Topological analysis of plasmid chromatin from yeast and mammalian cells

Yeast has proven to be a powerful system for investigation of chromatin structure. However, the extent to which yeast chromatin can serve as a model for mammalian chromatin is limited by the significant number of differences that have been reported. To further investigate the structural relationship between the two chromatins, we have performed a DNA topological analysis of pRSSVO, a 5889 base-pair plasmid that can replicate in either yeast or mammalian cells. When grown in mammalian cells, pRSSVO contains an average of 33 negative supercoils, consistent with one nucleosome per 181 bp. This is close to the measured nucleosome repeat length of 190 bp. However, when grown in yeast cells, pRSSVO contains an average of only 23 negative supercoils, which is indicative of only one nucleosome per 256 bp. This is dramatically different from the measured nucleosome repeat length of 165 bp. To account for these observations, we suggest that yeast chromatin is composed of relatively short ordered arrays of nucleosomes with a repeat of 165 bp, separated by substantial gaps, possibly corresponding to regulatory regions.     

Gadd45a interacts with aurora-A and inhibits its kinase activity

Centrosome stability is required for successful mitosis in mammalian cells. Amplification of the centrosome leads to chromosomal missegregation and generation of aneuploidy, which are closely associated with cell transformation and tumorigenesis (Doxsey, S. J. (2001) Nat. Cell Biol. 3, E105-E108; Hinchcliffe, E. H., and Sluder, G. (2001) Genes Dev. 15, 1167-1181; Pihan, G. A., Purohit, A., Wallace, J., Malhotra, R., Liotta, L., and Doxsey, S. J. (2001) Cancer Res. 61, 2212-2219). However, there are currently limited insights into mechanism(s) for this critical biological event. Here we show that Gadd45a, a DNA damage-inducible protein that is regulated by tumor suppressors p53 and BRCA1, participates in the maintenance of centrosome stability. Mouse embryonic fibroblasts derived from gadd45a knock-out mice exhibit centrosome amplification (designated as increased centrosome numbers). Introduction of exogenous Gadd45a into mouse embryonic fibroblasts isolated from gadd45a-null mice substantially restored the normal centrosome profile. In contrast to p21(waf1/cip1), which ensures coordinated initiation of centrosome, Gadd45a had no significant effect on centrosome duplication in S phase. Interestingly Gadd45a was found to physically associate with Aurora-A protein kinase, whose deregulated expression results in centrosome abnormality. Furthermore Gadd45a was demonstrated to strongly inhibit Aurora-A kinase activity and to antagonize Aurora-A-induced centrosome amplification. These findings identify a novel mechanism for Gadd45a in the maintenance of centrosome stability and broaden understandings of p53- and BRCA1-regulated signaling pathways in maintaining genomic fidelity.     

一类潜伏期和染病期均传染且具非线性传染率的流行病模型

研究了一类潜伏期和染病期都传染的具非线性传染率的SEIS流行病模型,确定了各类平衡点存在的条件阈值,讨论了各平衡点的稳定性,揭示了潜伏期传染和染病期传染对流行病发展趋势的共同影响．     

MAPK-Activated Protein Kinase 2 Is Required for Mouse Meiotic Spindle Assembly and Kinetochore-Microtubule Attachment

MAPK-activated protein kinase 2 (MK2), a direct substrate of p38 MAPK, plays key roles in multiple physiological functions in mitosis. Here, we show for the first time the unique distribution pattern of MK2 in meiosis. Phospho-MK2 was localized on bipolar spindle minus ends and along the interstitial axes of homologous chromosomes extending over centromere regions and arm regions at metaphase of first meiosis (MI stage) in mouse oocytes. At metaphase of second meiosis (MII stage), p-MK2 was localized on the bipolar spindle minus ends and at the inner centromere region of sister chromatids as dots. Knockdown or inhibition of MK2 resulted in spindle defects. Spindles were surrounded by irregular nondisjunction chromosomes, which were arranged in an amphitelic or syntelic/monotelic manner, or chromosomes detached from the spindles. Kinetochore–microtubule attachments were impaired in MK2-deficient oocytes because spindle microtubules became unstable in response to cold treatment. In addition, homologous chromosome segregation and meiosis progression were inhibited in these oocytes. Our data suggest that MK2 may be essential for functional meiotic bipolar spindle formation, chromosome segregation and proper kinetochore–microtubule attachments.     

Respiration in Adipocytes is Inhibited by Reactive Oxygen Species

It is a desirable goal to stimulate fuel oxidation in adipocytes and shift the balance toward less fuel storage and more burning. To understand this regulatory process, respiration was measured in primary rat adipocytes, mitochondria, and fat‐fed mice. Maximum O₂ consumption, in vitro, was determined with a chemical uncoupler of oxidative phosphorylation (carbonylcyanide p‐trifluoromethoxyphenylhydrazone (FCCP)). The adenosine triphosphate/adenosine diphosphate (ATP/ADP) ratio was measured by luminescence. Mitochondria were localized by confocal microscopy with MitoTracker Green and their membrane potential (Δψ_M) measured using tetramethylrhodamine ethyl ester perchlorate (TMRE). The effect of N‐acetylcysteine (NAC) on respiration and body composition in vivo was assessed in mice. Addition of FCCP collapsed Δψ_M and decreased the ATP/ADP ratio. However, we demonstrated the same rate of adipocyte O₂ consumption in the absence or presence of fuels and FCCP. Respiration was only stimulated when reactive oxygen species (ROS) were scavenged by pyruvate or NAC: other fuels or fuel combinations had little effect. Importantly, the ROS scavenging role of pyruvate was not affected by rotenone, an inhibitor of mitochondrial complex I. In addition, mice that consumed NAC exhibited increased O₂ consumption and decreased body fat in vivo. These studies suggest for the first time that adipocyte O₂ consumption may be inhibited by ROS, because pyruvate and NAC stimulated respiration. ROS inhibition of O₂ consumption may explain the difficulty to identify effective strategies to increase fat burning in adipocytes. Stimulating fuel oxidation in adipocytes by decreasing ROS may provide a novel means to shift the balance from fuel storage to fuel burning.     

