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Takehito Ito

Publications and source records attributed to Takehito Ito.

7 recordsLinked to original sources

Input-output analysis of in vivo photoassimilate translocation using Positron-Emitting Tracer Imaging System (PETIS) data.

The Positron-Emitting Tracer Imaging System (PETIS) is introduced for monitoring the distribution of (11)C-labelled photoassimilates in Sorghum. The obtained two-dimensional image data were quantitatively analysed using a transfer function analysis approach. While one half of a Sorghum root in a split root system was treated with either 0, 100, or 500 mM NaCl dissolved in the nutrient solution, tracer images of the root halves and the lower stem section were recorded using PETIS. From the observed tracer levels, parameters were estimated, from which the mean speed of tracer transport and the proportion of tracer moved between specified image positions were deduced. Transport speed varied between 0.7 and 1.8 cm min(-1) with the difference depending on which part of the stem was involved. When data were collected in the lowest 0.5-1 cm of the stem, which included the point where the roots emerge, transport speed was less. Rapid changes in NaCl concentration, from 0 to 100 mM, resulted in short-term increases of assimilate import into the treated root. This response represented a transient osmotic effect, that was compensated for in the medium-term by osmotic adaptation. Higher concentrations of NaCl (500 mM) resulted in distinctly less photoassimilate transport into the treated root half. The present results agree with earlier observations, showing that transport of (11)C-labelled photoassimilates measured with the PETIS detector system can be quantified using the method of input-output analysis. It is worth noting that with the PETIS detector system, areas of interest do not need to be defined until after data collection. This means that unexpected behaviour of a plant organ will be seen, which is not necessarily the case with conventional detector systems looking at predefined areas of interest.

Biological Transport↗

Synthesis and evaluation of N-(5-fluoro-2-phenoxyphenyl)-N-(2-[(18)F]fluoromethoxy-d(2)-5-methoxybenzyl)acetamide: a deuterium-substituted radioligand for peripheral benzodiazepine receptor.

N-(5-Fluoro-2-phenoxyphenyl)-N-(2-[(18)F]fluoromethoxy-d(2)-5-methoxybenzyl)acetamide ([(18)F]2) is a potent ligand (IC(50): 1.71 nM) for peripheral benzodiazepine receptor (PBR). However, in vivo evaluation on rodents and primates showed that this ligand was unstable and rapidly metabolized to [(18)F]F(-) by defluorination of the [(18)F]fluoromethyl moiety. In this study, we designed a deuterium-substituted analogue, N-(5-fluoro-2-phenoxyphenyl)-N-(2-[(18)F]fluoromethoxy-d(2)-5-methoxybenzyl)acetamide ([(18)F]5) as a radioligand for PBR to reduce the in vivo metabolic rate of the non-deuterated [(18)F]2. The design principle was based on the hypothesis that the deuterium substitution may reduce the rate of defluorination initiated by cleavage of the C-H bond without altering the binding affinity for PBR. The non-radioactive 5 was prepared by reacting diiodomethane-d(2) (CD(2)I(2), 6) with a phenol precursor 7, followed by treatment with tetrabutylammonium fluoride. The ligand [(18)F]5 was synthesized by the alkylation of 7 with [(18)F]fluoromethyl iodide-d(2) ([(18)F]FCD(2)I, [(18)F]9). Compound 5 displayed a similar in vitro affinity to PBR (IC(50): 1.90 nM) with 2. In vivo evaluation demonstrated that [(18)F]5 was metabolized by defluorination to [(18)F]F(-) as a main radioactive component, but its metabolic rate was slower than that of [(18)F]2 in the brain of mice. The deuterium substitution decreased the radioactivity level of [(18)F]5 in the bone of mouse, augmented by the percentage of specific binding to PBR in the rat brain determined by ex vivo autoradiography. However, the PET image of [(18)F]5 for monkey brain showed high radioactivity in the brain and skull, suggesting a possible species difference between rodents and primates.

Acetamides↗

Improved quality control of [18F]FDG by HPLC with UV detection.

