Test System Stability and Natural Variability of a Lemna Gibba L. Bioassay

BACKGROUND: In ecotoxicological and environmental studies Lemna spp. are used as test organisms due to their small size, rapid predominantly vegetative reproduction, easy handling and high sensitivity to various chemicals. However, there is not much information available concerning spatial and temporal stability of experimental set-ups used for Lemna bioassays, though this is essential for interpretation and reliability of results. We therefore investigated stability and natural variability of a Lemna gibba bioassay assessing area-related and frond number-related growth rates under controlled laboratory conditions over about one year. METHODOLOGY/PRINCIPAL FINDINGS: Lemna gibba L. was grown in beakers with Steinberg medium for one week. Area-related and frond number-related growth rates (r(area) and r(num)) were determined with a non-destructive image processing system. To assess inter-experimental stability, 35 independent experiments were performed with 10 beakers each in the course of one year. We observed changes in growth rates by a factor of two over time. These did not correlate well with temperature or relative humidity in the growth chamber. In order to assess intra-experimental stability, we analysed six systematic negative control experiments (nontoxicant tests) with 96 replicate beakers each. Evaluation showed that the chosen experimental set-up was stable and did not produce false positive results. The coefficient of variation was lower for r(area) (2.99%) than for r(num) (4.27%). CONCLUSIONS/SIGNIFICANCE: It is hypothesised that the variations in growth rates over time under controlled conditions are partly due to endogenic periodicities in Lemna gibba. The relevance of these variations for toxicity investigations should be investigated more closely. Area-related growth rate seems to be more precise as non-destructive calculation parameter than number-related growth rate. Furthermore, we propose two new validity criteria for Lemna gibba bioassays: variability of average specific and section-by-section segmented growth rate, complementary to average specific growth rate as the only validity criterion existing in guidelines for duckweed bioassays

An Epstein-Barr Virus Anti-Apoptotic Protein Constitutively Expressed in Transformed Cells and Implicated in Burkitt Lymphomagenesis: The Wp/BHRF1 Link

Two factors contribute to Burkitt lymphoma (BL) pathogenesis, a chromosomal translocation leading to c-myc oncogene deregulation and infection with Epstein-Barr virus (EBV). Although the virus has B cell growth–transforming ability, this may not relate to its role in BL since many of the transforming proteins are not expressed in the tumor. Mounting evidence supports an alternative role, whereby EBV counteracts the high apoptotic sensitivity inherent to the c-myc–driven growth program. In that regard, a subset of BLs carry virus mutants in a novel form of latent infection that provides unusually strong resistance to apoptosis. Uniquely, these virus mutants use Wp (a viral promoter normally activated early in B cell transformation) and express a broader-than-usual range of latent antigens. Here, using an inducible system to express the candidate antigens, we show that this marked apoptosis resistance is mediated not by one of the extended range of EBNAs seen in Wp-restricted latency but by Wp-driven expression of the viral bcl2 homologue, BHRF1, a protein usually associated with the virus lytic cycle. Interestingly, this Wp/BHRF1 connection is not confined to Wp-restricted BLs but appears integral to normal B cell transformation by EBV. We find that the BHRF1 gene expression recently reported in newly infected B cells is temporally linked to Wp activation and the presence of W/BHRF1-spliced transcripts. Furthermore, just as Wp activity is never completely eclipsed in in vitro–transformed lines, low-level BHRF1 transcripts remain detectable in these cells long-term. Most importantly, recognition by BHRF1-specific T cells confirms that such lines continue to express the protein independently of any lytic cycle entry. This work therefore provides the first evidence that BHRF1, the EBV bcl2 homologue, is constitutively expressed as a latent protein in growth-transformed cells in vitro and, in the context of Wp-restricted BL, may contribute to virus-associated lymphomagenesis in vivo

Restricted Expression of Epstein-Barr Virus Latent Genes in Murine B Cells Derived from Embryonic Stem Cells

Background: Several human malignancies are associated with Epstein-Barr virus (EBV) and more than 95 % of the adult human population carries this virus lifelong. EBV efficiently infects human B cells and persists in this cellular compartment latently. EBV-infected B cells become activated and growth transformed, express a characteristic set of viral latent genes, and acquire the status of proliferating lymphoblastoid cell lines in vitro. Because EBV infects only primate cells, it has not been possible to establish a model of infection in immunocompetent rodents. Such a model would be most desirable in order to study EBV’s pathogenesis and latency in a suitable and amenable host. Methodology/Principal Findings: We stably introduced recombinant EBV genomes into mouse embryonic stem cells and induced their differentiation to B cells in vitro to develop the desired model. In vitro differentiated murine B cells maintained the EBV genomes but expression of viral genes was restricted to the latent membrane proteins (LMPs). In contrast to human B cells, EBV’s nuclear antigens (EBNAs) were not expressed detectably and growth transformed murine B cells did not arise in vitro. Aberrant splicing and premature termination of EBNA mRNAs most likely prevented the expression of EBNA genes required for B-cell transformation. Conclusions/Significance: Our findings indicate that fundamental differences in gene regulation between mouse and ma

