Investigation of the ASR family in foxtail millet and the role of ASR1 in drought/oxidative stress tolerance.

KEY MESSAGE: Six foxtail millet ASR genes were regulated by various stress-related signals. Overexpression of ASR1 increased drought and oxidative tolerance by controlling ROS homeostasis and regulating oxidation-related genes in tobacco plants. Abscisic acid stress ripening (ASR) proteins with ABA/WDS domains constituted a class of plant-specific transcription factors, playing important roles in plant development, growth and abiotic stress responses. However, only a few ASRs genes have been characterized in crop plants and none was reported so far in foxtail millet (Setaria italic), an important drought-tolerant crop and model bioenergy grain crop. In the present study, we identified six foxtail millet ASR genes. Gene structure, protein alignments and phylogenetic relationships were analyzed. Transcript expression patterns of ASR genes revealed that ASRs might play important roles in stress-related signaling and abiotic stress responses in diverse tissues in foxtail millet. Subcellular localization assays showed that SiASR1 localized in the nucleus. Overexpression of SiASR1 in tobacco remarkably increased tolerance to drought and oxidative stresses, as determined through developmental and physiological analyses of germination rate, root growth, survival rate, relative water content, ion leakage, chlorophyll content and antioxidant enzyme activities. Furthermore, expression of SiASR1 modulated the transcript levels of oxidation-related genes, including NtSOD, NtAPX, NtCAT, NtRbohA and NtRbohB, under drought and oxidative stress conditions. These results provide a foundation for evolutionary and functional characterization of the ASR gene family in foxtail millet.

Anticancer effect of icaritin on human lung cancer cells through inducing S phase cell cycle arrest and apoptosis.

Icaritin, a prenylflavonoid derivative from Epimedium Genus, has been shown to exhibit many pharmacological and biological activities. However, the function and the underlying mechanisms of icaritin in human non-small cell lung cancer have not been fully elucidated. The purpose of this study was to investigate the anticancer effects of icaritin on A549 cells and explore the underlying molecular mechanism. The cell viability after icaritin treatment was tested by MTT assay. The cell cycle distribution, apoptosis and reactive oxygen species (ROS) levels were analyzed by flow cytometry. The mRNA and protein expression levels of the genes involved in proliferation and apoptosis were respectively detected by RT-PCR and Western blotting. The results demonstrated that icaritin induced cell cycle arrest at S phase, and down-regulated the expression levels of S regulatory proteins such as Cyclin A and CDK2. Icaritin also induced cell apoptosis characterized by positive Hoechst 33258 staining, accumulation of the Annexin V-positive cells, increased ROS level and alteration in Bcl-2 family proteins expression. Moreover, icaritin induced sustained phosphorylation of ERK and p38 MAPK. These findings suggested that icaritin might be a new potent inhibitor by inducing S phase arrest and apoptosis in human lung carcinoma A549 cells.

Fucoidan induces apoptosis of HepG2 cells by down-regulating p-Stat3.

Fucoidan is one of the main bioactive components of polysaccharides. The current study was focused on the anti-tumor effects of fucoidan on human heptoma cell line HepG2 and the possible mechanisms. Fucoidan treatment resulted in cell cycle arrest and apoptosis of HepG2 cells in a dose-dependent manner detected by MTT assay, flow cytometry and fluorescent microscopy. The results of flow cytometric analysis revealed that fucoidan induced G2/M arrest in the cell cycle progression. Hoechst 33258 and Annexin V/PI staining results showed that the apoptotic cell number was increased, which was associated with a dose-dependent up-regulation of Bax and down-regulation of Bcl-2 and p-Stat3. In parallel, the up-regulation of p53 and the increase in reactive oxygen species were also observed, which may play important roles in the inhibition of HepG2 growth by fucoidan. In the meantime, Cyclin B1 and CDK1 were down-regulated by fucoidan treatment. Down-regulation of p-Stat3 by fucoidan resulted in apoptosis and an increase in ROS in response to fucoidan exposure. We therefore concluded that fucoidan induces apoptosis through the down-regulation of p-Stat3. These results suggest that fucoidan may be used as a novel anti-cancer agent for hepatocarcinoma.

