Characterization of an ammonium transporter in the oleaginous alga Chlorella protothecoides

A suppression subtractive hybridization cDNA library was used to screen the differently expressed (up-regulated) genes in the photosynthesis–fermentation approach (PFA) of Chlorella protothecoides cultivation. A total of 87 clones were obtained and sequenced, in which 78 clones were homologous to known genes in databases. Among them, the ammonium transporter gene (CpAMT1) was characterized in detail. Quantitative real-time PCR showed that the expression of CpAMT1 was significantly induced by PFA and correlated with lipid accumulation. The up-regulation of CpAMT1 was suppressed by glutamine, while the lipid biosynthesis was also inhibited. Further analysis showed that the expression of CpAMT1 was correlated with glutamine synthetase activity, suggesting that CpAMT1, along with glutamine synthetase/glutamate synthase, may be responsible for nitrogen sensing in C. protothecoides. Together, these results imply that the ammonium transporter CpAMT1 could be the initial sensor of nitrogen deficiency and channels the carbon excess toward lipid biosynthesis.     

Characterization of two novel family 12 xyloglucanases from the thermophilic Rhizomucor miehei

Two novel glycoside hydrolase (GH) family 12 xyloglucanase genes (designated RmXEG12A and RmXEG12B) were cloned from the thermophilic fungus Rhizomucor miehei. Both genes contained open reading frames of 729 bp encoding 242 amino acids. Their deduced amino acid sequences shared 68 % identity with each other and less than 60 % with other xyloglucanases. The two genes, without the sequences for the signal peptides, were cloned and successfully expressed in Escherichia coli as active xyloglucanases, designated RmXEG12A and RmXEG12B, with similar molecular masses—25.6 and 25.9 kDa, respectively. RmXEG12A showed optimal activity at pH?6.5 and 65 °C, RmXEG12B at pH?5.0 and 60 °C. Both recombinant xyloglucanases displayed very high specific activities, 6,681.4 and 3,092.2 U?mg^?1, respectively, toward tamarind xyloglucan, but no activity toward carboxymethylcellulose, Avicel, or p-nitrophenyl derivatives. The main products of tamarind xyloglucan hydrolysis by the two xyloglucanases were XXXG, XXLG/XLXG, and XLLG (where G is an unsubstituted β-d-Glc residue, X is a xylosylated β-d-Glc residue, and L is a β-d-Glc residue substituted by xylosyl-galactose).     

Enhanced aerobic granulation and nitrogen removal by the addition of zeolite powder in a sequencing batch reactor

The aim of this study was to evaluate the impact of zeolite powders on feasibility of rapid aerobic granulation in the column-type sequencing batch reactors. After 90 days' operation, aerobic granular sludge was formed in both reactors by altering influent chemical oxygen demand/nitrogen (COD/N) ratios. R1 with zeolite powders had better removal capabilities of COD and total nitrogen than R2, which was without zeolite powders. Mixed liquor volatile suspended solid concentrations of the two reactors were 7.36 and 5.45 g/L, while sludge volume index (SVI₃₀) values were 34.9 and 47.9 mg/L, respectively. The mean diameters of aerobic granular sludge in the above two reactors were 2.5 and 1.5 mm, respectively. Both reactors achieved the largest simultaneous nitrification and denitrification (SND) efficiency at an influent COD/N ratio of 8; however, R1 exhibited more excellent SND efficiency than R2. The obtained results could provide a novel technique for rapid aerobic granulation and N removal simultaneously, especially when treating nitrogen-rich industrial wastewater.     

Production of influenza H1N1 vaccine from MDCK cells using a novel disposable packed-bed bioreactor

A process for human influenza H1N1 virus vaccine production from Madin–Darby canine kidney (MDCK) cells using a novel packed-bed bioreactor is described in this report. The mini-bioreactor was used to study the relationship between cell density and glucose consumption rate and to optimize the infection parameters of the influenza H1N1 virus (A/New Caledonia/20/99). The MDCK cell culture and virus infection were then monitored in a disposable perfusion bioreactor (AmProtein Current Perfusion Bioreactor) with proportional–integral–derivative control of pH, dissolved O₂ (DO), agitation, and temperature. During 6 days of culture, the total cell number increased from 2.0?×?10⁹ to 3.2?×?10¹⁰ cells. The maximum virus titers of 768 hemagglutinin units/100 μL and 7.8?×?10⁷ 50 % tissue culture infectious doses/mL were obtained 3 days after infection. These results demonstrate that using a disposable perfusion bioreactor for large-scale cultivation of MDCK cells, which allows for the control of DO, pH, and other conditions, is a convenient and stable platform for industrial-scale production of influenza vaccines.     

