Relative Electrophoretic Migration Research Articles

A Semiempirical Approach for a Rapid Comprehensive Evaluation of the Electrophoretic Behaviors of Small Molecules in Free Zone Electrophoresis.

A phenomenological model is proposed for the evaluation of relative electrophoretic migration of charged substances present in mixtures and for the rapid pH optimization prior CZE method development. The simple and robust model is based on the Offord model that takes account of the chemical structure. The effective charge and the molecular mass of the molecule are needed; the charge can easily be calculated from pK a obtained from known sources or simulated with existing pK-calculation programs. A first example was chosen with the separation of hydroxy-s-triazines to illustrate the applicability of this simple approach for determination of the first buffer-pH conditions prior experimental method optimization when separation of different ions is needed. In a second example, the confirmation of aminoalcohols in the CZE method development of unsaturated hexahydro-triazines and oxasolidines.

Role for the Proapoptotic Factor BIM in Mediating Imatinib-induced Apoptosis in a c-KIT-dependent Gastrointestinal Stromal Tumor Cell Line

The c-KIT receptor tyrosine kinase is constitutively activated and oncogenic in the majority of gastrointestinal stromal tumors. The identification of selective inhibitors of c-KIT, such as imatinib, has provided a novel therapeutic approach in the treatment of this chemotherapy refractory tumor. However, despite the clinical importance of these findings and the potential it provides as a model system for understanding targeted therapy, this approach has not yielded curative outcomes in most patients, and the biochemical pathways connecting c-KIT inhibition to cell death are not completely understood. Here, we show that inhibition of c-KIT with imatinib in gastrointestinal stromal tumors (GISTs) triggered the up-regulation of the proapoptotic protein BIM via both transcriptional and post-translational mechanisms. The inhibition of c-KIT by imatinib increased levels of the dephosphorylated and deubiquitinated form of BIM as well as triggered the accumulation of the transcription factor FOXO3a on the BIM promoter to activate transcription of BIM mRNA. Furthermore, using RNA interference directed against BIM, we demonstrated that BIM knockdown attenuated the effects of imatinib, suggesting that BIM functionally contributes to imatinib-induced apoptosis in GIST. The identification and characterization of the pathways that mediate imatinib-induced cell death in GIST provide for a better understanding of targeted therapy and may facilitate the development of new therapeutic approaches to further exploit these pathways.

Oxidative stresses induced by glycoxidized human or bovine serum albumin on human monocytes

Oxidative stress and protein modifications are frequently observed in numerous disease states. Albumin, the major circulating protein in blood, can undergo increased glycoxidation in diabetes. Protein glycoxidation can lead to the formation of advanced glycoxidation end products, which induce various deleterious effects on cells. Herein, we report the effect of glucose or methylglyoxal-induced oxidative modifications on BSA or HSA protein structures and on THP1 monocyte physiology. The occurrence of oxidative modifications was found to be enhanced in glycoxidized BSA and HSA, after determination of their free thiol group content, relative electrophoretic migration, carbonyl content, and antioxidant activities. Cells treated with glycoxidized albumin exhibited an overgeneration of intracellular reactive oxygen species, impairments in proteasomal activities, enhancements in RAGE expression, and an accumulation of carbonylated proteins. These novel observations made in the presence of a range of modified BSA and HSA facilitate the comparison of the glycoxidation extent of albumin with the oxidative stress induced in cultured monocytes. Finally, this study reconfirms the influence of experimental conditions in which AGEs are generated and the concentration levels in experiments designed to mimic pathological conditions.

