Peptides (v.31, #1)
Editorial Board (CO2).
Antimicrobial mechanism of pore-forming protegrin peptides: 100 pores to kill E. coli by Dan Bolintineanu; Ehsan Hazrati; H. Ted Davis; Robert I. Lehrer; Yiannis N. Kaznessis (1-8).
Antimicrobial peptides (AMPs), important effector molecules of the innate immune system, also provide templates for designing novel antibiotics. Protegrin, an especially potent AMP found in porcine leukocytes, was recently shown to form octameric transmembrane pores. We have employed a combination of experiments and models spanning length scales from the atomistic to the cellular level in order to elucidate the microbicidal mechanism of protegrin. Comparison of the modeling and experimental data suggests that approximately 10–100 protegrin pores are necessary to explain the observed rates of potassium leakage and Escherichia coli death in exponential-phase bacteria. The kinetics of viability loss suggest that bacterial death results largely from uncontrolled ion exchange processes and decay of transmembrane potential. However, ion exchange processes alone cannot account for the experimentally observed cell swelling and osmotic lysis—a redundant “overkill” mechanism most likely to occur in locales with high protegrin concentrations. Although our study is limited to protegrin and E. coli, the timeline of events described herein is likely shared by other AMPs that act primarily by permeabilizing microbial membranes. This work provides many of the missing links in describing antimicrobial action, as well as providing a quantitative connection between several previous experimental and simulation studies of protegrin.
Keywords: Antimicrobial peptides; Mechanism of action; Multiscale model;
Inhibition of bactericidal activity is maintained in a mouse α-defensin precursor with proregion truncations by Sharel M. Figueredo; André J. Ouellette (9-15).
α-Defensin biosynthesis requires the proteolytic conversion of inactive precursors to microbicidal forms. In mouse Paneth cell pro-α-defensin proCrp4(20–92), anionic amino acids positioned near the proregion N-terminus inhibit proCrp4 activity by an apparent charge neutralization mechanism. Because most pro-α-defensins contain proregions of highly conserved chain length, we tested whether decreasing the distance between the inhibitory acidic residues of the proregion and the α-defensin component of the precursor would alter proCrp4 inhibition. Accordingly, two proCrp4 deletion variants, (Δ44–53)-proCrp4 and (Δ44–58)-proCrp4, truncated in a manner corresponding to deletions between MMP-7 cleavage sites, were prepared and assayed for bactericidal peptide activity. Consistent with the properties of full-length proCrp4(20–92), (Δ44–53)-proCrp4 and (Δ44–58)-proCrp4 were processed effectively by MMP-7, lacked bactericidal activity at high peptide levels over a 3 h exposure period, and failed to induce permeabilization of live Escherichia coli in vitro. Thus, bringing the inhibitory proregion domain into greater proximity with the Crp4 component of the precursor did not alter the activity of this pro-α-defensin. Therefore, the conserved distance that separates inhibitory acidic proregion residues from the Crp4 peptide is not critical to maintaining proCrp4(20–92) in an inactive state.
Keywords: Antimicrobial peptide; Precursor convertase; Microbicide; Paneth cell; Innate immunity;
Molecular cloning, over expression, and activity studies of a peptidic HIV-1 protease inhibitor: Designed synthetic gene to functional recombinant peptide by Vinod Vathipadiekal; Perunthottathu K. Umasankar; Milind S. Patole; Mala Rao (16-21).
The aspartic protease inhibitor (ATBI) purified from a Bacillus sp. is a potent inhibitor of several proteases including recombinant HIV-1 protease, pepsin, and fungal aspartic protease. In this study, we report the cloning, and over expression of a synthetic gene coding for ATBI in Escherichia coli and establish a purification protocol. The ATBI molecule consists of eleven amino acids and is peptidic in nature. We used the peptide sequence data of ATBI to synthesize complementary oligonucleotides, which were annealed and subsequently cloned in-frame with the gene for glutathione-S-transferase (GST). The expression of the resulting fusion protein was induced in E. coli BL21-A1 cells using arabinose. The recombinant peptide was purified using a reduced glutathione column, and cleaved with Factor Xa to remove the GST tag. The resultant product was further purified to homogeneity using RP-HPLC. Mass spectroscopy analysis revealed that the purified peptide had a molecular weight of 1186 Da which matches the theoretical molecular weight of the amino acids present in the synthetic gene. The recombinant peptide was found to be active in vitro against HIV-1 protease, pepsin, and fungal aspartic protease. The protocol described in this study may be used to clone pharmaceutically important peptide molecules.
Keywords: Gateway cloning; Synthetic gene; Inhibitor; Proteases; HIV-1 protease;
Cloning and functional characterization of a new antimicrobial peptide gene StCT1 from the venom of the scorpion Scorpiops tibetanus by Wenying Yuan; Luyang Cao; Yibao Ma; Panyong Mao; Weipeng Wang; Ruiming Zhao; Yingliang Wu; Zhijian Cao; Wenxin Li (22-26).
Scorpion has an innovative venom gland, which is an important determinant in contributing to its successful survival for more than 400 million years. Scorpion venom contains a diversity of bioactive peptides, which represent a tremendous hitherto unexplored resource for use in drug design and development. Here, StCT1, a new antimicrobial peptide gene, was screened and isolated from the venomous gland cDNA library of the scorpion Scorpiops tibetanus. The full-length cDNA of StCT1 is 369 nucleotides encoding the precursor that contains a putative 24-residue signal peptide, a presumed 14-residue mature peptide, and an uncommon 37-residue acidic propeptide at the C-terminus. The minimal inhibitory concentrations (MICs) of the synthetic StCT1 peptide against Staphylococcus aureus and Micrococcus luteus were 12.5 μg/ml and 100 μg/ml, respectively. The MICs of StCT1 against clinical antibiotics-resistant bacterial strains, were 50–250 μg/ml, 2–40 folds lower than those of penicillin. These results show that the antimicrobial peptide encoded by StCT1 gene from the venom of the scorpion S. tibetanus is a potential anti-infective polypeptide or lead compound, especially for treating antibiotics-resistant pathogens.
