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PubMed2004

Neurological proteins are not enriched for repetitive sequences.

Proteins associated with disease and development of the nervous system are thought to contain repetitive, simple sequences. However, genome-wide surveys for simple sequences within proteins have revealed that repetitive peptide sequences are the most frequent shared peptide segments among eukaryotic proteins, including those of Saccharomyces cerevisiae, which has few to no specialized developmental and neurological proteins. It is therefore of interest to determine if these specialized proteins have an excess of simple sequences when compared to other sets of compositionally similar proteins. We have determined the relative abundance of simple sequences within neurological proteins and find no excess of repetitive simple sequence within this class. In fact, polyglutamine repeats that are associated with many neurodegenerative diseases are no more abundant within neurological specialized proteins than within nonneurological collections of proteins. We also examined the codon composition of serine homopolymers to determine what forces may play a role in the evolution of extended homopolymers. Codon type homogeneity tends to be favored, suggesting replicative slippage instead of selection as the main force responsible for producing these homopolymers.

Alanine↗
PubMed1992

Substrate specificity of the Rad3 ATPase/DNA helicase of Saccharomyces cerevisiae and binding of Rad3 protein to nucleic acids.

Rad3 protein from the yeast Saccharomyces cerevisiae is a single-stranded DNA-dependent ATPase which catalyzes the unwinding of DNA.DNA duplexes. In the present studies we have demonstrated that the purified enzyme additionally catalyzes the displacement of RNA fragments annealed to complementary DNA. Quantitative comparisons using otherwise identical partially duplex DNA.DNA and DNA.RNA substrates indicate a significant preference for the latter. Competition for ATPase or DNA helicase activity by various homopolymers suggests that Rad3 protein does not discriminate between ribonucleotide and deoxyribonucleotide homopolymers with respect to binding. However, neither single-stranded RNA nor various ribonucleotide homopolymers supported the hydrolysis of nucleoside 5'-triphosphates. Additionally, Rad3 protein was unable to catalyze the displacement of oligo(dA) annealed to poly(U), suggesting that the catalytic domain of the enzyme is exquisitely sensitive to chemical and/or or conformational differences between DNA and RNA. Hence, it appears that Rad3 protein is not an RNA helicase.

Adenosine Triphosphatases↗
PubMedDate not supplied

[Reiterative poly(A) synthesis on oligonucleotide templates in an Escherichia coli RNA-polymerase system].

The poly(A) synthesis in the E. coli RNA-polymerase system on the oligothymidilates, oligouridilates and oligonucleotides d(5'-OH-CCATCTTTT-3'-OH) and d(5'-HO-TCTTTT-3'-OH) as templates was studied. The reaction was shown to proceed more intensively on the oligothymidilates compared to oligouridilates. The insertion of phosphate residues into 3' end of oligouridilates deteriorates properties of their templates though the length of the synthesized poly(A) is not changed in this case. The synthesis of poly(A) on the nonanucleotide d(CCATCTTTT) proceeds intensively, but there is no AMP incorporation on the hexanucleotide of similar structure. The author's data combined with certain literature results suppose that the template activity of the oligonucleotides containing homopolymer sequences is determined in the RNA-polymerase system in reiterative regime by two factors: by the total amount of oligonucleotide links and by the homopolymer block size. The total amount of links seems to be important for the oligonucleotide enzyme bind stability and must comprise not less than 6 charges of the phosphate groups. The homopolymer block size must be no less than of 4 links.

Base Sequence↗
PubMed1993

Production and characterization of recombinant heteropolymers of human ferritin H and L chains.

