Language：English   Publishing type：Research paper (scientific journal)  

Alpha-glucosidase is an important target for glycemic control with the aim of reducing the risk of type 2 diabetes (T2D). Green tea catechins have been reported to inhibit alpha-glucosidase activity as a potential beverage to control blood glucose levels. However, the effects of the daily infusion style of green tea on tea catechins and their activity remain unclear. In this study, the extraction efficiency of catechins was investigated for 12 green tea extracts (GTEs) infused with 70% ethanol (70% EtOH for 24 h, a favored solvent for catechin extraction), room temperature water infusion (RT H2O for 24 h, an easy way to drink tea), and hot water infusion (Hot H2O for 90 s, a standard way to drink tea). Eight catechins were quantified by HPLC, and the inhibitory effect of GTEs and their catechins on alpha-glucosidase was measured with both rat intestinal enzymes and human Caco-2 cells. The inhibitory mechanism was further analyzed in silico by docking catechins to human alpha-glucosidase using Molecular Operating Environment software. The results showed that total catechins and gallate catechins were efficiently extracted in the order of 70% EtOH, RT H2O, and Hot H2O, and the inhibitory activity against alpha-glucosidase also followed a similar order. Pearson correlation analysis indicated that the alpha-glucosidase inhibitory activity of GTEs was significantly positively correlated with the contents of total catechins, especially gallate catechins. Gallate catechins, such as EGCg and ECg, showed lower IC50 values than free catechins for the enzyme in both rats and humans. In silico simulation revealed that gallate catechins were bound to the different sites with free catechins, and the docking energy of gallate catechins was lower than that of free catechins. Taken together, our data indicated that the daily infusion style of green tea significantly impacted the extraction efficiency and alpha-glucosidase inhibitory activities of catechins, which will give us insight into the use of green tea catechins for glycemic control through efficient infusion.

DOI： 10.3390/foods12132611

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Functional Food Center  

Background: Eggs are an excellent  nutrient-dense food containing proteins, fats, carbohydrates, minerals, and vitamins. While many proteins are present in egg yolk, there arefew studies on their health benefits. Objective: The aim of this study was to explore the antioxidant peptides derived from the soluble protein fractions of egg yolk. Lipids and lipoproteins were removed with activated carbon and centrifugation after water dilution at acidic pH. Materials and Methods: Compared with 3.6 grams of protein in egg white, egg yolk contains 2.7 grams of protein in  a single large egg. The egg yolk soluble protein (EYsP) was digested with pepsin for 2h at pH 3.0 (P-EYsP), followed by digestion with trypsin (P-EYsP-T), α-chymotrypsin (P-EYsP-α), or both enzymes (P-EYsP-T/α). The most active digest was fractionated using sephacryl S-100 gel filtration, followed by reversed phase-HPLC of the most active fraction. The antioxidant activities were evaluated using a superoxide anion-generating system of xanthine oxidase, DPPH-scavenging assay, and yeast cells as an oxidative-stress tolerance cellular model. Results: The intact proteins (EYsP) showed antioxidant activity, but pepsin hydrolysate (P-EYsP) exhibited greater superoxide-scavenging activities than EYsP, while P-EYsP-T, P-EYsP-α and P-EYsP-T/α lacked activities. The P-EYsP and its subsequent proteases (P-EYsP-T, P-EYsP-α and P-EYsP-T/α) exhibited significant DPPH reduction, but P-EYsP-T, P-EYsP-α and P-EYsP-T/α exhibited the strongest DPPH reduction activities. MALDI-TOF-MS analysis revealed five major antioxidant peptides, two derived from yolk glycoprotein 40 (859 Da, 883Da), two from lipovitelline (901Da, 945 Da), and one from livetin (1089 Da). The peptides exhibited potent superoxide anion as well as DPPH scavenging activities and markedly enhanced the tolerance of yeast cells against peroxide-induced oxidative stress.Conclusion: The results show that these bioactive peptides hold a fascinating opportunity for their potential as nutraceuticals in prevention and combating oxidative stress-associated diseases.Keywords: Egg yolk proteins; bioactive peptides; antioxidant; superoxide-scavenging; DPPH-reduction; yeast tolerance

DOI： 10.31989/ffs.v3i6.1110

Other Link： https://www.ffhdj.com/index.php/FunctionalFoodScience/article/viewFile/1110/1851

Language：English   Publishing type：Research paper (scientific journal)  

