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1.
World J Microbiol Biotechnol ; 35(9): 133, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31432254

RESUMO

There is a significant increase in the discovery of new antimicrobial compounds in recent past to combat drug resistant pathogens. Members of the genus Bacillus and related genera have been screened extensively due to their ability to produce wide range of antimicrobial compounds. In this study, we have isolated and characterized a new antimicrobial peptide from a marine bacterium identified as Virgibacillus species. The low molecular mass and stability of the antimicrobial substance pointed towards the bacteriocinogenic nature of the compound. The RAST analysis of genome sequence showed presence of a putative bacteriocin biosynthetic cluster containing genes necessary for synthesis of a lanthipeptide. Translated amino acid sequence of mature C-terminal propeptide showed identity with salivaricin A (52.2%) and lacticin A (33.3%). Accordingly, the mass (2417 Da) obtained by MALDI analysis was in agreement with posttranslational modifications of the leader peptide to yield three methyl lanthionine rings and a disulfide bond between two free cysteine residues. The lanthipeptide was named as virgicin, which selectively inhibited the growth of Gram-positive bacteria and biofilm formation by Enterococcus faecalis. Inhibition of biofilm formation by E. faecalis was also observed in in vitro model experiments using hydroxyapatite discs. Thus, virgicin appears to be a promising new bacteriocin to control oral biofilm formation by selective pathogens.


Assuntos
Bacteriocinas/isolamento & purificação , Bacteriocinas/farmacologia , Enterococcus faecalis/efeitos dos fármacos , Enterococcus faecalis/crescimento & desenvolvimento , Peptídeos/isolamento & purificação , Peptídeos/farmacologia , Virgibacillus/metabolismo , Bacteriocinas/química , Bacteriocinas/genética , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Vias Biossintéticas/genética , Genoma Bacteriano , Peso Molecular , Família Multigênica , Peptídeos/química , Peptídeos/genética , Água do Mar/microbiologia , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Virgibacillus/classificação , Virgibacillus/isolamento & purificação
2.
Plant Foods Hum Nutr ; 74(3): 405-413, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31273642

RESUMO

The aim of this work was to evaluate the ability of broken rice, an underutilized industrial by-product, as a potential functional and health promoting ingredient. With this purpose, the ability to inhibit the angiotensin converting enzyme and renin of a rice protein hydrolyzate (RPH) obtained from a high-protein variety of broken rice (var. Nutriar FCAyF) was analyzed (IC50 = 0.87 and 2.7 mg/mL, respectively). RPH was separated by gel permeation chromatography and in a second purification step by RP-HPLC. The sequence of antihypertensive peptides presented in two RP-HPLC fractions was analyzed. Peptides capable of interacting with the active sites of both enzymes were identified. In this study, we demonstrate that the hydrolysis treatment improves functional and biological properties of rice proteins. Protein preparations obtained from a by-product of rice industry, such as broken rice, are a promising ingredient with potentially good biological properties.


Assuntos
Inibidores da Enzima Conversora de Angiotensina/farmacologia , Anti-Hipertensivos/isolamento & purificação , Oryza/química , Peptídeos/isolamento & purificação , Renina/antagonistas & inibidores , Anti-Hipertensivos/farmacologia , Cromatografia Líquida de Alta Pressão , Promoção da Saúde , Hidrólise , Simulação de Acoplamento Molecular , Peptídeos/farmacologia , Peptidil Dipeptidase A/metabolismo , Proteínas de Plantas/antagonistas & inibidores , Proteínas de Plantas/metabolismo , Renina/metabolismo
3.
J Agric Food Chem ; 67(32): 8855-8867, 2019 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-31343893

RESUMO

Abalone (Haliotis discus hannai) is a precious seafood in the market. It has been reported that biological active substances derived from abalone have anti-oxidative, anti-inflammatory, anti-bacterial, and anti-thrombosis potential. However, there were few studies to assess whether they have anti-cancer potential. In this study, we evaluated the anti-metastasis and anti-pro-angiogenic factors and mechanism of action of boiled abalone byproduct peptide (BABP, EMDEAQDPSEW) in human fibrosarcoma (HT1080) cells and human umbilical vein endothelial cells (HUVECs). The results demonstrated that BABP treatment significantly lowers migration and the invasion of HT1080 cells and HUVECs. BABP inhibits phorbol 12-myristate 13-acetate (PMA)-induced matrix metalloproteinase (MMP) expression and activity by blocking mitogen-activated protein kinases (MAPKs) and NF-κB signaling and hypoxia-induced vascular endothelial growth factor (VEGF) secretion and hypoxia inducible factor (HIF)-1α accumulation through suppressing the AKT/mTOR signal pathway. BABP treatment inhibits VEGF-induced VEGFR-2 expression and tube formation in HUVECs. The effect of BABP on anti-metastatic and anti-vascular activity in HT1080 cells and HUVECs revealed that BABP may be a potential pharmacophore for tumor therapy in the future.


