Peptides (v.29, #11)
Editorial Board (CO2).
Identification of a novel N-cadherin antagonist by Emmanuelle Devemy; Orest W. Blaschuk (1853-1861).
The cell adhesion molecule, N-cadherin plays a pivotal role in many biological and disease processes. Drugs that modulate N-cadherin function should therefore be useful therapeutic agents. We have used phage display technology to identify amino acid sequences capable of binding to N-cadherin. All of these sequences harbor a Trp residue in the second position from the N-terminus. A synthetic linear peptide containing one of these sequences, H-SWTLYTPSGQSK-NH2 was found to bind a chimeric protein composed of the N-cadherin ectodomain fused to the immunoglobulin G1 Fc fragment with an affinity (K D) of 10.7 μM, as determined by surface plasmon resonance. It also blocked the aggregation of beads coated with this chimeric protein. Furthermore, this peptide disrupted adhesion and tube formation by N-cadherin-expressing human umbilical vein endothelial cells in vitro. These observations suggest that N-cadherin antagonists have the potential of serving as anti-angiogenic agents. The peptide, H-SWTLYTPSGQSK-NH2 should prove useful for studies designed to evaluate N-cadherin function in various biological processes.
Keywords: N-cadherin antagonist; Peptide; Phage display; Endothelial cell;
A biologically active rhIGF-1 fusion accumulated in transgenic rice seeds can reduce blood glucose in diabetic mice via oral delivery by Tingting Xie; Qingchuan Qiu; Wei Zhang; Tingting Ning; Wei Yang; Congyi Zheng; Chuan Wang; Yingguo Zhu; Daichang Yang (1862-1870).
Human insulin-like growth factor 1(hIGF-1) is essential for cell proliferation and used therapeutically in treating various diseases including diabetes mellitus. Here, we present that a recombinant hIGF-1(rhIGF-1) was expressed fused with the C-terminus of a rice luminal binding protein and accumulated highly in rice seeds, reaching 6.8 ± 0.5% of total seed protein. The rhIGF-1 fusion was demonstrated to possess biological activity to stimulate cell proliferation. Importantly, the unprocessed transgenic seeds could significantly increase plasma rhIGF-1 level and reduce blood glucose of diabetic mice via oral delivery. Further studies suggested that transgenic seeds reduced blood glucose of diabetic mice by enhancing islet cells survival and increasing insulin secretion rather than increasing insulin sensitivity. These results indicated the potential of the novel fusion expression system in production and oral delivery of biologically active small peptides for diseases.
Keywords: Human insulin-like growth factor 1; Transgenic rice seeds; Blood glucose; Hyperglycemia; Diabetes mellitus;
First isolation of a novel thermostable antifungal peptide secreted by Aspergillus clavatus by Houda Skouri-Gargouri; Ali Gargouri (1871-1877).
A novel antifungal peptide produced by an indigenous fungal strain (VR) of Aspergillus clavatus was purified. The antifungal peptide was enriched in the supernatant after heat treatment at 70 °C. The thermostable character was exploited in the first purification step, as purified peptide was obtained after ultrafiltration and reverse phase-HPLC on C18 column application. The purified peptide named “AcAFP” for A. clavatus antifungal peptide, has molecular mass of 5773 Da determined by MALDI-ToF spectrometry. The N-terminal sequence showed a notable identity to the limited family of antifungal peptides produced by ascomycetes fungi. The AcAFP activity remains intact even after heat treatment at 100 °C for 1 h confirming its thermostability. It exhibits a strong inhibitory activity against mycelial growth of several serious human and plant pathogenic fungi: Fusariuym oxysporum, Fusarium solani, Aspergillus niger, Botrytis cinerea, Alternaria solani, whereas AcAFP did not affect yeast and bacterial growth.
Keywords: Aspergillus clavatus; Antifungal peptide; Protein purification; Thermostability;
Scope and limitations of the designer proline-rich antibacterial peptide dimer, A3-APO, alone or in synergy with conventional antibiotics by Marco Cassone; Paraskevi Vogiatzi; Raffaele La Montagna; Vanessa De Olivier Inacio; Predrag Cudic; John D. Wade; Laszlo Otvos (1878-1886).
The proline-rich antimicrobial peptide dimer, A3-APO, was designed based on a statistical analysis of native antibacterial peptide and protein sequences. Analysis of a series of structural analogs failed to identify any single or multiple amino acid modification or architectural changes that would significantly improve its potential as a clinical therapeutic. However, a single chain Chex1-Arg20 version, a natural in vivo metabolite, showed a 2 to 8-fold increase in activity against test Enterobacteriaceae strains. In addition to bacterial species close to Escherichia coli in phylogeny, A3-APO analogs were able to effectively kill Pseudomonas aeruginosa and Staphylococcus saprophyticus. Antibacterial efficacy analysis together with biochemical experiments provided further evidence for a multiple mode of action of A3-APO that includes binding and inhibition of the bacterial heat shock protein DnaK. Through inactivating of resistance enzymes, A3-APO was able to recover the lost activity of conventional antibiotics including chloramphenicol, β-lactams, sulfonamides or trimethoprim against multidrug resistant strains with partial or full synergy. However, the synergy appeared to be individual strain and small molecule drug combination-dependent.
Keywords: A3-APO; Synergy; Antimicrobial peptide; DnaK;
Antimicrobial peptides from the venoms of Vespa bicolor Fabricius by Wenhu Chen; Xinbo Yang; Xiaolong Yang; Lei Zhai; Zekuan Lu; Jingze Liu; Haining Yu (1887-1892).
