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N C N Perera, G I Godahewa, Jehee Lee
Mitogen-activated protein kinase (MAPK) is involved in the regulation of cellular events by mediating signal transduction pathways. MAPK1 is a member of the extracellular-signal regulated kinases (ERKs), playing roles in cell proliferation, differentiation, and development. This is mainly in response to growth factors, mitogens, and many environmental stresses. In the current study, we have characterized the structural features of a homolog of MAPK1 from disk abalone (AbMAPK1). Further, we have unraveled its expressional kinetics against different experimental pathogenic infections or related chemical stimulants...
October 17, 2016: Fish & Shellfish Immunology
Y Sun, Y X Wang, J Zhang, X W Yu, S S Ge, H Wang, J Dong, X H Guo, W Wang
Objective: To analyze and compare the gender specific structure profiles of immunoglobulin G(IgG) N-glycans and provide evidence to understand the distribution of the structure of IgG N-glycans in general population. Methods: A cluster sampling was conducted to randomly select 669 participants from Beijing Xuanwu Hospital during January-June 2012. Venous blood sample (5 ml) was collected from each participant with vacuum negative pressure tube containing EDTA. Plasma IgG N-glycome was determined using ultra-performance liquid chromatography (UPLC) on Waters BEH Glycan chromatography column and mass spectrometry after the separation, purification and labeling...
October 10, 2016: Zhonghua Liu Xing Bing Xue za Zhi, Zhonghua Liuxingbingxue Zazhi
Lisai Zhu, Haibing Lu, Yufeng Cao, Xiaochun Gai, Changming Guo, Yajing Liu, Jiaxu Liu, Xinping Wang
As one of the major pathogens, bovine viral diarrhea virus caused a significant economic loss to the livestock industry worldwide. Although BVDV infections have increasingly been reported in China in recent years, the molecular aspects of those BVDV strains were barely characterized. In this study, we reported the identification and characterization of a novel BVDV isolate designated as SD-15 from cattle, which is associated with an outbreak characterized by severe hemorrhagic and mucous diarrhea with high morbidity and mortality in Shandong, China...
2016: PloS One
Ying Zhang, Xi Wang, Dan Cui, Jun Zhu
The human whole saliva is a vital body fluid to study the physiology and pathology of oral cavity. As a powerful technique for biomarker discovery, mass spectrometry-based proteomic strategy has been introduced to saliva analysis and identified hundreds of proteins and N-glycosylation sites. However, there still lacks of quantitative analysis, which is necessary for biomarker screening and biological researches. In this study, we established an integrated workflow by the combination of stable isotope dimethyl labeling, HILIC enrichment and high resolution mass spectrometry for both quantification of global proteome and N-glycoproteome of human saliva from oral ulcer patients...
October 20, 2016: Proteomics
Lucia Pavlikova, Mario Seres, Denisa Imrichova, Milan Hano, Andrej Rusnak, Martina Zamorova, Jaroslav Katrlik, Albert Breier, Zdena Sulova
In P-gp-positive cell variants obtained from L1210 cells either by selection with vincristine (L1210/R) or by transfection with the human gene encoding P-gp (L1210/T), we have previously described cross-resistance to tunicamycin (TNM), a protein N-glycosylation inhibitor. Here we studied whether this cross-resistance also underlies P-gp-positive variants of human acute myeloid leukemia cells (AML) derived from SKM-1 and MOLM-13 cells (SKM-1/VCR, SKM-1/LEN, MOLM-13/VCR) by selection with vincristine (VCR) and lenalidomide (LEN)...
October 2016: General Physiology and Biophysics
Ju Yeon Lee, Hyun Kyoung Lee, Gun Wook Park, Heeyoun Hwang, Hoi Keun Jeong, Ki Na Yun, Eun Sun Ji, Kwang Hoe Kim, Jun Seok Kim, Jong Won Kim, Sung Ho Yun, Chi-Won Choi, Seung Il Kim, Jong-Sun Lim, Seul-Ki Jeong, Young-Ki Paik, Soo-Youn Lee, Jisook Park, Su Yeon Kim, Young-Jin Choi, Yong-In Kim, Jawon Seo, Je-Yoel Cho, Myoung Jin Oh, Nari Seo, Hyun Joo An, Jin Young Kim, Jong Shin Yoo
Glycoprotein conformations are complex and heterogeneous. Currently, site-specific characterization of glycopeptides is a challenge. Herein, we sought to establish an efficient method of N-glycoprotein characterization using mass spectrometry. Using alpha-1-acid glycoprotein (AGP) as a model N-glycoprotein, its tryptic N-glycopeptides were identified, and the reproducibility of the data was examined in seven laboratories running different liquid chromatography-mass tandem spectrometry (LC-MS/MS) platforms. We used three test samples and one blind sample to evaluate instrument performance with entire sample preparation workflow...
