The primary structure of human apolipoprotein (apo) B-48 has been deduced and shown by a combination of DNA excess hybridization, sequencing of tryptic peptides, cloned complementary DNAs, and intestinal messenger RNAs (mRNAs) to be the product of an intestinal mRNA with an in-frame UAA stop codon resulting from a C to U change in the codon CAA encoding Gln2153 in apoB-100 mRNA. The carboxyl-terminal Ile2152 of apoB-48 purified from chylous ascites fluid has apparently been cleaved from the initial translation product, leaving Met2151 as the new carboxyl-terminus. These data indicate that approximately 85% of the intestinal mRNAs terminate within approximately 0.1 to 1.0 kilobase downstream from the stop codon. The other approximately 15% have lengths similar to hepatic apoB-100 mRNA even though they have the same in-frame stop codon. The organ-specific introduction of a stop codon to a mRNA appears unprecedented and might have implications for cryptic polyadenylation signal recognition and RNA processing.

EnvZ, a histidine kinase/phosphatase in Escherichia coli, responds to the osmolarity changes in the medium by regulating the phosphorylation state of the transcription factor OmpR, which controls the expression levels of outer membrane porin proteins OmpF and OmpC. Although both ompR and envZ genes are located on the ompB locus under the control of the ompB promoter and transcribed as a single polycistronic mRNA, the expression of envZ is known to be significantly less than ompR. However, to date no accurate estimation for the amounts of EnvZ and OmpR in the cell has been carried out. Here we examined the levels of EnvZ and OmpR in the wild-type strain MC4100 by quantitative Western blot analysis using anti-OmpR and anti-EnvZc (cytoplasmic domain of EnvZ) antisera. It was observed that during exponential growth in L-broth medium there were ϳ3500 and 100 molecules per cell of OmpR and EnvZ, respectively. The levels of OmpR and EnvZ in MC4100 cells grown in a high osmolarity medium (nutrient broth with 20% sucrose) were about the same as those grown in L-broth, whereas they were 1.7-fold higher than those in a low osmolarity medium (nutrient broth). With His 10 -OmpR, we also determined that the K d value for the EnvZc-OmpR complex formation is 1.20 ؎ 0.17 M. On the basis of these results, the molecular mechanism of osmoregulation of ompF and ompC is discussed.

The structure of the human hepatic triglyceride lipase gene was determined from multiple cosmid clones. All the exons, exon-intron junctions, and 845 bp of the 5' and 254 bp of the 3' flanking DNA were sequenced. Comparison of the exon sequences to three previously published cDNA sequences revealed differences in the sequence of the codons for residues 133, 193, 202, and 234 that may represent sequence polymorphisms. By primer extension, hepatic lipase mRNA initiates at an adenine 77 bases upstream of the translation initiation site. The hepatic lipase gene spans over 60 kb containing 9 exons and 8 introns, the latter being all located within the region encoding the mature protein. The exons are all of average size (118-234 bp). Exon 1 encodes the signal peptide, exon 4, a region that binds to the lipoprotein substrate, and exon 5, an evolutionarily highly conserved region of potential catalytic function, and exons 6 and 9 encode sequences rich in basic amino acids thought to be important in anchoring the enzyme to the endothelial surface by interacting with acidic domains of the surface glycosaminoglycans. The human lipoprotein lipase gene has been recently reported to have an identical exon-intron organization containing the analogous structural domains [Deeb & Peng (1989) Biochemistry 28, 4131-4135]. Our observations strongly support the common evolutionary origin of these two lipolytic enzymes.

SummaryEnvZ is a sensory histidine kinase in Escherichia coli to regulate the phosphorylation of OmpR, its cognate response regulator, required for the expression of genes for outer membrane porin proteins. Here, we re-examined the recent paper Mattison and Kenney, in which the authors reported that phosphorylated OmpR (OmpR-P) is unable to bind to EnvZ, thus casting doubts on the role of the EnvZ phosphatase activity in vivo . Using an identical method, the K d value for the interaction of the fluorescein-labelled OmpR ( Fl -OmpR) with EnvZc was determined to be 1.96 ± ± ± ± 0.28 m m m m M. We demonstrated that OmpR-P as well as OmpR inhibited the interaction of Fl -OmpR with EnvZc. Their 50% inhibitory concentrations were 1.09 ± ± ± ± 0.25 m m m m M and 0.89 ± ± ± ± 0.14 m m m m M, respectively, under the conditions used. The interaction between His-10-OmpR and EnvZc was also inhibited almost equally with OmpR-P and OmpR. Fluorescein labelling of OmpR was highly heterogeneous as detected by mass spectrometry, even though it slightly affected the OmpR phosphorylation (kinase) and the dephosphorylation of OmpR-P (phosphatase), indicating that EnvZc is able to interact with Fl -OmpR or Fl -OmpR-P as well as with OmpR or OmpR-P as a substrate. We demonstrated that OmpR-P is able to interact with EnvZc with a similar affinity to OmpR and serves as an effective substrate for the EnvZ phosphatase. These findings support the hypothesis that osmotic signals regulate the level of the cellular concentration of OmpR-P by modulating the ratio of kinase to phosphatase activity of the bifunctional enzymatic activities of EnvZ.

The estrogen-related receptor a (ERRa) was one of the first orphan receptors identified through a search for genes encoding proteins related to the steroid nuclear receptor, Estrogen Receptor a (ERa). The physiological role of ERRa has not yet been established nor has a natural ligand been elucidated. Importantly, research indicates that ERRa may be a novel drug target to treat breast cancer and/ or metabolic disorders. A homogeneous time-resolved fluorescence (HTRF) assay has been developed to screen for ERRa-specific antagonists. This assay uses the ERR ligand binding domain and the coactivator interaction domain of Proliferator-activated Receptor g Coactivator-1a (PGC-1a) to examine the ability of compounds to antagonize the constitutive interaction between ERRa and the coactivator. A dissociationenhanced lanthanide fluorescence immunoassay (DELFIA) was also created to counter screen compounds identified in the HTRF screen. Here we report the discovery of high-affinity ERRa subtype selective antagonists. Additionally, a homology model of ERRa in an antagonist conformation has been developed and after subsequent docking studies, we offer a model showing the molecular determinants that suggest why our novel tri-cyclic antagonist, N-[(2Z)-3-(4,5-dihydro-1,3-thiazol-2-yl)-1,3-thiazolidin-2-yl idene]-5 H dibenzo [a,d][7]annulen-5-amine, binds to ERRa with high affinity but does not bind to either ERRb or ERRg. Drug Dev Res 69:203-218, 2008.