Two Distinct Dynamic Modes Subtend the Detection of Unexpected Sounds

The brain response to auditory novelty comprises two main EEG components: an early mismatch negativity and a late P300. Whereas the former has been proposed to reflect a prediction error, the latter is often associated with working memory updating. Interestingly, these two proposals predict fundamentally different dynamics: prediction errors are thought to propagate serially through several distinct brain areas, while working memory supposes that activity is sustained over time within a stable set of brain areas. Here we test this temporal dissociation by showing how the generalization of brain activity patterns across time can characterize the dynamics of the underlying neural processes. This method is applied to magnetoencephalography (MEG) recordings acquired from healthy participants who were presented with two types of auditory novelty. Following our predictions, the results show that the mismatch evoked by a local novelty leads to the sequential recruitment of distinct and short-lived patterns of brain activity. In sharp contrast, the global novelty evoked by an unexpected sequence of five sounds elicits a sustained state of brain activity that lasts for several hundreds of milliseconds. The present results highlight how MEG combined with multivariate pattern analyses can characterize the dynamics of human cortical processes.     

Phorbol 12-myristate, 13-acetate potentiates the action of the calcium ionophore in stimulating arachidonic acid release and production of phosphatidic acid in rabbit neutrophils

The addition of the tumor-promoting phorbol 12-myristate, 13-acetate to rabbit neutrophils greatly potentiates the effect of the calcium ionophore A23187 on [3H]-arachidonic acid release and [32P]-phosphatidic acid generation. At 5 X 10(-8) M A23187, the addition of 20 ng/ml PMA potentiates the action of the ionophore on [3H]-arachidonic acid release by 5-fold. At 5 X 10(-7) M A23187, PMA enhances [32P]-phosphatidic acid production by 1.5-fold. Incubation of the neutrophils with 5 X 10(-7) M ionophore for two minutes causes a significant increase in the [32P] phosphatidic acid production but does not affect the levels of [32P]-phosphatidylinositol or [32P]-phosphatidylinositol 4,5 bis-phosphate. In addition, increasing the sodium chloride concentrations in the suspending medium causes an increase in the level of phosphatidylinositol 4,5 bis-phosphate. These results suggest that the phorbol ester either acting directly or through the activation of protein kinase C modulates significantly the activities of the various forms of phospholipases, particularly A2, and/or increases the availability or amounts of their substrates.     

Stimulation by chemotactic factor of actin association with the cytoskeleton in rabbit neutrophils. Effects of calcium and cytochalasin B

The amounts of actin and myosin in rabbit neutrophils expressed as micrograms/10(6) cells are 5.6 +/- 0.75 and 0.56 +/- 0.08, respectively. The average value of the total actin in rabbit neutrophils under unstimulated conditions is distributed between Triton X-100 soluble fraction (74 +/- 7%) and Triton X-100 insoluble fraction (26 +/- 3%). The Triton X-100 soluble and insoluble fractions will be referred to as the cytoplasmic and the cytoskeletal components. When the cells are stimulated by the chemotactic factor formyl-Met-Leu-Phe the amount of actin associated with the cytoskeleton increases to 73.7 +/- 6% of the total cell actin. This increase is rapid, dose-dependent and mediated through fMet-Leu-Phe receptors. Neither the time course of the response nor the dose-response curve is affected by the removal of calcium from the suspending medium. Calcium ions at concentrations greater than 10(-7) M added after Triton X-100 extraction dissociate actin from the cytoskeleton. Calcium at 1.9 microM added after Triton X-100 extraction reduces the amount of cytoskeletal actin under control and stimulated conditions to 10.3 +/- 0.9 and 33 +/- 1.5% of the total cell actin, respectively. The average value of the total myosin in rabbit neutrophils under unstimulated conditions is distributed between the cytosol (32 +/- 10%) and the cytoskeleton (68 +/- 18%). When neutrophils are stimulated with the chemotactic factor fMet-Leu-Phe the amount of myosin associated with the cytoskeleton does not increase significantly. Cytochalasin B decreases cytoskeletal actin and myosin and causes a shift in the amount of actin and myosin from the cytoskeleton to the cytoplasm both under fMet-Leu-Phe-stimulated and control conditions. In the presence of 1.6 mM extracellular Ca2+ and cytochalasin B (5 micrograms/ml) the amount of actin associated with the cytoskeleton under control and stimulated conditions is reduced to 13 +/- 2.2 and 10.2 +/- 3.5% of total cell actin, and that of myosin is reduced to 50.2 +/- 14 and 2.3 +/- 0.8% of the total cell myosin. The effect of cytochalasin B on actin does not depend on the time of its addition relative to that of fMet-Leu-Phe and is more pronounced in the presence of Ca2+. These results are discussed in terms of the roles of cytochalasin B and calcium in the overall mechanism of neutrophil degranulation induced by chemotactic factors.     

