高温和衰老对光合膜结构与功能影响的研究

本部分研究以菠菜和水稻为材料，比较系统的研究了高温对类囊体膜、PSII颗粒、PSII外周捕光天线LHCII、PSII核心复合物和PSII反应中心等不同层次膜蛋白结构与功能的影响，以探讨高温对光合膜蛋白的伤害机理。其主要结果如下： 1．类囊体膜结构与功能的完整性对于维持PSII的结构与功能在高温胁迫下的稳定性具有重要作用。当类囊体膜的完整性受到破坏，当与PSI有关的一系列保护机制失去作用时，PSII对高温胁迫的敏感性会大大加强。 2．虽然PSI的功能在高温下保持相对稳定，但PSI的结构在高温胁迫下并不稳定。本文的研究发现LHCI对高温非常敏感，在中度高温胁迫下就开始降解，但PSI的核心在高温下比较稳定，所以PSI介导的电子传递活性仍然维持在较高水平。 3．高温胁迫会对PSII的结构和功能产生多重破坏。这个过程首先应该是放氧复合体的失活：其次是反应中心的可逆失活;接下来可能是核心天线CP43和CP47的失活导致捕光天线同反应中心的能量传递受阻;再下来是QA到QR电子传递的受阻、反应中心的不可逆失活、捕光效率下降等过程;最后是大范围色素蛋白的变性和失活，PSII的结构和功能遭到彻底破坏。 4．高湿胁迫下Fo显著升高，Fo的升高的原因可能源于少量捕光天线同反应中心的分离和反应中心的失活。 5．高等植物体的类囊体膜中存在多种Chla和Chlb的光谱吸收形式。这些代表不同的光谱吸收形式的组分在高温胁迫下表现出不同程度的降解，其中C678 和C684组分降解最快。这些不同的光谱吸收形式组分可能以不同的比例存在于每一种色素蛋白复合物中。 6．LHCII的结构与功能对于维持PSII结构与功能的热稳定性具有重要作用，LHCII完全缺失的水稻突变体VG28及分离纯化的PSII核心复介物都人大增强了对高温的敏感性。但一种LHCII减少的水稻突变体249-Mutant,反而增加了PSII的热稳定性，进一步的研究表明，类囊体膜中 LHCII本身含量的多少对PSII热稳定性的影响不足决定性的，关键性因素可能主要取决于 LHCII含量改变而引起的膜脂组成和膜脂不饱和程度的改变，以及由膜脂变化引起的PSII放氧复合体结构与功能的变化。 7．本研究首次发现，高温可以促使分离纯化的LHCII的红区吸收光谱发生显著红移，而680nm处的荧光发射降低，长波长荧光组分大大增强。绿胶电泳表明中度高温胁迫能够诱导LHCII产生寡聚体，这种寡聚体可能在调节能量耗散方面具有重要生理意义：而严重高温胁迫下LHCII倾向于聚合产生大分子的非活性聚集体。 8．分离纯化的反应中心对高温非常敏感，各种色素的结构和功能在轻度高温胁迫下就开始受到破坏和抑制，各种色素变性和降解的顺序由快到慢是：P680>Pheo>Chla>β-Car。反应中心的多肽组分在高温胁迫下显著减少，Dl和D2减少的原因可能归因于高温胁迫导致大分子聚合物的产生，D2的减少显著快于D1的减少。

Genome-wide high-resolution mapping and functional analysis of DNA methylation in arabidopsis.

Cytosine methylation is important for transposon silencing and epigenetic regulation of endogenous genes, although the extent to which this DNA modification functions to regulate the genome is still unknown. Here we report the first comprehensive DNA methylation map of an entire genome, at 35 base pair resolution, using the flowering plant Arabidopsis thaliana as a model. We find that pericentromeric heterochromatin, repetitive sequences, and regions producing small interfering RNAs are heavily methylated. Unexpectedly, over one-third of expressed genes contain methylation within transcribed regions, whereas only approximately 5% of genes show methylation within promoter regions. Interestingly, genes methylated in transcribed regions are highly expressed and constitutively active, whereas promoter-methylated genes show a greater degree of tissue-specific expression. Whole-genome tiling-array transcriptional profiling of DNA methyltransferase null mutants identified hundreds of genes and intergenic noncoding RNAs with altered expression levels, many of which may be epigenetically controlled by DNA methylation.

