Isolation and characterization of two sorbitol transporter gene promoters in micropropagated apple plants (Malus?×?domestica) regulated by drought stress

The role of polyol transporters in stress tolerance in plants have been elucidated by many studies. Sorbitol transporter genes MdSOT3, MdSOT4 and MdSOT5 in apple plants, which are important for sorbitol loading and unloading, are regulated by drought stress. To further confirm the role of sorbitol transporters in stress tolerance, the constructs harboring MdSOT3 and MdSOT5 genes were introduced into wild type Arabidopsis plants (Col-0) and the Arabidopsis transformed with MdSOT3 or MdSOT5 performed higher drought stress tolerance compared to WT. In order to further understand how sorbitol transporters are involved in drought tolerance in apple plants, upstream regions of sorbitol transporter genes were isolated from apple plant source leaves by Anchored PCR from genomic DNA obtained, and then were used to drive expression of the GUS reporter in tobacco plants. The results showed that the longest fragments of MdSOT3 and MdSOT5 promoters induced the highest GUS activity under drought stress conditions. Additionally, fragments of these promoters that contain cis-acting elements known to be involved in stress response also induced GUS activity under drought stress. Taken together, our data suggest that increased MdSOT3 and MdSOT5 activity, through cis-acting elements in the promoters of these genes, play important roles in imparting tolerance to drought in micropropagated apple plants.     

Allelopathic impact of artificially applied coumarin on <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f.sp. <Emphasis Type="Italic">niveum</Emphasis>

Watermelon production is threatened by fusarium wilt caused by Fusarium oxysporum f.sp. niveum (FON) in continuous cultivation system. Some elements, mainly allelochemicals, released from living roots or decayed plants might be associated with the disease. The purpose of this work was to evaluate the possible impact of coumarin, one kind of watermelon allelochemical, on FON. Furthermore, possible new mechanisms might be investigated during the ecological interactions of plant-microbe. Results showed that coumarin strongly inhibited growth of FON leading to a decrease in its biomass, dry weight of mycelia of FON in a liquid culture. The dry weight was decreased by 62.9% compared with control. The hyphal growth of FON on plates was stopped at high (>400 mg l⁻¹) concentrations of coumarin. At 320 mg l⁻¹, sporulation and enzyme activities of FON were also severely suppressed by coumarin. The yield of conidia, and the activities of proteinase, cellulase, and amylase were reduced by 98.9%, 79.7%, 29.8% and 15.9% respectively. However, conidial germination and mycotoxin (MT) production of FON were greatly stimulated, being increased by 55.7% and 14.9 fold at 320 mg l⁻¹ respectively. We conclude that coumarin acted as an allelochemical substance to inhibit growth and pathogenic enzyme activities of FON but to stimulate mycotoxin production and conidial germination. It was suggested that coumarin acted as a signal transduction element bridging plant and pathogen in the process of plant-microbe interactions.     

Expression and purification of an FGF9 fusion protein in E. coli,and the effects of the FGF9 subfamily on human hepatocellular carcinoma cell proliferation and migration

Fibroblast growth factor (FGF) 9 has oncogenic activity and plays an important role in the development of ovarian, lung, prostate, and gastric cancers. In the present study, with the aim of reducing the cost of utilizing growth factors in cancer research, a simple and efficient method for the preparation of recombinant human (rh)FGF9 in Escherichia coli was established. The rhFGF9 fusion protein (6 × His-TEV-rhFGF9) and the native protein released by tobacco etch virus (TEV) protease were obtained using a Ni-NTA system, with > 95% purity. Both purified forms of rhFGF9, with and without fusion tags, significantly stimulated the proliferation of NIH3T3 cells. The FGF9 subfamily, including FGF9, FGF16, and FGF20, in addition to rhFGF16, rhFGF9, and rhFGF20, were shown to stimulate the proliferation and migration of HuH7 human hepatocellular carcinoma (HCC) cells. Mechanistic studies revealed that the stimulation of HuH7 cell proliferation and migration with rhFGF9 and rhFGF20 were associated with the activation of the extracellular signal-regulated kinase (ERK) and nuclear factor κB (NF-κB) pathways and matrix metalloproteinase-26 (MMP26). Inhibition of the ERK and NF-κB pathways blocked cell migration, and NF-κB was demonstrated to be regulated by ERK. Therefore, the present study demonstrates a simple method for the preparation of biologically active rhFGF9 protein. Furthermore, the results indicate that exogenous rhFGF9- and rhFGF20-activated ERK/NF-κB signal transduction pathways play important roles in the regulation of HCC cell proliferation and migration, and this discovery helps to find the potential for new solutions of the treatment of liver cancer.

