Peptides (v.31, #8)
Editorial Board (CO2).
Japan Announcement (III).
Rice seed ER-derived protein body as an efficient delivery vehicle for oral tolerogenic peptides by Hidenori Takagi; Takachika Hiroi; Sakiko Hirose; Lijun Yang; Fumio Takaiwa (1421-1425).
Mucosal delivery of peptide/protein therapeutics via the oral route is a desirable strategy in human immunotherapy. A key step for enhancing the bioavailability of orally administered therapeutics is to protect them from enzymatic digestion in the gastrointestinal tract. Here, we generated transgenic rice seeds accumulating allergen-derived T cell epitopes, a model tolerogen for the control of pollen allergy, in either ER-derived protein body-I (PB-I) or protein storage vacuole protein body-II (PB-II). Compared with PB-II-localized or chemically synthesized forms, PB-I-localized T cell epitopes showed higher resistance to enzymatic digestion in simulated gastric fluid. Moreover, the dose of T cell epitope required for suppression of allergen-specific IgE in mice was about 20-fold lower when fed in PB-I localized form than when unprotected. These findings demonstrate the potential of bioencapsulation in PB-I for broad applications as a viable strategy to achieve efficient mucosal delivery of oral peptide/protein therapeutics.
Keywords: Allergy immunotherapy; Mucosal delivery vehicle; Oral therapeutics; Rice protein body;
Identification of E. dysenterica laxative peptide: A novel strategy in the treatment of chronic constipation and irritable bowel syndrome by T.B. Lima; O.N. Silva; J.T.A. Oliveira; I.M. Vasconcelos; F.B. Scalabrin; T.L. Rocha; M.F. Grossi-de-Sá; L.P. Silva; R.V. Guadagnin; B.F. Quirino; C.F.S. Castro; E. Leonardecz; O.L. Franco (1426-1433).
Plants have contributed over the years to the discovery of various pharmacological products. Amongst the enormous diversity of herbs with remarkable medicinal use and further pharmacological potential, here in this report we evaluated pulp extracts from Eugenia dysenterica fruits and further identified the active principle involved in such laxative activity in rats. For protein isolation, fruits were macerated with an extraction solution following precipitation with (NH4)2SO4 (100%). After dialysis, the peptide was applied onto a reversed-phase semi-preparative HPLC column, and the major fraction was eluted with 26% and 66% acetonitrile. The evaluation of molecular masses by MALDI-TOF and Tris/Tricine SDS-PAGE of HPLC fractions showed the presence of a major peptide with approximately 7 kDa. The N-terminal amino acid peptide sequence was determined and showed no similarity to other proteins deposited in the Data Bank. Peptide from E. dysenterica was able to enhance rats’ intestinal motility by approximately 20.8%, probably being responsible for laxative activity. Moreover, these proteins were non-toxic to mammals, as observed in histopathology and hemolytic analyses. In conclusion, results here reported indicate that, in the near future, proteins synthesized by E. dysenterica fruits could be utilized in the development of novel biotechnological pharmaceutics with laxative properties for use in chronic constipation and irritable bowel syndrome treatment.
Keywords: E. dysenterica; Laxative; Hemolytic active; Bioactive peptides;
Isolation and characterization of cytotoxic cyclotides from Viola tricolor by Jun Tang; Conan K. Wang; Xulin Pan; He Yan; Guangzhi Zeng; Wenyan Xu; Wenjun He; Norelle L. Daly; David J. Craik; Ninghua Tan (1434-1440).
Many plants of the Violaceae plant family have been used in traditional remedies, and these plants often contain cyclotides, a particular type of plant cyclopeptide that is distinguished by a cyclic cystine knot motif. In general, bioactive plant cyclopeptides are interesting candidates for drug development. In the current study, a suite of 14 cyclotides, which includes seven novel cyclotides [vitri B, C, D, E, F, varv Hm, and He], together with seven known cyclotides [varv A, D, E, F, H, vitri A, and cycloviolacin O2], was isolated from Viola tricolor, a common flower. A chromatography-based method was used to isolate the cyclotides, which were characterized using tandem mass spectrometry and NMR spectroscopy. Several of the cyclotides showed cytotoxic activities against five cancer cell lines, U251, MDA-MB-231, A549, DU145, and BEL-7402. Three cyclotides, vitri A, vitri F, and cycloviolacin O2, were the most cytotoxic. The cytotoxic activity of the cyclotides did not correlate well with their hemolytic activity, indicating that different interactions, most likely with membranes, are involved for cytotoxic and hemolytic activities. Homology modeling of the structures was used in deriving structure–activity relationships.
Keywords: Cyclotides; Viola tricolor; Cytotoxic activity; vitri B–F; varv Hm; varv He;
NMR solution structure of poliovirus uridylyated peptide linked to the genome (VPgpU) by Catherine H. Schein; Numan Oezguen; Gerbrand J. van der Heden van Noort; Dmitri V. Filippov; Aniko Paul; Eric Kumar; Werner Braun (1441-1448).
Picornaviruses have a 22–24 amino acid peptide, VPg, bound covalently at the 5′ end of their RNA, that is essential for replication. VPgs are uridylylated at a conserved tyrosine to form VPgpU, the primer of RNA synthesis by the viral polymerase. This first complete structure for any uridylylated VPg, of poliovirus type 1 (PV1)-VPgpU, shows that conserved amino acids in VPg stabilize the bound UMP, with the uridine atoms involved in base pairing and chain elongation projected outward. Comparing this structure to PV1-VPg and partial structures of VPg/VPgpU from other picornaviruses suggests that enteroviral polymerases require a more stable VPg structure than does the distantly related aphthovirus, foot and mouth disease virus (FMDV). The glutamine residue at the C-terminus of PV1-VPgpU lies in back of the uridine base and may stabilize its position during chain elongation and/or contribute to base specificity. Under in vivo-like conditions with the authentic cre(2C) hairpin RNA and Mg2+, 5-methylUTP cannot compete with UTP for VPg uridylyation in an in vitro uridylyation assay, but both nucleotides are equally incorporated by PV1-polymerase with Mn2+ and a poly-A RNA template. This indicates the 5 position is recognized under in vivo conditions. The compact VPgpU structure docks within the active site cavity of the PV-polymerase, close to the position seen for the fragment of FMDV-VPgpU with its polymerase. This structure could aid in design of novel enterovirus inhibitors, and stabilization upon uridylylation may also be pertinent for post-translational uridylylation reactions that underlie other biological processes.
