Identification of selective neuropeptide Y2 peptide agonists

Activation of the NPY2 receptor to reduce appetite while avoiding stimulation of the NPY1 and NPY5 receptors that induce feeding provides a pharmaceutical approach to modulate food intake. The naturally occurring peptide PYY(3-36) is a nonselective NPY1, NPY2, and NPY5 agonist. N-terminal truncation of PYY to abrogate affinity for the NPY1 and NPY5 receptors and subsequent N-terminal modification with aminobenzoic analogs to restore NPY2 receptor potency results in a series of highly selective NPY2 receptor peptide agonists.     

Glucosylceramides are critical for cell‐type differentiation and organogenesis,but not for cell viability in Arabidopsis

Glucosylceramides (GlcCer), glucose‐conjugated sphingolipids, are major components of the endomembrane system and plasma membrane in most eukaryotic cells. Yet the quantitative significance and cellular functions of GlcCer are not well characterized in plants and other multi‐organ eukaryotes. To address this, we examined Arabidopsis lines that were lacking or deficient in GlcCer by insertional disruption or by RNA interference (RNAi) suppression of the single gene for GlcCer synthase (GCS, At2g19880), the enzyme that catalyzes GlcCer synthesis. Null mutants for GCS (designated ‘gcs‐1’) were viable as seedlings, albeit strongly reduced in size, and failed to develop beyond the seedling stage. Heterozygous plants harboring the insertion allele exhibited reduced transmission through the male gametophyte. Undifferentiated calli generated from gcs‐1 seedlings and lacking GlcCer proliferated in a manner similar to calli from wild‐type plants. However, gcs‐1 calli, in contrast to wild‐type calli, were unable to develop organs on differentiation media. Consistent with a role for GlcCer in organ‐specific cell differentiation, calli from gcs‐1 mutants formed roots and leaves on media supplemented with the glucosylated sphingosine glucopsychosine, which was readily converted to GlcCer independent of GCS. Underlying these phenotypes, gcs‐1 cells had altered Golgi morphology and fewer cisternae per Golgi apparatus relative to wild‐type cells, indicative of protein trafficking defects. Despite seedling lethality in the null mutant, GCS RNAi suppression lines with ≤2% of wild‐type GlcCer levels were viable and fertile. Collectively, these results indicate that GlcCer are essential for cell‐type differentiation and organogenesis, and plant cells produce amounts of GlcCer in excess of that required for normal development.     

Nox2 B-loop peptide, Nox2ds, specifically inhibits the NADPH oxidase Nox2

In recent years, reactive oxygen species (ROS) derived from the vascular isoforms of NADPH oxidase, Nox1, Nox2, and Nox4, have been implicated in many cardiovascular pathologies. As a result, the selective inhibition of these isoforms is an area of intense current investigation. In this study, we postulated that Nox2ds, a peptidic inhibitor that mimics a sequence in the cytosolic B-loop of Nox2, would inhibit ROS production by the Nox2-, but not the Nox1- and Nox4-oxidase systems. To test our hypothesis, the inhibitory activity of Nox2ds was assessed in cell-free assays using reconstituted systems expressing the Nox2-, canonical or hybrid Nox1-, or Nox4-oxidase. Our findings demonstrate that Nox2ds, but not its scrambled control, potently inhibited superoxide (O₂^•−) production in the Nox2 cell-free system, as assessed by the cytochrome c assay. Electron paramagnetic resonance confirmed that Nox2ds inhibits O₂^•− production by Nox2 oxidase. In contrast, Nox2ds did not inhibit ROS production by either Nox1- or Nox4-oxidase. These findings demonstrate that Nox2ds is a selective inhibitor of Nox2-oxidase and support its utility to elucidate the role of Nox2 in organ pathophysiology and its potential as a therapeutic agent.     