Intronically encoded siRNAs improve dynamic range of mammalian gene regulation systems and toggle switch

Applications of conditional gene expression, whether for therapeutic or basic research purposes, are increasingly requiring mammalian gene control systems that exhibit far tighter control properties. While numerous approaches have been used to improve the widely used Tet-regulatory system, many applications, particularly with respect to the engineering of synthetic gene networks, will require a broader range of tightly performing gene control systems. Here, a generically applicable approach is described that utilizes intronically encoded siRNA on the relevant transregulator construct, and siRNA sequence-specific tags on the reporter construct, to minimize basal gene activity in the off-state of a range of common gene control systems. To demonstrate tight control of residual expression the approach was successfully used to conditionally express the toxic proteins RipDD and Linamarase. The intronic siRNA concept was also extended to create a new generation of compact, single-vector, autoinducible siRNA vectors. Finally, using improved regulation systems a mammalian epigenetic toggle switch was engineered that exhibited superior in vitro and in vivo induction characteristics in mice compared to the equivalent non-intronic system

NIR, an inhibitor of histone acetyltransferases, regulates transcription factor TAp63 and is controlled by the cell cycle

p63 is a sequence-specific transcription factor that regulates epithelial stem cell maintenance and epithelial differentiation. In addition, the TAp63 isoform with an N-terminal transactivation domain functions as an inducer of apoptosis during the development of sympathetic neurons. Previous work has indicated that the co-activator and histone acetyltransferase (HAT), p300, can bind to TAp63 and stimulate TAp63-dependent transcription of the p21Cip1 gene. Novel INHAT Repressor (NIR) is an inhibitor of HAT. Here, we report that the central portion of NIR binds to the transactivation domain and the C-terminal oligomerization domain of TAp63. NIR is highly expressed in G2/M phase of the cell cycle and only weakly expressed in G1/S. Furthermore, except during mitosis, NIR is predominantly localized in the nucleolus; only a small portion co-localizes with TAp63 in the nucleoplasm and at the p21 gene promoter. Consistent with NIR acting as a repressor, the induced translocation of NIR from the nucleolus into the nucleoplasm resulted in the inhibition of TAp63-dependent transactivation of p21. Conversely, knockdown of NIR by RNAi stimulated p21 transcription in the presence of TAp63. Thus, NIR is a cell-cycle-controlled, novel negative regulator of TAp63. The low levels of nucleoplasmic NIR might act as a buffer toward potentially toxic TAp63

Super-Genotype: Global Monoclonality Defies the Odds of Nature

The ability to respond to natural selection under novel conditions is critical for the establishment and persistence of introduced alien species and their ability to become invasive. Here we correlated neutral and quantitative genetic diversity of the weed Pennisetum setaceum Forsk. Chiov. (Poaceae) with differing global (North American and African) patterns of invasiveness and compared this diversity to native range populations. Numerous molecular markers indicate complete monoclonality within and among all of these areas (FST = 0.0) and is supported by extreme low quantitative trait variance (QST = 0.00065–0.00952). The results support the general-purpose-genotype hypothesis that can tolerate all environmental variation. However, a single global genotype and widespread invasiveness under numerous environmental conditions suggests a super-genotype. The super-genotype described here likely evolved high levels of plasticity in response to fluctuating environmental conditions during the Early to Mid Holocene. During the Late Holocene, when environmental conditions were predominantly constant but extremely inclement, strong selection resulted in only a few surviving genotypes

C-terminal diversity within the p53 family accounts for differences in DNA binding and transcriptional activity

