Peptides (v.31, #7)

A fowlicidin-1 analog protects mice from lethal infections induced by methicillin-resistant Staphylococcus aureus by Yugendar R. Bommineni; Mallika Achanta; Justin Alexander; Lakshmi T. Sunkara; Jerry W. Ritchey; Guolong Zhang (1225-1230).
Fowlicidin-1 is a newly identified α-helical cathelicidin host defense peptide. We have shown that fowlicidin-1 possesses potent antibacterial activity, but also displays considerable toxicity toward mammalian cells. To further identify fowlicidin-1 analog(s) with enhanced therapeutic potential, a series of amino-terminal truncation analogs were synthesized and functionally evaluated. Relative to the full-length peptide, fowl-1(6-26), an analog with omission of five amino-terminal amino acid residues, maintained the antibacterial potency against a range of Gram-negative and Gram-positive bacteria including antibiotic-resistant strains. Fowl-1(6-26)-NH2, a carboxyl-terminal amidated form of fowl-1(6-26), retained the antibacterial activity for a minimum of 2 h in the presence of 100% serum. In addition, an intraperitoneal administration of 10 mg/kg of fowl-1(6-26)-NH2 led to a 50% increase in the survival of neutropenic mice over a 7-day period from a lethal dose of methicillin-resistant Staphylococcus aureus (MRSA), concomitant with a reduction in the bacterial titer in both peritoneal fluids and spleens of mice 24 h post-infection. Fowl-1(6-26)-NH2 at 20 μM was further found to suppress lipopolysaccharide-mediated production of TNF-α and nitric oxide in macrophages by 77% and 96%, respectively. Therefore, with potent endotoxin-neutralizing and bactericidal activities, fowlicidin-1(6-26)-NH2, may have strong therapeutic potential for drug-resistant infections and sepsis.
Keywords: Antimicrobial host defense peptides; Cathelicidin; Antimicrobial resistance; Methicillin-resistant Staphylococcus aureus (MRSA); Sepsis;

S-thanatin enhances the efficacy of tigecycline in an experimental rat model of polymicrobial peritonitis by Oscar Cirioni; Guoqiu Wu; Linxian Li; Fiorenza Orlando; Carmela Silvestri; Roberto Ghiselli; Zilong Shen; Alessandro Scalise; Eleonora Gabrielli; Daniele Scuppa; Chiara Romiti; Mauro Provinciali; Mario Guerrieri; Andrea Giacometti (1231-1236).
We investigated the efficacy of the peptide s-thanatin alone and in combination with tigecycline in an animal model of sepsis induced by cecal ligation and puncture. Adult male Wistar rats were randomized to receive intravenously isotonic sodium chloride solution, 5 mg/kg s-thanatin, 2 mg/kg tigecycline, 5 mg/kg s-thanatin combined with 2 mg/kg tigecycline. The experiment was also performed with administration of the drugs 360 min after the surgical procedure to better investigate the clinical situation where there is an interval between the onset of sepsis and the initiation of therapy. Lethality, bacterial growth in blood, peritoneum, spleen and liver, and NO indices were evaluated. All compounds reduced the lethality when compared to control. In all experiments, the compounds reduced significantly bacterial growth and lethality compared with saline treatment. Treatment with s-thanatin resulted in significant decrease in plasma NO levels compared to tigecycline and control group. The combination between s-thanatin and tigecycline proved to be the most effective treatment in reducing all variables measured. S-thanatin may have potential therapeutic usefulness alone and when associated to tigecycline in polymicrobial peritonitis.
Keywords: Antimicrobial peptides; S-thanatin; Tigecycline; Polymicrobial peritonitis;

Plantaricin A, a peptide pheromone produced by Lactobacillus plantarum, permeabilizes the cell membrane of both normal and cancerous lymphocytes and neuronal cells by Sverre L. Sand; Camilla Oppegård; Shinya Ohara; Toshio Iijima; Soheil Naderi; Heidi K. Blomhoff; Jon Nissen-Meyer; Olav Sand (1237-1244).
Antimicrobial peptides produced by multicellular organisms protect against pathogenic microorganisms, whereas such peptides produced by bacteria provide an ecological advantage over competitors. Certain antimicrobial peptides of metazoan origin are also toxic to eukaryotic cells, with preference for a variety of cancerous cells. Plantaricin A (PlnA) is a peptide pheromone with membrane permeabilizing strain-specific antibacterial activity, produced by Lactobacillus plantarum C11. Recently, we have reported that PlnA also permeabilizes cancerous rat pituitary cells (GH4 cells), whereas normal rat anterior pituitary cells are resistant. To investigate if preferential effect on cancerous cells is a general feature of PlnA, we have studied effects of the peptide on normal and cancerous lymphocytes and neuronal cells. Normal human B and T cells, Reh cells (from human B cell leukemia), and Jurkat cells (from human T cell leukemia) were studied by flow cytometry to detect morphological changes (scatter) and viability (propidium iodide uptake), and by patch clamp recordings to monitor membrane conductance. Ca2+ imaging based on a combination of fluo-4 and fura-red was used to monitor PlnA-induced membrane permeabilization in normal rat cortical neurons and glial cells, PC12 cells (from a rat adrenal chromaffin tumor), and murine N2A cells (from a spinal cord tumor). All the tested cell types were affected by 10–100 μM PlnA, whereas concentrations below 10 μM had no significant effect. We conclude that normal and cancerous lymphocytes and neuronal cells show similar sensitivity to PlnA.
Keywords: Plantaricin A (PlnA); Lactobacillus plantarum; Membrane permeabilization; Cell lysis; Lymphocytes; Neuronal cells;

