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Subphthalocyaninatoboron(III) was applied for the first time as a novel marker-tag of testosterone (Subpc-test) for the binding analysis with human serum albumin (HSA) at a liquid/liquid interface. The binding interaction of Subpc-test with HSA at the n-hexane/water interface was studied by UV-visible absorption and circular dichroism (CD) spectroscopy combined with a centrifugal liquid membrane cell at different pHs of aqueous solutions. Complementary studies by a high-speed stirring experiment and an interfacial tension measurement were also performed to characterize the interfacial adsorptivity of Subpc-test and HSA molecules, respectively. The n-hexane solution of Subpc-test showed no optical chirality, but the contact with the aqueous solution of HSA induced its optical chirality, clearly suggesting the formation of Subpc-test/HSA complexes at the n-hexane/water interface. Furthermore, pH profiles of CD signals showed that the interaction between Subpc-test and HSA was very sensitive to the neutral-to-base transition (N-B transition). Interfacial formation of the Subpc-test/HSA complex was studied in the presence of the site-selective ligand, furosemide (site I) or cefaclor (site II). The experimental results suggested that Subpc-test is bound to site I of the HSA molecule at the n-hexane/water interface. This technique using subphthalocyanine as a tag molecule and the liquid/liquid interface as a two-dimensional nanoreaction field will be useful for the evaluation of the interaction between drugs or hormones and proteins.

Antibody binding to bovine serum albumin (BSA) and human serum albumin (HSA) immobilized onto gold nanoparticles was studied by means of localized surface plasmon resonance (LSPR) spectroscopy. Amine-modified glass was prepared by self-assembly of amine-terminated silane on substrate, and gold (Au) nanoparticles were deposited on the amine-modified glass substrate. Au nanoparticles deposited on the glass surface were functionalized by BSA and HSA. BSA immobilization was confirmed by LSPR spectroscopy in conjunction with surface-enhanced Raman scattering spectroscopy. Then, LSPR response attributable to the binding of anti-BSA and anti-HSA to BSA- and HSA-functionalized Au nanoparticles, respectively, was examined. Anti-HSA at levels larger than approximately 10 nM could be detected by HSA-immobilized chips with LSPR optical response, which was saturated at concentrations greater than approximately 650 nM of anti-HSA.

A new principle of the magnetophoretic velocity modulation mass analysis of microparticles, which can determine simultaneously the mass and magnetic susceptibility of a single microparticle, has been proposed, and the measurement system was constructed by applying a magnetophoretic force on a falling microparticle through a magnetic field gradient in an atmosphere. A polystyrene microparticle as a test particle adsorbed on a glass plate was selectively knocked off by a pulsed Nd:YAG laser impact into a narrow gap of pole pieces of permanent magnets having a magnetic field gradient with a maximum intensity of 850 T2 m(-1). The falling particle was irradiated by a He-Ne laser, and the scattered light was detected through a slit array mask as a function of time. A bundle of spiked signals of scattered light intensity was analyzed to obtain velocities, which gave acceleration and deceleration of the falling particle. On the basis of the equation of motion under the magnetic field gradient, the mass and magnetic susceptibility of the test particle were reasonably determined.

A new principle to measure the magnetic susceptibility of a solution was proposed; it utilized the displacement of solution surface caused by the magnetic force applied to the solution. The interfacial displacement was measured with the sensitivity of 5 nm from a change of the interference fringes, so called Newton Ring, generated from the solution surface and an upper surface of a small plano-convex lens on the bottom wall of a cell. The surface displacement measurement was carried out in a homemade cell attached by one or two small Nd-Fe-B magnets. The performance of this method was investigated by the measurements of magnetic susceptibility of manganese(II) chloride solution and some lanthanide(III) solutions. An excellent linear relationship between the magnetic susceptibility and the interfacial displacement was observed for each solution system. Then, it was demonstrated that this relationship could be used for the determination of the magnetic susceptibility of solutions or the concentration of a solute by using the manganese(II) solution as a standard solution.

A new analytical technique, spinning microtube fluorometry (SMF), was developed and applied to the study of interfacial hydrolysis of 5-dodecanoylaminofluorescein di-beta-D: -galactopyranoside (C12FDG) by beta-galactosidase (beta-gal) in the toluene-water system. The nonfluorescent lactone form of C12FDG in the toluene phase was converted at the interface to 5-dodecanoylaminofluorescein (C12F), which was fluorescent in the aqueous phase as a dianion at pH 7.3, though some part of C12F was extracted into the toluene phase as its nonfluorescent lactone form. The distribution ratios of C12FDG and C12F at pH 7.3 were determined as 1.4 x 10(2) and 1.97, respectively. The interfacial adsorption constants from the toluene phase to the interface at pH 7.3 were 4.8 x 10(-4) and 1.7 x 10(-2) dm for C12FDG and C12F, respectively. The kinetic experiments with the SMF method concluded that the rate-determining step of the enzymatic hydrolysis at the interface and in the aqueous phase was the 1:1 reaction of C12FDG and beta-gal and that the hydrolysis reaction rate constant at the interface at pH 7.3 was 1.84 x 10(3) M(-1)s(-1), almost equal to that in the aqueous solution, 1.76 x 10(3) M(-1)s(-1). Finally, the SMF method revealed that the contribution of the interfacial reaction to the overall hydrolysis reaction rate of the toluene-water system was as high as 97%.

