endogenous CC10 expression were detected by Gestational Day 16 in both lobar and segmental bronchi. By the time of birth, CC10 transcripts were expressed at high levels in the trachea and at all levels of the bronchial tree; endogenous SP-C mRNA was restricted to epithelial cells of the terminal alveolar saccules; and SP-C-CAT expression was now detected in both alveolar and bronchiolar epithelial cells. These results indicate that (1) cis-acting regulatory elements of the human SP-C gene can direct high levels of foreign gene expression to epithelial cells of the embryonic mouse lung; (2) expression of the human SP-C-CAT chimeric gene is developmentally regulated, exhibiting a morphogenic expression pattern similar, but not identical, to that of the endogenous murine SP-C gene; (3) the embryonic expression of endogenous SP-C and chimeric SP-C-CAT transcripts identifies progenitor cells of the distal respiratory epithelium; and (4) differentiation of bronchial epithelium is coincident with loss of SP-C expression and subsequent acquisition of CC10 expression in proximal regions of the developing bronchial tubules.

Effects of tissue factor, PAR-2 and MMP-9 expression on human breast cancer cell line MCF-7 invasion.

PubMed

Lin, Zeng-Mao; Zhao, Jian-Xin; Duan, Xue-Ning; Zhang, Lan-Bo; Ye, Jing-Ming; Xu, Ling; Liu, Yin-Hua

2014-01-01

This study aimed to explore the expression of tissue factor (TF), protease activated receptor-2 (PAR-2), and matrix metalloproteinase-9 (MMP-9) in the MCF-7 breast cancer cell line and influence on invasiveness. Stable MCF-7 cells transfected with TF cDNA and with TF ShRNA were established. TF, PAR-2, and MMP-9 protein expression was analyzed using indirect immunofluorescence and invasiveness was evaluated using a cell invasion test. Effects of an exogenous PAR-2 agonist were also examined. TF protein expression significantly differed between the TF cDNA and TF ShRNA groups. MMP-9 protein expression was significantly correlated with TF protein expression, but PAR-2 protein expression was unaffected. The PAR- 2 agonist significantly enhanced MMP-9 expression and slightly increased TF and PAR-2 expression in the TF ShRNA group, but did not significantly affect protein expression in MCF-7 cells transfected with TF cDNA. TF and MMP-9 expression was positively correlated with the invasiveness of tumor cells. TF, PAR-2, and MMP-9 affect invasiveness of MCF-7 cells. TF may increase MMP-9 expression by activating PAR-2.

Safe and stable noninvasive focal gene delivery to the mammalian brain following focused ultrasound.

PubMed

Stavarache, Mihaela A; Petersen, Nicholas; Jurgens, Eric M; Milstein, Elizabeth R; Rosenfeld, Zachary B; Ballon, Douglas J; Kaplitt, Michael G

2018-04-27

OBJECTIVE Surgical infusion of gene therapy vectors has provided opportunities for biological manipulation of specific brain circuits in both animal models and human patients. Transient focal opening of the blood-brain barrier (BBB) by MR-guided focused ultrasound (MRgFUS) raises the possibility of noninvasive CNS gene therapy to target precise brain regions. However, variable efficiency and short follow-up of studies to date, along with recent suggestions of the potential for immune reactions following MRgFUS BBB disruption, all raise questions regarding the viability of this approach for clinical translation. The objective of the current study was to evaluate the efficiency, safety, and long-term stability of MRgFUS-mediated noninvasive gene therapy in the mammalian brain. METHODS Focused ultrasound under the control of MRI, in combination with microbubbles consisting of albumin-coated gas microspheres, was applied to rat striatum, followed by intravenous infusion of an adeno-associated virus serotype 1/2 (AAV1/2) vector expressing green fluorescent protein (GFP) as a marker. Following recovery, animals were followed from several hours up to 15 months. Immunostaining for GFP quantified transduction efficiency and stability of expression. Quantification of neuronal markers was used to determine histological safety over time, while inflammatory markers were examined for evidence of immune responses. RESULTS Transitory disruption of the BBB by MRgFUS resulted in efficient delivery of the AAV1/2 vector to the targeted rodent striatum, with 50%-75% of striatal neurons transduced on average. GFP transgene expression appeared to be stable over extended periods of time, from 2 weeks to 6 months, with evidence of ongoing stable expression as long as 16 months in a smaller cohort of animals. No evidence of substantial toxicity, tissue injury, or neuronal loss was observed. While transient inflammation from BBB disruption alone was noted for the first few days, consistent with prior observations, no evidence of brain inflammation was observed from 2 weeks to 6 months following MRgFUS BBB opening, despite delivery of a virus and expression of a foreign protein in target neurons. CONCLUSIONS This study demonstrates that transitory BBB disruption using MRgFUS can be a safe and efficient method for site-specific delivery of viral vectors to the brain, raising the potential for noninvasive focal human gene therapy for neurological disorders.

Recombinant expression of extracellular domain of mutant Epidermal Growth Factor Receptor in prokaryotic and baculovirus expression systems.

PubMed

Vettath, Sunitha Kodengil; Shivashankar, Gaganashree; Menon, Krishnakumar N; Vijayachandran, Lakshmi S

2018-04-15

Epidermal Growth Factor Receptor variant III (EGFRvIII) is a tumor specific antigen detected in various tumors including gliomas, breast cancer, lung cancer, head and neck squamous cell carcinoma (HNSCC). Screening of EGFRvIII targeting drug molecules can be accelerated by developing drug screening platforms using recombinantly expressed protein. Choice of expression system is one of the major factors deciding the success of recombinant expression of a protein. In our study, we have tried to express and purify the extracellular domain (ECD) of this highly unstable protein using bacterial and baculovirus expression systems to select the expression system suited for our purpose. Even though the protein was successfully expressed in prokaryotic system, purification could be done only under denaturing conditions. But in the baculovirus expression system, the protein was expressed in soluble form and could be purified under native conditions, with single step of purification. Based on our results, we conclude that insect cells are better choice over E. coli cells for expressing EGFRvIII ECD in soluble form. This study provides insights for other researchers involved in expression of similar unstable membrane proteins, on selecting the best expression system and challenges involved. Copyright © 2018 Elsevier B.V. All rights reserved.

Instability of monoclonal myeloma protein may be identified as susceptibility to penetration and binding by newly synthesized Congo red derivatives.

PubMed

Spólnik, Paweł; Konieczny, Leszek; Piekarska, Barbara; Rybarska, Janina; Stopa, Barbara; Zemanek, Grzegorz; Król, Marcin; Roterman, Irena

2004-06-01

Monoclonal myeloma proteins often have an abnormal, unstable structure, and tend to aggregate with fatal clinical consequences. A method for early clinical identification of this aggregation tendency is impatiently awaited. This work proposes the use of supramolecular dyes as specific ligands to reveal protein instability. Disclosure of excessive polypeptide chain flexibility in unstable monoclonal proteins, leading to increased susceptibility to penetration by foreign compounds, appeared possible when new supramolecular Congo red-derived dyes with different protein-binding capabilities were used for complexation. Two basic protein instability levels, local and global, were differentiated by comparing the extent of protein loading with dye and the subsequent electrophoretic migration rate of the complexes. A simple electrophoretic test is proposed for assessment of the instability of monoclonal proteins in clinical conditions.

Proteins related to the functions of fibroblast-like synoviocytes identified by proteomic analysis.

PubMed

Zhang, Hui; Fan, Lie Ying; Zong, Ming; Sun, Li Shan; Lu, Liu

2012-01-01

It is well known that the fibroblast-like synoviocytes (FLS) play a key role in pathogenesis of rheumatoid arthritis (RA). This study was performed to separate the differentially expressed proteins of FLS from the patients with RA or osteoarthritis (OA) by two-dimensional electrophoresis (2-DE), and found proteins associated with the functions of FLS by mass spectrometry (MS). Total proteins were extracted and quantified from the primary cultured FLS from patients of RA (n=8) or OA (n=6). Proteins were separated by high-resolution 2-DE, and identified the differentially expressed proteins by MS. Western blot analyses was used to validated the expression of candidate proteins. The mRNA of these proteins was detected by semi-quantitative fluorescent PCR. There are 1147 protein spots from RA and 1324 protein spots from OA showed on 2-DE graphs, respectively. We have selected 84 protein spots for MS analysis, and 27 protein spots were successfully identified. We have found that protein isoaspartyl methyltransferase (PIMT) and pirin (iron-binding nuclear protein, PIR) with lower expression in RA, and thioredoxin 1(Trx-1) only expressed in RA may be associated with functions of FLS. Western Blot confirmed the expression of PIMT and pirin lower in RA, and Trx-1 expressed only in RA. The results of semi-quantitative fluorescent PCR are also consistent with 2-DE graphs. PIMT, pirin and Trx-1 affect the functions of FLS in some style and can be the drug targets of RA.

Unintended changes in protein expression revealed by proteomic analysis of seeds from transgenic pea expressing a bean alpha-amylase inhibitor gene.

PubMed

Chen, Hancai; Bodulovic, Greg; Hall, Prudence J; Moore, Andy; Higgins, Thomas J V; Djordjevic, Michael A; Rolfe, Barry G

2009-09-01

Seeds of genetically modified (GM) peas (Pisum sativum L.) expressing the gene for alpha-amylase inhibitor-1 (alphaAI1) from the common bean (Phaseolus vulgaris L. cv. Tendergreen) exhibit resistance to the pea weevil (Bruchus pisorum). A proteomic analysis was carried out to compare seeds from GM pea lines expressing the bean alphaAI1 protein and the corresponding alphaAI1-free segregating lines and non-GM parental line to identify unintended alterations to the proteome of GM peas due to the introduction of the gene for alphaAI1. Proteomic analysis showed that in addition to the presence of alphaAI1, 33 other proteins were differentially accumulated in the alphaAI1-expressing GM lines compared with their non-GM parental line and these were grouped into five expression classes. Among these 33 proteins, only three were found to be associated with the expression of alphaAI1 in the GM pea lines. The accumulation of the remaining 30 proteins appears to be associated with Agrobacterium-mediated transformation events. Sixteen proteins were identified after MALDI-TOF-TOF analysis. About 56% of the identified proteins with altered accumulation in the GM pea were storage proteins including legumin, vicilin or convicilin, phaseolin, cupin and valosin-containing protein. Two proteins were uniquely expressed in the alphaAI1-expressing GM lines and one new protein was present in both the alphaAI1-expressing GM lines and their alphaAI1-free segregating lines, suggesting that both transgenesis and transformation events led to demonstrable changes in the proteomes of the GM lines tested.

The expression of Argonaute2 and related microRNA biogenesis proteins in normal and hypoxic trophoblasts.

PubMed

Donker, Rogier B; Mouillet, Jean-François; Nelson, D Michael; Sadovsky, Yoel

2007-04-01

Endogenous microRNAs (miRNAs) post-transcriptionally regulate mRNA and protein expression during tissue development and function. Whereas adaptation to environmental insults are tightly regulated in human tissues, the role of miRNAs and miRNA biogenesis proteins in this context is inadequately explored. We sought to analyse the expression of the key RNAi enzyme Argonaute2 (Ago2) and other miRNA biogenesis proteins in human trophoblasts during differentiation and in hypoxic environment. Using an in vitro analysis of primary term human trophoblasts, we identified the expression of the core miRNA biogenesis proteins in human villous trophoblasts, with expression levels unaffected by cellular differentiation. We found that the miRNA biosynthetic pathway was functional and produced miRNAs, with miR-93 up-regulated and miR-424 down-regulated in hypoxic environment. In contrast, hypoxia did not alter the expression of key miRNA machinery proteins. The pivotal miRNA processing enzyme Ago2, along with its interacting protein DP103, were expressed in normal placentas as well as in placentas from pregnancies complicated by placental hypoperfusion that resulted in fetal growth restriction. Ago2 and DP103 co-immunoprecipitated, and did not limit trophoblast response to hypoxic stress. We concluded that the core miRNA machinery proteins are expressed and functional in human trophoblasts. The influence of hypoxia on the expression of a subset of placental miRNA species is unlikely to reflect altered expression of key miRNA biogenesis proteins.

Proteomic Profiling for Identification of Novel Biomarkers Differentially Expressed in Human Ovaries from Polycystic Ovary Syndrome Patients

PubMed Central

Li, Li; Zhang, Jiangyu; Deng, Qingshan; Li, Jieming; Li, Zhengfen; Xiao, Yao; Hu, Shuiwang; Li, Tiantian; Tan, Qiuxiao; Li, Xiaofang; Luo, Bingshu; Mo, Hui

2016-01-01

Objectives To identify differential protein expression pattern associated with polycystic ovary syndrome (PCOS). Methods Twenty women were recruited for the study, ten with PCOS as a test group and ten without PCOS as a control group. Differential in-gel electrophoresis (DIGE) analysis and mass spectroscopy were employed to identify proteins that were differentially expressed between the PCOS and normal ovaries. The differentially expressed proteins were further validated by western blot (WB) and immunohistochemistry (IHC). Results DIGE analysis revealed eighteen differentially expressed proteins in the PCOS ovaries of which thirteen were upregulated, and five downregulated. WB and IHC confirmed the differential expression of membrane-associated progesterone receptor component 1 (PGRMC1), retinol-binding protein 1 (RBP1), heat shock protein 90B1, calmodulin 1, annexin A6, and tropomyosin 2. Also, WB analysis revealed significantly (P<0.05) higher expression of PGRMC1 and RBP1 in PCOS ovaries as compared to the normal ovaries. The differential expression of the proteins was also validated by IHC. Conclusions The present study identified novel differentially expressed proteins in the ovarian tissues of women with PCOS that can serve as potential biomarkers for the diagnosis and development of novel therapeutics for the treatment of PCOS using molecular interventions. PMID:27846214

Proteomic Profiling for Identification of Novel Biomarkers Differentially Expressed in Human Ovaries from Polycystic Ovary Syndrome Patients.

PubMed

Li, Li; Zhang, Jiangyu; Deng, Qingshan; Li, Jieming; Li, Zhengfen; Xiao, Yao; Hu, Shuiwang; Li, Tiantian; Tan, Qiuxiao; Li, Xiaofang; Luo, Bingshu; Mo, Hui

2016-01-01

To identify differential protein expression pattern associated with polycystic ovary syndrome (PCOS). Twenty women were recruited for the study, ten with PCOS as a test group and ten without PCOS as a control group. Differential in-gel electrophoresis (DIGE) analysis and mass spectroscopy were employed to identify proteins that were differentially expressed between the PCOS and normal ovaries. The differentially expressed proteins were further validated by western blot (WB) and immunohistochemistry (IHC). DIGE analysis revealed eighteen differentially expressed proteins in the PCOS ovaries of which thirteen were upregulated, and five downregulated. WB and IHC confirmed the differential expression of membrane-associated progesterone receptor component 1 (PGRMC1), retinol-binding protein 1 (RBP1), heat shock protein 90B1, calmodulin 1, annexin A6, and tropomyosin 2. Also, WB analysis revealed significantly (P<0.05) higher expression of PGRMC1 and RBP1 in PCOS ovaries as compared to the normal ovaries. The differential expression of the proteins was also validated by IHC. The present study identified novel differentially expressed proteins in the ovarian tissues of women with PCOS that can serve as potential biomarkers for the diagnosis and development of novel therapeutics for the treatment of PCOS using molecular interventions.

Tula hantavirus L protein is a 250 kDa perinuclear membrane-associated protein.

