Bead Column Research Articles

Differential mobility spectrometry of chlorocarbons with a micro-fabricated drift tube.

Chlorocarbons were ionized through gas phase chemistry at ambient pressure in air and resultant ions were characterized using a micro-fabricated drift tube with differential mobility spectrometry (DMS). Positive and negative product ions were characterized simultaneously in a single drift tube equipped with a 3 mCi (63)Ni ion source at 50 degrees C and drift gas of air with 1 ppm moisture. Scans of compensation voltage for most chlorocarbons produced differential mobility spectra with Cl(-) as the sole product ion and a few chlorocarbons produced adduct ions, M (.-) Cl(-). Detection limits were approximately 20-80 pg for gas chromatography-DMS measurements. Chlorocarbons also yielded positive ions through chemical ionization in air and differential mobility spectra showed peaks with characteristic compensation voltages for each substance. Field dependence of mobility was determined for positive and negative ions of each substance and confirmed characteristic behavior for each ion. A DMS analyzer with a membrane inlet was used to continuously monitor effluent from columns of bentonite or synthetic silica beads to determine breakthrough volumes of individual chlorocarbons. These findings suggest a potential of DMS for monitoring subsurface environments either on site or perhaps in situ.

Effect of Particle Size Uniformity on Solute Dispersion in Unsaturated Glass Beads Columns

Effect of Particle Size Uniformity on Solute Dispersion in Unsaturated Glass Beads Columns

Direct association between PU.1 and MeCP2 that recruits mSin3A-HDAC complex for PU.1-mediated transcriptional repression.

PU.1, a member of the Ets family of transcription factors, is implicated in hematopoietic cell differentiation through its interactions with other transcriptional factors and cofactors. To identify a novel protein(s) binding to PU.1, we carried out affinity purification using a column of Glutathione-Sepharose beads bound to GST-PU.1 fusion protein and isolated several individual proteins using murine erythroleukemia (MEL) cell extracts. Sequence analysis of these proteins revealed that one was MeCP2 a methyl CpG binding protein. GST-pull-down assay and immunoprecipitation assay showed that PU.1 bound directly to MeCP2 via its Ets domain and MeCP2 bound to PU.1 via either its amino terminal domain or trans-repression domain. MeCP2 repressed transcriptional activity of PU.1 on a reporter construct with trimerized PU.1 binding sites. This downregulation was recovered in the presence of histone deacetylase inhibitor, trichostatin A (TSA). MeCP2 was integrated in PU.1-mSin3A-HDAC complex but not in PU.1-CBP complex. Chromatin immunoprecipitation (ChIP) assays showed that PU.1 and MeCP2 were collocated at the PU.1 binding site on the reporter construct and the PU.1 binding site of the intervening sequence 2 (IVS2) region in the intron of the beta-globin gene, which has been proposed to regulate expression of the gene, in undifferentiated MEL cells. The complex disappeared from the region during the course of erythroid differentiation of MEL cells. Our results suggest that MeCP2 acts as a corepressor of PU.1 probably due to facilitating complex formation with mSin3A and HDACs.

Mechanisms of platelet retention in the collagen-coated–bead column

Although the glass-bead column has been used to measure platelet adhesion, whether platelet interaction with glass beads represents physiologic processes remains unsettled. In an attempt to obtain more physiologic platelet responses, plastic beads coated with type I collagen have been recently developed to replace glass beads. In this study, we analyzed the factors responsible for platelet retention in the collagen-coated–bead column and investigated its possible clinical applications. We pumped citrated whole-blood samples into columns at a fixed speed with an injection pump and calculated platelet-retention rates by measuring platelet counts before and after passage through the columns. The platelet-retention rates, which were highly reproducible with samples from healthy donors, were reduced in a patient with glycoprotein (GP) VI deficiency but not in patients with type III von Willebrand disease. Anti-GPIIb/IIIa antibody and GRGDS peptide markedly inhibited platelet retention, whereas inhibition of the GPIb–von Willebrand factor or GPIa/IIa-collagen interaction had no effect. Data on the effects of various antiplatelet agents (including the antithrombin agent argatroban, prostacyclin, acetylsalicylic acid, and the ADP scavenger creatine phosphate/creatine phosphokinase) support the usefulness of this assay method in clinical application. Our findings suggest that GPVI and GPIIb/IIIa but not the GPIb–von Willebrand factor interaction are mainly involved in platelet retention in this column.

