Low and high molecular weight poly(L-lysine)s/poly(L-lysine)-DNA complexes initiate mitochondrial-mediated apoptosis differently

Poly(L-lysine)s, PLLs, are commonly used for DNA compaction and cell transfection. We report that, although PLLs of low (2.9 kDa), L-PLL, and high (27.4 kDa), H-PLL, Mw in free form and DNA-complexed cannot only cause rapid plasma membrane damage in human cell lines, phosphatidylserine "scrambling" and loss of membrane integrity, but later (24 h) initiate stress-induced cell death via mitochondrial permeabilization without the involvement of processed caspase-2. Mitochondrially mediated apoptosis was confirmed by detection of cytochrome c (Cyt c) release, activation of caspases-9 and -3, and subsequent changes in mitochondrial membrane potential. Plasma membrane damage and apoptosis were most prominent with H-PLL. Cytoplasmic level of Cyt c was more elevated following H-PLL treatment, but unlike L-PLL case, inhibition of Bax channel-forming activity reduced the extent of Cyt c release from mitochondria by half. Inhibition of Bax channel-forming activity had no modulatory effect on L-PLL-mediated Cyt c release. Further, functional studies of isolated mitochondria indicate that H-PLL, but not L-PLL, can directly induce Cyt c release, membrane depolarization, and a progressive decline in the rate of uncoupled respiration. Combined, our data suggest that H-PLL and L-PLL are capable of initiating mitochondrially mediated apoptosis differently. The observed PLL-mediated late-phase apoptosis may provide an explanation for previously reported transient gene expression associated with PLL-based transfection vectors. The importance of our data in relation to design of novel and safer cationic non-viral vectors for human gene therapy is discussed.     

Geometrical constraints limiting the poly(ADP‐ribose) conformation investigated by molecular dynamics simulation

Poly(ADP‐ribosylation) is a post‐transductional modification that regulates protein's function. Most of the proteins subjected to this control mechanism belong to machineries involved in DNA damage repair, or DNA interacting proteins. Poly(ADP‐ribose) polymers are long chains of even 100 monomer length that can be branched at several positions but, not withstanding its importance, nothing is known concerning its structure. To understand, which are the geometrical parameters that confer to the polymer the structural constraints that determine its interaction with the target proteins, we have performed molecular dynamics of three chains of different length, made by 5, 25, and 30 units, the last one being branched. Analysis of the simulations allowed us to identify the main intra‐ and inter‐monomer dihedral angles that govern the structure of the polymer that however, does not reach a unique definite conformation. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 78–86, 2014.     

A molecular simulation of the compatibility of chitosan and poly(vinyl pyrrolidone)

The compatibility of chitosan (CS) and poly(vinyl pyrrolidone) was investigated by molecular dynamic (MD) simulations using the Flory–Huggins theory. The specific interactions in blends were studied by the radial distribution function (RDF). The Flory–Huggins interaction parameter, χ, was calculated at 298 K to assess the blend compatibility at different component ratios in the polymers. Miscibility was observed for blends with more than 50% of CS in the molar fraction, while immiscibility was prevalent at the molar fraction of CS between 10 and 50% of CS. Miscibility between poly(N-vinyl-2-pyrrolidone) (PVP) and CS polymers is attributed to the hydrogen bond formation of the –C = O group of PVP and the –CH₂OH groups of CS. This was further confirmed by MD simulations of RDFs for groups or atoms that are involved in interactions. These results are correlated well to obtain more realistic information on interactions involved as a function of blend composition.     

Helix-helix transitions in DNA: fibre X-ray study of the particular cases poly(dG-dC) · poly(dG-dC) and poly(dA) · 2poly(dT)

