Purification, identification, and computational analysis of xanthine oxidase inhibitory peptides from kidney bean.

Hyperuricemia is related to plenty of diseases, seriously damaging human health. Current clinical drugs used to treat hyperuricemia have many adverse effects. In this study, kidney bean hydrolysate (KBH) was found to exert high xanthine oxidase inhibitory (XOI) activity. Compared to KBH (50.31 ± 2.73%), XOI activities of three fractions (Mw <5 kDa, Mw <3 kDa, Mw  < 1 kDa) by ultrafiltration were higher and increased to 58.58 ± 3.57%, 59.34 ± 1.78%, and 55.05 ± 5.00%, respectively (P < 0.05). A total of 69 peptides were identified by HPLC-ESI-MS/MS and analyzed binding affinities with XO with the help of molecular docking. AVDSLVPIGR, DWYDIK, LDNLLR, ISPIPVLK, ISSLEMTR showed well binding affinities with XO and DWYDIK presented the highest XOI activity (68.63 ± 5.07%) among five synthetic peptides (P < 0.05). Additionally, visual analysis results indicated that DWYDIK was pushed into the hydrophobic channel and formed hydrogen bonds with pivotal amino acids of xanthine oxidase. Overall, KBH could be a promising candidate as anti- hyperuricemia functional food. PRACTICAL APPLICATION: This research initially revealed that kidney bean peptides could significantly inhibit the activity of xanthine oxidase, indicating kidney bean peptides could be a treatment for hyperuricemia. Kidney bean peptides may have commercial potentials as a safer alternative with few side effects to drugs.

Enzymatic preparation of Crassostrea oyster peptides and their promoting effect on male hormone production.

ETHNOPHARMACOLOGICAL RELEVANCE: Crassostrea gigas Thunberg and other oysters have been traditionally used in China as folk remedies to invigorate the kidney and as natural aphrodisiacs to combat male impotence. AIM OF THE STUDY: Erectile dysfunction (ED) has become a major health problem for the global ageing population. The aim of this study is therefore to evaluate the effect of peptide-rich preparations from C. gigas oysters on ED and related conditions as increasing evidence suggests that peptides are important bioactive components of marine remedies and seafood. MATERIALS AND METHODS: Crassostrea oyster peptide (COP) preparations COP1, COP2 and COP3 were obtained from C. gigas oysters by trypsin, papain or sequential trypsin-papain digestion, respectively. The contents of testosterone, cyclic adenosine monophosphate (cAMP) and nitric oxide (NO) and the activity of nitric oxide synthase (NOS) in mice and/or cells were measured by enzyme-linked immunosorbent assays. Real-time PCR was used to assess the expression of genes associated with sex hormone secretion pathways. The model animal Caenorhabditis elegans was also used to analyze the gene expression of a conserved steroidogenic enzyme. In silico analysis of constituent peptides was performed using bioinformatic tools based on public databases. RESULTS: The peptide-rich preparation COP3, in which >95% peptides were <3000 Da, was found to increase the contents of male mouse serum testosterone and cAMP, both of which are known to play important roles in erectile function, and to increase the activity of mouse penile NOS, which is closely associated with ED. Further investigation using mouse Leydig-derived TM3 cells demonstrates that COP3 was able to stimulate the production of testosterone as well as NO, a pivotal mediator of penile erection. Real-time PCR analysis reveals that COP3 up-regulated the expression of Areg and Acvr2b, the genes known to promote sex hormone secretion, but not Fst, a gene involved in suppressing follicle-stimulating hormone release. Furthermore, COP3 was also shown to up-regulate the expression of let-767, a well-conserved C. elegans gene encoding a protein homologous to human 17-ß-hydroxysteroid dehydrogenases. Preliminary bioinformatic analysis using the peptide sequences in COP3 cryptome identified 19 prospective motifs, each of which occurred in more than 10 peptides. CONCLUSIONS: In this paper, Crassostrea oyster peptides were prepared by enzymatic hydrolysis and were found for the first time to increase ED-associated biochemical as well as molecular biology parameters. These results may help to explain the ethnopharmacological use of oysters and provide an important insight into the potentials of oyster peptides in overcoming ED-related health issues.