Keywords: Uridylylation; Post-translational modification; Picornaviruses; Disordered structures; Enteroviruses; Polymerase priming mechanism; RNA editing; Antiviral compounds; Flexibility;
Evaluation of the epinecidin-1 peptide as an active ingredient in cleaning solutions against pathogens by Chieh-Yu Pan; Venugopal Rajanbabu; Jyh-Yih Chen; Guor Mour Her; Fan-Hua Nan (1449-1458).
We tested the activity of epinecidin-1, a novel antimicrobial peptide structurally related to pleurocidin, in commercial cleaning solutions stored at 4 and 25 °C for 7 and 14 days. The peptide's activities against Enterococcus faecalis, Escherichia coli, Klebsiella oxytoca, Pseudomonas aeruginosa, Staphylococcus aureus, Propionibacterium acnes, and Candida albicans were measured in a minimum inhibitory concentration (MIC) determination, minimal bactericidal concentration (MBC) determination, disk diffusion test, and a count of the bacterial numbers. Exposure to epinecidn-1 in a cleaning solution following MIC value comparisons in the disk diffusion test and counts of bacterial numbers after 16, 24, 48, and 72 h suggested that bacterial numbers were much lower than those treated with only commercial cleaning solutions for all bacteria. The efficacy of the antimicrobial activities of inhibiting bacterial numbers by epinecidin-1 in cleaning solutions at a low pH and a low temperature was not affected. Given its simple structure and antimicrobial activity, epinecidin-1 may be a useful component of microbicides designed to prevent pathogen infections and/or remediate abnormal vaginal or skin flora.
Keywords: Epinecidin-1; Cleaning solution; Lytic peptide; Gram-negative bacteria; Gram-positive bacteria;
Synthesis, characterization, antimicrobial activity and LPS-interaction properties of SB041, a novel dendrimeric peptide with antimicrobial properties by Michela Bruschi; Giovanna Pirri; Andrea Giuliani; Silvia Fabiole Nicoletto; Izabela Baster; Mariano Andrea Scorciapino; Mariano Casu; Andrea C. Rinaldi (1459-1467).
Multimeric peptides offer several advantages with respect to their monomeric counterparts, as increased activity and greater stability to peptidases and proteases. SB041 is a novel antimicrobial peptide with dendrimeric structure; it is a tetramer of pyrEKKIRVRLSA linked by a lysine core, with an amino valeric acid chain. Here, we report on its synthesis, NMR characterization, antimicrobial activity, and LPS-interaction properties. The peptide was especially active against Gram-negative strains, with a potency comparable (on molar basis) to that of lipopeptides colistin and polymixin B, but it also displayed some activity against selected Gram-positive strains. Following these indications, we investigated the efficacy of SB041 in binding Escherichia coli and Pseudomonas aeruginosa LPS in vitro and counteracting its biological effects in RAW-Blue™ cells, derived from RAW 264.7 macrophages. SB041 strongly bound purified LPS, especially that of E. coli, as proved by fluorescent displacement assay, and readily penetrated into LPS monolayers. However, the killing activity of SB041 against E. coli was not inhibited by increasing concentrations of LPS added to the medium. Checking the SB041 effect on LPS-induced activation of pattern recognition receptors (PRRs) in Raw-Blue cells revealed that while the peptide gave a statistically significant decrease in PRRs stimulation when RAW-Blue cells were challenged with P. aeruginosa LPS, the same was not seen when E. coli LPS was used to activate innate immune defense-like responses. Thus, as previously seen for other antimicrobial peptides, also for SB041 binding to LPS did not translate necessarily into LPS-neutralizing activity, suggesting that SB041–LPS interactions must be of complex nature.
Keywords: Peptidomimetics; Synthetic peptides; LPS; Endotoxin; Binding; Neutralization; Sepsis; NMR;
Peptides derived from the human laminin α4 and α5 chains exhibit antimicrobial activity by Ilknur Senyürek; Gerd Klein; Hubert Kalbacher; Martin Deeg; Birgit Schittek (1468-1472).
Laminins are a family of heterotrimeric extracellular matrix glycoproteins in the basement membrane of different tissues and are composed of α, β, and γ chains. In mammals, five different α chains, three β chains, and three γ chains have been identified that assemble into 15 different laminins. Each α-chain possesses a C-terminal globular domain which can be subdivided into the five subdomains LG1–LG5. LG1–LG3 modules are connected to LG4–LG5 by a linker domain which is known to be sensitive to proteolytic processing. Here, we show that peptides derived from the human laminin α4 and α5 chain, exhibit a dose-dependent antimicrobial activity against gram-positive and gram-negative bacteria. Furthermore, we show that these peptides permeabilize the bacterial membrane and are able to bind to bacterial DNA. Interestingly, the ability to kill the microorganisms correlated with their ability to bind to heparin. These data suggest that extracellular matrix components are able to protect the respective tissues from invading pathogens and are part of the host defense response.
Keywords: Antimicrobial peptides; Heparin binding; Laminin; LG module;
Identification of a novel melittin isoform from Africanized Apis mellifera venom by Juliana Mozer Sciani; Rafael Marques-Porto; Airton Lourenço; Ricardo de Oliveira Orsi; Rui Seabra Ferreira Junior; Benedito Barraviera; Daniel Carvalho Pimenta (1473-1479).
