Background:The ␣-hemoglobin-derived peptide RVDPVNFKLLSH was found to interact with cannabinoid CB 1 receptors. Results: We generated mAbs against RVDPVNFKLLSH and identified a new family of endogenous peptides (Pepcans) that act as negative allosteric modulators at CB 1 receptors. Conclusion: Allosteric ligands for CB 1 receptors are present in the brain. Significance: Pepcans are a novel class of endogenous modulators of endocannabinoid signaling.

Osmoregulated porin gene expression in Escherichia coli is controlled by the two-component regulatory system EnvZ and OmpR. EnvZ, the osmosensor, is an inner membrane protein and a histidine kinase. EnvZ phosphorylates OmpR, a cytoplasmic DNA-binding protein, on an aspartyl residue. Phospho-OmpR binds to the promoters of the porin genes to regulate the expression of ompF and ompC.We describe the use of limited proteolysis by trypsin and ion spray mass spectrometry to characterize phospho-OmpR and the conformational changes that occur upon phosphorylation. Our results are consistent with a two-domain structure for OmpR, an N-terminal phosphorylation domain joined to a C-terminal DNA-binding domain by a flexible linker region. In the presence of acetyl phosphate, OmpR is phosphorylated at only one site. Phosphorylation induces a conformational change that is transmitted to the C-terminal domain via the central linker. Previous genetic analysis identified a region in the C-terminal domain that is required for transcriptional activation. Our results indicate that this region is within a surface-exposed loop. We propose that this loop contacts the a subunit of RNA polymerase to activate transcription. Mass spectrometry also reveals an unusual dephosphorylated form of OmpR, the potential significance of which is discussed.

Kinetic interactions of ␤-lactam antibiotics such as penicillin-G and cefotaxime with normal, penicillin-susceptible PBP2x from Streptococcus pneumoniae and a penicillin-resistant PBP2x (PBP2x R ) from a resistant clinical isolate (CS109) of the bacterium have been extensively characterized using electrospray mass spectrometry coupled with a fast reaction (quench flow) technique. Kinetic evidence for a two-step acylation of PBP2x by penicillin-G has been demonstrated, and the dissociation constant, K d of 0.9 mM, and the acylation rate constant, k 2 of 180 s ؊1 , have been determined for the first time. The millimolar range K d implies that the ␤-lactam fits to the active site pocket of the penicillinsensitive PBP rather poorly, whereas the extremely fast k 2 value indicates that this step contributes most of the binding affinity of the ␤-lactam. R comes from the decreased (ϳ300-fold) k 2 . Kinetic studies of cefotaxime acylation of the two PBP2x proteins confirmed all of the above findings. Deacylation rate constants (k 3 ) for the third step of the interactions were determined to be 8 ؋ 10 ؊6 s ؊1 for penicilloyl-PBP2x and 5.7 ؋ 10 ؊4 s ؊1 for penicilloylPBP2x R , corresponding to over 70-fold increase of the deacylation rate for the resistant PBP2x R . Similarly, over 80-fold enhancement of the deacylation rate was found for cefotaxime-PBP2x R complex (k 3 ‫؍‬ 3 ؋ 10 ؊4 s ؊1 ) as compared with that of cefotaxime-PBP2x complex (3.5 ؋ 10 ؊6 s ؊1 ). This is the first time that such a significant increase of k 3 values was found for a ␤-lactamresistant penicillin-binding protein. These data indicate that the deacylation step also plays a role, which is much more important than previously thought, in PBP2x R resistance to ␤-lactams.Penicillin-binding proteins (PBPs) 1 are enzymes involved in the final reactions of bacterial peptidoglycan synthesis and are the targets of ␤-lactam antibiotics, which exert their action by acylating an active site serine of PBPs (1-4). Streptococcus pneumoniae, a major human pathogen of the upper respiratory tract, contains five high molecular mass (HMM, with molecular mass over 60 kDa), essential PBPs (5-7). Among them, PBP2x and PBP2b have been identified as the primary ␤-lactam resistance determinants based on genetic and biochemical evidence (6, 8 -10). Such resistance in PBP2x is largely because of the development of altered or mosaic forms of the PBP as a result of mutation, genetic exchange, and recombinational events (11,12). Kinetic interaction of PBPs with ␤-lactams has been a subject of investigation since the elucidation of these proteins as the targets of the ␤-lactam antibiotics (1, 13-15). Such studies may shed light on the mechanism of action and resistance at the molecular or enzymatic level, which should help in the design of better ␤-lactams or non-␤-lactam PBP inhibitors to confront bacterial resistance to ␤-lactam antibiotics. It is generally accepted that the interaction of a ␤-lactam (I) with a PBP follows a three-step reaction mechanism (13) as shown in Scheme ...