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Hong Qi, Aiping Li
The present study aimed to investigate the effects of JWA knockout (JWA-/- ) on malignant transformation of murine embryonic fibroblast (MEF) cells using a conditional JWA-/- mouse model. Once MEF cells were prepared, the potential role of JWA-/- on proliferation, migration, invasion and colony formation of MEF cells was investigated by cytological examination. The effects of JWA-/- on the regulation and protein expression levels of epithelial-mesenchymal transition (EMT)-related proteins in MEF cells, including poly(ADP-ribose) polymerase-1 (PARP-1), vimentin, β-catenin and E-cadherin, were investigated using western blot analysis...
April 2018: Experimental and Therapeutic Medicine
Jolanta Sroka, Eliza Zimolag, Slawomir Lasota, Wlodzimierz Korohoda, Zbigniew Madeja
Electrotaxis plays an important role during embryogenesis, inflammation, wound healing, and tumour metastasis. However, the mechanisms at play during electrotaxis are still poorly understood. Therefore intensive studies on signaling pathways involved in this phenomenon should be carried out. In this chapter, we described an experimental system for studying electrotaxis of Amoeba proteus, mouse embryonic fibroblasts (MEF), Walker carcinosarcoma cells WC256, and bone marrow adherent cells (BMAC).
2018: Methods in Molecular Biology
Yuqian Jiang, Enshun Xu, Jin Zhang, Mingyi Chen, Elsa Flores, Xinbin Chen
The RNA-binding protein Rbm38 is a target of p63 tumor suppressor and can in-turn repress p63 expression via mRNA stability. Thus, Rbm38 and p63 form a negative feedback loop. To investigate the biological significance of the Rbm38-p63 loop in vivo, a cohort of WT, Rbm38-/ - , TAp63+/- , and Rbm38-/- ;TAp63+/- mice were generated and monitored throughout their lifespan. While mice deficient in Rbm38 or TAp63 alone died mostly from spontaneous tumors, compound Rbm38-/- ;TAp63+/- mice had an extended lifespan along with reduced tumor incidence...
March 9, 2018: Oncogene
G Hawa, Linda Sonnleitner, A Missbichler, A Prinz, G Bauer, C Mauracher
Although Enzyme Linked Immuno Sorbent Assay (ELISA) technology is approaching it's 45th year of existence since first described in 1971, it is still the main diagnostic tool in clinical research and routine diagnostics. However, despite its broad usage it suffers from some drawbacks, limiting its use especially in more advanced assay formats like multiplexing platforms, point of care devices or protein arrays. Those limitations result from the need for an enzyme label, a soluble enzyme substrate, washing steps (multiplexing, point care, arrays) and in some cases also insufficient sensitivity, because the majority of circulating proteins and thus potential biomarkers may be found in lowor sub-picomolar concentrations...
March 5, 2018: Analytical Biochemistry
Johanna Fitz, Tobias Neumann, Rushad Pavri
Spt5 is a highly conserved RNA polymerase II (Pol II)-associated pausing and elongation factor. However, its impact on global elongation and Pol II processivity in mammalian cells has not been clarified. Here, we show that depleting Spt5 in mouse embryonic fibroblasts (MEFs) does not cause global elongation defects or decreased elongation rates. Instead, in Spt5-depleted cells, a fraction of Pol II molecules are dislodged during elongation, thus decreasing the number of Pol II complexes that complete the transcription cycle...
March 7, 2018: EMBO Journal
Tatyana Danyukova, Khandsuren Ariunbat, Melanie Thelen, Nahal Brocke-Ahmadinejad, Sara E Mole, Stephan Storch
Defects in the MFSD8 gene encoding the lysosomal membrane protein CLN7 lead to CLN7 disease, a neurodegenerative lysosomal storage disorder belonging to the group of neuronal ceroid lipofuscinoses (NCLs). Here we have performed a SILAC-based quantitative analysis of the lysosomal proteome using Cln7-deficient mouse embryonic fibroblasts (MEFs) from a Cln7 knockout (ko) mouse model. From 3335 different proteins identified, we detected 56 soluble lysosomal proteins and 29 highly abundant lysosomal membrane proteins...
March 5, 2018: Human Molecular Genetics
Esra Dilek, Sema Caglar, Kübra Erdogan, Bülent Caglar, Onur Sahin
In this study, the effects of four novel mononuclear palladium(II) and platinum(II) complexes on the activity of human serum paraoxanase1 were examined. First, four novel mononuclear palladium(II) and platinum(II) complexes were synthesized with a nitrogen donor ligand 1-(2-aminoethyl)pyrrolidine and nonsteroidal anti-inflammatory drugs diclofenac, mefenamic acid. These complexes were characterized by spectroscopic, thermal, and elemental analyses. The crystal structures of complex [Pd(2-amepyr)2 ](dicl)2 1 and [Pd(2-amepyr)2 ](mef)2 3 were determined by X-ray crystallography...
