Human HELQ regulates DNA end resection at DNA double-strand breaks and stalled replication forks.

Following a DNA double strand break (DSB), several nucleases and helicases coordinate to generate single-stranded DNA (ssDNA) with 3' free ends, facilitating precise DNA repair by homologous recombination (HR). The same nucleases can act on stalled replication forks, promoting nascent DNA degradation and fork instability. Interestingly, some HR factors, such as CtIP and BRCA1, have opposite regulatory effects on the two processes, promoting end resection at DSB but inhibiting the degradation of nascent DNA on stalled forks. However, the reason why nuclease actions are regulated by different mechanisms in two DNA metabolism is poorly understood. We show that human HELQ acts as a DNA end resection regulator, with opposing activities on DNA end resection at DSBs and on stalled forks as seen for other regulators. Mechanistically, HELQ helicase activity is required for EXO1-mediated DSB end resection, while ssDNA-binding capacity of HELQ is required for its recruitment to stalled forks, facilitating fork protection and preventing chromosome aberrations caused by replication stress. Here, HELQ synergizes with CtIP but not BRCA1 or BRCA2 to protect stalled forks. These findings reveal an unanticipated role of HELQ in regulating DNA end resection at DSB and stalled forks, which is important for maintaining genome stability.

Dynamic switching of crotonylation to ubiquitination of H2A at lysine 119 attenuates transcription-replication conflicts caused by replication stress.

The reversible post-translational modification (PTM) of proteins plays an important role in many cellular processes. Lysine crotonylation (Kcr) is a newly identified PTM, but its functional significance remains unclear. Here, we found that Kcr is involved in the replication stress response. We show that crotonylation of histone H2A at lysine 119 (H2AK119) and ubiquitination of H2AK119 are reversibly regulated by replication stress. Decrotonylation of H2AK119 by SIRT1 is a prerequisite for subsequent ubiquitination of H2AK119 by BMI1. Accumulation of ubiquitinated H2AK119 at reversed replication forks leads to the release of RNA Polymerase II and transcription repression in the vicinity of stalled replication forks. These effects attenuate transcription-replication conflicts (TRCs) and TRC-associated R-loop formation and DNA double-strand breaks. These findings suggest that decrotonylation and ubiquitination of H2A at lysine 119 act together to resolve replication stress-induced TRCs and protect genome stability.

Angiotensin-I-Converting Enzyme (ACE)-Inhibitory Peptides from the Collagens of Monkfish (Lophius litulon) Swim Bladders: Isolation, Characterization, Molecular Docking Analysis and Activity Evaluation.

The objective of this study was to isolate and characterize collagen and angiotensin-I-converting enzyme (ACE)-inhibitory (ACEi) peptides from the swim bladders of monkfish (Lophius litulon). Therefore, acid-soluble collagen (ASC-M) and pepsin-soluble collagen (PSC-M) with yields of 4.27 ± 0.22% and 9.54 ± 0.51%, respectively, were extracted from monkfish swim bladders using acid and enzyme methods. The ASC-M and PSC-M contained Gly (325.2 and 314.9 residues/1000 residues, respectively) as the major amino acid, but they had low imino acid content (192.5 and 188.6 residues/1000 residues, respectively) in comparison with collagen from calf skins (CSC) (216.6 residues/1000 residues). The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns and ultraviolet (UV) absorption spectrums of ASC-M and PSC-M illustrated that they were mainly composed of type I collagen. Subsequently, three ACEi peptides were isolated from a PSC-M hydrolysate prepared via a double-enzyme system (alcalase + neutrase) and identified as SEGPK (MHP6), FDGPY (MHP7) and SPGPW (MHP9), with molecular weights of 516.5, 597.6 and 542.6 Da, respectively. SEGPK, FDGPY and SPGPW displayed remarkable anti-ACE activity, with IC50 values of 0.63, 0.94 and 0.71 mg/mL, respectively. Additionally, a molecular docking assay demonstrated that the affinities of SEGPK, FDGPY and SPGPW with ACE were -7.3, -10.9 and -9.4 kcal/mol, respectively. The remarkable ACEi activity of SEGPK, FDGPY and SPGPW was due to their connection with the active pockets and/or sites of ACE via hydrogen bonding, hydrophobic interaction and electrostatic force. Moreover, SEGPK, FDGPY and SPGPW could protect HUVECs by controlling levels of nitric oxide (NO) and endothelin-1 (ET-1). Therefore, this work provides an effective means for the preparation of collagens and novel ACEi peptides from monkfish swim bladders, and the prepared ACEi peptides, including SEGPK, FDGPY and SPGPW, could serve as natural functional components in the development of health care products to control hypertension.