Assuntos
Inibidores da Angiogênese/farmacologia , Antineoplásicos/farmacologia , Gastrópodes/química , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Peptídeos/farmacologia , Resíduos/análise , Inibidores da Angiogênese/química , Inibidores da Angiogênese/isolamento & purificação , Animais , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Metástase Neoplásica , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Neovascularização Patológica/fisiopatologia , Peptídeos/química , Peptídeos/isolamento & purificação , Frutos do Mar/análise , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
4.
World J Microbiol Biotechnol ; 35(6): 92, 2019 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-31187317

RESUMO

Polyketides and peptides obtained from actinobacteria are important therapeutic compounds which include front line antibiotics and anticancer drugs. Many screening programs are directed towards isolation of bioactive compounds from these organisms but the chances of finding novel antimicrobial leads among common actinobacteria are fast dwindling. As a result, the focus has shifted to the members of less exploited genera of rare actinobacteria. Three isolates, MMS8, MMS16 and KCR3 found to be potent polyketide and peptide producers were identified by 16S rRNA gene sequencing and their sequences deposited in the GenBank under the accession numbers MG407702, MG372012 and MG430204 respectively. MMS8 identified as Micromonospora auratinigra, yielded one potent compound determined to be chloroanthraquinone with an minimum inhibitory concentration (MIC) of 8 µg/ml against Bacillus subtilis and an IC50 value of 10 µg/ml and 4 µg/ml against HeLa and IMR cell lines respectively. This is the first report of the production of chloroanthraquinone by M. auratinigra. MMS16, identified as a member of the family Micromonosporaceae, yielded a potent compound MMS16B analyzed to be a novel bafilomycin analogue. The MIC of the compound was found to be 7 µg/ml against B.subtilis and IC50 value against HeLa and IMR was observed to be 9 µg/ml and 14 µg/ml respectively. MMS16B was also found to exhibit anti-quorum sensing (AQS) activity at sublethal concentrations. KCR3 identified as Kocuria kristinae yielded a novel antimicrobial peptide with antibacterial, antifungal and AQS activity. To the best of our knowledge, no antimicrobial activity has ever been reported from K. kristinae.


Assuntos
Actinobacteria/metabolismo , Peptídeos/metabolismo , Policetídeos/metabolismo , Actinobacteria/genética , Actinobacteria/isolamento & purificação , Animais , Antibacterianos/metabolismo , Anti-Infecciosos/metabolismo , Anti-Infecciosos/farmacologia , Antifúngicos/metabolismo , Antifúngicos/farmacologia , Antineoplásicos/metabolismo , Bacillus subtilis/efeitos dos fármacos , Linhagem Celular , Testes de Sensibilidade Microbiana , Micromonospora/genética , Micromonospora/isolamento & purificação , Micromonospora/metabolismo , Peptídeos/isolamento & purificação , Peptídeos/farmacologia , Policetídeos/isolamento & purificação , Policetídeos/farmacologia , RNA Ribossômico 16S/genética
5.
J Agric Food Chem ; 67(24): 6757-6764, 2019 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-31184153

RESUMO

In the present study we purified and identified peptides from broccoli protein hydrolysates and evaluated their angiotensin-converting enzyme (ACE) inhibitory activity in vitro and hypotensive effect in vivo. Three ACE inhibitory peptides were isolated and identified as IPPAYTK, LVLPGELAK, and TFQGPPHGIQVER, and their inhibitory IC50 values were 23.5, 184.0, and 3.4 µM, respectively. We then investigated the effect of gastrointestinal digestion on ACE inhibitory activity. We detected almost two times the ACE inhibitory activity of the peptide LVLPGELAK following simulated digestion than prior to digestion. LVLPGE and LAK, two novel peptides exhibiting high ACE inhibitory activity, were discovered following digestion and possessed IC50 values of 13.5 and 48.0 µM, respectively. The hypotensive effect of the peptides was assessed after oral administration to spontaneous hypertensive rats (SHRs). We found that LVLPGE and LAK demonstrated a significant hypotensive effect in vivo. Protein from broccoli may thus constitute a potential antihypertensive peptide source.


