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https://www.readbyqxmd.com/read/28342833/a-high-density-cho-s-transient-transfection-system-comparison-of-expicho-and-expi293
#1
Nina K Jain, Susan Barkowski-Clark, Richard Altman, Krista Johnson, Fang Sun, Jonathan Zmuda, Chao Yan Liu, Adriana Kita, Ryan Schulz, Alyssa Neill, Robert Ballinger, Rekha Patel, Jian Liu, Alinafe Mpanda, Brian Huta, Henry Chiou, Walter Voegtli, Tadas Panavas
Chinese Hamster Ovary (CHO) cells are the principal mammalian host used for stable cell line generation and biotherapeutic protein production. Until recently, production of milligrams to grams of protein in CHO transient systems was challenging. As such, Human Embryonic Kidney (HEK293) cells are the most common mammalian cell type used for transient transfection. The post-translational modifications (PTMs) of a protein are dictated in part by the cell line used for expression, and changes in PTMs have been shown to affect both the activity and biophysical properties of proteins...
March 22, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28341602/cytosolic-yb-1-and-nsun2-are-the-only-proteins-recognizing-specific-motifs-present-in-mrnas-enriched-in-exosomes
#2
Olga A Kossinova, Alexander V Gopanenko, Svetlana N Tamkovich, Olga A Krasheninina, Alexey E Tupikin, Elena Kiseleva, Darya D Yanshina, Alexey A Malygin, Alia G Ven'yaminova, Marsel R Kabilov, Galina G Karpova
Exosomes, membranous vesicles secreted by various cells, are involved in intercellular communication and carry vast repertoires of RNAs and proteins. Processes mediating RNA sorting into exosomes are currently poorly understood. Using bioinformatics approaches, three structural motifs ACCAGCCU, CAGUGAGC and UAAUCCCA have been discovered as enriched in exosomal mRNAs and long noncoding RNAs. Here, utilizing short RNA hairpins, each containing one of the motifs, in a pull-down assay of cytosolic extract of human embryonic kidney 293 (HEK293) cells, we prove that multifunctional RNA-binding protein YB-1 specifically interacts with all three motifs, whereas methyltransferase NSUN2 recognizes only the motif CAGUGAGC...
March 21, 2017: Biochimica et Biophysica Acta
https://www.readbyqxmd.com/read/28341401/discovery-of-2-aminophenyl-methanol-as-a-new-molecular-chaperone-that-rescues-the-localization-of-p123s-mutant-pendrin-stably-expressed-in-hek293-cells
#3
Wataru Nabeyama, Kenji Ishihara, Hyun Seung Ban, Hiroshi Wada, Hiroyuki Nakamura
Pendred syndrome is the most common form of syndromic deafness. It is associated with a mutation in the SLC26A4 gene that encodes pendrin, which is thought to maintain the ion concentration of endolymph in the inner ear most likely by acting as a chloride/bicarbonate transporter. Mutations in the SLC26A4 gene are responsible for sensorineural hearing loss. In this study, we established a stable HEK293 cell line expressing P123S mutant pendrin and developed screening methods for compounds that show pharmacological chaperone activity by image analysis using CellInsight™...
March 14, 2017: Bioorganic & Medicinal Chemistry
https://www.readbyqxmd.com/read/28338530/pharmacologic-characterizations-of-a-p2x7-receptor-specific-radioligand-11c-gsk1482160-for-neuroinflammatory-response
#4
Junbin Han, Hui Liu, Chunling Liu, Hongjun Jin, Joel S Perlmutter, Terrance M Egan, Zhude Tu
OBJECTIVE: The P2X7 receptor (P2X7R) is a key regulatory element in the neuroinflammatory cascade that provides a promising target for imaging neuroinflammation. GSK1482160, a P2X7R modulator with nanomolar binding affinity and high selectivity, has been successfully radiolabeled and utilized for imaging P2X7 levels in a mouse model of lipopolysaccharide-induced systemic inflammation. In the current study, we further characterized its binding profile and determined whether [C]GSK1482160 can detect changes in P2X7R expression in a rodent model of multiple sclerosis...
