Peptides (v.30, #8)
Editorial Board (CO2).
Characterization of two novel polyfunctional mastoparan peptides from the venom of the social wasp Polybia paulista by Bibiana Monson de Souza; Alessandra Vaso Rodrigues da Silva; Virginia Maria Ferreira Resende; Helen Andrade Arcuri; Marcia Perez dos Santos Cabrera; João Ruggiero Neto; Mario Sergio Palma (1387-1395).
Hymenoptera venoms are complex mixtures of biochemically and pharmacologically active components such as biogenic amines, peptides and proteins. Polycationic peptides generally constitute the largest group of Hymenoptera venom toxins, and the mastoparans constitute the most abundant and important class of peptides in the venom of social wasps. These toxins are responsible for histamine release from mast cells, serotonin from platelets, and catecholamines and adenylic acids from adrenal chromafin cells. The present work reports the structural and functional characterization of two novel mastoparan peptides identified from the venom of the neotropical social wasp Polybia paulista. The mastoparans Polybia-MP-II and -III were purified, sequenced and synthesized on solid phase using Fmoc chemistry and the synthetic peptides used for structural and functional characterizations. Polybia-MP-II and -III are tetradecapeptides, amidated at their C-termini, and form amphipathic α-helical conformations under membrane-mimetic conditions. Both peptides were polyfunctional, causing pronounced cell lysis of rat mast cells and erythrocytes, in addition to having antimicrobial activity against both Gram-positive and Gram-negative bacteria.
Keywords: Wasp venom; Toxins; Mastoparan; Peptide–membrane interaction; Cell lysis;
Identification, by RT-PCR, of four novel T-1-superfamily conotoxins from the vermivorous snail Conus spurius from the Gulf of Mexico by Roberto Zamora-Bustillos; Manuel B. Aguilar; Andrés Falcón; Edgar P. Heimer de la Cotera (1396-1404).
cDNA was prepared from the venom duct of a single Conus spurius specimen collected near the coast of Campeche, Mexico. From it, PCR products were generated, sequenced, and predicted to encode eight distinct precursors of T-1-conotoxins. These precursors contain five different mature toxins, of which four are novel and one (sr5a) has been previously purified and characterized from the venom of this species. Three of the novel toxins are very similar to sr5a: two have one amino acid substitution at position 8, whereas the other is predicted to have one additional residue at the C-terminus; the fourth toxin has five amino acid substitutions and is predicted to have two additional residues at the C-terminus. In general, the precursors include a 22-residue signal peptide, a 24-residue “pro” region, and a 13- to 16-residue mature toxin region; however, the C-termini of two mature toxin regions are predicted to be altered by post-translational processing. Three precursors lack, in the same positions, 15 amino acid residues included in the “pre” (one residue) and “pro” (14 residues) regions, which suggests the existence of an exon encoding the last signal peptide residue and the first 14 residues of the “pro” region. Phylogenetic analysis indicates that the T-1-conotoxin precursors and mature toxins of C. spurius are more similar to certain precursors and toxins from molluscivorous Conus species than to any precursors and toxins from vermivorous cones. The results reported here will be useful for synthesizing the novel toxins in order to identify their molecular targets.
Keywords: Conoidea; Conidae; Conus spurius; T-conotoxin; cDNA cloning; Conotoxin precursor;
A novel antifungal peptide designed from the primary structure of a natural antimicrobial peptide purified from Argopecten purpuratus hemocytes by Gloria Arenas; Fanny Guzmán; Constanza Cárdenas; Luis Mercado; Sergio H. Marshall (1405-1411).
We have isolated and purified a natural antimicrobial peptide from Argopecten purpuratus hemocytes. 47 residues were determined from its primary structure representing the N-terminal of the complete sequence. This peptide of 5100.78 Da was chemically synthesized and named Ap. The peptide has 25% of hydrophobic amino acids with a net charge of +1, and partial homology with known active antimicrobial peptides. Based on that sequence, a new peptide was designed and modeled to increase hydrophobicity and cationicity. The designed 30-residue peptide was chemically synthesized resulting in a novel 38% hydrophobic molecule named peptide Ap-S, with a net charge of +5 and 3028 Da. A secondary structure was shown by circular dichroism, thus exposing a hydrophobic epitope toward the N-terminus and a hydrophilic one toward the C-terminus, improving amphipathicity. Ap-S was much more active than the parental Ap. Ap-S up to 100 μM has no cytotoxic effect against fish cell line CHSE-214. We demonstrated that the chemical modification of a natural peptide and the chemical synthesis of derived molecules may be a powerful tool for obtaining substitutes to conventional antibiotics, displaying the many advantages of antimicrobial peptides and overcoming the limitations of natural peptides for large-scale production and application, such as the low specific activity and the minute amounts recovered in vivo. This peptide may have a relevant application in aquaculture by controlling Saprolegna sp., a parasitic pathogen fungus that attacks the culture of fish in different stages of their growth, from egg to adult.
Keywords: Antimicrobial peptides; Scallop hemocytes; Chemical design; Chemical synthesis; In vitro antifungal activity;
Mapping and identification of the region and secondary structure required for the maturation of the nukacin ISK-1 prepeptide by Jun-ichi Nagao; Yoshiko Morinaga; Mohammad R. Islam; Sikder M. Asaduzzaman; Yuji Aso; Jiro Nakayama; Kenji Sonomoto (1412-1420).
