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https://www.readbyqxmd.com/read/28828655/analysis-of-extracellular-vesicles-using-fluorescence-nanoparticle-tracking-analysis
#1
Pauline Carnell-Morris, Dionne Tannetta, Agnieszka Siupa, Patrick Hole, Rebecca Dragovic
Fluorescence nanoparticle tracking analysis (fl-NTA) allows for accurate sizing, counting, and phenotyping of extracellular vesicles (EV). Here, we present two protocols for the analysis of EVs using fl-NTA, highlighting the potential pitfalls and challenges. The first protocol utilizes CellMask Orange™ (CMO) as a general membrane marker to label EVs derived from plasma. The second protocol describes the use of a Qdot-conjugated antibody to identify syncytiotrophoblast (STB)-derived EVs. "Standard" preparations of STB-derived EVs enriched for either microvesicles (STBMV) or exosomes (STBEX), containing a known amount of EV positive for the STB specific antigen placental alkaline phosphatase (PLAP), were also used to optimize fl-NTA camera settings...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28818618/ctenopharyngodon-idella-p53-mediates-between-nf-%C3%AE%C2%BAb-and-pkr-at-the-transcriptional-level
#2
Qingli Huang, Dingkun Xie, Huiling Mao, Haizhou Wang, Zhen Wu, Keyi Huang, Yiqi Wan, Qun Xu, Chengyu Hu
p53, NF-κB and PKR are well-known to be involved in antiviral response. Although p53 has been reported in fish, its role in the regulation of NF-κB and PKR is not well understood. Here, we cloned and characterized the full length of cDNA sequence of grass carp (Ctenopharyngodon idella) p53 (Cip53) and its promoter sequence. The full length cDNA of Cip53 was 1879 bp with an ORF of 1116 bp encoding a polypeptide of 371 amino acids. Phylogenetic tree analysis revealed that Cip53 shares high homology with Dario rerio p53 (Drp53)...
August 14, 2017: Fish & Shellfish Immunology
https://www.readbyqxmd.com/read/28818427/molecular-cloning-and-functional-characterization-of-nadph-dependent-cytochrome-p450-reductase-from-the-green-microalga-botryococcus-braunii-b-race
#3
Chung-Yau Tsou, Shigeki Matsunaga, Shigeru Okada
The green microalga Botryococcus braunii of the B race accumulates various lipophilic compounds containing a 10,11-oxidosqualene epoxide moiety in addition to large amounts of triterpene hydrocarbons. While 2,3-squalene epoxidases have already been isolated and characterized from the alga, the enzyme that catalyzes the 10,11-epoxidation of squalene has remained elusive. In order to obtain a molecular tool to explore a 10,11-squalene epoxidase, cDNA cloning of an NADPH-dependent cytochrome P450 reductase (CPR) that is required by both squalene epoxidases and cytochrome P450 enzymes was carried out...
August 14, 2017: Journal of Bioscience and Bioengineering
https://www.readbyqxmd.com/read/28811265/molecular-cloning-heterologous-expression-and-functional-characterization-of-gamma-tocopherol-methyl-transferase-%C3%AE-tmt-from-glycine-max
#4
Kalpana Tewari, Anil Dahuja, Archana Sachdev, Kishwar Ali, Vaibhav Kumar, Amresh Kumar, Sweta Kumari
γ-Tocopherol methyltransferase (γ-TMT) (EC 2.1.1.95) is the last enzyme in the tocopherol biosynthetic pathway and it catalyzes the conversion of γ-tocopherol into α-tocopherol, the nutritionally significant and most bioactive form of vitamin E. Although the γ-TMT gene has been successfully overexpressed in many crops to enhance their α-tocopherol content but still only few attempts have been made to uncover its structural, functional and regulation aspects at protein level. In this study, we have cloned the complete 909bp coding sequence of Glycine max γ-TMT (Gm γ-TMT) gene that encodes the corresponding protein comprising of 302 amino acid residues...
