X-ray diffraction by amorphous polymers: models for biological systems

The x-ray scattering method has been used to investigate the structure in two amorphous crosslinked polymers which are regarded as test systems to establish the power of the method as applied to amorphous biologically significant polymer associations. It is shown that structural information can be determined about the rigid regions within the polymer systems, i.e., those regions held in particular configuration by stereochemical effects. Models of such regions extending over distances with dimensions of ca 18 Å are proposed for an Araldite polymer and for crosslinked poly (methyl/butyl methacrylate). The results allow some general statements about the usefulness and limitations of the amorphous x-ray method.     

A novel nanocomposite film prepared from crosslinked cellulosic whiskers

Cellulose whiskers are increasingly being used as a reinforcing phase in polymer systems and their use is a growing area of importance in bionanocomposite research. Although the reinforcing effect of cellulose whiskers has been studied in various polymers, the impact of crosslinking cellulose whiskers has not been explored so far. This work deals with the development of novel cellulose nanocomposites, wherein the cellulose nanowhiskers are crosslinked with poly(methyl vinyl ether-co-maleic acid) and poly(ethylene glycol). The morphology of the nanocomposite was studied using atomic force microscopy (AFM), which revealed a network structure embedded in a continuous phase. The water sorption studies demonstrated that the crosslinked nanocomposites are capable of absorbing up to ～900% water and have potential to be used as hydrogels.     

Kinetic studies of the interaction of methyl orange with poly(L-lysine) by stopped-flow with circular dichroism detection

The interaction of methyl orange with poly(L -lysine) was studied kinetically by the stopped-flow technique with CD detection, as well as by static CD titration experiments. In the static experiments, the differences observed in the polymer-to-dye ratio dependences of the CD spectra and absorption spectra suggested at least two kinds of bound states of the methyl orange attached to the polymer. The kinetic experiments using the stopped-flow apparatus, however, revealed four distinct reaction processes. The reaction mechanism was elucidated from the concentration dependence of the time constant for each process as follows: the first process was attributed to the bimolecular binding step of methyl orange to the side chain of poly(L -lysine), the second and third process were ascribed to the intramolecular reaction of the polymer–dye complex, and the fourth process was found to be the intermolecular aggregation of the polymer–dye complex. The origin of the stacking of methyl orange on poly(L -lysine) is discussed on the basis of the characteristics of signal amplitudes obtained from the kinetic experiments for these processes.     

Review: Selective ligand-exchange adsorbents prepared by template polymerization

Highly selective ligand-exchange absorbents have been prepared by template polymerization, a process in which the target molecule serves as a template for assembly of specific recognition sites. In an effort to develop materials suitable for chromatographic separations, thin coatings of the selctive templated polymers have been grafted to two reactive macroporous supports, poly(trimethylolpropane trimethacrylate) (TRIM), and propylmethacrylate-derivatized silica beads. The precursor polymer prepared from the trifunctioal TRIM monomer is macroporous and highly crosslinked, providing a stable structure for surface grafting. The TRIM precursor polymer and various surface-grafted copolymers have been characterized by scanning electron microscopy (SEM) and IR, (13)C NMR, and XPS spectroscopic techniques. Composite adsorbents have also been prepared using propylmethacrylate-modified silica particles. While equilibrium rebinding selectivites for both types of surface-templated materials are similar to those reported previously for bulk-polymerized template polymers, the composite materials are far better suited to chromatographic separatios. Highly similar bis-imidazole substrates can be separated by ligand-exchange chromatography on these new templated adsorbents. (c) 1995 John Wiley & Sons, Inc.     

