bluuebunny/crossref_metadata_2025 huggingface dataset:
Murine Bone Marrow Mesenchymal Stem Cells Cause Mature Dendritic Cells to Promote T‐Cell Tolerance
Authors: Wang Q. , Sun B. , Wang D. , Ji Y. , Kong Q. , Wang G. , Wang J. , Zhao W. , Jin L. , Li H.
Published: August 2010

Bone mesenchymal stem cells (BMSC) are attractive not only in regenerative medicine, but also for the treatment of autoimmune diseases and graft‐versus‐host disease. BMSC also play a role in enabling alloantigen tolerance. An in‐depth mechanistic understanding of this phenomenon of tolerance could lead to novel cell‐based therapies for autoimmune disease. We demonstrate here that co‐culture of mature dendritic cells (DC) with BMSC in a transwell system (BMSC‐DC) downregulated expression of the maturation marker, CD83 and CD80/86 co‐stimulatory molecules on DC, while increasing their endocytic activity. This resulted in defective antigen presentation and co‐stimulatory capacity of mature DC. Functionally, BMSC‐DC have impaired T‐cell stimulatory activity in a mixed lymphocyte reaction and orchestrate a shift from predominantly pro‐inflammatory T‐helper (Th)‐1 to anti‐inflammatory Th2 cells. While the expression of MHC II, CD80 and CD86 were upregulated on BMSC co‐cultured with DC, these BMSC lacked the ability to stimulate T‐cell proliferation. Taken together, these data suggest that the interaction between BMSC and DC modulates the immunoregulatory function of these cells in a coordinated manner, effectively skewing the immune response towards T‐cell tolerance.
A peptide derivative (1‐70 fragment) of protein SV‐IV accelerates human blood coagulation in vitro by selective inhibition of the heparin‐induced antithrombin III activation process
Authors: MICCO B. DI , STIUSO P. , COLONNA G. , PORTA R. , MARFHESE M. , SCHININA M.E. , MACALELLO M.A. , METAFORA S.
Published: August 2010

The effect of two peptide derivatives of the rat SV‐IV (SV‐IV/A: 1–70 fragment; SV‐IV/B: 71–90 fragment) on human blood coagulation was investigated. The SV‐IV/A fragment was found to possess the same procoagulant activity of the native protein, whereas SV‐IV/B retained only a very small fraction of the activity. The results obtained strongly suggest that the procoagulant activity of SV‐IV/A is due, like SV‐IV, to a selective inhibition of the antithrombin III (AT III) activation process induced by heparin, an essential cofactor of AT III. The main data supporting this hypothesis are the following: I) the concentration of active serum AT III decreases when SV‐IV/A is present in the clotting system; 2) the plasma treatment with SV‐IV/A reduces the concentration of AT III, but not that of other plasma serine protease inhibitors; 3) the recalcification time (RT) of the plasma treated with a rabbit anti‐AT III polyclonal antiserum is not modified by SV‐IV/A; 4) the presence of SV‐IV/A in a reaction mixture containing pure fibrinogen, α‐thrombin, heparin. and AT III neutralizes the thrombin inhibition induced by AT III; 5) the concentration of the thrombin‐AT III complexes, occurring in sera obtained from CaCl2‐coagulated plasma, is markedly reduced in the presence of SV‐IV/A; 6) appropriate concentrations of heparin neutralize the inhibitory effect of either SV‐IV/A or SV‐IV on the AT III activity in vitro.
α‐Amino acids derived from ornithine as building blocks for peptide synthesis
Authors: LESCRINIER THEO , BUSSON ROGER , WINTER HANS DE , HENDRIX CHRIS , JANSSEN GERARD , PANNECOUQUE CHRISTOPHE , ROZENSKI JEF , AERSCHOT ARTHUR VAN , HERDEWIJN PIET
Published: August 2010

Recently great interest has arisen in the synthesis of combinatorial libraries, and this technology provides a significant partner to contemporary strategies in rational design and lead discovery. By simple combination of a given set of building blocks, high numbers of different molecules are produced simultaneously, increasing the possibility of discovery of a lead compound in a limited time. One direction of research in this field focuses on the synthesis of libraries composed of modified amino acids. Here, the synthesis and characteristics of some building blocks derived from ornithine are described. The synthesis is based on the acylation/sulfonation of the copper complex of ornithine by aroyl and arylsulfonyl chlorides exemplified by 2‐thiophenecarbonyl chloride, p‐toluenesulfonyl chloride and 8‐quinolinesulfonyl chloride. To evaluate the potential use of these modified α‐amino acids as component in an oligopeptide library, all three derivatives were incorporated in a hexapeptide with a random sequence using a standard coupling procedure (DIC/HOBt/DIEA). Depending upon the acidity of the amido hydrogen on the δ‐nitrogen, competition between intramolecular cyclization and peptide bond formation was observed. The higher the acidity, the more pronounced is this side reaction. Coupling conditions for peptide formation were optimized so that the newly described amino acid based building blocks are suitable for incorporation into libraries consisting of unnatural amino acids. The outlined procedures open up a broad avenue of possibilities for creation of diversity into peptidic libraries.
Studies on the peptide corresponding to residues 34–47 of bovine factor X
Authors: KING ADRIENE D. , HUH NAM‐WON , PEDERSEN LEE G. , HISKEY RICHARD G.
Published: August 2010

Calcium binding studies of a 14‐residue peptide corresponding to the 37–46 sequence of bovine factor X were performed using calcium ion selective electrode titrations and equilibrium dialysis. The presence of γ‐carboxyglutamic acid residues at positions 36 and 40 coupled with the assumption that the peptide would bind calcium ions also prompted an investigation of possible secondary conformational changes in the peptide by use of circular dichroism spectroscopy. Equilibrium dialysis revealed a single relatively weak calcium binding site (log Ka= 2.39); an ion selective electrode experiment confirmed this result (log Ka= 2.17). The peptide maintained a random coil conformation throughout the calcium ion titrations as measured by circular dichroism.
X‐Ray structure of Tyr‐D‐Tic‐Phe‐Phe‐NH2 (D‐TIPP‐NH2), a highly potent μ‐receptor selective opioid agonist. Comparisons with proposed model structures
Authors: FLIPPEN‐ANDERSON JUDITH L. , DESCHAMPS JEFFREY R. , GEORGE CLIFFORD , REDDY P. ANANTHA , LEWIN ANITA H. , BRINE GEORGE A. , SHELDRICK GEORGE , NIKIFOROVICH GREGORY
Published: August 2010

