Bovine liver slices combined with an androgen transcriptional activation assay: an in-vitro model to study the metabolism and bioactivity of steroids

Previously we described the properties of a rapid and robust yeast androgen bioassay for detection of androgenic anabolic compounds, validated it, and showed its added value for several practical applications. However, biotransformation of potent steroids into inactive metabolites, or vice versa, is not included in this screening assay. Within this context, animal-friendly in-vitro cellular systems resembling species-specific metabolism can be of value. We therefore investigated the metabolic capacity of precision-cut slices of bovine liver using 17β-testosterone (T) as a model compound, because this is an established standard compound for assessing the metabolic capacity of such cellular systems. However, this is the first time that slice metabolism has been combined with bioactivity measurements. Moreover, this study also involves bioactivation of inactive prohormones, for example dehydroepiandrosterone (DHEA) and esters of T, and although medium extracts are normally analyzed by HPLC, here the metabolites formed were identified with more certainty by ultra-performance liquid chromatography time-of-flight mass spectrometry (UPLC–TOFMS) with accurate mass measurement. Metabolism of T resulted mainly in the formation of the less potent phase I metabolites 4-androstene-3,17-dione (4-AD), the hydroxy-T metabolites 6α, 6β, 15β, and 16α-OH-T, and the phase II metabolite T-glucuronide. As a consequence the overall androgenic activity, as determined by the yeast androgen bioassay, decreased. In order to address the usefulness of bovine liver slices for activation of inactive steroids, liver slices were exposed to DHEA and two esters of T. This resulted in an increase of androgenic activity, because of the formation of 4-AD and T

Recombinant cell bioassays for the detection of (gluco)corticosteroids and endocrine-disrupting potencies of several environmental PCB contaminants

Sensitive and robust bioassays for glucocorticoids are very useful for the pharmaceutical industry, environmental scientists and veterinary control. Here, a recombinant yeast cell was constructed that expresses the human glucocorticoid receptor alpha and a green fluorescent reporter protein in response to glucocorticoids. Both the receptor construct and the reporter construct were stably integrated into the yeast genome. The correct and specific functioning of this yeast glucocorticoid bioassay was studied by exposures to cortisol and other related compounds and critically compared to a GR-CALUX bioassay based on a human bone cell. Although less sensitive, the new yeast glucocorticoid bioassay showed sensitivity towards all (gluco)corticoids tested, with the following order in relative potencies: budesonide >> corticosterone > dexamethasone > cortisol = betamethasone > prednisolone > aldosterone. Hormone representatives for other hormone nuclear receptors, like 17β-estradiol for the oestrogen receptor, 5α-dihydrotestosterone for the androgen receptor and progesterone for the progesterone receptor, showed no clear agonistic responses, whilst some polychlorinated biphenyls were clearly able to interfere with the GR activity

Intestinal barrier function in response to abundant or depleted mucosal glutathione in Salmonella-infected rats

ABSTRACT: BACKGROUND: Glutathione, the main antioxidant of intestinal epithelial cells, is suggested to play an important role in gut barrier function and prevention of inflammation-related oxidative damage as induced by acute bacterial infection. Most studies on intestinal glutathione focus on oxidative stress reduction without considering functional disease outcome. Our aim was to determine whether depletion or maintenance of intestinal glutathione changes susceptibility of rats to Salmonella infection and associated inflammation. Rats were fed a control diet or the same diet supplemented with buthionine sulfoximine (BSO; glutathione depletion) or cystine (glutathione maintenance). Inert chromium ethylenediamine-tetraacetic acid (CrEDTA) was added to the diets to quantify intestinal permeability. At day 4 after oral gavage with Salmonella enteritidis (or saline for non-infected controls), Salmonella translocation was determined by culturing extra-intestinal organs. Liver and ileal mucosa were collected for analyses of glutathione, inflammation markers and oxidative damage. Faeces was collected to quantify diarrhoea. RESULTS: Glutathione depletion aggravated ileal inflammation after infection as indicated by increased levels of mucosal myeloperoxidase and interleukin-1beta. Remarkably, intestinal permeability and Salmonella translocation were not increased. Cystine supplementation maintained glutathione in the intestinal mucosa but inflammation and oxidative damage were not diminished. Nevertheless, cystine reduced intestinal permeability and Salmonella translocation. CONCLUSIONS: Despite increased infection-induced mucosal inflammation upon glutathione depletion, this tripeptide does not play a role in intestinal permeability, bacterial translocation and diarrhoea. On the other hand, cystine enhances gut barrier function by a mechanism unlikely to be related to glutathione

