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protein DNA binding emsa

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https://read.qxmd.com/read/30738927/rapid-and-efficient-purification-of-drosophila-homeodomain-transcription-factors-for-biophysical-characterization
#1
Rachel Orlomoski, Aaron Bogle, Jeanmarie Loss, Rylee Simons, Jacqueline M Dresch, Robert A Drewell, Donald E Spratt
Homeodomain transcription factors (HD TFs) are a large class of evolutionarily conserved DNA binding proteins that contain a basic 60-amino acid region required for binding to specific DNA sites. In Drosophila melanogaster, many of these HD TFs are expressed in the early embryo and control transcription of target genes in development through interaction with cis-regulatory modules. Previous studies where some of the Drosophila HD TFs were purified required the use of strong denaturants (i.e. 6M urea) and multiple chromatography columns, making the downstream biochemical examination of the isolated protein difficult...
February 7, 2019: Protein Expression and Purification
https://read.qxmd.com/read/30698287/detection-of-rna-protein-interactions-using-a-highly-sensitive-non-radioactive-electrophoretic-mobility-shift-assay
#2
Gerasimos Daras, Anastasios Alatzas, Dikran Tsitsekian, Dimitris Templalexis, Stamatis Rigas, Polydefkis Hatzopoulos
Electrophoretic mobility shift assay (EMSA) is a sensitive technique useful in the identification and characterization of protein interactors with nucleic acids. This assay provides an efficient method to study DNA or RNA binding proteins and to identify nucleic acid substrates. The specific interaction plays important roles in many biological processes such as transcription, translation, splicing and global gene expression. In this article, we have modified the EMSA technique and developed a non-radioactive straightforward method to study and determine RNA-protein interactions...
January 30, 2019: Electrophoresis
https://read.qxmd.com/read/30599895/andrographis-paniculata-diterpenoids-and-ethanolic-extract-inhibit-tnf%C3%AE-induced-icam-1-expression-in-ea-hy926-cells
#3
Hung-Chih Lin, Chien-Chun Li, Ya-Chen Yang, Tzu-Hsuan Chiu, Kai-Li Liu, Chong-Kuei Lii, Haw-Wen Chen
BACKGROUND: Andrographis paniculata (A. paniculata), a traditional herb in Southeastern Asia, is used to treat inflammation-mediated diseases. PURPOSE: The two major bioactive diterpenoids in A. paniculata are andrographolide (AND) and 14-deoxy-11,12-didehydroandrographolide (deAND). Because of the anti-inflammatory evidence for AND, we hypothesized that deAND might possess similar potency for inhibiting monocyte adhesion to the vascular endothelium, which is a critical event for atherosclerotic lesion formation...
January 2019: Phytomedicine: International Journal of Phytotherapy and Phytopharmacology
https://read.qxmd.com/read/30599067/transcriptional-repression-of-the-ectodomain-sheddase-adam10-by-tbx2-and-potential-implication-for-alzheimer-s-disease
#4
Sven Reinhardt, Florian Schuck, Nicolai Stoye, Tobias Hartmann, Marcus O W Grimm, Gert Pflugfelder, Kristina Endres
BACKGROUND: The ADAM10-mediated cleavage of transmembrane proteins regulates cellular processes such as proliferation or migration. Substrate cleavage by ADAM10 has also been implicated in pathological situations such as cancer or Morbus Alzheimer. Therefore, identifying endogenous molecules, which modulate the amount and consequently the activity of ADAM10, might contribute to a deeper understanding of the enzyme's role in both, physiology and pathology. METHOD: To elucidate the underlying cellular mechanism of the TBX2-mediated repression of ADAM10 gene expression, we performed overexpression, RNAi-mediated knockdown and pharmacological inhibition studies in the human neuroblastoma cell line SH-SY5Y...
