Biomedical subjects

H Wenschuh

Publications and source records attributed to H Wenschuh.

18 recordsLinked to original sources

PubMed2002

Role of endothelial MCP-1 in monocyte adhesion to inflamed human endothelium under physiological flow.

Monocyte chemoattractant protein-1 (MCP-1) is an essential chemokine involved in monocyte traffic across endo- and epithelial barriers both in vitro and in vivo. However, the contribution of endothelial MCP-1 signaling via its CCR2 receptor in monocyte adhesion to inflamed endothelium under flow is incompletely understood. A sensitive flow chamber assay was used to assess monocyte adhesion to TNF-alpha-activated primary human pulmonary artery endothelial cells (HPAEC) during physiological shear stress. Monocyte adhesion was markedly reduced ( approximately 45%) when HPAEC-derived MCP-1 was either neutralized with anti-MCP-1 mAb or inhibited by translational arrest of MCP-1 mRNA transcripts with MCP-1 antisense oligomers. Corresponding efficacy was observed for blockade of monocyte CCR2 receptor function by anti-CCR2 mAb or MCP-1 antagonists (9-76 analog). The impact of endothelial MCP-1 on monocyte-HPAEC adhesion occurred via beta(2)-integrin but not via beta(1)-integrin adhesion pathways. In this line, pretreatment of monocytes with MCP-1 but not RANTES provoked a rapid and transient neoepitope 24 expression on beta(2)-integrin alpha-chains, as analyzed by increased reporter mAb24 binding. Collectively, our data show an important cross talk of endothelial MCP-1 with monocyte CCR2 effecting monocyte firm adhesion to inflamed HPAEC under physiological flow conditions.

Antibodies, Monoclonal↗

PubMed2001

Sequential nucleophilic substitution on halogenated triazines, pyrimidines, and purines: a novel approach to cyclic peptidomimetics.

A novel concept for the synthesis of macrocyclic peptidomimetics which incorporate heteroaromatic units is reported. The method involves sequential SNAr reactions of orthogonally protected amino groups of peptides and other linear oligomers on halogenated heterocycles such as 2,4,6-trichloro[1,3,5]triazine, 2,4,6-trichloropyrimidine, 4,6-dichloro-5-nitropyrimidine, and 2,6,8-trichloro-7-methylpurine. The scope of this novel solid-phase approach was systematically evaluated by means of the SPOT-synthesis methodology on planar cellulose membranes. Besides the question of the accessibility of different ring sizes and the compatibility with protecting groups of commonly used amino acids, the applicability of the technique toward different halogenated heteroaromatics and peptidomimetics was studied. It was found that the procedure is well suited to assemble a wide variety of cyclic peptidomimetics differing in both size (11- to 37-membered rings) and chemical nature of the assembled backbones.

Amino Acid Sequence↗

PubMed2001

Phosphopeptide analysis by positive and negative ion matrix-assisted laser desorption/ionization mass spectrometry.

This article describes a simple procedure for the detection of phosphorylated peptides by comparable positive and negative ion mode matrix-assisted laser desorption/ionization mass spectrometry measurements. Based on studies with phosphorylated peptides (EAIXAAPFAK, X = pS, pT, pY) and their corresponding non-phosphorylated analogs, it was found that phosphopeptides, which are characterized by a low ionization efficiency in the positive ion mode, exhibit drastically increased signal intensities in the negative ion mode compared to their non-phosphorylated analogs. The effect was successfully used to identify phosphorylated sequences of the commonly used phosphoprotein standards, protein kinase A and beta-casein, by peptide mass fingerprint analyses of the corresponding Lys C and trypsin digests using both (positive and negative) ion modes. The comparison of positive and negative ion spectra of a given protein digest (relative intensity([M - H]-)/relative intensity([M + H]+)) can be used to identify any phosphopeptides present which may then be separated and analyzed further.

Caseins↗

PubMed2001

2D relayed anisotropy correlation NMR: characterization of the 13C' chemical shift tensor orientation in the peptide plane of the dipeptide AibAib.

