Manganese(II)-oxidizing Bacillus spores in Guaymas Basin hydrothermal sediments and plumes

Microbial oxidation and precipitation of manganese at deep-sea hydrothermal vents are important oceanic biogeochemical processes, yet nothing is known about the types of microorganisms or mechanisms involved. Here we report isolation of a number of diverse spore-forming Mn(II)-oxidizing Bacillus species from Guaymas Basin, a deep-sea hydrothermal vent environment in the Gulf of California, where rapid microbially mediated Mn(II) oxidation was previously observed. mnxG multicopper oxidase genes involved in Mn(II) oxidation were amplified from all Mn(II)-oxidizing Bacillus spores isolated, suggesting that a copper-mediated mechanism of Mn(II) oxidation could be important at deep-sea hydrothermal vents. Phylogenetic analysis of 16S rRNA and mnxG genes revealed that while many of the deep-sea Mn(II)-oxidizing Bacillus species are very closely related to previously recognized isolates from coastal sediments, other organisms represent novel strains and clusters. The growth and Mn(II) oxidation properties of these Bacillus species suggest that in hydrothermal sediments they are likely present as spores that are active in oxidizing Mn(II) as it emerges from the seafloor.     

Dissection of GA 20-oxidase members affecting tomato morphology by RNAi-mediated silencing

GA 20-oxidase is a key enzyme involved in gibberellin (GA) biosynthesis. In tomato, the GA 20-oxidase gene family consists of three members: GA20ox1, GA20ox2, and GA20ox3. To investigate the roles of these three genes in regulating plant growth and development, we used RNA interference technology to generate three kinds of transgenic tomato plants with suppressed expression of each three individual genes. Suppression of GA20ox1 or GA20ox2 resulted in shorter stems, a decreased length of internodes, and small dark green leaves while plants with decreased expression of GA20ox3 had no visible changes on stems and leaves. The plants of the three transgenic lines can flower and set fruits normally, but the seeds from these plants germinated slower than that from the normal plants. Decreased levels of endogenous GAs were detected in the apex of the three transgenic lines. These results demonstrate that the three GA 20-oxidase genes play different roles in the control of plan vegetative growth, but show no effects on flower and fruit development.Equal contribution authors: J. Xiao and H. Li.     

青藏高原地区根田鼠脾脏重量及迟发性超敏反应的季节性变化

动物的免疫功能不仅与外部环境条件有密切关系,而且可能在种群调节中有重要作用.为探讨青藏高原地区根田鼠免疫功能的季节性变化,我们在中国科学院海北高寒草甸生态系统定位站地区,从2006年5月至2007年4月,逐月捕获根田鼠,测定其脾脏重量及二硝基氟苯诱导的迟发性超敏反应.冷季1(2006年9～10月)个体的脾脏指数及迟发性超敏反应与暖季(2006年4～8月)个体之间无显著差异,但冷季2(2006年11月至2007年1月)和冷季3(2007月2～4月)个体脾脏指数、以及冷季3个体的迟发性超敏反应均显著低于冷季1和暖季个体,表明根田鼠免疫功能随冷季而显著降低.此外,冷季2不同年龄个体间的脾脏指数呈显著差异.我们认为,青藏高原冷季低温是影响根田鼠免疫功能季节性变化的主要因素,且冷季2不同年龄个体免疫功能的差异对根田鼠种群自动调节具有重要的作用.     

Organization of metabolic pathways in vastus lateralis of patients with chronic obstructive pulmonary disease

The objective of this study was to determine whether patients with chronic obstructive lung disease (COPD) display differences in organization of the metabolic pathways and segments involved in energy supply compared with healthy control subjects. Metabolic pathway potential, based on the measurement of the maximal activity (V(max)) of representative enzymes, was assessed in tissue extracted from the vastus lateralis in seven patients with COPD (age 67 +/- 4 yr; FEV(1)/FVC = 44 +/- 3%, where FEV(1) is forced expiratory volume in 1 s and FVC is forced vital capacity; means +/- SE) and nine healthy age-matched controls (age 68 +/- 2 yr; FEV(1)/FVC = 75 +/- 2%). Compared with control, the COPD patients displayed lower (P < 0.05) V(max) (mol.kg protein(-1).h(-1)) for cytochrome c oxidase (COX; 21.2 +/- 2.0 vs. 28.7 +/- 2.2) and 3-hydroxyacyl-CoA dehydrogenase (HADH; 2.54 +/- 0.14 vs. 3.74 +/- 0.12) but not citrate synthase (CS; 2.20 +/- 0.16 vs. 3.19 +/- 0.5). While no differences between groups were observed in V(max) for creatine phosphokinase, phosphorylase (PHOSPH), phosphofructokinase (PFK), pyruvate kinase, and lactate dehydrogenase, hexokinase (HEX) was elevated in COPD (P < 0.05). Enzyme activity ratios were higher (P < 0.05) for HEX/CS, HEX/COX, PHOSPH/HADH and PFK/HADH in COPD compared with control. It is concluded that COPD patients exhibit a reduced potential for both the electron transport system and fat oxidation and an increased potential for glucose phosphorylation while the potential for glycogenolysis and glycolysis remains normal. A comparison of enzyme ratios indicated greater potentials for glucose phosphorylation relative to the citric acid cycle and the electron transport chain and glycogenolysis and glycolysis relative to beta-oxidation.     

