Structural mimicry of two cytochrome b(562) interhelical loops using macrocycles constrained by oxazoles and thiazoles

A major chemical challenge is the structural mimicry of discontinuous protein surfaces brought into close proximity through polypeptide folding. We report the design, synthesis, and solution structure of a highly functionalized saddle-shaped macrocyclic scaffold, constrained by oxazoles and thiazoles,upporting two short peptide loops projecting orthogonally from the same face of the scaffold. This structural mimetic of two interhelical loops of cytochrome b(562) illustrates a promising approach to structurally mimicking discontinuous loops of proteins.

Effect of osmolytes on the interaction of flavin adenine dinucleotide with muscle glycogen phosphorylase b

The effect of three osmolytes, trimethylamine N-oxide (TMAO), betaine and proline, on the interaction of muscle glycogen phosphorylase b with allosteric inhibitor FAD has been examined. In the absence of osmolyte, the interaction is described by a single intrinsic dissociation constant (17.8 muM) for two equivalent and independent binding sites on the dimeric enzyme. However, the addition of osmolytes gives rise to sigmoidal dependencies of fractional enzyme-site saturation upon free inhibitor concentration. The source of this cooperativity has been shown by difference sedimentation velocity to be an osmolyte-mediated isomerization of phosphorylase b to a smaller dimeric state with decreased affinity for FAD. These results thus have substantiated a previous inference that the tendency for osmolyte-enhanced self-association of dimeric glycogen phosphorylase b in the presence of AMP was being countered by the corresponding effect of molecular crowding on an isomerization of dimer to a smaller, nonassociating state. (C) 2004 Elsevier Ltd. Inc. All rights reserved.

NF-κB mediates proteolysis-inducing factor induced protein degradation and expression of the ubiquitin-proteasome system in skeletal muscle

Loss of skeletal muscle in cancer cachexia has a negative effect on both morbidity and mortality. The role of nuclear factor-κB (NF-κB) in regulating muscle protein degradation and expression of the ubiquitin-proteasome proteolytic pathway in response to a tumour cachectic factor, proteolysis-inducing factor (PIF), has been studied by creating stable, transdominant-negative, muscle cell lines. Murine C2C12 myoblasts were transfected with plasmids with a CMV promoter that had mutations at the serine phosphorylation sites required for degradation of I-κB��, an NF-κB inhibitory protein, and allowed to differentiate into myotubes. Proteolysis-inducing factor induced degradation of I-κB��, nuclear accumulation of NF-κB and an increase in luciferase reporter gene activity in myotubes containing wild-type, but not mutant, I-κB��, proteins. Proteolysis-inducing factor also induced total protein degradation and loss of the myofibrillar protein myosin in myotubes containing wild-type, but not mutant, plasmids at the same concentrations as those causing activation of NF-κB. Proteolysis-inducing factor also induced increased expression of the ubiquitin-proteasome pathway, as determined by 'chymotrypsin-like' enzyme activity, the predominant proteolytic activity of the β-subunits of the proteasome, protein expression of 20S α-subunits and the 19S subunits MSSI and p42, as well as the ubiquitin conjugating enzyme, E214k, in cells containing wild-type, but not mutant, I-κB��. The ability of mutant I-κB�� to inhibit PIF-induced protein degradation, as well as expression of the ubiquitin-proteasome pathway, confirms that both of these responses depend on initiation of transcription by NF-κB. © 2005 Cancer Research UK.

Increased expression of the ubiquitin-proteasome pathway in murine myotubes by proteolysis-inducing factor (PIF) is associated with activation of the transcription factor NF-kappa B

Proteolysis-inducing factor (PIF), isolated from a cachexia-inducing murine tumour, has been shown to stimulate protein breakdown in C 2C12 myotubes. The effect was attenuated by the specific proteasome inhibitor lactacystin and there was an elevation of proteasome 'chymotrypsin-like' enzyme activity and expression of 205 proteasome α-subunits at concentrations of PIF between 2 and 16 nM. Higher concentrations of PIF had no effect. The action of PIF was attenuated by eicosapentaenoic acid (EPA) (50 μM). At a concentration of 4 nM, PIF induced a transient decrease in IκB�� levels after 30 min incubation, while no effect was seen at 20 nM PIF. The level of IκB��, an NF-κB inhibitory protein, returned to normal after 60 min. Depletion of IκB�� from the cytosol was not seen in myotubes pretreated with EPA, suggesting that the NF-κB/IκB complex was stabilised. At concentrations between 2 and 8 nM, PIF stimulated an increased nuclear migration of NF-κB, which was not seen in myotubes pretreated with EPA. The PIF-induced increase in chymotrypsin-like enzyme activity was also attenuated by the NF-κB inhibitor peptide SN50, suggesting that NF-κB may be involved in the PIF-induced increase in proteasome expression. The results further suggest that EPA may attenuate protein degradation induced by PIF, at least partly, by preventing NF-κB accumulation in the nucleus. © 2003 Cancer Research UK.