An approach to the determination of the orientation of the carbonyl chemical shift (CS) tensor in a 13C-15N-1H dipolar coupled spin network is proposed. The method involves the measurement of the Euler angles of the 13C'-15N and 15N-1H dipolar vectors in the 13C' CS tensor principal axes system, respectively, via a 13C-15N REDOR experiment and by a 2D relayed anisotropy correlation of the 13C' CSA (omega2) and 15N-1H dipolar interaction (omega1). Via numerical simulations the sensitivity of the omega1 cross sections of the 2D spectrum to the Euler angles of the 15N-1H bond vector in the 13C' CSA frame is shown. Employing the procedure outlined in this work, we have determined the orientation of the 13C' CS tensor in the peptide plane of the dipeptide AibAib-NH2 (Aib = alpha-aminoisobutyric acid). The Euler angles are found to be (chiCN, psiCN) = (34 degrees +/- 2 degrees, 88 degrees +/- 2 degrees) and (chiNH, psiNH) = (90 degrees +/- 10 degrees, 80 degrees +/- 10 degrees). From the measured Euler angles it is seen that the sigma33 and sigma22 components of the 13C' CS tensor approximately lie in the peptide plane.

Aminoisobutyric Acids↗

PubMed2001

Synthesis and biological evaluation of destruxin A and related analogs.

This report describes the development of an efficient solid-phase synthesis protocol and adaptation of reported solution phase procedures for the synthesis of the cyclic depsihexapeptide destruxin A and related analogs. The solid-phase method described is based on standard Fmoc peptide chemistry, including a new synthetic method for the assembly of the depsi bond-containing unit. In order to select analogs of destruxin A for synthesis and evaluation of insecticidal activity, the work of Hellberg et al., describing a set of Z-descriptors for amino acid side-chains comparing their physicochemical properties, was utilized. Destruxin A and 27 different analogs with structural variations in four residues were synthesized and insecticidal activity was evaluated via injections into tobacco budworm (Heliothis virescens) larvae. Several destruxin A analogs were found to be at least as potent as the native compound.

Animals↗

PubMed2000

Coherent membrane supports for parallel microsynthesis and screening of bioactive peptides.

Since its invention the SPOT-synthesis methodology has become one of the most efficient strategies for the miniaturized assembly of large numbers of peptides. The combination of a facile synthetic method with high throughput solid- and solution-phase screening assays qualifies the SPOT-technique as a valuable tool in biomedical research. Recent developments such as the introduction of novel polymeric surfaces, new linker and cleavage strategies as well as automated robot systems extended the scope of practical chemical reactions that can be accommodated as well as the numbers of compounds obtainable by this technique. Thus, highly complex spatially addressed compound arrays have become accessible. Together with the introduction of novel screening assays, the method is excellently suited to elucidate recognition events on the molecular level.

Adsorption↗

PubMed2000

Human T-cell response to meningococcal immunoglobulin A1 protease associated alpha-proteins.

A unique feature of the immunoglobulin A1 (IgA1) protease from pathogenic Neisseriae, i.e. N. meningitidis and N. gonorrhoeae, is its co-secretion with an amphipathic a-protein. Polymerase chain reaction (PCR) analysis of the respective iga(alpha). gene region in 48 meningococcal strains revealed that this protein domain is conserved throughout all isolates in four different principal variants. Despite strain-dependent size and sequence variations, sequence analysis showed common structural characteristics. More than 80% of the amino acid sequence of all a-proteins is dependent on the five amino acids Q, E, A, K and R, resulting in a pI> 10. The sequences are highly conserved at the N-terminus and the C-terminus and contain long amphipathic alpha-helical stretches. These stretches have a strong probability of forming coiled coil conformations and comprise short repetitive sequence modules with pronounced similarities to T-cell epitopes. We therefore analyzed the T-cell response of 20 volunteer blood donors to four peptides, representing such predicted epitopes, and a recombinant meningococcal alpha-protein. Sixteen donors reacted against at least one peptide after culture of peripheral blood mononuclear cells in interleukin (IL)-2-rich medium, while two individuals showed a positive reaction only against an IgA1 protease-derived control peptide. From one donor, we established and maintained T-cell clones specific for purified alpha-protein. Characterization of the T-cell clones revealed a CD3- and a CD4-positive phenotype and the secretion of IL-2 and interferon-gamma (IFN-gamma),

Amino Acid Sequence↗

PubMed2000

The NMR solution structure of the ion channel peptaibol chrysospermin C bound to dodecylphosphocholine micelles.

