Inhibition of DNA restrictive endonucleases and Taq DNA polymerase by trimalonic acid C60

Activities of trimalonic acid fullerene (TMA C_60) on DNA restrictive enzymatic reaction were investigated by using two restrictive endonucleases Hind III and EcoR I and plasmid pEGFP-N1 with single restric-tive site for both enzymes. Meanwhile, TMA C60 was also tested to clarify its effects on polymerase chain reaction (PCR) with the catalyst of Taq DNA polymerase and the template of plasmid pEGFP-N1. The products from restrictive reactions or PCR were detected by agarose gel electrophoresis. It was found that the product amounts from restrictive reactions or PCR decreased significantly with addition of TMA C60. The inhibition by TMA C60 was dose-dependent and IC50 values for reactions of Hind III, EcoR I and PCR were 16.3, 6.0 and 6.0 μmol/L, respectively. Addition of two scavengers of reactive oxygen species (ROS), L-ascorbic acid-2-phosphate ester magnesium and sodium azide at the con-centrations of 2―10 mmol/L did not antagonize the activities of TMA C60 against PCR and two restrictive reactions. However, increase of Taq DNA polymerase amounts in PCR system antagonized the activities of TMA C60. These data implied that TMA C60 was able to inhibit the activities of the three above-mentioned enzymes involved in DNA metabolism, and that this inhibition probably did not correlate to ROS.     

Kinetic studies on the irreversible inhibition of restriction endonucleasePst I by site-specific inhibitors

The irreversible modifying effects onPst I of several inhibitors have been studied with the irreversible inhibition kinetic theory of single substrate reaction provided by Tsou, C. L. Pyridoxal phosphate (PLP), p-chloromercuribenzoic acid (PCMB), diisopropyl fluorophosphate (DEP), 2,3-diacetyl (DAC) and N-ethyl-5-phenylisoxazoliun-3′-sulfonate (woodward's reagent K, WRK) modify the lysine, cysine, serteine, arginine and carboxyl groups of the protein molecule respectively. These five inhibitors have been found to inhibit both the prime activity and star activity ofPst I. Used with the irreversible inhibition theory, the apparent inhibition rate constant,A and the microcosmic inhibition rate constants,k ₊₀ andk′ ₊₀ of every inhibitor were calculated. We also found that their inhibition effects belong to the noncompetitive irreversible inhibition. Results show that among the groups to be modified, some have nothing to do with the combination with the substrate, and some may have, but any of them isn't the only factor involved in the specific binding. Despite all this, they may take part in the catalysis of enzyme or have important effects on maintaining the active structure of enzyme molecules. Furthermore, serine and arginine residues are related to the alteration ofPst I conformation and then influence the ability ofPst I recognizing and incising DNA specifically. Foundation item: Supported by the Research Fund for the Doctoral Program of Higher Education (1999048601) Biography: Zou Guo-lin (1947-), male, Professor, research direction: enzymology.     

Study on the specific interaction between angiogenin and aptamer by atomic force microscopy （AFM）

The specific interaction between angiogenin and aptamer has been investigated by using AFM. The specificity of the interaction is revealed by comparing the binding probability of aptamer to other elements in a series of control experiments. The results have shown that there is specific interaction force between angiogenin and aptamer. Moreover, the single molecular pull-off force between angiogenin and aptamer has also been determined using the Poisson statistical method to be 133.7±11.7 pN. These findings obtained are helpful to the better revelation of recognition mechanism between angiogenin and aptamer, which provided basis for further understanding the inhibition of the aptamer to angiogenic activity.     

Quantitative structure-activity relationships of selected phenols with non-monotonic dose-response curves

Particular non-monotonic dose-response curves of many endocrine disrupting chemicals （EDCs） suggest the existence of diverse toxicity mechanisms at different dose levels. As a result, the biological activities of EDCs cannot be simply exhibited by unique EC50/LD50 values, and the quantitative structure-activity relationship （QSAR） analysis for non-monotonic dose-response relationship becomes an unknown field in the environmental science. In this paper, nine phenols with inverted U-shaped dose-response curves in lymphocyte proliferation test of Carassius auratus were selected. The binding interactions between the phenols and several typical EDCs-related receptors were then explored in a molecular simulation study. The estrogen receptor （ER）, androgen receptor （AR）, thyroid hormone receptor （TR）, bacterial O2 sensing FixL protein （FixL）, aryl hydrocarbon receptor （AhR）, and the peroxisome proliferator-activated receptor （PPAR） were the target receptors in the study. Linear regression QSAR models for the low and high exposure levels of the compounds were developed separately. The results indicated that the lymphocyte proliferation in the low-dose range might involve ER-mediated process, while the proliferation inhibition in the high dose range was dominated by the acute toxicity of phenols due to receptor occupancy and cell damage.     

