Peptides (v.27, #6)

The effect of tripeptide tyroserleutide (YSL) on animal models of hepatocarcinoma by Zhi Yao; Rong Lu; Jing Jia; Pengpeng Zhao; Jing Yang; Minna Zheng; Jing Lu; Mengjue Jin; Haixian Yang; Wenyuan Gao (1167-1172).
This study aimed to observe the effects of tyroserleutide (tyrosyl-seryl-leucine, YSL) on the survival time of mice transplanted with the ascitic fluid-type hepatocarcinoma H22, as well as the inhibitory effect of tyroserleutide on the human hepatocarcinoma Bel-7402 that was transplanted into nude mice. At doses of 80, 20 and 5 μg/kg/d, tyroserleutide significantly prolonged the survival of mice transplanted with H22 tumor cells, producing survival rates of 89%, 39% and 49%, respectively, which were statistically significantly different from the saline group (P  < 0.05). YSL, at doses of 80, 160 and 320 μg/kg/d significantly inhibited the growth of the human hepatocarcinoma Bel-7402 tumor in nude mice, producing inhibition of 40%, 64% and 59%, respectively; this inhibition was significantly greater than that by saline (P  < 0.05). HE staining and electron microscopy of the pathological changes of the tumor in nude mice showed that YSL changed the structure Bel-7402 tumor cells that were transplanted into nude mice, and also induced tumor cell apoptosis and necrosis, which could be a mechanism by which YSL inhibits the tumor growth in animal models.
Keywords: Tyroserleutide (tyrosyl-seryl-leucine, YSL); H22 model; Nude mice; Hepatocarcinoma Bel-7402; Antitumor; Apoptosis;

Isolation and characterization of a novel platelet aggregation inhibitory peptide from the medicinal mushroom, Inonotus obliquus by Kwang Wook Hyun; Seung Chan Jeong; Dae Hyoung Lee; Jeong Sik Park; Jong Soo Lee (1173-1178).
This study describes the extraction and characterization of a platelet aggregation inhibitory peptide from Inonotus obliquus. Ethanol extract from I. obliquus ASI 74006 mycelia showed the highest platelet aggregation inhibitory activity (81.2%). The maximum platelet aggregation inhibitory activity was found when the mycelia of I. obliquus ASI 74006 was extracted with ethanol at 80 °C for 12 h. The platelet aggregation inhibitor was purified by systematic solvent fractionation, ultrafiltration, Sephadex G-10 column chromatography, and reverse-phase HPLC. The purified platelet aggregation inhibitor is a novel tripeptide with a molecular mass of 365 Da, having a sequence of Trp-Gly-Cys. The purified platelet aggregation inhibitor also showed high platelet aggregation inhibitory activity in Institute of Cancer Research (ICR) mice.
Keywords: Platelet aggregation inhibitor; Peptide; Inonotus obliquus;

Design of genetically modified soybean proglycinin A1aB1b with multiple copies of bioactive peptide sequences by Krisna Prak; Yukie Maruyama; Nobuyuki Maruyama; Shigeru Utsumi (1179-1186).
The peptide IIAEK derived from β-lactoglobulin has a hypocholesterolemic activity greater than that of β-sitosterol. To create food proteins with multiple copies of this valuable peptide sequence, we introduced tandem multimers of the nucleotide sequence encoding the peptide into DNA regions corresponding to the five variable regions of soybean glycinin A1aB1b subunit, and expressed the mutants in Escherichia coli. The expression level and solubility of the five mutants, each containing four IIAEK sequences in each of the variable regions, were compared. Overall, the expression level and solubility of the mutants with four IIAEK sequences in the variable regions IV and V were the best followed by II > III > I. Further, introduction of the fifth IIAEK sequence to the variable region IV did not decrease expression level and solubility. Increasing the number of IIAEK to 7 and 10 slightly decreased expression level, while their solubility decreased to as low as 40 and 1%, respectively. Various mutations were combined to get a mutant containing as many IIAEK sequences as possible. Some of the resulting mutants were expressed in the soluble form. The mutant containing eight IIAEK from the combination of variable regions IV and V (IV-4 + V-4) showed the best balance of the expression level and solubility, followed by the combination of variable regions II and III (II-4 + III-4). The soluble fractions of these mutants were purified by hydrophobic, gel filtration and ion-exchange column chromatography. Yields of IIAEK peptide released by in vitro digestion with trypsin from both mutants were around 80%. This is the first report that a large amount of a physiologically active peptide could be introduced into food protein.
Keywords: Peptide; Cholesterol; β-Lactoglobulin; Glycinin; Soybean;

Antimicrobial activity of glycosidase inhibitory protein isolated from Cyphomandra betacea Sendt. fruit by Roxana M. Ordóñez; Adriana A.L. Ordóñez; Jorge E. Sayago; María I. Nieva Moreno; María I. Isla (1187-1191).
Broad-spectrum antimicrobial activity of an invertase inhibitory protein (IIP) isolated from Cyphomandra betacea ripe fruits is documented. Minimal inhibitory concentration (MIC) values were determined by agar macrodilution and broth microdilution assays. This IIP inhibited the growth of xylophagous and phytopatogenic fungi (Ganoderma applanatum, Schizophyllum commune, Lenzites elegans, Pycnoporus sanguineous, Penicillium notatum, Aspergillus niger, Phomopsis sojae and Fusarium mango) and phytopathogenic bacteria (Xanthomonas campestris pvar vesicatoria CECT 792, Pseudomonas solanacearum CECT 125, Pseudomonas corrugata CECT 124, Pseudomonas syringae pv. syringae and Erwinia carotovora var carotovora). The IIP concentration required to completely inhibit the growth of all studied fungi ranged from 7.8 to 62.5 μg/ml. Phytopatogenic bacteria were the most sensitive, with MIC values between 7.8 and 31.25 μg/ml. Antifungal and antibacterial activities can be associated with their ability to inhibit hydrolytic enzymes. Our results indicate the possible participation of IIP in the plant defense mechanism and its potential application as a biocontrol agent against phytopathogenic fungi and bacteria.
Keywords: Glycosidases inhibitory protein; Antibacterial activity; Antifungal activity; Pathogenesis related protein; Cyphomandra betacea Sendt. fruit;

Molecular dynamics simulations of three related helical antimicrobial peptides have been carried out in zwitterionic diphosphocholine (DPC) micelles and anionic sodiumdodecylsulfate (SDS) micelles. These systems can be considered as model mammalian and bacterial membrane interfaces, respectively. The goal of this study is to dissect the differences in peptide composition which make the mutant peptides (novispirin-G10 and novispirin-T7) less toxic than the parent peptide ovispirin (OVIS), although all three peptides have highly antibacterial properties. Compared to G10 and T7, OVIS inserts deepest into the DPC micelle. This correlates well with the lesser toxicity of G10 and T7. There is strong evidence which suggests that synergistic binding of hydrophobic residues drives binding of OVIS to the micelle. The helical content of G10 and T7 is reduced in the presence of DPC, and this leads to less amphipathic peptide structures, which bind weakly to the micelle. Simulations in SDS were carried out to compare the influence of membrane electrostatics on peptide structure. All three peptides bound strongly to SDS, and retained helical form. This corresponds well with their equally potent antibacterial properties. Based on the simulations, we argue that secondary structure stability often leads to toxic properties. We also propose that G10 and T7 operate by the carpet mechanism of cell lysis. Toxicity of peptides operating by the carpet mechanism can be attenuated by reducing the peptide helical content. The simulations successfully capture experimental binding states, and the different depths of binding of the three peptides to the two micelles correlate with their antibacterial and toxic properties.
Keywords: Ovispirin-1; Molecular dynamics simulations; DPC micelle; Peptide–membrane interactions; SDS micelle; Secondary structure induction; AMP activity; AMP toxicity; AMP;

In vitro biological activities of magainin alone or in combination with nisin by Lidia Cruz-Chamorro; María A. Puertollano; Elena Puertollano; Gerardo Álvarez de Cienfuegos; Manuel A. de Pablo (1201-1209).
Antimicrobial peptides have received increasing attention not only as potential candidates to their administration as antimicrobial agents, but also as potential drugs applied in cancer therapy. Here, we have examined the action of both nisin and magainin on human promyelocytic leukemia HL-60 cells. Cells were cultured in presence of either nisin or magainin 1 as well as in combination with both nisin and magainin 1. Results have revealed that magainin, but not nisin, produces a loss of cell viability in HL-60 cells, and a minor increase of hemolysis, whereas it is not responsible for cell membrane disruption and lactate dehydrogenase (LDH) leakage. In addition, magainin is involved in a significant generation of reactive oxygen species (ROS), as well as in an augment of caspase-3 activity. Magainin-induced apoptosis was verified by DNA fragmentation and annexin V-FITC/propidium iodide (PI) staining of the cells. Promotion of cell death by magainin occurs via cytochrome c release accompanied by a substantial increase of proteasome activity. These results underline the importance of magainin as a drug capable of exerting an in vitro antitumoral activity by triggering apoptosis.
Keywords: Magainin; Nisin; Apoptosis; Annexin-V; Cytochrome c; Caspase-3; Reactive oxygen species; Proteasome activity;

