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Yun Kyoung Ryu, Reilley P Mathena, Sanghee Lim, Minhye Kwak, Michael Xu, Cyrus D Mintz
BACKGROUND: Early postnatal exposure to general anesthetic agents causes a lasting impairment in learning and memory in animal models. One hypothesis to explain this finding is that exposure to anesthetic agents during critical points in neural development disrupts the formation of brain circuitry. Here, we explore the effects of sevoflurane on the neuronal growth cone, a specialization at the growing end of axons and dendrites that is responsible for the targeted growth that underlies connectivity between neurons...
October 2016: Journal of Neurosurgical Anesthesiology
Jaime I Davila, Numrah M Fadra, Xiaoke Wang, Amber M McDonald, Asha A Nair, Barbara R Crusan, Xianglin Wu, Joseph H Blommel, Jin Jen, Kandelaria M Rumilla, Robert B Jenkins, Umut Aypar, Eric W Klee, Benjamin R Kipp, Kevin C Halling
BACKGROUND: RNA-seq is a well-established method for studying the transcriptome. Popular methods for library preparation in RNA-seq such as Illumina TruSeq® RNA v2 kit use a poly-A pulldown strategy. Such methods can cause loss of coverage at the 5' end of genes, impacting the ability to detect fusions when used on degraded samples. The goal of this study was to quantify the effects RNA degradation has on fusion detection when using poly-A selected mRNA and to identify the variables involved in this process...
October 20, 2016: BMC Genomics
Seiji Takeuchi, Shintaro Iwama, Hiroshi Takagi, Atsushi Kiyota, Kohtaro Nakashima, Hisakazu Izumida, Haruki Fujisawa, Naoko Iwata, Hidetaka Suga, Takashi Watanabe, Kozo Kaibuchi, Yutaka Oiso, Hiroshi Arima, Yoshihisa Sugimura
Arginine vasopressin (AVP) is secreted via exocytosis; however, the precise molecular mechanism underlying the exocytosis of AVP remains to be elucidated. To better understand the mechanisms of AVP secretion, in our study we have identified proteins that bind with a 25 kDa synaptosomal-associated protein (SNAP25). SNAP25 plays a crucial role in exocytosis, in the posterior pituitary. Embryonic stem (ES) cell-derived AVP neurons were established to investigate the functions of the identified proteins. Using glutathione S-transferase (GST)-pulldown assays and proteomic analyses, we identified tomosyn-1 (syntaxin-binding protein 5) as a SNAP25-binding protein in the posterior pituitary...
2016: PloS One
Samuel Génier, Jade Degrandmaison, Pierrick Moreau, Pascale Labrecque, Terence E Hébert, Jean-Luc Parent
Mechanisms that prevent aggregation and promote folding of nascent G protein-coupled receptors (GPCRs) remain poorly understood. We identified CCT7 as an interacting partner of the β-isoform of thromboxane A2 receptor (TPβ) by yeast two-hybrid screening. CCT7 coimmunoprecipitated with overexpressed TPβ and β2-adrenergic receptor (β2AR) in HEK 293 cells, but also with endogenous β2AR. CCT7 depletion by siRNA reduced total and cell surface expression of both receptors, and caused redistribution of the receptors to juxta-nuclear aggresomes, significantly moreso for TPβ than β2AR...
October 5, 2016: Molecular Biology of the Cell
Josué Flores-Kim, Andrew J Darwin
: The Phage shock protein (Psp) system is a widely conserved cell envelope stress response that is essential for the virulence of some bacteria, including Yersinia enterocolitica Recruitment of PspA by the inner membrane PspB•PspC complex characterizes the activated state of this response. The PspB•PspC complex has been proposed to be a stress-responsive switch, changing from an OFF to an ON state in response to an inducing stimulus. In the OFF state, PspA cannot access its binding site in the C-terminal cytoplasmic domain of PspC (PspC(CT)) because this site is bound to PspB...
