bluuebunny/crossref_metadata_2025 huggingface dataset:
Novel galanin receptor ligands
Authors: POOGA MARGUS , JURÉUS ANDERS , REZAEI KHADIJEH , HASANVAN HOMA , SAAR KÜLLIKI , KASK KALEV , KJELLÉN PETER , LAND TIIT , HALONEN JUHA , MÄEORG UNO , URI ASKO , SOLYOM SANDOR , BARTFAI TAMAS , LANGEL ÜLO
Published: August 2010

Galanin is a neuroendocrine peptide which is 29/30 amino acids in length and is recognised by G‐protein‐coupled central nervous system receptors via its N‐terminus. We synthesised several galanin receptor ligands and fragments around C‐terminal extensions of galanin(l‐13) to yield chimeric peptides with C‐terminals corresponding to bioactive peptides like bradykinin(2‐9), mastoparan, neuropeptide Y(25‐36) or substance P(5‐11), respectively. We also synthesised short galanin analogs in which galanin(l‐13) was C‐terminally elongated with Lys14; different pharmacologically active small molecules were then attached to the ε‐amino group of Lys14. Several cysteine‐substituted linear and ring closed analogs of galanin(l‐9) and galanin(l‐16) were also synthesised. The equilibrium binding constants for these peptides at hypothalamic galanin receptors were determined and found in the subnanomolar to micromolar range. The large number of peptides and their binding affinities presented here permit structure‐activity relationship analysis of peptide‐type ligands to galanin receptors.
Stabilization of amphipathic α‐helical and β‐helical conformations in synthetic peptides in the presence and absence of ionic interactions
Authors: LAZO N. D. , DOWNING DONALD T.
Published: August 2010

The role of ionic interactions in stabilizing amphipathic α‐helices was studied in the synthetic peptide Ac‐NLEELKKKLEELKG‐NH2 (NLEKG14), potentially stabilized by attraction between complementary ions in successive turns of the helix, and in the peptide Ac‐NLEELEEELEELEG‐NH2 (NLEG14), in which no side‐chain ionic attractions are possible. At a pH below the pKa of glutamate, NLEG14 had a higher helix content than NLEKG14. At pH 3 to pH 10, the helicity of NLEKG14 did not change, whereas NLEG14 was converted to random coil at pH 7. The role of ionic interactions in stabilizing the conformation of β‐structures was studied in the synthetic peptides Ac‐KLKLKLELELELG‐NH2 (KLEG13) and Ac‐ELELELELELELG‐NH2 (ELG13). At a pH below the pKa of glutamate, ELG13 had a higher β‐content than KLEG13, as judged by their dichroic spectra, but at higher pH, ELG13 was converted to random coil, whereas KLEG13 retained a predominantly β‐conformation. At pH 7, high NaCl concentration produced a significant increase in the α‐helix content of NLEKG14, converted NLEG14 from random coil to α‐helix and converted ELG13 from random coil to β‐conformation. Overall, the results demonstrate that ionic attraction between side‐chains plays a lesser role than hydrogen bonding and hydrophobic effects in stabilizing the α‐ and β‐conformations exhibited by these amphipathic peptides.
UV‐difference and CD spectroscopy studies on juvenile hormone binding to its carrier protein
Authors: KRZYZANOWSKA DOROTA , LISOWSKI MAREK , KOCHMAN MARIAN
Published: August 2010

Juvenile hormone binding protein (JHBP) from hemolymph of Galleria mellonella is a single‐chain glycoprotein of molecular mass near 25,880 (1) containing no Trp residues. The fourth derivative of the protein absorption spectrum shows the characteristic vibrational components of the phenylalanine spectrum within the range 240–270 nm. At longer wavelengths two main bands 0–0 and 0+800 cm−1 appear, caused by vibrational levels of electronic transition, π→π*, in the tyrosine residues, with maxima at 279 nm and 286 nm, respectively. Two intersection points of the second derivative absorption band with abscissa at about 288.8 nm and 283 nm were analysed for estimation of the environment polarity of Tyr residues in the JHBP molecule. The results obtained suggest that JHBP contains at least two classes of Tyr residues with very apolar environment, similar to that found in azurine. In the JHBP‐JH complex only one class of Tyr residues located in a very apolar environment was found, and a small perturbation of disulphide bridges was deduced from the UV‐difference spectrum. Ligand perturbation appears as a minimum at 243 nm of the UV‐difference spectrum. Comparison of the circular dichroism (CD) spectra for free JHBP with the CD spectra for the JHBP‐JH complex monitored in the far‐UV (190–240 nm) region indicates rather small differences in the secondary structure of the protein. Although ligand binding induces distinct changes in the near‐UV (250–300 nm) region of the CD spectrum of JHBP, it is apparent where both Tyr and Phe residues contribute.
Interaction of α‐helical peptides with phospholipid membrane: effects of chain length and hydrophobicity of peptides
Authors: OHMORI NAOYA , NIIDOME TAKURO , HATAKEYAMA TOMOMITSU , MIHARA HISAKAZU , AOYAGI HARUHIKO
Published: August 2010

To investigate the interaction of amphiphilic α‐helical peptides with phospholipid membranes, we synthesized Ac‐(Leu‐Ala‐Arg‐Leu)3‐NHcH3 (43) and three derivatives, in which the chain length and the size of the hydrophobic region of the peptides were different from each other. These peptides formed an α‐helical structure in the presence of vesicles. In the membrane‐perturbation measurement, only 43 showed a strong membrane‐perturbation activity below phase‐transition temperature (25°C), but above phase‐transition temperature (50°C), most peptides showed similar strong activities. On the other hand, in membrane‐fusion measurement the long peptides, e.g., Ac‐(Leu‐Ala‐Arg‐Leu)3‐(Leu‐Arg‐Ala‐Leu)3‐NHCH3, had strong activities at low peptide concentrations at 25°C. The present study indicated a parallel relationship did not always exist between membrane fusion and perturbation caused by peptides.
Design, synthesis and CD4 binding studies of a fluorescent analogue of a peptide that enhances HIV‐1 infectivity
Authors: DETTIN M. , SCARINCI C. , ZANOTTO C. , CABRELLE A. , ROSSI A. DE , BELLO C. DI
Published: August 2010

