Study of the cellular mechanism of Sunitinib mediated inactivation of activated hepatic stellate cells and its implications in angiogenesis

The development of hepatocellular carcinomas from malignant hepatocytes is frequently associated with intra- and peritumoral accumulation of connective tissue arising from activated hepatic stellate cells (HSC). Inhibition of receptor tyrosine kinase (RTK) signaling showed promise in the treatment of hepatocellular carcinoma. However, there is a lack of knowledge about the effects of RTK inhibitors on the tumor supportive cells. We performed in vitro experiments to study whether Sunitinib, a platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) RTKs' inhibitor, could block both activated HSC functions and angiogenesis and thus prevent the progression of cirrhotic liver to hepatocellular carcinoma. In immortalized human activated HSC LX-2, treatment with Sunitinib 100 nM blocked collagen synthesis by 47%, as assessed by Sirius Red staining, attenuated HSC contraction by 65%, and reduced cell migration by 28% as evaluated using a Boyden's chamber, without affecting cell viability, measured by Trypan blue staining, and apoptosis, measured by propidium iodide (PI) incorporation assay. Our data revealed that Sunitinib treatment blocked the transdifferentiation of primary human HSC (hHSC) to activated myofibroblast-like cells by 65% without affecting hHSC apoptosis and migration. In in vitro angiogenic assays, Sunitinib 100 nM reduced endothelial cells (EC) ring formation by 46% and tube formation by 68%, and decreased vascular sprouting in aorta ring assay and angiogenesis in vascular bed of chick embryo. In conclusion, the present study demonstrates that the RTK inhibitor Sunitinib blocks the activation of HSC and angiogenesis suggesting its potential as a drug candidate in pathological conditions like liver fibrosis and hepatocellular carcinoma.

Activated carbon may have undesired side effects for testing allelopathy in invasive plants

Activated carbon has become a widely used tool to investigate root-mediated allelopathy of plants, especially in plant invasion biology, because it adsorbs and thereby neutralizes root exudates. Allelopathy has been a controversially debated phenomenon for years, which revived in plant invasion biology as one possible reason for the success of invasive plants. Noxious plant exudates may harm other plants and provide an advantage to the allelopathic plant. However, root exudates are not always toxic, but may stimulate the microbial community and change nutrient availability in the rhizosphere. In a greenhouse experiment, we investigated the interacting effects of activated carbon, arbuscular mycorrhiza and plant competition between the invasive Senecio inaequidens and the native Artemisia vulgaris. Furthermore, we tested whether activated carbon showed any undesired effects by directly affecting mycorrhiza or soil chemistry. Contrary to the expectation, S. inaequidens was a weak competitor and we could not support the idea that allelopathy was involved in the competition. Activated carbon led to a considerable increase in the aboveground biomass production and reduced the infection with arbuscular mycorrhiza of both plant species. We expected that arbuscular mycorrhiza promotes plant growth by increasing nutrient availability, but we found the contrary when activated carbon was added. Chemical analyses of the substrate showed, that adding activated carbon resulted in a strong increase in plant available phosphate and in a decrease of the C(organic)/N(total) ration both of which suggest stimulated microbial activity. Thus, activated carbon not only reduced potential allelopathic effects, but substantially changed the chemistry of the substrate. These results show that activated carbon should be handled with great care in ecological experiments on allelopathy because of possible confounding effects on the soil community.

Stimulation of monocytes by placental microparticles involves toll-like receptors and nuclear factor kappa-light-chain-enhancer of activated B cells.

Human pregnancy is accompanied by a mild systemic inflammatory response, which includes the activation of monocytes circulating in maternal blood. This response is exaggerated in preeclampsia, a placental-dependent disorder specific to human pregnancies. We and others showed that placental syncytiotrophoblast membrane microparticles (STBM) generated in vitro from normal placentas stimulated peripheral blood monocytes, which suggest a contribution of STBM to the systemic maternal inflammation. Here, we analyzed the inflammatory potential of STBM prepared from preeclamptic placentas on primary monocytes and investigated the mode of action in vitro. STBM generated in vitro by placental villous explants of normal or preeclamptic placentas were co-incubated with human peripheral blood monocytes. In some cases, inhibitors of specific cellular functions or signaling pathways were used. The analysis of the monocytic response was performed by flow cytometry, enzyme-linked immunoassays, real-time PCR, and fluorescence microscopy. STBM derived from preeclamptic placentas up-regulated the cell surface expression of CD54, and stimulated the secretion of the pro-inflammatory interleukin (IL)-6 and IL-8 in a similar, dose-dependent manner as did STBM prepared from normal placentas. STBM bound to the cell surface of monocytes, but phagocytosis was not necessary for activation. STBM-induced cytokine secretion was impaired in the presence of inhibitors of toll-like receptor (TLR) signaling or when nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation was blocked. Our results suggest that the inflammatory reaction in monocytes may be initiated by the interaction of STBM with TLRs, which in turn signal through NF-κB to mediate the transcription of genes coding for pro-inflammatory factors.