PubMed

Kukkonen, Sami K J; Vaheri, Antti; Plyusnin, Alexander

2004-05-01

The complete open reading frame of Tula hantavirus (TULV) L RNA was cloned in three parts. The middle third (nt 2191-4344) could be expressed in E. coli and was used to immunize rabbits. The resultant antiserum was then used to immunoblot concentrated TULV and infected Vero E6 cells. The L protein of a hantavirus was detected, for the first time, in infected cells and was found to be expressed as a single protein with an apparent molecular mass of 250 kDa in both virions and infected cells. Using the antiserum, the expression level of the L protein was followed and image analysis of immunoblots indicated that there were 10(4) copies per cell at the peak level of expression. The antiserum was also used to detect the L protein in cell fractionation studies. In cells infected with TULV and cells expressing recombinant L, the protein pelleted with the microsomal membrane fraction. The membrane association was confirmed with membrane flotation assays. To visualize L protein localization in cells, a fusion protein of L and enhanced green fluorescent protein, L-EGFP, was expressed in Vero E6 cells with a plasmid-driven T7 expression system. L-EGFP localized in the perinuclear region where it had partial co-localization with the Golgi matrix protein GM130 and the TULV nucleocapsid protein.

Transient expression and cellular localization of recombinant proteins in cultured insect cells

USDA-ARS?s Scientific Manuscript database

Heterologous protein expression systems are used for production of recombinant proteins, interpretation of cellular trafficking/localization, and for the determination of biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for ...

An improved conjugate vaccine technology; induction of antibody responses to the tumor vasculature.

PubMed

Huijbers, Elisabeth J M; van Beijnum, Judy R; Lê, Chung T; Langman, Sofya; Nowak-Sliwinska, Patrycja; Mayo, Kevin H; Griffioen, Arjan W

2018-05-17

The induction of an antibody response against self-antigens requires a conjugate vaccine technology, where the self-antigen is conjugated to a foreign protein sequence, and the co-application of a potent adjuvant. The choice of this foreign sequence is crucial as a very strong antibody response towards it may compromise the anti-self immune response. Here, we aimed to optimize the conjugate design for application of vaccination against the tumor vasculature, using two different approaches. First, the immunogenicity of the previously employed bacterial thioredoxin (TRX) was reduced by using a truncated from (TRXtr). Second, the Escherichia coli proteome was scrutinized to identify alternative proteins, based on immunogenicity and potency to increase solubility, suitable for use in a conjugate vaccine. This technology was used for vaccination against a marker of the tumor vasculature, the well-known extra domain B (EDB) of fibronectin. We demonstrate that engineering of the foreign sequence of a conjugate vaccine can significantly improve antibody production. The TRXtr construct outperformed the one containing full-length TRX, for the production of anti-self antibodies to EDB. In addition, efficient tumor growth inhibition was observed with the new TRXtr-EDB vaccine. Microvessel density was decreased and enhanced leukocyte infiltration was observed, indicative of an active immune response directed against the tumor vasculature. Summarizing, we have identified a truncated form of the foreign antigen TRX that can improve conjugate vaccine technology for induction of anti-self antibody titers. This technology was named Immuno-Boost (I-Boost). Our findings are important for the clinical development of cancer vaccines directed against self antigens, e.g. the ones selectively found in the tumor vasculature. Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.

[Tripartite-motif protein 25 and pyruvate kinase M2 protein expression in non-small cell lung cancer].

PubMed

Jing, Huai-Zhi; Qiu, Feng; Chen, Shi-Zhi; Su, Lin; Qu, Can

2015-03-01

To investigate the expression of tripartite-motif protein 25 (TRIM25) and pyruvate kinase M2 (PKM2) protein in non-small cell lung cancer (NSCLC) and explore their role in the occurrence and progression of NSCLC. The expressions of TRIM25 and PKM2 protein were detected in 60 NSCLC specimens and 20 adjacent normal lung tissue (>5 cm from the lesions) with immunofluorescence histochemical method and in 10 fresh specimens of NSCLC with Western blotting. The results were analyzed in relation with the clinicopathological features of the patients. The positivity rates of TRIM25 expression was 45% in the 60 lung carcinoma specimens, significantly higher than that in the 20 normal lung tissues (10%, P=0.005). TRIM25 protein was expressed in 28.6% of lung adenocarcinoma tissues and in 59.4% of squamous carcinoma tissues (P=0.017). TRIM25 protein expression was positively correlated with the TNM stages and lymph node metastasis of NSCLC (P<0.05). The expressions of PKM2 protein in 60 cases of lung carcinoma was 73.3%,while in 20 cases of normal lung tissues the expressions was 30%(P=0.001). The positivity rates of PKM2 expression differed significantly between lung adenocarcinoma and squamous carcinoma (57.1% vs 87.5%, P=0.008). An inverse correlation was noted between TRIM25 and PKM2 expressions (P=0.026). TRIM25 and PKM2 protein may participate in the occurrence and progression of NSCLC, and their expressions are inversely correlated.

[Expression of Ki-67 and P53 protein in oral squamous cell carcinoma and its clinical significance].

PubMed

He, Wei; Xiao, Yan; Chen, Wei-min

2015-04-01

To investigate the clinical and pathological features and its relationship with the expression of Ki-67 and p53 protein in oral squamous cell carcinoma. Immunohistochemical SP staining method was used to quantify the protein expression levels of Ki-67 and p53 protein in 10 cases of normal oral mucosa, 16 cases of oral leukoplakia (OLK) tissue, and 48 cases of oral squamous cell carcinoma. The relationship of the expression of Ki-67 and p53 protein to clinical and pathological data was analyzed, and SPSS17.0 software package was used for statistical analysis. The positive expression rate of Ki-67 protein in normal oral mucosa, oral leukoplakia and oral squamous cell carcinoma was 30%, 56.3% and 79.2%, respectively; The positive expression rate of p53 was 0%, 43.8%, and 70.8%, respectively; Ki-67 and p53 expression had significant difference among normal oral mucosa, oral leukoplakia and oral squamous cell carcinoma (P<0.05); The expression of Ki-67 protein was significantly elevated with tumor stage, differentiation and cervical lymph node metastasis (P<0.05); The expression of p53 protein was significantly related to the degree of tumor differentiation (P<0.05); The expression of Ki-67 and p53 was positively correlated in oral squamous cell carcinoma (P<0.05). The high expression of Ki-67 and p53 protein in oral squamous cell carcinoma tissues may play an important role in the development of oral squamous cell carcinoma.

Plasmids for variable expression of proteins targeted to the mitochondrial matrix or intermembrane space.

PubMed

Newman, Laura E; Schiavon, Cara; Kahn, Richard A

2016-01-01

We describe the construction and uses of a series of plasmids for directing expression to varied levels of exogenous proteins targeted to the mitochondrial matrix or intermembrane space. We found that the level of protein expression achieved, the kinetics of expression and mitochondrial import, and half-life after import can each vary with the protein examined. These factors should be considered when directing localization of an exogenous protein to mitochondria for rescue, proteomics, or other approaches. We describe the construction of a collection of plasmids for varied expression of proteins targeted to the mitochondrial matrix or intermembrane space, using previously defined targeting sequences and strength CMV promoters. The limited size of these compartments makes them particularly vulnerable to artifacts from over-expression. We found that different proteins display different kinetics of expression and import that should be considered when analyzing results from this approach. Finally, this collection of plasmids has been deposited in the Addgene plasmid repository to facilitate the ready access and use of these tools.

Uncovering Suitable Reference Proteins for Expression Studies in Human Adipose Tissue with Relevance to Obesity

PubMed Central

Pérez-Pérez, Rafael; López, Juan A.; García-Santos, Eva; Camafeita, Emilio; Gómez-Serrano, María; Ortega-Delgado, Francisco J.; Ricart, Wifredo; Fernández-Real, José M.; Peral, Belén

2012-01-01

Background Protein expression studies based on the two major intra-abdominal human fat depots, the subcutaneous and the omental fat, can shed light into the mechanisms involved in obesity and its co-morbidities. Here we address, for the first time, the identification and validation of reference proteins for data standardization, which are essential for accurate comparison of protein levels in expression studies based on fat from obese and non-obese individuals. Methodology and Findings To uncover adipose tissue proteins equally expressed either in omental and subcutaneous fat depots (study 1) or in omental fat from non-obese and obese individuals (study 2), we have reanalyzed our previously published data based on two-dimensional fluorescence difference gel electrophoresis. Twenty-four proteins (12 in study 1 and 12 in study 2) with similar expression levels in all conditions tested were selected and identified by mass spectrometry. Immunoblotting analysis was used to confirm in adipose tissue the expression pattern of the potential reference proteins and three proteins were validated: PARK7, ENOA and FAA. Western Blot analysis was also used to test customary loading control proteins. ENOA, PARK7 and the customary loading control protein Beta-actin showed steady expression profiles in fat from non-obese and obese individuals, whilst FAA maintained steady expression levels across paired omental and subcutaneous fat samples. Conclusions ENOA, PARK7 and Beta-actin are proper reference standards in obesity studies based on omental fat, whilst FAA is the best loading control for the comparative analysis of omental and subcutaneous adipose tissues either in obese and non-obese subjects. Neither customary loading control proteins GAPDH and TBB5 nor CALX are adequate standards in differential expression studies on adipose tissue. The use of the proposed reference proteins will facilitate the adequate analysis of proteins differentially expressed in the context of obesity, an aim difficult to achieve before this study. PMID:22272336

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.

Screening Fusion Tags for Improved Recombinant Protein Expression in E. coli with the Expresso® Solubility and Expression Screening System.

PubMed

Steinmetz, Eric J; Auldridge, Michele E

2017-11-01

The simplicity, speed, and low cost of bacterial culture make E. coli the system of choice for most initial trials of recombinant protein expression. However, many heterologous proteins are either poorly expressed in bacteria, or are produced as incorrectly folded, insoluble aggregates that lack the activity of the native protein. In many cases, fusion to a partner protein can allow for improved expression and/or solubility of a difficult target protein. Although several different fusion partners have gained favor, none are universally effective, and identifying the one that best improves soluble expression of a given target protein is an empirical process. This unit presents a strategy for parallel screening of fusion partners for enhanced expression or solubility. The Expresso® Solubility and Expression Screening System includes a panel of seven distinct fusion partners and utilizes an extremely simple cloning strategy to enable rapid screening and identification of the most effective fusion partner. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Insect cells-baculovirus system for the production of difficult to express proteins.

PubMed

Osz-Papai, Judit; Radu, Laura; Abdulrahman, Wassim; Kolb-Cheynel, Isabelle; Troffer-Charlier, Nathalie; Birck, Catherine; Poterszman, Arnaud

2015-01-01

The production of sufficient quantities of homogenous protein not only is an essential prelude for structural investigations but also represents a rate-limiting step for many human functional studies. Although technologies for expression of recombinant proteins and complexes have been improved tremendously, in many cases, protein production remains a challenge and can be associated with considerable investment. This chapter describes simple and efficient protocols for expression screening and optimization of protein production in insect cells using the baculovirus expression system. We describe the procedure, starting from the cloning of a gene of interest into an expression transfer baculovirus vector, followed by generation of the recombinant virus by homologous recombination, evaluation of protein expression, and scale-up. Handling of insect cell cultures and preparation of bacmid for co-transfection are also detailed.

Evaluation of Beauveria bassiana infection in the hemolymph serum proteins of the housefly, Musca domestica L. (Diptera: Muscidae).

PubMed

Mishra, Sapna; Kumar, Peeyush; Malik, Anushree

2017-11-01

Beauveria bassiana plays a prominent role in biocontrol of houseflies, Musca domestica (L.). Thus, a deeper insight into immune response of M. domestica during B. bassiana infection was warranted to assist the production of more efficient mycoinsecticides. The present study investigates changes in protein profile of M. domestica hemolymph serum post B. bassiana infection using two-dimensional difference gel electrophoresis (2D-DIGE) followed by identification of selected proteins by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). The non-infected or control group of flies showed an expression of 54 proteins, while M. domestica infected with B. bassiana expressed a total of 68 hemolymph serum proteins. Thirty three proteins were expressed in both groups of houseflies, whereas 35 proteins were exclusively expressed in infected flies and 21 proteins were exclusively expressed in control flies. Among the 33 proteins which were expressed in both groups of houseflies, 17 proteins showed downregulation, while16 proteins were upregulated in the infected flies compared to the non-infected ones. The results from this study are expected to facilitate better understanding of insect's immune response mechanism.

Distinct Protein Expression Profiles of Solid-Pseudopapillary Neoplasms of the Pancreas.

PubMed

Park, Minhee; Lim, Jong-Sun; Lee, Hyoung-Joo; Na, Keun; Lee, Min Jung; Kang, Chang Moo; Paik, Young-Ki; Kim, Hoguen

2015-08-07

Solid-pseudopapillary neoplasm (SPN) is an uncommon pancreatic tumor with mutation in CTNNB1 and distinct clinical and pathological features. We compared the proteomic profiles of SPN to mRNA expression. Pooled SPNs and pooled non-neoplastic pancreatic tissues were examined with high-resolution mass spectrometry. We identified 329 (150 up-regulated and 179 down-regulated) differentially expressed proteins in SPN. We identified 191 proteins (58.1% of the 329 dysregulated proteins) with the same expression tendencies in SPN based on mRNA data. Many overexpressed proteins were related to signaling pathways known to be activated in SPNs. We found that several proteins involved in Wnt signaling, including DKK4 and β-catenin, and proteins that bind β-catenin, such as FUS and NONO, were up-regulated in SPNs. Molecules involved in glycolysis, including PKM2, ENO2, and HK1, were overexpressed in accordance to their mRNA levels. In summary, SPN showed (1) distinct protein expression changes that correlated with mRNA expression, (2) overexpression of Wnt signaling proteins and proteins that bind directly to β-catenin, and (3) overexpression of proteins involved in metabolism. These findings may help develop early diagnostic biomarkers and molecular targets.

A Generic Protocol for Intracellular Expression of Recombinant Proteins in Bacillus subtilis.

PubMed

Phan, Trang; Huynh, Phuong; Truong, Tuom; Nguyen, Hoang

2017-01-01

Bacillus subtilis (B. subtilis) is a potential and attractive host for the production of recombinant proteins. Different expression systems for B. subtilis have been developed recently, and various target proteins have been recombinantly synthesized and purified using this host. In this chapter, we introduce a generic protocol to express a recombinant protein in B. subtilis. It includes protocols for (1) using our typical expression vector (plasmid pHT254) to introduce a target gene, (2) transformation of the target vector into B. subtilis, and (3) evaluation of the actual expression of a recombinant protein.

[Expression of c-jun protein after experimental rat brain concussion].

PubMed

Wang, Feng; Li, Yong-hong

2010-02-01

To observe e-jun protein expression after rat brain concussion and explore the forensic pathologic markers following brain concussion. Fifty-five rats were randomly divided into brain concussion group and control group. The expression of c-jun protein was observed by immunohistochemistry. There were weak positive expression of c-jun protein in control group. In brain concussion group, however, some neutrons showed positive expression of c-jun protein at 15 min after brain concussion, and reach to the peak at 3 h after brain concussion. The research results suggest that detection of c-jun protein could be a marker to determine brain concussion and estimate injury time after brain concussion.

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. Copyright © 2015 Elsevier Inc. All rights reserved.

Expression of bovine non-classical major histocompatibility complex class 1 proteins in mouse P815 and human K562 cells

USDA-ARS?s Scientific Manuscript database

Major histocompatibility complex class I (MHC-I) proteins can be expressed as cell surface or secreted proteins. To investigate whether bovine non-classical MHC-I proteins are expressed as cell surface or secreted proteins, and to assess the reactivity pattern of monoclonal antibodies with non-class...

Long-term Dietary Macronutrients and Hepatic Gene Expression in Aging Mice.

