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At least 1,747 records · Page 97Linked to original sources
PubMed2004

Recombinant protein purification from pea.

To assess the suitability of transgenic peas as a host for protein production from the perspective of ease of recovery, a strain containing recombinant beta-glucuronidase with poly(histidine) tail (GUSH6) was evaluated for solubility of the target protein in relation to native components (proteins, carbohydrates, and phenolics). Recovery of the recombinant GUSH6 from aqueous extracts by immobilized metal affinity chromatography with coupled Co(2+) yielded a nearly pure product with IDA (enrichment factor (EF) = 260) or NTA (EF = 200) resin. Single-step recoveries were also possible by isoelectric precipitation (EF = 4), polyelectrolyte precipitation (EF = 1.5), and anion-exchange chromatography (EF = 3.1), but enrichment factors were low.

Chemical Fractionation↗
PubMed2004

Murine leukemia virus clearance by flocculation and microfiltration.

Clearance of murine leukemia virus from CHO cell suspensions by flocculation and microfiltration was investigated. Murine leukemia virus is a retrovirus that is recommended by the U.S. Food and Drug Administration for validating clearance of retrovirus-like particles. Due to biosafety considerations, an amphotropic murine leukemia virus vector (A-MLV) that is incapable of self-replication was used. Further, A-MLV is incapable of infecting CHO cells, thus ensuring that infection of the CHO cells in the feed did not result in a reduced virus titer in the permeate. The virus vector contains the gene for the enhanced green fluorescent protein (EGFP) to facilitate assaying for infectious virus particles. The virus particles are 80-130 nm in size. The feed streams were flocculated using a cationic polyelectrolyte. Microfiltration was conducted using 0.1 and 0.65 microm pore size hollow fiber membranes. The level of virus clearance in the permeate was determined. For the 0.1 microm pore size membranes a 1,000-fold reduction in the virus titer in the permeate was observed for feed streams consisting of A-MLV, A-MLV plus flocculant, A-MLV plus CHO cells, and A-MLV plus flocculant and CHO cells. While the flocculant had little effect on the level of virus clearance in the permeate for 0.1 microm pore size membranes, it did lead to higher permeate fluxes for the CHO cell feed streams. Virus clearance experiments conducted with 0.65 microm pore size membranes indicate little clearance of A-MLV from the permeate in the absence of flocculant. However, in the presence of flocculant the level of virus clearance in the permeate was similar to that observed for 0.1 microm pore size membranes. The results obtained here indicate that significant clearance of A-MLV is possible during tangential flow microfiltration. Addition of a flocculant is essential if the membrane pore size is greater than the diameter of the virus particles. Flocculation of the feed stream leads to an increase in the permeate flux.

Animals↗
PubMed2005

Stabilization of glucose oxidase in alginate microspheres with photoreactive diazoresin nanofilm coatings.

The nanoassembly and photo-crosslinking of diazo-resin (DAR) coatings on small alginate microspheres for stable enzyme entrapment is described. Multilayer nanofilms of DAR with poly(styrene sulfonate) (PSS) were used in an effort to stabilize the encapsulation of glucose oxidase enzyme for biosensor applications. The activity and physical encapsulation of the trapped enzyme were measured over 24 weeks to compare the effectiveness of nanofilm coatings and crosslinking for stabilization. Uncoated spheres exhibited rapid loss of activity, retaining only 20% of initial activity after one week, and a dramatic reduction in effective activity over 24 weeks, whereas the uncrosslinked and crosslinked {DAR/PSS}-coated spheres retained more than 50% of their initial activity after 4 weeks, which remained stable even after 24 weeks for the two and three bilayer films. Nanofilms comprising more polyelectrolyte layers maintained higher overall activity compared to films of the same composition but fewer layers, and crosslinking the films increased retention of activity over uncrosslinked films after 24 weeks. These findings demonstrate that enzyme immobilization and stabilization can be achieved by using simple modifications to the layer-by-layer self-assembly technique.

Adsorption↗
PubMed2006

Development and validation of a rapid reversed-phase HPLC method for the determination of insulin from nanoparticulate systems.