Sweet as honey, bitter as bile: Mitochondriotoxic peptides and other therapeutic proteins isolated from animal tissues, for dealing with mitochondrial apoptosis.

Mitochondria are subcellular organelles involved in cell metabolism and cell life-cycle. Their role in apoptosis regulation makes them an interesting target of new drugs for dealing with cancer or rare diseases. Several peptides and proteins isolated from animal and plant sources are known for their therapeutic properties and have been tested on cancer cell-lines and xenograft murine models, highlighting their ability in inducing cell-death by triggering mitochondrial apoptosis. Some of those molecules have been even approved as drugs. Conversely, many other bioactive compounds are still under investigation for their proapoptotic properties. In this review we report about a group of peptides, isolated from animal venoms, with potential therapeutic properties related to their ability in triggering mitochondrial apoptosis. This class of compounds is known with different names, such as mitochondriotoxins or mitocans.

Complementary multiple hydrogen-bond-based magnetic composite microspheres for high coverage and efficient phosphopeptide enrichment in bio-samples.

Due to the number of phosphorylation sites, mono- and multiple-phosphopeptides exhibit significantly different biological effects. Therefore, comprehensive profiles of mono- and multiple-phosphopeptides are vital for the analysis of these biological and pathological processes. However, the most commonly used affinity materials based on metal oxide affinity chromatography (MOAC) show stronger selectivity toward mono-phosphopeptides, thus losing most information on multiple-phosphopeptides. Herein, we report polymer functionalized magnetic nanocomposite microspheres as an ideal platform to efficiently enrich both mono- and multiple-phosphopeptides from complex biological samples. Driven by complementary multiple hydrogen bonding interactions, the composite microspheres exhibited remarkable performance for phosphopeptide enrichment from model proteins and real bio-samples. Excellent selectivity (the molar ratio of nonphosphopeptides/phosphopeptides was 5000 : 1), high enrichment sensitivity (2 fmol) and coverage, as well as high capture rates of multiple-phosphopeptides revealed their great potential in comprehensive phosphoproteomics studies. More importantly, we successfully captured the cancer related phosphopeptides (from the phosphoprotein Stathmin-1) and identified their relevant phosphorylation sites from oral carcinoma patients' saliva and tissue lysate, demonstrating the potential of this material for phosphorylated disease marker detection and discovery.

Separation of Bioactive Small Molecules, Peptides from Natural Sources and Proteins from Microbes by Preparative Isoelectric Focusing (IEF) Method.

Natural products derived from plants and microbes are a rich source of bioactive molecules. Prior to their use, the active molecules from complex extracts must be purified for downstream applications. There are various chromatographic methods available for this purpose yet not all labs can afford high performance methods and isolation from complex biological samples can be difficult. Here we demonstrate that preparative liquid-phase isoelectric focusing (IEF) can separate molecules, including small molecules and peptides from complex plant extracts, based on their isoelectric points (pI). We have used the method for complex biological sample fractionation and characterization. As a proof of concept, we fractionated a Gymnema sylvestre plant extract, isolating a family of terpenoid saponin small molecules and a peptide. We also demonstrated effective microbial protein separation using the Candida albicans fungus as a model system.

Basidiosins A and B: Cyclopentapeptides from the entomophthoralean fungus Basidiobolus meristosporus.

Two new cyclopentapeptides, basidiosins A and B (1 and 2) were isolated from the mycelia extracts of entomophthoralean fungus Basidiobolus meristosporus RCEF 4516. The structures were determined based on spectroscopic methods, and the absolute config urations were assigned by Marfey's method on their acid hydrolyzates. Compounds 1 and 2 were identified as cyclo(L-Thr-L-Leu- L-Ile-D-Tyr-D-Thr) and cyclo(L-Thr-L-Leu-L-Val-D-Val-D-Ser), respectively. They were evaluated for the biological activities including antibacterial, antifungal and antioxidative activities. Furthermore, the biosynthetic pathway of 1 was proposed by bioinformatic analysis. This is the first study on the isolation of natural products from Basidiobolus fungus.