Malaria is a mosquito-borne infectious disease that is widespread in developing countries. Malaria vaccines are important in efforts to eradicate malaria; however, vaccines are usually administered by injection, which requires medical personnel and has a risk of causing infection. Transdermal vaccines can be administered without damaging the skin and thus are ideal for the prevention of malaria. However, the stratum corneum forms a "brick and mortar" like structure in which stratum corneum cells are embedded in a hydrophobic matrix composed of lipids, which strongly inhibits the permeation of hydrophilic substances. In the present study, we designed a transdermal vaccine against vivax malaria using a solid-in-oil (S/O) dispersion. The S/O dispersion of a transmission blocking vaccine candidate, Pvs25 from Plasmodium vivax, showed higher skin penetration than that of the aqueous solution. Mice immunized with the S/O dispersion generated antibodies at similar titers as the mice immunized by injection, over the mid- to long-term. These results provide information for the development of transdermally administered malaria vaccines toward the eradication of malaria.

DOI： 10.1016/j.xphs.2022.10.031

Scopus

PubMed

Publishing type：Research paper (scientific journal)   Publisher：Maad Rayan Publishing Company  

Background: Camel milk has been recognized for its health benefits since ancient times and has recently been attracting increasing attention as a form of medical treatment for diverse human diseases. Studies on the health benefits of camel milk attributed its medicinal effects to nutritional status, but the molecular mechanisms of proteins involved in such effects remain unknown. Objectives: The aim of this study was to explore the anticancer properties of camel milk proteins (CMPs). Methods: CMPs were fractionated into camel casein proteins (CCPs) and camel whey proteins (CWPs). The CWP exhibited the most potent anticancer activity against colon (HCT-116) and breast (MCF-7) cancer cells. The CWP was further fractionated into cationic and anionic proteins using HiTrap cationic (SP-XL) and anionic (QFF) exchange columns. Results: QFF-bound proteins (QFF-B) exhibited the strongest anticancer activities against both cancer cells. QFF-B proteins produced three peaks (P1~P3) on RP-HPLC, whereas P3 showed superior anticancer activity. The cytotoxic effects of CWP and QFF-B proteins are associated with increased production of intracellular ROS and subsequent apoptosis in both cancer cells. MALDI-TOF-MS identified lactophorin, glycation-dependent cell adhesion molecule1 (GlyCAM-1), and its three driven fragments as dominant peptides in QFF-B, while RP-HPLC-P3 contained two of them with molecular masses of 8080.3 and 9395.6 Da. The two peptides, both derived from the C-terminal of lactophorin, were the most representative peptides in the most active protein fractions (QFF-B and RP-HPLC-P3). Conclusion: The results highlight for the first time that lactophorin is the major anti-cancer ingredient in camel milk and its unique C-terminal peptides present potential candidacy as anticancer agents in nutraceutical and pharmacological applications.

DOI： 10.34172/ajmb.2022.01

Language：English   Publishing type：Research paper (scientific journal)  

Lactoferrin (LF) is a multifunctional protein with a broad spectrum of antimicrobial activities. In this study, we investigated the antimicrobial activity of LF against the potato common scab pathogen Streptomyces scabiei, which causes severe damage to potato tubers. LF derived from bovine (bLF) had much higher activity against S. scabiei than human LF. The minimal inhibitory concentration of bLF was 3.9 μM. The effects of both apo-bLF (iron-free) and holo-bLF (iron-saturated) on S. scabiei were not different. Bovine lactoferricin (LFcinB), a short peptide with a length of 25 amino acid residues located in the N-terminal region of bLF, showed antimicrobial activity against S. scabiei, similar to that of bLF. These results indicated that the antimicrobial activity of bLF against S. scabiei cannot be attributed to its iron-chelating effect but to the bioactivity of its peptides. When S. scabiei was treated with the fusion protein of mCherry-LFcinB (red fluorescent protein) expressed in Escherichia coli, the pseudohyphal cells instantly glowed, indicating that the peptide electrostatically binds to the surface of S. scabiei. An assay of synthetic peptides, with modified number of arginine (Arg) and tryptophan (Trp) residues based on the antimicrobial center (RRWQWR) of LFcinB showed that Trp residues are implicated in the antimicrobial activity against S. scabiei; however, Arg residues are also necessary to carry Trp residues to the cell surface to fully exert its activity. Although the single amino acid effect of Trp had low activity, Trp derivatives showed much higher activity against S. scabiei, suggesting that the derivatives effectively bind to the cell surface (cell membrane) by themselves without a carrier. Thus, amino acid derivatives might be considered effective and alternative antimicrobial substances.

