Simvastatin potentiates TNF-alpha-induced apoptosis through the down-regulation of NF-kappaB-dependent antiapoptotic gene products: role of IkappaBalpha kinase and TGF-beta-activated kinase-1

Numerous recent reports suggest that statins (hydroxy-3-methylglutaryl-CoA reductase inhibitors) exhibit potential to suppress tumorigenesis through a mechanism that is not fully understood. Therefore, in this article, we investigated the effects of simvastatin on TNF-alpha-induced cell signaling. We found that simvastatin potentiated the apoptosis induced by TNF-alpha as indicated by intracellular esterase activity, caspase activation, TUNEL, and annexin V staining. This effect of simvastatin correlated with down-regulation of various gene products that mediate cell proliferation (cyclin D1 and cyclooxygenase-2), cell survival (Bcl-2, Bcl-x(L), cellular FLIP, inhibitor of apoptosis protein 1, inhibitor of apoptosis protein 2, and survivin), invasion (matrix mellatoproteinase-9 and ICAM-1), and angiogenesis (vascular endothelial growth factor); all known to be regulated by the NF-kappaB. We found that simvastatin inhibited TNF-alpha-induced NF-kappaB activation, and l-mevalonate reversed the suppressive effect, indicating the role of hydroxy-3-methylglutaryl-CoA reductase. Simvastatin suppressed not only the inducible but also the constitutive NF-kappaB activation. Simvastatin inhibited TNF-alpha-induced IkappaBalpha kinase activation, which led to inhibition of IkappaBalpha phosphorylation and degradation, suppression of p65 phosphorylation, and translocation to the nucleus. NF-kappaB-dependent reporter gene expression induced by TNF-alpha, TNFR1, TNFR-associated death domain protein, TNFR-associated factor 2, TGF-beta-activated kinase 1, receptor-interacting protein, NF-kappaB-inducing kinase, and IkappaB kinase beta was abolished by simvastatin. Overall, our results provide novel insight into the role of simvastatin in potentially preventing and treating cancer through modulation of IkappaB kinase and NF-kappaB-regulated gene products.     

Heritability of brachydactyly type A3 in children, adolescents, and young adults from an endogamous population in eastern Nepal

Brachymesophalangia-V (BMP-V), a short and broad middle phalanx of the fifth digit, is the most common of all skeletal anomalies of the hand. When this feature appears alone, it is clinically known as brachydactyly type A3 (BDA3). A high prevalence of BDA3 has been observed among the children of the Jirel ethnic group in eastern Nepal. As part of the Jiri Growth Study, a hand-wrist radiograph is taken annually of each child to assess skeletal development. For this study the most recent radiographs of 1,357 Jirel children, adolescents, and young adults (676 boys, 681 girls), age 3-20 years, were examined for the presence or absence of BDA3, to report the prevalence and estimate the heritability of BDA3 in the Jirel population. The overall prevalence of BDA3 in this sample was 10.5% (12.9% of the males and 8.9% of the females were classified as BDA3 affected). The additive genetic heritability of BDA3 was statistically significant in this sample (h2 +/- SE = 0.87 +/- 0.16, p < 0.0001). This study is the first to estimate the prevalence and heritability of BDA3 in a large South Asian family-based sample.     

Evaluation of immune response to bovine rotavirus following oral and intraperitoneal inoculation in mice