Assuntos
Inibidores da Enzima Conversora de Angiotensina/administração & dosagem , Anti-Hipertensivos/administração & dosagem , Brassica/química , Hipertensão/tratamento farmacológico , Peptídeos/administração & dosagem , Proteínas de Plantas/química , Administração Oral , Inibidores da Enzima Conversora de Angiotensina/química , Inibidores da Enzima Conversora de Angiotensina/isolamento & purificação , Animais , Anti-Hipertensivos/química , Anti-Hipertensivos/isolamento & purificação , Pressão Sanguínea/efeitos dos fármacos , Feminino , Humanos , Hipertensão/enzimologia , Hipertensão/fisiopatologia , Mapeamento de Peptídeos , Peptídeos/isolamento & purificação , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/metabolismo , Hidrolisados de Proteína/química , Ratos , Ratos Endogâmicos SHR
6.
J Agric Food Chem ; 67(29): 8149-8159, 2019 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-31246442

RESUMO

Ganoderma lucidum (G. lucidum) has been widely used in Asia to treat hypertension, but the active substances responsible for its antihypertensive effects remain unclear. Using the well-established angiotensin I-converting enzyme (ACE) as a target, we identified three ACE inhibitory peptides (ACEIPs), Gln-Leu-Val-Pro (QLVP), Gln-Asp-Val-Leu (QDVL), and Gln-Leu-Asp-Leu (QLDL), which account for the antihypertensive activity of G. lucidum. Notably, QLVP worked in a mixed-type manner against ACE with an IC50 value of 127.9 µmol/L. Molecular dynamics simulation suggested that the potent charge energy of QLVP, which interacted with Gln242 and Lys472 of ACE via a hydrogen bond and a salt bridge, potentially contributed to ACE inhibitory activity. Moreover, QLVP markedly activated angiotensin I-mediated phosphorylation of endothelial nitric oxide synthase in human umbilical vein endothelial cells and partly reduced mRNA and protein expression of the vasoconstrictor factor endothelin-1. This is the first report of the antihypertensive activity of small ACEIPs originating from G. lucidum mycelia, paving the way for the possible application of these peptides as potent drug candidates for treating hypertension.


Assuntos
Anti-Hipertensivos/química , Anti-Hipertensivos/isolamento & purificação , Peptídeos/química , Peptídeos/isolamento & purificação , Reishi/química , Sequência de Aminoácidos , Inibidores da Enzima Conversora de Angiotensina/química , Inibidores da Enzima Conversora de Angiotensina/isolamento & purificação , Linhagem Celular , Humanos , Cinética , Espectrometria de Massas , Simulação de Dinâmica Molecular , Micélio/química , Mapeamento de Peptídeos , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/metabolismo
7.
J Agric Food Chem ; 67(27): 7650-7659, 2019 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-31241944

RESUMO

Neutrase-hydrolysates hydrolyzed from mulberry leaf proteins were separated by ion exchange chromatography, gel filtration chromatography, and semipreparative reverse-phase HPLC. Purified fractions were analyzed for their radical scavenging activity, hemolysis inhibition ability, and cellular antioxidant activity (CAA). Three new antioxidant peptides, P1 (SVL, 317 Da), P2 (EAVQ, 445 Da), and P3 (RDY, 452 Da), were obtained from the most active HPLC fraction (R1) and identified using UPLC-QTOF-MS. These three peptides were then synthesized, and their antioxidant activities were analyzed. P1 and P2 had no ability to inhibit hemolysis of erythrocytes but did show antioxidant activity on HepG2 cells. P3 showed the highest hemolysis inhibition ability (92%) and CAA value (2204 µM QE/100 g peptide). The Tyr residues at the C-terminal region play an important role in the antioxidant activity in P3. Thus, the natural peptide R1 and synthesized P3 could be used as antioxidants and might be promising components of functional foods.


Assuntos
Antioxidantes/farmacologia , Hemólise/efeitos dos fármacos , Morus/química , Peptídeos/farmacologia , Folhas de Planta/química , Proteínas de Plantas/metabolismo , Cromatografia Líquida de Alta Pressão , Células Hep G2 , Humanos , Hidrólise , Fígado/efeitos dos fármacos , Metaloendopeptidases/metabolismo , Peso Molecular , Peptídeos/química , Peptídeos/isolamento & purificação
8.
Mar Drugs ; 17(5)2019 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-31058809

