Defining the Molecular Basis for the First Potent and Selective Orthosteric Agonists of the FFA2 Free Fatty Acid Receptor

FFA2 is a G protein-coupled receptor that responds to short chain fatty acids and has generated interest as a therapeutic target for metabolic and inflammatory conditions. However, definition of its functions has been slowed by a dearth of selective ligands that can distinguish it from the closely related FFA3. At present, the only selective ligands described for FFA2 suffer from poor potency, altered signaling due to allosteric modes of action, or a lack of function at non-human orthologs of the receptor. To address the need for novel selective ligands, we synthesized two compounds potentially having FFA2 activity and examined the molecular basis of their function. These compounds were confirmed to be potent and selective orthosteric FFA2 agonists. A combination of ligand structure-activity relationship, pharmacological analysis, homology modeling, species ortholog comparisons, and mutagenesis studies were then employed to define the molecular basis of selectivity and function of these ligands. From this, we identified key residues within both extracellular loop 2 and the transmembrane domain regions of FFA2 critical for ligand function. One of these ligands was active with reasonable potency at rodent orthologs of FFA2 and demonstrated the role of FFA2 in inhibition of lipolysis and glucagon-like peptide-1 secretion in murine-derived 3T3-L1 and STC-1 cell lines, respectively. Together, these findings describe the first potent and selective FFA2 orthosteric agonists and demonstrate key aspects of ligand interaction within the binding site of FFA2 that will be invaluable in future ligand development at this receptor.     

Phosphomannose isomerase inhibitors improve N-glycosylation in selected phosphomannomutase-deficient fibroblasts

Congenital disorders of glycosylation (CDG) are rare genetic disorders due to impaired glycosylation. The patients with subtypes CDG-Ia and CDG-Ib have mutations in the genes encoding phosphomannomutase 2 (PMM2) and phosphomannose isomerase (MPI or PMI), respectively. PMM2 (mannose 6-phosphate → mannose 1-phosphate) and MPI (mannose 6-phosphate ⇔ fructose 6-phosphate) deficiencies reduce the metabolic flux of mannose 6-phosphate (Man-6-P) into glycosylation, resulting in unoccupied N-glycosylation sites. Both PMM2 and MPI compete for the same substrate, Man-6-P. Daily mannose doses reverse most of the symptoms of MPI-deficient CDG-Ib patients. However, CDG-Ia patients do not benefit from mannose supplementation because >95% Man-6-P is catabolized by MPI. We hypothesized that inhibiting MPI enzymatic activity would provide more Man-6-P for glycosylation and possibly benefit CDG-Ia patients with residual PMM2 activity. Here we show that MLS0315771, a potent MPI inhibitor from the benzoisothiazolone series, diverts Man-6-P toward glycosylation in various cell lines including fibroblasts from CDG-Ia patients and improves N-glycosylation. Finally, we show that MLS0315771 increases mannose metabolic flux toward glycosylation in zebrafish embryos.     

Disruption of planktonic phosphorus cycling by ultraviolet radiation

Ultraviolet radiation (UVR) may alter phosphorous (P) cycling by plankton through changes in the acquisition and/or regeneration of dissolved P. However, to date an effect of UVR on the uptake of P has not been observed at ambient phosphate (PO₄ ³⁻) concentrations. This has lead to the conclusion that the uptake of P by plankton may be insensitive to UVR. Past research has been limited to a few individual systems, prolonged incubations in bags, or lab cultures. We suspect that experimentation with natural plankton assemblages across broader environmental and/or chemical gradients is required to appreciably understand how UVR may alter P kinetics. Therefore, our study aimed to determine the effect of UVR on the turnover time of the dissolved PO₄ ³⁻ pool, the regeneration of dissolved P, the turnover rate of particulate P, and on PO₄ ³⁻ concentrations in natural plankton assemblages across broad environmental and chemical gradients. Second we aimed to assess how UVR may alter phosphatase activity and, determine if a change in phosphatase activity under UVR irradiance is correlated with a change in P uptake as proposed in the literature. Studies were conducted on 18 thermally stratified or polymictic lakes located in Ontario and Saskatchewan, Canada. Lake water samples were exposed to one of three experimental treatments: control, photosynthetically active radiation (PAR), or photosynthetically active radiation plus ultraviolet radiation (PAR + UVR). Our study is the first to demonstrate that UVR exposure has the potential to alter P cycling at ambient (picomolar) PO₄ ³⁻ concentrations. We have demonstrated that the turnover time of the PO₄ ³⁻ pool increases under UVR irradiance (i.e., P uptake decreases), while the regeneration rate of dissolved P and turnover rate of planktonic P are generally not affected; with the net effect being an increase in steady state PO₄ ³⁻ concentration (ssPO₄ ³⁻). Alkaline phosphatase activity (APA) in the dissolved and particulate fractions was significantly reduced in PAR + UVR treatments, but unrelated to changes in P uptake. In summary, we have demonstrated that the cycling of P may be disrupted by UVR, with a decrease in the uptake of P and the accumulation of PO₄ ³⁻ in the dissolved pool. This, in turn may exacerbate planktonic P limitation, alter the nutrient stoichiometry of plankton and/or indirectly alter rates of primary production in limnetic systems.     