Protective Effects of Novel Antioxidant Peptide Purified from Alcalase Hydrolysate of Velvet Antler Against Oxidative Stress in Chang Liver Cells in Vitro and in a Zebrafish Model In Vivo.

Velvet antler has a long history in traditional medicine. It is also an important healthy ingredient in food as it is rich in protein. However, there has been no report about antioxidant peptides extracted from velvet antler by enzymatic hydrolysis. Thus, the objective of this study was to hydrolyze velvet antler using different commercial proteases (Acalase, Neutrase, trypsin, pepsin, and α-chymotrypsin). Antioxidant activities of different hydrolysates were investigated using peroxyl radical scavenging assay by electron spin resonance spectrometry. Among all enzymatic hydrolysates, Alcalase hydrolysate exhibited the highest peroxyl radical scavenging activity. Alcalase hydrolysate was then purified using ultrafiltration, gel filtration, and reverse-phase high performance liquid chromatography. The purified peptide was identified to be Trp-Asp-Val-Lys (tetrapeptide) with molecular weight of 547.29 Da by Q-TOF ESI mass spectroscopy. This purified peptide exhibited strong scavenging activity against peroxyl radical (IC50 value, 0.028 mg/mL). In addition, this tetrapeptide showed significant protection ability against AAPH-induced oxidative stress by inhibiting of reactive oxygen species (ROS) generation in Chang liver cells in vitro and in a zebrafish model in vivo. This research suggests that the tetrapeptide derived from Alcalase-proteolytic hydrolysate of velvet antler are excellent antioxidants and could be effectively applied as functional food ingredients and pharmaceuticals.

Silk peptide treatment potentiates natural killer cell activity in vitro and induces natural killer cell maturation and activation in mouse splenocytes.

Context: Silk peptide from cocoons of silkworm (Bombyx mori L., Bombycidae) has been employed as a biomedical material and exhibits various bioactivities, including immune-modulating activity. Objective: We analyzed whether silk peptide exerts direct modulating effects on NK cells using an NK cell line in vitro and ex vivo splenocytes. We also attempted to delineate the mechanism underlying the modulation. Material and methods: In vitro activity of silk peptide on NK cells was determined by measurement of cytolytic activity against K562 cells at an effector-to-target ratio of 5:1 after incubation of NK-92MI cells with silk peptide (0-2000 µg/mL) for 48 and 72 h. Ex vivo activity of silk peptide on mouse splenic NK cells was determined similarly by using YAC-1 cells. Results: Treatment of NK-92MI NK cells with silk peptide (500-2000 µg/mL) significantly increased cytolytic activity on target cells by 2- to 4-fold. The same concentrations (500-2000 µg/mL) of silk peptide treatment also significantly enhanced the cytolytic activity of splenic NK cells against YAC-1 cells. Silk peptide treatment of IL-2-stimulated splenocytes induced enhanced expression of Th1, 2 and 17 cytokines including TNF-α, IFN-γ, IL-6, IL-4 and IL-17. Finally, ex vivo treatment with silk peptide on mouse splenocytes significantly enhanced the degree of NK cell maturation in a dose-dependent manner from 3.49 to 23.79%. Discussion and conclusions: These findings suggest that silk peptide stimulates NK cells, thereby influencing systemic immune functions and improving natural immunity. Thus, silk peptide could be useful as a complementary therapy in cancer patients.

Development of wheat genotypes expressing a glutamine-specific endoprotease from barley and a prolyl endopeptidase from Flavobacterium meningosepticum or Pyrococcus furiosus as a potential remedy to celiac disease.

