Purification, Identification and Characterization of Antioxidant Peptides from Corn Silk Tryptic Hydrolysate: An Integrated In Vitro-In Silico Approach

Corn silk (CS) is an agro-by-product from corn cultivation. It is used in folk medicines in some countries, besides being commercialized as health-promoting supplements and beverages. Unlike CS-derived natural products, their bioactive peptides, particularly antioxidant peptides, are understudied. This study aimed to purify, identify and characterize antioxidant peptides from trypsin-hydrolyzed CS proteins. Purification was accomplished by membrane ultrafiltration, gel filtration chromatography, and strong-cation-exchange solid-phase extraction, guided by 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt radical cation (ABTS•+) scavenging, hydrogen peroxide scavenging, and lipid peroxidation inhibition assays. De novo sequencing identified 29 peptides (6–14 residues; 633–1518 Da). The peptides consisted of 33–86% hydrophobic and 10–67% basic residues. Molecular docking found MCFHHHFHK, VHFNKGKKR, and PVVWAAKR having the strongest affinity (−4.7 to −4.8 kcal/mol) to ABTS•+, via hydrogen bonds and hydrophobic interactions. Potential cellular mechanisms of the peptides were supported by their interactions with modulators of intracellular oxidant status: Kelch-like ECH-associated protein 1, myeloperoxidase, and xanthine oxidase. NDGPSR (Asn-Asp-Gly-Pro-Ser-Arg), the most promising peptide, showed stable binding to all three cellular targets, besides exhibiting low toxicity, low allergenicity, and cell-penetrating potential. Overall, CS peptides have potential application as natural antioxidant additives and functional food ingredients.

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Kinetic analyses of vasculogenesis inform mechanistic studies

AJP Cell Physiology ◽

10.1152/ajpcell.00367.2016 ◽

2017 ◽

Vol 312 (4) ◽

pp. C446-C458 ◽

Cited By ~ 1

Author(s):

Kaela M. Varberg ◽

Seth Winfree ◽

Chenghao Chu ◽

Wanzhu Tu ◽

Emily K. Blue ◽

...

Keyword(s):

Network Formation ◽

De Novo ◽

Structural Connectivity ◽

Maternal Diabetes ◽

Time Correlation ◽

Network Structures ◽

Cellular Mechanisms ◽

Kinetic Measurements ◽

Over Time

Vasculogenesis is a complex process by which endothelial stem and progenitor cells undergo de novo vessel formation. Quantitative assessment of vasculogenesis is a central readout of endothelial progenitor cell functionality. However, current assays lack kinetic measurements. To address this issue, new approaches were developed to quantitatively assess in vitro endothelial colony-forming cell (ECFC) network formation in real time. Eight parameters of network structure were quantified using novel Kinetic Analysis of Vasculogenesis (KAV) software. KAV assessment of structure complexity identified two phases of network formation. This observation guided the development of additional vasculogenic readouts. A tissue cytometry approach was established to quantify the frequency and localization of dividing ECFCs. Additionally, Fiji TrackMate was used to quantify ECFC displacement and speed at the single-cell level during network formation. These novel approaches were then implemented to identify how intrauterine exposure to maternal diabetes mellitus (DM) impairs fetal ECFC vasculogenesis. Fetal ECFCs exposed to maternal DM form fewer initial network structures, which are not stable over time. Correlation analyses demonstrated that ECFC samples with greater division in branches form fewer closed network structures. Additionally, reductions in average ECFC movement over time decrease structural connectivity. Identification of these novel phenotypes utilizing the newly established methodologies provides evidence for the cellular mechanisms contributing to aberrant ECFC vasculogenesis.

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Overexpression of quality control proteins reduces prion conversion in prion-infected cells

Journal of Biological Chemistry ◽

10.1074/jbc.ra118.002754 ◽

2018 ◽

Vol 293 (41) ◽

pp. 16069-16082 ◽

Cited By ~ 4

Author(s):

Simrika Thapa ◽

Basant Abdulrahman ◽

Dalia H. Abdelaziz ◽

Li Lu ◽

Manel Ben Aissa ◽

...

