Detection and validation of QTLs for milky-white grains caused by high temperature during the ripening period in Japonica rice.

The occurrence of chalky rice (Oryza sativa L.) grains caused by high temperature is a serious problem in rice production. Of the several kinds of chalky grains, milky-white grains are not well analyzed. The milky-white rice grain phenomenon is caused by genetic factors as well as environmental and nutritional conditions. To analyze the genetic control system for rice grain quality, we raised recombinant inbred lines from progeny produced from 'Tsukushiroman' (high temperature sensitive) and 'Chikushi 52' (high temperature tolerant) cultivars. Quantitative trait locus (QTL) analysis revealed that the QTL on chromosome 4, linked to the simple sequence repeat marker RM16424, contributed substantially to the occurrence of milky-white grains, as it was detected over two experimental years. To validate the effect of the QTL, we developed near isogenic lines that have the 'Chikushi 52' segment on the short arm of chromosome 4 in the 'Tsukushiroman' genetic background, and that had a lower milky-white grain ratio than that of 'Tsukushiroman' when exposed to high temperatures during the ripening period. These results suggest that the 'Chikushi 52' allele on chromosome 4 suppresses the occurrence of milky-white grains and improves rice grain quality under heat stress during the grain ripening period.

Nestedness in Arbuscular Mycorrhizal Fungal Communities along Soil pH Gradients in Early Primary Succession: Acid-Tolerant Fungi Are pH Generalists.

Soil acidity is a major constraint on plant productivity. Arbuscular mycorrhizal (AM) fungi support plant colonization in acidic soil, but soil acidity also constrains fungal growth and diversity. Fungi in extreme environments generally evolve towards specialists, suggesting that AM fungi in acidic soil are acidic-soil specialists. In our previous surveys, however, some AM fungi detected in strongly acidic soils could also be detected in a soil with moderate pH, which raised a hypothesis that the fungi in acidic soils are pH generalists. To test the hypothesis, we conducted a pH-manipulation experiment and also analyzed AM fungal distribution along a pH gradient in the field using a synthesized dataset of the previous and recent surveys. Rhizosphere soils of the generalist plant Miscanthus sinensis were collected both from a neutral soil and an acidic soil, and M. sinensis seedlings were grown at three different pH. For the analysis of field communities, rhizosphere soils of M. sinensis were collected from six field sites across Japan, which covered a soil pH range of 3.0-7.4, and subjected to soil trap culture. AM fungal community compositions were determined based on LSU rDNA sequences. In the pH-manipulation experiment the acidification of medium had a significant impact on the compositions of the community from the neutral soil, but the neutralization of the medium had no effect on those of the community from the acidic soil. Furthermore, the communities in lower -pH soils were subsets of (nested in) those in higher-pH soils. In the field communities a significant nestedness pattern was observed along the pH gradient. These observations suggest that the fungi in strongly acidic soils are pH generalists that occur not only in acidic soil but also in wide ranges of soil pH. Nestedness in AM fungal community along pH gradients may have important implications for plant community resilience and early primary succession after disturbance in acidic soils.

Detection of QTLs for white-back and basal-white grains caused by high temperature during ripening period in japonica rice.

There is increasing evidence that global warming affects the development of rice. High temperatures during ripening increase the ratio of undesirable chalky grains followed by deteriorating grain appearance quality. In order to detect quantitative trait loci (QTLs) controlling the occurrence of white-back and basal-white chalky grains of brown rice, QTL analysis was performed using recombinant inbred lines derived from a cross between two strains, 'Tsukushiroman' (sensitive to heat stress) and 'Chikushi 52' (tolerant of heat stress). The F7 and F8 lines were exposed to heat stress during the ripening period in two locations, Fukuoka and Kagoshima, in Japan. QTLs for white-back grains and basal-white grains were detected on chromosomes 1, 3, and 8, and those for basal-white grains were detected on chromosomes 2, 3, and 12. QTLs on chromosome 8 for white-back grains were shared in the plants grown in both locations. Near-isogenic lines (NILs), which harbored a segment from 'Chikushi 52' on chromosome 8 with the genetic background of 'Tsukushiroman', showed relatively lower ratios of white-back grains than 'Tsukushiroman'. Therefore, insertion of the 'Chikushi 52' genomic region of the QTL on chromosome 8 can improve the quality of rice when it is grown under heat stress conditions.

Green tea catechin, epigallocatechin-3-gallate, attenuates the cell viability of human non-small-cell lung cancer A549 cells via reducing Bcl-xL expression.

