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Biomedical subjects

T Kaneta

Publications and source records attributed to T Kaneta.

At least 19 recordsLinked to original sources

Molecular cloning and gene expression of a fibrillarin homolog of tobacco BY-2 cells.

Fibrillarin is known to play an important role in precursor ribosomal RNA processing and ribosome assembly. The present study describes a fibrillarin homolog gene isolated from tobacco BY-2 cells and its expression during the cell cycle. The cDNA for a fibrillarin homolog, named NtFib1, was first cloned in Nicotiana tabacum with degenerate primers. It encodes 314 amino acids and the deduced amino acid sequence has some highly conserved functional domains, such as the glycine and arginine-rich (GAR) domain for nucleolar localization and the RNA-binding motif. The C-terminal region is highly conserved and has 7 beta-sheets and 7 alpha-helices which are peculiar to fibrillarin. Thus, it is suggested that the fibrillarin homolog of this plant species functions in the same way as the fibrillarin already known from human and yeast cells. Northern blot analysis of BY-2 cells synchronized with aphidicolin or a combination of aphidicolin and propyzamide showed that the histone H4 gene was specifically expressed in the S phase but NtFib1 mRNA remained at high levels during the cell cycle. Examination of the localization of NtFib1 protein tagged with green-fluorescent protein (GFP) suggested that some persisting in the mitotic apparatus was eventually incorporated into reconstructed nucleoli in late telophase. Newly synthesized GFP-tagged NtFib1 protein in the cytoplasm was added to the recycled protein in early mitosis. Highly concentrated actinomycin D completely inhibited the transcription of genes coding for rRNA (rDNA) but did not significantly suppress the amount of either NtFib1 mRNA or protein, although the NtFib1 protein was reversibly dislocated from nucleoli. Although hypoxic shock completely prohibited rDNA transcription, NtFib1 mRNA remained at the same level as in the control experiment, even after the 4 h treatment. These results indicate that the transcription of NtFib1 mRNA is not related to rDNA transcription and NtFib1 mRNA is resistant to disrupting factors during the cell cycle.

Amino Acid Sequence↗

Comparative study of cellular structures implicated in gravisensing in statocytes of primary and lateral roots of Vigna angularis.

The cellular structures of statocytes implicated in gravisensing in primary and lateral roots of Vigna angularis were compared. The statocytes of lateral roots already had small amyloplasts immediately after they emerged from the primary root. Although these amyloplasts sedimented, the lateral roots showed much weaker gravitropism than primary roots, at least until they reached a length of about 30 mm. The nuclei were usually positioned in the upper end of the statocytes in both types of roots. Electron microscopic surveys showed that many tubular elements of endoplasmic reticulum (ER) were frequently localized in the lower end of the statocyte and they sometimes diverged or curved, suggesting that the ER forms a large reticulate complex. It is worth noting that statocytes with a large ER complex were found much more frequently in primary roots than in lateral roots. The amyloplasts were not always settled on this complex but were very frequently under it, especially in the primary roots. In lateral roots, they were usually localized under the ER complex when they were present. Thus, it is suggested that the differential development and organization of the amyloplast-ER complex system is involved in the differential gravitropism of the two types of roots.

Endoplasmic Reticulum↗

An "optical channel": a technique for the evaluation of biological cell elasticity.

The development of an optical channel, a new analytical technique for evaluating the elasticity of biological cells, is described in this study. Two types of erythrocyte cells, i.e., young and old cells, were examined via their introduction into a flowing medium, to which a laser beam was focused in the opposite direction. An erythrocyte cell is trapped in a laser beam by a gradient force, moves in the downstream direction, and is then elongated at the beam waist. The change in shape was measured directly using a microscope equipped with a charge-coupled-device camera. It is probable the main driving force for the cell deformation is a shear stress generated by a medium flow, since an estimate of the gradient force suggests that it is too small to change the shape of an erythrocyte. The average values of the elongation of young and old cells were 2.4+/-0.6 and 2.1+/-0.5, respectively. These values are in reasonably good agreement with values obtained using a rheoscope method. The deformation was measured without any physical contact to the solid surface, and therefore, damage to cells such as these are minimal.

Algorithms↗

Immunoassay for human serum albumin using capillary electrophoresis-semiconductor laser-induced fluorometry.

Capillary electrophoresis combined with semiconductor laser-induced fluorometry was applied to an immunoassay of human serum albumin. Human serum albumin was labeled with a fluorescent molecule (Cy5), which has an absorption maximum at 649 nm. The labeled albumin was purified by ultrafiltration in order to reduce signals, which are unreacted labeling reagent, product, and fragment products derived there from. After the purification, no signal for unreacted labeling reagent and fragment products was detectable in the electropherogram of the labeled albumin. The labeled albumin was then reacted with anti-albumin to form an immunocomplex, which was separated from the excess free albumin. The competitive immunoassay was used in the determination of human serum albumin in a controlled serum sample, using the labeled albumin. The obtained value was found to be 0.21 +/- 0.02 mg/ml, which is in good agreement with other known values.

