THE SURFACE-STRUCTURE OF A C(2 X-2) POTASSIUM OVERLAYER ON CO(1010)

The surface structure of the clean Co{1010BAR} surface and a c(2 x 2) potassium overlayer have been determined by quantitative low energy electron diffraction. The Co{1010BAR} sample has been shown to be laterally unreconstructed with the surface being uniquely terminated by an outermost closely packed double layer (dz12 = 0.68 angstrom). A damped oscillatory relaxation of the outermost three atomic layers occurs, with relaxations DELTA-dz12 = -6.5 +/- 2% and DELTA-dz23 = +1.0 +/- 2%.

The c(2 x 2) overlayer formed at a coverage of 0.5 ML was subjected to a full I-V analysis. A range of adsorption sites were tested including fourfold hollow, on-top, and both long and short bridge sites in combination with both "long" and "short" cobalt interlayer terminations. A clear preference was found for adsorption in the maximal coordination fourfold hollow site. No switching of surface termination occurs. The potassium adatoms reside in the [1210BAR] surface channels directly above second layer cobalt atoms with a potassium to outermost cobalt interlayer separation of 2.44 +/- 0.05 angstrom. Potassium-cobalt bond lengths of 3.40 +/- 0.05 and 3.12 +/- 0.05 angstrom between the four (one) outermost (second) layer nearest-neighbour substrate atoms suggests a potassium effective radius of 1.87 +/- 0.05 angstrom, somewhat smaller than the Pauling covalent radius and considerably larger than the ionic radius (1.38 angstrom). The alkali-surface bonding is thus predominantly "covalent"/"metallic".

Activation of human TLR4/MD-2 by hypoacylated lipopolysaccharide from a clinical isolate of Burkholderia cenocepacia

Lung infection by Burkholderia species, in particular B. cenocepacia, accelerates tissue damage and increase post-lung transplant mortality in cystic fibrosis patients. Host- microbes interplay largely depends on interactions between pathogen specific molecules and innate immune receptors such as the Toll-like receptor 4 (TLR4), which recognizes the lipid A moiety of the bacterial lipopolysaccharide (LPS). The human TLR4/MD-2 LPS receptor complex is strongly activated by hexa-acylated lipid A and poorly activated by underacylated lipid A. Here, we report that B. cenocepacia LPS strongly activates human TLR4/MD-2 despite its lipid A having only five acyl chains. Further, we show that aminoarabinose residues in lipid A contribute to TLR4-lipid A interactions, and experiments in a mouse model of LPS-induced endotoxic shock confirmed the pro- inflammatory potential of B. cenocepacia penta-acylated lipid A. Molecular modeling, combined with mutagenesis of TLR4-MD2 interactive surfaces, suggests that longer acyl chains and the aminoarabinose residues in the B. cenocepacia lipid A allow exposure of the fifth acyl chain on the surface of MD-2 enabling interactions with TLR4 and its dimerization. Our results provide a molecular model for activation of the human TLR4/MD- 2 complex by penta-acylated lipid A, explaining the ability of hypoacylated B. cenocepacia LPS to promote pro- inflammatory responses associated to the severe pathogenicity of this opportunistic bacterium.

The role of WNT singling activation and its blockage in the focal retinal lesion of Ccl2-/-/Cx3cr1-/- mice with and without rd8 background

Purpose: The canonical Wnt signaling is activated by retinal injury. Under disease conditions, the Wnt mediates inflammatory responses. Inflammation has been detected in age-related macular degeneration (AMD) retinas and Ccl2-/-/Cx3cr1-/- (DKO) mice with or without rd8 background, a model with progressive AMD-like lesions including focal photoreceptor/RPE degeneration and A2E accumulation. We evaluated the effects of Wnt-β-catenin activation and an antibody against LRP6, the co-receptor of Wnt on these two models.

