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PubMed1995

Malarial haemozoin/beta-haematin supports haem polymerization in the absence of protein.

Malarial parasites growing inside erythrocytes digest up to 80% of the host cell's haemoglobin within a lysosomal organelle, the digestive vacuole. They sequester the potentially toxic haem (Fe (II) protohaematoporphyrin) that is released during this process into an insoluble pigment called haemozoin, which consists of polymerized Fe (III) protohaematoporphyrin subunits. We have studied this process of haem polymerization, which was previously reported to be enzyme-mediated and the target of the quinoline antimalarial drugs chloroquine and quinine. Here we show that, rather than being enzyme-mediated, haem polymerization is actually a chemical process, dependent only on the presence of haem-derived material associated with haemozoin and not on protein. This discovery does not invalidate haem polymerization as a target for drug intervention and the mechanism by which haemozoin formation is initiated is still not understood, but our view of this process and of the action of choroquine must be reconsidered.

Animals↗
PubMed2004

Polymerization within a molecular-scale stereoregular template.

Enzymes efficiently synthesize biopolymers by organizing monomer units within regularly structured molecular-scale spaces and exploiting weak non-covalent interactions, such as hydrogen bonds, to control the polymerization process. This 'template' approach is both attractive and challenging for synthetic polymer synthesis, where structurally regulated molecular-scale spaces could in principle provide solid-phase reaction sites for precision polymerization. Previously, free-radical polymerization of methyl methacrylate in solutions containing stereoregular isotactic (it) or syndiotactic (st) poly(methyl methacrylate) (PMMA) has been shown to result in template synthesis of the opposite PMMA based on stereocomplex formation with van der Waals interactions. However, using the structure of a solid to determine the stereochemical structure of a polymer has not been satisfactorily achieved. Here we show that macromolecularly porous ultrathin films, fabricated by a single assembly step, can be used for the highly efficient stereoregular template polymerization of methacrylates through stereocomplex formation. This reaction mould accurately transfers its structural properties of stereoregularity, molecular weight and organization within the template to the new polymer.

Journal Article↗
PubMed2002

Microscopic analysis of polymerization dynamics with individual actin filaments.

The polymerization-depolymerization dynamics of actin is a key process in a variety of cellular functions. Many spectroscopic studies have been performed in solution, but studies on single actin filaments have just begun. Here, we show that the time course of polymerization of individual filaments consists of a polymerization phase and a subsequent steady-state phase. During the steady-state phase, a treadmilling process of elongation at the barbed end and shortening at the pointed end occurs, in which both components of the process proceed at approximately the same rate. The time correlation of length fluctuation of the filaments in the steady-state phase showed that the polymerization-depolymerization dynamics follow a diffusion (stochastic) process, which cannot be explained by simple association and dissociation of monomers at both ends of the filaments.

Actins↗
PubMed2001

Integration of microfabricated needle-type glucose sensor devices with a novel thin-film Ag/AgCl electrode and plasma-polymerized thin film: mass production techniques.

We developed an integrated array of needle-type biosensors employing a novel process of fabrication, comprising conventional semiconductor fabrication and micromachining technology. Amperometric sensing electrodes with plasma-polymerized films and a thin-film Ag/AgCl reference electrode were directly integrated on a glass substrate with thin-film process, e.g., sputtering. An enzyme was immobilized on the electrode via the plasma-polymerized film, which was deposited directly on the substrate using a dry process. The novel thin-film Ag/AgCl reference electrode showed stable potentials in concentrated chloride solutions for a long period. The plasma-polymerized film is considered to play an important role as an interfacial design between the sensing electrode and the immobilized enzyme considering that the film is extremely thin, adheres well to the substrate (electrode) and has a highly cross-linked network structure and functional groups, such as amino groups. The results showed increments of the sensor signal, probably because the plasma-polymerized film allowed a large amount of enzyme to be immobilized. The greatest advantage is that the process can permit the mass production of high-quality biosensors at a low cost.

Biosensing Techniques↗
PubMed2002

13C kinetic isotope effects in the copper(I)-mediated living radical polymerization of methyl methacrylate.

