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https://www.readbyqxmd.com/read/29342181/optimization-of-mneongreen-for-homo-sapiens-increases-its-fluorescent-intensity-in-mammalian-cells
#1
Emiko Tanida-Miyake, Masato Koike, Yasuo Uchiyama, Isei Tanida
Green fluorescent protein (GFP) is tremendously useful for investigating many cellular and intracellular events. The monomeric GFP mNeonGreen is about 3- to 5-times brighter than GFP and monomeric enhanced GFP and shows high photostability. The maturation half-time of mNeonGreen is about 3-fold faster than that of monomeric enhanced GFP. However, the cDNA sequence encoding mNeonGreen contains some codons that are rarely used in Homo sapiens. For better expression of mNeonGreen in human cells, we synthesized a human-optimized cDNA encoding mNeonGreen and generated an expression plasmid for humanized mNeonGreen under the control of the cytomegalovirus promoter...
2018: PloS One
https://www.readbyqxmd.com/read/29335600/alteration-in-fluidity-of-cell-plasma-membrane-in-huntington-disease-revealed-by-spectral-phasor-analysis
#2
Sara Sameni, Leonel Malacrida, Zhiqun Tan, Michelle A Digman
Huntington disease (HD) is a late-onset genetic neurodegenerative disorder caused by expansion of cytosine-adenine-guanine (CAG) trinucleotide in the exon 1 of the gene encoding the polyglutamine (polyQ). It has been shown that protein degradation and lipid metabolism is altered in HD. In many neurodegenerative disorders, impaired lipid homeostasis is one of the early events in the disease onset. Yet, little is known about how mutant huntingtin may affect phospholipids membrane fluidity. Here, we investigated how membrane fluidity in the living cells (differentiated PC12 and HEK293 cell lines) are affected using a hyperspectral imaging of widely used probes, LAURDAN...
January 15, 2018: Scientific Reports
https://www.readbyqxmd.com/read/29334734/a-method-for-selective-depletion-of-zn-ii-ions-from-complex-biological-media-and-evaluation-of-cellular-consequences-of-zn-ii-deficiency
#3
Christopher E R Richardson, Lisa S Cunden, Vincent L Butty, Elizabeth M Nolan, Stephen J Lippard, Matthew D Shoulders
We describe the preparation, evaluation, and application of an S100A12 protein-conjugated solid support, hereafter the "A12-resin," that can remove 99% of Zn(II) from complex biological solutions without significantly perturbing the concentrations of other metal ions. The A12-resin can be applied to selectively deplete Zn(II) from diverse tissue culture media and from other biological fluids, including human serum. To further demonstrate the utility of this approach, we investigated metabolic, transcriptomic, and metallomic responses of HEK293 cells cultured in medium depleted of Zn(II) using S100A12...
January 16, 2018: Journal of the American Chemical Society
https://www.readbyqxmd.com/read/29334397/recombinant-phosphatidylserine-binding-nanobodies-for-targeting-of-extracellular-vesicles-to-tumor-cells-a-plug-and-play-approach
#4
Sander A A Kooijmans, Jerney J J M Gitz-Francois, Raymond M Schiffelers, Pieter Vader
Extracellular vesicles (EVs) are increasingly being recognized as candidate drug delivery systems due to their ability to functionally transfer biological cargo between cells. However, manipulation of targeting properties of EVs through engineering of the producer cells can be challenging and time-consuming. As a novel approach to confer tumor targeting properties to isolated EVs, we generated recombinant fusion proteins of nanobodies against the epidermal growth factor receptor (EGFR) fused to phosphatidylserine (PS)-binding domains of lactadherin (C1C2)...
January 15, 2018: Nanoscale
https://www.readbyqxmd.com/read/29333591/differential-inhibition-of-nav1-7-and-neuropathic-pain-by-hybridoma-produced-and-recombinant-monoclonal-antibodies-that-target-nav1-7-differential-activities-of-nav1-7-targeting-monoclonal-antibodies
#5
Sangsu Bang, Jiho Yoo, Xingrui Gong, Di Liu, Qingjian Han, Xin Luo, Wonseok Chang, Gang Chen, Sang-Taek Im, Yong Ho Kim, Judith A Strong, Ma-Zhong Zhang, Jun-Ming Zhang, Seok-Yong Lee, Ru-Rong Ji
The voltage-gated Na+ channel subtype Nav1.7 is important for pain and itch in rodents and humans. We previously showed that a Nav1.7-targeting monoclonal antibody (SVmab) reduces Na+ currents and pain and itch responses in mice. Here, we investigated whether recombinant SVmab (rSVmab) binds to and blocks Nav1.7 similar to SVmab. ELISA tests revealed that SVmab was capable of binding to Nav1.7-expressing HEK293 cells, mouse DRG neurons, human nerve tissue, and the voltage-sensor domain II of Nav1.7. In contrast, rSVmab showed no or weak binding to Nav1...
