Functional and structural alterations induced by copper in xanthine oxidase

Xanthine oxidase （XO）, a key enzyme in purine metabolism, produces reactive oxygen species causing vascular injuries and chronic heart failure. Here, copper＇s ability to alter XO activity and structure was investigated in vitro after pre-incubation of the enzyme with increasing Cue＋ concentrations for various periods of time. The enzymatic activity was measured by following XO-catalyzed xanthine oxidation to uric acid under steady-state kinetics conditions. Structural alterations were assessed by electronic absorption, fluorescence, and circular dichroism spectroscopy. Results showed that Cu^2＋ either stimulated or inhibited XO activity, depending on metal concentration and preincubation length, the latter also determining the inhibition type. Cu^2＋-xo complex formation was characterized by modifications in XO electronic absorption bands, intrinsic fluorescence, and α-helical and β-sheet content. Apparent dissociation constant values implied high- and low-affinity Cu^2＋ binding sites in the vicinity of the enzyme＇s reactive centers. Data indicated that Cu^2＋ binding to high-affinity sites caused alterations around XO molybdenum and flavin adenine dinucleotide centers, changes in secondary structure, and moderate activity inhibition; binding to low affinity sites caused alterations around all XO reactive centers including FeS, changes in tertiary structure as reflected by alterations in spectral properties, and drastic activity inhibition. Stimulation was attributed to transient stabilization of XO optimal conformation. Results also emphasized the potential role of copper in the regulation of XO activity stemming from its binding properties.     

Molecular Characterization of a Dehydroascorbate Reductase from Pinus bungeana

Dehydroascorbate reductase （DHAR） plays a critical role in the ascorbate-glutathione recycling reaction for most higher plants. To date, studies on DHAR in higher plants have focused largely on Arabidopsis and agricultural plants, and there is virtually no information on the molecular characteristics of DHAR in gymnosperms. The present study reports the cloning and characteristics of a DHAR （PbDHAR） from a pine, Pinus bungeana Zucc. ex Endl. The PbDHAR gene encodes a protein of 215 amino acid residues with a calculated molecular mass of 24.26 kDa. The predicted 3-D structure of PbDHAR showed a typical glutathione S-transferase fold. Reverse transcripUon-polymerase chain reaction revealed that the PbDHAR was a constitutive expression gene in P. bungeana. The expression level of PbDHAR mRNA in P. bungeana seedlings did not show significant change under high temperature stress. The recombinant PbDHAR was overexpressed in Escherichia coil following purification with affinity chromatography. The recombinant PbDHAR exhibited enzymatic activity （19.84 i.mnol/min per mg） and high affinity （a Krn of 0.08 mM） towards the substrates dehydroascorbate （DHA）. Moreover, the recombinant PbDHAR was a thermostable enzyme, and retained 77% of its initial activity at 55℃. The present study is the first to provide a detailed molecular characterization of the DHAR in P. bungeana.     

Effect of site-specific PEGylation on the fibrinolytic activity,immunogenicity, and pharmacokinetics of staphylokinase

The bacterial plasminogen-activator staphylokinase （Sak） is a promising thrombolytic agent for treating acute myocar- dial infarction. To effectively reduce the immunogenicity of Sak while maintaining its fibrinolytic activity, site-specific PEGylation was performed in the present study. The che- moselective cysteine PEGylation site was selected within an immunodominant region （amino acid residues 71-87） using an in sUico approach. The PEGylated Sak variants pre- pared in this study showed a purity of 〉97.0%. PEGylation at Position 80 resulted in a Sak variant Sak（E80C-PEG） which has similar fibrinolytic activity and thermostability compared with the native recombinant staphylokinase （r-Sak）. The immunogenicity of Sak（E80C-PEG） in guinea pigs was greatly reduced compared with the native r-Sak. Furthermore, preliminary pharmacokinetic results sug- gested that the plasma clearance of Sak（E80C-PEG） from the blood stream of rabbit was significantly decreased com- pared with that of r-Sak, resulting in a 2.8-fold increase of initial half-life and a 3.8-fold increase of systemic availabil- ity. In summary, these results demonstrated that site-specif- ic PEGylation yielded a novel Sak variant Sak（E80C-PEG） with remarkable advantages over the unmodified Sak.     

