Simple Poly Research Articles

Aggregated polyglutamine peptides delivered to nuclei are toxic to mammalian cells

A number of observations point to the aggregation of expanded polyglutamine [poly(Q)]-containing proteins as playing a central role in the etiology of Huntington's disease (HD) and other expanded CAG-repeat diseases. Transfected cell and transgenic animal models provide some of this support, but irrefutable data on the cytotoxicity of poly(Q) aggregates is lacking. This may be due in part to difficulties in observing all aggregated states in these models, and in part to the inability to conclusively rule out the role of monomeric states of the poly(Q) protein. To address these questions, we produced aggregates of simple poly(Q) peptides in vitro and introduced them to mammalian cells in culture. We find that Cos-7 and PC-12 cells in culture readily take up aggregates of chemically synthesized poly(Q) peptides. Simple poly(Q) aggregates are localized to the cytoplasm and have little impact on cell viability. Aggregates of poly(Q) peptides containing a nuclear localization signal, however, are localized to nuclei and lead to dramatic cell death. Amyloid fibrils of a non-poly(Q) peptide are non-toxic, whether localized to the cytoplasm or nucleus. Nuclear localization of an aggregate of a short, Q(20), poly(Q) peptide is just as toxic as that of a long poly(Q) peptide, supporting the notion that the influence of poly(Q) repeat length on disease risk and age of onset is at the level of aggregation efficiency. The results support a direct role for poly(Q) aggregates in HD-related neurotoxicity.

Polyglutamine aggregation behavior in vitro supports a recruitment mechanism of cytotoxicity

In expanded CAG repeat diseases such as Huntington’s disease, proteins containing polyglutamine (poly(Gln)) sequences with repeat lengths of about 37 residues or more are associated with development of both disease symptoms and neuronal intranuclear inclusions (NIIs). Disease physiology in animal and cellular models does not always correlate with NII formation, however, and the mechanism by which aggregate formation might lead to cytotoxicity is unknown. To help evaluate various possible mechanisms, we determined the biophysical properties of a series of simple poly(Gln) peptides. The circular dichroism spectra of poly(Gln) peptides with repeat lengths of five, 15, 28 and 44 residues are all nearly identical and are consistent with a high degree of random coil structure, suggesting that the length-dependence of disease is not related to a conformational change in the monomeric states of expanded poly(Gln) sequences. In contrast, there is a dramatic increase in both the kinetics and the thermodynamic favorability of the spontaneous formation of ordered, amyloid-like aggregates for poly(Gln) peptides with repeat lengths of greater than 37 residues. At the same time, poly(Gln) peptides with repeat lengths in the 15–20 residue range, despite their poor abilities to support spontaneous, self-nucleated aggregation, are capable of efficiently adding to an already-formed aggregate. We also find that morphologically small, finely divided aggregates are much more efficient at recruiting poly(Gln) peptides than are large aggregates, suggesting a possible explanation for why disease pathology does not always correlate with the observable NII burden. Together, these data are consistent with a model for disease pathology in which critical cellular proteins possessing poly(Gln) sequences of modest length become inactivated when they are recruited into aggregates of an expanded poly(Gln) protein.

Synthesis and Electrochemical Behaviour of Vinylferrocene-Propylene Sulfide-Graft Copolymers

Poly(vinylferrocene)–graft–poly(propylene sulfide) copolymers have been synthesized and were used to form surface-modified gold electrodes (SMEs) by self-assembly and drop-evaporation techniques. The electrochemical behaviours and stabilities of the polymer films in the SMEs in 1 M H2SO4 media were investigated by cyclic voltammetry. The SMEs exhibited the expected electrochemical response and it was confirmed that the electrontransfer reactions occurred on the surface of the electrode. Repeated cycling of new SMEs indicated that films containing sulfur groups were more robust than similar SMEs based on simple poly(vinylferrocene). The stability of the SMEs increased with increasing proportions of propylene sulfide in the graft polymers.