Keywords: Scorpion; Venom; Antimicrobial peptides; Antibiotics-resistant;
Combining in silico transcriptome mining and biological mass spectrometry for neuropeptide discovery in the Pacific white shrimp Litopenaeus vannamei by Mingming Ma; Ashley L. Gard; Feng Xiang; Junhua Wang; Naveed Davoodian; Petra H. Lenz; Spencer R. Malecha; Andrew E. Christie; Lingjun Li (27-43).
The shrimp Litopenaeus vannamei is arguably the most important aquacultured crustacean, being the subject of a multi-billion dollar industry worldwide. To extend our knowledge of peptidergic control in this species, we conducted an investigation combining transcriptomics and mass spectrometry to identify its neuropeptides. Specifically, in silico searches of the L. vannamei EST database were conducted to identify putative prepro-hormone-encoding transcripts, with the mature peptides contained within the deduced precursors predicted via online software programs and homology to known isoforms. MALDI-FT mass spectrometry was used to screen tissue fragments and extracts via accurate mass measurements for the predicted peptides, as well as for known ones from other species. ESI-Q-TOF tandem mass spectrometry was used to de novo sequence peptides from tissue extracts. In total 120 peptides were characterized using this combined approach, including 5 identified both by transcriptomics and by mass spectrometry (e.g. pQTFQYSRGWTNamide, Arg7-corazonin, and pQDLDHVFLRFamide, a myosuppressin), 49 predicted via transcriptomics only (e.g. pQIRYHQCYFNPISCF and pQIRYHQCYFIPVSCF, two C-type allatostatins, and RYLPT, authentic proctolin), and 66 identified solely by mass spectrometry (e.g. the orcokinin NFDEIDRAGMGFA). While some of the characterized peptides were known L. vannamei isoforms (e.g. the pyrokinins DFAFSPRLamide and ADFAFNPRLamide), most were novel, either for this species (e.g. pEGFYSQRYamide, an RYamide) or in general (e.g. the tachykinin-related peptides APAGFLGMRamide, APSGFNGMRamide and APSGFLDMRamide). Collectively, our data not only expand greatly the number of known L. vannamei neuropeptides, but also provide a foundation for future investigations of the physiological roles played by them in this commercially important species.
Keywords: Functional genomics; Expressed sequence tag (EST); Matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS); Electrospray ionization quadrupole time-of-flight tandem mass spectrometry (ESI-Q-TOF MS/MS);
Isolation, characterization and anti-cancer activity of SK84, a novel glycine-rich antimicrobial peptide from Drosophila virilis by Jie Lu; Zheng-wang Chen (44-50).
We report herein the isolation and characterization of a novel glycine-rich antimicrobial peptide purified from the larvae of Drosophila virilis. A range of chromatographic methods was used for isolation and its antibacterial activity against Bacillus subtilis was employed to screen for the most active fractions. The peptide, termed SK84 due to its N-terminal serine, C-terminal lysine and a total of 84 residues, was completed sequenced using RT-PCR cDNA cloning. SK84 contains a high level of glycine (15.5%) and a hexaglycine cluster motif in the N-terminal part. SK84 displayed antibacterial activity against the tested Gram-positive bacteria (B. subtilis, Bacillus thuringiensis and Staphylococcus aureus), but had no effect on Gram-negative bacteria (Pseudomonas aeruginosa, Escherichia coli) and fungi (Saccharomyces cerevisiae, Candida albicans). SK84 had specific inhibitory effects on the proliferation of several cancer cell lines (Human leukemia THP-1, liver cancer HepG2, and breast cancer MCF-7 cells), but no hemolytic activity. The results from scanning electron microscopy observations revealed that SK84 killed THP-1 cells by destroying the cell membranes. Alignment results show that SK84 is a mature protein processed from the pseudoprotein GJ19999 from D. virilis, and is very similar to several pseudoproteins from different Drosophila species. Our results show that SK84 represents a novel glycine-rich peptide family in Drosophila species with antimicrobial and anti-cancer cell activities.
Keywords: Antimicrobial peptide; Glycine-rich peptide; Drosophila virilis; Anti-cancer-cell activity;
A novel role for the adipokine visfatin/pre-B cell colony-enhancing factor 1 in prostate carcinogenesis by Snehal T. Patel; Tina Mistry; James E.P. Brown; Janet E. Digby; Raghu Adya; Ken M. Desai; Harpal S. Randeva (51-57).
Adipose tissue is now well established as an endocrine organ and multiple hormones termed ‘adipokines’ are released from it. With the rapidly increasing obese population and the increased risk mortality from prostate cancer within the obese population we looked to investigate the role of the adipokine visfatin in LNCaP and PC3 prostate cancer cell lines. Using immunohistochemistry and immunocytochemistry we demonstrate visfatin expression in LNCaP (androgen-sensitive) and PC3 (androgen-insensitive) human prostate cancer cell lines as well as human prostate cancer tissue. Additionally, we show that visfatin increases PC3 cell proliferation and demonstrate the activation of the MAPKs ERK-1/2 and p38. Moreover we also demonstrate that visfatin promotes the expression and activity of MMP-2/9 which are important proteases involved in the breakdown of the extracellular matrix, suggesting a possible role for visfatin in prostate cancer metastases. These data suggest a contributory and multifunctional role for visfatin in prostate cancer progression, with particular relevance and emphasis in an obese population.
