Synergistic stimulation of gamma-glutamyl transpeptidase and alkaline phosphatase activities by retinoic acid and astroglial factors in immortalized rat brain microvessel endothelial cells

The immortalized rat brain microvessel endothelial cell line RBE4 was used to investigate the in vitro regulation of two blood-brain barrier specific enzymes, gamma-glutamyl transpeptidase (GTP) and alkaline phosphatase (ALP). The effects of bFGF, astroglial factors, and retinoic acid (a cell differentiation agent) on GTP and ALP activities were separately or simultaneously studied in order to define optimal culture conditions for induction of these two specific enzymes of the blood-brain barrier. In the present study, a phenotypically distinct subpopulation of endothelial cells has been shown to develop from confluent cobblestone monolayers of RBE4 immortalized cerebral endothelial cells. These distinct cells were present within multicellular aggregates and specifically exhibited GTP and ALP activities. Addition of bFGF, astroglial factors, or retinoic acid induced the formation of these three-dimensional structures and in consequence an increase in GTP and ALP activities. For retinoic acid and astroglial factors, this increase could also be explained by the stimulation of either GTP or ALP expression in the phenotypically distinct positive cells associated with aggregates. Simultaneous treatment with retinoic acid and astroglial factors had a synergistic effect on GTP and ALP expression and thus may allow these distinct cells to evolve toward a more differentiated state. Since such results were also obtained with physiological concentrations of retinoic acid, we suggest that addition of this agent might contribute to greater differentiation of cells in in vitro blood-brain barrier models where endothelial cells are cocultured with astrocytes. © 1996 Wiley-Liss, Inc.     

Environmental vibrio phage–bacteria interaction networks reflect the genetic structure of host populations

Phages depend on their bacterial hosts to replicate. The habitat, density and genetic diversity of host populations are therefore key factors in phage ecology, but our ability to explore their biology depends on the isolation of a diverse and representative collection of phages from different sources. Here, we compared two populations of marine bacterial hosts and their phages collected during a time series sampling program in an oyster farm. The population of Vibrio crassostreae, a species associated specifically to oysters, was genetically structured into clades of near clonal strains, leading to the isolation of closely related phages forming large modules in phage–bacterial infection networks. For Vibrio chagasii, which blooms in the water column, a lower number of closely related hosts and a higher diversity of isolated phages resulted in small modules in the phage–bacterial infection network. Over time, phage load was correlated with V. chagasii abundance, indicating a role of host blooms in driving phage abundance. Genetic experiments further demonstrated that these phage blooms can generate epigenetic and genetic variability that can counteract host defence systems. These results highlight the importance of considering both the environmental dynamics and the genetic structure of the host when interpreting phage–bacteria networks.     

Diversity increases with elevation: empidine dance flies (Diptera,Empididae) challenge a predominant pattern

Tropical mountain forests are important reservoirs of biodiversity. They are usually species‐rich and often support endemic species making them prime targets for conservation effort. The aim of this study was to investigate elevational patterns of species diversity and phenology to provide a meaningful understanding of insects’ spatio‐temporal distributions along a tropical gradient of elevation. Our study focuses on the Empidinae communities (Diptera, Empididae) from Doi Inthanon (north Thailand, gradient 400–2556 m asl), sampled during two entire years (2006–2007, 2014). This group of insects is more diverse in temperate localities than in the tropics and we found that: (1) increase in altitude and latitude has a similar effect, so that the diversity of our model increases with elevation; (2) the phenology is strongly influenced by seasonality with a peak of diversity occurring during the transition between the dry and rainy seasons; (3) there is no phenological shift in the diversity peak with elevations; and (4) the species composition changes along the altitudinal gradient and through the year (high beta diversity). The increase in diversity with increasing elevation and the peak of diversity occurring at the transitional period both strongly coincide with abiotic factors, the decreasing temperature and the arrival of the monsoon, respectively.     

Inter‐annual variability of fruit timing and quantity at Nouragues (French Guiana): insights from hierarchical Bayesian analyses

The timing and quantity of fruit production are major determinants of the functioning of a forest community, but simultaneous analyses of both are rare. We analyzed a ten‐year dataset (2001–2011) of fruit production for 45 tree and liana species from the Nouragues rain forest, French Guiana. We developed a hierarchical Bayesian approach to determine variation in the timing and quantity of fruit production. Our analysis accommodates missing censuses and quantifies variation at seasonal and inter‐annual scales. The fruiting peak of 22 of 45 species occurred during the peak of the rainy season, which is typical for central and eastern Amazon. The timing and quantity of fruit production varied substantially across years in most species, with greater variation in quantity than in timing. The timing of fruit production varied from continuously fruiting species to mast fruiting species that had two or more consecutive years without fruit production. Fully 40% of species were mast fruiting species. The seasonal timing and inter‐annual variation in fruiting were unrelated to seed dispersal mode across species. We saw no evidence for directional change in the level of fruit production, the timing of fruit production, or their variances; however, 10 yr is a short record for such analyses.     

Evolution of Exchangeable Copper and Relative Exchangeable Copper through the Course of Wilson's Disease in the Long Evans Cinnamon Rat

Background

Wilson''s disease (WD) is an inherited disorder of copper metabolism leading to liver failure and/or neurological impairment. Its diagnosis often remains difficult even with genetic testing. Relative exchangeable copper (REC) has recently been described as a reliable serum diagnostic marker for WD.