Vertebrate ferritins are iron storage proteins composed by 24 subunits of one or more types. The recombinant homopolymers of human ferritin H- and L-type chains differ in iron uptake and in physical stability, but the properties of heteropolymers with various proportions of H- and L-type chains cannot be predicted. Present study shows that unfolded human ferritin H- and L- type chains renature under similar conditions to form homopolymers indistinguishable from the native ones and that, when mixed, the unfolded H and L chains renature to form heteropolymers with restricted heterogeneity and with the expected H:L ratios. Seven of these ferritins with different H:L ratios were analyzed; electrophoretic mobility, immunological reactivity, and stability to guanidine denaturation varied as predicted, based on the homopolymers. In contrast, the rate of iron uptake, monitored by the variation of absorbance at 310 nm, increased in the ferritins that ranged in H chain content from 0 to 35%; further increments in H chains had no additional effect. This finding indicates that, under the present conditions, only a limited number of H chains are needed for the maximum rate of ferritin iron uptake. Variations of L- and H-type chains in vivo may thus have biological relevance.

Circular Dichroism↗
PubMed1996

Use of ammonium halides as co-matrices for matrix-assisted laser desorption/ionization studies of oligonucleotides.

Four ammonium salts, NH4F, NH4Cl, NH4Br and NH4I were tested as co-matrices for the matrix-assisted laser desorption/ionization (MALDI) analysis of a number of DNA homopolymers, including phosphorylated d(T)8, d(A)4, d(C)4 and non-phosphorylated d(G)4, using 2-amino-5-nitropyridine (ANP) matrix. In the present study, all the ammonium halides displayed significant enhancement effects on the signal intensities of the intact molecular ions of the DNA homopolymers. Among the halides used, NH4F was found to exhibit the greatest enhancement effects. By comparison with results obtained using the corresponding isomorphous potassium halides as co-matrices, it is postulated that both the cationic and anionic portions of the co-matrix molecule play important roles in the desorption/ionization of the oligonucleotides under typical MALDI conditions. It was also demonstrated that the ANP matrix exhibits a strong inhibitory effect on the formation of alkali-metal adducts in oligonucleotide analysis.

DNA, Single-Stranded↗
PubMed1999

Mice with disrupted midsized and heavy neurofilament genes lack axonal neurofilaments but have unaltered numbers of axonal microtubules.

Mammalian neurofilaments are assembled from the light (NF-L), midsized (NF-M), and heavy (NF-H) neurofilament proteins. While NF-M and NF-H cannot self-assemble into homopolymers, the data concerning NF-L has been more contradictory. In vitro bovine, porcine, and murine NF-L can homopolymerize in the absence of other subunits. However, in vivo studies suggest that neither rat nor mouse NF-L can form filaments when transfected alone into cells lacking endogenous intermediate filaments. By contrast, human NF-L forms homopolymers in similar cell lines. Recently we generated mice with null mutations in the NF-M and NF-H genes. To determine if mouse NF-L can homopolymerize in mouse axons, NF-M and NF-H null mutants were bred to create a line of double mutant animals. Here we show that axons in NF-M/H double mutant animals are largely devoid of 10-nm filaments. Instead, the axoplasm is transformed to a microtubule-based cytoskeleton-although the lack of any increase in tubulin levels per unit length of nerve or of increases in microtubule numbers relative to myelin sheath thickness argues that microtubules are not increased in response to the loss of neurofilaments. Thus in vivo rodent neurofilaments are obligate heteropolymers requiring NF-L plus either NF-M or NF-H to form a filamentous network.

Animals↗
PubMed1999

Complement activation by PEO-grafted glass surfaces.

