Transduction of cultured fish cells with recombinant baculoviruses.

PubMed

Leisy, Douglas J; Lewis, Teresa D; Leong, Jo-Ann C; Rohrmann, George F

2003-05-01

Five fish cell lines were tested for their ability to be transduced by Ac-CAlacZ, a recombinant baculovirus that is capable of expressing a beta-galactosidase reporter gene from the CAG promoter (consisting of a cytomegalovirus enhancer element, a chicken actin promoter and rabbit beta-globin termination sequences). TO (Tilapia ovary), EPC (carp), CHH-1 (Chum salmon heart fibroblast) and CHSE-214 (chinook salmon embryo) cells were transducible, as demonstrated by an in situ beta-galactosidase assay, whereas RTG-2 (rainbow trout gonad) cells were not. The EPC cell line was used for more detailed studies on baculovirus transduction. The transduction frequency was found to be higher at 28 degrees C than at 21 degrees C. Addition of the histone deacetylase inhibitor sodium butyrate increased the number of blue cells detected 5- to 7-fold. The m.o.i. was positively correlated with transduction frequency, although the relationship did not appear to be strictly linear, as has been observed with mammalian cells. The temperature at which baculoviruses were adsorbed to EPC cells did not affect levels of beta-galactosidase expression. We also examined expression levels of beta-galactosidase in EPC cells after infection with a baculovirus construct that overexpresses the vesicular stomatitis virus G protein and displays it on the virion surface. Expression levels with this virus were approximately 15-fold higher than were observed with Ac-CAlacZ.

Global Screening of Antiviral Genes that Suppress Baculovirus Transgene Expression in Mammalian Cells.

PubMed

Wang, Chia-Hung; Naik, Nenavath Gopal; Liao, Lin-Li; Wei, Sung-Chan; Chao, Yu-Chan

2017-09-15

Although baculovirus has been used as a safe and convenient gene delivery vector in mammalian cells, baculovirus-mediated transgene expression is less effective in various mammalian cell lines. Identification of the negative regulators in host cells is necessary to improve baculovirus-based expression systems. Here, we performed high-throughput shRNA library screening, targeting 176 antiviral innate immune genes, and identified 43 host restriction factor genes in a human A549 lung carcinoma cell line. Among them, suppression of receptor interaction protein kinase 1 (RIP1, also known as RIPK1) significantly increased baculoviral transgene expression without resulting in significant cell death. Silencing of RIP1 did not affect viral entry or cell viability, but it did inhibit nuclear translocation of the IRF3 and NF-κB transcription factors. Also, activation of downstream signaling mediators (such as TBK1 and IRF7) was affected, and subsequent interferon and cytokine gene expression levels were abolished. Further, Necrostatin-1 (Nec-1)-an inhibitor of RIP1 kinase activity-dramatically increased baculoviral transgene expression in RIP1-silenced cells. Using baculovirus as a model system, this study presents an initial investigation of large numbers of human cell antiviral innate immune response factors against a "nonadaptive virus." In addition, our study has made baculovirus a more efficient gene transfer vector for some of the most frequently used mammalian cell systems.

Baculovirus enhancins and their role in viral pathogenicity. Chapter 9

Treesearch

James M. Slavicek

2012-01-01

Baculoviruses are a large group of viruses pathogenic to arthropods, primarily insects from the order Lepidoptera and also insects in the orders Hymenoptera and Diptera. Baculoviruses have been used to control insect pests on agricultural crops and forests around the world. Efforts have been ongoing for the last two decades to develop strains of baculoviruses with...

Generation of Envelope-Modified Baculoviruses for Gene Delivery into Mammalian Cells.

PubMed

Hofmann, Christian

2016-01-01

Genetically modified baculoviruses can efficiently deliver and express genes in mammalian cells. The major prerequisite for the expression of a gene transferred by baculovirus is its control by a promoter that is active in mammalian cells. This chapter describes methods for producing second generation baculovirus vectors through modification of their envelope. Envelope modified baculoviruses offer additional new applications of the system, such as their use in in vivo gene delivery, targeting, and vaccination. Methods of generating a recombinant baculovirus vector with a modified envelope and its amplification and purification, including technical scale production, are discussed. A variety of notes give clues regarding specific technical procedures. Finally, methods to analyze the virus and transduction procedures are presented.

Expression of the Bacillus anthracis protective antigen gene by baculovirus and vaccinia virus recombinants.

PubMed Central

Iacono-Connors, L C; Schmaljohn, C S; Dalrymple, J M

1990-01-01

The gene encoding Bacillus anthracis protective antigen (PA) was modified by site-directed mutagenesis, subcloned into baculovirus and vaccinia virus plasmid transfer vectors (pAcYM1 and pSC-11, respectively), and inserted via homologous recombinations into baculovirus Autographa californica nuclear polyhedrosis virus or vaccinia virus (strains WR and Connaught). Expression of PA was detected in both systems by immunofluorescence assays with antisera from rabbits immunized with B. anthracis PA. Western blot (immunoblot) analysis showed that the expressed product of both systems was slightly larger (86 kilodaltons) than B. anthracis-produced PA (83.5 kilodaltons). Analysis of trypsin digests of virus-expressed and authentic PA suggested that the size difference was due to the presence of a signal sequence remaining with the virus-expressed protein. Immunization of mice with either recombinant baculovirus-infected Spodoptera frugiperda cells or with vaccinia virus recombinants elicited a high-titer, anti-PA antibody response. Images PMID:2105271

Baculovirus: an Insect-derived Vector for Diverse Gene Transfer Applications

PubMed Central

Airenne, Kari J; Hu, Yu-Chen; Kost, Thomas A; Smith, Richard H; Kotin, Robert M; Ono, Chikako; Matsuura, Yoshiharu; Wang, Shu; Ylä-Herttuala, Seppo

2013-01-01

Insect-derived baculoviruses have emerged as versatile and safe workhorses of biotechnology. Baculovirus expression vectors (BEVs) have been applied widely for crop and forest protection, as well as safe tools for recombinant protein production in insect cells. However, BEVs ability to efficiently transduce noninsect cells is still relatively poorly recognized despite the fact that efficient baculovirus-mediated in vitro and ex vivo gene delivery into dormant and dividing vertebrate cells of diverse origin has been described convincingly by many authors. Preliminary proof of therapeutic potential has also been established in preclinical studies. This review summarizes the advantages and current status of baculovirus-mediated gene delivery. Stem cell transduction, preclinical animal studies, tissue engineering, vaccination, cancer gene therapy, viral vector production, and drug discovery are covered. PMID:23439502

[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.

Improved replication of the baculovirus Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) in vitro using proteins from Lonomia obliqua hemolymph.

PubMed

Sousa, Álvaro P B; Moraes, Roberto H P; Mendonça, Ronaldo Z

2015-03-01

The baculovirus Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV), a member of the family Baculoviridae, has been widely applied as a biopesticide for the control of the velvetbean caterpillar, a pest of soybean crop field. Baculoviruses are considered safe and efficient agents for this purpose, because they do not infect vertebrates, being safe for the health of humans and animals, as well as to the environment. The objective of this work was to identify proteins obtained from Lonomia obliqua hemolymph with potential application in the optimization of baculovirus AgMNPV replication in Sf9 insect cell culture. In this work the improvement of the cell culture and viral replication of the AgMNPV baculovirus was observed when Grace medium was supplemented with 10 % (v/v) Fetal Bovine Serum (FBS), 1 % (v/v) hemolymph extract, or 3 % (v/v) of hemolymph fractions or hemolymph sub-fractions obtained by purifying hemolymph through High Performance Liquid Chromatography. Hemolymph presented a positive effect on the synthesis of polyhedra and enhanced baculovirus replication in Spodoptera frugiperda (Sf9) cells (TCID50/mL), and led to Sf9 cell culture improvement. Grace medium supplemented with 10 % (v/v) FBS and 1 % (v/v) hemolymph provided an increase of baculovirus replication, when the cells were infected with multiplicity of infection of 1. In this case, the baculovirus replication was 6,443.91 times greater than that obtained with the control: Grace medium supplemented with 10 % (v/v) FBS. In addition, this work suggests that hemolymph from L. obliqua could have an interesting application in biotechnology, due to an increase in the viability of the cells and virus replication.

Improved Production Efficiency of Virus-Like Particles by the Baculovirus Expression Vector System.

PubMed

López-Vidal, Javier; Gómez-Sebastián, Silvia; Bárcena, Juan; Nuñez, Maria del Carmen; Martínez-Alonso, Diego; Dudognon, Benoit; Guijarro, Eva; Escribano, José M

2015-01-01

Vaccines based on virus-like particles (VLPs) have proven effective in humans and animals. In this regard, the baculovirus expression vector system (BEVS) is one of the technologies of choice to generate such highly immunogenic vaccines. The extended use of these vaccines for human and animal populations is constrained because of high production costs, therefore a significant improvement in productivity is crucial to ensure their commercial viability. Here we describe the use of the previously described baculovirus expression cassette, called TB, to model the production of two VLP-forming vaccine antigens in insect cells. Capsid proteins from porcine circovirus type 2 (PCV2 Cap) and from the calicivirus that causes rabbit hemorrhagic disease (RHDV VP60) were expressed in insect cells using baculoviruses genetically engineered with the TB expression cassette. Productivity was compared to that obtained using standard counterpart vectors expressing the same proteins under the control of the polyhedrin promoter. Our results demonstrate that the use of the TB expression cassette increased the production yields of these vaccine antigens by around 300% with respect to the standard vectors. The recombinant proteins produced by TB-modified vectors were fully functional, forming VLPs identical in size and shape to those generated by the standard baculoviruses, as determined by electron microscopy analysis. The use of the TB expression cassette implies a simple modification of the baculovirus vectors that significantly improves the cost efficiency of VLP-based vaccine production, thereby facilitating the commercial viability and broad application of these vaccines for human and animal health.

Improved Production Efficiency of Virus-Like Particles by the Baculovirus Expression Vector System

PubMed Central

Bárcena, Juan; Nuñez, Maria del Carmen; Martínez-Alonso, Diego; Dudognon, Benoit; Guijarro, Eva; Escribano, José M.

2015-01-01

Vaccines based on virus-like particles (VLPs) have proven effective in humans and animals. In this regard, the baculovirus expression vector system (BEVS) is one of the technologies of choice to generate such highly immunogenic vaccines. The extended use of these vaccines for human and animal populations is constrained because of high production costs, therefore a significant improvement in productivity is crucial to ensure their commercial viability. Here we describe the use of the previously described baculovirus expression cassette, called TB, to model the production of two VLP-forming vaccine antigens in insect cells. Capsid proteins from porcine circovirus type 2 (PCV2 Cap) and from the calicivirus that causes rabbit hemorrhagic disease (RHDV VP60) were expressed in insect cells using baculoviruses genetically engineered with the TB expression cassette. Productivity was compared to that obtained using standard counterpart vectors expressing the same proteins under the control of the polyhedrin promoter. Our results demonstrate that the use of the TB expression cassette increased the production yields of these vaccine antigens by around 300% with respect to the standard vectors. The recombinant proteins produced by TB-modified vectors were fully functional, forming VLPs identical in size and shape to those generated by the standard baculoviruses, as determined by electron microscopy analysis. The use of the TB expression cassette implies a simple modification of the baculovirus vectors that significantly improves the cost efficiency of VLP-based vaccine production, thereby facilitating the commercial viability and broad application of these vaccines for human and animal health. PMID:26458221

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

PubMed Central

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

2015-01-01

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

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

PubMed

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

2015-01-21

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

Evaluation of the Insecticidal Efficacy of Wild Type and Recombinant Baculoviruses.

PubMed

Popham, Holly J R; Ellersieck, Mark R; Li, Huarong; Bonning, Bryony C

2016-01-01

A considerable amount of work has been undertaken to genetically enhance the efficacy of baculovirus insecticides. Following construction of a genetically altered baculovirus, laboratory bioassays are used to quantify various parameters of insecticidal activity such as the median lethal concentration (or dose) required to kill 50 % of infected larvae (LC50 or LD50), median survival of larvae infected (ST50), and feeding damage incurred by infected larvae. In this chapter, protocols are described for a variety of bioassays and the corresponding data analyses for assessment of the insecticidal activity of baculovirus insecticides.

Baculovirus LEF-11 nuclear localization signal is important for viral DNA replication.

PubMed

Chen, Tingting; Dong, Zhanqi; Hu, Nan; Hu, Zhigang; Dong, Feifan; Jiang, Yaming; Li, Jun; Chen, Peng; Lu, Cheng; Pan, Minhui

2017-06-15

Baculovirus LEF-11 is a small nuclear protein that is involved in viral late gene transcription and DNA replication. However, the characteristics of its nuclear localization signal and its impact on viral DNA replication are unknown. In the present study, systemic bioinformatics analysis showed that the baculovirus LEF-11 contains monopartite and bipartite classical nuclear localization signal sequences (cNLSs), which were also detected in a few alphabaculovirus species. Localization of representative LEF-11 proteins of four baculovirus genera indicated that the nuclear localization characteristics of baculovirus LEF-11 coincided with the predicted results. Moreover, Bombyx mori nucleopolyhedrovirus (BmNPV) LEF-11 could be transported into the nucleus during viral infection in the absence of a cNLSs. Further investigations demonstrated that the NLS of BmNPV LEF-11 is important for viral DNA replication. The findings of the present study indicate that the characteristics of the baculovirus LEF-11 protein and the NLS is essential to virus DNA replication and nuclear transport mechanisms. Copyright © 2017 Elsevier B.V. All rights reserved.

DEVELOPMENT OF AN IN SITU TOXICITY ASSAY SYSTEM USING RECOMBINANT BACULOVIRUSES. (R825433)

EPA Science Inventory

<p>A new method for experimentally analyzing the role of enzymes involved in metabolizing mutagenic, carcinogenic, or cytotoxic chemicals is described. Spodoptera fugiperda (SF-21) cells infected with recombinant baculoviruses are used for high level expression of one or m...

Co-expression of human cytochrome P4501A1 (CYP1A1) variants and human NADPH-cytochrome P450 reductase in the baculovirus/insect cell system.

PubMed

Schwarz, D; Kisselev, P; Honeck, H; Cascorbi, I; Schunck, W H; Roots, I

2001-06-01

1. Three human cytochrome P4501A1 (CYP1A1) variants, wild-type (CYP1A1.1), CYP1A1.2 (1462V) and CYP1A1.4 (T461N), were co-expressed with human NADPH-P450 reductase (OR) in Spodoptera frugiperda (Sf9) insect cells by baculovirus co-infection to elaborate a suitable system for studying the role of CYPA1 polymorphism in the metabolism of exogenous and endogenous substrates. 2. A wide range of conditions was examined to optimize co-expression with regard to such parameters as relative multiplicity of infection (MOI), time of harvest, haem precursor supplementation and post-translational stabilization. tinder optimized conditions, almost identical expression levels and molar OR/CYP1A1 ratios (20:1) were attained for all CYP1A1 variants. 3. Microsomes isolated from co-infected cells demonstrated ethoxyresorufin deethlylase activities (nmol/min(-1) nmol(-1) CYP1A1) of 16.0 (CYP1A1.1), 20.5 (CYP1A1.2) and 22.5 (CYP1A1.4). Pentoxyresorufin was dealkylated approximately 10-20 times slower with all enzyme variants. 4. All three CYP1A1 variants were active in metabolizing the precarcinogen benzo[a]pyrene (B[a]P), with wild-type enzyme showing the highest activity, followed by CYP1A1.4 (60%) and CYP1A1.2 (40%). Each variant produced all major metabolites including B[a]P-7,8-dihydrodiol, the precursor of the ultimate carcinogenic species. 5. These studies demonstrate that the baculovirus-mediated co-expression-by-co-infection approach all CYP1A1 variants yields functionally active enzyme systems with similar molar OR/CYP1A1 ratios, thus providing suitable preconditions to examine the metabolism of and environmental chemicals by the different CY1A1 variants.

The Genome of Gryllus bimaculatus Nudivirus Indicates an Ancient Diversification of Baculovirus-Related Nonoccluded Nudiviruses of Insects▿

PubMed Central

Wang, Yongjie; Kleespies, Regina G.; Huger, Alois M.; Jehle, Johannes A.

2007-01-01

The Gryllus bimaculatus nudivirus (GbNV) infects nymphs and adults of the cricket Gryllus bimaculatus (Orthoptera: Gryllidae). GbNV and other nudiviruses such as Heliothis zea nudivirus 1 (HzNV-1) and Oryctes rhinoceros nudivirus (OrNV) were previously called “nonoccluded baculoviruses” as they share some similar structural, genomic, and replication aspects with members of the family Baculoviridae. Their relationships to each other and to baculoviruses are elucidated by the sequence of the complete genome of GbNV, which is 96,944 bp, has an AT content of 72%, and potentially contains 98 predicted protein-coding open reading frames (ORFs). Forty-one ORFs of GbNV share sequence similarities with ORFs found in OrNV, HzNV-1, baculoviruses, and bacteria. Most notably, 15 GbNV ORFs are homologous to the baculovirus core genes, which are associated with transcription (lef-8, lef-9, lef-4, vlf-1, and lef-5), replication (dnapol), structural proteins (p74, pif-1, pif-2, pif-3, vp91, and odv-e56), and proteins of unknown function (38K, ac81, and 19kda). Homologues to these baculovirus core genes have been predicted in HzNV-1 as well. Six GbNV ORFs are homologous to nonconserved baculovirus genes dnaligase, helicase 2, rr1, rr2, iap-3, and desmoplakin. However, the remaining 57 ORFs revealed no homology or poor similarities to the current gene databases. No homologous repeat (hr) sequences but fourteen short direct repeat (dr) regions were detected in the GbNV genome. Gene content and sequence similarity suggest that the nudiviruses GbNV, HzNV-1, and OrNV form a monophyletic group of nonoccluded double-stranded DNA viruses, which separated from the baculovirus lineage before this radiated into dipteran-, hymenopteran-, and lepidopteran-specific clades of occluded nucleopolyhedroviruses and granuloviruses. The accumulated information on the GbNV genome suggests that nudiviruses form a highly diverse and phylogenetically ancient sister group of the baculoviruses, which have

[Expression of goat IL-18 mature protein in insect/baculovirus and determination of bioactivity of the recombinant protein].

PubMed

Wang, Ting-Ting; Wang, Xi-Hui; Fan, Zhong-Ling; Chen, Jin-Long; Cao, Bing-Lei; Kong, Na; Hu, Jing-Dong; Zhao, Hong-Kun

2011-02-01

To express goat IL-18 in insect/baculovirus and detect the bioactivity of the recombinant protein. The mature goat interleukin-18(gIL-18) gene was cloned into the baculovirus transfer vector pFastBac Dual, and then the resulting eukaryotic expression plasmid pFastBac Dual-gIL18 was transformed into DH10Bac, followed by the identification of Bacmid-gIL18 recombinat plosmid by three antibiotics and blue-white patch. Finally, the recombinant bacmid was transfected into sf9 insect cells by Cellfectin and the transfected cells were harvested at different times. Then the expressed protein was identified by SDS-PAGE, Western blot and bioactivity assay. The recombinant protein recognized and bound to its specific antibody. Bioactivity assay showed that the recombinant protein stimulated the proliferation of lymphocytes and induced IFN-γproduction in spleen lymphocytes. The mature gIL-18 protein has been expressed successfully in insect/baculovirus expression system, and have good immunogenicity and bioactivity. The study paves a way for application of gIL-18 as an immunomodulator or immune adjuvant.

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.

Construction of a recombinant baculovirus expressing swine hepatitis E Virus ORF2 and preliminary research on its immune effect.

PubMed

Yang, Z; Hu, Y; Yuan, P; Yang, Y; Wang, K; Xie, L Y; Huang, S L; Liu, J; Ran, L; Song, Z H

2018-03-01

In the swine hepatitis E virus (HEV), open reading frame 2 (ORF2) is rich in antigenic determinants and neutralizing epitopes that could induce immune protection. We chose the Bac-to-Bac® Baculovirus Expression System to express fragments containing the critical neutralizing antigenic sites within the HEV ORF2 protein of pigs to obtain a recombinant baculovirus. The fragment of swine HEV ORF2 region (1198-1881bp) was cloned into vector pFastBacTM. A recombinant baculovirus, rBacmid-ORF2, was obtained after transposition and transfection. The molecular mass of the recombinant protein was 26 kDa. Mice were immunized by the intraperitoneal and oral routes with cell lysates of recombinant baculovirus rBacmid-ORF2. Serum and feces of the mice were collected separately at 0, 14, 28, and 42 d after immunization and the antibody levels of IgG and secretory IgA against swine HEV were determined using an enzyme-linked immunosorbent assay. The results suggested that rBacmid-ORF2 induced antibodies of the humoral and mucosal immune responses in mice and that the oral route was significantly superior to the intraperitoneal route. This is the first study to demonstrate that that recombinant baculovirus swine HEV ORF2 could induce humoral and mucosal immune responses in mice. Copyright© by the Polish Academy of Sciences.

Utilizing the virus-induced blocking of apoptosis in an easy baculovirus titration method

PubMed Central

Niarchos, Athanasios; Lagoumintzis, George; Poulas, Konstantinos

2015-01-01

Baculovirus-mediated protein expression is a robust experimental technique for producing recombinant higher-eukaryotic proteins because it combines high yields with considerable post-translational modification capabilities. In this expression system, the determination of the titer of recombinant baculovirus stocks is important to achieve the correct multiplicity of infection for effective amplification of the virus and high expression of the target protein. To overcome the drawbacks of existing titration methods (e.g., plaque assay, real-time PCR), we present a simple and reliable assay that uses the ability of baculoviruses to block apoptosis in their host cells to accurately titrate virus samples. Briefly, after incubation with serial dilutions of baculovirus samples, Sf9 cells were UV irradiated and, after apoptosis induction, they were viewed via microscopy; the presence of cluster(s) of infected cells as islets indicated blocked apoptosis. Subsequently, baculovirus titers were calculated through the determination of the 50% endpoint dilution. The method is simple, inexpensive, and does not require unique laboratory equipment, consumables or expertise; moreover, it is versatile enough to be adapted for the titration of every virus species that can block apoptosis in any culturable host cells which undergo apoptosis under specific conditions. PMID:26490731

Bioengineered baculoviruses as new class of therapeutics using micro and nanotechnologies: principles, prospects and challenges.

PubMed

Paul, Arghya; Hasan, Anwarul; Rodes, Laetitia; Sangaralingam, Mugundhine; Prakash, Satya

2014-05-01

Designing a safe and efficient gene delivery system is required for success of gene therapy trials. Although a wide variety of viral, non-viral and polymeric nanoparticle based careers have been widely studied, the current gene delivery vehicles are limited by their suboptimal, non-specific therapeutic efficacy and acute immunological reactions, leading to unwanted side effects. Recently, there has been a growing interest in insect-cell-originated baculoviruses as gene delivery vehicles for diverse biomedical applications. Specifically, the emergence of diverse types of surface functionalized and bioengineered baculoviruses is posed to edge over currently available gene delivery vehicles. This is primarily because baculoviruses are comparatively non-pathogenic and non-toxic as they cannot replicate in mammalian cells and do not invoke any cytopathic effect. Moreover, emerging advanced studies in this direction have demonstrated that hybridizing the baculovirus surface with different kinds of bioactive therapeutic molecules, cell-specific targeting moieties, protective polymeric grafts and nanomaterials can significantly improve the preclinical efficacy of baculoviruses. This review presents a comprehensive overview of the recent advancements in the field of bioengineering and biotherapeutics to engineer baculovirus hybrids for tailored gene therapy, and articulates in detail the potential and challenges of these strategies for clinical realization. In addition, the article illustrates the rapid evolvement of microfluidic devices as a high throughput platform for optimizing baculovirus production and treatment conditions. Copyright © 2014 Elsevier B.V. All rights reserved.

Characterization of viral proteins of Oryctes baculovirus and comparison between two geographical isolates.

PubMed

Mohan, K S; Gopinathan, K P

1989-01-01

Bacilliform Oryctes baculovirus particles have been visualized in electron micrographs of midgut sections from virus infected Oryctes rhinoceros beetles. Morphologically the Indian isolate (Oryctes baculovirus, KI) resembled the previously reported Oryctes baculovirus, isolate PV505. The constituent proteins of baculovirus KI have been analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by Western blots using polyclonal antibodies raised against the complete viral particles, as probes. A total of forty eight viral proteins have been identified. Fourteen viral proteins were located on the viral envelope. Among the proteins constituting the nucleocapsid, three were located internally within the capsid. A 23.5 kDa protein was tightly associated with viral DNA in the nucleocapsid core. Two envelope and seven capsid proteins of KI and PV505 revealed differences in SDS-PAGE profiles and glycosylation patterns. Immunoblotting of KI and PV505 proteins with anti KI antiserum demonstrated antigenic differences between the two viral isolates.

The Env-like open reading frame of the baculovirus-integrated retrotransposon TED encodes a retrovirus-like envelope protein.

PubMed

Ozers, M S; Friesen, P D

1996-12-15

TED is a 7.5-kbp member of the gypsy family of retrotransposons that was first identified by its integration within the baculovirus DNA genome. This lepidopteran (moth) transposon contains three retrovirus-like genes, including functional gag and pol that yield reverse transcriptase-containing virus-like particles. To identify and characterize the product(s) of the third env-like open reading frame, TED ORF3 was expressed in homologous lepidopteran cells by using a baculovirus vector, vENV. Immunoblots and immunoprecipitations with antiserum raised against a bacterial ORF3-fusion protein detected two ORF3-encoded proteins, p68env and gp75env. On the basis of selective incorporation of [3H]mannose and inhibition of modification by tunicamycin which blocks N-linked glycosylation, gp75env is a glycoprotein derived from core precursor p68env. As predicted by the presence of a transmembrane domain near the carboxyl terminus, both p68env and gp75env were associated with heavy membranes of vENV-infected cells. Thus, TED ORF3 encodes a membrane glycoprotein with properties characteristic of retroviral env proteins. These data are consistent with the hypothesis that TED is an invertebrate retrovirus. Moreover, TED integration within the baculovirus genome provides an example of retroelement-mediated acquisition of host genes that may contribute to virus evolution.

BACULOVIRUS REPLICATION ALTERS HORMONE-REGULATED HOST DEVELOPMENT.

EPA Science Inventory

The baculovirus Lymantria dispar nuclear polyhedrosis virus interferes with insect larval development by altering the host's hormonal system. The level of haemolymph ecdysteroids, the insect moulting hormone, was found to be higher in virus-infected larvae than in uninfected cont...

Baculovirus Insecticides in Latin America: Historical Overview, Current Status and Future Perspectives

PubMed Central

Haase, Santiago; Sciocco-Cap, Alicia; Romanowski, Víctor

2015-01-01

Baculoviruses are known to regulate many insect populations in nature. Their host-specificity is very high, usually restricted to a single or a few closely related insect species. They are amongst the safest pesticides, with no or negligible effects on non-target organisms, including beneficial insects, vertebrates and plants. Baculovirus-based pesticides are compatible with integrated pest management strategies and the expansion of their application will significantly reduce the risks associated with the use of synthetic chemical insecticides. Several successful baculovirus-based pest control programs have taken place in Latin American countries. Sustainable agriculture (a trend promoted by state authorities in most Latin American countries) will benefit from the wider use of registered viral pesticides and new viral products that are in the process of registration and others in the applied research pipeline. The success of baculovirus-based control programs depends upon collaborative efforts among government and research institutions, growers associations, and private companies, which realize the importance of using strategies that protect human health and the environment at large. Initiatives to develop new regulations that promote the use of this type of ecological alternatives tailored to different local conditions and farming systems are underway. PMID:25941826

Baculovirus replication induces the expression of heat shock proteins in vivo and in vitro

USDA-ARS?s Scientific Manuscript database

A recent handful of studies have linked baculovirus infection with the induction of heat shock proteins, a highly conserved family of cytoprotective proteins. Here, we demonstrate baculovirus-stimulated upregulation of hsp70 transcription in the natural host, Helicoverpa zea. Larvae lethally infec...

Reaching the Melting Point: Degradative Enzymes and Protease Inhibitors Involved in Baculovirus Infection and Dissemination

PubMed Central

Ishimwe, Egide; Hodgson, Jeffrey J.; Clem, Rollie J.; Passarelli, A. Lorena

2015-01-01

Baculovirus infection of a host insect involves several steps, beginning with initiation of virus infection in the midgut, followed by dissemination of infection from the midgut to other tissues in the insect, and finally culminating in “melting” or liquefaction of the host, which allows for horizontal spread of infection to other insects. While all of the viral gene products are involved in ultimately reaching this dramatic infection endpoint, this review focuses on two particular types of baculovirus-encoded proteins: degradative enzymes and protease inhibitors. Neither of these types of proteins is commonly found in other virus families, but they both play important roles in baculovirus infection. The types of degradative enzymes and protease inhibitors encoded by baculoviruses are discussed, as are the roles of these proteins in the infection process. PMID:25724418

Covert Infection of Insects by Baculoviruses.

PubMed

Williams, Trevor; Virto, Cristina; Murillo, Rosa; Caballero, Primitivo

2017-01-01

Baculoviruses ( Baculoviridae ) are occluded DNA viruses that are lethal pathogens of the larval stages of some lepidopterans, mosquitoes, and sawflies (phytophagous Hymenoptera). These viruses have been developed as biological insecticides for control of insect pests and as expression vectors in biotechnological applications. Natural and laboratory populations frequently harbor covert infections by baculoviruses, often at a prevalence exceeding 50%. Covert infection can comprise either non-productive latency or sublethal infection involving low level production of virus progeny. Latency in cell culture systems involves the expression of a small subset of viral genes. In contrast, covert infection in lepidopterans is associated with differential infection of cell types, modulation of virus gene expression and avoidance of immune system clearance. The molecular basis for covert infection may reside in the regulation of host-virus interactions through the action of microRNAs (miRNA). Initial findings suggest that insect nudiviruses and vertebrate herpesviruses may provide useful analogous models for exploring the mechanisms of covert infection by baculoviruses. These pathogens adopt mixed-mode transmission strategies that depend on the relative fitness gains that accrue through vertical and horizontal transmission. This facilitates virus persistence when opportunities for horizontal transmission are limited and ensures virus dispersal in migratory host species. However, when host survival is threatened by environmental or physiological stressors, latent or persistent infections can be activated to produce lethal disease, followed by horizontal transmission. Covert infection has also been implicated in population level effects on host-pathogen dynamics due to the reduced reproductive capacity of infected females. We conclude that covert infections provide many opportunities to examine the complexity of insect-virus pathosystems at the organismal level and to explore

Covert Infection of Insects by Baculoviruses

PubMed Central

Williams, Trevor; Virto, Cristina; Murillo, Rosa; Caballero, Primitivo

2017-01-01

Baculoviruses (Baculoviridae) are occluded DNA viruses that are lethal pathogens of the larval stages of some lepidopterans, mosquitoes, and sawflies (phytophagous Hymenoptera). These viruses have been developed as biological insecticides for control of insect pests and as expression vectors in biotechnological applications. Natural and laboratory populations frequently harbor covert infections by baculoviruses, often at a prevalence exceeding 50%. Covert infection can comprise either non-productive latency or sublethal infection involving low level production of virus progeny. Latency in cell culture systems involves the expression of a small subset of viral genes. In contrast, covert infection in lepidopterans is associated with differential infection of cell types, modulation of virus gene expression and avoidance of immune system clearance. The molecular basis for covert infection may reside in the regulation of host–virus interactions through the action of microRNAs (miRNA). Initial findings suggest that insect nudiviruses and vertebrate herpesviruses may provide useful analogous models for exploring the mechanisms of covert infection by baculoviruses. These pathogens adopt mixed-mode transmission strategies that depend on the relative fitness gains that accrue through vertical and horizontal transmission. This facilitates virus persistence when opportunities for horizontal transmission are limited and ensures virus dispersal in migratory host species. However, when host survival is threatened by environmental or physiological stressors, latent or persistent infections can be activated to produce lethal disease, followed by horizontal transmission. Covert infection has also been implicated in population level effects on host–pathogen dynamics due to the reduced reproductive capacity of infected females. We conclude that covert infections provide many opportunities to examine the complexity of insect–virus pathosystems at the organismal level and to

Characterization of canine herpesvirus glycoprotein C expressed by a recombinant baculovirus in insect cells.

PubMed

Xuan, X; Maeda, K; Mikami, T; Otsuka, H

1996-12-01

The gene encoding the canine herpesvirus (CHV) glycoprotein C (gC) homologue has been identified by sequence homology analyses with other well studied herpesviruses. Previously, we have identified three CHV glycoproteins, gp145/112, gp80 and gp47 using a panel of monoclonal antibodies (MAbs). To determine which CHV glycoprotein corresponds to gC, a recombinant baculovirus which contains the putative CHV gC structural gene under the baculovirus polyhedrin promoter was constructed. The recombinant baculovirus expressed gC-related polypeptides (44-62 kDa), which reacted only with MAbs against CHV gp80, indicating that the previously identified CHV gp80 is the translation product of the gC gene. The baculovirus expressed gC was glycosylated and transported to the surface of infected cells. At least seven neutralizing epitopes were conserved on the gC produced in insect cells. It was found that the recombinant baculovirus infected cells adsorbed murine erythrocytes as is the case for CHV-infected cells. The hemadsorption activity was inhibited by heparin, indicating that the CHV gC binds to heparan sulfate on the surface of murine erythrocytes. Mice immunized with the recombinant gC produced strong neutralizing antibodies. Our results suggest that CHV gC produced in insect cells may be useful as a subunit vaccine to control CHV infections.

A baculovirus (Bombyx mori nuclear polyhedrosis virus) repeat element functions as a powerful constitutive enhancer in transfected insect cells.

PubMed

Lu, M; Farrell, P J; Johnson, R; Iatrou, K

1997-12-05

It has been previously reported that baculovirus homologous regions, the regions of baculovirus genomes that contain the origins of DNA replication, can augment the expression of a small number of baculovirus genes in vitro. We are now reporting that a region of the genome of Bombyx mori nuclear polyhedrosis virus (BmNPV) containing the homologous region 3 (HR3) acts as an enhancer for the promoter of a nonviral gene, the cytoplasmic actin gene of the silkmoth B. mori. Incorporation of the HR3 sequences of BmNPV into an actin promoter-based expression cassette results in an augmentation of transgene expression in transfected cells by two orders of magnitude relative to the control recombinant expression cassette. This increase is due to a corresponding increase in the rate of transcription from the actin promoter and not to replication of the expression cassette and occurs only when the HR3 element is linked to the expression cassette in cis. A comparable degree of enhancement in the activity of the silkworm actin promoter occurs also in heterologous lepidopteran cells. Concomitant supplementation of transfected cells with the BmIE1 trans-activator, which was previously shown to be capable of functioning in vitro as a transcriptional co-activator of the cytoplasmic actin gene promoter, results in more than a 1,000-fold increase in the level of expression of recombinant proteins placed under the control of the actin gene promoter. These findings provide the foundation for the development of a nonlytic insect cell expression system for continuous high-level expression of recombinant proteins. Such a system should provide levels of expression of recombinant proteins comparable to those obtained from baculovirus expression systems and should also have the additional advantage of continuous production in a cellular environment that, in contrast to that generated by a baculovirus infection, supports continuously proper posttranslational modifications of recombinant

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.

Construction of a highly efficient display system for baculovirus and its application on multigene co-display.

PubMed

Zheng, Hao; Wang, Xiong; Ren, Feifei; Zou, Shenglong; Feng, Min; Xu, Liangliang; Yao, Lunguang; Sun, Jingchen

2018-06-19

The classical baculovirus display system (BDS) has often recruited fields including gene delivery, gene therapy, and the genetic engineering of vaccines, as it is capable of presenting foreign polypeptides on the membranes of recombinant baculovirus through a transmembrane protein. However, classical BDS's high cost, complicated operation, low display efficiency and its inability to simultaneously display multiple gene products impede its practicality. In this study, we present a novel and highly efficient display system based on ires-dependent gp64 for rescuing gp64-null Bacmid of baculovirus construction without affecting the viral replication cycle, which we name the baculovirus multigene display system (BMDS). Laser scanning confocal microscopy demonstrated that eGFP, eYFP, and mCherry were translocated on the membrane of Spodoptera frugiperda 9 cell successfully as expected. Western blot analysis further confirmed the presence of the fluorescent proteins on the budded, mature viral particles. The results showed the display efficiency of target gene on cell surface is fourfold that of classical BDS. In addition, a recombinant baculovirus displaying three kinds of fluorescent proteins simultaneously was constructed, thereby demonstrating the effectiveness of BMDS as a co-display system.

Glycobiotechnology of the Insect Cell-Baculovirus Expression System Technology.

PubMed

Palomares, Laura A; Srivastava, Indresh K; Ramírez, Octavio T; Cox, Manon M J

2018-06-10

The insect cell-baculovirus expression system technology (BEST) has a prominent role in producing recombinant proteins to be used as research and diagnostic reagents and vaccines. The glycosylation profile of proteins produced by the BEST is composed predominantly of terminal mannose glycans, and, in Trichoplusia ni cell lines, core α3 fucosylation, a profile different to that in mammals. Insects contain all the enzymatic activities needed for complex N- and O-glycosylation and sialylation, although few reports of complex glycosylation and sialylation by the BEST exist. The insect cell line and culture conditions determine the glycosylation profile of proteins produced by the BEST. The promoter used, dissolved oxygen tension, presence of sugar precursors, bovine serum or hemolymph, temperature, and the time of harvest all influence glycosylation, although more research is needed. The lack of activity of glycosylation enzymes possibly results from the transcription regulation and stress imposed by baculovirus infection. To solve this limitation, the glycosylation pathway of insect cells has been engineered to produce complex sialylated glycans and to eliminate α3 fucosylation, either by generating transgenic cell lines or by using baculovirus vectors. These strategies have been successful. Complex glycosylation, sialylation, and inhibition of α3 fucosylation have been achieved, although the majority of glycans still have terminal mannose residues. The implication of insect glycosylation in the proteins produced by the BEST is discussed. Graphical Abstract.

Sleeping Beauty-baculovirus hybrid vectors for long-term gene expression in the eye.

PubMed

Turunen, Tytteli Anni Kaarina; Laakkonen, Johanna Päivikki; Alasaarela, Laura; Airenne, Kari Juhani; Ylä-Herttuala, Seppo

2014-01-01

A baculovirus vector is capable of efficiently transducing many nondiving and diving cell types. However, the potential of baculovirus is restricted for many gene delivery applications as a result of the transient gene expression that it mediates. The plasmid-based Sleeping Beauty (SB) transposon system integrates transgenes into target cell genome efficiently with a genomic integration pattern that is generally considered safer than the integration of many other integrating vectors; yet efficient delivery of therapeutic genes into cells of target tissues in vivo is a major challenge for nonviral gene therapy. In the present study, SB was introduced into baculovirus to obtain novel hybrid vectors that would combine the best features of the two vector systems (i.e. effective gene delivery and efficient integration into the genome), thus circumventing the major limitations of these vectors. We constructed and optimized SB-baculovirus hybrid vectors that bear either SB100x transposase or SB transposon in the forward or reverse orientations with respect to the viral backbone The functionality of the novel hybrid vectors was investigated in cell cultures and in a proof-of-concept study in the mouse eye. The hybrid vectors showed high and sustained transgene expression that remained stable and demonstrated no signs of decline during the 2 months follow-up in vitro. These results were verified in the mouse eye where persistent transgene expression was detected two months after intravitreal injection. Our results confirm that (i) SB-baculovirus hybrid vectors mediate long-term gene expression in vitro and in vivo, and (ii) the hybrid vectors are potential new tools for the treatment of ocular diseases. Copyright © 2014 John Wiley & Sons, Ltd.

How baculovirus polyhedra fit square pegs into round holes to robustly package viruses.

PubMed

Ji, Xiaoyun; Sutton, Geoff; Evans, Gwyndaf; Axford, Danny; Owen, Robin; Stuart, David I

2010-01-20

Natural protein crystals (polyhedra) armour certain viruses, allowing them to survive for years under hostile conditions. We have determined the structure of polyhedra of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), revealing a highly symmetrical covalently cross-braced robust lattice, the subunits of which possess a flexible adaptor enabling this supra-molecular assembly to specifically entrap massive baculoviruses. Inter-subunit chemical switches modulate the controlled release of virus particles in the unusual high pH environment of the target insect's gut. Surprisingly, the polyhedrin subunits are more similar to picornavirus coat proteins than to the polyhedrin of cytoplasmic polyhedrosis virus (CPV). It is, therefore, remarkable that both AcMNPV and CPV polyhedra possess identical crystal lattices and crystal symmetry. This crystalline arrangement must be particularly well suited to the functional requirements of the polyhedra and has been either preserved or re-selected during evolution. The use of flexible adaptors to generate a powerful system for packaging irregular particles is characteristic of the AcMNPV polyhedrin and may provide a vehicle to sequester a wide range of objects such as biological nano-particles.

Cloning and baculovirus expression of a desiccation stress gene from the beetle, Tenebrio molitor.

PubMed

Graham, L A; Bendena, W G; Walker, V K

1996-02-01

The cDNA sequence encoding a novel desiccation stress protein (dsp28) found in the hemolymph of the common yellow mealworm beetle, Tenebrio molitor, has been determined. The sequence encodes a 225 amino acid protein containing a 20 amino acid signal peptide. Dsp28 shows no significant similarity to any known nucleic acid or protein sequence. Levels of dsp28 mRNA were found to increase approx 5-fold following desiccation. Dsp28 cDNA has been cloned into a baculovirus expression vector and the expressed protein was compared to native dsp28. Both dsp28 expressed by recombinant baculovirus and native dsp28 are glycosylated and N-terminally processed. Although dsp28 is induced by cold in addition to desiccation stress, it does not contribute to the freezing point depression (thermal hysteresis) observed in Tenebrio hemolymph.

Effect of spray drying processing parameters on the insecticidal activity of two encapsulated formulations of baculovirus

USDA-ARS?s Scientific Manuscript database

The aim of this work was to evaluate the effect of spray dryer processing parameters on the process yield and insecticidal activity of baculovirus to support the development of this beneficial group of microbes as biopesticides. For each of two baculoviruses [granulovirus (GV) from Pieris rapae (L....

MacoNPV baculovirus midgut-specific gene expression during infection of the bertha armyworm, Mamestra configurata

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

Donly, B. Cameron, E-mail: Cam.Donly@agr.gc.ca

Baculoviruses have two forms, occlusion derived virus (ODV) which is responsible for primary infection in host midgut tissue and budded virus (BV), which infects all other host tissues during secondary infection. This study examined the primary infection by ODV of midgut cells of bertha armyworm Mamestra configurata fourth instar larvae and measured the expression of viral genes over a time course of infection. Both digital PCR and RNA sequencing methods showed the profile of transcription to be different from those produced by AcMNPV BV infection of in vitro cell cultures. This included having unique collections of genes expressed early, asmore » well as much greater late gene expression of p6.9 and much reduced expression of polh and p10. These differences likely reflect characteristics unique to the critical step of in vivo midgut cell infection, and provide insights into the processes that regulate viral gene expression in different host tissues. -- Highlights: •The transcriptome of MacoNPV ODV in larval midgut was measured by RNA-seq and digital PCR. •The earliest genes expressed included fusion protein, hoar, and me53. •p6.9 was highly expressed late but polH and p10 were less so. •These patterns are unique from BV of other baculoviruses in tissue culture cells.« less

N-TERMINALLY ELONGATED SpliInx2 AND SpliInx3 REDUCE BACULOVIRUS-TRIGGERED APOPTOSIS VIA HEMICHANNEL CLOSURE.

PubMed

Chen, Ya-Bin; Xiao, Wei; Li, Ming; Zhang, Yan; Yang, Yang; Hu, Jian-Sheng; Luo, Kai-Jun

2016-05-01

The hemichannel and gap junction channel are major portals for the release of factors responsible for the effects of apoptotic cells on the spread of apoptosis to neighboring cells and apoptotic corpse clearance, typically by phagocytes. The N-terminal cytoplasmic domain in the connexins, gap junction proteins in vertebrate, has been implicated in regulating channel closure. However, little is known about how the hemichannel close responds to apoptotic signaling transduction leading to the reduction of neighboring cellular apoptosis in an invertebrate. An insect Bac-to-Bac expression system, pFastBac(TM) HT A, allows us to construct an N-terminally elongated SpliInx2 (Nte-Inx2) and SpliInx3 (Nte-Inx3). Here, we demonstrated that recombinant baculovirus Bac-Nte-Inx2 (reBac-Net-Inx2) and Bac-Nte-Inx3 (reBac-Nte-Inx3) closed the endogenous hemichannel on the Sf9 cell surface. Importantly, primary baculovirus infections significantly caused early apoptosis, and this apoptosis was reduced by hemichannel-closed Sf9 cells at 24-h post-infection (PI). Although N-terminal-elongated residue led to the increase in the phosphorylated sites in both Nte-Inx2 and Nte-Inx3 and an additional transmembrane domain in Nte-Inx3, both the proteins localized on the cell surface, suggesting Nte-Inxs proteins could mediate hemichannel closure. Further supporting evidence showed that hemichannel closure was dependent on N-Inxs expressed by baculovirus polyhedrin promoter, which began to express at 18-24 h PI. These results identify an unconventional function of N-terminal-elongated innexins that could act as a plug to manipulate hemichannel closure and provide a mechanism connecting the effect of hemichannel closure directly to apoptotic signaling transduction from intracellular to extracellular compartment. © 2016 Wiley Periodicals, Inc.

Structural Organization of Baculovirus Occlusion Bodies and Protective Role of Multilayered Polyhedron Envelope Protein.

PubMed

Sajjan, Dayanand B; Hinchigeri, Shivayogeppa B

2016-03-01

Baculoviruses are the ingenious insect pathogens. Outside the host, baculovirus occlusion bodies (OB) provide stability to occlusion-derived viruses (ODV) embedded within. The OB is an organized structure, chiefly composed of proteins namely polyhedrin, polyhedron envelope protein (PEP) and P10. Currently, the structural organization of OB is poorly understood and the role of OB proteins in conferring the stability to ODV is unknown. Here we have shown that the assembly of polyhedrin unit cells into an OB is a rapid process; the PEP forms in multiple layers; the PEP layers predominantly contribute to ODV viability. Full-grown OBs (n = 36) were found to be 4.0 ± 1.0 µm in diameter and possessed a peculiar geometry of a truncated rhombic dodecahedron. The atomic force microscopy (AFM) study on the structure of OBs at different stages of growth in insect cells revealed polyhedrin assembly and thickness of PEP layers. The thickness of PEP layers at 53 h post-transfection (hpt) ranged from 56 to 80 nm. Mature PEP layers filled up approximately one third of the OB volume. The size of ODV nucleocapsid was found to be 433 ± 10 nm in length. The zeta potential and particle size distribution study of viruses revealed the protective role of PEP layers. The presence of a multilayered PEP confers a viable advantage to the baculoviruses compared to single-layered PEP. Thus, these findings may help in developing PEP layer-based biopolymers for protein-based nanodevices, nanoelectrodes and more stable biopesticides.

Baculovirus GP64-mediated entry into mammalian cells.

PubMed

Kataoka, Chikako; Kaname, Yuuki; Taguwa, Shuhei; Abe, Takayuki; Fukuhara, Takasuke; Tani, Hideki; Moriishi, Kohji; Matsuura, Yoshiharu

2012-03-01

The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) serves as an efficient viral vector, not only for abundant gene expression in insect cells, but also for gene delivery into mammalian cells. Lentivirus vectors pseudotyped with the baculovirus envelope glycoprotein GP64 have been shown to acquire more potent gene transduction than those with vesicular stomatitis virus (VSV) envelope glycoprotein G. However, there are conflicting hypotheses about the molecular mechanisms of the entry of AcMNPV. Moreover, the mechanisms of the entry of pseudotyped viruses bearing GP64 into mammalian cells are not well characterized. Determination of the entry mechanisms of AcMNPV and the pseudotyped viruses bearing GP64 is important for future development of viral vectors that can deliver genes into mammalian cells with greater efficiency and specificity. In this study, we generated three pseudotyped VSVs, NPVpv, VSVpv, and MLVpv, bearing envelope proteins of AcMNPV, VSV, and murine leukemia virus, respectively. Depletion of membrane cholesterol by treatment with methyl-β-cyclodextrin, which removes cholesterol from cellular membranes, inhibited GP64-mediated internalization in a dose-dependent manner but did not inhibit attachment to the cell surface. Treatment of cells with inhibitors or the expression of dominant-negative mutants for dynamin- and clathrin-mediated endocytosis abrogated the internalization of AcMNPV and NPVpv into mammalian cells, whereas inhibition of caveolin-mediated endocytosis did not. Furthermore, inhibition of macropinocytosis reduced GP64-mediated internalization. These results suggest that cholesterol in the plasma membrane, dynamin- and clathrin-dependent endocytosis, and macropinocytosis play crucial roles in the entry of viruses bearing baculovirus GP64 into mammalian cells.

Unconventional P-35S sequence identified in genetically modified maize

PubMed Central

Al-Hmoud, Nisreen; Al-Husseini, Nawar; Ibrahim-Alobaide, Mohammed A; Kübler, Eric; Farfoura, Mahmoud; Alobydi, Hytham; Al-Rousan, Hiyam

2014-01-01

The Cauliflower Mosaic Virus 35S promoter sequence, CaMV P-35S, is one of several commonly used genetic targets to detect genetically modified maize and is found in most GMOs. In this research we report the finding of an alternative P-35S sequence and its incidence in GM maize marketed in Jordan. The primer pair normally used to amplify a 123 bp DNA fragment of the CaMV P-35S promoter in GMOs also amplified a previously undetected alternative sequence of CaMV P-35S in GM maize samples which we term V3. The amplified V3 sequence comprises 386 base pairs and was not found in the standard wild-type maize, MON810 and MON 863 GM maize. The identified GM maize samples carrying the V3 sequence were found free of CaMV when compared with CaMV infected brown mustard sample. The data of sequence alignment analysis of the V3 genetic element showed 90% similarity with the matching P-35S sequence of the cauliflower mosaic virus isolate CabbB-JI and 99% similarity with matching P-35S sequences found in several binary plant vectors, of which the binary vector locus JQ693018 is one example. The current study showed an increase of 44% in the incidence of the identified 386 bp sequence in GM maize sold in Jordan’s markets during the period 2009 and 2012. PMID:24495911

Genetically-engineered baculovirus pesticides and their environmental safety

Treesearch

H. Alan Wood; Yu Zailin

1991-01-01

Baculoviruses such as the Lymantria dispar nuclear polyhedrosis virus (LdMNPV) are ecologically attractive alternatives to chemical insect pesticides but have a slow rate of control. To overcome this we have developed and are field testing an environmentally acceptable strategy which can be used for the introduction and expression of pesticide-...

Inhibition of melanization by serpin-5 and serpin-9 promotes baculovirus infection in cotton bollworm Helicoverpa armigera

PubMed Central

Wang, Manli; Wang, Xi; Yin, Mengyi; Wang, Qianran; Hu, Zhihong

2017-01-01

Melanization, an important insect defense mechanism, is mediated by clip-domain serine protease (cSP) cascades and is regulated by serpins. Here we show that proteolytic activation of prophenoloxidase (PPO) and PO-catalyzed melanization kill the baculovirus in vitro. Our quantitative proteomics and biochemical experiments revealed that baculovirus infection of the cotton bollworm, Helicoverpa armigera, reduced levels of most cascade members in the host hemolymph and PO activity. By contrast, serpin-9 and serpin-5 were sequentially upregulated after the viral infection. The H. armigera serpin-5 and serpin-9 regulate melanization by directly inhibiting their target proteases cSP4 and cSP6, respectively and cSP6 activates PPO purified from hemolymph. Furthermore, serpin-5/9-depleted insects exhibited high PO activities and showed resistance to baculovirus infection. Together, our results characterize a part of the melanization cascade in H. armigera, and suggest that natural insect virus baculovirus has evolved a distinct strategy to suppress the host immune system. PMID:28953952

How baculovirus polyhedra fit square pegs into round holes to robustly package viruses

PubMed Central

Ji, Xiaoyun; Sutton, Geoff; Evans, Gwyndaf; Axford, Danny; Owen, Robin; Stuart, David I

2010-01-01

Natural protein crystals (polyhedra) armour certain viruses, allowing them to survive for years under hostile conditions. We have determined the structure of polyhedra of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), revealing a highly symmetrical covalently cross-braced robust lattice, the subunits of which possess a flexible adaptor enabling this supra-molecular assembly to specifically entrap massive baculoviruses. Inter-subunit chemical switches modulate the controlled release of virus particles in the unusual high pH environment of the target insect's gut. Surprisingly, the polyhedrin subunits are more similar to picornavirus coat proteins than to the polyhedrin of cytoplasmic polyhedrosis virus (CPV). It is, therefore, remarkable that both AcMNPV and CPV polyhedra possess identical crystal lattices and crystal symmetry. This crystalline arrangement must be particularly well suited to the functional requirements of the polyhedra and has been either preserved or re-selected during evolution. The use of flexible adaptors to generate a powerful system for packaging irregular particles is characteristic of the AcMNPV polyhedrin and may provide a vehicle to sequester a wide range of objects such as biological nano-particles. PMID:19959989

A baculovirus-mediated strategy for full-length plant virus coat protein expression and purification.

PubMed

Ardisson-Araújo, Daniel Mendes Pereira; Rocha, Juliana Ribeiro; da Costa, Márcio Hedil Oliveira; Bocca, Anamélia Lorenzetti; Dusi, André Nepomuceno; de Oliveira Resende, Renato; Ribeiro, Bergmann Morais

2013-08-15

Garlic production is severely affected by virus infection, causing a decrease in productivity and quality. There are no virus-free cultivars and garlic-infecting viruses are difficult to purify, which make specific antibody production very laborious. Since high quality antisera against plant viruses are important tools for serological detection, we have developed a method to express and purify full-length plant virus coat proteins using baculovirus expression system and insects as bioreactors. In this work, we have fused the full-length coat protein (cp) gene from the Garlic Mite-borne Filamentous Virus (GarMbFV) to the 3'-end of the Polyhedrin (polh) gene of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). The recombinant baculovirus was amplified in insect cell culture and the virus was used to infect Spodoptera frugiperda larvae. Thus, the recombinant fused protein was easily purified from insect cadavers using sucrose gradient centrifugation and analyzed by Western Blotting. Interestingly, amorphous crystals were produced in the cytoplasm of cells infected with the recombinant virus containing the chimeric-protein gene but not in cells infected with the wild type and recombinant virus containing the hexa histidine tagged Polh. Moreover, the chimeric protein was used to immunize rats and generate antibodies against the target protein. The antiserum produced was able to detect plants infected with GarMbFV, which had been initially confirmed by RT-PCR. The expression of a plant virus full-length coat protein fused to the baculovirus Polyhedrin in recombinant baculovirus-infected insects was shown to produce high amounts of the recombinant protein which was easily purified and efficiently used to generate specific antibodies. Therefore, this strategy can potentially be used for the development of plant virus diagnostic kits for those viruses that are difficult to purify, are present in low titers or are present in mix infection in

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.

Enhanced effect of fluorescent whitening agent on peroral infection for recombinant baculovirus in the host Bombyx mori L.

PubMed

Wang, Bing; Shang, Jinyan; Liu, Xunli; Cui, Weizheng; Wu, Xiaofeng; Zhao, Na

2007-01-01

The low efficiency of the oral infectivity of recombinant polyhedrin-negative baculovirus is a major bottleneck in the application of the baculovirus expression system in the silkworm (Bombyx mori L). In this study, the effects of a fluorescent whitening agent on improving the oral infection for the recombinant Bombyx mori nuclear polyhedrosis virus in silkworm larva and their possible mechanism were investigated. The results showed that the peroral infection can be remarkably enhanced by adding VBL into the larval artificial diet. The maximum infection rate reached as high as 90% with the concentration of VBL (1%), which was then considered as optimal. The total protease activity and pH value of the larval intestinal juice were found to be lower when compared to the control, indicating an abnormal physiological change of the larval digestive system by VBL, which, in turn, resulted in improved peroral infection of recombinant virus.

Localization of VP28 on the baculovirus envelope and its immunogenicity against white spot syndrome virus in Penaeus monodon

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

Syed Musthaq, S.; Madhan, Selvaraj; Sahul Hameed, A.S.

2009-09-01

White spot syndrome virus (WSSV) is a large dsDNA virus responsible for white spot disease in shrimp and other crustaceans. VP28 is one of the major envelope proteins of WSSV and plays a crucial role in viral infection. In an effort to develop a vaccine against WSSV, we have constructed a recombinant baculovirus with an immediate early promoter 1 which expresses VP28 at an early stage of infection in insect cells. Baculovirus expressed rVP28 was able to maintain its structural and antigenic conformity as indicated by immunofluorescence assay and western blot analysis. Interestingly, our results with confocal microscopy revealed thatmore » rVP28 was able to localize on the plasma membrane of insect cells infected with recombinant baculovirus. In addition, we demonstrated with transmission electron microscopy that baculovirus successfully acquired rVP28 from the insect cell membrane via the budding process. Using this baculovirus displaying VP28 as a vaccine against WSSV, we observed a significantly higher survival rate of 86.3% and 73.5% of WSSV-infected shrimp at 3 and 15 days post vaccination respectively. Quantitative real-time PCR also indicated that the WSSV viral load in vaccinated shrimp was significantly reduced at 7 days post challenge. Furthermore, our RT-PCR and immunohistochemistry results demonstrated that the recombinant baculovirus was able to express VP28 in vivo in shrimp tissues. This study will be of considerable significance in elucidating the morphogenesis of WSSV and will pave the way for new generation vaccines against WSSV.« less

Soluble forms of the cell adhesion molecule L1 produced by insect and baculovirus-transduced mammalian cells enhance Schwann cell motility.

PubMed

Lavdas, Alexandros A; Efrose, Rodica; Douris, Vassilis; Gaitanou, Maria; Papastefanaki, Florentia; Swevers, Luc; Thomaidou, Dimitra; Iatrou, Kostas; Matsas, Rebecca

2010-12-01

For biotechnological applications, insect cell lines are primarily known as hosts for the baculovirus expression system that is capable to direct synthesis of high levels of recombinant proteins through use of powerful viral promoters. Here, we demonstrate the implementation of two alternative approaches based on the baculovirus system for production of a mammalian recombinant glycoprotein, comprising the extracellular part of the cell adhesion molecule L1, with potential important therapeutic applications in nervous system repair. In the first approach, the extracellular part of L1 bearing a myc tag is produced in permanently transformed insect cell lines and purified by affinity chromatography. In the second approach, recombinant baculoviruses that express L1-Fc chimeric protein, derived from fusion of the extracellular part of L1 with the Fc part of human IgG1, under the control of a mammalian promoter are used to infect mammalian HEK293 and primary Schwann cells. Both the extracellular part of L1 bearing a myc tag accumulating in the supernatants of insect cultures as well as L1-Fc secreted by transduced HEK293 or Schwann cells are capable of increasing the motility of Schwann cells with similar efficiency in a gap bridging bioassay. In addition, baculovirus-transduced Schwann cells show enhanced motility when grafted on organotypic cultures of neonatal brain slices while they retain their ability to myelinate CNS axons. This proof-of-concept that the migratory properties of myelin-forming cells can be modulated by recombinant protein produced in insect culture as well as by means of baculovirus-mediated adhesion molecule expression in mammalian cells may have beneficial applications in the field of CNS therapies. ©2010 The Authors. Journal of Neurochemistry © 2010 International Society for Neurochemistry.

Baculovirus-based genome editing in primary cells.

PubMed

Mansouri, Maysam; Ehsaei, Zahra; Taylor, Verdon; Berger, Philipp

2017-03-01

Genome editing in eukaryotes became easier in the last years with the development of nucleases that induce double strand breaks in DNA at user-defined sites. CRISPR/Cas9-based genome editing is currently one of the most powerful strategies. In the easiest case, a nuclease (e.g. Cas9) and a target defining guide RNA (gRNA) are transferred into a target cell. Non-homologous end joining (NHEJ) repair of the DNA break following Cas9 cleavage can lead to inactivation of the target gene. Specific repair or insertion of DNA with Homology Directed Repair (HDR) needs the simultaneous delivery of a repair template. Recombinant Lentivirus or Adenovirus genomes have enough capacity for a nuclease coding sequence and the gRNA but are usually too small to also carry large targeting constructs. We recently showed that a baculovirus-based multigene expression system (MultiPrime) can be used for genome editing in primary cells since it possesses the necessary capacity to carry the nuclease and gRNA expression constructs and the HDR targeting sequences. Here we present new Acceptor plasmids for MultiPrime that allow simplified cloning of baculoviruses for genome editing and we show their functionality in primary cells with limited life span and induced pluripotent stem cells (iPS). Copyright © 2017 Elsevier Inc. All rights reserved.

Expression of the hemagglutinin HA1 subunit of the equine influenza virus using a baculovirus expression system.

PubMed

Sguazza, Guillermo H; Fuentealba, Nadia A; Tizzano, Marco A; Galosi, Cecilia M; Pecoraro, Marcelo R

2013-01-01

Equine influenza virus is a leading cause of respiratory disease in horses worldwide. Disease prevention is by vaccination with inactivated whole virus vaccines. Most current influenza vaccines are generated in embryonated hens' eggs. Virions are harvested from allantoic fluid and chemically inactivated. Although this system has served well over the years, the use of eggs as the substrate for vaccine production has several well-recognized disadvantages (cost, egg supply, waste disposal and yield in eggs). The aim of this study was to evaluate a baculovirus system as a potential method for producing recombinant equine influenza hemagglutinin to be used as a vaccine. The hemagglutinin ectodomain (HA1 subunit) was cloned and expressed using a baculovirus expression vector. The expression was determined by SDS-PAGE and immunoblotting. A high yield, 20μg/ml of viral protein, was obtained from recombinant baculovirus-infected cells. The immune response in BALB/c mice was examined following rHA1 inoculation. Preliminary results show that recombinant hemagglutinin expressed from baculovirus elicits a strong antibody response in mice; therefore it could be used as an antigen for subunit vaccines and diagnostic tests. Copyright © 2013 Asociación Argentina de Microbiología. Publicado por Elsevier España. All rights reserved.

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.

