A glycolipid of hypervirulent tuberculosis strains that inhibits the innate immune response

Fifty million new infections with Mycobacterium tuberculosis occur annually, claiming 2-3 million lives from tuberculosis worldwide. Despite the apparent lack of significant genetic heterogeneity between strains of M. tuberculosis, there is mounting evidence that considerable heterogeneity exists in molecules important in disease pathogenesis. These differences may manifest in the ability of some isolates to modify the host cellular immune response, thereby contributing to the observed diversity of clinical outcomes. Here we describe the identification and functional relevance of a highly biologically active lipid species-a polyketide synthase-derived phenolic glycolipid (PGL) produced by a subset of M. tuberculosis isolates belonging to the W-Beijing family that show 'hyperlethality' in murine disease models. Disruption of PGL synthesis results in loss of this hypervirulent phenotype without significantly affecting bacterial load during disease. Loss of PGL was found to correlate with an increase in the release of the pro-inflammatory cytokines tumour-necrosis factor-alpha and interleukins 6 and 12 in vitro. Furthermore, the overproduction of PGL by M. tuberculosis or the addition of purified PGL to monocyte-derived macrophages was found to inhibit the release of these pro-inflammatory mediators in a dose-dependent manner.     

Molecular breeding of viruses

Genetic recombination is a major force driving the evolution of many viruses. Recombination between two copackaged retroviral genomes may occur at rates as high as 40% per replication cycle. This enables genetic information to be shuffled rapidly, leading to recombinants with new patterns of mutations and phenotypes. The in vitro process of DNA shuffling (molecular breeding) mimics this mechanism on a vastly parallel and accelerated scale. Multiple homologous parental sequences are recombined in parallel, leading to a diverse library of complex recombinants from which desired improvements can be selected. Different proteins and enzymes have been improved using DNA shuffling. We report here the first application of molecular breeding to viruses. A single round of shuffling envelope sequences from six murine leukaemia viruses (MLV) followed by selection yielded a chimaeric clone with a completely new tropism for Chinese Hamster Ovary (CHOK1) cells. The composition and properties of the selected clone indicated that this particular permutation of parental sequences cannot be readily attained by natural retroviral recombination. This example demonstrates that molecular breeding can enhance the inherently high evolutionary potential of retroviruses to obtain desired phenotypes. It can be an effective tool, when information is limited, to optimize viruses for gene therapy and vaccine applications when multiple complex functions must be simultaneously balanced.     

An inhibitor of Bcl-2 family proteins induces regression of solid tumours

Proteins in the Bcl-2 family are central regulators of programmed cell death, and members that inhibit apoptosis, such as Bcl-X(L) and Bcl-2, are overexpressed in many cancers and contribute to tumour initiation, progression and resistance to therapy. Bcl-X(L) expression correlates with chemo-resistance of tumour cell lines, and reductions in Bcl-2 increase sensitivity to anticancer drugs and enhance in vivo survival. The development of inhibitors of these proteins as potential anti-cancer therapeutics has been previously explored, but obtaining potent small-molecule inhibitors has proved difficult owing to the necessity of targeting a protein-protein interaction. Here, using nuclear magnetic resonance (NMR)-based screening, parallel synthesis and structure-based design, we have discovered ABT-737, a small-molecule inhibitor of the anti-apoptotic proteins Bcl-2, Bcl-X(L) and Bcl-w, with an affinity two to three orders of magnitude more potent than previously reported compounds. Mechanistic studies reveal that ABT-737 does not directly initiate the apoptotic process, but enhances the effects of death signals, displaying synergistic cytotoxicity with chemotherapeutics and radiation. ABT-737 exhibits single-agent-mechanism-based killing of cells from lymphoma and small-cell lung carcinoma lines, as well as primary patient-derived cells, and in animal models, ABT-737 improves survival, causes regression of established tumours, and produces cures in a high percentage of the mice.     

