Role of endocytosis and low pH in murine hepatitis virus strain A59 cell entry

Infection by the coronavirus mouse hepatitis virus strain A59 (MHV-A59) requires the release of the viral genome by fusion with the respective target membrane of the host cell. Fusion is mediated by the viral S protein. Here, the entry pathway of MHV-A59 into murine fibroblast cells was studied by independent approaches. Infection of cells assessed by plaque reduction assay was strongly inhibited by lysosomotropic compounds and substances that interfere with clathrin-dependent endocytosis, suggesting that MHV-A59 is taken up via endocytosis and delivered to acidic endosomal compartments. Infection was only slightly reduced in the presence of substances inhibiting proteases of endosomal compartments, precluding that the endocytic uptake is required to activate the fusion potential of the S protein by its cleavage. Fluorescence confocal microscopy of labeled MHV-A59 confirmed that virus is taken up via endocytosis. Bright labeling of intracellular compartments suggests their fusion with the viral envelope. No fusion with the plasma membrane was observed at neutral pH conditions. However, when virus was bound to cells and the pH was lowered to 5.0, we observed a strong labeling of the plasma membrane. Electron microscopy revealed low pH triggered conformational alterations of the S ectodomain. Very likely, these alterations are irreversible because low-pH treatment of viruses in the absence of target membranes caused an irreversible loss of the fusion activity. The results imply that endocytosis plays a major role in MHV-A59 infection and the acidic pH of the endosomal compartment triggers a conformational change of the S protein mediating fusion.     

ATP-driven MalK dimer closure and reopening and conformational changes of the "EAA" motifs are crucial for function of the maltose ATP-binding cassette transporter (MalFGK2)

We have investigated conformational changes of the purified maltose ATP-binding cassette transporter (MalFGK(2)) upon binding of ATP. The transport complex is composed of a heterodimer of the hydrophobic subunits MalF and MalG constituting the translocation pore and of a homodimer of MalK, representing the ATP-hydrolyzing subunit. Substrate is delivered to the transporter in complex with periplasmic maltose-binding protein (MalE). Cross-linking experiments with a variant containing an A85C mutation within the Q-loop of each MalK monomer indicated an ATP-induced shortening of the distance between both monomers. Cross-linking caused a substantial inhibition of MalE-maltose-stimulated ATPase activity. We further demonstrated that a mutation affecting the "catalytic carboxylate" (E159Q) locks the MalK dimer in the closed state, whereas a transporter containing the "ABC signature" mutation Q140K permanently resides in the resting state. Cross-linking experiments with variants containing the A85C mutation combined with cysteine substitutions in the conserved EAA motifs of MalF and MalG, respectively, revealed close proximity of these residues in the resting state. The formation of a MalK-MalG heterodimer remained unchanged upon the addition of ATP, indicating that MalG-EAA moves along with MalK during dimer closure. In contrast, the yield of MalK-MalF dimers was substantially reduced. This might be taken as further evidence for asymmetric functions of both EAA motifs. Cross-linking also caused inhibition of ATPase activity, suggesting that transporter function requires conformational changes of both EAA motifs. Together, our data support ATP-driven MalK dimer closure and reopening as crucial steps in the translocation cycle of the intact maltose transporter and are discussed with respect to a current model.     

The Perception of Dynamic and Static Facial Expressions of Happiness and Disgust Investigated by ERPs and fMRI Constrained Source Analysis

A recent functional magnetic resonance imaging (fMRI) study by our group demonstrated that dynamic emotional faces are more accurately recognized and evoked more widespread patterns of hemodynamic brain responses than static emotional faces. Based on this experimental design, the present study aimed at investigating the spatio-temporal processing of static and dynamic emotional facial expressions in 19 healthy women by means of multi-channel electroencephalography (EEG), event-related potentials (ERP) and fMRI-constrained regional source analyses. ERP analysis showed an increased amplitude of the LPP (late posterior positivity) over centro-parietal regions for static facial expressions of disgust compared to neutral faces. In addition, the LPP was more widespread and temporally prolonged for dynamic compared to static faces of disgust and happiness. fMRI constrained source analysis on static emotional face stimuli indicated the spatio-temporal modulation of predominantly posterior regional brain activation related to the visual processing stream for both emotional valences when compared to the neutral condition in the fusiform gyrus. The spatio-temporal processing of dynamic stimuli yielded enhanced source activity for emotional compared to neutral conditions in temporal (e.g., fusiform gyrus), and frontal regions (e.g., ventromedial prefrontal cortex, medial and inferior frontal cortex) in early and again in later time windows. The present data support the view that dynamic facial displays trigger more information reflected in complex neural networks, in particular because of their changing features potentially triggering sustained activation related to a continuing evaluation of those faces. A combined fMRI and EEG approach thus provides an advanced insight to the spatio-temporal characteristics of emotional face processing, by also revealing additional neural generators, not identifiable by the only use of an fMRI approach.     

