Biomedical subjects

M G Khaledi

Publications and source records attributed to M G Khaledi.

At least 19 recordsLinked to original sources

PubMed2001

Prediction of retention in micellar electrokinetic chromatography from solute structure. 1. Sodium dodecyl sulfate micelles.

Like other chromatographic techniques, retention factor, k, in micellar electrokinetic chromatography (MEKC) is directly related to solute partition coefficient and the chromatographic phase ratio as k = Kphi. Unlike conventional chromatography, however, the phase ratio and partition coefficient can be accurately determined in MEKC for a given micellar pseudostationary phase. This means that retention factor in MEKC can be predicted for solutes with known micelle-water partition coefficients without any prior experimentation. In this paper, the use of this simple relationship for prediction of retention behavior in MEKC is examined. The principle of additivity of functional group contribution to partitioning is used to calculate the micelle-water partition coefficient, Kmw, for SDS micellar pseudophase. The micellar substituent constants for 20 functional groups (training set) were determined. Using these substituent constants, the Kmw and retention factors for a group of 80 neutral solutes (test set) were predicted. The linear plot of predicted versus observed log k had an R2 = 0.97 and a slope equal to 1.01. It is shown that the retention times (thus chromatograms) in MEKC can be predicted from the calculated retention factors after only one initial experiment to measure teo and t(mc) under the experimental conditions.

Journal Article↗

PubMed2001

Quantitation of intracellular concentration of a delivered morpholino oligomer by capillary electrophoresis-laser- induced fluorescence: correlation with upregulation of luciferase gene expression.

Antisense oligonucleotides have shown great promise over the past several years as viable drugs to combat various forms of cancer and viral diseases. However, quantitative detection to monitor cellular association is difficult using conventional methods such as radiolabeling of the oligonucleotide or fluorescence confocal microscopy. In this paper quantitation of intracellular concentration of the morpholino oligonucleotide is investigated using capillary electrophoresis coupled with laser-induced fluorescence detection (CE-LIF). HeLa cells, which produce luciferase as the antisense oligomer enters the cell, were scrape-loaded with varying concentrations of the morpholino antisense. The intracellular antisense concentration measured by CE-LIF was found to correlate with those obtained with the cellular functional assay based on upregulation of luciferase. Intracellular concentrations of the antisense were found to be in the range of 6 to 29 nmol/g total cell protein, depending on the amounts that were scrape-loaded. To our best knowledge, this is the first reported quantitative correlation between delivered antisense concentration in a cell extract and the subsequent antisense upregulation of gene expression.

Cell Membrane↗

PubMed2001

Quantitative nuclear and cytoplasmic localization of antisense oligonucleotides by capillary electrophoresis with laser-induced fluorescence detection.

We demonstrate the use of simple extraction procedures to separate nuclear and cytoplasmic material from cell extracts, which have been scrape-loaded with a 2-O-methyl phosphorothioate antisense oligonucleotide. Separation and quantitation of the fluorescein-labeled antisense and the flourescein isothiocyanate (FITC)-dextran (molecular weight 40000) as an internal standard is done using capillary electrophoresis coupled with laser-induced fluorescence detection (CE-LIF). The bulky FITC-dextran is unable to penetrate the nuclear membrane thereby making it a quantitative indicator of any overlap between the nuclear and cytoplasmic materials during separation of the two phases. Using this procedure, the fluorescein-labeled phosphorothioate oligomer was quantitated at 4.1 x 10(-13) and 3.4x 10(-14) mol antisense/microg-total cellular protein in the nuclear and cytoplasmic extracts respectively following scrape-load delivery of the phosphorothioate to a batch of confluent HeLa cells at a concentration of 0.5 microM (5 x 10(-10) total moles of oligomer). Additionally, gene expression was monitored by measurement of the luciferase reporter protein activity. Scrape-load, spontaneous and liposomal delivery were investigated and compared for subcellular distribution of the oligomer and subsequent gene expression.

Cell Fractionation↗

PubMed2000

Evaluation of the binding between potential anti-HIV DNA-based drugs and viral envelope glycoprotein gp120 by capillary electrophoresis with laser-induced fluorescence detection.

