A single-rate context-dependent learning process underlies rapid adaptation to familiar object dynamics.

Motor learning has been extensively studied using dynamic (force-field) perturbations. These induce movement errors that result in adaptive changes to the motor commands. Several state-space models have been developed to explain how trial-by-trial errors drive the progressive adaptation observed in such studies. These models have been applied to adaptation involving novel dynamics, which typically occurs over tens to hundreds of trials, and which appears to be mediated by a dual-rate adaptation process. In contrast, when manipulating objects with familiar dynamics, subjects adapt rapidly within a few trials. Here, we apply state-space models to familiar dynamics, asking whether adaptation is mediated by a single-rate or dual-rate process. Previously, we reported a task in which subjects rotate an object with known dynamics. By presenting the object at different visual orientations, adaptation was shown to be context-specific, with limited generalization to novel orientations. Here we show that a multiple-context state-space model, with a generalization function tuned to visual object orientation, can reproduce the time-course of adaptation and de-adaptation as well as the observed context-dependent behavior. In contrast to the dual-rate process associated with novel dynamics, we show that a single-rate process mediates adaptation to familiar object dynamics. The model predicts that during exposure to the object across multiple orientations, there will be a degree of independence for adaptation and de-adaptation within each context, and that the states associated with all contexts will slowly de-adapt during exposure in one particular context. We confirm these predictions in two new experiments. Results of the current study thus highlight similarities and differences in the processes engaged during exposure to novel versus familiar dynamics. In both cases, adaptation is mediated by multiple context-specific representations. In the case of familiar object dynamics, however, the representations can be engaged based on visual context, and are updated by a single-rate process.

Adaptation to stable and unstable dynamics achieved by combined impedance control and inverse dynamics model.

This study compared adaptation in novel force fields where trajectories were initially either stable or unstable to elucidate the processes of learning novel skills and adapting to new environments. Subjects learned to move in a null force field (NF), which was unexpectedly changed either to a velocity-dependent force field (VF), which resulted in perturbed but stable hand trajectories, or a position-dependent divergent force field (DF), which resulted in unstable trajectories. With practice, subjects learned to compensate for the perturbations produced by both force fields. Adaptation was characterized by an initial increase in the activation of all muscles followed by a gradual reduction. The time course of the increase in activation was correlated with a reduction in hand-path error for the DF but not for the VF. Adaptation to the VF could have been achieved solely by formation of an inverse dynamics model and adaptation to the DF solely by impedance control. However, indices of learning, such as hand-path error, joint torque, and electromyographic activation and deactivation suggest that the CNS combined these processes during adaptation to both force fields. Our results suggest that during the early phase of learning there is an increase in endpoint stiffness that serves to reduce hand-path error and provides additional stability, regardless of whether the dynamics are stable or unstable. We suggest that the motor control system utilizes an inverse dynamics model to learn the mean dynamics and an impedance controller to assist in the formation of the inverse dynamics model and to generate needed stability.

Changing the responses of cortical neurons from sub- to suprathreshold using single spikes in vivo.

Action Potential (APs) patterns of sensory cortex neurons encode a variety of stimulus features, but how can a neuron change the feature to which it responds? Here, we show that in vivo a spike-timing-dependent plasticity (STDP) protocol-consisting of pairing a postsynaptic AP with visually driven presynaptic inputs-modifies a neurons' AP-response in a bidirectional way that depends on the relative AP-timing during pairing. Whereas postsynaptic APs repeatedly following presynaptic activation can convert subthreshold into suprathreshold responses, APs repeatedly preceding presynaptic activation reduce AP responses to visual stimulation. These changes were paralleled by restructuring of the neurons response to surround stimulus locations and membrane-potential time-course. Computational simulations could reproduce the observed subthreshold voltage changes only when presynaptic temporal jitter was included. Together this shows that STDP rules can modify output patterns of sensory neurons and the timing of single-APs plays a crucial role in sensory coding and plasticity.DOI:http://dx.doi.org/10.7554/eLife.00012.001.

The temporal evolution of feedback gains rapidly update to task demands.

