[Transcriptome profiling of high oleic peanut under low temperatureduring germination].

High oleic (HO) peanut (Arachishypogaea L.) oils benefit human health and industrial production due to its superior nutritional composition and thermo-oxidative stability. However, HO peanut is sensitive to cold stress especially during germination, which limits its distribution in low temperature areas. To understand the molecular mechanism of cold responses in HO peanuts at germination stage, four HO peanut varieties with different cold tolerance were selected in field experiments to analyze their genome-wide gene regulation under low temperatures. High-throughput sequencing and transcriptome analysis revealed a total of 139 429 unigenes. Among these, 3520 common differentially expressed genes (DEG) were detected between two groups of cold-tolerant and cold-sensitive peanuts, and the number of up-regulated genes was greater than that of down-regulated genes in the cold-tolerant peanuts. Gene ontology analysis indicates that the number of DEGs involved in cell membrane metabolism and integrity as well as proteins located in the cell periphery were significantly higher in the cold-tolerant peanuts. KEGG pathway analysis suggests that plant-pathogen interaction and plant hormone signal transduction pathway play important roles in cold tolerance. Four cold-induced genes, TIC(TIME FOR COFFEE), ATX3(histone-lysine N-methyltransferase ATX3-like), AGO4(argonaute 4-like), FER(FERONIA-like receptor protein kinase), and three transcription factor genes, bHLH(bHLH49-like transcription factor), MYB(MYB-related protein 3R-1-like)and EREB(Ethylene-responsive element binding factor 6)were selected to verify the expression profile via real-time quantitative PCR detection. The expression of TIC, ATX3, AGO4, bHLH, MYB and EREB significantly increased within 3 hours after low temperature stress, while the expression of FER significantlyincreased after 12 hours, suggesting that these genes responded to low temperature stress during peanut germination. This work not only sheds light on the transcriptional regulation of HO peanut under low-temperature stress during germination but also provides data resources for screening candidate genes in improving peanuts stress resistance.

[Isolation of the promoter region of HAK gene from Aeluropus littoralis and functional analysis in rice].

The AlHAK1 gene encoding a high-affinity K+ transporter was isolated from Aeluropus littoralis (Gouan) Parl, a graminaceous halophyte, and plays a crucial role in nutrition and ion homeostasis in plant cell. To investigate the regulation role of AlHAK1 on the transcriptional level, an about 1.3 kb 5'-flanking region of the AlHAK1 gene containing a putative promoter was cloned by genome walking method. Cis-regulatory elements analysis showed AlHAK1-promoter region contained typical TATA and CAAT boxes, and some growth and development relative motifs, as well as environmental re-sponsive elements. To reveal the function and regulating role, the AlHAK1 promoter was fused to the ß-glucuronidase (GUS) reporter gene in the pCAMBIA1301 vector and introduced into rice via Agrobacterium-mediated transformation. Histo-chemical staining indicated that the GUS expression directed by AlHAK1 promoter was observed in leaves, stems, roots, anther, lemma, and palea. GUS quantitative fluorometric analysis indicated that GUS activity directed by AlHAK1 promoter was lower than CaMV35S and Ubiquitin constitutive promoters; however, in the roots and stems the GUS activity was rela-tively high and displayed a tissue-specific expression pattern. Under ABA, high temperature or drought stress, the GUS activity directed by AlHAK1 promoter was inducible in the roots and stems, suggesting the elements of HSE (-682 bp) and MybBS (-1 268 bp) might play a role in the inducible regulation.

Identification of ATP synthase beta subunit (ATPB) on the cell surface as a non-small cell lung cancer (NSCLC) associated antigen.

