Cell surface ceramide controls translocation of transferrin receptor to clathrin-coated pits

Transferrin receptor mediates internalization of transferrin with bound ferric ions through the clathrin-dependent pathway. We found that binding of transferrin to the receptor induced rapid generation of cell surface ceramide which correlated with activation of acid, but not neutral, sphingomyelinase. At the onset of transferrin internalization both ceramide level and acid sphingomyelinase activity returned to their basic levels. Down-regulation of acid sphingomyelinase in cells with imipramine or silencing of the enzyme expression with siRNA stimulated transferrin internalization and inhibited its recycling. In these conditions colocalization of transferrin with clathrin was markedly reduced. Simultaneously, K⁺ depletion of cells which interfered with the assembly of clathrin-coated pits inhibited the uptake of transferrin much less efficiently than it did in control conditions. The down-regulation of acid sphingomyelinase activity led to the translocation of transferrin receptor to the raft fraction of the plasma membrane upon transferrin binding. The data suggest that lack of cell surface ceramide, generated in physiological conditions by acid sphingomyelinase during transferrin binding, enables internalization of transferrin/transferrin receptor complex by clathrin-independent pathway.     

碳纳米管作为siRNA转运载体的研究进展

随着人们对RNA干扰分子机理的研究愈加深入,siRNA作为一种新的基因治疗药物极有可能为人类攻克癌症等难以治愈的疾病带来希望。然而,目前在RNA干扰应用中遇到的最大挑战就是如何有效地将siRNA导入靶细胞且不致引起严重的细胞毒性。碳纳米管在药物传递和基因传递等生物医学领域的潜在应用受到广泛关注;但要实现碳纳米管在基因治疗领域的应用,碳纳米管的功能化是关键,也是近几年来研究的重点。综述近年来碳纳米管作为siRNA转运载体在基因治疗领域的研究进展。     

种子萌发的抑制调控机制

种子萌发是植物生命周期中一个重要的生理过程,激素作用、miRNA抑制、mRNA区域化、表观遗传调控等多个层次的分子抑制参与该过程的调控。赤霉素（解除抑制的激素）合成和失活的调控主要发生在转录水平,而脱落酸（引起抑制的激素）信号转导途径的调控则通过蛋白质抑制物的降解来实现。miRNA在转录后水平使其靶基因的mRNA降解,抑制种子的萌发;通过mRNA的区域化抑制与萌发相关基因的翻译属于另一层次的转录后抑制;小RNA介导的表观遗传机制也可能在种子萌发过程基因表达的协同调控中发挥重要作用。与分子水平的抑制类似,胚乳和种皮产生的机械抑制也很重要。     

小分子RNA在植物叶发育中的调控作用

叶发育是叶原基细胞有序的分裂、生长和分化的过程,受到植物激素和多个转录因子的严格调控.近年的研究表明,在叶片发育的过程中,小分子RNA是基因调控网络的重要组分.小分子RNA通过对其中一些转录因子的抑制作用,影响其表达水平和空间分布,维持叶的正常发育.本综述介绍了小分子RNA及其靶基因调控模块在叶片发生、 叶片形状、叶子极性发育和叶子衰老等过程中的调控作用,并展望了未来研究中新方向.     

Epidemiology in evolutionary time: the case of Wolbachia horizontal transmission between arthropod host species

Wolbachia are bacterial endosymbionts that manipulate the reproduction of their arthropod hosts. Although theory suggests that infections are frequently lost within host species due to the evolution of resistance, Wolbachia infect a huge number of species worldwide. This apparent paradox suggests that horizontal transmission between host species has been a key factor in shaping the global Wolbachia pandemic. Because Wolbachia infections are thus acquired and lost like any other infection, we use a standard epidemiological model to analyse Wolbachia horizontal transmission dynamics over evolutionary time. Conceptually modifying the model, we apply it not to transmission between individuals but between species. Because, on evolutionary timescales, infections spread frequently between closely related species and occasionally over large phylogenetic distances, we represent the set of host species as a small‐world network that satisfies both requirements. Our model reproduces the effect of basic epidemiological parameters, which demonstrates the validity of our approach. We find that the ratio between transmission rate and recovery rate is crucial for determining the proportion of infected species (incidence) and that, in a given host network, the incidence may still be increasing over evolutionary time. Our results also point to the importance of occasional transmission over long phylogenetic distances for the observed high incidence levels of Wolbachia. In conclusion, we are able to explain why Wolbachia are so abundant among arthropods, although selection for resistance within hosts often leads to infection loss. Furthermore, our unorthodox approach of using epidemiology in evolutionary time can be applied to all symbionts that use horizontal transmission to infect new hosts.     