Development of transgenic rice containing a mutated β subunit of soybean β-conglycinin for enhanced phagocytosis-stimulating activity by Takayasu Motoyama; Yoshiki Amari; Mary Rose Tandang-Silvas; Cerrone Cabanos; Aiko Kimura; Masaaki Yoshikawa; Fumio Takaiwa; Shigeru Utsumi; Nobuyuki Maruyama (1245-1250).
Improving the nutraceutical value of rice would positively impact the health and well-being of rice consumers worldwide. Based on the three-dimensional structure of soybean β-conglycinin, we designed a β subunit with a strong phagocytosis-stimulating activity (mβ subunit). Here, we describe the genetic modification and production of rice seeds containing the mβ subunit as part of our aim to develop a food material that promotes human health. The mβ subunit folded correctly and was accumulated in the protein body II of rice seeds at a level similar to wild-type β subunit. Mutant β subunit purified from transgenic rice seeds exhibited high phagocytosis-stimulating activity, demonstrating its potential value in enhancing the nutritional value of rice.
Keywords: Rice; Soybean; Bioactive peptides; Phagocytosis-stimulating activity;

Cell selectivity and anti-inflammatory activity of a Leu/Lys-rich α-helical model antimicrobial peptide and its diastereomeric peptides by Peng Wang; Yong Hai Nan; Sung-Tae Yang; Shin Won Kang; Yangmee Kim; Il-Seon Park; Kyung-Soo Hahm; Song Yub Shin (1251-1261).
To investigate the effect of the number and distribution of d-amino acids introduced into non-cell-selective α-helical antimicrobial peptides on the cell selectivity, protease stability and anti-inflammatory activity, we synthesized an 18-meric Leu/Lys-rich α-helical model peptide (K9L8W) and d-amino acid-containing diastereomeric peptides. Increasing in cell selectivity of the peptides was increased in parallel with increasing in the number of d-amino acids introduced. Despite having the same number of d-amino acids, D9-K9L8W-1 had better cell selectivity than D9-K9L8W-2, indicating that a dispersed distribution of d-amino acids in diastereomeric peptides is more effective for cell selectivity than their segregated distribution. D3-K9L8W-2, D6-K9L8W, D9-K9L8W-1 and D9-K9L8W-2 showed complete resistance to tryptic digestion. Furthermore, K9L8W and all of its diastereomeric peptides significantly inhibited nitric oxide (NO) production, inducible nitric oxide synthase (iNOS) mRNA expression and tumor necrosis factor-α (TNF-α) release in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells at a lower concentration than bactericidal concentration. The order of anti-inflammatory activity for the peptides was K9L8W ≈ D3-K9L8W-1 ≈ D3-K9L8W-2 ≈ D6-K9L8W ≈ D9-K9L8W-2 > D4-K9L8W > D9-K9L8W-1. Increasing in hydrophobicity or α-helicity of the peptides was more closely correlated with increasing in hemolytic activity and anti-inflammatory activity than antimicrobial and LPS-disaggregation activities. Collectively, we successfully developed several d-amino acid-containing antimicrobial peptides (D4-K9L8W, D6-K9L8W and D9-K9L8W-1) with good cell selectivity, protease stability and potent anti-inflammatory activity. These antimicrobial peptides could serve as templates for the development of peptide antibiotics for the treatment of sepsis, as well as microbial infection.
Keywords: Antimicrobial activity; Anti-inflammatory activity; Cell selectivity; Diastereomeric peptides; Hemolytic activity; Leu/Lys-rich model antimicrobial peptide;

Antimicrobial peptide of an anti-lipopolysaccharide factor modulates of the inflammatory response in RAW264.7 cells by Ming-Ching Lin; Shih-Bin Lin; Shang-Chun Lee; Ching-Chun Lin; Cho-Fat Hui; Jyh-Yih Chen (1262-1272).
In this study, to clarify the protective mechanism of a peptide from shrimp anti-lipopolysaccharide (LPS) factor (SALF) against endotoxin shock, we evaluated the effects of the SALF and LPS on the production and release of tumor necrosis factor (TNF)-αin vitro using the RAW264.7 murine macrophage cell line. Stimulation by LPS induced the production of inflammatory cytokines, and the SALF was able to modulate TNF-α production in LPS-stimulated RAW264.7 cells. Microarray studies revealed a transcriptional profile which was assessed in the presence or absence of the SALF by a quantitative real-time polymerase chain reaction. Pretreatment with the SALF significantly downregulated the expression of nuclear factor (NF)-κB in the presence of LPS. In contrast, pretreatment with the SALF significantly elevated the expressions of Anp32a, CLU, and SLPI, which are considered to be immune-related genes in the presence of LPS. Inhibitor studies suggested that the SALF's modulation of LPS-induced TNF-α production involved a complex mechanism with mitogen-activated protein kinase kinase, calcium, and protein kinase C. The data from this study, which imply that the SALF can suppress TNF-α production, suggest a role for the SALF in the defense mechanism which can potentially be applied to mammals for endotoxin treatment.
Keywords: Inflammatory response; RAW264.7 cells; Penaeus monodon; Anti-lipopolysaccharide factor;

