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https://www.readbyqxmd.com/read/27865780/rasins-genetically-encoded-intrabodies-of-activated-ras-proteins
#1
Mehmet Cetin, William E Evenson, Garret G Gross, Farzad Jalali-Yazdi, Daniel Krieger, Don Arnold, Terry T Takahashi, Richard W Roberts
K- and H-Ras are the most commonly mutated genes in human tumors and are critical for conferring and maintaining the oncogenic phenotype in tumors with poor prognoses. Here, we design genetically encoded antibody-like ligands (intrabodies) that recognize active, GTP-bound K- and H-Ras. These ligands, which use the 10th domain of human fibronectin as their scaffold, are stable inside cells and when fused with a fluorescent protein label constitutively active G12V mutant H-Ras. Primary selection of ligands against Ras with mRNA display resulted in an intrabody (termed RasIn1) that binds with a KD of 2...
November 16, 2016: Journal of Molecular Biology
https://www.readbyqxmd.com/read/27862752/improving-the-binding-affinity-of-in-vitro-evolved-cyclic-peptides-by-inserting-atoms-into-the-macrocycle-backbone
#2
Jonas Wilbs, Simon J Middendorp, Christian Heinis
Cyclic peptides binding to targets of interest can be generated efficiently with powerful in vitro display techniques, such as phage display or mRNA display. The cyclic peptide libraries screened with these methods are generated by altering in a combinatorial fashion the amino acid sequence of the peptides, the number of amino acids in the macrocycle rings, and the cyclization chemistry. A structural element that cannot easily be varied in the cyclic peptides is the backbone, which is built from amino acids, each of which contributes three atoms to the macrocyclic ring structure...
November 11, 2016: Chembiochem: a European Journal of Chemical Biology
https://www.readbyqxmd.com/read/27624596/endoplasmic-reticulum-directed-recombinant-mrna-displays-subcellular-localization-equal-to-endogenous-mrna-during-transient-expression-in-cho-cells
#3
Thomas Beuchert Kallehauge, Stefan Kol, Mikael Rørdam Andersen, Christian Kroun Damgaard, Gyun Min Lee, Helene Faustrup Kildegaard
When expressing pharmaceutical recombinant proteins in mammalian cells, the protein is commonly directed through the secretory pathway, in a signal peptide-dependent manner, to acquire specific post-translational modifications and to facilitate secretion into the culture medium. One key premise for this is the direction of the mRNA encoding the recombinant protein to the surface of the endoplasmic reticulum (ER) for subsequent protein translocation into the secretory pathway. To evaluate the efficiency of this process in Chinese hamster ovary (CHO) cells, the subcellular localization of recombinant mRNA encoding the therapeutic proteins, erythropoietin (EPO) and Rituximab, was determined...
October 2016: Biotechnology Journal
https://www.readbyqxmd.com/read/27576167/region-specific-alterations-of-a-to-i-rna-editing-of-serotonin-2c-receptor-in-the-cortex-of-suicides-with-major-depression
#4
D Weissmann, S van der Laan, M D Underwood, N Salvetat, L Cavarec, L Vincent, F Molina, J J Mann, V Arango, J F Pujol
Brain region-specific abnormalities in serotonergic transmission appear to underlie suicidal behavior. Alterations of RNA editing on the serotonin receptor 2C (HTR2C) pre-mRNA in the brain of suicides produce transcripts that attenuate 5-HT2CR signaling by impairing intracellular G-protein coupling and subsequent intracellular signal transduction. In brain, the distribution of RNA-editing enzymes catalyzing deamination (A-to-I modification) shows regional variation, including within the cerebral cortex. We tested the hypothesis that altered pre-mRNA 5-HT2CR receptor editing in suicide is region-specific...
2016: Translational Psychiatry
https://www.readbyqxmd.com/read/27465925/directed-evolution-of-scanning-unnatural-protease-resistant-supr-peptides-for-in-vivo-applications
#5
Stephen V Fiacco, Lindsay E Kelderhouse, Amanda Hardy, Yonatan Peleg, Biliang Hu, Argentina Ornelas, Peiying Yang, Seth T Gammon, Shannon M Howell, Pin Wang, Terry T Takahashi, Steven W Millward, Richard W Roberts
Peptides typically have poor biostabilities, and natural sequences cannot easily be converted into drug-like molecules without extensive medicinal chemistry. We have adapted mRNA display to drive the evolution of highly stable cyclic peptides while preserving target affinity. To do this, we incorporated an unnatural amino acid in an mRNA display library that was subjected to proteolysis prior to selection for function. The resulting "SUPR (scanning unnatural protease resistant) peptide" showed ≈500-fold improvement in serum stability (t1/2 =160 h) and up to 3700-fold improvement in protease resistance versus the parent sequence...
