Amplified Intergenic Locus Polymorphism as a Basis for Bacterial Typing of Listeria spp. and Escherichia coli

DNA-based methods are increasingly important for bacterial typing. The high number of polymorphic sites present among closely related bacterial genomes is the basis for the presented method. The method identifies multilocus genomic polymorphisms in intergenic regions termed AILP (amplified intergenic locus polymorphism). For each locus, a pair of unique PCR primers was designed to amplify an intergenic sequence from one open reading frame (ORF) to the adjacent ORF. Presence, absence, and size variation of the amplification products were identified and used as genetic markers for rapidly differentiating among strains. Polymorphism was evaluated using 18 AILP sites among 28 strains of Listeria monocytogenes and 6 strains of Listeria spp. and 30 AILP markers among 27 strains of Escherichia coli. Up to four alleles per locus were identified among Listeria strains, and up to six were identified among E. coli strains. In both species, more than half of the AILP sites revealed intraspecies polymorphism. The AILP data were applied to phylogenetic analysis among Listeria and E. coli strains. A clear distinction between L. monocytogenes and Listeria spp. was demonstrated. In addition, the method separated L. monocytogenes into the three known lineages and discriminated the most common virulent serotypic group, 4b. In E. coli, AILP analysis separated the known groups as well as the virulent O157:H7 isolates. These findings for both Listeria and E. coli are in agreement with other phylogenetic studies using molecular markers. The AILP method was found to be rapid, simple, reproducible, and a low-cost method for initial bacterial typing that could serve as a basis for epidemiological investigation.     

DAP5 promotes cap-independent translation of Bcl-2 and CDK1 to facilitate cell survival during mitosis

DAP5 is an eIF4G protein previously implicated in mediating cap-independent translation in response to cellular stresses. Here we report that DAP5 is crucial for continuous cell survival in nonstressed cells. The knockdown of endogenous DAP5 induced M phase-specific caspase-dependent apoptosis. Bcl-2 and CDK1 were identified by two independent screens as DAP5 translation targets. Notably, the activity of the Bcl-2 IRES was reduced in DAP5 knockdown cells and a selective shift of Bcl-2 mRNA toward light polysomal fractions was detected. Furthermore, a functional IRES was identified in the 5'UTR of CDK1. At the cellular level, attenuated translation of CDK1 by DAP5 knockdown decreased the phosphorylation of its M phase substrates. Ectopic expression of Bcl-2 or CDK1 proteins partially reduced the extent of caspase activation caused by DAP5 knockdown. Thus, DAP5 is necessary for maintaining cell survival during mitosis by promoting cap-independent translation of at least two prosurvival proteins.     

Apoptosis in fresh and cryopreserved cardiac valves of pig samples

To analyse the influence of cold ischemic time (CIT) (2–24 h) and of cryopreservation (liquid phase) on the viability of the valvular fibroblasts and in the presence of apoptosis. Cardiac valves from 10 pigs were evaluated by anatomo-pathological study of the wall, muscle and leaflet. At the same time, the presence of cellular death due to apoptosis was investigated in two ways; directly on tissue by Apodetec system and by two-colour flow cytometry assay analyzing a suspension of fibroblast from valve leaflets using Anexina V and propidium iodure (PI). We established three groups of samples to compare different experimental conditions: 2 h of ischemia (group 1), 24 h of ischemia (group 2), and a programme of cryopreservation (−1°C/min) after 2 h of ischemia, followed by storage in liquid nitrogen during a week and thawing was performed (group 3). The analysis of viabilities showed slight differences between all three groups. The results indicated CIT of 24 h undergoing more structural affectation than CIT of 2 h. Flow cytometry analysis did not show important differences between groups; however cryopreserved samples (group 3) slightly less viability and a higher percentage of death by apoptosis than group 1 and 2 using flow cytometry. Apoptosis was confirmed on tissue from all valves but mainly in samples of group 2 and group 3. In summary, the viability of the valves in the case of ischemic times of 2 h, 24 h or after cryopreservation/thawing differs slightly. The death of the cells is mainly mediated by necrosis and not by apoptosis.     

Return of Salmon-Derived Nutrients from the Riparian Zone to the Stream during a Storm in Southeastern Alaska

Spawning salmon deliver nutrients (salmon-derived nutrients, SDN) to natal watersheds that can be incorporated into terrestrial and aquatic food webs, potentially increasing ecosystem productivity. Peterson Creek, a coastal watershed in southeast Alaska that supports several species of anadromous fish, was sampled over the course of a storm during September 2006 to test the hypothesis that stormflows re-introduce stored SDN into the stream. We used stable isotopes and PARAFAC modeling of fluorescence excitation–emission spectroscopy to detect flushing of DOM from salmon carcasses in the riparian zone back into a spawning stream. During the early storm hydrograph, streamwater concentrations of NH₄–N and total dissolved phosphorus (TDP), the fluorescent protein tyrosine and the δ¹⁵N content of DOM peaked, followed by a rapid decrease during maximum stormflow. Although δ¹⁵N has previously been used to track SDN in riparian zones, the use of fluorescence spectroscopy provides an independent indicator that SDN are being returned from the riparian zone to the stream after a period of intermediate storage outside the stream channel. Our findings further demonstrate the utility of using both δ¹⁵N of streamwater DOM and fluorescence spectroscopy with PARAFAC modeling to monitor how the pool of streamwater DOM changes in spawning salmon streams.     

