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Biomedical subjects

E Kobatake

Publications and source records attributed to E Kobatake.

34 records · Page 2Linked to original sources

Electrically induced NGF production by astroglial cells.

A controlled culture system has been developed to induce nerve growth factor (NGF) production in astroglial cells that are cultured on an electrode surface. The electrode potential is alternatively modulated at an amplitude of 300 mV and a frequency of 10 Hz. The electric stimulation triggers NGF production and secretion. The mechanism of the electrically induced NGF production is discussed in association with protein kinase C (PKC) activation.

Animals↗

Bioluminescent detection of RNA with sequence-specificity using RNA binding protein-luciferase fusion protein.

A novel bifunctional reagent has been synthesized for RNA detection by genetic fusion of a sequence-specific RNA binding protein and firefly luciferase. The RNA binding protein used in this study recognizes the oligoribonucleotide rH4 which contains four (UUAGGG)-repeat sequence, while luciferase works as a bioluminescent marker. The constructed fusion protein exhibited both sequence-specific RNA binding and bioluminescent activities. The rH4 and rECGF, which has an unrelated sequence having the same length as rH4, were immobilized on a nylon membrane. The membrane was pre-treated by 5% bovine serum albumin and soaked into a fusion protein solution. Bioluminescent detection has successfully been performed at more than 50 pmol of rH4, so the detection limit using this protein was 50 pmol. However, no appreciable bioluminescence was induced by rECGF.

Base Sequence↗

Production of the chimeric-binding protein, maltose-binding protein-protein A, by gene fusion.

A fusion protein between maltose-binding protein (MBP) and staphylococcal protein A (SpA) was genetically produced. The gene fusion plasmid, pMALPA2, was constructed by inserting the protein A gene into an expression vector of maltose-binding protein in frame, and was expressed efficiently in Escherichia coli. The resulting fusion protein of molecular mass 65 kDa, retained the activity of both MBP and SpA (binding capability to amylose and immunoglobulin G). This chimeric-binding protein was used as an adhesive molecule for immobilization of antibodies to a solid-phase surface for enzyme immunoassay. An enzyme immunoassay was performed with the fusion protein, and human IgG was determined in the concentration range from 10(-4) to 10(-6) g ml-1.

ATP-Binding Cassette Transporters↗

Biosensing of benzene derivatives in the environment by luminescent Escherichia coli.

Sensitive and convenient biosensing of environmental pollutants has been developed by fusing a gene of firefly luciferase to the TOL plasmid. TOL plasmid of Pseudomonas putida encodes a series of enzymes for degradation of benzene and its derivatives. The expression of these enzymes is controlled with the regulating proteins xylR and xylS, whose promoters are activated in the presence of aromatic compounds. The structural gene of firefly luciferase, as a reporter enzyme, was inserted under the control of the promoter of xylS protein, and gene fusion plasmid pTSN316 was constructed. The recombinant Escherichia coli transformed with this plasmid was applied to the environmental biosensing of benzene derivatives. The expression of luciferase was induced in the presence of aromatic compounds and the lower detection limit for m-xylene was 5 microM.

Benzene↗

Electronically modulated biological functions of molecular interfaced enzymes and living cells.

Conducting polymer molecular interfaces have been implemented to modulate biological functions of fructose dehydrogenase, pyruvate oxidase and Saccharomyces cerevisiae at the electrode surface by adjustment of electrode potential. The enzyme activity of the polypyrrole-interfaced fructose dehydrogenase was electronically modulated by means of electron transfer between the enzyme and the electrode surface. The enzyme activity of polypyrrole-interfaced pyruvate oxidase was modulated by an electronically driven change of substrate concentration. The gene expression in polypyrrole-interfaced Saccharomyces cerevisiae was electronically induced by a change in the phosphate concentration.

Electrodes↗

Neuronal selectivities to complex object features in the ventral visual pathway of the macaque cerebral cortex.

1. To infer relative roles of cortical areas at different stages of the ventral visual pathway, we quantitatively examined visual responses of cells in V2, V4, the posterior part of the inferotemporal cortex (posterior IT), and the anterior part of the inferotemporal cortex (anterior IT), using anesthetized macaque monkeys. 2. The critical feature for the activation was first determined for each recorded cell by using a reduction method. We started from images of three-dimensional complex objects and simplified the image of effective stimuli step by step by eliminating a part of the features present in the image. The simplest feature that maximally activated the cell was determined as the critical feature. The response to the critical feature was then compared with responses of the same cell to a routine set of 32 simple stimuli, which included white and black bars of four different orientations and squares or spots of four different colors. 3. Cells that responded maximally to particular complex object features were found in posterior IT and V4 as well as in anterior IT. The cells in posterior IT and V4 were, however, different from the cells in anterior IT in that many of them responded to some extent to some simple features, that the size of the receptive field was small, and that they intermingled in single penetrations with cells that responded maximally to some simple features. The complex critical features in posterior IT and V4 varied; they consisted of complex shapes, combinations of a shape and texture, and combinations of a shape and color. 4. We suggest that local neuronal networks in V4 and posterior IT play an essential role in the formation of selective responses to complex object features.