We previously demonstrated that a 23‐amino‐acid peptide derived from the V3 loop of the surface glycoprotein of human immunodeficiency virus (HIV‐I) strain MN was able to bind soluble CD4 and to enhance HIV‐1 infection. Further studies suggested that the peptide/CD4 interaction induces an increase in both CD4 expression and CD4/gp120 binding affinity. To facilitate identification of the complementary binding site for the peptide on cellular CD4, we designed an analogue carrying a single fluorescein moiety. The synthesis of this modified analogue presented several problems because of the presence of several amino acids in the sequence carrying potentially reactive groups in their side‐chains, and the necessity of introducing only one marker per molecule in a position that would not affect biological activity. The side‐chain of Lys19 was selected because separate studies demonstrated that its substitution with an uncharged amino acid does not reduce the peptide's biological activity. We compared the merits of various synthetic protocols used to condense the fluorescent marker with the peptide. Biological assays indicated that the presence of the fluorescein moiety did not compromise peptide binding to CD4; furthermore, binding of the labeled analogue was not abolished by trypsin treatment, suggesting that the peptide may interact with both CD4 and additional trypsin‐resistant binding sites on the cell surface. Finally, we verified the preservation of HIV infection enhancing ability in the labeled peptide.
Investigation on the stability of the Dde protecting group used in peptide synthesis: migration to an unprotected lysine1
Authors: AUGUSTYNS KOEN , KRAAS WOLFGANG , JUNG GÜNTHER
Published: August 2010

An investigation of the stability of the Dde protecting group for amines, used in solid‐phase peptide synthesis, shows that an unprotected ε‐NH2 group of lysine can acquire the Dde protection from another ε‐NH2 group or from an α‐NH2 group. An unprotected α‐NH2, however, cannot remove Dde from an ε‐NH2 function. This migration takes place during Fmoc removal from the ε‐NH2 with piperidine and/or during the subsequent washing steps. The Dde migration is also possible in neat dimethylformamide by a direct nucleophilic attack of the free ε‐NH2 group. Addition of piperidine to the reaction medium accelerates the side reaction, probably because of the formation of an unstable piperidine‐Dde adduct. Dde migration can he prevented if the 9‐fluorenylmethyloxycarbonyl is cleaved with 1,8‐diazabicyclo[5,4,0]undec‐7‐ene for a short reaction time (2%, 3 × 3 min). Finally, this rearrangement is shown to occur both as an intra‐ and intermolecular reaction between peptides on the same resin bead.
Biologically active analogues of arginine vasopressin containing conformationally restricted dipeptide fragments
Authors: LAMMEK BERNARD , CZAJA MAŁGORZATA , DERDOWSKA IZABELA , ŁEMPICKA ELŻBIETA , SIKORA PIOTR , SZKRÓBKA WITOLD , TRZECIAK HENRYK I.
Published: August 2010

In this study we described the synthesis and pharmacological properties of five new analogues of arginine vasopressin (AVP). Four of these analogues contained ethylene‐bridged dipeptide Phe‐Phe in positions 2 and 3; one had two N‐Me‐Phe residues. All new peptides were tested for vasopressor and antidiuretic activities. We also estimated the uterotonic activities of these compounds in vitro. Three analogues were highly potent V1‐antagonists. One of them, namely [Cpa1,(Phe‐Phe)2,3,Val4]AVP, which seemed to not interact with either V2 and oxytocic receptors, was outstandingly selective. It is interesting that the high antipressor potency of our second peptide, [(N‐Me‐Phe)2,3]AVP, was achieved without modification of position 1. Our results open new possibilities for the design of very potent and selective V1‐antagonists of AVP.
Isolation and characterization of insulin in Russian sturgeon (Acipenser guldenstaedti)
Authors: RUSAKOV YU I. , MORIYAMA S. , BONDAREVA V. M. , KOLYCHEV A. P. , AMEMIYA Y. , YASUDA A. , KAWAUCHI H.
Published: August 2010

Insulin was isolated from the pancreas of Chondrostean fish, the Russian sturgeon, Acipenser guldenstaedti, by acid‐ethanol extraction followed by ion‐exchange and reverse‐phase high‐performance liquid chromatographies. The amino acid sequence determined by automated Edman degradation is as follows: A‐chain (21‐amino‐acid peptide), H‐Gly‐Ile‐Val‐Glu‐Gln‐Cys‐Cys‐His‐Ser‐Pro‐Cys‐Ser‐Leu‐Tyr‐Asp‐Leu‐Glu‐Asn‐Tyr‐Cys‐Asn‐OH; and B‐chain (31‐amino‐acid peptide), H‐Ala‐Ala‐Asn‐Gln‐His‐Leu‐Cys‐Gly‐Ser‐His‐Leu‐Val‐Glu‐Ala‐Leu‐Tyr‐Leu‐Val‐Cys‐Gly‐Glu‐Arg‐Gly‐Phe‐Phe‐Tyr‐Thr‐Pro‐Asn‐Lys‐Val‐OH. The sturgeon insulin appears to be identical with one of two forms of paddlefish insulin and differs from the other form by a single substitution in the A‐chain, Asp15: His15. The amino acid sequence of sturgeon insulin is more similar to the amino acid sequence of mammalian insulins than of other fish insulins. Sturgeon insulin showed parallel but weaker displacement than porcine insulin and pink salmon insulin in their respective radioimmunoassays and was less potent than porcine insulin in displacing radiolabeled porcine insulin bound to partially purified rat liver plasma membranes.
Synthesis and pharmacological evaluation of dimer derivatives of the bradykinin receptor antagonist HOE‐140
Authors: Daffix I. , Amblard M. , Bergé G. , Dodey P. , Pruneau D. , Paquet J.‐L. , Fouchet C. , Franck R.‐M. , Defrêne E. , Luccarini J.‐M. , Bélichard P. , Martinez J.
Published: August 2010