Antiproliferative effect of β-elemene in chemoresistant ovarian carcinoma cells is mediated through arrest of the cell cycle at the G2-M phase

Elemene is a natural antitumor plant drug. However, the effect of elemene on cell growth in ovarian cancer is unknown. In this study, we show that -elemene inhibited the proliferation of cisplatin-resistant human ovarian cancer cells and their parental cells, but had only a marginal effect in human ovary cells, indicating differential inhibitory effects on cell growth between ovarian cancer cells and normal ovary cells. We also demonstrated for the first time that -elemene markedly enhanced cisplatin-induced growth inhibition in resistant cells compared to sensitive cells. In addition, cell cycle analysis revealed a synergistic effect of -elemene and cisplatin on the induction of cell cycle G2-M arrest in our resistant ovarian carcinoma cells. Furthermore, we showed that treatment of these cells with both drugs downregulated cyclin B1 and Cdc2 expression, but elevated the levels of p53, p21waf1/cip1, p27kip1 and Gadd45. Finally, the combination of -elemene and cisplatin was found to increase the phosphorylation of Cdc2 and Cdc25C, which leads to a reduction in Cdc2-cyclin B1 activity. These novel findings suggest that -elemene sensitizes chemoresistant ovarian carcinoma cells to cisplatin-induced growth suppression partly through modulating the cell cycle G2 checkpoint and inducing cell cycle G2-M arrest, which lead to blockade of cell cycle progression.Received 19 January 2005; accepted 5 February 2005     

Non-apoptotic role of BID in inflammation and innate immunity

Innate immunity is a fundamental defence response that depends on evolutionarily conserved pattern recognition receptors for sensing infections or danger signals. Nucleotide-binding and oligomerization domain (NOD) proteins are cytosolic pattern-recognition receptors of paramount importance in the intestine, and their dysregulation is associated with inflammatory bowel disease. They sense peptidoglycans from commensal microorganisms and pathogens and coordinate signalling events that culminate in the induction of inflammation and anti-microbial responses. However, the signalling mechanisms involved in this process are not fully understood. Here, using genome-wide RNA interference, we identify candidate genes that modulate the NOD1 inflammatory response in intestinal epithelial cells. Our results reveal a significant crosstalk between innate immunity and apoptosis and identify BID, a BCL2 family protein, as a critical component of the inflammatory response. Colonocytes depleted of BID or macrophages from Bid(-/-) mice are markedly defective in cytokine production in response to NOD activation. Furthermore, Bid(-/-) mice are unresponsive to local or systemic exposure to NOD agonists or their protective effect in experimental colitis. Mechanistically, BID interacts with NOD1, NOD2 and the IκB kinase (IKK) complex, impacting NF-κB and extracellular signal-regulated kinase (ERK) signalling. Our results define a novel role of BID in inflammation and immunity independent of its apoptotic function, furthering the mounting evidence of evolutionary conservation between the mechanisms of apoptosis and immunity.     

Effects of habitat type and degradation on avian species richness in Great Basin riparian habitats

The overwhelming majority of bird species in the Great Basin region are found in riparian habitats. However, most previous research on the impact of change in habitat condition through degradation on these bird communities failed to account for the large intersite differences, in both habitat type and extent of degradation. We examined songbird communities in 4 riparian habitat types (meadows, willow-birch-, and aspen-dominated forest stands) during summers 1994 (last year of a 7-yr drought) and 1995 (following the 6th wettest winter recorded) in the Toiyabe Mountain Range of central Nevada. Habitat degradation significantly influenced bird species richness in riparian areas, but the impact was dependent upon habitat type. While meadow bird communities were affected adversely by habitat degradation, with significant drops in species richness on degraded sites, bird species richness in forested riparian habitats was consistently greater on degraded sites. Data for the 6 most common species seen during our study indicated that degradation may have influenced distribution of American Robins ( Turdus migratorius ) and Yellow Warblers ( Dendroica petechia ), but habitat type was the best predictor of abundance for House Wrens ( Troglodytes aedon ), Red-naped Sapsuckers ( Sphyrapicus nuchalis ), Warbling Vircos ( Vireo gilvus ), and Brewer's Blackbirds ( Euphagus cyanocephalus ). Avian species diversity in meadow habitats may be linked to moisture levels during specific times of the year. Diversity increased during the pre-migratory period of the dry year (1994) when compared with that of the breeding season, but was unchanged in the wet year (1995).