Monitoring and purification of proteins using bovine papillomavirus E2 epitope tags

We describe here the use of two newly mapped bovine papillomavirus type 1 (BPV-1) E2 protein epitopes as tags. We constructed several vector plasmids for overexpression as well as for moderate expression of single- or double-tagged proteins in either Escherichia coli or eukaryotic cells. The new tags were fused to several proteins, and the activity of the tagged proteins was tested in different assays. The tags were shown not to interfere with the function of these proteins in vivo and in vitro. Interaction of the monoclonal antibodies 3F12 and 1E2 with their respective epitopes was specific and had high affinity in a variety of conditions. We have demonstrated that the 3F12 antibody-epitope interaction tolerates high salt concentrations up to 2 M. This permits immunoprecipitation and immunopurification of the tagged proteins in high-salt buffers and reduction of the nonspecific binding of the contaminating proteins. We also provide a protocol for DNA binding and DNase I footprinting assays using the tagged, resin-bound DNA-binding proteins. The BPV-1 E2-derived tags can be recommended as useful tools for detection and purification of proteins.     

Effect of physicochemical and pharmacokinetic properties of barbiturates on the induction of drug metabolism

In searching for possible determinants of the ability of compounds to induce microsomal drug metabolizing enzymes we studied relationships between the relative inducing activity of eleven barbiturates and their lipid solubility and pharmacokinetic behaviour. Allyl derivatives of barbituric acid were poor inducers regardless of their lipid solubility or half-life. The ability of other barbiturate derivatives to induce drug metabolism was directly related to biological half-life or time/concentration area value and inversely correlated to lipid solubility. The results suggest that the ability of a given compound to induce drug metabolism is not only related to lipid solubility and pharmacokinetic characteristics but also to the nature of chemical groups present in the molecule.     

Response of Ligated Intestinal Loops in Chickens to the Enterotoxin of Clostridium perfringens

Approximately 45-cm length of jejunoileum of 7-week-old chickens was found to be responsive and suitable for testing the enterotoxin of Clostridium perfringens by the ligated intestinal loop technique. Injections of 20 to 30 mug of enterotoxin per loop caused positive response of fluid accumulation. Chickens were found to be more convenient and economical for this purpose than other laboratory and domestic animals.     

Neuronostatin, a novel peptide encoded by somatostatin gene, regulates cardiac contractile function and cardiomyocyte survival

Neuronostatin, a recently discovered peptide encoded by somatostatin gene, is involved in regulation of neuronal function, blood pressure, food intake, and drinking behavior. However, the biological effects of neuronostatin on cardiac myocytes are not known, and the intracellular signaling mechanisms induced by neuronostatin remain unidentified. We analyzed the effect of neuronostatin in isolated perfused rat hearts and in cultured primary cardiomyocytes. Neuronostatin infusion alone had no effect on left ventricular (LV) contractile function or on isoprenaline- or preload-induced increase in cardiac contractility. However, infusion of neuronostatin significantly decreased the positive inotropic response to endothelin-1 (ET-1). This was associated with an increase in phosphorylation of p38 mitogen-activated protein kinase and c-Jun N-terminal kinase (JNK). Treatment of both neonatal and adult cardiomyocytes with neuronostatin resulted in reduced cardiomyocyte viability. Inhibition of JNK further increased the neuronostatin-induced cell death. We conclude that neuronostatin regulates cardiac contractile function and cardiomyocyte survival. Receptors for neuronostatin need to be identified to further characterize the biological functions of the peptide.