The Pd(II)-2,2'-bis(diphenylphosphino)-1,1'-binaphthyl (BINAP)-mediated chiral assembly of thioether-derivatised phthalocyanatomagnesium(II) compounds (MgPc(SR)8, SR is benzylthio (SBz) or benzhydrylthio (Bh)) was formed in toluene and at the toluene/water interface, and investigated by means of UV-Vis absorption and circular dichroism (CD) spectroscopy combined with the centrifugal liquid-membrane (CLM) devise. Interfacial tension measurements indicated that, in the presence of PdSO4 in the aqueous phase, BINAP ligand adsorbed as a monolayer forming Pd(II)BINAP2+ complex at the toluene/water interface. UV-Vis absorption spectrum of MgPc(SR)8 in the Q-band region was blue-shifted in toluene upon addition of [Pd(II)BINAP]Cl2, but red-shifted at the toluene/water interface when Pd(II)BINAP2+ was formed at the interface. These results suggested that MgPc(SR)8-Pd(II)BINAP complex formed H-aggregate (face-to-face type) in toluene solution and J-aggregate (end-to-end type) at the toluene/water interface, respectively. Moreover, the specific bisignate CD spectral pattern in both systems indicated that the aggregates of MgPc(SR)8-Pd(II)BINAP complexes were chirally twisted, well controlled by the chirality of BINAP ligand. Very interestingly, the morphology of MgPc(SBz)8-Pd(II)BINAP and MgPc(SBh)8-Pd(II)BINAP aggregates formed at the toluene/water interface were significantly different as the rodlike and the ribbonlike crystalline structure, respectively, as observed by a scanning electron microscopy (SEM). On the basis of these experimental results, we proposed schematic molecular models of the chiral aggregates of MgPc(SR)8-Pd(II)BINAP complexes and demonstrated the specific role of the toluene/water interface.

Adsorption and domain formation of dodecanethiol (DT)-bound silver nanoparticles (SNPs) at the cyclohexane/water interface were studied by means of total internal reflection (TIR) light scattering microscopy and TIR surface-enhanced Raman scattering (SERS). By the TIR light scattering microscopy, the extent of the interfacial adsorption and domain formation of SNP was observed, which was produced by the reaction between citrate-reduced SNPs in the aqueous phase and DT in the cyclohexane phase. The Raman spectra of DT on SNP showed that the relative intensity ratios of gauche to trans conformers in the nu(C-S) band region decreased with the increase of the initial concentration of DT, suggesting the change from the liquidlike structure to the solidlike structure of the DT. The residue of the negative charges on the SNPs at the interface was detected by the resonance SERS (SERRS) peaks of the adsorbed cationic porphyrin, 5,10,15,20-tetrakis(N-methylpyridinium-4-yl)-21H,23H-porphine (TMPyP). The efficiency of the interfacial SNPs domains as a SERS substrate for TMPyP strongly depended on the adsorption state of the DT.

A Raman microprobe spectrometer that could be installed in the bore of a cryogen free super-conducting magnet (10 T) was designed and constructed for the investigation of the external magnetic field effect on the Raman spectra of molecular aggregates in solutions and at interfaces. The performance of the present instrument was demonstrated by measuring the magnetic field effect (0 - 10 T) on the resonance Raman spectra of diprotonated meso-tetra-(sulfonatophenyl)porphine aggregates in an acidic solution. The Raman shifts of the aggregates were not significantly influenced even in 10 T. However, the relative intensity of 1123 cm(-1) peak (nu(C(a)-N)) was interestingly enhanced about 20% under the magnetic fields higher than 2.5 T.

In mouse liver transplantation, tolerance is readily inducible. Recent studies have revealed that CD25+ CD4+ regulatory T cells play an important role in regulating various immune responses, including transplant tolerance. However, the contribution of these cells to tolerance in mouse liver transplantation has not been elucidated. We showed here that depletion of CD25+ CD4+ T cells increased proliferative response of CD4+ T cells and cytotoxic T lymphocyte induction of CD8+ T cells. Depletion of these cells in the recipient but not in the donor before liver transplantation caused rejection. Furthermore, the number of CD25+ CD4+ population and forkhead/winged helix transcription factor expression in liver mononuclear lymphocytes derived from tolerant mice were higher than those from grafts undergoing rejection. In conclusion, these results indicate that CD25+ CD4+ regulatory T cells in the recipient but not in the donor of liver transplantation are important for the tolerance induction., (Copyright 2006 AASLD)

Several subphthalocyanine derivatives that contain an alkoxo substituent as an axial ligand (RO-Subpc, R = 9-anthracenemethyl, benzyl, phenyl, 3,5-dimethylbenzyl, 3,5-dimethylphenyl, 4-methylbenzyl, and 4-methylphenyl) were synthesized. The formation of inclusion complexes of RO-Subpc with beta-CD in DMSO and at the toluene/water interface was investigated by UV/Vis absorption spectroscopy, induced circular dichroism (ICD), and nuclear magnetic resonance (NMR) measurements. Interfacial tension measurements suggested that beta-CD adsorbed as a monolayer at the toluene/water interface and probably orientated towards the toluene phase with its primary face. The 1:1 composition of beta-CD.RO-Subpc inclusion complexes was confirmed in DMSO and at the toluene/water interface for BzO-Subpc, PhO-Subpc, MeBzO-Subpc, and MePhO-Subpc. A 2:1 inclusion complex of AnO-Subpc formed in DMSO. The observed ICD spectra of beta-CDRO-Subpc inclusion complexes are discussed with respect to molecular modeling and the simulation based on Tinoco-Kirkwood theory. Interestingly, the ICD spectra of beta-CD.BzO-Subpc and beta-CD.MeBzO-Subpc inclusion complexes exhibited a negative sign in DMSO and a positive sign at the toluene/water interface. This reversal of the ICD sign strongly suggests a difference in the structure of the inclusion complexes: beta-CD at the interface formed the inclusion complex with its primary face, whereas the secondary face of beta-CD bound favorably to RO-Subpc in DMSO.