Characterization of the putative tryptophan synthase β-subunit from Mycobacterium tuberculosis

The increasing emergence of drug-resistant tuberculosis （TB） poses a serious threat to the control of this disease. It is in urgent need to develop new TB drugs. Tryptophan biosynthetic pathway plays an important role in the growth and replication of Mycobacterium tuberculosis （Mtb）. The β-subunit of tryptophan synthase （TrpB） catalyzes the last step of the tryptophan biosynthetic pathway, and it might be a potential target for TB drug design. In this study, we overexpressed, purified, and characterized the putative TrpB-encoding gene Rv1612 in Mtb H37Rv. Results showed that Mtb His-TrpB optimal enzymatic activity is at pH 7.8 with 0.15 M Na^＋ or 0.18 M Mg^2＋ at 37℃. Structure analysis indicated that Mtb TrpB exhibited a typical β/α barrel structure. The amino acid residues believed to interact with the enzyme cofactor pyridoxal-5＇-phosphate were predicted by homology modeling and structure alignment. The role of these residues in catalytic activity of the Mtb His-TrpB was confirmed by site-directed mutagenesis. These results provided reassuring structural information for drug design based on TrpB.     

贯叶连翘提取物对小鼠抗应激反应及运动力竭小鼠抗氧化活性的影响

目的：研究贯叶连翘提取物（HPLE）对小鼠抗应激反应及运动力竭小鼠抗氧化活性的影响。方法：ICR小鼠,雌雄各半,随机均分为生理盐水（NS）对照组及高、中、低剂量HPLE组。分别采用负重游泳、常压耐缺氧和耐低温实验,观察小鼠抗急性应激反应能力,HPLE组分别按1.5 g/kg、0.75 g/kg和0.38 g/kg,ig,Bid,连续给药7 d;NS组等体积生理盐水ig。另取ICR小鼠40只,雌雄各半。随机均分为NS对照组及HPLE高、中、低剂量组。分别按ig HPLE 1.5 g/kg、0.75 g/kg和0.38 g/kg,q.d;对照组：ig等量NS,qd。连续用药2周后,开始每天进行游泳训练0.5 h,到第21天后,游泳力竭后休息20 min,断头取血,制备血清,检测小鼠血清中超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-Px）活性和丙二醛（MDA）的含量变化。结果：HPLE可延长小鼠负重游泳时间,缺氧存活时间及提高耐低温能力,并能升高运动力竭小鼠血清中SOD和GSH-Px活性,降低MDA的含量。结论：HPLE可提高小鼠应激反应能力,并具有抗氧化活性。     

Effects of Glycerol on the Fluorescence Spectra and Chloroplast Ultrastructure of Phaeodactylum tricornutum （Bacillariophyta）

Responses of the photosynthetic activity of Phaeodactylum tricornutum （Bacillariophyta） to organic carbon glycerol were investigated. The growth rate, photosynthetic pigments, 77 K fluorescence spectra, and chloroplast ultrastructure of P. tricornutum were examined under photoautotrophic, mixotrophic, and photoheterotrophic conditions. The results showed that the specific growth rate was the fastest under mixotrophic conditions. The cell photosynthetic pigment content and values of Chl a/Chl c were reduced under mixotrophic and photoheterotrophic conditions. The value of carotenoid/Chl a was enhanced under mixotrophic conditions, but was decreased under photoheterotrophic conditions. In comparison with photoautotrophic conditions, the fluorescence emission peaks and fluorescence excitation peaks were not shifted. The relative fluorescence of photosystem （PS） Ⅰ and PS Ⅱ and the values of F685/F710 and F685/F738 were decreased. Chloroplast thylakoid pairs were less packed under mixotrophic and photoheterotrophic conditions. There was a strong correlation between degree of chloroplast thylakoid packing and the excitation energy kept in PS Ⅱ. These results suggested that the PS Ⅱ activity was reduced by glycerol under mixotrophic conditions, thereby leading to repression of the photosynthetic activity.     

Vegetation Mapping of the Mond Protected Area of Bushehr Province （South-west Iran）

Arid regions of the world occupy up to 35% of the earth＇s surface, the basis of various definitions of climatic conditions, vegetation types or potential for food production. Due to their high ecological value, monitoring of arid regions is necessary and modern vegetation studies can help in the conservation and management of these areas. The use of remote sensing for mapping of desert vegetation is difficult due to mixing of the spectral reflectance of bright desert soils with the weak spectral response of sparse vegetation. We studied the vegetation types in the semiarid to arid region of Mond Protected Area, south-west Iran, based on unsupervised classification of the Spot XS bands and then produced updated maps. Sixteen map units covering 12 vegetation types were recognized in the area based on both field works and satellite mapping. Halocnemum strobilaceum and Suaeda fruticosa vegetation types were the dominant types and Ephedra foliata, Salicornia europaea-Suaeda heterophylla vegetation types were the smallest. Vegetation coverage decreased sharply with the increase in salinity towards the coastal areas of the Persian Gulf. The highest vegetation coverage belonged to the riparian vegetation along the Mond River, which represents the northern boundary of the protected area. The location of vegetation types was studied on the separate soil and habitat diversity maps of the study area, which helped in final refinements of the vegetation map produced.     

