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Biomedical subjects

Volker Oberle

Publications and source records attributed to Volker Oberle.

10 recordsLinked to original sources
PubMed2006

Inhibition of platelet aggregation by grafting RGD and KGD sequences on the structural scaffold of small disulfide-rich proteins.

Disintegrins represent a group of disulfide-rich peptides ranging in size from 41 to over 80 residues and are antagonists of several integrin receptors. Disintegrins containing an RGD or KGD sequence are potent inhibitors of platelet aggregation as they block the binding of fibrinogen to alpha(IIb)beta(3) integrin. The high affinity binding to alpha(IIb)beta(3) in comparison to short linear peptides has been attributed to the localisation of the RGD or KGD sequence within a defined three-dimensional structure. Cystine knot microproteins are members of another family of small disulfide-rich peptides that consist of only 28-40 amino acid residues. They display numerous biological activities depending on the peptide sequence of loop regions that are fixed on a structural scaffold that is stabilised by three knot-forming disulfide bonds. In the present study we grafted RGD and KGD containing peptide sequences with seven and 11 amino acids, respectively, into two cystine knot microproteins, the trypsin inhibitor EETI-II and the melanocortin receptor binding domain of the human agouti-related protein AGRP, as well as into the small disintegrin obtustatin. The engineered proteins were much more potent to inhibit the fibrinogen binding, alpha(IIb)beta(3) activation and platelet aggregation when compared to the grafted peptides. Differences that were observed between the engineered proteins indicate the importance of the structural scaffold and the amino acids neighbouring the grafted peptide sequences.

Amino Acid Sequence↗
PubMed2006

Platelet-derived microvesicles induce differential gene expression in monocytic cells: a DNA microarray study.

Platelet-derived microvesicles (PMV) that are shed from the plasma membrane of activated platelets, expose various platelet-type antigens on their surface and are able to adhere to other blood cells and endothelial cells. There are several clinical conditions with markedly increased numbers of PMV, e.g. acute coronary syndrome, thrombotic microangiopathy and sepsis. To prove whether PMV may contribute to an inflammatory response we used DNA microarray technology to study the effect of PMV on gene expression in the prototypic monocytic cell line MonoMac 6 (MM6). PMV were generated by activating human platelets in plasma with collagen and subsequent removal of platelets and plasma by repeated centrifugation. MM6 were incubated for 2 h with PMV in a ratio corresponding to 75 platelets/cell, or saline as control. After RNA isolation, reverse transcription and fluorescence labelling, cDNA was hybridized on a medium density microarray comprising 5308 probes addressing 4868 transcripts of 4730 human genes relevant to inflammation, immune response and related processes. The formation of PMV-MM6 conjugates was associated with significant variations in gene expression, i.e. 93 genes were found to be differentially expressed (P < 0.001; q < 0.087). Among them, 47 genes with annotated transcripts and proteins were identified. Using Ingenuity Pathway Analysis, 37 of the differentially expressed genes were identified as parts of networks associated with functional pathways including cell-to-cell signalling, cellular growth and proliferation, regulation of gene expression and lipid metabolism. For sphingosine kinase-1 the increased expression could be confirmed exemplarily not only by RT-PCR but also on the enzyme activity level. The data indicate that PMV signal differential expression of inflammation-relevant genes in monocytic cells and may represent a novel link between hemostasis and inflammation.

Blood Platelets↗
PubMed2005

Transcriptional regulation of ADAMTS13.

The metalloproteinase ADAMTS13 cleaves VWF multimers instantaneously when they are released from endothelial cells. Absent or manifestly diminished proteolytic activity of ADAMTS13 results in the appearance and accumulation of ultralarge VWF multimers (ULVWFM) in plasma, characterised by the manifestation of Thrombotic Thrombocytopenic Purpura (TTP). Despite congenital defects, infections and the actions of drugs such as cyclosporine A, doxycycline and corticosteroids apparently are involved in its development. To examine the possibility of transcriptional regulation of ADAMTS13 activity, we analyzed RNA levels in various cell culture systems and the response to known and assumed modulators of gene expression. We demonstrate the expression of ADAMTS13 in liver homogenates and a parenchyma liver cell culture system Hep3B, supporting the hypothesis that liver is an important source of plasma ADAMTS13, whereas there was no alteration in gene expression after stimulation of liver cells with proinflammatory stimuli such as endotoxin, TNF-alpha, IL-6, IL-1beta as well as immuno-suppressive agents, such as cyclosporine A, a variety of steroids as well as doxycycline. Therefore, we analysed the ADAMTS13 gene for binding sites of transcription factors in silico and compared the data with those found in two sets of 24 genes considered either as differentially regulated by prototypic inflammatory regulation or as unvaried under various conditions. On the basis of these data, the promotor of ADAMTS13 features the characteristics of a gene, which remains unvaried under a variety of conditions. To our knowledge, the current data demonstrate for the first time, that an alteration in transcriptional activity is negligible in accounting for diminished proteolytic activity as observed under various experimental and, in particular, clinical conditions.

