The abundance of nitrogen cycle genes amoA and nifH depends on land-uses and soil types in South-Eastern Australia

Nitrogen is a critical nutrient in plant-based primary production systems, therefore measurements of N cycling by microorganisms may add value to agricultural soil monitoring programs. Bacterial-mediated nitrogen cycling was investigated in soils from two broad land-uses (managed and remnant vegetation) across different Soil Orders from three geomorphic zones in Victoria, Australia, by examining the abundance of the genes amoA and nifH using quantitative polymerase chain reaction (qPCR). The aim of the study was to identify parameters influencing bacterial populations possessing the genes nifH and amoA, and examine their distribution at a regional scale across different management treatments. The gene amoA was most abundant in the neutral to slightly alkaline surface soils from Calcarosols in North-West Victoria. There was a highly significant (P < 0.001) interaction between land-use and geomorphic zones in terms of the abundance of amoA. Detection of the gene nifH was site specific with low copy number (less than 100 copies per nanogram of DNA) observed for some strongly acidic surface soil sites in North-East Victoria (Dermosols) and South-West Victoria (Sodosols/Chromosols), while nifH was more abundant in selected Calcarosols of North-West Victoria. The gene amoA was detected across more sites than nifH and was strongly influenced by land-use, with almost consistently greater abundance in managed compared to remnant sites, particularly for North-West and South-West Victoria. The abundance of nifH was not related to land-use, with similar copy numbers observed for both managed and remnant sites at some locations. For the gene nifH, there was no significant interaction between land-use and geomorphic zones, between managed and remnant sites or between the three geomorphic zones. Regression tree analysis revealed a number of likely soil chemical and microbial variables which may act as drivers of gene abundance of amoA and nifH. Variables identified as drivers for amoA included pH, Olsen P, microbial biomass carbon, nitrate and total nitrogen while for nifH the variables were microbial biomass carbon, electrical conductivity, microbial biomass nitrogen, total nitrogen and total potassium. Measures of N cycling genes could be used as an additional indicator of soil health to assess potential ecosystem functions. The spatial scale of the current study demonstrates that a landscape approach may assist soil health monitoring programs by evaluating N cycle gene abundance in the context of the different microbial and chemical conditions related to Soil Order and land-use management.     

Production of lactoferricin and other cationic peptides from food grade bovine lactoferrin with various iron saturation levels

Purification of lactoferricin (Lfcin), a cationic antimicrobial peptide, was achieved by peptic digestion of food grade bovine lactoferrin (LF) followed by fractionation on an industrial grade cation exchange resin with stepwise salt gradient elution. The digest and eluted fractions were partially characterized by MALDI-ToF MS and N-terminal sequencing. A fraction eluted using phosphate buffer with 2.0 M NaCl contained predominantly two peptides with masses of 3196 and 3124 Da, which corresponded to the 26- and 25-amino acid peptides FKCRR WQWRM KKLGA PSITC VRRAF (A), containing the Lfcin sequence. Putative sequences of cationic peptides in other eluted fractions included FKNKS RSFQ, WRMKK LGAPS ITCVR RA, and GAPSI TCVRR AFALE CIRAI AEKKA. The iron saturation level of LF had no effect on the production of Lfcin. Nevertheless, the digestion of LF containing lower iron content led to the production of a higher quantity of low molecular weight cationic peptides. A two-step process using industrial grade cation exchange resin led to 35% recovery of Lfcin and also produced other cationic peptides with potential bioactive properties.     

