bluuebunny/crossref_metadata_2025 huggingface dataset:
Naive Human T‐Cells become Non‐Responsive towards Anti‐TNFα (Infliximab) Treatment In vitro if Co‐Stimulated through CD28
Authors: Gunnlaugsdottir B. , Skaftadottir I. , Ludviksson B. R.
Published: August 2010

Tumour necrosis factor α (TNFα) inhibitors are widely used successfully as immunomodulatory agents in various autoimmune diseases. They primarily target the direct pro‐inflammatory effect of TNFα. They have also been found to be critical for T‐cell viability and activation. In this study we evaluated the effect of infliximab treatment under different in vitro stimulatory conditions of naive human cord blood T‐cells and adult peripheral blood mononuclear cells (PBMC). PBMC and negatively selected cord blood naive human T‐cells were stimulated with αCD3 with or without αCD28 co‐stimulation. The role of in vitro infliximab treatment was evaluated in relation to transforming growth factor‐β1 (TGF‐β1) under the above different stimulatory conditions. Anti‐TNFα treatment with infliximab significantly suppressed proliferation of adult and cord blood T‐cells (P < 0.013) during suboptimal stimulatory conditions. Infliximab prevented division of naive CD4+ and CD8+ T‐cells and consequently also activation induced cell death, which was induced after three cell divisions. Interleukin (IL)‐2 secretion was significantly decreased during infliximab treatment of suboptimally stimulated T‐cells (P < 0.05) while TGF‐β1 levels were unchanged. Strikingly, the anti‐proliferative effect of infliximab was overcome by the administration of anti‐TGF‐β1 or by the addition of exogenous IL‐2. Interestingly, CD28 mediated co‐stimulation restored the proliferative response in a dose–responsive manner during infliximab treatment. Finally, exogenous TNFα administration during suboptimal stimulation reduced the inhibitory effect of TGF‐β1 upon proliferation (P < 0.03). These results demonstrate that anti‐TNFα treatment is primarily working upon T cells under low stimulatory conditions and probably through TGF‐β1.
The Impact of Hormone Replacement Therapy on Humoral and Cell‐mediated Immune Responses In Vivo in Post‐menopausal Women with Rheumatoid Arthritis
Authors: Forsblad d′Elia H. , Carlsten H.
Published: August 2010

It is well known that oestrogen has immunomodulatory properties. We have previously shown disease ameliorating effects of hormone replacement therapy (HRT) in post‐menopausal women with rheumatoid arthritis (RA). The aim of this study was to investigate the effects of HRT and the patients inflammatory state on humoral and cell‐mediated immune responses. Eighty‐eight post‐menopausal RA women were allocated to receive HRT (oestradiol and noretisterone acetate), vitamin D3 and calcium or vitamin D3 and calcium alone in a 2‐year randomized controlled trial. Immunoglobulins (IgM, IgG and IgA) in serum were measured by nephelometry and rheumatoid factor (RF) concentration by enzyme‐linked immunosorbent assay. Immunization with influenza vaccine was performed to quantitate humoral response to recall antigen and tuberculin skin test with purified protein derivative (PPD) to test T‐cell‐mediated immune response. These immune related measures were correlated with demographic and disease‐related variables. HRT during 2 years did not alter concentrations of Ig, RF, IgM response to influenza vaccine or the PPD reaction. The increase in IgM against influenza vaccine was significantly positively correlated with signs of disease activity; C‐reactive protein, disease activity score 28 and inversely with haemoglobin. In contrast, PPD reactivity was inversely associated with disease activity. In conclusion, long‐term HRT in RA does not influence Ig or autoantibody concentrations in serum and has no significant impact on humoral and cell‐mediated immune responses to recall antigens. Interestingly, high disease activity was associated to increased humoral but decreased cell‐mediated immune responses irrespectively of hormone treatment.
A quantitative structure‐activity relationship study of some substance P‐related peptides A multivariate approach using PLS and variable selection
Authors: NORINDER ULF , RIVERA CLAUDIO , UNDÉN ANDERS
Published: August 2010

Nine new analogues of substance P (SP) were designed using quantitative sequence‐activity models based on the amino acid z‐scales with PLS as the statistical method and the GOLPE procedure for variable selection. The nine SP analogues were synthesised by solid‐phase peptide synthesis and tested for affinity to the NK‐1 receptor from rat brain with radio receptor assay using [125I]‐Bolton‐Hunter substance P as labelled ligand. All of the new substance P analogues showed high affinities, with IC50 values of less than 0.8 nM. One analog, Lys‐Arg‐Ala‐Lys‐Phe‐Met‐Met‐Phe‐Phe‐Gly‐Leu‐Let‐NH2, showed a exceptional high affinity for the NK1 receptor, with IC50= 5 PM.
Conformational analysis of homochiral and heterochiral diprolines as β‐turn‐forming peptidomimetics: unsubstituted and substituted models
Authors: BAURES PAUL W. , OJALA WILLIAM H. , GLEASON WILLIAM B. , JOHNSON RODNEY L.
Published: August 2010

The effect of replacing one of the proline residues in either unsubstituted homochiral or heterochiral diproline segments with either a 2‐ or a 3‐substituted prolyl residue on the allowed conformation of the diproline template has been examined. In heterochiral (l‐d) diprolines, placement of a 2‐methyl‐d‐proline residue in the i+ 2 position and placement of either a cis‐ or trans‐3‐methyl‐l‐proline residue in the i+ 1 position results in substituted diproline peptides that adopt the same type II β‐turn conformation as that identified experimentally for the unsubstituted diproline peptides. In contrast, placement of a cis‐3‐methyl‐d‐proline residue in the i+ 1 position of a homochiral (d‐d) diproline peptide seems to promote a different conformation than that seen in the unsubstituted case, whereas the trans‐3‐methyl‐d‐proline residue seems to provide a stabilizing influence for the predicted type VI'β‐turn. The demonstrated ability of certain substituted diproline templates to adopt predictable conformations coupled with the development of asymmetric synthetic routes to both 2‐ and 3‐substituted prolyl residues capable of mimicking a variety of side chains should make these templates useful tools in designing specific turn mimics of biologically active molecules.
Variation of acidity constants of peptides in acetonitrile‐water mixtures with solvent composition: effect of preferential solvation
Authors: BARBOSA J. , HERNÁNDEZ‐CASSOU S. , SANZ‐NEBOT V. , TORO I.
Published: August 2010