The role of the extracellular loops of the CGRP receptor, a family B GPCR

The CGRP (calcitonin gene-related peptide) receptor is a family B GPCR (G-protein-coupled receptor). It consists of a GPCR, CLR (calcitonin receptor-like receptor) and an accessory protein, RAMP1 (receptor activity-modifying protein 1). RAMP1 is needed for CGRP binding and also cell-surface expression of CLR. There have been few systematic studies of the ECLs (extracellular loops) of family B GPCRs. However, they are likely to be especially important for the interaction of the N-termini of the peptide agonists that are the natural agonists for these receptors. We have carried out alanine scans on all three ECLs of CLR, as well as their associated juxtamembrane regions. Residues within all three loops influence CGRP binding and receptor activation. Mutation of Ala203 and Ala206 on ECL1 to leucine increased the affinity of CGRP. Residues at the top of TM (transmembrane) helices 2 and 3 influenced CGRP binding and receptor activation. L351A and E357A in TM6/ECL3 reduced receptor expression and may be needed for CLR association with RAMP1. ECL2 seems especially important for CLR function; of the 16 residues so far examined in this loop, eight residues reduce the potency of CGRP at stimulating cAMP production when mutated to alanine.

Identification of aspirin analogues that repress NF-κB signalling and demonstrate anti-proliferative activity towards colorectal cancer in vitro and in vivo

Substantial evidence indicates that aspirin and related non-steroidal anti-inflammatory drugs (NSAIDs) have potential as chemopreventative/therapeutic agents. However, these agents cannot be universally recommended for prevention purposes due to their potential side-effect profiles. Here, we compared the growth inhibitory and mechanistic activity of aspirin to two novel analogues, diaspirin (DiA) and fumaryl diaspirin (F-DiA). We found that the aspirin analogues inhibited cell proliferation and induced apoptosis of colorectal cancer cells at significantly lower doses than aspirin. Similar to aspirin, we found that an early response to the analogues was a reduction in levels of cyclin D1 and stimulation of the NF-κB pathway. This stimulation was associated with a significant reduction in basal levels of NF-κB transcriptional activity, in keeping with previous data for aspirin. However, in contrast to aspirin, DiA and F-DiA activity was not associated with nucleolar accumulation of RelA. For all assays, F-DiA had a more rapid and significant effect than DiA, identifying this agent as particularly active against colorectal cancer. Using a syngeneic colorectal tumour model in mice, we found that, while both agents significantly inhibited tumour growth in vivo, this effect was particularly pronounced for F-DiA. These data identify two compounds that are active against colorectal cancer in vitro and in vivo. They also identify a potential mechanism of action of these agents and shed light on the chemical structures that may be important for the antitumour effects of aspirin.

Species differential regulation of COX2 can be described by an NFκB-dependent logic AND gate

Cyclooxygenase 2 (COX2), a key regulatory enzyme of the prostaglandin/eicosanoid pathway, is an important target for anti-inflammatory therapy. It is highly induced by pro-inflammatory cytokines in a Nuclear factor kappa B (NFκB)-dependent manner. However, the mechanisms determining the amplitude and dynamics of this important pro-inflammatory event are poorly understood. Furthermore, there is significant difference between human and mouse COX2 expression in response to the inflammatory stimulus tumor necrosis factor alpha (TNFα). Here, we report the presence of a molecular logic AND gate composed of two NFκB response elements (NREs) which controls the expression of human COX2 in a switch-like manner. Combining quantitative kinetic modeling and thermostatistical analysis followed by experimental validation in iterative cycles, we show that the human COX2 expression machinery regulated by NFκB displays features of a logic AND gate. We propose that this provides a digital, noise-filtering mechanism for a tighter control of expression in response to TNFα, such that a threshold level of NFκB activation is required before the promoter becomes active and initiates transcription. This NFκB-regulated AND gate is absent in the mouse COX2 promoter, most likely contributing to its differential graded response in promoter activity and protein expression to TNFα. Our data suggest that the NFκB-regulated AND gate acts as a novel mechanism for controlling the expression of human COX2 to TNFα, and its absence in the mouse COX2 provides the foundation for further studies on understanding species-specific differential gene regulation.