Tyr‐D‐Tic‐Phe‐Phe‐NH2 (D‐TIPP), a linear tetrapeptide containing the conformationally restricted Tic residue (tetrahydroisoquinoline‐3‐carboxylic acid), is an opioid agonist which exhibits high affinity and selectivity for the μ‐receptor. Its conformational features have been studied using a combination of solid‐state (X‐ray) and modeling (molecular mechanics and Monte Carlo simulations) methods. The results of the X‐ray study showed two distinct conformers for D‐TIPP, with the main differences lying in the orientation of the Tyr side‐chain and the presence of both D‐Tic(+) and D‐Tic(—) conformations for the D‐Tic residue. The peptide backbone is folded and stablized by the formation of one intramolecular hydrogen bond. The modeling results also indicated a folded backbone for the peptide and both cis and trans conformers for the D‐Tic residue are found in the lowest‐energy structures. Comparison of the X‐ray and modeling results shows many similarities especially around the D‐Tic residue. © Munksgaard 1997.
Conformational stability of a type II'β‐turn motif in human growth hormone [6–13] peptide analogues at hydrophobic surfaces
Authors: LEE TZONG‐HSIEN , THOMPSON PHILIP E. , HEARN MILTON T.W. , AGUILAR MARIE‐ISABEL
Published: August 2010

The interactive properties of several peptides related to human growth hormone (hGH) [6–13] containing a type II'β‐turn motif have been investigated using reversed‐phase high‐performance liquid chromato‐graphy (RP‐HPLC). Various chromatographic parameters related to the hydrophobic interactive surface area and binding affinity were measured over the range of temperatures between 5 and 85 °C. Variations in these parameters were consistent with significant differences in the relative stability of the type II’β‐turn structures of these peptidomimetics. The effect of changes in peptide conformation were also investigated through the analysis of band‐broadening behaviour during the chromatographic process. Significant variations in bandwidth observed at discrete temperatures were related to the rate of interconversion between the type II’β‐turn and more extended conformers. These investigations further document the potential of RP‐HPLC for monitoring subtle changes in peptide secondary structure at hydrophobic interfaces. © Munksgaard 1997.
Conformational analysis of cyclo(2,9)‐Ac‐QCRSVEGSCG‐OH from the C‐terminal loop of human growth hormone
Authors: JOIS D.S. SEETHARAMA , CONRAD MATTHEW W. , CHAKRABARTI SOMA , SIAHAAN TERUNA J.
Published: August 2010

A 10 amino acid residue cyclic peptide, cyclo(2,9)‐Ac‐Glnl‐Cys2‐Arg3‐Ser4‐Val5‐Glu6‐Gly7‐Ser8‐Cys9‐Glyl0, from the C‐terminal region of human growth hormone (hGH) was synthesized and studied by 2D proton NMR and molecular dynamics (MD) simulations. The solubility of the peptide was low in water; hence, NMR studies were done in two solvent mixtures, water and deuterated dimethyl sulfoxide. NOE‐constrained molecular dynamics and MD simulations resulted in major and minor conformers in solution. The major conformer has a type I β‐turn at Glnl‐Cys2‐Arg3‐Ser4 and a loop structure around Glu6‐Gly7‐Ser8. Comparison of the conformation of this peptide with the peptide fragment 181‐190 in the intact hGH protein X‐ray crystal structure indicated that the synthetic peptide retains some structural similarity to the intact protein. Since the C‐terminal region is important in binding the hGH protein to its receptor, the conformation of the synthetic peptide could be useful in understanding the binding mechanism. © Munksgaard 1997.
Vitamin K‐dependent carboxylase
Authors: LARUE VALÉRY , GHARBI‐BENAROUS JOSYANE , ACHER FRANCINE , AZERAD ROBERT , GIRAULT JEAN‐PIERRE
Published: August 2010

The conformational analysis of four glutamic acid analogues containing a cyclopentyl or cyclohexyl ring, substituted in position 1 by a Boc‐protected amino group and a methyl ester group and in position 3 by a free carboxylate group (6–9), has been carried out in an aqueous environment, by 1H and 13C NMR spectroscopy, and molecular dynamics (MD). These compounds have been shown to be weak competitive inhibitors (Ki≅ 20–65 mM) of the vitamin K‐dependent carboxylation of Boc‐Glu‐OMe in rat liver microsomes independently of their ring size and stereochemical features. However, the cyclic trans isomers have been found more active than the cis ones and Boc‐trans‐C5‐OMe (9) is the most potent inhibitor in the series (cis and trans isomers are defined by the relative arrangement of the carboxyl functions).

Such cyclic glutamyl derivatives may provide valuable informations on the preferred bioactive conformations of synthetic glutamyl substrates at the active site of the carboxylase. In aqueous solution, the Boc‐cis‐ and trans‐C6 esters exhibit chair conformations with exclusively equatorial and axial substituent positions, while the Boc‐cis‐ and trans‐C5 compounds may display envelope E or ‘twist’ T conformations with the substituents in the following positions. equatorial, axial and isoclinal. For each compound, the conformations resulting from NMR and MD data were analyzed and classified according to the dihedral angles %1 and %2, the distances of functional groups, and the spatial charge distribution involving the free carboxyl group.

