Peptides (v.36, #2)

The antimicrobial activity of the appetite peptide hormone ghrelin by Christine Min; Kouji Ohta; Mikihito Kajiya; Tongbo Zhu; Kanika Sharma; Jane Shin; Hani Mawardi; Mohammed Howait; Josefine Hirschfeld; Laila Bahammam; Isao Ichimonji; Srinivas Ganta; Mansoor Amiji; Toshihisa Kawai (151-156).
► Ghrelin possesses antimicrobial activities against Gram(+) bacteria. ► Ghrelin's antimicrobial activities against Gram(−) bacteria seems to be minimal. ► Ghrelin's cationic property appears to be responsible for its antimicrobial action. ► Ghrelin loses its bactericidal activity in the physiological NaCl concentration.The present study examined the antimicrobial activity of the peptide ghrelin. Both major forms of ghrelin, acylated ghrelin (AG) and desacylated ghrelin (DAG), demonstrated the same degree of bactericidal activity against Gram-negative Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa), while bactericidal effects against Gram-positive Staphylococcus aureus (S. aureus) and Enterococcus faecalis (E. faecalis) were minimal or absent, respectively. To elucidate the bactericidal mechanism of AG and DAG against bacteria, we monitored the effect of the cationic peptides on the zeta potential of E. coli. Our results show that AG and DAG similarly quenched the negative surface charge of E. coli, suggesting that ghrelin-mediated bactericidal effects are influenced by charge-dependent binding and not by acyl modification. Like most cationic antimicrobial peptides (CAMPs), we also found that the antibacterial activity of AG was attenuated in physiological NaCl concentration (150 mM). Nonetheless, these findings indicate that both AG and DAG can act as CAMPs against Gram-negative bacteria.
Keywords: Ghrelin; Peptide; Escherichia coli; Pseudomonas aeruginosa; LL37; Antimicrobial activity;

► Ghrelin stimulates TRH-like peptide release in brain and peripheral tissues. ► 3-Trp-ghrelin inhibits TRH-like peptide release in brain and peripheral tissues. ► TRH-like peptides mediate opposing effects of aceylated and unacylated ghrelin.Ghrelin is not only a modulator of feeding and energy expenditure but also regulates reproductive functions, CNS development and mood. Obesity and major depression are growing public health concerns which may derive, in part, from dysregulation of ghrelin feedback at brain regions regulating feeding and mood. We and others have previously reported that thyrotropin-releasing hormone (TRH, pGlu-His-Pro-NH2) and TRH-like peptides (pGlu-X-Pro-NH2, where “X” can be any amino acid residue) have neuroprotective, antidepressant, anti-epileptic, analeptic, anti-ataxic, and anorectic properties. For this reason male Sprague-Dawley rats were injected ip with 0.1 mg/kg rat ghrelin or 0.9 mg/kg 3-Trp-rat ghrelin. Twelve brain regions: cerebellum, medulla oblongata, anterior cingulate, posterior cingulate, frontal cortex, nucleus accumbens, hypothalamus, entorhinal cortex, hippocampus, striatum, amygdala, piriform cortex and 5 peripheral tissues (adrenals, testes, epididymis, pancreas and prostate) were analyzed. Rapid and profound decreases in TRH and TRH-like peptide levels (increased release) occurred throughout brain and peripheral tissues following ip ghrelin. Because ghrelin is rapidly deacylated in vivo we also studied 3-Trp-ghrelin which cannot be deacylated. Significant increases in TRH and TRH-like peptide levels following 3-Trp-ghrelin, relative to those after ghrelin were observed in all brain regions except posterior cingulate and all peripheral tissues except prostate and testis. The rapid stimulation of TRH and TRH-like peptide release by ghrelin in contrast with the inhibition of such release by 3-Trp-TRH is consistent with TRH and TRH-like peptides modulating the downstream effects of both ghrelin and unacylated ghrelin.
Keywords: Ghrelin; TRH-like peptides; Rat; Limbic system;

Intranasal administration of PACAP: Uptake by brain and regional brain targeting with cyclodextrins by Naoko Nonaka; Susan A. Farr; Tomoya Nakamachi; John E. Morley; Masanori Nakamura; Seiji Shioda; William A. Banks (168-175).
► PACAP is taken up by hippocampus and hypothalamus after intranasal administration. ► Cyclodextrins can be used to target intranasal PACAP to specific brain regions. ► Intranasal PACAP improves memory in a mouse model of Alzheimer's disease.Pituitary adenylate cyclase activating polypeptide (PACAP) is a potent neurotrophic and neuroprotectant that is transported across the blood–brain barrier in amounts sufficient to affect brain function. However, its short half-life in blood makes it difficult to administer peripherally. Here, we determined whether the radioactively labeled 38 amino acid form of PACAP can enter the brain after intranasal (i.n.) administration. Occipital cortex and striatum were the regions with the highest uptake, peaking at levels of about 2–4% of the injected dose per gram of brain region. Inclusion of unlabeled PACAP greatly increased retention of I-PACAP by brain probably because of inhibition of the brain-to-blood efflux transporter for PACAP located at the blood–brain barrier. Sufficient amounts of PACAP could be delivered to the brain to affect function as shown by improvement of memory in aged SAMP8 mice, a model of Alzheimer's disease. We found that each of three cyclodextrins when included in the i.n. injection produced a unique distribution pattern of I-PACAP among brain regions. As examples, β-cyclodextrin greatly increased uptake by the occipital cortex and hypothalamus, α-cyclodextrin increased uptake by the olfactory bulb and decreased uptake by the occipital cortex and striatum, and (2-hydropropyl)-β-cyclodextrin increased uptake by the thalamus and decreased uptake by the striatum. These results show that therapeutic amounts of PACAP can be delivered to the brain by intranasal administration and that cyclodextrins may be useful in the therapeutic targeting of peptides to specific brain regions.
Keywords: Intranasal; Alzheimer's disease; Pituitary adenylate cyclase activating polypeptide; Blood–brain barrier; Memory; Cyclodextrin;

