Interaction of DNA polymerase β with GRIP1 during meiosis

DNA polymerase beta (pol beta) is an essential enzyme that has been shown to localize as discrete foci to the synaptonemal complex during meiosis in the mouse. To identify proteins that associate with pol beta during meiosis, we employed the yeast two-hybrid screen. Here we show that a multiple PDZ domain-containing protein, the glutamate receptor interacting protein 1 (GRIP1), interacts specifically with pol beta. The PDZ domain-containing proteins, including GRIP1, act as scaffolds to promote rapid and localized biochemical events that require the interaction of multiple proteins. GRIP1 localizes to discrete foci on meiotic bivalents of both spermatocyte and oocyte nuclei, and colocalizes with pol beta. Together, these findings provide evidence that GRIP1 interacts with pol beta during meiosis. Our findings are consistent with the possibility that GRIP1 acts as a scaffold to promote interaction between proteins that function during meiosis.     

Benzylfumaric, benzylmaleic, and Z- and E-phenylitaconic acids: synthesis, characterization, and correlation with a metabolite generated by Azoarcus tolulyticus Tol-4 during anaerobic toluene degradation.

E-Phenylitaconic acid has been isolated as a metabolite generated by Azoarcus tolulyticus Tol-4 along with benzylsuccinic acid during anaerobic degradation of toluene. Strain Tol-4 converted 1 to 2% of toluene carbon to E-phenylitaconate and benzylsuccinate (10:1). The identification of E-phenylitaconic acid was based on 1H nuclear magnetic resonance (NMR) characterization of degradation products derived from 13C-labeled toluene followed by comparison of spectroscopic and chromatographic data for the isolated, unlabeled metabolite with those for chemically synthesized benzylfumaric acid, benzylmaleic acid, E-phenylitaconic acid, and Z-phenylitaconic acid. Spectroscopic comparisons included 1H NMR, 13C NMR, and nuclear overhauser effect correlations. High-pressure liquid chromatography (HPLC) retention times and HPLC coinjections with synthetic dioic acids provided another reliable line of evidence for structure assignment. The formation of E-phenylitaconic acid differs from previous reports of benzylfumaric acid generation along with benzylsuccinic acid during anaerobic microbial degradation of toluene. This has important implications relevant to elaboration of the metabolic route for anaerobic toluene degradation by strain Tol-4 and related organisms. Similar amounts of E-phenylitaconic acid were also produced by seven other strains of A. tolulyticus.     

Phospholipid Composition of Venezuelan Equine Encephalitis Virus

Phospholipid analyses of Venezuelan equine encephalitis virus showed that virus propagated in L-cell monolayers had a higher sphingomyelin content and a lower phosphatidylcholine content than virus grown in chick fibroblast monolayers. Virus of L-cell origin also was found to possess greater thermal stability than virus derived from the chick fibroblast cell.     

Identification of a conserved interface between PUF and CPEB proteins

Members of the PUF (Pumilio and FBF) and CPEB (cytoplasmic polyadenylation element-binding) protein families collaborate to regulate mRNA expression throughout eukaryotes. Here, we focus on the physical interactions between members of these two families, concentrating on Caenorhabditis elegans FBF-2 and CPB-1. To localize the site of interaction on FBF-2, we identified conserved amino acids within C. elegans PUF proteins. Deletion of an extended loop containing several conserved residues abolished binding to CPB-1. We analyzed alanine substitutions at 13 individual amino acids in FBF-2, each identified via its conservation. Multiple single point mutations disrupted binding to CPB-1 but not to RNA. Position Tyr-479 was particularly critical as multiple substitutions to other amino acids at this position did not restore binding. The complex of FBF-2 and CPB-1 repressed translation of an mRNA containing an FBF binding element. Repression required both proteins and was disrupted by FBF-2 alleles that failed to bind CPB-1 or RNA. The equivalent loop in human PUM2 is required for binding to human CPEB3 in vitro, although the primary sequences of the human and C. elegans PUF proteins have diverged in that region. Our findings define a key region in PUF/CPEB interactions and imply a conserved platform through which PUF proteins interact with their protein partners.     

Design of a novel class of biphenyl CETP inhibitors

A new class of CETP inhibitors was designed and prepared. These compounds are potent both in vitro and in vivo. The most active compound (12d) has shown an ability to raise HDL significantly in transgenic mouse PD model.