Flavobacterial Community Structure in a Hardwater Rivulet and Adjacent Forest Soil,Harz Mountain,Germany

The great increase in the abundance and phylogenetic diversity of Flavobacterium spp. within a few hundred meters downstream of the discharge site of the Westerhöfer Bach, a hardwater rivulet, raised the question whether adjacent soil may serve as a reservoir of bacteria not detected in discharge water. To address this question, denaturing gradient gel electrophoresis (DGGE) analyses of the V3 region of Flavobacterium 16S rRNA genes were performed on DNA from nine soil samples and five rivulet sites. The resulting patterns were tested for the significance of differences between the sampling habitats using the nonparametric analysis of similarities and multidimensional scaling procedures. Even though both habitats were sampled in two consecutive years DGGE patterns of soil and downstream water samples showed significant overlap (R = 0.614). Sequencing of 57 DGGE bands resulted in 30 different sequences, which, on the basis of BLAST analyses, were between 96% and 100% similar to published clone, DGGE, and strain sequences from a wide range of different habitats. Forty-five percent of the highly similar sequences included those of isolates from the Westerhöfer Bach, while the other sequences were more closely related to clones and cultures from other habitats, especially agricultural soil. Based on these results we suggest that the increase in flavobacterial strain diversity and abundance in the rivulet may originate from soil microflora.     

Combretastatin-like chalcones as inhibitors of microtubule polymerisation. Part 2: Structure-based discovery of alpha-aryl chalcones

Tubulin is an important molecular target in cancer chemotherapy. Antimitotic agents able to bind to the protein are currently under study, commonly used in the clinic to treat a variety of cancers and/or exploited as probes to investigate the protein’s structure and function. Here we report the binding modes for a series of colchicinoids, combretastatin A4 and chalcones established from docking studies carried out on the structure of tubulin in complex with colchicine. The proposed models, in agreement with published biochemical data, show that combretastatin A4 binds to the colchicine site of β-tubulin and that chalcones assume an orientation similar to that of podophyllotoxin. The models can be used to design a new class of podophyllotoxin mimics, the α-aryl chalcones, capable of binding to the colchicine-binding site of β-tubulin with higher affinity.     

Carbonic anhydrase inhibitors. Inhibition studies of a coral secretory isoform by sulfonamides

The inhibition of a newly cloned coral carbonic anhydrase (CA, EC 4.2.1.1) has been investigated with a series of sulfonamides, including some clinically used derivatives (acetazolamide, methazolamide, ethoxzolamide, dichlorophenamide, dorzolamide, brinzolamide, benzolamide, and sulpiride, or indisulam, a compound in clinical development as antitumor drug), as well as the sulfamate antiepileptic topiramate. Some simple amino-/hydrazine-/hydroxy-substituted aromatic/heterocyclic sulfonamides have also been included in the study. All types of activity have been detected, with low potency inhibitors (K_Is in the range of 163–770 nM), or with medium potency inhibitors (K_Is in the range of 75.1–105 nM), whereas ethoxzolamide, several clinically used sulfonamides and heterocyclic compounds showed stronger potency, with K_Is in the range of 16–48.2 nM. These inhibitors may be useful to better understand the physiological role of the Stylophora pistillata CA (STPCA) in corals and its involvement in biomineralisation in this era of global warming.     

The small ubiquitin-like modifier (SUMO)-conjugating system of Toxoplasma gondii

SUMOylation, the reversible covalent attachment of small ubiquitin-like modifier (SUMO) peptides has emerged as an important regulator of target protein function. Here we show, by characterization of the Toxoplasma gondii SUMO pathway, that the SUMO conjugation system operates in apicomplexan parasites. A gene encoding the SUMO tag was discovered as were genes encoding the various enzymes required for SUMO processing, ligation and release. Various SUMO conjugates were immuno-detected and by means of a global proteomic-based approach, we identified several T. gondii SUMOylated proteins that reveal many diverse cellular processes in which the modification plays a role. More specifically, SUMO conjugates were seen at the tachyzoite surface in response to signaling generated by host cell contact at the time of invasion. Also, under tissue culture conditions that stimulate bradyzoite differentiation (alkaline pH), we observed the conjugates at the parasitophorous vacuole membrane. The labeling was also at the surface of the mature cysts isolated from parasite-infected mouse brain. Overall, the SUMO conjugation system appears to be a complex and functionally heterogeneous pathway for protein modification in T. gondii with initial data indicating that it is likely to play a putative role in host cell invasion and cyst genesis.     

Heterogeneous distribution of TRPC proteins in the embryonic cortex

The present study was initiated to gain some information about the tissue distribution of transient receptor potential proteins of C-type (TRPC), a family of voltage-independent cation channels, at the beginning of neurogenesis in the telencephalon of embryonic mice. The mRNAs of all known TRPCs (TRPC1–TRPC7) could be found in the cortex at E13. TRPC1, TRPC3 and TRPC5 were the main isoforms, whereas the mRNAs for TRPC2, TRPC4, TRPC6 and TRPC7 were less abundant. The distribution throughout the cortical wall of TRPC1, TRPC3 and TRPC6 was studied by means of immuno-histochemistry. The data collected pointed to a heterogeneous expression of the channels. Three groups were identified. The first one comprises TRPC1, specifically found in the preplate but only in some post-mitotic neurons. It was mainly observed in a subset of cells distinct from the Cajal-Retzius cells. The second group is composed of TRPC3. It was found in non-neuronal cells and also in dividing (5-bromo-2′-deoxyuridine-positive) cells, indicating that TRPC3 is present in precursor cells. The third group contains TRPC6 detected in neuronal and in dividing non-neuronal cells. Double immunostaining experiments showed that TRPC3-positive cells also express TRPC6. Collectively, this report highlights a specific TRPC expression pattern in the immature cortical wall. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. S. Boisseau and C. Kunert-Keil have contributed equally to this work.     