Viscometric and spectroscopic studies on the solution behaviour of hydrophobically modified cellulosic polymers

The solution properties of hydroxyethyl cellulose (HEC) and hydrophobically modified hydroxyethyl cellulose (HM-HEC) have been investigated by means of viscometric and spectroscopic techniques involving free radical and fluorescent probes. The greater viscosity of HM-HEC solutions above a critical polymer concentration (C_p) of approximately 0·2% has been interpreted in terms of the formation of a three-dimensional network structure in which the polymer chains are effectively crosslinked by the intermolecular association of neighbouring hydrophobic side chains. C_p is considerably less than the predicted polymer coil overlap concentration (C*) of approximately 1%.

The interaction of the polymers with an anionic surfactant, sodium dodecyl sulphate (SDS) has also been investigated. A mechanism involving the interaction of free surfactant with the regions of intermolecular hydrophobic association is suggested to account for the considerable differences in the rheological behaviour of the polymers in the presence of SDS.     

The mechanism of the drug induced partial displacement of methyl green from DNA

The displacement of methyl green, a dye bound ionically to double stranded DNA, has been suggested as a potential assay for drug-DNA interaction. The present investigation studies the equilibrium system of methyl green, its colorless carbinol form and DNA in the presence of salt (MgSO4), ethidium bromide, and quinine. The reversibility of the equilibrium, and the absence of sequestered sites or two modes of binding for methyl green are demonstrated.     

Hydroxamic acid-containing hydrogels for nonabsorbed iron chelation therapy: synthesis, characterization, and biological evaluation

Iron overload is a severe clinical condition and can be largely prevented by the use of iron-specific chelating agents. A successful iron chelator needs to be orally active, nontoxic, and selective. In this study, hydrogels containing pendant hydroxamic acid groups have been synthesized as potential nonabsorbed chelators for iron in the gastrointestinal tract. The synthetic method employed to introduce hydroxamic acid groups to polymer chains involved reaction of polymer gels based on N-acryloxysuccinimide, acryloyl chloride, and (2-hydroxyethyl)acrylate monomers with hydroxylamine. These hydroxamic acid-functionalized polymer gels swell favorably in water and effectively sequester iron. In vitro iron-binding properties of these hydrogels were evaluated from their binding isotherms by use of iron(II) alone and in the presence of other competing metal ions. These polymers bind iron over a broad pH range. The iron-binding properties of the polymers were found to depend on the concentration of hydroxamate groups on polymer chains. The in vivo iron-binding efficacy of the polymers was evaluated in rat as the animal model. The polymers prevented an increase in serum hemoglobin and hematocrit levels in the animals, thus suggesting the prevention of systemic absorption of dietary iron from the gastrointestinal tract. The animals also maintained normal body weight during the treatment period, indicating the absence of any apparent toxicity associated with these polymers.     

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.     

The isolated MUC5AC gene product from human ocular mucin displays intramolecular conformational heterogeneity

Atomic force microscopy (AFM) has been used to show that human ocular mucins contain at least three distinct polymer conformations, separable by isopycnic density gradient centrifugation. In this work we have used affinity purification against the anti(mucin peptide core) monoclonal antibody 45M1 to isolate MUC5AC gene products, a major component of human ocular mucins. AFM images confirm that the affinity-purified polymers adopt distinct conformations that coidentify with two of those observed in the parent population, and further reveal that these two different conformations can be present within the same polymer. AFM images of the complexes formed after incubation of 45M1 with the parent sample reveal different rates of binding to the two MUC5AC polymer types. The variability of gene products within a mucin population was revealed by analyzing the height distributions along the polymer contour and periodicities in distances between occupied antibody binding sites. AFM analysis of mucin polymers at the single molecule level provides new information about the genetic origins of individual polymers and the contributions of glycosylation to the physicochemical properties of mucins, which can be correlated with information obtained from biochemistry, antibody binding assays, and molecular biology techniques.     

Combinatorial methods in molecular imprinting

Molecular imprinting is a general method for synthesizing robust, network polymers with highly specific binding sites for small molecules. Recently, combinatorial and computational approaches have been employed to select an optimal molecularly imprinted polymer (MIP) formulation for a targeted analyte. The use of MIPs in the combinatorial field, specifically their use for screening libraries of small molecules, has also been developed.     