With a view to use mice as an experimental model for studying immune response to bovine rotavirus (BRV), the kinetics of humoral and cellular immune responses to BRV in mice were evaluated by immunizing through intraperitoneal and oral route with UK strain of BRV. Following immunization with BRV, anti-rotavirus antibodies was developed in mice. The mean log antibody titres as measured by ELISA in mice immunized by intraperitoneal route were significantly higher than those immunized by oral route. Significant cellular immune response was observed in BRV-immunized mice on stimulation with BRV antigen, as measured by lymphocyte proliferation assay. The thymidine uptake by splenic and mesenteric lymph-node cells of intraperitoneally immunized mice on stimulation with BRV was 21328 +/- 1225 and 739 +/- 55 CPM, respectively. The splenic cells showed significantly higher stimulation (stimulation index 12.98) as compared to those of mesenteric cells (stimulation index 1.57). Foot pad inoculation test showed maximum virus-specific delayed type hypersensitivity reaction at 24 hr post-challenge following primary immunization and at 18 hr post-challenge following secondary immunization. The results indicate that BRV immunization by intraperitoneal route generates more efficient immune response in mice than by oral route and this route may be used for immune response studies involving BRV infection.     

Dihydro-[1H]-quinolin-2-ones as retinoid X receptor (RXR) agonists for potential treatment of dyslipidemia

A number of RXR modulators with novel structural features were synthesized and screened in the functional assays. The synthesis and the structure-activity relationship within the series of compounds will be presented. Some in vivo data generated in the models for dyslipidemia and diabetes will also be presented.     

RXR-LXR heterodimer modulators for the potential treatment of dyslipidemia

A number of RXR agonists were synthesized and screened in functional assays. The synthesis and the structure-activity relationship (SAR) within the series of compounds will be presented. Some in vivo data in rodent models for dyslipidemia and diabetes will also be presented.     

Role of pro-oxidants and antioxidants in the anti-inflammatory and apoptotic effects of curcumin (diferuloylmethane)

Extensive research within the past half-century has indicated that curcumin (diferuloylmethane), a yellow pigment in curry powder, exhibits antioxidant, anti-inflammatory, and proapoptotic activities. We investigated whether the anti-inflammatory and proapoptotic activities assigned to curcumin are mediated through its prooxidant/antioxidant mechanism. We found that TNF-mediated NF-kappaB activation was inhibited by curcumin; and glutathione reversed the inhibition. Similarly, suppression of TNF-induced AKT activation by curcumin was also abrogated by glutathione. The reducing agent also counteracted the inhibitory effects of curcumin on TNF-induced NF-kappaB-regulated antiapoptotic (Bcl-2, Bcl-xL, IAP1), proliferative (cyclin D1), and proinflammatory (COX-2, iNOS, and MMP-9) gene products. The suppression of TNF-induced AP-1 activation by curcumin was also reversed by glutathione. Also, the direct proapoptotic effects of curcumin were inhibited by glutathione and potentiated by depletion of intracellular glutathione by buthionine sulfoximine. Moreover, curcumin induced the production of reactive oxygen species and modulated intracellular GSH levels. Quenchers of hydroxyl radicals, however, were ineffective in inhibiting curcumin-mediated NF-kappaB suppression. Further, N-acetylcysteine partially reversed the effect of curcumin. Based on these results we conclude that curcumin mediates its apoptotic and anti-inflammatory activities through modulation of the redox status of the cell.     

Membrane-Localized Extra-Large G Proteins and Gβγ of the Heterotrimeric G Proteins Form Functional Complexes Engaged in Plant Immunity in Arabidopsis