Black cohosh acts as a mixed competitive ligand and partial agonist of the serotonin receptor

Extracts of the rhizome of black cohosh [Actaea racemosa L., formerly called Cimicifuga racemosa (L.) Nutt.] were evaluated for potential mechanisms of action in the alleviation of menopausal hot flashes. Ovariectomized Sprague-Dawley rats were administered a 40% 2-propanol extract of black cohosh [4, 40, and 400 mg/(kg.day)] by gavage for 2 weeks with or without estradiol [50 microg/(kg.day)] to determine if black cohosh could act as an estrogen or antiestrogen on the basis of an increase in uterine weight or vaginal cellular cornification. No effects were observed on uterine weight or on vaginal cellular cornification in rats treated with black cohosh alone or in combination with 17beta-estradiol, indicating this black cohosh extract had no estrogenic or antiestrogenic properties in the ovariectomized rat model. To evaluate other potential pathways by which black cohosh might reduce menopausal hot flashes, serotonin activity was first assessed by the inhibition of radioligand binding to cell membrane preparations containing recombinant human serotonin receptor (5-HT) subtypes. A 40% 2-propanol extract of black cohosh was tested against 10 subtypes of the serotonin receptor, revealing the presence of compounds with strong binding to the 5-HT(1A), 5-HT(1D), and 5-HT(7) subtypes. Subsequent binding studies were carried out using 5-HT(1A) and 5-HT(7) receptors because of their association with the hypothalamus, which has been implicated in the generation of hot flashes. The black cohosh 40% 2-propanol extract inhibited [(3)H]lysergic acid diethylamide (LSD) binding to the human 5-HT(7) receptor (IC(50) = 2.4 +/- 0.4 microg/mL) with greater potency than binding of [(3)H]-8-hydroxy-2-(di-N-propylamino)tetralin to the rat 5-HT(1A) receptor (IC(50) = 13.9 +/- 0.6 microg/mL). Analysis of ligand binding data indicated that components of a black cohosh methanol extract functioned as a mixed competitive ligand of the 5-HT(7) receptor. In addition, a black cohosh methanol extract elevated cAMP levels in 293T-5-HT(7)-transfected HEK cells, suggesting the extract acted as a partial agonist at the receptor. The elevation in cAMP mediated by the black cohosh extract could be reversed in the presence of the antagonist methiothepin, indicating a receptor-mediated process. These data suggest that reductions in hot flashes in some women taking black cohosh may not be due to estrogenic properties. This study identifies other possible biological targets of black cohosh that could account for reported biological effects.     

Incidence of plasmid-linked antibiotic-heavy metal resistant enterics in water-sediment from agricultural and harbor sites

This study assesses the impact of antibiotics used in clinical and veterinary practices, on the incidence of antibiotic- heavy metal resistant enterics in fresh water and sediment from agricultural and harbor sample sites. A total of 848 bacterial strains of the familyEnterobacteriaceae was isolated from agricultural and lake harbor samples and identified to genus level. These were examined for antibiotic-heavy metal resistance. A select smaller number of these isolates were also examined for the presence of plasmids and ability to transfer antibiotic resistance via conjugation or transformation. More than 85 % of the 848 isolates from all four sites were resistant to Ph, Zn, and Co while 5.6% to 16% were resistant to Te and 2.4% to 5.7% to Hg. The ranking of metal resistance according to frequency was the same as the frequency of metal occurrence in sediment samples. Of the total isolates tested, 87% were resistant to six or more antibiotics and 74% were also simultaneously resistant to Co, Zn, and Ph. Testing the resistance of the water isolates to antibiotics used solely in animal husbandry-veterinary medicine indicated that 55.6% of the agricultural isolates possessed resistance to these antibiotics while only 31.9% of the isolates from harbor water showed resistance to the same antibiotics. Of 41 ampicillin resistant isolates examined, 16 (39%)were capable of transferring antibiotic-heavy resistance markers via conjugation. From this same group, plasmid DNA preparations were made. Of these latter preparations, 67% transformed recipientE. coli cells while 58% possessed discernible, often multiple plasmids when examined by gel electrophoresis.     