Abstract: The synthesis and pharmacological evaluation of dimer derivatives of the C‐terminal fragments of the potent bradykinin antagonist HOE‐140, linked through their N‐termini, were performed. The influence of peptide moiety length was studied using the succinyl moiety as a linker. Our attention focused on the dimer of the C‐terminal tetrapeptide of HOE‐140 (compound JMV 980), which displayed some inhibiting activity (IC50= 247 nM) for bradykinin B2 receptors. Unexpectedly, it was orally active in inhibiting bradykinin‐induced hypotension in the rat. Based on this tetrapeptide dimer model, we synthesized pseudotetrapeptide dimer bradykinin antagonists 29 and 33, which exhibited high affinity (Ki= 76 and 61 nM, respectively) for the human cloned B2 receptor. In addition, compound 29 inhibited bradykinin‐induced contraction of the human umbilical vein giving a pKB value of 6.45. Compounds 29 and 33 were selective toward B2, receptors because they did not bind to the cloned human B1 receptor up to 10 μM.
A synthetic calcium‐chelating l‐glutamyl‐L‐serine phosphate (Glu‐Ser.P) enhances calcium absorption in the rat
Authors: Galzigna L. , Domergue N. , Previero A.
Published: August 2010

Abstract: A new synthetic procedure was used to prepare l‐glutamyl‐L‐serine phosphate (Glu‐Ser.P) with a good yield. The phosphopeptide is able to chelate calcium ion as shown by conductimetric titration and through the use of a calcium‐specific electrode. Intestinal absorption of radioactive calcium is enhanced in rats fed 45Ca and Glu‐Ser.P.
Release of aqueous contents from phospholipid vesicles induced by cecropin A (1–8)‐magainin 2 (1–12) hybrid and its analogues
Authors: Kang J.H. , Shin S.Y. , Jang S.Y. , Lee M.K. , Hahm K.‐S.
Published: August 2010

Abstract: The membrane‐disrupting properties of cecropin A (1–8)‐magainin 2 (1–12) hybrid peptide, which has higher antitumor with less hemolytic activities than cecropin A (1–8)‐melittin (1–12), and its analogues were assessed by measuring the induced release of vesicle‐entrapped fluorescence probes. A model membrane was made of zwitterionic phospholipid (phosphatidylcholine) or the mixture of negatively and zwitterionic phospholipids (phosphatidylcholine and phosphatidylserine). The extent of leakage of the aqueous content of the phospholipid vesicles was found to have remarkable discrepancies according to the amphipathic nature of each analogue peptide. The entrapped high molecular weight solute (fluorescein‐labeled immunoglobulin G, 55 kDa) also was released by the analogue which had the largest hydrophobic region and the highest amphipathic score among peptides tested. As the result of the determination of the relationships between the membrane‐disrupting properties and the hydrophobicity values of peptides, it was found that the membrane‐disrupting activity increased according to increasing the hydrophobicity of the peptide. The tryptophan fluorescence emission spectra and CD spectra showed that on interaction with the phospholipid vesicle, the peptide acquired the ordered structure and α‐helical conformation by moving a tryptophan residue into the nonpolar environment of the phospholipid vesicle. These results suggest that the breakdown of the lipid bilayer was mediated by the α‐helical amphipathic structure of the peptide interacting with the lipid bilayers as well as the by the hydrophobicity of the peptide.
Conformational investigation of α,β‐dehydropeptides. IX. N‐Acetyl‐(E)‐α,β‐dehydrobutyrine N′‐methylamide: stereoelectronic properties from infrared and theoretical studies*
Authors: Broda M.A. , Rzeszotarska B. , Smełka L. , Pietrzyński G.
Published: August 2010

Abstract: : The Fourier transform infrared spectra of Ac‐(E)‐ΔAbu‐NHMe were analyzed to determine the predominant solution conformation (s) of this (E)‐α,β‐dehydropeptide‐related compound and the electron density perturbation in its amide groups. The measurements were performed in dichloromethane and acetonitrile in the region of mode vs (N–H), amide I, amide II and vs (Cα= Cβ). The equilibrium geometrical parameters, calculated by a method based on the density functional theory with the B3LYP functional and the 6–31G* basis set, were used to support spectroscopic interpretation and gain some deeper insight into the molecule. The experimental and theoretical data were compared with those of three previously described molecules: isomeric Ac‐(Z)‐ΔAbu‐NHMe, Ac‐ΔAla‐NHMe, which is deprived of any β‐substituent, and saturated species Ac‐Abu‐NHMe. The titled compound assumes two conformational states in equilibrium in the DCM solution. One conformer is extended almost fully and like Ac‐ΔAla‐NHMe is C5 hydrogen‐bonded. The other adopts a warped C5 structure similar to that of Ac‐(Z)‐ΔAbu‐NHMe. The C5 hydrogen bond, unlike the H‐bond in Ac‐ΔAla‐NHMe, is disrupted by acetonitrile. The resonance within the N‐terminal amide groups in either of the (E)‐ΔAbu conformers is not as well developed as the resonance in Ac‐Abu‐NHMe. However, these N‐terminal groups, compared with the other unsaturated compounds, constitute better resonance systems in each conformationally related couple: the C5 hydrogen‐bonded Ac‐(E)‐ΔAbu‐NHMe/Ac‐ΔAla‐NHMe and the warped C5 Ac‐(E)‐ΔAbu‐NHMe/Ac‐(Z)‐ΔAbu‐NHMe. The resonance within the C‐terminal groups of the latter couple apparently is similar, but less developed than the resonance in Ac‐Abu‐NHMe. The electron distribution within the C‐terminal group of the hydrogen‐bonded C5 (E)‐ΔAbu conformer apparently is determined mainly by the electron influx from the Cα= Cβ double bond.
Characterization of antigen‐antibody interactions using single substitution analogs and mixture‐based synthetic combinatorial libraries
Authors: Appel J.R. , Campbell G.D. , Buencamino J. , Houghten R.A. , Pinilla C.
Published: August 2010