A J-like protein influences fatty acid composition of chloroplast lipids in Arabidopsis

A comprehensive understanding of the lipid and fatty acid metabolic machinery is needed for optimizing production of oils and fatty acids for fuel, industrial feedstocks and nutritional improvement in plants. T-DNA mutants in the poorly annotated Arabidopsis thaliana gene At1g08640 were identified as containing moderately high levels (50-100%) of 16∶1Δ7 and 18∶1Δ9 leaf fatty acids and subtle decreases (5-30%) of 16∶3 and 18∶3 (http://www.plastid.msu.edu/). TLC separation of fatty acids in the leaf polar lipids revealed that the chloroplastic galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) were the main lipid types affected by this mutation. Analysis of the inferred amino acid sequence of At1g08640 predicted the presence of a transit peptide, three transmembrane domains and an N-terminal J-like domain, and the gene was named CJD1 for Chloroplast J-like Domain 1. GFP reporter experiments and in vitro chloroplast import assays demonstrated CJD1 is a chloroplast membrane protein. Screening of an Arabidopsis cDNA library by yeast-2-hybrid (Y2H) using the J-like domain of CJD1 as bait identified a plastidial inner envelope protein (Accumulation and Replication of Chloroplasts 6, ARC6) as the primary interacting partner in the Y2H assay. ARC6 plays a central role in chloroplast division and binds CJD1 via its own J-like domain along with an adjacent conserved region whose function is not fully known. These results provide a starting point for future investigations of how mutations in CJD1 affect lipid composition.     

Oxygen and carbon dioxide fluctuations in burrows of subterranean blind mole rats indicate tolerance to hypoxic-hypercapnic stresses

The composition of oxygen (O2), carbon dioxide (CO2), and soil humidity in the underground burrows from three species of the Israeli subterranean mole rat Spalax ehrenbergi superspecies were studied in their natural habitat. Two geographically close populations of each species from contrasting soil types were probed. Maximal CO2 levels (6.1%) and minimal O2 levels (7.2%) were recorded in northern Israel in the breeding mounds of S. carmeli in a flooded, poor drained field of heavy clay soil with very high volumetric water content. The patterns of gas fluctuations during the measurement period among the different Spalax species studied were similar. The more significant differentiation in gas levels was not among species, but between neighboring populations inhabiting heavy soils or light soils: O2 was lower and CO2 was higher in the heavy soils (clay and basaltic) compared to the relatively light soils (terra rossa and rendzina). The extreme values of gas concentration, which occurred during the rainy season, seemed to fluctuate with partial flooding of the tunnels, animal digging activity, and over-crowded breeding mounds inhabited by a nursing female and her offspring. The gas composition and soil water content in neighboring sites with different soil types indicated large differences in the levels of hypoxic-hypercapnic stress in different populations of the same species. A growing number of genes associated with hypoxic stress have been shown to exhibit structural and functional differences between the subterranean Spalax and the above-ground rat (Rattus norvegicus), probably reflecting the molecular adaptations that Spalax went through during 40 million years of evolution to survive efficiently in the severe fluctuations in gas composition in the underground habitat.     

Isosteric ramatroban analogs: selective and potent CRTH-2 antagonists

The chemoattractant receptor-homologous molecule expressed on T(H)2 cells (CRTH-2), also found on eosinophils and basophils, is a prostaglandin D2 receptor involved in the recruitment of these cell types during an inflammatory response. In this report, we describe the synthesis and optimization of a ramatroban isostere that is a selective and potent antagonist of CRTH-2 which may be useful in the treatment of certain diseases.     

A STEPSCAN Differential Scanning Calorimetry Study of the Thermal Behavior of Chocolate

STEPSCAN differential scanning calorimetry, a form of temperature-modulated differential scanning calorimetry (DSC), was used to study the thermal transitions occurring during the heating of chocolate of varying thermal histories. Conventional DSC thermograms acquired during heating of chocolate can be complex, with the observation of a series of overlapping endothermic and exothermic events. STEPSCAN DSC was used to deconvolute the total heat flow into reversing (rapid) and nonreversing (slow) components, which were assigned to melting and recrystallization events, respectively. Such a separation is usually difficult using conventional DSC. The recrystallization events were more pronounced in rapidly cooled samples where the polymorphic form V had been nucleated through tempering. Because of the presence of artifacts, STEPSCAN can only provide a crude separation of reversing and nonreversing signals in this system. The general applicability and limitations of STEPSCAN DSC as well as the effects of prenucleation and rate of cooling of chocolate are discussed.