Peptides (v.35, #2)
Editorial Board (CO2).
Gayle and Richard Olson prize pages (III-IV).
Nesfatin-130–59 but not the N- and C-terminal fragments, nesfatin-11–29 and nesfatin-160–82 injected intracerebroventricularly decreases dark phase food intake by increasing inter-meal intervals in mice by Andreas Stengel; Miriam Goebel-Stengel; Lixin Wang; Ikuo Kato; Masatomo Mori; Yvette Taché (143-148).
► Nesfatin-11–82 injected icv reduces dark phase feeding in mice. ► The mid-fragment nesfatin-130–59 dose-dependently reduces 4-h food intake. ► The reduction goes along with reduced meal frequency and longer inter-meal interval. ► Neither nesfatin-11–29 nor nesfatin-160–82 reduce dark phase food intake. ► Nesfatin-130–59 is the active core of nesfatin-11–82.Nesfatin-1 is an 82 amino acid N-terminal fragment of nucleobindin2 that was consistently shown to reduce dark phase food intake upon brain injection in rodents. We recently reported that nesfatin-11–82 injected intracerebroventricularly (icv) reduces dark phase feeding in mice. Moreover, intraperitoneal injection of mid-fragment nesfatin-1 (nesfatin-130–59) mimics the food intake-reducing effects of nesfatin-11–82, whereas N-terminal (nesfatin-11–29) and C-terminal fragments (nesfatin-160–82) did not. We therefore characterized the structure-activity relationship of nesfatin-1 injected icv to influence the dark phase meal pattern in mice. Mouse nesfatin-11–29, nesfatin-130–59, nesfatin-160–82 or vehicle was injected icv in freely fed C57Bl/6 mice immediately before the dark phase and food intake was monitored using an automated episodic feeding monitoring system. Nesfatin-130–59 (0.1, 0.3, 0.9 nmol/mouse) induced a dose-related reduction of 4-h food intake by 28%, 49% and 49% respectively resulting in a 23% decreased cumulative 24-h food intake compared to vehicle at the 0.3 nmol/mouse dose (p < 0.05). The peak reduction occurred during the 3rd (-96%) and 4th hour (-91%) post injection and was associated with a reduced meal frequency (0–4 h: −47%) and prolonged inter-meal intervals (3.1-times) compared to vehicle (p < 0.05), whereas meal size was not altered. In contrast, neither nesfatin-11–29 nor nesfatin-160–82 reduced dark phase food intake at equimolar doses although nesfatin-160–82 prolonged inter-meal intervals (1.7-times, p < 0.05). Nesfatin-130–59 is the active core of nesfatin-11–82 to induce satiety indicated by a reduced meal number during the first 4 h post injection. The delayed onset may be indicative of time required to modulate other hypothalamic and medullary networks regulating nocturnal feeding as established for nesfatin-1.
Keywords: Food intake; Meal pattern; Mouse; Nesfatin-1 fragments; Satiation; Satiety;
Evidence suggesting that ghrelin O-acyl transferase inhibitor acts at the hypothalamus to inhibit hypothalamo-pituitary-adrenocortical axis function in the rat by Marcin Rucinski; Agnieszka Ziolkowska; Marta Szyszka; Anna Hochol; Ludwik K. Malendowicz (149-159).
Production of n-octanoyl-modified ghrelin (GHREL), an active form of the peptide requires prohormone processing protease and GHREL O-acyltransferase (GOAT), as well as n-octanoic acid. Recently a selective GOAT antagonist (GO-CoA-Tat) was invented and this tool was used to study the possible role of endogenous GHREL in regulating HPA axis function in the rat. Administration of GOAT inhibitor (GOATi) resulted in a notable decrease in plasma ACTH, aldosterone and corticosterone concentrations at min 60 of experiment. Octanoic acid (OA) administration had no effect on levels of studied hormones. Plasma levels of unacylated and acylated GHREL remained unchanged for 60 min after either GOATi or OA administration. Under experimental conditions applied, no significant changes were observed in the levels of GOAT mRNA in hypothalamus, pituitary, adrenal and stomach fundus. After GOATi injection hypothalamic CRH mRNA levels were elevated at 30 min and pituitary POMC mRNA levels at 60 min. Both GOATi and OA lowered basal, but not K+-stimulated CRH release by hypothalamic explants and had no effect on basal or CRH-stimulated ACTH release by pituitary slices. Neither GOATi nor OA affected corticosterone secretion by freshly isolated or cultured rat adrenocortical cells. Thus, results of our study suggest that in the rat endogenous GHREL exerts tonic stimulating effect on hypothalamic CRH release. This effect could be demonstrated by administering rats with selected inhibitor of ghrelin O-acyltransferase, the enzyme responsible for GHREL acylation, a process which is absolutely required for both GHSR-1a binding and its central endocrine activities.
Keywords: Ghrelin O-acyl transferase inhibitor; GOAT; CRH; ACTH; Aldosterone; Corticosterone; Cell culture; rat;
Acute ghrelin administration reverses depressive-like behavior induced by bilateral olfactory bulbectomy in mice by Valeria Paola Carlini; Daniele Guilhermano Machado; Florencia Buteler; Marisa Ghersi; Marina F. Ponzio; Ana Carolina Martini; Helgi B. Schiöth; Marta Fiol de Cuneo; Ana Lúcia S. Rodrigues; Susana R. de Barioglio (160-165).
► Ghrelin reverses depressive-like behavior induced by bilateral olfactory bulbectomy in mice. ► Ghrelin produced antidepressant-like effect in tail suspension test. ► Ghrelin reverses hyperactivity induced by bilateral olfactory bulbectomy in mice.This study aims to examine the antidepressant-like action of Ghrelin (Ghr), a hormone synthesized predominantly by gastrointestinal endocrine cells and released during periods of negative energy balance, in two behavioral models: tail suspension test (TST), a predictive model of antidepressant activity, and the olfactory bulbectomy (OB), an established animal model of depression. The reduction in the immobility time in the TST was the parameter used to assess antidepressant-like effect of Ghr. The depressive-like behavior in olfactory bulbectomized mice was inferred through the increase in the immobility time in the TST and the hyperlocomotor activity in the open-field test. Ghr produced antidepressant-like effect in TST (0.3 nmol/μl, i.c.v.), and reversed OB-induced depressive-like behavior. In conclusion, these results provide clear evidence that an acute administration of ghrelin produce antidepressant-like effect in the TST and OB.
