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protein DNA binding emsa

Haojie Cao, Julio Villatoro-Hernandez, Ruud Detert Oude Weme, Elrike Frenzel, Oscar P Kuipers
Bacillus subtilis is extensively applied as a microorganism for the high-level production of heterologous proteins. Traditional strategies for increasing the productivity of this microbial cell factory generally focused on the targeted modification of rate-limiting components or steps. However, the longstanding problems of limited productivity of the expression host, metabolic burden and non-optimal nutrient intake, have not yet been completely solved to achieve significant production-strain improvements. To tackle this problem, we systematically rewired the regulatory networks of the global nitrogen and carbon metabolism by random mutagenesis of the pleiotropic transcriptional regulators CodY and CcpA, to allow for optimal nutrient intake, translating into significantly higher heterologous protein production yields...
August 7, 2018: Metabolic Engineering
Meiying Wu, Yuting Jiang, Mingyuan Liu, Yu Shang, Jing An
The amino polycyclic aromatic hydrocarbons (amino-PAHs) were frequently detected in PM2.5 , and it was suggested that they contributed to the harmful health effects associated with PM2.5 . However, the process through which amino-PAHs induce oxidative stress responses as well as the pro-inflammatory processes along with the associated mechanisms is still not well-known. In this study, oxidative stress level, Nrf2/ARE anti-oxidative defense responses, oxidative damages and cytokine expressions were investigated in the A549 cell line after it was treated with typical airborne amino-PAHs including 1-aminopyrene (1-AP) and 3-aminofluoranthene (3-AF)...
May 8, 2018: Toxicology Research
Emanuela Blanco, Wilma Sabetta, Donatella Danzi, Donatella Negro, Valentina Passeri, Antonino De Lisi, Francesco Paolocci, Gabriella Sonnante
Flavonoids are a well-studied group of secondary metabolites, belonging to the phenylpropanoid pathway. Flavonoids are known to exhibit health promoting effects such as antioxidant capacities, anti-cancer and anti-inflammatory activity. Globe artichoke is an important source of bioactive phenolic compounds, including flavonoids. To study the regulation of their biosynthesis, a R2R3-MYB transcription factor, CcMYB12 , was isolated from artichoke leaves. Phylogenetic analysis showed that this protein belongs to the MYB subgroup 7 (flavonol-specific MYB), which includes Arabidopsis AtMYB12, grapevine VvMYBF1, and tomato SlMYB12...
2018: Frontiers in Plant Science
Gaëtan Le-Bel, Claude J Giasson, Alexandre Deschambeault, Patrick Carrier, Lucie Germain, Sylvain L Guérin
Based on the use of tissue-cultured human corneal endothelial cells (HCECs), cell therapy is a very promising avenue in the treatment of corneal endothelial pathologies such as Fuchs' dystrophy, and post-surgical corneal edema. However, once in culture, HCECs rapidly lose their phenotypic and physiological characteristics, and are therefore unsuitable for the reconstruction of a functional endothelial monolayer. Expression of NFI, a transcription factor that can either function as an activator or a repressor of gene transcription, has never been examined in endothelial cells...
July 9, 2018: Experimental Eye Research
Duan Liu, Sisi Qin, Bamiki Ray, Krishna R Kalari, Liewei Wang, Richard M Weinshilboum
CYP1A1 expression can be up-regulated by the ligand-activated aryl hydrocarbon receptor (AHR). Based on prior observations with estrogen receptors and estrogen response elements, we tested the hypothesis that single-nucleotide polymorphisms (SNPs) mapping hundreds of base pairs (bp) from xenobiotic response elements (XREs) might influence AHR binding and subsequent gene expression. Specifically, we analyzed DNA sequences 5 Kb up- and down-stream of the CYP1A1 gene for putative XREs. SNPs located ±500 bp of these putative XREs were studied using a genomic data-rich human lymphoblastoid cell line (LCL) model system...