January 1, 2019: Cellular and Molecular Life Sciences: CMLS
https://read.qxmd.com/read/30589568/the-phosphorylatable-ser320-of-nf-ya-is-involved-in-dna-binding-of-the-nf-y-trimer
#5
Andrea Bernardini, Mariangela Lorenzo, Marco Nardini, Roberto Mantovani, Nerina Gnesutta
Nuclear factor Y (NF-Y) is a transcription factor trimer binding to the functionally important CCAAT box, present in promoters of growth-promoting and cell cycle-regulated genes. The regulatory nuclear factor YA (NF-YA) subunit confers sequence-specificity to the histone-like nuclear factor YB/YC dimer. NF-YA harbors 2 serines-Ser320 and Ser326-shown to be phosphorylated by cyclin-dependent kinase 2. High-throughput proteomics data indicate that they are phosphorylated in vivo. Specifically, Ser320 makes structural contacts with the DNA phosphate backbone; Ser320-P is the major NF-YA phosphorylation isoform following overexpression in HeLa cells, increasing upon mitotic arrest...
December 27, 2018: FASEB Journal: Official Publication of the Federation of American Societies for Experimental Biology
https://read.qxmd.com/read/30584709/-porcine-transcription-factor-ap-2%C3%AE-promotes-porcine-circovirus-type-2-replication-through-enhancing-the-activity-of-the-rep-gene-promoter
#6
Yue Wang, Dongfeng Song, Cui Lin, Jiarong Li, Shengnan Wang, Jinyan Gu, Jiyong Zhou
Several putative transcription factor binding sites (TFBSs) exist in the PCV2 rep gene promoter. To explore if porcine circovirus type 2 (PCV2) could regulate the viral replication by using these TFBSs, we conducted electrophoretic mobility shift assay (EMSA), DNA-pull down and liquid chromatography-tandem mass spectrometric (LC-MS/MS) assays. EMSA confirmed the binding activity of the rep gene promoter with nuclear proteins of host cells. DNA-pull down and LC-MS/MS identified the porcine transcription factor AP-2δ (poTFAP2δ) could bind the PCV2 rep gene promoter...
December 25, 2018: Sheng Wu Gong Cheng Xue Bao, Chinese Journal of Biotechnology
https://read.qxmd.com/read/30549224/tetrandrine-inhibits-human-brain-glioblastoma-multiforme-gbm-8401-cancer-cell-migration-and-invasion-in-vitro
#7
Yi-Wen Jiang, Hsin-Yu Cheng, Chao-Lin Kuo, Tzong-Der Way, Jin-Cherng Lien, Fu-Shin Chueh, Yun-Lian Lin, Jing-Gung Chung
Tetrandrine (TET) has been reported to induce anti-cancer activity in many human cancer cells and also to inhibit cancer cell migration and invasion. However, there are no reports to show TET inhibits cell migration and invasion in human brain glioblastoma multiforme GBM 8401 cells. In this study, we investigated the anti-metastasis effects of TET on GBM 8401 cells in vitro. Under sub-lethal concentrations (from 1, 5 up to 10 μM), TET significantly inhibited cell mobility, migration and invasion of GBM 8401 cells that were assayed by wound healing and Transwell assays...
December 13, 2018: Environmental Toxicology
https://read.qxmd.com/read/30498484/emrr-dependent-upregulation-of-the-efflux-pump-emrcab-contributes-to-antibiotic-resistance-in-chromobacterium-violaceum
#8
Kelly C M Barroso, Maristela Previato-Mello, Bianca B Batista, Juliana H Batista, José F da Silva Neto
Chromobacterium violaceum is an environmental Gram-negative bacterium that causes infections in humans. Treatment of C. violaceum infections is difficult and little is known about the mechanisms of antibiotic resistance in this bacterium. In this work, we identified mutations in the MarR family transcription factor EmrR and in the protein GyrA as key determinants of quinolone resistance in C. violaceum , and we defined EmrR as a repressor of the MFS-type efflux pump EmrCAB. Null deletion of emrR caused increased resistance to nalidixic acid, but not to other quinolones or antibiotics of different classes...
