Palmitoylation of the rat mu opioid receptor.

We examined whether the mu opioid receptor was palmitoylated and attempted to determine sites of palmitoylation. Following metabolic labeling with [3H]palmitic acid and immunoaffinity purification of the mu opioid receptor, SDS-PAGE and fluorography revealed a broad labeled band with Mr of approximately 80 kDa in CHO cells stably expressing the rat mu receptor, but not in CHO cells transfected with the vector alone, indicating that the mu receptor is palmitoylated. Activation of the receptor with morphine did not affect the extent of palmitoylation. Hydroxylamine or dithiothreitol treatment removed most of the radioactivity, demonstrating that [3H]palmitic acid is incorporated into Cys residue(s) via thioester bond(s). Surprisingly, mutations of the only two Cys residues in the C-terminal domain did not reduce [3H]palmitic acid incorporation significantly. Thus, unlike many G-protein coupled receptors, the palmitoylation site(s) of the rat mu opioid receptor do(es) not reside in the C-terminal domain.

Development of a live and highly attenuated Listeria monocytogenes-based vaccine for the treatment of Her2/neu-overexpressing cancers in human.

A chimeric human Her2/neu gene (ChHer2) harboring most of the known major histocompatibility complex class I epitopes of the HER2/neu oncogene was expressed as a fusion protein to a non-hemolytic fragment of listeriolysin O (LLO), by the highly attenuated Listeria vector LmddA, which lacks antibiotic selection markers and the ability to spread from cell-to-cell. This construct (ADXS31-164) was tested for immunogenicity and anti-tumor effects in mice. Despite being highly attenuated, ADXS31-164 proved to be efficacious in breaking immune tolerance toward the HER2/neu self-antigen. ADXS31-164 elicited strong T-cell immune responses in experimental animals. In tumors, ADXS31-164 caused a reduction in regulatory T cells (Treg) accompanied by an increase in the CD8(+)/Treg ratio. Comparison of this vaccine with the conventional antibiotic resistant Listeria vector (Lm-LLO-ChHer2) shows that ADXS31-164 is more efficacious in delaying tumor growth in Her2/neu transgenic animals. Because of its well-defined attenuation mechanism and independence from antibiotic selection markers, ADXS31-164 is potentially more suitable for human use. These results support the future clinical development of this vaccine for the treatment of HER2/neu-overexpressing malignancies, such as breast, colorectal and pancreatic cancers.

Transport pathways for therapeutic concentrations of lithium in rat liver.

Although both amiloride- and phloretin-sensitive Na(+)/Li(+) exchange activities have been reported in mammalian red blood cells, it is still unclear whether or not the two are mediated by the same pathway. Also, little is known about the relative contribution of these transport mechanisms to the entry of therapeutic concentrations of Li(+) (0.2-2 mm) into cells other than erythrocytes. Here, we describe characteristics of these transport systems in rat isolated hepatocytes in suspension. Uptake of Li(+) by hepatocytes, preloaded with Na(+) and incubated in the presence of ouabain and bumetanide, comprised three components. (a) An amiloride-sensitive component, with apparent K(m) 1.2 mm Li(+), V(max) 40 mumol. (kg dry wt. min)(-1), showed increased activity at low intracellular pH. The relationship of this component to the concentration of intracellular H(+) was curvilinear suggesting a modifier role of [H(+)](i). This system persisted in Na(+)-depleted cells, although with apparent K(m) 3.8 mm. (b) A phloretin-sensitive component, with K(m) 1.2 mm, V(max) 21 mumol. (kg. min)(-1), was unaffected by pH but was inactive in Na(+)-depleted cells. Phloretin inhibited Li(+) uptake and Na(+) efflux in parallel. (c) A residual uptake increased linearly with the external Li(+) concentration and represented an increasing proportion of the total uptake. The results strongly suggest that the amiloride-sensitive and the phloretin-sensitive Li(+) uptake in rat liver are mediated by two separate pathways which can be distinguished by their sensitivity to inhibitors and intracellular [H(+)].

Synthesis of hybrid molecules between heat-labile enterotoxin and cholera toxin B subunits: potential for use in a broad-spectrum vaccine.

Three variants of the cholera toxin B subunit (CTB) were generated by site-specific mutagenesis in which regions of the mature protein were altered to the composition found at the corresponding positions of the closely related B subunit of the heat-labile enterotoxin of enterotoxigenic Escherichia coli (LTB). The mutant proteins were expressed in Vibrio cholerae and purified from the growth medium. In the first of the mutant proteins, the first 25 amino acids corresponded to the sequence found in LTB, and in the second, changes were made at positions 94 and 95 of the mature protein. The third mutant protein combined the changes made in the first two. Analysis of the immunological properties of these novel proteins by using monoclonal antibodies and absorbed polyclonal antiserum demonstrated that they had acquired LTB-specific epitopes. Immunizations with the mutant proteins resulted in antisera containing LTB-specific as well as CTB-specific and cross-reactive antibodies. The sera were also found to be more strongly cross-reactive in the in vitro neutralization of both cholera toxin and heat-labile enterotoxin than were antisera raised against either CTB or LTB. The results suggest that such hybrid CTB-LTB proteins may be useful in a broad-spectrum vaccine against enterotoxin-induced diarrhea.

Structural basis for differential receptor binding of cholera and Escherichia coli heat-labile toxins: influence of heterologous amino acid substitutions in the cholera B-subunit.

The closely related B-subunits of cholera toxin (CTB) and Escherichia coli heat-labile enterotoxin (LTB) both bind strongly to GM1 ganglioside receptors but LTB can also bind to additional glycolipids and glycoproteins. A number of mutant CT B-subunits were generated by substituting CTB amino acids with those at the corresponding positions in LTB. These were used to investigate the influence of specific residues on receptor-binding specificity. A mutated CTB protein containing the first 25 residues of LTB in combination with LTB residues at positions 94 and 95, bound to the same extent as native LTB to both delipidized rabbit intestinal cell membranes, complex glycosphingolipids (polyglycosylceramides) and neolactotetraosylceramide, but not to non-GM1 intestinal glycosphingolipids. In contrast, when LTB amino acid substitutions in the 1-25 region were combined with those in the 75-83 region, a binding as strong as that of LTB to intestinal glycosphingolipids was observed. In addition, a mutant LTB with a single Gly-33-->Asp substitution that completely lacked affinity for both GM1 and non-GM1 glycosphingolipids could still bind to receptors in the intestinal cell membranes and to polyglycosylceramides. We conclude that the extra, non-GM1 receptors for LTB consist of both sialylated and non-sialylated glycoconjugates, and that the binding to either class of receptors is influenced by different amino acid residues within the protein.