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https://www.readbyqxmd.com/read/29782014/identification-of-cyclin-dependent-kinase-1-specific-phosphorylation-sites-by-an-in-vitro-kinase-assay
#1
Heying Cui, Kyle M Loftus, Crystal R Noell, Sozanne R Solmaz
Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; however, the number of identified targets for Cdk1, particularly in human cells is still low. The identification of Cdk1-specific phosphorylation sites is important, as they provide mechanistic insights into how Cdk1 controls the cell cycle. Cell cycle regulation is critical for faithful chromosome segregation, and defects in this complicated process lead to chromosomal aberrations and cancer...
May 3, 2018: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/29782011/a-preclinical-mouse-model-of-osteosarcoma-to-define-the-extracellular-vesicle-mediated-communication-between-tumor-and-mesenchymal-stem-cells
#2
Tonny Lagerweij, Maria Pérez-Lanzón, S Rubina Baglio
Within the tumor microenvironment, resident or recruited mesenchymal stem cells (MSCs) contribute to malignant progression in multiple cancer types. Under the influence of specific environmental signals, these adult stem cells can release paracrine mediators leading to accelerated tumor growth and metastasis. Defining the crosstalk between tumor and MSCs is of primary importance to understand the mechanisms underlying cancer progression and identify novel targets for therapeutic intervention. Cancer cells produce high amounts of extracellular vesicles (EVs), which can profoundly affect the behavior of target cells in the tumor microenvironment or at distant sites...
May 6, 2018: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/29779667/terminal-alkyne-biosynthesis-in-marine-microbes
#3
Xuejun Zhu, Wenjun Zhang
The terminal alkyne is a readily derivatized functionality valued for its diverse applications in material synthesis, pharmaceutical science, and chemical biology. The synthetic biology routes to terminal alkynes are highly desired and yet underexplored. Some marine natural products contain a terminal alkyne functionality, and the discovery of the biosynthetic gene clusters for jamaicamide B and carmabin A marked the beginning of a new era in the understanding and engineering of terminal alkyne biosynthesis...
2018: Methods in Enzymology
https://www.readbyqxmd.com/read/29779664/pks-nrps-enzymology-and-structural-biology-considerations-in-protein-production
#4
Meredith A Skiba, Finn P Maloney, Qingyun Dan, Amy E Fraley, Courtney C Aldrich, Janet L Smith, W Clay Brown
The structural diversity and complexity of marine natural products have made them a rich and productive source of new bioactive molecules for drug development. The identification of these new compounds has led to extensive study of the protein constituents of the biosynthetic pathways from the producing microbes. Essential processes in the dissection of biosynthesis have been the elucidation of catalytic functions and the determination of 3D structures for enzymes of the polyketide synthases and nonribosomal peptide synthetases that carry out individual reactions...
2018: Methods in Enzymology
https://www.readbyqxmd.com/read/29779489/rap1-downregulation-by-mir-320c-reduces-platelet-activation-in-ex-vivo-storage
#5
Chintamani Atreya, Neetu Dahiya
BACKGROUND: A small GTPase protein, the Ras-related protein 1 (RAP1), abundant in platelets is known to be activated following agonist induced platelet activation, suggesting that RAP1 downregulation could in turn reduce platelet activation in storage. Our objective of this study is to identify RAP1 regulating miRNAs and their role in platelet activation during storage. METHODS: We applied MS2-TRAP (tagged RNA affinity purification) methodology to enrich miRNAs that target the 3' untranslated region (3'UTR) of RAP1 mRNA in two mammalian cell lines followed by miRNA identification by microarray of total RNA samples enriched for miRNAs...
May 20, 2018: MicroRNA
https://www.readbyqxmd.com/read/29778875/purification-of-recombinant-trichodysplasia-spinulosa-associated-polyomavirus-vp1-derived-virus-like-particles-using-chromatographic-techniques
#6
Mindaugas Zaveckas, Karolis Goda, Danguole Ziogiene, Alma Gedvilaite
Trichodysplasia spinulosa-associated polyomavirus (TSPyV) has been linked to a rare and recently characterized skin disease occurring in immunocompromised patients. In analogy with other polyomaviruses, the major capsid protein VP1 of TSPyV can self-assemble into virus-like particles (VLPs). VLPs are increasingly applied for the vaccination and diagnostics. Mostly, non-scalable and labor intensive ultracentrifugation-based techniques are used for the purification of VLPs. In this work, we developed a purification procedure for TSPyV VP1 VLPs based on two chromatographic steps, ion-exchange monolith and core bead chromatography...