Activation of the complement system is one way in which the human body reacts to foreign materials that come in contact with blood. Poly(ethylene oxide) (PEO) has been used quite frequently to modify biomaterial surfaces to prevent protein adsorption and cell adhesion. Despite extensive use of PEO, however, PEO-induced complement activation has not been examined before. We examined the complement activation by PEO chains grafted to glass surfaces. PEO was grafted to trichlorovinylsilane-treated glass (TCVS-glass) by gamma-irradiation using PEO homopolymer, Pluronic F108 (PF108), and PEO-polybutadiene-PEO triblock copolymer (COP5000). Complement activation was assessed by measuring the plasma C3a level. Of the three polymers grafted (PEO, PF108, and COP5000), only PF108 showed significant increases in complement activation over controls. Complement C3a production on PF108-grafted glass was linearly dependent on surface concentration of grafted PF108. The C3a concentration increased from 46 ng/mL to 316 ng/mL as the surface PF108 concentration increased from 0-0.25 microg/cm(2). Kinetics of C3a generation by PF108-grafted surfaces show that 60% of the steady state C3a concentration was generated during the first hour of plasma exposure. When the same PF108-grafted glass surface was repeatedly exposed to fresh plasma, the amount of C3a generated decreased by 70% after the first exposure. This supports the "single-hit" mechanism in complement activation. PEO homopolymer did not activate complement in bulk solution, and, thus, it appears that C3a complement activation by PF108-grafted surfaces is due to the presence of poly(propylene oxide) units. Grafting of PEO using PEO-containing block copolymers requires examination of complement activating properties of the non-PEO segment.

Biocompatible Materials↗
PubMed1999

Comparison of thermal characteristics and degradation properties of epsilon-caprolactone copolymers.

The objective of this study was to enhance the degradation of the homopolymer of epsilon-caprolactone (epsilon-CL) through its copolymerization with a second lactone monomer: delta-valerolactone (delta-VL), gamma-butyrolactone (gamma-BL), gamma-valerolactone (gamma-VL), and gamma-caprolactone (gamma-CL). The influences of the molar ratio and the structure of the second lactone monomers on the molecular weight, melting temperature, crystallinity, and degradation rate of the copolymers were compared and elucidated. The enhancement of degradation of the epsilon-CL polymers was achieved by the addition of a second lactone monomer during the copolymerization process, which prominently depended on the initial molar ratio and the chemical structure of the monomers. The changes in the molecular weight and the melting temperature and the enthalpy of fusion of the copolymers were directly related to the initial molar ratio of two monomers, which further affected the magnitude of the enhancement of the polymer degradation. The degradation rates of copolymers of epsilon-CL/delta-VL with initial molar ratios of 75/25, 80/20, and 85/15% were 7.72, 3.60, and 2.05%/day, respectively. However, the degradation rate of the homopolymer of epsilon-CL was 1. 85%/day. The degradation rates of copolymers of epsilon-CL and three five-member ring lactone monomers were in the order of gamma-BL > gamma-VL > gamma-CL. The number of carbon atoms in the side chain of the lactone monomers determined the hydrophobicity of the polymers, which was not favorable for water penetration causing degradation.

Biocompatible Materials↗
PubMed2000

Thermoreversible copolymer gels for extracellular matrix.

To improve the properties of a reversible synthetic extracellular matrix based on a thermally reversible polymer, copolymers of N-isopropylacrylamide and acrylic acid were prepared in benzene with varying contents of acrylic acid (0 to 3%) and the thermal properties were evaluated. The poly(N-isopropylacrylamide) and copolymers made with acrylic acid had molecular weights from 0.8 to 1.7 x10(6) D. Differential scanning calorimetry (DSC) showed the high-molecular-weight acrylic acid copolymers had similar onset temperatures to the homopolymers, but the peak width was considerably increased with increasing acrylic acid content. DSC and cloud point measurements showed that polymers with 0 to 3% acrylic acid exhibit a lower critical solution temperature (LCST) transition between 30 degrees and 37 degrees C. In swelling studies, the homopolymer showed significant syneresis at temperatures above 31 degrees C. Copolymers with 1 and 1.5% showed syneresis beginning at 32 degrees and 37 degrees C, respectively. At 37 degrees C the copolymers with 1.5-3% acrylic acid showed little or no syneresis. Due to the high water content and a transition near physiologic conditions (below 37 degrees C), the polymers with 1.5-2.0% acrylic acid exhibited properties that would be useful in the development of a refillable synthetic extracellular matrix. Such a matrix could be applied to several cell types, including islets of Langerhans, for a biohybrid artificial pancreas.

Acrylamides↗
PubMed2000

Crystallization inhibition in solid dispersions of MK-0591 and poly(vinylpyrrolidone) polymers.