The lack of floral synthesis and emission of isoeugenol in Petunia axillaris subsp. parodii is due to a mutation in the isoeugenol synthase gene

Floral scent has been extensively investigated in plants of the South American genus Petunia. Flowers of Petunia integrifolia emit mostly benzaldehyde, while flowers of Petunia axillaris subsp. axillaris emit a mixture of volatile benzenoid and phenylpropanoid compounds that include isoeugenol and eugenol. Flowers of the artificial hybrid Petunia hybrida, a cross between P. integrifolia and P. axillaris, emit a similar spectrum of volatiles as P. axillaris subsp. axillaris. However, the flowers of P. axillaris subsp. parodii emit neither isoeugenol nor eugenol but contain high levels of dihydroconiferyl acetate in the petals, the main scent‐synthesizing and scent‐emitting organs. We recently showed that both isoeugenol and eugenol in P. hybrida are biosynthesized from coniferyl acetate in reactions catalyzed by isoeugenol synthase (PhIGS1) and eugenol synthase (PhEGS1), respectively, via a quinone methide‐like intermediate. Here we show that P. axillaris subsp. parodii has a functional EGS gene that is expressed in flowers, but its IGS gene contains a frame‐shift mutation that renders it inactive. Despite the presence of active EGS enzyme in P. axillaris subsp. parodii, in the absence of IGS activity the coniferyl acetate substrate is converted by an as yet unknown enzyme to dihydroconiferyl acetate. By contrast, suppressing the expression of PhIGS1 in P. hybrida by RNA interference also leads to a decrease in isoeugenol biosynthesis, but instead of the accumulation of dihydroconiferyl acetate, the flowers synthesize higher levels of eugenol.     

Weissella halotolerans W22 combines arginine deiminase and ornithine decarboxylation pathways and converts arginine to putrescine

Aims: To demonstrate that the meat food strain Weissella halotolerans combines an ornithine decarboxylation pathway and an arginine deiminase (ADI) pathway and is able to produce putrescine, a biogenic amine. Evidence is shown that these two pathways produce a proton motive force (PMF). Methods and Results: Internal pH in W. halotolerans was measured with the sensitive probe 2′,7′–bis‐(2‐carboxyethyl)‐5(and‐6)‐carboxyfluorescein. Membrane potential was measured with the fluorescent probe 3,3′‐dipropylthiocarbocyanine iodine. Arginine and ornithine transport studies were made under several conditions, using cells loaded or not loaded with the biogenic amine putrescine. ADI pathway caused an increase in ΔpH dependent on the activity of F₀F₁ATPase. Ornithine decarboxylation pathway generates both a ΔpH and a ΔΨ. Both these pathways lead to the generation of a PMF. Conclusions: Weissella halotolerans W22 combines an ADI pathway and an ornithine decarboxylation pathway, conducing to the production of the biogenic amine putrescine and of a PMF. Transport studies suggest the existence of a unique antiporter arginine/putrescine in this lactic acid bacteria strain. Significance and Impact of the Study: The coexistence of two different types of amino acid catabolic pathways, leading to the formation of a PMF, is shown for a Weissella strain for the first time. Moreover, a unique antiport arginine/putrescine is hypothesized to be present in this food strain.     

Detection of Molecular Paths Associated with Insulitis and Type 1 Diabetes in Non-Obese Diabetic Mouse

Recent clinical evidence suggests important role of lipid and amino acid metabolism in early pre-autoimmune stages of type 1 diabetes pathogenesis. We study the molecular paths associated with the incidence of insulitis and type 1 diabetes in the Non-Obese Diabetic (NOD) mouse model using available gene expression data from the pancreatic tissue from young pre-diabetic mice. We apply a graph-theoretic approach by using a modified color coding algorithm to detect optimal molecular paths associated with specific phenotypes in an integrated biological network encompassing heterogeneous interaction data types. In agreement with our recent clinical findings, we identified a path downregulated in early insulitis involving dihydroxyacetone phosphate acyltransferase (DHAPAT), a key regulator of ether phospholipid synthesis. The pathway involving serine/threonine-protein phosphatase (PP2A), an upstream regulator of lipid metabolism and insulin secretion, was found upregulated in early insulitis. Our findings provide further evidence for an important role of lipid metabolism in early stages of type 1 diabetes pathogenesis, as well as suggest that such dysregulation of lipids and related increased oxidative stress can be tracked to beta cells.