Synergistic action of nectins and cadherins generates the mosaic cellular pattern of the olfactory epithelium

In the olfactory epithelium (OE), olfactory cells (OCs) and supporting cells (SCs), which express different cadherins, are arranged in a characteristic mosaic pattern in which OCs are enclosed by SCs. However, the mechanism underlying this cellular patterning is unclear. Here, we show that the cellular pattern of the OE is established by cellular rearrangements during development. In the OE, OCs express nectin-2 and N-cadherin, and SCs express nectin-2, nectin-3, E-cadherin, and N-cadherin. Heterophilic trans-interaction between nectin-2 on OCs and nectin-3 on SCs preferentially recruits cadherin via α-catenin to heterotypic junctions, and the differential distributions of cadherins between junctions promote cellular intercalations, resulting in the formation of the mosaic pattern. These observations are confirmed by model cell systems, and various cellular patterns are generated by the combinatorial expression of nectins and cadherins. Collectively, the synergistic action of nectins and cadherins generates mosaic pattern, which cannot be achieved by a single mechanism.     

Medium chain fatty acids are selective peroxisome proliferator activated receptor (PPAR) γ activators and pan-PPAR partial agonists

Thiazolidinediones (TZDs) act through peroxisome proliferator activated receptor (PPAR) γ to increase insulin sensitivity in type 2 diabetes (T2DM), but deleterious effects of these ligands mean that selective modulators with improved clinical profiles are needed. We obtained a crystal structure of PPARγ ligand binding domain (LBD) and found that the ligand binding pocket (LBP) is occupied by bacterial medium chain fatty acids (MCFAs). We verified that MCFAs (C8-C10) bind the PPARγ LBD in vitro and showed that they are low-potency partial agonists that display assay-specific actions relative to TZDs; they act as very weak partial agonists in transfections with PPARγ LBD, stronger partial agonists with full length PPARγ and exhibit full blockade of PPARγ phosphorylation by cyclin-dependent kinase 5 (cdk5), linked to reversal of adipose tissue insulin resistance. MCFAs that bind PPARγ also antagonize TZD-dependent adipogenesis in vitro. X-ray structure B-factor analysis and molecular dynamics (MD) simulations suggest that MCFAs weakly stabilize C-terminal activation helix (H) 12 relative to TZDs and this effect is highly dependent on chain length. By contrast, MCFAs preferentially stabilize the H2-H3/β-sheet region and the helix (H) 11-H12 loop relative to TZDs and we propose that MCFA assay-specific actions are linked to their unique binding mode and suggest that it may be possible to identify selective PPARγ modulators with useful clinical profiles among natural products.     

Characterization of 4-O-methyl-ascochlorin-induced apoptosis in comparison with typical apoptotic inducers in human leukemia cell lines

Apoptosis can be induced by various stimuli such as the ligands of death receptors, chemotherapeutic drugs and irradiation. It is generally believed that chemotherapeutic drugs induce mitochondrial damage, cytochrome c release and activation of caspase-9, leading to apoptosis. Here, we found that an isoprenoid antibiotic, 4-O-methyl ascochlorin, significantly induces typical apoptotic events in Jurkat cells including the degradation of poly (ADP-ribose) polymerase, DNA fragmentation, activation of caspase-3, -9 and -8, and cytochrome c release from mitochondria. Similar to Fas stimulation, 4-O-methyl ascochlorin but not staurosporine, cycloheximide and actinomycin D, induced apoptosis in SKW6.4 cells, in which apoptosis is strongly dependent on death-inducing signaling-complex. Bcl-2 overexpression in Jurkat cells completely suppressed the apoptosis, but procaspase-9 processing was partially induced. A caspase-8 inhibitor, IETD-fmk, effectively suppressed poly (ADP-ribose) polymerase cleavage and cytochrome c release. However, 4-O-methyl ascochlorin induced apoptosis in Jurkat cells deficient of caspase-8 or Fas-associated death domain protein. These results suggest that 4-O-methyl ascochlorin induces apoptosis through the mechanism distinct from conventional apoptosis inducers.     

A new method for loading Bursaphelenchus xylophilus (Nematoda: Aphelenchoididae) on adult Monochamus alternatus (Coleoptera: Cerambycidae)

A new method was developed for loading the pinewood nematode, Bursaphelenchus xylophilus (Steiner et Buhrer) Nickle, on the beetle Monochamus alternatus Hope. Postdiapause beetle larvae were sterilized with 70 and 99.9% aqueous ethanol and placed singly in flasks where B. xylophilus reproduced on the fungus Ophiostoma minus (Hedgcock) H. et P. Sydow that had been grown on autoclaved barley grain and Pinus densifiora Sieb. et Zucc. wood chips. The fungus produced a large nematode population that developed to a high proportion of third-stage dispersal juveniles that molted to the fourth-stage dispersal juveniles. The survival rate was 80%, and the mean nematode load was 10,096. It took a mean of 5 wk to obtain the nematode-infested beetles after the initiation of nematode rearing.     

Structure-Based Virtual Screening and Discovery of New PPARδ/γ Dual Agonist and PPARδ and γ Agonists

Peroxisome proliferator-activated receptors (PPARs) are involved in the control of carbohydrate and lipid metabolism and are considered important targets to treat diabetes mellitus and metabolic syndrome. The available PPAR ligands have several side effects leading to health risks justifying the search for new bioactive ligands to activate the PPAR subtypes, in special PPARδ, the less studied PPAR isoform. Here, we used a structure-based virtual screening protocol in order to find out new PPAR ligands. From a lead-like subset of purchasable compounds, we identified 5 compounds with potential PPAR affinity and, from preliminary in vitro assays, 4 of them showed promising biological activity. Therefore, from our in silico and in vitro protocols, new PPAR ligands are potential candidates to treat metabolic diseases.     

