Peptides (v.22, #12)

Peptide analog of fibronectin that inhibits cell migration and ERK 1/2 activity by Bożena Szaniawska; Halina Trembacz; Joanna Miłoszewska; Andrzej W. Lipkowski; Aleksandra Misicka; Jerzy Ostrowski; Przemysław Janik (1949-1953).
Two analogs of the peptide mimicking the 1977–1991 C- terminal part of fibronectin have been synthesized and tested. AWLI simulated human fibronectin fragment 1977–1991, whereas AWLII hybridized to both RGD and 1977–1991 fragments. AWLI and AWLII peptides inhibited the migration of the ovarian carcinoma cell line OVP10 regardless of the presence RGD. AWLI peptide inhibited spontaneous and fibronectin-activated cell migration and ERK1/2 activity. Neither AWLI nor fibronectin induced changes in FAK proteins, as could be judged from Western blots. In conclusion, it seems that the C-terminal fragment of fibronectin inhibits ERK1/2-dependent (random) migration of ovarian carcinoma cells.
Keywords: Fibronectin; Migration; FAK; ERK;

Synergistic inhibitory activity of α- and β-LFA-1 peptides on LFA-1/ICAM-1 interaction by Helena Yusuf-Makagiansar; Irwan T Makagiansar; Yongbo Hu; Teruna J Siahaan (1955-1962).
Interactions of cell-adhesion molecule LFA-1 and its ligand ICAM-1 play important roles during immune and inflammatory responses. Critical residues of LFA-1 for ICAM-1 binding are known to be in the I-domain of the α-subunit and the I-like domain of the β-subunit. On the basis of our previous work demonstrating the inhibitory activity of I-domain cyclic peptide cLAB.L on LFA-1/ICAM-1 interaction, here we have explored the activity of I-like-domain peptide LBE on the binding mechanism of cLAB.L. LBE enhances cLAB.L binding to T-cells and epithelial cells. The adherence of T-cells to epithelial monolayers was suppressed by the two peptides. The addition of LBE to the monolayers prior to the addition cLAB.L produced a better inhibitory effect than the reverse procedure. LBE, but not cLAB.L, changes the ICAM-1 conformation, suggesting that LBE binds to ICAM-1 at sites that are distinct from these of cLAB.L and induces improved conformation in ICAM-1 for binding to cLAB.L.
Keywords: LFA-1; ICAM-1; Adhesion-molecule peptides; Autoimmune diseases; Inflammation; Drug targeting;

Comparative study on the effect of phosphorylated TCR ζ chain ITAM sequences on early activation events in Jurkat T cells by Violeta Chiţu; Roberta Fajka-Boja; Gábor K Tóth; Györgyi Váradi; Zoltán Hegedüs; András Frankó; Kinga Székely Szücs; Éva Monostori (1963-1971).
One of the main dilemma in T cell receptor (TCR) signal transduction is whether the presence of multiple Immunoreceptor Tyrosine-based Activation Motifs (ITAMs) within the TCR signaling module serves for signal amplification or signal distribution. To contribute to answer this question, we analyzed the effect of synthetic oligopeptides representing the three bi-phosphorylated ζ chain-ITAMs on the early signaling events in permeabilized leukemia T cells. Our main observations were as follows: 1/Stimulation of the cells with the bi-phosphorylated membrane proximal and central ITAMs (ζ (1)ypyp and ζ (2)ypyp, respectively) resulted in a strong phosphorylation of proteins with a similar pattern. In contrast, the membrane distal ITAM, ζ (3)ypyp had a reduced ability to promote tyrosine phosphorylation and failed to induce the phosphorylation of a number of proteins. 2/ The phospho-peptide induced tyrosine phosphorylation events were at least partially mediated by p56lck and Syk/ZAP70 protein tyrosine kinases as it was shown in p56lck and Syk/ZAP70 deficient Jurkat variants. 3/The patterns of the association of the adaptor protein, Grb2 with tyrosine phosphorylated proteins following cell stimulation with the bi-phosphorylated membrane proximal or the central ITAMs were similar, while the membrane distal ITAM was unable to induce any of these associations.Our data provide additional evidence that the three ζITAMs differ in their capacity to induce tyrosine phosphorylation of intracellular proteins in permeabilized T cells, depending to their primary sequence. The first and second ITAM sequences of the ζ chain may have similar but not totally overlapping functions. This conclusion results from their similar but not identical abilities to induce tyrosine phosphorylation and association of Grb-2 with intracellular phosphoproteins. In contrast, the third ITAM (ζ3) may have distinct functions since this peptide fails to induce tyrosine phosphorylation of a number of proteins compared to the other two ITAMs, and it is unable to induce either new association or the increase in the amount of Grb-2 associated phosphoproteins.
Keywords: Phospho-peptides; TCR ζ chain; ITAM; Tyrosine phosphorylation; Signal transduction;

Cells use a ZIP code system to sort newly synthesized proteins and deliver them wherever they are needed: into different internal compartments called organelles or even out of the cell altogether. One of the most essential features of the ZIP code system is the signal sequence or “address tag,” which is originally present in the N-terminal part of the protein and is trimmed away by the time it is secreted. Owing to the importance of signal peptides for understanding the molecular mechanisms of genetic diseases, reprogramming cells for gene therapy, and constructing new drugs for correcting a specific defect, it is highly desirable to develop a fast and accurate method to identify the signal peptides. In this paper, a scaled window model is proposed. Based on such a model as well as Markov chain theory, a new algorithm is formulated for predicting the signal peptides. Test results for the 1939 secretory proteins and 1440 non-secretary proteins have indicated that the new algorithm is particularly successful in the overall success rate, and hence can serve as a complementary tool to the existing algorithms for signal peptide prediction.
Keywords: “Zipcode” sequence; Markov chain; Secretory proteins; Non-secretory proteins; Discriminant function;

