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A Porphyra dioica protein extract was enzymatically hydrolysed and then fractionated using semi-preparative reverse-phase high performance chromatography. The hydrolysate and its fractions were tested for their oxygen radical absorbance capacity (ORAC) along with their angiotensin converting enzyme (ACE) and dipeptidyl peptidase IV (DPP-IV) inhibitory activities. The most potent fraction was analysed by liquid chromatography mass spectrometry. Eight peptide sequences were selected for synthesis based on their structure-activity criteria for bioactivity. Asp-Tyr-Tyr-Lys-Arg showed the highest ORAC activity (4.27 ± 0.15 μmol Trolox equivalent per μM). Thr-Tyr-Ile-Ala had the highest ACE inhibitory activity (IC50: 89.7 ± 7.10 μM). Tyr-Leu-Val-Ala was the only peptide showing DPP-IV inhibitory activity (IC50: 439 ± 44 μM). Apart from Asp-Tyr-Tyr-Lys-Arg and Thr-Tyr-Ile-Ala, which displayed increased ORAC activity, the bioactivities of the peptides were either maintained or decreased following in vitro simulated gastrointestinal digestion. The results indicate that P. dioica-derived peptides may have potential applications as health enhancing ingredients.

Whey protein concentrate (WPC) hydrolysates generated using Neutrase®, Alcalase® and Flavourzyme® and their associated ultrafiltration fractions inhibited angiotensin-1-converting enzyme activity (71.14 ± 1.05, 73.22 ± 0.99 and 51.52 ± 5.80% inhibition when assayed at 14.3 µg/mL). Incubation of human umbilical vein endothelial cells with 5 kDa permeates of Neutrase®- and Alcalase®-hydrolysed WPC for 48 h resulted in the beneficial differential expression of genes relevant to blood pressure control, as measured by microarray. Furthermore, real-time reverse transcriptase polymerase chain reaction demonstrated an upregulation of endothelial nitric oxide synthase (+2.14 ± 0.45 and 2.36 ± 0.27-fold) and down-regulation of endothelin-1 (−0.58 ± 0.09 and −0.82 ± 0.11-fold) following incubation with the 5 kDa permeates of Alcalase®- and Neutrase®-hydrolysates, respectively. Peptide sequences within the 5 kDa permeate of the Alcalase®-hydrolysed WPC were identified, many of which have previously been demonstrated as having ACE-inhibitory and/or other bioactivities. These WPC hydrolysates potentially represent sources of bioactive peptides for the beneficial regulation of endothelial cell function.

Summary Antioxidant and angiotensin converting enzyme (ACE) inhibitory peptides were identified in a 5 kDa ultrafiltration permeate of a whey protein hydrolysate generated at semi-pilot scale. Further laboratory scale ultrafiltration of this 5 kDa permeate resulted in a 0.65 kDa permeate with antioxidant, (1.11 ± 0.074 μmol TE per mg dry weight, oxygen radical absorbance capacity, ORAC) and ACE inhibitory (ACE IC50 0.215 ± 0.043 mg mL−1) activities. Semi-preparative (SP) reverse phase high-performance liquid chromatography (RP-HPLC) of the 0.65 kDa permeate resulted in a fraction (SP_F3) with a 4.4-fold increase in ORAC activity (4.83 ± 0.45 μmol TE mg dry weight) and a 1.3-fold increase in ACE inhibitory activity (84.35 ± 1.36% inhibition when assayed at 0.28 mg mL−1). Peptides within SP_F3 were identified using UPLC-ESI-MS/MS. Met-Pro-Ile had the highest ORAC activity (205.75 ± 12.08 μmol TE per mmol peptide) while Met-Ala-Ala and Val-Ala-Gly-Thr had the highest ACE inhibitory activities (IC50:515.50 ± 1.11 and 610.30 ± 2.41 μm, respectively).

