High pressure fourier transform infrared spectroscopy of poly(dA)poly(dT), poly(dA) and poly(dT)

The effect of hydrostatic pressure upon the DNA duplex, poly(dA)poly(dT), and its component single strands, poly(dA) and poly(dT) has been studied by fourier-transform infrared spectroscopy (FT-IR). The spectral data indicate that at 28 degrees C and pressures up to 12 kbar (1200 MPa) all three polymers retain the B conformation. Pressure causes the band at 967 cm(-1), arising from water-deoxyribose interactions, to shift to higher frequencies, a result consistent with increased hydration at elevated pressures. A larger pressure-induced frequency shift in this band is observed in the single stranded polymers than in the double stranded molecule, suggesting that the effect of pressure on the hydration of single strands may be greater than upon a double stranded complex. A pressure-dependent hypochromicity in the bands attributed to base stacking indicates that pressure facilitates the base stacking in the three polymers, in agreement with previous assessments of the importance of stacking in the stabilization of DNA secondary structure at ambient and high pressures.     

Electronic structures of collagen model polymers: (Gly-Pro)n, (Gly-Hyp)n, (Ala-Pro)n, (Ala-Hyp)n, (Gly-Pro-Gly)n, (Gly-Hyp-Gly)n, (Gly-Pro-Pro)n, and (Gly-Pro-Hyp)n

In order to investigate the relationship existing between the electronic structures of collagen and its biochemical functions in vivo, the semiempirical CNDO/2 SCF MO calculations were carried out on several model polymers of collagen, (Gly-Pro)_n, (Gly-Hyp)_n, (Ala-Pro)_n, (Ala-Hyp)_n, (Gly-Pro-Gly)_n, (Gly-Hyp-Gly)_n, (Gly-Pro-Pro)_n and (Gly-Pro-Hyp)_n. Geometries of the skeleton of these polymers were assumed to be the same as those of poly(l-proline) I (cis) and II (trans) and the calculations were performed only on infinite polymers in a single chain. The results show that the cis form is always more stable than the trans form for all the polymers treated. This energy difference between the cis and trans forms depends, for example, on the kind of amino acid residue, Gly or Ala, but this could not be seen in the Pro or Hyp residue. The flexibility or mobility of the collagen structure was explained using the energy difference between the cis and trans forms of the polymers, i.e. the cis-trans conversion of the collagen was discussed in connection with the energy difference. The reason why the collagen has the constitution of (Gly-Pro-Hyp)_n is briefly discussed.     

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.     

Design of mucoadhesive polymeric films based on blends of poly(acrylic acid) and (hydroxypropyl)cellulose

Mixing of aqueous solutions of poly(acrylic acid) and (hydroxypropyl)cellulose results in formation of hydrogen-bonded interpolymer complexes, which precipitate and do not allow preparation of homogeneous polymeric films by casting. In the present work the effect of pH on the complexation between poly(acrylic acid) and (hydroxypropyl)cellulose in solutions and miscibility of these polymers in solid state has been studied. The pH-induced complexation-miscibility-immiscibility transitions in the polymer mixtures have been observed. The optimal conditions for preparation of homogeneous polymeric films based on blends of these polymers have been found, and the possibility of radiation cross-linking of these materials has been demonstrated. Although the gamma-radiation treatment of solid polymeric blends was found to be inefficient, successful cross-linking was achieved by addition of N,N'-methylenebis(acrylamide). The mucoadhesive potential of both soluble and cross-linked films toward porcine buccal mucosa is evaluated. Soluble films adhered to mucosal tissues undergo dissolution within 30-110 min depending on the polymer ratio in the blend. Cross-linked films are retained on the mucosal surface for 10-40 min and then detach.     

Iodinated salicylate-based poly(anhydride-esters) as radiopaque biomaterials

Poly(anhydride-esters) based on iodinated versions of salicylic acid were synthesized via both melt-condensation and solution polymerization techniques to generate radiopaque biomaterials. The poly(anhydride-esters) from iodinated salicylates were highly X-ray opaque compared to poly(anhydride-esters) from salicylic acid. Molecular weight and Young's modulus of polymers prepared by melt-condensation were typically two-to-three times higher than polymers prepared by solution methods. The glass transition temperatures of the polymers were dependent on the iodine concentration; polymers containing more iodine had higher glass transition temperatures. Cytotoxicity studies using mouse fibroblasts indicated that iodinated salicylate-based poly(anhydride-esters) prepared by both polymerization methods are biocompatible with cells at low polymer concentrations (0.01 mg/mL).     