The dissociation constant values of a series of peptides in 5.54, 10, 16.30, 25.03 and 50% (w/w) acetonitrile‐water mixed solvents at 298.15 K were determined according to the criteria endorsed by IUPAC. A pronounced change in the acid‐base pK values of carboxylic, phenol and thiol groups was observed as the solvent was enriched in acetonitrile. By contrast, pK values of protonated amino‐terminal groups were influenced slightly as the solvent was enriched in acetonitrile, although continually increasing pK values were observed. The variation of the pK values obtained, over the whole composition range studied, was explained by taking into account the preferential solvation of electrolytes in acetonitrile‐water mixtures. To obtain pK values in all possible binary solvent acetonitrile‐water mixtures, relationships between pK values and different bulk properties were examined and the Linear Solvation Energy Relationships methodology was applied. The pKa values were then correlated with the Kamlet‐Taft, π, α and β solvatochromic parameters of acetonitrile‐water mixtures. The equations obtained allowed calculation of the pK values of peptides in acetonitrile‐water mixtures up to 50% (w/w) and thus permitted the acid‐base behavior of these substances in the widely used acetonitrile‐water media to be known.
Synthesis and biological properties of β‐MePhe3analogues of deltorphin I and dermenkephalin: influence of biased X1of Phe3residues on peptide recognition for δ‐opioid receptors
Authors: MISICKA ALEKSANDRA , CAVAGNERO SYLVIA , HORVATH ROBERT , DAVIS PEG , PORRECA FRANK , YAMAMURA HENRY I. , HRUBY VICTOR J.
Published: August 2010

Using the method of conformational constraint, we have designed and synthesized analogues of deltorphin I and dermenkephalin containing each of the four stereoisomers (2S, 3S: 2S, 3R; 2R, 3S; 2R, 3R) of the unusual amino acid ß‐methylphenylalanine in position three. The potency and selectivity of these analogues were evaluated by radioreceptor binding assays in the rat brain using [3H]CTOP (δ‐ligand) and [3H][p‐CβPhe4]DPDPE (β‐ligand), and by bioassay using the mouse vas deferens (β‐receptor assay) and guinea pig ileum (β‐receptor assay) assays. The substitution of a ß‐MePhe for Phe3in deltorphin I and dermenkephalin has a large and variable effect on the bioactivities of the synthesized analogues. The synthesized analogues are somewhat less potent than the native peptides. Both [(2S, 3R)‐ß‐MePhe3]deltorphin and [(2S, 3R)‐ß‐MePhe3]dermenkephalin are more selective, however, and interact essentially specifically with the receptor in the binding assays and bioassays. The bioassay datain vitroof the synthesized analogues of deltorphin I and dermenkephalin follow the same general trends as the receptor binding data. These results demonstrate that topographical modifications of the side‐chain conformation of critical structural moieties in a ligand can significantly modulate both the potency and receptor selectivity for ligands that have multiple sites of biological activity, and they illustrate that this approach has general application to peptide and peptidomimetic ligand design.
Hydrolytic conditions for the formation of open‐chain oligopeptides from cyclosporin A
Authors: MAGNI FULVIO , FERMO ISABELLA , ARCELLONI CINZIA , ARNOLDI LOLITA , PUPPO MARINA DEL , PARONI RITA
Published: August 2010

In this work we report data on the hydrolysis of CsA, allowing interpretation on the formation of previously identified open chain oligopeptides from CsA. © Munksgaard 1997.
L‐Ala‐substituted rat galanin analogs distinguish between hypothalamic and jejunal galanin receptor subtypes
Authors: JURÉUS ANDERS , LANGEL ÜLO , BARTFAI TAMAS
Published: August 2010

In order to explore which amino acids or which blocks of amino acids in the 29 amino acid neuropeptide galanin are important for recognition of the endogenous ligand by galanin receptor subtypes present in the jejunum and in the hypothalamus, respectively, we have carried out L‐Ala substitutions of individual amino acids or of blocks of amino acids in the rat galanin sequence and examined the binding of the obtained analogs to the rat hypothalamic and jejunal galanin receptor subtypes. This study reveals that the galanin sequence YLLGPH9–14is essential for recognition of galanin by both the rat hypothalamic and jejunal galanin receptor subtypes. Substitution of theN‐terminal amino acids, GWTL1–4, leads to total loss of affinity of galanin for both hypothalamic and jejunal galanin receptors. The α‐helicalC‐terminal amino acid (25–29) part of galanin has no greater influence on the affinity of galanin to the hypothalamic galanin receptor subtype. L‐Ala substitution of theC‐terminal amino acids of galanin KHGLT25–29shows, however, that thisC‐terminal motif is essential for the recognition by the jejunal galanin receptor subtype, whereas amino acids in the middle portion of galanin NSAG5–8are of importance for binding to the hypothalamic but not to the jejunal receptor. [Ala5–8] Galanin thus has a more than 100‐fold higher affinity to jejunal receptor than to the hypothalamic receptor, while [Ala25–29] galanin has a more than 100‐fold higher affinity for the hypothalamic than for jejunal galanin receptor subtypes. pH dependence of the galanin binding to these receptor subtypes is also different. © Munksgaard 1997.
Chemical synthesis and characterization of the epidermal growth factor‐like module of human complement protease Clr
Authors: HERNANDEZ JEAN‐FRANÇOIS , BERSCH BEATE , PÉTILLOT YVES , GAGNON JEAN , ARLAUD GÉRARD J.
Published: August 2010

