Biophysical characterization of quaternary pyridinium functionalized polynorbornenes for DNA complexation and their cellular interactions

Cationic polymers with hydrophobic side chains have gained great interest as DNA carriers since they form a compact complex with negatively charged DNA phosphate groups and interact with the cell membrane. Amphiphilic polyoxanorbornenes with different quaternary alkyl pyridinium side chains with ethyl‐p(OPy2) and hexyl units‐p(OPy6) bearing 10 kDa MWT were synthesized by living Ring‐Opening Metathesis Polymerization method. The physicochemical characteristics: critical micellar concentration, size distribution, surface charge, and condensation of polymer/DNA complex were investigated. Morphology of complexes was monitored by Atomic force microscopy. Cytotoxicity and interaction of these complexes with model lipid vesicles mimicking the cell membrane were examined. These polymers were enabled to form small sized complexes of DNA, which interact with model membrane vesicles. It was found that the nature of hydrophobicity of the homopolymers significantly impacts rates of DNA complexation and the surface charge of the resulting complexes. These results highlight the prospect of the further examinations of these polymers as gene carriers.     

Copolymerization of tubulin-colchicine complex and unliganded tubulin in a nonmicrotubular polymer

We have shown previously [Saltarelli, D., & Pantaloni, D. (1982) Biochemistry 21, 2996-3006] that the tubulin-colchicine complex is able to polymerize in vitro into peculiar "curly" polymers, under the solution conditions permitting polymerization of unliganded tubulin into microtubules. Here it is further demonstrated that unliganded tubulin can be incorporated into these "curly" polymers. The partial critical concentration of tubulin-colchicine is decreased upon incorporation of unliganded tubulin into the copolymer. GTP hydrolysis occurs on unliganded tubulin upon incorporation in the copolymer. Tubulin-podophyllotoxin does not copolymerize with tubulin-colchicine to form a large polymer but interacts with it, preventing tubulin-colchicine polymerization. The data have been analyzed within a model of random copolymerization of unliganded tubulin and tubulin-colchicine into "curly" polymers. A corollary is that unliganded tubulin is virtually able to self-assemble into curly polymers with a critical concentration 10-fold higher than the critical concentration found for microtubule assembly. Consequently, these peculiar tubulin homopolymers cannot be observed except as transients at high concentrations, or when microtubule assembly is inhibited. Kinetic measurements of the T-TC copolymerization process and associated GTP hydrolysis at different T/TC ratios provide supplementary information about some privileged interactions between tubulin and tubulin-colchicine molecules. A comprehensive phase diagram of the various possible polymers formed in the presence of tubulin and tubulin-colchicine is presented.     

Effect of second-sphere coordination 12. Adduct formation behavior of crown ethers with ammine complexes of ruthenium containing a protic ligand of other type than ammine

Adduct formation of pentaammineruthenium complexes involving a different type of protic ligand, such as imidazole, was investigated for a series of crown ethers with different ring size. Changes in redox potential and in absorption spectra of the complex were measured on addition of crown ether to the complex solution. The magnitude of the change in both properties is dependent on the ring size of crown ethers. ¹H-NMR spectra of the complex were measured in the presence of crown ethers in order to elucidate hydrogen bonding sites. The chemical shifts of NH proton of imidazol and ammine protons were measured at various concentrations of crown ethers. Adduct formation was discussed based on the features of dependences of those chemical shifts on crown ether concentration.     

Retrovirus-polymer complexes: study of the factors affecting the dose response of transduction

We have previously shown that complexes of Polybrene (PB), chondroitin sulfate C (CSC), and retrovirus transduce cells more efficiently than uncomplexed virus because the complexes are large and sediment, reaching the cells more rapidly than by diffusion. Transduction reaches a peak at equal weight concentrations of CSC and PB and declines when the dose of PB is higher or lower than CSC. We hypothesized that the nonlinear dose response of transduction was a complex function of the molecular characteristics of the polymers, cell viability, and the number of viruses incorporated into the complexes. To test this hypothesis, we formed complexes using an amphotropic retrovirus and several pairs of oppositely charged polymers and used them to transduce murine fibroblasts. We examined the effect of the type and concentration of polymers used on cell viability, the size and charge of the complexes, the number of viruses incorporated into the complexes, and virus binding and transduction. Transduction was enhanced (2.5- to 5.5-fold) regardless of which polymers were used and was maximized when the number of positive charge groups was in slight excess (15-28%) of the number of negative charge groups. Higher doses of cationic polymer were cytotoxic, whereas complexes formed with lower doses were smaller, contained fewer viruses, and sedimented more slowly. These results show that the dose response of transduction by virus-polymer complexes is nonlinear because excess cationic polymer is cytotoxic, whereas excess anionic polymer reduces the number of active viruses that are delivered to the cells.     

