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https://www.readbyqxmd.com/read/29777695/proteasome-mediated-protein-degradation-is-enhanced-by-fusion-ubiquitin-with-unstructured-degron
#1
Tomonao Inobe, Masayuki Tsukamoto, Miyuki Nozaki
Methods to induce proteasomal degradation of unwanted proteins are valuable in biomedical studies and thus receive increasing attention. For efficient degradation, the proteasome requires both a ubiquitin tag, which delivers substrates to the proteasome, and an unstructured region, where the proteasome engages the substrate for unfolding and degradation. We fused two degron components into a single molecule to create a fusion protein comprising ubiquitin and Rpn4-derived unstructured region. We demonstrated that the fusion protein retained its function to polyubiquitinate target proteins, thereby inducing more efficient proteasomal target degradation than wild-type ubiquitin in vitro and in cells...
May 16, 2018: Biochemical and Biophysical Research Communications
https://www.readbyqxmd.com/read/29775788/complete-genome-sequence-of-achromobacter-spanius-type-strain-dsm-23806t-a-pathogen-isolated-from-human-blood
#2
Gengmi Li, Li Yang, Tao Zhang, Xiaojiao Guo, Jian Qin, Yingjiang Cao, Qianhua Yang, Shumei You, Guoliang Yuan, Xianqi Wan, Jing Luo, Zhaoxiang Li, Lei Gao, Ying Liu, Kaifeng Jiang, Jiakui Zheng
OBJECTIVES: Achromobacter spanius is a new described, non-fermenting Gram-negative and coccoid pathogen that isolated from patient blood. In order to investigate the pathogenesis of this strain in genomic level, whole genome sequencing was carried out on this strain. METHODS: In this study, complete genome of the type strain of this species DSM 23806T was sequenced using single-molecule real-time (SMRT) DNA sequencing. RESULTS: Complete genome of DSM 23806T consists of one circular DNA chromosome of 6,425,783bp with a GC content of 64...
May 15, 2018: Journal of Global Antimicrobial Resistance
https://www.readbyqxmd.com/read/29775732/il4-10-fusion-protein-has-chondroprotective-anti-inflammatory-and-potentially-analgesic-effects-in-the-treatment-of-osteoarthritis
#3
C Steen-Louws, J Popov-Celeketic, S C Mastbergen, K Coeleveld, C E Hack, N Eijkelkamp, M Tryfonidou, S Spruijt, J A G van Roon, F P J G Lafeber
OBJECTIVE: Effective disease-modifying drugs for osteoarthritis (DMOAD) should preferably have chondroprotective, anti-inflammatory, and analgesic activity combined in a single molecule. We developed a fusion protein of IL4 and IL10 (IL4-10 FP), in which the biological activity of both cytokines is preserved. The present study evaluates the chondroprotective, anti-inflammatory, and analgesic activity of IL4-10 FP in in vitro and in vivo models of osteoarthritis. METHODS: Human osteoarthritic cartilage tissue and synovial tissue were cultured with IL4-10 FP...
May 15, 2018: Osteoarthritis and Cartilage
https://www.readbyqxmd.com/read/29775584/conformational-activation-of-argonaute-by-distinct-yet-coordinated-actions-of-the-hsp70-and-hsp90-chaperone-systems
#4
Kotaro Tsuboyama, Hisashi Tadakuma, Yukihide Tomari
Loading of small RNAs into Argonaute, the core protein in RNA silencing, requires the Hsp70/Hsp90 chaperone machinery. This machinery also activates many other clients, including steroid hormone receptors and kinases, but how their structures change during chaperone-dependent activation remains unclear. Here, we utilized single-molecule Förster resonance energy transfer (smFRET) to probe the conformational changes of Drosophila Ago2 mediated by the chaperone machinery. We found that empty Ago2 exists in various closed conformations...
May 17, 2018: Molecular Cell
https://www.readbyqxmd.com/read/29775583/correlating-transcription-initiation-and-conformational-changes-by-a-single-subunit-rna-polymerase-with-near-base-pair-resolution
#5
Hye Ran Koh, Rahul Roy, Maria Sorokina, Guo-Qing Tang, Divya Nandakumar, Smita S Patel, Taekjip Ha
We provide a comprehensive analysis of transcription in real time by T7 RNA Polymerase (RNAP) using single-molecule fluorescence resonance energy transfer by monitoring the entire life history of transcription initiation, including stepwise RNA synthesis with near base-pair resolution, abortive cycling, and transition into elongation. Kinetically branching pathways were observed for abortive initiation with an RNAP either recycling on the same promoter or exchanging with another RNAP from solution. We detected fast and slow populations of RNAP in their transition into elongation, consistent with the efficient and delayed promoter release, respectively, observed in ensemble studies...