Apis mellifera, the European honey bee, is perhaps the most studied insect in the Apidae family. Its venom is comprised basically of melittin, phospholipase A2, histamine, hyaluronidase, cathecolamines and serotonin. Some of these components have been associated to allergic reactions, among several other symptoms. On the other hand, bee mass-stinging is increasingly becoming a serious public health issue; therefore, the development of efficient serum-therapies has become necessary, with a consequent better characterization of the venom. In this work, we report the isolation and biochemical characterization of melittin-S, an isoform of melittin comprising a Ser residue at the 10th position, from the venom of Africanized A. mellifera. This peptide demonstrated to be less hemolytic than melittin and to adopt a less organized secondary structure, as assessed by circular dichroism spectroscopy. Melittin-S venom contents varied seasonally, and the maximum secretion occurred during the (southern) winter months. Data on the variation of the honey bee venom composition are necessary to guide future immunological studies, aiming for the development of an efficient anti-serum against Africanized A. mellifera venom and, consequently, an effective treatment for the victims of mass-stinging.
Keywords: Apis mellifera; Melittin; Venom; Seasonal variation; Peptides; Natural peptides;
Cloning and expression of genes enocoding antimicrobial peptides and bradykinin from the skin and brain of Oki Tago's brown frog, Rana tagoi okiensis by Shoro Tazato; J. Michael Conlon; Shawichi Iwamuro (1480-1487).
Previous studies led to the isolation from skin extracts of Oki Tago's brown frog, Rana tagoi okiensis of five antimicrobial peptides belonging to the brevinin-1 (brevinin-1TOa), temporin (temporin-TOa and -TOb), and ranatuerin-2 (ranatuerin-2TOa and -2TOb) families, and bradykinin (BK) identical to mammalian BK. Using the reverse-transcription polymerase chain reaction (RT-PCR), we have now cloned from skin total RNA preparations cDNAs encoding biosynthetic precursors of brevinin-1TOa and brevinin-1TOb (containing the substitution Gly1 → Val), temporin-TOa and -TOb, and ranatuerin-2TOa and -2TOb. In addition, three cDNA clones encoding preprobradykinins were obtained that contained either one, two, or three tandem repeats of the sequence of BK followed by the sequence of [Thr6]-BK. In tissue expression analyses, preprobrevinin-1, preprotemporin, and preproranatuerin-2 gene transcripts were detected at higher levels in brain compared with peripheral tissues (heart, small intestine, kidney, liver lung, skeletal muscle, stomach, and testis). RT-PCR of brain RNA resulted in the amplification of cDNAs encoding ranatuerin-2TOc and ranatuerin-2TOd that contained the amino acid substitutions Lys6 → Arg and Ala14 → Thr, respectively compared with ranatuerin-2TOb. cDNAs encoding preprobrevinin-1TOa and preprotemporin-TOa were amplified from brain RNA as well as a second preprotemporin cDNA that contained a 10-nucleotide insertion that introduced a frame shift resulting in a premature stop codon. A cDNA encoding a novel peptide, DK25 (DVNDLKNLCAKTHNLLPMCAMFGKK) was amplified from brain RNA but neither DK25 nor its putative post-translationally modified form, DF22-amide (DVNDLKNLCAKTHNLLPMCAMF·NH2) displayed antimicrobial or hemolytic activities.
Keywords: Antimicrobial peptide; Bradykinin; Frog brain; Frog skin; Rana tagoi okiensis;
Structure–function relationship of king cobra cathelicidin by Yong Zhang; Hui Zhao; Guo-Yu Yu; Xiao-Dong Liu; Ji-Hong Shen; Wen-Hui Lee; Yun Zhang (1488-1493).
King cobra cathelicidin (OH-CATH) is composed of 34 amino acid residues having strong antibacterial and very weak hemolytic activities as reported by us recently. OH-CATH can be served as a valuable template to develop novel therapeutic drugs. In this study, OH-CATH and six of its analogs were synthesized to explore their structure–function relationships based on their bactericidal and hemolytic activities. Experimental results of OH-CATH(3–34) and OH-CATH(5–34) indicated that the N-terminal 4 amino acid residues of OH-CATH played an important role on its hemolytic activity but had weak effects on its bactericidal activity. Among OH-CATH and its analogs, OH-CATH(5–34) had the lowest hemolytic activity while maintained strong antimicrobial activity. To evaluate its potential usage, the biological activities of OH-CATH(5–34) were compared with those of pexiganan. The bactericidal activity of OH-CATH(5–34) against 5 different species (11 laboratory strains) was 2–4 times stronger than that of pexiganan (4–16 μg/ml vs 8–32 μg/ml). Hemolytic activity of OH-CATH(5–34) against human erythrocytes was 0.69% while that of pexiganan was 16.5% at the dosage of 200 μg/ml. OH-CATH(5–34) showed very weak cytotoxic activities against primary rabbit ventricular endothelial cells and four human cancer cell lines whereas pexiganan showed strong cytotoxic activity against these five cell lines (IC50 = 20–90 μg/ml). The intravenous LD50 value of OH-CATH(5–34) on mice was 7-fold higher than that of pexiganan (175 mg/kg vs 25 mg/kg). Taken together, our results suggested that OH-CATH(5–34) should be considered as an excellent candidate for developing therapeutic drugs.
Keywords: Antimicrobial peptide; Cathelicidin; King cobra; Pexiganan;
The antimicrobial peptide cecropin A induces caspase-independent cell death in human promyelocytic leukemia cells by José Mª. Cerón; Judit Contreras-Moreno; Elena Puertollano; Gerardo Álvarez de Cienfuegos; María A. Puertollano; Manuel A. de Pablo (1494-1503).
Most antimicrobial peptides have been shown to have antitumoral activity. Cecropin A, a linear 37-residue antimicrobial polypeptide produced by the cecropia moth, has exhibited cytotoxicity in various human cancer cell lines and inhibitory effects on tumor growth. In this study, we investigated the apoptosis induced by cecropin A in the promyelocytic cell line HL-60. Treatment of cells with cecropin A was characterized by loss of viability in a dose-dependent manner, lactate dehydrogenase (LDH) leakage, and modest attenuation of lysosomal integrity measured by neutral red assay. An increase of reactive oxygen species (ROS) generation, DNA fragmentation, and phosphatidylserine externalization were quantified following cecropin A exposure at a concentration of 30 μM, whereas cecropin A-induced apoptosis was independent of caspase family members, because the activity of caspase-8 and -9 were irrelevant. Nevertheless, caspase-3 activity showed a significant increase at concentrations of 20–40 μM, but a considerable reduction at 50 μM. Flow cytometry analysis revealed a dissipation of the mitochondrial transmembrane potential (Δψ m), and the accumulation of cells at sub-G1 phase measured by FACS analysis of propidium iodide (PI) stained nuclei suggested induction of apoptosis. Morphological changes measured by Hoechst 33342 or acridine orange/ethidium bromide staining showed nuclear condensation, corroborating the apoptotic action of cecropin A. Overall, these data indicate that cecropin A is able to induce apoptosis in HL-60 cells through a signaling mechanism mediated by ROS, but independently of caspase activation.