► We cloned pig GnIH cDNA 442 bp and GPR147 cDNA 1003 bp. ► Tissue expression revealed that GnIH and GPR147 was mainly expressed in the brain. ► Fluctuations of GnIH and GPR147 expression were found during the pig estrous cycle. ► GnIH and GPR147 were mainly localized in the luteal cells, granulosa, and theca cells.Since its discovery, gonadotropin-inhibitory hormone (GnIH) has appeared to act as a key neuropeptide in the control of vertebrate reproduction. GnIH acts via the novel G protein-coupled receptor 147 (GPR147) to inhibit gonadotropin release and synthesis. To determine the physiological functions of GnIH in the pig, a study was conducted to clone and sequence the cDNA of the GnIH precursor and GPR147. Our results demonstrated that the cloned pig GnIH precursor cDNA encoded three LPXRF and that its receptor possessed typical transmembrane features. Subsequently, tissue expression studies revealed that GnIH was mainly expressed in the brain, corresponding largely with the tissue expression patterns of GPR147 in the pig. The expression patterns in the reproductive axis of the female pig across the estrous cycle were also systemically investigated. The hypothalamic levels of both GnIH and its receptor mRNA were lowest in estrus and peaked in the proestrus and diestrus phases. The highest pituitary GnIH mRNA level was detected in the metestrus, and its receptor displayed a somewhat similar pattern of expression to that of the ligand. However, the expression patterns of GnIH and GPR147 were negatively correlated in the ovary. Immunolocalization in the ovary during the estrous cycle revealed that the immunoreactivities of GnIH and GPR147 were mainly localized in the granulosa and theca cells of the antral follicles during proestrus and estrus and in the luteal cells during metestrus and diestrus. Taken together, this research provided molecular and morphological data for further study of GnIH in the pig.
Keywords: Gonadotropin-inhibitory hormone (GnIH); G protein-coupled receptor 147 (GPR147); Estrous cycle; Hypothalamus–pituitary–ovary axis;

► β-Casomorphin-7 amelioration a deterioration of the renal function in diabetic rats that is independent of a hyperglycemic effect. ► β-Casomorphin-7 treatment can reduce renal tubular fibrosis of diabetic rats. ► The protective efforts of β-casomorphin-7 on diabetic nephropathy may be associated, in part, with inhibiting the EMT of renal tubular epithelial cells.This study was designed to investigate the putative protective effect of β-casomorphin-7 on diabetic nephropathy in a rat model, and to explore the possible mechanism of this effect. SD rats were randomly divided into the following three groups: control group, diabetes group and β-casomorphin-7-treatment group. All rats were euthanized after 30 days with or without β-casomorphin-7 treatment. Biochemical parameters including blood glucose and renal function were quantified. The concentration of plasma TGF-β1 was measured by ELISA. Histopathological changes to the kidney were studied by Masson and Sirius red staining. Expressions of α-smooth muscle actin (α-SMA), E-cadherin, vimentin, cytokeratin19 and TGF-β1 mRNA in rat renal cortices were analyzed by real-time PCR. Changes in α-SMA and E-cadherin protein expression in rat renal cortices were quantified by Western blot. β-Casomorphin-7 treatment of diabetic rats reduced urinary glucose, urinary protein, serum creatinine, blood urinary nitrogen, plasma TGF-β1 and the ratio of kidney: body weight. Masson and Sirius red staining showed that β-casomorphin-7 treatment attenuated renal interstitial fibrosis in diabetic rats. Compared to the control rats, diabetic rats had elevated expressions of α-SMA, vimentin and TGF-β1 mRNA and α -SMA protein and decreased expression of E-cadherin and cytokeratin19 mRNA, and E-cadherin protein. β-Casomorphin-7 treatment of diabetic rats partially normalized these changes. Our results suggest that administration of β-casomorphin-7 attenuates renal interstitial fibrosis caused by diabetes. This protective effect may be associated, in part, with down regulation of epithelial–mesenchymal transition of renal tubular epithelial cells.
Keywords: Diabetic nephropathy; Epithelial–mesenchymal transdifferentiation; Renal histology; β-Casomorphin-7;

The natriuretic peptide time-course in end-stage heart failure patients supported by left ventricular assist device implant: Focus on NT-proCNP by M. Cabiati; R. Caruso; C. Caselli; M. Frigerio; T. Prescimone; O. Parodi; D. Giannessi; S. Del Ry (192-198).
► Ventricular assist devices (VAD) lead to a modification of the neurohormonal profile. ► To evaluate the effects of VAD on natriuretic peptides plasma levels. ► Nt-proCNP would be useful for identifying patients more likely to recover.This study aimed to evaluate left ventricular assist device (LVAD) effects on natriuretic peptide (NP) prohormone plasma levels in end-stage heart failure (HF) patients, especially NT-proCNP, in order to better characterize the NP system during hemodynamic recovery by LVAD. HF patients (n  = 17, NYHA III-IV) undergoing LVAD were studied: 6 died of multi-organ failure syndrome (NS) and 11 survived (S). Total sequential organ failure assessment (t-SOFA) score and blood samples were obtained at admission (T1) and at 24, 72 h and 1, 2, 4 weeks (T2–T6) after LVAD. In S, NT-proANP and NT-proCNP significantly increased at 24 h after implantation, reaching a reduction to basal levels at 4 weeks following LVAD [NT-proANP: T1 vs. T2 p  = 0.017, NT-proCNP: T1 vs. T2 p  = 0.028, T1 vs. T3 p  = 0.043]. Elevated NT-proBNP plasma levels were observed at all times. In NS, NP plasma levels sustained higher with respect to S. No statistical variation was observed for NT-proCNP and NT-proANP in S and NS while NT-proBNP reached significant differences at T4 in NS. Considering S + NS, only NT-proCNP strongly correlated with t-SOFA score at T1 (rho = 0.554, p  = 0.04) while subdividing patients NT-proCNP positively correlated in NS with t-SOFA score (rho = 0.988, p  = 0.002) only at T4. In NS a correlation between NT-proCNP and NT-proBNP at T1 was observed (rho = −0.900, p  = 0.037). Both IL-6 and TNF-alpha sustained higher in NS patients than in S; in particular, statistical significance was observed for IL-6. The study of new peptides, such as NT-proCNP, would provide additional information for identifying patients who are more likely to recover.
Keywords: Left ventricular assist device; Heart failure; t-SOFA score; NT-proANP; NT-proBNP; NT-proCNP;