PubMed

Gokarn, Rahul; Solon-Biet, Samantha M; Cogger, Victoria C; Cooney, Gregory J; Wahl, Devin; McMahon, Aisling C; Mitchell, James R; Mitchell, Sarah J; Hine, Christopher; de Cabo, Rafael; Raubenheimer, David; Simpson, Stephen J; Le Couteur, David G

2018-04-23

Nutrition influences both hepatic function and aging, but mechanisms are poorly understood. Here, the effects of lifelong, ad libitum-fed diets varying in macronutrients and energy on hepatic gene expression were studied. Gene expression was measured using Affymetrix mouse arrays in livers of 46 mice aged 15 months fed one of 25 diets varying in protein, carbohydrates, fat, and energy density from 3 weeks of age. Gene expression was almost entirely influenced by protein intake. Carbohydrate and fat intake had few effects on gene expression compared with protein. Pathways and processes associated with protein intake included those involved with mitochondrial function, metabolic signaling (PI3K-Akt, AMPK, mTOR) and metabolism of protein and amino acids. Protein intake had variable effects on genes associated with regulation of longevity and influenced by caloric restriction. Among the genes of interest with expression that were significantly associated with protein intake are Cth, Gls2, Igf1, and Nnmt, which were increased with higher protein intake, and Igf2bp2, Fgf21, Prkab2, and Mtor, which were increased with lower protein intake. Dietary protein has a powerful impact on hepatic gene expression in older mice, with some overlap with genes previously reported to be involved with regulation of longevity or caloric restriction.

46 CFR 515.31 - General duties.

Code of Federal Regulations, 2010 CFR

2010-10-01

... principal or shipper without the express written authority of such principal or shipper. (i) Accounting to... MARITIME COMMISSION REGULATIONS AFFECTING OCEAN SHIPPING IN FOREIGN COMMERCE LICENSING, FINANCIAL...) have been reported to the Commission in writing; and (2) are covered by increased financial...

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.

[Pichia pastoris as an expression system for recombinant protein production].

PubMed

Ciarkowska, Anna; Jakubowska, Anna

2013-01-01

Pichia pastoris has become increasingly popular as a host for recombinant protein production in recent years. P. pastoris is more cost effective and allows achieving higher expression levels than insect and mammalian cells. It also offers some significant advantages over E. coli expression systems, such as avoiding problems with proper protein folding. Also, P. pastoris as an eukaryotic organism can carry out posttranslational modifications of produced proteins. Additionally, P. pastoris can produce high levels of recombinant proteins in extracellular medium which simplifies protein purification. Having many advantages over other expression systems makes P. pastoris an organism of choice for industrial protein production.

Newborn Mouse Lens Proteome and Its Alteration by Lysine 6 Mutant Ubiquitin

PubMed Central

2015-01-01

Ubiquitin is a tag that often initiates degradation of proteins by the proteasome in the ubiquitin proteasome system. Targeted expression of K6W mutant ubiquitin (K6W-Ub) in the lens results in defects in lens development and cataract formation, suggesting critical functions for ubiquitin in lens. To study the developmental processes that require intact ubiquitin, we executed the most extensive characterization of the lens proteome to date. We quantified lens protein expression changes in multiple replicate pools of P1 wild-type and K6W-Ub-expressing mouse lenses. Lens proteins were digested with trypsin, peptides were separated using strong cation exchange and reversed-phase liquid chromatography, and tandem mass (MS/MS) spectra were collected with a linear ion trap. Transgenic mice that expressed low levels of K6W-Ub (low expressers) had normal, clear lenses at birth, whereas the lenses that expressed high levels of K6W-Ub (higher expressers) had abnormal lenses and cataracts at birth. A total of 2052 proteins were identified, of which 996 were reliably quantified and compared between wild-type and K6W-Ub transgenic mice. Consistent with a delayed developmental program, fiber-cell-specific proteins, such as γ-crystallins (γA, γB, γC, and γE), were down-regulated in K6W-Ub higher expressers. Up-regulated proteins were involved in energy metabolism, signal transduction, and proteolysis. The K6W-Ub low expressers exhibited delayed onset and milder cataract consistent with smaller changes in protein expression. Because lens protein expression changes occurred prior to lens morphological abnormalities and cataract formation in K6W-Ub low expressers, it appears that expression of K6W-Ub sets in motion a process of altered protein expression that results in developmental defects and cataract. PMID:24450463

Transporter Expression in Liver Tissue from Subjects with Alcoholic or Hepatitis C Cirrhosis Quantified by Targeted Quantitative Proteomics

PubMed Central

Wang, Li; Collins, Carol; Kelly, Edward J.; Chu, Xiaoyan; Ray, Adrian S.; Salphati, Laurent; Xiao, Guangqing; Lee, Caroline; Lai, Yurong; Liao, Mingxiang; Mathias, Anita; Evers, Raymond; Humphreys, William; Hop, Cornelis E. C. A.; Kumer, Sean C.

2016-01-01

Although data are available on the change of expression/activity of drug-metabolizing enzymes in liver cirrhosis patients, corresponding data on transporter protein expression are not available. Therefore, using quantitative targeted proteomics, we compared our previous data on noncirrhotic control livers (n = 36) with the protein expression of major hepatobiliary transporters, breast cancer resistance protein (BCRP), bile salt export pump (BSEP), multidrug and toxin extrusion protein 1 (MATE1), multidrug resistance–associated protein (MRP)2, MRP3, MRP4, sodium taurocholate–cotransporting polypeptide (NTCP), organic anion–transporting polypeptides (OATP)1B1, 1B3, 2B1, organic cation transporter 1 (OCT1), and P-glycoprotein (P-gp) in alcoholic (n = 27) and hepatitis C cirrhosis (n = 30) livers. Compared with control livers, the yield of membrane protein from alcoholic and hepatitis C cirrhosis livers was significantly reduced by 56 and 67%, respectively. The impact of liver cirrhosis on transporter protein expression was transporter-dependent. Generally, reduced protein expression (per gram of liver) was found in alcoholic cirrhosis livers versus control livers, with the exception that the expression of MRP3 was increased, whereas no change was observed for MATE1, MRP2, OATP2B1, and P-gp. In contrast, the impact of hepatitis C cirrhosis on protein expression of transporters (per gram of liver) was diverse, showing an increase (MATE1), decrease (BSEP, MRP2, NTCP, OATP1B3, OCT1, and P-gp), or no change (BCRP, MRP3, OATP1B1, and 2B1). The expression of hepatobiliary transporter protein differed in different diseases (alcoholic versus hepatitis C cirrhosis). Finally, incorporation of protein expression of OATP1B1 in alcoholic cirrhosis into the Simcyp physiologically based pharmacokinetics cirrhosis module improved prediction of the disposition of repaglinide in liver cirrhosis patients. These transporter expression data will be useful in the future to predict transporter-mediated drug disposition in liver cirrhosis patients. PMID:27543206

Optimised 'on demand' protein arraying from DNA by cell free expression with the 'DNA to Protein Array' (DAPA) technology.

PubMed

Schmidt, Ronny; Cook, Elizabeth A; Kastelic, Damjana; Taussig, Michael J; Stoevesandt, Oda

2013-08-02

We have previously described a protein arraying process based on cell free expression from DNA template arrays (DNA Array to Protein Array, DAPA). Here, we have investigated the influence of different array support coatings (Ni-NTA, Epoxy, 3D-Epoxy and Polyethylene glycol methacrylate (PEGMA)). Their optimal combination yields an increased amount of detected protein and an optimised spot morphology on the resulting protein array compared to the previously published protocol. The specificity of protein capture was improved using a tag-specific capture antibody on a protein repellent surface coating. The conditions for protein expression were optimised to yield the maximum amount of protein or the best detection results using specific monoclonal antibodies or a scaffold binder against the expressed targets. The optimised DAPA system was able to increase by threefold the expression of a representative model protein while conserving recognition by a specific antibody. The amount of expressed protein in DAPA was comparable to those of classically spotted protein arrays. Reaction conditions can be tailored to suit the application of interest. DAPA represents a cost effective, easy and convenient way of producing protein arrays on demand. The reported work is expected to facilitate the application of DAPA for personalized medicine and screening purposes. Copyright © 2013 Elsevier B.V. All rights reserved.

Insect Larvae: A New Platform to Produce Commercial Recombinant Proteins.

PubMed

Targovnik, Alexandra M; Arregui, Mariana B; Bracco, Lautaro F; Urtasun, Nicolas; Baieli, Maria F; Segura, Maria M; Simonella, Maria A; Fogar, Mariela; Wolman, Federico J; Cascone, Osvaldo; Miranda, Maria V

2016-01-01

In Biotechnology, the expression of recombinant proteins is a constantly growing field and different hosts are used for this purpose. Some valuable proteins cannot be produced using traditional systems. Insects from the order Lepidoptera infected with recombinant baculovirus have appeared as a good choice to express high levels of proteins, especially those with post-translational modifications. Lepidopteran insects, which are extensively distributed in the world, can be used as small protein factories, the new biofactories. Species like Bombyx mori (silkworm) have been analyzed in Asian countries to produce a great number of recombinant proteins for use in basic and applied science and industry. Many proteins expressed in this larva have been commercialized. Several recombinant proteins produced in silkworms have already been commercialized. On the other hand, species like Spodoptera frugiperda, Heliothis virescens, Rachiplusia nu, Helicoverpa zea and Trichoplusia ni are widely distributed in both the occidental world and Europe. The expression of recombinant proteins in larvae has the advantage of its low cost in comparison with insect cell cultures. A wide variety of recombinant proteins, including enzymes, hormones and vaccines, have been efficiently expressed with intact biological activity. The expression of pharmaceutically proteins, using insect larvae or cocoons, has become very attractive. This review describes the use of insect larvae as an alternative to produce commercial recombinant proteins.

Immunohistochemical Analysis of ATRX, IDH1 and p53 in Glioblastoma and Their Correlations with Patient Survival

PubMed Central

2016-01-01

Glioblastoma (GBM) can be classified into molecular subgroups, on the basis of biomarker expression. Here, we classified our cohort of 163 adult GBMs into molecular subgroups according to the expression of proteins encoded by genes of alpha thalassemia/mental retardation syndrome X-linked (ATRX), isocitrate dehydrogenase (IDH) and TP53. We focused on the survival rate of molecular subgroups, depending on each and various combination of these biomarkers. ATRX, IDH1 and p53 protein expression were evaluated immunohistochemically and Kaplan-Meier analysis were carried out in each group. A total of 15.3% of enrolled GBMs demonstrated loss of ATRX expression (ATRX-), 10.4% expressed an aberrant IDH1 R132H protein (IDH1+), and 48.4% exhibited p53 overexpression (p53+). Survival differences were statistically significant when single protein expression or different combinations of expression of these proteins were analyzed. In conclusion, in the case of single protein expression, the patients with each IDH1+, or ATRX-, or p53- GBMs showed better survival than patients with counterparts protein expressed GBMs. In the case of double protein pairs, the patients with ATRX-/p53-, ATRX-/IDH1+, and IDH1+/p53- GBMs revealed better survival than the patients with GBMs with the remained pairs. In the case of triple protein combinations, the patients with ATRX-/p53-/IDH+ showed statistically significant survival gain than the patients with remained combination of proteins-expression status. Therefore, these three biomarkers, individually and as a combination, can stratify GBMs into prognostically relevant subgroups and have strong prognostic values in adult GBMs. PMID:27478330

Development of a silicon limitation inducible expression system for recombinant protein production in the centric diatoms Thalassiosira pseudonana and Cyclotella cryptica

DOE PAGES

Shrestha, Roshan P.; Hildebrand, Mark

2017-08-17

An inducible promoter for recombinant protein expression provides substantial benefits because under induction conditions cellular energy and metabolic capability can be directed into protein synthesis. The most widely used inducible promoter for diatoms is for nitrate reductase, however, nitrogen metabolism is tied into diverse aspects of cellular function, and the induction response is not necessarily robust. Silicon limitation offers a means to eliminate energy and metabolic flux into cell division processes, with little other detrimental effect on cellular function, and a protein expression system that works under those conditions could be advantageous. In this study, we evaluate a number ofmore » promoters for recombinant protein expression induced by silicon limitation and repressed by the presence of silicon in the diatoms Thalassiosira pseudonana and Cyclotella cryptica. In addition to silicon limitation, we describe additional strategies to elevate recombinant protein expression level, including inclusion of the 5' fragment of the coding region of the native gene and reducing carbon flow into ancillary processes of pigment synthesis and formation of photosynthetic storage products. We achieved yields of eGFP to 1.8% of total soluble protein in C. cryptica, which is about 3.6-fold higher than that obtained with chloroplast expression and ninefold higher than nuclear expression in another well-established algal system. Our studies demonstrate that the combination of inducible promoter and other strategies can result in robust expression of recombinant protein in a nuclear-based expression system in diatoms under silicon limited conditions, separating the protein expression regime from growth processes and improving overall recombinant protein yields.« less

Development of a silicon limitation inducible expression system for recombinant protein production in the centric diatoms Thalassiosira pseudonana and Cyclotella cryptica

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shrestha, Roshan P.; Hildebrand, Mark

An inducible promoter for recombinant protein expression provides substantial benefits because under induction conditions cellular energy and metabolic capability can be directed into protein synthesis. The most widely used inducible promoter for diatoms is for nitrate reductase, however, nitrogen metabolism is tied into diverse aspects of cellular function, and the induction response is not necessarily robust. Silicon limitation offers a means to eliminate energy and metabolic flux into cell division processes, with little other detrimental effect on cellular function, and a protein expression system that works under those conditions could be advantageous. In this study, we evaluate a number ofmore » promoters for recombinant protein expression induced by silicon limitation and repressed by the presence of silicon in the diatoms Thalassiosira pseudonana and Cyclotella cryptica. In addition to silicon limitation, we describe additional strategies to elevate recombinant protein expression level, including inclusion of the 5' fragment of the coding region of the native gene and reducing carbon flow into ancillary processes of pigment synthesis and formation of photosynthetic storage products. We achieved yields of eGFP to 1.8% of total soluble protein in C. cryptica, which is about 3.6-fold higher than that obtained with chloroplast expression and ninefold higher than nuclear expression in another well-established algal system. Our studies demonstrate that the combination of inducible promoter and other strategies can result in robust expression of recombinant protein in a nuclear-based expression system in diatoms under silicon limited conditions, separating the protein expression regime from growth processes and improving overall recombinant protein yields.« less

Molecular cloning, sequence characterization and recombinant expression of Nanog gene in goat fibroblast cells using lentiviral based expression system.

PubMed

Singhal, Dinesh K; Singhal, Raxita; Malik, Hruda N; Kumar, Surender; Kumar, Sudarshan; Mohanty, Ashok K; Kaushik, Jai K; Malakar, Dhruba

2014-01-01

Nanog is a homeodomain containing protein which plays important roles in regulation of signaling pathways for maintenance and induction of pluripotency in stem cells. Because of its unique expression in stem cells it is also regarded as pluripotency marker. In this study goat Nanog (gNanog) gene has been amplified, cloned and characterized at sequence level with successful over-expression in CHO-K1 cell line using a lentiviral based system. gNanog ORF is 903 bp long which codes for Nanog protein of size 300 amino acids (aas). Complete nucleotide sequence shows some evolutionary mutation in goat in comparision to other species. Protein sequence of goat is highly similar to other species. Overall, gNanog nucleotide sequence and predicted protein sequence showed high similarity and minimum divergence with cattle (96 % identity/4 % divergence) and buffalo (94/5 %) while low similarity and high divergence with pig (84/15 %), human (81/23 %) and mouse (69/40 %) indicating evolutionary closeness of gNanog to cattle and buffalo. gNanog lentiviral expression construct was prepared for over-expression of Nanog gene in adult goat fibroblast cells. Lentiviral expression construct of Nanog enabled continuous protein expression for induction and maintenance of pluripotency. Western blotting revealed the expression of Nanog gene at protein level which supported that the lentiviral expression system is highly promising for Nanog protein expression in differentiated goat cell.

