Multiplexing of multicolor bioluminescence resonance energy transfer

Bioluminescence resonance energy transfer (BRET) is increasingly being used to monitor protein-protein interactions and cellular events in cells. However, the ability to monitor multiple events simultaneously is limited by the spectral properties of the existing BRET partners. Taking advantage of newly developed Renilla luciferases and blue-shifted fluorescent proteins (FPs), we explored the possibility of creating novel BRET configurations using a single luciferase substrate and distinct FPs. Three new (to our knowledge) BRET assays leading to distinct color bioluminescence emission were generated and validated. The spectral properties of two of the FPs used (enhanced blue (EB) FP2 and mAmetrine) and the selection of appropriate detection filters permitted the concomitant detection of two independent BRET signals, without cross-interference, in the same cells after addition of a unique substrate for Renilla luciferase-II, coelentrazine-400a. Using individual BRET-based biosensors to monitor the interaction between G-protein-coupled receptors and G-protein subunits or activation of different G-proteins along with the production of a second messenger, we established the proof of principle that two new BRET configurations can be multiplexed to simultaneously monitor two dependent or independent cellular events. The development of this new multiplexed BRET configuration opens the way for concomitant monitoring of various independent biological processes in living cells.     

A model on liquid penetration into soft material with application to needle-free jet injection

A mathematical model has been presented for a high speed liquid jet penetration into soft solid by a needle-free injection system. The model consists of a cylindrical column formed by the initial jet penetration and an expansion sphere due to continuous deposition of the liquid. By solving the equations of energy conservation and volume conservation, the penetration depth and the radius of the expansion sphere can be predicted. As an example, the calculation results were presented for a typical needle-free injection system into which a silicon rubber was injected into. The calculation results were compared with the experimental results.     

Identification and functional analysis of NOL7 nuclear and nucleolar localization signals

Background  

NOL7 is a candidate tumor suppressor that localizes to a chromosomal region 6p23. This locus is frequently lost in a number of malignancies, and consistent loss of NOL7 through loss of heterozygosity and decreased mRNA and protein expression has been observed in tumors and cell lines. Reintroduction of NOL7 into cells resulted in significant suppression of in vivo tumor growth and modulation of the angiogenic phenotype. Further, NOL7 was observed to localize to the nucleus and nucleolus of cells. However, the mechanisms regulating its subcellular localization have not been elucidated.     

野化培训大熊猫采食和人为砍伐对拐棍竹无性系种群更新的影响

亚高山竹类占据着野生大熊猫(Ailuropoda melanoleuca)食物组分的99%,竹子的生命周期与大熊猫的生活史密切相关,竹子的更新和生长直接影响着大熊猫的生存与保护.为了弄清大熊猫的采食利用和人为砍伐是否促进或制约竹子的繁殖更新,应用样方法、定位观察法,连续研究了卧龙自然保护区大熊猫栖息地内野化培训大熊猫采食、人为砍伐和对照样方中拐棍竹无性系种群的更新动态.结果表明,在相同种群数量和环境条件的基础上(p>0.05),被大熊猫采食竹子的比例为67.07%,致死率29.07%;人为砍伐竹子的比例为65.67%,致死率46.68%,可见与大熊猫采食相比,砍伐更为影响拐棍竹种群的生存.从出笋数量来看,不同处理方式有利于拐棍竹无性系种群的更新(p<0.05),尤其是人为砍伐措施大大提高了竹子的出笋率,但人为砍伐样方的竹笋质量(地径和株高生长)却远低于大熊猫采食和对照样方,未能达到大熊猫觅食的选择利用标准.不同年份之间,各种处理方式下的拐棍竹出笋数量波动较大(p=0.006～0.035),并随着恢复时间的延长,逐渐趋于相似(2007,p=0 825).不同处理方式之间,拐棍竹无性系种群的年补充率,各年份均具有显著性差异(p<0.05,除2007年外),年死亡率仅2003年和2004年有明显的差异(p<0.05),2005～2007年均不显著(p>0.05).拐棍竹无性系种群的年补充率与年死亡率之间表现出年补充率﹥年死亡率的格局(p<0.05),唯有大熊猫采食样方的2004年和2005年、人为砍伐和对照样方的2005年的年死亡率略高于年补充率(p>0.05),这表明拐棍竹无性系种群对大熊猫采食和人为砍伐具有无性系整合的补偿效应.     

Identification of three protein disulfide isomerase members from Haemaphysalis longicornis tick

Three genes encoding putative protein disulfide isomerase (PDI) were isolated from the Haemaphysalis longicornis EST database and designed as HlPDI-1, HlPDI-2, and HlPDI-3. All three PDI genes contain two typical PDI active sites CXXC and encode putative 435, 499, and 488 amino acids, respectively. The recombinant proteins expressed in Escherichia coli all show PDI activities, and the activities were inhibited by a PDI-specific inhibitor, zinc bacitracin. Western blot analysis and real-time PCR revealed that three HlPDIs were present in all the developmental stages of the tick as well as in the midgut, salivary glands, ovary, hemolymph, and fatbody of adult female ticks, but the three genes were expressed at the highest level in the egg stage. HlPDI-1 is expressed primarily in the ovary and secondarily in the salivary glands. HlPDI-2 and HlPDI-3 are expressed primarily in the salivary gland, suggesting that the PDI genes are important for tick biology, especially for egg development, and that they play distinct roles in different tissues. Blood feeding induced significantly increased expression of HlPDI-1 and HlPDI-3 in both partially fed nymphs and adults. Babesia gibsoni-infected larval ticks expressed HlPDI-1 and HlPDI-3 2.0 and 4.0 times higher than uninfected normal larval ticks, respectively. The results indicate that HlPDI-1 and HlPDI-3 might be involved in tick blood feeding and Babesia parasite infection in ticks.     