Hornets possess highly toxic venoms, which are rich in toxins, enzymes and biologically active peptides. Many bioactive substances have been identified from wasp venoms. Vespa mastoparan (MP-VBs) and Vespa chemotatic peptide presenting antimicrobial action (VESP-VBs) were purified and characterized from the venom of the wasp, Vespa bicolor Fabricius. The precursors encoding VESP-VBs and MP-VBs were cloned from the cDNA library of the venomous glands. Analyzed by FAB-MS, the amino acid sequence and molecular mass for VESP-VB1 were FMPIIGRLMSGSL and 1420.6, for MP-VB1 were INMKASAAVAKKLL and 1456.5, respectively. The primary structures of these peptides are homologous to those of chemotactic peptides and mastoparans isolated from other vespid venoms. These peptides showed strong antimicrobial activities against bacteria and fungi and induced mast cell degranulation, but displayed almost no hemolytic activity towards human blood red cells.
Keywords: Wasp venom; Vespa bicolor; Chemotactic peptide; Mastoparan; Antimicrobial peptide;
INN-toxin, a highly lethal peptide from the venom of Indian cobra (Naja naja) venom—Isolation, characterization and pharmacological actions by K.C. Ponnappa; Pushpa Saviour; N.B. Ramachandra; R. Manjunatha Kini; T. Veerabasappa Gowda (1893-1900).
A novel toxic polypeptide, INN-toxin, is purified from the venom of Naja naja using combination of gel-permeation and ion-exchange chromatography. It has a molecular mass of 6951.6 Da as determined by MALDI-TOF/MS and the N-terminal sequence of LKXNKLVPLF. It showed both neurotoxic as well as cytotoxic activities. INN-toxin is lethal to mice with a LD50 of 1.2 mg/kg body weight. IgY raised in chicks against basic peptide pool neutralized the toxicity of INN-toxin. INN-toxin did not inhibit cholinesterase activity. It is toxic to Ehrlich ascites tumor (EAT) cells, but it is not toxic to leukocyte culture. The toxin appears to be specific in its mode of action. Interaction of N-bromosuccinamide (NBS) with the peptide resulted in the modification of tryptophan residues and loss of lethal toxicity of INN-toxin.
Keywords: Postsynaptic neurotoxins; Cytotoxins; Ehrlich ascites tumor cells; Leukocytes; IgY; N-bromosuccinamide; Naja naja;
An insecticidal peptide from the theraposid Brachypelma smithi spider venom reveals common molecular features among spider species from different genera by Gerardo Corzo; Elia Diego-García; Herlinda Clement; Steve Peigneur; George Odell; Jan Tytgat; Lourival D. Possani; Alejandro Alagón (1901-1908).
The soluble venom of the Mexican theraposid spider Brachypelma smithi was screened for insecticidal peptides based on toxicity to house crickets. An insecticidal peptide, named Bs1 (which stands for Brachypelma smithi toxin 1) was obtained in homogeneous form after the soluble venom was fractionated using reverse-phase and cation-exchange chromatography. It contains 41 amino acids cross-linked by three disulfide bridges. Its sequence is similar to an insecticidal peptide isolated from the theraposid spider Ornithoctonus huwena from China, and another from the hexathelid spider Macrothele gigas from Japan, indicating that they are phylogenetically related. A cDNA library was prepared from the venomous glands of B. smithi and the gene that code for Bs1 was cloned. Sequence analysis of the nucleotides of Bs1 showed similarities to that of the hexathelid spider from Japan proving additional evidence for close genetic relationship between these spider peptides. The mRNAs of these toxins code for signal peptides that are processed at the segment rich in acidic and basic residues. Their C-terminal amino acids are amidated. However, they contain only a glycine residue at the most C-terminal position, without the presence of additional basic amino acid residues, normally required for post-translation processing of other toxins reported in the literature. The possible mechanism of action of Bs1 was investigated using several ion channels as putative receptors. Bs1 had minor, but significant effects on the Para/tipE insect ion channel, which could indirectly correlate with the observed lethal activity to crickets.
Keywords: Insecticidal; Peptide; cDNA; Spider toxin; Theraposid;
Identification and cardiotropic actions of brain/gut-derived tachykinin-related peptides (TRPs) from the American lobster Homarus americanus by Andrew E. Christie; Christopher R. Cashman; Jake S. Stevens; Christine M. Smith; Kristin M. Beale; Elizabeth A. Stemmler; Spencer J. Greenwood; David W. Towle; Patsy S. Dickinson (1909-1918).
Two tachykinin-related peptides (TRPs) are known in decapods, APSGFLGMRamide and TPSGFLGMRamide. The former peptide appears to be ubiquitously conserved in members of this taxon, while the latter has been suggested to be a genus (Cancer)- or infraorder (Brachyura)-specific isoform. Here, we characterized a cDNA from the American lobster Homarus americanus (infraorder Astacidea) that encodes both TRPs: six copies of APSGFLGMRamide and one of TPSGFLGMRamide. Mass spectral analyses of the H. americanus supraoesophageal ganglion (brain) and commissural ganglia confirmed the presence of both peptides in these neural tissues; both isoforms were also detected in the midgut. Physiological experiments showed that both APSGFLGMRamide and TPSGFLGMRamide are cardioactive in H. americanus, eliciting identical increases in both heart contraction frequency and amplitude. Collectively, our data represent the first genetic confirmation of TRPs in H. americanus and of TPSGFLGMRamide in any species, demonstrate that TPSGFLGMRamide is not restricted to brachyurans, and show that both this peptide and APSGFLGMRamide are brain-gut isoforms, the first peptides thus far confirmed to possess this dual tissue distribution in H. americanus. Our data also suggest a possible role for TRPs in modulating the output of the lobster heart.
