201 resultados para alkaline proteases


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Series of tellurite glasses were prepared by traditional melting method, the glass composition were changed and the different effects of glass modifier oxides(alkali metals and alkaline earth metal oxides) and glass intermediate (Y2O3, GeO2, Nb2O5, WO3) on the optical and spectroscopic properties of Er3+ doped tellurite glass were researched and compared. The infrared transmitting spectra, absorption spectra and fluorescence spectra were tested, the results showed that Nb2O5 and WO3 in the glass act as part of the body's role in the formation of the network, caused the reduction of transmitting range in infrared wavelength, which decrease the transmitting properties of tellurite glass. The introduction of high valence cationic ions, WO3 especially, can increase the FWHM of Er3+ for the increased polarization effect. With the decreasing of cationic field of glass modifier ions, the ion filed of the environment around Er3+ increased, subsequently, the role of ligand field polarization effect reduced, which makes the luminescence lifetime increase, and on the contrary the FWHM decrease monotonously.

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研究了碱金属氟化物对掺Yb3+氟磷玻璃的光谱性质和析晶稳定性能的影响。运用倒易法计算了Yb3+的发射截面。结果显示,LiF的引入对吸收和发射截面的提高作用较大并出现最佳引入量极值,其次为KF。碱金属氟化物的引入可提高二元体系的析晶稳定性能,使玻璃网络结构得到改善;拉曼光谱显示二元体系中引入YbF3后玻璃网络结构得到增强,而在引入碱金属氟化物的三元体系中掺杂YbF3后破坏了网络完整性,降低系统析晶稳定性能。

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研究了碱金属和碱土金属离子修饰的掺Er^3+氟磷酸盐玻璃的光谱性质,讨论了碱金属和碱土金属对铒氟磷玻璃的吸收和发射截面、荧光半高宽,Judd-Ofelt强度参数和上转换发光强度等光谱性质的影响,并与一些传统氧化物玻璃系统进行了比较.研究表明碱金属K^+和碱土金属Sr^2+掺杂高的玻璃更适宜用作光放大器基质.含12mol%K^+的氟磷玻璃展现出7.83×10^-21cm^2的高发射截面和最小的荧光上转换强度;含23mol%Sr^2+的氟磷玻璃则有7.58×10^-21cm^2的高发射截面、65nm的荧光半高

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Electrochemically active Polypyrrole (PPy) nano-fiber array device was fabricated via electrochemical deposition method using aluminum anodic oxide (AAO) membrane as template. After alkaline treatment electrochemically active PPy nano-fiber lost electrochemical activity, and became electrochemically inactive PPy. The electronic properties of PPy nano-fiber array devices were measured by means of a simple method. It was found that for an indium-tin oxide/electrochemically inactive PPy nano-fiber device, the conductivity of nano-fiber increased with the increase of voltage applied on the two terminals of nano-fiber. The electrochemical inactive PPy nano-fiber might be used as a nano-fiber switching diode. Both Au/electrochemically active PPy and Au/electrochemically inactive PPy nano-fiber devices demonstrate rectifying behavior, and might have been used for further application as nano-rectifiers. (c) 2005 Elsevier B.V. All tights reserved.

