Biomedical subjects

Eiry Kobatake

Publications and source records attributed to Eiry Kobatake.

31 records · Page 2Linked to original sources

PubMed2003

Design and construction of glutamine binding proteins with a self-adhering capability to unmodified hydrophobic surfaces as reagentless fluorescence sensing devices.

The chemically and genetically remodeling of proteins with ligand binding specificities can be utilized to synthesize various protein-based microsensors for detecting single biomolecules. Here, we describe the construction and characterization of fluorophore-labeled glutamine binding proteins (QBP) and derivatives coupled to the independently designed hydrophobic polypeptide (E12) that can adhere onto solid surfaces via hydrophobic interactions. The single cysteine mutant (N160C QBP) modified with the three environmentally sensitive fluorescent dyes (IAANS, acrylodan, and IANBD ester) showed increased changes in fluorescence intensity induced by glutamine binding. The use of these conjugates as reagentless fluorescence sensors enables us to determine the glutamine concentrations (0.1-50 microM) in homogeneous solution. The fusion of N160C QBP with E12, (Gly4-Ser)n spacers (GSn), and IANBD resulted in the novel fluorescence sensing elements having an adhering capability to hydrophobic surfaces of unmodified microplates. In ELISA and fluorescence experiments for the microplates treated with a series of the conjugates, IANBD-labeled N160C QBP-GS1-E12 displayed the best reproducibility in adhesion onto the hydrophobic surfaces and the precise correlation between fluorescence changes and glutamine concentrations. The performance of the biosensor-attached microplate for glutamine titrations demonstrated that the hydrophobic interaction of E12 with solid surfaces is useful for effective immobilization of proteins that need specific conformational movements in recognizing particular biomolecules. Therefore, the technique using E12 as a surface-linking domain for protein adhesion onto unmodified substrates could be applied effectively to prepare microplates/arrays for a wide variety of high-throughput assays on chemical and biological samples.

Biosensing Techniques↗

PubMed2003

Intracellular delivery of antibodies using TAT fusion protein A.

Internalization of antibodies into mammalian cells is a useful method for analyzing and regulating cellular function. In this study, we developed a novel method for the delivery of antibodies into cells using the TAT-fused protein. This fusion protein consists of two functional domains, the protein transduction domain of HIV-1 TAT and the B domain of staphylococcal protein A (SpA), which has an ability to bind to the IgG. The TAT-SpA fusion protein was mixed with fluorescence-labeled rabbit IgG and added to cells. The internalization of antibody was analyzed using confocal microscopy and flow cytometry in living cells. As a result, fluorescence-labeled IgG with the TAT-SpA fusion protein was observed intracellularly. Flow cytometry results demonstrated time course and dose dependence relationships of antibody internalization. These results suggest that the TAT-SpA fusion protein can be a useful reagent for the delivery of antibody into cells.

3T3-L1 Cells↗

PubMed2003

Development of immune cellular biosensing system for assessing chemicals on inducible nitric oxide synthase signaling activator.

An immune cellular biosensing system has been constructed to assess immunomodulating effects of chemicals. Production of nitric oxide in the immune cellular biosensing system was used as readout of an immune cellular response for assessing the immunomodulating effects of chemicals. The macrophage-like cell line RAW264.7, which has signaling pathways of inducible nitric oxide synthase, was employed in the cellular biosensing system. The immune cellular biosensing system consisted of a Pt counter electrode, an Ag/AgCl reference electrode, and a gold electrode onto which a polyion complex layer was coated to allow adherence of the RAW264.7 cells. As the results of evaluating effects of a polyion complex layer on cell viabilities by using WST-8 assay, the polyion complex layer did not affect RAW264.7 cells. The polyion-coated gold electrode could measure NO without the drawback of electrochemical interference that occurs with differential pulse voltammetry. The detection limit of the immune cellular biosensing system was 4.2 nM released NO as measured by double potential step chronoamperometry. The potent immune activating abilities of lipopolysaccharide and interferon-gamma could be assessed by the cellular biosensing system; NO release from cells was detected within 600 ms.

Adjuvants, Immunologic↗

PubMed2003

A functional study on polymorphism of the ATP-binding cassette transporter ABCG2: critical role of arginine-482 in methotrexate transport.