CtIP suppresses primary microRNA maturation and promotes metastasis of colon cancer cells in a xenograft mouse model.

miRNAs are important regulators of eukaryotic gene expression. The post-transcriptional maturation of miRNAs is controlled by the Drosha-DiGeorge syndrome critical region gene 8 (DGCR8) microprocessor. Dysregulation of miRNA biogenesis has been implicated in the pathogenesis of human diseases, including cancers. C-terminal-binding protein-interacting protein (CtIP) is a well-known DNA repair factor that promotes the processing of DNA double-strand break (DSB) to initiate homologous recombination-mediated DSB repair. However, it was unclear whether CtIP has other unknown cellular functions. Here, we aimed to uncover the roles of CtIP in miRNA maturation and cancer cell metastasis. We found that CtIP is a potential regulatory factor that suppresses the processing of miRNA primary transcripts (pri-miRNA). CtIP directly bound to both DGCR8 and pri-miRNAs through a conserved Sae2-like domain, reduced the binding of Drosha to DGCR8 and pri-miRNA substrate, and inhibited processing activity of Drosha complex. CtIP depletion significantly increased the expression levels of a subset of mature miRNAs, including miR-302 family members that are associated with tumor progression and metastasis in several cancer types. We also found that CtIP-inhibited miRNAs, such as miR-302 family members, are not crucial for DSB repair. However, increase of miR-302b levels or loss of CtIP function severely suppressed human colon cancer cell line tumor cell metastasis in a mouse xenograft model. These studies reveal a previously unrecognized mechanism of CtIP in miRNA processing and tumor metastasis that represents a new function of CtIP in cancer.

Preparation, Identification, Molecular Docking Study and Protective Function on HUVECs of Novel ACE Inhibitory Peptides from Protein Hydrolysate of Skipjack Tuna Muscle.

To prepare bioactive peptides with high angiotensin-I-converting enzyme (ACE)-inhibitory (ACEi) activity, Alcalase was selected from five kinds of protease for hydrolyzing Skipjack tuna (Katsuwonus pelamis) muscle, and its best hydrolysis conditions were optimized using single factor and response surface experiments. Then, the high ACEi protein hydrolysate (TMPH) of skipjack tuna muscle was prepared using Alcalase under the optimum conditions of enzyme dose 2.3%, enzymolysis temperature 56.2 °C, and pH 9.4, and its ACEi activity reached 72.71% at 1.0 mg/mL. Subsequently, six novel ACEi peptides were prepared from TMPH using ultrafiltration and chromatography methods and were identified as Ser-Pro (SP), Val-Asp-Arg-Tyr-Phe (VDRYF), Val-His-Gly-Val-Val (VHGVV), Tyr-Glu (YE), Phe-Glu-Met (FEM), and Phe-Trp-Arg-Val (FWRV), with molecular weights of 202.3, 698.9, 509.7, 310.4, 425.6, and 606.8 Da, respectively. SP and VDRYF displayed noticeable ACEi activity, with IC50 values of 0.06 ± 0.01 and 0.28 ± 0.03 mg/mL, respectively. Molecular docking analysis illustrated that the high ACEi activity of SP and VDRYF was attributed to effective interaction with the active sites/pockets of ACE by hydrogen bonding, electrostatic force, and hydrophobic interaction. Furthermore, SP and VDRYF could significantly up-regulate nitric oxide (NO) production and down-regulate endothelin-1 (ET-1) secretion in HUVECs after 24 h treatment, but also abolish the negative effect of 0.5 µM norepinephrine (NE) on the generation of NO and ET-1. Therefore, ACEi peptides derived from skipjack tuna (K. pelamis) muscle, especially SP and VDRYF, are beneficial components for functional food against hypertension and cardiovascular diseases.