Interfacial ion-association adsorption and aggregation of a water-soluble porphyrin, tetrakis(4-sulfonatephenyl)porphyrin (TPPS) diacid, which was promoted by a cationic cetyltrimethylammonium ion (CTA(+)), was studied by second harmonic generation (SHG) spectroscopy. Comparing the interfacial SH spectrum with the transmission absorption spectrum of TPPS in the aqueous solution elucidated the aggregation behavior of TPPS at the heptane/water interface. The time-dependent SHG spectra for TPPS aggregation and the interfacial tension lowering in the presence of CTA(+) were discussed on the basis of an electrostatic adsorption model. Then, it was suggested that TPPS diacid was highly concentrated by the ion-association with CTA(+) at the interface, which was the intermediate state before the final aggregated state.

The size determination and the size sorting of micrometer-sized particles were carried out by using a piezo-controlled nano-gap. The nano-gap was built by combining a plano-convex lens and a flat glass plate. The fabricated gap had a gradient, while the center of the lens was narrow and the outer side was wide. When the sample solution was injected into the gap, a meniscus force carried the sample particles toward the center of the lens with solvent evaporation; then the individual particle was trapped at the specific position where the particle diameter was equal to the gap distance. The size determination ability of this new method was certified by using the standard polystyrene particles and the size measurements of DNA molecules and yeast cells were demonstrated.

A chiral thioether-substituted phthalocyanine ((2,3,9,10,16,17,23,24-octakis-1-phenylethylthiophthalocyaninato)magnesium(II) [MgPc(SEtPh)8]) has been synthesized, which can be bound by soft-metal ions such as palladium(II) ion. Aggregates formed from MgPc(SEtPh)8 and Pd(II) in toluene and at the toluene/water interface were characterized by means of UV-vis absorption and circular dichroism (CD) spectrometries using centrifugal liquid-membrane (CLM) cell. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS) and scanning electron microscope (SEM) were used as complementary techniques. The toluene solution of MgPc(SEtPh)8 had no optical chirality. However, the addition of PdCl2 into the toluene solution of MgPc(SEtPh)8 induced optical chirality, which indicated the formation of a twisted H-type dimer (face-to-face fashion) of MgPc(SEtPh)8 bound by four PdCl2 in the bulk toluene solution. On the other hand, in the toluene/water interface, helical J-aggregate (head-to-tail fashion) of MgPc(SEtPh)8 bound by PdCl2 was formed, in which one J-aggregate unit contained five 1:2 complexes of MgPc(SEtPh)8-Pd(II) on average. On the basis of the experimental results and the exciton theory for optical chirality, a possible mechanism for the chiral aggregation of MgPc(SEtPh)8-Pd(II) complexes at the interface was proposed. In the present study, we demonstrated a novel strategy for the design of helical phthalocyanine aggregates using the liquid/liquid interface as a template.

The B cell antigen receptor (BCR)-mediated activation of IkappaB kinase (IKK) and nuclear factor-kappaB require protein kinase C (PKC)beta; however, the mechanism by which PKCbeta regulates IKK is unclear. Here, we demonstrate that another protein kinase, TGFbeta-activated kinase (TAK)1, is essential for IKK activation in response to BCR stimulation. TAK1 interacts with the phosphorylated CARMA1 (also known as caspase recruitment domain [CARD]11, Bimp3) and this interaction is mediated by PKCbeta. IKK is also recruited to the CARMA1-Bcl10-mucosal-associated lymphoid tissue 1 adaptor complex in a PKCbeta-dependent manner. Hence, our data suggest that phosphorylation of CARMA1, mediated by PKCbeta, brings two key protein kinases, TAK1 and IKK, into close proximity, thereby allowing TAK1 to phosphorylate IKK.

The differential expression of surface molecules on dendritic cells (DC) reflects their functional differences as immature and mature subsets. It is difficult, however, to characterize differences in surface expression by standard proteomic approaches, due mainly to the hydrophobic nature and low abundance of the individual proteins in question. We have established a method for obtaining high-yield plasmalemma preparations which contain surface molecules enriched more than 200-fold by coating cells with beads conjugated with antibody against a cell type-specific cell-surface molecule, followed by nitrogen cavitated disruption, magnetic separation, and density gradient ultracentrifugation. We identified and quantified 339 human monocyte-derived DC transmembrane proteins, including 33 previously uncharacterized molecules. Whereas 106 proteins were selectively expressed in immature cells or down-regulated after maturation, 191 proteins were selectively expressed in mature cells or up-regulated after maturation.

A new device to measure the dielectrophoretic mobility alpha of a single micro-particle in a micro-flow system has been proposed. From the velocity analysis of the micro-particle flowing between a pair of parabolic electrodes, the value of alpha could be obtained.

