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Biomedical subjects

Eiry Kobatake

Publications and source records attributed to Eiry Kobatake.

At least 19 recordsLinked to original sources
PubMed2006

Delivery of antibody-captured proteins into living cells using PTD-fused protein A.

Protein transduction domain (PTD)-mediated protein delivery into animal cells is a useful technique for regulating cellular functions. Proteins captured by antibodies were delivered into living cells using an antibody/PTD-fused protein A complex. As a model protein, fluorescent-modified antibodies, captured by their respective primary antibody, were analyzed by fluorescence-activated cell sorting (FACS) which showed that the fluorescent-modified antibodies were directly delivered into cells. Peroxidase, captured by its specific antibody, was also delivered into cells and retained its activity.

Animals↗
PubMed2006

Transduction of NeuroD2 protein induced neural cell differentiation.

NeuroD2, one of the neurospecific basic helix-loop-helix transcription factors, has the ability to induce neural differentiation in undifferentiated cells. In this paper, we show that transduction of NeuroD2 protein induced mouse neuroblastoma cell line N1E-115 into neural differentiation. NeuroD2 has two basic-rich domains, one is nuclear localization signal (NLS) and the other is basic region of basic helix-loop-helix (basic). We constructed some mutants of NeuroD2, ND2(Delta100-115) (lack of NLS), ND2(Delta123-134) (lack of basic) and ND2(Delta100-134) (lack of both NLS and basic) for transduction experiments. Using these proteins, we have shown that NLS region of NeuroD2 plays a role of protein transduction. Continuous addition of NeuroD2 protein resulted in N1E-115 cells adopting neural morphology after 4 days and Tau mRNA expression was increased. These results suggest that neural differentiation can be induced by direct addition of NeuroD2 protein.

Animals↗
PubMed2006

In vitro selection of zinc finger DNA-binding proteins through ribosome display.

DNA-binding proteins with sequence specificities have a variety of applications. To create novel functional DNA-binding proteins, in vivo selection methods have been developed. There are, however, crucial problems with such methods, e.g., limitation of library size and difficulty of expression of toxic proteins for the host cells. In order to overcome these problems, we developed a novel way to select DNA-binding proteins using an in vitro ribosome display technique. The three zinc finger DNA-binding protein libraries, based on a Zif268 containing randomized sequence in each finger, were prepared and transcribed to mRNA in vitro. The ternary ribosomal complexes, formed by mRNA, ribosome, and translated DNA-binding protein during translation in a rabbit reticulocyte in vitro translation system, were selected with biotinylated target DNA fragments bound to streptavidin magnetic beads. The extracted mRNAs from the selected complexes were amplified using reverse transcription PCR and then sequenced. This is the first report of the selection of DNA-binding proteins involving an in vitro ribosome display technique.

Animals↗
PubMed2006

Cell-surface-localized ATP detection with immobilized firefly luciferase.

We demonstrate the application of an assay technique for the detection of ATP efflux from the cell surface. Until recently, the firefly luciferase assay has conventionally been used to detect ATP release because of its high sensitivity and wide detection ability. However, since this technique can be used only to infer the amount of diffused ATP in bulk solutions, it does not accurately reflect the dynamic ATP flux at the cell membrane. We therefore constructed luciferase fusion proteins that could be immobilized onto the cell surface. However, the low activities and limited application ranges of these proteins prompted us to use biotinylated luciferase given its attributes of strong and stable localization. Furthermore, luciferase can be immobilized strongly onto the biotinylated cell surface via streptavidin-biotin binding and can thus be used to determine the dynamic release of ATP near the cell surface.

Adenosine Triphosphate↗
PubMed2006

Construction of epidermal growth factor fusion protein with cell adhesive activity.

A novel protein for controlling cellular functions was constructed by combining functional units of various proteins. The Arg-Gly-Asp (RGD) sequence functioning as a cell adhesive function, an epidermal growth factor (EGF) as a cell growth function, and a hydrophobic sequence (E12) as an efficient assembling function, were combined and incorporated into one molecule. The fusion protein, designated ERE-EGF, was produced in Escherichia coli and purified with affinity chromatography using a His-tag. The ERE-EGF coated on an unmodified hydrophobic surface of a cell-culture plate (through the hydrophobic E12 moiety) retained both cell adhesive activity (through the RGD sequence) and cell growth activity (through the EGF moiety).