Ubiquitous nature of prolamin proteins dubbed gluten from wheat and allied cereals imposes a major challenge in the treatment of celiac disease, an autoimmune disorder with no known treatment other than abstinence diet. Administration of hydrolytic glutenases as food supplement is an alternative to deliver the therapeutic agents directly to the small intestine, where sensitization of immune system and downstream reactions take place. The aim of the present research was to evaluate the capacity of wheat grain to express and store hydrolytic enzymes capable of gluten detoxification. For this purpose, wheat scutellar calli were biolistically transformed to generate plants expressing a combination of glutenase genes for prolamin detoxification. Digestion of prolamins with barley endoprotease B2 (EP-HvB2) combined with Flavobacterium meningosepticum prolyl endopeptidase (PE-FmPep) or Pyrococcus furiosus prolyl endopeptidase (PE-PfuPep) significantly reduced (up to 67%) the amount of the indigestible gluten peptides of all prolamin families tested. Seven of the 168 generated lines showed inheritance of transgene to the T2 generation. Reversed phase high-performance liquid chromatography of gluten extracts under simulated gastrointestinal conditions allowed the identification of five T2 lines that contained significantly reduced amounts of immunogenic, celiac disease-provoking gliadin peptides. These findings were complemented by the R5 ELISA test results where up to 72% reduction was observed in the content of immunogenic peptides. The developed wheat genotypes open new horizons for treating celiac disease by an intraluminal enzyme therapy without compromising their agronomical performance.

Peganum harmalapeptides (PhAMP) impede bacterial growth and biofilm formation in burn and surgical wound pathogens.

Many clinical-pathogens have developed resistance against known antibiotics and there is an urgent need for the discovery of novel antibiotics. In this study, low molecular weight peptides were isolated from seeds/leaves of 20 medicinal plants and tested for their antibacterial activity against laboratory strains of S. aureusand P. aeruginosa. Peptides isolated from Peganum harmala (PhAMP) exhibited maximum activity against laboratory strains. As clinical-isolates are more virulent and resistant to antibiotics, we tested the potential of PhAMP on these bacterial strains isolated from infected wounds. Pathogens isolated from burn-wounds (S. aureus, P. aeruginosa and K. pneumoniae) and surgical-wounds (P. aeruginosa and K. pneumoniae) exhibited zones of inhibition against PhAMP when tested by disc diffusion method. Biofilm formation of wound pathogens in the presence/absence of PhAMP was analyzed to check its effect. Surgical-wound pathogens and K. pneumoniae from burn-wound showed significant reduction in biofilm formation and planktonic bacteria. While biofilms of S. aureus and P. aeruginosa from burn-wound showed resistance against PhAMP. An effective antibiotic treatment should not only inhibit but should also disrupt already developed biofilms. PhAMP was very effective in the disruption of developed biofilm of all pathogens after 36 hours. This data unravels the potential of PhAMP as a novel, natural antibiotic against clinical-pathogens.

In silico identification of milk antihypertensive di- and tripeptides involved in angiotensin I-converting enzyme inhibitory activity.

Identification of bioactive milk peptides could improve food technology through improved selection of food supplements with a focus on antihypertensive properties. We hypothesized that angiotensin I-converting enzyme (ACE) inhibitory activities of milk di- and tripeptides could be predicted using 3-dimensional quantitative structure activity relationship methods and that these activities could be explained through evaluation of structural features (hydrogen bond donor/acceptor, hydrophobic, steric, and electrostatic) that are responsible for this bioactivity. We aimed to build comparative molecular field analysis (CoMFA) models combined with in silico digestion to predict the peptide sequences released from enzymatic digestion and to evaluate peptides without experimental data. Furthermore, molecular docking simulation was performed with the aim to evaluate structural features. Molecular docking simulations revealed that the most potent inhibitory peptides contain hydrophobic amino acids that enter deep into the hydrophobic pocket of the ACE active site and make interactions with its residues. CoMFA results point out favorable steric interactions and electronegativity at the C-terminus of the milk dipeptides. The CoMFA model appears to favor electropositive amino acids at the second place in tripeptides and electronegative interaction with Tyr520. Furthermore, predicted values of ACE inhibitory activity of dipeptides obtained by peptide cutter are relatively high, which recommend them for application as functional food supplements and natural alternatives to ACE inhibitory drugs. This research suggests that obtained 3-dimensional quantitative structure activity relationship models are able to successfully identify milk-derived di- and tripeptides that have significant antihypertensive activity and provide information for screening and design of novel ACE inhibitors that could be used as supplements in human nutrition.

Isolation and identification of anti-proliferative peptides from Spirulina platensis using three-step hydrolysis.