DOI： 10.1371/journal.pone.0264094

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Ticks are classified as hematophagous arthropods and transfer a variety of pathogens-such as viruses, bacteria, and protozoans-to vertebrate hosts during blood feeding. These transmitted pathogens cause infectious diseases that continue to affect both humans and animals worldwide. Chemical acaricides are commonly used for tick control to prevent infectious diseases. However, the continuous use of acaricides leads to the emergence of acaricide-resistant tick species; thus, alternative methods for tick control are necessary. Vaccination of vertebrate hosts with tick-derived molecules is considered to be a better alternative against ticks than chemical acaricides because ticks feed on host blood for several days and also concentrate the host blood with antibodies. On the other hand, the host's immune responses against pathogens mainly take two pathways-Th1 (cell-mediated immunity) and Th2 (humoral immunity) pathways. Thus, the vaccine can suggest which immune pathway is more important for vaccination. This chapter describes the procedures of immunizing laboratory animals-mice-with a recombinant tick protein for the preliminary evaluation of its potential as an anti-tick vaccine candidate. In addition, the method of evaluating the antigen-specific antibody production in the host using ELISA is described, as is the subsequent tick-infestation challenge for determining the effectiveness of vaccination.

DOI： 10.1007/978-1-0716-1888-2_19

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

The stability of proteins is an important factor for industrial and medical applications. Improving protein stability is one of the main subjects in protein engineering. In a previous study, we improved the stability of a four-helix bundle dimeric de novo protein (WA20) by five mutations. The stabilised mutant (H26L/G28S/N34L/V71L/E78L, SUWA) showed an extremely high denaturation midpoint temperature (Tm). Although SUWA is a remarkably hyperstable protein, in protein design and engineering, it is an attractive challenge to rationally explore more stable mutants. In this study, we predicted stabilising mutations of WA20 by in silico saturation mutagenesis and molecular dynamics simulation, and experimentally confirmed three stabilising mutations of WA20 (N22A, N22E, and H86K). The stability of a double mutant (N22A/H86K, rationally optimised WA20, ROWA) was greatly improved compared with WA20 (ΔTm = 10.6 °C). The model structures suggested that N22A enhances the stability of the α-helices and N22E and H86K contribute to salt-bridge formation for protein stabilisation. These mutations were also added to SUWA and improved its Tm. Remarkably, the most stable mutant of SUWA (N22E/H86K, rationally optimised SUWA, ROSA) showed the highest Tm (129.0 °C). These new thermostable mutants will be useful as a component of protein nanobuilding blocks to construct supramolecular protein complexes.

DOI： 10.1038/s41598-021-86952-2

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

The newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is causing a spread of coronavirus disease 2019 (COVID-19) globally. In order to end the COVID-19 pandemic, an effective vaccine against SARS-CoV-2 must be produced at low cost and disseminated worldwide. The spike (S) protein of coronaviruses plays a pivotal role in the infection to host cells. Therefore, targeting the S protein is one of the most rational approaches in developing vaccines and therapeutic agents. In this study, we optimized the expression of secreted trimerized S protein of SARS-CoV-2 using a silkworm-baculovirus expression vector system and evaluated its immunogenicity in mice. The results showed that the S protein forming the trimeric structure was the most stable when the chicken cartilage matrix protein was used as the trimeric motif and could be purified in large amounts from the serum of silkworm larvae. The purified S protein efficiently induced antigen-specific antibodies in mouse serum without adjuvant, but its ability to induce neutralizing antibodies was low. After examining several adjuvants, the use of Alum adjuvant was the most effective in inducing strong neutralizing antibody induction. We also examined the adjuvant effect of paramylon from Euglena gracilis when administered with the S protein. Our results highlight the effectiveness and suitable construct design of the S protein produced in silkworms for the subunit vaccine development against SARS-CoV-2.

DOI： 10.3389/fimmu.2021.803647

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Camel milk is traditionally considered to have medicinal characteristics that it has potential health benefits and could help to treat several illnesses. Particularly, it is closest to human breast milk and has high levels of nutrients and bioactive components. The aim of this study was to explore the antioxidant peptides derived from protein fractions of camel milk. Camel milk proteins (CMP) were fractionated into camel casein protein (CCP) and camel whey protein (CWP), which were hydrolyzed with pepsin to produce peptic digests P-CCP and P-CWP, respectively. RP-HPLC was used for fractionation of the peptides from the P-CCP and P-CWP. The antioxidant activities were evaluated using superoxide anion generating system of xanthine oxidase (XOD) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging assay. Active peptides were analyzed using matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) whereas a number of antioxidant peptides, with masses ranging from 913 to 2,951 Da, derived mainly from alpha-casein, lactophorin and lactoferrin, were identified. When yeast cells are used as a system for modeling mitochondrial disease, the peptides in caseins and whey fractions significantly enhanced the tolerance of yeast cells against peroxide-induced oxidative stress. The results show that both caseins and whey proteins of camel milk possess bioactive peptides with significant radical-scavenging activities and thus herald a fascinating opportunity for their potential as nutraceuticals or therapeutic peptides for prevention and treatment of oxidative stress-associated diseases.