2018: Frontiers in Microbiology
https://read.qxmd.com/read/30478236/the-marr-family-regulator-bmrr-is-involved-in-bile-tolerance-of-bifidobacterium-longum-bbmn68-via-controlling-the-expression-of-an-abc-transporter
#9
Qi Xu, Zhengyuan Zhai, Haoran An, Yang Yang, Jia Yin, Guohong Wang, Fazheng Ren, Yanling Hao
In order to colonize the human gastrointestinal tract and exert their beneficial effects, bifidobacteria must effectively cope with the toxic bile salts in the intestine, but the molecular mechanism underlying bile tolerance is poorly understood. In this study, heterologous expression of a MarR family transcriptional regulator BmrR significantly reduced ox-bile resistance of Lactococcus lactis NZ9000, suggesting that it might play a role in bile stress response. In silico analysis combined with RT-PCR assay demonstrated that bmrR was co-transcribed with bmrA and bmrB , which encoded multidrug resistance (MDR) ABC transporters...
November 26, 2018: Applied and Environmental Microbiology
https://read.qxmd.com/read/30466615/icariin-abrogates-osteoclast-formation-through-the-regulation-of-the-rankl-mediated-traf6-nf-%C3%AE%C2%BAb-erk-signaling-pathway-in-raw264-7-cells
#10
Buyun Kim, Ki Yong Lee, Byoungduck Park
BACKGROUND: Icariin is pharmacologically active prenylated flavonoid glycoside that has various biologic effects such as antioxidant, anticancer, and anti-inflammatory activities. In addition, icariin has been used in Chinese medicine for thousands of years to treat osteoporosis and it is still being used today. However, direct mechanism of icariin in the treatment of bone disease is not understood. PURPOSE: The purpose of this study is to investigate whether icariin influences RANKL-induced osteoclast formation in murine macrophages...
December 1, 2018: Phytomedicine: International Journal of Phytotherapy and Phytopharmacology
https://read.qxmd.com/read/30429222/bombyx-mori-transcription-factors-foxa-and-sage-divergently-regulate-the-expression-of-wing-cuticle-protein-gene-4-during-metamorphosis
#11
Qihao Hu, Zidan Zhu, Danhui Zhao, Baojuan Zeng, Sichun Zheng, Qisheng Song, Huimin Deng, Qili Feng
Stage-specific gene expression governs metamorphosis of the silkworm, Bombyx mori. B. mori wing cuticle protein gene 4 (BmWCP4) is an essential gene for wing disc development expressed specifically during pupation. BmWCP4 transcription is suppressed at the larval stage by unknown mechanisms, which we sought to elucidate here. Bioinformatics analysis predicted seven potential Forkhead box (Fox) cis-regulatory elements (CRE) in the BmWCP4 promoter region, and we found that Fox CRE6 contributes to suppression of BmWCP4 expression...
November 14, 2018: Journal of Biological Chemistry
https://read.qxmd.com/read/30426431/identification-of-proteins-interacting-with-single-nucleotide-polymorphisms-snps-by-dna-pull-down-assay
#12
Bhupinder Singh, Swapan K Nath
Single nucleotide polymorphisms (SNPs) are the most abundantly found common form of DNA variation in the human genome. Many genetic association studies have proved that some of these SNPs are involved in regulating several cellular/physiological processes ranging from gene regulation to disease development. Analysis of the protein complex that binds to these SNPs is a crucial step in studying the mechanisms by which gene expressions are regulated in cis- or trans-acting manner. Commonly used techniques to determine DNA-protein interaction, such as electrophoretic mobility shift assay (EMSA), have limited value for simultaneously analyzing a large number of proteins in the complex...