March 23, 2017: Nuclear Medicine Communications
https://www.readbyqxmd.com/read/28337320/discovery-and-assessment-of-atropisomers-of-%C3%A2-lesinurad
#5
Jianfei Wang, Wenqin Zeng, Shaohua Li, Liang Shen, Zhengxian Gu, Yang Zhang, Jian Li, Shuhui Chen, Xiangbo Jia
(+)- and (-)-Lesinurad were isolated as atropisomers from racemic lesinurad for the first time. No interconversion was observed between the two atropisomers under various conditions tested. The two atropisomers showed significant differences in hURAT1 highly expressed HEK293 cell-based inhibition assays, monkey pharmacokinetic studies, and in vitro human recombinant CYP2C9 stability studies. It was speculated that (+)-lesinurad might offer a better hyperuricemia/gout therapy than (-)-lesinurad or the racemate...
March 9, 2017: ACS Medicinal Chemistry Letters
https://www.readbyqxmd.com/read/28336549/eavk-segment-c-sequence-confers-ca-2-dependent-changes-to-the-kinetics-of-full-length-human-ano1
#6
Peter R Strege, Simon J Gibbons, Amelia Mazzone, Cheryl E Bernard, Arthur Beyder, Gianrico Farrugia
Anoctamin1 (Ano1, TMEM16A) is a calcium-activated chloride channel specifically expressed in interstitial cells of Cajal (ICC) of the gastrointestinal (GI) tract muscularis propria. Ano1 is necessary for normal electrical slow waves and ICC proliferation. The full length human Ano1 sequence includes an additional exon, exon "0," at the N-terminus. Ano1 with exon "0" (Ano1(0)) had a lower EC50 for intracellular calcium ([Ca(2+)]i) and faster chloride current (ICl) kinetics. The Ano1 alternative splice variant with segment "c" encoding exon 13 expresses on the first intracellular loop four additional amino acid residues, EAVK, which alter ICl at low [Ca(2+)]i Exon 13 is expressed in 75-100% of Ano1 transcripts in most human tissues but only 25% in human stomach...
March 23, 2017: American Journal of Physiology. Gastrointestinal and Liver Physiology
https://www.readbyqxmd.com/read/28336299/inhibitory-effect-of-crizotinib-on-creatinine-uptake-by-renal-secretory-transporter-oct2
#7
Hiroshi Arakawa, Saki Omote, Ikumi Tamai
Crizotinib, a tyrosine kinase inhibitor, exhibits some cases of an increase in serum creatinine levels. Creatinine is secreted by not only glomerular filtration, but also active secretion by organic cation transporters such as organic cation transporter 2 (OCT2). In the present study, we evaluated in vitro inhibitory effect of crizotinib on OCT2 by directly measuring creatinine uptake by OCT2. Co-incubation of crizotinib reduced uptake of [(14)C]creatinine by cultured HEK293 cells expressing OCT2 (HEK293/OCT2) in a concentration-dependent manner with IC50 values of 1...
March 20, 2017: Journal of Pharmaceutical Sciences
https://www.readbyqxmd.com/read/28334855/slc30a9-mutation-affecting-intracellular-zinc-homeostasis-causes-a-novel-cerebro-renal-syndrome
#8
Yonatan Perez, Zamir Shorer, Keren Liani-Leibson, Pauline Chabosseau, Rotem Kadir, Michael Volodarsky, Daniel Halperin, Shiran Barber-Zucker, Hanna Shalev, Ruth Schreiber, Libe Gradstein, Evgenia Gurevich, Raz Zarivach, Guy A Rutter, Daniel Landau, Ohad S Birk
A novel autosomal recessive cerebro-renal syndrome was identified in consanguineous Bedouin kindred: neurological deterioration was evident as of early age, progressing into severe intellectual disability, profound ataxia, camptocormia and oculomotor apraxia. Brain MRI was normal. Four of the six affected individuals also had early-onset nephropathy with features of tubulo-interstitial nephritis, hypertension and tendency for hyperkalemia, though none had rapid deterioration of renal function. Genome wide linkage analysis identified an ∼18 Mb disease-associated locus on chromosome 4 (maximal logarithm of odds score 4...
