Advantages of using different data sources in assessment of landscape change and its effect on visual scale

This paper presents a data-flexible indicator framework for analysis of visual landscape character; the VisuLands framework. The theory-based framework encompasses currently used indicators for visual assessment based on four different data sources: land cover data, aerial photographs, landscape photographs and field observations. This paper presents a study applying the VisuLands framework in analysis of landscape change and its effect on visual scale in a landscape in Southeast Sweden. The paper provides a critical assessment of the pros and cons of the approach. It identifies the advantages and disadvantages of using different data sources as well as the applicability and sensitivity of existing indicators in detecting visible landscape change. The results show that while some of the VisuLands indicators are relatively easily applied, others are more complex and demanding in terms of interpretation. The flexibility of the VisuLands framework makes it applicable and user-friendly as it helps meet the requirements and restrictions of the users. The assessment has shown that the different data sources complement each other and that applying indicators using various data sources, when available, will enhance the comprehensiveness of visual landscape assessment. The experience of this study is that the VisuLands framework is a useful tool in landscape analysis, monitoring and planning, which provides a repeatable, systematic and transparent approach with strong links to landscape theory.     

Proteolytic processing of the ovine prion protein in cell cultures

The cellular compartment and purpose of the proteolytic processing of the prion protein (PrP) are still under debate. We have studied ovine PrP constructs expressed in four cell lines; murine neuroblastoma cells (N2a), human neuroblastoma cells (SH-SY5Y), dog kidney epithelial cells (MDCK), and human furin-deficient colon cancer cells (LoVo). Cleavage of PrP in LoVo cells indicates that the processing is furin independent. Neither is it reduced by some inhibitors of lysosomal proteinases, proteasomes or zinc-metalloproteinases, but incubation with bafilomycin A1, an inhibitor of vacuolar H+/ATPases, increases the amount of uncleaved PrP in the apical medium of MDCK cells. Mutations affecting the putative cleavage site near amino acid 113 reveal that the cleavage is independent of primary structure at this site. Absence of glycosylphosphatidylinositol anchor and glycan modifications does not influence the proteolytic processing of PrP. Our data indicate that PrP is cleaved during transit to the cell membrane.     

