Concentrations of erythromycin and azithromycin in mature Chinook salmon Oncorhynchus tshawytscha after intraperitoneal injection, and in their progeny

A single dose (40 mg kg(-1)) of erythromycin or azithromycin dihydrate was injected intraperitoneally into maturing female fall Chinook salmon 12 to 32 d before spawning to observe the distribution, retention and clearance of the drugs in plasma, kidney, coelomic fluid and egg vitellin, and their persistence in alevins derived from these fish. Salmon administered prophylactic dosages of erythromycin as subadults were also included to investigate potential interactive effects of oral and injected treatments on reproductive performance and antibiotic clearance. Erythromycin was rapidly cleared from plasma and coelomic fluid, but was detected in the kidney (3.52 to 12.40 microg g(-1)) and egg vitellin (5.32 to 8.87 microg ml(-1)) of all fish at spawning. High, stable concentrations of azithromycin were detected in plasma (14.66 to 20.33 microg ml(-1)), kidney (43.16 to 59.96 microg g(-1)), coelomic fluid (2.52 to 5.50 microg ml(-1)) and egg vitellin (12.65 to 23.51 microg ml(-1)). Oral administration of erythromycin to subadult salmon did not significantly affect tissue concentrations of either erythromycin or azithromycin administered by prespawning injection. Reductions in the percentage of eggs that yielded live embryos at the eyed stage of development occurred among eggs derived from females that had received orally administered erythromycin as subadults. Erythromycin was not detected in unfed fry derived from adults injected with the drug prespawning, but azithromycin was present for more than 2 mo after the onset of exogenous feeding.     

Manipulation of microparticles for construction of array patterns by negative dielectrophoresis using multilayered array and grid electrodes

In this study, a useful method was developed to fabricate array patterns of microparticles not on electrode surfaces, but on arbitrary surfaces, using negative‐dielectrophoresis (n‐DEP). First, electrodes were designed and electric field simulations were performed to manipulate microparticles toward target areas. Based on the simulation results, multilayered array and grid (MLAG) electrodes, consisting of array electrodes surrounded by insulated regions and a grid electrode, were fabricated for the formation of localized, non‐uniform electric fields. The MLAG electrode was mounted to a target substrate in a face‐to‐face configuration with a spacer. When an AC voltage (4.60 V_rms and 1 MHz) was applied to the MLAG electrode, array patterns of 6 and 20 µm diameter microparticles were rapidly fabricated on the target substrate with ease. The results suggest that MLAG electrodes can be widely applied for the fabrication of biochips including cell arrays. Biotechnol. Bioeng. 2009; 104: 709–718 © 2009 Wiley Periodicals, Inc.     

A new electrochemical assay method for gene expression using HeLa cells with a secreted alkaline phosphatase (SEAP) reporter system

A new electrochemical assay for the detection of secreted alkaline phosphatase (SEAP) from transfectant HeLa cells is proposed using a microarray device and scanning electrochemical microscopy (SECM). The assay consists of two steps: the first is the incubation of a transfected cell in a microarray culture device covered with a substrate modified with anti-SEAP under physiological conditions without any additives. The array device consists of a 4 × 4 array of microwells having a size of 100 μm × 100 μm (diameter × depth). The second step is SECM measurement of secreted SEAP at the antibody-immobilized substrate. This assay ensures accuracy and intactness because the undesired influence of endogeneous ALP is eliminated and the transfected cells are incubated in a culture device under suitable conditions. We successfully detected the expression of SEAP from intact cells at the single-cell level using this assay. The system is useful as a cell-based gene-expression assay.     

Tacrolimus Inhibits the Revascularization of Isolated Pancreatic Islets

Aims

Immunosuppressive drugs could be crucial factors for a poor outcome after islet allotransplantation. Unlike rapamycin, the effects of tacrolimus, the current standard immunosuppressant used in islet transplantation, on graft revascularization remain unclear. We examined the effects of tacrolimus on islet revascularization using a highly sensitive imaging system, and analyzed the gene expression in transplanted islets by introducing laser microdissection techniques.

Methods

Islets isolated from C57BL/6-Tg (CAG-EGFP) mice were transplanted into the nonmetallic dorsal skinfold chamber on the recipients. Balb/c athymic mice were used as recipients and were divided into two groups: including a control group (n = 9) and tacrolimus-treated group (n = 7). The changes in the newly-formed vessels surrounding the islet grafts were imaged and semi-quantified using multi-photon laser-scanning microscopy and a Volocity system. Gene expression in transplanted islets was analyzed by the BioMark dynamic system.