Association of Ovavesicula popilliae (Microsporida: Ovavesiculidae) with winter mortality of larvae and reduced fecundity of female Japanese beetles (Coleoptera: Scarabaeidae)

Populations of Japanese beetle at sites in Michigan where Ovavesicula popilliae (Andreadis) was introduced in 1999 and 2000 were compared with nearby control sites from fall of 2005 through spring of 2008. Percent infection by O. popilliae and winter mortality of Japanese beetle were determined by sampling larvae in October and April from 12 golf holes on six courses in southeast Michigan and eight holes on four courses in southwest Michigan. Adult Japanese beetles were also collected from these golf courses in July and August of 2007 to determine the impact of O. popilliae-infection on egg development in females. In southeast Michigan, O. popilliae appeared to spread rapidly from the 100 m(2) plots where it was previously introduced to surrounding golf course holes between 2000 and 2006. However, data from southwest Michigan suggests that O. popilliae had already been introduced into the area. Regression analysis of data from all 20 golf course holes gives a significant relationship between percent infection of larvae with O. popilliae and winter mortality of Japanese beetle. Mean winter mortality of larvae around golf course holes where <10% were infected with O. popilliae was 24.7% compared with 41.7% mortality where 10-30% were infected, and 72.0% mortality where >30% were infected. Females infected with O. popilliae contained 50% fewer mature eggs than uninfected females. In addition, females from golf courses where all of the fairways and roughs were treated annually with imidacloprid contained 48% fewer mature eggs than females from golf courses where insecticides were only used on the fairways or not at all.     

Goodpasture antigen-binding protein (GPBP) directs myofibril formation: identification of intracellular downstream effector 130-kDa GPBP-interacting protein (GIP130)

Goodpasture antigen-binding protein-1 (GPBP-1) is an exportable non-conventional Ser/Thr kinase that regulates glomerular basement membrane collagen organization. Here we provide evidence that GPBP-1 accumulates in the cytoplasm of differentiating mouse myoblasts prior to myosin synthesis. Myoblasts deficient in GPBP-1 display defective myofibril formation, whereas myofibrils assemble with enhanced efficiency in those overexpressing GPBP-1. We also show that GPBP-1 targets the previously unidentified GIP130 (GPBP-interacting protein of 130 kDa), which binds to myosin and promotes its myofibrillar assembly. This report reveals that GPBP-1 directs myofibril formation, an observation that expands its reported role in supramolecular organization of structural proteins to the intracellular compartment.     

Arsenite interacts selectively with zinc finger proteins containing C3H1 or C4 motifs