Individuation of monoclonal anti-HPV16 E7 antibody linear peptide epitope by computational biology by Darja Kanduc; Alberta Lucchese; Abraham Mittelman (1981-1985).
We applied computational biology to identify the linear amino acid sequence recognized by a mouse monoclonal antibody raised against the full length HPV16 E7 oncoprotein. Computer-assisted search for the epitopic peptide used two parameters: the capability of E7 peptides to bind to MHC class II molecules, and the similarity level of the oncoprotein sequence to the mouse proteome. We report that anti-E7 mAb recognized the peptide having both high binding potential to MHC II molecules and low level of molecular mimicry to mouse proteome. Peptide ability to bind to MHC II molecules appears a necessary but not sufficient condition to determine peptide immunodominance, by needing to be supported by a low degree of peptide similarity to the host’s proteome.
Keywords: Epitope prediction; MHC binding potential; Molecular mimicry; Peptide immunodominance; Computational biology;

Mast cell degranulating (MCD) peptide was modified in its two disulfide bridges and in the two arginine residues in order to measure the ability of these analogs to induce histamine release from mast cells in vitro. Analogs prepared were [Ala3,15]MCD, [Ala5,19]MCD, [Orn16]MCD, and [Orn7,16]MCD. Their histamine-releasing activity was determined spectrofluorometrically with peritoneal mast cells. The monocyclic analogs in which the cysteine residues were replaced pairwise with alanine residues showed three-to ten-fold diminished histamine-releasing activity respectively, compared with the parent MCD peptide. Substantial increases in activity were observed where arginine residues were replaced by ornithines. The ornithine-mono substituted analog showed an almost six-fold increase and the ornithine-doubly substituted analog three-fold increase in histamine-releasing activity compared with the parent MCD peptide. The structural changes associated with these activities were followed by circular dichroism (CD) spectroscopy. Changes in the shape and ellipticity of the CD spectra reflected a role for the disulfide bonds and the two arginine residues in the overall conformation and biological activity of the molecule.
Keywords: MCD peptide analogs; Histamine; Mast cell; CD spectroscopy;

Mast cell degranulating peptide binds to RBL-2H3 mast cell receptors and inhibits IgE binding by Angeliki Buku; Joseph A Price; Milton Mendlowitz; Sandra Masur (1993-1998).
Fluorescent and biotinylated analogs of mast cell degranulating (MCD) peptide were synthesized and the labels fluoresceinisothiocyanate and N-hydroxysuccinimidobiotin were conjugated at position 1 in the MCD peptide sequence. The analogs with these moieties retained histamine-releasing activity as high as that of the parent MCD peptide in rat peritoneal mast cell assays. These labeled analogs were used in rat basophilic leukemia cells (RBL-2H3) to demonstrate by confocal microscopy and flow cytometry the specific binding of MCD peptide to mast cell receptors. Consequently MCD peptide was found to compete with and inhibit the binding of fluorescent IgE on RBL cells as monitored by flow cytometry. Thus MCD peptide may prove to be useful in the study of IgE receptor-bearing cells.
Keywords: Fluorescent and biotinylated MCD peptide; Flow cytometry; Confocal microscopy; Fluorescent IgE;

Endogenous fragment of hemoglobin, neokyotorphin, as cell growth factor 1 1 Abbreviations: FBS - fetal bovine serum; FALS - forward angle light scattering; MTT - 3-(4;5-dimethylthiazol-2-yl)-2.5-diphenyl tetrazolium bromide; SRB - sulforhodamine B. by Elena Yu. Blishchenko; Olga A. Kalinina; Olga V. Sazonova; Sergei V. Khaidukov; Natalya S. Egorova; Andrei Yu. Surovoy; Marina M. Philippova; Arpad A. Vass; Andrei A. Karelin; Vadim T. Ivanov (1999-2008).
It is shown that neokyotorphin (the α-globin fragment 137–141) stimulates proliferation of normal cells (murine embryonic fibroblasts, red bone marrow and spleen cells) and tumor cells (murine melanoma and transformed fibroblasts L929) in the absence or in the presence of fetal bovine serum. In contrast to serum deprivation conditions, the ability to potentiate L929 cell growth in the presence of fetal serum is strongly cell density dependent. The peptide also enhances the viability of L929 cells, murine embryonic fibroblasts and of the primary cultures of murine red bone marrow cells and splenocytes under serum-deprivation conditions for at least 72 h. The results of flow cytometry analysis suggest that the effect of neokyotorphin on survival of L929 cells in serum-free culture medium is due to maintenance of cell proliferation in the absence of growth factors. Along with cell cycle progression the peptide induces reversible reduction of L929 cell size.
Keywords: Neokyotorphin; Tissue-specific peptide pool; Tumor cells; Proliferation;

Synthetic β-endorphin-like peptide immunorphin binds to non-opioid receptors for β-endorphin on T lymphocytes by Elena V Navolotskaya; Natalia V Malkova; Tatyana A Zargarova; Tatyana N Lepikhova; Vladimir P Zav’yalov; Valery M Lipkin (2009-2013).
The synthetic decapeptide H-SLTCLVKGFY-OH (termed immunorphin) corresponding to the sequence 364–373 of the CH3 domain of human immunoglobulin G heavy chain was found to compete with [125I]β-endorphin for high-affinity receptors on T lymphocytes from the blood of healthy donors (K i = 0.6 nM). Besides immunorphin, its synthetic fragments H-Val-Lys-Gly-Phe-Tyr-OH (K i = 15 nM), H-Leu-Val-Lys-Gly-Phe-Tyr-OH (K i = 8.0 nM), H-Cys-Leu-Val-Lys-Gly-Phe-Tyr-OH (K i = 3.4 nM), H-Thr-Cys-Leu-Val-Lys-Gly-Phe-Tyr-OH (K i = 2.2 nM), H-Leu-Thr-Cys-Leu-Val-Lys-Gly-Phe-Tyr-OH (K i = 1.0 nM) possessed the ability to inhibit specific binding of [125I]β-endorphin to T lymphocytes. Tests of the specificity of the receptors revealed that they are not sensitive to naloxone and Met-enkephalin, i.e. they are not opioid receptors. K d values characterizing the specific binding of 125I- labeled immunorphin and its fragment H-Val-Lys-Gly-Phe-Tyr-OH to the receptors have been determined to be 7.4 nM and 36.3 nM, respectively.
Keywords: β-Endorphin; Receptor; Peptide; T lymphocytes;