Keywords: Homarus americanus; American lobster; Tachykinin-related peptide; cDNA; APSGFLGMRamide; TPSGFLGMRamide; Brain-gut peptide; Expressed sequence tag (EST); Matrix assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS); Heart; Cardioactive peptide;
Characterization of a conformationally sensitive TOAC spin-labeled substance P by Aaron M. Shafer; Clovis R. Nakaie; Xavier Deupi; Vicki J. Bennett; John C. Voss (1919-1929).
To probe the binding of a peptide agonist to a G-protein coupled receptor in native membranes, the spin-labeled amino acid analogue 4-amino-4-carboxy-2,2,6,6-tetramethylpiperidino-1-oxyl (TOAC) was substituted at either position 4 or 9 within the substance P peptide (RPKPQQFFGLM-NH2), a potent agonist of the neurokinin-1 receptor. The affinity of the 4-TOAC analog is comparable to the native peptide while the affinity of the 9-TOAC derivative is ∼250-fold lower. Both peptides activate receptor signaling, though the potency of the 9-TOAC peptide is substantially lower. The utility of these modified ligands for reporting conformational dynamics during the neurokinin-1 receptor activation was explored using EPR spectroscopy, which can determine the real-time dynamics of the TOAC nitroxides in solution. While the binding of both the 4-TOAC substance P and 9-TOAC substance P peptides to isolated cell membranes containing the neurokinin-1 receptor is detected, a bound signal for the 9-TOAC peptide is only obtained under conditions that maintain the receptor in its high-affinity binding state. In contrast, 4-TOAC substance P binding is observed by solution EPR under both low- and high-affinity receptor states, with evidence of a more strongly immobilized peptide in the presence of GDP. In addition, to better understand the conformational consequences of TOAC substitution into substance P as it relates to receptor binding and activation, atomistic models for both the 4- and 9-TOAC versions of the peptide were constructed, and the molecular dynamics calculated via simulated annealing to explore the influence of the TOAC substitutions on backbone structure.
Keywords: Substance P; GPCR; TOAC spin label; EPR; ESR;
Corticotropin-releasing hormone mediates α-melanocyte-stimulating hormone-induced anorexigenic action in goldfish by Kouhei Matsuda; Kenji Kojima; Sei-Ichi Shimakura; Kohei Wada; Keisuke Maruyama; Minoru Uchiyama; Sakae Kikuyama; Seiji Shioda (1930-1936).
α-Melanocyte-stimulating hormone (α-MSH) and corticotropin-releasing hormone (CRH) both suppress food intake, and the α-MSH- or CRH-signaling pathway has possible potency to mediate anorexigenic actions induced by most other neuropeptides in goldfish. Therefore, using specific receptor antagonists, we examined whether the anorexigenic actions of α-MSH and CRH mutually interact. The inhibitory effect of ICV injection of the α-MSH agonist, melanotan II (MT II), on food intake was abolished by treatment with a CRH 1/2 receptor antagonist, α-helical CRH(9–41), whereas the anorexigenic action of ICV-injected CRH was not affected by treatment with a melanocortin 4 receptor antagonist, HS024. This led us to investigate whether α-MSH-containing neurons in the goldfish brain have direct inputs to CRH-containing neurons, using confocal laser scanning microscopy. α-MSH- and CRH-like immunoreactivities were distributed throughout the brain, especially in the diencephalon. α-MSH-containing nerve fibers or endings lay in close apposition to CRH-containing neurons in a region of the hypothalamus, the nucleus posterioris periventricularis (NPPv). These results indicate that, in goldfish, α-MSH-induced anorexigenic action is mediated by the CRH-signaling pathway, and that CRH plays a crucial role in the regulation of feeding behavior as an integrated anorexigenic neuropeptide in this species.
Keywords: Goldfish; α-MSH; CRH; Anorexigenic action; Antagonists; Neuronal interaction;
Involvement of CRH receptors in urocortin-induced hyperthermia by G. Telegdy; A. Adamik (1937-1942).
The actions of individual corticotropin-releasing hormone (CRH) receptor (CRHR1 and CRHR2) were studied on the hyperthermia caused by urocortin 1, urocortin 2 and urocortin 3 in rats. Urocortin 1, urocortin 2 or urocortin 3 was injected into the lateral brain ventricle in conscious rats and the colon temperature was measured at different times following injection, up to 6 h. In order to study the possible role of CRH receptors, the animals were treated with a urocortins together with the urocortin receptor inhibitors CRF 9–41, antalarmin and astressin 2B to influence the action of urocortins in initiating hyperthermia. Urocortin 1 at a dose of 2 μg caused an increase in colon temperature, maximal action being observed in body temperature at 3 h. CRH 9–41 and antalarmin, CRHR1 receptor antagonists, prevented the urocortin-induced increase in colon temperature while astressin 2B (CRHR2 receptor antagonist) was ineffective. Urocortin 2 at a dose of 2 μg showed a byphasic action in increase in colon temperature having the first peak between 30 min and 1 h and the second peak at 4 h following treatment. CRF (9–41) and antalarmin was ineffective while astressin 2B fully blocked the action of urocortin 2. Urocortin 3 in a dose of l μg increased colon temperature; the maximal effect was observed at 2 h. CRF (9–41) and antalarmin was ineffective while astressin 2B fully blocked the action of urocortin 3. The results demonstrated that urocortin 1, 2 or 3 when injected into the lateral brain ventricle caused increases in body temperature is mediated by urocortin receptors. The action of urocortin 1 is mediated by CRHR1 receptor, while in the action of urocortin 2 and urocortin 3 CRHR2 receptor is involved.