A reversed-phase high-performance liquid chromatographic (HPLC) method has been developed and validated for the determination of insulin in nanoparticulate dosage forms. Its application for the development and characterization of insulin-loaded nanoparticulates composed of polyelectrolytes has also been carried out. A reversed-phase (RP) C18 column and gradient elution with a mobile phase composed of acetonitrile (ACN) and 0.1% aqueous trifluoroacetic acid (TFA) solution at a flow rate of 1 mL/min was used. Protein identification was made by UV detection at 214 nm. The gradient changed from 30:70 (ACN:TFA, v/v) to 40:60 (v/v) in 5 min followed by isocratic elution at 40:60 (v/v) for a further five minutes. The method was linear in the range of 1-100 microg/mL (R2 = 0.9996), specific with a good inter-day and intra-day precision based on relative standard deviation values (less than 3.80%). The recovery was between 98.86 and 100.88% and the detection and quantitation limits were 0.24 and 0.72 microg/mL, respectively. The method was further tested for the determination of the association efficiency of insulin to nanoparticulate carriers composed of alginate and chitosan, as well as its loading capacity for this protein. Encapsulant release under simulated gastrointestinal fluids was evaluated. The method can be used for development and characterization of insulin-loaded nanoparticles made from cross-linked chitosan-alginate.

Chromatography, High Pressure Liquid↗
PubMed2004

Spatial and temporal sequence of events in cell adhesion: from molecular recognition to focal adhesion assembly.

A new concept that attributes a pivotal role to the pericellular coat in the regulation of the early stages of cell adhesion is presented. Quick, adaptable, and transient adhesion through multiple cooperative weak interactions provides the cell with an additional level of modulation in the decision-making process that precedes the commitment to adhesion at a particular site. Hyaluronan emerges as a modulator of cell adhesion in certain cells, mediating binding or repulsion through its polyelectrolyte character, in addition to its chirality and molecular-recognition properties. The biophysical properties of hyaluronan as well as its ultrastructural organization are analyzed in relation to this proposed function.

Animals↗
PubMed2003

Organized nanostructured complexes of inorganic clusters and surfactants that exhibit thermal solid-state transformations.

Facile organization of the inorganic crown-shaped [Ni(3)P(3)S(12)](3-) ion (1) into room-temperature liquid-crystalline materials by complexation with double-tail ammonium surfactants is achieved by the ionic self-assembly (ISA) route. Small-angle X-ray diffraction, UV/Vis spectroscopy, and (31)P NMR analyses reveal that these complexes show an interesting solid-state structure transition. Upon heating, the inorganic crown species polymerizes to the inorganic polyelectrolyte infinity [NiPS(4)](-). This structural transition is reversible, and involves a solvent/dissolution cycle. The facile preparation and facile optional induction of phase and structural changes make these complexes candidates for a number of applications in which cooperative, metastable switching with sufficient contrast of optical and solid-state properties is required.

Journal Article↗
PubMed2006

Assembly-controlled biocompatible interface on a microchip: strategy to highly efficient proteolysis.

A biocompatible interface was constructed on a microchip by using the layer-by-layer (LBL) assembly of charged polysaccharides incorporating proteases for highly efficient proteolysis. The controlled assembly of natural polyelectrolytes and the enzyme-adsorption step were monitored by using a quartz-crystal microbalance and atomic force microscopy (AFM). Such a multilayer-assembled membrane provides a biocompatible interconnected network with high enzyme-loading capacity. The maximum digestion rate of the adsorbed trypsin in a microchannel was significantly accelerated to 1600 mM min(-1) microg(-1), compared with the tryptic digestion in solution. Based on the Langmuir isotherm model, the thermodynamic constant of adsorption K was calculated to be 1.6 x 10(5) M(-1) and the maximum adsorption loading Gammamax was 3.6 x 10(-6) mol m(-2), 30 times more than a monolayer of trypsin on the native surface. The tunable interface containing trypsin was employed to construct a microchip reactor for digestion of femtomoles of proteins and the produced peptides were analyzed by MALDI-TOF mass spectroscopy. The efficient on-chip proteolysis was obtained within a few seconds, and the identification of biological samples was feasible.

Biocompatible Materials↗
PubMed2003

Detection by circular dichroism of conformational transitions in pH and thermosensitive copolymers based on N-isopropylacrylamide and N-methacryloyl-L-leucine.