Charged Molecules Modulate the Volume Exclusion Effects Exerted by Crowders on FtsZ Polymerization

We have studied the influence of protein crowders, either combined or individually, on the GTP-induced FtsZ cooperative assembly, crucial for the formation of the dynamic septal ring and, hence, for bacterial division. It was earlier demonstrated that high concentrations of inert polymers like Ficoll 70, used to mimic the crowded cellular interior, favor the assembly of FtsZ into bundles with slow depolymerization. We have found, by fluorescence anisotropy together with light scattering measurements, that the presence of protein crowders increases the tendency of FtsZ to polymerize at micromolar magnesium concentration, being the effect larger with ovomucoid, a negatively charged protein. Neutral polymers and a positively charged protein also diminished the critical concentration of assembly, the extent of the effect being compatible with that expected according to pure volume exclusion models. FtsZ polymerization was also observed to be strongly promoted by a negatively charged polymer, DNA, and by some unrelated polymers like PEGs at concentrations below the crowding regime. The influence of mixed crowders mimicking the heterogeneity of the intracellular environment on the tendency of FtsZ to assemble was also studied and nonadditive effects were found to prevail. Far from exactly reproducing the bacterial cytoplasm environment, this approach serves as a simplified model illustrating how its intrinsically crowded and heterogeneous nature may modulate FtsZ assembly into a functional Z-ring.     

DNA sequence dependent binding modes of 4',6-diamidino-2-phenylindole (DAPI)

The interactions of DAPI with natural DNA and synthetic polymers have been investigated by hydrodynamic, DNase I footprinting, spectroscopic, binding, and kinetic methods. Footprinting results at low ratios (compound to base pair) are similar for DAPI and distamycin. At high ratios, however, GC regions are blocked from enzyme cleavage by DAPI but not by distamycin. Both poly[d(G-C)]2 and poly[d(A-T)]2 induce hypochromism and shifts of the DAPI absorption band to longer wavelengths, but the effects are larger with the GC polymer. NMR shifts of DAPI protons in the presence of excess AT and GC polymers are significantly different, upfield for GC and mixed small shifts for AT. The dissociation rate constants and effects of salt concentration on the rate constants are also quite different for the AT and the GC polymer complexes. The DAPI dissociation rate constant is larger with the GC polymer but is less sensitive to changes in salt concentration than with the AT complex. Binding of DAPI to the GC polymer and to poly[d(A-C)].poly[d(G-T)] exhibits slight negative cooperativity, characteristic of a neighbor-exclusion binding mode. DAPI binding to the AT polymer is unusually strong and exhibits significant positive cooperativity. DAPI has very different effects on the bleomycin-catalyzed cleavage of the AT and GC polymers, a strong inhibition with the AT polymer but enhanced cleavage with the GC polymer. All of these results are consistent with two totally different DNA binding modes for DAPI in regions containing consecutive AT base pairs versus regions containing GC or mixed GC and AT base pair sequences. The binding mode at AT sites has characteristics which are similar to those of the distamycin-AT complex, and all results are consistent with a cooperative, very strong minor groove binding mode. In GC and mixed-sequence regions the results are very similar to those observed with classical intercalators such as ethidium and indicate that DAPI intercalates in DNA sequences which do not contain at least three consecutive AT base pairs.     