Molecular cloning and characterization of V2-type receptor in two ray-finned fish, gray bichir, Polypterus senegalus and medaka, Oryzias latipes by Norifumi Konno; Mayumi Kurosawa; Hiroyuki Kaiya; Mikiya Miyazato; Kouhei Matsuda; Minoru Uchiyama (1273-1279).
In tetrapods, vasopressin (VP) and vasotocin (VT) are involved in various aspects of physiology and behavior including osmoregulation, cardiovascular function, reproduction, stress response and social behavior. Pharmacological and molecular studies have identified three types of VP/VT receptors, V1a-type (V1aR), V1b-type (V1bR) and V2-type (V2R). On the other hand, only V1aR has so far been identified in teleosts. In the present study, we successfully cloned V2Rs from two ray-finned fish, gray bichir and medaka. Phylogenetic analysis showed that the cloned receptors belong to the V2R group of lobe-finned fish and tetrapods. The amino acid sequences of bichir V2R and medaka V2R were high identity (60–65.5% and 53.2–80.9%, respectively) with other known V2R members, respectively. Reverse transcriptase PCR revealed that ray-finned fish V2R transcripts have been detected in various tissues including brain, gill, heart, liver, kidney and reproductive organs, suggesting that ray-finned fish V2R might mediate multiple functions of VT. In functional analysis, the cells transfected with the cloned receptors responded with the accumulation of intracellular cAMP in a concentration-dependent manner following VT stimulation, but not respond with [Ca2+]i. Furthermore, pretreatment with mammalian V2R antagonist (OPC-31260) to the cells transfected with medaka V2R significantly inhibited an increase of the VT-induced intracellular cAMP. These results suggest that ray-finned fish possess a functional V2R linked to adenylate cyclase and the cAMP signaling pathway as well as V2Rs of lobe-finned fish and tetrapods. Thus, the present study suggests that functional V2R evolved prior to the divergence of the ray- and lobe-finned fish lineages.
Keywords: Vasotocin; V2-type receptor; Ray-finned fish; Bichir; Medaka;

The first pacifastin elastase inhibitor characterized from a blood sucking animal by Renato de Marco; Diogo V. Lovato; Ricardo J.S. Torquato; Renan O. Clara; Diego S. Buarque; Aparecida S. Tanaka (1280-1286).
Pacifastin-like protease inhibitors belong to a recent classified protease inhibitor family and they are the smallest protease inhibitors described in animals. In this work, we purified and characterized, for the first time, two neutrophil elastase inhibitors belonging to the pacifastin family from the blood sucking insect Triatoma infestans eggs. The inhibitors showed the same N-terminal sequences, molecular masses of 4257 and 4024 Da by MALDI-TOF mass spectrometry and dissociation constants (Ki) for neutrophil elastase of 0.52 and 0.29 nM, respectively. Using a fat body cDNA library, we cloned a pacifastin precursor containing two protease inhibitor domains similar to locust pacifastins. The first pacifastin domain translated to T. infestans purified protein, named TIPI1. Recombinant TIPI1 expressed in Pichia pastoris system showed similar inhibitory activities compared to the native inhibitor. Its precursor, called TiPP1, is mainly expressed in fat body, and it is up-regulated after blood feeding. The immune challenges of 1a instar T. infestans nymph with bacteria or dsRNA strongly stimulated TiPP1 expression in fat body, suggesting a possible role of TiPP1 in T. infestans immunity. This work is the first to characterize a blood feeding insect pacifastin inhibitor.
Keywords: Pacifastin; Serine protease inhibitors; Hematophagous; Immune response;

Peptide sr11a from Conus spurius is a novel peptide blocker for Kv1 potassium channels by Manuel B. Aguilar; Liliana I. Pérez-Reyes; Zinaeli López; Edgar P. Heimer de la Cotera; Andrés Falcón; Cicerón Ayala; Marcelo Galván; Carolina Salvador; Laura I. Escobar (1287-1291).
More than a hundred conotoxins are known today and from them, only seven conopeptides have been identified to target voltage-gated potassium channels (Kv). Conotoxin sr11a belongs to the I2-superfamily which is characterized by four disulfide bridges and provokes muscle stiffness when injected intracranially in mice. The aim of this work was to test the biological activity of sr11a on recombinant voltage-gated Kv1 potassium channels expressed in Xenopus laevis oocytes. Peptide sr11a was purified by high-performance liquid chromatography from the venom of the vermivorous Conus spurius. We found that peptide sr11a inhibits the delayed rectifiers Kv1.2 and Kv1.6 but had not effect on the slowly inactivating Kv1.3 channel. The functional dyad composed of a basic Lys and a hydrophobic amino acid residue is a crucial structural element, regarding the binding properties and blocking activities of more than a hundred K+ channel toxins. Peptide sr11a does not contain Lys residues and then, it lacks the functional dyad. Molecular modeling of peptide sr11a reveals the presence of exposed basic residues of Arg and suggests that Arg17 and Arg29 are important on its biological activity.
Keywords: I2-superfamily conotoxin; Kv1 potassium channels; Conus; Vermivorous; Kv1.2; Kv1.6;