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本文对松嫩平原羊草地的群落结构及空间格局进行了分析,主要内容包含以下三个方面: 1、本文第一部分是对碱化草地植物群落分布的空间、环境因子分离。决定碱化草地植物物种在空间分布的因素可以大致分解成4个部分:(1)通过物种的生态位起作用的环境因子;(2)决定物种、群落间竞争激烈程度的空间距离因子;(3)环境和空间因子的交叉或耦合作用;(4)其它未知因子(如生物和随机因子)。本研究在1996年野外实地采样的基础上,运用DCA消势对应分析(Detrended Correspondence Analysis)对群落主要变化趋势及其与土壤环境因子之间的关系进行了分析。运用DCCA消势典范对应分析(Detrended Canonical Correspondence Analysis)对影响松嫩平原碱化草地群落结构季节动态的空间和环境因素进行了定量的分解。结果表明: 在影响群落分布的各因子中,环境因子独立约占40%,而环境一空间耦合因子占35%,空间因子独立约占3%,其它因子约占20%。在诸多因子中,土壤的盐渍化程度在整个生长季起着决定性作用,但土壤水分和氮素的作用因季节而变化。在干旱季节,植物生长的主要制约因子表现为土壤碱化度和土壤水份,而土壤氮素的作用处于次要地位。但在降雨较多、土壤湿润度较大的季节,土壤氮素的影响明显增强,成为仅次于土经度的决定性因素。 2、第二部分对东北松嫩平原碱化草地植物群落空间格局的分形性质进行了分析,分别用边长-面积指数和Korcak指数估计了斑块边界复杂性和斑块面积分布的分形维数。结果表明: 随放牧强度增加,占优势的羊草斑块的相对斑块化加剧,而斑块的边界在中度放牧时最不规则: 在水淹地,占优势的羊草斑块和次优势的碱茅斑块、獐茅斑块的斑块边界复杂性和斑块化程度比未水淹地都低,说明水降低了群落复合体内的异质性: 水淹地优势植物斑块的边长-面积指数和Korcak指数均低于次优势种,但在重度放牧地结果好相反,说明两种样地处于不同的演替阶段; 斑块边界复杂性符合同一尺度规律,在现有的面积范围以内没有尺度转换,而斑块面积分布则存在尺度转换点。对重牧地和水淹地的尺度分析表明,较小尺度上, 群落空间格局的相对斑块化程度较低,说明较小尺度上的空间格局要相对稳定一些,而较大尺度上则相反。可能原因是放牧干扰对大尺度的斑块影响更大。 3、对1989年开始围栏封育的样地10年来的空间格局变化进行时间序列分析,研究恢复演替过程中各个主要斑块类型的分布特征及动态,以及整个样地总体格局在演替系列中的变化。得出如下结果: 羊草是该区植被的优势种,其格局动态为:1989-1993年,羊草斑块总面积增加,斑块数目减少,相对斑块化指数降低,1994年后羊草的空间格局基本稳定。 羊草的斑块面积分布曲线在20平方米左右发生转折,即空间格局在此尺度发生尺度转换。不同尺度上斑块的相对斑块化指数及其在恢复演替中的变化趋势不同:小斑块的相对斑块化指数低,在1989-1993年期间增加,而后降低;大斑块的情况正好相反。说明羊草空间格局的主要变化之一是中等大小斑块的合并和数目减少,羊草斑块生长对格局的影响比斑块合并的影响要小。 獐茅斑块的格局动态为:1989-1994,斑块总面积、斑块数目、最大斑块面积增大,1995年开始降低。相对斑块化程度则先降后增。碱蓬斑块在1989-1993年间的格局动态与羊草正好相反:总面积减少,斑块数目增加,斑块面积大小的变化范围变窄,说明1993年之前,控制碱蓬斑块的主要生态学过程是其他物种的侵入,隔离原来较大的斑块,同时占据某些小斑块。1994年之后,碱蓬斑块的变化比较随机,其格局变化受到其他物种如杂草对策种虎尾草等的影响很大。 样地总体格局的变化为:1991年前,斑块种类和总斑块数目增加,斑块化程度随着增大;1993年后,斑块种类基本稳定下来;1991-1995,斑块化指数降低。在此期间,斑块数目增加,所以斑块化程度降低意味着斑块大小频率分布趋于均匀,大斑块被分裂,小斑块在长大,群落总体空间格局逐渐稳定。总体格局在三个尺度上有不同的自相似规律,这三个尺度分别为:La(a)<=1; 15.5。小尺度上相对斑块化程度低,但年际间变化较中尺度剧烈;中尺度相对斑块化程度变化较为缓,大尺度上,斑块分布一直趋于减少最大的斑块数目。总的变化趋势为,随恢复演替的深入,最大和最小的斑块数目都减少,处于中间尺度的斑块最多。

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Neurotrypsin is one of the extra-cellular serine proteases that are predominantly expressed in the brain and involved in neuronal development and function. Mutations in humans are associated with autosomal recessive non-syndromic mental retardation (MR). We studied the molecular evolution of neurotrypsin by sequencing the coding region of neurotrypsin in 11 representative non-human primate species covering great apes, lesser apes, Old World monkeys and New World monkeys. Our results demonstrated a strong functional constraint of neurotrypsin that was caused by strong purifying selection during primate evolution, an implication of an essential functional role of neurotrypsin in primate cognition. Further analysis indicated that the purifying selection was in fact acting on the SRCR domains of neurotrypsin, which mediate the binding activity of neurotrypsin to cell surface or extracellular proteins. In addition, by comparing primates with three other mammalian orders, we demonstrated that the absence of the first copy of the SRCR domain (exon 2 and 3) in mouse and rat was due to the deletion of this segment in the murine lineage. Copyright (C) 2005 S. Karger AG, Basel.

