Recombinant expression of rt-PA gene (encoding Reteplase) in gametophytes of the seaweed Laminaria japonica (Laminariales, Phaeophyta)

The life cycle of seaweed Laminaria japonica involves a generation alternation between diploid sporophyte and haploid gametophte. The expression of foreign genes in sporophte has been proved. In this research, the recombinant expression in gametophyte was investigated by particle bombardment with the rt-PA gene encoding the recombinant human tissue-type plasminogen activator (Reteplase), which is a thrombolytic agent for acute myocardial infarction (AMI). Transgenic gametophytes were selected by their resistance to herbicide phosphiothricin (PPT), and proliferated in an established bubble column photo-bioreactor. According to the results from quantitative ELISA, Southern blotting, and fibrin agarose plate assay (FAPA) for bioactivity, it was showed that the rt-PA gene had been integrated into the genome of gametophytes of L. japonica, and the expression product showed the expected bioactivity, implying the proper post-transcript modification in haploid gametophyte.

Structural, morphological and optical properties of ZnO thin films grown on gamma-LiAlO2 substrate by pulsed laser deposition

ZnO thin films were deposited on the substrates of (100) gamma-LiAlO2 at 400, 550 and 700 degrees C using pulsed laser deposition (PLD) with the fixed oxygen pressure of 20 Pa, respectively. When the substrate temperature is 400 degrees C, the grain size of the film is less than 1 mu m observed by Leitz microscope and measured by X-ray diffraction (XRD). As the substrate temperature increases to 550 degrees C, highly-preferred c-orientation and high-quality ZnO film can be attained. While the substrate temperature rises to 700 degrees C, more defects appears on the surface of film and the ZnO films become polycrystalline again possibly because more Li of the substrate diffused into the ZnO film at high substrate temperature. The photoluminescence (PL) spectra of ZnO films at room temperature show the blue emission peaks centered at 430 nm. We suggest that the blue emission corresponds to the electron transition from the level of interstitial Zn to the valence band. Meanwhile, the films grown on gamma-LiAlO2 (LAO) exhibit green emission centered at 540 nm, which seemed to be ascribed to excess zinc and/or oxygen vacancy in the ZnO films caused by diffusion of Li. from the substrates into the films during the deposition.

Characterization of a fibrinogen-clotting enzyme from Trimeresurus stejnegeri venom, and comparative study with other venom proteases

Trimeresurus stejnegeri venom, which contains TSV-PA (a specific plasminogen activator sharing 60-70% sequence homology with venom fibrinogen-clotting enzymes), also possesses fibrinogen-clotting activity in vitro. A fibrinogen-clotting enzyme (stejnobin) has been purified to homogeneity by gel filtration and ion-exchange chromatography on a Mono-Q column. It is a single-chain glycoprotein with a mol. wt of 44,000. The NH2-terminal amino acid sequence of stejnobin shows great homology with venom fibrinogen-clotting enzymes and TSV-PA. Like TSV-PA, stejnobin was able to hydrolyse several chromogenic substrates. Comparative study of substrate specificities of stejnobin and other venom proteases purified in our laboratory was carried out on five chromogenic substrates. Stejnobin clotted human fibrinogen with a specific activity of 122 NIH thrombin-equivalent units/mg protein. However, stejnobin did not act on other blood coagulation factors, such as factor X, prothrombin and plasminogen. Diisopropyl fluorophosphate and phenylmethanesulfonyl fluoride inhibited its activity, whereas ethylenediamine tetracetic acid had no effect on it, indicating that it is a serine protease. Although stejnobin showed strong immunological cross-reaction with polyclonal antibodies raised against TSV-PA, it was interesting to observe that, unlike the case of TSV-PA, these antibodies did not inhibit the amidolytic and fibrinogen-clotting activities of stejnobin. (C) 1998 Elsevier Science Ltd. All rights reserved.

THE POSSIBLE INVOLVEMENT OF TISSUE-TYPE PLASMINOGEN-ACTIVATOR IN LUTEOLYSIS OF RHESUS-MONKEY

Changes of plasminogen activators (PA) during different stages of development of the corpus luteum, and their possible physiological role in luteolysis were studied in rhesus monkeys. It was demonstrated for the first time that monkey corpus luteal cells not only produce PA, but that the function of the corpus luteum is also closely related to the activity of this enzyme system. Generally, the life span for a corpus luteum in monkey is approximately 14-16 days, its demise beginning thereafter. In the present study, we found that urokinase in the corpus luteum is higher on day 5 and day 10 after human chorionic gonadotrophin injection, while the tissue type (t) PA is mainly produced on day 13 when luteolysis may take place. Progesterone production remained high on day 5 and day 10 and decreased dramatically from day 13, indicating the important role of tPA but not urokinase (u) PA in suppressing luteal function. When purified tPA (but not uPA) monoclonal antibody was added to luteal cell culture to neutralize endogenously produced tPA activity, progesterone production in the cells was increased significantly. Interestingly, prolactin alone was capable of increasing PA production by luteal cells; prolactin together with luteinizing hormone, however, had a synergistic luteotrophic effect.