Salt-dependent co-operative interaction of histone H1 with linear DNA

The nature of the complexes formed between histone H1 and linear double-stranded DNA is dependent on ionic strength and on the H1 : DNA ratio. At an input ratio of less than about 60% (w/w) H1 : DNA, there is a sharp transition from non-co-operative to co-operative binding at a critical salt concentration that depends on the DNA size and is in the range 20 to 50 mM-NaCl. Above this critical ionic strength the H1 binds to only some of the DNA molecules leaving the rest free, as shown by sedimentation analysis. The ionic strength range over which this change in behaviour occurs is also that over which chromatin folding is induced. Above the salt concentration required for co-operative binding of H1 to DNA, but not below it, H1 molecules are in close proximity as shown by the formation of H1 polymers upon chemical cross-linking. The change in binding mode is not driven by the folding of the globular domain of H1, since this is already folded at low salt in the presence of DNA, as indicated by its resistance to tryptic digestion. The H1-DNA complexes at low salt, where H1 is bound distributively to all DNA molecules, contain thickened regions about 6 nm across interspersed with free DNA, as shown by electron microscopy. The complexes formed at higher salt through co-operative interactions are rods of relatively uniform width (11 to 15 nm) whose length is about 1.6 times shorter than that of the input DNA, or are circular if the DNA is long enough. They contain approximately 70% (w/w) H1 : DNA and several DNA molecules. These thick complexes can also be formed at low salt (15 mM-NaCl) when the H1 : DNA input ratio is sufficiently high (approximately 70%).     

Rapid formation and high diffusibility of actin-cofilin cofilaments at low pH.

Cofilin is a small actin-binding protein that is known to bind both F-actin and G-actin, severing the former. The interaction of cofilin with actin is pH-sensitive, F-actin being preferentially bound at low pH and G-actin at higher pH, within the physiological range. Diffusion coefficients of F-actin with cofilin were measured by the fluorescence recovery after photobleaching (FRAP) technique. This has the potential for simultaneous and direct measurement of average polymer length via the average diffusion coefficient of the polymers (DLM) as well as the fraction of polymerized actin, fLM, present in solution. In the range of cofilin-actin ratios up to 1 : 1 and at both pH 6.5 and pH 8.0, the diffusion coefficients of the polymers increased with the amount of cofilin present in the complex, in a co-operative manner to a plateau. We interpret this as indicating co-operative binding/severing and that filaments less than a certain length cannot be severed further. Under the conditions used here, filaments were found to be more motile at pH 6.5 than at pH 8.0. At pH 8.0, some actin is expected to be sequestered as ADP-actin-cofilin complexes, with the remaining actin being present as long slowly diffusing filaments. At pH 6.5, however, cofilin binds to F-actin to form short rapidly diffusing cofilaments. These filaments form very rapidly from cofilin-actin monomeric complexes, possibly indicating that this complex is able to polymerize without dissociation. These findings may be relevant to the nuclear import of actin-cofilin complexes.     

Potent trophic activity of spermidine supramolecular complexes in in vitro models

AIM:To test the growth-promoting activity of the polyamine spermidine bound to various polymeric compounds in supramolecular complexes.METHODS:A thiazolyl blue cell viability assay was used to determine the growth-promoting potency of spermidine-supramolecular complexes in a human skin fibroblast cell line exposed to spermidine and different spermidine-supramolecular complexes that were obtained by combining spermidine and polyanionic polymers or cyclodextrin.Reconstituted human vaginal epithelium was exposed to a specific spermidinesupramolecular complex,i.e.,spermidine-hyaluronan(HA)50,and cell proliferation was determined by Ki-67immunohistochemical detection.Transepithelial electrical resistance and histological analysis were also performed on reconstituted human vaginal epithelium to assess tissue integrity.RESULTS:The effect of spermidine and spermidinesupramolecular complexes was first tested in skin fi-broblasts.Spermidine displayed a reverse dose-related mode of activity with mmol/L growth inhibition,whereas 30%stimulation over basal levels was detected at mol/L and nmol/L levels.Novel spermidine-supramolecular complexes that formed between spermidine and polyanionic polymers,such as HA,alginate,and polymaleate,were then tested at variable spermidine concentrations and a fixed polymer level(0.1%w/v).Spermidine-supramolecular complexes stimulated the cell growth rate throughout the entire concentration range with maximal potency(up to 80%)at sub-mol/L levels.Similar results were obtained with spermidine-(-cyclodextrin),another type of spermidine-supramolecular complex.Moreover,the increased expression of Ki-67 in the reconstituted human vaginal epithelium exposed to spermidine-HA 50 showed that the mode of action behind the spermidine-supramolecular complexes was increased cell proliferation.Functional and morphological assessments of reconstituted human vaginal epithelium integrity did not show significant alterations after exposure to spermidine-HA,thus supporting its safety.CONCLUSION:Spermidine found in spermidine-supramolecular complexes displayed potentiated regenerative effects.Safety data on reconstituted human vaginal epithelium suggested that assessing spermidinesupramolecular complex efficacy in atrophic disorders is justified.     

