Pretreatment of donor islets with the Na(+) /Ca(2+) exchanger inhibitor improves the efficiency of islet transplantation.

Pancreatic islet transplantation is an attractive therapy for the treatment of insulin-dependent diabetes mellitus. However, the low efficiency of this procedure necessitating sequential transplantations of islets with the use of 2-3 donors for a single recipient, mainly due to the early loss of transplanted islets, hampers its clinical application. Previously, we have shown in mice that a large amount of HMGB1 is released from islets soon after their transplantation and that this triggers innate immune rejection with activation of DC, NKT cells and neutrophils to produce IFN-γ, ultimately leading to the early loss of transplanted islets. Thus, HMGB1 release plays an initial pivotal role in this process; however, its mechanism remains unclear. Here we demonstrate that release of HMGB1 from transplanted islets is due to hypoxic damage resulting from Ca(2+) influx into ß cells through the Na(+) /Ca(2+) exchanger (NCX). Moreover, the hypoxia-induced ß cell damage was prevented by pretreatment with an NCX-specific inhibitor prior to transplantation, resulting in protection and long-term survival of transplanted mouse and human islets when grafted into mice. These findings suggest a novel strategy with potentially great impact to improve the efficiency of islet transplantation in clinical settings by targeting donor islets rather than recipients.

Applications of magnetic and electromagnetic forces in micro-analytical systems.

Novel applications of magnetic fields in analytical chemistry have become a remarkable trend in the last two decades. Various magnetic forces have been employed for the migration, orientation, manipulation, and trapping of microparticles, and new analytical platforms for separating and detecting molecules have been proposed. Magnetic materials such as functional magnetic nanoparticles, magnetic nanocomposites, and specially designed magnetic solids and liquids have also been developed for analytical purposes. Numerous attractive applications of magnetic and electromagnetic forces on magnetic and non-magnetic materials have been studied, but fundamental studies to understand the working principles of magnetic forces have been challenging. These studies will form a new field of magneto-analytical science, which should be developed as an interdisciplinary field. In this review, essential pioneering works and recent attractive developments are presented.

Sequentially addressable dielectrophoretic array for high-throughput sorting of large-volume biological compartments.

Droplet microfluidics has become a powerful tool in precision medicine, green biotechnology, and cell therapy for single-cell analysis and selection by virtue of its ability to effectively confine cells. However, there remains a fundamental trade-off between droplet volume and sorting throughput, limiting the advantages of droplet microfluidics to small droplets (<10 pl) that are incompatible with long-term maintenance and growth of most cells. We present a sequentially addressable dielectrophoretic array (SADA) sorter to overcome this problem. The SADA sorter uses an on-chip array of electrodes activated and deactivated in a sequence synchronized to the speed and position of a passing target droplet to deliver an accumulated dielectrophoretic force and gently pull it in the direction of sorting in a high-speed flow. We use it to demonstrate large-droplet sorting with ~20-fold higher throughputs than conventional techniques and apply it to long-term single-cell analysis of Saccharomyces cerevisiae based on their growth rate.

Less invasive technique for EC-IC bypass.

BACKGROUND: We introduce less invasive technique for superficial temporal to middle cerebral artery (STA-MCA) anastomosis, and also described an innovative technique to preoperatively identify the recipient artery using three dimensional CT angiography (3D CTA). Objective. In a period of 28 months between January 2004 and April 2006, 39 EC-IC bypass were performed for hemodynamic compromised patients (including 9 patients with Moyamoya disease) using less invasive technique. METHODS: Operative technique is as follows: 1) A parietal or frontal branch of STA and cortical arteries could be identified on the original images of 3D CTA. The most suitable segment of both the artery provided as donor and recipient arteries for EC-IC bypass. The distance between the afore-mentioned segment of donor artery (STA) and the superior border of the helix were calculated. 2) A 5 cm linear skin incision on the STA, the center of which was the point measured on preoperative 3D CTA, was made. The temporal muscle was divided in the same fashion, and a 3 cm small craniotomy was made. The recipient artery could be identified on the center of the craniotomy. End-to-side anastomosis was performed in the usual way. RESULTS: Operation times were 115-172 min (mean 154 min) and intraoperative blood loss was 20-60 ml (mean 38 ml). All bypasses were patent on the post-operative 3D CTA. CONCLUSIONS: This technique for EC-IC bypass was less invasive and cosmetically excellent. 3D CTA provides useful information for planning of the less invasive EC-IC bypass.

