The production in vivo of microcin E492 with antibacterial activity depends on salmochelin and EntF

Microcin E492 is a channel-forming bacteriocin that is found in two forms, namely, a posttranslationally modified form obtained by the covalent linkage of salmochelin-like molecules to serine 84 and an unmodified form. The production of modified microcin E492 requires the synthesis of enterochelin, which is subsequently glycosylated by MceC and converted into salmochelin. mceC mutants produced inactive microcin E492, and this phenotype was reversed either by complementation with iroB from Salmonella enterica or by the addition of exogenous salmochelin. Cyclic salmochelin uptake by Escherichia coli occurred mainly through the outer membrane catecholate siderophore receptor Fiu. The production of inactive microcin E492 by mutants in entB and entC was reverted by the addition of the end product of the respective mutated pathway (2,3-dihydroxybenzoic acid and enterochelin/salmochelin, respectively), while mutants in entF did not produce active microcin E492 in the presence of enterochelin or salmochelin. The EntF adenylation domain was the only domain required for this microcin E492 maturation step. Inactivation of the enzymatic activity of this domain by site-directed mutagenesis did not prevent the synthesis of active microcin E492 in the presence of salmochelin, indicating that the adenylation activity is not essential for the function of EntF at this stage of microcin E492 maturation.     

Use of light and inorganic carbon acquisition by two morphotypes of Zostera noltii Hornem

The affinity for dissolved inorganic carbon (DIC) and the mechanisms to use HCO₃⁻ as a source of DIC for photosynthesis were investigated in two morphotypes of Zostera noltii Hornem. Both morphotypes were collected at Ria Formosa lagoon (Southern Portugal) at two different levels in the intertidal. Affinity for DIC at saturating photon fluence rate (PFR), estimated as photosynthetic conductance for CO₂ (g_p(CO2)), was reduced by 75% in the Z. noltii plants adapted to shade conditions (lower intertidal) in comparison to the sun morphotype (45×10⁻⁶ and 182×10⁻⁶ m s⁻¹, respectively), indicating that the plants acclimated to sun conditions (higher intertidal) had a higher capacity to use HCO₃⁻ as DIC source for photosynthesis. Since external carbonic anhydrase activity was negligible and a large inhibitory effect was produced by Tris buffer addition, this HCO₃⁻ use was attributed to the operation of H⁺ ATPases creating low pH zones in periplasmic space. The photosynthetic CO₂-flux supported for this mechanism was calculated to be 53 μmol O₂ m⁻² s⁻¹ in sun morphotype, about 80% out of maximum photosynthesis rate. In order to determine the possible photosynthetic energy cost of the HCO₃⁻ use, the effect of decreasing light on photosynthetic rates and g_p(CO2) was estimated. Photosynthetic conductance decreased in both morphotypes at non-saturating PFR. This dependence of g_p(CO2) on PFR indicated the existence of a positive interactive effect between DIC and PFR which was more pronounced in the shade morphotype since the ascending slope of O₂ evolution vs. PFR curves at limiting PFRs was reduced from 7.2 to 2.3 mmol O₂ mol photon⁻¹ at 4 and 0.5 mol m⁻³ of DIC, respectively.     

Understanding auditory distance estimation by humpback whales: a computational approach

Ranging, the ability to judge the distance to a sound source, depends on the presence of predictable patterns of attenuation. We measured long-range sound propagation in coastal waters to assess whether humpback whales might use frequency degradation cues to range singing whales. Two types of neural networks, a multi-layer and a single-layer perceptron, were trained to classify recorded sounds by distance traveled based on their frequency content. The multi-layer network successfully classified received sounds, demonstrating that the distorting effects of underwater propagation on frequency content provide sufficient cues to estimate source distance. Normalizing received sounds with respect to ambient noise levels increased the accuracy of distance estimates by single-layer perceptrons, indicating that familiarity with background noise can potentially improve a listening whale's ability to range. To assess whether frequency patterns predictive of source distance were likely to be perceived by whales, recordings were pre-processed using a computational model of the humpback whale's peripheral auditory system. Although signals processed with this model contained less information than the original recordings, neural networks trained with these physiologically based representations estimated source distance more accurately, suggesting that listening whales should be able to range singers using distance-dependent changes in frequency content.     

Characterization and functional recovery of a novel antimicrobial peptide (CECdir-CECret) from inclusion bodies after expression in Escherichia coli

CECdir-CECret is a novel non-toxic doublet 8.5 kDa peptide representing the natural coding sequence of the antimicrobial peptide Cecropin A from Drosophila melanogaster fused in-frame to its own inverted version. Expression of this cloned doublet peptide in Escherichia coli, yielded peptides that were mostly packaged into inclusion bodies. The new molecule was purified, solubilized and refolded, through a standard guanidine-based procedure. The recovered refolded peptides were then characterized by HPLC chromatography, MALDI-TOF-mass spectrometry and peptide sequencing, and finally evaluated for their antimicrobial potential. The novel doublet peptide CECdir-CECret, displays an enhanced in vitro antimicrobial activity and action spectrum in comparison to the monomer Cecropin A.     

