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PubMed1991

Adhesion of Escherichia coli on to a series of poly(methacrylates) differing in charge and hydrophobicity.

The adhesion of three Escherichia coli strains on to six poly(methacrylates) differing in hydrophobicity and surface charge was measured as a function of time under laminar flow conditions. Polymers used were poly(methyl methacrylate) (PMMA), poly(hydroxyethyl methacrylate) (PHEMA) and copolymers of MMA or HEMA with either 15% methacrylic acid (MAA) or 15% trimethylaminoethyl methacrylate-HCl salt (TMAEMA-Cl). Bacterial and polymer surfaces were characterized by means of water contact angles and zeta potentials. Both the sessile drop contact angles and the zeta potentials of the bacterial surfaces were significantly different. No significant differences in the sessile drop contact angles of the polymer surfaces were observed. Using the Wilhelmy plate technique large contact angle hysteresis was observed for the different polymer surfaces. Surfaces of copolymers with MAA had more negative zeta potentials than those of the corresponding homopolymers. Surfaces of copolymers with TMAEMA-Cl had positive zeta potentials. The highest numbers of adherent bacteria were found on materials with positive zeta potentials, irrespective of the bacterial strain used. Bacterial adhesion on to copolymers with MAA was less than on to the corresponding homopolymers. Bacterial equilibrium adhesion values correlate with the zeta potentials of the polymer surfaces (r greater than 0.85). On substrates with less negative zeta potentials high numbers of adhered bacteria were observed. Additionally, the equilibrium bacterial adhesion values could be related with receding contact angles of polymer surfaces with negative zeta potentials (r greater than 0.86). High equilibrium adhesion values were obtained for polymers with high contact angles. No correlation between the zeta potentials and contact angles of the bacteria with the adhesion values was found.

Bacterial Adhesion↗
PubMed1988

Specific dissociation of alpha B subunits from alpha-crystallin.

Exposure of bovine alpha-crystallin to 0.1 M glycine at pH 7 decreases the average molar mass of the protein from 700 to 420 kDa. When the pH is lowered to 2.5, in the same buffer, the alpha B chains specifically dissociate from the aggregates, leaving a particle of 290 kDa containing only alpha A chains. The decrease in the molar mass corresponds to the mass of the alpha B chains in the original aggregate. The pH-dependent dissociation is fully reversible. Similar changes were observed with rat and kangaroo alpha-crystallins but the dogfish protein was not affected. Sedimentation velocity analyses and fluorescence spectroscopy yielded a pK, for the dissociation, of 3.7 for alpha-crystallin and 4.0 for a homopolymer constructed from purified alpha B2 polypeptides. An alpha A2 homopolymer was virtually unaffected by the lowering of pH. The products from the dissociation were isolated and their properties studied by sedimentation analysis and acrylamide quenching of tryptophan fluorescence. The alpha B chains were found to be completely denatured, whereas the structure of the alpha A chains, in the 290 kDa, particle, were only slightly altered. Comparisons of the sequences of the various proteins examined suggested that decreased ionization of aspartic acid 127 in the alpha B chain was responsible for the specific dissociation of this polypeptide.

Animals↗
PubMed1990

Inhibition of plant calcium-dependent protein kinases by basic polypeptides.

Wheat embryo Ca2+-dependent protein kinase (CDPK) is inhibited by a variety of polypeptides including actin, gramicidin S, melittin, protamine, various histone preparations, histone H4 and by basic amino-acid homopolymers. Melittin (Ki 9 microM) is a non-competitive inhibitor of wheat germ CDPK and also inhibits wheat leaf CDPK and silver beet leaf CDPKs. Protamine inhibits wheat germ CDPK in an apparently competitive fashion (Ki 0.2 microM) and is also a potent, albeit less effective, inhibitor of the leaf CDPKs. Various basic amino-acid homopolymers are also potent, apparently competitive inhibitors of wheat embryo CDPK, namely poly(L-lysine) (IC50 2 nM), poly(L-ornithine) (IC50 3 nM) and poly(L-arginine) (IC50 17 nM) and also inhibit the leaf CDPKs, albeit at higher concentrations. Histone H4 and various calf thymus histone preparations inhibit wheat embryo CDPK in a fashion that is not competitive and calmodulin can substantially reverse such inhibition.