The voltage-dependent anion channel 1 (AtVDAC1) negatively regulates plant cold responses during germination and seedling development in Arabidopsis and interacts with calcium sensor CBL1.

The voltage-dependent anion channel (VDAC), a highly conserved major mitochondrial outer membrane protein, plays crucial roles in energy metabolism and metabolite transport. However, knowledge about the roles of the VDAC family in plants is limited. In this study, we investigated the expression pattern of VDAC1 in Arabidopsis and found that cold stress promoted the accumulation of VDAC1 transcripts in imbibed seeds and mature plants. Overexpression of VDAC1 reduced tolerance to cold stress in Arabidopsis. Phenotype analysis of VDAC1 T-DNA insertion mutant plants indicated that a vdac1 mutant line had faster germination kinetics under cold treatment and showed enhanced tolerance to freezing. The yeast two-hybrid system revealed that VDAC1 interacts with CBL1, a calcium sensor in plants. Like the vdac1, a cbl1 mutant also exhibited a higher seed germination rate. We conclude that both VDAC1 and CBL1 regulate cold stress responses during seed germination and plant development.

Fucoidan induces G1 phase arrest and apoptosis through caspases-dependent pathway and ROS induction in human breast cancer MCF-7 cells.

Fucoidan is an active component of seaweed, which inhibits proliferation and induces apoptosis of several tumor cells while the detailed mechanisms underlying this process are still not clear. In this study, the effect of Fucoidan on the proliferation and apoptosis of human breast cancer MCF-7 cells and the molecular mechanism of Fucoidan action were investigated. Viable cell number of MCF-7 cells was decreased by Fucoidan treatment in a dose-dependent manner as measured by MTT assay. Fucoidan treatment resulted in G1 phase arrest of MCF-7 cells as revealed by flow cytometry, which was associated with the decrease in the gene expression of cyclin D1 and CDK-4. Annexin V/PI staining results showed that the number of apoptotic cells was associated with regulation of cytochrome C, caspase-8, Bax and Bcl-2 at transcriptional and translational levels. Both morphologic observation and Hoechst 33258 assay results confirmed the pro-apoptotic effect of Fucoidan. Meanwhile, the ROS production was also increased by Fucoidan treatment, which suggested that Fucoidan induced oxidative damage in MCF-7 cells. The results of present study demonstrated that Fucoidan could induce G1 phase arrest and apoptosis in MCF-7 cells through regulating the cell cycle and apoptosis-related genes or proteins expression, and ROS generation is also involved in these processes.

Germacrone induces apoptosis in human hepatoma HepG2 cells through inhibition of the JAK2/STAT3 signalling pathway.

Previous studies have shown that STAT3 plays a vital role in the genesis and progression of cancer. In this study, we investigated the relationship between the JAK2/STAT3 signalling pathway and germacrone-induced apoptosis in HepG2 cells. HepG2 cells were incubated with germacrone for 24 h, the protein expression of p-STAT3, STAT3, p-JAK2 and JAK2 was detected by Western Blotting, and RT-PCR was used to determine the expression of STAT3, p53, Bcl-2 and Bax at transcriptional levels. Besides that, HepG2 cells were pre-treated with AG490 or IL-6 for 2 h, and then incubated with germacrone for 24 h. The expression of p-JAK2, JAK2, p-STAT3, STAT3, p53, Bax and Bcl-2 was detected by Western blotting. The activity of HepG2 cells was tested by MTT assay. The apoptosis of HepG2 cells and levels of reactive oxygen species (ROS) were flow cytometrically measured. The results showed that germacrone exposure decreased p-STAT3 and p-JAK2 and regulated expression of p53 and Bcl-2 family members at the same time. Moreover, IL-6 enhanced the activation of the JAK2/STAT3 signalling pathway and therefore attenuated the germacrone-induced apoptosis. Suppression of JAK2/STAT3 signalling pathway by AG490, an inhibitor of JAK2, resulted in apoptosis and an increase in ROS in response to germacrone exposure. We therefore conclude that germacrone induces apoptosis through the JAK2/STAT3 signalling pathway.

Overexpression of soybean GmCBL1 enhances abiotic stress tolerance and promotes hypocotyl elongation in Arabidopsis.

