Biomedical subjects

M Bienert

Publications and source records attributed to M Bienert.

At least 37 records · Page 2Linked to original sources

PubMed2000

[Nphe(1)]NC(1-13)NH(2) selectively antagonizes nociceptin/orphanin FQ-stimulated G-protein activation in rat brain.

[Phe(1)psi(CH(2)-NH)Gly(2)]noc/OFQ(1-13)-amide ([F/G]NC(1-13)NH(2)) and acetyl-RYYRIK-amide (Ac-RYYRIK-NH(2)), two peptidic ligands of the nociceptin/orphanin FQ (noc/OFQ) receptor, have been shown to exert both agonist and antagonist activity in different in vitro and in vivo systems. This is despite the observation that both peptides competitively antagonized the coupling of the activated receptor to G-proteins in brain preparations, measured in GTPgamma(35)S binding assays. In this study, [Nphe(1)]NC(1-13)-amide ([Nphe(1)]NC(1-13)NH(2)), a new noc/OFQ analog recently characterized as a pure and selective noc/OFQ receptor antagonist in several in vitro and in vivo assay systems, was shown to competitively inhibit the noc/OFQ-stimulated GTPgamma(35)S binding to rat cerebral cortex membranes with pA(2) of 7.76 (Schild analysis). This antagonism of noc/OFQ receptor G-protein coupling was selective because the peptide inhibited the noc/OFQ-evoked GTPgamma(35)S binding to rat brain membranes but not that evoked by selective agonists of the mu-, delta-, and kappa-opioid receptors. In rat cortical membranes, the effects of [F/G]NC(1-13)NH(2) and Ac-RYYRIK-NH(2) on the binding of GTPgamma(35)S were clearly differentiated from the effect of [Nphe(1)]NC(1-13)NH(2) when the concentration of GDP, competing with GTPgammaS for binding, was lowered from 100 microM (assay optimum) to 5 microM. At 5 microM GDP, the former peptides showed clear partial agonist activity, whereas [Nphe(1)]NC(1-13)NH(2) did not. These data indicate that only [Nphe(1)]NC(1-13)NH(2) was a pure antagonist of noc/OFQ receptor G-protein coupling. Furthermore, it is suggested that the variable behavior of [F/G]NC(1-13)NH(2) and Ac-RYYRIK-NH(2) (agonist, partial agonist, and antagonist) in different in vitro and in vivo systems may be explained by different partial GTP binding agonism and the existence of a GTP binding stimulus/response reserve (coupling reserve).

Amino Acid Sequence↗

PubMed1999

Noncovalent immobilized artificial membrane chromatography, an improved method for describing peptide-lipid bilayer interactions.

A promising approach in assessing hydrophobic peptide-membrane interactions is the use of reversed-phase high-performance liquid chromatography. The present study describes the preparation and properties of a noncovalent immobilized artificial membrane (noncovalent IAM) stationary phase. The noncovalent IAM phase was prepared by coating the C18 chains of a reversed-phase HPLC column with the phospholipid ditetradecanoyl-sn-glycero-3-phosphocholine. Lipid coating was achieved by pumping a lipid solution in water-2-propanol through the column. The formation of a bilayer-like structure on the chromatographic surface was confirmed by calculating the phospholipid surface density of the stationary phase. The surface density was determined to be approximately 1.95 mumol m-2, which is close to that of lipid vesicles. The coating was found to be stable in chromatographic elution systems containing less than 35% of acetonitrile. Employing this new technique, we determined interaction parameters of a set of helical antibacterial magainin-2-amide peptides with pairwise substitutions of adjacent amino acids by their D-enatiomers. The results demonstrate that the chromatographic retention behavior of peptides on noncovalent IAM stationary phase shows an excellent correlation with lipid affinities to phospholipid vesicles.

Chromatography, High Pressure Liquid↗

PubMed1999

Water-soluble beta-sheet models which self-assemble into fibrillar structures.

