Fine mapping of a major quantitative trait locus, qLG-9, that controls seed longevity in rice (Oryza sativa L.).

KEY MESSAGE: We fine-mapped a quantitative trait locus, qLG - 9, for seed longevity detected between Japonica-type and Indica-type cultivars. qLG - 9 was mapped in a 30-kb interval of the Nipponbare genome sequence. A quantitative trait locus, qLG-9, for seed longevity in rice has previously been detected on chromosome 9 by using backcross inbred lines derived from a cross between Japonica-type (Nipponbare) and Indica-type (Kasalath) cultivars. In the present study, the chromosomal location of qLG-9 was precisely determined by fine-scale mapping. Firstly, allelic difference in qLG-9 was verified by QTL analysis of an F2 population derived from a cross between Nipponbare and NKSL-1, in which a segment of Kasalath chromosome 9 was substituted in Nipponbare genetic background. Then, we selected F2 plants in which recombination had occurred near qLG-9 and performed F3 progeny testing on these plants to determine the genotype classes of qLG-9. Eventually, qLG-9 was mapped in a 30-kb interval (defined by two markers, CAPSb and CHPa12) of the Nipponbare genome sequence. This allowed us to nominate positional candidate genes of qLG-9. Additionally, we developed near-isogenic lines (NIL) for qLG-9 by marker-assisted selection. qLG-9 NIL showed significantly higher seed longevity than isogenic control of Nipponbare. These results will facilitate cloning of the gene(s) underlying qLG-9 as well as marker-assisted transfer of desirable genes for seed longevity improvement in rice.

Dose reconstruction from PET images in carbon ion therapy: a deconvolution approach.

Dose and range verification have become important tools to bring carbon ion therapy to a higher level of confidence in clinical applications. Positron emission tomography is among the most commonly used approaches for this purpose and relies on the creation of positron emitting nuclei in nuclear interactions of the primary ions with tissue. Predictions of these positron emitter distributions are usually obtained from time-consuming Monte Carlo simulations or measurements from previous treatment fractions, and their comparison to the current, measured image allows for treatment verification. Still, a direct comparison of planned and delivered dose would be highly desirable, since the dose is the quantity of interest in radiation therapy and its confirmation improves quality assurance in carbon ion therapy. In this work, we present a deconvolution approach to predict dose distributions from PET images in carbon ion therapy. Under the assumption that the one-dimensional PET distribution is described by a convolution of the depth dose distribution and a filter kernel, an evolutionary algorithm is introduced to perform the reverse step and predict the depth dose distribution from a measured PET distribution. Filter kernels are obtained from either a library or are created for any given situation on-the-fly, using predictions of the [Formula: see text]-decay and depth dose distributions, and the very same evolutionary algorithm. The applicability of this approach is demonstrated for monoenergetic and polyenergetic carbon ion irradiation of homogeneous and heterogeneous solid phantoms as well as a patient computed tomography image, using Monte Carlo simulated distributions and measured in-beam PET data. Carbon ion ranges are predicted within less than 0.5 mm and 1 mm deviation for simulated and measured distributions, respectively.

Experimental validation of the FLUKA Monte Carlo code for dose and [Formula: see text]-emitter predictions of radioactive ion beams.

In the context of hadrontherapy, whilst ions are capable of effectively destroying radio resistant, deep seated tumors, their treatment localization must be well assessed to ensure the sparing of surrounding healthy tissue and treatment effectiveness. Thus, range verification techniques, such as online positron-emission-tomography (PET) imaging, hold great potential in clinical practice, providing information on the in vivo beam range and consequent tumor targeting. Furthermore, [Formula: see text] emitting radioactive ions can be an asset in online PET imaging, depending on their half-life, compared to their stable counterparts. It is expected that using these radioactive ions the signal obtained by a PET apparatus during beam delivery will be greatly increased, and exhibit a better correlation to the Bragg Peak. To this end, FLUKA Monte Carlo particle transport and interaction code was used to evaluate, in terms of annihilation events at rest and dose, the figure of merit in using [Formula: see text] emitter, radioactive ion beams (RI [Formula: see text]). For this purpose, the simulation results were compared with experimental data obtained with an openPET prototype in various online PET acquisitions at the Heavy Ion Medical Accelerator in Chiba (HIMAC), in collaboration with colleagues from the National Institute of Radiological Sciences' (NIRS) Imaging Physics Team. The dosimetry performance evaluation with FLUKA benefits from its recent developments in fragmentation production models. The present work estimated that irradiations with RI [Formula: see text], produced via projectile fragmentation and their signal acquisition with state-of-the-art PET scanner, lead to nearly a factor of two more accurate definition of the signals' peak position. In addition to its more advantageous distribution shape, it was observed at least an order magnitude higher signal acquired from 11C and 15O irradiations, with respect to their stable counterparts.