Although extensive studies and remarkable progress have been made with Arabidopsis calcineurin B-like proteins (CBLs), knowledge of their functions in other plant species is still limited. Here we isolated gene GmCBL1 from soybean, a homolog of AtCBL1 in Arabidopsis. GmCBL1 was differentially induced by multiple abiotic stress and plant hormones, and its transcripts were abundant in seedlings and mature roots. We over-expressed GmCBL1 in Arabidopsis and found that it enhanced tolerances to both high salt and drought stresses in the transgenic plants. Overexpression of GmCBL1 also promoted hypocotyl elongation under light conditions. GmCBL1 may regulate stress tolerance through activation of stress-related genes, and may control hypocotyl development by altering the expression of gibberellin biosynthesis-related genes. This study identifies a putative soybean CBL gene that functions in both stress tolerance and light-dependent hypocotyl development.

A mutation in Arabidopsis BSK5 encoding a brassinosteroid-signaling kinase protein affects responses to salinity and abscisic acid.

As the most recently characterized group of plant hormones, brassinosteroids (BR) are involved in a number of physiological responses. Although many key components of the BR signaling pathway have been isolated and characterized, there is little information on detailed characterization of brassinosteroid-signaling kinase (BSK) proteins. In this study, Arabidopsis BSK5 was isolated and functionally analyzed. BSK5 transcripts were detected in various tissues, and were induced by abiotic stresses including salt and drought, as well as phytohormones of BR and abscisic acid (ABA). Arabidopsis loss-of-function mutant bsk5 exhibited sensitivity to salinity and ABA. Mutations of the BSK5 gene also altered the expression of several stress-regulated genes. We suggest that BSK5 responds to other signals as well as BR.

Isolation and characterization of an endosperm-specific promoter from wheat (Triticum aestivum L.).

Genes coding for avenin-like proteins (ALP) represent a new family of wheat storage protein genes. To find a wheat endosperm-specific promoter, a 1644-bp fragment upstream of the ALP type-B gene (GenBank accession number JN622144) was isolated. The important promoter elements of the ALP type-B gene were ascertained through sequence analysis which revealed that this fragment contains the TATA and CAAT boxes, which are important elements in gene expression. A prolamin box containing an endosperm motif and a GCN4-like motif (GLM) is present at about 300 bp upstream of the translation start site. The promoter sequence has two ESP-like elements and one of them is followed by an RY motif with the nucleotides CATG overlapping. The RY motif is considered the core functional sequence in a promoter. In an attempt to confirm the promoter activity, a series of 5'-deletions of the promoter were fused with the beta-glucuronidase (GUS) gene, and the constructs were stably introduced into tobacco plants. GUS staining confirmed that the AVL type-B promoter is an endosperm-specific promoter in tobacco seeds. Quantitative analysis of GUS expression in transgenic plants showed that even the shortest 5'-deletion, i.e. a 290-bp promoter sequence within the prolamin box, was sufficient to drive GUS expression in the endosperm. The highest expression level was found in transgenic plants containing the 5'-deletion vector construct pALP-8. This suggests that the ESP-like element overlapping with the RY motif may play a crucial role in the regulatory function of the promoter.

Fangchinoline induced G1/S arrest by modulating expression of p27, PCNA, and cyclin D in human prostate carcinoma cancer PC3 cells and tumor xenograft.

Prostate cancer (PCA) is the most common invasive malignancy and the second leading cause of cancer-related death in males. The present study investigated the effects of fangchinoline (Fan), an important compound in Stephania Tetradra S. Moore (Fenfangji) with pain-relieving, blood pressure-depressing, and antibiotic activities, on human PCA. It was found that Fan inhibited human prostate cancer cell lines (PC3) cell proliferation in a dose- and time-dependent manner. Studies of cell-cycle progression showed that the anti-proliferative effect of Fan was associated with an increase in the G1/S phase of PC3 cells. Western blot results indicated that Fan-induced G1/S phase arrest was mediated through inhibition of cyclin-regulated signaling pathways. Fan induced p27 expression and inhibited cyclin D and proliferating cell nuclear antigen (PCNA) expression in PC3 cells. Increased exposure time to Fan caused apoptosis of PC3 cells, which was associated with up-regulation of pro-apoptotic proteins Bax and caspase 3, and down-regulation of anti-apoptotic protein Bcl-2. Furthermore, Fan had anti-tumorigenic activity in vivo, including reduction of tumor volume and pro-apoptotic and anti-proliferative effects in a PC3 nude mouse xenograft. Taking all this together, it can be concluded that Fan is an effective anti-proliferative agent that modulates cell growth regulators in prostate cancer cells.

