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PubMed2002

Increased beta-actin and tubulin polymerization in regrowing axons: relationship to the conditioning lesion effect.

Spinal motor neurons of Sprague-Dawley rats were examined to determine which of the neuronal isoforms of actin (beta or gamma) upregulate following axon injury. In situ hybridization studies showed greater beta-actin mRNA levels but no change in gamma-actin mRNA levels-suggesting that axon regrowth utilizes beta-actin. We radiolabeled the newly synthesized actin and tubulin that are subsequently transported in the axon to the site of an axotomizing injury. This allowed us to evaluate changes in polymerization as new cytoskeletal elements approach the injury site. Previous studies had shown that the rate of the most rapid subcomponent of actin and tubulin transport (called SCb) accelerates following axotomy (J. Jacob and I. McQuarrie, J. Neurobiol. 22: 570-583, 1991). This rate increase is associated with an increased proportion of SCb tubulin and actin in polymer (vs monomer) form (J. Jacob and I. McQuarrie, J. Neurosci, Res. 43: 412-419, 1996). However, in that study newly synthesized proteins were radiolabeled at 7 days after axotomy-which is at the peak of increased protein synthesis. This time-course did not examine actin and tubulin that were already in transit in axons when the injury occurred. This actin and tubulin would enter the regrowing axons first. Here, we have radiolabeled newly synthesized proteins 3 days prior to axotomy. For beta-tubulin, the ratio of monomer to polymer was unaffected. For actin, the equilibrium shifted strongly toward polymerization. We conclude that the acceleration of axonal outgrowth seen after the second of two serial axotomies (the "conditioning lesion effect") is related to the ability of actin that is already in transit to polymerize in response to the first axotomy.

Actins↗
PubMed1998

A Comparative Study of Surface Fractality between Polymeric and Particulate Titania Aerogels

The surface fractality was studied for polymeric and particulate titania aerogels prepared via CO2 supercritical drying of titania lyogels. The surface fractal dimensions of these mesoporous aerogels were determined from their nitrogen adsorption isotherms by means of the Frenkel-Halsey-Hill method and the thermodynamic method. The results from these distinctly different methods have been found to be similar; the fractal surfaces of particulate aerogels are slightly more irregular than those of polymeric aerogels. The difference in the surface morphology between the two types of aerogels, observed with scanning electron microscopy, can be attributed to the difference between the proposed mechanisms for formation of polymeric and particulate lyogels. Copyright 1998 Academic Press. Copyright 1998Academic Press

Journal Article↗
PubMed1999

Encapsulation of Inorganic Particles by Dispersion Polymerization in Polar Media.

Following a previous work (Bourgeat-Lami, E., and Lang, J., J. Colloid Interface Sci. 197, 293 (1998)), encapsulation of silica beads has been achieved by dispersion polymerization of styrene in an aqueous ethanol medium using poly(N-vinyl pyrrolidone) as stabilizer. Silica beads, prepared according to the Stöber method, were coated prior to polymerization by grafting 3-(trimethoxysilyl)propyl methacrylate onto the surface. A great number of silica beads per composite particle were previously found using beads that had diameters between 49 and 120 nm. In the present work, larger silica beads with diameters between 191 and 629 nm are investigated. We demonstrate by transmission electron microscopy that, consequently, only a small number of silica beads are contained in the composite particles. By counting the composite particles containing precisely zero, one, two, three, four, and more than four silica beads, it clearly appears that the encapsulation of only one silica bead can be obtained simply by increasing the size of the beads. Under our experimental conditions, the optimal bead diameter for achieving composite particles containing only one silica bead turns out to be around 450 nm. We show that increasing the silica bead size above this value results in an increased number of composite particles without silica beads. In contrast, the number of composite particles with two, three, four, or more than four silica beads increases with decreasing silica bead size. In addition to the above variations in composition of the composite particles, changes in particle shapes were also observed as a function of the size of the silica beads and the styrene concentration in the polymerization medium. Hypotheses concerning these variations are presented. Copyright 1999 Academic Press.

