Search PubMed⌕ Search

Biomedical subjects

E Krause

Publications and source records attributed to E Krause.

At least 55 records · Page 3Linked to original sources

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↗

Effects of long term treatment with corticotropin releasing factor on corticotropic tumor cells in vitro.

hCRF inhibits proliferation of corticotropic tumor cells cultivated in serum-reduced medium via interaction with CRF-receptors. This effect was attenuated by the specific antagonist hCRF (9-41), but not by a variety of substances which are inhibitors of cAMP production or cAMP-dependent kinases, suggesting that the effect was not mediated via cAMP. The growth inhibiting effect of hCRH was developed after 4 h incubation, a longer hCRF treatment did not change the effect observed after 4 h. Simultaneously, after hCRF treatment for 4 days the cells were insensitive for ACTH release by hCRF stimulation despite of an increase in the number of secretory granules. The results show that the inhibition of proliferation of pituitary tumor cells by hCRF seems to be a rapid receptor-mediated process connected with morphological changes, but not mediated via activation of adenylate cyclase.

Animals↗

LOE 908 blocks delayed rectifier type potassium channels in PC12 cells and cortical neurons in culture.

The effects of (R,S)-(3,4-dihydro-6,7-dimethoxy-isoquinoline-1-yl)-2- phenyl-N,N-di-[2-(2,3,4-trimethoxyphenyl)ethyl]-acetamide (LOE 908) were studied on K+ currents in undifferentiated cells from a phaeochromocytoma cell line (PC12), in cortical neurons from rat in primary culture, in a rat blood lymphoma cell line (RBL-1) and in a kidney cell line (BHK21). In PC12 cells delayed rectifier K+ currents measured in the whole-cell mode of the patch clamp technique were almost completely blocked by 10 microM LOE 908. The IC50 value was 0.7 microM and the Hill coefficient 0.8. After washout of the inhibitor about 80% of the current recovered. In rat cortical neurons in primary culture LOE 908 inhibited tetraethylammonium (TEA, 10 mM)-sensitive delayed rectifying K+ currents (LOE 908: 1 microM, 61 +/- 25% inhibition; 10 microM 103 +/- 19% inhibition). In contrast to the inhibitory action of LOE 908 on delayed rectifying K+ currents, Ca(2+)-activated potassium currents in BHK21 cells were only inhibited by 25 +/- 5% (10 microM LOE 908, n = 5) and no effect of LOE 908 was found on inward-rectifying K+ currents in RBL-1 cells. We conclude that LOE 908 is a K+ channel blocker with selectivity for delayed outward rectifying K+ channels.

Acetamides↗

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↗

Poor correlation between microimmunofluorescence serology and polymerase chain reaction for detection of vascular Chlamydia pneumoniae infection in coronary artery disease patients.

Chlamydia pneumoniae has been associated to coronary artery disease by various methods including recovery of viable bacteria from plaques. The pathogenetical relevance of this is unclear but investigation of antichlamydial therapy in coronary arteriosclerosis is already in progress. The microimmunofluorescence test (MIF), the only species-specific serological assay available, might be considered useful in identifying patients with vascular chlamydial infection. However, this has never been systematically examined. We compared levels of C. pneumoniae antibodies in sera using MIF with direct detection of C. pneumoniae in coronary artery segments from 158 patients undergoing myocardial revascularization. A polymerase chain reaction (PCR) protocol, recently evaluated for use with vascular materials, detected C. pneumoniae infection in 34 patients. Correlation of serology and PCR was poor: in relation to PCR. MIF-IgG analysis had 21% sensitivity, 90% specificity, 37% positive predictive value, and 81% negative predictive value for detection of chlamydial presence. Thus, the MIF test currently appears not suitable to predict individual vascular C. pneumoniae infection.

Aged↗

Endovascular presence of Chlamydia pneumoniae DNA is a generalized phenomenon in atherosclerotic vascular disease.

The common respiratory pathogen Chlamydia pneumoniae has been implicated in the pathogenesis of coronary artery disease and acute myocardial infarction. In order to verify the endovascular presence of potentially viable chlamydia by detection of genomic DNA, we examined atherosclerotic arteries from various vascular regions using a C. pneumoniae specific nested polymerase chain reaction (PCR). The samples were obtained during surgical revascularization procedures or at autopsy. Chlamydial DNA was detected in 51/238 (21%) atherosclerotic samples. A total of 17 non-atherosclerotic control samples were PCR-negative. Chlamydial presence was detected in 36/140 (26%) vascular samples obtained at coronary revascularization procedures, in 9/61 (15%) samples from carotid artery stenosis, 3/17 (18%) samples from the aorta, and 3/20 (15%) iliac artery samples. Histomorphological discrimination of infected and non-infected arterial samples was not possible. Antichlamydial IgG and IgM response as examined by microimmunofluorescence assay did not aid identification of individual endovascular infection. C. pneumoniae is present in a significant proportion of atherosclerotic arteries. Its occurrence in atheromatous plaques is not limited to coronary arteries and may be considered indicative of an infectious component in atherosclerosis. However, it remains unclear whether chlamydia actually initiates atherosclerotic injury, facilitates its progression, or merely colonizes pre-existing atheromata.

Adult↗

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↗

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↗

Hair cycle-dependent expression of corticotropin-releasing factor (CRF) and CRF receptors in murine skin.