Brewer's spent grain (BSG) is a co-product of the brewing industry and is a potentially rich source of bioactive peptides. A BSG protein hydrolysate, manufactured at a semi-pilot scale, mediated significant hypotensive effects 6 h after ingestion in spontaneously hypertensive rats. This hydrolysate was fractionated and further analysed by UPLC-ESI-MS/MS for peptide identification. The peptides, IPY and LPY demonstrated the highest antioxidant activity (0.37 ± 0.02 and 0.34 ± 0.02 µmol Trolox equivalents µM peptide−1, respectively), IPLQP and LPLQP had the highest ACE inhibitory activity (IC50: 3.10 ± 0.60 and 3.17 ± 0.60 µM, respectively) while IPVP had the highest DPP-IV inhibitory activity (IC50: 38.67 ± 5.94 µM). Overall, peptides containing I had higher in vitro bioactivities when compared to peptides having L. Novel BSG peptides were identified with potential for application as naturally derived ingredients for the management of cardiovascular disease and type 2 diabetes.

peer-reviewed The full text of this article will not be available in ULIR until the embargo expires on the 21/01/2021 Twenty-two novel dipeptidyl peptidase-IV (DPP-IV) inhibitory peptides (with IC50 values

Summary The in vitro dipeptidyl peptidase-IV (DPP-IV) inhibitory activity of a Brewers’ spent grain protein-enriched isolate (BSG-PI) Alcalase™ hydrolysate (AlcH), which had previously been identified as a relatively potent angiotensin-converting enzyme (ACE) inhibitor, was determined. The half maximal DPP-IV inhibitory concentration (IC50) value of AlcH following 240-min digestion was 3.57 ± 0.19 mg mL−1. Ultrafiltration fractionation did not significantly increase the DPP-IV inhibitory activity of the AlcH fractions. Subjection of AlcH to simulated gastrointestinal digestion (SGID), which yielded SAlcH, resulted in a significant increase in DPP-IV inhibitory activity (P

peer-reviewed The full text of this article will not be available in ULIR until the embargo expires on the 31/12/2019 The in vitro bioactivity of 5 kDa ultrafiltration permeate fractions of casein hydrolysates produced using different enzymes were compared. Reverse phase ultra-performance liquid chromatography and gel permeation chromatography showed that the permeates had different physicochemical properties (molecular mass and degree of hydrolysis). The Flavourzyme® permeate had the highest activity in the 2,2’-azinobis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) assay. Cellular antioxidant and immunomodulatory assays showed that none of the permeates exhibited in vitro antioxidant activity, while all permeates significantly (P < 0.05) decreased interleukin-6 (IL-6) production in ConA-stimulated Jurkat T cells at 0.50% (w/v) and LPS-stimulated RAW264.7 cells at 0.05 and 0.50% (w/v). Three permeates, obtained using Flavourzyme®, Flavorpro Whey and trypsin, also significantly (P < 0.05) decreased IL-1β production at 0.05% (w/v) in RAW264.7 cells. Western blot analysis showed that all permeates significantly decreased the expression of the NF-κB subunit, p65, in RAW264.7 cells indicating that anti-inflammatory activity may be associated with this pathway.

Incubation of sodium caseinate (NaCN) and purified α-casein (αs-CN) with an Aspergillus niger derived prolyl endoproteinase (An-PEP) for 1, 2, 3, 4, 8 and 24 h resulted in the generation of potent angiotensin converting enzyme (ACE) inhibitory hydrolysates. An ACE IC50 of 21.1±5.1 μg/ml was obtained on incubation of An-PEP with NaCN for 4 h. Fractionation of the NaCN hydrolysates using 3 kDa centrifugal filters resulted in highly active permeate fractions, the most potent being obtained from the 3 h hydrolysate (ACE IC50=2.9±0.3 μg/ml). The hydrolytic specificity of An-PEP for purified α-CN was assessed using UPLC ESI MS/MS. The analysis confirmed An-PEP's cleavage preference for the C-terminal side of Pro and also confirmed that An-PEP has the ability to cleave at the C-terminal of Ala, Leu, Arg and His residues.

An alkaline extracted brewers' spent grain protein-enriched isolate (BSG-PI) was hydrolysed using Alcalase, Corolase PP, Flavourzyme and Promod 144MG, yielding Alc hydrolysate (H), CorH, FlavH and ProH, respectively. The degree of hydrolysis (DH) of the protein hydrolysates varied from 4.45% for ProH to 16.4% for CorH. The in vitro ACE inhibitory activity of the BSG-PI increased significantly following 15min incubations with Alcalase, Corolase PP and Flavourzyme. The 5kDa ultrafiltration permeates of FlavH and CorH resulted in lower ACE IC50 values than their respective hydrolysates. The bioactivity of the BSG-PI hydrolysates was retained after simulated gastrointestinal digestion (SGID) while SGID also resulted in the release of ACE inhibitory peptides from the BSG-PI and ProH. UPLC-MS/MS analysis resulted in the identification of 34 peptides. Of 12 synthesised peptides, IVY and ILDL were the most potent, having ACE IC50 values at 80.4±11.9 and 96.4±8.36μM, respectively.

