Copper-induced trafficking of the Cu-ATPases: A key mechanism for copper homeostasis

The Menkes protein (MNK) and Wilson protein (WND) are transmembrane, CPX-type Cu-ATPases with six metal binding sites (MBSs) in the N-terminal region containing the motif GMXCXXC. In cells cultured in low copper concentration MNK and WND localize to the transGolgi network but in high copper relocalize either to the plasma membrane (MNK) or a vesicular compartment (WND). In this paper we investigate the role of the MBSs in Cu-transport and trafficking. The copper transport activity of MBS mutants of MNK was determined by their ability to complement a strain of Saccharomyces cerevisiae deficient in CCC2 (ccc2), the yeast MNK/WND homologue. Mutants (CXXC to SXXS) of MBS1, MBS6, and MBSs1-3 were able to complement ccc2 while mutants of MBS4-6, MBS5-6 and all six MBS inactivated the protein. Each of the inactive mutants also failed to display Cu-induced trafficking suggesting a correlation between trafficking and transport activity. A similar correlation was found with mutants of MNK in which various MBSs were deleted, but two constructs with deletion of MBS5-6 were unable to traffic despite retaining 25% of copper transport activity. Chimeras in which the N-terminal MBSs of MNK were replaced with the corresponding MBSs of WND were used to investigate the region of the molecules that is responsible for the difference in Cu-trafficking of MNK and WND. The chimera which included the complete WND N-terminus localized to a vesicular compartment, similar to WND in elevated copper. Deletions of various MBSs of the WND N-terminus in the chimera indicate that a targeting signal in the region of MBS6 directs either WND/MNK or WND to a vesicular compartment of the cell.     

X-ray absorption and phase contrast imaging to study the interplay between plant roots and soil structure

Plant performance is, at least partly, linked to the location of roots with respect to soil structure features and the micro-environment surrounding roots. Measurements of root distributions from intact samples, using optical microscopy and field tracings have been partially successful but are imprecise and labour-intensive. Theoretically, X-ray computed micro-tomography represents an ideal solution for non-invasive imaging of plant roots and soil structure. However, before it becomes fast enough and affordable or easily accessible, there is still a need for a diagnostic tool to investigate root/soil interplay. Here, a method for detection of undisturbed plant roots and their immediate physical environment is presented. X-ray absorption and phase contrast imaging are combined to produce projection images of soil sections from which root distributions and soil structure can be analyzed. The clarity of roots on the X-ray film is sufficient to allow manual tracing on an acetate sheet fixed over the film. In its current version, the method suffers limitations mainly related to (i) the degree of subjectivity associated with manual tracing and (ii) the difficulty of separating live and dead roots. The method represents a simple and relatively inexpensive way to detect and quantify roots from intact samples and has scope for further improvements. In this paper, the main steps of the method, sampling, image acquisition and image processing are documented. The potential use of the method in an agronomic perspective is illustrated using surface and sub-surface soil samples from a controlled wheat trial. Quantitative characterization of root attributes, e.g. radius, length density, branching intensity and the complex interplay between roots and soil structure, is presented and discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

The use of 2,2′‐dithiobis(5‐nitropyridine) (DTNP) for deprotection and diselenide formation in protected selenocysteine‐containing peptides

In contrast to the large number of sidechain protecting groups available for cysteine derivatives in solid phase peptide synthesis, there is a striking paucity of analogous selenocysteine Se‐protecting groups in the literature. However, the growing interest in selenocysteine‐containing peptides and proteins requires a corresponding increase in availability of synthetic routes into these target molecules. It therefore becomes important to design new sidechain protection strategies for selenocysteine as well as multiple and novel deprotection chemistry for their removal. In this paper, we outline the synthesis of two new Fmoc selenocysteine derivatives [Fmoc‐Sec(Meb) and Fmoc‐Sec(Bzl)] to accompany the commercially available Fmoc‐Sec(Mob) derivative and incorporate them into two model peptides. Sec‐deprotection assays were carried out on these peptides using 2,2′‐dithiobis(5‐nitropyridine) (DTNP) conditions previously described by our group. The deprotective methodology was further evaluated as to its suitability towards mediating concurrent diselenide formation in oxytocin‐templated target peptides. Sec(Mob) and Sec(Meb) were found to be extremely labile to the DTNP conditions whether in the presence or absence of thioanisole, whereas Sec(Bzl) was robust to DTNP in the absence of thioanisole but quite labile in its presence. In multiple Sec‐containing model peptides, it was shown that bis‐Sec(Mob)‐containing systems spontaneously cyclize to the diselenide using 1 eq DTNP, whereas bis‐Sec(Meb) and Sec(Bzl) models required additional manipulation to induce cyclization. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Comparing Effects of Nutrients on Algal Biomass in Streams in Two Regions with Different Disturbance Regimes and with Applications for Developing Nutrient Criteria