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A specific blood coagulation factor X activator was purified from the venom of Ophiophagus hannah by gel filtration and two steps of FPLC Mono-Q column ion-exchange chromatography. It showed a single protein band both in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and alkaline polyacrylamide gel electrophoresis. The mol. wt was estimated to be 62,000 in non-reducing conditions and 64,500 in reducing conditions by SDS-PAGE. The isoelectric point was found to be pH 5.6. The enzyme had weak amidolytic activities toward CBS 65-25, but it showed no activities on S-2266, S-2302, thrombin substrate S-2238, plasmin substrate S-2251 or factor Xa substrate S-2222. It had no arginine esterase activity toward substrate benzoylarginine ethylester (BAEE). The enzyme activated factor X in vitro and the effect was absolutely Ca2+ dependent, with a Hill coefficient of 6.83. It could not activate prothrombin nor had any effect on fibrinogen and thus appeared to act specifically on factor X. The procoagulant activity of the enzyme was almost completely inhibited by serine protease inhibitors like PMSF, TPCK and soybean trypsin inhibitor; partially inhibited by L-cysteine. Metal chelator EDTA did not inhibit its procoagulant activity. These results suggest that the factor X activator from O. hannah venom is a serine protease.

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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.

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Rong Gao, Yun Zhang, Qing-Xiong Meng, Wen-Hui Lee, Dong-Sheng Li, Yu-liang Xiong and Wan-Yu Wang. Characterization of three fibrinogenolytic enzymes from Chinese green tree viper (Trimeresurus stejneger ) venom. Toxicon 36, 457-467, 1998.-From the venom of Chinese green tree viper (Trimeresurus stejnegeri), three distinct fibrinogenolytic enzymes: stejnefibrase-l, stejnefibrase-2 and stejnefibrase-3, were purified by gel filtration, ion-exchange chromatography and reverse-phase high-performance chromatograghy (HPLC). SDS-PAGE analysis of those three enzymes showed that they consisted of a single polypeptide chain with mel. wt of -50 000, 31 000 and 32 000, respectively. Like TSV-PA (a specific plasminogen activator) and stejnobin (a fibrinogen-clotting enzyme) purified from the same venom, stejnfibrase-1, -2 and -3 were able to hydrolyze several chromogenic substrate. On the other hand, different from TSV-PA. and stejnobin, stejnefibrase-l, -2 and -3 did not activate plasminogen and did not possess fibrinogen-clotting activity. The three purified enzymes directly degraded fibrinogen to small fragments and rendered it unclottable by thrombin. Stejnefibrase-2 degraded preferentially BE-chain while stejnefibrase-l and -3 cleaved concomitantly Ax and B beta-chains of fibrinogen. None of these proteases degraded the gamma-chain of fibrinogen. When correlated with the loss of clottability of fibrinogen, the most active enzyme was stejnefibrase-l. The activities of the three enzymes were inhibited by phenylmethylsulfonyl fluoride (PMSF) and p-nitrophenyl-p-guanidinobenzoate (NPGB), indicating that like TSV-PA and stejnobin, they are venom serine proteases. (C) 1998 Elsevier Science Ltd. All rights reserved.

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A novel peptide inhibitor (OGTI) of serine protease with a molecular weight of 1949.8, was purified from the skin secretion of the frog, Odorrana grahami. Of the tested serine proteases, OGTI only inhibited the hydrolysis activity of trypsin on synthetic chromogenic substrate. This precursor deduced from the cDNA sequence is composed of 70 amino acid residues. The mature OGTI contains 17 amino acid residues including a six-residue loop disulfided by two half-cysteines (AVNIPFKVHFRCKAAFC). In addition to its unique six-residue loop, the overall structure and precursor of OGTI are different from those of other serine protease inhibitors. It is also one of the smallest serine protease inhibitors ever found. (C) 2008 Elsevier Masson SAS. All rights reserved.