Clr is one of the two serine proteases of Cl, the first component of complement, in which it is associated in a calcium‐dependent manner to the homologous serine protease Cls. This interaction is mediated by the N‐terminal region of Clr, which comprises a single epidermal growth factor (EGF)‐like module containing the consensus sequence required for calcium binding, surrounded by two CUB modules. With a view to determine the structure of the EGF‐like module of Clr and evaluate its contribution to calcium binding, this module [Clr(123–175)] was synthesized by automated solid‐phase methodology using the Boc strategy. A first synthesis using the Boc‐His(Z) derivative gave very low yield, due to partial deprotection of His residues leading to chain termination by acetylation, and to insertion of glycine residues. This could be circumvented by using the Boc‐His(DNP) derivative and by condensation of appropriate glycine‐containing segments. The synthetic peptide was efficiently folded under redox conditions to the species with three correct disulfide bridges, as determined by mass spectrometry and N‐terminal sequence analyses of thermolytic fragments. The homogeneity of the synthetic peptide was assessed by reversed‐phase HPLC and electrospray mass spectrometry. One‐dimensional 1H NMR spectroscopic analysis provided evidence that the EGF‐like module had a well defined structure, and was able to bind calcium with an apparent Kd of 10 mM. This value, comparable to that found for the isolated EGF‐like modules of coagulation factors IX and X, is much higher than that measured for native Clr. As already proposed for factors IX and X, it is suggested that neighbouring module(s), most probably the N‐terminal CUB module, contribute(s) to the calcium binding site. © Munksgaard 1997.
Influence of L‐naphthylalanine in position 3 of AVP and its analogues on their pharmacological properties
Authors: LAMMEK B. , CZAJA M. , DERDOWSKA I. , REKOWSKI P. , TRZECIAK H.I. , SIKORA P. , SZKRÓBKA W. , STOJKO R. , KUPRYSZEWSKI G.
Published: August 2010

We describe the synthesis and pharmacological properties of two series of analogues: one which consists of three peptides having L‐1‐naphthylalanine in position 3 and the second composed of analogues substituted in position 3 with L‐2‐naphthylalanine. All peptides were tested in bioassays for pressor and antidiuretic activities. We also checked the uterotonic activityin vitro. We observed that the activity of counterparts in both series is, in two cases, strikingly different.

One of. the new analogues, [(L‐2‐Nal)3, (D‐Arg)8]VP is among the most potent antagonists of the vasopressor response to AVP. Moreover, it is the first potent V1antagonist devoid of antiuterotonic activity. This analogue was designed without modification of position 1, which was previously thought to be essential for substantial pressor antagonism. Two other peptides, [Mpa1, (L‐2‐Nal)3, (o‐Arg)8]VP and [Mpa1, (L‐1‐Nal)3, D‐Arg)8]VP, are highly potent V2agonists. The second analogue is highly selective. With the exception of [(L‐2‐Nal)3]AVP, which showed weak antioxytocic activity, (L‐Nal)3modification resulted in the almost complete removal of interaction of our analogues with oxytocic receptorsin vitro.

Our results suggest that position 3 in AVP and its analogues is important not only for binding and recognition as previously thought, but also for pressor, antidiuretic and uterotonic activities. We also assume that the hindering effect caused by bulky naphthyl moiety has a significant impact on the bioactive conformations of molecules which contain Nal residue, and can thus influence their interaction with V1, V2and oxytocic receptors. © Munksgaard 1997.
Use of Fmoc‐N‐(2‐hydroxy‐4‐methoxybenzyl) amino acids in peptide synthesis
Authors: ZENG WEIGUANG , REGAMEY PIERRE‐OLIVIER , ROSE KEITH , WANG YUZHOU , BAYER ERNST
Published: August 2010

The use of N, O‐bisFmoc‐N‐(2‐hydroxy‐4‐methoxybenzyl) amino acid derivatives in the synthesis of peptides with difficult sequences has already been described. With these amino acid derivatives the reversible protecting group 2‐hydroxy‐4‐methoxybenzyl (Hmb) for the backbone amide bonds of peptide chains is introduced, and thus the aggregation due to hydrogen‐bond interchain association is inhibited. This paper describes the synthesis and use of Fmoc‐N‐(2‐hydroxy‐4‐methoxybenzyl)amino acid derivatives as an alternative means of introducing Hmb backbone protection. These new monoFmoc derivatives were obtained in higher yield than the bisFmoc derivatives. Coupling yields to the amino peptide resin were the same as those obtained with bisFmoc derivatives, under the TBTU/HOBt/DIEA conditions. We also compared different syntheses of a difficult peptide with the Fmoc approach [triple coupling, capping, use of chaotropic agents, backbone protection using monoFmoc (Hmb)Ala] and with optimized Boc chemistry. Both the backbone protection and optimized Boc chemistry approaches gave the desired product in excellent yield and purity. © Munksgaard 1997.
Comparison of the photochemical behavior of four different photoactivatable probes
Authors: WEBER PETER J.A. , BECK‐SICKINGER ANNETTE G.
Published: August 2010

The most commonly used photoaffinity labeling probes are compared, which are aryl azides, aryl diazirines, α‐diazocarbonyls and benzophenone‐derivatives. The compounds were used under identical conditions and crosslinking efficiency, influence of water, irradiation requirements, and by‐products were investigated. Using the pentapeptide thymopentin (TP5) as a model system, we synthesized four analogues by solid‐phase peptide synthesis and partially N‐terminal modification to obtain [p‐(3‐trifluoromethyl)diazirinophenylalanine5] TP5, [p‐benzoylphenylalanine5] TP5, 4‐azidobenzoyl‐TP5 and 2‐diazo‐3, 3, 3‐trifluoropropionyl‐TP5. The peptides were characterized by HPLC and ion‐spray mass spectroscopy. Irradiation of the peptides with two different ultraviolet sources was carried out in water, n‐propanol and water/n‐propanol to imitate both hydrophobic and hydrophilic peptide/protein‐interactions as well as the influence of the aqueous environment. Analysis of the products with HPLC, ion‐spray MS, HPLC‐MS and HPLC‐CID‐MS revealed that (Tmd)Phe is a highly potent carbene‐precursor, which can be transformed easily into uniform crosslinking products by smooth photolysis. However, the electrophilic nature of the intermediate causes a high tendency to react with water molecules. The 4‐azidobenzoyl group showed comparable crosslinking efficiency, but the probability to create non‐uniform irradiation products (e.g. through rearrangement) is higher, whereas the reaction with water is less dominant. In contrast, Bpa was found to have an extremely low affinity to react with water, whereas prolonged UV irradiation is needed to get complete rearrangement into a variety of products. As the absorption band of α‐diazocarbonyls at around 350 nm possesses a low extinction coefficient, 2‐diazo‐3, 3, 3‐trifluoropropionyl‐TP5 could not be activated at all with the optimized irradiation conditions that we have chosen for our comparative studies. © Munksgaard 1997.
Urethane‐protected N‐carboxyanhydrides (UNCAs) as unique reactants for the study of intrinsic racemization tendencies in peptide synthesis
Authors: ROMOFF TODD T. , GOODMAN MURRAY
Published: August 2010