The prepeptide NukA of the lantibiotic nukacin ISK-1 consists of an N-terminal leader peptide followed by a propeptide moiety that undergoes post-translational modifications, that is, formation of unusual amino acids by NukM, cleavage of the leader peptide and transport by NukT to yield a mature peptide. To identify the region and conformation required for the maturation of prepeptide, we expressed a series of NukA mutants, mutants with the N-terminus-truncated leader peptide and site-directed mutants with conserved residues in the leader peptide of type A(II) lantibiotics, which were evaluated on the basis of the production of nukacin ISK-1. In addition, the secondary structure data of NukA mutants or fragments were obtained by circular dichroism spectra. The results indicated the importance of the α-helical leader peptide with hydrophobic and hydrophilic orientation consisting of the conserved residues in type A(II) lantibiotics. The expression data from various combinations of the chimeric prepeptides consisting of NukA and LctA (the prepeptide of lacticin 481, which shows high identity with NukA) further revealed that the amino acid difference at the C-terminus of the propeptide moiety between NukA and LctA, especially His at position 15 and Phe at position 19, was important for the maturation processes by the nukacin ISK-1 biosynthetic enzymes. Our findings suggest that the determinants in NukA were critically involved in the biosynthesis of nukacin ISK-1 and would thus be important for recognition by the nukacin ISK-1 biosynthetic enzymes.
Keywords: Lantibiotics; Post-translational modification; α-Helix; Chimeric peptide;
The membrane action mechanism of analogs of the antimicrobial peptide Buforin 2 by Gang Hao; Yong-Hui Shi; Ya-Li Tang; Guo-Wei Le (1421-1427).
Previously, the antimicrobial peptides BF2-A and BF2-B, two analogs of Buforin 2 that was hypothesized to kill bacteria by entering cells and binding nucleic acids, had been designed based on the structure–activity analysis of Buforin 2. In the present study, BF2-A and BF2-B were chemically synthesized and their activities and lipopolysaccharide affinity were assayed. To elucidate the mechanism of action with cytoplasmic membranes, we subsequently examined the membrane permeability of both peptides in detail. Both peptides showed stronger antimicrobial activities against a broad spectrum of microorganisms than their parent peptide. Interestingly, BF2-A did not cause significant membrane permeabilization for influx of ONPG into cells, and hardly caused the leakage of intracellular macromolecules, probably BF2-A slightly disturbed cell membrane causing the K+ leakage during peptide crossing phospholipids bilayer. Electron micrographs indicated that the cell membrane treated by BF2-A was still intact within 20 min. On the contrary, BF2-B obviously increased the outer and inner membrane permeability, even induced the slight leakage of macromolecules in the cytoplasm. The leakage of cytoplasmic contents was also demonstrated by the electron micrographs. The increase of membrane permeabilization explained why BF2-B displayed better antimicrobial activity and rapid killing kinetics than BF2-A.
Keywords: Antimicrobial peptide; Buforin 2; Analog; Membrane action; Mechanism;
A family of kassinatuerin-2 related peptides from the skin secretion of the African hyperoliid frog, Kassina maculata by Lei Wang; Mei Zhou; Stephanie McGrath; Tianbao Chen; Sean P. Gorman; Brian Walker; Chris Shaw (1428-1433).
We describe the isolation and structural characterization of a family of antimicrobial peptides related to kassinatuerin-2, from the skin secretion of the African hyperoliid frog, Kassina maculata. All four peptides, designated kassinatuerin-2Ma through Md, are C-terminally-amidated 20-mers with the consensus sequence – FX1GAIAAALPHVIX2AIKNAL – where X1 = L/F/V/I and X2 = S/N. All four peptides are encoded by precursors of 69 amino acids. Synthetic replicates of all kassinatuerin-2 related peptides displayed a potent inhibitory activity against Staphylococcus aureus with a minimal inhibitory concentration of 16 μM, at which concentration, however, they effected 18% haemolysis of horse erythrocytes after 2 h. Despite obvious membranolytic properties, all peptides were ineffective at inhibiting the growth of Escherichia coli at concentrations up to 200 μM and were relatively ineffective against Candida albicans (MIC 120 μM). The kassinatuerin-2 related peptides of K. maculata skin secretion thus possess a discrete antimicrobial and weak haemolytic activity in contrast to the prototype kassinatuerin-2 from the skin secretion of Kassina senegalensis.
Keywords: Amphibian; Mass spectrometry; Antimicrobial peptide; Cloning;
Apelin in goldfish (Carassius auratus): Cloning, distribution and role in appetite regulation by Hélène Volkoff; Jessica L. Wyatt (1434-1440).
Apelin is a recently discovered peptide produced by several tissues including brain and adipose tissue. In mammals and zebrafish, apelin regulates cardiovascular functions. Recent evidence in mammals suggest that apelin might also regulate food intake. In this study, we cloned a cDNA encoding apelin and examined apelin mRNA distribution within the brain and in peripheral tissues. We also assessed the effects of fasting on apelin brain mRNA abundance. Apelin mRNA was expressed throughout the brain as well as in several peripheral tissues including brain, spleen, heart and fat. Apelin mRNA abundance in both hypothalamus and telencephalon was significant higher in fasted fish than in fed fish. In order to further characterize apelin in goldfish, we assessed the effects of central (intracerebroventricular, icv) and peripheral (intraperitoneal, ip) injections of apelin-13 on food intake in goldfish. Apelin injected ip at a dose of 100 ng/g or icv at a dose of 10 ng/g induced a significant increase in food intake compared to saline-injected fish. Our results suggest that apelin acts as an orexigenic factor in goldfish. Its widespread distribution in the brain and the periphery also suggests that apelin might have multiple physiological regulating roles in fish.
