Oxygen supply strongly influences metabolic fluxes,the production of poly(3-hydroxybutyrate) and alginate,and the degree of acetylation of alginate in Azotobacter vinelandii

The aim of this study was to evaluate carbon flux in Azotobacter vinelandii using metabolic flux analysis (MFA) under high and low aeration conditions to achieve an improved understanding of how these changes could be related to alginate acetylation and PHB production. Changes in oxygen availability had a considerable impact on the metabolic fluxes and were reflected in the growth rate, the specific glucose consumption rate, and the alginate and PHB yields. The main differences at the metabolic flux level were observed in three important pathways. The first important difference was consistent with respiratory protection; an increase in the flux generated through the tricarboxylic acid (TCA) cycle for cultures grown under high aeration conditions (up to 2.61 times higher) was observed. In the second important difference, the fluxes generated through pyruvate dehydrogenase, phosphoenol pyruvate carboxykinase and pyruvate kinase, all of which are involved in acetyl-CoA metabolism, increased by 10, 43.9 and 17.5%, respectively, in cultures grown under low aeration conditions compared with those grown under high aeration conditions. These changes were related to alginate acetylation, which was 2.6 times higher in the cultures with limited oxygen, and the changes were also related to a drastic increase in PHB production. Finally, the glyoxylate shunt was active under both of the conditions that were tested, and a 2.79-fold increase was observed in cultures that were grown under the low aeration condition.     

Exchange of Cytosolic Content between T Cells and Tumor Cells Activates CD4 T Cells and Impedes Cancer Growth

Background

T cells are known to participate in the response to tumor cells and react with cytotoxicity and cytokine release. At the same time tumors established versatile mechanisms for silencing the immune responses. The interplay is far from being completely understood. In this study we show contacts between tumor cells and lymphocytes revealing novel characteristics in the interaction of T cells and cancer cells in a way not previously described.

Methods/ Findings

Experiments are based on the usage of a hydrophilic fluorescent dye that occurs free in the cytosol and thus transfer of fluorescent cytosol from one cell to the other can be observed using flow cytometry. Tumor cells from cell lines of different origin or primary hepatocellular carcinoma (HCC) cells were incubated with lymphocytes from human and mice. This exposure provoked a contact dependent uptake of tumor derived cytosol by lymphocytes – even in CD4⁺ T cells and murine B cells – which could not be detected after incubation of lymphocytes with healthy cells. The interaction was a direct one, not requiring the presence of accessory cells, but independent of cytotoxicity and TCR engagement.Electron microscopy disclosed 100-200nm large gaps in the cell membranes of connected cells which separated viable and revealed astonishing outcome. While the lymphocytes were induced to proliferate in a long term fashion, the tumor cells underwent a temporary break in cell division. The in vitro results were confirmed in vivo using a murine acute lymphoblastic leukemia (ALL) model. The arrest of tumor proliferation resulted in a significant prolonged survival of challenged mice.

Conclusions

The reported cell-cell contacts reveal new characteristics i.e. the enabling of cytosol flow between the cells including biological active proteins that influence the cell cycle and biological behaviour of the recipient cells. This adds a completely new aspect in tumor induced immunology.     

Pollen morphology of the Pavetteae (Rubiaceae,Ixoroideae) and its taxonomic significance

Pollen grains of the tribe Pavetteae (Rubiaceae, subfamily Ixoroideae) are examined using LM and SEM. Grains are 3‐ or 4‐colporate and (semi‐) tectate (in one Versteegia species atectate). Sexine patterns vary between perforate, microreticulate, reticulate, rugulate and striato‐reticulate. Supratectal elements are sometimes present. The variation in pollen morphology in the Pavetteae allows to recognize seven pollen types, the distribution of which is useful to evaluate generic delimitations and relationships within the tribe. Pollen characters corroborate the close relationships between the genera Coleactina, Dictyandra and Leptactina and between Homollea, Homolliella and Paracephaelis. All the genera of the tribe proved to be stenopalynous (the species examined possess the same pollen type), except Pavetta, Rutidea, Versteegia and Tarenna which are eurypalynous. In the huge genus Pavetta the existing infrageneric classification is supported pollen morphologically. Pollen morphology further indicates that the genus Tarenna is badly delimited and strongly in need of a revision. The small genus Versteegia is in need of further taxonomic and palynological study to understand the pollen morphological variation encountered here. At a higher rank, pollen morphology also does not contradict the recent division of the Pavetteae in the Ixoreae (a stenopalynous tribe with presumably primitive pollen) and the Pavetteae sensu stricto (eurypalynous).     

Morphologically Homogeneous Red Blood Cells Present a Heterogeneous Response to Hormonal Stimulation

Red blood cells (RBCs) are among the most intensively studied cells in natural history, elucidating numerous principles and ground-breaking knowledge in cell biology. Morphologically, RBCs are largely homogeneous, and most of the functional studies have been performed on large populations of cells, masking putative cellular variations. We studied human and mouse RBCs by live-cell video imaging, which allowed single cells to be followed over time. In particular we analysed functional responses to hormonal stimulation with lysophosphatidic acid (LPA), a signalling molecule occurring in blood plasma, with the Ca²⁺ sensor Fluo-4. Additionally, we developed an approach for analysing the Ca²⁺ responses of RBCs that allowed the quantitative characterization of single-cell signals. In RBCs, the LPA-induced Ca²⁺ influx showed substantial diversity in both kinetics and amplitude. Also the age-classification was determined for each particular RBC and consecutively analysed. While reticulocytes lack a Ca²⁺ response to LPA stimulation, old RBCs approaching clearance generated robust LPA-induced signals, which still displayed broad heterogeneity. Observing phospatidylserine exposure as an effector mechanism of intracellular Ca²⁺ revealed an even increased heterogeneity of RBC responses. The functional diversity of RBCs needs to be taken into account in future studies, which will increasingly require single-cell analysis approaches. The identified heterogeneity in RBC responses is important for the basic understanding of RBC signalling and their contribution to numerous diseases, especially with respect to Ca²⁺ influx and the associated pro-thrombotic activity.     

