Protein a Carrying Monosize PMMA Microbeads for the Removal of HlgG from Human Plasma

1996 ◽  
Vol 19 (5) ◽  
pp. 311-317 ◽  
Author(s):  
E. Pişkin ◽  
H. Ayhan ◽  
E.V. Bulmuş ◽  
A.Y. Rad ◽  
D. Falkenhagen ◽  
...  
Keyword(s):  
Human Plasma ◽  
Phase Inversion ◽  
Covalent Binding ◽  
Protein A ◽  
Periodate Oxidation ◽  
Hydroxyl Groups

Protein A-incorporated polymethylmethacrylate (PMMA) microbeads were investigated for specific removal of HlgG from human plasma. The microbeads were prepared by a phase inversion polymerization, and activated by periodate oxidation. Protein A was then incorporated by covalent binding onto these microbeads through hydroxyl groups coming from the stabilizer. The amount of incorporated protein A was controlled by the initial concentrations of protein A in the immobilization medium and pH. The maximum protein A immobilization of 0.615 mg protein A/g PMMA, was observed at a pH of 9.5 corresponding to an initial protein A concentration of 0.1 mg/ml. There was no HlgG adsorption onto the plain PMMA microbeads, while high HlgG adsorptions of up to 32 mg HlgG/g PMMA were achieved with human plasma.

scholarly journals Tracing Potential Covalent Inhibitors of an E3 Ubiquitin Ligase through Target-Focused Modelling

Molecules ◽  
2019 ◽  
Vol 24 (17) ◽  
pp. 3125 ◽  
Author(s):  
Imane Bjij ◽  
Pritika Ramharack ◽  
Shama Khan ◽  
Driss Cherqaoui ◽  
Mahmoud E. S. Soliman
Keyword(s):  
Ubiquitin Ligase ◽  
Covalent Binding ◽  
Protein A ◽  
E3 Ubiquitin Ligase ◽  
Pharmacophore Model ◽  
Biological Evaluation ◽  
Pharmacokinetic Analysis ◽  
Unsaturated Ester ◽  
Dynamic Simulations ◽  
Covalent Inhibitors

The Nedd4-1 E3 Ubiquitin ligase has been implicated in multiple disease conditions due its overexpression. Although the enzyme may be targeted both covalently and non-covalently, minimal studies provide effective inhibitors against it. Recently, research has focused on covalent inhibitors based on their characteristic, highly-selective warheads and ability to prevent drug resistance. This prompted us to screen for new covalent inhibitors of Nedd4-1 using a combination of computational approaches. However, this task proved challenging due to the limited number of electrophilic moieties available in virtual libraries. Therefore, we opted to divide an existing covalent Nedd4-1 inhibitor into two parts: a non-covalent binding group and a pre-selected α, β-unsaturated ester that forms the covalent linkage with the protein. A non-covalent pharmacophore model was built based on molecular interactions at the binding site. The pharmacophore was then subjected to virtual screening to identify structurally similar hit compounds. Multiple filtrations were implemented prior to selecting four hits, which were validated with a covalent conjugation and later assessed by molecular dynamic simulations. The results showed that, of the four hit molecules, Zinc00937975 exhibited advantageous molecular groups, allowing for favourable interactions with one of the characteristic cysteine residues. Predictive pharmacokinetic analysis further justified the compound as a potential lead molecule, prompting its recommendation for confirmatory biological evaluation. Our inhouse, refined, pharmacophore model approach serves as a robust method that will encourage screening for novel covalent inhibitors in drug discovery.

scholarly journals Metabolism and disposition of pyrotinib in healthy male volunteers: covalent binding with human plasma protein

2018 ◽  
Vol 40 (7) ◽  
pp. 980-988 ◽  
Author(s):  
Jian Meng ◽  
Xiao-yun Liu ◽  
Sheng Ma ◽  
Hua Zhang ◽  
Song-da Yu ◽  
...  
Keyword(s):  
Human Plasma ◽  
Plasma Protein ◽  
Covalent Binding ◽  
Healthy Male ◽  
Human Plasma Protein ◽  
Healthy Male Volunteers ◽  
Male Volunteers

scholarly journals Characterization of Ionic Liquid Lignins Isolated from Spruce Wood with 1-Butyl-3-methylimidazolium Acetate and Methyl Sulfate and Their Binary Mixtures with DMSO

Molecules ◽  
2020 ◽  
Vol 25 (11) ◽  
pp. 2479 ◽  
Author(s):  
Artyom V. Belesov ◽  
Anton V. Ladesov ◽  
Ilya I. Pikovskoi ◽  
Anna V. Faleva ◽  
Dmitry S. Kosyakov
Keyword(s):  
Ionic Liquids ◽  
Binary Mixtures ◽  
Plant Biomass ◽  
High Resolution Mass Spectrometry ◽  
Covalent Binding ◽  
Structural Features ◽  
Size Exclusion ◽  
Hydroxyl Groups ◽  
Spruce Wood