Methodology/Principal Findings

The aim of this study was to validate the use of REC in the Long Evans Cinnamon (LEC) rat, an animal model for WD, and to study its relevance under different conditions in comparison with conventional markers. Two groups of LEC rats and one group of Long-Evans (LE) rats were clinically and biologically monitored from 6 to 28 weeks of age. One group of LEC rats was given copper-free food. The other groups had normal food. Blood samples were collected each month and different serum markers for WD (namely ceruloplasmin oxidase activity, exchangeable copper (CuEXC), total serum copper and REC) and acute liver failure (serum transaminases and bilirubinemia) were tested. Every LEC rat under normal food developed acute liver failure (ALF), with 40% global mortality. Serum transaminases and bilirubinemia along with total serum copper and exchangeable copper levels increased with the onset of acute liver failure. A correlation was observed between CuEXC values and the severity of ALF. Cut-off values were different between young and adult rats and evolved because of age and/or liver failure. Only REC, with values >19%, was able to discriminate LEC groups from the LE control group at every time point in the study. REC sensitivity and specificity reached 100% in adults rats.

Conclusions/Significance

REC appears to be independent of demographic or clinical data in LEC rats. It is a very simple and reliable blood test for the diagnosis of copper toxicosis owing to a lack of ATP7B function. CuEXC can be used as an accurate biomarker of copper overload.     

Cellular and subcellular localization of calcium in gravistimulated corn roots

Summary Antimonate staining procedures and energy dispersive X-ray microanalytical techniques were used to determine the patterns of localization of calcium in nonstimulated and gravistimulated corn roots. In horizontally positioned roots within the region of the developing bend there was a change in the staining from that principally localized within cells of the stele to asymmetric staining within the vacuoles of the cortical cells along the upper root surface. There was little staining in the walls. The pattern observed is quite different from that seen in gravistimulated coleoptiles. Staining of mitochondria, plastids and Golgi stacks was seen in most cell types, but no asymmetry of staining was observed. In the rootcap where graviperception is thought to occur, there was little staining of any cellular organelles.     

Tightening of the ATP-binding sites induces the opening of P2X receptor channels

The opening of ligand-gated ion channels in response to agonist binding is a fundamental process in biology. In ATP-gated P2X receptors, little is known about the molecular events that couple ATP binding to channel opening. In this paper, we identify structural changes of the ATP site accompanying the P2X2 receptor activation by engineering extracellular zinc bridges at putative mobile regions as revealed by normal mode analysis. We provide evidence that tightening of the ATP sites shaped like open 'jaws' induces opening of the P2X ion channel. We show that ATP binding favours jaw tightening, whereas binding of a competitive antagonist prevents gating induced by this movement. Our data reveal the inherent dynamic of the binding jaw, and provide new structural insights into the mechanism of P2X receptor activation.     

Ultrastructural Changes of the Mitochondrion During the Life Cycle of Trypanosoma brucei

The mitochondrion is crucial for ATP generation by oxidative phosphorylation, among other processes. Cristae are invaginations of the mitochondrial inner membrane that house nearly all the macromolecular complexes that perform oxidative phosphorylation. The unicellular parasite Trypanosoma brucei undergoes during its life cycle extensive remodeling of its single mitochondrion, which reflects major changes in its energy metabolism. While the bloodstream form (BSF) generates ATP exclusively by substrate-level phosphorylation and has a morphologically highly reduced mitochondrion, the insect-dwelling procyclic form (PCF) performs oxidative phosphorylation and has an expanded and reticulated organelle. Here, we have performed high-resolution 3D reconstruction of BSF and PCF mitochondria, with a particular focus on their cristae. By measuring the volumes and surface areas of these structures in complete or nearly complete cells, we have found that mitochondrial cristae are more prominent in BSF than previously thought and their biogenesis seems to be maintained during the cell cycle. Furthermore, PCF cristae exhibit a surprising range of volumes in situ, implying that each crista is acting as an independent bioenergetic unit. Cristae appear to be particularly enriched in the region of the organelle between the nucleus and kinetoplast, the mitochondrial genome, suggesting this part has distinctive properties.     

Protein turnover and proliferation. Turnover kinetics associated with the elevation of 3T3-cell acid-proteinase activity and cessation of net protein gain.

1. At least 95% of the total protein of A31-3T3 cell cultures undergoes turnover. 2. First-order exponential kinetics were used to provide a crude approximation of averaged protein synthesis, Ks, degradation, Kd, and net accumulation, Ka, as cells ceased growth at near-confluent density in unchanged Dulbecco's medium containing 10% serum. The values of the relationship Ka = Ks - Kd were : 5%/h = 6%/h - 1%/h in growing cells, and 0%/h = 3%/h - 3%/h in steady-state resting cells. 3. As determined by comparison of the progress of protein synthesis and net protein accumulation, the time course of increase in protein degradation coincided with the onset of an increase in lysosomal proteinase activity and decrease in thymidine incorporation after approx. 2 days of exponential growth. 4. After acute serum deprivation, rapid increases in protein degradation of less than 1%/h could be superimposed on the prevailing degradation rate in either growing or resting cells. The results indicate that two proteolytic mechanisms can be distinguished on the basis of the kinetics of their alterations. A slow mechanism changes in relation to proliferative status and lysosomal enzyme elevation. A prompt mechanism, previously described by others, changes before changes in cell-cycle distribution or lysosomal proteinase activity. 5. When the serum concentration of growing cultures was decreased to 1% or 0.25%, then cessation of growth was accompanied by a lower steady-state protein turnover rate of 2.0%/h or 1.5%/h respectively. When growth ceased under conditions of overcrowded cultures, or severe nutrient insufficiency, protein turnover did not attain a final steady state, but declined continually into the death of the culture.