Si RNA inhibition of GRP58 associated with decrease in mitomycin C-induced DNA cross-linking and cytotoxicity

The anti-cancer drug mitomycin C is metabolically activated to bind and cross-link DNA. The cross-linking contributes significantly to the cytotoxicity. The complex chemical structure of mitomycin C allows its metabolism by several known (cytosolic NAD(P)H:quinone oxidoreductase and microsomal NADPH:cytochrome P450 reductase) and unknown enzymes. The identification of new enzymes/proteins that metabolize mitomycin C and like drugs is an area of significant research interest since these studies have direct implications in drug development and clinical usage. In the present studies, we have investigated a role of cytosolic glucose regulatory protein GRP58 in mitomycin C-induced DNA cross-linking and cytotoxicity. The control and GRP58 siRNA were transfected in human colon carcinoma HCT116 cells in culture. The transfection of GRP58 siRNA but not control siRNA significantly inhibited GRP58 in human colon carcinoma HCT116 cells. The inhibition of GRP58 led to decrease in mitomycin C-induced DNA cross-linking and cytotoxicity. These results establish a role of GRP58 in mitomycin C-induced DNA cross-linking and cytotoxicity. Site-directed mutagenesis of cysteines to serines in thioredoxin domains of GRP58 and cross-linking assays revealed that both N- and C-terminal thioredoxin domains are required for GRP58-mediated mitomycin C-induced DNA cross-linking. These results suggest that GRP58 might be an important target enzyme for further studies on mitomycin C and similar drug therapy.     

Small GTPase RacF2 affects sexual cell fusion and asexual development in Dictyostelium discoideum through the regulation of cell adhesion

Cells of Dictyostelium discoideum become sexually mature when submerged and in darkness, and fuse with opposite mating-type cells as gametes. The gene for a Rho GTPase, RacF2, is one of the extremely gamete-enriched genes (>100-fold) identified by us previously. Here, we isolated knockout, overexpression, constitutively active and dominant negative mutants of RacF2, and analyzed their phenotypes. These mutants showed anomalies in the extent of sexual cell fusion and asexual development as well as in EDTA-sensitive cell-cell adhesion. It is suggested that RacF2 controls the process of sexual and asexual development through the regulation of cellular adhesiveness. An analysis of the expression of all 18 rac family genes by real-time polymerase chain reaction revealed that four additional genes, rac1b, rac1c, racF1 and racG, were induced during maturation, suggesting their possible involvement in sexual cell interactions.     

Fibroblast growth factor-2 induces the activation of Src through Fes, which regulates focal adhesion disassembly

Cell migration is regulated by focal adhesion (FA) turnover. Fibroblast growth factor-2 (FGF-2) induces FA disassembly in the murine brain capillary endothelial cell line IBE, leading to FGF-2-directed chemotaxis. We previously showed that activation of Src and Fes by FGF-2 was involved in chemotaxis of IBE cells. In this study, we examined the interplay between Src and Fes. FGF-2 treatment decreased the number of FA in IBE cells, but not in cells expressing dominant-negative Fes (denoted KE5-15 cells). FGF-2 induced the activation of Src and subsequent binding to and phosphorylation of Cas in IBE cells, but not in KE5-15 cells. Focal adhesion kinase (FAK) activation and tyrosine phosphorylation by Src were also delayed in KE5-15 cells compared to parental cells. FGF-2 induced activation of Src within FA in IBE cells, but not in KE5-15 cells. Downregulation of Fes or FAK using small interfering RNA diminished Src activation by FGF-2 within FA. These findings suggest that activation of Fes by FGF-2 enhances FAK-dependent activation of Src within FA, promoting FGF-2-induced disassembly of focal adhesions.     

Silencing of the human microsomal glucose-6-phosphate translocase induces glioma cell death: potential new anticancer target for curcumin

G6P translocase (G6PT) is thought to play a crucial role in transducing intracellular signaling events in brain tumor-derived cancer cells. In this report, we investigated the contribution of G6PT to the control of U-87 brain tumor-derived glioma cell survival using small interfering RNA (siRNA)-mediated suppression of G6PT. Three siRNA constructs were generated and found to suppress up to 91% G6PT gene expression. Flow cytometry analysis of propidium iodide/annexin-V-stained cells indicated that silencing the G6PT gene induced necrosis and late apoptosis. The anticancer agent curcumin, also inhibited G6PT gene expression by more than 90% and triggered U-87 glioma cells death. Overexpression of recombinant G6PT rescued the cells from curcumin-induced cell death. Targeting G6PT expression may provide a new mechanistic rationale for the action of chemopreventive drugs and lead to the development of new anti-cancer strategies.     

Silencing of HIV by hairpin-loop-structured DNA oligonucleotide

We describe inhibition of HIV replication by a partially double-stranded 54mer oligodeoxynucleotide, ODN, which consists of an antisense strand targeting the highly conserved polypurine tract, PPT, of HIV, and a second strand, compatible with triple-helix formation. Upon treatment of HIV-infected cells with ODN early after infection no viral nucleic acids, syncytia or p24 viral antigen expression was observed. The ODN-mediated effect was highly sequence-specific. The ODN against HIV-IIIB was effective preferentially against its homologous PPT and less against the PPT of HIV-BaL differing in two of 24 nucleotides and vice versa. It may be interesting mechanistically as an antiviral drug.