An unusual peptide from Conus villepinii: Synthesis, solution structure, and cardioactivity by Alesia Miloslavina; Christina Ebert; Daniel Tietze; Oliver Ohlenschläger; Christoph Englert; Matthias Görlach; Diana Imhof (1292-1300).
The venom of marine cone snails contains a variety of conformationally constrained peptides utilized by the animal to capture prey. Besides numerous conotoxins, which are characterized by complex disulfide patterns, other peptides with only a single disulfide bridge were isolated from different conus species. Here, we report the synthesis, structure elucidation and biological evaluation of the novel C-terminally amidated decapeptide CCAP-vil, PFc[CNSFGC]YN-NH2, from Conus villepinii. The linear precursor peptide was generated by standard solid phase synthesis. Oxidation of the cysteine residues to yield the disulfide-bridged peptide was investigated under different conditions, including several ionic liquids (ILs) as new biocompatible reaction media. Among the examined ILs, 1-ethyl-3-methylimidazolium tosylate ([C2mim][OTs]) was most efficient for CCAP-vil oxidative folding, since oxidation occurred without any byproduct formation. The structure of CCAP-vil was determined by NMR methods in aqueous solution and revealed a loop structure adopting a type(I) β-turn between residues 4–7 imposed by the flanking disulfide bridge. The amino acid side chains of Pro1, Phe2, Phe6 and Tyr9 point in three directions away from the cyclic core into the solvent creating a rather hydrophobic surface of the molecule. Based on sequence homology to cardioactive peptides (CAPs) from gastropods and arthropods, such as PFc[CNAFTGC]-NH2 (CCAP), the influence of CCAP-vil on heart rate using zebrafish embryos was investigated. CCAP-vil reduced the heart rate immediately upon injection into the heart as well as upon indirect application indicating an opposite effect to the cardioaccelerating CCAP.
Keywords: Conopeptide; Oxidation; Ionic liquids; NMR solution structure; Cardioactivity; Zebrafish embryos;

Morphology of neuropeptide CNP2 modulation of heart activity in terrestrial snail by Nikolay Aseyev; Igor S. Zakharov; Pavel M. Balaban (1301-1308).
A family of neuropeptides called Command Neuron Peptides (CNPs) was described ten years ago as the protein products of the gene HCS2, specifically expressed in the identified interneurons of the nervous system of terrestrial snail (Helix lucorum L. and H. pomatia L.). Recently, the CNP-like peptides have been detected by immunochemistry and immunoblotting in nervous systems of representatives of different invertebrate phyla (Mollusca, Annelida, and Insecta). Still, the function of these peptides remains largely unknown. In Helix it is shown that CNPs: modulate the electrical activity of unidentified central neurons, modulate the pneumostome motoneurons, stimulate neural cones growth in neural cultures. Here, we describe for the first time the CNPs-immunoreactive neural fibers in walls of both auricle and ventricle of the snail heart. We show that application of the synthetic neuropeptide CNP2 (DYPRLamide) in perfusion saline affects heart rate and magnitude of beats in isolated snail heart. The results suggest that in Helix the Command Neuron Peptides could participate in neural modulation of cardiovascular system.
Keywords: Heart control; Command Neuron Peptide; Snail; Mollusk; Helix;

C-type natriuretic peptide effects on cardiovascular nitric oxide system in spontaneously hypertensive rats by Carolina Caniffi; Rosana Elesgaray; Mariela Gironacci; Cristina Arranz; María Ángeles Costa (1309-1318).
The aim was to study the effects of C-type natriuretic peptide (CNP) on mean arterial pressure (MAP) and the cardiovascular nitric oxide (NO) system in spontaneously hypertensive rats (SHR), and to investigate the signaling pathways involved in this interaction. SHR and WKY rats were infused with saline or CNP. MAP and nitrites and nitrates excretion (NO x ) were determined. Catalytic NO synthase (NOS) activity and endothelial (eNOS), neuronal (nNOS) and inducible NOS (iNOS) were measured in the heart and aorta artery. NOS activity induced by CNP was determined in presence of: iNOS or nNOS inhibitors, NPR-A/B natriuretic peptide receptors blocker and Gi protein and calmodulin inhibitors. CNP diminished MAP and increased NO x in both groups. Cardiovascular NOS activity was higher in SHR than in WKY. CNP increased NOS activity, but this activation was lower in SHR. CNP had no effect on NOS isoforms expression. iNOS and nNOS inhibitors did not modify CNP-induced NOS activity. NPR-A/B blockade induced no changes in NOS stimulation via CNP in both tissues. Cardiovascular NOS response to CNP was reduced by Gi protein and calmodulin inhibitors in both groups. CNP interacts with NPR-C receptors, activating Ca–calmodulin eNOS via Gi protein. NOS response to CNP is impaired in the heart and aorta of SHR. Alterations in the interaction between CNP and NO would be involved in the maintenance of high blood pressure in this model of hypertension.
Keywords: CNP; Spontaneously hypertensive rats; Nitric oxide synthase; Heart; Aorta artery;

Upregulation of ANP and NPR-C mRNA in the kidney and heart of eNOS knockout mice by Kuichang Yuan; Sun Young Kim; Young-Bin Oh; Jiahua Yu; Amin Shah; Byung Hyun Park; Suhn Hee Kim (1319-1325).
The aim of the present studywas to examine the question of whether the atrial natriuretic peptide (ANP) system is altered by endothelial nitric-oxide synthase (eNOS).Male eNOS-deficient mice (eNOS−/−) and wild type control mice (eNOS+/+, C57B1/6J) were used. Blood pressure was measured in anesthetized mice by tail cuff plethysmography and renal function was measured. Expression of ANP, natriuretic peptide receptor (NPR)-A, NPR-C, and tonicity-responsive enhancer binding protein (TonEBP) mRNA was determined by real-time PCR. Localization of 125I-ANP binding sites was measured using in vitro autoradiography.In eNOS−/− mice, systolic blood pressure increased and left ventricular hypertrophy was observed. Urine volume and osmolarity did not change. Expression of ANP markedly increased in the heart and kidney of eNOS−/− mice. Expression of NPR-A and NPR-C increased in the heart and tended to increase in the kidney of eNOS−/− mice. In the renal medulla in particular, increased expression of NPR-C was more prominent. Expression of TonEBP mRNA was markedly decreased in the renal medulla, but not in the renal cortex. Maximum binding capacity (Bmax) of ANP and C-ANP increased in the renal medulla in eNOS−/− mice.These results suggest that the eNOS-NO system may be partly involved in regulation of ANP, NPR-A, -C, and TonEBP mRNA expression in the kidney.
Keywords: Medulla; Osmolarity; Blood pressure; Nitric oxide; Natriuretic peptide receptor; TonEBP; Atrial natriuretic peptide; Renal function;

