B-Raf Inhibits Programmed Cell Death Downstream of Cytochrome c Release from Mitochondria by Activating the MEK/Erk Pathway

Growth factor-dependent kinases, such as phosphatidylinositol 3-kinase (PI 3-kinase) and Raf kinases, have been implicated in the suppression of apoptosis. We have recently established Rat-1 fibroblast cell lines overexpressing B-Raf, leading to activation of the MEK/Erk mitogen-activated protein kinase pathway. Overexpression of B-Raf confers resistance to apoptosis induced by growth factor withdrawal or PI 3-kinase inhibition. This is accompanied by constitutive activation of Erk without effects on the PI 3-kinase/Akt pathway. The activity of MEK is essential for cell survival mediated by B-Raf overexpression, since either treatment with the specific MEK inhibitor PD98059 or expression of a dominant inhibitory MEK mutant blocks the antiapoptotic activity of B-Raf. Activation of MEK is not only necessary but also sufficient for cell survival because overexpression of constitutively activated MEK, Ras, or Raf-1, like B-Raf, prevents apoptosis after growth factor deprivation. Overexpression of B-Raf did not interfere with the release of cytochrome c from mitochondria after growth factor deprivation. However, the addition of cytochrome c to cytosols of cells overexpressing B-Raf failed to induce caspase activation. It thus appears that the B-Raf/MEK/Erk pathway confers protection against apoptosis at the level of cytosolic caspase activation, downstream of the release of cytochrome c from mitochondria.     

‘Being Honest to my Science’: Reo Fortune and J.H.P. Murray, 1927-301

Reo Fortune worked on Tewara Island in the D'Entrecasteaux group in the Australian Territory of Papua from November 1927 to May 1928. His relations with the Lieutenant-Governor J.H.P. Murray and the government anthropologist F.E. Williams were problematic. In this paper I argue that Fortune's actions, along with increasing tension between Murray and Radcliffe-Brown, were sufficient for a change in Murray's attitude towards continued independent anthropological research in Papua. After 1930, despite his early support for colonial officers receiving training in anthropology, Murray sent no cadets or officers for training in the university. With the exceptions of Geza Roheim and Paul Wirz, both in 1930, Williams conducted all anthropological research in Papua until the outbreak of the Pacific War in December 1941.     

Radiation-induced Release of Transforming Growth Factor α Activates the Epidermal Growth Factor Receptor and Mitogen-activated Protein Kinase Pathway in Carcinoma Cells, Leading to Increased Proliferation and Protection from Radiation-induced Cell Death

Exposure of A431 squamous and MDA-MB-231 mammary carcinoma cells to ionizing radiation has been associated with short transient increases in epidermal growth factor receptor (EGFR) tyrosine phosphorylation and activation of the mitogen-activated protein kinase (MAPK) and c-Jun NH(2)-terminal kinase (JNK) pathways. Irradiation (2 Gy) of A431 and MDA-MB-231 cells caused immediate primary activations (0-10 min) of the EGFR and the MAPK and JNK pathways, which were surprisingly followed by later prolonged secondary activations (90-240 min). Primary and secondary activation of the EGFR was abolished by molecular inhibition of EGFR function. The primary and secondary activation of the MAPK pathway was abolished by molecular inhibition of either EGFR or Ras function. In contrast, molecular inhibition of EGFR function abolished the secondary but not the primary activation of the JNK pathway. Inhibition of tumor necrosis factor alpha receptor function by use of neutralizing monoclonal antibodies blunted primary activation of the JNK pathway. Addition of a neutralizing monoclonal antibody versus transforming growth factor alpha (TGFalpha) had no effect on the primary activation of either the EGFR or the MAPK and JNK pathways after irradiation but abolished the secondary activation of EGFR, MAPK, and JNK. Irradiation of cells increased pro-TGFalpha cleavage 120-180 min after exposure. In agreement with radiation-induced release of a soluble factor, activation of the EGFR and the MAPK and JNK pathways could be induced in nonirradiated cells by the transfer of media from irradiated cells 120 min after irradiation. The ability of the transferred media to cause MAPK and JNK activation was blocked when media were incubated with a neutralizing antibody to TGFalpha. Thus radiation causes primary and secondary activation of the EGFR and the MAPK and JNK pathways in autocrine-regulated carcinoma cells. Secondary activation of the EGFR and the MAPK and JNK pathways is dependent on radiation-induced cleavage and autocrine action of TGFalpha. Neutralization of TGFalpha function by an anti-TGFalpha antibody or inhibition of MAPK function by MEK1/2 inhibitors (PD98059 and U0126) radiosensitized A431 and MDA-MB-231 cells after irradiation in apoptosis, 3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), and clonogenic assays. These data demonstrate that disruption of the TGFalpha-EGFR-MAPK signaling module represents a strategy to decrease carcinoma cell growth and survival after irradiation.     