柿ξ-胡萝卜素脱氢酶基因的克隆与序列分析

通过对柿ζ-胡萝卜素脱氢酶(-ζCarotene desaturase,ZDS)基因的克隆及序列分析,旨在为改良柿果实品质奠定基础。根据GenBank中其他植物ZDS基因的保守序列设计了两条PCR扩增引物,以柿果实中所提取的RNA为模板,采用RACE技术扩增出一条约为500bp的条带。经过序列分析,其长578bp,不含内含子,具有部分开放阅读框(177bp)、终止密码子(TGA)和poly(A)尾巴(11bp),共编码58个氨基酸。该序列及其所编码的氨基酸与其他植物ζ-胡萝卜素脱氢酶基因均具有较高的同源性。该基因已提交GenBank数据库,登录号为GU075728。     

The selenium-rich C-terminal domain of mouse selenoprotein P is necessary for the supply of selenium to brain and testis but not for the maintenance of whole body selenium

Selenoprotein P (Sepp1) has two domains with respect to selenium content: the N-terminal, selenium-poor domain and the C-terminal, selenium-rich domain. To assess domain function, mice with deletion of the C-terminal domain have been produced and compared with Sepp1-/- and Sepp1+/+ mice. All mice studied were males fed a semipurified diet with defined selenium content. The Sepp1 protein in the plasma of mice with the C-terminal domain deleted was determined by mass spectrometry to terminate after serine 239 and thus was designated Sepp1Delta240-361. Plasma Sepp1 and selenium concentrations as well as glutathione peroxidase activity were determined in the three types of mice. Glutathione peroxidase and Sepp1Delta240-361 accounted for over 90% of the selenium in the plasma of Sepp1Delta240-361 mice. Calculations using results from Sepp1+/+ mice revealed that Sepp1, with a potential for containing 10 selenocysteine residues, contained an average of 5 selenium atoms per molecule, indicating that shortened and/or selenium-depleted forms of the protein were present in these wild-type mice. Sepp1Delta240-361 mice had low brain and testis selenium concentrations that were similar to those in Sepp1-/- mice but they better maintained their whole body selenium. Sepp1Delta240-361 mice had depressed fertility, even when they were fed a high selenium diet, and their spermatozoa were defective and morphologically indistinguishable from those of selenium-deficient mice. Neurological dysfunction and death occurred when Sepp1Delta240-361 mice were fed selenium-deficient diet. These phenotypes were similar to those of Sepp1-/- mice but had later onset or were less severe. The results of this study demonstrate that the C terminus of Sepp1 is critical for the maintenance of selenium in brain and testis but not for the maintenance of whole body selenium.     

Angiostatin production in cultivation of recombinant Pichia pastoris fed with mixed carbon sources

A recombinant strain of Pichia pastoris with a phenotype of Mut^S was used to produce angiostatin. Due to the low methanol consumption rate of this strain, both methanol and glycerol feedings, that produced oscillation in dissolved O₂ concentration, were used during the expression phase to improve cell growth and angiostatin expression. However, enhanced cell growth led to nitrogen limitation that suppressed further production of angiostatin, but addition of ammonia allowed angiostatin concentration to reach 108 mg l^–1 after an expression period of 96 h. The ratio of consumed glycerol to methanol of 1.5:1 (w/w) in the expression phase suggested that methanol played an important role in the metabolism of carbon sources.     

Spatial Characteristics and Change for Tree Species along the North East China Transect (NECT)

Spatial characteristics of sixteen tree species were analyzed by theinformation from 287 permanent plots in 1986 and 1994 on North East ChinaTransect (NECT). Some species expanded and some retracted theirdistribution extents. Betula costata andPhellodendron amurense spread most fast toward west andeast, respectively. All tolerant tree species extended their frontiers and allintolerant tree species retracted their frontiers except Betulaplatyphylla. The distribution area decreased for all species exceptBetula costata, Juglans mandshurica,Ulmus spp. and Fraxinusrhynchophylla.The patch sizes of Pinus koraiensis, Populusdavidiana, Phellodendron amurense,Juglans mandshurica, Fraxinusmandshurica, Betula dahurica,Picea spp., Abies nephrolepis andLarixolgensis decreased, however, the patch sizes of Quercusmongolica, Betula costata, Acermono, Tilia spp., Ulmusspp., Betula platyphylla and Fraxinusrhynchophylla increased. The frequency pattern of Populusdavidiana, Betula platyphylla,Fraxinus rhynchophylla and Betuladahurica changed significantly(p< 0.05). The dominance pattern ofPopulus davidiana, Tilia spp.,Juglans mandshurica, Betulaplatyphylla, Betula dahurica andAbiesnephrolepis changed significantly(p < 0.05). The spatial correlation betweenspecies changed, such as the spatial correlation between Larixolgensis and Betula platyphylla, Acermono and Ulmus spp. increased. The possiblecause of these changes might be climate change, disturbances and habitat loss.