March 7, 2018: Journal of Biochemical and Molecular Toxicology
Cinzia Antognelli, Eliana Trapani, Simona Delle Monache, Andrea Perrelli, Claudia Fornelli, Francesca Retta, Paola Cassoni, Vincenzo Nicola Talesa, Saverio Francesco Retta
This article contains additional data related to the original research article entitled "KRIT1 loss-of-function induces a chronic Nrf2-mediated adaptive homeostasis that sensitizes cells to oxidative stress: implication for Cerebral Cavernous Malformation disease" (Antognelli et al., 2017) [1]. Data were obtained by si-RNA-mediated gene silencing, qRT-PCR, immunoblotting, and immunohistochemistry studies, and enzymatic activity and apoptosis assays. Overall, they support, complement and extend original findings demonstrating that KRIT1 loss-of-function induces a redox-sensitive and JNK-dependent sustained upregulation of the master Nrf2 antioxidant defense pathway and its downstream target Glyoxalase 1 (Glo1), and a drop in intracellular levels of AP-modified Hsp70 and Hsp27 proteins, leading to a chronic adaptive redox homeostasis that sensitizes cells to oxidative DNA damage and apoptosis...
February 2018: Data in Brief
I-Wei Chang, Kai-Wen Liu, Marlon Ragunanan, Hong-Lin He, Yow-Ling Shiue, Shou-Chun Yu
Background: Due to the varying characteristics and conflicting outcomes on the overall survival of rectal cancer patients, many studies have been undertaken to determine various prognostic and predictive factors for the mainstay treatment of CCRT followed by surgery. Cancer cell motility contributes to tumor invasion, migration and eventually metastasis. However, the genes associated with cell motility (i.e., GO:0048870) have not been systemically evaluated in rectal cancers. Methods: A comparative analysis of gene expression profiles was applied to the transcriptomic dataset (GSE35452) with a focus on genes associated with cell motility (GO:0048870), where SERPINB5 was recognized as the most significantly up-regulated gene...
2018: International Journal of Medical Sciences
Vesela Encheva, Clémence Foltz, Ambrosius P Snijders, Eva-Maria Frickel
OBJECTIVE: The intracellular parasite Toxoplasma gondii can invade any nucleated cell residing inside a parasitophorous vacuole (PV). Upon infection, the cytokine interferon gamma (IFNγ) is produced and elicits host defence mechanisms able to recognise the PV and destroy the parasite. Hereby, Guanylate binding proteins, ubiquitin and the E3 ubiquitin ligases Tripartite Motif Containing 21 (TRIM21) and TNF receptor associated factor 6 are targeted to the murine PV leading to its destruction...
March 6, 2018: BMC Research Notes
Jiaqi Chu, Yuanyuan Fu, Jiecheng Xu, Xueping Zheng, Qianqian Gu, Xia Luo, Qi Dai, Shuxian Zhang, Peiqing Liu, Liang Hong, Min Li
Atg4 is essential for autophagosome formation and Atg8 recycle with the function of processing the precursor and the lipidated Atg8-family proteins. Abnormal autophagic activity is involved in a variety of pathophysiological diseases and ATG4B is of interest as a potential therapeutic target due to its key roles in autophagy process. So ATG4B inhibitors are highly needed. FMK-9a is the most potent inhibitor reported so far. In this study, we confirmed FMK-9a could suppress ATG4B activity in vitro and in cells, with an IC50 of 260 nM...
March 3, 2018: Archives of Biochemistry and Biophysics
John Marshall, Xiao-Zhong Zhou, Gang Chen, Su-Qing Yang, Ya Li, Yin Wang, Zhi-Qing Zhang, Qin Jiang, Lutz Birnbaumer, Cong Cao
Stress-related alterations in brain-derived neurotrophic factor (BDNF) expression, a neurotrophin that plays a key role in synaptic plasticity, are believed to contribute to the pathophysiology of depression. Here, we show that in a chronic mild stress (CMS) model of depression the Gαi1 and Gαi3 subunits of heterotrimeric G proteins are down-regulated in the hippocampus, a key limbic structure associated with major depressive disorder. We provide evidence that Gαi1 and Gαi3 (Gαi1/3) are required for the activation of TrkB downstream signaling pathways...
March 5, 2018: Proceedings of the National Academy of Sciences of the United States of America
Philip R Brauer, Jee Hun Kim, Humberto J Ochoa, Elizabeth R Stratton, Kathryn M Black, William Rosencrans, Eliza Stacey, Engda G Hagos
Kru¨ppel like factor 4 (KLF4) is a transcription factor that regulates genes related to differentiation and proliferation. KLF4 also plays a role in metastasis via epithelial to mesenchymal transition. Here, we investigate the function of Klf4 in migration and invasion using mouse embryonic fibroblasts and the RKO human colon cancer cell line. Compared to wild-type, cells lacking Klf4 exhibited increased migration-associated phenotypes. In addition, overexpression of Klf4 in Klf4-/- MEFs attenuated the presence of stress fibers to wild-type levels...