The helix-helix transitions which occur in poly(dG-dC) · poly(dG-dC) and in poly (dG-m⁵dC) · poly(dG-m⁵dC) are commonly assumed to be changes between the right-handed A- or B-DNA double helices and the left-handed Z-DNA structure. The mechanisms for such transconformations are highly improbable, especially when they are supposed to be active in long polynucleotide chains organised in semicrystalline fibres. The present alternative possibility assumes that rather than the Z-DNA it is a right-handed double helix (S-DNA) which actually takes part in these form transitions. Two molecular models of this S form, in good agreement with X-ray measurements, are proposed. They present alternating C(2′)-endo and C(3′)-endo sugar puckering like the “alternating B-DNA” put forward some years ago. Dihedral angles, sets of atomic coordinates and stereo views of the two S-DNA structures are given, together with curves of calculated diffracted intensities. Furthermore, we question the possibility of obtaining semicrystalline fibres with triple helices of poly(dA) · 2poly(dT) in a way which renders X-ray diffraction efficient. It is suggested that, up to now, only double helices of poly(dA) · poly(dT) can actually be observed by fibre X-ray diffraction measurements. Received: 30 March 1999 / Revised version: 30 June 1999 / Accepted: 30 June 1999     

pH response of conformation of poly(propylene imine) dendrimer in water: a molecular simulation study

This work presents the first molecular dynamics simulation of poly(propylene imine) (PPI) dendrimer in explicit water under various pH conditions. The sizes, shapes, surfaces/volumes and density profiles of the PPI dendrimer are analysed. The PPI dendrimer essentially approaches the perfect sphere under all pH conditions, and higher pH leads to more globular structure. The radius of gyration, solvent-accessible surface area (SASA) and solvent-excluded volume (SEV) are all found to increase significantly from high pH to neutral pH and to level thereafter until low pH, which illustrate the dramatic changes in the whole conformation of the dendrimer. These behaviours of the PPI dendrimer quite differ from those of polyamidoamine [Liu, et al. J. Am. Chem. Soc. 2009, 131, 2798–2799], which can be explained by the favourable interactions arising from the additional amide groups. The density profiles have also been calculated to confirm the shifts of density and back-folding of terminals and penetrations of water.     

Hydrogen bonding in stereoregular poly(methyl methacrylate)/poly(vinyl chloride) blends as studied by molecular dynamics simulations

In this work, two technically important polymer blends composed of isotactic poly (methyl methacrylate) (iPMMA) or syndiotactic poly (methyl methacrylate) (sPMMA) and isotactic poly (vinyl chloride) (iPVC) have been extensively investigated by molecular dynamics simulations. It is confirmed that sPMMA exhibits stronger interactions with iPVC than does iPMMA, and the non-conventional hydrogen bonds (HBs) occur between the two distinct components. Furthermore, the HBs in sPMMA/iPVC are more than those in iPMMA/iPVC, and the structural relaxation of HBs is closely associated with the backbone chain dynamics, which well explain the experimental trends in miscibility of the two systems and in glass transition temperature of single components. It should be noted that these results cannot be directly obtained by the experiments and single simulations, and the multiscale schemes used to prepare the initial all-atomistic configurations can play an important role. This work provides some key clues to improve the performance of polymer materials.     

Biodegradation of poly(l-lactide)

The biodegradation of poly(L-lactide) (PLA) is reviewed. The important role of actinomycetes in PLA degradation is emphasized. These PLA-degrading actinomycetes belong phylogenetically to the Pseudonocardiaceae family and related genera, including Amycolatopsis, Lentzea, Streptoalloteichus, Kibdelosporangium and Saccharothrix. A PLA-degrading enzyme purified from an isolated Amycolatopsis strain-41 has substrate specificity on PLA higher than proteinase K. The application of these strains and their enzymes can be effectively used for biological treatment of plastic wastes containing PLA.     

A kinetic analysis of cyanine selectivity: CCyan2 and Cyan40 intercalation into poly(dA-dT) x poly(dA-dT) and poly(dG-dC) x poly(dG-dC)