Keywords: Cecropin A; Antimicrobial peptides; Apoptosis; Necrosis; Cytotoxicity; Caspases; Reactive oxygen species;
A cationic amphiphilic peptide ABP-CM4 exhibits selective cytotoxicity against leukemia cells by Yu Qing Chen; Cui Min; Ming Sang; Yang Yang Han; Xiao Ma; Xiao Qing Xue; Shuang Quan Zhang (1504-1510).
Some cationic antibacterial peptides exhibit a broad spectrum of cytotoxic activity against cancer cells, which could provide a new class of anticancer drugs. In the present study, the anticancer activity of ABP-CM4, an antibacterial peptide from Bombyx mori, against leukemic cell lines THP-1, K562 and U937 was evaluated, and the cytotoxicity compared with the effects on non-cancerous mammalian cells, including peripheral blood mononuclear cells (PBMCs), HEK-293 and erythrocytes. ABP-CM4 reduced the number of viable cells of the leukemic cell lines after exposure for 24 h. The reduction was concentration dependent, and the IC50 values ranged from 14 to 18 μM. Conversely, ABP-CM4, even at 120 μM, exhibited no cytotoxicity toward HEK-293 or PBMCs, indicating that there was no significant effect on these two types of non-cancer cells. ABP-CM4 at a concentration of 200 μM had no hemolytic activity on mammalian erythrocytes. Together, these results suggested a selective cytotoxicity in leukemia cells. Flow cytometry demonstrated that the binding activity of ABP-CM4 to leukemia cells was much higher than that to HEK-293 or PBMCs, and there was almost no binding to erythrocytes. FITC-labeled ABP-CM4 molecules were examined under a confocal microscope and found to be concentrated at the surface of leukemia cells and changes of the cell membrane were determined by a cell permeability assay, which led us to the conclusion that ABP-CM4 could act at the cell membrane for its anticancer activity on leukemia cells. Collectively, our results indicated that ABP-CM4 has the potential for development as a novel antileukemic agent.
Keywords: ABP-CM4; Leukemia; Cytotoxicity; Selectivity; FITC-labeling;
Protein expression of urotensin II, urotensin-related peptide and their receptor in the lungs of patients with lymphangioleiomyomatosis by Arnold S. Kristof; Zhipeng You; Yin-Shan Han; Adel Giaid (1511-1516).
Urotensin II (UII) and urotensin-related peptide (URP) are vasoactive neuropeptides with wide ranges of action in the normal mammalian lung, including the control of smooth muscle cell proliferation. UII and URP exert their actions by binding to the G-protein coupled receptor-14 known as UT. Lymphangioleiomyomatosis (LAM) is a disease of progressive lung destruction resulting from the excessive growth of abnormal smooth muscle-like cells that exhibit markers of neural crest origin. LAM cells also exhibit inactivation of the tumor suppressor tuberin (TSC2), excessive activity of ‘mammalian target of rapamycin (mTOR), and dysregulated cell growth and proliferation. In the present study we examined the expression and distribution of UII and UT in the lungs of patients with LAM. There was abundant expression of UII, URP and UT proteins in the interstitial nodular lesions of patients with LAM. By immunohistochemistry, UII, URP and UT were co-localized with HMB45, a diagnostic marker of LAM. Immunoreactivity for UII, URP and UT was also evident over the pulmonary epithelium, pulmonary vasculature and inflammatory cells. Western blotting revealed the presence of greater UT expression in the lungs of patients with LAM compared to normal human lungs. UT expression correlated with mTOR activity, as indicated by increased phosphorylation of S6 in LAM samples. These findings demonstrate for the first time the presence of UII, URP and their receptor in the lesions of patients with LAM, and suggest a possible role in the pathogenesis of the disease.
Keywords: Immunohistochemistry; Western blotting; UT; Human;
Role of VPAC1 and VPAC2 in VIP mediated inhibition of rat pulmonary artery and aortic smooth muscle cell proliferation by Rose-Claire St. Hilaire; Subramanyam N. Murthy; Philip J. Kadowitz; James R. Jeter (1517-1522).
Recent studies have suggested the potential use of vasoactive intestinal peptide (VIP) in the treatment of pulmonary arterial hypertension (PAH). An understanding of the mechanism of action of VIP is important for the development of new therapies for PAH. The biological effects of VIP are mediated by two type II guanine nucleotide binding protein (G-protein)-coupled receptors VIP/PACAP (pituitary adenylate cyclase activating peptide) receptor type1 (VPAC1) and VIP/PACAP receptor type 2 (VPAC2). In the present study, the distribution and role of these receptors were investigated and compared in cultured smooth muscle cells from rat aorta and pulmonary artery, as well as in fixed tissue sections of the aorta and pulmonary artery. Western blot analysis, RT-PCR and immunohistochemistry showed the expression of both VIP receptors in tissue sections of the aorta and pulmonary artery as well as in cultured smooth muscle cells from these vessels. The application of a specific antagonist of VPAC1 resulted in a small release from VIP induced inhibition of cell proliferation. In contrast (VIP 6–28; 300 nM) which is an antagonist against both receptors resulted in a significant restoration of proliferation. The expression of cAMP was reduced in the presence of VIP 6–28 and slightly decreased by VPAC1 antagonist. These findings suggest a dual role for VPAC1 and VPAC2 receptors in mediating the antiproliferative effects of VIP with VPAC2 appearing to play a more dominant role.
