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NEMO/IKKγ is the regulatory subunit of the IκB Kinase (IKK) complex, required for the activation of the NF-κB pathway, which is involved in a variety of key processes, including immunity, inflammation, differentiation, and cell survival. Termination of NF-κB activity on specific -κB responsive genes, which is crucial for the resolution of inflammatory responses, can be achieved by direct degradation of the chromatin-bound NF-κB subunit RelA/p65, a process mediated by a protein complex that contains Copper Metabolism Murr1 Domain 1 (COMMD1). In this study, we identify COMMD7, another member of the COMMDs protein family, as a novel NEMO-interacting protein. We show that COMMD7 exerts an inhibitory effect on NF-κB activation upon TNFα stimulation. COMMD7 interacts with COMMD1 and together they cooperate to down-regulate NF-κB activity. Accordingly, termination of TNFα-induced NF-κB activity on the -κB responsive gene, Icam1, is defective in cells silenced for COMMD7 expression. Furthermore, this impairment is not greatly increased when we silence the expression of both COMMD7 and COMMD1 indicating that the two proteins participate in the same pathway of termination of TNFα-induced NF-κB activity. Importantly, we have demonstrated that COMMD7's binding to NEMO does not interfere with the binding to the IKKs, and that the disruption of the IKK complex through the use of the NBP competitor impairs the termination of NF-κB activity. We propose that an intact IKK complex is required for the termination of NF-κB-dependent transcription and that COMMD7 acts as a scaffold in the IKK-mediated NF-κB termination.

With the expansion of the production and applications of multiwalled carbon nanotubes (MWCNTs) in several industrial and science branches, the potential adverse effects on human health have attracted attention. Numerous studies have been conducted to evaluate how chemical functionalization may affect MWCNT effects; however, controversial data have been reported, showing either increased or reduced toxicity. In particular, the impact of carboxylation on MWCNT cytotoxicity is far from being completely understood. The aim of this work was the evaluation of the modifications induced by carboxylated-MWCNTs (MWCNTs-COOH) on cell surface and the study of cell-MWCNT-COOH interactions by means of field emission scanning electron microscope (FESEM). Human pulmonary epithelial cells (A549) were incubated with MWCNTs-COOH for different exposure times and concentrations (10 μg/mL for 1, 2, 4 h; 5, 10, 20 μg/mL for 24 h). At short incubation time, MWCNTs-COOH were easily observed associated with plasma membrane and in contact with microvilli. After 24 h exposure, FESEM analysis revealed that MWCNTs-COOH induced evident changes in the cellular surface in comparison to control cells: treated cells showed blebs, holes and a depletion of the microvilli density in association with structure modifications, such as widening and/or lengthening. In particular, an increase of cells showing holes and microvilli structure alterations was observed at 20 μg/mL concentration. FESEM analysis showed nanotube agglomerates, of different sizes, entering into the cell with two different mechanisms: inward bending of the membrane followed by nanotube sinking, and nanotube internalization directly through holes. The observed morphological microvilli modifications, induced by MWCNTs-COOH, could affect epithelial functions, such as the control of surfactant production and secretion, leading to pathological conditions, such as alveolar proteinosis. More detailed studies will be, however, necessary to examine in depth the effects induced by MWCNTs-COOH and, in particular, the timing of the MWCNT-COOH-cell interaction.

The aim of the study was to enhance the antioxidant properties of fish burgers with microencapsulated propolis. Spray-drying process was used to microencapsulate propolis (30 g in 100 mL of ethanol 70% v/v) by means of gum Arabic and Capsul in different ratios (1:6 for gum Arabic and Capsul and then 1:20 just for Capsul). Once defined the optimal microencapsulation conditions, an alcohol-free powder able to mask the strong odor of propolis was obtained, thus promoting a potential food application as source of phenolics and antioxidants. Specifically, 5% w/w of spray-dried propolis was incorporated in fish burgers. To improve their sensory properties, new ingredients such as potato flakes (3%, 5%, 7% and 10% w/w) and extra virgin olive oil (9% w/w) were tested and optimized to give a final fish product with good acceptability. Proper tests on burgers also demonstrated an effective increase of both phenolic content and antioxidant activity.