A T-jump investigation of the binding of Cyan40 [3-methyl-2-(1,2,6-trimethyl-4(1H)pyridinylidenmethyl)-benzothiazolium ion] and CCyan2 [3-methyl-2-[2-methyl-3-(3-methyl-2(3H)-benzothiazolylidene)-1-propenyl]-benzothiazolium ion] with poly(dA-dT) x poly(dA-dT) and poly(dG-dC) x poly(dG-dC) is performed at I = 0.1M (NaCl), 25 degrees C and pH 7. Two kinetic effects are observed for both systems. The binding process is discussed in terms of the sequence D + P <==> P,D <==> PD(I) <==> PD(II), which leads first to fast formation of a precursor complex P,D and then to a partially intercalated complex PD(I) which converts to the fully intercalate complex PD(II). Concerning CCyan2 the rate parameters depend on the polymer nature and their analysis shows that in the case of poly(dG-dC) x poly(dG-dC) the most stable bound form is the fully intercalated complex PD(II), whereas in the case of poly(dA-dT) x poly(dA-dT) the partially intercalated complex PD(I) is the most stable species. Concerning Cyan40, the rate parameters remain unchanged on going from A-T to G-C indicating that this dye is unselective.     

Microbial synthesis of polyhydroxybutyrate from glycerol: Gluconeogenesis, molecular weight and material properties of biopolyester

Glycerol is considered as an ideal feedstock for producing bioplastics via bacterial fermentation due to its ubiquity, low price, and high degree of reduction substrate. In this work, we study the yield and cause of limitation in poly(3‐hydroxybutyrate) (PHB) production from glycerol. Compared to glucose‐based PHB production, PHB produced by Cupriavidus necator grown on glycerol has a low productivity (0.92 g PHB/L/h) with a comparably low maximum specific growth rate of 0.11 h^?1. We found that C. necator can synthesize glucose from glycerol and that the lithotrophical utilization of glycerol (non‐fermentative substrate) or gluconeogenesis is an essential metabolic pathway for biosynthesis of cellular components. Here, we show that gluconeogenesis affects the reduction of cell mass, the productivity of biopolymer product, and the molecular chain size of intracellular PHB synthesized from glycerol by C. necator. We use NMR spectroscopy to show that the isolated PHB is capped by glycerol. We then characterized the physical properties of the isolated glycerol‐based PHB with differential scanning calorimetry and tensile tests. We found that although the final molecular weight of the glycerol‐based PHB is lower than those of glucose‐based and commercial PHB, the thermal and mechanical properties of the biopolymers are similar. Biotechnol. Bioeng. 2012; 109: 2808–2818. © 2012 Wiley Periodicals, Inc.     

Physicochemical properties of multicomponent poly(hydroxyalkanoates)

The properties of new five-component poly(hydroxyalkanoates) (PHA) formed by short-and medium-chain monomers synthesized by the bacterium Wautersia eutropha B5786 were studied by X-ray diffraction, IR spectroscopy, differential thermal analysis, and viscometry. The degree of crystallinity of PHA decreased from 72 to 57% as the molar fraction of hydroxyhexanoate increased from 2.5 to 18.0 mol%. The melting temperature (T _m) and decomposition temperature (T _d) of the multicomponent PHA are lower than those for poly(hydroxybutyrate), whose T _m and T _d are 168–170 and 260–265°C, respectively. Both parameters of the multicomponent PHA decrease to 156 and 252°C, respectively, as the hydroxyhexanoate mole fraction is raised. The effect of hydroxyhexanoate on the physicochemical properties of the PHA is similar to that of hydroxyvalerate observed previously.     

Modulation of immunogenicity of poly(sarcosine) displayed on various nanoparticle surfaces due to different physical properties

Poly(sarcosine) displayed on polymeric micelle is reported to trigger a T cell‐independent type2 reaction with B1a cells in the mice to produce IgM and IgG3 antibodies. In addition to polymeric micelle, three kinds of vesicles displaying poly(sarcosine) on surface were prepared here to evaluate the amounts and avidities of IgM and IgG3, which were produced in mice, to correlate them with physical properties of the molecular assemblies. The largest amount of IgM was produced after twice administrations of a polymeric micelle of 35 nm diameter ( G1 ). On the other hand, the production amount of IgG3 became the largest after twice administrations of G3 (vesicle of 229 nm diameter) or G4 (vesicle of 85 nm diameter). The augmented avidity of IgG3 after the twice administrations compared with that at the single administration was the highest with G3 . These differences in immune responses are discussed in terms of surface density of poly(sarcosine) chains, nanoparticle size, hydrophobic component of poly(L‐lactic acid) or (Leu‐ or Val‐Aib)_n, and membrane elasticity of the nanoparticles. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Rheology,oxygen transfer,and molecular weight characteristics of poly(glutamic acid) fermentation by Bacillus subtilis