Linker‐modified microemulsions for a variety of oils and surfactants

AbstractPreviously, we reported on the use of hydrophilic and lipophilic linker molecules to enhance the solubilization capacity of chlorinated hydrocarbons using sodium dihexyl sulfosuccinate (SDHS). In this work we extend the use of linker molecules to a wider range of oils and surfactants. The data show that the linker effect works for all the systems studied and that linker‐based systems are even more economical than surfactant‐only systems for more hydrophobic oils. Using a more hydrophobic surfactant, such as sodium bis(2‐ethyl)dihexyl sulfosuccinate (Aerosol‐OT), requires a formulation enriched with a hydrophilic linker, whereas the formulation for the more hydrophilic SDHS requires the use of a more lipophilic linker. By considering the properties and appearance of the formulation before contacting with the oil, and by evaluation the coalescence dynamics, we found that hydrophilic linker‐rich formulations were preferred. These formulations were tested as fabric pretreatments for removing motor oil and hexadecane from cotton, and as flushing solutions for glass bead columns contaminated with these oils. The cleaning performance of these linker‐based systems was superior to common surfactant and pretreatment formulations in the detergency tests, achieving more than 80% removal of motor oil and hexadecane trapped in the packed‐column flushing tests.

Systematic Approach to Quantify Adsorption and Biodegradation Capacities on Biological Activated Carbon Following Ozonation

The goal of this research was to develop a systematic approach to quantify adsorption and biodegradation capacities on biological activated carbon (BAC). The role of absorption and biodegradation on BAC was studied using a continuous column. Several media, i.e., granular activated carbon (GAC), seeded glass bead and seeded GAC, and a target compound (p-hydroxybenzoic acid) were selected. Before breakthrough, the effluent of the GAC column contained a small amount of p-hydroxybenzoic acid that contributed the greatest amount of organic carbon to the effluent of the glass bead column, which suggests that adsorption should be the prevailing mechanism for removal the p-hydroxybenzoic acid, and biodegradation should be responsible for reducing the ozonation intermediates. Also, the bioactivity approach (biomass respiration potential, BRP) of BAC can not only reveal the importance of biodegradation mechanisms for the intermediates of ozonation, but also quantify the extent of the adsorption or biodegradation reaction occurring on BAC.

Mechanisms of platelet retention in the collagen-coated bead columns

Mechanisms of platelet retention in the collagen-coated bead columns

Effect of depletion interactions on transport of colloidal particles in porous media

The influence of depletion interactions on the transport of micrometer-sized, negatively charged polystyrene latex particles through porous media was studied by analysis of particle breakthrough curves as a response to short-pulse particle injections to the inlet of a packed column of glass beads. The column outlet latex particle concentration profiles and the total amount of particles exiting the column were determined as a function of the concentration of small, silica nanoparticles in the solution and the bulk flow rate. Because of similar charges, the silica particles do not adsorb to either the latex particles or glass beads and thus induce an attractive depletion force between the latex particles and glass bead collectors. The total column outlet latex particle amount was calculated by integrating the measured breakthrough concentration curve and compared to the known amount of injected particles at the column inlet. It was found that the particle recovery was a decreasing function of the silica nanoparticle concentration and the carrier fluid residence time, and an increasing function of the velocity in the bed. In addition, removing the silica nanoparticles from the flowing solution caused a second outlet peak to appear, suggesting that some of the polystyrene particles were captured in secondary energy wells. The experimental data were interpreted using the predicted potential energy profile between a single particle and a glass bead, which was assumed to consist of electrostatic, van der Waals, and depletion components. The results indicate that secondary energy wells significantly affect particle transport behavior through porous media.

Immunoaffinity removal of xenoreactive antibodies using modified dialysis or microfiltration membranes.

Hyperacute rejection following xenogeneic transplantation in primates is mediated by naturally occurring IgM antibodies, which are specifically directed to alpha-Galactosyl residues on many nonprimate mammalian cells. Current approaches to remove these anti-alphaGal IgM include plasmapheresis followed by immunoaffinity adsorption on bead columns using synthetic Gal epitopes, which requires two pieces of complex equipment. In this study, we explored the use of immunoaffinity adsorption with hollow fiber microporous or dialysis membranes to which a synthetic alphaGal trisaccharide ligand is bound. Covalent attachment of ligand directly to the surface produced negligible binding, but use of long-chain polyamines as reactive spacers yielded binding densities for anti-alphaGal IgM as high as 89 mg/mL membrane volume in breakthrough curve experiments with microporous nylon membranes having an internal surface area of 4.2 m(2)/mL membrane volume. A crossflow microfilter fabricated from the membranes described in this study and having about 0.4 m(2) luminal surface area would be able to carry out plasma separation and immunoadsorption in a single device with a large excess of binding capacity to ensure that all plasma that filters across the device and is returned to a human patient is essentially free of anti-alphaGal IgM. We conclude that immunoaffinity removal of xenoreactive antibodies using microfiltration hollow fiber membranes is feasible and has potential advantages of efficiency and simplicity for clinical application.

