Unlabeled Poly Research Articles

Role of Chain Connectivity across an Interface on the Dynamics of a Nanostructured Block Copolymer.

Fluorescence labeling enables component- and location-specific measurements of the glass transition temperature (T_{g}) in complex polymer systems. Here we characterize the T_{g} of fluorescently labeled poly(methyl methacrylate) homopolymers (PMMA-py) blended at low concentrations into an unlabeled lamellar poly(n-butyl methacrylate-b-methyl methacrylate) diblock copolymer (PBMA-PMMA). In this system, the PMMA-py homopolymer is sequestered within the PMMA domains of the diblock copolymer and subject to soft confinement by the domains of the lower-T_{g} PBMA block, which lowers the homopolymer T_{g} by ∼5 K beyond the contribution of segmental mixing. In contrast to the PMMA block in the diblock copolymer, the PMMA-py homopolymer is not covalently bound to the interdomain interface. A comparison of T_{g} for the homopolymers in the blends to T_{g} for diblock copolymers with equivalent labeled segment density profiles reveals that the homopolymer's T_{g} is consistently ∼10 K higher than for diblock segments at the same location within the domain structure, highlighting the dominant contribution of a covalent bond across the interface to the perturbation of the chain dynamics in the block copolymer.

Monitoring the confinement of methylene blue in pyrene labeled poly(acrylic acid)

This work was devoted to investigate the aggregation of methylene blue (MB) with pyrene labeled poly(acrylic acid) (PAA25Py3) by using the measurements of UV–vis absorption, steady-state and time-resolved fluorescence. For comparison, the effect of unlabeled poly(acrylic acid) (PAA25) on the self-association of MB was also evaluated. The spectral positions of the absorption and emission bands of MB as well as the pyrene monomer and excimer emissions were used to assess the local changes of polymer/MB microenvironment. The binding mechanism and thermodynamic analysis for the interaction between MB and pyrene labeled poly(acrylic acid) were evaluated by fluorescence quenching measurements. Moreover, the strategy of switching of pyrene excimer/monomer emission was used to manage the self-aggregation of the pH-responsive fluorescent polymer with phenothiazinium dye. The UV–vis absorption and fluorescence results showed that low amounts of polymers provoked the self-association of MB. At high concentrations of polymer, the MB monomer is stabilized. The labeling of polymer with pyrene induced a dramatic changing of optical properties suggesting that a peculiar process occurred between PAA25Py3 and MB. At low quencher concentrations, both steady-state and time-resolved fluorescence data indicated a static quenching process. The thermodynamic parameters (∆H, ∆S and ∆G) and the efficiency of pyrene excimer formation revealed that the association of MB with the fluorescent polymer occurred spontaneously due to non-covalent hetero π-π stacking, hydrophobic and electrostatic interactions. The study highlights the key contribution of the π-π stacking interactions in the capturing of methylene blue in pyrene labeled poly(acrylic acid). The outcome may be useful in biomedical applications or in wastewater treatment.

A resonance Rayleigh scattering study of unlabeled and pyrene-labeled poly(acrylic acid) polymers

Three different unlabeled poly(acrylic acid) (PAA) polymers, with molecular weight of 2, 150 and 450 kg/mol, and the correspondent PAA labeled with pyrene (PAAMePy polymers) were investigated, in aqueous solution and in dioxane: water mixtures, using the resonance Rayleigh scattering (RRS) technique. The pH showed to strongly affect the polymer conformation and a decrease of the scattering intensity ( I RRS) was observed, which is consistent with an increase of the hydrodynamic radius, reflecting an expanded polymer conformation in solution. The obtained p K a values were found higher for the labeled by comparison with the corresponding unlabeled polymers. For the PAAMePy polymers the increment of the water content in dioxane:water mixtures leads to an increase of the I RRS intensity; however, for the unlabeled PAA polymers non-significant differences were observed. In general, for the fluorescently labeled polymers, a good agreement between the present RRS results and those previously obtained from fluorescence measurements could be found.