OsMAPK6 phosphorylates a zinc finger protein OsLIC to promote downstream OsWRKY30 for rice resistance to bacterial blight and leaf streak.

Rice OsLIC encoding a CCCH zinc finger transcription factor plays an important role in immunity. However, the immune signaling pathways that OsLIC-involved and the underlying mechanisms that OsLIC-conferred resistance against pathogens are largely unclear. Here, we show that OsLIC, as a substrate for OsMAPK6, negatively regulates resistance to Xanthomonas oryzae pv. oryzae (Xoo) and X. oryzae pv. oryzicola (Xoc) by directly suppressing OsWRKY30 transcription. Biochemical assays showed that OsLIC bound to OsWRKY30 promoter and suppressed its transcription. Genetic assays confirmed that the osilc knockout mutants and OsWRKY30-overexpressing plants exhibited enhanced resistance to Xoo and Xoc, knocking out OsWRKY30 in the oslic mutants attenuated the resistance against bacterial pathogens. OsMAPK6 physically interacted with and phosphorylated OsLIC leading to decreased OsLIC DNA-binding activity, therefore, overexpression of OsLIC partially suppressed OsMAPK6-mediated rice resistance. In addition, both OsMAPK6-phosphorylated activation of OsLIC and phosphorylation-mimic OsLIC5D had reduced DNA-binding activity towards OsWRKY30 promoter, thereby promoting OsWRKY30 transcription. Collectively, these results reveal that OsMAPK6-mediated phosphorylation of OsLIC positively regulates rice resistance to Xoo and Xoc by modulating OsWRKY30 transcription, suggesting that OsMAPK6-OsLIC-OsWRKY30 module is an immune signaling pathway in response to the bacterial pathogens.

PLK1 targets CtIP to promote microhomology-mediated end joining.

Proper DNA double-strand break (DSB) repair is essential for maintaining genome integrity. Microhomology-mediated end joining (MMEJ) is an error-prone repair mechanism, which introduces mutations at break sites and contributes to chromosomal translocations and telomere fusions, thus driving carcinogenesis. Mitotic kinases PLK1, CDK1 and Aurora A are important for supporting MMEJ and are often overexpressed in various tumors. However, the functional interplay between these kinases and MMEJ has not been explored. Here, we found that MMEJ is preferentially employed to fix DSBs in cells arrested in mitosis following nocodazole treatment. We further showed that the DSB repair factor CtIP is jointly phosphorylated by CDK1/Aurora A and PLK1. CDK1/Aurora A-mediated CtIP phosphorylation at serine 327 triggers CtIP binding to the PLK1 polo-box domain, which in turn facilitates PLK1 to phosphorylate CtIP mainly at serine 723. A PLK1 phosphor-mimic CtIP mutant fails to initiate extended end resection and is thus unable to mediate homologous recombination and the G2/M checkpoint but can mediate MMEJ. These data imply that PLK1 may target CtIP to promote error-prone MMEJ and inactivate the G2/M checkpoint. These findings have helped elucidate the oncogenic roles of these factors.

Antioxidant Peptides from Collagen Hydrolysate of Redlip Croaker (Pseudosciaena polyactis) Scales: Preparation, Characterization, and Cytoprotective Effects on H2O2-Damaged HepG2 Cells.

Bioactive peptides from fish collagens with antioxidant properties have become a topic of great interest for health, food, and processing/preservation industries. To explore the high-value utilized way of scales produced during the fish processing, collagen hydrolysates of redlip croaker (Pseudosciaena polyactis) scales were prepared using six different proteases, and the hydrolysate (RSCH) prepared using neutrase showed the highest degree of hydrolysis (21.36 ± 1.18%) and 2,2-diphenyl-1-picrylhydrazyl (DPPH·) radical scavenging activity (30.97 ± 1.56%) among the six hydrolysates. Subsequently, six antioxidant peptides were purified from RSCH using membrane ultrafiltration and serial chromatography, and their amino acid sequences were identified as DGPEGR, GPEGPMGLE, EGPFGPEG, YGPDGPTG, GFIGPTE, and IGPLGA with molecular masses of 629.61, 885.95, 788.96, 762.75, 733.80, and 526.61 Da, respectively. Among six collagen peptides, GPEGPMGLE, EGPFGPEG, and GFIGPTE exhibited the strongest scavenging activities on DPPH· radical (EC50 0.59, 0.37, and 0.45 mg/mL), hydroxyl radical (EC50 0.45, 0.33, and 0.32 mg/mL), and superoxide anion radical (EC50 0.62, 0.47, and 0.74 mg/mL). GPEGPMGLE, EGPFGPEG, and GFIGPTE showed high inhibiting ability on lipid peroxidation in a linoleic acid model system and protective activities on oxidation-damaged DNA. More importantly, GPEGPMGLE, EGPFGPEG, and GFIGPTE could protect HepG2 cells from H2O2-induced oxidative damage through decreasing the levels of reactive oxygen species (ROS) and MDA and activating intracellular antioxidant enzymes of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px). These results suggested that six collagen peptides (RCP1-RCP6), especially GPEGPMGLE, EGPFGPEG, and GFIGPTE, might serve as potential antioxidants applied in nutraceutical and pharmaceutical products.