Design of tandem genes cluster and expression vector for biosynthesis of soybean isoflavones.

A tandem gene cluster CHS-CHI-IFS (rIFS) for secondary metabolites of plant isoflavones was constructed by using the chalcone synthase (CHS), chalcone isomerase (CHI), and isoflavone synthase (IFS) (GenBank accession numbers EU526827, EU526829, EU526830) in a single recombination event with the pET22b vector. The resulting expression vector pET-rIFS was heterogeneously expressed. The highlights of the vector include ease of handling, high efficiency and universal application among diverse plant species. To the best of our knowledge, this is the first attempt at developing a novel method of constructing tandem gene cluster for future research involving secondary metabolism of isoflavones and isoflavones engineering.

[Secretory expression of PR39 following adeno-associated viral-encoding fusion gene transfer induces angiogenesis in hypoxia chick embryo].

OBJECTIVES: To investigate the impact of AAV-encoding NT4-TAT-His-PR39 fusion gene expression on HIF-1alpha level in ECV304 cultured under hypoxic condition (1%O(2)) and on angiogenesis in hypoxic chick embryo. METHODS: PR39 cDNA was connected with NT4, TAT, 6 x His cDNA by molecular biology methods. The recombinant AAV vector was obtained by three plasmid co-transfection in 293 cells. Then ECV304 were respectively infected with AAV-NT4-TAT-His-PR39, 6 x His expression and HIF-1alpha level in ECV304 were detected by immunocytochemistry. The chicken embryos were randomized into the AAV-PR39, EV and PBS groups (n = 10 each) subject to hypoxia (5%O(2), n = 15) or normoxia environments (n = 15), the vessel density of the chicken chorioallantoic membrane (CAM) were measured by Image Pro Plus (IPP) software. RESULTS: The expression of 6 x His protein was detected in AAV-PR39 infected ECV304 cells. HIF-1alpha protein activity was significantly increased in AAV-PR39 infected ECV304 underwent hypoxia compared to PBS and non-infected ECV304 groups (P < 0.05). The vessel density of chicken CAM in hypoxia environment but not in normoxia environment was also significantly higher in AAV-PR39 group than in EV group and PBS group (all P < 0.05). CONCLUSION: AAV-encoding NT4-TAT-His-PR39 fusion gene expression significantly increased HIF-1alpha level in ECV304 exposed to hypoxia and promoted angiogenesis in hypoxic chicken embryo.

[Application of enzyme-labeled probe in testing of transgenic plant].

Used alkaline-phosphatase-labeled DNA as a probe to examine the expression of foreign UidA gene in transgenic plants. Alkaline phosphatase coupled with polyethyleneimine (PEI) using P-benzoquine as the cross-linking reagent was covalently linked to single-stranded DNA via glutraldehyde. Such DNA-enzyme complexes were used as a probe for dot hybridization and Southern blot. After hybridization and incubation with a substrate solution, results can be observed directly in three to six hours and the results showed that it was a sensitive, specific, rapid, safe and economical probe. Dot hybridization analysis showed that the UidA gene was transformed into the target plants and southern blot showed that there were at least 5 copies of UidA gene in transgenic plants.

[Inheritance of the foreign gene 1Ax1 in transgenic wheat (Triticum aestivum L.) with gene cassettes lacking vector backbone sequences].

Transformation with gene minimal expression cassettes without vector backbone sequence is a new transformation technique. In the current research, the inheritance of the foreign 1Ax1 gene cassettes were analyzed in transgenic plants recovered with gene cassettes lacking vector backbone sequences. The results showed that linear 1Ax1 gene cassettes were stably expressed and separated at the rate of 3 to 1 in the seeds of T1 generation. It suggested the functional copies of 1Ax1 gene cassettes were integrated in one sites, abiding by Mendelian genetic law. It revealed firstly the feasibility of the transformation technique with gene cassettes, and proved the inheritance of transgene cassettes in subsequent generations.

