Suppression of steady-state, but not stimulus-induced NF-kappaB activity inhibits alphavirus-induced apoptosis.

Recent studies have established cell type- specific, proapoptotic, or antiapoptotic functions for the transcription factor NF-kappaB. In each of these studies, inhibitors of NF-kappaB activity have been present before the apoptotic stimulus, and so the role of stimulus- induced NF-kappaB activation in enhancing or inhibiting survival could not be directly assessed. Sindbis virus, an alphavirus, induces NF-kappaB activation and apoptosis in cultured cell lines. To address whether Sindbis virus- induced NF-kappaB activation is required for apoptosis, we used a chimeric Sindbis virus that expresses a superrepressor of NF-kappaB activity. Complete suppression of virus-induced NF-kappaB activity neither prevents nor potentiates Sindbis virus-induced apoptosis. In contrast, inhibition of NF-kappaB activity before infection inhibits Sindbis virus-induced apoptosis. Our results demonstrate that suppression of steady-state, but not stimulus-induced NF-kappaB activity, regulates expression of gene products required for Sindbis virus-induced death. Furthermore, we show that in the same cell line, NF-kappaB can be proapoptotic or antiapoptotic depending on the death stimulus. We propose that the role of NF-kappaB in regulating apoptosis is determined by the death stimulus and by the timing of modulating NF-kappaB activity relative to the death stimulus

Identification of host proteins interacting with Toxoplasma gondii GRA15 (TgGRA15) by yeast two-hybrid system

Background Toxoplasma gondii, an obligate intracellular protozoan parasite, possesses the remarkable ability to co-opt host cell machinery in order to maintain its intracellular survival. This parasite can modulate signaling pathways of its host through the secretion of polymorphic effector proteins localized in the rhoptry and dense granule organelles. One of such effectors is T. gondii type II-specific dense granule protein 15, TgGRA15, which activates NF-κB pathway. The aim of the present study was to identify the host interaction partner proteins of TgGRA15. Methods We screened a yeast two-hybrid mouse cDNA library using TgGRA15 as the bait. TgGRA15 (PRU strain, Type II) was cloned into the pGBKT7 vector and expressed in the Y2HGold yeast strain. Then, the bait protein expression was validated by western blotting analysis, followed by auto-activation and toxicity tests in comparison with control (Y2HGold yeast strain transformed with empty pGBKT7 vector). Results This screening led to the identification of mouse Luzp1 and AW209491 as host binding proteins that interact with TgGRA15. Luzp1 contains three nuclear localizing signals and is involved in regulating a subset of host non-coding RNA genes. Conclusions These findings reveal, for the first time, new host cell proteins interacting with TgGRA15. The identification of these cellular targets and the understanding of their contribution to the host-pathogen interaction may serve as the foundation for novel therapeutic and prevention strategies against T. gondii infection

Statistical Techniques Complement UML When Developing Domain Models of Complex Dynamical Biosystems

Computational modelling and simulation is increasingly being used to complement traditional wet-lab techniques when investigating the mechanistic behaviours of complex biological systems. In order to ensure computational models are fit for purpose, it is essential that the abstracted view of biology captured in the computational model, is clearly and unambiguously defined within a conceptual model of the biological domain (a domain model), that acts to accurately represent the biological system and to document the functional requirements for the resultant computational model. We present a domain model of the IL-1 stimulated NF-κB signalling pathway, which unambiguously defines the spatial, temporal and stochastic requirements for our future computational model. Through the development of this model, we observe that, in isolation, UML is not sufficient for the purpose of creating a domain model, and that a number of descriptive and multivariate statistical techniques provide complementary perspectives, in particular when modelling the heterogeneity of dynamics at the single-cell level. We believe this approach of using UML to define the structure and interactions within a complex system, along with statistics to define the stochastic and dynamic nature of complex systems, is crucial for ensuring that conceptual models of complex dynamical biosystems, which are developed using UML, are fit for purpose, and unambiguously define the functional requirements for the resultant computational model

Registered Replication Report: Dijksterhuis and van Knippenberg (1998)

Dijksterhuis and van Knippenberg (1998) reported that participants primed with a category associated with intelligence ("professor") subsequently performed 13% better on a trivia test than participants primed with a category associated with a lack of intelligence ("soccer hooligans"). In two unpublished replications of this study designed to verify the appropriate testing procedures, Dijksterhuis, van Knippenberg, and Holland observed a smaller difference between conditions (2%-3%) as well as a gender difference: Men showed the effect (9.3% and 7.6%), but women did not (0.3% and -0.3%). The procedure used in those replications served as the basis for this multilab Registered Replication Report. A total of 40 laboratories collected data for this project, and 23 of these laboratories met all inclusion criteria. Here we report the meta-analytic results for those 23 direct replications (total N = 4,493), which tested whether performance on a 30-item general-knowledge trivia task differed between these two priming conditions (results of supplementary analyses of the data from all 40 labs, N = 6,454, are also reported). We observed no overall difference in trivia performance between participants primed with the "professor" category and those primed with the "hooligan" category (0.14%) and no moderation by gender