MicroRNAs and other small RNAs enriched in the Arabidopsis RNA-dependent RNA polymerase-2 mutant.

The Arabidopsis genome contains a highly complex and abundant population of small RNAs, and many of the endogenous siRNAs are dependent on RNA-Dependent RNA Polymerase 2 (RDR2) for their biogenesis. By analyzing an rdr2 loss-of-function mutant using two different parallel sequencing technologies, MPSS and 454, we characterized the complement of miRNAs expressed in Arabidopsis inflorescence to considerable depth. Nearly all known miRNAs were enriched in this mutant and we identified 13 new miRNAs, all of which were relatively low abundance and constitute new families. Trans-acting siRNAs (ta-siRNAs) were even more highly enriched. Computational and gel blot analyses suggested that the minimal number of miRNAs in Arabidopsis is approximately 155. The size profile of small RNAs in rdr2 reflected enrichment of 21-nt miRNAs and other classes of siRNAs like ta-siRNAs, and a significant reduction in 24-nt heterochromatic siRNAs. Other classes of small RNAs were found to be RDR2-independent, particularly those derived from long inverted repeats and a subset of tandem repeats. The small RNA populations in other Arabidopsis small RNA biogenesis mutants were also examined; a dcl2/3/4 triple mutant showed a similar pattern to rdr2, whereas dcl1-7 and rdr6 showed reductions in miRNAs and ta-siRNAs consistent with their activities in the biogenesis of these types of small RNAs. Deep sequencing of mutants provides a genetic approach for the dissection and characterization of diverse small RNA populations and the identification of low abundance miRNAs.

Dynamic regulation of ARGONAUTE4 within multiple nuclear bodies in Arabidopsis thaliana.

DNA methylation directed by 24-nucleotide small RNAs involves the small RNA-binding protein ARGONAUTE4 (AGO4), and it was previously shown that AGO4 localizes to nucleolus-adjacent Cajal bodies, sites of snRNP complex maturation. Here we demonstrate that AGO4 also localizes to a second class of nuclear bodies, called AB-bodies, which are found immediately adjacent to condensed 45S ribosomal DNA (rDNA) sequences. AB-bodies also contain other proteins involved in RNA-directed DNA methylation including NRPD1b (a subunit of the RNA Polymerase IV complex, RNA PolIV), NRPD2 (a second subunit of this complex), and the DNA methyltransferase DRM2. These two classes of AGO4 bodies are structurally independent--disruption of one class does not affect the other--suggesting a dynamic regulation of AGO4 within two distinct nuclear compartments in Arabidopsis. Abolishing Cajal body formation in a coilin mutant reduced overall AGO4 protein levels, and coilin dicer-like3 double mutants showed a small decrease in DNA methylation beyond that seen in dicer-like3 single mutants, suggesting that Cajal bodies are required for a fully functioning DNA methylation system in Arabidopsis.

Tandem repeats upstream of the Arabidopsis endogene SDC recruit non-CG DNA methylation and initiate siRNA spreading.

Plants use siRNAs to target cytosine DNA methylation to both symmetrical CG and nonsymmetrical (CHG and CHH) sequence contexts. DNA methylation and siRNA clusters most frequently overlap with transposons in the Arabidopsis thaliana genome. However, a significant number of protein-coding genes also show promoter DNA methylation, and this can be used to silence their expression. Loss of the majority of non-CG DNA methylation in drm1 drm2 cmt3 triple mutants leads to developmental phenotypes. We identified the gene responsible for these phenotypes as SUPPRESSOR OF drm1 drm2 cmt3 (SDC), which encodes an F-box protein and possesses seven promoter tandem repeats. The SDC repeats show a unique silencing requirement for non-CG DNA methylation directed redundantly by histone methylation and siRNAs, and display spreading of siRNAs and methylation beyond the repeated region. In addition to revealing the complexity of DNA methylation control in A. thaliana, SDC has important implications for how plant genomes utilize gene silencing to repress endogenous genes.