Keywords: Apelin; Feeding; Goldfish; Cloning; Distribution; Food intake; RNA expression;
Cloning and characterization of rabbit neuropeptide Y receptor subtypes by Tatsuya Umeda; Akio Kanatani; Hisashi Iwaasa (1441-1447).
Neuropeptide Y (NPY), peptide YY (PYY) and pancreatic polypeptide (PP) are structurally related peptides that have numerous functions in both neural and endocrine signaling. These effects are mediated by the NPY receptor family and five members of this family have been cloned in mammals. To better characterize these receptor subtypes, we cloned and expressed the Y1, Y2, Y4 and Y5 receptor subtypes from the rabbit. Comparison of these sequences with human orthologs revealed that the Y1, Y2 and Y5 receptors have generally strong amino-acid sequence conservation, with 91–96% identity, while Y4 receptor showed relatively weak similarity with 82% identity, as with other species. Particularly in the transmembrane regions, Y1, Y2, and Y5 receptor subtypes showed remarkable conservation, with 98–99% amino acid identity. Competitive binding studies by NPY-family peptides and analogs showed that Y1, Y2 and Y5 receptors had similar pharmacological profiles between the respective rabbit and human receptor subtypes. Interestingly, all the tested peptides had a greater affinity for rabbit Y4 receptor than human Y4 receptor. These results suggest that rabbit and human Y1, Y2 and Y5 receptor subtypes are well conserved, whereas Y4 receptors are less well conserved.
Keywords: Neuropeptide Y; Peptide YY; Pancreatic polypeptide; G-protein-coupled receptor; Rabbit; Pharmacological profile;
Expression of prepro-ghrelin and related receptor genes in the rat adrenal gland and evidences that ghrelin exerts a potent stimulating effect on corticosterone secretion by cultured rat adrenocortical cells by Marcin Rucinski; Agnieszka Ziolkowska; Marianna Tyczewska; Ludwik K. Malendowicz (1448-1455).
The orexigenic peptide ghrelin (GHREL) and obestatin (OBS) originate from the same peptide precursor, preproghrelin (ppGHREL). Apart from orexigenic effect, GHREL also regulates neuroendocrine function. We investigated GHREL and OBS effects on corticosterone secretion by freshly isolated and cultured rat adrenocortical cells. Classic RT-PCR revealed the presence of ppGHREL, GHS-R1a, GPR39v1 and GPR39v2 and GOAT4 (ghrelin O-acyl transferase) mRNAs in rat adrenals and cultured for 4 days rat adrenocortical cells. Expression of ppGHREL, GHS-R1a, and GOAT genes was notably higher in the cortex than in medulla. High expression level of GOAT gene was found in the zona glomerulosa, while expression level of both GPR39v1 and GPR39v2 genes was similar in adrenal cortical zones and in medulla. In freshly isolated cells neither GHREL nor OBS had an effect on corticosteroid output. Prolonged exposure of cultured cells to GHREL resulted in a potent, comparable to ACTH, stimulating effect of GHREL on corticosterone secretion. Prolonged exposure to OBS was ineffective. Neither GHREL nor OBS had any effect on proliferation of studied cells, while ACTH notably lowered it. GHREL down regulated GHS-R1a gene expression while both ACTH and GHREL stimulated expression level of GPR39v1 gene. Expression of CYP11A1 gene was notably stimulated and that of StAR gene remained unaffected by ACTH or GHREL. Thus, our study is the first to demonstrate direct stimulating effect of GHREL on corticosterone output by cultured rat adrenocortical cells. This stimulating action differs from that evoked by ACTH and is not dependent on the presence of functional ACTH receptor.
Keywords: Ghrelin; Obestatin; Ghrelin receptor; Adrenal; Corticosterone; Cell culture; Rat;
The effects of PACAP and related peptides on leptin, soluble leptin receptor and resistin in normal condition and LPS-induced inflammation by Rongjie Yu; Shanshan Xie; Jiansu Chen; Ling Zhang; Yun Dai (1456-1459).
Leptin and resistin are adipokines considered as pro-inflammatory factors related to metabolic syndrome, inflammatory and/or autoimmune conditions. Pituitary adenylate cyclase activating peptide (PACAP) is a pleiotropic neuropeptide with anti-inflammatory properties. We investigated the influence of PACAP on the serum level of leptin, soluble leptin receptor (SLR) and resistin in ordinary and LPS-induced inflammatory conditions using PACAP38 and a series of selective agonist for each PACAP receptor types. It was found that PACAP exerted opposite effects on the leptin:SLR ratio and the serum resistin level. In ordinary condition, PACAP acted as a pro-inflammatory factor by increasing the leptin:SLR ratio and serum resistin level. But in LPS-induced acute inflammatory condition, PACAP not only antagonized the effects of LPS, but also even reversed the effects of LPS. In mice treated with LPS, co-treatment with PACAP decreased the serum leptin and resistin levels and increased the serum soluble leptin receptor level significantly. It was also found that, in ordinary condition, treatment with PAC1 agonist maxadilan induced marked increase in serum leptin, leptin:SLR ratios and resistin levels; while in LPS-induced inflammation, VPAC1 mediated much more anti-inflammatory and reversing-LPS effects of PACAP on leptin and resistin than PAC1 and VPAC2. It is concluded that different receptors mediates different effects of PACAP on leptin, SLR and resistin in non-inflammatory and LPS-induced inflammatory conditions.