Antioxidant Peptides from the Protein Hydrolysate of Monkfish (Lophius litulon) Muscle: Purification, Identification, and Cytoprotective Function on HepG2 Cells Damage by H2O2.

In the work, defatted muscle proteins of monkfish (Lophius litulon) were separately hydrolyzed by pepsin, trypsin, and in vitro gastrointestinal (GI) digestion methods, and antioxidant peptides were isolated from proteins hydrolysate of monkfish muscle using ultrafiltration and chromatography processes. The antioxidant activities of isolated peptides were evaluated using radical scavenging and lipid peroxidation assays and H2O2-induced model of HepG2 cells. In which, the cell viability, reactive oxygen species (ROS) content, and antioxidant enzymes and malondialdehyde (MDA) levels were measured for evaluating the protective extent on HepG2 cells damaged by H2O2. The results indicated that the hydrolysate (MPTH) prepared using in vitro GI digestion method showed the highest degree of hydrolysis (27.24 ± 1.57%) and scavenging activity on a 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical (44.54 ± 3.12%) and hydroxyl radical (41.32 ± 2.73%) at the concentration of 5 mg protein/mL among the three hydrolysates. Subsequently, thirteen antioxidant peptides (MMP-1 to MMP-13) were isolated from MPTH. According to their DPPH radical and hydroxyl radical scavenging activity, three peptides with the highest antioxidant activity were selected and identified as EDIVCW (MMP-4), MEPVW (MMP-7), and YWDAW (MMP-12) with molecular weights of 763.82, 660.75, and 739.75 Da, respectively. EDIVCW, MEPVW, and YWDAW showed high scavenging activities on DPPH radical (EC50 0.39, 0.62, and 0.51 mg/mL, respectively), hydroxyl radical (EC50 0.61, 0.38, and 0.32 mg/mL, respectively), and superoxide anion radical (EC50 0.76, 0.94, 0.48 mg/mL, respectively). EDIVCW and YWDAW showed equivalent inhibiting ability on lipid peroxidation with glutathione in the linoleic acid model system. Moreover, EDIVCW, MEPVW, and YWDAW had no cytotoxicity to HepG2 cells at the concentration of 100.0 µM and could concentration-dependently protect HepG2 cells from H2O2-induced oxidative damage through decreasing the levels of reactive oxygen species (ROS) and MDA and activating intracellular antioxidant enzymes of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px). These present results indicated that the protein hydrolysate and isolated antioxidant peptides from monkfish muscle, especially YWDAW could serve as powerful antioxidants applied in the treatment of some liver diseases and healthcare products associated with oxidative stress.

Bronchial lavage under fiberoptic bronchoscopy in the treatment of severe pulmonary infection.