In animals, heterotrimeric G proteins, comprising Gα, Gβ, and Gγ subunits, are molecular switches whose function tightly depends on Gα and Gβγ interaction. Intriguingly, in Arabidopsis (Arabidopsis thaliana), multiple defense responses involve Gβγ, but not Gα. We report here that the Gβγ dimer directly partners with extra-large G proteins (XLGs) to mediate plant immunity. Arabidopsis mutants deficient in XLGs, Gβ, and Gγ are similarly compromised in several pathogen defense responses, including disease development and production of reactive oxygen species. Genetic analysis of double, triple, and quadruple mutants confirmed that XLGs and Gβγ functionally interact in the same defense signaling pathways. In addition, mutations in XLG2 suppressed the seedling lethal and cell death phenotypes of BRASSINOSTEROID INSENSITIVE1-associated receptor kinase1-interacting receptor-like kinase1 mutants in an identical way as reported for Arabidopsis Gβ-deficient mutants. Yeast (Saccharomyces cerevisiae) three-hybrid and bimolecular fluorescent complementation assays revealed that XLG2 physically interacts with all three possible Gβγ dimers at the plasma membrane. Phylogenetic analysis indicated a close relationship between XLGs and plant Gα subunits, placing the divergence point at the dawn of land plant evolution. Based on these findings, we conclude that XLGs form functional complexes with Gβγ dimers, although the mechanism of action of these complexes, including activation/deactivation, must be radically different form the one used by the canonical Gα subunit and are not likely to share the same receptors. Accordingly, XLGs expand the repertoire of heterotrimeric G proteins in plants and reveal a higher level of diversity in heterotrimeric G protein signaling.Heterotrimeric GTP-binding proteins (G proteins), classically consisting of Gα, Gβ, and Gγ subunits, are essential signal transduction elements in most eukaryotes. In animals and fungi, ligand perception by G protein-coupled receptors leads to replacement of GDP with GTP in Gα, triggering activation of the heterotrimer (Li et al., 2007; Oldham and Hamm, 2008). Upon activation, GTP-bound Gα and Gβγ are released and interact with downstream effectors, thereby transmitting signals to multiple intracellular signaling cascades. Signaling terminates when the intrinsic GTPase activity of Gα hydrolyzes GTP to GDP and the inactive heterotrimer reforms at the receptor. The large diversity of mammalian Gα subunits confers specificity to the multiple signaling pathways mediated by G proteins (Wettschureck and Offermanns, 2005). Five distinct classes of Gα have been described in animals (Gαi, Gαq, Gαs, Gα12 and Gαv), with orthologs found in evolutionarily primitive organisms such as sponges (Oka et al., 2009). Humans possess four classes of Gα involving 23 functional isoforms encoded by 16 genes (McCudden et al., 2005), while only a single prototypical Gα is usually found per plant genome (Urano et al., 2013). Multiple copies of Gα are present in some species with recently duplicated genomes, such as soybean (Glycine max) with four Gα genes (Blanc and Wolfe, 2004; Bisht et al., 2011). In the model plant Arabidopsis (Arabidopsis thaliana), a prototypical Gα subunit (GPA1) is involved in a number of important processes, including cell proliferation (Ullah et al., 2001), inhibition of inward K⁺ channels and activation of anion channels in guard cells by mediating the abscisic acid pathway (Wang et al., 2001; Coursol et al., 2003), blue light responses (Warpeha et al., 2006, 2007), and germination and postgermination development (Chen et al., 2006; Pandey et al., 2006).It is well established that heterotrimeric G proteins play a fundamental role in plant innate immunity. In Arabidopsis, two different Gβγ dimers (Gβγ1 and Gβγ2) are generally considered to be the predominant elements in G protein defense signaling against a variety of fungal pathogens (Llorente et al., 2005; Trusov et al., 2006, 2007, 2009; Delgado-Cerezo et al., 2012; Torres et al., 2013). By contrast, these studies attributed a small or no role to Gα, because mutants deficient in Gα displayed only slightly increased resistance against the fungal pathogens (Llorente et al., 2005; Trusov et al., 2006; Torres et al., 2013). The Gβγ-mediated signaling also contributes to defense against a model bacterial pathogen Pseudomonas syringae, by participating in programmed cell death (PCD) and inducing reactive oxygen species (ROS) production in response to at least three pathogen-associated molecular patterns (PAMPs; Ishikawa, 2009; Liu et al., 2013; Torres et al., 2013). Gα is not involved in PCD or PAMP-triggered ROS production (Liu et al., 2013; Torres et al., 2013). Nonetheless, Arabidopsis Gα plays a positive role in defense against P. syringae, probably by mediating stomatal function and hence physically restricting bacterial entry to the leaf interior (Zhang et al., 2008; Zeng and He, 2010; Lee et al., 2013). Given the small contribution from Gα, the involvement of heterotrimeric G proteins in Arabidopsis resistance could be explained in two ways: either the Gβγ dimer acts independently from Gα, raising a question of how is it activated upon a pathogen attack, or Gα is replaced by another protein for heterotrimer formation.The Arabidopsis genome contains at least three genes encoding Gα-like proteins that have been classified as extra-large G proteins (XLGs; Lee and Assmann, 1999; Ding et al., 2008). XLGs comprise two structurally distinct regions. The C-terminal region is similar to the canonical Gα, containing the conserved helical and GTPase domains, while the N-terminal region is a stretch of approximately 400 amino acids including a putative nuclear localization signal (Ding et al., 2008). GTP binding and hydrolysis were confirmed for all three XLG proteins, although their enzymatic activities are very slow and require Ca²⁺ as a cofactor, whereas canonical Gα utilizes Mg²⁺ (Heo et al., 2012). Several other features differentiate XLGs from Gα subunits. Comparative analysis of XLG1 and Gα at the DNA level showed that the genes are organized in seven and 13 exons, respectively, without common splicing sites (Lee and Assmann, 1999). XLGs have been reported to localize to the nucleus (Ding et al., 2008). Analysis of knockout mutants revealed a nuclear function for XLG2, as it physically interacts with the Related To Vernalization1 (RTV1) protein, enhancing the DNA binding activity of RTV1 to floral integrator gene promoters and resulting in flowering initiation (Heo et al., 2012). Therefore, it appears that XLGs may act independently of G protein signaling. On the other hand, functional similarities between XLGs and the Arabidopsis Gβ subunit (AGB1) were also discovered. For instance, XLG3- and Gβ-deficient mutants were similarly impaired in root gravitropic responses (Pandey et al., 2008). Knockout of all three XLG genes caused increased root length, similarly to the Gβ-deficient mutant (Ding et al., 2008). Furthermore, as observed in Gβ-deficient mutants, xlg2 mutants displayed increased susceptibility to P. syringae, indicating a role in plant defense (Zhu et al., 2009). Nevertheless, a genetic analysis of the possible functional interaction between XLGs and Gβ has not been established.In this report, we performed in-depth genetic analyses to test the functional interaction between the three XLGs and Gβγ dimers during defense-related responses in Arabidopsis. We also examined physical interaction between XLG2 and the Gβγ dimers using yeast (Saccharomyces cerevisiae) three-hybrid (Y3H) and bimolecular fluorescent complementation (BiFC) assays. Our findings indicate that XLGs function as direct partners of Gβγ dimers in plant defense signaling. To estimate relatedness of XLGs and Gα proteins, we carried out a phylogenetic analysis. Based on our findings, we conclude that plant XLG proteins most probably originated from a canonical Gα subunit and retained prototypical interaction with Gβγ dimers. They function together with Gβγ in a number of processes including plant defense, although they most probably evolved activation/deactivation mechanisms very different from those of a prototypical Gα.     