Keywords: Ghrelin; Tail suspension test; Olfactory bulbectomy; Hyperactivity; Antidepressant;
Relationship between plasma leptin levels and clinical outcomes of pediatric traumatic brain injury by Chao Lin; Shou-Jiang Huang; Ning Wang; Zhi-Peng Shen (166-171).
► Plasma leptin levels increased in children with traumatic brain injury. ► Plasma leptin levels were highly associated with poor outcome in pediatric traumatic brain injury. ► Leptin may be a good prognostic factor for 6-month mortality and functional outcome in children with traumatic brain injury.High plasma leptin level has been associated with mortality after adult intracerebral hemorrhage. The present study was undertaken to investigate the plasma leptin concentrations in children with traumatic brain injury and to analyze the correlation of leptin with pediatric traumatic brain injury outcome. Plasma leptin concentration of eighty-nine healthy children and 142 children with acute severe traumatic brain injury was measured by enzyme-linked immunosorbent assay. Twenty-six patients (18.3%) died and 42 patients (29.6%) had an unfavorable outcome (Glasgow outcome scale score of 1–3) at 6 months after traumatic brain injury. Upon admission, plasma leptin level in patients was substantially higher than that in healthy controls. A forward stepwise logistic regression selected plasma leptin level as an independent predictor for 6-month mortality and unfavorable outcome of patients. A receiver operating characteristic curve analysis showed plasma leptin level better predicted 6-month mortality and unfavorable outcome. The prognostic value of leptin was similar to that of Glasgow Coma scale score for 6-month clinical outcomes. Thus, plasma leptin level represents a novel biomarker for predicting 6-month clinical outcome in children with traumatic brain injury.
Keywords: Leptin; Traumatic brain injury; Functional outcome; Mortality;
Inhibitory role of kinins on microglial nitric oxide and tumor necrosis factor-α production by Ben-Shmuel Sarit; Gera Lajos; Danon Abraham; Apte Ron; Fleisher-Berkovich Sigal (172-181).
► Three kinin agonists attenuate LPS-induced NO and TNF-α production in BV2 cells. ► Bradykinin reduced basal NO and TNF-α production in BV2 cells. ► Anti-inflammatory effects of bradykinin were confirmed in primary microglia. ► Three kinin agonists reduce LPS-induced iNOS and TNF-α protein and mRNA levels in BV2 cells. ► Bradykinin abolishes LPS-induced NF-κB pathway activation in BV2 cells.Brain inflammation is sustained by chronic activation of microglia and the over-production of pro-inflammatory cytokines and nitric oxide (NO), which in turn can be highly neurotoxic. Microglial activation can be regulated by neuropeptides such as bradykinin (BK) and other members of the kinin family. Kinins are well known inflammatory regulators outside the CNS. Although the kinin system is well distributed throughout the brain, the precise role of BK in the CNS is not yet clear. The aim of this study was to examine and characterize the effects of BK and related kinins on the production of NO and TNF-α in microglia. We found that BK and selective agonists for both B1 and B2 receptors, attenuated both NO and TNF-α levels in the media of BV2 microglial cells that had been stimulated with LPS. The effects of BK that were observed in BV2 cells were confirmed in primary neonatal rat microglial cells as well. In addition, all kinin agonists reduced the expression of iNOS and TNF-α protein and mRNA levels in LPS-stimulated BV2 cells. Also, while LPS activated the nuclear factor-κB (NF-κB) pathway, BK inhibited NF-κB activation by preventing degradation of the κB protein (IκB) inhibitor, abolishing translocation of p65 and p50 subunits to the nucleus and inhibiting NF-κB transcription activity. These results suggest a role for bradykinin in modulation of glial inflammation, as evidenced by attenuation of NO and TNF-α synthesis pathways in activated microglial cells.
Keywords: Bradykinin; Microglia; Nitric oxide; Tumor necrosis factor-α; Nuclear factor-κB;
Inhalable sustained-release formulation of long-acting vasoactive intestinal peptide derivative alleviates acute airway inflammation by Satomi Onoue; Takuya Matsui; Kazuki Kuriyama; Kumiko Ogawa; Yoshiki Kojo; Takahiro Mizumoto; Shin-ichiro Karaki; Atsukazu Kuwahara; Shizuo Yamada (182-189).
► VIP-loaded nanospheres showed a biphasic release pattern with 20% initial burst and a sustained release. ► New formulation exhibited high dispersibility and suitable size for inhalation therapy. ► Combination use of nanospheres and respirable system led to marked attenuation of airway inflammation in rats.The present study was undertaken to develop a respirable sustained-release powder (RP) formulation of long-acting VIP derivative, [Arg15, 20, 21, Leu17]-VIP-GRR (IK312532), using PLGA nanospheres (NS) with the aim of improving the duration of action. NS formulation of IK312532 (IK312532/NS) was prepared by an emulsion solvent diffusion method in oil, and a mixture of the IK312532/NS and erythritol was jet-milled and mixed with lactose carrier to obtain the IK312532/NS-RP. Physicochemical properties were characterized focusing on appearance, particle size, and drug release, and in vivo pharmacological effects were assessed in antigen-sensitized rats. The IK312532/NS with a diameter of 140 nm showed a biphasic release pattern in distilled water with ca. 20% initial burst for 30 min and a sustained slow release up to ca. 55% for 24 h. Laser diffraction analysis demonstrated that IK312532/NS-RP had fine dispersibility and suitable particle size for inhalation. In antigen-sensitized rats, insufflated IK312532/NS-RP (10 μg of IK312532/rat) could suppress increases of granulocyte recruitment and myeloperoxidase in pulmonary tissue for up to 24 h after antigen challenge, although IK312532-RP at the same dose was less effective with limited duration of action. From these findings, newly prepared IK312532/NS-RP might be of clinical importance in improving duration of action and medication compliance for treatment of airway inflammatory diseases.