Peptides with the ability to chelate dietary minerals have been reported to have potential as functional food ingredients. A collagen tryptic hydrolysate (CTH), previously shown to chelate iron, was further investigated for the presence of Ca, Fe and Cu chelating peptides. Sequential purification steps, including immobilised metal affinity chromatography (IMAC) and gel permeation chromatography (GPC) were employed for the separation of chelating peptides. GPC analysis showed that the mineral chelating peptides were mainly between 500 and 2000 Da. Subsequent identification was carried out using UPLC-ESI-QTOF MS/MS. Overall, 10 sequences were identified as potential chelating peptides. The Ca, Fe and Cu chelating activity of GPAGPHGPPG was 11.52±2.23 nmol/μmol, 1.71±0.17 nmol/μmol and 0.43±0.02 μmol/μmol, respectively. This study identifies collagen as a good source of peptides with potential applications as functional ingredients in the management of mineral deficiencies.

peer-reviewed Large quantities of low-value protein rich co-products, such as salmon skin and trimmings, are generated annually. These co-products can be upgraded to high-value functional ingredients. The aim of this study was to assess the antidiabetic potential of salmon skin gelatin and trimmings-derived protein hydrolysates in vitro. The gelatin hydrolysate generated with Alcalase 2.4L and Flavourzyme 500L exhibited significantly higher (p

Enzymatic hydrolysis of whey protein concentrate (WPC) was performed using Alcalase, Neutrase, Corolase PP or Flavourzyme. The hydrolysates were filtered through membranes having different molecular mass cut-offs. The hydrolysate fractions had significantly (P

The pH shift method was utilised for the recovery of proteins from salmon trimmings (ST), yielding 93% (w/w) protein. ST protein (STP) hydrolysates were generated with different enzyme preparations. STP incubated with Corolase PP for 1h (STP-C1) had the most potent angiotensin converting enzyme (ACE) and dipeptidyl peptidase IV (DPP-IV) inhibitory and oxygen radical absorbance capacity (ORAC) activities. Analysis of fractions of STP-C1 using UPLC-MS/MS identified sixteen peptides/amino acids. Tyr-Pro had the highest ACE inhibitory activity (ACE IC50=5.21±0.94μM). The highest DPP-IV inhibitory activity was found with the amino acid Tyr (DPP-IV IC50=75.15±0.84μM). Val-Pro had the highest ORAC activity (19.45±2.15μmol of TEg-1). To our knowledge, the peptides Gly-Pro-Ala-Val, Val-Cys, and Phe-Phe have not been previously identified to have the activities tested in this study. These results indicate that STP hydrolysates are potential sources of bioactive peptides.

The human prostacyclin receptor (hIP) undergoes agonist-induced internalization but the mechanisms regulating its intracellular trafficking and/or recycling to the plasma membrane are poorly understood. Herein, we conducted a yeast-two-hybrid screen to identify proteins interacting with the carboxyl terminal (C)-tail domain of the hIP and discovered a novel interaction with Rab11a. This interaction was confirmed by co-immunoprecipitations in mammalian HEK293 and was augmented by cicaprost stimulation. The hIP co-localized to Rab11-containing recycling endosomes in both HEK293 and endothelial EA.hy 926 cells in a time dependent manner following cicaprost stimulation. Moreover, over-expression of Rab11a significantly increased recycling of the hIP, while the dominant negative Rab11S25N impaired that recycling. Conversely, while the hIP co-localized to Rab4-positive endosomes in response to cicaprost, ectopic expression of Rab4a did not substantially affect overall recycling nor did Rab4a directly interact with the hIP. The specific interaction between the hIP and Rab11a was dependent on a 22 amino acid (Val299 – Gln320) sequence within its C-tail domain and was independent of isoprenylation of the hIP. This study elucidates a critical role for Rab11a in regulating trafficking of the hIP and has identified a novel Rab11 binding-domain (RBD) within its C-tail domain that is both necessary and sufficient to mediate interaction with Rab11a. Science Foundation Ireland Wellcome Trust ti, ab, ke,-SB 01/09/2011

