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PubMed1990

Teniposide, a topoisomerase II inhibitor, prevents chromosome condensation and separation but not decondensation in fertilized surf clam (Spisula solidissima) oocytes.

DNA topoisomerase II has been implicated in regulating chromosome interactions. We investigated the effects of the specific DNA topoisomerase II inhibitor, teniposide on nuclear events during oocyte maturation, fertilization, and early embryonic development of fertilized Spisula solidissima oocytes using DNA fluorescence. Teniposide treatment before fertilization not only inhibited chromosome separation during meiosis, but also blocked chromosome condensation during mitosis; however, sperm nuclear decondensation was unaffected. Chromosome separation was selectively blocked in oocytes treated with teniposide during either meiotic metaphase I or II indicating that topoisomerase II activity may be required during oocyte maturation. Teniposide treatment during meiosis also disrupted mitotic chromosome condensation. Chromosome separation during anaphase was unaffected in embryos treated with teniposide when the chromosomes were already condensed in metaphase of either first or second mitosis; however, chromosome condensation during the next mitosis was blocked. When interphase two- and four-cell embryos were exposed to topoisomerase II inhibitor, the subsequent mitosis proceeded normally in that the chromosomes condensed, separated, and decondensed; in contrast, chromosome condensation of the next mitosis was blocked. These observations suggest that in Spisula oocytes, topoisomerase II activity is required for chromosome separation during meiosis and condensation during mitosis, but is not involved in decondensation of the sperm nucleus, maternal chromosomes, and somatic chromatin.

Animals↗
PubMed1985

Schistosoma mansoni, S. japonicum, and S. haematobium: permeability to acidic amino acids and effect of separated and unseparated adults.

Permeability of the tegument of male and female Schistosoma mansoni was measured in vitro and a comparison was made between copulating and separated worms. In unpaired (separated) schistosomes, a carrier-mediated (selective) transport system for acidic amino acids was demonstrated. Males and females exhibited similar uptake rates for aspartate and glutamate. Half-saturation constants for aspartate (males, 0.035 +/- 0.008 mM; females, 0.026 +/- 0.006 mM) and glutamate (males, 0.010 +/- 0.007 mM; females, 0.015 +/- 0.004 mM) were determined for separated worms only. Time-course studies provided estimates of aspartate influx rates in males (7.3 pmol min-1 worm-1) and females (2.3 pmol min-1 worm-1). The most dramatic observation, however, was that, in copula, neither male nor female schistosomes took up acidic amino acids, but may have excluded these compounds. Thus, this ouabain-insensitive, mediated mechanism was operational only when the worms were unmated. In S. japonicum, no uptake of glutamate was observed in either mated or separated males and females. In S. haematobium, saturable uptake of aspartate was apparent in both mated and unmated males and females, indicating that species-specific differences in uptake of acidic amino acids existed. These studies indicate the need for cautious interpretation of data obtained from in vitro analyses of separated male and female mansonian schistosomes, and that such conditions may not reflect in vivo or in copula function.

Animals↗
PubMed1987

Improved separation of perch liver metallothionein by fast protein liquid chromatography.

The separation of metallothionein isoforms by Mono-Q column chromatography by fast protein liquid chromatography is described and compared to separation on a DEAE-Sephadex A-25 ion-exchange column. The separation of two isoforms of rainbow trout (Salmo gairdneri) metallothionein was possible by DEAE-Sephadex A-25 chromatography, while only one form of perch (Perca fluviatilis) metallothionein was obtained with this method. However, by applying FPLC on a Mono-Q column it was possible to separate two isoforms of perch metallothionein. When comparing the chromatographic behaviour of rainbow trout and perch metallothioneins it was found that the two isoforms of perch metallothionein were eluted closer together than the two rainbow trout metallothioneins. Both forms of perch and rainbow trout metallothioneins lacked aromatic amino acids and histidine, and had a high cystein content (30 mol%). The molecular weight was estimated to be 6000 for the apothioneins. The results from this study demonstrate the usefulness of FPLC to improve the resolution of metallothionein separations.

