Recombinant expression and in vitro activity identification of a bioactive peptide QUB2984 from skin secretion of Agalychnis callidryas / 中国组织工程研究

BACKGROUND:Frog active peptides have rich activities,such as antibacterial and anti-tumor,and are expected to solve the problem of antibiotic resistance. OBJECTIVE:The active peptide QUB2984 was discovered in the skin secretions of Agalychnis callidryas.Its structure and properties were simulated by bioinformatics.The peptide was synthesized,purified,and identified and its biological functions were investigated. METHODS:Agalychnis callidryas skin secretions were collected by electrostimulation.The sequence of QUB2984 was obtained through constructing a cDNA library with isolated mRNA.BLAST was used for peptide sequence alignment.Besides that,Iterative Threading ASSEmbly Refinement(I-TASSER)and HeliQuest tools were used for protein secondary structure simulation.It was synthesized by solid-phase peptide synthesis,purified by reverse-phase high-performance liquid chromatography,and structurally confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.The purified peptide was used to evaluate its biological activity.Its antibacterial effect was evaluated by the minimum inhibitory concentration method.Its cytotoxic effect was detected by MTT assay.Its safety was investigated by a hemolysis test. RESULTS AND CONCLUSION:(1)Peptide QUB2984 had basically α-spiral structure,with a relatively intact hydrophobic surface,and a certain destructive ability to biofilm.The third amino acid position of QUB2984 was composed of W and had a G-X-G structure.(2)The minimum inhibitory concentration of QUB2984 against gram-positive Staphylococcus aureus was 2 μmol/L,the minimum inhibitory concentration against gram-negative Escherichia coli was 2 μmol/L,and the minimum inhibitory concentration against the fungus Candida albicans was 8 μmol/L.(3)The active peptide QUB2984 had obvious inhibitory effect on human non-small cell lung cancer cells NCI-H838 at 10-5 mol/L concentration,and the hemolytic effect on horse red cells at 64 μmol/L concentration was 50%.(4)The results showed that QUB2984 had anti-bacterial and anti-cancer activity,and it had a positive charge of +3,which was conducive to contact with bacteria or cells.

Activation of M1 muscarinic acetylcholine receptors by proline-rich oligopeptide 7a (

Background: The bioactive peptides derived from snake venoms of the Viperidae family species have been promising as therapeutic candidates for neuroprotection due to their ability to prevent neuronal cell loss, injury, and death. Therefore, this study aimed to evaluate the cytoprotective effects of a synthetic proline-rich oligopeptide 7a (PRO-7a; <EDGPIPP) from Bothrops jararaca snake, on oxidative stress-induced toxicity in neuronal PC12 cells and astrocyte-like C6 cells. Methods: Both cells were pre-treated for four hours with different concentrations of PRO-7a, submitted to H2O2-induced damage for 20 h, and then the oxidative stress markers were analyzed. Also, two independent neuroprotective mechanisms were investigated: a) L-arginine metabolite generation via argininosuccinate synthetase (AsS) activity regulation to produce agmatine or polyamines with neuroprotective properties; b) M1 mAChR receptor subtype activation pathway to reduce oxidative stress and neuron injury. Results: PRO-7a was not cytoprotective in C6 cells, but potentiated the H2O2-induced damage to cell integrity at a concentration lower than 0.38 μM. However, PRO-7a at 1.56 µM, on the other hand, modified H2O2-induced toxicity in PC12 cells by restoring cell integrity, mitochondrial metabolism, ROS generation, and arginase indirect activity. The α-Methyl-DL-aspartic acid (MDLA) and L-NΩ-Nitroarginine methyl ester (L-Name), specific inhibitors of AsS and nitric oxide synthase (NOS), which catalyzes the synthesis of polyamines and NO from L-arginine, did not suppress PRO-7a-mediated cytoprotection against oxidative stress. It suggested that its mechanism is independent of the production of L-arginine metabolites with neuroprotective properties by increased AsS activity. On the other hand, the neuroprotective effect of PRO-7a was blocked in the presence of dicyclomine hydrochloride (DCH), an M1 mAChR antagonist. Conclusions: For the first time, this work provides evidence that PRO-7a-induced neuroprotection seems to be mediated through M1 mAChR activation in PC12 cells, which reduces oxidative stress independently of AsS activity and L-arginine bioavailability.(AU)