Projecting future expansion of invasive species: comparing and improving methodologies for species distribution modeling

Modeling the distributions of species, especially of invasive species in non‐native ranges, involves multiple challenges. Here, we developed some novel approaches to species distribution modeling aimed at reducing the influences of such challenges and improving the realism of projections. We estimated species–environment relationships for Parthenium hysterophorus L. (Asteraceae) with four modeling methods run with multiple scenarios of (i) sources of occurrences and geographically isolated background ranges for absences, (ii) approaches to drawing background (absence) points, and (iii) alternate sets of predictor variables. We further tested various quantitative metrics of model evaluation against biological insight. Model projections were very sensitive to the choice of training dataset. Model accuracy was much improved using a global dataset for model training, rather than restricting data input to the species’ native range. AUC score was a poor metric for model evaluation and, if used alone, was not a useful criterion for assessing model performance. Projections away from the sampled space (i.e., into areas of potential future invasion) were very different depending on the modeling methods used, raising questions about the reliability of ensemble projections. Generalized linear models gave very unrealistic projections far away from the training region. Models that efficiently fit the dominant pattern, but exclude highly local patterns in the dataset and capture interactions as they appear in data (e.g., boosted regression trees), improved generalization of the models. Biological knowledge of the species and its distribution was important in refining choices about the best set of projections. A post hoc test conducted on a new Parthenium dataset from Nepal validated excellent predictive performance of our ‘best’ model. We showed that vast stretches of currently uninvaded geographic areas on multiple continents harbor highly suitable habitats for parthenium. However, discrepancies between model predictions and parthenium invasion in Australia indicate successful management for this globally significant weed.     