Keywords: PACAP; Leptin; Resistin; LPS;
Effect of a gastrin-releasing peptide receptor antagonist and a proton pump inhibitor association in an animal model of gastritis by Fabricia Petronilho; João H. Araújo; Amanda V. Steckert; Gislaine T. Rezin; Gabriela K. Ferreira; Rafael Roesler; Gilberto Schwartsmann; Felipe Dal-Pizzol; Emilio L. Streck (1460-1465).
It has been proposed that reactive oxygen species play a causative role of gastric mucosal damage induced by increased gastric secretion. Gastrin-releasing peptide is a typical neuropeptide that stimulates acid secretion by release of gastrin. In the present work we have investigated the mechanism of indomethacin (IDM)-induced gastric ulcer caused by ROS and determined the effects of a selective gastrin-releasing peptide receptor antagonist, RC-3095, alone and in association with omeprazole (OM) and compared it with an established antioxidant compound N-acetyl cysteine (NAC). Adult male Wistar rats were pre-treated for 7 days with OM, RC-3095, NAC, both drugs and water (control). The animals were then submitted to fasting for 24 h; IDM was administered. Rats were killed 6 h after that and the stomachs were used for evaluation of macroscopic damage and oxidative stress parameters. Our results showed that IDM increased mitochondrial superoxide production; OM and RC-3095 alone did not prevent such effect, but the combination of these drugs was effective. TBARS assay revealed that IDM-induced lipid peroxidation in gastric tissue and that OM and RC-3095, alone or in combination, prevented this effect with superior action that NAC. Finally, we verified that IDM increased protein carbonyl content and that this effect was prevented RC-3095, alone or in combination with OM, being similar to standard antioxidant. The present results support the view that, besides the inhibition of acid secretion, the protective effects exerted by OM and RC-3095 against IDM-induced gastric damage can be ascribed to a reduction of gastric oxidative injury.
Keywords: Indomethacin; Gastritis; Oxidative stress; Omeprazole; RC-3095;
Amylin prevents TRAIL-mediated apoptotic effects of reserpine in the rat gastric mucosa by Giuseppina Cantarella; Giulia Di Benedetto; Giuseppa Martinez; Carla Loreto; Giuseppe Clementi; Antonio Cantarella; Agatina Prato; Renato Bernardini (1466-1472).
We have previously shown that amylin has a protective effect upon the damaged rat gastric mucosa via a cytokine-mediated mechanism. Here, the effects of amylin on the proapoptotic cytokine TNF-related-apoptosis-inducing-ligand (TRAIL) were tested in the rat gastric mucosa damaged by reserpine administration in vivo. Intraperitoneal administration of reserpine in adult male Sprague–Dawley rats resulted in increased TRAIL expression in the gastric mucosa. Immunohistochemistry showed that the TRAIL death-receptor 5 (DR5) was constitutively expressed by the mucosa cells. Western blot showed that pretreatment of reserpine-treated rats with amylin was associated with attenuated expression of TRAIL. In the same samples, we also investigated about TRAIL-related signaling and observed that activation of caspases-8 and -3 occurs in parallel to increased TRAIL expression in rats treated with reserpine. Similarly to the latter, activation of caspases was attenuated in rats pretreated with amylin. Treatment with reserpine was associated with increased expression of the proapoptotic protein Bax, whereas that of the antiapoptotic protein Bcl-2 was significantly decreased. Amylin prevented the effects of reserpine on these genes. Reserpine sets into motion mechanisms of apoptosis in the rat gastric mucosa, which appear mediated, at least in part, by TRAIL. In addition, TRAIL downstream signaling is activated along with subversion of gene expression related to apoptosis. Amylin was able to prevent detrimental effects of reserpine. Finally, amylin and related molecules may be envisioned as protective agent in gastric mucosa damage.
Keywords: Gastric mucosa; Ulcer; Reserpine; Apoptotic cytokine; Amylin;
Characterization of putative GRP- and NMB-receptor antagonist's interaction with human receptors by Nieves González; Samuel A. Mantey; Tapas K. Pradhan; Veronica Sancho; Terry W. Moody; David H. Coy; Robert T. Jensen (1473-1486).
The mammalian bombesin (Bn) peptides neuromedin B (NMB) and gastrin-releasing peptide (GRP) actions are mediated by two receptors (NMB-receptor, GRP-receptor) which are widely distributed in the GI tract and CNS. From primarily animal studies NMB/GRP-receptor activation has physiological/pathophysiological effects in the CNS and GI tract including stimulating of growth of cancers and normal tissues. Whereas these Bn-receptors’ effects have been extensively studied in nonhuman cells and animals, little is known of the physiological/pathological role(s) in humans, largely due to lack of potent antagonists. To address this issue we compared NMB/GRP-receptor affinity/potency of 10 chemical classes of putative antagonists (35 compounds) for human Bn-receptors by performing binding studies or assessing abilities to activate hGRP/hNMB-receptor [assessing phospholipase C activation] in four different cells containing native Bn-receptors or transfected receptors. From binding studies 23 were GRP-receptor-preferring, 4 were NMB-receptor, and 8 nonselective. For the hGRP-receptor-preferring analogues none showed hGRP-receptor agonist activity, but 13 were full or partial hNMB-receptor agonists at hNMB-receptors. For hNMB-receptor-preferring analogues none were agonists. Analogue #24 ([(3-Ph-Pr6), His7, d-Ala11, d-Pro13, Ψ(13-14), Phe14]Bn(6-14)NH2) and analogue #7 [d-Phe6, Leu13, Ψ(CH2NH), Cpa14]Bn(6-14) were the most potent (0.2–1.4 nM) and selective (>10,000-fold) for the hGRP-receptor with analogue #7.5 [d-Tpi6, Leu13, Ψ(CH2NH), Leu14]Bn(6-14)[RC-3095] (0.2–1.4 nM) slightly less selective. Analogue #34 (PD168368) had the highest affinity for hNMB-receptor (1.32–1.58 nM) and the greatest selectivity (2298–6952-fold) for the hNMB-receptor. These results demonstrate numerous putative hGRP/hNMB-receptor antagonists identified in nonhuman cells and/or animals have agonist activity at the hNMB-receptor, limiting their potential usefulness. However, a number were identified which were potent/selective for human Bn-receptors and should be useful for investigating their roles in human physiological/pathophysiological conditions.