Vaccinia Virus Intracellular Mature Virions Contain only One Lipid Membrane

Vaccinia virus (VV) morphogenesis commences with the formation of lipid crescents that grow into spherical immature virus (IV) and then infectious intracellular mature virus (IMV) particles. Early studies proposed that the lipid crescents were synthesized de novo and matured into IMV particles that contained a single lipid bilayer (S. Dales and E. H. Mosbach, Virology 35:564–583, 1968), but a more recent study reported that the lipid crescent was derived from membranes of the intermediate compartment (IC) and contained a double lipid bilayer (B. Sodiek et al., J. Cell Biol. 121:521–541, 1993). In the present study, we used high-resolution electron microscopy to reinvestigate the structures of the lipid crescents, IV, and IMV particles in order to determine if they contain one or two membranes. Examination of thin sections of Epon-embedded, VV-infected cells by use of a high-angular-tilt series of single sections, serial-section analysis, and high-resolution digital-image analysis detected only a single, 5-nm-thick lipid bilayer in virus crescents, IV, and IMV particles that is covered by a 8-nm-thick protein coat. In contrast, it was possible to discern tightly apposed cellular membranes, each 5 nm thick, in junctions between cells and in the myelin sheath of Schwann cells around neurons. Serial-section analysis and angular tilt analysis of sections detected no continuity between virus lipid crescents or IV particles and cellular membrane cisternae. Moreover, crescents were found to form at sites remote from IC membranes—namely, within the center of virus factories and within the nucleus—demonstrating that crescent formation can occur independently of IC membranes. These data leave unexplained the mechanism of single-membrane formation, but they have important implications with regard to the mechanism of entry of IMV and extracellular enveloped virus into cells; topologically, a one-to-one membrane fusion suffices for delivery of the IMV core into the cytoplasm. Consistent with this, we have demonstrated previously by confocal microscopy that uncoated virus cores within the cytoplasm lack the IMV surface protein D8L, and we show here that intracellular cores lack the surface protein coat and lipid membrane.     

Shift of Clinical Human Immunodeficiency Virus Type 1 Isolates from X4 to R5 and Prevention of Emergence of the Syncytium-Inducing Phenotype by Blockade of CXCR4

The emergence of X4 human immunodeficiency virus type 1 (HIV-1) strains in HIV-1-infected individuals has been associated with CD4(+) T-cell depletion, HIV-mediated CD8(+) cell apoptosis, and an impaired humoral response. The bicyclam AMD3100, a selective antagonist of CXCR4, selected for the outgrowth of R5 virus after cultivation of mixtures of the laboratory-adapted R5 (BaL) and X4 (NL4-3) HIV strains in the presence of the compound. The addition of AMD3100 to peripheral blood mononuclear cells infected with X4 or R5X4 clinical HIV isolates displaying the syncytium-inducing phenotype resulted in a complete suppression of X4 variants and a concomitant genotypic change in the V2 and V3 loops of the envelope gp120 glycoprotein. The recovered viruses corresponded genotypically and phenotypically to R5 variants in that they could no longer use CXCR4 as coreceptor or induce syncytium formation in MT-2 cells. Furthermore, the phenotype and genotype of a cloned R5 HIV-1 virus converted to those of the R5X4 virus after prolonged culture in lymphoid cells. However, these changes did not occur when the infected cells were cultured in the presence of AMD3100, despite low levels of virus replication. Our findings indicate that selective blockade of the CXCR4 receptor prevents the switch from the less pathogenic R5 HIV to the more pathogenic X4 HIV strains, a process that heralds the onset of AIDS. In this article, we show that it could be possible to redirect the evolution of HIV so as to impede the emergence of X4 strains or to change the phenotype of already-existing X4 isolates to R5.     