OBJECTIVE: To investigate the clinical efficacy of bronchial lavage under fiberoptic bronchoscopy in the treatment of severe pulmonary infection. METHODS: One hundred forty eight patients with severe pulmonary infection who were admitted to our hospital from October 2016 to December 2017 were included in this study. According to the random number table method, they were divided into a control group and an observation group with 79 patients each. The control group was given conventional treatment, while the observation group was given bronchoalveolar lavage with fiberoptic bronchoscopy on the basis of the treatment in the control group. The clinical efficacy of the two groups was compared, the duration of mechanical ventilation, antibiotic use and symptoms improvement of the two groups were recorded, and the respiratory mechanics parameters, serum procalcitonin (PCT) and transforming growth factor ß (TGF-ß) level were measured before and after treatment. RESULTS: The duration of mechanical ventilation, antibiotic use, respiratory failure correction, body temperature decline and white blood cell recovery in the observation group were significantly shorter than those in the control group (P<0.05). The total efficacy of the observation group was significantly higher than that of the control group (92.4% vs. 74.7%). The respiratory mechanics parameters of the two groups after treatment were higher than those before treatment (P<0.05) and the increase of the observation group was more obvious than that of the control group (P<0.05). The serum PCT and TGF-ß levels of the two groups after treatment were lower than those before treatment (P<0.05), and the decrease level in the observation group was more obvious (P<0.05). CONCLUSION: Bronchial lavage under fiberoptic bronchoscopy can improve the clinical efficacy, accelerate the improvement of clinical symptoms and respiratory mechanics parameters, significantly reduce the PCT and TGF-ß levels, and promote the rapid recovery of patients in the treatment of severe pulmonary infection.

Purification and Characterization of Antioxidant Peptides Derived from Protein Hydrolysate of the Marine Bivalve Mollusk Tergillarca granosa.

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.

Eight Collagen Peptides from Hydrolysate Fraction of Spanish Mackerel Skins: Isolation, Identification, and In Vitro Antioxidant Activity Evaluation.

A previous report indicated that collagen hydrolysate fraction (F7) from Spanish mackerel (Scomberomorous niphonius) skins showed high reducing power and radical scavenging activities on 2,2-Diphenyl-1-picrylhydrazyl (DPPH) (EC50 value of 1.57 mg/mL) and hydroxyl (EC50 value of 1.20 mg/mL). In this work, eight peptides were isolated from F7 and identified as Gly-Pro-Tyr (GPY, 335.31 Da), Gly-Pro-Thr-Gly-Glu (GPTGE, 459.47 Da), Pro-Phe-Gly-Pro-Asp (PFGPD, 531.52 Da), Gly-Pro-Thr-Gly-Ala-Lys (GPTGAKG, 586.65 Da), Pro-Tyr-Gly-Ala-Lys-Gly (PYGAKG, 591.69 Da), Gly-Ala-Thr-Gly-Pro-Gln-Gly (GATGPQG, 586.61 Da), Gly-Pro-Phe-Gly-Pro-Met (GPFGPM, 604.73 Da), and Tyr-Gly-Pro-Met (YGPM, 466.50 Da), respectively. Among them, PFGPD, PYGAKG, and YGPM exhibited strong radical scavenging activities on DPPH (EC50 values of 0.80, 3.02, and 0.72 mg/mL for PFGPD, PYGAKG, and YGPM, respectively), hydroxyl (EC50 values of 0.81, 0.66, and 0.88 mg/mL for PFGPD, PYGAKG, and YGPM, respectively), superoxide anion (EC50 values of 0.91, 0.80, and 0.73 mg/mL for PFGPD, PYGAKG, and YGPM, respectively), and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) cation (EC50 values of 0.86, 1.07, and 0.82 mg/mL for PFGPD, PYGAKG, and YGPM, respectively) in a positive concentration-activity relationship. Furthermore, PFGPD, PYGAKG, and YGPM could effectively reduce Fe3+ to Fe2+ and inhibit lipid peroxidation. Hence, eight collagen peptides from hydrolysate of Spanish mackerel skins might be served as antioxidant candidates for various industrial applications.

Preparation and Characterization of Gelatin and Antioxidant Peptides from Gelatin Hydrolysate of Skipjack Tuna (Katsuwonus pelamis) Bone Stimulated by in vitro Gastrointestinal Digestion.

