Fatty acid-binding to erythrocyte ghost membranes and transmembrane movement

Summary Palmitate binding to human erythrocyte ghost membranes has been investigated with ghost preparations suspended in 0.2% albumin solutions. Free unbound palmitate in the extracellular water phase was measured in equilibrium studies using albumin-filled acid loaded ghosts as small semipermeable bags. The apparent dissociation constant of binding to the membrane is 13.5 nM and the binding capacity 19 nmoles per 7.2 × 109 cells.The 0°C exchange efflux kinetics of palmitate from albumin-filled ghosts is described by a model, which provides estimates of the rate constant of membrane transfer, k₃ = 0.024 s^–1, independent of the molar ratio of palmitate to albumin () and of a mean dissociation rate constant of the palmitate-albumin complex, k₁ = 0.0015 s^–1 at 0.2, allowing for a heterogeneity of the palmitate binding to albumin.The values of a third kinetically determined dependent model constant, Q, the ratio of palmitate bound to the membrane inner surface to palmitate on intracellular albumin, are not different from the Q values obtained by equilibrium experiments.The temperature dependences of k₁ and k₃ in the interval 0°C to 15°C give activation energies of 96 and 103 kJ/mole, respectively. The 0°C exchange efflux increases about 2 fold in response to a rise of pH from 6 to 9. The results suggest a carrier mediated palmitate flux at low with a V_max about 2 pmoles min^–1 cm^–2 at 0°C pH 7.3.     

Fibrinogen distribution on surfaces and in organelles of ADP stimulated human blood platelets

The fibrinogen distribution in platelet organelles after ADP-stimulation was investigated with anti-human fibrinogen using protein A-gold applied to serial sections. Fibrinogen was detected in the so-called alpha-granules of platelets and also in granule protrusions which were observed after ADP-stimulation. The ends of these protrusions were formed as coated membranes and the tips were often in apposition to the surface connected membranes or the plasmalemma. At such places fusion events and hence signs of an exocytosis could be demonstrated by means of cryofixation and cryosubstitution. Examination of serial sections revealed fibrinogen on all these granule profiles. Surface connected membranes, free surfaces and the characteristic structure of the contact zones of aggregated platelets were also labelled by gold particles but less than anticipated. On the platelet surfaces and surface connected membranes fibrinogen was rarely demonstrable with ferritin-labelled anti-human fibrinogen on washed or thrombin-stimulated, almost fibrinogen free platelets. After addition of human fibrinogen to the thrombin stimulated and disaggregated platelets a part of the platelets aggregated spontaneously and formed characteristic contact zones. Anti-human fibrinogen was observed on the free surfaces, in filamentous bridges between the contact spaces and in a tubular surface connected membrane system with involvement of coated membranes at the central ends of these structures. The results indicate the following: all alpha-granules contain fibrinogen; after ADP-stimulation secretion takes place with involvement of coated membranes; during aggregation fibrinogen binds to platelet surfaces and forms contact spaces; fibrinogen is taken up by the surface connected system with involvement of coated membranes.     

Increased Activity of a Cationic Peroxidase Associated with an Incompatible Interaction Between Xanthomonas oryzae pv oryzae and Rice (Oryza sativa)

Rice (Oryza sativa L.) cultivar Cas 209 carries the gene Xa-10 for resistance to race 2 of Xanthomonas oryzae pv oryzae, the bacterial blight pathogen. When seedling leaves of Cas 209 plants were infiltrated with bacterial cell suspensions of strain PXO86^Rif (race 2, incompatible), total peroxidase activity in extracts from extracellular spaces increased almost threefold between 16 and 24 hours after inoculation. The increase in total peroxidase activity in extracellular extracts was correlated with the appearance of a 43-kilodalton peroxidase isoenzyme with an isoelectric point of 8.6. Increases in the activities of two anionic peroxidase isoenzymes also were associated with the incompatible interaction. Later during the interactions, total peroxidase activities increased in both compatible (cv Cas 209 infiltrated with race 1, PXO61^Sm) and control (Cas 209 infiltrated with water) treatments, but final activity levels were less than that observed in the incompatible combination. Similarly, the cationic peroxidase was detected in all three treatments by 48 hours after infiltration, but at reduced levels in compatible and water-infiltrated control treatments relative to the incompatible combination. Accumulation of this peroxidase in extracellular spaces thus may play a role in the defense response in cultivar Cas 209.     