Chrysospermin C is a 19-residue peptaibol capable of forming transmembrane ion channels in phospholipid bilayers. The conformation of chrysospermin C bound to dodecylphosphocholine micelles has been solved using heteronuclear NMR spectroscopy. Selective 15N-labeling and 13C-labeling of specific alpha-aminoisobutyric acid residues was used to obtain complete stereospecific assignments for all eight alpha-aminoisobutyric acid residues. Structures were calculated using 339 distance constraints and 40 angle constraints obtained from NMR data. The NMR structures superimpose with mean global rmsd values to the mean structure of 0. 27 A (backbone heavy atoms) and 0.42 A (all heavy atoms). Chrysospermin C bound to decylphosphocholine micelles displays two well-defined helices at the N-terminus (residues Phe1-Aib9) and C-terminus (Aib13-Trp-ol19). A slight bend preceding Pro14, i.e. encompassing residues 10-12, results in an angle of approximately 38 degrees between the mean axes of the two helical regions. The bend structure observed for chrysospermin C is compatible with the sequences of all 18 long peptaibols and may represent a common 'active' conformation. The structure of chrysospermin C shows clear hydrophobic and hydrophilic surfaces which would be appropriate for the formation of oligomeric ion channels.

Amino Acid Sequence↗

PubMed1999

The dominance of arginine-containing peptides in MALDI-derived tryptic mass fingerprints of proteins.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a powerful tool for mass finger-printing of peptide mixtures obtained after enzymatic ingel digestion of proteins separated by two-dimensional electrophoresis (2-DE). In the course of a proteome analysis of mycobacteria using mass spectrometric identification, it was found that 94% of the most intense MALDI-MS peaks denote peptides bearing arginine at the C-terminal end. The effect was demonstrated to be equally prominent using an equimolar mixture of the synthetic peptides known to be present in the tryptic digest of the mycobacterial 35 kDa antigen ("synthetic mass map"). In addition, several binary mixtures of synthetic peptides differing exclusively at the C terminus (Arg or Lys) were examined to rationalize the higher sensitivity toward arginine-containing peptides. The extent of the effect described depends on the matrix used and may facilitate a more reliable assignment of mass fingerprint data to protein sequences in databases.

Amino Acid Sequence↗

PubMed1999

An anion-selective analogue of the channel-forming peptide alamethicin.

The peptide alamethicin self-assembles to form helix bundle ion channels in membranes. Previous macroscopic measurements have shown that these channels are mildly cation-selective. Models indicate that a source of cation selectivity is a zone of partial negative charge toward the C-terminal end of the peptide. We synthesized an alamethicin derivative with a lysine in this zone (replacing the glutamine at position 18 in the sequence). Microscopic (single-channel) measurements demonstrate that dimeric alamethicin-lysine18 (alm-K18) forms mildly anion-selective channels under conditions where channels formed by the parent peptide are cation-selective. Long-range electrostatic interactions can explain the inversion of ion selectivity and the conductance properties of alamethicin channels.

Alamethicin↗

PubMed1999

Engineering charge selectivity in alamethicin channels.

The peptide alamethicin provides a system for engineering ion channel charge selectivity. To define alamethicin charge selectivity experimentally, we measured single-channel current-voltage relationships in KCl gradients using covalently linked peptide dimers. Two factors were found to contribute to the charge selectivity of these channels: (i) the ionic strength of the surrounding solutions; and (ii) the distribution of fixed charge on the peptide. Native alamethicin channels exhibited either cation selectivity or anion selectivity depending on which end of the channel was at the low salt side of the membrane. When the glutamine residue at position 18 in the sequence was replaced with a lysine residue, an anion-selective channel was obtained regardless of which end of the channel was at the low salt side of the membrane.

