Recent developments of capillary electrophoresis in seawater analysis.

Recent applications of capillary electrophoresis (CE) to the determination of various seawater analytes are critically examined, with the aim to reveal method's state-of-the-art and possible future research trends in the area. Given highly developed separation and detection methodology, emphasis is given to the most advantageous sample preconcentration strategies used to evolve the method's practical utility, particularly to low-level analytes. Analytical performance of CE applied to seawater analysis is illustrated with a selection of real-world applications published from 2006, from which it appears that the primary developmental tendency is presently confined to a transit from inorganic to organic seawater analytes.

High-sensitivity capillary gel electrophoretic analysis of DNA fragments on an electrophoresis microchip using electrokinetic injection with transient isotachophoretic preconcentration.

The research adopted a single-channel microchip as the probe, and focused electrokinetic injection combined with transient isotachophoresis preconcentration technique on capillary electrophoresis microchip to improve the analytical sensitivity of DNA fragments. The channel length, channel width and channel depth of the used microchip were 40.5 mm, and 110 and 50 microm, respectively. The separation was detected by CCD (charge-coupled device) (effective length=25 mm, 260 nm). A 1/100 diluted sample (0.2 mg/l of each DNA fragment) of commercially available stepladder DNA sample could be baseline separated in 120 s with S/N=2-5. Compared with conventional chip gel electrophoresis, the proposed method is ideally suited to improve the sensitivity of DNA analysis by chip electrophoresis.

Large vessel arteritis associated with human herpesvirus 6 infections.

A 9-month-old boy presented with chronic arteritis of the aorta and its major branches. The clinical manifestations at onset of his illness were compatible with those of Kawasaki syndrome. However, the febrile period lasted for 2 months despite various immunosuppressive therapies, and the levels of C-reactive protein remain high 18 months after onset. Elevated circulating immune complexes, decreased serum complement levels, hypergammaglobulinaemia and monoclonal gammopathy were observed. Active HHV-6 infection was shown by increased serum levels of antihuman herpesvirus-6 (HHV-6) IgG and IgM antibodies, and positive HHV-6 DNA in sera, peripheral blood mononuclear cells (PBMNC) and lymph nodes. HHV-6 was actively replicating in PBMNC and lymph nodes, as shown by the detection of transcripts for the virus structural antigen. These results suggest that large vessel arteritis can be associated with HHV-6 infection.

Change of migration time and separation window accompanied by field-enhanced sample stacking in capillary zone electrophoresis.

When field-enhanced sample stacking was used in capillary zone electrophoresis (CZE) analysis of cations, the decrease of migration time and the reduction of separation window was observed with increase of sample plug length. A simple equation expressing the migration velocity in the stacking process was derived to explain the above phenomenon. From experiments and theoretical consideration, we confirmed that this effect was caused by the higher potential gradient and larger eletroosmotic flow (EOF) mobility at the sample plug than those at the supporting electrolyte. A mathematical model appropriate for the computer simulation of such a system was studied considering the experimental results, and it was concluded that electroosmotic velocity (v(eof)) should be introduced to the equation of continuity as a constant.

Operational modes for transient isotachophoretic preconcentration-capillary zone electrophoresis and detectable concentration of rare earth ions.

Operational modes for transient isotachophoretic preconcentration capillary zone electrophoresis (tr-ITP-CZE) were studied by using 5 microM and 0.5 microM rare earth mixtures as analytes in comparison with field-enhanced sample stacking. After examination of several operational modes for tr-ITP, it was found that tr-ITP effectively occured even if both the leading electrolyte and the terminating electrolyte were injected after the sample plug. This was explained as the result of a field-enhanced stacking for both sample components and the leading and terminating ions. The observed theoretical plate numbers were 4-20 times higher than those obtained by normal stacking; and the estimated low limit of detectable concentration of rare-earth elements (REE) was ca. 0.1 microM which was 2.5 times lower compared to normal stacking. For the 0.5 microM sample, a concentration factor of 20 000 could be achieved after only tr-ITP.

Generation of an X-ray microbeam for spectromicroscopy at SPring-8 BL39XU.

A pair of elliptical mirrors (KB mirror) was designed and fabricated to realize an energy tunable x-ray microbeam for spectromicroscopy at SPring-8 BL39XU. As is commonly recognized, the obtainable beam size with the aspherical total reflection mirrors is strongly affected with the slope error of the mirror. Considering that the extremely high brilliance of the undulator radiation from the SPring-8, the small mirror size and the small mirror-to-focus distance were employed to minimize effects of the slope error. Preliminary evaluation of the KB mirror was carried out using 10 keV monochromatized undulator radiation. Alignment of the mirror was assisted by the beam monitor system composed of a scintillator and a CCD, and the beam size less than 5 microm can be easily achieved even when the source was fully used. The beam size obtained with this experiment was 2 x 4 microm2 with the photon flux of 1 x 10(10) photons/s. Smaller beam size may be expected with the use of intermediate slits. Characterization of trace elements with the spatial resolution will be carried out by using x-ray fluorescence (XRF) analysis and x-ray absorption fine structure (XAFS) measurements with XRF yield method.

