Regulation of Foxo-1 and the angiopoietin-2/Tie2 system by shear stress

Transcription factor Foxo-1 can be inactivated via Akt-mediated phosphorylation. Since shear stress activates Akt, we determined whether Foxo-1 and the Foxo-1-dependent, angiogenesis-related Ang-2/Tie2-system are influenced by shear stress in endothelial cells. Expression of Foxo-1 and its target genes p27Kip1 and Ang-2 was decreased under shear stress (6dyn/cm(2), 24h), nuclear exclusion of Foxo-1 by phosphorylation increased. eNOS and Tie2 were upregulated. No effects on Ang-1 expression were detected. In conclusion, Foxo-1 and Ang-2/Tie2 are part of the molecular response to shear stress, which may regulate angiogenesis.

Evaluation of the colorimetric VITEK 2 card for identification of gram-negative nonfermentative rods: comparison to 16S rRNA gene sequencing

Ninety strains of a collection of well-identified clinical isolates of gram-negative nonfermentative rods collected over a period of 5 years were evaluated using the new colorimetric VITEK 2 card. The VITEK 2 colorimetric system identified 53 (59%) of the isolates to the species level and 9 (10%) to the genus level; 28 (31%) isolates were misidentified. An algorithm combining the colorimetric VITEK 2 card and 16S rRNA gene sequencing for adequate identification of gram-negative nonfermentative rods was developed. According to this algorithm, any identification by the colorimetric VITEK 2 card other than Achromobacter xylosoxidans, Acinetobacter sp., Burkholderia cepacia complex, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia should be subjected to 16S rRNA gene sequencing when accurate identification of nonfermentative rods is of concern.

Monitoring protein-protein interactions between the mammalian integral membrane transporters and PDZ-interacting partners using a modified split-ubiquitin membrane yeast two-hybrid system

PDZ-binding motifs are found in the C-terminal tails of numerous integral membrane proteins where they mediate specific protein-protein interactions by binding to PDZ-containing proteins. Conventional yeast two-hybrid screens have been used to probe protein-protein interactions of these soluble C termini. However, to date no in vivo technology has been available to study interactions between the full-length integral membrane proteins and their cognate PDZ-interacting partners. We previously developed a split-ubiquitin membrane yeast two-hybrid (MYTH) system to test interactions between such integral membrane proteins by using a transcriptional output based on cleavage of a transcription factor from the C terminus of membrane-inserted baits. Here we modified MYTH to permit detection of C-terminal PDZ domain interactions by redirecting the transcription factor moiety from the C to the N terminus of a given integral membrane protein thus liberating their native C termini. We successfully applied this "MYTH 2.0" system to five different mammalian full-length renal transporters and identified novel PDZ domain-containing partners of the phosphate (NaPi-IIa) and sulfate (NaS1) transporters that would have otherwise not been detectable. Furthermore this assay was applied to locate the PDZ-binding domain on the NaS1 protein. We showed that the PDZ-binding domain for PDZK1 on NaS1 is upstream of its C terminus, whereas the two interacting proteins, NHERF-1 and NHERF-2, bind at a location closer to the N terminus of NaS1. Moreover NHERF-1 and NHERF-2 increased functional sulfate uptake in Xenopus oocytes when co-expressed with NaS1. Finally we used MYTH 2.0 to demonstrate that the NaPi-IIa transporter homodimerizes via protein-protein interactions within the lipid bilayer. In summary, our study establishes the MYTH 2.0 system as a novel tool for interactive proteomics studies of membrane protein complexes.

Barium isotope fractionation during witherite (BaCO3) dissolution, precipitation and at equilibrium

This study examines the behavior of Ba isotope fractionation between witherite and fluid during mineral dissolution, precipitation and at chemical equilibrium. Experiments were performed in batch reactors at 25 oC in 10-2 M NaCl solution where the pH was adjusted by continuous bubbling of a water saturated gas phase of CO2 or atmospheric air. During witherite dissolution no Ba isotope fractionation was observed between solid and fluid. In contrast, during witherite precipitation, caused by a pH increase, a preferential uptake of the lighter 134Ba isotopomer in the solid phase was observed. In this case, the isotope fractionation factor αwitherite-fluid is calculated to be 0.99993 ± 0.00004 (or Δ137/134Bawitherite-fluid ≈ -0.07 ± 0.04 ‰, 2sd). The most interesting feature of this study, however, is that after the attainment of chemical equilibrium, the Ba isotope composition of the aqueous phase is progressively becoming lighter, indicating a continuous exchange of Ba2+ ions between witherite and fluid. Mass balance calculations indicate that the detachment of Ba from the solid is not only restricted to the outer surface layer of the solid, but affects several (~7 unit cells) subsurface layers of the crystal. This observation comes in excellent agreement with the concept of a dynamic system at chemical equilibrium in a mineral-fluid system, denoting that the time required for the achievement of isotopic equilibrium in the witherite-fluid system is longer compared to that observed for chemical equilibrium. Overall, these results indicate that the isotopic composition of Ba bearing carbonates in natural environments may be altered due to changes in fluid composition without a net dissolution/precipitation to be observed.

Annexins as intracellular calcium sensors

The annexins are a multigene family of Ca(2+)- and charged phospholipid-binding proteins. Although they have been ascribed with diverse functions, there is no consensus about the role played by this family as a whole. We have mapped the Ca(2+)-induced translocations of four members of the annexin family and of two truncated annexins in live cells, and demonstrated that these proteins interact with the plasma membrane as well as with internal membrane systems in a highly coordinated manner. Annexin 2 was the most Ca(2+) sensitive of the studied proteins, followed by annexins 6, 4 and 1. The calcium sensitivity of annexin 2 increased further following co-expression with S100A10. Upon elevation of [Ca(2+)](i), annexins 2 and 6 translocated to the plasma membrane, whereas annexins 4 and 1 also became associated with intracellular membranes and the nuclear envelope. The NH(2)-terminus had a modulatory effect on plasma membrane binding: its truncation increased the Ca(2+) sensitivity of annexin 1, and decreased that of annexin 2. Given the fact that several annexins are present within any one cell, it is likely that they form a sophisticated [Ca(2+)] sensing system, with a regulatory influence on other signaling pathways.