Persistence of Escherichia coli, Salmonella choleraesuis, Aujeszky's Disease virus and Blue Eye Disease virus in ensilages based on the solid fraction of pig faeces

AIMS: This study was carried out to determine the survival time of Escherichia coli, Salmonella choleraesuis, Aujeszky's Disease virus and Blue Eye Disease virus in ensilages based on the solid fraction of pig faeces. METHODS AND RESULTS: The four micro-organisms were inoculated into microsilos based on the solid fraction of pig faeces, sorghum and molasses. They were left for 0, 7, 14, 28 and 56 days, after which the state of each microsilo was evaluated, and isolation of the inoculated agents was attempted. The four inoculated agents were isolated only on day 0 of ensilage. The viral agents were identified through the cytopathic effect and fluorescence. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: It is concluded that ensilages based on the solid fraction of pig faeces appear to reduce the risk of the transmission of the agents inoculated in this study and help to reduce the environmental impact by using the solid in animal feed.     

Regeneration of whole plants from apical meristems of Pinus radiata

A methodology to regenerate whole plants of Pinus radiata from apical meristems of 3- and 7-year-old trees was developed. Meristematic domes with two or three leaf primordia were excised from surface-sterilized branch tips of field-grown plants and cultured in LP medium with half strength macronutrients (1/2 LP) and full strength micronutrients. The early growth of meristems required approximately 12 weeks, including a recovery stage during the first 2 weeks. After 8 weeks, some meristems developed abnormal phenotypes and died during the subsequent stages of development. However, healthy meristems elongated and formed shoots when they were transferred to LP medium supplemented with MS vitamins, 30 mg l^–1 casein hydrolysate, and 0.4 g l^–1 agar plus 2.85 g l^–1 Gelrite. Meristems that developed vigorous shoots were used for rooting experiments when they were 2 cm in length. Whole plants were obtained after 5 days of root induction in water-agar medium containing 8.2 M IBA and 5.4 M NAA and 1 month culture in LP medium with 10 g l^–1 sucrose. Plants regenerated from meristems were further propagated by rooting of cuttings. Of the rooted cuttings, 10% were morphologically juvenile.     

Limited acclimation of photosynthesis to blue light in the seaweed Gracilaria tenuistipitata

Changes in photosynthetic capacity of the seaweed Gracilaria tenuistipitata Zhang et Xia acclimated to monochromatic blue light were studied. For this purpose, affinity for external inorganic carbon, light use efficiency, carbonic anhydrase (CA; EC 4.2.1.1) activity and content of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) were determined in thalli acclimated to 45 µmol m⁻² s⁻¹ of blue light. Thalli cultured in white light of the same photon fluence rate were used as a control. Lower maximal photosynthetic rates (i.e. at light and carbon saturation) were obtained in the thalli cultured in blue light. Apparently, this lower photosynthetic capacity was not due to differences in affinity and/or capacity for use of external dissolved inorganic carbon (DIC) since (1) CA activity did not change significantly and (2) similar values of photosynthetic conductance for DIC at alkaline pH were obtained (0.95 × 10⁻⁶ m s⁻¹). In addition, the pool size of Rubisco was not modified by the blue light treatment since there were no significant differences in Rubisco content between white (12.14% of soluble proteins) and blue light (12.13% of soluble proteins) treatments. In contrast, F _v/ F _m was increased by 11% and photosynthetic efficiency for oxygen production was reduced by 50% in blue light. This absence of correlation between quantum yields for maximum stable charge separation of photosystem II and oxygen evolution suggests that blue light promote changes in rates of photosynthetic electron flow.     

Can albuterol help resistance exercise attenuate unloading-induced bone loss?

Hind-limb-suspended rats incur attenuated bone loss with beta(2)-agonists, and humans note similar changes with concurrent resistance exercise. To examine if the beta(2)-agonist albuterol helps resistance exercise reduce unloading-induced bone loss, human subjects performed 40 days of unilateral limb suspension with their left legs, otherwise refraining from normal ambulatory activity. While performing left leg strength training 3 days.week(-1), subjects received a concurrent placebo or albuterol (16 mg.day(-1)) treatment. Left leg muscle and bone changes were analyzed with 2 x 2 analyses of covariance (ANCOVAs). Mechanical loading values were calculated from workouts and compared using a 2 x 5 analysis of variance (ANOVA) and a Tukey post hoc test. The resistance exercise-albuterol assignment evoked significant (p < 0.05) left leg bone mineral content (BMC) gains (+2.24%) after 40 days. During the final unloading days, the resistance exercise-placebo group's mechanical loading data declined (-13.91%) significantly (p < 0.05) versus initial values. A resistance exercise-albuterol assignment likely increased BMC by maintaining the mechanical loading stimulus.     

Generalization of 'same-different' classification abilities in bottlenosed dolphins

Two bottlenosed dolphins taught to classify pairs of three-dimensional objects as either same or different were tested with novel stimulus sets to determine how well their classification abilities would generalize. Both dolphins were immediately able to classify novel pairs of planar objects, differing only in shape, as same or different. When tested on sets of three objects consisting of either all different objects or of two identical objects and one different object, both dolphins proved to be able to classify 'all different' sets as different and 'not all different' sets as same, at levels significantly above chance. These data suggest that dolphins can use knowledge about similarity-based classification strategies gained from previous training to perform successfully in a variety of novel same-different classification tasks. Visual classificatory abilities of dolphins appear to be comparable to those that have been demonstrated in primates.