Purification of a histamine H3 receptor negatively coupled to phosphoinositide turnover in the human gastric cell line HGT1.

The histamine H3 receptor agonist (R)alpha-methylhistamine (MeHA) inhibited, in a nanomolar range, basal and carbachol-stimulated inositol phosphate formation in the human gastric tumoral cell line HGT1-clone 6. The inhibition was reversed by micromolar concentrations of the histamine H3 receptor antagonist thioperamide and was sensitive to cholera or pertussis toxin treatment. Using [3H]N alpha-MeHA as specific tracer, high affinity binding sites were demonstrated with a Bmax of 54 +/- 3 fmol/mg of protein and a KD of either 0.61 +/- 0.04 or 2.2 +/- 0.4 nM, in the absence or presence of 50 microM GTP[gamma]S, respectively. The binding sites were solubilized by Triton X-100 and prepurified by gel chromatography. They were separated from the histamine H2 receptor sites by filtration through Sepharose-famotidine and finally retained on Sepharose-thioperamide. The purified sites concentrated in one single silver-stained protein band of 70 kDa in SDS-polyacrylamide gel electrophoresis. They specifically bound [3H]N alpha-MeHA with a KD of 1.6 +/- 0.1 nM and a Bmax of 12,000 +/- 750 pmol/mg of protein. This corresponds to a 90,225-fold purification over cell lysate and a purity degree of 84%. Binding was competitively displaced by N alpha-MeHA (IC50 = 5.8 +/- 0.7 nM), (R) alpha-MeHA (IC50 = 9 +/- 1 nM), and thioperamide (IC50 = 85 +/- 10 nM), but not by famotidine (H2 antagonist) or by mepyramine (H1 antagonist). These findings provide the first evidence for solubilization, purification, and molecular mass characterization of the histamine H3 receptor protein and for the negative coupling of this receptor phosphatidylinositol turnover through a so far unidentified G protein.     

Viruses in extreme environments

The tolerance limits of extremophiles in term of temperature, pH, salinity, desiccation, hydrostatic pressure, radiation, anaerobiosis far exceed what can support non-extremophilic organisms. Like all other organisms, extremophiles serve as hosts for viral replication. Many lines of evidence suggest that viruses could no more be regarded as simple infectious “fragments of life” but on the contrary as one of the major components of the biosphere. The exploration of niches with seemingly harsh life conditions as hypersaline and soda lakes, Sahara desert, polar environments or hot acid springs and deep sea hydrothermal vents, permitted to track successfully the presence of viruses. Substantial populations of double-stranded DNA virus that can reach 10⁹ particles per milliliter were recorded. All these viral communities, with genome size ranging from 14 kb to 80 kb, seem to be genetically distinct, suggesting specific niche adaptation. Nevertheless, at this stage of the knowledge, very little is known of their origin, activity, or importance to the in situ microbial dynamics. The continuous attempts to isolate and to study viruses that thrive in extreme environments will be needed to address such questions. However, this topic appears to open a new window on an unexplored part of the viral world. Marc Le Romancer and Mélusine Gaillard contributed equally to this work.     

Black Spot of Globe Artichoke: a Calcium-deficiency Disorder

Experiments were performed with field-grown artichoke ( Cynara scolymus L.) cv. Capitan to determine whether the black spot disorder was associated with calcium deficiency in receptacles. Ageing of the plants and size of heads contributed to lower Ca levels of receptacles. Receptacles with lower amounts of Ca were more affected by the disorder. Globe artichoke infection by artichoke latent virus (ALV) and/or broad bean wilt virus strain french artichoke (BBWV-FA) resulted in decreases in both the relative water content (RWC) of leaves and the Ca content of receptacles, and in increased black spot incidence. In addition, a wind-screen and an anti-trans-pirant spray (composed of a wax emulsion and a spreader/sticker surfactant) increased the RWC of leaves and bracts and the Ca content of receptacles, and also significantly reduced the incidence of necrosed buds.
These data support the view that Ca deficiency is involved in the occurrence of black spot disorder.     

Deciphering the virus‐to‐prokaryote ratio (VPR): insights into virus–host relationships in a variety of ecosystems

The discovery of the numerical importance of viruses in a variety of (aquatic) ecosystems has changed our perception of their importance in microbial processes. Bacteria and Archaea undoubtedly represent the most abundant cellular life forms on Earth and past estimates of viral numbers (represented mainly by viruses infecting prokaryotes) have indicated abundances at least one order of magnitude higher than that of their cellular hosts. Such dominance has been reflected most often by the virus‐to‐prokaryote ratio (VPR), proposed as a proxy for the relationship between viral and prokaryotic communities. VPR values have been discussed in the literature to express viral numerical dominance (or absence of it) over their cellular hosts, but the ecological meaning and interpretation of this ratio has remained somewhat nebulous or contradictory. We gathered data from 210 publications (and additional unpublished data) on viral ecology with the aim of exploring VPR. The results are presented in three parts: the first consists of an overview of the minimal, maximal and calculated average VPR values in an extensive variety of different environments. Results indicate that VPR values fluctuate over six orders of magnitude, with variations observed within each ecosystem. The second part investigates the relationship between VPR and other indices, in order to assess whether VPR can provide insights into virus–host relationships. A positive relationship was found between VPR and viral abundance (VA), frequency of visibly infected cells (FVIC), burst size (BS), frequency of lysogenic cells (FLC) and chlorophyll a (Chl a) concentration. An inverse relationship was detected between VPR and prokaryotic abundance (PA) (in sediments), prokaryotic production (PP) and virus–host contact rates (VCR) as well as salinity and temperature. No significant relationship was found between VPR and viral production (VP), fraction of mortality from viral lysis (FMVL), viral decay rate (VDR), viral turnover (VT) or depth. Finally, we summarize our results by proposing two scenarios in two contrasting environments, based on current theories on viral ecology as well as the present results. We conclude that since VPR fluctuates in every habitat for different reasons, as it is linked to a multitude of factors related to virus–host dynamics, extreme caution should be used when inferring relationships between viruses and their hosts. Furthermore, we posit that the VPR is only useful in specific, controlled conditions, e.g. for the monitoring of fluctuations in viral and host abundance over time.     

