The effect of medium viscosity on actin polymerization

A comparative study of the influence of two factors--excluded volume effect, and medium viscosity--on actin polymerization was carried out. Dextrane--500 (1%), poly(ethylene)glycol--6000 (2.7%), Ficol (1.8%), methyl cellulose (0.04%), saccharose (10%), and glycerine (10%) were used for creating a viscous medium. The concentrations of these agents in actin solution made approximately the same viscosity commensurable with the cytoplasm viscosity. By this it was possible to bring conditions of actin polymerization in vitro closer to those in vivo. It is shown that the medium viscosity in contrast to the excluded volume effect, prolongs the nucleation phase during actin polymerization. A conclusion is made that in the cell both factors can be involved in the regulation of actin filament formation.     

The influence of poly(ethylene glycol) 6000 on spermine-induced aggregation of liposomes.

Poly(ethylene glycol) 6000 affected the aggregation of mixed liposomes induced by spermine. It lowered the concentration of spermine causing 50% maximal aggregation, accelerated the rate and increased the extent of aggregation. The effect was inversely proportional to the density of the acidic phospholipid in the vesicles. These effects were not due either to poly(ethylene glycol) 6000-induced permanent structural modification of the liposome or increased binding of spermine to the vesicles. These findings are discussed in relation to a decreased hydration force caused by the ability of poly(ethylene glycol) 6000 to alter the water of hydration of the phospholipid polar groups in the liposome.     

Immunological properties of uricase conjugated to neutral soluble polymers.

For a comparative study of immunological properties of protein-polymer conjugates, uricase was modified with (a) poly(N-vinylpyrrolidone) 6000 Da, (b) poly(N-acriloylmorpholine) 6000 Da, (c) branched monomethoxypoly(ethylene glycol) 10000 Da, and (d) linear monomethoxypoly(ethylene glycol) 5000 Da. Spectroscopic studies performed by UV, fluorescence, and circular dichroism did not show any relevant difference in protein conformation among the native and the conjugates. Immunological studies showed that both uricase antigenicity and immunogenicity were altered by polymer conjugation to an extent that depended upon the polymer composition; in particular, monomethoxypoly(ethylene glycol) 10000 Da remarkably reduced the protein antigenicity, while unexpectedly, the poly(N-vinylpyrrolidone) derivative presented higher antigenicity than the native protein. In Balb/c mice, the native protein elicited a rapid and intense immunoresponse whereas all the conjugates induced a lower production of anti-native uricase antibodies. The rank order of immunogenicity was native uricase > uricase-poly(N-vinylpyrrolidone) > or = uricase-poly(N-acriloylmorpholine) > uricase-monomethoxypoly(ethylene glycol) 5000 Da > uricase-monomethoxypoly(ethylene glycol) 10000 Da. The four conjugates also induced anti polymer immunoresponse. Anti poly(N-vinylpyrrolidone) and anti poly(N-acriloylmorpholine) antibodies were generated from the first immunization while low levels of anti polymer antibodies were found with both poly(ethylene glycol) conjugates only after the second immunization.     

New ground for organic catalysis: a ring-opening polymerization approach to hydrogels

Herein, we describe an organocatalytic living polymerization approach to network and subsequent hydrogel formation. Cyclic carbonate-functionalized macromolecules were ring-opened using an alcoholic initiator in the presence of an organic catalyst, amidine 1,8-diazabicyclo[5.4.0]undec-7-ene. A model reaction for the cross-linking identified monomer concentration-dependent reaction regimes, and enhanced kinetic control was demonstrated by introducing a co-monomer, trimethylene carbonate. The addition of the co-monomer facilitated near-quantitative conversion of monomer to polymer (>96%). Resulting poly(ethylene glycol) networks swell significantly in water, and an open co-continuous (water-gel) porous structure was observed by scanning electron microscopy. The organocatalytic ring-opening polymerization of cyclic carbonate functional macromonomers using alcoholic initiators provides a simple, efficient, and versatile approach to hydrogel networks.     

Catalytic activity of non-cross-linked microcrystals of aspartate aminotransferase in poly(ethylene glycol).