Chemoenzymatic synthesis of glycopolypeptides carrying alpha-Neu5Ac-(2-->3)-beta-D-Gal-(1-->3)-alpha-D-GalNAc, beta-D-Gal-(1-->3)-alpha-D-GalNAc, and related compounds and analysis of their specific interactions with lectins

Glycopolypeptide (1) carrying the beta-D-Gal-(1-->3)-alpha-D-GalNAc unit as a kind model of asialo-type mucin was synthesized through three steps: enzymatic synthesis of p-nitrophenyl disaccharide glycoside, reduction of the p-nitrophenyl group, and coupling of the amino group with the carboxyl group of poly(L-glutamic acid)s (PGA). In a similar manner, glycopolypeptides (2-7) carrying beta-D-Gal-(1-->3)-beta-D-GalNAc, beta-D-Gal-(1-->3)-beta-D-GlcNAc, beta-D-Gal-(1-->6)-alpha-D-GalNAc, beta-D-Gal-(1-->6)-beta-D-GalNAc, alpha-D-GalNAc, and beta-D-GalNAc, respectively, were synthesized as analogous polymers of polymer 1. Glycopolypeptides 8 and 9 as a mimic of sialo-type mucin were further prepared from polymers 1 and 2 as the acceptor of CMP-Neu5Ac by alpha2,3-(O)-sialyltransferase, respectively. Interactions of these glycopolypeptides with lectins were investigated with the double-diffusion test and the hemagglutination-inhibition assay and in terms of an optical biosensor based on surface plasmon resonance. Polymers 1 and 2 reacted strongly with peanut (Arachis hypogaea) agglutinin (PNA) and Agaricus bisporus agglutinin (ABA). On the other hand, polymers 8 and 9 through sialylation from polymers 1 and 2 reacted with ABA, but did not with PNA. Other polymers 3-7 did not show any reactivity for both the lectins. These results show that PNA acts precisely in an exo manner on the beta-D-Gal-(1-->3)-D-GalNAc sequence, while ABA acts in an endo manner. Polymers 6 and 7 substituted with GalNAc reacted strongly with soybean (Glycine max) agglutinin and Vicia villosa agglutinin B4, regardless of the configuration of the glycosidic linkage. The interaction of all polymers with Bauhinia purpurea agglutinin was much stronger than that of the corresponding sugars. Polymers 8 and 9 reacted with wheat germ (Triticum vulgaris) agglutinin (WGA), to which Neu5Ac residues are needed for binding, but polymers 1 and 2 did not. These sugar-substituted glycopolypeptides interacted specifically with the corresponding lectins. Furthermore, polymers 4-7 reacted with WGA, but the corresponding sugars did not. It suggests that the N-acetyl group along the PGA backbone has a cluster effect for WGA. The artificial glycopolypeptides were shown to be useful as tools and probes of carbohydrate recognition and modeling in the analysis of glycoprotein-lectin interactions.     

Immobilization of Candida rugosa lipase on poly(allyl glycidyl ether-co-ethylene glycol dimethacrylate) macroporous polymer particles

Macroporous polymer particles containing surface epoxy groups were synthesized for immobilization of Candida rugosa lipase (CRL). The effect of incorporation of two different sets of monomers [allyl glycidyl ether (AGE) and glycidyl methacrylate (GMA)] and the effect of crosslinking density on immobilization of lipase were studied. AGE-co-EGDM polymers gave higher binding and expression of lipase than GMA-co-EGDM polymers. Optimization of immobilization parameters was done with respect to immobilization time and enzyme loading. Amongst AGE-co-EGDM polymer series, AGE-150 polymer found to give maximum lipase activity yield and therefore evaluated for temperature, pH and storage stability. Under optimum conditions, AGE-150 polymer gave 78.40% of activity yield. Immobilized lipase on AGE-150 showed a broader pH, higher temperature and excellent storage stability.     