Hydrogen-Ion Concentration↗
PubMed1991

Nearly 80% of cystic fibrosis heterozygotes and 64% of couples at risk may be detected through a unique screening of four mutations by ASO reverse dot blot.

A technique allowing the simultaneous screening of the four main CF mutations in the French population (delta F508, delta I507, G542X, S549N) has been developed by means of allele sequence-specific oligonucleotide (ASO) reverse dot blot. Using a strategy proposed by R. K. Saïki et al. (1989, Proc. Natl. Acad. Sci. USA 86: 6230-6234) for HLA-DQA, the seven ASOs for normal and mutant CF alleles were given a homopolymer T tail with terminal deoxyribonucleotidyltransferase and then immobilized on a nylon membrane. T-tail homopolymers were preferentially bound to the nylon, leaving the specific ASO sequences free to hybridize with amplified and radiolabeled exons 10 and 11 of a patient. These exons were simultaneously coamplified by a multiplex PCR and radiolabeled by random priming. ASO reverse dot blot currently appears to be the most efficient, rapid, and economic means of screening the population for CF mutations. This screening can detect nearly 80% of carriers and 64% of couples at risk and could prevent the birth of CF-affected infants in these families.

Alleles↗
PubMed1991

Antibody labeling of bovine neurofilaments: implications on the structure of neurofilament sidearms.

We carried out immunolabeling studies of purified bovine spinal cord neurofilaments (NFs) and filaments reconstituted from several combinations of the NF triplet polypeptides, NF-H, NF-M, and NF-L. Six antibodies with known epitopes in either the rod domains or the tailpiece extensions of the NF triplet were used in these studies, and the immune complexes were visualized directly by the glycerol-spray, rotary shadowing technique, which permitted unambiguous identification of the NF sidearms. Antibodies directed against the tailpiece extensions of NF-H and NF-M labeled the sidearms of native NFs and reconstituted filaments containing those two polypeptides, but not the backbone of the filaments. Combining these two antibodies in the same labeling experiment resulted in more intense labeling than either of the antibodies alone, indicating that both NF-H and NF-M are capable of forming sidearms. The anti-NF-L tailpiece antibody recognized only a limited number of sites along native NFs, but labeled reconstituted NF-L homopolymers uniformly and heavily. This suggests that the NF-L tailpiece extension is relatively inaccessible in native filaments, but is accessible in reconstituted homopolymers. One possible explanation is that, in native NFs, the NF-H- and NF-M-containing sidearms curtailed antibody access to NF-L. A second possibility that is not mutually exclusive with the first is that, when both NF-L and another triplet polypeptide are present, they preferentially form heterodimers such that the NF-L tailpiece epitope becomes hidden. Taken collectively, and in combination with published structural information, our data are consistent with a subunit packing scheme in which an NF-L-containing dimer serves as the fundamental building block of most mammalian NFs, such that their sidearms consist of pairs of NF-H/NF-L, NF-M/NF-L, or NF-L/NF-L tailpiece extensions.

Amino Acid Sequence↗
PubMed2004

A continuous nonradioactive assay for RNA-dependent RNA polymerase activity.

