Biomedical subjects

Mikako Saito

Publications and source records attributed to Mikako Saito.

16 recordsLinked to original sources

PubMed2006

Semi-quantitative expression and knockdown of a target gene in single-cell mouse embryonic stem cells by high performance microinjection.

Interactions of multiple genes and associated factors are involved in the differentiation and de-differentiation of embryonic stem (ES) cells. Quantitative analysis of these genes and factors is essential for the elucidation of their mechanism. To meet this requirement, we have investigated various experimental conditions for high performance microinjection into mouse ES cells. A speedy and rhythmic operation was found to be important and was accomplished robotically by using a single-cell manipulation technique and XY-address registrable culture dishes. Among many experimental parameters, the tip size of an injection capillary, the pressure condition, and the DNA concentration in the injection capillary were of critical significance. Their optimum values were 0.5-0.8 microm, 0.7 kgf/cm(2) for 30 ms, and 1-100 ng/microl, respectively. Under these conditions, semi-quantitative control of the EGFP gene expression in mouse ES cells and its knockdown was successfully demonstrated.

Animals↗

PubMed2006

Characterization of NtChitIV, a class IV chitinase induced by beta-1,3-, 1,6-glucan elicitor from Alternaria alternata 102: Antagonistic effect of salicylic acid and methyl jasmonate on the induction of NtChitIV.

Tobacco BY-2 class IV chitinases (TBC-1, TBC-3) were rapidly and transiently induced by the beta-1,3-, 1,6-glucan elicitor from Alternaria alternata 102 (AaGlucan). The full-length cDNA and 5'-flanking region of a gene encoding class IV chitinases were isolated on the basis of the amino acid sequence of TBC-1. Sequence analysis indicated that NtChitIV encoded TBC-1, TBC-3, or both. Since purified TBC-1 and TBC-3 from BY-2 cells lack a chitin binding domain in the N-terminal region, these enzymes suggested to be derived from NtChitIV by post-translational proteolytic processing. The transcripts of NtChitIV accumulated rapidly within 1h after treatment with AaGlucan. Accumulation was maximal 3h after treatment. Reporter gene assays were used to analyze the promoter regions involved in the transcriptional control of NtChitIV, and these assays revealed that the 1.89-kb NtChitIV promoter was activated by AaGlucan but not by salicylic acid (SA) or methyl jasmonate (MeJA). The AaGlucan-induced transcriptional activation via 1.89-kb NtChitIV promoter was attenuated by pretreatment with SA or MeJA. These results suggest that NtChitIV expression is particularly induced by AaGlucan and that the AaGlucan-dependent signaling pathway is different from the SA- and MeJA-dependent signaling pathways.

Acetates↗

PubMed2006

Rapid evaluation of the efficacy of microbial cell removal from fabrics.

The efficacy of microbial cell removal (EMR) from fabrics is a practically important indicator for the evaluation of cleansers and detergents. EMR is expressed quantitatively by the relative number of viable cells remaining on a fabric swatch after the treatment with these reagents. In order to count the viable cells on the swatch directly and rapidly, we have developed a unique microscopic imaging system with an ultra-deep focusing range. Standard swatches of cotton fabric were inoculated with microorganisms such as Pseudomonas fluorescence, Staphylococcus aureus, or Candida albicans. After the incubation on an agar medium, each swatch was treated with a fluorescent glucose, 2-[N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl) amino]-2-deoxyglucose, to stain only viable cells. The images of every cell distributed within the surface layer with no greater than 130 microm thickness could be integrated into one image. Thus visualized cells could be counted automatically by a novel imaging program. Using a pair of cotton swatches (0.5 x 1.0 cm(2)) inoculated with C. albicans, EMR was evaluated quantitatively. Before washing, the total number of viable cells found on the observation area (3.8 x 10(-4 )cm(2)) was 288 cells. After washing with a test detergent, no cell (<1) was detected. For this case, EMR was given by the formula: log(288/<1)=greater than 2.5. The imaging and cell count of a test fabric could be performed within 1 h.

4-Chloro-7-nitrobenzofurazan↗

PubMed2006

Automatic positioning of a microinjector in mouse ES cells and rice protoplasts.

