Antifreeze protein produced endogenously in winter rye leaves

After cold acclimation, winter rye (Secale cereale L.) is able to withstand the formation of extracellular ice at freezing temperatures. We now show, for the first time, that cold-acclimated winter rye plants contain endogenously produced antifreeze protein. The protein was extracted from the apoplast of winter rye leaves, where ice forms during freezing. After partial purification, the protein was identified as antifreeze protein because it modified the normal growth pattern of ice crystals and depressed the freezing temperature of water noncolligatively.     

Development of cell surface saccharides on embryonic pancreatic cells

Using a battery of seven lectin-ferritin conjugates as probes for cell surface glycoconjugates, we have studied the pattern of plasmalemmal differentiation of cells in the embryonic rat pancreas from day 15 in utero to the early postpartum stage. Our results indicate that differentiation of plasmalemmal glycoconjugates on acinar, endocrine, and centroacinar cells is temporally correlated with development and is unique for each cell type, as indicated by lectin-ferritin binding. Specifically, (a) expression of adult cell surface saccharide phenotype can be detected on presumptive acinar cells as early as 15 d in utero, as indicated by soybean agglutinin binding, and precedes development of intracellular organelles characteristic of mature acinar cells; (b) maturation of the plasmalemma of acinar cells is reached after intracellular cytodifferentiation is completed, as indicated by appearance of Con A and fucoselectin binding sites only at day 19 of development; conversely, maturation of the endocrine cell plasmalemma is accompanied by "loss" (masking) of ricinus communis II agglutinin receptors; and (c) binding sites for fucose lectins and for soybean agglutinin are absent on endocrine and centroacinar cells at all stages examined. We conclude that acinar, centroacinar, and endocrine cells develop from a common progenitor cell(s) whose plasmalemmal carbohydrate composition resembles most closely that of the adult centroacinar cell. Finally, appearance of acinar lumina beginning at approximately 17 d in utero is accompanied by differenetiation of apical and basolateral plasmalemmal domains of epithelial cells, as indicated by enhanced binding of several lectin-ferritin conjugates to the apical plasmalemmal, a pattern that persists from this stage through adult life.     

Airway Microbiota in Severe Asthma and Relationship to Asthma Severity and Phenotypes

BackgroundThe lower airways harbor a community of bacterial species which is altered in asthma.ObjectivesWe examined whether the lower airway microbiota were related to measures of asthma severity.MethodsWe prospectively recruited 26 severe asthma, 18 non-severe asthma and 12 healthy subjects. DNA was extracted from induced sputum and PCR amplification of the V3-V5 region of bacterial 16S rRNA gene was performed.ResultsWe obtained 138,218 high quality sequences which were rarefied at 133 sequences/sample. Twenty OTUs had sequences ≥1% of total. There were marked differences in the distribution of Phyla between groups (P = 2.8x10^-118). Bacteroidetes and Fusobacteria were reduced in non-severe and severe asthmatic groups. Proteobacteria were more common in non-severe asthmatics compared to controls (OR = 2.26; 95% CI = 1.94–2.64) and Firmicutes were increased in severe asthmatics compared to controls (OR = 2.15; 95%CI = 1.89–2.45). Streptococcal OTUs amongst the Firmicutes were associated with recent onset asthma, rhinosinusitis and sputum eosinophilia.ConclusionsSputum microbiota in severe asthma differs from healthy controls and non-severe asthmatics, and is characterized by the presence of Streptococcus spp with eosinophilia. Whether these organisms are causative for the pathophysiology of asthma remains to be determined.     

Testing Three Species Distribution Modelling Strategies to Define Fish Assemblage Reference Conditions for Stream Bioassessment and Related Applications

Species distribution models are widely used for stream bioassessment, estimating changes in habitat suitability and identifying conservation priorities. We tested the accuracy of three modelling strategies (single species ensemble, multi-species response and community classification models) to predict fish assemblages at reference stream segments in coastal subtropical Australia. We aimed to evaluate each modelling strategy for consistency of predictor variable selection; determine which strategy is most suitable for stream bioassessment using fish indicators; and appraise which strategies best match other stream management applications. Five models, one single species ensemble, two multi-species response and two community classification models, were calibrated using fish species presence-absence data from 103 reference sites. Models were evaluated for generality and transferability through space and time using four external reference site datasets. Elevation and catchment slope were consistently identified as key correlates of fish assemblage composition among models. The community classification models had high omission error rates and contributed fewer taxa to the ‘expected’ component of the taxonomic completeness (O/E₅₀) index than the other strategies. This potentially decreases the model sensitivity for site impact assessment. The ensemble model accurately and precisely modelled O/E₅₀ for the training data, but produced biased predictions for the external datasets. The multi-species response models afforded relatively high accuracy and precision coupled with low bias across external datasets and had lower taxa omission rates than the community classification models. They inherently included rare, but predictable species while excluding species that were poorly modelled among all strategies. We suggest that the multi-species response modelling strategy is most suited to bioassessment using freshwater fish assemblages in our study area. At the species level, the ensemble model exhibited high sensitivity without reductions in specificity, relative to the other models. We suggest that this strategy is well suited to other non-bioassessment stream management applications, e.g., identifying priority areas for species conservation.     