September 2, 2016: Chembiochem: a European Journal of Chemical Biology
https://www.readbyqxmd.com/read/27464514/cloning-and-characterization-of-lxr-and-srebp1-and-their-potential-roles-in-regulation-of-lc-pufa-biosynthesis-in-rabbitfish-siganus-canaliculatus
#6
Qinghao Zhang, Cuihong You, Fang Liu, Wendi Zhu, Shuqi Wang, Dizhi Xie, Óscar Monroig, Douglas R Tocher, Yuanyou Li
Rabbitfish Siganus canaliculatus was the first marine teleost demonstrated to have the ability to biosynthesize C20-22 long-chain polyunsaturated fatty acid (LC-PUFA) from C18 PUFA precursors, which is generally absent or low in marine teleosts. Thus, understanding the molecular basis of LC-PUFA biosynthesis in rabbitfish will contribute to efforts aimed at optimizing LC-PUFA biosynthesis in teleosts, especially marine species. In the present study, the importance of the transcription factors liver X receptor (Lxr) and sterol regulatory element-binding protein 1 (Srebp1) in regulation of LC-PUFA biosynthesis in rabbitfish was investigated...
September 2016: Lipids
https://www.readbyqxmd.com/read/27411326/use-of-peptide-libraries-for-identification-and-optimization-of-novel-antimicrobial-peptides
#7
Martin Ashby, Asya Petkova, Jurnorain Gani, Ralf Mikut, Kai Hilpert
The increasing rates of resistance among bacteria and to a lesser extent fungi have resulted in an urgent need to find new molecules that hold therapeutic promise against multidrug-resistant strains. Antimicrobial peptides have proven very effective against a variety of multidrug-resistant bacteria. Additionally, the low levels of resistance reported towards these molecules are an attractive feature for antimicrobial drug development. Here we summarise information on diverse peptide libraries used to discover or to optimize antimicrobial peptides...
July 13, 2016: Current Topics in Medicinal Chemistry
https://www.readbyqxmd.com/read/27385640/peptide-synthesis-on-a-next-generation-dna-sequencing-platform
#8
Nina Svensen, Olve B Peersen, Samie R Jaffrey
Methods for displaying large numbers of peptides on solid surfaces are essential for high-throughput characterization of peptide function and binding properties. Here we describe a method for converting the >10(7) flow cell-bound clusters of identical DNA strands generated by the Illumina DNA sequencing technology into clusters of complementary RNA, and subsequently peptide clusters. We modified the flow-cell-bound primers with ribonucleotides thus enabling them to be used by poliovirus polymerase 3D(pol) ...
September 2, 2016: Chembiochem: a European Journal of Chemical Biology
https://www.readbyqxmd.com/read/27306919/library-based-display-technologies-where-do-we-stand
#9
REVIEW
Asier Galán, Lubos Comor, Anita Horvatić, Josipa Kuleš, Nicolas Guillemin, Vladimir Mrljak, Mangesh Bhide
Over the past two decades, library-based display technologies have been staggeringly optimized since their appearance in order to mimic the process of natural molecular evolution. Display technologies are essential for the isolation of specific high-affinity binding molecules (proteins, polypeptides, nucleic acids and others) for diagnostic and therapeutic applications in cancer, infectious diseases, autoimmune, neurodegenerative, inflammatory pathologies etc. Applications extend to other fields such as antibody and enzyme engineering, cell-free protein synthesis and the discovery of protein-protein interactions...
July 19, 2016: Molecular BioSystems
https://www.readbyqxmd.com/read/26914638/high-throughput-measurement-of-binding-kinetics-by-mrna-display-and-next-generation-sequencing
#10
Farzad Jalali-Yazdi, Lan Huong Lai, Terry T Takahashi, Richard W Roberts
There is great demand for high-throughput methods to characterize ligand affinity. By combining mRNA display with next-generation sequencing, we determined the kinetic on- and off-rates for over twenty thousand ligands without the need for synthesis or purification of individual members. Our results are reproducible and as accurate as those obtained with other methods of affinity measurement.
March 14, 2016: Angewandte Chemie
https://www.readbyqxmd.com/read/26797799/expression-of-the-anti-inflammatory-suppressor-of-cytokine-signaling-3-socs3-in-human-clear-cell-renal-cell-carcinoma
#11
Anja Urbschat, Svenja Stumpf, Jörg Hänze, Patrick Paulus, Thorsten J Maier, Christine Weipert, Rainer Hofmann, Axel Hegele
The oncogenic transcription factor signal transducer and activator of transcription 3 (STAT3) is a cytokine-activated transcription factor controlling inflammation, cell proliferation, survival, and differentiation in normal tissue as well as in tumor growth. One of its most important negative regulators is the suppressor of cytokine signaling 3 (SOCS3). Here, we analyzed SOCS3 and other tumor-associated local immune regulators in human clear cell renal cell carcinoma (ccRCC). Analyses were performed in tumor and adjacent tumor-free healthy renal tissue from 35 patients with ccRCC...
