muramidase and epigallocatechin-gallate

Lysozyme amyloid fibrils, the misfolding structures generated from natural state of lysozyme, are found to be related with non-neuropathic systemic amyloidosis. Therefore, inhibiting the formation of amyloid and disaggregating amyloid fibers are both effective strategies. Herein, we present a combination of Epigallocatechin-3-gallate (EGCG), imprinting technology and magnetic nanoparticles to obtain a kind of promising nanomaterials (MINs@EGCG) for amyloid inhibition, drug carrier and facile separation triple functions. We declared the efficacy of MINs@EGCG from two perspectives. For inhibition, Circular dichroism (CD) spectrum illustrated that the miss transition from α-helix structure to β-sheet could be blocked by MINs@EGCG, and the inhibition efficiency was higher than 80%. These results were further verified by Thioflavin T (ThT) analysis. For disaggregation and cleansing, the helical and highly periodic structure of amyloid fibrils could be converted into their counterparts by MINs@EGCG. Furthermore, with the aid of external magnetic field, the cleansing efficiency of counterparts-MINs@EGCG complex was up to 80%. Most importantly, bio-related experiments showed superior biocompatibility and anti-amyloid fibrils toxicity of MINs@EGCG, indicating the great potential of our system to work as an effective amyloidosis therapy platform.

Topics: Amyloid; Animals; Benzothiazoles; Catechin; Cell Line; Circular Dichroism; Magnetite Nanoparticles; Male; Mice; Muramidase

The aggregation process of peptides and proteins is of great relevance as it is associated with a wide range of highly debilitating disorders, including Alzheimer's and Parkinson's diseases. The natural product (-)-epigallocatechin-3-gallate (EGCG) can redirect this process away from amyloid fibrils and towards non-toxic oligomers. In this study we used nuclear magnetic resonance (NMR) spectroscopy to characterize the binding of EGCG to a set of natively structured and unstructured proteins. The results show that the binding process is dramatically dependent on the conformational properties of the protein involved, as EGCG interacts with different binding modes depending on the folding state of the protein. We used replica exchange molecular dynamics simulations to reproduce the trends observed in the NMR experiments, and analyzed the resulting samplings to identify the dominant direct interactions between EGCG and ordered and disordered proteins.

Topics: alpha-Synuclein; Catechin; Humans; Intrinsically Disordered Proteins; Muramidase; Protein Binding

Protein aggregation is a hallmark of several neurodegenerative diseases, including Alzheimer's and Parkinson's diseases. It has been shown that lysine residues play a key role in the formation of these aggregates. Thus, the ability to disrupt aggregate formation by covalently modifying lysine residues could lead to the discovery of therapeutically relevant antiamyloidogenesis compounds. Herein, we demonstrate that an ortho-iminoquinone (IQ) can be utilized to inhibit amyloid aggregation. Using alpha-synuclein and Aβ

Topics: alpha-Synuclein; Amyloid beta-Peptides; Animals; Catechin; Cell Survival; Cells, Cultured; Chickens; Dopaminergic Neurons; HEK293 Cells; Humans; Lysine; Methionine; Mice; Micrococcus luteus; Microtubule-Associated Proteins; Muramidase; Neuroprotective Agents; Oxidation-Reduction; Peptide Fragments; Protein Aggregation, Pathological; Quinones; Tyrosine 3-Monooxygenase