Urotensin II protects ischemic reperfusion injury of hearts through ROS and antioxidant pathway by Shan Gao; Young Bin Oh; Byung Mun Park; Woo Hyun Park; Suhn Hee Kim (199-205).
► hUII increased recovery percentage of post-ischemic ventricular function. ► The hydrogen peroxide activity and antioxidant enzyme expression were increased in hUII-treated hearts. ► The apoptotic protein expression was decreased in hUII-treated hearts. ► hUII has protective effects on I/R cardiac injury partly through activating antioxidant enzymes and reactive oxygen species.Urotensin II (UII) is a vasoactive peptide which is bound to a G protein-coupled receptor. UII and its receptor are upregulated in ischemic and chronic hypoxic myocardium, but the effect of UII on ischemic reperfusion (I/R) injury is still controversial. The aim of the present study was to investigate whether UII protects heart function against I/R injury. Global ischemia was performed using isolated perfused Langendorff hearts of Sprague–Dawley rats. Hearts were perfused with Krebs–Henseleit buffer for 20 min pre-ischemic period followed by a 20 min global ischemia and 50 min reperfusion. Pretreatment with UII (10 nM) for 10 min increased recovery percentage of the post-ischemic left ventricular developed pressure and ±dp/dt, and decreased post-ischemic left ventricular end-diastolic pressure as compared with I/R group. UII decreased infarct size and an increased lactate dehydrogenase level during reperfusion. Cardioprotective effects of UII were attenuated by pretreatment with UII receptor antagonist. The hydrogen peroxide activity was increased in UII-treated heart before ischemia. The Mn-SOD, catalase, heme oxygenase-1 and Bcl-2 levels were increased, and the Bax and caspase-9 levels were decreased in UII-treated hearts. These results suggest that UII has cardioprotective effects against I/R injury partly through activating antioxidant enzymes and reactive oxygen species.
Keywords: Urotensin II; Heart; Ischemia; Atrial natriuretic peptide; Apoptosis; Reactive oxygen species;

Vascular effects and electrolyte homeostasis of the natriuretic peptide isolated from Crotalus oreganus abyssus (North American Grand Canyon rattlesnake) venom by S.L. Da Silva; C.A. Dias-Junior; P.A. Baldasso; D.C.S. Damico; B.M.A. Carvalho; A. Garanto; G. Acosta; E. Oliveira; F. Albericio; A.M. Soares; S. Marangoni; R.R. Resende (206-212).
► We have isolated and characterized from the venom of the Crotalus oreganus abyssus new natriuretic peptides (NPs). ► The NP family comprises three members, ANP (atrial natriuretic peptide), BNP (b-type natriuretic peptide) and CNP (c-type natriuretic peptide), and has an important role in blood pressure regulation and electrolyte homeostasis. ► The novel NP is Coa_NP2 and presents an average molecular mass of 3419.88 Da and its amino acid sequence presents the loop region that is characteristic of natriuretic peptides. ► We demonstrate, herein, that Coa_NP2 produces a dose-dependent decrease in mean arterial pressure in rats, followed by significant increases in concentrations nitric oxide markers formation. Crotalus oreganus abyssus is a rattlesnake that is usually found in the Grand Canyon, United States of America. Knowledge regarding the composition of C. o. abyssus venom is scarce. New natriuretic peptides (NPs) have been isolated and characterized from the venoms of members of the Crotalinae family. The NP family comprises three members, ANP (atrial natriuretic peptide), BNP (b-type natriuretic peptide) and CNP (c-type natriuretic peptide), and has an important role in blood pressure regulation and electrolyte homeostasis. The aim of the present study was to characterize a novel natriuretic-like peptide (Coa_NP2), isolated from C. o. abyssus venom. The Coa_NP2 presents an average molecular mass of 3419.88 Da (theoretical average molecular mass 3418.94 Da, monoisotopic molecular mass 3416.66 Da and theoretical PI 7.78) and its amino acid sequence presents the loop region that is characteristic of natriuretic peptides. The peptide has 32 amino acids and its complete sequence is SYGISSGCFGLKLDRIGTMSGLGCWRLLQDSP. Coa_NP2 is a natriuretic peptide of the ANP/BNP-like family, since the carboxyterminal region of CNP has its own NP domain. We demonstrate, herein, that Coa_NP2 produces a dose-dependent decrease in mean arterial pressure in rats, followed by significant increases in concentrations of markers of nitric oxide formation measured in the plasma and vasorelaxation in a thoracic aortic ring bath. The structural and biological aspects confirm Coa_NP2 as a new natriuretic peptide, isolated from snake venom.
Keywords: Crotalus oreganus abyssus; Hypotensive agents; Snake venoms; Natriuretc peptides; Nitric oxide;

StCT2, a new antibacterial peptide characterized from the venom of the scorpion Scorpiops tibetanus by Luyang Cao; Zhongjie Li; Ruhong Zhang; Yingliang Wu; Wenxin Li; Zhijian Cao (213-220).
► StCT2, a new antimicrobial peptide, was characterized from the venomous gland cDNA library of the Scorpiops tibetanus. ► The MICs of StCT2 for Staphylococcus aureus were 6.25–25 μg/ml, including antibiotic-resistant strains such as methicillin resistant S. aureus. ► StCT2 was found to show high in vivo antimicrobial activity by an S. aureus infection mouse model. ► StCT2 exerted its antimicrobial activity via a rapid bactericidal mechanism.Bacterial infection poses an increasing threat to global public health and new types of antibacterial agents are urgently needed to respond to the threat. Scorpion venom contains series of bioactive peptides, among which antibacterial peptide is an important part. Herein, a new antimicrobial peptide StCT2 was characterized from the venomous gland cDNA library of the Scorpiops tibetanus. The full-length cDNA of StCT2 is 369 nucleotides encoding the precursor that contains a putative 24 residues signal peptide, a presumed 14 residues mature peptide, and a putative 37 residues acidic propeptide at the C-terminus. The minimal inhibition concentrations (MICs) of StCT2 for Staphylococcus aureus were 6.25–25 μg/ml, including antibiotic-resistant strains such as methicillin resistant S. aureus (MRSA). StCT2 was further found to show high in vivo antimicrobial activity by an S. aureus infection mouse model. StCT2 exerted its antimicrobial activity via a rapid bactericidal mechanism. Taken together, these results demonstrate the efficacy and general mechanism of StCT2 antimicrobial action and the therapeutic potential of StCT2 as a new antimicrobial peptide.
Keywords: Scorpion; Venom; Antimicrobial peptide; StCT2; Antibiotics-resistant;

