一种适于转基因水稻PCR检测的微量DNA快速提取法

对已报道的小麦基因组DNA快速提取方法的部分步骤进行了简化,在水稻上进行了尝试。结果表明,简化法提取的水稻基因组DNA完整性好,PCR扩增效果与试剂盒提取法无明显的差异,结果稳定可靠;而且整个提取过程操作简单、花费时间少,样品用量少,仅需5-10mg,适用于大规模转基因水稻的PCR检测。     

Trivalent Arsenic Inhibits the Functions of Chaperonin Complex

The exact molecular mechanisms by which the environmental pollutant arsenic works in biological systems are not completely understood. Using an unbiased chemogenomics approach in Saccharomyces cerevisiae, we found that mutants of the chaperonin complex TRiC and the functionally related prefoldin complex are all hypersensitive to arsenic compared to a wild-type strain. In contrast, mutants with impaired ribosome functions were highly arsenic resistant. These observations led us to hypothesize that arsenic might inhibit TRiC function, required for folding of actin, tubulin, and other proteins postsynthesis. Consistent with this hypothesis, we found that arsenic treatment distorted morphology of both actin and microtubule filaments. Moreover, arsenic impaired substrate folding by both bovine and archaeal TRiC complexes in vitro. These results together indicate that TRiC is a conserved target of arsenic inhibition in various biological systems.ARSENIC is a ubiquitous environmental pollutant that causes severe health problems in humans. It is also used as an effective therapeutic agent against various diseases and infections. Using advanced genomic tools in the model organism yeast and biochemical experiments, we demonstrated that arsenic disturbs functions of the chaperonin complex required for proper folding and maturation of a large number of proteins. This mechanism of action by arsenic is conserved in various biological systems ranging from archaeal bacteria to mammals. Such an understanding should help in exploring possible ways to overcome toxic effects caused by exposure to arsenic.Trivalent inorganic arsenic is among the most significant environmental hazards affecting the health of millions of people worldwide (Nordstrom 2002). Particularly, inorganic trivalent arsenic [As(III)] in underground drinking water and some mining environments is recognized as the cause of various cancers affecting the skin, lung, urinary tract, bladder, liver, and kidney (Tapio and Grosche 2006), as well as being implicated in several other disorders such as diabetes, hypertension, neuropathy, and vascular diseases (Tseng 2004). Interestingly, As(III) is also an effective therapeutic agent against cancer and human pathogens. A number of models have been proposed to explain the biological effects of As(III), including stimulation of reactive oxygen species (ROS) production (Miller et al. 2002; Tapio and Grosche 2006) and inhibition of tubulin polymerization (Ramirez et al. 1997; Li and Broome 1999). However, exactly how As(III) disturbs biological systems is still not clear.The eukaryotic chaperonin TRiC (TCP1-ring complex, also called CCT) is a ∼900-kDa complex consisting of two apposed heterooligomeric protein rings. Each ring, constituted by eight homologous subunits (encoded by the essential CCT1–CCT8 genes in budding yeast), contains a central cavity in which unfolded polypeptide substrates attain a properly folded state in an ATP-requiring reaction (Bukau and Horwich 1998; Gutsche et al. 1999). TRiC is required for the proper folding of an important subset of cytosolic proteins, including cytoskeleton components, cell cycle regulators, and tumor suppressor proteins (Spiess et al. 2004). Some of these protein substrates are themselves encoded by essential genes; thus TRiC is indispensable for eukaryotic cell survival. Many TRiC substrates are subunits of oligomeric complexes and their assembly into functional multisubunit complexes also requires TRiC (Spiess et al. 2004). Assembly of such macromolecular complexes in some cases eliminates the accumulation of toxic subunits such as free β-tubulin molecules, which can bind to γ-tubulin and thereby disrupt the formation of mitotic spindles in the yeast S. cerevisiae (Archer et al. 1995). Folding of yeast actin, α-tubulin, and β-tubulin and their oligomerization require TRiC and GimC (also known as prefoldin), a nonessential protein complex of six distinct but structurally related subunits of 13–23 kDa (Geiser et al. 1997; Vainberg et al. 1998). Mutational loss of GimC function substantially reduces actin and tubulin folding efficiency although it does not cause obvious growth defects in yeast. However, deletion of various GimC subunits strongly reduces the viability of conditional-lethal alleles of TRiC subunits under permissive conditions (Siegers et al. 1999).To elucidate the mechanisms of inorganic As(III)''s action(s) in a eukaryotic system, we first took an unbiased functional chemogenomics approach in yeast to systematically probe for the genetic determinants of arsenic sensitivity. These genetic and subsequent biochemical results point to the conclusion that As(III) inhibits the yeast TRiC complex. This mechanism of action is apparently conserved because the activities of both a mammalian TRiC complex and an archaeal TRiC-like chaperonin are significantly inhibited by arsenic in vitro. Given that mammalian TRiC and some of its substrates are implicated in tumor suppression, angiogenesis, and neuropathy (Lee et al. 2003; Spiess et al. 2004; Bouhouche et al. 2006), TRiC is likely an important protein mediator of As(III)''s effects on human health.     

