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https://www.readbyqxmd.com/read/28723271/development-of-three-orthogonal-assays-suitable-for-the-identification-and-qualification-of-pikfyve-inhibitors
#1
Kylie Fogarty, Mohammed Kashem, Andras Bauer, Alexandra Bernardino, Debra Brennan, Brian Cook, Neil Farrow, Teresa Molinaro, Richard Nelson
FYVE-type zinc finger-containing phosphoinositide kinase (PIKfyve) catalyzes the formation of phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) from phosphatidylinositol 3-phosphate (PI(3)P). PIKfyve has been implicated in multiple cellular processes, and its role in the regulation of toll-like receptor (TLR) pathways and the production of proinflammatory cytokines has sparked interest in developing small-molecule PIKfyve inhibitors as potential therapeutics to treat autoimmune and inflammatory diseases. We developed three orthogonal assays to identify and qualify small-molecule inhibitors of PIKfyve: (1) a purified component microfluidic enzyme assay that measures the conversion of fluorescently labeled PI(3)P to PI(3,5)P2 by purified recombinant full-length human 6His-PIKfyve (rPIKfyve); (2) an intracellular protein stabilization assay using the kinase domain of PIKfyve expressed in HEK293 cells; and (3) a cell-based functional assay that measures the production of interleukin (IL)-12p70 in human peripheral blood mononuclear cells stimulated with TLR agonists lipopolysaccharide and R848...
July 2017: Assay and Drug Development Technologies
https://www.readbyqxmd.com/read/28722259/l-ornithine-and-l-lysine-stimulate-gastrointestinal-motility-via-transient-receptor-potential-vanilloid-1
#2
Junya Nakato, Yee Yin Ho, Ryo Omae, Takafumi Mizushige, Kunitoshi Uchida, Makoto Tominaga, Minji Kim, Tsuyoshi Goto, Nobuyuki Takahashi, Teruo Kawada, Saori Akiduki, Ryuhei Kanamoto, Kousaku Ohinata
SCOPE: The gastrointestinal (GI) tract senses and responds to intraluminal nutrients and these interactions often affect GI functions. We found that, among basic amino acids, L-ornithine (Orn) and L-lysine (Lys) stimulated but L-arginine (Arg) suppressed GI motility after oral administration (24 mmol/kg) in mice (Orn and Lys, 14.3 and 26.4% promotion; Arg, 7.7% suppression). We investigated the mechanism of the action of Orn and Lys on GI motility. METHODS AND RESULTS: Orn-induced promotion of small intestinal transit was significantly inhibited (P<0...
July 18, 2017: Molecular Nutrition & Food Research
https://www.readbyqxmd.com/read/28717968/deletion-of-type-2-cannabinoid-receptor-induces-alzheimer-s-disease-like-tau-pathology-and-memory-impairment-through-ampk-gsk3%C3%AE-pathway
#3
Lin Wang, Bing-Jin Liu, Yun Cao, Wei-Qi Xu, Dong-Sheng Sun, Meng-Zhu Li, Fang-Xiao Shi, Man Li, Qing Tian, Jian-Zhi Wang, Xin-Wen Zhou
Although several studies have shown that type-2 cannabinoid receptor (CB2R) is involved in Alzheimer's disease (AD) pathology, the effects of CB2R on AD-like tau abnormal phosphorylation and its underlying mechanism remain unclear. Herein, we employed the CB2R(-/-) mice as the animal model to explore roles of CB2R in regulating tau phosphorylation and brain function. We found that CB2R(-/-) mice display AD-like tau hyperphosphorylation, hippocampus-dependent memory impairment, increase of GSK3β activity, decrease of AMPK and Sirt1 activity and mitochondria dysfunction...
July 17, 2017: Molecular Neurobiology
https://www.readbyqxmd.com/read/28714588/cellular-force-assay-detects-altered-contractility-caused-by-a-nephritis-associated-mutation-in-nonmuscle-myosin-iia
#4
Shota P Fukuda, Tsubasa S Matsui, Takafumi Ichikawa, Taichi Furukawa, Noriyuki Kioka, Shuichiro Fukushima, Shinji Deguchi
Recent progress in understanding the essential roles of mechanical forces in regulating various cellular processes expands the field of biology to one where interdisciplinary approaches with engineering techniques become indispensable. Contractile forces or contractility-inherently present in proliferative cells due to the activity of ubiquitous nonmuscle myosin II (NMII)-are one of such mechano-regulators, but because NMII works downstream of diverse signaling pathways, it is often difficult to predict how the inherent cellular forces change upon perturbations to particular molecules...