铜尾矿复垦地土壤动物群落的恢复

土壤动物在退化生态系统的恢复与重建中发挥着重要作用.为了解铜尾矿复垦地区土壤动物群落特征的变化规律,采用样带法和陷阱法对安徽铜陵市林冲铜尾矿复垦地及其外围林灌地土壤动物群落进行调查.样带法共获得土壤动物3 154只,隶属5门10纲,共计27个类群,优势类群为线虫纲、弹尾目和蜱螨目,常见类群为猛水蚤目和鞘翅目幼虫;陷阱法共获得土壤动物2 160只,主要有弹尾目、膜翅目、双尾目、蜘蛛目、倍足纲和寡毛纲等12类群.自铜尾矿复垦地中心到外围林灌地,土壤动物密度和丰度呈递增趋势;与外围林灌地相比,铜尾矿复垦地土壤动物群落多样性DG指数较低而Shannon-Wiener指数较高;与外围林灌地距离越大,与外围林灌地群落相似性系数越小;表聚性越明显.地表土壤动物从外围林灌地迁入铜尾矿复垦地的多度和丰富度随间隔距离的增加而不断减少,随调查时间的延长而增加,随外围林灌类型的变化而变化.上述结果表明,铜尾矿复垦地及其外围林灌地土壤动物群落之间具有明显的时空差异性和渐变性,这为外围林灌地土壤动物迁入铜尾矿复垦地定居提供了证据.     

Leukotriene D4 induces amyloid-β generation via CysLT1R-mediated NF-κB pathways in primary neurons

Although the pathogenesis of sporadic Alzheimer’s disease (AD) is not clearly understood, neuroinflammation has been known to play a role in the pathogenesis of AD. To investigate a functional link between the neuroinflammation and AD, the effect of leukotriene D4 (LTD4), an inflammatory lipid mediator, was studied on amyloid-β generation in vitro. Application of LTD4 to cell monolayers at concentrations up to 40 nM LTD4 caused increases in the Aβ releases. Concentrations ?40 nM LTD4 decreased neuronal viability. Application of 20 nM LTD4 caused a significant increase in Aβ generation, as assessed by ELISA or Western blotting, without significant cytotoxicity. At this concentration, exposure of neurons to LTD4 for 24 h produced maximal effect in the Aβ generation, and significant increases in the expressions of cysteinyl leukotriene 1 receptor (CysLT₁R) and activity of β- or γ-secretase with complete abrogation by the selective CysLT₁R antagonist pranlukast. Exposure of neurons to LTD4 for 1 h showed activation of NF-κB pathway, by assessing the levels of p65 or phospho-p65 in the nucleus, and either CysLT₁R antagonist pranlukast or NF-κB inhibitor PDTC prevented the nuclear translocation of p65 and the consequent phosphorylation. PDTC also inhibited LTD4-induced elevations of β- or γ-secretase activity and Aβ generation in vitro. Overall, our data show for the first time that LTD4 causes Aβ production by enhancement of β- or γ-secretase resulting from activation of CysLT₁R-mediated NF-κB signaling pathway. These findings provide a novel pathologic link between neuroinflammation and AD.     

8-Oxoguanine DNA glycosylase 1 (ogg1) maintains the function of cardiac progenitor cells during heart formation in zebrafish

Genomic damage may devastate the potential of progenitor cells and consequently impair early organogenesis. We found that ogg1, a key enzyme initiating the base-excision repair, was enriched in the embryonic heart in zebrafish. So far, little is known about DNA repair in cardiogenesis. Here, we addressed the critical role of ogg1 in cardiogenesis for the first time. ogg1 mainly expressed in the anterior lateral plate mesoderm (ALPM), the primary heart tube, and subsequently the embryonic myocardium by in situ hybridisation. Loss of ogg1 resulted in severe cardiac morphogenesis and functional abnormalities, including the short heart length, arrhythmia, decreased cardiomyocytes and nkx2.5⁺ cardiac progenitor cells. Moreover, the increased apoptosis and repressed proliferation of progenitor cells caused by ogg1 deficiency might contribute to the heart phenotype. The microarray analysis showed that the expression of genes involved in embryonic heart tube morphogenesis and heart structure were significantly changed due to the lack of ogg1. Among those, foxh1 is an important partner of ogg1 in the cardiac development in response to DNA damage. Our work demonstrates the requirement of ogg1 in cardiac progenitors and heart development in zebrafish. These findings may be helpful for understanding the aetiology of congenital cardiac deficits.     

