The virulence-associated protein HsvA from the fire blight pathogen Erwinia amylovora is a polyamine amidinotransferase.

Studies of virulence determinants in the bacterial phytopathogen Erwinia amylovora, the cause of devastating fire blight disease in apple and pear, have shown that HsvA, a putative amidinotransferase enzyme located in the Hrp pathogenicity island, is required for systemic infection in apple. However, the mechanism by which HsvA contributes to virulence is unclear. To investigate the role of HsvA in virulence, we carried out a series of biochemical and structural studies to characterize the amidinotransferase activity of HsvA. We found that HsvA displays a preference for linear aliphatic polyamines as the amidino acceptor substrate, especially for spermidine and putrescine (Km values of 33 µm and 3.9 mm, respectively). The three-dimensional structure, determined at 2.30 Å resolution using X-ray crystallography, revealed that the overall architecture of HsvA is similar to that of the human arginine-glycine amidinotransferase in the creatine biosynthesis pathway. The active site is located in the core of the protein at the base of a long, narrow substrate access channel. Specific amino acids near the entrance of the channel may serve as major determinants of the substrate specificity, including a glutamate residue at the rim of the channel entrance that appears to be positioned to interact with the distal primary amine in the putrescine substrate as well as the internal and distal amines in the spermidine substrate. These results suggest potential in vivo functions for HsvA as a virulence factor in fire blight and may also provide a basis for strategies to control fire blight by inhibiting HsvA activity.

Clarifying mammalian RISC assembly in vitro.

BACKGROUND: Argonaute, the core component of the RNA induced silencing complex (RISC), binds to mature miRNAs and regulates gene expression at transcriptional or post-transcriptional level. We recently reported that Argonaute 2 (Ago2) also assembles into complexes with miRNA precursors (pre-miRNAs). These Ago2:pre-miRNA complexes are catalytically active in vitro and constitute non-canonical RISCs. RESULTS: The use of pre-miRNAs as guides by Ago2 bypasses Dicer activity and complicates in vitro RISC reconstitution. In this work, we characterized Ago2:pre-miRNA complexes and identified RNAs that are targeted by miRNAs but not their corresponding pre-miRNAs. Using these target RNAs we were able to recapitulate in vitro pre-miRNA processing and canonical RISC loading, and define the minimal factors required for these processes. CONCLUSIONS: Our results indicate that Ago2 and Dicer are sufficient for processing and loading of miRNAs into RISC. Furthermore, our studies suggest that Ago2 binds primarily to the 5'- and alternatively, to the 3'-end of select pre-miRNAs.

Influence of Algae Age and Population on the Response to TiO2 Nanoparticles.

This work shows the influence of algae age (at the time of the exposure) and the initial algae population on the response of green algae Raphidocelis subcapitata to titanium dioxide nanoparticles (TiO2 NPs). The different algae age was obtained by changes in flow rate of continually stirred tank reactors prior to NP exposure. Increased algae age led to a decreased growth, variations in chlorophyll content, and an increased lipid peroxidation. Increased initial algae population (0.3-4.2 × 106 cells/mL) at a constant NP concentration (100 mg/L) caused a decline in the growth of algae. With increased initial algae population, the lipid peroxidation and chlorophyll both initially decreased and then increased. Lipid peroxidation had 4× the amount of the control at high and low initial population but, at mid-ranged initial population, had approximately half the control value. Chlorophyll a results also showed a similar trend. These results indicate that the physiological state of the algae is important for the toxicological effect of TiO2 NPs. The condition of algae and exposure regime must be considered in detail when assessing the toxicological response of NPs to algae.

The short-term toxic effects of TiO2nanoparticles toward bacteria through viability, cellular respiration, and lipid peroxidation.

To better understand the potential impacts of metal oxide nanoparticles (NPs) on Gram(+) Bacillus subtilis and Gram(-) Escherichia coli (K12) bacteria, eight different nanosized titanium dioxide (TiO2) suspensions with five different concentrations were used. Water quality parameters (pH, temperature, and ionic strength), light sources, and light intensities were also changed to achieve different environmental conditions. The photosensitive TiO2 NPs were found to be harmful to varying degrees under ambient conditions, with antibacterial activity increasing with primary particle sizes from 16 to 20 nm. The presence of light was a significant factor under most conditions tested, presumably due to its role in promoting generation of reactive oxygen species (ROS). However, bacterial growth inhibition was also observed under dark conditions and different water quality parameters, indicating that undetermined mechanisms additional to photocatalytic ROS production were responsible for toxicity. The results also indicated that nano-TiO2 particles in the absence and the presence of photoactivation induced lipid peroxidation and cellular respiration disruption.

Small molecule inhibition of RISC loading.

Argonaute proteins are the core components of the microRNP/RISC. The biogenesis and function of microRNAs and endo- and exo- siRNAs are regulated by Ago2, an Argonaute protein with RNA binding and nuclease activities. Currently, there are no in vitro assays suitable for large-scale screening of microRNP/RISC loading modulators. We describe a novel in vitro assay that is based on fluorescence polarization of TAMRA-labeled RNAs loaded to human Ago2. Using this assay, we identified potent small-molecule inhibitors of RISC loading, including aurintricarboxylic acid (IC(50) = 0.47 µM), suramin (IC(50) = 0.69 µM), and oxidopamine HCL (IC(50) = 1.61 µM). Small molecules identified by this biochemical screening assay also inhibited siRNA loading to endogenous Ago2 in cultured cells.