In this work, the combined effects of sodium lactate and modified atmosphere packaging (MAP) in extending the shelf life of a ready-to-cook fresh skewer, made up of raw pork chops and semi-dried vegetable mix (i.e. zucchini, peppers and tomatoes), were investigated. In the first experimental step, a sodium lactate solution was used to dip pork chops at three different concentrations: 20, 40 and 60% w/w. The second part of the work was focused on the use of MAP. In particular, the following MAPs were tested: MA1 (50%O2/30%CO2/20%N2), MA2 (70%O2/30%CO2), MA3 (30%O2/70%CO2) and MA4 (30%O2/30%CO2/40%N2). Finally, the optimal concentration of sodium lactate and the best gas composition were combined. The samples were stored at 4 °C; their microbial and sensory qualities were monitored along the entire observation period. The results indicate that the shelf life of the investigated ready-to-cook meal can be extended by approximately 83%, if compared with the control skewer packaged in air. The best preservation strategy is the combination of dipping of meat pieces in 40% sodium lactate solution and packaging under MA1. Copyright © 2014 John Wiley & Sons, Ltd.

PED, known also as PED/PEA-15, is a Death Effector Domain (DED)-family member of 15 kDa having a variety of effects on cell growth and metabolism [1,2]. PED has a broad anti-apoptotic action, being able to inhibit both the intrinsic and the extrinsic apoptotic pathways [3,4,5,6]. Inhibition of the extrinsic pathway is accomplished through its DED, which likely acts as a competitive inhibitor for pro-apoptotic molecules during the assembly of the death-inducing signaling complex (DISC) [1,7]. To data, although PED has been reported to be over-expressed in a number of different cancer types, such as gliomas, squamous carcinoma, breast, lung cancer, and B cells chronic lymphocytic leukemia, the mechanisms that regulate its expression have not been fully addressed [3,5,8,9]. PED interacts with different molecules, among these ERK 1/2, altering their nuclear localization by sequestering them into the cytosol [10] . PED is present in the cells in either an unphosphorylated or a phosphorylated form. Phosphorylation occurs at Ser104 by protein kinase C (PKC), and on Ser116 by AKT or calcium calmodulin kinase II (CamK II) [11,12]. Interestingly, PED may interact with ERK1/2 only in its unphosphorylated form and it is associated to an increased ERK 1/2 phosphorylation [12]. Based on its phosphorylation status, PED might play a role as either a tumor-promoter factor (in the phosphorylated form) or tumor-suppressor (in the unphosphorylated form) factor [12]. To get further insights on the role of PED in cancer, we aimed to find new PED interactors. Using Tandem Affinity Purification (TAP), we previously identified and characterized, among others, Rac1, a member of mammalian Rho GTPase protein family, as a PED-interacting protein. PED-Rac1 interaction resulted in the modulation of cell migration/invasion processes in lung cancer cells through ERK1/2 pathway [13]. Another attractive PED-interacting protein was Hsc70 (Heat Shock cognate protein of 70 KDa), a chaperone protein, reported to be involved in a multitude of housekeeping chaperoning functions including folding of nascent polypeptides, protein translocation across membranes, prevention of protein aggregation under stress conditions, disassembly of clathrin coated vesicles and also in chaperone-mediated autophagy (CMA) [14,18]. CMA is a uniquely selective form of autophagy by which specific cytosolic proteins are transported one-by-one across the lysosomal membrane for degradation [14,15]-]. CMA is constitutively active in many cell types, but it is maximally activated under stress conditions (inducible CMA) such as nutritional stress, starvation or cellular stresses leading to protein damage [14]. CMA is selective for a subset of cytosolic soluble proteins. The selectivity is determined by the presence of a recognition-targeting motif in the amino acid sequence of the substrate proteins. All CMA substrates contain in their amino acid sequence a pentapeptide motif biochemically related to KFERQ, known as the CMA targeting motif [17]. The KFERQ sequence is recognized in the cytosol by Hsc70. Hsc70 not only targets the CMA substrate to the lysosomal membrane, where it can interact with the CMA receptor the lysosome-associated protein type 2A (LAMP-2A), but, along with its cochaperones it likely facilitates substrate unfolding, which is require for substrate translocation across the lysosomal membrane [14,18, 21]. In this manuscript we describe the interaction of PED with Hsc70 and demonstrate that Hsc70 targets the unphosphorylated PED for CMA.