January 15, 2018: Neuroscience Bulletin
https://www.readbyqxmd.com/read/29332592/construction-of-an-m1gs-ribozyme-for-targeted-and-rapid-mrna-cleavage-application-on-the-ets-2-oncogene
#6
Chrisavgi Toumpeki, Dimitrios Anastasakis, Ioannis Panagoulias, Vassiliki Stamatopoulou, Tassos Georgakopoulos, Sofia Kallia-Raftopoulos, Athanasia Mouzaki, Denis Drainas
Background RNase P-mediated cleavage of target RNAs has been proposed as a promising tool for gene silencing. Ets-2 proto-oncogene controls the expression of a wide variety of genes involved in cancer and immunity. Objective Construction of a functional RNase P-based ribozyme (M1GS303) that targets Ets-2 mRNA Method The accessible sites for targeting of Ets-2 mRNA were identified by footprinting analysis. M1GS303 ribozyme was constructed by cloning. The activity of the ribozyme in the presence or absence of spiramysin in E...
January 11, 2018: Medicinal Chemistry
https://www.readbyqxmd.com/read/29331741/extracellular-acidification-induces-ros-and-mptp-mediated-death-in-hek293-cells
#7
José Teixeira, Farhan Basit, Herman G Swarts, Marleen Forkink, Paulo J Oliveira, Peter H G M Willems, Werner J H Koopman
The extracellular pH (pHe) is a key determinant of the cellular (micro)environment and needs to be maintained within strict boundaries to allow normal cell function. Here we used HEK293 cells to study the effects of pHe acidification (24h), induced by mitochondrial inhibitors (rotenone, antimycin A) and/or extracellular HCl addition. Lowering pHe from 7.2 to 5.8 reduced cell viability by 70% and was paralleled by a decrease in cytosolic pH (pHc), hyperpolarization of the mitochondrial membrane potential (Δψ), increased levels of hydroethidine-oxidizing ROS and stimulation of protein carbonylation...
December 30, 2017: Redox Biology
https://www.readbyqxmd.com/read/29330065/stargazin-differentially-modulates-ampakine-gating-kinetics-and-pharmacology
#8
Daniel P Radin, Yong-Xin Li, Gary Rogers, Richard Purcell, Arnold Lippa
It was previously reported that Stargazin (STG) enhances the surface expression of AMPA receptors, controls receptor gating and slows channel desensitization as an auxiliary subunit of the receptors. Ampakines are a class of AMPA receptor positive allosteric modulators that modify rates of transmitter binding, channel activity and desensitization parameters. As such, they have shown efficacy in animal models of neurodegenerative diseases, where excitatory synaptic transmission is compromised. Given the functional similarities between STG and ampakines, the current study sought to probe interactions between STG and ampakine gating properties...
January 9, 2018: Biochemical Pharmacology
https://www.readbyqxmd.com/read/29330041/acute-epileptiform-activity-induced-by-gabazine-involves-proteasomal-rather-than-lysosomal-degradation-of-kca2-2-channels
#9
Steffen Müller, Xiati Guli, Judith Hey, Anne Einsle, Daniela Pfanz, Victor Sudmann, Timo Kirschstein, Rüdiger Köhling
Voltage-independent, Ca2+-activated K+ channels (KCa2.2, previously named SK2) are typically activated during a train of action potentials, and hence, are powerful regulators of cellular excitability by generating an afterhyperpolarizing potential (AHP) following prolonged excitation. In the acute in vitro epilepsy model induced in hippocampal brain slice preparations by exposure to the GABAA receptor blocker gabazine (GZ), the AHP was previously shown to be significantly decreased. Here, we asked the question whether KCa2...
January 9, 2018: Neurobiology of Disease
https://www.readbyqxmd.com/read/29324347/design-synthesis-and-biological-evaluation-of-7h-pyrrolo-2-3-d-pyrimidin-4-amine-derivatives-as-selective-btk-inhibitors-with-improved-pharmacokinetic-properties-for-the-treatment-of-rheumatoid-arthritis
#10
Linhong He, Heying Pei, Chufeng Zhang, Mingfeng Shao, Dan Li, Mingli Tang, Taijing Wang, Xiaoxin Chen, Mingli Xiang, Lijuan Chen
Bruton's tyrosine kinase (Btk) is a Tec family kinase with a well-defined role in the B cell receptor (BCR) and Fcγ receptor (FcR) signaling pathways, which makes it a uniquely attractive target for the treatment of autoimmune diseases, such as rheumatoid arthritis (RA). We reported a series of compounds bearing 7H-pyrrolo [2,3-d]pyrimidin-4-amine scaffold that potently inhibited Btk in vitro. Analysis of the structure-activity relationships (SAR) and drug-like profiles led to the discovery of the optimal compound B16...