Keyword(s):

Quality Control ◽

De Novo ◽

Prion Diseases ◽

Neuroblastoma Cells ◽

Cellular Prion Protein ◽

Control Mechanisms ◽

Cellular Mechanisms ◽

Prion Propagation ◽

Infected Cells

Prion diseases are fatal infectious neurodegenerative disorders in humans and other animals and are caused by misfolding of the cellular prion protein (PrPC) into the pathological isoform PrPSc. These diseases have the potential to transmit within or between species, including zoonotic transmission to humans. Elucidating the molecular and cellular mechanisms underlying prion propagation and transmission is therefore critical for developing molecular strategies for disease intervention. We have shown previously that impaired quality control mechanisms directly influence prion propagation. In this study, we manipulated cellular quality control pathways in vitro by stably and transiently overexpressing selected quality control folding (ERp57) and cargo (VIP36) proteins and investigated the effects of this overexpression on prion propagation. We found that ERp57 or VIP36 overexpression in persistently prion-infected neuroblastoma cells significantly reduces the amount of PrPSc in immunoblots and prion-seeding activity in the real-time quaking-induced conversion (RT-QuIC) assay. Using different cell lines infected with various prion strains confirmed that this effect is not cell type– or prion strain–specific. Moreover, de novo prion infection revealed that the overexpression significantly reduced newly formed PrPSc in acutely infected cells. ERp57-overexpressing cells significantly overcame endoplasmic reticulum stress, as revealed by expression of lower levels of the stress markers BiP and CHOP, accompanied by a decrease in PrP aggregates. Furthermore, application of ERp57-expressing lentiviruses prolonged the survival of prion-infected mice. Taken together, improved cellular quality control via ERp57 or VIP36 overexpression impairs prion propagation and could be utilized as a potential therapeutic strategy.

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A microfluidic approach for sequential assembly of siRNA polyplexes with a defined structure-activity relationship

10.7717/peerj-matsci.1 ◽

2019 ◽

Vol 1 ◽

pp. e1 ◽

Cited By ~ 1

Author(s):

Dominik M. Loy ◽

Philipp M. Klein ◽

Rafał Krzysztoń ◽

Ulrich Lächelt ◽

Joachim O. Rädler ◽

...

Keyword(s):

Nucleic Acids ◽

Hydrophobic Interactions ◽

Solid Phase ◽

Transfection Efficiency ◽

Folate Receptor ◽

Endosomal Escape ◽

The Core ◽

Functional Components ◽

Lipid Anchor

Therapeutic nucleic acids provide versatile treatment options for hereditary or acquired diseases. Ionic complexes with basic polymers are frequently used to facilitate nucleic acid’s transport to intracellular target sites. Usually, these polyplexes are prepared manually by mixing two components: polyanionic nucleic acids and polycations. However, parameters such as internal structure, size, polydispersity and surface charge of the complexes sensitively affect pharmaceutical efficiency. Hence a controlled assembly is of paramount importance in order to ensure high product quality. In the current study, we present a microfluidic platform for controlled, sequential formulation of polyplexes. We use oligo-amidoamines (termed “oligomers”) with precise molecular weight and defined structure due to their solid phase supported synthesis. The assembly of the polyplexes was performed in a microfluidic chip in two steps employing a design of two successive Y junctions: first, siRNA and core oligomers were assembled into core polyplexes. These core oligomers possess compacting, stabilizing, and endosomal escape mediating motifs. Second, new functional motifs were mixed to the core particles and integrated into the core polyplex. The iterative assembly formed multi-component polyplexes in a highly controlled manner and enabled us to investigate structure-function relationships. We chose nanoparticle shielding polyethylene glycol (PEG) and cell targeting folic acid (termed “PEG-ligands”) as functional components. The PEG-ligands were coupled to lipid anchor oligomers via strain promoted azide—alkyne click chemistry. The lipid anchors feature four cholanic acids for inserting various PEG-ligands into the core polyplex by non-covalent hydrophobic interactions. These core—lipid anchor—PEG-ligand polyplexes containing folate as cell binding ligand were used to determine the optimal PEG-ligand length for transfecting folate receptor-expressing KB cells in vitro. We found that polyplexes with 20 mol % PEG-ligands (relative to ncore oligomer) showed optimal siRNA mediated gene knock-down when containing defined PEG domains of in sum 24 and 36 ethylene oxide repetitions, 12 EOs each from the lipid anchor and 12 or 24 EOs from the PEG-ligand, respectively. These results confirm that transfection efficiency depends on the linker length and stoichiometry and are consistent with previous findings using core—PEG-ligand polyplexes formed by click modification of azide-containing core polyplexes with aforementioned PEG-ligands. Hence, successive microfluidic assembly might be a potentially powerful route to create defined multi-component polyplexes with reduced batch-to-batch variability.