May 17, 2018: Molecular Cell
https://www.readbyqxmd.com/read/29775570/rapid-non-classical-effects-of-steroids-on-the-membrane-receptor-dynamics-and-downstream-signaling-in-neurons
#6
REVIEW
Klaudia Barabás, Soma Godó, Ferenc Lengyel, Dávid Ernszt, József Pál, István M Ábrahám
Although rapid effects of steroid hormones on membrane receptors and intracellular signaling molecules have been extensively studied in neurons, we are only beginning to understand the molecular mechanisms behind these non-classical steroid actions. Single molecule tracking (SMT) studies on live cells demonstrated that surface trafficking of membrane receptors determines their ligand binding properties and downstream signaling events. Recent findings suggest that one of the underlying mechanisms of non-classical steroid actions is the alteration of receptor movements on the membrane surface...
May 15, 2018: Hormones and Behavior
https://www.readbyqxmd.com/read/29775295/noncovalent-grafting-of-a-dy-iii-2-single-molecule-magnet-onto-chemically-modified-multiwalled-carbon-nanotubes
#7
Vassilis Tangoulis, Nikolia Lalioti, John Parthenios, Nikos Boukos, Ondřej Malina, Jiří Tuček, Radek Zbořil
While synthetic methods for the grafting of nanoparticles or photoactive molecules onto carbon nanotubes (CNTs) have been developed in the last years, a very limited number of reports have appeared on the grafting of single-molecule magnets (SMMs) onto CNTs. There are many potential causes, mainly focused on the fact that the attachment of molecules on surfaces remains not trivial and their magnetic properties are significantly affected upon attachment. Nevertheless, implementation of this particular type of hybrid material in demanding fields such as spintronic devices makes of utmost importance the investigation of new synthetic protocols for effective grafting...
May 18, 2018: Inorganic Chemistry
https://www.readbyqxmd.com/read/29775043/designing-a-dy-2-single-molecule-magnet-with-two-well-differentiated-relaxation-processes-by-using-a-nonsymmetric-bis-bidentate-bipyrimidine-n-oxide-ligand-a-comparison-with-mononuclear-counterparts
#8
Ismael F Díaz-Ortega, Juan Manuel Herrera, Daniel Aravena, Eliseo Ruiz, Tulika Gupta, Gopalan Rajaraman, H Nojiri, Enrique Colacio
Herein we report a dinuclear [(μ-mbpymNO){(tmh)3 Dy}2 ] (1) single-molecule magnet (SMM) showing two nonequivalent DyIII centers, which was rationally prepared from the reaction of Dy(tmh)3 moieties (tmh = 2,2,6,6-tetramethyl-3,5-heptanedionate) and the asymmetric bis-bidentate bridging ligand 4-methylbipyrimidine (mbpymNO). Depending on whether the DyIII ions coordinate to the N^O or N^N bidentate donor sets, the DyIII sites present a NO7 ( D2 d geometry) or N2 O6 ( D4 d ) coordination sphere. As a consequence, two different thermally activated magnetic relaxation processes are observed with anisotropy barriers of 47...
May 18, 2018: Inorganic Chemistry
https://www.readbyqxmd.com/read/29772810/determination-of-the-membrane-environment-of-cd59-in-living-cells
#9
Gergő Fülöp, Mario Brameshuber, Andreas M Arnold, Gerhard J Schütz, Eva Sevcsik
The organization and dynamics of proteins and lipids in the plasma membrane, and their role in membrane functionality, have been subject of a long-lasting debate. Specifically, it is unclear to what extent membrane proteins are affected by their immediate lipid environment and vice versa. Studies on model membranes and plasma membrane vesicles indicated preferences of proteins for lipid phases characterized by different acyl chain order; however, whether such phases do indeed exist in live cells is still not known...
May 17, 2018: Biomolecules
https://www.readbyqxmd.com/read/29772550/exploring-the-conformations-of-nitric-oxide-synthase-with-fluorescence
#10
David C Arnett, Sheila K Bailey, Carey K Johnson
Multi-domain oxidoreductases are a family of enzymes that catalyze oxidation-reduction reactions through a series of electron transfers. Efficient electron transfer requires a sequence of protein conformations that position electron donor and acceptor domains in close proximity to each other so that electron transfer can occur efficiently. An example is mammalian nitric oxide synthase (NOS), which consists of an N-terminal oxygenase domain containing heme and a C-terminal reductase domain containing NADPH/FAD and FMN subdomains...