Current assays for the activity of viral RNA-dependent RNA polymerases (RdRps) are inherently end-point measurements, often requiring the use of radiolabeled or chemically modified nucleotides to detect reaction products. In an effort to improve the characterization of polymerases that are essential to the life cycle of RNA viruses and develop antiviral therapies that target these enzymes, a continuous nonradioactive assay was developed to monitor the activity of RdRps by measuring the release of pyrophosphate (PP(i)) generated during nascent strand synthesis. A coupled-enzyme assay method based on the chemiluminescent detection of PP(i), using ATP sulfurylase and firefly luciferase, was adapted to monitor poliovirus 3D polymerase (3D(pol)) and the hepatitis C virus nonstructural protein 5B (NS5B) RdRp reactions. Light production was dependent on RdRp and sensitive to the concentration of oligonucleotide primer directing RNA synthesis. The assay system was found to be amenable to sensitive kinetic studies of RdRps, requiring only 6nM 3D(pol) to obtain a reliable estimate of the initial velocity in as little as 4 min. The assay can immediately accommodate the use of both homopolymer and heteropolymer RNA templates lacking uridylates and can be adapted to RNA templates containing uridine by substituting alpha-thio ATP for ATP. The low background signal produced by other NTPs can be corrected from no enzyme (RdRp) controls. The effect of RdRp/RNA template preincubation was assessed using NS5B and a homopolymer RNA template and a time-dependent increase of RdRp activity was observed. Progress curves for a chain terminator (3(')-deoxyguanosine 5(')-triphosphate) and an allosteric NS5B inhibitor demonstrated the predicted time- and dose-dependent reductions in signal. This assay should facilitate detailed kinetic studies of RdRps and their potential inhibitors using either standard or single-nucleotide approaches.

Allosteric Regulation↗
PubMed2006

Stability of casein mRNA is ensured by structural interactions between the 3'-untranslated region and poly(A) tail via the HuR and poly(A)-binding protein complex.

The maintenance of mRNA stability has emerged as a mechanism of post-transcriptional control. We demonstrate that beta-casein mRNA stability is dictated by the poly(A) tail and sequences in the 3'-UTR. An in vitro mRNA decay assay revealed that beta-casein mRNA with a long poly(A) tail had higher stability than that with a short poly(A) tail. The addition of poly(A) homopolymer and 3'-UTR cRNA as competitor induced rapid degradation of beta-casein mRNA. The interaction between full-length beta-casein mRNA and poly(A) homopolymer was inhibited by the addition of the 3'-UTR cRNA in the lysates, which indicates that one region of the 3'-UTR associates with the poly(A) tail through an RNA-protein interaction. The putative AU-rich element (ARE) is present at nt 897-905; deletion and mutation analysis showed that the ARE site was required for maintaining the stability of the beta-casein 3'-UTR. In the immunoprecipitation analysis, the poly(A)-binding protein (PABP) and the RNA-binding protein HuR were pulled down by 3'-UTR cRNA, and the absence of the ARE site reduced the binding of these proteins. These experiments further revealed that the rapid degradation of beta-casein mRNA was induced by incubation with HuR- and PABP-depleted RRLs. Collectively, our results suggest that beta-casein mRNA is protected from degradation by virtue of the structural interaction between the 3'-UTR and poly(A) tail via a protein complex of HuR and PABP.

3' Untranslated Regions↗
PubMed2005

Glucose permeable poly (dimethyl siloxane) poly (N-isopropyl acrylamide) interpenetrating networks as ophthalmic biomaterials.

Poly (dimethyl siloxane) (PDMS) has been widely used as a biomaterial in ophthalmic and other applications due to its good compatibility, high mechanical strength, excellent oxygen permeability and transparency. However, for use as an artificial cornea, contact lens and in other applications, modifications with hydrophilic functional groups or polymers are necessary to improve wettability for tear protein and mucin interactions and to improve glucose permeability for cellular health. Poly (N-isopropyl acrylamide) (PNIPAAM) is a biocompatible and hydrophilic polymer that has been extensively studied on controlled drug release applications due to its lower critical solution temperature (LCST) phenomenon. In the current work, a composite interpenetrating network (IPN) of PDMS and PNIPAAM was formed to generate polymers with oxygen and glucose permeability as well as improved wettability compared to PDMS homopolymers and greater mechanical strength than PNIPAAM homopolymers. Transparent vinyl and hydroxyl terminated PDMS/PNIPAAM IPNs (PDMS-V and PDMS-OH IPNs, respectively) were successfully synthesized. Transmission electron microscopy images verified the structure of the IPNs. Surface analysis suggested that PNIPAAM was present on the surface as well as in the bulk material. PDMS-OH IPNs generated from a PDMS-OH matrix cured in the presence of solvent had the highest glucose permeability at 10(-7)cm2/s, comparable to that of the native cornea. The LCST phenomenon remained in these materials, although changes were not as abrupt as with pure PNIPAAM. These results suggest that these materials may be further developed as ophthalmic biomaterials or for controlled drug-release applications.

