Identification of the Propionicin F Bacteriocin Immunity Gene (pcfI) and Development of a Food-Grade Cloning System for Propionibacterium freudenreichii

This report describes the first functional analysis of a bacteriocin immunity gene from Propionibacterium freudenreichii and its use as a selection marker for food-grade cloning. Cloning of the pcfI gene (previously orf5 [located as part of the pcfABC propionicin F operon]) rendered the sensitive host 1,000-fold more tolerant to the propionicin F bacteriocin. The physiochemical properties of the 127-residue large PcfI protein resemble those of membrane-bound immunity proteins from bacteriocin systems found in lactic acid bacteria. The high level of immunity conferred by pcfI allowed its use as a selection marker for plasmid transformation in P. freudenreichii. Electroporation of P. freudenreichii IFO12426 by use of the pcfI expression plasmid pSL102 and propionicin F selection (200 bacteriocin units/ml) yielded 10⁷ transformants/μg DNA. The 2.7-kb P. freudenreichii food-grade cloning vector pSL104 consists of the pLME108 replicon, a multiple cloning site, and pcfI expressed from the constitutive P_pampS promoter for selection. The pSL104 vector efficiently facilitated cloning of the propionicin T1 bacteriocin in P. freudenreichii. High-level propionicin T1 production (640 BU/ml) was obtained with the IFO12426 strain, and the food-grade propionicin T1 expression plasmid pSL106 was maintained by ~91% of the cells over 25 generations in the absence of selection. To the best of our knowledge this is the first report of an efficient cloning system that facilitates the generation of food-grade recombinant P. freudenreichii strains.     

Is the Scotia Sea a centre of Antarctic marine diversification? Some evidence of cryptic speciation in the circum-Antarctic bivalve <Emphasis Type="Italic">Lissarca notorcadensis</Emphasis> (Arcoidea: Philobryidae)

The bivalve Lissarca notorcadensis is one of the most abundant species in Antarctic waters and has colonised the entire Antarctic shelf and Scotia Sea Islands. Its brooding reproduction, low dispersal capabilities and epizoic lifestyle predict limited gene flow between geographically isolated populations. Relationships between specimens from seven regions in the Southern Ocean and outgroups were assessed with nuclear 28S rDNA and mitochondrial cytochrome oxidase subunit I (COI) genes. The 28S dataset indicate that while Lissarca appears to be a monophyletic genus, there is polyphyly between the Limopsidae and Philobryidae. Thirteen CO1 haplotypes were found, mostly unique to the sample regions, and two distinct lineages were distinguished. Specimens from the Weddell and Ross Sea form one lineage while individuals from the banks and islands of the Scotia Sea form the other. Within each lineage, further vicariance was observed forming six regionally isolated groups. Our results provide initial evidence for reproductively isolated populations of L. notorcadensis. The islands of the Scotia Sea appear to act as centres of speciation in the Southern Ocean.     

The structure of the Caenorhabditis elegans manganese superoxide dismutase MnSOD‐3‐azide complex

C. elegans MnSOD‐3 has been implicated in the longevity pathway and its mechanism of catalysis is relevant to the aging process and carcinogenesis. The structures of MnSOD‐3 provide unique crystallographic evidence of a dynamic region of the tetrameric interface (residues 41–54). We have determined the structure of the MnSOD‐3‐azide complex to 1.77‐Å resolution. Analysis of this complex shows that the substrate analog, azide, binds end‐on to the manganese center as a sixth ligand and that it ligates directly to a third and new solvent molecule also positioned within interacting distance to the His30 and Tyr34 residues of the substrate access funnel. This is the first structure of a eukaryotic MnSOD‐azide complex that demonstrates the extended, uninterrupted hydrogen‐bonded network that forms a proton relay incorporating three outer sphere solvent molecules, the substrate analog, the gateway residues, Gln142, and the solvent ligand. This configuration supports the formation and release of the hydrogen peroxide product in agreement with the 5‐6‐5 catalytic mechanism for MnSOD. The high product dissociation constant k₄ of MnSOD‐3 reflects low product inhibition making this enzyme efficient even at high levels of superoxide.     