Prognostic impact of MYC protein expression in central nervous system diffuse large B-cell lymphoma: comparison with MYC rearrangement and MYC mRNA expression.

PubMed

Son, Seung-Myoung; Ha, Sang-Yun; Yoo, Hae-Yong; Oh, Dongryul; Kim, Seok-Jin; Kim, Won-Seog; Ko, Young-Hyeh

2017-01-01

The prognostic role of MYC has been well documented in non-central nervous system diffuse large B-cell lymphoma; however, it remains controversial in central nervous system diffuse large B-cell lymphoma. To investigate the prognostic value of MYC, we analyzed the MYC protein expression by immunohistochemistry, mRNA expression by RNA in situ hybridization, and gene status by fluorescence in situ hybridization in 74 cases of central nervous system diffuse large B-cell lymphoma. Moreover, we examined the correlation between MYC translocation, mRNA expression, and protein expression. The mean percentage of MYC immunopositive cells was 49%. Using a 44% cutoff value, 49 (66%) cases showed MYC protein overexpression. The result of mRNA in situ hybridization using the RNA scope technology was obtained using the H-scoring system; the median value was 34.2. Using the cutoff value of 63.5, 16 (22%) cases showed MYC mRNA overexpression. MYC gene rearrangement was detected in five out of 68 (7%) cases. MYC translocation showed no statistically significant correlation with mRNA expression; however, all MYC translocation-positive cases showed MYC protein overexpression, with a higher mean percentage of MYC protein expression than that of translocation-negative cases (78 vs 48%, P=0.001). The level of MYC mRNA expression was moderately correlated with the level of MYC protein expression (P<0.001). The mean percentage of MYC protein expression in the high MYC mRNA group was higher than that in the low MYC mRNA group (70 vs 47%, P<0.001). A univariate analysis showed that age over 60 years, Eastern Cooperative Oncology Group (ECOG) performance status ≥2 and MYC protein overexpression were significantly associated with an increased risk of death. MYC translocation and MYC mRNA expression had no prognostic significance. On multivariate analysis, MYC protein overexpression and ECOG score retained prognostic significance.

Age-associated decrease in GDNF and its cognate receptor GFRα-1 protein expression in human skin.

PubMed

Adly, Mohamed A; Assaf, Hanan A; Hussein, Mahmoud Rezk Abdelwahed

2016-06-01

Glial cell line-derived neurotrophic factor (GDNF) and its cognate receptor (GFRα-1) are expressed in normal human skin. They are involved in murine hair follicle morphogenesis and cycling control. We hypothesize that 'GDNF and GFRα-1 protein expression in human skin undergoes age-associated alterations. To test our hypothesis, the expression of these proteins was examined in human skin specimens obtained from 30 healthy individuals representing three age groups: children (5-18 years), adults (19-60 years) and the elderly (61-81 years). Immunofluorescent and light microscopic immunohistologic analyses were performed using tyramide signal amplification and avidin-biotin complex staining methods respectively. GDNF mRNA expression was examined by RT-PCR analysis. GDNF mRNA and protein as well as GFRα-1 protein expressions were detected in normal human skin. We found significantly reduced epidermal expression of these proteins with ageing. In the epidermis, the expression was strong in the skin of children and declined gradually with ageing, being moderate in adults and weak in the elderly. In children and adults, the expression of both GDNF and GFRα-1 proteins was strongest in the stratum basale and decreased gradually towards the surface layers where it was completely absent in the stratum corneum. In the elderly, GDNF and GFRα-1 protein expression was confined to the stratum basale. In the dermis, both GDNF and GFRα-1 proteins had strong expressions in the fibroblasts, sweat glands, sebaceous glands, hair follicles and blood vessels regardless of the age. Thus there is a decrease in epidermal GDNF and GFRα-1 protein expression in normal human skin with ageing. Our findings suggest that the consequences of this is that GFRα-1-mediated signalling is altered during the ageing process. The clinical and therapeutic ramifications of these observations mandate further investigations. © 2016 The Authors. International Journal of Experimental Pathology © 2016 International Journal of Experimental Pathology.

Exocyst Complex Protein Expression in the Human Placenta

PubMed Central

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

2014-01-01

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

On the nature of in vivo requirements for rde-4 in RNAi and developmental pathways in C. elegans.

PubMed

Blanchard, Daniel; Parameswaran, Poornima; Lopez-Molina, Javier; Gent, Jonathan; Saynuk, Jamie Fleenor; Fire, Andrew

2011-01-01

C. elegans RDE-4 is a double-stranded RNA binding protein that has been shown to play a key role in response to foreign double-stranded RNA (dsRNA). We have used diverse tools for analysis of gene function to characterize the domain and organismal foci of RDE-4 action in C. elegans. First, we examined the focus of activity within the RDE-4 protein, by testing a series of RDE-4 deletion constructs for their ability to support dsRNA-triggered gene silencing. These assays indicated a molecular requirement for a linker region and the second dsRNA-binding domain of RDE-4, with ancillary contributions to function from the C and N terminal domains. Second, we used mosaic analysis to explore the cellular focus of action of RDE-4. These experiments indicated an ability of RDE-4 to function non-autonomously in foreign RNA responses. Third, we used growth under stressful conditions to search for evidence of an organismal focus of action for RDE-4 distinct from its role in response to foreign dsRNA. Propagation at high temperatures exposed a conditional requirement for RDE-4 for optimal growth and fertility, indicating at least under these conditions that RDE-4 can serve an essential role in C. elegans. © 2011 Landes Bioscience

The Ogaden Insurrection: Implications for Islamist Extremism and U.S. National Security

DTIC Science & Technology

2011-02-16

DISTRIBUTION A. Approved for public release: distribution unlimited i DISCLAIMER The views expressed in this academic research paper are those of...23 Terrence Lyons, “Avoiding Conflict in the Horn of Africa, U.S. Policy Toward Ethiopia and Eritrea,” Council on Foreign Relations, CSR No. 21...ballot box and [the EPRDF regards] the expression of differing views and interests as a form of betrayal,” and are therefore repressed. International

Expression of hypoxia-inducible 2 (HIG2) protein in uterine cancer.

PubMed

Nishimura, S; Tsuda, H; Nomura, H; Kataoka, F; Chiyoda, T; Tanaka, H; Tanaka, K; Susumu, N; Aoki, D

2011-01-01

For both cervical cancer (UCC) and endometrial cancer (EMC) there are no effective prognostic markers. In this study, we evaluated HIG2 protein expression in 332 uterine cancers (186 UCCs and 146 EMCs) and examined the relationship between HIG2 protein expression and clinical factors, including prognosis. Totally, HIG2 expression was detected in 58% of UCC and 66% of EMC. However, there was no significant relationship between HIG2 expression and age, clinical stage and histology in either UCC or EMC. In addition, HIG2 protein expression was not related to prognosis of UCC or EMC. The positivity rate of HIG2 protein was 56% and 61% in early-stage UCC and EMC, respectively and 67% in non-squamous cell carcinoma of UCC. The positivity rate of HIG2 protein was high even in early-stage UCC and EMC

Feeding and fasting controls liver expression of a regulator of G protein signaling (Rgs16) in periportal hepatocytes

PubMed Central

Huang, Jie; Pashkov, Victor; Kurrasch, Deborah M; Yu, Kan; Gold, Stephen J; Wilkie, Thomas M

2006-01-01

Background Heterotrimeric G protein signaling in liver helps maintain carbohydrate and lipid homeostasis. G protein signaling is activated by binding of extracellular ligands to G protein coupled receptors and inhibited inside cells by regulators of G protein signaling (RGS) proteins. RGS proteins are GTPase activating proteins, and thereby regulate Gi and/or Gq class G proteins. RGS gene expression can be induced by the ligands they feedback regulate, and RGS gene expression can be used to mark tissues and cell-types when and where Gi/q signaling occurs. We characterized the expression of mouse RGS genes in liver during fasting and refeeding to identify novel signaling pathways controlling changes in liver metabolism. Results Rgs16 is the only RGS gene that is diurnally regulated in liver of ad libitum fed mice. Rgs16 transcription, mRNA and protein are up regulated during fasting and rapidly down regulated after refeeding. Rgs16 is expressed in periportal hepatocytes, the oxygen-rich zone of the liver where lipolysis and gluconeogenesis predominates. Restricting feeding to 4 hr of the light phase entrained Rgs16 expression in liver but did not affect circadian regulation of Rgs16 expression in the suprachiasmatic nuclei (SCN). Conclusion Rgs16 is one of a subset of genes that is circadian regulated both in SCN and liver. Rgs16 mRNA expression in liver responds rapidly to changes in feeding schedule, coincident with key transcription factors controlling the circadian clock. Rgs16 expression can be used as a marker to identify and investigate novel G-protein mediated metabolic and circadian pathways, in specific zones within the liver. PMID:17123436

Structural and biochemical analysis of nuclease domain of clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 3 (Cas3).

PubMed

Mulepati, Sabin; Bailey, Scott

2011-09-09

RNA transcribed from clustered regularly interspaced short palindromic repeats (CRISPRs) protects many prokaryotes from invasion by foreign DNA such as viruses, conjugative plasmids, and transposable elements. Cas3 (CRISPR-associated protein 3) is essential for this CRISPR protection and is thought to mediate cleavage of the foreign DNA through its N-terminal histidine-aspartate (HD) domain. We report here the 1.8 Å crystal structure of the HD domain of Cas3 from Thermus thermophilus HB8. Structural and biochemical studies predict that this enzyme binds two metal ions at its active site. We also demonstrate that the single-stranded DNA endonuclease activity of this T. thermophilus domain is activated not by magnesium but by transition metal ions such as manganese and nickel. Structure-guided mutagenesis confirms the importance of the metal-binding residues for the nuclease activity and identifies other active site residues. Overall, these results provide a framework for understanding the role of Cas3 in the CRISPR system.

Transient Expression of Chimeric Fluorescent Reporter Proteins in Pollen Tubes to Study Protein Polar Secretion and Dynamics.

PubMed

Zhong, Guitao; Liu, Ronghe; Zhuang, Menglong; Wang, Hao

2017-01-01

Transient expression of chimeric fluorescent reporter proteins by biolistic bombardment is a quick and useful procedure for studying subcellular protein localization and dynamics in plants. It is especially beneficial in specific plant cells which are not suitable for protoplast-based and Agrobacterium-mediated protein transient expression. Polar protein secretion and vesicular trafficking play essential functions for cell polarization and tip growth. The growing pollen tube is regarded as an ideal model plant cell system to study the machinery and regulation of polar protein trafficking and targeting. A large amount of newly synthesized proteins are packed and polarly transported to the apical region to support the rapid and highly polarized tip growth. Here, we described a detailed step-by-step protocol for the transient expression of chimeric fluorescent reporter proteins in growing Arabidopsis and tobacco pollen tubes to study polar transportation logistics and mechanisms. In addition, we have optimized the Arabidopsis and tobacco in vitro pollen germination medium and the conditions to maximize the efficiency of protein expression. As a proof of concept, we have used this protocol to express actin microfilament and late endosomal fluorescent markers in Arabidopsis and tobacco pollen tubes.

Detection of recombinant human lactoferrin and lysozyme produced in a bitransgenic cow.

PubMed

Kaiser, Germán G; Mucci, Nicolás C; González, Vega; Sánchez, Lourdes; Parrón, José A; Pérez, María D; Calvo, Miguel; Aller, Juan F; Hozbor, Federico A; Mutto, Adrián A

2017-03-01

Lactoferrin and lysozyme are 2 glycoproteins with great antimicrobial activity, being part of the nonspecific defensive system of human milk, though their use in commercial products is difficult because human milk is a limited source. Therefore, many investigations have been carried out to produce those proteins in biological systems, such as bacteria, yeasts, or plants. Mammals seem to be more suitable as expression systems for human proteins, however, especially for those that are glycosylated. In the present study, we developed a bicistronic commercial vector containing a goat β-casein promoter and an internal ribosome entry site fragment between the human lactoferrin and human lysozyme genes to allow the introduction of both genes into bovine adult fibroblasts in a single transfection. Embryos were obtained by somatic cell nuclear transfer, and, after 6 transferences to recipients, 3 pregnancies and 1 viable bitransgenic calf were obtained. The presence of the vector was confirmed by fluorescent in situ hybridization of skin cells. At 13 mo of life and after artificial induction of lactation, both recombinant proteins were found in the colostrum and milk of the bitransgenic calf. Human lactoferrin concentration in the colostrum was 0.0098 mg/mL and that in milk was 0.011 mg/mL; human lysozyme concentration in the colostrum was 0.0022 mg/mL and that in milk was 0.0024 mg/mL. The molar concentration of both human proteins revealed no differences in protein production of the internal ribosome entry site upstream and downstream protein. The enzymatic activity of lysozyme in the transgenic milk was comparable to that of human milk, being 6 and 10 times higher than that of bovine lysozyme present in milk. This work represents an important step to obtain multiple proteins or enhance single protein production by using animal pharming and fewer regulatory and antibiotic-resistant foreign sequences, allowing the design of humanized milk with added biological value for newborn nutrition and development. Transgenic animals can offer a unique opportunity to the dairy industry, providing starting materials suitable to develop specific products with high added value. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Functional redundancy and/or ongoing pseudogenization among F-box protein genes expressed in Arabidopsis male gametophyte.

PubMed

Ikram, Sobia; Durandet, Monique; Vesa, Simona; Pereira, Serge; Guerche, Philippe; Bonhomme, Sandrine

2014-06-01

F-box protein genes family is one of the largest gene families in plants, with almost 700 predicted genes in the model plant Arabidopsis. F-box proteins are key components of the ubiquitin proteasome system that allows targeted protein degradation. Transcriptome analyses indicate that half of these F-box protein genes are found expressed in microspore and/or pollen, i.e., during male gametogenesis. To assess the role of F-box protein genes during this crucial developmental step, we selected 34 F-box protein genes recorded as highly and specifically expressed in pollen and isolated corresponding insertion mutants. We checked the expression level of each selected gene by RT-PCR and confirmed pollen expression for 25 genes, but specific expression for only 10 of the 34 F-box protein genes. In addition, we tested the expression level of selected F-box protein genes in 24 mutant lines and showed that 11 of them were null mutants. Transmission analysis of the mutations to the progeny showed that none of the single mutations was gametophytic lethal. These unaffected transmission efficiencies suggested leaky mutations or functional redundancy among F-box protein genes. Cytological observation of the gametophytes in the mutants confirmed these results. Combinations of mutations in F-box protein genes from the same subfamily did not lead to transmission defect either, further highlighting functional redundancy and/or a high proportion of pseudogenes among these F-box protein genes.

Modulating secretory pathway pH by proton channel co-expression can increase recombinant protein stability in plants.