Amino Acids↗
PubMed1991

Chromatographic separation of mixed peptides from amino acids in biological digests with volatile buffers.

Two chromatographic methods, capable of separating mixed peptides from contaminating amino acids in biological digests, are described. Both methods involve separation on copper-Chelex resin, but each uses a different set of elution buffers. When separation method 1 was applied to a commercially available proteolytic digest of casein, the free amino acid content was reduced from 26.0% to 0.5%. With an enzymic digest of 14C-labelled proteins derived from plant biomass, separation method 2 decreased the contaminating free amino acids from 20.3% to 1.9%. Since the separated peptides are eluted with volatile buffers, they are suitable as substrates for biological experiments.

Amino Acids↗
PubMed1992

Computer simulation as a tool for the rapid optimization of the high-performance liquid chromatographic separation of a tryptic digest of human growth hormone.

Computer simulation was used to optimize the separation of a tryptic digest of recombinant human growth hormone using reversed-phase high-performance liquid chromatography in a gradient mode. DryLab G/plus software modelled the retention behavior of the complex tryptic digest mixture as a function of gradient conditions, based on data from two experimental gradient runs. The theoretical optimum separation conditions were rapidly obtained and reproduced experimentally. Resolution did not simply increase as gradient steepness was decreased, rather, an intermediate gradient time provided maximum sample resolution. The simulation results also indicate that the method is reasonably rugged, with little change in the separation expected for different high-performance liquid chromatography systems, and changes in the separation can be compensated by a change in the gradient steepness. Computer simulation can also be useful to quickly reoptimize conditions for a new column, if it fails to provide the same separation.

Chromatography, High Pressure Liquid↗
PubMed1993

Separation and tryptic digest mapping of normal and variant haemoglobins by capillary electrophoresis.

Characterization of haemoglobin (Hb) through whole protein separations and tryptic digest mapping allows the identification of structural Hb variants which result in haemoglobinopathies. Tryptic digest mapping by conventional two-dimensional paper chromatograph-electrophoresis provides high resolution but requires 48 h, while gradient elution reversed-phase high-performance liquid chromatography (HPLC) is faster (1.5 h), there is decreased resolution. CE analysis provides a fast separation with high resolution. We have used CE to optimise the tryptic digestion of globin purified from normal human haemoglobin A and to analyze tryptic digests from normal Hb. The separations were optimised and peak identification performed using UV scanning detection. In the optimised tryptic digest separation up to 28 peaks could be resolved in < 20 min. These peaks were identified as far as possible and a high-resolution map of the digest was constructed. The optimised analytical conditions were used to observe the separation pattern obtained from normal adult haemoglobin (HbA), common variant haemoglobins and some rarer haemoglobin variants.

Adult↗
PubMed1993

Comparison of selectivities of reversed-phase high-performance liquid chromatography, capillary zone electrophoresis and micellar capillary electrophoresis in the separation of neurohypophyseal peptides and analogues.

A number of RP-HPLC systems, including those prescribed in several official monographs, have been evaluated for separating oxytocin, the vasopressins, some clinically important analogues and two additional neurohypophyseal nonapeptides. The separation has been compared with capillary zone electrophoresis and micellar electrophoresis in four micellar systems: cationic, anionic, zwitterionic and neutral. Complete separation was achieved by both RP-HPLC and micellar CE but the importance of charge as a major parameter of separation in CE confers a distinct and useful selectivity to micellar CE based separations.

Amino Acid Sequence↗
PubMed1993

Separation of DNA fragments by capillary electrophoresis using replaceable linear polyacrylamide matrices.

The use of low percent (1.5-6% T) replaceable linear polyacrylamide (LPA) network matrices for rapid separation of double-stranded DNA fragments was explored. Separations of fragments ranging from 20 to 23,000 base pairs were readily achieved. Typically, 4 x 10(6) theoretical plates/m were obtained in less than 30 min. Short separation times under 2 min were also possible, using the DNA intercalating dye, ethidium bromide, along with high electric fields. The high resolving power of linear polyacrylamide was demonstrated in the separation of two fragments which differ by a single base pair (123/124 base pairs) using 6% T LPA and ethidium bromide intercalation. This LPA composition allowed for the possible single base-pair resolution of dsDNA fragments up to 300 base pairs in length. Several concentrations of the linear polyacrylamide for different ranges of fragment lengths have been employed. In addition, replaceable LPA offers the advantage of a fresh separation matrix for each run, thus overcoming column stability problems and minimizing needs for sample cleanup. Electro-osmotic flow was substantially reduced using stable capillary coatings, which were required for obtaining high efficiencies and good reproducibility.