Acrylic Resins↗
PubMed2005

Graft copolymerization of methyl methacrylate onto Bombyx mori initiated by semiconductor-based photocatalyst.

Methyl methacrylate (MMA) was graft copolymerized onto Bombyx mori fibre (natural silk). The graft copolymerization was carried out by photopolymerization of MMA using semiconductor particles (CdS) as photocatalyst in the presence of visible light. The effect of additives like triethylamine (Et(3)N) and ethylene glycol on graft copolymerization was studied. We have achieved 2-10% graft conversion with 10-20% homopolymer formation. After removal of the homopolymer, the graft copolymer (grafted fibre) was characterized by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC). The chemical resistance of grafted fibre was compared with virgin one.

Animals↗
PubMed2004

Efficiency of polyethylenimines and polyethylenimine-graft-poly (ethylene glycol) block copolymers to protect oligonucleotides against enzymatic degradation.

Enzymatic instability of oligonucleotides (ON) is one of the major drawbacks of this new class of therapeutic agents. The development of safe, efficient delivery systems capable of stabilizing and protecting these molecules within the formulation, as well as during application, is a challenge in modern gene therapy. In the present study, polyethylenimine (PEI) of different molecular weights and PEGylated PEI block copolymers (PEI-g-PEG) were investigated with regard to their protective properties when complexes with chemically unmodified DNA (d-ON) and RNA (r-ON) oligonucleotides. PEI/ON complexes were incubated with different amounts of serum or nucleases. The influence of pH on the stability was studied and the integrity of the ON was determined by gel electrophoresis. The amount of stable ON within the gels was quantified via densitometric analysis. PEI homopolymers ranging from 800 to 2 kDa protected both types of ON very efficiently, whereas PEI 0.8 kDa demonstrated a slight decrease in protection. The PEGylated PEI derivatives generally protected ON as efficiently as the PEI homopolymers. In particular, the PEI-g-PEG derivative containing 100 PEG chains of 550 Da yielded the highest protection efficiency for both d-ON and r-ON. In general, the highest protection could be achieved at pH 6.7. The ratio of polymer and ON (N/P ratio) also had a great impact on ON stability with higher N/P ratios achieving a better protection. In conclusion, PEIs showed advantageous protective properties for ON. The results of this study offer indications for a rational design of PEI derivatives for the protection and the delivery of ON.

Enzyme Stability↗
PubMed2004

Investigation of the molecular interactions in a pMDI formulation by atomic force microscopy.

The forces of interaction between inhalable formoterol fumarate dihydrate particles, and the various components of a pressurised Metered Dose Inhaler (pMDI) (e.g. aluminium container, other drug particles and stabilising excipients) were investigated in isolation and combination in the model propellant 2H, 3H perfluoropentane (HPFP). The results obtained offer conclusive proof that the model systems tested have a degree of instability both in the presence and absence of polymer(s). The attractive forces measured following the addition of a mixed homopolymer solution may well be weak enough to be overcome by shaking. The use of homopolymers has inherent difficulties associated with it in the form of bridging interaction. Block or comb copolymers may well be a better option therefore, as they will reduce the possibility of bridging interactions. AFM offers great insight into the behaviour of HFA drug suspensions, and could play a vital role in the future development of suspension formulations.

Bronchodilator Agents↗
PubMed2005

A surface-mediated origin of the RNA world: biogenic activities of clay-adsorbed RNA molecules.