Nitrosylcobalamin Potentiates the Anti-Neoplastic Effects of Chemotherapeutic Agents via Suppression of Survival Signaling

Nitrosylcobalamin (NO-Cbl) is a chemotherapeutic pro-drug derived from vitamin B12 that preferentially delivers nitric oxide (NO) to tumor cells, based upon increased receptor expression. NO-Cbl induces Apo2L/TRAIL-mediated apoptosis and inhibits survival signaling in a variety of malignant cell lines. Chemotherapeutic agents often simultaneously induce an apoptotic signal and activation of NF-kappaB, which has the undesired effect of promoting cell survival. The specific aims of this study were to 1) measure the anti-tumor effects of NO-Cbl alone and in combination with conventional chemotherapeutic agents, and to 2) examine the mechanism of action of NO-Cbl as a single agent and in combination therapy.Using anti-proliferative assays, electrophoretic mobility shift assay (EMSA), immunoblot analysis and kinase assays, we demonstrate an increase in the effectiveness of chemotherapeutic agents in combination with NO-Cbl as a result of suppressed NF-kappaB activation.Eighteen chemotherapeutic agents were tested in combination with NO-Cbl, in thirteen malignant cell lines, resulting in a synergistic anti-proliferative effect in 78% of the combinations tested. NO-Cbl pre-treatment resulted in decreased NF-kappaB DNA binding activity, inhibition of IkappaB kinase (IKK) enzymatic activity, decreased AKT activation, increased caspase-8 and PARP cleavage, and decreased cellular XIAP protein levels.The use of NO-Cbl to inhibit survival signaling may enhance drug efficacy by preventing concomitant activation of NF-kappaB or AKT

Longitudinal plasma measures of trimethylamine N-Oxide and risk of atherosclerotic cardiovascular disease events in community-based older adults

Background Trimethylamine N‐oxide (TMAO) is a gut microbiota‐dependent metabolite of dietary choline, L‐carnitine, and phosphatidylcholine‐rich foods. On the basis of experimental studies and patients with prevalent disease, elevated plasma TMAO may increase risk of atherosclerotic cardiovascular disease (ASCVD). TMAO is also renally cleared and may interact with and causally contribute to renal dysfunction. Yet, how serial TMAO levels relate to incident and recurrent ASCVD in community‐based populations and the potential mediating or modifying role of renal function are not established. Methods and Results We investigated associations of serial measures of plasma TMAO, assessed at baseline and 7 years, with incident and recurrent ASCVD in a community‐based cohort of 4131 (incident) and 1449 (recurrent) older US adults. TMAO was measured using stable isotope dilution liquid chromatography–tandem mass spectrometry (laboratory coefficient of variation, <6%). Incident ASCVD (myocardial infarction, fatal coronary heart disease, stroke, sudden cardiac death, or other atherosclerotic death) was centrally adjudicated using medical records. Risk was assessed by multivariable Cox proportional hazards regression, including time‐varying demographics, lifestyle factors, medical history, laboratory measures, and dietary habits. Potential mediating effects and interaction by estimated glomerular filtration rate (eGFR) were assessed. During prospective follow‐up, 1766 incident and 897 recurrent ASCVD events occurred. After multivariable adjustment, higher levels of TMAO were associated with a higher risk of incident ASCVD, with extreme quintile hazard ratio (HR) compared with the lowest quintile=1.21 (95% CI, 1.02–1.42; P‐trend=0.029). This relationship appeared mediated or confounded by eGFR (eGFR‐adjusted HR, 1.07; 95% CI, 0.90–1.27), as well as modified by eGFR (P‐interaction <0.001). High levels of TMAO were associated with higher incidence of ASCVD in the presence of impaired renal function (eGFR <60 mL/min per 1.73 m2: HR, 1.56 [95% CI, 1.13–2.14]; P‐trend=0.007), but not normal or mildly reduced renal function (eGFR ≥60 mL/min per 1.73 m2: HR, 1.03 [95% CI, 0.85–1.25]; P‐trend=0.668). Among individuals with prior ASCVD, TMAO associated with higher risk of recurrent ASCVD (HR, 1.25 [95% CI, 1.01–1.56]; P‐trend=0.009), without significant modification by eGFR. Conclusions In this large community‐based cohort of older US adults, serial measures of TMAO were associated with higher risk of incident ASCVD, with apparent modification by presence of impaired renal function and with higher risk of recurrent ASCVD