Conversion of iodine to fluorine-18 based on iodinated chalcone and evaluation for β-amyloid PET imaging

In the amyloid cascade hypothesis, β-amyloid (Aβ) plaques is one of the major pathological biomarkers in the Alzheimer’s disease (AD) brain. We report the synthesis and evaluation of novel radiofluorinated chalcones, [¹⁸F]4-dimethylamino-4′-fluoro-chalcone ([¹⁸F]DMFC) and [¹⁸F]4′-fluoro-4-methylamino-chalcone ([¹⁸F]FMC), as Aβ imaging probes. The conversion of iodine directly introduced to the chalcone backbone into fluorine was successfully carried out by ¹⁸F-labeling via the corresponding boronate precursors, achieving the direct introduction of fluorine-18 into the chalcone backbone to prepare [¹⁸F]DMFC and [¹⁸F]FMC. In a biodistribution study using normal mice, [¹⁸F]DMFC and [¹⁸F]FMC showed a higher initial uptake (4.43 and 5.47% ID/g at 2?min postinjection, respectively) into and more rapid clearance (0.52 and 0.66% ID/g at 30?min postinjection, respectively) from the brain than a Food and Drug Administration (FDA)-approved Aβ imaging agent ([¹⁸F]Florbetapir), meaning the improvement of the probability of detecting Aβ plaques and the reduction of non-specific binding in the brain. In the in vitro binding studies using aggregates of recombinant Aβ peptides, [¹⁸F]DMFC and [¹⁸F]FMC showed high binding affinity to recombinant Aβ aggregates at the K_d values of 4.47 and 6.50?nM, respectively. In the in vitro autoradiography (ARG) experiment with AD brain sections, [¹⁸F]DMFC and [¹⁸F]FMC markedly accumulated only in a region with abundant Aβ plaques, indicating that they clearly recognized human Aβ plaques in vitro. These encouraging results suggest that [¹⁸F]DMFC and [¹⁸F]FMC may be promising PET probes for the detection of an amyloid pathology and the early diagnosis of AD with marked accuracy.     

Critical regions for assembly of vertebrate nonmuscle myosin II

Myosin II molecules assemble and form filaments through their C-terminal rod region, and the dynamic filament assembly-disassembly process of nonmuscle myosin II molecules is important for cellular activities. To estimate the critical region for filament formation of vertebrate nonmuscle myosin II, we assessed the solubility of a series of truncated recombinant rod fragments of nonmuscle myosin IIB at various concentrations of NaCl. A C-terminal 248-residue rod fragment (Asp 1729-Glu 1976) was shown by its solubility behavior to retain native assembly features, and two regions within it were found to be necessary for assembly: 35 amino acid residues from Asp 1729 to Thr 1763 and 39 amino acid residues from Ala 1875 to Ala 1913, the latter containing a sequence similar to the assembly competence domain (ACD) of skeletal muscle myosin. Fragments lacking either of the two regions were soluble at any NaCl concentration. We referred to these two regions as nonmuscle myosin ACD1 (nACD1) and nACD2, respectively. In addition, we constructed an alpha-helical coiled-coil model of the rod fragment, and found that a remarkable negative charge cluster (termed N1) and a positive charge cluster (termed P2) were present within nACD1 and nACD2, respectively, besides another positive charge cluster (termed P1) in the amino-terminal vicinity of nACD2. From these results, we propose two major electrostatic interactions that are essential for filament formation of nonmuscle myosin II: the antiparallel interaction between P2 and N1 which is essential for the nucleation step and the parallel interaction between P1 and N1 which is important for the elongation step.     

Fluvial transport of carbon along the river-to-ocean continuum and its potential impacts on a brackish water food web in the Iwaki River watershed, northern Japan

Riverine transport of dissolved inorganic carbon (DIC) from land to the ocean is an important carbon flux that influences the carbon budget at the watershed scale. However, the dynamics of DIC in an entire river network has remained unknown, especially in mountainous Japanese watersheds. We examined the effects of watershed land use and geology on the transports of inorganic carbon as well as weathered silica (Si) and calcium (Ca) in the Iwaki River system where agricultural and residential areas have developed in the middle and lower parts of the watershed. The concentration and stable carbon isotope ratios (δ¹³C) of DIC showed the longitudinal increase of ¹³C-depleted inorganic carbon along the river. As a result, most streams and rivers were supersaturated in dissolved CO₂ that will eventually be emitted to the atmosphere. The possible origin of ¹³C-depleted carbon is CO₂ derived from the decomposition of organic matter in agricultural and urban landscapes, as well as from in-stream respiration. In addition, agricultural and urban areas, respectively, exported the large amount of dissolved Si and Ca to the rivers, suggesting that CO₂ increased by respiration accelerates the chemical weathering of silicate and carbonate materials in soils, river sediments, and/or urban infrastructure. Furthermore, riverine bicarbonate flux is likely to enter shell carbonates of Corbicula japonica, an aragonitic bivalve, in the downstream brackish lake (Lake Jusan). These results revealed that the flux of DIC from the human-dominated watersheds is a key to understanding the carbon dynamics and food-web structure along the land-to-river-to-ocean continuum.