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Preparation and Identification of Antioxidative Peptides from Pacific Herring (Clupea pallasii) Protein

Molecules ◽

10.3390/molecules24101946 ◽

2019 ◽

Vol 24 (10) ◽

pp. 1946 ◽

Cited By ~ 1

Author(s):

Xueqin Wang ◽

Huahua Yu ◽

Ronge Xing ◽

Song Liu ◽

Xiaolin Chen ◽

...

Keyword(s):

Antioxidant Activity ◽

High Performance ◽

Antioxidant Activities ◽

Gel Filtration ◽

Pacific Herring ◽

Antioxidant Peptides ◽

Cellular Antioxidant Activity ◽

Clupea Pallasii ◽

Ic50 Values

The aim of this study was to isolate and purify antioxidative peptides from Pacific herring (Clupea pallasii) protein. Five enzymes (pepsin, trypsin, papain, flavourzyme, and neutrase) were used for protein hydrolysis, and Pacific herring protein hydrolysates (PHPH) were separated by ultrafiltration. The fraction with the molecular weight below 3500 Da exhibited the highest in vitro antioxidant activities and cellular antioxidant activity. The PHPH was isolated and purified by consecutive chromatographic methods including gel filtration chromatography and reverse high-performance liquid chromatography (RP-HPLC). The purified antioxidant peptides were identified as Leu-His-Asp-Glu-Leu-Thr (MW = 726.35 Da) and Lys-Glu-Glu-Lys-Phe-Glu (MW = 808.40 Da), and the IC50 values of cellular antioxidant activity were 1.19 ± 0.05 mg/mL and 1.04 ± 0.06 mg/mL. The results demonstrate that is possible to produce natural antioxidative peptides from Pacific herring protein via enzymatic hydrolysis and purification.

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DNA methylase from Pisum sativum

Biochemical Journal ◽

10.1042/bj2730469 ◽

1991 ◽

Vol 273 (2) ◽

pp. 469-475 ◽

Cited By ~ 15

Author(s):

H M I Yesufu ◽

A Hanley ◽

A Rinaldi ◽

R L P Adams

Keyword(s):

De Novo ◽

Specific Activity ◽

Gel Filtration ◽

Micrococcal Nuclease ◽

Double Stranded Dna ◽

Sds Page ◽

Low Salt ◽

Dna Methylase ◽

Single Enzyme

DNA methylase activity was detected in nuclei from pea shoots. The enzyme can only be extracted by low-salt treatment if the nuclei are pretreated with micrococcal nuclease. Only a single enzyme was detected, and it was purified to a specific activity of 1620 units/mg of protein. It has an Mr of 160,000 on gel filtration and SDS/PAGE. Pea DNA methylase methylates cytosine in all four dinucleotides, and this is interpreted to show that it acts on CNG trinucleotides. Although it shows a strong preference for hemi-methylated double-stranded DNA, it is also capable of methylation de novo. Homologous DNA is the best natural substrate. In vitro the enzyme interacts with DNA to form a salt-resistant complex with DNA that is stable for at least 4 h.

