Artículos de revistas: "Fast size exclusion liquid chromatography (FastSEC)"

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Im, Kyuhyun, Hae-woong Park, Sekyung Lee y Taihyun Chang. "Two-dimensional liquid chromatography analysis of synthetic polymers using fast size exclusion chromatography at high column temperature". Journal of Chromatography A 1216, n.º 21 (mayo de 2009): 4606–10. http://dx.doi.org/10.1016/j.chroma.2009.03.072.

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Wang, Xueqin, Huahua Yu, Ronge Xing y Pengcheng Li. "Characterization, Preparation, and Purification of Marine Bioactive Peptides". BioMed Research International 2017 (2017): 1–16. http://dx.doi.org/10.1155/2017/9746720.

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Marine bioactive peptides, as a source of unique bioactive compounds, are the focus of current research. They exert various biological roles, some of the most crucial of which are antioxidant activity, antimicrobial activity, anticancer activity, antihypertensive activity, anti-inflammatory activity, and so forth, and specific characteristics of the bioactivities are described. This review also describes various manufacturing techniques for marine bioactive peptides using organic synthesis, microwave assisted extraction, chemical hydrolysis, and enzymes hydrolysis. Finally, purification of marine bioactive peptides is described, including gel or size exclusion chromatography, ion-exchange column chromatography, and reversed-phase high-performance liquid chromatography, which are aimed at finding a fast, simple, and effective method to obtain the target peptides.

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Koellensperger, Gunda, Simon Daubert, Ralf Erdmann, Stephan Hann y Hanspeter Rottensteiner. "Characterisation of zinc-binding domains of peroxisomal RING finger proteins using size exclusion chromatography/inductively coupled plasma-mass spectrometry". Biological Chemistry 388, n.º 11 (1 de noviembre de 2007): 1209–14. http://dx.doi.org/10.1515/bc.2007.125.

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Abstract We determined the zinc binding stoichiometry of peroxisomal RING finger proteins by measuring sulfur/metal ratios using inductively coupled plasma-mass spectrometry coupled to size exclusion chromatography, a strategy that provides a fast and quantitative overview on the binding of metals in proteins. As a quality control, liquid chromatography-electrospray ionisation-time of flight-mass spectrometry was used to measure the molar masses of the intact proteins. The RING fingers of Pex2p, Pex10p, and Pex12p showed a stoichiometry of 2.0, 2.1, and 1.2 mol zinc/mol protein, respectively. Thus, Pex2p and Pex10p possess a typical RING domain with two coordinated zinc atoms, whereas that of Pex12p coordinates only a single zinc atom.

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Girardet, Jean-Michel, Franck Saulnier, Jean-Luc Gaillard, Jean-Paul Ramet y Gérard Humbert. "Camel (camelus dromedarius) milk PP3: evidence for an insertion in the amino-terminal sequence of the camel milk whey protein". Biochemistry and Cell Biology 78, n.º 1 (1 de febrero de 2000): 19–26. http://dx.doi.org/10.1139/o99-067.

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The camel (camelus dromedarius) milk proteose peptone 3 (PP3) was purified successively by size exclusion fast protein liquid chromatography and reversed phase high performance liquid chromatography and then characterized by amino acid residue composition determination and chemical microsequencing after CNBr or trypsin cleavages. In comparison with the previously reported structure of camel milk whey protein, the camel PP3 contains an insertion in the N-terminal region which has approximately 24 residues, whereas the remaining C-terminal regions of these two homologous proteins are essentially identical. The camel PP3 seems to contain a potential O-glycosylation site localized in this insertion and 2 or 3 phosphorylated serine residues. PP3 belongs to the glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1) family and could therefore play an immunological role in the camel or its suckling young.

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Mounicou, Sandra, Juris Meija y Joseph Caruso. "Preliminary studies on selenium-containing proteins in Brassica juncea by size exclusion chromatography and fast protein liquid chromatography coupled to ICP-MS". Analyst 129, n.º 2 (2004): 116. http://dx.doi.org/10.1039/b312960h.

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Labrecque, Jean, Pangala V. Bhat y André Lacroix. "Purification and partial characterization of a rat kidney aldehyde dehydrogenase that oxidizes retinal to retinoic acid". Biochemistry and Cell Biology 71, n.º 1-2 (1 de enero de 1993): 85–89. http://dx.doi.org/10.1139/o93-013.

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A NAD-dependent aldehyde dehydrogenase (EC 1.2.1.3) which catalyzes the oxidation of retinal to retinoic acid was purified to homogeneity from rat kidney by using Affi-Gel blue affinity chromatography and chromatofocusing, followed by Mono-Q anion-exchange chromatography. The apparent molecular weight of the native enzyme determined by size-exclusion fast protein liquid chromatography was 140 000. Sodium dodecyl sulfate - polyacrylamide gel electrophoresis gave a subunit molecular weight of 53 000. The isoelectric point as measured by chromatofocusing was 8.5. The enzyme also catalyzed the oxidation of acetaldehyde, but showed much lower Km value for the retinal substrate. We suggest that aldehyde dehydrogenase found in the kidney may be a specific retinal dehydrogenase, involved in vitamin A metabolism.Key words: aldehyde dehydrogenase, vitamin A, retinal, retinoic acid, kidney.

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Uversky, V. N. "Use of fast protein size-exclusion liquid chromatography to study the unfolding of proteins which denature through the molten globule". Biochemistry 32, n.º 48 (diciembre de 1993): 13288–98. http://dx.doi.org/10.1021/bi00211a042.

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Housley, David M., Jeremy L. Pinyon, Georg von Jonquieres, Chamini J. Perera, Michael Smout, Michael J. Liddell, Ernest A. Jennings, David Wilson y Gary D. Housley. "Australian Scorpion Hormurus waigiensis Venom Fractions Show Broad Bioactivity through Modulation of Bio-Impedance and Cytosolic Calcium". Biomolecules 10, n.º 4 (16 de abril de 2020): 617. http://dx.doi.org/10.3390/biom10040617.

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Scorpion venoms are a rich source of bioactive molecules, but characterisation of toxin peptides affecting cytosolic Ca2+, central to cell signalling and cell death, is limited. We undertook a functional screening of the venom of the Australian scorpion Hormurus waigiensis to determine the breadth of Ca2+ mobilisation. A human embryonic kidney (HEK293) cell line stably expressing the genetically encoded Ca2+ reporter GCaMP5G and the rabbit type 1 ryanodine receptor (RyR1) was developed as a biosensor. Size-exclusion Fast Protein Liquid Chromatography separated the venom into 53 fractions, constituting 12 chromatographic peaks. Liquid chromatography mass spectroscopy identified 182 distinct molecules with 3 to 63 components per peak. The molecular weights varied from 258 Da—13.6 kDa, with 53% under 1 kDa. The majority of the venom chromatographic peaks (tested as six venom pools) were found to reversibly modulate cell monolayer bioimpedance, detected using the xCELLigence platform (ACEA Biosciences). Confocal Ca2+ imaging showed 9/14 peak samples, with molecules spanning the molecular size range, increased cytosolic Ca2+ mobilization. H. waigiensis venom Ca2+ activity was correlated with changes in bio-impedance, reflecting multi-modal toxin actions on cell physiology across the venom proteome.

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Hu, Jing, Chris Weise, Christoph Böttcher, Hua Fan y Jian Yin. "Expression, purification and structural analysis of functional GABA transporter 1 using the baculovirus expression system". Beilstein Journal of Organic Chemistry 13 (11 de mayo de 2017): 874–82. http://dx.doi.org/10.3762/bjoc.13.88.

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The γ-aminobutyric acid (GABA) transporter 1 (GAT1) belongs to a family of Na+ and Cl−-coupled transport proteins and possesses 12 putative transmembrane domains. To perform structural analyses of the GAT1 protein, the GAT1/green fluorescent protein (GFP) fusion protein was functionally expressed in insect Sf9 cells by the BAC-TO-BAC® baculovirus expression system. A two-step procedure to purify the GAT1/GFP fusion protein from insect Sf9 cells has been established and involves immunoaffinity chromatography using self-prepared anti-GFP antibodies and size-exclusion fast protein liquid chromatography (SE-FPLC). A yield of 200–300 μg of the GAT1/GFP protein could be purified from 400–600 mL of infected Sf9 cells. The purified protein was analyzed by transmission electron microscopy (TEM), which revealed that the GAT1/GFP fusion protein was isolated in its monomeric form.