Study of protein-lipid interactions at the bovine serum albumin/oil interface by Raman microspectroscopy

The interface of 10 or 25% (w/v) bovine serum albumin (BSA), pH 7, buffered solution against mineral or corn oil was studied with a Raman microscope. A gradient of distribution of protein and oil at the interface was observed. The difference spectrum obtained by subtracting the spectrum of mineral or corn oil from that of the BSA/oil interface indicated interactions involving different functional groups of the BSA and the oil molecules. Against mineral oil, the BSA spectrum showed reduced intensity of the tryptophan band at 750 cm(-1) and reduced intensity ratio of the tyrosine doublet at 850-830 cm(-1), indicating changes in the microenvironment of these hydrophobic residues. A negative band at 2850 cm(-1) indicated the involvement of the CH groups in the mineral oil. However, the amide regions, normally assigned to protein secondary structure, were not significantly changed. When the spectrum of BSA was subtracted from the BSA/mineral oil interface spectrum, the resultant difference spectrum showed changes of symmetric and antisymmetric CCC stretches at 980 and 1071 cm(-1), respectively. In contrast, the difference spectrum of BSA/corn oil interface - BSA showed a decrease of CH(2) symmetric stretching at 2850 cm(-1) and a decrease of unsaturated fatty acid hydrocarbon chain stretch at 3010 cm(-1). Raman spectroscopy is a useful tool to study the nature of protein-lipid interactions.     

Competition for nitrogen between adult European beech and its offspring is reduced by avoidance strategy

Plant growth, reproduction, and biomass allocation may be affected differently by nitrogen availability depending on tree size and age. In this context, competition for limited N may be avoided by different strategies of N acquisition between different vegetation components (i.e., seedlings, mature trees, other woody and herbaceous understorey). This study investigated in a field experiment whether the competition for N between different vegetation components in beech forests was prevented via seasonal timing of N uptake and affected by microbial N use. For this purpose, a removal approach was used to study the seasonal effects on N uptake and N metabolites in adult beech trees and beech natural regeneration, as well as soil microbial processes of inorganic N production and utilisation. We found that the competition for N between beech natural regeneration and mature beech trees was reduced by seasonal avoidance strategies (“good parenting”) of N uptake regardless of the N sources used. In spring, organic and inorganic N uptake capacity was significantly higher in beech seedlings compared to adult beech trees, whereas in autumn mature beech trees showed the highest N uptake rates. Removal of vegetation components did not result in changes in soil microbial N processes in the course of the growing season. Thus, N resources released by the removal of vegetation components were marginal. This consistency in soil microbial N processes indicates that competition between plants and soil microorganisms for N was not avoided by timing of acquisition during the vegetation period, but existed during the entire growing season. In conclusion, N nutrition in the studied forest ecosystem seems to be optimally attuned to European beech.     

The race to x-ray microbeam and nanobeam science

X-ray microbeams are an emerging characterization tool with broad implications for science, ranging from materials structure and dynamics, to geophysics and environmental science, to biophysics and protein crystallography. We describe how submicrometer hard x-ray beams with the ability to penetrate tens to hundreds of micrometers into most materials and with the ability to determine local composition, chemistry, and (crystal) structure can characterize buried sample volumes and small samples in their natural or extreme environments. Beams less than 10 nanometers have already been demonstrated, and the practical limit for hard x-ray beam size, the limit to trace-element sensitivity, and the ultimate limitations associated with near-atomic structure determinations are the subject of ongoing research.     

Effects of feeding diets containing various dietary protein and lipid ratios on the growth performance and pigmentation of post-juvenile coho salmon Oncorhynchus kisutch reared in sea water

Six extruded dry diets formulated to contain one of two levels of digestible protein (37% or 44%) and one of three levels of digestible lipid (16%, 23% or 30%) on a dry weight basis and a seventh diet (commercial control) were used to feed triplicate groups of post‐juvenile coho salmon Oncorhynchus kisutch in sea water. Fish were fed to satiation twice daily for 168 days. Growth performances were monitored every 28 days. On day 168, samples were taken from each replicate group per dietary treatment for determinations of whole‐body and muscle proximate compositions. Fatty acid compositions and astaxanthin concentration in both the experimental diets and fish flesh were assessed by gas chromatography (GC) and high‐performance liquid chromatography (HPLC) respectively. Coho salmon fed diets containing the higher lipid levels (23–30%) exhibited improved feed efficiency, protein efficiency ratio, percentage protein deposition and percentage gross energy utilization. Higher protein content diets supported better growth than those that had lower protein content, but the former led to lowered protein efficiency ratio, percentage protein deposition and gross energy utilization. Fish fed the diets with high lipid levels (23% or 30%) also had higher astaxanthin content in raw flesh.     