SIMS depth profiling of deuterium labeled polymers in polymer multilayers

Thin planar polymer films are model systems for probing physical phenomena related to molecular confinement at polymer surfaces and polymer/polymer interfaces. Existing experimental techniques such as forward recoil spectrometry (FRES) and neutron reflectometry (NR) have been used extensively for analysis of these systems, although they suffer from relatively low depth resolution (FRES) or difficulties associated with inversion to real space (NR). In contrast, secondary ion mass spectrometry (SIMS) can provide real-space depth profiles of tracer labeled polymers directly with sufficient depth resolution for optimal analyses of these systems. Deuterated polystyrene (dPS) has been employed as the tracer polymer and has been embedded in a matrix of either unlabeled polystyrene (PS) or poly(cyclohexyl methacrylate) (PCHMA). These doped films have been placed on either poly(methyl methacrylate) (PMMA) or poly(2-vinyl pyridine) (P2VP) and thermally annealed. Varied analysis conditions for a magnetic sector SIMS instrument (CAMECA IMS-6f) were used to optimize the depth resolution and detection sensitivity while minimizing matrix effects and sample charging. Both Cs + and O 2 + primary ions have been used along with detection of negative and positive secondary ions, respectively. Impact energy and primary ion species have been shown to affect matrix secondary ion count rate for the various films studied.

Conformational Changes of Pyrene-Labeled Polyelectrolytes with pH: Effect of Hydrophobic Modifications

Structural changes of pyrene-labeled and unlabeled poly(maleic acid/octyl vinyl ether) (PMAOVE) and poly(maleic acid/methyl vinyl ether) (PMAMVE) with changes in pH have been investigated in this study. The changes in the photophysical properties of pyrene are interpreted to investigate uncoiling or swelling of the polymeric chains with pH. The vibrational fine structure of the pyrene fluorescence (I(3)/I(1)) and the ratio between excimer and monomer fluorescence (I(e)/I(m)) of both pyrene-labeled and unlabeled PMAMVE and PMAOVE suggest that, at pH 4, the polymers are in the coiled form and PMAOVE forms hydrophobic nanodomains. An increase in pH ionizes a number of COOH groups on both PMAMVE and PMAOVE, which leads to the stretching or swelling of the polymers.

DNA Elongation by the Human DNA Polymerase λ Polymerase and Terminal Transferase Activities Are Differentially Coordinated by Proliferating Cell Nuclear Antigen and Replication Protein A

DNA polymerase lambda contains template-dependent (DNA polymerase) and template-independent (terminal transferase) activities. In this study we enzymologically characterized the terminal transferase activity of polymerase lambda (pol lambda-tdt). Pol lambda-tdt activity was strongly influenced by the nature of the 3'-terminal sequence of the DNA substrate, and it required a single-stranded (ss) DNA 3'-overhang of about 9-12 nucleotides for optimal activity. The strong preference observed for pyrimidine versus purine nucleotide incorporation was found to be due, at least partially, to a steric block imposed by the residue Tyr-505 in the active site of pol lambda. Pol lambda-tdt was found to be able to elongate a 3'-ssDNA end by two alternative mechanisms: first, a template-independent one resulting in addition of 1 or 2 nucleotides, and second, a template-dependent one where a homopolymeric tract as short as 3 nucleotides at the 3'-end could be used as a template to direct DNA polymerization by a looping back mechanism. Furthermore repetitive cycles of DNA synthesis resulted in the expansion of such a short homopolymeric terminal sequence. Most importantly we found that the proliferating cell nuclear antigen was able to selectively block the looping back mechanism while stimulating the single terminal nucleotide addition. Finally replication protein A completely suppressed the transferase activity of pol lambda while stimulating the polymerase activity, suggesting that proliferating cell nuclear antigen and replication protein A can coordinate the polymerase and the terminal transferase activities of pol lambda.

Gel-based assay for ribonuclease H activity toward unlabeled poly(A)–poly(dT)

Gel-based assay for ribonuclease H activity toward unlabeled poly(A)–poly(dT)

Characterization of RNA determinants recognized by the arginine- and proline-rich region of Us11, a herpes simplex virus type 1-encoded double-stranded RNA binding protein that prevents PKR activation.