Temperature-sensitive gold-nanotube array membranes modified with poly（N-isopropylacrylamide）

A kind of temperature-sensitive nanotube array membrane was developed by modifying gold-nanotube array membranes with poly（N-isopropylacrylamide） （PNIPAm）. The permeation ability of the mem-branes at different temperatures was investigated using sodium fluorescein and quantum dots as probes. The results showed that the pore diameter of nanotube was changed due to the reversible response of PNIPAm-modified membranes to temperature, and then the permeation ability of the mem-branes was changed. The permeation of fluorescence probes was slow and even almost blocked at 25℃ （below the lower critical solution temperature, LCST）, since PNIPAm formed expanded structures and decreased the pore size. While at 40℃ （above the LCST）, the permeation was increased, since PNIPAm became compact structures and the pore diameter was increased. Furthermore, the permeation ability of the temperature-sensitive nanotube array membranes could be adjusted reversibly and it is possible to use the membranes in nanofluidic devices, nanogates, etc.     

Integrated Evaluation Model for Eco-Environmental Quality in Mountainous Region Based on Remote Sensing and GIS

Based on Remote Sensing （RS）, Geographic Information System （GIS）, and combining Principal Component Analysis, this paper designed a numerical integrated evaluation model for mountain eco-environment on the base of grid scale. Using this model, we evaluated the mountain eco-environmental quality in a case study area-the upper reaches of Minjiang River, and achieved a good result, which accorded well with the real condition. The study indicates that, the integrated evaluation model is suitable for multi-layer spatial factor computation, effectively lowing man＇s subjective influence in the evaluation process; treating the whole river basin as a system, the model shows full respect to the circulation of material and energy, synthetically embodies the determining impact of such natural condition as water-heat and landform, as well as human interference in natural eco-system; the evaluation result not only clearly presents mountainous vertical distribution features of input factors, but also provides a scientific and reliable thought for quantitatively evaluating mountain eco-environment.     

PEG、SDBS及其复配体系对蒙脱土水化膨胀的抑制作用

通过线性膨胀、粒度分布和Zeta电势的测定，研究了聚乙二醇(PEG)、十二烷基苯磺酸钠(SDBS)及其复配体系对蒙脱土水化膨胀和分散作用的影响．结果表明，PEG／SDBS复配体系具有显著的抑制蒙脱土水化膨胀和分散的能力，改善并降低了温度对蒙脱土水化膨胀和分散的影响；结果还表明，线性膨胀实验和粒度分布实验具有同一性;即抑制性越强，越容易使蒙脱土颗粒聚集．     

A two-layer real-time rate control algorithm based on lagrange optimum algorithm

Because of the iterative process in the rate control algorithm of H.264 draft, it is not suitable for real-time transmission. A two-layer real-time rate control algorithm based on Lagrange optimum algorithm is proposed. This algorithm consists of two layers, namely, the group of pictures (GOP) layer and the frame layer. The simulation results show that the proposed algorithm is competitive in terms of peak signal to noise ratio (PSNR), real bitrates and the Q-value among frames over the original algorithm. Foundation item: Supported by the National High-Technology Research and Development Program of China (863 Program) (2001AA132050-03) and the Key Project of Chinese Ministry of Education (211CERS-10)     

Combining docking and comparative molecular similarity indices analysis （COMSIA） to predict estrogen activity and probe molecular mechanisms of estrogen activity for estrogen compounds

Estrogen compounds are suspected of disrupting endocrine functions by mimicking natural hormones, and such compounds may pose a serious threat to the health of humans and wildlife. Close attention has been paid to the prediction and molecular mechanisms of estrogen activity for estrogen com- pounds. In this article, estrogen receptor a subtype （ERa） -based comparative molecular similarity indices analysis （COMSIA） was performed on 44 estrogen compounds with structural diversity to find out the structural relationship with the activity and to predict the activity. The model with the significant correlation and the best predictive power （R^2= 0.965, Q^2 LOO： 0.599, R^2 pred ： 0.825） was achieved. The COMSIA and docking results revealed the structural features for estrogen activity and key amino acid residues in binding pocket, and provided an insight into the interaction between the ligands and these amino acid residues.     