Keywords: Airway inflammation; Inhalation; PLGA; Sustained release; VIP derivative;
Divergent pathways for the angiotensin-(1–12) metabolism in the rat circulation and kidney by Brian M. Westwood; Mark C. Chappell (190-195).
► Serum ACE processes Ang-(1–12) to Ang II through the generation of Ang I. ► Serum renin does not metabolize Ang-(1–12) to shorter angiotensin peptides. ► Rat, human and mouse neprilysin process Ang-(1–12) to Ang-(1–7) and Ang-(1–4). ► Kinetic modeling does not favor Ang-(1–12) as the immediate precursor to Ang I.Evidence of endogenous angiotensin-(1–12) [Ang-(1–12)] may necessitate revision of the accepted view that Ang I is the immediate peptide product derived from the precursor protein angiotensinogen. As the processing of this peptide has not been fully elucidated, we characterized Ang-(1–12) metabolism in the serum and kidney of the mRen2.Lewis rat, a model of high circulating renin and ACE expression. A sensitive HPLC-based method to detect the metabolism ex vivo of low concentrations of 125I-labeled Ang-(1–12) was utilized. Ang-(1–12) processing to serum did not reveal the participation of renin; however, serum ACE readily converted Ang-(1–12) to Ang I with subsequent metabolism to Ang II. Ang I and Ang II forming activities for serum ACE were 102 ± 4 and 104 ± 3 fmol/ml/min serum (n = 3), respectively, and both products were abolished by the potent ACE inhibitor lisinopril. The metabolism of Ang-(1–12) in renal cortical membranes also revealed the formation of Ang I; however, the main products were Ang-(1–7) and Ang-(1–4) at 129 ± 9 and 310 ± 12 fmol/mg/min protein (n = 4), respectively. Neprilysin inhibition abolished these products and substantially reduced the overall metabolism of Ang-(1–12). Incubation of Ang-(1–12) with either human or mouse neprilysin revealed identical products. We conclude that endogenous Ang-(1–12) may contribute to the expression of biologically active angiotensins through a renin-independent pathway. The preferred route for Ang-(1–12) metabolism likely reflects the relative tissue content of ACE and neprilysin.
Keywords: Angiotensin-(1–12); Renin; ACE; Neprilysin; Ang-(1–7); Ang-(1–4); HPLC;
The cardiac expression of Mas receptor is responsive to different physiological and pathological stimuli by Marco Fabricio Dias-Peixoto; Anderson J. Ferreira; Pedro W.M. Almeida; Vinícius B.A. Braga; Danielle C.O. Coutinho; Dirceu S. Melo; Ary Gomes Filho; Marcos B. Melo; Leonardo Greco; Maria José Campagnole-Santos; Ricardo F. Lima; Robson A.S. Santos; Silvia Guatimosim (196-201).
► Mas expression in the heart is regulated according to the stage of the disease. ► Cardiac Mas expression is responsive to different pathological stimuli. ► Physical training is unable to induce changes in cardiac Mas expression.The Mas protooncogene encodes a G protein-coupled receptor that has been described as a functional receptor for the cardioprotective fragment of the renin-angiotensin system (RAS), Angiotensin (Ang)-(1-7). The aim of this current study was to evaluate the responsiveness of Mas expression in hearts during different physiological and pathological conditions in rats. Physical training was considered a physiological condition, while isoproterenol-induced hypertrophy, myocardial infarction and DOCA-salt model of hypertension were used as pathological models of heart injury. The expression of Mas was analyzed by western blotting. Although swim-trained rats presented significant cardiac hypertrophy, our physical training protocol was unable to induce changes in the expression of Mas. On the other hand, cardiac hypertrophy and damage elicited by isoproterenol treatment led to a reduction in Mas expression. Myocardial infarction also significantly decreased the expression of Mas after 21 days of myocardial ischemia. Additionally, Mas expression levels were increased in hearts of DOCA-salt rats. Our present data indicate that Mas expression is responsive to different pathological stimuli, thereby suggesting that Mas receptor is involved in the homeostasis of the heart, as well as in the establishment and progression of cardiac diseases.
Keywords: Mas receptor; Physical training; Isoproterenol treatment; Myocardial infarction; DOCA-salt rats;
Localization of calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1) in human gastrointestinal tract by Graeme S. Cottrell; Farzad Alemi; Jacob G. Kirkland; Eileen F. Grady; Carlos U. Corvera; Aditi Bhargava (202-211).
► CLR and RAMP1 co-localize in enteric neurons of the human gastrointestinal tract. ► CLR and RAMP1 are present in close proximity to their ligands CGRP and intermedin. ► CGRP and intermedin must be secreted in a paracrine manner in human gut to mediate their effects via their receptors.Calcitonin gene-related peptide (CGRP) exerts its diverse effects on vasodilation, nociception, secretion, and motor function through a heterodimeric receptor comprising of calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1). Despite the importance of CLR·RAMP1 in human disease, little is known about its distribution in the human gastrointestinal (GI) tract, where it participates in inflammation and pain. In this study, we determined that CLR and RAMP1 mRNAs are expressed in normal human stomach, ileum and colon by RT-PCR. We next characterized antibodies that we generated to rat CLR and RAMP1 in transfected HEK cells. Having characterized these antibodies in vitro, we then localized CLR-, RAMP1-, CGRP- and intermedin-immunoreactivity (IMD-IR) in various human GI segments. In the stomach, nerve bundles in the myenteric plexus and nerve fibers throughout the circular and longitudinal muscle had prominent CLR-IR. In the proximal colon and ileum, CLR was found in nerve varicosities of the myenteric plexus and surrounding submucosal neurons. Interestingly, CGRP expressing fibers did not co-localize, but were in close proximity to CLR. However, CLR and RAMP1, the two subunits of a functional CGRP receptor were clearly localized in myenteric plexus, where they may form functional cell-surface receptors. IMD, another member of calcitonin peptide family was also found in close proximity to CLR, and like CGRP, did not co-localize with either CLR or RAMP1 receptors. Thus, CGRP and IMD appear to be released locally, where they can mediate their effect on their receptors regulating diverse functions such as inflammation, pain and motility.