The herpes simplex virus Us11 gene product inhibits activation of the cellular PKR kinase and associates with a limited number of unrelated viral and cellular RNA molecules via a carboxyl-terminal 68-amino-acid segment rich in arginine and proline. To characterize the determinants underlying the recognition of an RNA target by Us11, we employed an in vitro selection technique to isolate RNA ligands that bind Us11 with high affinity from a population of molecules containing an internal randomized segment. Binding of Us11 to these RNA ligands is specific and appears to occur preferentially on conformational isoforms that possess a higher-order structure. While the addition of unlabeled poly(I. C) reduced binding of Us11 to a selected radiolabeled RNA, single-stranded homopolymers were not effective competitors. Us11 directly associates with poly(I. C), and inclusion of an unlabeled selected RNA in the reaction reduces poly(I. C) binding, while single-stranded RNA homopolymers have no effect. Finally, Us11 binds to defined, double-stranded RNA (dsRNA) molecules that exhibit greater sequence complexity. Binding to these dsRNA perfect duplexes displays a striking dependence on length, as 39-bp or shorter duplexes do not bind efficiently. Furthermore, this interaction is specific for dsRNA as opposed to dsDNA, implying that the Us11 RNA binding domain can distinguish nucleic acid duplexes containing 2' hydroxyl groups from those that do not. These results establish that Us11 is a dsRNA binding protein. The arginine- and proline-rich Us11 RNA binding domain is unrelated to known dsRNA binding elements and thus constitutes a unique recognition motif that interacts with dsRNA. The ability of Us11 to bind dsRNA may be important for inhibiting activation of the cellular PKR kinase in response to dsRNA.

Preparation of sensitive and specific oligonucleotide probes tailed using terminal transferase and dITP.

An oligonucleotide probe tailed with deoxyadenosine-5'-triphosphate or deoxythymine-5'-triphosphate is detectable with high sensitivity, but has a major drawback--the tail co-hybridizes specifically to complementary sequences. This can be a problem when screening cDNA clones that contain poly(dA) sequences. While it is possible to mask the cDNA tail with unlabeled poly(dA) or poly(A) oligonucleotides, false-positive clones are still produced because complete masking of extremely long (dA) tails is difficult. As a result, only cDNA clones that have extremely long poly(dA) sequences are often obtained by hybridization screening using tailed probes. In this report, we describe an oligonucleotide probe tailed with DIG-labeled nucleotide in combination with deoxyinosine-5'-triphosphate that was highly specific and sensitive to cDNAs. Terminal deoxynucleotidyl transferase efficiently adds dI nucleotides to the 3'-end. The dI of the tails did not pair with any nucleotides under stringent hybridization so that the specificity of hybridization assays remained high without affecting the sensitivity of the test. Colony hybridization experiments demonstrated that there were very few (1 of 80 tested) false positives using this technique. Its use may increase the accuracy of cDNA screening.

Void closure and interdiffusion in latex film formation by photon transmission and fluorescence methods

Steady state fluorescence (SSF) and photon transmission methods were used to study void closure and interdiffusion processes during film formation from hard latex particles. Latex films were prepared separately by annealing pyrene (P y) labeled and unlabeled poly (methyl methacrylate) (PMMA) particles above the glass transition temperature. Direct fluorescence emission of excited pyrene from labeled latex films was monitored as a function of annealing temperature to detect void closure and interdiffusion. The increase in fluorescence intensity against temperature was used to determine the activation energy for viscous flow (Δ H≅47 kcal/mol). The decrease in I op above the void closure temperature was used to produce the backbone activation energy (Δ E≅44 kcal/mol) for the interdiffusing chains. Unlabeled PMMA particles were used to prepare films for UVV measurements. Transmitted photon intensity from these films increased as the annealing temperature was increased. Monte Carlo simulations were performed for photon transmission through a rectangular lattice. The number of transmitted and scattered photons were calculated as a function of disappeared particle–particle interfaces. The increase in the transmitted photon intensity ( I tr) is attributed to the increase in `crossing density' at the junction surface. The backbone activation energy (Δ E) was measured and found to be around 33 kcal/mol for a diffusing polymer chain across the junction surface.