Carbon-13 kinetic isotope effects (KIEs) have been determined for free-radical and copper-mediated living radical polymerizations of methyl methacrylate at 60 degrees C. While free-radical polymerization shows only one primary 13C KIE, on the least-substituted double bond carbon (k12/k13 = 1.045), two significant KIEs are observed, one on each double bond carbon, for copper-mediated polymerization (k12/k13(H2C=) = 1.050, k12/k13(=C <) = 1.010), showing that copper-mediated living radical polymerization does not propagate via a simple free radical process.

Journal Article↗
PubMed2004

Micro patterning of cell and protein non-adhesive plasma polymerized coatings for biochip applications.

Micro scale patterning of bioactive surfaces is desirable for numerous biochip applications. Polyethyleneoxide-like (PEO-like) coating with non-fouling functionality has been deposited using low frequency AC plasma polymerization. The non-fouling properties of the coating were tested with human cells (HeLa) and fluorescence labeled proteins (isothiocyanate-labeled bovine serum albumin, i.e. FITC-BSA). The PEO-like coatings were fabricated by plasma polymerization of 12-crown-4 (ppCrown) with plasma polymerized hexene (ppHexene) as adhesion layer. The coatings were micro patterned using conventional cleanroom photolithography and lift-off. Single cell arrays showed sharp contrast in cell adhesion between the untreated glass surface and the ppCrown layer. Similarly, proteins adsorbed selectively to untreated glass but not to ppCrown. The simplicity of the lift-off technique and the sturdiness and versatility of the plasma-polymerized coatings, make this technology highly suitable for bio-MEMS and biochip applications, where patterned high contrast non-fouling surfaces are needed.

Adsorption↗
PubMed2004

Synthesis, structure and heterogeneous catalytic activities of Cu-containing polymeric compounds: anion effect and comparison of homogeneous vs. heterogeneous catalytic activity.

The structures of new polymeric compounds containing Cu(II) ions and btp (2,6-bis(N'-1,2,4-triazolyl)pyridine) ligands have been determined. The btp ligands bridge Cu(II) ions to form double zigzag chains, [Cu(ClO4)2(btp)2] 3 with perchlorate anions, and form single zigzag chains, [Cu(btp)(H2O)4](SO4).2H2O 4 with sulfate anions. The polymeric compound 3 was found to effectively catalyze the epoxide ring-opening reaction with methanol, while polymeric compound 4 was almost inactive with epoxides under the same conditions. The polymeric compound 3 showed an efficient catalytic activity and regioselective reactivity in the ring opening of epoxides and allowed reuse without a significant loss of activity through three runs with epoxides.

Journal Article↗
PubMed2005

Numerical simulation of polymerization in interdigital multilamination micromixers.

Free radical polymerization in microfluidic devices modeled with the help of numerical simulations is discussed. The simulation method used allows the simultaneous solvation of partial differential equations resulting from the hydrodynamics, thermal and mass transfer (convection, diffusion and chemical reaction). Three microfluidic devices are modeled, two interdigital multilamination micromixers respectively with a large and short focusing section, and a simple T-junction followed by a microtube reactor together considered as a bilamination micromixer with a large focusing section. The simulations show that in spite of the heat released by the polymerization reaction, the thermal transfer in such microfluidic devices is high enough to ensure isothermal conditions. Moreover, for low radial Peclet number, microfluidic devices with a large focusing section can achieve better control over the polymerization than a laboratory scale reactor as the polydispersity index obtained is very close to the theoretical limiting value. As the characteristic dimension of the microfluidic device increases, i.e. for high radial Peclet number, the reactive medium cannot be fully homogenized by the diffusion transport before leaving the system resulting in a high polydispersity index and a loss in the control of the polymerization.

Biological Transport↗
PubMed2005

Direct oxidation of polymeric substrates by multifunctional manganese peroxidase isoenzyme from Pleurotus ostreatus without redox mediators.

