Establishment of ''normal'' nervous cell lines after transfer of polyoma virus and adenovirus early genes into murine brain cells.

Brain cells from murine embryos were transfected with the polyoma virus large T or the adenovirus 5 EIA gene and, simultaneously, with the phosphotransferase coding NeoR gene. The efficiently transfected cells were selected for their resistance to Geneticin (G418) and their ability to clone at low cell density. Subsequently, most of the selected cells could be sub-cloned and continuously grown for 6-18 months so far. Their doubling time varied between 18 and 72 h. From independent transfections, more than one hundred cell lines were established. They did not exhibit a transformed phenotype, but subsequent transfection with the polyoma middle T gene induced their oncogenic transformation. The maintenance and expression of the transferred genes were verified. Most of the analyzed cell lines retained glial properties. These results suggest that the lines obtained as well as a further extension of this in vitro system should be of interest for the study of nervous cell interactions, differentiation and functions.     

Electron microscopic examination of subcellular fractions. I. The preparation of representative samples from suspensions of particles

A method is described for preparing, by filtration on Millipore filters, very thin (about 10 µ) pellicles of packed particles. These pellicles can be embedded in Epon for electron microscopic examination. They are also suitable for cytochemical assays. The method was used with various particulate fractions from rat liver. Its main advantages over the usual centrifugal packing techniques are that it produces heterogeneity solely in the direction perpendicular to the surface of the pellicle and that sections covering the whole depth of the pellicle can be photographed in a single field. It thus answers the essential criterion of random sampling and can be used for accurate quantitative evaluations.     

Crystal structure of the EF-hand parvalbumin at atomic resolution (0.91 A) and at low temperature (100 K). Evidence for conformational multistates within the hydrophobic core.

Several crystal structures of parvalbumin (Parv), a typical EF-hand protein, have been reported so far for different species with the best resolution achieving 1.5 A. Using a crystal grown under microgravity conditions, cryotechniques (100 K), and synchrotron radiation, it has now been possible to determine the crystal structure of the fully Ca2+-loaded form of pike (component pI 4.10) Parv.Ca2 at atomic resolution (0.91 A). The availability of such a high quality structure offers the opportunity to contribute to the definition of the validation tools useful for the refinement of protein crystal structures determined to lower resolution. Besides a better definition of most of the elements in the protein three-dimensional structure than in previous studies, the high accuracy thus achieved allows the detection of well-defined alternate conformations, which are observed for 16 residues out of 107 in total. Among them, six occupy an internal position within the hydrophobic core and converge toward two small buried cavities with a total volume of about 60 A3. There is no indication of any water molecule present in these cavities. It is probable that at temperatures of physiological conditions there is a dynamic interconversion between these alternate conformations in an energy-barrier dependent manner. Such motions for which the amplitudes are provided by the present study will be associated with a time-dependent remodeling of the void internal space as part of a slow dynamics regime (millisecond timescales) of the parvalbumin molecule. The relevance of such internal dynamics to function is discussed.     

Fragmentation-tree density representation for crystallographic modelling of bound ligands

The identification and modelling of ligands into macromolecular models is important for understanding molecule's function and for designing inhibitors to modulate its activities. We describe new algorithms for the automated building of ligands into electron density maps in crystal structure determination. Location of the ligand-binding site is achieved by matching numerical shape features describing the ligand to those of density clusters using a "fragmentation-tree" density representation. The ligand molecule is built using two distinct algorithms exploiting free atoms with inter-atomic connectivity and Metropolis-based optimisation of the conformational state of the ligand, producing an ensemble of structures from which the final model is derived. The method was validated on several thousand entries from the Protein Data Bank. In the majority of cases, the ligand-binding site could be correctly located and the ligand model built with a coordinate accuracy of better than 1 ?. We anticipate that the method will be of routine use to anyone modelling ligands, lead compounds or even compound fragments as part of protein functional analyses or drug design efforts.     

Prepartum vaginal/anorectal Group B Streptococcus screening: improvement of the enrichment step by the broth additive RambaQUICK StrepB

We used RambaQUICK StrepB in enrichment broth with samples taken from 31 pregnant women and compared its performance to that of standard enrichment media. RambaQUICK StrepB shortened the required incubation time and raised the sensitivity of streptococcal screening by 1.6 fold.     