Abstract: In an effort to use monoclonal antibodies (mAbs) as selective probes for early detection of breast cancer, the specificities of a number of antipeptide mAbs have been studied at the individual amino acid level using single substitution peptide analogs and peptide combinatorial libraries. In this study, the mapping results are presented for mAbl72‐12A4, which was raised against the haptenic peptide LGSGAFGTIYKG(C), corresponding to residues 138–149 of the oncogene v‐erbB. This peptide is homologous with a region in epidermal growth factor receptor (EGFR) and human oncogene c‐erbB‐2, and contains the ATP binding motif that is common among protein kinases. The substitution profile of this interaction correlated well with the results from the screening of hexa‐ and decapeptide positional scanning libraries. Based on the results of this mAb's specificity for the antigenic determinant (‐AFGTIYK‐), proteins that have sequence homology were found from a database search of human sequences. Thirty‐two unique peptide sequences, a majority of which was from protein kinases, were synthesized and tested for recognition by mAb 172–12A4. Eleven peptides had activities that differed from the original peptide by less than an order of magnitude, and the activities for 29 of the 32 (90%) could be accurately predicted based on the individual substitution analog results. While both epitope mapping approaches address the amino acid level of mAb specificity, positional scanning libraries offer an advantage of identifying the positional importance of each antigenic determinant residue without any prior knowledge of the mAb's specificity. The fine specificity mapping of peptide‐specific mAbs using the synthetic tools illustrated here will be useful for the development of immunodiagnostics that detect cancer‐related proteins in clinical samples.
Design of ligands for the purification of anti‐MUC1 antibodies by peptide epitope affinity chromatography
Authors: Murray A. , Spencer D.I.R. , Missailids S. , Denton G. , Price M.R.
Published: August 2010

Abstract: The fine specificity of epitope recognition of the anti‐MUCl mucin monoclonal antibody, C595 has been studied using solid‐phase replacement net (RNET) analysis. Two peptides (RAAP and RPPP) showed increased reactivity with C595 antibody compared with the native epitope (RPAP). These were synthesized as integral motifs within MUCl immunodominant peptides and analyzed by fluorescence quenching (FQ) and circular dichroism (CD). They were also tested as ligands for the purification of C595 antibody using epitope affinity chromatography. Affinity matrices were compared with respect to capacity, affinity, and quality of the purified product.

In FQ tests the native epitope peptide (APDTRPAPG) and the alanine substituted peptide had similar association constants when reacting with C595 antibody, whereas the proline substituted peptide (APDTRPPPG) had a higher association constant. This order of affinity for C595 was confirmed in chromatography experiments in which antibody was eluted from the former two peptide matrices at approximately the same point on the NaSCN elution gradient, whereas antibody was desorbed from APDTRPPPG at a higher NaSCN concentration. Circular dichroism analysis showed that the thermodynamically preferred conformation of these peptides in aqueous solution is the P‐II extended helix, the conformation preferred for an extended bound form of the peptide held by interactions with the peptide amides. The stronger binding peptide (APDTRPPPG) has the higher population of the P‐II helix in solution.

In conclusion, RNET analysis is useful in the rational design of peptide ligands so that the performance of affinity matrices may be regulated.
The hydrophobic pocket of 24p3 protein from mouse uterine luminal fluid: Fatty acid and retinol binding activity and predicted structural similarity to lipocalins
Authors: Chu S.‐T. , Lin H.‐J. , Huang H.‐L. , Chen Y.‐H.
Published: August 2010

Abstract: The conformation of 24p3 protein purified from mouse uterine luminal fluid was studied by circular dichroism spectroscopy in 200–300 nm. At pH 7.4, the spectrum in the UV region appears as one negative band with a minimum mean residue ellipticity of ‐3,600 deg.cm2.dmole−1 at 217 nm, suggesting a very low or no helical content, but a considerable amount of β‐form, β‐turn, and unordered form in the protein molecule. This agrees with the predicted secondary structures consisting of only one a‐helical segment of residues 150–163 and nine segments of residues 28–35, 50–60, 67–72, 78–86, 94–97, 106–114, 119–125, 136–140 and 166–172 in β‐forms, which would construct two orthonormal β‐sheets to form a less polar β‐barrel. The environments around Trp‐31 and Trp‐81 of this protein were studied by intrinsic fluorescence and solute quenching. They give an emission peak at 332 nm, and only about 21% of them are accessible to quenching by acrylamide. This together with their low accessibility to either CsCl or KI suggests that they are located in the less polar P‐barrel.