Journal Article↗
PubMed2001

Microstructured Polyacrylamide Hydrogels Prepared Via Inverse Microemulsion Polymerization.

The synthesis by a two-stage polymerization process of microstructured polyacrylamide hydrogels with large swelling capacity and improved mechanical properties is reported. First, crosslinked polyacrylamide particles of nanosize scale are made by inverse microemulsion polymerization. These particles are then dried and redispersed in an aqueous solution of acrylamide and polymerized in the presence of a crosslinking agent. The microstructured hydrogels, in contrast to transparent conventional polyacrylamide hydrogels, are translucid due to the presence of the dispersed particles. The swelling capacity of these hydrogels increases as the particle content increases and their Young and elastic moduli (at equilibrium swelling) diminish only slightly. Mechanical tests disclose that the microstructured hydrogels have larger Young moduli than conventional hydrogels with an identical degree of swelling. Copyright 2001 Academic Press.

Journal Article↗
PubMed2001

The Level of Hydrophobic Substitution and the Molecular Weight of Amphiphilic Poly-L-lysine-Based Polymers Strongly Affects Their Assembly into Polymeric Bilayer Vesicles.

With the aim of producing new materials for drug and gene delivery, the variables associated with the preparation of poly-L-lysine-based vesicles were investigated. Amphiphilic poly-L-lysine graft copolymers with varying levels of grafted methoxypolyethylene glycol (mPEG) and palmitic acid were synthesized using two-step grafting reactions of the macromonomer, mPEG-p-nitrophenyl carbonate (mPEG, MW=5,000), and palmitic acid N-hydroxysuccinimide ester onto poly-L-lysine hydrobromide (MW=4,000 and 19,600). Polymers were characterized by gel permeation chromatography/light scattering. (1)H NMR, and an assay for unreacted varepsilon-amino groups. Polymeric unilamellar vesicles were produced by probe sonication of the amphiphilic poly-L-lysine-based polymers in the presence of cholesterol. Vesicles were characterized by electron microscopy and photon correlation spectroscopy. Vesicle formation was favored by a low molecular weight and a low level of palmitoyl substitution. A vesicle formation index has been derived, F ~ H/L DP, where H is the %molar level of unreacted L-lysine units, L is the %molar level of substituted palmitoyl units, and DP is the square root of the degree of polymerization of the polymer. Additionally, the size of these vesicles may be controlled by controlling the initial molecular weight of the parent poly-L-lysine/resulting amphiphilic polymer. Hence, amphiphilic poly-L-lysine-based polymers of molecular weight=89,000 and 25,000 produced polymeric vesicles of z-average mean diameter 570 nm and 252 nm, respectively. Vesicle encapsulation efficiency for the hydrophilic macromolecule, fluorescein isothiocyanate-dextran (MW=4,400), increased with vesicle size. Copyright 2001 Academic Press.

Journal Article↗
PubMed1996

Roles of alpha 114 and beta 87 amino acid residues in the polymerization of hemoglobin S: implications for gene therapy.