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Alcaide-Hidalgo, Juan María, Miguel Romero, Juan Duarte y Eduardo López-Huertas. "Antihypertensive Effects of Virgin Olive Oil (Unfiltered) Low Molecular Weight Peptides with ACE Inhibitory Activity in Spontaneously Hypertensive Rats". Nutrients 12, n.º 1 (20 de enero de 2020): 271. http://dx.doi.org/10.3390/nu12010271.

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The low molecular weight peptide composition of virgin olive oil (VOO) is mostly unknown. We hypothesised that unfiltered VOO could possess low molecular weight peptides with antihypertensive activity. We produced unfiltered VOO and obtained a water-soluble peptide extract from it. The peptides were separated by size-exclusion using fast protein liquid chromatography, and the low molecular weight fraction was analysed by nanoscale liquid chromatography-Orbitrap coupled with tandem mass spectrometry and de novo sequencing. We selected 23 peptide sequences containing between 6 and 9 amino acids and molecular masses ranging 698–1017 Da. Those peptides were chemically synthesised and their angiotensin-converting enzyme (ACE) inhibitory activity was studied in vitro. Seven peptides showed a strong activity, with half maximal inhibitory concentration (IC50) <10 µm. The antihypertensive effects of the four most active synthesised ACE inhibitor peptides were studied in spontaneously hypertensive rats (SHR). Acute oral administration of synthetic peptides RDGGYCC and CCGNAVPQ showed antihypertensive activity in SHR. We conclude that unfiltered VOO naturally contains low molecular weight peptides with specific ACE inhibitory activity and antihypertensive effects in SHR.

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Vlaanderen, Ineke, Rienk Van Grondelle y Michael Bloemendal. "The Use of Fast Protein Liquid (Size Exclusion) Chromatography for the Fractionation of Crystallins and the Study of β-Crystallin Aggregation". Journal of Liquid Chromatography 16, n.º 2 (enero de 1993): 367–82. http://dx.doi.org/10.1080/10826079308020919.

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Zhang, Xinrong, Rita Cornelis, Jurgen De Kimpe, Louis Mees y Norbert Lameire. "Study of arsenic–protein binding in serum of patients on continuous ambulatory peritoneal dialysis". Clinical Chemistry 44, n.º 1 (1 de enero de 1998): 141–47. http://dx.doi.org/10.1093/clinchem/44.1.141.

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Abstract Arsenic (As) bound to serum proteins in patients on continuous ambulatory peritoneal dialysis (CAPD) was studied. A prior experiment by ultrafiltration showed that 5.57% of total As was bound to serum proteins for 14 CAPD patients. Further identification of the As species and protein molecules in serum of three CAPD patients with high As concentrations was carried out by combining the separation methods of size-exclusion, anion-exchange, and affinity fast-protein liquid chromatography, detected by hydride generation atomic absorption spectrometry. The results indicated that only inorganic As species are bound to serum proteins. Transferrin is the main carrier. The concentrations of As bound to proteins in serum for the three patients were 0.44 ± 0.12, 0.19 ± 0.09, and 0.59 ± 0.09 μg/L (n = 3), respectively.

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Mitrović, Bojan y Radmila Milačič. "Speciation of aluminium in forest soil extracts by size exclusion chromatography with UV and ICP-AES detection and cation exchange fast protein liquid chromatography with ETAAS detection". Science of The Total Environment 258, n.º 3 (septiembre de 2000): 183–94. http://dx.doi.org/10.1016/s0048-9697(00)00569-6.

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Noonin, Chadanat, Chompunoot Kapincharanon, Kanyarat Sueksakit, Rattiyaporn Kanlaya y Visith Thongboonkerd. "Application of tandem fast protein liquid chromatography to purify intact native monomeric/aggregated Tamm–Horsfall protein from human urine and systematic comparisons with diatomaceous earth adsorption and salt precipitation: yield, purity and time-consumption". Analytical Methods 13, n.º 30 (2021): 3359–67. http://dx.doi.org/10.1039/d1ay00922b.

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The efficiency of tandem FPLC (Mono Q anion-exchange/Superdex 200 size-exclusion) for purification of intact Tamm–Horsfall protein (uromodulin) from human urine was compared with two conventional methods, i.e., salt precipitation and DE adsorption.

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Lopez-Huertas, Eduardo y Juan M. Alcaide-Hidalgo. "Characterisation of Endogenous Peptides Present in Virgin Olive Oil". International Journal of Molecular Sciences 23, n.º 3 (2 de febrero de 2022): 1712. http://dx.doi.org/10.3390/ijms23031712.

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The low molecular weight peptide composition of virgin olive oil (VOO) is mostly unknown. We aimed to investigate the composition of the endogenous peptides present in VOO, the protein sources from which those peptides originate and their biological activities. A water-soluble extract containing peptides was obtained from VOO. The peptides were separated by size-exclusion using fast protein liquid chromatography, and the low molecular weight fraction (1600–700 kDa) was analysed by nanoscale liquid chromatography Orbitrap coupled with tandem mass spectrometry and de novo sequencing. Nineteen new peptides were identified by Peaks database algorithm, using the available Olea europaea (cv. Farga) genome database. Eight new peptides were also identified by Peaks de novo sequencing. The protein sources of the peptides detected in the database by Peaks DB were identified by BLAST-P search. Seed storage proteins were among the most frequent sources of VOO peptides. BIOPEP software was used to predict the biological activities of peptides and to simulate (in silico) the proteolytic activity of digestive enzymes on the detected peptide sequences. A selection of synthetic peptides was obtained for investigation of their bioactivities. Peptides VCGEAFGKA, NALLCSNS, CPANGFY, CCYSVY and DCHYFL possessed strong ACE-inhibitory and antioxidant activities in vitro. Antioxidant peptides could play a role in VOO quality.

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McIntosh, C. H., C. L. Tang, A. J. Malcolm, M. Ho, Y. N. Kwok y J. C. Brown. "Effect of a purified somatostatin monoclonal antibody and its Fab fragments on gastrin release". American Journal of Physiology-Gastrointestinal and Liver Physiology 260, n.º 3 (1 de marzo de 1991): G489—G498. http://dx.doi.org/10.1152/ajpgi.1991.260.3.g489.

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The effect of a purified somatostatin monoclonal antibody, SOMA 10, and its Fab fragments on gastrin release have been examined in the isolated perfused rat stomach. SOMA 10 was purified from ascites fluid by ammonium sulfate precipitation followed by hydroxylapatite (HAP) chromatography. This gave a preparation of greater than 90% purity, as assessed by fast affinity and size exclusion high-performance liquid chromatography (HPLC). HAP-purified SOMA 10 had a binding capacity of 760 mmol somatostatin/mol antibody, a dissociation constant of 2.2 nM, and recognized amino acid residues 5-12 of the somatostatin molecule. Fab fragments of SOMA 10 were prepared and purified by protein A affinity chromatography, and the purity assessed by HPLC and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Single passage perfusion of SOMA 10 (100 micrograms/ml) into the gastric vasculature caused a paradoxical decrease in basal gastrin release. However, recirculation of the antibody (20 micrograms/ml) caused an increase in cumulated gastrin release. Perfusion of Fab fragments of SOMA 10 (66 micrograms/ml) also increased gastrin secretion. These results support the suggestion that somatostatin exerts a tonic restraint on gastrin release and that penetration of the antibody to the site of somatostatin release is necessary for it to have an effect.

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Groeneveld, Gino, Ron Salome, Melissa N. Dunkle, Mubasher Bashir, Andrea F. G. Gargano, Matthias Pursch, Edwin P. C. Mes y Peter J. Schoenmakers. "Fast determination of functionality-type × molecular-weight distribution of propoxylates with varying numbers of hydroxyl end-groups using gradient–normal-phase liquid chromatography × ultra-high pressure size-exclusion chromatography". Journal of Chromatography A 1659 (diciembre de 2021): 462644. http://dx.doi.org/10.1016/j.chroma.2021.462644.

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Pasch, Harald, Lars-Christian Heinz, Tibor Macko y Wolf Hiller. "High-temperature gradient HPLC and LC-NMR for the analysis of complex polyolefins". Pure and Applied Chemistry 80, n.º 8 (1 de enero de 2008): 1747–62. http://dx.doi.org/10.1351/pac200880081747.