Organization and structure of NADH-dependent glutamate synthase gene from rice plants.

Genomic clones for NADH-dependent glutamate synthase (NADH-GOGAT; EC 1.4.1.14) were obtained from a genomic library of rice (Oryza sativa L. cv. Sasanishki). A genomic clone (lambdaOS42, 14 kb) covered an entire structural gene and a 3.7 kb 5'-upstream region from the first methionine. Another clone (lambdaOS23, 14 kb) contained a 2.8 kb 3'-downstream region from the stop codon. A 7047 bp long clone (lambdaOSR51) consisting of full length cDNA for NADH-GOGAT was isolated from a cDNA library prepared using mRNA from roots of rice seedlings treated with 1 mM NH4Cl for 12 h. The presumed transcribed region (11.7 kb) consisted of 23 exons separated by 22 introns. Rice NADH-GOGAT is synthesized as a 2166 amino acid protein with a molecular mass of 236.7 kDa that includes a 99 amino acid presequence. DNA gel blot analysis suggested that NADH-GOGAT occurred as a single gene in rice. Primer extension experiments map the transcription start of NADH-GOGAT to identical positions. The 3. 7 kb 5'-upstream region was able to transiently express a reporter gene in cultured rice cells. Putative motifs related to the regulation of NADH-GOGAT gene expression were looked for within the 5'-upstream region by database.

Okadaic Acid Mimics Nitrogen-Stimulated Transcription of the NADH-Glutamate Synthase Gene in Rice Cell Cultures.

Okadaic acid (OKA), a potent and specific inhibitor of protein serine/threonine phosphatases 1 and 2A, induced the accumulation of NADH-glutamate synthase (GOGAT) mRNA within 4 h in rice (Oryza sativa L.) cell cultures. In contrast to the transient accumulation of NADH-GOGAT mRNA by NH(4)(+), OKA caused a continuous accumulation for at least 24 h. The induction of NADH-GOGAT mRNA by OKA was not inhibited in the presence of methionine sulfoximine, which inhibited the NH(4)(+)-induced accumulation of mRNA. These results suggest that the OKA-sensitive protein phosphatase is involved in the regulation of NADH-GOGAT gene expression and probably plays a role in the signal transduction pathway downstream from NH(4)(+), although a signal transduction pathway other than that of nitrogen sensing could be responsible. Nuclear run-on assays demonstrated that the accumulation of NADH-GOGAT mRNA induced by the supply of either NH(4)(+) or OKA was mainly regulated at the transcription level. OKA effects were synergistic to the NH(4)(+)-induced expression of the NADH-GOGAT gene. In the presence of K-252a, a protein kinase inhibitor, the accumulation of NADH-GOGAT mRNA induced by either NH(4)(+) or OKA was reduced. The possible roles of protein phosphatases in the regulation of NADH-GOGAT gene expression are discussed.

Changes in the Content of Two Glutamate Synthase Proteins in Spikelets of Rice (Oryza sativa) Plants during Ripening.

