The contractile basis of amoeboid movement: V. The control of gelation, solation, and contraction in extracts from dictyostelium discoideum

Motile extracts have been prepared from Dictyostelium discoideum by homogenization and differential centrifugation at 4 degrees C in a stabilization solution (60). These extracts gelled on warming to 25 degrees Celsius and contracted in response to micromolar Ca++ or a pH in excess of 7.0. Optimal gelation occurred in a solution containing 2.5 mM ethylene glycol-bis (β-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA), 2.5 mM piperazine-N-N'-bis [2-ethane sulfonic acid] (PIPES), 1 mM MgC1(2), 1 mM ATP, and 20 mM KCI at ph 7.0 (relaxation solution), while micromolar levels of Ca++ inhibited gelation. Conditions that solated the gel elicited contraction of extracts containing myosin. This was true regardless of whether chemical (micromolar Ca++, pH >7.0, cytochalasin B, elevated concentrations of KCI, MgC1(2), and sucrose) or physical (pressure, mechanical stress, and cold) means were used to induce solation. Myosin was definitely required for contraction. During Ca++-or pH-elicited contraction: (a) actin, myosin, and a 95,000-dalton polypeptide were concentrated in the contracted extract; (b) the gelation activity was recovered in the material sqeezed out the contracting extract;(c) electron microscopy demonstrated that the number of free, recognizable F-actin filaments increased; (d) the actomyosin MgATPase activity was stimulated by 4- to 10-fold. In the absense of myosin the Dictyostelium extract did not contract, while gelation proceeded normally. During solation of the gel in the absense of myosin: (a) electron microscopy demonstrated that the number of free, recognizable F- actin filaments increased; (b) solation-dependent contraction of the extract and the Ca++-stimulated MgATPase activity were reconstituted by adding puried Dictyostelium myosin. Actin purified from the Dictyostelium extract did not gel (at 2 mg/ml), while low concentrations of actin (0.7-2 mg/ml) that contained several contaminating components underwent rapid Ca++ regulated gelation. These results indicated : (a) gelation in Dictyostelium extracts involves a specific Ca++-sensitive interaction between actin and several other components; (b) myosin is an absolute requirement for contraction of the extract; (c) actin-myosin interactions capable of producing force for movement are prevented in the gel, while solation of the gel by either physical or chemical means results in the release of F-actin capable of interaction with myosin and subsequent contraction. The effectiveness of physical agents in producting contraction suggests that the regulation of contraction by the gel is structural in nature.     

Development of rRNA-targeted probes for detection of Prorocentrum micans (Dinophyceae) using whole cell in situ hybridization

Prorocentrum is a common dinoflagellate genus along the Chinese seacoast, which frequently causes harmful algal blooms. Efforts to understand and prevent blooms caused by these harmful species require the development of methods for rapid and precise identification and quantification so that an adequate early warning of harmful algal blooms may be given. Here, we report the development and application of rRNA-targeted oligonucleotide probes for fluorescence in situ hybridization (FISH) to aid in the detection of Prorocentrum micans. The hypervariable D1–D2 regions of a large subunit rDNA of a strain isolated from East China Sea identified as P. micans were first sequenced to design species-specific probes. Analysis of sequences identified as P. micans and deposited in GenBank revealed significant base differences among them and phylogenetic analyses revealed multiple clades within the taxon P. micans. Thus, it is likely that more than one taxonomic and genetically distinct entity has been identified as P. micans, if not misidentified. A series of probes were identified to one of these clades and tested for their specificity. Second, whole cell in situ hybridization procedures were established and the optimal probes were screened among the candidate probes. Next, cross-reactivity was performed to test the specificity of the probes and the detection reliability under various culture conditions, including different nutrient levels, temperatures, and light intensities. Finally, an improved protocol for natural samples was applied to the field material. The designed rRNA-targeted probe was specific, showing no cross-reactivity with other microalgae. The optimized detection protocol could be completed within 1.5 h. All target cells were speculated to be identified during all stages of their whole growth cycle under different culture conditions because the difference in fluorescence intensities throughout the experiment was not significant (p?>?0.05). The cell densities determined by FISH and light microscopy (LM) were comparable, without any significant difference (p?>?0.05) between them. In general, the established FISH probe was promising for specific, rapid, precise detection of a selected set of P. micans in natural samples and served as a good detection model for other Prorocentrum in the future.     

吉林珲春自然保护区东北虎和东北豹及其有蹄类猎物的多度与分布

珲春国家级自然保护区是东北虎(Panthera tigris altaica)、东北豹(Panthera pardus orientalis)等濒危物种在中国的核心分布区。为了探究该区域野生动物的多度水平和空间分布, 了解人类干扰情况, 我们运用相对多度指数(relative abundance index, RAI)分析了2013年4-6月设置于此的83个红外相机位点的监测数据。红外相机的总捕获天数6,060 d, 共捕获10科18种野生哺乳动物, 其中鼬科4种, 猫科动物3种, 犬科、鹿科和松鼠科各2种, 猪科、熊科、麝科、猬科和兔科各1种。研究期间共拍摄到东北虎11只个体, 东北豹13只个体。从相对多度指数来看, 东北虎的相对多度(0.84)远高于东北豹(0.48), 它们的有蹄类猎物中梅花鹿(Cervus nippon)的相对多度最高(2.18), 其次为狍(Capreolus pygargus)(1.53)和野猪(Sus scrofa)(0.92)。人类活动和放牧的相对多度水平(分别为40.64和2.76)显著高于野生动物。在空间分布上, 东北虎和梅花鹿主要在保护区的核心区分布, 且与保护区社区共管区的多度水平差异显著, 而东北豹在不同功能区之间的分布差异不显著, 狍在保护区北部的多度水平较高, 但各功能区之间差异不显著, 野猪在社区共管区的多度水平显著高于核心区。可见, 核心区频繁的人类活动和放牧活动对野生动物的保护产生了影响, 未来应加强关于人类干扰对虎、豹种群及其有蹄类猎物的影响评估。     

