Machine learning prediction of electron density and temperature from He I line ratios.

We propose to utilize machine learning to predict the electron density, ne, and temperature, Te, from He I line intensity ratios. In this approach, training data consist of measured He I line ratios as input and ne and Te measured using other diagnostic(s) as desired output, which is a Langmuir probe in our study. Support vector machine regression analysis is, then, performed with the training data to develop a predictive model for ne and Te, separately. It is confirmed that ne and Te predicted using the developed models agree well with those from the Langmuir probe in the ranges of 0.28 × 1018 ≤ ne (m-3) ≤ 3.8 × 1018 and 3.2 ≤ Te (eV) ≤ 7.5. The developed models are, further, examined with an evaluation data, which are not included in the training data, and are found to well reproduce absolute values and radial profiles of probe-measured ne and Te.

Expression analysis of cellulose synthases that comprise the Type II complex in hybrid aspen.

Gene duplication in plants occurs via several different mechanisms, including whole genome duplication, and the copied genes acquire various forms and types. The cellulose synthase (CesA) family functions in cellulose synthesis complex (CSC) formation, which is involved in the synthesis of primary and secondary cell walls in plants. In the genome of Populus, 17 CesA have been annotated, and some of them appeared through whole genome duplication. The nucleotide sequence of the duplicated genes changed during subsequent evolution, and functional differentiation of genes might have occurred. To gain insight into the evolutionary fate of the duplicated CesA, expression analysis with quantitative reverse transcription polymerase chain reactions and promoter-reporter assays was performed on three duplicated gene pairs whose products have been reported to form a single CSC. Changes in expression of each gene at different developmental stages were detected and divergent expression patterns in different organs and tissues observed between the gene pairs. Among the tested genes, expression of PttCesA3-C was apparently lower than that of its counterpart, PttCesA3-D. The results suggest that the six CesA are approaching sub-functionalisation or non-functionalisation. Furthermore, the level of functionalisation may vary among the three pairs of genes, and functional specialisation of each CesA should have been achieved, at least partially, through differences in expression of genes.

Compact and high-particle-flux thermal-lithium-beam probe system for measurement of two-dimensional electron density profile.

A compact and high-particle-flux thermal-lithium-beam source for two-dimensional measurement of electron density profiles has been developed. The thermal-lithium-beam oven is heated by a carbon heater. In this system, the maximum particle flux of the thermal lithium beam was ~4 × 10(19) m(-2) s(-1) when the temperature of the thermal-lithium-beam oven was 900 K. The electron density profile was evaluated in the small tokamak device HYBTOK-II. The electron density profile was reconstructed using the thermal-lithium-beam probe data and this profile was consistent with the electron density profile measured with a Langmuir electrostatic probe. We confirm that the developed thermal-lithium-beam probe can be used to measure the two-dimensional electron density profile with high time and spatial resolutions.

Discovery of pinoresinol reductase genes in sphingomonads.

Bacterial genes for the degradation of major dilignols produced in lignifying xylem are expected to be useful tools for the structural modification of lignin in plants. For this purpose, we isolated pinZ involved in the conversion of pinoresinol from Sphingobium sp. strain SYK-6. pinZ showed 43-77% identity at amino acid level with bacterial NmrA-like proteins of unknown function, a subgroup of atypical short chain dehydrogenases/reductases, but revealed only 15-21% identity with plant pinoresinol/lariciresinol reductases. PinZ completely converted racemic pinoresinol to lariciresinol, showing a specific activity of 46±3 U/mg in the presence of NADPH at 30°C. In contrast, the activity for lariciresinol was negligible. This substrate preference is similar to a pinoresinol reductase, AtPrR1, of Arabidopsis thaliana; however, the specific activity of PinZ toward (±)-pinoresinol was significantly higher than that of AtPrR1. The role of pinZ and a pinZ ortholog of Novosphingobium aromaticivorans DSM 12444 were also characterized.

Measurement of ion and electron temperatures in plasma blobs by using an improved ion sensitive probe system and statistical analysis methods.

