Distribution and survival of faecal indicator bacteria in the sediments of the Bay of Vidy,Lake Geneva,Switzerland

The purpose of this study was to determine the concentrations and the horizontal distribution of faecal indicator bacteria (FIB) including Escherichia coli and Enterococcus sp. in the bottom sediments of the Bay of Vidy, City of Lausanne, Switzerland. A vertical distribution of FIB in sediments near the municipal wastewater treatment plant (WWTP) outlet was evaluated and their persistence in those sediments was monitored for a period of 90 days. High FIB levels were measured in the sediments sampled near the WWTP outlet pipe and the mouth of the Chamberonne River, at concentrations ranging between 10⁵ and 10⁷ CFU 100 g^?1. FIB levels at 10 cm depth in the sediments near the WWTP outlet pipe ranged between 10⁴ and 10⁵ CFU 100 g^?1, and were still detected in the top 6 cm after 90 days. Results of this study indicate that freshwater sediments of the Bay of Vidy constitute a reservoir of faecal indicator bacteria, which can persist in certain areas of the bay. Possible resuspension of FIB and pathogens may affect water quality and may increase health risks to sensitive populations during recreational activities. FIB survival in sediments for long periods is of considerable significance for the understanding of microbial pollution in water and for the management of risk at specific recreational coastal sites.     

PNA-binding glycans are expressed at high levels on horse mature and immature T lymphocytes and a subpopulation of B lymphocytes

In mammals, the binding of peanut agglutinin (PNA) on the plasma membrane defines subpopulations among lymphocytes from peripheral blood and lymphoid organs. PNA binds Gal 1,3GalNAc residues provided that they are not sialylated. Here, we studied the expression of PNA-binding glycans on healthy horse peripheral blood, thymus, lymph node and spleen lymphocytes. We first demonstrated the binding specificity of PNA for galactose residues by competition experiments and the inhibitory role of sialic acids in PNA binding by sialidase digestion. Unlike human and murine lymphocytes, all equine lymphocytes were found positive by flow cytometry analysis. Double-staining analyses showed that lymphocytes expressing high levels of PNA-binding glycans (PNA^high lymphocytes) were made up of the great majority of CD5⁺, CD4⁺ and CD8⁺ cells, and of 30 and 50% of sIg-bearing lymphocytes in peripheral blood and in lymph nodes or spleen, respectively. Lectin histochemistry suggested that lymph node germinal centres contained PNA^high B cells. Contrary to what is found in humans and mice, PNA staining intensity on CD5⁺, CD4⁺ and CD8⁺ cells did not differentiate immature from mature T lymphocytes in the equine thymus. The functional consequences of these differences are discussed. Published in 2005.     

Structural, biochemical, and phylogenetic analyses suggest that indole-3-acetic acid methyltransferase is an evolutionarily ancient member of the SABATH family

The plant SABATH protein family encompasses a group of related small-molecule methyltransferases (MTs) that catalyze the S-adenosyl-L-methionine-dependent methylation of natural chemicals encompassing widely divergent structures. Indole-3-acetic acid (IAA) methyltransferase (IAMT) is a member of the SABATH family that modulates IAA homeostasis in plant tissues through methylation of IAA's free carboxyl group. The crystal structure of Arabidopsis (Arabidopsis thaliana) IAMT (AtIAMT1) was determined and refined to 2.75 A resolution. The overall tertiary and quaternary structures closely resemble the two-domain bilobed monomer and the dimeric arrangement, respectively, previously observed for the related salicylic acid carboxyl methyltransferase from Clarkia breweri (CbSAMT). To further our understanding of the biological function and evolution of SABATHs, especially of IAMT, we analyzed the SABATH gene family in the rice (Oryza sativa) genome. Forty-one OsSABATH genes were identified. Expression analysis showed that more than one-half of the OsSABATH genes were transcribed in one or multiple organs. The OsSABATH gene most similar to AtIAMT1 is OsSABATH4. Escherichia coli-expressed OsSABATH4 protein displayed the highest level of catalytic activity toward IAA and was therefore named OsIAMT1. OsIAMT1 exhibited kinetic properties similar to AtIAMT1 and poplar IAMT (PtIAMT1). Structural modeling of OsIAMT1 and PtIAMT1 using the experimentally determined structure of AtIAMT1 reported here as a template revealed conserved structural features of IAMTs within the active-site cavity that are divergent from functionally distinct members of the SABATH family, such as CbSAMT. Phylogenetic analysis revealed that IAMTs from Arabidopsis, rice, and poplar (Populus spp.) form a monophyletic group. Thus, structural, biochemical, and phylogenetic evidence supports the hypothesis that IAMT is an evolutionarily ancient member of the SABATH family likely to play a critical role in IAA homeostasis across a wide range of plants.     