Enantiomeric analysis of baclofen analogs by capillary zone electrophoresis, using highly sulfated cyclodextrins: Inclusion ionization constant pKa determination

Using cyclodextrin-capillary zone electrophoresis (CD-CZE), baseline separation of baclofen phaclofen, saclofen, and hydroxy-saclofen, potent gamma-aminobutyric acid(B) (GABA(B)) agonist or antagonists was achieved. A method for the enantioresolution of those analogs of GABA was developed using anionic cyclodextrins (highly sulfated CD or highly S-CD) as chiral selectors and capillaries dynamically coated with polyethylene oxide (PEO). With charged CDs we observed good resolutions due to the large electrophoretic mobility of these chiral selectors opposite to the mobility of the solutes. The highly S-alpha-CD and S-beta-CD were found to be complementary and the most effective complexing agent, allowing good enantiomeric resolution in short runtimes. The complete resolution was obtained using 25 mM phosphate buffer at pH 2.5 containing 3% w/v of highly S-alpha-CD or S-beta-CD at 25 degrees C with an applied field of 0.30 kV/cm. The apparent binding constants of the inclusion complexes were evaluated and the migration order was determined. A comparison was possible to investigate the importance of the anionic group of the molecules in the separations. The pK(a) values were determined for all four compounds in order to explain relative electrophoretic migration of the solutes.

Inhibitory effects of rosa roxburghii tratt juice on in vitro oxidative modification of low density lipoprotein and on the macrophage growth and cellular cholesteryl ester accumulation induced by oxidized low density lipoprotein

Background: Rosa roxburghii tratt juice (RRTJ) administration has been shown to significantly ameliorate atherosclerotic diseases in cholesterol-fed animals. However, the mechanism for the antiatherogenic effect of RRTJ is not clear. Methods: We investigated the effects of RRTJ on in vitro oxidative modification of LDL and on LDL-induced macrophage growth and cellular cholesteryl ester (CE) accumulation. The effects of RRTJ on LDL oxidative modification were assessed by relative electrophoretic migration, thiobarbituric acid-reactive substance (TBARS) content, and the formation of conjugated dienes. The inhibition of RRTJ on oxidized LDL (Ox-LDL)-induced murine peritoneal macrophage growth was evaluated by a cell-counting assay and an MTT assay. The effect of RRTJ on Ox-LDL-induced cellular CE accumulation was examined after macrophages were incubated with Ox-LDL in the presence of RRTJ. To clarify the mechanism of the inhibitory effect of RRTJ on Ox-LDL-induced CE accumulation in macrophages, its capacity for cholesterol efflux from macrophage-derived foam cells were examined. Results: We showed that RRTJ significantly reduced LDL oxidative susceptibility. In addition, RRTJ effectively suppressed Ox-LDL-induced macrophage growth and especially Ox-LDL-induced CE accumulation in murine peritoneal macrophages by promoting cellular cholesterol efflux. Conclusion: These results indicated that RRTJ exerted its antiatherogenic effects largely due to its ability to inhibit the oxidative modification of LDL and to suppress the formation of foam cells.

Complement C4 phenotypes in patients with end-stage renal disease.

The phenotypes of complement C4 were determined by agarose gel electrophoresis in 130 patients with end-stage renal failure of various causes and compared with those of 140 healthy controls. C4 allotype frequencies did not differ between patients and controls. Null alleles of both isotypes C4A and C4B were increased, but also without reaching significance. In type 1 diabetics an increased frequency of C4AQ0 (25 vs. 11.8%, p < 0.05) was found. Patients with two null alleles were far more frequent in the group with insulin-dependent diabetes mellitus (25 vs. 3.6%, p < 0.01). We confirmed the presence of a previously described uremic variant of C4B1. Additional uremic variants of C4 were detected in uremic patients homozygous for C4A3, B2 and B3. The relative electrophoretic migration values of the uremic variants of C4A3, B1, B2 and B3 were 132.1 +/- 2.9, 35.8 +/- 1.5, 70.4 and 73.9. These variants appear early in the course of chronic renal failure and disappear after successful renal transplantation. Uremic variants are the only acquired C4 phenotypes known so far. How uremia causes these variants remains unclear, but probably involves carbamylation of the C4 molecule.