We have established that urethane‐protected N‐carboxyanhydrides (UNCAs) are uniquely suited for the study of intrinsic racemization tendencies in peptide synthesis. The UNCA allows epimerization only by the direct enolization pathway (proton abstraction from the α‐carbon) and does not decompose upon epimerization. A protocol employing the quantitative separation and analysis of enantiomeric N‐protected amino acid derivatives by chiral HPLC has been developed to measure the intrinsic rate of racemization of UNCAs under widely varying reaction conditions. The influence of the tertiary amine structure, UNCA side chain structure, and solvent were studied. The same protocol was employed to study the intrinsic rate of racemization of N‐protected activated amino acid intermediates generated via ‘onium‐type’ activating reagents. We have shown that the trends influencing the intrinsic rate of racemization of UNCAs are maintained under the conditions of in situ activations, and are consistent with the trends found in classical studies in the literature. The results are relevant to peptide synthesis both in solution and on solid phase. The intrinsic rate of racemization for any type of activation with any tertiary amine can be measured by this protocol. © Munksgaard 1997.
Structure‐activity studies of hydrophobic amino acid replacements at positions 9, 11 and 16 of glucagon
Authors: STURM NOEL S. , HUTZLER ANN MARIE , DAVID CLINTON S. , AZIZEH BASSEM Y. , TRIVEDI DEV , HRUBY VICTOR J.
Published: August 2010

We have designed and synthesized eight compounds 2‐9 which incorporate neutral, hydrophobic amino acid residues in positions 9, 11 and 16 of the glucagon molecule: (2) [desHis1,Va19,11e11,16] glucagon amide, (3) [desHis1,Val9,11,16]glucagon amide, (4) [desHis1,Va19,Leu11,16]glucagon amide, (5) [desHis1,Nle9,11e11,16]glucagon amide, (6) [desHis1,Nle9,Val11,16]glucagon amide, (7) [desHis1,Nle9,Leu11,16]glucagon amide, (8) [desHis1,Val9,Leu11,16,Lys17,18,Glu21]glucagon amide and (9) [desHis1,Nle9,Leu11,16,Lys17,18,Glu21]glucagon amide. The effect of neutral, hydrophobic residues at positions 9, 11 and 16 led to good binding to the glucagon receptor. Compared to glucagon (IC50= 1.5 nM), analogues 2‐9 were found to have IC50 values of 6.0, 6.0, 11.0, 9.0, 2.5, 2.8, 6.5 and 7.0 nM, respectively. When these compounds were tested for their ability to block adenylate cyclase (AC) activity, they were found to be antagonists having no stimulation of adenyl cyclase, with PA2, values of 6.15, 6.20, 6.30, 7.25, 6.10, 7.30, 6.25 and 7.25, respectively. © Munksgaard 1997.
Comparative evaluation of the synthesis and purification of transmembrane peptide fragments Rat bradykinin receptor fragment 64‐97 as model
Authors: OLIVEIRA E. , MIRANDA A. , ALBERICIO F. , ANDREU D. , PAIVA A.C.M. , NAKAIE C.R. , TOMINAGA M.
Published: August 2010

The 34‐residue peptide CTVAEIYLGNLAGADLILASGLPFWAITIANNFD (TM‐34), corresponding to the 64‐97 sequence of the rat bradykinin receptor, was selected as a model of hydrophobic transmembrane peptide segment for systematic study of synthesis and purification strategies. Application of conventional Boc/Bzl chemistry resulted in very low yield of the synthesis (around 4%) when DMF was used as the solvent for coupling reactions. As shorter resin‐bound fragments of TM‐34 showed improved swelling in 80% NMP DMSO, the synthesis was repeated in this mixed solvent and the yield increased to 12%. A comparative synthesis using optimized Fmoc chemistry and Fmoc‐(FmocHmb) derivatives of Ala and Leu to prevent aggregation did not provide any detectable TM‐34. Taken together, these results illustrate the synthetic problems associated with hydrophobic sequences, almost regardless of the chemistry used. As expected, the hydrophobicity of TM‐34 and of most of its minor fragments made them scarcely soluble in common solvents. Purification could be achieved by loading the crude materials dissolved in 90% AcOH onto a C4 HPLC column and eluting with a TFA/MeCN linear gradient. CD studies of the TM‐34 and of the shorter fragment with the 74‐97 sequence (TM‐24) showed a higher percentage of r‐helix structure for the latter. This suggests that the shorter sequence may better represent the correct transmembrane region of the second helix of the rat bradykinin receptor. © Munksgaard 1997.
Unexpected lability of cysteine acetamidomethyl thiol protecting group Tyrosine ring alkylation and disulfide bond formation upon acidolysis
Authors: ENGEBRETSEN MAY , AGNER ERIK , SANDOSHAM JESSIE , FISCHER PETER M.
Published: August 2010