The involvement of clay surfaces in the origin of the first genetic molecules on Earth has long been suggested. However, the formation of these polymers was not sufficient by itself to initiate the evolutionary process leading to the appearance of life. These macromolecules had to persist in primeval habitats so that their biological potentiality could be expressed. In this study, we assess the possibility of development of the RNA world on a clay substrate by investigating the capacity of different RNA molecules adsorbed/bound on the clay minerals montmorillonite (M) and kaolinite (K) to persist in the presence of a degrading agent (RNase-A), to interact specifically with complementary RNA strands, and to transmit the information contained in their nucleotide sequences. The RNase-A degradation of clay-adsorbed 23S rRNA from Escherichia coli was significantly slower (75-80%) than that observed for free rRNA, and the complete digestion of nucleic acid in the presence of clay was obtained in 2 vs. 1 h. Clay-adsorbed Poly[A] homopolymer was able to recognize the complementary Poly[U] homopolymer present in the surrounding water solution and to establish a specific interaction (association) with it, possibly leading to the formation of double strands. Reverse transcription and amplification (RT-PCR) amplification of free and clay-adsorbed 16S indicated that the presence of clay particles partially reduced the efficiency and processivity of reverse transcriptase but did not inhibit its activity, demonstrating that clay-adsorbed RNA is still available for enzymatic replication. These findings indicate that primordial genetic molecules adsorbed on clay minerals would have been protected against degrading agents present in the environment and would have been in the right conditions to undergo evolutionary processes.

Aluminum Silicates↗
PubMed2005

Synthesis of polyhydroxyalkanoates from different short-chain fatty acids by mixed cultures submitted to aerobic dynamic feeding.

The production of polyhydroxyalkanoates from acetate and propionate by two mixed cultures well adapted to each of these substrates was evaluated. Sludge fed with acetate (A), produced a homopolymer of hydroxybutyrate (HB), whereas sludge fed with propionate (P) produced a copolymer of HB and HV (hydoxyvalerate). Switching the substrate feeds, propionate to sludge A and acetate to culture P, a terpolymer of HB, HV and hydroxymethylvalerate (HMV) was obtained with culture A and a copolymer of P(HB/HV) by sludge P. Regardless of the population used, the polymer yield and productivity were much higher for acetate than for propionate. Feeding a mixture of acetate and propionate, in equal parts, to both cultures resulted in an increase of HV units produced per C mol of propionate consumed, relative to the situation where only propionate was used. The individual use of butyrate and valerate by culture A was also studied. Butyrate produced a homopolymer whereas valerate was stored as a terpolymer of P(HB/HV/HMV). The polymer yields on acetate and butyrate were higher than those on propionate and valerate. The polymer productivity was higher for acetate and propionate than for butyrate and valerate. Results showed that the polymer composition, and consequently the polymer properties, could be manipulated by varying the volatile fatty acid feed composition and/or the population.

Acetic Acid↗
PubMed2004

Synthesis of biodegradable multi-block copolymers of poly(L-lysine) and poly(ethylene glycol) as a non-viral gene carrier.

Biodegradable and non-toxic multi-block copolymers based on poly(L-lysine) and poly(ethylene glycol) were synthesized. Synthesized copolymers showed almost negligible cytotoxicity above 95% cell viability and transfection efficiency compared to the PLL homopolymer with molecular weight of 25,700. Biodegradation under physiological conditions revealed that the molecular weight of copolymers decreased to 20% of the initial molecular weight within 72 h. Transfection efficiencies of copolymers were not affected by the presence of serum, while that of PLL homopolymer decreased to the level of naked DNA in the presence of serum. Based on the results, the new copolymers are believed to be a potentially efficient carrier for the delivery of bioactive agents.

Cell Line, Transformed↗
PubMed2005

Investigation of pH-dependent DNA-metal ion interactions by surface plasmon resonance.

Ni(II) and Zn(II) M-DNA formation and denaturation of double-stranded DNA (dsDNA) by Cd(2+) were monitored by surface plasmon resonance (SPR). When exposed to immobilized 30 bp 50% GC dsDNA, Zn(2+) and Ni(2+) were found to give signals indicative of a conformational change at pH 8.5 but not 7.5, while Mg(2+) and Ca(2+) caused small changes at both pHs. The concentrations that gave 50% of the maximum responses were 0.06 and 0.50 mM for Zn(2+) and Ni(2+), respectively. At pH 8.5, Cd(2+) denatured over 40% of the dsDNA, while other metals denatured less than 5% of the DNA. Smaller pH-dependent signals were induced by Zn(2+), Ni(2+) or Cd(2+) with 50% GC single-stranded DNA (ssDNA), and with a homopolymer of d(T)30. Homopolymers d(A)30 and d(C)30 showed small signals that were largely independent of pH in the presence of Zn(2+) or Ni(2+).