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The synthesis and characterization of polyolefins continues to be one of the most important areas for academic and industrial polymer research. One consequence of the development of new "tailor-made" polyolefins is the need for new and improved analytical techniques for the analysis of polyolefins with respect to molar mass and chemical composition distribution. The present article briefly reviews different new and relevant techniques for polyolefin analysis. Crystallization analysis fractionation is a powerful new technique for the analysis of short-chain branching in linear low-density polyethylene (LLDPE) and the analysis of polyolefin blends and copolymers regarding chemical composition. For the fast analysis of the chemical composition distribution, a new high-temperature gradient high-performance liquid chromatography (HPLC) system has been developed. The efficiency of this system for the separation of various olefin copolymers is demonstrated. The correlation between molar mass and chemical composition can be accessed by on-line coupling of high-temperature size exclusion chromatography (HT-SEC) and 1H NMR spectroscopy. It is shown that the on-line NMR analysis of chromatographic fractions yields information on microstructure and tacticity in addition to molar mass and copolymer composition.

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Wachter, Franziska, Radoslaw Nowak y Eric Fischer. "Prediction of PP2A Holoenzymes Guided By the Structural Characterization of Methylation Independent PP2A Assembly". Blood 142, Supplement 1 (28 de noviembre de 2023): 2802. http://dx.doi.org/10.1182/blood-2023-172895.

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Introduction: The dysregulation of phosphorylation-dependent signaling is a hallmark of leukemogenesis. Kinase inhibitors have revolutionized the outcomes of patients with chronic myeloid leukemia. An orthogonal, but less explored way to target phosphorylation pathways is to activate phosphatases, such as the ubiquitously expressed protein- phosphatase 2A (PP2A). PP2A is an essential serine/threonine phosphatase that regulates various cellular processes, including cell growth, division, and differentiation. Dysregulation of PP2A has been linked to leukemia. PP2A is a modular multi-subunit enzyme: One scaffold subunit (A) binds to a catalytic subunit (C) to form a core AC heterodimer, which together with one of many regulatory (B) subunits forms the active enzyme. The regulatory (B) subunits are classified into 4 distinct subfamilies without significant homology. The combinatorial assembly of these subunits generates a wide range of PP2A complexes, resulting in diverse substrate specificity and subcellular localization. The detailed mechanism of PP2A regulation remains elusive and reports about a governing role of methylation of the carboxy-terminal Leucin of PP2A C are conflicting. Understanding the underlying molecular regulation of PP2A activity is of significant interest for future therapeutic development for acute myeloid leukemia. Methods: Recombinant PP2A was expressed in insect cells, and purified by affinity purification, anion exchange chromatography and size exclusion chromatography (SEC). Extensive quality control, including liquid chromatography-mass spectrometry, SDS-PAGE gel and fast protein liquid chromatography was performed. The degree of methylation of PP2A C Leucine 309 was confirmed by mass spectrometry. We validated protein stability with differential scanning fluorimetry. Pull-down assays and size exclusion chromatography characterized complex formation. A calorimetric assay using p-nitrophenyl phosphate as a substrate functionally assessed PP2A in vitro. Structural information was obtained by X-ray crystallography and AlphaFold Multimer. Results: Using in vitro biochemistry, including pull down and size exclusion chromatography, we demonstrate that methylation of the carboxy terminus of the PP2A C subunit is dispensable for PP2A assembly in vitro. Absence of methylation of Leucine 309 was confirmed by mass-spectrometry. We show by SEC that instead PP2A complex formation is dependent on B subunit concentration. Carboxy-terminal methylation of the PP2A C subunit was also not required for functional activity in a calorimetric assay using p-nitrophenyl phosphate as a substrate. PP2A formed spontaneously in vitro and was enzymatically active independent of methylation of Leucine 309. To corroborate these findings, we determined the X-ray crystal structure of the PP2A:PPP2B56E:PPP2C complex to 2.7 Å resolution with the unmethylated Leucine 309. Interestingly our experimental structure superimposed with an AlphaFold Multimer model of the PP2A trimer. Building on this finding, we predicted the models of all 64 possible PP2A complexes providing a framework for further study and structural analysis of PP2A complexes and their activation. These studies provide key information for the structural design and development of novel drugs for hematologic malignancies by targeting of selected PP2A complexes and clarified the role of Leucine 309 methylation in PP2A activation. Conclusion: Methylation of the carboxy-terminal Leucine 309 of the PP2A C subunitis dispensable for PP2A activity in vitro. Integrative structural biology studies, combining biochemistry, X-ray crystallography and AlphaFold Multimer provide a comprehensive map of PP2A complexes. These studies have the potential to accelerate the drug discovery process and unlock PP2A activation as a new therapeutic modality for leukemia.

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Wang, Ruizhi, Xiaojing Yang, Lingwen Cui, Hang Yin y Shaohua Xu. "Gels of Amyloid Fibers". Biomolecules 9, n.º 6 (30 de mayo de 2019): 210. http://dx.doi.org/10.3390/biom9060210.

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Protein self-assembly and formation of amyloid fibers is an early event of numerous human diseases. Continuous aggregation of amyloid fibers in vitro produces biogels, which led us to suspect that amyloid plaques and neurofibrillary tangles in Alzheimer’s disease are of biogels in nature. We applied atomic force microscopy, size exclusion chromatography, and differential scanning calorimetry to elucidate the gel’s structure, kinetics of gel formation, and melting point. We found that (1) lysozyme gelation occurs when the protein concentration is above 5 mg/mL; (2) nonfibrous protein concentration decreases and plateaus after three days of gel synthesis reaction; (3) colloidal lysozyme aggregates are detectable by both atomic force microscopy (AFM) and fast protein liquid chromatography (FPLC); (4) the gels are a three-dimensional (3D) network crosslinked by fibers coiling around each other; (5) the gels have a high melting point at around around 110 °C, which is weakly dependent on protein concentration; (6) the gels are conductive under an electric field, and (7) they form faster in the presence than in the absence of salt in the reaction buffer. The potential role of the gels formed by amyloid fibers in amyloidosis, particularly in Alzheimer’s disease was thoroughly discussed, as gels with increased viscosity, are known to restrict bulk flow and then circulation of ions and molecules.

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Brown, Nathan, Hartmut Zehender, Kamal Azzaoui, Ansgar Schuffenhauer, Lorenz M. Mayr y Edgar Jacoby. "A Chemoinformatics Analysis of Hit Lists Obtained from High-Throughput Affinity-Selection Screening". Journal of Biomolecular Screening 11, n.º 2 (16 de diciembre de 2005): 123–30. http://dx.doi.org/10.1177/1087057105283579.

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The high-throughput affinity-selection screening platform SpeedScreen was recently reported by the Novartis Institutes for BioMedical Research as a homogeneous, label-free screening technology with mass-spectrometry readout. SpeedScreen relies on the screening of compound mixtures with various target proteins and uses fast size-exclusion chromatography to separate target-bound from unbound substances. After disintegration of the target-binder complex, the binder molecules are identified by their molecular masses using liquid chromatography/mass spectrometry. The authors report an analysis of the molecular properties of hits obtained with SpeedScreen on 26 targets screened within the past few years at Novartis using this technology. Affinity-based SpeedScreen is a robust high-throughput screening technology that does not accumulate frequent hitters or potential covalent binders. The hits are representative of the most commonly identified scaffold classes observed for known drugs. Validated SpeedScreen hits tend to be enriched on more lipophilic and larger-molecular-weight compounds compared to the whole library. The potential for a reduced SpeedScreen screening set to be used in case only limited protein quantities are available is evaluated. Such a reduced compound set should also maximize the coverage of the high-performing regions of the chemical property and class spaces; chemoinformatics methods including genetic algorithms and divisive K-means clustering are used for this aim.

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Antczak, Andrzej, Jan Szadkowski, Dominika Szadkowska y Janusz Zawadzki. "Assessment of the effectiveness of liquid hot water and steam explosion pretreatments of fast-growing poplar (Populus trichocarpa) wood". Wood Science and Technology 56, n.º 1 (6 de diciembre de 2021): 87–109. http://dx.doi.org/10.1007/s00226-021-01350-1.