Alternative Translation Initiation Generates Cytoplasmic Sheep Prion Protein

Cytoplasmic localization of the prion protein (PrP) has been observed in different species and cell types. We have investigated this poorly understood phenomenon by expressing fusion proteins of sheep prion protein and green fluorescent protein (^GFPPrP) in N2a cells, with variable sequence context surrounding the start codon Met¹. ^GFPPrP expressed with the wild-type sequence was transported normally through the secretory pathway to the cell surface with acquisition of N-glycan groups, but two N-terminal fragments of ^GFPPrP were detected intracellularly, starting in frame from Met¹⁷. When ^GFPPrP was expressed with a compromised Kozak sequence (^GFPPrP*), dispersed intracellular fluorescence was observed. A similar switch from pericellular to intracellular PrP localization was seen when analogous constructs of sheep PrP, without inserted GFP, were expressed, showing that this phenomenon is not caused by the GFP tag. Western blotting revealed a reduction in glycosylated forms of ^GFPPrP*, whereas the N-terminal fragments starting from Met¹⁷ were still present. Formation of these N-terminal fragments was completely abolished when Met¹⁷ was replaced by Thr, indicating that leaky ribosomal scanning occurs for normal sheep PrP and that translation from Met¹⁷ is the cause of the aberrant cytoplasmic localization observed for a fraction of the protein. In contrast, the same phenomenon was not detected upon expression of similar constructs for mouse PrP. Analysis of samples from sheep brain allowed immunological detection of N-terminal PrP fragments, indicating that sheep PrP is subject to similar processing mechanisms in vivo.PrP^{C 2} is a cell surface glycoprotein with an essential role in the pathogenesis of transmissible neurodegenerative prion diseases (1, 2). According to the prion hypothesis, a misfolded, pathogenic form of the protein (PrP^Sc) is the sole constituent of transmissible prions (3, 4), but the molecular details and required environs for the misfolding are incompletely understood. As would be expected for a glycosylphosphatidylinositol-anchored protein with N-linked glycans, PrP^C is observed at the outer leaflet of the plasma membrane, the end point of the secretory route. The half-time at the plasma membrane is fairly short, because the protein may undergo shedding or endocytic internalization (5–9). Thus, PrP^C can be encountered throughout the secretory and endocytic routes and is also able to leave cells via exosomes derived from multivesicular endosomes (10). In agreement with this, studies of the subcellular distribution of PrP^C in mammalian brain have identified localization to the outer cell membrane, in the Golgi apparatus, and in endosomal vesicles (11, 12). However, others have found that PrP^C is not solely associated with membranes, but, in some subpopulations of neurons, is localized to the cytoplasm (13, 14). In line with the latter observations, transgenic mice expressing PrP carrying a C-terminal GFP tag demonstrated intense cytoplasmic fluorescence from a limited number (approximately 1%) of the neurons in certain brain areas, such as the hippocampus (15). Immunohistochemical detection of intracellular, possibly cytoplasmic, PrP has also been reported from large mononuclear cells in the gut wall of sheep (16) and from enteric neurons in mice (17). The recent observations of pronounced cytoplasmic aggregation of PrP in pancreatic β-cells of rats prone to development of diabetes mellitus provide a perplexing example of nonstandard PrP localization in non-neuronal cells (18).The flexibility observed in the subcellular localization of PrP^C has been suggested to be a requirement for normal functions of the protein (14, 19, 20), but how cytoplasmic and nuclear variants arise has not been established. Cytoplasmic PrP could be a result of retro-translocation from the endoplasmic reticulum (ER), as part of an unfolded protein response (21–23) or from attenuated ER import of PrP under conditions of lumenal stress in the ER (24, 25). The finding of intact ER-targeting signal sequences on cytoplasmic PrPs (25, 26) favors the latter mechanism, namely a reduced ER import of PrP, possibly caused by saturation of the ER translocation machinery or an overload of unfolded proteins within the ER. However, no signs of stress or pathology could be detected in neurons of wild-type mice expressing cytoplasmic PrP (14), which led to the suggestion that the cytoplasmic appearance of PrP could constitute a physiologically relevant, but minor, pathway for the protein.Forced cytoplasmic expression of PrP in transgenic mice (22) and in the nematode Caenorhabditis elegans (27) resulted in neurodegenerative disease, suggesting that toxic mislocalization of PrP could be part of the pathogenic mechanism in prion diseases (28). However, transgenic mice expressing cytoplasmic PrP, on a PrP-null background, developed cerebellar atrophy but were resistant to experimental prion infection (29), suggesting that cytoplasmic PrP is unlikely to serve as substrate for prion replication. Furthermore, data obtained from transgenic mice expressing an anchorless secretory PrP show that, although these mice accumulate PrP-containing amyloid plaques upon challenge with PrP^Sc, they fail to develop clinical prion disease (30). Thus, membrane-attached PrP appears to be a prerequisite for development of prion-derived neurodegeneration.In eukaryotes, ribosomes bind specifically to linear mRNAs carrying a 7-methylguanosine 5′-end cap and slide along the mRNA in the 5′ → 3′ direction until they encounter the first start codon (AUG), from which the protein translation starts exclusively. Therefore, eukaryotic mRNAs are generally monocistronic. However, deviations from this standard principle have been reported, in which protein translation is initiated at alternative start codons either up or downstream from the primary AUG. The best characterized mechanism is known as context-dependent leaky ribosomal scanning (LRS) (31). This cap-dependent mechanism is particularly operative when the optimal (5′-GCCRCCaugG-3′) sequence context surrounding the first AUG codon is compromised, most notably at positions R⁻³ (where R= purine, A or G, but optimally G) and G⁺⁴ (32, 33).In this work, we report that in a cell culture system, sheep PrP mRNA displays a tendency to allow alternative translation initiation through LRS. Met¹⁷ serves as an internal in-frame alternative start codon giving rise to PrP with a severely shortened ER-targeting peptide.Although the LRS mechanism is active in sheep PrP, it appears to occur much less in mouse PrP (34). The molecular explanation and possible pathophysiological relevance of these observations in relation to PrP function await further studies. Interestingly, during the review process of this paper, observations of cytoplasmic PrP similar to some of those described herein were reported for human and hamster PrP (35).     

