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Affinity chromatography

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https://www.readbyqxmd.com/read/28821619/dsc-e3-ligase-localization-to-the-golgi-requires-the-atpase-cdc48-and-cofactor-ufd1-for-activation-of-sterol-regulatory-element-binding-protein-in-fission-yeast
#1
Risa Burr, Diedre Ribbens, Sumana Raychaudhuri, Emerson V Stewart, Jason Ho, Peter J Espenshade
Sterol regulatory element-binding proteins (SREBPs) in the fission yeast Schizosaccharomyces pombe regulate lipid homeostasis and the hypoxic response under conditions of low sterol or oxygen availability. SREBPs are cleaved in the Golgi through the combined action of the Dsc E3 ligase complex, the rhomboid protease Rbd2, and the essential ATPases Associated with diverse cellular Activities (AAA+) ATPase Cdc48. The soluble SREBP N-terminal transcription factor domain is then released in the cytosol to enter the nucleus and regulate gene expression...
August 18, 2017: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/28821545/heterologous-production-of-thermophile-thermochromatium-tepidum-photosynthetic-reaction-centre-and-light-harvesting-1-complexes-in-the-mesophile-rhodobacter-sphaeroides-and-thermal-stabilities-of-a-hybrid-core-complex
#2
D Jun, V Huang, J T Beatty
The photosynthetic complexes of the thermophile Thermochromatium tepidum are of considerable interest in biohybrid solar cell applications because of the capability of thermophilic proteins to tolerate elevated temperatures. Synthetic operons encoding reaction centre (RC) and light harvesting 1 (LH1) pigment-protein complexes of T. tepidum were expressed in the mesophile Rhodobacter sphaeroides The T. tepidum RC (TRC) was assembled, and functional with the addition of menadione to populate the QA pocket. The production of the T...
August 18, 2017: Applied and Environmental Microbiology
https://www.readbyqxmd.com/read/28820984/identification-of-m-tuberculosis-antigens-in-the-sera-of-tuberculosis-patients-using-biomimetic-affinity-chromatography-in-conjunction-with-esi-cid-ms-ms
#3
Guorong Ma, Jinting Pan, Jincheng Han, Lina Gao, Shulin Zhang, Rongxiu Li
The profiling of abnormally-expressed proteins in host cells using mass spectrometry (MS) analysis is a classical approach for screening disease-associated biomarkers in clinical diagnosis. However, few pathogen-specific antigens can currently be detected in serum using this proteomic approach, since these are very low-abundant proteins that are easily masked by host high-abundant proteins. Identification of pathogen-specific antigens in the sera of tuberculosis patients is crucial for the clinical diagnosis of this infectious disease, especially in immune-compromised patients...
July 31, 2017: Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences
https://www.readbyqxmd.com/read/28820982/direct-analysis-no-sample-preparation-of-bioavailable-cortisol-in-human-plasma-by-weak-affinity-chromatography-wac
#4
Sten Ohlson, Jagjit Kaur, Manfred Raida, Ulf Niss, Tim Bengala, Chester Lee Drum, Bernhard Boehm, Anthony R Torres
Pre-analytical treatment of blood plasma is a time consuming and often rate limiting step in the workflow of LC/MS analysis. We present in this pilot study a new approach for quantitative LC/MS based on weak affinity chromatography (WAC) of crude plasma. The steroid hormone cortisol was selected as a clinically relevant biomarker, as it currently requires extensive pre-analytical preparation. A WAC unit with saturating, immobilized albumin as a prototypic weak binder was used in combination with an ion-funnel MS/MS detector to perform zonal affinity chromatography of cortisol directly from a plasma sample, followed by quantitative multiple reaction monitoring (MRM)...
July 21, 2017: Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences
https://www.readbyqxmd.com/read/28818618/ctenopharyngodon-idella-p53-mediates-between-nf-%C3%AE%C2%BAb-and-pkr-at-the-transcriptional-level
#5
Qingli Huang, Dingkun Xie, Huiling Mao, Haizhou Wang, Zhen Wu, Keyi Huang, Yiqi Wan, Qun Xu, Chengyu Hu
p53, NF-κB and PKR are well-known to be involved in antiviral response. Although p53 has been reported in fish, its role in the regulation of NF-κB and PKR is not well understood. Here, we cloned and characterized the full length of cDNA sequence of grass carp (Ctenopharyngodon idella) p53 (Cip53) and its promoter sequence. The full length cDNA of Cip53 was 1879 bp with an ORF of 1116 bp encoding a polypeptide of 371 amino acids. Phylogenetic tree analysis revealed that Cip53 shares high homology with Dario rerio p53 (Drp53)...
