Part I. Bcl-2 and Bcl-x(L) limit apoptosis upon infection with alphavirus vectors

Viral expression systems offer the ability to generate high levels of a particular protein within a relatively short period of time. In particular, alphavirus constructs based on Sindbis virus (SV) and Semliki Forest virus (SFV) are promising vehicles as they are cytoplasmic vectors with the potential for high expression levels. Two such alphavirus vectors were utilized during the current study to infect two commercially relevant cell lines, baby hamster kidney (BHK) and Chinese hamster ovary (CHO); the first was a fully competent SV derivative carrying the gene for chloramphenicol acetyltransferase (dsSV-CAT), while the second was a replication deficient SFV construct containing the human interleukin-12 (IL-12) p35 and p40 genes (SFV-IL-12). Since infection with these vectors induced apoptosis in both cell lines, the present effort was dedicated to determining the ability of anti-apoptosis genes to limit the cell death associated with these virus constructs. Infection with the dsSV-CAT vector resulted in the rapid death of BHK and CHO cells within 4 days, a phenomenon which was considerably delayed by stably overexpressing bcl-2 or bcl-x(L). In fact, cellular lifespans were doubled in both BHK-bcl2 and CHO-bclx(L) cells relative to the parental cell lines. Furthermore, the presence of these gene products provided increases of up to 2-fold in recombinant CAT production. Overexpression of bcl-2 and bcl-x(L) also altered the response of these cells upon infection with SFV-IL-12. While the parental cell lines were completely nonviable within 1 week, the BHK-bcl2, BHK-bclx(L), and CHO-bclx(L) cells each recovered from the infection, resuming exponential growth and regaining viabilities of over 90% by 9 days post-infection. Total IL-12 productivities were nearly doubled by Bcl-2 and Bcl-x(L) in the CHO cells, although this effect was apparently cell-line specific, as the native BHK cells were able to secrete more IL-12 than either of its transfected derivatives. Regardless, the presence of the anti-apoptosis genes allowed the production of IL-12 to be maintained, albeit at low levels, from each of the cell lines for the duration of the culture process. Therefore, overexpression of bcl-2 family members can have a significant impact on culture viabilities and recombinant protein production during alphavirus infections of mammalian cells.     

Mucosal challenge of Macaca nemestrina with simian immunodeficiency virus (SIV) following SIV nucleocapsid mutant DNA vaccination

A simian immunodeficiency virus (SIV)(Mne) DNA clone was constructed that produces viruses containing a four amino acid deletion in the second zinc finger of the nucleocapsid (NC) domain of the Gag polyprotein. Viruses produced from this clone, although non-infectious both in vitro and in vivo, complete a majority of the steps in a single retroviral infection cycle. Eight pig-tailed macaques (Macaca nemestrina) were inoculated intramuscularly and subcutaneously three times over the course of 24 weeks with the NC mutant expressing DNA. These macaques, and four controls, were then challenged mucosally (intrarectally) with the homologous virus (SIV Mne CL E11S) and monitored for evidence of infection and clinical disease. Prior to challenge, a measurable humoral immune response was noted in four of eight immunized macaques. After challenge, all 12 macaques became infected, although four immunized animals greatly restricted their viral replication, and one immunized animal that controlled replication remains antibody negative. No disease has been evidence during the 46-week period of monitoring after challenge.     

Inhibition of drug-induced Fas ligand transcription and apoptosis by Bcl-XL.