May 9, 2018: Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences
https://www.readbyqxmd.com/read/29778873/bioanalysis-of-ibrutinib-and-its-dihydrodiol-and-glutathione-cycle-metabolites-by-liquid-chromatography-tandem-mass-spectrometry
#7
J J M Rood, P J A Dormans, M J van Haren, J H M Schellens, J H Beijnen, R W Sparidans
Ibrutinib is a targeted covalent inhibitor frequently used for the treatment of various lymphomas. In addition to oxidative metabolism, it is metabolized through glutathione coupling. The quantitative insight into this kind of metabolism is scarce, and tools for quantitation are lacking. The non-oxidative metabolism could prove a more prominent role when oxidative metabolism is impaired. Also, in-vitro studies could over-estimate the effect of CYP450-inhibition. To gain quantitative insight into this relatively unknown biotransformation pathway of the drug we have developed a validated simple, fast and sensitive bio-analytical assay for ibrutinib, dihydrodiol-ibrutinib, and the glutathione, cysteinylglycine and cysteine conjugates of ibrutinib in human plasma...
May 11, 2018: Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences
https://www.readbyqxmd.com/read/29778445/calixarene-immobilized-monolithic-cryogels-for-preparative-protein-chromatography
#8
Idris Guven, Orhan Gezici, Mevlut Bayrakci, Massimo Morbidelli
New cation exchanger monolithic stationary phases were prepared by immobilization of three different calixarene derivatives (i.e. tetracarboxylate calix[4]arene, CLX-COO, tetrasulfonate calix[4]arene, CLX-SO3 , and tetraphosphonate calix[4]arene, CLX-PO4 ) onto a monolithic cryogel support (i.e. poly(2-hydroksyethylmethacrilate-co-glycidyl methacrylate, P) and investigated with respect to preparative protein chromatography. The obtained monoliths were characterized through various techniques such as FTIR spectroscopy, isoelectric point measurements, titrimetric analyses, and mercury intrusion porosimetry...
May 16, 2018: Journal of Chromatography. A
https://www.readbyqxmd.com/read/29777748/a-brief-practical-review-of-size-exclusion-chromatography-rules-of-thumb-limitations-and-troubleshooting
#9
REVIEW
Richard R Burgess
Through years of research, teaching, troubleshooting, and editing, I have developed a solid working understanding of many aspects of protein purification and protein biochemistry. I have also found that many researchers do not understand the basics, let alone the pitfalls, of the techniques they use. In the case of size exclusion chromatography (SEC), I summarize below some of the rules of thumb, suggestions, limitations and troubleshooting I have found most useful in helping my students and colleagues carry out successful SEC...
May 16, 2018: Protein Expression and Purification
https://www.readbyqxmd.com/read/29775706/purification-and-characterization-of-an-organic-solvent-tolerant-and-detergent-stable-lipase-from-haloferax-mediterranei-cncmm-50101
#10
Siham Akmoussi-Toumi, Souad Khemili-Talbi, Imen Ferioune, Salima Kebbouche-Gana
The present study investigates the purification and biochemical characterization of an extracellular lipase (HML) from Haloarchaea Haloferax mediterranei strain ATS1, isolated from the Sebkha (Medea, Algeria). The pure protein was obtained with ammonium sulfate precipitation (40-70%)-dialysis and UNO Q-6 FPLC, and characterized biochemically. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis revealed that the purified enzyme was a monomer, with a molecular mass of 45,011...
May 15, 2018: International Journal of Biological Macromolecules
https://www.readbyqxmd.com/read/29775704/alkaline-active-cyanide-dihydratase-of-flavobacterium-indicum-mtcc-6936-growth-optimization-purification-characterization-and-in-silico-analysis
#11
Virender Kumar, Vijay Kumar, Tek Chand Bhalla
The present work explores a rare cyanide dihydratase of Flavobacterium indicum MTCC 6936 for its potential of cyanide degradation. The enzyme is purified to 12 fold with a yield of 76%. SDS and native-PAGE analysis revealed that enzyme was monomer of 40 kDa size. The enzyme works well in mesophilic range at wide array of pH. The thermostability profile of cyanide dihydratase revealed that the enzyme is quite stable at 30 °C and 35 °C with half-life of 6 h 30 min and 5 h respectively. Km and Vmax for cyanide dihydratase of F...
