Longer Migration Time Research Articles

Monitoring of Deamidation and Lanthionine Formation in Recombinant Mugwort Allergen by Capillary Zone Electrophoresis (CZE)-UV and Transient Capillary Isotachophoresis-CZE-Electrospray Ionization-TOF-MS.

The response to thermal stress is an important parameter relevant for characterizing the biological activity and long-term stability of recombinant proteins, which may show irreversible, pH dependent structural changes under these conditions. We selected the recombinant pollen allergen of mugwort ( Artemisia vulgaris) rArt v 3.0201 as a relevant model to study structural changes due to thermal and pH stress by means of capillary zone electrophoresis (CZE)-UV and capillary zone electrophoresis (CZE)-electrospray ionization (ESI)-TOF-MS. Therefore, this recombinant protein was exposed to 95 °C under acidic (pH 3.4) and slightly alkaline (pH 7.3) conditions for up to 120 min. CZE-UV data showed a continuous degradation of the allergen accompanied by the gradual formation of several reaction products. Characterization of novel allergen variants occurring at longer migration times was done via CZE-ESI-TOF-MS using in-capillary transient capillary isotachophoresis (tCITP) preconcentration to facilitate the identification of minor variants. MS data revealed various modifications of rArt v 3.0201 in response to heating. Variants with deamidations and sulfur-related modifications including both yield and loss of sulfur were identified at increased migration times. Desulfurization produced allergen variants with up to four lanthionines that replaced initial disulfide bonds. In addition, mass spectra revealed shifts in the charge state distribution which indicate concomitant conformational alterations. Moreover, several low-abundant oxidized variants were identified. With extended thermal stress, the portfolio of variants increased and progressively shifted toward rArt v 3.0201 with high lanthionine content. The kinetics of conversion and the complexity of variant composition were pH dependent and increased under alkaline conditions.

Differences in electrophoretic behavior between linear and branched PEG-conjugated proteins.

The objective of this study was to characterize the differences in electrophoretic behavior between linear and branched PEG-conjugated proteins. Human growth hormone and alpha-lactalbumin modified by linear or branched PEGs with molecular weight of 10kDa were analyzed by SEC, MALDI-TOF MS, SDS-PAGE, and microchip CGE (MCGE). Chromatographic and mass spectrometric differences between the linear and branched PEG-proteins on SEC and MALDI-TOF MS were small, but their electrophoretic behaviors on SDS-PAGE and MCGE were significantly different. In particular, MCGE showed significant differences in the peak width and the migration times of linear and branched PEG-proteins, in which the branched PEG-proteins exhibited a narrower peak and longer migration time than the linear PEG-proteins. This phenomenon may explain the longer circulation half-life for the branched PEG-proteins observed in previously reported in vivo studies. Consequently, this study indicates that MCGE may be a valuable tool for differentiating linear and branched PEG-proteins.

A Floating Bridge Disrupts Seaward Migration and Increases Mortality of Steelhead Smolts in Hood Canal, Washington State

BackgroundHabitat modifications resulting from human transportation and power-generation infrastructure (e.g., roads, dams, bridges) can impede movement and alter natural migration patterns of aquatic animal populations, which may negatively affect survival and population viability. Full or partial barriers are especially problematic for migratory species whose life histories hinge on habitat connectivity.Methodology/Principal FindingsThe Hood Canal Bridge, a floating structure spanning the northern outlet of Hood Canal in Puget Sound, Washington, extends 3.6 meters underwater and forms a partial barrier for steelhead migrating from Hood Canal to the Pacific Ocean. We used acoustic telemetry to monitor migration behavior and mortality of steelhead smolts passing four receiver arrays and several single receivers within the Hood Canal, Puget Sound, and Strait of Juan de Fuca. Twenty-seven mortality events were detected within the vicinity of the Hood Canal Bridge, while only one mortality was recorded on the other 325 receivers deployed throughout the study area. Migrating steelhead smolts were detected at the Hood Canal Bridge array with greater frequency, on more receivers, and for longer durations than smolts migrating past three comparably configured arrays. Longer migration times and paths are likely to result in a higher density of smolts near the bridge in relation to other sites along the migration route, possibly inducing an aggregative predator response to steelhead smolts.Conclusions/SignificanceThis study provides strong evidence of substantial migration interference and increased mortality risk associated with the Hood Canal Bridge, and may partially explain low early marine survival rates observed in Hood Canal steelhead populations. Understanding where habitat modifications indirectly increase predation pressures on threatened populations helps inform potential approaches to mitigation.

