Aurora-A Kinase Is Essential for Bipolar Spindle Formation and Early Development

Aurora-A is a conserved kinase implicated in mitotic regulation and carcinogenesis. Aurora-A was previously implicated in mitotic entry and spindle assembly, although contradictory results prevented a clear understanding of the roles of Aurora-A in mammals. We developed a conditional null mutation in the mouse Aurora-A gene to investigate Aurora-A functions in primary cells ex vivo and in vivo. We show here that conditional Aurora-A ablation in cultured embryonic fibroblasts causes impaired mitotic entry and mitotic arrest with a profound defect in bipolar spindle formation. Germ line Aurora-A deficiency causes embryonic death at the blastocyst stage with pronounced cell proliferation failure, mitotic arrest, and monopolar spindle formation. Aurora-A deletion in mid-gestation embryos causes an increase in mitotic and apoptotic cells. These results indicate that murine Aurora-A facilitates, but is not absolutely required for, mitotic entry in murine embryonic fibroblasts and is essential for centrosome separation and bipolar spindle formation in vitro and in vivo. Aurora-A deletion increases apoptosis, suggesting that molecular therapies targeting Aurora-A may be effective in inducing tumor cell apoptosis. Aurora-A conditional mutant mice provide a valuable system for further defining Aurora-A functions and for predicting effects of Aurora-A therapeutic intervention.The equal partitioning of chromosomes at mitosis is critical for avoiding aneuploidy, a condition associated with spontaneous miscarriage, developmental disorders, and cancer (50). Mitosis requires coordinated completion of multiple events including nuclear envelope breakdown, chromosome condensation and congression to the metaphase plate, centrosome separation, spindle formation, chromosome-spindle attachment and error correction, sister chromatid separation, and cytokinesis. Multiple regulators, many of which are kinases, are required to ensure that each event is completed in a timely fashion and in the proper order (reviewed in reference 46). Although a number of mitotic kinases have been identified, their targets and the intricacies of mitotic signal transduction pathways are just beginning to be understood.The Aurora kinases are key mitotic regulators in eukaryotes (reviewed in reference 45). The Aurora family includes a single member in yeasts (Saccharomyces cerevisiae Ipl1p, Schizosaccharomyces pombe Ark1), two members each in Caenorhabditis elegans and Drosophila, and two or three members in vertebrates. Although originally given a variety of names, Aurora kinases in multicellular eukaryotes have subsequently been classified into A, B, and C groups based on patterns of mitotic subcellular localization and homology, which also appear to reflect functional distinctions (8, 46). Aurora-A kinases are observed at centrosomes and adjacent spindle fibers, and current evidence supports key roles in regulating protein localization and function at centrosomes, as well as regulation of the assembly, stability, and function of the mitotic spindle (reviewed in reference 43). Aurora-B kinases display “chromosomal passenger” localization, residing on mitotic chromosomes and subsequently moving to the spindle midzone after separation of sister chromatids. Aurora-B family members have been implicated in the regulation of kinetochore-spindle attachment, the spindle checkpoint, and cytokinesis (reviewed in references 1 and 8). Aurora-C kinases, which have only been identified in mammals, have a limited expression pattern and appear to have functions that overlap those of Aurora-B (7, 53).The human Aurora-A kinase (hAurA) was first identified because of its overexpression in cancer cell lines (5, 58). The hAurA gene (stk15) resides on chromosome 20q13, a region frequently amplified in human cancers (5, 58). hAurA has been dubbed an oncogene because of the fact that its overexpression transforms immortalized rodent fibroblasts (5, 70). Polymorphisms in hAurA are associated with an increased risk of colon cancer, while murine AurA (mAurA) polymorphisms confer