Cleavage and deprotection of the peptidyl resin H‐Asn‐Gly‐Gly‐Cys(Acm)‐Glu(OBut)‐Gln‐Tyr(But)‐Cys(Acm)‐Ser(But)‐Asp(OBut)‐[(p‐alkoxy)benzyloxy polystyrene resin] using standard conditions with various trifluoroacetic acid‐containing mixtures were found to result in partial removal of ordinarily acid‐stable S‐Acm groups. Thus, apart from the desired peptide H‐Asn‐Gly‐Gly‐Cys(Acm)‐Glu‐Gln‐Tyr‐Cys(Acm)‐Ser‐Asp‐OH, a disulfide‐cyclic peptide derivative was also isolated. Furthermore, it was found that in another major by‐product of the peptide resin cleavage the tyrosine side chain had been alkylated with an Acm group in a position ortho to the phenolic function. The formation of both by‐products could be suppressed by carrying out the cleavage/deprotection reaction at higher dilution and by inclusion of scavengers such as phenol. An authentic sample of the disulfide‐cyclic peptide was obtained by oxidation of H‐Asn‐Gly‐Gly‐Cys‐Glu‐Gln‐Tyr‐Cys‐Ser‐Asp‐OH using Ellman's reagent. © Munksgaard 1997.
Pegylated peptides V. Carboxy‐terminal PEGylated analogs of growth hormone‐releasing factor (GRF) display enhanced duration of biological activityin vivo
Authors: CAMPBELL R.M. , HEIMER E.P. , AHMAD M. , EISENBEIS H.G. , LAMBROS T.J. , LEE Y. , MILLER R.W. , STRICKER P.R. , FELIX A.M.
Published: August 2010

In the present study, human growth hormone‐releasing factor (hGRF) and analogs were successfully pegylated at the carboxy‐terminus using a novel solid‐ and solution‐phase strategy. Following synthesis, these pegylated hGRF analogs were evaluated for in vitro and in vivo biological activity. Specifically, hGRF(1‐29)‐NH2, [Ala15‐hGRF(1‐29)‐NH2, [desNH22Tyr1, o‐Ala2, Ala15‐hGRF(1‐29)‐NH2 and [His1, Val2, Gin8, Ala15, Leu27‐hGRF(1‐32)‐OH were each C‐terminally extended using a Gly‐Gly‐Cys‐NH2 spacer (previously demonstrated not to alter intrinsic biological activity), and then monopegylated via coupling to an activated dithiopyridyl‐PEG reagent. PEG moieties of 750, 2000, 5000 or 10000 molecular weight (MW) were examined to determine the effect of polymer weight on activity. Initial biological evaluations in vitro revealed that all C‐terminally pegylated hGRF analogs retained high growth hormone (GH)‐releasing potencies, regardless of the MW of PEG polymer employed. Two of these pegylated hGRF analogs, [desN2Tyr, D‐Ala2, Ala15‐hGRF(1‐29)‐Gly‐Gly‐Cys(NH2)‐S‐Nle‐ PEG5000and [His, Val2, Gin8, Ala15, Leu27‐hGRF(1‐32)‐Gly‐Cys(NH2)‐S‐Nle‐PEG5000, were subsequently evaluated in both pig and mouse models and found to be highly potent (in vivo potency range = 12‐55‐fold that of native hGRF). Relative to their non‐pegylated counterparts, these two pegylated hGRF analogs exhibited enhanced duration of activity. © Munksgaard 1997.
Histidine‐rich human salivary peptides are inhibitors of proprotein convertases furin and PC7 but act as substrates for PC1
Authors: BASAK AJOY , ERNST BLAIR , BREWER DYANNE , SEIDAH NABIL G. , MUNZER JON S. , LAZURE CLAUDE , LAJOIE GILLES A.
Published: August 2010

A 32 amino acid peptide called histatin‐3 (H3; 22% His) and its TV‐terminal 24 amino acid fragment histatin‐5 (H5, 33% His), are found in human saliva and possess powerful antimicrobial properties. These His‐rich peptides have been synthesized by Fmoc‐based solid‐phase chemistry. Their sequences are: DSHAKRHHGYKRKFHEKHHSHRGYRSNYLYDN (H3) and DSHAKRHHGYKRKFHEKHHSHRGY (H5). In addition, we also prepared two H5 and one H3 mutants. The H5 mutants were: DH5 (all amino acids in D configuration) and H5F (where all His are replaced by Phe at positions 3, 7, 8, 15, 18, 19, 21). The 9–24 segment of H3 with all the His at positions 15, 18, 19, 21 replaced by Tyr was also prepared (Δ1–8H3Y). The behavior of these five peptides was examined with three proprotein convertases (PC's) which possess cleavage specificity directed towards single and pairs of basic residues. These were: human (h)PCl, an endocrine and neural convertase, hfurin and rat (r)PC7, two widely expressed enzymes. All are serine endoproteases belonging to the kexin/subtilisin family. Our in vitro study revealed that H3 behaves as a substrate for PCI, being cleaved by this endoprotease primarily at a site carboxy terminal to the single Arg25 residue (HRGYR↓SN). On prolonged incubation some minor cleavage was also observed C‐terminal to the first Lys Arg6 pairs of basic amino acids namely at: HAKR↓HH, which contains a P4 as well as P'l and P'2 His residues. The second potential site YKRK12‐FH which does not have a P4 basic residues is not cleaved, even upon incubation with excess protease. PCI only poorly cleaves H5 at the same site mentioned above for H3, i.e., at HAKR↓HH. As expected, neither the D‐amino acid analogue (DH5), nor the Phe and Tyr mutant analogues of the long and short histatins, respectively, are cleaved at all.

