The Akt/PKB pathway is constitutively activated in Theileria-transformed leucocytes, but does not directly control constitutive NF-kappaB activation

The intracellular protozoan parasites Theileria parva and Theileria annulata transform leucocytes by interfering with host cell signal transduction pathways. They differ from tumour cells, however, in that the transformation process can be entirely reversed by elimination of the parasite from the host cell cytoplasm using a specific parasiticidal drug. We investigated the state of activation of Akt/PKB, a downstream target of PI3-K-generated phosphoinositides, in Theileria-transformed leucocytes. Akt/PKB is constitutively activated in a PI3-K- and parasite-dependent manner, as judged by the specific phosphorylation of key residues, in vitro kinase assays and its cellular distribution. In previous work, we demonstrated that the parasite induces constitutive activation of the transcription factor NF-kappaB, providing protection against spontaneous apoptosis that accompanies transformation. In a number of other systems, a link has been established between the PI3-K-Akt/PKB pathway and NF-kappaB activation, resulting in protection against apoptosis. In Theileria-transformed leucocytes, activation of the NF-kappaB and the PI3-K-Akt/PKB pathways are not directly linked. The PI3-K-Akt/PKB pathway does not contribute to the persistent induction of IkappaBalpha phosphorylation, NF-kappaB DNA-binding or transcriptional activity. We show that the two pathways are downregulated with different kinetics when the parasite is eliminated from the host cell cytoplasm and that NF-kappaB-dependent protection against apoptosis is not dependent on a functional PI3-K-Akt/PKB pathway. We also demonstrate that Akt/PKB contributes, at least in part, to the proliferation of Theileria-transformed T cells.

Characterization of cellular and molecular functions of the p21 -activated kinase, Shk1, in the fission yeast, Schizosaccharomyces pombe

The p21-activated kinase, Shk1, is an essential serine/threonine kinase required for normal cell polarity, proper mating response, and hyperosmotic stress response, in the fission yeast, Schizosaccharomyces pombe. This study has established a novel role for Shk1 as a microtubule regulator in fission yeast and, in addition, characterized a potential biological substrate of Shk1. Cells defective in Shk1 function were found to exhibit malformed interphase and mitotic microtubules, are hypersensitive to the microtubule disrupting drug thiabendazole (TBZ), and are cold sensitive for growth. Microtubule disruption by TBZ results in a significant reduction of Shk1 kinase activity, which is restored after cells are released from the drug, thus providing a correlation between Shk1 kinase activity and active microtubule polymerization. Consistent with a role for Shk1 as a microtubule regulator, GFP-Shk1 fusion proteins localize to interphase microtubules and mitotic microtubule spindles. Furthermore, loss of Tea1, a presumptive microtubule regulator in fission yeast, exacerbates the growth and microtubule defects of cells deficient in Shk1 function, and results in illicit Shk1 localization. Moreover, loss of the Cdc2 inhibitory kinase Wee1, which has been implicated as a mediator of the Shk1 pathway, leads to significant microtubule defects. Intriguingly, Wee1 protein levels are markedly reduced both by partial loss of Shk1 function and by treatment with TBZ. These results suggest that Shk1 is required for proper regulation of microtubule dynamics in fission yeast and may interact with Tea1 and Wee1 in this regulatory process. ^ To further understand Shk1 function in fission yeast, a yeast two-hybrid screen for proteins that interact with the Shk1 catalytic domain was performed. This screen led to the identification of a novel protein, Skb10 (for S&barbelow;hk1 k&barbelow;inase b&barbelow;inding protein 10). Coprecipitation experiments demonstrated that Skb10 associates with Shk1 in S. pombe cells. (Abstract shortened by UMI.) ^

Air-steam gasification of sewage sludge in a bubbling bed reactor: Effect of alumina as a primary catalyst