Ionic liquids (ILs) based on 1-butyl-3-methylimidazolium (bmim) cation have proved to be promising solvents for the fractionation of plant biomass with the production of cellulose and lignin. This study deals with the characterization of lignins isolated from coniferous (spruce) wood using [bmim]OAc and [bmim]MeSO4 ionic liquids and their binary mixtures with DMSO (80:20). Molecular weight distributions, functional composition, and structural features of IL lignins were studied by size-exclusion chromatography, NMR spectroscopy (1D and 2D) and atmospheric pressure photoionization high-resolution mass spectrometry. It was shown that the interaction of ILs with lignin leads to significant chemical changes in the biopolymer; a decrease in the degree of polymerization and in the content of free phenolic hydroxyl groups due to alkylation, the disappearance (in the case of [bmim]OAc) of carbonyl groups and a significant destruction of β-O-4 bonds. The chemical reactions between lignin and 1-butyl-3-methylidazolium cation with covalent binding of ionic liquids or products of their decomposition is evidenced by the presence of a large number of nitrogen-containing oligomers in IL lignins.

scholarly journals Evidence for shared epitopes within the ‘naked’ protein domains of human mucus glycoproteins. A study performed by using polyclonal antibodies and electron microscopy

1991 ◽  
Vol 274 (1) ◽  
pp. 293-296 ◽  
Author(s):  
J K Sheehan ◽  
R P Boot-Handford ◽  
E Chantler ◽  
I Carlstedt ◽  
D J Thornton
Keyword(s):  
Electron Microscopy ◽  
Polyclonal Antibodies ◽  
Protein A ◽  
Periodate Oxidation ◽  
Protein Domains ◽  
Disulphide Bonds ◽  
T Domain ◽  
Cryptic Epitopes ◽  
Shared Epitopes ◽  
Mucus Glycoproteins

Polyclonal antibodies were raised in rabbits towards reduced subunits of human cervical mucus glycoproteins. The reduced subunits almost completely inhibited the antiserum, whereas the intact mucins and the heavily glycosylated fragments obtained after digestion of reduced subunits with trypsin (T-domains) caused only partial inhibition. Periodate oxidation of intact mucins, reduced subunits and T-domains caused no effect on the antibody response, and fragments obtained by more extensive proteolysis of the reduced subunits (P-domains) showed no inhibitory activity. By using electron microscopy, antibodies from T-domain-adsorbed antisera were revealed as bound to cervical mucin reduced subunits, either directly or with colloidal gold-Protein A. Binding sites (100-150 nm apart) were observed at the ends and at internal positions of the reduced subunits. We conclude that the antibodies do not recognize carbohydrate structures but are directed to two kinds of protein epitopes, one shared by whole mucins, reduced subunits and T-domains, and the other specific to the reduced subunit fragment. The latter epitopes are ‘cryptic’ and are probably shielded within folded protein domains stabilized by disulphide bonds. Human bronchial, cervical, gastric and salivary mucus glycoproteins share some of these cryptic epitopes.

Endocytosis of Proteins by Salivary Gland Duct Cells

1987 ◽  
Vol 66 (2) ◽  
pp. 412-419 ◽  
Author(s):  
A.R. Hand ◽  
R. Coleman ◽  
M.R. Mazariegos ◽  
J. Lustmann ◽  
L.V. Lotti
Keyword(s):  
Protein A ◽  
Periodate Oxidation ◽  
Secretory Protein ◽  
Secretory Proteins ◽  
Cationized Ferritin ◽  
Cell Secretion ◽  
High Concentration ◽  
Thin Sections ◽  
Duct Cells ◽  
Rat Parotid

The ability of the intralobular duct cells of the rat parotid gland to take up protein from the lumen was examined by retrograde infusion of exogenous proteins and by immunogold localization of endogenous secretory proteins. Small amounts of native horseradish peroxidase (HRP) were taken up by intercalated and striated duct cells, and were present in small vesicles, multi vesicular bodies, and lysosomes. In contrast, HRP modified by periodate oxidation was avidly internalized by the duct cells and was present in large apical vacuoles that acquired lysosomal hydrolase activity. Native and cationized ferritin were taken up in a similar manner when infused at a high concentration (up to 10 mg/mL). At lower concentrations (0.3-1.0 mg/mL), endocytosis of cationized ferritin occurred mainly in small apical tubules and vesicles in striated duct cells. Little native ferritin was taken up at these concentrations. After stimulation of acinar cell secretion by isoproterenol, similar vacuoles were occasionally observed in both intercalated and striated duct cells. Labeling of thin sections with antibodies to amylase and to a 26,000-dalton secretory protein (protein B1), followed by protein A-gold, revealed the presence of these proteins in the vacuoles, indicating endocytosis of acinar secretory proteins by the duct cells. Although uptake of acinar proteins by duct cells occurs at a low rate in normal animals, previous work suggests that extensive endocytosis may occur in certain pathological conditions. This may be a mechanism for removing abnormal or modified proteins from saliva before it reaches the oral cavity.