Fas/Fas ligand system triggers apoptosis in many cell types. Bcl-XL overexpresion antagonizes Fas/Fas ligand-mediated cell death. The mechanism by which Bcl-XL influences Fas-mediated cell death is unclear. We have found that microtubule-damaging drugs (e.g. Paclitaxel) induce apoptosis in a Fas/FasL-dependent manner. Inhibition of Fas/FasL pathway by anti-FasL antibody, mutant Fas or a dominant negative FADD blocks paclitaxel-induced apoptosis. Paclitaxel induced apoptosis through activation of both caspase-8 and caspase-3. Overexpression of Bcl-XL leads to inhibition of paclitaxel-induced FasL expression and apoptosis. Bcl-XL prevents the nuclear translocation of NFAT (nuclear factor of activated T lymphocytes) by inhibiting the activation of calcineurin, a calcium-dependent phosphatase that must dephosphorylate NFAT for it to move to the nucleus. The loop domain in Bcl-XL can suppress the anti-apoptotic function of Bcl-XL and may be a target for regulatory post-translational modifications. Upon phosphorylation, Bcl-XL loses its ability to bind with calcineurin. Without NFAT nuclear translocation, the FasL gene is not transcribed. Thus, paclitaxel and other drugs that disturb microtubule function kill cells, at least in part, through the induction of FasL, and Bcl-XL-mediated resistance to these agents is related to failure to induce FasL expression.     

Specificity in the symbiotic association between fungus-growing ants and protective Pseudonocardia bacteria

Fungus-growing ants (tribe Attini) engage in a mutualism with a fungus that serves as the ants' primary food source, but successful fungus cultivation is threatened by microfungal parasites (genus Escovopsis). Actinobacteria (genus Pseudonocardia) associate with most of the phylogenetic diversity of fungus-growing ants; are typically maintained on the cuticle of workers; and infection experiments, bioassay challenges and chemical analyses support a role of Pseudonocardia in defence against Escovopsis through antibiotic production. Here we generate a two-gene phylogeny for Pseudonocardia associated with 124 fungus-growing ant colonies, evaluate patterns of ant-Pseudonocardia specificity and test Pseudonocardia antibiotic activity towards Escovopsis. We show that Pseudonocardia associated with fungus-growing ants are not monophyletic: the ants have acquired free-living strains over the evolutionary history of the association. Nevertheless, our analysis reveals a significant pattern of specificity between clades of Pseudonocardia and groups of related fungus-growing ants. Furthermore, antibiotic assays suggest that despite Escovopsis being generally susceptible to inhibition by diverse Actinobacteria, the ant-derived Pseudonocardia inhibit Escovopsis more strongly than they inhibit other fungi, and are better at inhibiting this pathogen than most environmental Pseudonocardia strains tested. Our findings support a model that many fungus-growing ants maintain specialized Pseudonocardia symbionts that help with garden defence.     

Interrogation of the protein-protein interactions between human BRCA2 BRC repeats and RAD51 reveals atomistic determinants of affinity

The breast cancer suppressor BRCA2 controls the recombinase RAD51 in the reactions that mediate homologous DNA recombination, an essential cellular process required for the error-free repair of DNA double-stranded breaks. The primary mode of interaction between BRCA2 and RAD51 is through the BRC repeats, which are ～35 residue peptide motifs that interact directly with RAD51 in vitro. Human BRCA2, like its mammalian orthologues, contains 8 BRC repeats whose sequence and spacing are evolutionarily conserved. Despite their sequence conservation, there is evidence that the different human BRC repeats have distinct capacities to bind RAD51. A previously published crystal structure reports the structural basis of the interaction between human BRC4 and the catalytic core domain of RAD51. However, no structural information is available regarding the binding of the remaining seven BRC repeats to RAD51, nor is it known why the BRC repeats show marked variation in binding affinity to RAD51 despite only subtle sequence variation. To address these issues, we have performed fluorescence polarisation assays to indirectly measure relative binding affinity, and applied computational simulations to interrogate the behaviour of the eight human BRC-RAD51 complexes, as well as a suite of BRC cancer-associated mutations. Our computational approaches encompass a range of techniques designed to link sequence variation with binding free energy. They include MM-PBSA and thermodynamic integration, which are based on classical force fields, and a recently developed approach to computing binding free energies from large-scale quantum mechanical first principles calculations with the linear-scaling density functional code onetep. Our findings not only reveal how sequence variation in the BRC repeats directly affects affinity with RAD51 and provide significant new insights into the control of RAD51 by human BRCA2, but also exemplify a palette of computational and experimental tools for the analysis of protein-protein interactions for chemical biology and molecular therapeutics.     