2019: Methods in Molecular Biology
https://read.qxmd.com/read/30391287/silencing-of-lncrna-steroid-receptor-rna-activator-attenuates-polycystic-ovary-syndrome-in-mice
#13
Yan Li, Wanqiu Zhao, Haixu Wang, Chen Chen, Dongmei Zhou, Shengnan Li, Xiaohong Zhang, Haibo Zhao, Dangxia Zhou, Biliang Chen
BACKGROUND: Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in women of reproductive age and has a prevalence of 1 in 15 women worldwide. This study aims to investigate the role of lncRNA SRA in the pathological processes of polycystic ovary syndrome (PCOS). METHODS: Twenty five-day old female C57BL/6 mice received subcutaneous injection of 60 mg/kg dehydroepiandrosterone for 20 days to induce PCOS. Lentivirus containing lncRNA SRA-specific shRNA was subcapsularly injected into the ovaries of PCOS mice...
November 1, 2018: Biochimie
https://read.qxmd.com/read/30352970/new-insight-into-the-molecular-mechanism-of-the-fut2-regulating-escherichia-coli-f18-resistance-in-weaned-piglets
#14
Zhengchang Wu, Haiyue Feng, Yue Cao, Yanjie Huang, Chaohui Dai, Shenglong Wu, Wenbin Bao
Escherichia coli ( E. coli ) F18 is the main pathogen responsible for post-weaning diarrhea (PWD) in piglets. Resistance to E. coli F18 depends on the expression of the cognate receptors in the intestinal epithelial cells. However, the molecular mechanism of E. coli F18 resistance in weaned piglets remains unclear. Here, we performed a comparative transcriptome study of the duodenal tissue from Sutai E. coli F18 sensitive and resistant pigs by RNA-seq, and pig α(1,2) fucosyltransferase 2 ( FUT2 ) was identified as a host differentially expressed gene controlling the E...
October 24, 2018: International Journal of Molecular Sciences
https://read.qxmd.com/read/30333961/telomeric-repeat-binding-factor-homologs-in-entamoeba-histolytica-new-clues-for-telomeric-research
#15
Francisco Javier Rendón-Gandarilla, Víctor Álvarez-Hernández, Elizabeth J Castañeda-Ortiz, Helios Cárdenas-Hernández, Rosa Elena Cárdenas-Guerra, Jesús Valdés, Abigail Betanzos, Bibiana Chávez-Munguía, Anel Lagunes-Guillen, Esther Orozco, Lilia López-Canovas, Elisa Azuara-Liceaga
Telomeric Repeat Binding Factors (TRFs) are architectural nuclear proteins with critical roles in telomere-length regulation, chromosome end protection and, fusion prevention, DNA damage detection, and senescence regulation. Entamoeba histolytica , the parasite responsible of human amoebiasis, harbors three homologs of human TRFs, based on sequence similarities to their Myb DNA binding domain. These proteins were dubbed EhTRF-like I, II and III. In this work, we revealed that EhTRF-like I and II share similarity with human TRF1, while EhTRF-like III shares similarity with human TRF2 by in silico approach...
2018: Frontiers in Cellular and Infection Microbiology
https://read.qxmd.com/read/30310263/activator-protein-1-contributes-to-the-nacl-induced-expression-of-vegf-and-plgf-in-rpe-cells
#16
Jana Kleiner, Margrit Hollborn, Peter Wiedemann, Andreas Bringmann
Purpose: Systemic hypertension is a risk factor of neovascular age-related macular degeneration; consumption of dietary salt resulting in extracellular hyperosmolarity is a main cause of hypertension. Extracellular hyperosmolarity was shown to induce expression of angiogenic growth factors, such as vascular endothelial growth factor (VEGF) and placental growth factor (PlGF), in RPE cells. The aim of the present study was to determine whether the hyperosmotic expression of growth factor genes in RPE cells is mediated by activator protein-1 (AP-1), and whether c-Fos and c-Jun genes are regulated by extracellular osmolarity...
