Novel mechanistic class of fatty acid amide hydrolase inhibitors with remarkable selectivity

Fatty acid amide hydrolase (FAAH) is an integral membrane enzyme that degrades the fatty acid amide family of signaling lipids, including the endocannabinoid anandamide. Genetic or pharmacological inactivation of FAAH leads to analgesic, anti-inflammatory, anxiolytic, and antidepressant phenotypes in rodents without showing the undesirable side effects observed with direct cannabinoid receptor agonists, indicating that FAAH may represent an attractive therapeutic target for treatment of pain, inflammation, and other central nervous system disorders. However, the FAAH inhibitors reported to date lack drug-like pharmacokinetic properties and/or selectivity. Herein we describe piperidine/piperazine ureas represented by N-phenyl-4-(quinolin-3-ylmethyl)piperidine-1-carboxamide (PF-750) and N-phenyl-4-(quinolin-2-ylmethyl)piperazine-1-carboxamide (PF-622) as a novel mechanistic class of FAAH inhibitors. PF-750 and PF-622 show higher in vitro potencies than previously established classes of FAAH inhibitors. Rather unexpectedly based on the high chemical stability of the urea functional group, PF-750 and PF-622 were found to inhibit FAAH in a time-dependent manner by covalently modifying the enzyme's active site serine nucleophile. Activity-based proteomic profiling revealed that PF-750 and PF-622 were completely selective for FAAH relative to other mammalian serine hydrolases. We hypothesize that this remarkable specificity derives, at least in part, from FAAH's special ability to function as a C(O)-N bond hydrolase, which distinguishes it from the vast majority of metabolic serine hydrolases in mammals that are restricted to hydrolyzing esters and/or thioesters. The piperidine/piperazine urea may thus represent a privileged chemical scaffold for the synthesis of FAAH inhibitors that display an unprecedented combination of potency and selectivity for use as potential analgesic and anxiolytic/antidepressant agents.     

Integration of genetic and genomic methods for identification of genes and gene variants encoding QTLs in the nonhuman primate

We have developed an integrated approach, using genetic and genomic methods, in conjunction with resources from the Southwest National Primate Research Center (SNPRC) baboon colony, for the identification of genes and their functional variants that encode quantitative trait loci (QTL). In addition, we use comparative genomic methods to overcome the paucity of baboon specific reagents and to augment translation of our findings in a nonhuman primate (NHP) to the human population. We are using the baboon as a model to study the genetics of cardiovascular disease (CVD). A key step for understanding gene–environment interactions in cardiovascular disease is the identification of genes and gene variants that influence CVD phenotypes. We have developed a sequential methodology that takes advantage of the SNPRC pedigreed baboon colony, the annotated human genome, and current genomic and bioinformatic tools. The process of functional polymorphism identification for genes encoding QTLs involves comparison of expression profiles for genes and predicted genes in the genomic region of the QTL for individuals discordant for the phenotypic trait mapping to the QTL. After comparison, genes of interest are prioritized, and functional polymorphisms are identified in candidate genes by genotyping and quantitative trait nucleotide analysis. This approach reduces the time and labor necessary to prioritize and identify genes and their polymorphisms influencing variation in a quantitative trait compared with traditional positional cloning methods.     

The 1-deoxy-d-xylulose 5-phosphate synthase gene co-localizes with a major QTL affecting monoterpene content in grapevine

Muscat flavor is a relevant trait both in winemaking and in fresh grape consumption. From a chemical point of view, it is strongly related to the accumulation of monoterpenes in berries. However, knowledge of the genetic mechanisms underlying its regulation is still limited. The objective of this study was to dissect the genetic determinism of aroma in grapevine by applying the analysis of quantitative trait loci (QTL) and the candidate gene (CG) approach. Two F₁ segregating progenies were evaluated through high-resolution gas chromatography–mass spectrometry (HRGC–MS) for the amounts of individual monoterpenes over 3 and 2 years. In the Italia × Big Perlon cross 34 CGs, chosen according to gene ontology (GO) terms, were placed on a complete map and tested for linkage with QTLs for linalool, nerol and geraniol levels. Two CGs mapped within a QTL for linalool content on LG 10. A third one co-localized with a major QTL for the level of the three monoterpenes on LG 5; this gene encodes 1-deoxy-d-xylulose 5-phosphate synthase (DXS), which is the first enzyme in the plastidial pathway of terpene biosynthesis. Depending on these findings, we report the first in silico analysis of grapevine DXS genes based on the whole genome sequence. Further research on the functional significance of these associations might help to understand the genetic control of Muscat flavor. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. J. Battilana and L. Costantini equally contributed to the work.     

Macrolide resistance genotypes and phenotypes among erythromycin-resistant clinical isolates of Staphylococcus aureus and coagulase-negative staphylococci, Italy

One hundred macrolide-resistant staphylococcal isolates from clinically relevant infections in Italy during a 19-month period were studied. Four distinct resistance phenotypes were observed using the triple-disk induction test (erythromycin, clindamycin, telithromycin): the cMLS_B phenotype (24 isolates); the iMLS_B phenotype (41 isolates); the MS phenotype (three isolates); and the iMTS phenotype (erythromycin-induced telithromycin resistance) (32 isolates). ermC and ermA genes predominated within erythromycin-resistant Staphylococcus aureus isolates with iMLS_B phenotype and cMLS_B phenotype, respectively. Among erythromycin-resistant CoNS isolates, half of the strains showed the iMTS or MS/ msrA association, and ermC gene predominated among isolates with MLS_B phenotype. By pulsed-field gel electrophoresis, high genetic heterogeneity was observed among the isolates studied. Both independent acquisition of macrolide resistance genes and spread of specific resistant clones were observed. Association between certain clonal types and specific types of infection could be detected. To our knowledge, this is the first report on characterization of erythromycin-resistant staphylococci in Italy.     