Citropin 1.1-treated central venous catheters improve the efficacy of hydrophobic antibiotics in the treatment of experimental staphylococcal catheter-related infection by Oscar Cirioni; Andrea Giacometti; Roberto Ghiselli; Wojciech Kamysz; Fiorenza Orlando; Federico Mocchegiani; Carmela Silvestri; Alberto Licci; Leonardo Chiodi; Jerzy Łukasiak; Vittorio Saba; Giorgio Scalise (1210-1216).
An in vitro antibiotic susceptibility assay for Staphylococcus aureus biofilms developed on 96-well polystyrene tissue culture plates was performed to elucidate the activity of citropin 1.1, rifampin and minocycline. Efficacy studies were performed in a rat model of staphylococcal CVC infection. Silastic catheters were implanted into the superior cava. Twenty-four hours after implantation the catheters were filled with citropin 1.1 (10 μg/mL). Thirty minutes later the rats were challenged via the CVC with 1.0 × 106 CFU of S. aureus strain Smith diffuse. Administration of antibiotics into the CVC (the antibiotic lock technique) began 24 h later. The study included: one control group (no CVC infection), one contaminated group that did not receive any antibiotic prophylaxis, one contaminated group that received citropin 1.1-treated CVC, two contaminated groups that received citropin 1.1-treated CVC plus rifampin and minocycline at concentrations equal to MBCs for adherent cells and 1024 μg/mL in a volume of 0.1 mL that filled the CVC and two contaminated groups that received rifampin or minocycline at the same concentrations. All catheters were explanted 7 days after implantation. Main outcome measures were: minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC), synergy studies, quantitative culture of the biofilm formed on the catheters and surrounding venous tissues, and quantitative peripheral blood cultures. MICs of conventional antibiotics against the bacteria in a biofilm were at least four-fold higher than against the freely growing planktonic cells. In contrast, when antibiotics were used on citropin 1.1 pre-treated cells they showed comparable activity against both biofilm and planktonic organisms. The in vivo studies show that when CVCs were pre-treated with citropin 1.1 or with a high dose of antibiotics, biofilm bacterial load was reduced from 107 to 103  CFU/mL and bacteremia reduced from 103 to 101  CFU/mL. When CVCs were treated both with citropin 1.1 and antibiotics, biofilm bacterial load was further reduced to 101  CFU/mL and bacteremia was not detected, suggesting 100% elimination of bacteremia and a log 6 reduction in biofilm load. Citropin 1.1 significantly reduces bacterial load and enhances the effect of hydrophobic antibiotics in the treatment of CVC-associated S. aureus infections.
Keywords: Citropin 1.1; CVC; Antibiotic-lock technique; Biofilm; Antimicrobial peptides;

Structural analysis of peptides that interact with Newcastle disease virus by Suet Lin Chia; Wen Siang Tan; Khozirah Shaari; Noorsaadah Abdul Rahman; Khatijah Yusoff; Seetharama D. Satyanarayanajois (1217-1225).
A peptide with the sequence CTLTTKLYC has previously been identified to inhibit the propagation of Newcastle disease virus (NDV) in embryonated chicken eggs and tissue culture. NDV has been classified into two main groups: the velogenic group, and mesogenic with lentogenic strains as the other group based on its dissociation constants. In this study the peptide, CTLTTKLYC, displayed on the pIII protein of a filamentous M13 phage was synthesized and mutated in order to identify the amino acid residues involved in the interactions with NDV. Mutations of C1 and K6 to A1 and A6 did not affect the binding significantly, but substitution of Y8 with A8 dramatically reduced the interaction. This suggests that Y8 plays an important role in the peptide–virus interaction. The three-dimensional structure of the peptide was determined using circular dichroism (CD), nuclear magnetic resonance (NMR), and molecular modeling. The peptide exhibited two possible conformers. One that consists of consecutive β-turns around T2–L3–T4–T5 and K6–L7–Y8–C9. The other conformer exhibited a β-hairpin bend type of structure with a bend around L3–T4–T5–K6.
Keywords: Alanine-substitution; β-Turn; Circular dichroism; Filamentous M13 bacteriophage; Molecular modeling; Mutagenesis; Newcastle disease virus;

N-ethylmaleimide-sensitive fusion protein (NSF) is an essential component for the neurotransmitter or neurohormone release apparatus present in all eukaryotic cells. Here, a new NSF orthologue was characterized from the cotton bollworm, Helicoverpa armigera (Har). Northern blot exhibited a high expression in larval brain. Southern analysis indicated that a single copy of the gene is present in a haploid genome. Using antibodies labeled with fluoresceins, we directly proved that NSF is co-localized with two crucial neurohormones, prothoracicotropic hormone and diapause hormone, both of which regulate insect development. These findings suggest that Har-NSF may be involved in regulating insect neurohormone release.
Keywords: N-ethylmaleimide-sensitive fusion protein; Neurosecretory cell; Neurohormone release; Fluorescence immunocytochemistry; Co-localization; Development;

Cloning and characterization of a novel calcium channel toxin-like gene BmCa1 from Chinese scorpion Mesobuthus martensii Karsch by Cao Zhijian; Xie Yun; Dai Chao; Zhu Shunyi; Yin Shijin; Wu Yingliang; Li Wenxin (1235-1240).
Many studies have been carried on peptides and genes encoding scorpion toxins from the venom of Mesobuthus martensii Karsch (synonym: Buthus martensii Karsch, BmK), such as Na+, K+ and Cl channel modulators. In this study, a novel calcium channel toxin-like gene BmCa1 was isolated and characterized from the venom of Mesobuthus martensii Karsch. First, a partial cDNA sequence of the Ca2+ channel toxin-like gene was identified by random sequencing method from a venomous gland cDNA library of Mesobuthus martensii Karsch. The full-length sequence of BmCa1 was then obtained by 5’RACE technique. The peptide deduced from BmCa1 precursor nucleotide sequence contains a 27-residue signal peptide and a 37-residue mature peptide. Although BmCa1 and other scorpion toxins are different at the gene and protein primary structure levels, BmCa1 has the same precursor nucleotide organization and cysteine arrangement as that of the first subfamily members of calcium channel scorpion toxins. Genomic DNA sequence of BmCa1 was also cloned by PCR. Sequence analysis showed that BmCa1 gene consists of three exons separated by two introns of 72 bp and 1076 bp in length, respectively. BmCa1 is the first calcium channel toxin-like gene cloned from the venom of Mesobuthus martensii Karsch and potentially represents a novel class of calcium channel toxins in scorpion venoms.
Keywords: Mesobuthus martensii Karsch; Calcium channel scorpion toxins; Genomic organization and structure;

Allatostatins are important regulatory neuropeptides that inhibit juvenile hormone (JH) biosynthesis by the corpora allata (CA) in insects. However, to date, the structure and expression of the gene encoding allatostatins have not been reported in any species other than insects. In this study, we used a combination of a semi-nested polymerase chain reaction (PCR) and screening of a central nervous system cDNA library of Macrobrachium rosenbergii to isolate and sequence a cDNA clone (2885 bp) encoding a 701 amino acid FGLamide allatostatin precursor polypeptide. This is the first reported allatostatin gene in crustacean. The deduced precursor was conceptually split into at least 35 FGLamide allatostatins at dibasic cleavage sites (Lys and Lys/Arg), far more than reported for any other known FGLamide allatostatin precursors from insects (13–14 allatostatins). Reverse transcription-polymerase chain reaction (RT-PCR) analysis demonstrated that the gene was expressed in the brain, gut, thoracic and abdominal ganglia, but not in the heart, muscle, ovary, gill, or hepatopancreas. Furthermore, developmentally-dependent expression of the gene was observed in the brain and thoracic ganglia of the prawn by using semi-quantitative RT-PCR analysis.
Keywords: Allatostatin; Molecular cloning; Semi-quantitative RT-PCR; Macrobrachium rosenbergii;

Production and characterization of recombinant vitellogenesis-inhibiting hormone from the American lobster Homarus americanus by Tsuyoshi Ohira; Takuji Okumura; Michio Suzuki; Yosuke Yajima; Naoaki Tsutsui; Marcy N. Wilder; Hiromichi Nagasawa (1251-1258).
Recombinant peptides related to vitellogenesis-inhibiting hormone (VIH) of the American lobster Homarus americanus were expressed in bacterial cells, and then purified after being allowed to refold. Biological activities of the recombinant VIHs having an amidated C-terminus (rHoa-VIH-amide) and a free carboxyl-terminus (rHoa-VIH-OH) were examined using an ovarian fragment incubation system derived from the kuruma prawn, Marsupenaeus japonicus. The rHoa-VIH-amide significantly reduced vitellogenin mRNA levels in the ovary, while rHoa-VIH-OH had no effect. This is the first report that describes the production of a crustacean VIH having biological activity and the importance of the C-terminal amidation for its vitellogenesis-inhibiting activity.
Keywords: American lobster; Carboxyl-terminal amide; Homarus americanus; Marsupenaeus japonicus; Vitellogenesis-inhibiting hormone;

Ovarian jelly-peptides (OJPs), a new family of regulatory peptides identified in the cephalopod Sepia officinalis by Benoît Bernay; Michèle Baudy-Floc’h; Jean Gagnon; Joël Henry (1259-1268).
In marine invertebrates, numerous water-borne peptides involved in reproductive behavior have been characterized. In this study, we focused on three ovarian water-borne peptides, released by full-grown oocytes (FGO) in the genital coelom and in the lumen of the oviduct in the cuttlefish Sepia officinalis. The first one (DQVKIVL), was characterized by the monitoring of HPLC purified fraction using a myotropic bioassay. Subsequently, a peptidomic approach consisting of a mass spectrometry comparative screening performed between the peptide content of FGO with that of FGO-conditioned medium, led to the identification of two additional water-borne peptides. The second peptide identified (DEVKIVL) was characterized by MS/MS and the primary structure of the third one (DEVKIVLD) was elucidated by a combination of Edman degradation, acid hydrolysis and MS/MS analysis. Sequence homology, tissue mapping and bioactivity demonstrate that these peptides belong to the same family. DQVKIVL-related-peptides strictly localized in the female genital tract modulate the whole female genital tract and the main nidamental gland contractions. Furthermore, these peptides form a jelly, when resuspended in water. This particular property could play an important role in the kinetics of peptide diffusion in the external medium. Thus, these regulatory peptides were named ovarian jelly-peptides (OJPs).
Keywords: Egg-laying; Egg capsule secretion; Myotropin; Invertebrates; Mass spectrometry; Peptidomic; Sepia officinalis;

The crustacean hyperglycemic hormones from an euryhaline crab Pachygrapsus marmoratus and a fresh water crab Potamon ibericum: Eyestalk and pericardial isoforms by Jean-Yves Toullec; Laetitia Serrano; Philippe Lopez; Daniel Soyez; Céline Spanings-Pierrot (1269-1280).
Keywords: Crustacean hyperglycemic hormone; X-organ; Pericardial organ; Brachyura; Heterotremata; Alternative splicing;