Animals↗

Bioluminescent immunoassay with a protein A-luciferase fusion protein.

Protein A and firefly luciferase were genetically fused and the resulting fusion protein was applied to a bioluminescent immunoassay. The gene fusion plasmid, pMALU2, was constructed by inserting the structural gene of luciferase into a protein A expression vector, and was expressed in Escherichia coli. The resulting fusion protein of molecular weight 91 kDa retained not only the enzymatic activity of luciferase but also the binding capability of protein A to the Fc region of immunoglobulin G (IgG). The bioluminescent immunoassay was performed with the fusion protein and human IgG was determined in the concentration range from 10(-3) to 10(-7) g/ml.

Escherichia coli↗

Control of feeding rhythm in the terrestrial slug Incilaria bilineata.

A modulatory neuron of feeding rhythm was newly identified in the buccal ganglia of the isolated central nervous system (CNS) of the terrestrial slug Incilaria bilineata. This neuron was termed the "feeding rhythm modulator" (FRM). Its morphological and electrical properties were compared with those of the MGC (metacerebral giant cell, a cerebral modulatory neuron of feeding rhythm). There was no direct connection between FRM and MGC. In order to investigate the control mechanism of the buccal central pattern generator, feeding rhythm was observed by varying the activities of MGC and FRM simultaneously. At a lower level of activity of MGC, feeding rhythm was not only sensitive to the activity of MGC but also to that of FRM. As the level of activity of MGC increased, feeding rhythm was exclusively controlled by the activity of MGC, and became unaffected by the activity of FRM. This indicates that cerebral neurons such as MGC primarily control feeding rhythm and modulate the contribution of FRM in a hierarchical manner.

Animals↗

Application of a fusion protein, metapyrocatechase/protein A, to an enzyme immunoassay.

A fusion protein of metapyrocatechase and protein A was genetically produced for demonstration of effective conjugation of an enzyme with a binding protein employed in enzyme immunoassay. Plasmid pMPRA3, constructed by inserting the protein A gene into a plasmid pMK12 vector derived directly from the structural gene of metapyrocatechase, was expressed in Escherichia coli. The resulting fusion protein was shown to have promising properties for use in enzyme immunoassays due to the specific binding of the protein A moiety to the Fc portion of immunoglobulin G and to the high amplification of enzyme. Bovine serum albumin, a model antigen, was successfully determined in the concentration range from 1 x 10(-3) to 1 x 10(-7) g/ml.

Catechol 2,3-Dioxygenase↗

Hyperproduction of a bifunctional hybrid protein, metapyrocatechase-protein A, by gene fusion.

A hybrid protein between metapyrocatechase and Staphylococcal protein A was produced by recombinant DNA techniques. A plasmid carrying the fusion gene that encodes the hybrid protein was constructed and expressed in E. coli. Over 70% of soluble proteins of the cell extracts was estimated to be the hybrid protein. This fusion protein is about 65,000. Both the IgG-binding activity of protein A and the metapyrocatechase activity were found in the hybrid protein. The optimum pH of metapyrocatechase in the fusion protein was at around 6.5 and Km was 1.3 X 10(-5) M. A simple immuno-enzymometric assay was developed for anti-BSA antibody using the fusion protein.

Amino Acid Sequence↗

Genetic analysis of developmental mechanisms in hydra. XIX. Stimulation of regeneration by injury in the regeneration-deficient mutant strain, reg-16.

A mutant strain of Hydra magnipapillata, reg-16, has a very low regenerative capacity. After head removal, it usually restores 10-20% of the original number of tentacles in 7 days. A procedure was found to markedly improve tentacle regeneration in this strain. The closed wound located at the apical regenerating tip of the decapitated polyp was gently reopened using a pair of forceps. Reg-16 polyps treated in this way at 24 and 48 h after head removal restored nearly all of the original number of tentacles in 7 days. A lateral tissue transplantation procedure was employed to examine the effect of wound reopening on the morphogenetic potential of decapitated reg-16 polyps. Wound reopening produced a significant increase in head activation level without producing a preceding decrease in head inhibition level. This and other observations suggest that the coupled activation-inhibition changes that normally occur after head removal from the wild-type hydra do not occur in this strain. Mechanisms responsible for the wound reopening effect and the absence of activation-inhibition coupling in the mutant strain reg-16 are discussed.