PubMed

Jutras, Philippe V; D'Aoust, Marc-André; Couture, Manon M-J; Vézina, Louis-Philippe; Goulet, Marie-Claire; Michaud, Dominique; Sainsbury, Frank

2015-09-01

Eukaryotic expression systems are used for the production of complex secreted proteins. However, recombinant proteins face considerable biochemical challenges along the secretory pathway, including proteolysis and pH variation between organelles. As the use of synthetic biology matures into solutions for protein production, various host-cell engineering approaches are being developed to ameliorate host-cell factors that can limit recombinant protein quality and yield. We report the potential of the influenza M2 ion channel as a novel tool to neutralize the pH in acidic subcellular compartments. Using transient expression in the plant host, Nicotiana benthamiana, we show that ion channel expression can significantly raise pH in the Golgi apparatus and that this can have a strong stabilizing effect on a fusion protein separated by an acid-susceptible linker peptide. We exemplify the utility of this effect in recombinant protein production using influenza hemagglutinin subtypes differentially stable at low pH; the expression of hemagglutinins prone to conformational change in mildly acidic conditions is considerably enhanced by M2 co-expression. The co-expression of a heterologous ion channel to stabilize acid-labile proteins and peptides represents a novel approach to increasing the yield and quality of secreted recombinant proteins in plants and, possibly, in other eukaryotic expression hosts. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Accumulation and processing of a recombinant protein designed as a cleavable fusion to the endogenous Rubisco LSU protein in Chlamydomonas chloroplast

PubMed Central

Muto, Machiko; Henry, Ryan E; Mayfield, Stephen P

2009-01-01

Background Expression of recombinant proteins in green algal chloroplast holds substantial promise as a platform for the production of human therapeutic proteins. A number of proteins have been expressed in the chloroplast of Chlamydomonas reinhardtii, including complex mammalian proteins, but many of these proteins accumulate to significantly lower levels than do endogenous chloroplast proteins. We examined if recombinant protein accumulation could be enhanced by genetically fusing the recombinant reporter protein, luciferase, to the carboxy-terminal end of an abundant endogenous protein, the large subunit of ribulose bisphosphate carboxylase (Rubisco LSU). Additionally, as recombinant proteins fused to endogenous proteins are of little clinical or commercial value, we explored the possibility of engineering our recombinant protein to be cleavable from the endogenous protein in vivo. This strategy would obviate the need for further in vitro processing steps in order to produce the desired recombinant protein. To achieve this, a native protein-processing site from preferredoxin (preFd) was placed between the Rubisco LSU and luciferase coding regions in the fusion protein construct. Results The luciferase from the fusion protein accumulated to significantly higher levels than luciferase expressed alone. By eliminating the endogenous Rubisco large subunit gene (rbcL), we achieved a further increase in luciferase accumulation with respect to luciferase expression in the WT background. Importantly, near-wild type levels of functional Rubisco holoenzyme were generated following the proteolytic removal of the fused luciferase, while luciferase activity for the fusion protein was almost ~33 times greater than luciferase expressed alone. These data demonstrate the utility of using fusion proteins to enhance recombinant protein accumulation in algal chloroplasts, and also show that engineered proteolytic processing sites can be used to liberate the exogenous protein from the endogenous fusion partner, allowing for the purification of the intended mature protein. Conclusion These results demonstrate the utility of fusion proteins in algal chloroplast as a method to increase accumulation of recombinant proteins that are difficult to express. Since Rubisco is ubiquitous to land plants and green algae, this strategy may also be applied to higher plant transgenic expression systems. PMID:19323825

TA-GC cloning: A new simple and versatile technique for the directional cloning of PCR products for recombinant protein expression.

PubMed

Niarchos, Athanasios; Siora, Anastasia; Konstantinou, Evangelia; Kalampoki, Vasiliki; Lagoumintzis, George; Poulas, Konstantinos

2017-01-01

During the last few decades, the recombinant protein expression finds more and more applications. The cloning of protein-coding genes into expression vectors is required to be directional for proper expression, and versatile in order to facilitate gene insertion in multiple different vectors for expression tests. In this study, the TA-GC cloning method is proposed, as a new, simple and efficient method for the directional cloning of protein-coding genes in expression vectors. The presented method features several advantages over existing methods, which tend to be relatively more labour intensive, inflexible or expensive. The proposed method relies on the complementarity between single A- and G-overhangs of the protein-coding gene, obtained after a short incubation with T4 DNA polymerase, and T and C overhangs of the novel vector pET-BccI, created after digestion with the restriction endonuclease BccI. The novel protein-expression vector pET-BccI also facilitates the screening of transformed colonies for recombinant transformants. Evaluation experiments of the proposed TA-GC cloning method showed that 81% of the transformed colonies contained recombinant pET-BccI plasmids, and 98% of the recombinant colonies expressed the desired protein. This demonstrates that TA-GC cloning could be a valuable method for cloning protein-coding genes in expression vectors.

TA-GC cloning: A new simple and versatile technique for the directional cloning of PCR products for recombinant protein expression

PubMed Central

Niarchos, Athanasios; Siora, Anastasia; Konstantinou, Evangelia; Kalampoki, Vasiliki; Poulas, Konstantinos

2017-01-01

During the last few decades, the recombinant protein expression finds more and more applications. The cloning of protein-coding genes into expression vectors is required to be directional for proper expression, and versatile in order to facilitate gene insertion in multiple different vectors for expression tests. In this study, the TA-GC cloning method is proposed, as a new, simple and efficient method for the directional cloning of protein-coding genes in expression vectors. The presented method features several advantages over existing methods, which tend to be relatively more labour intensive, inflexible or expensive. The proposed method relies on the complementarity between single A- and G-overhangs of the protein-coding gene, obtained after a short incubation with T4 DNA polymerase, and T and C overhangs of the novel vector pET-BccI, created after digestion with the restriction endonuclease BccI. The novel protein-expression vector pET-BccI also facilitates the screening of transformed colonies for recombinant transformants. Evaluation experiments of the proposed TA-GC cloning method showed that 81% of the transformed colonies contained recombinant pET-BccI plasmids, and 98% of the recombinant colonies expressed the desired protein. This demonstrates that TA-GC cloning could be a valuable method for cloning protein-coding genes in expression vectors. PMID:29091919

Interpretation of biological and mechanical variations between the Lowry versus Bradford method for protein quantification.

PubMed

Lu, Tzong-Shi; Yiao, Szu-Yu; Lim, Kenneth; Jensen, Roderick V; Hsiao, Li-Li

2010-07-01

The identification of differences in protein expression resulting from methodical variations is an essential component to the interpretation of true, biologically significant results. We used the Lowry and Bradford methods- two most commonly used methods for protein quantification, to assess whether differential protein expressions are a result of true biological or methodical variations. MATERIAL #ENTITYSTARTX00026; Differential protein expression patterns was assessed by western blot following protein quantification by the Lowry and Bradford methods. We have observed significant variations in protein concentrations following assessment with the Lowry versus Bradford methods, using identical samples. Greater variations in protein concentration readings were observed over time and in samples with higher concentrations, with the Bradford method. Identical samples quantified using both methods yielded significantly different expression patterns on Western blot. We show for the first time that methodical variations observed in these protein assay techniques, can potentially translate into differential protein expression patterns, that can be falsely taken to be biologically significant. Our study therefore highlights the pivotal need to carefully consider methodical approaches to protein quantification in techniques that report quantitative differences.

Honeybee (Apis mellifera ligustica) drone embryo proteomes.

PubMed

Li, Jianke; Fang, Yu; Zhang, Lan; Begna, Desalegn

2011-03-01

Little attention has been paid to the drone honeybee (Apis mellifera ligustica) which is a haploid individual carrying only the set of alleles that it inherits from its mother. Molecular mechanisms underlying drone embryogenesis are poorly understood. This study evaluated protein expression profiles of drone embryogenesis at embryonic ages of 24, 48 and 72h. More than 100 reproducible proteins were analyzed by mass spectrometry on 2D electrophoresis gels. Sixty-two proteins were significantly changed at the selected three experimental age points. Expression of the metabolic energy requirement-related protein peaked at the embryonic age of 48h, whereas development and metabolizing amino acid-related proteins expressed optimally at 72h. Cytoskeleton, protein folding and antioxidant-related proteins were highly expressed at 48 and 72h. Protein networks of the identified proteins were constructed and protein expressions were validated at the transcription level. This first proteomic study of drone embryogenesis in the honeybee may provide geneticists an exact timetable and candidate protein outline for further manipulations of drone stem cells. Crown Copyright © 2010. Published by Elsevier Ltd. All rights reserved.

TGF-β1, Ghrelin, Neurexin, and Neuroligin are predictive biomarkers for postoperative prognosis of laparoscopic surgery in children with Hirschsprung disease.

PubMed

Shangjie, Xiao; Xiaochun, Zhu; Wenyi, Yang; Wuping, Ge; Ying, Zhang; Qiuming, He; Huimin, Xia

2015-03-01

The study was set to analyze the predictive values of transforming growth factor β1 (TGF-β1), Ghrelin, Neurexin, and Neuroligin protein expression on postoperative prognosis of laparoscopic surgery in children with Hirschsprung disease. 281 cases of children with Hirschsprung disease, admitted into Guangdong Women and Children Hospital and Guangzhou women and children's medical center from March 2009 to March 2014, were treated with laparoscopic radical surgery for Hirschsprung disease. They were divided into the good and the poor prognosis groups according to their recuperation and complications. Protein expressions of TGF-β1, Ghrelin, Neurexin, and Neuroligin were prospectively analyzed. The correlations between the expressions of these proteins and the prognosis were analyzed. There were 129 cases of children with poor prognosis, accounting for 45.9 %. There were no significant differences in the expressions of TGF-β1 mRNA and proteins within the group in both the groups (p > 0.05). TGF-β1 mRNA and protein expressions of the poor prognosis group were significantly higher than those of the good prognosis group in each segment of intestine (p < 0.05). Protein detection results manifested that Ghrelin protein expression gradually increased along narrow segment, transitional segment, and expansion segment in both groups. Ghrelin protein expression of the poor prognosis group was significantly lower than that of the good prognosis group in each segment of intestine (p < 0.05). There were significant differences in the protein expressions of Neurexin and Neuroligin within the group. The protein expressions of Neurexin and Neuroligin in expansion segment were the highest. Neurexin and Neuroligin protein expressions of the poor prognosis group were significantly lower than those of the good prognosis group in each segment of intestine (p < 0.05). Increasing expression of TGF-β1 protein, decreasing expressions of Ghrelin, Neurexin, and Neuroligin proteins can induce the loss or dysfunction of ganglion cells in distal intestinal canal, which is closely correlated with the occurrences of adverse prognosis, such as increased intestinal peristalsis recovery time, increased complication rate etc., in children. It has a high value for predicting prognosis of children patients with Hirschsprung disease after surgical intervention.

Differential expression of the major immediate early gene of human cytomegalovirus.

PubMed

Tsutsui, Y; Nogami-Satake, T

1990-01-01

We prepared a murine monoclonal antibody reactive to a human cytomegalovirus (HCMV)-induced nuclear protein with an Mr of 68,000. Expression of the 68K protein was compared with the major immediate early (IE) 72K protein in various cell types after infection with HCMV or microinjection of plasmid DNA containing the major IE gene. The 68K nuclear protein was detected 2 to 3 h after appearance of the 72K protein in human embryonal lung (HEL) cells infected with HCMV. The 68K protein was distributed throughout the cytoplasm in the late phase of infection, while the 72K protein remained chiefly in the nucleus. The 68K protein was barely detected in the cells under IE conditions by immunoprecipitation, but, together with the 72K protein, it was expressed after microinjection of cloned DNA, containing only the major IE region (region 1), into the nuclei of HEL cells. The 72K protein was expressed in nuclei 2 h after microinjection, whereas the 68K protein was detected 4 to 5 h after the injection. The 68K protein was expressed after microinjection in non-permissive rodent fibroblasts or non-permissive transformed human cells in which these proteins were not expressed after viral infection. Immunoprecipitations after chase-labelling from IE conditions or after partial digestions suggested that the 68K protein is neither a degradation nor a modification product of the major IE 72K protein.

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.

Prognostic significance of MYCN gene amplification and protein expression in primary brain tumors: Astrocytoma and meningioma.

PubMed

Estiar, Mehrdad Asghari; Javan, Firouzeh; Zekri, Ali; Mehrazin, Masoud; Mehdipour, Parvin

2017-07-04

Astrocytoma and meningioma are the most common primary brain tumors. MYCN as a member of MYC proto-oncogenes has recently appeared as an attractive therapeutic target. Functions of MYCN are critical for growth of nervous system and neural differentiation. We examined MYCN amplification and protein expression in astrocytoma and meningioma cases. In this study, we used real-time PCR, FISH assay and flowcytometry to analyze DNA amplification and protein expression of MYCN. Among 30 samples of brain tumor, 14 cases (46.6%) revealed MYCN amplification. High-protein expression of MYCN was also observed in 43.3% of patients. There was a significant correlation between MYCN gene amplification and protein expression (r= 0.523; p= 0.003), interestingly five case showed discrepancy between the gene amplification and protein expression. Although MYCN amplification fails to show correlation with poor prognosis (p= 0.305), protein high-expression of MYCN significantly reduce disease-free survival (p= 0.019). Our results challenge the concept of the neural specificity of MYCN by demonstrating contribution of MYCN in meningioma. Moreover, this study highlights the importance of research at both level of DNA and protein, to determine the biological functions and medical impacts of MYCN.

Expressing the sense of Congress that China release democratic activist Liu Xiaobo from imprisonment.

THOMAS, 111th Congress

Rep. Minnick, Walter [D-ID-1

2009-06-12

Senate - 10/01/2009 Received in the Senate and referred to the Committee on Foreign Relations. (All Actions) Tracker: This bill has the status Agreed to in HouseHere are the steps for Status of Legislation:

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.

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.

Stable zymomonas mobilis xylose and arabinose fermenting strains

DOEpatents

Zhang, Min [Lakewood, CO; Chou, Yat-Chen [Taipei, TW

2008-04-08

The present invention briefly includes a transposon for stable insertion of foreign genes into a bacterial genome, comprising at least one operon having structural genes encoding enzymes selected from the group consisting of xylAxylB, araBAD and tal/tkt, and at least one promoter for expression of the structural genes in the bacterium, a pair of inverted insertion sequences, the operons contained inside the insertion sequences, and a transposase gene located outside of the insertion sequences. A plasmid shuttle vector for transformation of foreign genes into a bacterial genome, comprising at least one operon having structural genes encoding enzymes selected from the group consisting of xylAxylB, araBAD and tal/tkt, at least one promoter for expression of the structural genes in the bacterium, and at least two DNA fragments having homology with a gene in the bacterial genome to be transformed, is also provided.The transposon and shuttle vectors are useful in constructing significantly different Zymomonas mobilis strains, according to the present invention, which are useful in the conversion of the cellulose derived pentose sugars into fuels and chemicals, using traditional fermentation technology, because they are stable for expression in a non-selection medium.

High expression of 23 kDa protein of augmenter of liver regeneration (ALR) in human hepatocellular carcinoma

PubMed Central

Yu, Hai-Ying; Zhu, Man-Hua; Xiang, Dai-Rong; Li, Jun; Sheng, Ji-Fang

2014-01-01

Background Augmenter of liver regeneration (ALR) is an important polypeptide that participates in the process of liver regeneration. Two forms of ALR proteins are expressed in hepatocytes. Previous data have shown that ALR is essential for cell survival and has potential antimetastatic properties in hepatocellular carcinoma (HCC). Aims The study aimed to evaluate the expression levels of two forms of ALR proteins in HCC and their possible significance in HCC development. Methods Balb/c mouse monoclonal antibody against ALR protein was prepared in order to detect the ALR protein in HCC by Western blotting and immunohistochemistry. ALR mRNA expression levels were measured by real-time polymerase chain reaction in HCC tissues and compared to paracancerous liver tissues in 22 HCC patients. Results ALR mRNA expression in HCC liver tissues (1.51×106 copies/μL) was higher than in paracancerous tissues (1.04×104 copies/μL). ALR protein expression was also enhanced in HCC liver tissues. The enhanced ALR protein was shown to be 23 kDa by Western blotting. Immunohistochemical analysis showed that the 23 kDa ALR protein mainly existed in the hepatocyte cytosol. Conclusion The 23 kDa ALR protein was highly expressed in HCC and may play an important role in hepatocarcinogenesis. PMID:24940072

Tunable Control of an Escherichia coli Expression System for the Overproduction of Membrane Proteins by Titrated Expression of a Mutant lac Repressor.