The structures of four bombesins and their cloned precursor-encoding cDNAs from acid-solvated skin secretion of the European yellow-bellied toad, Bombina variegata by Bing Bai; Hui Wang; Yilu Xue; Youjia Wu; Mei Zhou; Minjie Wei; Tianbao Chen; Lei Wang; Chris Shaw (221-229).
► First demonstration of stability of both peptides and corresponding precursor-encoding mRNAs in long-term frozen (12 years) acid-solvated amphibian skin secretion. ► First demonstration of four different bombesins in the skin of a single amphibian species. ► First report of the novel analog, Asp2-, Phe4-SAP bombesin.Four different bombesins (bombesin, His6-bombesin, Phe13-bombesin and Asp2-, Phe4-SAP-bombesin) have been identified by a systematic sequencing study of peptides in reverse phase HPLC fractions of the skin secretion of the European yellow-bellied toad, Bombina variegata, that had been solvated in 0.1% (v/v) aqueous trifluoroacetic acid (TFA) and stored frozen at −20 °C for 12 years. By using a 3′- and 5′-RACE PCR strategy, the corresponding biosynthetic precursor-encoding cDNAs of all four peptides were cloned from a cDNA library made from the same long-term frozen, acid-solvated skin secretion sample following thawing and lyophilization. Canonical bombesin and His6-bombesin are classical bombesin sub-family members, whereas Phe13-bombesin and Asp2-, Phe4-SAP-bombesin, belong to the litorin/ranatensin sub-family of bombesin-like peptides (BLPs). Assignment of these peptides to respective sub-families, was based upon both their primary structural similarities and their comparative pharmacological activities. An interesting observation in this study, was that the nucleotide sequences of the open-reading frames of cloned cDNAs encoding bombesin and its His6-substituted analog, were identical except for a single base that was responsible for the change observed at the position 6 residue in the mature peptide from Asn to His. In contrast, the precursor cDNA nucleotide sequences encoding the Phe13-bombesins, exhibited 53 base differences. The pharmacological activities of synthetic replicates of each bombesin were compared using two different mammalian smooth muscle preparations and all four peptides were found to be active. However, there were significant differences in their relative potencies.
Keywords: Amphibian; Skin; Peptide; Bombesin; Smooth muscle; Cloning;

Mass spectrometric elucidation of the neuropeptidome of a crustacean neuroendocrine organ by Limei Hui; Feng Xiang; Yuzhuo Zhang; Lingjun Li (230-239).
► The first comprehensive report on discovery of neuropeptides in the pericardial organ of blue crab Callinectes sapidus. ► 130 peptides from 11 families including 44 novel ones were discovered and sequenced. ► A combination of multifaceted mass spectrometry (MS) approach and chemical derivatization was employed for peptidomic analysis. ► Our results lay the groundwork for future neuropeptide physiology studies in C. sapidus and other crustaceans.The blue crab Callinectes sapidus has been used as an experimental model organism for the study of regulation of cardiac activity and other physiological processes. Moreover, it is an economically and ecologically important crustacean species. However, there was no previous report on the characterization of its neuropeptidome. To fill in this gap, we employed multiple sample preparation methods including direct tissue profiling, crude tissue extraction and tissue extract fractionation by HPLC to obtain a complete description of the neuropeptidome of C. sapidus. Matrix-assisted laser desorption/ionization (MALDI)–Fourier transform mass spectrometry (FTMS) and MALDI-time-of-flight (TOF)/TOF were utilized initially to obtain a quick snapshot of the neuropeptide profile, and subsequently nanoflow liquid chromatography (nanoLC) coupled with electrospray ionization quadrupole time-of-flight (ESI-Q-TOF) tandem MS analysis of neuropeptide extracts was conducted for de novo sequencing. Simultaneously, the pericardial organ (PO) tissue extract was labeled by a novel N,N-dimethylated leucine (DiLeu) reagent, offering enhanced fragmentation efficiency of peptides. In total, 130 peptide sequences belonging to 11 known neuropeptide families including orcomyotropin, pyrokinin, allatostatin A (AST-A), allatostatin B (AST-B), FMRFamide-like peptides (FLPs), and orcokinin were identified. Among these 130 sequences, 44 are novel peptides and 86 are previously identified. Overall, our results lay the groundwork for future physiological studies of neuropeptides in C. sapidus and other crustaceans.
Keywords: Callinectes sapidus; Pericardial organ; De novo sequencing; Neuropeptidome; Neuropeptides; Chemical derivatization;

