Biomedical subjects

Roland Brock

Publications and source records attributed to Roland Brock.

30 records · Page 2Linked to original sources

PubMed2005

Lipopeptide structure determines TLR2 dependent cell activation level.

Bacterial lipoproteins/peptides are composed of di-O-acylated-S-(2,3-dihydroxypropyl)-cysteinyl residues N-terminally coupled to distinct polypeptides, which can be N-acylated with a third fatty acid. Using a synthetic lipopeptide library we characterized the contribution of the lipid portion to the TLR2 dependent pattern recognition. We found that the two ester bound fatty acid length threshold is beyond eight C atoms because almost no response was elicited by cellular challenge with analogues carrying shorter acyl chains in HEK293 cells expressing recombinant human TLR2. In contrast, the amide bound fatty acid is of lesser importance. While two ester-bound palmitic acids mediate a high stimulatory activity of the respective analogue, a lipopeptide carrying one amide-bound and another ester-bound palmitic acid molecule was inactive. In addition, species specific LP recognition through murine and human TLR2 depended on the length of the two ester bound fatty acid chains. In conclusion, our results indicate the responsibility of both ester bound acyl chains but not of the amide bound fatty acid molecule for the TLR dependent cellular recognition of canonical triacylated LP, as well as a requirement for a minimal acyl chain length. Thus they might support the explanation of specific immuno-stimulatory potentials of different microorganisms and provide a basis for rational design of TLR2 specific adjuvants mediating immune activation to distinct levels.

Acylation↗

PubMed2004

Synthetic lipopeptide adjuvants and Toll-like receptor 2--structure-activity relationships.

Bacterial lipoproteins and their synthetic analogues (sLP) are strong immune modulators of the early host responses after infection. Synthetic lipopeptides are strong adjuvants for the adaptive immune system. Lipoproteins and lipopeptides induce signalling in immune cells through Toll-like receptor-TLR2/TLR1 heterodimers. By screening a combinatorial lipohexapeptide amide collection in an in vitro IL-8 induction assay, we systematically evaluated the potential of 19 proteinogenic amino acids in the peptide moiety of Pam3Cys-lipopeptides to interact with TLR2. New Pam3Cys-lipopeptides with high activity were obtained. Different fatty acids were introduced to investigate the influence of the acyl moiety. Lipopeptides with modifications in the core structure of the unusual amino acid S-glycerylcysteine were synthesized and tested for IL-8 induction via TLR2.

Adjuvants, Immunologic↗

PubMed2004

A stepwise dissection of the intracellular fate of cationic cell-penetrating peptides.

The role of endosomal acidification and retrograde transport for the uptake of the highly basic cell-penetrating peptides penetratin, Tat, and oligoarginine was investigated. The effect of a panel of drugs that interfere with discrete steps of endocytosis or Golgi-mediated transport on uptake and cellular distribution of fluorescein-labeled peptide analogues was probed by confocal microscopy, flow cytometry, and fluorescence spectroscopy of whole cell lysates. The analyses were carried out in MC57 fibrosarcoma cells and in HeLa cells. While MC57 fibrosarcoma cells showed some vesicular fluorescence and a pronounced cytoplasmic fluorescence, in HeLa cells little cytoplasmic fluorescence was observed. In MC57 cells the inhibitors of endosomal acidification chloroquine and bafilomycin A1 abolished the release of the peptides into the cytoplasm. Release into the cytosol preserved endosomal integrity. In addition, cellular uptake of the peptides was inhibited by brefeldin A, a compound interfering with trafficking in the trans-Golgi network. In contrast, nordihydroguaiaretic acid, a drug that stimulates the rapid retrograde movement of both Golgi stacks and trans-Golgi network to the endoplasmic reticulum, promoted a cytoplasmic localization of Tat peptides in peptide-pulsed HeLa cells. The effects of these drugs on trafficking shared characteristics with those reported for the trafficking of plant and bacterial toxins, such as cholera toxin, which reach the cytoplasm by means of retrograde transport. A sequence comparison revealed a common stretch of 8-10 amino acids with high sequence homology to the Tat peptide. The structural and functional data therefore strongly suggest a common mechanism of import for cationic cell-penetrating peptides and the toxins.