Modularity and evolutionary constraints in a baculovirus gene regulatory network

PubMed Central

2013-01-01

Background The structure of regulatory networks remains an open question in our understanding of complex biological systems. Interactions during complete viral life cycles present unique opportunities to understand how host-parasite network take shape and behave. The Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) is a large double-stranded DNA virus, whose genome may encode for 152 open reading frames (ORFs). Here we present the analysis of the ordered cascade of the AgMNPV gene expression. Results We observed an earlier onset of the expression than previously reported for other baculoviruses, especially for genes involved in DNA replication. Most ORFs were expressed at higher levels in a more permissive host cell line. Genes with more than one copy in the genome had distinct expression profiles, which could indicate the acquisition of new functionalities. The transcription gene regulatory network (GRN) for 149 ORFs had a modular topology comprising five communities of highly interconnected nodes that separated key genes that are functionally related on different communities, possibly maximizing redundancy and GRN robustness by compartmentalization of important functions. Core conserved functions showed expression synchronicity, distinct GRN features and significantly less genetic diversity, consistent with evolutionary constraints imposed in key elements of biological systems. This reduced genetic diversity also had a positive correlation with the importance of the gene in our estimated GRN, supporting a relationship between phylogenetic data of baculovirus genes and network features inferred from expression data. We also observed that gene arrangement in overlapping transcripts was conserved among related baculoviruses, suggesting a principle of genome organization. Conclusions Albeit with a reduced number of nodes (149), the AgMNPV GRN had a topology and key characteristics similar to those observed in complex cellular organisms, which indicates

Iron levels change in larval Heliothis virescens tissues following baculovirus infection

USDA-ARS?s Scientific Manuscript database

Inductively-coupled plasma mass spectrometry (ICP-MS) and 59Fe radiotracers were used to investigate changes in levels of iron (Fe) in the tissues of Heliothis virescens following baculovirus infection. Fe concentrations were determined by ICP-MS in hemolymph collected from 4th instar larvae infect...

Overexpression of p35 in Min6 pancreatic beta cells induces a stressed neuron-like apoptosis.

PubMed

Zheng, Ya-Li; Hu, Ya-Fang; Zhang, Aiping; Wang, Wei; Li, Bo; Amin, Niranjana; Grant, Philip; Pant, Harish C

2010-12-15

Cdk5 activity has been implicated in brain development and the regulation of many neuronal processes. Recently, the expression of p35 and Cdk5 activity has been reported in pancreatic beta cells. Decreased Cdk5 activity enhanced glucose-stimulated insulin secretion. This suggests that Cdk5 may play an important role in the regulation of insulin secretion. To further understand how Cdk5 regulates insulin secretion in glucose-stimulated pancreatic β cells, we first confirmed the presence of a low level of p35 in pancreatic Min6 cells. Next, in a time-course experiment in high glucose (25 mM) we showed that endogenous p35 increased gradually accompanied by a 3-fold increase in Cdk5 activity by 16 h. Insulin secretion, however, doubled after 2 h followed by progressive downregulation, negatively correlated with Cdk5 activity. On the other hand, overexpression of p35 in these cells resulted in more than a three-fold increase in Cdk5 activity within 2 h coupled to a 50% reduction in insulin secretion in both high and low (3 mM) glucose. Most significantly, cells overexpressing p35, treated with high glucose for 4 h, showed induction of p25, the p35-derived truncated fragment which hyperactivates Cdk5 in neurons. As a result, insulin secretion was inhibited and cells became apoptotic. Roscovitine or co-infection of dominant negative Cdk5 (dnCdk5) with p35 increased insulin secretion and inhibited apoptosis. These results suggest that the model for deregulation and hyperactivation of Cdk5 in neurodegeneration may apply to the pathology seen in type 2 diabetes (T2DM). It is consistent with the view that Alzheimer's disease and T2DM are linked metabolically and pathologically by Cdk5 in a number of ways. Copyright © 2010. Published by Elsevier B.V.

Expression of the lef5 gene from Spodoptera exigua multiple nucleopolyhedrovirus contributes to the baculovirus stability in cell culture.

PubMed

Martínez-Solís, María; Jakubowska, Agata K; Herrero, Salvador

2017-10-01

Baculoviruses are a broad group of viruses infecting insects, predominately of the order Lepidoptera. They are used worldwide as biological insecticides and as expression vectors to produce recombinant proteins. Baculoviruses replicate in their host, although several cell lines have been developed for in vitro replication. Nevertheless, replication of baculoviruses in cell culture involves the generation of defective viruses with a decrease in productivity and virulence. Transcriptional studies of the Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) and the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) infective process revealed differences in the expression patterns when the virus replicated under in vitro (Se301 cells) or in vivo (S. exigua larvae) conditions. The late expression factor 5 (lef5) gene was found to be highly overexpressed when the virus replicates in larvae. To test the possible role of lef5 expression in viral stability, recombinant AcMNPV expressing the lef5 gene from SeMNPV (Se-lef5) was generated and its stability was monitored during successive infection passages in Sf21 cells by evaluating the loss of several essential and non-essential genes. The gfp transgene was more stable in those viruses expressing the Se-LEF5 protein and the GFP-defective viruses were accumulated at a lower level when compared to its control viruses, confirming the positive influence of lef5 in viral stability during the multiplication process. This work describes for the first time a viral factor involved in transgene stability when baculoviruses replicate in cell culture, opening new ways to facilitate the in vitro production of recombinant proteins using baculovirus.

Characterization of 5-HT₁A receptors and their complexes with G-proteins in budded baculovirus particles using fluorescence anisotropy of Bodipy-FL-NAN-190.

PubMed

Tõntson, Lauri; Kopanchuk, Sergei; Rinken, Ago

2014-02-01

Bodipy-FL-NAN-190 was found to be well suited for characterization of ligand binding to 5-HT1A receptors expressed in budded baculovirus particles, as binding is accompanied by large increases in fluorescence intensity and anisotropy. This ligand appears to bind rapidly (t1/2,ass<1 min), reversibly (t1/2,diss∼6 min) and has high affinity (Kd=0.30 ± 0.13 nM). This fluorescence anisotropy assay based on Bodipy-FL-NAN-190 binding to baculovirus particles was also a suitable assay system for the pharmacological characterization of non-labelled serotonergic ligands, as well as being sensitive to the presence of G-proteins and guanine nucleotides. Coexpression of αi subunits of human G-proteins in baculovirus particles resulted in the appearance of significantly greater proportion of nucleotide sensitive high affinity agonist binding sites. There were no significant differences between αi1 and αi3 subtypes, while ligand binding in the presence of αi2 had higher sensitivity to GDP and Mn(2+). Copyright © 2014 Elsevier Ltd. All rights reserved.

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

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

Combining stable insect cell lines with baculovirus-mediated expression for multi-HA influenza VLP production.

PubMed

Sequeira, Daniela P; Correia, Ricardo; Carrondo, Manuel J T; Roldão, António; Teixeira, Ana P; Alves, Paula M

2018-05-24

Safer and broadly protective vaccines are needed to cope with the continuous evolution of circulating influenza virus strains and promising approaches based on the expression of multiple hemagglutinins (HA) in a virus-like particle (VLP) have been proposed. However, expression of multiple genes in the same vector can lead to its instability due to tandem repetition of similar sequences. By combining stable with transient expression systems we can rationally distribute the number of genes to be expressed per platform and thus mitigate this risk. In this work, we developed a modular system comprising stable and baculovirus-mediated expression in insect cells for production of multi-HA influenza enveloped VLPs. First, a stable insect High Five cell population expressing two different HA proteins from subtype H3 was established. Infection of this cell population with a baculovirus vector encoding three other HA proteins from H3 subtype proved to be as competitive as traditional co-infection approaches in producing a pentavalent H3 VLP. Aiming at increasing HA expression, the stable insect cell population was infected at increasingly higher cell concentrations (CCI). However, cultures infected at CCI of 3×10 6 cells/mL showed lower HA titers per cell in comparison to standard CCI of 2×10 6 cells/mL, a phenomenon named "cell density effect". To lessen the negative impact of this phenomenon, a tailor-made refeed strategy was designed based on the exhaustion of key nutrients during cell growth. Noteworthy, cultures supplemented and infected at a CCI of 4×10 6 cells/mL showed comparable HA titers per cell to those of CCI of 2×10 6 cells/mL, thus leading to an increase of up to 4-fold in HA titers per mL. Scalability of the modular strategy herein proposed was successfully demonstrated in 2L stirred tank bioreactors with comparable HA protein levels observed between bioreactor and shake flasks cultures. Overall, this work demonstrates the suitability of combining stable

MEMBRANOUS LABYRINTH IN BACULOVIRUS-INFECTED CRUSTRACEAN CELLS: POSSIBLE ROLES IN VIRAL REPRODUCTION

EPA Science Inventory

The origins and morphogenesis of the membranous labyrinth (ML) in Baculovirus penaei (BP) infected cells of penaeid shrimps (Crustacea:Decapoda) are described. t is hypothesized that, because of the close parallel and concurrent development of the ML and virus reproduction, and o...

A nanobiohybrid complex of recombinant baculovirus and Tat/DNA nanoparticles for delivery of Ang-1 transgene in myocardial infarction therapy.

PubMed

Paul, Arghya; Binsalamah, Zyad M; Khan, Afshan A; Abbasia, Sana; Elias, Cynthia B; Shum-Tim, Dominique; Prakash, Satya

2011-11-01

The study aims to design a new gene delivery method utilizing the complementary strengths of baculovirus, such as relatively high transduction efficiency and easy scale-up, and non-viral nanodelivery systems, such as low immunogenicity. This formulation was developed by generating a self assembled binary complex of negatively charged baculovirus (Bac) and positively charged endosomolytic histidine rich Tat peptide/DNA nanoparticles (NP). The synergistic effect of this hybrid (Bac-NP) system to induce myocardial angiogenesis in acute myocardial infarction (AMI) model has been explored in this study, using Angiopoietin-1 (Ang-1) as the transgene carried by both vector components. Under optimal transduction conditions, Bac-NP(Ang1) showed 1.75 times higher and sustained Ang-1 expression in cardiomyocytes than Bac(Ang1), with significantly high angiogenic potential as confirmed by functional assays. For in vivo analysis, we intramyocardially delivered Bac-NP(Ang1) to AMI rat model. 3 weeks post AMI, data showed increase in capillary density (p < 0.01) and reduction in infarct sizes (p < 0.05) in Bac-NP(Ang1) compared to Bac(Ang1), NP(Ang1) and control groups due to enhanced myocardial Ang-1 expression at peri-infarct regions (1.65 times higher than Bac(Ang1)). Furthermore, the Bac-NP(Ang1) group showed significantly higher cardiac performance in echocardiography than Bac(Ang1) (44.2 ± 4.77% vs 37.46 ± 5.2%, p < 0.01), NP(Ang1) and the control group (32.26 ± 2.49% and 31.58 ± 2.26%). Collectively, this data demonstrates hybrid Bac-NP as a new and improved gene delivery system for therapeutic applications. Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.

Baculovirus AC102 Is a Nucleocapsid Protein That Is Crucial for Nuclear Actin Polymerization and Nucleocapsid Morphogenesis.

PubMed

Hepp, Susan E; Borgo, Gina M; Ticau, Simina; Ohkawa, Taro; Welch, Matthew D

2018-06-01

The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the type species of alphabaculoviruses, is an enveloped DNA virus that infects lepidopteran insects and is commonly known as a vector for protein expression and cell transduction. AcMNPV belongs to a diverse group of viral and bacterial pathogens that target the host cell actin cytoskeleton during infection. AcMNPV is unusual, however, in that it absolutely requires actin translocation into the nucleus early in infection and actin polymerization within the nucleus late in infection coincident with viral replication. Of the six viral factors that are sufficient, when coexpressed, to induce the nuclear localization of actin, only AC102 is essential for viral replication and the nuclear accumulation of actin. We therefore sought to better understand the role of AC102 in actin mobilization in the nucleus early and late in infection. Although AC102 was proposed to function early in infection, we found that AC102 is predominantly expressed as a late protein. In addition, we observed that AC102 is required for F-actin assembly in the nucleus during late infection, as well as for proper formation of viral replication structures and nucleocapsid morphogenesis. Finally, we found that AC102 is a nucleocapsid protein and a newly recognized member of a complex consisting of the viral proteins EC27, C42, and the actin polymerization protein P78/83. Taken together, our findings suggest that AC102 is necessary for nucleocapsid morphogenesis and actin assembly during late infection through its role as a component of the P78/83-C42-EC27-AC102 protein complex. IMPORTANCE The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is an important biotechnological tool for protein expression and cell transduction, and related nucleopolyhedroviruses are also used as environmentally benign insecticides. One impact of our work is to better understand the fundamental mechanisms through which Ac

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.

The silencing suppressor (NSs) protein of the plant virus Tomato spotted wilt virus enhances heterologous protein expression and baculovirus pathogenicity in cells and lepidopteran insects.

PubMed

de Oliveira, Virgínia Carla; da Silva Morgado, Fabricio; Ardisson-Araújo, Daniel Mendes Pereira; Resende, Renato Oliveira; Ribeiro, Bergmann Morais

2015-11-01

In this work, we showed that cell death induced by a recombinant (vAcNSs) Autographa californica multiple nucleopolyhedrovirus (AcMNPV) expressing the silencing suppressor (NSs) protein of Tomato spotted wilt virus (TSWV) was enhanced on permissive and semipermissive cell lines. The expression of a heterologous gene (firefly luciferase) during co-infection of insect cells with vAcNSs and a second recombinant baculovirus (vAgppolhfluc) was shown to increase when compared to single vAgppolhfluc infections. Furthermore, the vAcNSs mean time-to-death values were significantly lower than those for wild-type AcMNPV on larvae of Spodoptera frugiperda and Anticarsia gemmatalis. These results showed that the TSWV-NSs protein could efficiently increase heterologous protein expression in insect cells as well as baculovirus pathogenicity and virulence, probably by suppressing the gene-silencing machinery in insects.

A baculovirus dual expression vector derived from the Autographa californica nuclear polyhedrosis virus polyhedrin and p10 promoters: co-expression of two influenza virus genes in insect cells.

PubMed

Weyer, U; Possee, R D

1991-12-01

A baculovirus transfer vector, pAcUW3, was developed to facilitate the insertion of two influenza virus genes, those encoding the haemagglutinin (HA) and neuraminidase (NA) membrane glycoproteins, into the Autographa californica nuclear polyhedrosis virus genome in a single cotransfection experiment. The NA gene was inserted in place of the polyhedrin coding sequences under the control of the polyhedrin promoter, whereas the HA gene was placed under the control of a copy of the p10 promoter at a site upstream of and in opposite orientation to the polyhedrin promoter. After infection of Spodoptera frugiperda cells with the recombinant virus, AcUW3HANA, both HA and NA were expressed in the very late phase of infection and were shown to be functional in appropriate assays. Immunofluorescence assays demonstrated their localization at the surface of infected insect cells. The expression of both foreign genes in the recombinant virus was found to be stable for at least 12 passages in cell culture.

The p40 Subunit of Interleukin (IL)-12 Promotes Stabilization and Export of the p35 Subunit

PubMed Central

Jalah, Rashmi; Rosati, Margherita; Ganneru, Brunda; Pilkington, Guy R.; Valentin, Antonio; Kulkarni, Viraj; Bergamaschi, Cristina; Chowdhury, Bhabadeb; Zhang, Gen-Mu; Beach, Rachel Kelly; Alicea, Candido; Broderick, Kate E.; Sardesai, Niranjan Y.; Pavlakis, George N.; Felber, Barbara K.

2013-01-01

IL-12 is a 70-kDa heterodimeric cytokine composed of the p35 and p40 subunits. To maximize cytokine production from plasmid DNA, molecular steps controlling IL-12p70 biosynthesis at the posttranscriptional and posttranslational levels were investigated. We show that the combination of RNA/codon-optimized gene sequences and fine-tuning of the relative expression levels of the two subunits within a cell resulted in increased production of the IL-12p70 heterodimer. We found that the p40 subunit plays a critical role in enhancing the stability, intracellular trafficking, and export of the p35 subunit. This posttranslational regulation mediated by the p40 subunit is conserved in mammals. Based on these findings, dual gene expression vectors were generated, producing an optimal ratio of the two subunits, resulting in a ∼1 log increase in human, rhesus, and murine IL-12p70 production compared with vectors expressing the wild type sequences. Such optimized DNA plasmids also produced significantly higher levels of systemic bioactive IL-12 upon in vivo DNA delivery in mice compared with plasmids expressing the wild type sequences. A single therapeutic injection of an optimized murine IL-12 DNA plasmid showed significantly more potent control of tumor development in the B16 melanoma cancer model in mice. Therefore, the improved IL-12p70 DNA vectors have promising potential for in vivo use as molecular vaccine adjuvants and in cancer immunotherapy. PMID:23297419

Angiopoietin-1-expressing adipose stem cells genetically modified with baculovirus nanocomplex: investigation in rat heart with acute infarction.

PubMed

Paul, Arghya; Nayan, Madhur; Khan, Afshan Afsar; Shum-Tim, Dominique; Prakash, Satya

2012-01-01

The objective of this study was to develop angiopoietin-1 (Ang1)-expressing genetically modified human adipose tissue derived stem cells (hASCs) for myocardial therapy. For this, an efficient gene delivery system using recombinant baculovirus complexed with cell penetrating transactivating transcriptional activator TAT peptide/deoxyribonucleic acid nanoparticles (Bac-NP), through ionic interactions, was used. It was hypothesized that the hybrid Bac- NP(Ang1) system can efficiently transduce hASCs and induces favorable therapeutic effects when transplanted in vivo. To evaluate this hypothesis, a rat model with acute myocardial infarction and intramyocardially transplanted Ang1-expressing hASCs (hASC-Ang1), genetically modified by Bac-NP(Ang1), was used. Ang1 is a crucial pro-angiogenic factor for vascular maturation and neovasculogenesis. The released hAng1 from hASC-Ang1 demonstrated profound mitotic and anti-apoptotic activities on endothelial cells and cardiomyocytes. The transplanted hASC-Ang1 group showed higher cell retention compared to hASC and control groups. A significant increase in capillary density and reduction in infarct sizes were noted in the infarcted hearts with hASC-Ang1 treatment compared to infarcted hearts treated with hASC or the untreated group. Furthermore, the hASC-Ang1 group showed significantly higher cardiac performance in echocardiography (ejection fraction 46.28% ± 6.3%, P < 0.001 versus control, n = 8) than the hASC group (36.35% ± 5.7%, P < 0.01, n = 8), 28 days post-infarction. The study identified Bac-NP complex as an advanced gene delivery vehicle for stem cells and demonstrated its potential to treat ischemic heart disease with high therapeutic index for combined stem cell-gene therapy strategy.

Effect of different baculovirus inactivation procedures on the integrity and immunogenicity of porcine parvovirus-like particles.

PubMed

Rueda, P; Fominaya, J; Langeveld, J P; Bruschke, C; Vela, C; Casal, J I

2000-11-22

We have demonstrated earlier the usefulness of recombinant porcine parvovirus (PPV) virus-like particles (VLPs) as an efficient recombinant vaccine for PPV. Here, we have demonstrated that preparations of PPV VLPs could be contaminated by recombinant baculoviruses. Since these baculoviruses can be a problem for the registration and safety requirements of the recombinant vaccine, we have tested different baculovirus inactivation strategies, studying simultaneously the integrity and immunogenicity of the VLPs. These methods were pasteurization, treatment with detergents and alkylation with binary ethylenimine (BEI). The structural and functional integrity of the PPV VLPs after the inactivation treatments were analyzed by electron microscopy, hemagglutination, double antibody sandwich (DAS)-ELISA and immunogenicity studies. Binary ethylenimine and Triton X-100 inactivated particles maintained all the original structural and antigenic properties. In addition, PPV VLPs were subjected to size-exclusion chromatography to analyze the presence of VP2 monomers or any other contaminant. The resulting highly purified material was used as the standard of reference to quantify PPV VLPs in order to determine the dose of vaccine by DAS-ELISA. After immunization experiments in guinea pigs, the antibody titers obtained with all the inactivation procedures were very similar. Triton X-100 treatment was selected for further testing in animals because of the speed, simplicity and safety of the overall procedure.

Reduction of the infectivity of baculovirus stocks frozen at ultra-low temperature in serum-free media: The role of lipid emulsions.

PubMed

Eberhardt, Ignacio; Gioria, Verónica Viviana; Micheloud, Gabriela Analía; Claus, Juan Daniel

2016-11-01

The infectivity of stocks of baculoviruses produced in serum-free media is sensitive to freezing at ultra-low temperatures. The objective of this work was to elucidate the causes of such sensitivity, using as a model the freezing of stocks of Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV), a baculovirus widely employed as biological insecticide. Titers of supernatants of cell cultures infected with AgMNPV in four different serum-free media supplemented with lipid emulsions were reduced by 50 to 90% after six months freezing. By using a full factorial experiment, freezing and lipid emulsion, as well as the interaction between them, were identified as the main factors reducing the viral titer. The virucidal effect of the lipid emulsion was reproduced by one of their components, the surfactant Polysorbate 80. Damaged viral envelopes were observed by transmission electron microscopy in most particles frozen in a medium supplemented with lipid emulsion or Polysorbate 80. Additionally, Polysorbate 80 also affected the infectivity of AgMNPV stocks that were incubated at 27°C. The identification of the roles played by the lipid emulsion and Polysorbate 80 is not only a contribution to the understanding of the mechanisms underlying the inactivation of baculovirus stocks produced in serum-free media during storage at ultra-low temperature, but is also an input for the rational development of new procedures aimed at improving both the preservation of baculovirus stocks and the composition of culture media for the production of baculovirus-based bioproducts in insect cells. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1559-1569, 2016. © 2016 American Institute of Chemical Engineers.

TiF(4) and NaF at pH 1.2 but not at pH 3.5 are able to reduce dentin erosion.

PubMed

Wiegand, Annette; Magalhães, Ana Carolina; Sener, Beatrice; Waldheim, Elena; Attin, Thomas

2009-08-01

This study aimed to analyse and compare the protective effect of buffered (pH 3.5) and native (pH 1.2) TiF(4) in comparison to NaF solutions of same pH on dentin erosion. Bovine samples were pretreated with 1.50% TiF(4) or 2.02% NaF (both 0.48M F) solutions, each with a pH of 1.2 and 3.5. The control group received no fluoride pretreatment. Ten samples in each group were eroded with HCl (pH 2.6) for 10x60s. Erosion was analysed by determination of calcium release into the acid. Additionally, the surface and the elemental surface composition were examined by scanning electron microscopy (two samples in each group) and X-ray energy-dispersive spectroscopy in fluoridated but not eroded samples (six samples in each group). Cumulative calcium release (nmol/mm(2)) was statistically analysed by repeated measures ANOVA and one-way ANOVA at t=10min. TiF(4) and NaF at pH 1.2 decreased calcium release significantly, while TiF(4) and NaF at pH 3.5 were not effective. Samples treated with TiF(4) at pH 1.2 showed a significant increase of Ti, while NaF pretreatment increased F concentration significantly. TiF(4) at pH 1.2 led to the formation of globular precipitates occluding dentinal tubules, which could not be observed on samples treated with TiF(4) at pH 3.5. NaF at pH 1.2 but not at pH 3.5 induced the formation of surface precipitates covering dentinal tubules. Dentin erosion can be significantly reduced by TiF(4) and NaF at pH 1.2, but not at pH 3.5.

THE EFFECT OF BACULOVIRUS INFECTION ON ECDYSTEROID TITER IN GYPSY MOTH LARVAE (LYMANTRIA DISPAR).

EPA Science Inventory

Insect baculovirus carries a gene refered to as egt. This gene encodes an enzyme known as ecdysteroid UDP-glucosyl transferase which catalyzes the sugar conjugation of ecdysteroids. Using a gypsy moth embryonic cell line EGT activity of Lymantria dispar nuclear polyhedrosis virus...

Enzyme-linked immunosorbent assay using a recombinant baculovirus-expressed Bacillus anthracis protective antigen (PA): measurement of human anti-PA antibodies.

PubMed Central

Iacono-Connors, L C; Novak, J; Rossi, C; Mangiafico, J; Ksiazek, T

1994-01-01

We developed an antigen capture enzyme-linked immunosorbent assay (ELISA) which does not require purified protective antigen (PA) for detection of human antibodies to Bacillus anthracis PA. Lysates of Spodoptera frugiperda (Sf-9) cells infected with recombinant baculovirus containing the PA gene were used as the source of PA to develop the ELISA. Recombinant PA from crude Sf-9 cell lysates or PA purified from B. anthracis Sterne strain was captured by an anti-PA monoclonal antibody coated onto microtiter plates. We demonstrated that human serum antibody titers to PA were identical in the ELISA whether we used crude Sf-9 cell lysates containing recombinant baculovirus-expressed PA or purified Sterne PA. Finally, false-positive results observed in a direct ELISA were eliminated with this antigen capture ELISA. Thus, the antigen capture ELISA with crude preparations of baculovirus-expressed PA is reliable, safe, and inexpensive for determining anti-PA antibody levels in human sera. PMID:7496927

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.

Physical mapping of the genomic DNA of the Oryctes rhinoceros baculovirus, KI.

PubMed

Mohan, K S; Gopinathan, K P

1991-11-15

A non-occluded baculovirus, OBV-KI has been isolated from the insect pest, Oryctes rhinoceros. The viral genome is estimated to be 123 kb, with a G + C content of 43 mol% and no detectible methylated bases. A restriction map of the OBV-KI genome for BamHI, EcoRI, HindIII, PstI, SalI and XbaI has been constructed.

Contributions of immune responses to developmental resistance in Lymantria dispar challenged with baculovirus

Treesearch

James McNeil; Diana Cox-Foster; James Slavicek; Kelli Hoover

2010-01-01

How the innate immune system functions to defend insects from viruses is an emerging field of study. We examined the impact of melanized encapsulation, a component of innate immunity that integrates both cellular and humoral immune responses, on the success of the baculovirus Lymantria dispar multiple nucleocapsid nucleopolyhedrovirus (LdMNPV) in its...

Immune responses to baculovirus-displayed enterovirus 71 VP1 antigen.

PubMed

Kiener, Tanja K; Premanand, Balraj; Kwang, Jimmy

2013-04-01

The increased distribution and neurovirulence of enterovirus 71 is an important health threat for young children in Asia Pacific. Vaccine design has concentrated on inactivated virus with the most advanced undergoing Phase III clinical trials. By using a subunit vaccine approach, production costs could be reduced by lowering the need for biocontainment. In addition, novel mutations could be rapidly incorporated to reflect the emergence of new enterovirus 71 subgenogroups. To circumvent the problems associated with conventional subunit vaccines, the antigen can be displayed on a viral vector that conveys stability and facilitates purification. Additional advantages of viral-vectored subunit vaccines are their ability to stimulate the innate immune system by transducing cells and the possibility of oral or nasal delivery, which dispenses with the need for syringes and medical personnel. Baculovirus-displayed VP1 combines all these benefits with protection that is as efficient as inactivated virus.

The xeroderma pigmentosum group B protein ERCC3 produced in the baculovirus system exhibits DNA helicase activity.

PubMed Central

Ma, L; Siemssen, E D; Noteborn, H M; van der Eb, A J

1994-01-01

The XPB/ERCC3 gene corrects the nucleotide excision-repair defect in the human hereditary disease xeroderma pigmentosum group B and encodes the largest subunit of the basal transcription factor BTF2/TFIIH. The primary sequence of the XPB/ERCC3 protein features the hallmarks of seven helicase motifs found in many known and putative helicases or helicase-related proteins. Recently, the multiprotein BTF2/TFIIH complex has been found to be associated with DNA helicase activity. To explore the properties and functions of XPB/ERCC3, we have used the baculovirus/insect-cell expression system to produce recombinant protein. We report here the construction and analysis of recombinant baculovirus expressing XPB/ERCC3. The XPB/ERCC3 protein is synthesized at a relatively high level in baculovirus-infected insect cells. While the majority of XPB/ERCC3 end up in the insoluble fraction of insect cell lysates, a minor fraction of recombinant protein is present in soluble form which can be purified under native conditions. We have found that a DNA helicase activity is associated with the purified XPB/ERCC3 protein, suggesting that XPB/ERCC3 may function as a DNA helicase in local unwinding of DNA template both in the context of transcription and nucleotide excision repair. Images PMID:7937133

Protein Expression Profiles of Permissive, Semi-Permissive and Non-Permissive Cells Infected by Baculovirus

USDA-ARS?s Scientific Manuscript database

Amassing information on the in vitro protein expression of an insect host challenged by an entomopathogenic agent, such as a baculovirus, is paramount to an enhanced understanding of how host-pathogen interactions determine the success or failure of a pathogen. In this study, 2D-gel electrophoresis...

Characterization of a baculovirus lacking the DBP (DNA-binding protein) gene

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

Vanarsdall, Adam L.; Mikhailov, Victor S.; N.K. Koltzov Institute of Developmental Biology, Russian Academy of Sciences, Moscow 117808

2007-08-01

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) encodes two proteins that possess properties typical of single-stranded DNA-binding proteins (SSBs), late expression factor-3 (LEF-3), and a protein referred to as DNA-binding protein (DBP). Whereas LEF-3 is a multi-functional protein essential for viral DNA replication, transporting helicase into the nucleus, and forms a stable complex with the baculovirus alkaline nuclease, the role for DBP in baculovirus replication remains unclear. Therefore, to better understand the functional role of DBP in viral replication, a DBP knockout virus was generated from an AcMNPV bacmid and analyzed. The results of a growth curve analysis indicated that the dbpmore » knockout construct was unable to produce budded virus indicating that dbp is essential. The lack of DBP does not cause a general shutdown of the expression of viral genes, as was revealed by accumulation of early (LEF-3), late (VP39), and very late (P10) proteins in cells transfected with the dbp knockout construct. To investigate the role of DBP in DNA replication, a real-time PCR-based assay was employed and showed that, although viral DNA synthesis occurred in cells transfected with the dbp knockout, the levels were less than that of the control virus suggesting that DBP is required for normal levels of DNA synthesis or for stability of nascent viral DNA. In addition, analysis of the viral DNA replicated by the dbp knockout by using field inversion gel electrophoresis failed to detect the presence of genome-length DNA. Furthermore, analysis of DBP from infected cells indicated that similar to LEF-3, DBP was tightly bound to viral chromatin. Assessment of the cellular localization of DBP relative to replicated viral DNA by immunoelectron microscopy indicated that, at 24 h post-infection, DBP co-localized with nascent DNA at distinct electron-dense regions within the nucleus. Finally, immunoelectron microscopic analysis of cells transfected with the dbp

Baculovirus IE2 Stimulates the Expression of Heat Shock Proteins in Insect and Mammalian Cells to Facilitate Its Proper Functioning.

PubMed

Tung, Hsuan; Wei, Sung-Chan; Lo, Huei-Ru; Chao, Yu-Chan

2016-01-01

Baculoviruses have gained popularity as pest control agents and for protein production in insect systems. These viruses are also becoming popular for gene expression, tissue engineering and gene therapy in mammalian systems. Baculovirus infection triggers a heat shock response, and this response is crucial for its successful infection of host insect cells. However, the viral protein(s) or factor(s) that trigger this response are not yet clear. Previously, we revealed that IE2-an early gene product of the baculovirus-could form unique nuclear bodies for the strong trans-activation of various promoters in mammalian cells. Here, we purified IE2 nuclear bodies from Vero E6 cells and investigated the associated proteins by using mass spectrometry. Heat shock proteins (HSPs) were found to be one of the major IE2-associated proteins. Our experiments show that HSPs are greatly induced by IE2 and are crucial for the trans-activation function of IE2. Interestingly, blocking both heat shock protein expression and the proteasome pathway preserved the IE2 protein and its nuclear body structure, and revived its function. These observations reveal that HSPs do not function directly to assist the formation of the nuclear body structure, but may rather protect IE2 from proteasome degradation. Aside from functional studies in mammalian cells, we also show that HSPs were stimulated and required to determine IE2 protein levels, in insect cells infected with baculovirus. Upon inhibiting the expression of heat shock proteins, baculovirus IE2 was substantially suppressed, resulting in a significantly suppressed viral titer. Thus, we demonstrate a unique feature in that IE2 can function in both insect and non-host mammalian cells to stimulate HSPs, which may be associated with IE2 stabilization and lead to the protection of the its strong gene activation function in mammalian cells. On the other hand, during viral infection in insect cells, IE2 could also strongly stimulate HSPs and

A New theraphosid Spider Toxin Causes Early Insect Cell Death by Necrosis When Expressed In Vitro during Recombinant Baculovirus Infection

PubMed Central

Ardisson-Araújo, Daniel Mendes Pereira; Morgado, Fabrício Da Silva; Schwartz, Elisabeth Ferroni; Corzo, Gerardo; Ribeiro, Bergmann Morais

2013-01-01

Baculoviruses are the most studied insect viruses in the world and are used for biological control of agricultural and forest insect pests. They are also used as versatile vectors for expression of heterologous proteins. One of the major problems of their use as biopesticides is their slow speed to kill insects. Thus, to address this shortcoming, insect-specific neurotoxins from arachnids have been introduced into the baculovirus genome solely aiming to improve its virulence. In this work, an insecticide-like toxin gene was obtained from a cDNA derived from the venom glands of the theraphosid spider Brachypelma albiceps. The mature form of the peptide toxin (called Ba3) has a high content of basic amino acid residues, potential for three possible disulfide bonds, and a predicted three-stranded β-sheetDifferent constructions of the gene were engineered for recombinant baculovirus Autographa californica multiple nuclepolyhedrovirus (AcMNPV) expression. Five different forms of Ba3 were assessed; (1) the full-length sequence, (2) the pro-peptide and mature region, (3) only the mature region, and the mature region fused to an (4) insect or a (5) virus-derived signal peptide were inserted separately into the genome of the baculovirus. All the recombinant viruses induced cell death by necrosis earlier in infection relative to a control virus lacking the toxin gene. However, the recombinant virus containing the mature portion of the toxin gene induced a faster cell death than the other recombinants. We found that the toxin construct with the signal peptide and/or pro-peptide regions delayed the necrosis phenotype. When infected cells were subjected to ultrastructural analysis, the cells showed loss of plasma membrane integrity and structural changes in mitochondria before death. Our results suggest this use of baculovirus is a potential tool to help understand or to identify the effect of insect-specific toxic peptides when produced during infection of insect cells. PMID

Increased frequency of gestational and delivery-related complications in women of 35 years of age and above.

PubMed

Bereczky, L-K; Kiss, Sz-L; Szabó, B

2015-02-01

This retrospective study evaluated gestational and delivery-related characteristics focusing on women aged 35 and above (≥ 35 years). Data were collected on maternal (n = 8,407) and newborn records during a 4-year admission period (2008-11) at the County Emergency Hospital, Tîrgu-Mureş, Romania. The prevalence of preterm deliveries increased in all age groups, from 19.5% to 27.8% (p = 0.006) in mothers ≥ 35 years. Twinning rate showed a highly significant increase, being 2.6% in 2008 and 9.5% in 2011 (p = 0.005), while caesarean delivery incidence increased from 46.3% to 51.0% in women aged ≥ 35. Our study revealed a highly significant decrease of mean gestational age and mean fetal weight, as well as a higher incidence of comorbidities and pregnancy-related complications in those aged ≥ 35 years. We assume that comorbidities, maternal and fetal indications to perform caesarean section (CS), in the more mature age group, were a main determinant of the elective or iatrogenic preterm deliveries, which might have contributed to further complications; moreover, previous CSs were likely a promoting factor for further CSs.

Interferon induced protein 35 exacerbates H5N1 influenza disease through the expression of IL-12p40 homodimer.

PubMed

Gounder, Anshu P; Yokoyama, Christine C; Jarjour, Nicholas N; Bricker, Traci L; Edelson, Brian T; Boon, Adrianus C M

2018-04-01

Pro-inflammatory cytokinemia is a hallmark of highly pathogenic H5N1 influenza virus (IAV) disease yet little is known about the role of host proteins in modulating a pathogenic innate immune response. The host Interferon Induced Protein 35 (Ifi35) has been implicated in increased susceptibility to H5N1-IAV infection. Here, we show that Ifi35 deficiency leads to reduced morbidity in mouse models of highly pathogenic H5N1- and pandemic H1N1-IAV infection. Reduced weight loss in Ifi35-/- mice following H5N1-IAV challenge was associated with reduced cellular infiltration and decreased production of specific cytokines and chemokines including IL-12p40. Expression of Ifi35 by the hematopoietic cell compartment in bone-marrow chimeric mice contributed to increased immune cell recruitment and IL-12p40 production. In addition, Ifi35 deficient primary macrophages produce less IL-12p40 following TLR-3, TLR-4, and TLR-7 stimulation in vitro. Decreased levels of IL-12p40 and its homodimer, IL-12p80, were found in bronchoalveolar lavage fluid of H5N1-IAV infected Ifi35 deficient mice. Specific antibody blockade of IL-12p80 ameliorated weight loss and reduced cellular infiltration following H5N1-IAV infection in wild-type mice; suggesting that increased levels of IL-12p80 alters the immune response to promote inflammation and IAV disease. These data establish a role for Ifi35 in modulating cytokine production and exacerbating inflammation during IAV infection.

Phosphoinositide 5-phosphatase Fig 4p is required for both acute rise and subsequent fall in stress-induced phosphatidylinositol 3,5-bisphosphate levels.

PubMed

Duex, Jason E; Nau, Johnathan J; Kauffman, Emily J; Weisman, Lois S

2006-04-01

Phosphoinositide lipids regulate complex events via the recruitment of proteins to a specialized region of the membrane at a specific time. Precise control of both the synthesis and turnover of phosphoinositide lipids is integral to membrane trafficking, signal transduction, and cytoskeletal rearrangements. Little is known about the acute regulation of the levels of these signaling lipids. When Saccharomyces cerevisiae cells are treated with hyperosmotic medium the levels of phosphatidylinositol 3,5-bisphosphate (PI3,5P(2)) increase 20-fold. Here we show that this 20-fold increase is rapid and occurs within 5 min. Surprisingly, these elevated levels are transient. Fifteen minutes following hyperosmotic shock they decrease at a rapid rate, even though the cells remain in hyperosmotic medium. In parallel with the rapid increase in the levels of PI3,5P(2), vacuole volume decreases rapidly. Furthermore, concomitant with a return to basal levels of PI3,5P(2) vacuole volume is restored. We show that Fig 4p, consistent with its proposed role as a PI3,5P(2) 5-phosphatase, is required in vivo for this rapid return to basal levels of PI3,5P(2). Surprisingly, we find that Fig 4p is also required for the hyperosmotic shock-induced increase in PI3,5P(2) levels. These findings demonstrate that following hyperosmotic shock, large, transient changes occur in the levels of PI3,5P(2) and further suggest that Fig 4p is important in regulating both the acute rise and subsequent fall in PI3,5P(2) levels.

Direct studies of low-energy resonances in 31P(p, ) 28Si and 35Cl(p, )32S

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

Moazen, Brian; Matei, Catalin; Bardayan, Daniel W

2011-01-01

Low-energy resonances in 31P(p,a)28Si and 35Cl(p,a)32S were studied directly in order to gain a better understanding of reaction cycling in the Si-Ar region in novae, supernovae, and X-ray bursts. Resonance strengths at Ec:m = 600 and 622 keV in 31P(p,a)28Si were measured as well as the Ec:m: = 611 keV resonance in 35Cl(p,a)32S, the lowest energy that any resonance in this reaction has been observed. The strengths of these resonances were found to be lower than previously determined through indirect methods, resulting in weak cycling in the Si-Ar region.

Roles for PI(3,5)P2 in nutrient sensing through TORC1

PubMed Central

Jin, Natsuko; Mao, Kai; Jin, Yui; Tevzadze, Gela; Kauffman, Emily J.; Park, Sujin; Bridges, Dave; Loewith, Robbie; Saltiel, Alan R.; Klionsky, Daniel J.; Weisman, Lois S.

2014-01-01

TORC1, a conserved protein kinase, regulates cell growth in response to nutrients. Localization of mammalian TORC1 to lysosomes is essential for TORC1 activation. Phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2), an endosomal signaling lipid, is implicated in insulin-dependent stimulation of TORC1 activity in adipocytes. This raises the question of whether PI(3,5)P2 is an essential general regulator of TORC1. Moreover, the subcellular location where PI(3,5)P2 regulates TORC1 was not known. Here we report that PI(3,5)P2 is required for TORC1 activity in yeast and regulates TORC1 on the vacuole (lysosome). Furthermore, we show that the TORC1 substrate, Sch9 (a homologue of mammalian S6K), is recruited to the vacuole by direct interaction with PI(3,5)P2, where it is phosphorylated by TORC1. Of importance, we find that PI(3,5)P2 is required for multiple downstream pathways via TORC1-dependent phosphorylation of additional targets, including Atg13, the modification of which inhibits autophagy, and phosphorylation of Npr1, which releases its inhibitory function and allows nutrient-dependent endocytosis. These findings reveal PI(3,5)P2 as a general regulator of TORC1 and suggest that PI(3,5)P2 provides a platform for TORC1 signaling from lysosomes. PMID:24478451

DNA microarrays of baculovirus genomes: differential expression of viral genes in two susceptible insect cell lines.

PubMed

Yamagishi, J; Isobe, R; Takebuchi, T; Bando, H

2003-03-01

We describe, for the first time, the generation of a viral DNA chip for simultaneous expression measurements of nearly all known open reading frames (ORFs) in the best-studied members of the family Baculoviridae, Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Bombyx mori nucleopolyhedrovirus (BmNPV). In this study, a viral DNA chip (Ac-BmNPV chip) was fabricated and used to characterize the viral gene expression profile for AcMNPV in different cell types. The viral chip is composed of microarrays of viral DNA prepared by robotic deposition of PCR-amplified viral DNA fragments on glass for ORFs in the NPV genome. Viral gene expression was monitored by hybridization to the DNA fragment microarrays with fluorescently labeled cDNAs prepared from infected Spodoptera frugiperda, Sf9 cells and Trichoplusia ni, TnHigh-Five cells, the latter a major producer of baculovirus and recombinant proteins. A comparison of expression profiles of known ORFs in AcMNPV elucidated six genes (ORF150, p10, pk2, and three late gene expression factor genes lef-3, p35 and lef- 6) the expression of each of which was regulated differently in the two cell lines. Most of these genes are known to be closely involved in the viral life cycle such as in DNA replication, late gene expression and the release of polyhedra from infected cells. These results imply that the differential expression of these viral genes accounts for the differences in viral replication between these two cell lines. Thus, these fabricated microarrays of NPV DNA which allow a rapid analysis of gene expression at the viral genome level should greatly speed the functional analysis of large genomes of NPV.

Purification of functional baculovirus particles from silkworm larval hemolymph and their use as nanoparticles for the detection of human prorenin receptor (PRR) binding

PubMed Central

2011-01-01

Background Baculovirus, which has a width of 40 nm and a length of 250-300 nm, can display functional peptides, receptors and antigens on its surface by their fusion with a baculovirus envelop protein, GP64. In addition, some transmembrane proteins can be displayed without GP64 fusion, using the native transmembrane domains of the baculovirus. We used this functionality to display human prorenin receptor fused with GFPuv (GFPuv-hPRR) on the surface of silkworm Bombyx mori nucleopolyhedrovirus (BmNPV) and then tested whether these baculovirus particles could be used to detect protein-protein interactions. Results BmNPV displaying GFPuv-hPRR (BmNPV-GFPuv-hPRR) was purified from hemolymph by using Sephacryl S-1000 column chromatography in the presence of 0.01% Triton X-100. Its recovery was 86% and the final baculovirus particles number was 4.98 × 108 pfu. Based on the results of enzyme-linked immunosorbent assay (ELISA), 3.1% of the total proteins in BmNPV-GFPuv-hPRR were GFPuv-hPRR. This value was similar to that calculated from the result of western blot by a densitometry (2.7%). To determine whether BmNPV-GFPuv-hPRR particles were bound to human prorenin, ELISA results were compared with those from ELISAs using protease negative BmNPV displaying β1,3-N-acetylglucosaminyltransferase 2 fused with the gene encoding GFPuv (GGT2) (BmNPV-CP--GGT2) particles, which do not display hPRR on their surfaces. Conclusion The display of on the surface of the BmNPV particles will be useful for the detection of protein-protein interactions and the screening of inhibitors and drugs in their roles as nanobioparticles. PMID:21635720

Antitumor effects of a tumor cell vaccine expressing a membrane-bound form of the IL-12 p35 subunit.

PubMed

Lim, Ho Yong; Ju, Hee Young; Chung, Hee-Yong; Kim, Young Sang

2010-08-15

We investigated whether expression of the IL-12 p35 subunit in membrane-bound form in tumor cells enhanced their immunogenicity. Since p35 is only secreted when associated with the IL-12 p40 subunit, we generated tumor cells expressing membrane-bound forms of p35 and p40 as chimeras with the transmembrane/cytoplasmic region of TNFα (mbIL-12p35 and mbIL-12p40). The relevant vectors were transfected into MethA fibrosarcoma cells, and mbIL-12p35 or mbIL-12p40-expressing tumor clones were isolated and their ability to induce antitumor immunity studied. Cells of the mbIL-12p35 tumor clone induced CD69 expression and IFNγ production in purified CD8(+) T cells in vitro, and their in vivo tumorigenicity was reduced. Cells of the mbIL-12p40 tumor clone failed to show either of these activities. Mice that had rejected cells of the mbIL-12p35 tumor clone possessed systemic antitumor immunity to wild type tumor cells. The growth rate of mbIL-12p35 tumor cells was greater in CD8(+) T cell-depleted mice than in CD4(+) T-cell- and NK cell-depleted mice or normal mice, suggesting that CD8(+) T cells were mainly responsible for the antitumor immunity. These results indicate that expression of mbIL-12p35 on tumor cells enhances their immunogenicity by increasing their ability to activate CD8(+) T cells, possibly by direct priming.

MicroRNAome of Spodoptera frugiperda cells (Sf9) and its alteration following baculovirus infection.

PubMed

Mehrabadi, Mohammad; Hussain, Mazhar; Asgari, Sassan

2013-06-01

MicroRNAs (miRNAs) as small non-coding RNAs play important roles in many biological processes such as development, cell signalling and immune response. Studies also suggest that miRNAs are important in host-virus interactions where the host limits virus infection by differentially expressing miRNAs that target essential viral genes. Here, we identified conserved and new miRNAs from Spodoptera frugiperda cells (Sf9) using a combination of deep sequencing and bioinformatics as well as experimental approaches. S. frugiperda miRNAs share common features of miRNAs in other organisms, such as uracil (U) at the 5' end of miRNA. The 5' ends of the miRNAs were more conserved than the 3' ends, revealing evolutionary protection of the seed region in miRNAs. The predominant miRNAs were found to be conserved among arthropods. The majority of homologous miRNAs were found in Bombyx mori, with 76 of the 90 identified miRNAs. We found that seed shifting and arm switching have happened in this insect's miRNAs. Expression levels of the majority of miRNAs changed following baculovirus infection. Results revealed that baculovirus infection mainly led to an overall suppression of cellular miRNAs. We found four different genes being regulated by sfr-miR-184 at the post-transcriptional level. The data presented here further support conservation of miRNAs in insects and other organisms. In addition, the results reveal a differential expression of host miRNAs upon baculovirus infection, suggesting their potential roles in host-virus interactions. Seed shifting and arm switching happened during evolution of miRNAs in different insects and caused miRNA diversification, which led to changes in the target repository of miRNAs.

Laboratory and field evaluations for efficacy of a fast-killing baculovirus isolate from Spodoptera frugiperda

USDA-ARS?s Scientific Manuscript database

Three biopesticide parameters were evaluated for a fast-killing isolate (3AP2) Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV) and a wild-type isolate (Sf3) of the same baculovirus. Both isolates were evaluated for virus production using in vivo methods, for speed of kill based on bioas...

Electron Tomography and Simulation of Baculovirus Actin Comet Tails Support a Tethered Filament Model of Pathogen Propulsion

PubMed Central

Mueller, Jan; Pfanzelter, Julia; Winkler, Christoph; Narita, Akihiro; Le Clainche, Christophe; Nemethova, Maria; Carlier, Marie-France; Maeda, Yuichiro; Welch, Matthew D.; Ohkawa, Taro; Schmeiser, Christian; Resch, Guenter P.; Small, J. Victor

2014-01-01

Several pathogens induce propulsive actin comet tails in cells they invade to disseminate their infection. They achieve this by recruiting factors for actin nucleation, the Arp2/3 complex, and polymerization regulators from the host cytoplasm. Owing to limited information on the structural organization of actin comets and in particular the spatial arrangement of filaments engaged in propulsion, the underlying mechanism of pathogen movement is currently speculative and controversial. Using electron tomography we have resolved the three-dimensional architecture of actin comet tails propelling baculovirus, the smallest pathogen yet known to hijack the actin motile machinery. Comet tail geometry was also mimicked in mixtures of virus capsids with purified actin and a minimal inventory of actin regulators. We demonstrate that propulsion is based on the assembly of a fishbone-like array of actin filaments organized in subsets linked by branch junctions, with an average of four filaments pushing the virus at any one time. Using an energy-minimizing function we have simulated the structure of actin comet tails as well as the tracks adopted by baculovirus in infected cells in vivo. The results from the simulations rule out gel squeezing models of propulsion and support those in which actin filaments are continuously tethered during branch nucleation and polymerization. Since Listeria monocytogenes, Shigella flexneri, and Vaccinia virus among other pathogens use the same common toolbox of components as baculovirus to move, we suggest they share the same principles of actin organization and mode of propulsion. PMID:24453943

Electron tomography and simulation of baculovirus actin comet tails support a tethered filament model of pathogen propulsion.

PubMed

Mueller, Jan; Pfanzelter, Julia; Winkler, Christoph; Narita, Akihiro; Le Clainche, Christophe; Nemethova, Maria; Carlier, Marie-France; Maeda, Yuichiro; Welch, Matthew D; Ohkawa, Taro; Schmeiser, Christian; Resch, Guenter P; Small, J Victor

2014-01-01

Several pathogens induce propulsive actin comet tails in cells they invade to disseminate their infection. They achieve this by recruiting factors for actin nucleation, the Arp2/3 complex, and polymerization regulators from the host cytoplasm. Owing to limited information on the structural organization of actin comets and in particular the spatial arrangement of filaments engaged in propulsion, the underlying mechanism of pathogen movement is currently speculative and controversial. Using electron tomography we have resolved the three-dimensional architecture of actin comet tails propelling baculovirus, the smallest pathogen yet known to hijack the actin motile machinery. Comet tail geometry was also mimicked in mixtures of virus capsids with purified actin and a minimal inventory of actin regulators. We demonstrate that propulsion is based on the assembly of a fishbone-like array of actin filaments organized in subsets linked by branch junctions, with an average of four filaments pushing the virus at any one time. Using an energy-minimizing function we have simulated the structure of actin comet tails as well as the tracks adopted by baculovirus in infected cells in vivo. The results from the simulations rule out gel squeezing models of propulsion and support those in which actin filaments are continuously tethered during branch nucleation and polymerization. Since Listeria monocytogenes, Shigella flexneri, and Vaccinia virus among other pathogens use the same common toolbox of components as baculovirus to move, we suggest they share the same principles of actin organization and mode of propulsion.

Acetylcholinesterase of the sand fly, Phlebotomus papatasi (Scopoli): cDNA sequence, baculovirus expression, and biochemical properties

PubMed Central

2013-01-01

Background Millions of people and domestic animals around the world are affected by leishmaniasis, a disease caused by various species of flagellated protozoans in the genus Leishmania that are transmitted by several sand fly species. Insecticides are widely used for sand fly population control to try to reduce or interrupt Leishmania transmission. Zoonotic cutaneous leishmaniasis caused by L. major is vectored mainly by Phlebotomus papatasi (Scopoli) in Asia and Africa. Organophosphates comprise a class of insecticides used for sand fly control, which act through the inhibition of acetylcholinesterase (AChE) in the central nervous system. Point mutations producing an altered, insensitive AChE are a major mechanism of organophosphate resistance in insects and preliminary evidence for organophosphate-insensitive AChE has been reported in sand flies. This report describes the identification of complementary DNA for an AChE in P. papatasi and the biochemical characterization of recombinant P. papatasi AChE. Methods A P. papatasi Israeli strain laboratory colony was utilized to prepare total RNA utilized as template for RT-PCR amplification and sequencing of cDNA encoding acetylcholinesterase 1 using gene specific primers and 3’-5’-RACE. The cDNA was cloned into pBlueBac4.5/V5-His TOPO, and expressed by baculovirus in Sf21 insect cells in serum-free medium. Recombinant P. papatasi acetylcholinesterase was biochemically characterized using a modified Ellman’s assay in microplates. Results A 2309 nucleotide sequence of PpAChE1 cDNA [GenBank: JQ922267] of P. papatasi from a laboratory colony susceptible to insecticides is reported with 73-83% nucleotide identity to acetylcholinesterase mRNA sequences of Culex tritaeniorhynchus and Lutzomyia longipalpis, respectively. The P. papatasi cDNA ORF encoded a 710-amino acid protein [GenBank: AFP20868] exhibiting 85% amino acid identity with acetylcholinesterases of Cx. pipiens, Aedes aegypti, and 92% amino acid identity for

CRISPR-Cas9 vectors for genome editing and host engineering in the baculovirus-insect cell system.

PubMed

Mabashi-Asazuma, Hideaki; Jarvis, Donald L

2017-08-22

The baculovirus-insect cell system (BICS) has been widely used to produce many different recombinant proteins for basic research and is being used to produce several biologics approved for use in human or veterinary medicine. Early BICS were technically complex and constrained by the relatively primordial nature of insect cell protein glycosylation pathways. Since then, recombination has been used to modify baculovirus vectors-which has simplified the system-and transform insect cells, which has enhanced its protein glycosylation capabilities. Now, CRISPR-Cas9 tools for site-specific genome editing are needed to facilitate further improvements in the BICS. Thus, in this study, we used various insect U6 promoters to construct CRISPR-Cas9 vectors and assessed their utility for site-specific genome editing in two insect cell lines commonly used as hosts in the BICS. We demonstrate the use of CRISPR-Cas9 to edit an endogenous insect cell gene and alter protein glycosylation in the BICS.

The baculovirus-integrated retrotransposon TED encodes gag and pol proteins that assemble into viruslike particles with reverse transcriptase.

PubMed Central

Lerch, R A; Friesen, P D

1992-01-01

TED is a lepidopteran retrotransposon found inserted within the DNA genome of the Autographa californica nuclear polyhedrosis virus mutant, FP-D. To examine the proteins and functions encoded by this representative of the gypsy family of retrotransposons, the gag- and pol-like open reading frames (ORFs 1 and 2) were expressed in homologous lepidopteran cells by using recombinant baculovirus vectors. Expression of ORF 1 resulted in synthesis of an abundant TED-specific protein (Pr55gag) that assembled into viruslike particles with a diameter of 55 to 60 nm. Expression of ORF 2, requiring a -1 translational frameshift, resulted in synthesis of a protease that mediated cleavage of Pr55gag to generate p37, the major protein component of the resulting particles. Expression of ORF 2 also produced reverse transcriptase that associated with these particles. Both protease and reverse transcriptase activities mapped to domains within ORF 2 that contain sequence similarities with the corresponding functional domains of the pol gene of the vertebrate retroviruses. These results indicated that TED ORFs 1 and 2 functionally resemble the retrovirus gag and pol genes and demonstrated for the first time that an invertebrate member of the gypsy family of elements encodes active forms of the structural and enzymatic functions necessary for transposition via an RNA intermediate. TED integration within the baculovirus genome thus represents one of the first examples of transposon-mediated transfer of host-derived genes to an eukaryotic virus. Images PMID:1371168

Antigenic evaluation of a recombinant baculovirus-expressed Sarcocystis neurona SAG1 antigen.

PubMed

Gupta, G D; Lakritz, J; Saville, W J; Livingston, R S; Dubey, J P; Middleton, J R; Marsh, A E

2004-10-01

Sarcocystis neurona is the primary parasite associated with equine protozoal myeloencephalitis (EPM). This is a commonly diagnosed neurological disorder in the Americas that infects the central nervous system of horses. Current serologic assays utilize culture-derived parasites as antigen. This method requires large numbers of parasites to be grown in culture, which is labor intensive and time consuming. Also, a culture-derived whole-parasite preparation contains conserved antigens that could cross-react with antibodies against other Sarcocystis species and members of Sarcocystidae such as Neospora spp., Hammondia spp., and Toxoplasma gondii. Therefore, there is a need to develop an improved method for the detection of S. neurona-specific antibodies. The sera of infected horses react strongly to surface antigen 1 (SnSAG1), an approximately 29-kDa protein, in immunoblot analysis, suggesting that it is an immunodominant antigen. The SnSAG1 gene of S. neurona was cloned, and recombinant S. neurona SAG1 protein (rSnSAG1-Bac) was expressed with the use of a baculovirus system. By immunoblot analysis, the rSnSAG1-Bac antigen detected antibodies to S. neurona from naturally infected and experimentally inoculated equids, cats, rabbit, mice, and skunk. This is the first report of a baculovirus-expressed recombinant S. neurona antigen being used to detect anti-S. neurona antibodies in a variety of host species.

Zika Virus Baculovirus-Expressed Virus-Like Particles Induce Neutralizing Antibodies in Mice.

PubMed

Dai, Shiyu; Zhang, Tao; Zhang, Yanfang; Wang, Hualin; Deng, Fei

2018-06-01

The newly emerged mosquito-borne Zika virus (ZIKV) strains pose a global challenge owing to its ability to cause microcephaly and neurological disorders. Several ZIKV vaccine candidates have been proposed, including inactivated and live attenuated virus vaccines, vector-based vaccines, DNA and RNA vaccines. These have been shown to be efficacious in preclinical studies in mice and nonhuman primates, but their use will potentially be a threat to immunocompromised individuals and pregnant women. Virus-like particles (VLPs) are empty particles composed merely of viral proteins, which can serve as a safe and valuable tool for clinical prevention and treatment strategies. In this study, we used a new strategy to produce ZIKV VLPs based on the baculovirus expression system and demonstrated the feasibility of their use as a vaccine candidate. The pre-membrane (prM) and envelope (E) proteins were co-expressed in insect cells and self-assembled into particles similar to ZIKV. We found that the ZIKV VLPs could be quickly and easily prepared in large quantities using this system. The VLPs were shown to have good immunogenicity in immunized mice, as they stimulated high levels of virus neutralizing antibody titers, ZIKV-specific IgG titers and potent memory T cell responses. Thus, the baculovirus-based ZIKV VLP vaccine is a safe, effective and economical vaccine candidate for use against ZIKV.

CHARACTERIZATION OF THE GLYCOSYLATED ECDYSTEROIDS IN THE HEMOLYMPH OF BACULOVIRUS-INFECTED GYPSY MOTH LARVAE AND CELLS IN CULTURE

EPA Science Inventory

Fourth-instar gypsy moth (Lymantria dispar; Lepidoptera: Lymantriidae) larvae, infected with the gypsy moth baculovirus (LdNPV), show an elevated and prolonged extension of the hemolymph ecdysteroid titer peak associated with molting. The ecdysteroid immunoreactivity associated w...

Method for Salmonella concentration from water at pH 3.5, using micro-fiber glass filters.

PubMed Central

Block, J C; Rolland, D

1979-01-01

A method is described for the concentration of Salmonella from water. As is done with enterovirus, Salmonella bacteria were concentrated from water in two steps: by pH 3.5 adsorption on and pH 9.5 elution from 8-micron porosity micro-fiber glass filter tubes. This method worked in less than 30 min, and Salmonella typhimurium was inactivated only slightly in spite of rapid pH variations (pH 3.5 to 9.5). It was demonstrated that the retention by the filters stems from two phenomena: a low retention in the micro-fiber glass labyrinth for small filtered volumes, and a high retention by adsorption at pH 3.5 for any filtered volume (experiments done with 15- and 80-liter samples). Addition in tap water of trivalent ions like Al3+ did not increase Salmonella adsorption. In most of the trials, Salmonella recovery varied from 42 to 93%. Preliminary field investigations indicate that enterovirus and Salmonella may both be concentrated from the same water sample by this procedure. PMID:39501

The activity of phenoloxidase in haemolymph plasma is not a predictor of Lymantria dispar resistance to its baculovirus

PubMed Central

Kasianov, Nikita S.; Belousova, Irina A.; Pavlushin, Sergey V.; Dubovskiy, Ivan M.; Podgwaite, John D.; Bakhvalov, Stanislav A.

2017-01-01

Host innate immunity is one of the factors that determines the resistance of insects to their entomopathogens. In the research reported here we studied whether or not phenoloxidase (PO), a key enzyme in the melanogenesis component of humoral immunity of insects, plays a role in the protection of Lymantria dispar larvae from infection by L. dispar multiple nucleopolyhedrovirus. We studied two types of viral infection: overt and covert. The following lines of investigation were tested: i) the intravital individual estimation of baseline PO activity in haemolymph plasma followed by virus challenging; ii) the specific inhibition of PO activity in vivo by peroral treatment of infected larvae with phenylthiourea (PTU), a competitive inhibitor of PO; iii) the evaluation of PO activity in the haemolymph plasma after larval starvation. Starvation is a stress that activates the covert infection to an overt form. All of these experiments did not show a relationship between PO activity in haemolymph plasma of L. dispar larvae and larval susceptibility to baculovirus. Moreover, starvation-induced activation of covert viral infection to an overt form occurred in 70 percent of virus-carrying larvae against the background of a dramatic increase of PO activity in haemolymph plasma in the insects studied. Our conclusion is that in L. dispar larvae PO activity is not a predictor of host resistance to baculovirus. PMID:28854240

Characterization of an Sf-rhabdovirus-negative Spodoptera frugiperda cell line as an alternative host for recombinant protein production in the baculovirus-insect cell system.