The fusion of the human immunodeficiency virus (HIV) with the target cell was assisted by the interaction between the viral envelope glycoprotein HIV-1 gp120 and a chemokine receptor. Studies have shown that the efficiency of the binding depends on the presence of the V3 loop of the gp120 which is known to interact with polyanions, such as phosphorothioate oligodeoxynucleotides (Sd, potential anti-HIV drugs). In this study, capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) was used to systematically evaluate binding between Sd and HIV-1 gp120. A 25-mer fluorescently tagged phosphorothioate oligodeoxynucleotide (GEM) was employed as a probe to study this interaction. The dissociation constant (K(d)) between GEM and gp120 was determined to be 0.98 nM by Scatchard analysis. The competition constants (K(c)) of a set of Sd that compete with GEM for binding to gp120 were also determined. The results showed that the interaction had a strong dependence on the sulfur phosphorothioate backbone. Chain length and the sequence of Sd also affect the ability of binding to gp120. The ability to study the protein-drug binding in the solution with minimal sample consumption makes CE-LIF very attractive for biological studies.

Anti-HIV Agents↗

PubMed2000

Separation of acidic solutes by nonaqueous capillary electrophoresis in acetonitrile-based media. Combined effects of deprotonation and heteroconjugation.

Nonaqueous capillary electrophoresis (NACE) is a chemical separation technique that has grown in popularity over the past few years. In this report, we focus on the combination of heteroconjugation and deprotonation in the NACE separation of phenols using acetonitrile (ACN) as the buffer solvent. By preparing various dilute buffers consisting of carboxylic acids and tetrabutylammonium hydroxide in ACN, selectivity may be manipulated based on a solute's dissociation constant as well as its ability to form heterogeneous ions with the buffer components. ACN's low viscosity, coupled with its ability to allow for heteroconjugation, often leads to rapid and efficient separations that are not possible in aqueous media. In this report, equations are derived showing the dependence of mobility on various factors, including the pKa of the analyte, the pH and concentration of the buffer, and the analyte-buffer heteroconjugation constant (Kf). The validity of these equations is tested as several nitrophenols are separated at different pH values and concentrations. Using nonlinear regression, the Kf values for the heteroconjugate formation between the nitrophenols and several carboxylate anions are calculated. Also presented in this report are the NACE separations of the 19 chlorophenol congeners and the 11 priority pollutant phenols (used in US Environmental Protection Agency methods 604, 625/1625 and 8270B).

Acetonitriles↗

PubMed2000

Characterization of chemical selectivity in micellar electrokinetic chromatography. VI. Effects of surfactant counter-ion.

Linear solvation energy relationships and free energy of transfer data were used to evaluate the influence of the surfactant counter-ion on selectivity in micellar electrokinetic chromatography. It was determined that selectivity differences are dependent on the valency of the counter-ion but not the type of counter-ion. Monovalent surfactants, sodium dodecyl sulfate (SDS) and lithium dodecyl sulfate, have nearly identical selectivity behavior. The divalent surfactants, magnesium didodecyl sulfate and copper didodecyl sulfate also show very similar behavior. However, when the divalent counter-ion species is compared to SDS under similar conditions, significant differences are observed. Most notably, the utilization of divalent counter-ion species of dodecyl sulfate surfactants causes the micelles to become more hydrophobic and a weaker hydrogen bond donating pseudo-stationary phases. It is believed that the divalent counter-ions reduce the electrostatic repulsion between the surfactant head groups and therefore, increase the chain packing of the monomers in the micelle aggregates. This reduces the degree of hydration of the micellar palisade layer leading to a decreased ability of the micelle to participate in polar/polarizable and hydrogen bonding interactions with solute molecules.

Chromatography, Micellar Electrokinetic Capillary↗

PubMed2000

Statistical evaluation of linear solvation energy relationship models used to characterize chemical selectivity in micellar electrokinetic chromatography.

Characterization of retention and selectivity differences between surfactants in micellar electrokinetic chromatography (MEKC) using linear solvation energy relationships (LSERs) has been given a significant amount of attention in the last four years. This report evaluates the validity of using the two LSER models that have been used to fit retention in MEKC in the literature. The results and the fit of the revised model and parameters developed by Abraham and coworkers are compared to the original model developed by Kamlet, Taft, and coworkers. LSERs can generally only be used as a comparative tool to describe the selectivity differences between surfactant systems used in MEKC. With this in mind, it was determined that the results of both models essentially provide the same information about these differences. However, the revised model and parameters have been found to yield a statistically better fit of the MEKC retention data as well as providing more chemically sound LSER coefficients.

Chromatography, Micellar Electrokinetic Capillary↗

PubMed2000

Enantiomeric separations by nonaqueous capillary electrophoresis.

This paper reviews the recent advances in enantioseparations by nonaqueous capillary electrophoresis (NACE) and the effect of organic solvents on mobility of enantiomers, separation selectivity and resolution. In general, the enantioseparation systems in NACE are similar to those of aqueous capillary electrophoresis (CE) except pure organic solvents are used. The influence of important parameters such as concentration and type of chiral selectors, apparent pH, ionic strength, temperature, and control of electroosmotic flow is discussed. In addition, the reported applications of NACE separations of racemates are presented.

