Carbohydrate Concentration in Pine as Affected by Inoculation with Bursaphelenchus xylophilus

Pines responded to inoculation with Bursaphelenchus xylophilus by changes in reducing and nonreducing carbohydrate concentrations dependent on the pine species and the pathotype of B. xylophilus with which the trees were inoculated. Carbohydrate concentrations, in compatible pine-nematode pathotype combinations, decreased initially after inoculation and then increased slightly before decreasing to approximately 10% of the control levels as the seedlings wilted. In compatible nematode pathotype-pine species combinations, carbohydrate concentrations decreased and then increased as the nematode population densities declined.     

Post-contest behaviour in black-capped chickadees (<Emphasis Type="Italic">Poecile atricapillus</Emphasis>): loser displays,not victory displays,follow asymmetrical countersinging exchanges

Victory displays are behaviours that occur after the conclusion of a signaling contest, performed solely by the contest winner. Victory displays may reinforce the dominance of the winner either to the loser or to other conspecifics within signaling range. Victory displays are poorly studied despite the significant consequences that post-conflict behaviour may have on the individuals involved. We examined the period immediately following 50 territorial countersinging contests between males in 10 neighbourhoods of black-capped chickadees (Poecile atricapillus) of known dominance rank. We characterized the post-contest singing behaviour of chickadees and evaluated whether post-contest behaviour is consistent with victory displays. Using a 16-microphone acoustic location system to simultaneously record entire neighbourhoods of breeding chickadees, we isolated 50 dyadic countersinging contests and measured the vocal behaviour of the contestants in the minutes following each interaction. Eighty-six percent of contests were followed by a period of solo singing by one of the contestants, while 14% were followed by silence. The post-contest singer was most often the contestant who held a subordinate dominance position in the previous winter’s dominance hierarchy; dominant males performed post-contest song bouts significantly less often. Asymmetry in overlapping between contestants did not predict which bird sang a post-contest bout. However, in a significant majority of cases, the post-contest singer was pitch-matched by his opponent during the contest more than he pitch-matched his opponent. Our results indicate that male chickadees do not perform acoustic victory displays after countersinging contests. In contrast, the post-contest behaviour of territorial chickadees is more consistent with a “loser display”.     

Influenza H5N1 virus infection of polarized human alveolar epithelial cells and lung microvascular endothelial cells

Background

Highly pathogenic avian influenza (HPAI) H5N1 virus is entrenched in poultry in Asia and Africa and continues to infect humans zoonotically causing acute respiratory disease syndrome and death. There is evidence that the virus may sometimes spread beyond respiratory tract to cause disseminated infection. The primary target cell for HPAI H5N1 virus in human lung is the alveolar epithelial cell. Alveolar epithelium and its adjacent lung microvascular endothelium form host barriers to the initiation of infection and dissemination of influenza H5N1 infection in humans. These are polarized cells and the polarity of influenza virus entry and egress as well as the secretion of cytokines and chemokines from the virus infected cells are likely to be central to the pathogenesis of human H5N1 disease.

Aim

To study influenza A (H5N1) virus replication and host innate immune responses in polarized primary human alveolar epithelial cells and lung microvascular endothelial cells and its relevance to the pathogenesis of human H5N1 disease.

Methods

We use an in vitro model of polarized primary human alveolar epithelial cells and lung microvascular endothelial cells grown in transwell culture inserts to compare infection with influenza A subtype H1N1 and H5N1 viruses via the apical or basolateral surfaces.

Results

We demonstrate that both influenza H1N1 and H5N1 viruses efficiently infect alveolar epithelial cells from both apical and basolateral surface of the epithelium but release of newly formed virus is mainly from the apical side of the epithelium. In contrast, influenza H5N1 virus, but not H1N1 virus, efficiently infected polarized microvascular endothelial cells from both apical and basolateral aspects. This provides a mechanistic explanation for how H5N1 virus may infect the lung from systemic circulation. Epidemiological evidence has implicated ingestion of virus-contaminated foods as the source of infection in some instances and our data suggests that viremia, secondary to, for example, gastro-intestinal infection, can potentially lead to infection of the lung. HPAI H5N1 virus was a more potent inducer of cytokines (e.g. IP-10, RANTES, IL-6) in comparison to H1N1 virus in alveolar epithelial cells, and these virus-induced chemokines were secreted onto both the apical and basolateral aspects of the polarized alveolar epithelium.