May 15, 2018: International Journal of Biological Macromolecules
https://www.readbyqxmd.com/read/29773808/quantitative-sumo-proteomics-reveals-the-modulation-of-several-pml-nuclear-body-associated-proteins-and-an-anti-senescence-function-of-ubc9
#12
Francis P McManus, Véronique Bourdeau, Mariana Acevedo, Stéphane Lopes-Paciencia, Lian Mignacca, Frédéric Lamoliatte, John W Rojas Pino, Gerardo Ferbeyre, Pierre Thibault
Several regulators of SUMOylation have been previously linked to senescence but most targets of this modification in senescent cells remain unidentified. Using a two-step purification of a modified SUMO3, we profiled the SUMO proteome of senescent cells in a site-specific manner. We identified 25 SUMO sites on 23 proteins that were significantly regulated during senescence. Of note, most of these proteins were PML nuclear body (PML-NB) associated, which correlates with the increased number and size of PML-NBs observed in senescent cells...
May 17, 2018: Scientific Reports
https://www.readbyqxmd.com/read/29773106/-expression-and-purification-of-flt3-and-flt3-itd-mutated-proteins-in-hek293t-cells
#13
Fan Yang, Jiahui Du, Jie Wu, Puyu Yuan, Qian Su, Rong Han, Qiaocheng Qiu, Shengli Xue, Songbai Liu, Zhongwen Sun
Objective To construct the eukaryotic expression vectors of human fms related tyrosine kinase 3 (FLT3) gene and FLT3-internal tandem duplication (FLT3-ITD) mutants and purify the native proteins through immunoprecipitation from HEK293T cell lysates. Methods The cDNA fragments of FLT3wt and FLT3-ITD were amplified from bone marrow cells of healthy individuals and FLT3-ITD-mutated acute myeloid leukemia (AML) patients with specific primers, and the PCR products were cloned in CD530A-T2A-GFP expression vectors...
March 2018: Xi Bao Yu Fen Zi Mian Yi Xue za Zhi, Chinese Journal of Cellular and Molecular Immunology
https://www.readbyqxmd.com/read/29773100/-eukaryotic-expression-purification-of-human-igecepsilon2-4-protein-and-its-target-fishing
#14
Huihui Huang, Zheyan Wang, Zhenggen Yang, Zhen Li, Ping Wang, Wenqi Dong
Objective To construct eukaryotic expression vectors of human IgE heavy chain 2-4 region (IgECepsilon2-4) and purify the recombinant protein, and then capture its interacted proteins by surface plasmon resonance (SPR). Methods Three recombinant eukaryotic expression vectors of IgECepsilon2-4 containing different signal peptides were constructed and transiently transfected into HEK293FT suspension cells separately. The recombinant plasmid with the highest-level expression was selected to express the recombinant protein in a huge amount, and then the recombinant protein was purified by Ni-NTA affinity chromatography...
March 2018: Xi Bao Yu Fen Zi Mian Yi Xue za Zhi, Chinese Journal of Cellular and Molecular Immunology
https://www.readbyqxmd.com/read/29768452/tyrosine-nitration-of-cytosolic-peroxidase-is-probably-triggered-as-a-long-distance-signaling-response-in-sunflower-seedling-cotyledons-subjected-to-salt-stress
#15
Prachi Jain, Satish C Bhatla
Present work focuses on tissue and concentration-dependent effect of nitric oxide (NO) on the modulation of cytosolic peroxidase (POD; EC 1.11.1.7) activity in 2-day old etiolated sunflower (Helianthus annuus L.) seedlings. Exogenously supplied NO (in the form of sodium nitroprusside [SNP] or diethylenetriamine NONOate [DETA]; 125 to 500 μM) results in noteworthy enhancement in seedling growth in a concentration dependent manner irrespective of salt-stress and differentially affects POD activity in 2-day old seedling cotyledons...