Keywords: Human; Gastrin-releasing peptide; Neuromedin B; Bombesin-related peptide; Bombesin;
Electrophysiological effects of orexin/hypocretin on nucleus accumbens shell neurons in rats: An in vitro study by Katsuyuki Mukai; Juhyon Kim; Kazuki Nakajima; Yutaka Oomura; Matthew J. Wayner; Kazuo Sasaki (1487-1496).
Orexin-A (ORX-A) and orexin-B (ORX-B) play critical roles in the regulation of sleep–wakefulness, energy homeostasis, neuroendocrine system and autonomic functions. Although ORXs are also implicated in the reward process, their electrophysiological effects on neurons in the shell of nucleus accumbems (NAcSh) have not been described thoroughly. Therefore we examined the electrophysiological effects of ORXs on rat NAcSh neurons. Whole cell patch clamp recording in vitro revealed that ORX-A and ORX-B depolarize NAcSh neurons in normal and/or tetrodotoxin (TTX)-containing artificial cerebrospinal fluid (ACSF). The depolarization accompanied by a decrease of membrane resistance was concentration-dependent, and there was no significant difference between the two dose–response curves obtained by ORX-A and ORX-B. The ORX-B-induced depolarization was reduced in low-Na+, flufenamic acid-containing, and high-K+ TTX ACSFs, and completely abolished in low-Na+/high-K+ TTX ACSF. An inhibitor of the Na+/Ca2+ exchanger had no effect on the depolarization. The reversal potential obtained from I–V relationships before and during the ORX-B-induced depolarization in low-Na+ TTX ACSF was about −84 mV, and that obtained in TTX ACSF using patch pipettes with Cs+-containing internal solution was about −38 mV. These results suggest that ORXs directly depolarize NAcSh neurons via OX2 receptors and via a dual ionic mechanism including an increase of nonselective cationic conductance and a decrease of K+ conductance, and that NAcSh neurons are involved in the cellular mechanisms through which ORXs participate in the regulation of the reward process as well as feeding and arousal.
Keywords: Orexin/hypocretin; Nucleus accumbens shell; Reward; Nonselective cation channel; K+ channel;
Differential involvement of hippocampal vasoactive intestinal peptide in nociception of rats with a model of depression by Iren Belcheva; Margarita Ivanova; Roman Tashev; Stiliana Belcheva (1497-1501).
The effects of VIP microinjected unilaterally (left or right) into the hippocampal CA1 area at a dose of 10 and 100 ng or bilaterally (10 ng), on nociception of male Wistar rats with a model of depression (bilateral olfactory bulbectomy—OBX) were studied. Nociception was examined applying mechanical pressure on the left hind paw of the rat (analgesy-meter test). It was found that in OBX rats the pain threshold is increased. VIP showed differential effects depending on the side and dose of administration. The pain threshold after left-side microinjections of VIP into the hippocampal CA1 area of OBX rats was significantly higher than that after injections into right-side. There are no significant differences between right-side VIP-treated and OBX rats. Bilateral microinjections of VIP also exerted antinociceptive effect. These findings suggest that the hippocampal lateralized antinociceptive effect of VIP in OBX rats depends on the hemisphere of injection and suggest that VIP-ergic neurons in the hippocampal CA1 area may play differential role in nociception of rats with a model of depression.
Keywords: Vasoactive intestinal peptide; Olfactory bulbectomy; Hippocampus; Depression; Nociception;
His-Arg-Trp potently attenuates contracted tension of thoracic aorta of Sprague-Dawley rats through the suppression of extracellular Ca2+ influx by Mitsuru Tanaka; Shimpei Watanabe; Zhengquan Wang; Kiyoshi Matsumoto; Toshiro Matsui (1502-1507).
In the present study, we primarily attempted to identify di- and tri-peptides showing potent vasodilation in 1.0 μM phenylephrine-contracted thoracic aortas of Sprague-Dawley rats. Synthetic 15 Trp-His (WH) skeleton analogues were used for rat aorta ring's force measurements, since WH was found to be a vasoactive di-peptide so far. Among the synthesized peptides consisted of both His and Trp amino acid residues, His-Arg-Trp (HRW) was found to evoke the most potent vasodilation with an EC50 value of 1.2 ± 0.08 mM in an endothelium-independent manner, while no effect was evoked by a mixture of individual amino acids. In addition to the structure of tri-peptides-activity relationship, chemically modified HRW analogues, i.e., 1- or 3-methyl-His-Arg-Trp and His-citrulline-Trp demonstrated the structural importance of tri-peptide to evoke the vasoactivity as following factors: (1) Neutral imidazole and indole groups from His and Trp residues at N- and C-terminals, respectively and (2) basic amino acids at the middle position. In mitogen (10 μM angiotensin II or 50 μM Bay K8644)-stimulated vascular smooth muscle cells, vasoactive HRW (100 μM) caused significant [Ca2+] i reduction to an extent of >30%. Thus, our results suggest that HRW caused vasodilation action via an endothelium-independent mechanism which probably involves the suppression of extracellular Ca2+ influx through voltage-gated l-type Ca2+ channel.