PubMed

Maghodia, Ajay B; Geisler, Christoph; Jarvis, Donald L

2016-06-01

Cell lines derived from the fall armyworm, Spodoptera frugiperda (Sf), are widely used as hosts for recombinant protein production in the baculovirus-insect cell system (BICS). However, it was recently discovered that these cell lines are contaminated with a virus, now known as Sf-rhabdovirus [1]. The detection of this adventitious agent raised a potential safety issue that could adversely impact the BICS as a commercial recombinant protein production platform. Thus, we examined the properties of Sf-RVN, an Sf-rhabdovirus-negative Sf cell line, as a potential alternative host. Nested RT-PCR assays showed Sf-RVN cells had no detectable Sf-rhabdovirus over the course of 60 passages in continuous culture. The general properties of Sf-RVN cells, including their average growth rates, diameters, morphologies, and viabilities after baculovirus infection, were virtually identical to those of Sf9 cells. Baculovirus-infected Sf-RVN and Sf9 cells produced equivalent levels of three recombinant proteins, including an intracellular prokaryotic protein and two secreted eukaryotic glycoproteins, and provided similar N-glycosylation patterns. In fact, except for the absence of Sf-rhabdovirus, the only difference between Sf-RVN and Sf9 cells was SF-RVN produced higher levels of infectious baculovirus progeny. These results show Sf-RVN cells can be used as improved, alternative hosts to circumvent the potential safety hazard associated with the use of Sf-rhabdovirus-contaminated Sf cells for recombinant protein manufacturing with the BICS. Copyright © 2016 Elsevier Inc. All rights reserved.

Evolution in Oryctes baculovirus: rate and types of genomic change.

PubMed

Crawford, A M; Zelazny, B

1990-01-01

Three cloned strains of Oryctes baculovirus were released into a previously unexposed population of the host insect, the coconut palm rhinoceros beetle, Oryctes rhinoceros. The experiment was conducted on Meemu Atoll in the Maldive Islands. Viruses were isolated from the beetle population at 1 year, 1.75 years, and 4 years after release. No changes in genotype were observed in viruses isolated after 1 and 1.75 years. After 4 years, however, three types of genomic change had occurred. A recombinant derived from two of the released strains, an isolate containing a 100-bp insert, and one example of a point mutation were found in the 22 isolates examined.

PI(3,5)P2 controls endosomal branched actin dynamics by regulating cortactin–actin interactions

PubMed Central

Hong, Nan Hyung; Qi, Aidong

2015-01-01

Branched actin critically contributes to membrane trafficking by regulating membrane curvature, dynamics, fission, and transport. However, how actin dynamics are controlled at membranes is poorly understood. Here, we identify the branched actin regulator cortactin as a direct binding partner of phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) and demonstrate that their interaction promotes turnover of late endosomal actin. In vitro biochemical studies indicated that cortactin binds PI(3,5)P2 via its actin filament-binding region. Furthermore, PI(3,5)P2 competed with actin filaments for binding to cortactin, thereby antagonizing cortactin activity. These findings suggest that PI(3,5)P2 formation on endosomes may remove cortactin from endosome-associated branched actin. Indeed, inhibition of PI(3,5)P2 production led to cortactin accumulation and actin stabilization on Rab7+ endosomes. Conversely, inhibition of Arp2/3 complex activity greatly reduced cortactin localization to late endosomes. Knockdown of cortactin reversed PI(3,5)P2-inhibitor–induced actin accumulation and stabilization on endosomes. These data suggest a model in which PI(3,5)P2 binding removes cortactin from late endosomal branched actin networks and thereby promotes net actin turnover. PMID:26323691

Armored DNA in recombinant Baculoviruses as controls in molecular genetic assays.

PubMed

Freystetter, Andrea; Paar, Christian; Stekel, Herbert; Berg, Jörg

2017-10-01

The widespread use of molecular PCR-based assays in analytical and clinical laboratories brings about the need for test-specific, stable, and reliable external controls (EC) as well as standards and internal amplification controls (IC), in order to arrive at consistent test results. In addition, there is also a growing need to produce and provide stable, well-characterized molecular controls for quality assurance programs. In this study, we describe a novel approach to generate armored double-stranded DNA controls, which are encapsulated in baculovirus (BV) particles of the species Autographa californica multiple nucleopolyhedrovirus. We used the well-known BacPAK™ Baculovirus Expression System (Takara-Clontech), removed the polyhedrin promoter used for protein expression, and generated recombinant BV-armored DNAs. The obtained BV-armored DNAs were readily extracted by standard clinical DNA extraction methods, showed favorable linearity and performance in our clinical PCR assays, were resistant to DNase I digestion, and exhibited marked stability in human plasma and serum. BV-armored DNA ought to be used as ECs, quantification standards, and ICs in molecular assays, with the latter application allowing for the entire monitoring of clinical molecular assays for sample adequacy. BV-armored DNA may also be used to produce double-stranded DNA reference materials for, e.g., quality assurance programs. The ease to produce BV-armored DNA should make this approach feasible for a broad spectrum of molecular applications. Finally, as BV-armored DNAs are non-infectious to mammals, they may be even more conveniently shipped than clinical specimen.

[Demonstration, stabilization and purification of an intracapsid nucleoprotein structure of Baculovirus of Oryctes rhinoceros L].

PubMed

Monsarrat, P; Revet, B; Gourevitch, I

1975-11-10

The presence of a structurally organized nucleoproteic structure in the capsid of the Baculovirus of Oryctes rhinoceros L. is shown. This structure is stabilized under definite conditions described in detail in the paper. It possesses a rope-like structure of about 280 nm in length on 15 nm in diameter containing the DNA molecule. A basic protein is found in the virus.

Isolation of a baculovirus variant that exhibits enhanced polyhedra production stability during serial passage in cell culture

Treesearch

James M. Slavicek; Melissa J. Mercer; Mary Ellen Kelly; Nancy Hayes-Plazolles

1996-01-01

The formation of few polyhedra mutants during serial propagation of baculoviruses in cell culture encumbers commercial scale production in this system. A Lymantria dispar nuclear polyhedrosis virus (LdMNPV) variant (isolate A21-MPV) has been isolated and the traits of budded virus (BV) production, synthesis of polyhedra, the...

P35 and P22 Toxoplasma gondii antigens abbreviate regions to diagnose acquired toxoplasmosis during pregnancy: toward single-sample assays.

PubMed

Costa, Juan G; Peretti, Leandro E; García, Valeria S; Peverengo, Luz; González, Verónica D G; Gugliotta, Luis M; Dalla Fontana, Maria L; Lagier, Claudia M; Marcipar, Iván S

2017-03-01

P35 and P22 Toxoplasma gondii proteins are recognized by specific IgG at the early infection stage, making them ideal for acute toxoplasmosis pregnancy control. Both proteins have been studied to discriminate between acute and chronic toxoplasmosis. However, results were hardly comparable because different protein obtainment procedures led to different antigens, the reference panels used were not optimally typified, and avidity tests were either not performed or narrowly examined. We bioinformatically predicted P35 and P22 regions with the highest density of epitopes, and expressed them in pET32/BL21DE3 alternative expression system, obtaining the soluble proteins rP35a and rP22a. We assessed their diagnostic performance using pregnant woman serum samples typified as: not infected, NI (IgG-, IgM-), typical-chronic, TC (IgM-, IgG+), presumably acute, A (IgG+, IgM+, low-avidity IgG), and recently chronic, RC (IgG+, IgM+, high-avidity IgG). rP35a performed better than rP22a to differentiate A from RC, the areas under the curve (AUC) being 0.911 and 0.818, respectively. They, however, performed similarly to differentiate A from TC+RC (AUC: 0.915 and 0.907, respectively). rP35a and rP22a evaluation by avidity ELISA to discriminate A from RC rendered AUC values of 0.974 and 0.921, respectively. The indirect ELISA and avidity ELISA results analyzed in tandem were consistent with those obtained using commercial kits. rP35a and rP22a features suggest that, with complementary use, they could replace parasite lysate for toxoplasmosis infection screening and for acute toxoplasmosis diagnosis. Our proposal should be validated by a longitudinal study and may lead to a reliable toxoplasmosis pregnancy control, performing tests in only one serum sample.

Induction of an IAP antagonist in Culex quinquefasciatus larvae in response to infection by the baculovirus CuniNPV

USDA-ARS?s Scientific Manuscript database

CuniNPV is a member of the Dipteran–specific baculoviruses in the genus Deltabaculovirus that specifically infects mosquito larvae within the genus Culex while species of Aedes and Anopheles are refractory. Infections are restricted to the nuclei of larval midgut epithelial cells with transmission...

Construction of baculovirus expression vector of miRNAs and its expression in insect cells.

PubMed

Huang, Yong; Zou, Quan; Shen, Xing Jia; Yu, Xue Li; Wang, Zhan Bin; Cheng, Xiang Chao

2012-01-01

MicroRNAs (miRNAs) are endogenous small non-protein coding RNAs that play important regulatory roles in animals and plants by binding to target transcripts for cleavage or translational repression. The miR-9a is very conservative in animals from flies to humans. Studies indicated that miR-9a is involved in the regulation of neurogenesis in animals. In our study, the baculovirus expression system was used to transcribe a recombinant vector containing miR-9a for further analysis the function ofmiR-9a. The sequence ofpre-miR-9a from silkworm DNA was first cloned into the donor pFastBac. The enhanced green fluorescent protein (EGFP) was used as reporter gene. The recombinant donor plasmid pFastBac-miR-9a was transformed into E.coli DH10Bac/AcNPV forming Bacmid-9a which was transfected into insect cells with cational lipofectin. The transcription of mature miR-9a was detected by Real-time PCR. The results show the recombinant Bacmid-9a was successfully constructed and effectively transcribed miR-9a in infected Sf21 insect cells.

PI(3,5)P2 biosynthesis regulates oligodendrocyte differentiation by intrinsic and extrinsic mechanisms

PubMed Central

Mironova, Yevgeniya A; Lenk, Guy M; Lin, Jing-Ping; Lee, Seung Joon; Twiss, Jeffery L; Vaccari, Ilaria; Bolino, Alessandra; Havton, Leif A; Min, Sang H; Abrams, Charles S; Shrager, Peter; Meisler, Miriam H; Giger, Roman J

2016-01-01

Proper development of the CNS axon-glia unit requires bi-directional communication between axons and oligodendrocytes (OLs). We show that the signaling lipid phosphatidylinositol-3,5-bisphosphate [PI(3,5)P2] is required in neurons and in OLs for normal CNS myelination. In mice, mutations of Fig4, Pikfyve or Vac14, encoding key components of the PI(3,5)P2 biosynthetic complex, each lead to impaired OL maturation, severe CNS hypomyelination and delayed propagation of compound action potentials. Primary OLs deficient in Fig4 accumulate large LAMP1+ and Rab7+ vesicular structures and exhibit reduced membrane sheet expansion. PI(3,5)P2 deficiency leads to accumulation of myelin-associated glycoprotein (MAG) in LAMP1+perinuclear vesicles that fail to migrate to the nascent myelin sheet. Live-cell imaging of OLs after genetic or pharmacological inhibition of PI(3,5)P2 synthesis revealed impaired trafficking of plasma membrane-derived MAG through the endolysosomal system in primary cells and brain tissue. Collectively, our studies identify PI(3,5)P2 as a key regulator of myelin membrane trafficking and myelinogenesis. DOI: http://dx.doi.org/10.7554/eLife.13023.001 PMID:27008179

pPKCδ activates SC35 splicing factor during H9c2 myoblastic differentiation.

PubMed

Zara, Susi; Falconi, Mirella; Rapino, Monica; Zago, Michela; Orsini, Giovanna; Mazzotti, Giovanni; Cataldi, Amelia; Teti, Gabriella

2011-01-01

Although Protein Kinase C (PKC) isoforms' role in the neonatal and adult cardiac tissue development and ageing has been widely described "in vivo", the interaction of such enzymes with specific nuclear substrates needs to be investigated. The aim of our research has been the study of the expression, localization and interaction with the splicing factor SC35 of PKC isoforms (α, δ, ε, ζ) and their potential role in modulating the transcription machinery. H9c2 cells induced to myoblast differentiation in the presence of 1% Horse Serum (HS) have represented our experimental model. The expression of PKC isoforms, their distribution and interaction with SC35 have been evaluated by western blotting, co-immunoprecipitation and double gold immunolabeling for transmission and scanning electron microscopy. Our results show PKCδ as the most expressed isoform in differentiated cells. Surprisingly, the distribution of PKCδ and SC35 does not show any significant modification between 10%FBS and 1%HS treated samples and no co-localization is observed. Moreover the interaction between the phosphorylated form of PKCδ (pPKCδ) and SC35 increases, is distributed and co-localizes within the nucleus of differentiated H9c2. These data represent reasonable evidence of pPKCδ mediated SC35 splicing factor activation, suggesting its direct effect on transcription via interaction with the transcription machinery. Furthermore, this co-localization represents a crucial event resulting in downstream changes in transcription of components which determine the morphological modifications related to cardiomyoblast differentiated phenotype.

Patterns of Proteins that Associate with p53 or with p53 Binding Sites Present in the Ribosomal Gene Cluster and MDM2 (P2) Promoter

DTIC Science & Technology

2000-08-01

Spodoptera frugiperda (Sf21) cells were infected with a recombinant baculovirus expressing the wild-type human p53. 3-4 and 10-1 cells were grown at 37 ’C in...for further use. Spodoptera fugiperda (Sf21) cells were grown at 27 0C in TC-100 medium (GIBCO), supplemented with 10% of heat inactivated Fetal

Chaperokine function of recombinant Hsp72 produced in insect cells using a baculovirus expression system is retained.

PubMed

Zheng, Hongying; Nagaraja, Ganachari M; Kaur, Punit; Asea, Edwina E; Asea, Alexzander

2010-01-01

Extracellular heat shock protein 72 (Hsp72; inducible form of the 70-kDa heat shock protein) plays a critical role in innate and adaptive immune responses and has shown promise as an ideal adjuvant for the optimization of antigen-specific anti-tumor vaccines. Recent studies suggest that to correctly elucidate the mechanisms by which Hsp72 exerts its beneficial effects in vitro, great care must be taken to ensure that endotoxin by-products do not invalidate the findings. In this study, we have taken advantage of the baculovirus expression vector system for production of endotoxin-free recombinant Hsp72. The coding sequence of human hsp72 was recombined into the baculovirus immediately downstream of the strong polyhedron gene promoter. Ninety-six h post-infection of Sf9 insect cells with recombinant baculovirus, maximal levels of Hsp72 protein were detected. The recombinant human Hsp72 was purified by affinity chromatography from insect cells, and purity was confirmed by SDS-PAGE and mass spectrometry. The purified human recombinant Hsp72(bv) (Hsp72 produced using the BEVS) was demonstrated to have no endotoxin contamination and was shown to have stimulated potent calcium flux in the human monocytic cell line. Furthermore, recombinant Hsp72(bv) enhanced the tolerance of neuroblastoma cells to heat stress-induced cell death and displayed classical chaperokine functions including augmentation of inflammatory cytokine productions in mouse splenocytes. The production of functional, endotoxin-free recombinant human Hsp72(bv) in insect cells is inexpensive and convenient and eliminates the need of special procedures for endotoxin depletion. Endotoxin-free recombinant human Hsp72(bv) can now be used to unlock the important role Hsp72 plays in modulating immune function.

Chaperokine Function of Recombinant Hsp72 Produced in Insect Cells Using a Baculovirus Expression System Is Retained*

PubMed Central

Zheng, Hongying; Nagaraja, Ganachari M.; Kaur, Punit; Asea, Edwina E.; Asea, Alexzander

2010-01-01

Extracellular heat shock protein 72 (Hsp72; inducible form of the 70-kDa heat shock protein) plays a critical role in innate and adaptive immune responses and has shown promise as an ideal adjuvant for the optimization of antigen-specific anti-tumor vaccines. Recent studies suggest that to correctly elucidate the mechanisms by which Hsp72 exerts its beneficial effects in vitro, great care must be taken to ensure that endotoxin by-products do not invalidate the findings. In this study, we have taken advantage of the baculovirus expression vector system for production of endotoxin-free recombinant Hsp72. The coding sequence of human hsp72 was recombined into the baculovirus immediately downstream of the strong polyhedron gene promoter. Ninety-six h post-infection of Sf9 insect cells with recombinant baculovirus, maximal levels of Hsp72 protein were detected. The recombinant human Hsp72 was purified by affinity chromatography from insect cells, and purity was confirmed by SDS-PAGE and mass spectrometry. The purified human recombinant Hsp72bv (Hsp72 produced using the BEVS) was demonstrated to have no endotoxin contamination and was shown to have stimulated potent calcium flux in the human monocytic cell line. Furthermore, recombinant Hsp72bv enhanced the tolerance of neuroblastoma cells to heat stress-induced cell death and displayed classical chaperokine functions including augmentation of inflammatory cytokine productions in mouse splenocytes. The production of functional, endotoxin-free recombinant human Hsp72bv in insect cells is inexpensive and convenient and eliminates the need of special procedures for endotoxin depletion. Endotoxin-free recombinant human Hsp72bv can now be used to unlock the important role Hsp72 plays in modulating immune function. PMID:19861412

A Novel Ideal Radionuclide Imaging System for Non-invasively Cell Monitoring built on Baculovirus Backbone by Introducing Sleeping Beauty Transposon

PubMed Central

Lv, Jing; Pan, Yu; Ju, Huijun; Zhou, Jinxin; Cheng, Dengfeng; Shi, Hongcheng; Zhang, Yifan

2017-01-01

Sleeping Beauty (SB) transposon is an attractive tool in stable transgene integration both in vitro and in vivo; and we introduced SB transposon into recombinant sodium-iodide symporter baculovirus system (Bac-NIS system) to facilitate long-term expression of recombinant sodium-iodide symporter. In our study, two hybrid baculovirus systems (Bac-eGFP-SB-NeoR and Bac-NIS-SB-NeoR) were successfully constructed and used to infect U87 glioma cells. After G418 selection screening, the Bac-eGFP-SB-NeoR-U87 cells remained eGFP positive, at the 18th and 196th day post transfection (96.03 ± 0.21% and 97.43 ± 0.81%), while eGFP positive population declined significantly at 18 days in cells transfected with unmodified baculovirus construct. NIS gene expression by Bac-NIS-SB-NeoR-U87 cells was also maintained for 28 weeks as determined by radioiodine uptake assay, reverse transcription-polymerase chain reaction (RT-PCR) and Western Blot (WB) assay. When transplanted in mice, Bac-NIS-SB-NeoR-U87 cells also expressed NIS gene stably as monitored by SPECT imaging for 43 days until the tumor-bearing mice were sacrificed. Herein, we showed that incorporation of SB in Bac-NIS system (hybrid Bac-NIS-SB-NeoR) can achieve a long-term transgene expression and can improve radionuclide imaging in cell tracking and monitoring in vivo. PMID:28262785

Sigma-1 receptor regulates Tau phosphorylation and axon extension by shaping p35 turnover via myristic acid

PubMed Central

Tsai, Shang-Yi A.; Pokrass, Michael J.; Klauer, Neal R.; Nohara, Hiroshi; Su, Tsung-Ping

2015-01-01

Dysregulation of cyclin-dependent kinase 5 (cdk5) per relative concentrations of its activators p35 and p25 is implicated in neurodegenerative diseases. P35 has a short t½ and undergoes rapid proteasomal degradation in its membrane-bound myristoylated form. P35 is converted by calpain to p25, which, along with an extended t½, promotes aberrant activation of cdk5 and causes abnormal hyperphosphorylation of tau, thus leading to the formation of neurofibrillary tangles. The sigma-1 receptor (Sig-1R) is an endoplasmic reticulum chaperone that is implicated in neuronal survival. However, the specific role of the Sig-1R in neurodegeneration is unclear. Here we found that Sig-1Rs regulate proper tau phosphorylation and axon extension by promoting p35 turnover through the receptor’s interaction with myristic acid. In Sig-1R–KO neurons, a greater accumulation of p35 is seen, which results from neither elevated transcription of p35 nor disrupted calpain activity, but rather to the slower degradation of p35. In contrast, Sig-1R overexpression causes a decrease of p35. Sig-1R–KO neurons exhibit shorter axons with lower densities. Myristic acid is found here to bind Sig-1R as an agonist that causes the dissociation of Sig-1R from its cognate partner binding immunoglobulin protein. Remarkably, treatment of Sig-1R–KO neurons with exogenous myristic acid mitigates p35 accumulation, diminishes tau phosphorylation, and restores axon elongation. Our results define the involvement of Sig-1Rs in neurodegeneration and provide a mechanistic explanation that Sig-1Rs help maintain proper tau phosphorylation by potentially carrying and providing myristic acid to p35 for enhanced p35 degradation to circumvent the formation of overreactive cdk5/p25. PMID:25964330

Sigma-1 receptor regulates Tau phosphorylation and axon extension by shaping p35 turnover via myristic acid.

PubMed

Tsai, Shang-Yi A; Pokrass, Michael J; Klauer, Neal R; Nohara, Hiroshi; Su, Tsung-Ping

2015-05-26

Dysregulation of cyclin-dependent kinase 5 (cdk5) per relative concentrations of its activators p35 and p25 is implicated in neurodegenerative diseases. P35 has a short t½ and undergoes rapid proteasomal degradation in its membrane-bound myristoylated form. P35 is converted by calpain to p25, which, along with an extended t½, promotes aberrant activation of cdk5 and causes abnormal hyperphosphorylation of tau, thus leading to the formation of neurofibrillary tangles. The sigma-1 receptor (Sig-1R) is an endoplasmic reticulum chaperone that is implicated in neuronal survival. However, the specific role of the Sig-1R in neurodegeneration is unclear. Here we found that Sig-1Rs regulate proper tau phosphorylation and axon extension by promoting p35 turnover through the receptor's interaction with myristic acid. In Sig-1R-KO neurons, a greater accumulation of p35 is seen, which results from neither elevated transcription of p35 nor disrupted calpain activity, but rather to the slower degradation of p35. In contrast, Sig-1R overexpression causes a decrease of p35. Sig-1R-KO neurons exhibit shorter axons with lower densities. Myristic acid is found here to bind Sig-1R as an agonist that causes the dissociation of Sig-1R from its cognate partner binding immunoglobulin protein. Remarkably, treatment of Sig-1R-KO neurons with exogenous myristic acid mitigates p35 accumulation, diminishes tau phosphorylation, and restores axon elongation. Our results define the involvement of Sig-1Rs in neurodegeneration and provide a mechanistic explanation that Sig-1Rs help maintain proper tau phosphorylation by potentially carrying and providing myristic acid to p35 for enhanced p35 degradation to circumvent the formation of overreactive cdk5/p25.

Baculovirus-mediated expression of GPCRs in insect cells.

PubMed

Saarenpää, Tuulia; Jaakola, Veli-Pekka; Goldman, Adrian

2015-01-01

G-protein-coupled receptors (GPCRs) are a large family of seven transmembrane proteins that influence a considerable number of cellular events. For this reason, they are one of the most studied receptor types for their pharmacological and structural properties. Solving the structure of several GPCR receptor types has been possible using almost all expression systems, including Escherichia coli, yeast, mammalian, and insect cells. So far, however, most of the GPCR structures solved have been done using the baculovirus insect cell expression system. The reason for this is mainly due to cost-effectiveness, posttranslational modification efficiency, and overall effortless maintenance. The system has evolved so much that variables starting from vector type, purification tags, cell line, and growth conditions can be varied and optimized countless ways to suit the needs of new constructs. Here, we present the array of techniques that enable the rapid and efficient optimization of expression steps for maximal protein quality and quantity, including our emendations. © 2015 Elsevier Inc. All rights reserved.

Science, Politics, and Best Practice: 35 Years after Larry P.

ERIC Educational Resources Information Center

Frisby, Craig L.; Henry, Betty

2016-01-01

A little over 35 years have passed since the original "Larry P." decision was handed down in 1979 by Robert Peckham, a federal judge for the US District Court for the Northern District of California. The "Larry P. case" is a shorthand moniker that refers to a class action lawsuit, supported by the Bay Area Association of Black…

Increased copper toxicity in Saccharomyces cerevisiae lacking VPS35, a component of the retromer and monogenic Parkinson disease gene in humans.

PubMed

Sowada, Nadine; Stiller, Barbara; Kubisch, Christian

2016-08-05

The Saccharomyces cerevisiae gene VPS35 encodes a component of the retromer complex which is involved in vesicle transport from endosomes to the trans-Golgi network. Yeast and human VPS35 orthologs are highly conserved and mutations in human VPS35 cause an autosomal dominant form of late-onset Parkinson disease (PD). We now show that deletion of VPS35 in yeast (vps35Δ) leads to a dose-dependent growth defect towards copper. This increased sensitivity could be rescued by transformation with yeast wild-type VPS35 but not by the expression of a construct harboring the yeast equivalent (i.e. D686N) of the most commonly identified VPS35-associated PD mutation, p.D620N. In addition, we show that expression of one copy of α-synuclein, which is known to directly interact with copper, leads to a pronounced aggravation of copper toxicity in vps35Δ cells, thereby linking the regulation of copper homeostasis by Vps35p in yeast to one of the key molecules in PD pathophysiology. Copyright © 2016 Elsevier Inc. All rights reserved.

Investigation of InP/In0.65Ga0.35As metamorphic p-channel doped-channel field-effect transistor

NASA Astrophysics Data System (ADS)

Tsai, Jung-Hui

2016-07-01

In this article, the device mechanism and characteristics of InP/InGaAs metamorphic p-channel field-effect transistor (FET), which has a high indium mole fraction of InGaAs channel, grown on the GaAs substrate is demonstrated. The device was fabricated on the top of the InxGa1-xP graded metamorphic buffer layer, and the In0.65Ga0.35As pseudomorphic channel was employed to elevate the transistor performance. For the p-type FET, due to the considerably large valence band discontinuity at InP/In0.65Ga0.35As heterojunction and a relatively thin as well as heavily doped pseudomorphic In0.65Ga0.35As channel between two undoped InP layers, a maximum extrinsic transconductance of 27.3 mS/mm and a maximum saturation current density of -54.3 mA/mm are obtained. Consequently, the studied metamorphic FET is suitable for the development in signal amplification, integrated circuits, and low supply-voltage complementary logic inverters.

A silencing suppressor protein (NSs) of a tospovirus enhances baculovirus replication in permissive and semipermissive insect cell lines.

PubMed

Oliveira, Virgínia Carla; Bartasson, Lorrainy; de Castro, Maria Elita Batista; Corrêa, José Raimundo; Ribeiro, Bergmann Morais; Resende, Renato Oliveira

2011-01-01

The nonstructural protein (NSs) of the Tomato spotted wilt virus (TSWV) has been identified as an RNAi suppressor in plant cells. A recombinant Autographa californica multiple nucleopolyhedrovirus (AcMNPV) designated vAcNSs, containing the NSs gene under the control of the viral polyhedrin (polh) gene promoter, was constructed and the effects of NSs in permissive, semipermissive and nonpermissive insect cells to vAcNSs infection were evaluated. vAcNSs produced more budded virus when compared to wild type in semipermissive cells. Co-infection of vAcNSs with wild type baculoviruses clearly enhanced polyhedra production in all host cells. Confocal microscopy analysis showed that NSs accumulated in abundance in the cytoplasm of permissive and semipermissive cells. In contrast, high amounts of NSs were detected in the nuclei of nonpermissive cells. Co-infection of vAcNSs with a recombinant AcMNPV containing the enhanced green fluorescent protein (egfp) gene, significantly increased EGFP expression in semipermissive cells and in Anticarsia gemmatalis-hemocytes. Absence of small RNA molecules of egfp transcripts in this cell line and in a permissive cell line indicates the suppression of gene silencing activity. On the other hand, vAcNSs was not able to suppress RNAi in a nonpermissive cell line. Our data showed that NSs protein of TSWV facilitates baculovirus replication in different lepidopteran cell lines, and these results indicate that NSs could play a similar role during TSWV-infection in its thrips vector. Copyright © 2010 Elsevier B.V. All rights reserved.

Baculovirus vectors expressing F proteins in combination with virus-induced signaling adaptor (VISA) molecules confer protection against respiratory syncytial virus infection.

PubMed

Zhang, Yuan; Qiao, Lei; Hu, Xiao; Zhao, Kang; Zhang, Yanwen; Chai, Feng; Pan, Zishu

2016-01-04

Baculovirus has been exploited for use as a novel vaccine vector. To investigate the feasibility and efficacy of recombinant baculoviruses (rBVs) expressing respiratory syncytial virus (RSV) fusion (F) proteins, four constructs (Bac-tF/64, Bac-CF, Bac-CF/tF64 and Bac-CF/tF64-VISA) were generated. Bac-tF64 displays the F ectodomain (tF) on the envelope of rBVs, whereas Bac-CF expresses full-length F protein in transduced mammalian cells. Bac-CF/tF64 not only displays tF on the envelope but also expresses F in cells. Bac-CF/tF64-VISA comprises Bac-CF/tF64 harboring the virus-induced signaling adaptor (VISA) gene. After administration to BALB/c mice, all four vectors elicited RSV neutralizing antibody (Ab), systemic Ab (IgG, IgG1, and IgG2a), and cytokine responses. Compared with Bac-tF64, mice inoculated with Bac-CF and Bac-CF/tF64 exhibited an increased mixed Th1/Th2 cytokine response, increased ratios of IgG2a/IgG1 antibody responses, and reduced immunopathology upon RSV challenge. Intriguingly, co-expression of VISA reduced Th2 cytokine (IL-4, IL-5, and IL-10) production induced by Bac-CF/tF64, thus relieving lung pathology upon a subsequent RSV challenge. Our results indicated that the Bac-CF/tF64 vector incorporated with the VISA molecule may provide an effective vaccine strategy for protection against RSV. Copyright © 2015 Elsevier Ltd. All rights reserved.

Increase in number of helminth species from Dutch red foxes over a 35-year period.

PubMed

Franssen, Frits; Nijsse, Rolf; Mulder, Jaap; Cremers, Herman; Dam, Cecile; Takumi, Katsuhisa; van der Giessen, Joke

2014-04-03

The red fox (Vulpes vulpes) is host to a community of zoonotic and other helminth species. Tracking their community structure and dynamics over decades is one way to monitor the long term risk of parasitic infectious diseases relevant to public and veterinary health. We identified 17 helminth species from 136 foxes by mucosal scraping, centrifugal sedimentation/flotation and the washing and sieving technique. We applied rarefaction analysis to our samples and compared the resulting curve to the helminth community reported in literature 35 years ago. Fox helminth species significantly increased in number in the last 35 years (p-value <0.025). Toxascaris leonina, Mesocestoides litteratus, Trichuris vulpis and Angiostrongylus vasorum are four new veterinary-relevant species. The zoonotic fox tapeworm (E. multilocularis) was found outside the previously described endemic regions in the Netherlands. Helminth fauna in Dutch red foxes increased in biodiversity over the last three decades.

Identification of human cytochrome P450 2D6 as major enzyme involved in the O-demethylation of the designer drug p-methoxymethamphetamine.

PubMed

Staack, Roland F; Theobald, Denis S; Paul, Liane D; Springer, Dietmar; Kraemer, Thomas; Maurer, Hans H

2004-04-01

p-Methoxymethamphetamine (PMMA) is a new designer drug, listed in many countries as a controlled substance. Several fatalities have been attributed to the abuse of this designer drug. Previous in vivo studies using Wistar rats had shown that PMMA was metabolized mainly by O-demethylation. The aim of the study presented here was to identify the human hepatic cytochrome P450 (P450) enzymes involved in the biotransformation of PMMA to p-hydroxymethamphetamine. Baculovirus-infected insect cell microsomes, pooled human liver microsomes (pHLMs), and CYP2D6 poor-metabolizer genotype human liver microsomes (PM HLMs) were used for this purpose. Only CYP2D6 catalyzed O-demethylation. The apparent K(m) and V(max) values in baculovirus-infected insect cell microsomes were 4.6 +/- 1.0 microM and 92.0 +/- 3.7 pmol/min/pmol P450, respectively, and 42.0 +/- 4.0 microM and 412.5 +/- 10.8 pmol/min/mg protein in pHLMs. Inhibition studies with 1 microM quinidine showed significant inhibition of the metabolite formation (67.2 +/- 0.6%; p < 0.0001), and comparison of the metabolite formation between pHLMs and PM HLMs revealed significantly lower metabolite formation in the incubations with PM HLMs (87.3 +/- 1.1%; p < 0.0001). According to these studies, CYP2D6 is the major P450 involved in O-demethylation of PMMA.

p35 Regulates the CRM1-Dependent Nucleocytoplasmic Shuttling of Nuclear Hormone Receptor Coregulator-Interacting Factor 1 (NIF-1)

PubMed Central

Zhao, Xiao-Su; Fu, Wing-Yu; Chien, Winnie W. Y.; Li, Zhen; Fu, Amy K. Y.; Ip, Nancy Y.

2014-01-01

Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase, which plays critical roles in a wide spectrum of neuronal functions including neuronal survival, neurite outgrowth, and synapse development and plasticity. Cdk5 activity is controlled by its specific activators: p35 or p39. While knockout studies reveal that Cdk5/p35 is critical for neuronal migration during early brain development, functions of Cdk5/p35 have been unraveled through the identification of the interacting proteins of p35, most of which are Cdk5/p35 substrates. However, it remains unclear whether p35 can regulate neuronal functions independent of Cdk5 activity. Here, we report that a nuclear protein, nuclear hormone receptor coregulator (NRC)-interacting factor 1 (NIF-1), is a new interacting partner of p35. Interestingly, p35 regulates the functions of NIF-1 independent of Cdk5 activity. NIF-1 was initially discovered as a transcriptional regulator that enhances the transcriptional activity of nuclear hormone receptors. Our results show that p35 interacts with NIF-1 and regulates its nucleocytoplasmic trafficking via the nuclear export pathway. Furthermore, we identified a nuclear export signal on p35; mutation of this site or blockade of the CRM1/exportin-dependent nuclear export pathway resulted in the nuclear accumulation of p35. Intriguingly, blocking the nuclear export of p35 attenuated the nuclear accumulation of NIF-1. These findings reveal a new p35-dependent mechanism in transcriptional regulation that involves the nucleocytoplasmic shuttling of transcription regulators. PMID:25329792

p35 regulates the CRM1-dependent nucleocytoplasmic shuttling of nuclear hormone receptor coregulator-interacting factor 1 (NIF-1).

PubMed

Zhao, Xiao-Su; Fu, Wing-Yu; Chien, Winnie W Y; Li, Zhen; Fu, Amy K Y; Ip, Nancy Y

2014-01-01

Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase, which plays critical roles in a wide spectrum of neuronal functions including neuronal survival, neurite outgrowth, and synapse development and plasticity. Cdk5 activity is controlled by its specific activators: p35 or p39. While knockout studies reveal that Cdk5/p35 is critical for neuronal migration during early brain development, functions of Cdk5/p35 have been unraveled through the identification of the interacting proteins of p35, most of which are Cdk5/p35 substrates. However, it remains unclear whether p35 can regulate neuronal functions independent of Cdk5 activity. Here, we report that a nuclear protein, nuclear hormone receptor coregulator (NRC)-interacting factor 1 (NIF-1), is a new interacting partner of p35. Interestingly, p35 regulates the functions of NIF-1 independent of Cdk5 activity. NIF-1 was initially discovered as a transcriptional regulator that enhances the transcriptional activity of nuclear hormone receptors. Our results show that p35 interacts with NIF-1 and regulates its nucleocytoplasmic trafficking via the nuclear export pathway. Furthermore, we identified a nuclear export signal on p35; mutation of this site or blockade of the CRM1/exportin-dependent nuclear export pathway resulted in the nuclear accumulation of p35. Intriguingly, blocking the nuclear export of p35 attenuated the nuclear accumulation of NIF-1. These findings reveal a new p35-dependent mechanism in transcriptional regulation that involves the nucleocytoplasmic shuttling of transcription regulators.

Roles of silkworm endoplasmic reticulum chaperones in the secretion of recombinant proteins expressed by baculovirus system.

PubMed

Imai, Saki; Kusakabe, Takahiro; Xu, Jian; Li, Zhiqing; Shirai, Shintaro; Mon, Hiroaki; Morokuma, Daisuke; Lee, Jae Man

2015-11-01

Baculovirus expression vector system (BEVS) is widely used for production of recombinant eukaryotic proteins in insect larvae or cultured cells. BEVS has advantages over bacterial expression system in producing post-translationally modified secreted proteins. However, for some unknown reason, it is very difficult for insects to secrete sufficiently for certain proteins of interest. To understand the reasons why insect cells fail to secrete some kinds of recombinant proteins, we here employed three mammalian proteins as targets, EPO, HGF, and Wnt3A, with different secretion levels in BEVS and investigated their mRNA transcriptions from the viral genome, subcellular localizations, and interactions with silkworm ER chaperones. Moreover, we observed that no significantly influence on the secretion amounts of all three proteins when depleting or overexpressing most endogenous ER chaperone genes in cultured silkworm cells. However, among all detected ER chaperones, the depletion of BiP severely decreased the recombinant protein secretion in BEVS, indicating the possible central role of Bip in silkworm secretion pathway.

In vivo, Pikfyve generates PI(3,5)P2, which serves as both a signaling lipid and the major precursor for PI5P

PubMed Central

Zolov, Sergey N.; Bridges, Dave; Zhang, Yanling; Lee, Wei-Wei; Riehle, Ellen; Verma, Rakesh; Lenk, Guy M.; Converso-Baran, Kimber; Weide, Thomas; Albin, Roger L.; Saltiel, Alan R.; Meisler, Miriam H.; Russell, Mark W.; Weisman, Lois S.

2012-01-01

Mutations that cause defects in levels of the signaling lipid phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2] lead to profound neurodegeneration in mice. Moreover, mutations in human FIG4 predicted to lower PI(3,5)P2 levels underlie Charcot–Marie–Tooth type 4J neuropathy and are present in selected cases of amyotrophic lateral sclerosis. In yeast and mammals, PI(3,5)P2 is generated by a protein complex that includes the lipid kinase Fab1/Pikfyve, the scaffolding protein Vac14, and the lipid phosphatase Fig4. Fibroblasts cultured from Vac14−/− and Fig4−/− mouse mutants have a 50% reduction in the levels of PI(3,5)P2, suggesting that there may be PIKfyve-independent pathways that generate this lipid. Here, we characterize a Pikfyve gene-trap mouse (Pikfyveβ-geo/β-geo), a hypomorph with ∼10% of the normal level of Pikfyve protein. shRNA silencing of the residual Pikfyve transcript in fibroblasts demonstrated that Pikfyve is required to generate all of the PI(3,5)P2 pool. Surprisingly, Pikfyve also is responsible for nearly all of the phosphatidylinositol-5-phosphate (PI5P) pool. We show that PI5P is generated directly from PI(3,5)P2, likely via 3′-phosphatase activity. Analysis of tissues from the Pikfyveβ-geo/β-geo mouse mutants reveals that Pikfyve is critical in neural tissues, heart, lung, kidney, thymus, and spleen. Thus, PI(3,5)P2 and PI5P have major roles in multiple organs. Understanding the regulation of these lipids may provide insights into therapies for multiple diseases. PMID:23047693

Increase in number of helminth species from Dutch red foxes over a 35-year period

PubMed Central

2014-01-01

Background The red fox (Vulpes vulpes) is host to a community of zoonotic and other helminth species. Tracking their community structure and dynamics over decades is one way to monitor the long term risk of parasitic infectious diseases relevant to public and veterinary health. Methods We identified 17 helminth species from 136 foxes by mucosal scraping, centrifugal sedimentation/flotation and the washing and sieving technique. We applied rarefaction analysis to our samples and compared the resulting curve to the helminth community reported in literature 35 years ago. Results Fox helminth species significantly increased in number in the last 35 years (p-value <0.025). Toxascaris leonina, Mesocestoides litteratus, Trichuris vulpis and Angiostrongylus vasorum are four new veterinary-relevant species. The zoonotic fox tapeworm (E. multilocularis) was found outside the previously described endemic regions in the Netherlands. Conclusions Helminth fauna in Dutch red foxes increased in biodiversity over the last three decades. PMID:24708710

Functional dissociation between PIKfyve-synthesized PtdIns5P and PtdIns(3,5)P2 by means of the PIKfyve inhibitor YM201636

PubMed Central

Sbrissa, Diego; Ikonomov, Ognian C.; Filios, Catherine; Delvecchio, Khortnal

2012-01-01

PIKfyve is an essential mammalian lipid kinase with pleiotropic cellular functions whose genetic knockout in mice leads to preimplantation lethality. Despite several reports for PIKfyve-catalyzed synthesis of phosphatidylinositol 5-phosphate (PtdIns5P) along with phosphatidylinositol-3,5-biphosphate [PtdIns(3,5)P2] in vitro and in vivo, the role of the PIKfyve pathway in intracellular PtdIns5P production remains underappreciated and the function of the PIKfyve-synthesized PtdIns5P pool poorly characterized. Hence, the recently discovered potent PIKfyve-selective inhibitor, the YM201636 compound, has been solely tested for inhibiting PtdIns(3,5)P2 synthesis. Here, we have compared the in vitro and in vivo inhibitory potency of YM201636 toward PtdIns5P and PtdIns(3,5)P2. Unexpectedly, we observed that at low doses (10–25 nM), YM201636 inhibited preferentially PtdIns5P rather than PtdIns(3,5)P2 production in vitro, whereas at higher doses, the two products were similarly inhibited. In cellular contexts, YM201636 at 160 nM inhibited PtdIns5P synthesis twice more effectively compared with PtdIns(3,5)P2 synthesis. In 3T3L1 adipocytes, human embryonic kidney 293 and Chinese hamster ovary (CHO-T) cells, levels of PtdIns5P dropped by 62–71% of the corresponding untreated controls, whereas those of PtdIns(3,5)P2 fell by only 28–46%. The preferential inhibition of PtdIns5P versus PtdIns(3,5)P2 at low doses of YM201636 was explored to probe contributions of the PIKfyve-catalyzed PtdIns5P pool to insulin-induced actin stress fiber disassembly in CHO-T cells, GLUT4 translocation in 3T3L1 adipocytes, and induction of aberrant cellular vacuolation in these or other cell types. The results provide the first experimental evidence that the principal pathway for PtdIns5P intracellular production is through PIKfyve and that insulin effect on actin stress fiber disassembly is mediated entirely by the PIKfyve-produced PtdIns5P pool. PMID:22621786

Protective Efficacy of a Single Dose of Baculovirus Hemagglutinin-Based Vaccine in Chickens and Ducks Against Homologous and Heterologous H5N1 Virus Infections

PubMed Central

Park, Eun Hye; Song, Byung Min; Yum, Jung; Kim, Ji An; Oh, Seung Kyoo; Kim, Hyun Soo; Cho, Gil Jae

2014-01-01

Abstract Outbreaks of the highly pathogenic H5N1 virus in poultry and humans are ongoing. Vaccination is an efficient method for prevention of H5N1 infection. Using chickens and ducks, we assessed the efficacy of a vaccine comprising H5N1 hemagglutinin (HA) protein produced in a baculovirus expression system. The immunized chickens and ducks were protected against lethal infection by H5N1 in an antigen dose-dependent manner. Complete protection against homologous challenge and partial protection against heterologous challenge were achieved in chickens immunized with 5 μg HA protein and in ducks immunized with 10 μg HA protein. The IgG antibody subtype was mainly detected in the sera and tissues, including the lungs. The neuraminidase (NA) inhibition assay was negative in immunized chickens and ducks. Our results indicated that the expressed HA protein by baculovirus was immunogenic to both chickens and ducks, and the immunized chickens and ducks were protected from the lethal infections of highly pathogenic H5N1 influenza virus, though ducks required more HA protein than chickens to be protected. Also, baculovirus HA-vaccinated poultry can be differentiated from infected poultry by NA inhibition assay. PMID:25211640

Sec34p, a Protein Required for Vesicle Tethering to the Yeast Golgi Apparatus, Is in a Complex with Sec35p

PubMed Central

VanRheenen, Susan M.; Cao, Xiaochun; Sapperstein, Stephanie K.; Chiang, Elbert C.; Lupashin, Vladimir V.; Barlowe, Charles; Waters, M. Gerard

1999-01-01

A screen for mutants of Saccharomyces cerevisiae secretory pathway components previously yielded sec34, a mutant that accumulates numerous vesicles and fails to transport proteins from the ER to the Golgi complex at the restrictive temperature (Wuestehube, L.J., R. Duden, A. Eun, S. Hamamoto, P. Korn, R. Ram, and R. Schekman. 1996. Genetics. 142:393–406). We find that SEC34 encodes a novel protein of 93-kD, peripherally associated with membranes. The temperature-sensitive phenotype of sec34-2 is suppressed by the rab GTPase Ypt1p that functions early in the secretory pathway, or by the dominant form of the ER to Golgi complex target-SNARE (soluble N-ethylmaleimide sensitive fusion protein attachment protein receptor)–associated protein Sly1p, Sly1-20p. Weaker suppression is evident upon overexpression of genes encoding the vesicle tethering factor Uso1p or the vesicle-SNAREs Sec22p, Bet1p, or Ykt6p. This genetic suppression profile is similar to that of sec35-1, a mutant allele of a gene encoding an ER to Golgi vesicle tethering factor and, like Sec35p, Sec34p is required in vitro for vesicle tethering. sec34-2 and sec35-1 display a synthetic lethal interaction, a genetic result explained by the finding that Sec34p and Sec35p can interact by two-hybrid analysis. Fractionation of yeast cytosol indicates that Sec34p and Sec35p exist in an ∼750-kD protein complex. Finally, we describe RUD3, a novel gene identified through a genetic screen for multicopy suppressors of a mutation in USO1, which suppresses the sec34-2 mutation as well. PMID:10562277

Baculovirus-mediated vascular endothelial growth factor-D(ΔNΔC) gene transfer induces angiogenesis in rabbit skeletal muscle.

PubMed

Heikura, Tommi; Nieminen, Tiina; Roschier, Miia M; Karvinen, Henna; Kaikkonen, Minna U; Mähönen, Anssi J; Lesch, Hanna P; Rissanen, Tuomas T; Laitinen, Olli H; Airenne, Kari J; Ylä-Herttuala, Seppo

2012-01-01

Occluded arteries and ischemic tissues cannot always be treated by angioplasty, stenting or by-pass-surgery. Under such circumstances, viral gene therapy may be useful in inducing increased blood supply to ischemic area. There is evidence of improved blood flow in ischemic skeletal muscle and myocardium in both animal and human studies using adenoviral vascular endothelial growth factor (VEGF) gene therapy. However, the expression is transient and repeated gene transfers with the same vector are inefficient due to immune responses. Different baculoviral vectors pseudotyped with or without vesicular stomatitis virus glycoprotein (VSV-G) and/or carrying woodchuck hepatitis virus post-transcriptional regulatory element (Wpre) were tested both in vitro and in vivo. VEGF-D(ΔNΔC) was used as therapeutic transgene and lacZ as a control. In vivo efficacy was evaluated as capillary enlargement and transgene expression in New Zealand White (NZW) rabbit skeletal muscle. A statistically significant capillary enlargement was detected 6 days after gene transfer in transduced areas compared to the control gene transfers with baculovirus and adenovirus encoding β-galactosidase (lacZ). Substantially improved gene transfer efficiency was achieved with a modified baculovirus pseudotyped with VSV-G and carrying Wpre. Dose escalation experiments revealed that either too large volume or too many virus particles caused inflammation and necrosis in the target tissue, whereas 10(9) plaque forming units injected in multiple aliquots resulted in transgene expression with only mild immune reactions. We show the first evidence of biologically significant baculoviral gene transfer in skeletal muscle of NZW rabbits using VEGF-D(ΔNΔC) as a therapeutic transgene. Copyright © 2012 John Wiley & Sons, Ltd.

[Use of the recombinant baculovirus BacVP6C for the construction of an internal positive control of rotavirus C].

PubMed

Abid-Ayadi, I; Guix, S; Pintó, R M; Bosch, A

2011-06-01

Unlike group A, a few studies have interested other groups of the rotavirus, especially in Tunisia. The role of rotavirus C (RVC) infection is underestimated because of its sporadic nature. The aim of our study was to develop rapid diagnostic procedures of RVC by using an internal positive control of reverse transcription PCR (RT-PCR). The internal positive control (386pb) was designed from the recombinant baculovirus BacVP6C containing the full length cDNA of the Cowden strain gene 5 (1353pb). A fragment of 596pb was amplified by PCR using the BacVP6C DNA ds as template. Then, a central part of 210pb was deleted and the remaining fragment (386pb) was cloned into pGEM-3Zf(+) plasmid between SP6 and T7 RNA polymerase promoters. The obtained recombinant plasmid "pIAM1" was then used for the generation of the internal positive control by in vitro transcription. The sensibility of the RT-PCR was about 3.66×10(5) molecules of RNA/μl. The use of a shorter positive control, as compared to the wild type, allows increased specificity of the RT-PCR reaction, and could be used for efficient diagnostic and surveillance of RVC-caused diseases. Copyright © 2009 Elsevier Masson SAS. All rights reserved.

Regulating NETosis: Increasing pH Promotes NADPH Oxidase-Dependent NETosis

PubMed Central

Khan, Meraj A.; Philip, Lijy M.; Cheung, Guillaume; Vadakepeedika, Shawn; Grasemann, Hartmut; Sweezey, Neil; Palaniyar, Nades

2018-01-01

Neutrophils migrating from the blood (pH 7.35–7.45) into the surrounding tissues encounter changes in extracellular pH (pHe) conditions. Upon activation of NADPH oxidase 2 (Nox), neutrophils generate large amounts of H+ ions reducing the intracellular pH (pHi). Nevertheless, how extracellular pH regulates neutrophil extracellular trap (NET) formation (NETosis) is not clearly established. We hypothesized that increasing pH increases Nox-mediated production of reactive oxygen species (ROS) and neutrophil protease activity, stimulating NETosis. Here, we found that raising pHe (ranging from 6.6 to 7.8; every 0.2 units) increased pHi of both activated and resting neutrophils within 10–20 min (Seminaphtharhodafluor dual fluorescence measurements). Since Nox activity generates H+ ions, pHi is lower in neutrophils that are activated compared to resting. We also found that higher pH stimulated Nox-dependent ROS production (R123 generation; flow cytometry, plate reader assay, and imaging) during spontaneous and phorbol myristate acetate-induced NETosis (Sytox Green assays, immunoconfocal microscopy, and quantifying NETs). In neutrophils that are activated and not resting, higher pH stimulated histone H4 cleavage (Western blots) and NETosis. Raising pH increased Escherichia coli lipopolysaccharide-, Pseudomonas aeruginosa (Gram-negative)-, and Staphylococcus aureus (Gram-positive)-induced NETosis. Thus, higher pHe promoted Nox-dependent ROS production, protease activity, and NETosis; lower pH has the opposite effect. These studies provided mechanistic steps of pHe-mediated regulation of Nox-dependent NETosis. Raising pH either by sodium bicarbonate or Tris base (clinically known as Tris hydroxymethyl aminomethane, tromethamine, or THAM) increases NETosis. Each Tris molecule can bind 3H+ ions, whereas each bicarbonate HCO3− ion binds 1H+ ion. Therefore, the amount of Tris solution required to cause the same increase in pH level is less than that of equimolar

Regulating NETosis: Increasing pH Promotes NADPH Oxidase-Dependent NETosis.

PubMed

Khan, Meraj A; Philip, Lijy M; Cheung, Guillaume; Vadakepeedika, Shawn; Grasemann, Hartmut; Sweezey, Neil; Palaniyar, Nades

2018-01-01

Neutrophils migrating from the blood (pH 7.35-7.45) into the surrounding tissues encounter changes in extracellular pH (pH e ) conditions. Upon activation of NADPH oxidase 2 (Nox), neutrophils generate large amounts of H + ions reducing the intracellular pH (pH i ). Nevertheless, how extracellular pH regulates neutrophil extracellular trap (NET) formation (NETosis) is not clearly established. We hypothesized that increasing pH increases Nox-mediated production of reactive oxygen species (ROS) and neutrophil protease activity, stimulating NETosis. Here, we found that raising pH e (ranging from 6.6 to 7.8; every 0.2 units) increased pH i of both activated and resting neutrophils within 10-20 min (Seminaphtharhodafluor dual fluorescence measurements). Since Nox activity generates H + ions, pH i is lower in neutrophils that are activated compared to resting. We also found that higher pH stimulated Nox-dependent ROS production (R123 generation; flow cytometry, plate reader assay, and imaging) during spontaneous and phorbol myristate acetate-induced NETosis (Sytox Green assays, immunoconfocal microscopy, and quantifying NETs). In neutrophils that are activated and not resting, higher pH stimulated histone H4 cleavage (Western blots) and NETosis. Raising pH increased Escherichia coli lipopolysaccharide-, Pseudomonas aeruginosa (Gram-negative)-, and Staphylococcus aureus (Gram-positive)-induced NETosis. Thus, higher pH e promoted Nox-dependent ROS production, protease activity, and NETosis; lower pH has the opposite effect. These studies provided mechanistic steps of pH e -mediated regulation of Nox-dependent NETosis. Raising pH either by sodium bicarbonate or Tris base (clinically known as Tris hydroxymethyl aminomethane, tromethamine, or THAM) increases NETosis. Each Tris molecule can bind 3H + ions, whereas each bicarbonate HCO3 - ion binds 1H + ion. Therefore, the amount of Tris solution required to cause the same increase in pH level is less than that of equimolar

pH regulation in anoxic rice coleoptiles at pH 3.5: biochemical pHstats and net H+ influx in the absence and presence of NO3−

PubMed Central

Greenway, Hank; Kulichikhin, Konstantin Y.; Cawthray, Gregory R.; Colmer, Timothy D.

2012-01-01

During anoxia, cytoplasmic pH regulation is crucial. Mechanisms of pH regulation were studied in the coleoptile of rice exposed to anoxia and pH 3.5, resulting in H+ influx. Germinating rice seedlings survived a combination of anoxia and exposure to pH 3.5 for at least 4 d, although development was retarded and net K+ efflux was continuous. Further experiments used excised coleoptile tips (7–10 mm) in anoxia at pH 6.5 or 3.5, either without or with 0.2 mM NO3−, which distinguished two processes involved in pH regulation. Net H+ influx (μmol g−1 fresh weight h−1) for coleoptiles with NO3− was ∼1.55 over the first 24 h, being about twice that in the absence of NO3−, but then decreased to 0.5–0.9 as net NO3− uptake declined from ∼1.3 to 0.5, indicating reduced uptake via H+–NO3− symports. NO3− reduction presumably functioned as a biochemical pHstat. A second biochemical pHstat consisted of malate and succinate, and their concentrations decreased substantially with time after exposure to pH 3.5. In anoxic coleoptiles, K+ balancing the organic anions was effluxed to the medium as organic anions declined, and this efflux rate was independent of NO3− supply. Thus, biochemical pHstats and reduced net H+ influx across the plasma membrane are important features contributing to pH regulation in anoxia-tolerant rice coleoptiles at pH 3.5. PMID:22174442

Generation of PCV2 in PK15 cells transfected with recombinant baculovirus containing a 1.1 copy of the PCV2 genome.

PubMed

Cai, Jie; Xie, Xiaohong; Hu, Yi; Zhan, Yang; Yu, Wanting; Wang, Aibing; Wang, Naidong

2017-06-01

Porcine circovirus associated diseases (PCVAD) caused by PCV2 are responsible for severe economic losses in the swine industry. The mechanism of PCV2 replication has not been fully elucidated yet. PCV2 may be successfully rescued by means of either an infectious DNA clone containing the full length of the viral genomic DNA, or from PCV2-infected clinical tissues in PK15 cell culture. However, viruses harvested by both methods have low titres. In this study, PCV2 was prepared with a higher titre from PK15 cells infected by recombinant baculoviruses containing 1PCV2 (one stem-loop structure) or 1.1PCV2 (two stem-loop structure) genomic DNA copy. In addition, infectious DNA clones containing two stem-loop structures in either plasmid or baculovirus backbones are capable of generating a higher virus titre than the DNA clones with only one copy of stem-loop structure.

The production of multiprotein complexes in insect cells using the baculovirus expression system.

PubMed

Abdulrahman, Wassim; Radu, Laura; Garzoni, Frederic; Kolesnikova, Olga; Gupta, Kapil; Osz-Papai, Judit; Berger, Imre; Poterszman, Arnaud

2015-01-01

The production of a homogeneous protein sample in sufficient quantities is an essential prerequisite not only for structural investigations but represents also a rate-limiting step for many functional studies. In the cell, a large fraction of eukaryotic proteins exists as large multicomponent assemblies with many subunits, which act in concert to catalyze specific activities. Many of these complexes cannot be obtained from endogenous source material, so recombinant expression and reconstitution are then required to overcome this bottleneck. This chapter describes current strategies and protocols for the efficient production of multiprotein complexes in large quantities and of high quality, using the baculovirus/insect cell expression system.

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.

Tribological Characteristics of Submicron SiC(p)-GR(p)/Zn-35Al-1Mg Composites in Semisolidification Casting process

NASA Astrophysics Data System (ADS)

Liu, S.; Fang, Z. W.; Li, L. X.

2018-05-01

Uniform SiC(p)-GR(p)/Zn-35Al-1Mg composites were prepared by powder pressing and semisolidification stirring-casting process by adding submicron silicon carbide and graphite reinforcement particles in an aluminum-zinc alloy matrix. Micro Vickers hardness and microstructures of the novel composites were studied, and their wear properties and wear temperature were measured for different load and friction conditions. The results show that silicon carbide and graphite particles homogeneously mix in the matrix, while contained silicon carbide particles improve the matrix hardness to 8.4%, graphite improves the matrix hardness to 16.8%, but two of them, combined, reduce the matrix hardness to 7.6%; the rate of temperature rise of the zinc-aluminum matrix alloy is the highest than the other three composites and is up to 48.5° C/s at 1.69MPa. At 0.56MPa and sliding 26.4km, the graphite composite anti-wear effect is optimal, while at 1.13MPa, the wear resistance of silicon carbide and graphite compound particles is the best; in the other case of only silicon carbide particles, the wear resistance is increased to 35% at 1.69MPa and 26.4km, and its anti-wear effects are excellent.

Recombinant ELISA using baculovirus-expressed VP2 for detection of antibodies against canine parvovirus.

PubMed

Elia, Gabriella; Desario, Costantina; Pezzoni, Giulia; Camero, Michele; Brocchi, Emiliana; Decaro, Nicola; Martella, Vito; Buonavoglia, Canio

2012-09-01

The gene encoding the VP2 protein of canine parvovirus type 2 was expressed in an insect-baculovirus system. The recombinant (r) VP2 was similar antigenically/functionally to the native capsid protein as demonstrated by hemagglutination, Western blotting and hemagglutination inhibition test, using Canine parvovirus type-2 (CPV-2) positive sera. An enzyme-linked immunosorbent assay (ELISA) using the rVP2 was used for testing CPV-2 positive and negative sera from dogs and for determining the threshold of maternally derived antibodies interfering with successful vaccination of pups against CPV-2. Copyright © 2012 Elsevier B.V. All rights reserved.

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

Increased IL-35 producing Tregs and CD19+IL-35+ cells are associated with disease progression in leprosy patients.

PubMed

Tarique, Mohd; Saini, Chaman; Naqvi, Raza Ali; Khanna, Neena; Rao, D N

2017-03-01

The clinical forms of leprosy consist of a spectrum that reflects the host's immune response to the M. leprae; it provides an ideal model to study the host pathogen interaction and immunological dysregulation in humans. IL-10 and TGF-β producing Tregs are high in leprosy patients and responsible for immune suppression and M. leprae specific T cells anergy. In leprosy, involvement of IL-35 producing Tregs and Bregs remain unstudied. To study the role of IL-35 producing Tregs and Bregs in the human leprosy. Peripheral blood mononuclear cells from leprosy patients were isolated and stimulated with M. leprae antigen (MLCwA) for 48h. Intracellular cytokine IL-35 was evaluated in CD4 + CD25 + Tregs, CD19 + cells by FACS. Expression of PD-1 on CD4 + CD25 + Tregs, CD19 + cells and its ligand (PD-L1) on B cells, CD11c cells were evaluated by flow cytometry (FACS). Serum IL-35 level was estimated by ELISA. The frequency of IL-35 producing Tregs and Bregs cells were found to be high in leprosy patients (p<0.0001) as compared to healthy controls. These cells produced suppressive cytokine IL-35 which showed positive correlation with bacteriological index (BI) and TGF-β producing Tregs, indicating its suppressive nature. We found higher expression of PD-1 on Tregs, B cell and its ligand (PD-L1) on antigen presenting cells in leprosy patients. This study point out a shift in our understanding of the immunological features that mediate and regulate the immune suppression and the disease progression in leprosy patients with a new paradigm (IL-35 producing Tregs and Bregs) that is beyond TGF-β and IL-10 producing Treg cells. Copyright © 2016 Elsevier Ltd. All rights reserved.

Cloning, promoter analysis and expression in response to bacterial exposure of sea bass (Dicentrarchus labrax L.) interleukin-12 p40 and p35 subunits.

PubMed

Nascimento, Diana S; do Vale, Ana; Tomás, Ana M; Zou, Jun; Secombes, Christopher J; dos Santos, Nuno M S

2007-03-01

Interleukin-12 (IL-12) is a heterodimeric cytokine pivotal in resistance to microbial and viral infections. In the search for immunoregulatory genes in sea bass the genes for the two IL-12 subunits p40 and p35 were cloned and sequenced. Molecular characterization of these two genes was performed at both the cDNA and genomic levels. Sea bass IL-12 p40 and p35 conserve most cysteines involved in the intra-chain disulfide bonds of human IL-12 subunits as well as the important structural residues for human IL-12 heterodimerization. The gene organization of sea bass IL-12 p40 is similar to the human orthologue, whilst the sea bass IL-12 p35 gene structure, as reported for pufferfish, differs from the human one in containing an additional exon and lacking a second copy of a duplicated exon present in the mammalian genes. The promoter analysis of both sea bass and pufferfish IL-12 genes showed the presence of the main cis-acting elements involved in the transcriptional regulation of human and mouse orthologues. The involvement of IL-12 in sea bass anti-bacterial immune responses was demonstrated by investigating the expression profiles of IL-1beta, IL-12 p40 and p35 in the head-kidney and spleen following intraperitoneal injection of UV-killed and live Photobacterium damselae ssp. piscicida (Phdp). Finally, the importance of nuclear factor (NF)-kappaB on UV-killed Phdp-induced IL-12 p40 and p35 gene transcription was shown by the use of pyrrolidine dithiocarbamate (PDTC).

Baculovirus-vectored multistage Plasmodium vivax vaccine induces both protective and transmission-blocking immunities against transgenic rodent malaria parasites.

