檀香SaRbohA基因的克隆与吸器诱导因子响应分析

植物Rboh基因家族编码产生活性氧(ROS)的NADPH氧化酶。了解半寄生植物檀香(Santalum album Linn.)基因Rboh相关信息和表达特性,可为研究檀香SaRbohA基因通过活性氧信号(ROS)调控吸器发育的响应提供理论依据。该研究以全长转录组测序为基础,通过序列拼接设计合成基因特异引物,从根中克隆获得了1个檀香respiratory burst oxidase homolog(Rboh)基因cDNA全长,命名为SaRbohA。序列分析表明,该基因cDNA全长2 790 bp,编码929个氨基酸,分子量105.37 kD,理论等电点9.13,预测亚细胞定位于细胞膜。结构预测表明,SaRbohA具有6个跨膜结构域,在膜内侧的C端包含典型的NADPH结合结构域和FAD结合结构域, N端含有两个EF手性结构。序列比对分析表明,檀香SaRbohA与苹果MdRboh同源进化关系较近,相似度为63.65%。组织特异性表达分析表明,SaRbohA基因在茎中表达量最低,幼叶和茎尖中表达量较高,而在根中表达量最高。采用2, 6-二甲氧基对苯醌(寄生植物吸器诱导因子)处理,可以强烈诱导檀香SaRbohA基因的响应并伴随大量活性氧信号。研究推测,SaRbohA基因的在ROS信号介导的檀香吸器发育过程中起重要作用,且受化学诱导因子调控表达。     

Microbial enrichment and multiplexed microfiltration for accelerated detection of Salmonella in spinach

To attain Salmonella detection thresholds in spinach suspensions using enrichment media requires at least 24 hr. Separation and concentration of selected microorganisms via microfiltration and microfugation reduce time for sample preparation, especially when working with large volumes of vegetable suspensions. This facilitates accelerated detection of Salmonella in spinach suspensions, and may contribute to effectively monitoring this pathogen before it reaches the consumer. We report a microfiltration-based protocol for accelerated sample preparation to concentrate and recover ≤1 colony forming unit (CFU) Salmonella/g pathogen-free spinach. Store-bought samples of spinach and a spinach plant subjected to two environmental conditions (temperature and light exposure) during its production were tested. The overall procedure involves extraction with buffer, a short enrichment step, prefiltration using a nylon filter, crossflow hollow fiber microfiltration, and retentate centrifugation to bring microbial cells to detection levels. Based on 1 CFU Salmonella/g frozen spinach, and a Poisson distribution statistical analyses with 99% probability, we calculated that 3 hr of incubation, when followed by microfiltration, is sufficient to reach the 2 log concentration required for Salmonella detection within 7 hr. Longer enrichment times (5 hr or more) is needed for concentrations lower than 1 CFU Salmonella/g of ready to eat spinach. The recovered microbial cells were identified and confirmed as Salmonella using both polymerase chain reaction (PCR) and plating methods. Different environmental conditions tested during production did not affect Salmonella viability; this demonstrated the broad adaptability of Salmonella and emphasized the need for methods that enable efficient monitoring of production for the presence of this pathogen.     

Ticks and mosquitoes as vectors of Borrelia burgdorferi s. l. in the forested areas of Szczecin

The aim of the study was to determine the infection level of adult forms and larvae of ticks and mosquitoes with Borrelia burgdorferi in the forested areas of Szczecin. A total of 1699 ticks Ixodes ricinus, including 1422 nymphs, 277 adult forms and 2862 mosquito females representing the genera Aedes (89.6%) and Culex (10.4%) were collected between the years 2004 and 2005. A further 3746 larvae and 1596 pupae of Culex pipiens pipiens were colleted from water bodies. Borrelia burgdorferi s. l. was detected in the arthropods by the method of indirect immunofluorescence assay (IFA). A positive immunological reaction was detected in 16.6% of the adult forms and in 16.5% of the nymphs of Ixodes ricinus. Spirochetes were also detected in 1.7% of mosquito females, 3.2% of larvae and in 1.6% of pupae of Culex pipiens pipiens. The results of the present study confirm that contact with ticks constitutes the main risk of contracting Lyme disease, although mosquitoes play a role as vectors as well.     

Are transversion mutations better? A Mutagenesis Assistant Program analysis on P450 BM-3 heme domain

Directed evolution represents a versatile tool to tailor enzyme properties to needs in industrial applications and to understand structure-function relationships. Genetic diversity is commonly generated using error-prone PCR. Exploration of sequence space by random mutagenesis strongly favors transitions when enzyme-based mutagenesis methods are employed (Wong, T. S., Zhurina, D., Schwaneberg, U., Comb. Chem. High Throughput Screen. 2006, 9, 271-288). The genetic code has been organized in a manner that limits chemical diversity when a single transition mutation occurs in a codon (Wong, T. S., Roccatano, D., Schwaneberg, U., Biocatal. Biotransformation 2006, in press). Are transitions more beneficial than transversions for adapting biocatalysts to non-natural process conditions? In a statistical analysis performed with the Mutagenesis Assistant Program (MAP), we compared the consequences of transition and transversion bias on amino acid substitution patterns of the P450 BM-3 heme domain. For the analysis, we used a recently introduced benchmarking system consisting of a protein structure indicator, an amino acid diversity indicator with a codon diversity coefficient, and a chemical diversity indicator. A detailed analysis for the P450 BM-3 heme domain showed that an ideal transversion bias generates more diverse amino acid substitution patterns with a significantly different chemical composition than an ideal transition bias. Emphasis is given on the theoretical analysis with a brief discussion on potential implication of transition and transversion bias in directed evolution experiments.     