Granular computing as a tool of human data interaction - a cognitive cities use case

The article proposes granular computing as a theoretical, formal and methodological basis for the newly emerging research field of human–data interaction (HDI). We argue that the ability to represent and reason with information granules is a prerequisite for data legibility. As such, it allows for extending the research agenda of HDI to encompass the topic of collective intelligence amplification, which is seen as an opportunity of today’s increasingly pervasive computing environments. As an example of collective intelligence amplification in HDI, we introduce a collaborative urban planning use case in a cognitive city environment and show how an iterative process of user input and human-oriented automated data processing can support collective decision making. As a basis for automated human-oriented data processing, we use the spatial granular calculus of granular geometry.

Granular flow in polymineralic rocks bearing sheet silicates: New evidence from natural examples

Natural deformation in carbonate mylonites bearing sheet silicates occurs via a complex interaction of granular flow and solution transfer processes and involves continuous cycles of dissolution, grain boundary diffusion, nucleation and growth. In this way, new sheet silicates (a) nucleate within voids formed by grain boundary sliding of calcite grains. (b) grow, and (c) rotate towards the shear plane. As a consequence, small mica grains show a wide range of orientations with respect to the shear plane, but moderate to large grains are subparallel both to each other and to the shear plane. Increases of average grain sizes with increasing temperature of sheet silicates in mica-rich layers is more pronounced than in mica-poor layers. In the calcitic matrix however, sheet silicates can only grow via solution-precipitation and mass transfer processes. Therefore, the observed grain size variability indicates drastic differences in mass transfer behavior between the individual layers, which might be related to differences in the fluid flux. Based on these observations, a conceptual model for the microfabric evolution in sheet silicate bearing mylonites is presented. © 2001 Elsevier Science B.V. All rights reserved.

Granular layer in the periplasmic space of gram-positive bacteria and fine structures of Enterococcus gallinarum and Streptococcus gordonii septa revealed by cryo-electron microscopy of vitreous sections.

High-resolution structural information on optimally preserved bacterial cells can be obtained with cryo-electron microscopy of vitreous sections. With the help of this technique, the existence of a periplasmic space between the plasma membrane and the thick peptidoglycan layer of the gram-positive bacteria Bacillus subtilis and Staphylococcus aureus was recently shown. This raises questions about the mode of polymerization of peptidoglycan. In the present study, we report the structure of the cell envelope of three gram-positive bacteria (B. subtilis, Streptococcus gordonii, and Enterococcus gallinarum). In the three cases, a previously undescribed granular layer adjacent to the plasma membrane is found in the periplasmic space. In order to better understand how nascent peptidoglycan is incorporated into the mature peptidoglycan, we investigated cellular regions known to represent the sites of cell wall production. Each of these sites possesses a specific structure. We propose a hypothetic model of peptidoglycan polymerization that accommodates these differences: peptidoglycan precursors could be exported from the cytoplasm to the periplasmic space, where they could diffuse until they would interact with the interface between the granular layer and the thick peptidoglycan layer. They could then polymerize with mature peptidoglycan. We report cytoplasmic structures at the E. gallinarum septum that could be interpreted as cytoskeletal elements driving cell division (FtsZ ring). Although immunoelectron microscopy and fluorescence microscopy studies have demonstrated the septal and cytoplasmic localization of FtsZ, direct visualization of in situ FtsZ filaments has not been obtained in any electron microscopy study of fixed and dehydrated bacteria.

