Tocopherol Transfer Protein Sensitizes Prostate Cancer Cells to Vitamin E

Prostate cancer is a major cause of mortality in men in developed countries. It has been reported that the naturally occurring antioxidant α-tocopherol (vitamin E) attenuates prostate cancer cell proliferation in cultured cells and mouse models. We hypothesized that overexpression of the tocopherol transfer protein (TTP), a vitamin E-binding protein that regulates tocopherol status, will sensitize prostate cancer cells to the anti-proliferative actions of the vitamin. To test this notion, we manipulated the expression levels of TTP in cultured prostate cells (LNCaP, PC3, DU145, and RWPE-1) using overexpression and knockdown approaches. Treatment of cells with tocopherol caused a time- and dose-dependent inhibition of cell proliferation. Overexpression of TTP dramatically sensitized the cells to the apoptotic effects of α-tocopherol, whereas reduction (“knockdown”) of TTP expression resulted in resistance to the vitamin. TTP levels also augmented the inhibitory effects of vitamin E on proliferation in semi-solid medium. The sensitizing effects of TTP were paralleled by changes in the intracellular accumulation of a fluorescent analog of vitamin E and by a reduction in intracellular levels of reactive oxygen species and were not observed when a naturally occurring, ligand binding-defective mutant of TTP was used. We conclude that TTP sensitizes prostate cancer cells to the anti-proliferative effects of vitamin E and that this activity stems from the ability of protein to increase the intracellular accumulation of the antioxidant. These observations support the notion that individual changes in the expression level or activity of TTP may determine the responsiveness of prostate cancer patients to intervention strategies that utilize vitamin E.     

Intracellular nucleotides act as critical prosurvival factors by binding to cytochrome C and inhibiting apoptosome

Cytochrome c (CC)-initiated Apaf-1 apoptosome formation represents a key initiating event in apoptosis. This process can be reconstituted in vitro with the addition of CC and ATP or dATP to cell lysates. How physiological levels of nucleotides, normally at high mM concentrations, affect apoptosome activation remains unclear. Here we show that physiological levels of nucleotides inhibit the CC-initiated apoptosome formation and caspase-9 activation by directly binding to CC on several key lysine residues and thus preventing CC interaction with Apaf-1. We show that in various apoptotic systems caspase activation is preceded or accompanied by decreases in overall intracellular NTP pools. Microinjection of nucleotides inhibits whereas experimentally reducing NTP pools enhances both CC and apoptotic stimuli-induced cell death. Our results thus suggest that the intracellular nucleotides represent critical prosurvival factors by functioning as natural inhibitors of apoptosome formation and a barrier that cells must overcome the nucleotide barrier to undergo apoptosis cell death.     

Endoplasmic Reticulum Stress Pathway Required for Immune Homeostasis Is Neurally Controlled by Arrestin-1

In response to pathogen infection, the host innate immune system activates microbial killing pathways and cellular stress pathways that need to be balanced because insufficient or excessive immune responses have deleterious consequences. Recent studies demonstrate that two G protein-coupled receptors (GPCRs) in the nervous system of Caenorhabditis elegans control immune homeostasis. To investigate further how GPCR signaling controls immune homeostasis at the organismal level, we studied arrestin-1 (ARR-1), which is the only GPCR adaptor protein in C. elegans. The results indicate that ARR-1 is required for GPCR signaling in ASH, ASI, AQR, PQR, and URX neurons, which control the unfolded protein response and a p38 mitogen-activated protein kinase signaling pathway required for innate immunity. ARR-1 activity also controlled immunity through ADF chemosensory and AFD thermosensory neurons that regulate longevity. Furthermore, we found that although ARR-1 played a key role in the control of immunity by AFD thermosensory neurons, it did not control longevity through these cells. However, ARR-1 partially controlled longevity through ADF neurons.     

Mutational analysis of cutaneous squamous cell carcinomas and verrucal keratosis in patients taking BRAF inhibitors

B-RAF inhibitors (BRAFi) have been shown to improve rates of overall and progression-free survival in patients with stage IV metastatic melanoma positive for the BRAF V600E mutation. However, the main drawback is the development of verrucal keratosis (hyperkeratotic papules with verruca-like characteristics with benign histological findings) and cutaneous squamous cell carcinomas (cuSCC). We have found upstream mutations in RAS as well as PIK3CA in both verrucal keratosis and cuSCC. This suggests that verrucal keratosis is an early clinical presentation of cuSCC in patients on BRAFi.     