Preliminary Studies on Black Spot of Globe Artichoke

Studies were conducted to determine the cause of an apparently new disorder of globe artichoke ( Cynara scotymus L.) characterised by a localised black necrosis in the receptacle of the flower buds. The effects of single and mixed infections by artichoke latent potyvirus (ALV) and broad bean wilt virus – French artichoke (BBWV-FA) in buds with necrosis of three globe artichoke cultivars were studied. Virus-free plants developed diseased buds irrespective of whether the plants were 1 or 2 years old, but the presence of ALV and/or BBWV-FA significantly increased black spot occurrence on 2-year-old plants. Virus effects were dependent on cultivar, cv. Capitan being less susceptible to virus infection than cvs Camus de Bretagne and Castel. The head size of the globe artichoke was found to be related to the rate of necrosis, with a higher incidence for terminal buds.
ALV and BBWV-FA did not cause the disorder but favour its development probably by weakening plants through increased leaf transpiration. The results are discussed in relation to a possible physiological disorder exacerbated by the presence of ALV and/or BBWV-FA.     

Characterization of two DNA polymerases from the hyperthermophilic euryarchaeon Pyrococcus abyssi.

The complete genome sequence of the hyperthermophilic archaeon Pyrococcus abyssi revealed the presence of a family B DNA polymerase (Pol I) and a family D DNA polymerase (Pol II). To extend our knowledge about euryarchaeal DNA polymerases, we cloned the genes encoding these two enzymes and expressed them in Escherichia coli. The DNA polymerases (Pol I and Pol II) were purified to homogeneity and characterized. Pol I had a molecular mass of approximately 90 kDa, as estimated by SDS/PAGE. The optimum pH and Mg(2+) concentration of Pol I were 8.5-9.0 and 3 mm, respectively. Pol II is composed of two subunits that are encoded by two genes arranged in tandem on the P. abyssi genome. We cloned these genes and purified the Pol II DNA polymerase from an E. coli strain coexpressing the cloned genes. The optimum pH and Mg(2+) concentration of Pol II were 6.5 and 15-20 mm, respectively. Both P. abyssi Pol I and Pol II have associated 3'-->5' exonuclease activity although the exonuclease motifs usually found in DNA polymerases are absent in the archaeal family D DNA polymerase sequences. Sequence analysis has revealed that the small subunit of family D DNA polymerase and the Mre11 nucleases belong to the calcineurin-like phosphoesterase superfamily and that residues involved in catalysis and metal coordination in the Mre11 nuclease three-dimensional structure are strictly conserved in both families. One hypothesis is that the phosphoesterase domain of the small subunit is responsible for the 3'-->5' exonuclease activity of family D DNA polymerase. These results increase our understanding of euryarchaeal DNA polymerases and are of importance to push forward the complete understanding of the DNA replication in P. abyssi.     

Activation of Epstein-Barr virus/C3d receptor (gp140, CR2, CD21) on human B lymphoma cell surface triggers Cbl tyrosine phosphorylation, its association with p85 subunit, Crk-L and Syk and its dissociation with Vav

It is well established that CD21 activation on human B cell surface triggers B cell proliferation. We previously demonstrated that CD21 activation also triggers tyrosine phosphorylation of two components, p95 and p120, both interacting with SH2 domains of the p85 subunit of PI 3-kinase. We successively identified p95 as the nucleolin and the first signal transduction pathway specifically triggered by CD21 activation, i.e.: pp60Src activation, tyrosine phosphorylation of p95 nucleolin, its interaction with SH2 domains of p85 subunit and PI 3-kinase activation, followed by AKT-GSK-3 activations. We herein identified the p120 component as the protooncoprotein Cbl and the first steps associated to its activation. First, CD21 activation triggered Cbl tyrosine phosphorylation, which required c-Src kinase but not PI 3-kinase or Syk kinase activities. Involvement of Src kinase in this step was supported by inhibition of Cbl phosphorylation and its interactions with other components when cells were either preincubated with specific Src inhibitor or transfected with dominant-negative c-Src form. Second, once tyrosine phosphorylated, Cbl interacts with SH2 domains of p85 subunit, SH2 domains of Crk-L and with tyrosine phosphorylated Syk kinase. The third and unexpected feature was to found that, at the contrary of BCR or of CD19 (herein also analyzed for the first time), CD21 activation triggers dissociation of Cbl-Vav complex. Thus, these results provide the first molecular basis of a new signal transduction pathway specifically triggered by CD21 activation.