A role for cyclic nucleotide monophosphates in synaptic modulation by a crayfish neuropeptide by Amit Badhwar; Andrea D. Weston; Jillian B. Murray; A. Joffre Mercier (1281-1290).
DF2 (DRNFLRFamide), a FMRFamide-like peptide, has been shown to increase the amount of transmitter released at crayfish neuromuscular junctions. Here, we examined a possible role for the cyclic nucleotide monophosphates, cAMP and cGMP, in DF2's effects on synaptic transmission. The effects of DF2 on synaptic transmission were monitored by recording excitatory postsynaptic potentials (EPSPs) in the deep abdominal extensor muscles of the crayfish, Procambarus clarkii. A number of activators and inhibitors were used to determine whether or not cAMP, cGMP, protein kinase A (PKA) and protein kinase G (PKG) mediate the effect of this neuropeptide. Phosphodiesterase inhibitors, known to inhibit the breakdown of cAMP (IBMX) and/or cGMP (mdBAMQ), potentiate the effect of DF2 on synaptic transmission. Activators of PKA (Sp-cAMPS) and PKG (8-pCPT-cGMP) increase EPSP amplitude, mimicking the effects of DF2. Inhibitors of PKA (Rp-cAMPS) and PKG (Rp-8-pCPT-cGMPS) each block a portion of the response to the peptide, and when applied together these two inhibitors completely block the response. Taken together, these results indicate that cyclic nucleotides and cyclic nucleotide-dependent protein kinases are necessary components of the pathway underlying modulation by this neuropeptide.
Keywords: FMRFamide; Neuropeptide; Crustacean; Protein kinase A; Protein kinase G;

FMRFamide related peptide ligands activate the Caenorhabditis elegans orphan GPCR Y59H11AL.1 by Inge Mertens; Isabelle Clinckspoor; Tom Janssen; Ronald Nachman; Liliane Schoofs (1291-1296).
G-protein coupled receptors (GPCRs) are ancient molecules that can sense environmental and physiological signals. Currently, the majority of the predicted Caenorhabditis elegans GPCRs are orphan. Here, we describe the characterization of such an orphan C. elegans GPCR, which is categorized in the tachykinin-like group of receptors. Since the C. elegans genome predicts only one tachykinin-like peptide (SFDRMGGTEFGLM), which could not activate the receptor, we hypothesized that one or some of the numerous FMRFamide related peptides (FaRPs) could be the cognate ligands for this receptor. This hypothesis was based on the suggestion that RFamides may be ancestral neuropeptides, from which a lot of the amidated neuropeptides, including tachykinins, derived. Indeed, we found that the orphan receptor encoded by the Y59H11AL.1 gene is activated by several C. elegans neuropeptides, including SPMERSAMVRFamide. These peptides activate the receptor in a concentration-dependent way.
Keywords: G-protein coupled receptor (GPCR); FMRFamide related peptides (FaRPs); Neuropeptides; Caenorhabditis elegans; Reverse pharmacology;

In vitro and in vivo studies of dansylated compounds, the putative agonists and antagonists on neuropeptide FF receptors by Quan Fang; Jia Guo; Ya-li Peng; Min Chang; Feng He; Qiang Chen; Rui Wang (1297-1304).
To further evaluate the importance of C-terminal modification of neuropeptide FF (NPFF), in the present work, four dansylated NPFF analogues, including two putative agonists (dansyl-PQRFamide and dansyl-GSRFamide) and two putative antagonists (dansyl-PQRamide and dansyl-GSRamide), were synthesized and investigated to address their potencies and efficacies in a series of in vitro and in vivo assays. (1) In the isolated mouse colon bioassay, the four dansylated compounds showed agonistic profiles: both dansyl-GSRFamide (1–10 μM) and dansyl-GSRamide (1–10 μM) dose-dependently caused colonic contractions, which were attenuated by pretreatment with BIBP3226; dansyl-PQRFamide and dansyl-PQRamide evoked modest colonic contractions at a high dose of 50 μM. (2) In urethane-anaesthetized rats, both dansyl-PQRFamide (50–300 nmol/kg, i.v.) and dansyl-GSRFamide (15–50 nmol/kg, i.v.) dose-dependently increased the mean arterial pressure and heart rate in a manner similar to NPFF (50–300 nmol/kg, i.v.); on the contrary, the two putative antagonists (100–800 nmol/kg, i.v.) decreased blood pressure in a dose-dependent manner. All the results suggest that dansyl-PQRFamide and dansyl-GSRFamide are NPFF full agonists; in contrast, dansyl-GSRamide and dansyl-PQRamide behave as agonists in vitro and antagonists in vivo on NPFF receptors. The findings reveal that the C-terminal Phe might be a crucial residue to determine the efficacy. In addition, the novel analogue dansyl-GSRFamide may be developed as a highly potent agonist to investigate the NPFF system.
Keywords: Neuropeptide FF (NPFF); Dansylated compound; Colonic contraction; Mean arterial pressure; Heart rate;

Evidence from peptidomic analysis of skin secretions that the red-legged frogs, Rana aurora draytonii and Rana aurora aurora, are distinct species by J. Michael Conlon; Nadia Al-Ghafari; Laurent Coquet; Jérôme Leprince; Thierry Jouenne; Hubert Vaudry; Carlos Davidson (1305-1312).
The northern red-legged frog Rana aurora aurora and the California red-legged frog Rana aurora draytonii are traditionally classified together in the same species group. Ten peptides with antimicrobial activity were isolated from norepinephrine-stimulated skin secretions of R. aurora draytonii and purified to near homogeneity. The peptides were identified as belonging to the ranatuerin-2 family (two peptides), brevinin-1 family (four peptides), temporin family (three peptides), and a novel peptide, RV-23 (RIGVLLARLPKLFSLFKLMGKKV) that has limited structural similarity to the bee venom peptide, melittin. This distribution of peptides contrasts with that found previously in skin secretions from R. aurora aurora collected under the same conditions and at the same time of year (one ranatuerin-2 peptide, two brevinin-1 peptides, and one temporin peptide). The variation in amino acid sequences between corresponding R. aurora draytonii and R. aurora aurora peptides is comparable with the variation in sequences of orthologs from other members of the Amerana group of New World ranid frogs (Rana boylii, Rana muscosa, and Rana luteiventris). It is proposed, therefore, that the red-legged frogs should be regarded as separate species (R. aurora and R. draytonii) within the Amerana group rather than conspecific subspecies. The data emphasize that amino acid sequences of antimicrobial peptides in skin secretions may be used to infer taxonomic and phylogenetic relationships between species of ranid frogs.
Keywords: Peptidomics; Antimicrobial peptides; Rana aurora; Rana draytonii; Skin secretions;

Histamine-releasing and antimicrobial peptides from the skin secretions of the Dusky Gopher frog, Rana sevosa by Ciaren Graham; Stephen C. Richter; Stephen McClean; Edmund O’Kane; Peter R. Flatt; Chris Shaw (1313-1319).
Seven novel peptides were isolated from the skin secretions of the North American dusky gopher frog, Rana sevosa, on the basis of antimicrobial activity and histamine release from rat peritoneal mast cells. The peptides were purified to homogeneity using HPLC and characterized by electrospray ion-trap mass spectrometry, MALDI-TOF mass spectrometry and Edman sequencing. Bioinformatic analysis of primary structures revealed that the novel peptides could be assigned to four established families of ranid frog antimicrobial peptides, namely esculentin-1, esculentin-2, brevinin-1 and ranatuerin-2. The peptides were named in accordance with accepted terminology as ranatuerin 2SEa, etc., reflecting the peptide family name, the species of origin (SE for sevosa) and the isotype (a). Of major interest was the fact that brevinin 1SE displayed significant structural similarity to ponericin W5, an antibacterial venom peptide from the ant, Pachyconyla goeldii. This is a further example of amphibian skin defensive peptides showing striking structural similarities to peptides from insects. These data may shed some light on the functional biological relevance of defensive peptides that possess both antimicrobial and histamine-releasing activities.
Keywords: Antimicrobial; Histamine; HPLC; Peptidomics; Rana sevosa; Skin secretions; Mass spectrometry;

Characterization of NPY receptor subtypes Y2 and Y7 in rainbow trout Oncorhynchus mykiss by Tomas A. Larsson; Earl T. Larson; Robert Fredriksson; J Michael Conlon; Dan Larhammar (1320-1327).
We report the cloning and pharmacological characterization of two neuropeptide Y (NPY) receptor subtypes, Y2 and Y7, in rainbow trout (Oncorhynchus mykiss). These subtypes are approximately 50% identical to each other and belong to the Y2 subfamily of NPY receptors. The binding properties of the receptors were investigated after expression in human HEK-293 EBNA cells. Both receptors bound the three zebrafish peptides NPY, PYYa, and PYYb, as well as porcine NPY and PYY, with affinities in the nanomolar range that are similar to mammalian Y2. The affinity of the truncated porcine NPY fragments, NPY 13–36 and NPY 18–36 was markedly lower compared to mammalian and chicken Y2. This suggests that mammalian and chicken Y2 are unique among NPY receptors in their ability to bind truncated peptide fragments. The antagonist BIIE0246, developed for mammalian Y2, did not bind either of the two rainbow trout receptors. Our results support the proposed expansion of this gene family by duplications before the gnathostome radiation. They also reveal appreciable differences in the repertoire and characteristics of NPY receptors between fish and tetrapods stressing the importance of lineage-specific gene loss as well as sequence divergence after duplication.
Keywords: NPY; Y2; Y7; Rainbow trout; G-protein coupled receptor;

Characterization and localization of cocaine- and amphetamine-regulated transcript (CART) binding sites by Patrick A. Keller; Valérie Compan; Joël Bockaert; Jean-Paul Giacobino; Yves Charnay; Constantin Bouras; Françoise Assimacopoulos-Jeannet (1328-1334).
Cocaine- and amphetamine-regulated transcript (CART) is widely expressed in the brain and various endocrine tissues. CART is implicated in many physiological functions including food intake, drug reward, stress and nociception. No CART receptor has been identified yet. We fused CART(55-102) to the green fluorescent protein (GFP) and found that the ligand suppresses significantly food intake after intracerebroventricular (i.c.v.) injection in mice. Using this ligand, we show specific CART binding sites on HepG2 cells and hypothalamic dissociated cells. In brain sections, CART displaceable binding sites were observed on cell bodies mainly localized in hypothalamic periventricular areas.
Keywords: CART receptor; GFP; Food intake; Hypothalamus; HepG2;