Estimating and Analyzing Savannah Phenology with a Lagged Time Series Model

Savannah regions are predicted to undergo changes in precipitation patterns according to current climate change projections. This change will affect leaf phenology, which controls net primary productivity. It is of importance to study this since savannahs play an important role in the global carbon cycle due to their areal coverage and can have an effect on the food security in regions that depend on subsistence farming. In this study we investigate how soil moisture, mean annual precipitation, and day length control savannah phenology by developing a lagged time series model. The model uses climate data for 15 flux tower sites across four continents, and normalized difference vegetation index from satellite to optimize a statistical phenological model. We show that all three variables can be used to estimate savannah phenology on a global scale. However, it was not possible to create a simplified savannah model that works equally well for all sites on the global scale without inclusion of more site specific parameters. The simplified model showed no bias towards tree cover or between continents and resulted in a cross-validated r² of 0.6 and root mean squared error of 0.1. We therefore expect similar average results when applying the model to other savannah areas and further expect that it could be used to estimate the productivity of savannah regions.     

Selective oxidation of UDP-glucose to UDP-glucuronic acid using permeabilized <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis> expressing human UDP-glucose 6-dehydrogenase

Objectives

To use permeabilized cells of the fission yeast, Schizosaccharomyces pombe, that expresses human UDP-glucose 6-dehydrogenase (UGDH, EC 1.1.1.22), for the production of UDP-glucuronic acid from UDP-glucose.

Results

In cell extracts no activity was detected. Therefore, cells were permeabilized with 0.3 % (v/v) Triton X-100. After washing away all low molecular weight metabolites, the permeabilized cells were directly used as whole cell biocatalyst. Substrates were 5 mM UDP-glucose and 10 mM NAD⁺. Divalent cations were not added to the reaction medium as they promoted UDP-glucose hydrolysis. With this reaction system 5 mM UDP-glucose were converted into 5 mM UDP-glucuronic acid within 3 h.

Conclusions

Recombinant permeabilized cells of S. pombe can be used to synthesize UDP-glucuronic acid with 100 % yield and selectivity.

     

Maternal genomic variability of the wild boar (Sus scrofa) reveals the uniqueness of East‐Caucasian and Central Italian populations

The phylogeography of the European wild boar was mainly determined by postglacial recolonization patterns from Mediterranean refugia after the last ice age. Here we present the first analysis of SNP polymorphism within the complete mtDNA genome of West Russian (n = 8), European (n = 64), and North African (n = 5) wild boar. Our analyses provided evidence of unique lineages in the East‐Caucasian (Dagestan) region and in Central Italy. A phylogenetic analysis revealed that these lineages are basal to the other European mtDNA sequences. We also show close connection between the Western Siberian and Eastern European populations. Also, the North African samples were clustered with the Iberian population. Phylogenetic trees and migration modeling revealed a high proximity of Dagestan sequences to those of Central Italy and suggested possible gene flow between Western Asia and Southern Europe which was not directly related to Northern and Central European lineages. Our results support the presence of old maternal lineages in two Southern glacial refugia (i.e., Caucasus and the Italian peninsula), as a legacy of an ancient wave of colonization of Southern Europe from an Eastern origin.     

Automated maintenance of embryonic stem cell cultures

Embryonic stem cell (ESC) technology provides attractive perspectives for generating unlimited numbers of somatic cells for disease modeling and compound screening. A key prerequisite for these industrial applications are standardized and automated systems suitable for stem cell processing. Here we demonstrate that mouse and human ESC propagated by automated culture maintain their mean specific growth rates, their capacity for multi-germlayer differentiation, and the expression of the pluripotency-associated markers SSEA-1/Oct-4 and Tra-1-60/Tra-1-81/Oct-4, respectively. The feasibility of ESC culture automation may greatly facilitate the use of this versatile cell source for a variety of biomedical applications.     

Dystroglycan regulates structure, proliferation and differentiation of neuroepithelial cells in the developing vertebrate CNS

In the developing CNS alpha- and beta-dystroglycan are highly concentrated in the endfeet of radial neuroepithelial cells at the contact site to the basal lamina. We show that injection of anti-dystroglycan Fab fragments, knockdown of dystroglycan using RNAi, and overexpression of a dominant-negative dystroglycan protein by microelectroporation in neuroepithelial cells of the chick retina and optic tectum in vivo leads to the loss of their radial morphology, to hyperproliferation, to an increased number of postmitotic neurons, and to an altered distribution of several basally concentrated proteins. Moreover, these treatments also altered the oriented growth of axons from retinal ganglion cells and from tectal projection neurons. In contrast, expression of non-cleavable dystroglycan protein in neuroepithelial cells reduced their proliferation and their differentiation to postmitotic neurons. These results demonstrate that dystroglycan plays a key role in maintaining neuroepithelial cell morphology, and that interfering with dystroglycan function influences proliferation and differentiation of neuroepithelial cells. These data also suggest that an impaired dystroglycan function in neuroepithelial cells might be responsible for some of the severe brain abnormalities observed in certain forms of congenital muscular dystrophy.