RNAi, DRD1, and histone methylation actively target developmentally important Non-CG DNA methylation in Arabidopsis

Cytosine DNA methylation protects eukaryotic genomes by silencing transposons and harmful DNAs, but also regulates gene expression during normal development. Loss of CG methylation in the Arabidopsis thaliana met1 and ddm1 mutants causes varied and stochastic developmental defects that are often inherited independently of the original met1 or ddm1 mutation. Loss of non-CG methylation in plants with combined mutations in the DRM and CMT3 genes also causes a suite of developmental defects. We show here that the pleiotropic developmental defects of drm1 drm2 cmt3 triple mutant plants are fully recessive, and unlike phenotypes caused by met1 and ddm1, are not inherited independently of the drm and cmt3 mutations. Developmental phenotypes are also reversed when drm1 drm2 cmt3 plants are transformed with DRM2 or CMT3, implying that non-CG DNA methylation is efficiently re-established by sequence-specific signals. We provide evidence that these signals include RNA silencing though the 24-nucleotide short interfering RNA (siRNA) pathway as well as histone H3K9 methylation, both of which converge on the putative chromatin-remodeling protein DRD1. These signals act in at least three partially intersecting pathways that control the locus-specific patterning of non-CG methylation by the DRM2 and CMT3 methyltransferases. Our results suggest that non-CG DNA methylation that is inherited via a network of persistent targeting signals has been co-opted to regulate developmentally important genes. © 2006 Chan et al.

Bacterial flora of fresh and iced Indian mackerel (Rastrelliger kanagurta) and its response to chlortetracycline (CTC) treatment

Quantitative and qualitative studies on the bacterial flora of fresh Indian mackerel (Rastrelliger kanagurta) have been made. The total native flora as well as 5 ppm CTC insensitive flora of the fish showed variations with season. About 90% of the fresh fish flora was sensitive to 5 ppm CTC. The natural flora of the fresh fish consisted of Vibrios, Pseudomonas, Achromobacter, Flavobacterium, Corynebacteria, Micrococci, Bacillus and yeasts. In the CTC insensitive flora, Vibrios predominated followed by yeasts. The selection of bacterial genera during storage of the fish in ice and in 5 ppm CTC incorporated ice has also been investigated. At the time of spoilage, Pseudomonas was found to be the dominant flora of the fish stored in both types of ice.

Anti-HIV-1 property of trichosanthin correlates with its ribosome inactivating activity

Trichosanthin (TCS) is a type I ribosome inactivating (RI) protein possessing anti-tumor and antiviral activity, including human immunodeficiency virus (HIV). The mechanism of these actions is not entirely clear, but is generally attributed to its RI property. In order to study the relationship between the anti-HIV-1 activity of TCS and its RI activity, three TCS mutants with different RI activities were constructed by using site-directed mutagenesis. The anti-HIV-1 activities of the three mutants were tested in vitro. Results showed that two TCS mutants, namely TCSM((120-123)), TCSE160A/E189A, with the greatest decrease in RI activity, lost almost all of the anti-HIV activity and cytopathic effect. Another mutant TCSR122G, which exhibited a 160-fold decrease in RI activity, retained some anti-HIV activity. The results from this study suggested that RI activity of TCS may have significant contribution to its anti-HIV-1 property. (C) 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

Independency of anti-HIV-1 activity from ribosome-inactivating activity of trichosanthin

Trichosanthin (TCS) is a type I ribosome-inactivating (RI) protein possessing multiple biological and pharmacological activities. Its major action is inhibition of human immunodeficiency virus (HIV) replication but the mechanism is still elusive. All evidences showed that this action is related to its RI activity. Previous studies found that TCS mutants with reduced RI activity simultaneously lost some anti-HIV activity. In this study, an exception was demonstrated by two TCS mutants retaining almost all RI activity but were devoid of anti-HIV-1 activity. Five mutants were constructed by using site-directed mutagenesis with either deletion or addition of amino acids to the C-terminal sequence. Results showed that the RI activity of mutants with C-terminal deletion mutants (TCSC2, TCSC4, and TCSC14) decreased by 1.2-3.3-fold with parallel downshifting of its anti-HIV-1 activity (1.4-4.8-fold). Another two mutants, TCSC19aa and TCSKDEL having 19 amino acid extension and a KDEL signal sequence added to the C-terminal sequence, retained all RI activity but subsequently lost most of the anti-HIV-1 activity. These findings suggested that ribosome inactivation alone might not be adequate to explain the anti-HIV action of TCS. (C) 2003 Elsevier Science (USA). All rights reserved.