RESUMO

In the work, water-soluble proteins of red stingray (Dasyatis akajei) cartilages were extracted by guanidine hydrochloride and hydrolyzed using trypsin. Subsequently, four antioxidant peptides (RSHP-A, RSHP-B, RSHP-C, and RSHP-D) were isolated from the water-soluble protein hydrolysate while using ultrafiltration and chromatographic techniques, and the amino acid sequences of RSHP-A, RSHP-B, RSHP-C, and RSHP-D were identified as Val-Pro-Arg (VPR), Ile-Glu-Pro-His (IEPH), Leu-Glu-Glu--Glu-Glu (LEEEE), and Ile-Glu-Glu-Glu-Gln (IEEEQ), with molecular weights of 370.46 Da, 494.55 Da, 647.64 Da, and 646.66 Da, respectively. VPR, IEPH, LEEEE, and IEEEQ exhibited good scavenging activities on the DPPH radical (EC50 values of 4.61, 1.90, 3.69, and 4.01 mg/mL, respectively), hydroxyl radical (EC50 values of 0.77, 0.46, 0.70, and 1.30 mg/mL, respectively), superoxide anion radical (EC50 values of 0.08, 0.17, 0.15, and 0.16 mg/mL, respectively), and ABTS cation radical (EC50 values of 0.15, 0.11, 0.19, and 0.18 mg/mL, respectively). Among the four isolated antioxidant peptides, IEPH showed the strongest reducing power and lipid peroxidation inhibition activity, but LEEEE showed the highest Fe2+-chelating ability. The present results suggested that VPR, IEPH, LEEEE, and IEEEQ might have the possibility of being an antioxidant additive that is used in functional food and pharmaceuticals.


Assuntos
Antioxidantes/química , Antioxidantes/farmacologia , Peroxidação de Lipídeos/efeitos dos fármacos , Peptídeos/química , Peptídeos/farmacologia , Sequência de Aminoácidos , Animais , Antioxidantes/isolamento & purificação , Cartilagem/química , Radical Hidroxila , Peptídeos/isolamento & purificação , Hidrolisados de Proteína , Raias , Superóxidos
9.
Enzyme Microb Technol ; 127: 65-69, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31088619

RESUMO

Matrix metalloproteinases (MMPs) are zinc-dependent proteases involved in the degradation of extracellular matrix proteins. As one of the isoforms, MMP-1 breaks down collagen, and its activity is known to be important in wound healing. Its timely and adequate level of expression is pivotal because MMP-1 is also involved in the damage or aging of skins as well as in certain types of cancers. Thus, both assaying the MMP-1 activity and developing its inhibitors are of great importance. We here developed an in-house assay system that gave us the high degree of freedom in screening peptide inhibitors of MMP-1. The assay system utilized a circularly permutated fusion of ß-lactamase and its inhibitory protein through an MMP-1-sensitive linker so that the activity of MMP-1 could be translated into that of ß-lactamase. As a proof of concept, we applied the developed assay system to initial screens of MMP-1 inhibitors and successfully identified one lead peptide that inhibited the collagenase activity of the enzyme.


Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Metaloproteinase 1 da Matriz/análise , Inibidores de Metaloproteinases de Matriz/isolamento & purificação , Inibidores de Metaloproteinases de Matriz/farmacologia , Peptídeos/isolamento & purificação , Peptídeos/farmacologia
10.
Mar Drugs ; 17(5)2019 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-31035632

RESUMO

In this report, protein hydrolysate (TGH) of blood cockle (Tegillarca granosa) was prepared using a two-enzyme system (Alcalase treatment for 1.5 h following Neutrase treatment for 1.5 h). Subsequently, six antioxidant peptides were isolated from TGH using ultrafiltration and chromatography methods, and their amino acid sequences were identified as EPLSD, WLDPDG, MDLFTE, WPPD, EPVV, and CYIE with molecular weights of 559.55, 701.69, 754.81, 513.50, 442.48, and 526.57 Da, respectively. In which, MDLFTE and WPPD exhibited strong scavenging activities on DPPH radical (EC50 values of 0.53 ± 0.02 and 0.36 ± 0.02 mg/mL, respectively), hydroxy radical (EC50 values of 0.47 ± 0.03 and 0.38 ± 0.04 mg/mL, respectively), superoxide anion radical (EC50 values of 0.75 ± 0.04 and 0.46 ± 0.05 mg/mL, respectively), and ABTS cation radical (EC50 values of 0.96 ± 0.08 and 0.54 ± 0.03 mg/mL, respectively). Moreover, MDLFTE and WPPD showed high inhibiting ability on lipid peroxidation. However, MDLFTE and WPPD were unstable and could not retain strong antioxidant activity at high temperatures (>80 °C for 0.5 h), basic pH conditions (pH > 9 for 2.5 h), or during simulated GI digestion. In addition, the effect of simulated gastrointestinal digestion on TGP4 was significantly weaker than that on MDLFTE. Therefore, MDLFTE and WPPD may be more suitable for serving as nutraceutical candidates in isolated forms than as food ingredient candidates in functional foods and products.