Stimulatory effect of n-octanoylated ghrelin on locomotor activity in the goldfish, Carassius auratus by Kouhei Matsuda; Tohru Miura; Hiroyuki Kaiya; Keisuke Maruyama; Minoru Uchiyama; Kenji Kangawa; Seiji Shioda (1335-1340).
Ghrelin is implicated in growth and feeding regulation in fish. The influence of ghrelin on behavior has not been well studied and the physiological role of des-fatty acid modification of this peptide is unclear. Therefore, the effects of intracerebroventricular (ICV) and intraperitoneal (IP) administration of synthetic n-octanoylated (acyl) goldfish ghrelin and des-n-octanoylated (des-acyl) ghrelin on locomotor and orexigenic activity in the goldfish were examined. ICV administration of acyl ghrelin at doses of 1 and 2 pmol/g body weight (BW) and IP administration at 16 pmol/g BW both induced significant increases in locomotor activity during for 45–60 min after treatment. Cumulative food intake was significantly increased by ICV injection of acyl ghrelin at doses of 1 and 2 pmol/g BW and IP injection at 8 and 16 pmol/g BW during the 60-min post-treatment observation period. In contrast, ICV and IP administration of des-acyl ghrelin produced no changes in locomotor and orexigenic activity. We also analyzed fasting-induced changes in the expression of ghrelin mRNA in the brain and intestine using a real-time PCR method. The level of ghrelin mRNA in the intestine, but not in the brain, obtained from fish fasted for 7 days was significantly higher than that in fish that had been fed normally. These results suggest that, in the goldfish, acyl ghrelin, but not des-acyl ghrelin, stimulates locomotor activity and enhances food intake via central and peripheral pathways.
Keywords: Goldfish; Ghrelin; Locomotor activity; ICV and IP administration; Ghrelin mRNA; Orexigenic action;

Normal circadian variations in vasoactive intestinal polypeptide, somatostatin, cholecystokinin and pancreatic polypeptide were measured to determine if these alter with aging and to identify gastrointestinal regulatory hormones that might control the dramatic diurnal variation in the gastric cytoprotective trefoil protein TFF2. Plasma pancreatic polypeptide concentrations showed a marked diurnal rhythm (p  < 0.0001). Basal and postprandial pancreatic polypeptide concentrations increased with age (p  < 0.01). The timing of the diurnal rhythm was consistent with pancreatic polypeptide inhibiting TFF2 secretion and there was a negative association between pancreatic polypeptide and TFF2 concentrations (p  < 0.002). The much higher pancreatic polypeptide concentrations in older people will induce increased satiety that may contribute to ‘anorexia of ageing’. These results identify potential therapies for treatment of gastric mucosal morbidity and age-associated loss of appetite.
Keywords: Human TFF2; Mucosal protection; Pancreatic polypeptide; Satiety; Diurnal rhythm; Age;

Pituitary adenylate cyclase-activating peptide (PACAP) is a member of the glucagon family of peptides. Like other members, most notably glucagon-like peptide-1 (GLP-1), PACAP is rapidly degraded by dipeptidylpeptidase IV (DPP IV). This study investigated how degradation by DPP IV affected the insulinotropic activity of PACAP, and whether PACAP exerted acute antihyperglycemic properties in normal or ob/ob mice. DPP IV degradation of PACAP(1–27) over 18 h led to the formation of PACAP(3–27), PACAP(5–27) and ultimately PACAP(6–27). In contrast to 1.4–1.8-fold concentration-dependent stimulation of insulin secretion by PACAP(1–27), these peptide fragments lacked insulinotropic activity. While PACAP(1–27) and PACAP(1–38) generated significant insulin responses when given alone or together with glucose in ob/ob and normal mice, they also elevated plasma glucose. These actions were eliminated following degradation of the peptide by incubation with DPP IV. The hyperglycemic effects may be explained at least partly by a potent glucagon-releasing action in ob/ob and normal mice. In conclusion, PACAP is inactivated by DPP IV and despite insulin-releasing effects, its actions on glucagon secretion and glucose homeostasis do not make it a good therapeutic tool for the treatment of type 2 diabetes.
Keywords: PACAP; Diabetes; Dipeptidyl peptidase IV; Glucose homeostasis; Insulin secretion;

Intein-mediated rapid purification and characterization of a novel recombinant agonist for VPAC2 by Rong-jie Yu; Qiu-ling Xie; Yun Dai; Yuan Gao; Tian-hong Zhou; An Hong (1359-1366).
In order to obtain the recombinant VPAC2 agonist efficiently by intein-mediated single column purification, a gene encoding 32-amino acids peptide was designed, synthesized and cloned into Escherichia coli expression vector pKYB. The recombinant vector pKY-ROM was transferred into E. coli ER2566 cells and the target protein was over-expressed as a fusion to the N-terminus of a self-cleavable affinity tag. After the rMROM–intein–CBD fusion protein was purified by chitin-affinity chromatography, the self-cleavage activity of the intein was induced by β-mercaptoethanol and the rMROM with the homogeneity over 95% was released from the chitin-bound intein tag. The recombinant linear rMROM competitively displaced [125I] PACAP38 on VPAC2 with a half-maximal inhibitory concentration (IC50) of 60 ± 5 nM, whereas the IC50 of rMROM at human VPAC1 was observed up to 10 μM and no binding was detected at PAC1. rMROM stimulated the cAMP accumulation in Chinese hamster ovary (CHO) cells expressing the human VPAC2 with a half-maximal stimulatory concentration (EC50) of 0.6 nM, which was 500-fold less potent at VPAC1and had no activity on PAC1. An efficient production procedure of a novel recombinant VPAC2-selective agonist was established.
Keywords: Intein; Recombinant; VPAC2 agonist; Purification;

Pharmacology of the human CGRP1 receptor in Cos 7 cells by Richard J. Bailey; Debbie L. Hay (1367-1375).
Only limited pharmacological characterization of the CGRP1 receptor, a heterodimer of the calcitonin (CT) receptor-like receptor (CL) and receptor activity-modifying protein 1 has been performed in cells that do not endogenously express RAMP2. We characterized the receptor in RAMP-deficient Cos 7 cells by measuring cAMP responses following agonist treatment in the absence or presence of antagonists. Potent cAMP responses to human α-and β-CGRP (Cys(Et)2,7)hαCGRP and human adrenomedullin (AM) were observed. Adrenomedullin15–52 was also an effective agonist of the CGRP1 receptor but human and salmon calcitonin and rat amylin were only weak agonists. As expected, BIBN4096BS and CGRP8–37 were effective antagonists of the CGRP1 receptor. (Cys(Acm)2,7)hαCGRP also antagonized CGRP responses. Antagonists of related receptors were only weakly able to inhibit CGRP responses.
Keywords: BIBN4096BS; Calcitonin receptor-like receptor; CGRP; CGRP1 receptor; Receptor activity modifying protein;

The effects of intravenous (iv) adrenomedullin (AM) on gastric emptying were investigated in conscious rats. AM induced a maximal 50% inhibition of gastric emptying at a dose of 1.2 nmol/kg. AM was about two-fold less potent than α-calcitonin gene-related peptide (α-CGRP), which induced a similar 50% maximal inhibition of gastric emptying at 0.6 nmol/kg. Delayed gastric emptying induced by i.v. AM and α-CGRP was prevented by peripheral injection of the selective CGRP1 antagonist, CGRP8–37, and by pretreatment with indomethacin, while not altered by blockade of the sympathetic nervous system with propranolol. These data indicate that peripheral AM inhibits gastric emptying through the interaction with CGRP8–37-sensitive receptors, likely CGRP1 receptors, and the recruitment of prostaglandin-dependent mechanisms.
Keywords: Adrenomedullin; β-adrenergic receptors; Gastric emptying; CGRP; CGRP8–37; Prostaglandins;

Immunocytochemical localization of adrenomedullin 2/intermedin-like immunoreactivity in human hypothalamus, heart and kidney by Kazuhiro Takahashi; Kumi Kikuchi; Yutaka Maruyama; Tomoko Urabe; Kiichiro Nakajima; Hironobu Sasano; Yutaka Imai; Osamu Murakami; Kazuhito Totsune (1383-1389).
Adrenomedullin 2/intermedin (AM2/IMD) is a novel member of the calcitonin/calcitonin gene-related peptide (CGRP) peptide family. AM2/IMD has a vasodilator action, and antidiuretic and antinatriuretic effects in mice. The aim of the present study is to clarify immunolocalization of AM2/IMD in human hypothalamus, heart and kidney obtained at autopsy. Immunocytochemistry showed AM2/IMD-immunoreactive cell bodies in the paraventricular and supraoptic nuclei of human hypothalamus. Both parvocellular and magnocellular cells in the paravetricular nucleus are immunostained with AM2/IMD. Immunostaining of serial sections showed co-localization of AM2/IMD-like immunoreactivity and vasopressin in the paraventricular nucleus. Myocardial cells of the heart and renal tubular cells were positively immunostained with AM2/IMD, whereas neither renal glomeruli nor vasculature in the heart and kidney were immunostained. Reverse-transcriptase polymerase chain reaction confirmed expression of AM2/IMD mRNA in the brain, pituitary, heart and kidney. The present study has shown the wide expression of AM2/IMD in human hypothalamus, heart and kidney, raising the possibility that this novel peptide may be related to the central and peripheral regulation of the circulation and water–electrolyte metabolism.
Keywords: Adrenomedullin; Intermedin; Immunocytochemistry; Hypothalamus; Heart; Kidney;

Intermedin/adrenomedullin-2 dilates the rat pulmonary vascular bed: Dependence on CGRP receptors and nitric oxide release by H. Burak Kandilci; Bulent Gumusel; Anatolio Wasserman; Norman Witriol; Howard Lippton (1390-1396).
Intermedin/adrenomedullin-2 (IMD/AM2) is a 47 amino acid peptide formed by enzymatic degradation of preprointermedin. The present study was undertaken to investigate the effects of rat IMD (rIMD) in the isolated buffer perfused rat lung (IBPR) under resting conditions and under conditions of elevated pulmonary vasoconstrictor tone (PVT). Under resting conditions in the IBPR, rIMD had little or no activity. When PVT was actively increased by infusion of U46619, bolus injection of IMD decreased pulmonary arterial pressure (PAP) in a dose-dependent manner. Since the pulmonary perfusion rate and left atrial pressure were constant, these reductions in PAP directly reflect reductions in pulmonary vascular resistance (PVR). The pulmonary vasodilator response to rIMD, when compared to calcitonin gene-related peptide (CGRP) on a molar basis, was greater at the lowest and midrange doses. The degree of inhibition by CGRP8–37 on pulmonary vasodilator response to rIMD was significantly less when compared to CGRP. Pretreatment with l-nitro-arginine-methyl ester (l-NAME), unlike meclofenamate and glybenclamide, significantly reduced the pulmonary vasodilator responses to rIMD. rIMD administration induced cross-tachyphylaxis to the pulmonary vasodilator response to CGRP whereas CGRP administration did not alter the ability of rIMD to dilate the IBPR. Pulmonary vasodilator responses to repeated injections of rIMD did not undergo tachyphylaxis. The present data demonstrate rIMD possesses direct vasodilator activity in the rat pulmonary vascular bed. The present data suggest activation of CGRP1 receptors and release of nitric oxide (NO•) mediate the pulmonary vasodilator response to rIMD whereas cyclooxygenase products and KATP channels do not contribute to the pulmonary vasodilator response to rIMD. The ability of rIMD to induce heterologous desensitization of CGRP1 receptor activation, to retain much of its pulmonary vasodilator activity after inhibition of CGRP1 receptors, and to lack homologous desensitization together suggests the pulmonary, unlike the systemic, vasodilator response to rIMD may depend on other vasodilator mechanisms including receptors in the calcitonin-receptor-like-receptor (CRLR) family.
Keywords: Intermedin/adrenomedullin-2; Pulmonary vascular bed; Nitric oxide; CGRP receptors; Cross-tachyphylaxis;