Conclusion

The predilection of viruses for different routes of entry and egress from the infected cell is important in understanding the pathogenesis of influenza H5N1 infection and may help unravel the pathogenesis of human H5N1 disease.     

Development of microsatellite markers in the Australasian snake-necked turtle Chelodina rugusa and cross-species amplification

Seventeen microsatellite loci were developed for the snake-necked turtle, Chelodina rugosa (Ogilby, 1890). Sixteen of the loci were polymorphic but three of these loci had null alleles. One locus displayed linkage disequilibrium. These 17 markers were tested for amplification in eight congeneric species with varying success; 98% amplification in Chelodina burrungandjii, 72% in C. canni, 38% in C. expansa, 58% in C. longicollis, 67% in C. mccordi, 73% in C. oblonga, 81% in C. parkeri, and 68% in C. pritchardi. These microsatellite markers will be useful for population assignment, gene flow, mating systems and hybridization studies in the genus Chelodina.     

Influence of Exposure History on the Immunology and Development of Resistance to Human Schistosomiasis Mansoni

Background

Previous studies suggest that humans can acquire immunity to reinfection with schistosomes, most probably due to immunologic mechanisms acquired after exposure to dying schistosome worms.

Methodology/Principal Findings

We followed longitudinally two cohorts of adult males occupationally exposed to Schistosoma mansoni by washing cars (120 men) or harvesting sand (53 men) in Lake Victoria. Men were treated with praziquantel each time S. mansoni infection was detected. In car washers, a significant increase in resistance to reinfection, as measured by the number of cars washed between cure and reinfection, was observed after the car washers had experienced, on average, seven cures. In the car washers who developed resistance, the level of schistosome-specific IgE increased between baseline and the time at which development of resistance was first evidenced. In the sand harvesters, a significant increase in resistance, as measured by the number of days worked in the lake between cure and reinfection, was observed after only two cures. History of exposure to S. mansoni differed between the two cohorts, with the majority of sand harvesters being lifelong residents of a village endemic for S. mansoni and the majority of car washers having little exposure to the lake before they began washing cars. Immune responses at study entry were indicative of more recent infections in car washers and more chronic infections in sand harvesters.

Conclusions/Significance

Resistance to reinfection with S. mansoni can be acquired or augmented by adults after multiple rounds of reinfection and cure, but the rate at which resistance is acquired by this means depends on immunologic status and history of exposure to S. mansoni infection.     

Small-molecule inhibition of choline catabolism in Pseudomonas aeruginosa and other aerobic choline-catabolizing bacteria

Choline is abundant in association with eukaryotes and plays roles in osmoprotection, thermoprotection, and membrane biosynthesis in many bacteria. Aerobic catabolism of choline is widespread among soil proteobacteria, particularly those associated with eukaryotes. Catabolism of choline as a carbon, nitrogen, and/or energy source may play important roles in association with eukaryotes, including pathogenesis, symbioses, and nutrient cycling. We sought to generate choline analogues to study bacterial choline catabolism in vitro and in situ. Here we report the characterization of a choline analogue, propargylcholine, which inhibits choline catabolism at the level of Dgc enzyme-catalyzed dimethylglycine demethylation in Pseudomonas aeruginosa. We used genetic analyses and 13C nuclear magnetic resonance to demonstrate that propargylcholine is catabolized to its inhibitory form, propargylmethylglycine. Chemically synthesized propargylmethylglycine was also an inhibitor of growth on choline. Bioinformatic analysis suggests that there are genes encoding DgcA homologues in a variety of proteobacteria. We examined the broader utility of propargylcholine and propargylmethylglycine by assessing growth of other members of the proteobacteria that are known to grow on choline and possess putative DgcA homologues. Propargylcholine showed utility as a growth inhibitor in P. aeruginosa but did not inhibit growth in other proteobacteria tested. In contrast, propargylmethylglycine was able to inhibit choline-dependent growth in all tested proteobacteria, including Pseudomonas mendocina, Pseudomonas fluorescens, Pseudomonas putida, Burkholderia cepacia, Burkholderia ambifaria, and Sinorhizobium meliloti. We predict that chemical inhibitors of choline catabolism will be useful for studying this pathway in clinical and environmental isolates and could be a useful tool to study proteobacterial choline catabolism in situ.     