DNA↗
PubMed2004

Oligomeric assemblies of the Escherichia coli MalT transcriptional activator revealed by cryo-electron microscopy and image processing.

MalT, the dedicated transcriptional activator of the maltose regulon in Escherichia coli, is the prototype for a family of large (approximately 100 kDa) transcriptional activators. MalT self-association plays a key role in recognition of the target promoters, which contain several MalT sites that are cooperatively bound by the activator. The unliganded form of MalT is monomeric. The protein self-associates only in the presence of both ATP (or AMP-PNP, a non-hydrolysable analog of ATP) and maltotriose, the inducer. Here, we report cryo-electron microscopy analyses of MalT multimeric forms. We show that, in the presence of maltotriose and AMP-PNP, MalT associates into novel, polydisperse, curved homopolymers. The building block, corresponding to a MalT monomer, comprises an outer globular domain connected by a peduncle to an inner domain that mediates self-association. Image analyses highlight the significant conformational flexibility of these polymeric forms. In the presence of a DNA fragment containing a MalT-controlled promoter, malPp500, MalT forms homopolymers with a much smaller radius of curvature and a different conformation. We propose that MalT binding to the target promoters involves the assembly of a MalT homo-oligomer that is governed by the array of MalT sites present.

Cryoelectron Microscopy↗
PubMed2005

Loop entropy and cytochrome c stability.

The loop entropy model proposes that loop closure in a protein becomes entropically more costly as the length of the loop increases. A model protein, cytochrome c, is composed of four loops connecting five helices surrounding a heme-containing core. To test the loop entropy model a series of mutant proteins are constructed with (Gly)n or (Thr)n segments (n = 4-20) inserted between Gly23 and Gly24 of omega loop A of a pseudo wild-type reference protein. Scanning calorimetry shows that protein stability decreases as n increases in the (Gly)n or (Thr)n segment. The dependence of stability on loop length is analyzed with the loop entropy model. Fitting to the model gives a quantitative description of stability differences for the mutant proteins, but with a smaller power-dependence of the probability of loop closure (c-value) than expected from polymer theory. A possible explanation for the discrepancy is that thermodynamically unfavorable loop entropy is partially offset by interactions between the inserted homopolymer and flanking heteropolymer portions of the unfolded protein. The interactions may involve molecular crowding that favors coalescence of the heteropolymer at the insert site and thus closure of the homopolymer loops, possibly as an aspect of the folding code. This may allow use of loop insert mutants to assess the strength of the heteropolymer-encoded folding signals that facilitate loop closure at the insert site.

Amino Acid Sequence↗
PubMed2002

Biochemical observations on medium-chain-length polyhydroxyalkanoate biosynthesis and accumulation in Pseudomonas mendocina.

Certain Pseudomonads are capable of accumulating high levels of medium-chain-length polyhydroxyalkanates (PHAmcl) when grown with carbohydrates as the main carbon source. 3-OH acyl components of PHAmcl are derived from fatty acid synthase (FAS) and these components are accessed by action of 3-hydroxyacyl-acyl carrier protein (ACP)-coenzyme A (CoA) transferase (transacylase). However, little is known with regard to the time courses of 3-OH acyl component occurrence and of transacylase activity during PHAmcl induction. Also, little is known with regard to the coupling mechanism between FAS and PHAmcl synthesis or whether the FAS pathway itself is specialized in PHAmcl-producing cells. Our results with regard to the time course of formation of 3-OH acids, 3-OH acyl-ACPs, and PHAmcl are consistent with the view that transacylase provides the key link between FAS and PHAmcl synthase. They also suggest that FAS specialization is not a feature of the mechanism. Further, we observed the formation of a 3-OH 10:0 homopolymer early in the induction phase followed by later formation of a mixed polymer containing 3-OH 8:0 and 3-OH 12:0 in addition to 3-OH 10:0. Early occurrence of 3-OH 10:0-CoA transacylase activity was coincident with homopolymer formation.

Acetyltransferases↗