In China, a large amount of fish bones are produced during the processing of tuna cans production. For full use of those by-products, gelatin (STB-G) with a yield of 6.37 ± 0.64% was extracted from skipjack tuna (Katsuwonus pelamis) bone using water at 60 °C for 8 h. Amino acid analysis showed that STB-G contained Gly (340.3 residues/1000 residues) as the major amino acid and its imino acid content was 177.3 residues/1000 residues. Amino acid composition, SDS-PAGE, and Fourier transform infrared (FTIR) spectrum investigations confirmed that the physicochemical properties of STB-G were similar to those of type I collagen from skipjack tuna bone (STB-C), but partial high molecular weight components of STB-G were degraded during the extraction process, which induced that the gelatin was easier to be hydrolyzed by protease than mammalian gelatins and was suitable for preparation of hydrolysate. Therefore, STB-G was hydrolyzed under in vitro gastrointestinal digestion (pepsin-trypsin system) and five antioxidant peptides were purified from the resulted hydrolysate (STB-GH) and identified as GPDGR, GADIVA, GAPGPQMV, AGPK, and GAEGFIF, respectively. Among the gelatin hydrolysate, fractions, and isolated peptides, GADIVA and GAEGFIF exhibited the strongest scavenging activities on 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical (EC50 0.57 and 0.30 mg/mL), hydroxyl radical (EC50 0.25 and 0.32 mg/mL), superoxide anion radical (EC50 0.52 and 0.48 mg/mL), and 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical (EC50 0.41 and 0.21 mg/mL). Moreover, GADIVA and GAEGFIF showed a high inhibiting ability on lipid peroxidation in a linoleic acid model system. The strong activities of five isolated peptides profited by their small molecular sizes and the antioxidant amino acid residues in their sequences. These results suggested that five isolated peptides (STP1⁻STP5), especially GADIVA and GAEGFIF, might serve as potential antioxidants applied in health food industries.

Gelatin and Antioxidant Peptides from Gelatin Hydrolysate of Skipjack Tuna (Katsuwonus pelamis) Scales: Preparation, Identification and Activity Evaluation.

For full use of fish by-products, scale gelatin (TG) and antioxidant peptides (APs) of skipjack tuna (Katsuwonus pelamis) were prepared, and their properties were characterized using an amino acid analyzer, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Fourier transform infrared spectroscopy (FTIR), electrospray ionization mass spectrometers (ESI-MS), and radical scavenging assays. The results indicate that TG with a yield of 3.46 ± 0.27% contained Gly (327.9 ± 5.2 residues/1000 residues) as the major amino acid and its imino acid content was 196.1 residues/1000 residues. The structure of TG was more unstable than that of type I collagen from scales of skipjack tuna (TC) and TG was more suitable for preparation of hydrolysate by protease than mammalian gelatins. Therefore, TG was separately hydrolyzed under five proteases (pepsin, papain, trypsin, neutrase, and alcalase) and ten APs (TGP1-TGP10) were isolated from the alcalase-hydrolysate. Among them, TGP5, TGP7, and TGP9 with high antioxidant activity were identified as His-Gly-Pro-Hyp-Gly-Glu (TGP5), Asp-Gly-Pro-Lys-Gly-His (TGP7) and Met-Leu-Gly-Pro-Phe-Gly-Pro-Ser (TGP9), respectively. Furthermore, TGP5, TGP7, and TGP9 exhibited a high radical scavenging capability on 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical (EC50 values of 1.34, 0.54, and 0.67 mg/mL, respectively), hydroxyl radical (EC50 values of 1.03, 0.41, and 0.74 mg/mL, respectively), and superoxide anion radical (EC50 values of 1.19, 0.71, and 1.59 mg/mL, respectively). These results suggest that three APs (TGP5, TGP7, and TGP9), especially TGP7, have a strong antioxidant activity and could act as potential antioxidant ingredients applied in functional products.

Antioxidant Peptides from the Protein Hydrolysate of Spanish Mackerel (Scomberomorous niphonius) Muscle by in Vitro Gastrointestinal Digestion and Their In Vitro Activities.