Antidiabetic vanadium compound and membrane interfaces: interface-facilitated metal complex hydrolysis

The interactions of metabolites of the antidiabetic vanadium-containing drug bis(maltolato)oxovanadium(IV) (BMOV) with lipid interface model systems were investigated and the results were used to describe a potentially novel mechanism by which these compounds initiate membrane-receptor-mediated signal transduction. Specifically, spectroscopic studies probed the BMOV oxidation and hydrolysis product interaction with interfaces created from cetyltrimethylammonium bromide (CTAB) which mimics the positively charged head group on phosphatidylcholine. ¹H and ⁵¹V NMR spectroscopies were used to determine the location of the dioxobis(maltolato)oxovanadate(V) and the maltol ligand in micelles and reverse micelles by measuring changes in the chemical shift, signal linewidth, and species distribution. Both micelles and reverse micelles interacted similarly with the complex and the ligand, suggesting that interaction is strong as anticipated by Coulombic attraction between the positively charged lipid head group and the negatively charged complex and deprotonated ligand. The nature of the model system was confirmed using dynamic light scattering studies and conductivity measurements. Interactions of the complex/ligand above and below the critical micelle concentration of micelle formation were different, with much stronger interactions when CTAB was in the form of a micelle. Both the complex and the ligand penetrated the lipid interface and were located near the charged head group. These studies demonstrate that a lipid-like interface affects the stability of the complex and raise the possibility that ligand exchange at the interface may be important for the mode of action for these systems. Combined, these studies support recently reported in vivo observations of BMOV penetration into 3T3-L1 adipocyte membranes and increased translocation of a glucose transporter to the plasma membrane.     

Cryo-electron tomography of Marburg virus particles and their morphogenesis within infected cells

Several major human pathogens, including the filoviruses, paramyxoviruses, and rhabdoviruses, package their single-stranded RNA genomes within helical nucleocapsids, which bud through the plasma membrane of the infected cell to release enveloped virions. The virions are often heterogeneous in shape, which makes it difficult to study their structure and assembly mechanisms. We have applied cryo-electron tomography and sub-tomogram averaging methods to derive structures of Marburg virus, a highly pathogenic filovirus, both after release and during assembly within infected cells. The data demonstrate the potential of cryo-electron tomography methods to derive detailed structural information for intermediate steps in biological pathways within intact cells. We describe the location and arrangement of the viral proteins within the virion. We show that the N-terminal domain of the nucleoprotein contains the minimal assembly determinants for a helical nucleocapsid with variable number of proteins per turn. Lobes protruding from alternate interfaces between each nucleoprotein are formed by the C-terminal domain of the nucleoprotein, together with viral proteins VP24 and VP35. Each nucleoprotein packages six RNA bases. The nucleocapsid interacts in an unusual, flexible "Velcro-like" manner with the viral matrix protein VP40. Determination of the structures of assembly intermediates showed that the nucleocapsid has a defined orientation during transport and budding. Together the data show striking architectural homology between the nucleocapsid helix of rhabdoviruses and filoviruses, but unexpected, fundamental differences in the mechanisms by which the nucleocapsids are then assembled together with matrix proteins and initiate membrane envelopment to release infectious virions, suggesting that the viruses have evolved different solutions to these conserved assembly steps.