We have measured ion temperature as well as electron temperature in plasma blobs observed in a linear plasma device by using an improved ion sensitive probe. Current-voltage characteristics of the ion sensitive probe inside and outside plasma blobs were re-constructed with a conditional sampling method. It is clearly found that both ion and electron temperatures in plasma blobs decrease more slowly in a cross-field direction than those in a bulk plasma without plasma blobs.

Expression of a gene for Mn-peroxidase from Coriolus versicolor in transgenic tobacco generates potential tools for phytoremediation.

In efforts aimed at the detoxification of contaminated areas, plants have many advantages over bacteria and fungi. We are attempting to enhance the environmental decontamination functions of plants by transferring relevant genes from microorganisms. When the gene for Mn-peroxidase (MnP) from Coriolus versicolor was expressed in transgenic tobacco plants, one line (designated fMnP21) expressed MnP activity at levels 54-fold higher than in control lines. When undamaged roots of transgenic plants were applied to liquid medium supplemented with 250 microM pentachlorophenol (PCP), the decrease in the level of PCP in fMnP21 (86% reduction) was about 2-fold higher than that in control lines (38% reduction). Expression of the gene for MnP in the transgenic plants had no obvious negative effects on their vegetative and sexual growth. Our system should contribute to the development of novel methods for the removal of hazardous chemicals from contaminated environments using transgenic plants.

Pharmacological characterization of a novel 5-HT4 receptor agonist, TS-951, in vitro.

The pharmacological effect of a novel selective 5-HT4 receptor agonist, TS-951 (N-[endo-8-(3-hydroxypropyl)-8-azabicyclo[3.2.1]oct-3-yl]-1-isopropyl-2-oxo-1,2-dihydro-3-quinolinecarboxamide) was investigated in vitro. TS-951 potently inhibited specific [3H]GR113808 binding both in guinea-pig striatum and in mouse brain. The affinity of TS-951 for the 5-HT4 receptor was higher than those of other agonists, 5-HT, cisapride, mosapride and renzapride. On the longitudinal muscle of the guinea-pig ileum, TS-951 caused a concentration-dependent increase in the amplitude of electrically induced submaximal twitch contractions. On the longitudinal muscle of the guinea-pig distal colon, TS-951 also caused concentration-dependent contractions. TS-951 is a high-affinity, selective and potent 5-HT4 receptor agonist. This compound therefore can be considered as a useful pharmacological tool for investigating 5-HT4 receptor-mediated events.

Specific accumulation of polysaccharide-linked hydroxycinnamoyl esters in the cell walls of irregularly shaped and collapsed internode parenchyma cells of the dwarf rice mutant Fukei 71.

We examined a novel rice mutant, Fukei 71 (Oryza sativa L.), for alterations in the levels of hydroxycinnamoyl esters that are linked to cell wall polysaccharides and lignin units. In this mutant, a recessive mutation at a single locus caused the collapse of parenchyma cells in the internodes. Light microscopy revealed that the abnormal walls of internode parenchyma cells of Fukei 71 were stained by the Mäule reaction, which is specific for syringyl units in phenolic compounds. These walls were not stained by Wiesner's reagent (phloroglucinol-HCl), which reacts cinnamaldehyde in lignin. Levels of p-coumaric acid (PCA) and ferulic acid (FA) were apparently elevated in the abnormal tissue of the mutant. Western blotting analysis with antibodies specific for phenylalanine ammonia-lyase (PAL) revealed higher levels of PAL in the abnormal parenchyma tissue of Fukei 71 than in the parenchyma tissue of the parent cultivar Fujiminori. These results and the observation that PAL was produced at a greatly elevated level indicated that the phenylpropanoid pathway that leads to the biosynthesis of polysaccharide-linked FA and PCA was abnormally activated in the irregularly shaped and collapsed internode parenchyma cells, in which the biosynthesis of lignin is normally repressed.

Comparison of 26,27-hexafluoro-1 alpha,25-dihydroxyvitamin D3 and 1 alpha,25-dihydroxyvitamin D3 on the resorption of bone explants ex vivo.