PubMed

Mizutani, Masanori; Iyori, Mitsuhiro; Blagborough, Andrew M; Fukumoto, Shinya; Funatsu, Tomohiro; Sinden, Robert E; Yoshida, Shigeto

2014-10-01

A multistage malaria vaccine targeting the pre-erythrocytic and sexual stages of Plasmodium could effectively protect individuals against infection from mosquito bites and provide transmission-blocking (TB) activity against the sexual stages of the parasite, respectively. This strategy could help prevent malaria infections in individuals and, on a larger scale, prevent malaria transmission in communities of endemicity. Here, we describe the development of a multistage Plasmodium vivax vaccine which simultaneously expresses P. vivax circumsporozoite protein (PvCSP) and P25 (Pvs25) protein of this species as a fusion protein, thereby acting as a pre-erythrocytic vaccine and a TB vaccine, respectively. A new-concept vaccine platform based on the baculovirus dual-expression system (BDES) was evaluated. The BDES-Pvs25-PvCSP vaccine displayed correct folding of the Pvs25-PvCSP fusion protein on the viral envelope and was highly expressed upon transduction of mammalian cells in vitro. This vaccine induced high levels of antibodies to Pvs25 and PvCSP and elicited protective (43%) and TB (82%) efficacies against transgenic P. berghei parasites expressing the corresponding P. vivax antigens in mice. Our data indicate that our BDES, which functions as both a subunit and DNA vaccine, can offer a promising multistage vaccine capable of delivering a potent antimalarial pre-erythrocytic and TB response via a single immunization regimen. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Protein Expression in Insect and Mammalian Cells Using Baculoviruses in Wave Bioreactors.

PubMed

Kadwell, Sue H; Overton, Laurie K

2016-01-01

Many types of disposable bioreactors for protein expression in insect and mammalian cells are now available. They differ in design, capacity, and sensor options, with many selections available for either rocking platform, orbitally shaken, pneumatically mixed, or stirred-tank bioreactors lined with an integral disposable bag (Shukla and Gottschalk, Trends Biotechnol 31(3):147-154, 2013). WAVE Bioreactors™ were among the first disposable systems to be developed (Singh, Cytotechnology 30:149-158, 1999). Since their commercialization in 1999, Wave Bioreactors have become routinely used in many laboratories due to their ease of operation, limited utility requirements, and protein expression levels comparability to traditional stirred-tank bioreactors. Wave Bioreactors are designed to use a presterilized Cellbag™, which is attached to a rocking platform and inflated with filtered air provided by the bioreactor unit. The Cellbag can be filled with medium and cells and maintained at a set temperature. The rocking motion, which is adjusted through angle and rock speed settings, provides mixing of oxygen (and CO2, which is used to control pH in mammalian cell cultures) from the headspace created in the inflated Cellbag with the cell culture medium and cells. This rocking motion can be adjusted to prevent cell shear damage. Dissolved oxygen and pH can be monitored during scale-up, and samples can be easily removed to monitor other parameters. Insect and mammalian cells grow very well in Wave Bioreactors (Shukla and Gottschalk, Trends Biotechnol 31(3):147-154, 2013). Combining Wave Bioreactor cell growth capabilities with recombinant baculoviruses engineered for insect or mammalian cell expression has proven to be a powerful tool for rapid production of a wide range of proteins.

High-yield production of canine parvovirus virus-like particles in a baculovirus expression system.

PubMed

Jin, Hongli; Xia, Xiaohong; Liu, Bing; Fu, Yu; Chen, Xianping; Wang, Huihui; Xia, Zhenqiang

2016-03-01

An optimized VP2 gene from the current prevalent CPV strain (new CPV-2a) in China was expressed in a baculovirus expression system. It was found that the VP2 proteins assembled into virus-like particles (VLPs) with antigenic properties similar to those of natural CPV and with an especially high hemagglutination (HA) titer (1:2(20)). Dogs intramuscularly or orally immunized with VLPs produced antibodies against CPV with >1:80 hemagglutination inhibition (HI) units for at least 3 months. The CPV VLPs could be considered for use as a vaccine against CPV or as a platform for research on chimeric VLP vaccines against other diseases.

Development of a general method for detection and quantification of the P35S promoter based on assessment of existing methods

PubMed Central

Wu, Yuhua; Wang, Yulei; Li, Jun; Li, Wei; Zhang, Li; Li, Yunjing; Li, Xiaofei; Li, Jun; Zhu, Li; Wu, Gang

2014-01-01

The Cauliflower mosaic virus (CaMV) 35S promoter (P35S) is a commonly used target for detection of genetically modified organisms (GMOs). There are currently 24 reported detection methods, targeting different regions of the P35S promoter. Initial assessment revealed that due to the absence of primer binding sites in the P35S sequence, 19 of the 24 reported methods failed to detect P35S in MON88913 cotton, and the other two methods could only be applied to certain GMOs. The rest three reported methods were not suitable for measurement of P35S in some testing events, because SNPs in binding sites of the primer/probe would result in abnormal amplification plots and poor linear regression parameters. In this study, we discovered a conserved region in the P35S sequence through sequencing of P35S promoters from multiple transgenic events, and developed new qualitative and quantitative detection systems targeting this conserved region. The qualitative PCR could detect the P35S promoter in 23 unique GMO events with high specificity and sensitivity. The quantitative method was suitable for measurement of P35S promoter, exhibiting good agreement between the amount of template and Ct values for each testing event. This study provides a general P35S screening method, with greater coverage than existing methods. PMID:25483893

Expression and Self-Assembly in Baculovirus of Porcine Enteric Calicivirus Capsids into Virus-Like Particles and Their Use in an Enzyme-Linked Immunosorbent Assay for Antibody Detection in Swine

PubMed Central

Guo, Mingzhang; Qian, Yuan; Chang, Kyeong-Ok; Saif, Linda J.

2001-01-01

Porcine enteric calicivirus (PEC) causes diarrhea and intestinal lesions in pigs. PEC strain Cowden grows to low to moderate titers in cell culture but only with the addition of intestinal contents from uninfected gnotobiotic pigs (W. T. Flynn and L. J. Saif, J. Clin. Microbiol. 26:206–212, 1988; A. V. Parwani, W. T. Flynn, K. L. Gadfield, and L. J. Saif, Arch. Virol. 120:115–122, 1991). Cloning and sequence analysis of the PEC Cowden full-length genome revealed that it is most closely related genetically to the human Sapporo-like viruses. In this study, the complete PEC capsid gene was subcloned into the plasmid pBlueBac4.5 and the recombinant baculoviruses were identified by plaque assay and PCR. The PEC capsid protein was expressed in insect (Sf9) cells inoculated with the recombinant baculoviruses, and the recombinant capsid proteins self- assembled into virus-like particles (VLPs) that were released into the cell supernatant and purified by CsCl gradient centrifugation. The PEC VLPs had the same molecular mass (58 kDa) as the native virus capsid and reacted with pig hyperimmune and convalescent-phase sera to PEC Cowden in enzyme-linked immunosorbent assay (ELISA) and Western blotting. The PEC capsid VLPs were morphologically and antigenically similar to the native virus by immune electron microscopy. High titers (1:102,400 to 204,800) of PEC-specific antibodies were induced in guinea pigs inoculated with PEC VLPs, suggesting that the VLPs could be useful for future candidate PEC vaccines. A fixed-cell ELISA and VLP ELISA were developed to detect PEC serum antibodies in pigs. For the fixed-cell ELISA, Sf9 cells were infected with recombinant baculoviruses expressing PEC capsids, followed by cell fixation with formalin. For the VLP ELISA, the VLPs were used for the coating antigen. Our data indicate that both tests were rapid, specific, and reproducible and might be used for large-scale serological investigations of PEC antibodies in swine. PMID:11283075

Inducible Knockout of the Cyclin-Dependent Kinase 5 Activator p35 Alters Hippocampal Spatial Coding and Neuronal Excitability

PubMed Central

Kamiki, Eriko; Boehringer, Roman; Polygalov, Denis; Ohshima, Toshio; McHugh, Thomas J.

2018-01-01

p35 is an activating co-factor of Cyclin-dependent kinase 5 (Cdk5), a protein whose dysfunction has been implicated in a wide-range of neurological disorders including cognitive impairment and disease. Inducible deletion of the p35 gene in adult mice results in profound deficits in hippocampal-dependent spatial learning and synaptic physiology, however the impact of the loss of p35 function on hippocampal in vivo physiology and spatial coding remains unknown. Here, we recorded CA1 pyramidal cell activity in freely behaving p35 cKO and control mice and found that place cells in the mutant mice have elevated firing rates and impaired spatial coding, accompanied by changes in the temporal organization of spiking both during exploration and rest. These data shed light on the role of p35 in maintaining cellular and network excitability and provide a physiological correlate of the spatial learning deficits in these mice. PMID:29867369

Identification of binding domains in the herpes simplex virus type 1 small capsid protein pUL35 (VP26).

PubMed

Apcarian, Arin; Cunningham, Anthony L; Diefenbach, Russell J

2010-11-01

In this study, fragments of the small capsid protein pUL35 (VP26) from herpes simplex virus type 1 (HSV-1) were generated to identify binding domains for a number of known ligands. Analysis of the binding of dynein light chain subunits, DYNLT1 and DYNLT3, as well the HSV-1 structural proteins pUL19 (VP5) and pUL37 was then undertaken using the LexA yeast two-hybrid assay. The N-terminal half of pUL35, in particular residues 30-43, was identified as a common region for the binding of DYNLT1 and DYNLT3. Additional distinct regions in the C terminus of pUL35 also contribute to the binding of DYNLT1 and DYNLT3. In contrast, only the C-terminal half of pUL35 was found to mediate the binding of pUL19 and pUL37 through distinct regions. The relevance of this information to the role of pUL35 in viral transport and assembly is discussed.

Reversibly Switchable, pH-Dependent Peptide Ligand Binding via 3,5-Diiodotyrosine Substitutions.

PubMed

Ngambenjawong, Chayanon; Sylvestre, Meilyn; Gustafson, Heather H; Pineda, Julio Marco B; Pun, Suzie H

2018-04-20

Cell type-specific targeting ligands utilized in drug delivery applications typically recognize receptors that are overexpressed on the cells of interest. Nonetheless, these receptors may also be expressed, to varying extents, on off-target cells, contributing to unintended side effects. For the selectivity profile of targeting ligands in cancer therapy to be improved, stimuli-responsive masking of these ligands with acid-, redox-, or enzyme-cleavable molecules has been reported, whereby the targeting ligands are exposed in specific environments, e.g., acidic tumor hypoxia. One possible drawback of these systems lies in their one-time, permanent trigger, which enables the "demasked" ligands to bind off-target cells if released back into the systemic circulation. A promising strategy to address the aforementioned problem is to design ligands that show selective binding based on ionization state, which may be microenvironment-dependent. In this study, we report a systematic strategy to engineer low pH-selective targeting peptides using an M2 macrophage-targeting peptide (M2pep) as an example. 3,5-Diiodotyrosine mutagenesis into native tyrosine residues of M2pep confers pH-dependent binding behavior specific to acidic environment (pH 6) when the amino acid is protonated into the native tyrosine-like state. At physiological pH of 7.4, the hydroxyl group of 3,5-diiodotyrosine on the peptide is deprotonated leading to interruption of the peptide native binding property. Our engineered pH-responsive M2pep (Ac-Y-Î-Î) binds target M2 macrophages more selectively at pH 6 than at pH 7.4. In addition, 3,5-diiodotyrosine substitutions also improve serum stability of the peptide. Finally, we demonstrate pH-dependent reversibility in target binding via a postbinding peptide elution study. The strategy presented here should be applicable for engineering pH-dependent functionality of other targeting peptides with potential applications in physiology-dependent in vivo targeting

A soluble form of P74 can act as a per os infectivity factor to the autographa californica multiple nucleopolyhedrovirus

USDA-ARS?s Scientific Manuscript database

The baculovirus occlusion-derived virion (ODV) is required to spread virus infection among insect hosts via the per os route. The Autographa californica Multicapsid Nucleopolyhedrovirus (AcMNPV) P74 protein is an ODV envelope protein that is essential for ODVs to be infectious. P74 is anchored in ...

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.

DEPENDENCE OF ECDYSTEROID METABOLISM AND DEVELOPMENT IN HOST LARVAE ON THE TIME OF BACULOVIRUS INFECTION AND THE ACTIVITY OF THE UDP-GLUCOSYL TRANSFERASE GENE.

EPA Science Inventory

Infection of fourth-instar gypsy moth (Lymantria dispar, Lepidoptera: Lymantriidae) larvae with the wild-type (Wt) gypsy moth baculovirus, LdNPV on the first day post-molt, or infection of fifth instars on the fifth day post-molt, results in elevated ecdysteroid levels in both he...

The complete genome sequence of a third distinct baculovirus isolated from the true armyworm, Mythimna unipuncta, contains two copies of the lef-7 gene

USDA-ARS?s Scientific Manuscript database

A baculovirus isolate from a USDA Forest Service collection was examined by electron microscopy and analysis of its genome sequence. The isolate, formerly referred to as Pseudoletia (Mythimna) sp. nucleopolyhedrovirus #7 (MyspNPV#7), was determined by barcoding PCR to derive from the host species My...

Baculovirus directly activates murine NK cells via TLR9.

PubMed

Moriyama, T; Suzuki, T; Chang, M O; Kitajima, M; Takaku, H

2017-04-01

The importance of natural killer (NK) cells in innate immune responses against tumors or viral infections enhances the appeal of NK cell-based immunotherapeutic approaches. We have recently reported that baculovirus (BV)-infected dendritic cells (DCs; BV-DCs) induce antitumor immunity against established tumors in mice. These antitumor effects were CD8 + T-cell and NK cell dependent; however, they were found to be CD4 + T-cell independent. In this study, we investigated the involvement of Toll-like receptor 9 (TLR9) in the process of BV recognition by NK cells. We found that BV directly stimulated NK cells, induced the expression of the activation marker CD69 and promoted interferon-gamma (IFN-γ) production and cytotoxicity. Moreover, TLR9 knockout in mice (tlr9-/- NK cells) inhibited NK cell responses to BV, indicating that TLR9 may have a relevant role in the BV-induced upregulation of NK cell functions. Our data demonstrated for the first time that NK cells directly recognize BV via TLR9, which provides opportunities for the use of this technique as an effective tool for BV-based immunotherapies against malignancies.

Increased plasma viscosity in young women with polycystic ovary syndrome using an oral contraceptive containing 35 μg ethinyl estradiol and 2 mg cyproterone acetate.

PubMed

Markantes, George; Saltamavros, Alexandros D; Vervita, Vasiliki; Armeni, Anastasia K; Karela, Anastasia; Adonakis, George; Decavalas, George; Georgopoulos, Neoklis A

2011-12-01

To investigate the influence of 6 months of treatment with an oral contraceptive (OC) containing 35 μ g ethinyl estradiol and 2 mg cyproterone acetate on plasma viscosity (PV) in young women with polycystic ovary syndrome (PCOS). Patients with PCOS were assessed for PV before and after 6 months of treatment with an OC containing 35 μg ethinyl estradiol and 2 mg cyproterone acetate. PV was determined by a viscometer Type 53610/I SCHOTT-Instruments, Mainz at 37°C. Subjects were recruited from the Department of Obstetrics and Gynaecology, Division of Reproductive Endocrinology at the University Hospital of Patras, Greece. The study included 66 young women with PCOS. PV. In PCOS women as a whole, PV at baseline was 1.249 ± 0.049 mm(2)/s (n = 66). After 6 months of treatment with an OC containing 35 μg ethinyl estradiol and 2 mg cyproterone acetate, PV was increased to 1.268 ± 0.065 mm(2)/s (p = 0.038). The difference between PV before and after 6 months of treatment with an OC containing 35 μg ethinyl estradiol and 2 mg cyproterone acetate (Δviscosity) was 0.01864 ± 0.071452 mm(2)/s. ΔViscosity was related to ?fibrinogen (r = 0.270, p = 0.046), to Δhematocrit (r = 0.514, p = 0.09) and to Δtriglycerides (r = 0.292, p = 0.021). Young women with PCOS presented an increased PV under OC treatment with 35 μg ethinyl estradiol and 2 mg cyproterone acetate.

The T-cell antigen CD5 acts as a receptor and substrate for the protein-tyrosine kinase p56lck.

PubMed Central

Raab, M; Yamamoto, M; Rudd, C E

1994-01-01

CD5 is a T-cell-specific antigen which binds to the B-cell antigen CD72 and acts as a coreceptor in the stimulation of T-cell growth. CD5 associates with the T-cell receptor zeta chain (TcR zeta)/CD3 complex and is rapidly phosphosphorylated on tyrosine residues as a result of TcR zeta/CD3 ligation. However, despite this, the mechanism by which CD5 generates intracellular signals is unclear. In this study, we demonstrate that CD5 is coupled to the protein-tyrosine kinase p56lck and can act as a substrate for p56lck. Coexpression of CD5 with p56lck in the baculovirus expression system resulted in the phosphorylation of CD5 on tyrosine residues. Further, anti-CD5 and anti-p56lck coprecipitated each other in a variety of detergents, including Nonidet P-40 and Triton X-100. Anti-CD5 also precipitated the kinase from various T cells irrespective of the expression of TcR zeta/CD3 or CD4. No binding between p59fyn(T) and CD5 was detected in T cells. The binding of p56lck to CD5 induced a 10- to 15-fold increase in p56lck catalytic activity, as measured by in vitro kinase analysis. In vivo labelling with 32P(i) also showed a four- to fivefold increase in Y-394 occupancy in p56lck when associated with CD5. The use of glutathione S-transferase-Lck fusion proteins in precipitation analysis showed that the SH2 domain of p56lck could recognize CD5 as expressed in the baculovirus expression system. CD5 interaction with p56lck represents a novel variant of a receptor-kinase complex in which receptor can also serve as substrate. The CD5-p56lck interaction is likely to play roles in T-cell signalling and T-B collaboration. Images PMID:7513045

Characterization of self-assembled virus-like particles of dromedary camel hepatitis e virus generated by recombinant baculoviruses.

PubMed

Zhou, Xianfeng; Kataoka, Michiyo; Liu, Zheng; Takeda, Naokazu; Wakita, Takaji; Li, Tian-Cheng

2015-12-02

Dromedary camel hepatitis E virus (DcHEV), a novel hepatitis E virus, has been identified in dromedary camels in Dubai, United Arab Emirates. The antigenicity, pathogenicity and epidemiology of this virus have been unclear. Here we first used a recombinant baculovirus expression system to express the 13 and 111 N-terminus amino-acid-truncated DcHEV ORF2 protein in insect Tn5 cells, and we obtained two types of virus-like particles (VLPs) with densities of 1.300 g/cm(3) and 1.285 g/cm(3), respectively. The small VLPs (Dc4sVLPs) were estimated to be 24 nm in diameter, and were assembled by a protein with the molecular mass 53 kDa. The large VLPs (Dc3nVLPs and Dc4nVLPs) were 35 nm in diameter, and were assembled by a 64-kDa protein. An antigenic analysis demonstrated that DcHEV was cross-reactive with G1, G3-G6, ferret and rat HEVs, and DcHEV showed a stronger cross-reactivity to G1 G3-G6 HEV than it did to rat and ferret HEV. In addition, the antibody against DcHEV-LPs neutralized G1 and G3 HEV in a cell culture system, suggesting that the serotypes of these HEVs are identical. We also found that the amino acid residue Met-358 affects the small DcHEV-LPs assembly. Copyright © 2015 Elsevier B.V. All rights reserved.

Increasing Incidence of Plasmodium knowlesi Malaria following Control of P. falciparum and P. vivax Malaria in Sabah, Malaysia

PubMed Central

William, Timothy; Rahman, Hasan A.; Jelip, Jenarun; Ibrahim, Mohammad Y.; Menon, Jayaram; Grigg, Matthew J.; Yeo, Tsin W.; Anstey, Nicholas M.; Barber, Bridget E.

2013-01-01

Background The simian parasite Plasmodium knowlesi is a common cause of human malaria in Malaysian Borneo and threatens the prospect of malaria elimination. However, little is known about the emergence of P. knowlesi, particularly in Sabah. We reviewed Sabah Department of Health records to investigate the trend of each malaria species over time. Methods Reporting of microscopy-diagnosed malaria cases in Sabah is mandatory. We reviewed all available Department of Health malaria notification records from 1992–2011. Notifications of P. malariae and P. knowlesi were considered as a single group due to microscopic near-identity. Results From 1992–2011 total malaria notifications decreased dramatically, with P. falciparum peaking at 33,153 in 1994 and decreasing 55-fold to 605 in 2011, and P. vivax peaking at 15,857 in 1995 and decreasing 25-fold to 628 in 2011. Notifications of P. malariae/P. knowlesi also demonstrated a peak in the mid-1990s (614 in 1994) before decreasing to ≈100/year in the late 1990s/early 2000s. However, P. malariae/P. knowlesi notifications increased >10-fold between 2004 (n = 59) and 2011 (n = 703). In 1992 P. falciparum, P. vivax and P. malariae/P. knowlesi monoinfections accounted for 70%, 24% and 1% respectively of malaria notifications, compared to 30%, 31% and 35% in 2011. The increase in P. malariae/P. knowlesi notifications occurred state-wide, appearing to have begun in the southwest and progressed north-easterly. Conclusions A significant recent increase has occurred in P. knowlesi notifications following reduced transmission of the human Plasmodium species, and this trend threatens malaria elimination. Determination of transmission dynamics and risk factors for knowlesi malaria is required to guide measures to control this rising incidence. PMID:23359830

Essential function of VCP/p97 in infection cycle of the nucleopolyhedrovirus AcMNPV in Spodoptera frugiperda Sf9 cells.

PubMed

Lyupina, Yulia V; Erokhov, Pavel A; Kravchuk, Oksana I; Finoshin, Alexander D; Abaturova, Svetlana B; Orlova, Olga V; Beljelarskaya, Svetlana N; Kostyuchenko, Margarita V; Mikhailov, Victor S

2018-06-08

The protein VCP/p97 (also named CDC48 and TER94) belongs to a type II subfamily of the AAA+ATPases and controls cellular proteostasis by acting upstream of proteasomes in the ubiquitin-proteasome protein degradation pathway. The function of VCP/p97 in the baculovirus infection cycle in insect cells remains unknown. Here, we identified VCP/p97 in the fall armyworm Spodoptera frugiperda (Sf9) cells and analyzed the replication of the Autographa californica multiple nucleopolyhedrovirus, AcMNPV, in Sf9 cells in which the VCP/p97 function was inhibited. The specific allosteric inhibitor of the VCP/p97 ATPase activity, NMS-873, did not deplete VCP/p97 in infected cells but caused a dose-dependent inhibition of viral DNA synthesis and efficiently suppressed expression of viral proteins and production of budded virions. NMS-873 caused accumulation of ubiquitinated proteins in a manner similar to the inhibitor of proteasome activity, Bortezomib. This suggests the essential function of VCP/p97 in the baculovirus infection cycle might be associated, at least in part, with the ubiquitin-proteasome system. Copyright © 2018 Elsevier B.V. All rights reserved.

Effect of pH on whitening efficacy of 35% hydrogen peroxide and enamel microhardness.

PubMed

Jurema, Ana Luiza Barbosa; de Souza, Mauricio Yugo; Torres, Carlos Rocha Gomes; Borges, Alessandra Bühler; Caneppele, Taciana Marco Ferraz

2018-03-01

This study aimed to evaluate the effect of 35% hydrogen peroxide at different pH values and the degree of tooth staining on whitening efficacy and enamel microhardness. 90 enamel-dentin specimens were obtained from bovine incisors. They were randomly divided into 2 groups (n = 45), 1 group was immersed in a staining broth for 14 days, and another group was not stained and kept in distilled water at 37°C. Twenty-four hours after the staining procedure, each group was distributed into 3 subgroups that were whitened by 35% hydrogen peroxide with different pH values (5, 7, and 8.4) for 30 minutes. The color was measured at baseline and 7 days after whitening. Microhardness was measured at baseline, immediate, 24 hours, and 1 month after the whitening procedure. Data were submitted to 2-way analysis of variance (ANOVA) and the Tukey test for multiple comparisons for color analysis. Repeated measures ANOVA and the Tukey test were used to analyze microhardness data. The color change of the stained groups (ΔE 00  = 4.6) was significantly higher than that of the nonstained groups (ΔE 00  = 3.7). Microhardness value decreased significantly immediately after whitening for all subgroups and did not return to initial values. For each measurement time, microhardness was not significantly different among subgroups with different pH values. Despite the effectiveness of 35% hydrogen peroxide, changes on gel pH did not affect the whitening efficacy, and the enamel was superficially demineralized, regardless of pH values. Independently of the pH value of whitening gel, enamel undergoes superficial demineralization and with a reduction in superficial microhardness that does not return to the initial values. However, using hydrogen peroxide with different pH values does not alter the whitening effect. © 2018 Wiley Periodicals, Inc.

Comparison of two eukaryotic systems for the expression of VP6 protein of rotavirus specie A: transient gene expression in HEK293-T cells and insect cell-baculovirus system.

PubMed

da Silva Junior, Haroldo Cid; da Silva E Mouta Junior, Sérgio; de Mendonça, Marcos César Lima; de Souza Pereira, Mirian Claudia; da Rocha Nogueira, Alanderson; de Azevedo, Maria Luiza Borges; Leite, José Paulo Gagliardi; de Moraes, Márcia Terezinha Baroni

2012-09-01

The VP6 protein of rotavirus A (RVA) is a target antigen used for diagnostic assays and also for the development of new RVA vaccines. We have compared the expression of VP6 protein in human embryonic kidney (HEK293-T) cells with results obtained using a well-established insect cell-baculovirus system. The recombinant VP6 (rVP6) expressed in HEK293-T cells did not present degradation and also retained the ability to form trimers. In the insect cell-baculovirus system, rVP6 was expressed at higher levels and with protein degradation as well as partial loss of ability to form trimers was observed. Therefore, HEK293-T cells represent a less laborious alternative system than insect cells for expression of rVP6 from human RVA.

Development of a sensitive and specific indirect enzyme-linked immunosorbent assay based on a baculovirus recombinant antigen for detection of specific antibodies against Ehrlichia canis.

PubMed

López, Lissett; Venteo, Angel; Aguirre, Enara; García, Marga; Rodríguez, Majosé; Amusátegui, Inmaculada; Tesouro, Miguel A; Vela, Carmen; Sainz, Angel; Rueda, Paloma

2007-11-01

An indirect enzyme-linked immunosorbent assay (ELISA) based on baculovirus recombinant P30 protein of Ehrlichia canis and the 1BH4 anticanine IgG monoclonal antibody was developed and evaluated by examining a panel of 98 positive and 157 negative sera using the indirect fluorescent antibody (IFA) test as the reference technique. The P30-based ELISA appeared to be sensitive and specific (77.55% and 95.54%, respectively) when qualitative results (positive/negative) were compared with those of the IFA test; the coefficient of correlation (R) between the 2 tests was 0.833. Furthermore, it was possible to establish a mathematical formula for use in comparing the results of both techniques. These results indicate that recombinant P30 antigen-based ELISA is a suitable alternative of the IFA test for simple, consistent, and rapid serodiagnosis of canine ehrlichiosis. Moreover, the use of this recombinant protein as antigen offers a great advantage for antigen preparation in comparison with other techniques in which the whole E. canis organism is used as antigen.

The role of the PI(3,5)P2 kinase TbFab1 in endo/lysosomal trafficking in Trypanosoma brucei.

PubMed

Gilden, Julia K; Umaer, Khan; Kruzel, Emilia K; Hecht, Oliver; Correa, Renan O; Mansfield, John M; Bangs, James D

2017-06-01

Protein trafficking through endo/lysosomal compartments is critically important to the biology of the protozoan parasite Trypanosoma brucei, but the routes material may take to the lysosome, as well as the molecular factors regulating those routes, remain incompletely understood. Phosphoinositides are signaling phospholipids that regulate many trafficking events by recruiting specific effector proteins to discrete membrane subdomains. In this study, we investigate the role of one phosphoinositide, PI(3,5)P 2 in T. brucei. We find a low steady state level of PI(3,5)P 2 in bloodstream form parasites comparable to that of other organisms. RNAi knockdown of the putative PI(3)P-5 kinase TbFab1 decreases the PI(3,5)P 2 pool leading to rapid cell death. TbFab1 and PI(3,5)P 2 both localize strongly to late endo/lysosomes. While most trafficking functions were intact in TbFab1 deficient cells, including both endocytic and biosynthetic trafficking to the lysosome, lysosomal turnover of an endogenous ubiquitinylated membrane protein, ISG65, was completely blocked suggesting that TbFab1 plays a role in the ESCRT-mediated late endosomal/multivesicular body degradative pathways. Knockdown of a second component of PI(3,5)P 2 metabolism, the PI(3,5)P 2 phosphatase TbFig4, also resulted in delayed turnover of ISG65. Together, these results demonstrate an essential role for PI(3,5)P 2 in the turnover of ubiquitinylated membrane proteins and in trypanosome endomembrane biology. Copyright © 2017 Elsevier B.V. All rights reserved.

Comparative proteomics analysis of apoptotic Spodoptera frugiperda cells during p35 knockout Autographa californica multiple nucleopolyhedrovirus infection.

PubMed

Yu, Qian; Xiong, Youhua; Liu, Jianliang; Wang, Qin; Qiu, Yuanxin; Wen, Dongling

2016-06-01

Infection with Autographa californica multiple nucleopolyhedrovirus (AcMNPV) mutants lacking a functional p35 gene can induce host cell apoptosis, which provides the possibility to use the potential of these viruses in the biological control of pest insects. Nonetheless, the proteomics or the protein changes of Spodoptera frugiperda (Sf9) cells infected with p35 knockout AcMNPV have not yet been studied. To further improve the use of AcMNPV, we set out to analyze the protein composition and protein changes of Sf9 cells of different infection stages by isobaric tag for relative and absolute quantification (iTRAQ) techniques. A total of 4004 sf9 proteins were identified by iTRAQ. After comparation of the significantly expressed 483 proteins from p35koAcMNPV-infected Sf9 cells and the significantly expressed 413 proteins from wtAcMNPV-infected Sf9 cells, we found that 226 proteins were specific to p35koAcMNPV-infected Sf9 cells. The 226 proteins were categorized according to GO classification for insects and were categorized into: biological processes, molecular functions and cellular components. Of interest, the most up-regulated proteins related to Epstein-Barr virus infection, RNA transport, Calcium signaling pathway, cGMP-PKG signaling pathway, oxidative phosphorylation and N-Glycan biosynthesis. Determination of the protein changes in p35 knockout AcMNPV-infected Sf9 cells would facilitate the better use of this virus-host cell interaction in pest insect control and other related fields. Copyright © 2016 Elsevier Inc. All rights reserved.

Seroprevalence of sapovirus in dogs using baculovirus-expressed virus-like particles.

PubMed

Melegari, Irene; Marsilio, Fulvio; Di Profio, Federica; Sarchese, Vittorio; Massirio, Ivano; Palombieri, Andrea; D'Angelo, Anna Rita; Lanave, Gianvito; Diakoudi, Georgia; Cavalli, Alessandra; Martella, Vito; Di Martino, Barbara

2018-06-02

Caliciviruses of the Sapovirus genus have been recently detected in dogs. Canine sapoviruses (SaVs) have been identified in the stools of young or juvenile animals with gastro-enteric disease at low prevalence (2.0-2.2%), but whether they may have a role as enteric pathogens and to which extent dogs are exposed to SaVs remains unclear. Here, we report the expression in a baculovirus system of virus like-particles (VLPs) of a canine SaV strain, the prototype virus Bari/4076/2007/ITA. The recombinant antigen was used to develop an enzyme-linked immunosorbent assay (ELISA). By screening an age-stratified collection of serum samples from 516 dogs in Italy, IgG antibodies specific for the canine SaV VLPs were detected in 40.3% (208/516) of the sera. Also, as observed for SaV infection in humans, we observed a positive association between seropositivity and age, with the highest prevalence rates in dogs older than 4 years of age. Copyright © 2018 Elsevier B.V. All rights reserved.

Membrane Order Is a Key Regulator of Divalent Cation-Induced Clustering of PI(3,5)P2 and PI(4,5)P2.

PubMed

Sarmento, Maria J; Coutinho, Ana; Fedorov, Aleksander; Prieto, Manuel; Fernandes, Fábio

2017-10-31

Although the evidence for the presence of functionally important nanosized phosphorylated phosphoinositide (PIP)-rich domains within cellular membranes has accumulated, very limited information is available regarding the structural determinants for compartmentalization of these phospholipids. Here, we used a combination of fluorescence spectroscopy and microscopy techniques to characterize differences in divalent cation-induced clustering of PI(4,5)P 2 and PI(3,5)P 2 . Through these methodologies we were able to detect differences in divalent cation-induced clustering efficiency and cluster size. Ca 2+ -induced PI(4,5)P 2 clusters are shown to be significantly larger than the ones observed for PI(3,5)P 2 . Clustering of PI(4,5)P 2 is also detected at physiological concentrations of Mg 2+ , suggesting that in cellular membranes, these molecules are constitutively driven to clustering by the high intracellular concentration of divalent cations. Importantly, it is shown that lipid membrane order is a key factor in the regulation of clustering for both PIP isoforms, with a major impact on cluster sizes. Clustered PI(4,5)P 2 and PI(3,5)P 2 are observed to present considerably higher affinity for more ordered lipid phases than the monomeric species or than PI(4)P, possibly reflecting a more general tendency of clustered lipids for insertion into ordered domains. These results support a model for the description of the lateral organization of PIPs in cellular membranes, where both divalent cation interaction and membrane order are key modulators defining the lateral organization of these lipids.

Dissolution of fluorapatite by Pseudomonas fluorescens P35 resulting in fluorine release

USGS Publications Warehouse

Zhou, Jianping; Wang, Hongmei; Cravotta, Charles A.; Dong, Qiang; Xiang, Xing

2017-01-01

Chemical weathering of fluorine-bearing minerals is widely accepted as the main mechanism for the release of fluorine (F) to groundwater. Here, we propose a potential mechanism of F release via microbial dissolution of fluorapatite (Ca5(PO4)3F), which has been neglected previously. Batch culture experiments were conducted at 30°C with a phosphate-solubilizing bacteria strain, Pseudomonas fluorescens P35, and rock phosphates as the sole source of phosphate for microbial growth in parallel with abiotic controls. Rock phosphates consisted of 55–91% of fluorapatite and 5–10% of dolomite before microbial dissolution as indicated by X-ray diffraction (XRD). Mineral composition and morphology changed after microbial dissolution characterized by the disappearance of dolomite and the development of etched cavities on rock phosphate surfaces. The pH of media used was approximately 7.4 at the beginning and increased gradually to 7.7 in abiotic controls; with the inoculum, the pH decreased to acidic values of 3.7–3.8 after 27 h. Phosphate, calcium, and fluoride were released from the rock phosphate to the acidified medium. At 42 h, the concentration of F reached 8.1–10.3 mg L−1. The elevated F concentration was two times higher than the F levels in groundwater in regions diagnosed with fluorosis, and was toxic to the bacteria, as demonstrated by a precipitous decrease in live cells. Geochemical modeling demonstrated that the oxidation of glucose (the carbon source for microbial growth in the medium) to gluconic acid could decrease the pH to 3.7–3.8 and result in the dissolution of fluorapatite and dolomite. Dolomite and fluorapatite remained unsaturated, while concentrations of dissolved phosphorus (P), calcium (Ca), and F increased throughout the time course Fluorite reached saturation [saturation index (SI) 0.22–0.42] after 42 h in rock phosphate–amended biotic systems. However, fluorite was not detected in XRD patterns of the final residue from

Targeting p35/Cdk5 signalling via CIP-peptide promotes angiogenesis in hypoxia.

PubMed

Bosutti, Alessandra; Qi, Jie; Pennucci, Roberta; Bolton, David; Matou, Sabine; Ali, Kamela; Tsai, Li-Huei; Krupinski, Jerzy; Petcu, Eugene B; Montaner, Joan; Al Baradie, Raid; Caccuri, Francesca; Caruso, Arnaldo; Alessandri, Giulio; Kumar, Shant; Rodriguez, Cristina; Martinez-Gonzalez, Jose; Slevin, Mark

2013-01-01

Cyclin-dependent kinase-5 (Cdk5) is over-expressed in both neurons and microvessels in hypoxic regions of stroke tissue and has a significant pathological role following hyper-phosphorylation leading to calpain-induced cell death. Here, we have identified a critical role of Cdk5 in cytoskeleton/focal dynamics, wherein its activator, p35, redistributes along actin microfilaments of spreading cells co-localising with p(Tyr15)Cdk5, talin/integrin beta-1 at the lamellipodia in polarising cells. Cdk5 inhibition (roscovitine) resulted in actin-cytoskeleton disorganisation, prevention of protein co-localization and inhibition of movement. Cells expressing Cdk5 (D144N) kinase mutant, were unable to spread, migrate and form tube-like structures or sprouts, while Cdk5 wild-type over-expression showed enhanced motility and angiogenesis in vitro, which was maintained during hypoxia. Gene microarray studies demonstrated myocyte enhancer factor (MEF2C) as a substrate for Cdk5-mediated angiogenesis in vitro. MEF2C showed nuclear co-immunoprecipitation with Cdk5 and almost complete inhibition of differentiation and sprout formation following siRNA knock-down. In hypoxia, insertion of Cdk5/p25-inhibitory peptide (CIP) vector preserved and enhanced in vitro angiogenesis. These results demonstrate the existence of critical and complementary signalling pathways through Cdk5 and p35, and through which coordination is a required factor for successful angiogenesis in sustained hypoxic condition.

The Baculovirus-Expressed Binding Region of Plasmodium falciparum EBA-140 Ligand and Its Glycophorin C Binding Specificity

PubMed Central

Rydzak, Joanna; Kaczmarek, Radoslaw; Czerwinski, Marcin; Lukasiewicz, Jolanta; Tyborowska, Jolanta; Szewczyk, Boguslaw; Jaskiewicz, Ewa

2015-01-01

The erythrocyte binding ligand 140 (EBA-140) is a member of the Plasmodium falciparum DBL family of erythrocyte binding proteins, which are considered as prospective candidates for malaria vaccine development. The EBA-140 ligand is a paralogue of the well-characterized P. falciparum EBA-175 protein. They share homology of domain structure, including Region II, which consists of two homologous F1 and F2 domains and is responsible for ligand-erythrocyte receptor interaction during invasion. In this report we describe, for the first time, the glycophorin C specificity of the recombinant, baculovirus-expressed binding region (Region II) of P. falciparum EBA-140 ligand. It was found that the recombinant EBA-140 Region II binds to the endogenous and recombinant glycophorin C, but does not bind to Gerbich-type glycophorin C, neither normal nor recombinant, which lacks amino acid residues 36–63 of its polypeptide chain. Our results emphasize the crucial role of this glycophorin C region in EBA-140 ligand binding. Moreover, the EBA-140 Region II did not bind either to glycophorin D, the truncated form of glycophorin C lacking the N-glycan or to desialylated GPC. These results draw attention to the role of glycophorin C glycans in EBA-140 binding. The full identification of the EBA-140 binding site on glycophorin C molecule, consisting most likely of its glycans and peptide backbone, may help to design therapeutics or vaccines that target the erythrocyte binding merozoite ligands. PMID:25588042

Development of antiviral gene therapy for Monodon baculovirus using dsRNA loaded chitosan-dextran sulfate nanocapsule delivery system in Penaeus monodon post-larvae.

PubMed

Ramesh Kumar, D; Elumalai, Rajasegaran; Raichur, Ashok M; Sanjuktha, M; Rajan, J J; Alavandi, S V; Vijayan, K K; Poornima, M; Santiago, T C

2016-07-01

In the present study, a suitable carrier system was developed for the delivery of dsRNA into Penaeus monodon (P. monodon) post larvae to silence the Monodon baculovirus (MBV) structural gene of p74. The carrier system was developed by layer by layer adsorption of oppositely charged chitosan-dextran sulfate, on charged silica nanoparticles. The silica template was removedto produce multilayered hollow nanocapsules (CS-DS) that were utilized for dsRNA loading at an alkaline pH. The capsule's surface was modified by conjugating with shrimp feed for enhanced cellular uptake. In vivo cellular uptake of CS-DS/FITC loaded nanocapsules conjugated with feed was studied after oral administration into post-larvae. The results revealed that the encapsulated FITC was effectively delivered and exhibited a sustained release into the cytoplasm of shrimp post-larvae. The MBV challenge study for structural gene p74was conducted after 3-25 days of post infection (dpi) with respective CS-DS/dsRNA coated with feed. The results showed a significant survival rate of 86.63% and effective gene silencing in P. monodon. Our findings indicated that the delivery of dsRNA using shrimp feed coatedCS-DSnanocapsules could be a novel approach to prevent viral infections in shrimp. Copyright © 2016 Elsevier B.V. All rights reserved.

The phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2)-dependent Tup1 conversion (PIPTC) regulates metabolic reprogramming from glycolysis to gluconeogenesis.

PubMed

Han, Bong-Kwan; Emr, Scott D

2013-07-12

Glucose/carbon metabolism is a fundamental cellular process in living cells. In response to varying environments, eukaryotic cells reprogram their glucose/carbon metabolism between aerobic or anaerobic glycolysis, oxidative phosphorylation, and/or gluconeogenesis. The distinct type of glucose/carbon metabolism that a cell carries out has significant effects on the cell's proliferation and differentiation. However, it is poorly understood how the reprogramming of glucose/carbon metabolism is regulated. Here, we report a novel endosomal PI(3,5)P2 lipid-dependent regulatory mechanism that is required for metabolic reprogramming from glycolysis to gluconeogenesis in Saccharomyces cerevisiae. Certain gluconeogenesis genes, such as FBP1 (encoding fructose-1,6-bisphosphatase 1) and ICL1 (encoding isocitrate lyase 1) are under control of the Mig1 repressor and Cyc8-Tup1 corepressor complex. We previously identified the PI(3,5)P2-dependent Tup1 conversion (PIPTC), a mechanism to convert Cyc8-Tup1 corepressor to Cti6-Cyc8-Tup1 coactivator. We demonstrate that the PIPTC plays a critical role for transcriptional activation of FBP1 and ICL1. Furthermore, without the PIPTC, the Cat8 and Sip4 transcriptional activators cannot be efficiently recruited to the promoters of FBP1 and ICL1, suggesting a key role for the PIPTC in remodulating the chromatin architecture at the promoters. Our findings expand our understanding of the regulatory mechanisms for metabolic reprogramming in eukaryotes to include key regulation steps outside the nucleus. Given that Tup1 and the metabolic enzymes that control PI(3,5)P2 are highly conserved among eukaryotes, our findings may provide important insights toward understanding glucose/carbon metabolic reprogramming in other eukaryotes, including humans.

Temperature Map, "Bonneville Crater" (1:35 p.m.)

NASA Image and Video Library

2004-05-17

Rates of change in surface temperatures during a martian day indicate differences in particle size in and near "Bonneville Crater." This image is the third in a series of five with color-coded temperature information from different times of day. This one is from 1:35 p.m. local solar time at the site where NASA's Mars Exploration Rover Spirit is exploring Mars. Temperature information from Spirit's miniature thermal emission spectrometer is overlaid onto a view of the site from Spirit's panoramic camera. In this color-coded map, quicker reddening during the day suggests sand or dust. (Red is about 270 Kelvin or 27 degrees Fahrenheit.) An example of this is in the shallow depression in the right foreground. Areas that stay blue longer into the day have larger rocks. (Blue indicates about 230 Kelvin or minus 45 Degrees F.) An example is the rock in the left foreground. http://photojournal.jpl.nasa.gov/catalog/PIA05930

Dengue-1 Virus Envelope Glycoprotein Gene Expressed in Recombinant Baculovirus Elicits Virus-Neutralizing Antibody in Mice and Protects them from Virus Challenge

DTIC Science & Technology

1991-01-01

8217 terminus of E. When the recombinant virus was grown in Spodoptera frugiperda cells. about I mg of E antigen was made per 10’ cells. Recombinant E antigen...assay with DEN-I virus coprotein gene and its expression in Spodoptera hyperimmune mouse ascitic fluid. This heat-in- frugiperda cells activated...immunization, S. frugiperda cells infected with tion with BstNI (cuts at nucleotides 801 and recombinant baculovirus were pelleted. lysed by 2150). The

Phage display antibodies against ectromelia virus that neutralize variola virus: Selection and implementation for p35 neutralizing epitope mapping.

PubMed

Khlusevich, Yana; Matveev, Andrey; Baykov, Ivan; Bulychev, Leonid; Bormotov, Nikolai; Ilyichev, Ivan; Shevelev, Georgiy; Morozova, Vera; Pyshnyi, Dmitrii; Tikunova, Nina

2018-04-01

In this study, five phage display antibodies (pdAbs) against ectromelia virus (ECTV) were selected from vaccinia virus (VACV)-immune phage-display library of human single chain variable fragments (scFv). ELISA demonstrated that selected pdAbs could recognize ECTV, VACV, and cowpox virus (CPXV). Atomic force microscopy visualized binding of the pdAbs to VACV. Three of the selected pdAbs neutralized variola virus (VARV) in the plaque reduction neutralization test. Western blot analysis of ECTV, VARV, VACV, and CPXV proteins indicated that neutralizing pdAbs bound orthopoxvirus 35 kDa proteins, which are encoded by the open reading frames orthologous to the ORF H3L in VACV. The fully human antibody fh1A was constructed on the base of the VH and VL domains of pdAb, which demonstrated a dose-dependent inhibition of plaque formation after infection with VARV, VACV, and CPXV. To determine the p35 region responsible for binding to neutralizing pdAbs, a panel of truncated p35 proteins was designed and expressed in Escherichia coli cells, and a minimal p35 fragment recognized by selected neutralizing pdAbs was identified. In addition, peptide phage-display combinatorial libraries were applied to localize the epitope. The obtained data indicated that the epitope responsible for recognition by the neutralizing pdAbs is discontinuous and amino acid residues located within two p35 regions, 15-19 aa and 232-237 aa, are involved in binding with neutralizing anti-p35 antibodies. Copyright © 2018. Published by Elsevier B.V.

The Pacific White Shrimp β-actin Promoter: Functional Properties and the Potential Application for Transduction System Using Recombinant Baculovirus.

PubMed

Shi, Yingli; Xiang, Jianhai; Zhou, Guangzhou; Ron, Tetsuzan Benny; Tong, Hsin-I; Kang, Wen; Sun, Si; Lu, Yuanan

2016-06-01

A newly isolated Pacific white shrimp (Litopenaeus vannamei) beta-actin promoter SbaP and its derivative compact construct SbaP (ENX) have recently been demonstrated to promote ectopic gene expression in vitro and in vivo. To further explore the potential transduction application, this newly isolated shrimp promoter SbaP was comparatively tested with cytomegalovirus (CMV), simian virus 40 (SV40), polyhedrin (Polh), and white spot syndrome virus immediate early gene 1 (WSSV ie1) four constitutive promoters and a beta-actin promoter (TbaP) from tilapia fish to characterize its promoting function in eight different cell lines. Luciferase quantitation assays revealed that SbaP can drive luciferase gene expression in all eight cell lines including sf21 (insect), PAC2 (zebrafish), EPC (carp), CHSE-214 (chinook salmon), GSTEF (green sea turtle), MS-1 (monk seal), 293T (human), and HeLa (human), but at different levels. Comparative analysis revealed that the promoting activity of SbaP was lower (≤10-fold) than CMV but higher (2-20 folds) than Polh in most of these cell lines tested. Whereas, SbaP mediated luciferase expression in sf21 cells was over 20-fold higher than CMV, SV40, Polh, and TbaP promoter. Compared to the SbaP, SbaP (ENX), which was constructed on the basis of SbaP by deletion of two "negative" regulatory elements, exhibited no significant change of promoting activity in EPC and PAC2 cells, but a 5 and 16 % lower promoting effect in 293T and HeLa cells, respectively. Additionally, a recombinant baculovirus was constructed under the control of SbaP (ENX), and efficient promoter activity of newly generated baculoviral vector was detected both in vitro of infected sf21 cells and in vivo of injected indicator shrimp. These results warrant the potential application of SbaP, particularly SbaP (ENX) in ectopic gene expression in future.

[Pregnancy outcome in women over the age of 35].

PubMed

Dakov, T; Dimitrova, V; Todorov, T

2014-01-01

To assess pregnancy outcome in women aged > or =35, followed prospectively, and the relation between maternal age and the incidence of abortions after 12 gw (spontaneous and medical), preterm delivery, mode of delivery and congenital anomalies. 495 women from the Fetal Medicine Clinic of the State University Hospital "Maichin Dom" in Sofia were enrolled in the study between 02/2012 and 02/2014 with gestational age > or =12 completed weeks. The patients were devided in 3 groups according to age-- < or =34, between 35 and 39 and > or = 40 years of age. Pregnancy outcome was veirified from hospital records and phone intenrviews with the patients themselves. Data were processed with SPSS 13.0 statistical package. Descriptive and comparative analysis was performed after grouping according to one or more characteristics; p values <0.05 were considered as evidence of statistical significance for tested effects. 131/495 (26.5%) of the patients were < or =34 years of age, 254/495 (51.3%) were between 35 and 39 and 110/495 (22.2%) were > or =40 years of age. Maternal age > or =35 was associated with statistically significant increase of the total late abortion rate (spontaneous and medical abortions)--from 6.9% for those < or =34 yars of age to 11.3%--for the ones between 35-39 and 11.4%--for the ones > or =40 years. There was also statistically significant increase in the incidence of late medical abortions in the group > or =40 years of age (p=0.011). It was 2/124 (1.6%) for the group aged < or =34, 7/230 (3%)--in the group aged 35-39 and 9/100 (9.0%)--in the group > or =40 years of age. The difference in late spontaneous abortions rate was not statistically significant among the groups. There was statistically significant increase in the incidence of preterm deliveries (spontaneous and induced)--14/131 (10.7%) in the group aged < or =34, 52/208 (25.0%)--in the group between 35-39 and 20/93 (21.5%)--in the group > or =40 years of age (p=0.013). There was also a significant

The Phosphatidylinositol 3,5-Bisphosphate (PI(3,5)P2)-dependent Tup1 Conversion (PIPTC) Regulates Metabolic Reprogramming from Glycolysis to Gluconeogenesis*

PubMed Central

Han, Bong-Kwan; Emr, Scott D.

2013-01-01

Glucose/carbon metabolism is a fundamental cellular process in living cells. In response to varying environments, eukaryotic cells reprogram their glucose/carbon metabolism between aerobic or anaerobic glycolysis, oxidative phosphorylation, and/or gluconeogenesis. The distinct type of glucose/carbon metabolism that a cell carries out has significant effects on the cell's proliferation and differentiation. However, it is poorly understood how the reprogramming of glucose/carbon metabolism is regulated. Here, we report a novel endosomal PI(3,5)P2 lipid-dependent regulatory mechanism that is required for metabolic reprogramming from glycolysis to gluconeogenesis in Saccharomyces cerevisiae. Certain gluconeogenesis genes, such as FBP1 (encoding fructose-1,6-bisphosphatase 1) and ICL1 (encoding isocitrate lyase 1) are under control of the Mig1 repressor and Cyc8-Tup1 corepressor complex. We previously identified the PI(3,5)P2-dependent Tup1 conversion (PIPTC), a mechanism to convert Cyc8-Tup1 corepressor to Cti6-Cyc8-Tup1 coactivator. We demonstrate that the PIPTC plays a critical role for transcriptional activation of FBP1 and ICL1. Furthermore, without the PIPTC, the Cat8 and Sip4 transcriptional activators cannot be efficiently recruited to the promoters of FBP1 and ICL1, suggesting a key role for the PIPTC in remodulating the chromatin architecture at the promoters. Our findings expand our understanding of the regulatory mechanisms for metabolic reprogramming in eukaryotes to include key regulation steps outside the nucleus. Given that Tup1 and the metabolic enzymes that control PI(3,5)P2 are highly conserved among eukaryotes, our findings may provide important insights toward understanding glucose/carbon metabolic reprogramming in other eukaryotes, including humans. PMID:23733183

Bupivacaine-induced apoptosis independently of WDR35 expression in mouse neuroblastoma Neuro2a cells

PubMed Central

2012-01-01

Background Bupivacaine-induced neurotoxicity has been shown to occur through apoptosis. Recently, bupivacaine was shown to elicit reactive oxygen species (ROS) production and induce apoptosis accompanied by activation of p38 mitogen-activated protein kinase (MAPK) in a human neuroblastoma cell line. We have reported that WDR35, a WD40-repeat protein, may mediate apoptosis through caspase-3 activation. The present study was undertaken to test whether bupivacaine induces apoptosis in mouse neuroblastoma Neuro2a cells and to determine whether ROS, p38 MAPK, and WDR35 are involved. Results Our results showed that bupivacaine induced ROS generation and p38 MAPK activation in Neuro2a cells, resulting in apoptosis. Bupivacaine also increased WDR35 expression in a dose- and time-dependent manner. Hydrogen peroxide (H2O2) also increased WDR35 expression in Neuro2a cells. Antioxidant (EUK-8) and p38 MAPK inhibitor (SB202190) treatment attenuated the increase in caspase-3 activity, cell death and WDR35 expression induced by bupivacaine or H2O2. Although transfection of Neuro2a cells with WDR35 siRNA attenuated the bupivacaine- or H2O2-induced increase in expression of WDR35 mRNA and protein, in contrast to our previous studies, it did not inhibit the increase in caspase-3 activity in bupivacaine- or H2O2-treated cells. Conclusions In summary, our results indicated that bupivacaine induced apoptosis in Neuro2a cells. Bupivacaine induced ROS generation and p38 MAPK activation, resulting in an increase in WDR35 expression, in these cells. However, the increase in WDR35 expression may not be essential for the bupivacaine-induced apoptosis in Neuro2a cells. These results may suggest the existence of another mechanism of bupivacaine-induced apoptosis independent from WDR35 expression in Neuro2a cells. PMID:23227925

Phenotypic Variation in Overwinter Environmental Transmission of a Baculovirus and the Cost of Virulence.

PubMed

Fleming-Davies, Arietta E; Dwyer, Greg

2015-12-01

A pathogen's ability to persist in the environment is an ecologically important trait, and variation in this trait may promote coexistence of different pathogen strains. We asked whether naturally occurring isolates of the baculovirus that infects gypsy moth larvae varied in their overwinter environmental transmission and whether this variation was consistent with a trade-off or an upper limit to virulence that might promote pathogen diversity. We used experimental manipulations to replicate the natural overwinter infection process, using 16 field-collected isolates. Virus isolates varied substantially in the fraction of larvae infected, leading to differences in overwinter transmission rates. Furthermore, isolates that killed more larvae also had higher rates of early larval death in which no infectious particles were produced, consistent with a cost of high virulence. Our results thus support the existence of a cost that could impose an upper limit to virulence even in a highly virulent pathogen.

Truncated ALK derived from chromosomal translocation t(2;5)(p23;q35) binds to the SH3 domain of p85-PI3K.

PubMed

Polgar, Doris; Leisser, Christina; Maier, Susanne; Strasser, Stephan; Rüger, Beate; Dettke, Markus; Khorchide, Maya; Simonitsch, Ingrid; Cerni, Christa; Krupitza, Georg

2005-02-15

The chromosomal translocation t(2;5)(p23;q35) is associated with "Anaplastic large cell lymphomas" (ALCL), a Non Hodgkin Lymphoma occurring in childhood. The fusion of the tyrosine kinase gene-ALK (anaplastic lymphoma kinase) on chromosome 2p23 to the NPM (nucleophosmin/B23) gene on chromosome 5q35 results in a 80 kDa chimeric protein, which activates the "survival" kinase PI3K. However, the binding mechanism between truncated ALK and PI3K is poorly understood. Therefore, we attempted to elucidate the molecular interaction between ALK and the regulatory p85 subunit of PI3K. Here we provide evidence that the truncated ALK homodimer binds to the SH3 domain of p85. This finding may be useful for the development of a new target-specific intervention.

Receptor binding proteins of Listeria monocytogenes bacteriophages A118 and P35 recognize serovar-specific teichoic acids

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

Bielmann, Regula; Habann, Matthias; Eugster, Marcel R.

Adsorption of a bacteriophage to the host requires recognition of a cell wall-associated receptor by a receptor binding protein (RBP). This recognition is specific, and high affinity binding is essential for efficient virus attachment. The molecular details of phage adsorption to the Gram-positive cell are poorly understood. We present the first description of receptor binding proteins and a tail tip structure for the siphovirus group infecting Listeria monocytogenes. The host-range determining factors in two phages, A118 and P35 specific for L. monocytogenes serovar 1/2 have been determined. Two proteins were identified as RBPs in phage A118. Rhamnose residues in wallmore » teichoic acids represent the binding ligands for both proteins. In phage P35, protein gp16 could be identified as RBP and the role of both rhamnose and N-acetylglucosamine in phage adsorption was confirmed. Immunogold-labeling and transmission electron microscopy allowed the creation of a topological model of the A118 phage tail. - Highlights: • We present the first description of receptor binding proteins and a tail tip structure for the Siphovirus group infecting Listeria monocytogenes. • The host-range determining factors in two phages, A118 and P35 specific for L. monocytogenes serovar 1/2 have been determined. • Rhamnose residues in wall teichoic acids represent the binding ligands for both receptor binding proteins in phage A118. • Rhamnose and N-acetylglucosamine are required for adsorption of phage P35. • We preset a topological model of the A118 phage tail.« less

Dynamic heterogeneity in the folding/unfolding transitions of FiP35

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

Mori, Toshifumi, E-mail: mori@ims.ac.jp; Saito, Shinji, E-mail: shinji@ims.ac.jp

Molecular dynamics simulations have become an important tool in studying protein dynamics over the last few decades. Atomistic simulations on the order of micro- to milliseconds are becoming feasible and are used to study the state-of-the-art experiments in atomistic detail. Yet, analyzing the high-dimensional-long-temporal trajectory data is still a challenging task and sometimes leads to contradictory results depending on the analyses. To reveal the dynamic aspect of the trajectory, here we propose a simple approach which uses a time correlation function matrix and apply to the folding/unfolding trajectory of FiP35 WW domain [Shaw et al., Science 330, 341 (2010)]. Themore » approach successfully characterizes the slowest mode corresponding to the folding/unfolding transitions and determines the free energy barrier indicating that FiP35 is not an incipient downhill folder. The transition dynamics analysis further reveals that the folding/unfolding transition is highly heterogeneous, e.g., the transition path time varies by ∼100 fold. We identify two misfolded states and show that the dynamic heterogeneity in the folding/unfolding transitions originates from the trajectory being trapped in the misfolded and half-folded intermediate states rather than the diffusion driven by a thermal noise. The current results help reconcile the conflicting interpretations of the folding mechanism and highlight the complexity in the folding dynamics. This further motivates the need to understand the transition dynamics beyond a simple free energy picture using simulations and single-molecule experiments.« less

Depletion of a Drosophila homolog of yeast Sup35p disrupts spindle assembly, chromosome segregation, and cytokinesis during male meiosis.

PubMed

Basu, J; Williams, B C; Li, Z; Williams, E V; Goldberg, M L

1998-01-01

In the course of a genetic screen for male-sterile mutations in Drosophila affecting chromosome segregation during the meiotic divisions in spermatocytes, we identified the mutation dsup35(63D). Examination of mutant testes showed that chromosome misbehavior was a consequence of major disruptions in meiotic spindle assembly. These perturbations included problems in aster formation, separation, and migration around the nuclear envelope; aberrations in spindle organization and integrity; and disappearance of the ana/telophase central spindle, which in turn disrupts cytokinesis. The dsup35(63D) mutation is caused by a P element insertion that affects, specifically in the testis, the expression of a gene (dsup35) encoding the Drosophila homolog of the yeast Sup35p and Xenopus eRF3 proteins. These proteins are involved in the termination of polypeptide synthesis on ribosomes, but previous studies have suggested that Sup35p and closely related proteins of the same family also interact directly with microtubules. An affinity-purified antibody directed against the product of the dsup35 gene was prepared; interestingly, this antibody specifically labels primary spermatocytes in one or two discrete foci of unknown structure within the nucleoplasm. We discuss how depletion of the dsup35 gene product in spermatocytes might lead to the global disruptions in meiotic spindle assembly seen in mutant spermatocytes.

Pseudotyped baculovirus is an effective gene expression tool for studying molecular function during axolotl limb regeneration.

PubMed

Oliveira, Catarina R; Lemaitre, Regis; Murawala, Prayag; Tazaki, Akira; Drechsel, David N; Tanaka, Elly M

2018-01-15

Axolotls can regenerate complex structures through recruitment and remodeling of cells within mature tissues. Accessing the underlying mechanisms at a molecular resolution is crucial to understand how injury triggers regeneration and how it proceeds. However, gene transformation in adult tissues can be challenging. Here we characterize the use of pseudotyped baculovirus (BV) as an effective gene transfer method both for cells within mature limb tissue and within the blastema. These cells remain competent to participate in regeneration after transduction. We further characterize the effectiveness of BV for gene overexpression studies by overexpressing Shh in the blastema, which yields a high penetrance of classic polydactyly phenotypes. Overall, our work establishes BV as a powerful tool to access gene function in axolotl limb regeneration. Copyright © 2017 Elsevier Inc. All rights reserved.

Harvesting Chlorella vulgaris by natural increase in pH: effect of medium composition.

PubMed

Nguyen, Thi Dong Phuong; Frappart, Matthieu; Jaouen, Pascal; Pruvost, Jérémy; Bourseau, Patrick

2014-01-01

The freshwater microalga Chlorella vulgaris was harvested by autoflocculation resulting from the precipitation of magnesium or calcium compounds induced by a slow increase in pH in the absence of CO2 input. Autoflocculation was tested in two culture media with, respectively, ammonium (NH4+) and nitrate (NO3-) ions as nitrogen source. The culture pH increased because of photosynthesis and CO2 stripping. pH rose to 11 after 8 h in the NO3- medium, but did not exceed 9 in the NH4+ medium. No flocculation took place in any of the media. Autoflocculation tests were repeated in the NO(3-)-based culture medium by progressively increasing the concentrations of Ca2+ and Mg2+ until inorganic compounds precipitated and flocculated microalgae. The minimal concentrations for flocculation were found to be 120 mg Ca2 L(-1) and 1000 mg Mg2+ L(-1). These values were, respectively, 3.5 times and 20 times higher than those allowing flocculation by NaOH addition. Energy-dispersive X-ray spectroscopy, zeta potential measurement, and ionic chromatography suggest that the mechanisms involved are different. The rate of cell removal was close to 90% in both cases, but cells were more concentrated in the aggregates obtained by magnesium compound precipitation, with an estimated concentration close to 33 g (dry matter) L(-1), against 19 g L(-1) for calcium phosphates.