Preparation and evaluation of antioxidant peptides from ethanol-soluble proteins hydrolysate of Sphyrna lewini muscle by Bin Wang; Zhong-Rui Li; Chang-Feng Chi; Qi-Hong Zhang; Hong-Yu Luo (240-250).
► An efficient method was developed to acquire the ethanol-soluble proteins (EP). ► An efficient method was achieved to acquire the antioxidant hydrolysate (SEPH) of EP. ► Two peptides with high antioxidant activity were isolated from SEPH. ► The structures of peptides were determined as Trp-Asp-Arg and Pro-Tyr-Phe-Asn-Lys. ► The antioxidant activities of purified peptides were evaluated by six methods.To get high yield of ethanol-soluble proteins (EP) and the antioxidant peptides from Sphyrna lewini muscle, orthogonal experiments (L9(3)4) were applied to optimize the best extraction conditions and enzyme hydrolysis conditions. The yield of EP reached 5.903 ± 0.053% under the optimum conditions of ethanol concentration 90%, solvent to material ratio 20:1, extraction temperature of 40 °C and extraction time of 80 min. The antioxidant SEPH (EP hydrolysate of S. lewini muscle) was prepared by using papain under the optimum conditions of enzymolysis time 2 h, total enzyme dose 1.2%, enzymolysis temperature 50 °C and pH 6, and its DPPH radical scavenging activity reached 21.76 ± 0.42% at the concentration of 10 mg/ml. Two peptides (F42-3 and F42-5) were isolated from SEPH by using ultrafiltration, anion-exchange chromatography, gel filtration chromatography and RP-HPLC. The structures of F42-3 and F42-5 were identified as Trp-Asp-Arg and Pro-Tyr-Phe-Asn-Lys with molecular weights of 475.50 Da and 667.77 Da, respectively. F42-3 and F42-5 exhibited good scavenging activity on hydroxyl radical (EC50 0.15 mg/ml and 0.24 mg/ml), ABTS radical (EC50 0.34 mg/ml and 0.12 mg/ml), and superoxide anion radical (EC50 0.09 mg/ml and 0.11 mg/ml), but moderate DPPH radical (EC50 3.63 mg/ml and 4.11 mg/ml). F42-3 and F42-5 were also effectively against lipid peroxidation in the model system and peroxyl free radical scavenging in β-carotene linoleic acid assay. Their high activities were due to the smaller size and the presence of antioxidative amino acids within the peptide sequences.
Keywords: Sphyrna lewini; Ethanol-soluble protein; Hydrolysate; Antioxidant activity; Peptides;

Therapeutic peptide production in Drosophila by Dongkook Park; Xiaowen Hou; Jonathan V. Sweedler; Paul H. Taghert (251-256).
► We developed a novel platform to produce biologically active peptides in Drosophila. ► As a proof-of-concept, biologically active human insulin was successfully produced. ► This method could provide an useful alternative to commercial peptide production. ► This also has the great potentials for the peptide drug development.Bioactive peptides are important therapeutic drugs, yet conventional methods of peptide synthesis are challenged to meet increasing demand. We developed a novel and efficient means of metabolic engineering: therapeutic peptide production in Drosophila and as a proof of concept, we demonstrate production of fully matured human insulin. This in vivo system offers an innovative means to produce valuable bioactive peptides for therapies, its inherent flexibility facilitates drug development, and its ease of producing fully processed peptides simplifies metabolic engineering of new peptide products.
Keywords: Drosophila; Insulin; Theraputic peptides; DIMM;

Novel cationic antimicrobial peptide GW-H1 induced caspase-dependent apoptosis of hepatocellular carcinoma cell lines by Yi-Lin Sophia Chen; Jun-Hong Li; Chao-Yuan Yu; Ching-Ju Lin; Pai-Hsuan Chiu; Po-Wen Chen; Chai-Ching Lin; Wei-Jung Chen (257-265).
► Novel cationic amphipathic AMP GW-H1 has selective cytotoxicity against hepatocellular carcinoma (HCC) cell lines J5, Huh7, and Hep3B. ► GW-H1 induced caspase-dependent apoptosis of J5 HCC cell line. ► GW-H1 inhibited tumor growth of J5 xenografts in nude mice. ► GW-H1 possesses chemotherapeutic potential for the treatment of HCC.Due to its malignancy, the development of effective therapeutic strategies for hepatocellular carcinoma (HCC) is of urgent needs. Natural antimicrobial peptides (AMPs), also known as host defense peptides (HDPs), not only act as direct antimicrobial agents, but also represent important regulators of the innate immune system. It has been reported that cationic AMPs may exhibit cancer-selective toxicity. We have designed a series of novel AMPs with potent antimicrobial activity against a broad spectrum of bacterial pathogens. In the current study, we evaluate the antitumor potency of these AMPs toward HCC cell lines J5, Huh7, and Hep3B. Selected AMPs inhibit the viability of HCC cells in a dose-dependent fashion, while the normal 3T3 cells were significantly less susceptible to these AMPs. GW-H1 treatment (20 μM) of J5 cells for 24–72 h resulted in the induction of apoptosis, as revealed by flow cytometry (increased sub-G1 populations), and western blot analysis for the appearance of activated caspase-3, -7 and -9 cleavages. Two-dimensional gel electrophoresis was applied to further analyze the AMP-responsive protein profiles of HCC, down-regulation of Hsp27, phophoglycerate kinase 1 and triosephosphate isomerase indicated that GW-H1 may induce apoptosis, and further inhibit progression and metastasis of J5 HCC cells. FITC-labeled GW-H1 was found to attach to cell membrane initially, then translocated into the cytoplasm, and eventually membranous organelles or nucleus. GW-H1 induced a marked growth suppression of J5 xenografts in nude mice in a dose dependent manner. These findings provided support for future application of GW-H1 as potential therapeutic agent for HCC.
Keywords: Antimicrobial peptide; Anticancer; Hepatocellular carcinoma; Apoptosis; Caspase;

Discovery of novel antimicrobial peptides with unusual cysteine motifs in dandelion Taraxacum officinale Wigg. flowers by A.A. Astafieva; E.A. Rogozhin; T.I. Odintsova; N.V. Khadeeva; E.V. Grishin; Ts.A. Egorov (266-271).
► Three novel antimicrobial peptides were isolated from Taraxacum officinale flowers. ► Primary structures of these peptides were determined and it was shown that their molecules belong to new types of cystein-rich plant peptides. ► The peptides of current structure types were discovered only in dandelion flowers. ► The peptides were shown to display high antimicrobial activity both against fungal and bacterial pathogens in laboratory conditions.Three novel antimicrobial peptides designated ToAMP1, ToAMP2 and ToAMP3 were purified from Taraxacum officinale flowers. Their amino acid sequences were determined. The peptides are cationic and cysteine-rich and consist of 38, 44 and 42 amino acid residues for ToAMP1, ToAMP2 and ToAMP3, respectively. Importantly, according to cysteine motifs, the peptides are representatives of two novel previously unknown families of plant antimicrobial peptides. ToAMP1 and ToAMP2 share high sequence identity and belong to 6-Cys-containing antimicrobial peptides, while ToAMP3 is a member of a distinct 8-Cys family. The peptides were shown to display high antimicrobial activity both against fungal and bacterial pathogens, and therefore represent new promising molecules for biotechnological and medicinal applications.
Keywords: Plant antimicrobial peptides; Taraxacum officinale Wigg.; Plant immunity;

