Peptides (v.24, #8)

Selective toxicity of engineered lentivirus lytic peptides in a CF airway cell model by Shruti M. Phadke; Kazi Islam; Berthony Deslouches; Sunil A. Kapoor; Donna Beer Stolz; Simon C. Watkins; Ronald C. Montelaro; Joseph M. Pilewski; Timothy A. Mietzner (1099-1107).
Lentivirus lytic peptides (LLPs) are derived from HIV-1 and have antibacterial properties. LLP derivatives (eLLPs) were engineered for greater potency against Staphylococcus aureus (SA) and Pseudomonas aeruginosa (PA). Minimum bactericidal concentration (MBC) was determined in low and physiologic salt concentrations. MBC was decreased against SA and equivalent against PA in physiologic salt when compared to the parent compound LLP1. In a novel cystic fibrosis (CF) airway cell model, one derivative, WLSA5, reduced the number of adherent PA and only moderately affected CF cell viability. Overall, eLLPs are selectively toxic to bacteria and may be useful against CF airway infections.
Keywords: Antimicrobial peptide; Cystic fibrosis; Airway epithelial cells; Selective toxicity;

Analogs of RGDVY and GRGD peptides inhibit Mycobacterium kansasii phagocytosis by Ignacy Z. Siemion; Monika Gawłowska; Krzysztof Krajewski; Iwona Strug; Zbigniew Wieczorek (1109-1115).
Continuing our research on Mycobacteria kansasii phagocytosis inhibition, we have examined in that context three series of peptides derived from the RGDVY and GRGD sequences. It was found that the levels of the inhibitory activity depend on the amino acid composition as well as on the particular peptide sequence. Distinct inhibitory activity was found in the case of thymopentin (RKDVY), the active fragment of thymopoietin. In this case the Mycobacterium phagocytosis inhibition should be combined with general immunostimulatory activity of RKDVY peptide. Our examination of a series of GRGDV analogs with a successively prolonged oligo-Gly linker inserted into the peptide chain showed that the distance between the Arg and Asp residues required for such an activity should be about 9 Å.
Keywords: Antiadhesive peptides; Mycobacteria phagocytosis; Thymopentin; Splenopentin; HLA-DQ fragments; Fibronectin fragments;

Peptide products of the atrial gland are not water-borne reproductive pheromones during egg laying in Aplysia by David-B.G. Akalal; Scott F. Cummins; Sherry D. Painter; Gregg T. Nagle (1117-1122).
Mate attraction in Aplysia involves long-distance water-borne signaling via the secretion of the peptide pheromone attractin from the exocrine albumen gland during egg laying. Previous studies have shown that a second exocrine organ, the atrial gland, produces abundant egg-laying hormone (ELH) precursor-related peptides and mollusk-derived growth factor (MDGF), and crude extracts of the atrial gland are attractive in T-maze attraction assays. However, it is not known whether these peptides and proteins are secreted during egg laying. In this report, seawater eluates of freshly laid egg cordons were concentrated and fractionated by C18 RP-HPLC, and the resulting major peaks were examined by amino acid compositional analysis, microsequence analysis, and electrospray mass spectrometry. Concentrated egg cordon eluates were also examined by immunoblot analysis using anti-MDGF antisera as probe. The combined data demonstrated that the atrial gland of Aplysia californica does not secrete detectable levels of either ELH precursor-related peptides or MDGF during egg laying. Although the atrial gland is the last major exocrine organ to make contact with eggs before they are laid, the gland does not appear to secrete water-borne peptide pheromones during egg laying.
Keywords: ADA; Adenosine deaminase; Adenosine deaminase-related growth factor; Aplysia; Atrial gland; Egg-laying hormone; MDGF; Mollusk-derived growth factor;