Numerous references can be found in scientific literature regarding biomass gasification. However, there are few works related to sludge gasification. A study of sewage sludge gasification process in a bubbling fluidised bed gasifier on a laboratory scale is here reported. The aim was to find the optimum conditions for reducing the production of tars and gain more information on the influx of different operating variables in the products resulting from the gasification of this waste. The variables studied were the equivalence ratio (ER), the steam-biomass ratio (SB) and temperature. Specifically, the ER was varied from 0.2 to 0.4, the SB from 0 to 1 and the temperature from 750 °C (1023 K) to 850 °C (1123 K). Although it was observed that tar production could be considerably reduced (up to 72%) by optimising the gasification conditions, the effect of using alumina (aluminium oxide, of proven efficacy in destroying the tar produced in biomass gasification) as primary catalyst in air and air-steam mixture tests was also verified. The results show that by adding small quantities of alumina to the bed (10% by weight of fed sludge) considerable reductions in tar production can be obtained (up to 42%) improving, at the same time, the lower heating value (LHV) of the gas and carbon conversion.

Endogenous, hyperactive Rac3 controls proliferation of breast cancer cells by a p21-activated kinase-dependent pathway

Uncontrolled cell proliferation is a major feature of cancer. Experimental cellular models have implicated some members of the Rho GTPase family in this process. However, direct evidence for active Rho GTPases in tumors or cancer cell lines has never been provided. In this paper, we show that endogenous, hyperactive Rac3 is present in highly proliferative human breast cancer-derived cell lines and tumor tissues. Rac3 activity results from both its distinct subcellular localization at the membrane and altered regulatory factors affecting the guanine nucleotide state of Rac3. Associated with active Rac3 was deregulated, persistent kinase activity of two isoforms of the Rac effector p21-activated kinase (Pak) and of c-Jun N-terminal kinase (JNK). Introducing dominant-negative Rac3 and Pak1 fragments into a breast cancer cell line revealed that active Rac3 drives Pak and JNK kinase activities by two separate pathways. Only the Rac3–Pak pathway was critical for DNA synthesis, independently of JNK. These findings identify Rac3 as a consistently active Rho GTPase in human cancer cells and suggest an important role for Rac3 and Pak in tumor growth.

A potential role for the nuclear factor of activated T cells family of transcriptional regulatory proteins in adipogenesis

NFAT (nuclear factor of activated T cells) is a family of transcription factors implicated in the control of cytokine and early immune response gene expression. Recent studies have pointed to a role for NFAT proteins in gene regulation outside of the immune system. Herein we demonstrate that NFAT proteins are present in 3T3-L1 adipocytes and, upon fat cell differentiation, bind to and transactivate the promoter of the adipocyte-specific gene aP2. Further, fat cell differentiation is inhibited by cyclosporin A, a drug shown to prevent NFAT nuclear localization and hence function. Thus, these data suggest a role for NFAT transcription factors in the regulation of the aP2 gene and in the process of adipocyte differentiation.

Pro-apoptotic gene expression mediated by the p38 mitogen-activated protein kinase signal transduction pathway

Neurotrophic factor deprivation causes apoptosis by a mechanism that requires macromolecular synthesis. This fact suggests that gene expression is necessary to achieve cell death. To identify mRNA that is expressed in apoptotic cells we used subtractive hybridization with cDNA prepared from neuronal pheochromocytoma cells. Monoamine oxidase (MAO) expression was increased in cells during nerve growth factor withdrawal-induced apoptosis. The increased apoptosis and induction of MAO was prevented by inhibition of the p38 mitogen-activated protein (MAP) kinase pathway. MAO may contribute to the apoptotic process because inhibition of MAO activity suppressed cell death. Together, these data indicate that MAO may be a target of pro-apoptotic signal transduction by the p38 MAP kinase pathway.

An essential role for the interferon-inducible, double-stranded RNA-activated protein kinase PKR in the tumor necrosis factor-induced apoptosis in U937 cells.