The molar activity of crystalline mitochondrial aspartate aminotransferase is decreased to 10% of that of the enzyme in solution. The activity was measured in suspensions of non-cross-linked microcrystals (average dimensions 22 microns X 5 microns X 0.8 microns) in 30% (w/v) poly(ethylene glycol). Kinetic tests ruled out the possibility that diffusion of the substrate in the crystals is rate-limiting. The observed decrease in catalytic efficiency can be attributed exclusively to crystal-packing effects. A direct inhibition by poly(ethylene glycol) is excluded because poly(ethylene glycol), with average Mr 6000, cannot penetrate the liquid channels of the crystals, owing to its large Stokes radius. The crystals examined were triclinic and of the same habit as those used for high-resolution X-ray-crystallographic analysis [Ford, Eichele & Jansonius (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 2559-2563]. The catalytic competence of crystalline aspartate aminotransferase confirms the relevance of the spatial model of this protein for the elucidation of its mechanism of action.     

Enhancement of viral fusion by nonadsorbing polymers.

Nonadsorbing polymers such as dextran and poly(ethylene glycol) enhance binding as well as extents of fusion of influenza virus with erythrocytes. Kinetics and extent of viral membrane fusion were measured using an assay based on lipid mixing of a fluorescent dye. The effects of nonadsorbing polymers were in the concentration range from 0 to 10 wt%, far below the concentration required to overcome hydration repulsion forces. The enhancing effects were dependent on the molecular weight of nonadsorbing polymer, and only occurred at molecular weight > 1500; this links the phenomena we observe to the so-called "excluded volume effect" of nonadsorbing polymers. The time delay between triggering and the onset of influenza virus fusion was significantly reduced in the presence of nonadsorbing polymers. High molecular weight poly(ethylene glycol) also induced fusion of vesicular stomatitis virus with intact erythrocytes, which do not serve as target of vesicular stomatitis virus fusion in the absence of the polymer. The forces between membranes which determine rate-limiting processes in viral fusion and how they are affected by nonadsorbing polymers are discussed.     

Photocurable biodegradable liquid copolymers: synthesis of acrylate-end-capped trimethylene carbonate-based prepolymers, photocuring, and hydrolysis

Various photocurable liquid biodegradable trimethylene carbonate (TMC)-based (co)oligomers were prepared by ring-opening (co)polymerization of TMC with or without L-lactide (LL) using low molecular weight poly(ethylene glycol) (PEG) (mol wt 200, 600, or 1000) or trimethylolpropane (TMP) as an initiator. Resultant (co)oligomers were pastes, viscous liquids, or liquids at room temperature, depending on the monomer composition and monomer/initiator ratio. Liquid (co)oligomers were subsequently end-capped with acrylate groups. Upon visible-light irradiation in the presence of camphorquinone as a radical generator, rapid liquid-to-solid transformation occurred to produce photocured solid. The photocuring yield increased with photoirradiation time, photointensity, and camphorquinone concentration. The photocured polymers derived from low molecular weight PEG (PEG200) and TMP exhibited much reduced hydrolysis potential compared with PEG1000-derived polymers in terms of weight loss, water uptake, and swelling depth. Force-distance curve measurements by nanoindentation using atomic force microscopy clearly showed that Young's moduli of the photocured polymer films decreased with increasing hydrolysis time. Their potential biomedical applications are discussed.     

Bound-cation exchange affects the lag phase in actin polymerization

The delay or lag phase at the onset of polymerization of actin by neutral salt is generally attributed to an actin nucleation reaction. However, when nucleation is circumvented by the use of phalloidin-stabilized nuclei, a lag phase persists when Ca2+-containing actin is polymerized with MgCl2. Pretreatment of actin with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and/or Mg2+ shortens or eliminates this lag phase, suggesting that exchange of the actin-bound divalent cation occurs during this nucleation-independent lag phase. Measurement of the actin-bound cation initially and after brief incubation with EGTA/Mg2+ directly verifies that Mg2+ has replaced Ca2+ as the actin-bound cation, producing a highly polymerizable Mg2+-actin species. Bound-cation exchange prolongs the lag phase in actin polymerization and probably explains what has been termed the monomer activation step in actin polymerization.     

Controlling network structure in degradable thiol-acrylate biomaterials to tune mass loss behavior

Degradable thiol-acrylate materials were synthesized from the mixed-mode polymerization of a diacrylate poly(ethylene glycol) (PEG) monomer with thiol monomers of varying functionalities to control the final network structure, ultimately influencing the material's degradation behavior and properties. The influence of the concentration of thiol groups and monomer functionality on the mass loss profiles were examined experimentally and theoretically. Mass loss behavior was also predicted for networks with varying extents of cyclization, PEG molecular weight, and backbone chain length distributions. Experimental results indicate that increasing the thiol concentration from 10 to 50 mol % shifted the reverse gelation time from 35 to 8 days and the extent of mass loss at reverse gelation from 75 to 40%. Similarly, decreasing the thiol functionality from 4 to 1 shifted the reverse gelation time from 18 to 8 days and the mass loss extent at reverse gelation from 70 to 45%.     