Keywords: Prostate cancer; Visfatin; PBEF; Proliferation; MMP-2; MMP-9;
Structure–activity relationship of human liver-expressed antimicrobial peptide 2 by Agnès Hocquellet; Benoit Odaert; Charlotte Cabanne; Abdelmajid Noubhani; Wilfrid Dieryck; Gilles Joucla; Caroline Le Senechal; Milen Milenkov; Stéphane Chaignepain; Jean-Marie Schmitter; Stéphane Claverol; Xavier Santarelli; Erick J. Dufourc; Marc Bonneu; Bertrand Garbay; Patricia Costaglioli (58-66).
Liver-expressed antimicrobial peptide 2 (LEAP-2) is a 40-residue cationic peptide originally purified from human blood ultrafiltrate. The native peptide contains two disulfide bonds and is unique regarding its primary structure. Its biological role is not known but a previous study showed that chemically synthesized LEAP-2 exhibited in vitro antimicrobial activities against several Gram-positive bacteria. In order to determine its antimicrobial mode of action, we expressed human recombinant LEAP-2 in Escherichia coli. Circular dichroism spectroscopy and nuclear magnetic resonance analyses showed that the structure of the recombinant peptide was identical to that of the chemically synthesized and oxidized LEAP-2, with two disulfide bonds between Cys residues in relative 1–3 and 2–4 positions. Minimal inhibitory concentration (MIC) of the recombinant human LEAP-2 was determined by a conventional broth dilution assay. It was found to be bactericidal against Bacillus megaterium at a 200 μM concentration. Interestingly, the linear LEAP-2 had a greater antimicrobial activity with a MIC value of 12.5 μM, which was comparable to that of magainin2. SYTOX Green uptake was used to assess bacterial membrane integrity. Linear LEAP-2 and magainin2 permeabilized B. megaterium membranes with the same efficiency, whereas oxidized LEAP-2 did not induce stain uptake. Binding of the peptides to plasmid DNA was evaluated by gel retardation assays. The DNA-binding efficacy of linear LEAP-2 was three times higher than that of the peptide-containing disulfide bridges. Altogether, these results show that the secondary structure of human LEAP-2 has a profound impact on its antibacterial activity.
Keywords: Antimicrobial peptide; DNA-binding; Structure–activity relation; Disulfide bond;
Effect of carnosine against thioacetamide-induced liver cirrhosis in rat by A. Fatih Aydın; Zeynep Küskü-Kiraz; Semra Doğru-Abbasoğlu; Mine Güllüoğlu; Müjdat Uysal; Necla Koçak-Toker (67-71).
Carnosine (β-alanyl-l-histidine) is a dipeptide with antioxidant properties. Oxidative stress has been proposed to be involved in thioacetamide (TAA)-induced liver cirrhosis in rats, that is similar to human disease. In this study we aimed to investigate the role of carnosine on the development of TAA-induced cirrhosis. 200 mg TAA/kg body weight has been given i.p. twice a week for three months to female wistar rats. Another group received same dose of TAA in the same pattern plus 2 g carnosine/L of drinking water for three months. TAA administration resulted in hepatic fibrosis, significant increases in plasma transaminase activities as well as hepatic hydroxyproline and lipid peroxide levels, while liver glutathione (GSH) and superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) protein expressions and activities decreased. Carnosine was found to behave as an antioxidant reducing malondialdehyde (MDA) and diene conjugate (DC) levels although it was not effective on increased transaminase activities and decreased antioxidants. It also did not affect the histopathological changes observed in TAA group. Thus our findings indicate that carnosine appears to attenuate peroxidation as an antioxidant itself but does not seem to prevent the development of TAA-induced cirrhotic process.
Keywords: Thioacetamide; Carnosine; Cirrhosis; Oxidative stress; SOD; GSH-Px;
Inhalable powder formulation of a stabilized vasoactive intestinal peptide (VIP) derivative: Anti-inflammatory effect in experimental asthmatic rats by Shingen Misaka; Yosuke Aoki; Shin-ichiro Karaki; Atsukazu Kuwahara; Takahiro Mizumoto; Satomi Onoue; Shizuo Yamada (72-78).
Vasoactive intestinal peptide (VIP) exerts immunomodulating and anti-inflammatory activities through its specific receptors, such as VPAC1 and 2 receptors. Previously, a stabilized VIP derivative, [R15,20,21, L17]-VIP-GRR (IK312532), was proposed as a candidate of anti-asthma drug, and a dry powder inhaler system of IK312532 was also developed for inhalation therapy with minimal systemic side-effects. In the present study, the anti-inflammatory properties of IK312532 respirable powder (RP) were characterized in an asthma/COPD-like animal model, with the use of newly developed ovalbumin (OVA)-RP for lung inflammation. Marked inflammatory events in the lung were observed after OVA-RP challenge in rats as evidenced by significant increase of inflammatory biomarkers such as eosinophil peroxidase (EPO), myeloperoxidase (MPO) and lactate dehydrogenase (LDH). However, intratracheal administration of IK312532-RP led to significant attenuation of plasma EPO, MPO and LDH activities, as well as significant reduction of recruited inflammatory cells in BALF, especially macrophages and eosinophils. In the rats pretreated with IK312532-RP, histochemical examinations revealed that the inflammatory cells infiltrating to the lung and the epithelial wall thickness decreased significantly by 85% and 58%, respectively. Thus, inhalable powder formulation of IK312532 exerts its anti-inflammatory activity by suppressing granulocyte recruitment to the lung and epithelial hyperplasia, followed by the reduction of cytotoxic peroxidases.