October 19, 2016: Journal of Proteome Research
Nathan J Hare, Ling Y Lee, Ian Loke, Warwick J Britton, Bernadette M Saunders, Morten Thaysen-Andersen
Tuberculosis (TB) remains a prevalent and lethal infectious disease. The glycobiology associated with Mycobacterium tuberculosis infection of front-line alveolar macrophages is still unresolved. Herein, we investigated the regulation of protein N-glycosylation in human macrophages and their secreted microparticles (MPs) used for intercellular communication upon M. tb infection. LC-MS/MS-based proteomics and glycomics were performed to monitor the regulation of glycosylation enzymes and receptors and the N-glycome in in vitro-differentiated macrophages and in isolated MPs upon M...
October 19, 2016: Journal of Proteome Research
Peter L Stern, Richard Harrop
The natural history of a patient's cancer is often characterised by genetic diversity and sequential sweeps of clonal dominance. It is therefore not surprising that identifying the most appropriate tumour-associated antigen for targeted intervention is challenging. The 5T4 oncofoetal antigen was identified by searching for surface molecules shared between human trophoblast and cancer cells with the rationale that they may function to allow survival of the foetus as a semi-allograft in the mother or a tumour in its host...
October 18, 2016: Cancer Immunology, Immunotherapy: CII
Leena Liljedahl, Maiken Højgaard Pedersen, Jenny Norlin, James N McGuire, Peter James
BACKGROUND: Diabetic nephropathy (DN) is a late complication in both type 1 diabetes mellitus (T1DM) and T2DM. Already at an early stage of DN morphological changes occur at the cell surface and in the extracellular matrix where the majority of the proteins carry N-linked glycosylations. These glycosylated proteins are highly important in cell adhesion and cell-matrix processes but not much is known about how they change in DN or whether the distinct etiology of T1DM and T2DM could have an effect on their abundances...
2016: Clinical Proteomics
Stefanie Memmer, Sandra Weil, Steffen Beyer, Tobias Zöller, Eike Peters, Jessica Hartmann, Alexander Steinle, Joachim Koch
The natural cytotoxicity receptor (NCR) NKp30 (CD337) is a key player for NK cell immunosurveillance of infections and cancer. The molecular details of ligand recognition and its connection to CD3ζ signaling remain unsolved. Here, we show that the stalk domain (K129EHPQLGAGTVLLLR143) of NKp30 is very sensitve to sequence alterations, as mutations lead to impaired ligand binding and/or signaling capacity. Surprisingly, the stalk domains of NKp30 and NKp46, another NCR employing CD3ζ for signaling, were not exchangeable without drastic deficiencies in folding, plasma membrane targeting and/or ligand induced receptor signaling...
October 17, 2016: Journal of Biological Chemistry
Matthias Brunner, Jens Fricke, Paul Kroll, Christoph Herwig
Understanding process parameter interactions and their effects on mammalian cell cultivations is an essential requirement for robust process scale-up. Furthermore, knowledge of the relationship between the process parameters and the product critical quality attributes (CQAs) is necessary to satisfy quality by design guidelines. So far, mainly the effect of single parameters on CQAs was investigated. Here, we present a comprehensive study to investigate the interactions of scale-up relevant parameters as pH, pO2 and pCO2 on CHO cell physiology, process performance and CQAs, which was based on design of experiments and extended product quality analytics...
October 17, 2016: Bioprocess and Biosystems Engineering
Kazuki Kawahara, Shinya Yonogi, Ryota Munetomo, Hiroya Oki, Takuya Yoshida, Yuko Kumeda, Shigeaki Matsuda, Toshio Kodama, Tadayasu Ohkubo, Tetsuya Iida, Shota Nakamura
Binary enterotoxin of Clostridium perfringens (BEC), consisting of the components BECa and BECb, was recently identified as a novel enterotoxin produced by C. perfringens that causes acute gastroenteritis in humans. Although the detailed mechanism of cell intoxication by BEC remains to be defined, BECa shows both NAD(+)-glycohydrolase and actin ADP-ribosyltransferase activities in the presence of NAD(+). In this study, we determined the first crystal structure of BECa in its apo-state and in complex with NADH...
October 14, 2016: Biochemical and Biophysical Research Communications
Carol A Casey, Ganapati Bhat, Melissa S Holzapfel, Armen Petrosyan
BACKGROUND: It is known that ethanol (EtOH) and its metabolites have a negative effect on protein glycosylation. The fragmentation of the Golgi apparatus induced by alteration of the structure of largest Golgi matrix protein, giantin, is the major consequence of damaging effects of EtOH-metabolism on the Golgi; however, the link between this and abnormal glycosylation remains unknown. Because previously we have shown that Golgi morphology dictates glycosylation, we examined the effect EtOH administration has on function of Golgi residential enzymes involved in N-glycosylation...