A large geometric distortion in the first photointermediate of rhodopsin, determined by double-quantum solid-state NMR

Double-quantum magic-angle-spinning NMR experiments were performed on 11,12-C-13(2)-retinylidene-rhodopsin under illumination at low temperature, in order to characterize torsional angle changes at the C11-C12 photoisomerization site. The sample was illuminated in the NMR rotor at low temperature (similar to 120 K) in order to trap the primary photointermediate, bathorhodopsin. The NMR data are consistent with a strong torsional twist of the HCCH moiety at the isomerization site. Although the HCCH torsional twist was determined to be at least 40A degrees, it was not possible to quantify it more closely. The presence of a strong twist is in agreement with previous Raman observations. The energetic implications of this geometric distortion are discussed

Microhabitat competition between Iberian fish species and the endangered Júcar nase (Parachondrostoma arrigonis; Steindachner, 1866)

"This is an Accepted Manuscript of an article published by Taylor & Francis in Journal of Ecohydraulics on 24-01-2017, available online: https://www.tandfonline.com/doi/full/10.1080/24705357.2016.1276417"[EN] Competition with invasive species is recognized as having a major impact on biodiversity conservation. The upper part of the Cabriel River (Eastern Iberian Peninsula) harbours the most important population of the Júcar nase (Parachondrostoma arrigonis; Steindachner, 1866), a fish species in imminent danger of extinction. Currently, this species cohabits with several non-native species, such as the Iberian nase (Pseudochondrostoma polylepis; Steindachner, 1864) and the bermejuela (Achondrostoma arcasii; Steindachner, 1866). The potential habitat competition with these species was studied by analysing the spatial and temporal overlapping of suitable microhabitats. Generalized Additive Mixed Models (GAMMs) were developed to model microhabitat selection and these GAMMs were used to assess the habitat suitability (i.e. probability of presence) under several flows simulated with River2D. The Júcar nase will compete, spatially and temporally, for the few suitable microhabitats with bermejuela and, to a lesser extent, with small Iberian nase; conversely, large Iberian nase was of minor concern, due to increased differences in habitat preferences. This study represents an important assessment of potential competition and, therefore, these results might assist to better define future management practices in the upper part of the Cabriel River.This study was funded by the Spanish Ministry of Economy and Competitiveness through the SCARCE project (Consolider Ingenio 2010 CSD2009 00065); the Universitat Politècnica de València, through the project UPPTE/2012/294 [PAID 06 12]; it was also partially funded by the IMPADAPT project (CGL2013-48424-C2-1-R) with Spanish MINECO (Ministerio de Economía y Competitividad) and FEDER funds. The authors would like to thank the help of the Conselleria de Territori i Vivenda (Generalitat Valenciana) and the Confederación Hidrográfica del Júcar (Spanish government), which provided environmental data to Alfredo Ollero, and the two anonymous reviewers who first suggested the submission of the paper to a regular journal. Finally, we would like to thank TECNOMA S.A. for the development of the hydraulic model.Muñoz Mas, R.; Soares Costa, RM.; Alcaraz-Hernández, JD.; Martinez-Capel, F. (2017). Microhabitat competition between Iberian fish species and the endangered Júcar nase (Parachondrostoma arrigonis; Steindachner, 1866). Journal of Ecohydraulics. 