Ultrastructural identification of uncoated caveolin-independent early endocytic vehicles

Using quantitative light microscopy and a modified immunoelectron microscopic technique, we have characterized the entry pathway of the cholera toxin binding subunit (CTB) in primary embryonic fibroblasts. CTB trafficking to the Golgi complex was identical in caveolin-1 null (Cav1 -/-) mouse embryonic fibroblasts (MEFs) and wild-type (WT) MEFs. CTB entry in the Cav1 -/- MEFs was predominantly clathrin and dynamin independent but relatively cholesterol dependent. Immunoelectron microscopy was used to quantify budded and surface-connected caveoloe and to identify noncaveolar endocytic vehicles. In WT MEFs a small fraction of the total Cav1-positive structures were shown to bud from the plasma membrane (2 % per minute), and budding increased upon okadaic acid or lactosyl ceramide treatment. However, the major carriers involved in initial entry of CTB were identified as uncoated tubular or ring-shaped structures. These carriers contained GPI-anchored proteins and fluid phase markers and represented the major vehicles mediating CTB uptake in both WT and caveolae-null cells.

Hypermethylation of the 5 ' CpG island of the gene encoding the serine protease Testisin promotes its loss in testicular tumorigenesis

The Testisin gene (PRSS21) encodes a glycosylphosphatidylinositol (GPI)-linked serine protease that exhibits testis tissue-specific expression. Loss of Testisin has been implicated in testicular tumorigenesis, but its role in testis biology and tumorigenesis is not known. Here we have investigated the role of CpG methylation in Testisin gene inactivation and tested the hypothesis that Testisin may act as a tumour suppressor for testicular tumorigenesis. Using sequence analysis of bisulphite-treated genomic DNA, we find a strong relationship between hypermethylation of a 385 bp 50 CpG rich island of the Testisin gene, and silencing of the Testisin gene in a range of human tumour cell lines and in 100% (eight/eight) of testicular germ cell tumours. We show that treatment of Testisin-negative cell lines with demethylating agents and/or a histone deacetylase inhibitor results in reactivation of Testisin gene expression, implicating hypermethylation in Testisin gene silencing. Stable expression of Testisin in the Testisin-negative Tera-2 testicular cancer line suppressed tumorigenicity as revealed by inhibition of both anchorage-dependent cell growth and tumour formation in an SCID mouse model of testicular tumorigenesis. Together, these data show that loss of Testisin is caused, at least in part, by DNA hypermethylation and histone deacetylation, and suggest a tumour suppressor role for Testisin in testicular tumorigenesis.

Flotillins and the PHB domain protein family: Rafts, worms and anaesthetics

While our understanding of lipid microdomains has advanced in recent years, many aspects of their formation and dynamics are still unclear. In particular, the molecular determinants that facilitate the partitioning of integral membrane proteins into lipid raft domains are yet to be clarified. This review focuses on a family of raft-associated integral membrane proteins, termed flotillins, which belongs to a larger class of integral membrane proteins that carry an evolutionarily conserved domain called the prohibitin homology (PHB) domain. A number of studies now suggest that eucaryotic proteins carrying this domain have affinity for lipid raft domains. The PHB domain is carried by a diverse array of proteins including stomatin, podocin, the archetypal PHB protein, prohibitin, lower eucaryotic proteins such as the Dictyostelium discoideum proteins vacuolin A and vacuolin B and the Caenorhabditis elegans proteins unc-1, unc-24 and mec-2. The presence of this domain in some procaryotic proteins suggests that the PHB domain may constitute a primordial lipid recognition motif. Recent work has provided new insights into the trafficking and targeting of flotillin and other PHB domain proteins. While the function of this large family of proteins remains unclear, studies of the C. elegans PHB proteins suggest possible links to a class of volatile anaesthetics raising the possibility that these lipophilic agents could influence lipid raft domains. This review will discuss recent insights into the cell biology of flotillins and the large family of evolutionarily conserved PHB domain proteins.

Lipid rafts: contentious only from simplistic standpoints

The hypothesis that lipid rafts exist in plasma membranes and have crucial biological functions remains controversial. The lateral heterogeneity of proteins in the plasma membrane is undisputed, but the contribution of cholesterol-dependent lipid assemblies to this complex, non-random organization promotes vigorous debate. In the light of recent studies with model membranes, computational modelling and innovative cell biology, I propose an updated model of lipid rafts that readily accommodates diverse views on plasma-membrane micro-organization.

