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Mammalian cell culture

Hee Kyoung Chung, Shelley R Wang, Lan Xiao, Navneeta Rathor, Douglas J Turner, Peixin Yang, Myriam Gorospe, Jaladanki N Rao, Jian-Ying Wang
The mammalian intestinal epithelium is a rapidly self-renewing tissue in the body and its homeostasis depends on a dynamic balance among proliferation, migration, apoptosis, and differentiation of intestinal epithelial cells (IECs). The PP2A-associated protein α4 controls the activity and specificity of serine/threonine phosphatases and is thus implicated in many cellular processes. Here we investigated the mechanisms whereby α4 controls the homeostasis of the intestinal epithelium using a genetic approach...
March 19, 2018: Molecular and Cellular Biology
Jie Lei, Lei Zhao, Yujing Zhang, Yanfeng Wu, Yanbo Liu
BACKGROUND/AIMS: The mechanisms by which high glucose (HG) results in podocyte damage remains unclear. We investigated the potential role of endoplasmic reticulum (ER) stress and mTOR signaling in HG injured podocyte. METHODS: In cultured mouse podocytes, cellular apoptosis was assessed using FITC-Annexin V and propidium iodide staining followed by flow cytometry analysis. Apoptosis-related proteins as well as the ER stress and the mTOR signals were evaluated using immunoblot assay...
March 15, 2018: Cellular Physiology and Biochemistry
Grace H Hwang, Jessica L Hopkins, Philip W Jordan
Chromatin spread techniques have been widely used to assess the dynamic localization of various proteins during gametogenesis, particularly for spermatogenesis. These techniques allow for visualization of protein and DNA localization patterns during meiotic events such as homologous chromosome pairing, synapsis and DNA repair. While a few protocols have been described in the literature, general chromatin spread techniques using mammalian prophase oocytes are limited and difficult due to the timing of meiosis initiation in fetal ovaries...
February 26, 2018: Journal of Visualized Experiments: JoVE
Chie Ishikawa, Masachika Senba, Naoki Mori
Adult T-cell leukemia (ATL) is an aggressive type of malignancy caused by human T-cell leukemia virus type 1 (HTLV-1). In ATL, the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway is constitutively active, promoting cell proliferation, survival and chemoresistance. Thus, the PI3K signaling pathway is an attractive therapeutic target for ATL. In the present study, the effects of RAD001 (an mTOR inhibitor), NVP-BKM120 (a pan-PI3K inhibitor) and NVP-BEZ235 (a novel dual PI3K/mTOR inhibitor) on cultured HTLV-1-infected T-cell lines were compared...
April 2018: Oncology Letters
João Crispim Encarnação, Tim Schulte, Adnane Achour, Hanna Björkelund, Karl Andersson
Time-resolved analysis assays of receptor-ligand interactions are fundamental in basic research and drug discovery. Adequate methods are well developed for the analysis of recombinant proteins such as antibody-antigen interactions. However, assays for time-resolved ligand-binding processes on living cells are still rare, in particular within microbiology. In this report, the real-time cell-binding assay (RT-CBA) technology LigandTracer®, originally designed for mammalian cell culture, was extended to cover Gram-positive and Gram-negative bacteria...
March 17, 2018: Applied Microbiology and Biotechnology
Katia Wehbe, Marzia Vezzalini, Gianfelice Cinque
Mycoplasma contamination represents a significant problem to the culture of mammalian cells used for research as it can cause disastrous effects on eukaryotic cells by altering cellular parameters leading to unreliable experimental results. Mycoplasma cells are very small bacteria therefore they cannot be detected by visual inspection using a visible light microscope and, thus, can remain unnoticed in the cell cultures for long periods. The detection techniques used nowadays to reveal mycoplasma contamination are time consuming and expensive with each having significant drawbacks...
March 17, 2018: Analytical and Bioanalytical Chemistry
Martin Kornecki, Jochen Strube
Productivity improvements of mammalian cell culture in the production of recombinant proteins have been made by optimizing cell lines, media, and process operation. This led to enhanced titers and process robustness without increasing the cost of the upstream processing (USP); however, a downstream bottleneck remains. In terms of process control improvement, the process analytical technology (PAT) initiative, initiated by the American Food and Drug Administration (FDA), aims to measure, analyze, monitor, and ultimately control all important attributes of a bioprocess...
March 16, 2018: Bioengineering
Iram Iqbal Hejazi, Rashmin Khanam, Syed Hassan Mehdi, Abdul Roouf Bhat, M Moshahid Alam Rizvi, Sonu Chand Thakur, Fareeda Athar
Plant phytoconstituents have been a valuable source of clinically important anticancer agents. Antioxidant and anticancerous activity of plant Curculigo orchioides Gaertn were explored In vitro antioxidant activity, antioxidant enzyme activity of oxidatively stressed tissue, and cell culture studies on human cancer cell lines HepG2, HeLa and MCF-7 were carried out. Active plant fractions were subjected to GC-MS analysis and compounds selected on the basis of their abundance were screened in silico with the help of Auto Dock 4...