MDR1 P-glycoprotein transports endogenous opioid peptides by Ronald P.J Oude Elferink; James Zadina (2015-2020).
MDR1 P-glycoprotein is generally regarded as an efflux pump for amphipathic toxic compounds. The question remains, however, whether certain endogenous compounds are also substrates for this transporter. Certain peptides have been shown to interact with MDR1 Pgp as well and we have therefore investigated whether endogenous bioactive peptides are substrates. We demonstrate here that the synthetic μ-opioid peptide DAMGO is a good substrate for MDR1 Pgp. In view of its low interaction with the membrane it is an attractive ligand for measurement of MDR1 Pgp-mediated transport activity in membrane vesicles. Various linear peptides with amidated C-termini were found to inhibit MDR1 Pgp-mediated DAMGO transport. This group includes endogenous opioid peptides such as adrenorphin and endomorphin 1 and 2, as well as the neurokinin, Substance P. The latter bioactive peptides have a relatively high affinity for the transporter. Transport of endomorphin 1 and 2 could be directly demonstrated by the uptake of the radiolabeled opioid peptides in membrane vesicles from MDR1-transfected cells with a K m of 15 and 12 μM, respectively. This opens the possibility that MDR1 Pgp is involved in the elimination and/or tissue distribution of these bioactive peptides.
Keywords: P-glycoproteins; ABC transporters; Opioids; Opiates; Blood brain barrier; Bioactive peptides; Transport;

Analyzing the radiation of the proenkephalin gene in tetrapods: cloning of a Bombina orientalis proenkephalin cDNA by Robert M Dores; David Costantino; Jeannine Walnutt; Phillip B Danielson; Stephanie Lecaude (2021-2025).
Analyzing the Radiation of the Proenkephalin Gene in Tetrapods: Cloning of a Bombina orientalis Proenkephalin cDNA: A proenkephalin cDNA was cloned from the brain of the anuran amphibian, Bombina orientalis (Family: Discoglossidae). This cDNA is 1358 nucleotides in length, and contains an open reading frame that codes for 251 amino acids. Within the open reading frame there are seven opioid (YGGF) sequences. There were five Met-enkephalin (YGGFM) sequences that are flanked by sets of paired basic amino acid proteolytic cleavage sites and two C-terminally extended Met-enkephalin sequences: YGGFMRGY and YGGFMRF. No Leu-enkephalin sequences were found in B. orientalis proenkephalin. It was possible to align the amino acid sequences of proenkephalin from several vertebrate taxa (human, Australian lungfish, B. orientalis, Xenopus laevis, Spea multiplicatus) by inserting a minimum of nine gaps. This alignment was then used to analyze the corresponding nucleotides for each proenkephalin sequence using maximum likelihood. This analysis yielded a single tree. In this tree, the Australian lungfish sequence was the outgroup or the tetrapod ingroup. The amphibian sequences form a clade separate from the human sequence. The bootstrap value for the amphibian clade was 100%. Within the amphibian clade the Bombina sequence was the sister group to a clade composed of the X. laevis and S. multiplicatus sequences. The bootstrap value for the X. laevis/S. multiplicatus clade was 94%. Collectively, these data indicate that the sequence of Bombina proenkephalin may be more similar to the proposed ancestral anuran proenkephalin sequence, than either X. laevis or S. multiplicatus proenkephalin.
Keywords: Proenkephalin cDNA; Amphibian;

Cloning, expression and processing of the CP2 neuropeptide precursor of Aplysia by F.S Vilim; V Alexeeva; L.L Moroz; L Li; T.P Moroz; J.V Sweedler; K.R Weiss (2027-2038).
The cDNA sequence encoding the CP2 neuropeptide precursor is identified and encodes a single copy of the neuropeptide that is flanked by appropriate processing sites. The distribution of the CP2 precursor mRNA is described and matches the CP2-like immunoreactivity described previously. Single cell RT-PCR independently confirms the presence of CP2 precursor mRNA in selected neurons. MALDI-TOF MS is used to identify additional peptides derived from the CP2 precursor in neuronal somata and nerves, suggesting that the CP2 precursor may give rise to additional bioactive neuropeptides.
Keywords: Cerebral peptide 2; Neuropeptide; Aplysia californica; cDNA cloning; Peptide processing; In situ hybridization; Single cell RT-PCR; MALDI-TOF MS;

Differences between ranamargarin and other tachykinins in the stimulation of ion transport in frog skin by Claudio Lippe; Vito Bellantuono; Giuseppe Cassano; Angelo Quaranta; Concetta Ardizzone (2039-2044).
In frog skin, tachykinins stimulate ion transport by interaction with NK1-like receptors. The structural requirements of the peptide are the presence of the C-terminal sequence Phe-X-Gly-Leu-Met-NH2 and at least one Pro residue in the N-terminal sequence. In this paper, we demonstrate that the C-terminal amino acid must be amidated but it can be different from Met, and that the sequence cannot be longer or shorter than 11–12 amino acids. Unexpectedly, Ranamargarin (14 amino acids, no Pro residue) increased the short circuit current value by 48 ± 0.3%. On the basis of considerable experimental evidence, we suggest that Ranamargarin interacts with a receptor different from those of other tachykinins.
Keywords: Ranamargarin; Tachykinins; Ion Transport; Skin; Rana esculenta;

Coding region of the sorbin gene in different species by K Wahbi; J.P Magaud; D Pansu; M Descroix-Vagne (2045-2053).
The coding region of 153 amino-acid sorbin, isolated from porcine intestine has been cloned and sequenced in pig, human and rat. The coding region includes 459 bases comprising the 5′ region of 24 bases, the middle region named “sorbin-like sequence” (25–432) and the 3′ region (433–459). The peptidic C-terminal segment presents the biological activity: absorption of water and electrolytes from the intestine and gall-bladder. The cDNA homology between the three species was 95%. Three forms of mRNA were found, two major forms (6.5 and 8 Kb) and one minor (4.5 Kb).
Keywords: Human; Pig; Rat; Gene; Sorbin; Sorbin-like proteins; Enterochromaffin cells; Intestinal carcinoid tumor;