Molecular analysis of silver crucian carp (Carassius auratus gibelio Bloch) clones by SCAR markers

Random amplified polymorphic DNA (RAPD) molecular markers specific for one, two or three clones have been identified from five gynogenetic clones of silver crucian carp (Carassius auratus gibelio Bloch) using RAPD markers developed earlier. In this study, three RAPD markers (RA1-PA, RA2-EF and RA4-D) produced by Opj-1, and two RAPD DNA fragments (RA3-PAD and RA5-D) produced by Opj-7, were selected for molecular cloning and sequencing. Sequence data indicated that there were identical 801-bp nucleotide sequences in the shared marker RA1-PA cloned respectively from clones P and A, and the shared marker RA2-EF (which was cloned from clones E and F), were also of identical 958-by nucleotide sequences. The nucleotide sequences of the shared marker RA3-PAD fragments were also similar for 1181 by among clones P, A and D. The specific fragment RA4-D was composed of 628 bp, and the fragment RA5-D from clone D contained 385 nucleotides. According to the nucleotide sequences, we designed and synthesized five pairs of sequence characterized amplified regions (SCAR) primers to identify the specific fragments in these gynogenetic clones of silver crucian carp. Only individuals from clones P and A amplified a specific band using a pair of SCI-PA primers synthesized according to the marker RA1-PA sequences, whereas no products were detected in individuals from clones D, E and F. The PCR products amplified using SC2-EF and SC3-PAD primers were as expected. Furthermore, the pair of SC4-D primers amplified specific bands only in individuals from clone D, although weak bands could be produced in all individuals of the five clones when lower annealing temperatures were used. However, an additional pair of SC5-D primers designed from the RA5-D marker sequences could amplify a DNA band in individuals from clones P, A and D, and the same weak band was produced in clone E, whereas no products were detected in individuals from clone F. Searches in GenBank revealed that the 385-bp DNA fragment from RA5-D was homologous to the 5' end of gonadotropin I beta subunit 2 gene and growth hormone gene. No homologous sequences were found for other markers in GenBank. The SCAR markers identified in this study will offer a powerful, easy, and rapid method for discrimination of different clones and for genetic analyses that examine their origins and unique reproductive modes in crucian carp. Furthermore, they will likely benefit future selective breeding programs as reliable and reproducible molecular markers. (C) 2001 Elsevier Science B.V. All rights reserved.

Effect of Nozzle Temperature on the Performance of a 1 kW H2-N2 Arcjet Thruster

A 1 kW-class arcjet thruster was ¯red in a vacuum chamber at a pressure of 18 Pa. A gas mixture of H2 : N2 = 2.8 : 1.5 in volume at a total °ow rate of 4.3 slm was used as the propellant with an input power ¯xed at 860 W. The time-dependent thrust, nozzle temperature and inlet pressure of the propellant were measured simultaneously. Results showed that with the increase in nozzle temperature the thrust decreased and various losses increased. The physical mechanisms involved in these effects are discussed.

The influence of heat treatment on structures of the aggregated state of polyamide-1010

The structural parameters of the aggregated state of polyamide (PA)-1010 annealed at various temperatures were computed by means of the desmearing intensity from Smalt Angle X-ray Scattering (SAXS) measurements and by using the concept of the distance distribution function. The results indicated that the structural parameters of the aggregated state were strongly dependent upon heat treatment conditions and the maximum values of the structural parameters were obtained for the samples annealed at T = 175 degrees C. The particle size Z annealed at different temperature was ranged between 8.1-12.8nm, and the values of the distance distribution function P-max(Z) were obtained when Z = 4.0-6.8 nm. Using one dimension electron density correlation function (1D EDCF) method long period (L) and thickness of the lamellar (d(0)) were estimated and were found to increase with the increase of the degree of crystallinity.

