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We used DNA transfection and protein introduction techniques to investigate the pressure tolerance of cytoskeletal structures in pectoral fin cells derived from the deep-sea fish Simenchelys parasiticus (habitat depth, 366-2,630 m). The deep-sea fish cells have G418 resistance. The cell number increased until day 6 of cultivation and all cells had died by day 35 when cultured in 35-mm Petri dishes in medium containing G418. Enhanced yellow fluorescent protein-tagged human beta-actin (EYFP-actin) was stably expressed by 1 in 100,000 deep-sea fish cells. Because almost none of the EYFP-actin was incorporated into actin filaments of the cells, we replaced the relatively large EYFP tag with a chemical fluorescent compound and succeeded in incorporating fluorescently labeled rabbit actins into the deep-sea fish actin filaments. Most of the filament structure in the cells with rabbit actin inserted underwent depolymerization when subjected to pressure of 100 MPa for 20 min, in contrast to control cells. There were no differences in the tubulin filament structure between control cells and deep-sea fish cells with fluorescein-labeled bovine tubulin inserted after the application of pressure ranging from 40 to 100 MPa for 20 min.

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.

We investigated the pressure tolerance of deep-sea eel (Simenchelys parasiticus; habitat depth, 366-2,630 m) cells, conger eel (Conger myriaster) cells, and mouse 3T3-L1 cells. Although there were no living mouse 3T3-L1 and conger eel cells after 130 MPa (0.1 MPa = 1 bar) hydrostatic pressurization for 20 min, all deep-sea eel cells remained alive after being subjected to pressures up to 150 MPa for 20 min. Pressurization at 40 MPa for 20 min induced disruption of actin and tubulin filaments with profound cell-shape changes in the mouse and conger eel cells. In the deep-sea eel cells, microtubules and some actin filaments were disrupted after being subjected to hydrostatic pressure of 100 MPa and greater for 20 min. Conger eel cells were sensitive to pressure and did not grow at 10 MPa. Mouse 3T3-L1 cells grew faster under pressure of 5 MPa than at atmospheric pressure and stopped growing at 18 MPa. Deep-sea eel cells were capable of growth in pressures up to 25 MPa and stopped growing at 30 MPa. Deep-sea eel cells required 4 h at 20 MPa to finish the M phase, which was approximately fourfold the time required under atmospheric conditions.

Immunosensors and biochemical array detection systems based on electrochemical transducers have many advantages such as low detection limit, fast response, simple design and ease of miniaturization. However, further development of such sensors will depend on the availability of suitable substrates that can be converted by a labeling enzyme to an electrochemically active product. Here, we report the synthesis of 4-amino-1-naphthylphosphate and it’s application as a new substrate for alkaline phosphatase. The electrochemical and enzymatic properties of this compound were investigated and compared with the properties of other aromatic 1,4-dihydroxy and 1,4-hydroxy-amine derivatives. The product of the enzyme reaction was 4-aminonaphthol, which was rapidly converted in the presences of air to 1,4-iminonaphthoquinone. This compound could then be detected in an amperometric flow injection assay (AFIA) with −200 mV versus Ag/AgCl potential application. The analytical range for mouse IgG, in an alkaline phosphatase amplified sandwich immuoassay with amperometric detection, was 0.01–100 μg ml −1 .

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.

Many types of proteins express their functions with conformation change in various biosystems. For the construction of biosensing system consisting of such functional proteins, the development of techniques for assembling of proteins with retaining their functions even on a solid substrate is required. Calmodulin was selected as a model protein for molecular assembly. Calmodulin expresses modulating function by conformation change with calcium binding. In the present study, some cysteine residues were introduced at the N-terminus of calmodulin by genetic engineering, and the resulting engineered calmodulin could be assembled efficiently with oriented manner. The modulating function of calmodulin on a solid-phase surface was also demonstrated.

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.

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.

