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Supplemental Digital Content is available in the text.Bigelovin, a sesquiterpene lactone extracted from plant Inula helianthus aquatica, exhibited multiple interesting biological activities, including anti-inflammation, antiangiogenesis and cytotoxic action against cancer cells. In the present study, we found that Bigelovin reduced the viability of human colon cancer cells and induced their apoptosis in a time- and dose-dependent manner, with an IC50–5 μM. RNAseq and luciferase reporter analyses revealed that the nuclear factor kappa B (NF-κB) signaling was one of the most significantly inhibited pathways after Bigelovin treatment. Further systemic examination showed that exposure to Bigelovin resulted in ubiquitination and degradation of inhibitor of kappa-B kinase-beta (IKK-β) and decrease of IκB-α and p65 phosphorylation, which led to the downregulation of NF-κB-regulated genes expression. Moreover, enforced expression of exogenous IKK-β attenuated Bigelovin-induced NF-κB suppression and cell viability reduction. These results indicated that Bigelovin exerts a cytotoxic action against colon cancer cells through the induction of IKK-β degradation and consequently the inhibition of NF-κB signaling. Given the abnormal activation of NF-κB signaling in colorectal cancer (CRC) cells and the critical role of chronic inflammation in CRC development, it is conceivable that at least some colorectal cancer cells are addictive to NF-κB activation and targeting the pathway is an effective anti-CRC strategy.

The article discusses the applicability of the high-throughput screening approach, avidity-based extracellular interaction screen (AVEXIS) for identifying the various weak extracellular protein interactions. The analysis has been extremely useful in the development of new therapeutic techniques.

The degradation of mRNA is an important regulatory step in the control of gene expression. The level of each mRNA in a cell is a tightly regulated function of its rate of transcription, processing and degradation.

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The MAGE (Melanoma-associated antigen) protein family members are structurally related to each other by a MAGE-homology domain comprised of 2 winged helix motifs WH/A and WH/B. This family specifically evolved in placental mammals although single homologs designated NSE3 (non-SMC element) exist in most eukaryotes. NSE3, together with its partner proteins NSE1 and NSE4 form a tight subcomplex of the structural maintenance of chromosomes SMC5–6 complex. Previously, we showed that interactions of the WH/B motif of the MAGE proteins with their NSE4/EID partners are evolutionarily conserved (including the MAGEA1-NSE4 interaction). In contrast, the interaction of the WH/A motif of NSE3 with NSE1 diverged in the MAGE paralogs. We hypothesized that the MAGE paralogs acquired new RING-finger-containing partners through their evolution and form MAGE complexes reminiscent of NSE1-NSE3-NSE4 trimers. In this work, we employed the yeast 2-hybrid system to screen a human RING-finger protein library against several MAGE baits. We identified a number of potential MAGE-RING interactions and confirmed several of them (MDM4, PCGF6, RNF166, TRAF6, TRIM8, TRIM31, TRIM41) in co-immunoprecipitation experiments. Among these MAGE-RING pairs, we chose to examine MAGEA1-TRIM31 in detail and showed that both WH/A and WH/B motifs of MAGEA1 bind to the coiled-coil domain of TRIM31 and that MAGEA1 interaction stimulates TRIM31 ubiquitin-ligase activity. In addition, TRIM31 directly binds to NSE4, suggesting the existence of a TRIM31-MAGEA1-NSE4 complex reminiscent of the NSE1-NSE3-NSE4 trimer. These results suggest that MAGEA1 functions as a co-factor of TRIM31 ubiquitin-ligase and that the TRIM31-MAGEA1-NSE4 complex may have evolved from an ancestral NSE1-NSE3-NSE4 complex.