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Elucidation of Interaction between Whey Proteins and Proanthocyanidins and Its Protective Effects on Proanthocyanidins during In-Vitro Digestion and Storage

Molecules ◽

10.3390/molecules26185468 ◽

2021 ◽

Vol 26 (18) ◽

pp. 5468

Author(s):

Chenyu Tang ◽

Bing Tan ◽

Xiangjun Sun

Keyword(s):

Hydrophobic Interactions ◽

Retention Rate ◽

Whey Proteins ◽

In Vitro Digestion ◽

Molar Ratio ◽

Protective Effects ◽

Food Ingredients ◽

Oligomeric Proanthocyanidins ◽

And Storage

Whey proteins and oligomeric proanthocyanidins have nutritional value and are widely used in combination as food supplements. However, the effect of the interactions between proanthocyanidins and whey proteins on their stability has not been studied in depth. In this work, we aimed to characterize the interactions between β-Lactoglobulin (β-LG) and α-lactalbumin (α-LA) and oligomeric proanthocyanidins, including A1, A2, B1, B2, B3, and C1, using multi-spectroscopic and molecular docking methods. Fluorescence spectroscopic data revealed that all of the oligomeric proanthocyanidins quenched the intrinsic fluorescence of β-LG or α-LA by binding-related fluorescence quenching. Among the six oligomeric proanthocyanidins, A1 showed the strongest affinity for β-LG (Ka = 2.951 (±0.447) × 104 L∙mol−1) and α-LA (Ka = 1.472 (±0.236) × 105 L∙mol−1) at 297 K. β-LG/α-LA and proanthocyanidins can spontaneously form complexes, which are mainly induced by hydrophobic interactions, hydrogen bonds, and van der Waals forces. Fourier-transform infrared spectroscopy (FTIR) and circular dichroism spectroscopy showed that the secondary structures of the proteins were rearranged after binding to oligomeric proanthocyanidins. During in vitro gastrointestinal digestion, the recovery rate of A1 and A2 increased with the addition of WPI by 11.90% and 38.43%, respectively. The addition of WPI (molar ratio of 1:1) increased the retention rate of proanthocyanidins A1, A2, B1, B2, B3, and C1 during storage at room temperature by 14.01%, 23.14%, 30.09%, 62.67%, 47.92%, and 60.56%, respectively. These results are helpful for the promotion of protein–proanthocyanidin complexes as functional food ingredients in the food industry.

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A microfluidic approach for sequential assembly of siRNA polyplexes with defined structure – activity relationship

10.7287/peerj.preprints.27669v1 ◽

2019 ◽

Author(s):

Dominik M Loy ◽

Philipp M Klein ◽

Rafał Krzysztoń ◽

Ulrich Lächelt ◽

Joachim O Rädler ◽

...

Keyword(s):