Study of Macromolecular Chain Dynamics in Polymer Complexes by Time-Resolved Fluorescence Spectroscopy

Poly(methyl methacrylate)s labeled with the anthracene fluorophore were prepared by free radical, anionic, and coordination polymerization yielding atactic and syndiotactic polymers. Unlabeled isotactic poly(methyl methacrylate) was prepared by anionic polymerization. Time-resolved fluorescence spectroscopy was used to study polymer association in solution. The time-dependent decays of fluorescence anisotropy show that stereocomplexation causes an increase in rotational correlation times of anthracene fluorophores both embedded in the polymer backbone and attached at the end of the side chain of polymer molecules. The rotational correlation time of anthracene fluorophore in dimethylformamide as a part of stereocomplex is 11.9 and 30 ns in the side chain and embedded in the polymer backbone, respectively, and shorter than 3 ns in noncomplexing solvent.

Study of bulk morphology of polymer latex films using laser confocal fluorescence microscopy

Unlabeled poly(methyl methacrylate) (PMMA) and polystyrene (PSt) latex particles as well as covalently fluorescent dye labeled PMMA latex particles (FPMMA) were prepared by emulsifier-free emulsion polymerization and dispersion polymerization, respectively. The surface and bulk morphology of the polymer latex mixture (FPMMA/PMMA or FPMMA/PSt, weight ratio: 1/99) filmed by evaporation at room temperature was studied by laser confocal fluorescence microscopy (LCFM). It was concluded from the LCFM images that labeled PMMA domains obviously coagulated in PSt matrix not only on the surface but also in the inner layers of the latex film. Furthermore, PMMA was richer in the middle layers but poorer on the surface layer or the layers near to the substrate for the polymer latex films.

Polymer Films from Size Exclusion Chromatography Using A Solvent-Evaporation Interface: Analysis of Polymer Blends

Abstract Two solvent-evaporation interfaces were used for high-temperature and room-temperature size exclusion chromatography (SEC) to deposit fractions as dried polymer films on substrates. In the high-temperature SEC work, polymers of interest included polyethylene labeled with a fluorescent dye (4-nitrobenzo-2-oxa-1,3-diazole (NBD)), unlabeled polystyrene, unlabeled polypropylene, both individually and as blends of these polymers. Fourier-transform infrared (FTIR) spectroscopy, optical microscopy, laser confocal fluorescence microscopy, and fluorescence spectroscopy were used to analyze the films. FTIR spectroscopy results were readily affected by film morphologies. Resulting spectral inaccuracies included Christiansen distortion, wavy baselines, and changes in absorption band ratios. Laser confocal fluorescence microscopy results proved especially misleading with some labeled polyethylene failing to fluoresce. NBD-labeled poly(methyl methacrylate), unlabeled poly(methyl methacrylate), unlabeled polyst...

A novel type of molecular assembly: aggregates formed by molecules with polar chains in nonpolar solvents

The emission spectra and photodimerization of poly(ethylene glycol) oligomers with a naphthalene group as one terminal of each chain (N−P n ) in nonpolar solvents were investigated. The fluorescence spectra of N−P n in isopentane at temperatures below −20°C are dominated by excimer emission. The addition of unlabeled poly(ethylene glycol) to the solutions results in a reduction in the excimer emission and an enhancement of monomer emission. Fluorescence depolarization measurements at 77 K suggest the existence of significant energy migration. The glassy solutions show a strong delayed excimer fluorescence and triplet exxcimer phosphorescence. The photoirradiation yields ‘cubane-like’ photodimer of the naphthalene groups with high quantum yield. All these observations indicate the lipophobic interactions push N−P n chains together to form aggregates.

Poly(U)-binding protein inhibits Drosophila pre-5 S RNA 3'-exonuclease digestion

A approximately 50-kDa protein binds specifically to the 3' terminus of 135-nucleotide Drosophila pre-5 S RNA. Unlabeled poly(U) competes out protein binding and stimulates the activity of a 3'-exonuclease, which eventually degrades the substrate to 120 nucleotides, the size of mature 5 S RNA. In its RNA binding and UV cross-linking properties, the endogenous poly(U)-binding protein resembles human La, an autoantigen that binds the U > 3 3' ends of vertebrate RNA polymerase III primary transcripts. This protein appears to inhibit a 3' exonuclease and could protect 5 S RNA for faithful processing and transport.

RecA protein filaments bind two molecules of single-stranded DNA with off rates regulated by nucleotide cofactor.

