Ion-exchange Paper Research Articles

Ion-Exchange Based Immobilization of Chromogenic Reagents on Microfluidic Paper Analytical Devices.

Distance-based detection methods, as used in development of microfluidic paper analytical devices (μPADs), rely on the dynamic formation of a colored band along the length of the paper microfluidic channels. The color change is driven by the reaction of chromogenic reagents (typically water-insoluble) that are bound to the paper, thus not subject to being washed away by the sample flow along the detection channel. Here, we introduce the use of an anion-exchange filter paper (as a replacement for standard, unmodified filter paper) for distance-based detection in μPADs, in order to immobilize the water-soluble anionic reagents upon the paper detection channels based on ion-exchange interactions of the oppositely charged paper (protonated tertiary amine groups) and the anionic groups of the reagents. The ion-exchange (IE) paper was initially characterized and its properties were compared with standard cellulose paper. The IE paper was shown to be capable of strong retention of anionic reagents exhibiting acidic functional groups (carboxylic, sulfonic), which become deprotonated and negatively charged when in contact with the IE paper. The effect of the ionic strength (10-250 mM Cl-) and pH (1-13) on the immobilization of the investigated reagents were also determined. The IE-μPADs were then modified with anionic chromogenic reagents and applied to distance-based determination of total calcium (LOD = 0.03 mM) and total acidity (LOD = 2.5 mM) content in serum and wine samples, respectively. The detailed mechanisms of the developed assays on the IE paper are also discussed. We propose that IE-μPADs represent a useful new addition to the distance-based detection toolbox and considerably enhance the applicability of such a detection method.

Evaluation of contamination associated with current blood spot technology for determining the fatty acid status of individuals

A newly developed method for determining fatty acid status in samples of whole blood using collection papers was evaluated. Fresh blood was analyzed either directly or after being applied to a strip of blood collection paper. When compared with fresh blood, spotted blood samples exhibited a higher percentage of saturated fatty acids and lower percentage of polyunsaturated fatty acids, especially when the volume of blood spot was low, suggesting the presence of contaminants in the paper. This was confirmed by gas chromatography analysis of blank collection papers which produced peaks which corresponded with saturated fatty acids. Further studies revealed that latex gloves, aluminium bags and polyethylene bags also have the potential to contaminate blood spot samples during sample collection, processing, and storage. The choice of collection paper, gloves, and storage bags which contain the minimum amount of contaminants is therefore essential to reduce the impact of external contaminants on the blood spot fatty acid analysis results.Practical applications: This research shows that the choice of collection paper, gloves, and storage bags which contain the minimum amount of contaminants is essential to reduce the impact of external contaminants on the blood spot fatty acid analysis results. Moreover, this work shows the potential of using Whatman ion exchange paper in clinical trials that are aimed at measuring the fatty acids composition in dried blood spots, to provide more accurate results.Attempts to validate blood spot technology for determining the fatty acid status of individuals found that contaminants various sources may reduce the accuracy of fatty acid analysis results of blood spot samples. This study evaluates levels of contaminants in different collection papers to develop a method which would minimize the contaminant problem.

Protein Kinase Cθ C2 Domain Is a Phosphotyrosine Binding Module That Plays a Key Role in Its Activation

Protein kinase Cθ (PKCθ) is a novel PKC that plays a key role in T lymphocyte activation. To understand how PKCθ is regulated in T cells, we investigated the properties of its N-terminal C2 domain that functions as an autoinhibitory domain. Our measurements show that a Tyr(P)-containing peptide derived from CDCP1 binds the C2 domain of PKCθ with high affinity and activates the enzyme activity of the intact protein. The Tyr(P) peptide also binds the C2 domain of PKCδ tightly, but no enzyme activation was observed with PKCδ. Mutations of PKCθ-C2 residues involved in Tyr(P) binding abrogated the enzyme activation and association of PKCθ with Tyr-phosphorylated full-length CDCP1 and severely inhibited the T cell receptor/CD28-mediated activation of a PKCθ-dependent reporter gene in T cells. Collectively, these studies establish the C2 domain of PKCθ as a Tyr(P)-binding domain and suggest that the domain may play a major role in PKCθ activation via its Tyr(P) binding.