Electrophoresis, Capillary↗

Determination of inorganic anions in human saliva by zwitterionic micellar capillary electrophoresis.

Capillary electrophoresis (CE) using sulfobetaine-type zwitterionic micelles as the background electrolyte (BGE) has been used to determine inorganic anions in human saliva. The zwitterionic micelles resulted in unique migration behavior for the separation of inorganic anions. They also prevented adsorption of proteins on the inner wall of the capillary. These properties of the zwitterionic micelles enabled the direct determination of inorganic anions in human saliva. Three species of inorganic anions, NO2-, NO3-, and SCN-, were found in real samples and the analysis was achieved within 3 min. Direct UV-absorption was used as the detection method and the detection limits for these anions were 2.0, 1.0, and 5.0 micromol L(-1), respectively (0.09, 0.06, and 0.30 microg mL(-1)).

Anions↗

Migration behavior of alkylphenols, bisphenol A and bisphenol S studied by capillary electrophoresis using sulfated beta-cyclodextrin.

An application of capillary electrophoresis (CE) using sulfated beta-cyclodextrin (SCD) has been investigated for separating alkylphenols with different chain lengths, as well as bisphenol A and bisphenol S. In the absence of SCD in running buffer, all the phenols migrated at the same velocity as the electroosmotic flow (EOF), whereas the addition of SCD effectively led to the baseline separation of alkylphenols on the basis of the difference in the abilities to bind into the hydrophobic cavity of CD. The host-guest binding constants between analyte phenols and SCD were evaluated from Benesi-Hildebrand plots of the data obtained by two independent methods, CE and UV-visible measurements, demonstrating that the greater the hydrophobicity of the phenols, the larger the binding constants. The effects of organic solvents on the resolution for alkylphenols and bisphenols were also examined. This system using SCD was effective for the separation of 4-octylphenol and 4-nonylphenol isomers having longer alkyl chains.

Journal Article↗

Determination of motility forces of bovine sperm cells using an "optical funnel".

An optical funnel, a new technique for the evaluation of the force of a microorganism, was applied to the determination of the motility force of bovine sperm cells. In this approach, sperm cells, suspended in an aqueous solution, are introduced into a flow cell, to which radiation pressure is applied from the direction opposite to a medium flow. The sperm cell, which is moving in a stream, is captured by radiation pressure and forced to move to the position at which the force induced by the laser radiation is equal to the force induced by a medium flow. The sperm cell then escapes by its own power on the way to this equilibrium (entrapping) position. The radiation force increases with decreasing distance from the focal point, and as a result, the force of the sperm cell can be determined by measuring the position where the sperm cell escaped against the laser irradiation field. The motility force of the sperm cell was measured in aqueous solution at different pH values and potassium ion concentrations. It was possible to measure more than 250 sperm cells in 3 h. Thus, the optical funnel has potential for use as a rapid and repetitive means for the determination of the motility force of the sperm cell.

Algorithms↗

[Correlation of pulmonary perfusion volume analysis with pulmonary function in emphysema].

Pulmonary perfusion single photon emission tomography with 99mTc MAA was performed on 13 pulmonary emphysema patients and 6 controls. We calculated perfusion volume with lower 10%, 20%, 30%, 40% and 50% of the highest counts/boxels in the lung cut-off. And perfusion index (PI) was defined as follows; PI = ((A% cut-off volume) - (B% cut-off volume))/(A% cut-off volume); A and B take 10 to 50, A < B. The correlation of each PI and pulmonary function test results (FEV1, FEV1%, VC, VC%, FVC, FVC%, PaO2 and PaCO2) was examined. There were significant correlation between every PI and FEV1 or FEV1% (p < 0.05), and any PI had no significant correlation with other functional results. When A = 10 and B = 40, the PI showed the best correlation with FEV1 (r = 0.680) and FEV1% (r = 0.830). And the PI showed an increasing tendency along with the rise of the emphysema severity. The PI may have the clinical utility of the evaluation of pulmonary function. Moreover, we showed the lung CT painted the area where the uptake counts/boxels was more than 10% and less than 40% of the highest counts/boxels. This makes it easy to understand the severe emphysematous area.

Adult↗

Regulation of gibberellin biosynthesis genes during flower and early fruit development of tomato.