August 12, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28807436/simple-and-efficient-knockdown-of-his-tagged-proteins-by-ternary-molecules-consisting-of-a-his-tag-ligand-a-ubiquitin-ligase-ligand-and-a-cell-penetrating-peptide
#5
Takayuki Hattori, Koyo Okitsu, Norikazu Yamazaki, Nobumichi Ohoka, Norihito Shibata, Takashi Misawa, Masaaki Kurihara, Yosuke Demizu, Mikihiko Naito
We designed and synthesized hybrid molecules for a protein knockdown method based on the recognition of a His-tag fused to a protein of interest (POI). The synthesized target protein degradation inducers contained three functional moieties: a His-tag ligand (nickel nitrilotriacetic acid [Ni-NTA]), an E3 ligand (bestatin [BS] or MV1), and a carrier peptide (Tat or nonaarginine [R9]). The designed hybrid molecules, BS-Tat-Ni-NTA, MV1-Tat-Ni-NTA, BS-R9-Ni-NTA, and MV1-R9-Ni-NTA, efficiently degraded His-tagged cellular retinoic acid binding protein 2 via the ubiquitin-proteasome system (UPS)...
August 2, 2017: Bioorganic & Medicinal Chemistry Letters
https://www.readbyqxmd.com/read/28804597/size-and-concentration-analyses-of-extracellular-vesicles-by-nanoparticle-tracking-analysis-a-variation-study
#6
Beate Vestad, Alicia Llorente, Axl Neurauter, Santosh Phuyal, Bente Kierulf, Peter Kierulf, Tore Skotland, Kirsten Sandvig, Kari Bente F Haug, Reidun Øvstebø
Current methods for characterisation of extracellular vesicles (EVs) need further standardisation in order to obtain an acceptable level of data comparability. Size and concentration of EVs can be determined by nanoparticle tracking analysis (NTA). However, both the heterogeneity of EVs and the choice of instrument settings may cause an appreciable analytical variation. Intra-assay (within-day, n = 6) and inter-assay (day-to-day, n = 6) variations (coefficient of variation, % CV) of different preparations of EVs and artificial vesicles or beads were determined using two NanoSight NS500 instruments, located at different laboratories...
2017: Journal of Extracellular Vesicles
https://www.readbyqxmd.com/read/28801864/isolation-and-characterization-of-iron-chelators-from-turmeric-curcuma-longa-selective-metal-binding-by-curcuminoids
#7
Donald J Messner, Christine Surrago, Celia Fiordalisi, Wing Yin Chung, Kris V Kowdley
Iron overload disorders may be treated by chelation therapy. This study describes a novel method for isolating iron chelators from complex mixtures including plant extracts. We demonstrate the one-step isolation of curcuminoids from turmeric, the medicinal food spice derived from Curcuma longa. The method uses iron-nitrilotriacetic acid (NTA)-agarose, to which curcumin binds rapidly, specifically, and reversibly. Curcumin, demethoxycurcumin, and bisdemethoxycurcumin each bound iron-NTA-agarose with comparable affinities and a stoichiometry near 1...
August 11, 2017: Biometals: An International Journal on the Role of Metal Ions in Biology, Biochemistry, and Medicine
https://www.readbyqxmd.com/read/28794937/label-free-direct-electrical-detection-of-a-histidine-rich-protein-with-sub-femtomolar-sensitivity-using-an-organic-field-effect-transistor
#8
Tsukuru Minamiki, Yui Sasaki, Shizuo Tokito, Tsuyoshi Minami
There is a growing interest in achieving sensor systems to enable on-site testing of biomarkers. Herein, a new strategy for highly sensitive protein detection at sub-femtomolar levels without any labelling has been demonstrated by using an organic field-effect transistor (OFET). An artificial histidine-rich protein receptor (Ni(II)-nitrilotriacetic acid complex, Ni(II)-nta) functionalizes a detection portion (i.e. an extended-gate electrode) of the fabricated OFET device. The OFET responds electrically and selectively to a target analyte (bovine serum albumin), meaning that the binding processes at the Ni(II)-nta on the extended-gate electrode for the analyte affect the field-effect properties of the device...