Self-assembly of beta-sheet domains resulting in the formation of pathogenic, fibrillar protein aggregates (amyloids) is a characteristic feature of various medical disorders. These include neurodegenerative diseases, such as Alzheimer's, Huntington's, and Creutzfeldt-Jacob's. A significant problem in studying such aggregation processes is the poor solubility of these beta-sheet complexes. The present work describes water-soluble de novo beta-sheet peptides which self-assemble into fibrillar structures. The model peptides enable studies of the relationship between beta-sheet stability and association behavior. The peptides [DPKGDPKG-(VT)n-GKGDPKPD-NH2, n = 3-8] are composed of a central beta-sheet-forming domain (VT-sequence), and N- and C-terminal nonstructured octapeptide sequences which promote water solubility. Conformational analyses by circular dichroism and Fourier transform infrared spectroscopy indicate the influence of peptide length, D-amino acid substitution, and concentration on the ability of the peptides to form stable beta-sheet structures. The association behavior investigated by analytical ultracentrifugation and dynamic light scattering was found to correlate strongly with the stability of a beta-sheet conformation. Model peptides with n >/= 6 form stable, water-soluble beta-sheet complexes with molecular masses of more than 2000 kDa, which are organized in fibrillar structures. The fibrils examined by Congo Red staining and electron microscopy show some similarities with naturally occurring amyloid fibrils.

Amino Acid Sequence↗

PubMed1999

Structural requirements for cellular uptake of alpha-helical amphipathic peptides.

The structure of the cell-permeable alpha-helical amphipathic model peptide FLUOS-KLALKLALKALKAALKLA-NH2 (I) was modified stepwise with respect to its helix parameters hydrophobicity, hydrophobic moment and hydrophilic face as well as molecular size and charge. Cellular uptake and membrane destabilizing activity of the resulting peptides were studied using aortic endothelial cells and HPLC combined with CLSM. With the exceptions that a reduction of molecule size below 16 amino acid residues and the introduction of a negative net charge abolished uptake, none of the investigated structural parameters proved to be essential for the passage of these peptides across the plasma membrane. Membrane toxicity also showed no correlation to any of the parameters investigated and could be detected only at concentrations higher than 2 microM. These results implicate helical amphipathicity as the only essential structural requirement for the entry of such peptides into the cell interior, in accord with earlier studies. The pivotal role of helical amphipathicity was confirmed by uptake results obtained with two further pairs of amphipathic/non-amphipathic 18-mer peptides with different primary structure, net charge and helix parameters from I. The amphipathic counterparts were internalized into the cells to a comparable extent as I, whereas no cellular uptake could be detected for the non-amphipathic analogues. The mode of uptake remains unclear and involves both temperature-sensitive and -insensitive processes, indicating non-endocytic contributions.

Animals↗

PubMed1999

Antagonism by acetyl-RYYRIK-NH2 of G protein activation in rat brain preparations and of chronotropic effect on rat cardiomyocytes evoked by nociceptin/orphanin FQ.

For the further elucidation of the central functions of nociceptin/orphanin FQ (noc/OFQ), the endogenous ligand of the G protein-coupled opioid receptor-like receptor ORL1, centrally acting specific antagonists will be most helpful. In this study it was found that the hexapeptide acetyl-RYYRIK-NH2 (Ac-RYYRIK-NH2), described in literature as partial agonist on ORL1 transfected in CHO cells, antagonizes the stimulation of [35S]-GTPgammaS binding to G proteins by noc/OFQ in membranes and sections of rat brain. The antagonism of the peptide was competitive, of high affinity (Schild constant 6.58 nM), and specific for noc/OFQ in that the stimulation of GTP binding by agonists for the mu-, delta-, and kappa-opioid receptor was not inhibited. The hexapeptide also fully inhibited the chronotropic effect of noc/OFQ on neonatal rat cardiomyocytes. It is suggested that Ac-RYYRIK-NH2 may provide a promising starting point for in vivo tests for antagonism of the action of noc/OFQ and for the further development of highly active and specific antagonists.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗

PubMed1998

Cellular uptake of an alpha-helical amphipathic model peptide with the potential to deliver polar compounds into the cell interior non-endocytically.

Evidence that multiple, probably non-endocytic mechanisms are involved in the uptake into mammalian cells of the alpha-helical amphipathic model peptide FLUOS-KLALKLALKALKAALKLA-NH2 (I) is presented. Extensive cellular uptake of N-terminally GC-elongated derivatives of I, conjugated by disufide bridges to differently charged peptides, indicated that I-like model peptides might serve as vectors for intracellular delivery of polar bioactive compounds. The mode of the cellular internalization of I comprising energy-, temperature-, pH- and ion-dependent as well as -independent processes suggests analogy to that displayed by small unstructured peptides reported previously (Oehlke et al., Biochim. Biophys. Acta 1330 (1997) 50-60). The uptake behavior of I also showed analogy to that of several protein-derived helical peptide sequences, recently found to be capable of efficiently carrying tagged oligonucleotides and peptides directly into the cytosol of mammalian cells (Derossi et al., J. Biol. Chem. 269 (1994) 10444-10450; Lin et al., J. Biol. Chem. 270 (1995) 14255-14258; Fawell et al., Proc. Natl. Acad. Sci. USA 91 (1994) 664-668; Chaloin et al., Biochemistry 36 (1997) 11179-11187; Vives et al., J. Biol. Chem., 272 (1997) 16010-16017).

