Activation of the phagocyte NADPH oxidase involves a conformational change in Nox2. The effector in this process is p67phox and there is evidence for a change in the configuration of p67phox being required for binding to Nox2. To study this, we measured binding of p67phox to a library of Nox2 peptides and binding of NusA–Nox2 fusion proteins to p67phox. We found, serendipitously, that deletion of residues 259–279 in p67phox (p67phoxΔ(259–279)), endowed it with the ability to bind selectively to Nox2 peptide 369–383 (peptide 28). There was no binding to scrambled Nox2 peptide 28 and to Nox4 peptide 28. Binding was cysteine independent and resistant to reducing and alkylating agents. Truncations of peptide 28 revealed that the actual binding site consisted of residues 375–383. Binding of p67phoxΔ(259–279) to peptide 28 was mimicked by that of a (p67phox‐RacGTP) chimera. Both p67phoxΔ(259–279) and the (p67pho–RacGTP) chimera bound a NusA–Nox2 fusion protein, comprising residues 375–383. Specific single residue deletion mutants, within the p67phox sequence 259–279, were also bound to Nox2 peptide 28. Peptides synthesized to correspond to the 259–279 sequence in p67phox, were found to autobind p67phox, suggesting that an intramolecular bond exists in p67phox, one pole of which was located within residues 259–279. We conclude that “resting” p67phox exists in a “closed” conformation, generated by an intramolecular bond. Deletion of specific residues within the 259–279 sequence, in vitro, or interaction with RacGTP, in vivo, causes “opening” of the bond and results in binding of p67phox to a specific, previously unknown, site in Nox2.

Three-hybrid assays for the analysis of RNA^protein interactions in vivo are usually used, due to technical limitations, only for RNA baits that do not contain runs of four or more consecutive uridines. The present study provides the ¢rst example of a three-hybrid analysis of synthetic and natural uridinerich RNA sequences. The use of the three-hybrid assay enabled us to demonstrate a functional di¡erence between two closely related proteins, heterogeneous nuclear ribonucleoprotein C1 (hnRNP-C1) and hnRNP-C2. The hnRNP-C2 protein, an alternatively spliced variant of hnRNP-C1, contains an additional 13 amino acids between an RNA binding domain (RBD) and a basic leucine zipper-like motif (bZLM), also implied in RNA binding. This study shows that (i) for e⁄cient binding of hnRNP-C1/C2 to RNA, the context of the U-stretch is more important than the stretch itself; (ii) both the RBD and the bZLM bind RNA independently ; and (iii) the C2-related 13-amino acid insert enhances the speci¢city of either the RBD, the bZLM, or the full-length protein towards its ligand, allowing it to bind only the most high-a⁄nity sequences while discriminating against those that do not perfectly match this category. The three-hybrid system is a powerful tool to work out the functional signi¢cance of peptide 'modules' within RNA binding proteins generated by alternative splicing. ß

Background: Closed system drug-transfer devices (CSTD) allow the reconstitution of hazardous drugs into infusion bags, while preserving the sterility of the product and preventing the escape of liquids and aerosols into the environment. Air-cleaning technology CSTD is based on an activated carbon filter and a membrane which enable maintaining the drug sterile by filtration of air entering the vial during pressure equalization. Objective: The study aimed to investigate if an air-cleaning CSTD can prevent liquid viral contamination by human coronavirus OC43 (HCoV-OC43). Methods: Chemfort TM CSTD with (intact) or without (control) a Toxi-Guard system was used to transfer liquids between an IV bag and an empty vial (a total of 5 liquid transfers) inside a sealed glove box contaminated by HCoV-OC43 aerosols. In addition, the vial adaptor was challenged by direct spray of HCoV-OC43 solution on the septum and filter areas. HCoV-OC43 RNA was extracted from samples of the transferred liquid and compared between the devices with or without a Toxi-Guard system. Results: Use of a CSTD with the Toxi-Guard system resulted in non-detectable cycle threshold (CT) values, indicative of no detectable HCoV-OC43RNA in the transferred liquid, even when the septa and filter areas were directly sprayed with HCoV-OC43 stock solution. In contrast, use of the CSTD with no Toxi-Guard system resulted in a detectable CT value of the transferred liquid. Conclusions: Using Chemfort CSTD with integral Toxi-Guard technology can prevent the introduction of microbial and airborne contaminants into the fluid path, thus potentially protecting patients from infection.