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AbstractIn this paper, the influence of physicochemical pretreatment methods on the chemical composition, enzymatic hydrolysis efficiency and porosity of fast-growing Populus trichocarpa wood was compared. Among the pretreatment methods, the liquid hot water (LHW) and steam explosion (SE) were used, which were performed at three different temperatures (160 °C, 175 °C and 190 °C) and two residence times (15 min and 1 h). The chemical composition, enzymatic hydrolysis and porosity analysis were done for native wood and solid fraction obtained after LHW and SE pretreatments. The porosity analysis was performed by inverse size exclusion chromatography method. Additionally, inhibitors of hydrolysis and fermentation processes in the liquid and solid fractions obtained after pretreatments were examined. Based on the results, it was found that the tested pretreatments caused the greatest changes in the chemical content of hemicelluloses. It was found that after LHW and SE pretreatments up to 99.1% or 94.0%, respectively, of hemicelluloses were removed from the obtained solid fraction. Moreover, the LHW and SE processes greatly enhanced the enzymatic digestibility of fast-growing poplar wood. The highest glucose yield was achieved after 15 min of SE pretreatment at 190 °C and was 676.4 mg/g pretreated biomass, while in the case of xylose the highest value (119.3 mg/g pretreated biomass) was obtained after 15 min of LHW pretreatment at 160 °C. Generally, after SE pretreatment process, more inhibitors were formed, and a greater effect of porous structure development was noticed than after LHW pretreatment. Despite this difference, the average glucose contents and yields after enzymatic hydrolysis of pretreated biomass were generally similar regardless of the pretreatment used.

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Mounicou, Sandra, Joanna Szpunar, Ryszard Lobinski, Daniel Andrey y Christopher-John Blake. "Bioavailability of cadmium and lead in cocoa: comparison of extraction procedures prior to size-exclusion fast-flow liquid chromatography with inductively coupled plasma mass spectrometric detection (SEC-ICP-MS)". J. Anal. At. Spectrom. 17, n.º 8 (2002): 880–86. http://dx.doi.org/10.1039/b201639g.

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Ali, Syed Abid, Fozia Humayun, Iqra Munir, Shakil Ahmad, Zarrien Ayub, Habib Fatima, Lakht-e. Zehra y Muhammad Samee Haider. "Morphological, Molecular, Biochemical and Nutritional Characterization of Three Major Thais Species from the Sindh Coast of Pakistan". Open Food Science Journal 10, n.º 1 (31 de diciembre de 2018): 33–45. http://dx.doi.org/10.2174/1874256401810010033.

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Objective: The present study was conducted to investigate the biomass assessment, morphological and molecular identification, nutritive status and biochemical characterization of three major Thais species (T. bufo, T. hippocastanum and T. rudolphi) from the Sindh Coast, Pakistan. Methods: Samples were collected from Buleji and Paradise Point at the Sindh Coast. Species were identified morphologically as well as genetically by amplifying two mitochondrial 16S rDNA & Cytochrome Oxidase I (COI) and one nuclear (Histone H3) genes. Shell microstructure and chemistry were also studied by scanning electron microscopy and Energy Dispersive X-ray spectrometry (EDX). The body muscle was dissected and used for nutritional composition determination such as estimation of total protein, carbohydrates, lipids, protein fingerprinting by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Size-Exclusion - Fast Protein Liquid Chromatography (SEC-FPLC), amino acid and fatty acid analysis. Results: Nutritionally, the total protein was found to be the major content followed by carbohydrate and lipid in the three Thais sp. The presence of medicinally important hemocyanin as abundant hemolymph protein was confirmed via SDS-PAGE and SEC FPLC. Nine different types of fatty acids and a high concentration of essential amino acids were also determined. Conclusion: Our findings suggest that Thais sp. are nutritionally rich and can be consumed as a valuable marine resource to overcome the malnutrition problem in developing countries.

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Lee, Haneul, Su Jin Kang, Jimin Lee, Kyong Hwa Park y Won Jong Rhee. "Isolation and Characterization of Urinary Extracellular Vesicles from Healthy Donors and Patients with Castration-Resistant Prostate Cancer". International Journal of Molecular Sciences 23, n.º 13 (27 de junio de 2022): 7134. http://dx.doi.org/10.3390/ijms23137134.

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Prostate cancer (PCa) is the most commonly diagnosed malignancy among men in developed countries. The five-year survival rate for men diagnosed with early-stage PCa is approximately 100%, while it is less than 30% for castration-resistant PCa (CRPC). Currently, the detection of prostate-specific antigens as biomarkers for the prognosis of CRPC is criticized because of its low accuracy, high invasiveness, and high false-positive rate. Therefore, it is important to identify new biomarkers for prediction of CRPC progression. Extracellular vesicles (EVs) derived from tumors have been highlighted as potential markers for cancer diagnosis and prognosis. Specifically, urinary EVs directly reflect changes in the pathophysiological conditions of the urogenital system because it is exposed to prostatic secretions. Thus, detecting biomarkers in urinary EVs provides a promising approach for performing an accurate and non-invasive liquid biopsy for CPRC. In this study, we effectively isolated urinary EVs with low protein impurities using size-exclusion chromatography combined with ultrafiltration. After EV isolation and characterization, we evaluated the miRNAs in urinary EVs from healthy donors and patients with CRPC. The results indicated that miRNAs (miR-21-5p, miR-574-3p, and miR-6880-5p) could be used as potential biomarkers for the prognosis of CRPC. This analysis of urinary EVs contributes to the fast and convenient prognosis of diseases, including CRPC, in the clinical setting.

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Allouche, Rania, Zeeshan Hafeez, Annie Dary-Mourot, Magali Genay y Laurent Miclo. "Streptococcus thermophilus: A Source of Postbiotics Displaying Anti-Inflammatory Effects in THP 1 Macrophages". Molecules 29, n.º 7 (30 de marzo de 2024): 1552. http://dx.doi.org/10.3390/molecules29071552.

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In addition to traditional use in fermented dairy products, S. thermophilus also exhibits anti-inflammatory properties both in live and heat-inactivated form. Recent studies have highlighted that some hydrolysates from surface proteins of S. thermophilus could be responsible partially for overall anti-inflammatory activity of this bacterium. It was hypothesized that anti-inflammatory activity could also be attributed to peptides resulting from the digestion of intracellular proteins of S. thermophilus. Therefore, total intracellular proteins (TIP) from two phenotypically different strains, LMD-9 and CNRZ-21N, were recovered by sonication followed by ammonium sulphate precipitation. The molecular masses of the TIP of both strains were very close to each other as observed by SDS-PAGE. The TIP were fractionated by size exclusion fast protein liquid chromatography to obtain a 3–10 kDa intracellular protein (IP) fraction, which was then hydrolysed with pancreatic enzyme preparation, Corolase PP. The hydrolysed IP fraction from each strain exhibited anti-inflammatory activity by modulating pro-inflammatory mediators, particularly IL-1β in LPS-stimulated THP-1 macrophages. However, a decrease in IL-8 secretion was only observed with hydrolysed IP fraction from CNRZ-21N, indicating that strain could be an important parameter in obtaining active hydrolysates. Results showed that peptides from the 3–10 kDa IP fraction of S. thermophilus could therefore be considered as postbiotics with potential beneficial effects on human health. Thus, it can be used as a promising bioactive ingredient for the development of functional foods to prevent low-grade inflammation.

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Sinha, Madhur K., Traci Songer, Qiang Xiao, John H. Sloan, Jin Wang, Shaoquen Ji, William E. Alborn et al. "Analytical Validation and Biological Evaluation of a High–Molecular-Weight Adiponectin ELISA". Clinical Chemistry 53, n.º 12 (1 de diciembre de 2007): 2144–51. http://dx.doi.org/10.1373/clinchem.2007.090670.

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Abstract Background: Of the 3 circulating multimeric forms of adiponectin, the high–molecular-weight (HMW) form, as measured by size-exclusion and/or immunoblotting techniques, is a better index of insulin sensitivity for monitoring health and disease than is total adiponectin. We aimed to develop a simple ELISA to measure HMW adiponectin. Methods: We pretreated serum or plasma samples with digestion solution containing proteinase K (Millipore, ESDS). HMW (Millipore, EZHMWA-64K) and total adiponectin (Millipore, EZHADP-61K) concentrations were measured in treated and untreated samples, respectively, from 108 individuals and from 20 morbidly obese patients before and at 1, 3, 6, and 12 months after gastric-bypass surgery. Results: The ELISA has a dynamic range of 3–200 μg/L and a detection limit of 0.8 μg/L. Intraassay and interassay CVs were <4% and <10%, respectively. Sample-dilution curves paralleled the calibration curves. Fast protein liquid chromatography profiles of the proteinase K-treated samples revealed predominantly HMW adiponectin. Values for HMW adiponectin produced with this method are comparable with those obtained with Western blot analysis (y = 0.77x − 0.15; r = 0.96; n = 56). Body mass index (BMI)- and sex-related changes were more pronounced for HMW adiponectin and percentage of HMW adiponectin than for total adiponectin. HMW and total adiponectin increased after bypass surgery, but changes in HMW adiponectin were more pronounced and preceded changes in total adiponectin. Conclusion: This simple, rapid ELISA for HMW adiponectin recognizes the HMW isoform, produces results closely correlated with those obtained with Western blotting, and appears to better distinguish BMI-, sex-, and weight loss–associated differences than assays for total adiponectin.