Energy landscape of chelated uranyl: antibody interactions by dynamic force spectroscopy

We used dynamic force spectroscopy (DFS) to explore the energy landscape of interactions between a chelated uranyl compound and a monoclonal antibody raised against the uranyl-dicarboxy-phenanthroline complex. We estimated the potential energy barrier widths and the relevant thermodynamic rate constants along the dissociation coordinate. Using atomic force microscopy, four different experimental setups with or without the uranyl ion in the chelate ligand, we have distinguished specific and nonspecific binding in the binding affinity of the uranyl compound to the antibody. The force loading rates for our system were measured from 15 to 26,400 pN/s. The results showed two regimes in the plot of the most probable unbinding force versus the logarithm of the loading rate, revealing the presence of two (at least) activation barriers. Analyses of DFS suggest parallel multivalent binding present in either regime. We have also built a molecular model for the variable fragment of the antibody and used computational graphics to dock the chelated uranyl ion into the binding pocket. The structural analysis led us to hypothesize that the two regimes originate from two interaction modes: the first one corresponds to an energy barrier with a very narrow width of 0.5 +/- 0.2 A, inferring dissociation of the uranyl ion from its first coordination shell (Asp residue); the second one with a broader energy barrier width (3.9 +/- 0.3 A) infers the entire chelate compound dissociated from the antibody. Our study highlights the sensitivity of DFS experiments to dissect protein-metal compound interactions.     

Ligand-receptor and receptor-receptor interactions act in concert to activate signaling in the Drosophila toll pathway

In Drosophila, the signaling pathway mediated by the Toll receptor is critical for the establishment of embryonic dorso-ventral pattern and for innate immune responses to bacterial and fungal pathogens. Toll is activated by high affinity binding of the cytokine Sp?tzle, a dimeric ligand of the cystine knot family. In vertebrates, a related family of Toll-like receptors play a critical role in innate immune responses. Despite the importance of this family of receptors, little is known about the biochemical events that lead to receptor activation and signaling. Here, we show that Sp?tzle binds to the N-terminal region of Toll and, using biophysical methods, that the binding is complex. The two binding events that cause formation of the cross-linked complex are non-equivalent: the first Toll ectodomain binds Sp?tzle with an affinity 3-fold higher than the second molecule suggesting that pathway activation involves negative cooperativity. We further show that the Toll ectodomains are able to form low affinity dimers in solution and that juxtamembrane sequences of Toll are critical for the activation or derepression of the pathway. These results, taken together, suggest a mechanism of signal transduction that requires both ligand-receptor and receptor-receptor interactions.     

Free mRNA in excess upon polysome dissociation is a scaffold for protein multimerization to form stress granules

The sequence of events leading to stress granule assembly in stressed cells remains elusive. We show here, using isotope labeling and ion microprobe, that proportionally more RNA than proteins are present in stress granules than in surrounding cytoplasm. We further demonstrate that the delivery of single strand polynucleotides, mRNA and ssDNA, to the cytoplasm can trigger stress granule assembly. On the other hand, increasing the cytoplasmic level of mRNA-binding proteins like YB-1 can directly prevent the aggregation of mRNA by forming isolated mRNPs, as evidenced by atomic force microscopy. Interestingly, we also discovered that enucleated cells do form stress granules, demonstrating that the translocation to the cytoplasm of nuclear prion-like RNA-binding proteins like TIA-1 is dispensable for stress granule assembly. The results lead to an alternative view on stress granule formation based on the following sequence of events: after the massive dissociation of polysomes during stress, mRNA-stabilizing proteins like YB-1 are outnumbered by the burst of nonpolysomal mRNA. mRNA freed of ribosomes thus becomes accessible to mRNA-binding aggregation-prone proteins or misfolded proteins, which induces stress granule formation. Within the frame of this model, the shuttling of nuclear mRNA-stabilizing proteins to the cytoplasm could dissociate stress granules or prevent their assembly.     

Late ontogenetic development of adenosine A1 receptor coupling to associated G-proteins in guinea pig cerebellum but not forebrain

The ontogenetic profile of [³H]forskolin and [³H]cyclohexyladenosine ([³H]CHA) binding sites in guinea pig forebrain and cerebellum was investigated. G-protein interactions of these binding sites were also examined by analyzing 5-guanylylimidodiphosphate (Gpp(NH)p) interactions with [³H]CHA and [³H]forskolin binding. In forebrain, similar binding characteristics of [³H]CHA and [³H]forskolin binding are observed between the developmental stages E₃₆ (the earliest time point studied) through to adult (P₂₈, the latest time point studied), although transient increased binding of both ligands is observed just prior to birth. Scatchard analysis of binding isotherms reveal that this transient rise just prior to birth is due to an increase in the number of binding sites (B_max) with little or no change in receptor affinity (K_d) In contrast, in cerebellum both [³H]CHA and [³H]forskolin binding remains at a relatively low level until just prior to birth when a dramatic increase of binding of both ligands is observed which continues to increase up to P₂₈. Scatchard analysis of binding isotherms reveal that such changes in binding of both ligands are largely due to increases in B_max and not K_d, although Scatchard analysis of [³H]CHA binding to cerebellar E₅₁ membranes reveals an absence of higher affinity [³H]CHA binding sites. Gpp(NH)p did not affect [³H]forskolin binding. Gpp (NH)_p displacement profiles of [³H]CHA binding reveal a maximum (adult) inhibition of [³H]CHA binding (approximately 80% displacement) at all time points (E₃₆ through P₂₈) in forebrain membranes, but not in cerebellar membranes. In cerebellum, displacement of [³H]CHA binding by Gpp(NH)p is much greater after birth than before birth. These results suggest that in cerebellum, but not in forebrain, postnatal coupling of adenosine A₁ receptors to associated G-proteins is much more extensive than in the pre-natal period. The extent of this inferred coupling may also coincide with the ontogenetic appearance or presence of [³H]forskolin binding sites.Abbreviations [³H]CHA N⁶-Cyclohexyl-[³H]-Adenosine - Gpp(NH)p–5 Guanylylimidodiphosphate