Nucleic Acids ◽

Hydrophobic Interactions ◽

Solid Phase ◽

Transfection Efficiency ◽

Folate Receptor ◽

Endosomal Escape ◽

The Core ◽

Functional Components ◽

Lipid Anchor

Therapeutic nucleic acids provide versatile treatment options for hereditary or acquired diseases. Ionic complexes with basic polymers are frequently used to facilitate nucleic acid’s transport to intracellular target sites. Usually, these polyplexes are prepared manually by mixing two components: polyanionic nucleic acids and polycations. However, parameters such as internal structure, size, polydispersity and surface charge of the complexes sensitively affect pharmaceutical efficiency. Hence a controlled assembly is of paramount importance in order to ensure high product quality. In the current study, we present a microfluidic platform for controlled, sequential formulation of polyplexes. We use oligo-amidoamines (termed ‘oligomers’) with precise molecular weight and defined structure due to their solid phase supported synthesis. The assembly of the polyplexes was performed in a microfluidic chip in two steps employing a design of two successive Y junctions: first, siRNA and core oligomers were assembled into core polyplexes. These core oligomers possess compacting, stabilizing, and endosomal escape mediating motifs. Second, new functional motifs were mixed to the core particles and integrated into the core polyplex. The iterative assembly formed multi-component polyplexes in a highly controlled manner and enabled us to investigate structure - function relationships. We chose nanoparticle shielding PEG and cell targeting folic acid (termed ‘PEG-ligands’) as functional components. The PEG-ligands were coupled to lipid anchor oligomers via strain promoted azide – alkyne click chemistry. The lipid anchors feature four cholanic acids for inserting various PEG-ligands into the core polyplex by non-covalent hydrophobic interactions. These core - lipid anchor - PEG-ligand polyplexes containing folate as cell binding ligand were used to determine the optimal PEG-ligand length for transfecting folate receptor-expressing KB cells in vitro. We found that polyplexes with 20 mol % PEG-ligands (relative to ncore oligomer) showed optimal siRNA mediated gene knock-down when containing defined polyethylene glycol (PEG) domains of in sum 24 and 36 ethylene oxide (EO) repetitions. These results confirm that transfection efficiency depends on the linker length and stoichiometry and are consistent with previous findings using core - PEG-ligand polyplexes formed by click modification of azide-containing core polyplexes with DBCO-PEG-ligand. Hence successive microfluidic assembly might be a potentially powerful route to create defined multi-component polyplexes with reduced batch-to-batch variability.

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Synthesis of peptides by the solid-phase method. V. Substance P and analogs

Canadian Journal of Biochemistry ◽

10.1139/o80-036 ◽

1980 ◽

Vol 58 (4) ◽

pp. 272-280 ◽

Cited By ~ 23

Author(s):

A. Fournier ◽

R. Couture ◽

J. Magnan ◽

M. Gendreau ◽

D. Regoli ◽

...

Keyword(s):

Substance P ◽

Solid Phase ◽

Gel Filtration ◽

Paper Electrophoresis ◽

Exchange Chromatography ◽

Phase Method ◽

Elemental Analyses ◽

Solid Phase Method

We have synthesized a series of 12 analogs of the undecapeptide substance P in order to perform a structure–activity study of this peptide. In the present work, each residue was substituted by L-alanine, and the C-terminal amide was replaced by the free carboxyl in order to pinpoint biologically important side chains and functional groups. The synthesis of the analogs was carried out by the automatic solid-phase method. Couplings were performed by the symmetrical anhydride procedure. After cleavage with liquid HF, the peptides were purified by gel filtration and ion-exchange chromatography. Their purity was assessed by thin-layer chromatography, paper electrophoresis, amino acid and elemental analyses, and high pressure liquid chromatography. They were tested for biological activity in vitro on the ileum of the guinea pig, the mesenteric vein of the rabbit, and the vas deferens of the rat, and in vivo by measuring their effect on the blood pressure of the rat.

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A microfluidic approach for sequential assembly of siRNA polyplexes with defined structure – activity relationship

10.7287/peerj.preprints.27669 ◽

2019 ◽

Author(s):

Dominik M Loy ◽

Philipp M Klein ◽

Rafał Krzysztoń ◽

Ulrich Lächelt ◽

Joachim O Rädler ◽

...

Keyword(s):