For the full use of Spanish mackerel (Scomberomorous niphonius) muscle to produce antioxidant peptides, the proteins of Spanish mackerel muscle were separately hydrolyzed under five kinds of enzymes and in vitro gastrointestinal digestion, and antioxidant peptides were isolated from the protein hydrolysate using ultrafiltration and multiple chromatography methods. The results showed that the hydrolysate (SMPH) prepared using in vitro GI digestion showed the highest degree of hydrolysis (27.45 ± 1.76%) and DPPH radical scavenging activity (52.58 ± 2.68%) at the concentration of 10 mg protein/mL among the six protein hydrolysates, and 12 peptides (SMP-1 to SMP-12) were prepared from SMPH. Among them, SMP-3, SMP-7, SMP-10, and SMP-11 showed the higher DPPH radical scavenging activities and were identified as Pro-Glu-Leu-Asp-Trp (PELDW), Trp-Pro-Asp-His-Trp (WPDHW), and Phe-Gly-Tyr-Asp-Trp-Trp (FGYDWW), and Tyr-Leu-His-Phe-Trp (YLHFW), respectively. PELDW, WPDHW, FGYDWW, and YLHFW showed high scavenging activities on DPPH radical (EC50 1.53, 0.70, 0.53, and 0.97 mg/mL, respectively), hydroxyl radical (EC50 1.12, 0.38, 0.26, and 0.67 mg/mL, respectively), and superoxide anion radical (EC50 0.85, 0.49, 0.34, and 1.37 mg/mL, respectively). Moreover, PELDW, WPDHW, FGYDWW, and YLHFW could dose-dependently inhibit lipid peroxidation in the linoleic acid model system and protect plasmid DNA (pBR322DNA) against oxidative damage induced by H2O2 in the tested model systems. In addition, PELDW, WPDHW, FGYDWW, and YLHFW could retain their high activities when they were treated under a low temperature (<60 °C) and a moderate pH environment (pH 5-9). These present results indicate that the protein hydrolysate, fractions, and isolated peptides from Spanish mackerel muscle have strong antioxidant activity and might have the potential to be used in health food products.

Structure and Neuroprotective Effect of Polysaccharide from Viscera Autolysates of Squid Ommastrephes bartrami.

To explore bioactive polysaccharides from the byproducts of squid processing, a heteropolysaccharide, named SV2-1, was isolated from the viscera of squid Ommastrephes bartrami by autolysis, anion-exchange and gel-permeation chromatography and measured for its neuroprotective activity. It was a homogeneous polysaccharide with a molecular weight of 2.3 kDa by HPSEC analysis. SV2-1 contained glucuronic acid, galactosamine and fucose in the ratio of 1.0:1.1:1.2. Its structural characteristics were elucidated by methylation analysis, gas chromatography-mass spectrometry (GC-MS), and nuclear magnetic resonance (NMR). The backbone of SV2-1 was composed of alternant →4)-α-l-Fucp-(1→ and →3)-ß-d-GlcUA-(1→ Most of →4)-α-l-Fucp-(1→ (90%) was substituted by single α-d-GlcNAc as the branches. SV2-1 can protect against the death of PC12 induced by 6-OHDA, and effectively improves cell viability and reduces extracellular LDH release in PC12 cells after injury. Moreover, SV2-1 significantly increases SOD activity but decreases MDA levels.

Cytoprotective Effect of Antioxidant Pentapeptides from the Protein Hydrolysate of Swim Bladders of Miiuy Croaker (Miichthys miiuy) against H2O2-Mediated Human Umbilical Vein Endothelial Cell (HUVEC) Injury.

In our previous research, ten antioxidant pentapeptides including FYKWP, FTGMD, GFEPY, YLPYA, FPPYERRQ, GFYAA, FSGLR, FPYLRH, VPDDD, and GIEWA were identified from the hydrolysate of miiuy croaker (Miichthys miiuy) swim bladder. In this work, their protective function on H2O2-induced oxidative damage to human umbilical vein endothelial cells (HUVECs) was studied. Results indicated that there was no significant difference in the HUVEC viability between the normal group and the treated groups with the 10 pentapeptides at the concentration of 100 µM for 24 h (p < 0.05). Furthermore, FPYLRH of 100 µg/mL extremely significantly (p < 0.001) increased the viability (80.58% ± 5.01%) of HUVECs with H2O2-induced oxidative damage compared with that of the model group. The protective mechanism indicated that FPYLRH could extremely significantly (p < 0.001) increase the levels of superoxide dismutase (SOD) (211.36 ± 8.29 U/mg prot) and GSH-Px (53.06 ± 2.34 U/mg prot) and decrease the contents of reactive oxygen species (ROS) (139.1 ± 11.8% of control), malondialdehyde (MDA) (13.66 ± 0.71 nM/mg), and nitric oxide (NO) (4.36 ± 0.32 µM/L) at the concentration of 100 µM in HUVECs with H2O2-induced oxidative damage compared with those of the model group. In addition, FPYLRH dose-dependently protected DNA in oxidative damage HUVECs model. These results suggested that FPYLRH could significantly attenuate the H2O2-induced stress injury in HUVECs and might be used as a potential natural antioxidant in the functional food industries.

