CmTCP20 Plays a Key Role in Nitrate and Auxin Signaling-Regulated Lateral Root Development in Chrysanthemum.

Lateral root (LR) formation and development play a vital role in plant development by permitting the establishment of branched root systems. It is well known that nutrient availability controls LR development. Moreover, LR development is fine-tuned by a myriad of hormonal signals. Many transcription factors (TFs) participate in LR development. Here, we discuss the TFs involved in the nitrate and auxin signaling pathways and how these function in the regulation of LR formation and development in chrysanthemum. AtTCP20 is a plant-specific TF, which can modulate LR development in response to nitrate. The roles of CmTCP20 in LR development were identified by overexpression in chrysanthemum and heterologous expression in Arabidopsis. Overexpression of CmTCP20 significantly increased the number and average length of LRs compared with the wild type in chrysanthemum and Arabidopsis. We also found that CmTCP20 positively influenced auxin accumulation in the LRs at least partly by improving auxin biosynthesis, transport and response, thereby promoting LR development. Moreover, we found that CmTCP20 interacts with an auxin response factor, CmARF8, which also can be induced by nitrate and combined to proximal sites in the upstream promoter region of CmCYCB1;1 to positively regulate the cell cycle. The CmTCP20-CmARF8 heterodimer links nitrate and auxin signaling and converts cell-cycle signals to regulate LR initiation and growth.

Proteomic profiling of root system development proteins in chrysanthemum overexpressing the CmTCP20 gene.

Plant root systems ensure the efficient absorption of water and nutrients and provide anchoring into the soil. Although root systems are a highly plastic set of traits that vary both between and among species, the basic root system morphology is controlled by inherent genetic factors. TCP20 has been identified as a key regulator of root development in plants, and yet its underlying mechanism has not been fully elucidated, especially in chrysanthemum. We found that overexpression of the CmTCP20 gene promoted both adventitious and lateral root development in chrysanthemum. To get further insight into the molecular mechanisms controlling root system development, we conducted a study employing tandem mass tag proteomic to characterize the differential root system development proteomes from CmTCP20-overexpressing and wild-type chrysanthemum root samples. Of the proteins identified, 234 proteins were found to be differentially abundant (>1.5-fold cut off, p < 0.05) in CmTCP20-overexpressing versus wild-type chrysanthemum root samples. Functional enrichment analysis indicated that the CmTCP20 gene may participate in "phytohormone signal transduction". Our findings provide a valuable perspective on the mechanisms of both adventitious and lateral root development via CmTCP20 modulation at the proteome level in chrysanthemum.

New seco-milbemycins from Streptomyces bingchenggensis: fermentation, isolation and structure elucidation.

Two new seco-milbemycins have been isolated and characterized from the Streptomyces bingchenggensis. On the basis of detailed spectroscopic analysis and comparison with the reported data of milbemycin beta(14), their structures were established to be the seco-milbemycins of milbemycin beta(14). However, milbemycin beta(14) and the two new seco-milbemycins were not converted to each other during purification by silica gel column or storage in the MeOH extract at room temperature. The two new seco-milbemycins are the first discovered from milbemycin-producing microorganisms. They may play an important role in understanding and perfecting the proposed biosynthesis pathways of milbemycins.

[Hepatitis B virus X protein suppressing adriamycin-induced apoptosis of HepG2 cells].