Identification of cDNA encoding motilin related peptide/ghrelin precursor from dog fundus by Catherine Tomasetto; Corinne Wendling; Marie-Christine Rio; Pierre Poitras (2055-2059).
A novel protein expressed by entero-endocrine cells of the mouse stomach was named prepromotilin Related Peptide (ppMTLRP) since it shares sequence similarities with the prepromotilin (Tomasetto et al.). The mouse ppMTLRP was found identical to the rat precursor of ghrelin (ppghrelin), an endogenous ligand specific for the Growth Hormone Secretagogue receptor identified from rat stomach (Kojima et al.). In the present study the cDNA encoding the dog counterpart of ppMTLRP/Ghrelin has been isolated and sequenced. The dog ppMTLRP/Ghrelin cDNA showed scores of respectively 80% and 75% homology with its human and mouse counterparts. By translation of the dog ppMTLRP/Ghrelin cDNA sequences, two ORFs could be deduced encoding either a 117 amino acid ppMTLRP/Ghrelin or the deleted Gln14 ppMTLRP/Ghrelin, as it was also known in mouse, rat and man. The dog ppMTLRP/Ghrelin shared 91% similarity and 78% identity, and 89% similarity and 78% identity with the human and mouse ppMTLRP/Ghrelin proteins respectively. The best score of homology was found in the MTLRP/Ghrelin sequence itself. Indeed the dog MTLRP/Ghrelin peptide shared 100% similarity and 93% identity, and 96% identity and similarity, with the human and mouse MTLRP/Ghrelin. Using Northern blot analysis to study dog ppMTLRP/Ghrelin gene expression on dog adult gut tissues, maximal expression level was found in the stomach fundus and corpus, and no expression could be detected in the stomach antrum nor in the duodenum, jejunum, ileum, colon or liver. In conclusion, we have identified ppMTLRP/Ghrelin from dog, and found that it is highly conserved with man, mouse or rat. The expression pattern along the gastro-intestinal tract is similar to the expression pattern previously described in mouse.
Keywords: Regulatory peptides; GI hormones; GI motility; Obesity; Appetite regulation; Growth hormone;

C-type natriuretic peptide system in rabbit colon by Jong Hun Kim; Goan Hee Jeon; Sung Zoo Kim; Kyung Woo Cho; Suhn Hee Kim (2061-2068).
C-type natriuretic peptide (CNP) is mainly distributed in the brain and vascular endothelium and is considered to act as a local regulator in many tissues. The present study was aimed to determine the presence of CNP system and its biological function in rabbit colon. The serial dilution curves of tissue extracts were parallel to the standard curve of CNP-22. With gel permeation chromatography and reverse-phase HPLC, the major immunoreactive peak of CNP was observed at the same elution time corresponding to the synthetic CNP-53. The concentration of CNP in the mucosal layer of colon was 212.49 ± 30.44 pg/g tissue wet weight (n = 7), which was significantly higher than that in the muscular layer. The presence of CNP mRNA was also detected by RT-PCR and Southern blot analysis. Production of cGMP by the activation of particulate guanylyl cyclase stimulated by BNP and CNP was higher in membranes obtained from the muscular layer than from mucosal layer. More cGMP was produced by CNP than by ANP. Both natriuretic peptide receptor-A and -B mRNAs were detected by RT-PCR and specific binding sites to 125I-[Tyr0]-CNP-22 were mainly localized to the muscular layer. Synthetic CNP inhibited basal tension, frequency and amplitude of basal motility of taenia coli of the right colon. This study showing the presence of CNP system and its biological function in colon suggests that endogenous CNP synthesized in the mucosal layer may have a paracrine function as a local regulator of colonic motility.
Keywords: C-type natriuretic peptide; Colon; cGMP; Motility; Natriuretic peptide receptor; RT-PCR; Autoradiography;

Effects of endothelin-3 on intestinal ion transport by L.V González Bosc; M.P Majowicz; M.C Ortiz; N.A Vidal (2069-2075).
We investigated the effects of endothelin 3 (ET-3) on electrolyte transport in rat small intestine using a voltage clamp technique in Ussing’s chamber. ET-3 diminished potential difference (PD) and short circuit current (Isc). ET-3 did not affect PD or Isc in low Na+ and/or D-glucose-free medium. Phloridzine (an inhibitor of sodium-glucose cotransporter [SGLT1]) pretreatment abolished the effect of ET-3 on Isc. Methylene blue (a soluble guanylate cyclase inhibitor) or N-nitro-L-arginine methyl ester (a NOS inhibitor) pretreatment delayed the effect of ET-3 on PD and Isc. ET-3 enhanced NOS activity on enterocytes and systemic NO production. Then, ET-3 could inhibit SGLT1 with the participation of NO.
Keywords: Endothelin 3; Small intestine; Ion transport; Nitric oxide; SGLT1;

The effect of α-melanocyte stimulating hormone on endotoxin-induced intestinal injury by Tangül Şan; Berna K. Oktar; Emsal Salik; Feriha Ercan; İnci Alican (2077-2082).
We investigated the effect of α-melanocyte stimulating hormone (α-MSH) on endotoxin-induced intestinal inflammation and the role of nitric oxide and prostaglandins in this response. α-MSH treatment (25 μg/rat, intraperitoneally (i.p.); twice daily) reduced the severity of the lesions macroscopically and microscopically. This protective effect was found to be confined mainly to the distal ileum. These lesions were reversed by pretreatment with the non-selective COX inhibitor indomethacin (10 mg/kg, subcutaneously (s.c.)) but not by the selective COX-2 inhibitor nimesulide (3 mg/kg, s.c.), the NO donor sodium nitroprusside (4 mg/kg, i.v.) or the iNOS inhibitor dexamethasone (3 mg./kg, i.p.) at macroscopic level and reversed by Indo or Dex at microscopic level. Increased peroxidase activity -index of tissue neutrophil infiltration- in the distal ileum of LPS-treated rats was decreased by α-MSH and this effect was reversed by pretreatment with Indo. In conclusion, the neuropeptide α-MSH has a beneficial effect on endotoxin-induced distal intestinal lesions by a mechanism which probably involves nitric oxide and COX-1 derived prostaglandins.
Keywords: Endotoxemia; α-Melanocyte stimulating hormone; Neutrophils; Prostaglandins; Nitric oxide;