Keywords: Urocortins; Hyperthermia; CRH receptors; Fever;
Blood pressure regulation and cardiac autonomic control in mice overexpressing α- and γ-melanocyte stimulating hormone by Petteri Rinne; Janne Harjunpää; Mika Scheinin; Eriika Savontaus (1943-1952).
Melanocyte stimulating hormones (MSH) derived from pro-opiomelanocortin have been demonstrated to participate in the central regulation of cardiovascular functions. The aim of the present study was to elucidate the chronic effects of increased melanocortin activation on blood pressure regulation and autonomic nervous system function. We adapted telemetry to transgenic mice overexpressing α- and γ-MSH and measured blood pressure, heart rate and locomotor activity, and analyzed heart rate variability (HRV) in the frequency-domain as well as baroreflex function by the sequence technique. Transgenic (MSH-OE) mice had increased systolic blood pressure but their heart rate was similar to wild-type (WT) controls. The 24-h mean of systolic blood pressure was 132 ± 7 mmHg in MSH-OE and 113 ± 4 mmHg in WT mice. Locomotor activity was decreased in the MSH-OE mice. Furthermore, MSH-OE mice showed slower adaptation to mild environmental stress in terms of blood pressure changes. The low frequency (LF) power of HRV tended to be higher in MSH-OE mice compared to WT mice, without a difference in overall variability. The assessment of baroreflex function indicated enhanced baroreflex effectiveness and more frequent baroreflex operations in MSH-OE mice. Baseline heart rate, increased LF power of HRV and increased baroreflex activity may all reflect maintenance of baroreflex integrity and an increase in cardiac vagal activity to counteract the increased blood pressure. These results provide new evidence that long-term activation of the melanocortin system elevates blood pressure without increasing heart rate.
Keywords: Melanocortins; Telemetry; Autonomic nervous system; Heart rate variability; Baroreflex sensitivity;
Anterior pituitary pyroglutamyl peptidase II activity controls TRH-induced prolactin release by Raymundo Cruz; Miguel Angel Vargas; Rosa Maria Uribe; Isel Pascual; Ivan Lazcano; Athanasios Yiotakis; Magdalini Matziari; Patricia Joseph-Bravo; Jean-Louis Charli (1953-1964).
Ecto-peptidases modulate the action of peptides in the extracellular space. The relationship between peptide receptor and ecto-peptidase localization, and the physiological role of peptidases is poorly understood. Current evidence suggests that pyroglutamyl peptidase II (PPII) inactivates neuronally released thyrotropin-releasing hormone (TRH). The impact of PPII localization in the anterior pituitary on the endocrine activities of TRH is unknown. We have studied whether PPII influences TRH signaling in anterior pituitary cells in primary culture. In situ hybridization (ISH) experiments showed that PPII mRNA was expressed only in 5–6% of cells. ISH for PPII mRNA combined with immunocytochemistry for prolactin, β-thyrotropin, or growth hormone, showed that 66% of PPII mRNA expressing cells are lactotrophs, 34% somatotrophs while none are thyrotrophs. PPII activity was reduced using a specific phosphorothioate antisense oligodeoxynucleotide or inhibitors. Compared with mock or scrambled oligodeoxynucleotide-treated controls, knock-down of PPII expression by antisense targeting increased TRH-induced release of prolactin, but not of thyrotropin. Similar data were obtained with either a transition-state or a tight binding inhibitor. These results demonstrate that PPII expression in lactotrophs coincides with its ability to control prolactin release. It may play a specialized role in TRH signaling in the anterior pituitary. Anterior pituitary ecto-peptidases may fulfill unique functions associated with their restricted cell-specific expression.
Keywords: Thyrotropin; Growth hormone; TRH; Anterior pituitary; Prolactin; Pyroglutamyl peptidase;
Matrix metalloprotease selective peptide substrates cleavage within hydrogel matrices for cancer chemotherapy activation by Jovita R. Tauro; Bao-Shiang Lee; Syed S. Lateef; Richard A. Gemeinhart (1965-1973).
To utilize biologic mechanisms to elicit controlled release in response to disease, protease-sensitive devices have been created. Hydrogels were created with pendant peptide–drug complexes. For the matrix metalloproteases (MMPs) examined, a length of six amino acids greatly improved the specificity of the peptide (k cat/K m ∼ 2.4 ± 0.1 × 104 M−1 s−1) over shorter sequences (k cat/K m ∼ 4.4 ± 0.2 × 102 M−1 s−1). The peptides did not exhibit anti-proliferative effects upon cancer cells, and peptide–platinum complexes showed similar anti-proliferative effects upon the cancer cells compared to the free platinum drugs. Once the peptide–drug complex was incorporated into the hydrogels, the release was dependent upon the presence of MMP in the solution with approximately 35% of platinum released from hydrogels in the presence of MMP and only 10% without MMP in the week examined. The released drug exhibited the expected anti-proliferative activity over several days of incubation. The MMP selective drug delivery holds much potential for treatment of cancer and other diseases.
Keywords: Prodrug; Cancer; Hydrogel; Drug targeting; Drug activation;
Positive and negative modulation of peptidases by pro-inflammatory cytokines by Antonella Cavazza; Mario Marini; Giulio C. Spagnoli; L. Giorgio Roda (1974-1981).