2018: PloS One
https://www.readbyqxmd.com/read/29768182/enhanced-production-and-immunological-characterization-of-recombinant-west-nile-virus-envelope-domain-iii-protein
#16
Nagesh K Tripathi, Divyanshi Karothia, Ambuj Shrivastava, Swati Banger, Jyoti S Kumar
West Nile virus (WNV) is an emerging mosquito-borne virus which is responsible for severe and fatal encephalitis in humans and for which there is no licensed vaccine or therapeutic available to prevent infection. The envelope domain III protein (EDIII) of WNV was over-expressed in Escherichia coli and purified using a two-step chromatography process which included immobilized metal affinity chromatography and ion exchange chromatography. E. coli cells were grown in a bioreactor to high density using batch and fed-batch cultivation...
May 13, 2018: New Biotechnology
https://www.readbyqxmd.com/read/29768061/structural-re-positioning-in-silico-molecular-modelling-oxidative-degradation-and-biological-screening-of-linagliptin-as-adenosine-3-receptor-adora3-modulators-targeting-hepatocellular-carcinoma
#17
Bassam M Ayoub, Yasmeen M Attia, Mahmoud S Ahmed
Chemical entities with structural diversity were introduced as candidates targeting adenosine receptor with different clinical activities, containing 3,7-dihydro-1H-purine-2,6-dione, especially adenosine 3 receptors (ADORA3). Our initial approach started with pharmacophore screening of ADORA3 modulators; to choose linagliptin (LIN), approved anti-diabetic drug as Dipeptidyl peptidase-4 inhibitors, to be studied for its modulating effect towards ADORA3. This was followed by generation, purification, analytical method development, and structural elucidation of oxidative degraded product (DEG)...
December 2018: Journal of Enzyme Inhibition and Medicinal Chemistry
https://www.readbyqxmd.com/read/29767339/simultaneous-decolorization-and-deproteinization-of-%C3%AE-%C3%AF-dodecanedioic-acid-fermentation-broth-by-integrated-ultrafiltration-and-adsorption-treatments
#18
Weifeng Cao, Yujue Wang, Jianquan Luo, Junxiang Yin, Yinhua Wan
α,ω-Dicarboxylic acids (DC) are versatile chemical intermediates with different chain length. For biosynthesis of DC, to obtain the highly pure product via crystallization, it is required to remove pigments and proteins in fermentation broth. However, a trade-off between decolorization/deproteinization ratio and DC recovery during the purification process was found, which impeded DC production by fermentation. When ultrafiltration (UF) was applied to treat α,ω-dodecanedioic acid (DC12 ) broth, 93.4% of DC12 recovery, 80...
May 16, 2018: Bioprocess and Biosystems Engineering
https://www.readbyqxmd.com/read/29765998/cloning-expression-purification-and-cd-analysis-of-recombinant-human-betatrophin
#19
Samaneh Gholami, Nematolah Gheibi, Reza Falak, Koorosh Goodarzvand Chegini
Background: Betatrophin is a member of the angiopoietin-like (ANGPTL) family that has been implicated in both triglyceride and glucose metabolism. The physiological functions and molecular targets of this protein remain largely unknown; hence, a purified available protein would aid study of the exact role of betatrophin in lipid or glucose metabolism. Methods: In this study, we cloned the full-length cDNA of betatrophin from a human liver cDNA library. Betatrophin was expressed in the pET-21b- E...
April 2018: Reports of Biochemistry & Molecular Biology
https://www.readbyqxmd.com/read/29765994/heterologous-expression-purification-and-characterization-of-the-hspx-ppe44-and-esxv-proteins-of-mycobacterium-tuberculosis
#20
Yousef Amini, Mohsen Tafaghodi, Saeid Amel Jamehdar, Zahra Meshkat, Bagher Moradi, Mojtaba Sankian
Background: Subunit vaccines are appropriate vaccine candidates for the prevention of some infections. In this study, three immunogenic proteins of Mycobacterium tuberculosis , including HspX, Ppe44, and EsxV as a new construction, were expressed alone and as a fusion protein to develop a new vaccine candidate against tuberculosis infection. Methods: To make the fusion protein, the three genes were linked together by AEAAAKEAAAKA linkers and inserted into pET21b and pET32b vectors...
April 2018: Reports of Biochemistry & Molecular Biology
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