Effects of magnesium ions on two EMCV IRES key activity fragments

Using NMR magnetization transfer from water and ammonia-catalyzed exchange of the imino protons, changes have been monitored in base-pair kinetics induced by Mg^2 + in two key activity fragments r(CACCUGGCGACAGGUG) and r(GGCCAAAAGCC) of the encephalomyocarditis virus internal ribosome entry site. For r(CACCUGGCGACAGGUG), the addition of Mg^2 + reveals two types of base-pairs: r(U⁵⁴⁵·A) and r(G⁵⁴⁶·C), in the first category, have lifetimes only slightly higher in the presence of Mg^2 +, whereas their dissociation constants are substantially reduced. This behavior has been termed proximal. The base-pairs r(G⁵⁵³·C) and r(G⁵⁵⁴·C), in the second category, have lifetimes substantially higher in the presence of Mg^2 +, whereas their dissociation constants remain almost constant. This behavior has been termed distal. Mg^2 + has a specific effect on r(CACCUGGCGACAGGUG), the magnitude of which is progressively modulated from the proximal region of the 16-mer towards its distal region. For r(GGCCAAAAGCC), an intermediate behavior is found for base-pairs r(G⁵⁶⁵·C) and r(G⁵⁷²·C). Their lifetimes are slightly higher in the presence of Mg^2 + and their dissociation constants are significantly lower, a behavior resembling that of the 16-mer proximal region. These results indicate that Mg^2 + diffusively moves around r(GGCCAAAAGCC).     

3-Amino-6-methoxy-9-(2-hydroxyethylamino) acridine: A new fluorescent dye to detect mycoplasma contamination in cell cultures

A new fluorescent acridine orange derivative, 3-amino-6-methoxy-9-(2-hydroxyethylamino) acridine (AMHA), has been applied to Hela cells in order to set up appropriate conditions for the detection of mycoplasma contaminations. Since AMHA staining reveals intensely fluorescent nuclei and slight fluorescent cytoplasm, we can visualize and localize mycoplasma contamination on each cell. In combination with a shortened Chen's staining method (1977), AMHA should allow a better detection of mycoplasma in animal cell cultures than the well established Hoechst dye.     

Myosin Va binding to neurofilaments is essential for correct myosin Va distribution and transport and neurofilament density

The identification of molecular motors that modulate the neuronal cytoskeleton has been elusive. Here, we show that a molecular motor protein, myosin Va, is present in high proportions in the cytoskeleton of mouse CNS and peripheral nerves. Immunoelectron microscopy, coimmunoprecipitation, and blot overlay analyses demonstrate that myosin Va in axons associates with neurofilaments, and that the NF-L subunit is its major ligand. A physiological association is indicated by observations that the level of myosin Va is reduced in axons of NF-L-null mice lacking neurofilaments and increased in mice overexpressing NF-L, but unchanged in NF-H-null mice. In vivo pulse-labeled myosin Va advances along axons at slow transport rates overlapping with those of neurofilament proteins and actin, both of which coimmunoprecipitate with myosin Va. Eliminating neurofilaments from mice selectively accelerates myosin Va translocation and redistributes myosin Va to the actin-rich subaxolemma and membranous organelles. Finally, peripheral axons of dilute-lethal mice, lacking functional myosin Va, display selectively increased neurofilament number and levels of neurofilament proteins without altering axon caliber. These results identify myosin Va as a neurofilament-associated protein, and show that this association is essential to establish the normal distribution, axonal transport, and content of myosin Va, and the proper numbers of neurofilaments in axons.     

Phylogenetic, genomic organization and expression analysis of hydrophobin genes in the ectomycorrhizal basidiomycete Laccaria bicolor