Effect of the solvent on recognition properties of molecularly imprinted polymer specific for ochratoxin A

A molecularly imprinted polymer specific for the mycotoxin ochratoxin A has been synthesised using a non-covalent approach. The polymer has shown an excellent affinity and specificity for the target template in aqueous solutions. The binding experiments, NMR study and molecular modelling have proven that the template recognition by polymer originates from the shape complementarity of binding sites. The binding mechanism is critically depended on factors that affect the polymer conformation. Thus the variation in buffer concentration, pH and presence of organic solvent, which affect the polymer swelling or shrinking, had a profound effect on the polymer recognition properties.     

Effect of residual side chain blocking groups and side chain charge on polypeptide conformation in aqueous solution

The circular dichroism (CD) spectrum of poly-L-lysine and poly-L -glutamic acid has been investigated in the presence of a small percent of side-chain blocking groups. The blocking groups benzyl, methyl, and carbobenzoxy show qualitatively similar effects. Less than five mole percent of aromatic blocking groups alters the CD spectrum. Consequently, unsuspected blocking groups may account for the variation observed in CD spectra of these polymers. A weak CD band at 235–240 nm was observed for the disordered unblocked polymer even in the absence of electrolyte. Viscosity data indicate that in salt-free solutions these chains at neutral pH still behave as random coils though with reduced conformational freedom, in contrast to some polyelectrolytes which behave as rigid rods in the absence of electrolyte. The viscosity data bring into question the relevance of isolated molecule conformational calculations to experimental CD spectra.     

Direct observation of single MuB polymers: evidence for a DNA-dependent conformational change for generating an active target complex

MuB, an ATP-dependent DNA binding protein, is critical for selection of target sites on the host chromosome during Mu transposition. We have developed a system for observing the behavior of single MuB polymers bound to an immobilized molecule of DNA. We show that the individual polymers display a broad distribution of disassembly rates and exhibit regional variations in DNA binding. Additionally, ATP hydrolysis was obligatorily coupled to dissociation of MuB subunits from the DNA during polymer disassembly. We propose a model in which the formation of an active target complex is mediated by a conformational change within the MuB polymer that is influenced by the sequence of the DNA.     

Development of molecularly imprinted polymers as tailored templates for the solid-state [2+2] photodimerization

In this study, a molecularly imprinted polymer (MIP) was prepared to selectively template the [2+2] photodimerization of trans-1,2-bis(4-pyridyl)ethylene. First, an MIP selective for rctt-tetrakis(4-pyridyl)cyclobutane, which is the [2+2] photodimerization product of trans-1,2-bis(4-pyridyl)ethylene, was prepared from methacrylic acid (MAA) and ethylene glycol dimethacrylate (EGDMA). The non-covalent MIP showed enhanced affinity for both the templating agent, rctt-tetrakis(4-pyridyl)cyclobutane, and the alkene precursor, trans-1,2-bis(4-pyridyl)ethylene. The solid-state photodimerization reaction proceeded in significantly higher yields in the presence of the MIP. Control reactions carried out in the absence of polymer gave no product, and reactions carried out in the presence of a non-imprinted polymer and an MIP imprinted with a different template, 3-hydroxymethylpyridine, gave much lower yields of the cyclobutane photodimerization product. The outcome of the MIP-templated photodimerization reaction was strongly influenced by the binding site heterogeneity of the non-covalently imprinted polymers. For example, higher yields were observed with decreasing olefin loadings levels on the MIPs. This binding site heterogeneity was characterized via application of the Freundlich binding model to the experimentally measured binding isotherms. These confirmed that the non-covalent MIPs had very few high-affinity binding sites, which greatly limits the capacity and ultimately the utility of these materials as templates in synthetic organic applications.     