increased susceptibility to experimentally induced skin tumors (14). The mAurA gene is frequently amplified in radiation-induced lymphomas from p53 heterozygous mice, while loss of one mAurA allele has been observed in lymphomas from p53-null mice (41). Thus, aberrant AurA expression is associated with tumorigenesis, suggesting that insight into AurA functions will lead to a better understanding of tumorigenesis mechanisms.A number of experimental observations suggest that AurA kinases are required for normal centrosome maturation and bipolar spindle assembly. The AurA ortholog in Drosophila melanogaster (Aurora) was identified in a screen for mutations that impact the centrosome cycle (21). Syncytial embryos from hypomorphic Aurora mutant females display a variety of mitotic abnormalities resulting from a failure to separate centrosomes. Aurora-null flies die at the larval stage with characteristic monopolar spindles and circular chromosome arrays in larval neuroblasts. Such monopolar spindles arise from failed centrosome separation (21). Subsequent studies of Drosophila Aurora mutant alleles revealed additional defects in centrosome maturation (including a failure to localize transforming acidic coiled-coil protein, centrosomin, and γ-tubulin at centrosomes) and in asymmetric localization of Numb protein in sensory organ precursor cells (3, 17). Similar to the case in Drosophila, disruption of the C. elegans AurA ortholog AIR-1 by RNA interference (RNAi) or mutation causes defects in centrosome maturation and monopolar spindle formation. Centrosomes undergo normal separation but collapse, leading to monopolar spindle formation (16, 24, 56). Studies of the Xenopus AurA homolog pEg2 revealed similar phenotypes after overexpression of kinase-dead mutants, antibody-mediated inhibition, or immunodepletion (18, 19, 38, 52). Furthermore, Xenopus AurA has been shown to interact with and phosphorylate Eg5, a mitotic kinesin required for bipolar spindle formation, suggesting a possible mechanism by which AurA could influence bipolar spindle formation and/or stabilization (19). Thus, existing reports from these systems are quite consistent in implicating AurA in centrosome separation and function.In contrast to the systems described above, published reports of RNAi-mediated reduction of AurA expression in mammalian cell lines have contained conflicting results about the role of AurA in mitotic entry, bipolar spindle formation, and mitotic progression. AurA RNAi in HeLa cells was reported to block or delay mitotic entry, prompting the conclusion that AurA is essential for mitotic commitment in mammalian cells (27, 36). In contrast, other AurA RNAi studies showed accumulation of mitotic cells with monopolar spindles (12, 20, 67). These discrepancies call into question the functional conservation of AurA in mammals and highlight a need for additional studies to definitively address the roles of AurA. This is particularly critical for understanding the roles of AurA in cancer and for projecting possible effects of AurA inhibitors currently in development as anticancer agents. We used gene targeting in mouse embryonic stem (ES) cells to produce a conditional null allele at the AurA locus. Here we describe cellular phenotypes of AurA deletion in primary cells in vitro and developmental phenotypes of AurA mutant mice. We show that AurA deletion in primary embryonic fibroblasts causes delayed mitotic entry with accumulation of cells in early prophase, consistent with a role for AurA in mitotic entry. Nevertheless, AurA-deficient cells that enter prometaphase arrest with monopolar spindles and eventually exit mitosis without segregating their chromosomes. Prolonged culture of AurA-deficient cells leads to polyploidy with abnormal nuclear structure. Germ line AurA deficiency causes embryonic death at the blastocyst stage with mitotic arrest and monopolar spindle formation, while AurA deletion in mid-gestation embryos causes an increased mitotic index and increased apoptosis. Together, our findings indicate that AurA is required for timely mitotic entry and bipolar spindle formation in vitro and in vivo.     