Methods: anti-LRP6 antibody (2F1, 1 μl of 5 μg/μL) was intravitreally injected into the right eyes in 3 separate experiments (DKOrd8, N=35; DKO, N=10). The left eyes were injected with mouse IgG as controls. Fundoscopy was taken before injection and sequentially monthly after injection. Two months after injection, light-adapted ERG responses were recorded; then the eyes were harvested for histopathology, the determination of retinal A2E, and molecular analysis. The microarray of ocular mRNA of 92 Wnt genes was compared between the treated and the control eyes. The phosphorylated types of LRP6 and β-catenin and endogenous forms of the proteins were assayed by Western blotting.

Results: For DKOrd8 mice, the fundus showed a slower progression or alleviation of retinal lesions in the right eyes as compared to the left eyes. Among 35 pairs of eyes, 26 (74.3%) were improved, 7 (20%) stayed the same and 2 (5.7%) remained progressing. Histology confirmed the clinical observation. Light-adapted ERG of the treated eyes exhibited larger amplitudes compared to control eyes (n=6), with greater improvements under UV light stimulus. There was a significantly lower A2E in the treated eyes compared to controls. Microarray of 92 Wnt genes expression pattern was similar in both eyes. Western blotting indicated local administration of 2F1 antibody to suppress the activation of Wnt pathway in the retina. For DKO mice, the treatment improved ERG but less effect on RPE degeneration.

Conclusions: The canonical Wnt signaling plays a role in the focal retina lesion of both DKOrd8 and DKO mice; and intravitreal anti-LRP6 antibody might be neuroprotective via deactivation of canonical Wnt pathway.

Selective induction of apoptosis by the pyrrolo-1,5-benzoxazepine 7-[[dimethylcarbamoyl]oxy]-6-(2-naphthyl)pyrrolo-[2,1-d] (1,5)-benzoxazepine (PBOX-6) in Leukemia cells occurs via the c-Jun NH2-terminal kinase-dependent phosphorylation and inactivation of Bcl-2 and Bcl-XL

Overexpression of the Bcl-2 proto-oncogene in tumor cells confers resistance against chemotherapeutic drugs. In this study, we describe how the novel pyrrolo-1,5-benzoxazepine compound 7-[[dimethylcarbamoyl]oxy]-6-(2-naphthyl)pyrrolo-[2,1-d] (1,5)-benzoxazepine (PBOX-6) selectively induces apoptosis in Bcl-2-overexpressing cancer cells, whereas it shows no cytotoxic effect on normal peripheral blood mononuclear cells. PBOX-6 overcomes Bcl-2-mediated resistance to apoptosis in chronic myelogenous leukemia (CML) K562 cells by the time- and dose-dependent phosphorylation and inactivation of antiapoptotic Bcl-2 family members Bcl-2 and Bcl-XL. PBOX-6 also induces Bcl-2 phosphorylation and apoptosis in wild-type T leukemia CEM cells and cells overexpressing Bcl-2. This is in contrast to chemotherapeutic agents such as etoposide, actinomycin D, and ultraviolet irradiation, whereby overexpression of Bcl-2 confers resistance against apoptosis. In addition, PBOX-6 induces Bcl-2 phosphorylation and apoptosis in wild-type Jurkat acute lymphoblastic leukemia cells and cells overexpressing Bcl-2. However, Jurkat cells containing a Bcl-2 triple mutant, whereby the principal Bcl-2 phosphorylation sites are mutated to alanine, demonstrate resistance against Bcl-2 phosphorylation and apoptosis. PBOX-6 also induces the early and transient activation of c-Jun NH2-terminal kinase (JNK) in CEM cells. Inhibition of JNK activity prevents Bcl-2 phosphorylation and apoptosis, implicating JNK in the upstream signaling pathway leading to Bcl-2 phosphorylation. Collectively, these findings identify Bcl-2 phosphorylation and inactivation as a critical step in the apoptotic pathway induced by PBOX-6 and highlight its potential as an effective antileukemic agent.