Counterion Distribution in the Coronal Layer of Polyelectrolyte Diblock Copolymer Micelles

With a view to describe the structural arrangement of the tetramethylammonium (TMA + ) counterions in simple salt-free aqueous poly(styrene-block-acrylic acid) (PS(20)-b-PA(85)) solutions, the partial structure factors pertaining to PS-PS, PS-TMA, and TMA-TMA density correlations were obtained with small-angle neutron scattering and isotopic labeling of the counterion. The contributions to the scattering related to the PA-blocks are blanked by contrast matching in water. The copolymers self-assemble with an aggregation number 100 into spherical micelles made of a PS-block core, surrounded by a coronal layer formed by the PA-blocks. At the present level of ionization (50%), the PA chains are almost fully stretched in the radial direction away from the core. A comparison of the counterion involved partial structure factors with the previously reported relevant structure factors pertaining to the PA-blocks shows that the counterion distribution along the radius is very close to the one for the segments of the corona-forming blocks. Within a 10% error margin, all counterions are trapped in the coronal layer, and there is no indication for charge annealing effects and/or counterion migration toward the outer corona region.

A composite polyadenylation signal with TATA box function.

A variant polyadenylation signal, which is conserved and employed by mammalian hepadnaviruses, has a sequence resembling that of the TATA box. We report here that this composite box manifests all the promoter characteristics. It binds effectively TATA-binding protein with TFIIB and TFIIA in a synergistic manner. This capacity, however, is lost when the box is converted to a canonical and simple poly(A) signal. Furthermore, we show that it has promoter activity and supports transcription of reporter genes preferentially in liver-derived cells, a characteristic behavior of the hepatitis B virus (HBV) promoters. In addition, we show that the HBV noncanonical poly(A) signal supports transcription initiation from the viral genome, suggesting that it is a genuine promoter, possibly of the polymerase/reverse transcriptase gene. Finally, we found that this deviant poly(A) signal is crucial for HBV replication since a viral mutant with a canonical poly(A) box is impaired in replication. Our data, therefore, raise the interesting and novel possibility that a composite poly(A) box might have a dual function. At the level of DNA it functions as a promoter to initiate transcription, whereas at the level of RNA it serves as a poly(A) signal to process RNA. An interesting outcome of this strategy of gene expression is that it provides a novel mechanism for the synthesis of an approximately genome length transcript.

Effect of stretching on the molecular conformation of oligo (ethylene oxide): a theoretical study

The force-extension measurements on simple poly(ethylene glycol) molecules by Oesterfelt et al in different solvents can be quantitatively explained based on ab initio quantum mechanical calculations of the potential energy surfaces of the polymer segments in vacuum and in the solvated form. The proper statistical mechanical calculations of the force-extension relation, both for isothermal-isochoric and isothermal-isobaric conditions (the latter appropriate to the experimental set-up), demonstrate, that in the low-force regime transitions from the amorphous to the helical conformers, and in the high-force regime stretching of the helical to the planar `all-trans' conformer occur. The presence of water affects all but the `all-trans' conformer by shortening and stiffening.

MALDI-TOF in the Characterizations of Dendritic−Linear Block Copolymers and Stars

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry was utilized to study simple poly(ethylene glycol)s (PEG) and a series of amphiphilic copolymers prepared from PEG and dendritic molecules. For the amphiphilic copolymers with branched dendritic structures, MALDI-TOF spectrometry affords more accurate molecular weight data than the conventional gel-permeation chromatography (GPC). For mass lower than 10 000, the molecular weight distribution of the polymer is well-resolved into individual peaks. Using MALDI-TOF in the linear mode, copolymers with molecular masses of up to 43 000 Da were analyzed. For various dendrons attached to the same PEG, a good correlation was observed between calculated and measured data for the expected incremental increase as a function of dendrimer generation. End-group analysis using MALDI-TOF mass spectrometry proved very useful for the analysis of polymers with relatively low molecular weights. The experimental results agree well with the calculated masses of selected oligomers. Such end-group analysis can differentiate between the AB dendritic−linear diblocks, ABA triblocks, and linear PEG. These analyses support our earlier finding that the Williamson ether synthesis utilized in the PEG−dendron coupling reaction indeed converts all of the PEG to the desired block copolymer products.