     

KPC1 expression and essential role after acute spinal cord injury in adult rat

KPC1 (Kip1 ubiquitylation-promoting complex 1) is the catalytic subunit of the ubiquitin ligase KPC, which regulates the degradation of the cyclin-dependent kinase inhibitor p27^kip1 at the G1 phase of the cell cycle. To elucidate the expression and role of KPC1 in nervous system lesion and repair, we performed an acute spinal cord contusion injury (SCI) model in adult rats. Western blot analysis showed a significant up-regulation of KPC1 and a concomitant down-regulation of p27^kip1 following spinal injury. Immunohistochemistry and immunofluorescence revealed wide expression of KPC1 in the spinal cord, including expression in neurons and astrocytes. After injury, KPC1 expression was increased predominantly in astrocytes, which highly expressed PCNA, a marker for proliferating cells. Co-immunoprecipitation demonstrated increased interactions between p27^kip1 and KPC1 4 days after injury. To understand whether KPC1 plays a role in astrocyte proliferation, we applied LPS to induce astrocyte proliferation in vitro. Western blot analysis demonstrated that p27^kip1 expression was negatively correlated with KPC1 expression following LPS stimulation. Immunofluorescence analysis showed subcellular localizations of p27^kip1 and KPC1 were also changed following the stimulation of astrocytes with LPS. These results suggest that KPC1 is related to the down-regulation of p27^kip1; this event may be involved in the proliferation of astrocytes after SCI.     

IL-1beta suppresses prolonged Akt activation and expression of E2F-1 and cyclin A in breast cancer cells

Cell cycle aberrations occurring at the G(1)/S checkpoint often lead to uncontrolled cell proliferation and tumor growth. We recently demonstrated that IL-1beta inhibits insulin-like growth factor (IGF)-I-induced cell proliferation by preventing cells from entering the S phase of the cell cycle, leading to G(0)/G(1) arrest. Notably, IL-1beta suppresses the ability of the IGF-I receptor tyrosine kinase to phosphorylate its major docking protein, insulin receptor substrate-1, in MCF-7 breast carcinoma cells. In this study, we extend this juxtamembrane cross-talk between cytokine and growth factor receptors to downstream cell cycle machinery. IL-1beta reduces the ability of IGF-I to activate Cdk2 and to induce E2F-1, cyclin A, and cyclin A-dependent phosphorylation of a retinoblastoma tumor suppressor substrate. Long-term activation of the phosphatidylinositol 3-kinase/Akt signaling pathway, but not the mammalian target of rapamycin or mitogen-activated protein kinase pathways, is required for IGF-I to hyperphosphorylate retinoblastoma and to cause accumulation of E2F-1 and cyclin A. In the absence of IGF-I to induce Akt activation and cell cycle progression, IL-1beta has no effect. IL-1beta induces p21(Cip1/Waf1), which may contribute to its inhibition of IGF-I-activated Cdk2. Collectively, these data establish a novel mechanism by which prolonged Akt phosphorylation serves as a convergent target for both IGF-I and IL-1beta; stimulation by growth factors such as IGF-I promotes G(1)-S phase progression, whereas IL-1beta antagonizes IGF-I-induced Akt phosphorylation to induce cytostasis. In this manner, Akt serves as a critical bridge that links proximal receptor signaling events to more distal cell cycle machinery.     

Helicobacter pylori (H. pylori) molecular signature in conjunctival mucosa-associated lymphoid tissue (MALT) lymphoma

Conjunctival mucosa-associated lymphoid tissue (MALT) lymphoma is an extranodal marginal zone B-cell lymphoma that is characterized by an exaggerated clonal expansion of B cells, which implicate a pathological proliferative response to antigen(s) including bacteria. Helicobacter pylori (H. pylori) infection is recognized as one of the causative agents of gastric MALT lymphoma; however, it has not been reported in extra gastric MALT lymphoma. We studied 5 patients (4 adults and 1 child) with salmon-colored conjunctival lesions. One patient also had a history of abnormal bone marrow biopsy a year earlier with lymphoid aggregates involving 5% of the overall bone marrow. The conjunctival lesions of the 5 patients were biopsied. Histopathological diagnoses were consistent with conjunctival MALT lymphoma. Lymphoma and normal conjunctival cells were microdissected using laser capture microscopy or manual techniques. DNA was extracted and subjected to PCR amplification using H. pylori gene-specific primers from the urease B and vac/m2 gene. Cells from chronic conjunctivitis (normal lymphocytes), conjunctival human T-cell lymphotropic virus type-1/adult T-cell leukemia/lymphoma (HTLV-1/ATL), and orbital B-cell lymphoma were also microdissected, processed and analyzed. PCR amplification and Southern blot hybridization demonstrated H. pylori DNA in the conjunctival MALT lymphoma cells of 4/5 cases. The negative case was the one with a history of abnormal bone marrow. In contrast, H. pylori gene was not detected in normal conjunctival cells from the cases of MALT lymphoma or the lymphocytes, ATL and orbital B-lymphoma cells from the controls. These data suggest that H. pylori may play a role in conjunctival MALT lymphoma.