Case study and application of process analytical technology (PAT) towards bioprocessing: Use of tryptophan fluorescence as at‐line tool for making pooling decisions for process chromatography

Process analytical technology (PAT) has been gaining momentum in the biopharmaceutical community due to the potential for continuous real time quality assurance resulting in improved operational control and compliance. Two imperatives for implementing any PAT tool are that “variability is managed by the process” and “product quality attributes can be accurately and reliably predicted over the design space established for materials used, process parameters, manufacturing, environmental, and other conditions.” Recently, we have been examining the feasibility of applying different analytical tools to bioprocessing unit operations. We have previously demonstarted that commercially available online‐high performance liquid chromatography and ultra performance liquid chromatography systems can be used for analysis that can facilitate real‐time decisions for column pooling based on product quality attributes (Rathore et al., 2008 a,b). In this article, we review an at‐line tool that can be used for pooling of process chromatography columns. We have demonstrated that our tryptophan fluorescence method offers a feasible approach and meets the requirements of a PAT application. It is significantly faster than the alternative of fractionation, offline analysis followed by pooling. Although the method as presented here is not an online method, this technique may offer better resolution for certain applications and may be a more optimal approach as it is very conducive to implementation in a manufacturing environment. This technique is also amenable to be used as an online tool via front face fluorescence measurements done concurrently with product concentration determination by UV. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Proteomic analysis of hepatic ischemia/reperfusion injury and ischemic preconditioning in mice revealed the protective role of ATP5β

Hepatic ischemia/reperfusion (I/R) injury is an inevitable consequence during liver surgery. Ischemic preconditioning (IPC) has been shown to protect the livers from I/R injury, partially mediated by preservation of hepatic ATP contents. However, the precise molecular mechanisms of these events remain poorly elucidated. In this study, liver proteomes of the mice subjected to I/R injury pretreated with or without IPC were analyzed using 2‐DE combined with MALDI‐TOF/TOF mass analysis. Twenty proteins showing more than 1.5‐fold difference were identified in the livers upon I/R injury. Among these proteins, four proteins were further regulated by IPC when compared with nonpretreated controls. One of these proteins, ATP synthase β subunit (ATP5β) catalyzes the rate‐limiting step of ATP formation. The expression level of ATP5β, which was further validated by Western blot analysis, was significantly decreased upon I/R injury while turned over by IPC pretreatment. Change pattern of hepatic ATP corresponded with that of ATP5β expression, indicating that increasing hepatic ATP5β expression might be a reason for ATP‐preserving effect of IPC. In summary, this study provided new clues for understanding the mechanisms of IPC against I/R injury. The protective role of ATP5β might give evidences for developing new therapeutic approaches against hepatic I/R injury.     

Proteomic characterization of Phragmites communis in ecotypes of swamp and desert dune

Phragmites communis Trin. (common reed) is a recognized model plant for studying its adaptation to contrasting and harsh environments. To understand the inherent molecular basis for its remarkable resistance to combined stresses, we performed a comprehensive proteomic analysis of the leaf proteins from two ecotypes, i.e. swamp and desert dune, naturally growing in the desert region of northwestern China. First, a proteome reference map of Phragmites was established based on the swamp ecotype. Proteins were resolved by 2‐D/SDS‐PAGE and identified by MALDI‐TOF/TOF MS. In total, 177 spots were identified corresponding to 51 proteins. The major proteins identified are proteins involved in photosynthesis, glutathione and ascorbic acid metabolism as well as protein synthesis and quality control. Second, the 2‐DE profiles of the two ecotypes were compared quantitatively via DIGE analysis. Compared with swamp ecotype, 51 proteins spots are higher‐expressed and 58 protein spots are lower‐expressed by twofold or more in desert dune ecotype. Major differences were found for the proteins involved in light reaction of photosynthesis, protein biosynthesis and quality control and antioxidative reactions. The physiological significance of such differences is discussed in the context of a flow of complex events in relation to plant adaptation to combined environmental stresses.     

Semi-interpenetrating polymer network hydrogels based on water-soluble N-carboxylethyl chitosan and photopolymerized poly (2-hydroxyethyl methacrylate)

Semi-interpenetrating polymer network (semi-IPN) hydrogels were prepared by UV irradiation of water-soluble N-carboxylethyl chitosan (CECS) and 2-hydroxyethyl methacrylate (HEMA) aqueous solutions in the presence of D-2959 as photoinitiator. Hydrogels were characterized by using scanning electron microscopy (SEM), thermal gravimetric analysis (TGA) and X-ray diffractometry (XRD). SEM showed that semi-IPN hydrogels displayed porous surface and therefore had high surface area. XRD indicated that CECS/poly (HEMA) semi-IPN hydrogels had amorphous structure. The thermal stability and equilibrium degree of swelling improved obviously with increase of CECS content. Differential scanning calorimetry (DSC) indicated that free water content increased with increase of CECS content while bonded water content decreased. Cytotoxicity results suggested that semi-IPN hydrogels had good biocompatibility. From these preliminary evaluations, it is possible to conclude that these materials have potential applications in the biomedical field.     

