Decreased intracellular superoxide levels activate Sindbis virus-induced apoptosis

Infection of many cultured cell types with Sindbis virus (SV), an alphavirus, triggers apoptosis through a commonly utilized caspase activation pathway. However, the upstream signals by which SV activates downstream apoptotic effectors, including caspases, remain unclear. Here we report that in AT-3 prostate carcinoma cells, SV infection decreases superoxide (O-2) levels within minutes of infection as monitored by an aconitase activity assay. This SV-induced decrease in O-2 levels appears to activate or modulate cell death, as a recombinant SV expressing the O-2 scavenging enzyme, copper/zinc superoxide dismutase (SOD), potentiates SV-induced apoptosis. A recombinant SV expressing a mutant form of SOD, which has reduced SOD activity, has no effect. The potentiation of SV-induced apoptosis by wild type SOD is because of its ability to scavenge intracellular O-2 rather than its ability to promote the generation of hydrogen peroxide. Pyruvate, a peroxide scavenger, does not affect the ability of wild type SOD to potentiate cell death; and increasing the intracellular catalase activity via a recombinant SV vector has no effect on SV-induced apoptosis. Moreover, increasing intracellular O-2 by treatment of 3T3 cells with paraquat protects them from SV-induced death. Altogether, our results suggest that SV may activate apoptosis by reducing intracellular superoxide levels and define a novel redox signaling pathway by which viruses can trigger cell death.     

Aven and Bcl-xL enhance protection against apoptosis for mammalian cells exposed to various culture conditions

A balance between proliferation and cell death is critical for achieving desirable high cell densities in mammalian cell culture. In this study, we evaluate a recently discovered anti-apoptotic gene, aven, and examine its effectiveness alone and in combination with a member of the Bcl-2 family, bcl-xL. The commercially popular cell line, Chinese hamster ovary (CHO), was genetically modified to constitutively express aven, bcl-xL, and the two genes in combination. Cells were exposed to several model insults that simulate severe bioreactor environments, including serum deprivation, spent medium, and Sindbis virus infection, as well as staurosporine, a known chemical inducer of apoptosis. CHO cells exhibited DNA fragmentation, a hallmark of apoptosis, after exposure to these model insults. After exposure to serum deprivation, 4- and 5-day spent medium, and staurosporine, cells expressing Aven provided limited protection against cell death when compared with the protection afforded by cells expressing Bcl-xL alone. However, the highest survival levels for all insults were achieved when Aven was expressed in combination with Bcl-xL. In fact, Aven appeared to act synergistically to enhance the protective function of Bcl-xL for several insults, because the protective function of the two genes expressed together in one cell line often exceeded the additive protective levels of each anti-apoptosis gene expressed alone. Surprisingly, Aven expression provided a mildly pro-apoptotic response in CHO isolates infected with Sindbis virus. However, CHO cells expressing both Bcl-xL and Aven showed protection against Sindbis virus infection due to the inhibitory properties of the bcl-xL anti-apoptosis gene. This study shows that combinatorial anti-apoptosis cell engineering strategies may be the most effective mechanisms for providing extended protection against cell death in mammalian cell culture.     

Directed walk designs for dose-response problems with competing failure modes

We examine adaptive allocation designs for the problem of determining the optimal therapeutic dose for subjects in early-phase clinical trials. A subject can fail due to lack of efficacy or due to a toxic reaction. Successful subjects will have both a positive response and no toxic side effects. Thus, we seek to maximize the product of the nontoxicity and efficacy dose-response curves. We are interested in sampling rules that perform well along several criteria, including the ethical criterion that, as often as possible, experimental subjects be treated at or close to the maximum in question. Statistically, we wish to identify the optimum dose with high probability at the close of the experiment. Here, we propose designs that combine new allocation policies, directed walks, with new smoothed shape-constrained curve-fitting techniques. These are compared with a variety of other curve-fitting techniques and with up-and-down and equal allocation rules.     

Viral versus cellular BCL-2 proteins

All gamma herpesviruses and a few other viruses encode at least one homologue of the mammalian cell death inhibitor BCL-2. Gamma herpesviruses are associated with human and animal lymphoid and epithelial tumours. However, the role of these viral BCL-2 homologues in the virus replication cycle or in human disease is not known, though recent developments show progress in this area. The structure of viral BCL-2 family protein, KSBcl-2, is similar to that of cellular family members, but viral BCL-2 proteins differ functionally from the cellular proteins, apparently escaping the regulatory mechanisms to which their cellular counterparts are subjected. Thus, exploring the biochemical and biological functions of the viral BCL-2 family proteins will increase our understanding of their role in virus infections and will undoubtedly teach us something about their cellular kin.     