Three novel recombinant mutants of sickle hemoglobin (Hb S, beta 6Glu-->Val) have been constructed to assess the role of proline at alpha 114 and threonine at beta 87 in the polymerization of deoxygenated Hb S. Using the hemoglobin expression system (pHE2) designed in our laboratory, four plasmids were expressed separately in Escherichia coli to produce the four recombinant hemoglobins: r Hb S (beta 6Glu-->Val); r Hb S-Chiapas (beta 6Glu-->Val, alpha 114Pro-->Arg); r Hb S-D-Ibadan (beta 6Glu-->Val, beta 87Thr-->Lys); and r Hb S-Chiapas-D-Ibadan (beta 6Glu-->Val, alpha 114Pro-->Arg, beta 87Thr-->Lys). The structural features of these four recombinant hemoglobins were analyzed by proton nuclear magnetic resonance spectroscopy, and were found to be similar to those of human normal adult hemoglobin (Hb A) under identical conditions. The recombinant hemoglobins were further investigated by measuring the oxygen-binding properties, which were found to be comparable to those of Hb A. Delay-time gelation studies of the three mutants of r Hb S were carried out in 1.8 M potassium phosphate (pH 7.34) by a temperature jump from 4 degrees C to 30 degrees C and an increase in delay time over that of r Hb S was observed, as well as an overall decrease in the polymerization of these three mutants of Hb S. A more detailed and quantitative investigation has also been carried out to determine the equilibrium solubility (Csat) in 0.1 M potassium phosphate (pH 7.35) at 25 degrees C of the three Hb S mutants as well as of mixtures of these mutants with Hb S versus mixtures of fetal hemoglobin (Hb F) and Hb A with Hb S. The inhibition of polymerization demonstrated in these experiments suggests that the interactions involving the two amino acid residues alpha 114Pro and beta 87Thr are very important to the formation of Hb S polymer, and modification of these amino acids results in an anti-sickling potential. Of particular interest is the inhibitory effect of alpha 114Pro-->Arg, which offers a novel opportunity to use an alpha-chain construct, in addition to a beta-chain construct in the same vector, in gene therapy for sickle cell anemia, with the objective of modifying a larger number of hemoglobin tetramers at a given level of expression.

Adult↗
PubMed2000

Polymerization, three-dimensional structure and mechanical properties of Ddictyostelium versus rabbit muscle actin filaments.

To assess more systematically functional differences among non-muscle and muscle actins and the effect of specific mutations on their function, we compared actin from Dictyostelium discoideum (D-actin) with actin from rabbit skeletal muscle (R-actin) with respect to the formation of filaments, their three-dimensional structure and mechanical properties. With Mg(2+) occupying the single high-affinity divalent cation-binding site, the course of polymerization is very similar for the two types of actin. In contrast, when Ca(2+ )is bound, D-actin exhibits a significantly longer lag phase at the onset of polymerization than R-actin. Crossover spacing and helical screw angle of negatively stained filaments are similar for D and R-F-actin filaments, irrespective of the tightly bound divalent cation. However, three-dimensional helical reconstructions reveal that the intersubunit contacts along the two long-pitch helical strands of D-(Ca)F-actin filaments are more tenuous compared to those in R-(Ca)F-actin filaments. D-(Mg)F-actin filaments on the other hand exhibit more massive contacts between the two long-pitch helical strands than R-(Mg)F-actin filaments. Moreover, in contrast to the structure of R-F-actin filaments which is not significantly modulated by the divalent cation, the intersubunit contacts both along and between the two long-pitch helical strands are weaker in D-(Ca)F-actin compared to D-(Mg)F-actin filaments. Consistent with these structural differences, D-(Ca)F-actin filaments were significantly more flexible than D-(Mg)F-actin. Taken together, this work documents that despite being highly conserved, muscle and non-muscle actins exhibit subtle differences in terms of their polymerization behavior, and the three-dimensional structure and mechanical properties of their F-actin filaments which, in turn, may account for their functional diversity.

Actin Cytoskeleton↗
PubMed1998

Rotational Motion of a Solute Molecule in a Highly Viscous Liquid Studied by 13C NMR: 1,3-Dibromoadamantane in Polymeric Chlorotrifluoroethene

The viscosity-dependent retarding effect of a polymeric solvent on the rotation of small solute molecules is investigated by 13C NMR relaxation measurements. It is found that the relaxation data of 1,3-dibromoadamantane in highly viscous polymeric chlorotrifluoroethene can be explained neither by isotropic nor by realistic anisotropic tumbling in a single environment. The experimental data are rationalized in terms of fast exchange between at least two environments with correlation times differing by up to two orders of magnitude. The study shows that a uniform retardation of molecular tumbling by a polymeric solvent, desirable for shifting the NMR observation window in studies of intramolecular mobility, is not always feasible. Copyright 1998 Academic Press.