Keywords: Asthma; Chronic obstructive pulmonary disease; Dry powder inhaler; Vasoactive intestinal peptide;
SHP-1, a novel peptide isolated from seahorse inhibits collagen release through the suppression of collagenases 1 and 3, nitric oxide products regulated by NF-κB/p38 kinase by BoMi Ryu; Zhong-Ji Qian; Se-Kwon Kim (79-87).
Considerable efforts have been taken to identify natural peptides as potential bioactive substances. In this study, novel peptide (SHP-1) derived from seahorse (Hippocampus, Syngnathidae) hydrolysate was explored for its inhibitory effects on collagen release in arthritis with the investigation of its underlying mechanism of action. The efficacy of SHP-1 was determined on cartilage protective effects such as inhibition of collagen and GAG release. SHP-1 was able to suppress not only the expression of collagenases 1 and 3, but also the production of NO via down-regulation of iNOS. However, it presented an irrelevant effect on the level of GAG release in chondrocytic and osteoblastic cells. Inhibition of collagen release by SHP-1 is associated with restraining the phosphorylation of NF-κB and p38 kinase cascade. Therefore, it could be suggested that SHP-1 has a potential to be used in arthritis treatment.
Keywords: SHP-1; Collagen; Arthritis; MMP-1; MMP-13; NO; NF-κB/p38 kinase;
Endogenous opioid peptide responses to opioid and anti-inflammatory medications following eccentric exercise-induced muscle damage by William J. Kraemer; Michael F. Joseph; Jeff S. Volek; Jay R. Hoffman; Nicholas A. Ratamess; Robert U. Newton; Maren S. Fragala; Duncan N. French; Martyn A. Rubin; Timothy P. Scheett; Michael R. McGuigan; Gwendolyn A. Thomas; Ana L. Gomez; Keijo Häkkinen; Carl M. Maresh (88-93).
To determine the effects of Vicoprofen®, Ibuprofen, and a placebo on the responses of endogenous opioid peptides following eccentric exercise-induced muscle damage 36 healthy men (age: 22.8 years; height: 178.8 ± 6.2 cm; body mass: 78.9 ± 13.7 kg; body fat: 15.8 ± 6.5%) volunteered to participate in the study. Each participant was evaluated for pain 24 h post and randomly assigned to an experimental group: VIC (Vicoprofen®), IBU (Ibuprofen), or P (placebo). Medication was given four times daily (i.e., VIC (hydrocodone bitartrate 7.5 mg with Ibuprofen 200 mg) and IBU 200 mg). Blood was obtained at rest and at 0, 24, 48, 72, 96 and 120 h following the eccentric exercise damage protocol. No significant changes for B-END were observed in the resting values over the recovery period among any of the treatment conditions. Conversely for plasma P-F, VIC and IBU had significantly (P < 0.05) higher plasma concentrations of P-F above placebo at 24, 48, 72, and 96 and 120 h with VIC higher than IBU and placebo conditions at 48, 72, 96, and 120 h. Significant resting elevations were observed for P-F from pre-exercise at 48, 72, 96, and 120 h for VIC; at 72 and 96 h for IBU and no changes in the placebo treatment. Less tissue damage (MRI analyses), improved physical function as well as reduced pain was observed for the VIC condition over IBU and placebo. These data indicate that exogenous medications appear to be differentially stimulating the peripheral (adrenal medulla) opioid neuroendocrine responses as measured by plasma concentrations.
Keywords: Opiate-like; Recovery; Proenkephalin; β-Endorphin; Peptide F; Stress;
Effects of Endokinin A/B and Endokinin C/D on the modulation of pain in mice by Yinliang Yang; Shouliang Dong (94-100).
Endokinins are novel tachykinins encoded on the human TAC4 and consist of Endokinin A (EKA), B (EKB), C (EKC) and D (EKD). To date, the function of Endokinins in pain processing was not fully understood. Therefore the aim of this study was to investigate the effects of Endokinin A/B (EKA/B, the common C-terminal decapeptide in EKA and EKB) and Endokinin C/D (EKC/D, the common C-terminal duodecapeptide in EKC and EKD) on pain modulation at supraspinal level in mice. Intracerebroventricular (i.c.v.) administration of EKA/B (1, 3, 12, 20 nmol/mouse) dose dependently induced potent analgesic effect. This effect could be fully antagonized by SR140333B but not SR48968C or SR142801. Naloxone could also block the analgesic effect, suggesting that this analgesic effect is related to opioid receptors. However, i.c.v. administration of EKA/B (10, 30, 100 pmol/mouse) caused hyperalgesic effect significantly, with a “U” shape curve. Interestingly, the hyperalgesic effect induced by EKA/B could be attenuated by SR140333B, SR142801 but not SR48968C. I.c.v. administration of EKC/D (1, 3, 12, 20 nmol/mouse) also dose dependently induced analgesic effect, which could not be blocked by SR48968C or SR142801 or naloxone. But to our astonishment, it could be significantly enhanced by SR140333B. More interestingly, the hyperalgesic effect induced by EKA/B could be significantly attenuated by EKC/D. In addition, the analgesic effect induced by co-administration of EKA/B and EKC/D was much less stronger than the effect of either EKA/B or EKC/D.
Keywords: Endokinins; Tachykinin; SR140333B; SR48968C; SR142801; Antinociception;
Human astrocytic bradykinin B2 receptor modulates zymosan-induced cytokine expression in 1321N1 cells by Donghoon Kim; Suk-Hee Cho; Jong-So Kim; Su-Hyun Jo; Sung Joong Lee; Kyong-Tai Kim; Se-Young Choi (101-107).