July 17, 2017: Development, Growth & Differentiation
https://www.readbyqxmd.com/read/28712913/acute-ochratoxin-a-exposure-induces-inflammation-and-apoptosis-in-human-embryonic-kidney-hek293-cells
#5
Shanel Raghubeer, Savania Nagiah, Anil A Chuturgoon
Ochratoxin A (OTA), a common contaminant of grain and fruit and known carcinogen, has been linked to impaired antioxidant response and cellular repair. The effect of OTA on inflammation in cells has not been explored. This study investigated OTA's influence on inflammatory mediators using a range of OTA concentrations (0.5 μM (sub-IC50), 1.2 μm (IC50) and 2 μm (supra-IC50)) on human embryonic kidney (HEK293) cells over 24hr. The markers of inflammation in HEK293 cells were evaluated using the following techniques: western blotting (phosphorylated (p-)NFκB (Ser536), p-IKK (Ser176/180) and p-p53 (Ser392), total NFκB, IKK, IκBα and p53), luminometry (caspases 1, 3/7, 8, 9, ATP) and ELISA to determine IL-1β levels...
July 14, 2017: Toxicon: Official Journal of the International Society on Toxinology
https://www.readbyqxmd.com/read/28711067/pa-6-inhibits-inward-rectifier-currents-carried-by-v93i-and-d172n-gain-of-function-kir2-1-channels-but-increases-channel-protein-expression
#6
Yuan Ji, Marlieke G Veldhuis, Jantien Zandvoort, Fee L Romunde, Marien J C Houtman, Karen Duran, Gijs van Haaften, Eva-Maria Zangerl-Plessl, Hiroki Takanari, Anna Stary-Weinzinger, Marcel A G van der Heyden
BACKGROUND: The inward rectifier potassium current IK1 contributes to a stable resting membrane potential and phase 3 repolarization of the cardiac action potential. KCNJ2 gain-of-function mutations V93I and D172N associate with increased IK1, short QT syndrome type 3 and congenital atrial fibrillation. Pentamidine-Analogue 6 (PA-6) is an efficient (IC50 = 14 nM with inside-out patch clamp methodology) and specific IK1 inhibitor that interacts with the cytoplasmic pore region of the KIR2...
July 15, 2017: Journal of Biomedical Science
https://www.readbyqxmd.com/read/28711019/photo-catalytic-anti-bacterial-and-anti-cancer-properties-of-phyto-mediated-synthesis-of-silver-nanoparticles-from-artemisia-tournefortiana-rchb-extract
#7
Fahimeh Baghbani-Arani, Rabee Movagharnia, Alireza Sharifian, Soheil Salehi, Seyed Ataollah Sadat Shandiz
Metal nanoparticles have largely been investigated due to their potential medicinal activities. This study demonstrates the biological properties of green-synthesized silver nanoparticles (AgNPs) by using Artemisia tournefortiana Rchb ethanol extract. Instrumentations such as ultraviolet-visible spectra analysis, high-resolution transmission electron microscopy, scanning electron microscopy, energy-dispersive X-ray analysis, X-ray diffraction, and Fourier transform infrared spectroscopy were used to reveal the synthesized AgNPs...
July 6, 2017: Journal of Photochemistry and Photobiology. B, Biology
https://www.readbyqxmd.com/read/28710851/generation-of-apoptosis-resistant-hek293-cells-with-crispr-cas-mediated-quadruple-gene-knockout-for-improved-protein-and-virus-production
#8
Weifeng Zhang, Dan Xiao, Linlin Shan, Junli Zhao, Qinwen Mao, Haibin Xia
Apoptosis has important functions during pathophysiologic processes. However, from a biopharmaceutical point of view, active apoptosis of host cells is undesirable during viral packaging or protein expression, because it decreases the efficiency of viral or protein production. Here we used the CRISPR/Cas technique to knock out four pro-apoptotic genes, Caspase3, Caspase6, Caspase7 and AIF1, in HEK293 cells, and successfully produced an apoptosis-resistant cell line. Furthermore, this cell line showed higher expression levels of pro-apoptotic proteins and higher packaging efficiency for the virus carrying these proteins than control HEK293 cells...
