Functional Differences between BHRF1, the Epstein-Barr Virus-Encoded Bcl-2 Homologue, and Bcl-2 in Human Epithelial Cells

BHRF1, a component of the restricted early antigen complex of the Epstein-Barr virus lytic cycle, encodes a 17-kDa protein with both sequence and functional homology to the antiapoptotic Bcl-2 oncogene. Recent work has suggested that BHRF1 behaves like Bcl-2 in protecting cells from apoptosis induced by a range of stimuli. In this study, the effect of BHRF1 and Bcl-2 on the growth and differentiation of the SCC12F human epithelial cell line was examined. The levels of stable transfected BHRF1 expression achievable in SCC12F cells was consistently lower than that obtained with Bcl-2. While both BHRF1 and Bcl-2 inhibited epithelial differentiation, the effect of Bcl-2 was more pronounced, resulting in an almost complete blockade of differentiation in organotypic raft cultures. However, BHRF1-expressing SCC12F cells proliferated at a much higher rate than SCC12F cells expressing Bcl-2, and this effect was supported by cell cycle analysis which demonstrated that BHRF1, but not Bcl-2, promotes rapid transit through the cell cycle. These data highlight important differences between BHRF1 and Bcl-2 and suggest that BHRF1 may function to promote the survival and proliferation of lytically infected cells. The proliferative properties of BHRF1 described in this study, together with the demonstration that other oncogenic gamma herpesviruses encode Bcl-2 homologues, suggests that these proteins may serve to increase the susceptibility of virus-infected cells to oncogenic transformation, thereby contributing to the development of virus-associated tumors.     

Effects of variation in egg size and hatching date on survival of Lesser Scaup Aythya affinis ducklings

The consequences of avian egg-size variation on offspring quality and survival remain unclear. We evaluated the effects of egg-size and hatch-date variation on survival of Lesser Scaup Aythya affinis ducklings in the wild. Duckling mass at hatching increased significantly with increasing egg size. Ducklings from larger eggs survived better than those from smaller eggs. We suspect that ducklings from larger eggs survived better because of advantages associated with larger or more efficient utilization of nutrient reserves, or both. We were unable to detect any within-clutch differences in egg size of survivors and non-survivors, nor any consistent direction in the difference in egg size between survivors and nonsurvivors within clutches. This suggests that within-clutch variation may be insufficient to have survival consequences for offspring. In addition, ducklings that hatched later in the breeding season had a higher probability of survival. We suggest a food-dependent hypothesis as an explanation for the seasonally increasing survival and for later nest initiation of Lesser Scaup compared with other North American ducks.     

Staurosporine Induces Programmed Cell Death in Embryonic Neurons and Activation of the Ceramide Pathway

Abstract: We activated the death pathway in embryonic chick cerebral hemisphere neuron (E7CH) cultures with staurosporine (0.1–1.0 µ M ) and observed the morphological changes, DNA laddering patterns, and DNA fragmentation (determined by Hoechst 33258 dye) associated with apoptosis. N -Acylsphingosine (C2-ceramide), a soluble ceramide analogue, was also able to induce apoptosis in these cells with the same characteristics and in the same time frame. We then observed that staurosporine was effective in inducing hydrolysis of sphingomyelin to ceramide as measured by a threefold increase in ceramide mass and increased incorporation of [³H]-palmitate into ceramide, concurrent with activating the cell death program. Furthermore, the coaddition of a specific ceramidase inhibitor, oleoylethanolamine (15 µ M ), enhanced the formation of ceramide as well as the degree of DNA fragmentation and cell death. Exogenous addition of sphingomyelinase activated the death pathway whereas ceramide glycanase did not, and inhibitors of sphingomyelin or protein synthesis failed to block this type of killing. Our data suggest that the formation of ceramide from sphingomyelin is a key event in staurosporine-induced and potentially all programmed cell death.     

CYCLOSPORIN A INHIBITS THE IN VIVO PRODUCTION OF INTERLEUKIN-1β AND TUMOUR NECROSIS FACTOR α, BUT NOT INTERLEUKIN-6, BY A T-CELL-INDEPENDENT MECHANISM

The effect of cyclosporin A (CsA) on cytokine production in the tissue chamber model of acute inflammation was investigated. CsA caused a dose-related inhibition of interleukin 1β (IL-1β) production in both normal and athymic mice, confirming earlier conclusions that this effect is not T cell dependent (ED₅₀s 40 and 53 mg/kg p.o., respectively).Tumour necrosis factor alpha (TNF-α) levels were similarly affected with ED₅₀s of 40 and 58 mg/kg p.o. for normal and athymic mice, respectively. By contrast, CsA inhibited interleukin 6 (IL-6) production only in normal mice (ED₅₀27 mg/kg p.o.)Differences in the absolute production of the three cytokines in normal and athymic mice were also noted. IL-1β and IL-6 levels were two-fold higher in athymic mice, while for TNF-α, there was no difference between the two groups.The present findings support the authors' original hypothesis, that the inhibitory mechanism of CsA on IL-1β is not mediated via T cells. The same mechanism also seems responsible for the inhibition of TNF-α production, but not for IL-6, where inhibition by CsA appears to require the presence of T cells.     