Novel chloro-subphthalocyaninatoboron()(Subpc) derivatives with six thioether at the 2 and 3 positions of peripheral benzene (Subpc(SR)) such as chloro-[2,3,9,10,16,17-hexakis-ethylthio-subphthalocyaninato]boron()(Subpc(SEt)) and chloro-[2,3,9,10,16,17-hexakis-benzylthio-subphthalocyaninato]boron()(Subpc(SBz)) were synthesized. The interfacial adsorption of Subpc and Subpc(SR), and the interfacial aggregation of Subpc(SR)-Pd() complexes in the toluene-water system have been investigated by means of high-speed stirring (HSS) spectrometry, centrifugal liquid-membrane (CLM) spectrometry, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). Characteristically orientated structures of Subpc(SR) molecules and the aggregates of Subpc(SR)-Pd() complexes at the toluene-water interface have been suggested depending on the structures of Et and Bz at the periphery. The interfacial aggregation was observed only in the presence of palladium(), in which one aggregate unit contained at least seven 1 : 2 complexes of Subpc(SR)-Pd(). Interestingly, the absorption spectra of the interfacial aggregates of Subpc(SEt)-Pd() and Subpc(SBz)-Pd() complexes were very similar to the previously reported spectra of μ-oxo dimer and binuclear molecule of subphthalocyanine, respectively, suggesting the formation of "face-to-face" or H-aggregate for Subpc(SEt)-Pd() and "head-to-tail" or J-aggregate for Subpc(SBz)-Pd(), respectively, at the toluene-water interface.

Valpha14 NKT cells exhibit various immune regulatory properties in vivo, but their precise mechanisms remain to be solved. In this study, we demonstrate the mechanisms of generation of regulatory dendritic cells (DCs) by stimulation of Valpha14 NKT cells in vivo. After repeated injection of alpha-galactosylceramide (alpha-GalCer) into mice, splenic DCs acquired properties of regulatory DCs in IL-10-dependent fashion, such as nonmatured phenotypes and increased IL-10 but reduced IL-12 production. The unique cytokine profile in these DCs appears to be regulated by ERK1/2 and IkappaB(NS). These DCs also showed an ability to suppress the development of experimental allergic encephalomyelitis by generating IL-10-producing regulatory CD4 T cells in vivo. These findings contribute to explaining how Valpha14 NKT cells regulate the immune responses in vivo.

It is well-documented that certain chemokines or their receptors play important roles in the graft rejection. However, the roles of chemokines and their receptors in the maintenance of transplantation tolerance remain unclear. In this study, we demonstrate that blocking of the interaction between the chemokine receptor, CXCR6, highly expressed on V alpha14+ NKT cells and its ligand, CXCL16, resulted in the failure to maintain graft tolerance and thus in the induction of acceleration of graft rejection. In a mouse transplant tolerance model, the expression of CXCL16 was up-regulated in the tolerated allografts, and anti-CXCL16 mAb inhibited intragraft accumulation of NKT cells. In vitro experiments further showed that blocking of CXCL16/CXCR6 interaction significantly affected not only chemotaxis but also cell adhesion of NKT cells. These results demonstrate the unique role of CXCL16 and CXCR6 molecules in the maintenance of cardiac allograft tolerance mediated by NKT cells.

A new principle for the chromatographic micro-separation of micrometer-sized particles in liquid has been invented by switching the electromagnetophoretic force in a capillary flow system. The principle is the combination of the Stokes force by the bulk flow and the adsorption-desorption force on a capillary inner surface controlled by an electromagnetophoretic buoyancy generated by an alternative current and a homogenous magnetic field. The observed retention profiles of test micro-particles was explained by the "zigzag" migration model mainly depended on particle size and their adsorption force to the capillary wall. By this method, we could succeed to separate polystyrene particles of 10 microm and 20 microm in diameter dispersed in 1 M KCl solution containing 0.01% Triton X-100 using only 1 mm long fused-silica capillary under 10T.

Two-photon excitation fluorescence microscopy was combined with the two-phase microflow system in order to measure the fast interfacial reaction rate at liquid/liquid interfaces. The lactone cleavage kinetics of octadecylrhodamine B (C(18)RB) at the toluene/water and heptane/water interfaces was studied by this new method. The organic solution containing the nonfluorescent lactone of C(18)RB was made to flow as an inner flow with an aqueous outer sheath flow. The diameter of the inner flow was <20 microm. A focused fundamental beam of a Ti:sapphire pulse laser of 780 nm was irradiated to the interface, and emitted fluorescence from the fluorescent product was detected by a charge-coupled device (CCD) camera or a streakscope. The increase in the concentration of the fluorescent form of C(18)RB was measured along the interface of the inner flow of the toluene/water and heptane/water systems for 80 micros just after the contact of two phases. The analysis made by the time-dependent Langmuir adsorption model with the aid of the digital simulation method gave the cleavage reaction rate constants of the lactone form of C(18)RB at the liquid/liquid interfaces.

Schnurri (Shn) is a large zinc finger protein implicated in cell growth, signal transduction, and lymphocyte development. Vertebrates possess at least three Shn orthologues (Shn-1, Shn-2, and Shn-3), which appear to act within the bone morphogenetic protein, transforming growth factor beta, and activin signaling pathways. However, the physiological functions of the Shn proteins remain largely unknown. In Shn-2-deficient mice, mature peripheral T cells exhibited normal anti-T cell receptor-induced proliferation, although there was dramatic enhancement in the differentiation into T helper type (Th)2 cells and a marginal effect on Th1 cell differentiation. Shn-2-deficient developing Th2 cells showed constitutive activation of nuclear factor kappaB (NF-kappaB) and enhanced GATA3 induction. Shn-2 was able to compete with p50 NF-kappaB for binding to a consensus NF-kappaB motif and inhibit NF-kappaB-driven promoter activity. Thus, Shn-2 plays a crucial role in the control of Th2 cell differentiation by regulating NF-kappaB function.