On Limiting Case of the Stein-Weiss Type Inequality for the B-Riesz Potentials

Mathematics Subject Classification: Primary 42B20, 42B25, 42B35

Compilation of downward flux observations from sediment trap deployments in the Atlantic Ocean - Contribution to EURO-BASIN's Data integration

We provide a compilation of downward fluxes (total mass, POC, PON, BSiO2, CaCO3, PIC and lithogenic/terrigenous fluxes) from over 6000 sediment trap measurements distributed in the Atlantic Ocean, from 30 degree North to 49 degree South, and covering the period 1982-2011. Data from the Mediterranean Sea are also included. Data were compiled from different sources: data repositories (BCO-DMO, PANGAEA), time series sites (BATS, CARIACO), published scientific papers and/or personal communications from PI's. All sources are specifed in the data set. Data from the World Ocean Atlas 2009 were extracted to provide each flux observation with contextual environmental data, such as temperature, salinity, oxygen (concentration, AOU and percentage saturation), nitrate, phosphate and silicate.

Immunohistochemical expression of nuclear factor B, matrix metalloproteinase 9, and endoglin (CD105) in odontogenic keratocysts, dentigerous cysts, and radicular cysts

OBJECTIVE: The aim of this study was to compare the immunohistochemical expression of nuclear factor κB (NF-κB), matrix metalloproteinase 9 (MMP-9), and CD105 in odontogenic keratocysts (OKCs), dentigerous cysts (DCs), and radicular cysts (RCs). STUDY DESIGN: Twenty cases of OKCs, 20 DCs, and 20 RCs were analyzed. A labeling index (LI), which expresses the percentage of NF-κB-stained nuclei, was calculated for the analysis of NF-κB expression. Expression of MMP-9 in the epithelium and in the capsule of each lesion was scored as 0 (<10% stained cells), 1 (10%-50% stained cells), or 2 (>50% stained cells). In addition, MMP-9 immunostaining was analyzed in endothelial cells of vessels with a conspicuous lumen. The angiogenic index was determined based on the number of anti-CD105 antibody-stained microvessels. RESULTS: In the epithelial component, the NF-κB LI was higher in OKCs than in DCs and RCs (P < .001). Analysis of MMP-9 expression in the epithelial component showed a predominance of score 2 in OKCs (90%), DCs (70%), and RCs (65%; P = .159). Evaluation of the NF-κB LI according to the expression of MMP-9 in the epithelial lining revealed no significant difference between lesions (P = .282). In the fibrous capsule, the highest percentage of MMP-9-stained cells (score 2) was observed in OKCs (P = .100). Analysis of the expression of MMP-9 in the vessels of odontogenic cysts showed a predominance of score 2 in OKCs (80%) and RCs (50%) and of score 1 in DCs (75%; P = .002). Mean microvessel count was high in RCs (16.9), followed by DCs (12.1) and OKCs (10.0; P = .163). No significant difference in microvessel count according to the expression of MMP-9 was observed between groups (P = .689). CONCLUSIONS: The results suggest that the more aggressive biologic behavior of OKCs is related to the higher expression of MMP-9 and NF-κB in those lesions. The differences in the biologic behavior of the lesions studied do not seem to be associated with the angiogenic index.

18-Fluorodeoxyglucose positron emission tomography/computed tomography in the management of aggressive non-Hodgkin's B-cell lymphoma

18-Fluorodeoxyglucose (FDG-PET/CT) is an established imaging modality that has been proven to be of benefit in the management of aggressive B-cell non-Hodgkin's lymphoma, such as diffuse large B-cell lymphoma and advanced stage follicular lymphoma. The combination of anatomic and functional imaging afforded by FDG-PET/CT has led to superior sensitivity and specificity in the primary staging, restaging, and assessment of response to treatment of hematological malignancies when compared to FDG-PET and CT alone. The use of FDG-PET/CT for post treatment surveillance imaging remains controversial, and further study is needed to ascertain whether this modality is cost effective and appropriate for use in this setting.

Pax3 expression enhances PDGF-B-induced brainstem gliomagenesis and characterizes a subset of brainstem glioma.