In contrast to the above findings for hPCl, the convertase hfurin did not cleave any of the five synthetic peptides. Instead, H3 and H5 were found to be moderately potent inhibitors of the furin‐mediated cleavage of the pentapeptide pGlu‐Arg‐Thr‐Lys‐Arg‐MCA fluorogenic substrate. This inhibition was reversible and competitive, with an estimated inhibition constant Ki of 1.98 μM for H3 and 2.98 μM for H5. The other analogs exhibited only a moderate to weak inhibition of furin, suggesting that substitution of all His by aromatic residues (Phe or Tyr) drastically reduces their inhibitory potency. When tested against rPC7, H3 exhibited almost identical inhibition profile with a measured Ki of 2.4 μM. The partial sequence identity of H3 to the inhibitory pro‐peptide of furin and PC7 provides a rationale for our observation. © Munksgaard 1997.
Preparation of a multiple antigen glycopeptide (MAG) carrying the Tn antigen. A possible approach to a synthetic carbohydrate vaccine
Authors: BAY SYLVIE , LO‐MAN RICHARD , OSINAGA EDUARDO , NAKADA HIROSHI , LECLERC CLAUDE , CANTACUZENE DANIELE
Published: August 2010

The glycosidic tumor‐associated Tn antigen was conjugated to a lysine backbone containing a helper T‐cell epitope in order to activate immune responses specific for some types of carcinomas. As opposed to traditional protein conjugates, this multiple antigen glycopeptide (MAG) offers the advantages of the lack of immunogenicity of the polylysine core and of accurate chemical definition. The MAG construction was assembled by conventional solid‐phase peptide synthesis. The analysis of its antigenicity demonstrated that the Tn antigen on the MAG is recognized by Tn‐specific monoclonal antibodies. © Munksgaard 1997.
Hydrophobically induced conformation in ovine corticotropin‐releasing hormone
Authors: ROTHEMUND SVEN , KRAUSE EBERHARD , BEYERMANN MICHAEL , BIENERT MICHAEL
Published: August 2010

Multiple peptide synthesis has been applied for the simultaneous synthesis of systematic replacement sets of model peptides which varied in length from 18 to 36 residues and ovine corticotropin‐releasing hormone (oCRH), a 41‐residue receptor‐binding peptide. The peptides were utilized to analyze the capability of the stationary phase during RP‐HPLC to induce secondary structure in long‐chain linear peptides. Double D‐amino acid replacement studies demonstrate that nonamphipathic helical domains can be recognized, even in the presence of highly amphipathic domains. On the other hand, systematic alteration of hydrophobicity at each residue along the sequence by methionine and methionine sulfoxide replacements results in characteristic pattern of HPLC retention‐time differences, which is shown to provide a useful method to probe hydrophobic surface regions in helical peptides. Both amino acid replacement strategies were successfully applied to characterize the hydrophobically induced structure of oCRH. Although an a‐helix is formed from residues 6 to 32, the N‐terminal residues 1–5 and the C‐terminal region 33–41 do not show any regular structure. The helical domain from residues 12 to 20 is highly amphipathic.
Protein splicing: estimation of the rate of O‐N and S‐N acyl rearrangements, the last step of the splicing process
Authors: SHAO YANG , PAULUS ENRY
Published: August 2010

The last step in the sequence of reactions that lead to protein splicing is the intramolecular O‐N or S‐N acyl rearrangement of the ester or thioester linkage, respectively, between the two exteins and hydrolysis of the aminosuccinimide residue at the C‐terminus of intein. This paper presents data on the rates of O‐N and S‐N acyl rearrangements of two model depsipeptides as a function of pH and temperature. The rates of rearrangement of both the oxygen ester and the thioester depsipeptide increased strikingly with pH, with the thioester being about 103 times more reactive at pH 5.5, and had a relatively low dependence on temperature, indicative of a low activating energy. The rates of O‐N and S‐N acyl rearrangement of these two model depsipeptides greatly exceed the rate of protein splicing, explaining why the last step of protein splicing can occur without catalysis by the intein.
Modulation of dimerization by residues distant from the interface in bovine neurophysin‐II
Authors: ZHENG CHANGSHENG , PEYTON DAVID , BRESLOW ESTHER
Published: August 2010

The crystal structure of bovine neurophysin‐II in its liganded state (Chen et al. [1991]. Proc. Natl. Acad. Sci. USA88, 4240‐4244) indicates that the 1–6 sequence has a disordered conformation, lacks noncovalent contacts to other regions of the protein and is distant from the monomer‐monomer interface. Cleavage of the 1–6 sequence by Staphylococcus protease V8 yielded a protein that, for the first time, crystallized in both liganded and unliganded states. Insights into the role of the 1–6 sequence in the unliganded state were obtained by NMR and related biophysical comparisons of the native and des‐1–6 proteins. NMR spectra demonstrated that the environment and/or conformation of residues in the 1–6 sequence differed in liganded and unliganded states. Additionally, the unliganded des‐1–6 protein exhibited a dimerization constant four to five times that of the native protein, potentially accounting for the observation that its peptide affinity was also increased. NMR studies further indicated that the increased dimerization constant of the des‐1–6 protein correlated with the presence in the native protein of two isoenergetic forms of the monomer, in contrast to only a single form in the des‐1–6 protein, as evidenced by signals from an internal dimerization‐sensitive a‐proton. Thus, the 1–6 sequence reduces the dimerization constant by stabilization of an alternative monomer conformation. A second product of Staphylococcus protease V8 digestion of the native protein was identified as the des‐1–6 protein with an internal clip after binding site residue Glu‐47, the clip presumably breaking the short 3, 10 helix that most directly connects the interface to the binding site. This product, although unable to bind peptide, retained the dimerization constant of the des‐1–6 protein, suggesting a lack of importance of the helix in dimerization and contrasting with the effects of the 1–6 sequence. A model is proposed in which the 1–6 sequence stabilizes the second conformation of the unliganded monomer via interactions affecting the loop region that separates the two neurophysin domains and which has been shown to influence neurophysin self‐association.
A novel algorithm for the coupling control in solid‐phase peptide synthesis
Authors: BAGNO ANDREA , BICCIATO SILVIO , DETTIN MONICA , BELLO CARLO DI
Published: August 2010

