Rational design of a nonpeptide general chemical scaffold for reversible inhibition of PDZ domain interactions.

Novel small molecules were designed to specifically target the ligand-binding pocket of a PDZ domain. Iterative molecular docking and modeling allowed the design of an indole scaffold 10a as a reversible inhibitor of ligand binding. The 10a scaffold inhibited the interaction between MAGI-3 and PTEN and showed cellular activities that are consistent with the inhibition of NHERF-1 function.

Type I PDZ ligands are sufficient to promote rapid recycling of G Protein-coupled receptors independent of binding to N-ethylmaleimide-sensitive factor.

Molecular sorting of G protein-coupled receptors (GPCRs) between divergent recycling and lysosomal pathways determines the functional consequences of agonist-induced endocytosis. The carboxyl-terminal cytoplasmic domain of the beta2 adrenergic receptor (beta2AR) mediates both PDZ binding to Na+/H+ exchanger regulatory factor/ezrin/radixin/moesin-binding phosphoprotein of 50 kDa (NHERF/EBP50) family proteins and non-PDZ binding to the N-ethylmaleimide-sensitive factor (NSF). We have investigated whether PDZ interaction(s) are actually sufficient to promote rapid recycling of endocytosed receptors and, if so, whether PDZ-mediated sorting is restricted to the beta2AR tail or to sequences that bind NHERF/EBP50. The trafficking effects of short (10 residue) sequences differing in PDZ and NSF binding properties were examined using chimeric mutant receptors. The recycling activity of the beta2AR-derived tail sequence was not blocked by a point mutation that selectively disrupts binding to NSF, and naturally occurring PDZ ligand sequences were identified that do not bind detectably to NSF yet function as strong recycling signals. The carboxyl-terminal cytoplasmic domain of the beta1-adrenergic receptor, which does not bind either to NSF or NHERF/EBP50 and interacts selectively with a distinct group of PDZ proteins, promoted rapid recycling of chimeric mutant receptors with efficiency similarly high as that of the beta2AR tail. These results indicate that PDZ domain-mediated protein interactions are sufficient to promote rapid recycling of GPCRs, independent of binding to NSF. They also suggest that PDZ-directed recycling is a rather general mechanism of GPCR regulation, which is not restricted to a single GPCR, and may involve additional PDZ domain-containing protein(s) besides NHERF/EBP50.

An outbreak of Escherichia coli O157:H7 infections among visitors to a dairy farm.

BACKGROUND: Outbreaks of Escherichia coli O157:H7 infections have involved direct transmission from animals and their environment to humans. We describe an outbreak among visitors to a Pennsylvania dairy and petting farm that provides public access to animals. METHODS: We conducted both a case-control study among visitors to a farm to identify risk factors for infection and a household survey to determine the rates of diarrheal illness among these visitors. We performed an extensive environmental study to identify sources of E. coli O157:H7 on the farm. RESULTS: Fifty-one patients with confirmed or suspected E. coli O157:H7 infection were enrolled in the case-control study. The median age of the patients was four years, and the hemolytic-uremic syndrome developed in eight. Contact with calves and their environment was associated with an increased risk of infection, whereas hand washing was protective. The household survey indicated that visitors to the farm during the outbreak had higher than expected rates of diarrhea. Environmental studies showed that 28 of the 216 cattle on the farm (13 percent) were colonized with E. coli O157:H7 that had the same distinct pattern on pulsed-field gel electrophoresis that was found in isolates from the patients. This organism was also recovered from surfaces that were accessible to the public. CONCLUSIONS: In a large outbreak of E. coli O157:H7 infections among visitors to a dairy farm, predominantly children, high rates of carriage of E. coli O157:H7 among calves and young cattle most likely resulted in contamination of both the animals' hides and the environment.