Az ökológiai lábnyom ökonómiája

Örömmel és büszkeséggel adjuk közre a Budapesti Corvinus Egyetem Fenntarthatósági Indikátorok Kutatóközpontjának első kötetét, amely az ökológiai lábnyomhoz kapcsolódó kutatásainkat foglalja össze. Az ökológiai lábnyomot lehet szeretni vagy kritizálni, de kevés kutató közömbös iránta. Jellemzően nagy lelkesedéssel vagy kritikai dühvel közelít hozzá a tudomány és a szakpolitika, és csak ritkán teszik fel azt a kérdést, hogy melyek azok a kérdések, amelyek jól megválaszolhatóak a segítségével, és melyek azok a kérdések, ahol alkalmazása sarlatánság vagy bűn. Lelkesedést és kritikát félretéve objektíven állíthatjuk, hogy az ökológiai lábnyom mind a laikusok között, mind pedig a tudományos világban terjed. Épp olyan népszerű a Google-on és Facebookon, mint a tudományos világban − A Web of Science-en vagy a Google-on végzett felméréseink azt mutatják, hogy míg több kompozit indikátor napja –HDI, ISEW GPI, ESI ¬− leáldozóban van, addig az ökológiai lábnyomra történő hivatkozások száma rendületlenül nő, még a legigényesebb, impakt faktoros tudományos közleményekben is. Ha többet nem is, de azt mindenképpen igazolja népszerűsége, hogy ez az indikátor „tud” valamit, amit az elődei nem tudtak, versenytársaihoz képest használhatóbbnak mutatkozott egyes problémák megválaszolására. Lehet tehát szeretni vagy nem szeretni, az igazi kérdés az, mely problémákat célszerű e mutató felhasználásával megközelíteni. E kötet reményeink szerint ebbe az irányba mutat. A kötet tanulmányainak túlnyomó része egy 1000 fős reprezentatív felmérés eredményeire támaszkodva mutatja be a magyar lakosság ökológiai lábnyomát, és annak összefüggéseit különböző gazdasági, társadalmi és pszichológiai tényezőkkel. Ezeket mintegy három elméleti fejezet fogja keretbe, melyek az ökológiai lábnyom elméletével és kritikájával, az energialábnyom csökkentése során tapasztalható visszapattanó hatással, illetve az ökológiai lábnyom−eltartóképesség dinamikájával foglalkoznak a Meadows-modellekből vett asszociációra támaszkodva. A kutatás „mellékterméke” egy ökológiai lábnyom kalkulátor, amely összhangban van a Global Footprint Network adataival és metodikájával, a reprezentatív felmérés eredményeire támaszkodva hoztuk létre, és konzisztens a KSH fogyasztási statisztika adataival is. Ebben a minőségében az országban egyedülálló.

Investigation of molecular mechanisms of LKB1 function in Dictyostelium development and stress responses and the function of superoxide dismutase C (SodC) in chemotaxis

The serine/threonine kinase LKB1 is a regulator of critical events including development and stress responses in metazoans. The current study was undertaken to determine the function of LKB1 in Dictyostelium . During multicellular development and in response to stress insult, an apparent increase in the DdLKB1 kinase activity was observed. Depletion of DdLKB1 with a knockdown construct led to aberrant development; a severe reduction in prespore cell differentiation and a precocious induction of prestalk cells, which were reminiscent of cells lacking GSK3, a well known cell-fate switch. Furthermore, DdLKB1 depleted cells displayed lower GSK3 activity than wild type cells in response to cAMP stimulation during development and failed to activate AMPK, a well known LKB1 target in mammals, in response to cAMP and stress insults. These results suggest that DdLKB1 positively regulates both GSK3 and AMPK during Dictyostelium development, and DdLKB1 is necessary for AMPK activation during stress response regulation. No apparent GSK3 activation was observed in response to stress insults. Spatial and temporal regulation of phosphatidylinositol-(3,4,5)-triphosphate (PIP3) along the membrane of polarized cells is important for efficient chemotaxis. A REMI screen for PIP3 suppressors in the absence of stimulation led to the identification of SodC as PIP3 regulator. Consistent with their higher PIP3 levels, sodC− cells showed defects in chemotaxis and exhibited higher intra-cellular superoxide levels. Protein localization studies along with observations from GPI specific PI-PLC treatment of wild-type cells suggested that SodC is a GPI anchored outer-membrane protein. SodC showed superoxide dismutase activity in vitro, and motility defects of sodC− cells can be rescued by expressing the intact SodC but not by the mutant SodC, which has point mutations that affect its dismutase function. Treatment of sodC− cells with LY294002, a pharmacological inhibitor of PI3K, partially rescued the polarization and chemoattractant sensing defects but not motility defects. Consistent with increased intracellular superoxide levels, sodC − cells also exhibited higher basal Ras activity, an upstream regulator of PI3K, which can be suppressed by a cell permeable superoxide scavenger, XTT, indicating that SodC is important in regulation of intracellular superoxide levels thereby regulating the Ras activity and PIP3 levels at the membrane.

