A molecular dynamics study of reovirus attachment protein sigma1 reveals conformational changes in sigma1 structure

Molecular dynamics simulations were performed using the recently determined crystal structure of the reovirus attachment protein, sigma1. These studies were conducted to improve an understanding of two unique features of sigma1 structure: the protonation state of Asp(345), which is buried in the sigma1 trimer interface, and the flexibility of the protein at a defined region below the receptor-binding head domain. Three copies of aspartic acids Asp(345) and Asp(346) cluster in a solvent-inaccessible and hydrophobic region at the sigma1 trimer interface. These residues are hypothesized to mediate conformational changes in sigma1 during viral attachment or cell entry. Our results indicate that protonation of Asp(345) is essential to the integrity of the trimeric structure seen by x-ray crystallography, whereas deprotonation induces structural changes that destabilize the trimer interface. This finding was confirmed by electrostatic calculations using the finite difference Poisson-Boltzmann method. Earlier studies show that sigma1 can exist in retracted and extended conformations on the viral surface. Since protonated Asp(345) is necessary to form a stable, extended trimer, our results suggest that protonation of Asp(345) may allow for a structural transition from a partially detrimerized molecule to the fully formed trimer seen in the crystal structure. Additional studies were conducted to quantify the previously observed flexibility of sigma1 at a defined region below the receptor-binding head domain. Increased mobility was observed for three polar residues (Ser(291), Thr(292), and Ser(293)) located within an insertion between the second and third beta-spiral repeats of the crystallized portion of the sigma1 tail. These amino acids interact with water molecules of the solvent bulk and are responsible for oscillating movement of the head of approximately 50 degrees during 5 ns of simulations. This flexibility may facilitate viral attachment and also function in cell entry and disassembly. These findings provide new insights about the conformational dynamics of sigma1 that likely underlie the initiation of the reovirus infectious cycle.     

CzcR-CzcS, a two-component system involved in heavy metal and carbapenem resistance in Pseudomonas aeruginosa

Pseudomonas aeruginosa is an environmental bacterium involved in mineralization of organic matter. It is also an opportunistic pathogen able to cause serious infections in immunocompromised hosts. As such, it is exposed to xenobiotics including solvents, heavy metals, and antimicrobials. We studied the response of P. aeruginosa upon exposure to heavy metals or antibiotics to investigate whether common regulatory mechanisms govern resistance to both types of compounds. We showed that sublethal zinc concentrations induced resistance to zinc, cadmium, and cobalt, while lethal zinc concentrations selected mutants constitutively resistant to these heavy metals. Both zinc-induced and stable zinc-resistant strains were also resistant to the carbapenem antibiotic imipenem. On the other hand, only 20% of clones selected on imipenem were also resistant to zinc. Heavy metal resistance in the mutants could be correlated by quantitative real time PCR with increased expression of the heavy metal efflux pump CzcCBA and its cognate two-component regulator genes czcR-czcS. Western blot analysis revealed reduced expression of the basic amino acid and carbapenem-specific OprD porin in all imipenem-resistant mutants. Sequencing of the czcR-czcS DNA region in eight independent zinc- and imipenem-resistant mutants revealed the presence of the same V194L mutation in the CzcS sensor protein. Overexpression in a susceptible wild type strain of the mutated CzsS protein, but not of the wild type form, resulted in decreased oprD and increased czcC expression. We further show that zinc is released from latex urinary catheters into urine in amounts sufficient to induce carbapenem resistance in P. aeruginosa, possibly compromising treatment of urinary tract infections by this class of antibiotics.     

A novel adaptation of the integrin PSI domain revealed from its crystal structure

Integrin beta-subunits contain an N-terminal PSI (for plexin-semaphorin-integrin) domain that contributes to integrin activation and harbors the PI(A) alloantigen associated with immune thrombocytopenias and susceptibility to sudden cardiac death. Here we report the crystal structure of PSI in the context of the crystallized alphaVbeta3 ectodomain. The integrin PSI forms a two-stranded antiparallel beta-sheet flanked by two short helices; its long interstrand loop houses Pl(A) and may face the EGF2 domain. The integrin PSI contains four cysteine pairs connected in a 1-4, 2-8, 3-6, 5-7 pattern. An unexpected feature of the structure is that the final, eighth cysteine is located C-terminal to the Ig-like hybrid domain and is thus separated by the hybrid domain from the other seven cysteines of PSI. This architecture may be relevant to the evolution of integrins and should help refine the current models of integrin activation.     