The human prostacyclin receptor (hIP) undergoes rapid agonist-induced internalization by largely unknown mechanism(s). Herein the involvement of Rab5 in regulating cicaprost-induced internalization of the hIP expressed in human embryonic kidney 293 cells was investigated. Over-expression of Rab5a significantly increased agonist-induced hIP internalization. Additionally, the hIP co-localized to Rab5a-containing endocytic vesicles in response to cicaprost stimulation and there was a coincident net translocation of Rab5 from the cytosol/soluble fraction of the cell. Co-immunoprecipitation studies confirmed a direct physical interaction between the hIP and Rab5a that was augmented by cicaprost. Whilst the dominant negative Rab5aS34N did not show decreased interaction with the hIP or fully impair internalization, it prevented hIP sorting to endocytic vesicles. Moreover, the GTPase deficient Rab5aQ79L significantly increased internalization and co-localized with the hIP in enlarged endocytic vesicles. While deletion of the carboxyl terminal (C)-tail domain of the hIP did not inhibit agonist-induced internalization, co-localization or co-immunoprecipitation with Rab5a per se, receptor trafficking was altered suggesting that it contains structural determinant(s) for hIP sorting post Rab5-mediated endocytosis. Taken together, data herein and in endothelial EA.hy 926 cells demonstrate a direct role for Rab5a in agonist-internalization and trafficking of the hIP and increases knowledge of the factors regulating prostacyclin signaling.

Milk proteins and milk protein-derived peptides have been widely studied for their health enhancing properties. This chapter presents the updated scientific knowledge on the bioactive properties of milk protein-derived peptides. The different bioactive properties which have been attributed to milk protein-derived peptides are discussed. These include mineral binding, cardioprotective, antidiabetic, satiating, opioid, antimicrobial, immunomodulatory, anticancer and antioxidant activities. The structure-function relationship is presented for the aforementioned bioactive properties based on current scientific knowledge. For each bioactive property, the data obtained in vitro is discussed, followed by an analysis of the information obtained from animal and human intervention studies. To date, most studies have been conducted in vitro. However, an increasing number of in vivo studies testing the efficacy of milk protein-derived peptides are being conducted. In certain instances, the in vivo studies have confirmed the bioactivity of specific milk protein-derived peptides or milk protein hydrolysates. However, conflicting data still exist in the scientific literature, which demonstrates that the bioactive properties observed in vitro do not always translate in vivo. Detailed knowledge of the peptide sequences responsible for the bioactive properties, together with a better understanding of the bioavailability and stability of these peptides in vivo may help to enhance the development of milk protein hydrolysates with health promoting capabilities in humans. Ultimately, this may lead to the approval of health claims by the relevant regulatory agencies.

Thromboxane (TX) A(2) plays a central role in hemostasis, regulating platelet activation status and vascular tone. We have recently established that the TP beta isoform of the human TXA(2) receptor (TP) undergoes rapid, agonist-induced homologous desensitization of signalling largely through a G protein-coupled receptor kinase (GRK) 2/3-dependent mechanism with a lesser role for protein kinase (PK) C. Herein, we investigated the mechanism of desensitization of signalling by the TP alpha isoform. TP alpha undergoes profound agonist-induced desensitization of signalling (intracellular calcium mobilization and inositol 1,4,5 trisphosphate generation) in response to the TXA(2) mimetic U46619 but, unlike that of TP beta, this is independent of GRKs. Similar to TP beta, TP alpha undergoes partial agonist-induced desensitization that occurs through a GF 109203X-sensitive, PKC mechanism where Ser(145) within intracellular domain (IC)(2) represents the key phospho-target. TP alpha also undergoes more profound sustained PKC- and PKG-dependent desensitization where Thr(337) and Ser(331), respectively, within its unique C-tail domain were identified as the phospho-targets. Desensitization was impaired by the nitric oxide synthase (NOS), soluble guanylyl cyclase (sGC) and PKG inhibitors L-NAME, LY 83583 and KT5823, respectively, indicating that homologous desensitization of TP alpha involves nitric oxide generation and signalling. Consistent with this, U46619 led to rapid phosphorylation/activation of endogenous eNOS. Collectively, data herein suggest a mechanism whereby agonist-induced PKC phosphorylation of Ser(145) partially and transiently impairs TP alpha signalling while PKG- and PKC-phosphorylation at both Ser(331) and Thr(337), respectively, within its C-tail domain profoundly desensitizes TP alpha, effectively terminating its signalling. Hence, in addition to the agonist-mediated PKC feedback mechanism, U46619-activation of the NOS/sGC/PKG pathway plays a significant role in inducing homologous desensitization of TP alpha.