Prefrontal cortex and somatosensory cortex in tactile crossmodal association: an independent component analysis of ERP recordings

Our previous studies on scalp-recorded event-related potentials (ERPs) showed that somatosensory N140 evoked by a tactile vibration in working memory tasks was enhanced when human subjects expected a coming visual stimulus that had been paired with the tactile stimulus. The results suggested that such enhancement represented the cortical activities involved in tactile-visual crossmodal association. In the present study, we further hypothesized that the enhancement represented the neural activities in somatosensory and frontal cortices in the crossmodal association. By applying independent component analysis (ICA) to the ERP data, we found independent components (ICs) located in the medial prefrontal cortex (around the anterior cingulate cortex, ACC) and the primary somatosensory cortex (SI). The activity represented by the IC in SI cortex showed enhancement in expectation of the visual stimulus. Such differential activity thus suggested the participation of SI cortex in the task-related crossmodal association. Further, the coherence analysis and the Granger causality spectral analysis of the ICs showed that SI cortex appeared to cooperate with ACC in attention and perception of the tactile stimulus in crossmodal association. The results of our study support with new evidence an important idea in cortical neurophysiology: higher cognitive operations develop from the modality-specific sensory cortices (in the present study, SI cortex) that are involved in sensation and perception of various stimuli.     

An effective sample preparation approach for screening the anticancer compound piceatannol using HPLC coupled with UV and fluorescence detection

Piceatannol, compared with the renowned resveratrol, is a better anticancer agent and a superior agent with other biological activities. However, as there are only few plants reported to contain minute quantity of piceatannol, the scarcity of sources greatly impedes the piceatannol-related researches. To explore new sources of piceatannol, we established a sample preparation approach for screening the piceatannol in plants using HPLC-UV-fluorescence detection. When the HPLC is coupled with UV and fluorescence detectors, the decrease of signals in interferences and increase of signal in piceatannol in the fluorescence chromatogram mark clearly the position of the piceatannol peak; ultimately, it allows identification without standards. In this study, we systematically evaluated the factors affecting the extraction efficiency of piceatannol in sample preparation. Of the sample preparation strategies studied, direct solvent extraction and liquid nitrogen treatment followed by solvent extraction gave satisfactory recoveries for both piceatannol and resveratrol. These approaches avoided time-consuming lyophilization procedure. In addition, all procedures must be done in the dark to avoid negative impact of irradiation from fluorescence light on the recoveries of piceatannol and resveratrol. With the present method, we re-examined the plants previously claimed to contain only resveratrol for their piceatannol contents. The species examined included Polygonum cuspidatum, Arachis hypogaea, Vitis thunbergii, and Ampelopsis brevipedunculaata. The results showed, for the first time, all these plants contain piceatannol. The finding implies that the resveratrol-containing plants may also contain piceatannol. The results demonstrate the feasibility of these sample preparation approaches and may further the understanding for the distribution of piceatannol in plants.     

NAMPT regulates PKM2 nuclear location through 14-3-3ζ: Conferring resistance to tamoxifen in breast cancer

The resistance against tamoxifen therapy has become one of the major obstacles in the clinical treatment of breast cancer. Nicotinamide phosphoribosyltransferase (NAMPT) is an essential enzyme catalyzing nicotinamide adenine dinucleotide biosynthesis and is important for tumor metabolism. The study here sought to explore the effect of NAMPT on breast cancer survival with tamoxifen conditioning. We found that NAMPT was highly expressed in breast cancer cells compared with normal mammary epithelial cells. Inhibition of NAMPT by FK866 inhibited cell viability and aggravated apoptosis in cancer cells treated with 4-hydroxytamoxifen. NAMPT overexpression upregulated 14-3-3ζ expression. Knockdown of 14-3-3ζ reduced cell survival and promoted apoptosis. Activation of Akt signaling, rather than ERK1/2 pathway, is responsible for 14-3-3ζ regulation by NAMPT overexpression. Furthermore, NAMPT overexpression led to PKM2 accumulation in the cell nucleus and could be dampened by 14-3-3ζ inhibition. In addition, NAMPT overexpression promoted xenografted tumor growth and apoptosis in nude mice, while 14-3-3ζ inhibition attenuated its effect. Collectively, our data demonstrate that NAMPT contributes to tamoxifen resistance through regulation of 14-3-3ζ expression and PKM2 translocation.     

The growth of the very large CD8+ T cell clones in older mice is controlled by cytokines

Older humans and mice frequently contain very large clones of CD8(+) T cells. In mice these cells are phenotypically very similar to memory CD8(+) T cells. Like memory CD8(+) T cells, most members of the clones are in continuous slow division, apparently independently of Ag stimulation. Proliferation of the CD8(+) clonal T cells is inhibited in mice treated with Ab to the IL-2R beta-chain that blocks signaling by either IL-2 or IL-15. However, inhibition of IL-2 increases the numbers of dividing clonal cells. Therefore, like normal memory CD8(+) T cells, expansion of the clones is driven by IL-15 and inhibited by IL-2 and is probably limited by the amounts of IL-15 and IL-2 present in the host. Control by these two cytokines may account for the fact that, although the clones can be very large, they do not overwhelm or kill their hosts. Nevertheless the clonal cells compete successfully with normal memory CD8(+) T cells for growth. Perhaps the clonal cells use IL-15 more effectively or are more resistant to the inhibitory effects of IL-2. Thus they might affect the immune response of their hosts by competing for factors that stimulate and inhibit normal CD8(+) memory T cells.