Low-dose inhalation of an endothelin-A receptor antagonist in experimental acute lung injury: ET-1 plasma concentration and pulmonary inflammation

Inhalation of endothelin (ET)-A receptor antagonists has been shown to improve gas exchange in experimental acute lung injury (ALI) but may induce side effects by increasing circulating ET-1 levels. We investigated whether the inhaled ET(A) receptor antagonist, LU-135252, at low doses, improves gas exchange without affecting ET-1 plasma concentrations and lung injury in an animal model of ALI. Twenty-two piglets were examined in a prospective, randomized, controlled study. In anesthetized animals, ALI was induced by surfactant depletion. Animals received either LU-135252 at a dose of 0.3 mg/kg during 20 mins (LU group; n = 11), or nebulization of saline buffer (control group; n = 11). The Mann-Whitney U test was used to compare groups (P < 0.05). In the LU group, arterial partial pressure of oxygen (PaO2) and mean pulmonary artery pressure (MPAP) improved compared with the control group (PaO2, 319 +/- 44 mm Hg vs. 57 +/- 3 mm Hg; MPAP, 32 +/- 2 mm Hg vs. 41 +/- 2 mm Hg; values at 6 hrs after induction of ALI; P < 0.05). Mean arterial pressure and cardiac output were not different between groups. ET-1 plasma concentrations increased from 0.96 +/- 0.06 fmol/ml after induction of ALI to a maximum of 1.17 +/- 0.09 fmol/ml at 3 hrs after ALI onset in the LU group and did not differ significantly from the control group (1.21 +/- 0.08 fmol/ml, not significant). On histologic examination, we found no differences in total lung injury score between groups. However, the LU group revealed significantly reduced interstitial inflammation and hemorrhage (P < 0.05 vs. control group). In this animal model of ALI, inhalation of LU-135252 at a dose of 0.3 mg/kg induced a significant and sustained improvement in gas exchange, whereas there were no changes in ET-1 plasma concentrations. Furthermore, our data indicate a trend toward decreased pulmonary inflammation in the group receiving the inhaled ET(A) receptor antagonist.     

Endothelin-1 influences the efficacy of inhaled nitric oxide in experimental acute lung injury

Beneficial effects of inhaled nitric oxide (iNO) on arterial oxygenation in acute lung injury (ALI) suggest the presence of vasoconstriction in ventilated lung regions and this may be influenced by endothelin-1 (ET-1). We studied a possible interaction between ET-1 and iNO in experimental ALI. Sixteen piglets were anesthetized and mechanically ventilated (inspired O2 fraction, 1.0). After induction of ALI by surfactant depletion, animals were randomly assigned to either inhale 30 ppm NO (iNO group, n = 8), or to receive no further intervention (controls, n = 8). Measurements were performed during the following 4 hrs. In all animals, induction of ALI significantly decreased arterial oxygen tension (PaO2) from 569 +/- 15 (prelavage) to 58 +/- 3 mm Hg. Inhaled NO significantly increased PaO2 when compared with controls (iNO group: 265 +/- 51 mm Hg; controls: 50 +/- 4 mm Hg, values at 4 hrs, P < 0.01). Prelavage ET-1 plasma levels were comparable between groups (iNO: 0.74 +/- 0.03, controls: 0.71 +/- 0.03 fmol/ml, NS). During the protocol, the ET-1 levels increased and were different at 3 hrs (iNO: 0.93 +/- 0.06, controls: 1.25 +/- 0.09 fmol/ml; P < 0.05). PaO2 changes induced by iNO revealed a moderate and significant correlation with ET-1 plasma levels (R = 0.548, P = 0.001). Our data suggest that endogenous ET-1 production influences the efficacy of iNO in ALI. Furthermore, iNO reduced ET-1 plasma levels, possibly indicating anti-inflammatory properties of iNO in the early phase of ALI.     

Insights into Peroxisome Function from the Structure of PEX3 in Complex with a Soluble Fragment of PEX19

The human peroxins PEX3 and PEX19 play a central role in peroxisomal membrane biogenesis. The membrane-anchored PEX3 serves as the receptor for cytosolic PEX19, which in turn recognizes newly synthesized peroxisomal membrane proteins. After delivering these proteins to the peroxisomal membrane, PEX19 is recycled to the cytosol. The molecular mechanisms underlying these processes are not well understood. Here, we report the crystal structure of the cytosolic domain of PEX3 in complex with a PEX19-derived peptide. PEX3 adopts a novel fold that is best described as a large helical bundle. A hydrophobic groove at the membrane-distal end of PEX3 engages the PEX19 peptide with nanomolar affinity. Mutagenesis experiments identify phenylalanine 29 in PEX19 as critical for this interaction. Because key PEX3 residues involved in complex formation are highly conserved across species, the observed binding mechanism is of general biological relevance.     