December 28, 2017: European Journal of Medicinal Chemistry
https://www.readbyqxmd.com/read/29317818/antibacterial-mechanisms-of-a-novel-type-picosecond-laser-generated-silver-titanium-nanoparticles-and-their-toxicity-to-human-cells
#11
Peri Korshed, Lin Li, Zhu Liu, Aleksandr Mironov, Tao Wang
In this study, we explored the antibacterial mechanisms for a novel type of Ag-TiO2 compound nanoparticles (NPs) produced from an Ag-TiO2 alloy using a picosecond laser and evaluated the toxicity of the Ag-TiO2 NPs to a range of human cell types. Transmission electron microscopy was used to determine the morphology, shapes, and size distribution of the laser-generated Ag-TiO2 NPs. UV-visible spectrometer was used to confirm the shift of light absorbance of the NPs toward visible light wavelength. Results showed that the laser-generated Ag-TiO2 NPs had significant antibacterial activities against both Gram-negative and Gram-positive bacterial strains, including Escherichia coli, Pseudomonas aeruginosa, and the methicillin-resistant Staphylococcus aureus...
2018: International Journal of Nanomedicine
https://www.readbyqxmd.com/read/29317744/a-role-for-divalent-metal-transporter-dmt1-in-mitochondrial-uptake-of-iron-and-manganese
#12
Natascha A Wolff, Michael D Garrick, Lin Zhao, Laura M Garrick, Andrew J Ghio, Frank Thévenod
Much of iron and manganese metabolism occurs in mitochondria. Uptake of redox-active iron must be tightly controlled, but little is known about how metal ions enter mitochondria. Recently, we established that the divalent metal transporter 1 (DMT1) is present in the outer mitochondrial membrane (OMM). Therefore we asked if it mediates Fe2+ and Mn2+ influx. Mitochondria were isolated from HEK293 cells permanently transfected with inducible rat DMT1 isoform 1 A/+IRE (HEK293-rDMT1). Fe2+-induced quenching of the dye PhenGreen™SK (PGSK) occurred in two phases, one of which reflected OMM DMT1 with stronger Fe2+ uptake after DMT1 overexpression...
January 9, 2018: Scientific Reports
https://www.readbyqxmd.com/read/29317492/a-protein-interaction-network-centered-on-leucine-rich-repeats-and-immunoglobulin-like-domains-1-lrig1-regulates-growth-factor-receptors
#13
Mahmood Faraz, Carl Herdenberg, Camilla Holmlund, Roger Henriksson, Håkan Hedman
Leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) is a tumor suppressor and a negative regulator of several receptor tyrosine kinases (RTKs). The molecular mechanisms by which LRIG1 mediates its tumor suppressor effects and regulates RTKs remain incompletely understood. Here, we performed a yeast two-hybrid screen to identify novel LRIG1-interacting proteins and mined data from the BioPlex (biophysical interactions of ORFeome-based complexes) protein interaction data repository. The putative LRIG1 interactors identified in the screen were functionally evaluated using a triple co-transfection system in which HEK293 cells were co-transfected with platelet-derived growth factor receptor alpha (PDGFRA), LRIG1, and shRNAs against the identified LRIG1 interactors...
January 9, 2018: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/29317166/synthesis-in-vitro-antimalarial-activities-and-cytotoxicities-of-amino-artemisinin-ferrocene-derivatives
#14
Christo de Lange, Dina Coertzen, Frans J Smit, Johannes F Wentzel, Ho Ning Wong, Lyn-Marie Birkholtz, Richard K Haynes, David D N'Da
Novel derivatives bearing a ferrocene attached via a piperazine linker to C-10 of the artemisinin nucleus were prepared from dihydroartemisinin and screened against chloroquine (CQ) sensitive NF54 and CQ resistant K1 and W2 strains of Plasmodium falciparum (Pf) parasites. The overall aim is to imprint oxidant (from the artemisinin) and redox (from the ferrocene) activities. In a preliminary assessment, these compounds were shown to possess activities in the low nM range with the most active being compound 6 with IC50 values of 2...
December 26, 2017: Bioorganic & Medicinal Chemistry Letters
https://www.readbyqxmd.com/read/29314777/concomitant-reduction-of-lactate-and-ammonia-accumulation-in-fed-batch-cultures-impact-on-glycoprotein-production-and-quality
#15
Eric Karengera, Anna Robotham, John Kelly, Yves Durocher, Gregory De Crescenzo, Olivier Henry
Lactate and ammonia accumulation is a major factor limiting the performance of fed-batch strategies for mammalian cell culture processes. In addition to the detrimental effects of these by-products on production yield, ammonia also contributes to recombinant glycoprotein quality deterioration. In this study, we tackled the accumulation of these two inhibiting metabolic wastes by culturing in glutamine-free fed-batch cultures an engineered HEK293 cell line displaying an improved central carbon metabolism. Batch cultures highlighted the ability of PYC2-overexpressing HEK293 cells to grow and sustain a relatively high viability in absence of glutamine without prior adaptation to the culture medium...