Development of genomic simple sequence repeat markers for linseed using next-generation sequencing technology

Linseed (Linum usitatissimum L.) is regarded as a cash crop of tomorrow because of the presence of nutraceutically important ??-linolenic acid (ALA) and lignan. However, only limited breeding progress has been made in this crop, mainly due to the lack of sufficient genetic and genomic resources. Among these, simple sequence repeats (SSR) are useful DNA markers for diversity analysis, genetic mapping and tagging traits because of their co-dominant and highly polymorphic nature. In order to develop SSR markers for linseed, we used three microsatellite isolation methods, viz., PCR Isolation of Microsatellite Arrays (PIMA), 5??-anchored PCR method, and Fast Isolation by AFLP of Sequences COntaining repeats (FIASCO). The amplified products from these methods were pooled and sequenced using the 454 GS-FLX platform. A total of 36,332 reads were obtained, which assembled into 2,183 contigs and 2,509 singlets. The contigs and the singlets contained 1,842 microsatellite motifs, with dinucleotide motifs as the most abundant repeat type (54%) followed by trinucleotide motifs (44%). Based on this, 290 SSR markers were designed, 52 of which were evaluated using a panel of 27 diverse linseed genotypes. Among the three enrichment methods, the 5??-anchored PCR method was most efficient for isolation of microsatellites, while FIASCO was most efficient for developing SSR markers. We show the utility of next-generation sequencing technology for efficiently discovering a large number of microsatellite markers in non-model plants.     

Marburg Virus VP35 Can Both Fully Coat the Backbone and Cap the Ends of dsRNA for Interferon Antagonism

Filoviruses, including Marburg virus (MARV) and Ebola virus (EBOV), cause fatal hemorrhagic fever in humans and non-human primates. All filoviruses encode a unique multi-functional protein termed VP35. The C-terminal double-stranded (ds)RNA-binding domain (RBD) of VP35 has been implicated in interferon antagonism and immune evasion. Crystal structures of the VP35 RBD from two ebolaviruses have previously demonstrated that the viral protein caps the ends of dsRNA. However, it is not yet understood how the expanses of dsRNA backbone, between the ends, are masked from immune surveillance during filovirus infection. Here, we report the crystal structure of MARV VP35 RBD bound to dsRNA. In the crystal structure, molecules of dsRNA stack end-to-end to form a pseudo-continuous oligonucleotide. This oligonucleotide is continuously and completely coated along its sugar-phosphate backbone by the MARV VP35 RBD. Analysis of dsRNA binding by dot-blot and isothermal titration calorimetry reveals that multiple copies of MARV VP35 RBD can indeed bind the dsRNA sugar-phosphate backbone in a cooperative manner in solution. Further, MARV VP35 RBD can also cap the ends of the dsRNA in solution, although this arrangement was not captured in crystals. Together, these studies suggest that MARV VP35 can both coat the backbone and cap the ends, and that for MARV, coating of the dsRNA backbone may be an essential mechanism by which dsRNA is masked from backbone-sensing immune surveillance molecules.     

Cardiovascular GO Annotation Initiative Year 1 Report: Why Cardiovascular GO?

Gene Ontology (GO) vocabularies are an established standard for linking functional information to genes and gene products (www.geneontology.org/). A recent collaboration between University College London and the European Bioinformatics Institute is providing GO annotation to human cardiovascular-associated genes (http://www.ucl.ac.uk/medicine/cardiovascular-genetics/geneontology.html). This report outlines the aims of this collaboration and summarizes how the cardiovascular community can help improve the quality and quantity of GO annotations. This new initiative is funded by the British Heart Foundation and fully supported by the GO Consortium.     

Efficient parallel synthesis of macrocyclic peptidomimetics

A new method for solid phase parallel synthesis of chemically and conformationally diverse macrocyclic peptidomimetics is reported. A key feature of the method is access to broad chemical and conformational diversity. Synthesis and mechanistic studies on the macrocyclization step are reported.