The capacity of pro-inflammatory cytokines to modulate proteolysis was analyzed by liquid chromatography using human fibroblasts as cell model and enzyme source, and the immunodominant epitope gp100280–288 (YLEPGPVTA) as substrate. The measurements made after fibroblast pre-incubation with either IL-1, TNF, or IL-6 plus its soluble receptors have been compared with those made with un-stimulated fibroblasts. The results obtained suggest an uneven association of cytokine treatment with substrate degradation, and with a prevailingly positive – but also negative – association with release of smaller peptides and free amino acids. Data obtained by separately measuring these two groups of by-products indicate that, after IL-1 cell pre-treatment, the velocity of formation of both groups of by-products increased, resulting in a net increase of substrate degradation. After TNF and IL-6 pre-treatment, the increase of one group was compensated by a decrease of the other group; specifically, the compensation was only partial for TNF, and overall substrate hydrolysis increased. In the case of IL-6, the increase of free amino acids was almost exactly compensated by a reduction of peptidic by-products, resulting in a negligible increase of substrate hydrolysis. In addition, the existence of reaction time-related modifications in the apparent velocity of substrate degradation and formation of by-products, allows hypothesizing different effects of cytokines on the enzymes degrading the substrate with different time constants. Taken together, these data can be interpreted as indicating different, positive and negative, effects of the three cytokines on the individual enzymes expressed by fibroblasts and capable of degrading peptidic substrates.
Keywords: Cytokines; Proteolysis; Human fibroblasts;
The multiple T-maze in vivo testing of the neuroprotective effect of humanin analogues by Gabriela Kunešová; Jan Hlaváček; Jiří Patočka; Alexandra Evangelou; Christos Zikos; Dimitra Benaki; Maria Paravatou-Petsotas; Maria Pelecanou; Evangelia Livaniou; Jirina Slaninova (1982-1987).
Humanin (HN) and its analogues have been shown to protect cells against death induced by various Alzheimer's disease (AD) genes and amyloid-β-peptides in vitro; the analogues [Gly14]-HN and colivelin have also been shown to be potent in reversing learning and memory impairment induced by scopolamine or quinuclidinyl benzilate (QNB) in mice or rats in vivo using the Y-maze or multiple T-maze tests. This paper describes the activity of new peptides of the HN family, after i.p. administration, on QNB-induced impairment of spatial memory in the multiple T-maze test in rats. The following peptides have been studied: HN analogues truncated either on the C- or N-terminus, or analogues having a tert-Leu in place of Leu in the central part of the molecule, the active HN core PAGASRLLLLTGEIDLP (RG-PAGA) and its analogues having three or five leucines instead of four, and finally the recently described hybrid peptide colivelin (i.e. a peptide having the activity-dependent neurotrophic factor SALLRSIPA attached to the N-terminus of the active RG-PAGA) and its des-Leu- and plus-Leu-analogues. While the truncated analogues and most of the tert-Leu containing analogues were devoid of activity, the analogues of the RG-PAGA were active, i.e. they reversed the impairment of spatial memory irrespective of the number of Leu present in their sequence. The highest activity was shown by colivelin and its des-Leu-analogue. These results demonstrate the potential of HN analogues in the modulation of the cholinergic system, which plays an important role in the cognitive deficits associated with AD and other neurodegenerative diseases.
Keywords: Humanin-like peptides; Colivelin; Rat; Memory; Alzheimer's disease; Quinuclidinyl benzilate;
Cryptic peptide derived from the rat neuropeptide FF precursor affects G-proteins linked to opioid receptors in the rat brain by Piotr Suder; Dominika Nawrat; Adam Bielawski; Agnieszka Zelek-Molik; Hana Raoof; Tomasz Dylag; Jolanta Kotlinska; Irena Nalepa; Jerzy Silberring (1988-1993).
Recently, we reported the discovery of a novel amino acid sequence derived from the NPFF precursor NAWGPWSKEQLSPQA, which blocked the expression of conditioned place preference induced by morphine and reversed the antinociceptive activity of morphine (5 mg/kg, s.c.) in the tail-immersion test in rats. Here, we name it as NPNA (Neuropeptide NA from its flanking amino acid residues). The synthetic peptide influenced the expression of mRNA coding for Gα(i1), (i2), and (i3) subunits. The results provide further evidence that yet another bioactive sequence might be present within the NPFF precursor.
Keywords: G-proteins; Opioids; GPCR; NPFF; mRNA; Metabolism;
Central administration of the RFamide peptides, QRFP-26 and QRFP-43, increases high fat food intake in rats by Stefany D. Primeaux; Christine Blackmon; Maria J. Barnes; H. Douglas Braymer; George A. Bray (1994-2000).
Pyrogultamylated arginine-phenylalanineamide peptide (QRFP) is strongly conserved across species and is a member of the family of RFamide-related peptides, with the motif Arg-Phe-NH2 at the C-terminal end. The precursor peptide for QRFP generates a 26-amino acid peptide (QRFP-26) and a 43-amino acid peptide (QRFP-43), both of which bind to the G protein-coupled receptor, GPR103. Recently, QRFP has been characterized in rats, mice and humans and has been reported to have orexigenic properties. In rodents, prepro-QRFP mRNA is expressed in localized regions of the mediobasal hypothalamus, a region implicated in feeding behavior. Increased intake of a high fat diet contributes to increased weight gain and obesity. Therefore, the current experiments investigated the effects of QRFP administration in rats and the effects of a high fat diet on prepro-QRFP mRNA and GPR103 receptor mRNA levels. Intracerebroventricular administration of QRFP-26 (3.0 nM, 5.0 nM) and QRFP-43 (1.0 nM, 3.0 nM) dose-dependently increased 1 h, 2 h, and 4 h cumulative intake of high fat (55% fat), but not low fat (10% fat) diet. In Experiment 2, hypothalamic prepro-QRFP mRNA levels and GPR103 receptor mRNA levels were measured in rats fed a high fat or a low fat diet for 21 days. Prepro-QRFP mRNA was significantly increased in the ventromedial nucleus/arcuate nucleus of the hypothalamus of rats fed a high fat diet compared to those fed a low fat diet, while GPR103 mRNA levels were unchanged. These findings suggest that QRFP is a regulator of dietary fat intake and is influenced by the intake of a high fat diet.