Nucleic Acids ◽

Hydrophobic Interactions ◽

Solid Phase ◽

Transfection Efficiency ◽

Folate Receptor ◽

Endosomal Escape ◽

The Core ◽

Functional Components ◽

Lipid Anchor

Therapeutic nucleic acids provide versatile treatment options for hereditary or acquired diseases. Ionic complexes with basic polymers are frequently used to facilitate nucleic acid’s transport to intracellular target sites. Usually, these polyplexes are prepared manually by mixing two components: polyanionic nucleic acids and polycations. However, parameters such as internal structure, size, polydispersity and surface charge of the complexes sensitively affect pharmaceutical efficiency. Hence a controlled assembly is of paramount importance in order to ensure high product quality. In the current study, we present a microfluidic platform for controlled, sequential formulation of polyplexes. We use oligo-amidoamines (termed ‘oligomers’) with precise molecular weight and defined structure due to their solid phase supported synthesis. The assembly of the polyplexes was performed in a microfluidic chip in two steps employing a design of two successive Y junctions: first, siRNA and core oligomers were assembled into core polyplexes. These core oligomers possess compacting, stabilizing, and endosomal escape mediating motifs. Second, new functional motifs were mixed to the core particles and integrated into the core polyplex. The iterative assembly formed multi-component polyplexes in a highly controlled manner and enabled us to investigate structure - function relationships. We chose nanoparticle shielding PEG and cell targeting folic acid (termed ‘PEG-ligands’) as functional components. The PEG-ligands were coupled to lipid anchor oligomers via strain promoted azide – alkyne click chemistry. The lipid anchors feature four cholanic acids for inserting various PEG-ligands into the core polyplex by non-covalent hydrophobic interactions. These core - lipid anchor - PEG-ligand polyplexes containing folate as cell binding ligand were used to determine the optimal PEG-ligand length for transfecting folate receptor-expressing KB cells in vitro. We found that polyplexes with 20 mol % PEG-ligands (relative to ncore oligomer) showed optimal siRNA mediated gene knock-down when containing defined polyethylene glycol (PEG) domains of in sum 24 and 36 ethylene oxide (EO) repetitions. These results confirm that transfection efficiency depends on the linker length and stoichiometry and are consistent with previous findings using core - PEG-ligand polyplexes formed by click modification of azide-containing core polyplexes with DBCO-PEG-ligand. Hence successive microfluidic assembly might be a potentially powerful route to create defined multi-component polyplexes with reduced batch-to-batch variability.

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Anti-Obesity Effects of Microalgae

International Journal of Molecular Sciences ◽

10.3390/ijms21010041 ◽

2019 ◽

Vol 21 (1) ◽

pp. 41 ◽

Cited By ~ 1

Author(s):

Saioa Gómez-Zorita ◽

Jenifer Trepiana ◽

Maitane González-Arceo ◽

Leixuri Aguirre ◽

Iñaki Milton-Laskibar ◽

...

Keyword(s):

Adipose Tissue ◽

De Novo ◽

De Novo Lipogenesis ◽

In Vivo Studies ◽

Acid Oxidation ◽

Low Grade ◽

Food Ingredients ◽

Brown Adipose

In recent years, microalgae have attracted great interest for their potential applications in nutraceutical and pharmaceutical industry as an interesting source of bioactive medicinal products and food ingredients with anti-oxidant, anti-inflammatory, anti-cancer, and anti-microbial properties. One potential application for bioactive microalgae compounds is obesity treatment. This review gathers together in vitro and in vivo studies which address the anti-obesity effects of microalgae extracts. The scientific literature supplies evidence supporting an anti-obesity effect of several microalgae: Euglena gracilis, Phaeodactylum tricornutum, Spirulina maxima, Spirulina platensis, or Nitzschia laevis. Regarding the mechanisms of action, microalgae can inhibit pre-adipocyte differentiation and reduce de novo lipogenesis and triglyceride (TG) assembly, thus limiting TG accumulation. Increased lipolysis and fatty acid oxidation can also be observed. Finally, microalgae can induce increased energy expenditure via thermogenesis activation in brown adipose tissue, and browning in white adipose tissue. Along with the reduction in body fat accumulation, other hallmarks of individuals with obesity, such as enhanced plasma lipid levels, insulin resistance, diabetes, or systemic low-grade inflammation are also improved by microalgae treatment. Not only the anti-obesity effect of microalgae but also the improvement of several comorbidities, previously observed in preclinical studies, has been confirmed in clinical trials.

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