To probe the role of nucleotide cofactor in the binding of single-stranded DNA to recA protein, we have developed a sedimentation assay using 5'-labeled 32P-poly(dT).recA.poly(dT) complexes sediment quantitatively when centrifuged at 100,000 x g for 45 min, whereas free poly(dT) remains in the supernatant. In the presence of ATP, between 6 and 7 bases cosediment per recA monomer; but when ADP is present or in the absence of added nucleotide cofactor, only 3-3.5 bases/recA monomer cosediment. In competition experiments in which recA.32P-poly(dT) complexes are incubated with unlabeled poly(dT), we again find 3-3.5 bases of labeled poly(dT) cosedimenting per recA monomer when no nucleotide cofactor is present. However, when the same experiment is performed with ATP, only half of the expected 6-7 bases of labeled poly(dT) remain bound to the DNA, demonstrating that half of the poly(dT) in the complex exchanges rapidly with free poly(dT), whereas the other half equilibrates slowly, like poly(dT) in the absence of nucleotide. The rate of exchange of the second more tightly bound poly(dT) is accelerated when ADP is present. Our observations are rationalized by a model in which each recA protein helical filament binds two strands of poly(dT) with a stoichiometry of 3-3.5 bases/recA monomer/strand.

Degradable Polymers. I. Synthesis, Characterization, and Long-Term in Vitro Degradation of a14C-Labeled Aliphatic Polyester

Abstract Poly(tetramethylene adipate) has been synthesized with and without 14C labeling at different molecular weights. Degradation tests of the labeled powder inoculated with microorganisms showed a decrease in molecular weight (GPC) and first an increase and later a decrease of crystallinity (DSC). These changes also occurred in the inoculated material, and the results indicate that the degradation process was abiotic hydrolysis which first takes place in the amorphous region. The radioactivity measurements showed that the microorganisms are able to degrade further the oligomers formed during the hydrolysis. The rate of degradation is related to the molecular weight. Unlabeled poly(tetramethylene adipate) was extruded and cold drawn to yield oriented fibers. These fibers retained more than 50% of the original tensile strength during 100 days of abiotic in vitro hydrolysis.

Neoantigen of the polymerized ninth component of complement. Characterization of a monoclonal antibody and immunohistochemical localization in renal disease.

A monoclonal antibody to a neoantigen of the C9 portion of the membrane attack complex (MAC) of human complement has been developed and characterized. The distribution of this neoantigen was assessed by indirect immunofluorescence microscopy in nephritic and nonnephritic renal diseases. The antibody (Poly C9-MA) reacted on enzyme-linked immunosorbent assay (ELISA) with a determinant in complement-activated serum that was undetectable in normal human serum (NHS). Zymosan particles incubated in NHS had positive immunofluorescent staining with Poly C9-MA; however, binding of Poly C9-MA was not observed with zymosan particles incubated in sera deficient in individual complement components C3, C5, C6, C7, C8, or C9. Reconstitution of C9-deficient sera with purified C9 restored the fluorescence with Poly C9-MA. Poly C9-MA reacted positively by ELISA in a dose-dependent manner with purified MC5b-9 solubilized from membranes of antibody-coated sheep erythrocytes treated with NHS but not with intermediate complement complexes. Poly C9-MA also reacted in a dose-dependent manner on ELISA and in a radioimmunoassay with polymerized C9 (37 degrees C, 64 h) (poly C9) but not with monomeric C9. Increasing amounts of either unlabeled poly C9 or purified MC5b-9 inhibited the 125I-poly C9 RIA in an identical manner. These studies demonstrate that Poly C9-MA recognizes a neoantigen of C9 common to both the MAC and to poly C9. By immunofluorescence, Poly C9-MA reacted minimally with normal kidney tissue in juxtaglomerular loci, the mesangial stalk, and vessel walls. Poly C9-MA stained kidney tissue from patients with glomerulonephritis in a pattern similar to that seen with polyclonal anti-human C3. In tissue from patients with nonnephritic renal disease--diabetes, hypertension, and obstructive uropathy--Poly C9-MA was strongly reactive in the mesangial stalk and juxtaglomerular regions, tubular basement membranes, and vascular walls. Poly C9-MA binding was especially prominent in areas of advanced tissue injury. Poly C9-MA frequently stained loci where C3 was either minimally present or absent. These studies provide strong evidence for complement activation not only in nephritic but also in nonnephritic renal diseases.