A reduced number of conformational families were found to be in qualitative agreement with NMR and MD data. These results are discussed in relation with the carboxylase inhibitory activity of the analogues, and a spatial disposition of the glutamyl side chain that could be recognized by the carboxylase is deduced. © Munksgaard 1997.
Synthesis and antibacterial action of cecropin and proline‐arginine‐rich peptides from pig intestine
Authors: VUNNAM SATYANARAYANA , JUVVADI PADMAJA , MERRIFIELD R. B.
Published: August 2010

Dedicated to Professor Koji Nakanishi on the occasion of his 70th birthday.

Two antimicrobial peptides, cecropin P1 (CPl), with a C‐terminal carboxyl group, and PR‐39, with an amidated C‐terminus, are found in the small intestine of the pig. Each is active against both Gram‐positive and Gram‐negative bacteria. We have synthesized these peptides and several analogs, including the d‐enantiomers and the retro sequences, each with a free or acetylated amino terminus. The CPI amide was also prepared.

The retro CP1 peptides were much less active than the parent CPl peptide, confirming the importance of sequence or the amide bond and helix dipole direction, and the Nα‐acetyl peptides were also less active, indicating that a free amino terminus is essential for high activity. The ratio of the lethal concentration of L/D isomers of CP1 is less than 1 for Gram‐negative, but greater than 1 for Gram‐positive bacteria. PR‐39 showed no significant chiral selectivity toward Escherichia coli, Bacillus subtilis and Streptococcus pyogenes, but the l/d ratio was high for Pseudomonas aeruginosa (66), and very high for Staphylococcus aureus (>1000). In the latter case the lethal concentration for the d‐isomer was 0.57 μ whereas this organism was quite resistant to the l‐isomer (>600μ). Thus the enantiomers of CP1 and PR‐39 are not equally active for all species. In a plate assay with a very small log‐phase inoculum of Staph. aureus, D‐PR‐39 produced a clear zone of killing surrounded by a zone of stimulated growth. After prolonged incubation the two zones became one clear zone. Addition of D‐PR‐39 to the wells of a dense turbid plate of growing cells showed a cleared zone for each of the test organisms, indicating that PR‐39 lyses the bacteria rather than simply inhibiting their multiplication. © Munksgaard 1997.
Design of active analogues of a 15‐residue peptide using D‐optimal design, QSAR and a combinatorial search algorithm
Authors: MEE ROGER P. , AUTON TIM R. , MORGAN PHILLIP J.
Published: August 2010

This report describes the rational design of novel analogues of a 15‐residue antibacterial peptide CAMELO. A constrained D‐optimal design was carried out to derive a training set of 60 analogues. Partial least squares (PLS) models describing quantitative structure‐activity relationships (QSARs) were initially derived for the peptides using two published and one novel parameter set. The novel ‘Design parameters’ were based on key structural features identified in hypothetical models of the mechanisms by which peptides interact with cell membranes. In an extension of the PLS method, influence statistics were used to decrease the weighting of compounds having a large effect on model predictions. A combinatorial search algorithm was developed which used PLS models as predictors to select a test set of 39 peptides with high predicted potencies. Within this set, the most potent analogue CAMEL135, which contained seven point mutations from CAMEL0, was identified. For a panel of 24 bacteria, the mean MIC value of CAMEL135 was approximately half of that for CAMEL0. For the parameter sets tested, covariance functions derived from Z‐scales gave highest Q2‐values for the training set, whilst the model using the the ‘Design parameters’ gave least error when predicting the activity of the test set. The predictive ability of a third published set of peptide parameters was found to compare favourably with that of the parameters used in the design. Analysis of the PLS models indicates that hydrophobicity and amphipathicity are the most important features influencing activity for this class of compound. © Munksgaard 1997.
SPPS of difficult sequences
Authors: DETTIN MONICA , PEGORARO STEFANO , ROVERO PAOLO , BICCIATO SILVIO , BAGNO ANDREA , DI BELLO CARLO
Published: August 2010

The H‐Ala‐Arg‐(Ala)6‐Lys‐OH sequence is a biologically interesting ‘difficult sequence’ presenting Nα‐Fmoc deprotection and coupling problems. Different chemical conditions and synthetic strategies have been tested in order to overcome the problems due to sequence‐dependent interactions. In particular, it was confirmed that different solvents in the deprotection step did not provide any significant improvement, but the use of a more efficient base in the deprotection mixture avoided insufficient unblocking of Nα‐protecting group; problems due to partial coupling in the last steps of the synthesis were solved by double coupling techniques. Moreover, the synthesis of the model peptide was carried out using both ‘continuous flow’ and ‘batch’ techniques. The present results demontrate that on‐line monitoring of the deprotection step by absorbance measurements represents a very effective tool to detect the onset of internal aggregations during the synthesis. © Munksgaard 1997.
Palmitate and stearate binding to human serum albumin
Authors: VORUM HENRIK , FISKER KIM , HONORÉ BENT
Published: August 2010

Multiple binding equilibria of two apparently insoluble ligands, palmitate and stearate, to defatted human serum albumin were studied in a 66 mM sodium phosphate buffer (pH 7.4) at 37 °C, by determination of dialytic exchange rates of ligands among identical equilibrium solutions.

