Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron (dgn)-destabilized Green Fluorescent Protein (GFP)-based Reporter Protein

Proteasome is the main intracellular organelle involved in the proteolytic degradation of abnormal, misfolded, damaged or oxidized proteins ^{1, 2}. Maintenance of proteasome activity was implicated in many key cellular processes, like cell''s stress response ³, cell cycle regulation and cellular differentiation ⁴ or in immune system response ⁵. The dysfunction of the ubiquitin-proteasome system has been related to the development of tumors and neurodegenerative diseases ^{4, 6}. Additionally, a decrease in proteasome activity was found as a feature of cellular senescence and organismal aging ^{7, 8, 9, 10}. Here, we present a method to measure ubiquitin-proteasome activity in living cells using a GFP-dgn fusion protein. To be able to monitor ubiquitin-proteasome activity in living primary cells, complementary DNA constructs coding for a green fluorescent protein (GFP)–dgn fusion protein (GFP–dgn, unstable) and a variant carrying a frameshift mutation (GFP–dgnFS, stable ¹¹) are inserted in lentiviral expression vectors. We prefer this technique over traditional transfection techniques because it guarantees a very high transfection efficiency independent of the cell type or the age of the donor. The difference between fluorescence displayed by the GFP–dgnFS (stable) protein and the destabilized protein (GFP-dgn) in the absence or presence of proteasome inhibitor can be used to estimate ubiquitin-proteasome activity in each particular cell strain. These differences can be monitored by epifluorescence microscopy or can be measured by flow cytometry.     

Biological availability and humic properties of dissolved organic carbon in Lake Balaton (Hungary)

The nursery role of the Mondego estuary for marine fish species was studied between June 2003 and May 2004. The spatial and temporal distribution and abundance patterns of 0-group Dicentrarchus labrax (Linnaeus, 1758), Platichthys flesus (Linnaeus, 1758) and Solea solea (Linnaeus, 1758) were analyzed based on monthly sampling surveys in five stations along the estuarine gradient. Fishing took place during the night at low water of spring tides, using a 2 m beam trawl. The spatial patterns of estuarine colonization were different according to species. D. labrax showed a wider distribution, but the main nursery ground was the same as for S. solea. Highest densities of S. solea juveniles were found in oligohaline areas, with muddy bottoms and high benthic invertebrates availability, while P. flesus occurred mainly in the sandy uppermost areas. D. labrax was found in both these areas. Fish abundance in the estuary mainly reflected seasonal changes.     

Where Are Socioeconomically Deprived Immigrants Located in Chile? A Spatial Analysis of Census Data Using an Index of Multiple Deprivation from the Last Three Decades (1992-2012)

Introduction and Purpose of the Study

Immigrants in Chile have diverse characteristics and include socioeconomically deprived populations. The location of socioeconomically deprived immigrants is important for the development of public policy intelligence at the local and national levels but their areas of residence have not been mapped in Chile. This study explored the spatial distribution of socioeconomic deprivation among immigrants in Chile, 1992–2012, and compared it to the total population.

Material and Methods

Areas with socioeconomically deprived populations were identified with a deprivation index which we developed modelled upon the Index of Multiple Deprivation (IMD) for England. Our IMD was based upon the indicators of unemployment, low educational level (primary) and disability from Census data at county level for the three decades 1992, 2002 and 2012, for 332, 339 and 343 counties respectively. We developed two versions of the IMD one based on disadvantage among the total population and another focused upon the circumstances of immigrants only. We generated a spatial representation of the IMD using GIS, for the overall IMD score and for each dimension of the index, separately. We also compared the immigrants´ IMD to the total population´s IMD using Pearson´s correlation test.

Results

Results showed that socioeconomically deprived immigrants tended to be concentrated in counties in the northern and central area of Chile, in particular within the Metropolitan Region of Santiago. These were the same counties where there was the greatest concentration of socioeconomic deprivation for the total population during the same time periods. Since 1992 there have been significant change in the location of the socioeconomically deprived populations within the Metropolitan Region of Santiago with the highest IMD scores for both the total population and immigrants becoming increasingly concentrated in the central and eastern counties of the Region.

Conclusion

This is the first study analysing the spatial distribution of socioeconomic deprivation among international immigrants and the total population in a Latin American country. Findings could inform policy makers about location of areas of higher need of social protection in Chile, for both immigrants and the total resident population in the country.     

A Disjunct Argentine Ant Metacolony in Macaronesia and Southwestern Europe

The Argentine ant, Linepithema humile, originally from South America, is now a major pest in many parts of the world with Mediterranean-like climates. Earlier research indicated that southwestern European L. humile populations are segregated into two distinct supercolonies, the ‘Main’ supercolony extending through Portugal, Spain, southern France, and Italy, and the ‘Catalonian’ supercolony in eastern Spain. Both supercolonies are unicolonial, with workers showing no aggression towards members of the same supercolony, but severe aggression towards members of the other supercolony. Here we evaluated the behavioral relationships among non-native L. humile populations on the Macaronesian islands of Madeira and the Azores and non-native populations in southwestern Europe. We conducted aggression assays among L. humile workers from Madeira, the Azores, and the two southwestern European supercolonies. We found no aggressive interactions among any combination of workers from Madeira, the Azores, and the Main supercolony. However, workers from Madeira and the Azores always fought aggressively with workers from the Catalonian supercolony. Thus, the populations of L. humile in Madeira and the Azores appear to be unicolonial and act as if they belong to the Main supercolony of southwestern Europe. This set of geographically separated but mutually compatible supercolonies, which we term a ‘metacolony,’ appears to descend from one supercolony. Historical evidence suggests that the first L. humile population in the greater Mediterranean region was established in Madeira and that propagules from the dominant supercolony in Madeira gave rise to the dominant supercolonies in to other parts of the region.     