A novel pressure-stat aquarium system has been developed for the study of living deep-sea multicellular organisms under high hydrostatic pressure. The system is designed for operation by submersibles and captures deep-sea organisms with a suction servomotor. The system can maintain an inner pressure of 20 MPa (1 bar ≈0.1 MPa ) even when the submersible surfaces from the deepest sea bottom. The system can control the pressure range up to 20 MPa within a fluctuation of ±0.1∼0.2 MPa by exchanging seawater. A specimen of the deep-sea fish Ebinania brephocephala collected by trawl net was kept alive under pressure in the pressure-stat aquarium system for 64 days. Oxygen consumption rate of a deep-sea fish E. brephocephala (total length 12.9 cm , wet weight 61.1 g ) was calculated as 0.10 μl O 2 / mg wet weight/h. Based on the calculation, two deep-sea fishes of the same size could be kept alive in the system. We also succeeded in maintaining the deep-sea fish Zoarcidae sp. captured by the submersible Shinkai 2000 at a depth of 1171 m in Sagami Bay, Japan.

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.

We found that extremely high hydrostatic pressure stresses induced a variety of cytokines production in normal human dermal fibroblasts. Normal human dermal fibroblasts were found to survive and were active in producing interleukin (IL) -6, -8, and monocyte chemoattractant protein-1 (MCP-1) under extremely high hydrostatic pressure, up to 70 MPa (=690.8 atm=713.8 kgf/cm2). 70 MPa pressure application extremely enhanced IL-6 and IL-8 secretions (about 130 folds) without transcriptional enhancement. Although induction of IL-1alpha, IL-1beta, and IL-12 mRNAs was appeared under high hydrostatic pressure condition, no translation of IL-1alpha, IL-1beta, and IL-12 proteins was found. Extracellular accumulation of constitutively produced MCP-1 was down regulated in the pressure-applied fibroblasts in the absence of transcriptional repression. These results indicated that hydrostatic pressure stresses triggered post-transcriptional regulation mechanisms that modulated the cytokines production.

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.

For evaluating the physiological status of cells, astringent response network was used. Fluorescence from intact E. coli, which has a plasmid encoding the green fluorescence protein (GFP) under the regulation of rpoS promoter, was monitored. Comparison of the response of different E. coli strains demonstrated an essential role of ppGpp in the expression of GFP, as it activated the rpoS promoter. The physiological status of intact cells, that depends on ppGpp accumulation in response to the nutritional status such as amino acid starvation, could therefore be monitored by measuring fluorescent intensity using this reporter gene.

Estradiol-displayed bioaffinity beads binding to the anti-estradiol antibody attached via the protein A-coated mica surface were examined by atomic force microscopy (AFM). The amount of specifically bound beads on the surface was directly proportional to the concentration of free estradiol in solution. Estradiol from 10 ng ml−1 to 10 μg ml−1 could be determined. This suggested that direct counting of bioaffnity beads by AFM can be used to detect specific ligand for the target protein.

The immunosensing system coupled with a disposable amperometric glucose oxidase (GOD) sensor was developed for the determination of α-fetoprotein (AFP). The combination of the simple, portable, and low cost electrochemical measurement with the specific and sensitive enzyme-linked immunosorbent assay (ELISA) indicates a promising means to establish an immunosensing system. The results proved that the established immunosensing system was capable of detecting AFP in the low concentration value of ng ml −1 . The assay took 20 min to be completed using an electrode and pre-coated microtiter plate with anti-AFP. The assay revealed linearity for AFP in the response range from 10 −8 to 10 −5 g ml −1 . On the basis of these results, the immunosensing system offered excellent performance within 20 min compared with typical ELISA which took several hours.

The immobilization of antibody molecules is a key step in fabrication of immunosensors. We have exploited biosynthetic lipid-tagging to provide a stable and oriented immobilization of antibody molecules on lipid membranes. In the present study, we prepared lipid-tagged antibody/phospholipid monolayers using Langmuir–Blodgett (LB) technique, and direct spreading from immunoliposomes. The formation and properties of the monolayers and the kinetics of antigen binding were studied by using fluorescence microscopy and atomic force microscopy (AFM). The results indicate formation of a stable film which was able to interact specifically with the antigen. The antigen binding was accompanied by (micro) aggregation of the antibody–antigen complexes. This approach may be suitable for immunosensor applications.

A novel flow injection analytical system with a screen-printed enzyme sensor incorporating Os-complex mediator has been developed. A newly created glucose biosensor was fabricated using thick-film technique and co-immobilization of glucose oxidase and Os-complex on a permeable membrane. The composition of the enzyme and mediator in the membrane and the analytical conditions were optimized. The flow injection electrochemical system showed that glucose was detected linearly in the concentration range from 0.1 to 10 mM with a detection limit of 0.03 mM by 50 μl of sample injection, giving a throughout of about 40 samples per hour. The glucose biosensors retained their constant response after more than 100 injections and storage over a month. The designed electrochemical flow system with a disposable biosensor is suitable for automatic and rapid determination of glucose.