This paper describes a new method for the evaluation of conductimetric data collected during the in‐line monitoring of the coupling step in solid‐phase peptide synthesis. The control scheme relies on a feed‐forward artificial neural network algorithm which can predict the final yield of the reaction within its initial 5 min by analyzing the conductivity signal profile. The yield values predicted by the artificial neural network algorithm result in good accordance with the data obtained by the commonly used ninhydrin test. © Munksgaard 1997.
Position one analogs of the Saccharomyces cerevisiae tridecapeptide pheromone
Authors: ZHANG Y. LARRY , LU HUI‐FEN , BECKER JEFFREY M. , NAIDER FRED
Published: August 2010

Analogs of theSaccharomyces cerevisiaeα‐mating factor [WHWLQLKPGQPMY]., in which a variety of residues replaced Trp were synthesized and assayed for biological activity and receptor affinity. Analogs containing Gly or Leu or many different aromatic residues in position 1 of the peptide exhibited bioactivity in a growth arrest assay slightly greater than, or equal to, that of the parent pheromone, whereas the Glu and Lys analogs exhibited significantly lower bioactivity. Analogs with an aromatic replacement at position 1 had 3‐ to 6‐fold lower receptor affinity than the parent peptide, whereas analogs with a hydrophilic residue at the N‐terminus exhibited large reductions in receptor affinity with the peptide with Glu in position I showing a 120‐fold reduction.Nα‐Acetylation had little effect on bioactivity but lowered receptor affinity by 20‐ to 40‐fold. Amidation of the carboxyl terminus resulted in a 10‐fold decrease in activity and a 160‐fold decrease in receptor affinity. These results indicate that the α‐factor receptor has a large hydrophobic binding pocket, possibly containing a negatively charged side‐chain, which interacts with the N‐terminus of a‐factor. The lack of correlation between activity and binding of several analogs suggests that small residues near the N‐terminus of a‐factor may be very efficient in triggering isomerization of the receptor to its activated state in the first step of the signal transduction pathway. © Munksgaard 1997.
Conformational investigation of α, β‐dehydropeptides. VIII.*N‐Acetyl‐α, β‐dehydroamino acid N'‐methylamides: conformation and electron density perturbation from infrared and theoretical studies
Authors: BRODA MALGORZATA A. , RZESZOTARSKA BARBARA , SMELKA LESZEK , ROSPENK MARIA
Published: August 2010

The Fourier transform infrared spectra are analyzed in the regions of vs(N‐H), amide I, amide II and vs(Cα=Cβ) bands for a series of Ac‐ΔXaa‐NHMe, where ΔXaa =ΔAla, (Z)‐ΔAbu, (Z)‐ΔLeu, (Z)‐ΔPhe and AVal, to determine the predominant solution conformation of these α, β‐dehydropeptide‐related molecules and the electron distribution perturbation in their amide bonds. The measurements were performed in dichloromethane (DCM). To confirm and rationalize the assignments, the spectra of the respective series of saturated Ac‐Xaa‐NHMe, recorded in DCM, and the spectra of these two series of unsaturated and saturated compounds, recorded in acetonitrile, were examined. To help interpret the spectroscopic results, the equilibrium geometrical parameters for some selected amides were used. These were optimized with ab initio methods in the 6‐31G** basis set. Each of the dehydroamides studied adopted a C5 structure, which in Ac‐ΔAla‐NHMe is fully extended and accompanied by the strong C5 hydrogen bond. Interaction with the Cα=Cβ bond lessened the amidic resonance within each of the flanking amide groups. The N‐terminal C=O bond was noticeably shorter, both amide bonds were longer than the corresponding bonds in the saturated entities and the N‐terminal amide system was distorted. Ac‐ΔAla‐NHMe constituted an exception. Its C‐terminal amide bond was shorter than the standard one and both amide systems were prototypically planar.
Reengineering a type II β‐turn as a potential helix nucleator. Part I. Crystal structure of Boc‐Val‐Pro‐(D)Asp‐Asp‐Val‐OMe monohydrate
Authors: FABIOLA.G FELCY , PATTABHI VASANTHA , RAJU E. B. , DURANI S.
Published: August 2010

The crystal structure of Boc‐Val1‐Pro2‐(D)Asp3‐Asp4‐Val5‐OMe is described as a type II β‐turn reengineered into a potential helix nucleator. (o)Asp3 in the peptide is responsible for the configurationally guided LD chiral type n β‐turn centered at Pro2‐(D)Asp3, as well as the partially developed LL chiral type I β‐turn centered at Asp4‐Val5 by acceptance of a conformation nucleating H‐bond from Val5NH to its carboxylic oxygen.
Primary structures of proteins characterized by proteinase K digestion and matrix‐assisted laser desorption/ionization mass spectrometry
Authors: YANG HOUJUN , AMFT MATTHIAS , GRUNDWÜRMER JOHANN M. , LI XIAOLING , GROTEMEYER JÜRGEN
Published: August 2010

To improve the sequence ions of a protein in matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS), proteinase K was used to digest the protein followed by MALDI‐MS characterization of the peptide fragments. The primary structures of three proteins, insulin B chain, cytochrome c and lysozyme, were determined by this method. A series of peptide fragments including those differentiated by one residue can be produced from the protein by using proteinase K digestion, thus providing support to the protein sequence. The peptide fragments liberated from proteinase K proteolysis of the insulin B chain allow the protein to be partially sequenced. Furthermore, some of the residues are double or triple checked by generating a variety of fragments. The same method was used to investigate cytochrome c and lysozyme denaturated in 3 M guanidine hydrochloride. The success of the method relies on the intrinsic properties of proteinase K and accurate determination of the peptide fragments by MALDI‐MS.
Comparison of the solution conformations of a human immunodeficiency virus peptidomimetic and its retro‐inverso isomer using H NMR spectroscopy
Authors: HIGGINS KERRY A. , BICKNELL WENDY , KEAH HOOI HONG , HEARN MILTON T W
Published: August 2010