Nitrogen accumulation in the apical spikelets on the primary branches of the main stem of rice plants have been studied during the ripening process (0-35 d after flowering). The level of NADH-dependent glutamate synthase (GOGAT) protein and activity increased 4- and 6-fold, respectively, in the first 15 d after flowering. Maximum levels of NADH-GOGAT were found at that time when the spikelets had just begun to increase in dry weight and to accumulate storage proteins. Subsequently, both the level of NADH-GOGAT protein and its activity in spikelets declined rapidly. Although changes in ferredoxin (Fd)-dependent GOGAT paralleled changes in NADH-GOGAT, the relative abundance of NADH-GOGAT protein in the spikelets was about 3 times higher than that of Fd-GOGAT from 5 to 15 d after flowering. When the chaff (lemma and palea) was separated from the spikelets 10 d after the flowering, 16% of the NADH-GOGAT protein was found in the chaff and 84% in the young grain tissues (endosperm, testae, aleurone tissues, and embryo). On the other hand, Fd-GOGAT protein was distributed 52% in the chaff and 48% in the young grain tissues in spikelets of the same age. Activity of NADP-isocitrate dehydrogenase, which may generate the 2-oxoglutarate required for the GOGAT reactions, was much higher than that of total GOGAT activities on a spikelet basis during the ripening process. These results suggest that in rice plants NADH-GOGAT is responsible for the synthesis of glutamate from the glutamine that is transported from senescing tissues to the spikelets.

Quantitative intercellular localization of NADH-dependent glutamate synthase protein in different types of root cells in rice plants

The quantitative analysis with immunogold-electron microscopy using a single-affinity-purified anti-NADH-glutamate synthase (GOGAT) immunoglobulin G (IgG) as the primary antibody showed that the NADH-GOGAT protein was present in various forms of plastids in the cells of the epidermis and exodermis, in the cortex parenchyma, and in the vascular parenchyma of root tips (<10 mm) of rice (Oryza sativa) seedlings supplied with 1 mM NH4+ for 24 h. The values of the mean immunolabeling density of plastids were almost equal among these different cell types in the roots. However, the number of plastids per individual cell type was not identical, and some parts of the cells in the epidermis and exodermis contained large numbers of plastids that were heavily immunolabeled. Although there was an indication of labeling in the mitochondria using the single-affinity-purified anti-NADH-GOGAT IgG, this was not confirmed when a twice-affinity-purified IgG was used, indicating an exclusively plastidial location of the NADH-GOGAT protein in rice roots. These results, together with previous work from our laboratory (K. Ishiyama, T. Hayakawa, and T. Yamaya [1998] Planta 204: 288-294), suggest that the assimilation of exogeneously supplied NH4+ ions is primarily via the cytosolic glutamine synthetase/plastidial NADH-GOGAT cycle in specific regions of the epidermis and exodermis in rice roots. We also discuss the role of the NADH-GOGAT protein in vascular parenchyma cells.

Effect of the intermittent prophylactic treatment of the central nervous system in adults with acute leukemia.

Nineteen patients with acute leukemia, who achieved complete remission between January, 1980 and March, 1983, were given 10 mg of methotrexate (MTX) and 20 mg of prednisolone (PSL) intrathecally at 1st, 3rd, 6th, 10th and 15th month after the termination of consolidation therapy followed by the same dose twice a year, when the intensification therapy was being performed. During the observation period of 29 to 68 months, none developed central nervous system (CNS) leukemia. On the other hand, the incidence of CNS leukemia in patients given the prophylactic intrathecal treatment once or twice just after the complete remission was 35% (7/20) compared with 24% (8/33) in patients without treatment. No statistical difference was observed in these groups. The intrathecal administration of MTX and PSL is easy to perform and no adverse reaction was observed in our series. It is concluded that the intermittent prophylaxis with MTX and PSL is of benefit in preventing CNS leukemia in adults patients with acute leukemia.

High-resolution image reconstruction method for time-of-flight positron emission tomography.

We present a new image reconstruction method for time-of-flight positron emission tomography (TOF-PET). The TOF-PET measurement system is modelled using the continuous-discrete mapping model, and images are reconstructed using an algebraic technique. The proposed method can produce images with better spatial resolution than conventional methods based on the filtered backprojection method. Numerical simulation results show that accurate modelling of the measurement system improves the spatial resolution and the contrast recovery, while the utilization of TOF information improves the signal-to-noise ratios of images.

[An intensification therapy of adults acute leukemia].