Thermal denaturation of chromatin and lysine copolymer-DNA complexes. Effects of ethylene glycol.

Chromatin was thermally denatured in the presence and absence of 1M ethylene glycol using a technique whereby both the hyperchromism and ellipticity are monitored simultaneously. Model complexes containing poly(L -Lys) or poly(L -Lys, L -Ala, Gly) and DNA were similarly melted in order to assist in interpreting the chromatin results. In both cases a general pattern emerged whereby ethylene glycol perturbed the resultant melting profile, showing increased hyperchromicity, ellipticity, and premelt slope. In addition, ethylene glycol destabilizes and reduces the high melting region of polypeptide-bound DNA and the extent of higher ordered structure in model complexes and chromatin. These results emphasize the importance of hydrophobic forces in polypeptide–polypeptide and polypeptide–DNA interactions.     

Osmoelastic coupling in biological structures: formation of parallel bundles of actin filaments in a crystalline-like structure caused by osmotic stress

A high molecular weight inert molecule, poly(ethylene glycol) (PEG), or a soluble protein, ovalbumin, causes parallel bundles of actin filaments in a crystalline-like structure under physiological conditions of ionic compositions and pH. The bundle formation depends on the molecular weight of PEG, and a larger molecular weight of PEG can make the bundle at a lower concentration. Actin bundle formation has a discrete dependence on the concentration of PEG. The light scattering following PEG-induced bundle formation increased abruptly at 4.5% (w/w) PEG 6000, while at concentrations less than or equal to 4.0% (w/w) no increase was observed. Labeling actin filaments with heavy meromyosin indicated that the polarity of the filament in the bundle is random. The PEG-induced bundle formation depends on the ionic strength of the solutions and also the concentration of the filament, showing that a higher concentration of PEG was required at lower ionic strength or a lower concentration of the filament. The results described above cannot be explained on the basis of the postulation that the direct binding of PEG molecules to the actin filaments may cause bundle formation. Alternatively, the mechanism can be explained reasonably by the theory of osmoelastic coupling based on preferential exclusion of PEG molecules from the filament surface. High molecular weight molecules such as PEG should be preferentially excluded from the region adjacent to the actin filaments (exclusion layer) by steric hindrance, thereby making imbalance of osmolarity between the bulk and the exclusion layer. This imbalance puts an osmotic stress on the actin filament.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rationalization of the effects of compatible solutes on protein stability in terms of thermodynamic nonideality.

Inhibition by compatible solutes such as proline and glycine betaine of the rate of coagulation, at 60 degrees C, of bovine serum albumin in 0.1 M acetate buffer, pH 5, is used as a model system to substantiate the concept that the production of high concentrations of osmolytes by plants and other organisms in response to stress (e.g., drought) results in stabilization of native enzyme structures via nonspecific excluded volume effects. The paradoxical situation whereby this effect of compatible solutes counters to some extent the protein-precipitating effect of poly(ethylene glycol) is also seemingly resolved.     

Thermal stability of proteins in the presence of poly(ethylene glycols)

Thermal unfolding of ribonuclease, lysozyme, chymotrypsinogen, and beta-lactoglobulin was studied in the absence or presence of poly(ethylene glycols). The unfolding curves were fitted to a two-state model by a nonlinear least-squares program to obtain values of delta H, delta S, and the melting temperature Tm. A decrease in thermal transition temperature was observed in the presence of poly(ethylene glycol) for all of the protein systems studied. The magnitude of such a decrease depends on the particular protein and the molecular size of poly(ethylene glycol) employed. A linear relation can be established between the magnitude of the decrease in transition temperature and the average hydrophobicity of these proteins; namely, the largest observable decrease is associated with the protein of the highest hydrophobicity. Further analysis of the thermal unfolding data reveals that poly(ethylene glycols) significantly effect the relation between delta H degrees of unfolding and temperature for all the proteins studied. For beta-lactoglobulin, a plot of delta H versus Tm indicates a change in slope from a negative to a positive value, thus implying a change in delta Cp in thermal unfolding caused by the presence of poly(ethylene glycols). Results from solvent-protein interaction studies indicate that at high temperature poly(ethylene glycol) 1000 preferentially interacts with the denatured state of protein but is excluded from the native state at low temperature. These observations are consistent with the fact that poly(ethylene glycols) are hydrophobic in nature and will interact favorably with the hydrophobic side chains exposed upon unfolding; thus, it leads to a lowering of thermal transition temperature.     