Preparation of soluble recombinant T cell receptor alpha chain by using a calmodulin fusion expression system.

We have isolated a full length T cell receptor alpha chain (TCR alpha) cDNA derived from a bee venom phospholipase A2-specific mouse suppressor T cell hybridoma. A bacterial fusion expression system was constructed using rat calmodulin as a fusion partner for production of soluble TCR alpha. In this system, calmodulin-TCR alpha fusion protein was expressed at a high level in the soluble fraction of bacterial cell lysate, and could be purified by binding of calmodulin portion of the protein to phenyl-Sepharose. Using this system, fusion proteins containing a TCR alpha peptide corresponding to the complete extracellular region, V alpha-J alpha region or C alpha extracellular region were isolated. TCR alpha peptides were then released from the fusion proteins by digestion with thrombin which recognizes a linker sequence between calmodulin portion and TCR alpha segment. Polyclonal antibodies against constant region of TCR alpha chain (C alpha) were obtained by immunization of rabbits with the recombinant C alpha peptide. ELISA for TCR protein was established by using the polyclonal antibodies and the monoclonal antibody specific for C alpha region.

Effect of nitric oxide on the recovery of the hypofunctional periodontal ligament.

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.

Effect of stirring on the ion-association extraction of copper and zinc 4,7-diphenyl-1,10-phenanthroline complexes.

The effect of high-speed stirring on the extraction of 4,7-diphenyl-1,10-phenanthroline (L) complexes into chloroform was examined for the copper(II)/perchlorate, copper(II)/chloride and zinc(II)/perchlorate systems. A significant reversible decrease in the organic phase concentration in all the systems was caused by stirring, but most evidently in the copper/perchlorate system, where the effect depended on the stirring rate, concentration of L, and metal-ion concentration. The phenomena are primarily explained in terms of an interfacial adsorption of the extracted complex, according to the Langmuir adsorption isotherm.

Interfacial reaction in the synergistic extraction rate of Ni(II) with dithizone and 1,10-phenanthroline.

The kinetic synergistic effect of 1,10-phenanthroline (phen) on the extraction rate of Ni(II) with dithizone (HDz) into chloroform was studied by means of a high-speed stirring method combined with photodiode-array spectrophotometry. The initial extraction rate of the adduct complex NiDz(2)phen depended upon the concentrations of both HDz and phen, suggesting the formation of NiDzphen(+) as the rate-controlling step. When [HDz] < [phen], the initial extraction of NiDz(2)phen competed with the formation of an intermediate complex, which was adsorbed at the interface and assigned most probably to NiDzphen(+)(2). The intermediate complex was gradually converted to NiDz(2)phen at a later stage of the extraction. The rate constants for the formation and consumption of the intermediate were determined, and the kinetic mechanism in the synergistic extraction was discussed.

Direct spectrophotometric measurements of acid-catalyzed complexation of palladium(II) with 2-(5-bromo-2-pyridylazo)-5-diethylaminophenol at the heptane/water interface by a centrifugal liquid membrane method.

The centrifugal liquid membrane (CLM) method, designed for the rapid sample injection, was applied to the kinetic study of the complexation of palladium(II) with 2-(5-bromo-2-pyridylazo)-5-diethyaminophenol (5-Br-PADAP) in the heptane/water system. The formation rates of Pd(II)-5-Br-PADAP complex, which existed only at the heptane/water interface, could be directly measured by the CLM method combined with transmission spectrophotometry. We found that the formation rates of Pd(II)-5-Br-PADAP complex were accelerated by the protonation of 5-Br-PADAP at the diethylamino-group that did not coordinate to Pd(II) ion and that the rate constant for the reaction of protonated 5-Br-PADAP at the interface was close to that in the aqueous phase. The present study demonstrated that the CLM method was easily applicable for the measurements of relatively fast interfacial reactions.

Treatment of cerebral aneurysms: surgical clipping and coil embolization.