Nine bradykinin-related peptides were identified in Phyllomedusa sauvagei skin secretion using QTOF MS/MS fragmentation sequencing. The major peptides were (Thr6)-bradykinin, (Hyp3, Thr6)-bradykinin, (Thr6)-phyllokinin and (Hyp3, Thr6)-phyllokinin. The phyllokinins occurred in both sulfated and non-sulfated forms. All (Thr6)-substituted bradykinins/phyllokinins could be generated from a common precursor by differential post-translational processing and modification. The open-reading frame of the cloned precursor cDNA consisted of 62 amino acid residues with a single bradykinin/phyllokinin coding sequence located at the C-terminus. Structural features included a Glu-Arg processing site at the N-terminus of the bradykinin/phyllokinin domain and the absence of an acidic amino acid residue adjacent to the C-terminal Tyr residue in the phyllokinins. However, the neutral amino acid residue (Ile) at position −1 and the basic amino acid residue (Arg) at position −2 from the Tyr residue, constitute a sulfation motif previously identified only in a protochordean.
Keywords: Bradykinin; Phyllokinin; Amphibian; Mass spectrometry; cDNA cloning;

Kinins are important mediators of cardiovascular homeostasis, inflammation and nociception. Bradykinin (BK) B1 receptors (BKB1-R) are over-expressed in pathological conditions including diabetes, and were reported to play a role in hyperglycemia, renal abnormalities, and altered vascular permeability associated with type 1 diabetes. Recent studies from our laboratory demonstrated that BKB1-R are implicated in streptozotocin (STZ)-diabetes-mediated hyperalgesia, since acute administration of the selective BKB1-R antagonists significantly and dose-dependently inhibited such hyperalgesic activity. In the present study, we examined the effect of chronic treatment of STZ-diabetic mice with the selective BKB1-R agonist desArg9bradykinin (DBK) and two specific antagonists R-715 and R-954, on diabetic hyperalgesia. Diabetes was induced in male CD-1 mice by injecting a single high dose of STZ (200 mg/kg, i.p.) and nociception was assessed using the hot plate, plantar stimulation, tail immersion and tail flick tests. Drugs were injected i.p. twice daily for 7 days, starting 4 days after STZ. We showed that chronically administered R-715 (400 μg/kg) and R-954 (200 μg/kg), significantly attenuated the hyperalgesic effect developed in STZ-diabetic mice as measured by the four thermal nociceptive tests. Further, chronic treatment with DBK (400 μg/kg) produced a marked potentiation of the hyperalgesic activity, an effect that was reversed by both R-715 and R-954. The results from this chronic study confirm a pivotal role of the BKB1-R in the development of STZ-diabetic hyperalgesia and suggest a novel approach to the treatment of this short-term diabetic complication using BKB1-R antagonists.
Keywords: Type 1 diabetes; Hyperalgesia; Kinins; Bradykinin B1 receptor; des-arg9bradykinin; Bradykinin B1 receptor antagonists; R-715; R-954;

The Bradykinin B2 receptor is required for full expression of renal COX-2 and renin by John D. Imig; Xueying Zhao; Sheyla R. Orengo; Susana Dipp; Samir S. El-Dahr (1141-1147).
Angiotensin converting enzyme (ACE) inhibition leads to increased levels of bradykinin, cyclooxygenase-2 (COX-2), and renin. Since bradykinin stimulates prostaglandin release, renin synthesis may be regulated through a kinin-COX-2 pathway. To test this hypothesis, we examined the impact of bradykinin B2 receptor (B2R) gene disruption in mice on kidney COX-2 and renin gene expression. Kidney COX-2 mRNA and protein levels were significantly lower in B2R−/− mice by 40–50%. On the other hand, renal COX-1 levels were similar in B2R−/− and +/+ mice. Renal renin protein was 61% lower in B2R−/− compared to B2R+/+ mice. This was accompanied by a significant reduction in renin mRNA levels in B2R−/− mice. Likewise, intrarenal angiotensin I levels were significantly lower in B2R−/− mice compared to B2R+/+ mice. In contrast, kidney angiotensin II levels were not different and averaged 261±16 and 266±15 fmol/g in B2R+/+ and B2R−/− mice, respectively. Kidney angiotensinogen, AT1 receptor and ACE activity were not different between B2R+/+ and B2R−/− mice. The results of these studies demonstrate suppression of renal renin synthesis in mice lacking the bradykinin B2R and support the notion that B2R regulation of COX-2 participates in the steady-state control of renin gene expression.
Keywords: Kallikrein–kinin system; Prostaglandins; Angiotensin; Kidney;