Combined cation-exchange and extraction chromatographic method of pre-concentration and concomitant separation of Cu(II) with high molecular mass liquid cation exchanger after its online detection

A selective method has been developed for the extraction chromatographic trace level separation of Cu(II) with Versatic 10 (liquid cation exchanger) coated on silanised silica gel (SSG-V10). Cu(II) has been extracted from 0.1 M acetate buffer at the range of pH 4.0–5.5. The effects of foreign ions, pH, flow-rate, stripping agents on extraction and elution have been investigated. Exchange capacity of the prepared exchanger at different temperatures with respect to Cu(II) has been determined. The extraction equilibrium constant ( K ex) and different standard thermodynamic parameters have also been calculated by temperature variation method. Positive value of Δ H (7.98 kJ mol −1) and Δ S (0.1916 kJ mol −1) and negative value of Δ G (−49.16 kJ mol −1) indicated that the process was endothermic, entropy gaining and spontaneous. Preconcentration factor was optimized at 74.7 ± 0.2 and the desorption constants K desorption 1(1.4 × 10 −2) and K desorption 2(9.8 × 10 −2) were determined. The effect of pH on R f values in ion exchange paper chromatography has been investigated. In order to investigate the sorption isotherm, two equilibrium models, the Freundlich and Langmuir isotherms, were analyzed. Cu(II) has been separated from synthetic binary and multi-component mixtures containing various metal ions associated with it in ores and alloy samples. The method effectively permits sequential separation of Cu(II) from synthetic quaternary mixture containing its congeners Bi(III), Sn(II), Hg(II) and Cu(II), Cd(II), Pb(II) of same analytical group. The method was found effective for the selective detection, removal and recovery of Cu(II) from industrial waste and standard alloy samples following its preconcentration on the column. A plausible mechanism for the extraction of Cu(II) has been suggested.

The determination of epitetracycline and tetracycline by ion-exchange paper chromatography and its application to human urine and serum.

Abstract A simple quantitative chromatographic method is described for the separation of epitetracycline and tetracycline in pharmaceutical preparations. The method is applicable qualitatively to human serum and quantitatively to the biological determination of tetracycline in urine. Whatman's modified cellulose phosphate cation-exchange paper is developed by the descending technique using 0·1 per cent w/v ammonium chloride solution. The spots are located as yellow fluorescent spots under ultra-violet light and ammonia vapour, eluted in 10 per cent w/v ammonium chloride, and the extinction determined at 356 mμ. The method is not suitable for the separation of chlortetracycline or oxytetracycline from tetracycline.

A note on the paper chromatographic separation of codeine, morphine and nalorphine.

Abstract Codeine has been separated from a mixture of codeine, morphine and nalorphine on Whatman ET20 ion-exchange paper, in 5 min. A mixture of morphine and nalorphine has been separated in 90 min. by horizontal circular reversed phase paper chromatography. Using ascending chromatography at 86° on tributyrin-treated paper, an unequivocal resolution of a mixture of morphine, nalorphine and codeine has been achieved within 20 min.

Splice Variants of the Dual Specificity Tyrosine Phosphorylation-regulated Kinase 4 (DYRK4) Differ in Their Subcellular Localization and Catalytic Activity

Dual specificity tyrosine phosphorylation-regulated kinases, DYRKs, are a family of conserved protein kinases that play key roles in the regulation of cell differentiation, proliferation, and survival. Of the five mammalian DYRKs, DYRK4 is the least studied family member. Here, we show that several splice variants of DYRK4 are expressed in tissue-specific patterns and that these variants have distinct functional capacities. One of these variants contains a nuclear localization signal in its extended N terminus that mediates its interaction with importin α3 and α5 and that is capable of targeting a heterologous protein to the nucleus. Consequently, the nucleocytoplasmic mobility of this variant differs from that of a shorter isoform in live cell imaging experiments. Other splicing events affect the catalytic domain, including a three-amino acid deletion within subdomain XI that markedly reduces the enzymatic activity of DYRK4. We also show that autophosphorylation of a tyrosine residue within the activation loop is necessary for full DYRK4 kinase activity, a defining feature of the DYRK family. Finally, by comparing the phosphorylation of an array of 720 peptides, we show that DYRK1A, DYRK2, and DYRK4 differ in their target recognition sequence and that preference for an arginine residue at position P -3 is a feature of DYRK1A but not of DYRK2 and DYRK4. Therefore, we highlight the use of subcellular localization as an important regulatory mechanism for DYRK proteins, and we propose that substrate specificity could be a source of functional diversity among DYRKs.