Establishing a baseline of psychometric estimates of measurement of intercultural competence of Ukrainian faculty: Setting the stage for domestic and global intercultural understanding

Post-Soviet Ukraine is in a time of upheaval and transition. Internal relations between pro-Western and pro-Russian supporters have deteriorated in the light of recent political events of Euro Revolution, Russia's occupation of the Crimean peninsula, and the militant confrontations in the southeastern regions of the country. In the light of these developments, intercultural competence is greatly needed to alleviate domestic tensions and enable effective intercultural communication with the representatives of different cultures within the country and beyond its borders.^ This study established a baseline of psychometric estimates of intercultural competence of Ukrainian higher education faculty. A sample of 276 professors of different academic majors from one university in Western Ukraine participated in the research. The Global Perspective Inventory (GPI; Merrill, Braskamp, & Braskamp, 2012) was chosen as a research instrument to measure intercultural competence of the faculty members. The GPI takes into account cognitive, intrapersonal, and interpersonal domains, each of which contains two scales reflective of theories of cultural development and intercultural communication – Cognitive-Knowing, Cognitive-Knowledge, Intrapersonal-Identity, Intrapersonal-Affect, Interpersonal-Social Responsibility, and Interpersonal-Social Interaction. Because the research instrument has neither been previously used as a measure of intercultural competence, nor administered in Ukraine, it was cross-validated using a Table of Specification (Newman, Lim, & Pineda, 2013) and two sets of factor analyses. As a result, a modified version of the GPI was created for use in Ukraine.^ Multiple linear regression analyses were used to test relationships between the participants' GPI scores on intercultural competence, and several independent variables that consisted of academic discipline, intercultural experience, and how long the participants taught at the university. The analyses determined a positive relationship between the scores on three out of six scales of the original version and two out of five scales of the modified version of the GPI and all the independent variables simultaneously. The relationship between the faculty responses on the six scales of both GPI versions and the independent variables controlling for each other produced mixed results. A unique role of intercultural professional development in predicting intercultural competence was discussed.^

Undergraduate Students’ Perceptions of Study Abroad and Their Level of Achievement of Global Learning Outcomes

This study expanded on current research on study abroad and global learning, using the Global Perspective Inventory (GPI), and conducted at Florida International University (FIU) in Miami, FL. The GPI assesses the holistic development of a global perspective in higher education within three domains and their respective FIU-determined equivalents: cognitive (global awareness), intrapersonal (global perspective), and interpersonal (global engagement). The main purpose of this study was to assess FIU’s undergraduate students’ perceptions of study abroad on their level of achievement of global awareness, global perspective, and global engagement. The secondary purpose was to determine how the students described their study abroad experience and achievement of global learning. The research design for this study consisted of parallel mixed methods. The quantitative component was an ex post facto with hypothesis design, using a pretest/posttest nonequivalent group methodology. FIU undergraduates (N=147) who studied abroad for one semester or more completed the GPI pre- and post-tests. Descriptive statistics and paired t-tests were conducted to compare the means. The interviews included 10 students, and were analyzed through Structural coding, Saldaña’s In Vivo coding, and Value coding. Quantitative analyses indicated positive changes in the students’ global awareness and global perspective. These analyses also showed that the FIU students achieved higher post-test means on all the domains of the GPI compared to other studies. Qualitative analyses showed that the students’ experiences incorporated all three global learning outcomes, most notably global awareness and perspective.

Investigation of molecular mechanisms of lkb1 function in dictyostelium development and stress responses and the function of superoxide dismutase c (sodc) in chemotaxis