Amino Acid Sequence↗
PubMed2006

Fabrication of an antibody microwell array with self-adhering antibody binding protein.

One of the promising methods of preparing antibody arrays is immobilizing antibodies with protein A or protein G, each of which binds specifically to the heavy chain constant (Fc) region of immunoglobulin G (IgG). In this system, antibody immobilization efficiency depends on the number of active Fc binding proteins that need to be immobilized on the surface. Here we have designed and constructed an Fc binding protein with a self-adhering ability that can be immobilized on the hydrophobic surface by simple adsorption. It consists of an Fc binding domain of protein G (G3) and hydrophobic domain of elastin (E72). Direct observation revealed its self-adhering ability on the hydrophobic surface. The enzyme-linked immunosorbent assay (ELISA) showed that it retained antibody binding ability on the surface. The antibody array model was prepared on a hydrophobic microwell glass slide with E72G3, which specifically detect the antigen with a sevenfold greater sensitivity than the G3-treated slide. These results suggest that the E72G3 is useful for simple and effective immobilization of antibodies and can be used to fabricate any immuno devices.

Animals↗
PubMed2006

Identification of cysteine residues critically involved in homodimer formation and protein expression of human ATP-binding cassette transporter ABCG2: a new approach using the flp recombinase system.

Since ABCG2 is a half-transporter in the ATP-binding cassette (ABC) transporter family, it has been suspected that ABCG2 functions as a homodimer. In the present study, we have investigated the molecular mechanism underlying homodimer formation of ABCG2. Based on the amino acid sequence of ABCG2, three cysteine residues (Cys592, Cys603, and Cys608) are expected to exist in the extracellular loop. To identify a cysteine residue(s) required for homodimer formation, we have substituted those cysteine residues to glycine by site-directed mutagenesis and stably expressed the resulting variants in Flp-In-293 cells. Substitution of the amino acid at position 603 from cysteine to glycine (C603G) completely diminished homodimer formation, whereas substitution of both Cys592 and Cys608 to glycine residues (C592G/C608G) had no effect on homodimer formation. These results strongly suggest that Cys603 is prerequisite for homodimer formation of ABCG2 via a disulfide bond. On the other hand, immunohistochemistry experiments revealed that the C592G/C608G variant is mainly located in intracellular compartments. The C592G/C608G variant exhibited lower activity of ATP-dependent methotrexate (MTX) transport, and its expression did not confer Flp-In-293 cells resistance to SN-38 or mitoxantrone. Cys592 and Cys608 appear to be important for intracellular sorting of the de novo synthesized ABCG2 protein to the plasma membrane. Taken together, cysteine residues in the extra-cellular loop are considered to play pivotal roles in homodimer formation and plasma membrane localization of ABCG2.

ATP Binding Cassette Transporter, Subfamily G, Mem↗
PubMed2005

Activity-based in vitro selection of T4 DNA ligase.

Recent in vitro methodologies for selection and directed evolution of proteins have concentrated not only on proteins with affinity such as single-chain antibody but also on enzymes. We developed a display technology for selection of T4 DNA ligase on ribosome because an in vitro selection method for DNA ligase had never been developed. The 3' end of mRNA encoding the gene of active or inactive T4 DNA ligase-spacer peptide fusion protein was hybridized to dsDNA fragments with cohesive ends, the substrate of T4 DNA ligase. After in vitro translation of the mRNA-dsDNA complex in a rabbit reticulocyte system, a mRNA-dsDNA-ribosome-ligase complex was produced. T4 DNA ligase enzyme displayed on a ribosome, through addition of a spacer peptide, is able to react with dsDNA in the complex. The complex expressing active ligase was biotinylated by ligation with another biotinylated dsDNA probe and selected with streptavidin-coated magnetic beads. We effectively selected active T4 DNA ligase from a small amount of protein. The gene of the active T4 DNA ligase was enriched 40 times from a mixture of active and inactive genes using this selection strategy. This ribosomal display strategy may have high potential to be useful for selection of other enzymes associated with DNA.

Catalysis↗
PubMed2005

Glucose oxidase assisted homogeneous electrochemical receptor binding assay for drug screening.

