Peptides (v.73, #C)

The membrane-active amphibian peptide caerin 1.8 inhibits fibril formation of amyloid β1-42 by Yanqin Liu; Tianfang Wang; Antonio N. Calabrese; John A. Carver; Scott F. Cummins; John H. Bowie (1-6).
Inhibition of fibril formation from amyloid beta 1-42 peptide by caerin 1.8 [1GLFKVLGSV10AKHLLPHVVP20VIAEKL(NH2)]. Thioflavin T fluorescence assay.Display OmittedThe amphibian host-defense peptide caerin 1.8 [1GLFKVLGSV10AKHLLPHVVP20VIAEKL(NH2)] inhibits fibril formation of amyloid β 1−42 [1DAEFRHDSG10YEVHHQKLVF20FAEDVGSNKG30AIIGLMVGGV40VIA] [Aβ42] (the major precursor of the extracellular fibrillar deposits of Alzheimer's disease). Some truncated forms of caerin 1.8 also inhibit fibril formation of Aβ42. For example, caerin 1.8 (1–13) [1GLFKVLGSV10AKHL(NH2) and caerin 1.8 (22–25) [KVLGSV10AKHLLPHVVP20VIAEKL(NH2)] show 85% and 75% respectively of the inhibition activity of the parent caerin 1.8. The synthetic peptide KLVFFKKKKKK is a known inhibitor of Aβ42 fibril formation, and was used as a standard in this study. Caerin 1.8 is the more effective fibril inhibitor. IC50 values (±15%) are caerin 1.8 (75 μM) and KLVFFKKKKKK (370 μM). MALDI mass spectrometry shows the presence of a small peak corresponding to a protonated 1:1 adduct [caerin 1.8/Aβ42]H + . Molecular dynamics simulation suggests that both hydrogen bonding and hydrophobic interactions between Aβ42 and caerin 1.8 facilitate the formation of a 1:1 complex in water. Fibril formation from Aβ42 has been proposed to be based around the 16KLVF20F region of Aβ42; this region in the 1:1 complex is partially blocked from attachment of a further molecule of Aβ42.
Keywords: Alzheimer’s disease; Amyloid beta 1-42 (Aβ42); Amyloid fibrils; Caerin 1.8, an Aβ42 fibrilisation inhibitor; MALDI mass spectrometry: 1:1 adduct; [Aβ42/caerin 1.8]H+; MD simulation of 1:1 adduct in water;

Up-regulation of the kinin B2 receptor pathway modulates the TGF-β/Smad signaling cascade to reduce renal fibrosis induced by albumin by Areli Cárdenas; Javiera Campos; Pamela Ehrenfeld; Sergio Mezzano; Marta Ruiz-Ortega; Carlos D. Figueroa; Leopoldo Ardiles (7-19).
Display OmittedThe presence of high protein levels in the glomerular filtrate plays an important role in renal fibrosis, a disorder that justifies the use of animal models of experimental proteinuria. Such models have proved useful as tools in the study of the pathogenesis of chronic, progressive renal disease. Since bradykinin and the kinin B2 receptor (B2R) belong to a renoprotective system with mechanisms still unclarified, we investigated its anti-fibrotic role in the in vivo rat model of overload proteinuria. Upon up-regulating the kinin system by a high potassium diet we observed reduction of tubulointerstitial fibrosis, decreased renal expression of α-smooth muscle actin (α-SMA) and vimentin, reduced Smad3 phosphorylation and increase of Smad7. These cellular and molecular effects were reversed by HOE-140, a specific B2R antagonist. In vitro experiments, performed on a cell line of proximal tubular epithelial cells, showed that high concentrations of albumin induced expression of mesenchymal biomarkers, in concomitance with increases in TGF-β1 mRNA and its functionally active peptide, TGF-β1. Stimulation of the tubule cells by bradykinin inhibited the albumin-induced changes, namely α-SMA and vimentin were reduced, and cytokeratin recovered together with increase in Smad7 levels and decrease in type II TGF-β1 receptor, TGF-β1 mRNA and its active fragment. The protective changes produced by bradykinin in vitro were blocked by HOE-140.The development of stable bradykinin analogues and/or up-regulation of the B2R signaling pathway may prove value in the management of chronic renal fibrosis in progressive proteinuric renal diseases.
Keywords: Kinins; Proteinuria; Smad proteins; Transforming growth factor beta1; Fibrosis; Kidney;