Site-directed PEGylation of trichosanthin retained its anti-HIV activity with reduced potency in vitro

Trichosanthin (TCS) was the first ribosome inactivating protein found to possess anti-HIV-1 activity. Phase I/II clinical trial of this compound had been done. Antigenicity and short plasma half-life were the major side effects preventing further clinical trial. Modification of TCS is therefore necessary to revive the interest to develop this compound as an anti-HIV agent. Three potential antigenic sites (Ser-7, Lys-173, and Gln-219) were identified by computer modeling. Through site-directed mutagenesis, these three antigenic amino acids were mutated to a cysteine residue resulting in 3 TCS mutants, namely S7C, K173C, and Q219C. These mutants were further coupled to polyethylene glycol with a molecular size of 20 kDa (PEG) via the cysteine residue. This produced another three TCS derivatives, namely PEG(20)k-S7C, PEG(20)k-K173C, and PEG(20)k-Q219C. PEGylation had been widely used recently to decrease immunogenicity by masking the antigenic sites and prolong plasma half-life by expanding the molecular size. The in vitro anti-HIV-1 activity of these mutants and derivatives was tested. Results showed that the anti-HIV-1 activity of S7C, K173C, and Q219C was decreased by about 1.5- to 5.5-fold with slightly lower cytotoxicity. On the other hand, PEGylation produced larger decrease (20- to 30-fold) in anti-HIV activity. Cytotoxicity was, however, weakened only slightly by about 3-fold. The in vitro study showed that the anti-HIV activity of PEGylated TCS was retained with reduced potency. The in vivo activity is expected to have only slightly changed due to other beneficial effects like prolonged half-life. (C) 2004 Elsevier Inc. All rights reserved.

Synchronization in climate dynamics and other extended systems

Synchronization is now well established as representing coherent behaviour between two or more otherwise autonomous nonlinear systems subject to some degree of coupling. Such behaviour has mainly been studied to date, however, in relatively low-dimensional discrete systems or networks. But the possibility of similar kinds of behaviour in continuous or extended spatiotemporal systems has many potential practical implications, especially in various areas of geophysics. We review here a range of cyclically varying phenomena within the Earth's climate system for which there may be some evidence or indication of the possibility of synchronized behaviour, albeit perhaps imperfect or highly intermittent. The exploitation of this approach is still at a relatively early stage within climate science and dynamics, in which the climate system is regarded as a hierarchy of many coupled sub-systems with complex nonlinear feedbacks and forcings. The possibility of synchronization between climate oscillations (global or local) and a predictable external forcing raises important questions of how models of such phenomena can be validated and verified, since the resulting response may be relatively insensitive to the details of the model being synchronized. The use of laboratory analogues may therefore have an important role to play in the study of natural systems that can only be observed and for which controlled experiments are impossible. We go on to demonstrate that synchronization can be observed in the laboratory, even in weakly coupled fluid dynamical systems that may serve as direct analogues of the behaviour of major components of the Earth's climate system. The potential implications and observability of these effects in the long-term climate variability of the Earth is further discussed. © 2010 Springer-Verlag Berlin Heidelberg.

Passive control of combustion instability in a low emissions aeroderivative gas turbine

This paper describes the conceptual ideas, the theoretical validation, the laboratory testing and the field trials of a recently patented fuel-air mixing device for use in high-pressure ratio, low emissions, gaseous-fueled gas turbines. By making the fuel-air mixing process insensitive to pressure fluctuations in the combustion chamber, it is possible to avoid the common problem of positive feedback between mixture strength and the unsteady combustion process. More specifically, a mixing duct has been designed such that fuel-air ratio fluctuations over a wide range of frequencies can be damped out by passive design means. By scaling the design in such a way that the range of damped frequencies covers the frequency spectrum of the acoustic modes in the combustor, the instability mechanism can be removed. After systematic development, this design philosophy was successfully applied to a 35:1 pressure ratio aeroderivative gas turbine yielding very low noise levels and very competitive NOx and CO measurements. The development of the new premixer is described from conceptual origins through analytic and CFD evaluation to laboratory testing and final field trials. Also included in this paper are comments about the practical issues of mixing, flashback resistance and autoignition.