Bradykinin is an important modulator of the neurons and glial cells of the nervous system. Bradykinin secreted from neurons affects astrocytic functions such as neurovascular coupling and astrocytic cytokine production. In human astrocytes, however, the detailed mechanism of bradykinin-mediated modulation of astrocytic functions has not yet been fully elucidated. Here, we report the functional expression of the bradykinin B2 receptor and its modulation of zymosan-induced cytokine expression in human astrocytoma 1321N1 cells. Bradykinin increased cytosolic [Ca2+] in a concentration-dependent manner, whereas [des-Arg10] kallidin (an agonist of the B1 receptor) did not have this effect. Bradykinin also triggered intracellular InsP3 production. Pretreating the cells with HOE140 (icatibant acetate, a B2 receptor antagonist) inhibited the bradykinin-induced increase in cytosolic [Ca2+] and InsP3 production. In contrast, [des-Arg10]HOE140 (a B1 receptor antagonist) did not show any inhibitory effect. Bradykinin increased the zymosan-induced expression of TNF-α, and interleukin 1β (IL-1β) but did not affect the expression of interleukin 6 (IL-6) or interleukin 10 (IL-10). Interestingly, a cyclooxygenase-2 specific inhibitor blocked the bradykinin-induced effect. In contrast to the result in human glioma cells, bradykinin inhibits the zymosan-induced expression of TNF-α and IL-1β in rat astrocytes, which shows a species-dependent manner. These data suggest that bradykinin B2 receptors are expressed in human astrocytoma cells and that they modulate the expression pattern of inflammatory cytokines.
Keywords: Bradykinin; Toll-like receptor; Astrocyte; Cytokine; Phospholipase C;
Neurokinin-2 receptor antagonism in medial septum influences temporal-order memory for objects and forebrain cholinergic activity by S. Schäble; J.P. Huston; M.L. Brandao; E. Dere; M.A. de Souza Silva (108-115).
In the mammalian brain the neurokinin NK2 receptors are predominantly located in the hippocampus, thalamus, septum and frontal cortex. It has been shown that administration of the NK2 receptor agonist, neurokinin A (NKA), into the medial septum of rats increases extracellular levels of acetylcholine (ACh) in the hippocampus and that NK2 receptor antagonism blocks this increase. Therefore, given the prominent role of hippocampal ACh in information processing, we hypothesized that NK2 receptor antagonism in the medial septum would negatively affect learning and memory via its influence on the cholinergic neurons of the basal forebrain. We investigated the action of local application of the peptidic NK2 receptor antagonist, Bz-Ala-Ala-D-Trp-Phe-D-Pro-Pro-Nle-NH (1, 10 and 100 pmol), into the medial septum on object memory for temporal order and spatial location using an object novelty paradigm. By means of in vivo microdialysis and HPLC analyses, we also examined the influence of NK2 receptor antagonism in the medial septum on ACh in major cholinergic projection areas of the basal forebrain, namely, hippocampus, frontal cortex and amygdala. Results: Injection of vehicle alone into the medial septum impaired memory for temporal order and spatial location of objects. Application of 1 pmol of the NK2 receptor antagonist partially reversed this deficit by reinstating memory for temporal order. Injection of 10 pmol of the NK2 receptor antagonist into the medial septum decreased levels of ACh in the hippocampus (at 30 min post-injection), and frontal cortex (at 30 and 80 min post-injection) in comparison to vehicle. However, this apparent decrease was the result of the blockade of a saline-induced increase in ACh levels.
Keywords: Neurokinin; NK2; Acetylcholine; In vivo microdialysis; Medial septal area; Temporal-order memory; Place memory; Object memory; Vehicle effects;
The peptide NDP-MSH induces phenotype changes in the heart that resemble ischemic preconditioning by Anna Catania; Caterina Lonati; Andrea Sordi; Patrizia Leonardi; Andrea Carlin; Stefano Gatti (116-122).
α-Melanocyte-stimulating hormone (α-MSH) is a pro-opiomelanocortin (POMC)-derived peptide that exerts multiple protective effects on host cells. Previous investigations showed that treatment with α-MSH or synthetic melanocortin agonists reduces heart damage in reperfusion injury and transplantation. The aim of this preclinical research was to determine whether melanocortin treatment induces preconditioning-like cardioprotection. In particular, the plan was to assess whether melanocortin administration causes phenotype changes similar to those induced by repetitive ischemic events. The idea was conceived because both ischemic preconditioning and melanocortin signaling largely depend on cAMP response element binding protein (CREB) phosphorylation. Rats received single i.v. injections of 750 μg/kg of the α-MSH analogue Nle4,DPhe7-α-MSH (NDP-MSH) or saline and were sacrificed at 0.5, 1, 3, or 5 h. Western blot analysis showed that rat hearts expressed melanocortin 1 receptor (MC1R) protein. Treatment with NDP-MSH was associated with early and marked increase in interleukin 6 (IL-6) mRNA. This was followed by signal transducer and activator of transcription 3 (STAT3) phosphorylation and induction of suppressor of cytokine signaling 3 (SOCS3). There were no changes in expression of other cytokines of the IL-6 family. Expression of IL-10, IL-1β, and TNF-α was likewise unaltered. In hearts of rats treated with NDP-MSH there was increased expression of the orphan nuclear receptor Nur77. The data indicate that NDP-MSH induces phenotype changes that closely resemble ischemic preconditioning and likely contribute to its established protection against reperfusion injury. In addition, the increased expression of Nur77 and SOCS3 could be part of a broader anti-inflammatory effect.