Responses of stream algal biomass to nutrient enrichment were studied in two regions where differences in hydrologic variability cause great differences in herbivory. Around northwestern Kentucky (KY) hydrologic variability constrains invertebrate biomass and their effects on algae, but hydrologic stability in Michigan (MI) streams permits accrual of high herbivore densities and herbivory of benthic algae. Multiple indicators of algal biomass and nutrient availability were measured in 104 streams with repeated sampling at each site over a 2−month period. Many measures of algal biomass and nutrient availability were positively correlated in both regions, however the amount of variation explained varied with measures of biomass and nutrient concentration and with region. Indicators of diatom biomass were higher in KY than MI, but were not related to nutrient concentrations in either region. Chl a and % area of substratum covered by Cladophora were positively correlated to nutrient concentrations in both regions. Cladophora responded significantly more to nutrients in MI than KY. Total phosphorus (TP) and total nitrogen (TN) explained similar amounts of variation in algal biomass, and not significantly more variation in biomass than dissolved nutrient concentrations. Low N:P ratios in the benthic algae indicated N as well as P may be limiting their accrual. Most observed responses in benthic algal biomass occurred in nutrient concentrations between 10 and 30 μg TP l⁻¹ and between 400 and 1000 μg TN l⁻¹.     

Sources of variation in response intervals after prostaglandin treatment in mares with functional corpora lutea.

Sixty-two non-cycling mares were classified according to the size of largest follicles at the time of treatment with Prostalene, an analogue of prostaglandin (PG) F-2 alpha. Although oestrus occurred in only 77.4% of mares, 98.4% ovulated at an average of 6.8 days after treatment. Greatest variance of interval to ovulation was observed in mares having follicles greater than or equal to 40 mm at the time of treatment. This was due to regression of large follicles about one-third of the time and later ovulation of a succeeding follicle. This resulted also in greatest uncertainty of prediction of ovulation time based on ovarian palpation. Ninety foaling mares were given Prostalene at various days following the first ovulation post partum. Ovulation less than 6 days after treatment was strongly associated with the presence of a large follicle on the day of treatment. Otherwise most (72%) ovulations occurred 6--10 days after treatment. The distribution of interovulatory periods resulting from Prostalene on Day 6 after ovulation differed from that of Day 8 treatment.     

Differences in airway responsiveness to leukotriene D4 in allergic sheep with and without late bronchial responses

Allergic sheep respond to inhaled antigen with either acute and late bronchial obstructions (dual responders) or only acute bronchoconstriction (acute responders). In this study we tested the hypothesis that one factor which may distinguish between these two populations is the difference in sensitivity to a specific mediator of airway anaphylaxis, leukotriene (LT) D₄ (a major component of slow reacting substance of anaphylaxis). We postulated that if the hypothesis was correct than dual responders should demonstrate increased airway responses to inhaled LTD₄ and that this increased responsiveness should also be reflected by a more severe response to inhaled antigen. To test this we used animals from both groups with the same degree of non-specific airway responsiveness to carbachol and determined their airway responses to controlled inhlation challenges with synthetic LTD₄ and antigen. Airway responsiveness to carbachol was determined by measuring the change in specific lung resistance (SR_L) to increasing concentrations of carbachol aerosol, and then identifying, by linear interpolation, the provocative carbachol concentration which produced a 150% increase (PC₁₅₀) in SR_L. Airway responses to LTD₄, and antigen were determined by measuring the percentage change in SR_L after a controlled inhlation challenge with either aerosol. Airway responsiveness to carbachol was not different between the two groups. There was, however, a difference (p<0.05) in the airway response to the same dose of LTD₄ in the two groups. Dual responders showed a 297±72% increase in SR_L as compared to a 90±13% increase in SR_L in the acute responders. Dual responders also showed a greater immediate and more prolonged response to antigen than did acute responders. These results suggest that increased responsiveness to LTD₄ may be one factor which may distinguish dual responders from acute responders.     

Status of Golden Eagle Aquila chrysaetos in Britain in 2015

Capsule: A complete survey of Golden Eagles Aquila chrysaetos in Britain in 2015 found that the population had increased by 15% since 2003 to 508 territorial pairs.

Aims: The survey aimed to investigate the population size, distribution and breeding success of Golden Eagles in Britain, and to compare results with similar surveys since the early 1980s.

Methods: Every home range was visited on a minimum of three occasions between January and August 2015. First, to look for eagles or signs of their presence (January–March), then to look for evidence of breeding or further checks for occupation (April–June) and finally to record productivity of nesting pairs (July–August).

Results: The figure of 508 territorial pairs represents a 15% increase in the population from 442 pairs in 2003. The proportion of home ranges occupied was 70%. The largest increases in the proportion of occupied home ranges were in south-central Highlands (71%), northern moors and flows (38%) and northwest Highlands (29%), with modest increases of up to 10% in the other regions. Productivity was lower in 2015 than in 2003, and there was significant variation in breeding success between regions.

Conclusion: The British Golden Eagle population has increased since 2003, although the species is absent from England and Wales. The population now meets the abundance target identified to define favourable conservation status in Scotland, and while home range occupancy has increased there is regional variation, with some regions falling below the target levels. A combination of increased annual monitoring and tagging of eagles, as well as the introduction of new legislation, may serve as effective deterrents against persecution of eagles thus facilitating this population increase. However, concerns remain over low levels of home range occupancy particularly in the east Highlands, but also the proportion of sub-adult pairs holding territory in that region and in the south-central Highlands. Persecution associated with grouse moor management has been highlighted as a particular population constraint in both of these areas.