The synthesis and degradation of poly(a)-containing mRNAs in mouse neuroblastoma cells treated with dibutyryl cAMP or with Ro20-1724.

Abstract: The relative rates of synthesis and turnover of poly(A)‐containing mRNAs were determined in the S‐20 (cholinergic) and NIE‐115 (adrenergic) neuroblastoma clones after 3 days treatment with dibutyryl cAMP or the phos‐phodiesterase inhibitor N‐4‐(3‐butoxy‐4‐methoxybenzyl)‐2‐imidazolidinone (Ro20‐1724) in an attempt to correlate transcriptional and posttranscriptional changes with observed cAMP‐induced changes in the differentiated state. Treatment of S‐20 cells with dibutyryl cAMP for 3 days caused a fourfold increase in intracellular cAMP and a 30% decrease in growth rate compared with the levels in control cells. However, there was no difference in the synthesis of the poly(A)‐containing RNAs, relative to the ribosomal RNAs, after either 2‐ or 18‐h labeling with 3H‐adenosine. There was also no increase in the steady‐state mRNA levels. Treatment with Ro20‐1724 caused only a transient twofold increase in intracellular cAMP level when S‐20 cells were plated at a low density and no increase at higher densities although growth was inhibited by 60%. There was, again, no increase in the relative synthesis of poly(A)‐mRNAs with 2‐h label in cells treated with Ro20‐1724, but there was a twofold increase after labeling for 18 h. Optical density measurements, however, showed that this increased incorporation of 3H‐adenosine did not result in an increase in the absolute amount of unlabeled poly(A)‐containing mRNAs. NIE‐115 cells showed an 8‐fold increase in intracellular cAMP after 3 days of treatment with Ro20‐1724 and an 80% inhibition of growth rate. However, the relative rates of synthesis of the poly(A)‐containing mRNAs were identical to those obtained using S‐20 cells. These results show that neither increased intracellular cAMP levels nor growth inhibition necessarily result in a relative increase in the synthesis of the poly(A)‐containing mRNAs in these neuroblastoma clones. There was no change in the synthesis or processing of the poly(A) regions of the mRNAs in the S‐20 control and treated cells. In contrast to results with other cells, the poly(A) profiles were heterogeneous, even after 1 hour of labeling. The average size of the poly(A) region decreased similarly in control and drug‐treated cells after 1, 2 and 18 h labeling.

Proteins of Small Subunits of Rat Liver Ribosomes that Interact with Poly(U)

(1) When rat liver 40 S ribosomal proteins in 6 M urea were were mixed with poly(U) at an appropriate ratio, a precipitate was formed which was also insoluble in the sample solution for two-dimensional acrylamide gel electrophoresis. Analyses by two-dimensional acrylamide gel electrophoresis showed that S7 and S10 proteins (according to our numbering system) had disappeared selectively from the fraction soluble in 6 M urea. These two proteins were present in the fraction insoluble in 6 M urea, and became soluble in the sample solution after treating it with RNase. The results suggest that S7 and S10 proteins have strong affinities for poly(U). When rat liver 40 S subunits were incubated with poly(U), similar results were obtained. (2) After incubation of 40 S subunits with [3H]poly(U) and then with unlabeled poly(U), UV irradiation cross-linked poly(U) to the protein moiety of the 40 S subunit. When the protein fraction insoluble in the sample solution for two-dimensional electrophoresis was prepared from 40 S subunits cross-linked to poly(U) and then subjected to two-dimensional acrylamide gel electrophoresis after RNase treatment, S7 and S10 proteins were detected on the gel. In addition to the S7 protein spot, a triangular area spreading from the spot to the origin contained radioactivity. The results suggest that poly(U) is cross-linked to S7 protein and oligo(U) fragments bound to S7 protein affect its electrophoretic mobility. (3) Ribosomal proteins were prepared from 40 S subunits cross-linked to carrier-free [3H]poly(U) and analyzed by three-dimensional acrylamide gel electrophoresis (Terao, K. & Ogata, K. (1975) Biochim. Biophys. Acta 402, 214--229) after RNase treatment. It was found that S7, S6, and S15 proteins are cross-linked to poly(U). From the results of the present and preceding experiments it is concluded that S7 is the poly(U)-binding protein. The possibility that other proteins in 40 S ribosomal subunits interact with poly(U) is discussed.