Keywords: Vasoactive intestinal peptide; Proliferation; VPAC1; VPAC2;
Chronic urotensin II receptor antagonist treatment does not alter hypertrophy or fibrosis in a rat model of pressure-overload hypertrophy by Andrew R. Kompa; Bing H. Wang; Arintaya Phrommintikul; Pei Y. Ho; Darren J. Kelly; David J. Behm; Stephen A. Douglas; Henry Krum (1523-1530).
Urotensin II (UII) is a potential mediator in the pathogenesis of cardiovascular disease, and inhibition of its actions at the urotensin receptor (UT) has been shown to improve cardiac function and structural changes of the myocardium in a model of myocardial infarction. In this study we utilized a model of pressure-overload hypertrophy induced by abdominal aortic constriction (AAC) which resulted in hypertrophy, increased fibrosis and impaired diastolic and systolic function. These changes were associated with a 4-fold increase in UII protein expression in the myocardium. Treatment of animals with a selective UT (SB-657510) antagonist for 20 weeks at a dose of 1500 ppm did not improve cardiac function as assessed by echocardiography and pressure–volume loop analysis, nor did it inhibit left ventricular hypertrophy or fibrosis. We hypothesize that other neurohumoral pathways may have a greater involvement in the pathogenesis of this model. Targeting the UII system appears to be insufficient to observe a beneficial outcome.
Keywords: Urotensin II receptor antagonist; Pressure-overload hypertrophy; Cardiac fibrosis; Left ventricular hypertrophy; Cardiac function; Echocardiography;
Natriuretic peptides and right atrial fibrosis in patients with paroxysmal versus persistent atrial fibrillation by Hailong Cao; Lei Xue; Yanhu Wu; Hongtai Ma; Liang Chen; Xiaowei Wang; Quan Zhu; Ninghuang Dai; Yijiang Chen (1531-1539).
Natriuretic peptides (NPs) are excellent diagnostic and prognostic markers of heart failure, but their roles in atrial fibrillation (AF), particularly of isolated cardiac valvular origin, are unclear. We assessed the mRNA and protein content of pro-atrial natriuretic peptide (pro-ANP) and pro-brain natriuretic peptide (pro-BNP) in right atrial appendages (RAAs) and their N-terminal fragments (nt-proANP and nt-proBNP) in the plasma of 30 patients with paroxysmal AF (PaAF) and 40 patients with persistent AF (PeAF) matched with 34 patients in sinus rhythm (SR) undergoing isolated valvular replacement. To explore the underlying mechanism, fibrosis related examinations were simultaneously carried out in RAAs. Unexpectedly, atrial expression of pro-NPs mRNA was notably augmented in the PaAF subgroup, but not so pronounced in the PeAF subgroup. Atrial content of pro-NPs proteins and plasma nt-proNPs, between which surprisingly strong positive correlations were found (pro-ANP and nt-proANP: r = 0.918, p < 0.001; pro-BNP and nt-proBNP: r = 0.913, p < 0.001), were increased analogously in PaAF and PeAF subgroups. We identified significantly increasing gradients of atrial collagen volume fraction (CVF), levels of collagen I and III in the SR, PaAF and PeAF groups, and convincing negative linear correlations between CVF, levels of collagen I and III, and atrial transcripts of pro-NPs. These findings suggest that the discordance between transcripts and protein contents of pro-NPs was possibly due to the more outstanding atrial fibrosis in PeAF, and that circulating nt-proNPs levels could reflect the corresponding atrial pro-NPs contents in this report.
Keywords: Natriuretic peptides; Atrial fibrosis; Atrial fibrillation; Valvular replacement;
Regional vascular response to ProAngiotensin-12 (PA12) through the rat arterial system by H.C. Prosser; A.M. Richards; M.E. Forster; C.J. Pemberton (1540-1545).
ProAngiotensin-12 (PA12) is the most recent peptide to be identified as a functional component of the renin–angiotensin system (RAS). PA12 is reported to constrict rat coronary arteries and the aorta, dependent upon angiotensin II-converting enzyme 1 (ACE1) and chymase. The current study employed myography to determine the direct vascular effects of PA12 on a range of isolated rat arteries extending from the core to periphery. PA12 significantly constricted the descending thoracic aorta, right and left common carotid arteries, abdominal aorta and superior mesenteric artery, with little effect on the femoral and renal arteries. AngII was found to produce similar responses to PA12 when administered at the same dose. A potency gradient in response to PA12 was clearly apparent, with vessels in closest proximity to the heart responding with the greatest constriction; while constrictive potency was lost further form the heart. Inhibition of ACE1 and chymase both significantly attenuated PA12-induced vasoconstriction, with chymostatin displaying lesser potency. We postulate ACE1 primarily regulates RAS activity within the circulation, while chymase may have an important role in local, tissue-based RAS activity.
Keywords: Angiotensin-converting enzyme; Chymase; Myography; ProAngiotensin-12; Rat vasculature;
A ligand-based approach to investigate the expression and function of angiotensin converting enzyme in intact human umbilical vein endothelial cells by Gérémy Abdull Koumbadinga; Marie-Thérèse Bawolak; Emilie Marceau; Albert Adam; Lajos Gera; François Marceau (1546-1554).
Angiotensin converting enzyme (ACE) is a drug target and an effective bradykinin (BK)-inactivating ectopeptidase. We exploited a recently described [3H]enalaprilat binding assay to quantify the full dynamic range of ACE expression in intact human umbilical vein endothelial cells (HUVECs) stimulated with known or novel modulators of ACE expression. Further, the affinities for ACE of a set of physiological substrates were determined using the same assay. BK has the highest affinity (K i 525 nM) among known substrates to displace [3H]enalaprilat binding from ACE. Tumor necrosis factor (TNF)-α repressed the expression of ACE in HUVECs while phorbol 12-myristate 13-acetate (PMA) upregulated it in 24 h (∼12-fold dynamic range by [3H]enalaprilat binding, corroborated by ACE immunoblotting). Intermediate levels of ACE expression were seen in cells stimulated with both PMA and a cytokine. In contrast, high glucose, insulin or EGF failed to affect ACE expression. The effect of TNF-α was abated by etanercept, the IKK2 inhibitor TPCA-1, or a p38 inhibitor while that of PMA was reduced by inhibitors of PKC isoforms sensitive to phorbol esters and calcium. The short-term PKC- and MEK1-dependent increase of c-Fos expression was best correlated to PMA-induced ACE upregulation. The [3H]enalaprilat binding assay applied to HUVECs supports that ACE is a particularly active kininase and that endothelial ACE expression is dynamically and specifically regulated. This has potential importance in inflammatory diseases and diabetes.