March 12, 2018: Food and Chemical Toxicology
Sheun Oshinbolu, Rachana Shah, Gary Finka, Mike Molloy, Mark Uden, Daniel G Bracewell
BACKGROUND: A current challenge in bioprocessing is the ability to analyse critical quality attributes such as aggregation without prior purification. This study evaluated the use of fluorescent dyes (Bis-ANS, SYPRO Orange, Thioflavin T and ProteoStat) to characterise mAb aggregates in Chinese hamster ovary clarified cultures. RESULTS: The null and mAb culture supernatants showed an increase in fluorescence intensity over the duration of the culture. The null cultures on day 14 saw a rapid increase in fluorescence intensity; day 10 to day 14, Bis-ANS and Thioflavin T had average increases of 21% and 48%, respectively, whereas ProteoStat and SYPRO Orange showed an average increase of 60%...
March 2018: Journal of Chemical Technology and Biotechnology
Jiajia Xi, Qian Huang, Lei Wang, Xiaodong Ma, Qipan Deng, Munish Kumar, Zhiyuan Zhou, Ling Li, Zhaoyang Zeng, Ken H Young, Mingzhi Zhang, Yong Li
MicroRNA-21 (miR-21) is one of the most abundant microRNAs in mammalian cells. It has been intensively studied for its role in regulating apoptosis and oncogenic transformation. However, the impact of miR-21 on host anti-tumor immunity remains unknown. Tumor-associated macrophages are a major leukocyte type that infiltrates tumors and predominantly develops into immunosuppressive, tumor-promoting M2-like macrophages. In contrast, the pro-inflammatory M1-like macrophages have tumoricidal activity. In this study, we show that genetic deficiency of miR-21 promotes the polarization of macrophages toward an M1-like phenotype in vivo and in vitro in the presence of tumor cells; thus it confers host mice with enhanced anti-tumor immunity...
March 15, 2018: Oncogene
Ernst R Werner, Markus A Keller, Sabrina Sailer, Daniele Seppi, Georg Golderer, Gabriele Werner-Felmayer, Raphael A Zoeller, Katrin Watschinger
Plasmanylethanolamine desaturase (E.C. introduces the 1-prime double bond into plasmalogens, one of the most abundant phospholipids in the human body. This labile membrane enzyme has not been purified and its coding sequence is unknown. Previous assays for this enzyme used radiolabeled substrates followed by multistep processing. We describe here a straight-forward method for the quantification of plasmanylethanolamine desaturase in enzyme incubation mixtures using pyrene-labeled substrates and reversed phase high performance liquid chromatography (HPLC) with fluorescence detection...
March 14, 2018: Journal of Lipid Research
Seiichi Uchiyama, Toshikazu Tsuji, Kyoko Kawamoto, Kentaro Okano, Eiko Fukatsu, Takahiro Noro, Kumiko Ikado, Sayuri Yamada, Yuka Shibata, Teruyuki Hayashi, Noriko Inada, Masaru Kato, Hideki Koizumi, Hidetoshi Tokuyama
A cationic fluorescent nanogel thermometer based on thermo-responsive N-isopropylacrylamide and environment-sensitive benzothiadiazole was developed with a new azo compound bearing imidazolium rings as the first cationic radical initiator. This cationic fluorescent nanogel thermometer showed an excellent ability to enter live mammalian cells in a short incubation period (10 min), a high sensitivity to temperature variations in live cells (temperature resolution of 0.02-0.84 °C in the range 2040 °C), and remarkable non-cytotoxicity, which permitted ordinary cell proliferation and even differentiation of primary cultured cells...
March 14, 2018: Angewandte Chemie
Peter Sykora, Ylenia Chiari, Andrew Heaton, Nickolas Moreno, Scott Glaberman, Robert W Sobol
DNA damage has been linked to genomic instability and the progressive breakdown of cellular and organismal homeostasis, leading to the onset of disease and reduced longevity. Insults to DNA from endogenous sources include base deamination, base hydrolysis, base alkylation, and metabolism-induced oxidative damage that can lead to single-strand and double-strand DNA breaks. Alternatively, exposure to environmental pollutants, radiation or ultra-violet light, can also contribute to exogenously derived DNA damage...