Combined cation-exchange and extraction chromatographic method of preconcentration and concomitant separation of bismuth(III) with high molecular mass liquid cation exchanger

A selective method has been developed for the extraction chromatographic separation of Bi III with Versatic 10 coated on silanized silica gel (SSG). Bi III has been quantitatively extracted from 0.1 M acetate buffer at the range of pH 5.0–5.5. The result showed that the solution pH, influent volume, flow-rate and solution temperature would affect the sorption of Bi III. The sorbed Bi III was eluted with 0.1 M H 2SO 4. The extractor system has got good values of exchange capacity (1.42 meq. of H + g −1 of dry exchanger at 25 °C), break-through capacity (19.75 mg g −1 at pH 5.5) and column efficiencies (300) with respect to Bi III. The positive value of Δ H (12.63 kJ mol −1) and Δ S (0.271 kJ mol −1 K −1) and negative value of Δ G (−68.241 kJ mol −1) indicated that the sorption process was endothermic, entropy gaining and spontaneous in nature. P.F. has been optimized at 76.4 ± 0.3 and the desorption constants K desorption (8 × 10 −3) and K ′ desorption (1.4 × 10 −1) have been determined. R f values and selectivity factors for diverse metal ions with respect to that of Bi III have been determined by ion-exchange paper chromatography. Bi III has been separated from synthetic mixtures containing its congeners and other metal ions associated with it in ores and alloy samples. The method was found effective for removal of Bi III from different water samples. A plausible mechanism for the extraction of Bi III has been suggested.

Extraction chromatographic method of preconcentration and separation of lead (II) with high molecular mass liquid cation exchanger

A selective method has been developed for the extraction chromatographic separation of lead (II) with Versatic 911 (liquid cation exchanger) coated on silanised silica gel. Lead (II) has been extracted from 0.1 M acetate buffer at the range of pH 4.5–6.5. The effects of pH, flow-rate, stripping agents on extraction and elution have been investigated. Exchange capacity of the prepared exchanger at different temperatures with respect to lead (II) has been determined. The extraction equilibrium constant ( K ex) and different standard thermodynamic parameters have also been calculated by temperature variation method. The effect of pH on R f values in ion exchange paper chromatography has been investigated. In order to investigate the sorption isotherm, two equilibrium models, the Freundlich and Langmuir isotherms, were analyzed. Lead (II) has been separated from synthetic binary and multi-component mixtures containing various metal ions associated with it in ores and alloy samples. The method effectively permits sequential separation of lead (II) from synthetic quaternary mixture containing its congeners Cu (II), Sn (II), Hg (II) and Cu (II), Cd (II), Bi (III) of the same analytical group. The method was found effective for removal and recovery of lead (II) from industrial waste and standard alloy samples following its preconcentration on the column. A plausible mechanism for the extraction of lead (II) has been suggested.

Identification of Acidic Amino Acid Residues in the Protein Kinase Cα V5 Domain That Contribute to Its Insensitivity to Diacylglycerol