The bacterial cytoskeleton modulates motility, type 3 secretion, and colonization in Salmonella.

Although there have been great advances in our understanding of the bacterial cytoskeleton, major gaps remain in our knowledge of its importance to virulence. In this study we have explored the contribution of the bacterial cytoskeleton to the ability of Salmonella to express and assemble virulence factors and cause disease. The bacterial actin-like protein MreB polymerises into helical filaments and interacts with other cytoskeletal elements including MreC to control cell-shape. As mreB appears to be an essential gene, we have constructed a viable ΔmreC depletion mutant in Salmonella. Using a broad range of independent biochemical, fluorescence and phenotypic screens we provide evidence that the Salmonella pathogenicity island-1 type three secretion system (SPI1-T3SS) and flagella systems are down-regulated in the absence of MreC. In contrast the SPI-2 T3SS appears to remain functional. The phenotypes have been further validated using a chemical genetic approach to disrupt the functionality of MreB. Although the fitness of ΔmreC is reduced in vivo, we observed that this defect does not completely abrogate the ability of Salmonella to cause disease systemically. By forcing on expression of flagella and SPI-1 T3SS in trans with the master regulators FlhDC and HilA, it is clear that the cytoskeleton is dispensable for the assembly of these structures but essential for their expression. As two-component systems are involved in sensing and adapting to environmental and cell surface signals, we have constructed and screened a panel of such mutants and identified the sensor kinase RcsC as a key phenotypic regulator in ΔmreC. Further genetic analysis revealed the importance of the Rcs two-component system in modulating the expression of these virulence factors. Collectively, these results suggest that expression of virulence genes might be directly coordinated with cytoskeletal integrity, and this regulation is mediated by the two-component system sensor kinase RcsC.

Frictional flow of damp granular material in a conical centrifuge

An analysis is given of velocity and pressure-dependent sliding flow of a thin layer of damp granular material in a spinning cone. Integral momentum equations for steady state, axisymmetric flow are derived using a boundary layer approximation. These reduce to two coupled first-order differential equations for the radial and circumferential sliding velocities. The influence of viscosity and friction coefficients and inlet boundary conditions is explored by presentation of a range of numerical results. In the absence of any interfacial shear traction the flow would, with increasing radial and circumferential slip, follow a trajectory from inlet according to conservation of angular momentum and kinetic energy. Increasing viscosity or friction reduces circumferential slip and, in general, increases the residence time of a particle in the cone. The residence time is practically insensitive to the inlet velocity. However, if the cone angle is very close to the friction angle then the residence time is extremely sensitive to the relative magnitude of these angles. © 2011 Authors.

The use of spatial impulse responses to characterise flexible forming processes with mobile tools

A novel test method for the characterisation of flexible forming processes is proposed and applied to four flexible forming processes: Incremental Sheet Forming (ISF), conventional spinning, the English wheel and power hammer. The proposed method is developed in analogy with time-domain control engineering, where a system is characterised by its impulse response. The spatial impulse response is used to characterise the change in workpiece deformation created by a process, but has also been applied with a strain spectrogram, as a novel way to characterise a process and the physical effect it has on the workpiece. Physical and numerical trials to study the effects of process and material parameters on spatial impulse response lead to three main conclusions. Incremental sheet forming is particularly sensitive to process parameters. The English wheel and power hammer are strongly similar and largely insensitive to both process and material parameters. Spinning develops in two stages and is sensitive to most process parameters, but insensitive to prior deformation. Finally, the proposed method could be applied to modelling, classification of existing and novel processes, product-process matching and closed-loop control of flexible forming processes. © 2012 Elsevier B.V.