Hydrophobic compounds such as fatty acids, retinoids, and cholesteryl oleate in the protein solution diminish the protein fluorescence. Analysis of the fluorescence data suggests that the protein has a binding site for hydrophobic ligand. The association constants for the complex formation are 1.03 × 106M−1, 1.92 × 105M−1, 2.38 × 105M−1 or 1.25 × 105M−1for cholesteryl oleate, oleic acid, retinol, or retinoic acid at pH 7.4. Analysis of the equilibrium binding data from binding assay using [H3]‐retinol and [H3]‐retinoic acid reveals a singular type of retinoid‐binding site in the protein with the association constant of 4.92 × 105M−1 and 1.17 × 105M−1 for retinol and retinoic acid, respectively. Trp‐31 or/ and Trp‐81 is in or very near the binding site and the gross conformation of protein changes considerably as the formation of protein‐ligand complex.
The contryphans, a d‐trvptophan‐containing family of Conus peptides: interconversion between conformers
Authors: JACOBSEN RICHARD , JIMENEZ ELSIE C. , GRILLEY MICHELLE , WATKINS MAREN , HILLYARD DAVID , CRUZ LOURDES J. , OLIVERA BALDOMERO M.
Published: August 2010

We previously characterized contryphan‐R, a d‐tryptophan‐containing octapeptide from the venom of Conus radiatus. In this study, we present evidence that the contryphan family of peptides is widely distributed in venoms of the fish‐hunting cone snails. We purified, synthesized and characterized contryphan‐Sm from Conus stercusmuscarum venom, and obtained molecular evidence for the existence of a third peptide, contryphan‐P from Conus purpurascens venom ducts. The sequences of these three contryphans showed identity in seven of eight amino acids and a conserved pattern of post‐translational modification. We also demonstrate that contryphan‐Sm equilibrates between two distinct conformational states.
Chemical modification monitored by electrospray mass spectrometry: a rapid and simple method for identifying and studying functional residues in enzymes
Authors: KRELL TINO , CHACKREWARTHY SUREKA , PITT ANDREW R. , ELWELL ANDREW , COGGINS JOHN R.
Published: August 2010

A simple method to identify functional amino acids in enzymes is described. This method is based on the mass spectrometric detection of molecular weight changes as the consequence of chemical modification of enzymes with group‐specific reagents. Here we report the use of phenylglyoxal, trinitrobenzene sulfonic acid, tetranitromethane and diethylpyrocarbonate to identify functional amino acid residues. Precise information is obtained about the stoichiometry of reaction, and a relationship between the loss of enzyme activity and the amount of chemical modification is easily established. Modification sites are located by proteolytic digestion of the modified enzyme, followed by peptide mapping based on high‐pressure liquid chromatography using an electrospray mass spectrometer as an on‐line detector. In comparison with more conventional methods, protein modification is monitored directly without the need to use radioactively or spectrally labelled reagents. The methodology is limited only by the stability of the chemically modified species produced. The method has been used to characterise the active sites of several shikimate pathway enzymes, and the results obtained have been confirmed by site‐directed mutagenesis and X‐ray crystallography.
Bufokinin: a substance P‐related peptide from the gut of the toad, Bufo marinus with high binding affinity but low selectivity for mammalian tachykinin receptors
Authors: CONLON J. MICHAEL , WARNER FIONA J. , BURCHER ELIZABETH
Published: August 2010

A tachykinin peptide, termed bufokinin, was isolated in pure form from an extract of the intestine of the toad,Bufo marinus, and its primary structure was established as: Lys‐Pro‐Arg‐Pro‐Asp‐Gln‐Phe‐Tyr‐Gly‐Leu‐Met.NH2. This sequence was confirmed by chemical synthesis and shows four amino acid substitutions (Arg → Lys,Lys3→ Arg,Gln5→ Asp and Phe8→ Tyr) compared with substance P. Binding parameters for synthetic bufokinin and mammalian tachykinins were compared using receptor‐selective radioligands and crude membranes from rat tissues enriched in the NK‐1 (submandibular gland), NK‐2 (stomach fundus) and NK‐3 (brain) receptors. In terms of inhibiting the binding of the selective radioligands, bufokinin(Kd=0.3 nM) was 1.8‐fold more potent than substance P at the rat NK‐1 site, but it was only 2‐fold less potent(Kd=2.8 nM) than neurokinin A at the NK‐2 site and only 2‐fold less potent(Kd= 48 nM) than neurokinin B at the NK‐3 site. Thus, bufokinin shows relatively high affinity but lack of selectivity for all three tachykinin binding sites in rat tissues.
Antigenicity of linear and cyclic peptides mimicking the disulfide loops in HIV‐2 envelope glycoprotein: synthesis, reoxidation and purification
Authors: JRAD BESMA BELHADJ , BAHRAOUI ELMOSTAFA
Published: August 2010

The external envelope glycoprotein (gp125) of human immunodeficiency virus type 2 (HIV‐2) contains 22 cysteine residues. The positions of the 11 disulfide bridges in HIV‐2 gp125 were determined by analogy with the experimental position of the disulfide bonds found in the gp120 of HIV‐I. Peptides expected to mimic all 11 disulfide‐bonded domains containing from 13 to 47 amino acids were synthesized by the solid‐phase method according to 9‐fluorenylmethoxycarbonyl strategy, except for peptide 5, which was assembled according to t‐butoxycarbonyl (Boc) strategy. Analysis of all the crude peptides showed that the expected peptides were obtained with good yields, between 75% and 85%. Peptides were purified further by high‐performance liquid chromatography (HPLC) on an Aquapore RPC30 C8 column. Peptide homogeneity was more than 90%. For each peptide, linear peptides (L) were SH‐iodoacetamidated, whereas cyclization of peptides (C) was performed by air oxidation. Oxidation kinetics was followed with the Ellman test and HPLC. Cyclic peptides were purified by HPLC and characterized by fast atom bombardment mass spectrometry. This analysis showed that a small quantity (˜10%) of dimeric peptides (2 and 8) and cyclic peptides containing oxidized methionine or tryptophan residues (4, 9 and 10) were formed. To assess the relevance of conformation for the antigenicity of disulfide‐bonded loops of HIV‐2 gp125, the antigenicity of linear and cyclic peptides was tested against a set of 76 HIV‐2 positive human sera by enzyme‐linked immunosorbent assay. Peptides 2, 4 and 9, mimicking the VI, V2 and V3 regions of the external envelope glycoprotein (gp 125) of HIV‐2, were the most highly reactive with HIV‐2 positive human sera tested at the dilution of 150. Cyclic peptides generally were recognized more than linear peptides, as shown by their greater inhibition (2 to 10 times more) of antigen‐antibody complexes. Structure‐antigenicity of peptide V3, the most reactive peptide (75% of the HIV‐2 positive sera tested), was analyzed further. Cyclic peptide 9C had a higher affinity for anti‐gpl25 antibodies than linear peptide 9L. In addition, circular dichroism showed that linear and cyclic peptides 9 had a similar structure, but when analyzed in aqueous solution or in trifluoroethanol (TFE), the structural difference shown with antibodies was not confirmed. No significant difference was observed between the antigenicity of linear and cyclic peptides 1, 8 and 11, mimicking the C1, C2 and C4 regions of HIV‐l gp125. These peptides were weakly reactive with HIV‐2 positive sera. This result agrees with the low immunogenicity of conserved regions. © Munksgaard 1998.
Solid‐phase synthesis and characterization of carcinoembryonic antigen (CEA) domains
Authors: Kaplan B.E. , Hefta L.J. , II R.C. Blake , Swiderek K.M. , Shively J.E.
Published: August 2010