A 2-channel (2C) microinjector was prepared by pulling a glass capillary with a theta-shaped cross section. One channel was used as a potential measuring electrode (MeaE) and the other was used as an electrophoretic introduction electrode (IntE). The 2C microinjector was propelled by an oil pressure manipulator driven by a pulse motor, while the MeaE output was recorded continuously. When the 2C microinjector penetrated the cell membrane of a mouse ES cell or a rice protoplast, the output potential changed sharply. The differential of this potential change was used as a stop signal for the pulse motor. Thus, the microinjector was correctly positioned in the cell without losing cell viability. Its success rate was 73% and 84% for ES cells and rice protoplasts, respectively. After the positioning of the microinjector in the cell, Lucifer yellow (LY) was introduced via IntE. Under these conditions, the rate of viable cells was 16% and 62% for ES cells and rice protoplasts, respectively.

Animals↗

PubMed2006

Novel beta-1,3-, 1,6-oligoglucan elicitor from Alternaria alternata 102 for defense responses in tobacco.

A novel elicitor that induces chitinases in tobacco BY-2 cells was isolated from Alternaria alternata 102. Six other fungi, including A. alternata IFO 6587, could not induce, or weakly induce chitinase activity. The purified elicitor was soluble in 75% methanol and showed the chitinase-inducing activity when applied at concentrations of as low as 25 ng x mL(-1). Structural determination by methylation analysis, reducing-end analysis, MALDI-TOF/MS, and NMR spectroscopy indicated that the elicitor was a mixture of beta-1,3-, 1,6-oligoglucans mostly with a degree of polymerization of between 8 and 17. Periodate oxidation of the elicitor suggested that the 1,6-linked and nonreducing terminal residues are essential for the elicitor activity. Further analysis of the elicitor responses in BY-2 cells indicated that the activity of this beta-1,3-, 1,6-glucan elicitor was about 1000 times more potent than that of laminarin, which is a known elicitor of defense responses in tobacco. Analyzing the expression of defense-related genes indicated that a phenylalanine ammonia-lyase gene and a coumaroyl-CoA O-methyltransferase gene were transiently expressed by this beta-1,3-, 1,6-glucan elicitor. The elicitor induced a weak oxidative burst but did not induce cell death in the BY-2 cells. In the tissue of tobacco plants, this beta-1,3-, 1,6-glucan elicitor induced the expression of basic PR-3 genes, the phenylpropanoid pathway genes, and the sesquiterpenoid pathway genes. In comparison with laminarin and laminarin sulfate, which are reported to be potent elicitors of defense responses in tobacco, the expression pattern of genes induced by the purified beta-1,3-, 1,6-glucan elicitor was more similar to that induced by laminarin than to that induced by laminarin sulfate.

Alternaria↗

PubMed2006

Rapid separation and counting of viable microbial cells in food by nonculture method with bioplorer, a focusing-free microscopic apparatus with a novel cell separation unit.

A nonculture method utilizing a novel apparatus, the bioplorer, was developed. The bioplorer is composed of an efficient cell separation unit, a focusing-free microscopic device, and an image analysis program. A meat or vegetable suspension is poured into the cell separation funnel, and insoluble matter in the sample suspension is trapped by prefilters. Microbial cells passing through the two prefilters are then trapped by the membrane filter (pore size, 0.4 microm). Trapped cells are double-stained with 4',6'-diamidino-2-phenylindole and propidium iodide, and the membrane filter is removed and set on the focusing-free microscope. A fluorescent image is then recorded. Total numbers of viable and dead cells on the membrane filter can thus be determined automatically. One assay can be performed within 10 min, which is much faster than the culture method. The results obtained with both the nonculture method and the culture method for meat and vegetable samples were highly correlated (r = 0.953 to 0.998). This method is feasible for the practical purpose of food safety control.

Bacteria↗

PubMed2004

High throughput easy microinjection with a single-cell manipulation supporting robot.

A single-cell manipulation supporting robot (SMSR) has been developed for the high throughput and easy microinjection. Its concept is to let an experimenter concentrate his/her attention only on the microinjection by facilitating other associated works. SMSR was applied to the microinjection into rice protoplasts and mouse embryonic stem (ES) cells. The microinjection into these cells is exceptionally difficult than usual animal cells such as fibroblasts. In the case of rice protoplast, for example, non-stop microinjection into 100 cells could be done within 1h that was 17-times faster than that of the robot-less work. The success rate was 7-8% that was same level obtained by the robot-less work. The present results indicate that SMSR is a useful machine for the microinjection of specific genes and proteins in living cells to analyze their respective functions, which is an urgent and important subject in the post-genome era.