Amino Acid Sequence↗

PubMed1998

Functional modifications of alamethicin ion channels by substitution of glutamine 7, glycine 11 and proline 14.

Alamethicin is a 20 amino acid, potentially helical peptaibol which forms voltage-dependent ion channels in bilayer systems. Two aspects of alamethicin structure have been suggested to be of particular functional significance for stabilization of alamethicin channels. (i) Proline 14 inducing a helix kink is together with glycine at position 11 responsible for an appropriate orientation of the molecules in the conducting associates. (ii) Glutamine 7 lining the channel interior is assumed to stabilize the channel structure by forming inter-helix hydrogen bonds. The functional importance of these residues was probed in macroscopic and single-channel experiments with alamethicin analogs containing polar, side chain bearing residues at position 11 (glutamine, asparagine) or at position 14 (glutamine). In order to investigate the crucial role of glutamine 7 for the stabilization of channel aggregates, this residue was substituted by alanine. The conformation of the lipid bound peptides was determined by circular dichroism spectroscopy. The results show that glutamine 7, glycine 11 and proline 14 are not essential for channel formation but substitution of any residue reduced the number of conductance levels and significantly reduced their lifetimes. Channel stabilization by the introduction of residues with potential hydrogen bonding capacity at positions 11 and 14 was not observed. Differences in the conformation of the lipid bound peptides, their orientation in the bilayer and their affinity for the lipid membrane appear thus to contribute to the modulation of functional properties.

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 degradation of corticotropin-releasing factor by enzymes of the rat brain studied by liquid chromatography-mass spectrometry.

The corticotropin-releasing factor (CRF; 41 amino acid residues) is a major regulatory peptide in the response to stress and is distributed over many regions of the brain. We have studied the enzymatic degradation of CRF and related peptides by the CRF-degrading enzyme(s) of the rat brain (CRF-DA) by liquid-chromatographic-mass spectrometric technique and by online tandem mass spectrometric experiments. Peptide fragments of the human/rat CRF (1-41) generated by the CRF-DA of the particulate cell fraction were separated and structurally assigned. Major sites of enzymatic attack were identified at the P1 positions Ser1, Thr11 , His13, Leu15, Arg23, Arg35, and Lys36 with Leu15 as the site of primary cleavage. The CRF-DA was shown to be dominated by a metalloendopeptidase activity inhibited by O-phenanthroline and EDTA. The cytosolic fraction generated a similar degradation pattern with a pronounced cleavage at the Arg35 position.

Animals↗

PubMed1998

Mechanism of peptide-induced mast cell degranulation. Translocation and patch-clamp studies.

Substance P and other polycationic peptides are thought to stimulate mast cell degranulation via direct activation of G proteins. We investigated the ability of extracellularly applied substance P to translocate into mast cells and the ability of intracellularly applied substance P to stimulate degranulation. In addition, we studied by reverse transcription--PCR whether substance P-specific receptors are present in the mast cell membrane. To study translocation, a biologically active and enzymatically stable fluorescent analogue of substance P was synthesized. A rapid, substance P receptor- and energy-independent uptake of this peptide into pertussis toxin-treated and -untreated mast cells was demonstrated using confocal laser scanning microscopy. The peptide was shown to localize preferentially on or inside the mast cell granules using electron microscopic autoradiography with 125I-labeled all-D substance P and 3H-labeled substance P. Cell membrane capacitance measurements using the patch-clamp technique demonstrated that intracellularly applied substance P induced calcium transients and activated mast cell exocytosis with a time delay that depended on peptide concentration (delay of 100-500 s at concentrations of substance P from 50 to 5 microM). Degranulation in response to intracellularly applied substance P was inhibited by GDPbetaS and pertussis toxin, suggesting that substance P acts via G protein activation. These results support the recently proposed model of a receptor-independent mechanism of peptide-induced mast cell degranulation, which assumes a direct interaction of peptides with G protein alpha subunits subsequent to their translocation across the plasma membrane.