Bacteriophage phi X 174↗
PubMed1993

Separation of estrogens and rodenticides using capillary electrophoresis with aqueous-methanolic buffers.

Capillary electrophoresis (CE) has proven to be an efficient method for the separation of various charged and neutral analytes. For analytes having limited solubility in water, the CE mode of separation has been micellar electrokinetic capillary chromatography (MECC). However, another approach is the direct addition of an organic solvent to a non-MECC CE separation system. Walbroehl and Jorgenson and also Balchunas and Sepaniak have reported on the use of organics in CE but the focus of their work was using MECC to separate small organic compounds. This work examines the use of aqueous-methanolic buffers in non-MECC CE separations of estrogens and rodenticides.

Buffers↗
PubMed1994

Separation of metallothionein isoforms by micellar electrokinetic capillary chromatography.

Current techniques for the separation and quantification of metallothionein isoforms have limited value for routine analysis. Isoforms having a similar charge have been separated successfully using reversed-phase HPLC but this technique suffers from a slow sample turnover time. The use of the surfactant sodium dodecyl sulphate for the separation of metallothionein isoforms by micellar electrokinetic capillary chromatography (MECC) is described. The charge-different isoforms MT-1 and MT-2 from rats, rabbits and sheep were separated within 9-12 min. In addition, a varying degree of heterogeneity was observed in purified samples of human MT-1, rat MT-2, rabbit MT-1, rabbit MT-2 and sheep MT-1. The behaviour of chicken MT was different from that of any other species. The separation of sheep liver extracts indicated the potential of MECC as the basis for a quantitative assay for both charge-different and charge-similar metallothionein isoforms.

Animals↗
PubMed1995

Chiral separation of MDL 73,005EF enantiomers using an alpha 1-acid glycoprotein column.

The enantiomeric separation of MDL 73,005EF (racemic mixture of two enantiomers) has been accomplished using a Chiral AGP (alpha 1-acid glycoprotein) column. The enantiomers are baseline resolved with a runtime of less than 10 min. This separation is used to quantitate the enantiomers present in bulk drug samples, tabletted formulations, and drug used in pharmacological and toxicological studies. Variables found to have an effect on the enantiomeric separation were studied and include: mobile phase ionic strength, type and concentration of organic modifier added to the mobile phase, mobile phase, mobile phase pH, column temperature and the amount of analyte injected. The enantiomeric separation was optimized on the Chiral AGP column based on the effects that each variable had on the separation. Calibration curves for a standard were linear over a range of 0.24 to 61.2 micrograms/g with a correlation coefficient of better than 0.999. A detection limit of 0.012 micrograms/g and a quantitation limit of 0.24 micrograms/g were also found.

Chromatography, High Pressure Liquid↗
PubMed1995

Capillary zone electrophoresis at subzero temperatures. I. Separation of the cis and trans conformers of small peptides.