Ischemic reperfusion injury in rabbit spinal cord: protective effect of superoxide dismutase on neurological recovery and spinal infarction

The potential role of superoxide dismutase (SOD), a specific superoxide anion radical scavenger, in treating spinal cord ischemia was investigated in rabbits subjected to aortic occlusion for 20 min. SOD treatment, targeted to the early reperfusion period, reduced both motor dysfunction and incidence of spinal infarcts at 7 days after ischemia. Present results suggest that oxygen-derived free radicals play a role in the pathogenesis of infarcts developing in the spinal cord after ischemia and reperfusion injuries.     

Bundles of Spider Silk,Braided into Sutures,Resist Basic Cyclic Tests: Potential Use for Flexor Tendon Repair

Repair success for injuries to the flexor tendon in the hand is often limited by the in vivo behaviour of the suture used for repair. Common problems associated with the choice of suture material include increased risk of infection, foreign body reactions, and inappropriate mechanical responses, particularly decreases in mechanical properties over time. Improved suture materials are therefore needed. As high-performance materials with excellent tensile strength, spider silk fibres are an extremely promising candidate for use in surgical sutures. However, the mechanical behaviour of sutures comprised of individual silk fibres braided together has not been thoroughly investigated. In the present study, we characterise the maximum tensile strength, stress, strain, elastic modulus, and fatigue response of silk sutures produced using different braiding methods to investigate the influence of braiding on the tensile properties of the sutures. The mechanical properties of conventional surgical sutures are also characterised to assess whether silk offers any advantages over conventional suture materials. The results demonstrate that braiding single spider silk fibres together produces strong sutures with excellent fatigue behaviour; the braided silk sutures exhibited tensile strengths comparable to those of conventional sutures and no loss of strength over 1000 fatigue cycles. In addition, the braiding technique had a significant influence on the tensile properties of the braided silk sutures. These results suggest that braided spider silk could be suitable for use as sutures in flexor tendon repair, providing similar tensile behaviour and improved fatigue properties compared with conventional suture materials.     

Problems,successes and challenges for the application of dispersion-corrected density-functional theory combined with dispersion-based implicit solvent models to large-scale hydrophobic self-assembly and polymorphism

The recent advent of dispersion-corrected density-functional theory (DFT) methods allows for quantitative modelling of molecular self-assembly processes, and we consider what is required to develop applications to the formation of large self-assembled monolayers (SAMs) on hydrophobic surfaces from organic solution. Focus is on application of the D3 dispersion correction of Grimme combined with the solvent dispersion model of Floris, Tomasi and Pascual–Ahuir to simulate observed scanning-tunnelling microscopy (STM) images of various polymorphs of tetraalkylporphyrin SAMs on highly oriented pyrolytic graphite surfaces. The most significant problem is identified as the need to treat SAM structures that are incommensurate with those of the substrate, providing a challenge to the use of traditional periodic-imaging boundary techniques. Using nearby commensurate lattices introduces non-systematic errors into calculated lattice constants and free energies of SAM formation that are larger than experimental uncertainties and polymorph differences. Developing non-periodic methods for polymorph interface simulation also remains a challenge. Despite these problems, existing methods can be used to interpret STM images and SAM atomic structures, distinguishing between multiple feasible polymorph types. They also provide critical insight into the factors controlling polymorphism. All this stems from a delicate balance that the intermolecular D3 and solvent Floris, Tomasi and Pascual–Ahuir corrections provide. Combined optimised treatments should yield fully quantitative approaches in the future.     