26,27-Hexafluoro-1 alpha,25-dihydroxyvitamin D3 [F6-1,25-(OH)2D3] is more potent than 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] in stimulating bone resorption in vitro and in vivo. The reason why F6-1,25(OH)2D3 is more active remains unclear. To clarify the relationship between the bone-resorbing activity of each vitamin D3 analogue and the metabolism of each analogue, in the present study, we used an ex vivo method that was established by Reynolds et al (Calcif Tissue Res, 1974, 15, 333-339). The effect of F6-1,25(OH)2D3 or 1,25(OH)2D3 on 45Ca release from parietal bones, prepared at 3, 14 and 24 h after injection of 1.9, 3.8, 7.6 or 15.2 pmol vitamin D analog/g body weight, was examined. F6-1,25(OH)2D3 was more potent than 1,25(OH)2D3 during each in vivo time period. 1,25(OH)2D3 at 3 h after the injection was more active compared to the control (no injection of 1,25(OH)2D3) but not at 14 and 24 h. The radioactivity of the bones after the injection of [3H]-F6-1,25(OH)2D3 was retained even at 24 h. In the case of [3H]-1,25(OH)2D3, the radioactivity of bones decreased with an increase in the in vivo period. In a HPLC analysis of the lipid extract of bone homogenate, [3H]-F6-1,25(OH)2D3 alone was detected at 3 h after the injection and both [3H]-F6-1,25(OH)2D3 and [3H]-26,27-hexafluoro-1 alpha, 23S,25-trihydroxyvitamin D3 [F6-1,23,25(OH)3D3] were detected at 14 and 24 h after the injection. [3H]-1,25(OH)2D3 was highly detected at 3 h after the injection, but it decreased with an increase in the in vivo period. In the ex vivo test, the activity of F6-1,23,25(OH)3D3 was less than that of F6-1,25(OH)2D3 but similar to that of 1,25(OH)2D3. The present study indicates that F6-1,25(OH)2D3 is more active and more long-lasting than 1,25(OH)2D3 in the ex vivo method. A higher potency of F6-1,25(OH)2D3 is explained, at least partly, by the results that the amounts of both F6-1,25(OH)2D3 and its active metabolite, F6-1,23,25(OH)3D3, in the bones are higher than that of 1,25(OH)2D3, and that F6-1,25(OH)2D3 and its metabolite are retained in bones longer than 1,25(OH)2D3.

Cloning of a thermostable ascorbate oxidase gene from Acremonium sp. HI-25 and modification of the azide sensitivity of the enzyme by site-directed mutagenesis.

A gene encoding a thermostable ascorbate oxidase (ASOM) was cloned from Acremonium sp. HI-25 and sequenced. The gene comprised 1709 bp and was interrupted by a single intron of 57 bp. ASOM consisted of 551 amino acids including a signal peptide with a molecular mass of 61200, and contained four histidine-rich regions with high sequence homology to the corresponding regions of other multicopper oxidases. The ASOM gene was expressed in Aspergillus nidulans under the Aspergillus oryzae Taka-amylase A gene promoter. The recombinant enzyme (An-ASOM) exhibited almost the same enzymatic properties as ASOM. The ASOM gene was mutated by site-directed mutagenesis with reference to the amino acid sequences of plant enzymes to generate enzymes with altered azide sensitivity. Site-directed mutagenesis at the trinuclear active copper site resulted in an increase in azide resistance; the Ala465Leu and Phe463Trp/Ala465Leu mutants exhibited approximately 10 and 20% increases in azide resistance, respectively.

Structural Characterization of Modified Lignin in Transgenic Tobacco Plants in Which the Activity of 4-Coumarate:Coenzyme A Ligase Is Depressed.

Transgenic tobacco (Nicotiana tabacum L.) plants in which the activity of 4-coumarate:coenzyme A ligase is very low contain a novel lignin in their xylem. Details of changes in hydroxycinnamic acids bound to cell walls and in the structure of the novel lignin were identified by base hydrolysis, alkaline nitrobenzene oxidation, pyrolysis-gas chromatography, and 13C-nuclear magnetic resonance analysis. In the brownish tissue of the transgenic plants, the levels of three hydroxycinnamic acids, p-coumaric, ferulic, and sinapic, which were bound to cell walls, were apparently increased as a result of down-regulation of the expression of the gene for 4-coumarate:coenzyme A ligase. Some of these hydroxycinnamic acids were linked to cell walls via ester and ether linkages. The accumulation of hydroxycinnamic acids also induced an increase in the level of condensed units in the novel lignin of the brownish tissue. Our data indicate that the behavior of some of the incorporated hydroxycinnamic acids resembles lignin monomers in the brownish tissue, and their accumulation results in dramatic changes in the biosynthesis of lignin in transgenic plants.