Immunohistochemical mapping of adrenomedullin in the human medulla oblongata by Veronica Macchi; Andrea Porzionato; Anna Sandra Belloni; Carla Stecco; Anna Parenti; Raffaele De Caro (1397-1404).
We studied by immunocytochemistry the expression of adrenomedullin (AM) in the human medulla oblongata, sampled from 13 adult subjects (mean age: 38 years), whose medical history was negative for neurological and neurovascular pathologies. Immunoreactive neurons were found in the medulla oblongata with statistically significant differences among the various nuclei (one-way ANOVA, P  < 0.001). The hypoglossal nucleus showed higher AM expression than that of the spinal tract of the trigeminal nerve (P  < 0.05), solitary tract nucleus (P  < 0.05), nucleus intercalatus (P  < 0.05), and area postrema (P  < 0.05). The arcuate nucleus and inferior olivary nuclear complex showed lower AM expression than the hypoglossal nucleus (P  < 0.001), vestibular nuclei (P  < 0.01), cuneate and gracile nuclei (P  < 0.05), lateral column of the reticular formation (P  < 0.05), and nucleus ambiguous (P  < 0.05). Furthermore the nuclei were grouped with reference to their function, into somatic sensitive nuclei, somatic motor nuclei, visceral nuclei, reticular formation, and nuclei involved in cerebellar functions. The ANOVA revealed statistically significant differences (P  < 0.001) in mean AM scores among the different groups. Nuclei involved in cerebellar function showed the lowest mean AM score (P  < 0.05). The difference in AM score between somatic motor nuclei and visceral nuclei was also statistically significant (P  < 0.05). Widespread AM immunoreactivity in the nuclei of the medulla oblongata may account for the role of the peptide in neuronal function and regulation of regional blood flow. Differences in the expression of AM in the nuclei studied indicate the different involvement of AM in neurotransmission and neuromodulation.
Keywords: Adrenomedullin; Medulla oblongata; Immunohistochemistry; Human;

Identification of adipocyte differentiation-related regulatory element for adrenomedullin gene repression (ADRE-AR) in 3T3-L1 cells by Yin Li; Yan Zhang; Kazumichi Furuyama; Satoru Yokoyama; Kazuhisa Takeda; Shigeki Shibahara; Kazuhiro Takahashi (1405-1414).
Adrenomedullin (AM), a potent vasodilator peptide, has been suggested to act against cardiovascular complications and insulin resistance in the metabolic syndrome. We have already reported the AM gene repression in the early phase of adipocyte differentiation of NIH 3T3-L1 cells. Here we show adipocyte differentiation-related regulatory element for AM gene repression (ADRE-AR) in 36-bp region (−2135/−2100) of the AM gene. 3T3-L1 cells were induced to differentiate to adipocytes by insulin, dexamethasone and 3-isobutyl-1-methylxanthine. On the third day of differentiation, the promoter function was analyzed using the reporter plasmids, which contain the promoter region of AM gene (−4616/+108) in pGL3-basic luciferase reporter vector. The promoter activity decreased to about 20% in 3T3-L1 adipocytes when compared with 3T3-L1 preadipocytes, and a 36-bp region (−2135 to −2100) upstream from the transcription initiation site of the AM gene was necessary for higher AM gene expression in preadipocytes. This 36-bp ADRE-AR contains three copies of G/AAAA sequence (5′-GAAATGAAAGTAAAA-3′) (−2124/−2110), which are conserved between mouse and human, and the introduction of mutations in each copy of G/AAAA sequence decreased the promoter activity in preadipocytes and adipocytes. Electrophoretic mobility shift assay showed that the full-length ADRE-AR was specifically bound by a certain nuclear protein(s). The present study has raised the possibility that ADRE-AR may play important roles in the AM gene expression in preadipocytes, and that the AM gene may be repressed through the ADRE-AR in adipocytes.
Keywords: Adrenomedullin; Adipocytes; Differentiation; Gene; Expression;

Uncorrected obesity is often accompanied by ventricular contractile dysfunction, elevation of the lipotoxic mediator ceramide and the obesity gene product leptin. Both ceramide and leptin participate in the regulation of cardiac function and are speculated to play roles in obesity-related cardiac dysfunctions. The purpose of this study was to examine the effect of ceramide on leptin-elicited cardiac contractile response. Adult rat left ventricular myocytes were incubated for 24 h with low (5 nM) or high (50 nM) concentration of leptin in the absence or presence of the active ceramide analog C2-dihydroceramide (25 μM). Contractile and intracellular Ca2+ properties were evaluated using an IonOptix MyoCam® system including peak shortening (PS), maximal velocity of shortening/relengthening (±dL/dt), time-to-PS (TPS), time-to-90% relengthening (TR90), intracellular Ca2+ rise (Δ[Ca2+]) and intracellular Ca2+ decay. While ceramide did not elicit any effect on cell mechanics and intracellular Ca2+ transients, it sensitized leptin-induced effects on myocyte shortening and intracellular Ca2+ transients. In the absence of ceramide, 5 nM leptin had no effect on cell mechanics while 50 nM depressed PS, ±dL/dt, Δ[Ca2+] and prolonged TR90. With ceramide co-incubation, 5 nM leptin depressed PS, ±dL/dt, Δ[Ca2+] and prolonged TR90 whereas 50 nM leptin-elicited effects on PS, ±dL/dt, Δ[Ca2+] and TR90 were significantly potentiated in addition to slowing intracellular Ca2+ decay. In summary, our data demonstrated that ceramide sensitizes cardiac depressive effects of leptin and may contribute to hyperleptinemia-related cardiac contractile dysfunction.
Keywords: Leptin; Ceramide; Cardiac; Myocyte shortening; Intracellular Ca2+ transients;

Effects of leptin on memory processing by Susan A. Farr; William A. Banks; John E. Morley (1420-1425).
Leptin is a peptide hormone secreted by adipose tissue. Studies have shown that leptin crosses the blood–brain barrier (BBB) by a saturable transport system where it acts within the hypothalamus to regulate food intake and energy expenditure. Leptin also acts in the hippocampus where it facilitates the induction of long-term potentiation and enhances NMDA receptor-mediated transmission. This suggests that leptin plays a role in learning and memory. Obese mice and rats, which have leptin receptor deficiency, have impaired spatial learning. In disease states such as diabetes, humans and animals develop leptin resistance at the BBB. This suggests that low leptin levels in the brain may be involved in cognitive deficits associated with diabetes. In the current study, the effects of leptin on post-training memory processing in CD-1 mice were examined. Mice were trained in T-maze footshock avoidance and step down inhibitory avoidance. Immediately after training, mice received bilateral injections of leptin into the hippocampus. Retention was tested 1 week later in the T-maze and 1 day later in step down inhibitory avoidance. Leptin administration improved retention of T-maze footshock avoidance and step down inhibitory avoidance. Leptin administered 24 h after T-maze training did not improve retention when tested 1 week after training. SAMP8 mice at 12 months of age have elevated amyloid-beta protein and impaired learning and memory. We examined the effect of leptin on memory processing in the hippocampus of 4 and 12 months old SAMP8 mice. Leptin improved retention in both 4 and 12 months old SAMP8 mice; 12 month SAMP8 mice required a lower dose to improve memory compared to 4 months SAMP8 mice. The current results indicate that leptin in the hippocampus is involved in memory processing and suggests that low levels of leptin may be involved in cognitive deficits seen in disease states where leptin transport into the CNS is compromised.
Keywords: Hippocampus; Leptin; Memory; Mouse; SAMP8;

Different skeletal regional response to continuous brain infusion of leptin in the rat by F. Guidobono; F. Pagani; V. Sibilia; C. Netti; N. Lattuada; D. Rapetti; E. Mrak; I. Villa; F. Cavani; L. Bertoni; C. Palumbo; M. Ferretti; G. Marotti; A. Rubinacci (1426-1433).
This study was designed to evaluate whether or not continuous intracerebroventricular infusion of leptin (1.5 μg/rat/24 h, for 28 days) produced different regional response on the skeleton of growing rats. Leptin reduce the accretion of total femoral bone mineral content (BMC) and density (BMD). This effect was related to a reduction of metaphyseal femur as no changes were detected in the diaphysis. Despite the reduced accretion in the volumetric of both femur and tibia compared to controls, leptin had no significant effects on the lumbar vertebrae. Urine deoxypyrydinoline and serum osteocalcin remained more elevated in the leptin-treated group as compared to controls. The results demonstrate that long-term central infusion of leptin activates bone remodeling with a negative balance. Leptin induces distinct responses in the different structure of bone and in the axial and appendicular skeleton.
Keywords: Leptin; Planar bone mineral density; DXA; Volumetric bone mineral density; pQCT; Osteocalcin; Deoxypyridinoline;