Tissue culture for the deep-sea clam Calyptogena soyoae (C. soyoae) has been examined. Mantle tissue was cultured in Dulbecco's modified Eagle medium that was prepared using artificial seawater supplemented with fetal bovine serum (FBS) and the body fluid of C. soyoae. The mantle cells were viable in culture for at least 13 days at 4 degrees C and atmospheric pressure on a polylysine-coated dish, although no cells attached in the body fluid-free culture medium. It was found that mantle cells synthesized DNA and seemed to proliferate under atmospheric conditions.

A novel strategy for designing and synthesizing extremely thermostable biologically active proteins is proposed. The design concept is based on combining a rigid and extremely hydrophobic peptide unit with a biologically active peptide unit. The cell adhesive peptide sequence, Arg-Gly-Asp (RGD), as a functional peptide unit was incorporated into the elastin-based rigid polyhexapeptide, whose repeating unit is Ala-Pro-Gly-Val-Gly-Val (APGVGV). The designed fusion gene was expressed in E. coli, and the resulting protein, designated ER4, was purified with affinity chromatography. The ER4-coated cell culture plate showed sufficient cell adhesive activity through the RGD sequence on the surface of ER4. The thermostability of ER4 was demonstrated by estimating the remaining cell adhesive activity after autoclaving at 120 degrees C for 20 min, and it retained over 90% of cell adhesive activity compared with native ER4.

The gene expression plasmid pMALU5 for the fusion protein of protein A (SpA) with a complete sequence of firefly luciferase (Luc) was constructed. The fused gene was expressed in Escherichia coli, and the resulting SpA–Luc fusion protein was purified by one-step affinity chromatography on IgG–Sepharose. The protein retained both activities: IgG binding capability of protein A and enzymatic activity of luciferase. Blotting analyses were performed with the fusion protein to determine a tumor marker of α-fetoprotein (AFP). AFP was detected at the lowest detection limit of 5 pg by dot blotting and Western blotting. The SpA–Luc fusion protein provides a highly selective, sensitive, and versatile marker for blotting analyses.

The single-chain antibody against 2-phenyloxazolone was modified with lipid molecules at its amino-terminus by genetic engineering. The engineered lipid-tagged antibody molecules were incorporated into liposomes consisting of phosphatidylcholine, and the immunoassay systems were constructed by the resulting immunoliposomes. One immunoassay system was based on fluoroimmunoassay. A competitive fluoroimmunoassay for caproic acid conjugate of 2-phenyloxazolone as a model antigen was performed with the calboxyfluoresceine-encapsulated immunoliposomes. Antigen could be determined in the concentration range from 10−7 to 10−9 M. The other system was based on quartz crystal microbalance (QCM). The immunoliposomes were competitively reacted with analyte to the hapten-immobilized surface of a crystal plate. The frequency change was observed by injection of the mixture of the immunoliposomes and analyte in a concentration dependent manner. In this competitive QCM assay, antigen could be measured in the concentration range from 10−5 to 10−8 M. Furthermore, direct observation of the immunoliposomes on the hapten-coated solid-surface by atomic force microscopy (AFM) was also performed in this study.

α-Chymotrypsin (Chy) was entrapped in polytetrafluoroethylene (PTFE) particles. The entrapped enzyme showed twofold catalytic activity for amino acid ester hydrolysis in aqueous solution than free enzyme. The Chy/PTFE particles also catalyzed the peptide synthesis in aqueous solution with a yield of 14%. Both the synthetic and the hydrolytic activities of the entrapped enzyme were enhanced as compared with the free enzyme. The PTFE matrix should provide the enzyme molecules by creating a hydrophobic environment which results in enhanced peptide synthesis in aqueous solution.

Mouse astroglial cells, which were cultured on an electrode, were found responsive to an electric stimulation of sine wave potential in enhancing hsp70 mRNA resulting from an activation of hsp70 gene expression. On the basis of this finding, electrically responsive cells were established by transfecting mouse 3T3-L1 cells with a constructed plasmid encoding hsp70 promoter and the firefly luciferase gene. A stable cell line has been established through selection of heat-stimulated luciferase expression. A 1-h electric stimulation of the cells resulted in activation of luciferase expression, which was confirmed to produce an increase in light emission. The sequential pattern of the electrically stimulated expression of luciferase was found different from that of the heat stimulation. Furthermore, the promoter was activated depending on the potential and duration of the stimulation applied. Consequently, the electric stimulation has proven effective on activating hspP70 promoter. This cell line is feasible in expressing the gene of interest by electrical stimulation, which lead us to construct environment responsive cells in general.