Molecularly imprinted polymer sensors for detection in the gas, liquid, and vapor phase

Fast, reliable, and inexpensive analytical techniques for detection of airborne chemical warfare agents are desperately needed. Recent advances in the field of molecularly imprinted polymers have created synthetic nanomaterials that can sensitively and selectively detect these materials in aqueous environments, but thus far, they have not been demonstrated to work for detection of vapors. The imprinted polymers function by mimicking the function of biological receptors. They can provide high sensitivity and selectivity but, unlike their biological counterparts, maintain excellent thermal and mechanical stability. The traditional imprinted polymer approach is further enhanced in this work by the addition of a luminescent europium that has been introduced into the polymers to provide enhanced chemical affinity as well as a method for signal transduction to indicate the binding event. The europium in these polymers is so sensitive to the bound target; it can distinguish between species differing by a single methyl group. The imprinted polymer technology is fiber optic-based making it inexpensive and easily integratable with commercially available miniature fiber optic spectrometer technologies to provide a shoebox size device. In this work, we will describe efforts to apply these sensors for detection of airborne materials and vapors. Successful application of this technology will provide accurate low level vapor detection of chemical agents or pesticides with little to no false positives.     

Reduction-degradable linear cationic polymers as gene carriers prepared by Cu(I)-catalyzed azide-alkyne cycloaddition

Linear reduction-degradable cationic polymers with different secondary amine densities (S2 and S3) and their nonreducible counterparts (C2 and C3) were synthesized by Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) step-growth polymerization of the dialkyne-oligoamine monomers and the diazide monomers. These polymers were studied with a goal of developing a set of new gene carriers. The buffering capacity and DNA binding ability of these polymers were evaluated by acid-base titration, gel retardation, and ethidium bromide (EB) exclusion assay. The polymers with lower amine density exhibit a weaker DNA-binding ability but a stronger buffering capacity in the range of pH 5.1 and 7.4. Particle size and zeta-potential measurements demonstrate that the polymers with higher amine density condense pDNA to form polyplexes with smaller sizes, while the disulfide bond in the backbone shows a negative effect on the condensing capability of the polymers, resulting in the formation of polyplexes with large size and nearly neutral surface. The reduction-sensitive polyplexes formed by polymer S2 or S3 can be disrupted by dithiothreitol (DTT) to release free DNA, which has been proven by the combination of gel retardation, EB exclusion assay, particles sizing, and zeta potential measurements. Cell viability measurements by MTT assay demonstrate that the reduction-degradable polymers (S2 and S3) have little cytotoxicity while the nonreducible polymers (C2 and C3) show obvious cytotoxicity, in particular, at high N/P ratios. In vitro transfection efficiencies of these polymers were evaluated using EGFP and luciferase plasmids as the reporter genes. Polymers S3 and S2 show much higher efficiencies than the nonreducible polymers C3 and C2 in the absence of 10% serum; unexpectedly, the lowest transfection efficiency has been observed for polymer S3 in the presence of serum.     

Using protein templates to direct the formation of thin-film polymer surfaces

Protein imprinted electrodes formed by the cyclic voltammetric deposition of conductive polymers, on screen-printed platinum supports, in the presence of target proteins have been fabricated. An initial layer of polypyrrole was used as a supporting polymer layer, upon which were formed two layers of polyaminophenylboronic acid. The first of these layers was non-imprinted and formed a barrier between the polypyrrole and the outer layer, which was deposited in the presence of a protein template (lysozyme or cytochrome c). After protein extraction, re-binding of the template proteins to their respective imprinted electrodes showed a distinct two-phase binding profile; whereas, binding to control polymers, made in the same way but without the addition of protein templates, showed progressive binding typical of non-specific recognition. Reductions in the observed current transmission due to bonding to the polymer surface of non-conductive protein have been used as a measure of re-binding. It was found that when challenged with 1 part per million protein in solution, the current reductions for the lysozyme and cytochrome c imprinted electrodes were 30.3 and 66.2%, respectively, compared to 4.5 and 29.9% for their respective control electrodes. All measurements carried out at -0.1 V with Ag/AgCl reference.     