August 2017: ChemistryOpen
https://www.readbyqxmd.com/read/28791349/ubiquitin-specific-peptidase-4-stabilizes-interferon-regulatory-factor-protein-and-promotes-its-function-to-facilitate-interleukin-4-expression-in-t-helper-type-2-cells
#9
Zhixiang Guo, Peng Xu, Shangqing Ge, Chengxin Zhang, Xiaoyan Zheng, Jinguo Xu, Zhuang Liu, Bin Li, Shenglin Ge
We speculated that ubiquitin specific peptidase 4 (USP4) may deubiquitinate interferon regulatory factor 4 (IRF4) and affect T helper type 2 (Th2) cell function. This study aimed to validate this hypothesis. Here, the interaction between USP4 and IRF4 were analyzed by co-immunoprecipitation assay. The deubiquitin effect of USP4 on IRF4 was analyzed by the Ni-NTA pull down assay. Luciferase reporter gene constructs were used to analyze the effects of USP4, IRF4 and nuclear factor of activated T cell-2 (NFATc2) on the activation of the interleukin-4 (IL-4) promoter...
August 3, 2017: International Journal of Molecular Medicine
https://www.readbyqxmd.com/read/28783894/expression-and-purification-of-transmembrane-protein-mere-from-mercury-resistant-bacillus-cereus
#10
Aatif Amin, Zakia Latif, Stanley J Opella
Mercury resistant (Hg(R)) bacteria were isolated from heavy metal polluted wastewater and soil collected near to tanneries of district Kasur, Pakistan. Bacterial isolates AZ-1, AZ-2 and AZ-3 showed resistance up to 40 μg/mL against mercuric chloride (HgCl2). 16S rDNA ribotyping and phylogenetic analysis were performed for the characterization of selected isolates as Bacillus sp. AZ-1 (KT270477), Bacillus cereus AZ-2 (KT270478) and Bacillus cereus AZ-3 (KT270479). Phylogenetic relationship on the basis of merA nucleotide sequence confirmed 51-100% homology with the corresponding region of the merA gene of already reported mercury resistant Gram positive bacteria...
August 8, 2017: Journal of Microbiology and Biotechnology
https://www.readbyqxmd.com/read/28778061/studies-on-the-interaction-of-polylactid-based-planar-and-nanoparticular-biomaterials-with-serum-albumin-and-fibrinogen
#11
Katharina Wulf, Ulrike Tschierschky, Thomas Eickner, Niels Grabow, Ralf G Mundkowski
Knowledge about the interactions of biomaterials with biological matrices is still poor however crucial to future development in terms of how to cope with immunomodulatory adverse effects and to control the integration of implants into tissue or the targeting of nano devices. The appropriate design of a material depends on reliable data about the influence of oligomer or polymer configuration and dispersity as well as surface properties including chemical modifications. This work addressed the interaction of four polylactide (PLA) resomers, a well-established, biodegradable biomaterial, and two physiologically relevant proteins, which are ubiquitous in human serum - albumin (HSA) and fibrinogen (HFG)...
July 8, 2017: Colloids and Surfaces. B, Biointerfaces
https://www.readbyqxmd.com/read/28762261/self-templating-construction-of-hollow-amorphous-comos4-nanotube-array-toward-efficient-hydrogen-evolution-electrocatalysis-at-neutral-ph
#12
Xuping Sun
Environmentally friendly electrochemical hydrogen production needs the development of earth-abundant catalyst materials for the hydrogen evolution reaction with high activity and durability at neutral pH. In this communication, we describe the self-templating construction of hollow amorphous CoMoS4 nanotube array on carbon cloth (CoMoS4 NTA/CC) via hydrothermal treating of Co(OH)F nanowire array on CC in (NH4)2MoS4 solution. When used as a 3D electrode for hydrogen evolution electrocatalysis, the resulting CoMoS4 NTA/CC demonstrates superior catalytic activity and strong long-term electrochemical durability in 1...
