Nitrate reductase deficient cell lines from diploid cell cultures and lethal mutant M2 plants of Arabidopsis thaliana

Summary Cell suspensions of diploid Arabidopsis thaliana were screened for resistance to chlorate on a medium with ammonium nitrate as the nitrogen source, and after plating on filters to increase the plating efficiency. Thirty-nine lines were selected, four of which were still resistant after two years of subculturing on non-selective medium. Of the latter lines three were nitrate reductase deficient but exhibited some residual nitrate reductase activity; the fourth line showed a high level of enzyme activity. Screening M2-seeds for callus production on selective medium with amino acids as the nitrogen source and chlorate revealed resistant calli in 17 out of 483 M2-groups. Nine well-growing lines, all but one (G3) exhibiting no detectable in vivo nitrate reductase activity, were classified as defective in the cofactor. Two lines (G1 and G3) could be analysed genetically at the plant level. Chlorate resistance was monogenic and recessive. Sucrose gradient fractionation of callus extracts of G1 revealed that a complete enzyme molecule can be assembled. Nitrate reductase activity in G1 could partly be restored by excess molybdenum. It is suggested that G1 is disturbed in the catalytic properties of the cofactor. It appeared that G1 is neither allelic with another molybdenum repairable mutant (B73) nor with another cofactor mutant (B25). Wilting of intact G1 plants could be ascribed to non-closing stomata.     

Red deer (<Emphasis Type="Italic">Cervus elaphus</Emphasis>) avoid mountain biking trails

Human outdoor activities commonly affect animal behaviour. Ungulates often avoid roads and trails and increase their avoidance with expanding and intensifying human recreational activity. Recently, mountain biking has become increasingly popular in many regions in Norway, but we still have limited knowledge about how mountain biking may affect wildlife. In this study, we used pellet-group counts and camera traps to study the effect of mountain biking on red deer abundance in Kaupanger, Norway. Pellet-group counts decreased close to biking trails, and it appeared that red deer avoided using areas within 40 m of such trails. Camera trap data showed that there was a tendency for deer abundance to decrease with increasing human activity (trail width) during daytime. Males reacted more strongly to this increasing activity than did females. Even with a small-scale approach, focusing on within-stand effects, we found that red deer were affected by mountain bikers and avoided areas close to biking trails. Our findings imply that any further increase in mountain biking may result in greater avoidance and, thus, less suitable habitat for red deer in forest areas.     

PD 142676 (CI 1002), a novel anticholinesterase and muscarinic antagonist

Inhibition of brain acetylcholinesterase (AChE) can provide relief from the cognitive loss associated with Alzheimer's disease (AD). However, unwanted peripheral side effects often limit the usefulness of the available anticholinesterases. Recently, we identified a dihydroquinazoline compound, PD 142676 (CI 1002) that is a potent anticholinesterase and a functional muscarinic antagonist at higher concentrations. Peripherally, PD 14276, unlike other anticholinesterases, inhibits gastrointestinal motility in rats, an effect consistent with its muscarinic antagonist properties. Centrally, the compound acts as a cholinomimetic. In rats, PD 142676, decreases core body temperature. It also increases neocortical arousal, as measured by quantitative electroencephalography, and cortical acetylcholine levels, measured by in vivo microdialysis. The compound improves the performance of C57/B10j mice in a water maze task and of aged rhesus monkeys in a delayed match-to-sample task involving short-term memory. The combined effect of AChE inhibition and muscarinic antagonism distinguishes PD 142676 from other anticholinesterases and may be useful in treating the cognitive dysfunction of AD and produce fewer peripheral side effects.     

Vegetation analysis in the coastal dune ecosystem of the Western Egyptian Desert

In the frame work of the SAMDENE project part of the coastal dune vegetation of the Western Desert of Egypt was studied using various field methods and multivariate analysis techniques. The results were compared with those of an earlier study carried out three years before, predating the protective fencing of the area. In the field methods cover-abundance estimates and frequency data were most efficient, while PCA showed to be the most powerful analysis technique. Three years of protective fencing had resulted in a slightly more homogeneous vegetation pattern in the area.     