Keywords: CGRP; Intermedin; Adrenomedullin 2; Myenteric plexus; Primary afferent;
Hepcidin protects against lipopolysaccharide-induced liver injury in a mouse model of obstructive jaundice by Ying-Hsien Huang; Ya-Ling Yang; Mao-Meng Tiao; Ho-Chang Kuo; Li-Tung Huang; Jiin-Haur Chuang (212-217).
► We examined the recombinant hepcidin is effective in alleviating cholestasis-induced liver injury and mortality in mice with superimposed sepsis. ► Hepcidin pretreatment significantly reduced hepatic proinflammatory cytokine expression and liver injury in obstructive jaundice mice receiving LPS. ► Enhanced autophagy and reduced apoptosis may account for the protective effects of hepcidin.Obstructive jaundice (OJ) increases the risk of liver injury and sepsis, leading to increased mortality. Cholestatic liver injury is associated with a downregulation of hepcidin expression levels. In fact, hepcidin has an important antimicrobial effect, especially against Escherichia coli. It is unknown whether supplementing recombinant hepcidin is effective in alleviating cholestasis-induced liver injury and mortality in mice with superimposed sepsis. A mouse model of cholestasis was developed using extrahepatic bile duct ligation for 3 days. In addition, sepsis due to E. coli 0111:B4 lipopolysaccharide (LPS) was induced in the model. The serum levels of total bilirubin, AST, ALT, and LDH and the mRNA levels of IL-1β, TNF-α, and MCP-1 in the liver were significantly higher in the OJ mice receiving LPS than in the sham-operated mice receiving LPS. Compared to the OJ mice receiving LPS, the hepcidin-pretreated OJ mice receiving LPS showed a significant decrease in the above mentioned parameters, as well as a reversal in the downregulation of LC3B-II and upregulation of cleaved caspase-3; this, in turn, led to significantly decreased lethality in 24 h. In conclusion, these results indicate that hepcidin pretreatment significantly reduced hepatic proinflammatory cytokine expression and liver injury, leading to reduced early lethality in OJ mice receiving LPS. Enhanced autophagy and reduced apoptosis may account for the protective effects of hepcidin.
Keywords: Obstructive jaundice; LPS; Hepcidin; Apoptosis; Autophagy;
Differential changes in Substance P, VIP as well as neprilysin levels in patients with gastritis or ulcer by Nuray Erin; Sema Türker; Özlem Elpek; Bülent Yıldırım (218-224).
Display Omitted► Neuronal SP decreased in gastritis and it further decreased in the presence of H. pylori. ► VIP and NEP in both disease-involved and uninvolved mucosa decreased in gastritis or ulcer. ► Decreased levels of VIP and SP fragments might be responsible from chronic gastritis. ► VIP analogs might be used in chronic gastritis and ulcer. ► Decreased SP levels in H. pylori (+) patients may predispose to malignancy.The protective effect of capsaicin-sensitive sensory nerve (CSSN) activation was recently demonstrated in human gastric mucosa. We here examined changes in neuropeptides, specifically Substance P (SP), calcitonin-gene related peptide (CGRP) and vasoactive intestinal peptide (VIP) in patients with chronic gastritis or ulcer. Furthermore changes in neprilysin levels, which hydrolyse these neuropeptides, were determined. Gastric biopsies were obtained from both lesion- and normal-appearing mucosa of 57 patients. The presence of H. pylori infection was verified with rapid urease assay. Neuronal and non-neuronal levels of SP, VIP, CGRP and neprilysin activity were determined in freshly frozen biopsies. Immunohistochemical localization of neprilysin was performed in 30 paraffin embedded specimens. We here found that neuronal SP levels decreased significantly in normally appearing mucosa of patients with gastritis while levels of non-neuronal SP increased in diseased areas of gastritis and ulcer. The presence of H. pylori led to further decreases of SP levels. The content of VIP in both disease-involved and uninvolved mucosa, and expression of neprilysin, markedly decreased in patients with gastritis or ulcer. Since VIP, as well as SP fragments, formed following hydrolysis with neprilysin is recognized to have gastroprotective effects, decreased levels of VIP, SP and neprilysin may predispose to cellular damage.
Keywords: Sensory neurons; Capsaicin; VIP; Substance P;
Effects of the antimicrobial peptide cecropin AD on performance and intestinal health in weaned piglets challenged with Escherichia coli by Shudan Wu; Fengrui Zhang; Zhimin Huang; Hong Liu; Chunyuan Xie; Jiang Zhang; Philip A. Thacker; Shiyan Qiao (225-230).
► Twenty-four weaned barrows were used to determine the effects of a dietary antimicrobial peptide in weaned pigs challenged with E. coli. ► We checked changes in growth performance, diarrhea, intestinal health and immune response pre and post-chanllenge. ► Antimicrobial peptides cecropin AD appeared to mediate immune status to improve the performance and reduce diarrhea incidence after E. coli challenge. ► Antimicrobial peptides have potential for use as alternatives to antibiotics in diets fed to weaned piglets.This study was conducted to determine the effects of the antimicrobial peptide cecropin on performance and intestinal health in piglets. Newly weaned barrows were randomly assigned to one of three treatments (n = 8), including a corn-soybean basal diet or similar diets supplemented with antibiotics (100 mg/kg kitasamycin plus 800 mg/kg colistin sulfate) or 400 mg/kg cecropin AD. On day 13, all piglets were orally challenged with 109 CFU/mL of Escherichia coli K88. On day 19, all piglets were euthanized and sampled. Before challenge, piglets fed antibiotics had greater weight gain, feed efficiency, nitrogen and energy retention than the control (P < 0.05). E. coli challenge decreased weight gain, feed intake and feed efficiency for the control piglets (P < 0.05) but not for the antibiotic or cecropin AD treated piglets. The incidence of diarrhea post-challenge in the antibiotic and cecropin AD treatments decreased compared with the control piglets. The total viable counts of cecal E. coli were lower while the Lactobacilli counts were higher in the antibiotic and cecropin AD treatments compared with the control (P < 0.05). Cecropin AD treatment decreased total aerobes while increasing total anaerobes in the ileum (P < 0.05). A higher villus height to crypt depth ratio in the jejunum and ileum as well as a deeper crypt depth in the jejunum and higher villus height in the ileum were observed in piglets fed antibiotics or cecropin AD compared with control piglets (P < 0.05). Piglets fed the control diet had lower levels of secretory IgA in their jejunum and lower serum IgA, IgG, interleukin-1β and interleukin-6 compared with the other treatments (P < 0.05). Overall, these data suggest that cecropin AD enhances pig performance through increasing immune status and nitrogen and energy retention as well as reducing intestinal pathogens in weaned piglets.