Urotensin II induction of adult cardiomyocytes hypertrophy involves the Akt/GSK-3β signaling pathway by D. Gruson; A. Ginion; N. Decroly; P. Lause; J.L. Vanoverschelde; J.M. Ketelslegers; L. Bertrand; J.P. Thissen (1326-1333).
Urotensin II (UII) a potent vasoactive peptide is upregulated in the failing heart and promotes cardiomyocytes hypertrophy, in particular through mitogen-activated protein kinases. However, the regulation by UII of GSK-3β, a recognized pivotal signaling element of cardiac hypertrophy has not yet been documented. We therefore investigated in adult cardiomyocytes, if UII phosphorylates GSK-3β and Akt, one of its upstream regulators and stabilizes β-catenin, a GSK-3β dependent nuclear transcriptional co-activator. Primary cultures of adult rat cardiomyocytes were stimulated for 48 h with UII. Cell size and protein/DNA contents were determined. Phosphorylated and total forms of Akt, GSK-3β and the total amount of β-catenin were quantified by western blot. The responses of cardiomyocytes to UII were also evaluated after pretreatment with the chemical phosphatidyl-inositol-3-kinase inhibitor, LY294002, and urantide, a competitive UII receptor antagonist. UII increased cell size and the protein/DNA ratio, consistent with a hypertrophic response. UII also increased phosphorylation of Akt and its downstream target GSK-3β. β-Catenin protein levels were increased. All of these effects of UII were prevented by LY294002, and urantide. The UII-induced adult cardiomyocytes hypertrophy involves the Akt/GSK-3β signaling pathways and is accompanied by the stabilization of the β-catenin. All these effects are abolished by competitive inhibition of the UII receptor, consistent with new therapeutic perspectives for heart failure treatment.
Keywords: Urotensin II; Cardiomyocytes; Heart failure; Hypertrophy; Akt;

Regulation of angiotensin converting enzyme II by angiotensin peptides in human cardiofibroblasts by Chih-Sheng Lin; Chun-Hsu Pan; Cheng-Hao Wen; Tzu-Hui Yang; Tang-Ching Kuan (1334-1340).
Numerous studies have suggested that angiotensin peptides modulate the expression of angiotensin converting enzyme II (ACE2) in the cardiovascular system, but the molecular mechanisms remain poorly understood. In the present study, human cardiac fibroblasts (HCF) were used to test the regulatory effects of angiotensin II (Ang II) and angiotensin-(1–7) [Ang-(1–7)] on ACE2 expression. The results show that Ang II upregulates ACE2 expression. This action is modulated through activation of Ang II type 1 receptor (AT1R). Ang II-mediated ACE2 upregulation was blocked by antagonists of AT1R and ERK–MAPK signaling pathways. Additionally, Ang-(1–7) increased ACE2 expression, and this upregulation was inhibited by Ang-(1–7) Mas receptor blockade. Our results further reveal that the activation of p-ERK1/2 proteins plays a critical role in upregulating ACE2 in Ang-(1–7)-stimulated HCF cells. This effect occurs independently of the Ang II–AT1R signaling pathway. In conclusion, we propose that Ang II-upregulated ACE2 may increase Ang-(1–7) formation from Ang II, and that ACE2 expression is further enhanced by Ang-(1–7) in a positive feedback loop.
Keywords: Angiotensin II; Angiotensin-(1–7); Angiotensin converting enzyme II; Human cardiofibroblasts;

Oxytocin protects rat heart against ischemia–reperfusion injury via pathway involving mitochondrial ATP-dependent potassium channel by Ali Mohammad Alizadeh; Mahdieh Faghihi; Hamid Reza Sadeghipour; Fahimeh MohammadGhasemi; Alireza Imani; Fariba Houshmand; Vahid Khori (1341-1345).
Cardiac preconditioning represents the most potent and consistently reproducible method of rescuing heart tissue from undergoing irreversible ischemic damage. One of the major goals of the current cardiovascular research is to identify a reliable cardioprotective intervention that can salvage ischemic myocardium. The aim of the present study is to evaluate the oxytocin (OT)-induced cardioprotection and the signaling pathway involved with mitochondrial ATP-dependent potassium (mitoKATP) channel in the anesthetized rat heart. Animals were divided into six groups (n  = 6): (1) IR; hearts were subjected to 25 min ischemia and 120 min reperfusion, (2) OT; oxytocin was administered (0.03 μg/kg i.p.) 25 min prior to ischemia, (3) ATO+OT; atosiban (ATO) was used as an OT-selective receptor antagonist (1.5 μg/kg i.p.) 10 min prior to OT administration, (4) ATO; atosiban was used 35 min prior to ischemia, (5) 5HD + OT; 5-hydroxydecanoic acid (5HD) was used as a specific inhibitor of mitoKATP channel (10 mg/kg i.v.) 10 min prior to OT administration, (6) 5HD; 5HD was used 35 min prior to ischemia. Then infarct size, ventricular arrhythmia and creatine kinase-MB isoenzyme (CK-MB) plasma level were measured. Hemodynamic parameters were recorded throughout the experiment. OT administration significantly decreased infarct size, CK-MB plasma level, severity and incidence of ventricular arrhythmia as compared to IR group. Administration of atosiban and 5HD abolished the cardiopreconditioning effect of OT. This study demonstrates that cardioprotective effects of OT are mediated through opening the mitoKATP channels.
Keywords: Ischemia; Reperfusion; Atosiban; Oxytocin; 5HD;