July 6, 2018: Drug Metabolism and Disposition: the Biological Fate of Chemicals
David I Bernstein, Zana L Lummus, Banu Kesavalu, Jianbo Yao, Leah Kottyan, Daniel Miller, André Cartier, Maria-Jesús Cruz, Catherine Lemiere, Xavier Muñoz, Santiago Quirce, Susan Tarlo, Joaquin Sastre, Louis Philippe Boulet, Matthew T Weirauch, Kenneth Kaufman
BACKGROUND: Isocyanates are major causes of occupational asthma, but susceptibility and mechanisms of diisocyanate-induced asthma (DA) remain uncertain. OBJECTIVE: The aim of this study was to identify DA-associated functional genetic variants through next-generation sequencing (NGS), bioinformatics, and functional assays. METHODS: NGS was performed in 91 workers with DA. Fourteen loci with known DA-associated single nucleotide polymorphisms (SNPs) were sequenced and compared with data from 238 unexposed subjects...
June 30, 2018: Journal of Allergy and Clinical Immunology
Kenji Ohe, Shinsuke Miyajima, Tomoko Tanaka, Yuriko Hamaguchi, Yoshihiro Harada, Yuta Horita, Yuki Beppu, Fumiaki Ito, Takafumi Yamasaki, Hiroki Terai, Masayoshi Mori, Yusuke Murata, Makito Tanabe, Ichiro Abe, Kenji Ashida, Kunihisa Kobayashi, Munechika Enjoji, Takashi Nomiyama, Toshihiko Yanase, Nobuhiro Harada, Toshiaki Utsumi, Akila Mayeda
Objectives: The high-mobility group A protein 1a (HMGA1a) protein is known as a transcription factor that binds to DNA, but recent studies have shown it exerts novel functions through RNA-binding. We were prompted to decipher the mechanism of HMGA1a-induced alternative splicing of the estrogen receptor alpha (ERα) that we recently reported would alter tamoxifen sensitivity in MCF-7 TAMR1 cells. Methods: Endogenous expression of full length ERα66 and its isoform ERα46 were evaluated in MCF-7 breast cancer cells by transient expression of HMGA1a and an RNA decoy (2'-O-methylated RNA of the HMGA1a RNA-binding site) that binds to HMGA1a...
2018: Frontiers in Molecular Biosciences
Yezi Zou, Zhiquan Chen, Jie Li, Wenyan Gong, Lei Zhang, Futian Xu, Lihao Chen, Peiqing Liu, Heqing Huang
Background: Progestin and adipoQ receptor 3 (PAQR3), is a Golgi-anchored membrane protein containing seven transmembrane helices. It has been demonstrated that PAQR3 mediates insulin resistance, glucose and lipid metabolism, and inflammation. In addition, kidney inflammatory fibrosis is an important pathological feature of diabetic nephropathy (DN). Therefore, we aimed to investigate the role of PAQR3 in diabetic kidney fibrosis as well as inflammation in DN. Object: The effect of PAQR3 on NF-κB signaling pathway, expressions of fibronectin (FN) and intercellular adhesion molecule-1 (ICAM-1) in glomerular mesangial cells (GMCs) cultured by high glucose (HG) were examined...
2018: Frontiers in Endocrinology
Marina Piscopo, Marco Trifuoggi, Rosaria Notariale, Sofiane Labar, Jacopo Troisi, Antonella Giarra, Dea Rabbito, Raffaela Puoti, Domenico de Benedictis, Maria Violetta Brundo, Adriana Basile, Katrina V Good, Juan Ausió, Giulia Guerriero
Here we report the industrial pollution effects due to cadmium on the reproductive health of Mytilus galloprovincialis. Mussels were removed from the biofouling of a Conatex panel after one year exposition at a polluted site near a disposal metallurgical factory. A high cadmium bioaccumulation was observed in the testis of mussels housed at the polluted site, with respect to a control site, as determined by inductively coupled plasma-mass spectrometry, along with a 10 fold increase in metallothionein 20 kDa gene (mt20) expression levels determined by qPCR...