Matrix metalloproteinases (MMPs) constitute a family of zinc-dependent proteolytic enzymes, which degrade several components of extracellular matrix, in arthritic synovial cells. In cultured synovial fibroblasts, both nitric oxide (NO) and reactive oxygen species (ROS) are potent inducers of MMPs production. PEP1261, a tetrapeptide derivative used in this study, corresponds to residues of 39–42 human lactoferrin. The parent protein lactoferrin is able to inhibit the production of free radicals in rheumatoid joints and it regulates many aspects of inflammation. This study is aimed to examine the effects of PEP1261 on MMP-2 production in the presence of nitric oxide donor in cultured synovial fibroblasts from collagen-induced arthritic rats. PEP1261 affects a significant reduction in nitrite levels as well as in MMP-2 production in SNAP stimulated synovial fibroblasts and this is validated by gelatin zymography and immunoblot analysis. Furthermore, RTPCR analysis has demonstrated that PEP1261 inhibits MMP-2 mRNA expression in SNAP treated synovial fibroblasts. The results of this study suggest that PEP1261 possesses antiarthritic activity by inhibiting nitrite levels as well as MMP-2 expression better than control peptides viz., KRDS and RGDS.
Keywords: Lactoferrin; Tetrapeptide; Nitric oxide; Matrix metalloproteinase; Synovial fibroblasts; Collagen-induced arthritis;

The MC3 receptor binding affinity of melanocortins correlates with the nitric oxide production inhibition in mice brain inflammation model by Ruta Muceniece; Liga Zvejniece; Edgars Liepinsh; Olga Kirjanova; Larisa Baumane; Ramona Petrovska; Felikss Mutulis; Ilze Mutule; Ivars Kalvinsh; Jarl E.S. Wikberg; Maija Dambrova (1443-1450).
Melanocortins possess strong anti-inflammatory effects acting in the central nervous system via inhibition of the production of nitric oxide (NO) during brain inflammation. To shed more light into the role of melanocortin (MC) receptor subtypes involved we synthesized and evaluated some novel peptides, modified in the melanocyte-stimulating hormone (MSH) core structure, natural MCs and known MC receptor selective peptides – MS05, MS06. Since the study included both selective, high affinity binders and the novel peptides, it was possible to do the correlation analysis of binding activities and the NO induction-related anti-inflammatory effect of the peptides. β-MSH, γ1-MSH, γ2-MSH, α-MSH, MS05, Ac-MS06 and Ac-[Ser12]MS06 caused dose dependent inhibition of the lipopolysaccharide (LPS)-induced increase of NO overproduction in the mice forebrain whereas MSH core modified peptides Ac-[Asp9,Ser12]MS06, [Asp9]α-MSH and [Asp16]β-MSH were devoid of this effect in doses up to 10 nmol per mouse. When the minimal effective dose required for inhibition of NO production was correlated with the in vitro binding activity to MC receptor subtypes a strong and significant correlation was found for the MC3 receptor (r  = 0.90; p  = 0.0008), whereas weak correlation was present for the other receptors. Our results suggest that the MC3 receptor is the major player in mediating the anti-inflammatory activity of MCs in the central nervous system.
Keywords: Melanocortins; MC receptors; Brain inflammation; Nitric oxide;

Anxiety-like behavior induced by IL-1β is modulated by α-MSH through central melanocortin-4 receptors by Andrea Beatríz Cragnolini; Helgi Birgir Schiöth; Teresa Nieves Scimonelli (1451-1456).
The proinflammatory cytokine interleukin-1β (IL-1β) influences neuroendocrine activity and produces other effects, including fever and behavioral changes such as anxiety. The melanocortin neuropeptides, such as alpha-melanocyte-stimulating hormone (α-MSH), antagonize many actions of IL-1, including fever, anorexia and hypothalamic-pituitary-adrenal (HPA) axis activation through specific melanocortin receptors (MC-R) in the central nervous system. The objective of the present study was to establish the effect of MSH peptides on IL-1β-induced anxiety-like behavior and the melanocortin receptors involved. We evaluated the effects of intracerebroventricular (i.c.v.) administration of IL-1β (30 ng) and melanocortin receptor agonists: α-MSH, an MC3/MC4-R agonist (0.2 μg) or γ-MSH, an MC3-R agonist (2 μg) or HS014, an MC4-R antagonist (2 μg), on an elevated plus-maze (EPM) test. Injection of IL-1β induced an anxiogenic-like response, as indicated by reduced open arms entries and time spent on open arms. The administration of α-MSH reversed IL-1β-induced anxiety with co-administration of HS014 inhibiting the effect of α-MSH. However, the associated treatment with γ-MSH did not affect the anxiety response to IL-1β. These data suggest that α-MSH, through central MC4-R can modulate the anxiety-like behavior induced by IL-1β.
Keywords: IL-1β; α-MSH; γ-MSH; Melanocortin receptors; Anxiety-like behavior;

Elevated maternal cortisol early in pregnancy predicts third trimester levels of placental corticotropin releasing hormone (CRH): Priming the placental clock by Curt A. Sandman; Laura Glynn; Christine Dunkel Schetter; Pathik Wadhwa; Thomas Garite; Aleksandra Chicz-DeMet; Calvin Hobel (1457-1463).
The purposes of this study were to determine the intervals when placental corticotrophic-releasing hormone (CRH) was most responsive to maternal cortisol. A sample of 203 women each were evaluated at 15, 19, 25 and 31 weeks gestation and followed to term. Placental CRH and maternal adrenocorticotropin hormone (ACTH), B-endorphin and cortisol were determined from plasma. CRH levels increased faster and were higher in women who delivered preterm compared with women who delivered at term (F 3,603  = 5.73, p  < .001). Simple effects indicated that CRH levels only at 31 weeks predicted preterm birth (F 1,201  = 5.53, p  = .02). Levels of cortisol were higher in women who delivered preterm at 15 weeks gestation (F 1,201  = 4.45, p  = .03) with a similar trend at 19 weeks gestation. Hierarchical regression suggested that the influence on birth outcome of maternal cortisol early in pregnancy was mediated by its influence on placental CRH at 31 weeks. Elevated cortisol at 15 weeks predicted the surge in placental CRH at 31 weeks (R  = .49, d.f. = 1,199, F change  = 61.78, p  < .0001). Every unit of change in cortisol (μg/dl) at 15 weeks was associated with a 34 unit change of CRH (pg/ml) at 31 weeks. These findings suggested that early detection of stress signals by the placenta stimulated the subsequent release of CRH and resulted in increased risk for preterm delivery.
Keywords: CRH; Cortisol; Preterm; Pregnancy; Stress; HPA;

Endogenous expression and in vitro study of CRF-related peptides and CRF receptors in the rat gastric antrum by Christophe Porcher; André Peinnequin; Sonia Pellissier; Julien Meregnani; Valérie Sinniger; Frédéric Canini; Bruno Bonaz (1464-1475).
In vivo studies suggest that corticotrophin-releasing factor (CRF) and CRF-like peptides, urocortin 1 (UCN 1) and UCN 2, inhibit gastric emptying and stimulate colonic motility through CRF2 and CRF1 receptors, respectively. We evaluated expression and functions of CRF, UCN 1, UCN 2 and CRF1 and CRF2 receptors in the rat gastric antrum. Tissues were processed for immunohistochemistry and real-time quantitative reverse transcription polymerase chain reaction (RT-PCR). In vitro studies were performed to test the functional significance of CRF, UCN 1 and UCN 2. Some experiments were realized in the presence of specific CRF1 or CRF2 receptors antagonists. CRF1 and CRF2 receptors-like immunoreactivity (CRF1 and CRF2 receptors-LI) was localized in fibers and neurons of the myenteric ganglia. CRF1 and CRF2 receptors-LI was also found in nerve fibers distributed in the muscle layers. CRF- and UCN 1-LI was observed in neuronal cell bodies of the myenteric ganglia and in numerous nerve fibers running parallel to smooth muscle cells. Quantitative RT-PCR demonstrated UCN 2, CRF1 and CRF2 receptors expressions in both muscle layers and mucosa of the gastric antrum. Functional studies showed that CRF, UCN 1 and UCN 2 decreased antral phasic contractions. CRF1 receptor antagonist (CP-154,526) did not block CRF-like peptides-induced inhibition of antral motility. In contrast, a CRF2 receptor antagonist (Astressin2-B) blocked the effects of CRF-like peptides on the antral muscle contractions. These results demonstrate (1) the presence of CRF, UCN and CRF1 and CRF2 receptors in the rat gastric antrum; (2) that, in vitro, CRF-like peptides inhibit phasic contractions of the antrum through CRF2 receptor. These results strongly suggest that CRF-like peptides play a major role in the regulatory mechanisms that underlie the neural control of gastric motility through CRF2 receptor.
Keywords: Urocortin 1; Urocortin 2; CRF; CRF receptors; CRF receptor antagonists; Stomach;

A tryptic hydrolysate from bovine milk αS1-casein improves sleep in rats subjected to chronic mild stress by Benjamin Guesdon; Michaël Messaoudi; Catherine Lefranc-Millot; Gilles Fromentin; Daniel Tomé; Patrick C. Even (1476-1482).
The putative effects of a tryptic bovine αS1-casein hydrolysate on stress-induced sleep disorders were investigated and their possible link with typical blood stress parameters such as plasma corticosterone concentrations and glycaemia was assessed. Rats were subjected to chronic stress in the form of environmental disturbances, while receiving an oral administration of the αS1-casein hydrolysate (CH). Chronic stress significantly reduced sleep duration in control rats during the first 2 days of the stress period, but stress-induced sleep disturbance was prevented in CH-treated rats. Indeed, CH administration allowed the maintenance of slow wave sleep (SWS) duration and even a slight increase in paradoxical sleep (PS) duration in treated rats. Results on plasma corticosterone concentrations and on glycemia values were inconclusive with respect to the implication of the HPA axis in this study. However, the protective effect of the αS1-casein hydrolysate on sleep during exposure to our chronic mild stress conditions may be mediated by modulation of the central adrenergic response.
Keywords: Sleep; Stress; αS1-Casein hydrolysate; Corticosterone;

Stress and pain responses in rats lacking CCK1 receptors by I. Hurwitz; O. Malkesman; Y. Stern; M. Schroeder; Y. Lavi-Avnon; M. Shayit; Y. Shavit; G. Wolf; R. Yirmiya; A. Weller (1483-1489).
CCK involvement in stress- and pain-responsiveness was examined by studying the behavior of infant (11–12-days-old) and adult OLETF rats that do not express CCK1 receptors. Infant odor- and texture-preferences were also assessed. We hypothesized that OLETF rats will show behavioral patterns similar to those previously observed after CCK1 antagonist administration. Rate of separation-induced ultrasonic vocalization was significantly greater in OLETF compared to controls, in two separate studies. Infant pups of the two strains did not differ in odor- and texture-preference tests. OLETF rats showed consistently longer hot-plate paw-lift (as infants, in two separate studies) and paw-lick (as adults) latencies. Summary: OLETF pups vocalized in isolation more than controls and showed relative hypoalgesic responses, evident also in adulthood, in concordance with the pharmacological literature.
Keywords: USV; Pain; Anxiety; Natural preference; CCK; OLETF rats;