Antioxidants↗

PubMed2004

Chemolabile cellular microarrays for screening small molecules and peptides.

Microarrays that mediate the uptake of small molecules into living cells are described. Tissue culture cells were seeded onto glass substrates functionalized locally with fluorescently labelled test substances. In order to enable a localized transfer of substances after contact of cells with the substrate, substances were immobilized on the surface either by non-covalent interactions or chemolabile linker groups. These chemolabile linker groups were incorporated into covalently immobilized compounds. Different ester linkages were evaluated as chemolabile linker groups. As model compounds, esters of the carboxy group of a cysteine with the hydroxy groups of carboxyfluorescein-labelled serine amide and tyrosine amide residues or the thiol group of another fluorescein-labelled cysteine amide were generated. Covalent immobilization occurred on maleimide-functionalized glass cover slips. The surface functionalization and release kinetics were assessed by confocal laser scanning microscopy. The fastest release was obtained for the phenolic tyrosine ester. Alternatively, fluorescently labelled peptides were immobilized by non-covalent interactions on glass and on a hydrogel matrix. In order to increase the efficiency of cellular uptake, peptides were N-terminally extended with a cell-penetrating peptide. Uptake of these peptides into cells was confined to the functionalized spots, and was specific for peptides extended with the cell-penetrating peptide.

Amino Acid Sequence↗

PubMed2003

Determination of binding constants on microarrays with confocal fluorescence detection.

Confocal laser scanning microscopy was employed for the determination of binding constants of receptor-ligand interactions in a microarray format. Protocols for a localized immobilization of amine containing substances on glass via GOPTS (3-glycidyloxypropyl)trimethoxysilane) were optimized with respect to the detection of ligand binding by fluorescence. Compatibility with miniaturization by nanopipetting devices was ensured during all steps. The interaction of the tripeptide L-Lys-D-Ala-D-Ala with vancomycin immobilized on glass served as a model. To minimize consumption of ligand, binding constants were determined by stepwise titration of binding sites. The binding constant of the unlabeled ligand was determined by competitive titration with a fluorescently labeled analogue. The determined binding constants agreed well with those determined by other techniques, previously. Labeled ligand bound stronger than the unlabeled one. This difference was dye-dependent. Still, binding was specific for the tripeptide moiety confirming that ligand and fluorescent analogue competed for the same binding sites these results validate the determination of binding constants by competitive titration. The protocols established for confocal fluorescence detection are applicable to axially resolved detection modalities and screening for unlabeled ligands by competitive titration in general.

Journal Article↗

PubMed2003

Quantitative image analysis of cellular protein translocation induced by magnetic microspheres: application to the EGF receptor.

BACKGROUND: The molecular analysis of intracellular signal transduction requires technologies that address quantitatively the activation of signaling proteins and formation of molecular complexes without disrupting cellular integrity. METHODS: Cells expressing the epidermal growth factor receptor (EGFR) in its endogenous form or fused to green fluorescent protein were incubated with 1-microm microspheres covalently functionalized with EGF. The disposition of the plasma membrane about the microspheres was analyzed by high-resolution confocal microscopy in combination with computational resolution enhancement and optimized fixation procedures. Receptor activation and translocation of signaling proteins to the microspheres was quantitated by image processing protocols for recovering the microsphere-associated fluorescence and the fluorescence in the local environment. RESULTS: EGF-functionalized microspheres were internalized in an activation-dependent manner similar to that of the soluble growth factor. The correlation of receptor activation and recruitment of a signaling protein was analyzed quantitatively by isolating immunofluorescence signals from the microspheres and from their immediate environment. CONCLUSIONS: The microsphere-based approach provides a quantitative analysis of cellular signal transduction with subcellular resolution under conditions maintaining cellular integrity. The analysis of signaling-induced (co)localization of proteins around a microsphere complements other technologies directly probing for molecular interactions such as fluorescence resonance energy transfer.