June 1, 2018: Frontiers in Bioscience (Landmark Edition)
https://www.readbyqxmd.com/read/29771534/nitroxyl-modified-tobacco-mosaic-virus-as-a-metal-free-high-relaxivity-mri-and-epr-active-superoxide-sensor
#11
Madushani Dharmarwardana, Andre F Martins, Zhuo Chen, Philip Palacios, Chance M Nowak, Raymond P Welch, Shaobo Li, Michael Andrew Luzuriaga, Leonidas Bleris, Brad S Pierce, A Dean Sherry, Jeremiah J Gassensmith
Superoxide overproduction is known to occur in multiple disease states requiring critical care yet non-invasive detection of superoxide in deep tissue remains a challenge. Herein, we report a metal-free magnetic resonance imaging (MRI) and electron paramagnetic resonance (EPR) active contrast agent prepared by "click conjugating" paramagnetic organic radical contrast agents (ORCAs) to the surface of tobacco mosaic virus (TMV). While ORCAs are known to be reduced in vivo to an MRI/EPR silent state, their oxidation is facilitated specifically by reactive oxygen species-in particular superoxide-and are largely unaffected by peroxides and molecular oxygen...
May 17, 2018: Molecular Pharmaceutics
https://www.readbyqxmd.com/read/29771496/what-can-pleiotropic-proteins-in-innate-immunity-teach-us-about-bioconjugation-and-molecular-design
#12
Michelle W Lee, Ernest Y Lee, Gerard C L Wong
A common bioengineering strategy to add function to a given molecule is by conjugation of a new moiety onto that molecule. Adding multiple functions in this way becomes increasingly challenging and leads to composite molecules with larger molecular weights. In this review, we attempt to gain a new perspective by looking at this problem in reverse, by examining nature's strategies of multiplexing different functions into the same pleiotropic molecule using emerging analysis techniques such as machine learning...
May 17, 2018: Bioconjugate Chemistry
https://www.readbyqxmd.com/read/29770394/a-neutral-auxiliary-ligand-enhanced-dysprosium-iii-single-molecule-magnet
#13
Xiao-Xiang Chen, Fang Ma, Mei-Xing Xu, Jin-Cheng Bi, Hao-Ling Sun, Bing-Wu Wang, Song Gao
An approximately equatorial three-coordinated dysprosium(iii) complex DyL2[N(SiMe3)2] (L = bis(2-(2,5-dimethyl-1H-pyrrol-1-yl)ethyl)amine) with an additional neutral auxiliary pyrrole ligand was synthesised and structurally characterized. The magnetic property measurement shows that it is an equatorial charge distributed field-induced single-molecule magnet. Ab initio calculations reveal that the neutral pyrrole ligand plays an important role in stabilizing the ground doublet and thus enhancing the magnetic anisotropy...
May 17, 2018: Dalton Transactions: An International Journal of Inorganic Chemistry
https://www.readbyqxmd.com/read/29769736/trna-tracking-for-direct-measurements-of-protein-synthesis-kinetics-in-live-cells
#14
Ivan L Volkov, Martin Lindén, Javier Aguirre Rivera, Ka-Weng Ieong, Mikhail Metelev, Johan Elf, Magnus Johansson
Our ability to directly relate results from test-tube biochemical experiments to the kinetics in living cells is very limited. Here we present experimental and analytical tools to directly study the kinetics of fast biochemical reactions in live cells. Dye-labeled molecules are electroporated into bacterial cells and tracked using super-resolved single-molecule microscopy. Trajectories are analyzed by machine-learning algorithms to directly monitor transitions between bound and free states. In particular, we measure the dwell time of tRNAs on ribosomes, and hence achieve direct measurements of translation rates inside living cells at codon resolution...
May 16, 2018: Nature Chemical Biology
https://www.readbyqxmd.com/read/29769290/assessment-of-an-organ-specific-de-novo-transcriptome-of-the-nematode-trap-crop-solanum-sisymbriifolium
#15
Alexander Q Wixom, N Carol Casavant, Joseph C Kuhl, Fangming Xiao, Louise-Marie Dandurand, Allan B Caplan
Solanum sisymbriifolium , also known as "Litchi Tomato" or "Sticky Nightshade," is an undomesticated and poorly researched plant related to potato and tomato. Unlike the latter species, S. sisymbriifolium induces eggs of the cyst nematode, Globodera pallida , to hatch and migrate into its roots, but then arrests further nematode maturation. In order to provide researchers with a partial blueprint of its genetic make-up so that the mechanism of this response might be identified, we used single molecule real time (SMRT) sequencing to compile a high quality de novo transcriptome of 41,189 unigenes drawn from individually sequenced bud, root, stem, and leaf RNA populations...