Bioactive properties of milk proteins in humans: A review by Alice B. Nongonierma; Richard J. FitzGerald (20-34).
Many studies have demonstrated that milk protein consumption has benefits in terms of promoting human health. This review assesses the intervention studies which have evaluated potential health enhancing effects in humans following the ingestion of milk proteins. The impact of milk protein ingestion has been studied to asses their satiating, hypotensive, antimicrobial, anti-inflammatory, anticancer, antioxidant and insulinotropic properties as well as their impact on morphological modifications (e.g., muscle and fat mass) in humans. Consistent health promoting effects appear to have been observed in certain instances (i.e., muscle protein synthesis, insulinotropic and hypotensive activity). However, controversial outcomes have also been reported (i.e., antimicrobial, anti-inflammatory, anticancer and antioxidant properties). Several factors including interindividual differences, the timing of protein ingestion as well as the potency of the active components may explain these differences. In addition, processing conditions have been reported, in certain instances, to affect milk protein structure and therefore modify their bioactive potential. It is thought that the health promoting properties of milk proteins are linked to the release of bioactive peptides (BAPs) during gastrointestinal digestion. There is a need for further research to develop a more in-depth understanding on the possible mechanisms involved in the observed physiological effects. In addition, more carefully controlled and appropriately powered human intervention studies are required to demonstrate the health enhancing properties of milk proteins in humans.
Keywords: Milk proteins; Human studies; Bioactive peptides; Processing;

Leptin signaling in the hypothalamus is critical for normal food intake and body weight regulation. Cumulative evidence suggests that besides the signal transducer and activator of transcription-3 (STAT3) pathway, several non-STAT3 pathways including the phosphodiesterase-3B (PDE3B) pathway mediate leptin signaling in the hypothalamus. We have shown that PDE3B is localized in various hypothalamic sites implicated in the regulation of energy homeostasis and that the anorectic and body weight reducing effects of leptin are mediated by the activation of PDE3B. It is still unknown if PDE3B is expressed in the long form of the leptin-receptor (ObRb)-expressing neurons in the hypothalamus and whether leptin induces STAT3 activation in PDE3B-expressing neurons. In this study, we examined co-localization of PDE3B with ObRb neurons in various hypothalamic nuclei in ObRb-GFP mice that were treated with leptin (5 mg/kg, ip) for 2 h. Results showed that most of the ObRb neurons in the arcuate nucleus (ARC, 93%), ventromedial nucleus (VMN, 94%), dorsomedial nucleus (DMN, 95%), ventral premammillary nucleus (PMv, 97%) and lateral hypothalamus (LH, 97%) co-expressed PDE3B. We next examined co-localization of p-STAT3 and PDE3B in the hypothalamus in C57BL6 mice that were treated with leptin (5 mg/kg, ip) for 1 h. The results showed that almost all p-STAT3 positive neurons in different hypothalamic nuclei including ARC, VMN, DMN, LH and PMv areas expressed PDE3B. These results suggest the possibility for a direct role for the PDE3B pathway in mediating leptin action in the hypothalamus.
Keywords: Phosphodiesterase-3B; ObRb; p-STAT3; Hypothalamus; Leptin;

Neurotensin enhances glutamatergic EPSCs in VTA neurons by acting on different neurotensin receptors by Poulomee Bose; Pierre-Paul Rompré; Richard A. Warren (43-50).
Neurotensin (NT) is an endogenous neuropeptide that modulates dopamine and glutamate neurotransmission in several limbic regions innervated by neurons located in the ventral tegmental area (VTA). While several studies showed that NT exerted a direct modulation on VTA dopamine neurons less is known about its role in the modulation of glutamatergic neurotransmission in this region. The present study was aimed at characterising the effects of NT on glutamate-mediated responses in different populations of VTA neurons. Using whole cell patch clamp recording technique in horizontal rat brain slices, we measured the amplitude of glutamatergic excitatory post-synaptic currents (EPSCs) evoked by electrical stimulation of VTA afferents before and after application of different concentrations of NT1-13 or its C-terminal fragment, NT8-13. Neurons were classified as either I h + or I h based on the presence or absence of a hyperpolarisation activated cationic current (I h). We found that NT1-13 and NT8-13 produced comparable concentration dependent increase in the amplitude of EPSCs in both I h + and I h neurons. In I h + neurons, the enhancement effect of NT8-13 was blocked by both antagonists, while in I h neurons it was blocked by the NTS1/NTS2 antagonist, SR142948A, but not the preferred NTS1 antagonist, SR48692. In as much as I h neurons are non-dopaminergic neurons and I h + neurons represent both dopamine and non-dopamine neurons, we can conclude that NT enhances glutamatergic mediated responses in dopamine, and in a subset of non-dopamine, neurons by acting respectively on NTS1 and an NT receptor other than NTS1.
Keywords: Neurotensin; Ventral tegmental area; Glutamatergic EPSCs; SR142948A; SR48692;