Endothelin-1 is a potent regulator in vivo in vascular calcification and in vitro in calcification of vascular smooth muscle cells by Sheng Ying Wu; Bao Hong Zhang; Chun Shui Pan; Hong Feng Jiang; Yong Zheng Pang; Chao Shu Tang; Yong Fen Qi (1149-1156).
We observed changes of endothelin content and endothelin mRNA in vivo in vascular calcification and in vitro in calcification of vascular smooth muscle cells to explore the role of endothelin in vascular calcification. Calcification model in vivo was induced by administration of Vitamin D3 plus nicotine. Calcification of vascular smooth muscle cells (VSMCs) was induced by β-glycerophosphate. Endothelin content was measured by using radioimmunoassay. Endothelin mRNA amount was determined by using competitive quantitative RT-PCR. The results showed that calcium content, 45 Ca 2+ uptake and alkaline phosphatase (ALP) activity were increased in calcified VSMCs, compared with controls, but were decreased, compared with calcified VSMCs plus BQ123 group. The endothelin content in the medium and endothelin mRNA in VSMCs were elevated by 35 and 120% (P<0.05), respectively, compared with those normal VSMCs. Calcium content, 45 Ca 2+ accumulation and ALP activity in calcified arteries increased by 5.0-, 1.4-, and 1.4-fold. The endothelin levels in plasma and aorta as well as the amount of endothelin mRNA in calcified aorta were increased by 102, 103, and 22%, respectively, compared with control group. However, calcium content, 45 Ca 2+ uptake and ALP activity in VDN plus bosentan group was 33, 36.7, and 40.4% lower than those in VDN group. These results indicated an upregulated endothelin gene expression as well as an increased production of endothelin in calcified aorta and VSMCs with BQ123 and bosentan significantly reducing vascular calcification. This suggested that endothelin might be involved in pathogenesis of vascular calcification.
Keywords: Aorta; Endothelin-1; Vitamin D; Calcification; Vascular smooth muscle cell;

Mercury contamination of rat amylin mimics vasoactivity and cytotoxic effects by Heiko A. Golpon; Annette Püchner; Lothar Schmidt; Hartmut Jungclas; Peter Barth; Tobias Welte; Peter V. Wichert; Christian O. Feddersen (1157-1162).
Rat amylin differs from human amylin (hIAPP) in that it lacks a fibril-forming capacity. As a consequence, toxic effects have been reported for human but not for rat amylin. This report demonstrates how a mercury contamination of commercial rat amylin imitates peptide-related vasoactive and cytotoxic effects on preparations of isolated blood vessels. The source of mercury contamination was believed related to the peptide synthesis. Thiol groups of cysteine-containing peptides are often protected by acetamidomethyl (Acm) which is cleaved by mercuric acetate.
Keywords: Amylin; Mercury; Vasoactivity; Nitric oxide;

Before parturition the uterine cervix undergoes a ripening process (“softens” and dilates) to allow passage of the fetus at term. The exact mechanism(s) responsible for cervical ripening are unknown, though a role for peptidergic sensory neurons is emerging. Previous work demonstrated that administration of substance P (SP) to ovariectomized rats caused events associated with cervical ripening, that production of SP in cervix-related dorsal root ganglion (DRG) is estrogen responsive, and that release of SP from neurons terminating in the cervix and spinal cord peaks prior to parturition. The present study was designed to test the hypothesis that calcitonin gene-related peptide (CGRP), a neuropeptide co-stored with SP in many sensory neurons, undergoes changes with pregnancy and hormonal environment. Immunohistochemistry, in situ hybridization, reverse transcriptase–polymerase chain reaction (RT–PCR) and radioimmunoassay (RIA) were used to investigate CGRP in L6-S1 DRG, spinal cord and cervix during pregnancy and the role of estrogen in CGRP synthesis. CGRP-immunoreactive primary sensory neurons expressed estrogen receptors (ER-α and ER-β). In the cervix, CGRP concentrations decreased, but in the L6-S1 DRG and the spinal cord segments, CGRP levels increased, with peak effects observed at day 20 of gestation. CGRP mRNA synthesis increased in DRG over pregnancy. Sensory neurons of ovariectomized rats treated with estrogen showed increased CGRP mRNA synthesis in a dose-related manner, an effect blocked by the ER antagonist ICI 182 780. From these results, we postulate that synthesis of CGRP in L6-S1 DRG and utilization in the cervix increase over pregnancy and this synthesis is the under influence of the estrogen–ER system. Collectively, these data are consistent with the hypothesis that CGRP plays a role in cervical ripening and, consequently in the birth process.
Keywords: Parturition; Estrogen; Estrogen receptor; Sensory neurons; Neurogenic inflammation; Pregnant rat;