Keywords: Tri-peptide; Vasodilation; Rat aorta; Hypertension; l-type Ca2+ channel;
Differential mRNA splicing and precursor processing of neurokinin B in neuroendocrine tissues by Nigel M. Page; Donald W. Morrish; Nicola J. Weston-Bell (1508-1513).
The tachykinin neurokinin B which is encoded on the tachykinin 3 precursor, has prominent roles in both neuronal and endocrine systems, yet little is known about its evolution, potential splice variants and the manner in which it is processed. Here, we deduce the diversity within the vertebrate tachykinin 3 precursors, and identify novel tachykinin 3 splice variants and precursors. A total of 35 different tachykinin 3 precursors were identified in mammals, birds and reptiles. Nine additional alternatively spliced tachykinin 3 mRNA transcripts were also discovered in humans leading to the formation of three tachykinin 3 precursors (named α, β and γ tachykinin 3), but no novel tachykinin. γ tachykinin 3, albeit rarer, was not found to encode neurokinin B. Differential processing of the tachykinin 3 precursor in the human placenta leads to the formation of potential NH2-terminally extended forms of neurokinin B. Moreover, we found increased proteolytic cleavage of the tachykinin 3 precursor during the pregnancy syndrome of pre-eclampsia. We have established neurokinin B to be an evolutionarily conserved peptide, nonetheless the significance of the three different tachykinin 3 precursors is not clear, but could represent an evolutionarily redundant splicing mechanism once employed by an ancestral gene that encoded two tachykinins. Our results indicate that differential mRNA splicing and precursor processing is likely to play an important role in differentiating the actions of the tachykinin 3 gene products in both neuronal and endocrine tissues.
Keywords: Neurokinin; Tachykinin; Splice variant; Precursor; Pre-eclampsia;
Human hemokinin-1 and human hemokinin-1(4–11), mammalian tachykinin peptides, suppress proliferation and induce differentiation in HL-60 cells by You-Li Zhao; Yan Tao; Cai-Yun Fu; Zi-Qing Kong; Qiang Chen; Rui Wang (1514-1522).
Human hemokinin-1 (h HK-1) and its truncated form h HK-1(4–11) are mammalian tachykinin peptides encoded by the TAC4 gene identified in human, and the biological functions of these peptides have not been well investigated. The tachykinins have shown immuno-regulatory activities in humans. In the present study, we investigated the effects of h HK-1 and h HK-1(4–11) on the proliferation and differentiation of a human promyelocyte leukemia cell line, HL-60. It is noteworthy that h HK-1 (1–300 μM) displayed inhibitory effects on the proliferation of HL-60 cells in a dose- and time-dependent manner. The effect of suppressing proliferation induced by these peptides was accompanied by an accumulation of cell cycle in the S phase. Moreover, this peptide induced differentiation of HL-60 cells by significantly increasing the NBT-reduction activity. The effects induced by h HK-1(4–11) on HL-60 cells were similar to that of h HK-1, indicating that it is the active fragment of h HK-1. However these effects induced by h HK-1 or h HK-1(4–11) were not antagonized by the NK1 receptor antagonist SR140333 or the NK2 receptor antagonist SR48968. All the results indicated that h HK-1 and h HK-1(4–11) were able to significantly inhibit proliferation and induce differentiation and S phase arrest of a human promyelocyte leukemia cell line HL-60, which may not be mediated through the activation of classical tachykinin NK1 receptors and tachykinin NK2 receptors. Our observations also implied that h HK-1 and h HK-1(4–11) could act as immunomodulatory factors in cancer chemotherapy.
Keywords: h HK-1 and h HK-1(4–11); HL-60; Differentiation; Proliferation inhibition; SR140333 and SR48968;
Constructing bioactive peptides with pH-dependent activities by Zhigang Tu; Melanie Volk; Khushali Shah; Kevin Clerkin; Jun F. Liang (1523-1528).
Many bioactive peptides are featured by their arginine and lysine rich contents. In this study, lysine and arginine residues in lytic peptides were selectively replaced by histidines. Although resulting histidine-containing lytic peptides had decreased activity, they did show pH-dependent cytotoxicity. The activity of the constructed histidine-containing lytic peptides increased 2–8 times as the solution pH changed from 7.4 to 5.5. More importantly, these histidine-containing peptides maintain the same cell killing mechanism as their parent peptides by causing cell lysis. Both the activity and pH-sensitivity of histidine-containing peptides are tunable by adjusting histidine substitution numbers and positions. This study has presented a general strategy to create bioactive peptides with desired pH-sensitivity to meet the needs of various applications such as cancer treatments.
Keywords: Peptide; pH sensitivity; Histidine; Bioactive; Cell lysis;
Identification of a stable chemerin analog with potent activity toward ChemR23 by Ken Shimamura; Masao Matsuda; Yasuhisa Miyamoto; Ryo Yoshimoto; Toru Seo; Shigeru Tokita (1529-1538).