Keywords: Bradykinin; Angiotensin converting enzyme; Endothelial cells; Regulation of expression; Enalaprilat;
The structure of helokinestatin-5 and its biosynthetic precursor from Gila monster (Heloderma suspectum) venom: Evidence for helokinestatin antagonism of bradykinin-induced relaxation of rat tail artery smooth muscle by Yao Zhang; Lei Wang; Mei Zhou; Zhihao Zhou; Xiaole Chen; Tianbao Chen; HangFai Kwok; Craig Ivanyi; Chris Shaw (1555-1561).
Here we report the primary structure of a novel peptide, named helokinestatin-5 (VPPPLQMPLIPR), from the venom of the Gila monster (Heloderma suspectum). Helokinestatin-5 differs in structure from helokinestatin-3 by deletion of a single prolyl residue in the N-terminally located polyproline region. Two different biosynthetic precursors were consistently cloned from a venom-derived cDNA library. The first encoded helokinestatins 1–4 and a single copy of C-type natriuretic peptide, as previously described, whereas the second was virtually identical, lacking only a single prolyl codon as found in the mature attenuated helokinestatin-5 peptide. Helokinestatins 1–3 and 5 were synthesized by solid-phase fmoc chemistry and each synthetic replicate was found to antagonize the relaxation effect induced by bradykinin on rat tail artery smooth muscle. Helokinestatins thus represent a novel family of vasoactive peptides from the venom of helodermatid lizards.
Keywords: Lizard; Venom; Bradykinin; Antagonist; Mass spectrometry; Molecular cloning;
Isolation and potential immunological characterization of TPSGLVY, a novel bursal septpeptide isolated from the bursa of Fabricius by Xiuli Feng; Xiaodong Su; Fangquan Wang; Jianchao Wei; Fengjuan Wang; Ruibing Cao; Bin Zhou; Xiang Mao; Qisheng Zheng; Puyan Chen (1562-1568).
The bursa of Fabricius is central immune organ unique to birds, and the extract is immunocompetent in stimulating B cell differentiation and enhancing antibody production. However, except for bursin, the active peptides from the bursa of Fabricius are little reported. In the paper, a novel bursal septpeptide (BSP-II) with the amino acids sequence of TPSGLVY was identified and similar to the MGC53864 protein of Gallus gallus. We investigated the effects of BSP-II on the immune response in terms of the antibodies titers (IgG1 and IgG2α), the levels of interferon-γ and interleukin-4 cytokines, spleen cell lymphocyte proliferation, and the T-lymphocyte subtype composition. It was noteworthy that BSP-II potentiates the Th1 and Th2-type immune responses in dose-dependent manner. BSP-II had specific enhancing effects on the hybridoma SP2/0 cell proliferation at two different serum concentrations (20% and 5%), but had no connection with the dose of BSP-II. The antibody secreting level of hybridoma SP2/0 cells rose in 5% and 20% serum when the concentrations of BSP-II increased. Also, BSP-II had effect on the viabilities of tumor cells (Hela and SP2/0). All the results indicated that BSP-II was able to significantly induce various immune responses and involved in the cell viability of different tumor cell lines. Our observations implied that BSP-II might be a novel biological active factor from the bursa of Fabricius with immunomodulatory activities.
Keywords: Second bursal septpeptide (BSP-II); Immune response; Hybridoma SP2/0; Tumor cell viability;
Pharmacology of putative selective hBRS-3 receptor agonists for human bombesin receptors (BnR): Affinities, potencies and selectivity in multiple native and BnR transfected cells by Veronica Sancho; Terry W. Moody; Samuel A. Mantey; Alessia Di Florio; Hirotsugu Uehara; David H. Coy; Robert T. Jensen (1569-1578).
The orphan receptor, bombesin receptor subtype-3(BRS-3) is a G-protein-coupled receptor classified in the bombesin (Bn) receptor family because of its high homology (47–51%) with other members of this family [gastrin-releasing peptide receptor [GRPR] and neuromedin B receptor [NMBR]]. There is increasing interest in BRS-3, because primarily from receptor knockout studies, it seems important in energy metabolism, glucose control, insulin secretion, motility and tumor growth. Pharmacological tools to study the role of BRS-3 in physiology/pathophysiology are limited because the natural ligand is unknown and BRS-3 has low affinity for all naturally occurring Bn-related peptides. However, a few years ago a synthetic high-affinity agonist [dTyr6,βAla11,Phe13,Nle14]Bn-(6-14) was described but was nonselective for BRS-3 over other Bn receptors. Based on this peptide, in various studies a number of putative selective, high-potency hBRS-3 agonists were described, however the results on their selectivity are conflicting in a number of cases. The purpose of the present study was to thoroughly study the pharmacology of four of the most select/potent putative hBRS-3 agonists (#2–4, 16a). Each was studied in multiple well-characterized Bn receptor-transfected cells and native Bn receptor bearing cells, using binding studies, alterations in cellular signaling (PLC, PKD) and changes in cellular function(growth). Two peptides (#2, #3) had nM affinities/potencies for hBRS-3, peptide #4 had low affinity/potency, and peptide #16a very low (>3000 nM). Peptide#3 had the highest selectivity for hBRS-3 (100-fold), whereas #2, 4 had lower selectivity. Peptide #16a's selectivity could not be determined because of its low affinity/potencies for all hBn receptors. These results show that peptide #3 is the preferred hBRS-3 agonist for studies at present, although its selectivity of only 100-fold may limit its utility in some cases. This study underscores the importance of full pharmacological characterization of newly reported selective agonists.
Keywords: Bombesin; Gastrin-releasing peptide; Neuromedin B; Agonist; Receptor;
Cytosolic calcium elevation induced by orexin/hypocretin in granule cell domain cells of the rat cochlear nucleus in vitro by Yuki Nakamura; Shinya Miura; Takashi Yoshida; Juhyon Kim; Kazuo Sasaki (1579-1588).