Interaction of neuropeptide Y and corticotropin-releasing factor signaling pathways in AR-5 amygdalar cells by S Sheriff; F.M Dautzenberg; J.J Mulchahey; M Pisarska; R.L Hauger; W.T Chance; A Balasubramaniam; J.W Kasckow (2083-2089).
Corticotropin-releasing factor (CRF) is a 41 amino acid neuropeptide which is involved in the stress response. CRF and neuropeptide Y (NPY) produce reciprocal effects on anxiety in the central nucleus of the amygdala. The molecular mechanisms of possible CRF-NPY interactions in regulating anxiety behavior is not known. In the central nervous system, the action of NPY leads to inhibition of cAMP production while CRF is known to stimulate levels of cAMP in the brain. Consequently, we hypothesized that NPY may antagonize anxiety-like behavior by counter-regulating CRF-stimulated cAMP accumulation and activation of the protein kinase A pathway. We have engineered an immortalized amygdalar cell line (AR-5 cells) which express via RT-PCR, the CRF, Y1 and Y5 NPY receptor. In addition, in these cells CRF treatment results in significant concentration-dependent increases in cAMP production. Furthermore, incubation of 3 μM CRF with increasing concentrations of NPY was able to significantly inhibit the increases in cAMP compared to that observed with 3 μM CRF treatment alone. These findings suggest that CRF and NPY may counter-regulate each other in amygdalar neurons via reciprocal effects on the protein kinase A pathway.
Keywords: Amygdala; Corticotropin-releasing factor; Neuropeptide Y; cAMP;

EEP is a tripeptide structurally similar to thyrotropin releasing hormone (TRH) and, like TRH, it is found in the mammalian brain. TRH has been found to increase in brain regions after seizures and to be neuroprotective. EEP has also been shown to increase in brain regions following seizure activity. We therefore sought to determine whether the similarities between these two peptides might be extended to include neuroprotection. Both TRH and EEP were found to be neuroprotective in vitro against an excitotoxic insult. Interestingly, the two tripeptides appeared to have different mechanisms of action. Even though EEP was as much as four times more neuroprotective than TRH, its ability to reduce glutamate-stimulated increases in intraneuronal Ca2+ was about half that of TRH.
Keywords: EEP; TRH; Glutamate; Neurotoxicity; Neuroprotection; Calcium;

Isolation and characterization of serum procalcitonin from patients with sepsis by Wolfgang Weglöhner; Joachim Struck; Christina Fischer-Schulz; Nils G Morgenthaler; Albrecht Otto; Claude Bohuon; Andreas Bergmann (2099-2103).
Procalcitonin (PCT) is one of the precursors in the synthesis of calcitonin in thyroidal C-cells and other neuroendocrine cells. PCT and other calcitonin precursors are elevated in the serum of many conditions leading to systemic inflammatory response syndrome. The measurement of PCT in patients suffering from severe bacterial infections is a useful tool for the diagnosis of sepsis. Furthermore, therapeutic decisions are often based on the increase or decline of serum PCT levels. PCT was reported to have 116 amino acids. The aim of our study was the determination of the primary structure of serum PCT from septic patients. Sera containing high PCT-concentrations (>100 ng/ml) were collected from 22 patients with severe sepsis and were pooled for further purification (12.7 μg total concentration of PCT). Pooled PCT was purified on a CT 21-immunoaffinity column, further purified by reversed phase HPLC, and the resulting pure PCT was digested with endoproteinase Asp-N. N-terminal Edman sequencing showed that the first two amino acids (Ala-Pro) of the proposed pro-peptide were missing. Further analyses by MALDI-TOF mass spectroscopy resulted in a distinct mass signal of 12640 Da ± 0.1%, which is in concordance with the theoretical molecular weight of the N-terminal truncated form (12628 Da). As opposed to previous suggestions, we could not detect any chemical modifications of PCT. In summary, we could demonstrate that PCT in the serum of septic patients is a peptide of only 114 amino acids, instead of the predicted 116 amino acids, lacking the N-terminal dipeptide Ala-Pro. This information on the primary structure of PCT might help in further studies on the physiological role of PCT during sepsis.
Keywords: Procalcitonin; PCT; Dipeptidyl peptidase IV; DPIV; Primary structure;

Alzheimer’s disease (AD) and frontotemporal dementia (FTD) are two prevalent neurodegenerative disorders for which the causes are unknown, except in rare familial cases. Several changes in neuropeptide levels as measured by radioimmunoassay (RIA) have been observed in these illnesses. Somatostatin (SOM) levels in cerebrospinal fluid (CSF) are consistently decreased in AD and FTD. Neuropeptide Y (NPY) levels are decreased in AD, but normal in FTD. Galanin (GAL) levels increase with the duration of illness in AD patients. The majority of studies of neuropeptides in CSF have not been verified by HPLC. The observed decrease in a neuropeptide level as measured by RIA may therefore reflect an altered synthesis or extracellular processing, resulting in neuropeptide fragments that may or may not be detected by RIA. Matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-MS) has been shown to be a powerful technique in the analysis of biological materials without any pre-treatment, by detecting peptides and proteins at a specific mass-to-charge (m/z) ratio. We studied the processing of the neuropeptides NPY , NPY , SOM and GAL in the cerebrospinal fluid of patients with AD (n = 3), FTD (n = 3) and controls (n = 2) using MALDI-MS. We found that considerable inter-individual variability exists in the rate of neuropeptide metabolism in CSF, as well as the number of peptide fragments formed. Certain patients showed differences in the processing of specific neuropeptides, relative to other patients and controls. This analysis of the metabolic processing of neuropeptides in CSF yielded a large amount of data for each individual studied. Further studies are required to determine the changes in neuropeptide processing that can be associated with AD and FTD. With further investigations using MALDI-MS analysis, it may be possible to identify a neuropeptide fragment or processing enzyme that can be correlated to these disease states.
Keywords: Alzheimer’s disease; Frontotemporal dementia; Cerebrospinal fluid; Neuropeptide Y; Somatostatin; Galanin; Mass spectrometry;