This electron microscopic study aimed at investigating effects of oral astringent stimuli on the enamel pellicle's morphology.. Pellicles were formed in situ within 30min on bovine enamel slabs, fixed to individuals' upper jaw splints. The pellicle-coated specimens were immersed in vitro in seven diverse astringent solutions and subsequently analyzed by scanning electron microscopy (SEM), energy dispersive X-ray (EDX) spectroscopy, as well as transmission electron microscopy (TEM). Four biocompatible astringents, namely the polyphenol epigallocatechin gallate, the metal salt iron(III) sulfate, the basic protein lysozyme, and the aminopolysaccharide chitosan, were additionally applied in situ. After rinsing the oral cavity with these compounds, the pellicle's ultrastructure was imaged by SEM and TEM, respectively. Untreated pellicle samples served as controls.. Exposure to polyphenols and lysozyme induced particularly thicker and electron-denser pellicles in comparison to the control pellicle with similar characteristics in vitro and in situ. In contrast, acidic chitosan and metal salt solutions, respectively, revealed minor pellicle alterations. The incorporation of Fe and Al into the pellicles treated with the corresponding inorganic salts was verified by EDX analysis.. Astringent-induced pellicle modifications were for the first time visualized by TEM. The ultrastructural alterations of the dental pellicle may partly explain the tooth-roughening effect caused by oral astringent stimuli.. Astringents might modify the pellicle's protective properties against dental erosion, attrition, as well as bacterial adhesion, and by this means may influence tooth health. The findings may thus be particularly relevant for preventive dentistry.

Topics: Adult; Aluminum Chloride; Aluminum Compounds; Animals; Astringents; Bacterial Adhesion; Catechin; Cattle; Chitosan; Chlorides; Dental Enamel; Dental Pellicle; Ferric Compounds; Humans; Materials Testing; Microscopy, Electron, Scanning; Microscopy, Electron, Transmission; Mouth; Muramidase; Polyphenols; Preventive Dentistry; Salivary Proteins and Peptides; Spectrometry, X-Ray Emission; Surface Properties; Time Factors; Tooth Attrition; Tooth Erosion

Epigallocatechin gallate (EGCG), the most abundant flavanoid in green tea, is currently being evaluated in the clinic due to its benefits in the treatment of amyloid disorders. Its anti-amyloidogenic effect has been attributed to direct interaction of the intact molecule with misfolded polypeptides. In addition, antioxidant activity is also involved in the anti-amyloidogenic role. The detailed molecular mechanism is still unclear and requires further investigation. In the present study, the kinetics of EGCG oxidation and the anti-amyloidogenic effect of the resultant oxidation substances have been examined. The results indicate that EGCG degrades in a medium at pH 8.0 with a half-life less than 2h. By utilizing lysozyme as an in vitro model, the oxidized EGCG demonstrates a more potent anti-amyloidogenic capacity than the intact molecule, as shown by ThT and ANS fluorescence, TEM determination, and hemolytic assay. The oxidized EGCG also has a stronger disruptive effect on preformed fibrils than the native form. Ascorbic acid eliminates the disruptive role of native EGCG on the fibrils, suggesting that oxidation is a prerequisite in fibril disruption. The results of this work demonstrate that oxidized EGCG plays a more important role than the intact molecule in anti-amyloidogenic activity. These insights into the action of EGCG may provide a novel route to understand the anti-amyloidogenic activity of natural polyphenols.

Topics: Amyloidogenic Proteins; Animals; Antioxidants; Ascorbic Acid; Catechin; Chickens; Half-Life; Humans; Hydrogen-Ion Concentration; Hydrolysis; Kinetics; Muramidase; Oxidation-Reduction; Solutions