One of the greatest challenges of the post-genomic era is the construction of a more comprehensive human protein interaction map. While this process may take many years to complete, the development of stringent high throughput techniques and the emergence of complementary assays mean that the aim of building a detailed binary map of the human interactome is now a very realistic goal. In particular, methods which facilitate the analysis of large numbers of membrane-protein interactions mean that it will be possible to construct more extensive networks, which in turn provide new insights into the functional connectivity between intra- and extra-cellular processes. This is important as many therapeutic strategies are designed to elicit effects via ‘tractable’ cell-surface proteins. Therefore, the construction of maps depicting the complexity of trans-cellular communication networks will not only improve our understanding of physiological processes, it will also aid the design of rational therapeutic strategies, with fewer potential side effects. This review aims to provide a basic insight into the approaches currently being used to construct binary human protein interaction networks, with particular reference to newer techniques, which have the potential to extend network coverage and aid the conditional annotation of interactome-scale protein interaction maps.

Yeast two-hybrid analysis is a valuable approach to the discovery and characterization of protein interactions. We have developed vectors that can indicate the presence of an insert when used in two-hybrid bait and prey construction by gap repair cloning. The strategy uses a recombination cloning site flanked by sequences encoding the GAL4 activation and binding domains. After gap repair cloning in standard hosts carrying an ADE2 reporter gene, disruption of GAL4 by an insert can be identified by the development of red colony color, while empty vector plasmids produce white colonies. Function in yeast two-hybrid applications was initially validated using known interacting proteins in pair-wise analyses, and subsequently, the bait vectors were used in library screens with the mouse Mad212 and human Mccd1 proteins, identifying a number of putative new interactions for these proteins. These vectors should facilitate high-throughput yeast two-hybrid screens in which large numbers of bait and prey constructs may be required.

The inhibitory κB (IκB)-like (IκBL) gene is located within the Class III region of the MHC on human chromosome 6. Previous analysis of the predicted amino acid sequence of the human IκBL protein revealed three putative functional domains; 2–3 ankyrin repeat sequences, which are similar to the second and third ankyrin repeats of the nuclear factor κB (NF-κB) protein; three PEST sequence motifs (a sequence that is rich in proline, serine, aspartic acid and threonine residues), which are also found in other IκB family members; and a C-terminal leucine zipper-like motif. In the present study we have identified a novel bipartite motif, which is required for nuclear localization of the IκBL protein. Analyses of IκBL-specific transcripts revealed the existence of a widely expressed spliced variant form of IκBL (IκBLsv1), which lacks the amino acid sequence GELEDEWQEVMGRFE (where single-letter amino-acid notation has been used). Interestingly, translation of IκBL mRNA in vivo was found to initiate predominantly from the second available methionine, thereby resulting in the disruption of the predicted N-terminal PEST sequence. Also, transient expression of T7 epitope-tagged IκBL and IκBLsv1 proteins in mammalian cells showed that both proteins were targeted to the nucleus, where they accumulate in nuclear speckles. To define the protein domains required for nuclear import and subnuclear localization, a complementary set of deletion mutants and enhanced green fluorescent protein—IκBL domain fusions were expressed in mammalian cells. Data from these experiments show that a combination of the ankyrin-repeat region and an adjacent arginine-rich sequence are necessary and sufficient for both nuclear import and speckle localization.

The consequence of redirecting the vaccinia virus (VV) B5R protein to the endoplasmic reticulum (ER) has been investigated by the addition of an ER retrieval signal KKSL (K2X2) to the B5R C-terminus. This mutant B5R gene and a version of the gene with the inactive ER retrieval sequence KKSLAL (K2X4) were inserted into the thymidine kinase locus of a VV mutant lacking the B5R gene, vΔB5R. Similar levels of B5R protein were made by each virus, but the B5R-K2X2protein remained sensitive to endoglycosidase H and colocalised with protein disulphide isomerase in the ER. In contrast, the B5R-K2X4protein colocalised with 1,4-galactosyltransferase in the trans-Golgi network. Electron microscopy revealed that even when the B5R protein was redirected to the ER, intracellular mature virus particles were wrapped by cellular membranes to form intracellular enveloped virus particles, although more incompletely wrapped particles were evident compared with wild type. These intracellular enveloped virus particles were, however, unable to efficiently induce the polymerisation of actin and the plaque size formed by vB5R-K2X2was small. Nevertheless, the amount and specific infectivity of EEV produced by vB5R-K2X2were similar to those of wild type, despite the dramatic reduction in the amount of B5R protein present in vB5R-K2X2EEV.