Recent theoretical frameworks such as optimal feedback control suggest that feedback gains should modulate throughout a movement and be tuned to task demands. Here we measured the visuomotor feedback gain throughout the course of movements made to "near" or "far" targets in human subjects. The visuomotor gain showed a systematic modulation over the time course of the reach, with the gain peaking at the middle of the movement and dropping rapidly as the target is approached. This modulation depends primarily on the proportion of the movement remaining, rather than hand position, suggesting that the modulation is sensitive to task demands. Model-predictive control suggests that the gains should be continuously recomputed throughout a movement. To test this, we investigated whether feedback gains update when the task goal is altered during a movement, that is when the target of the reach jumped. We measured the visuomotor gain either simultaneously with the jump or 100 ms after the jump. The visuomotor gain nonspecifically reduced for all target jumps when measured synchronously with the jump. However, the visuomotor gain 100 ms later showed an appropriate modulation for the revised task goal by increasing for jumps that increased the distance to the target and reducing for jumps that decreased the distance. We conclude that visuomotor feedback gain shows a temporal evolution related to task demands and that this evolution can be flexibly recomputed within 100 ms to accommodate online modifications to task goals.

Medaka vasa is required for migration but not survival of primordial germ cells

Vasa is essential for germline development. However, the precise processes in which vasa involves vary considerably in diverse animal phyla. Here we show that vasa is required for primordial germ cell (PGC) migration in the medakafish. vasa knockdown by two morpholinos led to the PGC migration defect that was rescued by coinjection of Vasa RNA. Interestingly, Vasa knockdown did not alter the PGC number, identity, proliferation and motility even at ectopic locations. We established a cell culture system for tracing PGCs at the single cell level in vitro. In this culture system, control and morpholino-injected gastrulae produced the same PGC number and the same time course of PGC survival. importantly, vasa-depleted PGCs in culture had similar motility and locomotion to normal PGCs. Expression patterns of wt1a, sdf1b and cxcT4b in migratory tissues remained unchanged by Vasa knockdown. By chimera formation we show that PGCs from vasa-depleted blastulae failed to migrate properly in the normal environment, whereas control PGCs migrated normally in vasa-disrupted embryos. Furthermore, ectopic PGCs in vasa-depleted embryos also retained all the PGC properties examined. Taken together, medaka vasa is cell-autonomously required for PGC migration, but dispensable to PGC proliferation, motility, identity and survival. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Evaluation of estrogenic activities and mechanism of action of perfluorinated chemicals determined by vitellogenin induction in primary cultured tilapia hepatocytes

Perfluorochemicals (PFCs) are emerging persistent organic pollutants (POPs) and are widely present in the environment, wildlife and humans. Recently, reports have suggested that PFCs may have endocrine-disrupting activities. In the present study, we have developed a non-competitive enzyme-linked immunosorbent assay (ELISA) method to investigate estrogenic activities of selected PFCs using vitellogenin (VTG) induction in primary cultured hepatocytes of freshwater male tilapia (Oreochromis niloticus). Cultured hepatocytes were exposed to various concentrations of perfluorooctanyl sulfonate (PFOS), pentadecafluorooctanoic acid (PFOA), 1H, 1H, 2H, 2H-nonafluoro-1-hexanol (4:2 FTOH), 1H, 1H, 2H, 2H-perfluorooctanol (6:2 FTOH) and 1H, 1H, 2H, 2H-perfluoro-1-decanol (8:2 FTOH) for 48h, while 17 beta-estradiol (E2) and 4-nonylphenol (4-NP) were used as positive controls. A dose-dependent induction of VTG was observed in E2-, 4-NP-, PFOS-, PFOA- and 6:2 FrOH-treated cells, whereas VTG levels remained unchanged in the 4:2 FTOH and 8:2 FTOH exposure groups at the concentrations tested. The estimated 48-h EC50 values for E2,4-NP, PFOS, PFOA and 6:2 FTOH were 4.7 x 10(-7), 7.1 x 10(-6), 1.5 x 10(-5), 2.9 x 10(-5) and 2.8 x 10(-5) M, respectively. In the time-course study, significant VTG induction took place at 24 h (E2), 6 It (4-NP), 48 It (PFOS), 48 It (PFOA), 72 It (4:2 FTOH), 12 h (6:2 FTOH), 72 h (8:2 FTOH), and increased further after 96 It of exposure. Co-exposure to binary mixtures of individual PFCs and E2 for 48 It significantly inhibited E2-induced hepatocellular VTG production in a dose-dependent manner except for 4:2 FTOH. The estimated 48-h IC50 (concentration of a compound that elicits 50% inhibition of maximally E2-induced VTG) values for PFOS, PFOA, 6:2 FTOH and 8:2 FTOH were 3.1 x 10(-7), 5.1 X 10(-7), 1.1 X 10(-6) and 7.5 x 10(-7) M, respectively. In order to further investigate the estrogenic mechanism of PFCs, the hepatocytes were co-exposed to binary mixtures of individual chemicals (E2,4-NP, PFOS, PFOA and 6:2 FTOH) and the known estrogen receptor inhibitor tamoxifen for 48 h; tamoxifen significantly inhibited the ability of these chemicals to stimulate vitellogenesis. The overall results demonstrated that PFOS, PFOA and FTOHs have estrogenic activities and that exposure to a combination of E2 and PFCs produced anti-estrogenic effects. The results of the estrogen receptor inhibition assay further suggested that the estrogenic effect of PFCs may be mediated by the estrogen receptor pathway in primary cultured tilapia hepatocytes. (c) 2007 Elsevier B.V. All rights reserved.