The cis-trans conformers of two dipeptides, Phe-Pro and Leu-Pro, and two opioid heptapeptides containing one or two proline residues were separated by capillary zone electrophoresis (CZE) in borate buffer at low temperatures down to -17 degrees C. At temperatures near ambient, the relaxation time of the cis-trans isomerization is on the time-scale of minutes for the dipeptides and thus commensurate with the migration times in CZE under usual operating conditions. The conformers of both dipeptides could be separated with baseline resolution below 10 degrees C in neat aqueous 100 mM sodium borate (pH 8.4). The conformer peaks on the electropherograms were identified by using authentic samples of the cis and trans forms of Phe-Pro and Leu-Pro that were obtained by reversed-phase HPLC at 0 degree C, validated by NMR spectroscopy and stored in liquid nitrogen. The interplay of the electrophoretic migration and on-column isomerization reaction in CZE of Phe-Pro under various conditions was analyzed in the light of the Damköhler number (Da). The results showed that besides employing low temperature increasing the voltage and/or decreasing the capillary length also reduce the magnitude of Da to bring about the separation of interconverting species. In this work the use of low temperature in this work was preferred due to the experimental simplicity. The separation of cis-trans conformers of two opioid heptapeptides was carried out by CZE at subzero temperatures with aqueous sodium borate containing 23% (v/v) glycerol at pH* 11.3 as measured with a glass electrode. The two conformers of Tyr-Pro-Phe-Asp-Val-Val-Gly-NH2 were baseline separated at -12 degrees C and the four conformers of Tyr-Pro-Phe-Gly-Tyr-Pro-Ser-NH2 due to the presence of two peptidyl-proline bonds in the molecule, were also resolved at -12 degrees C. From the electrophoretic mobilities, the hydrodynamic radii of the cis-trans conformers of the dipeptides Phe-Pro and Leu-Pro were estimated. In both cases, the trans isomers had 1.3 times greater Stokes radii than the cis conformers. This agrees with the observed migration order and molecular modeling results. The hydrodynamic radii of the Phe-Pro conformers were smaller than those of the Leu-Pro isomers despite the lower molecular mass of the latter. The results demonstrate that CZE is suitable for measuring certain molecular properties and suggest that the methods introduced here are applicable to the study of other systems of interconverting conformers.

Amino Acid Sequence↗
PubMed1996

Separation of enantiomers of drugs by capillary electrophoresis. III. Beta-cyclodextrin as chiral solvating agent.

Enantiomer separation by capillary zone electrophoresis was studied for a set of 34 chiral drugs. Keeping the concentration of beta-cyclodextrin as a chiral solvating agent as constant as possible led to the separation of seven enantiomeric pairs. Carvedilol, Tetryzoline, Tropicamide and Zopiclone gave a baseline separation, Chlorphenamine, Ketamine, and Orciprenaline a partial separation. Statistical analysis revealed that the best separation factors were observed for a medium degree of interaction with the cyclodextrin. A theory explaining this effect provides a helpful guideline for further optimization.

Azabicyclo Compounds↗
PubMed1996

Preparative separation of pyrrolizidine alkaloids by high-speed counter-current chromatography.

We have applied a high-speed counter-current chromatography (CCC) technique to the separation and purification of pyrrolizidine alkaloids from Amsinckia tessellata, Symphytum spp., Trichodesma incanum (Boraginaceae), and Senecio douglasii var. longilobus (Asteraceae). Alkaloidal fractions were separated in a solvent system composed of a chloroform mobile phase and 0.2 M potassium phosphate buffer, of an optimum pH, as the stationary phase. Up to 800 mg of sample could be successfully separated in a single run, with excellent resolution of alkaloids. Lycopsamine and several of its acetylated derivatives were resolved from alkaloidal fractions of Amsinckia and Symphytum. However, diastereomeric pairs such as 7-acetyl-lycopsamine and 7-acetyl-intermedine, could not be separated. The presence of diastereoisomers was determined by gas chromatography-mass spectrometry. Trichodesma contained predominantly trichodesmine, which was resolved from a small quantity of incanine. we report the electron impact mass spectrum of incanine for the first time. Resolving power of CCC was sufficient to separate the closely related alkaloids senecionine and seneciphylline from Senecio, in addition to florosenine and retrorsine, Pyrrolizidine alkaloid compositions of the four species, determined by mass spectral techniques, were consistent with literature, except for the lack of riddelliine and the presence of the otonecine-based florosenine in Senecio douglasii var. longilobus.

Chromatography, Liquid↗
PubMed1996

Enantiomer separation of drugs by electrokinetic chromatography.