ABSTRACTVaccinia virus (VV) induces two forms of cell motility: cell migration, which is dependent on the expression of early genes, and the formation of cellular projections, which requires the expression of late genes. The need for viral gene expression prior to cell motility suggests that VV proteins may affect how infected cells interact with the extracellular matrix. To address this, we have analyzed changes in cell-matrix adhesion after infection of BS-C-1 cells with VV. Whereas uninfected cells round up and detach from the culture flask in the presence of EGTA, infected cells remain attached to the culture flask with a stellate morphology. Ca2+-independent cell-matrix adhesion was evident by 10 h postinfection, after the onset of cell motility but before the formation of virus-induced cellular projections. Progression to Ca2+-independent adhesion required the expression of late viral genes but not the formation of intracellular enveloped virus particles or intracellular actin tails. Analyses of specific matrix proteins identified vitronectin and fibronectin as optimal ligands for Ca2+-independent adhesion and the formation of cellular projections. Adhesion to fibronectin was mediated via RGD motifs alone and was not inhibited by 500 µg of heparin/ml. Kistrin, a disintegrin which binds preferentially to the avß3 (vitronectin/fibronectin) receptor inhibited the formation of cellular projections without disrupting preformed matrix interactions. Finally, we show that Ca2+-independent cell-matrix adhesion is a dynamic process which mediates changes in the morphology of VV-infected cells and uninfected cells which exhibit a transformed phenotype.

The vaccinia virus gene A38L encodes a highly hydrophobic protein with amino acid similarity to mammalian integrin-associated protein (IAP). In this report we have identified the A38L protein of strain Western Reserve (WR), defined its membrane topology, and analyzed its role in virus production and virulence. An antiserum raised against an A38L peptide identified the A38L gene product as a 33-kDa protein which is expressed at low levels during virus infection. A serum from a rabbit previously infected with WR virus recognized the A38L protein, thus confirming that the A38L gene is expressedin vivo.Using a coupledin vitro-translation/membrane-translocation system the 33-kDa protein was shown to be a membrane-associated and glycosylated form of a 29-kDa polypeptide precursor. The membrane topology of the A38L protein was defined by its glycosylation and protease sensitivity when associated with microsomal membranes. The N-terminal immunoglobulin-like variable domain was protected from exogenous protease and was therefore in the lumen of the vesicle, whereas the C-terminus was sensitive and therefore cytoplasmic. A38L deletion and revertant viruses were constructed and were used to study the involvement of A38L in virus assembly, release, and virulence. Deletion of the A38L gene caused a slight reduction in virus plaque size but did not affect the production of intracellular mature virus or extracellular enveloped virus particles in tissue culture cells nor the virulence of the virus in the murine intranasal model. The A38L protein therefore possesses similar sequence and membrane topology to the mammalian IAP protein but is not required for virus particle production or virulence.

Three patients with anomalous left main coronary artery arising from the pulmonary artery were treated. The subclavian artery was used to construct a two coronary system. Intraoperative blood flow studies demonstrated increased total blood flow to the myocardium in all cases. Preoperative and postoperative angiograms demonstrated growth of the vessels and anastomoses, which confirmed previous findings from our laboratory.