Shrinkage of Gobiocypris rarus, Procypris rabaudi, and Sinilabeo rendahli preserved in formalin

Length measurements of preserved fishes are necessary in many types of fish surveys because logistics often do not allow for fish measurement immediately after catch. If the fixative causes significant shrinkage, then the preserved lengths cannot be directly used to indicate accurate live lengths. The objective of this study was to determine how preservation in formalin affects standard length of Gobiocypris rarus larvae (24-day-old and newly hatched), larval Procypris rabaudi (4-day-old), and larval Sinilabeo rendahli (12-day-old). Fishes were measured (to nearest 0.01 mm) and individually fixed in the appropriate formalin solution (2.5% or 5.0% formalin), then re-measured at 0.5, 1, 3, 7, 14, 30, 45 and 75 days after preservation to follow the time course of shrinkage. Most of the shrinkage occurred within the first half day after preservation. The 5.0% formalin caused a higher relative shrinkage rate than did the 2.5% solution; however, the difference was not statistically significant. In G. rarus, initial shrinkage of newly hatched larvae was higher than that of 24-day-old larvae.

Primary cultured cells as sensitive in vitro model for assessment of toxicants-comparison to hepatocytes and gill epithelia

In an effort to develop cultured cell models for toxicity screening and environmental biomonitoring, we compared primary cultured gill epithelia and hepatocytes from freshwater tilapia (Oreochromis niloticus) to assess their sensitivity to AhR agonist toxicants. Epithelia were cultured on permeable supports (terephthalate membranes, "filters") and bathed on the apical with waterborne toxicants (pseudo in vivo asymmetrical culture conditions). Hepatocytes were cultured in multi-well plates and exposed to toxicants in culture medium. Cytochrome P4501A (measured as 7-Ethoxyresorufin-O-deethylase, EROD) was selected as a biomarker. For cultured gill epithelia, the integrity of the epithelia remained unchanged on exposure to model toxicants, such as 1,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), benzo(a)pyrene B[a]P, polychlorinated biphenyl (PCB) mixture (Aroclor 1254), and polybrominated diphenyl ether (PBDE) mixture (DE71). A good concentration-dependent response of EROD activity was clearly observed in both cultured gill epithelia and hepatocytes. The time-course response of EROD was measured as early as 3 h, and was maximal after 6 h of exposure to TCDD, B [alp and Aroclor 1254. The estimated 6 h EC50 for TCDD, B [a]P, and Aroclor 1254 was 1.2x10(-9), 5.7x10(-8) and 6.6x10(-6) M. For the cultured hepatocytes, time-course study showed that a significant induction of EROD took place at 18 h, and the maximal induction of EROD was observed at 24 h after exposure. The estimated 24 It EC50 for TCDD, B[a]P, and Aroclor 1254 was 1.4x10(-9), 8.1x10(-8) and 7.3x10(-6) M. There was no induction or inhibition of EROD in DE71 exposure to both gill epithelia and hepatocytes. The results show that cultured gill epithelia more rapidly induce EROD and are slightly more sensitive than cultured hepatocytes, and could be used as a rapid and sensitive tool for screening chemicals and monitoring environmental AhR agonist toxicants. (c) 2006 Elsevier B.V. All rights reserved.

