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.

Osteogenesis coordinated in C3H10T1/2 cells by adipogenesis-dependent BMP-2 expression system.

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.

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.

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.

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.

Two types of electrochemical nitric oxide (NO) sensing systems with heat-denatured Cyt C and radical scavenger PTIO.

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.

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.

Self-assembling of glutathione S-transferase/calmodulin fusion protein on chemically modified gold surface.

The fusion protein technique was used to prepare an artificial polyfunctional protein from calmodulin (CaM) and glutathione S-transferase (GST). The fusion protein was designed, expressed, and then assembled to the glutathione self-assembled gold surface. The protein assembly was confirmed through enzyme binding assay and enzyme immunoassay. Specific binding of the fusion protein to glutathione self-assembled on the gold surface was assessed via a quartz crystal microbalance (QCM). The fusion protein was reversibly adsorbed and desorbed by the competitive binding of glutathione present in a solution, thus showing that the binding of the fusion protein was specific and had a highly oriented molecular configuration.

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.

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.

Electrically stimulated induction of hsp70 gene expression in mouse astroglia and fibroblast cells.

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.

Assembling of engineered IgG-binding protein on gold surface for highly oriented antibody immobilization.

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.

Gene expression in the electrically stimulated differentiation of PC12 cells.

Cell differentiation of PC12 cells was electrically induced to grow neurites in the absence of nerve growth factor (NGF) on the electrode surface, of which potential was modulated by a rectangular wave of potential. The electric stimulation induced the c-fos expression which is essential for cell differentiation. Non-specific calcium channel blocker, lanthanum ion, inhibited the electrically induced differentiation, while NGF-induced differentiation was not suppressed. An L-type calcium channel blocker, nifedipine, also inhibited the electrically induced calcium influx and c-fos expression. Moreover, a stretch-activated (SA) channel blocker, gadolinium ion, inhibited the electrically stimulated differentiation by blocking the calcium influx, but gave no prominent effects on the potassium ion-induced differentiation. Chelerythrine, a specific protein kinase C (PKC) inhibitor, almost inhibited the cell differentiation by the electric stimulation but not by the NGF treatment. These results indicate that the alternative potential may stimulate cell differentiation through a PKC cascade.

Site-directed lipid modification of IgG-binding protein by intracellular bacterial lipoprotein process.

IgG-binding protein was genetically expressed and lipid-modified in a site-directed manner in Escherichia coli. The DNA sequence encoding the signal peptide and the nine N-terminal amino acid residues of the major lipoprotein of E. coli (lpp) was fused to the sequence of B-domain which was one of the IgG binding domains of Staphylococcal Protein A (SpA). The N-terminal cysteine residue of the resulting protein was enzymatically linked with lipids in the bacterial membrane. The lipid-modified protein was translocated at the bacterial membrane in a manner similar to native bacterial lipoprotein, and it was purified with IgG-Sepharose by affinity chromatography. The lipid modified proteins (lppB1 and lppB5) showed a similar IgG binding activity to unmodified proteins, which was estimated by competitive ELISA. Proteoliposomes of lipid modified proteins were prepared in an elegant fashion so that the IgG binding site should be properly oriented on the surface of an individual liposome by anchoring the lipid-tail into the hydrophobic layer of the liposome membrane. As compared with the unmodified one, the lipid modified protein incorporated into the proteoliposome exhibited higher IgG binding activity.

Electrically induced neurite outgrowth of PC12 cells on the electrode surface.

Morphological differentiation of PC12 cells cultured on an indium-tin oxide (ITO) electrode has been induced to grow neurites in the absence of nerve growth factor (NGF) by electrical stimulation. Rectangular pulse wave potentials were applied to the electrode at amplitudes of 200 mV and 400 mV with frequencies of 50 Hz, 500 Hz, and 1 kHz. The PC12 cells differentiated most prominently at 200 mV with 100 Hz. No statistically significant differences were observed among the electrically induced neurite lengths. The electrically induced differentiation was completely inhibited by a blockade of calcium influx using LaCl3. This indicates that repeated potential shift in the vicinity of a cellular membrane may stimulate morphological response, probably through calcium ion channels.

Stabilization and translation of immobilized mRNA on latex beads for cell-free protein synthesis system.

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.

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.

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.

Translation of immobilized genetic information by yeast cell-free protein synthesizing system.

A cell-free protein-synthesizing system for the purpose of specific genetic information translation was constructed: ribosome, t-RNA, and enzymes extracted from yeast cells were combined with an immobilized mRNA. The soluble fraction mixed with energy sources and amino acids was incubated with the immobilized mRNA such as poly(U), yeast mRNA, and myeloma mRNA to incorporate [(3)H]phenylalanine into polypeptides of de novo synthesis. By supplying amino acids to these protein-synthesizing systems, amino acid incorporation was ascertained. Lower efficiency of the incorporation in the immobilized system than that of the homogeneous system was mainly attributed to the heterogeneous reaction where the mass-transfer process is diffusion limited. Results obtained show the possibility of a system for specific translation of a desired genetic code.

Effects of shape-discrimination training on the selectivity of inferotemporal cells in adult monkeys.

Through extensive training, humans can become "visual experts, " able to visually distinguish subtle differences among similar objects with greater ease than those who are untrained. To understand the neural mechanisms behind this acquired discrimination ability, adult monkeys were fully trained to discriminate 28 moderately complex shapes. The training effects on the stimulus selectivity of cells in area TE of the inferotemporal cortex were then examined in anesthetized preparations. Area TE represents a later stage of the ventral visual cortical pathway that is known to mediate visual object discrimination and recognition. The recordings from the trained monkeys and untrained controls showed that the proportion of TE cells responsive to some member of the 28 stimuli was significantly greater in the trained monkeys than that in the control monkeys. Cell responses recorded from the trained monkeys were not sharply tuned to single training stimuli, but rather broadly covered several training stimuli. The distances among the training stimuli in the response space spanned by responses of the recorded TE cells were significantly greater in the trained monkeys than those in the control monkeys. The subset of training stimuli to which individual cells responded differed from cell to cell with only partial overlaps, suggesting that the cells responded to features common to several stimuli. These results are consistent with a model in which visual expertise is acquired through the development of differential responses by inferotemporal cells to the images of relevant objects.