The serine/threonine kinase LKB1 is a regulator of critical events including development and stress responses in metazoans. The current study was undertaken to determine the function of LKB1 in Dictyostelium. During multicellular development and in response to stress insult, an apparent increase in the DdLKB1 kinase activity was observed. Depletion of DdLKB1 with a knockdown construct led to aberrant development; a severe reduction in prespore cell differentiation and a precocious induction of prestalk cells, which were reminiscent of cells lacking GSK3, a well known cell-fate switch. Furthermore, DdLKB1 depleted cells displayed lower GSK3 activity than wild type cells in response to cAMP stimulation during development and failed to activate AMPK, a well known LKB1 target in mammals, in response to cAMP and stress insults. These results suggest that DdLKB1 positively regulates both GSK3 and AMPK during Dictyostelium development, and DdLKB1 is necessary for AMPK activation during stress response regulation. No apparent GSK3 activation was observed in response to stress insults. Spatial and temporal regulation of phosphatidylinositol-(3,4,5)-triphosphate (PIP3) along the membrane of polarized cells is important for efficient chemotaxis. A REMI screen for PIP3 suppressors in the absence of stimulation led to the identification of SodC as PIP3 regulator. Consistent with their higher PIP3 levels, sodC- cells showed defects in chemotaxis and exhibited higher intra-cellular superoxide levels. Protein localization studies along with observations from GPI specific PI-PLC treatment of wild-type cells suggested that SodC is a GPI anchored outer-membrane protein. SodC showed superoxide dismutase activity in vitro, and motility defects of sodC- cells can be rescued by expressing the intact SodC but not by the mutant SodC, which has point mutations that affect its dismutase function. Treatment of sodC- cells with LY294002, a pharmacological inhibitor of PI3K, partially rescued the polarization and chemoattractant sensing defects but not motility defects. Consistent with increased intracellular superoxide levels, sodC- cells also exhibited higher basal Ras activity, an upstream regulator of PI3K, which can be suppressed by a cell permeable superoxide scavenger, XTT, indicating that SodC is important in regulation of intracellular superoxide levels thereby regulating the Ras activity and PIP3 levels at the membrane.

Composition of sediments from the Arabian Sea

Thirty seven deep-sea sediment cores from the Arabian Sea were studied geochemically (49 major and trace elements) for four time slices during the Holocene and the last glacial, and in one high sedimentation rate core (century scale resolution) to detect tracers of past variations in the intensity of the atmospheric monsoon circulation and its hydrographic expression in the ocean surface. This geochemical multi-tracer approach, coupled with additional information on the grain size composition of the clastic fraction, the bulk carbonate and biogenic opal contents makes it possible to characterize the sedimentological regime in detail. Sediments characterized by a specific elemental composition (enrichment) originated from the following sources: river suspensions from the Tapti and Narbada, draining the Indian Deccan traps (Ti, Sr); Indus sediments and dust from Rajasthan and Pakistan (Rb, Cs); dust from Iran and the Persian Gulf (Al, Cr); dust from central Arabia (Mg); dust from East Africa and the Red Sea (Zr/Hf, Ti/Al). Corg, Cd, Zn, Ba, Pb, U, and the HREE are associated with the intensity of upwelling in the western Arabian Sea, but only those patterns that are consistently reproduced by all of these elements can be directly linked with the intensity of the southwest monsoon. Relying on information from a single element can be misleading, as each element is affected by various other processes than upwelling intensity and nutrient content of surface water alone. The application of the geochemical multi-tracer approach indicates that the intensity of the southwest monsoon was low during the LGM, declined to a minimum from 15,000-13,000 14C year BP, intensified slightly at the end of this interval, was almost stable during the Bölling, Alleröd and the Younger Dryas, but then intensified in two abrupt successions at the end of the Younger Dryas (9900 14C year BP) and especially in a second event during the early Holocene (8800 14C year BP). Dust discharge by northwesterly winds from Arabia exhibited a similar evolution, but followed an opposite course: high during the LGM with two primary sources-the central Arabian desert and the dry Persian Gulf region. Dust discharge from both regions reached a pronounced maximum at 15,000-13,000 14C year. At the end of this interval, however, the dust plumes from the Persian Gulf area ceased dramatically, whereas dust discharge from central Arabia decreased only slightly. Dust discharge from East Africa and the Red Sea increased synchronously with the two major events of southwest monsoon intensification as recorded in the nutrient content of surface waters. In addition to the tracers of past dust flux and surface water nutrient content, the geochemical multi-tracer approach provides information on the history of deep sea ventilation (Mo, S), which was much lower during the last glacial maximum than during the Holocene. The multi-tracer approach-i.e. a few sedimentological parameters plus a set of geochemical tracers widely available from various multi-element analysis techniques-is a highly applicable technique for studying the complex sedimentation patterns of an ocean basin, and, specifically in the case of the Arabian Sea, can even reveal the seasonal structure of climate change.

Database of the GLAMAP project

A uniform chronology for foraminifera-based sea surface temperature records has been established in more than 120 sediment cores obtained from the equatorial and eastern Atlantic up to the Arctic Ocean. The chronostratigraphy of the last 30,000 years is mainly based on published d18O records and 14C ages from accelerator mass spectrometry, converted into calendar-year ages. The high-precision age control provides the database necessary for the uniform reconstruction of the climate interval of the Last Glacial Maximum within the GLAMAP-2000 project.