February 9, 2017: Brain: a Journal of Neurology
https://www.readbyqxmd.com/read/28334417/pharmacovigilance-database-search-discloses-clc-k-channels-as-a-novel-target-of-the-at1-receptor-blockers-valsartan-and-olmesartan
#9
Paola Imbrici, Domenico Tricarico, Giuseppe Felice Mangiatordi, Orazio Nicolotti, Marcello Diego Lograno, Diana Conte, Antonella Liantonio
BACKGROUND AND PURPOSE: Human ClC-K chloride channels are highly attractive targets for drug discovery for their physiological role and association with genetic disorders. These channels are pivotal in the kidney as they control chloride reabsorption and water diuresis. In addition, loss-of-function mutations of CLCKNB and BSND genes cause Bartter's syndrome (BS), whereas CLCNKA and CLCKNB gain-of-function polymorphisms predispose to a rare form of salt sensitive hypertension. Both disorders lack a personalized therapy that is in most cases only symptomatic...
March 23, 2017: British Journal of Pharmacology
https://www.readbyqxmd.com/read/28331181/multi-walled-carbon-nanotubes-act-as-a-chemokine-and-recruit-macrophages-by-activating-the-plc-ip3-crac-channel-signaling-pathway
#10
Hui Li, Xiao-Qiu Tan, Li Yan, Bo Zeng, Jie Meng, Hai-Yan Xu, Ji-Min Cao
The impact of nanomaterials on immune cells is gaining attention but is not well documented. Here, we report a novel stimulating effect of carboxylated multi-walled carbon nanotubes (c-MWCNTs) on the migration of macrophages and uncover the underlying mechanisms, especially the upstream signaling, using a series of techniques including transwell migration assay, patch clamp, ELISA and confocal microscopy. c-MWCNTs dramatically stimulated the migration of RAW264.7 macrophages when endocytosed, and this effect was abolished by inhibiting phospholipase C (PLC) with U-73122, antagonizing the IP3 receptor with 2-APB, and blocking calcium release-activated calcium (CRAC) channels with SK&F96365...
March 22, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28330959/predominantly-myalgic-phenotype-caused-by-the-c-3466g-a-p-a1156t-mutation-in-scn4a-gene
#11
Johanna Palmio, Satu Sandell, Michael G Hanna, Roope Männikkö, Sini Penttilä, Bjarne Udd
OBJECTIVE: To characterize the clinical phenotype in patients with p.A1156T sodium channel mutation. METHODS: Twenty-nine Finnish patients identified with the c.3466G>A p.A1156T mutation in the SCN4A gene were extensively examined. In a subsequent study, 63 patients with similar myalgic phenotype and with negative results in myotonic dystrophy type 2 genetic screening (DM2-neg group) and 93 patients diagnosed with fibromyalgia were screened for the mutation. Functional consequences of the p...
March 22, 2017: Neurology
https://www.readbyqxmd.com/read/28327575/analysis-of-exome-data-for-4293-trios-suggests-gpi-anchor-biogenesis-defects-are-a-rare-cause-of-developmental-disorders
#12
Alistair T Pagnamenta, Yoshiko Murakami, John M Taylor, Consuelo Anzilotti, Malcolm F Howard, Venessa Miller, Diana S Johnson, Shereen Tadros, Sahar Mansour, I Karen Temple, Rachel Firth, Elisabeth Rosser, Rachel E Harrison, Bronwen Kerr, Niko Popitsch, Taroh Kinoshita, Jenny C Taylor, Usha Kini
Over 150 different proteins attach to the plasma membrane using glycosylphosphatidylinositol (GPI) anchors. Mutations in 18 genes that encode components of GPI-anchor biogenesis result in a phenotypic spectrum that includes learning disability, epilepsy, microcephaly, congenital malformations and mild dysmorphic features. To determine the incidence of GPI-anchor defects, we analysed the exome data from 4293 parent-child trios recruited to the Deciphering Developmental Disorders (DDD) study. All probands recruited had a neurodevelopmental disorder...