A Recombination Hotspot in a Schizophrenia-Associated Region of GABRB2

Background

Schizophrenia is a major disorder with complex genetic mechanisms. Earlier, population genetic studies revealed the occurrence of strong positive selection in the GABRB2 gene encoding the β₂ subunit of GABA_A receptors, within a segment of 3,551 bp harboring twenty-nine single nucleotide polymorphisms (SNPs) and containing schizophrenia-associated SNPs and haplotypes.

Methodology/Principal Findings

In the present study, the possible occurrence of recombination in this ‘S1–S29’ segment was assessed. The occurrence of hotspot recombination was indicated by high resolution recombination rate estimation, haplotype diversity, abundance of rare haplotypes, recurrent mutations and torsos in haplotype networks, and experimental haplotyping of somatic and sperm DNA. The sub-segment distribution of relative recombination strength, measured by the ratio of haplotype diversity (H_d) over mutation rate (θ), was indicative of a human specific Alu-Yi6 insertion serving as a central recombining sequence facilitating homologous recombination. Local anomalous DNA conformation attributable to the Alu-Yi6 element, as suggested by enhanced DNase I sensitivity and obstruction to DNA sequencing, could be a contributing factor of the increased sequence diversity. Linkage disequilibrium (LD) analysis yielded prominent low LD points that supported ongoing recombination. LD contrast revealed significant dissimilarity between control and schizophrenic cohorts. Among the large array of inferred haplotypes, H26 and H73 were identified to be protective, and H19 and H81 risk-conferring, toward the development of schizophrenia.

Conclusions/Significance

The co-occurrence of hotspot recombination and positive selection in the S1–S29 segment of GABRB2 has provided a plausible contribution to the molecular genetics mechanisms for schizophrenia. The present findings therefore suggest that genome regions characterized by the co-occurrence of positive selection and hotspot recombination, two interacting factors both affecting genetic diversity, merit close scrutiny with respect to the etiology of common complex disorders.     

HLA Class I Restriction as a Possible Driving Force for Chikungunya Evolution

After two decades of quiescence, epidemic resurgence of Chikungunya fever (CHIKF) was reported in Africa, several islands in the Indian Ocean, South-East Asia and the Pacific causing unprecedented morbidity with some cases of fatality. Early phylogenetic analyses based on partial sequences of Chikungunya virus (CHIKV) have led to speculation that the virus behind recent epidemics may result in greater pathogenicity. To understand the reasons for these new epidemics, we first performed extensive analyses of existing CHIKV sequences from its introduction in 1952 to 2009. Our results revealed the existence of a continuous genotypic lineage, suggesting selective pressure is active in CHIKV evolution. We further showed that CHIKV is undergoing mild positive selection, and that site-specific mutations may be driven by cell-mediated immune pressure, with occasional changes that resulted in the loss of human leukocyte antigen (HLA) class I-restricting elements. These findings provide a basis to understand Chikungunya virus evolution and reveal the power of post-genomic analyses to understand CHIKV and other viral epidemiology. Such an approach is useful for studying the impact of host immunity on pathogen evolution, and may help identify appropriate antigens suitable for subunit vaccine formulations.     

Lovastatin Inhibits VEGFR and AKT Activation: Synergistic Cytotoxicity in Combination with VEGFR Inhibitors

Background

In a recent study, we demonstrated the ability of lovastatin, a potent inhibitor of mevalonate synthesis, to inhibit the function of the epidermal growth factor receptor (EGFR). Lovastatin attenuated ligand-induced receptor activation and downstream signaling through the PI3K/AKT pathway. Combining lovastatin with gefitinib, a potent EGFR inhibitor, induced synergistic cytotoxicity in a variety of tumor derived cell lines. The vascular endothelial growth factor receptor (VEGFR) and EGFR share similar activation, internalization and downstream signaling characteristics.

Methodology/Principal Findings

The VEGFRs, particularly VEGFR-2 (KDR, Flt-1), play important roles in regulating tumor angiogenesis by promoting endothelial cell proliferation, survival and migration. Certain tumors, such as malignant mesothelioma (MM), also express both the VEGF ligand and VEGFRs that act in an autocrine loop to directly stimulate tumor cell growth and survival. In this study, we have shown that lovastatin inhibits ligand-induced VEGFR-2 activation through inhibition of receptor internalization and also inhibits VEGF activation of AKT in human umbilical vein endothelial cells (HUVEC) and H28 MM cells employing immunofluorescence and Western blotting. Combinations of lovastatin and a VEGFR-2 inhibitor showed more robust AKT inhibition than either agent alone in the H28 MM cell line. Furthermore, combining 5 µM lovastatin treatment, a therapeutically relevant dose, with two different VEGFR-2 inhibitors in HUVEC and the H28 and H2052 mesothelioma derived cell lines demonstrated synergistic cytotoxicity as demonstrated by MTT cell viability and flow cytometric analyses.

Conclusions/Significance

These results highlight a novel mechanism by which lovastatin can regulate VEGFR-2 function and a potential therapeutic approach for MM through combining statins with VEGFR-2 inhibitors.