Keywords: α-Melanocyte-stimulating hormone (α-MSH); Nle4,DPhe7-α-MSH (NDP-MSH) melanocortin 1 receptor (MC1R); Reperfusion injury; Ischemic preconditioning; Signal transducer and activator of transcription (STAT)3; Interleukin 6 (IL-6); Suppressor of cytokine signaling (SOCS)3; Nur77;
The metabolic phenotype of SCD1-deficient mice is independent of melanin-concentrating hormone by Melissa B. Glier; Pavlos Pissios; Sandra L. Babich; Marcia L.E. Macdonald; Michael R. Hayden; Eleftheria Maratos-Flier; William T. Gibson (123-129).
We propose that deletion of pro-melanin-concentrating hormone (pMCH) would increase energy expenditure and further improve glucose tolerance in mice lacking stearoyl-coA desaturase-1 (SCD1). To test our hypothesis, we bred and metabolically challenged Pmch−/−; Scd1−/− double-knockout mice, with comparison to Pmch−/− mice; Scd1−/− mice and C57Bl/6J controls. Deletion of both Pmch and Scd1 increased both food intake and energy expenditure relative to control mice. Pmch−/−; Scd1−/− double-knockout mice had improved glucose tolerance relative to control mice. The majority of the metabolic effects were contributed by inactivation of the Scd1 gene. We conclude that the increased food intake and increased energy expenditure of Scd1−/− mice are independent of the neuropeptide melanin-concentrating hormone.
Keywords: Stearoyl-CoA desturase-1; Energy metabolism; Melanin-concentrating hormone; Indirect calorimetry;
Importance of extracellular loop one of the neuropeptide S receptor for biogenesis and function by Stewart D. Clark; Ha T. Tran; Joanne Zeng; Rainer K. Reinscheid (130-138).
Neuropeptide S (NPS) is the endogenous ligand of a formerly orphan G protein-coupled receptor (GPCR). The NPS receptor (NPSR) belongs to the subfamily of peptide GPCRs and is widely expressed in the brain. NPS promotes arousal and induces anxiolytic-like effects after central administration in rodents. Previously, we have reported that the N107I polymorphism in the human NPS receptor results in a gain-of-function characterized by an increase in agonist potency without changing agonist binding affinity. We have extended our findings by investigating pharmacological and biochemical consequences of mutations in the vicinity of position 107. Alanine substitutions were made for D105 and N101, and stable clones were analyzed for agonist-induced changes of intracellular Ca2+. Receptor protein expression was monitored by Western blot and flow cytometry. The mutation D105A produced receptors that have a ∼200-fold higher EC50 despite elevated total receptor protein and surface expression compared to cell lines expressing the parental receptor NPSR-N107. The mutation N101A resulted in slightly reduced agonist potency without affecting the ability of the protein to form functional receptors. Stable NPSR-A101 clones show little expression of the fully glycosylated form. However, NPSR-A101 receptors are expressed on the cell surface and are functional, suggesting that full glycosylation is not required for receptor function. Our studies suggest that N-linked glycosylation is not important for receptor biogenesis or function, and that residue D105 might be critical for receptor binding.
Keywords: Neuropeptide S; GPCR; Extracellular loop one; Biogenesis; Mutagenesis; Pharmacological chaperone;
Effect of anorexinergic peptides, cholecystokinin (CCK) and cocaine and amphetamine regulated transcript (CART) peptide, on the activity of neurons in hypothalamic structures of C57Bl/6 mice involved in the food intake regulation by Zdeno Pirnik; Jana Maixnerová; Resha Matyšková; Darja Koutová; Blanka Železná; Lenka Maletínská; Alexander Kiss (139-144).
The hypothalamus plays an important role in food consumption, receiving information about energy balance via hormonal, metabolic, and neural inputs. Its neurons produce neuropeptides influencing energy balance. Especially important to regulation of food consumption are certain hypothalamic structures, including the arcuate (ARC) and ventromedial (VMN) nuclei and the lateral hypothalamic area (LHA). We determined the impact of cholecystokinin (CCK) and cocaine and amphetamine regulated transcript (CART) peptide, on activity of ARC and VMN neurons and hypocretin (Hcrt) synthesizing neurons in LHA. ARC is an integrative nucleus regulating food consumption, VMN is considered to be a satiety centre, and LHA a hunger sensing centre. After overnight fasting, male C57Bl/6 mice received intraperitoneal injection of (i.p.) saline (SAL) or CCK (4 μg/kg) or intracerebroventricular injection of (i.c.v.) CART peptide (0.1 μg/mice) or CCK (i.p.) followed by CART peptide (i.c.v.) 5 min later. Sixty minutes later, the presence of Fos or Fos/Hcrt immunostaining indicated activity of ARC and VMN neurons, as well as of Hcrt cells in LHA. CCK alone did not influence neuronal activity in any of the nuclei studied. CART peptide stimulated neurons in ARC and VMN (p < 0.01) but decreased Hcrt neuronal activity in LHA (p < 0.05). Co-administration of both peptides synergistically stimulated ARC neurons (p < 0.01) and asynergistically inhibited LHA Hcrt neurons (p < 0.01). Results indicate that CCK may modify the effect of CART peptide and thus substantially influence activity of neurons in hypothalamic structures involved in regulation of food intake.
Keywords: Cholecystokinin; CART; Hypocretin; Fos peptide; Arcuate nucleus; Ventromedial nucleus; Lateral hypothalamic area;
Glucocorticoids increase NPY gene expression in the arcuate nucleus by inhibiting mTOR signaling in rat hypothalamic organotypic cultures by Hiroshi Shimizu; Hiroshi Arima; Yoshiharu Ozawa; Minemori Watanabe; Ryoichi Banno; Yoshihisa Sugimura; Nobuaki Ozaki; Hiroshi Nagasaki; Yutaka Oiso (145-149).