Carcinoembryonic antigen (CEA), a 180,000 dalton cell surface glycoprotein expressed on tumors of the colon, breast, ovary, and lung, has seven predicted immunoglobulin‐like domains (N‐A1‐B1‐A2‐B2‐A3‐B3), most of which are recognized by distinct monoclonal antibodies. To study the individual domains, we have prepared several of the domains (N, A3, B3, and A3‐B3) by solid‐phase peptide synthesis. The syntheses were performed by the Fmoc method using single couplings, elevated temperatures for both the coupling and deblocking reactions, and a flexible solvent system for the coupling reactions. The syntheses were accomplished on an in‐house built synthesizer which allowed for temperature control and flexible solvent control during the course of the coupling reactions. Due to the large size of the peptides (84‐184 residues), it was anticipated that the overall purity of the final product would not exceed 60% even for an average coupling yield of 99.5%. Therefore, several of the peptides were synthesized with a His6“tail” at the amino terminus, allowing for purification on a Ni‐NTA chelate column. For the most part, the purified peptides exhibited single sharp peaks by RP‐HPLC, migrated at their expected molecular weights by gel permeation chromatography, gave correct masses by electrospray ionization or matrix‐assisted laser desorption ionization time of flight mass spectrometry, gave the expected amino acid analyses, N‐terminal sequences, and tryptic maps, and bound their appropriate monoclonal antibodies. The N‐domain was extremely hydrophobic, requiring 6m guanidinium hydrochloride for solubilization, the A3 domain was soluble in dilute acid, and the B3 domain had an intermediate solubility. The affinity constants of the A3 domain and several mutants (also made by peptide synthesis) are reported, along with characterization of the 178 amino acid two‐domain peptide, A3‐B3. Although there is no evidence for proper folding of these domains by NMR, their ability to bind monoclonal antibodies with high affinity suggests that this is a plausible approach for producing individual domains of CEA.
Properties of HIV envelope expressed in the presence of SPC3, an Env‐derived peptide drug under phase II clinical trials
Authors: Barbouche R. , Fenouillet E. , Papandréou M.‐J. , Kiény M.P. , Sabatier J M.
Published: August 2010

A multibranched peptide construct (SPC3) derived from the conserved sequence of the third variable domain (V3) of the human immunodeficiency virus (HIV) envelope (Env) inhibits HIV infectivity. It is being tested in phase II clinical trials (FDA protocol 257A). Because some Env‐derived peptides inhibit HIV infectivity through alteration of Env biosynthetic pathway, we studied whether SPC3 displays its activity through interference with Env biosynthesis or with its functions at the membrane. Syncytium formation was impaired when human CD4+ cells expressed recombinant HIV Env in the presence of SPC3. This inhibition was not due to an effect of SPC3 on the amount of Env expressed at the cell membrane. As assessed using antibodies, the conformation of the receptor binding site and of V3 presented on membrane Env was not affected by the presence of SPC3 during biosynthesis. Finally, despite the ability of SPC3 to bind to CD4+ cell membrane, SPC3 did not interfere with Env binding to CD4. These data suggest that SPC3 interferes with the infection process at a post‐CD4 binding step, and not with the folding of Env.
The use of synthetic peptides in the design of a consensus sequence vaccine for Pseudomonas aeruginosa
Authors: Cachia P.J. , Glasier L.M.G. , Hodgins R.R.W. , Wong W.Y. , Irvin R.T. , Hodges R.S.
Published: August 2010

Pseudomonas aeruginosa employs pili to mediate adherence to epithelial cell surfaces. Research has shown that the C‐terminal region of the pilin monomer contains the epithelial cell binding domain, which is semiconserved in seven different strains of this bacterium. Antibodies to this region of the pilin molecule are also able to block and prevent the infection process. As there is a degree of sequence and structural homology in the C‐terminal region and all strains examined have been shown to bind to the same cell surface receptor, we reasoned that it should be possible to produce a synthetic peptide consensus sequence which would provide cross‐reactive antiserum from a single peptide immunogen inhibiting the adherence of the known strains of P. aeruginosa.

In this article we examine the cross‐reactivity of five rabbit polyclonal antisera. One has been raised against the cell‐surface receptor binding domain of native PAK strain pilin (residues 128‐144) while the others have been raised to analogues of this region. Analysis of the cross‐reactivity of these antisera, using competitive ELISA assay, has shown that it is possible to manipulate the amino acid sequence of a peptide immunogen to generate antiserum, which exhibits enhanced cross‐reactivity to various strains of P. aeruginosa. Furthermore, when this peptide is conjugated to tetanus toxoid and used to vaccinate mice it provided cross‐reactive protection against heterologous challenge with PAO strain bacteria.