OBJECTIVES: To investigate the effect of hepatitis B virus X protein (HBx) on adriamycin-induced apoptosis of hepatocellular carcinoma cells. METHODS: HBx gene fragment was amplified from subtype adr HBV plasmid by PCR, and inserted into Hind III and Kpn I sites of green fluorescent protein (GFP) eukaryotic expression vector pEGFP-C1 to construct recombinant pGFP/HBx. The pEGFP-C1 and pGFP-HBx were introduced into HepG2 cells by Lipofectamine 2000 to obtain HepG2 cells expressing GFP. GFP-HBx fusion protein was selected using G418. The expression of HBx gene was demonstrated by RT-PCR analysis. HepG2, HepG2/GFP and HepG2/GFP-HBx cells were treated with adriamycin (2.5 microg/ml), and apoptosis of the cells was determined by their morphological changes, trypan blue exclusion, and flow cytometry analysis. RESULTS: Under a fluorescence microscope, visible expression of GFP and GFP-HBx fusion proteins were observed in HepG2/GFP and HepG2/GFP-HBx cells, even after growing over 70 generations. RT-PCR analysis showed that HBx gene was expressed in HepG2/GFP-HBx cells. Trypan blue exclusion showed adriamycin induced time-dependent cell death in HepG2 and HepG2/GFP cells while no significant cell death was observed in HepG2/GFP-HBx cells. Flow cytometry analysis showed that apoptosis rates in HepG2/GFP-HBx (3.94%) cells were significantly lower than those in HepG2 (59.03%) and HepG2/GFP cells (61.38%) at 36 hours after the adriamycin treatment (P < 0.01). No significant differences of apoptosis rates of HepG2/GFP-HBx (3.94%) and of the untreated cells (2.12%, 2.78%, 2.55%) (P > 0.05) were observed. CONCLUSION: A HepG2 cell line expressing GFP and GFP-HBx fusion proteins was successfully established. HBV X protein blocks adriamycin-induced apoptosis of these HepG2 cells.

[Effects of Chinese herbs for cool-moistening and freeing collaterals on serum gastrin and surface electrogastrogram in patients of diabetes mellitus with gastroparesis].

OBJECTIVE: To explore the effect of Chinese herbs (CH) for cool-moistening and freeing collaterals on gastro-dynamic disturbance in patients of diabetes mellitus type 2 with gastroparesis (DM-GP). METHODS: Fifty-three patients of DM-GP were enrolled and treated with CH (n = 28) and Cisapride (n = 25) respectively for 4 weeks, the changes of gastrin and electro-gastrogram (EGG) before and after treatment were observed. RESULTS: After treatment, the EGG improved significantly, showing the rhythm significantly improved, and level of serum gastrin lowered significantly, as compared with those before treatment, the difference was significant (P<0.01), but insignificant difference was found between the two groups. Fifteen patients in each group were followed-up afar stopping medication for 3 months, recurrence occurred in 1 patient of CH treated group, and 2 patients of Cisapride treated group. No adverse reaction was found in the rest patients. CONCLUSION: CH could obviously improve the gastro-intestinal motility and hormones abnormality.

[Hepatitis B virus X protein suppresses adriamycin-induced apoptosis of hepatocellular carcinoma cells and expression of p53 and PTEN].

OBJECTIVE: To investigate the effect of hepatitis B virus X protein (HBx) on adriamycin-induced apoptosis of hepatocellular carcinoma cells and the expressions of p53 and PTEN. METHODS: HepG2, HepG2/GFP, and HepG2/GFP-HBx cells were treated with adriamycin (2.5 microg/ml), and the apoptotic cell death was determined by observing the morphological changes and flow cytometry. The expressions of p53 and PTEN mRNA in the 3 cells were detected by RT-PCR, and the expressions of p53 and PTEN protein were analyzed by Western blotting. RESULTS: Adriamycin induced significant cell death in HepG2 and HepG2/GFP cells, which became rounded, shrunk, and detached after the treatment; but no significant cell death occurred in HepG2/GFP-HBx cells. Flow cytometry analysis showed that the apoptotic rate was significantly lower in HepG2/GFP-HBx cells (3.94%) than in HepG2 (59.03%) and HepG2/GFP cells (61.38%) at 36 h after the treatment (P<0.001), while no significant difference was observed between HepG2/GFP-HBx (3.94%) and the control cells (2.12%, 2.78%, and 2.55%) (P>0.05). RT-PCR showed lowered expression of PTEN mRNA in HepG2/GFP-HBx cells as compared to that in HepG2 and HepG2/GFP cells, while no significant difference was noted in p53 mRNA. Western blot analysis showed that PTEN protein decreased while p53 protein remain unchanged in HepG2/GFP-HBx cells. CONCLUSION: HBx suppresses adriamycin-induced apoptosis of HepG2 cells and PTEN expression. The inhibitory effect of HBx on the cell apoptosis may be related to the inhibition of p53-PTEN pathway.