Seasonal dynamic changes in bacterial compositions in the Inner Mongolia desert steppe

Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) technique was employed to examine the seasonal dynamic changes in bacterial community composition in the Inner Mongolia desert steppe using specific primers F954 and R1369. Bright and reproducible bands were sequenced, and the phylogenic tree was constructed. The results show that the bacterial community composition changed between different seasons. The specific bands were different between the sampling sites with light and heavy levels of degraded grassland. Three main types of bacteria constituting the microbial community in the Inner Mongolia desert steppe belonged to the α, γ andgd-sub phyla of Proteobacteria, Bacteroidetes and Acidobacteria. The unculturable bacteria accounted for 69% of the whole bacterial community of the Inner Mongolia desert steppe.     

Enhanced sensitivity to oxidative stress in transgenic tobacco plants with decreased glutathione reductase activity leads to a decrease in ascorbate pool and ascorbate redox state

To investigate the possible mechanisms of glutathione reductase (GR) in protecting against oxidative stress, we obtained transgenic tobacco (Nicotiana tabacum) plants with 30–70% decreased GR activity by using a gene encoding tobacco chloroplastic GR for the RNAi construct. We investigated the responses of wild type and transgenic plants to oxidative stress induced by application of methyl viologen in vivo. Analyses of CO₂ assimilation, maximal efficiency of photosystem II photochemistry, leaf bleaching, and oxidative damage to lipids demonstrated that transgenic plants exhibited enhanced sensitivity to oxidative stress. Under oxidative stress, there was a greater decrease in reduced to oxidized glutathione ratio but a greater increase in reduced glutathione in transgenic plants than in wild type plants. In addition, transgenic plants showed a greater decrease in reduced ascorbate and reduced to oxidized ascorbate ratio than wild type plants. However, there were neither differences in the levels of NADP and NADPH and in the total foliar activities of monodehydroascorbate reductase and dehydroascorbate reductase between wild type and transgenic plant. MV treatment induced an increase in the activities of GR, ascorbate peroxidase, superoxide dismutase, and catalase. Furthermore, accumulation of H₂O₂ in chloroplasts was observed in transgenic plants but not in wild type plants. Our results suggest that capacity for regeneration of glutathione by GR plays an important role in protecting against oxidative stress by maintaining ascorbate pool and ascorbate redox state.     

野老鹳草DNA的提取方法及RAPD分析

目的：以野老鹳草（Geranium carolinianum L．）的茎、叶为材料,研究野老鹳草DNA的提取方法及RAPD的分析。方法：采用CATB法、高盐低pH法和SDS法分别提取野老鹳草的基因纽DNA,并对3种方法进行了一些改进。通过RAPD分析所提取的DNA,比较所用的提取方法。结果：比较DNA产量、质量等,确定了高盐低pH法较佳。结论：干燥的材料和新鲜的材料均可提取得到DNA,高盐低pH法提取的效果优于CTAB和SDS法。     

The mouse frizzled 8 receptor is expressed in anterior organizer tissues

Wnt signaling has been shown to be important for axis formation in vertebrates. However, no Wnt ligand or receptor has been shown to be specifically expressed in all the organizer tissues in the mouse embryo. Here we report that the mouse frizzled 8 (mfz8) gene, a Wnt receptor, is expressed in the anterior visceral endoderm (AVE) and the anterior primitive streak, which have been shown to possess organizer activity. mFz8 is also expressed in the descendents of the anterior streak that comprise the anterior mesendoderm (AME) at midgastrulation, with subsequent expression in the anterior neurectoderm, which is specified and patterned by the AVE and AME. Thus, mfz8 is specifically expressed in the organizer tissues that establish the anterior-posterior axis in the mouse embryo.