Activated protein C inhibits lipopolysaccharide-induced nuclear translocation of nuclear factor kappaB (NF-kappaB) and tumour necrosis factor alpha (TNF-alpha) production in the THP-1 monocytic cell line

Activated protein C (APC) protects against sepsis in animal models and inhibits the lipopolysacharide (LPS)-induced elaboration of proinflammatory cytokines from monocytes. The molecular mechanism responsible for this property is unknown. We assessed the effect of APC on LPS-induced tumour necrosis factor alpha (TNF-alpha) production and on the activation of the central proinflammatory transcription factor nuclear factor-kappaB (NF-kappaB) in a THP-1 cell line. Cells were preincubated with varying concentrations of APC (200 microg/ml, 100 microg/ml and 20 microg/ml) before addition of LPS (100 ng/ml and 10 microg/ml). APC inhibited LPS-induced production of TNF-alpha both in the presence and absence of fetal calf serum (FCS), although the effect was less marked with 10% FCS. APC also inhibited LPS-induced activation of NF-kappaB, with APC (200 microg/ml) abolishing the effect of LPS (100 ng/ml). The ability of APC to inhibit LPS-induced translocation of NF-kappaB is likely to be a significant event given the critical role of the latter in the host inflammatory response.

HES6 drives a critical AR transcriptional programme to induce castration-resistant prostate cancer through activation of an E2F1-mediated cell cycle network

Castrate-resistant prostate cancer (CRPC) is poorly characterized and heterogeneous and while the androgen receptor (AR) is of singular importance, other factors such as c-Myc and the E2F family also play a role in later stage disease. HES6 is a transcription co-factor associated with stem cell characteristics in neural tissue. Here we show that HES6 is up-regulated in aggressive human prostate cancer and drives castration-resistant tumour growth in the absence of ligand binding by enhancing the transcriptional activity of the AR, which is preferentially directed to a regulatory network enriched for transcription factors such as E2F1. In the clinical setting, we have uncovered a HES6-associated signature that predicts poor outcome in prostate cancer, which can be pharmacologically targeted by inhibition of PLK1 with restoration of sensitivity to castration. We have therefore shown for the first time the critical role of HES6 in the development of CRPC and identified its potential in patient-specific therapeutic strategies.

Thiol isomerases negatively regulate the cellular shedding activity of ADAM17

ADAM17 (where ADAM is 'a disintegrin and metalloproteinase') can rapidly modulate cell-surface signalling events by the proteolytic release of soluble forms of proligands for cellular receptors. Many regulatory pathways affect the ADAM17 sheddase activity, but the mechanisms for the activation are still not clear. We have utilized a cell-based ADAM17 assay to show that thiol isomerases, specifically PDI (protein disulfide isomerase), could be responsible for maintaining ADAM17 in an inactive form. Down-regulation of thiol isomerases, by changes in the redox environment (for instance as elicited by phorbol ester modulation of mitochondrial reactive oxygen species) markedly enhanced ADAM17 activation. On the basis of ELISA binding studies with novel fragment antibodies against ADAM17 we propose that isomerization of the disulfide bonds in ADAM17, and the subsequent conformational changes, form the basis for the modulation of ADAM17 activity. The shuffling of disulfide bond patterns in ADAMs has been suggested by a number of recent adamalysin crystal structures, with distinct disulfide bond patterns altering the relative orientations of the domains. Such a mechanism is rapid and reversible, and the role of thiol isomerases should be investigated further as a potential factor in the redox regulation of ADAM17.

TNF alpha-Induced p38MAPK Activation Regulates TRPA1 and TRPV4 Activity in Odontoblast-Like Cells

The transient receptor potential (TRP) channels are unique cellular sensors that are widely expressed in many neuronal and nonneuronal cells. Among the TRP family members, TRPA1 and TRPV4 are emerging as candidate mechanosensitive channels that play a pivotal role in inflammatory pain and mechanical hyperalgesia. Odontoblasts are nonneuronal cells that possess many of the features of mechanosensitive cells and mediate important defense and sensory functions. However, the effect of inflammation on the activity of the odontoblast's mechanosensitive channels remains unknown. By using immunohistochemistry and calcium microfluorimetry, we showed that odontoblast-like cells express TRPA1 and TRPV4 and that these channels were activated by hypotonicity-induced membrane stretch. Short treatment of odontoblast-like cells with tumor necrosis factor (TNF)-α enhanced TRPA1 and TRPV4 responses to their chemical agonists and membrane stretch. This enhanced channel activity was accompanied by phospho-p38 mitogen-activated protein kinase (MAPK) expression. Treatment of cells with the p38 inhibitor SB202190 reduced TNF-α effects, suggesting modulation of channel activity via p38 MAPK. In addition, TNF-α treatment also resulted in an up-regulation of TRPA1 expression but down-regulation of TRPV4. Unlike TRPV4, enhanced TRPA1 expression was also evident in dental pulp of carious compared with noncarious teeth. SB202190 treatment significantly reduced TNF-α-induced TRPA1 expression, suggesting a role for p38 MAPK signaling in modulating both the transcriptional and non-transcriptional regulation of TRP channels in odontoblasts.