树突状细胞体外分化成熟的时程、mi/mRNA 表达谱分析和共生菌影响的研究

树突状细胞（dendritic cells, DC）作为机体天然免疫和获得性免疫反应的桥梁和枢纽，发挥着重要的启动和调控作用。随着体外诱导方法的建立和生物学技术的进步，有关DC 的基础生物学研究得到了快速的发展，在诱导方法、个体发生及基因表达和调控等方面，涌现出很多新的、未解的关键问题。同时，随着对粘膜免疫机理研究的深入，DC 在粘膜生态环境中的功能和影响，渐已成为免疫学研究前沿领域中的热点和要点。在本研究中，为了确定DC 体外分化成熟的最短时程，同时为了研究DC 分化成熟相关的基因表达调控，我们建立了快速的DC 体外诱导方法，分析了体外快速诱导 DC 的mi/mRNA 表达谱。此外，在原始分离的女性生殖道共生乳酸杆菌的基础上，以THP-1作为DC 前体细胞的细胞系模型，开展了女性生殖道共生乳酸杆菌刺激活化 THP-1 的研究，希望能够为乳酸杆菌作为生殖道粘膜免疫疫苗的应用提供理论基础。首先，采用外周血单个核细胞（peripheral blood mononuclear cells, PBMC）来源的CD14+细胞为DC 前体，经过GM-CSF 和IL-4 的刺激，1-6 天后得到未成熟DC （immature dendritic cells, iDC），并经成熟因子（TNF-α, IL-1β, IL-6 与PGE2）诱导 1-2 天后，获得成熟DC（mature dendritic cells, mDC）。经过比较和分析，明确了完全分化和成熟各2 天，即“2+2”，为DC 诱导分化的最佳和最短时程，从而证实和建立了DC 体外快速诱导的体系和方法。该方法获得的iDC 与mDC，具有与传统的“6+2” 方法获得的DC 相同的形态与表型，而且，利用该方法获得的DC 总数高于“1+1”， “1+2”与“6+2”的方法，为DC 的生物学研究提供了基础数据。我们进而采用芯片技术，对体外快速分化成熟的DC 进行了mi/mRNA 表达谱分析，确定了DC 不同分化发育阶段特征性的mi/mRNA 表达差异。结果发现，与CD14+ 单核细胞即DC 前体相比，iDC 与mDC 之间具有更加相近的mi/mRNA 表达方式。 miRNA 表达谱分析则表明，不同的miRNA 表达与DC 的不同分化和发育阶段相关。而且，位于同一基因簇内的miRNA，呈现协同表达的情况。特别值得注意的是，本研究发现了在DC 的某些发育阶段特异表达的miRNA，它们在DC 发育过程中的功能，还未得到诠释，它们在DC 某些分化阶段的特异表达，提示了DC 各分化阶段的相关性与特异性。结合mRNA 表达谱分析，我们发现miRNA 的表达与其目的基因的表达在mRNA 水平呈现负相关的特性。同时，免疫相关mRNA 与miRNA 在DC 体外不同发育阶段的表达亦呈现差异，其中，miRNA（如hsa-miR-181a, hsa-miR-223, hsa-miR-155, hsa-miR-146, hsa-miR-106a 与hsa-miR-20a 等）与mRNA（如ALM1 等）参与了特定的与免疫相关的GO（Gene Ontology）与通路（Pathway），提示这些miRNA 与mRNA 可能通过不同的方式调节控制着DC 的体外诱导过程。在有关粘膜生态环境中DC 的分化、成熟及其功能影响的研究中，我们首先通过各种乳酸杆菌鉴定方法的综合应用，确定了6 种原始分离的女性生殖道主要共生乳酸杆菌：发酵乳酸杆菌（L.Fermentum）、约氏乳酸杆菌（L.Johnsonni）、卷曲乳酸杆菌（L.Crispatus）、革氏乳酸杆菌（L.Gasseri）、詹氏乳酸杆菌（L.Jensenii）与德氏乳酸杆菌（L.Delbrueckii ）。其中，德氏乳酸杆菌（L.Delbrueckii）和发酵乳酸杆菌（L.Fermentum）具有较高的产H2O2 的能力。在此基础上，我们在与THP-1 的共同培养体系中，将乳酸杆菌对DC 前体的作用和影响进行了比较和研究。结果发现，L.Crispatus 在分离的各原始菌株中，具有最强的刺激THP-1 活化的能力，而且，在相同刺激比例下，L.Crispatus 活菌具有比死菌更强的免疫刺激能力，表现为明显上调THP-1 细胞表面标志CD40、CD80、CD86、 CD1a、CCR6 与CD324 的表达水平，同时可诱导活化THP-1 上调表达Th1 型细胞因子。通过FITC-Dextran 吞噬实验，我们发现，经过L.Crispatus 刺激的THP-1 细胞，其吞噬外来抗原的能力明显下降，但尚未检测到经过活化的THP-1 细胞刺激T 细胞增殖的能力。通过流式细胞术分析的方法，我们检测了TLR1、TLR2、TLR4 与TLR6 在不同的刺激分化阶段的表达水平，结果表明，THP-1 主要通过TLR2 与TLR6 识别女性生殖道L.Crispatus。综上所述，本研究首先通过对DC 体外分化成熟的最短时程的分析，确立了快速诱导DC 的最佳方法，进而利用芯片技术，研究了快速诱导DC 的mi/mRNA 表达谱，揭示了DC 体外分化发育过程中可能的调控途径，为进一步研究DC 的基础生物学提供了恰当的模型和具有指向性的线索。同时，通过与DC 前体THP-1 的共同培养体系，证实了生殖道共生乳酸杆菌的免疫调节作用，为以乳酸杆菌为载体的生殖道粘膜免疫疫苗的研究和应用提供了实验依据