August 14, 2017: Fish & Shellfish Immunology
https://www.readbyqxmd.com/read/28818427/molecular-cloning-and-functional-characterization-of-nadph-dependent-cytochrome-p450-reductase-from-the-green-microalga-botryococcus-braunii-b-race
#6
Chung-Yau Tsou, Shigeki Matsunaga, Shigeru Okada
The green microalga Botryococcus braunii of the B race accumulates various lipophilic compounds containing a 10,11-oxidosqualene epoxide moiety in addition to large amounts of triterpene hydrocarbons. While 2,3-squalene epoxidases have already been isolated and characterized from the alga, the enzyme that catalyzes the 10,11-epoxidation of squalene has remained elusive. In order to obtain a molecular tool to explore a 10,11-squalene epoxidase, cDNA cloning of an NADPH-dependent cytochrome P450 reductase (CPR) that is required by both squalene epoxidases and cytochrome P450 enzymes was carried out...
August 14, 2017: Journal of Bioscience and Bioengineering
https://www.readbyqxmd.com/read/28816641/preparation-of-recombinant-human-thymic-stromal-lymphopoietin-protein
#7
Yanlu Zhang, Liehua Wu, Jingai Yu, JianFeng Mei, Yu Yi, JianShu Chen, Guoqing Ying
Human thymic stromal lymphopoietin (hTSLP) protein plays a central role in inflammation. Characterizing properties of hTSLP requires a recombinant overexpression system that produces correctly folded, active hTSLP. In this report, an efficient overexpression system for production of hTSLP was developed. We constructed expression plasmids of the full-length hTslp gene with or without the signal peptide and transformed the plasmids into E. coli. The design of the recombinant proteins included an N-terminal His-tag, which facilitated purification...
August 17, 2017: Preparative Biochemistry & Biotechnology
https://www.readbyqxmd.com/read/28816534/seven-familial-dysalbuminemic-hyperthyroxinemia-cases-in-three-unrelated-japanese-families-and-high-performance-liquid-chromatography-analysis-of-the-thyroxine-binding-profile
#8
Hidekazu Nagano, Yoshinori Nakagawa, Naofumi Ishikawa, Yuya Watanabe, Yui Miyabayashi, Akitoshi Nakayama, Masanori Fujimoto, Eri Komai, Akina Shiga, Ai Tamura, Takashi Kono, Tomoko Takiguchi, Seiichiro Higuchi, Ikki Sakuma, Naoko Hashimoto, Sawako Suzuki, Hisashi Koide, Kotaro Yokote, Tomoaki Tanaka
OBJECTIVE: Familial dysalbuminemic hyperthyroxinemia (FDH) is caused by abnormal human serum albumin (HSA) with an increased thyroxine (T4) affinity leading to euthyroid hyperthyroxinemia. One- and two-step immunoassay of serum samples from FDH patients (e.g., Japanese patients) with the HSA R218P mutation can yield false-positive free thyroxine (FT4) results. Therefore, it is difficult to distinguish FDH from syndrome of inappropriate secretion of thyroid-stimulating hormone (e.g., syndrome of resistance to thyroid hormone, thyroid-stimulating hormone [TSH]-producing pituitary adenoma), even when multiple assays are used...
August 17, 2017: Endocrine Practice
https://www.readbyqxmd.com/read/28815507/using-purified-tyrosine-site-specific-recombinases-in-vitro-to-rapidly-construct-and-diversify-metabolic-pathways
#9
Wei Liu, Laura R Tuck, Jon Marles Wright, Yizhi Cai
The site-specific recombinase Cre was previously reported to have in vitro activity. Here, we describe the method of purifying two new tyrosine site-specific recombinases VCre and Dre along with Cre by nickel affinity chromatography. We proved the in vitro function of the VCre and Dre on their respective conditional recombination sites. We also developed a methodology to one-step construct and optimize the productivity of a biosynthetic pathway through the combinatorial integration of promoters into a plasmid-encoded pathway by simply incubating a DNA mixture with recombinase system at 37 °C in vitro...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28812241/plasma-glycoproteomic-study-of-therapeutic-hypothermia-reveals-novel-markers-predicting-neurologic-outcome-post-cardiac-arrest
#10
Wenjun Deng, Jing Cao, Lei Chen, David McMullin, James L Januzzi, Ferdinando S Buonanno, Eng H Lo, MingMing Ning
Therapeutic hypothermia (TH) is a neuroprotective treatment post-cardiac arrest but is grossly underutilized. After TH induction, traditional biomarkers and parameters can no long predict clinical outcome due to a lack of understanding of hypothermic response. Innovative approaches to better understand the clinical effect of TH will help to prognosticate outcome and expand beneficial population. Protein glycosylation is an important extracellular post-translational modification, regulating various extracellular signaling pathways...