Characterization of the priming effects of human granulocyte-macrophage colony-stimulating factor on human neutrophil leukotriene synthesis

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) is an in vitro and in vivo stimulator of human bone marrow myelomonocytic precursor cells and mature granulocyte and macrophage effector cells. We have compared the effect of GM-CSF on the synthesis of 5-lipoxygenase products induced by the chemotactic peptide fMet-Leu-Phe and the calcium ionophore A23187 in human neutrophils. Although GM-CSF alone did not stimulate detectable synthesis of products of the 5-lipoxygenase pathway, pre-incubation of neutrophils with 200 pM GM-CSF for 1 hour at 23 degrees C enhanced synthesis of leukotriene B4, its all-trans isomers and omega-oxidation products, and 5-hydroxyeicosatetraenoic acid in response to both the calcium ionophore A23187 (1.5 microM), and the chemotactic peptide fMet-Leu-Phe (0.1 microM). This priming effect of GM-CSF was maximal after a 60 min incubation at 23 degrees C, or after a 30 min preincubation at 37 degrees C. The effect of GM-CSF was maximal using a concentration of 1 nM. Enhancement of the leukotriene synthesis stimulated by A23187 was only observed when the cells were stimulated by the ionophore for periods of 3 minutes or less. In contrast, the enhancing effect of GM-CSF was still apparent when cells were exposed to fMet-Leu-Phe for as long as 15 minutes. Furthermore, the enhancing effect of GM-CSF was ablated when neutrophils were stimulated with A23187 and exogenous arachidonic acid. However, co-addition of exogenous arachidonic acid with fMet-Leu-Phe did not entirely mask the effect of GM-CSF. Possible mechanisms of action of GM-CSF are discussed.     

Calcium mobilization, actin polymerization and right-angle light scatter responses to leukotriene B4, 12(R)- and 12(S)-hydroxyeicosatetraenoic acid in human neutrophils

The presence of microgram quantities of 12(R)-hydroxyeicosatetraenoic acid (12(R)HETE) in psoriatic scales has been reported. 12(R)HETE has been found to be 5-10 times more potent than its isomer 12(S)HETE in inducing neutrophil locomotion and aggregation. In this study, the ability of these two eicosanoids to elicit a mobilization of calcium and a polymerization of actin was examined and compared to that of leukotriene B4. These two cell-biochemical assays were chosen in view of the likelihood that they are relevant to the motile functions of the neutrophils. 12(R)HETE was found to induce an increase in the cytoplasmic level of free calcium and in the amount of polymerized actin. 12(S)HETE also raised the level of free calcium, though to a lesser extent than 12(R)HETE, but did not induce a detectable polymerization of actin. Leukotriene B4 was more active, on the basis of concentration and maximal response, than either 12(R)HETE or 12(S)HETE. The activity of 12(R)HETE detected and reported in this communication provides support for the suggestion that this eicosanoid may play a significant role in the pathogenesis of the inflammatory reactions in psoriasis.     

Chemoattractant-induced cytoplasmic pH changes and cytoskeletal reorganization in human neutrophils. Relationship to the stimulated calcium transients and oxidative burst

The relationships between the chemotactic factor-stimulated mobilization of calcium, activation of the NADPH-oxidase, changes in cytosolic pH, and in the level of polymerized actin in human neutrophils have been examined. The approach taken was to use intracellular calcium chelators, and pharmacologic modulators (both positive and negative) of the NADPH-oxidase to measure the aforementioned responses under conditions where the calcium transients were abrogated and/or the generation of superoxide anions was either inhibited or augmented. The decrease in cytosolic pH induced by chemoattractants was inhibited by the calcium chelator BAPTA and by the diglyceride kinase inhibitor 6-[2-(4-[(4-fluorophenyl)phenylmethylene]-1-piperidinylethyl ]-7-methyl-5H-thiazolo[3,2-alpha]pyriimidin-5-one (R59022) (this latter compound enhanced the oxidative response of the cells). Furthermore, a specific inhibitor of the NADPH-oxidase (diphenyleneiodonium) had no significant effect on the cytosolic acidification induced by FMLP or leukotriene B4. These results indicate that the initiation of the cytosolic acidification induced by chemotactic factors is a calcium-dependent event that is not directly linked to the activation of the NADPH-oxidase. In contrast, the stimulated polymerization of actin was insensitive to BAPTA, R59022, and diphenyleneiodonium. Thus, neither the calcium transients nor the oxidative burst play a signaling role in the initiation of actin polymerization elicited by chemoattractants. These data indicate that additional investigations are needed to uncover the biochemical basis of the signals initiated in human neutrophils by chemotactic factors that lead to the polymerization of actin and to the cytosolic acidification.     

