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https://www.readbyqxmd.com/read/28109905/cloning-genetic-engineering-and-characterization-of-tmof-expressed-in-saccharomyces-cerevisiae-to-control-larval-mosquitoes
#1
Borovsky Dov, Sabine Nauewelaers, Charles A Powell, Robert G Shatters
Trypsin modulating oostatic factor, a decapaptide isolated from the ovaries of A. aegypti, is the physiological factor that terminates the trypsin biosynthesis after the blood meal. Earlier results obtained from feeding mosquito larvae and injecting female mosquitoes with TMOF show that trypsin biosynthesis and egg development are inhibited, indicating that TMOF traverses the gut epithelial cells and modulates trypsin biosynthesis, making it a potential larvacidal peptide hormone. Therefore, TMOF and TMOF green fluorescent protein (GFP) fusion protein with a trypsin cleavage site, allowing TMOF release in the larval gut, were expressed in S...
January 18, 2017: Journal of Insect Physiology
https://www.readbyqxmd.com/read/28109886/truncated-prion-protein-prp226-a-structural-view-on-its-role-in-amyloid-disease
#2
Valerija Kovač, Blaž Zupančič, Gregor Ilc, Janez Plavec, Vladka Čurin Šerbec
In the brain of patients with transmissible spongiform encephalopathies, besides PrP(Sc) aggregates, deposition of truncated PrP molecules was described. Jansen et al. reported two clinical cases with deposition of C-terminally truncated PrP, one of them ending with Tyr226. We have previously described the discovery of monoclonal antibody V5B2 that selectively recognizes this version of the prion protein, which we called PrP226*. Using monoclonal antibody V5B2 we showed that accumulation of PrP226* is characteristic for most types of human and animal TSEs...
January 18, 2017: Biochemical and Biophysical Research Communications
https://www.readbyqxmd.com/read/28109682/label-free-checkerboard-assay-to-determine-overlapping-epitopes-of-ebola-virus-vp-40-antibodies-using-surface-plasmon-resonance
#3
George P Anderson, Jinny L Liu, Dan Zabetakis, Patricia M Legler, Ellen R Goldman
Immunoassay formats, in which antibodies provide sensitivity and specificity, are often utilized to provide rapid and simple diagnostic tests. Surface plasmon resonance is frequently used to evaluate the suitability of antibodies by determining binding kinetics to agents or surrogate antigens. We used SPR to evaluate a number of commercial monoclonal antibodies as well as single domain antibodies produced in-house. All the antibodies targeted the Ebola virus viral protein 40 (VP40). We determined the ability of each antibody to bind to immobilized VP40, and ensured they did not bind Ebola glycoprotein or the nucleoprotein...
January 18, 2017: Journal of Immunological Methods
https://www.readbyqxmd.com/read/28109583/detection-of-recombinant-human-lactoferrin-and-lysozyme-produced-in-a-bitransgenic-cow
#4
Germán G Kaiser, Nicolás C Mucci, Vega González, Lourdes Sánchez, José A Parrón, María D Pérez, Miguel Calvo, Juan F Aller, Federico A Hozbor, Adrián A Mutto
Lactoferrin and lysozyme are 2 glycoproteins with great antimicrobial activity, being part of the nonspecific defensive system of human milk, though their use in commercial products is difficult because human milk is a limited source. Therefore, many investigations have been carried out to produce those proteins in biological systems, such as bacteria, yeasts, or plants. Mammals seem to be more suitable as expression systems for human proteins, however, especially for those that are glycosylated. In the present study, a bicistronic commercial vector containing a goat β-casein promoter and an internal ribosome entry site fragment between the human lactoferrin and human lysozyme genes was developed to allow the introduction of both genes into bovine adult fibroblasts in a single transfection...
January 18, 2017: Journal of Dairy Science
https://www.readbyqxmd.com/read/28109434/application-of-a-thioredoxin-trapping-mutant-for-analysis-of-the-cellular-nitrosoproteome
#5
M Benhar
Nitric oxide influences a wide range of cellular functions through S-nitrosylation, a redox-dependent posttranslational protein modification that involves attachment of a nitroso moiety to a reactive thiol group. Over the past two decades, S-nitrosylation has emerged as a ubiquitous mechanism for controlling the activity, subcellular localization, and molecular interactions of proteins, thereby influencing many cellular processes. In addition, recent studies have indicated that aberrant S-nitrosylation may lead to cellular dysfunction and damage...