Role of invasive <Emphasis Type="Italic">Melilotus officinalis</Emphasis> in two native plant communities

This study examines the impact of the exotic nitrogen-fixing legume Melilotus officinalis (L.) Lam. on native and exotic species cover in two Great Plains ecosystems in Badlands National Park, South Dakota. Melilotus is still widely planted and its effects on native ecosystems are not well studied. Melilotus could have direct effects on native plants, such as through competition or facilitation. Alternatively, Melilotus may have indirect effects on natives, e.g., by favoring exotic species which in turn have a negative effect on native species. This study examined these interactions across a 4-year period in two contrasting vegetation types: Badlands sparse vegetation and western wheatgrass (Pascopyrum smithii) mixed-grass prairie. Structural equation models were used to analyze the pathways through which Melilotus, native species, and other exotic species interact over a series of 2-year time steps. Melilotus can affect native and exotic species both in the current year and in the years after its death (a lag effect). A lag effect is possible because the death of a Melilotus plant can leave an open, potentially nitrogen-enriched site on the landscape. The results showed that the relationship between Melilotus and native and exotic species varied depending on the habitat and the year. In Badlands sparse vegetation, there was a consistent, strong, and positive relationship between Melilotus cover and native and exotic species cover suggesting that Melilotus is acting as a nurse plant and facilitating the growth of other species. In contrast, in western wheatgrass prairie, Melilotus was acting as a weak competitor and had no consistent effect on other species. In both habitats, there was little evidence for a direct lag effect of Melilotus on other species. Together, these results suggest both facilitative and competitive roles for Melilotus, depending on the vegetation type it invades.     

Volume regulation mechanisms in Rana castebeiana cardiac tissue under hyperosmotic stress

Volume changes of cardiac tissue under hyperosmotic stress in Rana catesbeiana were characterized by the identification of the osmolytes involved and the possible regulatory processes activated by both abrupt and gradual changes in media osmolality (from 220 to 280mosmol/kg H(2)O). Slices of R. catesbeiana cardiac tissue were subjected to hyperosmotic shock, and total tissue Na(+), K(+), Cl(-) and ninhydrin-positive substances were measured. Volume changes were also induced in the presence of transport inhibitors to identify osmolyte pathways. The results show a maximum volume loss to 90.86+/-0.73% of the original volume (measured as 9% decrease in wet weight) during abrupt hyperosmotic shock. However, during a gradual osmotic challenge the volume was never significantly different from that of the control. During both types of hyperosmotic shock, we observed an increase in Na(+) but no significant change in Cl(-) contents. Additionally, we found no change in ninhydrin-positive substances during any osmotic challenge. Pharmacological analyses suggest the involvement of the Na(+)/H(+) exchanger, and perhaps the HCO(3)(-)/Cl(-) exchanger. There is indirect evidence for decrease in Na(+)/K(+)-ATPase activity. The Na(+) fluxes seem to result from Mg(2+) signaling, as saline rich in Mg(2+) enhances the regulatory volume increase, followed by a higher intracellular Na(+) content. The volume maintenance mechanisms activated during the gradual osmotic change are similar to that activated by abrupt osmotic shock.     

The irreversible binding of amyloid peptide substrates to insulin-degrading enzyme: A biological perspective

Insulin-degrading enzyme (IDE) is a conserved Zn²⁺metalloendopeptidase involved in insulin degradation and in the maintenance of brain steady-state levels of amyloid β peptide (Aβ) of Alzheimer''s disease (AD). Our recent demonstration that IDE and Aβ are capable of forming a stoichiometric and extremely stable complex raises several intriguing possibilities regarding the role of this unique protein-peptide interaction in physiological and pathological conditions. These include a protective cellular function of IDE as a “dead-end chaperone” alternative to its proteolytic activity and the potential impact of the irreversible binding of Aβ to IDE upon its role as a varicella zoster virus receptor. In a pathological context, the implications for insulin signaling and its relationship to AD pathogenesis are discussed. Moreover, our findings warrant further research regarding a possible general and novel interaction between amyloidogenic peptides and other Zn²⁺metallopeptidases with an IDE-like fold and a substrate conformation-dependent recognition mechanism.Key words: amyloid, insulin-degrading enzyme, peptides, alzheimer''s disease, irreversible binding, metalloproteases     

Mutating the tight-dimer interface of dihydrodipicolinate synthase disrupts the enzyme quaternary structure: toward a monomeric enzyme

Dihydrodipicolinate synthase (DHDPS) is a tetrameric enzyme that is the first enzyme unique to the ( S)-lysine biosynthetic pathway in plants and bacteria. Previous studies have looked at the important role of Tyr107, an amino acid residue located at the tight-dimer interface between two monomers, in participating in a catalytic triad of residues during catalysis. In this study, we examine the importance of this residue in determining the quaternary structure of the DHDPS enzyme. The Tyr107 residue was mutated to tryptophan, and structural, biophysical, and kinetic studies were carried out on the mutant enzyme. These revealed that while the solid-state structure of the mutant enzyme was largely unchanged, as judged by X-ray crystallography, it exists as a mixture of primarily monomer and tetramer in solution, as determined by analytical ultracentrifugation, size-exclusion chromatography, and mass spectrometry. The catalytic ability of the DHDPS enzyme was reduced by the mutation, which also allowed the adventitious binding of alpha-ketoglutarate to the active site. A reduction in the apparent melting temperature of the mutant enzyme was observed. Thus, the tetrameric quaternary structure of DHDPS is critical to controlling specificity, heat stability, and intrinsic activity.