Expression of the class 1 outer-membrane protein of Neisseria meningitidis in Escherichia coli and purification using a self-cleavable affinity tag

The class 1 protein (PorA) is a major component of the outer membrane of Neisseria meningitidis and functions as a cationic porin. The protein is particularly effective in generating a bactericidal immune response following infection and is therefore under investigation as a potential antigen for inclusion in new meningococcal vaccines. Studies on the vaccine potential of PorA would be facilitated by the production of pure protein, free from other components of the meningococcal outer membrane. In the current study, PorA was expressed from the heterologous host Escherichia coli as a C-terminal fusion to an inducible protein-splicing element (intein) with an N-terminal chitin-binding domain (CBD) (IMPACT-TWIN system). The CBD acted as an affinity tag and allowed binding of the fusion protein to a chitin bead column, after which self-cleavage of the intein at its C-terminus was induced, resulting in the release of mature PorA. Cleavage of the fusion protein was temperature- and time-dependent, and was optimal at pH 7.0 after 5 days of storage at 4 °C. Efficient cleavage was also dependent on the addition of a minimal amino acid sequence (Gly–Arg–Ala) to the N-terminus of the mature PorA protein. This represented a significant improvement on the large N-terminal sequences introduced by other expression systems previously used to prepare recombinant PorA, and the yields of PorA purified with the IMPACT-TWIN system were similar. Thus, the IMPACT-TWIN system provides a facile method for producing recombinant PorA and may also be useful for the production of other bacterial outer-membrane proteins for vaccine studies.

Self-activating factor X derivative fused to the C-terminus of a cellulose-binding module: Production and properties.

In this work, a new derivative of FX was engineered. It comprises a cellulose-binding module (CBM) fused to the N-terminus of the truncated light chain (E2FX) of FX and a hexahistidine tag (H6) fused to the C-terminus of the heavy chain. The sequence LTR at the site of cleavage of the activation peptide from the N-terminus of the heavy chain is changed to IEGR to render the derivative self-activating. However, N-linked glycans on the CBM of the derivative blocked its binding to cellulose and those on the activation peptide slowed its activation. Therefore, the sites of N-linked glycosylation on the CBM and on the activation peptide were eliminated by mutation. The final derivative can be produced in good yield by cultured mammalian cells. It is purified easily with Ni(2+)-agarose, it is self-activating, and it can be immobilized on cellulose. When immobilized on a column of cellulose beads, the activated derivative retains approximately 80% of its initial activity after 30 days of continuous hydrolysis of a fusion protein substrate. Under these conditions of operation, the effective substrate:enzyme ratio is >10(4).

Surface modification of nonporous glass beads with chitosan and their adsorption property for transition metal ions

A new hybrid material that adsorbs transition metal ions was prepared by immobilizing chitosan on the surface of nonporous glass beads. The glass beads, prepared by etching in aqueous NaOH at 100°C, were first reacted with γ-aminopropyltriethoxysilane (APES) to introduce amino groups on the surface. Subsequently, the resulting aminated beads were treated with glutaraldehyde at 25°C to change the amino groups into aldehyde groups. Finally, chitosan of average molecular weight 40,000 was introduced via the aldehyde groups through a Schiff's reaction. After complete acid-hydrolysis of the immobilized chitosan, the Svennerholm method for glucosamine analysis showed that 0.3% (w/w) chitosan had been successfully introduced on the glass beads. Atomic absorption spectroscopic analysis of eluants of a column of the chitosan-modified glass beads showed that metal ions such as Cu 2+, Ag +, Pb 2+, Fe 3+, and Cd 2+ were more than 90% entrapped on a column of beads prepared in this manner.