Hydrophobins are morphogenetic, small secreted hydrophobic fungal proteins produced in response to changing development and environmental conditions. These proteins are important in the interaction between certain fungi and their hosts. In mutualistic ectomycorrhizal fungi several hydrophobins form a subclass of mycorrhizal-induced small secreted proteins that are likely to be critical in the formation of the symbiotic interface with host root cells. In this study, two genomes of the ectomycorrhizal basidiomycete Laccaria bicolor strains S238N-H82 (from North America) and 81306 (from Europe) were surveyed to construct a comprehensive genome-wide inventory of hydrophobins and to explore their characteristics and roles during host colonization. The S238N-H82 L. bicolor hydrophobin gene family is composed of 12 genes while the 81306 strain encodes nine hydrophobins, all corresponding to class I hydrophobins. The three extra hydrophobin genes encoded by the S238N-H82 genome likely arose via gene duplication and are bordered by transposon rich regions. Expression profiles of the hydrophobin genes of L. bicolor varied greatly depending on life stage (e.g. free living mycelium vs. root colonization) and on the host root environment. We conclude from this study that the complex diversity and range of expression profiles of the Laccaria hydrophobin multi-gene family have likely been a selective advantage for this mutualist in colonizing a wide range of host plants.     

Centrosome positioning in polarized cells: Common themes and variations

The centrosome position is tightly regulated during the cell cycle and during differentiated cellular functions. Because centrosome organizes the microtubule network to coordinate both intracellular organization and cell signaling, centrosome positioning is crucial to determine either the axis of cell division, the direction of cell migration or the polarized immune response of lymphocytes. Since alteration of centrosome positioning seems to promote cell transformation and tumor spreading, the molecular mechanisms controlling centrosome movement in response to extracellular and intracellular cues are under intense investigation. Evolutionary conserved pathways involving polarity proteins and cytoskeletal rearrangements are emerging as common regulators of centrosome positioning in a wide variety of cellular contexts.     

Co-culture of human fibroblasts,smooth muscle and endothelial cells promotes osteopontin induction in hypoxia

Arteriovenous fistulas (AVFs) are the preferred vascular access for haemodialysis of patients suffering from end-stage renal disease, a worldwide public health problem. However, they are prone to a high rate of failure due to neointimal hyperplasia and stenosis. This study aimed to determine if osteopontin (OPN) was induced in hypoxia and if OPN could be responsible for driving AVF failure. Identification of new factors that participate in remodelling of AVFs is a challenge. Three cell lines representing the cells of the three layers of the walls of arteries and veins, fibroblasts, smooth muscle cells and endothelial cells, were tested in mono- and co-culture in vitro for OPN expression and secretion in normoxia compared to hypoxia after silencing the hypoxia-inducible factors (HIF-1α, HIF-2α and HIF-1/2α) with siRNA or after treatment with an inhibitor of NF-kB. None of the cells in mono-culture showed OPN induction in hypoxia, whereas cells in co-culture secreted OPN in hypoxia. The changes in oxygenation that occur during AVF maturation up-regulate secretion of OPN through cell-cell interactions between the different cell layers that form AVF, and in turn, these promote endothelial cell proliferation and could participate in neointimal hyperplasia.     

The distribution of phosphorylation sites among identified proteolytic fragments of mammalian neurofilaments

Neurofilaments were treated with chymotrypsin or with Staphylococcus aureus V8 protease (V8 protease) and the proteolytic fragments in soluble and particulate centrifugal fractions were identified by immune blotting, using antibodies raised against the Mr = 68,000 (P68), 145,000 (P145), and 200,000 (P200) subunits. The data provide further evidence that each of the three subunits has a different disposition within the filament. A Mr = 160,000 fragment of P200, which may correspond to the side arm projections on neurofilaments, was released into solution by chymotrypsin. In contrast, the proteolytic fragments of P68 and P145 were recovered mainly in the particulate centrifugal fraction, indicating that the two subunits are more closely associated with the filament backbone. Proteolytic cleavage studies on neurofilaments that were 32P-labeled in vivo indicated that the phosphorylated domains in P200 and P145 are localized in a restricted segment of each subunit, which occurs between the chymotryptic and V8 protease cleavage sites. No 32P was associated with the bulk of chymotryptic fragments, which are found in the particulate fraction, are about 40,000 daltons in size, and derive from all three neurofilament subunits. Most of the phosphorylation sites in neurofilaments are peripherally located in the projection domain of P200, suggesting that phosphorylation may modulate interactions between neurofilaments and other neuronal components.     