Ecotoxicological Evaluation of a Bench-Scale Bioslurry Treating Explosives-Spiked Soil

The ecotoxicological effects of four bioslurry reactors treating 2,4,6-trinitotoluene (TNT)- and 1,3,5-trinitro-1,3,5-triazacyclohexane (RDX)-spiked soil were evaluated. A control bioslurry reactor was used to assess the endogenous toxicity of the bioslurry operation conditions. A battery of ecotoxicity tests was used: Microtox, green algae growth inhibition, bacterial genotoxicity and mutagenicity, and earthworm mortality and growth inhibition. Bioslurry soluble and solid phases were separated by centrifugation in order to identify toxicity and possible toxicants associated with each phase. Microtox toxicity values were initially very high in both bioslurry reactors spiked with TNT, in relation with TNT concentration. Initial toxicity was also detected by algal growth inhibition, earthworm lethality, genotoxicity and mutagenicity tests. An endogenous toxicity was detected in the control bioreactor using the Microtox and the SOS Chromotest. The soluble phase of the control bioslurry was genotoxic, suggesting that some potentially genotoxic agents were induced in the bioslurry samples. At the end of the bioremediation treatment, data showed that toxicity was reduced using all of the bioassays, except for earthworm lethality and growth inhibition tests in both RDX-spiked bioslurries. This study demonstrates the usefulness of a battery of toxicity tests to monitor bioremediation processes.     

1,25-Dihydroxyvitamin D3 inhibits parathyroid hormone-related peptide mRNA expression in fetal rat long bones in culture

Summary When fetal rat long bones are incubated in the presence of 10⁻⁸ M 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], steady-state parathyroid hormone-related peptide (PTHrP) mRNA levels are decreased. This decrease is temporary: it is observed as soon as after 3 h of exposure and reaches a nadir after 6 h. At that time, PTHrP mRNA levels are significantly lower in the experimental than in the control bones. However the inhibitory effect vanishes after 24 h, despite continuous exposure to 1,25(OH)₂D₃ for even 48 h. This is the first report showing that PTHrP mRNA expression can be regulated in rat fetal long bones in vitro by 1,25(OH)₂D₃.     

Stage-specific transforming genes of human and mouse B- and T-lymphocyte neoplasms

DNAs of 20 B- and T-lymphocyte neoplasms of human and mouse origin induced transformation of NIH/3T3 cells with high efficiencies, indicating that these neoplasms contained activated transforming genes that were detectable by transfection. Analysis of the susceptibility of the transforming activities of lymphocyte-neoplasm DNAs to digestion with restriction endonucleases indicated that the same or closely related transforming genes were activated in independent neoplasms representative of the same stage of normal cell differentiation. However, different transforming genes were activated in neoplasms representative of different stages of normal B- and T-lymphocyte differentiation. These results indicate that specific transforming genes are activated in neoplasms of discrete stages of differentiation within these cell lineages.     

Restored oyster reefs and living shorelines can augment predator trophic dynamics

Coastal habitat loss has led to declines in many higher trophic level predators. These declines can be mitigated through habitat restoration, which putatively enhances predator populations and trophic dynamics by creating foraging opportunities in reestablished habitat, but this lacks empirical support. Here, we assess the prediction restored coastal habitat supports sportfish feeding relationships similar to natural habitat, at restored oyster reefs (Crassostrea virginica) and living shoreline habitats in Florida, U.S.A. Stable isotopes and gut contents were analyzed from young sportfish (i.e. predatory fish targeted by anglers) collected at control and restored sites for up to 3 years. The influence of habitat features, predator size, and prey availability on carbon and nitrogen isotope values were examined using a model species (mangrove/gray snapper [Lutjanus griseus]). In summary, sportfish species had distinct isotopic values, consuming similar prey but in different proportions between habitats. Prey abundance and richness, and reef height were influential predictors of L. griseus stable isotope values, with restored reefs contributing to their diet more than controls according to Bayesian mixing models. The trophic niche area of L. griseus and their predominant prey achieved trophic equivalence between restored and natural oyster reefs within 1 year following restoration. Stabilized living shorelines attained trophic equivalency to natural shorelines, but may take longer to accrue prey for some species. These findings generate fundamental ecological knowledge and actionable science, including targets, timelines, and indicator species, that natural resource managers and restoration practitioners can use to assess trophic structure and success of coastal habitat restoration.