第十四章 WIA-PA:用于过程自动化的工业无线网络系统结构与通信规范

概要介绍工业无线网络的标准化进程,详细介绍了用于过程自动化的工业无线网络WIA规范的网络构成、拓扑结构、协议体系和关键技术,并将WIA-PA与WirelessHART、ISASP100两大工业无线标准进行了比较分析。

PROFIBUS PA通信协议软件开发

随着控制技术、计算机技术、通信技术的飞速发展，现场总线技术正逐渐取代集散控制技术。PROFIBUS现场总线技术作为应用最为广泛的现场总线技术之一，截止至2008年8月，其安装节点已经超过2500万个，市场份额超过20%。 本文首先深入研究了现场总线国际标准IEC61158-Type3(PROFIBUS标准)，分析了PFOFIBUS 现场总线的层次结构模型、各层功能及层间通信关系，然后介绍了PROFIBUS PA的功能块模型，包括块的构成、分类、参数详解，以及把功能块模型映射到模块化DP从站设备的规则等。然后本文采用uC/OS-II嵌入式实时操作系统作为软件平台，应用瑞萨公司M16C/62P系列MCU与中科院沈阳自动化研究所自主研发的FBC0409现场总线通信控制器作为硬件平台，开发了支持PROFIBUS DPV0和DP V1的通信协议栈，并根据PA应用行规(V3.01)编写了相应的功能块程序，并且进行了验证。 本文给出了PROFIBUS DPV0、DPV1实现过程中的主要数据和功能定义，介绍了各层任务的创建初始化，处理过程，绘制了相关的软件流程图。本文给出了编写物理块、功能块、变换块和设备管理的参数定义和读写函数的方法。

A NOVEL PLASMINOGEN-ACTIVATOR FROM SNAKE-VENOM - PURIFICATION, CHARACTERIZATION, AND MOLECULAR-CLONING

A novel plasminogen activator from Trimeresurus stejnegeri venom (TSV-PA) has been identified and purified to homogeneity. It is a single chain glycoprotein with an apparent molecular weight of 33,000 and an isoelectric point of pH 5.2. It specifically activates plasminogen through an enzymatic reaction. The activation of human native GIu-plasminogen by TSV-PA is due to a single cleavage of the molecule at the peptide bond Arg(561)-Val-(562). Purified TSV-PA, which catalyzes the hydrolysis of several tripeptide p-nitroanilide substrates, does not activate nor degrade prothrombin, factor X, or protein C and does not clot fibrinogen nor show fibrino(geno)lytic activity in the absence of plasminogen. The activity of TSV-PA was readily inhibited by phenylmethanesulfonyl fluoride and by p-nitrophenyl-p-guanidinobenzoate. Oligonucleotide primers designed on the basis of the N-terminal and the internal peptide sequences of TSV-PA were used for the amplification of cDNA fragments by polymerase chain reaction. This allowed the cloning of a full-length cDNA encoding TSV-PA from a cDNA library prepared from the venom glands. The deduced complete amino acid sequence of TSV-PA indicates that the mature TSV-PA protein is composed of 234 amino acids and contains a single potential N-gIycosylation site at Asn(1G1). The sequence of TSV-PA exhibits a high degree of sequence identity with other snake venom proteases: 66% with the protein C activator from Aghistrodon contortrix contortrix venom, 63% with batroxobin, and 60% with the factor V activator from Russell's viper venom. On the other hand, TSV-PA shows only 21-23% sequence similarity with the catalytic domains of u-PA and t-PA. Furthermore, TSV-PA lacks the sequence site that has been demonstrated to be responsible for the interaction of t-PA (KHRR) and u-PA (RRHR) with plasminogen activator inhibitor type 1.

Trimeresurus stejnegeri snake venom plasminogen activator - Site-directed mutagenesis and molecular modeling

The specific plasminogen activator from Trimeresurus stejnegeri venom (TSV-PA) is a serine proteinase presenting 23% sequence identity with the proteinase domain of tissue type plasminogen activator, and 63% with batroxobin, a fibrinogen clotting enzyme from Bothrops atrox venom that does not activate plasminogen. TSV-PA contains six disulfide bonds and has been successfully overexpressed in Escherichia coli (Zhang, Y., Wisner, A., Xiong, Y. L,, and Bon, C, (1995) J. Biol. Chem. 270, 10246-10255), To identify the functional domains of TSV-PA, we focused on three short peptide fragments of TSV-PA showing important sequence differences with batroxobin and other venom serine proteinases. Molecular modeling shows that these sequences are located in surface loop regions, one of which is next to the catalytic site, When these sequences were replaced in TSV-PA by the equivalent batroxobin residues none generated either fibrinogen-clotting or direct fibrinogenolytic activity, Two of the replacements had little effect in general and are not critical to the specificity of TSV-PA for plasminogen. Nevertheless, the third replacement, produced by the conversion of the sequence DDE 96a-98 to NVI, significantly increased the K-m for some tripeptide chromogenic substrates and resulted in undetectable plasminogen activation, indicating the key role that the sequence plays in substrate recognition by the enzyme.