Simple poly(p-xylylene)s as precursor polymers for poly(p-phenylenevinylene)s and segmented poly(p-phenylenevinylene)s: synthesis, polymer analogous reactions, and thermal and optical properties

The preparation of a high molecular weight soluble derivative of poly(p-xylylene) by vapor phase pyrolysis followed by chemical vapor deposition (CVD) and its dehydrogenation with 1,2-dichloro-3,4-dicyanoquinone (DDQ) to yield the corresponding derivative of poly(phenylenevinylene) is reported. Derivatives of poly(phenylenevinylene)s with nonconjugated segments were prepared by partial dehydrogenation with substoichiometric amounts of DDQ. The thermal and optical properties of segmented and nonsegmented poly(p-phenylenevinylene)s are compared as well as their electroluminescence properties with particular focus on the quantum yields.

Simple poly(p-xylylene)s as precursor polymers for poly(p-phenylenevinylene)s and segmented poly(p-phenylenevinylene)s: synthesis, polymer analogous reactions, and thermal and optical properties

The preparation of a high molecular weight soluble derivative of poly(p-xylylene) by vapor phase pyrolysis followed by chemical vapor deposition (CVD) and its dehydrogenation with 1,2-dichloro-3,4-dicyanoquinone (DDQ) to yield the corresponding derivative of poly(phenylenevinylene) is reported. Derivatives of poly(phenylenevinylene)s with nonconjugated segments were prepared by partial dehydrogenation with substoichiometric amounts of DDQ. The thermal and optical properties of segmented and nonsegmented poly(p-phenylenevinylene)s are compared as well as their electroluminescence properties with particular focus on the quantum yields.

Irregularities of distributions with respect to polytopes

In the first part of the paper we show that the L2-discrepancy with respect to squares is of the same order of magnitude as the usual L2- discrepancy for point distributions in the K-dimensional torus. In the second part we adapt this method to obtain a generalization of Roth's [7] lower bound (log N)(k-1)/2 (for the usual discrepancy) to the discrepancy with respect to homothetic simple convex poly topes.

Isothermal lamellar thickening and the distribution of thermal stability in spherulitic isotactic poly(4-methylpentene-1)

The thickening of polymer lamellae which occurs when a melt-crystallized sample is heated within its melting range has been investigated for the morphologically simple polymer poly(4-methylpentene-1) using transmission electron microscopy following permanganic etching. It is shown that, even for an isothermally crystallized sample, the lamellar melting point varies repetitively and systematically with position in the spherulitic texture, being highest for dominant lamellae then progressively decreasing because, it is suggested, of increasing constraints during crystallization. Two thickening processes have been distinguished experimentally. At the beginning of the melting range, the least stable lamellae melt and, given time, recrystallize at greater thicknesses. However, the usual interpretation of a doubly peaked melting endotherm in terms of original and transformed lamellae is shown to be oversimplified. At a higher annealing temperature, between the two peaks of the melting endotherm, a population of lamellae forms which is never melted yet increases its average thickness in the solid state logarithmically with time by a second process operative for all lamellae.

Dynamic light scattering study of poly(di-n-butyl itaconate) in the glass transition region

Dynamic light scattering (DLS) studies of several polymers were carried out in the bulk state at temperatures just above the glass transition using a multi-tau correlator. The principal focus was to characterize the viscoelastic relaxation spectrum of poly(di-n-butyl itaconate) (PdnBI), a thermorheologically complex polymer with a pronounced secondary relaxation process due to motions of the alkyl side chains. These data were compared with corresponding observations of two thermorheologically simple polymers, polystyrene (PS), and poly(vinyl acetate) (PVAc) whose DLS properties have been previously investigated. The complete DLS spectra of PS and PVAc can be obtained in a single multi-tau record and are reasonably consistent with earlier literature data, provided appropriate scaling to the glass transition temperature is made. The DLS spectrum of PdnBI requires splicing of two multi-tau records and thus exhibits a wider distribution of relaxation times than those of PS and PVAc. The shape of the PdnBI spectrum appears to be independent of temperature suggesting that there is a strong coupling of the side- and main-chain motions in the glass transition region.