Improving understanding of early behavioral indicators of lumbosacral disease in working dogs using 3D visualization of skeletal movements during working tasks: Feasibility study

An improved understanding of the early behavioral indicators of lumbosacral disease in working dogs may allow earlier interventions and help reduce premature retirement because of disability. However, recognition of early behavioral indicators can be challenging in stoic, high-drive working dogs because they often mask clinical signs. The purpose of this feasibility study was to develop a technique for visualizing canine skeletal movements during working tasks and to describe veterinary clinical specialist opinions on the utility of the visualization technique. Three detection-trained police dogs with a recent history of working task deficits and suspected lumbosacral disease were recruited for the study. Conventional and motion capture video recordings were acquired as dogs performed walking and search high working tasks. Whole-body computed tomography (CT) scans were acquired using clinical multislice CT scanners. Image data from motion capture recordings and whole-body CT scans were analyzed and merged. Three-dimensional (3D) computer animation video clips of skeletal movements were created for each dog and each task, using multiple viewing angle perspectives. Interactive meetings with veterinary clinical specialist reviewers were used to refine point placements for the final renderings. Veterinary clinical specialists reviewed final 3D animation movie clips and recorded their opinions on the utility for the visualization technique. Veterinary clinical specialists reported that the computer animations helped them recognize behavioral characteristics that they had not initially noticed in physical examinations. Potential applications for this visualization technique include creating educational training aids for veterinary students, owners, and handlers; assisting veterinarians in planning rehabilitative treatments; and assisting researchers in developing computer models for biomechanical analyses. Future controlled prospective studies are needed in a large number of normal and affected working dogs to improve accuracy of the visualization technique and test the effect of the technique on observer performance.     

Effect of a novel topical diester glucocorticoid spray on immediate- and late-phase cutaneous allergic reactions in Maltese–beagle atopic dogs: a placebo-controlled study

The inhibitory effect of 0.0584% hydrocortisone aceponate spray on immediate- and late-phase skin reactions and the duration of inhibition after medication withdrawal were studied in 10 Maltese-beagle atopic dogs. All subjects were sprayed on axillary and inguinal regions and on one randomly chosen side of the thorax once daily for 14 (phase 1) or 7 days (phase 2). Intradermal injections (IDT) of histamine and anticanine IgE antiserum were performed bilaterally on the thorax before, 7 and 14 days after treatment. During phase 2, IDT was performed once weekly for 5 weeks. Each IDT was evaluated by an investigator blinded to the site of active treatment. Skin biopsies of 24-h anti-IgE-associated late-phase reactions were collected from both thoracic sides before and 14 days after treatment to determine the number of inflammatory cells and dermal thickness. Phase 1 : Histamine and anti-IgE-induced global wheal scores at treated sites were significantly lower after 7 and 14 days with negative reactions present in >90% of dogs. Late-phase reactions at both sides were also significantly decreased compared with that at baseline, and this was associated with reduced inflammatory cell influx. Moreover, a significant decrease in dermal thickness was recorded at treated sides after 14 days. Phase 2 : Histamine reactions became positive at untreated sides in all dogs 2 weeks after treatment. In conclusion, the 0.0584% hydrocortisone aceponate spray significantly decreased immediate- and late-phase IDT reactions, and prolonged application caused skin atrophy at treated sites. A 2-week withdrawal period prior to IDT is proposed.