FGF3 attached to a phosholipid membrane anchor gains a high transforming capacity. Implications of microdomains for FGF3 cell transformation

NIH3T3 cells transformed by mouse FGF3-cDNA (DMI cells) selected for their ability to grow as anchorage-independent colonies in soft agar and in defined medium lacking growth factors exhibit a highly transformed phenotype. We have used dominant negative (DN) fibroblast growth factor (FGF) receptor 2 (FGFR2) isoforms to block the FGF response in DMI cells. When the DN-FGFR was expressed in DMI cells, their transformed phenotype can be reverted. The truncated FGFR2(IIIb), the high affinity FGFR for FGF3, is significantly more efficient at reverting the transformed phenotype as the IIIc isoform, reaffirming the notion that the affinity of the ligand to the DN-FGFR2 isoform determines the effect. Heparin or heparan sulfate displaces FGF3 from binding sites on the cell surface inhibiting the growth of DMI cells and reverts the transformed phenotype (). However, the presence of heparin is necessary to induce a mitogenic response in NIH3T3 cells when stimulated with soluble purified mouse FGF3. We have investigated the importance of cell surface binding of FGF3 for its ability to transform NIH3T3 cells by creating an FGF3 mutant anchored to the membrane via glycosylphosphatidylinositol (GPI). The GPI anchor renders the cell surface association of FGF3 independent from binding to heparan sulfate-proteoglycan of the cell surface membrane. Attachment of a GPI anchor to FGF3 also confers a much higher transforming potential to the growth factor. Even more, the purified GPI-attached FGF3 is as much transforming as the secreted protein acting in an autocrine mode. Because NIH3T3 cells do not express the high affinity tyrosine kinase FGF receptors for FGF3, these findings suggest that FGF3 attached to GPI-linked heparan sulfate-proteoglycan may have a broader biological activity as when bound to transmembrane or soluble heparan sulfate-proteoglycan.     

Dynamics of human DNA topoisomerases IIalpha and IIbeta in living cells

DNA topoisomerase (topo) II catalyses topological genomic changes essential for many DNA metabolic processes. It is also regarded as a structural component of the nuclear matrix in interphase and the mitotic chromosome scaffold. Mammals have two isoforms (alpha and beta) with similar properties in vitro. Here, we investigated their properties in living and proliferating cells, stably expressing biofluorescent chimera of the human isozymes. Topo IIalpha and IIbeta behaved similarly in interphase but differently in mitosis, where only topo IIalpha was chromosome associated to a major part. During interphase, both isozymes joined in nucleolar reassembly and accumulated in nucleoli, which seemed not to involve catalytic DNA turnover because treatment with teniposide (stabilizing covalent catalytic DNA intermediates of topo II) relocated the bulk of the enzymes from the nucleoli to nucleoplasmic granules. Photobleaching revealed that the entire complement of both isozymes was completely mobile and free to exchange between nuclear subcompartments in interphase. In chromosomes, topo IIalpha was also completely mobile and had a uniform distribution. However, hypotonic cell lysis triggered an axial pattern. These observations suggest that topo II is not an immobile, structural component of the chromosomal scaffold or the interphase karyoskeleton, but rather a dynamic interaction partner of such structures.     

SEARCH: a system for evaluation and archiving of radiation accidents based on case histories

Overexposure of humans to ionizing radiation has occurred worldwide in the past and will surely occur again in the future. In order to allow an effective radiation accident management, it is consequently necessary to be prepared for such emergency situations and to improve means and ways to help people suffering from radiation-induced health impairments. Such approaches should rely on knowledge and experience gained from previous radiation incidents. A prerequisite for any scientific evaluation and comparison of information related to radiation accidents is to collect data in a standardized way. Therefore, the SEARCH database (System for Evaluation and Archiving of Radiation accidents based on Case Histories) has been developed in our department and implemented as an Oracle 8.0 database containing to date more than 800 case histories. The use of this registry is so far limited to active contributors and requires each contributor to sign a cooperation agreement. More information is available under http://www.faw.uni-ulm.de/radmed/. Received: 7 February 2000 / Accepted: 8 May 2000