ADAM Proteins↗
PubMed2004

Efficient transfer of chromosome-based DNA constructs into mammalian cells.

Artificial chromosomes, engineered minichromosomes and other chromosome-based DNA constructs are promising new vectors for use in gene therapy, protein production and transgenics. However, a major drawback in the application of chromosome-based DNA is the lack of a suitable and convenient procedure for large-scale cellular introduction, which is particularly frustrated by their size (1 by 2 microm). Here we present a method to transfer Artificial Chromosome Expression systems (ACEs) into mammalian cells, which relies on a combined approach of using cationic amphiphiles and high frequency ultrasound. Thus, when cells were preincubated with liposomes consisting of the cationic lipid SAINT-2 and the phospholipid dioleoylphosphatidylethanolamine (molar ratio 1:1), followed by ultrasound, ACEs could be introduced into mammalian cells, which resulted in the expression of ACEs-harbored reporter genes, such as Green Fluorescent Protein. Depending on cell type, transfection efficiencies ranged from 12% to 53%. Interestingly, no detectable delivery occurred when cells were treated alone with either ultrasound or liposomes. Evidence is provided, based on cellular entry of differently sized beads and trypan-blue permeation, which supports a mechanism in which integration of the lipids creates unstable membrane domains, which are particularly prone to ultrasound-induced pore formation. Time- and temperature-dependent experiments indicate that these pores display a transient stability. Hence, following ultrasound, the pores disappear as a function of time as suggested by a time-window for ACEs entry, and trypan blue exclusion, 80% of the cells becoming stained immediately following ultrasound, dropping to approximately 20% after 30 min. Co-expression of different genes in conjunction with fluorescence in situ hybridization (FISH) analysis indicates that the current procedure provides a means to introduce functionally active artificial chromosomes into eukaryotic cells.

Animals↗
PubMed2004

Size-dependent internalization of particles via the pathways of clathrin- and caveolae-mediated endocytosis.

Non-phagocytic eukaryotic cells can internalize particles <1 microm in size, encompassing pathogens, liposomes for drug delivery or lipoplexes applied in gene delivery. In the present study, we have investigated the effect of particle size on the pathway of entry and subsequent intracellular fate in non-phagocytic B16 cells, using a range of fluorescent latex beads of defined sizes (50-1000 nm). Our data reveal that particles as large as 500 nm were internalized by cells via an energy-dependent process. With an increase in size (50-500 nm), cholesterol depletion increased the efficiency of inhibition of uptake. The processing of the smaller particles was significantly perturbed upon microtubule disruption, while displaying a negligible effect on that of the 500 nm beads. Inhibitor and co-localization studies revealed that the mechanism by which the beads were internalized, and their subsequent intracellular routing, was strongly dependent on particle size. Internalization of microspheres with a diameter <200 nm involved clathrin-coated pits. With increasing size, a shift to a mechanism that relied on caveolae-mediated internalization became apparent, which became the predominant pathway of entry for particles of 500 nm in size. At these conditions, delivery to the lysosomes was no longer apparent. The data indicate that the size itself of (ligand-devoid) particles can determine the pathway of entry. The clathrin-mediated pathway of endocytosis shows an upper size limit for internalization of approx. 200 nm, and kinetic parameters may determine the almost exclusive internalization of such particles along this pathway rather than via caveolae.

Animals↗
PubMed2004

Platelet-derived microvesicles transfer tissue factor to monocytes but not to neutrophils.

Activated platelets are known to adhere to both blood monocytes and neutrophils, and this adhesion is mainly mediated by the surface exposure of the platelet granule protein CD62P. Platelets as well as platelet-derived microvesicles (PMV) have also been shown to contain and to transfer tissue factor (TF), the most important initiator of intravascular thrombin and fibrin formation, to monocytes. However, the role of neutrophils for gathering platelet-derived TF is controversial. Here we studied the interaction of PMV with monocytes and neutrophils using a whole blood system. Platelet-rich plasma (PRP) obtained from citrated human blood was incubated with collagen (5 microg/ml, 15 min) and the platelets were removed by centrifugation (5 min at 5000 x g). After incubating the PMV-containing plasma for further 30 min with a sediment of red and white bloods cells that had been obtained after PRP preparation, monocytes and neutrophils were analysed by flow cytometry for the surface exposure of the platelet-specific antigen CD42a and TF. Compared to a control with non-activated PRP, there was a significant increase in the number of both CD42a-positive monocytes and neutrophils. In contrast, there was no change in the number of TF-positive neutrophils, but a more than 2-fold increase in the number of TF-positive monocytes. The changes in CD42a on monocytes and neutrophils as well as the changes in TF on monocytes could be significantly reduced by an anti-CD62P antibody or by removal of PMV from the plasma samples. The data indicate that the transfer of TF to monocytes is not simply an CD62P-mediated adhesion of platelets or PMV to monocytes, but may involve other not yet identified mechanisms.