Animals↗

PubMed2004

Functional analysis of a rice putative voltage-dependent Ca2+ channel, OsTPC1, expressed in yeast cells lacking its homologous gene CCH1.

We isolated a cDNA (OsTPC1) from rice that was homologous to AtTPC1, a putative voltage-dependent Ca(2+) channel (VDCC) gene of Arabidopsis thaliana. The hydropathy profile of its deduced amino acid sequence showed significant structural features of the alpha 1-subunit of animal VDCCs. Functional analysis using a heterologous yeast expression system showed that OsTPC1 facilitates Ca(2+) permeation. The K(m) value for Ca(2+) of OsTPC1, 47.5 micro M, was comparable to that of intrinsic CCH1, a candidate VDCC in yeast. Ca(2+) permeation by OsTPC1 was inhibited by verapamil, a VDCC blocker. These findings indicate for the first time that OsTPC1 is a putative VDCC in rice.

Amino Acid Sequence↗

PubMed2004

Cell-lytic activity of tobacco BY-2 induced by a fungal elicitor from alternaria alternata attributed to the expression of a class I beta-1,3-glucanase gene.

Stress-induced cell-lytic activity was found in tobacco BY-2 cells treated with various stresses. Among 14 stresses, an elicitor fraction isolated from Alternaria alternata showed the highest inducing activity. Cell-lytic activity increased for 72 h even in the control sample, treated with distilled water, and several isozymes of beta-1,3-glucanases and chitinases were found to be involved in it. In contrast, cell-lytic activity in BY-2 cells treated with a fungal elicitor reached a higher level after 60 h. The principal enzymes specifically involved in this stress-induced portion are speculated to be basic beta-1,3-glucanases. A class I beta-1,3-glucanase gene (glu1) was found to be the specific gene for the stress-induced cell-lytic activity. Its expression became observable at 24 h, and the intensity reached a maximum at about 60-72 h. The glu1 was thus assigned as a late gene. Its role in the stress response is discussed in conjunction with earlier genes such as chitinases.

Gene Expression Regulation↗

PubMed2004

Production of a cloned mouse by nuclear transfer from a fetal fibroblast cell of a mouse closed colony strain.

We have tested a closed colony mouse strain as a source for nuclei donors to determine differences in cloning efficiency. When donor nuclei were isolated from fetal fibroblast cells and injected into recipient oocytes from closed colony mice (ICR), reconstructed oocytes developed to full term and the success rate of cloning was 0.1%. This result indicates that cloning efficiency does not depend on the cell type. The body weight of the cloned mouse was lighter than controls, and the lifetime of the cloned mouse was the average for a mouse. These results contradict commonly-held views on cloning.

Animals↗

PubMed2003

Expression of rice chitinase gene triggered by the direct injection of Ca2+.

In response to an elicitor, the Ca2+-dependent fluorescence (Fluo-3-Ca2+) increased transiently and then the expression of the chitinase gene (chi) followed. The gene expression was detected by Northern analysis. The deletion of Ca2+ from the medium or the addition of a Ca2+ channel blocker, verapamil, to the medium caused no gene expression, which supported the key role of Ca2+ in the signaling towards the chi expression. Then the Ca2+-injection experiment was done in order to investigate if it could trigger the chi expression. The plasmid pCHI-GFP (promoter: chi, reporter: green fluorescent protein gene (gfp)) was injected into the single-protoplasts, then after 1 day of incubation at 25 degrees C, 100 microM CaCl2 was injected into the same cells. After successive incubation for 1 day, 41 out of 85 cells showed the gene expression. The injection of 100 microM MgCl2, however, caused no gene expression. Therefore, Ca2+ could induce the chi of rice in the absence of the elicitor stimulus.

Calcium↗

PubMed2003

Isolation of H+ -translocating ATPase in tonoplast of Tradescantia virginiana L. leaf cells.