Nitric oxide (NO(*)) has many characteristics including cytotoxicity, radiosensitization and anti-angiogenesis, which make it an attractive molecule for use in cancer therapy. We have investigated the use of iNOS gene transfer, driven by both a constitutive (CMV) and X-ray inducible (WAF1) promoter, for generating high concentrations of NO(*) within tumour cells. We have combined this treatment with radiation to exploit the radiosensitizing properties of this molecule. Transfection of murine RIF-1 tumour cells in vitro with the iNOS constructs resulted in increased iNOS protein levels. Under hypoxic conditions cells were radiosensitized by delivery of both constructs so that these treatments effectively eliminated the radioresistance observed under hypoxic conditions. In vivo transfer of the CMV/iNOS construct by direct tumour injection resulted in a delay (4.2 days) in tumour growth compared with untreated controls. This was equivalent to the effect of 20 Gy X-rays alone. Combination of CMV/iNOS gene transfer with 20 Gy X-rays resulted in a dramatic 19.8 day growth delay compared with controls. Tumours treated with the CMV/iNOS showed large areas of necrosis and abundant apoptosis. We believe that iNOS gene transfer has the potential to be a highly effective treatment in combination with radiotherapy.

Numerous low molecular mass bioactive peptides (BAPs) can be generated during the hydrolysis of bovine milk proteins. Low molecular mass BAP sequences are less likely to be broken down by digestive enzymes and are thus more likely to be active in vivo. However, the identification of short peptides remains a challenge during mass spectrometry (MS) analysis due to issues with the transfer and over-fragmentation of low molecular mass ions. A method is described herein using time-of-flight ESI-MS/MS to effectively fragment and identify short peptides. This includes (a) short synthetic peptides, (b) short peptides within a defined hydrolysate sample, i.e. a prolyl endoproteinase hydrolysate of β-casein and (c) short peptides within a complex hydrolysate, i.e. a Corolase PP digest of sodium caseinate. The methodology may find widespread utilisation in the efficient identification of low molecular mass peptide sequences in food protein hydrolysates.

Antioxidant and angiotensin converting enzyme (ACE) inhibitory peptides were extracted and isolated from two different types of Thai traditional fermented shrimp pastes, Kapi Ta Dam (Kp-B6) and Kapi Ta Deang (Kp-R6). Compounds with masses less than 500Da were found to be predominantly presented in both extracts. Following fractionation with sequential anion exchange chromatography and solid phase extraction (C18 matrix), three dipeptides were identified. Ser-Val and Ile-Phe were shown to exhibit ACE inhibitory activity with IC50 values of 60.68±1.06 and 70.03±1.45μM, respectively. Trp-Pro was shown to have high 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) radical scavenging activity (EC50 17.52±0.46μM). These results indicate that Thai traditional fermented shrimp pastes are potential sources of bioactive peptides possessing ACE inhibitory and antioxidant activities.

Dipeptidyl peptidase (DPP)-IV inhibitory peptides were purified and identified from an aqueous Palmaria palmata protein extract hydrolysed with Corolase PP. The hydrolysate was fractionated by solid phase extraction (SPE) using a C18 matrix followed by semi-preparative reverse phase-high performance liquid chromatography (SP RP-HPLC). IC50 values of 1.47 ± 0.09, 0.54 ± 0.03 and 0.36 ± 0.03 mg/ml were obtained for the hydrolysate, the 25%--acetonitrile (ACN) SPE fraction and the most active SP RP-HPLC peptide fraction (SP RP-HPLC 25_F28), respectively. Thirteen peptide sequences were identified following UPLC-ESI MS/MS analysis of SP RP-HPLC 25_F28. Three novel DPP-IV inhibitory peptides, Ile-Leu-Ala-Pro, Leu-Leu-Ala-Pro and Met-Ala-Gly-Val-Asp-His-Ile, with IC50 values in the range 43-159 μM were identified. The results indicate that P. palmata derived peptides may have potential as functional food ingredients in the prevention and management of type 2 diabetes.