The N-terminal metabolite of the undecapeptide substance P (SP), substance P1–7 (SP1–7), is known to modulate nociception in the central nervous system (CNS) and often has opposite effects from SP. This study investigated the ability of SP1–7 to modulate the vasodilatation response to SP in anaesthetized rats under different injury conditions using a blister model of inflammation on the hind footpad. The results indicated that SP1–7 inhibited the vascular response to SP in a dose-dependent manner. The putative antagonists naloxone and d-Pro2-d-Phe7-SP1–7 (d-SP1–7) reversed the effect of SP1–7. d-SP1–7 improved the responsiveness to SP under chronic nerve injury, which suggests a role for endogenous SP1–7 in this model. SP1–7 did not inhibit the response to electrical stimulation of the sciatic nerve, which indicates that the heptapeptide interacts at a post-terminal binding site. The current results suggest that SP1–7 may have inhibitory properties in inflammation, analogous to its antinociceptive role in the central nervous system.
Keywords: Inflammation; Nerve injury; Opioid antagonist; Substance P (SP); Substance P fragments SP1–7, SP5–11 and SP9–11; Rats; Vasodilatation;

Proenkephalin peptide F immunoreactivity in different circulatory biocompartments after exercise by Jill A. Bush; Andrea M. Mastro; William J. Kraemer (1498-1506).
This study was the first study to examine the three circulatory biocompartments (plasma, white blood cell layer (WBC) and red blood cell layer (RBC)) and determine PF concentrations before and after exercise. Proenkephalin peptide F (PF) is an enkephalin-containing peptide found predominantly within the adrenal medulla. PF is co-packaged with epinephrine, and both can be co-secreted in response to similar stimuli. PF and epinephrine have shown immunomodulating properties. Ten healthy resistance trained men performed six sets of 10 RM squats with 2 min rest periods between sets and 10 healthy active men were matched and served as resting controls. Blood samples were obtained pre-exercise, immediately post-exercise and 15 min post-exercise and were analyzed for lactate, cortisol, epinephrine and biocompartmentalized PF. There was no change in resting control values measured across time within respective PF biocompartments and endocrine profile, indicating stability and technique validity of peptide F across the time period measured. As expected, the acute resistance exercise protocol caused an increase in lactate at 15 min post-exercise. Circulating epinephrine increased immediately post-exercise and returned to baseline during 15 min into recovery. Plasma PF increased immediate post-exercise and 15 min post-exercise, while WBC-PF and RBC-PF only increased at 15 min into recovery. For all time points tested, resting and exercise WBC-PF and RBC-PF concentrations were lower than plasma PF thus indicating a concentration difference across the three different biocompartments within the same blood sample. The presence of PF within all three biocompartments of whole blood may indicate the potential for biological transport and interactions with other cells in other biocompartments of the blood.
Keywords: Opioid peptides; Exercise; White blood cells; Epinephrine; Adrenal medulla;

Partial and full agonism in endomorphin derivatives: Comparison by null and operational model by András Z. Rónai; Mahmoud Al-Khrasani; Sándor Benyhe; Imre Lengyel; László Kocsis; György Orosz; Géza Tóth; Erzsébet Kató; László Tóthfalusi (1507-1513).
The partial μ-opioid receptor pool inactivation strategy in isolated mouse vas deferens was used to determine partial agonism of endomorphins and their analogs (endomorphin-1-ol, 2′,6′-dimethyltyrosine (Dmt)-endomorphin-1, endomorphin-2-ol and (d-Met2)-endomorphin-2) using morphine, normorphine, morphiceptin, (d-Ala2,MePhe4,Gly5-ol)-enkephalin (DAMGO) and its amide (DAMGA) as reference opioid agonists. Agonist affinities (K A) and efficacies were assessed both by the “null” and the “operational” method. The K A values determined by the two methods correlated significantly with each other and also with the displacing potencies against 3H-naloxone in the receptor binding assay in the presence of Na+. DAMGO and DAMGA were full agonist prototypes, morphine, endomorphin-1, endomorphin-1-ol, Dmt-endomorphin-1, endomorphin-2-ol and (d-Met2)-endomorphin-2 were found by both methods to be partial agonists whereas the parameters for normorphine, morphiceptin and endomorphin-2 were intermediate.
Keywords: Endomorphin derivatives; Partial agonism; Residual receptor fraction; β-Funaltrexamine; Mouse vas deferens; Null and operational model;

Role of endothelin (ETA) receptors in neonatal morphine withdrawal by Bhagya L. Puppala; Shaifali Bhalla; George Matwyshyn; Anil Gulati (1514-1519).
We have previously demonstrated role of central endothelin (ET) receptors in neonatal morphine tolerance. The present study was conducted to investigate involvement of central ET receptors in neonatal rat morphine withdrawal. The aim was to determine activation of G-proteins coupled to opioid and ET receptors by morphine and ET ligands in neonatal rat brains during morphine withdrawal. Pregnant female rats were rendered tolerant to morphine by chronic exposure to morphine pellets over 7 days. Withdrawal was induced on day 8 by removal of pellets. Rat pups were delivered by cesarean section 24 h after pellet removal. G-protein stimulation induced by morphine; ET-1; ETA receptor antagonist, BMS182874; and ETB receptor agonist, IRL1620, was determined in the brain of neonatal rats undergoing morphine withdrawal by [35S]GTPγS binding assay. Morphine-induced maximal stimulation of G-protein in morphine withdrawal group (83.60%) was significantly higher compared to placebo control group (66.81%). EC50 value for ET-1-induced G-protein stimulation during morphine withdrawal (170.60 nM) was higher than control (62.5 nM). BMS182874, did not stimulate GTP binding in control but significantly increased maximal stimulation of G-proteins in morphine withdrawal (86.07%, EC50  = 31.25 nM). IRL1620-induced stimulation of G-proteins was similar in control and morphine withdrawal. The present findings indicate involvement of central ETA receptors in neonatal morphine withdrawal.
Keywords: Morphine withdrawal; Central nervous system; Neonatal rats; Endothelin; BMS182874 (5-(dimethylamino)-N-(3,4-dimethyl-5-isoxazolyd)-1-naphthalenesulfonamide); IRL1620 ({N-Suc-[Glu9,Ala11,15]ET-1(8–21)}; [35S]GTPγS binding;

The cardiovascular responses to mu opioid agonist and antagonist in conscious normal and obese rats by Crystal Hill-Pryor; DaShawnda Lindsey; Karen Lapanowski; Joseph C. Dunbar (1520-1526).
Beta-endorphin decreases blood pressure in normal rats but increases blood pressure in obese rats. Since beta-endorphins can bind both mu opioid and kappa-opioid receptors we investigated the effect of a mu specific receptor agonist, d-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO) and a mu specific antagonist, d-Phe-Cys-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) on cardiovascular responses in conscious control and obese rats. Rats were also implanted with telemetry transmitters and intracerebroventricular (ICV) cannulas for recording and peptide administration. The mu agonist, DAMGO, increased blood pressure (BP) in control rats. DAMGO also increased BP in obese rats but only at high concentrations. The heart rate responses paralleled the MAP responses. CTAP, the mu antagonist, paradoxically increased the MAP in both control and obese rats. The responsiveness to the mu agonist and antagonist was greater in controls. In other animals the brains were excised and the ventral medial hypothalamic area removed and mu receptor expression determined using PCR. The expression of mu opioid receptors was increased in obese rats. We conclude that the mu opioids can stimulate cardiovascular responses, but the excitatory responsiveness was not increased in conscious obese rats.
Keywords: Opioid receptors; Obesity; Blood pressure; Fat diet;

Urotensin II (UII) is a potent vasoactive cyclic peptide thought to play a role in myocardial hypertrophy and remodelling. We therefore determined UII plasma levels in congestive heart failure (CHF) patients and its relationship with the severity of the disease and well-established markers of left ventricular function. UII was significantly higher in CHF patients (n  = 57) than in controls (n  = 48) [geometric mean (pg/ml), 95% PI: 1.32 (0.67–2.59) versus 0.84 (0.31–1.61), p  < 0.0001], was related to the functional class of the disease and correlated negatively with left ventricular ejection fraction (r  = −0.316, P  = 0.016). Furthermore, UII correlated significantly with Big-ET1 (r  = 0.32, p  = 0.03), BNP (r  = 0.42, p  = 0.005) but poorly with Nt-proANP (r  = 0.28, p  = 0.07). Our results suggest that UII could play a role in worsening the course of congestive heart failure and is associated with established markers of cardiovascular dysfunction.
Keywords: Urotensin II; Neurohormonal markers; Congestive heart failure;

Urotensin-II (U-II), a ligand for the G-protein-coupled receptor UT, has been characterized as the most potent mammalian vasoconstrictor identified to date. Although circulating levels of U-II are altered in lower species (e.g., fish) upon exposure to hypo-osmotic stress, little is known about the actions of this cyclic undecapeptide within the kidney, an organ that plays a pivotal role in the control of cardiovascular homeostasis, influencing both cardiac preload (plasma volume) and after load (peripheral resistance). The present study reports the identification of specific, high affinity [125I]hU-II binding sites in Sprague–Dawley rat kidney outer medulla by autoradiography and also through membrane radioligand binding (K d 1.9 ± 0.9 nM and B max 408 ± 47 amol mm−2 and K d 1.4 ± 0.3 nM and B max 51.3 ± 7.8 fmol mg−1 protein, respectively). Differences were observed in the binding characteristics within rat strains. Compared to the Sprague–Dawley, Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rat kidney outer medulla displayed low density <20 fmol mg−1 protein and low affinity (>1 μM) [125I]hU-II binding sites. Thus, the relative contribution of specific U-II binding sites to the physiological actions of U-II in the control of cardiorenal homeostasis is worthy of further investigation.
Keywords: Urotensin-II; UT; Autoradiography;