DOI： 10.1016/j.aninu.2018.05.004

PubMed

Other Link： https://www.ncbi.nlm.nih.gov/pubmed/30175255

Porcine circovirus type 2 (PCV2) is a primary causative agent of postweaningmultisystemic wasting syndrome (PMWS), which has a significant economic impact on the swine industry. The capsid protein (Cap) encoded by ORF2 of the viral genome has been used effectively as a vaccine against PCV2 infection. The Cap protein can spontaneously assemble into virus-like particles (VLPs) that are safe and highly immunogenic for vaccine applications. Several expression systems, including bacteria, yeast and insect cells, have been utilized to produce PCV2 VLPs. However, in some cases, the recombinant Cap (rCap) proteins produced in bacteria and yeast do not assemble spontaneously. In this study, we expressed rCap protein using a silkworm-baculovirus expression vector system (silkworm-BEVS) for mass production of PCV2 VLPs and established a simple three-step protocol for its purification from pupae: extraction by detergent, ammonium sulfate precipitation and anion exchange column chromatography. Size-exclusion chromatography (SEC) analysis and transmission electron microscope (TEM) observation showed that purified rCap proteins formed VLPs with a similar morphology to that of the original virus. Furthermore, the VLPs produced in silkworms were capable of inducing neutralizing antibodies against PCV2 in mice. Our results demonstrated that the silkworm system is a powerful tool for the production of PCV2 VLPs and will be useful for the development of a reliable and cost-effective PCV2 vaccine.

DOI： 10.1099/jgv.0.001087

PubMed

Ticks require blood feeding on vertebrate animals throughout their life cycle, and also concentrate the iron-containing blood, resulting in a high concentration of hydrogen peroxide (H2O2). High concentrations of H2O2 are harmful to organisms, due to their serious damage of macromolecules. Ticks have antioxidant enzymes, such as peroxiredoxins (Prxs), that scavenge H2O2. Prxs may have important roles in regulating the H2O2 concentration in ticks during blood feeding and oviposition. Moreover, Prxs are considered potential vaccine candidates in other parasites, such as Leishmania and Fasciola. In the present study, the efficacy of a tick Prx (HlPrx2) as a vaccine candidate antigen was evaluated. First, recombinant HlPrx2 (rHlPrx2) was expressed in Escherichia coli, and then, its purity and endotoxin levels were confirmed prior to administration. The rHlPrx2 proteins were of high purity with acceptably low endotoxin levels. Second, the ability of rHlPrx2 administration to stimulate mouse immunity was evaluated. The rHlPrx2 protein, with or without an adjuvant, could stimulate immunity in mice, especially the IgG1 of Th2 immune response. Using Western blot analysis, we also observed whether rHlPrx2-immunized mice sera could recognize native HlPrx2 protein in crude tick midgut proteins. Western blot analysis demonstrated that rHlPrx2-administrated mouse sera could detect the native HlPrx2. Finally, the effects of rHlPrx2 immunization in mice were studied using nymphal ticks. Although the challenged ticks were not affected by rHlPrx2 immunization, rHlPrx2 still might be considered as a vaccine candidate against ticks because of its high immunogenicity.