We demonstrate the presence and hair cycle-dependent expression of corticotropin-releasing factor (CRF) and CRF receptors (CRF-R) in C57BL/6 mouse skin. To correlate this with a physiological, developmentally controlled tissue remodeling process, we have analyzed CRF and CRF-R expression during defined stages of the murine hair cycle with its rhythmic changes between growth (anagen), regression (catagen), and resting (telogen). Using reversed-phase HPLC combined with two independent anti-CRF radioimmunoassays, we have identified CRF in murine skin. Maximal CRF levels were found in anagen III-IV skin, and minimal values were detected in catagen and telogen skin. By immunofluorescence, maximal CRF immunoreactivity (CRF-IR) was seen in the basal epidermis, nerve bundles of skin, the outer root sheath and matrix region of anagen IV-VI follicles, and in defined sections of their perifollicular neural network, whereas catagen and telogen skin displayed minimal CRF-IR. Using quantitative autoradiography and 125I-CRF as a tracer, high-affinity binding sites for CRF were detected in murine skin. The highest density of specific binding sites was detected in the panniculus carnosus, the epidermis, and the hair follicle. CRF-R type 1 (CRF-R1) IR was detected by immunohistology mainly in the outer root sheath, hair matrix, and dermal papilla of anagen VI follicles, as well as in the inner and outer root sheaths of early catagen follicles. CRF-R1 expression was also hair cycle dependent. Therefore, in normal murine skin, the CRF-CRF-R signaling system may operate as an additional neuroendocrine pathway regulating skin functions, possibly in the context of cutaneous stress responses.

Animals↗

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↗

Expression and characterization of a trpl homolog from rat.

Mammalian homologues of the Drosophila trp/trpl-gene-family code for "Ca(2+)-store-operated" channels. Here we describe the cloning and expression of a trp/trpl homologous gene from rat brain. The clone is named Rtrp3 because of its high homology to the recently described Htrp3 (Zhu et al (1996) Cell 85:661-671). Expression of Rtrp3 in the mammalian COS-1 cell line leads to 100% increase of capacitative Ca2+ entry as compared to the controls. This capacitative calcium entry can be completely blocked with La3+ (10 microM). We conclude that Rtrp3, a new member of the growing family of trp/trpl homologues in mammalians, is involved in "Ca(2+)-store-operated" Ca2+ entry into the cell.

Amino Acid Sequence↗

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↗

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↗

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↗

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↗

Travel advice: a study among Swiss and German general practitioners.

Travelling to tropical and subtropical destinations is common among European citizens. Many of them consult their general practitioners (GPs) for pre-travel advice. Little is known about the knowledge, the sources of information and the needs of physicians. One hundred and fifty Swiss and I50 German GPs giving travel advice were interviewed using pretested telephone interviews and questionnaires to assess their knowledge about travel advice to be given for 2 frequent holiday destinations (Kenya and Thailand), and to ask which information sources were available to them. Ninety-six per cent and 89%, respectively, of GPs in 2 neighbouring areas of Switzerland and Germany gave travel advice to their clients. In telephone interviews, standard recommendations on malaria medication, as approved by the national travel advice committees, were stated by 45 and 25% of Swiss GPs, and 22 and 9% of German GPs for Kenya and Thailand. The figures for correct advice on vaccination requirements were 23 and 47% for the Swiss GPs, and 2 and 25% for the German GPs. Half of the GPs wanted to consult their documents before giving advice. The main source of information used by Swiss GPs was the monthly updated Bulletin of the Federal Office of Public Health (BFOPH). A variety of different sources was recorded among German practitioners. Regular, concise information on travel advice tops the list of requested information material in both countries. The extent of correct pre-travel advice is unsatisfactory in both study groups. Use of standardized, regularly updated and readily available sources of information on travel advice for the GP could avoid uncertainties of both the provider and the recipient of advice.

Family Practice↗

Hydrophobically induced conformation in ovine corticotropin-releasing hormone.

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 alpha-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.

Amino Acid Sequence↗

Endovascular presence of Chlamydia pneumoniae in patients with hemodynamically effective carotid artery stenosis.

The bacterial pathogen Chlamydia pneumoniae has been associated with atherosclerosis. Recent studies have reported chlamydial antigen to be present in atherosclerotic coronary arteries, but this relation has not yet been widely accepted. In order to verify an endovascular presence of potentially viable chlamydiae by detection of genomic DNA, the authors examined atherosclerotic carotid arteries by using a C. pneumoniae-specific nested polymerase chain reaction. Chlamydial DNA was detected in 9 of 61 (15%) arterial samples obtained from therapy of hemodynamically effective cervical vascular stenosis. Chlamydial presence was limited to advanced atherosclerotic lesions (P < or = 0.02): tissues from the same arteries with early subendothelial lesions did not harbor the pathogen. Thus, an etiologic role of C. pneumoniae is more plausible for progression than for initiation of atherosclerotic lesions. Histomorphologic discrimination of infected and noninfected samples was not possible and serology was unrewarding in detecting the infected patients. Chlamydial occurrence in atheromatous plaques is apparently a general phenomenon of atherosclerosis not limited to coronary arteries. Endovascular presence of genomic DNA of a bacterial pathogen susceptible to antimicrobial agents encourages thought on a new approach to prevent progression of atherosclerosis in a substantial proportion of patients. However, an etiologic contribution of C. pneumoniae in the multifactorial process of atherogenesis yet remains to be demonstrated.

Aged↗