Tumor necrosis factor alpha (TNF-alpha) is well-characterized for its necrotic action against tumor cells; however, it has been increasingly associated with an apoptosis-inducing potential on target cells. While the signaling events and the actual cytolytic mechanism(s) for both TNF-alpha-induced necrosis and apoptosis remain to be fully elucidated, we report here on (i) the ability of TNF-alpha to induce apoptosis in the promonocytic U937 cells, (ii) the discovery of a cross-talk between the TNF-alpha and the interferon signaling pathways, and (iii) the pivotal role of interferon-inducible, double-stranded RNA-activated protein kinase (PKR) in the induction of apoptosis by TNF-alpha. Our data from microscopy studies, trypan blue exclusion staining, and apoptotic DNA ladder electrophoresis revealed that a subclone derived from U937 and carrying a PKR antisense expression vector was resistant to TNF-alpha-induced apoptosis. Further, TNF-alpha initiated a generalized RNA degradation process in which the participation of PKR was required. Finally, the PKR gene is a candidate "death gene" since overexpression of this gene could bring about apoptosis in U937 cells.

Down-regulation of NF-kappa B protein levels in activated human lymphocytes by 1,25-dihydroxyvitamin D3.

The effect of 1,25-dihydroxyvitamin D3 [1,25(OH)2)D3], a steroid hormone with immunomodulating properties, on nuclear factor kappa B (NF-kappa B) proteins was examined in in vitro activated normal human lymphocytes by Western blot analysis. Over a 72-hr period of activation, the expression of the 50-kDa NF-kappa B, p50, and its precursor, p105, was increased progressively. When cells were activated in the presence of 1,25(OH)2D3, the levels of the mature protein as well as its precursor were decreased. The effect of the hormone on the levels of p50 was demonstrable in the cytosolic and nuclear compartments; it required between 4 and 8 hr and was specific, as 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 were ineffective. Besides p50, 1,25(OH)2D3 decreased the levels of another NF-kappa B protein, namely c-rel. In addition, 1,25(OH)2D3 decreased the abundance of a specific DNA-protein complex formed upon incubation of nuclear extracts from activated lymphocytes with a labeled NF-kappa B DNA binding motif. Further, 1,25(OH)2D3 inhibited the transcriptional activity of NF-kappa B in Jurkat cells transiently transfected with a construct containing four tandem repeats of the NF-kappa B binding sequence of the immunoglobulin kappa light chain gene linked to the chloramphenicol acetyltransferase reporter gene. These observations demonstrate directly that there is de novo synthesis of NF-kappa B during human lymphocyte activation and suggest that this process is hormonally regulated.

Depletion-activated calcium current is inhibited by protein kinase in RBL-2H3 cells.

Whole-cell patch-clamp recordings and single-cell Ca2+ measurements were used to study the control of Ca2+ entry through the Ca2+ release-activated Ca2+ influx pathway (ICRAC) in rat basophilic leukemia cells. When intracellular inositol 1,4,5-trisphosphate (InsP3)-sensitive stores were depleted by dialyzing cells with high concentrations of InsP3, ICRAC inactivated only slightly in the absence of ATP. Inclusion of ATP accelerated inactivation 2-fold. The inactivation was increased further by the ATP analogue adenosine 5'-[gamma-thio]triphosphate, which is readily used by protein kinases, but not by 5'-adenylyl imidodiphosphate, another ATP analogue that is not used by kinases. Neither cyclic nucleotides nor inhibition of calmodulin or tyrosine kinase prevented the inactivation. Staurosporine and bisindolylmaleimide, protein kinase C inhibitors, reduced inactivation of ICRAC, whereas phorbol ester accelerated inactivation of the current. These results demonstrate that a protein kinase-mediated phosphorylation, probably through protein kinase C, inactivates ICRAC. Activation of the adenosine receptor (A3 type) in RBL cells did not evoke much Ca2+ influx or systematic activation of ICRAC. After protein kinase C was blocked, however, large ICRAC was observed in all cells and this was accompanied by large Ca2+ influx. The ability of a receptor to evoke Ca2+ entry is determined, at least in part, by protein kinase C. Antigen stimulation, which triggers secretion through a process that requires Ca2+ influx, activated ICRAC. The regulation of ICRAC by protein kinase will therefore have important consequences on cell functioning.