Bioactive Exopolysaccharides Reveal Camellia oleifera Infected by the Fungus Exobasidium gracile Could Have a Functional Use.

Camellia oleifera is an important Chinese commercial crop. Camellia oleifera can display abnormal leaves due to infection by the parasitic fungus Exobasidium gracile. Exobasidium gracile was isolated from infected leaves and used in fermentation, and exopolysaccharides EP0-1 and EP0.5-1 were purified from the fermentation broth. EP0-1 was an alkaline polysaccharide consisting mainly of the linkages α-d-Manp(1→, →2)-α-d-Manp(1→ and →6)-α-d-Manp(1→, →3)-α-d-Glcp(1→ and→4)-α-d-Glcp(1→, terminal ß-d-Galf, (1→5)-ß-d-Galf, and terminal ß-D-GlcN(1→. EP0.5-1 was an acidic galactofuranose-containing polysaccharide. It contained the linkages of α-d-Manp(1→, →2)-α-d-Manp(1→, →6)-α-d-Manp(1→,→2, 6)-α-d-Manp(1→, →4)-α-d-Glcp(1→, and →4)-α-d-GlcUA(1→. Galactofuranose linkages were composed of terminal ß-d-Galf, (1→6)-ß-d-Galf and (1→2)-ß-d-Galf. Exobasidium gracile exopolysaccharides displayed significant immunoregulatory activity by activating macrophages. This research indicates that infected leaves from Camellia oleifera including the exopolysaccharides produced by the parasitic fungus Exobasidium gracile by are worth further investigation as a functional product.

Bioactive Peptides from Cartilage Protein Hydrolysate of Spotless Smoothhound and Their Antioxidant Activity In Vitro.

In the experiment, crude proteins from spotless smoothhound (Mustelus griseus), cartilages were isolated by HCl-Guanidine buffer, and its hydrolysate was prepared using trypsin at pH 8.0, 40 °C with a total enzyme dose of 2.5%. Subsequently, three antioxidant peptides were purified from the hydrolysate using membrane ultrafiltration, anion-exchange chromatography, gel filtration chromatography, and reverse phase high-performance liquid chromatography. The amino acid sequences of isolated peptides were identified as Gly-Ala-Glu-Arg-Pro (MCPE-A); Gly-Glu-Arg-Glu-Ala-Asn-Val-Met (MCPE-B); and Ala-Glu-Val-Gly (MCPE-C) with molecular weights of 528.57, 905.00, and 374.40 Da, respectively, using protein amino acid sequence analyzer and mass spectrum. MCPE-A, MCPE-B and MCPE-C exhibited good scavenging activities on 2,2-diphenyl-1-picrylhydrazyl radicals (DPPH•) (EC50 3.73, 1.87, and 2.30 mg/mL, respectively), hydroxyl radicals (HO•) (EC50 0.25, 0.34, and 0.06 mg/mL, respectively), 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid radicals (ABTS⁺•) (EC50 0.10, 0.05, and 0.07 mg/mL, respectively) and superoxide anion radicals ( O 2 - •) (EC50 0.09, 0.33, and 0.18 mg/mL, respectively). MCPE-B showed similar inhibiting ability on lipid peroxidation with butylated hydroxytoluene (BHT) in a linoleic acid model system. Furthermore, MCPE-A, MCPE-B, and MCPE-C could protect H2O2-induced HepG2 cells from oxidative stress by decreasing the content of malonaldehyde (MDA) and increasing the levels of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and glutathione reductase (GSH-Rx). Glu, Gly, Met, and Pro in their sequences and low molecular weight could be attributed to the antioxidant activities of three isolated peptides. These results suggested that GAERP (MCPE-A), GEREANVM (MCPE-B), and AEVG (MCPE-C) from cartilage protein hydrolysate of spotless smoothhound might serve as potential antioxidants and be used in the pharmaceutical and health food industries.