The ecology of visual landscapes: Exploring the conceptual common ground of visual and ecological landscape indicators

This paper explores the conceptual common ground between visual and ecological landscape indicators. Indicators can be used to summarise complex information about landscape functions and the ability to extract a common set of indicators for analysing different landscape functions may provide valuable support for multiple-use planning.The development of landscape ecological indicators has been a very active research field that has resulted in a wide range of landscape metrics and composite indices with a strong conceptual base in landscape ecological principles. For the visual aspects of landscapes, however, this conceptual base is often missing and thus hindering progress in the development of indicators. This paper presents the results of an analysis of the correspondence between ecological indicators and visual indicators in order to explore whether there is common ground in both concept and operation. The conceptual level is presented for both ecological and visual indicators, as it is the concepts that define their aim and scope. We feel that transparency over this issue is crucial to the development and use of environmental indicators. The approach identified a candidate set of indicators that capture important aspects of both ecological and visual quality. The strength of the approach is that it forces us to focus on the identification of what we wish to indicate by means of landscape theory and assessment that are relevant to a specific landscape context. We believe that the approach presented here forms the basis for development of new methods for understanding the impact of landscape change in a management and planning context.     

Differences in the apical and basolateral pathways for glycosaminoglycan biosynthesis in Madin-Darby canine kidney cells

Serglycin with a green fluorescent protein tag (SG-GFP) expressed in epithelial Madin-Darby canine kidney cells is secreted mainly (85%) into the apical medium, but the glycosaminoglycan (GAG) chains on the SG-GFP protein core secreted basolaterally (15%) carry most of the sulfate added during biosynthesis (Tveit et al. (2005) J. Biol. Chem., 280, 29596-29603). Here we report further differences in apical and basolateral GAG synthesis. The less intensely sulfated chondroitin sulfate (CS) chains on apically secreted SG-GFP are longer than CS chains attached to basolateral SG-GFP, whereas the heparan sulfate (HS) chains are of similar lengths. When the supply of 3'-phosphoadenosine-5'-phosphosulfate (PAPS) is limited by chlorate treatment, the synthesis machinery maintains sulfation of HS chains on basolateral SG-GFP until it is inhibited at 50 mM chlorate, whereas basolateral CS chains lose sulfate already at 12.5 mM chlorate and become longer. Apically, incorporation of 35S-sulfate into CS is reduced to a lesser extent at higher chlorate concentrations than basolateral CS, although apical CS is less intensely sulfated than basolateral CS in control cells. Similar to what was found for basolateral HS, sulfation of apical HS was not reduced at chlorate concentrations below 50 mM. Also, protein-free, xyloside-based GAG chains secreted basolaterally are more intensely sulfated than their apical counterpart, supporting the view that separate apical and basolateral pathways exist for GAG synthesis and sulfation. Introduction of benzyl beta-d-xyloside (BX) to the GAG synthesis machinery reduces the apical secretion of SG-GFP dramatically and also the modification of SG-GFP by HS.     

An immunocytochemical method for assaying oestrogen receptors in breast cancers. A comparison with the steroid binding assay

The presence of oestrogen receptors was studied in 105 human breast carcinomas using monoclonal antibodies (Abbott ER-ICA kit). The oestrogen receptors of neoplastic cells were semiquantitatively measured and correlations were made to receptor values determined by a dextran-coated charcoal (DCC) steroid binding assay and to histological grade. Immunoreactive cells were found in about 2/3 of the tumours. Usually only a fraction of the cells within each tumour were immunoreactive, and the staining intensity varied among different cells. In general, well differentiated tumours had a greater proportion of immunoreactive cells than poorly differentiated ones. In most cases (65/98) a good agreement was found between the ER-ICA method and the DCC assay. However, in 33 cases discrepancies were demonstrated.