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.     

CD3-specific antibody-induced immune tolerance involves transforming growth factor-beta from phagocytes digesting apoptotic T cells

Intact CD3-specific antibody transiently depletes large numbers of T cells and subsequently induces long-term immune tolerance. The underlying mechanisms for the systemic tolerance, however, remain unclear. We show here that treatment of normal mice with intact antibody to CD3 increases systemic transforming growth factor-beta (TGF-beta) produced by phagocytes exposed to apoptotic T cells. Among the phagocytes, macrophages and immature dendritic cells (iDCs) secrete TGF-beta upon ingestion of apoptotic T cells, which induces CD4+Foxp3+ regulatory T cells in culture and contributes to immune tolerance mediated by CD3-specific antibody in vivo. In accordance with these results, depletion of macrophages and iDCs not only abrogates CD3-specific antibody-mediated prevention of myelin oligodendrocyte glycoprotein-induced acute experimental autoimmune encephalomyelitis (EAE), but also reverses the therapeutic effects of antibody to CD3 on established disease in a model of relapsing-remitting EAE. Thus, CD3-specific antibody-induced immune tolerance is associated with TGF-beta production in phagocytes involved in clearing apoptotic T cells, which suggests that apoptosis is linked to active suppression in immune tolerance.     

Superoxide flashes in single mitochondria

In quiescent cells, mitochondria are the primary source of reactive oxygen species (ROS), which are generated by leakiness of the electron transport chain (ETC). High levels of ROS can trigger cell death, whereas lower levels drive diverse and important cellular functions. We show here by employing a newly developed mitochondrial matrix-targeted superoxide indicator, that individual mitochondria undergo spontaneous bursts of superoxide generation, termed "superoxide flashes." Superoxide flashes occur randomly in space and time, exhibit all-or-none properties, and provide a vital source of superoxide production across many different cell types. Individual flashes are triggered by transient openings of the mitochondrial permeability transition pore stimulating superoxide production by the ETC. Furthermore, we observe a flurry of superoxide flash activity during reoxygenation of cardiomyocytes after hypoxia, which is inhibited by the cardioprotective compound adenosine. We propose that superoxide flashes could serve as a valuable biomarker for a wide variety of oxidative stress-related diseases.     

A novel cold-adapted phospholipase A(1) from Serratia sp. xjF1: Gene cloning, expression and characterization

The gene encoding a cold-adapted phospholipase A(1) (PLA(1)) from a psychrotrophic, glacier soil bacterium Serratia sp. xjF1 was cloned by two-step PCR (general PCR and TAIL-PCR). The full-length fragment comprised two open reading frames plA and plS. The gene product of plA encoding 320 amino acids with a molecular weight of 33.8kDa was identified as a phospholipase A(1). Its amino acid sequence exhibited the highest homology to PLA(1) of Serratia marcescens (71%). plS encoded a protein of 251 amino acids, which showed no enzymatic activity. The result of plA expression in Escherichia coli indicated that plS might improve the efficient expression of PLA(1) in E. coli. Furthermore, PLA(1) was functionally expressed in Pichia pastoris, yielding 41.8U/mL in a 3.7L fermentor. The purified recombinant phospholipase A(1) (rPLA(1)) had features typical of cold-adapted enzymes with a temperature optimum of 35°C and a maximum activity of 70% at 10°C. The rate of catalysis was optimal at pH 9.0 and the enzyme could be slightly activated by Ca(2+). This is the first report on gene isolation and expression of cold-adapted PLA(1).     

The crystal structure of human WD40 repeat-containing peptidylprolyl isomerase (PPWD1)

Cyclophilins comprise one of the three classes of peptidylprolyl isomerases found in all eukaryotic and prokaryotic organisms, as well as viruses. Many of the 17 annotated human cyclophilins contain the catalytic domain in tandem with other domains, and many of the specific functions of a particular cyclophilin or its associated domains remain unknown. The structure of the isomerase domain from a spliceosome-associated cyclophilin, PPWD1 (peptidylprolyl isomerase containing WD40 repeat), has been solved to 1.65 A. In the crystal, the N-terminus of one isomerase domain is bound in the active site of a neighboring isomerase molecule in a manner analogous to substrate. NMR solution studies show that this sequence binds to the active site of the cyclophilin, but cannot be turned over by the enzyme. A pseudo-substrate immediately N-terminal to the cyclophilin domain in PPWD1 could have wider implications for the function of this cyclophilin in the spliceosome, where it is located in human cells.