BACKGROUND: Antibody-based immunotherapy has achieved some success for cancer. But the main problem is that only a few tumor-associated antigens or therapeutic targets have been known to us so far. It is essential to identify more immunogenic antigens (especially cellular membrane markers) for tumor diagnosis and therapy. METHODS: The membrane proteins of lung adenocarcinoma cell line A549 were used to immunize the BALB/c mice. A monoclonal antibody 4E7 (McAb4E7) was produced with hybridoma technique. MTT cell proliferation assay was carried out to evaluate the inhibitory effect of McAb4E7 on A549 cells. Flow cytometric assay, immunohistochemistry, western blot and proteomic technologies based on 2-DE and mass spectrometry were employed to detect and identify the corresponding antigen of McAb4E7. RESULTS: The monoclonal antibody 4E7 (McAb4E7) specific against A549 cells was produced, which exhibited inhibitory effect on the proliferation of A549 cells. By the proteomic technologies, we identified that ATP synthase beta subunit (ATPB) was the corresponding antigen of McAb4E7. Then, flow cytometric analysis demonstrated the localization of the targeting antigen of McAb4E7 was on the A549 cells surface. Furthermore, immunohistochemistry showed that the antigen of McAb4E7 mainly aberrantly expressed in tumor cellular membrane in non-small cell lung cancer (NSCLC), but not in small cell lung cancer (SCLC). The rate of ectopic expressed ATPB in the cellular membrane in lung adenocarcinoma, squamous carcinoma and their adjacent nontumourous lung tissues was 71.88%, 66.67% and 25.81% respectively. CONCLUSION: In the present study, we identified that the ectopic ATPB in tumor cellular membrane was the non-small cell lung cancer (NSCLC) associated antigen. ATPB may be a potential biomarker and therapeutic target for the immunotherapy of NSCLC.

[Differentiation of erythroleukemia K562 cells induced by piperine].

BACKGROUND & OBJECTIVE: The most common haematological malignancy is leukaemia. Differentiation induction is considered as one of the effective therapies for leukemia. Piperine, an alkaloid extracted from piperaceae, has been reported to display a variety of pharmacological activities, including sedation, anti-inflammation and antitumor effects. This study was to investigate the effect of piperine on proliferation, differentiation and apoptosis of erythroleukemia K562 cells. METHODS: Inhibition of cell growth was determined by trypan blue exclusion test; cell cycle and cell apoptosis were analyzed by FACS; induction of cell differentiation was confirmed by morphological observation, nitroblue tetrazolium (NBT) reduction assay and measurements of CD33 and CD14 expressions. RESULTS: Piperine induced K562 cells to differentiate into macrophages/monocytes at 20 micromol/L or 40 micromol/L. After incubation with 40 mumol/L piperine for 3 d, the NBT reduction rate of K562 cells increased from (8.5+/-1.9)% to (76.7+/-5.3)%; after incubation with 20 mumol/L piperine for 3 d, the mean fluorescence intensity (MFI) of CD33 in K562 cells was decreased by 42.05% (P<0.01), whereas the MFI of CD14 was doubled (P<0.01). Piperine inhibited the proliferation of K562 cells in a dose-and time-dependent manner at a concentration of above 20 micromol/L. CONCLUSION: Piperine can induce K562 cells to differentiate into macrophages/monocytes.

Characterization and expression of a vacuolar Na(+)/H(+) antiporter gene from the monocot halophyte Aeluropus littoralis.

Plant vacuolar Na(+)/H(+) antiporter plays an important role in salt tolerance. In order to understand the molecular basis of vacuolar Na(+)/H(+) antiporter responded to salinity and reveal a possible role of salt tolerance in monocots, a vacuolar Na(+)/H(+) antiporter gene (AlNHX) was isolated by reverse transcription-PCR and RNA ligase mediated rapid amplification of cDNA ends (RLM-RACE) based on the homology from Aeluropus littoralis (Gouan) Parl, a graminaceous halophyte. The AlNHX sequence contained 2706bp with an open read frame of 1623bp and the deduced transcripts encoding 540 amino acids shared a high homology with those putative vacuolar Na(+)/H(+) antiporters of higher plants. AlNHX was predicted containing ten putative hydrophobic regions, which was different with AtNHX1 and OsNHX1. DNA gel blot analysis indicated that there were two or three copies of AlNHX in the A. littoralis genome. The increased transcript levels of AlNHX were much higher in roots than that in shoots under salt stress. In addition, overexpression of AlNHX in tobacco conferred high salt tolerance to the transgenic plants. The analysis of ion contents indicated that under high salt stress for one month, the transgenic plants compartmentalized more Na(+) in the roots and kept a relative high K(+)/Na(+) ratio in the leaves compared with wild-type plants.