The experimental data were analysed by a computerised curve fitting procedure using equilibrium equations for multiple binding of ligands, containing relative binding constants, valid whether the ligands are truly insoluble or are slightly soluble and irrespective of aggregation in aqueous solution. A best‐fit set of relative binding constants was found, and subsequently 30 sets of acceptable constants for each set of data in order to evaluate the variation. The data were first fitted by the relative Scatchard's equation, then by the relative, stoichiometric equation. Scatchard's equation is deduced on the presumption that cooperativity is absent while the stoichiometric equation is valid even when cooperativity is present. It was found with palmitate as well as with stearate that the two equations fitted the data equally well, and it was concluded that the observations were compatible with absence of cooperativity. The relative Scatchard binding constants were converted to relative, stoichiometric constants and it was found that the variations of the latter were slight. © Munksgaard 1997.
Crystal structures of the even and odd bolaform amino acid derivatives (11‐N‐[benzyloxycarbonyl‐L‐alanyl] aminoundecanoyl)‐L‐alanyl benzyl ester and (12‐N‐[benzyloxycarbonyl‐L‐alanyl]aminododecanoyl)‐L‐alanyl benzyl ester
Authors: SCHADE BORIS , HUBERT VERA , ANDRÉ CHRISTOPH , LUGER PETER , FUHRHOP JÜRGEN‐HINRICH
Published: August 2010

Reaction of two α,ω‐aminocarboxylic acids with N‐ and C‐protected alanine leads to bolaform compounds with two secondary amide groups on one end and one such group at the other end. Unsymmetric sheet‐like structures are formed in the crystals. (11‐N‐[Benzyloxycarbonyl‐L‐alanyl]aminoundecanoyl)‐L‐alanyl benzyl ester (1) and (12‐N‐[benzyloxycarbonyl‐L‐alanyl]aminododecanoyl)‐L‐anyl benzyl ester (2) form triclinic crystals (spacegroup P1, No.1) with a= 4.917, b= 5.614, c= 29.02 Å, α= 88.40, β= 93.50, γ= 100.21°, Z=1 (1) and a= 4.954, b= 5.613, c= 30.23 Å, α= 93.44, β= 90.07, γ= 104.15°, Z=1 (2). The crystal structures were solved via direct methods and refined to R= 0.040 (1) and 0,078 (2) from 2441 and 2125 reflections. © Munksgaard 1997.
Evaluation of laminin peptide fragments labeled with indium‐111 for the potential imaging of malignant tumors
Authors: MOKOTOFF M. , SWANSON D.P. , JONNALAGADDA S.S. , EPPERLY M.W. , BROWN M.L.
Published: August 2010

The laminin peptide fragments GYIGSR‐NH2 and CDPGYIGSR‐NH2 are known to bind to a 67‐kDa laminin receptor. This receptor is understood to be expressed at higher than normal levels in malignant tumor cells, particularly those of breast and colon carcinomas. Peptides DTPA‐GYIGSR‐NH2 (1), DTPA‐(GYIGSR‐NH2)2 (2), DTPA‐CDPGYIGSR‐NH2 (3), DTPA‐(CDPGYIGSR‐NH2)2 (4), and negative control DTPA‐GAGAGA‐NH2 (5) were prepared by solid‐phase peptide synthesis. All five DTPA‐conjugated peptides were subsequently radiolabeled with 111ln and their tissue distribution evaluated in mice bearing C3H tumors. 111In‐3 and 111In‐4 showed the highest specific tumor localization. These preliminary data support further study of radiolabeled peptide fragments for the potential detection of malignant tumors of the breast and other organs. © Munksgaard 1997.
Complex formation between a formyl peptide and 24p3 protein with a blocked N‐terminus of pyroglutamate
Authors: CHU SIN‐TAK , LIN HAN‐JIA , CHEN YEE‐HSIUNG
Published: August 2010

We have purified 24p3 protein from mouse uterine fluid (Biochem. J.316, 545–550, 1996). It is a 25.8‐kDa glycoprotein with a N‐blocked terminus. This work demonstrated the N‐blocked residue to be pyroglutamate, supporting the post‐translational cleavage site at Ala‐Gln in the precursor protein to generate a putative protein of 180 amino acid residues. Consequently, the two cysteines, Cys78 and Cys177, and the two tryptophans, Trp31 and Trp81, are assigned along the polypeptide chain. No free thiol group was detected in the protein. The presence of formyl‐Met‐Leu‐Phe in the protein solution causes a considerable decrease in the protein fluorescence due to Trp31 and Trp81. Analysis of the fluorescence data supports the idea that the protein can be complexed with the formyl peptide. The association constant for the complex formation is (4.8 ± 0.29) × 105 M‐1 at pH 7.4.
Identification of new autolytic sites of recombinant truncated mature human fibroblast stromelysin by mass spectrometry
Authors: QORONFLEH M. WALID , CHOWDHURY SWAPAN K. , ESHRAGHI JAMSHID , HO THAU , BRAKE PATRICIA G. , BANKS TRACEY , HUANG JAMES , PULVINO TRICIA , JONES BARRY N.
Published: August 2010

Stromelysin has been proposed to play a major role in the pathologic degradation of diseased cartilage of osteoarthritis and rheumatoid arthritis patients. A truncated, recombinant form of this enzyme, with the sequence Phe83 to Thr260 (mSL‐t), has been expressed and purified from E. coli to investigate its biochemical and biophysical properties, and to develop inhibitors for arthritis treatment. LC/ESI‐MS technique was utilized for the characterization of mSL‐t. The mass spectra of mSL‐t showed the presence of a number of different protein components in addition to the full‐length mSL‐t form. We have demonstrated that protein degradation arose from autolysis. Molecular weights determined by LC/ESI‐MS of these autolysis products allowed for the identification of new autolytic sites in mSL‐t. Furthermore, two strategies were undertaken to prepare mSL‐t free of degradation products. These include preparation of a mutant form of the enzyme in which Arg163 was substituted for Leu163 and purification of mSL‐t using affinity chromatography. The LC/ESI‐MS data of the mutant protein confirmed the Leu to Arg mutation. The affinity‐purified material showed only one LC peak in the LC/MS chromatograms, and the mass spectrum of the peak identified only the intact protein, demonstrating that the full‐length protein has been successfully separated from the autodegradation products and further autolysis of the enzyme has been prevented. © Munksgaard 1997.
New renin inhibitors containing novel analogues of statine
Authors: JONES D. MICHAEL , SUEIRAS‐DIAZ JAVIER , SZELKE MICHAEL , LECKIE BRENDA J. , BEATTIE SHEILA R , MORTON JAMES , NEIDLE STEPHEN , KURODA REIKO
Published: August 2010