Circular dichroism (CD) spectra in the region of 210-250 nm allow visualization of intrachain phase transition of pH- and thermosensitive polyelectrolytes. Indeed, in 0.001 M citrate and acetate buffers, at pH 4.0-5.5, aqueous solutions of a poly(N-isopropylacrylamide-co-N-methacryloyl-L-leucine) (NIPAAm-MALEU) copolymer containing 90.9 mol% of NIPAAm residues exhibit a well-defined sigmoidal increase in the CD signal at 220 nm with increasing temperature. This phenomenon is suggestive of a highly cooperative transition which occurs at lower temperatures compared to that observed by cloud point measurements. The change in the CD signal is less sharp at higher pH, indicating varying cooperativity with pH. For pH 6.0 and higher, no such phenomena are observed.

Journal Article↗
PubMed2003

3-D surface charges modulate protrusive and contractile contacts of chondrosarcoma cells.

Up to now, most of the studies addressing the critical roles played by protrusive and contractile cell-matrix contacts in cell adhesion, guidance, migration, matrix assembly, and activation of signaling molecules have been performed on two-dimensional surfaces. Here, we analysed the organization of chondrosarcoma cell contacts in a new three-dimensional environment made of titanium beads. Surface charges were modified by deposition of polyelectrolyte multilayer films built up by alternated polycations poly-(L-lysine) or poly(allylamine hydrochloride) and polyanions poly-(L-glutamic acid) or poly(sodium 4-styrenesulfonate). Negatively charged 3-D titanium surfaces amplified the occurrence and length of cell protrusions. These protrusions had pseudopod characteristics extended to 200 microm in length, growing off the substratum to distant beads. Pseudopod formation is inhibited by the exocytosis inhibitor concanamycin A and is triggered by a secreted factor. Chondrosarcoma cells adhering on uncoated or on negatively charged surfaces contained discrete focal spots of vinculin and actin cables. In cells plated onto these surfaces, phosphorylation of p44/42 MAPK/ERK was twofold increased. In contrast, no cytoskeletal vinculin and actin organization was observed when the surface was positively charged. These data suggest that chondrosarcoma cells adapt a more stable adhesion on uncoated or negatively charged surfaces. This point may be critical in tissue engineering strategies designed for cartilage repair.

Actins↗
PubMed2003

Overcharging in colloids: beyond the Poisson-Boltzmann approach.

A broad range of manufactured products and biological fluids are colliods. The ability to understand and control the processes (of scientific, technological and industrial interest) in which such colloids are involved relies upon a precise knowledge of the electrical double layer. The traditional approach to describing this ion cloud around colloidal particles has been the Gouy-Chapman model developed on the basis of the Poisson-Boltzmann equation. Since the early 1980s, however, more sophisticated theoretical treatments have revealed both quantitative and qualitative deficiencies in the Poisson-Boltzmann theory, particularly at high ionic strengths and/or high surface charge densities. This review deals with these novel approaches, which are mostly computer simulations and approximate integral equation theories based on the so-called primitive model. Special attention is paid to phenomena that cannot be accounted for by the classic theory as a result of neglecting ion size correlations, such as overcharging, namely, the counterion concentration in the immediate neighborhood of the surface is so large that the particle surface is overcompensated. Other illustrative examples are the nonmonotonic behavior of the electrostatic potential and attractive interactions between equally charged surfaces. These predictions are certainly remarkable and, on paper, they can have an effect on experimentally measurable quantities (for instance, electrophoretic mobility). Even so, these new approaches have scarcely been applied in practice. Thus a critical survey on the relevance of ion size correlation in real systems is also included. Overcharging of macroions can also be brought about by adsorption of oppositely charged polyelectrolytes. Noteworthy examples and theoretical approaches for them are also briefly reviewed.

Colloids↗
PubMed2006

Superquenching as a detector for microsphere-based flow cytometric assays.

BACKGROUND: Fluorescent conjugated polymers display high fluorescence quantum yields and enhanced sensitivity to quenching (superquenching) by oppositely charged quenchers through energy or electron transfer. Fluorescent polymers and their quenchers are used in bead-based biosensor applications where the polymers are coated on particles. In this work, we investigate a detection method that utilizes superquenching on microspheres, which can be used for flow cytometric assays. METHODS: Microspheres were coated with the fluorescent cationic polyelectrolyte poly(p-phenylene-ethynylene) (PPE), and its superquenching by 9,10-anthraquinone-2,6-disulfonic acid (AQS) was examined by fluorometric methods in presence and in absence of a barrier to superquenching in the form of an anionic lipid bilayer. RESULTS: Flow cytometry detected superquenching of PPE on microspheres (MS-PPE) by AQS where high levels of reduction in fluorescence were observed. Adding different concentrations of AQS to MS-PPE yielded a Stern-Volmer quenching constant of 0.8x10(6) M-1. While forming an anionic lipid bilayer around the MS-PPE acted as a barrier to superquenching by AQS, disrupting the lipid bilayer allowed superquenching to take place. CONCLUSIONS: The sensitivity of flow cytometry in detecting fluorescence of microspheres and the amplified quenching sensitivity of fluorescent conjugated polymers both offer advantages over other fluorometric methods and conventional quenching detection. This study used superquenching of fluorescent polymers as a new tool in flow cytometry, thus combining the advantages offered by both method and detector. In addition, we employed the formation and the disruption of a supported lipid bilayer in mediating superquenching to offer new biosensing applications.