Animals↗

PubMed1997

Evidence for extensive and non-specific translocation of oligopeptides across plasma membranes of mammalian cells.

After exposure of bovine aortic endothelial cells to various small peptides (tetra- to undeca-mer), extensive transport of the peptides across the plasma membrane was observed in the concentration range 10(-7) to 10(-2) M. The observed transport events, which contradict the generally anticipated poor permeability of peptides across plasma membranes, exhibited high complexity and showed no saturability up to a concentration of 10(-2) M. Evidence was found for the involvement of mdrp-like transporters as well as of energy-independent facilitated diffusion events. The peptide levels within the cells approximated those of the incubation solution within 30 min, indicating high capacity and velocity for the involved transport processes. Correspondingly, preloaded cells exported about 80% of the internalized peptide within 5 min at 37 degrees C. Analogous results were found after peptide exposure to several other mammalian cell types, indicating a more general importance of the transport phenomena described here. Our findings contradict the prevailing opinion that the often observed lack of activity of externally administered peptides against their targets within intact cells is accounted for primarily by poor cellular uptake and point to export processes counteracting the uptake to be more important in this context.

Animals↗

PubMed1997

Modulation of membrane activity of amphipathic, antibacterial peptides by slight modifications of the hydrophobic moment.

Starting from the sequences of magainin 2 analogs, peptides with slightly increased hydrophobic moment (mu) but retained other structural parameters were designed. Circular dichroism investigations revealed that all peptides adopt an alpha-helical conformation when bound to phospholipid vesicles. Analogs with increased mu were considerably more active in permeabilizing vesicles mainly composed of zwitterionic lipid. In addition, the antibacterial and hemolytic activities of these analogs were enhanced. Correlation of permeabilization and binding indicated that the activity increase is predominantly caused by an increased membrane affinity of the peptides due to strengthened hydrophobic interactions.

Anti-Infective Agents↗

PubMed1997

Influence of the angle subtended by the positively charged helix face on the membrane activity of amphipathic, antibacterial peptides.

To investigate the influence of the angle subtended by the positively charged helix face on membrane activity, six amphipathic alpha-helical peptides with angles between 80 degrees and 180 degrees, but with retained hydrophobicity, hydrophobic moment, and positive overall charge, were designed starting from the sequence of the antibacterial peptide magainin 2. CD investigations revealed that all analogs are in an alpha-helical conformation in vesicle suspension. The ability of the peptides to induce dye release from negatively charged phosphatidylglycerol (PG) vesicles decreased with increasing angle. However, peptides with a large angle of positively charged residues (140-180 degrees) exhibited a considerably higher permeabilizing activity at zwitterionic phosphatidylcholine (PC) and mixed PC/PG (3:1) vesicles than analogs with a small angle (80-120 degrees). In addition, analogs with large angles were more active in antibacterial and hemolytic assays. The antibacterial specificity of these analogs was decreased. Binding investigations showed that peptide binding is favored by a large angle and a high content of negatively charged phospholipid. In contrast, a small angle and a low negative membrane charge enhanced the membrane-permeabilizing efficiency of the bound peptide fraction. All analogs stabilized the bilayer phase of phosphatidylethanolamine over the inverted hexagonal phase. Therefore, a class L mechanism of permeabilization can be excluded. Furthermore, the analogs do not act by the induction of positive curvature strain or by a "carpet-like" mechanism. Our results are in accordance with a pore mechanism: The membrane-permeabilizing efficiency of analogs with enhanced angle of positively charged residues is reduced due to electrostatic repulsion between adjacent helices within the pore, thus resulting in a decreased pore-forming probability and/or pore destabilization.

Amino Acid Sequence↗

PubMed1997

Extensive cellular uptake into endothelial cells of an amphipathic beta-sheet forming peptide.

Extensive internalization into endothelial cells has been found for a water soluble amphipathic 26-mer beta-sheet peptide (FLUOS-DPKGDPKGVTVTVTVTVTGKGDPKPD-NH2; VT5). With the D-Val13,D-Thr14 di-D-amino acid analog of VT5 (DD-VT5), exhibiting an identical primary structure but no propensity to adopt a beta-sheet conformation, only about 5% of the cellular uptake of VT5 was found. The mechanism of entry of VT5 into the cells remained unclear, but proved to be energy, temperature and pH dependent and, therefore, clearly distinct from that reported for helical amphipathic peptides. No detectable cytotoxicity, high solubility in water and the found extensive entry into endothelial cells make VT5 appear a good lead for developing new types of vectors for delivering oligonucleotides and peptides into intact cells.