2(1):3-15. https://doi.org/10.1080/24705357.2016.1276417S31521Alcaraz, C., Carmona-Catot, G., Risueño, P., Perea, S., Pérez, C., Doadrio, I., & Aparicio, E. (2014). Assessing population status of Parachondrostoma arrigonis (Steindachner, 1866), threats and conservation perspectives. Environmental Biology of Fishes, 98(1), 443-455. doi:10.1007/s10641-014-0274-3ALMEIDA, D., & GROSSMAN, G. D. (2012). Utility of direct observational methods for assessing competitive interactions between non-native and native freshwater fishes. Fisheries Management and Ecology, 19(2), 157-166. doi:10.1111/j.1365-2400.2012.00847.xAlmeida, D., Merino-Aguirre, R., Vilizzi, L., & Copp, G. H. (2014). Interspecific Aggressive Behaviour of Invasive Pumpkinseed Lepomis gibbosus in Iberian Fresh Waters. PLoS ONE, 9(2), e88038. doi:10.1371/journal.pone.0088038Anderson, D. R., Burnham, K. P., & Thompson, W. L. (2000). Null Hypothesis Testing: Problems, Prevalence, and an Alternative. The Journal of Wildlife Management, 64(4), 912. doi:10.2307/3803199Aparicio, E., Vargas, M. J., Olmo, J. M., & de Sostoa, A. (2000). Environmental Biology of Fishes, 59(1), 11-19. doi:10.1023/a:1007618517557Arlot, S., & Celisse, A. (2010). A survey of cross-validation procedures for model selection. Statistics Surveys, 4(0), 40-79. doi:10.1214/09-ss054Austin, M. (2007). Species distribution models and ecological theory: A critical assessment and some possible new approaches. Ecological Modelling, 200(1-2), 1-19. doi:10.1016/j.ecolmodel.2006.07.005Baltz, D. M., Vondracek, B., Brown, L. R., & Moyle, P. B. (1991). Seasonal Changes in Microhabitat Selection by Rainbow Trout in a Small Stream. Transactions of the American Fisheries Society, 120(2), 166-176. doi:10.1577/1548-8659(1991)1202.3.co;2Barbet-Massin, M., Jiguet, F., Albert, C. H., & Thuiller, W. (2012). Selecting pseudo-absences for species distribution models: how, where and how many? Methods in Ecology and Evolution, 3(2), 327-338. doi:10.1111/j.2041-210x.2011.00172.xBeakes, M. P., Moore, J. W., Retford, N., Brown, R., Merz, J. E., & Sogard, S. M. (2012). EVALUATING STATISTICAL APPROACHES TO QUANTIFYING JUVENILE CHINOOK SALMON HABITAT IN A REGULATED CALIFORNIA RIVER. River Research and Applications, 30(2), 180-191. doi:10.1002/rra.2632BROOK, B., SODHI, N., & BRADSHAW, C. (2008). Synergies among extinction drivers under global change. Trends in Ecology & Evolution, 23(8), 453-460. doi:10.1016/j.tree.2008.03.011Brosse, S., Laffaille, P., Gabas, S., & Lek, S. (2001). Is scuba sampling a relevant method to study fish microhabitat in lakes? Examples and comparisons for three European species. Ecology of Freshwater Fish, 10(3), 138-146. doi:10.1034/j.1600-0633.2001.100303.xCLAVERO, M. (2011). Assessing the risk of freshwater fish introductions into the Iberian Peninsula. Freshwater Biology, 56(10), 2145-2155. doi:10.1111/j.1365-2427.2011.02642.xCollares-Pereira, M. J., & Coelho, M. M. (1983). Biometrical analysis of Chondrostoma polylepis x Rutilus arcasi natural hybrids (Osteichthyes-Cypriniformes-Cyprinidae). Journal of Fish Biology, 23(5), 495-509. doi:10.1111/j.1095-8649.1983.tb02930.xCosta, R. M. S., Martínez-Capel, F., Muñoz-Mas, R., Alcaraz-Hernández, J. D., & Garófano-Gómez, V. (2011). HABITAT SUITABILITY MODELLING AT MESOHABITAT SCALE AND EFFECTS OF DAM OPERATION ON THE ENDANGERED JúCAR NASE, PARACHONDROSTOMA ARRIGONIS (RIVER CABRIEL, SPAIN). River Research and