2017: Methods in Enzymology
https://www.readbyqxmd.com/read/28109425/beyond-the-natural-proteome-nondegenerate-saturation-mutagenesis-methodologies-and-advantages
#6
M M Ferreira Amaral, L Frigotto, A V Hine
Beyond the natural proteome, high-throughput mutagenesis offers the protein engineer an opportunity to "tweak" the wild-type activity of a protein to create a recombinant protein with required attributes. Of the various approaches available, saturation mutagenesis is one of the core techniques employed by protein engineers, and in recent times, nondegenerate saturation mutagenesis is emerging as the approach of choice. This review compares the current methodologies available for conducting nondegenerate saturation mutagenesis with traditional, degenerate saturation and briefly outlines the options available for screening the resulting libraries, to discover a novel protein with the required activity and/or specificity...
2017: Methods in Enzymology
https://www.readbyqxmd.com/read/28109330/the-structure-of-mammalian-prions-and-their-aggregates
#7
E Vázquez-Fernández, H S Young, J R Requena, H Wille
Prion diseases, such as Creutzfeldt-Jakob disease in humans, bovine spongiform encephalopathy in cattle, chronic wasting disease in cervids (i.e., deer, elk, moose, and reindeer), and sheep scrapie, are caused by the misfolding of the cellular prion protein (PrP(C)) into a disease-causing conformer (PrP(Sc)). PrP(C) is a normal, GPI-anchored protein that is expressed on the surface of neurons and other cell types. The structure of PrP(C) is well understood, based on studies of recombinant PrP, which closely mimics the structure of native PrP(C)...
2017: International Review of Cell and Molecular Biology
https://www.readbyqxmd.com/read/28109155/-preparation-and-application-of-rabbit-anti-mouse-setd8-polyclonal-antibody
#8
Haitao Ma, Jiaqian Guo, Jing Xia, Changmin Niu, Xueyi Shen, Hongya Sun, Ying Zheng
Objective To purify the recombinant Setd8 protein and prepare rabbit anti-mouse Setd8 polyclonal antibody. Methods The recombinant plasmid pET-30a-Setd8 was constructed by double enzyme digestion and linkage, and then transformed into E.coli BL21. The expression of the target protein was induced by IPTG and the expression product was purified by Ni-NTA affinity chromatograph. The purified protein was used to immunize New Zealand white rabbits to produce polyclonal antibody. The titer and specificity of the antibody were identified by ELISA, Western blotting and immunohistochemistry...
February 2017: Xi Bao Yu Fen Zi Mian Yi Xue za Zhi, Chinese Journal of Cellular and Molecular Immunology
https://www.readbyqxmd.com/read/28109146/-cloning-expression-and-activity-identification-of-human-innate-immune-protein-apolipoprotein-b-mrna-editing-enzyme-catalytic-subunit-3a-apobec3a
#9
Shan Cheng, Liyan Cao, Juan Du, Wenyong Wang, Yanhai Guo
Objective To construct the expression vector of apolipoprotein B mRNA editing enzyme catalytic subunit 3A (APOBEC3A), express APOBEC3A in eukaryotic cells and identify its cytosine deaminase activity. Methods The APOBEC3A gene was obtained by PCR and inserted into the eukaryotic expression vector pcDNA3.0(+). The recombinant vector pcDNA3.0-APOBEC3A was then transfected into HEK293T and HepG2 cells after confirmed by DNA sequencing. The recombinant protein was purified by Ni-NTA HisBind affinity column. Western blot analysis was used to detect the expression of APOBEC3A protein...
February 2017: Xi Bao Yu Fen Zi Mian Yi Xue za Zhi, Chinese Journal of Cellular and Molecular Immunology
https://www.readbyqxmd.com/read/28109042/expression-and-characterization-of-a-codon-optimized-blood-coagulation-factor-viii
#10
S A Shestopal, J-J Hao, E Karnaukhova, Y Liang, M V Ovanesov, M Lin, J H Kurasawa, T K Lee, J H McVey, A G Sarafanov
BACKGROUND: Production of recombinant factor VIII (FVIII) is challenging due to its low expression. It was previously shown that codon-optimization of a B domain-deleted FVIII (BDD-FVIII) cDNA resulted in increased protein expression. However, it is well-recognized that synonymous mutations may affect the protein structure and function. OBJECTIVES: To compare biochemical properties of a BDD-FVIII expressed from codon-optimized (CO) and the wild-type (WT) cDNAs. METHODS: Each variant of the BDD-FVIII was expressed in several independent CHO cell lines, generated using a lentiviral platform...
January 21, 2017: Journal of Thrombosis and Haemostasis: JTH
https://www.readbyqxmd.com/read/28109028/breadth-of-t-cell-responses-after-immunization-with-adenovirus-vectors-encoding-ancestral-antigens-or-polyvalent-papillomavirus-antigens
#11
Emeline Ragonnaud, Anders Gorm Pedersen, Peter Johannes Holst
Oncogenic human papillomaviruses (HPVs) are in most cases eliminated by intervention of T cells. As many other pathogens, these oncogenic HPVs belong to an ancient and diverse virus family. Therefore, we found it relevant to investigate the potential and limitations of inducing a broad response - either by inducing cross-reactive T cells or by administering a polyvalent vaccine. To test these strategies, we designed 3 ancestral and 2 circulating sequences based on the two domains of the E1 and E2 proteins of papillomaviruses (PVs) that exhibit the highest degree of conservation in comparison to the other PV proteins...