When the water-in-oil (w/o) microemulsion droplets including the Co(III)-pyridylazo complex as the photo-absorber were irradiated with a continuous-wave Ar(+) ion laser (514.5 nm), we have observed unexpected phenomenon that photo-absorbing microemulsion droplets in water repeated the expansion and the sudden contraction during the laser photophoretic migration. The period of the expansion-contraction cycle was inversely proportional to both the concentration of the complex and the irradiated laser power and was independent of the initial size of the droplet. The mechanism of the periodic motion of the droplet was investigated by local temperature measurement and Raman microscope spectroscopy. It was suggested that the first step was the phase separation of the bicontinuous microemulsion droplet into the normal w/o microemulsion outer phase and the aqueous inner phase in the droplet, which was caused by the laser-induced temperature gradient inside the droplet. Subsequently, an expansion of the inner aqueous phase was induced by the percolation of the external water by thermo-osmosis, which was caused by the laser-induced temperature gradient between the inside and the outside of the microemulsion liquid membrane of the droplet. When the liquid membrane became thinner to a critical thickness, the inner aqueous phase was released and the droplet shrank into the original size. The proposed mechanism can give an account of the unique cyclical motion.

A Newton-ring micro-particle sorter was constructed from a small convex lens and a flat glass. The sub-micrometer gap between them was controlled by a piezo-actuator and utilized for the fractionation of very small amounts of micro-particles in liquid.

The complex formation of Cu(II) and Fe(II) with a hydrophobic ligand, 5-(octadecyloxy)-2-(2-thiazolylazo)phenol (TARC18), was investigated in the heptane/water system by the high-speed stirring spectrometry and the micro-two phase flow ESI/MS method. At first, the dissociation constant of TARC18 at the heptane/water interface was determined as pKa = 7.11. The interfacial complexation of the ligand with Cu(II) and Fe(II) under stirred conditions progressed with an increase of the pH. The experimental results showed that a 1:1 complex of Cu(II) and TARC18 was formed at the interface, but was hardly extracted into the heptane phase. On the other hand, the 1:2 complex of Fe(II) with TARC18 formed at the interface was significantly extracted into the heptane phase. The extraction constants and interfacial complex formation constants were estimated for the two systems from the experimental results, and all of the reaction schemes, including the interfacial reactions, were elucidated.

The direct measurement of the circular dichroism (CD) spectra of liquid/liquid interface has been achieved for the first time by the centrifugal liquid membrane (CLM) method combined with a conventional CD spectropolarimetry. In the sample chamber of the CD spectropolarimeter, a cylindrical glass cell containing small amounts of organic and aqueous phases was rotated at ca. 7000 rpm to generate a two-phase liquid membrane with a high specific interfacial area. The CD spectra of the J-aggregate of protonated 5,10,15,20-tetraphenylporphyrin formed at the toluene/sulfuric acid interface in the rotating cell have been measured. The results demonstrated the novelty and advantages of this method.

A microscopic system for the observation of reactions at a liquid-liquid interface was established, to which strong magnetic fields (0-0.4 T) could be applied with permanent magnets. In situ observation for the interfacial extraction of fluorescent and paramagnetic Eu(III) ion with 2-thenoyltrifluoroacetone (Htta) in dodecane was carried out. In the presence of oxalate (ox2-), micro-aggregates of Eu(III)-tta-ox complexes were generated in the aqueous phase before its extraction. When the micro-aggregates diffused to the dodecane-water interface, Eu(tta)3 was extracted with excess Htta in the dodecane phase. The microextraction process of the aggregates was observed as random flashes of Eu(tta)3 fluorescence at the dodecane-water interface. The single flash contained about 10(-16)-10(-14) mol of Eu(III). An application of magnetic fields made the flash frequency increase, which corresponded to an enhancement of interfacial Eu(III) extraction rate. The enhancement effect was attributable to the magnetophoresis of the paramagnetic microaggregates to the dodecane-water interface.

We propose a novel principle of velocity mass analysis of a micro-particle using magnetophoretic force. The new method can determine the mass of a particle from its magnetophoretic velocity change in a high magnetic field gradient in a low viscous medium such as air. In the present study, the new principle was demonstrated by the magnetophoretic acceleration of an aqueous manganese(II) chloride micro-droplet and the deceleration of a water micro-droplet in the atmosphere. The observed velocity change was analyzed taking into account the mass of the droplet through the acceleration term of the equation of motion. The experimental results proved that the inertia force in the magnetophoretic velocity of a micro-particle could be detected in air. The present method provided an innovative mass analysis method without any ionization of the sample.

Oleate-stabilized silver colloids of 5-nm-diameter were adsorbed to a toluene/water interface, and surface enhanced Raman scattering (SERS) spectra from these colloids were measured under the total internal reflection (TIR) condition. From the observed spectra, we examined the states of oleate ions and toluene molecules on silver colloids at the liquid/liquid interfacial region. The TIR-SERS spectra of oleate ions showed stronger peaks of the carboxylate group and the ethylene group than those of alkyl chains. From these results, it was found that the oleate ions were adsorbed on the silver surface in two different ways at the liquid/liquid interface; the carboxylate group adsorbed in the organic phase side, while the ethylene group adsorbed in the aqueous phase side. The shifts of the toluene in the interfacial SERS spectra were identical to those of bulk toluene, though the relative intensities among the peaks were not same. This result suggested that the toluene was adsorbed with a weak interaction, but was significantly enhanced by the local electromagnetic field at the colloid surface.

Magnetophoretic velocimetry detected the spin transition of a single Co-Fe Prussian Blue analogous micro-crystal in water induced by a single-shot pulse laser.

