Plant Protective and Growth Promoting Effects of Seed Endophytes in Soybean Plants.

Seed-borne diseases reduce not only the seed germination and seedling growth but also seed quality, resulting in the significant yield loss in crop production. Plant seed harbors diverse microbes termed endophytes other than pathogens inside it. However, their roles and application to agricultures were rarely understood and explored to date. Recently, we had isolated from soybean seeds culturable endophytes exhibiting in-vitro antagonistic activities against common bacterial and fungal seed-borne pathogens. In this study, we evaluated effects of seed treatment with endophytes on plant growth and protection against the common seed-borne pathogens: four fungal pathogens (Cercospora sojina, C. kikuchii, Septoria glycines, Diaporthe eres) and two bacterial pathogens (Xanthomonas axonopodis pv. glycines, Pseudomonas syringae pv. tabaci). Our experiments showed that treatment of soybean seeds with seed endophytes clearly offer protection against seed-borne pathogens. We also found that some of the endophytes promote plant growth in addition to the disease suppression. Taken together, our results demonstrate agricultural potential of seed endophytes in crop protection.

Synthesis of Benzotriazepin-1-ones via Decarboxylative [4 + 3]-Annulation of Isatoic Anhydrides with Nitrile Imines.

A novel and streamlined approach to synthesizing benzotriazepin-1-ones has been developed through a hexafluoroisopropanol-promoted decarboxylative cascade reaction between isatoic anhydrides and hydrazonoyl chloride. The [4 + 3] annulation of hexafluoroisopropyl 2-aminobenzoates with nitrile imines, generated in situ, is a key feature of this innovative reaction. This approach has offered a simple and efficient method for synthesizing a broad range of structurally intricate and highly functional benzotriazepinones.

Culturable Endophytes Associated with Soybean Seeds and Their Potential for Suppressing Seed-Borne Pathogens.

Seed-borne pathogens in crops reduce the seed germination rate and hamper seedling growth, leading to significant yield loss. Due to the growing concerns about environmental damage and the development of resistance to agrochemicals among pathogen populations, there is a strong demand for eco-friendly alternatives to synthetic chemicals in agriculture. It has been well established during the last few decades that plant seeds harbor diverse microbes, some of which are vertically transmitted and important for plant health and productivity. In this study, we isolated culturable endophytic bacteria and fungi from soybean seeds and evaluated their antagonistic activities against common bacterial and fungal seed-borne pathogens of soybean. A total of 87 bacterial isolates and 66 fungal isolates were obtained. Sequencing of 16S rDNA and internal transcribed spacer amplicon showed that these isolates correspond to 30 and 15 different species of bacteria and fungi, respectively. Our antibacterial and antifungal activity assay showed that four fungal species and nine bacterial species have the potential to suppress the growth of at least one seed-borne pathogen tested in the study. Among them, Pseudomonas koreensis appears to have strong antagonistic activities across all the pathogens. Our collection of soybean seed endophytes would be a valuable resource not only for studying biology and ecology of seed endophytes but also for practical deployment of seed endophytes toward crop protection.

The Bacillus zanthoxyli HS1 Strain Renders Vegetable Plants Resistant and Tolerant against Pathogen Infection and High Salinity Stress.

Various management systems are being broadly employed to minimize crop yield loss resulting from abiotic and biotic stresses. Here we introduce a Bacillus zanthoxyli HS1 strain as a potent candidate for managing manifold stresses on vegetable plants. Considering 16S rDNA sequence and biochemical characteristics, the strain is closely related to B. zanthoxyli. The B. zanthoxyli HS1's soil-drench confers disease resistance on tomato and paprika plants against infection with Ralstonia solanacearum and Phytophthora capsici, respectively. Root and shoot growths are also increased in B. zanthoxyli HS1-treated cabbage, cucumber, and tomato plants, compared with those in mock-treated plants, after application of high salinity solution. Moreover, the pretreatment of B. zanthoxyli HS1 on cabbage plants inhibits the degradation of chloroplast pigments caused by high salinity stresses, whereas the inhibitory effect is not observed in cucumber plants. These findings suggest that B. zanthoxyli HS1 stain inhibits disease development and confers tolerance to salinity stress on vegetable plants.

Modulation of transcription by the peroxisome proliferator-activated receptor delta--binding RNA aptamer in colon cancer cells.

Peroxisome proliferator-activated receptor delta (PPAR-delta), one of three PPAR subtypes, is a lipid-sensing nuclear receptor that has been implicated in multiple processes, including inflammation and cancer. To directly establish the role of PPAR-delta in colon cancer development and progression, we selected high-affinity RNA aptamers and expressed them in several colon cancer cell lines. Nuclear-expressed aptamers efficiently inhibited PPAR-delta-dependent transcription from a synthetic peroxisome proliferator response element-driven luciferase reporter. PPAR-delta-specific aptamers suppressed transcription from natural promoters of vascular endothelial cell growth factor-A and cyclooxygenase-2. Moreover, vascular endothelial cell growth factor-A and cyclooxygenase-2 mRNA levels were significantly reduced by the PPAR-delta-specific aptamers in colon cancer cells. Most significantly, HCT116 colon cancer cells with high-level expression of PPAR-delta-specific aptamers exhibited a striking loss of tumorigenic potential. Further study on these RNA aptamers could provide an opportunity to modulate PPAR-delta-mediated colon cancer development and progression. Taken together, our results establish an important role for PPAR-delta in transcription of tumor-promoting genes, which can be specifically modulated by high-affinity RNA intramers in colon cancer cells. The RNA intramers may be further developed as specific inhibitors for cancer therapeutic strategies.

beta-catenin regulates multiple steps of RNA metabolism as revealed by the RNA aptamer in colon cancer cells.