Identification of a novel bombesin bioactive peptide QUB2995 and its insulin secreting activity / 中华内分泌代谢杂志

Objective:To identify a novel bombesin bioactive peptide from the skin secretion of Hylarana Latouchii, and to explore its effect on insulin secretion in islet cells.Methods:The skin secretion from Hylarana Latouchii was extracted by electrical stimulation, and the single chain of bombesin peptide was cloned and sequenced. The peptide QUB2995 was synthesized via solid-phase synthesis, then purified using reversed-phase high performance liquid chromatography (HPLC). Matrix assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF) was applied to validate. QPCR and ELISA were used to probe the effect of QUB2995 on insulin secretion in MIN6 and INS-1 cells.Results:A novel bombesin peptide named QUB2995 (GAFGDFLKGAAKA GALKILSIAQCKLSGTC) was found in the skin secretion of Hylarana Latouchii through molecular cloning. The bioactive peptide could significantly promote the proliferation and insulin secretion from mouse islet MIN6 cells and rat islet INS-1 cells. The effect reached a climax at the concentration of 10 -5 mol/L. Conclusion:A novel bombesin bioactive peptide named QUB2995 was found from Hylarana Latouchii. It could significantly promote insulin secretion in MIN6 cells of mouse islets and INS-1 cells of rat islets, indicating its potential in the treatment of diabetes.

Physical characteristics of nanoliposomes prepared from hydrolyzed cannabis protein

The present study was conducted on the protein extracted from the pulp that obtained by cannabis oil-pressingand loading the bioactive peptides into nanoliposomes. Physical properties of empty and loaded nanoliposemes(average particle size, polydispersity index, and zeta potential) and encapsulation efficiency were evaluated.Moreover, the effect of storage condition (fridge and ambient temperature) on the physical stability and maintainingthe encapsulation efficiency in nanoliposomes loaded with peptides was investigated. Eventually, the effect ofpeptide loading on the nanoparticle chemical structure Fourier-transform infrared (FTIR) as well as the morphologyof nanocarriers scanning electron microscopy (SEM) was evaluated. Physical properties of nanoliposomes wereaffected by the hydrolyzed type. The average particle size and polydispersity index of nanoliposomes varied from79.5 to 101.5 nm and 0.234 to 0.326, depending on the type of loaded peptides. The zeta potential of nanoliposomeswas changed from −10.32 to −15.33 mV when loaded with the peptide obtained by enzymatic hydrolysis during300 min. The efficiency of nanoliposomal encapsulation varied from 90.7 to 81.4%. Thus, peptides with the highestdegree of hydrolysis had the least encapsulation efficiency. The evaluation of physical stability and the maintenanceof encapsulation efficiency indicated the highest stability of nanoliposomes, which were stored at fridge temperature.FTIR spectroscopy in empty nanoliposomes and those loaded with peptides implied the presence of peptides inthe polar regions of phosphatidylcholine, such as the internal regions of the liposomes and the formation of ioniccomplexes between them. Conversely, SEM images of the structural and superficial properties of nanoliposomesindicated the existence of dense and compact clusters of the spherical nanoparticles with flat surfaces.

Bioprospection of proteases from Halobacillus andaensis for bioactive peptide production from fish muscle protein