January 5, 2018: Biotechnology Progress
https://www.readbyqxmd.com/read/29311259/the-endosomal-trafficking-factors-corvet-and-escrt-suppress-plasma-membrane-residence-of-the-renal-outer-medullary-potassium-channel-romk
#16
Timothy D Mackie, Bo-Young Kim, Arohan R Subramanya, Daniel J Bain, Allyson F O'Donnell, Paul A Welling, Jeffrey L Brodsky
Protein trafficking can act as the primary regulatory mechanism for ion channels with high open probabilities, such as the renal outer medullary (ROMK) channel. ROMK, also known as Kir1.1 (KCNJ1), is the major route for potassium secretion into the pro-urine and plays an indispensable role in regulating serum potassium and urinary concentrations. However, the cellular machinery that regulates ROMK trafficking has not been fully defined. To identify regulators of the cell-surface population of ROMK, we expressed a pH-insensitive version of the channel in the budding yeast Saccharomyces cerevisiae...
January 8, 2018: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/29302759/regulation-of-the-human-fc-neonatal-receptor-alpha-chain-gene-fcgrt-by-microrna-3181
#17
Daniel C Ferguson, Javier G Blanco
PURPOSE: FCGRT encodes the alpha-chain component of the neonatal Fc receptor (FcRn). FcRn is critical for the trafficking of endogenous and exogenous IgG molecules and albumin in various tissues. Few regulators of FcRn expression have been identified. We investigated the epigenetic regulation of FcRn by two microRNAs (hsa-miR-3181 and hsa-miR-3136-3p) acting on FCGRT. METHODS: The binding of candidate microRNAs to the 3'-untranslated region of FCGRT was evaluated using luciferase reporter constructs in CHO cells...
January 4, 2018: Pharmaceutical Research
https://www.readbyqxmd.com/read/29302047/functional-and-association-analysis-of-an-amerindian-derived-population-specific-p-thr280met-variant-in-rbpjl-a-component-of-the-ptf1-complex
#18
Anup K Nair, Jeff R Sutherland, Michael Traurig, Paolo Piaggi, Peng Chen, Sayuko Kobes, Robert L Hanson, Clifton Bogardus, Leslie J Baier
PTF1 complex is critical for pancreatic development and maintenance of adult exocrine pancreas. As a part of our ongoing studies to identify genetic variation that contributes to type 2 diabetes (T2D) in American Indians, we analyzed variation in genes that form this complex, namely PTF1A, RBPJ, and its paralogue RBPJL. A c.839C>T (p.(Thr280Met)) variant (rs200998587:C>T, risk allele frequency = 0.03) in RBPJL, identified only in Amerindian-derived populations, associated with T2D (OR = 1.60[1...
January 4, 2018: European Journal of Human Genetics: EJHG
https://www.readbyqxmd.com/read/29301942/leucine-rich-repeat-containing-8b-protein-is-associated-with-the-endoplasmic-reticulum-ca2-%C3%A2-leak-in-hek293-cells-doi-%C3%A2-10-1242-jcs-203646
#19
Arijita Ghosh, Nitin Khandelwal, Arvind Kumar, Amal Kanti Bera
No abstract text is available yet for this article.
January 4, 2018: Journal of Cell Science
https://www.readbyqxmd.com/read/29301535/inhibition-of-histone-deacetylase-activity-rescues-inflammatory-cystic-fibrosis-lung-disease-by-modulating-innate-and-adaptive-immune-responses
#20
Manish Bodas, Steven Mazur, Taehong Min, Neeraj Vij
BACKGROUND: Chronic lung disease resulting from dysfunctional cystic fibrosis transmembrane conductance regulator (CFTR) and NFκB-mediated neutrophilic-inflammation forms the basis of CF-related mortality. Here we aimed to evaluate if HDAC inhibition controls Pseudomonas-aeruginosa-lipopolysaccharide (Pa-LPS) induced airway inflammation and CF-lung disease. METHODS: For in vitro experiments, HEK293-cells were transfected with IL-8 or NFκB-firefly luciferase, and SV40-renilla- luciferase reporter constructs or ΔF508-CFTR-pCEP, followed by treatment with suberoylanilide hydroxamic acid (SAHA), Trichostatin-A (TSA) and/or TNFα...
January 4, 2018: Respiratory Research
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