Amphiphilic cationic peptides mediate cell adhesion to plastic surfaces

Four amphiphilic peptides, each with net charges of +2 or more at neutrality and molecular weights under 4 kilodaltons, were found to mediate the adhesion of normal rat kidney fibroblasts to polystyrene surfaces. Two of these peptides, a model for calcitonin (peptide 1, MCT) and melittin (peptide 2, MEL), form amphiphilic alpha-helical structures at aqueous/nonpolar interfaces. The other two, a luteinizing hormone-releasing hormone model (peptide 3, LHM) and a platelet factor model (peptide 4, MPF) form beta-strand structures in amphiphilic environments. Although it contains only 10 residues, LHM mediated adhesion to surfaces coated with solutions containing as little as 10 pmoles/ml of peptide. All four of these peptides were capable of forming monolayers at air-buffer interfaces with collapse pressures greater than 20 dynes/cm. None of these four peptides contains the tetrapeptide sequence Arg-Gly-Asp-Ser, which has been associated with fibronectin-mediated cell adhesion. Ten polypeptides that also lacked the sequence Arg-Gly-Asp-Ser but were nonamphiphilic and/or had net charges less than +2 at neutrality were all incapable of mediating cell adhesion (Pierschbacher and Ruoslahti, 1984). The morphologies of NRK cells spread on polystyrene coated with peptide LHM resemble the morphologies on fibronectin-coated surfaces, whereas cells spread on surfaces coated with MCT or MEL exhibit strikingly different morphologies. The adhesiveness of MCT, MEL, LHM, and MPF implies that many amphiphilic cationic peptides could prove useful as well defined adhesive substrata for cell culture and for studies of the mechanism of cell adhesion.     

Functional alterations in beta''-actin from a KB cell mutant resistant to cytochalasin B

We recently described the isolation of mutant KB cells (Cyt 1 cells) resistant to the cytotoxic effect of cytochalasin B (CB). This mutant carried an altered beta-actin; i.e., beta'-actin (Toyama, S., and S. Toyama. 1984. Cell. 37:609-614). In the present study, we have examined the functional properties of actin in Cyt 1 cells. Our results showed that increased resistance of Cyt 1 cells to CB was reflected in altered properties of beta'-actin itself. This was shown directly by two findings. First, the polymerization of beta'-actin was more resistant than that of beta- or gamma-actin to the multiple effects of CB. Second, beta'-actin bound less CB than beta- or gamma-actin. The functional alteration of beta'-actin in Cyt 1 cells was further supported by the observation that, although treatment of KB cells with CB increased the pool of unpolymerized actin, the same treatment did not affect the pool of unpolymerized actin in Cyt 1 cells, and that microfilaments of Cyt 1 cells were more resistant to the disrupting action of CB than those of KB cells. These results strongly suggest that the primary site of action of CB on cell motility processes is actin.     

Characterization of membrane-bound spermidine dehydrogenase of Citrobacter freundii.

Spermidine dehydrogenase found in the membrane fraction of Citrobacter freundii IFO 12681 was solubilized with Triton X-100 and further purified to homogeneity. The properties of the membrane enzyme were almost identical to those obtained from the soluble fraction of the organism with respect to molecular and catalytic properties. Thus, binding properties of the enzyme to the bacterial membrane were checked. The ratio of enzyme activity found in the soluble fraction to the membrane fraction was dependent on salt concentration during cell disruption. A hydrophobic interaction was largely involved in anchoring the enzyme to the membrane fraction. Purified spermidine dehydrogenase from the soluble fraction was readily adsorbed into the membrane fraction in the presence of salt. Spermidine dehydrogenase appeared to be a membrane-bound enzyme localized in the cytoplasmic membranes in a manner that makes a partial release of the enzyme possible during mechanical cell disruption. When spermidine oxidation was done with the resting cells of C. freundii, a stoichiometric formation of two reaction products, 1,3-diaminopropane and gamma-aminobutyraldeyde, was observed without any lag time. These facts indicate that the enzyme is localized on the outer surface of the cytoplasmic membranes or in the periplasmic space of the organism.     