August 15, 2017: Translational Stroke Research
https://www.readbyqxmd.com/read/28810892/absence-of-cx3cr1-impairs-the-internalization-of-tau-by-microglia
#11
Marta Bolós, María Llorens-Martín, Juan Ramón Perea, Jerónimo Jurado-Arjona, Alberto Rábano, Félix Hernández, Jesús Avila
BACKGROUND: Extracellular Tau is toxic for neighboring cells, and it contributes to the progression of AD. The CX3CL1/CX3CR1 axis is an important neuron/microglia communication mechanism. METHODS: We studied Tau clearance by microglia both in vitro (microglia primary cultures treated with Cy5-Tau, affinity chromatography to study the binding of Tau to CX3CR1, and Tau-CX3CL1 competition assays) and in vivo (stereotaxic injection of Cy5-Tau into WT and CX3CR1(-/-) mice)...
August 15, 2017: Molecular Neurodegeneration
https://www.readbyqxmd.com/read/28808292/binding-of-the-helicobacter-pylori-oipa-causes-apoptosis-of-host-cells-via-modulation-of-bax-bcl-2-levels
#12
Omid Teymournejad, Ashraf Mohabati Mobarez, Zuhair Mohammad Hassan, Amin Talebi Bezmin Abadi
The H. pylori outer inflammatory protein A (OipA) is an outer membrane protein that contributes to gastric inflammation. OipA is believed to affect intra-cellular signalling and modulate the host signalling pathways. The aim of the current study was to clarify the role of OipA in H. pylori pathogenesis and its effect on host cell signalling pathways. To this end, the oipA gene was isolated and inserted into cloning and expression vectors. The recombinant plasmid was transferred into an expression host to produce OipA, which was subsequently purified by affinity chromatography and used for antibody production...
August 14, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28803056/analysis-of-microcystin-lr-and-nodularin-using-triple-quad-liquid-chromatography-tandem-mass-spectrometry-and-histopathology-in-experimental-fish
#13
Daljit Vudathala, Stephen Smith, Lester Khoo, David D Kuhn, Mary E Mainous, James Steadman, Lisa Murphy
Microcystins (MCs) are hepatotoxic cyanobacterial metabolites produced sporadically in aquatic environments under favorable environmental conditions. Affinity of these toxins to covalently bind with protein phosphatases poses a challenge in their detection. Lemieux oxidation to release 2-methyl-3-methoxy-4-phenylbutyric acid (MMPB), a common moiety to all MCs congeners, has been used in detection of these compounds, however a lack of sensitivity has limited the usefulness of the method. In this study, modifications of the oxidation and solid phase extraction procedures, combined with a sensitive LC/MS/MS (liquid chromatography/mass spectrometry) detection, have resulted in 25 ng/g method detection limits in both liver and plasma samples...
August 10, 2017: Toxicon: Official Journal of the International Society on Toxinology
https://www.readbyqxmd.com/read/28801948/a-proteomics-method-using-immunoaffinity-fluorogenic-derivatization-liquid-chromatography-tandem-mass-spectrometry-fd-lc-ms-ms-to-identify-a-set-of-interacting-proteins
#14
Katsunori Nakata, Ryoichi Saitoh, Masaki Ishigai, Kazuhiro Imai
Biological functions in organisms are usually controlled by a set of interacting proteins, and identifying the proteins that interact is useful for understanding the mechanism of the functions. Immunoprecipitation is a method that utilizes the affinity of an antibody to isolate and identify the proteins that have interacted in a biological sample. In this study, the FD-LC-MS/MS method, which involves fluorogenic derivatization followed by separation and quantification by HPLC and finally identification of proteins by HPLC-tandem mass spectrometry, was used to identify proteins in immunoprecipitated samples, using Heat shock protein 90 (HSP90) as a model of an interacting protein in HepaRG cells...
August 12, 2017: Biomedical Chromatography: BMC
https://www.readbyqxmd.com/read/28801830/cloning-and-expression-analysis-of-human-amelogenin-in-nicotiana-benthamiana-plants-by-means-of-a-transient-expression-system
#15
Mattia Pegoraro, Slavica Matić, Barbara Pergolizzi, Luca Iannarelli, Andrea M Rossi, Marco Morra, Emanuela Noris
Enamel is the covering tissue of teeth, made of regularly arranged hydroxyapatite crystals deposited on an organic matrix composed of 90% amelogenin that is completely degraded at the end of the enamel formation process. Amelogenin has a biomineralizing activity, forming nanoparticles or nanoribbons that guide hydroxyapatite deposit, and regenerative functions in bone and vascular tissue and in wound healing. Biotechnological products containing amelogenin seem to facilitate these processes. Here, we describe the production of human amelogenin in plants by transient transformation of Nicotiana benthamiana with constructs carrying synthetic genes with optimized human or plant codons...
