种衣剂17号包衣对小麦条锈菌发育影响的超微结构研究

本研究采用电镜技术研究了种衣剂17号对小麦条锈菌发育的影响。观察结果表明,该种衣剂引起病菌和寄主细胞内发生了一系列变化。病菌菌丝和吸器内脂肪粒和液泡明显增加;菌丝壁和吸器壁呈不规则加厚;菌丝分枝处无隔膜产生或隔膜畸形;有的吸器母细胞产生的畸形入侵栓,大都不能穿透寄主细胞壁,初生吸器外间质内沉积有染色较深的物质,次生吸器可产生多个不规则分枝,但不能扩张膨大;菌丝外渗的物质可能引起寄主细胞的坏死;大多数受侵寄主细胞可分泌形成较大的胼胝质,有时寄主细胞分泌的物质可将吸器体完全包围起来。上述结果表明,种衣剂17号不仅可直接作用于条锈菌,而且也可通过影响寄主而间接地影响病菌。     

Genome-wide CRISPR-Cas9 viability screen reveals genes involved in TNF-α-induced apoptosis of human umbilical vein endothelial cells

Tumor necrosis factor α (TNF-α), a pivotal cytokine in sepsis, protects the host against pathogens by promoting an inflammatory response while simultaneously inducing apoptosis of the vascular endothelium. Unfortunately, inhibitors targeting certain components of the TNF-α signaling pathway to reduce cellular apoptosis have failed to translate into clinical applications, partly due to the adverse effects of excessive immunosuppression. In an attempt to discover potential targets in the TNF-α signaling pathway to modulate moderate inflammation and apoptosis during the development of sepsis, we performed a pooled genome-wide CRISPR/Cas9 knockout screen in human umbilical vein endothelial cells (HUVECs). Tumor necrosis factor receptor superfamily member 1A (TNFRSF1A), B-cell lymphoma 2 (BCL2), Bcl2-associated death promoter (BAD), and NLR family member X1 (NLRX1) deficiencies were identified as the effective genetic suppressors of TNF-α cytotoxicity on a list of candidate regulators. CRISPR-mediated NLRX1 knockout conferred cellular resistance to challenge with TNF-α, and NLRX1 could be induced to colocalize with mitochondria following TNF-α stimulation. Thus, our work demonstrates the advantage of genome-scale screening with Cas9 and validates NLRX1 as a potential modulator of TNF-α-induced vascular endothelial apoptosis during sepsis.     

Leucine zipper domain is required for Kaposi sarcoma-associated herpesvirus (KSHV) K-bZIP protein to interact with histone deacetylase and is important for KSHV replication

The Kaposi sarcoma-associated herpesvirus (KSHV; or human herpesvirus-8)-encoded protein called K-bZIP (also named K8) was found to be multifunctional. In this study, we discovered that K-bZIP interacts with histone deacetylase (HDAC) 1/2 in 12-O-tetradecanoylphorbol-13-acetate-stimulated BCBL-1 lymphocyte cells. K-bZIP appears to repress HDAC activity through this interaction, which we determined to be independent of K-bZIP SUMOylation. We dissected the domains of K-bZIP and found that the leucine zipper (LZ) domain is essential for the interaction of K-bZIP and HDAC. In addition, we constructed a KSHV bacterial artificial chromosome (BAC) with LZ domain-deleted K-bZIP (KSHVdLZ) and transfected this mutated KSHV BAC DNA into HEK 293T cells. As a result, it was consistently found that K-bZIP without its LZ domain failed to interact with HDAC2. We also showed that the interaction between K-bZIP and HDAC is necessary for the inhibition of the lytic gene promoters (ORF50 and OriLyt) of KSHV by K-bZIP. Furthermore, we found that the LZ domain is also important for the interaction of K-bZIP with the promoters of ORF50 and OriLyt. Most interestingly, although it was found to have suppressive effects on the promoters of ORF50 and OriLyt, KSHVdLZ replicates at a significantly lower level than its BAC-derived revertant (KSHVdLZRev) or KSHVWT (BAC36) in HEK 293T cells. The defectiveness of KSHVdLZ replication can be partially rescued by siRNA against HDAC2. Our results suggest that the function of K-bZIP interaction with HDAC is two-layered. 1) K-bZIP inhibits HDAC activity generally so that KSHVdLZ replicates at a lower level than does KSHVWT. 2) K-bZIP can recruit HDAC to the promoters of OriLyt and ORF50 through interaction with HDAC for K-bZIP to have a temporary repressive effect on the two promoters.     