July 31, 2017: Chemistry: a European Journal
https://www.readbyqxmd.com/read/28751805/astaxanthin-ameliorates-ferric-nitrilotriacetate-induced-renal-oxidative-injury-in-rats
#13
Yasumasa Okazaki, Shigeru Okada, Shinya Toyokuni
Daily intake of vegetables can reduce the risk of cancer and lifestyle-related diseases. However, supplementary intake of β-carotene alone has been reported to increase the risk of lung cancer in male cigarette smokers and people who were exposed to asbestos. The mechanism of the antioxidative properties of carotenoids in vivo, especially under oxidative stress conditions, still remains unclear. To investigate the antioxidant properties of dietary compounds, we examined the effects of chemically modified astaxanthin (Ax-C-8) using a rat model of ferric nitrilotriacetate (Fe-NTA)-induced renal oxidative injury...
July 2017: Journal of Clinical Biochemistry and Nutrition
https://www.readbyqxmd.com/read/28750864/identification-of-a-new-diagnostic-antigen-for-glanders-using-immunoproteome-analysis
#14
Sudhir K Dohre, Aayushi Kamthan, Sandeep Singh, Syed Imteyaz Alam, Subodh Kumar
Glanders is a disease of horses, donkeys and mules. The causative agent Burkholderia mallei, is a biorisk group 3 pathogen and is also a biothreat agent. Simple and rapid diagnostic tool is essential for control of glanders. Using a proteomic approach and immunoblotting with equine sera, we identified 12 protein antigens that may have diagnostic potential. Various immunoreactive proteins e.g. GroEL, translation elongation factor Tu, elongation factor Ts, arginine deiminase, malate dehydrogenase, DNA directed RNA polymerase subunit alpha were identified on 2-dimentional immunoblots...
August 2017: Comparative Immunology, Microbiology and Infectious Diseases
https://www.readbyqxmd.com/read/28749328/desulfovibrio-da2_cueo-is-a-novel-multicopper-oxidase-with-cuprous-ferrous-and-phenol-oxidase-activity
#15
Stefano Mancini, Ranjeet Kumar, Veena Mishra, Marc Solioz
Desulfovibrio sp. A2 is a novel Gram-negative sulfate-reducing bacterium that was isolated from sediments of the Norilsk mining/smelting area in Russia. The organism possesses a monocistronic operon encoding a 71 kDa periplasmic multicopperoxidase, which we call DA2_CueO. Histidine-tagged DA2_CueO expressed from a plasmid in Escherichia coli and purified by Ni-NTA affinity chromatography oxidizes Cu+ and Fe2+, and exhibits phenol oxidase activity with 2,2-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid), 2,3-dihydroxybenzoic acid and 2,6-dimethoxyphenol as substrates, using O2 as the oxidant...
July 28, 2017: Microbiology
https://www.readbyqxmd.com/read/28748350/expression-of-adenylate-kinase-fused-mek1r4f-in-escherichia-coli-and-its-application-in-erk-phosphorylation
#16
Fang Yang, Hongtao Li
OBJECTIVE: To construct a highly expressed and active MEK1R4F (a constitutively active form of mitogen-activated protein kinase kinase 1) by fusion of soluble adenylate kinase (Adk) tag, resulting in Adk-MEK1R4F protein suitable for preparation of phosphorylated ERK. RESULTS: We fused the Adk to the N-terminus of MEK1R4F through overlapping PCR. The expression of Adk-MEK1R4F fusion protein increased ~10-fold in Escherichia coli, and was purified to 95% via two purification steps including Ni-NTA and Q Sepharose fast flow (QFF) chromatography...