Peptides from the scorpion Vaejovis punctatus with broad antimicrobial activity by Santos Ramírez-Carreto; Juana María Jiménez-Vargas; Bruno Rivas-Santiago; Gerardo Corzo; Lourival D. Possani; Baltazar Becerril; Ernesto Ortiz (51-59).
The antimicrobial potential of two new non-disulfide bound peptides, named VpAmp1.0 (LPFFLLSLIPSAISAIKKI, amidated) and VpAmp2.0 (FWGFLGKLAMKAVPSLIGGNKSSSK) is here reported. These are 19- and 25-aminoacid-long peptides with +2 and +4 net charges, respectively. Their sequences correspond to the predicted mature regions from longer precursors, putatively encoded by cDNAs derived from the venom glands of the Mexican scorpion Vaejovis punctatus. Both peptides were chemically synthesized and assayed against a variety of microorganisms, including pathogenic strains from clinical isolates and strains resistant to conventional antibiotics. Two shorter variants, named VpAmp1.1 (FFLLSLIPSAISAIKKI, amidated) and VpAmp2.1 (FWGFLGKLAMKAVPSLIGGNKK), were also synthesized and tested. The antimicrobial assays revealed that the four synthetic peptides effectively inhibit the growth of both Gram-positive (Staphylococcus aureus and Streptococcus agalactiaea) and Gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacteria, with MICs in the range of 2.5–24.0 μM; yeasts (Candida albicans and Candida glabrata) with MICs of 3.1–50.0 μM; and two clinically isolated strains of Mycobacterium tuberculosis-including a multi-drug resistant one- with MICs in the range of 4.8–30.5 μM. A comparison between the activities of the original peptides and their derivatives gives insight into the structural/functional role of their distinctive residues.
Keywords: Antimicrobial peptides; cDNA library; Hemolysis; Non-disulfide bound peptides; Scorpion; Therapeutic index;

Angiotensin (Ang)—a peptide that is part of the renin-angiotensin system-induces vasoconstriction and a subsequent increase in blood pressure; Ang peptides, especially AngII, can also act as potent pro-inflammatory mediators. Platelet-activating factor (PAF) is a potent phospholipid mediator that is implicated in many inflammatory diseases. In this study, we investigated the effects of Ang peptides (AngII, AngIII, and AngIV) on PAF-induced inflammatory activity. In experiments using a rat hind-paw oedema model, AngII markedly and dose-dependently attenuated the paw oedema induced by PAF. The inhibitory effects of AngIII and AngIV on PAF-induced paw oedema were lower than that of AngII. Two Ang receptors, the AT1 and AT2 receptors, did not affect the AngII-mediated attenuation of PAF-induced paw oedema. Moreover, intrinsic tyrosine fluorescence studies demonstrated that AngII, AngIII, and AngIV interact with PAF, and that their affinities were closely correlated with their inhibitory effects on PAF-induced rat paw oedema. Also, AngII interacted with metabolite/precursor of PAF (lyso-PAF), and an oxidized phospholipid, 1-palmitoyl-2-(5′-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC), which bears a marked structural resemblance to PAF. Furthermore, POVPC dose-dependently inhibited AngII-mediated attenuation of PAF-induced paw oedema. These results suggest that Ang peptides can attenuate PAF-induced inflammatory activity through binding to PAF and lyso-PAF in rats. Therefore, Ang peptides may be closely involved in the regulation of many inflammatory diseases caused by PAF.
Keywords: Angiotensin peptide; Platelet-activating factor; Interaction; Inflammation; Oxidized phospholipid;

