Dielectrophoresis (DEP) is the motion of a matter caused by polarization effects in a non-uniform electric field. Recently, studies on DEP are promoted in various medical fields, including separation and characterization of biological cells. It is very important but not easy to measure this motive force, so-called dielectrophoretic force (DEP force). In general, the DEP force generated by the designed electrodes is analyzed by the computer simulation that employs the finite element analysis method. However, it does not always calculate the correct DEP force. Therefore, we propose the method of measuring the DEP force accurately based on the null method. The experiment is conducted with the dielectrophoretic device (DEP device) in which microfabricated electrodes were formed and which contains the polystyrene particles exhibiting the negative DEP in distilled water in a non-uniform electric field. The displacement of the particle reaches a steady state 15 min. after the change of applied voltage v, angle θ or frequency f. The equilibrium state of the particle in a non-uniform electric field can be reached at any place by adjusting both of angles θ and φ, where θ is the “Pitch” angle and φ the “Yaw” angle for the device. The proposed method can measure the incredibly minute DEP force ranging from 25 fN (femto-Newton) at θ=5° to 298 fN at θ=90°, the accuracy of which is determined by the static friction and rolling friction between particles and the inner floor of the DEP device.

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Oxidative stress is implicated in a broad variety of chronic and acute diseases, including Alzheimer's disease, arteriosclerosis and diabetes. In order to study oxidative stress at the proteome level proteins were labeled with Cy2NHS (the N-hydroxyl succinimidyl ester derivative of a cyanine dye reactive with the ε-amino group of lysine residues), or with Cy3- and Cy5HZ (hydrazide derivatives of the dyes reactive with protein carbonyls) and analyzed by two-dimensional fluorescence difference gel electrophoresis (2D-DIGE). Two samples were used for testing the CyHZ labeling; one is low molecular mass marker proteins artificially oxidized by NaOCl and the other renal cortex extracts of 30-week-old diabetes model OLETF (Otsuka Long-Evans Tokushima Fatty) and control LETO (Long-Evans Tokushima Otsuka) rats. Spot volumes V_ni and contrasts c_ni of 2D-DIGE images were quantified following background subtraction, where the suffices n (=2, 3 or 5) and i (=1, 2, ...) represent the Cy-signal and the Cy2-detected spot identification numbers, respectively. The contrasts c_3i and c _5i were on average about 0.53 times as high as c_2i. Taking the contrast into account, a simple parameter q_ni was proposed for judging the quality of spot images. The difference in-gel analysis (DIA) algorithm should be used in combination with the quality parameters to identify statistical outliers as candidates for abnormally carbonylated proteins. Inspection of the background flatness around the candidate spots is also required before making a conjecture as to whether the candidate protein is abnormally oxidized.

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Urine cytology is the gold standard for detection of bladder cancer. However, it has poor sensitivity, and currently several diagnostic markers have high false-positive rates. One of the problems with bladder cancer is high locoregional recurrence after surgery. Therefore new diagnostic markers of bladder cancer are needed. We have conducted a comprehensive study of high molecular mass (HMM) protein expression in bladder cancer tissue by means of agarose two-dimensional gel electrophoresis (agarose 2-DE) followed by the analysis of liquid chromatography tandem mass spectroscopy. We successfully identified 53 proteins from cancer tissue and 33 proteins from normal mucosa in the HMM area of the 2DE gel, and they were classified as proteins of transcription, translation or the cytoskeleton. Among them, eight differentially regulated proteins were selected as new marker candidates, and we performed experiments to validate their use as diagnostic markers. The results of Western blotting showed that these proteins except for α spectrin were significantly upregulated in bladder cancer tissue, and one of them, structural maintenance of chromosomes protein 3 (SMC3), was specifically detected in urine samples from bladder cancer patients. On the other hand, the results of Immunohistochemistry showed that expression of neuroblast differentiation-associated protein AHNAK (AHNAK) was significantly increased and that of plectin-1 was significantly decreased in the bladder cancer specimens compared with normal bladder mucosa specimens. However, no significant difference was observed in other proteins. These results suggest that AHNAK and Plectin-1 would be the potential markers for urine cytology and SMC3 is possible to be that for diagnosis marker for urinalysis.

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We studied serum samples which showed alkaline phosphatase (ALP) activity at or near the origin of the polyacrylamide gel electrophoresis. 1) Sucrose monolaurate (final concentration, 1.7%) treatment produced a new ALP band (SM-ALP band). 2) The SM-ALP band disappeared from the original region with anti-human placental ALP antibody and appeared at the high molecular region. 3) ALP activity of SM-ALP band was completely inactivated by heat treatment (at 65°C for 10 min). 4) The molecular mass was estimated to be approximately 350 kDa by a 5-20% (linear gradient) polyacrylamide slab gel electrophoresis. We concluded that the SM-ALP band was intestinal ALP tetramer.

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Proteins from silkworm (Bombyx mori L.) silk glands, fat bodies, and hemolymph were analyzed by matrix assisted laser desorption ionization time-of-flight mass spectrometry using sinapinic acid as a matrix. Peak percentages were calculated for each peak from m/z 2,000 to m/z 20,000 against the sum of all observed peak intensities; hierarchical cluster analysis and heat map analysis were performed using R-statistics software. For the silk glands, clusters obtained from MALDI-TOF-MS analysis were consistented with the morphological classification. Hierarchical clustering using MALDI-TOF-MS data was considered applicable to the small and sequential biopsies. In hemolymph, obvious clusters were observed from the anterior part to posterior part, and the proteins in hemolymph were not considered homogeneous throughout the body. While the clusters obtained from fat body analysis were weaker than those of silk glands and hemolymph, three clusters were observed. Based on the protein profile, fat bodies were also considered as differentiated tissue.

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