DOI： 10.1007/s10493-018-0209-3

PubMed

Publisher：ELSEVIER SCIENCE BV  

Lysozyme is commonly found in spots where bacterial infections are most likely to enter the body. Earlier we found that lysozyme possesses five antimicrobial peptide motifs in its N-terminal region which can be generated by newborn pepsin. In this study, we explore the role of these peptides in the anti-inflammatory activity of lysozyme. The five peptides, helix1 (H1), helix2 (H2), H1 and H2 connected with a loop (HLH), H2 extended with either 2 β-strands (H2-S12) or 3 β-strands (H2-S13), were synthesized and examined for anti-inflammatory action. The five peptides dose-dependently decreased, to different degrees, expression of pro-inflammatory cytokines, TNF-α, IL-6 and IL-1β, in lipopolysaccharide (LPS)- or interferon-gamma (INF-γ)-stimulated mouse macrophage cells (RAW264.7). The HLH peptide and its individual helices (H1 and H2) were markedly the most potent anti-inflammatory. When macrophage cells were stimulated with live bacteria (E. coli), H1 peptide was the most powerful suppressor of TNF-α and IL-6 expression, providing evidence that the peptide is able to antagonize the pathogen-induced inflammatory response. Receptor binding assay and docking simulation provided evidence that H1 peptide bind specifically to the pocket for endotoxin binding of the toll-like receptor 4 (TLR-4) of macrophage. The results demonstrate, for the first time, the molecular basis of anti-inflammatory action of lysozyme that N-terminal helical peptides are the main contributors. This exciting finding offers new classes of therapeutic peptides with potential in the treatment of infection-induced inflammatory diseases.

DOI： 10.1016/j.ejps.2017.07.005

Web of Science

PubMed

Publisher：ELSEVIER SCIENCE BV  

Angiotensin-converting enzyme (ACE) plays a central role in blood pressure regulation by producing the vasoconstrictor angiotensin II. The inhibition of ACE with natural inhibitors, as alternatives to avoid the side effect of synthetic drugs, is a major target in the prevention of hypertension. In this study, we examined the separated caseins and whey proteins of goat milk for the presence of ACE inhibitory peptides. Digestion of isolated whey proteins and caseins of goat milk by gastric pepsin generated soluble hydrolysates exhibiting significant inhibition of ACE compared to weak inhibition by undigested proteins. The hydrolysates were fractionated by size exclusion chromatography, Sephacryl S-100 column, into four fractions (F1-F4). The late-eluting fraction (F4) of either whey or caseins exhibited greater ACE inhibition. Peptides in both F4 fractions, isolated by RP-HPLC, exhibited variable ACE inhibitory activities with the hydrophobic peptide peaks being the most potent ACE inhibitors. MALDI-TOF MS/MS resulted in identification of three potent ACE inhibitory peptides: PEQSLACQCL from beta-lactoglobulin (residues 113-122), QSLVYPFTGPI from beta-casein (residues 56-66), and ARHPHPHLSFM from kappa-casein (residues 96-106). The peptides from whey and caseins exert significant ACE inhibitory activities comparable to that of captopril, an antihypertensive drug, exhibiting IC50 values of 4.45 mu M and 4.27 mu M, respectively. The results introduce, for the first time, new potent ACE-inhibitory peptides that can be released by gastric pepsin of goat milk whey and caseins and thus may pave the way for their candidacy as anti-hypertensive bioactive peptides and prevention of associated disorders. (C) 2016 Production and hosting by Elsevier B.V. on behalf of Cairo University.

DOI： 10.1016/j.jare.2016.12.002

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier  

DOI： 10.1016/j.vaccine.2016.01.034

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：Japanese   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Publisher：日本衛生動物学会  

J-GLOBAL

J-GLOBAL

J-GLOBAL

J-GLOBAL

Publisher：(公社)日本獣医学会  

Event date： 2014.11

Language：Japanese  

Venue：てんぶすホール、沖縄  

国内学会

Event date： 2014.11

Language：Japanese  

Venue：てんぶすホール、沖縄  

国内学会

Event date： 2014.11

Language：Japanese  

Venue：てんぶすホール、沖縄  

国内学会

Event date： 2014.11

Language：Japanese  

Venue：鹿児島大学、鹿児島  

国内学会

Event date： 2014.11

Language：Japanese  

Venue：鹿児島大学、鹿児島  

国内学会

Event date： 2014.9

Language：Japanese  

Venue：北海道大学、北海道  

国内学会

Event date： 2014.3

Language：Japanese  

Venue：愛媛大学、愛媛  

国内学会

Event date： 2014.3

Language：Japanese  

Venue：愛媛大学、愛媛  

国内学会

Event date： 2013.12

Language：English  

Venue：幕張メッセ、千葉  

国際学会

Event date： 2013.10

Language：Japanese  

Venue：長崎ブリックホール、長崎  

国内学会

Event date： 2013.10

Language：Japanese  

Venue：長崎ブリックホール、長崎  

国内学会

Event date： 2013.10

Language：Japanese  

Venue：長崎ブリックホール、長崎  

国内学会

Event date： 2013.3

Language：Japanese  

Venue：東京  

国内学会

Event date： 2013.3

Language：Japanese  

Venue：東京  

国内学会

Event date： 2012.11

Language：Japanese  

Venue：神奈川  

国内学会

Event date： 2012.11

Language：Japanese  

Venue：神奈川  

国内学会