Chronic sugar intake dampens feeding-related activity of neurons synthesizing a satiety mediator, oxytocin by Anaya Mitra; Blake A. Gosnell; Helgi B. Schiöth; Martha K. Grace; Anica Klockars; Pawel K. Olszewski; Allen S. Levine (1346-1352).
Increased tone of orexigens mediating reward occurs upon repeated consumption of sweet foods. Interestingly, some of these reward orexigens, such as opioids, diminish activity of neurons synthesizing oxytocin, a nonapeptide that promotes satiety and feeding termination. It is not known, however, whether consumption-related activity of the central oxytocin system is modified under chronic sugar feeding reward itself. Therefore, we examined how chronic consumption of a rewarding high-sucrose (HS) vs. bland cornstarch (CS) diet affected the activity of oxytocin cells in the hypothalamus at the time of meal termination. Schedule-fed (2 h/day) rats received either a HS or CS powdered diet for 20 days. On the 21st day, they were given the same or the opposite diet, and food was removed after the main consummatory activity was completed. Animals were perfused 60 min after feeding termination and brains were immunostained for oxytocin and the marker of neuronal activity, c-Fos. The percentage of c-Fos-positive oxytocin cells in the hypothalamic paraventricular nucleus was significantly lower in rats chronically exposed to the HS than to the CS diet, regardless of which diet they received on the final day. A similar pattern was observed in the supraoptic nucleus. We conclude that the chronic rather than acute sucrose intake reduces activity of the anorexigenic oxytocin system. These findings indicate that chronic consumption of sugar blunts activity of pathways that mediate satiety. We speculate that a reduction in central satiety signaling precipitated by regular intake of foods high in sugar may lead to generalized overeating.
Keywords: Sucrose; Starch; Obesity; Palatability; Reward; Satiation; Oxytocin;

Exploration of structure–activity relationships at the two C-terminal residues of potent 11mer Glucagon-Like Peptide-1 receptor agonist peptides via parallel synthesis by Tasir S. Haque; Rogelio L. Martinez; Ving G. Lee; Douglas G. Riexinger; Ming Lei; Ming Feng; Barry Koplowitz; Claudio Mapelli; Christopher B. Cooper; Ge Zhang; Christine Huang; William R. Ewing; John Krupinski (1353-1360).
We report the identification of potent agonists of the Glucagon-Like Peptide-1 receptor (GLP-1R) via evaluation of two positional scanning libraries and a two-dimensional focused library, synthesized in part on SynPhase™ Lanterns. These compounds are 11 amino acid peptides containing several unnatural amino acids, including (in particular) analogs of biphenylalanine (Bip) at the two C-terminal positions. Typical activities of the most potent peptides in this class are in the picomolar range in an in vitro functional assay using human GLP-1 receptor.
Keywords: GLP-1R; Agonist; 11mer; Biphenylalanine; Combinatorial; Library; Parallel;

The aim of this study was to assess the effects of dietary leucine supplementation in lactating dams, particularly on energy homeostasis through signaling mechanisms in the central nervous system. Dams were fed ad libitum with standard diet during pregnancy (control dams) or supplemented with 2% leucine (leucine-supplemented dams) from delivery onwards. Food intake, body weight and composition were periodically recorded. Hypothalamus was collected at the end of lactation, and the expression of neuropeptide Y (NPY), agouti-related protein (AgRP) pro-opiomelanocortin (POMC), cocaine and amphetamine regulated transcript (CART), insulin receptor (InsR), ghrelin receptor (GSHR), melanocortin receptor (MCR4), leptin receptor (Ob-Rb) and suppressor of cytokine signaling 3 (SOCS3) were analyzed. Dietary leucine supplementation to lactating rats increased plasma leucine by 56%, modulated body composition and contributed to a tendency of higher ratio of lean/fat mass content of dams during lactation, without affecting food intake, thermogenesis capacity or body or tissue/organs weights. No differences in body weight of offspring from control and leucine-supplemented dams were found. The expression of orexigenic peptides (NPY and AgRP) decreased in leucine-dams, whereas the expression of anorexigenic peptides (POMC and CART), the hypothalamic receptors of insulin, ghrelin, melanocortin and leptin and SOCS3 did not change by leucine supplementation. In conclusion, increased leucine intake during lactation may contribute to a healthier profile of body composition in dams, without compromising the growth and development of the progeny by a mechanism associated with lower expression of orexigenic neuropeptides in hypothalamus.
Keywords: Leucine supplementation; Lactation; NPY; AgRP; POMC; CART;

NAP (davunetide) enhances cognitive behavior in the STOP heterozygous mouse—A microtubule-deficient model of schizophrenia by Avia Merenlender-Wagner; Regina Pikman; Eliezer Giladi; Annie Andrieux; Illana Gozes (1368-1373).
NAP (generic name, davunetide) is an active fragment of activity-dependent neuroprotective protein (ADNP). ADNP−/− embryos exhibit CNS dysgenesis and die in utero. ADNP+/− mice survive but demonstrate cognitive dysfunction coupled with microtubule pathology. NAP treatment ameliorates, in part, ADNP-associated dysfunctions. The microtubule, stable tubule-only polypeptide (STOP) knockout mice were shown to provide a reliable model for schizophrenia. Here, STOP−/− as well as STOP+/− showed schizophrenia-like symptoms (hyperactivity) that were ameliorated by chronic treatment with the antipsychotic drug, clozapine. Daily intranasal NAP treatment significantly decreased hyperactivity in the STOP+/− mice and protected visual memory.
Keywords: Schizophrenia; Stable tubule-only polypeptide (STOP); Activity-dependent neuroprotective protein; NAP (davunetide); Hyperactivity; Visual memory;

