Molecular mechanism of hTid-1, the human homolog of Drosophila tumor suppressor l(2)Tid, in the regulation of NF-kappaB activity and suppression of tumor growth

hTid-1, a human homolog of the Drosophila tumor suppressor l(2)Tid and a novel DnaJ protein, regulates the activity of nuclear factor kappaB (NF-kappaB), but its mechanism is not established. We report here that hTid-1 strongly associated with the cytoplasmic protein complex of NF-kappaB-IkappaB through direct interaction with IkappaBalpha/beta and the IKKalpha/beta subunits of the IkappaB kinase complex. These interactions resulted in suppression of the IKK activity in a J-domain-dependent fashion and led to the cytoplasmic retention and enhanced stability of IkappaB. Overexpression of hTid-1 by using recombinant baculovirus or adenovirus led to inhibition of cell proliferation and induction of apoptosis of human osteosarcoma cells regardless of the p53 expression status. Adherent cultured cells transduced with Ad.hTid-1 detached from the dish surface. Morphological changes consistent with apoptosis and cell death were evident 48 h after Ad.EGFP-hTid-1 transduction. In contrast, cells transduced with Ad.EGFP or Ad.EGFP-hTd-1DeltaN100, a mutant that has the N-terminal J domain deletion and that lost suppressive activity on IKK, continued to proliferate. Similar data were obtained with A375 human melanoma cells. Ad.EGFP or Ad.EGFP-hTd-1DeltaN100 ex vivo-transduced A375 cells injected subcutaneously into nude mice produced growing tumors, whereas Ad.EGFP-hTid-1-transduced cells did not. Collectively, the data suggest that hTid-1 represses the activity of NF-kappaB through physical and functional interactions with the IKK complex and IkappaB and, in doing so, it modulates cell growth and death.     

Antisense reduction of thylakoidal ascorbate peroxidase in <Emphasis Type="Italic">Arabidopsis</Emphasis> enhances Paraquat-induced photooxidative stress and Nitric Oxide-induced cell death

The production and characterization of Arabidopsis plants containing a transgene in which the Arabidopsis tAPX is inserted in antisense orientation, is described. tAPX activity in these transgenic tAPX plants is around 50% of control level. The tAPX antisense plants are phenotypically indistinguishable from control plants under normal growth conditions; they show, however, enhanced sensitivity to the O₂^–-generating herbicide, Paraquat. Interestingly, the tAPX antisense plants show enhanced symptoms of damage when cell death is triggered through treatment with the nitric oxide-donor, SNP. These results are in accordance with the ones recently obtained with transgenic plants overexpressing tAPX; altogether, they suggest that tAPX, besides the known ROS scavenging role, is also involved in the fine changes of H₂O₂ concentration during signaling events.     

Activation of glucose transport during simulated ischemia in H9c2 cardiac myoblasts is mediated by protein kinase C isoforms

Glucose transport into cells may be regulated by a variety of conditions, including ischemia. We investigated whether some enzymes frequently involved in the metabolic adaptation to ischemia are also required for glucose transport activation. Ischemia was simulated by incubating during 3 h H9c2 cardiomyoblasts in a serum- and glucose-free medium in hypoxia. Under these conditions 2-deoxy-d-[2,6-³H]-glucose uptake was increased (57% above control levels, p < 0.0001) consistently with GLUT1 and GLUT4 translocation to sarcolemma. Tyrosine kinases inhibition via tyrphostin had no effect on glucose transport up-regulation induced by simulated ischemia. On the other hand, chelerythrine, a broad range inhibitor of protein kinase C isoforms, and rottlerin, an inhibitor of protein kinase C delta, completely prevented the stimulation of the transport rate. A lower activation of hexose uptake (19%, p < 0.001) followed also treatment with Gö6976, an inhibitor of conventional protein kinases C. Finally, PD98059-mediated inhibition of the phosphorylation of ERK 1/2, a downstream mitogen-activated protein kinase (MAPK), only partially reduced the activation of glucose transport induced by simulated ischemia (31%, p < 0.01), while SB203580, an inhibitor of p38 MAPK, did not exert any effect. These results indicate that stimulation of protein kinase C delta is strongly related to the up-regulation of glucose transport induced by simulated ischemia in cultured cardiomyoblasts and that conventional protein kinases C and ERK 1/2 are partially involved in the signalling pathways mediating this process.     

Inhibition of nitric oxide synthesis blocks the inhibitory response to capsaicin in intestinal circular muscle preparations from different species

Moderate concentrations of the sensory stimulant drug capsaicin caused relaxation in human and animal intestinal circular muscle preparations (guinea-pig proximal, mouse distal colon, human small intestine and appendix) in vitro. With the exception of the guinea-pig colon, the nitric oxide (NO) synthase inhibitor N(G)-nitro-L-arginine (L-NOARG; 10(-4) M) strongly inhibited the relaxant effect of capsaicin. Tetrodotoxin, an inhibitor of voltage-sensitive Na+ channels failed to significantly reduce the inhibitory effect of capsaicin in the guinea-pig colon, human ileum and appendix; it caused an approximately 50% reduction in the mouse colon. The relaxant effect of capsaicin was strongly reduced in colonic preparations from transient receptor potential vanilloid type (TRPV1) receptor knockout mice as compared to their wildtype controls. It is concluded that nitric oxide, possibly of sensory origin, is involved in the relaxant action of capsaicin in the circular muscle of the mouse and human intestine.     