Poly(glutamic acid) (PGA) is a water-soluble, biodegradable biopolymer that is produced by microbial fermentation. Recent research has shown that PGA can be used in drug delivery applications for the controlled release of paclitaxel (Taxol) in cancer treatment. A fundamental understanding of the key fermentation parameters is necessary to optimize the production and molecular weight characteristics of poly(glutamic acid) by Bacillus subtilis for paclitaxel and other applications of pharmaceuticals for controlled release. Because of its high molecular weight, PGA fermentation broths exhibit non-Newtonian rheology. In this article we present experimental results on the batch fermentation kinetics of PGA production, mass transfer of oxygen, specific oxygen uptake rate, broth rheology, and molecular weight characterization of the PGA biopolymer.     

Biosynthesis of poly(3-hydroxybutyrate) copolymers by Azotobacter chroococcum 7B: A precursor feeding strategy

A precursor feeding strategy for effective biopolymer producer strain Azotobacter chroococcum 7B was used to synthesize various poly(3-hydroxybutyrate) (PHB) copolymers. We performed experiments on biosynthesis of PHB copolymers by A. chroococcum 7B using various precursors: sucrose as the primary carbon source, various carboxylic acids and ethylene glycol (EG) derivatives [diethylene glycol (DEG), triethylene glycol (TEG), poly(ethylene glycol) (PEG) 300, PEG 400, PEG 1000] as additional carbon sources. We analyzed strain growth parameters including biomass and polymer yields as well as molecular weight and monomer composition of produced copolymers. We demonstrated that A. chroococcum 7B was able to synthesize copolymers using carboxylic acids with the length less than linear 6C, including poly(3-hydroxybutyrate-co-3-hydroxy-4-methylvalerate) (PHB-4MHV) using Y-shaped 6C 3-methylvaleric acid as precursor as well as EG-containing copolymers: PHB–DEG, PHB–TEG, PHB–PEG, and PHB–HV–PEG copolymers using short-chain PEGs (with n?≤?9) as precursors. It was shown that use of the additional carbon sources caused inhibition of cell growth, decrease in polymer yields, fall in polymer molecular weight, decrease in 3-hydroxyvalerate content in produced PHB–HV–PEG copolymer, and change in bacterial cells morphology that were depended on the nature of the precursors (carboxylic acids or EG derivatives) and the timing of its addition to the growth medium.     

Preparation and PCR-amplification properties of a novel amphiphilic poly(N-vinylpyrrolidone) (PVP) copolymer

A novel amphiphilic copolymer, P(NVP-co-TrpAMT) (9) was prepared, comprising hydrophilic N-vinylpyrrolidone (NVP; 8) and hydrophobic 'N-[(tert-butoxy)carbonyl]tryptophanamido-N'-methacryl thiourea' (TrpAMT; 7) segments. The amphiphilic copolymer 9 was characterized by (1)H-NMR, GPC-MALLS, TEM, and MTT assay. It has a critical micelle concentration (cmc) of 45.7 mg/l in aqueous solution, and good biocompatibility in vivo. According to TEM, the polymer is mostly present as spherical micelles in water, with a diameter of ca. 60-90 nm. In the presence of 0.1 mug/ml of 9, the PCR amplification of the GC-rich beta-actin was efficiently enhanced. Also, the fluorescence intensity of the reporter dye SYBR Green I was increased by 26% at the 14th cycle during real-time PCR of plasmid pUC18 DNA.     

A study of conformational stability of poly(L-alanine), poly(L-valine), and poly(L-alanine)/poly(L-valine) blends in the solid state by (13)C cross-polarization/magic angle spinning NMR

13C cross-polarization/magic angle spinning (CP/MAS) NMR and (1)H T(1rho) experiments of poly(L-alanine) (PLA), poly(L-valine) (PLV), and PLA/PLV blends have been carried out in order to elucidate the conformational stability of the polypeptides in the solid state. These were prepared by adding a trifluoroacetic acid (TFA) solution of the polymer with a 2.0 wt/wt % of sulfuric acid (H(2)SO(4)) to alkaline water. From these experimental results, it is clarified that the conformations of PLA and PLV in their blends are strongly influenced by intermolecular hydrogen-bonding interactions that cause their miscibility at the molecular level.     