BACKGROUND: Biologically active peptides produced from fish wastes are gaining attention because their health benefits. Proteases produced by halophilic microorganisms are considered as a source of active enzymes in high salt systems like fish residues. Hence, the aim of this study was the bioprospection of halophilic microorganisms for the production of proteases to prove their application for peptide production. RESULTS: Halophilic microorganisms were isolated from saline soils of Mexico and Bolivia. An enzymatic screening was carried out for the detection of lipases, esterases, pHB depolymerases, chitinases, and proteases. Most of the strains were able to produce lipases, esterases, and proteases, and larger hydrolysis halos were detected for protease activity. Halobacillus andaensis was selected to be studied for proteolytic activity production; the microorganism was able to grow on gelatin, yeast extract, skim milk, casein, peptone, fish muscle (Cyprinus carpio), and soy flour as protein sources, and among these sources, fish muscle protein was the best inducer of proteolytic activity, achieving a protease production of 571 U/mL. The extracellular protease was active at 50°C, pH 8, and 1.4 M NaCl and was inhibited by phenylmethylsulfonyl fluoride. The proteolytic activity of H. andaensis was used to hydrolyze fish muscle protein for peptide production. The peptides obtained showed a MW of 5.3 kDa and a radical scavenging ability of 10 to 30% on 2,2-diphenyl-1-picrylhydrazyl and 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) and a ferric reducing ability of plasma. Conclusion: The use of noncommercial extracellular protease produced by H. andaensis for biologically active peptide production using fish muscle as the protein source presents a great opportunity for high-value peptide production.

Purification and identification of novel cytotoxic oligopeptides from soft coral Sarcophyton glaucum / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Globally, peptide-based anticancer therapies have drawn much attention. Marine organisms are a reservoir of anticancer peptides that await discovery. In this study, we aimed to identify cytotoxic oligopeptides from Sarcophyton glaucum. Peptides were purified from among the S. glaucum hydrolysates produced by alcalase, chymotrypsin, papain, and trypsin, guided by a methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay on the human cervical cancer (HeLa) cell line for cytotoxicity evaluation. Purification techniques adopted were membrane ultrafiltration, gel filtration chromatography, solid phase extraction (SPE), and reversed-phase high-performance liquid chromatography (RP-HPLC). Purified peptides were identified by de novo peptide sequencing. From papain hydrolysate, three peptide sequences were identified: AGAPGG, AERQ, and RDTQ (428.45, 502.53, and 518.53 Da, respectively). Peptides synthesized from these sequences exhibited cytotoxicity on HeLa cells with median effect concentration (EC50) values of 8.6, 4.9, and 5.6 mmol/L, respectively, up to 5.8-fold stronger than the anticancer drug 5-fluorouracil. When tested at their respective EC50, AGAPGG, AERQ, and RDTQ showed only 16%, 25%, and 11% cytotoxicity to non-cancerous Hek293 cells, respectively. In conclusion, AERQ, AGAPGG, and RDTQ are promising candidates for future development as peptide-based anticancer drugs.

Purification and identification of novel cytotoxic oligopeptides from soft coral Sarcophyton glaucum / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Globally, peptide-based anticancer therapies have drawn much attention. Marine organisms are a reservoir of anticancer peptides that await discovery. In this study, we aimed to identify cytotoxic oligopeptides from Sarcophyton glaucum. Peptides were purified from among the S. glaucum hydrolysates produced by alcalase, chymotrypsin, papain, and trypsin, guided by a methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay on the human cervical cancer (HeLa) cell line for cytotoxicity evaluation. Purification techniques adopted were membrane ultrafiltration, gel filtration chromatography, solid phase extraction (SPE), and reversed-phase high-performance liquid chromatography (RP-HPLC). Purified peptides were identified by de novo peptide sequencing. From papain hydrolysate, three peptide sequences were identified: AGAPGG, AERQ, and RDTQ (428.45, 502.53, and 518.53 Da, respectively). Peptides synthesized from these sequences exhibited cytotoxicity on HeLa cells with median effect concentration (EC50) values of 8.6, 4.9, and 5.6 mmol/L, respectively, up to 5.8-fold stronger than the anticancer drug 5-fluorouracil. When tested at their respective EC50, AGAPGG, AERQ, and RDTQ showed only 16%, 25%, and 11% cytotoxicity to non-cancerous Hek293 cells, respectively. In conclusion, AERQ, AGAPGG, and RDTQ are promising candidates for future development as peptide-based anticancer drugs.