Biodegradable amphiphilic block copolymers bearing protected hydroxyl groups: synthesis and characterization

A new biodegradable amphiphilic block copolymer, poly(ethylene glycol)-b-poly(L-lactide-co-9-phenyl-2,4,8,10-tetraoxaspiro[5,5]undecan-3-one) [PEG-b-P(LA-co-PTO)], was successfully prepared by ring-opening polymerization (ROP) of L-lactide (LA) and functionalized carbonate monomer 9-phenyl-2,4,8,10-tetraozaspiro[5,5]undecan-3-one (PTO) in the presence of monohydroxyl poly(ethylene glycol) as macroinitiator using Sn(Oct)2 as catalyst. NMR, FT-IR, and GPC studies confirmed the copolymer structure. It could self-assemble into micelles in aqueous solution with critical micelle concentration (CMC) in the magnitude of mg/L, which changed with the composition of the copolymer. After catalytic hydrogenation, copolymers with active hydroxyl groups were obtained. Adhesion and proliferation of Vero cells on the copolymer films showed that the synthesized copolymers were good biocompatible materials. In vitro degradation of the copolymer before and after deprotection was investigated in the presence of proteinase K. The free hydroxyl groups on the copolymers were capable of further modification with biotin. This new amphiphilic block copolymer has great potential for both drug encapsulation and conjugate because of its low CMC and the presence of active hydroxyl groups.     

Synthesis of novel cationic poly(ethylene glycol) containing lipids.

In this paper, the synthesis of novel divalent cationic lipids with poly(ethylene glycol) segments is described. The lipids consist of an unsaturated double-chain hydrophobic moiety based on 3, 4-dihydroxy benzoic acid, attached to a hydrophilic poly(ethylene glycol) spacer which contains a divalent cationic end group. As poly(ethylene glycol) spacers monodisperse triethylene glycol and telechelic poly(ethylene glycol)s with an average degree of polymerization of 9, 23, and 45 were used. The divalent cationic end group was attached by coupling a protected dibasic amino acid to the PEG spacer and following cleavage of the protecting groups. These novel class of cationic lipids is of particular interest for nonviral gene delivery applications.     

Stimulation of ligninolytic enzyme production in Phanerochaete chrysosporium by polyoxyalkanes

AIMS: Poly(ethylene glycol) (PEG) and some substances similar to PEG in chemical structure were tested as stimulators of ligninolytic enzyme production in shaken culture of Phanerochaete chrysosporium. METHODS AND RESULTS: The substances that caused high enzymatic activity were linear polymers [poly(ethylene glycol), poly(propylene glycol), poly(butylene glycol) and poly(vinyl alcohol)] and cyclic polymers (crown ether). They can have terminal groups other than -OH [PEG (di)methyl ether, PEG sulphate, PEG derivative with the amino group and xanthate]. The maximum lignin peroxidase activities were compared with the surface pressure caused by the stimulator. Addition of polymers composed of charged monomer units did not increase the enzymatic activity and the fungi did not grow at all on addition of polymers having a fixed positive charge. CONCLUSIONS: Lignin peroxidase activity was increased after the addition of polymers with uncharged monomer units. It was higher and its maximum was reached in a shorter time on addition of polymers with higher molecular weights. SIGNIFICANCE AND IMPACT OF STUDY: Beside Tweens there are several polymers that stimulate ligninolytic enzyme production in shaken culture of P. chrysosporium. Their characteristics are: similarity to PEG in chemical structure, having uncharged monomer units and high molecular weight.     

Biopharmaceutical properties of uricase conjugated to neutral and amphiphilic polymers.

A comparative pharmacokinetic and biodistribution investigation of polymer-protein conjugates prepared with various amphiphilic polymers was carried out using uricase as a model. Four polymer-uricase derivatives have been obtained by covalent binding of a similar number of polymer chains of (a) linear poly(ethylene glycol) (Mw 5000 Da); (b) branched poly(ethylene glycol) (Mw 10 000 Da); (c) poly(N-vinylpyrrolidone) (Mw 6000 Da); (d) poly(N-acryloilmorpholine) (Mw 6000 Da). By intravenous administration to Balb/c mice, the conjugates displayed different pharmacokinetic and organ distribution behaviors. (1) The unmodified enzyme and the poly(N-vinylpyrrolidone) conjugate were the enzyme forms with the shortest and the longest permanence in blood respectively (mean residence time 45 and 4378 min). (2) Native uricase was found to localize soon after administration significantly in heart, lungs, and liver from where it was also rapidly cleared. (3) The poly(N-acryloilmorpholine) derivative showed the highest concentration levels in liver (up to 25.5% of the dose) and considerable accumulation took also place in the other considered organs. (4) Poly(N-vinylpyrrolidone)-uricase displayed a relevant tropism for liver but low uptake indexes were found for the other organs. (5) The branched poly(ethylene glycol) derivative accumulated preferentially in liver and spleen. (6) The linear poly(ethylene glycol) conjugate was, among the various uricase forms, the species with the lowest distribution levels in all the examined organs. (7) Finally, all the enzyme forms slowly disposed in kidneys with higher levels for the poly(N-acryloilmorpholine) derivative (15% after 2880 min) and unmodified uricase (14% after 1440 min).     

