Protein kinase C zeta phosphorylates a subset of selective sites of the NADPH oxidase component p47phox and participates in formyl peptide-mediated neutrophil respiratory burst

Generation of superoxide anion by the multiprotein complex NADPH phagocyte oxidase is accompanied by extensive phosphorylation of its 47-kDa protein component, p47(phox), a major cytosolic component of this oxidase. Protein kinase C zeta (PKC zeta), an atypical PKC isoform expressed abundantly in human polymorphonuclear leukocytes (PMN), translocates to the PMN plasma membrane upon stimulation by the chemoattractant fMLP. We investigated the role of PKC zeta in p47(phox) phosphorylation and in superoxide anion production by human PMN. In vitro incubation of recombinant p47(phox) with recombinant PKC zeta induced a time- and concentration-dependent phosphorylation of p47(phox) with an apparent K(m) value of 2 microM. Phosphopeptide mapping analysis of p47(phox) showed that PKC zeta phosphorylated fewer selective sites in comparison to "conventional" PKCs. Serine 303/304 and serine 315 were identified as targets of PKC zeta by site-directed mutagenesis. Stimulation of PMN by fMLP induced a rapid and sustained plasma membrane translocation of PKC zeta that correlated to that of p47(phox). A cell-permeant-specific peptide antagonist of PKC zeta inhibited both fMLP-induced phosphorylation of p47(phox) and its membrane translocation. The antagonist also inhibited the fMLP-induced production of oxidant (IC(50) of 10 microM), but not that induced by PMA. The inhibition of PKC zeta expression in HL-60 neutrophil-like cells using antisense oligonucleotides (5 and 10 microM) inhibited fMLP-promoted oxidant production (27 and 50%, respectively), but not that induced by PMA. In conclusion, p47(phox) is a substrate for PKC zeta and participates in the signaling cascade between fMLP receptors and NADPH oxidase activation.     

Stem cell factor and H2O2 induce GLUT1 translocation in M07e cells

This work aims to elucidate the mechanisms involved in the early activation of glucose transport in hematopoietic M07e cells by stem cell factor (SCF) and a reactive oxygen species (ROS) as H2O2. SCF and H2O2 increase Vmax for glucose transport; this enhancement is due to a higher content in GLUT1 in plasma membranes, possibly through a translocation from intracellular stores. Inhibitors of tyrosine kinases or phospholipase C (PLC) remove glucose transport enhancement and prevent translocation. The inhibitory effect of STI-571 suggests a role for c-kit tyrosine kinase on glucose transport activation not only by SCF, but also by H2O2. On the other hand, neither protein kinase C nor phosphoinositide-3-kinase appear to be involved in the acute activation of glucose transport. Our data suggest that i) in M07e cells, SCF and exogenous H2O2 elicit a short-term activation of glucose transport through a translocation of GLUT1 from intracellular stores to plasma membranes; ii) both stimuli could share at least some signaling pathways leading to glucose uptake activation, involving protein tyrosine kinases and PLC iii) H2O2 could act increasing the level of tyrosine phosphorylation through the inhibition of tyrosine phosphatases and mimicking the regulation role of endogenous ROS.     

Control of life, death, and differentiation in cultured midgut cells of the lepidopteran, Heliothis virescens

Summary Differentiated cells in the insect midgut depend on stem cells for renewal. We have immunologically identified Integrin β₁, a promotor of cell-cell adhesion that also induces signals mediating proliferation, differentiation, and apoptosis on the surfaces of culturedHeliothis virescens midgut cells; clusters of immunostained integrin β₁-like material, indicative of activated integrin, were detected on aggregating midgut columnar cells. Growth factor-like peptides (midgut differentiation factors 1 and 2 [MDF1 and MDF2]), isolated from conditioned medium containingManduca sexta midgut cells, may be representative of endogenous midgut signaling molecules. Exposing the cultured midgut cells toBacillus thuringiensis (Bt) toxin caused large numbers of mature differentiated cells to die, but the massive cell death simultaneously induced a 150–200% increase in the numbers of midgut stem and differentiating cells. However, after the toxin was washed out, the proportions of cell types returned to near-control levels within 2 d, indicating endogenous control of cell-population dynamics. MDF1 was detected immunologically in larger numbers of Bt-treated columnar cells than controls, confirming its role in inducing the differentiation of rapidly produced stem cells. However, other insect midgut factors regulating increased proliferation, differentiation, as well as inhibition of proliferation and adjustment of the ratio of cell types, remain to be discovered. Products mentioned in this article are not endorsed by the U.S. Department of Agriculture.     