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Gröschl, Michael, Hans G. Topf, Jörg Bohlender, Johannes Zenk, Sven Klussmann, Jörg Dötsch, Wolfgang Rascher y Manfred Rauh. "Identification of Ghrelin in Human Saliva: Production by the Salivary Glands and Potential Role in Proliferation of Oral Keratinocytes". Clinical Chemistry 51, n.º 6 (1 de junio de 2005): 997–1006. http://dx.doi.org/10.1373/clinchem.2004.040667.

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Abstract Background: We investigated whether ghrelin is present in human saliva, is produced by salivary glands, and physiologic consequences of these findings. Methods: Expression of ghrelin and specific receptor mRNA was determined by PCR. Proteins were identified by immunoblotting and size-exclusion fast protein liquid chromatography (FPLC) with consecutive RIA. Specific RIAs were used for quantification of salivary total and bioactive ghrelin. Distribution of ghrelin was investigated by immunohistochemistry in cryosections of the salivary glands. The effect of ghrelin on incorporation of 5-bromo-2′-deoxyuridine as a measure of cell proliferation was investigated in primary oral keratinocytes. Results:Ghrelin is produced by the salivary glands. The hormone was identified in saliva and glands by immunoblotting and by FPLC fractionation of saliva. Immunohistochemistry demonstrated ghrelin distribution in the salivary glands. The receptor was also produced by the glands and by oral keratinocytes and was shown to be functional. Comparison of total ghrelin values for healthy individuals (body mass index, 18–27 kg/m2) showed significantly lower concentrations in saliva than in serum (P <0.01). The correlation between both matrices was r2 = 0.56 (P <0.001) with a negative correlation to body mass index (r2 = 0.314; P <0.01). Bioactive acylated ghrelin was also present in saliva. Incubation of keratinocytes with ghrelin led to significantly increased cell proliferation (P <0.001). This effect could be completely suppressed by co-incubation with NOX-B11 (50 nmol/L), a novel specific inhibitor of acylated ghrelin. Conclusions: Ghrelin in saliva is produced and released by salivary glands. The effect of ghrelin on oral cell proliferation adds to the pro-proliferative action of other salivary growth factors.

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Benomar, Souhila, Sanaa Yahia, Faiza Dehiba, Natalia Guillen, Maria Jesús Rodriguez-Yoldi, Jesús Osada y Ahmed Boualga. "Differential antioxidative and hypocholesterolemic responses to two fish protein hydrolysates (Sardina pilchardus and Boops boops) in cholesterol-fed rats". Nutrition & Food Science 45, n.º 3 (11 de mayo de 2015): 448–66. http://dx.doi.org/10.1108/nfs-11-2014-0096.

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Purpose – The purpose of this study was to evaluate the antioxidant and hypocholesterolemic activities of sardine and bogue protein hydrolysates in cholesterol-fed rats. Design/methodology/approach – In total, 18 male Wistar rats (220 ± 10 g) fed 20 per cent casein, 1 per cent cholesterol and 0.5 per cent cholic acid were divided into three groups and received a daily gavage of 250 mg of sardine (SPH) or bogue (BPH) protein hydrolysates for 30 days. The third group, named control group (CG), received in the same conditions water. Lipoproteins were fractionated by size-exclusion fast protein liquid chromatography, and serum lipids, apolipoproteins and lipoproteins were assayed. Findings – In SPH and BPH groups, serum total cholesterol concentrations were −66 per cent lower than in CG. This corresponded to the decreased very low-density lipoprotein-C in the former groups. Moreover, BPH treatment reduced low-density lipoprotein-C compared with CG and SPH groups. Compared with CG, serum phospholipids were reduced by SPH and BPH. Furthermore, BPH increased significantly APOA4 and sphingomyelin but lowered phosphatidylcholine. In the latter group, serum lecithin cholesterol acyltransferase activity was +23 per cent higher, but with SPH, this activity was −35 per cent reduced compared with CG. Apolipoprotein A-I contents were similar in the three groups. Compared with CG, hydroperoxide and lipid peroxidation contents in serum and lipoprotein fractions were reduced by SPH and BPH. Compared with CG, serum superoxide dismutase and glutathione peroxidase activities were increased in the treated groups, particularly in the BPH group. Originality/value – These results suggest that sardine protein hydrolysates and particularly those of bogue could be a very useful natural compound to prevent hypercholesterolemia by both improving the lipid profile and modulating oxidative stress in cholesterol-fed rats.

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Hao, Xiaolei, Shu-Ting Chen, Yu-Hsuan Chiao, Hideto Matsuyama, Xianghong Qian y Ranil Wickramasinghe. "Aggregate Removal by Responsive Electrospun Membrane based Hydrophobic Interaction Chromatography". Chemie Ingenieur Technik, 14 de febrero de 2024. http://dx.doi.org/10.1002/cite.202300144.

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AbstractPoly(N‐vinylcaprolactam) (PVCL), a salt responsive ligand, was grafted from electrospun cellulose nanofiber membranes by atom transfer radical polymerization (ATRP). The membranes were used for hydrophobic interaction chromatography. The modified PVCL‐cellulose membranes were used to separate BSA dimers from monomers as well as monoclonal antibody (mAb) aggregates. Fast protein liquid chromatography (FPLC) was used for protein separation and fraction collection. High performance liquid chromatography with a size exclusion column (HPLC‐SEC) analysis confirmed the separation of monomers and aggregates from both BSA and the mAb. The results suggest that use of a responsive ligand that changes its conformation with ionic strength could improve the performance of hydrophobic interaction chromatography enabling the separation of compounds based on slight differences in hydrophobicity.

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Bellotti, Cristina, Kristina Lang, Nataliya Kuplennik, Alejandro Sosnik y Robert Steinfeld. "High-grade extracellular vesicles preparation by combined size-exclusion and affinity chromatography". Scientific Reports 11, n.º 1 (18 de mayo de 2021). http://dx.doi.org/10.1038/s41598-021-90022-y.

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AbstractExtracellular vesicles (EVs) have recently gained growing interest for their diagnostic and therapeutic potential. Despite this, few protocols have been reported for the isolation of EVs with preserved biological function. Most EV purification methods include a precipitation step that results in aggregation of vesicles and most available techniques do not efficiently separate the various types of EVs such as exosomes and ectosomes, which are involved in distinct biological processes. For this reason, we developed a new two-step fast performance liquid chromatography (FPLC) protocol for purification of large numbers of EVs. The method comprises size exclusion chromatography followed by immobilized metal affinity chromatography, which is enabled by expression of poly-histidine tagged folate receptor α in the parental cells. Characterisation and comparison of the EVs obtained by this method to EVs purified by differential centrifugation, currently the most common method to isolate EVs, demonstrated higher purity and more selective enrichment of exosomes in EV preparations using our FPLC method, as assessed by comparison of marker proteins and density distribution. Our studies reveal new possibilities for the isolation of defined subpopulations of EVs with preserved biological function that can easily be upscaled for production of larger amounts of EVs.

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Zhao, Lei, Lin Li, Guoqin Liu, Ling Chen, Xiaoxi Li y Bing Li. "Fractional Separation and Estimation of Mark-Houwink-Sakurada Equation Parameters for Gluten". International Journal of Food Engineering 8, n.º 1 (16 de mayo de 2012). http://dx.doi.org/10.1515/1556-3758.2667.

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Fractional separation of gluten using the fast protein liquid chromatography (FPLC) and a numerical method for determination of Mark-Houwink-Sakurada (MHS) equation were studied. With 1% SDS buffer as solvent, the gluten was disassembled into two components: SDS soluble (SDS-S-G) and SDS insoluble gluten proteins (SDS-IS-G). The average molecular weight (Mn, Mw and Mz) and intrinsic viscosity [η] were measured by the multi-angle laser light scattering (MALLS) in conjunction with a size-exclusion chromatography (SEC) and viscosimetry. MHS equations for SDS soluble gluten and SDS insoluble gluten proteins were established as: (SDS-S-G) [η]=1.7459×10-2Mv0.6933=1.7459×10-2qMHSMw0.6933=1.6677×10-2Mw0.6933 (SDS-IS-G) [η]=7.5682×10-3Mv0.7323=7.5682×10-3qMHSMw0.7323=7.2079×10-3Mw0.7323 where qMHS was the polydispersity correction factor.