A novel ternary nanogel based on the self-assembly of hyaluronic acid-epigallocatechin gallate conjugates (HA-EGCG), linear polyethylenimine (PEI) and Granzyme B (GzmB) in an aqueous environment was developed for the targeted intracellular delivery of GzmB into cancer cells. Lysozyme-encapsulated HA-EGCG nanogels were first prepared and characterized. HA-EGCG nanogels exhibited smaller particle sizes and a more homogeneous size distribution than the HA counterpart. Fluorescence quenching and lysozyme activity studies revealed that EGCG moieties facilitated protein binding through physical interactions and led to the formation of stable nanogels. When CD44-overexpressing HCT-116 colon cancer cells were treated with GzmB-encapsulated HA-EGCG nanogels in vitro, a significant cytotoxic effect was observed. Caspase assays and intracellular trafficking studies confirmed that cell death was due to apoptosis triggered by the delivery of GzmB to the cytosol of those cells. In comparison, little cytotoxic effect was observed in CD44-deficient cells treated with GzmB-encapsulated HA-EGCG nanogels. This study highlights the potential utility of HA-EGCG as effective intracellular protein carriers for targeted cancer therapy.. Intracellularly activated cytotoxic proteins can be used to kill cancer cells but viable carriers for such proteins are lacking. In this work, we developed novel nanogels based on selfassembly of hyaluronic acid (HA)-(-)-epigallocatechin-3-gallate (EGCG) conjugates, linear polyethylenemine (PEI) and the cytotoxic protein Granzyme B (GzmB) for the intracellular delivery of GzmB for cancer therapy. HA was exploited for its ability to target CD44 which are overexpressed in many types of cancer cells, while EGCG, the main component of green tea catechins, was chosen for its ability to bind to proteins. Characterization studies showed that EGCG facilitated protein complexation through physical interactions and led to the formation of stable nanogels. HA-EGCG nanogels were able to achieve CD44 targeted killing of HCT-116 cancer cells by delivering GzmB into the cytosol of these cells. We believe that the applications of the HA-EGCG nanogels can be expanded to the intracellular delivery of other cytotoxic protein drugs for cancer therapy.

Topics: Animals; Catechin; Cell Survival; Chickens; Dimerization; Drug Delivery Systems; Dynamic Light Scattering; Flow Cytometry; Granzymes; HCT116 Cells; Hep G2 Cells; Humans; Hyaluronan Receptors; Hyaluronic Acid; Intracellular Space; Muramidase; Nanogels; Polyethylene Glycols; Polyethyleneimine; Spectrometry, Fluorescence; Tea

The main scope of the present study was to analyze the membrane interaction of members of different classes of polyphenols, i.e. resveratrol, naringenin, epigallocatechin gallate and enterodiol, in model systems of different compositions and phase states. In addition, the possible association between membrane affinity and membrane protection against both lipid oxidation and bilayer-disruptive compounds was studied. Gibbs monolayer experiments indicated that even though polyphenols showed poor surface activity, it readily interacted with lipid films. Actually, a preferential interaction with expanded monolayers was observed, while condensed and cholesterol-containing monolayers decreased the affinity of these phenolic compounds. On the other hand, fluorescence anisotropy studies showed that polyphenols were able to modulate membrane order degree, but again this effect was dependent on the cholesterol concentration and membrane phase state. In fact, cholesterol induced a surface rather than deep into the hydrophobic core localization of phenolic compounds in the membranes. In general, the polyphenolic molecules tested had a better antioxidant activity when they were allowed to get inserted into the bilayers, i.e. in cholesterol-free membranes. On the other hand, a membrane-protective effect against bilayer permeabilizing activity of lysozyme, particularly in the presence of cholesterol, could be assessed. It can be hypothesized that phenolic compounds may protect membrane integrity by loosely covering the surface of lipid vesicles, once cholesterol push them off from the membrane hydrophobic core. However, this cholesterol-driven distribution may lead to a reduced antioxidant activity of linoleic acid double bonds.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Antioxidants; Catechin; Cholesterol; Dimyristoylphosphatidylcholine; Flavanones; Fluorescence Polarization; Hydrophobic and Hydrophilic Interactions; Lignans; Linoleic Acid; Lipid Bilayers; Lipid Peroxidation; Liposomes; Muramidase; Reactive Oxygen Species; Resveratrol; Stilbenes; Surface Properties

Numerous studies demonstrate that natural polyphenols can inhibit amyloid formation and disrupt preformed amyloid fibrils. In the present study, the fibril-disruptive effects of epigallocatechin-3-gallate (EGCG) were examined using lysozyme as a model protein. The results indicated that EGCG dose dependently inhibited lysozyme fibrillation and modified the peptide chains with quinonoid moieties under acidic conditions, as measured by ThT fluorescence, transmission electron microscopy, and an NBT-staining assay. Moreover, EGCG transformed the preformed lysozyme fibrils to amorphous aggregates through quinopeptide formation. The thiol blocker, N-ethylmaleimide, inhibited the disruptive effect of EGCG on preformed fibrils, suggesting that thiol groups are the binding sites for EGCG. We propose that the formation of quinone intermediates via oxidation and subsequent binding to lysozyme chains are the main processes driving the inhibition of amyloid formation and disruption of preformed fibrils by EGCG. The information presented in this study may provide fresh insight into the link between the antioxidant capacity and anti-amyloid activity of polyphenols.