Synthesis of beta-cyclodextrin and starch based polymers for sorption of azo dyes from aqueous solutions

Three beta-cyclodextrin (polymers 1-3) and a starch-based (polymer 4) polymers were synthesized using hexamethylene diisocyanate (HMDI) as a cross-linking agent in dry dimethylformamide and used as a sorbent for the removal of some selected azo dyes from aqueous solutions. The cross-linked polymers were characterized by Fourier transform infrared spectroscopy, thermogravimetric and differential scanning calorimetric analysis. Results of sorption showed that cyclodextrin and starch based polymers can be effectively used as a sorbent for the removal of anionic azo dyes. The Influence of the amide groups and the chemical structure of azo dyes are also studied. Results of sorption experiments showed that these adsorbent exhibited high sorption capacities toward Direct Violent 51 (80% for polymer 1, 69% for polymer 2, 70% for polymer 3 and 78% for polymer 4). The sorption capacity of dyes on the polymers was dependent on the presence of sulfonate groups of the anionic dyes. In order to explain the results an adsorption mechanism mainly physical adsorption and interactions such as hydrogen bonding, ion-exchange due to the nature of the polymer network and the formation of an inclusion complex due to the beta-CD molecules through host-guest interaction is proposed.     

Energetics of the cooperative assembly of cell division protein FtsZ and the nucleotide hydrolysis switch

FtsZ is the first protein recruited to the bacterial division site, where it forms the cytokinetic Z ring. We have determined the functional energetics of FtsZ assembly, employing FtsZ from the thermophilic Archaea Methanococcus jannaschii bound to GTP, GMPCPP, GDP, or GMPCP, under different solution conditions. FtsZ oligomerizes in a magnesium-insensitive manner. FtsZ cooperatively assembles with magnesium and GTP or GMPCPP into large polymers, following a nucleated condensation polymerization mechanism, under nucleotide hydrolyzing and non-hydrolyzing conditions. The effect of temperature on the critical concentration indicates polymer elongation with an apparent heat capacity change of -800 +/- 100 cal mol-1 K-1 and positive enthalpy and entropy changes, compatible with axial hydrophobic contacts of each FtsZ in the polymer, and predicts optimal polymer stability near 75 degrees C. Assembly entails the binding of one medium affinity magnesium ion and the uptake of one proton per FtsZ. Interestingly, GDP- or GMPCP-liganded FtsZ cooperatively form helically curved polymers, with an elongation only 1-2 kcal mol-1 more unfavorable than the straight polymers formed with nucleotide triphosphate, suggesting a physiological requirement for FtsZ polymerization inhibitors. This GTP hydrolysis switch should provide the basic properties for FtsZ polymer disassembly and its functional dynamics.     

Heterogenized polymetallic catalysts Part II. Oxidation of 3,5-di-t-butylphenol by Cu(II), Fe(III) and Pd(II) complexed to a polyphenylene polymer containing ß-di- and triketone surface ligands; an improved catalyst system

Polyphenylene polymer preparation involves the cyclic trimerization polymerization of acetylated methyl benzoate with diacetyl benzene. Since the methyl benzoate groups do not take part in the polymerization they are present in high concentration. The ß-diketone ligands were placed on the surface by reaction of the methylbenzoate group with base and a methyl ketone and the triketone by reaction with base to give the ß-triketone. The ß-triketones can bind two metal ions in a known geometry that is suitable for bimetallic catalysis of the rapid polyelectron oxidation of catechols. The final catalytic surfaces were prepared by treating the chemically modified polymer with copper(II), iron(II) and palladium(II) acetonitrile complexes with non-coordinating BF₄⁻ as the anion. Since the metal ions contain no strongly coordinating ligand, they are very reactive species. These surfaces catalyzed the rapid air oxidation of 3,5-di-tert-butylcatechol (DTBC). The diketone surfaces gave only 3,5-di-tert-butyl-o-quinone (DTBQ) while the triketone surfaces gave ring-cleaved products, confirming the special catalytic effect of the triketone surface. Also, only the triketone catalysts showed any activity for ring cleavage oxidation of DTBQ. These catalysts were much more reactive than previous ones using the same polyphenylene polymer but without the methyl benzoate groups. With these polymers the di- and triketone groups were placed on the surface by chemical modification of the unpolymerized acetyl groups.     