Alterations in the biosynthesis of lignin in transgenic plants with chimeric genes for 4-coumarate: coenzyme A ligase.

The introduction of chimeric sense and antisense gene constructs for 4-coumarate:coenzyme A ligase into tobacco plants caused the reduction of the 4CL activity in the transgenic plants. In the transgenic plants, the cell walls of the xylem tissue in stems were brown and the molecular structure of lignin in the colored cell walls was dramatically different from that in the control plants. Analysis with different types of stain revealed that levels of cinnamyl aldehyde residues and syringyl units in lignin were depressed in the brownish cell walls. Furthermore, the lignin content in colored tissue was lower than that in the normal tissue. Our results indicate that 4CL has important roles in the determination of the composition and the amount of lignin in tobacco plants.

Isolation and analysis of cinnamic acid 4-hydroxylase homologous genes from a hybrid aspen, Populus kitakamiensis.

Cinnamic acid 4-hydroxylase (CA4H) is the second enzyme involved in phenylpropanoid biosynthesis and is a member of the cytochrome P-450 superfamily. Three CA4H homologous genes, cyp73A, cyp73b, and cyp73c, and a cDNA clone of cyp73a were isolated from a genomic library and a cDNA library of a hybrid aspen; Populus kitakamiensis, and were characterized. They might be interrupted by two introns each. cyp73a and cyp73b were very similar to each other not only in coding regions but also in non-coding regions. Southern blot analysis showed that four homologous genes for CA4H constructed a small gene family in the diploid genome of P. kitakamiensis. In the promoter regions, there were many common cis-element-like sequences in phenylpropanoid biosynthesis genes.

Inhibition of multidrug resistance by a new staurosporine derivative, NA-382, in vitro and in vivo.

The effects of a newly synthesized compound, N-ethoxycarbonyl-7-oxo-staurosporine (NA-382), on multidrug resistance in tumor cells were investigated. Protein kinase-inhibitory activity of NA-382 was lower but more selective to Ca2+/phospholipid-dependent protein kinase than that of staurosporine. NA-382 at noncytotoxic concentrations effectively reversed in vitro multidrug resistance of Adriamycin-resistant P388 (P388/ADR) cells, without influencing the drug sensitivity of sensitive P388 cells. NA-382 inhibited extrusion of vinblastine (VBL) and increased intracellular accumulation of VBL, more in P388/ADR cells than in sensitive P388 cells, with higher potency than staurosporine. This compound also reduced VBL resistance of other multidrug-resistant cell lines, AH66 and K562/ADR, by inhibiting VBL efflux and promoting VBL accumulation. NA-382 also dose dependently potentiated the effects of VBL and Adriamycin in P388/ADR-bearing mice. The toxicity of staurosporine was too high to use the combination with VBL in vitro and in vivo. NA-382 accumulated VBL in P388/ADR cells even after desensitization of Ca2+/phospholipid-dependent protein kinase by treatment with 12-O-tetradecanoylphorbol-13-acetate and 18 h, while being suppressed by 12-O-tetradecanoylphorbol-13-acetate added simultaneously or shortly before NA-382. Both staurosporine and NA-382 inhibited the photolabeling of [3H]azidopine on M(r) 140,000 P-glycoprotein in the plasma membrane from P388/ADR cells. These results indicate that this new staurosporine analogue, NA-382, reverses multidrug resistance by directly inhibiting the drug binding to P-glycoprotein, but not by Ca2+/phospholipid-dependent protein kinase inhibitory action.

Effect of staurosporine derivatives on protein kinase activity and vinblastine accumulation in mouse leukaemia P388/ADR cells.