Keywords: Antimicrobial peptides; Antibiotics; Weaned piglets;
Lysine substitutions convert a bacterial-agglutinating peptide into a bactericidal peptide that retains anti-lipopolysaccharide activity and low hemolytic activity by Mahsa Abdolhosseini; Seshagiri R. Nandula; Jonathan Song; Helmut Hirt; Sven-Ulrik Gorr (231-238).
► Lysine substitution converts an agglutinating peptide to a bactericidal peptide. ► The peptide retains activity in saliva and in vivo. ► The peptide exhibits anti-lipopolysaccharide activity in vitro and in vivo. ► A serine hydroxyl is necessary for anti-LPS activity but not bactericidal activity. ► The peptide has low hemolytic activity.GL13NH2 is a bacteria-agglutinating peptide derived from the sequence of the salivary protein parotid secretory protein (PSP, BPIFA2, SPLUNC2, C20orf70). The peptide agglutinates both Gram negative and Gram positive bacteria, and shows anti-lipopolysaccharide activity in vitro and in vivo. However, GL13NH2 does not exhibit bactericidal activity. To generate a more cationic peptide with potential bactericidal activity, three amino acid residues were replaced with lysine residues to generate the peptide GL13K. In this report, the antibacterial and anti-inflammatory activities of GL13K were characterized. GL13K had lost the ability to agglutinate bacteria but gained bactericidal activity. Substitution of individual amino acids in GL13K with alanine did not restore bacterial agglutination. GL13K was bactericidal against Pseudomonas aeruginosa, Streptococcus gordonii and Escherichia coli but not Porphyromonas gingivalis. Unlike the agglutinating activity of GL13NH2, the bactericidal activity of GL13K against P. aeruginosa was retained in the presence of saliva. Both GL13NH2 and GL13K exhibited anti-lipopolysaccharide activity. In GL13K, this activity appeared to depend on a serine hydroxyl group. GL13K protected mice from lipopolysaccharide-induced sepsis and the peptide exhibited a low level of hemolysis, suggesting that it may be suitable for in vivo application.
Keywords: Agglutination; Bactericidal; BPIFA2; Hemolysis; Lipopolysaccharide; LPS;
Prokaryotic selectivity and LPS-neutralizing activity of short antimicrobial peptides designed from the human antimicrobial peptide LL-37 by Yong Hai Nan; Jeong-Kyu Bang; Binu Jacob; Il-Seon Park; Song Yub Shin (239-247).
► The proper hydrophobicity of antimicrobial peptides is crucial to exert the amalgamated property of LPS-neutralizing activity and prokaryotic selectivity. ► Analog a4-W2 showed more improved prokaryotic selectivity compared to LL-37. ► Analog a4-W2 displayed LPS-neutralizing activity comparable to that of LL-37. ► The effective site of Trp-substitution for high LPS-neutralizing activity of α-helical antimicrobial peptides is the amphipathic interface in the α-helical wheel projection.To develop novel antimicrobial peptides (AMPs) with shorter lengths, improved prokaryotic selectivity and retained lipolysaccharide (LPS)-neutralizing activity compared to human cathelicidin AMP, LL-37, a series of amino acid-substituted analogs based on IG-19 (residues 13–31 of LL-37) were synthesized. Among the IG-19 analogs, the analog a4 showed the highest prokaryotic selectivity, but much lower LPS-neutralizing activity compared to parental LL-37. The analogs, a5, a6, a7 and a8 with higher hydrophobicity displayed LPS-neutralizing activity comparable to that of LL-37, but much lesser prokaryotic selectivity. These results indicate that the proper hydrophobicity of the peptides is crucial to exert the amalgamated property of LPS-neutralizing activity and prokaryotic selectivity. Furthermore, to increase LPS-neutralizing activity of the analog a4 without a remarkable decrease in prokaryotic selectivity, we synthesized Trp-substituted analogs (a4-W1 and a4-W2), in which Phe5 or Phe15 of a4 is replaced by Trp. Despite their same prokaryotic selectivity, a4-W2 displayed much higher LPS-neutralizing activity compared to a4-W1. When compared with parental LL-37, a4-W2 showed retained LPS-neutralizing activity and 2.8-fold enhanced prokaryotic selectivity. These results suggest that the effective site for Trp-substitution when designing novel AMPs with higher LPS-neutralizing activity, without a remarkable reduction in prokaryotic selectivity, is the amphipathic interface between the end of the hydrophilic side and the start of the hydrophobic side rather than the central position of the hydrophobic side in their α-helical wheel projection. Taken together, the analog a4-W2 can serve as a promising template for the development of therapeutic agents for the treatment of endotoxic shock and bacterial infection.
Keywords: Human antimicrobial peptide LL-37; Hydrophobicity; LPS-neutralizing activity; Prokaryotic selectivity; Tryptophan-substitution;
Novel polymyxin derivatives are less cytotoxic than polymyxin B to renal proximal tubular cells by Marie-Paule Mingeot-Leclercq; Paul M. Tulkens; Sophie Denamur; Timo Vaara; Martti Vaara (248-252).