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Snake venom Kunitz/BPTI members are good tools for understanding of structure-functional relationship between serine proteases and their inhibitors. A novel dual Kunitz/BPTI serine proteinase inhibitor named OH-TCI (trypsin- and chymotrypsin-dual inhibito

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A novel kinin-releasing and fibrin (ogen)olytic enzyme termed jerdonase was purified to homogeneity from the venom of Trimeresurus jerdonii by DEAE Sephadex A-50 anion exchange, Sephadex G-100 (superfine) gel filtration and reverse-phase high performance liquid chromatography (RP-HPLC). Jerdonase migrated as a single band with an approximate molecular weight of 55 kD under the reduced conditions and 53 kD under the non-reduced conditions. The enzyme was a glycoprotein containing 35.8% neutral carbohydrate. The N-terminal amino acid sequence of jerdonase was determined to be IIGGDECNINEHPFLVALYDA, which showed high sequence identity to other snake venom serine proteases. Jerdonase catalyzed the hydrolysis of BAEE, S-2238 and S-2302, which was inhibited by phenymethylsulfonyl fluoride (PMSF), but not affected by ethylenediaminetetraacetic acid (EDTA). Jerdonase preferentially cleaved the Aalpha-chain of human fibrinogen with lower activity towards Bbeta-chain. Moreover, the enzyme hydrolyzed bovine low-molecular-mass kininogen and releasing bradykinin. In conclusion, all results indicated that jerdonase was a multifunctional venom serine protease.

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The trypsin-like serine protease (Tryp_SPc) family is ubiquitous in animals and plays diverse roles, especially in the digestive system, in different phyla. In the mosquito, some Tryp_SPc proteases make important contributions to the digestion of the bloo

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In the present study, five homologous feeder cell lines were developed for the culture and maintenance of rhesus monkey embryonic stem cells (rESCs). Monkey ear skin fibroblasts (MESFs), monkey oviductal fibroblasts (MOFs), monkey follicular granulosa fibroblast-like (MFG) cells, monkey follicular granulosa epithelium-like (MFGE) cells, and clonally derived fibroblasts from MESF (CMESFs) were established and compared with the ability of mouse embryonic fibroblasts (MEFs) to support rESC growth. MESF, MOF, MFG, and CMESF cells, but not MFGE cells, were as good as or better than MEFs in supporting undifferentiated growth while maintaining the differentiation potential of the rESCs. In an effort to understand the unique properties of supportive feeder cells, expression levels for a number of candidate genes were examined. MOF, MESF, and MEF cells highly expressed leukemia inhibitory factor, ciliary neurotrophic factor, basic fibroblast growth factor, stem cell factor, transforming growth factor PI, bone morphogenetic protein 4, and WNT3A, whereas WNT2, WNT4, and WNT5A were downregulated, compared with MFGE cells. Additionally, all monkey feeder cell lines expressed Dkk1 and LRP6, antagonists of the WNT signaling pathway, but not WNT1, WNT8B, or Dkk2. rESCs grown on homologous feeders maintained normal karyotypes, displayed the characteristics of ESCs, including morphology, alkaline phosphatase, Oct4, the cell surface markers stage-specific embryonic antigen (SSEA)-3, SSEA-4, tumor-related antigen (TRA)-1-60, and TRA-1-81, and formed cystic embryoid bodies in vitro that included differentiated cells representing the three major germ layers. These results indicate that the four homologous feeder cell lines can be used to support the undifferentiated growth and maintenance of pluripotency in rESCs.

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Previous studies have shown that the maintenance and proliferation of undifferentiated rhesus monkey embryonic stem (rES) cells requires medium supplemented with fetal bovine serum (FBS). Due to the uncharacterized composition and variation in serum nature, the present study aimed to replace the serum-containing medium with a serum-free medium in the rES cell culture. The results showed that after the initial 48-h culture in the routinely used serum-containing medium, rES cells can grow and proliferate for a prolonged period in the serum-free medium composed of DMEM supplemented with a cocktail of BSA, IGF-1, TGF-alpha, bFGF, aFGF, estradiol, and progesterone. rES cells cultured in the serum-free medium maintained high level of alkaline phosphatase activity and OCT4 level. There was no indication of differentiation as judged by the marker gene expression of all three embryonic germ layers and trophoblast. In addition, serum-free culture would not affect the passage capacity and differentiation potential of rES cells. This work will facilitate the future study of induced differentiation of rES cells and other applications.