The protein kinase C (PKC) isoforms are maintained in an inactive and closed conformation by intramolecular interactions. Upon activation these are disrupted by activators, binding proteins and cellular membrane. We have seen that autophosphorylation of two sites in the C-terminal V5 domain is crucial to keep PKC alpha insensitive to the activator diacylglycerol, which presumably is caused by a masking of the diacylglycerol-binding C1a domain. Here we demonstrate that the diacylglycerol sensitivity of the PKC beta isoforms also is suppressed by autophosphorylation of the V5 sites. To analyze conformational differences, a fusion protein ECFP-PKC alpha-EYFP was expressed in cells and the FRET signal was analyzed. The analogous mutant with autophosphorylation sites exchanged for alanine gave rise to a substantially lower FRET signal than wild-type PKC alpha indicating a conformational difference elicited by the mutations. Expression of the isolated PKC alpha V5 domain led to increased diacylglycerol sensitivity of PKC alpha. We identified acidic residues in the V5 domain that, when mutated to alanines or lysines, rendered PKC alpha sensitive to diacylglycerol. Furthermore, mutation to glutamate of four lysines in a lysine-rich cluster in the C2 domain gave a similar effect. Simultaneous reversal of the charges of the acidic residues in the V5 and the lysines in the C2 domain gave rise to a PKC alpha that was insensitive to diacylglycerol. We propose that these structures participate in an intramolecular interaction that maintains PKC alpha in a closed conformation. The disruption of this interaction leads to an unmasking of the C1a domain and thereby increased diacylglycerol sensitivity of PKC alpha.

PDE7A1, a cAMP-specific Phosphodiesterase, Inhibits cAMP-dependent Protein Kinase by a Direct Interaction with C

The N-terminal regulatory region of the high affinity cAMP-specific phosphodiesterase, PDE7A1, contains two copies of the cAMP-dependent kinase (PKA) pseudosubstrate site RRGAI. In betaTC3 insulinoma cells, PDE7A1 co-localizes with PKA II in the Golgi-centrosome region. The roles PDE7A1 and its regulatory region play in cAMP signaling were examined by studying interactions with PKA subunits. PDE7A1 associates with the dissociated C subunit of PKA (C), but does not bind tetrameric PKA holoenzyme. High affinity binding of C by PDE7A1 inhibits kinase activity in vitro (IC50 = 0.5 nm). The domain containing PKA pseudosubstrate sites at the N terminus of PDE7A1 mediates complex formation with C. The PDE7A1 N-terminal repeat region inhibits C activity in CHO-K1 cells and also suppresses C dependent, cAMP-independent, physiological responses in yeast. Thus, PDE7A1 possesses a non-catalytic activity that can contribute to the termination of cAMP signals via direct inhibition of C. This study identifies a novel inhibitor of PKA and a non-catalytic affect of a cyclic nucleotide phosphodiesterase.

Roles of Ionic Residues of the C1 Domain in Protein Kinase C-α Activation and the Origin of Phosphatidylserine Specificity

On the basis of extensive structure-function studies of protein kinase C-alpha (PKC-alpha), we have proposed an activation mechanism for conventional PKCs in which the C2 domain and the C1 domain interact sequentially with membranes (Medkova, M., and Cho, W. (1999) J. Biol. Chem. 274, 19852-19861). To further elucidate the interactions between the C1 and C2 domains during PKC activation and the origin of phosphatidylserine specificity, we mutated several charged residues in two C1 domains (C1a and C1b) of PKC-alpha. We then measured the membrane binding affinities, activities, and monolayer penetration of these mutants. Results indicate that cationic residues of the C1a domain, most notably Arg(77), interact nonspecifically with anionic phospholipids prior to the membrane penetration of hydrophobic residues. The mutation of a single aspartate (Asp(55)) in the C1a domain to Ala or Lys resulted in dramatically reduced phosphatidylserine specificity in vesicle binding, activity, and monolayer penetration. In particular, D55A showed much higher vesicle affinity, activity, and monolayer penetration power than wild type under nonactivating conditions, i.e. with phosphatidylglycerol and in the absence of Ca(2+), indicating that Asp(55) is involved in the tethering of the C1a domain to another part of PKC-alpha, which keeps it in an inactive conformation at the resting state. Based on these results, we propose a refined model for the activation of conventional PKC, in which phosphatidylserine specifically disrupts the C1a domain tethering by competing with Asp(55), which then leads to membrane penetration and diacylglycerol binding of the C1a domain and PKC activation.