Neuropeptide FF (NPFF) exhibited anti-/pro-opioid effects when centrally injected. It was proved to bind to its own receptors, namely NPFF1 and NPFF2 receptors, but did not bind to opioid receptors. In our previous study, we found that i.c.v. injected NPFF suppressed morphine-induced conditioned place preference (CPP) in rats, which indicated that NPFF may play a role in the modulation of morphine-induced reward. In the present study, we further investigated the action site of NPFF to attenuate morphine-induced reward. Bilateral intra-VTA (ventral tegmental area) and intra-NAc (nucleus accumbens) injections of NPFF both blocked the CPP caused by morphine in rats. This suggests that NPFF may act at both VTA and NAc to inhibit the sensitization of the mesocorticolimbic dopaminergic pathway. Neurochemical analyses support that NPFF could be acting through the inhibition of the mesocorticolimbic dopaminergic activity increased by morphine. We also determined the distribution of NPFF receptors in rat brains. Our results showed that both NPFF receptors were abundantly expressed in VTA but with less content in NAc. In fluorescent immunohistochemical staining, our results revealed that NPFF1 and NPFF2 receptors could be expressed at the TH (tyrosine hydroxylase)- or GAD67 (glutamic acid decarboxylase-67)-positive neurons in VTA, whereas some of them were present in the negative neurons. This implied a possible function of NPFF to modulate dopaminergic neurons directly and a possible indirect action of NPFF on GABAergic neurons to modulate dopamine release. Taken together, our study should be helpful for clarifying the possible mechanisms of NPFF system to modulate morphine-induced reward.
Keywords: Morphine; Neuropeptide FF (NPFF); Mesolimbic pathway; Conditioned place preference (CPP); Dopamine; GABA;

We made Drosophila which express the mu opioid receptor under control of UAS in order to inactivate neurons or neuroendocrine cells expressing this receptor with opioid agonists. However, while exposing flies expressing the mu opioid receptor in the SIFamide neurons to opioid agonists was expected to induce male-male courtship behavior, this did not occur. Furthermore, flies which expressed the mu opioid receptor in the AKH or corazonin endocrine cells increased rather than decreased trehalose levels and this was independent of opioid agonists. When the mu opioid receptor is expressed in AKH endocrine cells whole body glycogen also increases, which is no longer the case if the expression of the AKH gene is suppressed by RNAi. It appears that mu opioid receptors expressed in AKH or corazonin endocrine cells are constitutively active and facilitate release of neurohormones. The simultaneous increase in both glycogen and trehalose in these flies suggested that they consumed more food. Indeed, when normally fed males are offered sucrose, those that express this receptor in AKH cells consumed more sucrose, suggesting that AKH increases the motivation to feed. These pharmacological effects of the mu opioid receptor are not limited to neuroendocrine cells; expressing it in the fat body also leads to an increase in trehalose. Thus in Drosophila the mu opioid receptors appear to change the base line activity in the cells in which it is expressed, not unlike to what has been found in transgenic mice expressing receptors activated solely by synthetic ligands with significant constitutive activity.
Keywords: SIFamide; Corazonin; RASSL; RNAi;

Analytical characterization and comparison of the blood–brain barrier permeability of eight opioid peptides by Sylvia Van Dorpe; Antita Adriaens; Ingeborgh Polis; Kathelijne Peremans; Jan Van Bocxlaer; Bart De Spiegeleer (1390-1399).
Opioid drugs, including the newly developed peptides, should penetrate the blood–brain barrier (BBB) for pain management activity. Although BBB transport is fragmentarily described for some μ-opioid peptides, a complete and comparative overview is currently lacking. In this study, the BBB transport of eight opioid peptides (EM-1, EM-2, CTAP, CTOP, DAMGO, dermorphin, TAPP and TAPS) is described and compared. In addition, the metabolic stability in plasma and brain was evaluated. The highest influx rate was obtained for dermorphin (K in  = 2.18 μl/(g × min)), followed by smaller rates for EM-1, EM-2 and TAPP (K in  = 1.06–1.14 μl/(g × min)). Negligible influx was observed for DAMGO, CTOP and TAPS (K in  = 0.18–0.40 μl/(g × min)) and no influx for CTAP. Capillary depletion revealed that all peptides reached brain parenchyma for over 75%. Efflux was shown for TAPP (t 1/2  = 2.82 min) and to a lesser extent for EM-1, EM-2 and DAMGO (t 1/2  = 10.66–21.98 min), while no significant efflux was observed for the other peptides. All peptides were stable in mouse plasma and brain, with generally higher stability in brain, except for EM-1 and EM-2 which showed plasma half-life stabilities of a few minutes only.
Keywords: Blood–brain barrier; Metabolism; Opioid receptor; Peptides;