► The tricationic NAB compounds were compared to the pentacationicpolymyxin B (PMB). ► To electroporated cells, the NAB compounds were 8–32 fold less toxic than PMB. ► Without electroporation, PMB was toxic at 0.5 mM while NABs were inert even at 1 mM. ► The toxicity manifested as necrosis, no apoptosis was observed.The emergence of very multiresistant Gram-negative bacterial strains has reinstated polymyxins (polymyxin B, colistin), pentacationic lipopeptides, in the therapy, in spite of their nephrotoxicity. Extensive tubular reabsorption concentrates polymyxin in proximal tubular cells. The novel polymyxin derivatives NAB739, NAB7061 and NAB741 have their cyclic part identical to that of polymyxin B, but their side chain consists of uncharged octanoyl-threonyl-d-serinyl, octanoyl-threonyl-aminobutyryl, and acetyl-threonyl-d-serinyl respectively. In this study, we compared the toxicities of NAB739, NAB7061 and NAB741 with that of polymyxin B by using the porcine renal proximal tubular cell line LLC-PK1 electroporated or incubated with the selected compound. Both the ability to cause cell necrosis (quantified as the leakage of lactate dehydrogenase) and the ability to cause apoptosis (as quantified by counting apoptotic nuclei) were assessed. In electroporated cells, polymyxin B induced total (>85%) necrosis of the cells at 0.016 mM, whereas an approx. 8-fold concentration of NAB739 and NAB7961 and an approx. 32-fold concentration of NAB741 was required for the same effect. In cells treated without electroporation (incubated), polymyxin B elicited a marked degree (approx. 50%) of necrosis at 0.5 mM, whereas the NAB compounds were inert even at 1 mM. Neither polymyxin B nor the NAB compounds induced apoptosis.
Keywords: NAB739; NAB7061; NAB741; Gram-negative bacteria; Nephrotoxicity; Polymyxin B;
Two novel antimicrobial peptides purified from the symbiotic bacteria Xenorhabdus budapestensis NMC-10 by Yao Xiao; Fanlu Meng; Dewen Qiu; Xiufen Yang (253-260).
► Symbiotic bacteria Xenorhabdus associated with entomopathogenic nematodes produced broad-spectrum antibiotics. ► Two novel antimicrobial peptides were purified from Xenorhabdus budapestensis NMC-10. ► Two peptides are linear peptides and with low similarity to published peptide sequences. ► Synthetic peptides inhibit a wide range of plant pathogens and show potential application in plant disease control.Symbiotic bacteria, which are carried in the intestinal vesicle of the infective stage of juvenile entomopathogenic nematodes, produce broad-spectrum antibiotics. In this study, we aimed to isolate the antimicrobial peptides from the culture of the entomopathogenic bacterium Xenorhabdus budapestensis NMC-10. By screening chromatography columns and optimizing flow rate, pH, salinity and other purification conditions, we identified the final purification procedures which consisted of Q ion-exchange chromatography, gel filtration chromatography and two-step reverse-phase chromatography. Two novel antimicrobial peptides were identified via Q-TOF-TOF and de novo sequencing, and designated as GP-19 and EP-20. Both natural and synthetic peptides demonstrated broad-spectrum antimicrobial activities. The synthetic GP-19 peptide was active against Verticillium dahlia with EC50 values of 17.54 μg/ml and highly inhibited the growth of a variety of bacteria, while the synthetic EP-20 peptide was highly active against Phytophthora capsici with EC50 values of 3.14 μg/ml.
Keywords: Xenorhandus; Antimicrobial peptide; Chromatography; Solid phase synthesis; Antimicrobial activity;
In vitro and in vivo studies on the antioxidant activity of fish peptide isolated from the croaker (Otolithes ruber) muscle protein hydrolysate by R.A. Nazeer; N.S. Sampath Kumar; R. Jai Ganesh (261-268).
Characterization of purified antioxidant peptide from croaker muscle protein hydrolysate using ESI-MS has confirmed the mass of peptide as 861.6 Da. This peptide was further ionized by loading into ESI MS/MS and the fragments were manually calculated and sequence was identified as Lys-Thr-Phe-Cys-Gly-Arg-His.Display Omitted► Simple procedure was used to purify the peptide. ► Peptides had reducing, metal chelating ability besides scavenging radicals. ► It is successful in inhibiting the lipid peroxidation and DNA damage. ► Its ability to enhance in vivo antioxidant enzymes was proved. ► Peptide acted as a shield against ethanol induced free radicals in Wistar rats.Peptide from croaker (Otolithes ruber) muscle protein hydrolysate was purified, characterized and evaluated for its in vitro and in vivo antioxidant activity. Results showed that purified peptide contained the amino acid sequence as Lys-Thr-Phe-Cys-Gly-Arg-His (861.6 Da), which were expected to contribute to its antioxidant activities. This peptide efficiently quenched 1,1-diphenyl-2-picrylhydrazyl (DPPH) and hydroxyl radicals (84.5 ± 1.2 and 62.4 ± 2.9%), and successfully inhibits the lipid peroxidation and DNA damage and proven to be a potent antioxidant at different in vitro systems. It also improved the endogenous cellular antioxidant enzymes in Wistar rat by increasing the activities of catalase (CAT), glutathione-S-transferase (GST) and superoxide dismutase (SOD) after supplementation of the peptide (283.6 ± 7.25, 4.3 ± 0.78 and 28.42 ± 1.97) compared to the negative control (196.4 ± 5.65, 1.3 ± 0.45 and 15.1 ± 0.35). Therefore, croaker muscle peptide can increase an endurance capacity and facilitate recovery from oxidative stress.
Keywords: Otolithes ruber; Antioxidant; Lipid peroxidation; DNA damage; Peptide;
The hymenochirins: A family of host-defense peptides from the Congo dwarf clawed frog Hymenochirus boettgeri (Pipidae) by Milena Mechkarska; Manju Prajeep; Laurent Coquet; Jérôme Leprince; Thierry Jouenne; Hubert Vaudry; Jay D. King; J. Michael Conlon (269-275).