Although the idea of homogeneous electrochemical immunoassay using antibody and an electroactive modified antigen as a probe looks to be very useful for high-throughput drug screening, there have been few reports. One reason for this is the difficulty experienced making an electroactive probe, because the introduction of electroactive compounds to antigens often interferes with the antigen-antibody interaction. To apply a homogeneous electrochemical assay to drug screening, we have designed new probes referring to the information of immobilization on beads which could identify the drug receptor. FK506 (also called Tacrolimus), immunosuppressive agent is modified with ferrocene derivatives as an electron mediator between glucose oxidase and an electrode, at a non-obstructing part. One of the probes still indicated the electrochemical activity as a mediator and had the specific binding capability for FKBP12 (FK506 binding protein). The current decrease in response to the additional FKBP12, detected with constant voltage amperometry using the probe, was observed within 5 min. Then, free FK506 as a leader drug, rapamycin and cyclosporine A as unknown drugs were used as a model for drug screening. Since the order of response currents at the same concentration of each drug reflected their binding constants, it was shown that binding capacity of an unknown drug candidate could be estimated by comparison of response currents between the leader drug and the unknown drug candidate. Thus, this glucose oxidase assisted homogeneous electrochemical drug-receptor binding assay has been proved to be a useful tool for drug screening.

Biosensing Techniques↗
PubMed2005

Method for detection of specific nucleic acids by recombinant protein with fluorescent resonance energy transfer.

Detection of specific nucleic acids is important to understand cellular mechanisms and functions of gene regulation. Here, we demonstrated a novel method to detect specific nucleic acids using recombinant protein and oligonucleotides. A recombinant protein YRGnC-11ad, which has a Rev-peptide between enhanced yellow fluorescent protein (EYFP) and enhanced cyan fluorescent protein (ECFP) was constructed and expressed in HeLa cells. Rev-peptide, which corresponds to amino acids 34-50 of the HIV-1 Rev protein, indicates disordered structure in solution but forms alpha-helical and elongated conformation upon binding to Rev response element RNA (RRE-RNA) and Rev-aptamer, respectively. We confirmed that YRGnC-11ad could specifically bind to RRE-RNA and Rev-aptamer in cell lysate, and fluorescent resonance energy transfer (FRET) signal was changed upon binding following the conformational change of Rev-peptide. To utilize this FRET signal change toward the detection of specific nucleic acids, we split the RRE-RNA sequence and connected to the complementary oligonucleotide for target nucleic acids. When each two oligonucleotides hybridized to an adjacent region of target nucleic acids correctly, a Rev-peptide binding site was reformed on the hybridized complex. And we could confirm that YRGnC-11ad recombinant protein indicated FRET increase upon binding to the hybridized complex in cell lysate. These results suggest that the recombinant protein probe is available for specific nucleic acid detection.

Fluorescence Resonance Energy Transfer↗
PubMed2005

Electrochemical evaluation of cellular physiological status under stress in Escherichia coli with the rpoS-lacZ reporter gene.

We developed an electrochemical detection method for evaluating cellular physiological status based on the stringent response as a means to monitor cell viability. A reporter plasmid was constructed by inserting the beta-galactosidase gene (lacZ) under the control of the rpoS promoter, and then used to transform E. coli cells. Electrochemical responses from the products catalyzed by beta-galactosidase expressed by these E. coli cells were detected using the chronoamperometric technique in a nondestructive manner. Comparisons of response currents between the relA-positive strain and relA-negative strain revealed that increases in these currents were caused by the stringent response due to the stressful alcoholic environment, and thus as a model of stressful cultivating conditions. The current was proportional to the beta-galactosidase activity assayed by a conventional method that required the destruction of cells. The cellular physiological status, which depends on the stringent response as a viability marker, therefore, could then be evaluated online with a current using the rpoS-lacZ reporter gene in the relA-positive strain without pretreatment.

Bacterial Proteins↗
PubMed2004

Translational control by internal ribosome entry site in Saccharomyces cerevisiae.

To confirm the active involvement of the internal ribosome entry site (IRES)-dependent translation in living eukaryotes, Saccharomyces cerevisiae and HAP4 IRES were used for in vitro and in vivo experiments. Since HAP4 protein might be required for activating mRNAs transcription in yeast cells when they are released from catabolite repression, the translational efficiency of HAP4 mRNA is presumed to increase under such a condition. The in vitro experiment showed clearly that the translational mechanism was shifted from the cap-dependent to the completely IRES-dependent translation when the yeast cells were derepressed from catabolite repression. From in vivo experiment, it was confirmed that the IRES-dependent translational efficiency was in a low level at the beginning of the stationary growth phase, and was enhanced at the glucose-exhausted phase. These results indicate that yeast cells on the catabolite derepressed condition could get a large amount of HAP4 protein for the completely IRES-dependent translation, while the IRES-dependent translational efficiency is increased. It has been proven that IRES functions directing the initiation of translation in living yeast cells.