August 11, 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/28800497/glycoconjugate-probes-containing-a-core-fucosylated-n-glycan-trisaccharide-for-fucose-lectin-identification-and-purification
#16
Deqin Cai, Chaochao Xun, Feng Tang, Xiaobo Tian, Liyun Yang, Kan Ding, Wenzhe Li, Zhiping Le, Wei Huang
Glyco-PAMAM dendrimers and glyco-agarose beads bearing a core-fucosylated N-glycan trisaccharide GlcNAcβ1,4(Fucα1,6)GlcNAc or a non-fucose disaccharide GlcNAcβ1,4GlcNAc were successfully synthesized and characterized by monosaccharide analysis with HPAEC-PAD technique. These glycoconjugates as fucose lectin probes were applied in fucose-specific lectin detection and purification. The model fucose lectin AAL indicated binding activity with the FITC-labeled PAMAM carrying core-fucose trisaccharide. An affinity chromatography column stuffed with the agarose beads carrying core-fucosylated trisaccharide exhibited a good specificity in purification of AAL than non-fucose disaccharide agarose beads...
August 3, 2017: Carbohydrate Research
https://www.readbyqxmd.com/read/28800364/expression-optimization-of-a-cell-membrane-penetrating-human-papillomavirus-type-16-therapeutic-vaccine-candidate-in-nicotiana-benthamiana
#17
Romana J R Yanez, Renate Lamprecht, Milaid Granadillo, Brandon Weber, Isis Torrens, Edward P Rybicki, Inga I Hitzeroth
High-risk human papillomaviruses (hr-HPVs) cause cervical cancer, the fourth most common cancer in women worldwide. A HPV-16 candidate therapeutic vaccine, LALF32-51-E7, was developed by fusing a modified E7 protein to a bacterial cell-penetrating peptide (LALF): this elicited both tumour protection and regression in pre-clinical immunization studies. In the current study, we investigated the potential for producing LALF32-51-E7 in a plant expression system by evaluating the effect of subcellular localization and usage of different expression vectors and gene silencing suppressors...
2017: PloS One
https://www.readbyqxmd.com/read/28797938/bihc-a-t-cell-engaging-bispecific-recombinant-antibody-has-potent-cytotoxic-activity-against-her2-tumor-cells
#18
Jieyu Xing, Limin Lin, Jing Li, Jiayu Liu, Changhua Zhou, Haitao Pan, Rui Shu, Bin Dong, Donglin Cao, Qing Li, Zhong Wang
Among different cancer immunotherapy approaches, bispecific antibodies (BsAbs) are of great interest due to their ability to recruit immune cells to kill tumor cells directly. Various BsAbs against Her2 tumor cells have been proposed with potent cytotoxic activities. However, most of these formats require extensive processing to obtain heterodimeric bispecific antibodies. In this study, we describe a bispecific antibody, BiHC (bispecific Her2-CD3 antibody), constructed with a single-domain anti-Her2 and a single-chain Fv (variable fragment) of anti-CD3 in an IgG-like format...
August 7, 2017: Translational Oncology
https://www.readbyqxmd.com/read/28795574/molecularly-imprinted-porous-monolithic-materials-from-melamine-formaldehyde-for-selective-trapping-of-phosphopeptides
#19
Mingquan Liu, Tri Minh Tran, Ahmed Awad Abbas Elhaj, Silje Bøen Torsetnes, Ole Nørregaard Jensen, Börje Sellergren, Knut Irgum
Thirty-five melamine-formaldehyde (MF) monolithic materials with bimodal pore distributions were synthesized in fused silica capillaries by catalyst-free polycondensation, starting with an aqueous MF precondensate, using acetonitrile as macroporogen and a variety of aliphatic polyethers and triblock copolymeric surfactants as porogens and mesoporogens, respectively. By varying the prepolymer composition and the type and molecular weights of the polymeric porogen components, a library of porous monolithic materials were produced, covering a range of meso- and macroporous properties...
August 10, 2017: Analytical Chemistry
https://www.readbyqxmd.com/read/28795493/-molecularly-imprinted-chromatography-fails-to-distinguish-homeopatic-remedy-from-placebo
#20
Roman Kaliszan, Małgorzata Waszczuk-Jankowska, Danuta Siluk, Bartosz Wielgomas
It was sensation when Dr. J. Beneviste reported alleged experimental biological evidence of the water memory effect (E. Dayenas et al. Nature 333, 816-818, 1988; https://doi.org/10.1038/333816a0). These experiments have never been reproduced in controlled conditions, however. Instead, the hypothesis that water can be imprinted with the memory of past solutes was ruled out (J. Maddox et al. Nature 334, 287-290, 1988; https://doi.org/10.1038/334287a0). At present, so-called "molecular imprinting" is a technology of preparing polymer matrices as affinity-based analyte separation media (J...
August 10, 2017: Journal of Separation Science
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