March 22, 2017: European Journal of Human Genetics: EJHG
https://www.readbyqxmd.com/read/28326637/purinergic-receptors-p2rx4-and-p2rx7-in-familial-multiple-sclerosis
#13
A Dessa Sadovnick, Ben J Gu, Anthony L Traboulsee, Cecily Q Bernales, Mary Encarnacion, Irene M Yee, Maria G Criscuoli, Xin Huang, Amber Ou, Carol J Milligan, Steven Petrou, James S Wiley, Carles Vilariño-Güell
Genetic variants in the purinergic receptors P2RX4 and P2RX7 have been shown to affect susceptibility to multiple sclerosis (MS). In this study we set out to evaluate whether rare coding variants of major effect could also be identified in these purinergic receptors. Sequencing analysis of P2RX4 and P2RX7 in 193 MS patients and 100 controls led to the identification of a rare three variant haplotype (P2RX7 rs140915863:C>T (p.T205M), P2RX7 rs201921967:A>G (p.N361S) and P2RX4 rs765866317:G>A (p.G135S)) segregating with disease in a multi-incident family with six family members diagnosed with MS (LOD = 3...
March 21, 2017: Human Mutation
https://www.readbyqxmd.com/read/28326459/preparation-and-evaluation-of-plga-coated-capsaicin-magnetic-nanoparticles
#14
Mrudhula Baskaran, Padmamalini Baskaran, Navamoney Arulsamy, Baskaran Thyagarajan
PURPOSE: Drugs used in the treatment of diseases can cause several unwanted systemic side effects. A site-specific drug delivery system can eliminate such consequences by delivering drugs to certain target areas of the body where therapeutic effects are required. Here we present the preparation and evaluation of magnetic nanoparticles of capsaicin, the active ingredient in chili peppers, coated with poly-L-lactide co-glycolide (PLGA), a FDA-approved biodegradable bioavailable polymer...
March 21, 2017: Pharmaceutical Research
https://www.readbyqxmd.com/read/28324659/optimization-of-cell-based-cdna-microarray-conditions-for-gene-functional-studies-in-hek293-cells
#15
Hi Chul Kim, Jin Yeong Heo, Tae-Kyu Lee, Ssang-Goo Cho, Yong-Jun Kwon
Since the cell-based cDNA microarray (CBCM) technique has been a useful tool for gain-of-function studies, many investigators have used CBCMs to identify interesting genes. However, this method requires better-established conditions to ensure high reverse transfection efficiency without cross-contamination. Therefore, we optimized CBCM techniques through various means. We determined that Lipofectamine 2000 was the most appropriate transfection reagent by evaluating eight commercialized reagents, and we determined that the most effective concentrations for printing solution constituents were 0...
March 1, 2017: SLAS Discov
https://www.readbyqxmd.com/read/28324065/neurod-factors-discriminate-mineralocorticoid-from-glucocorticoid-receptor-dna-binding-in-the-male-rat-brain
#16
Lisa T C M van Weert, Jacobus C Buurstede, Ahmed Mahfouz, Pamela S M Braakhuis, J Annelies E Polman, Hetty C M Sips, Benno Roozendaal, Judit Balog, E Ronald de Kloet, Nicole A Datson, Onno C Meijer
In the limbic brain, mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs) both function as receptors for the naturally occurring glucocorticoids (corticosterone/cortisol), but mediate distinct effects on cellular physiology via transcriptional mechanisms. The transcriptional basis for specificity of these MR- versus GR-mediated effects is unknown. To address this conundrum we have identified the extent of MR/GR DNA binding selectivity in the rat hippocampus using chromatin immunoprecipitation followed by sequencing (ChIP-seq)...