Gene Expression Regulation, Fungal↗
PubMed2004

Blockage of RNA polymerase II at a cyclobutane pyrimidine dimer and 6-4 photoproduct.

The blockage of transcription elongation by RNA polymerase II (pol II) at a DNA damage site on the transcribed strand triggers a transcription-coupled DNA repair (TCR), which rapidly removes DNA damage on the transcribed strand of the expressed gene and allows the resumption of transcription. To analyze the effect of UV-induced DNA damage on transcription elongation, an in vitro transcription elongation system using pol II and oligo(dC)-tailed templates containing a cyclobutane pyrimidine dimer (CPD) or 6-4 photoproduct (6-4PP) at a specific site was employed. The results showed that pol II incorporated nucleotides opposite the CPD and 6-4PP and then stalled. Pol II formed a stable ternary complex consisting of pol II, the DNA damage template, and the nascent transcript. Furthermore, atomic force microscopy imaging revealed that pol II stalled at the damaged region. These findings may provide the basis for analysis of the initiation step of TCR.

DNA↗
PubMed2004

On-chip biosensing of estrogen receptor-alpha at single molecular level.

A novel method for detecting interaction between DNA and DNA-binding protein at single molecular level has been proposed. In this study, estrogen receptor-alpha (ER-alpha) was used for biosensing as the proof-example. A 518 bp-long (ca. 176 nm) DNA probe labeled with streptavidin at its 5'-terminus was prepared by inserting a consensus oligonucleotide sequence that binds to ER-alpha. A solution containing ER-alpha was dropped onto the Ni-treated mica substrate on which the DNA prove was previously immobilized, and it was observed by AFM. Specific binding of ER-alpha could be observed by measuring the distance between the site where binding occur, to the streptavidin label.

Binding Sites↗
PubMed2004

Combined effect of electrical stimulation and cisplatin in HeLa cell death.

The combined effect of electrical stimulation and cisplatin administration on HeLa cells was investigated. The combination of electric potentials (-0.5 V to 0.5 V) with 10-Hz frequency and 1000 ng/mL cisplatin decreased cancer cell viability by 32% and was more effective than either treatment given alone. Combined treatment with cisplatin and electrical stimulation also increased the number of apoptotic cells. It is shown that the efficacy of cisplatin was enhanced in electrically stimulated HeLa cells, and the addition of electrical stimulation amplified the chemotherapeutic effect of cisplatin in cervical cancer cells.

Antineoplastic Agents↗
PubMed2004

The construction of endothelial cellular biosensing system for the control of blood pressure drugs.

In order to assess blood pressure control drugs, the endothelial cellular biosensing system for assessing blood pressure control drugs was constructed. This system consists of human umbilical vein endothelial cells (HUVEC) on a polyion-coated gold electrode, a platinum counter electrode and an Ag/AgCl reference electrode. Nitric oxide (NO) as an indicator of blood vessel relaxation was detected with a polyion-coated electrode in the system. The NO detection limit of this electrode was 8.4 nM by differential pulse voltammetry (DPV). The drugs of blood pressure control (acetylcholine chloride (AcChCl), NOC 7 and NG-monomethyl-L-arginine (L-NMMA)) were assessed with this endothelial cellular biosensing system. One milli molar of AcChCl make NO released from HUVEC stimulated by activating endothelial nitric oxide synthase (eNOS) in HUVEC. In the case of 5 mM of L-NMMA, NO releasing was inhibited by inhibiting eNOS activation by 1 mM of AcChCl. NOC 7 immediately released NO regardless of eNOS activation in endothelial cells.

Antihypertensive Agents↗
PubMed2004

Construction of streptavidin-luciferase fusion protein for ATP sensing with fixed form.

A fusion protein consisting of streptavidin and firefly luciferase was constructed to establish an accurate measuring technique of local ATP concentration. The fusion protein retained the binding ability of streptavidin and enzymatic activity of luciferase. Also, it could detect the concentration of antigens and could determine nanomolar concentrations of ATP in its fixed form via interactions with biotin-conjugated antibodies.

Adenosine Triphosphate↗