The solution conformations of the all l‐α‐peptide 1 and the corresponding retro‐all d‐α‐peptide 2, two 20‐meric peptides which generate antibodies that cross‐react with the gp120 envelop protein of human immunodeficiency virus‐1 (HIV‐1), have been investigated by high‐field H NMR spectroscopy. Complete sequential and inter‐residue interaction assignments were made from the 2D NMR spectra acquired at 500 MHz and 600 MHz in 40% deuterotrifluoroethanol (d3‐TFE)/H2O at pH 2.3, and in 300 mm sodium dodecyl sulphate (SDS) in 100% D2O or 90% H2O/10% D2O at pH 2.6. Based on analysis of the nuclear Overhauser effect (NOE) and amide exchange data, peptide 1 and its retro‐inverso isomer 2 in the polar solvent environment of 40% d3‐TFE/H2O at pH 2.3 show very similar topological features. However, in the relatively non‐polar 300 mM SDS micellar environment, peptides 1 and 2 exhibit differences in their solution structures in terms of the amide backbone and side‐chain orientations. In particular, under the SDS micellar condition, peptide 1 maintains much of the secondary structure observed for this 20‐mer peptide in 40% d3‐TFE/H2O, pH 2.3, whereas peptide 2 adopts a more extended structure. These NMR results provide the first confirmation that the secondary structure of the all l‐α‐peptide 1 is maintained in both polar and non‐polar environments, whereas the secondary structure and topology of the notionally equivalent retro‐inverso isomer depends more on the solvent conditions. These results with the all l‐α‐peptide 1 and its retro‐inverso isomer 2 provide important insight into the conformational influences of the C‐ and N‐end group with l‐α‐ and retro‐l‐α‐isomer pairs in non‐polar environments, and thus have general relevance to the design of bioactive retro‐inverso peptidomimetic analogues related to immunogenic or hormonal peptides.
Structure and other chemical characterizations of gila toxin, a lethal toxin from lizard venom
Authors: DATTA GEETA , TU ANTHONY T.
Published: August 2010

The complete primary structure of a lethal toxin, horridum toxin, from the venom of the lizard, Heloderma horridum horridum, was determined by Edman degradation. The amino acid sequence was deduced by overlapping peptide fragments generated by chemical and enzymatic digestions. Horridum toxin causes hemorrhage in internal organs and particularly in the eye, leading to exophthalmia, an effect that has not been observed for other toxins. It is a glycoprotein with a total of 210 residues. Examination of the primary sequence revealed that horridum toxin has considerable homology to tissue‐type kallikrein and trypsin. Furthermore, synthetic substrates for trypsin, such as tosyl‐l‐arginine methyl ester, benzoyl‐l‐arginine ethyl ester and other p‐nitroanilide substrates, were hydrolyzed. The toxin released bradykinin upon hydrolysis of kininogen. This enzymatic behavior is similar to that of plasma kallikrein; however, the presence of a characteristic “kallikrein‐like” loop at 91‐100 (GTIYNCNYVN) in the primary structure and other features similar to tissue kallikrein suggest that horridum toxin is more like tissue kallikrein. This toxin degraded all three chains of fibrinogen but did not form a clot, which suggests that it is different from thrombin. Moreover, it differs from another lethal factor from H. horridum horridum, gila toxin, which has 245 amino acid residues and does not cause exophthalmia.
Potent in vivo inhibitors of rat renin: Analogues of human and rat angiotensinogen sequences containing different classes of pseudodipeptides at the scissile site*
Authors: SUEIRAS‐DIAZ JAVIER , JONES D. MICHAEL , SZELKE MICHAEL , LECKIE BRENDA J. , BEATTIE SHEILA R. , BEATTIE C. , MORTON JAMES J.
Published: August 2010

Using solid‐phase methodology we have synthesised peptides based on the 8–14 or 6–14 human and rat angiotensinogen sequences, containing the following different isosteric units at the P1‐P1’cleavage site: Leu‐Ψ[CH2NH]Leu; Leu‐[CH(OH)CH2]Val; Leu‐Ψ[CH(OH)CH2]Leu and Leu‐Ψ[CH(NH2)CH2]Val. In vitro, peptide Piv‐His‐Pro‐Phe‐His‐Leu‐Ψ[CH(OH)CH2]Leu‐Tyr‐Tyr‐Ser‐NH2(XXI) is the most potent inhibitor of rat plasma renin reported having an IC50 of 0.21 nM; it is a much weaker inhibitor of human renin (IC50 45 nM). Peptide Boc‐His‐Pro‐Phe‐His‐Leu‐Ψ[CH(OH)CH2] Leu‐Val‐Ile‐His‐NH2 (XX) was a highly effective inhibitor of rat renin in vivo. When infused (1 mg/kg/h) into two‐kidney, one‐clip chronic renal hypertensive rats, it lowered blood pressure and suppressed both plasma renin and angiotensin II. When given as a bolus (1 mg/kg) there was a divergence between the rapid rebound of renin levels and blood pressure, which remained suppressed. These results indicate that potent in vivo inhibitors of rat renin could be useful not only in examining the role of circulating renin but also in elucidating the equally important involvement of extracirculatory renin pools.
Secondary structure formation in N‐substituted peptides*
Authors: MÖHLE KERSTIN , HOFMANN HANS‐JÖRG
Published: August 2010

A systematic conformational analysis on several model peptides with N‐substituted amino acids was performed on the basis of ab initio MO theory at the HF/6‐31G* and HF/3‐21G levels with inclusion of solvation effects to study the influence of N‐substitution on the formation of typically secondary structural elements, e.g. β sheets, helices and turns. The conformational flexibility of some structures was examined by means of molecular dynamics simulations in the gas phase and in solution. The results show a restriction of the conformational flexibility of the peptide chain after introduction of an N‐substituted amino acid, N‐substitution makes β sheet formation more difficult. Several consecutive N‐substituted amino acids in a sequence lead to conformers different from those found on the energy hypersurface of the corresponding N‐unsubstituted peptides. There is a strong tendency to form periodically helical conformations, e.g. the polyglycine II or the α helix, which can be extended over several N‐substituted amino acid residues. As long as 1 → 4 hydrogen bond formation remains possible, the major types of β turns can be formed with a distinct preference for the βII and βIa turns. The β turn in particular is considerably destabilized.