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Zheng, Jack Jingyuan, Joanne K. Agus, Brian V. Hong, Xinyu Tang, Christopher H. Rhodes, Hannah E. Houts, Chenghao Zhu et al. "Isolation of HDL by sequential flotation ultracentrifugation followed by size exclusion chromatography reveals size-based enrichment of HDL-associated proteins". Scientific Reports 11, n.º 1 (9 de agosto de 2021). http://dx.doi.org/10.1038/s41598-021-95451-3.

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AbstractHigh-density lipoprotein (HDL) particles have multiple beneficial and cardioprotective roles, yet our understanding of their full structural and functional repertoire is limited due to challenges in separating HDL particles from contaminating plasma proteins and other lipid-carrying particles that overlap HDL in size and/or density. Here we describe a method for isolating HDL particles using a combination of sequential flotation density ultracentrifugation and fast protein liquid chromatography with a size exclusion column. Purity was visualized by polyacrylamide gel electrophoresis and verified by proteomics, while size and structural integrity were confirmed by transmission electron microscopy. This HDL isolation method can be used to isolate a high yield of purified HDL from a low starting plasma volume for functional analyses. This method also enables investigators to select their specific HDL fraction of interest: from the least inclusive but highest purity HDL fraction eluting in the middle of the HDL peak, to pooling all of the fractions to capture the breadth of HDL particles in the original plasma sample. We show that certain proteins such as lecithin cholesterol acyltransferase (LCAT), phospholipid transfer protein (PLTP), and clusterin (CLUS) are enriched in large HDL particles whereas proteins such as alpha-2HS-glycoprotein (A2HSG), alpha-1 antitrypsin (A1AT), and vitamin D binding protein (VDBP) are enriched or found exclusively in small HDL particles.

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Romero, Iván García, Mubasher Ahmed Bashir y Harald Pasch. "Determination of chemical composition and molar mass distribution of random styrene-ethyl acrylate copolymers by high-throughput liquid chromatography". e-Polymers 5, n.º 1 (1 de diciembre de 2005). http://dx.doi.org/10.1515/epoly.2005.5.1.832.

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AbstractSynthesis and characterization of random copolymers by spectroscopic and chromatographic techniques are normally slow and expensive because most measurements are conducted consecutively and no high-throughput techniques are used. In the present work parallel synthesis and high-throughput analytical techniques for random styrene-ethyl acrylate copolymers were developed. With combinatorial methods, high-throughput instruments and a better design of experiments, the molar mass and chemical composition analyses were carried out simultaneously with time savings of more than 90% as compared to conventional set-ups. The chemical heterogeneity of high-conversion styrene-ethyl acrylate copolymers was determined successfully by fast gradient HPLC. Determination of the average composition was carried out via selective UV detection in size exclusion chromatography. The results were verified by 1H NMR and calculations based on the copolymerisation parameters. Differential scanning calorimetry and optical microscopy techniques were used for determination of the phase separation behaviour of the copolymers and correlated with the chemical heterogeneity.

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Romero, Iván García y Harald Pasch. "High-throughput screening of the chemical composition distribution of random styrene-butyl acrylate copolymers". e-Polymers 5, n.º 1 (1 de diciembre de 2005). http://dx.doi.org/10.1515/epoly.2005.5.1.433.

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AbstractThe development of high-throughput liquid chromatographic techniques for the analysis of styrene-butyl acrylate (SBA) copolymers is discussed. The analysis time in size-exclusion chromatography (SEC) can be reduced to about 3 min per sample when high-throughput SEC columns and high flow rates are used. In gradient HPLC, small columns with improved separation efficiencies can be applied. The time requirements can be decreased to less than 2 min per sample. Using the high-throughput HPLC technique, the chemical composition distribution of high-conversion SBA copolymers can be analyzed in a fast and efficient way. The calibration of HPLC separation is conducted by coupling the HPLC system with FTIR through the LC-transform interface. A comparison of the chemical compositions of the copolymers obtained by 1H NMR, off-line FTIR and coupled HPLCFTIR verifies the accuracy of the high-throughput copolymer analysis approach.

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Duan, Zhijian, Pui Yan Ho, Joeseph Jilek, Neelu Batra, Ralph DeVere White, Theodore Wun, Primo Lara y Aiming Yu. "A multidimensional FPLC approach for the purification of recombinant non‐coding RNAs". FASEB Journal 31, S1 (abril de 2017). http://dx.doi.org/10.1096/fasebj.31.1_supplement.823.2.

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Translational and clinical research on RNA therapeutics for the treatment of cancer diseases requires the use of high‐purity RNA agents. Here we present a new, single‐run, multidimensional fast performance liquid chromatography (MD‐FPLC) approach for the purification of non‐coding RNAs that are bioengineered in bacteria using a novel tRNA/pre‐miRNA carrier we have invented recently. Total RNAs are separated on a combination of columns including size exclusion, weak anion exchange and strong anion exchange in a single run using a multidimensional NGC Chromatography System. The MD‐FPLC workflow is fast and highly reproducible. This MD‐FPLC platform is reliable, allowing a rapid and consistent production of multi‐milligrams of biological RNAs in a single run and offering an overall yield of 10–15% (target RNA/total RNAs). The purified ncRNAs are highly homogenous (> 97%, determined by a HPLC assay) and have minimal endotoxin activities (< 20 EU/mg RNA). In addition, the resultant biologic RNA products (e.g., let‐7c, miR‐34a and miR‐124a) are active in the regulation of target gene expression (e.g., BCL‐2/BCL‐xL, CDK6 and STAT3, respectively) and suppression of human cancer cell proliferation. Therefore, this MD‐FPLC approach offers a robust platform for the production of high‐purity biologic RNAs for research and development.Support or Funding InformationThis study was supported by grants R01GM133888, U01CA175315, and P30CA093373 from NIH.

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Krutul, Donata, Jan Szadkowski, Eva Výbohová, Viera Kučerová, Iveta Čabalová, Andrzej Antczak, Dominika Szadkowska, Michał Drożdżek y Janusz Zawadzki. "Effect of steam explosion pretreatment on chosen saccharides yield and cellulose structure from fast-growing poplar (Populus deltoides × maximowiczii) wood". Wood Science and Technology, 12 de febrero de 2024. http://dx.doi.org/10.1007/s00226-024-01532-7.

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AbstractThe aim of this study was to determine the changes occurring in the wood cellulose of the fast-growing poplar (Populus deltoides × maximowiczii) under the influence of steam explosion (SE) pretreatment. Cellulose from native wood and after pretreatment at 160 and 205 °C was isolated. Cellulose polymerization degree by size exclusion chromatography (SEC) and cellulose crystallinity index by Fourier transform infrared spectroscopy-attenuated total reflectance (FTIR-ATR) were determined. The profiles of sugars in the native wood and in the solid fraction after pretreatment (using the acid hydrolysis method) were also determined. In addition, the profile of monosaccharides in the liquid fraction obtained after steam explosion and in the liquid fraction after acid hydrolysis of the oligosaccharides were investigated using high-performance liquid chromatography (HPLC). This allowed to determine the change in the yield of hexoses and pentoses in the studied material.The behavior of cellulose in wood subjected to steam explosion at 160 and 205 °C and isolated by the Kürschner–Hoffer method was studied by determining the absorption bands of FTIR-ATR spectra. The lateral order index (LOI) of cellulose was calculated from the ratio of the intensity of the corresponding absorption bands A1422/A896 cm−1. Total crystallinity index (TCI) of cellulose was calculated from the ratio of the intensity of absorption bands A1372/A2900 cm−1. TCI of Kürschner-Hoffer cellulose isolated from wood subjected to steam explosion at 160 and 205 °C decreased by 5.6 and 5.0%, respectively, with regard to the applied temperature. LOI increased in cellulose isolated from wood subjected to steam explosion at 160 °C (by 0.7%) and at 205 °C (by 19.2%) in relation to the index of cellulose isolated from native wood. Kürschner–Hoffer cellulose isolated from wood subjected to steam explosion at 160 and 205 °C exhibited, respectively, a reduced degree of polymerization of about 11% and about 8%. Polydispersity index in Kürschner–Hoffer cellulose was 1% lower after both pretreatments than native sample.

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Shersher, Elena, Mohini Lahiry, Annamil Alvarez-Trotta, Giulia Diluvio, David J. Robbins, Ramin Shiekhattar y Anthony J. Capobianco. "NACK and INTEGRATOR act coordinately to activate Notch-mediated transcription in tumorigenesis". Cell Communication and Signaling 19, n.º 1 (22 de septiembre de 2021). http://dx.doi.org/10.1186/s12964-021-00776-1.