The mammalian target of rapamycin (mTOR) has been implicated in the regulation of physiological functions such as cell growth and proliferation, and glucocorticoids reportedly inhibit mTOR signaling in peripheral tissues. Recent studies suggest that the mTOR signaling in the hypothalamus plays a critical role in maintaining energy homeostasis. In this study, we examined whether the mTOR signaling in the hypothalamus is involved in the regulation of neuropeptide Y (Npy) gene expression in the arcuate nucleus by glucocorticoids. In the hypothalamic organotypic cultures, the incubation with rapamycin significantly inhibited the mTOR signaling which was shown by decreases in the levels of phosphorylated p70S6K1 and S6. Similar to the action of the mTOR inhibitor rapamycin, dexamethasone (DEX), a synthetic glucocorticoid, also inhibited the mTOR signaling in the hypothalamic explants. Analyses of the explants with in situ hybridization demonstrated that the DEX or rapamycin alone significantly increased Npy gene expression in the arcuate nucleus, but that there were no additive effects of DEX and rapamycin on the expression. These data suggest that glucocorticoids upregulate the Npy gene expression in the arcuate nucleus by inhibiting mTOR signaling, at least in part.
Keywords: Mammalian target of rapamycin; Glucocorticoids; Neuropeptide Y;
Effect of intracerebroventricular infusion of neurotensin in glucose-dependent insulinotropic peptide secretion in dogs by Maria P. Yavropoulou; Kalliopi Kotsa; Isaak Kesisoglou; Anna Gotzamani-Psarakou; John G. Yovos (150-154).
GIP is a major physiological component of the enteroinsular axis. Several researchers have pointed to a neural regulation of GIP secretion. We have previously studied the effect of intracerebroventricular (icv) infusion of insulin, NPY and bombesin in the regulation of GIP secretion. The aim of the present study is to evaluate a possible role of neurotensin in neural regulation of GIP secretion. Thirty-two adult dogs were used in this study. In a dose–response study (experiment 1) we used 3 different doses of neurotensin (25, 50 and 100 μg) in a bolus icv infusion. In experiment 2 the animals received a bolus icv infusion of 50 μg neurotensin and an equivalent amount of artificial cerebrospinal fluid (aCSF) at 1-week interval. In experiment 3 the animals received a continuous icv infusion of neurotensin at a constant rate of 1 μg/kg/h and aCSF over a 3-h period, at 1-week interval. In experiment 4 the experiment of group 3 was repeated with a simultaneous intraduodenal infusion of a glucose load through the Mann-Bollman fistula. Plasma levels of glucose, insulin and GIP were assayed. Results: Bolus and continuous icv infusion of neurotensin produced a significant increase in glucose, GIP and insulin levels. In the 4th experiment icv infusion of neurotensin produced a more prominent increase of glucose and insulin levels compare to infusion of aCSF. GIP levels were lower after infusion of neurotensin compared to aCSF. Conclusions: Our data suggest a differential effect of neurotensin on GIP secretion, dependent on the energy load.
Keywords: Neurotensin; Neural regulation; GIP secretion; Insulin; Icv infusion;
Chronic hyperleptinemia induces resistance to acute natriuretic and NO-mimetic effects of leptin by Jerzy Bełtowski; Grażyna Wójcicka; Anna Jamroz-Wiśniewska; Andrzej Wojtak (155-163).
Apart from controlling energy balance, leptin, secreted by adipose tissue, is also involved in the regulation of cardiovascular function. Previous studies have demonstrated that acutely administered leptin stimulates natriuresis and vascular nitric oxide (NO) production and that these effects are impaired in obese animals. However, the mechanism of resistance to leptin is not clear. Because obesity is associated with chronically elevated leptin, we examined if long-term hyperleptinemia impairs acute effects of leptin on sodium excretion and NO production in the absence of obesity. Hyperleptinemia was induced in lean rats by administration of exogenous leptin at a dose of 0.5 mg/kg/day for 7 days, and then acute effect of leptin (1 mg/kg i.v.) was studied under general anesthesia. Leptin increased fractional sodium excretion and decreased Na+,K+-ATPase activity in the renal medulla. In addition, leptin increased the level of NO metabolites and cyclic GMP in plasma and aortic wall. These acute effects of leptin were impaired in hyperleptinemic animals. In both control and hyperleptinemic groups the effect of leptin on Na+ excretion and renal Na+,K+-ATPase was abolished by phosphoinositide 3-kinase (PI3K) inhibitor, wortmannin, but not by protein kinase B/Akt inhibitor, triciribine,. In contrast, acute effect of leptin on NO metabolites and cGMP was abolished by triciribine but not by wortmannin. Leptin stimulated Akt phosphorylation at Ser473 in aortic tissue but not in the kidney, and this effect was comparable in control and hyperleptinemic groups. These results suggest that hyperleptinemia may mediate “renal” and “vascular” leptin resistance observed in obesity.
Keywords: Leptin; Obesity; Arterial hypertension; Nitric oxide; Cyclic GMP; Na+,K+-ATPase; Natriuresis;
Urotensin II stimulates high frequency-induced ANP secretion via PLC-PI 3K-PKC pathway by Shan Gao; Amin Shah; Young Bin Oh; Woo Hyun Park; Suhn Hee Kim (164-169).