Arsenic inhibits DNA repair and enhances the genotoxicity of DNA-damaging agents such as benzo[a]pyrene and ultraviolet radiation. Arsenic interaction with DNA repair proteins containing functional zinc finger motifs is one proposed mechanism to account for these observations. Here, we report that arsenite binds to both CCHC DNA-binding zinc fingers of the DNA repair protein PARP-1 (poly(ADP-ribose) polymerase-1). Furthermore, trivalent arsenite coordinated with all three cysteine residues as demonstrated by MS/MS. MALDI-TOF-MS analysis of peptides harboring site-directed substitutions of cysteine with histidine residues within the PARP-1 zinc finger revealed that arsenite bound to peptides containing three or four cysteine residues, but not to peptides with two cysteines, demonstrating arsenite binding selectivity. This finding was not unique to PARP-1; arsenite did not bind to a peptide representing the CCHH zinc finger of the DNA repair protein aprataxin, but did bind to an aprataxin peptide mutated to a CCHC zinc finger. To investigate the impact of arsenite on PARP-1 zinc finger function, we measured the zinc content and DNA-binding capacity of PARP-1 immunoprecipitated from arsenite-exposed cells. PARP-1 zinc content and DNA binding were decreased by 76 and 80%, respectively, compared with protein isolated from untreated cells. We observed comparable decreases in zinc content for XPA (xeroderma pigmentosum group A) protein (CCCC zinc finger), but not SP-1 (specificity protein-1) or aprataxin (CCHH zinc finger). These findings demonstrate that PARP-1 is a direct molecular target of arsenite and that arsenite interacts selectively with zinc finger motifs containing three or more cysteine residues.     

Attitudes toward Olfaction: A Cross-regional Study

This study aimed to determine whether there are regional influences on attitudes toward olfaction. A total of 1082 participants aged 21-50 years from 4 different regions (Mexican, Korean, Czech, and German) were asked to rate general attitudes toward olfaction in everyday life. To examine affective attitudes to odors (i.e., pleasantness), participants were also asked to list 3 odors as being the most pleasant or unpleasant, respectively. Next, the mentioned odor names were attributed to 1 of 4 main categories: "Food & Drink," "Social relationship," "Nature," and "Civilization" and the distribution of these categories was compared across regions. Mexicans were significantly different to the other regions in their general attitudes toward olfaction. In addition, in all regions, in comparison with men, women indicated a higher interest in the sense of smell. Moreover, a significant positive correlation was present between individuals' self-rating of olfactory sensitivity and general attitudes toward olfaction. Finally, there were significant cross-regional differences in affective attitudes toward specific categories of odors. In conclusion, our findings support and extend the notion that regions affect attitudes toward the olfactory world.     

Exercise-induced oxidative stress in humans: cause and consequences

The observation that muscular exercise is associated with oxidative stress in humans was first reported over 30 years ago. Since this initial report, numerous studies have confirmed that prolonged or high-intensity exercise results in oxidative damage to macromolecules in both blood and skeletal muscle. Although the primary tissue(s) responsible for reactive oxygen species (ROS) production during exercise remains a topic of debate, compelling evidence indicates that muscular activity promotes oxidant production in contracting skeletal muscle fibers. Mitochondria, NADPH oxidase, PLA₂-dependent processes, and xanthine oxidase have all been postulated to contribute to contraction-induced ROS production in muscle but the primary site of contraction-induced ROS production in muscle fibers remains unclear. Nonetheless, contraction-induced ROS generation has been shown to play an important physiological function in the regulation of both muscle force production and contraction-induced adaptive responses of muscle fibers to exercise training. Although knowledge in the field of exercise and oxidative stress has grown markedly during the past 30 years, this area continues to expand and there is much more to be learned about the role of ROS as signaling molecules in skeletal muscle.     

3-[(Imidazolidin-2-yl)imino]indazole ligands with selectivity for the α(2)-adrenoceptor compared to the imidazoline I(1) receptor

A series of 3-[(4,5-dihydroimidazolidin-2-yl)imino]indazoles has been synthesized as positional analogues of marsanidine, a highly selective ??₂-adrenoceptor ligand. Parent compound 4a and its 4-chloro (4c) and 4-methyl (4d) derivatives display ??₂-adrenoceptor affinity at nanomolar concentrations (K_i = 39.4, 15.9 and 22.6 nM, respectively) and relatively high ??₂/I₁ selectivity ratios of 82, 115 and 690, respectively. Evidence was obtained that these compounds act as partial agonists at ??_2A-adrenoceptors. Compound 4d with intrinsic activity comparable with that of marsanidine, but lower than that of clonidine, elicited pronounced cardiovascular effects in anesthetized rats at doses as low as 0.01 mg/kg iv