Between January 1980 and March 1983, a study was conducted on the effects of intensification therapy in 20 adult acute leukemia patients who had achieved complete remission with induction therapy. Intensification therapy consisted of cyclic administration of six combination therapies given at gradually longer intervals, using daunorubicin, cytosine arabinoside, 6-mercaptopurine and prednisolone (DCMP), cyclocytidine (DCyMP), vincristine (DCVP), behenoyl-ara-c (BHAC-DMP), aclacinomycin (BHAC-AMP) and (ACM-MP). Six combinations were given sequentially at one-month intervals, at 2-, 3-, 4-, 5- and eventually 6-month intervals, until 5-year survival. The median remission duration was 38 months for AML, and 17 months for ALL. The median survival was 66 months for AML, and 37 months for ALL. The five year survival rate was 50%. Nine of the 20 patients are still alive. Methotrexate and prednisolone were administered intrathecally for prophylaxis of CNS leukemia on Day 4 for each intensification therapy. There was no CNS leukemia. This intensification protocol was shown to be effective in improving the prognosis of adults acute leukemia.

Resistance to acetohydroxamate acquired by slow adaptive increases in urease in cultured tobacco cells.

Urease activity of tobacco XD cells (1U cells) had undergone a 4-fold increase (4U cells) during a year of growth on urea (Skokut and Filner 1980 Plant Phvsiol 65: 995-1003). A clone of 4U cells gave rise to 12U cells during another year of growth on urea. The doubling time of 12U cells on urea is 2.2 days, compared to about 4 days for 1U cells, while 1U and 12U cells double in 2 days on nitrate. Acetohydroxamic acid (AHA), a specific inhibitor/reversible inactivator of jack bean urease, affects tobacco cell urease similarly. Fifty per cent inhibition of growth by AHA occurred at 20 micromolar in 1U cells growing on urea and at 165 micromolar in 12U cells growing on urea, but at 600 micromolar for either 1U or 12U cells growing on nitrate. When 12U cells were grown on urea with 100 micromolar AHA, extractable urease activity decreased 80% within 2.5 hours and remained at this level for 2 weeks; the doubling time increased to 3.7 days, and intracellular urea rose 2-fold, compared to 12U cells grown on urea without AHA. Urease of 12U cells inactivated by AHA in vivo could be reactivated to its pre-AHA level by incubation at 30 C after extraction and separation from free AHA. AHA inhibited incorporation of (15)N from [(15)N]urea into Kjeldahl nitrogen in the cells, in spite of the increased intracellular urea. These results indicate that AHA acts primarily by inhibiting urease action, rather than by inhibition of formation of urease protein or of uptake of urea. Because 12U cells are 8 times more tolerant of AHA than 1U cells, it is likely that growth on urea in the presence of AHA should select strongly for cells with high urease.

Activation of nitrate reductase by extracts from corn scutella.

NADH-nitrate reductase (NR) from the primary leaves and root tips of corn seedlings (var. W64A x W182E) were activated by extracts from corn scutella. The activator extracted in potassium phosphate buffer (pH 7.5) or 80% (v/v) ethanol and fractionated by Dowex 1 (acetate) and Dowex 50 (H(+)) resins was recovered in the cationic fraction. The activator was not detected in extracts from shoots, roots, or endosperm of the seedlings. It activated the nitrate-induced cytochrome c reductase of NR complex but had slight inhibitory effects on the activities of FMNH(2)-NR and reduced methylviologen-NR. In addition the activator inhibited the activities of purified NR-inactivating proteins from corn roots (var. Wf9 x 38-11) and rice cell cultures.

Cellular compartmentation of ammonium assimilation in rice and barley.