The Rice Transcription Factor WRKY53 Suppresses Herbivore-Induced Defenses by Acting as a Negative Feedback Modulator of Mitogen-Activated Protein Kinase Activity

The mechanisms by which herbivore-attacked plants activate their defenses are well studied. By contrast, little is known about the regulatory mechanisms that allow them to control their defensive investment and avoid a defensive overshoot. We characterized a rice (Oryza sativa) WRKY gene, OsWRKY53, whose expression is rapidly induced upon wounding and induced in a delayed fashion upon attack by the striped stem borer (SSB) Chilo suppressalis. The transcript levels of OsWRKY53 are independent of endogenous jasmonic acid but positively regulated by the mitogen-activated protein kinases OsMPK3/OsMPK6. OsWRKY53 physically interacts with OsMPK3/OsMPK6 and suppresses their activity in vitro. By consequence, it modulates the expression of defensive, MPK-regulated WRKYs and thereby reduces jasmonic acid, jasmonoyl-isoleucine, and ethylene induction. This phytohormonal reconfiguration is associated with a reduction in trypsin protease inhibitor activity and improved SSB performance. OsWRKY53 is also shown to be a negative regulator of plant growth. Taken together, these results show that OsWRKY53 functions as a negative feedback modulator of MPK3/MPK6 and thereby acts as an early suppressor of induced defenses. OsWRKY53 therefore enables rice plants to control the magnitude of their defensive investment during early signaling.

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.

INHIBITION OF THE MITOCHONDRIAL RESPIRATION OF TUMOR CELLS BY SOLUBLE FACTORS RELEASED BY ACTIVATED MACROPHAGES (CYTOTOXICITY, ELECTRON TRANSPORT, MONOCYTE)

Particular interest has been directed towards the macrophage as a primary antineoplastic cell due to its tumoricidal properties in vitro and the observation that an inverse relationship exists between the number of macrophages infiltrating a tumor and metastatic potential. The mechanism of macrophage-mediated injury of tumor cells remains unknown. Recently, it has been shown that injured tumor cells have defective mitochondrial respiration. Our studies have shown that activated macrophages can release soluble factors which can alter tumor cell respiration.^ The effects of a conditioned supernatant (CS) from cultures of activated macrophages on tumor cell (TC) mitochondrial respiration was studied. CS was obtained by incubation of BCG-elicited, murine peritoneal macrophage with RPMI-1640 supplemented with 10% FCS and 50 ng/ml bacterial endotoxin. This CS was used to treat cultures of EMT-6 TC for 24 hours. Mitochondrial respiration was measured polarigraphically using a Clark-type oxygen electrode. Cell growth rate was assessed by ('3)H-Thymidine incorporation. Exposure of EMT-6 TC to CS resulted in the inhibition of malate and succinate oxidation 76.6% and 72.9%, respectively. While cytochrome oxidase activity was decreased 61.1%. This inhibition was accompanied by a 98.8% inhibition of DNA synthesis (('3)H-Thymidine incorporation). Inhibition was dose-related with a 21.3% inhibition of succinate oxidase from a 0.3 ml dose of CS and a 50% inhibition with 1.0 mls. Chromatography of CS on Sephacryl S-200 resulted in isolation of an 80,000 and a 55,000 dalton component which contained the respiration inhibiting activity (RIF). These factors were distinct from a 120,000 dalton cytolytic factor determined by bioassay on Actinomycin-D treated L929 cells. RIF activity was also distinct from several other cytostatic factors but was itself associated with 2 peaks of cytostatic activity. Characterization of the RIF activity showed that it was destroyed by trypsin and heat (100(DEGREES)C, 5 min). It was stable over a broad range of pH (4-9) and its production was inhibited by cycloheximide. The RIF did not have a direct effect on isolated mitochondria of TC nor did it induce the formation of a stable intracellular toxin for mitochondria.^ In conclusion, activated macrophages synthesize and secrete an 80,000 and a 55,000 dalton protein which inhibits the mitochondrial metabolism of TC. These factors induce a cytostatic but not a cytolytic effect on TC.^ The macrophage plays a role in the control of normal and tumor cell growth and in tissue involution. Inhibition of respiration may be one mechanism used by macrophages to control cell growth.^