The specific sequence in a linearlized double-stranded DNA target has been identified at a single-molecular level by atomic force microscopy (AFM). This was accomplished using RecA-coated, single-stranded DNA probes which were paired with a specific complementary DNA sequence in a linear double-stranded DNA target by strand-exchange reaction at a homologous sequence site with target DNA. The sites of interaction between the nucleoprotein filaments and the double-stranded DNA targets were directly visualized by AFM in solution containing 4 mM magnesium acetate. Measurements of the position of RecA-coated probes paired to individual target DNA showed that DNA probes specifically paired at their corresponding homologous target sequences. Strand exchange promoted by RecA and the visualization by AFM provided a rapid and efficient way to identify homologous sequence on a single-molecule target DNA.

A series of novel substituted-1,10-phenanthroline complexes of osmium and ruthenium have been synthesized and characterized electrochemically with respect to their ability to act as electron transfer mediator for glucose oxidase. These complexes exhibit high second-order rate constants for reduced glucose oxidase in the order of 10 6 –10 7 M −1 s −1 . The complexes bearing electron-donating substituents give a significantly higher rate constant with glucose oxidase and lower redox potential than those bearing electron-accepting substituents. 4,7-Dimethy-1,10-phenanthroline and 3,4,7,8-tetramethy-1,10-phenanthroline complexes of osmium have been characterized in details as mediator for glucose oxidase. The application of the family of mediators in amperometric biosensors seems feasible.

A novel tissue engineering for bone formation has been proposed, to make osteoblast differentiation balanced by transfecting the mesenchymal stem cells with a gene encoding human bone morphogenetic protein-2 (hBMP-2) under the control of adipocyte specific lipoprotein lipase (LPL) promoter. Due to the promoter specificity, the initiation of BMP transcription is dependent on adipogenesis. For 14-day culture in the presence of ascorbic acid (asc) and beta-glycerophosphate (gly), nontransfected mouse embryonic fibroblast C3H10T1/2 (10T1/2) cells showed extensive accumulation of lipid droplets and adipocyte specific enzyme glycerol-3-phosphate dehydrogenase (G3PDH) mRNA expression, but exhibited neither BMP-2 expression, high alkaline phosphatase (ALP) activity which reflects osteoblast phenotype. On the other hand, transfected 10T1/2 cells showed hBMP-2 expression, high ALP activity and low level of G3PDH. mRNA expression accompanied with minimal lipid droplets. These results indicate that 10T1/2 cells are proved to be differentiated with maintaining coordinated balance of adipogenesis and osteogenesis, when they are transfected by the gene encoding hBMP-2 under the control of LPL promoter.

A novel cap-independent translation has been performed where the ribosome entry is regulated by the 5′-noncoding region (NCR) of L-A virus mRNA. Despite L-A virus mRNA containing neither cap structure nor a poly(A) tail, the reconstructed mRNA encoding the 5′ NCR of L-A virus mRNA and a reporter gene (luciferase) was translated, in yeast lysate, 60 times more efficiently than control mRNA. The 5′ NCR from L-A virus was effective in regulating the recruitment of ribosome in vitro. A possible mechanism in Saccharomyces cerevisiae is also suggested, whereby the ribosome entry is regulated by the 5′ NCR of L-A virus mRNA.

The B-domain, which is one of IgG-binding domains of staphylococcal protein A, was repeated five times and a cysteine residue was introduced at its C-terminus by a genetic engineering technique. The resulting protein, designated B5C1, retained the same IgG-binding activity as native protein A. The B5C1 was assembled on a gold plate surface by utilizing a strong affinity between thiol of cysteine and a gold surface. IgG-binding activity of B5C1 on a gold surface was much higher than that of physically adsorbed B5, which lacks cysteine residue. Furthermore, antigen-binding activity of immobilized antibody molecules through the use of assembled B5C1 on a gold surface was about 4.3 times higher than that of physically adsorbed antibody molecules. Immobilization of highly oriented antibody molecules was realized with the engineered IgG-binding protein.