固定化红酵母细胞合成L-苯丙氨酸研究

In this paper, bioconversion of trans-cinnamic acid(t-Ca)to L-phenylalanine (L-phe) has been investigated by using immobilized yeast cells with induced L-phe Ammonia-lyase(PAL, EC.4.3.1.5) as biocatalysts. The contents are the following. (1) Thirty strains of yeasts, including two genera (Rhodotorula, Sporobolomyces), six species (R. glutinis R. minuta,R.rubra,R.sineses,R.roseus and S.salmonicolor)were screened for their ability to converse the substrates, t-Ca and ammonia, to the product, L-phe, by using yeast cells as biocatalyst, and primary evaluation for PAL activity of the selected strains was investigated. From the results of the screening experiments, it was found that 22 strains were able to produce L-phe from t-Ca with the range of conversion yield from 2% to 67%. Studies on PAL formation time course during cultivation show that the maximum PAL activity of several different strains ranges from 2.3 to 14.4×10-3U/mg cell dry weight. The biomass of tested strains at their maximum enzyme activity is also greatly varied. (2)One of the selected strains, R. rubra as 2.166, was used for immobilized cells as biocatalysts to produce L-phe. The optimum conversion conditions and effective stablization agents were investigated. The results shown that polyacrylamide gel was chosen as a suitable matrix for immobilization of the yeast cells, and it can retain 88% of the PAL activity in the reverse direction at the following reactive conditions: [t-Ca]: 34mM. [NH4OH]: 6.OM.PH10.00, temperature: 30℃. (3) The effects of various kinds of effectors on the production of L-phe were also examined. Membrane permeabilizing agents can stimulate L-phe synthesis, but make the stability of PAL decline greatly. Polyalchoholic agents and glutamic acid were very effective for the stabilization of PAL. At the presence of glutamic acid (5%), the half life of L-phe productivity with the immobilized cells was extended to 192 hours, which was much higher than most of that having been reproted, while the half life of resting cells was only about 15 hours. (4) Use of initial velocity studies on the kinetics of enzyme-catalized reaction indicated that the apparent Km value was 13.0mM for the immobilized cells, and 4.8mM for the resting cells. Thermostability of the immobilized cells was better than the resting cells. Fluid bed bioreactor is more effective than batch bioreator in prolonging the thermostability of the biocatalysts. (5) CGA- 688 resin column chromatographic procedure was employed in the isolation and purification of L-phe, t-Ca and other substances from the reactire mixture. (6) Preparative-scale production of L-phe on a level of gram amount by immobilized cells from the culture broth of R. rubra AS2.166 allowed for the conversion yield with 30%. The characteristic physico-chemical criteria (including melting point, optical activity, elements analysis, IR, NMR) are the same with the standard L-phe. 本文报告了利用诱导的苯丙氨酸解氨酶 (PAL.EC.4.3.1.5)催化反式肉桂酸(t-Ca)氨加 成制备L-苯丙氨酸(L-phe)的研究，主要内容为：(1) 我们搜集了三十株酵母菌株，利用全细胞转化t-Ca生成L-phe的能力进行了直 接筛选，并对其PAL活性水平进行了初步评估研究。研究结果表明，其中22株酵母具有转化t-Ca生产L-phe的能力，它们包括 Rhodotorula glutinis,R.rubra, R.sineses 和Sporobolomyces roseus 的菌株，转化率在2-67%。细胞生长和PAL形成过程的研究 表明，不同菌株PAL最大活力在2.3-14.4×10-3U/mg 细胞干重，达到最大PAL活性时各株酵母的生长情况也极不一致。(2) 利用筛 选出的一株深红酵母R.rubra AS2.166 作为供试菌株，研究了细胞固定化条件下生物转化的最适条件及PAL在固定化条件下的稳定 性。结果表明以聚丙烯酰胺凝胶包埋法较为理想，能使细胞合成L-phe活力保持88%，最适t-Ca浓度为34mM，最适NH4OH浓度为6M,最 适PH10.0，最适温度45℃。(3) 多种效应物对L-phe 合成的影响研究表明：表面活性剂能刺激L-phe的合成，但使PAL稳定性下降。 多羟基化合物及Glu对PAL的稳定十分有效在有Glu存在下，能使固定化细胞合成L-phe的半寿期达192小时左右，高于大部分现已报 导的固定化结果。(4) 用初速度法研究了深红酵母AS2.166中PAL的酶促反应特征，测得固定化细胞对t-Ca的表观米氏常数Km为 13.0mM，全细胞为4.8mM，细胞固定后热稳定性提高。(5) 建立了适合低浓度分离纯化产物与底物的聚苯乙烯大孔树脂柱层析技术 ，能使L-phe与t-Ca及产物混合物中其它成分有效分开。(6) 利用固定化的R.rubra AS2.166细胞所做的制备实验能够使L-phe的产 率达到30%左右，其主要的理化指标(包括熔点、比旋光度、元素分析、IR、NMR等)与标准L-phe一致。