Subcellular fractionation of rat liver homogenates using two-polymer phase systems in a toroidal-coil centrifuge.

The principal organelles of rat liver homogenates were fractionated by two-phase partition chromatography using toroidal-coil centrifugation with a mixture of dextran T 500 and poly(ethylene glycol) 6000 in 0.26 M-sucrose containing 10 mM-sodium phosphate/phosphoric acid buffer, pH 7.4. The effects of varying the following parameters on organelle elution profiles, as reflected by their marker-enzyme activities, were studied: centrifuge speed; the composition and relative proportion of dextran-rich and poly(ethylene glycol)-rich phases in the eluent; flow rate; sample volume; homogenate concentration; helix diameter; tubing bore and the number of loops in the coil. Optimal resolution of the organelles was achieved with a toroidal coil of internal diameter 1.07 mm with a 4.55 mm helix diameter on a 0.42 m-diameter rotor running at 1000 rev./min. The eluent was prepared by combining, in a ratio of 93:7 (v/v), the poly(ethylene glycol)-rich upper phase and dextran-rich lower phase obtained from a phase mixture containing 3.3% (w/w) dextran and 5.4% (w/w) poly(ethylene glycol). The flow rate of the eluent was 14ml/h. Optimal conditions for separation of the organelles were evaluated. Resolution of plasma membrane and lysosomes was achieved. Separation of endoplasmic reticulum, which showed marked heterogeneity, from plasma membrane was also demonstrated. DNA and marker enzymes for peroxisomes, mitochondria and cytosol showed distinct elution profiles.     

Calcium control of Saccharomyces cerevisiae actin assembly.

Low levels of Ca2+ dramatically influence the polymerization of Saccharomyces cerevisiae actin in KCl. The apparent critical concentration for polymerization (C infinity) increases eightfold in the presence of 0.1 mM Ca2+. This effect is rapidly reversed by the addition of ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid or of 0.1 mM Mg2+. Furthermore, the addition of Ca2+ to polymerized actin causes a reversible increase in the apparent C infinity. In the presence of Ca2+, at actin concentrations below the apparent C infinity, particles of 15 to 50 nm in diameter are seen instead of filaments. These particles are separated from soluble actin when Ca2+-treated filamentous actin is sedimented at high speed; both the soluble and particulate fractions retain Ca2+-sensitive polymerization. The Ca2+ effect is S. cerevisiae actin-specific: the C infinity for rabbit muscle actin is not affected by the presence of Ca2+ and S. cerevisiae actin. Ca2+ may act directly on S. cerevisiae actin to control the assembly state in vivo.     

Detection and characterization of a novel factor that stimulates DNA polymerase alpha

A novel factor that stimulates DNA polymerase alpha activity on poly(dA) X oligo(dT) has been identified and partially purified from mouse FM3A cells. The assay system for the factor contained poly(ethylene glycol) 6000. The activities of DNA polymerase alpha on poly(dA) X oligo(dT) in the presence and absence of the stimulating factor were increased greatly by the addition of poly(ethylene glycol). Stimulation by the factor was observed at all the primer to template ratios tested from 0.01 to 0.3. The highest activity was observed at the ratio of 0.05, corresponding to about 3.3 primers on one template in the presence of the factor. The concentration of DNA polymerase alpha used in the assay affected the stimulation by the factor, and the stimulation became more prominent at concentrations of the enzyme lower than 0.04 unit per assay. The stimulating factor lowered the Km value of DNA polymerase alpha for the template-primer, though they had no effect on the Km value for dTTP substrate. The results of product analysis suggested that the stimulation by the factor is mainly due to the increase in the initiation frequency of DNA synthesis from the primers. The stimulating factor specifically stimulated DNA polymerase alpha but not DNA polymerases beta and gamma. Furthermore, the factor formed a complex with DNA polymerase alpha under a certain condition.