Using rat brain slice preparations, we examined the effect of orexin on cytosolic Ca2+ concentrations ([Ca2+]i) in the granule cell domain (GCD) cells of the cochlear nucleus that carry non-auditory information to the dorsal cochlear nucleus. Application of orexin concentration-dependently increased [Ca2+]i, and in two thirds of GCD cells these increases persisted in the presence of tetrodotoxin. There was no significant difference between the dose–response curve for orexin-A and that for orexin-B. Extracellular Ca2+ removal abolished the [Ca2+]i elevation induced by orexin-B, whereas depletion of intracellular Ca2+ stores had no effect. The orexin-B-induced elevation of [Ca2+]i was not blocked by inhibitors of reverse-mode Na+/Ca2+ exchanger (NCX) and nonselective cation channel, whereas it was blocked by lowering the extracellular Na+ or by applying inhibitors of forward-mode NCX and voltage-gated R- and T-type Ca2+ channels. The ORX-B-induced increase in [Ca2+]i was also blocked by inhibitors of adenylcyclase (AC) and protein kinase A (PKA), but not by inhibitors of phosphatidylcholine-specific and phosphatidylinositol-specific phospholipase C. In electrophysiological experiments using whole-cell patch clamp recordings, half of GCD cells were depolarized by orexin-B, and the depolarization was abolished by a forward-mode NCX inhibitor. These results suggest that orexin increases [Ca2+]i postsynaptically via orexin 2 receptors, and the increase in [Ca2+]i is induced via the AC–PKA–forward-mode NCX-membrane depolarization-mediated activation of voltage-gated R- and T-type Ca2+ channels. The results further support the hypothesis that the orexin system participates in integrating neural systems that are involved in arousal, sensory processing, energy homeostasis and autonomic function.
Keywords: Orexin; Cytosolic Ca2+ concentration; Granule cell domain; Cochlear nucleus; Na+/Ca2+ exchanger; Ni2+-sensitive Ca2+ channel; Adenylcyclase; Protein kinase A; Sensory processing;
Interactions between orexin–immunoreactive fibers and adrenaline or noradrenaline-expressing neurons of the lower brainstem in rats and mice by Nela Puskás; Rege Sugárka Papp; Katalin Gallatz; Miklós Palkovits (1589-1597).
Orexins are expressed in neurons of the dorsolateral hypothalamus and their axons widely distribute throughout the central nervous system. The noradrenergic cell groups of the lower brainstem belong to the targets of these orexin projections. Double immunostainings for orexin and phenylethanolamine N-methyltransferase (PNMT), as well as orexin and tyrosine hydroxylase (TH) were applied to demonstrate the orexinergic innervation of catecholamine cell groups in the lower brainstem of the mouse and the rat. In various densities, networks of orexin-positive fibers and terminals were present on neurons of each adrenaline (C1, C2, C3) and noradrenaline (locus coeruleus, A1, A2, A4, A5 and A7) cell groups. The most dense networks of orexin fibers and terminals were detected in the locus coeruleus, the subcoeruleus area, and in the nucleus of the solitary tract. By using confocal microscope to analyze triple immunostainings we could detect that about two-third of the orexin-PNMT or orexin-TH immunopositive close contacts contained synaptophysin (a presynapse-specific protein) in the C1, C2 and C3 adrenaline, or in the A1, A2 noradrenaline cell groups, respectively. Orexin–immunopositive axons in the C1, C2, as well as A1, A2 and A6 cell groups have been examined by an electron microscope. Relatively few asymmetrical (excitatory) synaptic contacts could be demonstrated between PNMT- or TH-positive dendrites and orexin terminals, although the vast majority of orexin-positive axons was located in juxtaposition to PNMT- or TH-positive neurons.
Keywords: Orexin; Tyrosine hydroxylase; Phenylethanolamine N-methyltransferase; Adrenaline cell groups; Medullary noradrenaline cell groups;
Sexual dimorphic evolution of metabolic programming in non-genetic non-alimentary mild metabolic syndrome model in mice depends on feed-back mechanisms integrity for pro-opiomelanocortin-derived endogenous substances by Stefano Loizzo; Stefano Vella; Alberto Loizzo; Andrea Fortuna; Antonella Di Biase; Serafina Salvati; Giovanni V. Frajese; Vincent Agrapart; Rafael Ramirez Morales; Santi Spampinato; Gabriele Campana; Anna Capasso; Gabriella Galietta; Irene Guarino; Stefania Carta; Ciriaco Carru; Angelo Zinellu; Giovanni Ghirlanda; Giuseppe Seghieri; Paolo Renzi; Flavia Franconi (1598-1605).
Previously, we showed that our post-natal handling model induces pro-opiomelanocortin-derived (POMC) endogenous systems alterations in male mice at weaning. These alterations last up to adult age, and are at the basis of adult hormonal and metabolic conditions similar to mild metabolic syndrome/type-2 diabetes. Here, we evaluate how sex influences post-natal programming in these metabolic conditions. Subjects are adult control (non-handled) female (NHF) and male (NHM) CD-1 mice; adult post-natal handled female (HF) and male (HM) mice. Handling consists of daily maternal separation (10 min) plus sham injection, from birth to weaning (21 days). In adult handled males (90-days old) we find not only POMC-derived hormones alterations (enhanced basal plasma corticosterone (+91%) and ACTH (+109%)) but also overweight (+5.4%), fasting hyperglycemia (+40%), hypertriglyceridemia (+21%), enhanced brain mRNA expression of hydroxysteroid(11-β)dehydrogenase type-1 (HSD11B1) (+49%), and decreased mRNA-HSD11B2 (−39%). Conversely, uric acid, creatinine, HDL(C), total cholesterol, glucose and insulin incremental area under-the-curve are not affected. In females, post-natal handling does not produce both hormonal and dysmetabolic diabetes-like changes; but handling enhances n3- and n6-poly-unsaturated, and decreases saturated fatty acids content in erythrocyte membrane composition in HF versus NHF. In conclusion, for the first time we show that female sex in mice exerts effective protection against the hypothalamus–pituitary–adrenal homeostasis disruption induced by our post-natal handling model on POMC cleavage products; endocrine disruption is in turn responsible for altered metabolic programming in male mice. The role of sex hormones is still to be elucidated.