Research progress of bioactive peptide monocyte locomotion inhibitory factor / 药学实践杂志

Monocyte locomotion inhibitory factor(MLIF)was an anti‐inflammatory pentapeptide produced by Entamoeba histolytica .In vivo and in vitro study showed that MLIF displayed anti‐inflammatory and immune protection effects and MLIF had protective effects on rheumatoid arthritis ,nerve injury ,myocardial ischemia ,cerebral ischemia and Alzheimer′s disease . Studies had shown that MLIF regulated inflammatory response and immune protection through NF‐κB and MAPK signal path‐ways .The sources and biological activities of MLIF were reviewed in this paper .

Effect of biofunctionalized implant surface on osseointegration: a histomorphometric study in dogs

Among the different properties that influence bone apposition around implants, the chemical or biochemical composition of implant surface may interfere on its acceptance by the surrounding bone. The aim of this study was to investigate if a biofunctionalization of implant surface influences the bone apposition in a dog model and to compare it with other surfaces, such as a microstructured created by the grit-blasting/acid-etching process. Eight young adult male mongrel dogs had the bilateral mandibular premolars extracted and each one received 6 implants after 12 weeks, totaling 48 implants in the experiment. Four groups of implants were formed with the same microrough topography with or without some kind of biofunctionalization treatment. After histomorphometric analysis, it was observed that the modified microstructured surface with a "low concentration of the bioactive peptide" provided a higher adjacent bone density (54.6 percent) when compared to the other groups (microstructured + HA coating = 46.0 percent, microstructured only = 45.3 percent and microstructured + "high concentration of the bioactive peptide" = 40.7 percent), but this difference was not statistically significant. In conclusion, biofunctionalization of the implant surface might interfere in the bone apposition around implants, especially in terms of bone density. Different concentrations of bioactive peptide lead to different results.

Entre as diferentes propriedades de uma superfície capazes de influenciar a deposição óssea ao redor de implantes, a composição química e bioquímica pode atuar no reconhecimento do tecido ósseo circundante. O presente trabalho investigou a influência da biofuncionalização de superfícies de implante na deposição óssea ao redor dos mesmos em um modelo animal, comparando-as com outras superfícies, como a microtexturizada obtida pelo processo de jateamento e ataque ácido. Metodologicamente, os pré-molares mandibulares bilaterais de 8 cães foram extraídos e após 12 semanas foram instalados 6 implantes em cada cão, constituindo uma amostra de 48 implantes. Dos 4 grupos experimentais de diferentes superfícies, todos continham a mesma microtopografia rugosa, porém possuindo ou não alguma biofuncionalização. A análise histomorfométrica revelou que a superfície microtexturizada modificada pela adição de baixa concentração peptídica obteve uma maior densidade óssea adjacente (54,6 por cento) quando comparada aos outros grupos (microtexturizada + HA = 46 por cento, somente microtexturizada = 45,3 por cento e microtexturizada com adição de alta concentração peptídica = 40,7 por cento), no entanto estas diferenças numéricas não foram estatisticamente significantes. Dentro deste contexto, conclui-se que a biofuncionalização da superfície de implantes pode interferir na aposição óssea, em particular na densidade óssea, e que diferentes concentrações peptídicas podem conduzir a diferentes resultados.

Purification and Characterization of ?-AE Protein Expressed in E.coli / 中国生物工程杂志

Many bioactive peptides from neural and endocrine tissue are amidated at C-terminals,which is essential for their activities.The ?-amide comes from post-translational modification that is catalyzed by ?-AE (?-amidating enzyme) or PAM (pepdilylglycine ?-amidating monooxygenase).The gene encoding ?-AE was amplified with PCR and cloned into the plasmid pET-30a.After the recombinant plasmid pET-A was transformed into E.coli BL21,the ?-AE was expressed and purified by the Ni2+affinity chromatography,which has the ability catalyzing Dns-Tyr-Val-Gly to Dns-Tyr-Val-NH2.It identified that the recombinant protein producing by E.coli BL21 is ?-AE,which will benefit for studies of amidation at the C-terminals of peptides.