ABSTRACTMany viruses induce profound changes in cell metabolism and function. Here we show that vaccinia virus induces two distinct forms of cell movement. Virus-induced cell migration was demonstrated by an in vitro wound healing assay in which infected cells migrated independently into the wound area while uninfected cells remained relatively static. Time-lapse microscopy showed that the maximal rate of migration occurred between 9 and 12 h postinfection. Virus-induced cell migration was inhibited by preinactivation of viral particles with trioxsalen and UV light or by the addition of cycloheximide but not by addition of cytosine arabinoside or rifampin. The expression of early viral genes is therefore necessary and sufficient to induce cell migration. Following migration, infected cells developed projections up to 160 µm in length which had growth-cone-like structures and were frequently branched. Time-lapse video microscopy showed that these projections were formed by extension and condensation of lamellipodia from the cell body. Formation of extensions was dependent on late gene expression but not the production of intracellular enveloped (IEV) particles. The requirements for virus-induced cell migration and for the formation of extensions therefore differ from each other and are distinct from the polymerization of actin tails on IEV particles. These data show that poxviruses encode genes which control different aspects of cell motility and thus represent a useful model system to study and dissect cell movement.

ABSTRACTVaccinia virus produces two morphologically distinct forms of infectious virus, termed intracellular mature virus (IMV) and extracellular enveloped virus (EEV). EEV is important for virus dissemination within a host and has different surface proteins which bind to cell receptors different from those used by IMV. Six genes are known to encode EEV-specific proteins. One of these, B5R, encodes a 42-kDa glycoprotein with amino acid similarity to members of the complement control protein superfamily and contains four copies of a 50- to 70-amino-acid repeat called the short consensus repeat (SCR). Deletion of B5R causes a small-plaque phenotype, a 10-fold reduction in EEV formation, and virus attenuation in vivo. In this study, we inserted mutated versions of the B5R gene lacking different combinations of the SCRs into a virus deletion mutant lacking the B5R gene. The resultant viruses each formed small plaques only slightly larger than those of the deletion mutant; however, the virus containing only SCR 1 formed plaques slightly larger than those of viruses with SCRs 1 and 2 or SCRs 1, 2, and 3. All of these viruses produced approximately 50-fold more infectious EEV than wild-type virus and formed comet-shaped plaques under liquid overlay. Despite producing more EEV, the mutant viruses were unable to induce the polymerization of actin on intracellular virus particles. The implications of these results for our understanding of EEV formation, release, and infectivity are discussed.

ABSTRACT A breath‐test has been developed for the detection of gastric infection with Campylobacter pylori.Urea that is labelled with carbon 14 is administered to a fasting patient and the patient's breath is sampled for radioactivity over the following 30 minutes. If C. pyloriis present in the patient's stomach, urease activity causes hydrolysis of the urea and the 14C is absorbed as carbon dioxide. This carbon dioxide enters the patient's bicarbonate pool and eventually is excreted in the breath. The results are expressed as a percentage of the administered dose/mmol carbon dioxide x kg body weight. Sixty‐three patients who were undergoing endoscopy were studied. The radioactivity in exhaled breath which was sampled within five minutes of 14C‐urea administration was attributed to the presence of urease enzyme in mouth organisms and was discounted. The time–radioactivity curves for breath samples from five to 30 minutes after the administration of 14C‐urea gave an excellent separation between subjects with negative results of the examination of gastric‐biopsy samples and patients with microbiological and histological evidence of infection with C. pylori.The area under the time–radioactivity curve at between five and 30 minutes after the administration of 14C‐urea in 24 patients with negative microbiological results was 6.9±4.4 area units; in 35 of 39 patients with positive microbiological results, this area was greater than 40 area units. Measured against the results of the microbiological examination of gastric‐biopsy samples, the sensitivity of breath‐testing was 90% and the specificity was 100%. Measured against the results of histological examination for the presence of C. pyloriinfection, breath‐testing had a sensitivity of 94% and a specificity of 93%. A positive breath‐test result also correlated well (P=0.0001) with the serological antibody test‐result. The role of non‐invasive tests — enzyme‐linked immunosorbent assays and 14C‐urea breath‐testing — in the management of gastritis and peptic ulcer disease is discussed. We consider that the 14C‐urea breath‐test has an important role in the noninvasive confirmation of gastric infection with C. pyloriand in the follow‐up of patients after treatment.