July 15, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/28710025/inhibitory-effects-of-tetramethylpyrazine-on-pain-transmission-of-trigeminal-neuralgia-in-cci-ion-rats
#9
Wei Xiong, Mengxia Tan, Lingkun He, Xiaoyan Ou, Youhong Jin, Guo Yang, Liping Huang, Yulin Shen, Shu Guan, Changshui Xu, Guilin Li, Shuangmei Liu, Hong Xu, Shangdong Liang, Yun Gao
Tetramethylpyrazine (TMP) has anti-inflammatory effects and is used to treat cerebral ischemic injury, but the mechanism of TMP on neural protection is not clear. Trigeminal neuralgia (TN) is a facial pain syndrome that is characterized by paroxysmal, shock-like pain attacks located in the somatosensory distribution of the trigeminal nerve. P2X3 receptor plays a crucial role in facilitating pain transmission. The present study investigates the effects of TMP on trigeminal neuralgia transmission mediated by P2X3 receptor of the trigeminal ganglia (TG)...
July 11, 2017: Brain Research Bulletin
https://www.readbyqxmd.com/read/28707163/characterization-of-snps-in-the-dopamine-%C3%AE-hydroxylase-gene-providing-new-insights-into-its-structure-function-relationship
#10
Toyanji Joseph Punchaichira, Sanjay Kumar Dey, Anirban Mukhopadhyay, Suman Kundu, B K Thelma
Dopamine-β-hydroxylase (DBH, EC 1.14.17.1), an oxido-reductase that catalyses the conversion of dopamine to norepinephrine, is largely expressed in sympathetic neurons and adrenal medulla. Several regulatory and structural variants in DBH associated with various neuropsychiatric, cardiovascular diseases and a few that may determine enzyme activity have also been identified. Due to paucity of studies on functional characterization of DBH variants, its structure-function relationship is poorly understood. The purpose of the study was to characterize five non-synonymous (ns) variants that were prioritized either based on previous association studies or Sorting Tolerant From Intolerant (SIFT) algorithm...
July 13, 2017: Neurogenetics
https://www.readbyqxmd.com/read/28705041/ionizing-radiation-induces-epha2-s897-phosphorylation-in-a-mek-erk-rsk-dependent-manner
#11
Paul R Graves, Shaun U Din, Mark Ashamalla, Hani Ashamalla, Thomas S K Gilbert, Lee M Graves
PURPOSE: The EphA2 tyrosine kinase is frequently overexpressed in human tumors that are also treated with radiation. However, few studies have examined the effect of radiation on the EphA2 receptor itself. The purpose of this project was to investigate the impact of radiation on EphA2 to better understand mechanisms of radioresistance. MATERIALS AND METHODS: Cell lines were exposed to X-rays and assayed for changes in EphA2 protein levels and phosphorylation over time by Western blotting...
July 14, 2017: International Journal of Radiation Biology
https://www.readbyqxmd.com/read/28704682/mutagenic-potential-of-hypoxanthine-in-live-human-cells
#12
Stephen DeVito, Jordan Woodrick, Linze Song, Rabindra Roy
Hypoxanthine (Hx) is a major DNA lesion generated by deamination of adenine during chronic inflammatory conditions, which is an underlying cause of various diseases including cancer of colon, liver, pancreas, bladder and stomach. There is evidence that deamination of DNA bases induces mutations, but no study has directly linked Hx accumulation to mutagenesis and strand-specific mutations yet in human cells. Using a site-specific mutagenesis approach, we report the first direct evidence of mutation potential and pattern of Hx in live human cells...