Activated carbon fibre monoliths

Activated carbon fibre monoliths were prepared by physical activation of carbon fibre monoliths derived from two kinds of pitch-based carbon fibre (CF) (carbon fibres from a coal tar pitch and carbon fibres derived from a petroleum pitch). The monoliths were conformed using a coal tar pitch binder. The carbon fibre monoliths and the activated carbon fibre monoliths were studied by scanning electron microscopy (SEM) and gas adsorption (i.e. N2 at 77 K and CO2 at 273 K). The results obtained reveal that monoliths perform a good activation process that produce a quite high development of microporosity (BET surface areas around 2600 m2/g and N2 micropore volume of 1.23 cm3/g). On the other hand, it is remarkable that the activation process used allow to easily control the degree of activation and hence to select the adsorption capacities of the activated carbon fibre monoliths.

NaOH and KOH for preparing activated carbons used in energy and environmental applications

Alkaline hydroxides, especially sodium and potassium hydroxides, are multi-million-ton per annum commodities and strong chemical bases that have large scale applications. Some of them are related with their consequent ability to degrade most materials, depending on the temperature used. As an example, these chemicals are involved in the manufacture of pulp and paper, textiles, biodiesels, soaps and detergents, acid gases removal (e.g., SO2) and others, as well as in many organic synthesis processes. Sodium and potassium hydroxides are strong and corrosive bases, but they are also very stable chemicals that can melt without decomposition, NaOH at 318ºC, and KOH at 360ºC. Hence, they can react with most materials, even with relatively inert ones such as carbon materials. Thus, at temperatures higher than 360ºC these melted hydroxides easily react with most types of carbon-containing raw materials (coals, lignocellulosic materials, pitches, etc.), as well as with most pure carbon materials (carbon fibers, carbon nanofibers and carbon nanotubes). This reaction occurs via a solid-liquid redox reaction in which both hydroxides (NaOH or KOH) are converted to the following main products: hydrogen, alkaline metals and alkaline carbonates, as a result of the carbon precursor oxidation. By controlling this reaction, and after a suitable washing process, good quality activated carbons (ACs), a classical type of porous materials, can be prepared. Such carbon activation by hydroxides, known since long time ago, continues to be under research due to the unique properties of the resulting activated carbons. They have promising high porosity developments and interesting pore size distributions. These two properties are important for new applications such as gas storage (e.g., natural gas or hydrogen), capture, storage and transport of carbon dioxide, electricity storage demands (EDLC-supercapacitors-) or pollution control. Because these applications require new and superior quality activated carbons, there is no doubt that among the different existing activating processes, the one based on the chemical reaction between the carbon precursor and the alkaline hydroxide (NaOH or KOH) gives the best activation results. The present article covers different aspects of the activation by hydroxides, including the characteristics of the resulting activated carbons and their performance in some environment-related applications. The following topics are discussed: i) variables of the preparation method, such as the nature of the hydroxide, the type of carbon precursor, the hydroxide/carbon precursor ratio, the mixing procedure of carbon precursor and hydroxide (impregnation of the precursor with a hydroxide solution or mixing both, hydroxide and carbon precursor, as solids), or the temperature and time of the reaction are discussed, analyzing their effect on the resulting porosity; ii) analysis of the main reactions occurring during the activation process, iii) comparative analysis of the porosity development obtained from different activation processes (e.g., CO2, steam, phosphoric acid and hydroxides activation); and iv) performance of the prepared activated carbon materials on a few applications, such as VOC removal, electricity and gas storages.