Gibberellins (GAs) are essential for the development of fertile flowers in tomato, and may also be required immediately after fertilization. In the GA-biosynthetic pathway, the reactions catalyzed by GA 20-oxidases have been implicated as site of regulation. To study the regulation of GA biosynthesis in flower and early fruit development, we isolated three tomato GA 20-oxidase cDNA clones, Le20ox-1, -2 and -3. The three genes showed different organ-specific patterns of mRNA accumulation. Analysis of the transcript levels of the three GA 20-oxidase genes, as well as those of copalyl diphosphate synthase (LeCPS) and GA 3 beta-hydroxylase (Le3OH-2) during flower bud and early fruit development, revealed temporally distinct patterns of mRNA accumulation. Up until anthesis, transcripts were observed for LeCPS, Le20ox-1, -2 and Le3OH-2, with an accumulation of Le20ox-1 mRNA. In contrast to the high level of Le3OH-2 transcripts in the fully open flower, mRNA levels of Le20ox-1, -2 and LeCPS were reduced at this stage. After anthesis, LeCPS and Le20ox-1 transcripts increased again. In addition, Le20ox-3transcripts increased whereas the transcripts of Le3OH-2 decreased to an undetectable level. In situ hybridization results demonstrated that during early stages of bud development, Le20ox-2 transcripts were localized in the tapetum and placenta. The presented results supply novel data about localization of GA biosynthesis gene transcripts, and indicate that transcript levels of GA biosynthesis genes are all highly regulated during flower bud development.

Alkyl and Aryl Transferases↗

[Compartment analysis of 123I-iomazenil brain SPECT in patients with moyamoya disease].

We investigated 11 patients with moyamoya disease about 123I-Iomazenil kinetics in the brain using three-compartment, two-parameter model. The transition rate constant (K1) from the blood to the brain and the binding potential (BP) of the benzodiazepine to the receptors were calculated for every ROI (right and left side of cerebellum, frontal lobe, parietal lobe, occipital lobe and temporal lobe; 10 ROIs a case). The K1 value correlated with BP value significantly, but not so closely (r = 0.639). And there is no significant difference in BP value among low-K1 group (mean (of K1) -S.D. < or = K1 < or = mean) and high-K1 group (mean < K1 < or = mean + S.D.). This means that CBF and BP do not correlate closely in the average Moyamoya disease patients. And we showed a case with IMP/IMZ discrepancy. The nerve cell in the hypoperfused area which has almost normal BP value is ischemic but viable. IMZ-SPECT presents an important information about the viability of the hypoperfused area in Moyamoya disease patients' brain.

Adult↗

S-adenosyl-L-methionine:L-methionine S-methyltransferase from germinating barley. Purification and localization.

S-Adenosyl-L-methionine:L-methionine S-methyltransferase (MMT) catalyzes the synthesis of S-methyl-L-methionine (SMM) from L-methionine and S-adenosyl-L-methionine. SMM content increases during barley (Hordeum vulgare L.) germination. Elucidating the role of this compound is important from both a fundamental and a technological standpoint, because SMM is the precursor of dimethylsulfide, a biogenic source of atmospheric S and an undesired component in beer. We present a simple purification scheme for the MMT from barley consisting of 10% to 25% polyethylene glycol fractionation, anion-exchange chromatography on diethylaminoethyl-Sepharose, and affinity chromatography on adenosine-agarose. A final activity yield of 23% and a 2765-fold purification factor were obtained. After digestion of the protein with protease, the amino acid sequence of a major peptide was determined and used to produce a synthetic peptide. A polyclonal antibody was raised against this synthetic peptide conjugated to activated keyhole limpet hemocyanin. The antibody recognized the 115-kD denatured MMT protein and native MMT. During barley germination, both the specific activity and the amount of MMT protein increased. MMT-specific activity was found to be higher in the root and shoot than in the endosperm. MMT could be localized by an immunohistochemical approach in the shoot, scutellum, and aleurone cells but not in the root or endosperm (including aleurone).

Amino Acid Sequence↗

Theory of optical chromatography.

To evaluate the performance of optical chromatography, a number of equations are theoretically derived using a ray-optics model. These mathematical formalisms are experimentally verified by determining the relationship between the velocity of motion of a polystyrene bead with respect to the intensity of an applied radiation force under the condition where there exists no applied fluid flow. The force is confirmed to be at a maximum at the focal point and to decrease with increasing distance from this position. The radiation force is verified to be proportional to the square of the particle size when the particle diameter is much smaller than the beam diameter. In addition, the radiation force is ascertained to be proportional to the laser power. These results are in excellent agreement with the proposed theoretical model, which is based on ray optics. Furthermore, by analogy with conventional chromatography, fundamental parameters such as retention distance, selectivity, theoretical plate number, and resolution are calculated, and optimum conditions for chromatographic separation are discussed. Based on the results obtained, the dynamic range can be extended by increasing laser power and decreasing flow rate. Peak broadening is primarily caused by variations in laser power and flow rate of the medium for large particles (< 1 microm). It is possible, in theory, to distinguish particles whose diameters differ by less than 1% for particles with a diameter larger than 1 microm. Three sizes of polystyrene beads are well separated at a flow rate of 20 microm s(-1) and a laser power of 700 mW. This technique is also applied to the separation of human erythrocytes. Two fractions, one consisting of cells ranging from 1.5 to 2.4 microm in diameter and another consisting of cells ranging from 3.5 to 5.7 microm in diameter, are observed. Optical chromatography is useful for separation and size measurement of particles and biological cells.