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Abstract Background Notch signaling drives many aspects of neoplastic phenotype. Here, we report that the Integrator complex (INT) is a new component of the Notch transcriptional supercomplex. Together with Notch Activation Complex Kinase (NACK), INT activates Notch1 target genes by driving RNA polymerase II (RNAPII)-dependent transcription, leading to tumorigenesis. Methods Size exclusion chromatography and CBF-1/RBPJ/Suppressor of Hairless/Lag-1 (CSL)-DNA affinity fast protein liquid chromatography (FPLC) was used to purify Notch/CSL-dependent complexes for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Chromatin immunoprecipitation (ChIP) and quantitative polymerase chain reaction (qPCR) were performed to investigate transcriptional regulation of Notch target genes. Transfection of Notch Ternary Complex components into HEK293T cells was used as a recapitulation assay to study Notch-mediated transcriptional mechanisms. Gene knockdown was achieved via RNA interference and the effects of protein depletion on esophageal adenocarcinoma (EAC) proliferation were determined via a colony formation assay and murine xenografts. Western blotting was used to examine expression of INT subunits in EAC cells and evaluate apoptotic proteins upon INT subunit 11 knockdown (INTS11 KD). Gene KD effects were further explored via flow cytometry. Results We identified the INT complex as part of the Notch transcriptional supercomplex. INT, together with NACK, activates Notch-mediated transcription. While NACK is required for the recruitment of RNAPII to a Notch-dependent promoter, the INT complex is essential for RNAPII phosphorylated at serine 5 (RNAPII-S5P), leading to transcriptional activation. Furthermore, INT subunits are overexpressed in EAC cells and INTS11 KD results in G2/M cell cycle arrest, apoptosis, and cell growth arrest in EAC. Conclusions This study identifies the INT complex as a novel co-factor in Notch-mediated transcription that together with NACK activates Notch target genes and leads to cancer cell proliferation.

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Wu, Yuanzheng, Hetong Yang y Hyun-Jae Shin. "Expression and Self Assembly of Cowpea Chlorotic Mottle Virus Capsid Proteins inPichia pastorisand Encapsulation of Fluorescent Myoglobin". MRS Proceedings 1317 (2011). http://dx.doi.org/10.1557/opl.2011.138.

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Abstract:Cowpea chlorotic mottle virus (CCMV) has been a model system for virus studies for over 40 years and now is considered to be a perfect candidate as nanoplatform for applications in materials science and medicine. The ability of CCMV to self assemblein vitrointo virus-like particles (VLPs) or capsids makes an ideal reaction vessel for nanomaterial synthesis and entrapment. Here we report expression of codon optimized CCMV coat protein inPichia pastorisand production of self assembled CCMV VLPs by large-scale fermentation. CCMV coat protein gene (573 bp) was synthesized according to codon preference ofP. pastorisand cloned into pPICZA vector. The recombinant plasmid pPICZA-CP was transformed intoP. pastorisGS115 by electroporation. The resulting yeast colonies were screened by PCR and analyzed for protein expression by SDS-PAGE. After large-scale fermentation CCMV coat protein yields reached 4.8 g L−1. The CCMV VLPs were purified by modified PEG precipitation followed by cesium chloride density gradient ultracentrifugation, and then analyzed by size exclusion fast performance liquid chromatography (FPLC), UV spectrometry and transmission electron microscopy. Myoglobin was used as a model protein to be encapsulated in CCMV VLPs. The fluorescence spectroscopy showed that inclusion of myoglobin had occurred. The results indicated the production of CCMV capsids byP. pastorisfermentation now available for utilization in pharmacology or nanotechnology fields.

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Chen, Y., J. Zhao, W. Zhang, T. Zhao, Q. Zhang, G. Mao, W. Feng, Q. Li, L. Yang y X. Wu. "Purification of novel polypeptides from bee pupae and their immunomodulatory activity in vivo and in vitro". Journal of Insects as Food and Feed, 27 de marzo de 2022, 1–16. http://dx.doi.org/10.3920/jiff2021.0190.

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Edible insects have been proposed as an understudied food whose cultivation could be increased with global population growth. However, the bioactivators and bioactivities of bee pupae are poorly studied. In this paper, the active ingredients of bee pupa powder were analysed, and novel bee pupa polypeptides (BPP) were obtained through protein hydrolysis with alkaline protease. Two purified polypeptide components (BPP-21 and BPP-22) were isolated and purified on a diethylaminoethyl-sepharose fast flow column and a Sephadex G-25 column, and identified using size exclusion chromatography-high performance liquid chromatography and amino acid composition analyses. Due to its higher cell proliferation activity, BPP-22 was selected for further study of its immunomodulatory activity and mechanism in vivo and in vitro. The analysis of immunomodulatory activity showed that BPP-22 significantly increased the body weight growth rate, organ index, macrophage phagocytosis, delayed-type hypersensitivity reaction, cytokine level (interleukin (IL)-2 and interferon (IFN)-γ), immunoglobulin (Ig) levels (IgA, IgG, and IgM), and routine blood indexes in cyclophosphamide-treated immunosuppressed mice (P<0.01). Mechanistic research in RAW264.7 cells showed that BPP-22 might promote the secretion of cytokines (IL-2, tumour necrosis factor-α and IFN-γ) and the production of nitric oxide by increasing homologous mRNA expression and could exert immunomodulatory activity by increasing the phosphorylation of ERK and p38, and modulating the expression of intranuclear transcription factors (EIK-1, MEF-2 and CREB) in the MAPK signalling pathway. These findings are helpful for promoting the application of bee pupae as potential immunomodulatory agents and protein supplements.

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Zhai, Lu, Xiaohao Xu, Jiangzeng Liu, Chenxu Jing, Xinzhao Yang, Daqing Zhao, Rui Jiang y Li-Wei Sun. "A Novel Biochemical Study of Anti-Dermal Fibroblast Replicative Senescence Potential of Panax Notoginseng Oligosaccharides". Frontiers in Pharmacology 12 (30 de junio de 2021). http://dx.doi.org/10.3389/fphar.2021.690538.

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Dermal fibroblast replicative senescence that often occurs in aging skin is characterized by loss of cell proliferative capacity, cell cycle arrest, decreased cell elongation, and decreased synthesis of dermal extracellular matrix (ECM) components. Although Panax notoginseng is known for its effectiveness in alleviating many age-related degenerative diseases, few studies have evaluated P. notoginseng components for efficacy or mechanisms of action in delaying cell replicative senescence. In this study, P. notoginseng oligosaccharides (PNO) were isolated using a stepwise purification procedure involving water extraction and alcohol precipitation followed by DEAE Sepharose Fast Flow column chromatography, preparative high performance liquid chromatography, and size-exclusion chromatography. Monosaccharides detected in PNO constituents included mannose, galactose, and sorbitose in relative molar proportions of 14.2:12.3:1, respectively, aligning with PNO absorption spectrum results resembling typical known spectra for sugars. In vitro, PNO treatment of replicative senescent NIH-3T3 fibroblasts significantly promoted cell vitality, inhibited SA-β-galactosidase (SA-β-Gal) activity, and reduced p16 and p21 protein-level expression. Moreover, PNO treatment of senescent fibroblasts led to a lower proportion of G1 phase cells and higher proportion of S phase cells, while also inducing aging NIH-3T3 cells to migrate and synthesize collagen-I (CoL-I). Mechanistically, PNO treatment up-regulated expression of proliferating cell nuclear antigen (PCNA), cyclin E, cyclin D1, and cyclin-dependent kinase 4 (CDK4) proteins and promoted phosphorylation of MEK, p38, and ERK1/2 to trigger cell cycle progression. Additionally, PNO treatment also up-regulated protein-level expression of TGF-β1 and levels of p-Smad2/3, p-FAK, and p-Pax to trigger CoL-I synthesis and cell migration. Taken together, these findings demonstrate that oligosaccharides purified from P. notoginseng could reverse fibroblast replicative senescence by promoting fibroblast cell proliferation, migration, and CoL-I production.

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Adenowo, Abiola Fatimah, Priscilla Masamba, Ndibonani Kebonang Qokoyi, Babatunji Emmanuel Oyinloye y Abidemi Paul Kappo. "Recombinant Expression and Biophysical Characterization of a Druggable Schistosoma mansoni Universal Stress G4LZI3 Protein". Advanced Pharmaceutical Bulletin, 31 de enero de 2021. http://dx.doi.org/10.34172/apb.2022.035.