Biodosimetry estimate for high-LET irradiation

The purpose of this paper is to prepare for an easy and reliable biodosimeter protocol for radiation accidents involving high-linear energy transfer (LET) exposure. Human peripheral blood lymphocytes were irradiated using carbon ions (LET: 34.6 keV mu m(-1)), and the chromosome aberrations induced were analyzed using both a conventional colcemid block method and a calyculin A induced premature chromosome condensation (PCC) method. At a lower dose range (0-4 Gy), the measured dicentric (dics) and centric ring chromosomes (cRings) provided reasonable dose information. At higher doses (8 Gy), however, the frequency of dics and cRings was not suitable for dose estimation. Instead, we found that the number of Giemsa-stained drug-induced G2 prematurely condensed chromosomes (G2-PCC) can be used for dose estimation, since the total chromosome number (including fragments) was linearly correlated with radiation dose (r = 0.99). The ratio of the longest and the shortest chromosome length of the drug-induced G2-PCCs increased with radiation dose in a linear-quadratic manner (r = 0.96), which indicates that this ratio can also be used to estimate radiation doses. Obviously, it is easier to establish the dose response curve using the PCC technique than using the conventional metaphase chromosome method. It is assumed that combining the ratio of the longest and the shortest chromosome length with analysis of the total chromosome number might be a valuable tool for rapid and precise dose estimation for victims of radiation accidents.

NMR and pattern recognition studies on the acute biochemical effects of Lu(NO3)(3)

Pattern recognition methods were applied to the analysis of 600 MHz H-1 NMR spectra of urine from rats dosed with compounds that induced organ-specific damage in the liver and kidney. Male Wistar rats were separated into groups (n=4) and each was treated with one of following compounds: HgCl2, CCl4, Lu(NO3)(3) and Changle (a kind of rare earth complex mixed with La, Ce, Pr and Nd). Urine samples from the rats dosed with HgCl2, CCl4 and Lu(NO3)(3) were collected over a 24 h time course and the samples from the rats administrated with Changle were gained after 3 months. These samples were measured by 600 MHz NMR spectroscopy. Each spectrum was data-processed to provide 223 intensity-related descriptors of spectra. Urine spectral data corresponding to the time intervals, 0-8 h (HgCl2 and CCl4), 4-8 (Lu(NO3)(3)) h and 90 d (Changle) were analyzed using principal component analysis (PCA). Successful classification of the toxicity and biochemical effects of Lu(NO3)(3) was achieved.