A Ficus carica L. latex proteinase preparation was investigated for its ability to produce antioxidant hydrolysates/peptides from bovine casein (CN). The Oxygen Radical Absorbance Capacity (ORAC) values for NaCN and β-CN hydrolysates ranged from 0.06 to 0.18, and from 0.51 to 1.19μmol Trolox equivalents/mg freeze-dried sample, respectively. Gel permeation HPLC showed that the β-CN hydrolysate with a degree of hydrolysis of 21% had 65% of peptide material with a molecular mass

The contribution of endothelial cell growth to angiogenesis has been widely studied; however, the involvement of pericytes is less well documented, especially in human tumors. In this study we aimed to quantify and assess the prognostic significance of pericyte coverage, the extent of hypoxia, and microvessel density (MVD) in normal bladder mucosa and urothelial carcinoma. Antibody to alpha-smooth muscle actin was used to assess the distribution of pericytes (mural/smooth muscle cells) in the microvessels of normal human bladder (n = 4) mucosa and in urothelial carcinoma (n = 47) samples; this was quantitated using microvessel pericyte index (MPI). The MVD was measured using two different methods (n = 47) and hypoxia was assessed using glucose transporter-1 (Glut-1) staining (n = 30). There was a 70% reduction in MPI in urothelial carcinomas compared to normal bladder mucosa (p0.0012); MPI did not correlate with tumor stage or grade. Ta and T1 superficial tumors were divided into two groups with a MPI of15% or15%. Progression-free survival was significantly shorter for tumors with MPI15% (p = 0.0036). MVD had no prognostic value using either evaluation method. Glut-1 immunoreactivity was not prognostic in superficial urothelial carcinoma samples. Tumors with a higher MPI showed a greater Glut-1 immunoreactivity (p = 0.0051). Microvessels in urothelial carcinoma have a considerable loss of pericyte coverage compared to normal bladder mucosa. The data from this preliminary study indicate that progression-free survival was shorter in patients whose superficial tumors had higher pericyte coverage of the microvessels. This may be due to increased levels of hypoxia, as demonstrated by a significant increase in Glut-1 staining.

In humans, thromboxane (TX) A2 signals through two receptor isoforms, thromboxane receptor (TP)alpha and TPbeta, which are transcriptionally regulated by distinct promoters, Prm1 and Prm3, respectively, within the single TP gene. The aim of the current study was to investigate the ability of the endogenous peroxisome proliferator-activated receptor (PPAR)gamma ligand 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) to regulate expression of the human TP gene and to ascertain its potential effects on the individual TPalpha and TPbeta isoforms. 15d-PGJ2 suppressed Prm3 transcriptional activity and TPbeta mRNA expression in the platelet progenitor megakaryocytic human erythroleukemia (HEL) 92.1.7 cell line but had no effect on Prm1 or Prm2 activity or on TPalpha mRNA expression. 15d-PGJ2 also resulted in reductions in the overall level of TP protein expression and TP-mediated intracellular calcium mobilization in HEL cells. 15d-PGJ2 suppression of Prm3 transcriptional activity and TPbeta mRNA expression was found to occur through a novel mechanism involving direct binding of PPARgamma-retinoic acid X receptor (RXR) heterodimers to a PPARgamma response element (PPRE) composed of two imperfect hexameric direct repeat (DR) sequences centred at -159 and -148, respectively, spaced by five nucleotides (DR5). These data provide direct evidence for the role of PPARgamma in the regulation of human TP gene expression within the vasculature and point to further critical differences in the modes of transcriptional regulation of TPalpha and TPbeta in humans. Moreover, these data highlight a further link between enhanced risk of cardiovascular disease in diabetes mellitus associated with increased synthesis and action of thromboxane A2 (TXA2).