Nuclear beta-catenin forms a transcription complex with TCF-4, which is implicated in colon cancer development and progression. Recently, we and others have shown that beta-catenin could be a regulator of RNA splicing and it also stabilizes the cyclooxygenase-2 (COX-2) mRNA. Here, we further explored the role of beta-catenin in the RNA metabolism in colon cancer cells. To specifically modulate the subcellular functions of beta-catenin, we expressed the RNA aptamer in the form of RNA intramers with unique cellular localizations. The nucleus-expressed RNA intramer proved to be effective in reducing the protein-protein interaction between beta-catenin and TCF-4, thus shown to be a specific regulator of beta-catenin-activated transcription. It could also regulate the alternative splicing of E1A minigene in diverse colon cancer cell lines. In addition, we tested whether beta-catenin could stabilize any other mRNAs and found that cyclin D1 mRNA was also bound and stabilized by beta-catenin. Significantly, the cytoplasm-expressed RNA intramer reverted the beta-catenin-induced COX-2 and cyclin D1 mRNA stabilization. We show here that beta-catenin regulated multiple steps of RNA metabolism in colon cancer cells and might be the protein factor coordinating RNA metabolism. We suggest that the RNA intramers could provide useful ways for inhibiting beta-catenin-mediated transcription and RNA metabolism, which might further enhance the antitumorigenic effects of these molecules in colon cancer cells.

Arsenic-induced dysfunction in relaxation of blood vessels.

Several epidemiological studies have suggested that exposure to arsenic is strongly correlated with the development of cardiovascular diseases such as hypertension. To determine whether arsenic affects vasomotor tone in blood vessels, we investigated the effect of arsenic on vasorelaxation using isolated rat aortic rings in an organ-bath system. Treatment with arsenite inhibited acetylcholine-induced relaxation of the aortic rings in a concentration-dependent manner, whereas several other arsenic species did not have any effect. Consistent with these findings, the levels of guanosine 3',5'-cyclic monophosphate (cGMP) in the aortic rings were significantly reduced by arsenite treatment. In cultured human aortic endothelial cells, treatment with arsenite resulted in a concentration-dependent inhibition of endothelial nitric oxide synthase (eNOS). In addition, higher concentrations of arsenite decreased the relaxation induced by sodium nitroprusside (an NO donor) and 8-Br-cGMP (a cGMP analog) in aortic rings without endothelium. These in vitro results indicate that arsenite is capable of suppressing relaxation in blood vessels by inhibiting eNOS activity in endothelial cells and by impairing the relaxation machinery in smooth muscle cells. In vivo studies revealed that the reduction of blood pressure by acetylcholine infusion was significantly suppressed after arsenite was administered intravenously to rats. These data suggest that an impairment of vasomotor tone due to arsenite exposure may be a contributing factor in the development of cardiovascular disease.

In vitro pharmacodynamics of CKD-602 in HT-29 cells.

CKD-602 (7-[2-(N-isopropylamino)ethyl]-(20S)-camptothecin) is a recently-developed synthetic camptothecin analogue and currently under clinical development by Chong Kun Dang Pharm (Seoul, Korea). CKD-602 showed potent topoisomerase inhibitory activity in vitro and broad antitumor activity against various human tumor cells in vitro and in vivo in animal models. This study describes the pharmacodynamics of the immediate and delayed cytotoxicity induced by CKD-602 in a human colorectal adenocarcinoma cell line, HT-29, and its intracellular drug accumulation by HPLC. The present study was designed to address whether the higher activity of CKD-602 with prolonged exposure is due to delayed exhibition of cytotoxicity and/or an accumulation of antiproliferative effect on continuous drug exposure. The drug uptake study was performed to determine whether the delayed cytotoxicity is due to a slow drug accumulation in cells. CKD-602 produced a cytotoxicity that was exhibited immediately after treatment (immediate effect) and after treatment had been terminated (delayed effect). Both the immediate and delayed effects of CKD-602 showed a time dependent decrease in IC50 values. Drug uptake was biphasic and the second equilibrium level was obtained as early as at 24 hr, indicating that the cumulative and delayed antitumor effects of CKD-602 were not due to slow drug uptake. On the other hand, CKD-602 treatment was sufficient to induce delayed cytotoxicity after 4 hr, however, longer treatment (>24 hr) enhanced its cytotoxicity due to the intracellular accumulation of the drug, which requires 24 hr to reach maximum equilibrium concentration. In addition, Cn x T=h analysis (n=0.481) indicated that increased exposure times may contribute more to the overall antitumor activity of CKD-602 than drug concentration. Additional studies to determine the details of the intracellular uptake kinetics (e.g., concentration dependency and retention studies) are needed in order to identify the optimal treatment schedules for the successful clinical development of CKD-602.

Methamphetamine-induced apoptosis in a CNS-derived catecholaminergic cell line.

Methamphetamine (METH) causes neurotoxic damages to the dopaminergic system in mammals, but whether it exerts toxicity to dopamine cells in culture has not been fully explored. In order to develop an in vitro model of METH-induced dopamine neurotoxicity toward more systemical examination of the mechanism, we investigated METH toxicity in a clonal dopamine producing cell line (CATH.a). We show in the present study that METH produces a time- and dose-dependent increase in cell death via a process similar to apoptosis. The METH toxicity seems to be produced by oxidative stress, as it was attenuated by the antioxidant glutathione, and to involve dopamine because dopamine release and synthesis inhibitors attenuated the toxicity. This catecholaminergic cell line derived from the central nervous system may become a useful in vitro model to elucidate the mechanism underlying the METH-induced dopaminergic neuronal damage.