July 2016: Tumour Biology: the Journal of the International Society for Oncodevelopmental Biology and Medicine
https://www.readbyqxmd.com/read/26711234/pure-mrna-display-for-in-vitro-selection-of-single-chain-antibodies
#12
Yu Nagumo, Kei Fujiwara, Kenichi Horisawa, Hiroshi Yanagawa, Nobuhide Doi
mRNA display is a method to form a covalent linkage between a cell-free synthesized protein (phenotype) and its encoding mRNA (genotype) through puromycin for in vitro selection of proteins. Although a wheat germ cell-free translation system has been previously used in our mRNA display system, a protein synthesis using recombinant elements (PURE) system is a more attractive approach because it contains no endogenous nucleases and proteases and is optimized for folding of antibodies with disulphide bonds. However, when we used the PURE system for mRNA display of single-chain Fv (scFv) antibodies, the formation efficiency of the mRNA-protein conjugates was quite low...
May 2016: Journal of Biochemistry
https://www.readbyqxmd.com/read/26683999/silent-encoding-of-chemical-post-translational-modifications-in-phage-displayed-libraries
#13
Katrina F Tjhung, Pavel I Kitov, Simon Ng, Elena N Kitova, Lu Deng, John S Klassen, Ratmir Derda
In vitro selection of chemically modified peptide libraries presented on phage, while a powerful technology, is limited to one chemical post-translational modification (cPTM) per library. We use unique combinations of redundant codons to encode cPTMs with "silent barcodes" to trace multiple modifications within a mixed modified library. As a proof of concept, we produced phage-displayed peptide libraries Ser-[X]4-Gly-Gly-Gly, with Gly and Ser encoded using unique combinations of codons (TCA-[X]4-GGAGGAGGA, AGT-[X]4-GGTGGTGGT, etc...
January 13, 2016: Journal of the American Chemical Society
https://www.readbyqxmd.com/read/26388988/integrated-transcriptional-profiling-and-genomic-analyses-reveal-rpn2-and-hmgb1-as-promising-biomarkers-in-colorectal-cancer
#14
Jialing Zhang, Bin Yan, Stephan Stanislaw Späth, Hu Qun, Shaleeka Cornelius, Daogang Guan, Jiaofang Shao, Koichi Hagiwara, Carter Van Waes, Zhong Chen, Xiulan Su, Yongyi Bi
Colorectal cancer (CRC) is a heterogeneous disease that is associated with a gradual accumulation of genetic and epigenetic alterations. Among all CRC stages, stage II tumors are highly heterogeneous with a high relapse rate in about 20-25 % of stage II CRC patients following surgery. Thus, a comprehensive analysis of gene signatures to identify aggressive and metastatic phenotypes in stage II CRC is desired for a more accurate disease classification and outcome prediction. By utilizing a Cancer Array, containing 440 oncogenes and tumor suppressors to profile mRNA expression, we identified a larger number of differentially expressed genes in poorly differentiated stage II colorectal adenocarcinoma tissues, compared to their matched normal tissues...
2015: Cell & Bioscience
https://www.readbyqxmd.com/read/26388492/papep-a-small-peptide-derived-from-human-pancreatitis-associated-protein-attenuates-corneal-inflammation-in-vivo-and-in-vitro-through-the-ikk%C3%AE-%C3%AE-i%C3%AE%C2%BAb%C3%AE-nf-%C3%AE%C2%BAb-signaling-pathway
#15
Shaopin Zhu, Xun Xu, Kun Liu, Qing Gu, Xiaolu Yang
Keratitis is a worldwide sight-threatening disease. Current drugs generate various adverse effects. Large molecules hardly penetrate ocular tissues. Small peptides derived from endogenous protein display certain advantages. Previously we indentified a novel peptide (PAPep) from human pancreatitis-associated protein (PAP), a protein with protective effect against inflammatory diseases. To further examine the effect of PAPep on inflammatory disease and expand its scope of potential clinical application, especially in keratitis, we tested the effect of PAPep on various aspects of lipopolysaccharide (LPS)--induced corneal inflammation in vivo and in vitro...