The Evolution Pathway of Ammonia-Oxidizing Archaea Shaped by Major Geological Events

Primordial nitrification processes have been studied extensively using geochemical approaches, but the biological origination of nitrification remains unclear. Ammonia-oxidizing archaea (AOA) are widely distributed nitrifiers and implement the rate-limiting step in nitrification. They are hypothesized to have been important players in the global nitrogen cycle in Earth’s early history. We performed systematic phylogenomic and marker gene analyses to elucidate the diversification timeline of AOA evolution. Our results suggested that the AOA ancestor experienced terrestrial geothermal environments at ∼1,165 Ma (1,928–880 Ma), and gradually evolved into mesophilic soil at ∼652 Ma (767–554 Ma) before diversifying into marine settings at ∼509 Ma (629–412 Ma) and later into shallow and deep oceans, respectively. Corroborated by geochemical evidence and modeling, the timing of key diversification nodes can be linked to the global magmatism and glaciation associated with the assembly and breakup of the supercontinent Rodinia, and the later oxygenation of the deep ocean. Results of this integrated study shed light on the geological forces that may have shaped the evolutionary pathways of the AOA, which played an important role in the ancient global nitrogen cycle.     

A concise and practical synthesis of antigenic globotriose, alpha-D-Gal-(1-->4)-beta-D-Gal-(1-->4)-beta-D-Glc

A concise and practical synthesis of the antigenic globotriose, alpha-D-Gal-(1-->4)-beta-D-Gal-(1-->4)-beta-D-Glc (13), was achieved by coupling of a monosaccharide donor, 3-O-allyl-2-O-benzoyl-4,6-O-benzylidene-alpha-D-galactopyranosyl trichloroacetimidate (4) with a disaccharide acceptor, p-methoxyphenyl 2,3,6-tri-O-benzoyl-beta-D-galactopyranosyl-(1-->4)-2,3,6-tri-O-benzoyl-beta-D-glucopyranoside (8), followed by deprotection. In spite of the existence of a C-2-ester substituent capable of neighboring-group participation in the donor, the coupling gave exclusively the alpha-linkage in satisfactory yield. The acceptor 8 was readily obtained from selective 3-O-benzoylation of the galactosyl ring of p-methoxyphenyl 2,6-di-O-benzoyl-beta-D-galactopyranosyl-(1-->4)-2,3,6-tri-O-benzoyl-beta-D-glucopyranoside (7), which was prepared from p-methoxyphenyl beta-D-lactoside (5) via isopropylidenation, benzoylation, and deisopropylidenation. Donor 4 was obtained from p-methoxylphenyl 3-O-allyl-2,4,6-tri-O-benzoyl-beta-D-galactopyranoside (1) via selective 4,6-di-O-debenzoylation, oxidative removal of 1-O-MP, benzylidenation, and trichloroacetimidate formation.     

Pre-B cell leukemia homeobox 1 is associated with lupus susceptibility in mice and humans

Sle1a.1 is part of the Sle1 susceptibility locus, which has the strongest association with lupus nephritis in the NZM2410 mouse model. In this study, we show that Sle1a.1 results in the production of activated and autoreactive CD4(+) T cells. Additionally, Sle1a.1 expression reduces the peripheral regulatory T cell pool, as well as induces a defective response of CD4(+) T cells to the retinoic acid expansion of TGF-β-induced regulatory T cells. At the molecular level, Sle1a.1 corresponds to an increased expression of a novel splice isoform of Pbx1, Pbx1-d. Pbx1-d overexpression is sufficient to induce an activated/inflammatory phenotype in Jurkat T cells and to decrease their apoptotic response to retinoic acid. PBX1-d is expressed more frequently in the CD4(+) T cells from lupus patients than from healthy controls, and its presence correlates with an increased central memory T cell population. These findings indicate that Pbx1 is a novel lupus susceptibility gene that regulates T cell activation and tolerance.     