Determination of 1-aminocycopropane-1-carboxylic acid (ACC) to assess the effects of ACC deaminase-containing bacteria on roots of canola seedlings

Previously, it was proposed that plant growth-promoting bacteria that possess the enzyme, 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, can reduce the amount of ethylene produced by a plant and thereby promote root elongation. To test this model, canola seeds were imbibed in the presence of the chemical ethylene inhibitor, 2-aminoethoxyvinyl glycine (AVG), various strains of plant growth-promoting bacteria, and a psychrophilic bacterium containing an ACC deaminase gene on a broad host range plasmid. The extent of root elongation and levels of ACC, the immediate precursor of ethylene, were measured in the canola seedling roots. A modification of the Waters AccQ.Tag Amino Acid Analysis Method was used to quantify ACC in the root extracts. It was found that, in the presence of the ethylene inhibitor, AVG, or any one of several ACC deaminase-containing strains of bacteria, the growth of canola seedling roots was enhanced and the ACC levels in these roots were lowered.     

Adenine phosphoribosyltransferase isoforms of Arabidopsis and their potential contributions to adenine and cytokinin metabolism

Adenine phosphoribosyltransferase (APT; EC 2.4.2.7) is a constitutively expressed enzyme involved in the one-step salvage of adenine to AMP. The Arabidopsis thaliana genome contains five sequences annotated as encoding APT or APT-like enzymes. Three of these have now been cloned, over-expressed and compared using kinetic analyses. At a cytosolic pH, all bind adenine efficiently based on their K_m values (0.8–2.6 µ M ), although APT1 metabolizes adenine at a rate 31–53 times faster than APT2 and APT3, respectively. Since APT also has a possible role in the interconversion of cytokinin bases to nucleotides, we characterized the activity of each isoform on zeatin, isopentenyladenine and benzyladenine. Based on their K_m values, APT2 and APT3 had much higher affinities than APT1 for all three cytokinins (15–440 µ M for APT2 and 3 vs. 1.8–2.5 m M for APT1); conversely the V_max values for APT2 and APT3 on these CK substrates showed the opposite trend, being 4- to 19-fold lower than those of APT1. Anti-peptide antibodies for APT1, APT2, and APT3 were prepared and used to examine the subcellular localization of each isoform. Based on these results, APT1 and APT3 appear to be cytosolic, while the localization of APT2 was inconclusive although sequence analysis implies that APT2 is also cytosolic. Each isoform was modelled against the crystal structure of APT from Leishmania donovani , and structural differences in substrate specificity-determining domains have been found. The estimated kinetic activities of these APTs suggest that they contribute primarily to adenine recycling, although an involvement in cytokinin interconversion cannot be discounted.     

Engineered viruses to select genes encoding secreted and membrane-bound proteins in mammalian cells

We have developed a functional genomics tool to identify the subset of cDNAs encoding secreted and membrane-bound proteins within a library (the ‘secretome’). A Sindbis virus replicon was engineered such that the envelope protein precursor no longer enters the secretory pathway. cDNA fragments were fused to the mutant precursor and expression screened for their ability to restore membrane localization of envelope proteins. In this way, recombinant replicons were released within infectious viral particles only if the cDNA fragment they contain encodes a secretory signal. By using engineered viral replicons to selectively export cDNAs of interest in the culture medium, the methodology reported here efficiently filters genetic information in mammalian cells without the need to select individual clones. This adaptation of the ‘signal trap’ strategy is highly sensitive (1/200 000) and efficient. Indeed, of the 2546 inserts that were retrieved after screening various libraries, more than 97% contained a putative signal peptide. These 2473 clones encoded 419 unique cDNAs, of which 77% were previously annotated. Of the 94 cDNAs encoding proteins of unknown function, 24% either had no match in databases or contained a secretory signal that could not be predicted from electronic data.