Nociceptin/orphanin FQ prevents ethanol-induced gastric lesions in the rat

Nociceptin/orphanin FQ (N/OFQ), the endogenous ligand of the NOP receptor, exerts a variety of effects on the gastrointestinal tract. The present study was aimed at evaluating the possible implication of N/OFQ in the maintenance of gastric mucosal integrity. N/OFQ was given either centrally or peripherally 30 min prior to intragastric administration (i.g.) of 1 ml/rat of ethanol (either 25% or 50%, v/v), which produces macroscopically visible gastric lesions. Intracerebroventricular (i.c.v.) injection of 2 microg/rat of N/OFQ significantly reduced lesions caused by 50% ethanol, while 1 microg/rat was enough to significantly reduce lesions caused by 25% ethanol. Intracerebroventricular injection of 5 microg/rat of the selective NOP receptor antagonist, UFP-101, completely reversed the protective effect of N/OFQ, 1 or 4 microg/rat against 25% or 50% ethanol, respectively. The intraperitoneal (i.p.) injection of N/OFQ produced a significant reduction of lesions induced by 50% ethanol, the peak effect being observed at 10 microg/kg. Intraperitoneal pretreatment with UFP-101, 120 microg/kg, completely abolished the protective effect of peripherally injected N/OFQ. Therefore, N/OFQ acts both centrally and peripherally as a protective agent against ethanol-induced gastric lesions, and its effect is mediated by NOP receptors.     

Experimental demonstration that mammalian oocytes are not selective towards X- or Y-bearing sperm

Mammalian oocytes are thought to be neutral as for X- or Y-bearing sperm selection is concerned, and penetration of an oocyte by an X- or a Y-bearing sperm is considered a random event. This assumption is mainly based on a posteriori evidences of a nearly equal sex ratio at birth, but it has never been experimentally demonstrated. We have designed a simple experiment, which allowed the penetration of an oocyte by more than one sperm and the further sexing by PCR of each single pronucleus present within the ooplasm. For the first time, we provide experimental evidence that mammalian oocytes do not play a selecting role since a single oocyte may be simultaneously fertilised by both X- and Y-bearing sperm.     

Spectral signal-to-noise ratio and resolution assessment of 3D reconstructions

Measuring the quality of three-dimensional (3D) reconstructed biological macromolecules by transmission electron microscopy is still an open problem. In this article, we extend the applicability of the spectral signal-to-noise ratio (SSNR) to the evaluation of 3D volumes reconstructed with any reconstruction algorithm. The basis of the method is to measure the consistency between the data and a corresponding set of reprojections computed for the reconstructed 3D map. The idiosyncrasies of the reconstruction algorithm are taken explicitly into account by performing a noise-only reconstruction. This results in the definition of a 3D SSNR which provides an objective indicator of the quality of the 3D reconstruction. Furthermore, the information to build the SSNR can be used to produce a volumetric SSNR (VSSNR). Our method overcomes the need to divide the data set in two. It also provides a direct measure of the performance of the reconstruction algorithm itself; this latter information is typically not available with the standard resolution methods which are primarily focused on reproducibility alone.     

Dynamic partitioning into lipid rafts controls the endo-exocytic cycle of the alphaL/beta2 integrin, LFA-1, during leukocyte chemotaxis

Cell migration entails the dynamic redistribution of adhesion receptors from the cell rear toward the cell front, where they form new protrusions and adhesions. This process may involve regulated endo-exocytosis of integrins. Here we show that in primary neutrophils unengaged alphaL/beta2 integrin (LFA-1) is internalized and rapidly recycled upon chemoattractant stimulation via a clathrin-independent, cholesterol-sensitive pathway involving dynamic partitioning into detergent-resistant membranes (DRM). Persistent DRM association is required for recycling of the internalized receptor because 1) >90% of endocytosed LFA-1 is associated with DRM, and a large fraction of the internalized receptor colocalizes intracellularly with markers of DRM and the recycling endocytic compartment; 2) a recycling-defective mutant (alphaL/beta2Y735A) dissociates rapidly from DRM upon being endocytosed and is subsequently diverted into a late endosomal pathway; and 3) a dominant negative Rab11 mutant (Rab11S25N) induces intracellular accumulation of endocytosed alphaL/beta2 and prevents its enrichment in chemoattractant-induced lamellipodia. Notably, chemokine-induced migration of neutrophils over immobilized ICAM-1 is abrogated by cholesterol-sequestering agents. We propose that DRM-associated endocytosis allows efficient retrieval of integrins, as they detach from their ligands, followed by polarized recycling to areas of the plasma membrane, such as lamellipodia, where they establish new adhesive interactions and promote outside-in signaling events.