The synthesis and the use of a new substrate, 4-hydroxy-naphthyl-1-phosphate (HNP), for the amperometric detection of alkaline phosphatase activity is described. The product of the enzymatic hydrolysis of HNP was dihydroxy naphthalene (DHN). DHN was rapidly oxidized in air to give naphthoquinone (NQ), which was measured in amperometric flow injection analysis (AFIA) at 300 mV versus Ag/AgCl. DHN standards could be measured at a 60 nM detection limit. There was a linear response to the enzyme with a 300 fM detection limit, which was equivalent to 6 attomole for each injection. The measurement range for human IgG, in an alkaline phosphatase amplified sandwich immunoassay with amperometric, was 1–1000 ng/ml.

New in vitro RNA synthesis has been performed with an L-A virus nanoparticles, in which the gene and polymerase are integrated. The specific recognition sequence (packaging site) of L-A virus was inserted within a gene of interest. Based on the intrinsic replication cycle, the exogenous RNA with the packaging site was encapsulated by an empty L-A virus nanoparticle. The packaging site worked as a recognition site even for exogenous RNAs. The recognized RNA was replicated to dsRNA, and was then transcribed by empty L-A virus nanoparticles. These results indicate that empty L-A virus nanoparticles recognize an exogenous RNA with the packaging site and synthesize RNA in vitro.

A highly sensitive electrochemical luminescence (ECL) method has been developed for the determination of thiamine based on electrochemically oxidizing thiamine with rhodamine B as a sensitizer. A weak ECL signal of thiamine was electrochemically generated on a platinum electrode in an alkaline solution and strongly enhanced in the presence of rhodamine B. Under the condition of 0.10 mM rhodamine B, 6 mM CTMAB and 0.36 M sodium hydroxide, the response to the concentration of thiamine is linear range from 0.1 μg ml −1 to 2 μg ml −1 and a detection limit of 0.08 μg ml −1 can be achieved. This method has been successfully applied to the determination of thiamine in pharmaceutical preparations. The energy transfer ECL scheme is also postulated.

A disposable creatinine sensor has been developed using a thick-film carbon electrode for hydrogen peroxide. Carbon paste containing 10% Pt powder was metal mask printed on a plastic plate, and irradiated by UV light under pure oxygen atmosphere for activation of thick-film electrodes. Thin layer of three different enzymes involving creatininase, creatinase, and sarcosine oxidase was fabricated beside the printed electrode to form a creatinine sensor. A drop of a test solution automatically runs through a microchannel reaching the enzyme layer and then the enzymes quickly become solubilized and disperse throughout the volume of the microchannel. The detection of hydrogen peroxide at 500 mV vs. Ag/AgCl serves as the analytical signal. The sensor shows a linear response range for creatinine between 0.2 and 2 mM in a pH 7.5 buffered solution with 0.1 M phosphate and 0.1 M KCl. The oxygen plasma treatment was found effective on improving the voltammetric behavior of the thick-film electrodes.

In order to construct intelligent materials consisting of functional proteins, the assembling techniques of proteins with retaining their functions even on a solid surfaces are required. Calmodulin, which expresses modulating function by conformation change with calcium binding, was selected as a model protein for molecular assembling. Calmodulin was genetically engineered for self-assembling on the gold surface with an oriented manner by introducing cysteine residues at the N-terminus of calmodulin. The engineered calmodulin retained Ca2+-responsive modulating function of enzyme activity as native calmodulin. Enzyme immunoassay and quartz crystal microbalance revealed that the engineered calmodulin was efficiently assembled on the gold surface in an acidic buffer solution as compared with native calmodulin. Genetic engineering is discussed to be effective on modifying proteins to be self-assembled on solid matrices.

The stability of immobilized mRNA against ribonucleases was investigated in a cell-free protein synthesis system. The plasmid-encoding protein A with the 20-mer poly(A) tail under the control of T7 promoter was constructed, and the corresponding mRNA was synthesized by T7 RNA polymerase reaction. The resulting mRNA was immobilized on oligo(dT)-immobilized latex beads by hybridization utilizing the poly(A) tail of mRNA at the 3'-terminus. The mRNA was stabilized against three types of nucleases (3'-OH exonuclease, 5'-OH exonuclease, and endonuclease) by immobilization. Translation of immobilized mRNA with a continuous-flow cell-free protein-synthesizing system from Saccharomyces cerevisiae was ascertained. Reusability of the immobilized mRNA as genetic information was also examined.