Molecular Characterization of the Calvin Cycle Enzyme Phosphoribulokinase in the Stramenopile Alga Vaucheria litorea and the Plastid Hosting Mollusc Elysia chlorotica

Phosphoribulokinase （PRK）, a nuclear-encoded plastid-localized enzyme unique to the photosynthetic carbon reduction （Calvin） cycle, was cloned and characterized from the stramenopile alga Vaucheria litorea. This alga is the source of plastids for the mollusc （sea slug） Elysia chlorotica which enable the animal to survive for months solely by photoautotrophic CO2 fixation. The 1633-bp V. litorea prk gene was cloned and the coding region, found to be interrupted by four introns, encodes a 405-amino acid protein. This protein contains the typical bipartite target sequence expected of nuclearencoded proteins that are directed to complex （i.e. four membrane-bound） algal plastids. De novo synthesis of PRK and enzyme activity were detected in E. chlorotica in spite of having been starved of V. litorea for several months. Unlike the algal enzyme, PRK in the sea slug did not exhibit redox regulation. Two copies of partial PRK-encoding genes were isolated from both sea slug and aposymbiotic sea slug egg DNA using PCR. Each copy contains the nucleotide region spanning exon 1 and part of exon 2 of V litorea prk, including the bipartite targeting peptide. However, the larger prk fragment also includes intron 1. The exon and intron sequences of prk in E. chlorotica and V/itorea are nearly identical. These data suggest that PRK is differentially regulated in V. litorea and E. chlorotica and at least a portion of the V. litorea nuclear PRK gene is present in sea slugs that have been starved for several months.     

Oxygation Enhances Growth,Gas Exchange and Salt Tolerance of Vegetable Soybean and Cotton in a Saline Vertisol

Impacts of salinity become severe when the soil is deficient in oxygen. OxygaUon （using aerated water for subsurface drip irrigation of crop） could minimize the impact of salinity on plants under oxygen-limiting soil environments. Pot experiments were conducted to evaluate the effects of oxygation （12% air volume/volume of water） on vegetable soybean （moderately salt tolerant） and cotton （salt tolerant） in a salinized vertisol at 2, 8, 14, 20 dS/m ECe. In vegetable soybean, oxygation increased above ground biomass yield and water use efficiency （WUE） by 13% and 22%, respectively, compared with the control. Higher yield with oxygation was accompanied by greater plant height and stem diameter and reduced specific leaf area and leaf Na^＋ and CI^- concentrations. In cotton, oxygation increased lint yield and WUE by 18% and 16%, respectively, compared with the control, and was accompanied by greater canopy light interception, plant height and stem diameter. Oxygation also led to a greater rate of photosynthesis, higher relative water content in the leaf, reduced crop water stress index and lower leaf water potential. It did not, however, affect leaf Na^＋ or CI^- concentration. Oxygation invariably increased, whereas salinity reduced the K^＋： Na^＋ ratio in the leaves of both species. Oxygation improved yield and WUE performance of salt tolerant and moderately tolerant crops under saline soil environments, and this may have a significant impact for irrigated agriculture where saline soils pose constraints to crop production.     

Identification,expression, and characterization of the highly conserved D-xylose isomerase in animals

D-xylose is a necessary sugar for animals. The xylanase from a mollusk, Ampullaria crossean, was previously reported by our laboratory. This xylanase can degrade the xylan into D-xylose. But there is still a gap in our knowledge on its metabolic pathway. The question is how does the xylose enter the pentose pathway？ With the help of genomic databases and bioinformatic tools, we found that some animals, such as bacteria, have a highly conserved D-xylose isomerase （EC 5.3.1.5）. The xyiose isomerase from a sea squirt, Ciona intestinali, was heterogeneously expressed in Escherichia coli and purified to confirm its function. The recombinant enzyme had good thermal stability in the presence of Mg^2＋. At the optimum temperature and optimum pH environment, its specific activity on D-xylose was 0.331 μmol/mg/min. This enzyme exists broadly in many animals, but it disappeared in the genome of Amphibia-like Xenopus laevis. Its sequence was highly conserved. The xylose isomerases from animals are very interesting proteins for the study of evolution.     