The fate of micronucleated cells post X-irradiation detected by live cell imaging

Micronuclei are closely related to DNA damage. The presence of micronuclei in mammalian cells is a common phenomenon post ionizing radiation. The level of micronucleation in tumor cells has been used to predict prognosis after radiotherapy in many cancers. In order to understand how irradiation-induced micronuclei affect cell fate, we performed extensive long-term live cell imaging on X-irradiated nasopharyngeal carcinoma (NPC) cells. To visualize the dynamics of micronuclei more clearly, chromosomes were stably labeled with red fluorescent protein (RFP) by targeting to human histone H2B. Initially, significantly more micronuclei were observed in radiosensitive cells than in radioresistant cells post irradiation. Additionally, cells with micronuclei were found to be more likely to die or undergo cell cycle arrest when compared with micronucleus-free cells after irradiation, and the more micronuclei the cells contained the more likely they would die or undergo arrest. Moreover, micronucleated cells showed predisposition to produce daughter cells with micronuclei through chromosome lagging. Fluorescence in situ hybridization using human pan-centromeric probes revealed that about 70% of these micronuclei and lagging chromosomes did not contain centromeric signals. Finally, DNA damage was more severe and p38 stress kinase activity was higher in micronucleated cells than in micronucleus-free cells as shown by phospho-H2AX and phospho-p38 immunofluorescence staining. Altogether, our observations indicated that the presence of micronuclei coupled with activated DNA damage response could compromise the proliferation capacity of irradiated cells, providing the evidence and justification for using micronucleus index as a valuable biomarker of radiosensitivity.     

Regulation of asymmetrical cytokinesis by cAMP during meiosis I in mouse oocytes

Mammalian oocytes undergo an asymmetrical first meiotic division, extruding half of their chromosomes in a small polar body to preserve maternal resources for embryonic development. To divide asymmetrically, mammalian oocytes relocate chromosomes from the center of the cell to the cortex, but little is known about the underlying mechanisms. Here, we show that upon the elevation of intracellular cAMP level, mouse oocytes produced two daughter cells with similar sizes. This symmetrical cell division could be rescued by the inhibition of PKA, a cAMP-dependent protein kinase. Live cell imaging revealed that a symmetrically localized cleavage furrow resulted in symmetrical cell division. Detailed analyses demonstrated that symmetrically localized cleavage furrows were caused by the inappropriate central positioning of chromosome clusters at anaphase onset, indicating that chromosome cluster migration was impaired. Notably, high intracellular cAMP reduced myosin II activity, and the microinjection of phospho-myosin II antibody into the oocytes impeded chromosome migration and promoted symmetrical cell division. Our results support the hypothesis that cAMP plays a role in regulating asymmetrical cell division by modulating myosin II activity during mouse oocyte meiosis I, providing a novel insight into the regulation of female gamete formation in mammals.     