VPs (versatile peroxidases) sharing the functions of LiP (lignin peroxidase) and MnP (manganese peroxidase) have been described in basidiomycetous fungi Pleurotus and Bjerkandera. Despite the importance of this enzyme in polymer degradation, its reactivity with polymeric substrates remains poorly understood. In the present study, we first report that, unlike LiP, VP from Pleurotus ostreatus directly oxidized two polymeric substrates, bovine pancreatic RNase and Poly R-478, through a long-range electron pathway without redox mediators. P. ostreatus produces several MnP isoenzymes, including the multifunctional enzyme MnP2 (VP) and a typical MnP isoenzyme MnP3. MnP2 (VP) depolymerized a polymeric azo dye, Poly R-478, to complete its catalytic cycle. Reduction of the oxidized intermediates of MnP2 (VP) to its resting state was also observed for RNase. RNase inhibited the oxidation of VA (veratryl alcohol) in a competitive manner. Blocking of the exposed tryptophan by N-bromosuccinimide inhibited the oxidation of RNase and VA by MnP2 (VP), but its Mn2+-oxidizing activity was retained, suggesting that Trp-170 exposed on an enzyme surface is a substrate-binding site both for VA and the polymeric substrates. The direct oxidation of RNase and Poly R by MnP2 (VP) is in sharp contrast with redox mediator-dependent oxidation of these polymers by LiP from Phanerochaete chrysosporium. Molecular modelling of MnP2 (VP) revealed that the differences in the dependence on redox mediators in polymer oxidation by MnP2 (VP) and LiP were explained by the anionic microenvironment surrounding the exposed tryptophan.

Anthraquinones↗
PubMed1994

ADP-ribosylation of the GTP-binding protein Rho by Clostridium limosum exoenzyme affects basal, but not N-formyl-peptide-stimulated, actin polymerization in human myeloid leukaemic (HL60) cells.

Treatment of human myeloid leukaemic (HL60) cells with Clostridium limosum exoenzyme, which inactivates the small GTP-binding protein Rho by ADP-ribosylation, decreased the basal F-actin content. Inhibition of F-actin occurred after long-term treatment (24 h) of intact HL60 cells or after introduction of the toxin by electropermeabilization in a toxin-concentration-dependent manner. Concomitantly with the decrease in the basal F-actin content, the GTP-binding protein Rho was ADP-ribosylated in intact cells. However, Clostridium limosum toxin had no inhibitory effect on N-formyl-peptide-induced actin polymerization. Moreover, the relative N-formyl-peptide-stimulated polymerization was substantially enhanced in cells treated with Clostridium limosum transferase. In contrast with Clostridium limosum exoenzyme, component C21 of the Clostridium botulinum C2 toxin, which ADP-ribosylates G-actin, depolymerized basal F-actin and inhibited N-formyl-peptide-induced actin polymerization in electropermeabilized HL60 cells. These findings indicate that Rho proteins are involved in the basal, but not the ligand-evoked, actin polymerization in HL60 cells.

ADP Ribose Transferases↗
PubMed2002

Immediate GTP hydrolysis upon FtsZ polymerization.

To understand the polymerization dynamics of FtsZ, a bacterial cell division protein similar to tubulin, insight is required into the nature of the nucleotide bound to the polymerized protein. In a previous study, we showed that the FtsZ polymers contain mostly GDP. A recent study challenged this result, suggesting that the polymerized FtsZ is in a GTP-bound state. Here, we show that, when radiolabelled [gamma-32P]-GTP is used to polymerize FtsZ, GTP is hydrolysed instantaneously. The FtsZ polymer contains both GDP and the radiolabelled inorganic phosphate.

Bacterial Proteins↗
PubMed2001

Defined sequence segments of the small heat shock proteins HSP25 and alphaB-crystallin inhibit actin polymerization.

The interaction of small heat shock proteins (sHSPs) with the actin cytoskeleton has been described and some members of this family, e.g. chicken and murine HSP25 (HSP27), inhibit the polymerization of actin in vitro. To analyse the molecular basis of this interaction, we synthesized a set of overlapping peptides covering the complete sequence of murine HSP25 and tested the effect of these peptides on actin polymerization in vitro by fluorescence spectroscopy and electron microscopy. Two peptides comprising the sequences W43 to R57 (peptide 6) and I92 to N106 (peptide 11) of HSP25 were found to be potent inhibitors of actin polymerization. Phosphorylation of N-terminally extended peptide 11 at serine residues known to be phosphorylated in vivo resulted in decline of their inhibitory activity. Interestingly, peptides derived from the homologous peptide 11 sequence of murine alphaB-crystallin showed the same behaviour. The results suggest that both HSP25 and alphaB-crystallin have the potential to inhibit actin polymerization and that this activity is regulated by phosphorylation.

Actins↗
PubMed2000

Combined glomerular deposition of polymeric rat IgA and IgG aggravates renal inflammation.