This review surveys the enantiomer separation of drugs by electrokinetic chromatography (EKC). EKC is one option of capillary electrophoretic (CE) techniques, which permits the separation of electrically neutral drugs. In enantiomer separation by EKC, ionic pseudo-stationary phases such as chiral micelles and proteins, which can migrate with its electrophoretic mobility and can interact with the solutes, are employed as chiral selectors. Addition of electrically neutral chiral selectors such as cyclodextrins and precolumn conversion of solutes into diastereomers are also successful in EKC. A brief theory of separation and modes of EKC enantiomer separation are described with some typical applications.

Chromatography↗
PubMed1996

Enantiomer separation of basic drugs by capillary electrophoresis using ionic and neutral polysaccharides as chiral selectors.

The enantiomer separation of basic drugs by capillary electrophoresis was investigated employing two types of polysaccharides as chiral selectors. One type consists of electrically neutral polysaccharides such as dextran and dextrin, in which the separation mode is capillary zone electrophoresis (CZE), and ionic drugs are suitable for this mode. The other consists of ionic polysaccharides such as chondroitin sulfate C. Chondroitin sulfates are known as mucopolysaccharides and natural components, being charged, linear, sulfated polysaccharides of high mass. Therefore, the latter approach can be classified as affinity electrokinetic chromatography (AEKC). Both ionic and neutral drugs can be separated by AEKC owing to its separation principle. The separation of enantiomers of some basic drugs such as diltiazem and trimetoquinol was investigated with both CZE and AEKC employing polysaccharides as mentioned above and the enantioselectivity was compared. A brief mechanism of enantiorecognition by polysaccharides is also described.

Chondroitin Sulfates↗
PubMed1996

Investigation of enantioselective separation of quinolonecarboxylic acids by capillary zone electrophoresis using vancomycin as a chiral selector.

When a chiral selector that is a pharmaceutical compound is added to the separation buffer in capillary electrophoresis, the enantioselectivity and the mobility of analytes which interact with that chiral selector may be altered. The changes in enantioselectivity and mobility of the analyte are a function of the strength of the affinity interaction, which depends on the structure of each. The macrocyclic antibiotic vancomycin contains a variety of functionalities that are known to be useful for enantioselective interactions (e.g., hydrogen bonding groups, hydrophobic pockets, aromatic groups, amide linkages). Capillary electrophoresis with vancomycin as a buffer additive was used to separate the enantiomers of different compounds. In this study, the chiral separation of quinolonecarboxylic acids that exhibit marked antibacterial activity and of related compounds was achieved by capillary electrophoresis using vancomycin. The correlations between the separation parameters and analyte structures were investigated. The molecular interaction, which is based on the differences of structure, and the effect of experimental parameters on the enantioselective separation between the quinolonecarboxylic acids and vancomycin are discussed.

Anti-Infective Agents↗
PubMed1996

Separation and determination of amino acids, creatinine, bioactive amines and nucleic acid bases by dual-mode gradient ion-pair chromatography.

A simple and fast high-performance liquid chromatography method for the analysis of amino acids and biological bases such as creatinine was developed using a technique termed "dual-mode gradient ion-pair chromatography". A butyl-silica reversed-phase column and water-acetonitrile eluent containing sodium dodecyl sulfate (SDS) and perchloric acid were used for the separation. A concentration gradient of acetonitrile from 15 to 35% provided a good separation of such organic cations. Since change in concentration of acetonitrile causes change in distribution equilibrium of SDS between mobile and stationary phases, a complete regeneration of the column to the initial state is required for the reproducible separations. Completion of the reequilibrium was indicated by a system peak appearing in the UV chromatogram and by conductivity measurements. The formation mechanism of the system peak was revealed. A flow-rate gradient from 1 to 2 ml/min was introduced in addition to the concentration gradient to shorten the cycle time of the chromatography. More than twenty kinds of amino acids, creatine and creatinine were simultaneously separated within 50 min and the cycle time was 80 min including the reequilibration time. A post-column derivatization fluorescence detection system was usable as well as UV detection. This elution system was also useful for the separation of bioactive amines and nucleic acid bases. The developed method was applied to the simultaneous determination of urinary creatinine and diagnostic amino acids due to inherited metabolic disorders.

Acetonitriles↗