Topics: Amyloid; Animals; Catechin; Molecular Structure; Muramidase; Peptides; Polyphenols; Protein Aggregation, Pathological

Green tea polyphenols (GTPs) are found to be potent inhibitors of amyloid fibril formation. We report the effective inhibitory property of (-)-epicatechin gallate (ECG) during the alkali-salt induced fibrillogenesis of hen egg white lysozyme (HEWL) at 37 °C. Spectroscopic techniques such as fluorescence, circular dichroism and microscopic images show that (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG), and (-)-epigallocatechin gallate (EGCG) show moderate inhibition of fibrillation with ECG as the most potent polyphenol. Aromatic interactions, hydrophobic interactions, the radical scavenging activity and autoxidation of polyphenols are likely to be the major reasons for ECG being the most effective inhibitor.

Topics: Alkalies; Animals; Benzothiazoles; Catechin; Chickens; Circular Dichroism; Guanosine Triphosphate; Hydrogen-Ion Concentration; Kinetics; Microscopy, Fluorescence; Muramidase; Nephelometry and Turbidimetry; Protein Structure, Secondary; Salts; Thiazoles; Time Factors; Tryptophan

The interaction of lysozyme (Lyz)-conjugated silver (Ag) nanoparticles with (-)-epigallocatechin gallate (EGCG), one of the major components of green tea, has been investigated. Interaction of a protein with ligand/drug molecules perturbs the conformation of secondary and tertiary structures of the protein. We have demonstrated the conformational changes in the tertiary structures of the Lyz molecules on EGCG binding using surface-enhanced Raman scattering (SERS) and circular dichroism (CD) spectroscopic measurements. From the analysis of the amide I band of Lyz in SERS and CD spectra, the site of interaction of EGCG with protein molecules in Lyz-conjugated Ag particles has been identified. Spectroscopic evidence for the conformational response of Trp62 and Trp63, in the β-domain of the protein, to the binding of EGCG has been discussed.

Topics: Catechin; Circular Dichroism; Colloids; Metal Nanoparticles; Models, Molecular; Muramidase; Protein Binding; Protein Conformation; Silver; Spectrum Analysis, Raman

Epigallocatechin-3-gallate (EGCG), a very potent antioxidant derived from green tea, was compared with vitamin E in terms of its effects on antioxidant defense and immune response of rainbow trout, by means of a feeding trial of eight weeks. Two of the experimental diets were supplemented with EGCG at either 20 or 100 mg kg(-1) diet (which contained only 30% of the intended levels) and the third was provided with 100 mg kg(-1) vitamin E but not EGCG. The control diet was not supplemented with the test components. Observation of tissue levels indicated that the high amount of EGCG helped to increase the availability of the lipid-soluble antioxidant vitamin E. The lower levels of lipid hydroperoxide in the liver of fish fed the higher amount of EGCG suggested that it was an effective antioxidant. Considering the immune indices, EGCG and vitamin E at 100 mg (actual amounts 31.9 and 94.1 mg kg(-1) diet, respectively) had identical capabilities in improving phagocytic activity and controlling hydrogen peroxide production by leucocytes. However, EGCG could possibly be more effective at enhancing serum lysozyme activity and the alternative complement activity. This work revealed the potential of EGCG as an antioxidant and an immunostimulant for rainbow trout, at least at the inclusion level of 32 mg kg(-1) diet.

Topics: Analysis of Variance; Animals; Antioxidants; Catechin; Complement Pathway, Alternative; Dietary Supplements; Flow Cytometry; Lipid Peroxides; Liver; Muramidase; Oncorhynchus mykiss; Reactive Oxygen Species; Superoxide Dismutase; Vitamin E