Solid‐phase methodology has been used to synthesize a series of peptides based on the N‐terminal sequence of human angiotensinogen in which statine (Sta) or the novel analogues (3S, 4S)‐3, 4‐diamino‐ or (3J?, 45)‐3, 4‐diamino‐6‐methylheptanoic acid (Ads or R‐Ads) and (3S, 4S)‐4‐amino‐3‐aminomethyl‐ or (3R, 4S)‐4‐amino‐3‐aminomethyl‐6‐methylheptanoic acid (Amd or R‐Amd) replace either residue 10 or both residues 10–11 at the P1‐P'1 cleavage site. The synthesis of these novel analogues of statine together with biological results on the inhibition of human and rat renin by peptides derived from them is reported. The absolute stereochemistry of the (3S, 4S) Ads was determined by an X‐ray crystallographic analysis of its Nγ‐Boc, Nβ‐Z, R(+)‐1‐methyl benzamide derivative. Peptide Boc‐His‐Pro‐Phe‐His‐Sta‐Val‐Ile‐His‐NH2 (VI) is the best inhibitor of human renin containing Sta at position 10. However, peptides containing Ads and Amd gave better rat renin inhibitors than the corresponding Sta‐containing peptides. Peptide Boc‐His‐Pro‐Phe‐His‐Ads‐Val‐Ile‐His‐NH2 (VII) having Ads at position 10 had an IC50 of 12 nM against rat renin. Although Sta has come to be accepted as an isosteric replacement for a dipeptide unit rather than for a single amino acid residue, in our series of inhibitors Sta is more effective when replacing only the amino acid at position 10 in the natural angiotensinogen sequence. None of the peptides gave any effect in vivo in a hypertensive rat model.
Synthetic peptide vaccines: palmitoylation of peptide antigens by a thioester bond increases immunogenicity
Authors: BEEKMAN NICO J.C.M. , SCHAAPER WIM M.M. , TESSER GODEFRIDUS I. , DALSGAARD KRISTIAN , KAMSTRUP SØREN , LANGEVELD JAN P.M. , BOSHUIZEN RONALD S. , MELOEN ROB H.
Published: August 2010

Synthetic peptides have frequently been used to immunize animals. However, peptides less than about 20 to 30 amino acids long are poor immunogens. In general, to increase its immunogenicity, the presentation of the peptide should be improved, and molecular weight needs to be increased. Many attempts have been made to couple peptide immunogens to different carrier proteins [e.g. keyhole limpet haemocyanin (KLH) or ovalbumin]. This leads to very complex structures, however. We used a controlled conjugation of a peptide to a single long‐chain fatty acid like palmitic acid by a thioester or an amide bond. It was found that these S‐palmitoylated peptides were much more immunogenic than N‐palmitoylated peptides and at least similar to KLH‐conjugated peptides with respect to appearance and magnitude of induced antibodies (canine parvovirus) or immunocastration effect (gonadotropin‐releasing hormone). For chemical synthesis of thioesters, we established conditions for solution and solid‐phase synthesis. In both phases, Cys(SBut) could only be deprotected efficiently using phosphines, and S‐acylation was accomplished using standard coupling at pH 5. We speculate that, in vivo, the presence of an appropriate fatty acid chain, chemically linked through a labile thioester bond, greatly enhances immunogenicity, because it represents a favourable substrate for cleavage by cellular thioesterases in cells of the immune system.
Comparison of solution properties of human and rat ciliary neurotrophic factor
Authors: NARHI LINDA O. , ROSENFELD ROBERT , SHIMAMOTO GRANT , LEE RICHARD , HAWKINS NESSA , LI TIANSHENG , PHILO JOHN , WEN JIE , ARAKAWA TSUTOMU
Published: August 2010

The solution structure and stability of rat and human ciliary neurotrophic factor (CNTF) were examined by circular dichroism (CD), Fourier transform infrared (FTIR) and fluorescence spectroscopy and sedimentation equilibrium analyses. The secondary structure of both proteins, as assessed by CD and FTIR, consists primarily of α‐helix, consistent with CNTF being a member of the four‐helical bundle family of cytokines and neurokines, with rat CNTF containing slightly less helix (about 10% less) and slightly more disordered structure. The environment of the tyrosine and tryptophan residues, assessed by intrinsic fluorescence emission spectroscopy, appears to be the same in both proteins. Binding of anilinonaphthalene sulfonate is identical for both proteins, indicating that these two proteins have similar surface hydrophobicities in the native state. The thermal stability of the human CNTF is significantly less than that of the rat CNTF, yet their stabilities to guanidine HCl‐induced denaturation are equivalent. This apparent discrepancy in stability between the two proteins may be explained by solubility differences upon thermal unfolding. Although the human protein precipitates as it is denatured by heat, the rat protein does not. It thus appears that the unfolded state of human CNTF is less soluble and more prone to aggregation than that of the rat protein upon heating, although their conformational stability is similar. Both proteins remain largely folded at pH 3.0. Sedimentation equilibrium analysis demonstrates that both rat and human CNTF exist primarily as monomers; however, significant dimer formation occurs as the protein concentrations are increased to greater than 3 mg/mL, particularly in the presence of ammonium sulfate.
Mixtures of trifluoroethanol or hexafluoroisopropanol and dimethylformamide are not of general applicability for peptide condensations catalyzed by trypsin
Authors: BEMQUERER MARCELO P. , LIRIA CLEBER W. , KITAGAWA KOUKI , MIRANDA M. TERÊSA M. , TOMINAGA MINEKO
Published: August 2010