June 28, 2017: Mutation Research
https://www.readbyqxmd.com/read/28703801/lncrna-miat-functions-as-a-competing-endogenous-rna-to-upregulate-dapk2-by-sponging-mir-22-3p-in-diabetic-cardiomyopathy
#13
Xiang Zhou, Wei Zhang, Mengchao Jin, Jianchang Chen, Weiting Xu, Xiangqing Kong
We previously established a rat model of diabetic cardiomyopathy (DCM) and found that the expression of long non-coding RNA myocardial infarction-associated transcript (MIAT) was significantly upregulated. The present study was aimed to determine the pathologic role of MIAT in the development of DCM. MIAT knockdown was found to reduce cardiomyocyte apoptosis and improve left ventricular function in diabetic rats. High glucose could increase MIAT expression and induce apoptosis in cultured neonatal cardiomyocytes...
July 13, 2017: Cell Death & Disease
https://www.readbyqxmd.com/read/28702714/immunodeficiency-in-two-female-patients-with-incontinentia-pigmenti-with-heterozygous-nemo-mutation-diagnosed-by-lps-unresponsiveness
#14
Hidenori Ohnishi, Yuka Kishimoto, Tomohide Taguchi, Norio Kawamoto, Mina Nakama, Tomoki Kawai, Manabu Nakayama, Osamu Ohara, Kenji Orii, Toshiyuki Fukao
PURPOSE: Anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID) is caused by mutations in the NF-κB essential modulator (NEMO) or NF-κB inhibitor, alpha (IKBA) genes. A heterozygous NEMO mutation causes incontinentia pigmenti (IP) in females, while a hemizygous hypomorphic mutation of NEMO causes EDA-ID in males. In general, immunodeficiency is not shown in IP patients. Here, we investigated two female patients with IP and immunodeficiency. METHODS: The patients were initially suspected to have IRAK4 deficiency and Mendelian susceptibility to mycobacterial disease, respectively, because of recurrent pneumonia with delayed umbilical cord detachment or disseminated mycobacterial infectious disease...
July 12, 2017: Journal of Clinical Immunology
https://www.readbyqxmd.com/read/28702328/ask1-map3k5-is-transcriptionally-upregulated-by-e2f1-in-adipose-tissue-in-obesity-molecularly-defining-a-human-dys-metabolic-obese-phenotype
#15
Yulia Haim, Matthias Blüher, Daniel Konrad, Nir Goldstein, Nora Klöting, Ilana Harman-Boehm, Boris Kirshtein, Doron Ginsberg, Tanya Tarnovscki, Yftach Gepner, Iris Shai, Assaf Rudich
OBJECTIVE: Obesity variably disrupts human health, but molecular-based patients' health-risk stratification is limited. Adipose tissue (AT) stresses may link obesity with metabolic dysfunction, but how they signal in humans remains poorly-characterized. We hypothesized that a transcriptional AT stress-signaling cascade involving E2F1 and ASK1 (MAP3K5) molecularly defines high-risk obese subtype. METHODS: ASK1 expression in human AT biopsies was determined by real-time PCR analysis, and chromatin immunoprecipitation (ChIP) adopted to AT explants was used to evaluate the binding of E2F1 to the ASK1 promoter...
July 2017: Molecular Metabolism
https://www.readbyqxmd.com/read/28701349/actin-cytoskeleton-dependent-regulation-of-corticotropin-releasing-factor-receptor-heteromers
#16
Burcu Hasdemir, Shilpi Mahajan, Juan Oses-Prieto, Shreya Chand, Michael Woolley, Alma Burlingame, Dimitris K Grammatopoulos, Aditi Bhargava
Stress responses are highly nuanced and variable, but how this diversity is achieved by modulating receptor function, is largely unknown. Corticotropin-releasing factor receptors (CRFRs), class B GPCRs, are pivotal in mediating stress responses. Here we show that the two known CRFRs interact to form heteromeric complexes in HEK293 cells co-expressing both CRFRs and in vivo in mouse pancreas. Co-immunoprecipitation and mass spectrometry confirmed the presence of both CRF1R and CRF2βR, along with actin in these heteromeric complexes...