A CD determination of the alpha-helix contents of the coat proteins of four filamentous bacteriophages: fd, IKe, Pf1, and Pf3.

The CD spectra of four filamentous bacteriophages--fd, IKe, Pf1, and Pf3--were analyzed to determine the alpha-helix contents of their major coat proteins. Measured spectra included the 192-nm band so that analyses could be carried out over the full wavelength range of the reference spectra for protein secondary structures available (a) from globular proteins [J.T. Yang, C.S.C. Wu, and H.M. Martinez (1986) Methods in Enzymology 130, 208-269] and (b) from poly(L-lysine) [N. Greenfield and G.D. Fasman (1960) Biochemistry 8, 4108-4116]. Extended analyses were also performed with the addition of the spectrum of a model beta-turn to the Greenfield and Fasman reference set, with the spectrum of a short alpha-helix in the Yang et al. reference set, and with an estimate of the spectrum of Trp added to both reference sets. The reference set based on the simple poly(L-lysine) polypeptide, plus a spectrum of a model beta-turn or of Trp, gave reasonably good fits to the measured spectra for all four phages and yielded the largest percentages of alpha-helix. The class I phages--fd and IKe--had large percentages of alpha-helix of 98 +/- 2 and 97 +/- 5%, respectively, while the two class II phages--Pf1 and Pf3--had similar but smaller alpha-helix contents of 83 +/- 6 and 84 +/- 2, respectively. While these alpha-helix contents were within the ranges previously reported from CD spectra of these phages in solution, they were more precise, and they indicated that the coat proteins of the intact phages have CD spectra that are probably modeled better by the reference spectra of polypeptides than by those of globular proteins.

The polythiophene paradox

Cyclic voltammetry and constant current oxidation show that polythiophene is not stable at potentials used for its synthesis. In contrast to bithiophene electropolymerization of thiophene is very concentration dependent. Even at high monomer concentrations yields for polythiophene are less than 60% of those for polybithiophene (corrected for the bigger monomer). UV/VIS and IR spectra for neutral and overoxidized polythiophene or polybithiophene are almost identical. From this we conclude that the polymer from thiophene electrooxidation is not the simple poly(2,5-thienylene) but a mixture of that and its overoxidation product.

Titrations of non-ionic surfactants with sodium tetraphenylborate using simple potentiometric sensors

A method is described for the determination of non-ionic surfactants containing poly(oxyethylene) chains with sodium tetraphenylborate, based on the precipitation of ternary compounds in the presence of bivalent metal ions with an ionic radius greater than 100 pm [barium(II) salts preferred]. Titrations were monitored potentiometrically with a simple poly(vinyl chloride) membrane coated aluminium wire electrode plasticised with 2,4-dinitrophenyl octyl ether. The value of the potentiometric titration break depended on the length of the poly(oxyethylene) chains and on the type of cation present in excess. Stoicheiometric ratios (number of oxyethylene units per titrant molecule) could be corrected for compounds containing shorter chains on the basis of high-performance liquid chromatographic analyses. The results of 52 sets of titrations indicate that an average of 5.2 oxyethylene units correspond to one tetraphenylborate ion, i.e., 10.4 units to each Ba2+ ion. The possible conformation of the ternary complexes is discussed. For the analysis of detergents or other samples of practical importance, the establishment of empirical titration equivalents is recommended.