Stepwise prediction and statistical screening: B-cell epitopes on neuraminidase of human avian H5N1 virus

The B-cell epitopes of virus are associated with the antiviral drug and the vaccine screening. As the nucleotide sequences of neuraminidase （NA） of stain GD-01-06 were sequenced, we predicted the a-helix and β-fold structure and the indexes of the flexible regions of secondary structure of NA with methods of the Hydrophilicity plot by Kyte-Doolittle, the Surface probability plot by Emini and the An- tigenic index by Jameson-Wolf, and then screened statistically the parameters to predict B-cell epi- topes by the Hierarchical cluster and the Bivariate correlation and the quartiles with SPSS 13.0. The impact of variation of amino acids in NA on its epitopes was analyzed. The predictive results were evaluated by Wu＇s Antigenic Index and SWISS-MODEL. We found that the most possible epitopes on NA were located within or nearby its N-terminal Nos. 120--137, 81--84, 408--415, 273--282, 429--432, 356--368, 46--55, 146--155, 341 --350 and 198--209, which were the dominant regions of NA epitopes. Peptide 120--137 including the glycoprotein domain （NGT126 128） was first chosen as the B-cell epitopes on NA. NA in H5N1 strain isolated after 2003 lacked in No. 53 amino acid （I）, resulting in an increase in the surface flexible region of NA in GD-01-06 and an enlargement to their epitope regions （VEP48-48→ VEPISNTNFL46-55）. Conclusively, prediction of the B-cell epitopes on the NA based on multiple pa- rameters is useful for researches on the molecular immunology and drug screening and immuno-prophylaxis. A deletion of No. 53 amino acid （I） in NA in strain GD-01-06 might increase its anti- genicity.     

Engineering <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis> into biosensor to monitor radioactivity and genotoxicity in environment

Based on a genetically modified radioresistant bacteria Deinococcus radiodurans, we constructed a real time whole cell biosensor to monitor radioactivity and genotoxicity in highly radioactive environment. The enhanced green fluorescence protein （eGFP） was fused to the promoter of the crucial DNA damage-inducible recA gene from D. radiodurans, and the consequent DNA fragment （PrecA-egfp） carried by plasmid was introduced into D. radiodurans R1 strain to obtain the biosensor strain DRG300. This engineered strain can express eGFP protein and generate fluorescence in induction of the recA gene promoter. Based on the correlation between fluorescence intensity and protein expression level in live D. radiodurans cells, we discovered that the fluorescence induction of strain DRG300 responds in a remarkable dose-dependent manner when treated with DNA damage sources such as gamma radiation and mitomycin C. It is encouraging to find the widely detective range and high sensitivity of this reconstructed strain comparing with other whole cell biosensors in former reports. These results suggest that the strain DRG300 is a potential whole cell biosensor to construct a detective system to monitor the biological hazards of radioactive and toxic pollutants in environment in real time.     

Study the ethanol SI/HCCI combustion mode transition by using the fast thermal manasement system

In this paper the ethanol homogeneous charge compression ignition （HCCI） is achieved in a modified single cylinder engine by means of a self-developed fast thermal management system （FTMS）, and the ethanol SI/HCCI operation regions are defined. It can be concluded that the thermal efficiency is higher and the NOx emission is lower in the HCCI operation region. In addition, the maximum NOx emission drops by 98%. The ethanol SI/HCCI combustion mode transition is conducted in different conditions near the SI/HCCI operation boundaries. It is likely to realize the transition by the utilization of FTMS. However, it is impossible to complete the transition within one operating cycle under current operation conditions. There are fluctuations in engine speed and brake mean effective pressure during the transition process. In order to reduce the fluctuations during the transition, the initial work concerning the effects of the spark ignition on the transition smoothness is carried out and the investigation indicates that the engine speed and brake mean effective pressure fluctuations cannot be eradicated only through spark ignition. Therefore, the control strategies combined with other factors should be further optimized.     

4,5-二氨基-2-硫脲嘧啶膜氯化钠溶液中对铜缓蚀性能的研究

通过自组装方法将4,5-二氨基-2-硫脲嘧啶(MPD)分子吸附在铜(Cu)表面,应用电化学极化、电化学阻抗谱(EIS)和拉曼光谱方法,研究其在质量分数为3.5%的氯化钠(NaCl)溶液中的缓蚀能力.在最佳装配条件下,MPD膜的最大缓蚀效率达到98.1%.拉曼光谱研究表明:MPD分子通过N_9-H_(10)和S_7-H_8吸附在Cu表面上.     