Clinical and epidemiological studies have indicated that the consumption of green tea has a number of beneficial effects on health. Epigallocatechin-3-gallate (EGCg), the major polyphenolic compound present in green tea, has received much attention as an active ingredient. Among the numerous promising profiles of EGCg, the present study focused on the anticancer effects. Apoptosis induced by EGCg and subsequent cell growth suppression have been demonstrated in a number of cell culture studies. However, the underlying mechanism of apoptotic cell death remains unclear. Thus, the aim of the present study was to identify the major molecule that mediates proapoptotic cell death by EGCg. The effect of EGCg on cell proliferation and the induction of mRNA that modulates apoptotic cell death was evaluated in the A549 human non-small-cell lung cancer cell line. In addition, morphological changes were assessed by microscopy in A549 cells that had been treated with 100 µM EGCg for 24 h. The MTT assay revealed that cell proliferation was significantly reduced by EGCg in a dose-dependent manner (3-100 µM). The mRNA expression level of B-cell lymphoma-extra large (Bcl-xL) was decreased in A549 cells following 24 h incubation with 100 µM EGCg. Therefore, the results indicated that the inhibition of cell proliferation by EGCg may be achieved via suppressing the expression of the cell death-inhibiting gene, Bcl-xL.

Simultaneous detection of green tea catechins and gallic acid in human serum after ingestion of green tea tablets using ion-pair high-performance liquid chromatography with electrochemical detection.

We developed an analytical method for the simultaneous determination of tea catechins and gallic acid (GA) in human serum using ion-pair high-performance liquid chromatography (HPLC) with electrochemical detection. GA was measured to estimate the amount of gallate moiety produced by degradation of gallated catechins ((-)-epicatechin-3-gallate, ECG; (-)-epigallocatechin-3-gallate, EGCG). Ethyl gallate was adopted as an internal standard to correct for the extraction efficiency. To maximize extraction efficiency, a hydrophobic polytetrafluoroethylene (PTFE) filter was selected for pre-treatment prior to separation. HPLC separation was performed using a C18 reversed-phase column with a gradient mobile phase of phosphate buffer (pH 2.5) containing tetrahexylammonium hydrogensulfate as an ion-pair reagent. Using this method, (-)-epicatechin (EC), (-)-epigallocatechin (EGC), ECG, EGCG, ethyl gallate, and GA were detected as single peaks. The resolution values for target analytes were 4.0-13.0 and the mean values of the absolute recoveries of catechins and GA were 77.3-93.9%. The detection limits for catechins and GA in serum were 0.4-3.1ng/mL. The serum catechin levels of eight healthy volunteers after ingestion of a single dose of green tea tablets were measured using this method. The concentration of total catechins (free+conjugated forms) in serum peaked 60min after ingestion. From these results, this method is thought to enable the simultaneous quantification of GA, the hydrolysis product of gallated catechins, and target catechins, and to be sufficiently sensitive for pharmacokinetic studies of catechins following oral administration of green tea.

Reduction of oxidative stress in liver cancer patients by oral green tea polyphenol tablets during hepatic arterial infusion chemotherapy.

Hepatic arterial infusion chemotherapy (HAI) using an implanted port system is the standard regimen for primary and metastatic liver cancers (MLCs). However, there have been few studies concerning HAI-induced oxidative stress and damage to the liver or other organs. The aim of the present study was to investigate the ability of green tea polyphenols (GTPs) to reduce the oxidative stress or increase the biological antioxidative potential in HAI-treated patients. A total of 19 patients with inoperable hepatocellular carcinoma (HCC) or MLC from colorectal malignancy were eligible for HAI with cisplatin (CDDP) and 5-fluorouracil (5FU). The study subjects were randomly assigned to either a 3 or a 6 oral GTP tablets per day group. Each tablet had a GTP content equivalent to 79 mg of epigallocatechin-3-gallate. The oxidative stress was assessed by measuring the levels of derivatives of reactive oxygen metabolites (d-ROMs) and the biological antioxidative potential (BAP) values in patient plasma using the Free Radical Analytical System 4 (FRAS4), and correlating the results with clinical laboratory data for the patients. The levels of d-ROMs were significantly reduced by the oral intake of 6 GTP tablets for 6-9 months (P=0.0463) but were not significantly reduced by the oral intake of 3 GTP tablets daily. BAP values remained constant in the 3 and 6 tablet groups for 6-9 months during the follow-up study. The total serum bilirubin (T-bil) levels increased significantly at 3 (P=0.028) and 9 (P=0.0151) months and the red blood cell (RBC) count decreased at 6 months (P=0.0458) after intake for the 6 GTP tablet group. Alkaline phosphatase (ALP) levels increased significantly at 9 months (P=0.0298). Cholinesterase (ChE) decreased significantly at 9 (P= 0.0127) and 12 (P= 0.0207) months after intake for the 3 GTP tablet group. The results indicate that the daily intake of 6 GTP tablets containing 474 mg polyphenols significantly reduces HAI-induced oxidative stress in HCC or MLC patients while the antioxidative potentials of the patients remain constant.