SUMMARY: The present series provides a balanced overview of the treatment of aneurysms in surgical clipping and coil embolization. Between January 2004 and March 2006, 76 consecutive patients with cerebral aneurysms underwent endovascular embolization and/or surgical clipping. Of these, 42 patients suffered an aneurysmal subarachnoid hemorrhage (SAH), while the remaining 34 patients had nonruptured cerebral aneurysms. Of the 23 surgically treated patients, 17 (73.9%) achieved a favorable outcome. Of the 19 patients who underwent endovascular embolization, 12 (63.2%) achieved a favorable outcome. Three patients (15.8%) who underwent endovascular embolization needed to undergo re-treatments, while no re-treatment was needed in the surgically treated patients. Of the 34 nonruptured aneurysms, 12 (35.3%) were treated using surgical clipping, while 22 (64.7%) underwent endovascular embolization. The complication rates of the two treatment modalities demonstrated no significant difference. A combined microsurgical-endovascular team approach is thus considered to provide the most effective means to achieve favorable outcomes for patients with cerebral aneurysms.

Surface-enhanced Raman spectroscopy of dodecanethiol-bound silver nanoparticles at the liquid/liquid 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.

Two-phase stopped-flow measurement of the protonation of tetraphenylporphyrin at the liquid-liquid interface.

The formation rate of the protonated form of tetraphenylporphyrin (TPP) in a dispersed two-phase system composed of dodecane and aqueous trichloroacetic acid (TCA) was studied by means of a stopped-flow method. The protonation reaction took place at the liquid-liquid interface, and the diprotonated TPP (H(2)TPP(2+)) formed was adsorbed there. In order to determine the rate-determining process, changes in absorbance at the absorption maximum wavelengths of TPP and H(2)TPP(2+) were analyzed. The obtained rate constant for the decrease of TPP in the organic phase, 21 ± 2 s(-1), was in agreement with that for the increase of diprotonated TPP at the interface, 20 ± 3 s(-1). The observed rate constants did not show any dependence on concentrations of both TPP and the acid. The experimental results suggested the rate-determining step to be the molecular diffusion process of TPP in the stagnant layer in the organic phase side at the liquid-liquid interface, and the thickness of the stagnant layer was estimated as 1.4 × 10(-4) cm.

Dielectrophoresis of microbioparticles in water with planar and capillary quadrupole electrodes.

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.

Magnetophoretic velocimetry of manganese(II) in a single emulsion droplet at the femtomole level.

We developed a new experimental technique named magnetophoretic velocimetry to determine a small amount of paramagnetic species in a single microdroplet. The magnetophoretic velocity of an aqueous droplet containing paramagnetic metal ion dispersed in an organic medium could response to a very small amount of the metal ion under an inhomogeneous magnetic field. The paramagnetic droplet (2 approximately 8 microm diam) used as a test sample in this study was the aqueous droplet of manganese(II) chloride dispersed in ethylbenzoate whose density was nearly equal to water. A pair of small Nd-Fe-B magnets placed with a gap of 400 microm generated an inhomogeneous magnetic field between the edges, at which the product of the magnetic flux density and the gradient, B(dB/dx), was as high as 410 T2 m(-1). When a silica capillary containing the emulsion was inserted into the gap between the magnets, the magnetophoretic migration of the droplets was observed with a video microscope. The magnetophoretic velocity divided by the squared radius of the droplet was proportional to the MnCl2 concentration in the droplet, as predicted by a theoretical calculation. The estimated detection limit in this simple method was lower than 10(-16) mol for manganese(II).

Nitric oxide synthase expression is increased by occlusal force in rat periodontal ligament.

OBJECTIVES: The goal of this study was to investigate the role of nitric oxide synthase (NOS) in occlusal force-induced signal transduction in rat periodontal ligament (PDL). DESIGN: Rats were fitted with a bite plate and a metal cap to the maxillary and mandibular incisors, respectively, to eliminate the occlusal forces on rat molars. One group was sacrificed at 7 days (exclusion group), while the remaining rats had their appliances removed to reestablish molar occlusal contact (reload group) and were sacrificed 7 days thereafter. Another group of rats (normal group) were left completely untreated. Frozen cross sections of the upper first molars were stained with NADPH-diaphorase to quantify NOS activity. The distal sides of the disto-palatal roots of the upper first molars were examined, and the number and the area of stained cells in the PDL were measured. RESULTS: In the normal group, NOS expression was detected in blood vessels, monocyte-macrophages, fibroblastic cells and osteoclastic cells. NOS expression was lower in the exclusion group when compared with the normal group or the reload group (p < 0.05), and the exclusion group exhibited occluded blood vessels and a narrowing of PDL. In contrast, in the reload group the PDL and blood vessel structure had recovered and NOS expression was increased to the level of the controls. CONCLUSION: Occlusal force resulted in increased NOS expression. NO may mediate changes in PDL structure in response to occlusal force.