Applications, 28(6), 740-752. doi:10.1002/rra.1598Dal Pozzolo A, Caelen O, Bontempi G. 2015. unbalanced: Racing for unbalanced methods selection. R package version 2.0.Elith, J., & Leathwick, J. R. (2009). Species Distribution Models: Ecological Explanation and Prediction Across Space and Time. Annual Review of Ecology, Evolution, and Systematics, 40(1), 677-697. doi:10.1146/annurev.ecolsys.110308.120159Elvira, B., & Almodovar, A. (2001). Freshwater fish introductions in Spain: facts and figures at the beginning of the 21st century. Journal of Fish Biology, 59(sa), 323-331. doi:10.1111/j.1095-8649.2001.tb01393.xElvira, B., & Almodóvar, A. (2006). Threatened fishes of the world: Chondrostoma arrigonis (Steindachner, 1866) (Cyprinidae). Environmental Biology of Fishes, 81(1), 27-28. doi:10.1007/s10641-006-9172-7Friedman, J. H. (2001). machine. The Annals of Statistics, 29(5), 1189-1232. doi:10.1214/aos/1013203451Fukuda, S., De Baets, B., Waegeman, W., Verwaeren, J., & Mouton, A. M. (2013). Habitat prediction and knowledge extraction for spawning European grayling (Thymallus thymallus L.) using a broad range of species distribution models. Environmental Modelling & Software, 47, 1-6. doi:10.1016/j.envsoft.2013.04.005Girard, V., Monti, D., Valade, P., Lamouroux, N., Mallet, J.-P., & Grondin, H. (2013). HYDRAULIC PREFERENCES OF SHRIMPS AND FISHES IN TROPICAL INSULAR RIVERS. River Research and Applications, 30(6), 766-779. doi:10.1002/rra.2675Gozlan, R. E., Britton, J. R., Cowx, I., & Copp, G. H. (2010). Current knowledge on non-native freshwater fish introductions. Journal of Fish Biology, 76(4), 751-786. doi:10.1111/j.1095-8649.2010.02566.xGuay, J. C., Boisclair, D., Rioux, D., Leclerc, M., Lapointe, M., & Legendre, P. (2000). Development and validation of numerical habitat models for juveniles of Atlantic salmon (Salmo salar). Canadian Journal of Fisheries and Aquatic Sciences, 57(10), 2065-2075. doi:10.1139/f00-162Guisan, A., Graham, C. H., Elith, J., & Huettmann, F. (2007). Sensitivity of predictive species distribution models to change in grain size. Diversity and Distributions, 13(3), 332-340. doi:10.1111/j.1472-4642.2007.00342.xHeggenes, J., Brabrand, Åg., & Saltveit, S. (1990). Comparison of Three Methods for Studies of Stream Habitat Use by Young Brown Trout and Atlantic Salmon. Transactions of the American Fisheries Society, 119(1), 101-111. doi:10.1577/1548-8659(1990)1192.3.co;2Jowett, I. G., & Davey, A. J. H. (2007). A Comparison of Composite Habitat Suitability Indices and Generalized Additive Models of Invertebrate Abundance and Fish Presence–Habitat Availability. Transactions of the American Fisheries Society, 136(2), 428-444. doi:10.1577/t06-104.1Jowett, I. G., & Duncan, M. J. (2012). Effectiveness of 1D and 2D hydraulic models for instream habitat analysis in a braided river. Ecological Engineering, 48, 92-100. doi:10.1016/j.ecoleng.2011.06.036Laurikkala, J. (2001). Improving Identification of Difficult Small Classes by Balancing Class Distribution. Lecture Notes in Computer Science, 63-66. doi:10.1007/3-540-48229-6_9Leunda, P. (2010). Impacts of non-native fishes on Iberian freshwater ichthyofauna: current knowledge and gaps. Aquatic Invasions, 5(3), 239-262. doi:10.3391/ai.2010.5.3.03Lin, X., & Zhang, D. (1999). Inference in generalized additive mixed modelsby using smoothing splines. Journal of the Royal Statistical Society: Series B (Statistical Methodology), 61(2), 381-400. doi:10.1111/1467-9868.00183Liu, C., Berry, P. M., Dawson, T. P., & Pearson, R. G. (2005). Selecting