The results of these experiments are analyzed, and the applicability of our hypothesis and the implications of this approach to the design of a strain‐independent consensus vaccine for immunization against Pseudomonas aeruginosa are discussed.
Carboxy‐terminal extension stabilizes the topological stereoisomers of guanylin
Authors: Schulz A. , Escher S. , Marx U.C. , Meyer M. , Rösch P. , Forssmann W‐G. , Adermann K.
Published: August 2010

The peptide hormone guanylin constitutes two topological stereoisomers, which are connected through an equilibrium of interconversion. To investigate the importance of amino acid residues in the central region between the inner cysteines and at the carboxy terminus for this isomerism, synthetic derivatives of guanylin were compared by HPLC, 2D 1H NMR spectroscopy and by their guanylyl cyclase‐C (GC‐C)‐activating potency. An increase in the central sterical bulk by introduction of diiodo‐Tyr9 had virtually no effect on the isomerization kinetics. Compared to guanylin, carboxy‐terminal amidation did not affect the equilibrium between the two isoforms either. In contrast, two significantly stabilized isomers were obtained by extending the carboxy terminus of guanylin with one additional leucine resembling the characteristic of human uroguanylin isomers. This effect was intensified by a further Lys‐Lys extension, thus revealing that the conformational exchange between the guanylin isomers is dependent on the extent of the sterical hindrance in the carboxyterminal region of this peptide. Demonstrated by 2D NMR spectroscopy, the separated isomers of the carboxy‐terminally extended derivatives of guanylin exhibit unambiguously closely related structures as found originally for guanylin isomers, which are only detectable as a mixture. Because only one of the stabilized guanylin isomers activates guanylyl cyclase‐C, the three‐dimensional structure of the GC‐C‐activating guanylin isomer is now defined. The stabilized isoforms of guanylin described in this study represent suitable tools for the separate functional investigation of the GC‐C‐agonistic isomer of guanylin as well as of its isomeric counterpart.
Influence of preformed α‐helix and α‐helix induction on the activity of cationic antimicrobial peptides
Authors: Houston M.E. , Kondejewski L.H. , Karunaratne D.N. , Gough M. , Fidai S. , Hodges R.S. , Hancock R.E.W.
Published: August 2010

One prominent class of cationic antibacterial peptides comprises the α‐helical class, which is unstructured in free solution but folds into an amphipathic α‐helix upon insertion into the membranes of target cells. To investigate the importance of α‐helicity and its induction on interaction with membranes, a series of peptides was constructed based on a hybrid of moth cecropin (amino acids 1‐8) and bee melittin (amino acids 1‐18) peptides. The new peptides were predicted to have a high tendency to form α‐helices or to have preformed α‐helices by virtue of construction of a lactam bridge between glutamate and lysine side‐chains at positions i and i+ 4 at various locations along the primary sequence. In two examples where the use of lactam bridge constraints induced and stabilized α‐helical structure in benign (aqueous buffer) and/or hydrophobic medium, there was a decrease in antibacterial activity relative to the linear counterparts. Thus the preformation of α‐helix in solution was not necessarily beneficial to antimicrobial activity. In the one case where the lactam bridge did result in increased antibacterial activity (lower minimal inhibitory concentration values) it did not increase α‐helical content in benign or hydrophobic medium. Broadly speaking, good activity of the peptides against Pseudomonas aeruginosa correlated best (r2= 0.88) with a helican parameter which was calculated as the induction of α‐helix in α membrane‐mimicking environment divided by the α‐helix formation under benign conditions. Interestingly, the activity of the lactam bridge peptide constructs correlated in part with alterations in bacterial outer or cytoplasmic membrane permeability.
Identification of critical residues of staphylococcal enterotoxin B for lymphomonocyte proliferation and cytokine production
Authors: Stefano A. Di , Paulesu L. , Niccolai N. , Scarselli M. , Soldani P. , Neri P.
Published: August 2010

Superantigens bind to major histocompatibility complex class II molecules and stimulate large numbers of T cells expressing particular Vβ elements of the T‐cell receptor. Staphylococcal enterotoxin B (SEB) is a bacterial superantigen that causes food poisoning and toxic‐shock syndrome. The toxicity of SEB is thought to be mediated by T‐cell stimulation and cytokine production. Different regions of the SEB molecule are important for mitogenic activity. To identify critical residues of SEB in the region 124‐154, which competitively inhibits the mitogenic activity of the toxin, we used the synthetic peptide approach and alanine scanning mutagenesis as a probe. We synthesized eight peptides with alanine replacement of all residues in the SEB sequence 131‐138 and tested them for the capacity to inhibit both SEB‐induced proliferation of human lymphomonocytes and the production of tumor necrosis factor α and interferon γ. Mutation to alanine of the residue Thr 133 improved the inhibition of SEB‐induced proliferation and cytokine production, whereas the substitution of Ser 131 also increased the inhibition, albeit to a lesser degree. The peptide obtained by substitution of Val 136 with alanine was unable to inhibit SEB‐induced proliferation and cytokine production, suggesting that Val 136 is essential for mitogenic activity. Thus hydrophobic interactions apparently are very important for mitogenic activity. The identification of critical residues in this active site in the SEB and the computer modeling based on crystal X‐ray data contribute to a better understanding of the molecular mechanism of the superantigen and may be useful for therapeutical applications.
Isolation and characterization of a group of oligopeptides related to oxidized glutathione from the root ofPanax ginseng
Authors: Chen Z.‐K. , Fan C.‐X. , Ye Y.‐H. , Yang L. , Jiang Q. , Xing Q.‐Y.
Published: August 2010