Flow fractionation of microparticles under a dielectrophoretic field in a quadrupole electrode capillary.

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.

Association of the "major histocompatibility complex subregion" I-J determinant with bioactive glycosylation-inhibiting factor.

Murine suppressor T-cell hybridoma cells (231F1) secrete not only bioactive glycosylation-inhibiting factor (GIF) but also an inactive peptide comparable to bioactive GIF peptide in its molecular size and reactivity with anti-GIF; the amino acid sequence of the inactive peptide is identical to that of the bioactive homologue. The inactive GIF peptide in culture supernatant of both the 231F1 cells and a stable transfectant of human GIF cDNA in the murine suppressor T hybridoma selectively bound to Affi-Gel 10, whereas bioactive GIF peptides from the same sources failed to bind to the gel. The inactive cytosolic human GIF from the stable transfectant and Escherichia coli-derived recombinant human GIF also had affinity for Affi-Gel 10. Both the bioactive murine GIF peptide from the suppressor T hybridoma and bioactive recombinant human GIF from the stable transfectant bound to the anti-I-J monoclonal antibody H6 coupled to Affi-Gel. However, bioactive hGIF produced by a stable transfectant of human GIF cDNA in BMT10 cells failed to be retained in H6-coupled Affi-Gel. These results indicate that the I-J specificity is determined by the cell source of the GIF peptide and that the I-J determinant recognized by monoclonal antibody H6 does not represent a part of the primary amino acid sequence of GIF. It appears that the epitope is generated by a posttranslational modification of the peptide.

C-terminal identification of AD74, a proteolytic product of Enterococcus faecalis aggregation substance: application of liquid chromatography/mass spectrometry.

Sexual aggregation involved in conjugative transfer of Enterococcus faecalis plasmid pAD1 is enhanced by the sex pheromone cAD1, which is excreted from recipient cells. A membrane-anchored 137 kDa protein is a pAD1-encoded aggregation substance designated asal, which is responsible for cell-cell contact and leads to the aggregation of cells. An AD74 protein is a proteolytic product corresponding to the N-terminal half of asal. The C-terminal of AD74 was identified as lysine at position 510 (K-510) by liquid chromatography/mass spectrometry (LC/MS): it indicates that asal is cleaved specifically between K-510 and G-511.

Conversion of inactive glycosylation inhibiting factor to bioactive derivatives by modification of a SH group.

Escherichia coli-derived recombinant human glycosylation inhibiting factor (rhGIF) contains three cysteine residues (Cys-57, -60, and -81). All SH groups in the cysteine residues are free, and the GIF molecule had no biologic activity. Carboxymethylation of the SH group of Cys-60 in the molecule resulted in the generation of bioactivity, although the activity of the carboxymethylated GIF was 10- to 20-fold less than that of suppressor T cell (Ts)-derived GIF. However, treatment of the inactive rhGIF with ethylmercurithiosalicylate or 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) resulted in the generation of derivatives whose bioactivity was comparable to that of the Ts-derived bioactive GIF. The activity of these derivatives was lost by treatment with DTT. Isolation and chemical analysis of the DTNB-treated GIF derivative revealed that binding the 5-thio-2-nitrobenzoic acid group with Cys-60 was responsible for the generation of the highly bioactive derivative. Inactive cytosolic GIF from mammalian cells could also be converted to bioactive derivative by treatment with the SH reagent, while Ts-derived bioactive GIF was inactivated by DTT. These results, together with an x-ray crystal structure of GIF molecules, strongly suggest that the generation of bioactivity of GIF in Ts cells is due to posttranslational modifications that result in conformational changes in the molecule.