Secretory and vascular effects of adrenomedullin in gastric ulcer: role of CGRP- and adrenomedullin-receptors by Salvatore Salomone; Antonina Caruso; Giuseppa Martinez; Vincenza Maria Cutuli; Agata Prato; Alfredo Bianchi; Matilde Amico-Roxas; Giuseppe Clementi (1175-1180).
Adrenomedullin prevents damage of gastric mucosa in either reserpine-treated or pylorus-ligated rats. Pre-treatment with CGRP8–37 resulted in a decrease of the gastro-protective effect of adrenomedullin in both models and reversed the inhibitory effect of adrenomedullin on gastric acid output in the pylorus-ligated rats. These adrenomedullin actions were less effectively modified by pre-treatment with adrenomedullin22–52. These data suggest that the anti-ulcer effect of adrenomedullin is mainly related to its anti-secretory action, presumably mediated through CGRP-receptors.
Keywords: Adrenomedullin; Adrenomedullin22–52; CGRP8–37; Gastric ulcer; Gastric artery; Anti-secretory effect;

Gastroprotective effect of leptin on gastric mucosal injury induced by ischemia–reperfusion is related to gastric histamine content in rats by Nilüfer Erkasap; Kubilay Uzuner; Mustafa Serteser; Tülay Köken; Yasemin Aydın (1181-1187).
Leptin has cytoprotective effect to gastric mucosal injury in rats. We aimed to test the hypothesis that leptin induced histamine is involved in the prevention of ischemia–reperfusion (I/R) induced gastric mucosal injury in rats. At the end of the 30 min celiac artery occlusion and 12 h reperfusion process, serum and gastric tissue samples were taken from three group of rats to measure oxidative status, histamine levels and for histological examinations. Leptin decreased ulcer and polymorphonuclear leukocyte (PMNL) index, and serum malondialdehyde (MDA) and protein carbonyl content but increased gastric tissue histamine levels. We concluded that leptin exerts a protective effect on gastric mucosa to I/R induced gastric injury probably through increasing tissue histamine content which, in turn, maintain the gastric mucosal blood flow.
Keywords: Gastric ischemia–reperfusion; Leptin; Histamine;

Neurotensin (NT) produces behavioral and physiological effects, including analgesia and hypotheria, when administered into the CNS. Fischer and Lewis rats exhibit differential behavioral responses to central NT receptor activation. To further characterize these differences, we assessed central NT-induced analgesia and hypothermia in independent groups of rats from each strain. Fischer and Lewis rats showed a similar dose-orderly analgesic response in a hot-plate test. Such an isosensitivity was not observed for NT-induced hypothermia. Although NT produced a dose-orderly decrease in mean rectal temperature in both strains, the magnitude of the hypothermic response was significantly smaller in Fischer than in Lewis rats. These findings provide further evidence of genetic differences in central neurotensinergeric neurotransmission in these two strains.
Keywords: Analgesia; Fischer; Hypothermia; Lewis; Neurotensin; Peptide transmitters; Strain differences;

