[Callus distraction and bone transport in the treatment of bone defects]. / Kallusdistraktion und Segmenttransport zur Behandlung von Knochendefekten.

BACKGROUND: The method of "callus distraction" is the only technique which spontaneously produces vascularized bone within the surrounding soft tissues during lengthening reconstructive procedures. Remodeling of the regenerate bone to specific mechanical load can be influenced by the surgeon. In principle, there is no limit to the amount of new bone formation which can be created; this vascularized bone is both resistant to infection and can be created to replace resected infected bone. This is an important prerequisite for the successful treatment of large bone defects. TECHNIQUE: The ring fixator is still a standard tool if no radiological control is available in the operating theater, or in other less sophisticated environments. Over the last 30 years, however, the development of motorized, external and fully implantable systems has made it possible to achieve a significant increase in device implementation, which goes far beyond the standard. RESULTS: High-performance, reliable, custom-made external and fully implantable systems are cost intensive and require special surgical skills, which can only be ensured at specialized centers. However, the complication-free treatment results justify the effort both for the patient and, ultimately, for the cost bearers.

Cations induce shape remodeling of negatively charged phospholipid membranes.

The divalent cation Ca2+ is a key component in many cell signaling and membrane trafficking pathways. Ca2+ signal transduction commonly occurs through interaction with protein partners. However, in this study we show a novel mechanism by which Ca2+ may impact membrane structure. We find an asymmetric concentration of Ca2+ across the membrane triggers deformation of membranes containing negatively charged lipids such as phosphatidylserine (PS) and phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2). Membrane invaginations in vesicles were observed forming away from the leaflet with higher Ca2+ concentration, showing that Ca2+ induces negative curvature. We hypothesize that the negative curvature is produced by Ca2+-induced clustering of PS and PI(4,5)P2. In support of this notion, we find that Ca2+-induced membrane deformation is stronger for membranes containing PI(4,5)P2, which is known to more readily cluster in the presence of Ca2+. The observed Ca2+-induced membrane deformation is strongly influenced by Na+ ions. A high symmetric [Na+] across the membrane reduces Ca2+ binding by electrostatic shielding, inhibiting Ca2+-induced membrane deformation. An asymmetric [Na+] across the membrane, however, can either oppose or support Ca2+-induced deformation, depending on the direction of the gradient in [Na+]. At a sufficiently high asymmetric Na+ concentration it can impact membrane structure in the absence of Ca2+. We propose that Ca2+ works in concert with curvature generating proteins to modulate membrane curvature and shape transitions. This novel structural impact of Ca2+ could be important for Ca2+-dependent cellular processes that involve the creation of membrane curvature, including exocytosis, invadopodia, and cell motility.

Radiosensitizing effect of epothilone B on human epithelial cancer cells.

BACKGROUND: A combined modality treatment employing radiation and chemotherapy plays a central role in the management of solid tumors. In our study, we examined the cytotoxic and radiosensitive effect of the microtubule stabilizer epothilone B on two human epithelial tumor cell lines in vitro and its influence on the microtubule assembly. METHODS: Cancer cells were treated with epothilone B in proliferation assays and in combination with radiation in colony-forming assays. For the analysis of ionizing radiation-induced DNA damage and the influence of the drug on its repair a γH2AX foci assay was used. To determine the effect of epothilone B on the microtubule assembly in cells and on purified tubulin, immunofluorescence staining and tubulin polymerization assay, respectively, were conducted. RESULTS: Epothilone B induced a concentration- and application-dependent antiproliferative effect on the cells, with IC(50) values in the low nanomolar range. Colony forming assays showed a synergistic radiosensitive effect on both cell lines which was dependent on incubation time and applied concentration of epothilone B. The γH2AX assays demonstrated that ionizing radiation combined with the drug resulted in a concentration-dependent increase in the number of double-strand breaks and suggested a reduction in DNA repair capacity. Epothilone B produced enhanced microtubule bundling and abnormal spindle formation as revealed by immunofluorescence microscopy and caused microtubule formation from purified tubulin. CONCLUSION: The results of this study showed that epothilone B displays cytotoxic antitumor activity at low nanomolar concentrations and also enhances the radiation response in the tumor cells tested; this may be induced by a reduced DNA repair capacity triggered by epothilone B. It was also demonstrated that epothilone B in fact targets microtubules in a more effective manner than paclitaxel.

Temperature-dependent phase behavior and protein partitioning in giant plasma membrane vesicles.

Liquid-ordered (Lo) and liquid-disordered (Ld) phase coexistence has been suggested to partition the plasma membrane of biological cells into lateral compartments, allowing for enrichment or depletion of functionally relevant molecules. This dynamic partitioning might be involved in fine-tuning cellular signaling fidelity through coupling to the plasma membrane protein and lipid composition. In earlier work, giant plasma membrane vesicles, obtained by chemically induced blebbing from cultured cells, were observed to reversibly phase segregate at temperatures significantly below 37 degrees C. In this contribution, we compare the temperature dependence of fluid phase segregation in HeLa and rat basophilic leukemia (RBL) cells. We find an essentially monotonic temperature dependence of the number of phase-separated vesicles in both cell types. We also observe a strikingly broad distribution of phase transition temperatures in both cell types. The binding of peripheral proteins, such as cholera toxin subunit B (CTB), as well as Annexin V, is observed to modulate phase transition temperatures, indicating that peripheral protein binding may be a regulator for lateral heterogeneity in vivo. The partitioning of numerous signal protein anchors and full length proteins is investigated. We find Lo phase partitioning for several proteins assumed in the literature to be membrane raft associated, but observe deviations from this expectation for other proteins, including caveolin-1.