Keywords: Pro-opiomelanocortin (POMC); ACTH; Corticosterone; Hypothalamus–pituitary–adrenal axis (HPA) upset; Brain–body interaction; Gender; Post-natal handling;
A comparative study of morphine stimulation and biphalin inhibition of human glioblastoma T98G cell proliferation in vitro by Marzena Lazarczyk; Ewa Matyja; Andrzej W. Lipkowski (1606-1612).
Biphalin is a new type of opioid peptide analog with high analgesic potency that is over 1000-fold greater than morphine. Because of its less addictive nature, biphalin has been suggested as a prospective new analgesic drug. Its high analgesic activity may be related to synergic interaction with all three types of opioid receptors (μ, δ, and κ). Earlier data implicating involvement of opioid receptors, particularly MOR (μ opioid receptor) and KOR (κ opioid receptor), in cell cycle regulation prompted us to investigate the effect of biphalin and morphine on human glioma T98G cell proliferation in vitro. We have documented an inhibitory effect of biphalin on tumor cell growth related to a decreased proliferation rate, decline of cell ability to form colonies, and modulation of the Ki-67 proliferation index. Morphine displayed the opposite effect and triggered stimulation of T98G cell proliferation. Our experiments have shown that biphalin might constitute an alternative solution for morphine application in anti-pain and anti-cancer therapy.
Keywords: Biphalin; Morphine; Opioid receptor; Glioblastoma;
Characterization of intrathecally administered hemokinin-1-induced nociceptive behaviors in mice by Chizuko Watanabe; Hirokazu Mizoguchi; Akihiko Yonezawa; Shinobu Sakurada (1613-1616).
Hemokinin-1 is a novel mammalian tachykinin cloned from mouse bone marrow. At present, pharmacological profile and physiological role of hemokinin-1 are still unclear. In the present study, we found that intrathecal (i.t.) administration of hemokinin-1 (0.00625–1.6 nmol) induced nociceptive responses consisting of scratching, biting and licking, which resemble substance P-induced behavioral responses in mice. The behaviors evoked by low-dose of hemokinin-1 (0.0125 nmol) were dose-dependently inhibited by i.t. co-administration of CP-99,994, a non-peptidic tachykinin NK1 receptor antagonist, whereas high-dose of hemokinin-1 (0.1 nmol)-induced behaviors were not affected. Moreover, sendide, a peptidic tachykinin NK1 receptor antagonist, failed to reduce the behavioral responses of both low- and high-dose of hemokinin-1. In contrast, substance P-induced behaviors were completely suppressed by both CP-99,994 and sendide. These results suggest that hemokinin-1 plays an important role in pain transmission at spinal cord. Moreover, the mechanism of hemokinin-1-induced nociceptive behaviors may be dose-dependent, and distinct from substance P-induced nociceptive behaviors.
Keywords: Hemokinin-1; Tachykinin; Nociceptive behavior; Pain; Spinal cord;
Synthesis and biological evaluation of novel peripherally active morphiceptin analogs by Katarzyna Gach; Jean Claude do-Rego; Jakub Fichna; Martin Storr; Dick Delbro; Geza Toth; Anna Janecka (1617-1624).
Morphiceptin (Tyr-Pro-Phe-Pro-NH2), a tetrapeptide present in the enzymatic digest of bovine β-casein, is a selective ligand of the μ-opioid receptor. In the present study, we describe the synthesis of a series of novel morphiceptin analogs modified in positions 1–3. Two of the obtained analogs, [Dmt1, d-Ala2, d-1-Nal3]morphiceptin and [Dmt1, d-NMeAla2, d-1-Nal3]morphiceptin (Dmt—2′,6′-dimethyltyrosine and d-1-Nal—3-(1-naphthyl)-d-alanine)) displayed very high μ-receptor affinity, resistance to enzymatic degradation, and remarkable supraspinally mediated analgesia, as shown in the hot-plate test after intracerebroventricular but not intravenous administration, which indicated that they could not cross the blood–brain barrier. Therefore, these two analogs were further tested in vitro and in vivo towards their possible peripheral analgesic activity and inhibitory effect on gastrointestinal (GI) motility. We report that both peptides showed strong antinociceptive effect in the writhing test after intraperitoneal administration, inhibited smooth muscle contractility in vitro and GI motility in vivo. Taken together, these findings indicate that the novel morphiceptin analogs which induce peripheral, but not central antinociception, inhibit GI transit, and possess exceptional metabolic stability, may provide an interesting approach to the development of peripherally restricted agents for the treatment of GI motility disorders, such as diarrhea or diarrhea-predominant irritable bowel syndrome.
Keywords: Solid-phase peptide synthesis; Binding studies; Opioid receptors; Hot-plate test; Gastrointestinal transit;
Cholecystokinin in plasma and cerebrospinal fluid—A study in healthy young women by Kristina Lundberg; Susanne Hilke; Conny Nordin; Elvar Theodorsson; Ann Josefsson (1625-1628).
Cholecystokinin (CCK) is widely distributed in the brain and is known to affect behavioral and physiological functions including anxiety and pain. The expression of CCK has been shown to be regulated by estrogen and to vary during the estrous cycle in rat brain. In the present study CCK was determined in plasma from 25 healthy women (age 25.0 ± 3.5) during the menstrual cycle, in the late luteal phase and in the follicular phase. In the follicular phase, a lumbar puncture was performed at the same time that a plasma sample was taken in 15 subjects. The participants had fasted and were nicotine-free for at least 8 h preceding the sampling. We compared CCK-like immunoreactivity (CCK-LI) in plasma from 25 subjects in the late luteal phase (LLP) and the follicular phase (FP) and found that there was no difference during the menstrual cycle (n = 25, R 2 = 89.60%, p = n.s.). In the follicular phase no significant difference was found between CCK-LI in plasma and in cerebrospinal fluid (CSF) collected at the same time (n = 15, R 2 = 55.32%, p = n.s.).