CeO2-promoted Ni/activated carbon catalysts for the water–gas shift (WGS) reaction

The low temperature water–gas shift (WGS) reaction has been studied over carbon-supported nickel catalysts promoted by ceria. To this end, cerium oxide has been dispersed (at different loadings: 10, 20, 30 and 40 wt.%) on the activated carbon surface with the aim of obtaining small ceria particles and a highly available surface area. Furthermore, carbon- and ceria-supported nickel catalysts have also been studied as references. A combination of N2 adsorption analysis, powder X-ray diffraction, temperature-programmed reduction with H2, X-ray photoelectron spectroscopy and TEM analysis were used to characterize the Ni–CeO2 interactions and the CeO2 dispersion over the activated carbon support. Catalysts were tested in the low temperature WGS reaction with two different feed gas mixtures: the idealized one (with only CO and H2O) and a slightly harder one (with CO, CO2, H2, and H2O). The obtained results show that there is a clear effect of the ceria loading on the catalytic activity. In both cases, catalysts with 20 and 10 wt.% CeO2 were the most active materials at low temperature. On the other hand, Ni/C shows a lower activity, this assessing the determinant role of ceria in this reaction. Methane, a product of side reactions, was observed in very low amounts, when CO2 and H2 were included in the WGS feed. Nevertheless, our data indicate that the methanation process is mainly due to CO2, and no CO consumption via methanation takes place at the relevant WGS temperatures. Finally, a stability test was carried out, obtaining CO conversions greater than 40% after 150 h of reaction.

Removal of 8-quinolinecarboxylic acid pesticide from aqueous solution by adsorption on activated montmorillonites

Sodium montmorillonite (Na-M), acidic montmorillonite (H-M), and organo-acidic montmorillonite (Org-H-M) were applied to remove the herbicide 8-quinolinecarboxylic acid (8-QCA). The montmorillonites containing adsorbed 8-QCA were investigated by transmission electron microscopy, FT-IR spectroscopy, X-ray diffraction analysis, X-ray fluorescence thermogravimetric analysis, and physical adsorption of gases. Experiments showed that the amount of adsorbed 8-QCA increased at lower pH, reaching a maximum at pH 2. The adsorption kinetics was found to follow the pseudo-second-order kinetic model. The Langmuir model provided the best correlation of experimental data for adsorption equilibria. The adsorption of 8-QCA decreased in the order Org-H-M > H-M > Na-M. Isotherms were also used to obtain the thermodynamic parameters. The negative values of ΔG indicated the spontaneous nature of the adsorption process.

Influence of sludge retention time on filtration performance and biomass characteristics in a hollow fiber membrane bioreactor

In this study, the filtration process and the biomass characteristics in a laboratory-scale submerged membrane bioreactor (MBR) equipped with a hollow fiber (HF) microfiltration membrane were studied at different solid retention times (SRT). The MBR was fed by synthetic wastewater and the organic loading rate (OLR) was 0.5, 0.2, 0.1, and 0.08 kg COD kg VSS−1 d−1 for 10, 30, 60, and 90 days of SRT, respectively. The hydraulic retention time was 8.4 h and the permeate flux was 6 L m−2 h−1(LMH). Data analysis confirmed that at all the studied SRTs, the HF-MBR operated very good obtaining of high quality permeates. Chemical Oxygen Demand (COD) removal efficiencies were higher than 95%. The best filtration performance was reached at SRT of 30 d. On the other hand, the respirometric analysis showed that biomass was more active and there was more biomass production at low SRTs. The concentration of soluble extracellular polymeric substances (EPS) decreased with increasing SRT. A decrease of soluble EPS caused a decrease of membrane fouling rate, decreasing the frequency of chemical cleanings. The floc size decreased with SRT increasing. At high SRTs, there was more friction among particles due to the increase of the cellular density and the flocs broke decreasing their size.

Enhanced removal of 8-quinolinecarboxylic acid in an activated carbon cloth by electroadsorption in aqueous solution

The effect of the electrochemical treatment (potentiostatic treatment in a filter-press electrochemical cell) on the adsorption capacity of an activated carbon cloth (ACC) was analyzed in relation with the removal of 8-quinolinecarboxylic acid pollutant from water. The adsorption capacity of an ACC is quantitatively improved in the presence of an electric field (electroadsorption process) reaching values of 96% in comparison to 55% in absence of applied potential. In addition, the cathodic treatment results in higher removal efficiencies than the anodic treatment. The enhanced adsorption capacity has been proved to be irreversible, since the removed compound remains adsorbed after switching the applied potential. The kinetics of the adsorption processes is also improved by the presence of an applied potential.