This review describes immunolocalization studies of the tissue and cellular location of glutamine synthetase (GS; EC 6.3.1.2) and glutamate synthase (Fd GOGAT; EC 1.4.7.1 and NADH-GOGAT; EC 1.4.1.14) proteins in roots and leaves of rice (Oryza sativa L.) and barley (Hordeum vulgare L.). In rice, cytosolic GS (GS1) protein was distributed homogeneously through all cells of the root. NADH GOGAT protein was strongly induced and its cellular location altered by ammonium treatment, becoming concentrated within the epidermal and exodermal cells. Fd GOGAT protein location changed with root development, from a widespread distribution in young cells to becoming concentrated within the central cylinder as cells matured. Plastid GS protein was barely detectable in rice roots, but was the major isoform in leaves, being present in the mesophyll and parenchyma sheath cells. GS1 was specific to the vascular bundle, as was NADH GOGAT, whereas Fd GOGAT was primarily found in mesophyll cells. In barley roots, GS1 protein was found in the cortical and vascular parenchyma and its concentration was highest in N-deficient seedlings. Plastid GS protein was detected in both cortical and vascular cells, where different plastid forms, containing different concentrations of GS protein, were identified. In barley leaves, GS2 protein was detected in the mesophyll chloroplasts and GS1 was found in the mesophyll and vascular cells. N nutrition strongly influenced this distribution, with a marked increase in GS1 concentration in the vascular cells in response to nitrate and ammonium, and an increase in mesophyll GS2 concentration in nitrate-grown seedlings. Fd GOGAT protein was found in both the mesophyll and vascular plastids. These localization studies show that the GS/GOGAT cycle is highly compartmentalized at both the subcellular and cellular levels. Reasons for this compartmentation, and the roles of each isoform, are discussed.

Expression of NADH-dependent glutamate synthase protein in the epidermis and exodermis of rice roots in response to the supply of ammonium ions.

The mRNA and protein for NADH-dependent glutamate synthase (NADH-GOGAT; EC 1.4.1.14) in root tips of rice (Oryza sativa L. cv. Sasanishiki) plants increases dramatically within 12 h of supplying a low concentration (> 0.05 mM) of ammonium ions (T. Yamaya et al., 1995, Plant Cell Physiol 36: 1197-1204). To identify the specific cells which are responsible for this rapid increase, the cellular localization of NADH-GOGAT protein was investigated immunocytologically with an affinity-purified anti-NADH-GOGAT immunoglobulin G. When root tips (> 1 mm) of rice seedlings which had been grown for 26 d in water were immuno-stained, signals for the NADH-GOGAT protein were detected in the central cylinder, in the apical meristem, and in the primordia of the secondary roots, Signals for ferredoxin-dependent GOGAT (Fd-GOGAT; EC 1.4.7.1) protein were also seen in the same three areas. When the roots were supplied with 1 mM ammonium ions for 24 h, there were strong signals for the NADH-GOGAT protein in two cell layers of the root surface, i.e. epidermis and exodermis, in addition to the cells giving signals in the absence of ammonium ions. The supply of ammonium ions was less effective on the profile of signals for Fd-GOGAT. Although the supply of ammonium ions had less effect on the expression of cytosolic glutamine synthetase (GS; EC 6.3.1.2), this enzyme was also found to be located in the epidermis and exodermis, as well as in the central cylinder and cortex. The results indicate that NADH-GOGAT, coupled to the cytosolic GS reaction, is probably important for the assimilation of ammonium ions in the two cell layers of the root surface.

Stimulation of Mitochondrial Calcium Uptake by Light during Growth of Corn Shoots.

Ca(2+) uptake in mitochondrial fractions, isolated on Percoll discontinuous density gradients, from light- and dark-grown corn (Zea mays L. var W64A x W182E) shoots was characterized by dual wavelength spectroscopy and the Ca(2+)-sensitive dye murexide. In light-grown seedlings, the rate of mitochondrial Ca(2+) uptake was about 40 nanomoles per minute per milligram of mitochondrial protein. A portion of the Ca(2+) uptake required an exogenous supply of ATP (65%) while the remaining 35% was the respiratory substrate-dependent reaction. Ruthenium red (2 micromolar) completely inhibited both ATP- and substrate-dependent reactions. There was no detectable Ca(2+) efflux from the mitochondria with the inhibitor. When the mitochondrial fraction was prepared from the dark-grown shoots, the rate of uptake, in particular the ATP-dependent reaction, was greatly reduced. The dark treatment caused a reduction in mitochondrial Ca content which is largely due to the reduction of Ca associated with the mitochondrial membrane rather than to a reduction of Ca in the soluble matrix.