Keywords: Prepro-QRFP mRNA; GPR103 receptor; High fat diet; Real-time PCR;
Acute and chronic responses associated with adrenomedullin administration in experimental colitis by E. Talero; S. Sánchez-Fidalgo; C. Alarcón de la Lastra; M. Illanes; J.R. Calvo; V. Motilva (2001-2012).
Adrenomedullin (AM) is a 52 amino acid peptide and member of the calcitonin gene-related peptide (CGRP) super family. Given that AM has emerged as a potential immuno-regulatory and anti-inflammatory agent in various experimental models, this study has deepened into its possible therapeutic effect in intestinal inflammation analyzing the responses in both acute and chronic (14 and 21 days) phases of TNBS-induced colitis in rats. In the acute model, AM treatment reduced the incidence of diarrhea and the severity of colonic damage, and improved the survival rate at the three doses assayed (50, 100, and 200 ng/kg animal). AM administration was able to reduce the early production of TNF-α and collaborated to maintaining basal levels of IFN-γ and IL-10. In the chronic studies the peptide attenuated the extent of the damage with lesser incidence of weight loss and diarrhea (50 and 100 ng/kg animal). Cellular neutrophil infiltration, with the subsequent increase in myeloperoxidase (MPO) levels caused by TNBS, was reduced after chronic AM administration. The peptide played a role in the evolution of Th1/Th2 cytokines balance and chronic disease recuperation: levels of proinflammatory TNF-α and IFN-γ decreased and anti-inflammatory IL-10 increased significantly. Cyclooxygenase-2 (COX-2) and nitric oxide synthase (iNOS) protein expression were not modified by AM administration, although a reduction of nitric oxide (NO) production could be detected in the chronic model. These results support a role of AM as an anti-inflammatory factor with beneficial effects in intestinal inflammatory colitis.
Keywords: Adrenomedullin; Regulatory peptides; Intestinal inflammation; Colitis; IBD;
Adrenomedullin stimulates angiogenic response in cultured human vascular endothelial cells: Involvement of the vascular endothelial growth factor receptor 2 by Diego Guidolin; Giovanna Albertin; Raffaella Spinazzi; Elisa Sorato; Alessandra Mascarin; Donatella Cavallo; Michele Antonello; Domenico Ribatti (2013-2023).
In recent years, evidence has accumulated that many endogenous peptides play an important regulatory role in angiogenesis by modulating endothelial cell behavior. Adrenomedullin (AM), one such factor, was previously shown to exert a clearcut proangiogenic effect in vitro when tested on specialized human endothelial cells, such as HUVECs and immortalized endothelial cell lines. In the present study we used normal adult vascular endothelial cells isolated from human saphenous vein to analyze in vitro the role of AM, related to both early (increased cell proliferation) and late (differentiation and self-organization into capillary-like structures) angiogenic events and their relationship with the vascular endothelial growth factor (VEGF) signaling cascade. The results indicated that also in this endothelial cell phenotype AM promoted cell proliferation and differentiation into cord-like structures. These actions resulted specific and were mediated by the binding of AM to its AM1 (CRLR/RAMP2) receptor. Neither the administration of a VEGF receptor 2 (VEGFR-2) antagonist nor the downregulation of VEGF production by gene silencing were able to suppress the proangiogenic effect of AM. However, when the experiments were performed in the presence of SU5416 (a selective inhibitor of the VEGFR-2 receptor at the level of the intra-cellular tyrosine kinase domain) the proangiogenic effect of AM was abolished. This result suggests that in vascular endothelial cells the binding of AM to its AM1 receptor could trigger a transactivation of the VEGFR-2 receptor, leading to a signaling cascade inducing proangiogenic events in the cells.
Keywords: Adrenomedullin; Angiogenesis; Human vascular endothelial cells; Receptor transactivation; VEGF; VEGF receptors;
Regulation of steroidogenesis by atrial natriuretic peptide (ANP) in the rat testis: Differential involvement of GC-A and C receptors by Virgínia Mara Pereira; Amilton P. Raposo Costa; Alzira Amelia Martins Rosa-e-Silva; Maria Aparecida Ribeiro Vieira; Adelina Martha dos Reis (2024-2032).
Previous studies have established a stimulatory effect of natriuretic peptides (NP) on testosterone production in mouse Leydig cells as intense as that of LH. Chronic administration of ANP in mice, on the other side, reduced testosterone levels. So, the understanding of the role of ANP on testicular steroidogenesis has been impaired by discrepant findings. The aim of the present study was to clarify the physiological role of ANP in the rat testis steroidogenesis using a model that preserves the interactions between testis cells and a medium devoid of any circulating factors that could interfere with testosterone production. First, ANP was immunolocalized in the interstitial compartment of the rat testis, mainly in Leydig cells. We also determined the presence of ANP and both GC-A (guanylyl cyclase A) and C receptors by real-time PCR in testis. Perfusion in vitro of testis with ANP (1 and 3 × 10−7 M) stimulated testosterone production in a time- and dose-dependent manner. On the other side, testosterone secretion induced by LH was blunted by ANP. Similar effect was obtained using the specific C receptor ligand, cANF, indicating the involvement of C receptor in such response. In conclusion, ANP stimulated testosterone production in the rat testis perfused in vitro but decreased testosterone production LH-induced, effect that seems to involve C receptor. To this extent, our results suggest the existence of a local and complex peptidergic system in the rat testis, involving ANP and its receptors that could importantly modulate the androgen biosynthesis.