Molecular cloning and characterisation of prophenoloxidase (ProPO) cDNA from Fenneropenaeus chinensis and its transcription injected by Vibrio anguillarum

The prophenoloxidase(ProPO) gene was cloned from haemocytes of Chinese shrimp Fenneropenaeus chinensis by Rapid Amplification Complementary DNA Ends (RACE) method. The full-length cDNA of prophenoloxidase gene consists of 3040 bp with a 2061 bp Open Reading Frame (ORF), encoding 686 amino acids. Phylogenetic analysis revealed that it belongs to insect-type invertebrate prophenoloxidase gene family. To understand ProPO reaction for pathogeny's challenge in shrimp, the expressions of ProPO in different tissues were studied by real-time PCR after challenged by Vibrio anguillarum. The results showed that the expression level of ProPO gene in haemocytes was highest among three studied tissues including haemocytes, lymphoid organ and hepatopancreas. The time-course change of ProPO mRNA levels in challenge experiment showed that ProPO mRNA transcripts had the biggest change extent in lymphoid organ.

Molecular cloning and characterization of a catalase gene from Zhikong scallop Chlamys farreri

Catalase is one of the central enzymes involved in scavenging the high level of reactive oxygen species (ROS) by degradation of hydrogen peroxide to oxygen and water. The full-length catalase cDNA of Zhikong scallop Chlamys farreri (denoted as CfCAT) was identified from hemocytes by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) approaches. The nucleotide sequence of CfCAT cDNA consisted of 3146 bp with a 5' UTR of 103 bp, an unusually long 31 UTR of 1519 bp with a canonical polyadenylation signal sequence AATAAA and a potyA tail, and an open reading frame (ORF) of 1521 bp encoding a polypeptide of 507 amino acids with predicted molecular weight of 57.5 kDa. The deduced amino acid sequence of CfCAT has significant homology to catalases from animals, plants and bacteria. Several highly conserved motifs including the proximal heme-ligand signature sequence RLFSYNDTH, the proximal active site signature FNRERIPERVVHAKGGGA, and the three catalytic amino acid residues of His(72), Asn(145) and Tyr(355) were identified in the deduced amino acid sequence of CfCAT. The CfCAT was demonstrated to be a peroxisomal glycoprotein with two potential glycosylation sites and a peroxisome targeting signal of ANL that was consistent with human, mouse and rat catalases. The time-course expression of CfCAT in hemocytes was measured by quantitative real-time PCR. The expression of CfCAT increased gradually and reached the highest point at 12 h post-Vibrio infection, then recovered to the original level at 24 h. All these results indicate that CfCAT, a constitutive and inducible protein, is a member of the catalase family and is involved in the process against ROS in scallop. (c) 2007 Published by Elsevier Ltd.

The first molluscan TCTP in Venerupis philippinarum: Molecular cloning and expression analysis

Translationally controlled tumor protein (TCTP) is one of the abundant and ubiquitously expressed proteins in metazoans In the present study, the first molluscan TCTP (denoted as VpTCTP) was identified from Venerupis philippinarum haemocytes by EST and RACE approaches The full-length cDNA of VpTCTP consisted of 1148 nucleotides with an open-reading frame of 555 bp encoding 184 amino acids The deduced amino acid sequence of VpTCTP shared high similarity with TCTPs from other species, indicating that VpTCTP should be a new member of TCTP family Several highly conserved motifs, including 5'terminal ologopyrimidine (5'TOP) starting sequence and rich AU and AUUT elements in 3'UTR, were also identified in VpTCTP The tissue and temporal expression of VpTCTP after Vi boo anguillarum challenge was recorded by quantitative real-time RT-PCR. VpTCTP transcript could be detected in all examined tissues with the highest expression level in haemocytes and the lowest in hepatopancreas Concerning the time-course expression in haemocytes, the relative expression of VpTCTP mRNA was down-regulated sharply from 6 h to 12 h post-infection. Then, the expression level was obviously up-regulated and reached 3.4-fold to that in the control group at 48 h post challenge As time progressed, the expression of VpTCTP recovered to the original level at 96 h. All these results indicated that VpTCTP was an acute-phase protein involved in the Immune response of V philippinarum (C) 2010 Elsevier Ltd. All rights reserved.