Sensitive Assay for Mitochondrial DNA Polymerase γ

The mitochondrial DNA polymerase gamma is the principal polymerase required for mitochondrial DNA replication. Primary or secondary deficiencies in the activity of DNA polymerase gamma may lead to mitochondrial DNA depletion. We describe a sensitive and robust clinical assay for this enzyme. The assay was performed on mitochondria isolated from skeletal muscle biopsies. High-molecular weight polynucleotide reaction products were captured on ion-exchange paper, examined qualitatively by autoradiography, and quantified by scintillation counting. Kinetic analysis of DNA polymerase gamma by this method showed a K(m) for dTTP of 1.43 micromol/L and a K(i) for azidothymidine triphosphate of 0.861 micromol/L. The assay was linear from 0.1 to 2 microgram of mitochondrial protein. The detection limit was 30 units (30 fmol dTMP incorporated in 30 min). The linear dynamic range was three orders of magnitude; 30-30 000 units. Imprecision (CV) was 6.4% within day and 12% between days. Application of this assay to a mixed population of 38 patients referred for evaluation of mitochondrial disease revealed a distribution with a range of 0-2506 U/microgram, reflecting extensive biologic variation among patients with neuromuscular disease. This assay provides a useful adjunct to current laboratory methods for the evaluation of patients with suspected mitochondrial DNA depletion syndromes.

Development and production of 81Rb/81mKr radionuclide generator in NPI

A project of 81Rb production and 81Rb/81mKr generator manufacturing is described. 81Rb nuclide is produced on U-120M cyclotron via Kr(p, 2n)Rb reaction using pressurised gaseous target. Generator of a “dry” type is based on the sorption of 81Rb on an ion-exchange paper from which the daughter 81mKr is eluted by air. Parameters of targetry and generator assembly are given. Generator which will be manufactured under pharmaceutical “clean” conditions is intended for lung ventilation studies in nuclear medicine.

Evaluation of silver substrates for surface-enhanced Raman detection of drugs banned in sport practices

Five different forms of surface-enhanced Raman active silver have been investigated. These forms include colloidal silver prepared by tetrahydroborate or citrate reduction, colloidal silver supported on ion-exchange paper or on cellulose paper and acid etched silver foils. These substrates have been evaluated using nine drugs as model compounds, which are known to be SERS-active on at least one of these substrates. A comparative study of short and long-term signal stability and limits of detection is discussed. The analysis of binary and ternary mixtures of drugs on the different substrates is discussed in terms of adsorptivity and detection power.

The detection of neomycin phosphotransferase activity in plant extracts.

AbstractThe selectable marker gene so far most frequently used in plant transformation experiments is the well-known nptII or neomycin phosphotransferase II gene, also referred to as aph(3′)II or aminoglycoside phosphotransferase II (1). Derived from the bacterial transposon Tn5, the gene product (E.C.2.7.1.95) transfers the γ phosphate group of ATP to a specific hydroxyl group of aminoglycosidic antibiotics. In this way, it detoxifies neomycin, kanamycin, G418, as well as paromomycin. Plants expressing the enzyme can tolerate certain concentrations of these antibiotics which lead to bleaching and growth inhibition in nontransformed plants. In many cases, kanamycin sulfate is a suitable additive for selective plant media, but which of the selective agents results in the most reliable distinction between sensitive and resistant cells must be evaluated for the specific plant material under investigation. Although quantification of gene expression and its tissue specificity in plants are routinely determined by the use of the β-glucuronidase (GUS) reporter gene, there are still some applications where the level of gene expression originating from the selectable marker is of interest. Several protocols for in vitro assays of NPTII activity have been published (2–11). All are based on the transfer of the 32P-labeled γ phosphate group from ATP to kanamycin (2), with the drawback that other phosphorylation reactions result in additional labeled products. Therefore, assays specific for neomycin phosphotransferase require a protein separation prior to the reaction and/or a separation of the reaction products. Protein separation has been achieved by gel filtration (3), nondenaturing gel electrophoresis (4), phenol extraction (9), or immunoaffinity purification (10). Reaction products can be distinguished by paper chromatography (3), thin-layer chromatography (7), or by the strong binding of the positively charged kanamycin to phosphocellulose ion-exchange paper (2,4,5,8–11) in combination with passage through nitrocellulose (6). As usual, each procedure encompasses a certain combination of benefits and obstacles. Immunopurification of the NPTII enzyme (10) makes the assay sensitive and specific, but requires some additional handling and expensive chemicals. If the molecular weight of the NPTII protein is of importance, e.g., in translational fusion markers (12), the sensitive assay using electrophoretic separation followed by an in situ reaction and subsequent blotting of the reaction products (4) can be applied, but this requires time and substantial amounts of radioactivity and limits the sample number. A recently published protocol describes a qualitative assay for NPTII activity without the necessity to prepare plant extracts and with the potential to analyze GUS activity in parallel (11).KeywordsNeomycin PhosphotransferaseKanamycin SulfateImmunoaffinity PurificationNontransformed PlantDisposable CuvetsThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Direct assay method for guanosine 5′-monophosphate reductase activity