A conventional high-performance liquid chromatographic (HPLC) method for the analysis of 2-fluoro-2-deoxy-d-glucose (FDG) and 2-deoxy-2-chloro-d-glucose (ClDG) in [18F]FDG preparations is described. This method was based on a postcolumn derivatization with 2-cyanoacetamide (2-CA) and UV detection. FDG and ClDG were separated on a normal-phase column using acetonitrile/water as the mobile phase. The eluate was mixed with 2-CA in sodium borate buffer solution at the outlet of a PTFE coil (10 m x 0.5 mm id) from the column, and the reaction was carried out at 100 degrees C during the passage through the coil. The UV absorbance of the resultant product was monitored at 276 nm. Under optimum conditions, the detection limits [signal-to-noise (S/N) ratio=3] for FDG and ClDG were 0.31 and 0.17 microg/ml for a 20-microl injection volume, respectively, and the linearity ranges were 0.5-100 microg/ml for both compounds. The intra- and interday reproducibilities were better than 2.2% [relative standard deviation (R.S.D.)]. This HPLC separation procedure is also useful for determining the radiochemical purity of [18F]FDG preparations since it allows the analysis of 2-[18F]fluoro-1,3,4,6-tetra-O-acetyl-d-glucose ([18F]TAG), partially hydrolyzed [18F]TAG and [18F]F-. This method can be used at many positron emission tomography (PET) facilities since it does not require an expensive, sophisticated electrochemical detector.

Chromatography, High Pressure Liquid↗

Nerve growth factor-induced expression of the GTP cyclohydrolase I gene via Ras/MEK pathway in PC12D cells.

Neurotrophins are essential for the development and survival of the catecholaminergic neurons. GTP cyclohydrolase I (GCH) is the first and rate-limiting enzyme in the biosynthesis of 5,6,7,8-tertahydrobiopterin (BH4), the required cofactor for tyrosine hydroxylase. Previously, we reported that TH requires the Ras/mitogen-activated protein kinase kinase (MEK) pathway for its induction by nerve growth factor (NGF). Here, we examined intracellular signals required for NGF-induced expression of the GCH gene in PC12D cells. The activity of GCH was increased up to 5-fold after the NGF treatment, and the increase was repressed by pretreatment with U0126, an MEK1/2 inhibitor, but not with protein kinase A (PKA), phosphoinositide 3-kinase (PI3K), p38 mitogen-activated protein kinase (MAPK), and c-Jun NH2-terminal kinase (JNK) inhibitors. Induction of GCH mRNA by NGF was also abolished by pretreatment with U0126. The human GCH promoter activity was significantly enhanced by NGF treatment. Deletion analysis showed that the 465-bp 5'-flanking region is responsible for NGF-enhanced promoter activity. These data suggest that the Ras-MEK pathway is required for coordinate expression of the GCH and TH genes induced by neurotrophins.

5' Flanking Region↗

Development of a new radioligand, N-(5-fluoro-2-phenoxyphenyl)-N-(2-[18F]fluoroethyl-5-methoxybenzyl)acetamide, for pet imaging of peripheral benzodiazepine receptor in primate brain.

To develop a positron emission tomography (PET) ligand for imaging the 'peripheral benzodiazepine receptor' (PBR) in brain and elucidating the relationship between PBR and brain diseases, four analogues (4-7) of N-(2,5-dimethoxybenzyl)-N-(5-fluoro-2-phenoxyphenyl)acetamide (2) were synthesized and evaluated as ligands for PBR. Of these compounds, fluoromethyl (4) and fluoroethyl (5) analogues had similar or higher affinities for PBR than the parent compound 2 (K(i) = 0.16 nM for PBR in rat brain sections). Iodomethyl analogue 6 displayed a moderate affinity, whereas tosyloxyethyl analogue 7 had weak affinity. Radiolabeling was performed for the fluoroalkyl analogues 4 and 5 using fluorine-18 ((18)F, beta(+); 96.7%, T(1/2) = 109.8 min). Ligands [(18)F]4 and [(18)F]5 were respectively synthesized by the alkylation of desmethyl precursor 3 with [(18)F]fluoromethyl iodide ([(18)F]8) and 2-[(18)F]fluoroethyl bromide ([(18)F]9). The distribution patterns of [(18)F]4 and [(18)F]5 in mice were consistent with the known distribution of PBR. However, compared with [(18)F]5, [(18)F]4 displayed a high uptake in the bone of mice. The PET image of [(18)F]4 for monkey brain also showed significant radioactivity in the bone, suggesting that this ligand was unstable for in vivo defluorination and was not a useful PET ligand. Ligand [(18)F]5 displayed a high uptake in monkey brain especially in the occipital cortex, a region with richer PBR than the other regions in the brain. The radioactivity level of [(18)F]5 in monkey brain was 1.5 times higher than that of [(11)C]2, and 6 times higher than that of (R)-(1-(2-chlorophenyl)-N-[(11)C]methyl,N-(1-methylpropyl)isoquinoline ([(11)C]1). Moreover, the in vivo binding of [(18)F]5 was significantly inhibited by PBR-selective 2 or 1, indicating that the binding of [(18)F]5 in the monkey brain was mainly due to PBR. Metabolite analysis revealed that [(18)F]4 was rapidly metabolized by defluorination to [(18)F]F(-) in the plasma and brain of mice, whereas [(18)F]5 was metabolized by debenzylation to a polar product [(18)F]13 only in the plasma. No radioactive metabolite of [(18)F]5 was detected in the mouse brain. The biological data indicate that [(18)F]5 is a useful PET ligand for PBR and is currently used for imaging PBR in human brain.

