Light-speed genomics

A pico-scale reaction system serves as the basis for a new generation of high-throughput sequencing machines that promise to bring genome-center power down to the laboratory level.     

Coke and chips--a winning combination?

An aptamer-based electronic sensor for the detection of cocaine demonstrates the capabilities of a sensitive and generalizable new platform for small-molecule recognition.     

A unique spatial arrangement of the snRNPs within the native spliceosome emerges from in silico studies

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Highlights? We present a computational procedure for fast matching of thousands of conformers ? We obtained a unique model localizing the snRNPs within the native spliceosome ? The 5 snRNPs are accommodated mostly within the large subunit of the spliceosome ? The large spliceosomal cavity emerges as the site of mRNA binding and splicing     

Dominant thermodynamic role of the third independent receptor binding site in the receptor-associated protein RAP.

The 39 kDa receptor-associated protein (RAP) is a three-domain escort protein in the secretory pathway for several members of the low-density lipoprotein receptor (LDLR) family of endocytic receptors, including the LDLR-related protein (LRP). The minimal functional unit of LRP required for efficient binding to RAP is composed of complement-type repeat (CR)-domain pairs, located in clusters on the extracellular part of LRP. Here we investigate the binding of full-length RAP and isolated RAP domains 1-3 to an ubiquitin-fused CR-domain pair consisting of the fifth and sixth CR domains of LRP (U-CR56). As shown by isothermal titration calorimetric analysis of simple RAP domains as well as adjoined RAP domains, all three RAP domains bind to this CR-domain pair in a noncooperative way. The binding of U-CR56 to RAP domains 1 and 2 is (at room temperature) enthalpically driven with an entropy penalty (K(D) = 2.77 x 10(-6) M and 1.85 x 10(-5) M, respectively), whereas RAP domain 3 binds with a substantially lower enthalpy, but is favored due to a positive entropic contribution (K(D) = 1.71 x 10(-7) M). The heat capacity change for complex formation between RAP domain 1 and the CR-domain pair is -1.65 kJ K(-1) mol(-1). There is an indication of a conformational change in RAP domain 3 upon binding in the surface plasmon resonance analysis of the interaction. The different mechanisms of binding to RAP domains 1 and 3 are further substantiated by the different effects on binding of mutations of the Asp and Trp residues in the LRP CR5 or CR6 domains, which are important for the recognition of several ligands.     

Increased IRP1 and IRP2 RNA binding activity accompanies a reduction of the labile iron pool in HFE-expressing cells.

Iron regulatory proteins (IRPs), the cytosolic proteins involved in the maintenance of cellular iron homeostasis, bind to stem loop structures found in the mRNA of key proteins involved iron uptake, storage, and metabolism and regulate the expression of these proteins in response to changes in cellular iron needs. We have shown previously that HFE-expressing fWTHFE/tTA HeLa cells have slightly increased transferrin receptor levels and dramatically reduced ferritin levels when compared to the same clonal cell line without HFE (Gross et al., 1998, J Biol Chem 273:22068-22074). While HFE does not alter transferrin receptor trafficking or non-transferrin mediated iron uptake, it does specifically reduce (55)Fe uptake from transferrin (Roy et al., 1999, J Biol Chem 274:9022-9028). In this report, we show that IRP RNA binding activity is increased by up to 5-fold in HFE-expressing cells through the activation of both IRP isoforms. Calcein measurements show a 45% decrease in the intracellular labile iron pool in HFE-expressing cells, which is in keeping with the IRP activation. These results all point to the direct effect of the interaction of HFE with transferrin receptor in lowering the intracellular labile iron pool and establishing a new set point for iron regulation within the cell.     

Phylogenetic comparisons of the branched-chain alpha-ketoacid dehydrogenase complex

1. Antibodies against the E1b and E2b components of bovine branched-chain alpha-ketoacid (BCKA) dehydrogenase (BCKAD) complex completely inhibited BCKA oxidation in mammalian and avian mitochondria. BCKA oxidation by salmonid mitochondria was less affected and the enzyme from Pseudomonas putida was unaffected. 2. In rodents, anti-E1b E2b IgG inhibited oxidation of all three BCKA in a similar dose-dependent manner: oxidation of alpha-ketobutyrate and alpha-keto-y-methiolbutyrate was also partially inhibited. 3. Except for the salmonid BCKAD, a similar Mr for the E2b and E1b alpha proteins was observed in these species. 4. After digestion with V-8 protease similar immunoreactive peptides were observed for the human and rodent complex.