January 21, 2017: Scandinavian Journal of Immunology
https://www.readbyqxmd.com/read/28108731/loss-of-endothelial-cell-specific-molecule-1-promotes-the-tumorigenicity-and-metastasis-of-prostate-cancer-cells-through-regulation-of-the-timp-1-mmp-9-expression
#12
Chien-Min Chen, Chu-Liang Lin, Hui-Ling Chiou, Shu-Ching Hsieh, Chia-Liang Lin, Chun-Wen Cheng, Chia-Hung Hung, Jen-Pi Tsai, Yi-Hsien Hsieh
The Endothelial cell specific molecule-1 (ESM1) protein has been involved in proliferation and metastatic progression in multiple tumors. However, there are no studies regarding the mechanism of ESM1 in prostate cancer. We found that ESM1 knockdown in prostate cancer cells resulted in increased cell proliferation and colony formation ability response evidenced by decreased expression of p21 and increased expression of cyclin D1 in prostate cancer cells. Moreover, we revealed that knockdown ESM1 also induced the epithelial-mesenchymal transition (EMT), motility and invasiveness in accordance with the upregulated the MMP-9 expression, while downregulated the TIMP-1 expression...
January 17, 2017: Oncotarget
https://www.readbyqxmd.com/read/28108661/a-structure-function-analysis-of-the-yeast-elg1-protein-reveals-the-importance-of-pcna-unloading-in-genome-stability-maintenance
#13
Keren Shemesh, Marek Sebesta, Martin Pacesa, Soumitra Sau, Alex Bronstein, Oren Parnas, Batia Liefshitz, Česlovas Venclovas, Lumir Krejci, Martin Kupiec
The sliding clamp, PCNA, plays a central role in DNA replication and repair. In the moving replication fork, PCNA is present at the leading strand and at each of the Okazaki fragments that are formed on the lagging strand. PCNA enhances the processivity of the replicative polymerases and provides a landing platform for other proteins and enzymes. The loading of the clamp onto DNA is performed by the Replication Factor C (RFC) complex, whereas its unloading can be carried out by an RFC-like complex containing Elg1...
January 20, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/28108607/insulin-like-growth-factor-binding-protein-1-could-improve-glucose-regulation-and-insulin-sensitivity-through-its-rgd-domain
#14
Natalie J Haywood, Paul A Cordell, Kar Yeun Tang, Natallia Makova, Nadira Y Yuldasheva, Helen Imrie, Hema Viswambharan, Alexander F Bruns, Richard M Cubbon, Mark T Kearney, Stephen B Wheatcroft
Low circulating levels of insulin-like growth factor binding protein 1 (IGFBP-1) are associated with insulin resistance and predict the development of type 2 diabetes. IGFBP-1 can affect cellular functions independently of IGF binding through an Arg-Gly-Asp (RGD) integrin-binding motif. Whether causal mechanisms underlie the favorable association of high IGFBP-1 levels with insulin sensitivity and whether these could be exploited therapeutically remain unexplored. We used recombinant IGFBP-1 and a synthetic RGD-containing hexapeptide in complementary in vitro signaling assays and in vivo metabolic profiling in obese mice to investigate the effects of IGFBP-1 and its RGD domain on insulin sensitivity, insulin secretion, and whole-body glucose regulation...
February 2017: Diabetes
https://www.readbyqxmd.com/read/28108587/meiotic-crossing-over-in-maize-knob-heterochromatin
#15
Stephen M Stack, Lindsay A Shearer, Leslie Lohmiller, Lorinda K Anderson
There is ample evidence that crossing over is suppressed in heterochromatin associated with centromeres and nucleolus organizers (NORs). This characteristic has been attributed to all heterochromatin, but the generalization may not be justified. To investigate the relation of crossing over to heterochromatin that is not associated with centromeres or NORs, we used a combination of fluorescence in situ hybridization of the maize 180 bp knob repeat to show the locations of knob heterochromatin and fluorescent immunolocalization of MLH1 protein and AFD1 protein to show the locations of MLH1 foci on maize synaptonemal complexes (SCs ~ pachytene chromosomes)...