RNA-Activated Protein Kinase, PKR

The double-stranded RNA (dsRNA) activated protein kinase, PKR, is one of the several enzymes induced by interferons and a key molecule mediating the antiviral effects of interferons. PKR contain an N-terminal, double-stranded RNA binding domain (dsRBD), which has two tandem copies of the motifs (dsRBM I and dsRBM II). Upon binding to viral dsRNA, PKR is activated via autophosphorylation. Activated PKR has several substrates; one of the examples is eukaryotic translation initiation factor 2 (eIF2a). The phosphorylation of eIF2a leads to the termination of cell growth by inhibiting protein synthesis in response to viral infection. The objective of this project was to characterize the dsRBM I and define the dsRNA binding using biophysical methods. First, the dsRBM I gene was cloned from a pET-28b to a pET-11a expression plasmid. N-terminal poly-histidine tags on pET-28b are for affinity purification; however, these tags can alter the structure and function of proteins, thus the gene of dsRBM I was transferred into the plasmid without tags (pET-11a) and expressed as a native protein. The dsRBM I was transformed into and expressed by Rosetta DE3plyS expression cells. Purification was done by FPLC using a Sepharose IEX ion exchange followed by Heparin affinity column; yielding pure protein was assayed by PAGE. Analytical Ultracentrifugation, Sedimentation Velocity, was used to characterize free solution association state and hydrodynamic properties of the protein. The slight decrease in S-value with concentration is due to the hydrodynamic non-ideality. No self association was observed. The obtained molecule weight was 10,079 Da. The calculated sedimentation constant at zero concentration at 20°C in water was 1.23 and its friction coefficient was 3.575 ´ 10-8. The frictional ratio of sphere and dsRBM I became 1.30. Therefore, dsRBM I must be non-globular and more asymmetric shape. Isolated dsRBM I exhibits the same tertiary fold as compared to context in the full domain but it exhibited weaker binding affinity than full domain to a 20 bp dsRNA. However, when the conditions allowed for its saturation, dsRBM I to 20 bp dsRNA has similar stoichiometry as full dsRBD.

p21 activated kinase 5 links multiple GTPase signaling pathways at mitochondria

The p21-activated kinase 5 (PAK5) is a serine/threonine protein kinase associated with the group 2 subfamily of PAKs. Although our understanding about PAK5 is very limited, it is receiving increasing interest due to its tissue specific expression pattern and important signaling properties. PAK5 is highly expressed in brain. Its overexpression induces neurite outgrowth in neuroblastoma cells and promotes survival in fibroblasts. ^ The serine/threonine protein kinase Raf-1 is an essential mediator of Ras-dependent signaling that controls the ERK/MAPK pathway. In contrast to PAK5, Raf-1 has been the subject of intensive investigation. However due to the complexity of its activation mechanism, the biological inputs controlling Raf-1 activation are not fully understood. ^ PAKs 1-3 are the known kinases responsible for phosphorylation of Raf-1 on serine 338, which is a crucial phosphorylation site for Raf-1 activation. However, dominant negative versions of these kinases do not block EGF-induced Raf-1 activation, indicating that other kinases may regulate the phosphorylation of Raf-1 on serine 338. ^ This thesis work was initiated to test whether the group 2 PAKs 4, 5 and 6 are responsible for EGF-induced Raf-1 activation. We found that PAK5, and to a lesser extent PAK4, can activate Raf-1 in cells. Our studies thereafter focused on PAK5. With the progress of our study we found that PAK5 does not significantly stimulate serine 338 phosphorylation of Triton X-100 soluble Raf-1. PAK5, however, constitutively and specifically associates with Raf-1 and targets it to a Triton X-100 insoluble, mitochondrial compartment, where PAK5 phosphorylates serine 338 of Raf-1. We further demonstrated that endogenous PAK5 and Raf-1 colocalize in Hela cells at the mitochondrial outer membrane. In addition, we found that the mitochondria-targeting of PAK5 is determined by its C-terminal kinase domain plus the upstream proximal region, and facilitated by the N-terminal p21 binding domain. We also demonstrated that Rho GTPases Cdc42 and RhoD associate with and regulate the subcellular localization of PAK5. Taken together, this work suggests that the mitochondria-targeting of PAK5 may link Ras and Rho GTPase-mediated signaling pathways, and sheds light on aspects of PAK5 signaling that may be important for regulating neuronal homeostasis. ^