Biological Transport↗
PubMed2002

Phase behavior of cationic amphiphiles and their mixtures with helper lipid influences lipoplex shape, DNA translocation, and transfection efficiency.

Cationic lipids are widely used for gene transfection, but their mechanism of action is still poorly understood. To improve this knowledge, a structure-function study was carried out with two pyridinium-based lipid analogs with identical headgroups but differing in alkyl chain (un)saturation, i.e., SAINT-2 (diC18:1) and SAINT-5 (diC18:0). Although both amphiphiles display transfection activity per se, DOPE strongly promotes SAINT-2-mediated transfection, but not that of SAINT-5, despite the fact that DOPE effectively facilitates plasmid dissociation from either lipoplex. This difference appears to correlate with membrane stiffness, dictated by the cationic lipid packing in the donor liposomes, which governs the kinetics of lipid recruitment by the plasmid upon lipoplex assembly. Because of its interaction with the relatively rigid SAINT-5 membranes, the plasmid becomes inappropriately condensed, which results in formation of structurally deformed lipoplexes. This structural deformation does not affect its cellular uptake but, rather, hampers plasmid translocation across endosomal and/or nuclear membranes. This is inferred from the observation that both lipoplexes effectively translocate much smaller oligonucleotides into cells. In fact, SAINT-5/DOPE-mediated transfection is greatly improved when, before lipoplex assembly, the plasmid is stabilized by condensation with polylysine. The results emphasize a role of the structural shape of the plasmid in gaining cytosolic/nuclear access. Moreover, it has been proposed that such a translocation is promoted when the lipoplex adopts the hexagonal phase, and data are presented that demonstrate that the lamellar SAINT-5/DOPE lipoplex adopts such a phase after its interaction with acidic phospholipid-containing membranes.

Animals↗
PubMed2002

Fluorescent lipid probes: some properties and applications (a review).

Odd as it may seem, experimental challenges in lipid research are often hampered by the simplicity of the lipid structure. Since, as in protein research, mutants or overexpression of lipids are not realistic, a considerable amount of lipid research relies on the use of tagged lipid analogues. However, given the size of an average lipid molecule, special care is needed for the selection of probes, since if the size and intramolecular localization of the probe is not specifically taken into account, it may dramatically affect the properties of the lipids. The latter is particularly important in cell biological studies of lipid trafficking and sorting, where the probed lipid should resemble its natural counterpart as closely as possible. On the other hand, for biophysical applications, these considerations may be less critical. Here we provide a brief overview of the application of several lipid probes in cell biological and biophysical research, and critically analyze their validity in the various fields.

Animals↗
PubMedDate not supplied

The influence of physicochemical parameters on the efficacy of non-viral DNA transfection complexes: a comparative study.

Various polycationic vehicles have been developed to facilitate the transfer of foreign DNA into mammalian cells. Structure-activity studies suggested that biophysical properties, such as size, charge, and morphology of the resulting DNA complexes determine transfection efficiency within one class of vector. To investigate the general validity of these criteria, we studied the efficacy of a variety of DNA delivery vehicles including liposomes (DOTAP, SAINT2) with and without helper lipid (DOPE), the polymer polyethyleneimine (PEI), and cationic nanoparticles (Si26H, PLGA/chitosan) in a comparative manner. Sizes of the DNA complexes varied between 100 and 500 nm for PEI polyplexes and DOTAP/DOPE lipoplexes, respectively. The zeta potential was positive for PEI, Si26H, and DOTAP based complexes, while it was neutral for SAINT2-DNA complexes and negative for PLGA/chitosan-DNA complexes. The latter finding was elucidated by AFM, showing a layer of DNA adsorbed onto the nanoparticles. Transfection activity was negligible for PLGA/chitosan nanospheres, moderate for Si26H nanospheres and high for all other complexes, PEI being the most active carrier. The liposomal preparations were of low (DOTAP) or moderate (SAINT2) stability in serum, resulting in a pronounced reduction of gene expression, which was partially restored by the addition of chloroquine. In conclusion, transfection efficiency (i) seems to require a positive or neutral zeta potential, (ii) is depending on size, e.g., is higher for smaller particles, and (iii) requires a vector that is stable in serum.

Animals↗