Low-dose methotrexate inhibits lung metastasis and lengthens survival in rat osteosarcoma.

Lung metastasis is the most crucial event affecting the treatment of osteosarcoma and is dependent on tumor angiogenesis. To improve the prognosis for patients with osteosarcoma, prevention of lung metastasis is essential. Low-dose methotrexate is a useful drug for treating a variety of diseases. Low-dose methotrexate reportedly plays a role in antiangiogenesis for the synovial blood vessels in rheumatoid arthritis. However, whether low-dose methotrexate is correlated with tumor angiogenesis and metastasis is unclear. We investigated the inhibitory effect of methotrexate on lung metastasis in a rat osteosarcoma cell line with high metastatic potential, S-SLM. Two weeks after inoculation of S-SLM cells into male Fischer 344 rats, low-dose methotrexate (1.2 mg/kg once or twice a week) or saline was intraperitonealy injected for 4 weeks and the antimetastatic effect was evaluated. Low-dose methotrexate significantly reduced the number of lung metastatic nodules and the wet weight of the lungs. Immunohistochemical staining showed a decrease in microvessel density in the metastatic nodules. We also evaluated the effect of methotrexate on the proliferation of endothelial cells and S-SLM osteosarcoma cells in vitro. Methotrexate significantly inhibited the proliferation of endothelial cells at a lower concentration than that of S-SLM osteosarcoma cells. These data suggest that low-dose methotrexate inhibited lung metastasis of osteosarcoma through its antiangiogenic activity. Our results indicate that low-dose methotrexate is a promising drug for tumor dormancy therapy in patients with osteosarcoma and lung metastasis.

Telomerase activity and human telomerase reverse transcriptase mRNA expression are correlated with clinical aggressiveness in soft tissue tumors.

BACKGROUND: Telomerase is a ribonucleoprotein enzyme that extends telomere specific repeats on the ends of chromosomes. Telomerase activity has been detected frequently in various types of human tumors and has been associated with cell immortality and oncogenesis. Human telomerase reverse transcriptase (hTERT), a telomerase catalytic subunit, reportedly regulates telomerase activity. Little is known about telomerase activity and hTERT mRNA expression in soft tissue tumors. The objective of this study was to clarify the correlation between these two parameters and clinical aggressiveness in soft tissue tumors. METHODS: In 41 surgically resected soft tissue tumors, telomerase activity was measured by the fluorescence-based telomeric repeat-amplification protocol and hTERT mRNA expression was determined by real-time polymerase chain reaction. RESULTS: Telomerase activity was detected in 52% of sarcomas and in none of the benign soft tissue tumors (P < 0.05). Telomerase activity was found in 77% of 13 locally recurrent sarcomas and in 89% of 9 sarcomas with distant metastasis. The frequency of the presence of telomerase activity in those tumors was significantly greater compared with the frequency of telomerase activity in the other sarcomas (P < 0.05 and P < 0.01, respectively). All telomerase positive sarcomas expressed hTERT mRNA. The mean level of hTERT mRNA expression in sarcomas was significantly greater compared with the mean hTERT mRNA expression level in benign tumors (P < 0.05) and in locally recurrent sarcomas compared with primary sarcomas (P < 0.001). CONCLUSIONS: The results of the current study suggest that the detection of telomerase activity and the level of hTERT mRNA expression are useful markers for evaluating the clinical aggressiveness in soft tissue tumors.

Suppression of tumor growth and pulmonary metastasis in murine osteosarcoma using gene therapy.

We evaluated the effect of gene therapy in the murine osteosarcoma cell line, LM8, which preferentially metastasizes to the lungs. LM8 cells were transduced with the gene for a herpes simplex virus thymidine kinase (HSV-tk) or Escherichia coli beta-galactosidase (lacZ). We investigated the cytotoxicity of LM8 cells bearing an HSV-tk gene after treatment with ganciclovir (GCV). LM8 cells bearing an HSV-tk gene were more sensitive than non-transduced cells. The remarkable inhibition of tumor growth and pulmonary metastases was confirmed in vivo. Our findings indicated that GCV kills tumor cells transduced with HSV-tk in vitro and in vivo.