VEGF mRNA expressed in microvessels of neonatal and adult rat cerebral cortex

We measured mRNA levels of vascular endothelial growth factor (VEGF) and its Flk-1/KDR receptor in isolated cerebral cortical microvessels and in the cerebral cortex of neonatal (1 week) and adult (11 week) rats using reverse transcription-polymerase chain reaction (RT-PCR). Cerebral microvessels were isolated by density centrifugation, mesh filtration and passage through glass bead columns. The dominant cell types in this preparation are endothelial cells and pericytes. Among the four isoforms of VEGF mRNA expressed in these tissues, VEGF 165 was dominant (67% higher than VEGF 189 or VEGF 206). All isoforms of VEGF were higher in adult cortical microvessels than in cortical homogenates. In isolated microvessels, VEGF mRNA for all isoforms combined was 70% higher in the neonate than in the adult. VEGF receptor Flk-1/KDR mRNA was also present in cortical microvessels and was higher in neonatal than in adult microvessels. The results suggest that VEGF is normally expressed in cerebral microvessels of both neonates and adults. Whether the source of VEGF is the endothelial cell or pericyte, will determine if VEGF has autocrine or paracrine actions. The results also support the hypothesis that microvascular cell turnover continues in the adult brain.

Affinity purification and characterization of the Escherichia coli molecular chaperones.

The molecular chaperones are a group of proteins that are effective in vitro and in vivo folding aids and show a well-documented affinity for proteins lacking tertiary structure. The molecular chaperones were induced from lon(-) Escherichia coli mutants, affinity purified with an immobilized beta-casein column, and assayed for refolding activity with thermally and chemically denatured carbonic anhydrase B (CAB). Chaperones were induced with three treatments: heat shock at 39 degrees C, heat shock 42 degrees C, and alcohol shock with 3% ethanol (v/v). Lysates were applied to an immobilized beta-casein (30 mg/g beads) column. After removing nonspecifically bound proteins with 1 M NaCl, the molecular chaperones were eluted with cold water or 1 mM Mg-ATP. The cold water and Mg-ATP eluates were analyzed by SDS-PAGE. Western analysis identified five E. coli molecular chaperones including DnaK, DnaJ, GrpE, GroEL, and GroES. The purity of eluted chaperones was 58% with cold water and 100% with Mg-ATP. Refolding denatured CAB in the presence of Mg-ATP resulted in a 97% recovery of heat-denatured CAB and a 68% recovery of chemically denatured CAB. The use of affinity matrices for the chaperone purification which are effective as in vitro folding aids will be presented.

Evaluation by ELISA of anisakis simplex larval antigen purified by affinity chromatography.

In order to improve the specificity and sensitivity of the techniques for the human anisakidosis diagnosis, a method of affinity chromatography for the purification of species-specific antigens from Anisakis simplex third-stage larvae (L3) has been developed. New Zealand rabbits were immunized with A. simplex or Ascaris suum antigens or inoculated with Toxocara canis embryonated eggs. The IgG specific antibodies were isolated by means of protein A-Sepharose CL-4B beads columns. IgG anti-A. simplex and -A. suum were coupled to CNBr-activated Sepharose 4B. For the purification of the larval A. simplex antigens, these were loaded into the anti-A. simplex column and bound antigens eluted. For the elimination of the epitopes responsible for the cross-reactions, the A. simplex specific proteins were loaded into the anti-A. suum column. To prove the specificity of the isolated proteins, immunochemical analyses by polyacrylamide gel electrophoresis were carried out. Further, we studied the different responses by ELISA to the different antigenic preparations of A. simplex used, observing their capability of discriminating among the different antisera raised in rabbits (anti-A. simplex, anti-A. suum, anti-T. canis). The discriminatory capability with the anti-T. canis antisera was good using the larval A. simplex crude extract (CE) antigen. When larval A. simplex CE antigen was loaded into a CNBr-activated Sepharose 4B coupled to IgG from rabbits immunized with A. simplex CE antigen, its capability for discriminate between A. simplex and A. suum was improved, increasing in the case of T. canis. The best results were obtained using larval A. simplex CE antigen loaded into a CNBr-activated Sepharose 4B coupled to IgG from rabbits immunized with adult A. suum CE antigen. When we compared the different serum dilution and antigenic concentration, we selected the working serum dilution of (1/4)00 and 1 microg/ml of antigenic concentration.

UV-irradiated DNA matrixes selectively bind endocrine disruptors with a planar structure.