Study on cellular internalization of poly（vinyldiaminotriazine）-based hydrogen bonding type non-viral transgene vector

Previously we successfully prepared poly（vinyldiaminotriazine）（PVDT）-based non-viral vectors which complexed piasmid DNA via hydrogen bonding with adenine-thymine base pairs, in this report, surface charges and complex sizes of this system were further examined. The results showed that PVDT-based polymer could cover surface charges of DNA resulting in slightly negative or neutral complexes. It was also found that the complex sizes were governed by two events： the aggregation induced by the instability of neutral particles, and more compact complexes produced by PVDT-based polymers. In the study of cellular uptake, chlorpromazine and filipin III were used to inhibit clathrin-and caveolae-mediated endocytosis, respectively. We found that PVDT-based systems were transported into cells via a non-clathrin, non-caveolae mediated endocytosis. This special process was studied by temperature inhibition and kinetics assays. It was revealed that such a pathway was characterized by （i） a more en- ergy dependent process and （ii） a much slow transfection-effective internalization.     

Studies of new EST-SSRs derived from Gossypium barbadense

Existing cotton EST-SSR markers are mostly derived from Gossypium arboreum and Gossypium hir-sutum, but EST-SSR markers from Gossypium barbadense are scarce. One hundred and nineteen EST-SSRs were developed based on 98 unique ESTs from a cDNA library constructed in our laboratory using developing fibers from G. barbadense cv. Pima3-79. Among the SSRs, trinucleotide AAG appeared at a high frequency of 11.76%. 36 accessions (consisting of 13 diploids of the A genome, 11 diploids of the D genome and 12 allotetraploids of the AD genome) were employed to test new EST-SSRs. 76 EST-SSRs were successfully amplified, and 313 polymorphic fragments were yielded, with an average of 4.11 fragments per primer pair. The PIC ranged from 0.17 to 0.95 with an average of 0.53. Based on Jaccard’s genetic similarity coefficient, these 36 accessions were clustered into three groups. 21 EST-SSRs exhibited polymorphisms in BC1 population ((Emian22 × Pima3-79) × Emian22), 24 polymor- phic loci were generated, while 22 of the 24 polymorphic loci were integrated with our interspecific BC1 backbone genetic linkage map, and anchored in 12 chromosomes. This study effectively proved that EST-SSRs from G. barbadense are valuable for genetic diversity analysis and genetic mapping.     

Molecular identification and phylosenetic position of Cuora yunnanensis

The Yunnan box turtle （Cuora yunnanensis, Boulenger, 1906）, which has drawn much attention in conservation biology, was regarded as extinct since it was previously known only from 12 specimens collected in Yunnan, China, before 1908. Recently, live specimens have been discovered which are suggested to be C. yunnanensis. To determine whether the newly discovered specimens are really C. yunnanensis, we have established a molecular phylogeny, with a 1725-bp fragment of mitochondrial DNA, using samples from three live individuals of C. yunnanensis, together with sequence data from a museum specimen of C. yunnanensis （MNHN 1907.10） and other members of the genus Cuora. We found that the three newly discovered individuals and the old museum specimen of C. yunnanensis are very similar both in morphology and in mitochondrial DNA sequence, suggesting that the three new individuals are the very C. yunnanensis, and thus the species is not extinct. Our phylogenetic analysis also demonstrates that C. yunnanensis is not of recent hybrid origin, but rather represents a distinct evolutionary lineage.     

铜离子和聚乙二醇辛基苯基醚(OP)对钢的缓蚀协同效应

 用失重法研究了在盐酸介质中,金属铜离子(II)和非离子表面活性剂聚乙二醇辛基苯基醚(OP)对钢的缓蚀协同作用,讨论了产生缓蚀协同作用的原因.     