2018: Molecular Vision
https://read.qxmd.com/read/30278355/a-novel-transcription-factor-rwdd1-and-its-sumoylation-inhibit-the-expression-of-sqr-a-key-gene-of-mitochondrial-sulfide-metabolism-in-urechis-unicinctus
#17
Xueyu Li, Xiaolong Liu, Zhenkui Qin, Maokai Wei, Xitan Hou, Tingting Zhang, Zhifeng Zhang
Sulfide-quinone oxidoreductase (SQR) is a key enzyme of sulfide metabolism in metazoans, and responsible for oxidizing sulfide into thiosulfate and transmitting the generated electrons to the ubiquinone. It has been revealed that the sqr mRNA level increases significantly in echiuran worm Urechis unicinctus exposed to sulfide, and HSF1, NF1 and Sp1 have been verified to participate in its transcriptional regulation. In this study, we obtained 23 potential transcription factors interacting possibly with the proximal region (-391 to +50) of sqr promoter, and focused on the RWD domain-containing 1 (Rwdd1), a protein with the maximum number of clones in yeast one-hybrid (Y1H) screening, to investigate its transcriptional regulation to U...
November 2018: Aquatic Toxicology
https://read.qxmd.com/read/30278286/potential-in-vitro-and-ex-vivo-targeting-of-bzip53-involved-in-stress-response-and-seed-maturation-in-arabidopsis-thaliana-by-five-designed-peptide-inhibitors
#18
Prateek Jain, Koushik Shah, Vikas Rishi
Basic leucine zipper (bZIP) transcription factors (TFs) are eukaryote-specific proteins that bind to DNA as a homodimer or heterodimer and regulate gene expression. They are involved in several biological processes in plants; therefore inhibiting bZIP-DNA binding activity by targeting protein-protein interface is an attractive proposition with aspects of both basic and applied biology. Here, we describe the equilibrium and kinetic interactions studies of a designed peptide inhibitor A-ZIP53 and its four variants with the bZIP53 protein, a key regulator of seed maturation phase and stress response in Arabidopsis...
December 2018: Biochimica et biophysica acta. Proteins and proteomics
https://read.qxmd.com/read/30266832/structural-and-functional-characterization-of-the-transcriptional-regulator-rv3488-of-mycobacterium-tuberculosis-h37rv
#19
Meera Kumari, Ravi Kant Pal, Alok K Mishra, Sarita Tripathi, Bichitra Kumar Biswal, Kishore K Srivastava, Ashish Arora
Rv3488 of Mycobacterium tuberculosis H37Rv has been assigned to the phenolic acid decarboxylase repressor (PadR) family of transcriptional regulators that play key roles in multi-drug resistance and virulence of prokaryotes. The binding of cadmium, zinc, and several other metals to Rv3488 was discovered and characterized by ITC to be an exothermic process. Crystal structures of apo-Rv3488 and Rv3488 in complex with cadmium or zinc ions were determined by X-ray crystallography. Structure of Rv3488 revealed a dimeric protein with N-terminal winged-helix-turn-helix DNA-binding domains composed of helices α1, α2, α3, strand β1 and β2, with the dimerization interface being formed of helices α4 and α1...
September 28, 2018: Biochemical Journal
https://read.qxmd.com/read/30243709/coordinate-regulation-by-transcription-factors-and-dna-methylation-in-the-core-promoter-region-of-sirt6-in-bovine-adipocytes
#20
Jie-Yun Hong, Chu-Gang Mei, Shi-Jun Li, Hong-Bao Wang, Chun-Ping Zhao, Lin-Sen Zan
Sirtuin6 (SIRT6) is an ADP-ribosyltransferase and NAD+ -dependent deacylase of acetyl groups and long-chain fatty acyl groups, and has been shown as a regulator of insulin secretion, glucose metabolism, lipid metabolism, and cancer. In this study, we determined that the bovine SIRT6 showed higher levels of mRNA expression in the testis, longissimus thoracis, and subcutaneous fat tissue. To elucidate the molecular regulation mechanism of bovine SIRT6 expression, we obtained a 2-kb fragment containing the 5'-regulatory region, and the functional proximal minimal promoter of bovine SIRT6 was identified in the -472/-73 bp region...
September 21, 2018: Archives of Biochemistry and Biophysics
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