PubMed

Kim, Seong Keun; Lee, Dae-Hee; Kim, Oh Cheol; Kim, Jihyun F; Yoon, Sung Ho

2017-09-15

Most inducible expression systems suffer from growth defects, leaky basal induction, and inhomogeneous expression levels within a host cell population. These difficulties are most prominent with the overproduction of membrane proteins that are toxic to host cells. Here, we developed an Escherichia coli inducible expression system for membrane protein production based on titrated expression of a mutant lac repressor (mLacI). Performance of the mLacI inducible system was evaluated in conjunction with commonly used lac operator-based expression vectors using a T7 or tac promoter. Remarkably, expression of a target gene can be titrated by the dose-dependent addition of l-rhamnose, and the expression levels were homogeneous in the cell population. The developed system was successfully applied to overexpress three membrane proteins that were otherwise difficult to produce in E. coli. This gene expression control system can be easily applied to a broad range of existing protein expression systems and should be useful in constructing genetic circuits that require precise output signals.

Differences in protein expression among five species of stream stonefly (Plecoptera) along a latitudinal gradient in Japan.

PubMed

Gamboa, Maribet; Tsuchiya, Maria Claret; Matsumoto, Suguru; Iwata, Hisato; Watanabe, Kozo

2017-11-01

Proteome variation among natural populations along an environmental gradient may provide insights into how the biological functions of species are related to their local adaptation. We investigated protein expression in five stream stonefly species from four geographic regions along a latitudinal gradient in Japan with varying climatic conditions. The extracted proteins were separated by two-dimensional gel electrophoresis and identified by matrix-assisted laser desorption/ionization of time-of-flight (MALDI TOF/TOF), yielding 446 proteins. Low interspecies variation in the proteome profiles was observed among five species within geographical regions, presumably due to the co-occurring species sharing the environments. However, large spatial variations in protein expression were found among four geographic regions, suggesting strong regulation of protein expression in heterogeneous environments, where the spatial variations were positively correlated with water temperature. We identified 21 unique proteins expressed specifically in a geographical region and six common proteins expressed throughout all regions. In warmer regions, metabolic proteins were upregulated, whereas proteins related to cold stress, the photoperiod, and mating were downregulated. Oxygen-related and energy-production proteins were upregulated in colder regions with higher altitudes. Thus, our proteomic approach is useful for identifying and understanding important biological functions related to local adaptations by populations of stoneflies. © 2017 Wiley Periodicals, Inc.

The Interrelationship between Promoter Strength, Gene Expression, and Growth Rate

PubMed Central

Klesmith, Justin R.; Detwiler, Emily E.; Tomek, Kyle J.; Whitehead, Timothy A.

2014-01-01

In exponentially growing bacteria, expression of heterologous protein impedes cellular growth rates. Quantitative understanding of the relationship between expression and growth rate will advance our ability to forward engineer bacteria, important for metabolic engineering and synthetic biology applications. Recently, a work described a scaling model based on optimal allocation of ribosomes for protein translation. This model quantitatively predicts a linear relationship between microbial growth rate and heterologous protein expression with no free parameters. With the aim of validating this model, we have rigorously quantified the fitness cost of gene expression by using a library of synthetic constitutive promoters to drive expression of two separate proteins (eGFP and amiE) in E. coli in different strains and growth media. In all cases, we demonstrate that the fitness cost is consistent with the previous findings. We expand upon the previous theory by introducing a simple promoter activity model to quantitatively predict how basal promoter strength relates to growth rate and protein expression. We then estimate the amount of protein expression needed to support high flux through a heterologous metabolic pathway and predict the sizable fitness cost associated with enzyme production. This work has broad implications across applied biological sciences because it allows for prediction of the interplay between promoter strength, protein expression, and the resulting cost to microbial growth rates. PMID:25286161

BPI-fold (BPIF) containing/plunc protein expression in human fetal major and minor salivary glands.

PubMed

Alves, Daniel Berretta Moreira; Bingle, Lynne; Bingle, Colin David; Lourenço, Silvia Vanessa; Silva, Andréia Aparecida; Pereira, Débora Lima; Vargas, Pablo Agustin

2017-01-16

The aim of this study was to determine expression, not previously described, of PLUNC (palate, lung, and nasal epithelium clone) (BPI-fold containing) proteins in major and minor salivary glands from very early fetal tissue to the end of the second trimester and thus gain further insight into the function of these proteins. Early fetal heads, and major and minor salivary glands were collected retrospectively and glands were classified according to morphodifferentiation stage. Expression of BPI-fold containing proteins was localized through immunohistochemistry. BPIFA2, the major BPI-fold containing protein in adult salivary glands, was detected only in the laryngeal pharynx; the lack of staining in salivary glands suggested salivary expression is either very late in development or is only in adult tissues. Early expression of BPIFA1 was seen in the trachea and nasal cavity with salivary gland expression only seen in late morphodifferentiation stages. BPIFB1 was seen in early neural tissue and at later stages in submandibular and sublingual glands. BPIFA1 is significantly expressed in early fetal oral tissue but BPIFB1 has extremely limited expression and the major salivary BPIF protein (BPIFA2) is not produced in fetal development. Further studies, with more sensitive techniques, will confirm the expression pattern and enable a better understanding of embryonic BPIF protein function.

[Arf6, RalA and BIRC5 protein expression in non small cell lung cancer].

PubMed

Knizhnik, A V; Kovaleva, O B; Laktionov, K K; Mochal'nikova, V V; Komel'kov, A V; Chevkina, E M; Zborovskaia, I B

2011-01-01

Evaluation of tumor markers expression pattern which determines individual progression parameters is one of the major topics in molecular oncopathology research. This work presents research on expression analysis of several Ras-Ral associated signal transduction pathway proteins (Arf6, RalA and BIRC5) in accordance with clinical criteria in non small cell lung cancer patients. Using Western-blot analysis and RT-PCR Arf6, RalA and BIRC5 expression has been analyzed in parallel in 53 non small cell lung cancer samples of different origin. Arf6 protein expression was elevated in 55% non small cell lung cancer tumor samples in comparison with normal tissue. In the group of squamous cell lung cancer Arf6 expression elevation was observed more often. RalA protein expression was decreased in comparison to normal tissue samples in 64% of non small cell lung cancer regardless to morphological structure. Correlation between RalA protein expression decrease and absence of regional metastases was revealed for squamous cell lung cancer. BIRC5 protein expression in tumor samples versus corresponding normal tissue was 1.3 times more often elevated in the squamous cell lung cancer group (in 76% tumor samples). At the same time elevation of BIRC5 expression was fixed only in 63% of adenocarcinoma tumor samples. A statistically significant decrease (p = 0.0158) of RalA protein expression and increase (p = 0.0498) of Arf6 protein expression in comparison with normal tissue was found for T1-2N0M0 and T1-2N1-2M0 groups of squamous cell lung cancer correspondingly.

MUC1-ARF-A Novel MUC1 Protein That Resides in the Nucleus and Is Expressed by Alternate Reading Frame Translation of MUC1 mRNA.

PubMed

Chalick, Michael; Jacobi, Oded; Pichinuk, Edward; Garbar, Christian; Bensussan, Armand; Meeker, Alan; Ziv, Ravit; Zehavi, Tania; Smorodinsky, Nechama I; Hilkens, John; Hanisch, Franz-Georg; Rubinstein, Daniel B; Wreschner, Daniel H

2016-01-01

Translation of mRNA in alternate reading frames (ARF) is a naturally occurring process heretofore underappreciated as a generator of protein diversity. The MUC1 gene encodes MUC1-TM, a signal-transducing trans-membrane protein highly expressed in human malignancies. Here we show that an AUG codon downstream to the MUC1-TM initiation codon initiates an alternate reading frame thereby generating a novel protein, MUC1-ARF. MUC1-ARF, like its MUC1-TM 'parent' protein, contains a tandem repeat (VNTR) domain. However, the amino acid sequence of the MUC1-ARF tandem repeat as well as N- and C- sequences flanking it differ entirely from those of MUC1-TM. In vitro protein synthesis assays and extensive immunohistochemical as well as western blot analyses with MUC1-ARF specific monoclonal antibodies confirmed MUC1-ARF expression. Rather than being expressed at the cell membrane like MUC1-TM, immunostaining showed that MUC1-ARF protein localizes mainly in the nucleus: Immunohistochemical analyses of MUC1-expressing tissues demonstrated MUC1-ARF expression in the nuclei of secretory luminal epithelial cells. MUC1-ARF expression varies in different malignancies. While the malignant epithelial cells of pancreatic cancer show limited expression, in breast cancer tissue MUC1-ARF demonstrates strong nuclear expression. Proinflammatory cytokines upregulate expression of MUC1-ARF protein and co-immunoprecipitation analyses demonstrate association of MUC1-ARF with SH3 domain-containing proteins. Mass spectrometry performed on proteins coprecipitating with MUC1-ARF demonstrated Glucose-6-phosphate 1-dehydrogenase (G6PD) and Dynamin 2 (DNM2). These studies not only reveal that the MUC1 gene generates a previously unidentified MUC1-ARF protein, they also show that just like its 'parent' MUC1-TM protein, MUC1-ARF is apparently linked to signaling and malignancy, yet a definitive link to these processes and the roles it plays awaits a precise identification of its molecular functions. Comprising at least 524 amino acids, MUC1-ARF is, furthermore, the longest ARF protein heretofore described.

MUC1-ARF—A Novel MUC1 Protein That Resides in the Nucleus and Is Expressed by Alternate Reading Frame Translation of MUC1 mRNA

PubMed Central

Pichinuk, Edward; Garbar, Christian; Bensussan, Armand; Meeker, Alan; Ziv, Ravit; Zehavi, Tania; Smorodinsky, Nechama I.; Hilkens, John; Hanisch, Franz-Georg; Rubinstein, Daniel B.; Wreschner, Daniel H.

2016-01-01

Translation of mRNA in alternate reading frames (ARF) is a naturally occurring process heretofore underappreciated as a generator of protein diversity. The MUC1 gene encodes MUC1-TM, a signal-transducing trans-membrane protein highly expressed in human malignancies. Here we show that an AUG codon downstream to the MUC1-TM initiation codon initiates an alternate reading frame thereby generating a novel protein, MUC1-ARF. MUC1-ARF, like its MUC1-TM 'parent’ protein, contains a tandem repeat (VNTR) domain. However, the amino acid sequence of the MUC1-ARF tandem repeat as well as N- and C- sequences flanking it differ entirely from those of MUC1-TM. In vitro protein synthesis assays and extensive immunohistochemical as well as western blot analyses with MUC1-ARF specific monoclonal antibodies confirmed MUC1-ARF expression. Rather than being expressed at the cell membrane like MUC1-TM, immunostaining showed that MUC1-ARF protein localizes mainly in the nucleus: Immunohistochemical analyses of MUC1-expressing tissues demonstrated MUC1-ARF expression in the nuclei of secretory luminal epithelial cells. MUC1-ARF expression varies in different malignancies. While the malignant epithelial cells of pancreatic cancer show limited expression, in breast cancer tissue MUC1-ARF demonstrates strong nuclear expression. Proinflammatory cytokines upregulate expression of MUC1-ARF protein and co-immunoprecipitation analyses demonstrate association of MUC1-ARF with SH3 domain-containing proteins. Mass spectrometry performed on proteins coprecipitating with MUC1-ARF demonstrated Glucose-6-phosphate 1-dehydrogenase (G6PD) and Dynamin 2 (DNM2). These studies not only reveal that the MUC1 gene generates a previously unidentified MUC1-ARF protein, they also show that just like its ‘parent’ MUC1-TM protein, MUC1-ARF is apparently linked to signaling and malignancy, yet a definitive link to these processes and the roles it plays awaits a precise identification of its molecular functions. Comprising at least 524 amino acids, MUC1-ARF is, furthermore, the longest ARF protein heretofore described. PMID:27768738

Sialotranscriptomics of Rhipicephalus zambeziensis reveals intricate expression profiles of secretory proteins and suggests tight temporal transcriptional regulation during blood-feeding.

PubMed

de Castro, Minique Hilda; de Klerk, Daniel; Pienaar, Ronel; Rees, D Jasper G; Mans, Ben J

2017-08-10

Ticks secrete a diverse mixture of secretory proteins into the host to evade its immune response and facilitate blood-feeding, making secretory proteins attractive targets for the production of recombinant anti-tick vaccines. The largely neglected tick species, Rhipicephalus zambeziensis, is an efficient vector of Theileria parva in southern Africa but its available sequence information is limited. Next generation sequencing has advanced sequence availability for ticks in recent years and has assisted the characterisation of secretory proteins. This study focused on the de novo assembly and annotation of the salivary gland transcriptome of R. zambeziensis and the temporal expression of secretory protein transcripts in female and male ticks, before the onset of feeding and during early and late feeding. The sialotranscriptome of R. zambeziensis yielded 23,631 transcripts from which 13,584 non-redundant proteins were predicted. Eighty-six percent of these contained a predicted start and stop codon and were estimated to be putatively full-length proteins. A fifth (2569) of the predicted proteins were annotated as putative secretory proteins and explained 52% of the expression in the transcriptome. Expression analyses revealed that 2832 transcripts were differentially expressed among feeding time points and 1209 between the tick sexes. The expression analyses further indicated that 57% of the annotated secretory protein transcripts were differentially expressed. Dynamic expression profiles of secretory protein transcripts were observed during feeding of female ticks. Whereby a number of transcripts were upregulated during early feeding, presumably for feeding site establishment and then during late feeding, 52% of these were downregulated, indicating that transcripts were required at specific feeding stages. This suggested that secretory proteins are under stringent transcriptional regulation that fine-tunes their expression in salivary glands during feeding. No open reading frames were predicted for 7947 transcripts. This class represented 17% of the differentially expressed transcripts, suggesting a potential transcriptional regulatory function of long non-coding RNA in tick blood-feeding. The assembled sialotranscriptome greatly expands the sequence availability of R. zambeziensis, assists in our understanding of the transcription of secretory proteins during blood-feeding and will be a valuable resource for future vaccine candidate selection.

Bovine papillomavirus type 4 L1 gene transfection in a Drosophila S2 cell expression system: absence of L1 protein expression

PubMed Central

Góes, Luiz Gustavo Bentim; de Freitas, Antonio Carlos; Ferraz, Oilita Pereira; Rieger, Tania Tassinari; dos Santos, José Ferreira; Pereira, Alexandre; Beçak, Willy; Lindsey, Charles J.; de Cassia Stocco, Rita

2008-01-01

The development of a bovine papillomavirus (BPV) vaccine is an outstanding challenge. BPV protein L1 gene transfection in the Drosophila melanogaster S2 cell expression system failed to produce L1 protein notwithstanding correct L1 gene insertion. Severe genetic inbalance in the host cell line, including cytogenetic alterations, may account for the lack of protein expression. PMID:24031166

Regulation of DREAM Expression by Group I mGluR

PubMed Central

Lee, Jinu; Kim, Insook; Oh, So Ra; Ko, Suk Jin; Lim, Mi Kyung; Kim, Dong Goo

2011-01-01

DREAM (downstream regulatory element antagonistic modulator) is a calcium-binding protein that regulates dynorphin expression, promotes potassium channel surface expression, and enhances presenilin processing in an expression level-dependent manner. However, no molecular mechanism has yet explained how protein levels of DREAM are regulated. Here we identified group I mGluR (mGluR1/5) as a positive regulator of DREAM protein expression. Overexpression of mGluR1/5 increased the cellular level of DREAM. Up-regulation of DREAM resulted in increased DREAM protein in both the nucleus and cytoplasm, where the protein acts as a transcriptional repressor and a modulator of its interacting proteins, respectively. DHPG (3,5-dihydroxyphenylglycine), a group I mGluR agonist, also up-regulated DREAM expression in cortical neurons. These results suggest that group I mGluR is the first identified receptor that may regulate DREAM activity in neurons. PMID:21660149

Use of a protein engineering strategy to overcome limitations in the production of "Difficult to Express" recombinant proteins.