An Abundant Dysfunctional Apolipoprotein A1 in Human Atheroma

Recent studies have indicated that high-density lipoproteins (HDLs) and their major structural protein, apolipoprotein A1 (apoA1), recovered from human atheroma are dysfunctional and are extensively oxidized by myeloperoxidase (MPO). In vitro oxidation of either apoA1 or HDL particles by MPO impairs their cholesterol acceptor function. Here, using phage display affinity maturation, we developed a high-affinity monoclonal antibody that specifically recognizes both apoA1 and HDL that have been modified by the MPO-H2O2-Cl− system. An oxindolyl alanine (2-OH-Trp) moiety at Trp72 of apoA1 is the immunogenic epitope. Mutagenesis studies confirmed a critical role for apoA1 Trp72 in MPO-mediated inhibition of the ATP-binding cassette transporter A1 (ABCA1)-dependent cholesterol acceptor activity of apoA1 in vitro and in vivo. ApoA1 containing a 2-OH-Trp72 group (oxTrp72-apoA1) is in low abundance within the circulation but accounts for 20% of the apoA1 in atherosclerosis-laden arteries. OxTrp72-apoA1 recovered from human atheroma or plasma is lipid poor, virtually devoid of cholesterol acceptor activity and demonstrated both a potent proinflammatory activity on endothelial cells and an impaired HDL biogenesis activity in vivo. Elevated oxTrp72-apoA1 levels in subjects presenting to a cardiology clinic (n = 627) were associated with increased cardiovascular disease risk. Circulating oxTrp72-apoA1 levels may serve as a way to monitor a proatherogenic process in the artery wall

Loss of Metal Ions, Disulfide Reduction and Mutations Related to Familial ALS Promote Formation of Amyloid-Like Aggregates from Superoxide Dismutase

Mutations in the gene encoding Cu-Zn superoxide dismutase (SOD1) are one of the causes of familial amyotrophic lateral sclerosis (FALS). Fibrillar inclusions containing SOD1 and SOD1 inclusions that bind the amyloid-specific dye thioflavin S have been found in neurons of transgenic mice expressing mutant SOD1. Therefore, the formation of amyloid fibrils from human SOD1 was investigated. When agitated at acidic pH in the presence of low concentrations of guanidine or acetonitrile, metalated SOD1 formed fibrillar material which bound both thioflavin T and Congo red and had circular dichroism and infrared spectra characteristic of amyloid. While metalated SOD1 did not form amyloid-like aggregates at neutral pH, either removing metals from SOD1 with its intramolecular disulfide bond intact or reducing the intramolecular disulfide bond of metalated SOD1 was sufficient to promote formation of these aggregates. SOD1 formed amyloid-like aggregates both with and without intermolecular disulfide bonds, depending on the incubation conditions, and a mutant SOD1 lacking free sulfhydryl groups (AS-SOD1) formed amyloid-like aggregates at neutral pH under reducing conditions. ALS mutations enhanced the ability of disulfide-reduced SOD1 to form amyloid-like aggregates, and apo-AS-SOD1 formed amyloid-like aggregates at pH 7 only when an ALS mutation was also present. These results indicate that some mutations related to ALS promote formation of amyloid-like aggregates by facilitating the loss of metals and/or by making the intramolecular disulfide bond more susceptible to reduction, thus allowing the conversion of SOD1 to a form that aggregates to form resembling amyloid. Furthermore, the occurrence of amyloid-like aggregates per se does not depend on forming intermolecular disulfide bonds, and multiple forms of such aggregates can be produced from SOD1

Protandim, a Fundamentally New Antioxidant Approach in Chemoprevention Using Mouse Two-Stage Skin Carcinogenesis as a Model

Oxidative stress is an important contributor to cancer development. Consistent with that, antioxidant enzymes have been demonstrated to suppress tumorigenesis when being elevated both in vitro and in vivo, making induction of these enzymes a more potent approach for cancer prevention. Protandim, a well-defined combination of widely studied medicinal plants, has been shown to induce superoxide dismutase (SOD) and catalase activities and reduce superoxide generation and lipid peroxidation in healthy human subjects. To investigate whether Protandim can suppress tumor formation by a dietary approach, a two-stage mouse skin carcinogenesis study was performed. At the end of the study, the mice on a Protandim-containing basal diet had similar body weight compared with those on the basal diet, which indicated no overt toxicity by Protandim. After three weeks on the diets, there was a significant increase in the expression levels of SOD and catalase, in addition to the increases in SOD activities. Importantly, at the end of the carcinogenesis study, both skin tumor incidence and multiplicity were reduced in the mice on the Protandim diet by 33% and 57% respectively, compared with those on basal diet. Biochemical and histological studies revealed that the Protandim diet suppressed tumor promoter-induced oxidative stress (evidenced by reduction of protein carbonyl levels), cell proliferation (evidenced by reduction of skin hyperplasia and suppression of PKC/JNK/Jun pathway), and inflammation (evidenced by reduction of ICAM-1/VCAM-1 expression, NF-κB binding activity, and nuclear p65/p50 levels). Overall, induction of antioxidant enzymes by Protandim may serve as a practical and potent approach for cancer prevention