Overexpression of the ATP-binding cassette transporter ABCG2 reportedly causes multidrug resistance, whereas altered drug-resistance profiles and substrate specificity are implicated for certain variant forms of ABCG2. At least three variant forms of ABCG2 have been hitherto documented on the basis of their amino acid moieties (i.e., arginine, glycine and threonine) at position 482. In the present study we have generated those ABCG2 variants by site-directed mutagenesis and expressed them in HEK-293 cells. Exogenous expression of the Arg(482), Gly(482), and Thr(482) variant forms of ABCG2 conferred HEK-293 cell resistance toward mitoxantrone 15-, 47- and 54-fold, respectively, as compared with mock-transfected HEK-293 cells. The transport activity of those variants was examined by using plasma-membrane vesicles prepared from ABCG2-overexpressing HEK-293 cells. [Arg(482)]ABCG2 transports [(3)H]methotrexate in an ATP-dependent manner; however, no transport activity was observed with the other variants (Gly(482) and Thr(482)). Transport of methotrexate by [Arg(482)]ABCG2 was significantly inhibited by mitoxantrone, doxorubicin and rhodamine 123, but not by S -octylglutathione. Furthermore, ABCG2 was found to exist in the plasma membrane as a homodimer bound via cysteinyl disulphide bond(s). Treatment with mercaptoethanol decreased its apparent molecular mass from 140 to 70 kDa. Nevertheless, ATP-dependent transport of methotrexate by [Arg(482)]ABCG2 was little affected by such mercaptoethanol treatment. It is concluded that Arg(482) is a critical amino acid moiety in the substrate specificity and transport of ABCG2 for certain drugs, such as methotrexate.

ATP Binding Cassette Transporter, Subfamily G, Mem↗

PubMed2003

A biosensing system based on extracellular potential recording of ligand-gated ion channel function overexpressed in insect cells.

We have used outer cell potential measurement to record agonist-dependent cellular responses in cells engineered to express ligand-gated ion channels and grown on a microelectrode surface. Application of glutamate, a natural agonist, induced a complex and robust potentiometric response in cells expressing homomeric GluR-D glutamate receptor, but not in nonexpressing control cells. The response consisted of an initial decrease in outer potential followed by a transient increase and was not obtained for other amino acids devoid of agonist activity at glutamate receptors. Furthermore, the pharmacological agonist of the GluR-D receptor, kainate, also produced the potentiometric response whereas 6-cyano-7-nitroquinoxaline-2,3-dione, a competitive antagonist, was not active in itself but attenuated the responses to glutamate. The time course of the measured changes was slow, which may be partially due to the ligand being applied by free diffusion but may also reflect a contribution by secondary changes in the behavior of the cells. This novel approach should be applicable to other ligand-gated ion channels and holds promise as a cell-based biosensor for high-throughput drug screening and other applications.

Animals↗

PubMed2003

Induction of neural differentiation by electrically stimulated gene expression of NeuroD2.

Regulation of cell differentiation is an important assignment for cellular engineering. One of the techniques for regulation is gene transfection into undifferentiated cells. Transient expression of NeuroD2, one of neural bHLH transcription factors, converted mouse N1E-115 neuroblastoma cells into differentiated neurons. The regulation of neural bHLH expression should be a novel strategy for cell differentiation. In this study, we tried to regulate neural differentiation by NeuroD2 gene inserted under the control of heat shock protein-70 (HSP) promoter, which can be activated by electrical stimulation. Mouse neuroblastoma cell line, N1E-115, was stably transfected with expression vector containing mouse NeuroD2 cDNA under HSP promoter. Transfected cells were cultured on the electrode surface and applied electrical stimulation. After stimulation, NeuroD2 expression was induced, and transfected cells adopt a neuronal morphology at 3 days after stimulation. These results suggest that neural differentiation can be induced by electrically stimulated gene expression of NeuroD2.

Animals↗

PubMed2003

Cellular biosensing system for assessing immunomodulating effects on the inducible nitric oxide synthase (iNOS) cascade.

A cellular biosensing system has been constructed to assess the biological safety/toxicity of chemicals. Detection of nitric oxide (NO) by the cellular biosensing system was used as a readout for assessing the immunomodulating effects of various chemicals, because some are known to induce NO synthase (iNOS) activity thereby increasing NO production. The macrophage-like cell line, RAW264.7, was cultured on the electrode coated with a polyion complex layer. The potent immune activating abilities of lipopolysaccharide could be verified by the cellular biosensing system: NO release from cells was detected within 600 ms by double potential step chronoamperometry.

Adjuvants, Immunologic↗

PubMed2003

The secretory response through electric stimulation of differentiated PC12 rat pheochromocytoma cells transfected with neuropeptide Y fused with enhanced green fluorescent protein.

Exocytosis in pheochromocytoma cells was induced by electric stimulation. To chase the movement of vesicles by electric stimulation, dense-core secretory vesicles were visualized by expression of the fusion protein between neuropeptide Y and enhanced green fluorescent protein (EGFP) in these differentiated PC12 rat pheochromocytoma cells. When the cells were stimulated with constant voltage potential at -300 mV, the movement of dense-core secretory vesicles could be regulated.