The tonoplast of Tradescantia virginiana L. was prepared from leaf cells and then solubilized with deoxycholate (DOC) and n-octyl-beta-D-glucoside (n-OG). Three major polypeptides (68, 60, 16 kDa) and several other minor components were isolated. These polypeptides were reconstituted in soybean phospholipids (asolectin). The H(+) pump activity was investigated with the reconstituted system as well as with the tonoplast. In both cases, the quinacrine-fluorescence quenching was observed in the presence of ATP-Mg(2+), indicating the H(+) pumping. The H(+) pump activity was inhibited by gramicidin D, a channel-forming ionophore, and by KNO(3), an inhibitor specific to tonoplast-type (V-type) H(+)-ATPase.

Enzyme Activation↗

PubMed2003

Viable cell detection by the combined use of fluorescent glucose and fluorescent glycine.

The combined use of a fluorescent glucose (2NBDG) and a fluorescent glycine (NBD-Gly) was tried for the detection of viable cells of significant foodborne pathogenic strains in addition to several Escherichia coli strains and coliforms. Thirty-five out of 41 strains showed marked uptake of 2NBDG but 6 strains were not able to take in 2NBDG. Five out of these 6 strains showed NBD-Gly uptake.

4-Chloro-7-nitrobenzofurazan↗

PubMed2002

Single-cell viability assessment with a novel spectro-imaging system.

Single-cell viability assessment by means of plural dye probes require the spectral and temporal analysis of microscopic images of the test cells. To meet this requirement, we have developed a simple and compact spectro-imaging system using an image slicer and a grism. The image slicer was made of a bundle of 100 optical fibers. The field of view is divided into 10 x 10 sections. The spectral data of each section could be recorded every 5 s in the range from 400 to 800 nm at 5 nm resolution. The viability changes of yeast or tobacco single-cells were measured with this system. Using BY-2 cells, for example, the response to a chemical stress of saponin was measured by means of two fluorescent probes. The spectral-spatial-temporal data of fluorescein and DNA bound ethidium bromide provided us with useful information about the dynamic change of cell membrane permeability from which the cell viability was assessed.

Amphotericin B↗

PubMed2002

Single-cell imaging of the Ca2+ influx into bovine endothelial cells occurring in response to an alternating electric stimulus.

The electric control of cellular functions via Ca2+ was formerly suggested. From this viewpoint, the involvement of a Ca2+ channel was studied using bovine fetal arterial endothelial (BFAE) cells in which P2X4, an ATP-operated and fluid shear stress sensitive Ca2+ channel, exists predominantly. An electric stimulus (sine wave, 10 Hz, 10 VPP, 30 s) caused a marked influx of Ca2+ into BFAE cells from an extracellular solution. The magnitude of the [Ca2+]i change increased with a decrease in the frequency in the range from 100 Hz to 5 Hz. Regarding the pathway of this Ca2+ influx, single-cell imaging and an ATP depletion experiment strongly suggested the involvement of a pathway different from P2X4. This pathway was thought to be a non-specific one, because typical Ca2+ channel blockers, such as verapamil, Gd3+, and Co2+, could not inhibit the Ca2+ influx.

Animals↗

PubMed2002

Simultaneous imaging of multiple fluorescent probes in bio-cells.

In order to obtain the full spectrum from 400 to 800 nm of each pixel of a microscopic image, a unique spectro-imaging system was developed using an image slicer. The image slicer is composed of 100 photo fibers which are arranged in a matrix of 10 x 10 at the entrance and 100 x 1 at the exit. A line of this 100 signals is passed through a glism and projected onto a CCD. This system was applied to the fluorescent imaging of bio-cells. One of the demonstrative examples was simultaneous measurements of the Ca2+ concentration and the pH using of respective fluorescent probes. An electric signal was applied to BY-2 protoplasts and the fluorescent spectrum from 500 nm to 800 nm was measured every 5 s. The spectrum of the BY-2 protoplasts changed in response to the electric signal and the Ca2+ concentration, and the pH changes could be monitored. The wavelength resolution was satisfactory, but the space resolution was still rough in comparison with the usual microscopic systems. Notwithstanding these conditions, we could obtain discrete data from more than several tens of sites in a single-cell or a chain of several cells.

Calcium↗