TcUBP1 is a trypanosome cytoplasmic RNA-binding protein containing a single, conserved RNA-recognition motif (RRM) domain involved in selective destabilization of U-rich mRNAs such as the Trypanosoma cruzi small mucin gene family mRNA, TcSMUG. TcUBP1 binds specific transcripts in vivo and co-localizes in the perinuclear part of the cell with components of the mRNA-stability determinant pathway such as poly(A)-binding protein 1 (PABP1) and TcUBP2, a closely related RRM-containing protein. In TcUBP proteins, the RRM domain is flanked by N-terminal Gln-rich and C-terminal Gly-Gln-rich extensions, which are involved in protein-protein interactions. In this work, we determined the solution structure of the TcUBP1 RRM domain by nuclear magnetic resonance (NMR) spectroscopy. The domain has a characteristic betaalphabetabetaalphabeta fold, consisting of a beta sheet composed of four antiparallel betastrands and two alpha helices packed against one face of the beta sheet. A unique aspect of TcUBP1 is the participation of a beta hairpin (beta4-beta5) in the beta sheet, resulting in an enlarged RNA-binding surface. Detailed analysis of the TcUBP1 interaction with a short single-stranded RNA derived from the 3' UTR of TcSMUG was carried out by titration experiments using both NMR spectroscopy and isothermal titration calorimetry. This analysis revealed that amino acids located within the beta hairpin (beta4-beta5) contribute to complex formation. This enlarged protein-RNA interface could compensate for the lack of additional RNA-binding domains in TcUBP1, as observed in many other RRM-containing proteins. The structure of TcUBP1 reveals new aspects of single RRM-RNA interactions and insight into how N- and C-terminal extensions can contribute to RNA binding.

In vertebrates, the positioning of the internal organs relative to the midline is asymmetric and evolutionarily conserved. A number of molecules have been shown to play critical roles in left-right patterning. Using representational difference analysis to identify genes that are differentially expressed on the left and right sides of the chick embryo, we cloned chick Claudin-1, an integral component of epithelial tight junctions. Here, we demonstrate that retroviral overexpression of Claudin-1, but not Claudin-3, on the right side of the chick embryo between HH stages 4 and 7 randomizes the direction of heart looping. This effect was not observed when Claudin-1 was overexpressed on the left side of the embryo. A small, but reproducible, induction of Nodal expression in the perinodal region on the right side of the embryo was noted in embryos that were injected with Claudin-1 retroviral particles on their right sides. However, no changes in Lefty, Pitx2 or cSnR expression were observed. In addition, Flectin expression remained higher in the left dorsal mesocardial folds of embryos with leftwardly looped hearts resulting from Claudin-1 overexpression on the right side of the embryo. We demonstrated that Claudin-1's C-terminal cytoplasmic tail is essential for this effect: mutation of a PKC phosphorylation site in the Claudin-1 C-terminal cytoplasmic domain at threonine-206 eliminates Claudin-1's ability to randomize the direction of heart looping. Taken together, our data provide evidence that appropriate expression of the tight junction protein Claudin-1 is required for normal heart looping and suggest that phosphorylation of its cytoplasmic tail is responsible for mediating this function. q

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The phosphoenolpyruvate-dependent carbohydrate transport system (PTS) couples uptake with phosphorylation of a variety of carbohydrates in prokaryotes. In this multienzyme complex, the enzyme II (EII), a carbohydrate-specific permease, is constituted of two cytoplasmic domains, IIA and IIB, and a transmembrane channel IIC domain. Among the five families of EIIs identified in Escherichia coli, the galactitol-specific transporter (II gat ) belongs to the glucitol family and is structurally the least wellcharacterized. Here, we used nuclear magnetic resonance (NMR) spectroscopy to solve the threedimensional structure of the IIB subunit (GatB). GatB consists of a central four-stranded parallel b-sheet flanked by a-helices on both sides; the active site cysteine of GatB is located at the beginning of an unstructured loop between b1 and a1 that folds into a P-loop-like structure. This structural arrangement shows similarities with other IIB subunits but also with mammalian low molecular weight protein tyrosine phosphatases (LMW PTPase) and arsenate reductase (ArsC). An NMR titration was performed to identify the GatA-interacting residues.

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The purpose of this study was to investigate the physicomechanical and dissolution properties of tablets containing controlled-release pellets prepared by a hot-melt extrusion and spheronization process. A powder blend of anhydrous theophylline, Eudragit Preparation 4135 F, and functional excipients was melt-extruded, pelletized, and then spheronized. The pellets were compressed into tablets using forces of 5, 10, 15, and 20 kN. Tablet diluents included microcrystalline cellulose, a mixture of spray-dried lactose and microcrystalline cellulose, modified food starch, and soy polysaccharides. The effective porosity of the compressed pellets was measured using mercury porosimetry and helium pycnometry, while the surface area was determined using Brunauer, Emmett, and Teller (BET) analysis. The disintegration time, hardness, and friability of compacts were determined. Drug release studies were performed according to USP 27 Apparatus 3 guidelines in 250 mL of medium (pH 1.0, 3.0, 5.0, 6.8, and 7.4) 37 degrees C and 20 dpm. Samples were analyzed by high pressure-liquid chromatography (HPLC). Effective porosity and surface area determinations of the melt-extruded pellets were not influenced by compression. The percent of theophylline released from rapidly disintegrating tablets was not affected by compression force or excipient selection, but tablets with prolonged disintegration times exhibited delayed drug release in acidic media. However, dissolution profiles of uncompressed pellets and all compacts were identical after transition from 0.1 N HCl to media increasing in pH from 3.0 to 7.4. Furthermore, pellet to filler excipient ratio and filler excipient selection did not influence the rate of drug release from compacts.