The effects of poly(vinylpyrrolidone) (PVP) molecular weight, composition, and content on the crystallization of a model drug, MK-0591 (Form I), were investigated. Solid dispersions of crystalline MK-0591 with PVP homopolymers of different molecular weights (2500-1 x 10(6) g/mol) and with a copolymer containing poly(vinyl acetate) (PVA), (PVP/VA, 60:40, 5.8 x 10(4) g/mol) were prepared by the solvent method. MK-0591 in the solid dispersions was found to be X-ray amorphous. One glass transition temperature (T(g)) was observed suggesting drug-polymer miscibility. The T(g) values were higher than predicted by the Gordon-Taylor equation, indicating drug-polymer interactions. The extent of crystallization inhibition increased with PVP molecular weight and, for a comparable PVP molecular weight, the homopolymer was more effective in the crystallization inhibition of the drug than the copolymer. The first onset temperature of crystallization (T(c)(obs)) increased with polymer content. The T(c)(obs) values (normalized to polymer content) were a function of the difference between the T(g) of the polymer and drug. For PVP K-90, K-30, and K-17 dispersions, the T(c)(obs) values increased proportionally to the T(g) of the dispersions. However, for PVP K-12 and PVP/VA, the increase in T(c)(obs) values corresponded to a small decrease in the T(g) values of the dispersions. This result suggests that additional factors other than the reduction in mobility affect the crystallization behavior of MK-0591 in the solid dispersions, such as specific interactions. By Fourier transform-infrared spectroscopy, changes in the carbonyl-stretching band of PVP in the solid dispersions were observed. The existence of an ion-dipole interaction between COO(-)Na(+) of the drug and the cyclic amide group of PVP was postulated.

Crystallization↗
PubMed1989

Hydration of B-DNA: comparison between the water network around poly(dG).poly(dC) and poly(dG-dC).poly(dG-dC) on the basis of Monte Carlo computations.

A computational method is elaborated for studying the water environment around regular polynucleotide duplexes; it allows rigorous structural information on the hydration shell of DNA to be obtained. The crucial aspect of this Monte Carlo simulation is the use of periodical boundary conditions. The output data consists of local maxima of water density in the space near the DNA molecule and the properties of one- and two-membered water bridges as function of pairs of polar groups of DNA. In the present paper the results for poly(dG).poly(dC) and poly(dG-dC).poly(dG-dC) are presented. The differences in their hydration shells are of a purely structural nature and are caused by the symmetry of the polar groups of the polymers under study, the symmetry being reflected by the hydration shell. The homopolymer duplex hydration shell mirrors the mononucleotide repeat. The water molecules contacting the polynucleotide in the minor groove are located nearly in the plane midway between the planes of successive base pairs. One water molecule per base pair forms a water bridge facing two polar groups of bases from adjacent base pairs and on different strands making a "spine"-like structure. In contrast, the major groove hydration is stabilized exclusively by two-membered water bridges; the water molecules deepest in the groove are concentrated near the plane of the corresponding base pair. The alternating polymer is characterized by a marked dyad symmetry of the hydration shell corresponding to the axis between two successive base pairs. The minor groove hydration of the dCpdG step resembles the characteristic features of the homopolymer, but the bridge between the O2 oxygens of the other base-stacking type is formed by two water molecules. The major groove hydration is characterized by high probability of one-membered water bridges and by localization of a water molecule on the dyad axis of the dGpdC step. The found structural elements are discussed as reasonable invariants of a dynamic hydration shell.

DNA↗
PubMed2005

Adsorption of poly(ethylene glycol)-modified ribonuclease A to a poly(lactide-co-glycolide) surface.