Mammalian PIK3C3/VPS34: the key to autophagic processing in liver and heart

PIK3C3/Vps34 is the class III PtdIns3K that is evolutionarily conserved from yeast to mammals. Its central role in mammalian autophagy has been suggested through the use of pharmacological inhibitors and the study of its binding partners. However, the precise role of PIK3C3 in mammals is not clear. Using mouse strains that allow tissue-specific deletion of PIK3C3, we have described an essential role of PIK3C3 in regulating autophagy, and liver and heart function.     

Synthesis of Partially Graphitic Ordered Mesoporous Carbons with High Surface Areas

Graphitic carbons with ordered mesostructure and high surface areas (of great interest in applications such as energy storage) have been synthesized by a direct triblock‐copolymer‐templating method. Pluronic F127 is used as a structure‐directing agent, with a low‐molecular‐weight phenolic resol as a carbon source, ferric oxide as a catalyst, and silica as an additive. Inorganic oxides can be completely eliminated from the carbon. Small‐angle XRD and N₂ sorption analysis show that the resultant carbon materials possess an ordered 2D hexagonal mesostructure, uniform bimodal mesopores (about 1.5 and 6 nm), high surface area (～1300 m²/g), and large pore volumes (～1.50 cm³/g) after low‐temperature pyrolysis (900 °C). All surface areas come from mesopores. Wide‐angle XRD patterns demonstrate that the presence of the ferric oxide catalyst and the silica additive lead to a marked enhancement of graphitic ordering in the framework. Raman spectra provide evidence of the increased content of graphitic sp² carbon structures. Transmission electron microscopy images confirm that numerous domains in the ordered mesostructures are composed of characteristic graphitic carbon nanostructures. The evolution of the graphitic structure is dependent on the temperature and the concentrations of the silica additive, and ferric oxide catalyst. Electrochemical measurements performed on this graphitic mesoporous carbon when used as an electrode material for an electrochemical double layer capacitor shows rectangular‐shaped cyclic voltammetry curves over a wide range of scan rates, even up to 200 mV/s, with a large capacitance of 155 F/g in KOH electrolyte. This method can be widely applied to the synthesis of graphitized carbon nanostructures.     

Adjuvant Effects of L. acidophilus LW1 on Immune Responses to the Foot-and-Mouth Disease Virus DNA Vaccine in Mice

The adjuvant effects of Lactobacillus acidophilus on DNA vaccination are not fully understood. It has been hypothesized that swine-derived Lactobacillus acidophilus SW1 (LASW1) could function as an immune adjuvant to enhance antigen-specific immune responses after foot-and-mouth disease (FMD) DNA vaccination in mice. To evaluate the effect of oral LASW1 on the immune response to a DNA vaccine (pRC/CMV-vp1) harboring FMD VP1 gene, anti-FMDV antibody and its isotypes, T-cell proliferation, and cytokine detection were investigated. The results showed that LASW1 was able to enhance FMDV-specific antibody levels and FMDV-neutralizing antibodies. After a booster vaccine, the anti-FMDV antibody titers and FMDV-neutralizing antibodies levels induced by pRC/CMV-vp1 were higher in mice treated with LSAW1 than in the group immunized with pRC/CMV-vp1 alone (the control). Using T-cell proliferation, the stimulation index of the LASW1 group was significantly higher in response to ConA and 146S antigen (P<0.05) than in the control group. Importantly, higher concentrations of IFN-γ and IFN-γ-producing cells were also observed in splenocytes isolated from the experimental LASW1 mice, indicating that INF-γ secretion is important to the immune response to LASW1. The results indicate that LASW1 is a promising immune adjuvant in DNA vaccination against FMD when administrated orally.     

中国圈养林麝微卫星DNA多样性研究

本文利用13对微卫星标记,对我国3个核心种源地（巴山、秦岭、川西高原）圈养林麝种群进行遗传多样性和遗传结构分析。在167份样品中共检测到142个等位基因（Na）,每个位点等位基因数介于7~16,均值为10.92,平均有效等位基因数（Ne）为6.3730,期望杂合度（He）和观测杂合度（Ho）均值分别为0.8302和0.3897。这些圈养林麝种群遗传多样性水平较高,但较低的观测杂合度表明圈养群体存在近交现象。两两群体间的Fst 值和AMOVA分析结果均表明种群之间分化程度不明显。群体遗传结构分析显示,全部样本聚为3个遗传簇（最佳K值=3）,其主体与3个地理来源相符,但种群间存在基因渗透现象。本研究中的秦岭种群遗传变异最为丰富,可以作为种质改良的基因池。