scholarly journals Efficient Refolding of Aggregation-prone Citrate Synthase by Polyol Osmolytes

2005 ◽  
Vol 280 (16) ◽  
pp. 15553-15560 ◽  
Author(s):  
Rajesh Mishra ◽  
Robert Seckler ◽  
Rajiv Bhat
Keyword(s):  
Small Molecules ◽  
Molecular Chaperones ◽  
Rational Design ◽  
Protein Production ◽  
Citrate Synthase ◽  
Protein A ◽  
Complete Recovery ◽  
Hydroxyl Groups ◽  
Native State

Efficient refolding of proteins and prevention of their aggregation during folding are of vital importance in recombinant protein production and in finding cures for several diseases. We have used citrate synthase (CS) as a model to understand the mechanism of aggregation during refolding and its prevention using several known structure-stabilizing cosolvent additives of the polyol series. Interestingly, no parallel correlation between the folding effect and the general stabilizing effect exerted by polyols was observed. Although increasing concentrations of polyols increased protein stability in general, the refolding yields for CS decreased at higher polyol concentrations, with erythritol reducing the folding yields at all concentrations tested. Among the various polyols used, glycerol was the most effective in enhancing the CS refolding yield, and a complete recovery of enzymatic activity was obtained at 7mglycerol and 10 μg/ml protein, a result superior to the action of the molecular chaperones GroEL and GroESin vitro. A good correlation between the refolding yields and the suppression of protein aggregation by glycerol was observed, with no aggregation detected at 7m. The polyols prevented the aggregation of CS depending on the number of hydroxyl groups in them. Stopped-flow fluorescence kinetics experiments suggested that polyols, including glycerol, act very early in the refolding process, as no fast and slow phases were detectable. The results conclusively demonstrate that both the thermodynamic and kinetic aspects are critical in the folding process and that all structure-stabilizing molecules need not always help in productive folding to the native state. These findings are important for the rational design of small molecules for efficient refolding of various aggregation-prone proteins of commercial and medical relevance.

scholarly journals Zwitterionic Acetylated Cellulose Nanofibrils

Molecules ◽  
2019 ◽  
Vol 24 (17) ◽  
pp. 3147 ◽  
Author(s):  
Jowan Rostami ◽  
Aji P. Mathew ◽  
Ulrica Edlund
Keyword(s):  
Acetic Acid ◽  
Schiff Base ◽  
Reaction Time ◽  
Structural Changes ◽  
Glacial Acetic Acid ◽  
Cellulose Nanofibrils ◽  
Periodate Oxidation ◽  
Hydroxyl Groups ◽  
Borohydride Reduction ◽  
Short Reaction Time

A strategy is devised to synthesize zwitterionic acetylated cellulose nanofibrils (CNF). The strategy included acetylation, periodate oxidation, Schiff base reaction, borohydride reduction, and a quaternary ammonium reaction. Acetylation was performed in glacial acetic acid with a short reaction time of 90 min, yielding, on average, mono-acetylated CNF with hydroxyl groups available for further modification. The products from each step were characterized by FTIR spectroscopy, ζ-potential, SEM-EDS, AFM, and titration to track and verify the structural changes along the sequential modification route.

scholarly journals ALKALINE BISMUTH REAGENT FOR HIGH RESOLUTION ULTRASTRUCTURAL DEMONSTRATION OF PERIODATE-REACTIVE SITES

1972 ◽  
Vol 20 (12) ◽  
pp. 995-1005 ◽  
Author(s):  
STERLING K. AINSWORTH ◽  
MORRIS J. KARNOVSKY ◽  
SUSUMU ITO
Keyword(s):  
High Resolution ◽  
Periodic Acid ◽  
Periodate Oxidation ◽  
Ultrastructural Localization ◽  
Blocking Agent ◽  
Hydroxyl Groups ◽  
Electron Microscopic ◽  
Periodic Acid Schiff ◽  
Thin Sections ◽  
Bismuth Subnitrate

A simple technique is described for the ultrastructural localization of periodate-reactive mucosubstances and polysaccharides containing 1,2-glycols in thin sections of routinely fixed tissues. In this method the sugar residues are oxidized by periodic acid and the resulting aldehydes presumably reduce chelated bismuth subnitrate to metallic bismuth which then appears as a fine electron-opaque precipitate at the sites of the reducing sugars. The periodic acid-alkaline bismuth procedure provides a high resolution electron microscopic technique for demonstrating tissue sites of periodate-engendered groups very similar to the light microscopic periodic acid-Schiff reaction. The reaction can be prevented by the omission of periodate oxidation or alkaline bismuth subnitrate and by aldehyde blockage with the blocking agent, m-aminophenol. However, glycogen stains markedly without prior periodate oxidation, presumably through chelation of bismuth by hydroxyl groups. Other structures which stain without prior periodate oxidation are liver lysosomal dense bodies and, occasionally, ribosomes.

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