Artoindonesianins Q-T,four isoprenylated flavones from Artocarpus champeden Spreng. (Moraceae)

Four isoprenylated flavones, artoindonesianins Q-T, were isolated from the heartwood of Artocarpus champeden Roxb. The structures of these compounds were elucidated on the basis of their spectroscopic data.     

Stem cells from midguts of Lepidopteran larvae: clues to the regulation of stem cell fate

Previously, we showed that isolated stem cells from midguts of Heliothis virescens can be induced to multiply in response to a multiplication protein (MP) isolated from pupal fat body, or to differentiate to larval types of mature midgut cells in response to either of 4 differentiation factors (MDFs) isolated from larval midgut cell-conditioned medium or pupal hemolymph. In this work, we show that the responses to MDF-2 and MP in H. virescens stem cells decayed at different time intervals, implying that the receptors or response cascades for stem cell differentiation and multiplication may be different. However, the processes appeared to be linked, since conditioned medium and MDF-2 prevented the action of MP on stem cells; MP by itself appeared to repress stem cell differentiation. Epidermal growth factor, retinoic acid, and platelet-derived growth factor induced isolated midgut stem cells of H. virescens and Lymantria dispar to multiply and to differentiate to mature midgut cells characteristic of prepupal, pupal, and adult lepidopteran midgut epithelium, and to squamous-like cells and scales not characteristic of midgut tissue instead of the larval types of mature midgut epithelium induced by the MDFs. Midgut stem cells appear to be multipotent and their various differentiated fates can be influenced by several growth factors.     

Suppressing Manganese Dissolution from Lithium Manganese Oxide Spinel Cathodes with Single‐Layer Graphene

Spinel‐structured LiMn₂O₄ (LMO) is a desirable cathode material for Li‐ion batteries due to its low cost, abundance, and high power capability. However, LMO suffers from limited cycle life that is triggered by manganese dissolution into the electrolyte during electrochemical cycling. Here, it is shown that single‐layer graphene coatings suppress manganese dissolution, thus enhancing the performance and lifetime of LMO cathodes. Relative to lithium cells with uncoated LMO cathodes, cells with graphene‐coated LMO cathodes provide improved capacity retention with enhanced cycling stability. X‐ray photoelectron spectroscopy reveals that graphene coatings inhibit manganese depletion from the LMO surface. Additionally, transmission electron microscopy demonstrates that a stable solid electrolyte interphase is formed on graphene, which screens the LMO from direct contact with the electrolyte. Density functional theory calculations provide two mechanisms for the role of graphene in the suppression of manganese dissolution. First, common defects in single‐layer graphene are found to allow the transport of lithium while concurrently acting as barriers for manganese diffusion. Second, graphene can chemically interact with Mn³⁺ at the LMO electrode surface, promoting an oxidation state change to Mn⁴⁺, which suppresses dissolution.     

A new method for the extraction of fetal ECG from the dependent abdominal signals using blind source separation and adaptive noise cancellation techniques

Background

The electrocardiogram (ECG) is a diagnostic tool that records the electrical activity of the heart, and depicts it as a series of graph-like tracings, or waves. Being able to interpret these details allows diagnosis of a wide range of heart problems. Fetal electrocardiogram (FECG) extraction has an important impact in medical diagnostics during the mother pregnancy period. Since the observed FECG signals are often mixed with the maternal ECG (MECG) and the noise induced by the movement of electrodes or by mother motion, the separation process of the ECG signal sources from the observed data becomes quite complicated. One of its complexity is when the ECG sources are dependent, thus, in this paper we introduce a new approach of blind source separation (BSS) in the noisy context for both independent and dependent ECG signal source. This approach consist in denoising the observed ECG signals using a bilateral total variation (BTV) filter; then minimizing the Kullbak-Leibler divergence between copula densities to separate the FECG signal from the MECG one.

Results

We present simulation results illustrating the performance of our proposed method. We will consider many examples of independent/dependent source component signals. The results will be compared with those of the classical method called independent component analysis (ICA) under the same conditions. The accuracy of source estimation is evaluated through a criterion, called again the signal-to-noise-ratio (SNR). The first experiment shows that our proposed method gives accurate estimation of sources in the standard case of independent components, with performance around 27 dB in term of SNR. In the second experiment, we show the capability of the proposed algorithm to successfully separate two noisy mixtures of dependent source components - with classical criterion devoted to the independent case - fails, and that our method is able to deal with the dependent case with good performance.