PubMed

Hussain, Hirra; Fisher, David I; Abbott, W Mark; Roth, Robert G; Dickson, Alan J

2017-10-01

Certain recombinant proteins are deemed "difficult to express" in mammalian expression systems requiring significant cell and/or process engineering to abrogate expression bottlenecks. With increasing demand for the production of recombinant proteins in mammalian cells, low protein yields can have significant consequences for industrial processes. To investigate the molecular mechanisms that restrict expression of recombinant proteins, naturally secreted model proteins were analyzed from the tissue inhibitors of metalloproteinase (TIMP) protein family. In particular, TIMP-2 and TIMP-3 were subjected to detailed study. TIMP proteins share significant sequence homology (∼50% identity and ∼70% similarity in amino acid sequence). However, they show marked differences in secretion in mammalian expression systems despite this extensive sequence homology. Using these two proteins as models, this study characterized the molecular mechanisms responsible for poor recombinant protein production. Our results reveal that both TIMP-2 and TIMP-3 are detectable at mRNA and protein level within the cell but only TIMP-2 is secreted effectively into the extracellular medium. Analysis of protein localization and the nature of intracellular protein suggest TIMP-3 is severely limited in its post-translational processing. To overcome this challenge, modification of the TIMP-3 sequence to include a furin protease-cleavable pro-sequence resulted in secretion of the modified TIMP-3 protein, however, incomplete processing was observed. Based on the TIMP-3 data, the protein engineering approach was optimized and successfully applied in combination with cell engineering, the overexpression of furin, to another member of the TIMP protein family (the poorly expressed TIMP-4). Use of the described protein engineering strategy resulted in successful secretion of poorly (TIMP-4) and non-secreted (TIMP-3) targets, and presents a novel strategy to enhance the production of "difficult" recombinant targets. Biotechnol. Bioeng. 2017;114: 2348-2359. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Fusagene vectors: a novel strategy for the expression of multiple genes from a single cistron.

PubMed

Gäken, J; Jiang, J; Daniel, K; van Berkel, E; Hughes, C; Kuiper, M; Darling, D; Tavassoli, M; Galea-Lauri, J; Ford, K; Kemeny, M; Russell, S; Farzaneh, F

2000-12-01

Transduction of cells with multiple genes, allowing their stable and co-ordinated expression, is difficult with the available methodologies. A method has been developed for expression of multiple gene products, as fusion proteins, from a single cistron. The encoded proteins are post-synthetically cleaved and processed into each of their constituent proteins as individual, biologically active factors. Specifically, linkers encoding cleavage sites for the Golgi expressed endoprotease, furin, have been incorporated between in-frame cDNA sequences encoding different secreted or membrane bound proteins. With this strategy we have developed expression vectors encoding multiple proteins (IL-2 and B7.1, IL-4 and B7.1, IL-4 and IL-2, IL-12 p40 and p35, and IL-12 p40, p35 and IL-2 ). Transduction and analysis of over 100 individual clones, derived from murine and human tumour cell lines, demonstrate the efficient expression and biological activity of each of the encoded proteins. Fusagene vectors enable the co-ordinated expression of multiple gene products from a single, monocistronic, expression cassette.

Identification of Fur, Aconitase, and Other Proteins Expressed by Mycobacterium tuberculosis under Conditions of Low and High Concentrations of Iron by Combined Two-Dimensional Gel Electrophoresis and Mass Spectrometry

PubMed Central

Wong, Diane K.; Lee, Bai-Yu; Horwitz, Marcus A.; Gibson, Bradford W.

1999-01-01

Iron plays a critical role in the pathophysiology of Mycobacterium tuberculosis. To gain a better understanding of iron regulation by this organism, we have used two-dimensional (2-D) gel electrophoresis, mass spectrometry, and database searching to study protein expression in M. tuberculosis under conditions of high and low iron concentration. Proteins in cellular extracts from M. tuberculosis Erdman strain grown under low-iron (1 μM) and high-iron (70 μM) conditions were separated by 2-D polyacrylamide gel electrophoresis, which allowed high-resolution separation of several hundred proteins, as visualized by Coomassie staining. The expression of at least 15 proteins was induced, and the expression of at least 12 proteins was decreased under low-iron conditions. In-gel trypsin digestion was performed on these differentially expressed proteins, and the digestion mixtures were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry to determine the molecular masses of the resulting tryptic peptides. Partial sequence data on some of the peptides were obtained by using after source decay and/or collision-induced dissociation. The fragmentation data were used to search computerized peptide mass and protein sequence databases for known proteins. Ten iron-regulated proteins were identified, including Fur and aconitase proteins, both of which are known to be regulated by iron in other bacterial systems. Our study shows that, where large protein sequence databases are available from genomic studies, the combined use of 2-D gel electrophoresis, mass spectrometry, and database searching to analyze proteins expressed under defined environmental conditions is a powerful tool for identifying expressed proteins and their physiologic relevance. PMID:9864233

Autoimmunity and asthma: The dirt on the hygiene hypothesis.

PubMed

Mannie, Mark D

2010-04-01

Self peptides shape T-cell development through selectional processes in the thymus and secondary lymphoid organs to promote a diverse and balanced repertoire of conventional and regulatory T cells. Foreign proteins and their derivative peptides permeate our mucosal tissues to constitute another diverse array of peptides that may specify and diversify the mucosal T-cell repertoire. Indeed, the distinction between self peptides and environmental foreign peptides may be academic if both are present constantly within the body. The premise here is that the plethora of foreign peptides, present ubiquitously in our environment and body, form homeostatic niches to foster highly diversified repertoires of conventional and regulatory T cells that recognize persistent environmental peptides as self. Highly diversified repertoires that recognize myriads of self and environmental foreign peptides as homeostatic ligands may be critical for adaptive distinctions of friend or foe in mucosal tissues. The change from our agrarian past to the highly sterile environments of today may adversely impact the diversity and concentrations of foreign peptides that shape the mucosal T-cell repertoire. Various hygiene hypotheses postulate that the lack of factors such as infectious pathogens, innate receptor engagement or Th1 bias is key to the marked increase in immunological disease in modern society. In this version of the hygiene hypothesis, highly diverse constellations of innocuous environmental peptides are postulated to be the critical factor for immune balance and homeostasis.

Preventing Food Allergies by Tricking Dendritic Cells

USDA-ARS?s Scientific Manuscript database

Food allergies are adverse responses to components (usually proteins) within the foods we eat, which result in a self-damaging response from our immune system. A myriad of cellular and molecular components are involved in the decision to tolerate or respond to foreign molecules that pass through the...

Protein Expression Profile using Two-Dimensional Gel Analysis in Squamous Cervical Cancer Patients

PubMed Central

Bae, Su-Mi; Min, Hyun-Jin; Ding, Guo Hua; Kwak, Sun-Young; Cho, Young-Lae; Nam, Kye-Hyun; Park, Choong Hak; Kim, Yong-Wan; Kim, Chong-Kook; Han, Byoung-Don; Lee, Young-Joo; Kim, Do Kang

2006-01-01

Purpose Screening in cervical cancer is now progressing to discover candidate genes and proteins that may serve as biological markers and that play a role in tumor progression. We examined the protein expression patterns of the squamous cell carcinoma (SCC) tissues from Korean women with using two- dimensional polyacrylamide gel electrophoresis (2-DE) and matrix assisted laser desorption/ionization-time of flight (MALDI- TOF) mass spectrometer. Materials and Methods Normal cervix and SCC tissues were solubilized and 2-DE was performed using pH 3~10 linear IPG strips of 17 cm length. The protein expression was evaluated using PDQuest 2-D software™. The differentially expressed protein spots were identified with a MALDI-TOF mass spectrometer, and the peptide mass spectra identifications were performed using the Mascot program and by searching the Swiss-prot or NCBInr databases. Results A total of 35 proteins were detected in SCC. 17 proteins were up-regulated and 18 proteins weredown-regulated. Among the proteins that were identified, 12 proteins (pigment epithelium derived factor, annexin A2 and A5, keratin 19 and 20, heat shock protein 27, smooth muscle protein 22 alpha, α-enolase, squamous cell carcinoma antigen 1 and 2, glutathione S-transferase and apolipoprotein a1) were protein previously known to be involved in tumor, and 21 proteins were newly identified in this study. Conclusion 2-DE offers the total protein expression profiles of SCC tissues; further characterization of these differentially expressed proteins will give a chance to identify the badly needed tumor-specific diagnostic markers for SCC. PMID:19771267

Clinicopathologic significance of minichromosome maintenance protein 2 and Tat-interacting protein 30 expression in benign and malignant lesions of the gallbladder.

PubMed

Liu, Dong-cai; Yang, Zhu-lin

2011-11-01

Gallbladder cancers are aggressive tumors with a poor prognosis and high mortality rate. To find specific biological markers for early diagnosis and prognosis and to develop possible alternative treatment strategies, we examined minichromosome maintenance protein 2 (MCM2) and Tat-interacting protein 30 (TIP30) expression in 108 gallbladder adenocarcinomas, 15 gallbladder polyps, 35 chronic cholecystitis tissues, and 46 peritumoral tissues using immunohistochemistry. Expression of MCM2 was significantly higher in adenocarcinomas than in peritumoral tissues (χ² = 8.41; P < .01), adenomatous polyps (χ² = 6.81; P < .01), and chronic cholecystitis (χ² = 21.00; P < .01). In contrast, Tat-interacting protein 30 expression was significantly less in adenocarcinomas than in peritumoral tissues (χ² = 13.26; P < .01), adenomatous polyps (χ² = 4.76; P < .05), and chronic cholecystitis (χ² = 18.93; P < .01). The benign lesions in gallbladder epithelium with positive MCM2 or negative Tat-interacting protein 30 expression showed moderate to severe atypical hyperplasia. Expression of MCM2 and absence of Tat-interacting protein 30 were significantly associated with poor differentiation, large tumor mass, lymph node metastasis, and invasion of adenocarcinoma. Univariate Kaplan-Meier analysis showed that either elevated MCM2 (P = .006) or lowered Tat-interacting protein 30 (P = .006) expression was closely associated with shorter overall survival. Multivariate Cox regression analysis revealed that expression of MCM2 (P = .007) or nonexpression of Tat-interacting protein 30 (P = .009) was an independent predictor of a poor prognosis in adenocarcinoma. Our results suggest that overexpression of MCM2 or loss of expression of Tat-interacting protein 30 is closely related to carcinogenesis, progression, biological behavior, and prognosis of gallbladder adenocarcinoma. Copyright © 2011 Elsevier Inc. All rights reserved.

Enteric glial-derived S100B protein stimulates nitric oxide production in celiac disease.

PubMed

Esposito, Giuseppe; Cirillo, Carla; Sarnelli, Giovanni; De Filippis, Daniele; D'Armiento, Francesco Paolo; Rocco, Alba; Nardone, Gerardo; Petruzzelli, Raffaella; Grosso, Michela; Izzo, Paola; Iuvone, Teresa; Cuomo, Rosario

2007-09-01

Enteric glia participates to the homeostasis of the gastrointestinal tract. In the central nervous system, increased expression of astroglial-derived S100B protein has been associated with the onset and maintaining of inflammation. The role of enteric glial-derived S100B protein in gastrointestinal inflammation has never been investigated in humans. In this study, we evaluated the expression of S100B and its relationship with nitric oxide production in celiac disease. Duodenal biopsy specimens from untreated and on gluten-free diet patients with celiac disease and controls were respectively processed for S100B and inducible nitric oxide synthase (iNOS) protein expression and nitrite production. To evaluate the direct involvement of S100B in the inflammation, control biopsy specimens were exposed to exogenous S100B, and iNOS protein expression and nitrite production were measured. We also tested gliadin induction of S100B-dependent inflammation in cultured biopsy specimens deriving from on gluten-free diet patients in the absence or presence of the specific S100B antibody. S100B messenger RNA and protein expression, iNOS protein expression, and nitrite production were significantly increased in untreated patients but not in on gluten-free diet patients vs controls. Addition of S100B to control biopsy specimens resulted in a significant increase of iNOS protein expression and nitrite production. In celiac disease patients but not in controls biopsy specimens, gliadin challenge significantly increased S100B messenger RNA and protein expression, iNOS protein expression, and nitrite production, but these effects were completely inhibited by S100B antibody. Enteric glial-derived S100B is increased in the duodenum of patients with celiac disease and plays a role in nitric oxide production.

Quantitative proteomic analysis of the Salmonella-lettuce interaction

PubMed Central

Zhang, Yuping; Nandakumar, Renu; Bartelt-Hunt, Shannon L; Snow, Daniel D; Hodges, Laurie; Li, Xu

2014-01-01

Human pathogens can internalize food crops through root and surface uptake and persist inside crop plants. The goal of the study was to elucidate the global modulation of bacteria and plant protein expression after Salmonella internalizes lettuce. A quantitative proteomic approach was used to analyse the protein expression of Salmonella enterica serovar Infantis and lettuce cultivar Green Salad Bowl 24 h after infiltrating S. Infantis into lettuce leaves. Among the 50 differentially expressed proteins identified by comparing internalized S. Infantis against S. Infantis grown in Luria Broth, proteins involved in glycolysis were down-regulated, while one protein involved in ascorbate uptake was up-regulated. Stress response proteins, especially antioxidant proteins, were up-regulated. The modulation in protein expression suggested that internalized S. Infantis might utilize ascorbate as a carbon source and require multiple stress response proteins to cope with stresses encountered in plants. On the other hand, among the 20 differentially expressed lettuce proteins, proteins involved in defense response to bacteria were up-regulated. Moreover, the secreted effector PipB2 of S. Infantis and R proteins of lettuce were induced after bacterial internalization into lettuce leaves, indicating human pathogen S. Infantis triggered the defense mechanisms of lettuce, which normally responds to plant pathogens. PMID:24512637

Differentially expressed proteins among normal cervix, cervical intraepithelial neoplasia and cervical squamous cell carcinoma.