January 20, 2017: Genetics
https://www.readbyqxmd.com/read/28108401/constitutive-expression-and-characterization-of-a-surface-srs-ncsrs67-protein-of-neospora-caninum-with-no-orthologue-in-toxoplasma-gondii
#16
Marcos Alexandre Bezerra, Luiz Miguel Pereira, Aline Bononi, Carla Agostino Biella, Luciana Baroni, Leticia Pollo-Oliveira, Ana Patrícia Yatsuda
Neospora caninum is a parasite of the Apicomplexa phylum responsible for abortion and losses of fertility in cattle. As part of its intracellular cycle, the first interaction of the parasite with the target cell is performed with the surface proteins known as the SRS superfamily (Surface Antigen Glycoprotein - Related Sequences). SAG related or SRS proteins have been a target of intense research due to its immunodominant pattern, exhibiting potential as diagnostic and/or vaccine candidates. The aim of this study was the cloning, expression and characterization of the gene NcSRS67 of N...
January 17, 2017: Parasitology International
https://www.readbyqxmd.com/read/28108341/molecular-cloning-genomic-structure-polymorphism-analysis-and-recombinant-expression-of-a-%C3%AE-1-antitrypsin-like-gene-from-swamp-eel-monopterus-albus
#17
Wei Li, Quanhe Wang, Shaobin Li, Ao Jiang, Wenxiu Sun
Alpha-1-antitrypsin (AAT) is a highly polymorphic glycoprotein antiprotease, involved in the regulation of human immune response. Beyond some genomic characterization and a few protein characterizations, the function of teleost AAT remains uncertain. In this study we cloned an AAT-like gene from a swamp eel liver identifying four exons and three introns, and the full-length cDNA. The elucidated swamp eel AAT amino acid sequence showed high homology with known AATs from other teleosts. The swamp eel AAT was examined both in ten healthy tissues and in four bacterially-stimulated tissues resulting in up-regulation of swamp eel AAT at different times...
January 17, 2017: Fish & Shellfish Immunology
https://www.readbyqxmd.com/read/28108183/evaluation-of-multiplex-assay-platforms-for-detection-of-influenza-hemagglutinin-subtype-specific-antibody-responses-influenza-ha-subtype-specific-antibody-detection-platform
#18
Zhu-Nan Li, Kimberly M Weber, Rebecca A Limmer, Bobbi J Horne, James Stevens, Joy Schwerzmann, Jens Wrammert, Megan McCausland, Andrew J Phipps, Kathy Hancock, Daniel B Jernigan, Min Levine, Jacqueline M Katz, Joseph D Miller
Influenza hemagglutination inhibition (HI) and virus microneutralization assays (MN) are widely used for seroprevalence studies. However, these assays have limited field portability and are difficult to fully automate for high throughput laboratory testing. To address these issues, three multiplex influenza subtype-specific antibody detection assays were developed using recombinant hemagglutinin antigens in combination with Chembio, Luminex(®), and ForteBio(®) platforms. Assay sensitivity, specificity, and subtype cross-reactivity were evaluated using a panel of well characterized human sera...
January 17, 2017: Journal of Virological Methods
https://www.readbyqxmd.com/read/28107808/in-situ-release-of-vegf-enhances-osteogenesis-in-3d-porous-scaffolds-engineered-with-osterix-modified-adipose-derived-stem-cells
#19
Wan-Lin Xu, Hui-Shan Ong, Yun Zhu, Sheng-Wen Liu, Li-Min Liu, Kai-Hua Zhou, Zeng-Qi Xu, Jun Gao, Yan Zhang, Jin-Hai Ye, Wen-Jun Yang
Adipose-derived stem cells (ADSCs) can differentiate into various cell types and thus have great potential for regenerative medicine. Herein, rat ADSCs were isolated; transduced with lentiviruses expressing Osterix (Osx), a transcriptional factor essential for osteogenesis. Osx overexpression up-regulated key osteogenesis-related genes, such as special AT-rich binding protein 2, alkaline phosphatase, osteocalcin and osteopontin, at both the mRNA and protein levels. In addition, mineral nodule formation and alkaline phosphatase activity were enhanced in Osx-overexpressing ADSCs...
January 20, 2017: Tissue Engineering. Part A
https://www.readbyqxmd.com/read/28107434/anti-mrka-monoclonal-antibodies-reveal-distinct-structural-and-antigenic-features-of-mrka
#20
Qun Wang, Yan Chen, Romana Cvitkovic, Meghan E Pennini, Chew Shun Chang, Mark Pelletier, Jessica Bonnell, Adem C Koksal, Herren Wu, William F Dall'Acqua, C Kendall Stover, Xiaodong Xiao
Antibody therapy against antibiotics resistant Klebsiella pneumoniae infections represents a promising strategy, the success of which depends critically on the ability to identify appropriate antibody targets. Using a target-agnostic strategy, we recently discovered MrkA as a potential antibody target and vaccine antigen. Interestingly, the anti-MrkA monoclonal antibodies isolated through phage display and hybridoma platforms all recognize an overlapping epitope, which opens up important questions including whether monoclonal antibodies targeting different MrkA epitopes can be generated and if they possess different protective profiles...
2017: PloS One
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