The design principle of a thermostable functional protein has been proposed by demonstrating genetic engineering synthesis of a thermostable cell adhesion protein. The cell adhesive peptide sequence, Arg-Gly-Asp (RGD), was incorporated into the elastin-based polyhexapeptide, whose repeating unit is Ala-Pro-Gly-Val-Gly-Val (APGVGV). The resulting protein possesses cell adhesion activity approximately 80% of fibronectin. After autoclaving at 120 °C for 20 min, the protein retained over 90% of cell adhesion activity, while the activity of autoclaved fibronectin decreased to 50%.

A flow injection method for the determination of catecholamines based on the inhibition of the intensity of chemiluminescence from the luminol–hypochlorite system is described. The hypochlorite was electrogenerated on-line by constant current electrolysis, resulting in the elimination of the instability of hypochlorite solution prepared from commercially available sodium hypochlorite. The detection limits are 6×10 −10 g ml −1 for dopamine, 8×10 −10 g ml −1 for adrenaline and 8×10 −10 g ml −1 for isoprenaline. Linear ranges are 1×10 −9 –2×10 −8 g ml −1 for dopamine, 2×10 −9 –2×10 −8 g ml −1 for adrenaline, 2×10 −9 –2×10 −8 g ml −1 for isoprenaline. The relative standard deviations are lower than 2.8%. The proposed method has been applied to the determination of catecholamines in pharmaceutical injections with satisfactory results.

Glucose oxidase (GOD) complexed with Cu[II] was deposited on the electrode surface with retention of enzyme activity by an electrochemical reduction of Cu[II]. The deposited GOD showed an apparently reversible electron transfer to an electrode through the coordinated Cu. The GOD/Cu electrode responded to glucose in anodic current due to an electrochemical oxidation of enzymatically generated FADH. In conclusion, the electron transfer was mediated by bound Cu which was situated at an active center of GOD. The GOD/Cu system provided an “atomic interface” to perform a smooth electron transfer between a flavoenzyme and an electrode.

Two types of protein engineering have been developed for self-assembling antibody (IgG) molecules in an oriented manner. The first is to tag a cysteine group to Protein A which has a specific affinity to the Fc part of IgG. The cysteine-tagged Protein A was self-assembled on the gold surface, which was followed by self-assembling of IgG to face the antigen recognition sites to the solution phase. The second is concerned with tailing a single chain antibody by lipid at the one terminal and the hexahistidinyl group at the other. The lipid and histidine-tailed single chain antibody was embedded in a liposome to make the antigen recognition site appear on the outsphere, which was immobilized on the Ni-treated mica surface through chelating of the histidine tail. The individual liposome was clearly imaged by a tapping mode of AFM.

To develop an in situ NO sensing system for primarily biological and medical uses, two types of NO sensing materials, which may be coupled with an electrochemical reaction for signal transduction, have been investigated. Heat-denatured Cyt C and a radical scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetra methyl imidazoline-1-oxyl-3-oxide (C-PTIO) were found to be effective and were incorporated into electrochemical sensing systems. Heat-denatured Cyt C deposited on a 4-mercaptopyridine modified gold electrode responded to NO with an increase of cathodic current through electrochemical reduction of Cyt C (Fe3+), when the electrode potential was controlled at 0 mV vs Ag/AgCl. The dynamic range of the sensing system was 0.5-4 microM. The sensing system with C-PTIO exhibited an anodic output in response to NO at 0.7 V vs Ag/AgCl, and showed a wider dynamic range from 0.05 to 100 microM.

Gene engineering has been successfully applied to develop biosensing material, which includes an enzyme-labeled binding protein for enzyme immunosensing, a lipid-tagged single chain antibody for immunosensing and an environmentally responsive gene-bearing cell for environment sensing. Protein A was labeled with firefly luciferase according to the gene engineering to fuse both proteins. A single chain antibody was genetically tagged with a lipid chain at a specific site by enzymatic modification of a preprotein in a transmembrane process within a bacterial cell. A recombinant plamid bearing xyl R protein and firefly luciferase genes with Ps promoter was transfected to E. coli to make the cell responsive to environmentally hazardous compounds such as xylene in emitting light.