Biosensor analysis of dynamics of interleukin 5 receptor subunit beta(c) interaction with IL5:IL5R(alpha) complexes

To gain insight into IL5 receptor subunit recruitment mechanism, and in particular the experimentally elusive pathway for assembly of signaling subunit beta(c), we constructed a soluble beta(c) ectodomain (s(beta)(c)) and developed an optical biosensor assay to measure its binding kinetics. Functionally active s(beta)(c) was anchored via a C-terminal His tag to immobilized anti-His monoclonal antibodies on the sensor surface. Using this surface, we quantitated for the first time direct binding of s(beta)(c) to IL5R(alpha) complexed to either wild-type or single-chain IL5. Binding was much weaker if at all with either R(alpha) or IL5 alone. Kinetic evaluation revealed a moderate affinity (0.2-1 microM) and relatively fast off rate for the s(beta)(c) interaction with IL5:R(alpha) complexes. The data support a model in which beta(c) recruitment occurs with preformed IL5:R(alpha) complex. Dissociation kinetics analysis suggests that the IL5-alpha-beta(c) complex is relatively short-lived. Overall, this study solidifies a model of sequential recruitment of receptor subunits by IL5, provides a novel biosensor binding assay of beta(c) recruitment dynamics, and sets the stage for more advanced characterization of the roles of structural elements within R(alpha), beta(c), and cytokines of the IL5/IL3/GM-CSF family in receptor recruitment and activation.

c-Kit+ progenitors generate vascular cells for tissue-engineered grafts through modulation of the Wnt/Klf4 pathway

The development of decellularised scaffolds for small diameter vascular grafts is hampered by their limited patency, due to the lack of luminal cell coverage by endothelial cells (EC) and to the low tone of the vessel due to absence of a contractile smooth muscle cells (SMC). In this study, we identify a population of vascular progenitor c-Kit+/Sca-1- cells available in large numbers and derived from immuno-privileged embryonic stem cells (ESCs). We also define an efficient and controlled differentiation protocol yielding fully to differentiated ECs and SMCs in sufficient numbers to allow the repopulation of a tissue engineered vascular graft. When seeded ex vivo on a decellularised vessel, c-Kit+/Sca-1-derived cells recapitulated the native vessel structure and upon in vivo implantation in the mouse, markedly reduced neointima formation and mortality, restoring functional vascularisation. We showed that Krüppel-like transcription factor 4 (Klf4) regulates the choice of differentiation pathway of these cells through β-catenin activation and was itself regulated by the canonical Wnt pathway activator lithium chloride. Our data show that ESC-derived c-Kit+/Sca-1-cells can be differentiated through a Klf4/β-catenin dependent pathway and are a suitable source of vascular progenitors for the creation of superior tissue-engineered vessels from decellularised scaffolds.

Persistent endothelial activation and inflammation after Plasmodium falciparum Infection in Malawian children

Endothelial dysregulation is central to the pathogenesis of acute Plasmodium falciparum infection. It has been assumed that this dysregulation resolves rapidly after treatment, but this return to normality has been neither demonstrated nor quantified. We therefore measured a panel of plasma endothelial markers acutely and in convalescence in Malawian children with uncomplicated or cerebral malaria. Evidence of persistent endothelial activation and inflammation, indicated by increased plasma levels of soluble intracellular adhesion molecule 1, angiopoetin 2, and C-reactive protein, were observed at 1 month follow-up visits. These vascular changes may represent a previously unrecognized contributor to ongoing malaria-associated morbidity and mortality.