Azobenzene-modified poly(l-glutamic acid) (AZOPLGA): its conformational and photodynamic properties

Azobenzene-modified poly(l-glutamic acid) (AZOPLGA) polymers with 22 and 35 mol % of azo chromophores in the side chains have been synthesized by condensing 4-methoxy-4'-aminoazobenzene and poly(l-glutamic acid). These polymers have been characterized by NMR, FT-IR, and UV-visible spectroscopic techniques. The conformational features of the polymer backbone chains in the films that were cast from the polymer solutions prepared in different solvents have been investigated by circular dichroism spectroscopy. Experimental data suggested that the thermal cis-trans relaxation and photoinduced birefringence, which are related to the azo chromophores in the side chains of polymer, are not affected by the conformations of polymer backbones. However, the modulations of the surface relief gratings, the result of photoinduced mass transport process, recorded on these polymers are sensitive to polymer main chain conformation, as well as the degree of functionalization.     

Purification and characterization of poly(ADP-ribose) glycohydrolase. Different modes of action on large and small poly(ADP-ribose)

Poly(ADP-ribose) glycohydrolase was purified approximately 74,000-fold to apparent homogeneity from calf thymus with a yield of 3.2%. The enzyme was a monomeric protein of Mr = 59,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The action of glycohydrolase on poly(ADP-ribose) was exoglycosidic in the direction of adenosine terminus----ribose terminus; radioactive ADP-ribose monomers were immediately produced from evenly labeled poly(ADP-ribose), but not from the polymer labeled selectively at the ribose terminus. The enzymatic degradation of large poly(ADP-ribose) (greater than 20 ADP-ribose residues) proceeded in a biphasic as well as bimodal manner. In the early and rapid phase, the enzyme degraded part of large polymers successively, leaving the remainder completely intact, and accumulated ADP-ribose monomers and small polymers of the size less than half of original polymers, indicating that the enzyme action was processive up to a certain extent. In the late and 20-fold slower phase, by contrast, the enzyme degraded the accumulated small polymers gradually and evenly, i.e. in a nonprocessive manner. The Km for large polymers was approximately 100-fold lower than that for small polymers. Similar rates and processivities were observed with large and small polymers bound to various proteins. These results suggested that the glycohydrolase may regulate differentially the levels of large and small poly(ADP-ribose) in the cell.     

The helix-coil transitions of poly (dA)-poly (dT) and poly (dA-dT)-poly (dA-dT)

Helix–coil transition curves are calculated for poly (dA) poly(dT) and poly (dA-dT) poly (dA-dT) using the integral equation approach of Goel and Montroll.⁵ The transitions are described by the loop entropy model with the exponent of the loop entropy factor, k, remaining an arbitrary constant. The theoretical calculations are compared with experimental transition curves of the two polymers. Results indicate that the stacking energies for these two polymers differ by about 1 kcal/mole of base pairs. Also, a fit between theory and experiment was not possible for k > 1.70.     

Facile synthesis of multiamino vinyl poly(amino acid)s for promising bioapplications

We presented a general and facile strategy to prepare biocompatible multiamino polymers. Series of new monomers were synthesized by esterification of 2-hydroxyethyl methacrylate (HEMA) and Boc-amino acids, such as Boc-l-phenylalanine, Boc-glycine, Boc-l-alanine, Boc-l-valine, and Boc-l-lysine. Subsequent vinyl polymerization of monomers gave rise to vinyl poly(amino acid)s with a side primary amino group at each unit if deprotected. Both atom transfer radical polymerization (ATRP) and conventional free radical polymerization (FRP) were employed to prepare the multiamino polymers. A well controlled effect upon molecular weight with the standard first-order kinetics was achieved in cases of ATRP, and high molecular weight polymers were obtained via FRP. MTT assay showed that cell survival rates for the multiamino polymers were almost maintained above 90% and that their cytotoxicities were much lower than that of linear PEI (PEI 25000). Zeta potential measurements demonstrated that the vinyl poly(amino acid)s are electropositive, and AFM measurements showed that the vinyl poly(amino acid)s could tightly condense DNA into granular structures at a suitable concentration. The combination of facile availability, controlled productivity, low cytotoxicity and strong binding ability with DNA promises the great potential of the novel multiamino polymers in bioapplications.     

Synthesis of poly(ADP-ribose)-agarose beads: an affinity resin for studying (ADP-ribose)n-protein interactions.

