Nod factor perception during infection thread growth fine-tunes nodulation

Bacterial nodulation factors (NFs) are essential signaling molecules for the initiation of a nitrogen-fixing symbiosis in legumes. NFs are perceived by the plant and trigger both local and distant responses, such as curling of root hairs and cortical cell divisions. In addition to their requirement at the start, NFs are produced by bacteria that reside within infection threads. To analyze the role of NFs at later infection stages, several phases of nodulation were studied by detailed light and electron microscopy after coinoculation of adventitious root primordia of Sesbania rostrata with a mixture of Azorhizobium caulinodans mutants ORS571-V44 and ORS571-X15. These mutants are deficient in NF production or surface polysaccharide synthesis, respectively, but they can complement each other, resulting in functional nodules occupied by ORS571-V44. The lack of NFs within the infection threads was confirmed by the absence of expression of an early NF-induced marker, leghemoglobin 6 of S. rostrata. NF production within the infection threads is shown to be necessary for proper infection thread growth and for synchronization of nodule formation with bacterial invasion. However, local production of NFs by bacteria that are taken up by the plant cells at the stage of bacteroid formation is not required for correct symbiosome development.     

Amphiphilic polyether branched molecules to increase the circulation time of cationic particles

The preparation, physicochemical and biological properties of amphiphilic polyether branched molecules is described. These 'bunch shaped' molecules when inserted into cationic liposomes/DNA complexes have shown efficient surface charge shielding. As a consequence they efficiently inhibited the non specific interactions with blood components and significantly enhanced circulation time of the particles in the blood track. Formulations containing these molecules compared positively with those containing PEG lipids, providing a 5-fold increase in circulation time.     

Kinetic evidence is consistent with the rocker-switch mechanism of membrane transport by GlpT

Secondary active transport of substrate across the cell membrane is crucial to many cellular and physiological processes. The crystal structure of one member of the secondary active transporter family, the sn-glycerol-3-phosphate (G3P) transporter (GlpT) of the inner membrane of Escherichia coli, suggests a mechanism for substrate translocation across the membrane that involves a rocker-switch-type movement of the protein. This rocker-switch mechanism makes two specific predictions with respect to kinetic behavior: the transport rate increases with the temperature, whereas the binding affinity of the transporter to a substrate is temperature-independent. In this work, we directly tested these two predictions by transport kinetics and substrate-binding experiments, integrating the data on this single system into a coherent set of observations. The transport kinetics of the physiologically relevant G3P-phosphate antiport reaction were characterized at different temperatures using both E. coli whole cells and GlpT reconstituted into proteoliposomes. Substrate-binding affinity of the transporter was measured using tryptophan fluorescence quenching in detergent solution. Indeed, the substrate transport velocity of GlpT increased dramatically with temperature. In contrast, neither the apparent Michaelis constant (Km) nor the apparent substrate-binding dissociation constant (Kd) showed temperature dependence. Moreover, GlpT-catalyzed G3P translocation exhibited a completely linear Arrhenius function with an activation energy of 35.2 kJ mol-1 for the transporter reconstituted into proteoliposomes, suggesting that the substrate-loaded transporter is delicately poised between the inward- and outward-facing conformations. When these results are taken together, they are in agreement with a rocker-switch mechanism for GlpT.     

Nanoarchaea: representatives of a novel archaeal phylum or a fast-evolving euryarchaeal lineage related to Thermococcales?

Background  

Cultivable archaeal species are assigned to two phyla - the Crenarchaeota and the Euryarchaeota - by a number of important genetic differences, and this ancient split is strongly supported by phylogenetic analysis. The recently described hyperthermophile Nanoarchaeum equitans, harboring the smallest cellular genome ever sequenced (480 kb), has been suggested as the representative of a new phylum - the Nanoarchaeota - that would have diverged before the Crenarchaeota/Euryarchaeota split. Confirming the phylogenetic position of N. equitans is thus crucial for deciphering the history of the archaeal domain.     

Passage of murine scrapie prion protein across the mouse vascular blood-brain barrier

Prions are the infectious agents associated with transmissible spongiform encephalopathies and are composed mainly of a misfolded form of the endogenous prion protein. Prion protein must enter the brain to produce disease. Previous work has emphasized various mechanisms which partially bypass the blood-brain barrier (BBB). Here, we used the brain perfusion method to directly assess the ability of mouse scrapie protein (PrP(SC)) to cross the mouse BBB independent of the influences of neural pathways or circulating immune cells. We found that PrP(SC) oligomers rapidly crossed the BBB without disrupting it with a unidirectional influx rate of about 4.4microl/g-min. HPLC and capillary depletion confirmed that PrP(SC) crossed the entire width of the capillary wall to enter brain parenchyma. PrP(SC) also entered the cerebrospinal fluid (CSF) compartment. These results show that a prion protein can cross the intact BBB to enter both the parenchymal and CSF compartments of the brain.     