January 24, 2017: Endocrinology
https://www.readbyqxmd.com/read/28323990/galactose-supplementation-in-tmem165-cdg-patients-rescues-the-glycosylation-defects
#17
Willy Morelle, Sven Potelle, Peter Witters, Sunnie Wong, Leslie Climer, Vladimir Lupashin, Gert Matthijs, Therese Gadomski, Jaak Jaeken, David Cassiman, Eva Morava, François Foulquier
Context: TMEM165 deficiency is a severe multisystem disease that manifests with metabolic, endocrine and skeletal involvement. It leads to one type of congenital disorders of glycosylation (CDG), a rapidly growing group of inherited diseases where the glycosylation process is altered. Patients have decreased galactosylation by serum glycan analysis. There are over one hundred CDGs but only specific types are treatable. Objective: Galactose has been shown to be beneficial in other CDG types with abnormal galactosylation...
January 9, 2017: Journal of Clinical Endocrinology and Metabolism
https://www.readbyqxmd.com/read/28323029/the-dopamine-d3-receptor-antagonists-pg01037-ngb2904-sb277011a-and-u99194-reverse-abcg2-transporter-mediated-drug%C3%A2-resistance-in-cancer-cell-lines
#18
Noor Hussein, Haneen Amawi, Chandrabose Karthikeyan, F Scott Hall, Roopali Mittal, Piyush Trivedi, Charles R Ashby, Amit K Tiwari
The ATP - binding cassette (ABC) family G2 (ABCG2) transporters are known to produce multidrug resistance (MDR) in cancer, thereby limiting the clinical response to chemotherapy. Molecular modeling data indicated that certain dopamine (DA) D3 receptor antagonists had a significant binding affinity for ABCG2 transporter. Therefore, in this in vitro study, we determined the effect of the D3 receptor antagonists PG01037, NGB2904, SB277011A, and U99194 on MDR resulting from the overexpression of ABCG2 transporters...
March 18, 2017: Cancer Letters
https://www.readbyqxmd.com/read/28322744/basal-ryanodine-receptor-activity-suppresses-autophagic-flux
#19
Tim Vervliet, Isabel Pintelon, Kirsten Welkenhuyzen, Martin D Bootman, Hiroko Bannai, Katsuhiko Mikoshiba, Wim Martinet, Nael Nadif Kasri, Jan B Parys, Geert Bultynck
The inositol 1,4,5-trisphosphate receptors (IP3Rs) and intracellular Ca(2+) signaling are critically involved in regulating different steps of autophagy, a lysosomal degradation pathway. The ryanodine receptors (RyR), intracellular Ca(2+)-release channels mainly expressed in excitable cell types including muscle and neurons, have however not yet been extensively studied in relation to autophagy. Yet, aberrant expression and excessive activity of RyRs in these tissues has been implicated in the onset of several diseases including Alzheimer's disease, where impaired autophagy regulation contributes to the pathology...
March 16, 2017: Biochemical Pharmacology
https://www.readbyqxmd.com/read/28322559/identification-of-novel-inhibitors-of-leishmania-donovani-%C3%AE-glutamylcysteine-synthetase-using-structure-based-virtual-screening-docking-molecular-dynamics-simulation-and-in-vitro-studies
#20
Pragati Agnihotri, Arjun Kumar Mishra, Shikha Mishra, Vijay Kumar Sirohi, Amogh A Sahasrabuddhe, J Venkatesh Pratap
Trypansomatids maintain their redox balance by the trypanothione based redox system, enzymes of which exhibit differences from mammalian homologues. γ-glutamylcysteine synthetase (Gcs) is an essential enzyme of this pathway performing the first and rate limiting step. L-buthionine-S,R-sulfoximine (BSO), a specific inhibitor of Gcs induces toxicity in hosts infected with T. brucei underlining for novel Gcs inhibitors. The present study reports identification of Ld Gcs inhibitors using computational approaches and their experimental validation...
March 21, 2017: Journal of Chemical Information and Modeling
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