Polymers of ADP-ribose bind chromatosomal histones in solution and may play a role in chromatin accessibility in vivo. We have enzymatically synthesized a poly(ADP-ribose) affinity resin to further characterize binding of nuclear proteins to ADP-ribose polymers. NAD+- and (ADP-ribose)-derivatized agarose beads were recognized as polymer acceptors by the nuclear enzyme poly(ADP-ribose) polymerase. This polymerase elongated the existing ligands by successive addition of exogenously available ADP-ribose residues to form polymers covalently linked to the agarose beads. Poly(ADP-ribose) formation on the beads was dependent on incubation time and the mode of ligand attachment to the agarose. The resulting poly(ADP-ribose)-derivatized agarose beads possessed polymers which closely resembled those modifying the ADP-ribose polymerase by the automodification reaction. Fractionation of rat liver nuclear lysate over the poly(ADP-ribose) resin revealed a strong affinity of H1 for ADP-ribose polymers, thereby supporting a role for poly(ADP-ribose) in chromatin functions. Poly(ADP-ribose)-agarose beads are extremely stable and will be useful not only for affinity studies, but also for mechanistic studies involving polymer elongation and catabolism.     

Anti-Candida and mode of action of two newly synthesized polymers: a modified poly (methylmethacrylate-co-vinylbenzoylchloride) and a modified linear poly (chloroethylvinylether-co-vinylbenzoylchloride) with special reference to Candida albicans and Candida tropicalis

Polymeric antimicrobial agents represent a new and important direction that is developing in the field of antimicrobial agents. Antimicrobial activity of two newly synthesized polymers: a modified poly (methylmethacrylate-co-vinylbenzoylchloride) and a modified linear poly (chloroethylvinylether-co-vinylbenzoylchloride) have been investigated and found to be active. Both polymers have showed a broad antimicrobial activity against C. albicans and C. tropicalis. Minimal inhibitory concentrations (MIC's) for poly (methylmethacrylate-co-vinylbenzoyl chloride) were 100, 75 and 100 microg/ml in case of C. albicans (ATCC 2091), C. albicans (SC5314) and C. tropicalis, respectively. However, polycholoroethylvinylether-covinylbenzoylchloride inhibited C. albicans (ATCC 2091), C. albicans (SC5314) and C. tropicalis with minimum inhibitory concentration values (MIC's) of 150 microg/ml against the three tested Candida strains. Mode of action studies of both polymers on the medically important yeasts, C. albicans and C. tropicalis revealed that poly (methylmethacrylate-co-vinylbenzoylchloride) induced cytotoxicity, DNA damage, and altered cell permeability and morphology, which was manifested as aggregated and swollen yeast cells (C. albicans ATCC 2091) by fluorescent microscopy examination. Poly (chloroethylvinylether-co-vinylbenzoylchloride) increased cell permeability, and respiration for C. albicans and C. tropicalis. The tested polymers at 50 microg/ml had pronounced effects on C. albicans and C. tropicalis cell wall phosphopeptidomannane, proteins, sugars and phosphorus. Generally, the two polymers proved effective against the tested microorganisms, but growth inhibitory effect varied according to the composition of the polymer active group. Many investigators consider polymeric antimicrobial agents as a potential new approach for enhancing the efficiency of some existing antimicrobial agents, including prolonged activity, reduce their toxicity, as well as reduce the environmental issues associated with product use.     

Synthesis and cytotoxicity of salicylate-based poly(anhydride esters)

This paper describes the synthesis and cytotoxicity of poly(anhydride esters) that are composed of several salicylate derivatives, including halogenated salicylates, aminosalicylates, salicylsalicylic acid, and thiolsalicylic acid. The incorporation of these nonsteroidal antiinflammatory drugs (NSAIDs) into a biodegradable polymer backbone yields drug-based polymers that have potential for a variety of applications. The poly(anhydride esters) were synthesized by melt condensation polymerization. The halogenated salicylate derivatives yielded the highest molecular polymers as well as the highest glass transition temperatures. All polymers displayed in vitro degradation lag times from 1 to 3 days, depending on the water solubility of the salicylate derivative. Cell viability and proliferation were determined with L929 fibroblast cells in serum-containing medium to assess the polymer cytotoxicities, which varied as a function of the saliyclate chemistry. Cell morphology was normal for most of the polymers evaluated.     

In vitro assessment of acetic-acid-soluble proteins (glutenin) toxicity in celiac disease

Acetic-acid-soluble storage proteins from gluten of the bread wheat cv. Sprint 3 were fractionated by adsorption chromatography on 2000 Å controlled-pore glass (CPG) beads, and glutenin polymers with molecular mass higher than 10⁷ Da and free from monomeric gliadins were recovered. The glutenin polymers were found to consist of high-molecular-weight (HMW) and low-molecular-weight (LMW) glutenin subunits. Peptic-tryptic (PT) digests of glutenins were examined for their agglutination activity on human myelogenous leukemia K 562(S) cells, agglutination being strongly correlated with toxicity for the celiac intestine. The peptide fraction at a concentration of 1 g/L of culture medium was able to agglutinate 30% of K 562(S) cells, suggesting a moderate toxic effect. This toxicity may be accounted for by homologies in amino acid sequences between glutenin subunits and α/β-and γ-gliadins. © 1997 John Wiley & Sons, Inc.     