Nano-cerium-element-doped titanium dioxide induces apoptosis of Bel 7402 human hepatoma cells in the presence of visible light.

AIM: To investigate the apoptotic effect of photoexcited titanium dioxide (TiO(2)) nanoparticles in the presence of visible light on human hepatoma cell line (Bel 7402) and to study the underlying mechanism. METHODS: Cerium-element-doped titanium dioxide nanoparticles were prepared by impregnation method. Bel 7402 human hepatoma cells were cultured in RPMI 1640 medium in a humidified incubator with 50 mL/L CO(2) at 37 centigrade. A 15 W fluorescent lamp with continuous wavelength light was used as light source in the photocatalytic test. Fluorescence morphology and agarose gel eletrophoresis pattern were performed to analyze apoptotic cells. RESULTS: The Ce (IV)-doped TiO(2) nanoparticles displayed their superiority. The adsorption edge shifted to the 400-450 nm region. With visible light illuminated for 10 min, 10 microg/cm(3) Ce (IV)-doped TiO(2) induced micronuclei and significant apoptosis in 4 and 24 h, respectively. Hochest 33258 staining of the fixed cells revealed typical apoptotic structures (apoptotic bodies), agarose gel electrophoresis showed typical DNA ladder pattern in treated cells but not in untreated ones. CONCLUSION: Ce (IV) doped TiO(2) nanoparticles can induce apoptosis of Bel 7402 human hepatoma cells in the presence of visible light.

Observation of DNA damage of human hepatoma cells irradiated by heavy ions using comet assay.

AIM: Now many countries have developed cancer therapy with heavy ions, especially in GSI (Gesellschaft f r Schwerionenforschung mbH, Darmstadt, Germany), remarkable results have obtained, but due to the complexity of particle track structure, the basic theory still needs further researching. In this paper, the genotoxic effects of heavy ions irradiation on SMMC-7721 cells were measured using the single cell gel electrophoresis (comet assay). The information about the DNA damage made by other radiations such as X-ray, gamma-ray, UV and fast neutron irradiation is very plentiful, while little work have been done on the heavy ions so far. Hereby we tried to detect the reaction of liver cancer cells to heavy ion using comet assay, meanwhile to establish a database for clinic therapy of cancer with the heavy ions. METHODS: The human hepatoma cells were chosen as the test cell line irradiated by 80 Mev/u (20)Ne(10+) on HIRFL (China), the radiation-doses were 0, 0.5, 1, 2, 4 and 8 Gy, and then comet assay was used immediately to detect the DNA damages, 100-150 cells per dose-sample (30-50 cells were randomly observed at constant depth of the gel). The tail length and the quantity of the cells with the tail were put down. EXCEL was used for statistical analysis. RESULTS: We obtained clear images by comet assay and found that SMMC-7721 cells were all damaged apparently from the dose 0.5 Gy to 8 Gy (t-test: P<0.001, vs control). The tail length and tail moment increased as the doses increased, and the number of cells with tails increased with increasing doses. When doses were higher than 2 Gy, nearly 100 % cells were damaged. Furthermore, both tail length and tail moment, showed linear equation. CONCLUSION: From the clear comet assay images, our experiment proves comet assay can be used to measure DNA damages by heavy ions. Meanwhile DNA damages have a positive correlation with the dose changes of heavy ions and SMMC-7721 cells have a great radiosensitivity to (20)Ne(10+). Different reactions to the change of doses indicate that comet assay is a useful tool to detect DNA damage induced by heavy ions.