October 17, 2016: Alcoholism, Clinical and Experimental Research
Keisuke Yoshihara, Minh Nhat Le, Koo Nagasawa, Hiroyuki Tsukagoshi, Hien Anh Nguyen, Michiko Toizumi, Hiroyuki Moriuchi, Masahiro Hashizume, Koya Ariyoshi, Duc Anh Dang, Hirokazu Kimura, Lay-Myint Yoshida
We performed molecular evolutionary analyses of the G gene C-terminal 3rd hypervariable region of RSV-A genotypes NA1 and ON1 strains from the paediatric acute respiratory infection patients in central Vietnam during the 2010-2012 study period. Time-scaled phylogenetic analyses were performed using Bayesian Markov Chain Monte Carlo (MCMC) method, and pairwise distances (p-distances) were calculated. Bayesian Skyline Plot (BSP) was constructed to analyze the time-trend relative genetic diversity of central Vietnam RSV-A strains...
October 14, 2016: Infection, Genetics and Evolution
Anna Agakova, Frano Vučković, Lucija Klarić, Gordan Lauc, Felix Agakov
Ultra-performance liquid chromatography (UPLC) is the established technology for accurate analysis of IgG Fc N-glycosylation due to its superior sensitivity, resolution, speed, and its capability to provide branch-specific information of glycan species. Correct and cost-efficient preprocessing of chromatographic data is the major prerequisite for subsequent analyses ranging from inference of structural isomers to biomarker discovery and prediction of humoral immune response from characterized changes in glycosylation...
2017: Methods in Molecular Biology
Tamara Pavić, Olga Gornik
Glycosylation of membrane proteins plays a crucial role in various physiological events, including intercellular recognition and intermolecular interactions on the cell surface (Gornik et al., Biochim Biophys Acta 1820:1318-1326, 2012). To study composition and function of N-glycans on membrane proteins one has to have an efficient and reproducible analytical method, which includes protein extraction and analysis of glycans. In this chapter we provide an analytical approach that includes cloud-point extraction (CPE) of total membrane proteins with the non-ionic detergent Triton X-114 and subsequent analysis of their N-glycans using hydrophilic interaction liquid chromatography (HILIC)-UPLC/HPLC...
2017: Methods in Molecular Biology
Stephanie Holst, Gabi W van Pelt, Wilma E Mesker, Rob A Tollenaar, Ana I Belo, Irma van Die, Yoann Rombouts, Manfred Wuhrer
The current protocols for glycomic analysis of cells often require a large quantity of material (5-20 million cells). In order to analyze the N-glycosylation from small amounts of cells (≤1 million) as obtained from, for example, primary cell lines or cell sorting, and in a higher throughput approach, we set up a robust 96-well format PVDF-membrane based N-glycan release protocol followed by linkage-specific sialic acid stabilization, cleanup, and MALDI-TOF-MS analysis. We further evaluated the influence of PNGase F incubation time on the N-glycan profile...
2017: Methods in Molecular Biology
David Falck, Bas C Jansen, Noortje de Haan, Manfred Wuhrer
This chapter contains a nanoscale liquid chromatography-mass spectrometry method for the glycoform profiling of the conserved Fc N-glycosylation site of monoclonal and polyclonal immunoglobulin G (IgG). It describes in detail LaCyTools, a program for automated data (pre-)processing of the obtained LC-MS data. The minimal sample preparation necessary is explained as well as an optional method for affinity purification of (polyclonal) antibodies from serum or plasma.After (optional) affinity purification, the pure IgG is cleaved with trypsin...
2017: Methods in Molecular Biology
Ramanujam Saravanan, Muthuvel Bharathselvi, Aravind C Babu, Jyothirmay Biswas, Konerirajapuram N Sulochana
PURPOSE: Eales' disease (ED), an enigmatic inflammatory disease affects peripheral retinal veins and thereby vision, in males. This study was aimed at identifying and deciphering the role of a novel 88 kDa protein reported in the serum and vitreous of patients with ED. EXPERIMENTAL DESIGN: The purified 88 kDa protein was identified by UPLC coupled ESI-QTOF-MS. The identified proteins were quantified in the serum from 20 ED patients and controls (age and sex matched), respectively by ELISA...
October 14, 2016: Proteomics. Clinical Applications
Eun Jae Kim, Jun Hyuck Lee, Sung Gu Lee, Se Jong Han
To survive in a sub-zero environment, polar organisms produce ice-binding proteins (IBPs). These IBPs prevent the formation of large intracellular ice crystals, which may be fatal to the organism. Recently, a recombinant FfIBP (an IBP from Flavobacterium frigoris PS1) was cloned and produced in Pichia pastoris using fed-batch fermentation with methanol feeding. In this study, we demonstrate that FfIBP produced by P. pastoris has a glycosylation site, which diminishes the thermal hysteresis (TH) activity of FfIBP...
October 13, 2016: Preparative Biochemistry & Biotechnology
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