Six γ‐glutamyl oligopeptides were isolated for the first time from aqueous methanol extracts ofPanax ginsengroot by using column chromatography on ion‐exchange resin, gel filtration and reverse‐phase high‐performance liquid chromatography. Their structures had been established with the methods of amino acid analysis, N‐terminal, C‐terminal determination and double‐coupling sequence analysis. They were: P‐I (N‐γ‐glutamylcystinyl‐bis‐glycine), P‐II (γ‐glutamylcysteinylglycine disulfide, oxidized glutathione), P‐III (N,N′‐bis‐γ‐glutamylcystinylglycine), P‐IV (γ‐glutamylcysteinylglycinamide disulfide), P‐V (N‐γ‐glutamylglycylcysteine disulfide), P‐VI (γ‐glutamylarginine); five of them are related to oxidized glutathione. The structures were further confirmed by the chemical synthesis. As far as we know, P‐V (N‐γ‐glutamylglycylcysteine disulfide) is a new biologically active peptide which exhibits somnogenic effect and is more potent than that of P‐II.
Combined solid‐phase/solution synthesis of a 31‐residue vasoactive intestinal peptide analog: general method for repetitive coupling of fragments without isolation and purification of intermediates
Authors: Felix A.M. , Zhao Z. , Lambros T. , Ahmad M. , Liu W. , Daniewski A. , Michalewsky J. , Heimer E.P
Published: August 2010

A novel analog of vasoactive intestinal peptide (VIP) has been reported which exhibits high potency and enhanced duration of in vivo biological activity. This VIP analog, cyclo‐(Lys21‐Asp25)‐Ac[Glu8,Lys12,Nle17,Ala19, Asp25,Leu26,Lys27,28,Gly29,30,Thr31]‐VIP, which also has a lactam bridge, has been reported to have relaxant effects that are significantly more potent than other β‐agonists such as salbutamol and salmeterol. Because it has potential use for the treatment of bronchial asthma in humans, various convergent syntheses were evaluated to enable the economic preparation of large quantities of this medium‐sized hentriacontapeptide. From these studies we developed a combined solid‐phase/solution synthesis which uses four protected fragments (each prepared by solid‐phase synthesis with highly acid‐labile resins) possessing Nα‐9‐fluorenylmethyloxycarbonyl and side‐chain tert‐butyl protection. Only equivalent amounts of each fragment were required to achieve near‐quantitative coupling reactions using N‐[(1H‐benzotriazol‐1‐yl)(dimethylamino)methylene]‐N‐methylmethanaminium hexafluorophosphate N‐oxide/N‐hydroxybenzotriazole. All reagents and side products were removed at each stage by simple extraction procedures. Final deprotection was carried out with 90% trifluoroacetic acid. Under these conditions only low levels of epimerization were observed (<2%). These diastereoisomers and other trace impurities were removed from the product in a single purification by preparative high‐performance liquid chromatography. The procedure has been scaled up (10‐g scale) and the final product obtained in an overall (nonoptimized) yield of 24%. This procedure for the repetitive coupling of fragments, without isolation of intermediates, may be generally applicable for the economic synthesis of other medium‐sized and longer peptides.
Interaction of gonadotropin‐releasing hormone and its agonist analogs with Ca2+ in a nonpolar milieu. Correlation with biopotencies
Authors: Ananthanarayanan V. S. , Salehian O. , Bumble K. S.
Published: August 2010

Extracellular Ca2+ is necessary for the action of gonadotropin‐releasing hormone (GnRH). Assuming that this partly because of the interaction of the hormone with the relatively abundant extracellular Ca2+ in the low dielectric milieu of the bilayer plasma membrane, we studied the interaction of GnRH and five of its agonist analogs with Ca2+ under membrane‐mimetic conditions. The peptides used, in increasing order of their reported gonadotropin‐releasing activities, were: des‐amide GnRH (or GnRH‐OH); [Ala6]GnRH; [d‐Ala6]GnRH; des‐Gly10[d‐Ala6,Pro9‐NHEt]GnRH and, des‐Gly10[d‐Trp6,Pro9‐NHEt]GnRH. Changes in the far‐UV CD and fluorescence spectra of these peptides in trifluoroethanol were used to monitor conformational changes and obtain the Ca2+‐binding isotherms. The data show that GnRH and its active analogs contain two Ca2+ binding sites, whereas the inactive analogs have only one. The extent of Ca2+ binding by the agonist peptides paralleled their biological potency ranking. The superactive analog des‐Gly10[d‐Trp6,Pro9‐NHEt]GnRH exhibited the ability to transport Ca2+ ions across large unilamellar vesicles of dimyristoylphosphatidylcholine. Our study shows that significant differences among the GnRH and its analog peptides, suggestive of differences in their conformations, are manifested only in the presence of Ca2+. This observation would provide a basis for understanding GnRH action in terms of the hormone's interaction with Ca2+ in the lipid milieu.
Amino acids with aryl‐keto function in their side chains
Authors: Bednaiek M.A.
Published: August 2010

Amino acids with aryl‐keto function in their side‐chains were obtained in excellent yields in the reaction of ω‐carboxyamino acids with liquid HF in the presence of aromatic compounds susceptible to electrophilic substitution, such as anisole, 2‐methoxybiphenyl, butyl phenyl ether or 1,3‐dimethoxybenzene. The new amino acids were converted smoothly into N‐tert‐ butyloxycarbonyl or W‐fluorenylmethoxycarbonyl derivatives and then incorporated into peptides by conventional coupling methods. During the coupling step, no formation of cyclic Schiff bases was observed for aryl‐keto amino acids derived from dl‐α‐aminopimelic acid and from l‐α‐aminosuberic acid. In the crude products, truncated peptides terminated at the keto amino acids were not detected by liquid chromatography‐mass spectrometry.