Anthraquinones↗
PubMed1991

Immobilized pH gradients (IPG) simulator--an additional step in pH gradient engineering: I. Linear pH gradients.

A new computer program, called immobilized pH gradients (IPG) simulator, is proposed for calculating and optimizing any recipe for use in isoelectric focusing in immobilized pH gradients. Unlike our previous monoprotic electrolyte gradient simulation (MGS) and polyelectrolyte gradient simulation (PGS) programs, based on minimizing CV(beta), the present program has a target function the minimization of the quadratic moment around zero of the residuals (mu 2). With this algorithm it is possible to formulate IPG recipes which have deviations from linearity well below 1% of the given pH interval (a limit set with the previous MGS and PGS programs), in fact, as small as 0.1-0.2% (in pH units). The new simulator performs 2-3 times better than the previous ones in the pH 4-10 range, and is absolutely necessary when working outside this range, at extreme pH values, where CV(beta) cannot work against the buffering power of bulk water, thus generating pH recipes with huge deviations from linearity. In the latter cases, mu 2 performs 10 times better than CV(beta). When utilizing strong titrants for extended pH intervals, the "all or none" rule has been discovered: such titrants should always be used in tandem, since omission of one of the two at either the acidic or basic extremes produces strongly distorted pH profiles. Our new, most powerful simulator also contains equations for creating nonlinear gradients, notably: concave and convex exponentials and sigmoidal (see the companion paper: Righetti, P. G. and Tonani, C., Electrophoresis 1991, 12, 1021-1027).

Algorithms↗
PubMed1993

A new concept for separating nucleic acids by electrophoresis in solution using hybrid synthetic end labelled-nucleic acid molecules.

Analogy between the symmetry breaking of the electrical driving force and the opposing friction force in gels using pulsed electric fields is made with the corresponding effect for polyelectrolyte coils in solution related to the molecular weight-independent charge density. The synthesis of hybrid molecules to break the symmetry of constant charge density is proposed, in which standard polypeptide end labels are attached to nucleic acid fragments. The combinatorial chemical library scheme of Brenner and Lerner (Proc. Natl. Acad. Sci. USA 1992, 89, 5381-5383), making use of hybrid molecules for another purpose, is mentioned, with the suggestion that for electrophoresis in solution a neutral end-labelled polypeptide chain would give the largest molecular weight and time-dependent change in the charge density for pulsed fields.

Electrophoresis↗
PubMed1993

DNA electrophoresis in polymer solutions: Ogston sieving, reptation and constraint release.

The electrophoresis of long polyelectrolytes is considered theoretically, with special attention to duplex DNA. We first discuss quantitative approaches to determine unambiguously the entanglement properties of polymer solutions. Following an idea proposed by Grossman and Soane, we then assume that the "mesh" size of the solution plays the role of a dynamic "pore size" in order to apply theories for gel electrophoresis. In the framework of the Ogston model, we predict that duplex DNA up to 1 kb or more should be separable in dilute (i.e. nonentangled) solutions of high molecular weight polymers. In an entangled solution, and for DNA larger than the pore size, we use a recently developed fluctuation-reptation model to predict the range of sizes in which separation should be possible as a function of electric field E and pore size zeta b. For zeta b larger than the Kuhn length of DNA, we predict a separation up to a size N*scaling as E-1 zeta b-1. For zeta b smaller than the Kuhn length, two different regimes are expected. For small electric fields (typically of the order of 10 V/cm), N*should be proportional to E-1 zeta b-3/2, whereas for high electric fields such as encountered in capillary electrophoresis, we expect that N*is proportional to E-2/5 zeta b-12/5. These predictions are qualitatively different from earlier ones. Finally, we demonstrate that the finite lifetime of the "pores" in an entangled solution (as opposed to a gel) may lead to a new migration mechanism by constraint release, which is not size-dependent.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemical Phenomena↗
PubMed1994

Gel electrophoresis of end-labeled DNA. II. Dynamics and detrapping in pulsed fields.