微小RNA与肺癌耐药

肺癌细胞对化疗药物产生耐药性是目前肺癌化疗过程中遇到的主要问题。微小RNA（miRNA）是一类内源性非编码短链小分子RNA,它能调节细胞生长、凋亡和信号转导。miRNA的多态性与药物代谢和耐药形成密切相关,异常表达的miRNA对预测肺癌化疗药物敏感性有重要作用。调节特异miRNA的表达,将为克服肺癌耐药和选择个体化治疗开辟新的途径。     

Acetylation-dependent regulation of Skp2 function

Aberrant Skp2 signaling has been implicated as a driving event in tumorigenesis. Although the underlying molecular mechanisms remain elusive, cytoplasmic Skp2 correlates with more aggressive forms of breast and prostate cancers. Here, we report that Skp2 is acetylated by p300 at K68 and K71, which is a process that can be antagonized by the SIRT3 deacetylase. Inactivation of SIRT3 leads to elevated Skp2 acetylation, which leads to increased Skp2 stability through impairment of the Cdh1-mediated proteolysis pathway. As a result, Skp2 oncogenic function is increased, whereby cells expressing an acetylation-mimetic mutant display enhanced cellular proliferation and tumorigenesis in vivo. Moreover, acetylation of Skp2 in the nuclear localization signal (NLS) promotes its cytoplasmic retention, and cytoplasmic Skp2 enhances cellular migration through ubiquitination and destruction of E-cadherin. Thus, our study identifies an acetylation-dependent regulatory mechanism governing Skp2 oncogenic function and provides insight into how cytoplasmic Skp2 controls cellular migration.     

Complete Genome Sequence of the Pyrene-Degrading Bacterium Cycloclasticus sp. Strain P1

Cycloclasticus sp. strain P1 was isolated from deep-sea sediments of the Pacific Ocean and characterized as a unique bacterium in the degradation of pyrene, a four-ring polycyclic aromatic hydrocarbon (PAH). Here we report the complete genome of P1 and genes associated with PAH degradation.     

CCR5 antagonist TD-0680 uses a novel mechanism for enhanced potency against HIV-1 entry, cell-mediated infection, and a resistant variant

Regardless of the route of transmission, R5-tropic HIV-1 predominates early in infection, rendering C-C chemokine receptor type 5 (CCR5) antagonists as attractive agents not only for antiretroviral therapy but also for prevention. Here, we report the specificity, potency, and underlying mechanism of action of a novel small molecule CCR5 antagonist, TD-0680. TD-0680 displayed the greatest potency against a diverse group of R5-tropic HIV-1 and SIV strains when compared with its prodrug, TD-0232, the Food and Drug Administration-approved CCR5 antagonist Maraviroc, and TAK-779, with EC(50) values in the subnanomolar range (0.09-2.29 nm). Importantly, TD-0680 was equally potent at blocking envelope-mediated cell-cell fusion and cell-mediated viral transmission as well as the replication of a TAK-779/Maraviroc-resistant HIV-1 variant. Interestingly, TD-0232 and TD-0680 functioned differently despite binding to a similar transmembrane pocket of CCR5. Site-directed mutagenesis, drug combination, and antibody blocking assays identified a novel mechanism of action of TD-0680. In addition to binding to the transmembrane pocket, the unique exo configuration of this molecule protrudes and sterically blocks access to the extracellular loop 2 (ECL2) region of CCR5, thereby interrupting the interaction between virus and its co-receptor more effectively. This mechanism of action was supported by the observations of similar TD-0680 potency against CD4-dependent and -independent SIV strains and by molecular docking analysis using a CCR5 model. TD-0680, therefore, merits development as an anti-HIV-1 agent for therapeutic purposes and/or as a topical microbicide for the prevention of sexual transmission of R5-tropic HIV-1.