Isotachophoretic separation behavior of rare-earth EDTA chelates and analysis of minor rare-earth elements in an iron ore by bidirectional isotachophoresis-particle-induced X-ray emission.

Mobilities of 16 anions of rare-earth-EDTA 1:1 chelate (RE-EDTAs) were isotachophoretically measured by using two leading electrolytes (pH 3.6 and 6.0) in order to assess their separation behavior. The leading electrolyte was 20 mM hydrochloric acid. The pH of the solution was adjusted to 3.6 by adding beta-alanine and to 6.0 by adding histidine. The obtained mobilities were very close to each other in the range 20.1x10(-5)-21.9x10(-5) cm2 V(-1) s(-1) with the minimum mobilities for Pr-EDTA and Nd-EDTA for pH 3.6 and 6.0, respectively, and pH dependence was hardly observed. On the basis of the above knowledge. minor rare-earth elements in a standard iron ore sample were determined as RE-EDTAs by bidirectional isotachophoresis-particle-induced X-ray emission (PIXE), where the Fe(II) matrix digested by alkali fusion was separated as Fe(II)Phen3(2+) (phen = 1,10-phenanthroline). Since 5% of the total iron was still detected as Fe(III)EDTA- and might disturb PIXE analysis of RE-EDTA-, itaconic acid was used as the spacer for Fe(III)EDTA- and RE-EDTA-. The fractions of RE-EDTA- were successfully analyzed off-line by a multielemental analytical method, PIXE [analytical result (3.62% (w/w) as RE2O3]; the nominal value was 3.37% (w/w) as RExOy.

Standardization of capillary zone electropherograms obtained by using field enhanced sample stacking.

Migration times in a capillary zone electropherogram obtained by using the field enhanced sample stacking technique are strongly affected by the injected sample volume. That is, the migration times significantly decrease with the increase of the sample volume. To avoid inaccurate qualitative analysis due to the above phenomena, the time axis of the electropherograms was converted into an effective mobility axis using our conversion method taking account of the temperature increase in the separation tube and relaxation of the potential gradient of the separation field. After the conversion, accurate qualitative analysis was possible in spite of drastic change of the migration time, suggesting our conversion method could be successfully used for the standardization of electropherograms obtained even by using the stacking effect. The cause of the decrease of the migration time in the stacking process was briefly discussed.

New method for standardization of electropherograms obtained in capillary zone electrophoresis.

A new method for standardization of electropherograms obtained by capillary zone electrophoresis was proposed, where the migration time axis was replaced by the effective mobility axis. The mobility increase due to temperature increase by Joule heating and the relaxation effect of the potential gradient were eliminated successfully by introducing a temperature coefficient for mobility expression and a delay time, respectively. The precision of the mobility evaluated by the proposed conversion methods was evaluated for a model sample. By using the conversion method, almost the same electropherograms could be obtained even from the electropherograms originally obtained by using different hardware conditions.

Electropherogram of capillary zone electrophoresis with effective mobility axis as a transverse axis and its analytical utility. I. Transformation applying the hypothetical electroosmotic flow.

For capillary zone electrophoresis, a new method of transformation from migration time to effective mobility was proposed, in which the mobility increase due to Joule heating and the relaxation effect of the potential gradient were eliminated successfully. The precision of the mobility evaluated by the proposed transformation was discussed in relation to the analysis of rare earth ions. By using the transformation, almost the same pherograms could be obtained even from the pherograms obtained originally at different applied voltages.

Free-flow isotachophoresis under micro-gravity.

When free flow electrophoresis (FFE) is applied to the separation and purification process under microgravity, higher separability is expected than that under usual 1 G environment, because sedimentation of samples and heat convection can be neglected. Although FFE under micro-gravity is a very important technique for efficient separation of macromolecules because of the above merits, FFE unit (FFEU) should be more sophisticated to give stable operation, high separability, high through-put and high separation efficiency. In this study, fundamental research to achieve FFE with such high performance was done by using a commercial FFEU. The research contains (1) the quantitative study on the separation mode of FFE and separation efficiency, (2) the feasibility analysis of free-flow bidirectional isotachophoresis (FFBITP) which enables separation of cations anions simultaneously by isotachophoresis (ITP) and zone electrophoresis (ZE), (3) the optimization of hardware factors such as thickness of the separation chamber which may cause deterioration of separation due to heat convection, and (4) the analysis of hardware factors depending on the long-time operation. The ultimate goal of this study is to obtain fundamental data for the development of FFE for the international space station. In this paper, the experimental results obtained in a reduced-gravity aircraft was mainly.