Mutagenesis of Ser24 of Cytochrome b559 α Subunit Affects PSII Acitivies in Chlamydomonas reinhardtii

In order to study the functions of cytochrome b559 (Cyt b559) in photosystem two (PSII) activity, mutant S24F of Chlamydomonas reinhardtii was constructed using site directed mutagenesis, in which Serine24 (Ser24) locating downstream of Histidine23 (His23) in α subunit of Cyt b559 was replaced by Phenylalanine (Phe). Physiological and biochemical analysis showed that mutant S24F could be grown photoautotrophically or photoheterotrophically. However, their growth rate was slower either on HSM or TAP medium than that of the control; Analysis of PSII activity revealed that its oxygen evolution was about 71% of wild type (WT); The Photochemical efficiency of PSII (Fv/Fm) of S24F was reduced 0.23 compared with WT; S24F was more sensitive to strong light irradiance than the wild type; Furthermore, SDS-PAGE and Western-blotting analysis indicated that the expression levels of α subunit of Cyt b559, LHCII and PsbO of S24F were a little less than those of the wild type. Overall, these data suggests that Ser24 plays a significant role in making Cyt b559 structure maintain PSII complex activity of oxygen evolution although it is not directly bound to heme group.     

Molecular simulation study of the binding mechanism of [α-PTi<Subscript>2</Subscript>W<Subscript>10</Subscript>O<Subscript>40</Subscript>]<Superscript>7−</Superscript> for its promising broad-spectrum inhibitory activity to FluV-A neuraminidase

Polyoxometalate (POM) has promising antiviral activities. It shows broad-spectrum inhibiting ability, high efficiency, and low tox-icity. Experimental assays show that titanium containing polyoxotungstates have anti-influenza-virus activity. In this paper, the bind-ing mechanisms of five isomers of di-Ti-substituted polyoxotungstate, [α-1,2-PTi₂W₁₀O₄₀]^7– (α-1,2), [α-1,6-PTi₂W₁₀O₄₀]^7– (α-1,6), [α-1,5-PTi₂W₁₀O₄₀]^7– (α-1,5), [α-1,4-PTi₂W₁₀O₄₀]^7– (α-1,4) and [α-1,11-PTi₂W₁₀O₄₀]^7– (α-1,11), to five subtypes of influenza virus A neuraminidase (FluV-A NA) were investigated in the context of aqueous solution by using molecular docking and molecular dynamics studies. The results show that the isomer α-1,2 is superior to other isomers as a potential inhibitor to neuraminidase. The positively charged arginine residues around the active site of NA could be induced by negatively charged POM to adapt themselves and could form salt bridge interactions and hydrogen bond interactions with POM. The binding free energies of POM/NA complexes range from –5.36 to –8.31 kcal mol^–1. The electrostatic interactions are found to be the driving force during the binding process of POM to NA. The conformational analysis shows that POM tends to bind primarily with N1 and N8 at the edge of the active pocket, which causes the conformational change of the pincers structure comprising residue 347 and loop 150. Whereas, the active pockets of N2, N9 and N4 are found to be more spacious, which allows POM to enter into the active pockets directly and anchor there firmly. This study shows that negatively charged ligand as POM could induce the reorganization of the active site of NA and highlights POM as a promising inhibitor to NA despite the ever increasing mutants of NA.     

Molecular characterization of H1N1 influenza A viruses from human cases in North America

Subtypes of H1N1 influenza virus can be found in humans in North America, while they are also associated with the infection of swine. Characterization of the genotypes of viral strains in human populations is important to understand the source and distribution of viral strains. Genomic and protein sequences of 10 isolates of the 2009 outbreak of influenza A （H1N1） virus in North America were obtained from GenBank database. To characterize the genotypes of these viruses, phylogenetic trees of genes PB2, PB1, PA, HA, NP, NA, NS and M were constructed by Phylip3.67 program and N-Linked glycosylation sites of HA, NA, PB2, NS1 and M2 proteins were analyzed online by NetNGlyc1.0 program. Phylogenetic analysis indicated that these isolates are virtually identical but may be recombinant viruses because their genomic fragments come from different viruses. The isolates also contain a characteristic lowly pathogenic amino acid motif at their HA cleavage sites （IPSIQSR↓GL）, and an E residue at position 627 of the PB2 protein which shows its high affinity to humans. The homologous model of M proteins showed that the viruses had obtained the ability of anti-amantadine due to the mutation at the drug-sensitive site, while sequence analysis of NA proteins indicated that the viruses are still susceptible to the neuraminidase inhibitor drug （i.e. oseltamivir and zanamivir） because no mutations have been observed. Our results strongly suggested that the viruses responsible for the 2009 outbreaks of influenza A （H1N1） virus have the ability to cross species barriers to infect human and mammalian animals based on molecular analysis. These findings may further facilitate the therapy and prevention of possible transmission from North America to other countries.