High-grade Brainstem Glioma (BSG), also known as Diffuse Intrinsic Pontine Glioma (DIPG), is an incurable pediatric brain cancer. Increasing evidence supports the existence of regional differences in gliomagenesis such that BSG is considered a distinct disease from glioma of the cerebral cortex (CG). In an effort to elucidate unique characteristics of BSG, we conducted expression analysis of mouse PDGF-B-driven BSG and CG initiated in Nestin progenitor cells and identified a short list of expression changes specific to the brainstem gliomagenesis process, including abnormal upregulation of paired box 3 (Pax3). In the neonatal mouse brain, Pax3 expression marks a subset of brainstem progenitor cells, while it is absent from the cerebral cortex, mirroring its regional expression in glioma. Ectopic expression of Pax3 in normal brainstem progenitors in vitro shows that Pax3 inhibits apoptosis. Pax3-induced inhibition of apoptosis is p53-dependent, however, and in the absence of p53, Pax3 promotes proliferation of brainstem progenitors. In vivo, Pax3 enhances PDGF-B-driven gliomagenesis by shortening tumor latency and increasing tumor penetrance and grade, in a region-specific manner, while loss of Pax3 function extends survival of PDGF-B-driven;p53-deficient BSG-bearing mice by 33%. Importantly, Pax3 is regionally expressed in human glioma as well, with high PAX3 mRNA characterizing 40% of human BSG, revealing a subset of tumors that significantly associates with PDGFRA alterations, amplifications of cell cycle regulatory genes, and is exclusive of ACVR1 mutations. Collectively, these data suggest that regional Pax3 expression not only marks a novel subset of BSG but also contributes to PDGF-B-induced brainstem gliomagenesis.

EXAMINATION OF VALPROIC ACID-INDUCED PERTURBATIONS TO DNA REPAIR, NF-ΚB SIGNALING AND CBP/P300 TRANSCRIPTIONAL CO-ACTIVATORS

Exposure to the antiepileptic drug valproic acid (VPA) is associated with an increased risk of congenital malformations including heart, skeletal and most frequently neural tube defects. Although the mechanisms contributing to its teratogenesis are not well understood, VPA was previously shown to increase homologous recombination (HR)-mediated DNA repair and decrease protein expression of the transcription factor NF-κB/p65. The studies in this thesis utilized in vivo and in vitro models to evaluate the expression of HR mediators, investigate the implications of decreased p65 including DNA binding and transcriptional activation, and the expression and histone acetyltransferase activity of Cbp/p300 with an aim to provide mechanistic insight into VPA-mediated alterations. The first study demonstrated that following maternal administration of VPA, mouse embryonic mRNA expression of HR mediators Rad51, Brca1 and Brca2 exhibited temporal and tissue-specific alterations. Protein expression of Rad51 was similarly altered and preceded increased cleavage of caspase-3 and PARP; indicative of apoptosis. The second study confirms previous findings of decreased total cellular p65 protein using P19 cells, but is the first to demonstrate that nuclear p65 protein is unchanged. NF-κB DNA binding was decreased following VPA exposure and maybe mediated by decreased p50 protein, which dimerizes with p65 prior to DNA binding. Transcriptional activity of NF-κB was also increased with VPA exposure which was not due to increased p65 phosphorylation at Ser276. Furthermore, the transcriptional activation capacity was unaffected by VPA exposure as combined exposure to VPA and TNFα additively increased NF-κB activity. The third study demonstrated that VPA exposure in P19 cells decreased Cbp/p300 total cellular and nuclear protein attributed primarily to ubiquitin proteasome-mediated degradation. Histone acetyltransferase (HAT) activity of p300 was decreased proportionately to nuclear protein following VPA exposure. Inhibition of Cbp/p300 HAT activity decreased p65 total cellular protein, increased caspase-3 cleavage and ROS similar to VPA exposures. Furthermore, pre-treatment with the antioxidant enzyme catalase attenuated the increase in caspase-3 cleavage, but not p65 protein. Overall, this thesis demonstrates that VPA exposure impacts the expression and activity of the transcription factor NF-κB and transcriptional co-activators/HATs Cbp/p300, which has implications for downstream VPA targets including Rad51, Brca1 and Brca2.

Sedimentology of a Middle Jurassic Base-of-Slope environment, Cutri Formation, Mallorca

The Cutri Formation’s, type location, exposed in the NW of Mallorca, Spain has previously been described by Álvaro et al., (1989) and further interpreted by Abbots (1989) unpublished PhD thesis as a base-of-slope carbonate apron. Incorporating new field and laboratory analysis this paper enhances this interpretation. From this analysis, it can be shown without reasonable doubt that the Cutri Formation was deposited in a carbonate base-of-slope environment on the palaeowindward side of a Mid-Jurassic Tethyan platform. Key evidence such as laterally extensive exposures, abundant deposits of calciturbidtes and debris flows amongst hemipelagic deposits strongly support this interpretation.