A high-yield, rapid and non-denaturing purification protocol for baculovirus recombinant wildtype pS3 is described. Gel-filtration chromatography and chemical cross-linking experiments indicated that purified pS3 assembles into multimeric forms ranging from tetramer to higher oligomers. A gel-mobility-shift assay and protein-DNA cross-linking studies demonstrated that purified baculovirus recombinant pS3 binds to consensus DNA target as a dimer but that additional pS3 molecules may then associate with the preformed pS3-dimer-DNA complexes to form larger pS3 DNA complexes. These observations suggest that the pS3 tetramers and higher oligomers that form the minimal pS3 association in solution dissociate upon DNA binding to form pS3 dimer-DNA complexes. Binding of the mAB PAb 421 to the oligomerization-promoting domain on pS3 stimulated sequentially formation of both pS3-dimer-DNA and larger pS3 -DNA complexes. This observation suggests that factors may exist in vivo that could participate in the formation and the stabilization of the various pS3-DNA complexes. Further characterization of the purified pS3 revealed that the protein possesses highly reactive cysteine residues. We show that intrachain disulfide bonds form within the purified pS3 molecules during storage in the absence of reducing agent. Zn2+ binding to pS3 protect sulfhydryl groups from oxidation. Cysteine oxidation by intramolecular disulfide-bond formation did not modify the wild-type immunoreactive phenotype of the pS3 protein but totally inhibited its DNA-binding activities. The oxidation of the pS3 cysteine residues was also observed for nuclear pS3 in baculovirus-infected insect cells. The redox status of the nuclear pS3 regulates its DNA-binding activity in vitro confirming the essential role of the reduced state of cysteine residues in pS3 for detectable DNA-binding activity. pS3 protein is a product of a tumour-supressor gene and contributes to growth arrest of cells (for reviews on the tumour-suppressor activity of pS3 see Vogelstein and Kinzler, 1992;Zambetti and Levine, 1993). Most human tumours have an abnormal or inactivated pS3 protein. Mutations are multiple and are mostly single missense point mutations within the conserved coding sequences. Mutations result in conformational changes in the protein, first revealed by differences in immunoreactivity toward the monoclonal antibodies wild-type specific PAb 246 and mutant specific PAb 240 (Yewdell et al., 1986;Gannon et al., 1990). Furthermore, mutant pS3 have lost or reduced DNA-binding activity (ElDeiry et al., 1992;Friedman et al., 1993). The understanding at the molecular level of the differences in immunoreactivities and activities between wild-type pS3 and mutant pS3 requires a better characterization of the biochemical and conformational properties of the proteins. Such studies are vital steps for further development of therapeutic strategies designed to restore tumour-suppressor activity of the protein in transformed cells.

BackgroundHomeostatic turnover of the extracellular matrix conditions the structure and function of the healthy lung. In lung transplantation, long-term management remains limited by chronic lung allograft dysfunction, an umbrella term used for a heterogeneous entity ultimately associated with pathological airway and/or parenchyma remodeling.ObjectiveThis study assessed whether the local cross-talk between the pulmonary microbiota and host cells is a key determinant in the control of lower airway remodeling posttransplantation.MethodsMicrobiota DNA and host total RNA were isolated from 189 bronchoalveolar lavages obtained from 116 patients post lung transplantation. Expression of a set of 11 genes encoding either matrix components or factors involved in matrix synthesis or degradation (anabolic and catabolic remodeling, respectively) was quantified by real-time quantitative PCR. Microbiota composition was characterized using 16S ribosomal RNA gene sequencing and culture.ResultsWe identified 4 host gene expression profiles, among which catabolic remodeling, associated with high expression of metallopeptidase-7, -9, and -12, diverged from anabolic remodeling linked to maximal thrombospondin and platelet-derived growth factor D expression. While catabolic remodeling aligned with a microbiota dominated by proinflammatory bacteria (eg, Staphylococcus, Pseudomonas, and Corynebacterium), anabolic remodeling was linked to typical members of the healthy steady state (eg, Prevotella, Streptococcus, and Veillonella). Mechanistic assays provided direct evidence that these bacteria can impact host macrophage-fibroblast activation and matrix deposition.ConclusionsHost-microbes interplay potentially determines remodeling activities in the transplanted lung, highlighting new therapeutic opportunities to ultimately improve long-term lung transplant outcome.

Background. The poor correlation between allergen-specific immunoglobulin E (asIgE) and clinical signs of allergy in helminth infected populations suggests that helminth infections could protect against allergy by uncoupling asIgE from its effector mechanisms. We investigated this hypothesis in Ugandan schoolchildren coinfected with Schistosoma mansoni and hookworm.Methods. Skin prick test (SPT) sensitivity to house dust mite allergen (HDM) and current wheeze were assessed pre-anthelmintic treatment. Nonspecific (anti-IgE), helminth-specific, and HDM-allergen-specific basophil histamine release (HR), plus helminth- and HDM-specific IgE and IgG4 responses were measured pre- and post-treatment.Results. Nonspecific- and helminth-specific-HR, and associations between helminth-specific IgE and helminth-specific HR increased post-treatment. Hookworm infection appeared to modify the relationship between circulating levels of HDM-IgE and HR: a significant positive association was observed among children without detectable hookworm infection, but no association was observed among infected children. In addition, hookworm infection was associated with a significantly reduced risk of wheeze, and IgG4 to somatic adult hookworm antigen with a reduced risk of HDM-SPT sensitivity. There was no evidence for S. mansoni infection having a similar suppressive effect on HDM-HR or symptoms of allergy.Conclusions. Basophil responsiveness appears suppressed during chronic helminth infection; at least in hookworm infection, this suppression may protect against allergy.

No abstract