PRASAD, C. Role of Endogenous Cyclo(His-Pro) in Voluntary Alcohol Consumption by C57BL Mice. PEPTIDES —-. Cyclo (His-Pro) or CHP is a cyclic dipeptide endogenous to the brain of a variety of animal species including man. Administration of exogenous peptide to rodents has been shown to exhibit a variety of biological activities some of which appear to be mediated via a dopaminergic mechanism. Since a hypodopaminergic state has been associated with excessive drinking in animal models as well as man, we have explored the potential role of CHP in alcohol-preferring C57BL mice. The results of this study show that the level of CHP, a peptide that mimics dopamine in many of its pharmacologic actions, is lower in brains of alcohol-preferring C57BL mice compared to alcohol non-preferring DBA2 mice. Furthermore, administration of exogenous CHP to C57BL mice caused a pronounced decrease in their voluntary alcohol consumption. In conclusion, endogenous CHP may play a role in risk for developing excessive alcohol use by modulating central dopaminergic tone.
Keywords: Cyclo (His-Pro); Histidyl-proline diketopiperazine; Voluntary alcohol intake; C57BL mice; Alcoholism; Alcohol; Alcohol preference;

Effects of peripheral and central administration of GHRH on feeding in aging LOU rats by Christelle Veyrat-Durebex; Pierrette Gaudreau; Stéphane Boghossian; Josette Alliot (2119-2126).
In aging LOU rats, a decreased protein intake is restored by GH administration. To study the contribution of GHRH to macronutrient selection, hGHRH NH2 was administered sc. (1 mg/kg B.W./day/14 days) or icv. (4 and 40 pmol/rat) to 11-, 19-, 24- and 28-month-old rats. Sc. administration induced a decreased food and lipid intakes from 24 months of age and a transient stimulation of protein intake in 19-month-old and older low protein eaters (<10% protein/total intake). Icv. administration induced decreased food and lipid intakes in all age groups. These results suggest that GHRH may regulate feeding through pituitary and/or hypothalamic GHRH receptor mechanisms.
Keywords: Growth hormone-releasing hormone; Protein intake; Lipid intake; Feeding behavior; Aging; LOU/C/jall rats;

Multiple-time regression analysis has been used to study the influx of radiolabeled peptides and polypeptides across the blood-brain barrier (BBB). This study used both tritiated and iodinated leptin to clarify several issues associated with these measurements. Recombinant murine leptin was radiolabeled with 3H by derivatization or with 125I by the iodobead method and each studied separately in mice. Intact 3H-leptin had a higher apparent influx rate from blood to brain than did intact 125I-leptin, correlating with its higher proportion of reversible association with the capillary lumen that would misleadingly appear to reflect entry. Yet the majority of 3H-leptin and 125I-leptin reached brain parenchyma. There was no significant difference in the influx rate between cerebral cortex and the subcortical regions, thus ruling out a predominant contribution of simple diffusion through the circumventricular organs or choroid plexuses outside the BBB. The influx of radiolabeled leptin, especially 125I-leptin, was decreased by excess unlabeled leptin, supporting the presence of a saturable transport system for leptin at the BBB. To identify the specificity of the transport system and determine whether it is shared by 3H-leptin and 125I-leptin, these radioactively labeled leptins were heat-denatured. Denaturation had no effect on the fast influx of 3H-leptin, but abolished the entry of 125I-leptin into brain; excess denatured leptin failed to inhibit the influx of either 3H-leptin or 125I-leptin. This indicates that the conformation of 125I-leptin is similar to that of native unlabeled leptin, so that iodination would be the better choice for investigating the interaction of leptin with the BBB. However, 3H-leptin can use the same transport system, as shown by inhibition of its influx by unlabeled leptin, whereas the derivatization procedure altered its biophysical properties such that its non-saturated influx was greatly enhanced. Finally, the rapid influx of radioactively labeled leptin contrasted greatly with that of the reference compounds 99mTc-albumin and 3H-inulin which had no significant penetration of the BBB. Thus, with additional considerations such as stability and interactions with the vasculature, multiple-time regression analysis is sensitive and selective for study of the penetration of peptides across the BBB.
Keywords: Blood-brain barrier; Peptides; Polypeptides; Leptin; Tritiation;

Interactions among challenges of hydromineral balance, angiotensin-converting enzyme, and cystine aminopeptidase by Paulo Flávio Silveira; Jon Irazusta; Javier Gil; Naiara Agirregoitia; Luı́s Casis (2137-2144).
Enzymatic cleavage of some peptides could be included among the mechanisms of water-electrolyte homeostasis. To test this hypothesis, the angiotensin-converting activity (ACE) of plasma and the L-cystine-di-β-naphthylamidase activity (CAP) of plasma and of soluble and particulate fractions from different areas of the central nervous system (CNS) were investigated in rats submitted to treatments eliciting hydromineral imbalance. CAP in the CNS was unchanged by hydromineral challenges. The correlations observed between plasma osmolality and CAP, and plasma CAP and ACE suggested a contribution of these activities to the restoration of basal water-electrolyte and blood pressure conditions through the hydrolysis of vasopressin, oxytocin, angiotensin I and bradykinin.
Keywords: Angiotensin-converting enzyme; Kininase II; ACE; Cystine aminopeptidase; Oxytocinase; Vasopressinase; CAP; Hydromineral balance; Water-electrolyte homeostasis; Blood pressure;

Role of Asp297 of the AT2 receptor in high-affinity binding to different peptide ligands by Dieter Knowle; Jayson Kurfis; Narasaiah Gavini; Lakshmidevi Pulakat (2145-2149).
To determine how ligand-receptor interaction is affected by the charges of the amino acids at position 2 of the ligands and position 297 of the AT2 receptor, we generated the Asp297Lys mutant of AT2 and a ligand SarAsp2Ile. Asp297Lys mutant lost affinity to Ang II and SarIle however retained partial affinity to 125I-CGP42112A. The SarAsp2Ile had high affinity to Asp297Lys (IC503.5nM) and partial affinity to the AT2 (IC5015nM). Therefore, not only the charge, but also the length of the side arms of the amino acids at position 2 of the ligand and position 297 of the receptor affect their interaction.
Keywords: Angiotensin II; AT2 receptor; Phospholipase C; Xenopus oocytes; Third extracellular loop; Transmembrane domain;