The Response Difference of Mitochondria in Recalcitrant Antiaris toxicaria Axes and Orthodox Zea mays Embryos to Dehydration Injury

Long-term preservation of recalcitrant seeds is very difficult because the physiological basis on their desiccation sensitivity is poorly understood. Survival of Antiaris toxicaria axes rapidly decreased and that of immature maize embryos very slowly decreased with dehydration. To understand their different responses to dehydration, we examined the changes in mitochondria activity during dehydration. Although activities of cytochrome （Cyt） c oxidase and malate dehydrogenase of the A. toxicaria axis and maize embryo mitochondria decreased with dehydration, the parameters of maize embryo mitochondria were much higher than those of A. toxicaria, showing that the damage was more severe for the A. toxicaria axis mitochondria than for those of maize embryo. The state I and III respiration of the A. toxicaria axis mitochondria were higher than those of maize embryo, the former rapidly decreased, and the latter slowly decreased with dehydration. The proportion of Cyt c pathway to state III respiration for the A. toxicaria axis mitochondria was low and rapidly decreased with dehydration, and the proportion of alternative oxidase pathway was high and slightly increased with dehydration. In contrast, the proportion of Cyt c pathway for maize embryo mitochondria was high, and that of alternative oxidase pathway was low. Both pathways decreased slowly with dehydration.     

The Translational Apparatus of Plastids and Its Role in Plant Development

Chloroplasts （plastids） possess a genome and their own machinery to express it. Translation in plastids occurs on bacterial-type 70S ribosomes utilizing a set of tRNAs that is entirely encoded in the plastid genome. In recent years, the components of the chloroplast translational apparatus have been intensely studied by proteomic approaches and by reverse genetics in the model systems tobacco （plastid-encoded components） and Arabidopsis （nucleus-encoded components）. This work has provided important new insights into the structure, function, and biogenesis of chloroplast ribosomes, and also has shed fresh light on the molecular mechanisms of the translation process in plastids. In addition, mutants affected in plastid translation have yielded strong genetic evidence for chloroplast genes and gene products influencing plant develop- ment at various levels, presumably via retrograde signaling pathway（s）. In this review, we describe recent progress with the functional analysis of components of the chloroplast translational machinery and discuss the currently available evidence that supports a significant impact of plastid translational activity on plant anatomy and morphology.     

Chloroplast Proteomics and the Compartmentation of Plastidial Isoprenoid Biosynthetic Pathways

Recent advances in the proteomic field have allowed high-throughput experiments to be conducted on chloroplast samples. Many proteomic investigations have focused on either whole chloroplast or sub-plastidial fractions. To date, the Plant Protein Database （PPDB, Sun et al., 2009） presents the most exhaustive chloroplast proteome available online. However, the accurate localization of many proteins that were identified in different sub-plastidial compartments remains hypothetical. Ferro et al. （2009） went a step further into the knowledge of Arabidopsis thaliana chloroplast proteins with regards to their accurate localization within the chloroplast by using a semi-quantitative proteomic approach known as spectral counting. Their proteomic strategy was based on the accurate mass and time tags （AMT） database approach and they built up AT_CHLORO, a comprehensive chloroplast proteome database with sub-plastidial localization and curated information on envelope proteins. Comparing these two extensive databases, we focus here on about 100 enzymes involved in the synthesis of chloroplast-specific isoprenoids. Well known pathways （i.e. compartmentation of the methyl erythritol phosphate biosynthetic pathway, of tetrapyrroles and chlorophyll biosynthesis and breakdown within chloroplasts） validate the spectral counting-based strategy. The same strategy was then used to identify the precise localization of the biosynthesis of carotenoids and prenylquinones within chloroplasts （i.e. in envelope membranes, stroma, and/or thylakoids） that remains unclear until now.     

Response of Chinese Wampee Axes and Maize Embryos to Dehydration at Different Rates

Survival of wampee （Clausena lansium Skeels） axes and maize （Zea mays L.） embryos decreased with rapid and slow dehydration. Damage of wampee axes by rapid dehydration was much less than by slow dehydration, and that was contrary to maize embryos. The malondialdehyde contents of wampee axes and maize embryos rapidly increased with dehydration, those of wampee axes were lower during rapid dehydration than during slow dehydration, and those of maize embryos were higher during rapid dehydration than during slow dehydration. Activities of superoxide dismutase （SOD）, ascorbate peroxidase （APX） and catalase （CAT） of wampee axes markedly increased during the early phase of dehydration, and then rapidly decreased, and those of rapidly dehydrated axes were higher than those of slow dehydrated axes when they were dehydrated to low water contents. Activities of SOD and APX of maize embryos notable decreased with dehydration. There were higher SOD activities and lower APX activities of slowly dehydrated maize embryos compared with rapidly dehydrated maize embryos. CAT activities of maize embryos markedly increased during the early phase of dehydration, and then decreased, and those of slowly dehydrated embryos were higher than those of rapidly dehydrated embryos during the late phase of dehydration.     