P. falciparum RH5‐Basigin interaction induces changes in the cytoskeleton of the host RBC

The successful invasion of Plasmodium is an essential step in their life cycle. The parasite reticulocyte‐binding protein homologues (RHs) and erythrocyte‐binding like proteins are two families involved in the invasion leading to merozoite‐red blood cell (RBC) junction formation. Ca²⁺ signaling has been shown to play a critical role in the invasion. RHs have been linked to Ca²⁺ signaling, which triggers the erythrocyte‐binding like proteins release ahead of junction formation, consistent with RHs performing an initial sensing function in identifying suitable RBCs. RH5, the only essential RHs, is a highly promising vaccine candidate. RH5‐basigin interaction is essential for merozoite invasion and also important in determining host tropism. Here, we show that RH5 has a distinct function from the other RHs. We show that RH5‐Basigin interaction on its own triggers a Ca²⁺ signal in the RBC resulting in changes in RBC cytoskeletal proteins phosphorylation and overall alterations in RBC cytoskeleton architecture. Antibodies targeting RH5 that block the signal prevent invasion before junction formation consistent with the Ca²⁺ signal in the RBC leading to rearrangement of the cytoskeleton required for invasion. This work provides the first time a functional context for the essential role of RH5 and will now open up new avenues to target merozoite invasion.     

Baroreflex control of lumbar and renal sympathetic nerve activity in conscious rats

This study compared the baroreflex control of lumbar and renal sympathetic nerve activity (SNA) in conscious rats. Arterial pressure (AP) and lumbar and renal SNA were simultaneously recorded in six freely behaving rats. Pharmacological estimates of lumbar and renal sympathetic baroreflex sensitivity (BRS) were obtained by means of the sequential intravenous administration of sodium nitroprusside and phenylephrine. Sympathetic BRS was significantly (P < 0.05) lower for lumbar [3.0 +/- 0.4 normalized units (NU)/mmHg] than for renal (7.6 +/- 0.6 NU/mmHg) SNA. During a 219-min baseline period, spontaneous lumbar and renal BRS were continuously assessed by computing the gain of the transfer function relating AP and SNA at heart rate frequency over consecutive 61.4-s periods. The transfer gain was considered only when coherence between AP and SNA significantly differed from zero, which was verified in 99 +/- 1 and 96 +/- 3% of cases for lumbar and renal SNA, respectively. When averaged over the entire baseline period, spontaneous BRS was significantly (P < 0.05) lower for lumbar (1.3 +/- 0.2 NU/mmHg) than for renal (2.3 +/- 0.3 NU/mmHg) SNA. For both SNAs, spontaneous BRS showed marked fluctuations (variation coefficients were 26 +/- 2 and 28 +/- 2% for lumbar and renal SNA, respectively). These fluctuations were positively correlated in five of six rats (R = 0.44 +/- 0.06; n = 204 +/- 8; P < 0.0001). We conclude that in conscious rats, the baroreflex control of lumbar and renal SNA shows quantitative differences but is modulated in a mostly coordinated way.     

The crystal structure of the plexin-semaphorin-integrin domain/hybrid domain/I-EGF1 segment from the human integrin beta2 subunit at 1.8-A resolution

Integrins are modular (alphabeta) heterodimeric proteins that mediate cell adhesion and convey signals across the plasma membrane. Interdomain motions play a key role in signal transduction by propagating structural changes through the molecule, thus controlling the activation state and adhesive properties of the integrin. We expressed a soluble fragment of the human integrin beta2 subunit comprising the plexin-semaphorin-integrin domain (PSI)/hybrid domain/I-EGF1 fragment and present its crystal structure at 1.8-A resolution. The structure reveals an elongated molecule with a rigid architecture stabilized by nine disulfide bridges. The PSI domain is located centrally and participates in the formation of extended interfaces with the hybrid domain and I-EGF1 domains, respectively. The hybrid domain/PSI interface involves the burial of an Arg residue, and contacts between PSI and I-EGF1 are mainly mediated by well conserved Arg and Trp residues. Conservation of key interacting residues across the various integrin beta subunits sequences suggests that our structure represents a good model for the entire integrin family. Superposition with the integrin beta3 receptor in its bent conformation suggests that an articulation point is present at the linkage between its I-EGF1 and I-EGF2 modules and underlines the importance of this region for the control of integrin-mediated cell adhesion.