Full-length and truncated human BCL2 lacking the entire C-terminal hydrophobic domain have been overexpressed in Spodoptera frugiperda insect cells with the baculovirus expression system. Immunoblot analysis with BCL2-specific antibodies revealed that both full-length and truncated BCL2 are expressed as multiple immunoreactive species, suggesting posttranslational modifications. The expression of the full-length but not the truncated BCL2 extended the survival of baculovirus-infected cells by preventing virusinduced DNA cleavage. This result is consistent with the reported protective effect of BCL2 against apoptosis in mammalian lymphocytes and suggests a conserved function in evolution. Subcellular fractionation and indirect immunofluorescence studies in intact cells demonstrated that the recombinant full-length and truncated BCL2 proteins were expressed predominantly as nuclear membrane-associated proteins. These results imply that BCL2 must utilize hydrophobic domains other than the deleted domain for its association with the subcellular membranes. Metabolic labeling of insect cells expressing the full-length and the truncated form of BCL2 with 32p, demonstrated that BCL2 is a phosphoprotein.The protooncogene BCL2 has been isolated and characterized as a result of its involvement with the t(14;18) chromosomal translocation in follicular lymphomas (1-5). The t(14;18) translocation places BCL2 under the transcriptional regulation of the immunoglobulin heavy-chain locus, causing the deregulation ofBCL2 expression (4, 5). The BCL2 protein is a 26-kDa membrane-associated cytoplasmic protein (6-8). It is different from other protooncogene products in that it seems to function in enhancing the survival of hematopoietic cells of T and B origins rather than promoting their growth (9-15). The ability of BCL2 to enhance the survival of these cells is related to its ability to block programmed cell death, or apoptosis, initiated by several factors. BCL2 overexpression in pre-B lymphocytes inhibits apoptosis following cytokine deprivation (8,9,14) or glucocorticoid treatment (16). Recently, two independent studies (17, 18) have also shown that expression of BCL2 in immature thymocytes of transgenic mice protects them from glucocorticoid-, radiation-, and anti-CD3-induced apoptosis. BCL2 upregulation also inhibits apoptosis of Epstein-Barr virus-infected B-cell lines (19). The mechanism by which BCL2 inhibits apoptosis is not known.To gain insight into the structure and mechanism of action of BCL2, we overexpressed full-length and truncated BCL2 in insect cells with the baculovirus expression system. Because of the low abundance of BCL2 in lymphocytes, overexpression of this protein in Sf9 insect cells will avail large quantities of protein for structural, functional, and biochemical analysis. MATERIALS AND METHODSCell Culture. Sf9 insect cells were grown in TNM-FH medium (20, 21). Cells were maintained and infected either as monolayers or in suspension in a rotary shaker. When cells were grown in suspension the me...

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Human exposure to nano- and microplastics (NMPs) has raised major societal concerns, yet no framework to assess the risks of NMPs for human health exists. A substantial proportion of plastic produced worldwide is not properly disposed and persists in the environment for decades while degrading. Plastic degradation generates a size continuum of fragments, including nano- and microplastic particles, with numerous associated environmental pollutants and plastic additives, and microbial communities colonising their surfaces. The ubiquitous presence of NMPs, their availability for uptake by organisms and their potential to act as vectors for toxicants and pathogens render risk assessment a priority on the political agenda at the global level. We provide a new, fully integrated risk assessment framework tailored to the specificities of NMPs, enabling an assessment of current and future human health risks from NMPs. The framework consists of four novel paradigms to the traditional risk assessment methodology. These paradigms deal with techniques in NMP analysis, gaps in empirical data, theoretical and modelling approaches and stakeholder engagement. Within the proposed framework, we propose how we can use research experiences gained so far to carry out the different steps of the assessment process, and we define priorities for further research.

Pseudomonas aeruginosa is an important pathogen that has been implicated in a number of serious conditions in humans and animals. Studies regarding the efficacy of antimicrobial drugs against virulent strains of P. aeruginosa in human and animal hosts have demonstrated that common therapeutic options are sometimes ineffective. In captive animals, in particular reptiles, literature on this topic is scarce. In this study, a 6-yr-old monitor lizard, Varanus niloticus, died suddenly without previous symptoms. Postmortem examination and bacteriologic analysis of necropsy samples revealed severe fibrinonecrotic enteritis caused by P. aeruginosa. The isolate presented a typical resistance profile, showing resistance to amoxycillin-clavulanic acid, ampicillin, cephotaxime, and sulphamethoxazole-trimethoprim. Virulence characterization demonstrated that the bacterium was able to express hemolysins, DNases, gelatinases, and lipases and had biofilm-forming ability. This study highlights the importance of surveillance for antimicrobial resistance and virulence traits in captive animals, which may present a challenge to practitioners.