2018: Acta Biochimica Polonica
Akira Kitamura, Ai Shibasaki, Kayo Takeda, Ryoji Suno, Masataka Kinjo
Normal function and abnormal aggregation of transactivation response (TAR) DNA/RNA-binding protein 43 kDa (TDP-43) are directly associated with the lethal genetic diseases: cystic fibrosis, amyotrophic lateral sclerosis (ALS), and frontotemporal lobar degeneration (FTLD). The binding of TDP-43 to single-stranded DNA (ssDNA) or RNA is involved in transcriptional repression, regulation of RNA splicing, and RNA stabilization. Equilibrium dissociation constants ( K d ) of TDP-43 and ssDNA or RNA have been determined using various methods; however, methods that can measure K d with high sensitivity in a short time using a small amount of TDP-43 in solution would be advantageous...
July 2018: Biochemistry and Biophysics Reports
Farah Hanan Abu Hanifiah, Siti Nor Akmar Abdullah, Ashida Othman, Noor Azmi Shaharuddin, Halimi Mohd Saud, Hasnul Abdul Hakim Hasnulhadi, Umaiyal Munusamy
TAAAAT and a novel motif, GCTTCA found in the oil palm stearoyl-ACP desaturase (SAD1) promoter are involved in regulating mesocarp-specific expression. Two key fatty acid biosynthetic genes, stearoyl-ACP desaturase (SAD1), and acyl-carrier protein (ACP3) in Elaeis guineensis (oil palm) showed high level of expression during the period of oil synthesis in the mesocarp [12-19 weeks after anthesis (w.a.a.)] and kernel (12-15 w.a.a.). Both genes are expressed in spear leaves at much lower levels and the expression increased by 1...
August 2018: Plant Cell Reports
Takashi Honda, Daichi Morimoto, Yoshihiko Sako, Takashi Yoshida
Previously, we showed that DNA replication and cell division in toxic cyanobacterium Microcystis aeruginosa are coordinated by transcriptional regulation of cell division gene ftsZ and that an unknown protein specifically bound upstream of ftsZ (BpFz; DNA-binding protein to an upstream site of ftsZ) during successful DNA replication and cell division. Here, we purified BpFz from M. aeruginosa strain NIES-298 using DNA-affinity chromatography and gel-slicing combined with gel electrophoresis mobility shift assay (EMSA)...
August 2018: Marine Biotechnology
Kyle K Biggar, Kenneth B Storey
In many cases, the DNA-binding activity of a transcription factor does not change, while its transcriptional activity is greatly influenced by the make-up of bound proteins. In this study, we assessed the protein composition and DNA-binding ability of the E2F transcription factor complex to provide insight into cell cycle control in an anoxia tolerant turtle through the use of a modified ELISA protocol. This modification also permits the use of custom DNA probes that are tailored to a specific DNA binding region, introducing the ability to design capture probes for non-model organisms...
2018: PeerJ
Park Ju Ho, Jang Jun Sung, Kim Ki Cheon, Hong Jin Tae
BACKGROUND: Centella asiatica phytosome (CA phytosome) has potent antioxidant and anti-inflammatory properties. However, its anti-dermatitic effect has not yet been reported. PURPOSE: We investigated the effects of CA phytosome on inflammatory reponses by macrophages in an atopic dermatitis (AD) mouse model. STUDY DESIGN: The effects of CA phytosome on atopic dermatitis were examined by using phthalic anhydride (PA)-induced AD mouse model and RAW 264...
April 1, 2018: Phytomedicine: International Journal of Phytotherapy and Phytopharmacology
Hsin-Pai Li, Chen-Ching Peng, Chih-Ching Wu, Chien-Hsun Chen, Meng-Jhe Shih, Mei-Yuan Huang, Yi-Ru Lai, Yung-Li Chen, Ting-Wen Chen, Petrus Tang, Yu-Sun Chang, Kai-Ping Chang, Cheng-Lung Hsu
BACKGROUND: Aberrant hypermethylation of cellular genes is a common phenomenon to inactivate genes and promote tumorigenesis in nasopharyngeal carcinoma (NPC). METHODS: Methyl binding domain (MBD)-ChIP sequencing of NPC cells, microarray data of NPC biopsies and gene ontology analysis were conducted to identify a potential tumor suppressor gene CLDN11 that was both hypermethylated and downregulated in NPC. Bisulfite sequencing, qRT-PCR, immunohistochemistry staining of the NPC clinical samples and addition of methylation inhibitor, 5'azacytidine, in NPC cells were performed to verify the correlation between DNA hypermethylation and expression of CLDN11...