Activation of p38 MAP kinase by asbestos in rat mesothelial cells is mediated by oxidative stress

Asbestos fibers are biopersistent particles that are capable of stimulating chronic inflammatory responses in the pleura of exposed individuals. Exposure of pleural mesothelial cells, the progenitor cell of malignant mesothelioma, to asbestos induces an array of cellular responses. The present studies investigated whether the p38 mitogen-activated protein kinase cascade was induced under asbestos-exposed conditions. p38 plays a vital role in the response to stressful stimuli and enables the cell to enter an inflammatory state characterized by cytokine production. Western blot and in vitro kinase assays showed increases in dual phosphorylation and actual activity of p38 after exposure to fibrous and nonfibrous (milled) crocidolite; in contrast, polystyrene beads and iron (III) oxide had no such effects. In common with other asbestos-induced events, this was shown to be an oxidative stress-sensitive effect, inasmuch as preincubation with N-acetyl-L-cysteine or -tocopherol (vitamin E) ameliorated the effect. The present studies show that p38 activity is important for crocidolite-induced activator protein-1 DNA binding, inasmuch as an inhibitor of p38, SB-203580, reduced this activity. Crocidolite-induced cytotoxicity was also reduced with SB-203580, indicating a role for p38 in asbestos-mediated cell death. Our studies suggest that p38 activity could be a crucial factor in the chronic immune response elicited by asbestos and may represent a target for future pharmacological intervention.     

Impact of DNA ligase IV on the fidelity of end joining in human cells

A DNA ligase IV (LIG4)-null human pre-B cell line and human cell lines with hypomorphic mutations in LIG4 are significantly impaired in the frequency and fidelity of end joining using an in vivo plasmid assay. Analysis of the null line demonstrates the existence of an error-prone DNA ligase IV-independent rejoining mechanism in mammalian cells. Analysis of lines with hypomorphic mutations demonstrates that residual DNA ligase IV activity, which is sufficient to promote efficient end joining, nevertheless can result in decreased fidelity of rejoining. Thus, DNA ligase IV is an important factor influencing the fidelity of end joining in vivo. The LIG4-defective cell lines also showed impaired end joining in an in vitro assay using cell-free extracts. Elevated degradation of the terminal nucleotide was observed in a LIG4-defective line, and addition of the DNA ligase IV–XRCC4 complex restored end protection. End protection by DNA ligase IV was not dependent upon ligation. Finally, using purified proteins, we demonstrate that DNA ligase IV–XRCC4 is able to protect DNA ends from degradation by T7 exonuclease. Thus, the ability of DNA ligase IV–XRCC4 to protect DNA ends may contribute to the ability of DNA ligase IV to promote accurate rejoining in vivo.     

Calcium dynamics in the failing heart: restoration by beta-adrenergic receptor blockade

Changes in calcium (Ca2+) regulation contribute to loss of contractile function in dilated cardiomyopathy. Clinical treatment using beta-adrenergic receptor antagonists (beta-blockers) slows deterioration of cardiac function in end-stage heart failure patients; however, the effects of beta-blocker treatment on Ca2+ dynamics in the failing heart are unknown. To address this issue, tropomodulin-overexpressing transgenic (TOT) mice, which suffer from dilated cardiomyopathy, were treated with a nonselective beta-receptor blocker (5 mg. kg-1. day-1 propranolol) for 2 wk. Ca2+ dynamics in isolated cardiomyocytes of TOT mice significantly improved after treatment compared with untreated TOT mice. Frequency-dependent diastolic and Ca2+ transient amplitudes were returned to normal in propranolol-treated TOT mice and but not in untreated TOT mice. Ca2+ kinetic measurements of time to peak and time decay of the caffeine-induced Ca2+ transient to 50% relaxation were also normalized. Immunoblot analysis of untreated TOT heart samples showed a 3.6-fold reduction of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), whereas Na+/Ca2+ exchanger (NCX) concentrations were increased 2.6-fold relative to nontransgenic samples. Propranolol treatment of TOT mice reversed the alterations in SERCA and NCX protein levels but not potassium channels. Although restoration of Ca2+ dynamics occurred within 2 wk of beta-blockade treatment, evidence of functional improvement in cardiac contractility assessed by echocardiography took 10 wk to materialize. These results demonstrate that beta-adrenergic blockade restores Ca2+ dynamics and normalizes expression of Ca2+-handling proteins, eventually leading to improved hemodynamic function in cardiomyopathic hearts.