Fluence-Response Dynamics of the UV-Induced SOS Response in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Bacteria in nature often suffer sudden stresses, such as ultraviolet (UV) irradiation, nutrient deprivation, and chemotoxins that would cause DNA damage and DNA replication failure, which in turn trigger SOS response. According to the strength and duration of the stress, the SOS system not only repairs DNA damage but also induces mutagenesis, so as to adapt to the changing environment. The key proteins in charge of mutagenesis are UmuD and UmuD’. In this paper, we quantitatively measure the growth rate and cellular levels of proteins UmuD and UmuD’ in Escherichia coli after various fluences of UV irradiation. To compare with the experimental observations, an ordinary differential equation model is built to describe the SOS response. Considering the fact that the DNA lesions affect cellular protein production and replication origination, the simulation results fit well with the experimental data. Our results show how the fluence of UV irradiation determines the dynamics of the inducing signal and the mutation frequency of the cell. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A novel fluorescent probe for the detection of nitric oxide in vitro and in vivo

Fluorescence imaging of nitric oxide (NO) in vitro and in vivo is essential to developing our understanding of the role of nitric oxide in biology and medicine. Current probes such as diaminofluorescein depend on reactions with oxidized NO products, but not with nitric oxide directly, and this limits their applicability. Here we report the formation of an imaging probe for nitric oxide by coordinating the highly fluorescent chemical 4-methoxy-2-(1H-naphtho[2,3-d]imidazol-2-yl)phenol (MNIP) with Cu(II). The coordination compound MNIP-Cu reacts rapidly and specifically with nitric oxide to generate a product with blue fluorescence that can be used in vitro and in vivo. In the present study MNIP-Cu was used to reveal nitric oxide produced by inducible nitric oxide synthase in lipopolysaccharide (LPS)-activated macrophages (Raw 264.7 cells) and by endothelial nitric oxide synthase in endothelial cells (HUVEC). MNIP-Cu was also used to evaluate the distribution of nitric oxide synthesis in a model of acute liver injury induced by LPS and d-galactosamine in mice. The results demonstrate that MNIP-Cu can act as a novel fluorescent probe for nitric oxide and has many potential applications in biomedical research.     

Electrochemical DNA biosensor for the detection of interaction between di[azino-di(5,6-azafluorene)-kappa2-NN'] dibromicoppers and DNA

A new Cu(II) complex CuL(2)Br(2) (L = azino-di(5,6-azafluorene)-kappa(2)-NN') was synthesized, and a new method of electrochemical probe has been proposed for the determination of hepatitis B virus (HBV) based on its interaction with [CuL(2)](2+). This ligand, containing functional groups, as well as planar aromatic domains, is capable of binding to double-stranded DNA (dsDNA) more efficiently than to single-stranded DNA (ssDNA). Emphasis has been placed on the elucidation of the nature of the interaction by electrochemical techniques. The electroactive [CuL(2)](2+) could be employed as an electrochemical indicator to detect hybridization events in DNA biosensors. These biosensors have been constructed by immobilization of a probe DNA sequence from HBV onto glassy carbon electrode (GCE). After hybridization with the complementary target sequence, [CuL(2)](2+) was accumulated within the dsDNA layer. Electrochemical detection was performed by differential pulse voltammetry over the potential range. Using this approach, complementary target sequences of HBV can be quantified over the range of 1.74 x 10(-9) to 3.45 x 10(-7) M, with a detection limit of 8.32 x 10(-10) M and a linear correlation coefficient of 0.9936.In addition, this approach is capable of detecting hybridization of complementary sequences containing one or three mismatched bases.     

The spatiotemporal pattern of Src activation at lipid rafts revealed by diffusion-corrected FRET imaging

Genetically encoded biosensors based on fluorescence resonance energy transfer (FRET) have been widely applied to visualize the molecular activity in live cells with high spatiotemporal resolution. However, the rapid diffusion of biosensor proteins hinders a precise reconstruction of the actual molecular activation map. Based on fluorescence recovery after photobleaching (FRAP) experiments, we have developed a finite element (FE) method to analyze, simulate, and subtract the diffusion effect of mobile biosensors. This method has been applied to analyze the mobility of Src FRET biosensors engineered to reside at different subcompartments in live cells. The results indicate that the Src biosensor located in the cytoplasm moves 4-8 folds faster (0.93+/-0.06 microm(2)/sec) than those anchored on different compartments in plasma membrane (at lipid raft: 0.11+/-0.01 microm(2)/sec and outside: 0.18+/-0.02 microm(2)/sec). The mobility of biosensor at lipid rafts is slower than that outside of lipid rafts and is dominated by two-dimensional diffusion. When this diffusion effect was subtracted from the FRET ratio images, high Src activity at lipid rafts was observed at clustered regions proximal to the cell periphery, which remained relatively stationary upon epidermal growth factor (EGF) stimulation. This result suggests that EGF induced a Src activation at lipid rafts with well-coordinated spatiotemporal patterns. Our FE-based method also provides an integrated platform of image analysis for studying molecular mobility and reconstructing the spatiotemporal activation maps of signaling molecules in live cells.     

Mechanism of gate opening in the 20S proteasome by the proteasomal ATPases

Substrates enter the cylindrical 20S proteasome through a gated channel that is regulated by the ATPases in the 19S regulatory particle in eukaryotes or the homologous PAN ATPase complex in archaea. These ATPases contain a conserved C-terminal hydrophobic-tyrosine-X (HbYX) motif that triggers gate opening upon ATP binding. Using cryo-electron microscopy, we identified the sites in the archaeal 20S where PAN's C-terminal residues bind and determined the structures of the gate in its closed and open forms. Peptides containing the HbYX motif bind to 20S in the pockets between neighboring alpha subunits where they interact with conserved residues required for gate opening. This interaction induces a rotation in the alpha subunits and displacement of a reverse-turn loop that stabilizes the open-gate conformation. This mechanism differs from that of PA26/28, which lacks the HbYX motif and does not cause alpha subunit rotation. These findings demonstrated how the ATPases' C termini function to facilitate substrate entry.