Electrophoresis, Capillary↗

PubMed2000

Congeneric behavior in estimations of octanol-water partition coefficients by micellar electrokinetic chromatography.

Linear Solvation Energy Relationships (LSERs) are used to explain the congeneric behavior observed when using Micellar Electrokinetic Chromatography (MEKC) to estimate the octanol-water partition coefficient scale of solute hydrophobicity. Such studies provide useful insights about the nature of solute interactions that are responsible for the sources of congeneric relationships between MEKC retention and log Po/w. It was determined that solute dipolarity/polarizability and hydrogen-bonding character play the most important roles in the congeneric behavior observed for many surfactant systems. The individual dipolarity/polarizability and hydrogen-bonding contributions to the free energy of transfer were also investigated.

Chromatography, Micellar Electrokinetic Capillary↗

PubMed2000

Calculation of electrophoretic mobilities in water-organic modifier mixtures in capillary electrophoresis.

In order to correlate/predict electrophoretic mobility data in the mixture of water+organic modifier four equations have been presented and examined. The experimental mobilities of five analytes were determined in a water-methanol mixture. These data have been used to assess the accuracy and predictability of the models. Also, some previously published mobility data in water-organic modifier mixtures has been employed for further evaluation of the models. The models produced accurate results and the means of percentage deviations were in the range of 0.66-1.30.

Electrophoresis, Capillary↗

PubMed2000

Influence of ester and amide-containing surfactant headgroups on selectivity in micellar electrokinetic chromatography.

The selectivity differences between six anionic surfactants in micellar electrokinetic chromatography (MEKC) are presented and the structural influence of the surfactant head-group is investigated. It was determined that the surfactant structure can have a significant impact on retention and selectivity. Linear solvation energy relationships (LSERs) are used to study the role of solute size, polarity/polarizability, and hydrogen bonding characteristics in determining retention and selectivity. While both the solute size and hydrogen bond accepting ability were found to be the most important factors in solute retention, the hydrogen bonding characteristic of the solutes have the largest influence on selectivity differences between surfactants.

Amides↗

PubMed2000

Detection and sequencing of phosphopeptides affinity bound to immobilized metal ion beads by matrix-assisted laser desorption/ionization mass spectrometry.

Consecutive enzymatic reactions of analytes which are affinity bound to immobilized metal ion beads with subsequent direct analysis of the products by matrix-assisted laser desorption/ionization mass spectrometry have been used for detecting phosphorylation sites. The usefulness of this method was demonstrated by analyzing two commercially available phosphoproteins, beta-casein and alpha-casein, as well as one phosphopeptide from a kinase reaction mixture. Agarose loaded with either Fe3+ or Ga3+ was used to isolate phosphopeptides from the protein digest. Results from using either metal ion were complementary. Less overall suppression effect was achieved when Ga3+-loaded agarose was used to isolate phosphopeptides. The selectivity for monophosphorylated peptides, however, was better with Fe3+-loaded agarose. This technique is easy to use and has the ability to analyze extremely complicated phosphopeptide mixtures. Moreover, it eliminates the need for prior high-performance liquid chromatography separation or radiolabeling, thus greatly simplifying the sample preparation.

Amino Acid Sequence↗

PubMed1999

Efficiency studies in nonaqueous capillary electrophoresis.

Nonaqueous capillary electrophoresis (NACE) is a relatively new area with several advantages that include enhanced efficiency and improved detection sensitivity. The goal of this study was to investigate the influence of NACE compared to aqueous CE on the separation efficiency of oligosaccharides. The applied voltage and buffer concentration were optimized for the aqueous and nonaqueous buffer media to minimize the band broadening effects of Joule heating and electrophoretic dispersion. At the optimized conditions a 1.5-fold enhancement in efficiency was obtained with the nonaqueous buffer medium.

Buffers↗

PubMed1999

Direct analysis of the products of sequential cleavages of peptides and proteins affinity-bound to immobilized metal ion beads by matrix-assisted laser desorption/ionization mass spectrometry.

Consecutive enzymatic reactions on analytes affinity-bound to immobilized metal ion beads with subsequent direct analysis of the products by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry have been used for detecting protein synthesis errors occuring at the N-terminus. The usefulness of this method was demonstrated by analyzing two commercially available recombinant HIV proteins with affinity tags at the N-terminus, and histatin-5, a peptide with multiple histidine residues. The high specificity, sensitivity, and speed of analysis make this method especially useful in obtaining N-terminal sequencing information of histidine-tagged recombinant proteins.