► DMS decreased gut motility and cardiac contractility dose dependently. ► Truncated and Y-[Bpa2] DMS analogs were increased agonists in pupal and adult heart. ► Adult, pupal, and larval heart and gut active cores were developmental- and tissue specific. ► Y-[Bpax] DMS activity and binding differed on a developmental- and tissue-specific level. ► DMS-specific antisera stained unique cells and fibers that innervated gut or heart.Myosuppressin peptides dramatically diminish contractions of the gut and heart. Thus, delineating mechanisms involved in myosuppressin signaling may provide insight into peptidergic control of muscle contractility. Drosophila myosuppressin (DMS, TDVDHVFLRFamide) structure–activity relationship (SAR) was investigated to identify an antagonist and explore signaling. Alanyl-substituted, N-terminal truncated, and modified amino acid analogs identified residues and peptide length required for activity. Immunochemistry independently provided insight into myosuppressin mechanisms. DMS decreased gut motility and cardiac contractility dose dependently; the different effective concentrations at half maximal-response were indicative of tissue-specific mechanisms. Replacement of aspartic acid 2 (D2) generated an analog with different developmental- and tissue-specific effects; [A2] DMS mimicked DMS in adult gut (100% inhibition), yet decreased larval gut contractions by only 32% with increased potency in pupal heart (126% inhibition). The DMS active core differed across development and in tissues; adult (DHVFLRFamide) and larval gut (TDVDHVFLRFamide), and adult (VFLRFamide) and pupal heart (VFLRFamide). Substitution of D2 and D4 with a modified amino acid, p-benzoyl-phenylalanine, produced developmental- and tissue-specific antagonists. In the presence of protease inhibitors, DMS and VFLRFamide were more effective in adult gut, but lower or unchanged in pupal heart compared to peptide or analog alone, respectively. DMS-specific antisera stained neurons that innervated the gut or heart. This study describes novel antagonists and data to identify developmental- and tissue-specific mechanisms underlying the pleotropic effects of myosuppressin in muscle physiology.
Keywords: DMS; FMRFamide; Gut; Heart; RFRP-1;

► Determination of PAC1 expression in normal RGC-5 cells and after UVB irradiation. C*HSDGIC* and PACAP promoted the proliferation of RGC-5 cells. C*HSDGIC* pretreatment attenuates UVB-induced apoptosis in RGC-5 cells.Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide that confers potent neurotrophic and neuroprotective effects. Cyclopeptide C*HSDGIC* (CHC), which results from the cyclization of PACAP (1–5) with disulfide, has been demonstrated to represent a potent agonist for the PACAP-specific receptor PAC1 which mediates the majority of PACAP's effects. In this study, the expression of PAC1 in a rat retinal ganglion cell line (RGC-5) was confirmed using a western blot analysis, and it was determined that CHC promoted the proliferation of RGC-5 cells using the cell counting kit-8 (CCK8) assay and flow cytometry. Furthermore, the treatment of CHC attenuated the decrease of cell viability in cells exposed to UVB irradiation. Flow cytometry and a JC-1 assay revealed that the CHC treatment protected the RGC-5 cells against UVB-induced apoptosis. In addition, similar to PACAP, the anti-apoptotic effect of CHC was related to the down-regulation of caspase-3. In summary, these results demonstrate for the first time that PAC1 is present in RGC-5 cells and that CHC, a cyclopeptide from PACAP, promotes RGC-5 cell proliferation and attenuates UVB-induced apoptosis.
Keywords: C*HSDGIC*; RGC-5; Apoptosis; PAC1; PACAP;

GE-25-like immunoreactivity in the rat eye by Katrin Lorenz; Oliver W. Gramlich; Franz H. Grus; Daniela Ehrlich; Christian Humpel; Marina Nogalo; Reiner Fischer-Colbrie; Nikolaos E. Bechrakis; Rosa Hattmannstorfer; Josef Troger (286-291).
► GE-25 is a neuropeptide generated in vivo by proteolytic processing of chromogranin A. ► In the retina and trigeminal ganglion GE-25-like immunoreactivity appeared as uncleaved intact chromogranin A. ► GE-25-like immunoreactivity was found to be distinctly distributed throughout the rat eye. ► The peptide has been localized to small and medium-sized ganglion cells in the rat trigeminal ganglion. ► In the retina, immunoreactivity has been localized to glia which is atypical for neuropeptides.This study aimed to investigate the presence and distribution of the chromogranin A-derived peptide GE-25 in the rat eye. The molecular form detected by the GE-25 antiserum was evaluated in the rat trigeminal ganglion, retina and remaining tissues of the rat eye by means of Western blots and the distribution pattern of GE-25-like immunoreactivity was studied in the rat eye and rat trigeminal ganglion by immunofluorescence. One single band of approximately 70 kDa was stained in the trigeminal ganglion and retina which represents the uncleaved intact chromogranin A indicating that the proteolytic processing of chromogranin A to GE-25 is limited in these tissues. Sparse GE-25-like immunoreactive nerve fibers were visualized in the corneal stroma, at the limbus around blood vessels, in the sphincter and dilator muscle and stroma of the iris, in the stroma of the ciliary body and ciliary processes and in the stroma and around blood vessels in the choroid. This distribution pattern is characteristic for neuropeptides whereas the presence of immunoreactivity in the corneal endothelium and in Müller glia in the retina is atypical. GE-25-like immunoreactivity was found in small to medium-sized ganglion cells in the rat trigeminal ganglion clearly indicating that the nerve fibers in the rat eye are of sensory origin. The colocalization of GE-25-immunoreactivity with SP-immunoreactivity in the rat ciliary body is in agreement with the presumption of the sensory nature of the innervation of the anterior segment of the eye by GE-25.
Keywords: GE-25; Retina; Eye; Trigeminal ganglion;