Highly potent fluorescent analogues of the opioid peptide [Dmt1]DALDA by Irena Berezowska; Nga N Chung; Carole Lemieux; Bogumil Zelent; Hazel H Szeto; Peter W Schiller (1195-1200).
H-Dmt-d-Arg-Phe-Lys-NH2 (Dmt=2′,6′-dimethyltyrosine) ([Dmt1]DALDA) is a highly potent and selective μ opioid peptide agonist capable of producing an antinociceptive effect after systemic administration. Fluorescent analogues of [Dmt1]DALDA containing either β-dansyl-l-α,β-diaminopropionic acid [Dap(dns)] or β-anthraniloyl-l-α,β-diaminopropionic acid [Dap(atn)] in place of Lys4 were synthesized. Both analogues retained subnanomolar μ opioid receptor binding affinity, very high μ opioid agonist activity in the guinea pig ileum assay and extraordinarily high antinociceptive activity in the mouse tail-flick test (intrathecal administration). The maxima of the fluorescence emission spectra recorded in Tris–HCl buffer (pH 6.6) indicated a completely aqueous environment of the fluorophore in both peptides. The high fluorescence quantum yield (ϕ=0.358) of the [Dap(atn)4] analogue was particularly remarkable. These fluorescent [Dmt1]DALDA analogues represent valuable pharmacological tools for various applications, including studies on the binding to receptors and other biopolymers, cellular uptake and intracellular distribution, and tissue distribution.
Keywords: [Dmt1]DALDA; Opioid peptides; Fluorescent opioid peptide analogues; Fluorescence spectroscopy; Fluorescence quantum yield; Opioid activity profile in vitro; Antinociception;

In vitro metabolism of LVV-Hemorphin-7 by renal cytosol and purified prolyl endopeptidase by I. Fruitier-Arnaudin; M. Cohen; C. Coitoux; J.-M. Piot (1201-1206).
The metabolism of LVVH7, an endogenous peptide obtained by cathepsin D hydrolysis of the β chain of hemoglobin, was studied, in vitro, in the presence of cytosol of rat kidney and compared with angiotensin IV. High metabolic activity was found against these two peptides (half life time <2 min) in this subcellular fraction. The main products of LVVH7 metabolism by renal cytosol are VVH7, H7 and LVVH6 suggesting both aminopeptidase and carboxypeptidase activities. The use of PEP inhibitor in kidney cytosol permitted to demonstrate the major role of prolyl endopeptidase (PEP) in LVVH7 degradation. This fact was reinforced by a kinetic study investigated with purified enzyme (K m/V max about 238 mM−1  s−1 and close to that observed for angiotensin related peptides).
Keywords: Hemorphin; Peptide hydrolysis; Metabolism; Homogenate; Renal cytosol; Subcellular fractions; Prolyl endopeptidase;

Development of an IL-6 antagonist peptide that induces apoptosis in 7TD1 cells by Rossella Manfredini; Elena Tenedini; Michela Siena; Enrico Tagliafico; Monica Montanari; Alexis Grande; Tommaso Zanocco-Marani; Cristina Poligani; Roberta Zini; Claudia Gemelli; Anna Bergamaschi; Tatiana Vignudelli; Francesca De Rienzo; Pier Giuseppe De Benedetti; Maria Cristina Menziani; Sergio Ferrari (1207-1220).
Interleukin-6 (IL-6) is a pleiotropic cytokine involved in the regulation of proliferation and differentiation of hematopoietic cells and in the pathogenesis of many diseases, including multiple myeloma. This study pursues a way to interfere with IL-6 pathway in an attempt to modulate its biological activity. Here we describe the rational design and biological evaluation of peptides able to antagonize the murine IL-6 activity by interfering with IL-6 Receptor alpha in 7TD1 cells, a IL-6-dependent B-cell line. Of the peptide tested, only Guess 4a is capable of interfering with IL-6 transducing pathway, therefore inducing growth arrest and apoptosis of 7TD1 cells.
Keywords: Interleukin-6; Interleukin-6 receptor; Molecular modelling; Computational simulation; Antagonist peptides; Apoptosis; Multiple myeloma;

Identification of a binding site for the anti-inflammatory tripeptide feG by Ronald D Mathison; Joseph S Davison; Essam Metwally (1221-1230).
The mechanism of action of feG, an anti-inflammatory peptide, was explored using data mining, molecular modeling, and enzymatic techniques. The molecular coordinates of protein kinase A (PKA) were used to create six virtual isoforms of protein kinase C (PKCα, βI, βII, δ, ι, and ζ). With in silico techniques a binding site for feG was identified on PKCβI that correlated significantly with a biological activity, the inhibition of intestinal anaphylaxis. Since feG selectively increased the binding of a PKCβI antibody, it is proposed that this peptide inhibits the reassociation of the hydrophobic tail of PKCβI with its binding site and prevents the enzyme from assuming an inactive conformation.
Keywords: Molecular modeling; Data mining; Protein kinase C; Hydrophobic motif; In silico binding; GRAMM; Anaphylaxis; Allergy; Intestine; Neutrophils;