Urotensin II (U-II) and its receptor are coexpressed in the heart and show various cardiovascular functions. However, the relationship between U-II and cardiac hormone atrial natriuretic peptide (ANP) is still unknown. The aim of the present study is to test whether U-II affects ANP secretion using in vitro perfusion experiments and in vivo studies. Human U-II (hU-II) (10−11, 5 × 10−11, 10−10, 5 × 10−10 M) stimulated ANP secretion from isolated perfused rat atria paced with high frequency (6.0 Hz). However, atrial contractility and translocation of extracellular fluid (ECF) did not change. An increase in ANP secretion by rat U-II was similar to that by hU-II; however, urotensin-related peptide showed no significant effect on ANP secretion. Pretreatment with urotensin receptor antagonist and inhibitor for phospholipase C (PLC), phosphoinositide 3-kinase (PI3K), or protein kinase C (PKC) attenuated hU-II-induced ANP secretion from atria paced with high frequency, but an inhibitor for inositol triphosphate did not. Intravenous infusion of hU-II at a dose of 2.5 μM for 20 min increased plasma ANP level, along with increased heart rate and pulse pressure in anesthetized rats. Therefore, we suggest that U-II stimulates high stimulation frequency-induced ANP secretion partly through the urotensin receptor and the PLC/PI3K/PKC pathway.
Keywords: Urotensin II; ANP; Signal transduction; Heart; Blood pressure; Heart rate;
Mapping interaction sites within the N-terminus of the calcitonin gene-related peptide receptor; the role of residues 23–60 of the calcitonin receptor-like receptor by James Barwell; Philip S. Miller; Dan Donnelly; David R. Poyner (170-176).
The calcitonin receptor-like receptor (CLR) acts as a receptor for the calcitonin gene-related peptide (CGRP) but in order to recognize CGRP, it must form a complex with an accessory protein, receptor activity modifying protein 1 (RAMP1). Identifying the protein/protein and protein/ligand interfaces in this unusual complex would aid drug design. The role of the extreme N-terminus of CLR (Glu23-Ala60) was examined by an alanine scan and the results were interpreted with the help of a molecular model. The potency of CGRP at stimulating cAMP production was reduced at Leu41Ala, Gln45Ala, Cys48Ala and Tyr49Ala; furthermore, CGRP-induced receptor internalization at all of these receptors was also impaired. Ile32Ala, Gly35Ala and Thr37Ala all increased CGRP potency. CGRP specific binding was abolished at Leu41Ala, Ala44Leu, Cys48Ala and Tyr49Ala. There was significant impairment of cell surface expression of Gln45Ala, Cys48Ala and Tyr49Ala. Cys48 takes part in a highly conserved disulfide bond and is probably needed for correct folding of CLR. The model suggests that Gln45 and Tyr49 mediate their effects by interacting with RAMP1 whereas Leu41 and Ala44 are likely to be involved in binding CGRP. Ile32, Gly35 and Thr37 form a separate cluster of residues which modulate CGRP binding. The results from this study may be applicable to other family B GPCRs which can associate with RAMPs.
Keywords: CLR; CGRP; RAMP1; Family B GPCRs; Molecular modelling; Alanine scan;
Two short d-Phe-containing cysteine-free conopeptides from Conus marmoreus by Ling Zhang; Xiaoxia Shao; Chengwu Chi; Chunguang Wang (177-179).
The cysteine-free conopeptides are naturally occurring components of the venom of cone snails and have been relatively less investigated than the cysteine-containing conopeptides. In this work, we used thiol-exchange chromatography to isolate cysteine-free conopeptides from the venom of Conus marmoreus. The full-length previously reported conomarphin and two novel shortened forms of it were found in the cysteine-free conopeptide fraction. The two shortened conomarphins also contain a d-Phe and a hydroxylated proline residue, suggesting complex post-translational modifications of these conopeptides. In particular, the shorter conomarphin fragment shares significant sequence similarity with conophans. This work demonstrated that thiol-exchange chromatography is a useful approach to isolate cysteine-free conopeptides from venom cocktail. The newly identified conopeptides further revealed the complexity of post-translational processing and the high diversity of conotoxins.
NPR-C is expressed in the cholinergic and dopaminergic amacrine cells in the rat retina by Essam Mohamed Abdelalim; Ikuo Tooyama (180-183).
Natriuretic peptide receptor C (NPR-C) is known to bind all natriuretic peptides with similar affinity. Given their biological role it is interesting that natriuretic peptides and their activated guanylate cyclases (NPR-A and NPR-B) are expressed in retinal amacrine cells. The purpose of this study is to examine the presence of NPR-C in the rat retina and its relationship to cholinergic and dopaminergic amacrine cells using immunofluorescence techniques. NPR-C immunoreactivity was found in several layers of the retina including the ganglion cell layer (GCL), inner nuclear layer (INL), outer plexiform layer (OPL), and inner segments of photoreceptors (IS). Immunofluorescence double-labeling showed the co-localization of NPR-C with tyrosine hydroxylase, a marker of dopaminergic cells, and with choline acetyltransferase (ChAT), a marker of cholinergic cells. These data suggest that natriuretic peptides may play a role in maintaining the retinal functions via interaction with NPR-C.
Keywords: NPR-C; Choline acetyltransferase; Tyrosine hydroxylase; Retina; ACs; Immunofluorescence;
Recent developments in liposomes, microparticles and nanoparticles for protein and peptide drug delivery by Mei Lin Tan; P.F.M. Choong; C.R. Dass (184-193).
Proteins and peptides are increasingly recognized as potential leads for the development of new therapeutics for a variety of human ailments. Due to their relatively specific mode of action, proteins and peptides can be administered at relatively low doses for therapeutic effects. As natural biological products, these low doses reduce the risk otherwise caused by other small molecular drugs or larger charged molecules. Unfortunately, their therapeutic potential and clinical application is frequently hampered by various obstacles to their successful delivery. This review discusses the recent developments in the fields of liposome, microparticle and nanoparticle pertinent to protein and peptide delivery covering those systems tested and/or validated in vivo.
Keywords: Drug delivery systems; Proteins; Peptides; Therapy;