thresholds of occurrence in the prediction of species distributions. Ecography, 28(3), 385-393. doi:10.1111/j.0906-7590.2005.03957.xMaceda-Veiga, A. (2012). Towards the conservation of freshwater fish: Iberian Rivers as an example of threats and management practices. Reviews in Fish Biology and Fisheries, 23(1), 1-22. doi:10.1007/s11160-012-9275-5Maggini, R., Lehmann, A., Zimmermann, N. E., & Guisan, A. (2006). Improving generalized regression analysis for the spatial prediction of forest communities. Journal of Biogeography, 33(10), 1729-1749. doi:10.1111/j.1365-2699.2006.01465.xMarr, S. M., Olden, J. D., Leprieur, F., Arismendi, I., Ćaleta, M., Morgan, D. L., … García-Berthou, E. (2013). A global assessment of freshwater fish introductions in mediterranean-climate regions. Hydrobiologia, 719(1), 317-329. doi:10.1007/s10750-013-1486-9MARTÍNEZ-CAPEL, F., GARCÍA DE JALÓN, D., WERENITZKY, D., BAEZA, D., & RODILLA-ALAMÁ, M. (2009). Microhabitat use by three endemic Iberian cyprinids in Mediterranean rivers (Tagus River Basin, Spain). Fisheries Management and Ecology, 16(1), 52-60. doi:10.1111/j.1365-2400.2008.00645.xMouton, A. M., Alcaraz-Hernández, J. D., De Baets, B., Goethals, P. L. M., & Martínez-Capel, F. (2011). Data-driven fuzzy habitat suitability models for brown trout in Spanish Mediterranean rivers. Environmental Modelling & Software, 26(5), 615-622. doi:10.1016/j.envsoft.2010.12.001Mouton, A. M., De Baets, B., & Goethals, P. L. M. (2010). Ecological relevance of performance criteria for species distribution models. Ecological Modelling, 221(16), 1995-2002. doi:10.1016/j.ecolmodel.2010.04.017Muñoz-Mas, R., Fukuda, S., Vezza, P., & Martínez-Capel, F. (2016). Comparing four methods for decision-tree induction: A case study on the invasive Iberian gudgeon ( Gobio lozanoi ; Doadrio and Madeira, 2004). Ecological Informatics, 34, 22-34. doi:10.1016/j.ecoinf.2016.04.011Muñoz-Mas, R., Lopez-Nicolas, A., Martínez-Capel, F., & Pulido-Velazquez, M. (2016). Shifts in the suitable habitat available for brown trout (Salmo trutta L.) under short-term climate change scenarios. Science of The Total Environment, 544, 686-700. doi:10.1016/j.scitotenv.2015.11.147Muñoz-Mas, R., Martínez-Capel, F., Garófano-Gómez, V., & Mouton, A. M. (2014). Application of Probabilistic Neural Networks to microhabitat suitability modelling for adult brown trout (Salmo trutta L.) in Iberian rivers. Environmental Modelling & Software, 59, 30-43. doi:10.1016/j.envsoft.2014.05.003Muñoz-Mas, R., Martínez-Capel, F., Schneider, M., & Mouton, A. M. (2012). Assessment of brown trout habitat suitability in the Jucar River Basin (SPAIN): Comparison of data-driven approaches with fuzzy-logic models and univariate suitability curves. Science of The Total Environment, 440, 123-131. doi:10.1016/j.scitotenv.2012.07.074Muñoz-Mas, R., Papadaki, C., Martínez-Capel, F., Zogaris, S., Ntoanidis, L., & Dimitriou, E. (2016). Generalized additive and fuzzy models in environmental flow assessment: A comparison employing the West Balkan trout (Salmo farioides; Karaman, 1938). Ecological Engineering, 91, 365-377. doi:10.1016/j.ecoleng.2016.03.009Olaya-Marín, E. J., Martínez-Capel, F., Soares Costa, R. M., & Alcaraz-Hernández, J. D. (2012). Modelling native fish richness to evaluate the effects of hydromorphological changes and river restoration (Júcar River Basin, Spain). Science of The Total Environment, 440, 95-105. doi:10.1016/j.scitotenv.2012.07.093Paredes-Arquiola, J., Solera, A., Martinez-Capel, F., Momblanch, A., & Andreu, J. (2014). Integrating