The complexation mechanism and aggregate formation of bis[2-(5-bromo-2-pyridylazo)-5-diethylaminophenolate] iron(II) and iron(III) complexes at the heptane-water interface were studied spectrophotometrically by the high-speed stirring method and the centrifugal liquid membrane method. Furthermore, the reduction process of the Fe(III) complex with ascorbic acid at the interface was spectrophotometrically observed. The chemical compositions of the interfacial aggregate of complexes have been proved by the X-ray photoelectron spectroscopy. The aggregation of the complex at the interface was observed as a red-shifted, very strong and narrower absorption band with respect to the absorption band of the monomer complex. The aggregate of Fe(III) complex showed more shifted spectrum than that of Fe(II) complex, which proposed the larger aggregation number of Fe(III) aggregate (n = 8) than that of Fe(II) aggregate (n = 3). The obtained rate constants of interfacial aggregation were smaller than rate constants of interfacial monomer complexation, because the formation of aggregate required the assembly of the monomers., (Copyright 2004 Elsevier Inc.)

The electromagnetophoretic migration of micro-particles in a capillary flow system was demonstrated using a homogeneous magnetic field applied at right angles to an electric current. We utilized a high-magnetic-field of 10 T for observing this phenomenon. When the direction of the electric current was alternatively changed, polystyrene latex particles in a flowing aqueous medium migrated zigzag affected by a Lorentz force exerted on the medium. Carbon particles also migrated in the same manner with polystyrene particles. Further, we tried the electromagnetophoretic migration of biological particles, such as yeasts and human red blood cells. The migration velocity component perpendicular to the flow was proportional to both the electric current and the magnetic flux density. These results proved that the dominant force of the zigzag migration was an electromagnetophoretic buoyancy generated in the flowing medium. Moreover, it was found that the force exerted on the particles in the magnetic field of 10 T was sufficient for the desorption of particles adsorbed on the capillary wall.

The magnetic field-induced migration of particles in liquids is a highly-promising technique for the micro-separation analysis of bioparticles, such as cells and large DNA. Here, new methods that make use of magnetophoresis and electromagnetophoresis to induce the migration of microparticles in liquids are briefly reviewed. Magnetic force and Lorentz force are utilized in the new methods. Some typical examples of the use of these methods are described, and the advantages of using a superconducting magnet for them are demonstrated.

The relationship between occlusal stimuli and a hypofunctional periodontal ligament (PDL) structure has been reported, though changes in occlusal recovery conditions were still unclear. Nitric oxide (NO) produced by NO synthase (NOS) is considered a factor for vascular and immune system control, and it increases according to mechanical stimuli. The objective of this study was to examine the relationship between NOS and occlusal stimuli in PDL by comparing hypofunction with occlusal recovery. The study focused on the expression of endothelial NOS (eNOS) and inducible NOS (iNOS). Their expression significantly decreased in occlusal hypofunction compared with the control group and increased close to normal in an occlusal recovery group. The change in the immunopositive area was more dramatic than the immunopositive cell number. Moreover, the rate of iNOS increase was higher than that of eNOS. This study suggests that NO plays an important role in the recovery of the hypofunctional PDL.

Background and Purpose: A significant proportion of patients with lacunar infarctions experience neurologic deterioration after onset. However, no clinical examination has been established for prediction of the progress of symptoms. To determine the hemodynamic predictors of such progression, we performed perfusion CT to quantitatively assess cerebral blood flow (CBF), cerebral blood volume (CBV), and mean transit time (MTT) of patients with lacunar infarctions in the territory of the lenticulostriate artery., Methods: We performed MR imaging and perfusion CT of 26 patients with lacunar infarction within 24 hr after onset. On the CBF map on perfusion CT scans, a round small region of interest was set at the region, with decreased CBF in the territory of the lenticulostriate artery (region of interest 1). Another region of interest was set in the mirror position to region of interest 1 in the contralateral hemisphere (region of interest 2). Using these two regions of interest, CBF, CBV, and MTT were measured. All patients underwent neurologic and MR imaging follow-up while receiving equivalent medical treatment., Results: Neurologic deterioration after onset was shown in 13 patients (progress group), whereas no neurologic deterioration was shown in the other 13 patients (control group). In the progress group, lacunar infarctions were enlarged on follow-up MR images. The ratio of region of interest 1/region of interest 2 showed significantly lower CBF and higher MTT in the progress group than in the control group., Conclusions: These results suggest that progressive lacunar infarction in the territory of the lenticulostriate artery could be predicted with a higher MTT ratio (>1.26) and a lower CBF ratio (<0.76) on perfusion CT scans obtained within 24 hr after onset.

The recent development of new migration methods of micro-particles in liquids using various external fields is reviewed. The combination of a laser scattering force and a photothermal effect produced photothermal-conversion laser-photophoresis. A dielectric field generated in a planer or a capillary quadrupole electrode realized dielectrophoresis. Using a micrometer-scaled magnetic field gradient, the "Magnetophoretic velocimetry" of micro-particles was invented. Furthermore, the Lorentz force generated by combining an electric field and a magnetic field was utilized for electromagnetophoresis. These new methods were overlooked and the advantages in analytical use were discussed.