Conclusions

In this work, we focus specifically on the separation of the ECG signal sources taken from skin two electrodes located on a pregnant woman’s body. The ECG separation is interpreted as a noisy linear BSS problem with instantaneous mixtures. Firstly, a denoising step is required to reduce the noise due to motion artifacts using a BTV filter as a very effective one-pass filter for denoising. Then, we use the Kullbak-Leibler divergence between copula densities to separate the fetal heart rate from the mother one, for both independent and dependent cases.

     

Connecting the Dots: The Effects of Macromolecular Crowding on Cell Physiology

The physicochemical properties of cellular environments with a high macromolecular content have been systematically characterized to explain differences observed in the diffusion coefficients, kinetics parameters, and thermodynamic properties of proteins inside and outside of cells. However, much less attention has been given to the effects of macromolecular crowding on cell physiology. Here, we review recent findings that shed some light on the role of crowding in various cellular processes, such as reduction of biochemical activities, structural reorganization of the cytoplasm, cytoplasm fluidity, and cellular dormancy. We conclude by presenting some unresolved problems that require the attention of biophysicists, biochemists, and cell physiologists. Although it is still underappreciated, macromolecular crowding plays a critical role in life as we know it.     

Plutonium(IV) reduction by the metal-reducing bacteria Geobacter metallireducens GS15 and Shewanella oneidensis MR1

The bacterial reduction of actinides has been suggested as a possible remedial strategy for actinide-contaminated environments, and the bacterial reduction of Pu(VI/V) has the potential to produce highly insoluble Pu(IV) solid phases. However, the behavior of plutonium with regard to bacterial reduction is more complex than for other actinides because it is possible for Pu(IV) to be further reduced to Pu(III), which is relatively more soluble than Pu(IV). This work investigates the ability of the metal-reducing bacteria Geobacter metallireducens GS15 and Shewanella oneidensis MR1 to enzymatically reduce freshly precipitated amorphous Pu(IV) (OH)(4) [Pu(IV)(OH)(4(am))] and soluble Pu(IV)(EDTA). In cell suspensions without added complexing ligands, minor Pu(III) production was observed in cultures containing S. oneidensis, but little or no Pu(III) production was observed in cultures containing G. metallireducens. In the presence of EDTA, most of the Pu(IV)(OH)(4(am)) present was reduced to Pu(III) and remained soluble in cell suspensions of both S. oneidensis and G. metallireducens. When soluble Pu(IV)(EDTA) was provided as the terminal electron acceptor, cell suspensions of both S. oneidensis and G. metallireducens rapidly reduced Pu(IV)(EDTA) to Pu(III)(EDTA) with nearly complete reduction within 20 to 40 min, depending on the initial concentration. Neither bacterium was able to use Pu(IV) (in any of the forms used) as a terminal electron acceptor to support growth. These results have significant implications for the potential remediation of plutonium and suggest that strongly reducing environments where complexing ligands are present may produce soluble forms of reduced Pu species.     

Gene expression profiling defines ATP as a key regulator of human dendritic cell functions

Extracellular ATP and PGE2 are two cAMP-elevating agents inducing semimaturation of human monocyte-derived dendritic cells (MoDCs). We have extensively compared the gene expression profiles induced by adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS) and PGE2 in human MoDCs using microarray technology. At 6 h of stimulation, ATPgammaS initiated an impressive expression profile compared with that of PGE2 (1125 genes compared with 133 genes, respectively) but after 24 h the number of genes regulated by ATPgammaS or PGE2 was more comparable. Many target genes involved in inflammation have been identified and validated by quantitative RT-PCR experiments. We have then focused on novel ATPgammaS and PGE2 target genes in MoDCs including CSF-1, MCP-4/CCL13 chemokine, vascular endothelial growth factor-A, and neuropilin-1. ATPgammaS strongly down-regulated CSF-1 receptor mRNA and CSF-1 secretion, which are involved in monocyte and dendritic cell (DC) differentiation. Additionally, ATPgammaS down-regulated several chemokines involved in monocyte and DC migration including CCL2/MCP-1, CCL3/MIP-1alpha, CCL4/MIP-1beta, CCL8/MCP-2, and CCL13/MCP-4. Interestingly, vascular endothelial growth factor A, a major angiogenic factor displaying immunosuppressive properties, was secreted by MoDCs in response to ATPgammaS, ATP, or PGE2, alone or in synergy with LPS. Finally, flow cytometry experiments have demonstrated that ATPgammaS, ATP, and PGE2 down-regulate neuropilin-1, a receptor playing inter alia an important role in the activation of T lymphocytes by DCs. Our data give an extensive overview of the genes regulated by ATPgammaS and PGE2 in MoDCs and an important insight into the therapeutic potential of ATP- and PGE2-treated human DCs.