The adapter protein SLP-76 mediates "outside-in" integrin signaling and function in T cells.

The adapter protein SH2 domain-containing leukocyte protein of 76 kDa (SLP-76) is an essential mediator of signaling from the T-cell antigen receptor (TCR). We report here that SLP-76 also mediates signaling downstream of integrins in T cells and that SLP-76-deficient T cells fail to support adhesion to integrin ligands. In response to both TCR and integrin stimulation, SLP-76 relocalizes to surface microclusters that colocalize with phosphorylated signaling proteins. Disruption of SLP-76 recruitment to the protein named LAT (linker for activation of T cells) inhibits SLP-76 clustering downstream of the TCR but not downstream of integrins. Conversely, an SLP-76 mutant unable to bind ADAP (adhesion and degranulation-promoting adapter protein) forms clusters following TCR but not integrin engagement and fails to support T-cell adhesion to integrin ligands. These findings demonstrate that SLP-76 relocalizes to integrin-initiated signaling complexes by a mechanism different from that employed during TCR signaling and that SLP-76 relocalization corresponds to SLP-76-dependent integrin function in T cells.

Sorting of lipids and proteins in membrane curvature gradients.

The sorting of lipids and proteins in cellular trafficking pathways is a process of central importance in maintaining compartmentalization in eukaryotic cells. However, the mechanisms behind these sorting phenomena are currently far from being understood. Among several mechanistic suggestions, membrane curvature has been invoked as a means to segregate lipids and proteins in cellular sorting centers. To assess this hypothesis, we investigate the sorting of lipid analog dye trace components between highly curved tubular membranes and essentially flat membranes of giant unilamellar vesicles. Our experimental findings indicate that intracellular lipid sorting, contrary to frequent assumptions, is unlikely to occur by lipids fitting into membrane regions of appropriate curvature. This observation is explained in the framework of statistical mechanical lattice models that show that entropy, rather than curvature energy, dominates lipid distribution in the absence of strongly preferential lateral intermolecular interactions. Combined with previous findings of curvature induced phase segregation, we conclude that lipid cooperativity is required to enable efficient sorting. In contrast to lipid analog dyes, the peripheral membrane binding protein Cholera toxin subunit B is effectively curvature-sorted. The sorting of Cholera toxin subunit B is rationalized by statistical models. We discuss the implications of our findings for intracellular sorting mechanisms.

Membrane elasticity in giant vesicles with fluid phase coexistence.

Biological membranes are known to contain compositional heterogeneities, often termed rafts, with distinguishable composition and function, and these heterogeneities participate in vigorous transport processes. Membrane lipid phase coexistence is expected to modulate these processes through the differing mechanical properties of the bulk domains and line tension at phase boundaries. In this contribution, we compare the predictions from a shape theory derived for vesicles with fluid phase coexistence to the geometry of giant unilamellar vesicles with coexisting liquid-disordered (L(d)) and liquid-ordered (L(o)) phases. We find a bending modulus for the L(o) phase higher than that of the L(d) phase and a saddle-splay (Gauss) modulus difference with the Gauss modulus of the L(o) phase being more negative than the L(d) phase. The Gauss modulus critically influences membrane processes that change topology, such as vesicle fission or fusion, and could therefore be of significant biological relevance in heterogeneous membranes. Our observations of experimental vesicle geometries being modulated by Gaussian curvature moduli differences confirm the prediction by the theory of Juelicher and Lipowsky.

Crosslinking a lipid raft component triggers liquid ordered-liquid disordered phase separation in model plasma membranes.

The mechanisms by which a cell uses and adapts its functional membrane organization are poorly understood and are the subject of ongoing investigation and discussion. Here, we study one proposed mechanism: the crosslinking of membrane components. In immune cell signaling (and other membrane-associated processes), a small change in the clustering of specific membrane proteins can lead to large-scale reorganizations that involve numerous other membrane components. We have investigated the large-scale physical effect of crosslinking a minor membrane component, the ganglioside GM1, in simple lipid models of the plasma membrane containing sphingomyelin, cholesterol, and phosphatidylcholine. We observe that crosslinking GM1 can cause uniform membranes to phase-separate into large, coexistent liquid ordered and liquid disordered membrane domains. We also find that this lipid separation causes a dramatic redistribution of a transmembrane peptide, consistent with a raft model of membrane organization. These experiments demonstrate a mechanism that could contribute to the effects of crosslinking observed in cellular processes: Domains induced by clustering a small number of proteins or lipids might rapidly reorganize many other membrane proteins.

Fusion of small unilamellar vesicles onto laterally mixed self-assembled monolayers of thiolipopeptides.