Exact and user-friendly kinetic analysis of the two-step rapid equilibrium Michaelis-Menten mechanism

Most enzyme kinetic experiments are carried out under pseudo-first-order conditions, that is, when one of the reactant species (the enzyme or the substrate) is in a large excess of the other species. More accurate kinetic information about the system can be gained without the restrictions of the pseudo-first-order conditions. We present a practical and general method of analysis of the common two-step rapid equilibrium Michaelis-Menten mechanism. The formalism is exact in that it does not involve any other approximations such as the steady-state, limitations on the reactant concentrations or on reaction times. We apply this method to the global analysis of kinetic progress curves for bovine alkaline phosphatase assays carried out under both pseudo-first-order and pseudo-second-order conditions.     

Phosphorylation of Collapsin Response Mediator Protein 2 on Tyr-479 Regulates CXCL12-induced T Lymphocyte Migration

In the central nervous system, collapsin response mediator protein 2 (CRMP2) is a transducer protein that supports the semaphorin-induced guidance of axons toward their cognate target. However, we previously showed that CRMP2 is also expressed in immune cells and plays a crucial role in T lymphocyte migration. Here we further investigated the molecular mechanisms underlying CRMP2 function in chemokine-directed T-cell motility. Examining Jurkat T-cells treated with the chemokine CXCL12, we found that 1) CXCL12 induces a dynamic re-localization of CRMP2 to uropod, the flexible structure of migrating lymphocyte, and increases its binding to the cytoskeletal protein vimentin; 2) CXCL12 decreases phosphorylation of the glycogen synthase kinase-3β-targeted residues CRMP2-Thr-509/514; and 3) tyrosine Tyr-479 is a new phosphorylation CRMP2 residue and a target for the Src-family kinase Yes. Moreover, phospho-Tyr-479 increased under CXCL12 signaling while phospho-Thr-509/514 decreased. The functional importance of this tyrosine phosphorylation was demonstrated by Y479F mutation that strongly reduced CXCL12-mediated T-cell polarization and motility as tested in a transmigration model and on neural tissue. We propose that differential phosphorylation by glycogen synthase kinase-3β and Yes modulates the contribution of CRMP2 to cytoskeletal reorganization during chemokine-directed T-cell migration. In addition to providing a novel mechanism for T lymphocyte motility, our findings reveal CRMP2 as a transducer of chemokine signaling.T lymphocyte migration is the basis of major immune functions such as responses to infection and inflammation, as well as normal recirculation through the lymphoid organs. Indeed, the role of T-cells depends strongly on their ability to travel between organs via the blood and lymph and to move rapidly within these tissues, by extravasation (1). This latter function is dependent on extracellular signals, among which chemokines play a major role.Chemokines form a superfamily of small proteins that orchestrate lymphocyte polarization and migration (2). These proteins exert their functions by binding specific seven-transmembrane-domain G-protein-coupled receptors on the T-cell surface (3). T-lymphocytes exposed to chemokines, in a soluble or surface-bound gradient, develop a polarized shape, extending at the front, an F-actin-rich lamellipodium, which constitutes the leading edge, and a trailing edge or uropod in which both the microtubule and vimentin networks are retracted during migration. Although F-actin has the well known function of producing the mechanical forces required to generate movement (4), the role of microtubules and vimentin in T-cell migration requires further investigation.Cytoskeletal remodeling is of key importance in migrating cells (5) and is one of the functions carried out by the chemokine stromal cell-derived factor-1α, also named CXCL12. In association with its cognate receptor CXCR4, CXCL12 is a potent chemoattractant for mature T-cells and monocytes (6). Following ligand recognition and binding, CXCR4 signaling starts with the activation of G proteins, followed by various signaling cascade effectors, including MAP² kinases, phosphoinositide 3-kinase, and phospholipase Cγ (7). Although this intracellular signaling cascade has not been completely elucidated, the Src family non-receptor tyrosine kinase Lck and the Syk kinase ZAP-70 have emerged as the main candidates for delivering the input signal following CXCR4 activation (8). Thus, tyrosine kinase activity appears as a central step in CXCR4-dependent chemotaxis.While searching for molecules involved in T-cell motility, we recently identified collapsin response mediator protein 2 (CRMP2) (9), a protein first described in the context of neuronal growth cone advance (10, 11). We demonstrated that CRMP2 regulated both T-cell polarization and spontaneous/chemokine-induced migration of T-lymphocytes. Moreover, CRMP2 was found at the uropod of motile T-cells and has the ability to bind cytoskeletal elements, including vimentin. A correlation between CRMP2 expression levels and cell migratory rates toward a chemokine gradient, including CXCL12, was demonstrated by overexpression and knockdown experiments in T-cells (9). In addition, we recently reported that, in mouse model of neuroinflammation, elevated CRMP2 expression in T lymphocytes correlated with their elevated migratory rates and their ability to target the central nervous system (12). The importance of CXCL12 in the central nervous system and its implication in the pathogenesis of central nervous system disorders, including neuroinflammatory diseases, are well documented (review in Ref. 13). Thus, the aim of the present study was to determine whether and how CRMP2 participates in the transduction pathway induced by CXCL12 on T lymphocytes.