Intensive inhibition of hTERT expression by a ribozyme induces rapid apoptosis of cancer cells through a telomere length-independent pathway.

Restoration of telomerase activity is essential for most of the malignancies. Telomerase reverse transcriptase (TERT) is the key component of telomerase. In this study, we designed a hammerhead ribozyme against human telomerase reverse transcriptase (hTERT) and observed its growth inhibition and pro-apoptosis effects on cancer cells. The efficiency of this ribozyme was verified in in vitro cleavage experiment. A recombinant retrovirus was constructed to transduce the ribozyme to telomerase positive colon carcinoma cell line SW480 and gastric carcinoma cell line SGC7901. We found that the ribozyme could strongly inhibit hTERT expression and telomerase activity, resulting in rapid apoptosis of cancer cells. Shortening of telomere and replicative senescence were not observed before cell death, indicating intensive inhibition of hTERT expression can induce apoptosis by some mechanism(s) except telomere shortening and replicative senescence. This study suggests that hTERT exerts a direct antiapoptotic function in cancer cells. Anti-hTERT ribozyme might be a potential means in the therapy of telomerase-positive malignancies.

[Fusion of TGF-beta 1 antigenic determinant and HBV core antigen as an anti-TGF-beta 1 vaccine].

OBJECTIVES: To examine the expression and purification of the TGFbeta1 vaccine from prokaryotic expression system and to determine the antigenicity of the fusion protein of recombinant vector pET28a/ HBcAg1-71-TGFbeta132-HBcAg89-144. METHODS: The reconstructed vector pGEMEX-1/CTC was subcloned to pET28a and transformed into E. coli BL21 (DE3). The recombinant 6xHis- HBcAg1-71- TGFbeta132- HBcAg89-144 was to be expressed after induction by IPTG and purified with Ni-NTA-His affinity chromatography. The detection of the formation of core-like particles was done under an electron microscope and of their antigenity by using ELISA and Western blot. RESULTS: A 2.46 x 10(4) protein was obtained by optimizing the conditions for both expression and purification. The protein had the TGFbeta1 antigenicity but not a HBc antigenity and the formed core-like particles were bigger than natural core particles. CONCLUSION: The recombinant fusion protein in the prokaryotic expressed system can be used as an anti-TGFbeta1 vaccine to inhibit hepatic fibrosis.

The soybean GmDi19-5 interacts with GmLEA3.1 and increases sensitivity of transgenic plants to abiotic stresses.

Drought-induced (Di19) proteins played important roles in plant growth, development, and abiotic stress responses. In the present study, a total of seven Di19 genes were identified in soybean. Each soybean Di19 gene showed specific responses to salt, drought, oxidative, and ABA stresses based on expression profiles. With a relatively higher transcript level among Di19 members under four stress treatments, GmDi19-5 was selected for detailed analysis. Inhibitor assays revealed that ABA inhibitor (Fluridone) or H2O2 inhibitor (DMTU) was involved in the drought- or salt-induced transcription of GmDi19-5. The GUS activity driven by the GmDi19-5 promoter was induced by salt, PEG, ABA, and MV treatments and tended to be accumulated in the vascular bundles and young leaves. A subcellular localization assay showed that GmDi19-5 protein localized in the nucleus. Further investigation showed that GmDi19-5 protein was involved in the interaction with GmLEA3.1. Overexpression of GmDi19-5 increased sensitivity of transgenic Arabidopsis plants to salt, drought, oxidative, and ABA stresses and regulated expression of several ABA/stress-associated genes. This present investigation showed that GmDi19-5 functioned as a negative factor under abiotic stresses and was involved in ABA and SOS signaling pathway by altering transcription of stress-associated genes.

Design of a ribozyme targeting human telomerase reverse transcriptase and cloning of it's gene.