Cell Separation↗

Application of optical chromatography to immunoassay.

Optical chromatography, a new separation technique involving the use of a radiation force and a medium flow, is used for trace analysis of protein. Two polystyrene beads, coated with antibody (anti-mouse IgG), are combined in the presence of an antigen (mouse IgG). The bound (B) and free (F) beads are readily separated by optical chromatography, and the B/F ratio can be correlated with the concentration of antigen (protein). Nanomolar concentrations of protein can be measured by this technique. The rates of the forward and reverse immunological reactions were independently determined by measuring the time of formation and dissociation, respectively, of the immunobeads.

Animals↗

Gibberellin A3 causes a decrease in the accumulation of mRNA for ACC oxidase and in the activity of the enzyme in azuki bean (Vigna angularis) epicotyls.

Differential screening, aimed at the isolation of cDNA clones of mRNAs whose accumulation is influenced by GA3, resulted in the isolation of a cDNA clone of an mRNA whose level was decreased by GA3 in segments of epicotyls of Vigna angularis. The putative protein encoded by this cDNA resembled the 1-aminocyclopropane-1-carboxylate oxidases (ACC oxidases) identified in other plant species (about 80% homology at the amino acid level). Thus, the corresponding gene was designated AB-ACO1 (azuki bean ACC oxidase). GA3 also decreased the activity of ACC oxidase in azuki bean epicotyls, but it did not decrease the rate of ethylene evolution. In fact, GA3 increased the rate of ethylene evolution and the level of ACC. Thus, GA3 seemed to increase the production of ethylene by promoting the synthesis of ACC.

Amino Acid Oxidoreductases↗

[Effects of combination chemoradiotherapy with daily low-dose CDDP for esophageal cancer--results of a randomized trial].

Between 1994 and 1996, 24 patients with esophageal carcinoma were entered in a randomized clinical trial comparing radiotherapy (RT) only (arm A) versus RT and a daily low dose of CDDP (5 mg/m2) (arm B). For arm B group, CDDP was injected within 30 min after irradiation. All patients were irradiated with a total dose of 60 Gy /30fr. at first, and some patients were boosted. An overall response rate of 75.0% (complete response rate of 25.0%) was observed in arm A and 91.7% (complete response rate of 16.7%) in arm B, respectively. The survival rate at 1 year was 23.8% in arm A and 40.0% in arm B. Median survival time was 7 months in arm A and 9 months in arm B. Toxicity was acceptable and no significant difference was noted between arm A and arm B.

Aged↗

Indirect detection of aromatic hydrocarbons by semiconductor laser fluorometry in micellar electrokinetic chromatography.

A semiconductor laser is used as an exciting light source in indirect fluorescence detection of neutral samples separated by micellar electrokinetic chromatography. A surfactant, tetradecyltrimethylammonium chloride, is used to form micelles, which act as a pseudostationary phase in the chromatographic separation. This surfactant has a positive charge, thus preventing adsorption of a visualizing agent, oxazine 750 (which contains a positive charge), to the capillary wall (which is negatively charged). The detection mechanism is based on the exclusion of the fluorophore, which is located in the hydrophilic part of the micelle, by a hydrophilic sample. The fluorescence intensity is reduced when the fluorophore is released into the aqueous solution. Separations of several aromatic compounds are demonstrated, and the parameters affecting the selectivity are discussed.

Chromatography↗

Effect of organic modifier on resolution of hydrophobic compounds by micellar electrokinetic chromatography.

Three organic modifiers, methanol, acetonitrile, and dimethylformamide, were used as organic modifiers to improve the resolution of hydrophobic compounds in micellar electrokinetic chromatography. The capacity factor decreased with increasing concentration of organic modifier. Acetonitrile and dimethylformamide were more effective modifiers to decrease the capacity factor than methanol. Maximum resolution was obtained when the capacity factor was reduced to about 2. Methanol enhanced the resolution for less hydrophobic compounds, while acetonitrile and dimethylformamide were better modifiers to improve the resolution for strongly hydrophobic compounds.

Acetonitriles↗