Expression levels in oligonucleotide microarray experiments depend on a potentially large number of factors, for example, treatment conditions, different probes, different arrays, and so on. To dissect the effects of these factors on expression levels, fixed-effects ANOVA methods have previously been proposed. Because we are not necessarily interested in estimating the specific effects of different probes and arrays, we propose to treat these as random effects. Then we only need to estimate their means and variances but not the effect of each of their levels; that is, we can work with a much reduced number of parameters and, consequently, higher precision for estimating expression levels. Thus, we developed a mixed-effects ANOVA model with some random and some fixed effects. It automatically accounts for local normalization between different arrays and for background correction. The method was applied to each of the 6,584 genes investigated in a microarray experiment on two mouse cell lines, PA6/S and PA6/8, where PA6/S enhances proliferation of Pre B cells in vitro but PA6/8 does not. To detect a set of differentially expressed genes (multiple testing problem), we applied the method of controlling the false discovery rate (FDR), which successfully identified 207 genes with significantly different expression levels.
Metabotropic glutamate (mGlu) receptors are a family of G-protein-coupled receptors that play central roles as modulators of both glutamatergic and other major neurotransmitter systems in CNS. Using molecular modeling, site-directed mutagenesis, [3H]LY354740 binding, [35S]GTPγS binding, and activation of GIRK current, we have been able to identify residues crucial for the binding of LY354740 and glutamate to rat mGlu2 receptors. Several of the crucial residues located in the binding site (Arg-57, Tyr-144, Tyr-216, Asp-295) have not been identified previously. We propose that the γ-carboxyl group of LY354740 forms H-bonds to Arg-57, whereas the α-carboxyl group forms an H-bond with the hydroxyl group of Ser-145. The α-amino group of LY354740 forms H-bonds to Asp-295 and to the side-chain hydroxyl group of Thr-168. In addition, Tyr-144 may establish a hydrophobic (C-H/π)–interaction with the bicyclo-hexane ring of LY354740. Furthermore, the mutation of residues Ser-148 and Arg-183, which are too remote for a direct interaction, affected the ligand affinity dramatically. These results suggest that Ser-148 and Arg-183 may be important for the 3D structure and/or are involved in closure of the domain. Finally, Asp-146, which is also remote from the binding site, was shown to be involved in the differential binding affinity of [3H]LY354740 for mGlu2 versus mGlu3 receptors. All the mGlu receptors except mGlu2 are activated by Ca2+ and have serine instead of aspartic acid at this position, which suggests a critical role of this aspartic acid residue in the binding properties of this unique receptor.
Kynureninase (L‐kynurenine hydrolase), a pyridoxal‐5′‐phosphate‐(pyridoxal‐ P )‐dependent enzyme, catalyses the cleavage of L‐kynurenine and L‐3‐hydroxykynurenine into anthranilic and 3‐hydroxyanthranilic acids, respectively. In this report, we describe the isolation of a cDNA clone encoding human kynureninase. Degenerate oligonucleotides designed from the amino acid sequences of peptides from rat liver kynureninase, were used as primers for reverse‐transcription PCR of rat kidney RNA. The resulting rat cDNA product was then used to screen a human hepatoma cell line (Hep G 2 ) cDNA library. Analysis of a positive cDNA clone showed the presence of an insert of 1651 nucleotides containing an open reading frame coding for a protein of 456 amino acids (theoretical molecular mass = 52357 Da). The predicted amino acid sequence of human kynureninase displayed high similarity to that reported for the rat enzyme and to a Saccharomyces cerevisiae gene product putatively ascribed to kynureninase. Profile analysis of kynureninase primary structure indicated the presence of a pyridoxal‐ P ‐binding site consensus sequence assigned to class‐V aminotransferases, with Lys276 being the residue binding the cofactor. RNA blot analysis of human tissues, including brain, showed the presence of an ≈2.0‐kb mRNA species in all tissues tested. A second mRNA species (≈2.6 kb) was also detected in some tissues. After transfection of HEK‐293 cells with the cDNA coding for kynureninase, the K m , values of l ‐kynurenine and dl ‐3‐hydroxykynurenine for the recombinant enzyme were 671±37 μM and 13.2±2.0 μM, respectively.