October 3, 2016: Journal of Bacteriology
Jianjun Luo, Xiexiong Deng, Christopher Buehl, Xinjing Xu, Min-Hao Kuo
To ensure genome stability during cell division, all chromosomes must attach to spindles emanating from the opposite spindle pole bodies before segregation. The tension between sister chromatids generated by the poleward pulling force is an integral part of chromosome bi-orientation. In budding yeast, the residue Gly44 of histone H3 is critical for retaining the conserved Shugoshin protein Sgo1p at the pericentromeres for monitoring the tension status during mitosis. Studies carried out in this work showed that Lys42, Gly44, and Thr45 of H3 form the core of a tension sensing motif, TSM...
September 26, 2016: Genetics
Jayanta K Chakrabarty, Aishwarya G Naik, Michael B Fessler, Gerhard R Munske, Saiful M Chowdhury
Chemical cross-linking and mass spectrometry are now widely used to analyze large-scale protein-protein interactions. The major challenge in cross-linking approaches is the complexity of the mass spectrometric data. New approaches are required that can identify cross-linked peptides with high-confidence and establish a user-friendly analysis protocol for the biomedical scientific community. Here, we introduce a novel cross-linker that can be selectively cleaved in the gas phase using two differential tandem mass-spectrometric fragmentation methods, such as collision-induced or electron transfer dissociation (CID and ETD)...
October 18, 2016: Analytical Chemistry
Sheng Shen, Han Liu, Yueqi Wang, Jiwen Wang, Xiaolin Ni, Zhilong Ai, Hongtao Pan, Houbao Liu, Yebo Shao
Deleted in malignant brain tumors 1 (DMBT1) is deleted during cancer progression and as a potential tumor-suppressor gene in various types of cancer. However, its role in Gallbladder cancer remains poorly understood. DMBT1 has low-expression and deletion of copy number were detected in normal tissues and GBC cancer tissues by qRT-PCR. Knockdown of DMBT1 increased migration and invasion and overexpressed DMBT1 impaired migration and invasion in GBC cells. We also evaluated the molecular mechanism of DMBT1 by RNA sequencing and GSEA analysis...
September 14, 2016: Oncotarget
Gang Li, Pierre Cunin, Di Wu, Dorothée Diogo, Yu Yang, Yukinori Okada, Robert M Plenge, Peter A Nigrovic
Understanding the implications of genome-wide association studies (GWAS) for disease biology requires both identification of causal variants and definition of how these variants alter gene function. The non-coding triallelic dinucleotide polymorphism CCR6DNP is associated with risk for rheumatoid arthritis, and is considered likely causal because allelic variation correlates with expression of the chemokine receptor CCR6. Using transcription activator-like effector nuclease (TALEN) gene editing, we confirmed that CCR6DNP regulates CCR6...
September 2016: PLoS Genetics
Matthew Dean, Adam Lassak, Anna Wilk, Adriana Zapata, Luis Marrero, Patricia Molina, Krzysztof Reiss
Ethanol plays a detrimental role in the development of the brain. Multiple studies have shown that ethanol inhibits insulin-like growth factor I receptor (IGF-IR) function. Because the IGF-IR contributes to brain development by supporting neural growth, survival, and differentiation, we sought to determine the molecular mechanism(s) involved in ethanol's effects on this membrane-associated tyrosine kinase. Using multiple neuronal cell types, we performed Western blot, immunoprecipitation, and GST-pulldowns following acute (1 - 24 hours) or chronic (3 weeks) treatment with ethanol...