Three new species of Pristionchus (Nematoda: Diplogastridae) show morphological divergence through evolutionary intermediates of a novel feeding‐structure polymorphism

Developmental plasticity is often correlated with diversity and has been proposed as a facilitator of phenotypic novelty. Yet how a dimorphism arises or how additional morphs are added is not understood, and few systems provide experimental insight into the evolution of polyphenisms. Because plasticity correlates with structural diversity in Pristionchus nematodes, studies in this group can test the role of plasticity in facilitating novelty. Here, we describe three new species, Pristionchus fukushimae sp. nov. , Pristionchus hoplostomus sp. nov. , and the hermaphroditic Pristionchus triformis sp. nov. , which are characterized by a novel polymorphism in their mouthparts. In addition to showing the canonical mouth dimorphism of diplogastrid nematodes, comprising a stenostomatous (‘narrow‐mouthed’) and a eurystomatous (‘wide‐mouthed’) form, the new species exhibit forms with six, 12, or intermediate numbers of cheilostomatal plates. Correlated with this polymorphism is another trait that varies among species: whereas divisions between plates are complete in P. triformis sp. nov. , which is biased towards a novel ‘megastomatous’ form comprising 12 complete plates, the homologous divisions in the other new species are partial and of variable length. In a reconstruction of character evolution, a phylogeny inferred from 26 ribosomal protein genes and a partial small subunit rRNA gene supported the megastomatous form of P. triformis sp. nov. as the derived end of a series of split‐plate forms. Although split‐plate forms were normally only observed in eurystomatous nematodes, a single 12‐plated stenostomatous individual of P. hoplostomus sp. nov. was also observed, suggesting independence of the two types of mouth plasticity. By introducing these new species to the Pristionchus model system, this study provides further insight into the evolution of polymorphisms and their evolutionary intermediates. © 2013 The Linnean Society of London     

Aerobic exercise alone results in clinically significant weight loss for men and women: Midwest exercise trial 2

Exercise is recommended by public health agencies for weight management; however, the role of exercise is generally considered secondary to energy restriction. Few studies exist that have verified completion of exercise, measured the energy expenditure of exercise, and prescribed exercise with equivalent energy expenditure across individuals and genders.

Objective:

The objective of this study was to evaluate aerobic exercise, without energy restriction, on weight loss in sedentary overweight and obese men and women.

Design and Methods:

This investigation was a randomized, controlled, efficacy trial in 141 overweight and obese participants (body mass index, 31.0 ± 4.6 kg/m²; age 22.6 ± 3.9 years). Participants were randomized (2:2:1 ratio) to exercise at either 400 kcal/session or 600 kcal/session or to a nonexercise control. Exercise was supervised, 5 days/week, for 10 months. All participants were instructed to maintain usual ad libitum diets. Because of the efficacy design, completion of ≥90% of exercise sessions was an a priori definition of per protocol, and these participants were included in the analysis.

Results:

Weight loss from baseline to 10 months for the 400 and 600 kcal/session groups was 3.9 ± 4.9 kg (4.3%) and 5.2 ± 5.6 kg (5.7%), respectively, compared with weight gain for controls of 0.5 ± 3.5 kg (0.5%) (P < 0.05). Differences for weight loss from baseline to 10 months between the exercise groups and differences between men and women within groups were not statistically significant.

Conclusions:

Supervised exercise, with equivalent energy expenditure, results in clinically significant weight loss with no significant difference between men and women.     

Fractionation of serum components using nanoporous substrates

Numerous previously uncharacterized molecules resident within the low molecular weight circulatory proteome may provide a picture of the ongoing pathophysiology of an organism. Recently, proteomic signatures composed of low molecular weight molecules have been identified using mass spectrometry combined with bioinformatic algorithms. Attempts to sequence and identify the molecules that underpin the fingerprints are currently underway. The finding that many of these low molecular weight molecules may exist bound to circulating carrier proteins affords a new opportunity for fractionation and separation techniques prior to mass spectrometry-based analysis. In this study we demonstrate a method whereby nanoporous substrates may be used for the facile and reproducible fractionation and selective binding of the serum-based biomarker material, including subcellular proteins found within the serum. Aminopropyl-coated nanoporous silicon, when exposed to serum, can deplete serum of proteins and yield a serum with a distinct, altered MS profile. Additionally, aminopropyl-coated, nanoporous controlled-pore glass beads are able to bind a subset of serum proteins and release them with stringent elution. The eluted proteins have distinct MS profiles, gel electrophoresis profiles, and differential peptide sequence identities, which vary based on the size of the nanopores. These material surfaces could be employed in strategies for the harvesting and preservation of labile and carrier-protein-bound molecules in the blood.     