Goose parvovirus structural proteins expressed by recombinant baculoviruses self-assemble into virus-like particles with strong immunogenicity in goose

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

Ju, Huanyu; Wei, Na; Wang, Qian

Highlights: {yields} All three capsid proteins can be expressed in insect cells in baculovirus expression system. {yields} All three recombinant proteins were spontaneously self-assemble into virus-like particles whose size and appearance were similar to those of native purified GPV virions. {yields} The immunogenicity of GPV-VLPs was better than commercial inactivated vaccine and attenuated vaccine. -- Abstract: Goose parvovirus (GPV), a small non-enveloped ssDNA virus, can cause Derzsy's disease, and three capsid proteins of VP1, VP2, and VP3 are encoded by an overlapping nucleotide sequence. However, little is known on whether recombinant viral proteins (VPs) could spontaneously assemble into virus-like particlesmore » (VLPs) in insect cells and whether these VLPs could retain their immunoreactivity and immunogenicity in susceptible geese. To address these issues, genes for these GPV VPs were amplified by PCR, and the recombinant VPs proteins were expressed in insect cells using a baculovirus expression system for the characterization of their structures, immunoreactivity, and immunogenicity. The rVP1, rVP2, and rVP3 expressed in Sf9 cells were detected by anti-GPV sera, anti-VP3 sera, and anti-His antibodies, respectively. Electron microscopy revealed that these rVPs spontaneously assembled into VLPs in insect cells, similar to that of the purified wild-type GPV virions. In addition, vaccination with individual types of VLPs, particularly with the rVP2-VLPs, induced higher titers of antibodies and neutralized different strains of GPVs in primary goose and duck embryo fibroblast cells in vitro. These data indicated that these VLPs retained immunoreactivity and had strong immunogenicity in susceptible geese. Therefore, our findings may provide a framework for development of new vaccines for the prevention of Derzsy's disease and vehicles for the delivery of drugs.« less

Reversal of Trimethyltin-Induced Learning and Memory Deficits by 3,5-Dicaffeoylquinic Acid.

PubMed

Kang, Jin Yong; Park, Seon Kyeong; Guo, Tian Jiao; Ha, Jeong Su; Lee, Du Sang; Kim, Jong Min; Lee, Uk; Kim, Dae Ok; Heo, Ho Jin

2016-01-01

The antiamnesic effect of 3,5-dicaffeoylquinic acid (3,5-diCQA) as the main phenolic compound in Artemisia argyi H. extract on cognitive dysfunction induced by trimethyltin (TMT) (7.1  μ g/kg of body weight; intraperitoneal injection) was investigated in order to assess its ameliorating function in mice. In several behavioral tests, namely, the Y-maze, passive avoidance, and Morris water maze (MWM) test, 3,5-diCQA significantly ameliorated learning and memory deficits. After the behavioral tests, brain tissues from the mice were analyzed to characterize the basis of the neuroprotective effect. Acetylcholine (ACh) levels increased, whereas the activity of acetylcholinesterase (AChE) decreased upon administration of 3,5-diCQA. In addition, 3,5-diCQA effectively protected against an increase in malondialdehyde (MDA) content, an increase in the oxidized glutathione (GSH) ratio, and a decline of total superoxide dismutase (SOD) level. 3,5-diCQA may prevent neuronal apoptosis through the protection of mitochondrial activities and the repression of apoptotic signaling molecules such as p-Akt, BAX, and p-tau (Ser 404).

Reversal of Trimethyltin-Induced Learning and Memory Deficits by 3,5-Dicaffeoylquinic Acid

PubMed Central

Kang, Jin Yong; Park, Seon Kyeong; Guo, Tian Jiao; Ha, Jeong Su; Lee, Du Sang; Kim, Jong Min; Lee, Uk; Kim, Dae Ok

2016-01-01

The antiamnesic effect of 3,5-dicaffeoylquinic acid (3,5-diCQA) as the main phenolic compound in Artemisia argyi H. extract on cognitive dysfunction induced by trimethyltin (TMT) (7.1 μg/kg of body weight; intraperitoneal injection) was investigated in order to assess its ameliorating function in mice. In several behavioral tests, namely, the Y-maze, passive avoidance, and Morris water maze (MWM) test, 3,5-diCQA significantly ameliorated learning and memory deficits. After the behavioral tests, brain tissues from the mice were analyzed to characterize the basis of the neuroprotective effect. Acetylcholine (ACh) levels increased, whereas the activity of acetylcholinesterase (AChE) decreased upon administration of 3,5-diCQA. In addition, 3,5-diCQA effectively protected against an increase in malondialdehyde (MDA) content, an increase in the oxidized glutathione (GSH) ratio, and a decline of total superoxide dismutase (SOD) level. 3,5-diCQA may prevent neuronal apoptosis through the protection of mitochondrial activities and the repression of apoptotic signaling molecules such as p-Akt, BAX, and p-tau (Ser 404). PMID:28105250

Immunoexpression of p16 in uterine leiomyomas with infarct-type necrosis: an analysis of 35 cases.

PubMed

Ip, Philip P; Lim, Diana; Cheung, Annie N Y; Oliva, Esther

2017-11-01

Uterine leiomyosarcomas frequently show p16 immunoexpression. However, p16 may also be expressed in some benign leiomyoma variants such as leiomyomas with bizarre nuclei and cellular leiomyomas, limiting its utility as a biomarker to distinguish between benign and malignant neoplasms. We investigated p16 expression in leiomyomas with infarct-type necrosis, tumours which may sometimes be misinterpreted as smooth muscle tumours of uncertain malignant potential or even leiomyosarcoma on conventional light microscopy. p16 immunostaining was performed on 35 leiomyomas with infarct-type necrosis and the staining pattern was analysed. Staining was classified as absent, scattered/isolated, <33-, 33-66- or >66%-positive cells, and was assessed in the areas immediately surrounding and distant from the infarct. The median age of patients was 44 years. Seventeen had hormonal/non-hormonal drugs and three were pregnant. The median tumour size was 7.25 cm. The mean mitotic count was 0.9/10 high-power fields. Only one tumour had multifocal mild nuclear atypia. Positive p16 was noted in 34 of 35 (97.2%) tumours. It was typically patchy, and was concentrated in areas immediately surrounding the necrosis. Distant from the necrosis, p16 positivity was seen predominantly in scattered/isolated cells. One tumour without any worrisome microscopic features showed diffuse p16 positivity throughout. Median follow-up was 55 months, and none of the patients experienced any recurrence. p16 expression in benign uterine smooth muscle tumours with infarct-type necrosis is common. The staining is particularly concentrated adjacent to areas of necrosis. It is important to be aware of this potential pitfall when interpreting p16 expression. © 2017 John Wiley & Sons Ltd.

Kinetic comparison of tissue non-specific and placental human alkaline phosphatases expressed in baculovirus infected cells: application to screening for Down's syndrome.

PubMed

Denier, Colette C; Brisson-Lougarre, Andrée A; Biasini, Ghislaine G; Grozdea, Jean J; Fournier, Didier D

2002-01-01

In humans, there are four alkaline phosphatases, and each form exhibits a characteristic pattern of tissue distribution. The availability of an easy method to reveal their activity has resulted in large amount of data reporting correlations between variations in activity and illnesses. For example, alkaline phosphatase from neutrophils of mothers pregnant with a trisomy 21 fetus (Down's syndrome) displays significant differences both in its biochemical and immunological properties, and in its affinity for some specific inhibitors. To analyse these differences, the biochemical characteristics of two isozymes (non specific and placental alkaline phosphatases) were expressed in baculovirus infected cells. Comparative analysis of the two proteins allowed us to estimate the kinetic constants of denaturation and sensitivity to two inhibitors (L-p-bromotetramisole and thiophosphate), allowing better discrimination between the two enzymes. These parameters were then used to estimate the ratio of the two isoenzymes in neutrophils of pregnant mothers with or without a trisomy 21 fetus. It appeared that the placental isozyme represented 13% of the total activity of neutrophils of non pregnant women. This proportion did not significantly increase with normal pregnancy. By contrast, in pregnancies with trisomy 21 fetus, the proportion reached 60-80% of activity. Over-expression of the placental isozyme compared with the tissue-nonspecific form in neutrophils of mother with a trisomy 21 fetus may explain why the characteristics of the alkaline phosphatase in these cells is different from normal. Application of this knowledge could improve the potential of using alkaline phosphatase measurements to screen for Down's syndrome.

Kinetic comparison of tissue non-specific and placental human alkaline phosphatases expressed in baculovirus infected cells: application to screening for Down's syndrome

PubMed Central

Denier, Colette C; Brisson-Lougarre, Andrée A; Biasini, Ghislaine G; Grozdea, Jean J; Fournier, Didier D

2002-01-01

Background In humans, there are four alkaline phosphatases, and each form exibits a characteristic pattern of tissue distribution. The availability of an easy method to reveal their activity has resulted in large amount of data reporting correlations between variations in activity and illnesses. For example, alkaline phosphatase from neutrophils of mothers pregnent with a trisomy 21 fetus (Down's syndrome) displays significant differences both in its biochemical and immunological properties, and in its affinity for some specific inhibitors. Results To analyse these differences, the biochemical characteristics of two isozymes (non specific and placental alkaline phosphatases) were expressed in baculovirus infected cells. Comparative analysis of the two proteins allowed us to estimate the kinetic constants of denaturation and sensitivity to two inhibitors (L-p-bromotetramisole and thiophosphate), allowing better discrimination between the two enzymes. These parameters were then used to estimate the ratio of the two isoenzymes in neutrophils of pregnant mothers with or without a trisomy 21 fetus. It appeared that the placental isozyme represented 13% of the total activity of neutrophils of non pregnant women. This proportion did not significantly increase with normal pregnancy. By contrast, in pregnancies with trisomy 21 fetus, the proportion reached 60–80% of activity. Conclusion Over-expression of the placental isozyme compared with the tissue-nonspecific form in neutrophils of mother with a trisomy 21 fetus may explain why the characteristics of the alkaline phosphatase in these cells is different from normal. Application of this knowledge could improve the potential of using alkaline phosphatase measurements to screen for Down's syndrome. PMID:11818032

Reduced expression of IL-12 p35 by SJL/J macrophages responding to Theiler's virus infection is associated with constitutive activation of IRF-3

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

Dahlberg, Angela; Auble, Mark R.; Petro, Thomas M.

2006-09-30

Macrophages responding to viral infections may contribute to autoimmune demyelinating diseases (ADD). Macrophages from ADD-susceptible SJL/J mice responding to Theiler's Virus (TMEV) infection, the TLR7 agonist loxoribine, or the TLR4 agonist-LPS expressed less IL-12 p35 but more IL-12/23 p40 and IFN-{beta} than macrophages from ADD-resistant B10.S mice. While expression of IRF-1 and -7 was similar between B10.S and SJL/J TMEV-infected macrophages, SJL/J but not B10.S macrophages exhibited constitutively active IRF-3. In contrast to overexpressed IRF-1, IRF-5, and IRF-7, which stimulated p35 promoter reporter activity, overexpressed IRF-3 repressed p35 promoter activity in response to TMEV infection, loxoribine, IFN-{gamma}/LPS, but not IFN-{gamma}more » alone. IRF-3 lessened but did not eliminate IRF-1-stimulated p35 promoter activity. Repression by IRF-3 required bp -172 to -122 of the p35 promoter. The data suggest that pre-activated IRF-3 is a major factor in the differences in IL-12 production between B10.S and SJL/J macrophages responding to TMEV.« less

Baculovirus expression of the avian paramyxovirus 2 HN gene for diagnostic applications.

PubMed

Choi, Kang-Seuk; Kye, Soo-Jeong; Kim, Ji-Ye; Seul, Hee-Jeong; Lee, Hee-Soo; Kwon, Hyuk-Moo; Sung, Haan-Woo

2014-03-01

Avian paramyxovirus 2 (APMV-2) infections are associated with respiratory diseases in poultry worldwide. The hemagglutination inhibition (HI) test is a useful tool for surveillance and monitoring of this virus. In this study, full-length hemagglutinin (HN) gene of APMV-2 was chemically synthesized based on its published sequence, cloned and expressed in Spodoptera frugiperda insect cells using recombinant baculoviruses. The biological, antigenic and immunogenic properties of the expressed protein were evaluated to assess its ability to produce diagnostic reagents for HI testing. Recombinant APMV-2 HN protein showed two distinct bands with molecular masses of 64 and 75kDa, which showed hemagglutination (HA) and neuraminidase activities, respectively. The recombinant HN (rHN) protein extracted from infected cells produced high HA titers (2(13) per 25μL). HA activity of the protein was inhibited by APMV-2 antiserum, although there were weak cross reactions with other APMV serotype antisera. The rHN protein induced high titers of APMV-2-specific antibodies in immunized chickens based on the HI test. These results indicated that recombinant APMV-2 HN protein is a useful alternative to the APMV-2 antigen in HI assays. Copyright © 2013 Elsevier B.V. All rights reserved.

Plant based dietary supplement increases urinary pH

PubMed Central

Berardi, John M; Logan, Alan C; Rao, A Venket

2008-01-01

Background Research has demonstrated that the net acid load of the typical Western diet has the potential to influence many aspects of human health, including osteoporosis risk/progression; obesity; cardiovascular disease risk/progression; and overall well-being. As urinary pH provides a reliable surrogate measure for dietary acid load, this study examined whether a plant-based dietary supplement, one marketed to increase alkalinity, impacts urinary pH as advertised. Methods Using pH test strips, the urinary pH of 34 healthy men and women (33.9 +/- 1.57 y, 79.3 +/- 3.1 kg) was measured for seven days to establish a baseline urinary pH without supplementation. After this initial baseline period, urinary pH was measured for an additional 14 days while participants ingested the plant-based nutritional supplement. At the end of the investigation, pH values at baseline and during the treatment period were compared to determine the efficacy of the supplement. Results Mean urinary pH statistically increased (p = 0.03) with the plant-based dietary supplement. Mean urinary pH was 6.07 +/- 0.04 during the baseline period and increased to 6.21 +/- 0.03 during the first week of treatment and to 6.27 +/- 0.06 during the second week of treatment. Conclusion Supplementation with a plant-based dietary product for at least seven days increases urinary pH, potentially increasing the alkalinity of the body. PMID:18990209

The supplementation of low-P diets with microbial 6-phytase expressed in Aspergillus oryzae increases P and Ca digestibility in growing pigs.

PubMed

Torrallardona, D; Salvadó, R; Broz, J

2012-12-01

A trial was conducted to evaluate the dose response of a novel microbial 6-phytase expressed in Aspergillus oryzae (Ronozyme HiPhos; DSM Nutritional Products, Basel, Switzerland) in pigs. Forty-eight individually housed pigs (Landrace × Pietrain; 52 kg BW; 24 males and 24 females) were distributed among 6 experimental treatments consisting of a low-P diet (3.5 g P/kg; 1.1 g digestible P/kg), which was supplemented with 500, 1000, 2000, or 4000 units of phytase activity/kg, and a standard-P diet (4.5 g P/kg; 1.8 g digestible P/kg) that was supplemented with CaHPO(4). After 17 d, fresh feces were sampled from all pigs and the apparent total tract digestibility of DM, OM, ash, P, and Ca was measured using TiO(2) as indigestible marker. Blood samples were also obtained from each pig and serum was analyzed for P and Ca concentrations. The nonsupplemented low-P diet increased Ca and reduced P blood serum concentrations (P < 0.05) relative to the standard-P diet (10.8 vs. 10.2 and 6.7 vs. 7.7 mg/dL, respectively). Phytase supplementation of the low-P diet reduced Ca (from 10.8 to 9.9 mg/dL; linear, P < 0.001) and increased P concentrations (from 6.7 to 8.0 mg/dL; linear and quadratic, P < 0.001) in serum and reduced P concentration in feces (from 13.7 to 7.6 g/kg DM; linear and quadratic, P < 0.001). Phytase improved the total tract digestibility of P (from 29.0 to 62.3%; linear and quadratic, P < 0.001 and P < 0.05), Ca (from 54.0 to 75.7%; quadratic, P < 0.01), and ash (from 46.2 to 57.7%; quadratic, P < 0.01). In conclusion, the microbial 6-phytase tested improves the apparent total tract digestibility of P in growing pigs and reduces P excretion in feces in a dose-dependent manner.

Intracellular Trafficking of Baculovirus Particles: A Quantitative Study of the HearNPV/HzAM1 Cell and AcMNPV/Sf9 Cell Systems.

PubMed

Matindoost, Leila; Nielsen, Lars K; Reid, Steve

2015-05-05

To replace the in vivo production of baculovirus-based biopesticides with a more convenient in vitro produced product, the limitations imposed by in vitro production have to be solved. One of the main problems is the low titer of HearNPV budded virions (BV) in vitro as the use of low BV titer stocks can result in non-homogenous infections resulting in multiple virus replication cycles during scale up that leads to low Occlusion Body yields. Here we investigate the baculovirus traffic in subcellular fractions of host cells throughout infection with an emphasis on AcMNPV/Sf9 and HearNPV/HzAM1 systems distinguished as "good" and "bad" BV producers, respectively. qPCR quantification of viral DNA in the nucleus, cytoplasm and extracellular fractions demonstrated that although the HearNPV/HzAM1 system produces twice the amount of vDNA as the AcMNPV/Sf9 system, its percentage of BV to total progeny vDNA was lower. vDNA egress from the nucleus to the cytoplasm is sufficient in both systems, however, a higher percentage of vDNA in the HearNPV/HzAM1 system remain in the cytoplasm and do not bud out of the cells compared to the AcMNPV/Sf9 system. In both systems more than 75% of the vDNA produced in the nuclear fraction go unused, without budding or being encapsulated in OBs showing the capacity for improvements that could result from the engineering of the virus/cell line systems to achieve better productivities for both BV and OB yields.

Does Cholecystectomy Increase the Esophageal Alkaline Reflux? Evaluation by Impedance-pH Technique.

PubMed

Uyanikoglu, Ahmet; Akyuz, Filiz; Ermis, Fatih; Arici, Serpil; Bas, Gurhan; Cakirca, Mustafa; Baran, Bulent; Mungan, Zeynel

2012-04-01

The aim of this study is to investigate the reflux patterns in patients with galbladder stone and the change of reflux patterns after cholecystectomy in such patients. Fourteen patients with cholecystolithiasis and a control group including 10 healthy control subjects were enrolled in this prospective study. Demographical findings, reflux symptom score scale and 24-hour impedance pH values of the 14 cholecystolithiasis cases and the control group were evaluated. The impedance pH study was repeated 3 months after cholecystectomy. Age, gender, and BMI were not different between the two groups. Total and supine weakly alkaline reflux time (%) (1.0 vs 22.5, P = 0.028; 201.85 vs 9.65, P = 0.012), the longest episodes of total, upright and supine weakly alkaline reflux mediums (11 vs 2, P = 0.025; 8.5 vs 1.0, P = 0.035; 3 vs 0, P = 0.027), total and supine weakly alkaline reflux time in minutes (287.35 vs 75.10, P = 0.022; 62.5 vs 1.4, P = 0.017), the number of alkaline reflux episodes (162.5 vs 72.5, P = 0.022) were decreased with statistical significance. No statistically significant difference was found in the comparison of symptoms between the subjects in the control group and the patients with cholecystolithiasis, in preoperative, postoperative and postcholecystectomy status. Significant reflux symptoms did not occur after cholecystectomy. Post cholecystectomy weakly alkaline reflux was decreased, but it was determined that acid reflux increased after cholecystectomy by impedance pH-metry in the study group.

Cell-cycle-dependent three-dimensional redistribution of nuclear proteins, P 120, pKi-67, and SC 35 splicing factor, in the presence of the topoisomerase I inhibitor camptothecin.

PubMed

Elias, Emmanuel; Lalun, Nathalie; Lorenzato, Marianne; Blache, Laurent; Chelidze, Pavel; O'Donohue, Marie-Françoise; Ploton, Dominique; Bobichon, Hélène

2003-11-15

Topoisomerase I (Topo I) is mostly known for its role in DNA relaxation, which is required for duplication and transcription. Topo I acts as a protein kinase mainly directed to the mRNA splicing factor SC35. Camptothecin is one of the specific Topo I inhibitors and is effective on the two functions of the enzyme. In this study we demonstrated that treatment of KB cells with camptothecin for only 30 min induced the 3D reorganization and redistribution of three proteins involved in the nucleus machinery, P 120, pKi-67, and SC 35, and this occurred in a cell cycle-dependent manner. Our data were obtained from confocal microscopic studies after immunolabeling, 3D reconstruction, and measurement of the nuclear components volumes. In the presence of camptothecin, P 120, which occupied the nucleolar volume, lost its reticulation and pKi-67 was redistributed within the nucleoplasm and even into the cytoplasm. Finally, for SC 35 the fusion of its dots into bigger volumes was observed specifically during the G1 phase. Variations of volumes were also observed for the nucleolus and for the nucleus. These results pointed out that, depending on the cell cycle phase, Topo I functions were selective toward the three different proteins.

Partial wave analysis of the reaction p(3.5 GeV) + p → pK + Λ to search for the "ppK –" bound state

DOE PAGES

Agakishiev, G.; Arnold, O.; Belver, D.; ...

2015-01-26

Employing the Bonn–Gatchina partial wave analysis framework (PWA), we have analyzed HADES data of the reaction p(3.5GeV) + p → pK +Λ. This reaction might contain information about the kaonic cluster “ppK -” (with quantum numbers J P=0 - and total isospin I =1/2) via its decay into pΛ. Due to interference effects in our coherent description of the data, a hypothetical K ¯NN (or, specifically “ppK -”) cluster signal need not necessarily show up as a pronounced feature (e.g. a peak) in an invariant mass spectrum like pΛ. Our PWA analysis includes a variety of resonant and non-resonant intermediatemore » states and delivers a good description of our data (various angular distributions and two-hadron invariant mass spectra) without a contribution of a K ¯NN cluster. At a confidence level of CL s=95% such a cluster cannot contribute more than 2–12% to the total cross section with a pK + Λ final state, which translates into a production cross-section between 0.7 μb and 4.2 μb, respectively. The range of the upper limit depends on the assumed cluster mass, width and production process.« less

Share 35 Changes Center Level Liver Acceptance Practices

PubMed Central

Goldberg, David S.; Levine, Matthew; Karp, Seth; Gilroy, Richard; Peter, L

2017-01-01

Share 35 was implemented to provide improved access to organs for patients with MELD scores ≥35. However, little is known about the impact of Share 35 on organ offer acceptance rates. We evaluated all liver offers to adult patients that were ultimately transplanted between 1/1/2011–12/31/2015. The analyses focused on patients ranked in the top five positions of a given match run, and used multi-level mixed-effects models, clustering on individual waitlist candidate and transplant center. There was a significant interaction between Share 35 era and MELD category (p<0.001). Comparing offers to MELD score ≥35 patients, offers post-Share 35 were 36% less likely to be accepted compared to offers to MELD score ≥35 patients pre-Share 35 (adjusted OR: 0.64). There was no clinically meaningful difference in the DRI of livers that were declined for patients with an allocation MELD score ≥35 in the pre- vs post-Share 35 era. Organ offer acceptance rates for patients with an allocation MELD≥35 decreased in every region post-Share 35; the magnitude of these changes was bigger in regions 2, 3, 4, 5, 6, 7, and 11, compared to regions 8 and 9 that had regional sharing in place pre-Share 35. There were significant changes in organ offer acceptance rates at the center level pre- vs post-Share 35, and these changes varied across centers (p<0.001). Conclusions In liver transplant candidates achieving a MELD score ≥35, liver acceptance of offers declined significantly after implementation of Share 35. The alterations in behavior at the center level suggest that practice patterns changed as a direct result of Share 35. Changes in organ acceptance under even broader organ sharing (redistricting) would likely be even greater, posing major logistical and operational challenges, while potentially increasing discard rates, thus decreasing the total number of transplant nationally. PMID:28240804

Nqrs Data for C26H35Br2CuP2 (Subst. No. 1603)

NASA Astrophysics Data System (ADS)

Chihara, H.; Nakamura, N.

This document is part of Subvolume B 'Substances Containing C10H16 … Zn' of Volume 48 'Nuclear Quadrupole Resonance Spectroscopy Data' of Landolt-Börnstein - Group III 'Condensed Matter'. It contains an extract of Section '3.2 Data tables' of the Chapter '3 Nuclear quadrupole resonance data' providing the NQRS data for C26H35Br2CuP2 (Subst. No. 1603)

A common pathway for p10 and calyx proteins in progressive stages of polyhedron envelope assembly in AcMNPV-infected Spodoptera frugiperda larvae.

PubMed

Lee, S Y; Poloumienko, A; Belfry, S; Qu, X; Chen, W; MacAfee, N; Morin, B; Lucarotti, C; Krause, M

1996-01-01

The assembly of the polyhedron envelope in baculovirus-infected cells has been the subject of several studies, yet it is still poorly understood. We have used immunogold-labelled antibodies to two baculovirus proteins, p10 and calyx (also referred to as polyhedron envelope protein or PEP), to follow envelope assembly in AcMNPV-infected tissues of Spodoptera frugiperda larvae. We show that, in wild type virus, both proteins colocalize in fibrillar structures and associated electron-dense spacers which progress to encircle the polyhedra, as well as in completed polyhedron envelopes. In cells infected with polyhedrin-negative (PH-) viruses, an unusual proliferation of these spacers was observed suggesting a deregulatory event in the envelope assembly process. Results of Northern and Western blot analysis revealed that synthesis of P10 and calyx mRNA and proteins in PH- AcMNPV is unaffected as compared to wild type virus. Taken together, the observed physical and compositional connection between fibrillar structures, spacers and polyhedron envelopes, as well as the abnormal appearance of the spacers in PH- mutants, provide further evidence in support of a cooperative role of these structures in the assembly of the polyhedron envelope.

Deuteration and selective labeling of alanine methyl groups of β2-adrenergic receptor expressed in a baculovirus-insect cell expression system.

PubMed

Kofuku, Yutaka; Yokomizo, Tomoki; Imai, Shunsuke; Shiraishi, Yutaro; Natsume, Mei; Itoh, Hiroaki; Inoue, Masayuki; Nakata, Kunio; Igarashi, Shunsuke; Yamaguchi, Hideyuki; Mizukoshi, Toshimi; Suzuki, Ei-Ichiro; Ueda, Takumi; Shimada, Ichio

2018-03-08

G protein-coupled receptors (GPCRs) exist in equilibrium between multiple conformations, and their populations and exchange rates determine their functions. However, analyses of the conformational dynamics of GPCRs in lipid bilayers are still challenging, because methods for observations of NMR signals of large proteins expressed in a baculovirus-insect cell expression system (BVES) are limited. Here, we report a method to incorporate methyl- 13 C 1 H 3 -labeled alanine with > 45% efficiency in highly deuterated proteins expressed in BVES. Application of the method to the NMR observations of β 2 -adrenergic receptor in micelles and in nanodiscs revealed the ligand-induced conformational differences throughout the transmembrane region of the GPCR.

Use of baculovirus expression system for generation of virus-like particles: successes and challenges.

PubMed

Liu, Fuxiao; Wu, Xiaodong; Li, Lin; Liu, Zengshan; Wang, Zhiliang

2013-08-01

The baculovirus expression system (BES) has been one of the versatile platforms for the production of recombinant proteins requiring multiple post-translational modifications, such as folding, oligomerization, phosphorylation, glycosylation, acylation, disulfide bond formation and proteolytic cleavage. Advances in recombinant DNA technology have facilitated application of the BES, and made it possible to express multiple proteins simultaneously in a single infection and to produce multimeric proteins sharing functional similarity with their natural analogs. Therefore, the BES has been used for the production of recombinant proteins and the construction of virus-like particles (VLPs), as well as for the development of subunit vaccines, including VLP-based vaccines. The VLP, which consists of one or more structural proteins but no viral genome, resembles the authentic virion but cannot replicate in cells. The high-quality recombinant protein expression and post-translational modifications obtained with the BES, along with its capacity to produce multiple proteins, imply that it is ideally suited to VLP production. In this article, we critically review the pros and cons of using the BES as a platform to produce both enveloped and non-enveloped VLPs. Copyright © 2013 Elsevier Inc. All rights reserved.

Convergent regulation of the lysosomal two-pore channel-2 by Mg2+, NAADP, PI(3,5)P2 and multiple protein kinases

PubMed Central

Jha, Archana; Ahuja, Malini; Patel, Sandip; Brailoiu, Eugen; Muallem, Shmuel

2014-01-01

Lysosomal Ca2+ homeostasis is implicated in disease and controls many lysosomal functions. A key in understanding lysosomal Ca2+ signaling was the discovery of the two-pore channels (TPCs) and their potential activation by NAADP. Recent work concluded that the TPCs function as a PI(3,5)P2 activated channels regulated by mTORC1, but not by NAADP. Here, we identified Mg2+ and the MAPKs, JNK and P38 as novel regulators of TPC2. Cytoplasmic Mg2+ specifically inhibited TPC2 outward current, whereas lysosomal Mg2+ partially inhibited both outward and inward currents in a lysosomal lumen pH-dependent manner. Under controlled Mg2+, TPC2 is readily activated by NAADP with channel properties identical to those in response to PI(3,5)P2. Moreover, TPC2 is robustly regulated by P38 and JNK. Notably, NAADP-mediated Ca2+ release in intact cells is regulated by Mg2+, PI(3,5)P2, and P38/JNK kinases, thus paralleling regulation of TPC2 currents. Our data affirm a key role for TPC2 in NAADP-mediated Ca2+ signaling and link this pathway to Mg2+ homeostasis and MAP kinases, pointing to roles for lysosomal Ca2+ in cell growth, inflammation and cancer. PMID:24502975

First measurement of the 34S(p ,γ )35Cl reaction rate through indirect methods for presolar nova grains

NASA Astrophysics Data System (ADS)

Gillespie, S. A.; Parikh, A.; Barton, C. J.; Faestermann, T.; José, J.; Hertenberger, R.; Wirth, H.-F.; de Séréville, N.; Riley, J. E.; Williams, M.

2017-08-01

Sulphur isotopic ratio measurements may help to establish the astrophysical sites in which certain presolar grains were formed. Nova model predictions of the 34S/32S ratio are, however, unreliable due to the lack of an experimental 34S(p ,γ )35Cl reaction rate. To this end, we have measured the 34S(3He,d )35Cl reaction at 20 MeV using a high resolution quadrupole-dipole-dipole-dipole magnetic spectrograph. Twenty-two levels over 6.2 MeV 35Cl) <7.4 MeV were identified, ten of which were previously unobserved. Proton-transfer spectroscopic factors have been measured for the first time over the energy range relevant for novae. With this new spectroscopic information a new 34S(p ,γ )35Cl reaction rate has been determined using a Monte Carlo method. Hydrodynamic nova model calculations have been performed using this new reaction rate. These models show that remaining uncertainties in the 34S(p ,γ ) rate affect nucleosynthesis predictions by less than a factor of 1.4, and predict a 34S/32S isotopic ratio of 0.014-0.017. Since recent type II supernova models predict 34S/32S=0.026 -0.053 , the 34S/32S isotopic ratio may be used, in conjunction with other isotopic signatures, to distinguish presolar grains from oxygen-neon nova and type II supernova origin. Our results address a key nuclear physics uncertainty on which recent considerations discounting the nova origin of several grains depend.

In 0.35Ga 0.65P light-emitting diodes grown by gas-source MBE

NASA Astrophysics Data System (ADS)

Masselink, W. Ted; Zachau, Martin

1993-02-01

This paper describes the growth and optical characteristics of In yGa 1- yP with 0.3< y<0.5, and the LED operation of p-i-n structures in the same materials system. The InGaP is grown using gas-source molecular beam epitaxy (GSMBE). The non-lattice-matched In yGa 1- yP grown on GaAs using GSMBE has a specularly smooth surface morphology through the use of unique strained-layer superlattice (SLS) buffer. We have measured the luminescence, luminescence excitation, and Raman spectra of these undoped films and observe strong excitonic luminescence over the entire composition range investigated. The band gap derived from the luminescence excitation spectra corresponds to that of a fully relaxed InGaP film with no residual strain, which is confirmed by the Raman measurements. Light-emitting diodes with peak (300 K) emission centered at less than 590 nm have been fabricated from p-i-n junctions in In 0.35Ga 0.65P. This alloy is close to that with the largest direct band gap in the In yGa 1- y P system and has lattice mismatch from the GaAs substrate of 1%.

Identification and molecular characterization of the Choristoneura fumiferana multicapsid nucleopolyhedrovirus genomic region encoding the regulatory genes pkip, p47, lef-12, and gta.

PubMed

Lapointe, R; Back, D W; Ding, Q; Carstens, E B

2000-05-25

Choristoneura fumiferana multicapsid nucleopolyhedrovirus (CfMNPV) is a baculovirus pathogenic to spruce budworm, the most damaging insect pest in Canadian forestry. CfMNPV is less virulent to its host insect and its replication cycle is slower than the baculovirus type species Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) but the basis of these characteristics is not known. We have now identified, localized, and determined the sequence of the region of CfMNPV carrying potentially important regulatory genes including p47, lef-12, gta, and pkip. DNA database searches revealed that this region of CfMNPV is most closely related to the homologous OpMNPV genes. Transcription analysis demonstrated that CfMNPV P47 is encoded by a 1.6-kb transcript, LEF-12 is encoded by a 2.6-kb transcript, and GTA is encoded by a 2.1-kb transcript. Transcripts for these genes were detectable at 6 h postinfection but all of them showed a burst in expression levels between 12 and 24 h postinfection corresponding to the time of initiation of CfMNPV DNA replication. A polyclonal antibody, raised against CfMNPV P47, detected a nuclear 43-kDa polypeptide from 12 to 72 h postinfection, demonstrating that the CfMNPV p47 gene product is first expressed at a time corresponding to the burst of transcriptional activity between the early and the late phases. Both AcMNPV and CfMNPV P47 translocate to the nucleus of infected cells. Copyright 2000 Academic Press.

The genome sequence of Condylorrhiza vestigialis NPV, a novel baculovirus for the control of the Alamo moth on Populus spp. in Brazil.

PubMed

Castro, Maria Elita B; Melo, Fernando L; Tagliari, Marina; Inglis, Peter W; Craveiro, Saluana R; Ribeiro, Zilda Maria A; Ribeiro, Bergmann M; Báo, Sônia N

2017-09-01

Condylorrhiza vestigialis (Lepidoptera: Cambridae), commonly known as the Brazilian poplar moth or Alamo moth, is a serious defoliating pest of poplar, a crop of great economic importance for the production of wood, fiber, biofuel and other biomaterials as well as its significant ecological and environmental value. The complete genome sequence of a new alphabaculovirus isolated from C. vestigialis was determined and analyzed. Condylorrhiza vestigialis nucleopolyhedrovirus (CoveNPV) has a circular double-stranded DNA genome of 125,767bp with a GC content of 42.9%. One hundred and thirty-eight putative open reading frames were identified and annotated in the CoveNPV genome, including 38 core genes and 9 bros. Four homologous regions (hrs), a feature common to most baculoviruses, and 19 perfect and imperfect direct repeats (drs) were found. Phylogenetic analysis confirmed that CoveNPV is a Group I Alphabaculovirus and is most closely related to Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) and Choristoneura fumiferana DEF multiple nucleopolyhedrovirus CfDEFMNPV. The gp37 gene was not detected in the CoveNPV genome, although this gene is found in many NPVs. Two other common NPV genes, chitinase (v-chiA) and cathepsin (v-cath), that are responsible for host insect liquefaction and melanization, were also absent, where phylogenetic analysis suggests that the loss these genes occurred in the common ancestor of AgMNPV, CfDEFMNPV and CoveNPV, with subsequent reacquisition of these genes by CfDEFMNPV. The molecular biology and genetics of CoveNPV was formerly very little known and our expectation is that the findings presented here should accelerate research on this baculovirus, which will facilitate the use of CoveNPV in integrated pest management programs in Poplar crops. Copyright © 2017 Elsevier Inc. All rights reserved.

Membrane fusion between baculovirus budded virus-enveloped particles and giant liposomes generated using a droplet-transfer method for the incorporation of recombinant membrane proteins.

PubMed

Nishigami, Misako; Mori, Takaaki; Tomita, Masahiro; Takiguchi, Kingo; Tsumoto, Kanta

2017-07-01

Giant proteoliposomes are generally useful as artificial cell membranes in biochemical and biophysical studies, and various procedures for their preparation have been reported. We present here a novel preparation technique that involves the combination of i) cell-sized lipid vesicles (giant unilamellar vesicles, GUVs) that are generated using the droplet-transfer method, where lipid monolayer-coated water-in-oil microemulsion droplets interact with oil/water interfaces to form enclosed bilayer vesicles, and ii) budded viruses (BVs) of baculovirus (Autographa californica nucleopolyhedrovirus) that express recombinant transmembrane proteins on their envelopes. GP64, a fusogenic glycoprotein on viral envelopes, is activated by weak acids and is thought to cause membrane fusion with liposomes. Using confocal laser scanning microscopy (CLSM), we observed that the single giant liposomes fused with octadecyl rhodamine B chloride (R18)-labeled wild-type BV envelopes with moderate leakage of entrapped soluble compounds (calcein), and the fusion profile depended on the pH of the exterior solution: membrane fusion occurred at pH ∼4-5. We further demonstrated that recombinant transmembrane proteins, a red fluorescent protein (RFP)-tagged GPCR (corticotropin-releasing hormone receptor 1, CRHR1) and envelope protein GP64 could be partly incorporated into membranes of the individual giant liposomes with a reduction of the pH value, though there were also some immobile fluorescent spots observed on their circumferences. This combination may be useful for preparing giant proteoliposomes containing the desired membranes and inner phases. Copyright © 2017 Elsevier B.V. All rights reserved.

Incorporation of adenylate cyclase into membranes of giant liposomes using membrane fusion with recombinant baculovirus-budded virus particles.

PubMed

Mori, Takaaki; Kamiya, Koki; Tomita, Masahiro; Yoshimura, Tetsuro; Tsumoto, Kanta

2014-06-01

Recombinant transmembrane adenylate cyclase (AC) was incorporated into membranes of giant liposomes using membrane fusion between liposomes and baculovirus-budded virus (BV). AC genes were constructed into transfer vectors in a form fused with fluorescent protein or polyhistidine at the C-terminus. The recombinant BVs were collected by ultracentrifugation and AC expression was verified using western blotting. The BVs and giant liposomes generated using gentle hydration were fused under acidic conditions; the incorporation of AC into giant liposomes was demonstrated by confocal laser scanning microscopy through the emission of fluorescence from their membranes. The AC-expressing BVs were also fused with liposomes containing the substrate (ATP) with/without a specific inhibitor (SQ 22536). An enzyme immunoassay on extracts of the sample demonstrated that cAMP was produced inside the liposomes. This procedure facilitates direct introduction of large transmembrane proteins into artificial membranes without solubilization.

Association between serum interleukin-35 levels and severity of acute pancreatitis

PubMed Central

Zhang, Yi-Li; Zhou, Xiu-Yun; Guo, Xian-Yang; Tu, Jun-Wei

2015-01-01

Inflammatory cytokines have been reported to be associated with pathogenesis of acute pancreatitis. The aim of this study was to measure the serum IL-35 levels in patients with acute pancreatitis and analyze the relationship between IL-35 levels and the disease severity. Thirty-two patients with acute pancreatitis and 32 healthy control subjects were included into the study. The serum levels of IL-35 were measured by enzyme-linked immunosorbent assay upon admission and the following seven days. The relationships with severity and etiology during the clinical course were analyzed. Serum IL-35 levels in patients with acute pancreatitis at the time of admission (5.25±0.37 ng/mL) were significantly higher than those in healthy controls (1.93±0.16 ng/mL, P<0.001). Moreover, serum IL-35 levels in patients with severe attacks (7.15±0.48 ng/mL) were significantly higher than those with moderately severe attacks (5.14±0.49 ng/mL, P=0.01) and mild attacks (3.69±0.53 ng/mL, P<0.001). However, there was no significant difference of serum IL-35 levels among patients with acute pancreatitis due of alcohol, gallstone and idiopathy. In addition, the peak serum concentrations of IL-35 were on day 1 after admission. Our results demonstrate that increased serum IL-35 levels may be related to the inflammatory response in patients with acute pancreatitis, suggesting that IL-35 may be used for a potential biomarker of acute pancreatitis. PMID:26221286

Gender Trends in Radiology Authorship: A 35-Year Analysis.

PubMed

Piper, Crystal L; Scheel, John R; Lee, Christoph I; Forman, Howard P

2016-01-01

The purpose of this study was to describe trends over time in female authorship in the radiology literature and to investigate the tendency of female first authors to publish with female senior authors. Data on the gender of academic physician authors based in the United States for all major articles published in three general radiology journals--Radiology, AJR, and Academic Radiology--were collected and analyzed for the years 1978, 1988, 1998, 2008, and 2013. Multivariate logistic regression was used to identify significant trends over time, and a chi-square test of independence was performed to determine significant relations between the genders of first and senior authors. The gender of 4182 of 4217 (99.17%) authors with MD degrees was determined. The proportion of original research articles published by women as first authors increased from 8.33% in 1978 to 32.35% in 2013 (p < 0.001). The proportion of original research articles with women as senior authors increased from 6.75% in 1978 to 21.90% in 2013 (p < 0.001). Female first and senior authorship increased significantly over time (first author, p < 0.001; senior author, p < 0.001). There was a statistically significant relation between the genders of first and senior authors of original research articles and guest editorials (p < 0.001). Over 35 years, there was a statistically significant upward linear trend of female physician participation in authorship of academic radiology literature. Female first authors were more likely to publish with female senior authors.

Expression and purification of the matrix protein of Nipah virus in baculovirus insect cell system.

PubMed

Masoomi Dezfooli, Seyedehsara; Tan, Wen Siang; Tey, Beng Ti; Ooi, Chien Wei; Hussain, Siti Aslina

2016-01-01

Nipah virus (NiV) causes fatal respiratory illness and encephalitis in humans and animals. The matrix (M) protein of NiV plays an important role in the viral assembly and budding process. Thus, an access to the NiV M protein is vital to the design of viral antigens as diagnostic reagents. In this study, recombinant DNA technology was successfully adopted in the cloning and expression of NiV M protein. A recombinant expression cassette (baculovirus expression vector) was used to encode an N-terminally His-tagged NiV M protein in insect cells. A time-course study demonstrated that the highest yield of recombinant M protein (400-500 μg) was expressed from 107 infected cells 3 days after infection. A single-step purification method based on metal ion affinity chromatography was established to purify the NiV M protein, which successfully yielded a purity level of 95.67% and a purification factor of 3.39. The Western blotting and enzyme-linked immunosorbent assay (ELISA) showed that the purified recombinant M protein (48 kDa) was antigenic and reacted strongly with the serum of a NiV infected pig. © 2015 American Institute of Chemical Engineers.

An eight-year epidemiologic study based on baculovirus-expressed type-specific spike proteins for the differentiation of type I and II feline coronavirus infections

PubMed Central

2014-01-01

Background Feline infectious peritonitis (FIP) is a fatal disease caused by feline coronavirus (FCoV). FCoVs are divided into two serotypes with markedly different infection rates among cat populations around the world. A baculovirus-expressed type-specific domain of the spike proteins of FCoV was used to survey the infection of the two viruses over the past eight years in Taiwan. Results An immunofluorescence assay based on cells infected with the recombinant viruses that was capable of distinguishing between the two types of viral infection was established. A total of 833 cases from a teaching hospital was surveyed for prevalence of different FCoV infections. Infection of the type I FCoV was dominant, with a seropositive rate of 70.4%, whereas 3.5% of cats were infected with the type II FCoV. In most cases, results derived from serotyping and genotyping were highly agreeable. However, 16.7% (4/24) FIP cats and 9.8% (6/61) clinically healthy cats were found to possess antibodies against both viruses. Moreover, most of the cats (84.6%, 22/26) infected with a genotypic untypable virus bearing a type I FCoV antibody. Conclusion A relatively simple serotyping method to distinguish between two types of FCoV infection was developed. Based on this method, two types of FCoV infection in Taiwan was first carried out. Type I FCoV was found to be predominant compared with type II virus. Results derived from serotyping and genotyping support our current understanding of evolution of disease-related FCoV and transmission of FIP. PMID:25123112

The Role of Biotransformation and Oxidative Stress in 3,5-Dichloroaniline (3,5-DCA) Induced Nephrotoxicity in Isolated Renal Cortical Cells from Male Fischer 344 Rats

PubMed Central

Racine, Christopher R.; Ferguson, Travis; Preston, Debbie; Ward, Dakota; Ball, John; Anestis, Dianne; Valentovic, Monica; Rankin, Gary O.

2016-01-01

Among the mono- and dichloroanilines, 3,5-Dichloroaniline (3,5-DCA) is the most potent nephrotoxicant in vivo and in vitro. However, the role of renal biotransformation in 3,5-DCA induced nephrotoxicity is unknown. The current study was designed to determine the in vitro nephrotoxic potential of 3,5-DCA in isolated renal cortical cells (IRCC) obtained from male Fischer 344 rats, and the role of renal bioactivation and oxidative stress in 3,5-DCA nephrotoxicity. IRCC (~4 million cells/ml) from male rats were exposed to 3,5-DCA (0-1.0 mM) for up to 120 min. In IRCC, 3,5-DCA was cytotoxic at 1.0 mM by 60 min as evidenced by the increased release of lactate dehydrogenase (LDH), but 120 min was required for 3,5-DCA 0.5 mM to increase LDH release. In subsequent studies, IRCC were exposed to a pretreatment (antioxidant or enzyme inhibitor) prior to exposure to 3,5-DCA (1.0 mM) for 90 min. Cytotoxicity induced by 3,5-DCA was attenuated by pretreatment with inhibitors of flavin-containing monooxygenase (FMO; methimazole, N-octylamine), cytochrome P450 (CYP; piperonyl butoxide, metyrapone), or peroxidase (indomethacin, mercaptosuccinate) enzymes. Use of more selective CYP inhibitors suggested that the CYP 2C family contributed to 3,5-DCA bioactivation. Antioxidants (glutathione, N-acetyl-L-cysteine, α-tocopherol, ascorbate, pyruvate) also attenuated 3,5-DCA nephrotoxicity, but oxidized glutathione levels and the oxidized/reduced glutathione ratios were not increased. These results indicate that 3,5-DCA may be activated via several renal enzyme systems to toxic metabolites, and that free radicals, but not oxidative stress, contribute to 3,5-DCA induced nephrotoxicity in vitro. PMID:26808022

Role of the IL-12/IL-35 balance in patients with Sjögren syndrome.

PubMed

Fogel, Olivier; Rivière, Elodie; Seror, Raphaèle; Nocturne, Gaetane; Boudaoud, Saida; Ly, Bineta; Gottenberg, Jacques-Eric; Le Guern, Véronique; Dubost, Jean-Jacques; Nititham, Joanne; Taylor, Kimberly E; Chanson, Philippe; Dieudé, Philippe; Criswell, Lindsey A; Jagla, Bernd; Thai, Alice; Mingueneau, Michael; Mariette, Xavier; Miceli-Richard, Corinne

2017-09-12

An interferon signature is involved in the pathogenesis of primary Sjögren syndrome (pSS), but whether the signature is type 1 or type 2 remains controversial. Mouse models and genetic studies suggest the involvement of T H 1 and type 2 interferon pathways. Likewise, polymorphisms of the IL-12A gene (IL12A), which encodes for IL-12p35, have been associated with pSS. The IL-12p35 subunit is shared by 2 heterodimers: IL-12 and IL-35. We sought to confirm genetic association of the IL12A polymorphism and pSS and elucidate involvement of the IL-12/IL-35 balance in patients with pSS by using functional studies. The genetic study involved 673 patients with pSS from 2 French pSS cohorts and 585 healthy French control subjects. Functional studies were performed on sorted monocytes, irrespective of whether they were stimulated. IL12A mRNA expression and IL-12 and IL-35 protein levels were assessed by using quantitative RT-PCR and ELISA and a multiplex kit for IL-35 and IL-12, respectively. We confirmed association of the IL12A rs485497 polymorphism and pSS and found an increased serum protein level of IL-12p70 in patients with pSS carrying the risk allele (P = .016). Serum levels of IL-12p70 were greater in patients than control subjects (P = .0001), especially in patients with more active disease (P = .05); conversely, IL-35 levels were decreased in patients (P = .0001), especially in patients with more active disease (P = .05). In blood cellular subsets both IL12p35 and EBV-induced gene protein 3 (EBI3) mRNAs were detected only in B cells, with a trend toward a lower level among patients with pSS. Our findings emphasize involvement of the IL-12/IL-35 balance in the pathogenesis of pSS. Serum IL-35 levels were associated with low disease activity, in contrast with serum IL-12p70 levels, which were associated with more active disease. Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Enhanced Expression of WD Repeat-Containing Protein 35 via CaMKK/AMPK Activation in Bupivacaine-Treated Neuro2a Cells

PubMed Central

Huang, Lei; Kondo, Fumio; Gosho, Masahiko; Feng, Guo-Gang; Harato, Misako; Xia, Zhong-yuan; Ishikawa, Naohisa; Fujiwara, Yoshihiro; Okada, Shoshiro

2014-01-01

We previously reported that bupivacaine induces reactive oxygen species (ROS) generation, p38 mitogen-activated protein kinase (MAPK) activation and nuclear factor-kappa B activation, resulting in an increase in expression of WD repeat-containing protein 35 (WDR35) in mouse neuroblastoma Neuro2a cells. However, the identity of signaling upstream of p38 MAPK pathways to WDR35 expression remains unclear. It has been shown that AMP-activated protein kinase (AMPK) can activate p38 MAPK through diverse mechanisms. In addition, several kinases acting upstream of AMPK have been identified including Ca2+/calmodulin-dependent protein kinase kinase (CaMKK). Recent studies reported that AMPK may be involved in bupivacaine-induced cytotoxicity in Schwann cells and in human neuroblastoma SH-SY5Y cells. The present study was undertaken to test whether CaMKK and AMPK are involved in bupivacaine-induced WDR35 expression in Neuro2a cells. Our results showed that bupivacaine induced activation of AMPK and p38 MAPK in Neuro2a cells. The AMPK inhibitors, compound C and iodotubercidin, attenuated the bupivacaine-induced activation of AMPK and p38 MAPK, resulting in an inhibition of the bupivacaine-induced increase in WDR35 expression. Treatment with the CaMKK inhibitor STO-609 also attenuated the bupivacaine-induced activation of AMPK and p38 MAPK, resulting in an inhibition of the bupivacaine-induced increase in WDR35 expression. These results suggest that bupivacaine activates AMPK and p38 MAPK via CaMKK in Neuro2a cells, and that the CaMKK/AMPK/p38 MAPK pathway is involved in regulating WDR35 expression. PMID:24859235

Transition metal complexes of 2-amino-3,5-dihalopyridines: Syntheses, structures and magnetic properties of (3,5-diCAPH)2CuX4 and (3,5-diBAPH)2CuX4.

PubMed

Tremelling, Grant W; Foxman, Bruce M; Landee, Christopher P; Turnbull, Mark M; Willett, Roger D

2009-12-21

A family of bis(2-amino-3,5-dihalopyridinium)tetrahalocuprate(II) compounds has been synthesized, including (3,5-diCAPH)2CuCl4 (1), (3,5-diCAPH)2CuBr4 (2), (3,5-diBAPH)2CuCl4 (3), and (3,5-diBAPH)2CuBr4 (4) [3,5-diCAPH = 2-amino-3,5-dichloropyridinium; 3,5-diBAPH = 2-amino-3,5-dibromopyridinium]. These complexes have been analyzed through single crystal X-ray diffraction and temperature dependent magnetic susceptibility. Compound 1 crystallizes in the P-1 space group and the tetrachlorocuprate ion is best described as possessing a distorted square planar geometry. Compounds 2-4 are structurally similar and crystallized in the P2(1)/n, P2(1)/c, and P2(1)/n space groups respectively. The tetrahalocuprate ions are best described as distorted tetrahedra. All four compounds show antiferromagnetic interactions and were fit to the uniform chain Heisenberg model with resulting 2J/kB values of -11.71(2) K, -2.21(1) K, -12.43 (2) K, and -1.36(1) K, respectively. The exchange values correlate well with the two-halide exchange pathway parameters. The unusual observation that the chloride complexes show stronger magnetic exchange than the bromide complexes provides strong support that the exchange can be strongly dependent upon the Cu-X...X angles and Cu-X...X-Cu torsion angles.

Phosphatidylinositol 3,5-Bisphosphate-Rich Membrane Domains in Endosomes and Lysosomes.

PubMed

Takatori, Sho; Tatematsu, Tsuyako; Cheng, Jinglei; Matsumoto, Jun; Akano, Takuya; Fujimoto, Toyoshi

2016-02-01

Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2 ) has critical functions in endosomes and lysosomes. We developed a method to define nanoscale distribution of PtdIns(3,5)P2 using freeze-fracture electron microscopy. GST-ATG18-4×FLAG was used to label PtdIns(3,5)P2 and its binding to phosphatidylinositol 3-phosphate (PtdIns(3)P) was blocked by an excess of the p40(phox) PX domain. In yeast exposed to hyperosmotic stress, PtdIns(3,5)P2 was concentrated in intramembrane particle (IMP)-deficient domains in the vacuolar membrane, which made close contact with adjacent membranes. The IMP-deficient domain was also enriched with PtdIns(3)P, but was deficient in Vph1p, a liquid-disordered domain marker. In yeast lacking either PtdIns(3,5)P2 or its effector, Atg18p, the IMP-deficient, PtdIns(3)P-rich membranes were folded tightly to make abnormal tubular structures, thus showing where the vacuolar fragmentation process is arrested when PtdIns(3,5)P2 metabolism is defective. In HeLa cells, PtdIns(3,5)P2 was significantly enriched in the vesicular domain of RAB5- and RAB7-positive endosome/lysosomes of the tubulo-vesicular morphology. This biased distribution of PtdIns(3,5)P2 was also observed using fluorescence microscopy, which further showed enrichment of a retromer component, VPS35, in the tubular domain. This is the first report to show segregation of PtdIns(3,5)P2 -rich and -deficient domains in endosome/lysosomes, which should be important for endosome/lysosome functionality. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Substance P is increased in patients with sickle cell disease and associated with haemolysis and hydroxycarbamide use

PubMed Central

Brandow, Amanda M.; Wandersee, Nancy J.; Dasgupta, Mahua; Hoffmann, Raymond G.; Hillery, Cheryl A.; Stucky, Cheryl L.; Panepinto, Julie A.

2016-01-01

Summary Sickle cell disease (SCD) pain transitions from acute to chronic for unknown reasons. Chronic elevation of the pain neurotransmitter substance P (SP) sensitizes pain nociceptors. We evaluated SP levels in controls and SCD patients during baseline and acute pain and investigated associations between SP and age, gender, pain history, haemolysis and hydroxycarbamide (also termed hydroxyurea) use. Plasma SP levels were measured using enzyme-linked immunosorbent assay. Independent samples t-test compared SP levels between: 1) SCD baseline and controls and 2) SCD baseline and acute pain. Multivariate linear regression determined associations between SP and age, gender, pain history and hydroxycarbamide use. Spearman correlation determined an association between SP and haemolysis. We enrolled 35 African American controls, 25 SCD baseline and 12 SCD pain patients. SCD patients were 7-19 years old. Mean±standard deviation SP level (pg/ml) in SCD baseline was higher than controls (32.4±11.6 vs. 22.9±7.6, P=0.0009). SP in SCD pain was higher than baseline (78.1±43.4 vs. 32.4±11.6, P=0.004). Haemolysis correlated with increased SP: Hb (r=−0.7, P=0.0002), reticulocyte count (r=0.61, P=0.0016), bilirubin (r=0.68, P=0.0216), LDH (r=0.62, P=0.0332), aspartate aminotransferase (r=0.68, P=0.003). Patients taking hydroxycarbamide had increased SP (β=29.2, P=0.007). SP could be a mediator of or marker for pain sensitization in SCD and a biomarker and/or target for novel pain treatment. PMID:27539682

Comparison of Causes of Death After Heart Transplantation in Patients With Left Ventricular Ejection Fractions ≤35% Versus >35.

PubMed

Birati, Edo Y; Mathelier, Hansie; Molina, Maria; Hanff, Thomas C; Mazurek, Jeremy A; Atluri, Pavan; Acker, Michael A; Rame, J Eduardo; Margulies, Kenneth B; Goldberg, Lee R; Jessup, Mariell

2016-04-15

Sudden cardiac death (SCD) is a common cause of death in the general population, occurring in 300,000 to 350,000 people in the United States alone. Currently, there are no data supporting implantable cardioverter-defibrillator therapy in patients who underwent orthotopic heart transplant (OHT) with low left ventricular ejection fraction (LVEF). In this retrospective study, we included all patients who underwent primary OHT at our institution from 2007 to 2013. We compared the cause of death in patients who underwent OHT and evaluated the correlation of the cause of death and the patients' LVEF. Our objectives were to determine whether patients who underwent OHT with LVEF <35% are at increased risk for SCD compared with those who underwent OHT with normal LVEF. To summarize our results, a total of 345 patients were included in our study (mean age 50 ± 14 years, 68% men). The mean follow-up was 1,260 ± 698 days. Forty patients (11.5%) died >6 months after OHT. Surviving patients had higher LVEF compared with deceased patients (64 ± 7% and 50 ± 24%, respectively, p ≤0.001). In all, 10 (25%) of the deceased patients died suddenly, 9 (23%) from sepsis, and 8 (20%) from malignancy. Of the 11 deceased patients with LVEF ≤35%, 2 patients (18%) died suddenly compared with 9 SCDs among the 29 deceased patients (31%) with LVEF >35% (p = 0.54). In conclusion, patients who underwent OHT who died were more likely to have LVEF <35%, and a quarter of the deceased patients who underwent OHT died suddenly. A reduced LVEF was not associated with an increased risk of SCD. Copyright © 2016 Elsevier Inc. All rights reserved.

Correlation between IL-17A/F, IL-23, IL-35 and IL-12/-23 (p40) levels in peripheral blood lymphocyte cultures and disease activity in Behcet's patients.

PubMed

Sonmez, Cemile; Yucel, Aysegul Atak; Yesil, Turan Hilmi; Kucuk, Hamit; Sezgin, Berna; Mercan, Ridvan; Yucel, Ahmet Eftal; Demirel, Gulderen Yanikkaya

2018-03-20

Behcet's disease is a chronic multisystemic disease with remissions and relapses. Several studies have shown that immune mechanisms play an important role in the development of the disease. In order to assess the association of disease activity with IL-17A/F, IL-23, IL-12/23 (p40) and IL-35 expression, we aimed to investigate production of these cytokines in peripheral blood mononuclear cells (PBMCs) from Behcet's patients and normal controls. Furthermore, we included Systemic Lupus Erythematosus (SLE) as disease control to evaluate the specificity of our data for immunopathogenesis of BD. Totally 15 active, 15 inactive Behcet's patients, 12 active and 12 inactive SLE patients and 12 healthy volunteers were enrolled in the study. Peripheral blood mononuclear cells were separated, lymphocyte cultures were performed and IL-17A/F, IL-12/23 p(40), IL-23, IL-35 cytokine levels were measured by ELISA in culture supernatants in the presence or absence of phytohemagglutinin (PHA) on time-dependent manner. IL-17 A/F levels increased parallel to IL-23 levels in Behcet's and SLE patients. Compared to healthy controls, IL-17 A/F levels were higher in active Behcet's and SLE patients; on the contrary, levels of IL-35 were lower. IL-17A/F, IL-12/23 (p40) and IL-23 levels were detectable most frequently in active Behcet's patients followed by active SLE patients. Our results indicate that IL-17 A/F, IL-23 and IL-12/23 (p40) may play role in the immunopathogenesis of BD so as Th17 and Th1 cell responses. Since IL-35 levels were lower in active Behcet's patients compared to inactive patients and healthy controls, there may be a plasticity between Th17 and Treg cells according to the state of disease activity.

Proteolytic Processing and Assembly of gag and gag-pol Proteins of TED, a Baculovirus-Associated Retrotransposon of the Gypsy Family

PubMed Central

Hajek, Kathryn L.; Friesen, Paul D.

1998-01-01

TED (transposable element D) is an env-containing member of the gypsy family of retrotransposons that represents a possible retrovirus of invertebrates. This lepidopteran (moth) retroelement contains gag and pol genes that encode proteins capable of forming viruslike particles (VLP) with reverse transcriptase. Since VLP are likely intermediates in TED transposition, we investigated the roles of gag and pol in TED capsid assembly and maturation. By using constructed baculovirus vectors and TED Gag-specific antiserum, we show that the principal translation product of gag (Pr55gag) is cleaved to produce a single VLP structural protein, p37gag. Replacement of Asp436 within the retrovirus-like active site of the pol-encoded protease (PR) abolished Pr55gag cleavage and demonstrated the requirement for PR in capsid processing. As shown by expression of an in-frame fusion of TED gag and pol, PR is derived from the Gag-Pol polyprotein Pr195gag-pol. The PR cleavage site within Pr55gag was mapped to a position near the junction of a basic, nucleocapsid-like domain and a C-terminal acidic domain. Once released by cleavage, the C-terminal fragment was not detected. This acidic fragment was dispensable for VLP assembly, as demonstrated by the formation of VLP by C-terminal Pr55gag truncation proteins and replacement of the acidic domain with a heterologous protein. In contrast, C-terminal deletions that extended into the adjacent nucleocapsid-like domain of Pr55gag abolished VLP recovery and demonstrated that this central region contributes to VLP assembly or stability, or both. Collectively, these data suggest that the single TED protein p37gag provides both capsid and nucleocapsid functions. TED may therefore use a simple processing strategy for VLP assembly and genome packaging. PMID:9765414

Proteolytic processing and assembly of gag and gag-pol proteins of TED, a baculovirus-associated retrotransposon of the gypsy family.