A key role for tryptophan 84 in receptor activity-modifying protein 1 in the amylin 1 receptor by Joseph J. Gingell; Tao Qi; Richard J. Bailey; Debbie L. Hay (1400-1404).
Amylin (Amy) receptors are complexes of the calcitonin receptor with receptor activity-modifying proteins. RAMP1 with the calcitonin receptor forms the AMY1 receptor; the insert negative isoform of the calcitonin receptor in this complex makes the AMY1(a) receptor. This receptor has high affinity for Amy and the related peptide calcitonin gene-related peptide (CGRP). Amy is a peptide that has a role in lowering blood glucose levels and therefore its receptors represent potential drug targets for the treatment of diabetes. It has been suggested that the peptides bind in a pocket formed between the long N-termini of the calcitonin receptor and RAMP1, although very few residues in either component have been assigned specific roles. Based on the crystal structure of the RAMP1 N-terminus, the RAMP1 residues Arg67, Asp71, Glu78, Trp74 and Trp84 were identified as potentially having a role in peptide binding. Here, Arg67, Asp71, Glu78 and Trp84 were individually mutated to alanine and the function of mutant AMY1(a) receptors was determined using a cAMP assay. ELISA was used to measure cell surface expression and western blotting for total expression. Mutation of Arg67, Asp71 and Glu78 had no significant effect on Amy or CGRP potency, cell surface or total expression. Trp84Ala, however, resulted in a significant reduction in agonist potency and cell surface expression. Interestingly, a Trp84Phe substitution was able to restore some of this function, without restoring cell surface expression, suggesting that the residue may be important for peptide interactions. The data reveal the importance of Trp84 in the AMY1(a) receptor.

Expression of (pro)renin receptor in human kidneys with end-stage kidney disease due to diabetic nephropathy by Kazuhiro Takahashi; Hajime Yamamoto; Takuo Hirose; Keisuke Hiraishi; Itaru Shoji; Akiko Shibasaki; Ichiro Kato; Kiriko Kaneko; Hironobu Sasano; Fumitoshi Satoh; Kazuhito Totsune (1405-1408).
(Pro)renin receptor ((P)RR), a specific receptor for renin and prorenin, is a 350 amino-acid protein with a single transmembrane domain and may play important pathophysiological roles in diabetic nephropathy. The aim of the present study is to clarify the expression of (P)RR in the kidney with end-stage renal disease due to diabetic nephropathy. The kidney tissues were obtained at autopsy from patients with and without Type 2 diabetes mellitus (n  = 5 without diabetes mellitus; and n  = 8 with diabetes mellitus). Immunocytochemistry showed that (P)RR was mainly expressed in the tubular cells and collecting duct cells of the kidney without diabetic nephropathy. Cells in glomeruli were very weakly and sporadically immunostained for (P)RR. Vascular smooth muscle cells and endothelial cells were very weakly or were not immunostained for (P)RR. Adipocytes in the adipose tissue around the kidney were positively immunostained for (P)RR. Immunostaining pattern of (P)RR in the kidney with diabetic nephropathy was similar to that without diabetic nephropathy. However, most notably, (P)RR immunostaining in the tubular cells and collecting duct cells was clearly and frequently more strongly observed in the kidney with diabetic nephropathy up to the end-stage renal disease. The present study has raised the possibility that (P)RR expressed in the diabetic kidney may play a pathophysiological role in angiotensin I generation and renal fibrosis found in end-stage renal disease.

The anti-Epstein-Barr Virus (EBV) myristoylated-peptide (M.W. 916.2 Da) is a natural product isolated from Heliothis virescens insect larval hemolymph (blood) that essentially has no cytotoxicity against human foreskin fibroblast cells. A (3 methyl only) version (M.W. 902.2 Da) of the structure was synthesized and tested for in vitro anti-EBV activity and cytotoxicity. The N-terminal end is lipophilic and used to get the compound across the cell membrane. The C-terminal end with its ring-shaped structures is likely used to inhibit DNA synthesis. The synthetic compound inhibited DNA synthesis/replication of EBV in Akata cells (B-lymphocyte from Burkitt's lymphoma patient) in in vitro tissue culture. A DNA hybridization assay for anti-EBV activity using the Akata B-cell and two cytotoxicity assays using human foreskin fibroblast cells were done with the synthetic peptide. Effective concentration (EC90) at 20 μM inhibited viral replication by 90%. The EBV, known as Human Herpesvirus-4 (HHV-4) of the Herpesviridae family, has been described as a cancer-promoting double-stranded DNA virus that may also be involved in autoimmune disease. There are no antiviral drugs in clinical use for diseases caused by the EBV.

Atrial natriuretic peptide and oxidative stress by Paolo De Vito; Sandra Incerpi; Jens Z. Pedersen; Paolo Luly (1412-1419).
Atrial natriuretic peptide (ANP) is a hormone, produced mainly by cardiomyocytes, with a major role in cardiovascular homeostatic mechanisms such as natriuresis and vasodilation, which serve to regulate blood pressure. However, ANP also acts as an autocrine/paracrine factor on other targets such as kidney, lung, thymus, liver and the immune system. ANP participates in the regulation of cell growth and proliferation, and evidence is accumulating that these effects are associated with the generation of reactive oxygen species (ROS). In vascular cells and cardiomyocytes ANP stimulates the antioxidant defense, but in other systems such as hepatoblastoma and macrophages ANP may produce either antioxidant or prooxidant effects, depending on experimental conditions and cell context. At present very little is known on the relationship between ANP and ROS production in the normal homeostatic processes or during the development of cardiovascular diseases and cancer. Our current knowledge of the role of ANP in signaling pathways leading to the generation of intracellular messengers such as diacylglycerol (DAG), and guanosine 3′-5′-cyclic monophosphate has been examined in order to clarify the mechanisms by which the hormone may counteract or contribute to the potentially dangerous effects of free radicals.
Keywords: ANP; Reactive oxygen species; Superoxide; Antioxidant;