Dual target strategy: combining distinct non‐dopaminergic treatments reduces neuronal cell loss and synergistically modulates l‐DOPA‐induced rotational behavior in a rodent model of Parkinson's disease

The glutamate metabotropic receptor 5 (mGluR5) and the adenosine A_2A receptor (A_2AR) represent major non‐dopaminergic therapeutic targets in Parkinson's disease (PD) to improve motor symptoms and slow down/revert disease progression. The 6‐hydroxydopamine rat model of PD was used to determine/compare the neuroprotective and behavioral impacts of single and combined administration of one mGluR5 antagonist, 2‐methyl‐6‐(phenylethynyl)pyridine (MPEP), and two A_2AR antagonists, (E)‐phosphoric acid mono‐[3‐[8‐[2‐(3‐methoxyphenyl)vinyl]‐7‐methyl‐2,6‐dioxo‐1‐prop‐2‐ynyl‐1,2,6,7‐tetrahydropurin‐3‐yl]propyl] (MSX‐3) and 8‐ethoxy‐9‐ethyladenine (ANR 94). Chronic treatment with MPEP or MSX‐3 alone, but not with ANR 94, reduced the toxin‐induced loss of dopaminergic neurons in the substantia nigra pars compacta. Combining MSX‐3 and MPEP further improved the neuroprotective effect of either antagonists. At the behavioral level, ANR 94 and MSX‐3 given alone significantly potentiated l ‐DOPA‐induced turning behavior. Combination of either A_2AR antagonists with MPEP synergistically increased L‐DOPA‐induced turning. This effect was dose‐dependent and required subthreshold drug concentration, which per se had no motor stimulating effect. Our findings suggest that co‐treatment with A_2AR and mGluR5 antagonists provides better therapeutic benefits than those produced by either drug alone. Our study sheds some light on the efficacy and advantages of combined non‐dopaminergic PD treatment using low drug concentration and establishes the basis for in‐depth studies to identify optimal doses at which these drugs reach highest efficacy.

     

Submergence of black ash logs to control emerald ash borer and preserve wood for American Indian basketmaking

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Detection of E. coli O157:H7 by immunomagnetic separation coupled with fluorescence immunoassay

Conventional culture-based methods for detection of E. coli O157:H7 in foods and water sources are time-consuming, and results can be ambiguous, requiring further confirmation by biochemical testing and PCR. A rapid immunoassay prior to cultivation to identify presumptive positive sample would save considerable time and resources. Immunomagnetic separation (IMS) techniques are routinely used for isolation of E. coli O157:H7 from enriched food and water samples, typically in conjunction with cultural detection followed by biochemical and serological confirmation. In this study, we developed a new method that combines IMS with fluorescence immunoassay, termed immunomagnetic fluorescence assay (IMFA), for the detection of E. coli O157:H7. E. coli O157:H7 cells were first captured by anti-O157 antibody-coated magnetic beads and then recognized by a fluorescent detector antibody, forming an immunosandwich complex. This complex was subsequently dissociated for measurement of fluorescence intensity with Signalyte™-II spectrofluorometer. Experiments were conducted to evaluate both linearity and sensitivity of the assay. Capture efficiencies were greater than 98%, as determined by cultural plating and quantitative real-time PCR, when cell concentrations were <10⁵ cells/mL. Capture efficiency decreased at higher cell concentrations, due to the limitation of bead binding capacity. At lower cell concentrations (10–10⁴ cells/mL), the fluorescence intensity of dissociated Cy5 solution was highly correlated with E. coli 157:H7 cell concentrations. The detection limit was 10 CFU per mL of water. The assay can be completed in less than 3 h since enrichment is not required, as compared to existing techniques that typically require a 24 h incubation for pre-enrichment, followed by confirmatory tests.     

Interaction of angio-associated migratory cell protein with the TPα and TPβ isoforms of the human thromboxane A₂ receptor

In humans, thromboxane (TX) A₂ signals through the TPα and TPβ isoforms of its G-protein coupled TXA₂ receptor (TP) to mediate a host of (patho)physiologic responses. Herein, angio-associated migratory cell protein (AAMP) was identified as a novel interacting partner of both TPα and TPβ through an interaction dependent on common (residues 312-328) and unique (residues 366-392 of TPβ) sequences within their carboxyl-terminal (C)-tail domains. While the interaction was constitutive in mammalian cells, agonist-stimulation of TPα/TPβ led to a transient dissociation of AAMP from immune complexes which coincided with a transient redistribution of AAMP from its localization in an intracellular fibrous network. Although the GTPase RhoA is a downstream effector of both AAMP and the TPs, AAMP did not influence TP-mediated RhoA or vice versa. Small interfering RNA (siRNA)-mediated disruption of AAMP expression decreased migration of primary human coronary artery smooth muscle cells (1° hCoASMCs). Moreover, siRNA-disruption of AAMP significantly impaired 1° hCoASMC migration in the presence of the TXA₂ mimetic U46619 but did not affect VEGF-mediated cell migration. Given their roles within the vasculature, the identification of a specific interaction between TPα/TPβ and AAMP is likely to have substantial functional implications for vascular pathologies in which they are both implicated.