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1.
Cyclin-dependent kinase 1 (Cdk1) is thought to trigger centrosome separation in late G2 phase by phosphorylating the motor protein Eg5 at Thr927. However, the precise control mechanism of centrosome separation remains to be understood. Here, we report that in G2 phase polo-like kinase 1 (Plk1) can trigger centrosome separation independently of Cdk1. We find that Plk1 is required for both C-Nap1 displacement and for Eg5 localization on the centrosome. Moreover, Cdk2 compensates for Cdk1, and phosphorylates Eg5 at Thr927. Nevertheless, Plk1-driven centrosome separation is slow and staggering, while Cdk1 triggers fast movement of the centrosomes. We find that actin-dependent Eg5-opposing forces slow down separation in G2 phase. Strikingly, actin depolymerization, as well as destabilization of interphase microtubules (MTs), is sufficient to remove this obstruction and to speed up Plk1-dependent separation. Conversely, MT stabilization in mitosis slows down Cdk1-dependent centrosome movement. Our findings implicate the modulation of MT stability in G2 and M phase as a regulatory element in the control of centrosome separation.  相似文献
2.
The synthesis and differential antiproliferative activity of monastrol (1a), oxo-monastrol (1b) and eight oxygenated derivatives 3a,b-6a,b on seven human cancer cell lines are described. For all evaluated cell lines, monastrol (1a) was shown to be more active than its oxo-analogue, except for HT-29 cell line, suggesting the importance of the sulfur atom for the antiproliferative activity. Monastrol (1a) and the thio-derivatives 3a, 4a and 6a displayed relevant antiproliferative properties with 3,4-methylenedioxy derivative 6a being approximately more than 30 times more potent than monastrol (1a) against colon cancer (HT-29) cell line.  相似文献
3.
HsEg5 has microtubule-activated ATPase activity and plays essential roles in bipolar spindle formation. Because HsEg5 is validated as an attractive cancer target, in vitro biochemical assays have been developed for identifying compounds with high inhibitory activity. Several compounds, including quinazoline ring-containing compounds, have been identified and are currently in clinical trials. Although considerable progress has been made during recent years, limitations of HsEg5 in vitro screening assays still reside in two main aspects. First, colorimetric-based assays exhibit relatively low sensitivity and limited dynamic range that are unable to accurately measure compounds with nanomolar potencies. Second, current fluorescence assays are relatively low throughput without "mix and read" homogeneous features. In this study, we describe a sensitive fluorescence-based assay for HsEg5-specific inhibitors. By coupling several enzymes' activities, the release of ADP was measured quantitatively through red fluorescent resorufin. The Km for ATP hydrolysis in this assay was calculated as 23 microM. The known HsEg5 inhibitors CK0106023 and CK0238273 gave IC50 values of 9.8 and 30.6 nM, respectively. Our fluorescence assay has a 20-fold increase in sensitivity with broader dynamic range when compared with a colorimetric assay. We further automated this assay for high-throughput screening with a Z' factor of 0.8.  相似文献
4.
Eg5 is a mitotic kinesin that plays a crucial role in the formation of bipolar mitotic spindles, by hydrolyzing ATP to push apart anti-parallel microtubules. Dimethylenastron is potent specific small molecule inhibitor of Eg5. The mechanism by which dimethylenastron inhibits Eg5 function remains unclear. By comparing with enastron, here we report that dimethylenastron prevents the growth of pancreatic and lung cancer cells more effectively, by halting mitotic progression and triggering apoptosis. We analyze their interactions with ADP-bound Eg5 crystal structure, and find that dimethylenastron binds Eg5 motor domain with higher affinity. In addition, dimethylenastron allosterically blocks the conformational change of the “sandwich”-like ADP-binding pocket more effectively. We subsequently use biochemical approach to reveal that dimethylenastron slows ADP release more significantly than enastron. These data thus provide biological, structural and mechanistic insights into the potent inhibitory activity of dimethylenastron.  相似文献
5.
Radial glia serve as the resident neural stem cells in the embryonic vertebrate nervous system, and their proliferation must be tightly regulated to generate the correct number of neuronal and glial cell progeny in the neural tube. During a forward genetic screen, we recently identified a zebrafish mutant in the kif11 loci that displayed a significant increase in radial glial cell bodies at the ventricular zone of the spinal cord. Kif11, also known as Eg5, is a kinesin-related, plus-end directed motor protein responsible for stabilizing and separating the bipolar mitotic spindle. We show here that Gfap+ radial glial cells express kif11 in the ventricular zone and floor plate. Loss of Kif11 by mutation or pharmacological inhibition with S-trityl-l-cysteine (STLC) results in monoastral spindle formation in radial glial cells, which is characteristic of mitotic arrest. We show that M-phase radial glia accumulate over time at the ventricular zone in kif11 mutants and STLC treated embryos. Mathematical modeling of the radial glial accumulation in kif11 mutants not only confirmed an ~226× delay in mitotic exit (likely a mitotic arrest), but also predicted two modes of increased cell death. These modeling predictions were supported by an increase in the apoptosis marker, anti-activated Caspase-3, which was also found to be inversely proportional to a decrease in cell proliferation. In addition, treatment with STLC at different stages of neural development uncovered two critical periods that most significantly require Kif11 function for stem cell progression through mitosis. We also show that loss of Kif11 function causes specific reductions in oligodendroglia and secondary interneurons and motorneurons, suggesting these later born populations require proper radial glia division. Despite these alterations to cell cycle dynamics, survival, and neurogenesis, we document unchanged cell densities within the neural tube in kif11 mutants, suggesting that a mechanism of compensatory regulation may exist to maintain overall proportions in the neural tube. We propose a model in which Kif11 normally functions during mitotic spindle formation to facilitate the progression of radial glia through mitosis, which leads to the maturation of progeny into specific secondary neuronal and glial lineages in the developing neural tube.  相似文献
6.