Mixtures of a good hydrogen bond donor, 2,2,2‐trifluoroethanol (TFE) or 1,1,1,3,3,3‐hexafluroisopropanol, and an acceptor, dimethylformamide (DMF) (1:1,v/v), containing 4% buffer have been described as adequate solvent systems for trypsin‐catalyzed peptide fragment condensations [Mihara et al. (1993) Int. J. Pept. Protein Res.41, 405]. Thus, we decided to study the behaviour of trypsin in such solvent systems. We investigated whether this protease would efficiently catalyze condensations between fragments derived from an analogue of the gp‐41 capsid protein of HIV virus or from cholecystokinin‐22. None of the reactions carried out yielded the desired condensation products. However, when Fmoc‐NLQNLDPSHR‐OH and cholecystokinin‐12 (H‐ISDRDYMGWMDF‐NH2) were used as substrates, the last had its R‐D peptide bond hydrolyzed producing cholecystokinin‐8. The proteolytic activity of this enzyme measured against a fluorogenic peptide derivative was 50 times lower in DMF/TFE containing 5% of aqueous phase than in buffer. Steady‐state fluorescence studies in DMF/TFE buffer were performed to examine the structure of this protease in these media. Steady‐state spectra obtained with increasing proportions of these two organic solvents in buffer showed that the emission intensities built up. Quenching studies with iodide revealed that the Io/I ratio (where Io and I are the fluorescence emission intensities in the absence and presence of quencher, respectively) changed from 1.2 in aqueous media to 2.2 in DMF/TFE (1:1, v/v) containing 11% 0.2 m Tris‐HCI buffer, pH 8.0, for 0.5 m iodide. The complete data indicated a higher exposure of tryptophan residues to the quencher in organic media, probably because of the partial unfolding of the enzyme.
Identification of a melanocyte growth‐inhibiting tripeptide and determination of its structure
Authors: GEMBITSKY D. S. , REICHELT K. L. , HAAKONSEN P. , PAULSEN J. E. , ELGJO K.
Published: August 2010

The function and proliferation of melanocyte cell populations are influenced by a wide range of hormones and growth factors. Local cell renewal after sudden melanocyte loss appears to be regulated according to a negative feedback principle, however. In accordance with this assumption, we have examined growth‐inhibitory activity in water extracts of cultured non‐transformed melanocytes and melanoma cells (B16 cells). The extracts were fractionated by gel filtration on Sephadex G‐25 and Fractogel MG 2000, by ion‐exchange chromatography on DEAE‐cellulose and Dowex 50 and by reverse‐phase high‐performance liquid chromatography on Bondesil and Partisil columns. Two peptides were isolated with the structures pyroGlu‐Phe‐GlyNH2 and pyroGlu‐Cys‐GlyNH2 as revealed by mass spectrometry, peptide sequencing and amino acid analysis. The two peptides were synthesized and tested for the ability to inhibit the growth of melanocyte cultures. Only pyroGlu‐Phe‐GlyNH2 was inhibitory. The dose‐response curve was bell‐shaped with maximum inhibition around 10−15 M. The melanocyte tripeptide thus appears to be a new member of a group of N‐substituted growth‐regulating oligopeptides found in other tissues.
Chemical ligation of unprotected peptides directly from a solid support
Authors: CAMARERO JULIO A. , COTTON GRAHAM J. , ADEVA ALBERTO , MUIR TOM W.
Published: August 2010

In this article we describe a new, convenient procedure to carry out intramolecular (cyclization) and intermolecular native chemical ligations of unprotected peptides directly from a solid support. Our solid‐phase ligation approach eliminates the need to manipulate peptide „thioacid and peptide αthioester intermediates in aqueous solution before the ligation step, thereby leading to a reduction in handling losses and significantly increasing the overall efficiency of the chemical ligation strategy. A key step in our ligation scheme is the ability to generate fully unprotected peptides tethered to a solid support through an ”thioester linkage. This can be achieved efficiently using optimized Boc‐solid‐phase peptide synthesis on a 3‐mercaptopropionamide‐polyethylene glycol‐poly‐(N,N‐dimethylacrylamide) copolymer support (HS‐PEGA). Once the synthesis is complete, the fully protected peptide „thioester resin is treated with HF to give the corresponding fully unprotected peptide „thioester resin. Using this procedure several polypeptides ranging from 15 to 47 residues were synthesized successfully. These peptide‐resins were then used to perform both intramolecular (head‐to‐tail cyclizations) and intermolecular solid‐phase ligations. The intramolecular solid‐phase ligations proceeded much faster than their intermolecular counterparts, but in both cases the reactions were observed to be remarkably clean. The presence of aromatic thiol cofactors significantly accelerated the relatively slow intermolecular ligations. This novel methodology was then extended to provide a general method for performing sequential intermolecular ligations, allowing easy access to much larger polypeptide and protein systems.
A peptide of nine amino acid residues from α‐sarcin cytotoxin is a membrane‐perturbing structure
Authors: MANCHEÑO JOSÉ M. , POZO ALVARO MARTÍNEZ DEL , ALBAR JUAN P. , OÑADERRA MERCEDES , GAVILANES JOSÉ G.
Published: August 2010

A water‐soluble synthetic peptide with only nine amino acid residues, comprising the 131–139 sequence region of the cytotoxic protein α‐sarcin (secreted by the mold Aspergillus giganteus), interacts with large unilamellar vesicles composed of acid phospholipids. It promotes lipid mixing between bilayers and leakage of vesicle aqueous contents, and it also abolishes the phospholipid phase transition. Other larger peptides containing such an amino acid sequence also produce these effects. These peptides acquire α‐helical conformation in the presence of trifluoroethanol, but display β‐strand conformation in the presence of sodium dodecyl sulfate. The interaction of these peptides with the lipid vesicles also results in β‐structure. The obtained data are discussed in terms of the involvement of the 131–139 stretch of α‐sarcin in its interaction with lipid membranes.
Comparison of the binding of α‐helical and β‐sheet peptides to a hydrophobic surface
Authors: STEER DAVID L. , THOMPSON PHILIP E. , BLONDELLE SYLVIE E. , HOUGHTEN RICHARD A. , AGUILAR MARIE‐ISABEL
Published: August 2010