Strand-specific transcription of the simple sequences poly[(dG-dT):(dC-dA)] and poly[(dG-dA):(dC-dT)] in D. melanogaster embryos revealed by S 1 nuclease treatment

The transcription of different strands of the simple sequences poly[(dG-dT):(dC-dA)] and poly[(dG-dA):(dT-dC)] in Drosophila melanogaster embryos was studied. After selective labelling of one strand, the polymers were hybridized with immobilized RNA and hybrids were treated on filters with S 1 nuclease. Appropriate control has shown that this treatment destroys sandwich-like complexes and leaves only true hybrids. Transcription of poly[(dG-dA):(dT-dC)] proved to be nearly symmetric, while poly(dG-dT) is transcribed 3-times more intensively than poly(dA-dC). Our approach allowed us to estimate the relative content of simple sequences in RNA.

Nuclear proteins from Drosophila melanogaster embryos which specifically bind to simple homopolymeric sequences poly [(dT-dG).(dC-dA)

The binding of nuclear proteins from Drosophila melanogaster embryos to simple homopolymeric DNA sequences was studied. Nuclear proteins were electrophoresed, transferred onto nitrocellulose and incubated with labelled synthetic homopolymers or natural fragment containing simple sequences. Several protein bands were found in the 65-72 KDa region, which specifically bind both poly [(dG-dT).(dA-dC)] and a natural fragment containing 40 bp of this sequence. These proteins do not bind to homopolymers poly [(dA).(dT)] and poly [(dG-dA).(dC-dT)], or other foreign DNAs.

Simple repetitive sequences in the genomes of archaebacteria

Stretches of simple sequences poly(dG-dT) · poly(dC-dA) · poly(dG-dA) · poly(dC-dT) · poly(dG) · poly(dC) and poly(dA) · poly(dT), the occurrence of which is a characteristic feature of eukaryotic genomes, are found in the genomes of archaebacteria Halobacterium halobium and Sulfolobus acidocaldarius. In S. acidocaldarius these sequences constitute a considerable portion of the genome; they belong to a class of repetitive sequences dispersed throughout the genome, being transcribed and found in RNAs of different lengths.

Chemical and biological evaluation of heparinized poly(amido-amine) grafted polyurethane

By a simple process poly(amido-amine) chains have been grafted onto the surface of polyurethane. The poly(amido-amine) was found to be able to complex heparin by electrostatic interaction. Heparin can be released only at pH > 10 with NaOH solution. The heparin adsorbing capacity of the material was biologically tested, and the anticoagulant activity of the heparinized polyurethane was demonstrated.

The simple repeat poly(dT-dG).poly(dC-dA) common to eukaryotes is absent from eubacteria and archaebacteria and rare in protozoans.

Genomic DNA from a wide variety of prokaryotic and eukaryotic organisms has been assayed for the simple repeat sequence poly(dT-dG).poly(dC-dA) by Southern blotting and DNA slot blot hybridizations. Consistent with findings of others, we have found the simple alternating sequence to be present in multiple copies in all organisms in the animal kingdom (e.g., mammals, reptiles, amphibians, fish, crustaceans, insects, jellyfish, nematodes). The TG element was also found in lower eukaryotes (Saccharomyces cerevisiae, Neurospora crassa, and Dictyostelium discoideum) and at a much lower frequency in protozoans (Oxytricha fallux and Tetrahymena thermophila). The sequence was also repeated in high copy number in a higher plant (Zea mays) as well as at very high levels in a unicellular green alga (Chlamydomonas reinhardi). Although the copy number of the repeat per haploid genome was generally proportional to genome size, there was a greater-than-1,000-fold variation in the number of (TG)25/100-kb genomic DNA. By contrast, no eu-or archaebacterium--including Myxococcus xanthus, whose life cycle is very similar to that of the slime mold Dictyostelium discoideum, and Halobacter volcanii, whose genome contains other repeated sequences--was found whose genomic DNA contained this sequence in detectable amounts. A computer search also failed to find the TG element in human mitochondrial DNA.