Mu-opioid receptor splice variants: sex-dependent regulation by chronic morphine.

The gene encoding the mu-opioid receptor (MOR) generates a remarkable diversity of subtypes, the functional significance of which remains largely unknown. The structure of MOR could be a critical determinant of MOR functionality and its adaptations to chronic morphine exposure. As MOR antinociception has sexually dimorphic dimensions, we determined the influence of sex, stage of estrus cycle, and chronic systemic morphine on levels of MOR splice variant mRNA in rat spinal cord. Chronic systemic morphine influenced the spinal expression of mRNA encoding rMOR-1B2 and rMOR-1C1 in a profoundly sex-dependent fashion. In males, chronic morphine resulted in a twofold increase in expression levels of rMOR-1B2 and rMOR-1C1 mRNA. This effect of chronic morphine was completely absent in females. Increased density of MOR protein in spinal cord of males accompanied the chronic morphine-induced increase in MOR variant mRNA, suggesting that it reflected an increase in corresponding receptor protein. These results suggest that tolerance/dependence results, at least in part, from different adaptational strategies in males and females. The signaling consequences of the unique composition of the C-terminus tip of rMOR-1C1 and rMOR-1B2 could point the way to defining the molecular components of sex-dependent tolerance and withdrawal mechanisms. Chronic systemic morphine increases levels of mRNA encoding two splice variants of mu-opioid receptor (MOR), MOR-1B2 and MOR-1C1, variants differing from rMOR-1 in their C-terminal (and phosphorylation sites therein) and thus possibly signaling sequelae. This adaptation is sex-specific. It occurs in the spinal cord of males, but not females, indicating the importance of sex-specific mechanisms for and treatments of tolerance and addiction.

Antibodies in proteomics I: generating antibodies.

The explosion in genome sequencing, and in subsequent DNA array experiments, has provided extensive information on gene sequence, organization and expression. This has resulted in a desire to perform similarly broad experiments on all the proteins encoded by a genome. Panels of specific antibodies, or other binding ligands, will be essential tools in this endeavour. Because traditional immunization will be unlikely to generate antibodies in sufficient quantity, and of the required quality and reproducibility, in vitro selection methods will probably be used. This review--the first of two--examines the strategies available for in vitro antibody selection. The second review discusses the adaptation of these methods to high throughput and the uses to which antibodies, once derived, can be put.

Antibodies in proteomics II: screening, high-throughput characterization and downstream applications.

There are many ways in which the use of antibodies and antibody selection can be improved and developed for high-throughput characterization. Standard protocols, such as immunoprecipitation, western blotting and immunofluorescence, can be used with antibody fragments generated by display technologies. Together with novel approaches, such as antibody chips and intracellular immunization, these methods will yield useful proteomic data following adaptation of the protocols for increased reliability and robustness. To date, most work has focused on the use of standard, well-characterized commercial antibodies. Such protocols need to be adapted for broader use, for example, with antibody fragments or other binders generated by display technologies, because it is unlikely that traditional approaches will provide the required throughput.

Predicting antigenic peptides suitable for the selection of phage antibodies.

Selection from phage antibody libraries can be considered to be an in vitro immune system in which the antibody response is reduced to the bare minimum of antigen recognition. Using selections of antibodies on peptides from a phage antibody library, we investigated what constitutes peptide antigenicity in the context of the antibody-protein binding site. We selected polyclonal antibodies in a high throughput format against 44% of 90 overlapping peptides derived from three different proteins. Of these, 33% of peptides (epitopic peptides) were able to select antibodies that recognized the protein from which the peptides were derived. Although no algorithm was able to predict all epitopic peptides, solvent accessibility was the best predictor in this cell-free antibody selection context. We subsequently applied solvent accessibility to successfully predict epitopic peptides from p53 and Znf217, and showed that such peptide selected single-chain antibodies were able to recognize soluble p53 in ELISA and Znf217 in a western blot. This is likely to have considerable utility in functional genomics and proteomics where it should be possible to select antibodies against gene products on the basis of deduced amino acid sequence in a high throughput fashion.