May 16, 2018: G3: Genes—Genomes—Genetics
https://www.readbyqxmd.com/read/29767997/dissecting-conformational-dynamics-modulated-enzyme-catalysis-with-single-molecule-fret
#16
Shaowen Wu, Jianwei Liu, Wenning Wang
Conformational changes of enzyme proteins are often coupled with the catalytic reaction and modulate the enzyme activity. Single molecule technology is a powerful tool to study the mechanism of enzyme catalysis in these complicated cases. However, the chemical reaction cycles and conformational changes could not be monitored simultaneously in a single molecule detection experiment, resulting in some key kinetic parameters unresolved. Here, we describe a method to extract all kinetic parameters from comprehensive single molecule FRET (smFRET) measurements and model analysis...
May 16, 2018: Journal of Physical Chemistry. B
https://www.readbyqxmd.com/read/29767969/single-molecule-imaging-reveals-conformational-manipulation-of-holliday-junction-dna-by-the-junction-processing-protein-ruva
#17
Dalton Gibbs, Soma Dhakal
Interactions between DNA and motor proteins regulate nearly all biological functions of DNA such as gene expression, DNA replication and repair, and transcription. During the late stages of homologous recombination (HR), the Escherichia coli recombination machinery, RuvABC, resolves the four-way DNA motifs called Holliday junctions (HJs) that are formed during exchange of nucleotide sequences between two homologous duplex DNA. Although the formation of RuvA-HJ complex is known to be the first critical step in the RuvABC pathway, the mechanism for the binding interaction between RuvA and HJ has remained elusive...
May 16, 2018: Biochemistry
https://www.readbyqxmd.com/read/29767965/bifunctional-chimera-that-coordinately-targets-human-immunodeficiency-virus-1-hiv-1-envelope-gp120-and-host-cell-ccr5-co-receptor-at-the-virus-cell-interface
#18
Adel A Rashad, Li-Rui Song, Andrew P Holmes, Kriti Acharya, Shiyu Zhang, Zhi-Long Wang, Ebony Gary, Xin Xie, Vanessa Pirrone, Michele A Kutzler, Ya-Qiu Long, Irwin Chaiken
To address the urgent need for new agents to reduce the global occurrence and spread of AIDS, we investigated the underlying hypothesis that antagonists of HIV-1 Envelope (Env) gp120 and host cell co-receptor (CoR) proteins can be covalently joined into bifunctional synergistic combinations that will improve antiviral. A synthetic protocol was established to covalently combine a CCR5 small molecule antagonist and a gp120 peptide triazole antagonist to form the bifunctional chimera. Importantly, the chimeric inhibitor preserved the specific targeting properties of the two separate chimera components, and at the same time exhibited low to sub-nanomolar potencies, in inhibiting cell infection by different pseudoviruses, that were substantially greater than those of a non-covalent mixture of individual components...
May 16, 2018: Journal of Medicinal Chemistry
https://www.readbyqxmd.com/read/29767959/stabilization-of-immobilized-enzymes-via-the-chaperone-like-activity-of-mixed-lipid-bilayers
#19
Andres F Chaparro Sosa, Daniel F Kienle, Rebecca M Falatach, Jessica Flanagan, Joel L Kaar, Daniel K Schwartz
Biomimetic lipid bilayers represent intriguing materials for enzyme immobilization, which is critical for many biotechnological applications. Here, through the creation of mixed lipid bilayers, the retention of immobilized enzyme structure and catalytic activity are dramatically enhanced. The enhancement in the retention of enzyme structure, which correlated with an increase in enzyme activity, is observed using dynamic single-molecule (SM) fluorescence methods. The results of SM analysis specifically show that lipid bilayers composed of mixtures of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG) stabilize the folded state of nitroreductase (NfsB) increasing the rate of re-folding relative to unfolding of enzyme molecules on the bilayer surface...
May 16, 2018: ACS Applied Materials & Interfaces
https://www.readbyqxmd.com/read/29767365/current-and-future-methods-for-mrna-analysis-a-drive-toward-single-molecule-sequencing
#20
Anthony Bayega, Somayyeh Fahiminiya, Spyros Oikonomopoulos, Jiannis Ragoussis
The transcriptome encompasses a range of species including messenger RNA, and other noncoding RNA such as rRNA, tRNA, and short and long noncoding RNAs. Due to the huge role played by mRNA in development and disease, several methods have been developed to sequence and characterize mRNA, with RNA sequencing (RNA-Seq) emerging as the current method of choice particularly for large high-throughput studies. Short-read RNA-Seq which involves sequencing of short cDNA fragments and computationally assembling them to reconstruct the transcriptome, or aligning them to a reference is the most widely used approach...
2018: Methods in Molecular Biology
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