DNA has unique chemical properties and is readily purified from salmon milts and shellfish gonads. DNA has few commercial uses and is generally discarded as an industrial waste. Recently, we prepared water-insoluble and nuclease-resistant DNA-films by UV irradiation. The DNA-films removed DNA-intercalating compounds. Here, we immobilized double-stranded DNA onto porous glass beads by UV irradiation and prepared DNA-immobilized glass bead columns. The DNA-immobilized columns effectively accumulated more DNA-intercalating materials than the DNA-films. The DNA-immobilized columns bound endocrine disruptors with a planar structure, such as dioxin-derivatives, polychlorobiphenyl (PCB)-derivatives, and benzo[a]pyrene. Bisphenol A and diethylstilbestrol, which lack a planar structure, did not bind to the DNA-immobilized columns. These results suggest that DNA-immobilized glass bead columns could selectively remove chemical compounds with a planar structure by intercalation into the double-stranded DNA. DNA-immobilized glass beads and DNA-films prepared by UV irradiation have the potential to serve as useful biomaterials for medical, engineering, and environmental applications.

113Sn-113mIn generator with a glass beads column

The adsorption characteristics of 113Sn(IV) and 113mIn(III) on glass beads from NaCl solutions have been studied. On the basis of these studies, 113Sn-113mIn generator has been prepared by adsorbing 113Sn on the glass beads column. 113mIn has been eluted by the 0.16M NaCl solution with pH 3.0, remaining 113Sn adsorbed on the glass beads. The yield of 113mIn has been about 73% in the first 6 ml of eluate, while the breakthrough of 113Sn has been about 0.042%.

Identification of reddish alcoholic beverages by GC/MS using aroma components as indicators

A method to identify reddish alcoholic beverages used for dissolving stimulant drugs was devised using aroma components as indicators. Thirteen brands of beverages including red wines, blueberry liquors, raspberry liquors, strawberry liquor, a mixture of red wine and blueberry juice and a mixture of red wine and grape juice were examined. Aroma components in each sample were effectively concentrated with a porous polymer beads column (Porapak Q ®) and were analyzed by gas chromatography/mass spectrometry (GC/MS). Specific aroma components were detected in each beverage and reddish alcoholic beverages were successfully classified into five groups using five aroma components as indicators. The present method should prove to be useful in criminal investigations.

Purification of Anisakis simplex antigen by affinity chromatography.

In order to improve the specificity and sensitivity of the techniques for the diagnosis of human anisakidosis, a method of affinity chromatography for the purification of species-specific antigens from Anisakis simplex third-stage larvae (L3) has been developed. New Zealand rabbits were immunized with A. simplex or Ascaris suum antigens or inoculated with Toxocara canis embryonated eggs. The IgG-specific antibodies were isolated by means of protein A-Sepharose CL-4B bead columns. IgG anti-Anisakis simplex, anti-Ascaris suum and anti-T. canis were coupled to CNBr-activated Sepharose 4B. For the purification of the larval Anisakis simplex antigens, it was loaded into the anti-A. simplex column and bound antigens were eluted. For the elimination of the epitopes responsible for the cross-reactions, the A. simplex-specific proteins were loaded into the anti-Ascaris suum and anti- T. canis columns. To prove the specificity of the isolated proteins, immunochemical analyses by polyacrylamide gel electrophoresis and immunoblotting were carried out. Likewise, immunoaffinity columns were prepared using specific IgG from patients with Anisakis simplex sensitization, previously diagnosed by fluoro-enzymo-immunoassay. The protein patterns of antigen after purification by the human columns were similar to those obtained using the rabbit columns.

Rapid two-step purification of a recombinant mouse Fab fragment expressed in Escherichia coli.

We report a rapid, large-scale process for the purification of a recombinant Fab fragment specific for the tobacco mosaic virus coat protein (Fab57P). The fragment is expressed periplasmically in Escherichia coli. The expression level was optimized in 0.3-L fermentors. The highest levels were obtained using the following conditions: (1) low postinduction temperature (21 degrees C), (2) combined use of two beta-lactam antibiotics (carbenicillin and ampicillin), (3) IPTG concentration 0.1 mM, (4) regulated pH 7.2, (5) 17-h induction time, and (6) conditions that reduce mechanical stress. Optimized large-scale fermentations were done in 15- and 300-L capacity fermentors. The recombinant Fab fragment was purified by two chromatographic steps. After disruption of the bacteria using an APV Gaulin homogenizer, the crude E. coli homogenate was directly applied, without centrifugation, to an SP Sepharose Big Beads column. The recombinant Fab fragment was eluted as a single peak in a sodium chloride gradient. The fragment was further purified by affinity adsorption to a column packed with Epoxy-activated Sepharose 6B to which the antigen peptide NH(2)-CGS YNR GSF SQS SGLV-CONH(2) had been coupled through its N-terminal cysteine. The purified Fab57P fragment showed one band in SDS-PAGE. The overall purification yield was 35%.