Amino Acid Sequence↗

PubMed1999

Non-aqueous capillary electrophoresis chiral separations with sulfated beta-cyclodextrin.

This paper reports the application of an anionic cyclodextrin (CD), sulfated beta-cyclodextrin with a degree of substitution of four (beta-CD-(SO4-)4, in chiral separations of pharmaceutical enantiomers by non-aqueous capillary electrophoresis (NACE). Upon complexation with the anionic CD, electrophoretic mobilities of the basic enantiomers decreased, however, both separation selectivity and resolution were enhanced. The advantage of NACE chiral separations over the aqueous CE with the charged CD is that higher electric field strength and higher ionic strength could be applied due to the characteristics of the solvent formamide. The higher ionic strength leads to stacking of peaks and reduces the electrodispersion caused by the mobility mismatch between beta-CD-(SO4-)4-analyte complexes and the co-ions in the running buffer. As a result, better peak shapes and higher separation efficiency were obtained. Comparing with NACE chiral separations with neutral CDs, lower concentration of beta-CD-(SO4-)4 was needed due to the fact that the electrostatic attraction caused stronger binding between beta-CD-(SO4-)4 and the enantiomers. The effects of the experimental parameters, such as concentration of the CD, apparent pH (pH*), degree of substitutions of the CDs, percentage of water in mixed solvent systems, and type of solvents were also studied.

Cyclodextrins↗

PubMed1999

Steroid analysis in single cells by capillary electrophoresis with collinear laser-induced fluorescence detection.

Capillary electrophoresis with collinear laser-induced fluorescence detection was used for the analysis of steroids in single R2C cells. Progesterone secretion was monitored from cultured cells and subsequently detected in single cells. Mass detection limit of 10(-18) mol for dansylated steroids was achieved with the 325-nm line of a helium-cadmium laser. Dansylhydrazine proved to be an effective fluorescent tag for derivatization of steroids outside and inside the biological cell. Fluorescence microscopy indicates that a dimethyl sulfoxide-containing physiological buffer was sufficient to incorporate the tag inside the cell for subsequent steroid derivatization.

Animals↗

PubMed1999

Characterization of chemical selectivity in micellar electrokinetic chromatography. 4. Effect of surfactant headgroup.

The influence of surfactant headgroups on migration behavior in micellar electrokinetic chromatography is examined. Using linear solvation energy relationships (LSER) and functional group selectivity studies, the effect of six anionic headgroups on chemical selectivity is characterized. The sodium dodecyl surfactants of the sulfate [SO4-], sulfonate [SO3-], carboxylate [CO2-], carbonyl valine [OC(O)NHCH(CH(CH3)2)CO2-], and sulfoacetate [OC(O)CH2SO3-] anions are investigated. Solute size and the hydrogen-bond-donating ability of the micellar phase play the most significant roles in solute retention in all of the surfactants studied. While solute-micelle hydrogen bonding plays a dominant role in the observed selectivity, the dipolarity and polarizability of the micellar phase also have a small influence. The results also suggest that the hydrogen-bond-accepting ability for surfactants is inversely proportional to the proton acidity (pKa) of its headgroup. The observed hydrogen-bond-donating ability and dipolarity of surfactant systems are believed to be a result of the water that resides near the micelle surface.

Chromatography, Micellar Electrokinetic Capillary↗

PubMed1999

Expression and analysis of green fluorescent proteins in human embryonic kidney cells by capillary electrophoresis.

The green fluorescent protein (GFP) has attracted much interest as a reporter for gene expression. In this paper, application of capillary electrophoresis with laser-induced fluorescent (CE-LIF) for quantitation of green fluorescence protein in cellular extracts and single cells is investigated. The S65T mutant form of GFP protein was successfully expressed in human embryonic kidney (HEK293) cells, and its production was confirmed by fluorescence microscopy and CE-LIF. The mass limit of detection for the mutant S65T was 5.3 x 10(-20) mol, which was better than that for the wild-type GFP by a factor of six. Detection of a small amount of GFP is difficult by conventional techniques such as fluorescent microscopy due to interference from cell autofluorescence at low GFP concentrations. The HEK293 cells were transfected with the GFP plasmid that produced S65T-GFP. Transient production of S65T protein was detected 2 h after the transfection and reached a maximum after 48 h. The protein concentration began to decrease significantly after 96 h. Single cell analysis of HEK293 cells after transfection with GFP plasmid indicate a nonuniform production of S65T-GFP protein among cells.

Cell Line↗