Amino Acid Sequence↗

PubMed1997

Peptide hydrophobicity controls the activity and selectivity of magainin 2 amide in interaction with membranes.

The magainins are antibacterial peptides from the skin of Xenopus laevis. They show a broad range of activity against prokaryotic cells but lyse eukaryotic cells poorly. To elucidate the influence of peptide hydrophobicity on membrane activity and selectivity, we designed and synthesized analogs of magainin 2 amide with slightly varying hydrophobicities but retained hydrophobic moment, peptide charge, and angle subtended by the hydrophilic helix region. Circular dichroism investigations of the peptides revealed that all peptides investigated adopt an alpha-helical conformation when bound to phospholipid vesicles. Dye-releasing experiments from vesicles of phosphatidylglycerol (PG) showed that the membrane-permeabilizing activity of the analogs is not influenced by peptide hydrophobicity. In contrast, the permeability-enhancing activity on vesicles bearing high amounts of phosphatidylcholine (PC) increases drastically with enhanced peptide hydrophobicity, resulting in a reduced selectivity of more hydrophobic analogs for negatively charged membranes. Likewise, the peptide affinity to PC-rich membranes increases in the order of hydrophobicity. Correlation of peptide binding and membrane permeabilization of PC/PG (3:1) vesicles revealed that the observed differences in peptide activity on membranes of low negative surface charge are mainly caused by the different binding affinities. The antibacterial and hemolytic activity of the peptides increases with enhanced hydrophobicity. A strong correlation was found between the hemolytic effect and the bilayer-permeabilizing activity against PC-rich vesicles. Whereas the antibacterial specificity of the more hydrophobic analogs is retained for Escherichia coli, the specificity for Pseudomonas aeruginosa decreases with increasing hydrophobicity.

Anti-Bacterial Agents↗

PubMed1997

The tendency of magainin to associate upon binding to phospholipid bilayers.

Fluorescence energy transfer (FET) from [Trp16]-magainin-2-amide (Trp-Mag) and [D-Ala15,D-Trp16]magainin-2-amide (DD-Trp-Mag) to N(alpha)-dansyl-magainin-2-amide (DNS-Mag) was used to study the association of magainin 2 analogs bound to phosphatidylglycerol vesicles. As shown by circular dichroism and fluorescence spectroscopy, the all-L-analogs exist in a helical conformation and are completely bound to the lipid membrane. The observed FET between Trp-Mag and DNS-Mag is rather small and increases with the DNS-Mag surface concentration. The experimentally determined transfer efficiency is lower than predicted for monomeric magainin analogs randomly distributed exclusively at the outer leaflet of lipid vesicles. These observations can be explained by two different models of spatial distribution for the monomeric magainin analogs. The first model takes into account translocation of magainin which might result in a uniform distribution of magainin at the inner and outer vesicle leaflets. The second model assumes that at least one shell of lipids exists between two magainin molecules, thus reducing the probability of direct contact. Both models explain the measured FET without any contribution of stable associates of magainin analogs. Furthermore, for Trp-Mag and DD-Trp-Mag, an identical energy transfer efficiency was observed, although the nonhelical double-D substituted analog should have a significantly reduced association tendency resulting in decreased FET. Our conclusion that the observed FET is not the result of magainin association is confirmed by the equivalence of the measured energy transfer efficiencies.

Anti-Infective Agents↗

PubMed1997

Hydrophobicity, hydrophobic moment and angle subtended by charged residues modulate antibacterial and haemolytic activity of amphipathic helical peptides.

The hydrophobicity (H), hydrophobic moment (mu) and the angle subtended by the positively charged helix face (phi) of a set of model and magainin 2 amide peptides with conserved charge and helix propensity have been shown to be effective modulators of antibacterial and haemolytic activity. Except peptides of low hydrophobicity which are inactive, changing the parameters has little influence on the activity against Gram-negative bacteria, thus revealing the dominance of electrostatic interactions for the effect. However, the increase of H, mu and phi substantially enhances haemolytic and Gram-positive antibacterial activity and is related to a reduction of peptide specificity for Gram-negative bacteria.

Amino Acid Sequence↗

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↗