Pollarding and a possible explanation of the neolithic elmfall

Sections of two pollarded parkelms (Ulmus glabra) from Damsgaard, Bergen, west Norway have been studied. Changes in annual ring-width are attributed partly to management, namely pollarding, and partly to pathogenic attacks, probably by Ceratocystis ulmi. The oldest attack on the trees dates back to 1826: so far the oldest known record of Dutch elm disease in Norway. Pollarding may be an important factor in attacks by the pathogen within parkelms. A possible relationship between pollarding, the pathogen and the Neolithic elmfall is suggested.     

Cure of osteopetrosis by injection of allogenic bone marrow into "op" rats treated with cyclosporin A

The cure of osteopetrosis by allogenic bone marrow injection has been obtained in "op" mutant rat kept under cyclosporin A treatment. This immunosuppressive agent able to prevent the rejection of transplanted cells does not impair the propriety of these cells to restore the bone resorption process in this severe osteopetrotic form.     

Effects of colcemid and taxol on microtubules and intermediate filaments in chick embryo fibroblasts

Reports on how changes in microtubule (MT) distribution or polymerization affect the distribution of intermediate filaments (IFs) differ. Therefore, we have used cytoimmunofluorescence techniques and electron microscopy to systematically examine and compare the arrangements of MTs and IFs in cultures of chick embryo fibroblasts under the following conditions: at different times during the cell cycle, in the presence of Colcemid or of taxol, in the presence of both drugs in succession or simultaneously in varying ratios, and during recovery from treatment with Colcemid or taxol. We have found that depolymerization of MTs by 1 microM Colcemid resulted in the rapid formation of massive IF-cables, structures distinct from "collapsed IFs" or "juxtanuclear coils." Neither the rapid formation of IF-cables nor their dispersion during recovery required protein synthesis. Cells treated with 10 microM taxol rapidly formed MT-bundles, as well as aggregates of intertwining IFs, termed "IF-skeins." MT-bundles and IF-skeins displayed strikingly complementary distributions. This reciprocal distribution of packed MTs and IFs was also obvious in untreated anaphase and telophase cells. When 10 microM taxol and 1 microM Colcemid were applied simultaneously, the complementary distributions of MT-bundles and IF-skeins mimicked those in taxol alone. This ability of taxol to block Colcemid's effects was concentration dependent. Decreasing the taxol: Colcemid ratio allowed the depolymerization of MTs, which correlated with the formation of IF-cables.     

The catalytic mechanism of bovine intestinal 5'-nucleotide phosphodiesterase. pH and inhibition studies

Extensive kinetic studies of bovine intestinal 5'-nucleotide phosphodiesterase as a function of pH have confirmed and amplified the catalytic mechanism previously proposed on the basis of isolation of a covalent phosphorylated intermediate (Landt, M., and Butler, L.G. (1978) Biochemistry 17, 4130-4135). An enzyme-ionizing group with apparent pKa = 6.85 controls the rate-determining step. Electrostatic interactions between anionic substrate and two or more ionic groups on the enzyme have a major role in substrate binding. Binding of strongly inhibitory 5'-AMP is controlled by an ionizing group, probably on the enzyme, with pKa less than or equal to 5.9. At pH 6.0, imidazole is a classic uncompetitive inhibitor, in agreement with independent evidence that it stabilizes the covalent intermediate form of the enzyme. KI values for phosphonate analogs, which are competitive inhibitors, indicate that phosphodiesterase binds its products and product analogs more strongly than it binds substrate analogs. Some of the results presented here can be interpreted as indicating that 5'-nucleotide phosphodiesterase is the evolutionary precursor of alkaline phosphatase, with which it has many structural and catalytic properties in common, and which is found in relatively large amounts in the same tissue.