Signaling pathways of a structural analogue of apelin-12 involved in myocardial protection against ischemia/reperfusion injury by Oleg I. Pisarenko; Valentin S. Shulzhenko; Irina M. Studneva; Larisa I. Serebryakova; Yulia A. Pelogeykina; Oxana M. Veselova (67-76).
Exogenously administered chemically modified apelin-12 (MA) has been shown to exhibit protective effects in myocardial ischemia/reperfusion (I/R) injury. They include reduction of ROS formation, cell death and cardiometabolic abnormalities. The aim of the present study was to explore the role of the underlying signaling mechanisms involved in cardioprotection afforded by MA. Isolated perfused working rat hearts subjected to global ischemia and anaesthetized rats in vivo exposed to LAD coronary artery occlusion were used. Myocardial infarct size, cell membrane damage, cardiac dysfunction and metabolic state of the heart were used as indices of I/R injury at the end of reperfusion. Administration of specific inhibitors of MEK1/2, PI3K, NO synthase (NOS) or the mitochondrial ATP-sensitive K+ (mito KATP) channels (UO126, LY294002, L-NAME or 5-hydroxydecanoate, respectively) reduced protective efficacy of MA in both models of I/R injury. This was evidenced by abrogation of infarct size limitation, deterioration of cardiac function recovery, and attenuation of metabolic restoration and sarcolemmal integrity. An enhancement of functional and metabolic recovery in isolated reperfused hearts treated with MA was suppressed by U-73122, chelerythrine, amiloride or KB-R7943 (inhibitors of phospholipase С (PLC), protein kinase C (PKC), Na+/H+ or Na+/Ca2+ exchange, respectively). Additionally, co-infusion of MA with amiloride or L-NAME reduced the integrity of cell membranes at early reperfusion compared with the effect of peptide alone. In conclusion, cardioprotection with MA is mediated by signaling via PLC and survival kinases, PKC, PI3K, and MEK1/2, with activation of downstream targets, NOS and mito KATP channels, and the sarcolemmal Na+/H+ and Na+/Ca2+ exchangers.
Keywords: Structural analogue of apelin-12; Signal transduction; Ischemia/reperfusion injury; Cardioprotection;

Ghrelin, an endogenous ligand of the growth hormone secretagogue receptor (GHSR), has been found to stimulate angiogenesis in vivo and in vitro. However, the effect and the corresponding mechanisms of ghrelin on impaired myocardial angiogenesis in diabetic and myocardial infarction (MI) rat model are still unknown.In the present study, adult SD rats were randomly divided into 4 groups: control, DM, DM + ghrelin, DM + ghrelin + [D-Lys3]-GHRP-6 groups. DM was induced by streptozotocin (STZ) 60 mg/kg body weight. 12 weeks post STZ injection all groups were subjected to MI, which was induced by ligation left anterior descending artery (LAD). Ghrelin and [D-Lys3]-GHRP-6 were administered via intraperitoneal injection at the doses 200 μg/kg and 50 mg/kg for 4 weeks, respectively. Left ventricular function, microvascular density (MVD), myocardial infarct size, the expression of hypoxia-inducible factor (HIF1α), vascular endothelial growth factor (VEGF), fetal liver kinase-1 (Flk-1) and fms-like tyrosine kinase-1 (Flt-1), AMPK and endothelial nitric oxide synthase (eNOS) phosphorylation were examined.Compared with the DM group, left ventricular ejection fraction (LVEF), fractional shortening (FS), and MVD were increased, whereas myocardial infarct size decreased remarkably in DM + ghrelin group. For the mechanism study, we found that ghrelin promoted the HIF1α, VEGF, Flk-1 and Flt-1 expression, AMPK and eNOS phosphorylation in diabetic rats. However, the above biochemical events in ghrelin treated diabetic rats were completely inhibited by GHSR-1a blocker [D-Lys3]-GHRP-6.These results suggest that administration of ghrelin ameliorates impaired angiogenesis in diabetic MI rats. And these beneficial effects derive from regulating GHSR1a-mediated AMPK/eNOS signal pathway by upregulating of HIF1α, VEGF and its receptors Flk-1, Flt-1 expressions.
Keywords: Ghrelin; GHSR; Angiogenesis; Diabetes; Myocardial infarction;