PubMed

Zhao, Q; He, Y; Wang, X-L; Zhang, Y-X; Wu, Y-M

2015-08-01

To explore the differentially expressed proteins in normal cervix, cervical intraepithelial neoplasia (CIN) and cervical squamous cell carcinoma (CSCC) tissues by differential proteomics technique. Cervical tissues (including normal cervix, CIN and CSCC) were collected in Department of Gynecologic Oncology of Beijing Obstetrics and Gynecology Hospital. Two-dimensional fluorescence difference in gel electrophoresis (2-D DIGE) and DeCyder software were used to detect the differentially expressed proteins. Matrix-assisted laser desorption/ionization-time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS) was used to identify the differentially expressed proteins. Western blot (WB) and immunohistochemistry (IHC) were performed to validate the expressions of selected proteins among normal cervix, CIN and CSCC. 2-D DIGE images with high resolution and good repeatability were obtained. Forty-six differentially expressed proteins (27 up-regulated and 19 down-regulated) were differentially expressed among the normal cervix, CIN and CSCC. 26 proteins were successfully identified by MALDI-TOF/TOF MS. S100A9 (S100 calcium-binding protein A9) was the most significantly up-regulated protein. Eukaryotic elongation factor 1-alpha-1 (eEF1A1) was the most significantly down-regulated protein. Pyruvate kinase isozymes M2 (PKM2) was both up-regulated and down-regulated. The results of WB showed that with the increase in the severity of cervical lesions, the expression of S100A9 protein was significantly increased among the three groups (P = 0.010). The expression of eEF1A1 was reduced but without significant difference (P = 0.861). The expression of PKM2 was significantly reduced (P = 0.000). IHC showed that protein S100A9 was mainly expressed in the cytoplasm, and its positive expression rate was 20.0 % in normal cervix, 70.0 % in CIN and 100.0 % in CSCC, with a significant difference among them (P = 0.006). eEF1A1 was mainly expressed in the cell plasma, and its positive expression rate was 70.0 % in normal cervix, 73.3 % in CIN and 60.0 % in CSCC tissues, without significant difference among them (P = 0.758). PKM2 was mainly expressed in the cell nuclei, and its positive expression rate was 100.0 % in normal cervix, 93.3 % in CIN and 75.0 % in CSCC tissues, showing a difference close to statistical significance (P = 0.059) among them. There are differentially expressed proteins among normal cervix, CIN and CSCC. S100A9, eEF1A1 and PKM2 may become candidate markers for early diagnosis of cervical cancer and new targets for therapy. It also provides a basis for further studies of the mechanism for CIN developing to CSCC.

Role of ID Proteins in BMP4 Inhibition of Profibrotic Effects of TGF-β2 in Human TM Cells.

PubMed

Mody, Avani A; Wordinger, Robert J; Clark, Abbot F

2017-02-01

Increased expression of TGF-β2 in primary open-angle glaucoma (POAG) aqueous humor (AH) and trabecular meshwork (TM) causes deposition of extracellular matrix (ECM) in the TM and elevated IOP. Bone morphogenetic proteins (BMPs) regulate TGF-β2-induced ECM production. The underlying mechanism for BMP4 inhibition of TGF-β2-induced fibrosis remains undetermined. Bone morphogenic protein 4 induces inhibitor of DNA binding proteins (ID1, ID3), which suppress transcription factor activities to regulate gene expression. Our study will determine whether ID1and ID3 proteins are downstream targets of BMP4, which attenuates TGF-β2 induction of ECM proteins in TM cells. Primary human TM cells were treated with BMP4, and ID1 and ID3 mRNA, and protein expression was determined by quantitative PCR (Q-PCR) and Western immunoblotting. Intracellular ID1 and ID3 protein localization was studied by immunocytochemistry. Transformed human TM cells (GTM3 cells) were transfected with ID1 or ID3 expression vectors to determine their potential inhibitory effects on TGF-β2-induced fibronectin and plasminogen activator inhibitor-I (PAI-1) protein expression. Basal expression of ID1-3 was detected in primary human TM cells. Bone morphogenic protein 4 significantly induced early expression of ID1 and ID3 mRNA (P < 0.05) and protein in primary TM cells, and a BMP receptor inhibitor blocked this induction. Overexpression of ID1 and ID3 significantly inhibited TGF-β2-induced expression of fibronectin and PAI-1 in TM cells (P < 0.01). Bone morphogenic protein 4 induced ID1 and ID3 expression suppresses TGF-β2 profibrotic activity in human TM cells. In the future, targeting specific regulators may control the TGF-β2 profibrotic effects on the TM, leading to disease modifying IOP lowering therapies.

Prostacyclin synthase expression and epigenetic regulation in nonsmall cell lung cancer.

PubMed

Cathcart, Mary-Clare; Gray, Steven G; Baird, Anne-Marie; Boyle, Elaine; Gately, Kathy; Kay, Elaine; Cummins, Robert; Pidgeon, Graham P; O'Byrne, Kenneth J

2011-11-15

Prostacyclin synthase (PGIS) metabolizes prostaglandin H(2), into prostacyclin. This study aimed to determine the expression profile of PGIS in nonsmall cell lung cancer (NSCLC) and examine potential mechanisms involved in PGIS regulation. PGIS expression was examined in human NSCLC and matched controls by reverse transcriptase polymerase chain reaction (RT-PCR), Western analysis, and immunohistochemistry. A 204-patient NSCLC tissue microarray was stained for PGIS and cyclooxygenase 2 (COX2) expression. Staining intensity was correlated with clinical parameters. Epigenetic mechanisms underpinning PGIS promoter expression were examined using RT-PCR, methylation-specific PCR, and chromatin immunoprecipitation analysis. PGIS expression was reduced/absent in human NSCLC protein samples (P < .0001), but not mRNA relative to matched controls. PGIS tissue expression was higher in squamous cell carcinoma (P = .004) and in male patients (P < .05). No significant correlation of PGIS or COX2 expression with overall patient survival was observed, although COX2 was prognostic for short-term (2-year) survival (P < .001). PGIS mRNA expression was regulated by DNA CpG methylation and histone acetylation in NSCLC cell lines, with chromatin remodeling taking place directly at the PGIS gene. PGIS mRNA expression was increased by both demethylation agents and histone deacetylase inhibitors. Protein levels were unaffected by demethylation agents, whereas PGIS protein stability was negatively affected by histone deacetylase inhibitors. PGIS protein expression is reduced in NSCLC, and does not correlate with overall patient survival. PGIS expression is regulated through epigenetic mechanisms. Differences in expression patterns between mRNA and protein levels suggest that PGIS expression and protein stability are regulated post-translationally. PGIS protein stability may have an important therapeutic role in NSCLC. Copyright © 2011 American Cancer Society.

Pathogenicity of Different Rabies Virus Variants Inversely Correlates with Apoptosis and Rabies Virus Glycoprotein Expression in Infected Primary Neuron Cultures

PubMed Central

Morimoto, Kinjiro; Hooper, D. Craig; Spitsin, Sergei; Koprowski, Hilary; Dietzschold, Bernhard

1999-01-01

The mouse-adapted rabies virus strain CVS-24 has stable variants, CVS-B2c and CVS-N2c, which differ greatly in their pathogenicity for normal adult mice and in their ability to infect nonneuronal cells. The glycoprotein (G protein), which has previously been implicated in rabies virus pathogenicity, shows substantial structural differences between these variants. Although prior studies have identified antigenic site III of the G protein as the major pathogenicity determinant, CVS-B2c and CVS-N2c do not vary at this site. The possibility that pathogenicity is inversely related to G protein expression levels is suggested by the finding that CVS-B2c, the less pathogenic variant, expresses at least fourfold-higher levels of G protein than CVS-N2c in infected neurons. Although there is some difference between CVS-B2c- and CVS-N2c-infected neurons in G protein mRNA expression levels, the differential expression of G protein appears to be largely determined by posttranslational mechanisms that affect G protein stability. Pulse-chase experiments indicated that the G protein of CVS-B2c is degraded more slowly than that of CVS-N2c. The accumulation of G protein correlated with the induction of programmed cell death in CVS-B2c-infected neurons. The extent of apoptosis was considerably lower in CVS-N2c-infected neurons, where G protein expression was minimal. While nucleoprotein (N protein) expression levels were similar in neurons infected with either variant, the transport of N protein into neuronal processes was strongly inhibited in CVS-B2c-infected cells. Thus, downregulation of G protein expression in neuronal cells evidently contributes to rabies virus pathogenesis by preventing apoptosis and the apparently associated failure of the axonal transport of N protein. PMID:9847357

Downregulation of miRNA-30c and miR-203a is associated with hepatitis C virus core protein-induced epithelial–mesenchymal transition in normal hepatocytes and hepatocellular carcinoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Dongjing; Wu, Jilin, E-mail: 6296082@qq.com; Liu, Meizhou

Hepatitis C virus (HCV) Core protein has been demonstrated to induce epithelial–mesenchymal transition (EMT) and is associated with cancer progression of hepatocellular carcinoma (HCC). However, how the Core protein regulates EMT is still unclear. In this study, HCV Core protein was overexpressed by an adenovirus. The protein levels of EMT markers were measured by Western blot. The xenograft animal model was established by inoculation of HepG2 cells. Results showed that ectopic expression of HCV core protein induced EMT in L02 hepatocytes and HepG2 tumor cells by upregulating vimentin, Sanl1, and Snal2 expression and downregulating E-cadherin expression. Moreover, Core protein downregulatedmore » miR-30c and miR-203a levels in L02 and HepG2 cells, but artificial expression of miR-30c and miR-203a reversed Core protein-induced EMT. Further analysis showed that ectopic expression of HCV core protein stimulated cell proliferation, inhibited apoptosis, and increased cell migration, whereas artificial expression of miR-30c and miR-203a significantly reversed the role of Core protein in these cell functions in L02 and HepG2 cells. In the HepG2 xenograft tumor models, artificial expression of miR-30c and miR-203a inhibited EMT and tumor growth. Moreover, L02 cells overexpressing Core protein can form tumors in nude mice. In HCC patients, HCV infection significantly shortened patients' survival time, and loss of miR-30c and miR-203 expression correlated with poor survival. In conclusion, HCV core protein downregulates miR-30c and miR-203a expression, which results in activation of EMT in normal hepatocytes and HCC tumor cells. The Core protein-activated-EMT is involved in the carcinogenesis and progression of HCC. Loss of miR-30c and miR-203a expression is a marker for the poor prognosis of HCC. - Highlights: • HCV core protein downregulates miR-30c and miR-203a expression. • Downregulation of miR-30c and miR-203a activates EMT. • Activated-EMT is involved in the carcinogenesis and progression of HCC. • Loss of miR-30c and miR-203a expression is a marker for the poor prognosis of HCC.« less

Proteins Differentially Expressed in the Pancreas of Hepatic Alcohol Dehydrogenase-Deficient Deer Mice Fed Ethanol For 3 Months.

PubMed

Bhopale, Kamlesh K; Amer, Samir M; Kaphalia, Lata; Soman, Kizhake V; Wiktorowicz, John E; Shakeel Ansari, Ghulam A; Kaphalia, Bhupendra S

2017-07-01

The aim of this study was to identify differentially expressed proteins in the pancreatic tissue of hepatic alcohol dehydrogenase-deficient deer mice fed ethanol to understand metabolic basis and mechanism of alcoholic chronic pancreatitis. Mice were fed liquid diet containing 3.5 g% ethanol daily for 3 months, and differentially expressed pancreatic proteins were identified by protein separation using 2-dimensional gel electrophoresis and identification by mass spectrometry. Nineteen differentially expressed proteins were identified by applying criteria established for protein identification in proteomics. An increased abundance was found for ribosome-binding protein 1, 60S ribosomal protein L31-like isoform 1, histone 4, calcium, and adenosine triphosphate (ATP) binding proteins and the proteins involved in antiapoptotic processes and endoplasmic reticulum function, stress, and/or homeostasis. Low abundance was found for endoA cytokeratin, 40S ribosomal protein SA, amylase 2b isoform precursor, serum albumin, and ATP synthase subunit β and the proteins involved in cell motility, structure, and conformation. Chronic ethanol feeding in alcohol dehydrogenase-deficient deer mice differentially expresses pancreatic functional and structural proteins, which can be used to develop biomarker(s) of alcoholic chronic pancreatitis, particularly amylase 2b precursor, and 60 kDa heat shock protein and those involved in ATP synthesis and blood osmotic pressure.

Exposure to an extremely low-frequency electromagnetic field only slightly modifies the proteome of Chromobacterium violaceumATCC 12472.

PubMed

Baraúna, Rafael A; Santos, Agenor V; Graças, Diego A; Santos, Daniel M; Ghilardi, Rubens; Pimenta, Adriano M C; Carepo, Marta S P; Schneider, Maria P C; Silva, Artur

2015-05-01

Several studies of the physiological responses of different organisms exposed to extremely low-frequency electromagnetic fields (ELF-EMF) have been described. In this work, we report the minimal effects of in situ exposure to ELF-EMF on the global protein expression of Chromobacterium violaceum using a gel-based proteomic approach. The protein expression profile was only slightly altered, with five differentially expressed proteins detected in the exposed cultures; two of these proteins (DNA-binding stress protein, Dps, and alcohol dehydrogenase) were identified by MS/MS. The enhanced expression of Dps possibly helped to prevent physical damage to DNA. Although small, the changes in protein expression observed here were probably beneficial in helping the bacteria to adapt to the stress generated by the electromagnetic field.

Stomatin-like protein 2 is overexpressed in epithelial ovarian cancer and predicts poor patient survival.

PubMed

Sun, Fei; Ding, Wen; He, Jie-Hua; Wang, Xiao-Jing; Ma, Ze-Biao; Li, Yan-Fang

2015-10-20

Stomatin-like protein 2 (SLP-2, also known as STOML2) is a stomatin homologue of uncertain function. SLP-2 overexpression has been suggested to be associated with cancer progression, resulting in adverse clinical outcomes in patients. Our study aim to investigate SLP-2 expression in epithelial ovarian cancer cells and its correlation with patient survival. SLP-2 mRNA and protein expression levels were analysed in five epithelial ovarian cancer cell lines and normal ovarian epithelial cells using real-time PCR and western blotting analysis. SLP-2 expression was investigated in eight matched-pair samples of epithelial ovarian cancer and adjacent noncancerous tissues from the same patients. Using immunohistochemistry, we examined the protein expression of paraffin-embedded specimens from 140 patients with epithelial ovarian cancer, 20 cases with borderline ovarian tumours, 20 cases with benign ovarian tumours, and 20 cases with normal ovarian tissues. Statistical analyses were applied to evaluate the clinicopathological significance of SLP-2 expression. SLP-2 mRNA and protein expression levels were significantly up-regulated in epithelial ovarian cancer cell lines and cancer tissues compared with normal ovarian epithelial cells and adjacent noncancerous ovarian tissues. Immunohistochemistry analysis revealed that the relative overexpression of SLP-2 was detected in 73.6 % (103/140) of the epithelial ovarian cancer specimens, 45.0 % (9/20) of the borderline ovarian specimens, 30.0 % (6/20) of the benign ovarian specimens and none of the normal ovarian specimens. SLP-2 protein expression in epithelial ovarian cancer was significantly correlated with the tumour stage (P < 0.001). Epithelial ovarian cancer patients with higher SLP-2 protein expression levels had shorter progress free survival and overall survival times compared to patients with lower SLP-2 protein expression levels. Multivariate analyses showed that SLP-2 expression levels were an independent prognostic factor for survival in epithelial ovarian cancer patients. SLP-2 mRNA and proteins were overexpressed in epithelial ovarian cancer tissues. SLP-2 protein overexpression was associated with advanced stage disease. Patients with higher SLP-2 protein expression had shorter progress free survival and poor overall survival times. Thus, SLP-2 protein expression was an independent prognostic factor for patients with epithelial ovarian cancer.

Cloning and expression of Tenebrio molitor antifreeze protein in Escherichia coli.

PubMed

Yue, Chang-Wu; Zhang, Yi-Zheng

2009-03-01

A novel antifreeze protein cDNA was cloned by RT-PCR from the larva of the yellow mealworm Tenebrio molitor. The coding fragment of 339 bp encodes a protein of 112 amino acid residues and was fused to the expression vectors pET32a and pTWIN1. The resulted expression plasmids were transformed into Escherischia coli strains BL21 (DE3), ER2566, and Origami B (DE3), respectively. Several strategies were used for expression of the highly disulfide-bonded beta-helix-contained protein with the activity of antifreeze in different expression systems. A protocol for production of refolded and active T. molitor antifreeze protein in bacteria was obtained.