AIM: To design a hammerhead ribozyme targeting human telomerase reverse transcriptase (hTERT) and clone it's gene for future use in the study of tumor gene therapy. METHODS: Using the software RNA structure, the secondary structure of hTERT mRNA was predicted and the cleavage site of ribozyme was selected. A hammerhead ribozyme targeting this site was designed and bimolecular fold between the ribozyme and hTERT was predicted. The DNA encoding the ribozyme was synthesized and cloned into pGEMEX-1 and the sequence of the ribozyme gene was confirmed by DNA sequencing. RESULTS: Triplet GUC at 1742 of hTERT mRNA was chosen as the cleavage site of the ribozyme. The designed ribozyme was comprised of 22nt catalytic core and 17nt flanking sequence. Computer-aided prediction suggested that the ribozyme and hTERT mRNA could cofold into a proper conformation. Endonuclease restriction and DNA sequencing confirmed the correct insertion of the ribozyme gene into the vector pGEMEX-1. CONCLUSION: This fundamental work of successful designing and cloning of an anti-hTERT hammerhead ribozyme has paved the way for further study of inhibiting tumor cell growth by cleaving hTERT mRNA with ribozyme.

A novel role for Arabidopsis CBL1 in affecting plant responses to glucose and gibberellin during germination and seedling development.

Glucose and phytohormones such as abscisic acid (ABA), ethylene, and gibberellin (GA) coordinately regulate germination and seedling development. However, there is still inadequate evidence to link their molecular roles in affecting plant responses. Calcium acts as a second messenger in a diverse range of signal transduction pathways. As calcium sensors unique to plants, calcineurin B-like (CBL) proteins are well known to modulate abiotic stress responses. In this study, it was found that CBL1 was induced by glucose in Arabidopsis. Loss-of-function mutant cbl1 exhibited hypersensitivity to glucose and paclobutrazol, a GA biosynthetic inhibitor. Several sugar-responsive and GA biosynthetic gene expressions were altered in the cbl1 mutant. CBL1 protein physically interacted with AKINß1, the regulatory ß subunit of the SnRK1 complex which has a central role in sugar signaling. Our results indicate a novel role for CBL1 in modulating responses to glucose and GA signals.

[Construction of a general AAV vector regulated by minimal and artificial hypoxic-responsive element].

AIM: To synthesize the minimal and artificial HRE, and to insert it into the anterior extremity of CMV promoter of a AAV plasmid, and then to construct the AAV regulated by hypoxic-responsive element which was introduced into 293 cell by method of Ca3(PO4)2 using three plasmids. Thus obtaining the adenoassociated virus vector regulated by hypoxic-responsive element was possibly used for gene therapy in ischemia angiocardiopathy and cerebrovascular disease. METHODS: Artificially synthesize the 36 bp nucleotide sequences of four connection in series HIF-binding sites A/GCGTG(4×HBS)and a 35 bp nucleotide sequences spacing inserted into anterior extremity of CMV promoter TATA Box, then amplified by PCR. The cDNA fragment was confirmed to be right by DNA sequencing. Molecular biology routine method was used to construct a AAV vector regulated by minimal hypoxic-responsive element after the normal CMV promoter in AAV vector was replaced by the CMV promoter included minimal hypoxic-responsive element. Then, NT4-6His-PR39 fusogenic peptide was inserted into MCS of the plasmid, the recombinant AAV vector was obtained by three plasmid co-transfection in 293 cells, in which we can also investigate the expression of 6×His using immunochemistry in hypoxia environment. RESULTS: Artificial HRE was inserted into anterior extremity of CMV promoter and there was a correct spacing between the HRE and the TATA-box. The DNA sequencing and restriction enzyme digestion results indicated that the AAV regulated by hypoxic-responsive element was successfully constructed. Compared to the control group, the expressions of 6×His was significantly increased in the experimental groups in hypoxia environment, which confirmed that the AAV effectually regulated by the minimal HRE was inserted into anterior extremity of CMV promoter. CONCLUSION: The HRE is inserted into anterior extremity of CMV promoter to lack incision enzyme recognition site by PCR. And eukaryotic expression vector regulated by hypoxic-responsive is constructed. The AAV effectually regulated by the minimal HRE inserted into anterior extremity of CMV promoter. The vector is successfully constructed and it has important theoretical and practical value in the synteresis and therapy of ischemia angiocardiopathy and cerebrovascular disease.