Methods for Analysis of Protein Glutathionylation and their Application to Photosynthetic Organisms

Protein S-glutathionylation, the reversible formation of a mixed-disulfide between glutathione and protein thiols, is involved in protection of protein cysteines from irreversible oxidation, but also in protein redox regulation. Recent studies have implicated S-glutathionylation as a cellular response to oxidative/nitrosative stress, likely playing an important role in signaling. Considering the potential importance of glutathionylation, a number of methods have been developed for identifying proteins undergoing glutathionylation. These methods, ranging from analysis of purified proteins in vitro to large-scale proteomic analyses in vivo, allowed identification of nearly 200 targets in mammals. By contrast, the number of known glutathionylated proteins is more limited in photosynthetic organisms, although they are severely exposed to oxidative stress. The aim of this review is to detail the methods available for identification and analysis of glutathionylated proteins in vivo and in vitro. The advantages and drawbacks of each technique will be discussed as well as their application to photosynthetic organisms. Furthermore, an overview of known glutathionylated proteins in photosynthetic organisms is provided and the physiological importance of this post-translational modification is discussed.     

The Earliest Normal Flower from Liaoning Province,China

The early evolution of angiosperms has been a focus of intensive research for more than a century. The Yixian Formation in western Liaoning yields one of the earliest angiosperm macrofioras. Despite multitudes of angiosperm fossils uncovered, including Archaefructus and Sinocarpus, no bona fide normal flower has been dated to 125 Ma （mega-annum） or older. Here we report Callianthus dilae gen. et sp. nov. from the Yixian Formation （Early Cretaceous） in western Liaoning, China as the earliest normal flower known to date. The flower demonstrates a typical floral organization, including tepals, androecium, and gynoecium. The tepals are spatulate with parallel veins. The stamens have a slender filament, a globular anther, bristles at the anther apex, and in situ round-triangular pollen grains. The gynoecium is composed of two stylate carpels enclosed in a fleshy envelope, and develops into a ＂hip＂ when mature. Since the well-accepted history of angiosperms is not much longer than 125 Ma, Callianthus together with Chaoyangia, Archaefructus and Sinocarpus from the Yixian Formation demonstrate a surprisingly high diversity of angiosperms, implying a history of angiosperms much longer than currently accepted.     

Histological and Ultrastructural Observation Reveals Significant Cellular Differences between Agrobacterium Transformed Embryogenic and Non-embryogenic Calli of Cotton

Over the past few decades genetic engineering has been applied to improve cotton breeding. Agrobacterium medicated transformation is nowadays widely used as an efficient approach to introduce exogenous genes into cotton for genetically modified organisms. However, it still needs to be improved for better transformation efficiency and higher embryogenic callus induction ratios. To research further the difference of mechanisms for morphogenesis between embryogenic callus and non-embryogenic callus, we carried out a systematical study on the histological and cellular ultrastructure of Agrobacterium transformed calli. Results showed that the embryogenic callus developed nodule-like structures, which were formed by small, tightly packed, hemispherical cells. The surface of some embryogenic callus was covered with a fibrilar-like structure named extracellular matrix. The cells of embryogenic calli had similar morphological characteristics. Organelles of embryogenic callus cells were located near the nucleus, and chloroplasts degraded to proplastid-like structures with some starch grains. In contrast, the non-embryogenic calli were covered by oval or sphere cells or small clusters of cells. It was observed that cells had vacuolation of cytoplasm and plastids with a well organized endomembrane system. This study aims to understand the mechanisms of embryogenic callus morphogenesis and to improve the efficiency of cotton transformation in future.     

XPA A23G polymorphism is associated with the elevated response to platinum-based chemotherapy in advanced non-small cell lung cancer

DNA repair capacity （DRC） is correlated with sensitivity of cancer cells toward platinum-based chemotherapy. We hypothesize that genetic polymorphisms in DNA repair gene XPA （xeroderma pigmentosum group A） and XPG （xeroderma pigmentosum group G） （ERCC5, excision repair cross-complementation group 5）, which result in inter-individual differences in DNA repair efficiency, may predict clinical response to platinum agents in advanced non-small cell lung cancer （NSCLC） patients. In this study, we find that the A → G change of XPA A23G polymorphism significantly increased response to platinum-based chemotherapy. Polymorphism in XPG His46His was associated with a decreased treatment response, but was not statistically significant.