Regulatory effects of fibroblast growth factor-8 and tumor necrosis factor-α on osteoblast marker expression induced by bone morphogenetic protein-2 by Takayuki Katsuyama; Fumio Otsuka; Tomohiro Terasaka; Kenichi Inagaki; Mariko Takano-Narazaki; Yoshinori Matsumoto; Ken-Ei Sada; Hirofumi Makino (88-94).
BMP induces osteoblast differentiation, whereas a key proinflammatory cytokine, TNF-α, causes inflammatory bone damage shown in rheumatoid arthritis. FGF molecules are known to be involved in bone homeostasis. However, the effects of FGF-8 on osteoblast differentiation and the interaction between FGF-8, BMPs and TNF-α have yet to be clarified. Here we investigated the effects of FGF-8 in relation to TNF-α actions on BMP-2-induced osteoblast marker expression using myoblast cell line C2C12, osteoblast precursor cell line MC3T3-E1 and rat calvarial osteoblasts. It was revealed that FGF-8 inhibited BMP-2-induced expression of osteoblast differentiation markers, including Runx2, osteocalcin, alkaline phosphatase, type-1 collagen and osterix, in a concentration-dependent manner. The inhibitory effects of FGF-8 on BMP-induced osteoblast differentiation and Smad1/5/8 activation were enhanced in the presence of TNF-α action. FGF-8 also inhibited BMP-2-induced expression of Wnt5a, which activates a non-canonical Wnt signaling pathway. FGF-8 had no significant influence on the expression levels of TNF receptors, while FGF-8 suppressed the expression of inhibitory Smad6 and Smad7, suggesting a possible feedback activity through FGF to BMP receptor (BMPR) signaling. Of note, inhibition of ERK activity and FGF receptor (FGFR)-dependent protein kinase, but not JNK or NFκB pathway, suppressed the FGF-8 actions on BMP-induced osteoblast differentiation. FGF-8 was revealed to suppress BMP-induced osteoblast differentiation through the ERK pathway and the effects were enhanced by TNF-α. Given the finding that FGF-8 expression was increased in synovial tissues of rheumatoid arthritis, the functional interaction between FGFR and BMPR signaling may be involved in the development process of inflammatory bone damage.
Keywords: Bone morphogenetic protein (BMP); Fibroblast growth factor (FGF)-8; Osteoblast; Osteogenesis; Tumor necrosis factor (TNF)-α;

Increased food intake with oxyntomodulin analogues by Samantha L. Price; James S. Minnion; Stephen R. Bloom (95-100).
Oxyntomodulin analogues offer a novel treatment for obesity. However during analogue screening in a rat model increased food intake was consistently observed. To further investigate this finding, a series of representative analogues (OXM14 and OXM15) and their Glu-3 equivalents (OXM14E3 and OXM15E3) were administered to rats for 7 days and food intake and bodyweight measurements taken. To investigate the role of glucagon receptor activation glutamate (Glu/E) was substituted at amino acid position 3. GLP-1 and glucagon receptor efficacy of the oxyntomodulin analogues and their Glu-3 counterparts were measured at the rat receptors in vitro. Doses of 25 n mol/kg of OXM14 and OXM15 increased food intake by up to 20%. Bodyweight was not significantly increased. Food intake was not increased with the Glu-3 peptides, indicating that a glucagon receptor mechanism may be responsible for the increase in food intake.
Keywords: Oxyntomodulin; Glucagon; GLP-1; cAMP;

Hydrolyzed infant formulas serve as appropriate nutritional sources for infants afflicted with cow’s milk allergy, and milk proteins in hydrolyzed formulas are industrially hydrolyzed extensively or partially. To investigate whether industrial hydrolysis may modulate the digestive trajectory of milk proteins, thereby releasing different profiles of bioactive peptides compared with standard formulas, both standard and hydrolyzed formulas were subjected to in vitro digestion and formation of bioactive peptides were compared. One standard, one extensively hydrolyzed, and one partially hydrolyzed infant formula were digested in vitro with pepsin and pancreatin, taking into account the higher gastric pH of infants, and the digesta were subjected to peptidomic analysis. The standard formula released a larger variety of bioactive peptides than from the hydrolyzed formulas, indicating that industrial hydrolysis of milk proteins may generally attenuate their indigenous bioactivities such as antibacterial, immuno-regulatory, and anti-oxidative activities. Conversely, industrial hydrolysis may facilitate the formation of bioactive peptides from hydrophobic proteins/regions such as β-LG and the “strategic zone” of β-CN, which encrypt bioactive peptides including a dipeptidyl dipeptidase-4-inhibitory, hypocholesterolemic, and opioid peptides. Infants fed hydrolyzed infant formulas may be influenced by milk protein-derived bioactive peptides in a manner different from those fed standard formula.
Keywords: Bioactive peptide; Infant formula; In vitro digestion; Milk protein; Peptidomic technique;

Corrigendum to “Effects of urotensin II on intracellular pH regulation in cultured human internal mammary artery smooth muscle cells” Peptides 56 (2014) 173–182 by Yi-Ting Tsai; Chung-Yi Lee; Chih-Chin Hsu; Chung-Yi Chang; Ming-Kai Hsueh; Eagle Yi-Kung Huang; Chien-Sung Tsai; Shih-Hurng Loh (106).