Assuntos
Organismos Aquáticos , Bivalves , Depuradores de Radicais Livres/farmacologia , Peptídeos/farmacologia , Hidrolisados de Proteína/química , Sequência de Aminoácidos , Animais , Suplementos Nutricionais , Depuradores de Radicais Livres/química , Depuradores de Radicais Livres/isolamento & purificação , Alimento Funcional , Temperatura Alta , Concentração de Íons de Hidrogênio , Peroxidação de Lipídeos/efeitos dos fármacos , Peptídeos/química , Peptídeos/isolamento & purificação , Hidrolisados de Proteína/isolamento & purificação
11.
J Chromatogr A ; 1600: 158-166, 2019 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-31040030

RESUMO

This study is concerned with a chromatography-based approach (Immobilized Metal Ion Affinity Chromatography) for the recovery of gallium binding peptide sequences from a recombinant phage display library. The here described methods apply the fundamental knowledge and methods of separation science and meet thereby the key requirement of the phage display technique of precise separation of target-binding bacteriophage clones from non-interacting bacteriophage during the biopanning. During the chromatopanning called process, a total of 101 bacteriophage clones were identified of which in subsequent binding experiments, phage clones expressing the peptide sequences TMHHAAIAHPPH, SQALSTSRQDLR and HTQHIQSDDHLA were characterized to bind >10 fold better to a target that presents immobilized gallium ions than control phage, displaying no peptide sequence. The performance of biopanning experiments in chromatographic systems is particularly suitable for demanding targets such as trivalent metal ions. We found, that the selection process benefits immensely from the stable immobilization of the target metal ions during the entire biopanning process as well as the complete recovery of well interacting bacteriophage clones. Among others, this was possible due to an enhanced monitoring of process conditions and fractionation of eluates.


Assuntos
Técnicas de Química Analítica/métodos , Cromatografia de Afinidade , Gálio/química , Peptídeos/química , Sequência de Aminoácidos , Biblioteca de Peptídeos , Peptídeos/isolamento & purificação
12.
J Agric Food Chem ; 67(20): 5772-5781, 2019 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-31046268

RESUMO

This study aimed to purify and identify antioxidant peptides from the low-molecular-weight fraction (SPH-I, MW < 3 kDa) of Alcalase-hydrolyzed soybean ( Glycine max L.) hydrolysate and further evaluate the cytoprotective effects of synthesized peptides against oxidative stress in human intestinal Caco-2 cells. After purification by gel filtration chromatography and reversed-phase HPLC, four major peptides were sequenced by nano-LC-ESI-MS/MS as VVFVDRL (847 Da, SPH-IA), VIYVVDLR (976 Da, SPH-IB), IYVVDLR (877 Da, SPH-IC), and IYVFVR (795 Da, SPH-ID). The antioxidant peptides were synthesized and displayed desirable DPPH radical-scavenging activity (from 16.5 ± 0.5 to 20.3 ± 1.0 µM Trolox equivalent (TE)/µM), ABTS•+ radical-scavenging activity (from 3.42 ± 0.2 to 4.24 ± 0.4 mM TE/µM), ORAC (from 143 ± 2.1 to 171 ± 4.8 µM TE/µM), and FRAP (from 54.7 ± 1.2 to 79.0 ± 0.6 mM Fe2+/µM). Moreover, the synthesized peptides protected Caco-2 cells against H2O2-induced oxidative damage via significantly downregulating intracellular ROS generation and lipid peroxidation ( p < 0.05). Additionally, SPH-IC and SPH-ID statistically upregulated total reduced glutathione synthesis, enhanced activities of catalase and glutathione reductase, and suppressed ROS-mediated inflammatory responses via inhibiting interleukin-8 secretion ( p < 0.05).


Assuntos
Antioxidantes/química , Antioxidantes/farmacologia , Peptídeos/química , Peptídeos/farmacologia , Substâncias Protetoras/química , Substâncias Protetoras/farmacologia , Soja/química , Antioxidantes/isolamento & purificação , Biocatálise , Células CACO-2 , Sobrevivência Celular/efeitos dos fármacos , Glutationa/metabolismo , Humanos , Peróxido de Hidrogênio/toxicidade , Hidrólise , Intestinos/citologia , Intestinos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Peptídeos/isolamento & purificação , Substâncias Protetoras/isolamento & purificação , Subtilisinas/química
13.
Phytochemistry ; 164: 136-143, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31128493

RESUMO

The increasing resistance of pathogenic fungi to conventional antifungal therapies is a major global health concern. Currently, antifungal peptides are receiving increasing attention as suitable candidates for antifungal drug discovery. In the present study, an antifungal peptide was isolated from Satureja khuzistanica by reverse phase-HPLC column and sequenced by de novo sequencing and Edman degradation. The peptide cytotoxicity on human red blood cells and HEK293 cells was assessed using hemolytic and MTT assays. The purified peptide had 25 amino acids with pI and net charge equal to 9.31 and + 2, respectively. According to the systematic nomenclature, this peptide was named Skh-AMP1. The peptide showed strong antifungal activity against pathogenic species of Aspergillus and Candida with MIC values of 19.8-23.4 µM and MFC values of 39.6-58.5 µM. Molecular modeling analysis predicted a α-helix conformation for Skh-AMP1 and the probable hydrophilic residues and hydrophobic regions in the peptide structure which may responsible for its antifungal activity. Skh-AMP1 preserved its stability at the pH of 7 and 8 and the temperatures of 30 and 40 °C. The peptide showed negligible hemolytic activity in the range of 0.19-2.1% at the concentrations of 3.6-72 µM. It has no obvious cytotoxicity against HEK293 cells at the MIC of 25.2 µM for the fungal growth. All together, these properties make Skh-AMP1 as a previously undescribed peptide a promising potential therapeutic agent to combat immerging fungal infections.