A sensitive and simple micromethod for the accurate measurement of GMP reductase (EC 1.6.6.8) activity in crude extracts is described. The reaction product of [8- 14C]IMP was separated from the substrate [8- 14C]GMP by descending chromatography on Whatman DE81 ion-exchange paper. This separation method provides an analysis of the possible interfering reactions, such as the metabolic conversion of the substrate GMP to GDP, GTP, and/or guanosine, and guanine and the loss of the product IMP to inosine, hypoxanthine, and other metabolites. Low blank values (70–90 cpm) were obtained consistently with this assay because the IMP spot moves faster than the GMP spot. The major advantages of this method are direct measurement of GMP reductase activity in crude extracts, high sensitivity (with a limit of detection of less than 10 pmol of IMP production), high reproducibility (< ± 5%), and capability to measure activity in small samples (9 μg protein).

Thyrotropin-Releasing Hormone-Degrading Enzyme in Human Serum Is Classified as Type II of Pyroglutamyl Aminopeptidase: Influence of Thyroid Status

The characteristics of thyrotropin-releasing hormone (TRH)-degrading enzyme in human serum were studied. Serum was incubated in 0.1 M phosphate buffer containing [proline-3H]TRH at 37 degrees C. A thin layer chromatography analysis of TRH degradation did not show any radioactive peak located in an acid TRH position, but apparent radioactive peaks corresponding to His-Pro and His-ProNH2 occurred in the presence of p-hydroxymercuriphenyl sulfonic acid, an inhibitor of proline dipeptidase. With ion exchange paper chromatography, the formation of 3H-labeled His-Pro and His-ProNH2 was estimated as an end point in the measurement of pyroglutamyl aminopeptidase (pGlu-peptidase) activity. An assay using p-hydroxymercuriphenyl sulfonic acid was developed to sensitively quantitate the pGlu-peptidase. Neither bacitracin nor p-chloromercuribenzoic acid increased the activity of pGlu-peptidase. The addition of EDTA, dithiothreitol, and o-phenanthroline significantly inhibited pGlu-peptidase activity, but neither iodoacetamide nor ethylmaleimide altered its activity. The pGlu-peptidase had a stereotypic specificity for the tripeptide, pGlu-His-ProNH2 of TRH, and its Km was 44.9 microM. The pGlu-peptidase activity was not changed by either hyper- or hypothyroidism. The present data indicate that a TRH-degrading enzyme in human serum possesses a nature identical to type II of pGlu-peptidase which is not altered by thyroid status.

An automated microprocessor controlled system for the production of a new type of81Rb−81mKr generator

Routine production of81Rb, utilizing the82Kr(p, 2n)81Rb reaction with natural krypton gas is described. Development and construction of a fully automated gas target system controlled by microprocess or is presented. A new type of81Rb−81mKr generator based on the sorption of81Rb on ion-exchange paper was developed and investigated in operation. It may be used either for gas-phase or for liquid elution of81mKr for different medical applications.

A method for determination of glucose 1,6-bisphosphatase.

A sensitive and specific method to measure glucose 1,6-bisphosphatase activity, which allows the identification of the reaction products is described. [U-14 C]Glucose 1,6-P2, synthesized by the glucose 1-P kinase activity of phosphofructokinase, is used as substrate. The reaction products are separated and identified by chromatography on ion-exchange paper.