BACKGROUND: IgA nephropathy (IgAN) is characterized by deposition in the glomerular mesangium of IgA together with C3, C5b-9, and properdin. IgG deposition as a risk factor in IgAN was recently confirmed by a long-term follow-up of patients with IgAN. We previously reported on an acute model of IgA-mediated glomerular inflammation in Wistar rats. METHODS: To investigate the effect of the combination of IgA and IgG on glomerular injury, Wistar rats were injected with a minimum dose of rat IgG in the presence or absence of a subnephritogenic dose of polymeric rat IgA. Subsequently, glomerular complement activation, influx of inflammatory cells, proteinuria, and hematuria were assessed. RESULTS: Administration of IgG to the rats resulted in maximal proteinuria of 20.3 +/- 12.1 mg/24 h on day 2 and an absence of overt glomerular inflammation. Administration of polymeric rat IgA antibodies to rats resulted in hematuria with a moderate mesangial complement deposition. In the combination group, however, glomerular deposition of C5b-9 was dramatically increased. This was accompanied by increased proteinuria as compared with rats receiving IgA or IgG antibody injections alone on day 7. Microhematuria occurred in rats receiving either polymeric rat IgA or IgG alone or the combination. While both rat IgG and polymeric IgA induced minor mesangial cell (MC) proliferation and MC lysis, the combination resulted in a pronounced, significant increased percentage of aneurysm formation on day 7 after injection. CONCLUSIONS: We conclude that in this model of IgA-induced glomerulopathy, a selective, complement-dependent glomerular inflammation is induced in Wistar rats by glomerular codeposition of rat isotypic monoclonal antibodies.

Animals↗
PubMed2001

Charge-dependent binding of polymeric IgA1 to human mesangial cells in IgA nephropathy.

BACKGROUND: IgA nephropathy (IgAN) is characterized by raised serum IgA1 and predominant mesangial IgA1 deposits of polymeric nature. The mechanism of polymeric IgA1 (pIgA1) deposition in the kidney mesangium is poorly understood in IgAN. It has been suggested that increased sialic acid content and anionic charge of the pIgA1 molecules may be operational in the IgA1 deposition in human mesangial cells (HMCs). The present study examined the binding of pIgA1 with different surface charges to HMCs. The binding characteristics of IgA1 to HMCs in the presence of polycation (poly-L-lysine) or polyanion (heparin) were also investigated. METHODS: IgA1 was purified in sera from patients with IgAN and from healthy controls by jacalin affinity chromatography. IgA1 was further separated into pIgA1 and monomeric IgA1 (mIgA1) by fast protein liquid chromatography (FPLC). pIgA1 or mIgA1 with different net charges on their surface were resolved by ion exchange chromatography (IEC) with a Mono Q column. The binding characteristics of pIgA1 and mIgA1 to HMCs in the presence or absence of polycation or polyanion were examined by flow cytometry. RESULTS: In patients with IgAN, the absolute amount of mIgA1 and pIgA1 is significantly higher than that of healthy controls (P < 0. 001). There was significant increase in binding of pIgA1 from patients with IgAN to HMC and cell lysate. pIgA1 that interacted strongly with the ion exchanger also bound more to HMCs when compared with IgA1 interacted weakly with the ion exchanger (P < 0. 001). The anionic charged pIgA1 from patients was significantly higher than that of healthy controls (P < 0.001). Preincubation with poly-L-lysine increased the binding of pIgA1 to HMCs. The binding of pIgA1 to HMCs was decreased by preincubation with heparin. CONCLUSIONS: The binding of IgA to HMCs is charge dependent. Polymeric IgA with the highest net negative charge binds more to HMCs. Preincubation with polyanion decreased the binding of polymeric IgA to HMCs. These results suggest an important role for anionic charge in IgA1 deposition onto the kidney mesangial cells.

Adult↗
PubMed2002

Sickle Hb polymerization in RBC components from donors with sickle cell trait prevents effective WBC reduction by filtration.