Assuntos
Antifúngicos/farmacologia , Aspergillus fumigatus/efeitos dos fármacos , Peptídeos/farmacologia , Folhas de Planta/química , Satureja/química , Antifúngicos/química , Antifúngicos/isolamento & purificação , Aspergillus fumigatus/crescimento & desenvolvimento , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Testes de Sensibilidade Microbiana , Conformação Molecular , Peptídeos/química , Peptídeos/isolamento & purificação , Relação Estrutura-Atividade
14.
Artigo em Inglês | MEDLINE | ID: mdl-31132734

RESUMO

The technique of Solid-Phase Extraction (SPE) is widely used in various fields to concentrate samples and the search for tools to improve recoveries remains of outmost importance. The use of polymer based cartridges has become prevailing in a broad range of fields to enrich peptides from biological matrices. However, the existing SPE protocols are characterized by disparity. Ion-pairing (IP) reagents are commonly used in chromatographic applications, but their combination with SPE is less known. The aim of this study was to evaluate various SPE loading conditions, including the use of IP reagents, to improve the recoveries of nine selected peptide molecules. Control of pH and the use of IP reagents were found to be crucial to improve the enrichment of the peptides, especially cationic peptides, for which an up to ten-fold increase was observed. The practical potential of the presented theoretical findings were verified by employing IP-SPE for the development of an efficient extraction method for the doping relevant peptide Synacthen. The general proof of principle was obtained by analysis of excretion study urine samples and validation was performed with focus on the limit of detection (20 pg/ml) and recovery (37%).


Assuntos
Peptídeos/isolamento & purificação , Peptídeos/urina , Extração em Fase Sólida/métodos , Cromatografia Líquida de Alta Pressão , Humanos , Concentração de Íons de Hidrogênio , Limite de Detecção , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem
15.
J Chromatogr A ; 1598: 92-100, 2019 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-30961963

RESUMO

A strategy for determining a suitable solvent gradient in silico in preparative peptide separations is presented. The strategy utilizes a machine-learning-based method, called ELUDE, for peptide retention time predictions based on the amino acid sequences of the peptides. A suitable gradient is calculated according to linear solvent strength theory by predicting the retention times of the peptides being purified at three different gradient slopes. The advantage of this strategy is that fewer experiments are needed to develop a purification method, making it useful for labs conducting many separations but with limited resources for method development. The preparative separation of met-enkephalin and leu-enkephalin was used as model solutes on two stationary phases: XBridge C18 and CSH C18. The ELUDE algorithm contains a support vector regression and is pre-trained, meaning that only 10-50 peptides are needed to calibrate a model for a certain stationary phase and gradient. The calibration is done once and the model can then be used for new peptides similar in size to those in the calibration set. We found that the accuracy of the retention time predictions is good enough to usefully estimate a suitable gradient and that it was possible to compare the selectivity on different stationary phases in silico. The absolute relative errors in retention time for the predicted gradients were 4.2% and 3.7% for met-enkephalin and leu-enkephalin, respectively, on the XBridge C18 column and 2.0% and 2.8% on the CSH C18 column. The predicted retention times were also used as initial values for adsorption isotherm parameter determination, facilitating the numerical calculation of overloaded elution profiles. Changing the trifluoroacetic acid (TFA) concentration from 0.05% to 0.15% in the eluent did not seriously affect the error in the retention time predictions for the XBridge C18 column, an increase of 1.0 min (in retention factor, 1.3). For the CSH C18 column the error was, on average, 2.6 times larger. This indicates that the model needs to be recalibrated when changing the TFA concentration for the CSH column. Studying possible scale-up complications from UHPLC to HPLC such as pressure, viscous heating (i.e., temperature gradients), and stationary-phase properties (e.g., packing heterogeneity and surface chemistry) revealed that all these factors were minor to negligible. The pressure effect had the largest effect on the retention, but increased retention by only 3%. In the presented case, method development can therefore proceed using UHPLC and then be robustly transferred to HPLC.