Keywords: Atrial natriuretic peptide; Steroidogenesis; Rat testis; Testosterone; C receptor;
Crosstalk between the signaling pathways triggered by angiotensin II and adenosine in the renal proximal tubules: Implications for modulation of Na+-ATPase activity by C.P. Gomes; L.R. Leão-Ferreira; A.A.S. Pinheiro; E. Gomes-Quintana; M. Wengert; A.G. Lopes; C. Caruso-Neves (2033-2038).
We have previously demonstrated that adenosine (Ado) reverses the stimulatory effect of angiotensin II (Ang II) on Na+-ATPase activity via the A2A receptor. In this work, the molecular mechanism involved in Ado-induced shutdown in the signaling pathway triggered by 10−8 M Ang II was investigated. It was observed that: (1) both 10−12 M PMA (a PKC activator) and 5 × 10−8 M U73122 (an inhibitor of PI-PLCβ) prevent the reversion effect induced by 10−6 M Ado (only observed in the presence of 10−6 M DPCPX (an A1 receptor antagonist)) on Ang II-stimulated Na+-ATPase and PKC activities; (2) Ang II-stimulated PKC activity was reversed by 10−6 M forskolin (an adenylyl cyclase activator) or 10−8 M PKA inhibitory peptide and 10−8 M DMPX (an A2 receptor-selective antagonist). Considering that PMA prevents the inhibitory effect of Ado on Ang II-stimulated Na+-ATPase and PKC activities, it is likely that the PMA-induced effect, i.e. PKC activation, is downstream of the target for Ado-induced reversion of Ang II stimulation of Na+-ATPase activity. We investigated the hypothesis that PI-PLCβ could be the target for Ado-induced PKA activation. Our data demonstrate that Ang II-stimulated PI-PLCβ activity was reversed by Ado or 10−7 M cAMP; the reversibility of the Ado-induced effect was prevented by either DMPX or PKA inhibitory peptide. These data demonstrate that Ado-induced PKA activation reduces Ang II-induced stimulation of PI-PLCβ.
Keywords: Na+-ATPase; Adenosine; Angiotensin II; PLC; PKA; Proximal tubule;
Endothelin-3-dependent pulmonary vasoconstriction in monocrotaline-induced pulmonary arterial hypertension by Stéphanie Sauvageau; Eric Thorin; Louis Villeneuve; Jocelyn Dupuis (2039-2045).
Blockade of the endothelin (ET) system is beneficial in pulmonary arterial hypertension (PAH). The contribution of ET-3 and its interactions with ET receptors have never been evaluated in the monocrotaline (MCT)-induced model of PAH. Vasoreactivity of pulmonary arteries was investigated; ET-3 localization was determined by confocal imaging and gene expression of prepro-ET-3 quantified using RT-PCR. ET-3 plasma levels tended to increase in PAH. ET-3 localized in the media of pulmonary arteries, where gene expression of prepro-ET-3 was reduced in PAH. ET-3 induced similar pulmonary vasoconstrictions in sham and PAH rats. In sham rats, the ETA antagonist A-147627 (10 nmol/l) significantly reduced the maximal response to ET-3 (E max 77 ± 1 to 46 ± 2%, mean ± S.E.M., P < 0.001), while the ETB antagonist A-192621 (1 μmol/l) reduced the sensitivity (EC50 21 ± 7 to 59 ± 16 nmol/l, P < 0.05) without affecting E max. The combination of both antagonists completely abolished ET-3-induced pulmonary vasoconstriction. In PAH, the ETA antagonist further reduced the maximal response to ET-3 and shifted the EC50 (E max 23 ± 2%, P < 0.001, EC50 104 ± 24 nmol/l, P < 0.05), while the ETB antagonist only shifted the EC50 (123 ± 36 nmol/l, P < 0.05) without affecting the E max. In PAH, dual ET receptor inhibition did not further reduce constriction compared to selective ETA inhibition. ET-3 significantly contributes to pulmonary vasoconstriction by activating the ETB at low concentration, and the ETA at high concentration. The increased inhibitory effect of the ETA antagonist in PAH suggests that the contribution of ETB to ET-3-induced vasoconstriction is reduced. Although ET-3 is a potent pulmonary vasoconstrictor in PAH, its potential pathophysiologic contribution remains uncertain.
Keywords: Endothelial receptors; Endothelins; Pulmonary circulation; Vasoactive agents;
Ghrelin induces proliferation in human aortic endothelial cells via ERK1/2 and PI3K/Akt activation by Fabio Rossi; Antonella Castelli; Maria J. Bianco; Cora Bertone; Marina Brama; Vittorio Santiemma (2046-2051).