Chemerin is a novel peptide that was identified as a natural ligand for ChemR23. As it has been reported to be involved in the regulation of immune responses and adipogenesis, chemerin may have a variety of physiological functions. Chemerin is synthesized as a precursor (prochemerin) and is proteolytically activated and inactivated in sequential steps, which control its physiological roles in a coordinated manner. Chemerin-9 (chemerin148–156) was previously identified as the smallest peptide with low nanomolar potency. However, like mature chemerin, chemerin-9 is rapidly degraded and inactivated in plasma, which has limited the use of chemerin-9 in in vivo experiments. In order to identify stable chemerin analogs that facilitate in vivo studies, we synthesized a series of chemerin-9 analogs and examined intrinsic activity and metabolic stability. We identified an agonistic and metabolically stable chemerin-9 analog (d-Tyr147-[d-Ser151, d-Ala154, Tic155]chemerin148–156) that shows enhanced plasma exposure with prolonged half-life in mice upon intraperitoneal administration. Improvement of metabolic stability resulted in a reduction in the plasma free fatty acid levels in fasted mice, which cannot be accomplished by unstable-mouse chemerin-9. This reduction in plasma free fatty acids reflects the anti-lipolysis activity of chemerin-9 and analogs in mouse primary adipocytes. The discovery of a metabolically stable chemerin analog will facilitate investigation of the pharmacological roles of chemerin in vivo. Moreover, this stable chemerin analog might provide new therapeutic approaches to inflammatory diseases such as asthma and metabolic disorders such as obesity and diabetes where ChemR23 activation may be of benefit.
Keywords: Chemerin; Adipokine; Peptide tool;
Identification of a novel dual E- and N-cadherin antagonist by Emmanuelle Devemy; Orest W. Blaschuk (1539-1547).
E- and N-cadherin are related calcium-dependent cell adhesion molecules that exert an influence over multiple biological and disease processes. Antagonists of these cadherins can therefore be envisaged as therapeutically useful drugs. We have used phage display technology to discover such antagonists. A peptide phage library was screened against a chimeric protein composed of the human E-cadherin ectodomain fused to the Fc fragment of human immunoglobulin G1 (E-cad/Fc). All of the phage clones that were isolated also bound a chimeric protein composed of the human N-cadherin ectodomain fused to the Fc fragment of human immunoglobulin G1 (N-cad/Fc). A peptide displayed by several of the isolated phage clones was synthesized (H-SWELYYPLRANL-NH2) and found to bind both E- and N-cad/Fc chimeric proteins with affinities (K D) of 9.4 μM and 323 nM, respectively, as judged by surface plasmon resonance spectroscopy. This peptide was also capable of blocking the aggregation of E- and N-cad/Fc chimeric protein-coated beads, as well as the aggregation of MCF-7 and MDA-MB435 human breast cancer cells (these cells express E- and N-cadherin, respectively). Finally, we showed that the peptide disrupted MCF-7 and MDA-MB435 cell monolayers. The peptide, H-SWELYYPLRANL-NH2 thus proved to be a biologically active, dual E- and N-cadherin antagonist. Such an antagonist has application in a wide variety of biological contexts.
Keywords: E- and N-cadherin antagonist; Peptide; Phage display;
Transduction of adenovirus vectors modified with cell-penetrating peptides by Yusuke Eto; Yasuo Yoshioka; Ratima Asavatanabodee; Shinya Kida; Mitsuko Maeda; Yohei Mukai; Hiroyuki Mizuguchi; Koichi Kawasaki; Naoki Okada; Shinsaku Nakagawa (1548-1552).
Adenovirus vectors (Advs) are widely used for basic and clinical research because of their high transduction efficiency. However, they are poorly transduced into cells lacking the primary adenovirus receptor, the coxsackievirus and adenovirus receptor (CAR). Here, we generated Adv conjugated with cell-penetrating peptides (CPPs), such as Tat, octaarginine (R8) or proline-rich (Pro) peptides, and compared the transduction properties of these constructs. We constructed the Advs conjugated to the CPPs (CPP–Adv) by chemical conjugation. The CPP-conjugated Advs created with optimal modification ratios led to gene expression 1–2 log orders higher than unmodified Adv in CAR-negative cells. Tat-Adv and R8-Adv were taken up into the cells mainly through macropinocytosis, independently of the CAR. In addition, the cellular uptake of Tat-Adv was highly dependent on heparan sulfate on the cell surface, whereas that of R8-Adv was dependent on chondroitin sulfate B. These data suggest that the use of CPP-Advs with different cellular uptake pathways might create new methods for the delivery of Adv. The results obtained in this research encourage the use of CPP-peptide-modified Advs as an attractive tool for transducing cells and as useful platform vectors for gene therapy and basic research.
Keywords: Adenovirus vector; Cell-penetrating peptide; Chemical conjugation; Gene therapy; Macropinocytosis;
Scorpion toxin of Androctonus australis garzonii induces neuropeptide Y release via bradykinin stimulation in rat atria and kidneys by Hayet Soualmia; Joëlle Eurin; Yasmina Djeridane (1553-1556).
The ability of scorpion toxins to produce hemodynamic alterations is well documented but all mediators implied in cardiovascular disturbances are not known. In the present investigation we studied the effect of North African Androctonus australis garzonii scorpion toxin on neuropeptide Y (NPY) release from rat atria and kidneys by a perifusion system in vitro. To further understand the mechanisms of the scorpion toxin action on NPY release, the effects of icatibant (HOE 140, a selective bradykinin-B2 receptor antagonist), tetrodotoxin (TTX, a sodium channel antagonist) and diltiazem (a calcium channel antagonist), and the effect of the scorpion toxin on bradykinin (BK, a potent vasorelaxant peptide of the kinin group) release were studied in both tissues. We showed that the scorpion toxin (10−6 M) increased the NPY release from both atria (35%) and kidneys (40%). This increase was significantly (p < 0.001) inhibited by HOE 140 (10−5 M). The scorpion toxin (10−6 M) enhanced BK secretion in both atria (52%) and kidneys (55%). Diltiazem (10−5 M) and TTX (10−5 M) decreased by 45–75% NPY levels induced by scorpion toxin in both organs. The results show that A. australis garzonii scorpion toxin stimulates NPY release from both rat atria and kidneys, and suggest that the toxin induces NPY release via BK stimulation through B2 receptors. This effect appears to involve calcium and sodium channel activation.