The cytokine thrombopoietin (TPO), the ligand for the hematopoietic receptor c-Mpl, acts as a primary regulator of megakaryocytopoiesis and platelet production. We have determined the crystal structure of the receptor-binding domain of human TPO (hTPO(163)) to a 2.5-A resolution by complexation with a neutralizing Fab fragment. The backbone structure of hTPO(163) has an antiparallel four-helix bundle fold. The neutralizing Fab mainly recognizes the C-D crossover loop containing the species invariant residue Q111. Titration calorimetric experiments show that hTPO(163) interacts with soluble c-Mpl containing the extracellular cytokine receptor homology domains with 1:2 stoichiometry with the binding constants of 3.3 x 10(9) M(-1) and 1.1 x 10(6) M(-1). The presence of the neutralizing Fab did not inhibit binding of hTPO(163) to soluble c-Mpl fragments, but the lower-affinity binding disappeared. Together with prior genetic data, these define the structure-function relationships in TPO and the activation scheme of c-Mpl.

The interfacial adsorption and dimerization behavior of tetraphenylporphinatomanganese(III)chroride (Mn(TPP)Cl) in the toluene/water two phase system was studied. The absorption spectra of the porphyrin complex in the organic phase were blue shifted with increasing of pH in the aqueous phase, suggesting the formation of the mu-oxo dimer. The interfacial species in these systems were examined by means of UV/vis external reflection (ER) spectrophotometry. ER spectral maxima were also blue shifted with the increase in pH, whereas the shape of the ER spectra in the basic conditions was different from that of the mu-oxo dimer. The observed results suggest that the hydroxide-coordinated monomer, Mn(TPP)OH, was generated from the Mn(TPP)(H2O)+ at the interface under the basic conditions and that was further converted to the mu-oxo dimer, which was soluble into the toluene phase.

A new monitoring system for bacterial communities involving dimethylarsinic acid (DMAA) decomposition was provided by combining the MPN (Most Probable Number) method and RFLP (restriction-fragment-length polymorphism analysis). The abundance of DMAA decomposing bacteria was estimated by the MPN method using a bacterial culture medium, which included DMAA as the sole carbon source, indicating bacterial cell densities of 1700 cells/ml in Lake Kahokugata and 330 cells/ml in Lake Kibagata. After isolating the dominant bacteria using agar plates, the isolates were classified into some genotype groups by RFLP analysis using 16S rDNA sequences. Classification of the RFLP analysis indicated that 14 isolates of Lake Kahokugata were classified into 6 types, which included 2 dominant types related to genus Pseudomonas, while 8 isolates of Lake Kibagata displayed 6 types including one or two isolates. Moreover, the RFLP types were unique for each lake, suggesting that DMAA decomposing bacteria were specific for the aquatic environment related to the arsenic cycle. The activities of DMAA decomposition mostly matched with the RFLP type category of the isolates. Accordingly, combining the MPN method with the RFLP analysis will play an important role in elucidating the distributions and dynamics of the DMAA-decomposing bacterial community.

Dielectrophoresis of single microbioparticles was measured in a planar quadrupole microelectrode (50 mum or 65 mum in working area radius) with a microscope. Carbon and polystyrene microparticles, yeast cells and DNA molecules (about 40 kbp) were adopted as a sample. Their dielectrophoretic mobilities were analysed quantitatively with their intrinsic and surface conductivity, their permittivities and their sizes as well as the conductivity and permittivity of aqueous media. Using the dielectrophoretic mobilities obtained with the planar quadrupole microelectrode, some instances of the separation performance between the microparticles were demonstrated with a fabricated capillary quadrupole microelectrode (82.5 mum in bore radius) under the field flow fractionation regime.

By using an improved apparatus for the observation of magnetophoresis, the magnetophoretic velocity of 2-fluorotoluene droplets including lauric acid was measured in aqueous dysprosium(III) solution. The magnetophoretic velocity of pure 2-fluorotoluene droplets was proportional to the square of the radius. On the other hand, the velocity of the organic droplets including lauric acid in the dysprosium(III) solution showed a deviation from the square radius relationship, more remarkably in smaller droplets than 2 microm in radius. These results indicated that the dysprosium(III)-laurate complex was formed at the liquid-liquid interface. This study is the first report on the detection of the interfacial complex by the magnetophoresis of the microdroplet.

Crystal structure of 2-(5-bromo-2-pyridylazo)-5-diethylaminophenolatochloropalladium(II) (PdLCl) has been determined by X-ray diffraction. A crystal of PdLCl was constructed by two structural isomers of PdLCl molecules, which were particularly different in the positions of the carbon atoms at the diethylamino group, having cis-like or trans-like conformation for the carbon atoms. The molecules of PdLCl in the crystal were warped by a steric hindrance of the diethylamino group.

Objective: We report the first application and usefulness of image transfer through a mobile telephone, which can be used between the hospital and another location., Methods: With this system, brain computed tomographic scans, magnetic resonance images, and angiograms obtained from more than 100 patients in the emergency department were transferred and diagnostically analyzed via a mobile phone. The mobile phone had a 110,000-pixel digital camera and a built-in thin-film transistor liquid crystal display. We reviewed the distribution of pathological characteristics on the transferred images and compared them with the diagnoses made using full-scale original film., Results: This system of transferring images was useful in all cases for correct early diagnosis and early treatment. The quality of magnetic resonance images received was better than that of computed tomographic scans. Hemorrhage provided better contrast than infarction for allowing easy identification. Cerebral angiography revealed small aneurysms if the target area was focused properly. Image quality was sufficient for interpretation despite the small dimensions of the monitor. Ease of operation and portability were both satisfactory. The mean time from commencement of image uptake to complete reception was 2 to 3 minutes., Conclusion: The mobile phone system is adequately useful for early diagnosis and initiation of treatment in emergent cases. This is attributable to its low cost and ease of handling for sending images to remote areas and between hospitals, despite the small dimensions of the monitor. Better-quality image transfer will be realized through an advanced mobile phone system based on the new International Mobile Telecommunication 2000 standards, which also will be useful in the development of telemedicine and telecare.