March 14, 2018: Environmental and Molecular Mutagenesis
Karissa L Cross, Payal Chirania, Weili Xiong, Clifford J Beall, James G Elkins, Richard J Giannone, Ann L Griffen, Adam M Guss, Robert L Hettich, Snehal S Joshi, Elaine M Mokrzan, Roman K Martin, Igor B Zhulin, Eugene J Leys, Mircea Podar
The human oral microbiota encompasses representatives of many bacterial lineages that have not yet been cultured. Here we describe the isolation and characterization of previously uncultured Desulfobulbus oralis , the first human-associated representative of its genus. As mammalian-associated microbes rarely have free-living close relatives, D. oralis provides opportunities to study how bacteria adapt and evolve within a host. This sulfate-reducing deltaproteobacterium has adapted to the human oral subgingival niche by curtailing its physiological repertoire, losing some biosynthetic abilities and metabolic independence, and by dramatically reducing environmental sensing and signaling capabilities...
March 13, 2018: MBio
Jaebong Jang, Sooyoung Chung, Youjeong Choi, Hye Young Lim, Yeongeon Son, Sung Kook Chun, Gi Hoon Son, Kyungjin Kim, Young-Ger Suh, Jong-Wha Jung
AIMS: We have previously identified a chemical scaffold possessing 2-ethoxypropanoic acid (designated as KS15) that directly binds to the C-terminal region of cryptochromes (CRYs: CRY1 and CRY2) and enhances E-box-mediated transcription. However, it is still unclear how KS15 impairs the feedback actions of the CRYs and which chemical moieties are functionally important for its actions. MAIN METHODS: The E-box-mediated transcriptional activities were mainly used to examine the effects of KS15 and its derivatives...
March 10, 2018: Life Sciences
Yuanyuan Weng, Xueyu Fan, Yongfeng Bai, Siwei Wang, Hui Huang, Huimin Yang, Jin Zhu, Feng Zhang
The metabolism of cancer cells is highly plastic. Cancer cells can change their preference for nutrient uptake under nutrient stress. Fructose is one of the most common carbohydrates in diet and its metabolism is also involved in the development and progression of tumors. GLUT5, encoded by SLC2A5 , is the specific fructose transporter in mammalian cells. In this study, we found that SLC2A5 is significantly upregulated in lung adenocarcinoma (LUAD) patients and overexpression of SLC2A5 is highly correlated with poor prognosis of LUAD patients...
December 2018: Cell Death Discovery
Asif Elahi, Subrata Sabui, Nell N Narasappa, Sudhanshu Agrawal, Nils W Lambrecht, Anshu Agrawal, Hamid M Said
Biotin (vitamin B7) is essential for human health because of its involvement, as a cofactor, in a variety of critical cellular metabolic reactions. Previous studies have shown that biotin deficiency enhances inflammation, and certain chronic inflammatory diseases are associated with biotin deficiency; however, the mechanisms that mediate the association between biotin status and inflammation are not well understood. In this study, we examined the effect of biotin deficiency on human CD4+ T cell responses to determine their role in biotin deficiency-associated inflammation...
March 12, 2018: Journal of Immunology: Official Journal of the American Association of Immunologists
U Kandalam, N Ledra, H Laubach, K V Venkatachalam
BACKGROUND AND OBJECTIVES: Pathogenic infections caused by Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia can result in the production of volatile sulfur compounds (VSC's) and other toxic compounds from methionine catabolism that can lead to halitosis and periodontitis. Our aim is to block the activity of methionine gammalyase-deaminase (Mgld) of methionine catabolism to prevent halitosis/periodontitis. DESIGNS: Cloned, expressed, Mgld protein was tested for purity by SDS-PAGE and western blotting...
March 1, 2018: Archives of Oral Biology
Ji Tu, Wentian Li, Shuai Li, Wei Liu, Yukun Zhang, Xinghuo Wu, Rongjin Luo, Wenbin Hua, Kun Wang, Yu Song, Liang Kang, Wen Yang, Shuhua Yang, Cao Yang
BACKGROUND/AIMS: Intervertebral disc degeneration (IDD) is a pathological process that is the primary cause of low back pain and is potentially mediated by compromised stress defense. Sestrins (Sesn) promote cell survival under stress conditions and regulate AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) signaling. Here, we investigated the expression of Sesn in normal and degraded nucleus pulposus (NP) cells and its potential roles during IDD pathogenesis...
March 2, 2018: Cellular Physiology and Biochemistry
Aditi Jain, Jafar Hasan, Perumal Arumugam Desingu, Nagalingam R Sundaresan, Kaushik Chatterjee
Neonatal cardiomyocytes cultured on flat surfaces are commonly used as a model to study cardiac failure of diverse origin. A major drawback of such a system is that the cardiomyocytes do not exhibit alignment, organization and calcium transients, similar to the native heart. Therefore, there is a need to develop in vitro platforms that recapitulate the cellular microenvironment of the murine heart as organotypic models to study cardiovascular diseases. In this study, we report an engineered platform that mimics cardiac cell organization and function of the heart...
February 27, 2018: Colloids and Surfaces. B, Biointerfaces
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