July 12, 2017: Molecular Biology of the Cell
https://www.readbyqxmd.com/read/28699756/incorporation-of-a-biguanide-scaffold-enhances-drug-uptake-by-organic-cation-transporters-1-and-2
#17
Obinna N Obianom, Ana L Coutinho, Wei Yang, Hong Yang, Fengtian Xue, Yan Shu
Membrane transporters play a significant role in the transport of many endogenous and exogenous compounds. The knowledge of transporter substrate requirements has allowed further development of drugs that utilize them to ensure tissue permeation. In this study, we demonstrate that inclusion of a biguanide functionality can potentiate uptake by the organic cation transporters organic cation transporters 1 and 2 (OCT1 and OCT2). We synthesized 18 pairs of structurally diverse compounds, each pair consisting of a parent amino compound and its biguanide analog; and then assessed their cellular uptake in HEK293 cells overexpressing human OCT1 or OCT2...
July 12, 2017: Molecular Pharmaceutics
https://www.readbyqxmd.com/read/28699740/design-synthesis-and-evaluation-of-the-highly-selective-and-potent-g-protein-coupled-receptor-kinase-2-grk2-inhibitor-for-the-potential-treatment-of-heart-failure
#18
Tomohiro Okawa, Yoshio Aramaki, Mitsuo Yamamoto, Toshitake Kobayashi, Shoji Fukumoto, Yukio Toyoda, Tsutomu Henta, Akito Hata, Shota Ikeda, Manami Kaneko, Isaac D Hoffman, Bi-Ching Sang, Hua Zou, Tetsuji Kawamoto
A novel class of therapeutic drug candidates for heart failure, highly potent and selective GRK2 inhibitors, exhibit potentiation of β-adrenergic signaling in vitro studies. Hydrazone derivative 5 and 1,2,4-triazole derivative 24a were identified as hit compounds by HTS. New scaffold generation and SAR studies of all parts resulted in a 4-methyl-1,2,4-triazole derivative with an N-benzylcarboxamide moiety with highly potent activity towards GRK2, and selectivity over other kinases. In terms of subtype selectivity, these compounds showed enough selectivity against GRK1, 5, 6, 7 with almost equipotent inhibition to GRK3...
July 12, 2017: Journal of Medicinal Chemistry
https://www.readbyqxmd.com/read/28698187/the-stoichiometry-%C3%AE-4-3-%C3%AE-2-2-of-the-nicotinic-acetylcholine-receptor-predominates-in-the-rat-motor-cortex
#19
Kristen E DeDominicis, Niaz Sahibzada, Thao T Olson, Yingxian Xiao, Barry B Wolfe, Kenneth J Kellar, Robert P Yasuda
The α4β2 nicotinic acetylcholine receptor (nAChR) is important in normal CNS physiology and in mediating several of the pharmacological effects of nicotine on cognition, attention, and affective states. It is also the likely receptor that mediates nicotine addiction. This receptor assembles in two distinct stoichiometries: (α4)2(β2)3 and (α4)3(β2)2 that are referred to as high (HS) and low (LS) sensitivity nAChRs, respectively, based on a difference in the potency of acetylcholine to activate them. The physiological and pharmacological differences between these two receptor subtypes have been described in heterologous expression systems...
July 11, 2017: Molecular Pharmacology
https://www.readbyqxmd.com/read/28696213/ca-2-regulation-of-cav3-3-t-type-ca-2-channel-is-mediated-by-calmodulin
#20
Narae Lee, Sua Jeong, Kang-Chang Kim, Jin-Ah Kim, Jin-Yong Park, Ho-Won Kang, Edward Perez-Reyes, Jung-Ha Lee
Numerous investigations reported that increases of internal Ca(2+) (Ca(2+)i) pivotally regulate high voltage-activated (HVA) Ca(2+) channels via calmodulin (CaM). However, it is largely elusive that Ca(2+)i can regulate low voltage-activated T-type Ca(2+) channels. Using whole cell patch clamp, we compared the biophysical properties of Ca(2+) current through T-type Ca(2+) channel Cav3.1, Cav3.2, or Cav3.3 stably expressed in HEK293 cells between internal solutions containing 27 nM and l μM free Ca(2+) Both activation and inactivation kinetics of Cav3...
July 10, 2017: Molecular Pharmacology
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