► Five host-defense peptides, termed hymenochirins, were isolated from skin secretions of Hymenochirus boettgeri (Pipidae). ► The peptides show little structural similarity with host-defense peptides from Xenopus and Silurana species. ► The hymenochirins display broad-spectrum antimicrobial and low hemolytic activities.Skin secretions of frogs from the subfamily Xenopodinae (Xenopus + Silurana) within the family Pipidae are a rich source of antimicrobial peptides with therapeutic potential but species from the sister taxon Hymenochirus in the subfamily Pipinae (Hymenochirus + Pseudhymenochirus + Pipa) have not been investigated. Peptidomic analysis of norepinephrine-stimulated skin secretions from two distinct populations of the Congo dwarf clawed frog Hymenochirus boettgeri (Tornier, 1896) has led to identification of five structurally related peptides with broad-spectrum antimicrobial activity. Hymenochirin-1B (IKLSPETKDNLKKVLKGAIKGAIAVAKMV.NH2) is C-terminally α-amidated whereas hymenochirins-2B–5B have the general structure XKIPX2VKDTLKKVAKGX2SX2AGAX3.COOH. Hymenochirin-3B (IKIPAVVKDTLKKVAKGVLSAVAGALTQ) was the most abundant peptide in the secretions. The hymenochirins show very low structural similarity with the antimicrobial peptides isolated from skin secretions of Silurana tropicalis and Xenopus laevis consistent with the proposed ancient divergence of the Pipinae and Xenopodinae. Synthetic replicates of hymenochirin-1B–4B inhibit the growth of Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Staphylococcus aureus (MIC in the range 10–40 μM) and Candida albicans (MIC = 80 μM). The peptides display relatively weak hemolytic activity against human erythrocytes (LC50 in the range 160 to >300 μM).
Keywords: Antimicrobial peptide; Frog skin; Pipinae; Hymenochirus; Xenopodinae; Hymenochirin;
Inhibitory and antimicrobial activities of OGTI and HV-BBI peptides, fragments and analogs derived from amphibian skin by Dawid Dębowski; Rafał Łukajtis; Anna Łęgowska; Natalia Karna; Michał Pikuła; Magdalena Wysocka; Irena Maliszewska; Marcin Sieńczyk; Adam Lesner; Krzysztof Rolka (276-284).
► OGTI do not display trypsin inhibitory activity as reported previously. ► OGTI exerts antimicrobial activity toward Staphylococcus epidermitis. ► HV-BBI can be the starting compound to design inhibitors of particular enzyme. ► HV-BBI and its analogs display dual properties: inhibitory and antibacterial.A series of linear and cyclic fragments and analogs of two peptides (OGTI and HV-BBI) isolated from skin secretions of frogs were synthesized by the solid-phase method. Their inhibitory activity against several serine proteinases: bovine β-trypsin, bovine α-chymotypsin, human leukocyte elastase and cathepsin G from human neutrophils, was investigated together with evaluation of their antimicrobial activities against Gram-negative bacteria (Escherichia coli) and Gram-positive species isolated from patients (Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus sp., Streptococcus sp.). The cytotoxicity of the selected peptides toward an immortal human skin fibroblast cell line was also determined. Three peptides: HV-BBI, its truncated fragment HV-BBI(3–18) and its analog [Phe8]HV-BBI can be considered as bifunctional compounds with inhibitory as well as antibacterial properties. OGTI, although it did not display trypsin inhibitory activity as previously reported in the literature, exerted antimicrobial activity toward S. epidermidis. In addition, under our experimental conditions, this peptide did not show cytotoxicity.
Keywords: Inhibitor; Serine proteinases; Peptomer; Peptide; Amphibian skin;
Novel antimicrobial peptides isolated from the skin secretions of Hainan odorous frog, Odorrana hainanensis by Hui Wang; Zhijun Yu; Yuhong Hu; Fengjiao Li; Limeng Liu; Hongyuan Zheng; Hao Meng; Shujie Yang; Xiaolong Yang; Jingze Liu (285-290).
► 7 antimicrobial peptides belonging to 5 families were identified from Odorrana hainanensis. ► Three antimicrobial peptide, temporin-HN1, temporin-HN2 and brevinin-1HN1 were purified from O. hainanensis. ► Each of these peptides was selected for the functional analysis, and displayed different antibacterial and hemolytic activity. ► Temporin-HN1, temporin-HN2 and brevinin-2HS2 display weak hemolytic activity in vitro.Long time geographical isolation of Hainan Island from the China continent has resulted in appearance of many novel frog species. As one of them, Hainan odorous frog, Odorrana hainanensis possesses some special antimicrobial peptides distinct from those found in other Odorrana. In this study, three antimicrobial peptides have been purified and characterized from the skin secretion of O. hainanensis. With the similarity to the temporin family, two peptides are characterized by amidated C-terminals, so they are named as temporin-HN1 (AILTTLANWARKFL-NH2) and temporin-HN2 (NILNTIINLAKKIL-NH2). The third antimicrobial peptide belongs to the brevinin-1 family which is widely distributed in Eurasian ranids, and thus, it is named as brevinin-1HN1 (FLPLIASLAANFVPKIFCKITKKC). Furthermore, after sequencing 68 clones, eight cDNAs encoding antimicrobial peptide precursors were cloned from the skin-derived cDNA library of O. hainanensis. These eight cDNAs can encode seven mature antimicrobial peptides including the above three, as well as brevinin-1V, brevinin-2HS2, odorranain-A6, and odorranain-B1. Twelve different species of microorganisms were chosen, including Gram-positive, Gram-negative and fungi, to test the antimicrobial activities of temporin-HN1, temporin-HN2, brevinin-1HN1, brevinin-1V, and brevinin-2HS2. The result shows that, in addition to their activities against Gram-positive bacteria, temporin-HN1 and temporin-HN2 also possess activities against some Gram-negative bacteria and fungi. However, the two antimicrobial peptides, brevinin-1HN1 and brevinin-1V of the brevinin-1 family have stronger antimicrobial activities than temporin-HN1 and temporin-HN2 of the temporin family. Brevinin-1HN1 possesses activity against Staphylococcus aureus (ATCC25923), Rhodococcus rhodochrous X15, and Slime mould 090223 at the concentration of 1.2 μM.
Keywords: Amphibian; Odorrana hainanensis; Frog skin; Antimicrobial peptide;
Antimicrobial/cytolytic peptides from the venom of the North African scorpion, Androctonus amoreuxi: Biochemical and functional characterization of natural peptides and a single site-substituted analog by Ammar Almaaytah; Mei Zhou; Lei Wang; Tianbao Chen; Brian Walker; Chris Shaw (291-299).