Selectivity in Enrichment of cAMP-dependent Protein Kinase Regulatory Subunits Type I and Type II and Their Interactors Using Modified cAMP Affinity Resins

cAMP regulates cellular functions primarily by activating PKA. The involvement of PKAs in various signaling pathways occurring simultaneously in different cellular compartments necessitates stringent spatial and temporal regulation. This specificity is largely achieved by binding of PKA to protein scaffolds, whereby a distinct group of proteins called A kinase anchoring proteins (AKAPs) play a dominant role. AKAPs are a diverse family of proteins that all bind via a small PKA binding domain to the regulatory subunits of PKA. The binding affinities between PKA and several AKAPs can be different for different isoforms of the regulatory subunits of PKA. Here we employ a combination of affinity chromatography and mass spectrometry-based quantitative proteomics to investigate specificity in PKA-AKAP interactions. Three different immobilized cAMP analogs were used to enrich for PKA and its interacting proteins from several systems; HEK293 and RCC10 cells and rat lung and testis tissues. Stable isotope labeling was used to confidently identify and differentially quantify target proteins and their preferential binding affinity for the three different cAMP analogs. We were able to enrich all four isoforms of the regulatory subunits of PKA and concomitantly identify more than 10 AKAPs. A selective enrichment of the PKA RI isoforms could be achieved; which allowed us to unravel which AKAPs bind preferentially to the RI or RII regulatory domains of PKA. Of the twelve AKAPs detected, seven preferentially bound to RII, whereas the remaining five displayed at least dual specificity with a potential preference for RI. For some of these AKAPs our data provide the first insights into their specificity.cAMP is an ubiquitous second messenger that transduces signals from a variety of hormones, neurotransmitters, and inflammatory mediators to regulate a large number of key cellular processes. cAMP can influence cell growth, differentiation, and movement as well as regulating specialized actions unique to specific cell types. The principal target of cAMP is cAMP-dependent protein kinase (PKA)¹. Several other proteins such as cyclic nucleotide gated ion channels (1), phosphodiesterases (PDE) (2), and guanine nucleotide exchange factors (Epac) (3) bind cAMP. Interestingly, localized pools of cAMP regulate defined physiological events. It appears that for such events a supramolecular complex is required that comprises of the appropriate effector system together with signal termination enzymes such as PDEs and phosphatases that are sequestered by scaffolding proteins (4). Some of the best described scaffolding proteins are the so-called A-kinase anchoring proteins (AKAPs), which all bind specifically to the N-terminal dimerization domain of the PKA regulatory domain. The organization of a few of these individual supramolecular complexes containing PKA/AKAPs/PDE etc. has been described (4); numerous more of such complexes are expected to exist.The regulatory domains of mammalian PKAs exist in several isoforms such as RIα, RIβ, RIIα, and RIIβ, which are all encoded by separate genes. The two major isoforms i.e. RI and RII differ in molecular weight, isoelectric point, amino acid sequence, phosphorylation status, tissue distribution, and sub-cellular localization. RI and RII subunits are known to bind to AKAPs with distinct levels of affinity adding another level of intracellular organization for PKA and also facilitating the diversity of the cAMP-mediated signal transduction pathways. Although the PKA-R isoforms differ in functionality, they share a similar overall organization i.e. a dimerization domain, the catalytic subunits inhibitor region, and two cAMP binding domains. The two cAMP binding domains differ in cAMP binding kinetics and are known as site A and site B, respectively (5). Both sites share considerable sequence identity, as a result of a tandem gene duplication, and have conserved phosphate binding cassettes that can be considered as signature motif for cAMP binding. The relative orientation of these two sites is nonetheless, quite different in RI and RII. Additionally, A and B sites have different binding affinity to cAMP derivatives. Site A has a preference for N6-substituted analogs whereas site B is preferred by C2- and C8-substituted analogs (6).PKA has been studied extensively (7, 8). One of the important goals therein is to develop different cAMP analogs that can result in specific binding, activation, and/or inhibition for each individual cAMP interaction site of the RI and RII isoforms (9). This can help to decipher in detail specific cyclic nucleotide signaling pathways (10). To fully interpret such pathways, analogs should ideally not cross-activate (or inhibit) with other cAMP-regulated proteins such as the before mentioned PDEs, Epac, cyclic nucleotide gated ion channels, and the cGMP-dependent protein kinase (PKG). Although the latter is mainly activated by cGMP, it also binds to cAMP (11, 12). It has been suggested that cGMP and cAMP can cross-activate their respective kinases (13). This cross-talk between PKA and PKG hampers, to some degree, the study of these proteins individually, as dissecting the individual pathways of PKA and PKG requires specific binders, activators, or inhibitors (9, 14). Compared with PKA, PKG is involved in quite different signaling pathways, such as the well characterized nitric oxide-mediated relaxation of smooth muscle cells (15).The development of synthetic cAMP and cGMP analogs as tools to unravel specific signal transduction pathways requires the sensitive identification and characterization of their cyclic nucleotide interacting proteins. These proteins are typically relatively low abundant, and therefore specific enrichment techniques are essential to study these so-called cyclic-nucleotide interactomes. In recent years such affinity enrichment techniques have been coupled to sensitive mass spectrometric identification of the enriched proteins, nowadays often referred to as chemical proteomics. Chemical proteomics using small molecules as baits, i.e. messenger molecules, drugs, or metabolites, becomes more and more widely used to selectively isolate target proteins from whole cell lysates enabling the analysis of protein subcomplexes and/or signaling pathways (16, 17).In the present study we compare the properties of three cAMP analogs immobilized individually on agarose beads, for enrichment, isolation, and detection of cyclic nucleotide interacting proteins and their interaction partners, like AKAPs, directly from a crude lysate of cells and tissue. To quantify differential affinity, we use a common strategy in proteomics, namely stable isotope labeling, whereby we introduce the label via reductive amination (18–20). Most interestingly, a very selective enrichment of PKA RI isoforms can be achieved by using cAMP-agarose beads in which the hydroxyl group at the 2′ position on the ribose was replaced with a methoxyl group. This allows us to distinguish, which AKAPs bind preferentially to the RI or RII isoforms. Therefore, this approach provides an elegant tool to further decipher specific cyclic nucleotide signaling pathways.     