Inhibitors of phosphatidylinositol 3'-kinases promote mitotic cell death in HeLa cells

The phosphatidylinositol 3-kinase (PI3K) pathway plays an important role in many biological processes, including cell cycle progression, cell growth, survival, actin rearrangement and migration, and intracellular vesicular transport. However, the involvement of the PI3K pathway in the regulation of mitotic cell death remains unclear. In this study, we treated HeLa cells with the PI3K inhibitors, 3-methyladenine (3-MA, as well as a widely used autophagy inhibitor) and wortmannin to examine their effects on cell fates using live cell imaging. Treatment with 3-MA decreased cell viability in a time- and dose-dependent manner and was associated with caspase-3 activation. Interestingly, 3-MA-induced cell death was not affected by RNA interference-mediated knockdown (KD) of beclin1 (an essential protein for autophagy) in HeLa cells, or by deletion of atg5 (an essential autophagy gene) in mouse embryonic fibroblasts (MEFs). These data indicate that cell death induced by 3-MA occurs independently of its ability to inhibit autophagy. The results from live cell imaging studies showed that the inhibition of PI3Ks increased the occurrence of lagging chromosomes and cell cycle arrest and cell death in prometaphase. Furthermore, PI3K inhibitors promoted nocodazole-induced mitotic cell death and reduced mitotic slippage. Overexpression of Akt (the downstream target of PI3K) antagonized PI3K inhibitor-induced mitotic cell death and promoted nocodazole-induced mitotic slippage. These results suggest a novel role for the PI3K pathway in regulating mitotic progression and preventing mitotic cell death and provide justification for the use of PI3K inhibitors in combination with anti-mitotic drugs to combat cancer.     

Activation of p90 ribosomal S6 kinase by ORF45 of Kaposi's sarcoma-associated herpesvirus and its role in viral lytic replication

The extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway is essential for infection by a variety of viruses. The p90 ribosomal S6 kinases (RSKs) are direct substrates of ERK and functional mediators of ERK MAPK signaling, but their roles in viral infection have never been examined. We demonstrate that ORF45 of Kaposi's sarcoma-associated herpesvirus (KSHV) interacts with RSK1 and RSK2 and strongly stimulates their kinase activities. The activation of RSK by ORF45 is correlated with ERK activation but does not require MEK. We further demonstrate that RSK1/RSK2 is activated during KSHV primary infection and reactivation from latency; a subset of RSK1/RSK2 is present in the viral replication compartment in the nucleus. Depletion of RSK1/RSK2 by small interfering RNA or the specific inhibitor BI-D1870 suppresses KSHV lytic gene expression and progeny virion production, suggesting an essential role of RSK1/RSK2 in KSHV lytic replication.     

Kaposi's sarcoma-associated herpesvirus ori-Lyt-dependent DNA replication: involvement of host cellular factors

Herpesvirus lytic DNA replication requires both the cis-acting element, the origin, and trans-acting factors, including virally encoded origin-binding protein, DNA replication enzymes, and auxiliary factors. Two lytic DNA replication origins (ori-Lyt) of Kaposi's sarcoma-associated herpesvirus (KSHV) have been identified, and two virally encoded proteins, namely, RTA and K8, have been shown to bind to the origins. In this study, we sought to identify cellular factors that associate with ori-Lyt by using DNA affinity purification and mass spectrometry. This approach led to identification of several cellular proteins that bind to KSHV ori-Lyt. They include topoisomerases (Topo) I and II, MSH2/6, RecQL, poly(ADP-ribose) polymerase I (PARP-1), DNA-PK, Ku86/70 autoantigens, and scaffold attachment factor A (SAF-A). RecQL appears to associate with prereplication complexes and be recruited to ori-Lyt through RTA and K8. Topoisomerases, MSH2, PARP-1, DNA-PK, and Ku86 were not detected in prereplication complexes but were present in replication initiation complexes on ori-Lyt. All these cellular proteins accumulate in viral replication compartments in the nucleus, indicating that these proteins may have a role in viral replication. Topo I and II appear to be essential for viral DNA replication as inhibition of their activities with specific inhibitors (camptothecin and ellipticine) blocked ori-Lyt-dependent DNA replication. Furthermore, inhibition of PARP-1 with chemical inhibitors (3-aminobenzamide and niacinamide) resulted in decreased ori-Lyt-dependent DNA replication, whereas hydroxyurea, which raises PARP-1 activity, caused an increase in the DNA replication, suggesting a positive role for PARP-1 in KSHV lytic DNA replication.