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Polyclonal antibodies that had been raised against particular PDI (protein disulphide-isomerase) family proteins did not cross-react with other PDI family proteins. To evade immune tolerance to the important self-motif Cys-Xaa-Xaa-Cys, which is present in PDI family proteins, we used the phage display library [established by Griffiths, Williams, Hartley, Tomlinson, Waterhouse, Crosby, Kontermann, Jones, Low, Allison et al. (1994) EMBO J. 13, 3245-3260] to isolate successfully the phage antibodies that can cross-react with human and bovine PDIs, human P5, human PDI-related protein and yeast PDI. By measuring the binding of scFv (single-chain antibody fragment of variable region) to synthetic peptides and to mutants of PDI family proteins in a surface plasmon resonance apparatus, we identified clones that recognized sequences containing the CGHC motif or the CGHCK sequence. By using the isolated phage antibodies, we demonstrated for the first time that a lysine residue following the CXXC motif significantly increases the isomerase activities of PDI family proteins. Moreover, we demonstrated that the affinity of isolated scFvs for mutant PDI family proteins is proportional to the isomerase activities of their active sites.

The T-box transcription factors T-bet and eomesodermin (Eomes) have been shown to regulate the lineage-specific expression of interferon-c (IFN-c). However, in contrast to T-bet, the role of Eomes in the expression of IFN-c remains unclear. In this study, we investigated the Eomes-dependent expression of IFN-c in the mouse thymoma BW5147 and EL4 cells, which do not express T-bet or Eomes. The ectopic expression of Eomes induced BW5147 and EL4 cells to produce IFN-c in response to phorbol 12-myristate 13-acetate (PMA) and ionomycin (IM). In BW5147 cells, Eomes augmented luciferase activity driven by the Ifng promoter encoding from -2500 to +113 bp; however, it was not increased by a stimulation with PMA and IM. A chromatin immunoprecipitation assay showed that Eomes bound to the Ifng promoter and conserved noncoding sequence (CNS) -22 kb across the Ifng locus with high efficacy in BW5147 cells. Moreover, Eomes increased permissive histone modifications in the Ifng promoter and multiple CNSs. The stimulation with PMA and IM greatly augmented Eomes binding to CNS-54, CNS-34, CNS+19 and CNS+30, which was inhibited by FK506. These results indicated that Eomes bound to the Ifng promoter and multiple CNSs in stimulationdependent and stimulation-independent manners.

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Background and objectiveIt has been reported that physicians spend about 50% of their time in the outpatient department doing nonface-to-face patient work, such as charting and desk activities. Work outside of patient consultations is often considered a burden. In this study, we aimed to examine whether the use of pre-consultation tablet-based questionnaires for first-visit diabetic patients had any impact on the time spent for consultation. MethodsThe sole participant was a diabetologist with more than 20 years of experience. The time spent in the clinic was compared via a single-case experimental design (ABAB) using paper-and tablet-based questionnaires for a total of 20 first-visit diabetic patients. ResultsThe median pre-clinical time without patients was significantly shorter in the tablet group than in the paper group (two minutes and 45 seconds vs. five minutes and 39 seconds; p=0.003). The median clinical time with patients in the tablet group was significantly longer than that in the paper group (19 minutes and 37 seconds vs. 11 minutes and 25 seconds; p=0.026). The total clinical time was not significantly different between the two groups (p=0.25). ConclusionsOur results suggest that tablet-based pre-consultation questionnaires may have an impact on the allocation of time for medical examinations and improve the quality of diabetes care.