Immunomodulatory roles and functional analysis of pre-B lymphocyte DT40 cells with the bursal-derived BSP-II treatment by Xiu-Li Feng; Bin Zhou; Rui-Bing Cao; Qing-Tao Liu; Ke Liu; Xiao-Dong Liu; Yuan-Peng Zhang; Li Huang; Xiang-Bo Ji; Jun Luo; Gaiping Zhang; Pu-Yan Chen (292-298).
Display Omitted► BSP-II induced strong humoral immune responses in immunized chicks. ► BSP-II induced DT40 cell viability at the reachable concentrations. ► Numerous genes expression were regulated after BSP-II treatment. ► Pathway analysis was performed to analysis BSP-II-regulated genes. ► RT-qPCR was used to confirm the microarray expression data.The bursa of Fabricius, the acknowledged central humoral immune organ, is vital to B cell differentiation. However, the regulatory function of the bursal-derived peptide on avian B cell proliferation has not been reported. BSP-II is a recently reported bursal-derived bioactive peptide. In this paper, 75 days-old chicks were twice subcutaneously immunized with BSP-II and inactivated avian influenza virus (AIV, H9N2 strain). It was proved that BSP-II induced a strongly AIV-specific HI antibody production in the immunized chicks. Also, BSP-II could enhance avian pre-B lymphocyte DT40 cell viability. To investigate the global patterns of gene expression in DT40 cells after BSP-II treatment, gene microarray was carried out. It was identified that the differentially expressed genes were involved in various pathways, of which six pathways were associated with signaling transductions, including ErbB signaling, MAPK signaling, Toll-like receptor signaling, Notch signaling, mTOR signaling, and Wnt signaling. Finally, RT-qPCR was used to confirm the microarray expression data. These results indicated the molecular basis of pre-B lymphocyte viability with BSP-II treatment, which provided a potential mechanism of the bursa of Fabricius on pre-B lymphocyte viability, differentiation, and development. These results are valid for the mechanism of the bursa of Fabricius on B lymphocytes development.
Keywords: Bursal-derived BSP-II; Immunization experiment; Pre-B lymphocyte DT40 cell; Gene microarray; Functional analysis;

Neuromedin B stimulates proliferation of mouse chondrogenic cell line ATDC5 by Hiroki Saito; Ryuji Ikeda; Kazuhiko Inoue; Sayaka Nagata; Kazuo Kitamura; Naoto Minamino; Kenji Kangawa; Atsuro Miyata (299-302).
► NMB and its receptor (NMB-R) were expressed in mouse chondrogenic cell line, ATDC5. ► Differentiation of ATDC5 cells transiently increased gene expression of NMB on day 4. ► NMB-R expression was decreased on days 7 and 14 in differentiation of ATDC5 cells. ► NMB significantly induced ATDC5 proliferation, but did not affect its differentiation.Neuromedin B (NMB), which was originally isolated from porcine spinal cord, is a mammalian bombesin-related peptide that exerts various physiological effects. Previously, we observed expression of NMB in rib cartilage from chicken. Here, we report the initial attempt to elucidate the role of NMB in cartilage. We used RT-PCR to measure the expression of NMB and its receptor (NMB-R) in mouse chondrogenic cell line ATDC5. During chondrogenic differentiation of ATDC5 cells, NMB mRNA transiently increased on day 4 and then decreased on day 14, whereas NMB-R mRNA decreased on days 7 and 14. We also characterized immunoreactive NMB in ATDC5 culture medium using a combination of specific radioimmunoassay (RIA) and reverse phase-high performance liquid chromatography (RP-HPLC). Furthermore, using the WST-8 assay, we demonstrated that NMB significantly induced ATDC5 proliferation; this was inhibited by NMB-R antagonist, BIM 23127. These results implicate that NMB is involved in cartilage development, either in an autocrine or paracrine manner.
Keywords: Neuromedin B; Neuromedin B receptor; ATDC5 cells; Differentiation; Proliferation;

Importance of the disulfide bridges in the antibacterial activity of human hepcidin by Agnès Hocquellet; Caroline le Senechal; Bertrand Garbay (303-307).
► The absence of disulfide bridges prevents antibacterial activity of human hepcidin. ► Hepcidin 25 does not kill bacteria by destroying their membranes. ► The disulfide bridges of hepcidin are mandatory for binding on DNA.Hepcidin was first identified as an antimicrobial peptide present in human serum and urine. It was later demonstrated that hepcidin is the long sought hormone that regulates iron homeostasis in mammals. The native peptide of 25 amino acids (Hepc25) contains four disulfide bridges that maintain a β-hairpin motif. The aim of the present study was to assess whether the intramolecular disulfide bridges are necessary for Hepc25 antimicrobial activity. We show that a synthetic peptide corresponding to human Hepc25, and which contains the four disulfide bridges, has an antibacterial activity against several strains of Gram-positive and Gram-negative bacteria. On the contrary, a synthetic peptide where all cysteines were replaced by alanines (Hepc25-Ala) had no detectable activity against the same strains of bacteria. In a further step, the mode of action of Hepc25 on Escherichia coli was studied. SYTOX Green uptake was used to assess bacterial membrane integrity. No permeabilization of the membrane was observed with Hepc25, indicating that this peptide does not kill bacteria by destroying their membranes. Gel retardation assay showed that the Hepc25 binds to DNA with high efficiency, and that this binding ability is dependent on the presence of the intramolecular disulfide bridges. Reduction of Hepc25 or replacement of the eight cysteines by alanine residues led to peptides that were no longer able to bind DNA in the in vitro assay. Altogether, these results demonstrate that Hepc25 should adopt a three-dimensional structure stabilized by the intramolecular disulfide bridges in order to have antibacterial activity.
Keywords: Antimicrobial peptide; DNA-binding; Hepcidin; Disulfide-bridges;