Degradation of poly(3-hydroxybutyrate) by soil streptomycetes

The ability of 64 soil streptomycetes to degrade poly(3-hydroxybutyrate) [P(3HB)] was evaluated on Pridham and Lyons mineral salts agar medium overlayered with the same medium containing 0.2% P(3HB). The streptomycete isolates were grown on this overlayered medium and the degradation was detected by the formation of clear zone surrounding the growth. Four potent degrader isolates identified as species of Streptomyces were selected. Degradation of P(3HB) by these isolates was studied for a period of 8 days. The rate of degradation increased with increase in concentration of P(3HB) in the medium while it decreased with the supplementation of readily utili- zable carbon sources like glucose, fructose and sucrose. All four isolates also degraded the copolymer of 3-hydroxybutyrate and 3-hydroxyvalerate [P(3HB–co–3HV)] in solid medium but to a lesser extent. However, the isolates were equally efficient in degrading P(3HB) in liquid medium.     

聚3-羟基丁酸酯（PHB）生物降解过程的研究

利用DS9701菌株对聚3-羟基丁酸酯(PHB)膜进行降解,对降解到不同程度的PHB膜采用扫描电子显微镜观察其表面形态结构的变化,并对其降解产物进行分析测定.结果表明,PHB的生物降解首先发生在PHB表面的非晶部分,随后结晶部分开始降解,并且降解首先发生在球晶的中心部分.DS9701菌株所产生的PHB解聚酶主要降解PHB的第二个酯键,降解产物为二聚体.     

Conformational transitions and hydration of poly d (A-T) · poly d (A-T) in fibers

Conformational transitions of poly d(A-T) · poly d (A-T) have been studied by fiber X-ray diffraction and measurement of fiber dimensions. Results obtained for the D-A-B and D-B transitions are presented and analyzed. For all these form transitions, cooperativity effects are observed for the variation of the rise per nucleotide versus the relative humidity. Detailed information about hydration of the polynucleotide during form transitions and the numbers of water molecules per nucleotide necessary to stabilize the different helical conformations are presented. Offprint requests to: S. Premilat     

Investigation of the role of hydrophilic chain length in amphiphilic perfluoropolyether/poly(ethylene glycol) networks: towards high-performance antifouling coatings

The facile preparation of amphiphilic network coatings having a hydrophobic dimethacryloxy-functionalized perfluoropolyether (PFPE-DMA; M _w = 1500 g mol^?1) crosslinked with hydrophilic monomethacryloxy functionalized poly(ethylene glycol) macromonomers (PEG-MA; M _w = 300, 475, 1100 g mol^?1), intended as non-toxic high-performance marine coatings exhibiting antifouling characteristics is demonstrated. The PFPE-DMA was found to be miscible with the PEG-MA. Photo-cured blends of these materials containing 10 wt% of PEG-MA oligomers did not swell significantly in water. PFPE-DMA crosslinked with the highest molecular weight PEG oligomer (ie PEG1100) deterred settlement (attachment) of algal cells and cypris larvae of barnacles compared to a PFPE control coating. Dynamic mechanical analysis of these networks revealed a flexible material. Preferential segregation of the PEG segments at the polymer/air interface resulted in enhanced antifouling performance. The cured amphiphilic PFPE/PEG films showed decreased advancing and receding contact angles with increasing PEG chain length. In particular, the PFPE/PEG1100 network had a much lower advancing contact angle than static contact angle, suggesting that the PEG1100 segments diffuse to the polymer/water interface quickly. The preferential interfacial aggregation of the larger PEG segments enables the coating surface to have a substantially enhanced resistance to settlement of spores of the green seaweed Ulva, cells of the diatom Navicula and cypris larvae of the barnacle Balanus amphitrite as well as low adhesion of sporelings (young plants) of Ulva, adhesion being lower than to a polydimethyl elastomer, Silastic T2.