Active immunization with amyloid-beta 1-42 impairs memory performance through TLR2/4-dependent activation of the innate immune system

Active immunization with amyloid-β (Aβ) peptide 1-42 reverses amyloid plaque deposition in the CNS of patients with Alzheimer's disease and in amyloid precursor protein transgenic mice. However, this treatment may also cause severe, life-threatening meningoencephalitis. Physiological responses to immunization with Aβ(1-42) are poorly understood. In this study, we characterized cognitive and immunological consequences of Aβ(1-42)/CFA immunization in C57BL/6 mice. In contrast to mice immunized with myelin oligodendrocyte glycoprotein (MOG)(35-55)/CFA or CFA alone, Aβ(1-42)/CFA immunization resulted in impaired exploratory activity, habituation learning, and spatial-learning abilities in the open field. As morphological substrate of this neurocognitive phenotype, we identified a disseminated, nonfocal immune cell infiltrate in the CNS of Aβ(1-42)/CFA-immunized animals. In contrast to MOG(35-55)/CFA and PBS/CFA controls, the majority of infiltrating cells in Aβ(1-42)/CFA-immunized mice were CD11b(+)CD14(+) and CD45(high), indicating their blood-borne monocyte/macrophage origin. Immunization with Aβ(1-42)/CFA was significantly more potent than immunization with MOG(35-55)/CFA or CFA alone in activating macrophages in the secondary lymphoid compartment and peripheral tissues. Studies with TLR2/4-deficient mice revealed that the TLR2/4 pathway mediated the Aβ(1-42)-dependent proinflammatory cytokine release from cells of the innate immune system. In line with this, TLR2/4 knockout mice were protected from cognitive impairment upon immunization with Aβ(1-42)/CFA. Thus, this study identifies adjuvant effects of Aβ(1-42), which result in a clinically relevant neurocognitive phenotype highlighting potential risks of Aβ immunotherapy.     

Targeting XIAP for the treatment of malignancy

X-linked inhibitor of apoptosis protein (XIAP) is a member of the inhibitor of apoptosis proteins family of caspase inhibitors that selectively binds and inhibits caspases-3, -7 and -9, but not caspase-8. As such, XIAP blocks a substantial portion of the apoptosis pathway and is an attractive target for novel therapeutic agents for the treatment of malignancy. Antisense oligonucleotides directed against XIAP are effective in vitro and are currently being evaluated in clinical trials. Small molecule XIAP inhibitors that target the baculovirus IAP repeat (BIR) 2 or BIR 3 domain are in preclinical development and are advancing toward the clinic. This review will discuss the progress being made in developing antisense and small-molecule XIAP inhibitors.     

Soil seed banks near rubbing trees indicate dispersal of plant species into forests by wild boar

Current knowledge about processes that generate long-distance dispersal of plants is still limited despite its importance for persistence of populations and colonization of new potential habitats. Today wild large mammals are presumed to be important vectors for long-distance transport of diaspores within and between European temperate forest patches, and in particular wild boars recently came into focus. Here we use a specific habit of wild boar, i.e. wallowing in mud and subsequent rubbing against trees, to evaluate epizoochorous dispersal of vascular plant diaspores. We present soil seed bank data from 27 rubbing trees versus 27 control trees from seven forest areas in Germany. The mean number of viable seeds and the plant species number were higher in soil samples near rubbing trees compared with control trees. Ten of the 20 most frequent species were more frequent, and many species exclusively appeared in the soil samples near rubbing trees. The large number of plant species and seeds – more than 1000 per tree – in the soils near rubbing trees is difficult to explain unless the majority were dispersed by wild boar. Hooked and bristly diaspores, i.e. those adapted to epizoochory, were more frequent; however, many species with unspecialized diaspores occurred exclusively near rubbing trees. As opposed to plant species closely tied to forests species which occur both in forest and open vegetation and non-forest species were more frequent near rubbing trees compared with controls. These findings are consistent with previous studies on diaspore loads in the coats and hooves of shot wild boars. However, our method allows to identify the transport of diaspores from the open landscape into forest stands, where they might especially emerge after disturbance, and a clustered distribution of epizoochorically dispersed seeds. Moreover, accumulation of seeds of wetness indicators near rubbing trees demonstrates directed dispersal of plant species inhabiting wet places among remote wallows.