Animals↗

Bioluminescent monitoring of intracellular ATP during fermentation.

The luciferase gene was introduced as a probe into a cell in order to develop a bioluminescent monitoring of intracellular ATP during fermentation. Two plasmids were constructed with two types of promoters. One was pLac-Luc, which had the luciferase gene under the lac promoter to be expressed at a high level. The other was pTet-Luc, which had the luciferase gene under the tetracycline promoter to be stably expressed. A threonine-overproducing strain of Escherichia coli (No. 29-4) was transformed with each plasmid. The recombinant E. coli strains were characterized in their growth, threonine production and luciferase expression. The bioluminescence produced intracellularly from expressed luciferase was detected during fermentation -in a non-destructive manner. The bioluminescent intensity reflected both intracellular ATP and luciferase levels, and the results indicate that stable expression of a luciferase reporter is essential for monitoring intracellular ATP.

Adenosine Triphosphate↗

Genetically synthesized antibody-binding protein self-assembled on hydrophobic matrix.

A unique antibody-binding protein, (E12B2)n, was genetically synthesized, which was characterized by a hydrophobic peptide, E12, at one terminus and an antibody-binding peptide, B2, at the other. It was clarified by atomic force microscopy (AFM) imaging that this protein was efficiently self-assembled on a hydrophobic solid surface. (E12B2)n self-assembled on a microplate exhibited an excellent performance of antibody-binding affinity. The proposed design of antibody-binding protein seems promising in immobilizing antibody molecules on hydrophobic solid surfaces.

Antibodies↗

Ca(2+)-responsive extensible monolayer membrane of calmodulin-albumin conjugate.

A Ca(2+)-responsive monolayer protein membrane was prepared by developing calmodulin and bovine serum albumin at the air-water interface and by conjugating them with a bifunctional agent. In the case of the BSA monolayer, complex formation with Mg2+ generated a larger change in surface pressure than that with Ca2+. On the other hand, a drastic change in surface pressure was observed for the conjugated thin membrane associated with Ca2+ than that associated with Mg2+. Due to a drastic change in the conformation of calmodulin, the conjugated protein film changes its morphology (STM image), depending on Ca2+ concentration: the extended structure in the presence of Ca2+ transforms to a shrinked structure in the absence of Ca2+. The largest surface pressure change was detected when calmodulin was mixed with an equimolar amount of bovine serum albumin.

Calcium↗

Calcium responsive two-dimensional molecular assembling of lipid-conjugated calmodulin.

Calmodulin (CaM), a calcium ion sensitive protein, was conjugated with dioctadecyldimethylammonium bromide and subsequently assembled into a monolayer at the air-water interface using the LB method. The lipid-conjugated calmodulin (LCC) retains its calcium sensitivity, determined from the changes in the area-pressure isotherm of the monolayer obtained at the air-water interface. The functionality of this protein assembly was characterized by the activation of phosphodiesterase (PDE), a CaM responsive enzyme. The enzyme activity of PDE coupled with LCC at the air-water interface was measured by using the enzymatic method. It was found that LCC retained its enzyme activity modulating function of calmodulin, which is triggered by calcium ions. This characteristic plays an important role in fabricating molecular assembly of proteins which have a cooperative interaction at the molecular level.

3',5'-Cyclic-AMP Phosphodiesterases↗

On-off switching of enzymatic reaction by recombinant calmodulin on a solid-phase matrix.

A fusion protein consisting of human calmodulin (CaM) and glutathione S-transferase (GST) was produced by gene fusion. The fusion protein was overexpressed in Escherichia coli as a soluble form and purified with one-step affinity chromatography using glutathione-Sepharose. The protein had the modulating activity of CaM and the binding capability to glutathione of GST. Phosphodiesterase, which is a CaM dependent enzyme, was activated by the fusion protein, with the Ca2+ level equal to the level equivalent to a native CaM. Furthermore, CaM could be immobilized on a solid-phase matrix through the use of GST moiety while its modulating activity was retained. Phosphodiesterase activity was switched on and off by the immobilized CaM with or without Ca2+, and repeated use of CaM was demonstrated.

3',5'-Cyclic-AMP Phosphodiesterases↗