December 2018: Cell Communication & Adhesion
Simon Lagies, Roman Pichler, Michael M Kaminski, Manuel Schlimpert, Gerd Walz, Soeren S Lienkamp, Bernd Kammerer
Fibroblasts can be directly reprogrammed to induced renal tubular epithelial cells (iRECs) using four transcription factors. These engineered cells may be used for disease modeling, cell replacement therapy or drug and toxicity testing. Direct reprogramming induces drastic changes in the transcriptional landscape, protein expression, morphological and functional properties of cells. However, how the metabolome is changed by reprogramming and to what degree it resembles the target cell type remains unknown. Using untargeted gas chromatography-mass spectrometry (GC-MS) and targeted liquid chromatography-MS, we characterized the metabolome of mouse embryonic fibroblasts (MEFs), iRECs, mIMCD-3 cells, and whole kidneys...
March 1, 2018: Scientific Reports
Xuemei Wan, Jeffrey D Serrill, Ian R Humphreys, Michelle Tan, Kerry L McPhail, Ian G Ganley, Jane E Ishmael
Our understanding of autophagy and lysosomal function has been greatly enhanced by the discovery of natural product structures that can serve as chemical probes to reveal new patterns of signal transduction in cells. Coibamide A is a cytotoxic marine natural product that induces mTOR-independent autophagy as an adaptive stress response that precedes cell death. Autophagy-related (ATG) protein 5 (ATG5) is required for coibamide-induced autophagy but not required for coibamide-induced apoptosis. Using wild-type and autophagy-deficient mouse embryonic fibroblasts (MEFs) we demonstrate that coibamide-induced toxicity is delayed in ATG5-/- cells relative to ATG5+/+ cells...
March 1, 2018: Marine Drugs
Xiaoyu Si, Chihao Shao, Jing Li, Shuting Jia, Wenru Tang, Jihong Zhang, Julun Yang, Xiaoming Wu, Ying Luo
Werner syndrome (WS) is a rare autosomal recessive progeria disease with genetic instability/cancer predisposition, thus a good model in understanding aging related carcinogenesis. Telomere dysfunction induced cellular senescence is essential in the manifestation of the WS phenotype. Our previous data has shown that p21 (encoded by Cdkn1a gene) could induce cellular senescence and suppress cellular growth of ALT (alternative lengthening of telomere) tumors derived from WS, suggested that p21 might play a key role in maintaining senescence of WS cells...
2018: International Journal of Biological Sciences
Joshua D Bryant, Shannon R Sweeney, Enrique Sentandreu, Minhye Shin, Helene Ipas, Blerta Xhemalce, Jessica Momb, Stefano Tiziani, Dean R Appling
One-carbon (1C) metabolism is a universal folate-dependent pathway essential for de novo purine and thymidylate synthesis, amino acid interconversion, universal methyl-donor production, and regeneration of redox cofactors. Homozygous deletion of the 1C pathway gene Mthfd1l encoding methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1-like, which catalyzes mitochondrial formate production from 10-formyltetrahydrofolate, results in 100% penetrant embryonic neural tube defects (NTDs), underscoring the central role of mitochondrially derived formate in embryonic development and providing a mechanistic link between folate and NTDs...
February 26, 2018: Journal of Biological Chemistry
Yongbo Lu, Suzan A Kamel-El Sayed, Kun Wang, LeAnn M Tiede-Lewis, Michael A Grillo, Patricia A Veno, Vladimir Dusevich, Charlotte L Phillips, Lynda F Bonewald, Sarah L Dallas
Type I collagen is the most abundant extracellular matrix protein in bone and other connective tissues and plays key roles in normal and pathological bone formation as well as in connective tissue disorders and fibrosis. Although much is known about the collagen biosynthetic pathway and its regulatory steps, the mechanisms by which it is assembled extracellularly are less clear. We have generated GFPtpz and mCherry-tagged collagen fusion constructs for live imaging of type I collagen assembly by replacing the α2(I)-procollagen N-terminal propeptide with GFPtpz or mCherry...
February 20, 2018: Journal of Bone and Mineral Research: the Official Journal of the American Society for Bone and Mineral Research
G Yeryomenko
To improve the effectiveness of diagnosis and prediction of adverse asthma comorbid conditions based on research of clinical manifestations features, functional disorders of the airways and endothelial dysfunction. 79 patients were selected for the study. All the patients were divided into 3 groups: group 1 - patients with asthma (n-22); group II (n-24) - patients with asthma + diabetes mellitus type 2; group III (n-33) - patients with asthma + obesity + arterial hypertension (AH) and control group (n-17) - healthy people...
January 2018: Georgian Medical News
Christian Murer, Lars Christian Huber, Thomas Kurowski, Astrid Hirt, Cécile A Robinson, Urs Bürgi, Christian Benden
AIMS OF THE STUDY: Cystic fibrosis is the most common genetic disorder in Caucasians. The combination of the cystic fibrosis transmembrane conductance regulator (CFTR) corrector lumacaftor / potentiator ivacaftor (LUM/IVA) has been shown to increase forced expiratory volume in 1 second (FEV1) moderately, but predominantly reduce acute exacerbation rate (AER) in Phe508del homozygous cystic fibrosis patients; however, patients with FEV1 <40% predicted were excluded from studies. We used LUM/IVA on a "compassionate use" basis in cystic fibrosis patients with end-stage pulmonary disease...
February 16, 2018: Swiss Medical Weekly
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