Animals↗

PubMed2003

Modulation of neuronal activity by the endogenous pentapeptide QYNAD.

Inflammation and demyelination both contribute to the neurological deficits characteristic of multiple sclerosis. Neurological dysfunctions are attributable to inflammatory demyelination and, in addition, to soluble factors such as nitric oxide, cytokines and antibodies. QYNAD, an endogenous pentapeptide identified in the cerebrospinal fluid of patients with demyelinating disorders, has been proposed to promote axonal dysfunction by blocking sodium channels. The present study aimed at characterizing the properties of QYNAD in acutely isolated thalamic neurons in vitro. QYNAD, but not a scrambled peptide (NYDQA), blocked sodium channels in neurons by shifting the steady-state inactivation to more negative potentials. Blocking properties followed a dose-response curve with a maximum effect at 10 microm. A fluorescently labelled QYNAD analogue with retained biological activity specifically stained thalamic neurons, positive for type II sodium channels, thus demonstrating the specificity of QYNAD binding. Our study confirms and extends previous observations describing QYNAD as a potent sodium channel-blocking agent. These data as well as our preliminary observations in in vivo experiments in an animal model of inflammatory CNS demyelination warrant further in vivo studies in order to clarify the exact pathogenetic role of QYNAD in inflammatory neurological diseases.

Anesthetics, Local↗

PubMed2002

Lipoconjugates for the noncovalent generation of microarrays in biochemical and cellular assays.

The generation of microarrays by functionalization of hydrophobic glass surfaces with conjugates of triacylated lipophilic end-groups and with a peptide or hapten as a test substance is presented. Immobilization on the hydrophobic surfaces through the triacylated anchor group is fully orthogonal to the reactivity of functional groups within the test substances. The technique is therefore free of risk that reactions of these functional groups may influence the biological activity of the test compounds in screening applications. In addition, no preactivation of either the surface or the compounds is required. Reagents and substrates may be stored at ambient conditions for long periods of time. The lipoconjugates are administered from aqueous solution enabling automated nanopipetting down to spot dimensions of 100 microm across. The microstructures are stable with respect to the conditions of biochemical assays and applications in cell biology. Due to the hydrophobicity of the nonfunctionalized surfaces, standard blocking protocols used in microtiter-plate testing can be employed, thereby inhibiting nonspecific binding of assay reagents. Generation of these microstructures on hydrophobic glass slides or coverslips enables highly sensitive multichannel read-outs with high-resolution fluorescence microscopy.

Antigens, Surface↗

PubMed2002

A quantitative validation of fluorophore-labelled cell-permeable peptide conjugates: fluorophore and cargo dependence of import.

Cell-permeable peptides were evaluated for a quantitatively controlled import of small molecules. The dependence of the import efficiency on the fluorophore, on the position of the fluorophore as well as on the nature of the cargo were addressed. Cellular uptake was quantitated by flow cytometry and fluorescence correlation microscopy (FCM). Fluorophores with different spectral characteristics, covering the whole visible spectral range, were selected in order to enable the simultaneous detection of several cell-permeable peptide constructs. The transcytosis sequences were based either on the sequence of the Antennapedia homeodomain protein (AntpHD)-derived penetratin peptide or the Kaposi fibroblast growth factor (FGF)-derived membrane translocating sequence (MTS)-peptide. In general, the AntpHD-derived peptides had a three- to fourfold higher import efficiency than the MTS-derived peptides. In spite of the very different physicochemical characteristics of the fluorophores, the import efficiencies for analogues labelled at different positions within the sequence of the import peptides showed a strong positive correlation. However, even for peptide cargos of very similar size, pronounced differences in import efficiency were observed. The use of cell-permeable peptide/cargo constructs for intracellular analyses of structure-function relationships therefore requires the determination of the intracellular concentrations for each construct individually.