September 8, 2016: Journal of Cellular Physiology
Jinghan Wang, Shucheng Chen, Yajin Liao, Enyu Zhang, Shuo Feng, Shaoxiong Yu, Lian-Feng Li, Wen-Rui He, Yongfeng Li, Yuzi Luo, Yuan Sun, Mo Zhou, Xiao Wang, Muhammad Munir, Su Li, Hua-Ji Qiu
: Mitogen-activated protein kinase kinase/extracellular regulated kinase (MEK1/2/ERK1/2) cascade is involved in the replication of several members of the Flaviviridae family including hepatitis C virus and dengue virus. The effects of the cascade on the replication of classical swine fever virus (CSFV), a fatal pestivirus of pigs, remain unknown. In this study, MEK2 was identified as a novel binding partner of the E2 protein of CSFV using yeast two-hybrid screening. The E2-MEK2 interaction was confirmed by glutathione S-transferase pulldown, coimmunoprecipitation, and laser confocal microscopy assays...
September 7, 2016: Journal of Virology
Kousuke Uranishi, Tadayuki Akagi, Hiroshi Koide, Takashi Yokota
Estrogen-related receptor beta (Esrrb) is expressed in embryonic stem (ES) cells and is involved in self-renewal ability and pluripotency. Previously, we found that Dax1 is associated with Esrrb and represses its transcriptional activity. Further, the disruption of the Dax1-Esrrb interaction increases the expression of the extra-embryonic endoderm marker Gata6 in ES cells. Here, we investigated the influences of Esrrb and Dax1 on Gata6 expression. Esrrb overexpression in ES cells induced endogenous Gata6 mRNA and Gata6 promoter activity...
September 30, 2016: Biochemical and Biophysical Research Communications
Yufeng Tian, Grzegorz Gawlak, Xinyong Tian, Alok S Shah, Nicolene Sarich, Sandra Citi, Anna A Birukova
Agonist-induced activation of Rho GTPase signaling leads to endothelial cell (EC) permeability and may culminate to pulmonary edema, a devastating complication of acute lung injury. Cingulin is an adaptor protein first discovered in epithelium and involved in the organization of the tight junctions. This study investigated the role of cingulin in control of agonist-induced lung EC permeability via interaction with RhoA-specific activator GEF-H1. SiRNA-induced cingulin knockdown augmented thrombin-induced EC permeability monitored by measurements of transendothelial electrical resistance and endothelial cell permeability for macromolecules...
September 2, 2016: Journal of Biological Chemistry
Johannes von Burstin, Friedrich Bachhuber, Mariel Paul, Roland M Schmid, Anil K Rustgi
Pancreatic ductal adenocarcinoma (PDAC) remains one of the most deadly types of cancer, and the majority of pancreatic cancer deaths is caused by metastasis. Therapeutic options for systemic disease are limited, in particular due to the heterogeneous events leading to tumor dissemination. Previous studies highlighted an association of the homeodomain transcription factor MEIS1 with a ductal phenotype in pancreatic tissue architecture. Using immunohistochemistry, we demonstrate that MEIS1 is expressed in aberrant duct structures of Ela-TGFα transgenic mice as well as in pancreatic intraepithelial neoplasia (PanINs), primary PDAC, and metastatic disease in Ptf1a(Cre/+) ;LSL-Kras(G12D/+) mice...
September 1, 2016: Molecular Carcinogenesis
Emily Chen, Meng S Choy, Katalin Petrényi, Zoltán Kónya, Ferenc Erdődi, Viktor Dombrádi, Wolfgang Peti, Rebecca Page
UNLABELLED: The opportunistic pathogen Candida is one of the most common causes of nosocomial bloodstream infections. Because candidemia is associated with high mortality rates and because the incidences of multidrug-resistant Candida are increasing, efforts to identify novel targets for the development of potent antifungals are warranted. Here, we describe the structure and function of the first member of a family of protein phosphatases that is specific to fungi, protein phosphatase Z1 (PPZ1) from Candida albicans We show that PPZ1 not only is active but also is as susceptible to inhibition by the cyclic peptide inhibitor microcystin-LR as its most similar human homolog, protein phosphatase 1α (PP1α [GLC7 in the yeast Saccharomyces cerevisiae])...