A theory for field-inversion gel electrophoresis of a flexible polyelectrolyte bearing an uncharged bulky label at one end is described, and the evolution of the mobility with chain length, field strength, friction of the label, and the duration of the forward and reverse pulses is predicted. A new critical size, Ndetrap, is introduced, and its value calculated. It increases roughly linearly with the duration of the reverse pulses. Chains smaller than Ndetrap are detrapped by reverse pulses, and may have a high mobility, whereas chains larger than Ndetrap are not trapped, and have a very small mobility. This leads to an increase of the mobility (as compared with constant field) in a given range of sizes, and to a strong selectivity around Ndetrap. Depending on the parameters, numerous other effects, including a secondary mobility plateau and band inversion, may appear. The corresponding regimes are predicted and discussed. All predictions are qualitatively consistent with available experimental data. We use them to suggest efficient conditions for the development of pulsed-field trapping electrophoresis, a possible tool for improved DNA sequencing. In particular, we recommend using a ramping of pulse times, with a constant ratio of forward to reverse time in the range 3 to 5.

DNA↗
PubMed1995

Trapping gel electrophoresis of end-labeled DNA: an analytical model for mobility and diffusion.

As shown by Ulanovsky, Drouin and Gilbert (Nature 1990, 343, 190-192), the gel electrophoretic migration of DNA is severely reduced by steric trapping when streptavidin is attached to one end of the polyelectrolyte. We present a model that allows us to calculate both the mobility and the diffusion coefficient, hence the resolution factor of the resulting separation. We compare our results to those of Défontaines and Viovy (Electrophoresis 1993, 14, 8-17) and we show that the averages over the molecular conformations must be done carefully. We also show that trapping increases diffusion substantially and that this makes constant-field trapping electrophoresis incapable of increasing the number of bases read per sequencing run. Finally, we conclude that severe trapping may lead to highly anomalous transport behavior where one cannot define a velocity or a diffusion constant.

Bacterial Proteins↗
PubMed1996

Electrophoretic mobility of composite objects in free solution: application to DNA separation.

We propose here a simple procedure to estimate the electrophoretic mobility of composite objects obtained by linking two charged subunits, stressing that this electrophoretic mobility is in general different from the ratio of the total charge to the total friction coefficient. We focus especially on the situation where at least one of the subunits is a polyelectrolyte. Our remarks in particular correct the existing theoretical analysis of separation capabilities of devices in which a buoy (sphere, protein, polymer) is attached to a DNA chain. We also predict that, in some cases, the direction of electrophoretic motion can be reversed by increasing the electric field amplitude.

DNA↗
PubMed1996

Protein electrophoresis in polyacrylamide gels with templated pores.

An approach is described for the synthesis of nanostructured hydrogels with defined size channels or pores for separations of biological macromolecules. Polyacrylamide gels (15-20% acrylamide) were cast in the presence of high concentrations (5%-28%) of hydrophilic, macromolecular cosolutes either semirigid, rod-like polyelectrolytes (short fragments of DNA or xanthan) or spherical micelles of sodium dodecyl sulfate (SDS). Polyanionic cosolutes were then removed by a combination of diffusion and electrophoresis. These processes are expected to yield gels with 'templated' channels having dimensions near those of the double helical polymers (diameters approximately 2-3 nm, lengths of 50-200 nm) or pores near the size of the spherical SDS micelles (approximately 4-5 nm). This hypothesis was tested by comparing the relative electrophoretic mobilities of proteins (3400 to 43,000 Da) complexed with SDS on templated and conventional gels. Differences in electrophoretic mobilities were observed between all templated gels and control normal gels, demonstrating that the templating process altered the polyacrylamide network. No evidence was found for phase separation of cosolutes from the polyacrylamide network during polymerization. Templated pores are expected to enhance the mobilities of molecules in a particular size range relative to smaller and larger molecules. Gels templated with DNA or xanthan (8-10% final concentration) exhibited little size selectivity, but selectivity was observed for gels templated with SDS varied with polyacrylamide concentration in a manner consistent with the creation of templated pores approximately the size of SDS micelles and larger than the average pore size in a surrounding polyacrylamide network.

Acrylamide↗