IL-6 expression induced by adenosine A2b receptor stimulation in U373 MG cells depends on p38 mitogen activated kinase and protein kinase C

Adenosine binds to a class of G-protein coupled receptors, which are further distinguished as A(1), A(2a), A(2b) and A(3) adenosine receptors. As we have shown earlier, the stable adenosine analogue NECA (N6-(R)-phenylisopropyladenosine) stimulates IL-6 expression in the human astrocytoma cell line U373 MG via the A(2b) receptor. The mechanism by which NECA promotes astrocytic IL-6 expression has not been identified. By using various inhibitors of signal transduction, we found that p38 mitogen-activated protein kinases (MAPK) activation (inhibitor SB202190), but not extracellular signal-regulated kinase (ERK) (PD98059) and c-jun N-terminal kinase (JNK)(SP600125), is essential in the NECA-induced signalling cascade that leads to the increase in IL-6 synthesis in U373 MG cells. Results obtained with protein kinase C (PKC) inhibitors that have different substrate specificities, indicated that the PKC delta and epsilon isoforms are also involved in adenosine receptor A(2b) dependent upregulation of IL-6 expression. This is supported by the fact that NECA induced the activation of PKC delta and epsilon in U373 MG cells.     

Neomycin induces high-affinity agonist binding of G-protein-coupled receptors

Neomycin, an inositol-phospholipid-binding aminoglycoside antibiotic, is known to interfere with signal transduction mechanisms involving phospholipase C as effector enzyme. In this study, we report that neomycin can also markedly influence agonist binding of G-protein-coupled receptors. In membranes of differentiated human leukemia cells (HL 60 cells), neomycin (0.1-10 mM) was found to induce high-affinity binding of the chemotactic tripeptide, N-formyl-methionylleucylphenylalanine (fMet-Leu-Phe), to its receptor sites in a manner similar to magnesium. Gentamycin and streptomycin, two other aminoglycoside antibiotics, were as potent and as effective as neomycin or magnesium in inducing high-affinity agonist receptor binding. Pretreatment of the cells with pertussis toxin reduced the effects of magnesium and neomycin on agonist receptor binding likewise. In contrast, magnesium but not neomycin largely enhanced the potency of guanine nucleotides, particularly of GTP and its analog, guanosine-5'-O-(3-thiotriphosphate), to reduce fMet-Leu-Phe receptor binding, while maximal inhibition of agonist receptor binding by guanine nucleotides was identical with magnesium and neomycin. Furthermore, neomycin could not replace magnesium in providing stimulation of HL 60 membrane high-affinity GTPase by fMet-Leu-Phe. In close agreement to these findings on the pertussis-toxin-sensitive Gi-protein-coupled formyl peptide receptors, neomycin in a manner similar to magnesium induced high-affinity agonist binding of Gs-protein-coupled beta-adrenoceptors. Similar to formyl peptide receptor binding, high-affinity binding of isoproterenol to beta-adrenoceptors in guinea pig lung membranes induced by magnesium and neomycin was inhibited by the GTP analog, guanosine-5'-O-(3-thiotriphosphate), to a similar maximal extent but with an about 100-fold higher potency in the presence of magnesium than in the presence of neomycin. The data presented thus indicate that neomycin and other aminoglycoside antibiotics can mimic the action of magnesium (or other divalent cations) in inducing high-affinity agonist binding of Gi- and Gs-protein-coupled receptors, but not in inducing subsequent G-protein activation by guanosine triphosphates. The data, furthermore, suggest that neomycin by this selective action will be a powerful tool to dissect the multiple sites of magnesium's action in the agonist receptor-G-protein interaction.     

Expression,secretion and surface display of a human alkaline phosphatase by the ciliate <Emphasis Type="Italic">Tetrahymena thermophila</Emphasis>

Background  

Tetrahymena thermophila possesses many attributes that render it an attractive host for the expression of recombinant proteins. Surface proteins from the parasites Ichthyophthirius multifiliis and Plasmodium falciparum and avian influenza virus antigen H5N1 were displayed on the cell membrane of this ciliate. Furthermore, it has been demonstrated that T. thermophila is also able to produce a functional human DNase I. The present study investigates the heterologous expression of the functional human intestinal alkaline phosphatase (hiAP) using T. thermophila and thereby presents a powerful tool for the optimization of the ciliate-based expression system.