The induction and stabilisation of secondary structure for a series of amphipathic α‐helical and β‐sheet peptides upon their binding to lipid‐like surfaces has been characterised by reversed phase high‐performance liquid chromatography (RP‐HPLC). In addition, a series of peptides which have been shown to switch from β‐sheet to α‐helical conformation upon transfer from a polar to a non‐polar solution environment also have been studied. Binding parameters related to the hydrophobic contact area and affinity for immobilised C18 chains were determined at temperatures that ranged from 5 to 85°C, allowing conformational transitions for the peptides during surface adsorption to be monitored. The results demonstrated that all peptides which adopt secondary structure in solution also exhibited large changes in their interactive properties. Overall, this study demonstrates that the hydrophobic face of each amphipathic peptide dominates the binding process and that hydrophobic interactions are a major factor controlling the surface induction of secondary structure.
Lanthionine macrocyclization by in situ activation of serine
Authors: MAYER JOHN P. , ZHANG JINGWEN , GROEGER STEFAN , LIU CHUAN‐FA , JAROSINSKI MARK A.
Published: August 2010

The present report details a straightforward, solid‐phase approach to cyclolanthionine peptides. After stepwise assembly of the linear sequence and transformation of a single exposed serine to bromoalanine using P(Ph)3/CBr4, the detritilation of a cysteine side‐chain sets the stage for a base‐promoted macrocyclization. The entire procedure can be carried out in a solid‐phase vessel using conventional 9‐fluorenylmethyloxycarbonyl/tert‐butyl‐based chemistry and is amenable to automated format. The utility of this novel procedure is demonstrated by the synthesis of two previously reported lanthionine‐containing cyclic peptides.
Effect of omeprazole treatment on plasma concentrations of the gastric peptides, xenin, gastrin and somatostatin, and of pepsinogen
Authors: Stoschus B. , Hamscher G. , Ikonomou S. , Partoulas G. , Eberle C. , Sauerbruch T. , Feurle G.E.
Published: August 2010

Abstract: The peptide xenin 25 is a gastric mucosal constituent like gastrin, somatostatin and pepsinogen. Gastrin and pepsinogen plasma concentrations increase when the secretion of gastric acid is reduced by proton pump inhibitors. In the present investigation, treatment with omeprazole led to an increase in fasting and postprandial plasma concentrations of xenin, gastrin and pepsinogens A and C (P < 0.05, in each instance), whereas somatostatin plasma levels remained unchanged. Because subcutaneous injection of pentagastrin did not raise xenin plasma concentrations, a direct effect of gastrin on xenin production seems unlikely. This study indicates that xenin plasma concentrations are regulated by intragastric pH, as are those of gastrin and pepsinogen.
Preparation of head‐to‐tail cyclic peptides via side‐chain attachment: Implications for library synthesis
Authors: Romanovskis P. , Spatola A.F.
Published: August 2010

Abstract: : Cyclic peptide mixtures represent a promising approach for drug lead discovery. Diversity can be expanded via changes in ring size, amide bond replacements, d‐ and l‐natural and unnatural amino acids, and by the incorporation of β‐turn mimics and related conformational constraints. We expand the scope of our side‐chain attachment/resin‐bound cyclization strategy first developed using aspartic acid, asparagine, and related acid‐based attachments. In this report, we describe the preparation and use of Boc‐Lys‐OFm, Boc‐Orn‐OFm, and Boc‐Dab‐OFm. The synthetic strategies were verified by the independent synthesis of two small RGD‐based cyclic mixtures, cyclo(Pro‐Xxx‐Lys‐Arg‐Gly‐Asp). where Xxx = Ala, Ser, Leu, Tyr, using Boc‐Asp‐OFm, and then Boc‐Lys‐OFm for the initial resin‐bound side‐chain linkage. A new orthogonal synthetic approach to cyclic peptides, based on the use of the para‐nitrobenzyl (ONB) ester for a‐carboxyl protection (selectively cleaved via SnCl2 reduction), was developed. It led to the successful synthesis of individual cyclic pentapeptides using serine (Boc‐Ser‐ONB), and tyrosine (Fmoc‐Tyr‐ONB) as the side chain‐linked residues.
Direct anticancer activity of gonadotropin‐releasing hormone‐III
Authors: Lovas S. , Páyi I. , Vincze B. , Horvath J. , Kovács M. , Mezö I. , Tóth G. , Tepán I. , Murphy R.F.
Published: August 2010

Abstract: In previous studies GnRH‐III, a variant of the hypothalamic neurohormone GnRH, was isolated from the brain of the sea lamprey and structurally characterized. GnRH‐III is a hypothalamic neurohormone in both female and male sea lampreys. In the present work biological activities of GnRH‐III in mammalian systems were examined. In superfused rat pituitary cells, GnRH‐III at 1 nM to 100 nM neither induced LH‐secretion nor inhibited the LH‐secretion elicited by native GnRH and elicited LH release only at 1 μM. At high dose (500 μg/day) in vim, GnRH‐III behaved as a GnRH agonist, though, it was 1000‐fold less active than ovurelin. The in vitro and in vivo results were in good agreement in showing that GnRH‐III is only a weak agonist of the endocrine activity of GnRH. GnRH‐III specifically bound to receptors on cancer cells and recognized not only the high‐, but also the low‐affinity binding sites. GnRH‐III significantly suppressed growth of human cancer cells which have GnRH receptors. The inhibitory effect of GnRH‐III on growth of cancer cells was specific and direct since the peptide did not have endocrine activity in the concentration range found to be effective in anticancer assays. GnRH‐III inhibited equally the growth of ER‐positive and ‐negative breast and TeR‐positive and ‐negative prostate cells.
Structure‐function analysis of a series of glucagon‐like peptide‐1 analogs
Authors: Parker J.C. , Andrews K.M. , Rescek D.M. , Massefski W. , Andrews G.C. , Contillo L.G. , Stevenson R.W. , Singleton D.H. , Suleske R.T.
Published: August 2010