Abstract P1-01-14: Intraoperative Injection of Technetium-99m- Dextran 500 for the Identification of Sentinel Lymph Nodes in Breast Cancer

Abstract Background: The intraoperative injection of radiocolloid before a sentinel node biopsy (SLN) in breast cancer has been recently described.The advantages of this approach would be precluding the performance of a scintigram, cost reduction and elimination of pain and anxiety related to the preoperative procedure. Objectives: To evaluate the efficacy of intraoperative injection of Dextran 500-99m-Tc and to analyze whether different radiocolloid doses, breast volume and Body Mass Index (BMI) influence the migration time to the axilla. Material and Methods: This is a prospective study of 74 SLN biopsies of breast cancer patients, stages T1N0 and T2N0. After anesthesia induction, patients underwent subareolar dermal injection of 0.5 to 1.5 mCi (18 to 55 MBq) of filtered Dextran 500-99m-Tc (0.22µl) in a volume of 5 ml along with 2 ml of blue dye. Results: The probe identification rate was 98% (73/74 cases) and the medium radiocolloid dose used was 0.97 mCi ± 0.22. The radiocolloid migration time to the axila was 10 min and 43 s (± 5:43 s). An average of 1.66 lymphnodes were identified through the use of radiocolloid and 1.53 through the use of bue dye. Different radiocolloid doses did not modify migration time to SLN (p=0.73). However, larger breast volumes were associated with longer migration time to the axilla (Pearson Correlation 0.393 p< 0.01). The same correlation was observed with BMI (Pearson Correlation 0.469 p< 0.01). Discussion: The intraoperative injection of Dextran 500-99m-Tc is efficient in the identification of SLN in breast patients. Higher BMI and larger breasts determine longer migration time, however injection of larger doses of radiocolloid do not reduce transit time to the axilla. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P1-01-14.

Separation of DNA-containing organelles fromToxoplasma gondiiby CZE

Toxoplasma gondii and other members of the family Apicomplexa have two organelles, in addition to the nucleus, that contain DNA. Herein is reported the separation of the DNA-carrying organelles from T. gondii tachyzoites, i.e. the mitochondrion and the apicoplast, by CZE. The cells were stained with SYTO9, a dye that exhibit fluorescence when interacting with double stranded nucleic acids (e.g. DNA) and disrupted by nitrogen cavitation. Following careful removal of the heavier cellular material, the remaining lysate was injected on a CE instrument and the DNA-containing organelles were detected by LIF. The mitochondrion had longer migration time than the apicoplast, and the migration times were comparable in the replicates. This method should potentially also work for other members of the Apicomplexa including Plasmodium falciparum.

CE of Small DNA Fragments Using Linear Polyacrylamide Matrices

Mutations at codons 248 and 249 of p53 gene showed a relatively high incidence in gastric cancer patients. Development of novel methods for the detection of codon mutations is of great importance for gastric cancer research. Studies have showed that the separation matrix can significantly influence the separation efficiency and resolution of small DNA fragments in CE. In order to achieve baseline separation of PCR-amplified products of small DNA fragments from gastric cancer tissue, linear polyacrylamides (LPA I and LPAII) were designed and synthesized in the current study. LPAI and LPAII were used as separation matrixes to separate small size fragments (less than 70 bp) of pBR322/BsuRI DNA Marker and the separation conditions were optimized. Optimum separations were performed at 25 kV in reversed-polarity mode with capillary temperature set at 15 °C. The signal of DNA fragments was detected using laser-induced fluorescence detector, with an argon ion laser as the excitation source that emits at 488 nm. A 520 nm bandpass filter was used as an emission cut-off filter. The resolution of small DNA fragments was higher when LPAI was used as separation matrix compared to LPAII, accompanied with longer migration time. The results indicated that LPAI as separation matrix was more efficient for the separation of small DNA fragments (less than 70 bp) than other LPAs. A rapid and sensitive analysis method for the separation and detection of small DNA fragments (less than 70 bp) was established in this study. The method was successfully applied to detect the mutations at codons 248 and 249 in p53 gene from gastric cancer tissues.

Direct chiral resolution of tartaric acid by ion-pair capillary electrophoresis using an aqueous background electrolyte with (1R,2R)-(-)-1,2-diaminocyclohexane as a chiral counterion.

Chiral resolution of native DL-tartaric acid was achieved by ion-pair capillary electrophoresis (CE) using an aqueous-ethanol background electrolyte with (1R,2R)-(-)-1,2-diaminocyclohexane (R-DACH) as a chiral counterion. Factors affecting chiral resolution and migration time of tartaric acid were studied. By increasing the viscosity of the background electrolyte and the ion-pair formation, using organic solvents with a lower relative dielectric constant, resulted in a longer migration time. The optimum conditions for both high resolution and short migration time of tartaric acid were found to be a mixture of 65% v/v ethanol and 35% v/v aqueous solution containing 30 mM R-DACH and 75 mM phosphoric acid (pH 5.1) with an applied voltage of -30 kV at 25 degrees C, using direct detection at 200 nm. By using this system, the resolution (Rs) of racemic tartaric acid was approximately 1. The electrophoretic patterns of tartaric and malic acids suggest that two carboxyl groups and two hydroxyl groups of tartaric acid are associated with the enantioseparation of tartaric acid by the proposed CE method.