Pituitary adenylate cyclase-activating peptide inhibits neutrophil chemotaxis by Johan Kinhult; Rolf Uddman; Marti Laan; Anders Lindén; Lars-Olaf Cardell (2151-2154).
Pituitary adenylate cyclase-activating peptide 38 (PACAP 38) is a neuropeptide that displays several biological effects of interest in the context of airway diseases such as asthma and chronic obstructive pulmonary disease. These effects include inhibition of airway and vascular smooth muscle tone as well as modulation of inflammatory cell activity. However, little is known about the effect of PACAP on granulocytes. The present study was designed to investigate if PACAP and the closely related peptide vasoactive intestinal peptide (VIP) could affect neutrophil migration. A standard 48 well chemotaxis chamber was used to assess the effects of PACAP on N-Formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)-induced neutrophil chemotaxis and spontaneous random migration. PACAP 38 and VIP inhibited fMLP-induced human neutrophil chemotaxis. Furthermore, both peptides also exhibited a dose-related trend toward inhibiting the spontaneous, unstimulated migration of neutrophils. Since enhanced cell migration in cell chamber systems is reported to correlate with increased invasive properties in vivo, the presented inhibitory effects of PACAP 38 on neutrophil chemotaxis, supports the idea of an anti-inflammatory role for PACAP. This together with the well documented bronchodilatory capacity of PACAP might indicate a role for PACAP-agonists in future treatment of asthma and other inflammatory airway diseases.
Keywords: PACAP; VIP; Neutrophils; Chemotaxis; Random migration;

Characterization of functional VIP/PACAP receptors in the human erythroleukemic HEL cell line by Roger Lema-Kisoka; Nathalie Hayez; Ingrid Langer; Patrick Robberecht; Eric Sariban; Christine Delporte (2155-2162).
R. LEMA-KISOKA, N. HAYEZ, I. LANGER, P. ROBBERECHT, E. SARIBAN AND C. DELPORTE. Characterization of functional VIP/PACAP receptors in the human erythroleukemic HEL cell line. PEPTIDES. The presence of VIP/PACAP receptors was investigated on the human erythroleukemic cell line HEL. Specific binding of [125I]-PACAP or [125I]-VIP on HEL cells or membranes was very low and did not allow to perform competition curves. At 37°C PACAP transiently increased cAMP levels in the presence of the non-specific phosphodiesterase inhibitor IBMX, suggesting rapid desensitization. Kinetic studies revealed that optimal conditions to measure the EC50 of PACAP(1–27) were 10 min at 20°C. Under those conditions, PACAP-related peptides increased cAMP levels with EC50 in agreement with the pharmacological profile of the VPAC1 receptor subtype: PACAP = VIP > [K15, R16, L27]VIP(1–7)/GRF(8–27) = [R16]ChSn (two VPAC1 agonists) ≫ helodermin = secretin. RO 25–1553, a selective activator of VPAC2 receptor was inactive at 1 μM. Dose-response curves of VPAC1 agonist molecules (PACAP, VIP, [K15, R16, L27]VIP(1–7)/GRF(8–27), [R16]ChSn) were shifted to the right by the VPAC1 receptor antagonist [AcHis1, D-Phe2, Lys15, Leu17]VIP(3–7)/GRF(8–27), with a Ki of 3 ± 1 nM (n = 3). The presence of VPAC1 receptor mRNA was confirmed by RT-PCR. Preincubation with PACAP or PMA showed that VPAC1 receptors underwent homologous and heterologous desensitization.This study provides the first evidence for the expression of functional VPAC1 receptors undergoing rapid desensitization in HEL cells.
Keywords: VIP; PACAP receptors; Erythroleukemia; Desensitization; Cyclic AMP;

Study on the hypothalamic factors mediating the inhibitory effect of PACAP38 on ovulation by O Kántor; J Molnár; A Heinzlmann; A Arimura; Zs Fürst; K Köves (2163-2168).

Radioiodination of the stable metabolic fragment of bradykinin, RPPGF by Thomas A Morinelli; G.Patrick Meier; Kevin L Schey; Harry S Margolius (2169-2174).
Arg-Pro-Pro-Gly-Phe (RPPGF, BK[1–5]), is a stable metabolite of the peptide hormone bradykinin. Considering the short half-life of bradykinin (BK, ∼15 secs), RPPGF has been used as a marker for BK’s endogenous generation. A lack of a radioiodinated RPPGF has precluded the development of a radioimmunoassay for this peptide. The present study describes a two-step reaction that allows for the incorporation of 125I into the aromatic ring of the phenylalanine of RPPGF. This radioiodinated analog is recognized by an antibody to RPPGF, demonstrating its utility for the development of a radioimmunoassay for measurements of RPPGF, a stable metabolic product of bradykinin.
Keywords: Bradykinin; Radioiodination; Phenylalanine; RPPGF; BK[1–5]; Deamination of aryl-amines;

The effect of food deprivation and experimental diabetes on orexin and NPY mRNA levels by Irné Swart; J Michael Overton; Thomas A Houpt (2175-2179).
Although exogenous orexin can induce feeding, reports of increased orexin gene expression after caloric manipulations have been inconsistent. We hypothesized that orexin gene expression is increased only by extreme negative energy balance challenges. We measured hypothalamic orexin and NPY mRNA by in situ hybridization and orexin-A immunoreactivity in rats after food deprivation, streptozotocin-induced diabetes, and combined deprivation and diabetes. Neither food deprivation, nor diabetes, nor the combination affected orexin mRNA levels, although orexin-A immunoreactivity was increased by diabetes. NPY mRNA levels were increased by either treatment. These results suggest that increased orexin gene expression is not a consistent correlate of negative energy balance challenges.
Keywords: Negative energy balance; Hypothalamus; Orexin-A immunoreactivity;