Antimicrobial peptides: Clinical relevance and therapeutic implications by Fabiano Pinheiro da Silva; Marcel Cerqueira César Machado (308-314).
► Antimicrobial peptides (AMPs) are a large group of compounds produced by multicellular organisms from the vegetal and animal kingdoms. ► Despite the antimicrobial effects, cathelicidins also have immunomodulatory properties. LL-37 is strongly induced by vitamin D, but their effects are paradoxically divergent in cancer biology. ► It is unclear how AMPs are able to induce cell signaling. Models include direct receptor-binding, but they can also induce ion fluxes in a receptor-independent manner. ► AMPs have been proposed as novel therapeutic agents in complex diseases. Here, we explore this scenario.Antimicrobial peptides (AMPs) are molecules that provide protection against environmental pathogens, acting against a large number of microorganisms, including bacteria, fungi, yeast, virus and others. Two major groups of antimicrobial peptides are found in humans: cathelicidins and defensins. Recently, several studies have furnished information that besides their role in infection diseases, antimicrobial peptides play a role in diseases as diverse as inflammatory disorders, autoimmunity and cancer. Here, we discuss the role of antimicrobial peptides and vitamin D have in such complex diseases and propose their use should be more explored in the diagnosis and treatment of such conditions.
Keywords: Cathelicidins; Defensins; Vitamin D; Autoimmunity, Infection and cancer;

Antimicrobial peptide control of pathogenic microorganisms of the oral cavity: A review of the literature by Bruno Rocha da Silva; Victor Aragão Abreu de Freitas; Luiz Gonzaga Nascimento-Neto; Victor Alves Carneiro; Francisco Vassiliepe Sousa Arruda; Andréa Silvia Walter de Aguiar; Benildo Sousa Cavada; Edson Holanda Teixeira (315-321).
► There was detected a large increase in the number of publications on the use of antimicrobial peptides. ► Highlights the focus on oral cavity AMPs in the most recently published studies. ► Many studies are focused on the discovery of new methods of bacterial control. ► The evaluation of the use of AMPs on microorganisms in biofilms multispecies is necessary.Antimicrobial peptides, molecules produced in many different organisms, have high biocidal activity against several microorganisms. However, several questions about these molecules remain unclear. Therefore, this report details a systematic survey of the literature on the use of antimicrobial peptides against oral pathogens and indicates which peptides and microorganisms are most extensively studied. Articles were located using the PubMed and Science Direct databases with the following inclusion criteria: publication date between 2002 and 2011; keywords “biofilm OR biological film OR biological layer OR bacterial growth” AND “peptide” AND “oral cavity OR mouth OR buccal mucosa OR oral mucosa OR mouth mucosa”; and abstract in English. A total of 73 articles were selected after refinement of the data. An increase in publications focusing on the use of antimicrobial peptides against oral microorganisms was observed. In addition, the peptides produced by cells of the oral mucosa (defensins, LL-37 and histatins) as well as Streptococcus mutans (among cariogenic bacteria) and Porphyromonas gingivalis (among periodontal bacteria) were the most studied subjects. It was concluded that the use of antimicrobial peptides as a tool for microbial control is of increasing importance, likely due to its widespread use, mechanism of action, and low rates of bacterial resistance.
Keywords: Antimicrobial peptide; Biofilm; Dental caries; Periodontal disease;

Biotic stress resistance in agriculture through antimicrobial peptides by Sarika; M.A. Iquebal; Anil Rai (322-330).
► AMPs which is part of innate immunity in response to microbial challenges is reviewed. ► Distribution and classification of AMPs are described with recent statistics. ► Role of AMPs in biotechnological research is discussed. ► Specialized AMP databases and prediction servers available till date are discussed.Antimicrobial peptides (AMPs) are the hosts’ defense molecules against microbial pathogens and gaining extensive research attention worldwide. These have been reported to play vital role of host innate immunity in response to microbial challenges. AMPs can be used as a natural antibiotic as an alternative of their chemical counterpart for protection of plants/animals against diseases. There are a number of sources of AMPs including prokaryotic and eukaryotic organisms and are present, both in vertebrates and invertebrates. AMPs can be classified as cationic or anionic, based on net charges. Large number of databases and tools are available in the public domain which can be used for development of new genetically modified disease resistant varieties/breeds for agricultural production. The results of the biotechnological research as well as genetic engineering related to AMPs have shown high potential for reduction of economic losses of agricultural produce due to pathogens.In this article, an attempt has been made to introduce the role of AMPs in relation to plants and animals. Their functional and structural characteristics have been described in terms of its role in agriculture. Different sources of AMPs and importance of these sources has been reviewed in terms of its availability. This article also reviews the bioinformatics resources including different database tools and algorithms available in public domain. References of promising biotechnology research in relation to AMPs, prospects of AMPs for further development of genetically modified varieties/breeds are highlighted. AMPs are valuable resource for students, researchers, educators and medical and industrial personnel.
Keywords: Agriculture; Antimicrobial peptides; Bioengineering; Innate immunity; Transgenic;

Galanin and its receptors: A novel strategy for appetite control and obesity therapy by Penghua Fang; Mei Yu; Lili Guo; Ping Bo; Zhenwen Zhang; Mingyi Shi (331-339).
► Galanin is useful as a clinical measure to identify individuals at risk for obesity. ► Galanin via GalR1, not GalR2/3 to accelerate food intake and to increase body weight. ► Galanin-based therapies offering a safer approach to combat the obesity epidemic.The rapid increase in the prevalence of overweight and obesity is becoming an important health problem. Overweight and obesity may cause several metabolic complications, including type 2 diabetes mellitus, hyperlipidemia, high cholesterol, coronary artery disease as well as hypertension. Prevention and treatment of obesity will benefit the treatment of these related diseases. Current strategies for treatment of obesity are not adequately effective and are frequently companied with many side effects. Thus, new ways to treat obesity are urgently needed. Galanin is undoubtedly involved in the regulation of food intake and body weight. The aim of this review is to provide up-to-date knowledge concerning the roles of central and peripheral galanin as well as its receptors in the regulation of metabolism, obesity and appetite. We also highlight the mechanisms of galanin and its receptors in experimental obesity, trying to establish a novel anti-obesity strategy.
Keywords: Galanin; Appetite; Obesity;

Corrigendum to “Purification and characterization of a novel antimicrobial peptide from Brevibacillus laterosporus strain A60” [Peptides 33(2) (2012) 206–211] by Jing Zhao; Lihua Guo; Hongmei Zeng; Xiufen Yang; Jingjing Yuan; Huaixing Shi; Yehui Xiong; Mingjia Chen; Lei Han; Dewen Qiu (340).