Large-scale candidate gene analysis of HDL particle features

Background

HDL cholesterol (HDL-C) is an established marker of cardiovascular risk with significant genetic determination. However, HDL particles are not homogenous, and refined HDL phenotyping may improve insight into regulation of HDL metabolism. We therefore assessed HDL particles by NMR spectroscopy and conducted a large-scale candidate gene association analysis.

Methodology/Principal Findings

We measured plasma HDL-C and determined mean HDL particle size and particle number by NMR spectroscopy in 2024 individuals from 512 British Caucasian families. Genotypes were 49,094 SNPs in >2,100 cardiometabolic candidate genes/loci as represented on the HumanCVD BeadChip version 2. False discovery rates (FDR) were calculated to account for multiple testing. Analyses on classical HDL-C revealed significant associations (FDR<0.05) only for CETP (cholesteryl ester transfer protein; lead SNP rs3764261: p = 5.6*10⁻¹⁵) and SGCD (sarcoglycan delta; rs6877118: p = 8.6*10⁻⁶). In contrast, analysis with HDL mean particle size yielded additional associations in LIPC (hepatic lipase; rs261332: p = 6.1*10⁻⁹), PLTP (phospholipid transfer protein, rs4810479: p = 1.7*10⁻⁸) and FBLN5 (fibulin-5; rs2246416: p = 6.2*10⁻⁶). The associations of SGCD and Fibulin-5 with HDL particle size could not be replicated in PROCARDIS (n = 3,078) and/or the Women''s Genome Health Study (n = 23,170).

Conclusions

We show that refined HDL phenotyping by NMR spectroscopy can detect known genes of HDL metabolism better than analyses on HDL-C.     

Microbial community composition in sediments resists perturbation by nutrient enrichment

Functional redundancy in bacterial communities is expected to allow microbial assemblages to survive perturbation by allowing continuity in function despite compositional changes in communities. Recent evidence suggests, however, that microbial communities change both composition and function as a result of disturbance. We present evidence for a third response: resistance. We examined microbial community response to perturbation caused by nutrient enrichment in salt marsh sediments using deep pyrosequencing of 16S rRNA and functional gene microarrays targeting the nirS gene. Composition of the microbial community, as demonstrated by both genes, was unaffected by significant variations in external nutrient supply in our sampling locations, despite demonstrable and diverse nutrient-induced changes in many aspects of marsh ecology. The lack of response to external forcing demonstrates a remarkable uncoupling between microbial composition and ecosystem-level biogeochemical processes and suggests that sediment microbial communities are able to resist some forms of perturbation.     

Evidence that the species barrier of human immunodeficiency virus-1 does not extend to uptake by the blood--brain barrier: comparison of mouse and human brain microvessels

HIV-1 within the CNS produces a neuroAIDS syndrome and may act as a reservoir for reinfection of the peripheral tissues. Study of how HIV-1 crosses the blood-brain barrier (BBB) has been hampered by the lack of nonprimate animal models. However, BBB transport of HIV-1 does not involve any of the known steps conferring species specificity, including binding to CD4 receptors. In vivo and in vitro studies show that HIV-1 and its glycoprotein coat, gp120, are taken up and transported across the BBB of the mouse. Here, we compared the ability of gp120 and HIV-1 to be taken up by isolated brain microvessels (IBM) freshly isolated from mice, from post-mortem human brain, and from mice that had been treated in a manner analogous to the human material (mouse post-mortem). Freshly isolated mouse IBM took up more gp120 and HIV-1 than the human or mouse post-mortem cells. We found no difference between the ability of mouse post-mortem and human IBM to take up either gp120 or HIV-1. Wheatgerm agglutinin has been previously shown to stimulate gp120 and HIV-1 uptake by the BBB; here, it stimulated the uptake of gp120 and of HIV-1 by both mouse post-mortem and human IBM, although stimulated uptake was greatest for fresh mouse IBM. These results show that the mouse can be used to study the initial phases of HIV-1 uptake by the BBB.