Acetamides↗

Proposal of blood-collecting needle approach to semi-invasive method.

Many diabetic patients carry a portable self-monitoring of blood glucose (SMBG)-analyzer in order to collect their own blood and examine their glucose levels; this allows them to determine such factors as insulin dose, diet and exercise to stay healthy. However, the test causes physical and mental stress for the subjects. The authors aim to develop a semi-invasive blood-collecting needle which does not need a power source for the pump mechanism. In this study, we fabricated a capillary action needle that can collect the blood sample automatically. A blood-collecting needle was fabricated from 25 gage sized medical needle (diameter of 0.5 mm, stainless steel) by cutting process, and it had a half-opened crevice in the tip. In order to evaluate the physical characteristics of the blood-collecting needle, the relationship between the size and suction time and/or suction volume were measured using an isotonic sodium chloride solution, whole rabbit blood, and whole human blood with anticoagulant. Next, in order to evaluate the degree of invasion, the diameters of erythema in auricles of rabbits were observed for 2 days using a CCD camera-type microscope. The mean suction time of the isotonic sodium chloride solution and the whole rabbit blood were 1.5 s (n = 10) and 9.0 s (n = 5), respectively. Selection of a suitable size of the blood-collecting needle enabled the collection of 0.1 microL of whole human blood in 10 s. Moreover, it was shown, by comparing the observed diameter of the erythema, that the invasiveness of the blood-collecting needle was smaller than for commercial needles of the equal diameter. It became clear that this fulfils the fundamental functions of a semi-invasive blood-collecting needle.

Adult↗

Quick and reversible inhibition of soybean root nodule growth by nitrate involves a decrease in sucrose supply to nodules.

The upper part of a nodulated soybean root hydroponically cultured in a glass bottle was monitored using a computer microscope under controlled environmental conditions, and the diameter of individual nodules was measured from 10-24 d after planting. The diameter of a root nodule attached to the primary root increased from 1 mm to 6 mm for 2 weeks under nitrogen-free conditions. The increase in diameter of the nodules was almost completely stopped after 1 d of supplying 5 mM nitrate, and was due to the cessation of nodule cell expansion. However, nodule growth quickly returned to the normal growth rate following withdrawal of nitrate from the solution. The reversible depression of nodule growth by nitrate was similar to the restriction of photoassimilate supply by continuous dark treatment for 2 d followed by normal light/dark conditions. In addition, the inhibitory effect of nitrate was partially alleviated by the addition of 3% (w/v) sucrose to the medium. Plant leaves were exposed to (11)C or (14)C-labelled carbon dioxide to investigate the effects of 5 mM nitrate on the translocation and distribution of photosynthates to nodules and roots. Supplying 5 mM nitrate stimulated the translocation rate and the distribution of labelled C in nitrate-fed parts of the roots. However, the (14)C partitioning to nodules decreased from 9% to 4% of total (14)C under conditions of 5 mM nitrate supply. These results indicate that the decrease in photoassimilate supply to nodules may be involved in the quick and reversible nitrate inhibition of soybean nodule growth.

Biological Transport↗