Evaluation of CHROMagar™ StrepB agar, an aerobic chromogenic medium for prepartum vaginal/rectal Group B Streptococcus screening

An aerobic chromogenic medium, CHROMagar™ StrepB agar, designed for isolation of group B Streptococci, was evaluated on 285 prepartum vaginal/rectal swabs from pregnant women. After overnight enrichment in Todd-Hewitt broth containing 15 μg/ml nalidixic acid and 10 μg/ml colistin, sensitivities were respectively 79% on day 1 and 92% on day 2, and significantly higher than those achieved by blood agar (40% and 58%) and colimycin-nalidixic-acid agar (82% on day 2).     

Hierarchical control of dopamine neuron-firing patterns by nicotinic receptors

Nicotine elicits dopamine release by stimulating nicotinic acetylcholine receptors (nAChRs) on dopaminergic neurons. However, a modulation of these neurons by endogenous acetylcholine has not been described. We recorded, in vivo, the spontaneous activity of dopaminergic neurons in the VTA of anaesthetized wt and nAChR knockout mice and their response to nicotine injections. Deleting alpha7 or beta2 subunits modified the spontaneous firing patterns, demonstrating their direct stimulation by endogenous acetylcholine. Quantitative analysis further revealed four principal modes of firing, each depending on the expression of particular nAChR subunits and presenting unique responses to nicotine. The prominent role of the beta2 subunit was further confirmed by its selective lentiviral reexpression in the VTA. These data suggest a hierarchical control of dopaminergic neuron firing patterns by nAChRs: activation of beta2*-nAChR switches cells from a resting to an excited state, whereas activation of alpha7*-nAChRs finely tunes the latter state but only once beta2*-nAChRs have been activated.     

Forward to the north: two Euro-Mediterranean bumblebee species now cross the Arctic Circle

In recent decades, several animal and plant species have been in regression (population size decrease and geographical distribution shrinking). This loss of biodiversity can be due to various factors such as the destruction and fragmentation of habitat, urban development, pesticides or climate change. However, some species benefit from these changes and expand their distribution. Here we report observations (in 2013 and 2014) of two Euro-Mediterranean bumblebee species: Bombus terrestris for the first time and Bombus lapidarius, north of the Arctic Circle in Fennoscandia.     

Cell volume changes during rapid temperature shifts

The effect of a rapid temperature increase on the volume of different types of cells was investigated. Experiments were carried out using continuous microscopic image analysis. Volume variation of yeast cells, yeast spheroplasts and human leukaemia cells was measured during the transient phase after a thermal shift. The thermal shift was found to induce rapid increase in cell volume for cells lacking a cell wall (yeast spheroplasts and human leukaemia cells). This increase in cell volume is assumed to be a main cause of the heat shock-induced cell death. A theoretical mechanistic model that explains the behaviour of these cells is finally proposed.     

In plants, 3-o-methylglucose is phosphorylated by hexokinase but not perceived as a sugar

In plants, sugars are the main respiratory substrates and important signaling molecules in the regulation of carbon metabolism. Sugar signaling studies suggested that sugar sensing involves several key components, among them hexokinase (HXK). Although the sensing mechanism of HXK is unknown, several experiments support the hypothesis that hexose phosphorylation is a determining factor. Glucose (Glc) analogs transported into cells but not phosphorylated are frequently used to test this hypothesis, among them 3-O-methyl-Glc (3-OMG). The aim of the present work was to investigate the effects and fate of 3-OMG in heterotrophic plant cells. Measurements of respiration rates, protein and metabolite contents, and protease activities and amounts showed that 3-OMG is not a respiratory substrate and does not contribute to biosynthesis. Proteolysis and lipolysis are induced in 3-OMG-fed maize (Zea mays L. cv DEA) roots in the same way as in sugar-starved organs. However, contrary to the generally accepted idea, phosphorous and carbon nuclear magnetic resonance experiments and enzymatic assays prove that 3-OMG is phosphorylated to 3-OMG-6-phosphate, which accumulates in the cells. Insofar as plant HXK is involved in sugar sensing, these findings are discussed on the basis of the kinetic properties because the catalytic efficiency of HXK isolated from maize root tips is five orders of magnitude lower for 3-OMG than for Glc and Man.