Comparisons between hog intestinal peroxidase and bovine lactoperoxidase-compound I formation and inhibition by benzhydroxamic acid.

The bihelical polydeoxyribonucleotides DNA and poly (deoxyadenylate-deoxythymidylate form at least two distinct complexes with the dye Hoechst 33258. The nonfluorescent complex formed at low polymer/dye ratios is replaced at high polymer/dye ratios by an intensely fluorescent complex. The transition is accompanied by pronounced changes in circular dichroism and absorption spectra and may be interpreted in terms of a noncooperative replacement of dye molecules bound in proximity by isolated molecules of bound dye. In the case of the bihelical polyribonucleotides the transition exhibits positive cooperativity and major differences from the deoxyribose polymers exist in the circular dichroism spectra, suggesting a different geometry for the complex species.     

Strongylocentrotus purpuratus spindle tubulin. I. Characteristics of its polymerization and depolymerization in vitro

Tubulin was extracted from spindles isolated from embryos of the sea urchin Strongylocentrotus purpuratus, repolymerized in vitro, and purified through three cycles of temperature-dependent assembly and disassembly. In addition to the tubulin, these preparations contain a protein of 80 kdaltons and a small but variable amount of actin. At 37 degrees C, the tubulin polymerizes with a critical concentration of 0.15-0.2 mg/ml into smooth-walled polymers which contain predominantly 14 protofilaments. Removal of the 80 kdalton protein and the actin by DEAE-chromatography does not change the critical concentration for polymerization. At 15 degrees C, which is within the range of physiological temperatures for S. purpuratus embryos, the spindle tubulin will self-assemble, but the rate of total polymer formation is very slow, requiring hours in the test tube. This rate can be increased by shearing the polymerizing microtubules, creating more ends for assembly, indicating that the slow rate of polymer formation is due to a slow rate of self-initiation. If spindle tubulin is polymerized at 37 degrees C and then lowered to 15 degrees C, some polymer will be retained, the percentage of which depends on the protein concentration. These results demonstrate that spindle tubulin from S. purpuratus will assemble at 37 degrees C with a low critical concentration for polymerization in the absence of detectable MAPs and will self-assemble and maintain steady state levels of polymer at physiological temperatures.     

Informational symmetry breaking between proteins and nucleic acids.

Anti-symmetry of the information-processing mechanisms between proteins and nucleic acids is generalized to informational symmetry breaking between a genetic polymer and an anti-genetic polymer so as not to depend on particular chemical species. In a genetic polymer, e.g. nucleic acids, any sequence can form a closed double-stranded structure with a specific partner sequence. On the other hand, in an anti-genetic polymer, e.g. proteins, a chain could fold to an open multi-stranded structure and reinterpretation of the genetic information through slides or shifts between stacked strands could be induced by external perturbations. The possibility of the informational symmetry breaking by hierarchical organization of a single chemical species, i.e. polypeptides as a genetic polymer and nucleic acids as an anti-genetic polymer, is examined. The informational functions of genetic polymers and anti-genetic polymers in a complex mixture of macromolecules are characterized.     

Homology in Trivaline Complex Formation with dsDNA and ssRNA

Abstract

It has been shown by the equilibrium dialysis that at a polyU concentration above the “critical” one, the complete polymer saturation with trivaline reaches approximately 0.7 trivaline molecules per one phosphate group. i.e. at these conditions peptide dimer occupies on polyU a site of three bases (phosphates) in length. The trivaline complex with polyU at a concentration lower than the “critical” one does not reveal any noticeable fluorescence, but has rather significant positive linear dichroism at 265 and 330 nm. The trivaline-nucleic acids complex has a significant fluorescence at any dsDNA concentration while with polyU it is only so at a concentration above the “critical” one. Electron microscopy has shown that at a rather high concentration of dsDNA molecules in solution a “biduplex” structure undergoes an additional stage of compaction, during which the extended particles more than 30 nm in diameter are formed.