Nitric oxide-dependent penile erection in mice lacking neuronal nitric oxide synthase.

BACKGROUND: Nitric oxide (NO) has been implicated as a mediator of penile erection, because the neuronal isoform of NO synthase (NOS) is localized to the penile innervation and NOS inhibitors selectively block erections. NO can also be formed by two other NOS isoforms derived from distinct genes, inducible NOS (iNOS) and endothelial NOS (eNOS). To clarify the source of NO in penile function, we have examined mice with targeted deletion of the nNOS gene (nNOS- mice). MATERIALS AND METHODS: Mating behavior, electrophysiologically induced penile erection, isolated erectile tissue isometric tension, and eNOS localization by immunohistochemistry and Western blot were performed on nNOS- mice and wild-type controls. RESULTS: Both intact animal penile erections and isolated erectile tissue function are maintained in nNOS mice, in agreement with demonstrated normal sexual behaviors, but is stereospecifically blocked by the NOS inhibitor, L-nitroarginine methyl ester (L-NAME). eNOS is abundantly present in endothelium of penile vasculature and sinusoidal endothelium within the corpora cavemosa, with levels that are significantly higher in nNOS- mice than in wild-type controls. CONCLUSIONS: eNOS mediates NO-dependent penile erection in nNOS- animals and normal penile erection. These data clarify the role of nitric oxide in penile erection and may have implications for therapeutic agents with selective effects on NOS isoforms.     

Neurovirulent simian immunodeficiency virus infection induces neuronal, endothelial, and glial apoptosis.

BACKGROUND: Studies of human immunodeficiency virus type 1 (HIV-1) associated dementia have shown neuronal loss in discrete areas. The presence and mechanism of neuronal death, however, has remained quite elusive. One mechanism of cell death, apoptosis, has been clearly demonstrated outside the central nervous system (CNS) in HIV-1 infection but has not been firmly established within the CNS. Therefore, we set out to ascertain whether neuronal cell loss in simian immunodeficiency virus (SIV) encephalitis, an animal model of HIV-1-associated dementia, is a result of apoptosis. MATERIALS AND METHODS: With the aid of an in situ technique for identifying the 3'-OH ends of newly fragmented DNA characteristic of apoptosis, in conjunction with specific detected morphological criteria via light microscopy, we have examined encephalitic and nonencephalitic brains of macaques infected with a neurovirulent, neuroendotheliotropic strain of SIV to see if virus is spatially associated with apoptosis of neurons and non-neuronal cell types. RESULTS: We demonstrate the presence of DNA damage, indicative of apoptosis, in neurons, endothelial cells, and glial cells of the CNS of SIV-infected macaques. Furthermore, we observe an association between the localization of cells with significant DNA fragmentation and perivascular inflammatory cell infiltrates containing SIV-infected macrophages and multinucleated giant cells. Quantitative analysis reveals significantly more cells with DNA fragmentation in the CNS of macaques infected with neurovirulent, neuroendotheliotropic SIV strains as compared with strictly lymphocyte-tropic SIV strains and SIV negative controls. CONCLUSIONS: Our findings of apoptosis in SIV-infected CNS may potentially lead to a better understanding of the AIDS dementia complex, ultimately providing a basis for better treatments.     

The diverse spectroscopic properties of ferrous cytochrome P-450-CAM ligand complexes

The UV-visible absorption, magnetic circular dichroism (MCD) and CD spectral characteristics of a variety of low spin ferrous P-450-ligand complexes have been carefully determined in order to establish whether all such complexes are hyperporphyrins as previously suggested in the literature. Two general spectral classes are found to occur. Complexes in the first class are, indeed, hyperporphyrin in nature, with pi-acceptor ligands such as CO, NO, phosphine, nitrosoalkanes and isocyanides trans to cysteinate. Individual, but minor, variations in the spectral properties of the hyperporphyrins suggest that subclasses exist, wherein the nature of the trans ligand to thiolate affects the orbital overlap pattern and thus the observed spectra. Adducts in the second spectral class, which have sigma-donor nitrogen and sulfur ligands, also have the red-shifted Soret absorption maximum but are spectrally distinct in all other respects from the hyperporphyrins. Comparison of the MCD spectra of the second category to those of ferrous cytochromes b5, c, and P-420 suggests that the axial cysteinate ligand is still present in the nonhyper ferrous P-450 species. Thus, the combination of a strongly electron-donating cysteinate ligand and a trans sigma-donor, not the orbital mixing mechanism, is most likely the origin of the red-shifted Soret absorption maximum of nonhyper ferrous P-450 ligand complexes. Further, the nature of the total electronic interactions between both axial ligands and the heme iron of ferrous P-450 and not solely the cysteinate ligand determines whether the ligand complexes will be of the hyper or nonhyperporphyrin category. These findings are strengthened by the simultaneous use of three different spectroscopic techniques; together they provide a more detailed explanation for the unusual spectroscopic properties of cytochrome P-450.