A triangle lattice model that predicts transmembrane helix configuration using a polar jigsaw puzzle.

We developed a method of predicting the tertiary structures of seven transmembrane helical proteins in triangle lattice models, assuming that the configuration of helices is stabilized by polar interactions. Triangle lattice models having 12 or 11 nearest neighbor pairs were used as general templates of a seven-helix system, then the orientation angles of all helices were varied at intervals of 15 degrees. The polar interaction energy for all possible positions of each helix was estimated using the calculated polar indices of transmembrane helices. An automated system was constructed and applied to bacteriorhodopsin, a typical membrane protein with seven transmembrane helices. The predicted optimal and actual structures were similar. The top 100 predicted helical configurations indicated that the helix-triangle, CFG, occurred at the highest frequency. In fact, this helix-triangle of bacteriorhodopsin forms an active proton-pumping site, suggesting that the present method can identify functionally important helices in membrane proteins. The possibility of studying the structure change of bacteriorhodopsin during the functional process by this method is discussed, and may serve to explain the experimental structures of photointermediate states.

Physicochemical factors for discriminating between soluble and membrane proteins: hydrophobicity of helical segments and protein length.

The average hydrophobicity of a polypeptide segment is considered to be the most important factor in the formation of transmembrane helices, and the partitioning of the most hydrophobic (MH) segment into the alternative nonpolar environment, a membrane or hydrophobic core of a globular protein may determine the type of protein produced. In order to elucidate the importance of the MH segment in determining which of the two types of protein results from a given amino acid sequence, we statistically studied the characteristics of MH helices, longer than 19 residues in length, in 97 membrane proteins whose three-dimensional structure or topology is known, as well as 397 soluble proteins selected from the Protein Data Bank. The average hydrophobicity of MH helices in membrane proteins had a characteristic relationship with the length of the protein. All MH helices in membrane proteins that were longer than 500 residues had a hydrophobicity greater than 1.75 (Kyte and Doolittle scale), while the MH helices in membrane proteins smaller than 100 residues could be as hydrophilic as 0.1. The possibility of developing a method to discriminate membrane proteins from soluble ones, based on the effect of size on the type of protein produced, is discussed.

Proportion of membrane proteins in proteomes of 15 single-cell organisms analyzed by the SOSUI prediction system.

A software system, SOSUI, was previously developed for discriminating between soluble and membrane proteins and predicting transmembrane regions (Hirokawa et al., Bioinformatics, 14 (1998) 378-379). The performance of the system was 99% for the discrimination between two types of proteins and 96% for the prediction of transmembrane helices. When all of the amino acid sequences from 15 single-cell organisms were analyzed by SOSUI, the proportion of predicted polytopic membrane proteins showed an almost constant value of 15-20%, irrespective of the total genome size. However, single-cell organisms appeared to be categorized in terms of the preference of the number of transmembrane segments: species with small genomes were characterized by a significant peak at a helix number of approximately six or seven; species with large genomes showed a peak at 10 or 11 helices; and species with intermediate genome sizes showed a monotonous decrease of the population of membrane proteins against the number of transmembrane helices.

SOSUI: classification and secondary structure prediction system for membrane proteins.

UNLABELLED: The system SOSUI for the discrimination of membrane proteins and soluble ones together with the prediction of transmembrane helices was developed, in which the accuracy of the classification of proteins was 99% and the corresponding value for the transmembrane helix prediction was 97%. AVAILABILITY: The system SOSUI is available through internet access: http://www.tuat.ac.jp/mitaku/sosui/. CONTACT: sosui@biophys.bio.tuat. ac.jp.

A continuum theory for the prediction of lateral and rotational positioning of alpha-helices in membrane proteins: bacteriorhodopsin.

We have developed a new method for the prediction of the lateral and the rotational positioning of transmembrane helices, based upon the present status of knowledge about the dominant interaction of the tertiary structure formation. The basic assumption about the interaction is that the interhelix binding is due to the polar interactions and that very short extramembrane loop segments restrict the relative position of the helices. Another assumption is made for the simplification of the prediction that a helix may be regarded as a continuum rod having polar interaction fields around it. The polar interaction field is calculated by a probe helix method, using a copolymer of serine and alanine as probe helices. The lateral position of helices is determined by the strength of the interhelix binding estimated from the polar interaction field together with the length of linking loop segments. The rotational positioning is determined by the polar interaction field, assuming the optimum lateral configuration. The structural change due to the binding of a prosthetic group is calculated, fixing the rotational freedom of a helix that is connected to the prosthetic group. Applying this method to bacteriorhodopsin, the optimum lateral and rotational positioning of transmembrane helices that are very similar to the experimental configuration was obtained. This method was implemented by a software system, which was developed for this work, and automatic calculation became possible for membrane proteins comprised of several transmembrane helices.