Journal Article↗
PubMedDate not supplied

Intermediate filament protein polymerization: molecular analysis of Drosophila nuclear lamin head-to-tail binding.

Polymerization of intermediate filament proteins results from interactions among several distinct binding sites on the constituent proteins. Nuclear lamin head-to-tail polymers arise from one such interaction. We studied this binding using Drosophila lamin Dm0-derived fragments containing either the NH2-terminal or COOH-terminal binding site with a combination of co-immunoprecipitation, yeast two-hybrid, analytical ultracentrifugation, and electron microscopic assays. Fragment binding and full-length lamin head-to-tail polymerization were similar to each other in morphology, buffer requirements, and inhibition after phosphorylation with cdc2 kinase. Deletion analysis localized the binding sites to the ends of the rod domain that are highly conserved among all intermediate filament proteins. Point mutants, defective in binding, were isolated. Two were identical to point mutations in specific human keratin genes known to affect keratin assembly and to cause genetic skin diseases. Results further indicate that the binding sites only function in specific sequence contexts and that binding can be modulated by elements outside the binding sites (like the cdc2 kinase phosphorylation site). Our data indicate that one type of interaction in intermediate filament protein polymerization is the longitudinal binding of dimers via the conserved end segments of the coiled-coil rod domain.

Amino Acid Sequence↗
PubMed1993

Purification and characterization of recombinant polymeric hemoglobin P1 of Glycera dibranchiata.

The apoprotein of component P1 of the polymeric fraction of the intracellular hemoglobin of the marine polychaete Glycera dibranchiata has been expressed at a high level in Escherichia coli. The expressed globin was reconstituted with heme and purified. The N-terminal sequence of the recombinant P1 is identical to the cDNA-derived sequence of cloned P1 (Zafar et al., Biochem. Biophys. Acta, 1041, 117-123, 1990). Gel filtration, SDS-PAGE, optical spectra over the range 200-650 nm, and circular dichroism over the range 200-250 nm of the purified recombinant P1 were very similar to the polymeric fraction of native Glycera hemoglobin. The molar ellipticity at 222 nm provided an estimate of 77% for the alpha-helical content of the recombinant P1, in excellent agreement with that calculated from the crystal structure of Glycera monomeric component M-II. Although the oxygen binding affinity of the recombinant P1 is higher than that of the polymeric fraction of Glycera hemoglobin (3-4 torr vs 7-13 torr), which consists of at least six different single-chain hemoglobins, the Hill coefficient is lower (1.0-1.2 vs 1.2-1.4).

Animals↗
PubMed1994

Focal contact assembly through cytoskeletal polymerization: steady state analysis.

For many cell types, initial receptor-mediated attachment to a ligand-coated surface is followed by the formation of focal contacts--strong, specialized, discrete adhesive connections between cell and substrate in which receptors are clustered and simultaneously linked to extracellular ligand and cytoskeletal proteins. Since adhesion affects many aspects of cellular physiology including growth, differentiation, and motility, understanding the biochemical factors which regulate focal contact assembly should enhance our understanding of these phenomena. In this paper, we present a mathematical model to examine how receptor-ligand, receptor-cytoskeleton, and cytoskeleton-cytoskeleton interactions affect the formation of receptor clusters which serve as precursors to mature focal contacts. Receptor clustering is presumed to occur through self-recognition of cytoskeletal elements which induce the polymerization of ligand-receptor-cytoskeleton complexes. Polymerization only occurs when the ligand density is above a critical value and a decrease in the receptor-ligand affinity shifts the critical ligand density to higher values. While cytoskeletal protein expression and receptor-cytoskeleton affinity influence the concentration of monomeric complexes, the formation of polymeric ligand-receptor-cytoskeleton aggregates is most sensitive to changes in the self-association affinity between cytoskeletal proteins. We find that a 100-fold enhancement in the affinity between cytoskeletal elements can produce a substantial increase in the total fraction of adhesion receptors associated with focal contact precursors (from 5% to over 90%). Our results suggest that under physiological conditions, cellular control of focal contact assembly most likely occurs through modulation of specific cytoskeletal proteins to solidify cytoskeleton-cytoskeleton connections within precursor focal contact structures.