Assuntos
Técnicas de Química Analítica/métodos , Cromatografia de Fase Reversa , Aprendizado de Máquina , Peptídeos/isolamento & purificação , Adsorção , Peptídeos/química , Solventes/química , Temperatura Ambiente , Tempo , Ácido Trifluoracético/química
16.
Int J Mol Sci ; 20(7)2019 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-30965558

RESUMO

The growing integration of quality-by-design (QbD) concepts in biomanufacturing calls for a detailed and quantitative knowledge of the profile of impurities and their impact on the product safety and efficacy. Particularly valuable is the determination of the residual level of host cell proteins (HCPs) secreted, together with the product of interest, by the recombinant cells utilized for production. Though often referred to as a single impurity, HCPs comprise a variety of species with diverse abundance, size, function, and composition. The clearance of these impurities is a complex issue due to their cell line to cell line, product-to-product, and batch-to-batch variations. Improvements in HCP monitoring through proteomic-based methods have led to identification of a subset of "problematic" HCPs that are particularly challenging to remove, both at the product capture and product polishing steps, and compromise product stability and safety even at trace concentrations. This paper describes the development of synthetic peptide ligands capable of capturing a broad spectrum of Chinese hamster ovary (CHO) HCPs with a combination of peptide species that allow for advanced mixed-mode binding. Solid phase peptide libraries were screened for identification and characterization of peptides that capture CHO HCPs while showing minimal binding of human IgG, utilized here as a model product. Tetrameric and hexameric ligands featuring either multipolar or hydrophobic/positive amino acid compositions were found to be the most effective. Tetrameric multipolar ligands exhibited the highest targeted binding ratio (ratio of HCP clearance over IgG loss), more than double that of commercial mixed-mode and anion exchange resins utilized by industry for IgG polishing. All peptide resins tested showed preferential binding to HCPs compared to IgG, indicating potential uses in flow-through mode or weak-partitioning-mode chromatography.


Assuntos
Peptídeos/isolamento & purificação , Animais , Células CHO , Cromatografia de Afinidade , Cromatografia Líquida , Cricetinae , Cricetulus , Humanos , Peptídeos/química , Proteômica/métodos
17.
J Sci Food Agric ; 99(11): 5112-5121, 2019 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-30982967

RESUMO

BACKGROUND: Salmon bones, a waste by-product from the salmon industry, were used as a protein hydrolysate source for the production of bioactive peptides. The aim of this work was to evaluate the potential antioxidant and anti-inflammatory properties of salmon bone protein hydrolysate (SBPH). RESULTS: Salmon bones were hydrolyzed by separately using one of four proteases (Alcalase, Favourzyme, Neutrase and papain) at various concentrations (10, 25 and 50 mg mL-1 ), where the SBPH derived from 10 mg mL-1 papain hydrolysis exhibited the highest nitric oxide (NO) radical scavenging activity. After ultrafiltration, the MW < 0.65 kDa fraction showed the strongest NO inhibitory activity and was further fractionated by gel filtration chromatography (G1 and G2 fractions) and reverse-phase high-performance liquid chromatographic fractionation of the G1 fraction, from which the three main peaks (H1, H2 and H3) were found to have a marked NO-inhibitory activity and their peptide sequences were determined. Moreover, the G1 fraction was shown to inhibit both the lipopolysaccharide (LPS)-induced NO production and the LPS-induced inducible NO synthase , interleukin-6, tumor necrosis factor-α and induced NO production and the LPSCOX-2 mRNA levels in RAW 264.7 cells. CONCLUSIONS: Salmon bones from the salmon fisheries and farming industry were utilized by enzymatic hydrolysis for the production of valuable peptides. The results of this study suggested that bioactive peptides derived from salmon bones would be alternative anti-inflammation materials in functional resources. © 2019 Society of Chemical Industry.


Assuntos
Anti-Inflamatórios/farmacologia , Osso e Ossos/química , Proteínas de Peixes/química , Depuradores de Radicais Livres/farmacologia , Hidrolisados de Proteína/farmacologia , Salmão , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/isolamento & purificação , Biocatálise , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Depuradores de Radicais Livres/química , Depuradores de Radicais Livres/isolamento & purificação , Hidrólise , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Óxido Nítrico/imunologia , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/imunologia , Peptídeo Hidrolases/química , Peptídeos/química , Peptídeos/isolamento & purificação , Peptídeos/farmacologia , Hidrolisados de Proteína/química , Células RAW 264.7
18.
Molecules ; 24(7)2019 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-30987412

