Primary antibody response to keyhole limpet hemocyanin in rat as a model for immunotoxicity evaluation.

To address current regulatory expectations on immunotoxicity testing of new chemicals, we describe an animal model that measures the primary antibody response to the T-cell dependent antigen, keyhole limpet hemocyanin (KLH). Single immunization with KLH by either footpad (300microg/rat) or intravenous (300microg/kg) route in Sprague Dawley rats resulted in increased germinal center formation in the spleen and a robust anti-KLH IgM (70-388microg/ml) and IgG (230-470microg/ml) antibody response with peak detection on Days 5 and 14 post-immunization, respectively. Subcutaneous immunization with KLH (300microg/kg) resulted in a much weaker anti-KLH IgM and IgG (< or =20microg/ml) antibody response with no detectable increase in splenic germinal center formation. The utility of a rat KLH immunization model in detecting immunosuppression was evaluated with the known immunosuppressive drugs: cyclosporin, azathioprine and prednisolone. Rats, treated with drug at a maximum tolerated dose, were immunized with KLH by footpad or intravenous injection and serum samples were collected at various intervals up to 2 weeks post-immunization. Additional study parameters included terminal body weight, hematology and/or histopathology. All three drugs inhibited the IgM (60%) and IgG (> or =90%) antibody responses in the absence of overt toxicity based on evaluation of the standard toxicology parameters. In conclusion, measurement of a rat primary antibody response to KLH by ELISA is a reliable and readily standardized method for assessing immunotoxicity of pharmaceuticals.

Structure of Rev-erbalpha bound to N-CoR reveals a unique mechanism of nuclear receptor-co-repressor interaction.

Repression of gene transcription by the nuclear receptor Rev-erbalpha plays an integral role in the core molecular circadian clock. We report the crystal structure of a nuclear receptor-co-repressor (N-CoR) interaction domain 1 (ID1) peptide bound to truncated human Rev-erbalpha ligand-binding domain (LBD). The ID1 peptide forms an unprecedented antiparallel beta-sheet with Rev-erbalpha, as well as an alpha-helix similar to that seen in nuclear receptor ID2 crystal structures but out of register by four residues. Comparison with the structure of Rev-erbbeta bound to heme indicates that ID1 peptide and heme induce substantially different conformational changes in the LBD. Although heme is involved in Rev-erb repression, the structure suggests that Rev-erbalpha could also mediate repression via ID1 binding in the absence of heme. The previously uncharacterized secondary structure induced by ID1 peptide binding advances our understanding of nuclear receptor-co-repressor interactions.

Crystallization of protein-ligand complexes.

Obtaining diffraction-quality crystals has long been a bottleneck in solving the three-dimensional structures of proteins. Often proteins may be stabilized when they are complexed with a substrate, nucleic acid, cofactor or small molecule. These ligands, on the other hand, have the potential to induce significant conformational changes to the protein and ab initio screening may be required to find a new crystal form. This paper presents an overview of strategies in the following areas for obtaining crystals of protein-ligand complexes: (i) co-expression of the protein with the ligands of interest, (ii) use of the ligands during protein purification, (iii) cocrystallization and (iv) soaks.

Structural analyses reveal phosphatidyl inositols as ligands for the NR5 orphan receptors SF-1 and LRH-1.

Vertebrate members of the nuclear receptor NR5A subfamily, which includes steroidogenic factor 1 (SF-1) and liver receptor homolog 1 (LRH-1), regulate crucial aspects of development, endocrine homeostasis, and metabolism. Mouse LRH-1 is believed to be a ligand-independent transcription factor with a large and empty hydrophobic pocket. Here we present structural and biochemical data for three other NR5A members-mouse and human SF-1 and human LRH-1-which reveal that these receptors bind phosphatidyl inositol second messengers and that ligand binding is required for maximal activity. Evolutionary analysis of structure-function relationships across the SF-1/LRH-1 subfamily indicates that ligand binding is the ancestral state of NR5A receptors and was uniquely diminished or altered in the rodent LRH-1 lineage. We propose that phospholipids regulate gene expression by directly binding to NR5A nuclear receptors.