BAK alters neuronal excitability and can switch from anti- to pro-death function during postnatal development

BAK is a pro-apoptotic BCL-2 family protein that localizes to mitochondria. Here we evaluate the function of BAK in several mouse models of neuronal injury including neuronotropic Sindbis virus infection, Parkinson's disease, ischemia/stroke, and seizure. BAK promotes or inhibits neuronal death depending on the specific death stimulus, neuron subtype, and stage of postnatal development. BAK protects neurons from excitotoxicity and virus infection in the hippocampus. As mice mature, BAK is converted from anti- to pro-death function in virus-infected spinal cord neurons. In addition to regulating cell death, BAK also protects mice from kainate-induced seizures, suggesting a possible role in regulating synaptic activity. BAK can alter neurotransmitter release in a direction consistent with its protective effects on neurons and mice. These findings suggest that BAK inhibits cell death by modifying neuronal excitability.     

Sites,mechanism of action and lack of reversibility of primate lentivirus inactivation by preferential covalent modification of virion internal proteins

By exploiting the intrinsic chemistry of retroviruses, we have developed a novel method for generating whole inactivated virion vaccine immunogens with functional envelope glycoproteins. The method takes advantage of the fact that the internal proteins of retroviruses are adapted to the intracellular (reducing) environment, and have cysteine residues present in thiol-form (S-H), while the surface proteins of retroviruses (the envelope glycoproteins SU and TM) are adapted to the (oxidizing) environment of the extracellular milieu, and have their cysteines present as disulfides (S-S). Treatment of retroviral virions with appropriate mild oxidizing agents thus results in preferential covalent modification and functional inactivation of key S-H-containing internal viral proteins, such as the nucleocapsid (NC) protein, that are required for infectivity, while the envelope glycoproteins with their disulfide bonded cysteines remain unaffected. This treatment thus results in virions that do not retain detectable infectivity, but preserves the conformational and functional integrity of the envelope glycoproteins. We have extensively used the disulfide reagent 2,2'-dithiodipyridine (aldrithiol-2, AT-2) to inactivate HIV and SIV via this mechanism and such inactivated virions appear to be a promising vaccine immunogen based on macaque studies. We have biochemically characterized the targets and mechanisms of inactivation involved in AT-2 treatment of virions, and investigated the kinetics of inactivation. Although extremely unlikely under physiological conditions, reversibility of this type of inactivation is a theoretical concern. We have therefore conducted a series of in vitro experiments, in cell free systems and in cell culture, to evaluate this possibility. The results indicate that as judged by both biochemical and biological (infectivity) criteria, inactivation by AT-2 does not appear to be reversible under conditions likely to obtain in vivo.     

Bax-type apoptotic proteins porate pure lipid bilayers through a mechanism sensitive to intrinsic monolayer curvature

During apoptosis, Bax-type proteins permeabilize the outer mitochondrial membrane to release intermembrane apoptogenic factors into the cytosol via a poorly understood mechanism. We have proposed that Bax and DeltaN76Bcl-x(L) (the Bax-like cleavage fragment of Bcl-x(L)) function by forming pores that are at least partially composed of lipids (lipidic pore formation). Since the membrane monolayer must bend during lipidic pore formation, we here explore the effect of intrinsic membrane monolayer curvature on pore formation. Nonlamellar lipids with positive intrinsic curvature such as lysophospholipids promoted membrane permeabilization, whereas nonlamellar lipids with negative intrinsic curvature such as diacylglycerol and phosphatidylethanolamine inhibited membrane permeabilization. The differential effects of nonlamellar lipids on membrane permeabilization were not correlated with lipid-induced changes in membrane binding or insertion of Bax or DeltaN76Bcl-x(L). Altogether, these results are consistent with a model whereby Bax-type proteins change the bending propensity of the membrane to form pores comprised at least in part of lipids in a structure of net positive monolayer curvature.     

Worldwide distribution of Nitrosococcus oceani,a marine ammonia-oxidizing gamma-proteobacterium,detected by PCR and sequencing of 16S rRNA and amoA genes

Diversity of cultured ammonia-oxidizing bacteria in the gamma-subdivision of the Proteobacteria was investigated by using strains isolated from various parts of the world ocean. All the strains were very similar to each other on the basis of the sequences of both the 16S rRNA and ammonia monooxygenase genes and could be characterized as a single species. Sequences were also cloned directly from environmental DNA from coastal Pacific and Atlantic sites, and these sequences represented the first Nitrosococcus oceani-like sequences obtained directly from the ocean. Most of the environmental sequences clustered tightly with those of the cultivated strains, but some sequences could represent new species of NITROSOCOCCUS: These findings imply that organisms similar to the cultivated N. oceani strains have a worldwide distribution.