Amino Acid Sequence↗
PubMed1980

Effects of the principal hydroxy-metabolites of benzene on microtubule polymerization.

The principal hydroxy-metabolites of benzene - phenol, catechol and hydroquinone - possess characteristics and produce toxicity similar to those reported for certain inhibitors of microtubule polymerization. In this study we examined the effects of phenol, catechol and hydroquinone on purified microtubule polymerization and the decay of tubulin-colchicine binding activity. Hydroquinone, but not catechol or phenol, inhibited microtubule polymerization and accelerated the decay of tubulin-colchicine binding activity. The latter effect was shown to be dependent on the concentration of GTP. Hydroquinone did not directly complex with GTP or ATP but bound to the high molecular weight fraction of tubulin. concentration ratios of hydroquinone to tubulin resulting in altered activity were low, suggesting a specific interaction, presumably at the tubulin-GTP binding site. The acceleration of tubulin-colchicine binding activity decay was completely prevented under anaerobic conditions, indicative of an oxidative mechanism. These studies suggest that hydroquinone, which auto-oxidizes, may interfere with microtubule function, nucleotide binding or both and that this mechanism may be involved in eliciting the wide range of cytoskeletal-related abnormalities observed in cells exposed to benzene in vivo or its metabolites in vitro.

Animals↗
PubMed1989

Acute experimental glomerulonephritis induced by the glomerular deposition of circulating polymeric IgA-concanavalin A complexes.

The perfusion of polymeric or secretory IgA-Concanavalin A complexes into the aorta of rats led to a mannose-dependent binding of both IgA and lectin to the glomerular capillary wall, as shown by double immunolocalization experiments, by quantitative analysis of the amount of radiolabelled complexes bound per g of kidney, and by blocking experiments with the corresponding carbohydrate. Rats injected with amounts of those complexes as low as 500 micrograms developed, one hour later, a focal and segmental proliferative glomerulonephritis characterized by the deposition of injected complexes and of rat C3 and rat fibrin/fibrinogen in most glomeruli; focal thrombosis and small areas of necrosis in 10 to 15% of glomeruli, confined to the periphery of a single lobule of the tuft and segmental infiltration of these glomeruli by polymorphonuclear leucocytes and platelets. At the same time, many mesangial cells exhibited a hyperactive appearance, and red blood cells were noted in tubular lumens. In contrast, rats similarly injected with either monomeric IgA-ConA complexes, multimeric or secretory IgA-peanut agglutinin complexes or polymeric or monomeric IgA aggregates of comparable apparent molecular weight did not develop obvious glomerular lesions within one hour. The data indicate that performed polymeric IgA-ConA complexes can specifically bind to glomerular structures in vivo and trigger acute glomerular lesions locally, analogous to those observed in some glomerular diseases associated with a cryoglobulinaemia.

Acute Disease↗
PubMed1990

Hyperammonemia induces polymerization of brain tubulin.

Rats were made hyperammonemic by feeding them a diet containing ammonium acetate. The tubulin content in their brain increased greater than or equal to 30% after 20 days on the diet. All the increase was found in polymerized tubulin; no increase in free tubulin was noted. When rats on the ammonium diet were then fed the standard diet, the tubulin increased slightly on the first day but decreased markedly on the second day, reaching control values on the third day. It should be noted that brain tubulin synthesis, was not reduced on the first day of feeding the standard diet but was markedly inhibited (to approximately 40% of control) on the second day, returning to control values on the third day. On the first day of refeeding there is a remarkable disassembly of microtubules with a large, proportional increase (approximately 50%) of free tubulin. Both free and polymerized tubulin levels returned to control values on the third day. These results indicate that in hyperammonemia changes in the degree of polymerization of tubulin preceded those in tubulin synthesis.