Sequencing of antisense DNA analogues by capillary gel electrophoresis with laser-induced fluorescence detection

A method using capillary gel electrophoresis with laser-induced fluorescence detection is described which permits complete sequence determination of antisense DNA analogues of unknown sequence. This method, originally created as a tool to confirm the sequence of antisense oligonucleotides being developed as therapeutic drugs, utilizes data collected under a range of experimental conditions described by the Ogston model as applied to gel electrophoresis. A linear relationship independent of experimental conditions between the relative electrophoretic migration time and the oligonucleotide base number was observed and is shown to be consistent with a simplified version of this model and can be used to facilitate the sequence determination.

Electrophoretic resolution of monosaccharide enantiomers in borate-oligosaccharide complexation media

Monosaccharide enantiomers have been directly separated by capillary electroplroresis as complexes with borate and linear or cyclic dextrins of the general poly(gly-(1 -4)-α-D-Glu) structure. Their relative electrophoretic migration and their separation selectivity were mainly influenced by the type and concentration of the chiral additive, the concentration of boric acid, and the chemical nature of a fluorescence-tagging (derivatizing) agent. The analytes were detected by laser-induced fluorescence. All sugar enantromers were resolved using β-cyclodextrin as the electrolyte additive. Their migration order could be adjusted by a type of chiral additive, a derivatizing agent, or the surface property (chemical modification) of the separation capillary

The Genesis and Clinical Significance of Creatine Kinase Isoforms

Two of the three major isoenzymes of creatine kinase (CK), MM and MB, undergo postsynthetic modification resulting in the creation of five isoforms. On the basis of their relative electrophoretic migration, the isoforms are designated MM3, MM2, MM1 MB2, and MB1 (from most cathodal to most anodal). CK isoform formation is a naturally occurring phenomenon mediated by carboxypeptidase (CPase) N. CPase N cleaves the carboxy-terminal amino acid, lysine, from the M subunits of MM3 to form MM2 and MM1 and from the M subunit of MB2 to form MBV MM3 and MB2 are tissue isoforms, and MM2, MM1, and MB1 are formed after the tissue isoforms are released into the circulation. The CK isoforms can be used as markers for confirmation of an acute myocardial infarction (AMI) and for successful reperfusion of AMI patients treated with thrombolytic therapy. Both MM3 and MB2 are significantly increased in the blood shortly after an AMI. Total MM3 and MB2 and the MM3/MM1 and MB2/MBl ratios are all elevated subsequent to AMI and prior to total CK and CK-MB levels. The CK-MB isoforms are preferable to the MM isoforms for diagnosing AMI and reperfusion because they are more myocardial tissue specific. The CK isoforms can be assayed using electrophoresis, isoelectric focusing, chromatofocusing, highperformance liquid chromatography, and immunoinhibition. Rapid, precise, and sensitive electrophoresis procedures allow CK isoform analysis to be used for diagnosis and treatment of patients as early as 2 to 6 hours after AMI.

Relative Electrophoretic Migration Distances for the Classification of C4 Allotypes

For the definition of common C4 allotypes relative electrophoretic migration (RM) values were determined. A set of standard C4 variants were investigated by prolonged agarose gel electrophoresis and subsequent immunofixation with specific antiserum. RM distances were measured by laser densitometry. Using an arbitrary standard of 100 units for the migration distance between the C4B 1 and C4A 3 bands a total deviation of +/- 6.45% in more than 108 single determinations was found. The common C4 alleles used for standardization were C4A*6, C4A*4, C4A*3, C4A*2, C4B*5, C4B*3, C4B*2, C4B*1. In addition, side by side comparison and admixture of known variants will be necessary for the differentiation of some of the closely migrating allotypes. RM values are now available for eight alleles frequently found in all populations for future comparison and designation of newly discovered C4 allotypes.