Amino Acid Sequence↗

PubMed2002

Intracellular concentration measurements in adherent cells: a comparison of import efficiencies of cell-permeable peptides.

A protocol was developed for performing intracellular concentration measurements in flat adherent tissue culture cells by fluorescence correlation microscopy (FCM). Determination of the number of molecules in the confocal detection volume had to account for background fluorescence caused by molecules adsorbed to the surface of the measurement chamber. Such a background signal leads to a decrease in the amplitude of the autocorrelation function, and thereby to the calculation of an erroneously high number of molecules. Because of the spatial heterogeneity of the background intensity, a method was devised by which its contribution to the total fluorescence could be determined directly from each individual autocorrelation measurement. This method was applied to a comparison of the import efficiencies of different cell-permeable peptides at nanomolar concentrations. The Antennapedia homeodomain-derived peptide penetratin was imported about three times as efficient as the basic fibroblast growth factor-derived MTS peptide. Both peptides equilibrated between cytoplasm and nucleus. A relatively high mobility of these molecules inside the cells indicated that they may be rapidly degraded by cytosolic proteases. Based on these results, it will be possible to determine intracellular concentrations of inhibitors linked to import peptides directly by FCM at nanomolar concentrations and to optimise such constructs for proteolytic stability.

Carrier Proteins↗

PubMedDate not supplied

Extending the applicability of carboxyfluorescein in solid-phase synthesis.

Optimized coupling protocols are presented for the efficient and automated generation of carboxyfluorescein-labeled peptides. Side products, generated when applying earlier protocols for the in situ activation of carboxyfluorescein, were eliminated by a simple procedure, yielding highly pure fluorescent peptides and minimizing postsynthesis workup. For the cost-efficient labeling of large compound collections, coupling protocols were developed reducing the amount of coupling reagent and fluorophore. To enable further chemical derivatization of carboxyfluorescein-labeled peptides in solid-phase synthesis, the on-resin introduction of the trityl group was devised as a protecting group strategy for carboxyfluorescein. This protecting group strategy was exploited for the synthesis of peptides labeled with two different fluorescent dyes, essential tools for bioanalytical applications based on fluorescence resonance energy transfer (FRET). Tritylation and optimized labeling conditions led to the development of a fluorescein-preloaded resin for the automated synthesis of fluorescein-labeled compound collections with uniform labeling yields.

Fluoresceins↗

PubMedDate not supplied

Structure property analysis of pentamethine indocyanine dyes: identification of a new dye for life science applications.

A collection of nine pentamethine indocyanine dyes was synthesized, and the photophysical characteristics relevant to applications in cell biology and single molecule detection were analyzed in detail. Substituents at the aromatic system covering the auxochromic series and substitutions in the polymethine chain were investigated with respect to absorption and emission spectra, fluorescence lifetimes, fluorescence quantum yields, and fluorescence autocorrelations. Substitutions in the polymethine chain increased the nonradiative energy dissipation of the excited singlet state and decreased the fluorescence quantum yield, relative to the unsubstituted compound. For substituents at the aromatic rings the fluorescence quantum yield negatively correlates with the position of the substituents in the auxochromic series -SO(3)(-), -H, -F, -CH(3). Compounds with sulfonic acid groups or halogen atoms attached to the indolenine systems had the highest fluorescence quantum yields. The compound S0387 had nearly 70% of the quantum yield of Cy5 and comparable photostability. The free carboxylic acid of S0387 was attached to peptides in high yield and purity by established procedures of solid-phase synthesis. The dye-labeled peptides did not aggregate or bind to tissue culture cells and proteins unspecifically. The indocyanine dye S0387 is therefore an attractive new fluorophore for in vitro and cell-based detection of receptor ligand interaction at nanomolar concentrations by flow cytometry, fluorescence correlation spectroscopy, and laser scanning microscopy.

Algorithms↗