Desialylated N-CAM and chromatin-derived acidic peptide effects in the hypothalamus by Chiara Di Nisio; Luigi Brunetti; Diana L. Esposito; Lucia Recinella; Giustino Orlando; Barbara Michelotto; Michele Vacca (1231-1236).
Chromatin-derived acidic peptides (ACPs) have been shown to acutely modulate hypothalamic catecholamine release. To investigate whether this effect is mediated through membrane polysialylated neural-cell adhesion molecule (PSA-N-CAM), we pretreated rat hypothalamic synaptosomes with neuraminidase enzyme, which partially cleaves sialic acid residues from N-CAM, and perfused them with ACP-1 (Asp-Asp-Ser-Asp-Glu-Glu-Asn) or a more lipophilic derivative, ACP-2 ([Ala-Ile-Ser-Pro]-Asp-Asp-Ser-Asp-Glu-Glu-Asn). We have found that neuraminidase completely abolish the inhibitory effect of ACP-1 on dopamine release, while the inhibitory activity of ACP-1 on norepinephrine release is partially lost. On the other hand, ACP-2 inhibition of dopamine release is not modified by neuraminidase pretreatment.
Keywords: Hypothalamus; Catecholamine; Dopamine; Norepinephrine; Chromatin-derived acidic peptides; Neuraminidase; N-CAM; Sialic acid;

Localization of carboxypeptidase A-like enzyme in rat kidney by Rajko Igić; Sandra Garber; Marin Sekosan; Renata A Urbanska; Rahim Behnia (1237-1240).
In this study we demonstrate that carboxypeptidase A (CPA)-like enzyme is expressed in rat kidney. The major metabolites of angiotensin (Ang) I by the rat renal mesangial cell extract at 37 °C, pH 7.4, were Ang 1–9 and Ang II. Quinaprilat did not influence the formation of Ang 1–9, but it inhibited formation of Ang II. The formation of Ang 1–9 was inhibited by potato carboxypeptidase inhibitor, 1,10-phenanthroline or EDTA. Lowering the pH from 7.4 to 4.0 also inhibited the formation of this nonapeptide. These findings suggest that a metallocarboxypeptidase is responsible for Ang 1–9 production. Using monoclonal antibodies to CPA, Western blot showed the presence of CPA-like enzyme in the extracts prepared from the mesangial cells or kidney cortex of the rat. Immunohistochemistry showed that CPA-like enzyme is localized in the mesangial glomerular cells and adventitia of kidney blood vessels, whereas it was absent in the renal tubules. Our data suggest that a CPA-like enzyme could be added to a repertoire of enzymes present in the rat mesangial cells and adventitia of renal blood vessels.
Keywords: Angiotensin I; Angiotensin 1–9; Carboxypeptidase A; Kidney; Mesangial cells; Adventitia; Immunohistochemistry; Rat;

Endogenous opiates and behavior: 2002 by Richard J. Bodnar; Maria M. Hadjimarkou (1241-1302).
This paper is the twenty-fifth consecutive installment of the annual review of research concerning the endogenous opioid system, now spanning over a quarter-century of research. It summarizes papers published during 2002 that studied the behavioral effects of molecular, pharmacological and genetic manipulation of opioid peptides, opioid receptors, opioid agonists and opioid antagonists. The particular topics that continue to be covered include the molecular-biochemical effects and neurochemical localization studies of endogenous opioids and their receptors related to behavior (Section 2), and the roles of these opioid peptides and receptors in pain and analgesia (Section 3); stress and social status (Section 4); tolerance and dependence (Section 5); learning and memory (Section 6); eating and drinking (Section 7); alcohol and drugs of abuse (Section 8); sexual activity and hormones, pregnancy, development and endocrinology (Section 9); mental illness and mood (Section 10); seizures and neurologic disorders (Section 11); electrical-related activity and neurophysiology (Section 12); general activity and locomotion (Section 13); gastrointestinal, renal and hepatic functions (Section 14); cardiovascular responses (Section 15); respiration and thermoregulation (Section 16); and immunological responses (Section 17).
Keywords: Endogenous opiates; Behavior; Opioid peptides;