Results

The revascularization process was completed within 14 days after pancreatic islet transplantation at subcutaneous sites. The newly-formed vascular volume surrounding the transplanted islets in the tacrolimus-treated group was significantly less than that in the control group (p<0.05). Although the expression of Vegfa (p<0.05) and Ccnd1 (p<0.05) was significantly upregulated in the tacrolimus-treated group compared with that of the control group, no differences were observed between the groups in terms of other types of gene expression.

Conclusions

The present study demonstrates that tacrolimus inhibits the revascularization of isolated pancreatic islets without affecting the characteristics of the transplanted grafts. Further refinements of this immunosuppressive regimen, especially regarding the revascularization of islet grafts, could improve the outcome of islet allotransplantation.     

A microvoltammetric study of permeation of ferrocene derivatives through a planer bilayer lipid membrane

Permeability of ferrocene derivatives through a planer bilayer lipid membrane (BLM) was examined by an electrochemical method using microelectrodes. Location of the microelectrode tip inside the unstirred layer enables the detection of electroactive substances permeating the membrane without unstirred layer perturbation.     

Detection of pesticide residues using an immunodevice based on negative dielectrophoresis

The detection of atrazine using a novel optical immunosensing technique based on negative dielectrophoresis (n-DEP) in microfluidic channels is described. Atrazine is a toxic triazine herbicide within the most frequently used. Polystyrene microparticles (6 microm diameters) modified with bovine serum albumin conjugated with atrazine (atrazine-BSA) were manipulated and captured when subjected to intense n-DEP electric fields. Specifically, particles were trapped when AC voltages with amplitudes of 10 V(peak) and frequencies over 1 MHz were applied to the electrodes. The immunological reaction occurring on the particles for detecting atrazine is based on an indirect competitive assay using a secondary anti-mouse immunogloburin G (IgG) antibody labeled with fluorescein isothiocyanate. The microfluidic device, with three-dimensional microelectrodes, was fabricated comprising two caged areas, allowing two simultaneous measurements inside the same microfluidic channel. The performance of this n-DEP immunosensing technique was evaluated using wine samples. The immunodevice showed a limit of detection for atrazine in buffer samples of 0.11 microgL(-1) and in pre-treated wine samples of 6.8 microg L(-1); these detection limits are lower than the maximum residue level (MRL) established by the European Community for residues of this herbicide in wine (50 microg L(-1)). This methodology offers great promise for rapid, simple, cost effective, and on-site analysis of biological, foods and beverages, and environmental samples.     

Dendritic cells undergo rapid apoptosis in vitro during antigen-specific interaction with CD4+ T cells

The terminal fate of dendritic cells (DC) remains relatively uncertain. In this study, we tested the hypothesis that DC undergo apoptosis after Ag-specific interaction with T cells. When splenic DC isolated from BALB/c mice were cocultured with HDK-1 T cells (a keyhole limpet hemocyanin (KLH)-specific CD4+ Th1 clone) in the presence of KLH, they showed conspicuous cell death as measured by propidium iodide (PI) uptake and chromatin condensation, whereas they remained relatively intact when incubated with either T cells or KLH alone. Likewise, the long term DC line XS52, which was established from BALB/c mouse epidermis, also died rapidly (within 2 h), and they exhibited characteristic DNA laddering when cocultured with HDK-1 T cells in the presence of KLH. RT-PCR and FACS analyses revealed the expression of CD95 (Fas) by XS52 DC and of CD95 ligand (CD95L) (Fas ligand) by activated HDK-1 T cells, suggesting a functional role for these molecules. In fact, anti-CD95L mAb inhibited partially (50%) T cell-mediated XS52 cell death, and coupling of surface CD95 with anti-CD95 mAb triggered significant XS52 cell death, but only in the presence of cycloheximide. Thus, ligation of CD95 (on DC) with CD95L (on T cells) is one, but not the only, mechanism by which T cells induce DC death. Finally, DC isolated from the CD95-deficient mice were found to be significantly more efficient than DC from control mice in their capacity to induce delayed type hypersensitivity responses in vivo. We propose that T cell-induced DC apoptosis serves as a unique down-regulatory mechanism that prevents the interminable activation of T cells by Ag-bearing DC.