Abstract:We have used NMR in conjunction with measurements of functional bioactivity to define the receptor‐binding structure of glucagon‐like peptide‐1 (GLP‐1.) Identification of the important residues for binding was accomplished by the substitution of amino acids at sites that seemed likely, from an examination of the amino acid sequence and from previously published observations, to be important in the three‐dimensional (3D) structure of the molecule. Identification of the receptor‐bound conformation of GLP‐1, because it is a flexible peptide, required constraint of the peptide backbone into a predetermined 3D structure. Constraint was achieved by the introduction of disulfide bonds and specific side chain‐side chain cross‐links. The biological relevance of the synthetic structure of each rigidified peptide was assessed by measurement of its ability to bind to the receptor present on RINm5F cells and to elicit a functional response, cyclic AMP production. NMR solution structures were obtained for the most biologically relevant of these analogs. The results of this study indicated that the residues necessary for the biological activity of GLP‐1 occupy approximately three equally‐spaced regions of the peptide 3D structure, at the corners of an equilateral triangle whose sides are, at a minimum estimate, 12–15 Å.
Conformational and topological requirements of cell‐permeable peptide function
Authors: DU CAIGAN , YAO SONGYI , ROJAS MAURICIO , LIN YAO‐ZHONG
Published: August 2010

Cell‐permeable peptide import recently was developed to deliver synthetic peptides into living cells for studying intracellular protein functions. This import process is mediated by an N‐terminal carrier sequence which is the hydrophobic region of a signal peptide. In this study, the conformational consequence of the interaction of cell‐permeable peptides with different mimetic membrane environments was investigated by circular dichroism analysis. We showed that cell‐permeable peptides adopted a‐helical structures in sodium dodecyl sulfate (SDS) micelles or aqueous trifluoroethanol (TFE). The potency of these peptides in forming helical structures is higher in an amphiphilic environment (SDS) than in a hydrophobic environment (TFE), suggesting that some hydrophilic molecules associated with the cell membrane may be involved in peptide import. We also studied topological requirements of cell‐permeable peptide function. We demonstrated that peptides containing the carrier sequence in their C‐termini can also be imported into cells efficiently. This important discovery can avoid repetitious synthesis of the membrane‐translocating sequence for peptides with different functional cargoes and is potentially useful for developing a cell‐permeable peptide library. Finally, we showed that, when a retro version of the carrier sequence was used, the peptide lost its translocating ability despite retaining a high content of a‐helical structure in mimetic membrane environments. This suggests that the propensity of peptides to adopt a helical conformation is required but not sufficient for cellular import and that other structural factors such as the side‐chain topology of the carrier sequence are also important. Our studies together contribute to the more rational design of useful cell‐permeable peptides. © Munksgaard 1998.
Charcoal surface‐assisted catalysis of intramolecular disulfide bond formation in peptides
Authors: VOLKMER‐ENGERT RUDOLF , LANDGRAF CHRISTIANE , SCHNEIDER‐MERGENER JENS
Published: August 2010

A mild and highly efficient method for intramolecular disulfide bond formation in peptides mediated by charcoal has been developed. Completion of the charcoal‐assisted catalysis of disulfide bond formation took less than 6 h, testing a series of peptides with ring sizes varying from 2 to 17 amino acids. Kinetic studies revealed that the formation of small and large intramolecular disulfide cycles especially was accelerated significantly by the aid of charcoal as compared with dimethyl sulfoxide‐ and air‐mediated cyclization. Oxygen adsorbed onto the charcoal surface is a prerequisite for disulfide bond formation. Thermodynamic studies showed that cyclization is accelerated by reduction of entropy of the peptides, most likely because of transient adsorption to the charcoal surface, thus resulting in a lower activation energy. © Munksgaard 1998.
The receptor‐bound conformation of H‐Tyr‐Tic‐(Phe‐Phe)‐OH‐related δ‐opioid antagonists contains all trans peptide bonds
Authors: WILKES BRIAN C. , NGUYEN THI M.‐D. , WELTROWSKA GRAZYNA , CARPENTER KATHARINE A. , LEMIEUX CAROLE , CHUNG NGA N. , SCHILLER PETER W.
Published: August 2010

Two different models for the receptor‐bound conformation of δ‐opioid peptide antagonists containing the N‐terminal dipeptide segment H‐Tyr‐Tic (Tic = 1,2,3,4‐tetrahydroisoquinoline‐3‐carboxylic acid) have been proposed. Both models are based on spatial overlap of the Tyr and Tic2 aromatic rings and N‐terminal amino group with the corresponding aromatic rings and nitrogen atom of the nonpeptide δ‐antagonist naltrindole. However, in one model the peptide bond between the Tyr and Tic2 residues assumes the trans conformation, whereas in the other it is in the cis conformation. To distinguish between these two models, we prepared the two peptides H‐Tyrψ[CH2NH]. Tic‐Phe‐Phe‐OH and H‐Tyrψ[CH2NH]. MeTic‐Phe‐Phe‐OH (MeTic = 3‐methyl‐1,2,3,4‐tetrahydroisoquinoline‐3‐carboxylic acid) in which a cis peptide bond between the Tyr and Tic (or MeTic) residues is sterically forbidden. Both compounds turned out to be moderately potent δ‐opioid antagonists in the mouse vas deferens assay. A molecular mechanics study performed with both peptides resulted in low‐energy conformations in which the torsional angle (“ω1”) of the reduced peptide bond between Tyr and Tic (or MeTic) had a value of 180°(trans conformation) and which were in good agreement with the proposed model with all trans peptide bonds. Furthermore, this study confirmed that neither of these two peptides could assume low‐energy conformations in which “ω1” had a value of 0°(cis conformation). Conformers with that same bond in the gauche‐ conformation (“ω1”= ‐60“) were also identified, but were higher in energy and showed no spatial overlap with naltrindole. On the basis of these results it is concluded that the receptor‐bound conformation of δ‐peptide antagonists containing an N‐terminal H‐Tyr‐Tic‐dipeptide segment must have all trans peptide bonds. © Munksgaard 1998.