A simultaneous measurement technique for determining the migration velocity of a micrometer-sized particle in a capillary and the adsorption force to the inner surface of the capillary has been proposed. This technique is based on an electromagnetophoretic force being exerted on a micro-particle in an electrolyte solution, which is governed mainly by the electromagnetic buoyancy, when a homogeneous magnetic field is applied at a right angle to the electric current through the medium. By the electromagnetic buoyancy, micro-particles such as polystyrene, carbon and yeast were migrated perpendicular to the direction of the electric current and reached a fused-silica wall. A switching of the current direction could desorb the particle from the wall, and allowed to calculate the detaching force from the desorbing current. The migration velocity normalized to the size in the magnetic field of 10 T was increased in the order of yeast, carbon and polystyrene, while reflecting the decreasing order of the apparent conductivity of the particles. The desorption force could be measured up to 1 nN with a sensitivity of pN. The observed interaction forces of polystyrene and carbon were in the range of 250-600 pN with large deviations.

The dielectrophoretic (DEP) behavior of individual yeast cells (5-7 microm in diameter) in aqueous media was observed in a fabricated planar quadrupole microelectrode with a working area of 100 microm in diameter by an optical microscope. The yeast cells migrated in the radial direction in the working area. The DEP velocity of the cells increased as they approached the electrode. The DEP trajectory of the cells was analyzed with a theoretical equation derived previously, and the dielectrophoretic mobility was determined. The dielectrophoretic mobility was found to be affected by the viability of cells, the conductivity of the medium, and the binding of lectin protein (concanavalin A) to the cell surface. These DEP behaviors were analyzed based on the permittivities and conductivities of the cell interior and wall, and those of the medium.

An experimental system for magnetophoretic velocimetry, which could determine the volume magnetic susceptibility of a single particle dispersed in a liquid phase from a magnetophoretic velocity, has been developed. A micrometer-sized high-gradient magnetic field could be generated in a capillary by a pair of iron pole pieces in a superconducting magnet (10 T). The magnetophoretic behavior of a single particle in a capillary flow system was investigated under the inhomogeneous magnetic field. From the magnetophoretic velocity of a polystyrene latex particle dispersed in a MnCl2 aqueous solution, the product of the magnetic flux density and the gradient, B(dB/dx), was determined as a function of the position along the capillary. The maximum value of B(dB/dx) was 4.7 x 10(4) T2 m(-1), which was approximately 100 times higher than that obtained by two Nd-Fe-B permanent magnets (0.4 T). Organic droplets extracting manganese(II) with 2-thenoyltrifluoroacetone and tri-n-octylphosphine oxide from MnCl2 solution were used as test samples. The difference of the volume magnetic susceptibility between the droplet and the medium could be determined from the magnetophoretic velocity. This method allowed us to continuously measure a volume magnetic susceptibility of 10-6 level for a picoliter droplet and to determine manganese(II) in the single droplet at the attomole level.

The performance of a capillary magnetophoretic device was improved by enhancing the magnetic field gradient using a pair of small iron tips attached to the Nd-Fe-B magnets. The magnetophoretic intensity, B(dB/dx), was determined as a function of distance along the gap between the tips from the magnetophoretic velocity of a 3 microm polystyrene microparticle in 0.6 M manganese(II) chloride solution. The maximum intensity was increased 4.5 times by the attached iron pieces. The magnetophoresis of a single human blood cell in 0.1 M manganese(II) solution was studied by this method and its magnetic susceptibility was estimated. Magnetophoretic trapping of red blood cells was demonstrated under counter-current flow conditions in the capillary.

Thrombopoietin (TPO) is a cytokine which primarily stimulates megakaryocytopoiesis and thrombopoiesis. The functional domain of TPO (TPO(163)) consisting of the N-terminal 163 amino acids was prepared and crystallized. Since the crystallization of TPO(163) was unsuccessful using the standard screening methods, a Fab fragment derived from a neutralizing monoclonal antibody was used for crystallization. It was found that the TPO(163)-Fab complex crystallized reproducibly in 0.1 M potassium phosphate buffer pH 6.0 containing 20-25% polyethylene glycol 4000. Thin crystals (0.2 x 0.2 x 0.02 mm) grew in two space groups: P2(1), with unit-cell parameters a = 133.20, b = 46.71, c = 191.47 A, beta = 90.24 degrees, and C2, with unit-cell parameters a = 131.71, b = 46.48, c = 184.63 A, beta = 90.42 degrees. The results of a molecular-replacement analysis indicate that the Fab molecules interact with each other and provide a suitable interface for crystallization.

A technique of fractionation for microparticles was proposed that utilized a unique combination of a dielectrophoretic (DEP) field generated by a quadrupole electrode and a laminar flow in a capillary of 82.5 microm in radius. The fabricated capillary possessed four platinum wires in its inside wall as a quadrupole electrode. In a nonuniform electric field generated by the quadrupole electrode, microparticles, such as polystyrene and carbon, in water experienced DEP forces in the radial direction. When a sample solution was pumped in, an ideal laminar flow perpendicular to the DEP force was formed inside the capillary. The microparticles dynamically migrated by the DEP force across the laminar flow while they were carried by the flow. A theoretical model taking the DEP force and the laminar flow pattern into account predicted the elution profiles of the single microparticles quantitatively. The elution times of the microparticles depended on the dielectric properties and the sizes of the microparticles, as well as the voltage and frequency of the applied alternating current.