Keywords: Scorpion toxin; Neuropeptide Y; Bradykinin; Atrium; Kidney;
PdT-2: A novel myotropic Type-2 tryptophyllin from the skin secretion of the Mexican giant leaf frog, Pachymedusa dacnicolor by Lei Wang; Mei Zhou; Tianbao Chen; Brian Walker; Chris Shaw (1557-1561).
Amphibian skin secretions represent a unique resource for the discovery of new bioactive peptides. Here we report the isolation, structural and functional characterization of a novel heptapeptide amide, DMSPPWHamide, from the defensive skin secretion of the Mexican giant leaf frog, Pachymedusa dacnicolor. This peptide is of unique primary structure and has been classified as a member of the rather heterogenous tryptophyllin-2 (T-2) family of amphibian skin peptides and named P. dacnicolor Tryptophyllin-2 (PdT-2) in accordance. PdT-2 is the first Type 2-tryptophyllin to possess discrete bioactivity. Both natural and synthetic replicates of the peptide were found to contract the smooth muscle of rat urinary bladder, the latter displaying an EC50 of 4 nM.
An overview of the mosaic bacteriocin pln loci from Lactobacillus plantarum by Dzung B. Diep; Daniel Straume; Morten Kjos; Carmen Torres; Ingolf F. Nes (1562-1574).
The pln locus responsible for bacteriocin biosynthesis in Lactobacillus plantarum C11 was first unraveled about 15 years ago and since then different strains of L. plantarum (NC8, WCFS1, J23 and J51) have been found to harbor mosaic pln loci in their genomes. Each locus is of 18–19 kb and contains 22–25 genes organized into 5–6 operons. Together these strains produce four different class IIb two-peptide bacteriocins, plantaricins EF, JK, NC8 and J51 and a pheromone peptide plantaricin A with antimicrobial activity. Their production has been found to be regulated through a quorum-sensing based network consisting of a secreted peptide pheromone, a membrane-located sensor and one or two transcription regulators. The individual loci each contain a set of semi-conserved regulated promoters with subtle differences necessary for the regulators to regulate their promoter activity individually with respect to timing and strength. These subtle differences in the promoters are highly conserved across the different pln loci, in a functionally related manner. In this review we will discuss various aspects of these bacteriocin loci with special focus on their mosaic genetic composition, gene regulation and mode of action. We also present a novel pln locus containing a transposon of the MULE superfamily, a mobile element which has not been described in L. plantarum before.
Keywords: Bacteriocin; Plantaricin; Probiotics; Antimicrobial peptide;
The renin–angiotensin system, adrenomedullins and urotensin II in the kidney: Possible renoprotection via the kidney peptide systems by Kazuhiro Takahashi; Takuo Hirose; Nobuyoshi Mori; Ryo Morimoto; Masahiro Kohzuki; Yutaka Imai; Kazuhito Totsune (1575-1585).
The incidence of chronic kidney disease, such as diabetic nephropathy, is increasing throughout the world. Many biologically active peptides play important roles in the kidney. The classical example is the renin–angiotensin system (RAS). Angiotensin II plays critical roles in the progression of chronic kidney disease through its vasoconstrictor action, stimulatory action on cell proliferation, and reactive oxygen-generating activity. A renin inhibitor, aliskiren, has recently been shown to be a clinically effective drug to reduce proteinuria in patients with diabetic nephropathy. (Pro)renin receptor, a specific receptor for renin and prorenin, was newly identified as a member of the RAS. When bound to prorenin, (pro)renin receptor activates the angiotensin I-generating activity of prorenin in the absence of cleavage of the prosegment, and directly stimulates the pathway of mitogen-activated protein kinase independently from the RAS. The kidney peptides that antagonize the intrarenal RAS may have renoprotective actions. Adrenomedullins, potent vasodilator peptides, have been shown to have renoprotective actions. On the other hand, urotensin II, a potent vasoconstrictor peptide, may promote the renal dysfunction in chronic kidney disease together with the renal RAS. Thus, in addition to the renin inhibitor and (pro)renin receptor, adrenomedullins and urotensin II may be novel targets to develop therapeutic strategies against chronic kidney disease.
Keywords: Adrenomedullin; Angiotensin; Prorenin receptor; Urotensin;
The involvement of substance P in the induction of aggressive behavior by Eleni Katsouni; Pavlos Sakkas; Apostolos Zarros; Nikolina Skandali; Charis Liapi (1586-1591).
Aggression is a complex social behavior that involves a similarly complex neurochemical background. The involvement of substance P (SP) and its potent tachykinin receptor (NK1) in the induction of both defensive rage and predatory attack appears to be a consistent finding. However, an overall understanding of the nature of the SP involvement in the induction of aggressive behavior has not yet been fully achieved. The aim of this review is to summarize and present the current knowledge with regards to the role of SP in the induction of aggressive behavior and to synopsize: (a) its biochemical profile, and (b) the exact anatomical circuits through which it mediates all types of aggressive behavior. Future studies should seriously consider the potential use of this knowledge in their quest for the treatment of mood and anxiety disorders.
Keywords: Substance P; NK1 receptor; Aggression; Defensive rage; Aggressive attack; Medial amygdaloid nucleus; Medial and lateral hypothalamus; Periaqueductal grey;