PubMed

Hajek, K L; Friesen, P D

1998-11-01

TED (transposable element D) is an env-containing member of the gypsy family of retrotransposons that represents a possible retrovirus of invertebrates. This lepidopteran (moth) retroelement contains gag and pol genes that encode proteins capable of forming viruslike particles (VLP) with reverse transcriptase. Since VLP are likely intermediates in TED transposition, we investigated the roles of gag and pol in TED capsid assembly and maturation. By using constructed baculovirus vectors and TED Gag-specific antiserum, we show that the principal translation product of gag (Pr55(gag)) is cleaved to produce a single VLP structural protein, p37(gag). Replacement of Asp436 within the retrovirus-like active site of the pol-encoded protease (PR) abolished Pr55(gag) cleavage and demonstrated the requirement for PR in capsid processing. As shown by expression of an in-frame fusion of TED gag and pol, PR is derived from the Gag-Pol polyprotein Pr195(gag-pol). The PR cleavage site within Pr55(gag) was mapped to a position near the junction of a basic, nucleocapsid-like domain and a C-terminal acidic domain. Once released by cleavage, the C-terminal fragment was not detected. This acidic fragment was dispensable for VLP assembly, as demonstrated by the formation of VLP by C-terminal Pr55(gag) truncation proteins and replacement of the acidic domain with a heterologous protein. In contrast, C-terminal deletions that extended into the adjacent nucleocapsid-like domain of Pr55(gag) abolished VLP recovery and demonstrated that this central region contributes to VLP assembly or stability, or both. Collectively, these data suggest that the single TED protein p37(gag) provides both capsid and nucleocapsid functions. TED may therefore use a simple processing strategy for VLP assembly and genome packaging.

Bicarbonate Increases Tumor pH and Inhibits Spontaneous Metastases

PubMed Central

Robey, Ian F.; Baggett, Brenda K.; Kirkpatrick, Nathaniel D.; Roe, Denise J.; Dosescu, Julie; Sloane, Bonnie F.; Hashim, Arig Ibrahim; Morse, David L.; Raghunand, Natarajan; Gatenby, Robert A.; Gillies, Robert J.

2010-01-01

The external pH of solid tumors is acidic as a consequence of increased metabolism of glucose and poor perfusion. Acid pH has been shown to stimulate tumor cell invasion and metastasis in vitro and in cells before tail vein injection in vivo. The present study investigates whether inhibition of this tumor acidity will reduce the incidence of in vivo metastases. Here, we show that oral NaHCO3 selectively increased the pH of tumors and reduced the formation of spontaneous metastases in mouse models of metastatic breast cancer. This treatment regimen was shown to significantly increase the extracellular pH, but not the intracellular pH, of tumors by 31P magnetic resonance spectroscopy and the export of acid from growing tumors by fluorescence microscopy of tumors grown in window chambers. NaHCO3 therapy also reduced the rate of lymph node involvement, yet did not affect the levels of circulating tumor cells, suggesting that reduced organ metastases were not due to increased intravasation. In contrast, NaHCO3 therapy significantly reduced the formation of hepatic metastases following intrasplenic injection, suggesting that it did inhibit extravasation and colonization. In tail vein injections of alternative cancer models, bicarbonate had mixed results, inhibiting the formation of metastases from PC3M prostate cancer cells, but not those of B16 melanoma. Although the mechanism of this therapy is not known with certainty, low pH was shown to increase the release of active cathepsin B, an important matrix remodeling protease. PMID:19276390

The Pharmacological Chaperone Isofagomine Increases Activity of the Gaucher Disease L444P Mutant Form of β-Glucosidase

PubMed Central

Khanna, Richie; Benjamin, Elfrida R.; Pellegrino, Lee; Schilling, Adriane; Rigat, Brigitte A.; Soska, Rebecca; Nafar, Hadis; Ranes, Brian E.; Feng, Jessie; Lun, Yi; Powe, Allan C.; Palling, David J.; Wustman, Brandon A.; Schiffmann, Raphael; Mahuran, Don J.; Lockhart, David J.; Valenzano, Kenneth J.

2010-01-01

SUMMARY Gaucher disease is caused by mutations in the gene that encodes the lysosomal enzyme acid β-glucosidase (GCase). We have shown previously that the small molecule pharmacological chaperone isofagomine (IFG) binds and stabilizes N370S GCase, resulting in increased lysosomal trafficking and cellular activity. In this study, we investigated the effect of IFG on L444P GCase. Incubation of Gaucher patient-derived lymphoblastoid cell lines (LCLs) or fibroblasts with IFG led to approximately 3.5- and 1.3-fold increases in L444P GCase activity, respectively, as measured in cell lysates. The effect in fibroblasts was increased approximately 2-fold using glycoprotein-enrichment, GCase-immunocapture, or by incubating cells overnight in IFG-free media prior to assay, methods designed to maximize GCase activity by reducing IFG carryover and inhibition in the enzymatic assay. IFG incubation also increased the lysosomal trafficking and in situ activity of L444P GCase in intact cells, as measured by reduction in endogenous glucosylceramide levels. Importantly, this reduction was seen only following three-day incubation in IFG-free media, underscoring the importance of IFG removal to restore lysosomal GCase activity. In mice expressing murine L444P GCase, oral administration of IFG resulted in significant increases (2- to 5-fold) in GCase activity in disease-relevant tissues, including brain. Additionally, eight-week IFG administration significantly lowered plasma chitin III and IgG levels, and 24-week administration significantly reduced spleen and liver weights. Taken together, these data suggest that IFG can increase the lysosomal activity of L444P GCase in cells and tissues. Moreover, IFG is orally available and distributes into multiple tissues, including brain, and may thus merit therapeutic evaluation for patients with neuronopathic and non-neuronopathic Gaucher disease. PMID:20148966

Increasing dietary lysine increases final pH and decreases drip loss of broiler breast meat.

PubMed

Berri, C; Besnard, J; Relandeau, C

2008-03-01

Responses to increased dietary Lys concentrations were evaluated on 1,584 Ross 308 male broilers between 21 and 42 d of age housed according to 2 bird densities. The experimental design was composed of 8 factorial treatments: 2 bird densities (22 or 44 broilers/ 1.7 m(2) pen) x 4 true digestible (TD) Lys levels (0.83, 0.93, 1.03, and 1.13%). There were 6 repetitions per treatment. Birds were weighed individually at d 21 and 42. Feed consumption was recorded per pen. Body weight gain and feed conversion were calculated over the experimental period. Forty-eight broilers per treatment were dissected at 42 d of age. Final pH and drip loss during storage were measured on the pectoralis major. Density adversely affected feed intake (169 +/- 1 and 160 +/- 1 g/d with 22 and 44 birds per pen, respectively, P < 0.05), growth rate (97.4 +/- 0.5 and 91.0 +/- 0.7 g/d, P < 0.05), and feed conversion (1.730 +/- 0.008 and 1.760 +/- 0.006, P < 0.05). Except for feed intake, there was no interaction between the effects of bird density and dietary Lys. An increase in dietary TD Lys from 0.83 to 0.93% resulted in an increased growth rate (from 91.8 +/- 1.6 to 95.5 +/- 0.8 g/d, P < 0.05), improved feed conversion (from 1.783 +/- 0.008 to 1.742 +/- 0.009, P < 0.05), and increased breast meat yield (22.0 +/- 0.1% to 22.7 +/- 0.2%, P < 0.01). Performance and body composition traits were not significantly improved for concentrations of TD Lys higher than 0.93%. However, final breast pH increased from 0.83 up to 1.03% TD Lys in the diet (6.02 +/- 0.01 vs. 5.91 +/- 0.01, P < 0.05), and drip loss correlatively decreased (0.85 +/- 0.03% vs. 1.10 +/- 0.06, P < 0.05). This result opens new way of research for the definition of an amino acid requirement and on metabolic pathways involved in variations of breast muscle pH.

The components of shear stress affecting insect cells used with the baculovirus expression vector system.

PubMed

Weidner, Tobias; Druzinec, Damir; Mühlmann, Martina; Buchholz, Rainer; Czermak, Peter

2017-09-26

Insect-based expression platforms such as the baculovirus expression vector system (BEVS) are widely used for the laboratory- and industrial-scale production of recombinant proteins. Thereby, major drawbacks to gain high-quality proteins are the lytic infection cycle and the shear sensitivity of infected insect cells due to turbulence and aeration. Smaller bubbles were formerly assumed to be more harmful than larger ones, but we found that cell damage is also dependent on the concentration of protective agents such as Pluronic®. At the appropriate concentration, Pluronic forms a layer around air bubbles and hinders the attachment of cells, thus limiting the damage. In this context, we used microaeration to vary bubble sizes and confirmed that size is not the most important factor, but the total gas surface area in the reactor is. If the surface area exceeds a certain threshold, the concentration of Pluronic is no longer sufficient for cell protection. To investigate the significance of shear forces, a second study was carried out in which infected insect cells were cultivated in a hollow fiber module to protect them from shear forces. Both model studies revealed important aspects of the design and scale-up of BEVS processes for the production of recombinant proteins.

Interleukin-35 induces regulatory B cells that suppress autoimmune disease.

PubMed

Wang, Ren-Xi; Yu, Cheng-Rong; Dambuza, Ivy M; Mahdi, Rashid M; Dolinska, Monika B; Sergeev, Yuri V; Wingfield, Paul T; Kim, Sung-Hye; Egwuagu, Charles E

2014-06-01

Interleukin-10 (IL-10)-producing regulatory B (Breg) cells suppress autoimmune disease, and increased numbers of Breg cells prevent host defense to infection and promote tumor growth and metastasis by converting resting CD4(+) T cells to regulatory T (Treg) cells. The mechanisms mediating the induction and development of Breg cells remain unclear. Here we show that IL-35 induces Breg cells and promotes their conversion to a Breg subset that produces IL-35 as well as IL-10. Treatment of mice with IL-35 conferred protection from experimental autoimmune uveitis (EAU), and mice lacking IL-35 (p35 knockout (KO) mice) or defective in IL-35 signaling (IL-12Rβ2 KO mice) produced less Breg cells endogenously or after treatment with IL-35 and developed severe uveitis. Adoptive transfer of Breg cells induced by recombinant IL-35 suppressed EAU when transferred to mice with established disease, inhibiting pathogenic T helper type 17 (TH17) and TH1 cells while promoting Treg cell expansion. In B cells, IL-35 activates STAT1 and STAT3 through the IL-35 receptor comprising the IL-12Rβ2 and IL-27Rα subunits. As IL-35 also induced the conversion of human B cells into Breg cells, these findings suggest that IL-35 may be used to induce autologous Breg and IL-35(+) Breg cells and treat autoimmune and inflammatory disease.

Crimean-Congo Hemorrhagic Fever Virus Gn Bioinformatic Analysis and Construction of a Recombinant Bacmid in Order to Express Gn by Baculovirus Expression System

PubMed Central

Rahpeyma, Mehdi; Fotouhi, Fatemeh; Makvandi, Manouchehr; Ghadiri, Ata; Samarbaf-Zadeh, Alireza

2015-01-01

Background Crimean-Congo hemorrhagic fever virus (CCHFV) is a member of the nairovirus, a genus in the Bunyaviridae family, which causes a life threatening disease in human. Currently, there is no vaccine against CCHFV and detailed structural analysis of CCHFV proteins remains undefined. The CCHFV M RNA segment encodes two viral surface glycoproteins known as Gn and Gc. Viral glycoproteins can be considered as key targets for vaccine development. Objectives The current study aimed to investigate structural bioinformatics of CCHFV Gn protein and design a construct to make a recombinant bacmid to express by baculovirus system. Materials and Methods To express the Gn protein in insect cells that can be used as antigen in animal model vaccine studies. Bioinformatic analysis of CCHFV Gn protein was performed and designed a construct and cloned into pFastBacHTb vector and a recombinant Gn-bacmid was generated by Bac to Bac system. Results Primary, secondary, and 3D structure of CCHFV Gn were obtained and PCR reaction with M13 forward and reverse primers confirmed the generation of recombinant bacmid DNA harboring Gn coding region under polyhedron promoter. Conclusions Characterization of the detailed structure of CCHFV Gn by bioinformatics software provides the basis for development of new experiments and construction of a recombinant bacmid harboring CCHFV Gn, which is valuable for designing a recombinant vaccine against deadly pathogens like CCHFV. PMID:26862379

Crimean-Congo Hemorrhagic Fever Virus Gn Bioinformatic Analysis and Construction of a Recombinant Bacmid in Order to Express Gn by Baculovirus Expression System.

PubMed

Rahpeyma, Mehdi; Fotouhi, Fatemeh; Makvandi, Manouchehr; Ghadiri, Ata; Samarbaf-Zadeh, Alireza

2015-11-01

Crimean-Congo hemorrhagic fever virus (CCHFV) is a member of the nairovirus, a genus in the Bunyaviridae family, which causes a life threatening disease in human. Currently, there is no vaccine against CCHFV and detailed structural analysis of CCHFV proteins remains undefined. The CCHFV M RNA segment encodes two viral surface glycoproteins known as Gn and Gc. Viral glycoproteins can be considered as key targets for vaccine development. The current study aimed to investigate structural bioinformatics of CCHFV Gn protein and design a construct to make a recombinant bacmid to express by baculovirus system. To express the Gn protein in insect cells that can be used as antigen in animal model vaccine studies. Bioinformatic analysis of CCHFV Gn protein was performed and designed a construct and cloned into pFastBacHTb vector and a recombinant Gn-bacmid was generated by Bac to Bac system. Primary, secondary, and 3D structure of CCHFV Gn were obtained and PCR reaction with M13 forward and reverse primers confirmed the generation of recombinant bacmid DNA harboring Gn coding region under polyhedron promoter. Characterization of the detailed structure of CCHFV Gn by bioinformatics software provides the basis for development of new experiments and construction of a recombinant bacmid harboring CCHFV Gn, which is valuable for designing a recombinant vaccine against deadly pathogens like CCHFV.

Ebola virus requires phosphatidylinositol (3,5) bisphosphate production for efficient viral entry.

PubMed

Qiu, Shirley; Leung, Anders; Bo, Yuxia; Kozak, Robert A; Anand, Sai Priya; Warkentin, Corina; Salambanga, Fabiola D R; Cui, Jennifer; Kobinger, Gary; Kobasa, Darwyn; Côté, Marceline

2018-01-01

For entry, Ebola virus (EBOV) requires the interaction of its viral glycoprotein with the cellular protein Niemann-Pick C1 (NPC1) which resides in late endosomes and lysosomes. How EBOV is trafficked and delivered to NPC1 and whether this is positively regulated during entry remain unclear. Here, we show that the PIKfyve-ArPIKfyve-Sac3 cellular complex, which is involved in the metabolism of phosphatidylinositol (3,5) bisphosphate (PtdIns(3,5)P 2 ), is critical for EBOV infection. Although the expression of all subunits of the complex was required for efficient entry, PIKfyve kinase activity was specifically critical for entry by all pathogenic filoviruses. Inhibition of PIKfyve prevented colocalization of EBOV with NPC1 and led to virus accumulation in intracellular vesicles with characteristics of early endosomes. Importantly, genetically-encoded phosphoinositide probes revealed an increase in PtdIns(3,5)P 2 -positive vesicles in cells during EBOV entry. Taken together, our studies suggest that EBOV requires PtdIns(3,5)P 2 production in cells to promote efficient delivery to NPC1. Copyright © 2017 Elsevier Inc. All rights reserved.

Administration of 3,5-diiodothyronine (3,5-T2) causes central hypothyroidism and stimulates thyroid-sensitive tissues.

PubMed

Padron, Alvaro Souto; Neto, Ruy Andrade Louzada; Pantaleão, Thiago Urgal; de Souza dos Santos, Maria Carolina; Araujo, Renata Lopes; de Andrade, Bruno Moulin; da Silva Leandro, Monique; de Castro, João Pedro Saar Werneck; Ferreira, Andrea Claudia Freitas; de Carvalho, Denise Pires

2014-06-01

In general, 3,5-diiodothyronine (3,5-T2) increases the resting metabolic rate and oxygen consumption, exerting short-term beneficial metabolic effects on rats subjected to a high-fat diet. Our aim was to evaluate the effects of chronic 3,5-T2 administration on the hypothalamus-pituitary-thyroid axis, body mass gain, adipose tissue mass, and body oxygen consumption in Wistar rats from 3 to 6 months of age. The rats were treated daily with 3,5-T2 (25, 50, or 75 μg/100 g body weight, s.c.) for 90 days between the ages of 3 and 6 months. The administration of 3,5-T2 suppressed thyroid function, reducing not only thyroid iodide uptake but also thyroperoxidase, NADPH oxidase 4 (NOX4), and thyroid type 1 iodothyronine deiodinase (D1 (DIO1)) activities and expression levels, whereas the expression of the TSH receptor and dual oxidase (DUOX) were increased. Serum TSH, 3,3',5-triiodothyronine, and thyroxine were reduced in a 3,5-T2 dose-dependent manner, whereas oxygen consumption increased in these animals, indicating the direct action of 3,5-T2 on this physiological variable. Type 2 deiodinase activity increased in both the hypothalamus and the pituitary, and D1 activities in the liver and kidney were also increased in groups treated with 3,5-T2. Moreover, after 3 months of 3,5-T2 administration, body mass and retroperitoneal fat pad mass were significantly reduced, whereas the heart rate and mass were unchanged. Thus, 3,5-T2 acts as a direct stimulator of energy expenditure and reduces body mass gain; however, TSH suppression may develop secondary to 3,5-T2 administration. © 2014 The authors.

Self-assembly and release of peste des petits ruminants virus-like particles in an insect cell-baculovirus system and their immunogenicity in mice and goats.

PubMed

Li, Wenchao; Jin, Hongyan; Sui, Xiukun; Zhao, Zhanzhong; Yang, Chenghuai; Wang, Wenquan; Li, Junping; Li, Gang

2014-01-01

Peste des petits ruminants (PPR) is an acute, febrile, viral disease of small ruminants that has a significant economic impact. For many viral diseases, vaccination with virus-like particles (VLPs) has shown considerable promise as a prophylactic approach; however, the processes of assembly and release of peste des petits ruminants virus (PPRV) VLPs are not well characterized, and their immunogenicity in the host is unknown. In this study, VLPs of PPRV were generated in a baculovirus system through simultaneous expression of PPRV matrix (M) protein and hemaglutin in (H) or fusion (F) protein. The released VLPs showed morphology similar to that of the native virus particles. Subcutaneous injection of these VLPs (PPRV-H, PPRV-F) into mice and goats elicited PPRV-specific IgG production, increased the levels of virus neutralizing antibodies, and promoted lymphocyte proliferation. Without adjuvants, the immune response induced by the PPRV-H VLPs was comparable to that obtained using equivalent amounts of PPRV vaccine. Thus, our results demonstrated that VLPs containing PPRV M protein and H or F protein are potential "differentiating infected from vaccinated animals" (DIVA) vaccine candidates for the surveillance and eradication of PPR.

p62 improves AD-like pathology by increasing autophagy.

PubMed

Caccamo, A; Ferreira, E; Branca, C; Oddo, S

2017-06-01

The multifunctional protein p62 is associated with neuropathological inclusions in several neurodegenerative disorders, including frontotemporal lobar degeneration, amyotrophic lateral sclerosis and Alzheimer's disease (AD). Strong evidence shows that in AD, p62 immunoreactivity is associated with neurofibrillary tangles and is involved in tau degradation. However, it remains to be determined whether p62 also plays a role in regulating amyloid-β (Aβ) aggregation and degradation. Using a gene therapy approach, here we show that increasing brain p62 expression rescues cognitive deficits in APP/PS1 mice, a widely used animal model of AD. The cognitive improvement was associated with a decrease in Aβ levels and plaque load. Using complementary genetic and pharmacologic approaches, we found that the p62-mediated changes in Aβ were due to an increase in autophagy. To this end, we showed that removing the LC3-interacting region of p62, which facilitates p62-mediated selective autophagy, or blocking autophagy with a pharmacological inhibitor, was sufficient to prevent the decrease in Aβ. Overall, we believe these data provide the first direct in vivo evidence showing that p62 regulates Aβ turnover.

Reducing Ambiguities in Spectroscopic Factors with Combined Measurements and the 86Kr(d,p) Reaction at 35MeV/u

NASA Astrophysics Data System (ADS)

Walter, D.; Cizewski, J. A.; Baugher, T.; Ratkiewicz, A.; Manning, B.; Lonsdale, S. J.; Burcher, S.; Pain, S. D.; Chipps, K. A.; Nunes, F. M.; Ahn, S.; Baumann, T.; Bazin, D.; Pereira, J.; Williams, S.; Thompson, P.; Cerizza, G.; Thornsberry, C.; Jones, K. L.; Bardayan, D. W.; O'Malley, P. D.; Kozub, R. L.; Ota, S.

2015-10-01

Spectroscopic information for low-lying states above shell closures depends on the shape of the bound-state potential, which greatly affects the extracted spectroscopic factors. To mitigate this uncertainty, Mukhamedzhanov and Nunes have proposed a combined method; the external portion is fixed with a peripheral reaction, and is combined with a higher energy measurement with a larger contribution from the interior. This will constrain the single-particle ANC, and should enable spectroscopic factors to be deduced with uncertainties dominated by cross-section measurements rather than the bound-state potential. Published measurements of 86Kr(d,p) at 5.5 MeV/u were used for the external contribution of this reaction. An ANC analysis shows that the reaction is peripheral at this energy and the ANC has been extracted. At less-peripheral energies, 86Kr(d,p) at 35 MeV/u has been measured in inverse kinematics at the NSCL using the ORRUBA and SIDAR arrays of silicon strip detectors. Results of the ANC analysis and preliminary results from 86Kr(d,p) at 35 MeV/u will be presented. This work is supported in part by the NSF and the U.S. DOE.

42 CFR 35.35 - Unsalable articles.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 42 Public Health 1 2010-10-01 2010-10-01 false Unsalable articles. 35.35 Section 35.35 Public... HOSPITAL AND STATION MANAGEMENT Disposition of Articles Produced by Patients § 35.35 Unsalable articles. Articles having no commercial value shall be stored, destroyed, or otherwise disposed of as the officer in...

42 CFR 35.35 - Unsalable articles.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 42 Public Health 1 2014-10-01 2014-10-01 false Unsalable articles. 35.35 Section 35.35 Public... HOSPITAL AND STATION MANAGEMENT Disposition of Articles Produced by Patients § 35.35 Unsalable articles. Articles having no commercial value shall be stored, destroyed, or otherwise disposed of as the officer in...

42 CFR 35.35 - Unsalable articles.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 42 Public Health 1 2011-10-01 2011-10-01 false Unsalable articles. 35.35 Section 35.35 Public... HOSPITAL AND STATION MANAGEMENT Disposition of Articles Produced by Patients § 35.35 Unsalable articles. Articles having no commercial value shall be stored, destroyed, or otherwise disposed of as the officer in...

42 CFR 35.35 - Unsalable articles.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 42 Public Health 1 2013-10-01 2013-10-01 false Unsalable articles. 35.35 Section 35.35 Public... HOSPITAL AND STATION MANAGEMENT Disposition of Articles Produced by Patients § 35.35 Unsalable articles. Articles having no commercial value shall be stored, destroyed, or otherwise disposed of as the officer in...

42 CFR 35.35 - Unsalable articles.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 42 Public Health 1 2012-10-01 2012-10-01 false Unsalable articles. 35.35 Section 35.35 Public... HOSPITAL AND STATION MANAGEMENT Disposition of Articles Produced by Patients § 35.35 Unsalable articles. Articles having no commercial value shall be stored, destroyed, or otherwise disposed of as the officer in...

The 38K-Mediated Specific Dephosphorylation of the Viral Core Protein P6.9 Plays an Important Role in the Nucleocapsid Assembly of Autographa californica Multiple Nucleopolyhedrovirus.

PubMed

Lai, Qingying; Wu, Wenbi; Li, Ao; Wang, Wei; Yuan, Meijin; Yang, Kai

2018-05-01

Encapsidation of the viral genomes, leading to the assembly of the nucleocapsids to form infectious progeny virions, is a key step in many virus life cycles. Baculovirus nucleocapsid assembly is a complex process that involves many proteins. Our previous studies showed that the deletion of the core gene 38K ( ac98 ) interrupted the nucleocapsid assembly by producing capsid sheaths devoid of viral genomes by an unknown mechanism. All homologs of 38K contain conserved motifs of the haloacid dehalogenase superfamily, which are involved in phosphoryl transfer. The requirements of these motifs for nucleocapsid assembly, confirmed in the present study, suggest that 38K may be a functioning haloacid dehalogenase. P6.9 is also encoded by a core gene ( ac100 ) and is required for viral genome encapsidation. It has been reported that multiple phosphorylated species of P6.9 are present in virus-infected cells, while only an unphosphorylated species is detected in the budded virus. Therefore, whether 38K mediates the dephosphorylation of P6.9 was investigated. An additional phosphorylated species of P6.9 in 38K -deleted or -mutated virus-transfected cells was detected, and the dephosphorylated sites mediated by 38K were determined by mass spectrometry. To assess the effects of dephosphorylation of P6.9 mediated by 38K on virus replication, these sites were mutated to glutamic acids (phosphorylation-mimic mutant) or to alanines (phosphorylation-deficient mutant). Studies showed that the nucleocapsid assembly was interrupted in phosphorylation-mimic mutant virus-transfected cells. Taken together, our findings demonstrate that 38K mediates the dephosphorylation of specific sites at the C terminus of P6.9, which is essential for viral genome encapsidation. IMPORTANCE Genome packaging is a fundamental process in the virus life cycle, and viruses have different strategies to perform this step. For several double-stranded DNA (dsDNA) viruses, the procapsid is formed before genome

The 38K-Mediated Specific Dephosphorylation of the Viral Core Protein P6.9 Plays an Important Role in the Nucleocapsid Assembly of Autographa californica Multiple Nucleopolyhedrovirus

PubMed Central

Lai, Qingying; Li, Ao; Wang, Wei; Yuan, Meijin

2018-01-01

ABSTRACT Encapsidation of the viral genomes, leading to the assembly of the nucleocapsids to form infectious progeny virions, is a key step in many virus life cycles. Baculovirus nucleocapsid assembly is a complex process that involves many proteins. Our previous studies showed that the deletion of the core gene 38K (ac98) interrupted the nucleocapsid assembly by producing capsid sheaths devoid of viral genomes by an unknown mechanism. All homologs of 38K contain conserved motifs of the haloacid dehalogenase superfamily, which are involved in phosphoryl transfer. The requirements of these motifs for nucleocapsid assembly, confirmed in the present study, suggest that 38K may be a functioning haloacid dehalogenase. P6.9 is also encoded by a core gene (ac100) and is required for viral genome encapsidation. It has been reported that multiple phosphorylated species of P6.9 are present in virus-infected cells, while only an unphosphorylated species is detected in the budded virus. Therefore, whether 38K mediates the dephosphorylation of P6.9 was investigated. An additional phosphorylated species of P6.9 in 38K-deleted or -mutated virus-transfected cells was detected, and the dephosphorylated sites mediated by 38K were determined by mass spectrometry. To assess the effects of dephosphorylation of P6.9 mediated by 38K on virus replication, these sites were mutated to glutamic acids (phosphorylation-mimic mutant) or to alanines (phosphorylation-deficient mutant). Studies showed that the nucleocapsid assembly was interrupted in phosphorylation-mimic mutant virus-transfected cells. Taken together, our findings demonstrate that 38K mediates the dephosphorylation of specific sites at the C terminus of P6.9, which is essential for viral genome encapsidation. IMPORTANCE Genome packaging is a fundamental process in the virus life cycle, and viruses have different strategies to perform this step. For several double-stranded DNA (dsDNA) viruses, the procapsid is formed before genome

¹H-MAS-NMR chemical shifts in hydrogen-bonded complexes of chlorophenols (pentachlorophenol, 2,4,6-trichlorophenol, 2,6-dichlorophenol, 3,5-dichlorophenol, and p-chlorophenol) and amine, and H/D isotope effects on ¹H-MAS-NMR spectra.

PubMed

Honda, Hisashi

2013-04-22

Chemical shifts (CS) of the ¹H nucleus in N···H···O type hydrogen bonds (H-bond) were observed in some complexes between chlorophenols [pentachlorophenol (PCP), 2,4,6-tricholorophenol (TCP), 2,6-dichlorophenol (26DCP), 3,5-dichlorophenol (35DCP), and p-chlorophenol (pCP)] and nitrogen-base (N-Base) by solid-state high-resolution ¹H-NMR with the magic-angle-spinning (MAS) method. Employing N-Bases with a wide range of pKa values (0.65-10.75), ¹H-MAS-NMR CS values of bridging H atoms in H-bonds were obtained as a function of the N-Base's pKa. The result showed that the CS values were increased with increasing pKa values in a range of DpKa < 0 [DpKa = pKa(N-Base)-pKa(chlorophenols)] and decreased when DpKa > 2: The maximum CS values was recorded in the PCP (pKa = 5.26)-4-methylpyridine (6.03), TCP (6.59)-imidazole (6.99), 26DCP (7.02)-2-amino-4-methylpyridine (7.38), 35DCP (8.04)-4-dimethylaminopyridine (9.61), and pCP (9.47)-4-dimethylaminopyridine (9.61) complexes. The largest CS value of 18.6 ppm was recorded in TCP-imidazole crystals. In addition, H/D isotope effects on ¹H-MAS-NMR spectra were observed in PCP-2-amino-3-methylpyridine. Based on the results of CS simulation using a B3LYP/6-311+G** function, it can be explained that a little changes of the N-H length in H-bond contribute to the H/D isotope shift of the ¹H-MAS-NMR peaks.

Acidic pH increases airway surface liquid viscosity in cystic fibrosis

PubMed Central

Tang, Xiao Xiao; Ostedgaard, Lynda S.; Hoegger, Mark J.; Moninger, Thomas O.; Karp, Philip H.; McMenimen, James D.; Choudhury, Biswa; Varki, Ajit; Stoltz, David A.; Welsh, Michael J.

2016-01-01

Cystic fibrosis (CF) disrupts respiratory host defenses, allowing bacterial infection, inflammation, and mucus accumulation to progressively destroy the lungs. Our previous studies revealed that mucus with abnormal behavior impaired mucociliary transport in newborn CF piglets prior to the onset of secondary manifestations. To further investigate mucus abnormalities, here we studied airway surface liquid (ASL) collected from newborn piglets and ASL on cultured airway epithelia. Fluorescence recovery after photobleaching revealed that the viscosity of CF ASL was increased relative to that of non-CF ASL. CF ASL had a reduced pH, which was necessary and sufficient for genotype-dependent viscosity differences. The increased viscosity of CF ASL was not explained by pH-independent changes in HCO3– concentration, altered glycosylation, additional pH-induced disulfide bond formation, increased percentage of nonvolatile material, or increased sulfation. Treating acidic ASL with hypertonic saline or heparin largely reversed the increased viscosity, suggesting that acidic pH influences mucin electrostatic interactions. These findings link loss of cystic fibrosis transmembrane conductance regulator–dependent alkalinization to abnormal CF ASL. In addition, we found that increasing Ca2+ concentrations elevated ASL viscosity, in part, independently of pH. The results suggest that increasing pH, reducing Ca2+ concentration, and/or altering electrostatic interactions in ASL might benefit early CF. PMID:26808501

Expression of p24 gag protein of bovine leukemia virus in insect cells and its use in immunodetection of the disease.

PubMed

Larsen, Alejandra; Gonzalez, Ester Teresa; Serena, María Soledad; Echeverría, María Gabriela; Mortola, Eduardo

2013-06-01

Bovine leukemia is a common retroviral infection of cattle. The disease is characterized by a strong immunological response to several viral proteins, but the antibodies against p24 and gp51 are predominant. In this study, a recombinant baculovirus containing the gag gene p24 was constructed and the protein, used as antigen, analyzed by western blot and an indirect in-house rp24-ELISA test. This allowed detecting the presence of antibodies for bovine leukemia virus in a panel of cattle sera. The authentication of the protein expands its potential use for different medical applications, from improved diagnosis of the disease to source of antigens to be included in a subunit vaccine.

20 CFR 225.35 - When a PIA used in computing a retirement annuity can be increased for DRC's.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 20 Employees' Benefits 1 2010-04-01 2010-04-01 false When a PIA used in computing a retirement... Credits § 225.35 When a PIA used in computing a retirement annuity can be increased for DRC's. Delayed retirement credits earned at different times are added to the PIA used in computing a retirement annuity as...

18 CFR 35.35 - Transmission infrastructure investment.

Code of Federal Regulations, 2011 CFR

2011-04-01

... infrastructure investment. 35.35 Section 35.35 Conservation of Power and Water Resources FEDERAL ENERGY... AND TARIFFS Transmission Infrastructure Investment Provisions § 35.35 Transmission infrastructure investment. (a) Purpose. This section establishes rules for incentive-based (including performance-based...

Reduction of a 4q35-encoded nuclear envelope protein in muscle differentiation

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

Ostlund, Cecilia; Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, NY 10032; Guan, Tinglu

2009-11-13

Muscular dystrophy and peripheral neuropathy have been linked to mutations in genes encoding nuclear envelope proteins; however, the molecular mechanisms underlying these disorders remain unresolved. Nuclear envelope protein p19A is a protein of unknown function encoded by a gene at chromosome 4q35. p19A levels are significantly reduced in human muscle as cells differentiate from myoblasts to myotubes; however, its levels are not similarly reduced in all differentiation systems tested. Because 4q35 has been linked to facioscapulohumeral muscular dystrophy (FSHD) and some adjacent genes are reportedly misregulated in the disorder, levels of p19A were analyzed in muscle samples from patients withmore » FSHD. Although p19A was increased in most cases, an absolute correlation was not observed. Nonetheless, p19A downregulation in normal muscle differentiation suggests that in the cases where its gene is inappropriately re-activated it could affect muscle differentiation and contribute to disease pathology.« less

Coordinated Regulation of Intestinal Functions in C. elegans by LIN-35/Rb and SLR-2

PubMed Central

Kirienko, Natalia V.; McEnerney, John D. K.; Fay, David S.

2008-01-01

LIN-35 is the sole C. elegans representative of the pocket protein family, which includes the mammalian Retinoblastoma protein pRb and its paralogs p107 and p130. In addition to having a well-established and central role in cell cycle regulation, pocket proteins have been increasingly implicated in the control of critical and diverse developmental and cellular processes. To gain a greater understanding of the roles of pocket proteins during development, we have characterized a synthetic genetic interaction between lin-35 and slr-2, which we show encodes a C2H2-type Zn-finger protein. Whereas animals harboring single mutations in lin-35 or slr-2 are viable and fertile, lin-35; slr-2 double mutants arrest uniformly in early larval development without obvious morphological defects. Using a combination of approaches including transcriptome profiling, mosaic analysis, starvation assays, and expression analysis, we demonstrate that both LIN-35 and SLR-2 act in the intestine to regulate the expression of many genes required for normal nutrient utilization. These findings represent a novel role for pRb family members in the maintenance of organ function. Our studies also shed light on the mechanistic basis of genetic redundancy among transcriptional regulators and suggest that synthetic interactions may result from the synergistic misregulation of one or more common targets. PMID:18437219

Cannibalism and virus production in Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) larvae fed with two leaf substrates inoculated with Baculovirus spodoptera.

PubMed

Valicente, F H; Tuelher, E S; Pena, R C; Andreazza, R; Guimarães, M R F

2013-04-01

Cannibalism in the fall armyworm, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) (FAW), is a limiting factor in a baculovirus production system. To detect the impact of cannibalism, a two-step bioassay was conducted with different larval ages of FAW fed on two food sources (corn and castor bean leaves) contaminated with the S. frugiperda multiple-embedded nucleopolyhedrovirus. In a first bioassay, the food source affected the cannibalism, being higher for all larval ages tested (5-, 6- and 7-day-old larvae) in larvae fed on corn than on those fed on castor bean leaves. Larval mortality, weight equivalent and larval equivalents (LEs) per hectare decreased as the larval age increased. Larval weight, occlusion bodies (OBs)/larva and total OBs increased when the larval age increased. In a second bioassay, in which only 6- and 7-day-old larvae were used because of the performance in the first bioassay, the cannibalism rates were affected by the interaction between food sources and time of feeding (48 and 72 h), reaching the highest values for 6- and 7-day-old larvae fed on corn leaves for 72 h. Mortality of the FAW was affected by the interaction between food sources, larval age and time of feeding. The lowest mortalities were on 7-day-old larvae when they were fed on castor bean leaves for 48 and 72 h. Larval weight, OBs/larva, total OBs and LEs were affected by the interaction between food sources and larval age. A significant correlation was observed between larval weight and OBs/larva that fed on both food sources, suggesting that larval weight can be used to achieve a concentration to be sprayed in 1 ha.

Population increase and environmental deterioration correspond with microlithic innovations in South Asia ca. 35,000 years ago

PubMed Central

Petraglia, Michael; Clarkson, Christopher; Boivin, Nicole; Haslam, Michael; Korisettar, Ravi; Chaubey, Gyaneshwer; Ditchfield, Peter; Fuller, Dorian; James, Hannah; Jones, Sacha; Kivisild, Toomas; Koshy, Jinu; Lahr, Marta Mirazón; Metspalu, Mait; Roberts, Richard; Arnold, Lee

2009-01-01

Genetic studies of South Asia's population history have led to postulations of a significant and early population expansion in the subcontinent, dating to sometime in the Late Pleistocene. We evaluate this argument, based on new mtDNA analyses, and find evidence for significant demographic transition in the subcontinent, dating to 35–28 ka. We then examine the paleoenvironmental and, particularly, archaeological records for this time period and note that this putative demographic event coincides with a period of ecological and technological change in South Asia. We document the development of a new diminutive stone blade (microlithic) technology beginning at 35–30 ka, the first time that the precocity of this transition has been recognized across the subcontinent. We argue that the transition to microlithic technology may relate to changes in subsistence practices, as increasingly large and probably fragmented populations exploited resources in contracting favorable ecological zones just before the onset of full glacial conditions. PMID:19620737

Population increase and environmental deterioration correspond with microlithic innovations in South Asia ca. 35,000 years ago.

PubMed

Petraglia, Michael; Clarkson, Christopher; Boivin, Nicole; Haslam, Michael; Korisettar, Ravi; Chaubey, Gyaneshwer; Ditchfield, Peter; Fuller, Dorian; James, Hannah; Jones, Sacha; Kivisild, Toomas; Koshy, Jinu; Lahr, Marta Mirazón; Metspalu, Mait; Roberts, Richard; Arnold, Lee

2009-07-28

Genetic studies of South Asia's population history have led to postulations of a significant and early population expansion in the subcontinent, dating to sometime in the Late Pleistocene. We evaluate this argument, based on new mtDNA analyses, and find evidence for significant demographic transition in the subcontinent, dating to 35-28 ka. We then examine the paleoenvironmental and, particularly, archaeological records for this time period and note that this putative demographic event coincides with a period of ecological and technological change in South Asia. We document the development of a new diminutive stone blade (microlithic) technology beginning at 35-30 ka, the first time that the precocity of this transition has been recognized across the subcontinent. We argue that the transition to microlithic technology may relate to changes in subsistence practices, as increasingly large and probably fragmented populations exploited resources in contracting favorable ecological zones just before the onset of full glacial conditions.

Increased Optoelectronic Quality and Uniformity of Hydrogenated p-InP Thin Films

DOE PAGES

Wang, Hsin -Ping; Sutter-Fella, Carolin M.; Lobaccaro, Peter; ...

2016-06-08

The thin-film vapor–liquid–solid (TF-VLS) growth technique presents a promising route for high quality, scalable, and cost-effective InP thin films for optoelectronic devices. Toward this goal, careful optimization of material properties and device performance is of utmost interest. Here, we show that exposure of polycrystalline Zn-doped TF-VLS InP to a hydrogen plasma (in the following referred to as hydrogenation) results in improved optoelectronic quality as well as lateral optoelectronic uniformity. A combination of low temperature photoluminescence and transient photocurrent spectroscopy was used to analyze the energy position and relative density of defect states before and after hydrogenation. Notably, hydrogenation reduces themore » relative intragap defect density by 1 order of magnitude. As a metric to monitor lateral optoelectronic uniformity of polycrystalline TF-VLS InP, photoluminescence and electron beam induced current mapping reveal homogenization of the grain versus grain boundary upon hydrogenation. At the device level, we measured more than 260 TF-VLS InP solar cells before and after hydrogenation to verify the improved optoelectronic properties. Hydrogenation increased the average open-circuit voltage (V OC) of individual TF-VLS InP solar cells by up to 130 mV and reduced the variance in V OC for the analyzed devices.« less

Improved isolation and purification of functional human Fas receptor extracellular domain using baculovirus-silkworm expression system.

PubMed

Muraki, Michiro; Honda, Shinya

2011-11-01

To achieve an efficient isolation of human Fas receptor extracellular domain (hFasRECD), a fusion protein of hFasRECD with human IgG1 heavy chain Fc domain containing thrombin cleavage sequence at the junction site was overexpressed using baculovirus-silkworm larvae expression system. The hFasRECD part was separated from the fusion protein by the effective cleavage of the recognition site with bovine thrombin. Protein G column treatment of the reaction mixture and the subsequent cation-exchange chromatography provided purified hFasRECD with a final yield of 13.5mg from 25.0 ml silkworm hemolymph. The functional activity of the product was examined by size-exclusion chromatography analysis. The isolated hFasRECD less strongly interacted with human Fas ligand extracellular domain (hFasLECD) than the Fc domain-bridged counterpart, showing the contribution of antibody-like avidity in the latter case. The purified glycosylated hFasRECD presented several discrete bands in the disulphide-bridge non-reducing SDS-PAGE analysis, and virtually all of the components were considered to participate in the binding to hFasLECD. The attached glycans were susceptible to PNGase F digestion, but mostly resistant to Endo Hf digestion under denaturing conditions. One of the components exhibited a higher susceptibility to PNGase F digestion under non-denaturing conditions. Copyright © 2011 Elsevier Inc. All rights reserved.

Interleukin 35 (IL-35) and IL-37: Intestinal and peripheral expression by T and B regulatory cells in patients with Inflammatory Bowel Disease.

PubMed

Fonseca-Camarillo, Gabriela; Furuzawa-Carballeda, Janette; Yamamoto-Furusho, Jesús K

2015-10-01

The aim of the study was to characterize and to quantify peripheral and tissue. IL-35- and IL-37-producing cells in Inflammatory Bowel Disease (IBD) patients. We studied a total of 38 active UC, 31 inactive UC, 17 active CD, and 13 inactive CD and 50 non-inflamed tissues as control group. Gene expression was measured by real time polymerase chain reaction (RT-PCR) and protein expression was evaluated in tissue by immunohistochemistry and in peripheral blood mononuclear cells by flow cytometry. Higher levels of IL-35 was produced by intestinal regulatory B cells and circulating regulatory CD4(+) and CD8(+) T cells in active vs. inactive disease or healthy donors (P<0.05). The IL-37 was conspicuously synthesized by circulating B cells, active natural killer cells and monocytes. These results suggest that down-regulation of inflammation in active IBD patients might be based on the increased expression of IL-35 and IL-37. Copyright © 2015 Elsevier Ltd. All rights reserved.

Prevalence, diversity and co-occurrence of the white spot syndrome virus, monodon baculovirus and Penaeus stylirostris densovirus in wild populations of Penaeus monodon in the Philippines.

PubMed

Orosco, Fredmoore L; Lluisma, Arturo O

2017-08-09

The farming of the black tiger shrimp Penaeus monodon in the Philippines relies on wild broodstock. PCR was thus used to determine the prevalence of white spot syndrome virus (WSSV), monodon baculovirus (MBV) and Penaeus stylirostris densovirus (PstDV) in a total of 178 shrimp from 6 geographically disparate locations where broodstock are captured for use in hatcheries. PCR amplicons were also sequenced to identify phylogenetic relationships of the virus haplotypes detected. Shrimp from southeastern Luzon (Camarines Norte) had the highest prevalence of each of the 3 viruses and were frequently co-infected with 2 or more viruses. No viruses were detected in shrimp from northwestern Luzon (Pangasinan). MBV was most prevalent and PstDV strains displayed the most genetic diversity. WSSV was detected at 3 sites, and a VP28 gene sequence examined was invariant and consistent with strains found in many countries, including Thailand, China, Japan, Korea, Indonesia, Iran, Brazil and Mexico. WSSV open reading frame 94 gene sequence analysis identified location-specific repeat types. MBV sequences were dissimilar to haplotypes detected in India. PstDV sequences were diverse and included 2 lineages detected either in Australia or in the United States, Ecuador, Taiwan, China and Vietnam. The PCR data confirmed that WSSV, MBV and PstDV are endemic in P. monodon in the Philippines but that populations at some locations might remain free of infection.

Evaluation of the interaction of cyclin-dependent kinase 5 with activator p25 and with p25-derived inhibitor CIP.

PubMed

Cardone, Antonio; Albers, R Wayne; Sriram, Ram D; Pant, Harish C

2010-05-01

A high-affinity inhibitor protein called CIP, produced by small truncations of p35, was experimentally identified. P35 is a physiological activator of the cyclin-dependent kinase cdk5. P25 is derived from proteolytic truncation of p35 within "stressed" neurons, and it is associated with the hyperphosphorylation of specific neuronal proteins, typically occurring in neurodegenerative diseases such as Alzheimer's. Here, we report a study of the binding mechanisms of the cdk5-p25 and cdk5-CIP complexes. This provides a better understanding of the source of the inhibitory activity of the protein CIP. We use a geometry-based technique to test the hypothesis that p25's truncation increases the flexibility of CIP and thus prevents cdk5 from reaching its active conformation. Our study is based on a geometry-based alignment algorithm, which aligns two given protein conformations with respect to their interfaces. Our results support the flexibility hypothesis and will be used as a basis for targeted molecular dynamics simulations.

Bibliography on Cold Regions Science and Technology. Volume 35, Part 2

DTIC Science & Technology

1981-12-01

tailings under permafrost conditions [1171, pp.7, rusj 35-352 Peel . D.A. Short-term fluctuations in heavy metal concentrations in an- tarctic...I-", chi] 35.4026 Clencial lealurcs ol Banna (llacici /hang, \\ , cl ai. [1980. p 8-:7, ,lii] 35-4027 Measnicmenls and analvsis on the ice...the vicnnlv of Banna Cilaciei C.n \\ . el al. [USD. p I4b-15;. chi]’ 35.4035 Migrating subglacial channel ol Banna Cilaciei /hanu. X

Interleukin-35: Expanding Its Job Profile

PubMed Central

Sawant, Deepali V.; Hamilton, Kristia

2015-01-01

Counter-regulation afforded by specialized regulatory cell populations and immunosuppressive cytokines is critical for balancing immune outcome. The inhibitory potential of the established suppressive cytokines, IL-10 and TGFβ, has been well elucidated in diverse inflammatory scenarios in conjunction with their key roles in Treg development and function. Despite the early predictions for an immunomodulatory role for the Ebi3/p35 heterodimer in placental trophoblasts, IL-35 biology remained elusive until 2007 when it was established as a Treg-restricted inhibitory cytokine. Since then, Treg-derived IL-35 has been shown to exhibit its suppressive activities in a range of autoimmune diseases and cancer models. Recent studies are beginning to explore other cellular sources of IL-35, such as Bregs and CD8+ Tregs. Despite these new cellular sources and targets, the mode of IL-35 suppression remains restricted to inhibition of proliferation and induction of an IL-35-producing induced regulatory T cell population referred to as iTr35. In this review, we explore the early beginnings, status quo, and future prospects of IL-35 biology. The unparalleled opportunity of targeting multiple immunosuppressive populations (Tregs, Bregs, CD8+ Tregs) through IL-35 is highly exciting and offers tremendous promise from a translational standpoint, particularly for cancer immunotherapies. PMID:25919641

Hyperthyroidism results in increased glycolytic capacity in the rat heart. A 31P-NMR study.

PubMed

Seymour, A M; Eldar, H; Radda, G K

1990-11-12

We have investigated the metabolic adaptations that occur in the thyroxine-treated rat heart. Rats were made hyperthyroid by daily intra-peritoneal injections of thyroxine (35 micrograms/100 g body weight) over seven days. 31P-NMR investigations of isolated glucose-perfused isometric hearts showed that thyroxine treatment caused an increase in Pi (from 4.9 mumols.(g dry wt.)-1 in control hearts to 11.7 mumols.(g dry wt.)-1 in hyperthyroid hearts), a decrease in phosphocreatine (from 36.5 mumols.(g dry wt.)-1 to 21.8 mumols.(g dry wt.)-1) with no change in ATP or ADP concentrations under the same conditions of cardiac work. The unidirectional exchange flux Pi----ATP was measured by saturation transfer NMR in hyperthyroid rat hearts. This exchange (which has been shown to contain a significant glycolytic component) increased by 2.2-fold in thyroxine-treated hearts in comparison to control hearts (to 3.6 mumols.(g dry wt.)-1.s-1, from 1.6 mumols.(g dry wt.)-1.s-1). In parallel experiments, NMR analysis of extracts from hyperthyroid rat hearts showed significantly elevated levels of glucose 6-phosphate, and fructose 6-phosphate. Measurements of enzyme activities isolated from hyperthyroid and control tissue showed a 40% increase in phosphofructokinase activity. These data together with the increased concentration of Pi show that both glycolytic and glycogenolytic fluxes are increased in the hyperthyroid rat heart. This metabolic adaptation may be necessary to cope with the increased number and activity of Na+/K(+)-ATPase pumps that occur in response to thyroxine treatment.

Acute p-synephrine ingestion increases fat oxidation rate during exercise.

PubMed

Gutiérrez-Hellín, Jorge; Del Coso, Juan

2016-08-01

p-Synephrine is a protoalkaloid widely used in dietary supplements for weight management because of its purported thermogenic effects. However, there is a lack of scientific information about its effectiveness to increase fat metabolism during exercise. The purpose of this investigation was to determine the effects of an acute ingestion of p-synephrine on fat oxidation at rest and during exercise. In a double-blind, randomized and counterbalanced experimental design, 18 healthy subjects performed two acute experimental trials after the ingestion of p-synephrine (3 mg kg(-1) ) or after the ingestion of a placebo (cellulose). Energy expenditure and fat oxidation rates were measured by indirect calorimetry at rest and during a cycle ergometer ramp exercise test (increases of 25 W every 3 min) until volitional fatigue. In comparison with the placebo, the ingestion of p-synephrine did not change energy consumption (1.6 ± 0.3 vs. 1.6 ± 0.3 kcal min(-1) ; P = 0.69) or fat oxidation rate at rest (0.08 ± 0.02 vs. 0.10 ± 0.04 g min(-1) ; P = 0.15). However, the intake of p-synephrine moved the fat oxidation-exercise intensity curve upwards during the incremental exercise (P < 0.05) without affecting energy expenditure. Moreover, p-synephrine increased maximal fat oxidation rate (0.29 ± 0.15 vs. 0.40 ± 0.18 g min(-1) ; P = 0.01) during exercise although it did not affect the intensity at which maximal fat oxidation was achieved (55.8 ± 7.7 vs. 56.7 ± 8.2% VO2peak ; P = 0.51). The acute ingestion of p-synephrine increased the fat oxidation rate while it reduced the carbohydrate oxidation rate when exercising at low-to-moderate exercise intensities. © 2016 The British Pharmacological Society.

High IL-35 pleural expression in patients with tuberculous pleural effusion.

PubMed

Dong, Xuan; Yang, Jiong

2015-05-03

IL-35 is a novel anti-inflammatory and immunosuppressive cytokine primarily produced by Treg cells, and is involved in inflammatory diseases and autoimmune diseases. However, its roles in tuberculous pleural effusion (TPE) remain unknown. We aimed to investigate the potential involvement of IL-35 in TPE. Thirty TPE patients and 20 lung cancer patients with malignant pleural effusion (MPE) were recruited. Samples of pleural effusion (100 mL) were collected after traditional pleurocentesis. Blood was sampled from TPE patients. Mononuclear cells were isolated by Ficoll-Hypaque gradient. Proportions of Th1, Th17, and IL-35-producing cells were analyzed by flow cytometry. IL-35 was assessed by real-time RT-PCR, ELISA, and immunofluorescence. An ELISPOT assay was used to assess the effect of IL-35 on pleural effusion mononuclear cells (PEMCs). Proportions of IL-35-producing cells were higher in TPE compared with MPE (49.4±6.0 vs. 15.8±5.4%, P<0.001) and blood from TPE patients (49.4±6.0% vs. 16.6±3.1, P<0.001). IL-35, IL-17 and IFN-γ were elevated in TPE compared with MPE (all P<0.01). ELISPOT assay showed that IL-35 reduced the proportion of IFN-γ-producing CD4+ T cells in TPE. IL-35 mRNA expression was higher in TPE compared with MPE (P<0.001). Immunofluorescence showed that IL-35-positive cells were present in pleural tissues from TPE patients. Results suggest that there is an imbalance in IL-35 metabolism in TPE. However, further studies are required to assess the exact relationship with the immune system response to tuberculosis. IL-35 might play a role in TPE and might be targeted as a treatment for TPE.

46 CFR 35.20-35 - Whistling-T/ALL.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 46 Shipping 1 2011-10-01 2011-10-01 false Whistling-T/ALL. 35.20-35 Section 35.20-35 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY TANK VESSELS OPERATIONS Navigation § 35.20-35 Whistling—T/ALL. The unnecessary sounding of a vessel's whistle is prohibited within any harbor limits of the...

46 CFR 35.20-35 - Whistling-T/ALL.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 46 Shipping 1 2010-10-01 2010-10-01 false Whistling-T/ALL. 35.20-35 Section 35.20-35 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY TANK VESSELS OPERATIONS Navigation § 35.20-35 Whistling—T/ALL. The unnecessary sounding of a vessel's whistle is prohibited within any harbor limits of the...

46 CFR 35.35-75 - Emergencies-TB/ALL.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 46 Shipping 1 2011-10-01 2011-10-01 false Emergencies-TB/ALL. 35.35-75 Section 35.35-75 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY TANK VESSELS OPERATIONS Cargo Handling § 35.35-75 Emergencies—TB/ALL. In case of emergencies nothing in the regulations in this subchapter shall be construed as...

46 CFR 35.35-75 - Emergencies-TB/ALL.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 46 Shipping 1 2014-10-01 2014-10-01 false Emergencies-TB/ALL. 35.35-75 Section 35.35-75 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY TANK VESSELS OPERATIONS Cargo Handling § 35.35-75 Emergencies—TB/ALL. In case of emergencies nothing in the regulations in this subchapter shall be construed as...

46 CFR 35.35-75 - Emergencies-TB/ALL.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 46 Shipping 1 2010-10-01 2010-10-01 false Emergencies-TB/ALL. 35.35-75 Section 35.35-75 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY TANK VESSELS OPERATIONS Cargo Handling § 35.35-75 Emergencies—TB/ALL. In case of emergencies nothing in the regulations in this subchapter shall be construed as...

46 CFR 35.35-75 - Emergencies-TB/ALL.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 46 Shipping 1 2013-10-01 2013-10-01 false Emergencies-TB/ALL. 35.35-75 Section 35.35-75 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY TANK VESSELS OPERATIONS Cargo Handling § 35.35-75 Emergencies—TB/ALL. In case of emergencies nothing in the regulations in this subchapter shall be construed as...

46 CFR 35.35-75 - Emergencies-TB/ALL.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 46 Shipping 1 2012-10-01 2012-10-01 false Emergencies-TB/ALL. 35.35-75 Section 35.35-75 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY TANK VESSELS OPERATIONS Cargo Handling § 35.35-75 Emergencies—TB/ALL. In case of emergencies nothing in the regulations in this subchapter shall be construed as...

VPS35 Mutations in Parkinson Disease

PubMed Central

Vilariño-Güell, Carles; Wider, Christian; Ross, Owen A.; Dachsel, Justus C.; Kachergus, Jennifer M.; Lincoln, Sarah J.; Soto-Ortolaza, Alexandra I.; Cobb, Stephanie A.; Wilhoite, Greggory J.; Bacon, Justin A.; Behrouz, Bahareh; Melrose, Heather L.; Hentati, Emna; Puschmann, Andreas; Evans, Daniel M.; Conibear, Elizabeth; Wasserman, Wyeth W.; Aasly, Jan O.; Burkhard, Pierre R.; Djaldetti, Ruth; Ghika, Joseph; Hentati, Faycal; Krygowska-Wajs, Anna; Lynch, Tim; Melamed, Eldad; Rajput, Alex; Rajput, Ali H.; Solida, Alessandra; Wu, Ruey-Meei; Uitti, Ryan J.; Wszolek, Zbigniew K.; Vingerhoets, François; Farrer, Matthew J.

2011-01-01

The identification of genetic causes for Mendelian disorders has been based on the collection of multi-incident families, linkage analysis, and sequencing of genes in candidate intervals. This study describes the application of next-generation sequencing technologies to a Swiss kindred presenting with autosomal-dominant, late-onset Parkinson disease (PD). The family has tremor-predominant dopa-responsive parkinsonism with a mean onset of 50.6 ± 7.3 years. Exome analysis suggests that an aspartic-acid-to-asparagine mutation within vacuolar protein sorting 35 (VPS35 c.1858G>A; p.Asp620Asn) is the genetic determinant of disease. VPS35 is a central component of the retromer cargo-recognition complex, is critical for endosome-trans-golgi trafficking and membrane-protein recycling, and is evolutionarily highly conserved. VPS35 c.1858G>A was found in all affected members of the Swiss kindred and in three more families and one patient with sporadic PD, but it was not observed in 3,309 controls. Further sequencing of familial affected probands revealed only one other missense variant, VPS35 c.946C>T; (p.Pro316Ser), in a pedigree with one unaffected and two affected carriers, and thus the pathogenicity of this mutation remains uncertain. Retromer-mediated sorting and transport is best characterized for acid hydrolase receptors. However, the complex has many types of cargo and is involved in a diverse array of biologic pathways from developmental Wnt signaling to lysosome biogenesis. Our study implicates disruption of VPS35 and retromer-mediated trans-membrane protein sorting, rescue, and recycling in the neurodegenerative process leading to PD. PMID:21763482

PAMAM dendrimer-baculovirus nanocomplex for microencapsulated adipose stem cell-gene therapy: in vitro and in vivo functional assessment.

PubMed

Paul, Arghya; Shao, Wei; Abbasi, Sana; Shum-Tim, Dominique; Prakash, Satya

2012-09-04

The present study aims to develop a new stem cell based gene delivery system consisting of human adipose tissue derived stem cells (hASCs) genetically modified with self-assembled nanocomplex of recombinant baculovirus and PAMAM dendrimer (Bac-PAMAM) to overexpress the vascular endothelial growth factor (VEGF). Cells were enveloped into branched PEG surface functionalized polymeric microcapsules for efficient transplantation. In vitro analysis confirmed efficient transduction of hASCs expressing 7.65 ± 0.86 ng functionally active VEGF per 10(6) microencapsulated hASCs (ASC-VEGF). To determine the potential of the developed system, chronically infarcted rat hearts were treated with either empty microcapsules (MC), microencapsulated hASCs expressing MGFP reporter protein (MC+ASC-MGFP), or MC+ASC-VEGF, and analyzed for 10 weeks. Post-transplantation data confirmed higher myocardial VEGF expressions with significantly enhanced neovasculature in the MC+ASC-VEGF group. In addition, the cardiac performance, as measured by percentage ejection fraction, also improved significantly in the MC+ASC-VEGF group (48.6 ± 6.1%) compared to that in MC+ASC-MGFP (38.8 ± 5.3%) and MC groups (31.5 ± 3.3%). Collectively, these data demonstrate the feasibility of this system for improved stem cell therapy applications.

[The expression and clinical significance of IL-35 in periodontitis and oral lichen planus].

PubMed

Wang, Yu; Jin, Ying; Wang, Yi-Yue; Xu, Dan-Ni; Lin, Xiao-Ping

2016-08-01

To investigate the influence of IL-35 in the pathogenesis of periodontitis and oral lichen planus, and the correlation between periodontitis and oral lichen planus patients. The gingival crevicular fluid（GCF） and serum were collected from patients with oral lichen planus (n=20), periodontitis (n=20), periodontitis and oral lichen planus (n=20) and healthy controls (n=12). The patients' basic information, probing depth, clinical attachment loss, gingival index, sulcus bleeding index were measured and collected. The expression of IL-35 in GCF and serum was detected by ELISA. SPSS19.0 software package was used for statistical analysis. The concentration of IL-35 in patients with periodontitis and oral lichen planus was significantly higher than that of other groups(P<0.05). There was significantly positive correlation between IL-35 level and periodontal clinical parameters. Periodontitis and oral lichen planus may increase the concentration of IL-35 both in serum and GCF. The expression of IL-35 was positively correlated with periodontitis and oral lichen planus.

Structural analysis and localization of the carbohydrate moieties of a soluble human interferon gamma receptor produced in baculovirus-infected insect cells.

PubMed Central

Manneberg, M.; Friedlein, A.; Kurth, H.; Lahm, H. W.; Fountoulakis, M.

1994-01-01

A soluble form of the human interferon gamma receptor that is required for the identification of interferon gamma antagonists was expressed in baculovirus-infected insect cells. The protein carried N-linked carbohydrate and showed a heterogeneity on denaturing polyacrylamide gels. We investigated the utilization of the potential sites for N-linked glycosylation and the structure of the carbohydrate moieties of this soluble receptor. Amino acid sequence analysis and ion spray mass spectrometry revealed that of the five potential sites for N-linked glycosylation, Asn17 and Asn69 were always utilized, whereas Asn62 and Asn162 were utilized in approximately one-third of the protein population. Asn223 was never found to be glycosylated. The soluble receptor was treated with N-glycosidase F and the oligosaccharides released were analyzed by matrix-assisted laser desorption mass spectrometry, which showed that the protein carried six types of short carbohydrate chains. The predominant species was a hexasaccharide of molecular mass 1,039, containing a fucose subunit linked to the proximal N-acetylglucosamine residue: [formula: see text] PMID:8142896

GNPAT p.D519G is independently associated with markedly increased iron stores in HFE p.C282Y homozygotes.