During movement along microtubules, kinesin usually follows a track parallel to the axis of a single protofilament. The question arises what happens when kinesin encounters blockages. The present study describes the movement of kinesin labeled by 20-nm gold beads along immobilized microtubules artificially decorated with blocking proteins. To guarantee that exactly the kinesin-binding sites were occupied and to avoid steric effects exerted by large molecules, the KIF5A motor domain was used for blocking. After binding, the blockages were irreversibly cross-linked to the microtubules to make them non-exchangeable. Under such conditions, kinesin movement became a non-continuous one. As a rule, after temporary stopping the kinesin moved on without being released from the microtubule. The results strongly suggest a bypassing mechanism based on the postulation that kinesin changes to and continues movement along a neighbouring protofilament. Bypassing is considered to ensure an efficient long-distance transport of cellular cargoes by kinesins.  相似文献
7.
Eg5 is a kinesin whose inhibition leads to cycle arrest during mitosis, making it a potential therapeutic target in cancers. Circular dichroism and isothermal titration calorimetry of our pyrrolotriazine-4-one series of inhibitors with Eg5 motor domain revealed enhanced binding in the presence of adenosine 5′-diphosphate (ADP). Using this information, we studied the interaction of this series with ADP-Eg5 complexes using a thermal shift assay. We measured up to a 7 °C increase in the thermal melting (Tm) of Eg5 for an inhibitor that produced IC50 values of 60 and 130 nM in microtubule-dependent adenosine triphosphatase (ATPase) and cell-based cytotoxicity assays, respectively. In general, the inhibitor potency of the pyrrolotriazine-4-one series in in vitro biological assays correlated with the magnitude of the thermal stability enhancement of ADP-Eg5. The thermal shift assay also confirmed direct binding of Eg5 inhibitors identified in a high-throughput screen and demonstrated that the thermal shift assay is applicable to a range of chemotypes and can be useful in evaluating both potent (nM) and relatively weakly binding (μM) leads. Overall, the thermal shift assay was found to be an excellent biophysical method for evaluating direct binding of a large number of compounds to Eg5, and it complemented the catalytic assay screens by providing an alternative determination of inhibitor potency.  相似文献
8.
HsEg5 is an important mitotic kinesin responsible for bipolar spindle formation at early mitosis. A rich body of evidence shows that inhibition of HsEg5 can result in mitotic arrest followed by cellular apoptosis. Recently identified HsEg5 inhibitor, CK0238273, exhibits potent antitumor activity and is currently in clinical trial. Here we report the cocrystal structure of the motor domain of HsEg5 in complex with CK0238273 at a 2.15 Å resolution. Compared to the previously published HsEg5-Monastrol complex structure, CK0238273 shares the same induced-fit pocket with similar allosteric inhibitory mechanism. However, CK0238273 shows better fitting to the binding pocket with 65% increase of hydrophobic interaction area than that of Monastrol. Some unique hydrophilic interactions were also observed mostly between the phenyl ring and 8-chloro on quinazolinone of CK0238273 with ARG221 and GLY217. We believe that the combination of these interactions defines the superior potency and specificity of CK0238273.  相似文献
9.
Despite the high level of similarity in structural organisation of their motor domains and, consequently, in the mechanism of motility generation, kinesin-5 moves about 25-fold slower than conventional kinesin (kinesin-1). To elucidate the structural motifs contributing to velocity regulation, we expressed a set of Eg5- and KIF5A-based chimeric proteins with interchanged native neck linker and neck elements. Among them, the chimera consisting of the Eg5 catalytic core (residues 1-357) fused to the KIF5A linker and neck (residues 326-450) displayed increased velocity compared to the Eg5 control protein. This is the first evidence that the velocity of the slow-moving motor Eg5 can be elevated by insertion of neck linker and neck elements taken from a fast-moving motor. Whereas the complementary chimera composed of the KIF5A core (1-325) and the Eg5 linker and neck (358-513) was found to be immotile, insertion of the first half-KIF5A linker into this chimera restored motility. Our results indicate that the neck linker and the neck are involved not only in motility generation in general and in determination of movement direction, but also in velocity regulation.  相似文献
10.
Eg5 is a plus end directed kinesin related motor protein (KRP) previously shown to be involved in the assembly and maintenance of the mitotic spindle. KRPs are molecular motors capable of generating forces upon microtubules (MTs) in dividing cells and driving structural rearrangements necessary in the developing spindle. In vitro experiments demonstrate that loss of Eg5 results in cell cycle arrest and defective centrosome separation resulting in the development of monopolar spindles. Here we describe mice with a genetrap insertion in Eg5. Heterozygous mutant mice appear phenotypically normal. In contrast, embryos homozygous for the Eg5 null allele recovered at embryonic days 2.5-3.5 display signs of a proliferation defect as reduced cell numbers and failure of compaction and progression to the blastocyst stage was observed. These data, in conjunction with previous in vitro data, suggest that loss of Eg5 results in abnormal spindle structure, cell cycle arrest and thereby reduced cell proliferation of early cleavage pre-implantation embryos. These observations further support the conclusion that Eg5 is essential for cell division early in mouse development, and that maternal contribution may sustain the embryo through the maternal to zygotic transition at which point supplies of functional Eg5 are exhausted, preventing further cell cleavage.  相似文献
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