The alpha-glycosidic bonds of poly(ADP-ribose) are acid-labile.

The poly(ADP-ribosyl)ation system of higher eukaryotes produces multiple ADP-ribose polymers of distinct sizes which exhibit different binding affinities for histones. Although precipitation with trichloroacetic acid (TCA) is the standard procedure for isolation of poly(ADP-ribose) from biological material, we show here that poly(ADP-ribose) is not stable under acidic conditions. Storage of poly(ADP-ribose) as TCA pellets results in acid hydrolysis of polymers, the extent of which is dependent on storage time and temperature. The alpha-glycosidic, inter-residue bonds are the preferred sites of attack, thus reducing polymer sizes by integral numbers of ADP-ribose to yield artefactually more and smaller polymers than originally present. Therefore, poly(ADP-ribosyl)ation studies involving TCA precipitation, histone extraction with acids, or acidic incubations of ADP-ribose polymers must account for the impact of acids on resulting polymer populations.     

The Specificity of Antibodies Elicited by Poly(dG)-Poly(dC)

Abstract

Antibodies have been raised to the synthetic DNA polymer poly(dG)·poly(dC). These antibodies have the ability to distinguish this right-handed polymer from natural mixed sequence DNA, as well as from other right- and left-handed synthetic DNA polymers. They show reduced but measurable binding to synthetic polymers which contain various combinations of guanine and cytosine polynucleotides suggesting that both helical shape and sequence are recognized by this antiserum.     

A theoretical study of the structures of (Gly-Pro-Leu)n and (Gly-Leu-Pro)n

Theoretical conformation studies have been carried out for the polytripeptides (Gly-Pro-Leu)n and (Gly-Leu-Pro)n and the Fourier transforms of the structures have been calculated. X-ray powder patterns of these polymers had indicated that both these polymers take up coiled-coil triple-helical structures, but in the case of (Gly-Pro-Leu)n it was not clear whether the triple helix is formed by three parallel polypeptide chains or by a single chain folding back on itself (Scatturin et al 1975). Our studies show that both the polytripeptides can take up stereochemically satisfactory triple-helical structures with three parallel chains. There is also very good agreement between the calculated intensity distribution and that of the observed X-ray pattern, in each case.     

Modification of the Low Molecular Weight (LMW) Glutenin Composition of Transgenic Durum Wheat: Effects on Glutenin Polymer Size and Gluten Functionality

The low-molecular weight (LMW) glutenin subunits are major determinants of the viscoelasticity of durum wheat gluten, and therefore of its technological quality, with both quantitative effects and qualitative effects. We have modified the LMW glutenin subunit composition of the durum wheat cultivar Ofanto by expression of a transgene encoding a B-type LMW glutenin subunit and have carried out detailed analyses of two independent transformed lines in order to assess the effect of the transgene on the size distribution of the glutenin polymers and on their functional properties. In one line the expression of the transgene led to an increase in the amount of large glutenin polymers resulting in stronger and more stable dough. In the second line, however, the expression of the transgenic subunit was accompanied by decreased expression of endogenous LMW subunits with consequent detrimental effects on glutenin polymers and dough viscoelasticity. These results demonstrate that the LMW glutenin subunits contribute to the functional properties of wheat by influencing the amount and the distribution of glutenin polymers and indicate that either plant breeding or GM technology can be used to 'fine tune' the properties of durum wheat for different end uses by manipulating the amount and structures of individual LMW subunit proteins.     

聚乙烯醇的生物降解

聚乙烯醇(PVA)是较少的可溶于水并被生物降解的乙烯聚合物之一。研究表明,在受PVA污染的自然环境中存在着能降解PVA的微生物,并从中提取出了PVA降解酶。介绍了国内外研究聚乙烯醇生物降解的情况。分别讨论了聚乙烯醇被单一菌种、共生细菌和真菌降解过程中的生物化学和生理学特性,以及结构因素对聚乙烯醇生物降解的影响。这些研究促进了可有效生物降解的PVA类材料产品项目的发展。