Characterization of an<Emphasis Type="Italic">Eco</Emphasis>R 124I restriction-modification enzyme produced from a deleted form of the DNA-binding subunit,which results in a novel DNA specificity

We purified and characterized both the methyltransferase and the endonuclease containing the HsdS delta 50 subunit (type I restriction endonucleases are composed of three subunits--HsdR required for restriction, HsdM required for methylation and HsdS responsible for DNA recognition) produced from the deletion mutation hsdS delta 50 of the type IC R-M system EcoR 124I; this mutant subunit lacks the C-terminal 163 residues of HsdS and produces a novel DNA specificity. Analysis of the purified HsDs delta 50 subunit indicated that during purification it is subject to partial proteolysis resulting in removal of approximately 1 kDa of the polypeptide at the C-terminus. This proteolysis prevented the purification of further deletion mutants, which were determined as having a novel DNA specificity in vivo. After biochemical characterization of the mutant DNA methyltransferase (MTase) and restriction endonuclease we found only one difference comparing with the wild-type enzyme--a significantly higher binding affinity of the MTase for the two substrates of hemimethylated and fully methylated DNA. This indicates that MTase delta 50 is less able to discriminate the methylation status of the DNA during its binding. However, the mutant MTase still preferred hemimethylated DNA as the substrate for methylation. We fused the hsdM and hsdS delta 50 genes and showed that the HsdM-HsdS delta 50 fusion protein is capable of dimerization confirming the model for assembly of this deletion mutant.     

Iron Limitation Modulates Ocean Acidification Effects on Southern Ocean Phytoplankton Communities

The potential interactive effects of iron (Fe) limitation and Ocean Acidification in the Southern Ocean (SO) are largely unknown. Here we present results of a long-term incubation experiment investigating the combined effects of CO₂ and Fe availability on natural phytoplankton assemblages from the Weddell Sea, Antarctica. Active Chl a fluorescence measurements revealed that we successfully cultured phytoplankton under both Fe-depleted and Fe-enriched conditions. Fe treatments had significant effects on photosynthetic efficiency (F_v/F_m; 0.3 for Fe-depleted and 0.5 for Fe-enriched conditions), non-photochemical quenching (NPQ), and relative electron transport rates (rETR). pCO₂ treatments significantly affected NPQ and rETR, but had no effect on F_v/F_m. Under Fe limitation, increased pCO₂ had no influence on C fixation whereas under Fe enrichment, primary production increased with increasing pCO₂ levels. These CO₂-dependent changes in productivity under Fe-enriched conditions were accompanied by a pronounced taxonomic shift from weakly to heavily silicified diatoms (i.e. from Pseudo-nitzschia sp. to Fragilariopsis sp.). Under Fe-depleted conditions, this functional shift was absent and thinly silicified species dominated all pCO₂ treatments (Pseudo-nitzschia sp. and Synedropsis sp. for low and high pCO₂, respectively). Our results suggest that Ocean Acidification could increase primary productivity and the abundance of heavily silicified, fast sinking diatoms in Fe-enriched areas, both potentially leading to a stimulation of the biological pump. Over much of the SO, however, Fe limitation could restrict this possible CO₂ fertilization effect.     

Accommodating the bacterial decoding release factor as an alien protein among the RNAs at the active site of the ribosome

The decoding release factor （RF） triggers termination of protein synthesis by functionally mimicking a tRNA to span the decoding centre and the peptidyl transferase centre （PTC） of the ribosome. Structurally, it must fit into a site crafted for a tRNA and surrounded by five other RNAs, namely the adjacent peptidyl tRNA carrying the completed polypeptide, the mRNA and the three rRNAs. This is achieved by extending a structural domain from the body of the protein that results in a critical conformational change allowing it to contact the PTC. A structural model of the bacterial termination complex with the accommodated RF shows that it makes close contact with the first, second and third bases of the stop codon in the mRNA with two separate loops of structure＂ the anticodon loop and the loop at the tip of helix orS. The anticodon loop also makes contact with the base following the stop codon that is known to strongly influence termination efficiency. It confirms the close contact of domain 3 of the protein with the key RNA structures of the PTC. The mRNA signal for termination includes sequences upstream as well as downstream of the stop codon, and this may reflect structural restrictions for specific combinations of tRNA and RF to be bound onto the ribosome together. An unbiased SELEX approach has been investigated as a tool to identify potential rRNA-binding contacts of the bacterial RF in its different binding conformations within the active centre of the ribosome.     