Alamethicin↗

PubMed1998

Proline at position 14 of alamethicin is essential for hemolytic activity, catecholamine secretion from chromaffin cells and enhanced metabolic activity in endothelial cells.

Alamethicin is known to lyse different biological cells and to induce voltage dependent ion channels in lipid bilayers. A set of analogs with proline shifted from position 14 in the native peptide towards the N- and C-terminus was used to investigate the role of proline in: (i) alamethicin induced hemolysis of human red blood cells, (ii) stimulation of catecholamine secretion from bovine adrenal chromaffin cells and (iii) induction of metabolic activity in bovine aortic endothelial cells. Half maximal hemolytic activity was found at 30 microM alamethicin concentration, complete lysis occurred at 100 microM. The stimulation of catecholamine secretion in the presence of extracellular Ca2+ was concentration dependent up to 50 microM alamethicin. At this high concentration mild secretion was also found in the absence of Ca2+ indicating cell membrane damage. Alamethicin transiently stimulated the metabolic rate of endothelial cells in a concentration dependent mode up to 20 microM while the inhibition of metabolism at higher concentrations pointed to a toxic effect. The alamethicin analogs were completely inactive in all the biological assays. The effects correlated with a loss of dye release inducing activities on phosphatidylcholine vesicles and reduction of channel forming properties in lipid bilayers and were associated with modifications of membrane affinity rather than conformational changes of the peptides. The results indicate that proline at position 14 of the native peptide is essential for the interaction with different membrane systems.

Alamethicin↗

PubMed1998

The ease of peptide detection by matrix-assisted laser desorption/ionization mass spectrometry: the effect of secondary structure on signal intensity.

Several structurally well-characterized model peptides were used to examine the relationship between peptide structure and signal intensity in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). It was found that peptides displaying stable alpha-helical and beta-sheet structures show lower signal intensities than the corresponding analogs having disturbed secondary structures caused by substitution of two adjacent amino acids by their D isomers. Since such substitutions do not affect properties other than the secondary structure propensity, the differences observed are ascribed to this phenomenon or some related effect such as association. The results indicate that the formation of stable secondary structures in peptides may be a possible source of incomplete peptide mass fingerprints resulting from protein digestion and for difficulties in the quantitative evaluation of peptide mixtures via MALDI-MS.

Amino Acid Sequence↗

PubMed1998

A solution NMR study of the selectively 13C, 15N-labeled peptaibol chrysospermin C in methanol.

The conformation of the 19-residue peptaibol chrysospermin C in methanol has been investigated by NMR spectroscopy using selective 15N and 13C labeling of the alpha-aminoisobutyric acid (Aib) residues. Complete 1H and 13C sequential assignments, including stereospecific assignments for the heavily overlapped resonances from the two Cbeta methyl groups of the eight Aib residues, are reported for a peptaibol for the first time. An Aib residue followed by a Pro is an exception to previous suggestions regarding stereospecific assignment of the two Cbeta methyl groups of Aib residues. Local nuclear Overhauser effects and 3J(HNC') and 3J(HNCbeta) scalar couplings indicate that the phi angles of the Aib residues are restricted sterically to local conformations consistent with right-handed helices. Despite these constraints on the eight Aib residues, the NMR data for chrysospermin C in methanol are generally most consistent with an ensemble of transient conformations, including backbone conformations inconsistent with helical structures. Initial NMR measurements for chrysospermin C bound to micelles suggest structural and dynamic differences relative to alamethicin bound to micelles which may be related to differences in gating voltages for formation of ion channels.

Amino Acid Sequence↗

PubMed1997

Structure-function relationships in helix-bundle channels probed via total chemical synthesis of alamethicin dimers: effects of a Gln7 to Asn7 mutation.