Protein adsorption is a source of variability in the release profiles of therapeutic proteins from biodegradable microspheres. We employ optical reflectometry and total internal reflection fluorescence to explore the extent and kinetics of ribonuclease A (RNase A) adsorption to spin-cast films of poly(lactide-co-glycolide) (PLG) and, in particular, to determine how covalent grafting of polyethylene glycol (PEG) to RNase A affects adsorption. Adsorption kinetics on PLG surfaces are surface-limited for RNase A but transport-limited for unconjugated PEG homopolymers and for PEG-modified RNase A, indicating that PEG anchors the conjugates to the surface during the transport-limited regime. PEG modification of RNase A decreases the total number of adsorbed molecules per unit area but increases the areal surface coverage because the grafted PEG chains exclude additional surface area. Total internal reflection fluorescence-based exchange measurements show that there is no exchange between adsorbed and solution-phase protein molecules. This indicates an unusually tenacious adsorption. Streaming current measurements indicate that the zeta potential of the PLG surface becomes increasingly negative as the film is exposed to water for several weeks, as expected. Aging of the PLG surface results in increased adsorption of unmodified RNase A but decreased adsorption of unconjugated PEG homopolymers and of PEG-RNase A conjugates, relative to the extent of adsorption on freshly prepared PLG surfaces. Adsorption results correlate well with an increase in the rate, total extent and preservation of bioactivity of RNase A released from PLG microspheres for the PEG-modified version of RNase A.

Adsorption↗
PubMed1985

Antithrombogenicity and oxygen permeability of block and graft copolymers of polydimethylsiloxane and poly(alpha-amino acid).

A-B-A-type block copolymers consisting of poly(alpha-amino acid) as A component and polydimethylsiloxane as B component and graft copolymers consisting of polydimethylsiloxane as trunk polymer and poly(alpha-amino acid) as branch polymer were synthesized. gamma-Benzyl-L- or DL-glutamate, epsilon-benzyloxycarbonyl L-lysine, and sarcosine were used as alpha-amino acid. Different microphase-separated structures were found on the film surface according to the copolymer composition and the casting conditions. In vitro antithrombogenicity test showed higher antithrombogenicity of block or graft copolymers than homopolymers. The best antithrombogenicity was independent of the kind of alpha-amino acid and the degree of polymerization of copolymers. The best ratio was 65-75% in block copolymer and 40-50% in the case of graft copolymer. The oxygen permeability of block and graft copolymer film was intermediate between those of homopolymers and varied with changing the composition of the copolymer. These experiments showed that the microphase-separated structure on the film surface was most important both for the antithrombogenicity and oxygen permeability of these copolymer films.

Dimethylpolysiloxanes↗
PubMed1986

Suppression of platelet activity on microdomain surfaces of 2-hydroxyethyl methacrylate-polyether block copolymers.

Block copolymers constructed from chains of poly(2-hydroxyethyl methacrylate) (PHEMA) and either poly-ethyleneoxide (PEO) or poly-propyleneoxide (PPO) were synthesized. These block copolymers exhibited microdomain structure. Platelet adhesion on their surfaces was investigated by a column elution method to examine the effect of microdomain structure. The number of platelets adhered from whole blood was smaller for the block copolymer systems than for the homopolymers. Minimum points of platelet adhesion appeared at approximately 0.38 mol fraction of HEMA in the HEMA-PO system. Both block copolymer surfaces showed microdomains of alternate lamellar structure. Furthermore, the percent of platelets released from the column after incubation was investigated using PRP. In the case of homopolymers, released platelet percentages decreased with an increase of incubation time. Released platelet percentages from the block copolymers, however, were nearly constant with changing incubation time. These results show that HEMA-EO and HEMA-PO block copolymers had the ability to suppress both reversible and irreversible adhesion of platelets to their respective microdomain surfaces.

Animals↗
PubMed2003

Adhesion and growth of human Schwann cells on trimethylene carbonate (co)polymers.