C4 Reference Typing Report

Human C4 is most polymorphic at the protein level, distinction between allotypes of the C4A and C4B proteins resting on electrophoretic migration patterns and difference in hemolytic activity. The aim of the C4 reference typing has been the definition of reference variants, the assignment of rare variants, and the investigation of duplicated, deleted, or non-expressed and hybrid genes. Samples from 136 individuals, predominantly with known segregation, from 16 laboratories were investigated by standard electrophoretic techniques, for their relative hemolytic activity, reactivity with monoclonal antibodies and Rg/Ch reagents, alpha-, and beta-chain types, relative electrophoretic migration distance, as well as the C4/21-OH-TaqI RFLPs. The results were evaluated in three groups; they consisted in the definition of the eight most common C4 alleles, and the ten Rg/Ch standard phenotypes in group I. In group II twelve C4A and fourteen C4B duplications among 96 complotypes, as well as eighteen deleted/non-expressed C4A and twenty-two C4B alleles, and hybrid alleles were seen by correlation of lytic activity, electrophoretic mobility, and monoclonal and/or Rg/Ch reactivity. Group III consisted of the newly defined allotypes A 8, A 7, A 58, A 55, A 45, B 45, B 35, and B 22, furthermore of alleles subdividing the A 1/A 91, and the B 13/B 12/B 11 regions. The reference typing has allowed reclassification of the majority of described C4 allotypes and resulted in a revision of the C4 nomenclature.

Different conformations of ribosomal DNA in active and inactive chromatin in Xenopus laevis

The chromatin structure of the ribosomal DNA in Xenopus laevis was studied by micrococcal nuclease digestions of blood, liver and embryonic cell nuclei. We have found that BglI-restricted DNA from micrococcal nuclease-digested blood cell nuclei has an increased electrophoretic mobility compared to the undigested control. Micrococcal nuclease digestion of liver cell nuclei causes a very slight shift in mobility, only in the region of the spacer containing the “Bam Islands”. In contrast, the mobility of ribosomal DNA in chromatin of embryonic cells, under identical digestion conditions, remains unaffected by the nuclease activity. Denaturing gels or ligase action on the nuclease-treated DNA abolishes the differences in the electrophoretic mobility. Ionic strength and ethidium bromide influence the relative electrophoretic migration of the two DNA fragment populations, suggesting that secondary structure may play an important role in the observed phenomena. In addition, restriction analysis under native electrophoretic conditions of DNA prepared from blood, liver and embryonic cells shows that blood cell DNA restriction fragments always have a faster mobility than the corresponding fragments of liver and embryo cell DNA. We therefore propose that nicking activity by micrococcal nuclease modifies the electrophoretic mobility of an unusual DNA conformation, present in blood cell, and to a lesser extent, in liver cell ribosomal chromatin. A possible function for these structures is discussed. The differences of the ribosomal chromatin structures in adult and embryonic tissues may reflect the potential of the genes to be expressed.

Structure and conformation of low molecular weight pathogenic RNA from exocortis disease

The electrophoretic mobility of the low molecular weight pathogenic RNA from exocortis disease of citrus (CEV) is markedly affected by polyacrylamide gel concentrations. Molecular weight estimates of 100,000 or 50,000 are derived from relative electrophoretic migration in gels either less than 10% or greater than 10%, respectively. The target size of the biologically active unit is estimated at 110,000 daltons from inactivation by ionizing radiation. On the basis of bouyant density distribution as well as thermal and dimethyl sulfoxide denaturation, molecular structures of the pathogenic RNA are presented, and possible functional significance is discussed.

LACTIC DEHYDROGENASE ISOENZYMES IN HUMAN CANCER CELLS AND MALIGNANT EFFUSIONS.

Lactic dehydrogenase isoenzyme activity was investigated in seven human tumors and the corresponding tissues of origin by an adsorption-elution technique on Sephadex-DEAE®. Independent of the isoenzyme pattern of the tissue of origin, all malignant tissue samples showed an absolute and relative increase in the electrophoretically slow migrating fractions (y-isoenzymes). Isoenzyme distribution in cancer tissues approaches a uniform type with high total and high y-lactic dehydrogenase. Electrophoretic migration of benign and malignant effusions was investigated by agar gel electrophoresis and tétrazolium staining of lactic dehydrogenase. Relative electrophoretic migration was identical in sera of benign and malignant effusions in spite of the increase of the electrophoretically slow migrating isoenzymes. Exsudates of malignant