► The manuscript describes two novel antimicrobial/cytolytic peptides from the venom of a North African scorpion, Androctonus amoreuxi. ► The cDNAs encoding each peptide were successfully cloned from a cDNA library made from the same venom sample. ► A single site-substituted analog of the first peptide was found to be a more potent antimicrobial than the natural peptides and to have negligible hemolytic activity at effective concentrations. ► In addition, the analog was active against several tumor cell lines and endothelial cells – an activity not possessed by tye natural peptides at the highest concentrations tested.The venoms of scorpions are complex cocktails of polypeptide toxins that fall into two structural categories: those that contain cysteinyl residues with associated disulfide bridges and those that do not. As the majority of lethal toxins acting upon ion channels fall into the first category, most research has been focused there. Here we report the identification and structural characterization of two novel 18-mer antimicrobial peptides from the venom of the North African scorpion, Androctonus amoreuxi. Named AamAP1 and AamAP2, both peptides are C-terminally amidated and differ in primary structure at just two sites: Leu ⇒ Pro at position 2 and Phe ⇒ Ile at position 17. Synthetic replicates of both peptides exhibited a broad-spectrum of antimicrobial activity against a Gram-positive bacterium (Staphylococcus aureus), a Gram-negative bacterium (Escherichia coli) and a yeast (Candida albicans), at concentrations ranging between 20 μM and 150 μM. In this concentration range, both peptides produced significant degrees of hemolysis. A synthetic replicate of AamAP1 containing a single substitution (His ⇒ Lys) at position 8, generated a peptide (AamAP-S1) with enhanced antimicrobial potency (3–5 μM) against the three test organisms and within this concentration range, hemolytic effects were negligible. In addition, this His ⇒ Lys variant exhibited potent growth inhibitory activity (ID50 25–40 μm) against several human cancer cell lines and endothelial cells that was absent in both natural peptides. Natural bioactive peptide libraries, such as those that occur in scorpion venoms, thus constitute a unique source of novel lead compounds with drug development potential whose biological properties can be readily manipulated by simple synthetic chemical means.
Keywords: Peptide; Molecular cloning; Venom; Scorpion; Antimicrobial;
Helokinestatin-7 peptides from the venoms of Heloderma lizards by Chengbang Ma; Hui Wang; Yuxin Wu; Mei Zhou; Gemma Lowe; Lei Wang; Yingqi Zhang; Tianbao Chen; Chris Shaw (300-305).
► The paper describes two novel peptides, helokinestatin-7S and helokinestatin-7H. ► Helokinestatin-7S is from the venom of the Gila Monster (Heloderma suspectum). ► Helokinestatin-7H is from the venom of the Mexican beaded lizard (Heloderma horridum). ► Helokinestatin-7 peptides had different effects on rat tail artery smooth muscle.Helokinestatins 1–6 constitute a family of bradykinin antagonist peptides originally isolated from the venoms of the Gila Monster, Heloderma suspectum and the Mexican beaded lizard, Heloderma horridum. Here we report the identification, isolation and preliminary pharmacological characterization of two novel tridecapeptides, named helokinestatin-7S (FDDDSTELILEPR – 1550 Da) and helokinestatin-7H (FDDDSRKLILEPR – 1604 Da), whose primary structures were predicted from cDNAs cloned from venom libraries of respective Heloderma lizards. Computed molecular masses of putative helokinestatin-7 peptides were used as tools to locate these peptides in archived LC/MS fractions from respective venoms and sequences were confirmed by MS/MS fragmentation. A synthetic replicate of helokinestatin-7H was found to antagonize the relaxation effect of bradykinin on rat arterial smooth muscle but to have no measurable effects alone. In contrast, synthetic helokinestatin-7S was found to directly contract this preparation. Studies on related natural peptides with subtle differences in primary structure can provide the tools for structure/activity studies in pharmacological investigations directed toward unraveling the molecular basis of venom toxicity and for the evaluation of potential therapeutic leads.
Keywords: Lizard; Venom; Bradykinin; Antagonist; Mass spectrometry; Molecular cloning; Peptide;
Non-opioid nociceptive activity of human dynorphin mutants that cause neurodegenerative disorder spinocerebellar ataxia type 23 by Hiroyuki Watanabe; Hirokazu Mizoguchi; Dineke S. Verbeek; Alexander Kuzmin; Fred Nyberg; Oleg Krishtal; Shinobu Sakurada; Georgy Bakalkin (306-310).
► Dynorphin A-R6W induces nociceptive responses in the mouse spinal cord. ► Dynorphin A-R6W-induced nociception was diminished by morphine and MK-801. ► Dynorphin A-R6W produces nociception via activation of NMDA receptor in mice.We previously identified four missense mutations in the prodynorphin gene that cause human neurodegenerative disorder spinocerebellar ataxia type 23 (SCA23). Three mutations substitute Leu5, Arg6, and Arg9 to Ser (L5S), Trp (R6W) and Cys (R9C) in dynorphin A(1–17) (Dyn A), a peptide with both opioid activities and non-opioid neurodegenerative actions. It has been reported that Dyn A administered intrathecally (i.t.) in femtomolar doses into mice produces nociceptive behaviors consisting of hindlimb scratching along with biting and licking of the hindpaw and tail (SBL responses) through a non-opioid mechanism. We here evaluated the potential of the three mutant peptides to produce similar behaviors. Compared to the wild type (WT)-peptide, the relative potency of Dyn A R6W, L5S and R9C peptides for SBL responses was 50-, 33- and 2-fold higher, and Dyn A R6W and L5S induced the SBL responses at a 10–30-fold lower doses. Dyn A R6W was the most potent peptide. The SBL responses induced by Dyn A R6W were dose dependently inhibited by morphine (i.p.; 0.1–1 mg/kg) or MK-801, an NMDA ion channel blocker (i.t. co-administration; 5–7.5 nmol). CP-99,994, a tachykinin NK1 receptor antagonist (i.t. co-administration; 2 nmol) and naloxone (i.p.; 5 mg/kg) failed to block effects of Dyn A R6W. Thus, similarly to Dyn A WT, the SBL responses induced by Dyn A R6W may involve the NMDA receptor but are not mediated through the opioid and tachykinin NK1 receptors. Enhanced non-opioid excitatory activities of Dyn A mutants may underlie in part development of SCA23.
Keywords: Dynorphin A mutant; Nociception; Mice; Non-opioid activity; Neurodegeneration;