Alamethicin channels are prototypical helix bundles that may serve as tractable models for more complex protein ion channels. Solid-phase peptide synthesis of alamethicin analogues using FMOC-amino acid fluorides followed by chemical dimerization of these peptides facilitates structure-function studies of particular channel states in bilayer membranes. State 3 in particular, tentatively assigned to a hexameric helix bundle, is sufficiently long-lived that current-voltage measurements can be made during the lifetime of an individual channel opening. Molecular models of hexameric helix bundles, generated using restrained molecular dynamics with simulated annealing, indicate that a Gln7-->Asn7 (Q7-->N7) mutation will increase channel diameter locally. Experimentally, the conductance of state 3 of the N7-alm channel is found to be larger than that of the Q7-alm channel when ion flow is in the usual direction (cations entering the C-terminal end of the channel). When ion flow is in the opposite direction, no difference in the conductances of state 3 of Q7 and state 3 of N7 channels is observed. These results indicate that the effect of a change in pore diameter at position 7 is dependent on the magnitude of other barriers to permeation and that these barriers are voltage-dependent.

Alamethicin↗

PubMed1997

Influence of proline position upon the ion channel activity of alamethicin.

Alamethicin, a 20-residue peptaibol, induces voltage-dependent ion channels in lipid bilayers according to the barrel-stave model. To study relationships between the proline-14-induced kink region and the channel-forming behavior of the peptide, a set of alamethicin analogs with proline incorporated at positions 11, 12, 13, 14, 15, 16, and 17, respectively, as well as an analog with alanine instead of proline at position 14 were synthesized. Macroscopic conductance experiments show that the voltage dependence of the peptides is conserved although slightly influenced, but the apparent mean number of monomers forming the channels is significantly reduced when proline is not located at position 14. This is confirmed in single-channel experiments. The analogs with proline next to position 14 (i.e., 13, 15, 16) show stable conductance levels, but of reduced number, which follows the order Alam-P14 > Alam-P15 > Alam-P16 > Alam-P13. This reduction in the number of levels is connected with changes in the lifetime of the channels. Analogs with proline at position 11, 12, or 17 produce erratic, extremely short-lived current events that could not be resolved. The changes in functional properties are related to structural properties as probed by circular dichroism. The results indicate that proline at position 14 results in optimal channel activity, whereas channels formed by the analogs bearing proline at different positions are considerably less stable.

Alamethicin↗

PubMedDate not supplied

Spatially addressed synthesis of amino- and amino-oxy-substituted 1, 3,5-triazine arrays on polymeric membranes.

Effective spatially addressed parallel assembly of trisamino- and amino-oxy-1,3,5-triazines was achieved by applying the SPOT-synthesis technique on cellulose and polypropylene membranes. In addition to developing a suitable linker strategy and employing amines and phenolate ions as building blocks, a highly effective microwave-assisted nucleophilic substitution procedure at membrane-bound monochlorotriazines was developed. The 1,3, 5-triazines obtained could be cleaved in parallel from the solid support by TFA vapor to give compounds adsorbed on the membrane surface in a conserved spatially addressed format for analysis and screening. The reaction conditions developed were employed for the synthesis of 8000 cellulose-bound 1,3,5-triazines which were probed in parallel for binding to the anti-transforming growth factor-alpha monoclonal antibody Tab2 in order to identify epitope mimics.

Amines↗

PubMedDate not supplied

Mass spectrometric sequencing of synthetic peptides containing alpha, alpha-dialkylated amino acid residues by MALDI post-source decay analysis.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), a method well-suited for mass determination of biomolecules, has been used to analyze fragment ions generated by post-source decay (PSD) of synthetic peptaibols containing high proportions of the sterically hindered amino acids alpha-amino isobutyric acid (Aib) and isovaline (Iva). Since peptaibols do not have a free N-terminal amino group or side chains subject to protonation, the analyzed peptides saturnisporin SA III, trichotoxin A-50 and chrysospermin B were shown to provide preferred N-terminal and C-terminal a, b, and y fragments as sodium adduct. Additionally, a cleavage of the labile Aib-Probond was observed for all peptides investigated. The fragmentation pattern allowed confirmation of the primary structure and, therefore, demonstrated the usefulness of MALDI-PSD mass spectrometry for sequence analysis of the peptaibols.

Alkylation↗