Schematic models for the trivaline complexes and compact structures with dsDNA and ssRNA are propose     

Extraction of lipase from Aspergillus niger by insoluble complex formation with anionic and cationic polyelectrolytes

The insoluble complex formation between lipase from Aspergillus niger and the electrically charged polymers, polyacrylic acid (PAA), poly-vinil sulfonate (PVS) and chitosan (CHI), was studied by using turbidimetric and enzymatic methods on a commercial lyophilized (Ly) and a filtrate of solid culture medium (SCM). It could be shown that both electrostatic interactions as hydrophobic are involved in the formation of insoluble complexes. The kinetics of the complex formation were determined. Lipase enzymatic activity is maintained through time in the presence of polyelectrolytes.On the Ly the three polymers produced insoluble complex, with a stoichiometric ratio (polymer mass per mass of Ly from Aspergillus niger) of PAA/Ly: 0.035, PVS/Ly: 0.099 and CHI/Ly: 0.071 mg/mg Ly. For the anionic polyelectrolytes, the PAA presents slightly better results than PVS to be used when the protein concentration is similar to the lyophilized.The filtrate of the SCM has a total protein concentration much lower than commercial lyophilized. Working with CHI as cationic polymer a recovery of the activity in the re-dissolved precipitate higher than 80%, with purification factors greater than 3 were achieved, both at 8 and 20 °C. Therefore, this methodology could be used as a first step of purification.     

The role of subunit entropy in cooperative assembly. Nucleation of microtubules and other two-dimensional polymers.

The self-assembly and nucleation of two-dimensional polymers is described by a theory based on a model of rigid subunits and bonds and simple principles of thermodynamics. The key point in the theory is to separate as an explicit parameter the free energy, primarily attributed to the entropy of the free subunit, that is required to immobilize a subunit in the polymer. Quantitative relations for the association of a subunit forming a longitudinal bond, a lateral bone, or both together are obtained, which demonstrate the basis and magnitude of cooperativity. The same formalism leads to a quantitative estimate for th concentration of the small polymers that are important intermediates in nucleation. It is shown that, if the concentration of free subunits is below a certain "critical supersaturation," the concentration of some essential intermediates is too low to support any significant assembly and nucleation is blocked. If the subunit concentration is above the critical supersaturation, all of the small intermediates are sufficiently stable to form and grow spontaneously. The theory predicts a critical supersaturation of 3.5 to 7 (the ratio of subunit concentration to the equilibrium solubility) for parameters appropriate to assembly of the microtubule wall. Experimentally, nucleation and assembly of microtubules is obtained at somewhat lower concentrations, 1.5 to 3 times the equilibrium solubility. Special mechanisms that could stabilize small polymers and facilitate nucleation of microtubule assembly are suggested.     

Synthesis and characterization of biodegradable peptide-based polymers prepared by microwave-assisted click chemistry

In this study, the microwave-assisted copper(I)-catalyzed 1,3-dipolar cycloaddition reaction was used to synthesize peptide triazole-based polymers from two novel peptide-based monomers: azido-phenylalanyl-alanyl-lysyl-propargyl amide (1) and azido-phenylalanyl-alanyl-glycolyl-lysyl-propargyl amide (2). The selected monomers have sites for enzymatic degradation as well as for chemical hydrolysis to render the resulting polymer biodegradable. Depending on the monomer concentration in DMF, the molecular mass of the polymers could be tailored between 4.5 and 13.9 kDa (corresponding with 33-100 amino acid residues per polymer chain). As anticipated, both polymers can be enzymatically degraded by trypsin and chymotrypsin, whereas the ester bond in the polymer of 2 undergoes chemical hydrolysis under physiological conditions, as was shown by a ninhydrin-based colorimetric assay and MALDI-TOF analysis. In conclusion, the microwave-assisted copper(I)-catalyzed 1,3-dipolar cycloaddition reaction is an effective tool for synthesizing biodegradable peptide polymers, and it opens up new approaches toward the synthesis of (novel) designed biomedical materials.