BACKGROUND: Spirulina platensis is an excellent source of proteins (>60%) that can be hydrolyzed into bioactive peptides. RESULTS: In this study, whole proteins of Spirulina platensis were extracted and hydrolyzed using three gastrointestinal endopeptidases (pepsin, trypsin and chymotrypsin). Subsequently, gel filtration chromatography was employed to separate hydrolysates, and four fractions (Tr1-Tr4) were obtained. Among them, Tr2 showed the strongest anti-proliferation activities on three cancer cells (MCF-7, HepG-2 and SGC-7901), with IC50 values of <31.25, 36.42 and 48.25 µg mL-1 , respectively. Furthermore, a new peptide, HVLSRAPR, was identified from fraction Tr1. This peptide exhibited strong inhibition on HT-29 cancer cells with an IC50 value of 99.88 µg mL-1 . CONCLUSION: Taken together, these peptides possessed anti-proliferation activities on cancer cells and low cytotoxicity on normal cells, suggesting that they might serve as a natural anticancer agent for nutraceutical and pharmaceutical industries. © 2016 Society of Chemical Industry.

Affinity chromatography matrices for depletion and purification of casein glycomacropeptide from bovine whey.

Casein glycomacropeptide (CMP) is a 64- amino acid peptide found in cheese whey, which is released after κ-casein specific cleavage by chymosin. CMP lacks aromatic amino acids, a characteristic that makes it usable as a nutritional supplement for people with phenylketonuria. CMP consists of two nonglycosylated isoforms (aCMP A and aCMP B) and its different glycosylated forms (gCMP A and gCMP B). The most predominant carbohydrate of gCMP is N-acetylneuraminic acid (sialic acid). Here, we developed a CMP purification process based on the affinity of sialic acid for wheat germ agglutinin (WGA). After formation of chitosan beads and adsorption of WGA, the agglutinin was covalently attached with glutaraldehyde. Two matrices with different WGA density were assayed for CMP adsorption. Maximum adsorption capacities were calculated according to the Langmuir model from adsorption isotherms developed at pH 7.0, being 137.0 mg/g for the matrix with the best performance. In CMP reduction from whey, maximum removal percentage was 79% (specifically 33.7% of gCMP A and B, 75.8% of aCMP A, and 93.9% of aCMP B). The CMP was recovered as an aggregate with an overall yield of 64%. Therefore, the matrices developed are promising for CMP purification from cheese whey. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:171-180, 2017.

Hypocholesterolaemic Activity of Lupin Peptides: Investigation on the Crosstalk between Human Enterocytes and Hepatocytes Using a Co-Culture System Including Caco-2 and HepG2 Cells.

Literature indicates that peptic and tryptic peptides derived from the enzymatic hydrolysis of lupin protein are able to modulate cholesterol metabolism in human hepatic HepG2 cells and that part of these peptides are absorbed in a small intestine model based on differentiated human Caco-2 cells. In this paper, a co-culture system, including Caco-2 and HepG2 cells, was investigated with two objectives: (a) to verify whether cholesterol metabolism in HepG2 cells was modified by the peptides absorption through Caco-2 cells; (b) to investigate how lupin peptides influence cholesterol metabolism in Caco-2 cells. The experiments showed that the absorbed peptides, not only maintained their bioactivity on HepG2 cells, but that this activity was improved by the crosstalk of the two cells systems in co-culture. In addition, lupin peptides showed a positive influence on cholesterol metabolism in Caco-2 cells, decreasing the proprotein convertase subtilisin/kexin type 9 (PCSK9) secretion.

Generation of bioactive peptide hydrolysates from cattle plasma using plant and fungal proteases.

Four protease preparations from plant and fungal sources (papain, bromelain, FP400 and FPII) were used to hydrolyse plasma which was separated from slaughterhouse cattle blood. The o-phthaldialdehyde assay was used to follow the release of TCA-soluble peptides over a 24h period. Hydrolysis profiles were displayed using SDS-PAGE. The in vitro antioxidant and antimicrobial activities of the hydrolysates were determined. The results showed that hydrolysates of cattle plasma generated with fungal protease FPII had higher antioxidant activities. Overall than hydrolysates generated with papain, bromelain and FP400. None of the hydrolysates demonstrated antimicrobial activity. The FPII peptide hydrolysate was fractionated using gel permeation chromatography, OFFGEL isoelectric focusing and RP-HPLC. The RP-HPLC fraction with highest antioxidant activity contained 15 novel peptide sequences. The use of protease FPII to hydrolyse cattle plasma resulted in a hydrolysate with high antioxidant properties and unique peptide sequences.

