Electro-Enhancement of the Formation of Cell Clusters from Protoplasts of Tritium aestivum

Under the treatment of 0.5—2.0 μA direct electric currents, the number of cell clusters derived from wheat (Triticum aestivum L. ) protoplasts was dramatically increased. The effect was related to the time-length of the treatments, and appeared only when the timelength was more than the threshold. The threshold time decreased with increase of electric current intensity. The effect was not related to the starting time of the treatments in culture of the protoplasts.     

Interferon regulatory factor-1 as a positive regulator for high glucose-induced proliferation of vascular smooth muscle cells

High glucose-induced proliferation of vascular smooth muscle cells (VSMCs) plays an important role in the development of diabetic vascular diseases. However, molecular mediators responding for the proliferation of VSMCs remain to be determined. In this study, VSMCs were isolated from the rat thoracic aorta, and two cell models with Irf-1 knockdown and overexpression were established by transfecting cells with pGCsi-FU-Irf-1 and pGC-FU-Irf-1, respectively. Subsequently, high glucose was added to cells to induce proliferation. Proliferation assays were performed to see whether Irf-1 was involved in high glucose-induced proliferation of VSMCs. In addition, the expression of Irf-1 was detected in VSMCs stimulated with high glucose and the thoracic aorta of diabetic rats to confirm the relationship between Irf-1 expression and the proliferation of hyperglycemia-dependent VSMCs. The results showed that Irf-1 expression was significantly higher in the thoracic aorta of diabetic rats and VSMCs stimulated with high glucose than that in nondiabetic rats and untreated cells. Overexpression of Irf-1 accelerated the proliferation of VSMCs, and down-regulation of Irf-1 expression significantly depressed the proliferative ability of VSMCs under high-glucose conditions, indicating that Irf-1 was a positive regulator for high glucose-induced proliferation of VSMCs. It could be presumed that Irf-1 is associated with the accelerated proliferation of VSMCs in diabetic vascular diseases and may prove to be a potential target gene for disease treatment.     

Wogonin Induced Calreticulin/Annexin A1 Exposure Dictates the Immunogenicity of Cancer Cells in a PERK/AKT Dependent Manner

In response to ionizing irradiation and certain chemotherapeutic agents, dying tumor cells elicit a potent anticancer immune response. However, the potential effect of wogonin (5,7-dihydroxy-8-methoxyflavone) on cancer immunogenicity has not been studied. Here we demonstrated for the first time that wogonin elicits a potent antitumor immunity effect by inducing the translocation of calreticulin (CRT) and Annexin A1 to cell plasma membrane as well as the release of high-mobility group protein 1 (HMGB1) and ATP. Signal pathways involved in this process were studied. We found that wogonin-induced reactive oxygen species (ROS) production causes an endoplasmic reticulum (ER) stress response, including the phosphorylation of PERK (PKR-like endoplasmic reticulum kinase)/PKR (protein kinase R) and eIF2α (eukaryotic initiation factor 2α), which served as upstream signal for the activation of phosphoinositide 3-kinase (PI3K)/AKT, inducing calreticulin (CRT)/Annexin A1 cell membrane translocation. P22/CHP, a Ca²⁺-binding protein, was associated with CRT and was required for CRT translocation to cell membrane. The releases of HMGB1 and ATP from wogonin treated MFC cells, alone or together with other possible factors, activated dendritic cells and induced cytokine releases. In vivo study confirmed that immunization with wogonin-pretreated tumor cells vaccination significantly inhibited homoplastic grafted gastric tumor growth in mice and a possible inflammatory response was involved. In conclusion, the activation of PI3K pathway elicited by ER stress induced CRT/Annexin A1 translocation (“eat me” signal) and HMGB1 release, mediating wogonin-induced immunity of tumor cell vaccine. This indicated that wogonin is a novel effective candidate of immunotherapy against gastric tumor.     

Dendritic cells transduced with lentiviral vector targeting RelB gene using RNA interference induce hyporesponsiveness in memory CD4(+) T cells and naïve CD4(+) T cells

The ability of DCs to induce immune tolerance depends on its maturation status. RelB plays a pivotal role in DCs differentiation. A therapeutic protocol of DCs-based not only induces hyporesponsiveness in T(N)s, but also in alloreactive T(M)s is required. Thus, it is urgent to assess modulatory effects of RelB-silenced DCs on T(M)s and T(N)s. In this study, we constructed lentiviral vector which could efficiently silenced the RelB in DCs (DCs-miR RelB) to keep them immature. These DCs induced antigen-specific hyporesponsiveness in CD4(+) T(N)s. In contrast, upon re-stimulation with mature DCs, CD4(+) T(M)s primed by DCs-miR RelB maintained hyporesponsiveness in terms of proliferation and cytokine production. And these may be associated with micro155 and micro181a expression levels in T(M)s and T(N)s. These results may help developing the DCs-based therapeutical protocols by inducing hyporesponsiveness in CD4(+) T(N)s and T(M)s.     

Activation of protein kinase A and clustering of cell surface receptors by N-methyl-N'-nitro-N-nitrosoguanidine are independent of genomic DNA damage

Alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) induces cellular stress leading to chromosomal aberrations, mutations and cell death. Previous reports from our laboratory have shown that low concentration of MNNG induces untargeted mutation (UTM), which occurs on intact DNA in mammalian cells through changes in gene expression profile. It also causes the activation of cAMP-protein kinase A (PKA) and up-regulation of POL-beta, which is demonstrated to play a role in DNA repair system. In order to find out the possible initial signal involved in UTM, we try to investigate whether the activation of PKA-CREB signal pathway is closely related to DNA damage. Our data shows that the treatment of low concentration MNNG (0.2 microM) activates PKA-CREB pathway in a comparable level both in a nuclear and enucleated cell system. And similar to the cell response caused by UV, the clustering of cell surface receptors of epidermal growth factor (EGF) and tumor necrosis factor alpha (TNFalpha) was also observed in cells exposed to MNNG. It was further demonstrated that the clustering of the surface receptors is independent of the genomic DNA damage, as this phenomenon was also observed in enucleated cells. These observations indicate that the initiation of signal cascades induced by low concentration of MNNG might be associated with its interaction with cell surface receptors and/or direct activation of related signal proteins but not its DNA damaging property.