Pro-apoptotic cleavage products of Bcl-xL form cytochrome c-conducting pores in pure lipid membranes.

During apoptotic cell death, cells usually release apoptogenic proteins such as cytochrome c from the mitochondrial intermembrane space. If Bcl-2 family proteins induce such release by increasing outer mitochondrial membrane permeability, then the pro-apoptotic, but not anti-apoptotic activity of these proteins should correlate with their permeabilization of membranes to cytochrome c. Here, we tested this hypothesis using pro-survival full-length Bcl-x(L) and pro-death Bcl-x(L) cleavage products (DeltaN61Bcl-x(L) and DeltaN76Bcl-x(L)). Unlike Bcl-x(L), DeltaN61Bcl-x(L) and DeltaN76Bcl-x(L) caused the release of cytochrome c from mitochondria in vivo and in vitro. Recombinant DeltaN61Bcl-x(L) and DeltaN76Bcl-x(L), as well as Bcl-x(L), cleaved in situ by caspase 3-possessed intrinsic pore-forming activity as demonstrated by their ability to efficiently permeabilize pure lipid vesicles. Furthermore, only DeltaN61Bcl-x(L) and DeltaN76Bcl-x(L), but not Bcl-x(L), formed pores large enough to release cytochrome c and to destabilize planar lipid bilayer membranes through reduction of pore line tension. Because Bcl-x(L) and its C-terminal cleavage products bound similarly to lipid membranes and formed oligomers of the same size, neither lipid affinity nor protein-protein interactions appear to be solely responsible for the increased membrane-perturbing activity elicited by Bcl-x(L) cleavage. Taken together, these data are consistent with the hypothesis that Bax-like proteins oligomerize to form lipid-containing pores in the outer mitochondrial membrane, thereby releasing intermembrane apoptogenic factors into the cytosol.

Both carboxy- and amino-terminal domains of the vaccinia virus interferon resistance gene, E3L, are required for pathogenesis in a mouse model.

The vaccinia virus (VV) E3L gene is responsible for providing interferon (IFN) resistance and a broad host range to VV in cell culture. The E3L gene product contains two distinct domains. A conserved carboxy-terminal domain, which is required for the IFN resistance and broad host range of the virus, has been shown to bind double-stranded RNA (dsRNA) and inhibit the antiviral dsRNA-dependent protein kinase, PKR. The amino-terminal domain, while conserved among orthopoxviruses, is dispensable in cell culture. To study the role of E3L in whole-animal infections, WR strain VV recombinants either lacking E3L (VVDeltaE3L) or expressing an amino-terminal (VVE3LDelta83N) or carboxy-terminal (VVE3LDelta26C) truncation of E3L were constructed. Whereas wild-type VV had a 50% lethal dose of approximately 10(4) PFU after intranasal infection, and elicited severe weight loss and morbidity, VVDeltaE3L was apathogenic, leading to no death, weight loss, or morbidity. VVDeltaE3L was also apathogenic after intracranial injection. Although the amino-terminal domain of E3L is dispensable for infection of cells in culture, both the amino- and carboxy-terminal domains of E3L were required for full pathogenesis in intranasal infections. These results demonstrate that the entire E3L gene is required for pathogenesis in the mouse model.

Determination of albendazole-2-aminosulfone in bovine milk using high-performance liquid chromatography with fluorometric detection.

An analytical method for the determination of albendazole (ABZ) residues in bovine milk was developed using one of its major metabolites, albendazole-2-aminosulfone (ABZ2NH2) as the marker. The method involved acid hydrolysis of milk followed by liquid-liquid extraction and solid-liquid phase clean-up of the extract. A reversed-phase HPLC with fluorometric detection was used to quantitate the marker residue. The method exhibited a high degree of precision and good accuracy as demonstrated by a relative standard deviation (R.S.D.) < 5% for the replicate analyses and 91.8 to 104.1% recovery of the fortification level (25-200 ng/ml), respectively. The ratio of the concentrations of the marker and total residues in milk over a 36-120 h withdrawal period was found to be steady at 43.1 indicating a definite relationship between the marker and the total residues of ABZ. The analytical method was used successfully to determine total residues in milk of cattle treated with ABZ.