Chiral recognition of verapamil by cyclodextrins studied with capillary electrophoresis, NMR spectroscopy, and electrospray ionization mass spectrometry.

Capillary electrophoresis (CE) allows the observation of the opposite affinities of the enantiomers of (+/-)-verapamil [2-isopropyl-2,8-bis(3,4-dimethoxyphenyl)-6-methyl-6-azaoctannitrile+ ++, VP] toward beta-cyclodextrin (beta-CD) and heptakis(2,3, 6-tri-O-methyl)-beta-CD (TM-beta-CD). In addition, in the presence of beta-CD in the background electrolyte, longer migration times and lower separation factors were observed compared to TM-beta-CD. The binding constants of (+)- and (-)-VP with beta-CD and TM-beta-CD determined using (13)C NMR spectroscopy explain the results observed in CE. Electrospray ionization mass spectrometry (ESI-MS) was used as an alternative technique for the characterization of VP-CD complexes.

Analysis of Lactosylated β-Lactoglobulin by Capillary Electrophoresis

Variously lactosylated species of β-lactoglobulin AB were analysed by capillary electrophoresis at low pH and high pH with and without buffer additives. By simple capillary zone electrophoresis at either low or high pH the lactosylated forms had longer migration times than the native β-lactoglobulin, most likely due to their larger molecular size and lower pI. By micellar electrokinetic chromatography using SDS as detergent the lactosylated forms of β-lactoglobulin had shorter migration times than the native species, due to less interaction with the micelles. The methods used were also able to distinguish between more or less lactosylated forms of β-lactoglobulin. By all methods used, partial separation of at least five forms of β-lactoglobulin were obtained. However, baseline separation of β-lactoglobulin with varying number of lactose units attached was not obtained, probably due to the presence of both variants of β-lactoglobulin with various numbers of lactosyl groups at different sites.

Separation of pd(GC) 12 from pd(AT) 12 by free solution capillary electrophoresis

The two synthetic self complementary oligonucleotides pd(AT) 12 and pd(GC) 12 were separated by free solution capillary electrophoresis (CZE) using simple borate buffers. The effects of pH (7.5–9) and the concentration of the buffer (0.03–0.35 M) were investigated. Higher pH values and buffer concentrations led to better resolution and longer migration times, the pH having a more pronounced effect on the separation than the concentration of the buffer. It is proposed that the conformation and effective length of the oligonucleotides may have a role in their separation in free solution capillary electrophoresis.

Control of migration time window and selectivity in electrokinetic chromatography with mixed polymeric pseudostationary phases

Control of migration time windows and the improvement of separation were achieved by utilizing mixed polymeric pseudostationary phases in electrokinetic chromatography (EKC) in water–organic solvent mixtures. Polyallylamine (PAA)- supported pseudostationary phases, one with hexadecyl groups (PAA-C 16) with the shorter migration times and the other with decyl groups (PAA-C 10) with the longer migration times, enabled the control of migration time windows when used as a mixture. PAA-C 16 provided better separation for a region with relatively small k′ values as in typical micellar EKC, while PAA-C 10 gave better separation for a region with relatively large k′ values as in reversed-phase liquid chromatography, the mixture of the two PAA derivatives resulting in better overall separations for a wide range of hydrophobic compounds. The migration time of a solute is determined by the relative contribution of each pseudostationary phase to the partition of the solute in a mixed carrier system. A theoretical explanation was provided by assuming the contribution of the two polymeric pseudostationary phases working independently. The migration time of the effective (imaginary) pseudostationary phase was shown to be in between the actual migration times of the two carriers, and can be different for each solute depending upon the relative contribution of the two pseudostationary phases to the solute partition.

Stereoselective interaction of drug enantiomers with human serum transferrin in capillary zone electrophoresis (II).

Further studies on chiral resolution of drugs with different chemical structures by capillary zone electrophoresis using iron-free human serum transferrin are described. The substances passed a highly concentrated pseudo-stationary protein zone applied in a coated capillary and the possible chiral separation of the optical isomers was followed. Eighteen drugs with different structures were screened, and the enantiomers of clofedanol, buphenine, acebutolol and chlorphenamine were resolved. Several, but not all drugs, showed longer migration times while passing the protein zone, indicating an interaction with transferrin, although chiral resolution was not observed in all cases. The observations provided further information about the properties of the surface interaction sites of transferrin.