The direct ghrelin (Ghr) involvement in cardiovascular (CV) system homeostasis has been suggested by the expression of its receptor in CV tissues and by evidence that ghrelin mediates CV activities in animals and in humans. Moreover, low Ghr plasma levels have been reported in pathological conditions characterized by high cardiovascular risk. In the present study, we investigated Ghr effect on proliferation of human aortic endothelial cell (HAEC) and involved transduction pathways. Our results indicate that ghrelin elicited proliferation in a dose-dependent manner (EC50 about of 5 nmol/L) in cultured HAEC, and that this effect was inhibited by the receptor antagonist (D-Lys3)-GHRP-6. Western blot experiments documented an activation of external receptor activated kinases (ERK1/2) and Akt in a dose-dependent fashion, as well as involvement of the cAMP pathway in ERK1/2 phosphorylation. Experiments conducted with appropriate pharmacological inhibitors to investigate Ghr-induced HAEC proliferation confirmed the involvement of ERK1/2 and I3P/Akt pathways, as well as the role of AMP cyclase/PKA pathway in ERK1/2 phosphorylation. Our results indicate that Ghr promotes HAEC proliferation, and thus may be a protective factor against vascular damage. The low ghrelin serum levels reported in insulin-resistant states may not be able to effectively counteract endothelial cell injury.
Keywords: Ghrelin; Endothelial cell; Aorta; Proliferation;
Gene expression profiles of adipose tissue of obese rats after central administration of neuropeptide Y-Y5 receptor antisense oligodeoxynucleotides by cDNA microarrays by Jie Qiu; Yu-hui Ni; Rong-hua Chen; Chen-bo Ji; Feng Liu; Chun-mei Zhang; Chun-lin Gao; Xiao-hui Chen; Mei-ling Tong; Xia Chi; Xiao-yu Zhou; Xi-rong Guo (2052-2060).
To investigate the gene expression profiles of adipose tissue of obese rats after central administration of neuropeptide Y-Y5 receptor antisense oligodeoxynucleotides (ODNs), Y5 receptor antisense, mismatched ODNs or vehicle was intracerebroventricularly injected and cDNA microarrays were undertaken. Central administration of NPY-Y5 receptor antisense ODNs decreased food intake, body weight and serum insulin compared with both vehicle and mismatched ODNs. The average area of adipocytes both at retroperitoneal and epididymal adipose tissue were fall in antisense group while only the weight of the retroperitoneal fat pats was reduced in antisense group. cDNA microarrays containing 18,000 genes/Ests were used to investigate gene expression of adipose tissue. Autoradiographic analysis showed that 404, 81, and 34 genes were differently expressed over twofold, threefold, and fivefold, respectively. The analysis of gene expression profiles indicated that 332 genes were up-regulated and 187 genes were down-regulated in response to Y5 receptor antisense ODNs treatment. Different clusters of genes associated with apoptosis, signal transduction, energy metabolism, lipid metabolism, etc., such as FXR1, PHLDA1, MAEA, PIK3R1, ICAM2, PITPN, CALM2, CAMK2D, PKIA, DRD2, SLC25A14, CKB, AADAC, LIPA, ACOX3, FADS1, were concerned. Analysis of differentially expressed genes will help to understand the effects of Y5 receptor antisense ODNs therapy.
Keywords: Neuropeptide Y-Y5 receptor; Antisense oligodeoxynucleotides; Adipose tissue; cDNA microarrays; Gene expression; Obesity;
Effects of triglycerides, obesity, and starvation on ghrelin transport across the blood–brain barrier by William A. Banks; Basil O. Burney; Sandra M. Robinson (2061-2065).
Human ghrelin is transported across the blood–brain barrier (BBB) of normal mice. Here, we studied the effects of triglycerides, obesity, and starvation in retired breeder mice maintained on a high fat diet, mice age-matched to the retired breeders but maintained on normal chow, and 8-week-old mice maintained on breeder chow. The rate of ghrelin transport across the BBB was studied by both the intravenous administration method of multiple-time regression analysis and by the brain perfusion method. We found that (1) obese, aged mice lost the ability to transport intravenously administered ghrelin across the BBB, resulting in an inverse relation between body weight and ghrelin BBB permeability; (2) serum triglycerides promoted transport of intravenously administered ghrelin across the BBB, whereas epinephrine had no effect; (3) fasting tended to promote ghrelin transport across the BBB as most readily shown in brain perfusion studies; (4) evidence suggested that a serum factor promoted ghrelin transport in 8-week-old mice. Overall, these results show that serum factors and physiological states influence the rate at which ghrelin is transported across the blood–brain barrier.
Enzymatic degradation of endomorphins by Anna Janecka; Renata Staniszewska; Katarzyna Gach; Jakub Fichna (2066-2073).
Centrally acting plant opiates, such as morphine, are the most frequently used analgesics for the relief of severe pain, even though their undesired side effects are serious limitation to their usefulness. The search for new therapeutics that could replace morphine has been mainly focused on the development of peptide analogs or peptidomimetics with high selectivity for one receptor type and high bioavailability, that is good blood–brain barrier permeability and enzymatic stability. Drugs, in order to be effective, must be able to reach the target tissue and to remain metabolically stable to produce the desired effects. The study of naturally occurring peptides provides a rational and powerful approach in the design of peptide therapeutics. Endogenous opioid peptides, endomorphin-1 and endomorphin-2, are two potent and highly selective μ-opioid receptor agonists, discovered only a decade ago, which display potent analgesic activity. However, extensive studies on the possible use of endomorphins as analgesics instead of morphine met with failure due to their instability. This review deals with the recent investigations that allowed determine degradation pathways of endomorphins in vitro and in vivo and propose modifications that will lead to more stable analogs.
Keywords: Opioid peptides; Peptidases; Peptidase inhibitors;
A consistent nomenclature of antimicrobial peptides isolated from frogs of the subfamily Phyllomedusinae by Mohamed Amiche; Ali Ladram; Pierre Nicolas (2074-2082).
Keywords: Frogs; Phyllomedusinae; Antimicrobial peptide; Nomenclature;