Effects of central injection of angiotensin-converting-enzyme inhibitor and angiotensin type 1 receptor antagonist on the brain NF-κB and AP-1 activities of rats given LPS by Kana Watanabe; Makoto Taniguchi; Michio Miyoshi; Hideki Shimizu; Toshiaki Imoto; Kenzo Sato; Tatsuo Watanabe (1538-1546).
Angiotensin II (ANG II) activation of the angiotensin type 1 (AT1) receptor facilitates the production of brain interleukin-1β (IL-1β) and contributes to the induction of the fever following the intracerebroventricular (i.c.v.) injection of lipopolysaccharide (LPS). The purpose of the present study was to investigate whether proinflammatory transcription factors [nuclear factor-κB (NF-κB) and activator protein-1 (AP-1)] contribute to the ANG II-dependent production of cytokines within the brain. Interestingly, we found that a single i.c.v. injection of LPS had no effect on NF-κB and AP-1 activities in the hypothalamus, hippocampus, and cerebellum at either 1 or 3 h post-injection (except for a decrease in hypothalamic AP-1 activity at 1 h). Furthermore, both an angiotensin-converting-enzyme (ACE) inhibitor and an AT1 receptor antagonist enhanced (rather than reduced) the NF-κB and AP-1 activities in the hippocampus and/or cerebellum of rats given LPS. In contrast, an i.c.v. injection of ANG II increased the NF-κB activity in the hypothalamus. These results suggest that while “endogenous” ANG II exerts (via AT1 receptors) inhibitory effects on the activation of transcription factors in the brain of rats given LPS, a large dose of exogenous ANG II produces effects opposite to those induced by the presumably small amount of endogenous ANG II released locally by LPS. Our results seem not to support the idea that NF-κB and AP-1 play key roles in the ANG II-induced enhancement of the production of proinflammatory cytokines that is induced by LPS in the rat's brain.
Keywords: Angiotensin II; LPS; IL-1; NF-κB; AP-1;

Murine atrial HL-1 cells express highly active peptidylglycine α-amidating enzyme by William J. Driscoll; Diane Hill; Alexi Smalstig; Gregory P. Mueller (1547-1553).
Peptidylglycine-α-hydroxylating monooxygenase (PHM; EC catalyzes the rate limiting step in peptide α-amidation, a posttranslational modification that is essential for receptor recognition and signal transduction. Secretory granules of the cardiac atrium contain the highest natural concentration of PHM and clearly demonstrate regulation of PHM expression and activity. The HL-1 atrial myocyte cell line faithfully maintains the differentiated phenotype of native atrial cells and thus provides an in vitro model system for investigating the mechanisms that regulate PHM. We observed that the specific activity of PHM expressed in HL-1 cells is five times higher than that found in rat atrium. The increased activity of HL-1 cell PHM was not reflected by a difference in K m for peptide substrate, change in copper optimum, altered sensitivity to inactivation by suicide inhibitor or variance in response to limited proteolysis by trypsin. Additionally, mixing experiments indicated that the increased activity in HL-1 cells versus rat atrium was not due to a diffusible factor. Based upon these findings we propose that the increased V max of HL-1 cell PHM results from a structural or conformational difference that involves either differential posttranslational modification and/or a high affinity chaperone that serves to regulate enzymatic activity by protein–protein interaction. The mechanism involved may participate in physiologic regulation of PHM.
Keywords: Cardiac atrium; α-Amidating enzyme; Secretory granules; Atrial natriuretic peptide; HL-1 cells;

Studies on cleavage of DNA by N-phosphoryl branched peptides by Yuping Feng; Shengli Cao; Anshan Xiao; Wenjun Xie; Yanmei Li; Yufen Zhao (1554-1560).
It was found that N α,N ɛ-di[N-(O,O-diisopropyl)phosphoryl-l-leucy]-l-lysyl-methyl ester (1) and N α,N ɛ-di[N-(O,O-diisopropyl)phosphoryl-l-phenylalanyl]-l-lysyl-methyl ester (2) could cleave supercoiled DNA such as PUC19 efficiently in 40 mM Britton-Robinson buffer. The cleavage activities for both were investigated by agarose gel electrophoresis. The T4 ligase experiments implied that the cleavage of DNA occurs via a hydrolytic path. The results showed that the cleavage reaction of DNA is dependent on the value of pH and ionic strength in the solution. DNA cleavage is more efficient by N-phosphoryl branched peptide 2 than by N-phosphoryl branched peptide 1. The experiments also show that hydrolysis of DNA by N-phosphoryl branched peptide 1 was accelerated in the presence of Mg2+ or Zn2+ ions. The interactions of DNA with N-phosphoryl branched peptides were also characterized by melting temperature measurements and circular dichroism (CD) techniques. On the basis of experimental data, the possible mechanism of interactions between DNA with N-phosphoryl branched peptides was discussed.
Keywords: N-Phosphoryl branched peptide; DNA; Cleavage; Hydrolysis; Interaction;

Catabolism of the octadecaneuropeptide ODN by prolyl endopeptidase: Identification of an unusual cleavage site by Jérôme Leprince; David Cosquer; Gaëlle Bellemère; David Chatenet; Hélène Tollemer; Sylvie Jégou; Marie-Christine Tonon; Hubert Vaudry (1561-1569).
The octadecaneuropeptide ODN (QATVGDVNTDRPGLLDLK), a biologically active fragment of diazepam-binding inhibitor, exerts a number of behavioral and neurophysiological activities. The presence of a proline residue in the sequence of ODN led us to investigate the role of proline endopeptidase (PEP) in the catabolism of this neuropeptide. The effect of PEP on the breakdown of ODN and related analogs was studied by combining RP-HPLC analysis and MALDI-TOF MS characterization. Incubation of ODN with PEP generated two products, i.e. ODN3–18 and ODN5–18 which resulted from cleavage of the Ala-Thr and Val-Gly peptide bonds. S 17092, a specific PEP inhibitor, significantly reduced the PEP-induced cleavages of ODN. Similarly, [Ala2]OP showed S 17092-sensitive post-alanine cleavage, while [pGlu1]ODN and OP (ODN11–18) were not catabolized by the enzyme. For all these peptides, cleavage of the Pro-Gly peptide bond by PEP was never observed, even after prolonged incubation times. In contrast, PEP hydrolyzed human urotensin II at the canonical post-proline site. Collectively, these data suggest that the Ala2 residue is the preferential cleavage site of ODN and that the Pro-Gly bond of ODN is not hydrolyzed by PEP. In addition, this study reveals for the first time that the endoproteolytic activity of PEP can specifically take place after a valine moiety.
Keywords: Prolyl oligopeptidase; EC; PEP; Neuropeptide catabolism; Endozepines; Urotensin II;

The effect of heme oxygenase-1 induction by octreotide on radiation enteritis by Semra Doğru Abbasoğlu; Yeşim Erbil; Tunç Eren; Murat Giriş; Umut Barbaros; Rifat Yücel; Vakur Olgaç; Müjdat Uysal; Gülçin Toker (1570-1576).
Radiation enteritis occurs as a response to abdominal radiation, which can cause mucosal damage in the gastrointestinal mucosal epithelium. The small intestine is one of the most radiosensitive organs in the abdomen. The present study was undertaken to investigate the effect of octreotide (OCT) administration on heme oxygenase-1 (HO-1) expression of the radiation enteritis model. Rats received 50 mg/kg/day OCT for 4 days before irradiation and continued for 3 days after irradiation. Intestinal myeloperoxidase (MPO) activities, malondialdehyde (MDA) levels are indicators of oxidative damage while caspase-3 activities reveal apoptosis degree of the small intestine. At histological examination, the terminal ileum tissue was analyzed for morphological changes. Irradiation significantly increased the intestinal MPO and caspase-3 activities, MDA levels and HO-1 expression in comparison to sham control group. OCT treatment was associated with increased HO-1 expression and caspase-3 activity, decreased MPO activity and MDA levels. Histological examination revealed that the intestinal mucosal structure was preserved in the OCT treated group. OCT appears to have protective effects against radiation-induced intestinal damage. This protective effect is, in part, mediated by modification of the inflammatory response and the induction of HO-1 expression.
Keywords: Heme oxygenase-1; Octreotide; Irradiation; Apoptosis;

Rapid modulation of TRH-like peptides in rat brain by thyroid hormones by A. Eugene Pekary; Albert Sattin; Schetema A. Stevens (1577-1588).
Recent identification of membrane receptors for T4, T3, 3,5-T2, and 3-iodothyronamine that mediate rapid physiologic effects of thyroid hormones suggested that such receptors may supplement the regulation of TRH and TRH-like peptides by nuclear T3 receptors. For this reason 200 g male Sprague–Dawley rats received daily i.p. injections of PTU or T4. Levels of TRH and TRH-like peptides were measured 0, 2 h or 1, 2, 3, or 4 days later. Rapid increases or decreases in TRH and TRH-like peptide levels were observed in response to PTU and T4 treatments in various brain regions involved in mood regulation. Significant effects were measured within 2 h of T4 injection. Nuclear T3 receptor-mediated changes in gene expression altering translation, post-translational processing and constitutive release of peptides require more than 2 h. We conclude that non-genomic mechanisms may contribute to the psychiatric effects of thyroid disease and thyroid hormone adjuvant treatment for major depression.
Keywords: TRH; TRH-like peptides; Thyroid hormones; Depression; Limbic system;

Tissue distribution and plasma clearance of heparin-binding EGF-like growth factor (HB-EGF) in adult and newborn rats by Jiexiong Feng; Veela B. Mehta; Osama N. El-Assal; Dan Wu; Gail E. Besner (1589-1596).
Heparin-binding EGF-like growth factor (HB-EGF), a member of the epidermal growth factor (EGF) family, can protect intestinal epithelial cells from various forms of injury in vitro and attenuate intestinal ischemia/reperfusion damage in vivo. With the goal of eventual clinical use of HB-EGF to protect the intestines from injury in neonates, children, and adults, the pharmacokinetics and biodistribution of 125I-labeled HB-EGF were investigated. After intravenous bolus, HB-EGF had a distribution half-life of 0.8 min and an elimination half-life of 26.67 min. After gastric administration, the bioavailability was 7.8%, with a 2.38 h half-life in the absorption phase and an 11.13 h half-life in the elimination phase. After intravenous dosing, most radioactivity was found in the plasma, liver, kidneys, bile, and urine, whereas it was mainly distributed in the gastrointestinal tract after intragastric administration. The degradation of 125I-HB-EGF in plasma from newborn rats was lower than that in adult rats after gastric administration. This supports the feasibility of enteral administration of HB-EGF in the treatment of gastrointestinal diseases, including newborns afflicted with necrotizing enterocolitis.
Keywords: HB-EGF; Tissue distribution; Pharmacokinetics;