Anxiolytic activity and evaluation of potentially adverse effects of a bradykinin-related peptide isolated from a social wasp venom.

Anxiety disorders are major health problems in terms of costs stemming from sick leave, disabilities, healthcare and premature mortality. Despite the availability of classic anxiolytics, some anxiety disorders are still resistant to treatment, with higher rates of adverse effects. In this respect, several toxins isolated from arthropod venoms are useful in identifying new compounds to treat neurological disorders, particularly pathological anxiety. Thus, the aims of this study were to identify and characterize an anxiolytic peptide isolated from the venom of the social wasp Polybia paulista. The peptide was identified as Polisteskinin R, with nominal molecular mass [M+H](+)=1301Da and primary structure consisting of Ala-Arg-Arg-Pro-Pro-Gly-Phe-Thr-Pro-Phe-Arg-OH. The anxiolytic effect was tested using the elevated plus maze test. Moreover, adverse effects on the spontaneous behavior and motor coordination of animals were assessed using the open field and rotarod tests. Polisteskinin R induced a dose-dependent anxiolytic effect. Animals treated with the peptide and diazepam spent significantly more time into the open arms when compared to the groups treated with the vehicle and pentylenetetrazole. No significant differences in spontaneous behavior or motor coordination were observed between the groups, showing that the peptide was well tolerated. The interaction by agonists in both known BK receptors induces a variability of physiological effects; Polisteskinin R can act on these receptors, inducing modulatory activity and thus, attenuating anxiety behaviors. The results of this study demonstrated that the compound Polisteskinin R exerted potent anxiolytic effects and its analogues are promising candidates for experimental pharmacology.

A Review of Potential Marine-derived Hypotensive and Anti-obesity Peptides.

Bioactive peptides are food derived components, usually consisting of 3-20 amino acids, which are inactive when incorporated within their parent protein. Once liberated by enzymatic or chemical hydrolysis, during food processing and gastrointestinal transit, they can potentially provide an array of health benefits to the human body. Owing to an unprecedented increase in the worldwide incidence of obesity and hypertension, medical researchers are focusing on the hypotensive and anti-obesity properties of nutritionally derived bioactive peptides. The role of the renin-angiotensin system has long been established in the aetiology of metabolic diseases and hypertension. Targeting the renin-angiotensin system by inhibiting the activity of angiotensin-converting enzyme (ACE) and preventing the formation of angiotensin II can be a potential therapeutic approach to the treatment of hypertension and obesity. Fish-derived proteins and peptides can potentially be excellent sources of bioactive components, mainly as a source of ACE inhibitors. However, increased use of marine sources, poses an unsustainable burden on particular fish stocks, so, the underutilized fish species and by-products can be exploited for this purpose. This paper provides an overview of the techniques involved in the production, isolation, purification, and characterization of bioactive peptides from marine sources, as well as the evaluation of the ACE inhibitory (ACE-I) activity and bioavailability.

A comparison of the enzymatic properties of three recombinant isoforms of thrombolytic and antibacterial protein--Destabilase-Lysozyme from medicinal leech.