July 26, 2017: Biotechnology Letters
https://www.readbyqxmd.com/read/28745869/high-resolution-imaging-and-multiparametric-characterization-of-native-membranes-by-combining-confocal-microscopy-and-an-atomic-force-microscopy-based-toolbox
#17
Pawel R Laskowski, Moritz Pfreundschuh, Mirko Stauffer, Zöhre Ucurum, Dimitrios Fotiadis, Daniel J Müller
To understand how membrane proteins function requires characterizing their structure, assembly, and inter- and intramolecular interactions in physiologically relevant conditions. Conventionally, such multiparametric insight is revealed by applying different biophysical methods. Here we introduce the combination of confocal microscopy, force-distance curve-based (FD-based) atomic force microscopy (AFM), and single-molecule force spectroscopy (SMFS) for the identification of native membranes and the subsequent multiparametric analysis of their membrane proteins...
August 22, 2017: ACS Nano
https://www.readbyqxmd.com/read/28736549/cloning-expression-isotope-labeling-and-purification-of-transmembrane-protein-merf-from-mercury-resistant-enterobacter-sp-az-15-for-nmr-studies
#18
Aatif Amin, Zakia Latif
Mercury resistant (Hg(R)) Enterobacter sp. AZ-15 was isolated from heavy metal polluted industrial wastewater samples near to districts Kasur and Sheikhupura, Pakistan. 16S rDNA ribotyping and phylogentic analysis showed 98% homology with already reported Enterobacter species. The merF gene encoding transmembrane protein-MerF was amplified from genomic DNA and ligated into pET31b+ vector using restriction endonucleases, SphI and XhoI. The genetic codons of merF gene encoding cysteine residues were mutated into codons, translating into serine residues by site-directed mutagenesis...
2017: Frontiers in Microbiology
https://www.readbyqxmd.com/read/28735080/recombinant-truncated-e-protein-as-a-new-vaccine-candidate-against-nontypeable-h-%C3%A2-influenzae-its-expression-and-immunogenic-evaluation
#19
Ava Behrouzi, Saeid Bouzari, Farzam Vaziri, Abolfazl Fateh, Parviz Afrough, Atefeh Davoudi Vijeh Motlagh, Seyed Davar Siadat
Protein E (PE) is a conserved entity observed in both nontypeable Haemophilus influenzae (NTHi) and encapsulated H. influenzae. This is a small surface lipoprotein, consisting of only 160 amino acids, involved in the adhesion of H. influenzae to various types of epithelial cells. A 384-bp-long fragment from NTHi PE was cloned into the prokaryotic expression vector pBAD-gIIIA. The recombinant protein was expressed with arabinose and then purified by affinity purification on an Ni-NTA agarose matrix. BALB/c mice were immunized by subcutaneous injection with purified recombinant truncated PE mixed with an alum adjuvant...
July 19, 2017: Microbial Pathogenesis
https://www.readbyqxmd.com/read/28734839/recombinant-expression-of-ixolaris-a-kunitz-type-inhibitor-from-the-tick-salivary-gland-for-nmr-studies
#20
V S De Paula, F H S Silva, I M B Francischetti, R Q Monteiro, A P Valente
Ixolaris is an anticoagulant protein identified in the tick saliva of Ixodes scapularis. Ixolaris contains 2 Kunitz like domains and binds to Factor Xa or Factor X as a scaffold for inhibition of the Tissue Factor (TF)/Factor VIIa (FVIIa). In contrast to tissue factor pathway inhibitor (TFPI), however, Ixolaris does not bind to the active site cleft of FXa. Instead, complex formation is mediated by the FXa heparin-binding exosite. Due to its potent and long-lasting antithrombotic activity, Ixolaris is a promising agent for anticoagulant therapy...
July 19, 2017: Protein Expression and Purification
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