Animals↗

PubMed2002

Atomic force microscopy identification of transcription factor NFkappaB bound to streptavidin-pin-holding DNA probe.

A novel method for identifying DNA-binding proteins from image analysis using AFM was developed. Here, transcription factor NFkappaB, which a well-studied example of transcription activator proteins, was used as a target protein. 5'-biotinlynated double-stranded DNA probe was labeled site specifically through high affinity with streptavidin. When the biotinylated DNA fragments were incubated with the streptavidin at a 1:2 molar ratio of DNA:streptavidin, the overall efficiency of labeling was over 90%. The double-stranded DNA probes were immobilized on a mica surface by the adsorption of streptavidin that attached to the 5'-end of DNA and applied for selection of the target protein NFkappaB in solution and then AFM was used to image the DNA probe-NFkappaB complexes. The length of the distance between 5'-labeled streptavidin and NFkappaB bound on DNA probes from AFM images is 0.64, the normalized position of the NFkappaB binding site, and this result is in close agreement with the expected 299 and 167bp values.

Base Sequence↗

PubMed2002

Non-destructive monitoring of rpoS promoter activity as stress marker for evaluating cellular physiological status.

To monitor the extent of cellular physiological stress, the activity of the rpoS promoter was evaluated as a marker of the stress pathway. A reporter plasmid was constructed by inserting the GFPuv gene under the rpoS promoter and used to transform Escherichia coli cells. The fluorescence of the GFPuv protein was measured in intact cells in a non-destructive manner. The physiological status of the cells could be conveniently monitored using the rpoS-GFPuv reporter gene with respect to the cellular growth phase and to elevated ethanol and NaCl concentrations as two examples of environmental stress factors. Comparison of the response of different E. coli strains demonstrated an essential role of the relA gene in the induction of the rpoS-GFPuv reporter gene.

Alcohols↗

PubMed2002

Ribosome display for selection of active dihydrofolate reductase mutants using immobilized methotrexate on agarose beads.

Ribosome display was applied to the selection of an enzyme. As a model, we selected and amplified the dihydrofolate reductase (DHFR) gene by ribosome display utilizing a wheat germ cell-free protein synthesis system based on binding affinity to its substrate analog, methotrexate, immobilized on agarose beads. After three rounds of selection, the DHFR gene could be effectively selected and preferentially amplified from a small proportion in a mixture also containing competitive genes. Active enzymes were expressed and amplified and by sequence analysis, four mutants of DHFR were identified. These mutants showed as much activity as the wild-type enzyme.

Cell-Free System↗

PubMed2002

Direct atomic force microscopy visualization of integration host factor-induced DNA bending structure of the promoter regulatory region on the Pseudomonas TOL plasmid.

Atomic force microscopy (AFM) was used to analyze DNA bending induced by integration host factor (IHF). The direct AFM visualization of IHF-DNA complexes on the OP1 promoter regulatory regions on the Pseudomonas TOL plasmid showed that there was no intrinsic DNA bend in the OP1 promoter region, but a sharp DNA bend was induced by binding of IHF to the region between the upstream regulatory sequence and the promoter sequence. The DNA bending angles were distributed with a mean bend angle of 123 degrees. The IHF-DNA complexes were shown to bend at the IHF binding site giving rise to an asymmetric structure. These results provide direct evidence that IHF is required functionally for activation of OP1 transcription and support the DNA-loop model that the sharp DNA bend induced by binding of IHF facilitates the contact between RNA polymerase bound by the promoter sequence and XylR protein attached to the upstream sequence in the OP1 promoter.

Bacterial Proteins↗

PubMed2002

Cellular biosensing system for discovery of protein synthesis inhibitors with an electrochemical phosphate modulator to regulate the acid phosphatase gene expression of Saccharomyces cerevisiae.

A cellular biosensing system for screening protein synthesis inhibitors has been developed by linking an electrochemical phosphate modulator and matrix-immobilized yeast cells with an optical sensing device. To screen the protein synthesis inhibitors, yeast phosphatase gene regulating system has been employed by linking an electrochemical phosphate modulator. Since the yeast phosphatase gene coding gammaAPase is expressed, when the phosphate concentration in solution is lowered below the threshold, the gammaAPase production is triggered by lowering the phosphate concentration with the electrochemical phosphate modulator, and monitored continuously with the photometric device. The electrochemical phosphate modulator was assembled with matrix-immobilized yeast cells. The module could insert to ordinal cuvette to monitor the induced gammaAPase activity in an ordinal photometer. Using the system, induction profile of protein synthesis was easily observed and was affected remarkably by various protein synthesis inhibitors. This seems promising that the system can be applied for first screening process of de novo protein inhibitors. The cellular biosensing system seems promising in screening protein synthesis inhibitors.

Acid Phosphatase↗