PubMed

Barton, James C; Chen, Wen-Pin; Emond, Mary J; Phatak, Pradyumna D; Subramaniam, V Nathan; Adams, Paul C; Gurrin, Lyle C; Anderson, Gregory J; Ramm, Grant A; Powell, Lawrie W; Allen, Katrina J; Phillips, John D; Parker, Charles J; McLaren, Gordon D; McLaren, Christine E

2017-03-01

GNPAT p.D519G positivity is significantly increased in HFE p.C282Y homozygotes with markedly increased iron stores. We sought to determine associations of p.D519G and iron-related variables with iron stores in p.C282Y homozygotes. We defined markedly increased iron stores as serum ferritin >2247pmol/L (>1000μg/L) and either hepatic iron >236μmol/g dry weight or iron >10g by induction phlebotomy (men and women). We defined normal or mildly elevated iron stores as serum ferritin <674.1pmol/L (<300μg/L) or either age≥40y with iron ≤2.5g iron by induction phlebotomy or age≥50y with ≤3.0g iron by induction phlebotomy (men only). We compared participant subgroups using univariate methods. Using multivariable logistic regression, we evaluated associations of markedly increased iron stores with these variables: age; iron supplement use (dichotomous); whole blood units donated; erythrocyte units received as transfusion; daily alcohol consumption, g; and p.D519G positivity (heterozygosity or homozygosity). The mean age of 56 participants (94.6% men) was 55±10 (SD) y; 41 had markedly increased iron stores. Prevalences of swollen/tender 2nd/3rd metacarpophalangeal joints and elevated aspartate or alanine aminotransferase were significantly greater in participants with markedly increased iron stores. Only participants with markedly increased iron stores had cirrhosis. In multivariable analyses, p.D519G positivity was the only exposure variable significantly associated with markedly increased iron stores (odds ratio 9.9, 95% CI [1.6, 60.3], p=0.0126). GNPAT p.D519G is strongly associated with markedly increased iron stores in p.C282Y homozygotes after correction for age, iron-related variables, and alcohol consumption. Copyright © 2016 Elsevier Inc. All rights reserved.

Demonstration of a Sub-Millimeter Wave Integrated Circuit (S-MMIC) using InP HEMT with a 35-nm Gate

NASA Technical Reports Server (NTRS)

Deal, W. R.; Din, S.; Padilla, J.; Radisic, V.; Mei, G.; Yoshida, W.; Liu, P. S.; Uyeda, J.; Barsky, M.; Gaier, T.;

[The effect of edaravone on MAPKs signal pathway associated with Abeta(25-35) treatment in PC12 cells].

PubMed

Zhang, Gui-lian; Guo, Ying-ying; Zhang, Lei; Li, Ting-ting; Du, Yun; Yao, Li; Zhang, Wang-gang; Wu, Hai-qin; Ma, Zhu-lin

2015-03-01

To explore whether edaravone protects cells damage via mitogen-activated protein kinases (MAPKs) signal pathway, and which procedure of p38 be affected so as to add theories for AD pathogenesis and treatments. According to different drugs treated, PC12 cells in vitro were divided into four groups. Negative control group: cells were treated with media alone. AD model group: cells were treated with 30 pmol/L Abeta(25-35). Inhibitor control group: cells were treated with 10 micromol/L SB203580 Cp38 mitogen-activated protein kinase (p38) inhibitor], 10 micromol/L SP600125 [c-Jun NH2 terminal kinase (JNK) inhibitor], or 10 micromol/L PD98059 extracelular signal regulated kinase (ERK) inhibitor]. Low-dose, middle-dose and high-dose edaravone group: cells plated for 24 hours treated with 30 micromol/L Abeta(25-35) and co-treated with 20, 40, 80 micromol/L edaravone 3 hours, respectively. The morphology of the treated cells were observed, the p-p38, p-JNK and p-ERK proteins in each group were tested by the Western blot. The p38 mRNA were tested in each group above (only add SB203580 10 micromol/L in third group) by the real time PCR. (1) The p-p38 protein was significantly increased in model control group compared with that in negative control group (P<0.05). The p-p38 protein in the inhibitor group and edaravone groups was decreased significantly (P<0.05) when compared with that in model control group. The p-p38 proteins were significantly increased in the three edaravone groups compared with that in inhibiter control group (P<0.05). The p-p38 protein in middle-dose edaravone group was decreased compared with that in low-dose edaravone group (P<0.05). There was no relationship in dose-dependent manner about edaravone. Compared with three edaravone groups, the p-p38 protein was lower than it in high-dose edaravone & inhibiter group (P<0.05). (2) The p-JNK protein was significantly increased in model control group compared with that in negative control group (P<0.05). The p

The baculovirus expression vector system: A commercial manufacturing platform for viral vaccines and gene therapy vectors.

PubMed

Felberbaum, Rachael S

2015-05-01

The baculovirus expression vector system (BEVS) platform has become an established manufacturing platform for the production of viral vaccines and gene therapy vectors. Nine BEVS-derived products have been approved - four for human use (Cervarix(®), Provenge(®), Glybera(®) and Flublok(®)) and five for veterinary use (Porcilis(®) Pesti, BAYOVAC CSF E2(®), Circumvent(®) PCV, Ingelvac CircoFLEX(®) and Porcilis(®) PCV). The BEVS platform offers many advantages, including manufacturing speed, flexible product design, inherent safety and scalability. This combination of features and product approvals has previously attracted interest from academic researchers, and more recently from industry leaders, to utilize BEVS to develop next generation vaccines, vectors for gene therapy, and other biopharmaceutical complex proteins. In this review, we explore the BEVS platform, detailing how it works, platform features and limitations and important considerations for manufacturing and regulatory approval. To underscore the growth in opportunities for BEVS-derived products, we discuss the latest product developments in the gene therapy and influenza vaccine fields that follow in the wake of the recent product approvals of Glybera(®) and Flublok(®), respectively. We anticipate that the utility of the platform will expand even further as new BEVS-derived products attain licensure. Finally, we touch on some of the areas where new BEVS-derived products are likely to emerge. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Fullerene-like organization of HIV gag-protein shell in virus-like particles produced by recombinant baculovirus.

PubMed

Nermut, M V; Hockley, D J; Jowett, J B; Jones, I M; Garreau, M; Thomas, D

1994-01-01

Virus-like particles produced by a recombinant baculovirus containing the HIV gag gene were examined by negative staining after delipidization. This technique demonstrated that the gag-protein shell consisted of radially arranged short rods which formed a network of ring-like structures. Similar structures were observed at the plasma membrane of infected cells which had been opened by wet-cleaving. Occasionally five or six subunits were observed forming a ring. These findings suggest that the gag-encoded precursor (pr55) is a rod-like molecule about 34 A in diameter and 85 A in length. A protein cylinder of such dimensions would have a molecular weight of 56K. The center-to-center distance of two neighboring rings formed by the rods was 66 +/- 8 A (N = 200) by direct measurements and 65 A as obtained from averaged images. This morphology and these dimensions indicate that the virus-like particles contain the gag precursor in the form of a near-spherical "fullerene-like" icosahedral shell. Our data indicate that the triangulation number of the rings equals 63. However, since one rod of pr55 is shared by two rings, the number of copies of the precursor will be 1890 as opposed to 2522 if the molecules were closely packed. The particle diameter of 102 nm deduced from the proposed model was close to the diameter obtained from thin sections of low-temperature-embedded specimens (103-108 nm).

Splenda alters gut microflora and increases intestinal p-glycoprotein and cytochrome p-450 in male rats.

PubMed

Abou-Donia, Mohamed B; El-Masry, Eman M; Abdel-Rahman, Ali A; McLendon, Roger E; Schiffman, Susan S

2008-01-01

Splenda is comprised of the high-potency artificial sweetener sucralose (1.1%) and the fillers maltodextrin and glucose. Splenda was administered by oral gavage at 100, 300, 500, or 1000 mg/kg to male Sprague-Dawley rats for 12-wk, during which fecal samples were collected weekly for bacterial analysis and measurement of fecal pH. After 12-wk, half of the animals from each treatment group were sacrificed to determine the intestinal expression of the membrane efflux transporter P-glycoprotein (P-gp) and the cytochrome P-450 (CYP) metabolism system by Western blot. The remaining animals were allowed to recover for an additional 12-wk, and further assessments of fecal microflora, fecal pH, and expression of P-gp and CYP were determined. At the end of the 12-wk treatment period, the numbers of total anaerobes, bifidobacteria, lactobacilli, Bacteroides, clostridia, and total aerobic bacteria were significantly decreased; however, there was no significant treatment effect on enterobacteria. Splenda also increased fecal pH and enhanced the expression of P-gp by 2.43-fold, CYP3A4 by 2.51-fold, and CYP2D1 by 3.49-fold. Following the 12-wk recovery period, only the total anaerobes and bifidobacteria remained significantly depressed, whereas pH values, P-gp, and CYP3A4 and CYP2D1 remained elevated. These changes occurred at Splenda dosages that contained sucralose at 1.1-11 mg/kg (the US FDA Acceptable Daily Intake for sucralose is 5 mg/kg). Evidence indicates that a 12-wk administration of Splenda exerted numerous adverse effects, including (1) reduction in beneficial fecal microflora, (2) increased fecal pH, and (3) enhanced expression levels of P-gp, CYP3A4, and CYP2D1, which are known to limit the bioavailability of orally administered drugs.

Increased Arf/p53 activity in stem cells, aging and cancer.

PubMed

Carrasco-Garcia, Estefania; Moreno, Manuel; Moreno-Cugnon, Leire; Matheu, Ander

2017-04-01

Arf/p53 pathway protects the cells against DNA damage induced by acute stress. This characteristic is the responsible for its tumor suppressor activity. Moreover, it regulates the chronic type of stress associated with aging. This is the basis of its anti-aging activity. Indeed, increased gene dosage of Arf/p53 displays elongated longevity and delayed aging. At a cellular level, it has been recently shown that increased dosage of Arf/p53 delays age-associated stem cell exhaustion and the subsequent decline in tissue homeostasis and regeneration. However, p53 can also promote aging if constitutively activated. In this context, p53 reduces tissue regeneration, which correlates with premature exhaustion of stem cells. We discuss here the current evidence linking the Arf/p53 pathway to the processes of aging and cancer through stem cell regulation. © 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

Ingestion of Wheat Protein Increases In Vivo Muscle Protein Synthesis Rates in Healthy Older Men in a Randomized Trial.

PubMed

Gorissen, Stefan Hm; Horstman, Astrid Mh; Franssen, Rinske; Crombag, Julie Jr; Langer, Henning; Bierau, Jörgen; Respondek, Frederique; van Loon, Luc Jc

2016-09-01

Muscle mass maintenance is largely regulated by basal muscle protein synthesis and the capacity to stimulate muscle protein synthesis after food intake. The postprandial muscle protein synthetic response is modulated by the amount, source, and type of protein consumed. It has been suggested that plant-based proteins are less potent in stimulating postprandial muscle protein synthesis than animal-derived proteins. However, few data support this contention. We aimed to assess postprandial plasma amino acid concentrations and muscle protein synthesis rates after the ingestion of a substantial 35-g bolus of wheat protein hydrolysate compared with casein and whey protein. Sixty healthy older men [mean ± SEM age: 71 ± 1 y; body mass index (in kg/m(2)): 25.3 ± 0.3] received a primed continuous infusion of l-[ring-(13)C6]-phenylalanine and ingested 35 g wheat protein (n = 12), 35 g wheat protein hydrolysate (WPH-35; n = 12), 35 g micellar casein (MCas-35; n = 12), 35 g whey protein (Whey-35; n = 12), or 60 g wheat protein hydrolysate (WPH-60; n = 12). Plasma and muscle samples were collected at regular intervals. The postprandial increase in plasma essential amino acid concentrations was greater after ingesting Whey-35 (2.23 ± 0.07 mM) than after MCas-35 (1.53 ± 0.08 mM) and WPH-35 (1.50 ± 0.04 mM) (P < 0.01). Myofibrillar protein synthesis rates increased after ingesting MCas-35 (P < 0.01) and were higher after ingesting MCas-35 (0.050% ± 0.005%/h) than after WPH-35 (0.032% ± 0.004%/h) (P = 0.03). The postprandial increase in plasma leucine concentrations was greater after ingesting Whey-35 than after WPH-60 (peak value: 580 ± 18 compared with 378 ± 10 μM, respectively; P < 0.01), despite similar leucine contents (4.4 g leucine). Nevertheless, the ingestion of WPH-60 increased myofibrillar protein synthesis rates above basal rates (0.049% ± 0.007%/h; P = 0.02). The myofibrillar protein synthetic response to the ingestion of 35 g casein is greater than after an

The response of meat ducks from 15 to 35 d of age to gossypol from cottonseed meal.

PubMed

Zeng, Q F; Bai, P; Wang, J P; Ding, X M; Luo, Y H; Bai, S P; Xuan, Y; Su, Z W; Lin, S Q; Zhao, L J; Zhang, K Y

2015-06-01

The objective of this study was to investigate the responses of meat ducks of 15 to 35 d of age to free gossypol (FG) from cottonseed meal (CSM) and to establish the maximum limits of dietary FG concentration based on growth performance, blood parameters, and tissue residues of gossypol. Nine hundred 15-d-old ducks were randomly allocated to 5 treatments with 10 cages/treatment and 18 ducks/cage on the basis of BW. Five isonitrogenous and isocaloric experimental diets were formulated on a digestible amino acid basis to produce diets in which 0% (without FG), 25% (36 mg FG/kg), 50% (75 mg FG/kg), 75% (111 mg FG/kg), and 100% (153 mg FG/kg) of protein from soybean meal were replaced by that from CSM. Increasing dietary FG content, BW, and ADG decreased (linearly, P<0.05, except for ADG of days 29 to 35), and F/G linearly increased (P<0.05). At 35 d, blood hemoglobin, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration linearly decreased (P<0.05), while serum total protein, albumin, and globulin content linearly decreased (P<0.05), and the residue of gossypol in liver, kidney, heart, breast, and leg muscle linearly increased (P<0.001) with increases in dietary FG concentration. Ducks fed 36 mg FG/kg (5.83% CSM of diet) diet had a normal histological structure of liver, and muscle (breast and leg) had no residue of gossypol. The maximum limit of dietary FG concentration was estimated to range from a low of 36 mg/kg to maximize serum globulin concentration to a high of 124 mg/kg to minimize feed intake for 22 to 28 d on the basis of a quadratic broken-line model. © The Author 2015. Published by Oxford University Press on behalf of Poultry Science Association.

Global riverine N and P transport to ocean increased during the 20th century despite increased retention along the aquatic continuum

NASA Astrophysics Data System (ADS)

Beusen, Arthur H. W.; Bouwman, Alexander F.; Van Beek, Ludovicus P. H.; Mogollón, José M.; Middelburg, Jack J.

2016-04-01

Various human activities - including agriculture, water consumption, river damming, and aquaculture - have intensified over the last century. This has had a major impact on nitrogen (N) and phosphorus (P) cycling in global continental waters. In this study, we use a coupled nutrient-input-hydrology-in-stream nutrient retention model to quantitatively track the changes in the global freshwater N and P cycles over the 20th century. Our results suggest that, during this period, the global nutrient delivery to streams increased from 34 to 64 Tg N yr-1 and from 5 to 9 Tg P yr-1. Furthermore, in-stream retention and removal grew from 14 to 27 Tg N yr-1 and 3 to 5 Tg P yr-1. One of the major causes of increased retention is the growing number of reservoirs, which now account for 24 and 22 % of global N and P retention/removal in freshwater systems, respectively. This increase in nutrient retention could not balance the increase in nutrient delivery to rivers with the consequence that river nutrient transport to the ocean increased from 19 to 37 Tg N yr-1 and from 2 to 4 Tg P yr-1. Human activities have also led to a global increase in the molar N : P ratio in freshwater bodies.

Pseudomonas aeruginosa phage PaP1 DNA polymerase is an A-family DNA polymerase demonstrating ssDNA and dsDNA 3'-5' exonuclease activity.

PubMed

Liu, Binyan; Gu, Shiling; Liang, Nengsong; Xiong, Mei; Xue, Qizhen; Lu, Shuguang; Hu, Fuquan; Zhang, Huidong

2016-08-01

Most phages contain DNA polymerases, which are essential for DNA replication and propagation in infected host bacteria. However, our knowledge on phage-encoded DNA polymerases remains limited. This study investigated the function of a novel DNA polymerase of PaP1, which is the lytic phage of Pseudomonas aeruginosa. PaP1 encodes its sole DNA polymerase called Gp90 that was predicted as an A-family DNA polymerase with polymerase and 3'-5' exonuclease activities. The sequence of Gp90 is homologous but not identical to that of other A-family DNA polymerases, such as T7 DNA polymerases (Pol) and DNA Pol I. The purified Gp90 demonstrated a polymerase activity. The processivity of Gp90 in DNA replication and its efficiency in single-dNTP incorporation are similar to those of T7 Pol with processive thioredoxin (T7 Pol/trx). Gp90 can degrade ssDNA and dsDNA in 3'-5' direction at a similar rate, which is considerably lower than that of T7 Pol/trx. The optimized conditions for polymerization were a temperature of 37 °C and a buffer consisting of 40 mM Tris-HCl (pH 8.0), 30 mM MgCl2, and 200 mM NaCl. These studies on DNA polymerase encoded by PaP1 help advance our knowledge on phage-encoded DNA polymerases and elucidate PaP1 propagation in infected P. aeruginosa.

Remission of systemic lupus erythematosus disease activity with regulatory cytokine interleukin (IL)-35 in Murphy Roths Large (MRL)/lpr mice.

PubMed

Cai, Z; Wong, C K; Dong, J; Chu, M; Jiao, D; Kam, N W; Lam, C W K; Tam, L S

2015-08-01

The immunological mechanisms mediated by regulatory cytokine interleukin (IL)-35 are unclear in systemic lupus erythematosus (SLE). We investigated the frequency of CD4(+) CD25(+) forkhead box protein 3 (FoxP3)(+) regulatory T (Treg ) and IL-10(+) regulatory B (Breg ) cells and related immunoregulatory mechanisms in a female Murphy Roths Large (MRL)/lpr mouse model of spontaneous lupus-like disease, with or without IL-35 treatment. A remission of histopathology characteristics of lupus flare and nephritis was observed in the MRL/lpr mice upon IL-35 treatment. Accordingly, IL-35 and IL-35 receptor subunits (gp130 and IL-12Rβ2) and cytokines of MRL/lpr and BALB/c mice (normal controls) were measured. The increased anti-inflammatory cytokines and decreased proinflammatory cytokines were possibly associated with the restoration of Treg and Breg frequency in MRL/lpr mice with IL-35 treatment, compared to phosphate-buffered saline (PBS) treatment. mRNA expressions of Treg -related FoxP3, IL-35 subunit (p35 and EBI3) and soluble IL-35 receptor subunit (gp130 and IL12Rβ2) in splenic cells were up-regulated significantly in IL-35-treated mice. Compared with the PBS treatment group, IL-35-treated MRL/lpr mice showed an up-regulation of Treg -related genes and the activation of IL-35-related intracellular Janus kinase/signal transducer and activator of transcription signal pathways, thereby indicating the immunoregulatory role of IL-35 in SLE. These in vivo findings may provide a biochemical basis for further investigation of the regulatory mechanisms of IL-35 for the treatment of autoimmune-mediated inflammation. © 2015 British Society for Immunology.

Mitochondrial Impairment May Increase Cellular NAD(P)H: Resazurin Oxidoreductase Activity, Perturbing the NAD(P)H-Based Viability Assays.

PubMed

Aleshin, Vasily A; Artiukhov, Artem V; Oppermann, Henry; Kazantsev, Alexey V; Lukashev, Nikolay V; Bunik, Victoria I

2015-08-21

Cellular NAD(P)H-dependent oxidoreductase activity with artificial dyes (NAD(P)H-OR) is an indicator of viability, as the cellular redox state is important for biosynthesis and antioxidant defense. However, high NAD(P)H due to impaired mitochondrial oxidation, known as reductive stress, should increase NAD(P)H-OR yet perturb viability. To better understand this complex behavior, we assayed NAD(P)H-OR with resazurin (Alamar Blue) in glioblastoma cell lines U87 and T98G, treated with inhibitors of central metabolism, oxythiamin, and phosphonate analogs of 2-oxo acids. Targeting the thiamin diphosphate (ThDP)-dependent enzymes, the inhibitors are known to decrease the NAD(P)H production in the pentose phosphate shuttle and/or upon mitochondrial oxidation of 2-oxo acids. Nevertheless, the inhibitors elevated NAD(P)H-OR with resazurin in a time- and concentration-dependent manner, suggesting impaired NAD(P)H oxidation rather than increased viability. In particular, inhibition of the ThDP-dependent enzymes affects metabolism of malate, which mediates mitochondrial oxidation of cytosolic NAD(P)H. We showed that oxythiamin not only inhibited mitochondrial 2-oxo acid dehydrogenases, but also induced cell-specific changes in glutamate and malate dehydrogenases and/or malic enzyme. As a result, inhibition of the 2-oxo acid dehydrogenases compromises mitochondrial metabolism, with the dysregulated electron fluxes leading to increases in cellular NAD(P)H-OR. Perturbed mitochondrial oxidation of NAD(P)H may thus complicate the NAD(P)H-based viability assay.

46 CFR 35.35-5 - Electrical bonding-TB/ALL.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 46 Shipping 1 2013-10-01 2013-10-01 false Electrical bonding-TB/ALL. 35.35-5 Section 35.35-5 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY TANK VESSELS OPERATIONS Cargo Handling § 35.35-5 Electrical bonding—TB/ALL. The use of a vessel/shore bonding cable or wire is permissible only if...

46 CFR 35.40-35 - Rudder orders-TB/ALL.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 46 Shipping 1 2014-10-01 2014-10-01 false Rudder orders-TB/ALL. 35.40-35 Section 35.40-35 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY TANK VESSELS OPERATIONS Posting and Marking Requirements-TB/ALL § 35.40-35 Rudder orders—TB/ALL. At all steering stations, there shall be installed a suitable...

46 CFR 35.40-35 - Rudder orders-TB/ALL.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 46 Shipping 1 2013-10-01 2013-10-01 false Rudder orders-TB/ALL. 35.40-35 Section 35.40-35 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY TANK VESSELS OPERATIONS Posting and Marking Requirements-TB/ALL § 35.40-35 Rudder orders—TB/ALL. At all steering stations, there shall be installed a suitable...

46 CFR 35.40-35 - Rudder orders-TB/ALL.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 46 Shipping 1 2012-10-01 2012-10-01 false Rudder orders-TB/ALL. 35.40-35 Section 35.40-35 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY TANK VESSELS OPERATIONS Posting and Marking Requirements-TB/ALL § 35.40-35 Rudder orders—TB/ALL. At all steering stations, there shall be installed a suitable...

46 CFR 35.35-5 - Electrical bonding-TB/ALL.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 46 Shipping 1 2014-10-01 2014-10-01 false Electrical bonding-TB/ALL. 35.35-5 Section 35.35-5 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY TANK VESSELS OPERATIONS Cargo Handling § 35.35-5 Electrical bonding—TB/ALL. The use of a vessel/shore bonding cable or wire is permissible only if...

46 CFR 35.40-35 - Rudder orders-TB/ALL.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 46 Shipping 1 2010-10-01 2010-10-01 false Rudder orders-TB/ALL. 35.40-35 Section 35.40-35 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY TANK VESSELS OPERATIONS Posting and Marking Requirements-TB/ALL. § 35.40-35 Rudder orders—TB/ALL. At all steering stations, there shall be installed a suitable...

46 CFR 35.40-35 - Rudder orders-TB/ALL.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 46 Shipping 1 2011-10-01 2011-10-01 false Rudder orders-TB/ALL. 35.40-35 Section 35.40-35 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY TANK VESSELS OPERATIONS Posting and Marking Requirements-TB/ALL. § 35.40-35 Rudder orders—TB/ALL. At all steering stations, there shall be installed a suitable...

46 CFR 35.35-85 - Air compressors-TB/ALL.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 46 Shipping 1 2014-10-01 2014-10-01 false Air compressors-TB/ALL. 35.35-85 Section 35.35-85 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY TANK VESSELS OPERATIONS Cargo Handling § 35.35-85 Air compressors—TB/ALL. No person may operate, install, or reinstall an air compressor in a cargo area described...

46 CFR 35.35-85 - Air compressors-TB/ALL.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 46 Shipping 1 2012-10-01 2012-10-01 false Air compressors-TB/ALL. 35.35-85 Section 35.35-85 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY TANK VESSELS OPERATIONS Cargo Handling § 35.35-85 Air compressors—TB/ALL. No person may operate, install, or reinstall an air compressor in a cargo area described...

46 CFR 35.35-85 - Air compressors-TB/ALL.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 46 Shipping 1 2013-10-01 2013-10-01 false Air compressors-TB/ALL. 35.35-85 Section 35.35-85 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY TANK VESSELS OPERATIONS Cargo Handling § 35.35-85 Air compressors—TB/ALL. No person may operate, install, or reinstall an air compressor in a cargo area described...

46 CFR 35.35-85 - Air compressors-TB/ALL.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 46 Shipping 1 2010-10-01 2010-10-01 false Air compressors-TB/ALL. 35.35-85 Section 35.35-85 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY TANK VESSELS OPERATIONS Cargo Handling § 35.35-85 Air compressors—TB/ALL. No person may operate, install, or reinstall an air compressor in a cargo area described...

46 CFR 35.35-85 - Air compressors-TB/ALL.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 46 Shipping 1 2011-10-01 2011-10-01 false Air compressors-TB/ALL. 35.35-85 Section 35.35-85 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY TANK VESSELS OPERATIONS Cargo Handling § 35.35-85 Air compressors—TB/ALL. No person may operate, install, or reinstall an air compressor in a cargo area described...

Burning mechanism and regression rate of RX-35-AU and RX-35-AV as a function of HMX particle size measured by the hybrid closed bomb-strand burner

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

Tao, W.C.; Costantino, M.S.; Ornellas, D.L.

1990-04-01

In this study, the average surface regression rate of two HMX-based cast explosives, RX-35-AU and RX-35-AV, is measured to pressures above 750 MPa using a hybrid closed bomb-strand burner. The hybrid design allows the simultaneous measurement of pressure and regression rate over a large range of pressures in each experiment. Nitroglycerin/Triacetin (75/25) and polyethylene glycol (PEG) are used as the energetic plasticizer and polymeric binder, respectively, in both formulations. The HMX solids loading in each formulation is 50 wt %, consisting of a narrow particle size distribution of 6--8 {mu}m for RX-35-AU and 150--177 {mu}m for RX-35-AV. Of special interestmore » are the regression rate and burning mechanism as a function of the initial particle size distribution and the mechanical properties fo the cast explosives. In general, the regression rate for the larger particle size formulation, RX-35-AV, is two to three times faster compared to that for RX-35-AU. Up to 750 MPa and independent of the initial confinement pressure, RX-35-AU exhibits a planar burning mechanism with the regression rate obeying the classical aP{sup n} formalism. For RX-35-AV, however, the burning behavior is erratic for samples ignited at 200 MPa confinement pressure. At confinement pressures above 400 MPa, the regression exhibits more of a planar burning mechanism. The unstable combustion behavior for RX-35-AV at lower confinement pressures is related to several mechanisms: (1) an abrupt increase in surface area due to particle fracture and subsequent translation and rotation, resulting in debonding and creating porosity, (2) thixotropic'' separation of the binder and nitramine, causing the significantly greater fracture damage to the nitramine during the loading cycle, (3) microscopic damage to the nitramine crystals that increase its intrinsic burning rate. 12 refs., 8 figs., 2 tabs.« less

SfDronc, an initiator caspase involved in apoptosis in the fall armyworm Spodoptera frugiperda

PubMed Central

Huang, Ning; Civciristov, Srgjan; Hawkins, Christine J.; Clem, Rollie J.

2013-01-01

Initiator caspases are the first caspases that are activated following an apoptotic stimulus, and are responsible for cleaving and activating downstream effector caspases, which directly cause apoptosis. We have cloned a cDNA encoding an ortholog of the initiator caspase Dronc in the lepidopteran insect Spodoptera frugiperda. The SfDronc cDNA encodes a predicted protein of 447 amino acids with a molecular weight of 51 kDa. Overexpression of SfDronc induced apoptosis in Sf9 cells, while partial silencing of SfDronc expression in Sf9 cells reduced apoptosis induced by baculovirus infection or by treatment with UV or actinomycin D. Recombinant SfDronc exhibited several expected biochemical characteristics of an apoptotic initiator caspase: 1) SfDronc efficiently cleaved synthetic initiator caspase substrates, but had very little activity against effector caspase substrates; 2) mutation of a predicted cleavage site at position D340 blocked autoprocessing of recombinant SfDronc and reduced enzyme activity by approximately 10-fold; 3) SfDronc cleaved the effector caspase Sf-caspase-1 at the expected cleavage site, resulting in Sf-caspase-1 activation; and 4) SfDronc was strongly inhibited by the baculovirus caspase inhibitor SpliP49, but not by the related protein AcP35. These results indicate that SfDronc is an initiator caspase involved in caspase-dependent apoptosis in S. frugiperda, and as such is likely to be responsible for the initiator caspase activity in S. frugiperda cells known as Sf-caspase-X. PMID:23474489

Active Depletion of Host Cell Inhibitor-of-Apoptosis Proteins Triggers Apoptosis upon Baculovirus DNA Replication▿

PubMed Central

Vandergaast, Rianna; Schultz, Kimberly L. W.; Cerio, Rebecca J.; Friesen, Paul D.

2011-01-01

Apoptosis is an important antivirus defense by virtue of its impact on virus multiplication and pathogenesis. To define molecular mechanisms by which viruses are detected and the apoptotic response is initiated, we examined the antiviral role of host inhibitor-of-apoptosis (IAP) proteins in insect cells. We report here that the principal IAPs, DIAP1 and SfIAP, of the model insects Drosophila melanogaster and Spodoptera frugiperda, respectively, are rapidly depleted and thereby inactivated upon infection with the apoptosis-inducing baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). Virus-induced loss of these host IAPs triggered caspase activation and apoptotic death. Elevation of IAP levels by ectopic expression repressed caspase activation. Loss of host IAP in both species was triggered by AcMNPV DNA replication. By using selected inhibitors, we found that virus-induced IAP depletion was mediated in part by the proteasome but not by caspase cleavage. Consistent with this conclusion, mutagenic disruption of the SfIAP RING motif, which acts as an E3 ubiquitin ligase, stabilized SfIAP during infection. Importantly, SfIAP was also stabilized upon the removal of its 99-residue N-terminal leader, which serves as a critical determinant of IAP turnover. These data indicated that a host pathway initiated by virus DNA replication and acting through instability motifs embedded within IAP triggers IAP depletion and thereby causes apoptosis. Taken together, the results of our study suggest that host modulation of cellular IAP levels is a conserved mechanism by which insects mount an apoptotic antiviral response. Thus, host IAPs may function as critical sentinels of virus invasion in insects. PMID:21653668

Mitochondrial Impairment May Increase Cellular NAD(P)H: Resazurin Oxidoreductase Activity, Perturbing the NAD(P)H-Based Viability Assays

PubMed Central

Aleshin, Vasily A.; Artiukhov, Artem V.; Oppermann, Henry; Kazantsev, Alexey V.; Lukashev, Nikolay V.; Bunik, Victoria I.

2015-01-01

Cellular NAD(P)H-dependent oxidoreductase activity with artificial dyes (NAD(P)H-OR) is an indicator of viability, as the cellular redox state is important for biosynthesis and antioxidant defense. However, high NAD(P)H due to impaired mitochondrial oxidation, known as reductive stress, should increase NAD(P)H-OR yet perturb viability. To better understand this complex behavior, we assayed NAD(P)H-OR with resazurin (Alamar Blue) in glioblastoma cell lines U87 and T98G, treated with inhibitors of central metabolism, oxythiamin, and phosphonate analogs of 2-oxo acids. Targeting the thiamin diphosphate (ThDP)-dependent enzymes, the inhibitors are known to decrease the NAD(P)H production in the pentose phosphate shuttle and/or upon mitochondrial oxidation of 2-oxo acids. Nevertheless, the inhibitors elevated NAD(P)H-OR with resazurin in a time- and concentration-dependent manner, suggesting impaired NAD(P)H oxidation rather than increased viability. In particular, inhibition of the ThDP-dependent enzymes affects metabolism of malate, which mediates mitochondrial oxidation of cytosolic NAD(P)H. We showed that oxythiamin not only inhibited mitochondrial 2-oxo acid dehydrogenases, but also induced cell-specific changes in glutamate and malate dehydrogenases and/or malic enzyme. As a result, inhibition of the 2-oxo acid dehydrogenases compromises mitochondrial metabolism, with the dysregulated electron fluxes leading to increases in cellular NAD(P)H-OR. Perturbed mitochondrial oxidation of NAD(P)H may thus complicate the NAD(P)H-based viability assay. PMID:26308058

Expression of Clonorchis sinensis GIIIsPLA2 protein in baculovirus-infected insect cells and its overexpression facilitating epithelial-mesenchymal transition in Huh7 cells via AKT pathway.

PubMed

Shang, Mei; Xie, Zhizhi; Tang, Zeli; He, Lei; Wang, Xiaoyun; Wang, Caiqin; Wu, Yinjuan; Li, Ye; Zhao, Lu; Lv, Zhiyue; Wu, Zhongdao; Huang, Yan; Yu, Xinbing; Li, Xuerong

2017-04-01

Although prior studies confirmed that group III secretory phospholipase A 2 of Clonorchis sinensis (CsGIIIsPLA 2 ) had stimulating effect on liver fibrosis by binding to LX-2 cells, large-scale expression of recombinant protein and its function in the progression of hepatoma are worth exploring. Because of high productivity and low lipopolysaccharides (LPS) in the Sf9-baculovirus expression system, we firstly used this system to express the coding region of CsGIIIsPLA 2 . The molecular weight of recombinant CsGIIIsPLA 2 protein was about 34 kDa. Further investigation showed that most of the recombinant protein presented intracellular expression in Sf9 insect cell nucleus and could be detected only into cell debris, which made the protein purification and further functional study difficult. Therefore, to study the role of CsGIIIsPLA 2 in hepatocellular carcinoma (HCC) progression, CsGIIIsPLA 2 overexpression Huh7 cell model was applied. Cell proliferation, migration, and the expression level of epithelial-mesenchymal transition (EMT)-related molecules (E-cadherin, N-cadherin, α-catenin, Vimentin, p300, Snail, and Slug) along with possible mechanism were measured. The results indicated that CsGIIIsPLA 2 overexpression not only inhibited cell proliferation and promoted migration and EMT but also enhanced the phosphorylation of AKT in HCC cells. In conclusion, this study supported that CsGIIIsPLA 2 overexpression suppressed cell proliferation and induced EMT through the AKT pathway.

Toluene diisocyanate increases airway responsiveness to substance P and decreases airway neutral endopeptidase.

PubMed

Sheppard, D; Thompson, J E; Scypinski, L; Dusser, D; Nadel, J A; Borson, D B

1988-04-01

Substance P and related tachykinins contribute to the airway hyperresponsiveness caused by toluene diisocyanate (TDI) in guinea pigs. Neutral endopeptidase (NEP) is an important modulator of substance P-induced responses. To test the hypothesis that exposure to TDI would increase responsiveness to substance P by inhibiting activity of this enzyme, we determined the dose of substance P required to increase pulmonary resistance by 200% above baseline (PD200) before and after administration of the pharmacologic inhibitor phosphoramidon in guinea pigs studied 1 h after a 1-h exposure to air or 3 ppm TDI. TDI exposure increased responsiveness to substance P significantly. However, phosphoramidon caused a significantly greater leftward shift of the substance P dose-response curve in air-exposed animals than it did in TDI-exposed animals, so that after phosphoramidon, mean values of PD200 in animals exposed to air or TDI did not differ. Tracheal NEP activity was significantly less after exposure to TDI than after exposure to air, whereas activity in the esophagus was the same in both groups. These results suggest that TDI exposure increases the bronchoconstrictor responsiveness of guinea pigs to substance P, in large part through inhibition of airway NEP.

Toluene diisocyanate increases airway responsiveness to substance P and decreases airway neutral endopeptidase.

PubMed Central

Sheppard, D; Thompson, J E; Scypinski, L; Dusser, D; Nadel, J A; Borson, D B

1988-01-01

Substance P and related tachykinins contribute to the airway hyperresponsiveness caused by toluene diisocyanate (TDI) in guinea pigs. Neutral endopeptidase (NEP) is an important modulator of substance P-induced responses. To test the hypothesis that exposure to TDI would increase responsiveness to substance P by inhibiting activity of this enzyme, we determined the dose of substance P required to increase pulmonary resistance by 200% above baseline (PD200) before and after administration of the pharmacologic inhibitor phosphoramidon in guinea pigs studied 1 h after a 1-h exposure to air or 3 ppm TDI. TDI exposure increased responsiveness to substance P significantly. However, phosphoramidon caused a significantly greater leftward shift of the substance P dose-response curve in air-exposed animals than it did in TDI-exposed animals, so that after phosphoramidon, mean values of PD200 in animals exposed to air or TDI did not differ. Tracheal NEP activity was significantly less after exposure to TDI than after exposure to air, whereas activity in the esophagus was the same in both groups. These results suggest that TDI exposure increases the bronchoconstrictor responsiveness of guinea pigs to substance P, in large part through inhibition of airway NEP. PMID:2450892

[Low extracellular pH increases the persistent sodium current in guinea pig ventricular myocytes].

PubMed

Ma, Ji-Hua; Luo, An-Tao; Wang, Wei-Ping; Zhang, Pei-Hua

2007-04-25

Whole-cell and cell-attached patch-clamp techniques were used to record the changes of persistent sodium current (I(Na.P)) in ventricular myocytes of guinea pig to investigate the effect of low extracellular pH on I(Na.P) and its mechanism. The results showed that low extracellular pH (7.0, 6.8 and 6.5) obviously increased the amplitude of whole-cell I(Na.P) in a [H(+)] concentration-dependent manner. Under the condition of extracellular pH 6.5, I(Na.P) was markedly augmented from control (pH 7.4) value of (0.347+/-0.067) pA/pF to (0.817+/- 0.137) pA/pF (P<0.01, n=6), whereas the reducing agent dithiothreitiol (DTT, 1 mmol/L) reversed the increased IN(Na.P) from (0.817+/-0.137) pA/pF to (0.233+/-0.078) pA/pF (P<0.01 vs pH 6.5, n=6). Decreasing extracellular pH to 6.5 also increased the persistent sodium channel activity in cell-attached patches. The mean open probability and mean open time were increased from control value of 0.021+/-0.007 and (0.899+/-0.074) ms to 0.205+/-0.023 and (1.593+/-0.158) ms, respectively (both P<0.01, n=6), and such enhancement was reversed by application of 1 mmol/L DTT [to 0.019+/-0.005 and (0.868+/-0.190) ms, both P<0.01 vs pH 6.5, n=6]. Furthermore, protein kinase C (PKC) inhibitor bisindolylmaleimide (BIM, 5 micromol/L) reduced the enhanced mean open probability and mean open time at pH 6.5 from 0.214+/-0.024 and (1.634+/-0.137) ms to 0.025+/-0.006 and (0.914+/-0.070) ms, respectively (both P<0.01 vs pH 6.5, n=6). The results demonstrate that low extracellular pH markedly increases I(Na.P) in guinea pig ventricular myocytes, in which activation of PKC may be involved.

Development of a subunit vaccine for infectious pancreatic necrosis virus using a baculovirus insect/larvae system

USGS Publications Warehouse

Shivappa, R.B.; McAllister, P.E.; Edwards, G.H.; Santi, N.; Evensen, O.; Vakharia, V.N.; ,

2005-01-01

Various attempts to develop a vaccine against infectious pancreatic necrosis virus (IPNV) have not yielded consistent results. Thus, at present, no commercial vaccine is available that can be used with confidence to immunize fry of salmon and trout. We generated a cDNA clone of the large genome segment A of an IPNV Sp strain and expressed all structural protein genes in insect cells and larvae using a baculovirus expression system. Green fluorescent protein was also co-expressed as a reporter molecule. High yields of IPNV proteins were obtained and the structural proteins self assembled to form virus-like particles (VLPs). We tested the immunogenicity of the putative VLP antigen in immersion vaccine experiments (two concentrations) in rainbow trout (Oncorhynchus mykiss) fry, and by intraperitoneal immunisation of Atlantic salmon (Salmo salar) pre-smolts using an oil adjuvant formulation. Rainbow trout were challenged by immersion using either the Sp or the VR-299 strain of IPNV two or three weeks post-vaccination, while Atlantic salmon were bath challenged with Sp strain after two months, after parr-smolt transformation. In the rainbow trout fry challenged two weeks post-immunization, cumulative mortality rates three weeks post challenge were 14 % in the fry that had received the highest dose versus 8 % in the control groups. No indication of protection was seen in repeated trials using a lower dose of antigen and challenge three weeks post-immunisation. The cumulative mortality rate of intraperitoneally immunised Atlantic salmon post-smolts four weeks post challenge was lower (56 %) than in the control fish (77 %), showing a dose-response pattern.

[Mode of conception, acompanying medical disorders and complications in the second half of pregnancy in women over the age of 35].

PubMed

Dakov, T; Dimitrova, V; Maseva, A; Todorov, T

2014-01-01

To assess the impact of maternal age on the mode of conception, the incidence of accompanying medical disorders and past surgical procedures (gynecological and non-gynecological) and the complications in the second half of pregnancy (preeclampsia, placenta praevia, placental abruption, preterm delivery) in women ≥ 35 years, followed prospectively. Between 02/2012 - 02/2014 495 pregnant women of ≥ 12 weeks of gestation were enrolled in the study. The patients were admitted for different indications at the Fetal Medicine Clinic of the State University Hospital "Maichin Dom". They were divided in 3 age groups according to age: ≤ 34, 35-39 and ≥ 40 years. The information about pregnancy course and outcome was retrieved from the hospital records or obtained from the patents themselves by phone interviews. The following complications in the second half of pregnancy were analyzed: preeclampsia, placenta praevia, placental abruption, preterm delivery. The data were processed with SPSS 13.0. statistical package. Descriptive and comparative analysis was performed after grouping according to one or more characteristics; p values < 0.05 were considered as evidence of statistical significance for tested effects. The number of patients ≤ 34 years of age was 131/495 (26.5%), between 35-39 years--54/495 (51,.%) and >≥40 years --10/495 (22,.%). Conception by ART was significantly more frequent in women aged >3 5. Pregnancy occurred afterA RTi n 1,.% (2/131) of the women <3 4, in 8,.% (22/254) of those between 35-39 and in 10,.% (12/110) of those >≥ 0 years of age (p= O 0.08). The incidence of accompanying medical disorders (intemrnl, tumors, of the female genital system) was significantly higher in women of more advanced age (p< O 0.01). It was 36,.% (48/131) in those <3 4, 53,1% (135/254) --n the ones between 35-39 and 68,.% (75/110) --mong those >≥ 0 years of age. The increased incidence was mainly due to more frequent intemrnl (p= 0 ,.218) and female reproductive

Characterization of Toxoplasma gondii SAG2 Expressed in Insect Cells by Recombinant Baculovirus and Evaluation of Its Diagnostic Potential in an Enzyme-Linked Immunosorbent Assay

PubMed Central

Huang, Xiaohong; Xuan, Xuenan; Suzuki, Hiroshi; Sugimoto, Chihiro; Nagasawa, Hideyuki; Fujisaki, Kozo; Mikami, Takeshi; Igarashi, Ikuo

2002-01-01

A baculovirus carrying the SAG2 gene of Toxoplasma gondii was constructed, and recombinant SAG2 protein (S-rSAG2) was expressed in insect cells. S-rSAG2 was recognized by sera from cats and pigs infected with T. gondii. Mice immunized with S-rSAG2 produced high titers of specific immunoglobulin G2a (IgG2a) and IgG1 antibodies. In an indirect fluorescent antibody test, all mouse antisera against S-rSAG2 reacted strongly to the natural parasites, but those against rSAG2 expressed in Escherichia coli (E-rSAG2) only showed very weak reaction, although no markedly difference was found in the reaction to denatured antigen, T. gondii lysate, in Western blot analysis. The results suggest that S-rSAG2 is better than E-rSAG2 in both antigenicity and immunogenicity. Enzyme-linked immunosorbent assay (ELISA) with S-rSAG2 could differentiate clearly between sera from 30 specific-pathogen-free cats and 4 experimentally infected cats. Serum samples from domestic cats in Japan were tested by the ELISA and compared with a latex agglutination test (LAT) and ELISA with E-rSAG2. Of 187 samples, all 35 LAT-positive sera had strong reactions to S-rSAG2 and E-rSAG2. Of the 152 LAT-negative sera, 18 were positive in the ELISA with S-rSAG2, whereas only 2 were positive in the ELISA with E-rSAG2. Although there were significant correlations among the three methods, the ELISA with S-rSAG2 was more sensitive than the others, which could be attributed to the fact that S-rSAG2 shares some common conformational structure with the native antigen. The results suggest that S-rSAG2 would be a useful reagent for the detection of T. gondii infection in cats. PMID:12414772

Increased intracellular pH is necessary for adult epithelial and embryonic stem cell differentiation

PubMed Central

Azimova, Dinara R.

2016-01-01

Despite extensive knowledge about the transcriptional regulation of stem cell differentiation, less is known about the role of dynamic cytosolic cues. We report that an increase in intracellular pH (pHi) is necessary for the efficient differentiation of Drosophila adult follicle stem cells (FSCs) and mouse embryonic stem cells (mESCs). We show that pHi increases with differentiation from FSCs to prefollicle cells (pFCs) and follicle cells. Loss of the Drosophila Na+–H+ exchanger DNhe2 lowers pHi in differentiating cells, impairs pFC differentiation, disrupts germarium morphology, and decreases fecundity. In contrast, increasing pHi promotes excess pFC cell differentiation toward a polar/stalk cell fate through suppressing Hedgehog pathway activity. Increased pHi also occurs with mESC differentiation and, when prevented, attenuates spontaneous differentiation of naive cells, as determined by expression of microRNA clusters and stage-specific markers. Our findings reveal a previously unrecognized role of pHi dynamics for the differentiation of two distinct types of stem cell lineages, which opens new directions for understanding conserved regulatory mechanisms. PMID:27821494

Increasing Saturated Electron-Drift Velocity in Donor-Acceptor Doped pHEMT Heterostructures

NASA Astrophysics Data System (ADS)

Protasov, D. Yu.; Gulyaev, D. V.; Bakarov, A. K.; Toropov, A. I.; Erofeev, E. V.; Zhuravlev, K. S.

2018-03-01

Field dependences of the electron-drift velocity in typical pseudomorphic high-electron-mobility transistor (pHEMT) heteroepitaxial structures (HESs) and in those with donor-acceptor doped (DApHEMT) heterostructures with quantum-well (QW) depth increased by 0.8-0.9 eV with the aid of acceptor layers have been studied by a pulsed technique. It is established that the saturated electron-drift velocity in DA-pHEMT-HESs is 1.2-1.3 times greater than that in the usual pHEMT-HESs. The electroluminescence (EL) spectra of DA-pHEMT-HESs do not contain emission bands related to the recombination in widebandgap layers (QW barriers). The EL intensity in these HESs is not saturated with increasing electric field. This is indicative of a suppressed real-space transfer of hot electrons from QW to barrier layers, which accounts for the observed increase in the saturated electron-drift velocity.

40 CFR 35.910-3-35.910-4 - [Reserved

Code of Federal Regulations, 2014 CFR

2014-07-01

... 40 Protection of Environment 1 2014-07-01 2014-07-01 false [Reserved] 35.910-3-35.910-4 Section 35.910-3-35.910-4 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY GRANTS AND OTHER FEDERAL ASSISTANCE STATE AND LOCAL ASSISTANCE Grants for Construction of Treatment Works-Clean Water Act §§ 35.910-3...

40 CFR 35.910-3-35.910-4 - [Reserved

Code of Federal Regulations, 2012 CFR

2012-07-01

... 40 Protection of Environment 1 2012-07-01 2012-07-01 false [Reserved] 35.910-3-35.910-4 Section 35.910-3-35.910-4 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY GRANTS AND OTHER FEDERAL ASSISTANCE STATE AND LOCAL ASSISTANCE Grants for Construction of Treatment Works-Clean Water Act §§ 35.910-3...

40 CFR 35.910-3-35.910-4 - [Reserved

Code of Federal Regulations, 2013 CFR

2013-07-01

... 40 Protection of Environment 1 2013-07-01 2013-07-01 false [Reserved] 35.910-3-35.910-4 Section 35.910-3-35.910-4 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY GRANTS AND OTHER FEDERAL ASSISTANCE STATE AND LOCAL ASSISTANCE Grants for Construction of Treatment Works-Clean Water Act §§ 35.910-3...

40 CFR 35.910-3-35.910-4 - [Reserved

Code of Federal Regulations, 2011 CFR

2011-07-01

... 40 Protection of Environment 1 2011-07-01 2011-07-01 false [Reserved] 35.910-3-35.910-4 Section 35.910-3-35.910-4 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY GRANTS AND OTHER FEDERAL ASSISTANCE STATE AND LOCAL ASSISTANCE Grants for Construction of Treatment Works-Clean Water Act §§ 35.910-3...

40 CFR 35.910-3-35.910-4 - [Reserved

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 1 2010-07-01 2010-07-01 false [Reserved] 35.910-3-35.910-4 Section 35.910-3-35.910-4 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY GRANTS AND OTHER FEDERAL ASSISTANCE STATE AND LOCAL ASSISTANCE Grants for Construction of Treatment Works-Clean Water Act §§ 35.910-3...

Okadaic acid mediates p53 hyperphosphorylation and growth arrest in cells with wild-type p53 but increases aberrant mitoses in cells with non-functional p53.

PubMed

Milczarek, G J; Chen, W; Gupta, A; Martinez, J D; Bowden, G T

1999-06-01

The protein phosphatase inhibitor and tumor promoting agent okadaic acid (OA), has been shown previously to induce hyperphosphorylation of p53 protein, which in turn correlated with increased transactivation or apoptotic function. However, how the tumor promotion effects of OA relate to p53 tumor supressor function (or dysfunction) remain unclear. Rat embryonic fibroblasts harboring a temperature-sensitive mouse p53 transgene were treated with 50 nM doses of OA. At the wild-type permissive temperature this treatment resulted in: (i) the hyperphosphorylation of sites within tryptic peptides of the transactivation domain of p53; (ii) an increase in p53 affinity for a p21(waf1) promotor oligonucleotide; (iii) an increase in cellular steady state levels of p21(waf1) message; (iv) a G2/M cell cycle blockage in addition to the G1/S arrest previously associated with p53; and (v) no increased incidence of apoptosis. On the other hand, OA treatment at the mutated p53 permissive temperature resulted in a relatively high incidence of aberrant mitosis with no upregulation of p21(waf1) message. These results suggest that while wild-type p53 blocks the proliferative effects of OA through p21(waf1)-mediated growth arrest, cells with non-functional p53 cannot arrest and suffer relatively high levels of OA-mediated aberrant mitoses.

Putative endogenous filovirus VP35-like protein potentially functions as an IFN antagonist but not a polymerase cofactor.

PubMed

Kondoh, Tatsunari; Manzoor, Rashid; Nao, Naganori; Maruyama, Junki; Furuyama, Wakako; Miyamoto, Hiroko; Shigeno, Asako; Kuroda, Makoto; Matsuno, Keita; Fujikura, Daisuke; Kajihara, Masahiro; Yoshida, Reiko; Igarashi, Manabu; Takada, Ayato

2017-01-01

It has been proposed that some non-retroviral RNA virus genes are integrated into vertebrate genomes. Endogenous filovirus-like elements (EFLs) have been discovered in some mammalian genomes. However, their potential roles in ebolavirus infection are unclear. A filovirus VP35-like element (mlEFL35) is found in the little brown bat (Myotis lucifugus) genome. Putative mlEFL35-derived protein (mlEFL35p) contains nearly full-length amino acid sequences corresponding to ebolavirus VP35. Ebola virus VP35 has been shown to bind double-stranded RNA, leading to inhibition of type I interferon (IFN) production, and is also known as a viral polymerase cofactor that is essential for viral RNA transcription/replication. In this study, we transiently expressed mlEFL35p in human kidney cells and investigated its biological functions. We first found that mlEFL35p was coimmunoprecipitated with itself and ebolavirus VP35s but not with the viral nucleoprotein. Then the biological functions of mlEFL35p were analyzed by comparing it to ebolavirus VP35s. We found that the expression of mlEFL35p significantly inhibited human IFN-β promoter activity as well as VP35s. By contrast, expression of mlEFL35p did not support viral RNA transcription/replication and indeed slightly decrease the reporter gene expression in a minigenome assay. These results suggest that mlEFL35p potentially acts as an IFN antagonist but not a polymerase cofactor.

HLA-B35 Upregulates Endothelin-1 and Downregulates Endothelial Nitric Oxide Synthase via Endoplasmic Reticulum Stress Response in Endothelial Cells

PubMed Central

Lenna, Stefania; Townsend, Danyelle M.; Tan, Filemon K.; Kapanadze, Bagrat; Markiewicz, Malgorzata; Trojanowska, Maria; Scorza, Raffaella

2013-01-01

The presence of the HLA-B35 allele has emerged as an important risk factor for the development of isolated pulmonary hypertension in patients with scleroderma, however the mechanisms underlying this association have not been fully elucidated. The goal of our study was to determine the molecular mechanisms that mediate the biological effects of HLA-B35 in endothelial cells (ECs). Our data demonstrate that HLA-B35 expression at physiological levels via adenoviral vector resulted in significantly increased endothelin-1 (ET-1) and a significantly decreased endothelial NO synthase (eNOS), mRNA, and protein levels. Furthermore, HLA-B35 greatly upregulated expression of chaperones, including heat shock proteins (HSPs) HSP70 (HSPA1A and HSPA1B) and HSP40 (DNAJB1 and DNAJB9), suggesting that HLA-B35 induces the endoplasmic reticulum (ER) stress and unfolded protein response in ECs. Examination of selected mediators of the unfolded protein response, including H chain binding protein (BiP; GRP78), C/Ebp homologous protein (CHOP; GADD153), endoplasmic reticulum oxidase, and protein disulfide isomerase has revealed a consistent increase of BiP expression levels. Accordingly, thapsigargin, a known ER stress inducer, stimulated ET-1mRNAand protein levels in ECs. This study suggests that HLA-B35 could contribute to EC dysfunction via ER stress-mediated induction of ET-1 in patients with pulmonary hypertension. PMID:20335527

HLA-B35 upregulates endothelin-1 and downregulates endothelial nitric oxide synthase via endoplasmic reticulum stress response in endothelial cells.

PubMed

Lenna, Stefania; Townsend, Danyelle M; Tan, Filemon K; Kapanadze, Bagrat; Markiewicz, Malgorzata; Trojanowska, Maria; Scorza, Raffaella

2010-05-01

The presence of the HLA-B35 allele has emerged as an important risk factor for the development of isolated pulmonary hypertension in patients with scleroderma, however the mechanisms underlying this association have not been fully elucidated. The goal of our study was to determine the molecular mechanisms that mediate the biological effects of HLA-B35 in endothelial cells (ECs). Our data demonstrate that HLA-B35 expression at physiological levels via adenoviral vector resulted in significantly increased endothelin-1 (ET-1) and a significantly decreased endothelial NO synthase (eNOS), mRNA, and protein levels. Furthermore, HLA-B35 greatly upregulated expression of chaperones, including heat shock proteins (HSPs) HSP70 (HSPA1A and HSPA1B) and HSP40 (DNAJB1 and DNAJB9), suggesting that HLA-B35 induces the endoplasmic reticulum (ER) stress and unfolded protein response in ECs. Examination of selected mediators of the unfolded protein response, including H chain binding protein (BiP; GRP78), C/Ebp homologous protein (CHOP; GADD153), endoplasmic reticulum oxidase, and protein disulfide isomerase has revealed a consistent increase of BiP expression levels. Accordingly, thapsigargin, a known ER stress inducer, stimulated ET-1 mRNA and protein levels in ECs. This study suggests that HLA-B35 could contribute to EC dysfunction via ER stress-mediated induction of ET-1 in patients with pulmonary hypertension.

Nicergoline increases serum substance P levels in patients with an ischaemic stroke.

PubMed

Nishiyama, Yasuhiro; Abe, Arata; Ueda, Masayuki; Katsura, Ken-ichiro; Katayama, Yasuo

2010-01-01

Aspiration pneumonia is one of the most important complications following ischaemic stroke, and a leading cause of mortality in stroke patients. This is particularly prevalent in patients with involvement of the basal ganglia, which may be due to impaired neurotransmission through lack of production of substance P. Consecutive patients in the chronic stage, 1-3 months after cerebral ischaemic infarction, were assessed for basal ganglia involvement by magnetic resonance imaging. The patients were randomised to 4 weeks of treatment with (n = 25) or without (n = 25) nicergoline (15 mg t.i.d.). Serum concentration of substance P was measured by radioimmunoassay. At entry to the study, mean concentration of substance P was significantly (p < 0.001) lower in patients with bilateral basal ganglia lesions than in patients with no or unilateral basal ganglia involvement. Nicergoline administration caused a significant (p = 0.021) increase from baseline in mean substance P concentration. No significant change was seen in the nicergoline-untreated patients (p = 0.626). Among the patients who received nicergoline, 11 patients had bilateral basal ganglia involvement and there was no significant mean change in substance P in these patients, whereas there was a significant increase (p = 0.032) in the 14 nicergoline-treated patients with no or unilateral basal ganglia involvement. The present study suggests a possible effect of nicergoline to increase substance P level in ischaemic stroke patients with partial damage to basal ganglia, who have a decreased swallowing response and consequent risk of aspiration pneumonia. Further trials of nicergoline treatment in patients at risk for aspiration pneumonia are warranted. (c) 2009 S. Karger AG, Basel.

Increasing physically effective fiber content of dairy cow diets through forage proportion versus forage chop length: chewing and ruminal pH.

PubMed

Yang, W Z; Beauchemin, K A

2009-04-01

A study was conducted to evaluate whether the risk of acidosis in dairy cows can be lowered by increasing the physically effective fiber (peNDF) concentration of the diet, either through increased theoretical chop length of alfalfa silage or higher proportion of forage in the diet. The experiment was designed as a replicated 4 x 4 Latin square using 8 ruminally cannulated lactating dairy cows. Treatments were arranged in a 2 x 2 factorial design; 2 forage particle lengths (FPL) of alfalfa silage (short and long) were combined with low (35:65) and high (60:40) forage:concentrate (F:C) ratios [dry matter (DM) basis]. Dietary peNDF concentration (DM basis) was determined from the sum of the proportion of dietary DM retained either on the 2 sieves (8 and 19 mm) or on the 3 sieves (1.18, 8, and 19 mm) of the Penn State Particle Separator multiplied by the neutral detergent fiber concentration of the diet. The dietary peNDF concentrations were altered by changing the F:C or the FPL, and ranged from 10.7 to 17.5% using 2 sieves, or from 23.1 to 28.2% using 3 sieves. Intake of peNDF was increased by increasing FPL but not by increasing F:C ratio because of the reduction of DM intake at the higher F:C ratio. Chewing activity, including number of chews and chewing time, increased with increasing F:C ratio or FPL. Mean ruminal pH was elevated by 0.4 and 0.2 units with increasing F:C ratio and FPL, respectively. Lowering the F:C ratio decreased the duration that ruminal pH was below 5.8 (1.2 vs. 8 h/d). Increased F:C ratio or FPL reduced ruminal volatile fatty acids concentration from 137 to 122 or from 133 to 126 mM, respectively, whereas acetate:propionate ratio was increased from 2.55 to 3.46 with increasing F:C ratio. Dietary peNDF concentration measured using 2 sieves was correlated to chewing time (r = 0.57) and mean ruminal pH (r = 0.75), whereas dietary peNDF concentration measured using 3 sieves was correlated to mean ruminal pH (r = 0.83) and negatively correlated to

Optical Study of 2D Photonic Crystals in an InP/GaInAsP Slab Waveguide Structure

DTIC Science & Technology

2002-01-01

the values n,,,,. = 3.35 and n, ,, = 3.17 are assumed for the refraction index of GaInAsP and InP, respectively. The resulting structure is a multimode...contributes to increase out-of- plane scattering. On the other hand, when entering the PC, the hole pattern is felt as a low refractive index contrast...in an InP/GaInAsP step- index waveguide. Transmission (T) measurements through simple PC slabs and through one-dimensional (1D) Fabry-P6rot (FP

Update to Millstone 3 elevated pH tests

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

Bergmann, C.A.; Perock, J.D.; Hudson, M.J.B.

1995-03-01

In view of the potential radiological benefits of elevated coolant pH operation, Northwest Utilities (NU), in support of an EPRI-Westinghouse program, agreed to operate the Millstone 3 plant at the start of its second fuel cycle as a demonstration of the effect of elevated coolant pH on out-of-core radiation fields. Operating with an elevated pH is defined as operating with an average lithium concentration of 3.35 ppm, until reaching an end of cycle pH of 7.2 or 7.4. The plant operated during its second and third cycles with an elevated coolant pH. The end of cycle pH during the secondmore » cycle was 7.4, and 7.2 during the third cycle. (During the first cycle, operation was with a coordinated pH of 7.0). Evaluation of the dose rate trends in Millstone 3 after two cycles of elevated coolant pH operation concluded that an elevated coolant pH resulted in a 15 percent lower component dose rate compared to other plants that operated with coordinated pH 6.9. However, due to a possible increase in fuel clad corrosion, operation during cycle 4 was restricted to pH 6.9 coordinated chemistry, with the exception of the last two months during which the pH increased to 7.35. At the end of cycle 4 (EOC4), there was a greater increase in component and crud trap dose rates than expected. This paper reviews the radiological trends in the plant and discusses the potential causes for the increase in the dose rates at EOC4.« less

Global riverine N and P transport to ocean increased during the twentieth century despite increased retention along the aquatic continuum

NASA Astrophysics Data System (ADS)

Beusen, A. H. W.; Bouwman, A. F.; Van Beek, L. P. H.; Mogollón, J. M.; Middelburg, J. J.