Dual Gating Mechanism and Function of P2X7 Receptor Channels

The ATP-gated P2X7 receptor channel (P2X7R) operates as a cytolytic and apoptotic receptor but also controls sustained cellular responses, including cell growth and proliferation. However, it has not been clarified how the same receptor mediates such opposing effects. To address this question, we have combined electrophysiological, imaging, and mathematical studies using wild-type and mutant rat P2X7Rs. Activation of naïve (not previously stimulated) receptors by low agonist concentrations caused monophasic slow desensitizing currents and internalization of receptors without other changes in the cellular morphology, much like other P2XRs. In contrast, saturating agonist concentrations induced high-amplitude biphasic currents, reflecting pore dilation and causing rapid cell swelling and lysis. The existence of these two signaling patterns was accounted for using a revised Markov-state model that included, in addition to naïve and sensitized states, desensitized states. Occupancy of one or two ATP-binding sites of naïve receptors favored a slow transition to desensitized states, whereas occupancy of the third binding site favored a transition to sensitized/dilated states. Consistent with model predictions, nondilating P2X7R mutants always generated desensitizing currents. These results suggest that the level of saturation of the ligand binding sites determines the nature of the P2X7R gating and cellular actions.     

A modified metabolic model for mixed culture fermentation with energy conserving electron bifurcation reaction and metabolite transport energy

A modified metabolic model for mixed culture fermentation (MCF) is proposed with the consideration of an energy conserving electron bifurcation reaction and the transport energy of metabolites. The production of H₂ related to NADH/NAD⁺ and Fdred/Fdox is proposed to be divided in three processes in view of energy conserving electron bifurcation reaction. This assumption could fine‐tune the intracellular redox balance and regulate the distribution of metabolites. With respect to metabolite transport energy, the proton motive force is considered to be constant, while the transport rate coefficient is proposed to be proportional to the octanol–water partition coefficient. The modeling results for a glucose fermentation in a continuous stirred tank reactor show that the metabolite distribution is consistent with the literature: (1) acetate, butyrate, and ethanol are main products at acidic pH, while the production shifts to acetate and propionate at neutral and alkali pH; (2) the main products acetate, ethanol, and butyrate shift to ethanol at higher glucose concentration; (3) the changes for acetate and butyrate are following an increasing hydrogen partial pressure. The findings demonstrate that our modified model is more realistic than previous proposed model concepts. It also indicates that inclusion of an energy conserving electron bifurcation reaction and metabolite transport energy for MCF is sound in the viewpoint of biochemistry and physiology. Biotechnol. Bioeng. 2013; 110: 1884–1894. © 2013 Wiley Periodicals, Inc.     

2‐(Undecyloxy)‐ethanol is a major component of the male‐produced aggregation pheromone of Monochamus sutor

The small white‐marmorated longicorn beetle, Monochamus sutor (L.) (Coleoptera: Cerambycidae), is widely distributed throughout Europe and Asia. It is a potential vector of the pine wood nematode, Bursaphelenchus xylophilus (Steiner et Buhrer) Nickle, the causal agent of the devastating pine wilt disease. Volatiles were collected from both male and female beetles after maturation feeding. In analyses of these collections using gas chromatography (GC) coupled to mass spectrometry, a single male‐specific compound was detected and identified as 2‐(undecyloxy)‐ethanol. In analyses by GC coupled to electroantennography the only consistent responses from both female and male antennae were to this compound. Trapping tests were carried out in Spain, Sweden, and China. 2‐(Undecyloxy)‐ethanol was attractive to both male and female M. sutor beetles. A blend of the bark beetle pheromones ipsenol, ipsdienol, and 2‐methyl‐3‐buten‐2‐ol was also attractive to both sexes in Spain and Sweden, and further increased the attractiveness of the 2‐(undecyloxy)‐ethanol. The host plant volatiles α‐pinene, 3‐carene, and ethanol were weakly attractive, if at all, in all three countries and did not significantly increase the attractiveness of the blend of 2‐(undecyloxy)‐ethanol and bark beetle pheromones. 2‐(Undecyloxy)‐ethanol is thus proposed to be the major, if not only, component of the male‐produced aggregation pheromone of M. sutor, and its role is discussed. This compound has been reported as a pheromone of several other Monochamus species and is another example of the parsimony that seems to exist among the pheromones of many of the Cerambycidae. Traps baited with 2‐(undecyloxy)‐ethanol and bark beetle pheromones should be useful for monitoring and control of pine wilt disease, should M. sutor be proven to be a vector of the nematode.