Silvopastoralism in the Alps: Native plant species selection under different grazing pressure

To evaluate the suitability of wood pastures as a managing tool in subalpine regions it is essential to know more about the influence of grazing on the ground vegetation. This study assessed native plant species selection by cattle at different stocking rates, feeding habits and site preferences of cattle. Based on the results, conclusions concerning the value of silvopastoral systems in the Alps were drawn. A field study on six different wood pasture areas, grazed by cattle at different stocking rates, was accompanied by an experiment on three adjoining areas of 0.51 ha each, stocked with either three, six, or nine heifers. Plant species were recorded in plots of 20 cm × 20 cm before and after grazing, and the intensity of grazing on each species was assessed. At low stocking rates, grasses and tall species were most intensely grazed, while at higher stocking rates the intake of forbs and small species increased. Since no relationship was found between nutritional value and species preference, other factors such as accessibility of a plant seem to be important for the feeding preferences of cattle. The preference for grasses at low and medium stocking rates suggests that an increased growth of forbs might lead to an increase in plant species diversity.     

Fungal laccase, cellobiose dehydrogenase, and chemical mediators: combined actions for the decolorization of different classes of textile dyes

Dyes belonging to the mono-, di-, tri- and poly-azo as well as anthraquinonic and mono-azo Cr-complexed classes, chosen among the most utilized in textile applications, were employed for a comparative enzymatic decolorization study using the extracellular crude culture extracts from the white rot fungus Funalia (Trametes) trogii grown on different culture media and activators able to trigger different levels of expression of oxidizing enzymes: laccase and cellobiose dehydrogenase. Laccase containing extracts were capable to decolorize some dyes from all the different classes analyzed, whereas the recalcitrant dyes were subjected to the combined action of laccase and the chemical mediator HBT, or laccase plus cellobiose dehydrogenase. Correlations among the decolorization degree of the various dyes and their electronic and structural diversities were rationalized and discussed. The utilization of cellobiose dehydrogenase in support to the activity of laccase for the decolorization of azo textile dyes resulted in substantial increases in decolorization for all the refractory dyes proving to be a valid alternative to more expensive and less environmentally friendly chemical treatments of textile dyes wastes.     

Characterization of tachykinin NK2 receptor in the anterior pituitary gland

Tachykinins are a family of bioactive peptides that interact with three subtypes of receptors: NK1, NK2 and NK3. Substance P has greater affinity for NK1, and neurokinin A (NKA) for NK2 receptor subtype. Although only NK1 receptor has been characterized in the anterior pituitary gland, some evidence suggests the existence of NK2 receptors in this gland. Therefore, we investigated the presence of NK2 receptors in the anterior pituitary gland of male rats by radioligand binding studies using labeled SR48968, a non peptidic specific antagonist. [3H]SR48968 specific binding to cultured anterior pituitary cells was time-dependent and saturable, but with a lower affinity than previously reported values for cells expressing NK2 receptors. Unlabeled NKA inhibited only partially [(3)H]SR48968 specific binding to whole anterior pituitary cells. Since SR48968 is a non polar molecule, we performed experiments to discriminate surface from intracellular binding sites. SR48968 exhibited both surface and intracellular specific binding. Analysis of the surface-bound ligand indicated that [3H]SR48968 binds to one class of receptor with high affinity. Neurokinin A completely displaced [3H]SR48968 surface specific binding fitting to a two-site/two-state model with high and low affinity. Additionally, immunocytochemical studies showed that the NK2 receptor is expressed at least in a subset of lactotropes. These results demonstrate the presence of NK2 receptors in the anterior pituitary gland and suggest that NKA actions in this gland are mediated, at least in part, by the NK2 receptor subtype.     

Competing for blood: the ecology of parasite resource competition in human malaria–helminth co‐infections

Ecological theory suggests that co‐infecting parasite species can interact within hosts directly, via host immunity and/or via resource competition. In mice, competition for red blood cells (RBCs) between malaria and bloodsucking helminths can regulate malaria population dynamics, but the importance of RBC competition in human hosts was unknown. We analysed infection density (i.e. the concentration of parasites in infected hosts), from a 2‐year deworming study of over 4000 human subjects. After accounting for resource‐use differences among parasites, we find evidence of resource competition, priority effects and a competitive hierarchy within co‐infected individuals. For example reducing competition via deworming increased Plasmodium vivax densities 2.8‐fold, and this effect is limited to bloodsucking hookworms. Our ecological, resource‐based perspective sheds new light into decades of conflicting outcomes of malaria–helminth co‐infection studies with significant health and transmission consequences. Beyond blood, investigating within‐human resource competition may bring new insights for improving human health.