Priming of calcium mobilization in human neutrophils by granulocyte-macrophage colony-stimulating factor: Evidence for an involvement of phospholipase D-derived phosphatidic acid

Human neutrophils pre-incubated with granulocyte-macrophage-colony-stimulating factor (GM-CSF) exhibit an enhanced mobilization of calcium in response to secondary stimuli such as chemotactic factors. The mechanisms underlying this priming effect of GM-CSF were examined. It was first demonstrated that the additional calcium mobilized by chemotactic factors in GM-CSF-treated cells was derived from intracellular stores and was associated neither with an increased permeability to calcium nor with production of inositol 1,4,5-trisphosphate. These results indicated that GM-CSF called upon a novel mechanism in order to enhance the mobilization of calcium in human neutrophils. The growth factor has recently been shown to prime phospholipase D leading to an enhanced activation by chemotactic factors and an augmented production of phosphatidic acid. Furthermore the ability of exogenous phosphatidic acid to mobilize calcium in cell types other than neutrophils has been previously demonstrated. Therefore, we examined the potential involvement of phospholipase D in the priming of the calcium response by GM-CSF in human neutrophils. Inhibition of the production of the fMet-Leu-Phe-stimulated production of phosphatidic acid by ethanol or wortmannin had only marginal effects on the concurrent mobilization of calcium. However, the priming of the mobilization of calcium by GM-CSF was greatly decreased in cells treated with either ethanol or wortmannin. These results provide strong support for the hypothesis that the production of phosphatidic acid, which is enhanced in GM-CSF-treated cells, is linked to an increased mobilization of intracellular calcium. These results may have relevance to the mechanism of action of GM-CSF in mature haematopoeitic cells as well to the mitogenic activity of other growth factors.     

Sodium and calcium transport in cat red cells.

The transport of radioactive sodium and calcium in high sodium low potassium cat red blood cells has been studied under various experimental conditions. In these cells, calcium uptake was found to increase by 25-fold when cell volume was decreased from 1.0 normal to 0.55. Increasing cell volume from 1.0 to 1.1 normal was found to decrease calcium-uptake by 30%. N-ethylmaleimide, NEM, phloretin, colchicine, and vinblastine were found to inhibit Na-uptake by these cells. Among these four agents, phloretin was the most potent inhibitor of Na-influx. It appears that phloretin produces its effect through its interaction with the cell membrane and not with cell interior.     

Discovery of a Novel Human Pegivirus in Blood Associated with Hepatitis C Virus Co-Infection

Hepatitis C virus (HCV) and human pegivirus (HPgV), formerly GBV-C, are the only known human viruses in the Hepacivirus and Pegivirus genera, respectively, of the family Flaviviridae. We present the discovery of a second pegivirus, provisionally designated human pegivirus 2 (HPgV-2), by next-generation sequencing of plasma from an HCV-infected patient with multiple bloodborne exposures who died from sepsis of unknown etiology. HPgV-2 is highly divergent, situated on a deep phylogenetic branch in a clade that includes rodent and bat pegiviruses, with which it shares <32% amino acid identity. Molecular and serological tools were developed and validated for high-throughput screening of plasma samples, and a panel of 3 independent serological markers strongly correlated antibody responses with viral RNA positivity (99.9% negative predictive value). Discovery of 11 additional RNA-positive samples from a total of 2440 screened (0.45%) revealed 93–94% nucleotide identity between HPgV-2 strains. All 12 HPgV-2 RNA-positive cases were identified in individuals also testing positive for HCV RNA (12 of 983; 1.22%), including 2 samples co-infected with HIV, but HPgV-2 RNA was not detected in non-HCV-infected individuals (p<0.0001), including those singly infected by HIV (p = 0.0075) or HBV (p = 0.0077), nor in volunteer blood donors (p = 0.0082). Nine of the 12 (75%) HPgV-2 RNA positive samples were reactive for antibodies to viral serologic markers, whereas only 28 of 2,429 (1.15%) HPgV-2 RNA negative samples were seropositive. Longitudinal sampling in two individuals revealed that active HPgV-2 infection can persist in blood for at least 7 weeks, despite the presence of virus-specific antibodies. One individual harboring both HPgV-2 and HCV RNA was found to be seronegative for both viruses, suggesting a high likelihood of simultaneous acquisition of HCV and HPgV-2 infection from an acute co-transmission event. Taken together, our results indicate that HPgV-2 is a novel bloodborne infectious virus of humans and likely transmitted via the parenteral route.