Use of Humanised Rat Basophilic Leukaemia Cell Line RS-ATL8 for the Assessment of Allergenicity of Schistosoma mansoni Proteins

Background

Parasite-specific IgE is thought to correlate with protection against Schistosoma mansoni infection or re-infection. Only a few molecular targets of the IgE response in S. mansoni infection have been characterised. A better insight into the basic mechanisms of anti-parasite immunity could be gained from a genome-wide characterisation of such S. mansoni allergens. This would have repercussions on our understanding of allergy and the development of safe and efficacious vaccinations against helminthic parasites.

Methodology/Principal Findings

A complete medium- to high-throughput amenable workflow, including important quality controls, is described, which enables the rapid translation of S. mansoni proteins using wheat germ lysate and subsequent assessment of potential allergenicity with a humanised Rat Basophilic Leukemia (RBL) reporter cell line. Cell-free translation is completed within 90 minutes, generating sufficient amounts of parasitic protein for rapid screening of allergenicity without any need for purification. Antigenic integrity is demonstrated using Western Blotting. After overnight incubation with infected individuals'' serum, the RS-ATL8 reporter cell line is challenged with the complete wheat germ translation mixture and Luciferase activity measured, reporting cellular activation by the suspected allergen. The suitability of this system for characterization of novel S. mansoni allergens is demonstrated using well characterised plant and parasitic allergens such as Par j 2, SmTAL-1 and the IgE binding factor IPSE/alpha-1, expressed in wheat germ lysates and/or E. coli. SmTAL-1, but not SmTAL2 (used as a negative control), was able to activate the basophil reporter cell line.

Conclusion/Significance

This method offers an accessible way for assessment of potential allergenicity of anti-helminthic vaccine candidates and is suitable for medium- to high-throughput studies using infected individual sera. It is also suitable for the study of the basis of allergenicity of helminthic proteins.     

Transbilayer movement of Glc-P-dolichol and its function as a glucosyl donor: protein-mediated transport of a water-soluble analog into sealed ER vesicles from pig brain

The results described in the accompanying article support the model in which glucosylphosphoryldolichol (Glc-P-Dol) is synthesized on the cytoplasmic face of the ER, and functions as a glucosyl donor for three Glc-P-Dol:Glc0-2Man9-GlcNAc2-P-P-Dol glucosyltransferases (GlcTases) in the lumenal compartment. In this study, the enzymatic synthesis and structural characterization by NMR and electrospray-ionization tandem mass spectrometry of a series of water-soluble beta-Glc-P-Dol analogs containing 2-4 isoprene units with either the cis - or trans - stereoconfiguration in the beta-position are described. The water- soluble analogs were (1) used to examine the stereospecificity of the Glc-P-Dol:Glc0-2Man9GlcNAc2-P-P-Dol glucosyltransferases (GlcTases) and (2) tested as potential substrates for a membrane protein(s) mediating the transbilayer movement of Glc-P-Dol in sealed ER vesicles from rat liver and pig brain. The Glc-P-Dol-mediated GlcTases in pig brain microsomes utilized [3H]Glc-labeled Glc-P-Dol10, Glc-P-(omega, c )Dol15, Glc-P(omega, t,t )Dol20, and Glc-P-(omega, t,c )Dol20as glucosyl donors with [3H]Glc3Man9GlcNAc2-P-P-Dol the major product labeled in vitro. A preference was exhibited for C15-20 substrates containing an internal cis -isoprene unit in the beta-position. In addition, the water-soluble analog, Glc-P-Dol10, was shown to enter the lumenal compartment of sealed microsomal vesicles from rat liver and pig brain via a protein-mediated transport system enriched in the ER. The properties of the ER transport system have been characterized. Glc- P-Dol10was not transported into or adsorbed by synthetic PC-liposomes or bovine erythrocytes. The results of these studies indicate that (1) the internal cis -isoprene units are important for the utilization of Glc-P-Dol as a glucosyl donor and (2) the transport of the water- soluble analog may provide an experimental approach to assay the hypothetical "flippase" proposed to mediate the transbilayer movement of Glc-P-Dol from the cytoplasmic face of the ER to the lumenal monolayer.