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Purpose: Universal stress protein from S. mansoni, designated as G4LZI3, was previously hypothesised as a druggable target and vaccine candidate for human schistosomiasis. The purpose of this study is to characterize a purified recombinant G4LZI3 preliminarily for subsequent structural characterisation, which will provide baseline structural data for future functional studies for the discovery, design and development of new schistosomal drugs for the treatment, control and elimination of schistosomiasis. Methods: Restriction digest analysis of a GenScript-synthesised codon-optimised G4LZI3 gene construct was carried out to ascertain its integrity and size. Thereafter, the pQE30-G4LZI3 construct was transformed into an M15 bacterial expression host. Transformed cells were induced with isopropyl β-D-thiogalactoside for recombinant protein expression of an appreciable amount of pQE30-G4LZI3, which was subsequently purified with fast protein liquid chromatography and a size exclusion chromatographic purification scheme. Preliminary biophysical characterisation of the 6X His-tagged G4LZI3 was done to determine its secondary structure characteristics and protein stability. Results: A molecular weight protein of 20.3 kDa was confirmed subsequent to restriction digest analysis, while heterologous protein expression yielded a highly soluble and considerable amount of histidine-tagged G4LZI3 protein, which was successfully purified to homogeneity. Biophysical characterisation indicated that the protein was well folded, heat-stable, had the functional groups and secondary structure composition required and was thus amenable to further structural characterisation and determination. Conclusion: Biophysical characterisation of purified G4LZI3 showed that further structural studies can be embarked upon on the use of G4LZI3 as a druggable target and possibly a vaccine target against schistosomiasis via vaccinomics.

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Hernandez, Sonia Lisseth, Sabi Shrestha, Josh Beckhem y Walter L. Fast. "Investigation of Possible Inhibitors Against Multidrug‐Resistant New Delhi Metallo‐Beta Lactamase (NDM‐1)". FASEB Journal 31, S1 (abril de 2017). http://dx.doi.org/10.1096/fasebj.31.1_supplement.921.10.

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Since penicillin was introduced in the world of medicine, this antibiotic saved millions from infectious diseases. However, in the past decades, antimicrobial resistance has developed into a widespread public health threat causing high morbidity and mortality rates. New Delhi Metallo β‐lactamase (NDM‐1) is associated with many resistant nosocomial infections by organisms such as Klebsiella pneumoniae. This organism has become resistant by producing NDM‐1 which hydrolyzes the beta‐lactam ring found in beta‐lactam antibiotics. It is important to find novel inhibitors against New Delhi Metallo β‐lactamase (NDM‐1). In order to find potential small molecule inhibitors against NDM‐1, a combination of wet lab and virtual screening procedures were performed. Virtual screening was done to identify high scoring compounds using GOLD. Different virtual libraries were screened along with positive and negative controls. In the wet lab, competent BL21(DE3) bacteria cells were transformed with an expression plasmid containing the CDS of NDM‐1 and then expressed using IPTG and ZnSO4 to improve soluble yield. In addition, three methods of purification were performed which included nickel affinity chromatography with 6xHIS tag, ion exchange and size exclusion Fast Protein Liquid Chromatography (FPLC). The protein samples were characterized using SDS‐PAGE gel. Furthermore, protein activity of the enzyme was analyzed in the presence of potential inhibitors using Nitrocefin as a substrate in spectrophotometric assays. Differential Scanning Fluorimetry (DSF) was performed to analyze the binding impact of several potential inhibitors on the melting temperature of NDM‐1. Out of the 12 compounds, only 1 showed modest shifts to the melting curve. Any novel compounds found to inhibit NDM‐1 activity may lead to effective new inhibitors to overcome antibiotic resistance.

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Jin, Yunjung, Fuyao Li, Berkiye Sonoustoun, Naveen Chandra Kondru, Yuka A. Martens, Wenhui Qiao, Michael G. Heckman et al. "APOE4 exacerbates α-synuclein seeding activity and contributes to neurotoxicity in Alzheimer’s disease with Lewy body pathology". Acta Neuropathologica, 26 de abril de 2022. http://dx.doi.org/10.1007/s00401-022-02421-8.

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AbstractApproximately half of Alzheimer’s disease (AD) brains have concomitant Lewy pathology at autopsy, suggesting that α-synuclein (α-SYN) aggregation is a regulated event in the pathogenesis of AD. Genome-wide association studies revealed that the ε4 allele of the apolipoprotein E (APOE4) gene, the strongest genetic risk factor for AD, is also the most replicated genetic risk factor for Lewy body dementia (LBD), signifying an important role of APOE4 in both amyloid-β (Aβ) and α-SYN pathogenesis. How APOE4 modulates α-SYN aggregation in AD is unclear. In this study, we aimed to determine how α-SYN is associated with AD-related pathology and how APOE4 impacts α-SYN seeding and toxicity. We measured α-SYN levels and their association with other established AD-related markers in brain samples from autopsy-confirmed AD patients (N = 469), where 54% had concomitant LB pathology (AD + LB). We found significant correlations between the levels of α-SYN and those of Aβ40, Aβ42, tau and APOE, particularly in insoluble fractions of AD + LB. Using a real-time quaking-induced conversion (RT-QuIC) assay, we measured the seeding activity of soluble α-SYN and found that α-SYN seeding was exacerbated by APOE4 in the AD cohort, as well as a small cohort of autopsy-confirmed LBD brains with minimal Alzheimer type pathology. We further fractionated the soluble AD brain lysates by size exclusion chromatography (SEC) ran on fast protein liquid chromatography (FPLC) and identified the α-SYN species (~ 96 kDa) that showed the strongest seeding activity. Finally, using human induced pluripotent stem cell (iPSC)-derived neurons, we showed that amplified α-SYN aggregates from AD + LB brain of patients with APOE4 were highly toxic to neurons, whereas the same amount of α-SYN monomer was not toxic. Our findings suggest that the presence of LB pathology correlates with AD-related pathologies and that APOE4 exacerbates α-SYN seeding activity and neurotoxicity, providing mechanistic insight into how APOE4 affects α-SYN pathogenesis in AD.

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Jin, Yunjung, Fuyao Li, Berkiye Sonoustoun, Naveen Chandra Kondru, Yuka A. Martens, Wenhui Qiao, Michael G. Heckman et al. "APOE4 exacerbates α-synuclein seeding activity and contributes to neurotoxicity in Alzheimer’s disease with Lewy body pathology". Acta Neuropathologica, 26 de abril de 2022. http://dx.doi.org/10.1007/s00401-022-02421-8.

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AbstractApproximately half of Alzheimer’s disease (AD) brains have concomitant Lewy pathology at autopsy, suggesting that α-synuclein (α-SYN) aggregation is a regulated event in the pathogenesis of AD. Genome-wide association studies revealed that the ε4 allele of the apolipoprotein E (APOE4) gene, the strongest genetic risk factor for AD, is also the most replicated genetic risk factor for Lewy body dementia (LBD), signifying an important role of APOE4 in both amyloid-β (Aβ) and α-SYN pathogenesis. How APOE4 modulates α-SYN aggregation in AD is unclear. In this study, we aimed to determine how α-SYN is associated with AD-related pathology and how APOE4 impacts α-SYN seeding and toxicity. We measured α-SYN levels and their association with other established AD-related markers in brain samples from autopsy-confirmed AD patients (N = 469), where 54% had concomitant LB pathology (AD + LB). We found significant correlations between the levels of α-SYN and those of Aβ40, Aβ42, tau and APOE, particularly in insoluble fractions of AD + LB. Using a real-time quaking-induced conversion (RT-QuIC) assay, we measured the seeding activity of soluble α-SYN and found that α-SYN seeding was exacerbated by APOE4 in the AD cohort, as well as a small cohort of autopsy-confirmed LBD brains with minimal Alzheimer type pathology. We further fractionated the soluble AD brain lysates by size exclusion chromatography (SEC) ran on fast protein liquid chromatography (FPLC) and identified the α-SYN species (~ 96 kDa) that showed the strongest seeding activity. Finally, using human induced pluripotent stem cell (iPSC)-derived neurons, we showed that amplified α-SYN aggregates from AD + LB brain of patients with APOE4 were highly toxic to neurons, whereas the same amount of α-SYN monomer was not toxic. Our findings suggest that the presence of LB pathology correlates with AD-related pathologies and that APOE4 exacerbates α-SYN seeding activity and neurotoxicity, providing mechanistic insight into how APOE4 affects α-SYN pathogenesis in AD.

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