WIA网络与MODBUS网络互联设计与实现

本文首先介绍了WIA技术和Modbus协议的基本原理和结构,然后详细介绍了互联过程中使用的两种方式:透明传输方式和协议转换方式。分析了两种方式的适用范围和改造难度,并给出了实现案例。

磁层-电离层耦合现象的分析研究

The magnetosphere-ionosphere coupling is mainly manifested by the trans- porting processes of energy into the ionosphere , the energy is carried by solar wind and firstly accumulate at the magnetosphere, and the coupling processes also significantly include the interaction between the magnetosphere and ionosphere for mass and energy. At the quiet condition, energy is delivered by the large-scale convection of the geomagnetic field; the huge energy from solar wind bulk will be injected into and consumed at the near magnetosphere and ionosphere by the geomagnetic storm and substorm activities. Aurorae and FACs (Field-aligned currents) are the important phenomena in the coupling processes. In the present work, firstly, we analyze the activity characteristics of auroral precipitating particle, secondly, we study the distribution characters of large-scale field aligned currents (LS FACs) at storm-time using the observations from different satellites at different altitudes. Finally, we investigate the evolution of the geomagnetic field configuration at the nightside sector on the onset of the expansion phase in a substorm event, the substorm event happened at 0430UT to 0630UT on 8th Nov. 2004. The main results as follows： At the first, the data of the estimated power input (EPI) of auroral particles from NOAA/POES (Polar orbiting environmental satellite) for some 30 years have been analyzed. The variation tendencies of the EPI generally coincide with aa, AE and Dst indices. The annual variation of EPI shows equinox peaks and an asymmetric-activity with a higher peak in the winter-hemisphere than in the summer-hemisphere. The diurnal UT variations are different from north and south hemisphere: for north hemisphere, the peak appears at 1200UT, and the relative deviation is 22% to the daily average of the north hemisphere. For south hemisphere, the maximal deviation is 22% at 2000UT. So the diurnal variation of EPI is more dominant than the annual variation which maximal deviation is 3% to 12% for different seasons. Studies on correlations of the hourly average of EPI, Pa, with AE and Dst indices show a correlation coefficient r=0.74 of Pa and AE, and r=-0.55 of Pa and Dst. The hourly EPIs for north and south polar regions, NPa and SPa, show a north-south asymmetry with a higher correlation of SPa and AE (or Dst). Time delays of EPI with respect to magnetic indices are examined, the maximum correlation coefficient of Pa with AE (r=0.78) occurs when the time delay =0, suggesting a synchronous activity of auroral electrojet and auroral precipitating particles, while =1-2h, the correlation coefficient of Pa with Dst is maximum (r=0.57), suggesting that the activity of auroral particle precipitating may influence the ring current on some extent. Sencondly, we use the high-resolution magnetic field vector data of the CHAMP satellite to investigate the distribution of large-scale FACs during the great magnetic storm on 7th to 8th Nov. 2004. The results show that, whether in the northern or southern hemisphere, the number and density of large-scale FACs during the main-phase are more and bigger than these during the recover-phase, and the number of large-scale FACs in morning sector obviously is more than that in afternoon sector. In terms of the magnetic indices, we find that large-scale FACs in morning sector significantly affected by the substorm activities, while in afternoon sector the large-scale FACs mainly indicate the fluctuations of the ring-current in storm time. Accordingly to the former studies, similarly, we find that in the morning sector, the scale of the large-scale FACs move to the high-latitude region, and in the afternoon sector, large-scale FACs distinctly expand to the low-latitude region. During the time periods that the NOAA/POES auroral precipitating particle power data temporally correspond to the large-scale FACs, the more the power of auroral particle is, the more and bigger the number and density of FACs are. At the same time, we use the magnetic field vector data of POLAR obtain a good form of region 1, region 2, and three pieces of cusp FACs during a single transit at 1930UT-2006UT on 07th. And the characteristics of simultaneous electric field and energy particles observations on Polar are coincide with the five FACs pieces. Finally, by means of the observation of Cluster 4 and Goes 10、 Goes 12, we analyze the evolution process of the change of the magnetic field configuration at night sector at the expansion phase of a substorm event which happened during 0430UT to 0630UT on 8th Nov. 2004, we find that the times of the beginning of the polarizations of magnetic field are observed from Goes 10 to Goes 12 then to Cluster 4. So, at the synchronous orbit ( 6.6 RE) to 10RE distance scale of the neutral sheet, the current disruption spread tailward. Simultaneously, the strengthen of the FACs deduced from these satellites’ magnetic field observations are almost consistent with the times of polarizations, as well as the high energy particles injection and the electric field dominant variation. The onset times determined by the magnetic field polarizations from these satellites are all ahead of the onset time that confirmed from the auroral electrojet indices. So, these characters of different observations can be used as the criterions to determine the onset time for the substorms of such type as we studied.