Monolayers of the thiolipopeptide NH(2)-Cys-Ala-Ser-Ala-Ala-Ser-Ser-Ala-Pro-Ser-Ser-(Myr)Lys(Myr)-OH (III) were formed on gold surfaces by self-assembly, mixed with a lateral spacer of the same peptide composition, NH(2)-Cys-Ala-Ser-Ala-Ala-Ser-Ser-Ala-Pro-Ser-Ser-Lys-OH (I). Different mixing ratios were employed ranging from 0.1 to 1, corresponding to 10-100% thiolipopeptide. These self-assembled monolayers (SAMs) were then exposed to a suspension of liposomes with the aim of forming lipid bilayers as a function of the mixing ratio. A clear optimum with respect to homogeneity and electrical properties of the membranes was obtained in the middle region (0.5) of mixing ratio, as revealed by surface plasmon resonance spectroscopy, impedance spectroscopy, and fluorescence microscopy. The combination of these methods was shown to be a powerful tool, although a true lipid bilayer was not obtained. Instead, vesicle adsorption was shown to be the predominant process, and FRAP (fluorescence recovery after photobleaching) measurements showed that the films were not fluid on the micrometer length scale.

Proton transport through a peptide-tethered bilayer lipid membrane by the H(+)-ATP synthase from chloroplasts measured by impedance spectroscopy.

A lipid membrane was tethered to a gold film by a peptide spacer molecule terminated by a sulfhydryl group. Membranes were formed by fusion of liposomes prepared from egg phosphatidylcholine on self assembled monolayers of the thiolipopeptide Myr-Lys(Myr)-Ser-Ser-Pro-Ala-Ser-Ser-Ala-Ala-Ser-Ala-Cys-amide mixed with mercaptoethanol as a diluent molecule or lateral spacer. These mixed films, although not representing a perfect lipid bilayer, have been shown to retain the activity of incorporated H(+)-ATP synthases from chloroplasts in contrast to films prepared from the pure thiolipopeptide. The activity of the protein was demonstrated by impedance spectroscopy. The resistance decreased due to proton transport across the lipid film, which occurs as a consequence of adenosine triphosphate (ATP) hydrolysis. Several effects previously determined from kinetic measurements of the enzyme reconstituted in liposomes such as saturation with respect to the substrate (ATP), inhibition by venturicidin, activation by a positive potential pulse and increase of the proton current as a function of increasingly negative potentials have been confirmed also for this tethered membrane system. Changes in the impedance spectra due to the addition of ATP were fully reversible.

A conformationally-constrained MHC class II I-Ag7-derived peptide protects NOD mice from the development of diabetes.

Allele-specific peptide vaccination against disease-associated MHC class II molecules is a promising new strategy for modulating self-antigen presentation to autoreactive T cells in autoimmune diseases. To evaluate the potential of this approach for treatment of insulin-dependent diabetes mellitus (IDDM), we have designed a cyclic peptide vaccine, DiavaX, from the third hypervariable region of the beta-chain of the NOD mouse MHC class II I-Ag7. NOD mice were treated at 5 and 9 weeks of age with 100 microg DiavaX emulsified in alum, a control peptide in alum, or alum alone. At the end of the study, 87% of alum treated mice had developed diabetes, compared with only 28% of DiavaX-treated mice. None of the control peptides, including a linear I-Ag7, a scrambled cyclic I-Ag7, or an analogous cyclic I-Aspeptide, reduced the incidence of diabetes, demonstrating that the protective effect of DiavaX is conformationally dependent and both allele- and sequence-specific. DiavaX treatment did not cause any general immune suppression, but did induce peptide-specific antibodies and memory T cells. DiavaX-induced protection from diabetes was associated with the maintenance of a non-destructive islet-associated autoimmune response. These data indicate that a conformationally constrained peptide from the disease-associated MHC represents a potential vaccine candidate for the prevention of clinical IDDM.

[Magnetic resonance tomography in heart septal defects. Model studies on parameter optimizing]. / Magnetresonanz-Tomographie bei Herzscheidewanddefekten. Modelluntersuchungen zur Parameteroptimierung.

Various flow phenomena can occur in MR tomography of the heart. In the present study the influence of various examination parameters on the imaging of flow phenomena behind cardiac septum defects was investigated by means of a model. In the examination by fast field echo sequence (FFE) the contrast between poststenotic jet and surrounding area was most pronounced if short echo times (TE 10 ms), a narrow flip angle (d = 10 degrees) and layers of minor thickness (d = 5 mm) had been selected. The number of acquisitions and the flow compensation exercised only slight influence on contrast. Our results point to the possibility of obtaining quantitative information on cardiac septum defects if the influence of examination parameters on the imaging of flow phenomena is accurately known.

[MR angiography]. / MR-Angiographie.

MR angiography allows the imaging of vessels comparable with the vessels imaged with digital angiography. MR-angiograms were also generated by image subtraction. Basis are the different magnetic properties of moved and stationary spins. Contrast media are not required. In recent years the various techniques of image subtraction with the aim of good imaged vessels were developed. The most important are described in this paper.