water management, habitat modelling and water quality at the basin scale and environmental flow assessment: case study of the Tormes River, Spain. Hydrological Sciences Journal, 59(3-4), 878-889. doi:10.1080/02626667.2013.821573Platts, P. J., McClean, C. J., Lovett, J. C., & Marchant, R. (2008). Predicting tree distributions in an East African biodiversity hotspot: model selection, data bias and envelope uncertainty. Ecological Modelling, 218(1-2), 121-134. doi:10.1016/j.ecolmodel.2008.06.028Reyjol, Y., Hugueny, B., Pont, D., Bianco, P. G., Beier, U., Caiola, N., … Virbickas, T. (2007). Patterns in species richness and endemism of European freshwater fish. Global Ecology and Biogeography, 16(1), 65-75. doi:10.1111/j.1466-8238.2006.00264.xRibeiro, F., Elvira, B., Collares-Pereira, M. J., & Moyle, P. B. (2007). Life-history traits of non-native fishes in Iberian watersheds across several invasion stages: a first approach. Biological Invasions, 10(1), 89-102. doi:10.1007/s10530-007-9112-2RIBEIRO, F., & LEUNDA, P. M. (2012). Non-native fish impacts on Mediterranean freshwater ecosystems: current knowledge and research needs. Fisheries Management and Ecology, 19(2), 142-156. doi:10.1111/j.1365-2400.2011.00842.xRincon, P. A., Correas, A. M., Morcillo, F., Risueno, P., & Lobon-Cervia, J. (2002). Interaction between the introduced eastern mosquitofish and two autochthonous Spanish toothcarps. Journal of Fish Biology, 61(6), 1560-1585. doi:10.1111/j.1095-8649.2002.tb02498.xRobalo, J. I., Almada, V. C., Levy, A., & Doadrio, I. (2007). Re-examination and phylogeny of the genus Chondrostoma based on mitochondrial and nuclear data and the definition of 5 new genera. Molecular Phylogenetics and Evolution, 42(2), 362-372. doi:10.1016/j.ympev.2006.07.003Romão, F., Quintella, B. R., Pereira, T. J., & Almeida, P. R. (2011). Swimming performance of two Iberian cyprinids: the Tagus nase Pseudochondrostoma polylepis (Steindachner, 1864) and the bordallo Squalius carolitertii (Doadrio, 1988). Journal of Applied Ichthyology, 28(1), 26-30. doi:10.1111/j.1439-0426.2011.01882.xShiroyama, R., & Yoshimura, C. (2016). Assessing bluegill (Lepomis macrochirus) habitat suitability using partial dependence function combined with classification approaches. Ecological Informatics, 35, 9-18. doi:10.1016/j.ecoinf.2016.06.005Thomas, J. A., & Bovee, K. D. (1993). Application and testing of a procedure to evaluate transferability of habitat suitability criteria. Regulated Rivers: Research & Management, 8(3), 285-294. doi:10.1002/rrr.3450080307Vezza, P., Muñoz-Mas, R., Martinez-Capel, F., & Mouton, A. (2015). Random forests to evaluate biotic interactions in fish distribution models. Environmental Modelling & Software, 67, 173-183. doi:10.1016/j.envsoft.2015.01.005Vilizzi, L., Copp, G. H., & Roussel, J.-M. (2004). Assessing variation in suitability curves and electivity profiles in temporal studies of fish habitat use. River Research and Applications, 20(5), 605-618. doi:10.1002/rra.767Wood, S. N. (2004). Stable and Efficient Multiple Smoothing Parameter Estimation for Generalized Additive Models. Journal of the American Statistical Association, 99(467), 673-686. doi:10.1198/016214504000000980Wood, S. N. (2006). Generalized Additive Models. doi:10.1201/9781420010404Zuur, A. F., Ieno, E. N., Walker, N., Saveliev, A. A., & Smith, G. M. (2009). Mixed effects models and extensions in ecology with R. Statistics for Biology and Health. doi:10.1007/978-0-387-87458-