Traumatic Brain Injury in the Netherlands: Incidence,Costs and Disability-Adjusted Life Years

Objective

Traumatic brain injury (TBI) is a major cause of death and disability, leading to great personal suffering and huge costs to society. Integrated knowledge on epidemiology, economic consequences and disease burden of TBI is scarce but essential for optimizing healthcare policy and preventing TBI. This study aimed to estimate incidence, cost-of-illness and disability-adjusted life years (DALYs) of TBI in the Netherlands.

Methods

This study included data on all TBI patients who were treated at an Emergency Department (ED - National Injury Surveillance System), hospitalized (National Medical Registration), or died due to their injuries in the Netherlands between 2010–2012. Direct healthcare costs and indirect costs were determined using the incidence-based Dutch Burden of Injury Model. Disease burden was assessed by calculating years of life lost (YLL) owing to premature death, years lived with disability (YLD) and DALYs. Incidence, costs and disease burden were stratified by age and gender.

Results

TBI incidence was 213.6 per 100,000 person years. Total costs were €314.6 (USD $433.8) million per year and disease burden resulted in 171,200 DALYs (on average 7.1 DALYs per case). Men had highest mean costs per case (€19,540 versus €14,940), driven by indirect costs. 0–24-year-olds had high incidence and disease burden but low economic costs, whereas 25–64-year-olds had relatively low incidence but high economic costs. Patients aged 65+ had highest incidence, leading to considerable direct healthcare costs. 0–24-year-olds, men aged 25–64 years, traffic injury victims (especially bicyclists) and home and leisure injury victims (especially 0–5-year-old and elderly fallers) are identified as risk groups in TBI.

Conclusions

The economic and health consequences of TBI are substantial. The integrated approach of assessing incidence, costs and disease burden enables detection of important risk groups in TBI, development of prevention programs that target these risk groups and assessment of the benefits of these programs.     

Collagen stimulation of platelets induces a rapid spatial response of cAMP and cGMP signaling scaffolds

Intracellular communication is tightly regulated in both space and time. Spatiotemporal control is important to achieve a high level of specificity in both dimensions. For instance, cAMP-dependent kinase (PKA) attains spatial resolution by interacting with distinct members of the family of A-kinase anchoring proteins (AKAPs) that position PKA at specific loci within the cell. To control the cAMP induced signal in time, distinct signal terminators such as phosphodiesterases and phosphatases are often co-localized at the AKAP scaffold. In platelets, high levels of cAMP/cGMP maintain the resting state to allow free circulation. Exposure to collagen, for instance when the vessel is damaged, triggers platelet activation through initiation of the GPVI (glycoprotein VI)/FcRγ-chain forming the onset of a plethora of signaling pathways. Consequently overall intra-platelet cAMP and cGMP levels drop, however detail on how PKA, but also cGMP-dependent protein kinase (PKG) respond in relation to their localized signaling scaffolds is currently missing. To investigate this, we employed a quantitative chemical proteomics approach in activated human platelets enabling the specific enrichment of cAMP/cGMP signaling nodes. Our data reveal that within a few minutes several specific PKA and PKG signaling nodes respond significantly to the activating signal, whereas others do not, suggesting a rapid adaption of specific localized cAMP and cGMP pools to the stimulus. Using protein phosphorylation data gathered we touch upon the potential cross-talk between protein phosphorylation and signaling scaffold function as a general theme in platelet spatiotemporal control.