May 10, 2018: Journal of Experimental & Clinical Cancer Research: CR
Yongjian Qiao, Tiantian Tong, Jiao Xue, Wenjing Lin, Zixin Deng, Yi-Qiang Cheng, Dongqing Zhu
DepR, a LysR-type transcriptional regulator encoded by the last gene of the putative min operon (orf21-20-19-depR) located at the downstream region of the anticancer agent FK228 biosynthetic gene cluster in Chromobacterium violaceum No. 968, positively regulates the biosynthesis of FK228. In this work, the mechanism underlining this positive regulation was probed by multiple approaches. Electrophoretic mobility shift assay (EMSA) and DNase I footprinting assay (DIFA) identified a conserved 35-nt DNA segment in the orf21-orf22 intergenic region where the purified recombinant DepR binds to...
2018: PloS One
Neil O Robertson, Ngaio C Smith, Athina Manakas, Mahiar Mahjoub, Gordon McDonald, Ann H Kwan, Jacqueline M Matthews
Intrinsically disordered regions are highly represented among mammalian transcription factors, where they often contribute to the formation of multiprotein complexes that regulate gene expression. An example of this occurs with LIM-homeodomain (LIM-HD) proteins in the developing spinal cord. The LIM-HD protein LHX3 and the LIM-HD cofactor LDB1 form a binary complex that gives rise to interneurons, whereas in adjacent cell populations, LHX3 and LDB1 form a rearranged ternary complex with the LIM-HD protein ISL1, resulting in motor neurons...
May 1, 2018: Proceedings of the National Academy of Sciences of the United States of America
Stephanie J Attarian, Sandra L Leibel, Ping Yang, Danielle N Alfano, Brian P Hackett, F Sessions Cole, Aaron Hamvas
BackgroundMutations in the NK2 homeobox 1 (NKX2-1) gene are associated with lung disease in infants and children. We hypothesize that disruption of normal surfactant gene expression with these mutations contributes to the respiratory phenotypes observed.MethodsTo assess transactivational activity, cotransfection of luciferase reporter vectors containing surfactant protein B or C (SFTPB or SFTPC) promoters with NKX2-1 plasmids was performed and luciferase activity was measured. To assess the binding of mutated proteins to target DNA, electrophoretic mobility shift assays (EMSA) were performed using nuclear protein labeled with oligonucleotide probes representing NKX2-1 consensus binding sequences followed by gel electrophoresis...
April 11, 2018: Pediatric Research
Linda Ye, Lei Gu, Joseph Caprioli, Natik Piri
Rbpms (RNA-binding protein with multiple splicing) is a member of the RRM (RNA Recognition Motif) family of RNA-binding proteins, which is expressed as multiple alternatively spliced transcripts encoding different protein isoforms. We have shown earlier that Rbpms expression in the retina is restricted to retinal ganglion cells (RGCs), and have characterized this gene as a marker for RGCs. The aim of this study was to identify isoforms representing Rbpms in human retinas and to analyze its transcriptional regulation...
August 2018: Molecular Genetics and Genomics: MGG
Chin-Shiu Huang, Haw-Wen Chen, Tzu-Yu Lin, Ai-Hsuan Lin, Chong-Kuei Lii
ETHNOPHARMACOLOGICAL RELEVANCE: Shikonin, a naphthoquinone pigment abundant in the root of the Chinese herb Lithospermum erythrorhizon, has been widely used to treat inflammatory diseases for thousands of years. Whether shikonin changes drug metabolism remains unclear. AIM OF THE STUDY: We investigated whether shikonin modulates the expression of hepatic drug-metabolizing enzymes and transporters as well as the possible mechanisms of this action. MATERIALS AND METHODS: Primary hepatocytes isolated from Sprague-Dawley rats were treated with 0-2 μM shikonin and the protein and mRNA levels of drug-metabolizing enzymes and transporters as well as the activation of aryl hydrocarbon receptor (AhR) and NF-E2-related factor 2 (Nrf2) were determined...
April 24, 2018: Journal of Ethnopharmacology
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