Design and Applications of an Intelligent Financial Reporting and Auditing Agent with Net Knowledge (FRAANK)

This paper discusses the use of intelligent Internet agents as essential tools for automating financial analysis functions in the virtual world. Several important characteristic features of intelligent agents are discussed, including autonomy, communication ability, collaboration, and mobility. This paper focuses on developing a new intelligent agent called FRAANK – Financial Reporting and Auditing Agent with Net Knowledge. The prototype of FRAANK described in this paper provides intelligent access to, and processing and integration of rapidly changing financial information available from various sources on the Internet. FRAANK is an example of an agent that provides a value-added service that can be used for extracting data from natural text financial statements and converting them into XBRL-tagged statements, can potentially be utilized in an auditing practice, or used by investors and creditors in making their decisions

Proton gradient formation in early endosomes from proximal tubules

AbstractHeavy endosomes were isolated from proximal tubules using a combination of magnesium precipitation and wheat-germ agglutinin negative selection techniques. Two small GTPases (Rab4 and Rab5) known to be specifically present in early endosomes were identified in our preparations. Endosomal acidification was followed fluorimetrically using acridine orange. In presence of chloride ions and ATP, the formation of a proton gradient (ΔpH) was observed. This process is due to the activity of an electrogenic V-type ATPase present in the endosomal membrane since specific inhibitors bafilomycin and folimycin effectively prevented or eliminated endosomal acidification. In presence of chloride ions (Km = 30 mM) the formation of the proton gradient was optimal. Inhibitors of chloride channel activity such as DIDS and NPPB reduced acidification. The presence of sodium ions stimulated the dissipation of the proton gradient. This effect of sodium was abolished by amiloride derivative (MIA) but only when loaded into endosomes, indicating the presence of a physiologically oriented Na+/H+-exchanger in the endosomal membrane. Monensin restored the gradient dissipation. Thus three proteins (V-type ATPase, Cl−-channel, Na+/H+-exchanger) present in early endosomas isolated from proximal tubules may regulate the formation, maintenance and dissipation of the proton gradient

Contexting Koreans: Does the High/Low Model Work?

South Korea is assumed to be a high-context culture with extensive shared information and an emphasis on relationships in doing business. The follow ing study reported here tests this assumption and illustrates similarities and differences between Korean and American writers in an attempt to document language differences between high- and low- context societies. Data in the texts studied did not confirm the high/low contextfeatures expected. South Korean texts showed more similarities to than differences from the American texts, and the language features found suggest a more complex context situa tion than the high/low context model may be able to accommodate.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66563/2/10.1177_108056999806100403.pd