RESUMO

In this study, a 8.5-kDa antifungal peptide designated as BGAP was purified from the crude extract of the seeds of Brassica oleracea var. gongylodes by employing a protocol that comprised cation exchange chromatography on SP-Sepharose, cation exchange chromatography on Mono S and gel filtration chromatography on Superdex peptide. BGAP showed the highest amino acid sequence similarity to defensin peptides by mass spectrometric analysis. BGAP showed a broad spectrum of antifungal activity with a half maximal inhibitory concentration at 17.33 µg/mL, 12.37 µg/mL, 16.81 µg/mL, and 5.60 µg/mL toward Colletotrichum higginsianum, Exserohilum turcicum, Magnaporthe oryzae and Mycosphaerella arachidicola, respectively. The antifungal activity of BGAP remained stable (i) after heat treatment at 40-100 °C for 15 min; (ii) after exposure to solutions of pH 1-3 and 11-13 for 15 min; (iii) after incubation with solutions containing K⁺, Ca2+, Mg2+, Mn2+ or Fe3+ ions at the concentrations of 20-150 mmol/L for 2 h; and (iv) following treatment with 10% methyl alcohol, 10% ethanol, 10% isopropanol or 10% chloroform for 2 h. Fluorescence staining experiments showed that BGAP brought about an increase in cell membrane permeability, a rise in reactive oxygen species production, a decrease in mitochondrial membrane potential, and an accumulation of chitin at the hyphal tips of Mycosphaerella arachidicola.


Assuntos
Antifúngicos/química , Antifúngicos/farmacologia , Brassica/química , Peptídeos/química , Peptídeos/farmacologia , Sementes/química , Antifúngicos/isolamento & purificação , Concentração Inibidora 50 , Testes de Sensibilidade Microbiana , Peptídeos/isolamento & purificação , Fenótipo , Espécies Reativas de Oxigênio , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
19.
Int J Mol Sci ; 20(8)2019 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-31027239

RESUMO

BACKGROUND: Human epidermal growth factor (hEGF) has drawn intense research attention due to its potential ability to promote healing of serious injuries, such as cuts, burns, and diabetic ulcers. Although hEGF displays prospective clinical value, the growth factor is restricted to the treatment of chronic diabetic ulcers because of its high production cost. METHODS: Leguminous plant peanut (Arachis hypogaea L.) hairy roots contain relatively few toxic and harmful substances, and tested as an excellent production system for hEGF in our study. To explore the possibility of hEGF expression in peanut, hEGF overexpression hairy roots were obtained by infecting leaves with Agrobacterium rhizogenes R1601. RESULTS: The maximum transgenic hairy roots inducing rate was 82%. Protein purification and mass spectrometry assays showed that the protein expressed in peanut hairy roots was identified as hEGF. Furthermore, Methylthiazolyldiphenyl-tetrazolium bromide assay showed that hEGF promoted HL-7702 liver cells proliferation, which indicate that hEGF has biological activity and non-toxic on human cells. CONCLUSION: Our results demonstrate the capacity of peanut hairy root cultures as a controlled, sustainable, and scalable production system that can be induced to produce valued human proteins, such as hEGF.


Assuntos
Arachis/genética , Fator de Crescimento Epidérmico/genética , Fator de Crescimento Epidérmico/isolamento & purificação , Expressão Gênica , Sequência de Aminoácidos , Fator de Crescimento Epidérmico/química , Humanos , Peptídeos/química , Peptídeos/isolamento & purificação , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas
20.
Int J Mol Sci ; 20(8)2019 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-31009989

RESUMO

The valorization of by-products from natural organic sources is an international priority to respond to environmental and economic challenges. In this context, electrodialysis with filtration membrane (EDFM), a green and ultra-selective process, was used to separate peptides from salmon frame protein hydrolysate. For the first time, the simultaneous separation of peptides by three ultrafiltration membranes of different molecular-weight exclusion limits (50, 20, and 5 kDa) stacked in an electrodialysis system, allowed for the generation of specific cationic and anionic fractions with different molecular weight profiles and bioactivity responses. Significant decreases in peptide recovery, yield, and molecular weight (MW) range were observed in the recovery compartments depending on whether peptides had to cross one, two, or three ultrafiltration membranes. Moreover, the Cationic Recovery Compartment 1 fraction demonstrated the highest increase (42%) in glucose uptake on L6 muscle cells. While, in the anionic configuration, both Anionic Recovery Compartment 2 and Anionic Recovery Compartment 3 fractions presented a glucose uptake response in basal condition similar to the insulin control. Furthermore, Cationic Recovery Compartment 3 was found to contain inhibitory peptides. Finally, LC-MS analyses of the bioassay-guided bioactive fractions allowed us to identify 11 peptides from salmon by-products that are potentially responsible for the glucose uptake improvement.


Assuntos
Glucose/metabolismo , Peptídeos/isolamento & purificação , Hidrolisados de Proteína/isolamento & purificação , Salmão/metabolismo , Animais , Ânions , Cátions , Linhagem Celular , Diálise , Camundongos , Peso Molecular , Termodinâmica , Ultrafiltração
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