Ammonia↗
PubMed1994

In vivo and in vitro effects of diisopropyl phosphorofluoridate (DFP) on the rate of hen brain tubulin polymerization.

Diisopropyl phosphorofluoridate (DFP) produces organophosphorus ester-induced delayed neurotoxicity (OPIDN) in sensitive species. We have investigated the in vivo and in vitro effects of DFP on hen brain tubulin polymerization. Hens were treated with a single dose of DFP (1.7 mg/kg, sc.), and were sacrificed after 18-21 days. Tubulin from DFP-treated hen brains showed small but significant decrease (14.42%) in the rate of polymerization and 11.05% decrease in rise in O.D. at 340 nm in 30 min. DFP in vivo treatment also resulted in decreased concentration of tau and an enhanced concentration of two peptides (45 kDa, 35 kDa) in the brain supernatant. These peptides seemed to be the degradation products of MAP-2. The decrease in the rate of brain tubulin polymerization in treated hens is consistent with neurochemical alterations and the focal degeneration and aggregation of these filamentous structures in OPIDN.

Animals↗
PubMed1975

Polymerization of amino acid methyl esters via their copper complexes.

Polymerization of glycine methyl ester catalyzed by cupric ions in organic solvents yields oligoglycines with a degree of polymerization up to none. With a trifunctional amino acid, the yield and degree of polymerization were much lower. Extension of this reaction to an aqueous medium was not successful even when copper ions were complexed with substances like montmorillonite or fatty acids. The prebiotic significance of this reaction is discussed.

Amino Acids↗
PubMed1990

Effects of marginal zinc deficiency on microtubule polymerization in the developing rat brain.

One of the possible mechanisms that has been proposed to underlie the deleterious effects of zinc deficiency on brain development is an impairment in the normal formation of the cytoskeletal network. In the current study, in vivo microtubule polymerization was characterized in brain supernatant fluids, from 20-d-old pups whose dams were fed diets containing control (50 micrograms zinc/g) or marginal levels of zinc (10 micrograms zinc/g) throughout pregnancy and lactation. Pup brain and body weights were similar between the groups; however, plasma zinc concentrations were lower (27%) in pups fed the marginal zinc diet than in controls. Tubulin concentrations in 100,000 g brain supernates were similar between the groups; however, tubulin polymerization in the brain supernates was significantly lower in pups fed the marginal zinc diet compared to controls. Primarily, the early events of polymerization were affected; the lag period of the reaction was doubled, and the initial velocity was slower (26%) in supernates from pups fed the marginal zinc diet than in controls. These findings support the idea that some of the negative effects of marginal zinc deficiency on brain development and function may be mediated by an alteration in microtubule formation.

Animals↗
PubMed2001

Fractalkine induces chemotaxis and actin polymerization in human dendritic cells.

OBJECTIVE AND DESIGN: Dendritic cells (DCs) are considered as the principle initiators of immune responses by virtue of their ability to migrate into target sites, process antigens and activate naive T cells. Here, the chemotactic activity and intracellular signaling of fractalkine was analyzed and compared to well known chemotaxins. METHODS: The mRNA-expression of G protein-coupled CX3CR1 was analyzed by RT-PCR. Chemotaxis was measured in 48-well Boyden chambers and actin polymerization by flow cytometry. RESULTS: The mRNA-expression of CX3CR1 in immature and mature DCs was revealed. Fractalkine elicited actin polymerization and chemotaxis in a dose-dependent manner in DCs independent of their state of maturation. CONCLUSIONS: These results show that immature and mature DCs express mRNA for the CX3CRI and that fractalkine induces chemotaxis and migration associated actin polymerization in immature as well as in mature DCs, contrasting with the action of other chemokines such as RANTES or MIP-3beta which act only on distinct maturation states of DCs.

Actins↗