PED (phosphoprotein enriched in diabetes) is a death-effector domain (DED) family member with a broad anti-apoptotic action. PED inhibits the assembly of the death-inducing signalling complex (DISC) of death receptors following stimulation. Recently, we reported that the expression of PED is increased in breast cancer cells and determines the refractoriness of these cells to anticancer therapy. In the present study, we focused on the role of PED in non-small cell lung cancer (NSCLC), a tumour frequently characterized by evasion of apoptosis and drug resistance. Immunohistochemical analysis of a tissue microarray, containing 160 lung cancer samples, indicated that PED was strongly expressed in different lung tumour types. Western blotting performed with specimens from NSCLC-affected patients showed that PED was strongly up-regulated (>6 fold) in the areas of tumour compared to adjacent normal tissue. Furthermore, PED expression levels in NSCLC cell lines correlated with their resistance to tumour necrosis factor related apoptosis-inducing ligand (TRAIL)-induced cell death. The involvement of PED in the refractoriness to TRAIL-induced cell death was investigated by silencing PED expression in TRAIL-resistant NSCLC cells with small interfering (si) RNAs: transfection with PED siRNA, but not with cFLIP siRNA, sensitized cells to TRAIL-induced cell death. In conclusion, PED is specifically overexpressed in lung tumour tissue and contributes to TRAIL resistance.

During neuropathological conditions such as infections and degenerative diseases, astrocytes can be activated by infiltrating immune cells. Activated astrocytes can produce chemokines, cytokines and adhesion molecules. In this study, the production of IL-6 and adhesion molecules intercellular adhesion molecule-1 (ICAM-1), vascular cellular adhesion molecule-1 (VCAM-1) and E-selectin by human astroglioma cells stimulated with Gram-negative surface components was investigated. Haemophilus influenzae type b porin P2 and its selected active peptide, loop L7, were found to induce MEK1-MEK2/ mitogen-activated protein kinase phosphorylation in U87-MG cells as demonstrated by ELISA, and up-regulate cellular adhesion molecule and interleukin-6 (IL-6) production as shown by RT-PCR and ELISA. Using two potent and selective inhibitors of MEK activation by Raf-1 (PD-098059) and p38 (SB-203580), it was also demonstrated that both ERK1/2 and p38 pathways play key roles in the production of IL-6 as well as in ICAM-1, VCAM-1 and E-selectin expression by Hib porin.

Background ABT-737 is a BH3 mimetic small-molecule inhibitor that binds with high affinity to Bcl-2 to induce apoptosis in malignant cells and has shown promise as an effective anti-leukemic agent in pediatric preclinical tests. This study focuses on the effects of ABT-737 on leukemia cells with MLL rearrangement and identifies some of the biological correlates of its activity. Procedure Cells were cultured in the presence of increasing concentrations of ABT-737 alone or in combination with other agents. After 4 days in culture, cell growth inhibition was measured by Alamar blue assay. The expression and activation of potential intracellular targets of ABT-737 activity were determined by Western blot analysis. Results Significant Bcl-2 expression was detected in all infant leukemia cells investigated. ABT-737 induced cell death in all cell lines studied although the IC50 values differed somewhat between cell lines. Western blot analysis identified the effects of ABT-737 on survival and apoptosis-regulatory proteins PARP, caspase-8, and cytochrome-c. Drug combination studies indicated synergy with distinct anti-neoplastic agents, including the multi-tyrosine kinase inhibitor sunitinib. This effective drug synergy appears to be mediated by the combined inhibition of Bcl-2 and intracellular signaling pathways. Conclusions We describe the in vitro studies to demonstrate the activity and drug combinability of ABT-737 against MLL rearranged leukemia cells. In addition, identification of the molecular changes that occur in the presence of ABT-737 provides information regarding effective target validation and target modulation analyses in future clinical trials. Pediatr Blood Cancer 2011;56:353–360. © 2010 Wiley-Liss, Inc.