December 2015: Pharmacological Research: the Official Journal of the Italian Pharmacological Society
https://www.readbyqxmd.com/read/26387190/-effect-of-p65-gene-inhibited-by-sirna-on-differention-of-rat-marrow-mesenchymal-stem-cells-into-neurons
#16
Er-yi Zhao, Yan-jie Jia, Dai-mei Wang, Guo-qiang Wen, Wen-juan Guan, Li-jun Jing, Yi-dong Deng
OBJECTIVE: To investigate the effect of p65 gene inhibited by siRNA on neuronic differentiation in the marrow mesenchymal stem cells (MSCs). METHODS: The MSCs were transfected with Rn-p65-siRNA. Fasudil hydrochloride induced MSCs differentiating into neurons. The non-transfected group and negative control group (transfected with negative control siRNA marked by Cy3) were used as controls. The fluorescence expressed by transfected MSCs were observed under inverted fluorescence microscope at 24 h,48 h and 72 h after transfected with negative control siRNA...
May 2015: Chinese Journal of Applied Physiology
https://www.readbyqxmd.com/read/26386943/depletion-of-cardiac-14-3-3%C3%AE-protein-adversely-influences-pathologic-cardiac-remodeling-during-myocardial-infarction-after-coronary-artery-ligation-in-mice
#17
Remya Sreedhar, Somasundaram Arumugam, Rajarajan A Thandavarayan, Vijayasree V Giridharan, Vengadeshprabhu Karuppagounder, Vigneshwaran Pitchaimani, Rejina Afrin, Meilei Harima, Masahiko Nakamura, Kenji Suzuki, Narasimman Gurusamy, Prasanna Krishnamurthy, Kenichi Watanabe
BACKGROUND/OBJECTIVES: 14-3-3η protein, a dimeric phosphoserine-binding protein, provides protection against adverse cardiac remodeling during pressure-overload induced heart failure in mice. To identify its role in myocardial infarction (MI), we have used mice with cardio-specific expression of dominant-negative 14-3-3η protein mutant (DN14-3-3) and performed the surgical ligation of left anterior descending coronary artery. METHODS: We have performed echocardiography to assess cardiac function, protein expression analysis using Western blotting, mRNA expression by real time-reverse transcription polymerase chain reaction and histopathological analyses...
January 1, 2016: International Journal of Cardiology
https://www.readbyqxmd.com/read/26386462/hyperinsulinemia-caused-by-dexamethasone-treatment-is-associated-with-reduced-insulin-clearance-and-lower-hepatic-activity-of-insulin-degrading-enzyme
#18
André Otávio Peres Protzek, Luiz Fernando Rezende, José Maria Costa-Júnior, Sandra Mara Ferreira, Ana Paula Gameiro Cappelli, Flávia Maria Moura de Paula, Jane Cristina de Souza, Mirian Ayumi Kurauti, Everardo Magalhães Carneiro, Alex Rafacho, Antonio Carlos Boschero
OBJECTIVES: Glucocorticoid treatment induces insulin resistance (IR), which is counteracted by a compensatory hyperinsulinemia, due to increased pancreatic β-cell function. There is evidence for also reduced hepatic insulin clearance, but whether this correlates with altered activity of insulin-degrading enzyme (IDE) in the liver, is not fully understood. Here, we investigated whether hyperinsulinemia, in glucocorticoid-treated rodents, is associated with any alteration in the insulin clearance and activity of the IDE in the liver...
January 2016: Journal of Steroid Biochemistry and Molecular Biology
https://www.readbyqxmd.com/read/26383775/simira-a-tool-to-identify-coregulation-between-micrornas-and-rna-binding-proteins
#19
Martin Preusse, Carsten Marr, Sita Saunders, Daniel Maticzka, Heiko Lickert, Rolf Backofen, Fabian Theis
microRNAs and microRNA-independent RNA-binding proteins are 2 classes of post-transcriptional regulators that have been shown to cooperate in gene-expression regulation. We compared the genome-wide target sets of microRNAs and RBPs identified by recent CLIP-Seq technologies, finding that RBPs have distinct target sets and favor gene interaction network hubs. To identify microRNAs and RBPs with a similar functional context, we developed simiRa, a tool that compares enriched functional categories such as pathways and GO terms...
2015: RNA Biology
https://www.readbyqxmd.com/read/26381935/hypothalamic-pka-regulates-leptin-sensitivity-and-adiposity
#20
Linghai Yang, G Stanley McKnight
Mice lacking the RIIβ regulatory subunit of cyclic AMP-dependent protein kinase A (PKA) display reduced adiposity and resistance to diet-induced obesity. Here we show that RIIβ knockout (KO) mice have enhanced sensitivity to leptin's effects on both feeding and energy metabolism. After administration of a low dose of leptin, the duration of hypothalamic JAK/STAT3 signalling is increased, resulting in enhanced POMC mRNA induction. Consistent with the extended JAK/STAT3 activation, we find that the negative feedback regulator of leptin receptor signalling, Socs3, is inhibited in the hypothalamus of RIIβ KO mice...
September 18, 2015: Nature Communications
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