The p53 family is known as a family of transcription factors with functions in tumor suppression and development. Whereas the central DNA-binding domain is highly conserved among the three family members p53, p63 and p73, the C-terminal domains (CTDs) are diverse and subject to alternative splicing and post-translational modification. Here we demonstrate that the CTDs strongly influence DNA binding and transcriptional activity: while p53 and the p73 isoform p73γ have basic CTDs and form weak sequence-specific protein–DNA complexes, the major p73 isoforms have neutral CTDs and bind DNA strongly. A basic CTD has been previously shown to enable sliding along the DNA backbone and to facilitate the search for binding sites in the complex genome. Our experiments, however, reveal that a basic CTD also reduces protein–DNA complex stability, intranuclear mobility, promoter occupancy in vivo, target gene activation and induction of cell cycle arrest or apoptosis. A basic CTD therefore provides both positive and negative regulatory functions presumably to enable rapid switching of protein activity in response to stress. The different DNA-binding characteristics of the p53 family members could therefore reflect their predominant role in the cellular stress response (p53) or developmental processes (p73)

Latent Epstein-Barr Virus Can Inhibit Apoptosis in B Cells by Blocking the Induction of NOXA Expression

Latent Epstein-Barr virus (EBV) has been shown to protect Burkitt's lymphoma-derived B cells from apoptosis induced by agents that cause damage to DNA, in the context of mutant p53. This protection requires expression of the latency-associated nuclear proteins EBNA3A and EBNA3C and correlates with their ability to cooperate in the repression of the gene encoding the pro-apoptotic, BH3-only protein BIM. Here we confirm that latent EBV in B cells also inhibits apoptosis induced by two other agents – ionomycin and staurosporine – and show that these act by a distinct pathway that involves a p53-independent increase in expression of another pro-apoptotic, BH3-only protein, NOXA. Analyses employing a variety of B cells infected with naturally occurring EBV or B95.8 EBV-BAC recombinant mutants indicated that the block to NOXA induction does not depend on the well-characterized viral latency-associated genes (EBNAs 1, 2, 3A, 3B, 3C, the LMPs or the EBERs) or expression of BIM. Regulation of NOXA was shown to be at least partly at the level of mRNA and the requirement for NOXA to induce cell death in this context was demonstrated by NOXA-specific shRNA-mediated depletion experiments. Although recombinant EBV with a deletion removing the BHRF1 locus – that encodes the BCL2-homologue BHRF1 and three microRNAs – partially abrogates protection against ionomycin and staurosporine, the deletion has no effect on the EBV-mediated block to NOXA accumulation

Gammaherpesvirus Latency Accentuates EAE Pathogenesis: Relevance to Epstein-Barr Virus and Multiple Sclerosis

Epstein-Barr virus (EBV) has been identified as a putative environmental trigger of multiple sclerosis (MS), yet EBV's role in MS remains elusive. We utilized murine gamma herpesvirus 68 (γHV-68), the murine homolog to EBV, to examine how infection by a virus like EBV could enhance CNS autoimmunity. Mice latently infected with γHV-68 developed more severe EAE including heightened paralysis and mortality. Similar to MS, γHV-68EAE mice developed lesions composed of CD4 and CD8 T cells, macrophages and loss of myelin in the brain and spinal cord. Further, T cells from the CNS of γHV-68 EAE mice were primarily Th1, producing heightened levels of IFN-γ and T-bet accompanied by IL-17 suppression, whereas a Th17 response was observed in uninfected EAE mice. Clearly, γHV-68 latency polarizes the adaptive immune response, directs a heightened CNS pathology following EAE induction reminiscent of human MS and portrays a novel mechanism by which EBV likely influences MS and other autoimmune diseases

Stromal Interferon-γ Signaling and Cross-Presentation Are Required to Eliminate Antigen-Loss Variants of B Cell Lymphomas in Mice

To study mechanisms of T cell-mediated rejection of B cell lymphomas, we developed a murine lymphoma model wherein three potential rejection antigens, human c-MYC, chicken ovalbumin (OVA), and GFP are expressed. After transfer into wild-type mice 60–70% of systemically growing lymphomas expressing all three antigens were rejected; lymphomas expressing only human c-MYC protein were not rejected. OVA expressing lymphomas were infiltrated by T cells, showed MHC class I and II upregulation, and lost antigen expression, indicating immune escape. In contrast to wild-type recipients, 80–100% of STAT1-, IFN-γ-, or IFN-γ receptor-deficient recipients died of lymphoma, indicating that host IFN-γ signaling is critical for rejection. Lymphomas arising in IFN-γ- and IFN-γ-receptor-deficient mice had invariably lost antigen expression, suggesting that poor overall survival of these recipients was due to inefficient elimination of antigen-negative lymphoma variants. Antigen-dependent eradication of lymphoma cells in wild-type animals was dependent on cross-presentation of antigen by cells of the tumor stroma. These findings provide first evidence for an important role of the tumor stroma in T cell-mediated control of hematologic neoplasias and highlight the importance of incorporating stroma-targeting strategies into future immunotherapeutic approaches