BACKGROUND: Destabilase-Lysozyme (mlDL) is a multifunctional i-type enzyme that has been found in the secretions from the salivary glands of medicinal leeches. mlDL has been shown to exhibit isopeptidase, muramidase and antibacterial activity. This enzyme attracts interest because it expresses thrombolytic activity through isopeptidolysis of the ε-(γ-Glu)-Lys bonds that cross-link polypeptide chains in stabilised fibrin. To date, three isoforms of mlDL have been identified. The enzymatic properties of pure mlDL isoforms have not yet been described because only destabilase complexes containing other proteins could be isolated from the salivary gland secretion and because low product yield from the generation of recombinant proteins has made comprehensive testing difficult. RESULTS: In the present study, we optimised the procedures related to the expression, isolation and purification of active mlDL isoforms (mlDL-Ds1, mlDL-Ds2, mlDL-Ds3) using an Escherichia coli expression system, and we detected and compared their muramidase, lytic, isopeptidase and antimicrobial activities. After optimisation, the product yield was 30 mg per litre of culture. The data obtained in our study led to the suggestion that the recombinant mlDL isoforms isolated from inclusion bodies form stable oligomeric complexes. Analyses of the tested activities revealed that all isoforms exhibited almost identical patterns of pH and ionic strength effects on the activities. We determined that mlDL-Ds1, 2, 3 possessed non-enzymatic antibacterial activity independent of their muramidase activity. For the first time, we demonstrated the fibrinolytic activity of the recombinant mlDL and showed that only intact proteins possessed this activity, suggesting their enzymatic nature. CONCLUSIONS: The recombinant Destabilase-Lysozyme isoforms obtained in our study may be considered potential thrombolytic agents that act through a mechanism different from that of common thrombolytics.

Peptides derived from Rhopilema esculentum hydrolysate exhibit angiotensin converting enzyme (ACE) inhibitory and antioxidant abilities.

Jellyfish (Rhopilema esculentum) was hydrolyzed using alcalase, and two peptides with angiotensin-I-converting enzyme (ACE) inhibitory and antioxidant activities were purified by ultrafiltration and consecutive chromatographic methods. The amino acid sequences of the two peptides were identified as VKP (342 Da) and VKCFR (651 Da) by electrospray ionization tandem mass spectrometry. The IC50 values of ACE inhibitory activities of the two peptides were 1.3 µM and 34.5 µM, respectively. Molecular docking results suggested that VKP and VKCFR bind to ACE through coordinating with the active site Zn(II) atom. Free radical scavenging activity and protection against hydrogen peroxide (H2O2)-induced rat cerebral microvascular endothelial cell (RCMEC) injury were used to evaluate the antioxidant activities of the two peptides. As the results clearly showed that the peptides increased the superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-px) activities in RCMEC cells), it is proposed that the R. esculentum peptides exert significant antioxidant effects.

Screening the anti infectivity potentials of native N- and C-lobes derived from the camel lactoferrin against hepatitis C virus.

BACKGROUND: Hepatitis C virus (HCV) infection represents a worldwide health threat that still needs efficient protective vaccine and/or effective drug. The traditional medicine, such as camel milk, is heavily used by the large sector of HCV patients to control the infection due to the high cost of the available standard therapy. Camel milk contains lactoferrin, which plays an important and multifunctional role in innate immunity and specific host defense against microbial infection. Continuing the analysis of the effectiveness of camel lactoferrin against HCV, the current study aimed to separate and purify the native N- and C-lobes from the proteolytically cleaved camel lactoferrin (cLF) and to compare their in vitro activities against the HCV infection in Huh7.5 cells in order to determine the most active domain. METHODS: Lactoferrin and its digested N- and C-lobes were purified by Mono S 5/50 GL column and Superdex 200 5/150 column. The purified proteins were assessed through three venues: 1. To inhibit intracellular replication, HCV infected cells were treated with the proteins at different concentrations and time intervals; 2. The proteins were directly incubated with the viral particles (neutralization) and then such neutralized viruses were used to infect cells; 3. The cells were protected with proteins before exposure to the virus. The antiviral potentials of the cLf and its lobes were determined using three techniques: 1. RT-nested PCR, 2. Real-time PCR, and 3. Flow cytometry. RESULTS: N- and C-lobes were purified in two consecutive steps; using Mono-S and Superdex 200 columns. The molecular mass of N- and C-lobes was about 40 kDa. cLF and its lobes could prevent HCV entry into Huh 7.5 cells with activity reached 100% through direct interaction with the virus. The inhibition of intracellular viral replication by N-lobe is 2-fold and 3-fold more effective than that of the cLF and C-lobe, respectively. CONCLUSION: Generated native N- and C-lobes from camel lactoferrin demonstrated a range of noticeably different potentials against HCV cellular infectivity. The anti-HCV activities were sorted as N-lobe > cLf > C-lobe.