Towards high-throughput metabolomics using ultrahigh-field Fourier transform ion cyclotron resonance mass spectrometry

With unmatched mass resolution, mass accuracy, and exceptional detection sensitivity, Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FTICR-MS) has the potential to be a powerful new technique for high-throughput metabolomic analysis. In this study, we examine the properties of an ultrahigh-field 12-Tesla (12T) FTICR-MS for the identification and absolute quantitation of human plasma metabolites, and for the untargeted metabolic fingerprinting of inbred-strain mouse serum by direct infusion (DI). Using internal mass calibration (mass error ≤1 ppm), we determined the rational elemental compositions (incorporating unlimited C, H, N and O, and a maximum of two S, three P, two Na, and one K per formula) of approximately 250 out of 570 metabolite features detected in a 3-min infusion analysis of aqueous extract of human plasma, and were able to identify more than 100 metabolites. Using isotopically-labeled internal standards, we were able to obtain excellent calibration curves for the absolute quantitation of choline with sub-pmol sensitivity, using 500 times less sample than previous LC/MS analyses. Under optimized serum dilution conditions, chemical compounds spiked into mouse serum as metabolite mimics showed a linear response over a 600-fold concentration range. DI/FTICR-MS analysis of serum from 26 mice from 2 inbred strains, with and without acute trichloroethylene (TCE) treatment, gave a relative standard deviation (RSD) of 4.5%. Finally, we extended this method to the metabolomic fingerprinting of serum samples from 49 mice from 5 inbred strains involved in an acute alcohol toxicity study, using both positive and negative electrospray ionization (ESI). Using these samples, we demonstrated the utility of this method for high-throughput metabolomics, with more than 400 metabolites profiled in only 24 h. Our experiments demonstrate that DI/FTICR-MS is well-suited for high-throughput metabolomic analysis. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Functional and biochemical characterisation of the Escherichia coli major facilitator superfamily multidrug transporter MdtM

Multidrug resistance (MDR) occurs when bacteria simultaneously acquire resistance to a broad spectrum of structurally dissimilar compounds to which they have not previously been exposed. MDR is principally a consequence of the active transport of drugs out of the cell by proteins that are integral membrane transporters. We characterised and purified the putative Escherichia coli MDR transporter, MdtM, a 410 amino acid residue protein that belongs to the large and ubiquitous major facilitator superfamily. Functional characterisation of MdtM using growth inhibition and whole cell transport assays revealed its role in intrinsic resistance of E. coli cells to the antimicrobials ethidium bromide and chloramphenicol. Site-directed mutagenesis studies implied that the MdtM aspartate 22 residue and the highly conserved arginine at position 108 play a role in proton recognition. MdtM was homologously overexpressed and purified to homogeneity in dodecyl-β-D-maltopyranoside detergent solution and the oligomeric state and stability of the protein in a variety of detergent solutions was investigated using size-exclusion HPLC. Purified MdtM is monomeric and stable in dodecyl-β-D-maltopyranoside solution and binds chloramphenicol with nanomolar affinity in the same detergent. This work provides a firm foundation for structural studies on this class of multidrug transporter protein.     

Mapping functionally important motifs SPF and GGQ of the decoding release factor RF2 to the Escherichia coli ribosome by hydroxyl radical footprinting. Implications for macromolecular mimicry and structural changes in RF2

The function of the decoding release factor (RF) in translation termination is to couple cognate recognition of the stop codon in the mRNA with hydrolysis of the completed polypeptide from its covalently linked tRNA. For this to occur, the RF must interact with specific A-site components of the active centers within both the small and large ribosomal subunits. In this work, we have used directed hydroxyl radical footprinting to map the ribosomal binding site of the Escherichia coli class I release factor RF2, during translation termination. In the presence of the cognate UGA stop codon, residues flanking the universally conserved (250)GGQ(252) motif of RF2 were each shown to footprint to the large ribosomal subunit, specifically to conserved elements of the peptidyltransferase and GTPase-associated centers. In contrast, residues that flank the putative "peptide anticodon" of RF2, (205)SPF(207), were shown to make a footprint in the small ribosomal subunit at positions within well characterized 16 S rRNA motifs in the vicinity of the decoding center. Within the recently solved crystal structure of E. coli RF2, the GGQ and SPF motifs are separated by 23 A only, a distance that is incompatible with the observed cleavage sites that are up to 100 A apart. Our data suggest that RF2 may undergo gross conformational changes upon ribosome binding, the implications of which are discussed in terms of the mechanism of RF-mediated termination.     

Characteristics of Shelter-Relinquished Animals and Their Owners Compared With Animals and Their Owners in U.S. Pet-Owning Households

Animal shelters in the United States annually receive millions of relinquished dogs and cats, and risk factors for relinquishment are not fully understood. Investigators sponsored by the National Council on Pet Population Study and Policy interviewed people who relinquished dogs and cats at 12 shelters in four regions. We collected similar data from a sample of U.S. households with companion animals. Data collected included nonhuman animal characteristics such as age, sex, and frequency of selected behaviors. We also obtained data on keepers' (owners') age, sex, and level of education as well as their general knowledge of pet care and behavior. We found that relinquishment was associated with physical and behavioral characteristics of the animals and owner characteristics and knowledge. Relinquished animals were more likely to be intact, younger, and mixed bred. People relinquishing animals were significantly more likely to be men and younger than 35 years. Duration of ownership was significantly shorter for relinquished animals.