Effects of peptides on animal and human behavior: a review of studies published in the first twenty years of the journal Peptides a a Abbreviations: 5-HT, Serotonin; 5-HTP, 5-Hydroxytryptophan; 6-OHDA, 6-Hydroxydopamine; A18fa, Ala-Gly Glu-Gly Leu-Ser-Ser-Pro-Phe-Trp-Ser-Leu-Ala-Ala-Pro-Gln-Arg-Phe-NH2, Admin., Administration; Act., Activity; ACTH, Adrenocorticotropic Hormone; ANF, Atrial Natriuretic Factor; AT, Angiotensin; BBS, Bombesin; BUBUC, Tyr-D-Cys(StBu)-Gly Phe-Leu-Thr-(OtBu); CCK, Cholecystokinin; CCK-4, Cholecystokinin Tetrapeptide; Tetragastrin; CCK-8, Cholecystokinin Octapeptide; Sincalide; CGRP, Calcitonin Gene-Related Peptide; CRF, Corticotropin Releasing Factor; CTAP, D-Phe-Cys-Try-D-Trp-ORn-Thr-Pen-Thr-NH2; CTOP, D-Phe-Cys-Tyr-D-Tyr-Orn-Thr-NH2; DADELT II, [D-Ala2]Deltorphin; DADLE, D-Ala2-D-Leu5-Enkephalin; DAGO, D-Ala2-MePhe4-Gly-ol5-Enkephalin; DALA, D-Ala2-Methionine-enkephalinamide; DALCE, [D-Ala2,Leu5,Cys6]-Enkephalin; DAMA, D-Ala2-Met-Enkephalin Amide; DAME, [D-Ala2-Met5]Enkephalinamide; DAMCK, Tyr-D-Ala-Gly-(NMe)Phe-CH2Cl; DAMGO, Tyr-D-Ala-Gly-(NMe)Phe-Gly-ol; DDAVP, Desmopressin; 1-deamino-Cys-8-D-Arg-Vasopressin; DGAVP; Desglycinamide-arginine8-Vasopressin; DPEN [D-Pen2,D-Pen5]-Enkephalin; DPDPE, [D-Pen2,D-Pen5]-Enkephalin; DSIP, Delta Sleep-Inducing Peptide; EGF, Epidermal Growth Factor; F8Fa, Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2; Frag., Fragment; FMRFa, Phe-Met-Arg-Phe-amide; GHRH, Growth Hormone-Releasing Hormone; GR73632, D-Ala1-[L-Pro9,Me-Leu8]Substance P-(7–11); Ind., Induced; LHRH, Luteinizing Hormone Releasing Hormone; LLTNAM, Lys ψ(CH2NH)-Trp(Nps)-OMe; MCH, Melanin Concentrating Hormone; MIF-1, Melanotropin (MSH) Release-Inhibiting Factor-1; MSH, Melanocyte Stimulating Hormone; NT, Neurotensin; NP-, Neuropeptide-; OT, Oxytocin; Org2766, H-Met-(O2)-Glu-His-Phe-D-Lys-Phe-OH; PACAP, Pituitary Adenylate Cyclase-Activating Polypeptide; PG-KII, pGlu-Pro-Asn-Pro-Asp-Glu-Phe-Val-Gly-Leu-Met-NH2; PVN, Paraventricular Nucleus; SP, Substance P; TRH, Thyrotropin-Releasing Hormone; Tyr-MIF-1, Tyr-Pro-Leu-Gly-NH2; Tyr-W-MIF-1, Tyr-Pro-Trp-Gly NH2; VP, Vasopressin; VIP, Vasoactive Intestinal Peptide; YPLG, Tyr-Pro-Leu-Gly by Robert N McLay; Weihong Pan; Abba J Kastin (2181-2255).
This review catalogs effects of peptides on various aspects of animal and human behavior as published in the journal Peptides in its first twenty years. Topics covered include: activity levels, addiction behavior, ingestive behaviors, learning and memory-based behaviors, nociceptive behaviors, social and sexual behavior, and stereotyped and other behaviors. There are separate tables for these behaviors and a short introduction for each section.
Keywords: Peptides; Behavior; Activity; Addiction; Ingestion; Learning; Memory; Nociception; Sex; Stereotypy;

Endogenous opiates: 2000 by Anthony L. Vaccarino; Abba J. Kastin (2257-2328).
This paper is the twenty-third installment of the annual review of research concerning the opiate system. It summarizes papers published during 2000 that studied the behavioral effects of the opiate peptides and antagonists, excluding the purely analgesic effects, although stress-induced analgesia is included. The specific topics covered this year include stress; tolerance and dependence; learning, memory, and reward; eating and drinking; alcohol and other drugs of abuse; sexual activity, pregnancy, and development; mental illness and mood; seizures and other neurological disorders; electrical-related activity; general activity and locomotion; gastrointestinal, renal, and hepatic function; cardiovascular responses; respiration and thermoregulation; and immunological responses.
Keywords: Stress; Tolerance; Dependence; Learning; Memory; Reward; Eating; Drinking; Alcohol; Mental illness; Depression; Activity; Cardiovascular responses; Temperature; Respiration; Epilepsy; Sex; Immunology; Opiate; Peptide;

Peptide drug modifications to enhance bioavailability and blood-brain barrier permeability by Ken A Witt; Terrence J Gillespie; Jason D Huber; Richard D Egleton; Thomas P Davis (2329-2343).
Peptides have the potential to be potent pharmaceutical agents for the treatment of many central nervous system derived maladies. Unfortunately peptides are generally water-soluble compounds that will not enter the central nervous system, via passive diffusion, due to the existence of the blood-brain barrier. Peptides can also undergo metabolic deactivation by peptidases, thus further reducing their therapeutic benefits. In targeting peptides to the central nervous system consideration must be focused both on increasing bioavailability and enhancing brain uptake. To date multiple strategies have been examined with this focus. However, each strategy comes with its own complications and considerations. In this review we assess the strengths and weaknesses of many of the methods currently being examined to enhance peptide entry into the central nervous system.

Author index (2345-2349).

Keyword index (2351-2356).

Volume contents (2357-2372).