Inhibition by staurosporine derivatives of cyclic AMP-dependent protein kinase (A-kinase) and protein kinase C (C-kinase), and drug resistance has been investigated. The substitution of an acetyl or an ethoxycarbonyl group for the amine N-ethoxycarbonyl-7-oxostaurosporine moiety on the tetrahydropyran ring of staurosporine decreased inhibition of both protein kinases, but increased selectivity for C-kinase by further modification of the lactam moiety to the imide (NA-382). The activities of SF-2370 on protein kinases were decreased by decarboxylation and hydroxyalkylation. These staurosporine derivatives enhanced accumulation of vinblastine in adriamycin-resistant P388 (P388/ADR) cells in a dose-dependent manner. The potency for the drug accumulation of these compounds was correlated with their inhibitory activity on the drug efflux, but was not correlated with their activity on protein kinases. Staurosporine and NA-382, with high potency for vinblastine accumulation, inhibited the photolabelling of [3H]azidopine on 140 kDa P-glycoprotein in the plasma membrane. The tetrahydrofuran compounds and NA-357, which had low potency for the drug accumulation, hardly interacted with azidopine on P-glycoprotein. Most of these compounds were highly cytotoxic by themselves, and only NA-382 was less cytotoxic among them and completely reversed the vinblastine-resistance of P388/ADR cells at a non-cytotoxic concentration. These results suggest that staurosporine derivatives can enhance drug accumulation and inhibit drug resistance through their direct action on the P-glycoprotein.

Reduction of rat striatal thyrotropin-releasing hormone receptors produced by repeated methamphetamine administration.

It has been reported previously that repeated, but not continuous, administration of methamphetamine (MAP) to animals produces progressive and sustained enhancement of MAP-induced behavior (behavioral sensitization), which may be related to functional changes in central dopamine (DA) systems. To investigate the possible involvement of thyrotropin-releasing hormone (TRH), a neuromodulator of DA, both immunoreactive TRH (IR-TRH) levels and specific TRH binding were examined in rat brain regions after MAP administration either repeatedly (4 mg/kg intraperitoneally once a day for 14 consecutive days) or continuously (about 4 mg/kg/day for 13 consecutive days). Although no significant changes were observed in IR-TRH levels in any regions of the brain following repeated MAP injections, specific TRH binding in the striatum significantly decreased. Scatchard analysis revealed that the decrease was due to a reduction in the maximum number of binding sites (Bmax). Pretreatment with haloperidol prior to each MAP injection prevented this decrease. Continuous MAP administration had no effect on regional specific TRH binding. These results suggest that repeated MAP administration caused lasting dysfunction in the brain TRH system, which may be implicated in the behavioral sensitization.

[Effect of amygdaloid kindling on thyrotropin-releasing hormone, somatostatin, cholecystokinin and substance P contents of the amygdala/piriform cortex and hippocampus of rats].

Recently, several systems of neuropeptides have been demonstrated to have anticonvulsant action in some forms of epilepsy to some extent. However, considerably less knowledge has been taken to their involvement in convulsive disorders either with regard to the development, expression or control of seizures. In this study, therefore, we examined the influence of amygdaloid kindling, an experimental model of temporal lobe epilepsy, on thyrotropin-releasing hormone (TRH), somatostatin (SS), cholecystokinin (CCK) and substance P (SP) content in the amygdala/piriform cortex and hippocampus. Male Sprague-Dawley rats were implanted bipolar electrodes into the left amygdala under pentobarbital anesthesia. Daily kindling stimulation was made to the left amygdala with 1 sec, 60 Hz, 400 microA, until 5 consecutive fully kindled generalized convulsive seizures were elicited. Subsequently, amygdaloid kindled rats were decapitated 30 min, 24 hrs, 48 hrs, 7 days and 21 days after the last amygdaloid stimulation, and the amygdala/piriform cortex and hippocampus were dissected. Control animals only received chronic electrodes, but no stimulation was delivered. The immunoreactivity of TRH, SS, CCK and SP was examined by methods of specific radioimmunoassay. The TRH content in these two brain regions significantly increased 24 hrs after the last kindled convulsion. This increase became maximal 48 hrs after the last convulsion: about 3-fold and 4-fold of the control in the amygdala/piriform cortex and hippocampus, respectively. Such increases in the TRH content tended to persist for 7 days, but returned to the control level 21 days after the last convulsion.(ABSTRACT TRUNCATED AT 250 WORDS)