Gibberellic acid stabilises microtubules in maize suspension cells to cold and stimulates acetylation of alpha-tubulin.
Gibberellic acid is known to stabilise microtubules in plant organs against depolymerisation. We have now devised a simplified cell system for studying this. Pretreatment of a maize cell suspension with gibberellic acid for just 3 h stabilised protoplast microtubules against depolymerisation on ice. In other eukaryotes, acetylation of alpha-tubulin is known to correlate with microtubule stabilisation but this is not established in plants. By isolating the polymeric tubulin fraction from maize cytoskeletons and immunoblotting with the antibody 6-11B-1, we have demonstrated that gibberellic acid stimulates the acetylation of alpha-tubulin. This is the first demonstrated link between microtubule stabilisation and tubulin acetylation in higher plants. (+info)
The L1 major capsid protein of human papillomavirus type 11 recombinant virus-like particles interacts with heparin and cell-surface glycosaminoglycans on human keratinocytes.
The L1 major capsid protein of human papillomavirus (HPV) type 11, a 55-kDa polypeptide, forms particulate structures resembling native virus with an average particle diameter of 50-60 nm when expressed in the yeast Saccharomyces cerevisiae. We show in this report that these virus-like particles (VLPs) interact with heparin and with cell-surface glycosaminoglycans (GAGs) resembling heparin on keratinocytes and Chinese hamster ovary cells. The binding of VLPs to heparin is shown to exhibit an affinity comparable to that of other identified heparin-binding proteins. Immobilized heparin chromatography and surface plasmon resonance were used to show that this interaction can be specifically inhibited by free heparin and dextran sulfate and that the effectiveness of the inhibitor is related to its molecular weight and charge density. Sequence comparison of nine human L1 types revealed a conserved region of the carboxyl terminus containing clustered basic amino acids that bear resemblance to proposed heparin-binding motifs in unrelated proteins. Specific enzymatic cleavage of this region eliminated binding to both immobilized heparin and human keratinocyte (HaCaT) cells. Removal of heparan sulfate GAGs on keratinocytes by treatment with heparinase or heparitinase resulted in an 80-90% reduction of VLP binding, whereas treatment of cells with laminin, a substrate for alpha6 integrin receptors, provided minimal inhibition. Cells treated with chlorate or substituted beta-D-xylosides, resulting in undersulfation or secretion of GAG chains, also showed a reduced affinity for VLPs. Similarly, binding of VLPs to a Chinese hamster ovary cell mutant deficient in GAG synthesis was shown to be only 10% that observed for wild type cells. This report establishes for the first time that the carboxyl-terminal portion of HPV L1 interacts with heparin, and that this region appears to be crucial for interaction with the cell surface. (+info)
Lactic acid polymers as biodegradable carriers of fluoroquinolones: an in vitro study.
A biodegradable polymer of DL-dilactide that facilitates release of ciprofloxacin or pefloxacin at levels exceeding MICs for the causative microorganisms of chronic osteomyelitis is described. Duration and peak of release were found to depend on the molecular weight of the polymer. Its characteristics make it promising for treating chronic bone infections. (+info)
Quantitative study of polymer conformation and dynamics by single-particle tracking.
We present a new method for analyzing the dynamics of conformational fluctuations of individual flexible polymer molecules. In single-particle tracking (SPT), one end of the polymer molecule is tethered to an immobile substratum. A microsphere attached to the other end serves as an optical marker. The conformational fluctuations of the polymer molecule can be measured by optical microscopy via the motion of the microsphere. The bead-and-spring theory for polymer dynamics is further developed to account for the microsphere, and together the measurement and the theory yield quantitative information about molecular conformations and dynamics under nonperturbing conditions. Applying the method to measurements carried out on DNA molecules provides information complementary to recent studies of single DNA molecules under extensional force. Combining high precision measurements with the theoretical analysis presented here creates a powerful tool for studying conformational dynamics of biological and synthetic macromolecules at the single-molecule level. (+info)
Purification and characterization of rat hippocampal CA3-dendritic spines associated with mossy fiber terminals.
We report a revised and improved isolation procedure for CA3-dendritic spines, most of them still in association with mossy fiber terminals resulting in a 7.5-fold enrichment over nuclei and a 29-fold enrichment over myelin. Additionally, red blood cells, medullated fibers, mitochondria and small synaptosomes were significantly depleted. We show by high resolution electron microscopy that this subcellular fraction contains numerous dendritic spines with a rich ultrastructure, e.g. an intact spine apparatus, membranous organelles, free and membrane-bound polyribosomes, endocytic structures and mitochondria. This improved experimental system will allow us to study aspects of post-synaptic functions at the biochemical and molecular level. (+info)
Adaptation of bulk constitutive equations to insoluble monolayer collapse at the air-water interface.
A constitutive equation based on stress-strain models of bulk solids was adapted to relate the surface pressure, compression rate, and temperature of an insoluble monolayer of monodendrons during collapse at the air-water interface. A power law relation between compression rate and surface pressure and an Arrhenius temperature dependence of the steady-state creep rate were observed in data from compression rate and creep experiments in the collapse region. These relations were combined into a single constitutive equation to calculate the temperature dependence of the collapse pressure with a maximum error of 5 percent for temperatures ranging from 10 degrees to 25 degrees C. (+info)
Actin polymerization: Where the WASP stings.
How do extracellular signals induce actin polymerization, as required for many cellular responses? Key signal transducers, such as the small GTPases Cdc42 and Rac, have now been shown to link via proteins of the WASP family to the Arp2/3 complex, which nucleates actin polymerization. (+info)
Structure and anticoagulant activity of sulfated fucans. Comparison between the regular, repetitive, and linear fucans from echinoderms with the more heterogeneous and branched polymers from brown algae.
Sulfated fucans are among the most widely studied of all the sulfated polysaccharides of non-mammalian origin that exhibit biological activities in mammalian systems. Examples of these polysaccharides extracted from echinoderms have simple structures, composed of oligosaccharide repeating units within which the residues differ by specific patterns of sulfation among different species. In contrast the algal fucans may have some regular repeating structure but are clearly more heterogeneous when compared with the echinoderm fucans. The structures of the sulfated fucans from brown algae also vary from species to species. We compared the anticoagulant activity of the regular and repetitive fucans from echinoderms with that of the more heterogeneous fucans from three species of brown algae. Our results indicate that different structural features determine not only the anticoagulant potency of the sulfated fucans but also the mechanism by which they exert this activity. Thus, the branched fucans from brown algae are direct inhibitors of thrombin, whereas the linear fucans from echinoderms require the presence of antithrombin or heparin cofactor II for inhibition of thrombin, as reported for mammalian glycosaminoglycans. The linear sulfated fucans from echinoderms have an anticoagulant action resembling that of mammalian dermatan sulfate and a modest action through antithrombin. A single difference of one sulfate ester per tetrasaccharide repeating unit modifies the anticoagulant activity of the polysaccharide markedly. Possibly the spatial arrangements of sulfate esters in the repeating tetrasaccharide unit of the echinoderm fucan mimics the site in dermatan sulfate with high affinity for heparin cofactor II. (+info)