Seeding of artificial nerve grafts with Schwann cells is a promising strategy for bridging large nerve defects. The aim of the present study was to evaluate the adhesion and growth of human Schwann cells (HSCs) on 1,3-trimethylene carbonate (TMC) and epsilon-caprolactone copolymers, with the final goal of using these materials in the development of an artificial nerve graft. The adhesion, proliferation, and morphology of HSCs on copolymers containing 10 and 82 mol % of TMC and on the parent homopolymers were investigated. HSCs adhered faster and in greater numbers on the copolymer with 82 mol % of TMC and on the TMC homopolymer compared with the other (co)polymers. On all polymer films, cell adhesion was lower than on gelatin (positive control). Despite differences in cell adhesion, cells displayed exponential growth on all tested surfaces, with similar growth rates. Cell numbers doubled approximately every 3 days on all substrates. When the polymer films were coated with fibronectin, no significant differences in cell adhesion and proliferation were observed between coated polymer surfaces and gelatin. The results indicate that all tested materials support the adhesion and proliferation of HSCs and can in principle be used for the preparation of flexible and slowly degrading nerve guides.

Cell Adhesion↗
PubMed2005

Salicylic acid-derived poly(anhydride-esters) inhibit bone resorption and formation in vivo.

The objective of this study was to investigate the effects of a novel polymer that biodegrades into salicylic acid (SA) on the healing of critical sized long bone defects. Microspheres of the homopolymer, or a copolymer containing 50% less of the SA, were packed into 5-mm mid-diaphyseal defects in rat femurs. Control animals received collagen sponge implants. After 4 and 8 weeks of implantation, bone healing was evaluated using microradiography and quantitative histomorphometry. Four weeks postsurgery, significantly less new bone was formed in both of the polymer groups (p<0.038). Reduced bone loss was also noted with the polymers at this time, although it was not statistically significant. However, at 8 weeks postsurgery, a statistically significant reduction in bone loss was observed in both of the polymer groups compared with controls (p<0.0072). Both polymers seemed to elicit identical tissue responses because there were no differences detected between the homopolymer and copolymer materials at either time point. These results indicate that locally released SA can significantly reduce both bone loss and bone formation in this animal model.

Anhydrides↗
PubMed2006

Investigation into the erosion mechanism of salicylate-based poly(anhydride-esters).

Salicylate-based poly(anhydride-esters), collectively referred to as PolyAspirin, hydrolytically degrade into salicylic acid, a nonsteroidal anti-inflammatory drug (NSAID). The variations of the poly(anhydride-ester) investigated in this article are linked by adipic acid, suberic acid, or sebacic acid. To elucidate the erosion mechanism of these polymers, water uptake, mass loss, contact angle, and changes in device thickness were monitored as a function of in vitro degradation time. The polymers examined here appeared to primarily undergo surface erosion. The adipate homopolymer absorbed the most water and therefore swelled more than the other versions of the poly(anhydride-ester). Additionally, the adipate homopolymer eroded most quickly. Increasing the length of the linker moiety decreased the driving force for hydrolysis, which prolonged the lifetime of the polymer sample.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed2006

Cellular response to phase-separated blends of tyrosine-derived polycarbonates.

Two-dimensional thin films consisting of homopolymer and discrete compositional blends of tyrosine-derived polycarbonates were prepared and characterized in an effort to elucidate the nature of different cell responses that were measured in vitro. The structurally similar blends were found to phase separate after annealing with domain sizes dependent on the overall composition. The thin polymer films were characterized with the use of atomic force microscopy (AFM), water contact angles, and time-of-flight secondary ion mass spectrometry (TOF-SIMS) and significant changes in roughness were measured following the annealing process. Genetic expression profiles of interleukin-1beta and fibronectin in MC3T3-E1 osteoblasts and RAW 264.7 murine macrophages were measured at several time points, demonstrating the time and composition-dependent nature of the cell responses. Real-time reverse transcriptase polymerase chain reaction (RT-PCR) depicted upregulation of the fibronectin gene copy numbers in each of the blends relative to the homopolymers. Moreover, the interleukin-1beta expression profile was found to be compositionally dependent. The data suggest strongly that optimal composition and processing conditions can significantly affect the acute inflammatory and extracellular matrix production responses.

Animals↗