General structure-activity relationship for poly(glycoamidoamine)s: the effect of amine density on cytotoxicity and DNA delivery efficiency

Herein, two new series of poly(glycoamidoamine)s (branched and linear) have been synthesized by polycondensation. The polymer repeat units have been designed to contain D-glucaramide, meso-galactaramide, D-mannaramide, or L-tartaramide structures and five or six ethyleneamine units to investigate the amine density effects on the bioactivity as compared to a similar series of poly(glycoamidoamine)s previously described that contain four ethyleneamines. These delivery vehicles were created to examine the effects that the number of secondary amines in the polymer repeat unit and the polymer structure (branched and linear) have on plasmid DNA (pDNA) binding affinity, polyplex formation, cell viability, and gene expression in the absence and presence of serum in the culture medium. The results reveal that the new polymers with higher amine density in the repeat unit do not significantly enhance the transfection efficiency compared to that of previous models containing four ethyleneamines, but an increase in cytotoxicity is noticed. Linear polymers reveal higher pDNA neutralization efficacy, gene expression, and toxicity than the branched versions containing a similar chemical structure, which may be caused by a higher protonation of the amine groups. With these new vectors, some interesting trends emerged. The galactaramide and tartaramide analogues revealed higher delivery efficiency than the glucaramide and mannaramide structures. In addition, the branched and linear structures containing five ethyleneamines in the repeat unit formed polyplexes at higher N/P ratios, which had lower zeta potential and lower delivery efficacy than the analogues with six ethyleneamines, and also the linear structures generally revealed higher delivery efficiency and toxicity when compared to those of their branched analogues.     

Effects of DNA binding proteins on DNA methylation in vitro

The inheritance of DNA methylation patterns may play an important role in the stability of the differentiated state. We have therefore studied the inhibitory effects of DNA binding proteins on DNA methylation in vitro. Mouse L1210 cells grown in the presence of 5-azacytidine acquire hemimethylated sites in their DNA. Purified hemimethylated DNA accepted methyl groups from S-adenosyl-L-methionine in the presence of a crude maintenance methylase more readily than purified DNA isolated from cells not exposed to 5-azacytidine. On the other hand, chromatin fractions isolated from cells grown in the presence or absence of 5-azacytidine were poor substrates for the maintenance methylase irrespective of the number of hemimethylated sites present in the DNA. Inhibition of DNA methylation was shown to be associated primarily with chromatin proteins bound to DNA, and trypsinization of nuclei increased their methyl accepting abilities. Methyl acceptance was increased by salt extraction of chromosomal proteins. These data suggest that association of histones with DNA may play a role in the modulation of methylation patterns.     

Preparation of superabsorbent cellulosic hydrogels

In this work, milled softwood (SW) bleached kraft fibers were crosslinked by esterification with poly(vinyl methyl ether-co-maleic acid) (PVMEMA) and polyethylene glycol (PEG). The effects of fiber length, crosslinking reaction time, and dosage of PVMEMA on water absorption and retention value (WAARV) for the crosslinked fibers were determined. The results show that as the softwood fiber length is mechanically decreased from 2.41 to 0.50 mm and employing a weight ratio of fiber to polymers equivalent to 1.00:1.28 the WAARV increased from 86.50 to 189.20 g/g. Analysis of the crosslinked fibers by SEM and light microscope indicated that the polymers and fibers form a crosslinked fibrous matrix. FT-IR spectroscopy was employed to detect the ester linkage between PVMEMA and PEG/SW kraft pulp fibers. The results suggested that the ester crosslinked pulps exhibit excellent water absorbent properties and have the potential of utilizing milled bleached SW kraft fibers, such as refiner dust or pulp fines, for novel water absorbent applications.