2016: MBio
Van T T Huynh, Yun-Sook Lim, Si C Tran, Tu M Pham, Lam N Nguyen, Soon B Hwang
The propagation of hepatitis C virus (HCV) is highly dependent on host cellular factors. In order to identify the cellular factors involved in HCV propagation, we have previously performed protein microarray assays using the HCV nonstructural 5A (NS5A) protein as a probe. Out of ~9,000 host proteins immobilized in a microarray, approximately 90 cellular proteins were identified as HCV NS5A interacting partners. Of these candidates, we selected Abelson interactor 1 (Abi1) for further characterization. Binding of HCV NS5A to Abi1 was verified by both in vitro pulldown and coimmunoprecipitation assays...
August 22, 2016: Journal of Biological Chemistry
David Baillat, William K Russell, Eric J Wagner
The Integrator Complex (INT) is a large multi-subunit protein complex, containing at least 14 subunits and a host of associated factors. These protein components have been established through pulldowns of overexpressed epitope tagged subunits or by using antibodies raised against specific subunits. Here, we utilize CRISPR/Cas9 gene editing technology to introduce N-terminal FLAG epitope tags into the endogenous genes that encode Integrator subunit 4 and 11 within HEK293T cells. We provide specific details regarding design, approaches for facile screening, and our observed frequency of successful recombination...
December 2016: Protein Expression and Purification
Balaje Vijayaraghavan, Mohammed Hakim Jafferali, Ricardo A Figueroa, Einar Hallberg
Samp1 is a transmembrane protein of the inner nuclear membrane (INM), which interacts with the nuclear lamina and the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex in interphase and during mitosis, it localizes to the mitotic spindle. Samp1 was recently found to coprecipitate a protein complex containing Ran, a GTPase with fundamental regulatory functions both in interphase and in mitosis. To investigate the interaction between Samp1 and Ran in further detail, we have designed and expressed recombinant fusion proteins of the Chaetomium thermophilum homolog of Samp1 (Ct...
July 3, 2016: Nucleus
Andrea Di Francesco, Clara Di Germanio, Amaresh C Panda, Phu Huynh, Robert Peaden, Ignacio Navas-Enamorado, Paul Bastian, Elin Lehrmann, Alberto Diaz-Ruiz, David Ross, David Siegel, Jennifer L Martindale, Michel Bernier, Myriam Gorospe, Kotb Abdelmohsen, Rafael de Cabo
NAD(P)H: quinone oxidoreductase (NQO1) is essential for cell defense against reactive oxidative species, cancer, and metabolic stress. Recently, NQO1 was found in ribonucleoprotein (RNP) complexes, but NQO1-interacting mRNAs and the functional impact of such interactions are not known. Here, we used ribonucleoprotein immunoprecipitation (RIP) and microarray analysis to identify comprehensively the subset of NQO1 target mRNAs in human hepatoma HepG2 cells. One of its main targets, SERPINA1 mRNA, encodes the serine protease inhibitor α-1-antitrypsin, A1AT, which is associated with disorders including obesity-related metabolic inflammation, chronic obstructive pulmonary disease (COPD), liver cirrhosis and hepatocellular carcinoma...
August 8, 2016: Free Radical Biology & Medicine
F Ng, E Bødtkjer, M J Caulfield, S Ye
OBJECTIVE: Genomewide-association studies have identified a number of loci influencing blood pressure, one of which is located near SLC4A7 (lead SNP rs13082711). SLC4A7 encodes an electroneutral Na+, HCO3- co-transporter, NBCn1, which contributes to intracellular pH (pHi) regulation in a range of tissues, including vascular smooth muscle and endothelium. Additionally, the NBCn1 knockout mouse demonstrates an altered blood pressure phenotype. We investigated the potential influence of SLC4A7 genotype on DNA-protein interaction, gene expression, and pHi regulation...
September 2016: Journal of Hypertension
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