BACKGROUND: RBC components collected from donors with sickle cell trait frequently occlude WBC-reduction filters. In vitro, sickle trait RBCs have the potential for sickle Hb (Hb S) polymerization at low oxygen saturations and high Hb concentrations. STUDY DESIGN AND METHOD: To determine if the low pH and high osmolarity of the CP2D used in the collection contributed to filter failures, the filterability of sickle trait donor RBCs collected in CP2D was compared with RBCs from the same donors collected in heparin. RESULTS: Five of six sickle trait components collected in CP2D did not complete filtration, but all six RBC components collected in heparin filtered completely. RBC components collected in CP2D from four other sickle trait donors were divided in two, and one-half was treated with carbon monoxide to convert Hb S to its liganded form to prevent Hb S polymerization. All four carbon monoxide-treated components filtered within 9 minutes, but only one untreated component filtered completely. RBC components collected by apheresis contained less CP2D, and five of seven sickle trait apheresis components filtered completely; four of the five filtered rapidly (<15 min) and one filtered in 100 minutes. Hb oxygen saturation was greater in the four rapidly filtering apheresis RBC components (68 +/- 9%) than in the three that filtered slowly or incompletely (37 +/- 5%, p = 0.03). CONCLUSIONS: Hb S polymerization appears responsible for RBC WBC-reduction filter failures. Citrate anticoagulant and low oxygen saturation are responsible in part for Hb S polymerization in this setting.

Anticoagulants↗
PubMed2002

The effect of reactor geometry on frontal polymerization spin modes.

Using reactors of different sizes and geometries the dynamics of the frontal polymerization of 1,6-hexanediol diacrylate (HDDA) and pentaerythritol tetraacrylate (PETAC), with ammonium persulfate as the initiator were studied. For this system, the frontal polymerization exhibits complex behavior that depends on the ratio of the monomers. For a particular range of monomers concentration, the polymerization front becomes nonplanar, and spin modes appear. By varying the reactor diameter, we experimentally confirmed the expected shift of the system to a greater number of "hot spots" for larger diameters. For square test tubes a "zig-zag" mode was observed for the first time in frontal polymerization. We confirmed the viscosity-dependence of the spin mode instabilities. We also observed novel modes in cylinder-inside-cylinder reactors. Lastly, using a conical reactor with a continuously varying diameter, we observed what may be evidence for bistability depending on the direction of propagation. We discuss these finding in terms of the standard linear stability analysis for propagating fronts. (c) 2002 American Institute of Physics.

Journal Article↗
PubMed1999

Period-doubling behavior in frontal polymerization of multifunctional acrylates.

Front dynamics in the frontal polymerization of two multifunctional acrylate monomers, 1,6-hexanediol diacrylate (HDDA) and trimethylolpropane ethoxylate triacrylate (TMPTA), with Lupersol 231 [1,1-di(t-butylperoxy)-3,3,5-trimethylcyclohexane] as the initiator, are studied. In most frontal polymerization systems, the dynamics are associated with a planar front propagating through the sample. However, in some cases, front behavior can be altered: the front becomes nonplanar characterized by complex patterns like spin modes and pulsations. To determine how these periodic and aperiodic modes arise, reactant solutions consisting of HDDA diluted with diethyl phthalate (DEP) and TMPTA diluted with dimethyl sulfoxide (DMSO) were used in the study. In the study we reveal frontal behavior characteristic of period-doubling behavior, a doubling of spin heads that degenerate into an apparently chaotic mode. Also, a pulsating symmetric mode has been observed. These observations have a striking similarity to observations made in studies of self-propagating high-temperature synthesis (SHS) in which the addition of an inert diluent afforded a rich variety of dynamical behavior. The degree of cross-linking has also been found to be a bifurcation parameter. The energy of activation of multifunctional acrylate polymerization is a strong function of the degree of polymerization. By adding a monoacrylate (benzyl acrylate: BzAc), such that the front temperature was invariant, we observed a period-doubling bifurcation sequence through changes in the energy of activation, which has not been previously reported. (c) 1999 American Institute of Physics.

Journal Article↗
PubMed2004

Physical properties of sulfur near the polymerization transition.

Acoustical measurements, electron spin resonance, and Raman spectroscopy have been employed to probe sulfur over the temperature range 80-180 degrees C, which includes the polymerization transition and the supercooled liquid state. Acoustical properties (sound velocity, absorption, and impedance) have been studied with both longitudinal and transverse waves at frequencies between 500 kHz and 22 MHz. The results confirm that polymeric sulfur is a solution of long chain molecules in monomeric solvent, and that the polymerization transition is not a second-order phase transition, as was proposed theoretically. Sulfur is a viscous liquid, but not viscoelastic, both below and above the polymerization transition temperature. It is shown that the classical Navier-Stokes theory is not applicable to the sound absorption in liquid sulfur in the highly viscous state.

Journal Article↗