2015-12-01

Various human activities, including agriculture, water consumption, river damming, and aquaculture, have intensified over the last century. This has had a major impact on nitrogen (N) and phosphorus (P) cycling in global continental waters. In this study, we use a coupled nutrient-input, hydrology, in-stream nutrient retention model to quantitatively track the changes in the global freshwater N and P cycles over the 20th century. Our results suggest that, during this period, the global nutrient delivery to streams increased from 34 to 64 Tg N yr-1 and from 5 to 9 Tg N yr-1. Furthermore, in-stream retention and removal grew from 14 to 27 Tg N yr-1 and 3 to 5 Tg N yr-1. One of the major cause of increased retention is the growing number of reservoirs which now account for 24 and 22 % of global N and P retention/removal in freshwater systems, respectively. This increase in nutrient retention could not balance the increase in nutrient delivery to rivers with the consequence that river nutrient transport to the ocean increased from 19 to 37 Tg N yr-1 and from 2 to 4 Tg N yr-1. Human activities have also led to a global increase in the molar N : P ratio in freshwater bodies.

Putative endogenous filovirus VP35-like protein potentially functions as an IFN antagonist but not a polymerase cofactor

PubMed Central

Kondoh, Tatsunari; Manzoor, Rashid; Nao, Naganori; Maruyama, Junki; Furuyama, Wakako; Miyamoto, Hiroko; Shigeno, Asako; Kuroda, Makoto; Matsuno, Keita; Fujikura, Daisuke; Kajihara, Masahiro; Yoshida, Reiko; Igarashi, Manabu

2017-01-01

It has been proposed that some non-retroviral RNA virus genes are integrated into vertebrate genomes. Endogenous filovirus-like elements (EFLs) have been discovered in some mammalian genomes. However, their potential roles in ebolavirus infection are unclear. A filovirus VP35-like element (mlEFL35) is found in the little brown bat (Myotis lucifugus) genome. Putative mlEFL35-derived protein (mlEFL35p) contains nearly full-length amino acid sequences corresponding to ebolavirus VP35. Ebola virus VP35 has been shown to bind double-stranded RNA, leading to inhibition of type I interferon (IFN) production, and is also known as a viral polymerase cofactor that is essential for viral RNA transcription/replication. In this study, we transiently expressed mlEFL35p in human kidney cells and investigated its biological functions. We first found that mlEFL35p was coimmunoprecipitated with itself and ebolavirus VP35s but not with the viral nucleoprotein. Then the biological functions of mlEFL35p were analyzed by comparing it to ebolavirus VP35s. We found that the expression of mlEFL35p significantly inhibited human IFN-β promoter activity as well as VP35s. By contrast, expression of mlEFL35p did not support viral RNA transcription/replication and indeed slightly decrease the reporter gene expression in a minigenome assay. These results suggest that mlEFL35p potentially acts as an IFN antagonist but not a polymerase cofactor. PMID:29040311

F-35 Joint Strike Fighter Aircraft (F-35)

DTIC Science & Technology

2015-12-01

Selected Acquisition Report ( SAR ) RCS: DD-A&T(Q&A)823-198 F-35 Joint Strike Fighter Aircraft (F-35) As of FY 2017 President’s Budget Defense...Acquisition Management Information Retrieval (DAMIR) March 21, 2016 08:47:09 UNCLASSIFIED F-35 December 2015 SAR March 21, 2016 08:47:09 UNCLASSIFIED 2...Document OSD - Office of the Secretary of Defense O&S - Operating and Support PAUC - Program Acquisition Unit Cost F-35 December 2015 SAR March 21

The interaction of intracellular Mg2+ and pH on Cl- fluxes associated with intracellular pH regulation in barnacle muscle fibers

PubMed Central

1988-01-01

The intracellular dialysis technique was used to measure unidirectional Cl- fluxes and net acid extrusion by single muscle fibers from the giant barnacle. Decreasing pHi below normal levels of 7.35 stimulated both Cl- efflux and influx. These increases of Cl- fluxes were blocked by disulfonic acid stilbene derivatives such as SITS and DIDS. The SITS- sensitive Cl- efflux was sharply dependent upon pHi, increasing approximately 20-fold as pHi was decreased from 7.35 to 6.7. Under conditions of normal intracellular Mg2+ concentration, the apparent pKa for the activation of Cl- efflux was 7.0. We found that raising [Mg2+]i, but not [Mg2+]o, had a pronounced inhibitory effect on both SITS-sensitive unidirectional Cl- fluxes as well as on SITS-sensitive net acid extrusion. Increasing [Mg2+]i shifted the apparent pKa of Cl- efflux to a more acid value without affecting the maximal flux that could be attained. This relation between pHi and [Mg2+]i on SITS- sensitive Cl- efflux is consistent with a competition between H ions and Mg ions. We conclude that the SITS-inhibitable Cl- fluxes are mediated by the pHi-regulatory transport mechanism and that changes of intracellular Mg2+ levels can modify the activity of the pHi regulator/anion transporter. PMID:3392519

The ML1Nx2 Phosphatidylinositol 3,5-Bisphosphate Probe Shows Poor Selectivity in Cells.

PubMed

Hammond, Gerald R V; Takasuga, Shunsuke; Sasaki, Takehiko; Balla, Tamas

2015-01-01

Phosphatidylinositol (3,5)-bisphosphate (PtdIns(3,5)P2) is a quantitatively minor phospholipid in eukaryotic cells that plays a fundamental role in regulating endocytic membrane traffic. Despite its clear importance for cellular function and organism physiology, mechanistic details of its biology have so far not been fully elucidated. In part, this is due to a lack of experimental tools that specifically probe for PtdIns(3,5)P2 in cells to unambiguously identify its dynamics and site(s) of action. In this study, we have evaluated a recently reported PtdIns(3,5)P2 biosensor, GFP-ML1Nx2, for its veracity as such a probe. We report that, in live cells, the localization of this biosensor to sub-cellular compartments is largely independent of PtdIns(3,5)P2, as assessed after pharmacological, chemical genetic or genomic interventions that block the lipid's synthesis. We therefore conclude that it is unwise to interpret the localization of ML1Nx2 as a true and unbiased biosensor for PtdIns(3,5)P2.

Blockade of P-Glycoprotein Decreased the Disposition of Phenformin and Increased Plasma Lactate Level.

PubMed

Choi, Min-Koo; Song, Im-Sook

2016-03-01

This study aimed to investigate the in vivo relevance of P-glycoprotein (P-gp) in the pharmacokinetics and adverse effect of phenformin. To investigate the involvement of P-gp in the transport of phenformin, a bi-directional transport of phenformin was carried out in LLC-PK1 cells overexpressing P-gp, LLC-PK1-Pgp. Basal to apical transport of phenformin was 3.9-fold greater than apical to basal transport and became saturated with increasing phenformin concentration (2-75 μM) in LLC-PK1-Pgp, suggesting the involvement of P-gp in phenformin transport. Intrinsic clearance mediated by P-gp was 1.9 μL/min while passive diffusion clearance was 0.31 μL/min. Thus, P-gp contributed more to phenformin transport than passive diffusion. To investigate the contribution of P-gp on the pharmacokinetics and adverse effect of phenformin, the effects of verapamil, a P-gp inhibitor, on the pharmacokinetics of phenformin were also examined in rats. The plasma concentrations of phenformin were increased following oral administration of phenformin and intravenous verapamil infusion compared with those administerd phenformin alone. Pharmacokinetic parameters such as Cmax and AUC of phenformin increased and CL/F and Vss/F decreased as a consequence of verapamil treatment. These results suggested that P-gp blockade by verapamil may decrease the phenformin disposition and increase plasma phenformin concentrations. P-gp inhibition by verapamil treatment also increased plasma lactate concentration, which is a crucial adverse event of phenformin. In conclusion, P-gp may play an important role in phenformin transport process and, therefore, contribute to the modulation of pharmacokinetics of phenformin and onset of plasma lactate level.

Blockade of P-Glycoprotein Decreased the Disposition of Phenformin and Increased Plasma Lactate Level

PubMed Central

Choi, Min-Koo; Song, Im-Sook

2016-01-01

This study aimed to investigate the in vivo relevance of P-glycoprotein (P-gp) in the pharmacokinetics and adverse effect of phenformin. To investigate the involvement of P-gp in the transport of phenformin, a bi-directional transport of phenformin was carried out in LLC-PK1 cells overexpressing P-gp, LLC-PK1-Pgp. Basal to apical transport of phenformin was 3.9-fold greater than apical to basal transport and became saturated with increasing phenformin concentration (2–75 μM) in LLC-PK1-Pgp, suggesting the involvement of P-gp in phenformin transport. Intrinsic clearance mediated by P-gp was 1.9 μL/min while passive diffusion clearance was 0.31 μL/min. Thus, P-gp contributed more to phenformin transport than passive diffusion. To investigate the contribution of P-gp on the pharmacokinetics and adverse effect of phenformin, the effects of verapamil, a P-gp inhibitor, on the pharmacokinetics of phenformin were also examined in rats. The plasma concentrations of phenformin were increased following oral administration of phenformin and intravenous verapamil infusion compared with those administerd phenformin alone. Pharmacokinetic parameters such as Cmax and AUC of phenformin increased and CL/F and Vss/F decreased as a consequence of verapamil treatment. These results suggested that P-gp blockade by verapamil may decrease the phenformin disposition and increase plasma phenformin concentrations. P-gp inhibition by verapamil treatment also increased plasma lactate concentration, which is a crucial adverse event of phenformin. In conclusion, P-gp may play an important role in phenformin transport process and, therefore, contribute to the modulation of pharmacokinetics of phenformin and onset of plasma lactate level. PMID:26797108

Emerging role of IL-35 in inflammatory autoimmune diseases.

PubMed

Su, Lin-Chong; Liu, Xiao-Yan; Huang, An-Fang; Xu, Wang-Dong

2018-05-03

Interleukin 35 (IL-35) is the recently identified member of the IL-12 family of cytokines and provides the possibility to be a target for new therapies for autoimmune, inflammatory diseases. It is composed of an α chain (p35) and a β chain (EBI3). IL-35 mediates signaling by binding to its receptors, activates subsequent signaling pathways, and therefore, regulates the differentiation, function of T, B cells, macrophages, dendritic cells. Recent findings have shown abnormal expression of IL-35 in inflammatory autoimmune diseases, such as systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, type 1 diabetes, psoriasis, multiple sclerosis, autoimmune hepatitis, experimental autoimmune uveitis. In addition, functional analysis suggested that IL-35 is critical in the onset and development of these diseases. Therefore, the present study will systematically review what had been occurred regarding IL-35 in inflammatory autoimmune disease. The information collected will help to understand the biologic role of IL-35 in immune cells, and give information about the therapeutic potential of IL-35 in these diseases. Copyright © 2018. Published by Elsevier B.V.

Fasting mediated increase in p-BAD(ser155) and p-AKT(ser473) in the prefrontal cortex of mice.

PubMed

Pitchaimani, Vigneshwaran; Arumugam, Somasundaram; Thandavarayan, Rajarajan Amirthalingam; Karuppagounder, Vengadeshprabhu; Sreedhar, Remya; Afrin, Rejina; Harima, Meilei; Suzuki, Hiroshi; Miyashita, Shizuka; Nomoto, Mayumi; Sone, Hirohito; Suzuki, Kenji; Watanabe, Kenichi

2014-09-05

BAD-deficient mice and fasting have several common functional roles in seizures, beta-hydroxybutyrate (BHB) uptake in brain and alteration in counterregulatory hormonal regulation during hypoglycemia. Neuronal specific insulin receptor knockout (NIRKO) mice display impaired counterregulatory hormonal responses during hypoglycemia. In this study we investigated the fasting mediated expression of p-BAD(ser155) and p-AKT(ser473) in different regions of brain (prefrontal cortex, hippocampus, midbrain and hypothalamus). Fasting specifically increases p-BAD(ser155) and p-AKT(ser473) in prefrontal cortex and decreases in other regions of brain. Our results suggest that fasting may increase the uptake BHB by decreasing p-BAD(ser155) in the brain during hypoglycemia except prefrontal cortex and it uncovers specific functional area of p-BAD(ser155) and p-AKT(ser473) that may regulates counter regulatory hormonal response. Overall in support with previous findings, fasting mediated hypoglycemia activates prefrontal cortex insulin signaling which influences the hypothalamic paraventricular nucleus mediated activation of sympathoadrenal hormonal responses. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Protection of chickens from Newcastle disease with a recombinant baculovirus subunit vaccine expressing the fusion and hemagglutinin-neuraminidase proteins

PubMed Central

Lee, Youn-Jeong; Sung, Haan-Woo; Choi, Jun-Gu; Lee, Eun-Kyoung; Yoon, Hachung; Kim, Jae-Hong

2008-01-01

Recombinant baculoviruses containing the fusion (F) and hemagglutinin-neuraminidase (HN) glycoprotein gene of the viscerotropic velogenic (vv) Newcastle disease virus (NDV) isolate, Kr-005/00, and a lentogenic La Sota strain of the NDV were constructed in an attempt to develop an effective subunit vaccine to the recent epizootic vvNDV. The level of protection was determined by evaluating the clinical signs, mortality, and virus shedding from the oropharynx and cloaca of chickens after a challenge with vvNDV Kr-005/00. The recombinant ND F (rND F) and recombinant HN (rND HN) glycoproteins derived from the velogenic strain provided good protection against the clinical signs and mortality, showing a 0.00 PI value and 100% protection after a booster immunization. On the other hand, the combined rND F + HN glycoprotein derived from the velogenic strain induced complete protection (0.00 PI value and 100% protection) and significantly reduced the amount of virus shedding even after a single immunization. The rND F and rND HN glycoproteins derived from the velogenic strain had a slightly, but not significantly, greater protective effect than the lentogenic strain. These results suggest that the combined rND F + HN glycoprotein derived from vvNDV can be an ideal subunit marker vaccine candidate in chickens in a future ND eradication program. PMID:18716451

P and Ca digestibility is increased in broiler diets supplemented with the high-phytase HIGHPHY wheat.

PubMed

Scholey, D; Burton, E; Morgan, N; Sanni, C; Madsen, C K; Dionisio, G; Brinch-Pedersen, H

2017-09-01

Around 70% of total seed phosphorus is represented by phytate which must be hydrolysed to be bioavailable in non-ruminant diets. The limited endogenous phytase activity in non-ruminant animals make it common practice to add an exogenous phytase source to most poultry and pig feeds. The mature grain phytase activity (MGPA) of cereal seeds provides a route for the seeds themselves to contribute to phytate digestion, but MGPA varies considerably between species and most varieties in current use make negligible contributions. Currently, all phytases used for feed supplementation and transgenic improvement of MGPA are derived from microbial enzymes belonging to the group of histidine acid phosphatases (HAP). Cereals contain HAP phytases, but the bulk of MGPA can be attributed to phytases belonging to a completely different group of phosphatases, the purple acid phosphatases (PAPhy). In recent years, increased MGPAs were achieved in cisgenic barley holding extra copies of barley PAPhy and in the wheat HIGHPHY mutant, where MGPA was increased to ~6200 FTU/kg. In the present study, the effect of replacing 33%, 66% and 100% of a standard wheat with HIGHPHY wheat was compared with a control diet with and without 500 FTU of supplemental phytase. Diets were compared by evaluating broiler performance, ileal Ca and P digestibility and tibia development, using nine replicate pens of four birds per diet over 3 weeks from hatch. There were no differences between treatments in any tibia or bird performance parameters, indicating the control diet did not contain sufficiently low levels of phosphorus to distinguish effect of phytase addition. However, in a comparison of the two wheats, the ileal Ca and P digestibility coefficients for the 100% HIGHPHY wheat diets are 22.9% and 35.6% higher, respectively, than for the control diet, indicating the wheat PAPhy is functional in the broiler digestive tract. Furthermore, 33% HIGHPHY replacement of conventional wheat, significantly improved

46 CFR 35.35-5 - Electric bonding-TB/ALL.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 46 Shipping 1 2011-10-01 2011-10-01 false Electric bonding-TB/ALL. 35.35-5 Section 35.35-5... Electric bonding—TB/ALL. A tank vessel may be electrically connected to the shore piping, through which the cargo is to be transferred, prior to the connecting of a cargo hose. This electrical connection, if made...

46 CFR 35.35-5 - Electric bonding-TB/ALL.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 46 Shipping 1 2012-10-01 2012-10-01 false Electric bonding-TB/ALL. 35.35-5 Section 35.35-5... Electric bonding—TB/ALL. A tank vessel may be electrically connected to the shore piping, through which the cargo is to be transferred, prior to the connecting of a cargo hose. This electrical connection, if made...

46 CFR 35.35-5 - Electric bonding-TB/ALL.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 46 Shipping 1 2010-10-01 2010-10-01 false Electric bonding-TB/ALL. 35.35-5 Section 35.35-5... Electric bonding—TB/ALL. A tank vessel may be electrically connected to the shore piping, through which the cargo is to be transferred, prior to the connecting of a cargo hose. This electrical connection, if made...

Increased efficiency with surface texturing in ITO/InP solar cells

NASA Technical Reports Server (NTRS)

Jenkins, Phillip; Landis, Geoffrey A.; Fatemi, Navid; Li, Xiaonan; Scheiman, David; Bailey, Sheila

1992-01-01

Optimization of an InP solar cell with a V-grooved surface is discussed. Total internal reflection in the coverglass reduces surface reflection and can recover light reflected from the front metallization. Results from the first ITO/InP solar cells on low-angle V-grooved substrates are presented, showing a 5.8 percent increase in current.

Paclitaxel-induced peripheral neuropathy increases substance P release in rat spinal cord.

PubMed

Chiba, Terumasa; Oka, Yusuke; Kambe, Toshie; Koizumi, Naoya; Abe, Kenji; Kawakami, Kazuyoshi; Utsunomiya, Iku; Taguchi, Kyoji

2016-01-05

Peripheral neuropathy is a common adverse effect of paclitaxel treatment. The major dose-limiting side effect of paclitaxel is peripheral sensory neuropathy, which is characterized by painful paresthesia of the hands and feet. To analyze the contribution of substance P to the development of paclitaxel-induced mechanical hyperalgesia, substance P expression in the superficial layers of the rat spinal dorsal horn was analyzed after paclitaxel treatment. Behavioral assessment using the von Frey test and the paw thermal test showed that intraperitoneal administration of 2 and 4mg/kg paclitaxel induced mechanical allodynia/hyperalgesia and thermal hyperalgesia 7 and 14 days after treatment. Immunohistochemistry showed that paclitaxel (4mg/kg) treatment significantly increased substance P expression (37.6±3.7% on day 7, 43.6±4.6% on day 14) in the superficial layers of the spinal dorsal horn, whereas calcitonin gene-related peptide (CGRP) expression was unchanged. Moreover, paclitaxel (2 and 4mg/kg) treatment significantly increased substance P release in the spinal cord on day 14. These results suggest that paclitaxel treatment increases release of substance P, but not CGRP in the superficial layers of the spinal dorsal horn and may contribute to paclitaxel-induced painful peripheral neuropathy. Copyright © 2015 Elsevier B.V. All rights reserved.

LEDGF/p75 Deficiency Increases Deletions at the HIV-1 cDNA Ends.

PubMed

Bueno, Murilo T D; Reyes, Daniel; Llano, Manuel

2017-09-15

Processing of unintegrated linear HIV-1 cDNA by the host DNA repair system results in its degradation and/or circularization. As a consequence, deficient viral cDNA integration generally leads to an increase in the levels of HIV-1 cDNA circles containing one or two long terminal repeats (LTRs). Intriguingly, impaired HIV-1 integration in LEDGF/p75-deficient cells does not result in a correspondent increase in viral cDNA circles. We postulate that increased degradation of unintegrated linear viral cDNA in cells lacking the lens epithelium-derived growth factor (LEDGF/p75) account for this inconsistency. To evaluate this hypothesis, we characterized the nucleotide sequence spanning 2-LTR junctions isolated from LEDGF/p75-deficient and control cells. LEDGF/p75 deficiency resulted in a significant increase in the frequency of 2-LTRs harboring large deletions. Of note, these deletions were dependent on the 3' processing activity of integrase and were not originated by aberrant reverse transcription. Our findings suggest a novel role of LEDGF/p75 in protecting the unintegrated 3' processed linear HIV-1 cDNA from exonucleolytic degradation.

Tobacco Smoking and Increased Risk of Death and Progression for Patients With p16-Positive and p16-Negative Oropharyngeal Cancer

PubMed Central

Gillison, Maura L.; Zhang, Qiang; Jordan, Richard; Xiao, Weihong; Westra, William H.; Trotti, Andy; Spencer, Sharon; Harris, Jonathan; Chung, Christine H.; Ang, K. Kian

2012-01-01

Purpose Tobacco smoking is associated with oropharynx cancer survival, but to what extent cancer progression or death increases with increasing tobacco exposure is unknown. Patients and Methods Patients with oropharynx cancer enrolled onto a phase III trial of radiotherapy from 1991 to 1997 (Radiation Therapy Oncology Group [RTOG] 9003) or of chemoradiotherapy from 2002 to 2005 (RTOG 0129) were evaluated for tumor human papillomavirus status by a surrogate, p16 immunohistochemistry, and for tobacco exposure by a standardized questionnaire. Associations between tobacco exposure and overall survival (OS) and progression-free survival (PFS) were estimated by Cox proportional hazards models. Results Prevalence of p16-positive cancer was 39.5% among patients in RTOG 9003 and 68.0% in RTOG 0129. Median pack-years of tobacco smoking were lower among p16-positive than p16-negative patients in both trials (RTOG 9003: 29 v 45.9 pack-years; P = .02; RTOG 0129: 10 v 40 pack-years; P < .001). After adjustment for p16 and other factors, risk of progression (PFS) or death (OS) increased by 1% per pack-year (for both, hazard ratio [HR], 1.01; 95% CI, 1.00 to 1.01; P = .002) or 2% per year of smoking (for both, HR, 1.02; 95% CI, 1.01 to 1.03; P < .001) in both trials. In RTOG 9003, risk of death doubled (HR, 2.19; 95% CI, 1.46 to 3.28) among those who smoked during radiotherapy after accounting for pack-years and other factors, and risk of second primary tumors increased by 1.5% per pack-year (HR, 1.015; 95% CI, 1.005 to 1.026). Conclusion Risk of oropharyngeal cancer progression and death increases directly as a function of tobacco exposure at diagnosis and during therapy and is independent of tumor p16 status and treatment. PMID:22565003

Baculovirus-expressed vitamin D-binding protein-macrophage activating factor (DBP-maf) activates osteoclasts and binding of 25-hydroxyvitamin D(3) does not influence this activity.

PubMed

Swamy, N; Ghosh, S; Schneider, G B; Ray, R

2001-01-01

Vitamin D-binding protein (DBP) is a multi-functional serum protein that is converted to vitamin D-binding protein-macrophage activating factor (DBP-maf) by post-translational modification. DBP-maf is a new cytokine that mediates bone resorption by activating osteoclasts, which are responsible for resorption of bone. Defective osteoclast activation leads to disorders like osteopetrosis, characterized by excessive accumulation of bone mass. Previous studies demonstrated that two nonallelic mutations in the rat with osteopetrosis have independent defects in the cascade involved in the conversion of DBP to DBP-maf. The skeletal defects associated with osteopetrosis are corrected in these mutants with in vivo DBP-maf treatment. This study evaluates the effects of various forms of DBP-maf (native, recombinant, and 25-hydroxyvitamin D(3) bound) on osteoclast function in vitro in order to determine some of the structural requirements of this protein that relate to bone resorbing activities. Osteoclast activity was determined by evaluating pit formation using osteoclasts, isolated from the long bones of newborn rats, incubated on calcium phosphate coated, thin film, Ostologic MultiTest Slides. Incubation of osteoclasts with ex vivo generated native DBP-maf resulted in a dose dependent, statistically significant, activation of the osteoclasts. The activation was similar whether or not the vitamin D binding site of the DBP-maf was occupied. The level of activity in response to DBP-maf was greater than that elicited by optimal doses of other known stimulators (PTH and 1,25(OH(2)D(3)) of osteoclast function. Furthermore, another potent macrophage activating factor, interferon--gamma, had no effect on osteoclast activity. The activated form of a full length recombinant DBP, expressed in E. coli showed no activity in the in vitro assay. Contrary to this finding, baculovirus-expressed recombinant DBP-maf demonstrated significant osteoclast activating activity. The normal

Low pH increases the yield of exosome isolation.

PubMed

Ban, Jae-Jun; Lee, Mijung; Im, Wooseok; Kim, Manho

2015-05-22

Exosomes are the extracellular vesicles secreted by various cells. Exosomes mediate intercellular communication by delivering a variety of molecules between cells. Cancer cell derived exosomes seem to be related with tumor progression and metastasis. Tumor microenvironment is thought to be acidic and this low pH controls exosome physiology, leading to tumor progression. Despite the importance of microenvironmental pH on exosome, most of exosome studies have been performed without regard to pH. Therefore, the difference of exosome stability and yield of isolation by different pH need to be studied. In this research, we investigated the yield of total exosomal protein and RNA after incubation in acidic, neutral and alkaline conditioned medium. Representative exosome markers were investigated by western blot after incubation of exosomes in different pH. As a result, the concentrations of exosomal protein and nucleic acid were significantly increased after incubation in the acidic medium compared with neutral medium. The higher levels of exosome markers including CD9, CD63 and HSP70 were observed after incubation in an acidic environment. On the other hand, no exosomal protein, exosomal RNA and exosome markers have been detected after incubation in an alkaline condition. In summary, our results indicate that the acidic condition is the favorable environment for existence and isolation of exosomes. Copyright © 2015 Elsevier Inc. All rights reserved.

Evidence of Increase in Mortality After the Introduction of Diphtheria-Tetanus-Pertussis Vaccine to Children Aged 6-35 Months in Guinea-Bissau: A Time for Reflection?

PubMed

Aaby, Peter; Mogensen, Søren Wengel; Rodrigues, Amabelia; Benn, Christine S

2018-01-01

boys [HR = 1.71 (0.99-2.93)] (test for interaction p  = 0.27). Although having better nutritional status and being protected against three infections, 6-35 months old DTP-vaccinated children tended to have higher mortality than DTP-unvaccinated children. All studies of the introduction of DTP have found increased overall mortality.

Initial preclinical safety of non-replicating human endogenous retrovirus envelope protein-coated baculovirus vector-based vaccines against human papillomavirus.

PubMed

Han, Su-Eun; Kim, Mi-Gyeong; Lee, Soondong; Cho, Hee-Jeong; Byun, Youngro; Kim, Sujeong; Kim, Young Bong; Choi, Yongseok; Oh, Yu-Kyoung

2013-12-01

Human endogenous retrovirus (HERV) envelope protein-coated, baculovirus vector-based HPV 16 L1 (AcHERV-HPV16L1) is a non-replicating recombinant baculoviral vaccine. Here, we report an initial evaluation of the preclinical safety of AcHERV-HPV16L1 vaccine. In an acute toxicity study, a single administration of AcHERV-HPV16L1 DNA vaccine given intramuscularly (i.m.) to mice at a dose of 1 × 10(8) plaque-forming units (PFU) did not cause significant changes in body weight compared with vehicle-treated controls. It did cause a brief increase in the weights of some organs on day 15 post-treatment, but by day 30, all organ weights were not significantly different from those in the vehicle-treated control group. No hematological changes were observed on day 30 post-treatment. In a range-finding toxicity study with three doses of 1 × 10(7) , 2 × 10(7) and 5 × 10(7) PFU once daily for 5 days, the group treated with 5 × 10(7) PFU showed a transient decrease in the body weights from day 5 to day 15 post-treatment, but recovery to the levels similar to those in the vehicle-treated control group by post-treatment day 20. Organ weights were slightly higher for lymph nodes, spleen, thymus and liver after repeated dosing with 5 × 10(7) PFU on day 15, but had normalized by day 30. Moreover, repeated administration of AcHERV-HPV16L1 did not induce myosin-specific autoantibody in serum, and did not cause immune complex deposition or tissue damage at injection sites. Taken together, these results provide preliminary evidence of the preclinical safety of AcHERV-based HPV16L1 DNA vaccines in mice. Copyright © 2012 John Wiley & Sons, Ltd.

IL-35 promotes pancreas cancer growth through enhancement of proliferation and inhibition of apoptosis: evidence for a role as an autocrine growth factor.

PubMed

Nicholl, Michael B; Ledgewood, Chelsea L; Chen, Xuhui; Bai, Qian; Qin, Chenglu; Cook, Kathryn M; Herrick, Elizabeth J; Diaz-Arias, Alberto; Moore, Bradley J; Fang, Yujiang

2014-12-01

Interleukin-35 (IL-35), an IL-12 cytokine family member, mediates the immune inhibitory function of regulatory T cells (Treg). We assayed the presence of IL-35 in paraffin-embedded human pancreas cancer (PCAN) and unexpectedly found IL-35 was expressed mainly by epithelial derived PCAN cells, but not by Treg. We further examined the expression and effect of exogenous IL-35 in human PCAN cell lines and found IL-35 promoted growth and inhibited apoptosis in PCAN cell lines. IL-35 induced proliferation correlated with an increase in cyclin B, cyclin D, cdk2, and cdk4 and a decrease in p27 expression, while inhibition of apoptosis was associated with an increase in Bcl-2 and a decrease in TRAILR1. We conclude IL-35 is produced by PCAN in vivo and promotes PCAN cell line growth in vitro. These results might indicate an important new role for IL-35 as an autocrine growth factor in PCAN growth. Copyright © 2014 Elsevier Ltd. All rights reserved.

Positive Peer-Pressured Productivity (P-QUAD): Novel Use of Increased Transparency and a Weighted Lottery to Increase a Division's Academic Output.

PubMed

Pitt, Michael B; Furnival, Ronald A; Zhang, Lei; Weber-Main, Anne M; Raymond, Nancy C; Jacob, Abraham K

2017-03-01

Evaluate a dual incentive model combining positive peer pressure through increased transparency of peers' academic work with a weighted lottery where entries are earned based on degree of productivity. We developed a dual-incentive peer mentoring model, Positive Peer-Pressured Productivity (P-QUAD), for faculty in the Pediatric Hospital Medicine Division at the University of Minnesota Masonic Children's Hospital. This model provided relative value-based incentives, with points assigned to different scholarly activities (eg. 1 point for abstract submission, 2 points for poster presentation, 3 points for oral presentation, etc.). These points translated into to lottery tickets for a semi-annual drawing for monetary prizes. Productivity was compared among faculty for P-QUAD year to the preintervention year. Fifteen (83%) of 18 eligible faculty members participated. Overall annual productivity per faculty member as measured by total P-QUAD score increased from a median of 3 (interquartile range [IQR] 0-14) in the preintervention year to 4 (IQR 0-27) in the P-QUAD year (P = .051). Submissions and acceptances increased in all categories except posters which were unchanged. Annual abstract submissions per faculty member significantly increased from a median of 1 (IQR 0-2) to 2 (IQR 0-2; P = .047). Seventy-three percent (8 of 11) of post-survey respondents indicated that the financial incentive motivated them to submit academic work; 100% indicated that increased awareness of their peers' work was a motivator. The combination of increased awareness of peers' academic productivity and a weighted lottery financial incentive appears to be a useful model for stimulating academic productivity in early-career faculty. Copyright © 2016 Academic Pediatric Association. Published by Elsevier Inc. All rights reserved.

Development and Validation of a P-35S, T-nos, T-35S and P-FMV Tetraplex Real-time PCR Screening Method to Detect Regulatory Genes of Genetically Modified Organisms in Food.

PubMed

Eugster, Albert; Murmann, Petra; Kaenzig, Andre; Breitenmoser, Alda

2014-10-01

In routine analysis screening methods based on real-time PCR (polymerase chain reaction) are most commonly used for the detection of genetically modified (GM) plant material in food and feed. Screening tests are based on sequences frequently used for GM development, allowing the detection of a large number of GMOs (genetically modified organisms). Here, we describe the development and validation of a tetraplex real-time PCR screening assay comprising detection systems for the regulatory genes Cauliflower Mosaic Virus 35S promoter, Agrobacterium tumefaciens nos terminator, Cauliflower Mosaic Virus 35S terminator and Figwort Mosaic Virus 34S promoter. Three of the four primer and probe combinations have already been published elsewhere, whereas primers and probe for the 35S terminator have been developed in-house. Adjustment of primer and probe concentrations revealed a high PCR sensitivity with insignificant physical cross-talk between the four detection channels. The sensitivity of each PCR-system is sufficient to detect a GMO concentration as low as 0.05% of the containing respective element. The specificity of the described tetraplex is high when tested on DNA from GM maize, soy, rapeseed and tomato. We also demonstrate the robustness of the system by inter-laboratory tests. In conclusion, this method provides a sensitive and reliable screening procedure for the detection of the most frequently used regulatory elements present in GM crops either authorised or unauthorised for food.

Structural basis for selective inhibition of Cyclooxygenase-1 (COX-1) by diarylisoxazoles mofezolac and 3-(5-chlorofuran-2-yl)-5-methyl-4-phenylisoxazole (P6).

PubMed

Cingolani, Gino; Panella, Andrea; Perrone, Maria Grazia; Vitale, Paola; Di Mauro, Giuseppe; Fortuna, Cosimo G; Armen, Roger S; Ferorelli, Savina; Smith, William L; Scilimati, Antonio

2017-09-29

The diarylisoxazole molecular scaffold is found in several NSAIDs, especially those with high selectivity for COX-1. Here, we have determined the structural basis for COX-1 binding to two diarylisoxazoles: mofezolac, which is polar and ionizable, and 3-(5-chlorofuran-2-yl)-5-methyl-4-phenylisoxazole (P6) that has very low polarity. X-ray analysis of the crystal structures of COX-1 bound to mofezolac and 3-(5-chlorofuran-2-yl)-5-methyl-4-phenylisoxazole allowed the identification of specific binding determinants within the enzyme active site, relevant to generate structure/activity relationships for diarylisoxazole NSAIDs. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Chromosomal distribution of pTa-535, pTa-86, pTa-713, 35S rDNA repetitive sequences in interspecific hexaploid hybrids of common wheat (Triticum aestivum L.) and spelt (Triticum spelta L.)

PubMed Central

Duba, Adrian; Kwiatek, Michał; Wiśniewska, Halina; Wachowska, Urszula; Wiwart, Marian

2018-01-01

Fluorescent in situ hybridization (FISH) relies on fluorescent-labeled probes to detect specific DNA sequences in the genome, and it is widely used in cytogenetic analyses. The aim of this study was to determine the karyotype of T. aestivum and T. spelta hybrids and their parental components (three common wheat cultivars and five spelt breeding lines), to identify chromosomal aberrations in the evaluated wheat lines, and to analyze the distribution of polymorphisms of repetitive sequences in the examined hybrids. The FISH procedure was carried out with four DNA clones, pTa-86, pTa-535, pTa-713 and 35S rDNA used as probes. The observed polymorphisms between the investigated lines of common wheat, spelt and their hybrids was relatively low. However, differences were observed in the distribution of repetitive sequences on chromosomes 4A, 6A, 1B and 6B in selected hybrid genomes. The polymorphisms observed in common wheat and spelt hybrids carry valuable information for wheat breeders. The results of our study are also a valuable source of knowledge about genome organization and diversification in common wheat, spelt and their hybrids. The relevant information is essential for common wheat breeders, and it can contribute to breeding programs aimed at biodiversity preservation. PMID:29447228

Chromosomal distribution of pTa-535, pTa-86, pTa-713, 35S rDNA repetitive sequences in interspecific hexaploid hybrids of common wheat (Triticum aestivum L.) and spelt (Triticum spelta L.).

PubMed

Goriewa-Duba, Klaudia; Duba, Adrian; Kwiatek, Michał; Wiśniewska, Halina; Wachowska, Urszula; Wiwart, Marian

2018-01-01

Fluorescent in situ hybridization (FISH) relies on fluorescent-labeled probes to detect specific DNA sequences in the genome, and it is widely used in cytogenetic analyses. The aim of this study was to determine the karyotype of T. aestivum and T. spelta hybrids and their parental components (three common wheat cultivars and five spelt breeding lines), to identify chromosomal aberrations in the evaluated wheat lines, and to analyze the distribution of polymorphisms of repetitive sequences in the examined hybrids. The FISH procedure was carried out with four DNA clones, pTa-86, pTa-535, pTa-713 and 35S rDNA used as probes. The observed polymorphisms between the investigated lines of common wheat, spelt and their hybrids was relatively low. However, differences were observed in the distribution of repetitive sequences on chromosomes 4A, 6A, 1B and 6B in selected hybrid genomes. The polymorphisms observed in common wheat and spelt hybrids carry valuable information for wheat breeders. The results of our study are also a valuable source of knowledge about genome organization and diversification in common wheat, spelt and their hybrids. The relevant information is essential for common wheat breeders, and it can contribute to breeding programs aimed at biodiversity preservation.

40 CFR 35.635 - Maximum federal share.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 1 2010-07-01 2010-07-01 false Maximum federal share. 35.635 Section... (sections 319(h) and 518(f)) § 35.635 Maximum federal share. (a) The Regional Administrator may provide up... be provided from non-federal sources. (b) The Regional Administrator may increase the maximum federal...

40 CFR 35.685 - Maximum federal share.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 1 2010-07-01 2010-07-01 false Maximum federal share. 35.685 Section... (section 1443(b)) § 35.685 Maximum federal share. (a) The Regional Administrator may provide up to 75 percent of the approved work plan costs. (b) The Regional Administrator may increase the maximum federal...

A glass of water immediately increases gastric pH in healthy subjects.

PubMed

Karamanolis, George; Theofanidou, Ioanna; Yiasemidou, Marina; Giannoulis, Evangelos; Triantafyllou, Konstantinos; Ladas, Spiros D

2008-12-01

Onset of action of antisecretory agents is of pivotal importance for patients with gastroesophageal reflux disease (GERD) treated "on-demand." To study the acute effect of acid-inhibiting drugs and water administration on gastric pH. A cross-over study was performed in 12 H. pylori (-), healthy subjects (6 men; mean age: 26 years). A single oral dose of the following agents was received with a wash-out period between each study: a glass of water (200 ml), antacid, ranitidine, omeprazole, esomeprazole, and rabeprazole. Gastric pH was recorded for 6 h after drug intake. Water increased gastric pH >4 in 10/12 subjects after 1 min. The time (median) needed to pH >4 was for: antacid 2 min, ranitidine 50 min, omeprazole 171 min, esomeprazole 151 min, and rabeprazole 175 min. Gastric pH >4 lasted for 3 min after water and for 12 min after antacids; it remained >4 until the end of recording in: 4/12 subjects with ranitidine, 11/12 with rabeprazole, and all with omeprazole and esomeprazole. Water and antacid immediately increased gastric pH, while PPIs showed a delayed but prolonged effect compared to ranitidine.

SfDronc, an initiator caspase involved in apoptosis in the fall armyworm Spodoptera frugiperda.

PubMed

Huang, Ning; Civciristov, Srgjan; Hawkins, Christine J; Clem, Rollie J

2013-05-01

Initiator caspases are the first caspases that are activated following an apoptotic stimulus, and are responsible for cleaving and activating downstream effector caspases, which directly cause apoptosis. We have cloned a cDNA encoding an ortholog of the initiator caspase Dronc in the lepidopteran insect Spodoptera frugiperda. The SfDronc cDNA encodes a predicted protein of 447 amino acids with a molecular weight of 51 kDa. Overexpression of SfDronc induced apoptosis in Sf9 cells, while partial silencing of SfDronc expression in Sf9 cells reduced apoptosis induced by baculovirus infection or by treatment with UV or actinomycin D. Recombinant SfDronc exhibited several expected biochemical characteristics of an apoptotic initiator caspase: 1) SfDronc efficiently cleaved synthetic initiator caspase substrates, but had very little activity against effector caspase substrates; 2) mutation of a predicted cleavage site at position D340 blocked autoprocessing of recombinant SfDronc and reduced enzyme activity by approximately 10-fold; 3) SfDronc cleaved the effector caspase Sf-caspase-1 at the expected cleavage site, resulting in Sf-caspase-1 activation; and 4) SfDronc was strongly inhibited by the baculovirus caspase inhibitor SpliP49, but not by the related protein AcP35. These results indicate that SfDronc is an initiator caspase involved in caspase-dependent apoptosis in S. frugiperda, and as such is likely to be responsible for the initiator caspase activity in S. frugiperda cells known as Sf-caspase-X. Copyright © 2013 Elsevier Ltd. All rights reserved.

High glucose increases Cdk5 activity in podocytes via transforming growth factor-β1 signaling pathway

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

Zhang, Yue; Li, Hongbo; Hao, Jun

Podocytes are highly specialized and terminally differentiated glomerular cells that play a vital role in the development and progression of diabetic nephropathy (DN). Cyclin-dependent kinase 5 (Cdk5), who is an atypical but essential member of the Cdk family of proline-directed serine/threonine kinases, has been shown as a key regulator of podocyte differentiation, proliferation and morphology. Our previous studies demonstrated that the expression of Cdk5 was significantly increased in podocytes of diabetic rats, and was closely related with podocyte injury of DN. However, the mechanisms of how expression and activity of Cdk5 are regulated under the high glucose environment have notmore » yet been fully elucidated. In this study, we showed that high glucose up-regulated the expression of Cdk5 and its co-activator p35 with a concomitant increase in Cdk5 kinase activity in conditionally immortalized mouse podocytes in vitro. When exposed to 30 mM glucose, transforming growth factor-β1 (TGF-β1) was activated. Most importantly, we found that SB431542, the Tgfbr1 inhibitor, significantly decreased the expression of Cdk5 and p35 and Cdk5 kinase activity in high glucose-treated podocytes. Moreover, high glucose increased the expression of early growth response-1 (Egr-1) via TGF-β1-ERK1/2 pathway in podocytes and inhibition of Egr-1 by siRNA decreased p35 expression and Cdk5 kinase activity. Furthermore, inhibition of Cdk5 kinase activity effectively alleviated podocyte apoptosis induced by high glucose or TGF-β1. Thus, the TGF-β1-ERK1/2-Egr-1 signaling pathway may regulate the p35 expression and Cdk5 kinase activity in high glucose-treated podocytes, which contributes to podocyte injury of DN. - Highlights: • HG up-regulated the expression of Cdk5 and p35, and Cdk5 activity in podocytes. • HG activated TGF-β1 pathway and SB431542 inhibited Cdk5 expression and activity. • HG increased the expression of Egr-1 via TGF-β1-ERK1/2 pathway. • Inhibition of Egr

Prevalence of increased canine pancreas-specific lipase concentrations in young dogs with parvovirus enteritis.

PubMed

Kalli, Irida V; Adamama-Moraitou, Katerina K; Patsika, Michael N; Pardali, Dimitra; Steiner, Jörg M; Suchodolski, Jan S; Menexes, George; Brellou, Georgia D; Rallis, Timoleon S

2017-03-01

Pancreatic abnormalities during canine parvovirus (CPV) enteritis have not been studied prospectively. The objective of this study was to evaluate the diagnostic significance of canine serum pancreas-specific lipase (Spec cPL) concentration in dogs with CPV enteritis for the presence of acute pancreatitis (AP). Puppies with naturally occurring CPV enteritis were recruited and prospectively allocated into 2 groups according to normal or increased serum Spec cPL concentration. Clinical signs, laboratory findings, and pancreas-associated variables were compared between groups, and the impact of possible AP on disease course, duration of hospitalization, and outcome was assessed. Serum Spec cPL concentration in 35 puppies was above the upper limit of the RI in 17/35 (48.6%) dogs (Group A) and within the RI in 18 dogs (Group B). An increased serum lipase activity was present in 29/35 (82.9%) dogs, and Group A dogs had a higher serum lipase activity than Group B (P = .006). Serum Spec cPL in Group A dogs was positively correlated with serum lipase activity at the day of presentation (r = .667; P = .003) and day of discharge (r = .628; P = .007). No statistically significant difference was found between groups (P = .233) for the presence of systemic inflammatory response syndrome (SIRS) (6/17 or 35.3% dogs Group A, and 8/18 or 44.4% dogs Group B), the disease course, duration of hospitalization, or outcome between groups. Increased serum Spec cPL is relatively common in dogs with CPV enteritis. However, such increases do not seem to correlate with the outcome of disease. © 2017 American Society for Veterinary Clinical Pathology.

Inflammatory stimuli induce inhibitory S-nitrosylation of the deacetylase SIRT1 to increase acetylation and activation of p53 and p65.

PubMed

Shinozaki, Shohei; Chang, Kyungho; Sakai, Michihiro; Shimizu, Nobuyuki; Yamada, Marina; Tanaka, Tomokazu; Nakazawa, Harumasa; Ichinose, Fumito; Yamada, Yoshitsugu; Ishigami, Akihito; Ito, Hideki; Ouchi, Yasuyoshi; Starr, Marlene E; Saito, Hiroshi; Shimokado, Kentaro; Stamler, Jonathan S; Kaneki, Masao

2014-11-11

Inflammation increases the abundance of inducible nitric oxide synthase (iNOS), leading to enhanced production of nitric oxide (NO), which can modify proteins by S-nitrosylation. Enhanced NO production increases the activities of the transcription factors p53 and nuclear factor κB (NF-κB) in several models of disease-associated inflammation. S-nitrosylation inhibits the activity of the protein deacetylase SIRT1. SIRT1 limits apoptosis and inflammation by deacetylating p53 and p65 (also known as RelA), a subunit of NF-κB. We showed in multiple cultured mammalian cell lines that NO donors or inflammatory stimuli induced S-nitrosylation of SIRT1 within CXXC motifs, which inhibited SIRT1 by disrupting its ability to bind zinc. Inhibition of SIRT1 reduced deacetylation and promoted activation of p53 and p65, leading to apoptosis and increased expression of proinflammatory genes. In rodent models of systemic inflammation, Parkinson's disease, or aging-related muscular atrophy, S-nitrosylation of SIRT1 correlated with increased acetylation of p53 and p65 and activation of p53 and NF-κB target genes, suggesting that S-nitrosylation of SIRT1 may represent a proinflammatory switch common to many diseases and aging. Copyright © 2014, American Association for the Advancement of Science.

Inflammatory stimuli induce inhibitory S-nitrosylation of the deacetylase SIRT1 to increase acetylation and activation of p53 and p65

PubMed Central

Shinozaki, Shohei; Chang, Kyungho; Sakai, Michihiro; Shimizu, Nobuyuki; Yamada, Marina; Tanaka, Tomokazu; Nakazawa, Harumasa; Ichinose, Fumito; Yamada, Yoshitsugu; Ishigami, Akihito; Ito, Hideki; Ouchi, Yasuyoshi; Starr, Marlene E.; Saito, Hiroshi; Shimokado, Kentaro; Stamler, Jonathan S.; Kaneki, Masao

2015-01-01

Inflammation increases the abundance of inducible nitric oxide synthase (iNOS), leading to enhanced production of nitric oxide (NO), which can modify proteins by S-nitrosylation. Enhanced NO production increases the activities of the transcription factors p53 and nuclear factor κB (NF-κB) in several models of disease-associated inflammation. S-Nitrosylation inhibits the activity of the protein deacetylase SIRT1. SIRT1 limits apoptosis and inflammation by deacetylating p53 and p65 (also known as RelA), a subunit of NF-κB. We showed in multiple cultured mammalian cell lines that NO donors or inflammatory stimuli induced S-nitrosylation of SIRT1 within CXXC motifs, which inhibited SIRT1 by disrupting its ability to bind zinc. Inhibition of SIRT1 reduced deacetylation and promoted activation of p53 and p65, leading to apoptosis and increased expression of proinflammatory genes. In rodent models of systemic inflammation, Parkinson’s disease, or aging-related muscular atrophy, S-nitrosylation of SIRT1 correlated with increased acetylation of p53 and p65 and activation of p53 and NF-κB target genes, suggesting that S-nitrosylation of SIRT1 may represent a proinflammatory switch common to many diseases and aging. PMID:25389371

Plant genotype and induced defenses affect the productivity of an insect-killing obligate viral pathogen.

PubMed

Shikano, Ikkei; McCarthy, Elizabeth M; Elderd, Bret D; Hoover, Kelli

2017-09-01

Plant-mediated variations in the outcomes of host-pathogen interactions can strongly affect epizootics and the population dynamics of numerous species, including devastating agricultural pests such as the fall armyworm. Most studies of plant-mediated effects on insect pathogens focus on host mortality, but few have measured pathogen yield, which can affect whether or not an epizootic outbreak occurs. Insects challenged with baculoviruses on different plant species and parts can vary in levels of mortality and yield of infectious stages (occlusion bodies; OBs). We previously demonstrated that soybean genotypes and induced anti-herbivore defenses influence baculovirus infectivity. Here, we used a soybean genotype that strongly reduced baculovirus infectivity when virus was ingested on induced plants (Braxton) and another that did not reduce infectivity (Gasoy), to determine how soybean genotype and induced defenses influence OB yield and speed of kill. These are key fitness measures because baculoviruses are obligate-killing pathogens. We challenged fall armyworm, Spodoptera frugiperda, with the baculovirus S. frugiperda multi-nucleocapsid nucleopolyhedrovirus (SfMNPV) during short or long-term exposure to plant treatments (i.e., induced or non-induced genotypes). Caterpillars were either fed plant treatments only during virus ingestion (short-term exposure to foliage) or from the point of virus ingestion until death (long-term exposure). We found trade-offs of increasing OB yield with slower speed of kill and decreasing virus dose. OB yield increased more with longer time to death and decreased more with increasing virus dose after short-term feeding on Braxton compared with Gasoy. OB yield increased significantly more with time to death in larvae that fed until death on non-induced foliage than induced foliage. Moreover, fewer OBs per unit of host tissue were produced when larvae were fed induced foliage than non-induced foliage. These findings highlight the

Does the S.D.E.P. increase performance?

NASA Astrophysics Data System (ADS)

Syltebo, Andy

2003-05-01

Through the guidance of the program, "Physical Systems," at The Evergreen State College in Olympia Washington, Andy Syltebo will be investigating how the Surface Drive Enhancement Project will affect the performance of a planing hull powered by surface drive propulsion. A radio controlled model boat of the forementioned design is the prototype vehicle used for experimentation and analysis. The idea of this project revolves around harnessing the energy in the water of a rooster tail ejected from the wake of a surface drive propeller of a boat with a planing hull design. The Surface Drive Enhancement Project (S.D.E.P. for short) is an angled set of adjustable platforms placed in the path of the rooster tail. Theoretically, it experiences the normal force of the water on its surface which, through conservation of momentum, distributes a force on the boat, with which the S.D.E.P. is attached, in both the upwards and forwards directions. This design will be tested and documented to see if it increases forward velocity without sacrificing handling characteristics.

Full protection against African horsesickness (AHS) in horses induced by baculovirus-derived AHS virus serotype 4 VP2, VP5 and VP7.

PubMed

Martínez-Torrecuadrada, J L; Díaz-Laviada, M; Roy, P; Sánchez, C; Vela, C; Sánchez-Vizcaíno, J M; Casal, J I

1996-06-01

African horsesickness virus serotype 4 (AHSV-4) outer capsid protein VP2, or VP2 and VP5 plus inner capsid protein VP7, derived from single or dual recombinant baculovirus expression vectors were used in different combinations to immunize horses. When the proteins were purified by affinity chromatography, the combination of all three proteins induced low levels of neutralizing antibodies and conferred protection against virulent virus challenge. However, purified VP2 or VP2 and VP5 in the absence of VP7 failed to induce neutralizing antibodies and protection. Immunization with non-purified proteins enhanced the titres of neutralizing antibodies. Again, the combination of the three proteins was able to confer total protection to immunized horses, which showed absence of viraemia. The antigenicity of recombinant VP2 was analysed with a collection of 30 MAbs. Both purified and unpurified recombinant VP2 proteins showed different antigenic patterns in comparison to that of VP2 on virions. An immunization experiment with four more horses confirmed these results. The vaccine described here would not only prevent the disease, but would drastically reduce the propagation of the virus by vectors.

Construction of C35 gene bait recombinants and T47D cell cDNA library.

PubMed

Yin, Kun; Xu, Chao; Zhao, Gui-Hua; Liu, Ye; Xiao, Ting; Zhu, Song; Yan, Ge

2017-11-20

C35 is a novel tumor biomarker associated with metastasis progression. To investigate the interaction factors of C35 in its high expressed breast cancer cell lines, we constructed bait recombinant plasmids of C35 gene and T47D cell cDNA library for yeast two-hybrid screening. Full length C35 sequences were subcloned using RT-PCR from cDNA template extracted from T47D cells. Based on functional domain analysis, the full-length C35 1-348bp was also truncated into two fragments C351-153bp and C35154-348bp to avoid auto-activation. The three kinds of C35 genes were successfully amplified and inserted into pGBKT7 to construct bait recombinant plasmids pGBKT7-C351-348bp, pGBKT7-C351-153bp and pGBKT7-C35154-348bp, then transformed into Y187 yeast cells by the lithium acetate method. Auto-activation and toxicity of C35 baits were detected using nutritional deficient medium and X-α-Gal assays. The T47D cell ds cDNA was generated by SMART TM technology and the library was constructed using in vivo recombination-mediated cloning in the AH109 yeast strain using a pGADT7-Rec plasmid. The transformed Y187/pGBKT7-C351-348bp line was intensively inhibited while the truncated Y187/pGBKT7-C35 lines had no auto-activation and toxicity in yeast cells. The titer of established cDNA library was 2 × 10 7 pfu/mL with high transformation efficiency of 1.4 × 10 6 , and the insert size of ds cDNA was distributed homogeneously between 0.5-2.0 kb. Our research generated a T47D cell cDNA library with high titer, and the constructed two C35 "baits" contained a respective functional immunoreceptor tyrosine based activation motif (ITAM) and the conserved last four amino acids Cys-Ile-Leu-Val (CILV) motif, and therefore laid a foundation for screening the C35 interaction factors in a BC cell line.

Increased brain radioactivity by intranasal 32P-labeled siRNA dendriplexes within in situ-forming mucoadhesive gels

PubMed Central

Perez, Ana Paula; Mundiña-Weilenmann, Cecilia; Romero, Eder Lilia; Morilla, Maria Jose

2012-01-01

Background Molecules taken up by olfactory and trigeminal nerve neurons directly access the brain by the nose-to-brain pathway. In situ-forming mucoadhesive gels would increase the residence time of intranasal material, favoring the nose-to-brain delivery. In this first approach, brain radioactivity after intranasal administration of 32P-small interference RNA (siRNA) complexed with poly(amidoamine) G7 dendrimers (siRNA dendriplexes) within in situ-forming mucoadhesive gels, was determined. Materials 32P-siRNA dendriplexes were incorporated into in situ-forming mucoadhesive gels prepared by blending thermosensitive poloxamer (23% w/w) with mucoadhesive chitosan (1% w/w, PxChi) or carbopol (0.25% w/w, PxBCP). Rheological properties, radiolabel release profile, and local toxicity in rat nasal mucosa were determined. The best-suited formulation was intranasally administered to rats, and blood absorption and brain distribution of radioactivity were measured. Results The gelation temperature of both formulations was 23°C. The PxChi liquid showed non-Newtonian pseudoplastic behavior of high consistency and difficult manipulation, and the gel retained 100% of radiolabel after 150 minutes. The PxCBP liquid showed a Newtonian behavior of low viscosity and easy manipulation, while in the gel phase showed apparent viscosity similar to that of the mucus but higher than that of aqueous solution. The gel released 35% of radiolabel and the released material showed silencing activity in vitro. Three intranasal doses of dendriplexes in PxCBP gel did not damage the rat nasal mucosa. A combination of 32P-siRNA complexation with dendrimers, incorporation of the dendriplexes into PxCBP gel, and administration of two intranasal doses was necessary to achieve higher brain radioactivity than that achieved by intravenous dendriplexes or intranasal naked siRNA. Conclusion The increased radioactivity within the olfactory bulb suggested that the combination above mentioned favored the

14 CFR 35.35 - Centrifugal load tests.

Code of Federal Regulations, 2010 CFR

2010-01-01

... STANDARDS: PROPELLERS Tests and Inspections § 35.35 Centrifugal load tests. The applicant must demonstrate that a propeller complies with paragraphs (a), (b) and (c) of this section without evidence of failure, malfunction, or permanent deformation that would result in a major or hazardous propeller effect. When the...

14 CFR 35.35 - Centrifugal load tests.

Code of Federal Regulations, 2012 CFR

2012-01-01

... STANDARDS: PROPELLERS Tests and Inspections § 35.35 Centrifugal load tests. The applicant must demonstrate that a propeller complies with paragraphs (a), (b) and (c) of this section without evidence of failure, malfunction, or permanent deformation that would result in a major or hazardous propeller effect. When the...

14 CFR 35.35 - Centrifugal load tests.

Code of Federal Regulations, 2011 CFR

2011-01-01

... STANDARDS: PROPELLERS Tests and Inspections § 35.35 Centrifugal load tests. The applicant must demonstrate that a propeller complies with paragraphs (a), (b) and (c) of this section without evidence of failure, malfunction, or permanent deformation that would result in a major or hazardous propeller effect. When the...

14 CFR 35.35 - Centrifugal load tests.

Code of Federal Regulations, 2014 CFR

2014-01-01

... STANDARDS: PROPELLERS Tests and Inspections § 35.35 Centrifugal load tests. The applicant must demonstrate that a propeller complies with paragraphs (a), (b) and (c) of this section without evidence of failure, malfunction, or permanent deformation that would result in a major or hazardous propeller effect. When the...

14 CFR 35.35 - Centrifugal load tests.

Code of Federal Regulations, 2013 CFR

2013-01-01

... STANDARDS: PROPELLERS Tests and Inspections § 35.35 Centrifugal load tests. The applicant must demonstrate that a propeller complies with paragraphs (a), (b) and (c) of this section without evidence of failure, malfunction, or permanent deformation that would result in a major or hazardous propeller effect. When the...

Surgery Increases Survival in Patients With Gastrinoma

PubMed Central

Norton, Jeffrey A.; Fraker, Douglas L.; Alexander, H R.; Gibril, Fathia; Liewehr, David J.; Venzon, David J.; Jensen, Robert T.

2006-01-01

Objective: To determine whether the routine use of surgical exploration for gastrinoma resection/cure in 160 patients with Zollinger-Ellison syndrome (ZES) altered survival compared with 35 ZES patients who did not undergo surgery. Summary Background Data: The role of routine surgical exploration for resection/cure in patients with ZES has been controversial since the original description of this disease in 1955. This controversy continues today, not only because medical therapy for acid hypersecretion is so effective, but also in large part because no studies have shown an effect of tumor resection on survival. Methods: Long-term follow-up of 160 ZES patients who underwent routine surgery for gastrinoma/resection/cure was compared with 35 patients who had similar disease but did not undergo surgery for a variety of reasons. All patients had preoperative CT, MRI, ultrasound; if unclear, angiography and somatostatin receptor scintigraphy since 1994 to determine resectability. At surgery, all had the same standard ZES operation. All patients were evaluated yearly with imaging studies and disease activity studies. Results: The 35 nonsurgical patients did not differ from the 160 operated in clinical, laboratory, or tumor imaging results. The 2 groups did not differ in follow-up time since initial evaluation (range, 11.8–12 years). At surgery, 94% had a tumor removed, 51% were cured immediately, and 41% at last follow-up. Significantly more unoperated patients developed liver metastases (29% vs. 5%, P = 0.0002), died of any cause (54 vs. 21%, P = 0.0002), or died a disease-related death (23 vs. 1%, P < 0.00001). Survival plots showed operated patients had a better disease-related survival (P = 0.0012); however, there was no difference in non-disease-related survival. Fifteen-year disease-related survival was 98% for operated and 74% for unoperated (P = 0.0002). Conclusions: These results demonstrate that routine surgical exploration increases survival in patients with

Shuttling imbalance of MLF1 results in p53 instability and increases susceptibility to oncogenic transformation.

PubMed

Yoneda-Kato, Noriko; Kato, Jun-Ya

2008-01-01

Myeloid leukemia factor 1 (MLF1) stabilizes the activity of the tumor suppressor p53 by suppressing its E3 ubiquitin ligase, COP1, through a third component of the COP9 signalosome (CSN3). However, little is known about how MLF1 functions upstream of the CSN3-COP1-p53 pathway and how its deregulation by the formation of the fusion protein nucleophosmin (NPM)-MLF1, generated by t(3;5)(q25.1;q34) chromosomal translocation, leads to leukemogenesis. Here we show that MLF1 is a cytoplasmic-nuclear-shuttling protein and that its nucleolar localization on fusing with NPM prevents the full induction of p53 by both genotoxic and oncogenic cellular stress. The majority of MLF1 was located in the cytoplasm, but the treatment of cells with leptomycin B rapidly induced a nuclear accumulation of MLF1. A mutation of the nuclear export signal (NES) motif identified in the MLF1 sequence enhanced the antiproliferative activity of MLF1. The fusion of MLF1 with NPM translocated MLF1 to the nucleolus and abolished the growth-suppressing activity. The introduction of NPM-MLF1 into early-passage murine embryonic fibroblasts allowed the cells to escape from cellular senescence at a markedly earlier stage and induced neoplastic transformation in collaboration with the oncogenic form of Ras. Interestingly, disruption of the MLF1-derived NES sequence completely abolished the growth-promoting activity of NPM-MLF1 in murine fibroblasts and hematopoietic cells. Thus, our results provide important evidence that the shuttling of MLF1 is critical for the regulation of cell proliferation and a disturbance in the shuttling balance increases the cell's susceptibility to oncogenic transformation.

Magnetic properties of x(Fe2O3).(100-x)[P2O5.Li2O] and x(Fe2O3).(100-x)[P2O5.CaO] glass systems

NASA Astrophysics Data System (ADS)

Andronache, Constantin; Racolta, Dania; Ardelean, Gheorghe

2017-12-01

Magnetic properties of x(Fe2O3).(100-x)[P2O5 .Li2O] and x(Fe2O3).(100-x)[P2O5 .CaO] with 0 < x ≤ 50 mol % were investigated using magnetic susceptibility measurements. The both glass systems were prepared in the same condition. The valence states and the distribution of iron ions in the glass matrix depend on the Fe2O3 content. For the P2O5.CaO glass matrix with x≤35mol%, the data revealed iron ions as isolated or participating in dipole-dipole interaction. For x > 35 mol% an antiferromagnetic coupling is observed. For the P2O5.Li2O glass matrix, the iron ions behave magnetically similarly as in other oxide glasses, but concentration of Fe2O3 over which magnetic superexchange interactions occur is lower. The absolute magnitude of θp values increases when content of Fe2O3 are increased. If the content of the magnetic ions is increased in the glass, the exchange integral increased and as a result the magnitude of the θP increases.

Goose parvovirus structural proteins expressed by recombinant baculoviruses self-assemble into virus-like particles with strong immunogenicity in goose.

PubMed