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Aberrant DNA methylation is associated with oncogenesis of various human cancers, including pancreatic cancer (PC). PC is the seventh most common cancer, and obesity is a known high-risk factor. However, whether obesity influences DNA methylation in pancreatic exocrine cells and if this influences PC development remain unclear. Here, we performed an epigenome-wide analysis of isolated pancreatic exocrine cells obtained from mice with high-fat-diet-induced obesity (DIO). Using the Illumina Mouse Methylation BeadChip array (280K), we identified 316 differentially methylated regions (DMRs) that were enriched for cellular processes, such as DNA repair, transcription regulation, and cell proliferation, which confirmed obesity-related dysregulation of certain metabolic processes in the pancreatic cells in DIO mice. Comparing the DMRs with those in stage IB PC helped identify 82 overlapping DMRs. Three pathways including the cell hypertrophy pathway involving PLC, PKC, SMAD2/3, and TRKA; the metabolic control pathway involving CREB and AMPK; and the potassium regulation pathway involving K

Neuronal plasma membrane has been thought to retain a lot of lipid raft components which play important roles in the neural function. Although the biochemical analyses of lipid raft using brain tissues have been extensively carried out in the past 20 years, many of their experimental conditions do not coincide with those of standard neuroscience researches such as neurophysiology and neuropharmacology. Hence, the physiological methods for lipid raft analysis that can be compatible with general neuroscience have been required. Herein, we developed a system to physiologically analyze ganglioside GM1-enriched lipid rafts in brain tissues using the “Enzyme-Mediated Activation of Radical Sources (EMARS)” method that we reported (Kotani N. et al. Proc. Natl. Acad. Sci. U S A 105, 7405–7409 (2008)). The EMARS method was applied to acute brain slices prepared from mouse brains in aCSF solution using the EMARS probe, HRP-conjugated cholera toxin subunit B, which recognizes ganglioside GM1. The membrane molecules present in the GM1-enriched lipid rafts were then labeled with fluorescein under the physiological condition. The fluorescein-tagged lipid raft molecules called “EMARS products” distributed differentially among various parts of the brain. On the other hand, appreciable differences were not detected among segments along the longitudinal axis of the hippocampus. We further developed a device to label the lipid raft molecules in acute hippocampal slices under two different physiological conditions to detect dynamics of the lipid raft molecules during neural excitation. Using this device, several cell membrane molecules including Thy1, known as a lipid raft resident molecule in neurons, were confirmed by the EMARS method in living hippocampal slices.

It is unclear whether local anesthetics, such as tetracaine, and antipsychotics, such as phenothiazines, act on lipids or proteins. In Saccharomyces cerevisiae, these drugs inhibit growth, translation initiation, and actin polarization, and induce cell lysis at high concentrations. These activities are likely due to the cationic amphiphilic structure common to these agents. Although drug-induced translational inhibition is conserved in mammalian cells, other mechanisms, including the phosphorylation of eIF2α, a eukaryotic translational initiation factor, remain poorly understood. At a concentration of 10 mM, tetracaine rapidly inhibited translation initiation and lysed cells, whereas, at 2.5 mM, it slowly induced inhibition without lysis. The pat1 disruptant defective in mRNA decapping and the xrn1 disruptant defective in 5'-3' exoribonuclease were partially resistant to translational inhibition by tetracaine at each concentration, but the gcn2 disruptant defective in the eIF2α kinase was not. Phosphorylation of eIF2α was induced by 10 mM but not by 2.5 mM tetracaine, whereas processing bodies (P-bodies) were formed at 2.5 mM in Pat1-dependent and -independent manners. Therefore, administration of tetracaine inhibits translation initiation with P-body formation at both concentrations but acts via the Gcn2-eIF2α system only at the higher concentration. Because other local anesthetics and phenothiazines induced Pat1-dependent P-body formation, the mechanisms involved in translational inhibition by these cationic amphiphiles are similar. These results suggest that this dose-dependent biphasic translational inhibition by tetracaine results from an increase in membrane proteins that are indirectly inhibited by nonspecific interactions of cationic amphiphiles with membrane lipids.

Action mechanisms of anesthetics remain unclear because of difficulty in explaining how structurally different anesthetics cause similar effects. In Saccharomyces cerevisiae, local anesthetics and antipsychotic phenothiazines induced responses similar to those caused by glucose starvation, and they eventually inhibited cell growth. These drugs inhibited glucose uptake, but additional glucose conferred resistance to their effects; hence, the primary action of the drugs is to cause glucose starvation. In hxt0 strains with all hexose transporter (HXT) genes deleted, a strain harboring a single copy of HXT1 (HXT1s) was more sensitive to tetracaine than a strain harboring multiple copies (HXT1m), which indicates that quantitative reduction of HXT1 increases tetracaine sensitivity. However, additional glucose rather than the overexpression of HXT1/2 conferred tetracaine resistance to wild-type yeast; therefore, Hxts that actively transport hexoses apparently confer tetracaine resistance. Additional glucose alleviated sensitivity to local anesthetics and phenothiazines in the HXT1m strain but not the HXT1s strain; thus, the glucose-induced effects required a certain amount of Hxt1. At low concentrations, fluorescent phenothiazines were distributed in various membranes. At higher concentrations, they destroyed the membranes and thereby delocalized Hxt1-GFP from the plasma membrane, similar to local anesthetics. These results suggest that the aforementioned drugs affect various membrane targets via nonspecific interactions with membranes. However, the drugs preferentially inhibit the function of abundant Hxts, resulting in glucose starvation. When Hxts are scarce, this preference is lost, thereby mitigating the alleviation by additional glucose. These results provide a mechanism that explains how different compounds induce similar effects based on lipid theory.

We examined a novel rice mutant, Fukei 71 (Oryza sativa L.), for alterations in the levels of hydroxycinnamoyl esters that are linked to cell wall polysaccharides and lignin units. In this mutant, a recessive mutation at a single locus caused the collapse of parenchyma cells in the internodes. Light microscopy revealed that the abnormal walls of internode parenchyma cells of Fukei 71 were stained by the Mäule reaction, which is specific for syringyl units in phenolic compounds. These walls were not stained by Wiesner's reagent (phloroglucinol-HCl), which reacts cinnamaldehyde in lignin. Levels of p-coumaric acid (PCA) and ferulic acid (FA) were apparently elevated in the abnormal tissue of the mutant. Western blotting analysis with antibodies specific for phenylalanine ammonia-lyase (PAL) revealed higher levels of PAL in the abnormal parenchyma tissue of Fukei 71 than in the parenchyma tissue of the parent cultivar Fujiminori. These results and the observation that PAL was produced at a greatly elevated level indicated that the phenylpropanoid pathway that leads to the biosynthesis of polysaccharide-linked FA and PCA was abnormally activated in the irregularly shaped and collapsed internode parenchyma cells, in which the biosynthesis of lignin is normally repressed.

High osmolarity and glucose deprivation cause rapid shutdowns of both actin polarization and translation initiation in yeast. Like these stresses, administration of local anesthetics and of antipsychotic phenothiazines caused similar responses. All these drugs have amphiphilic structures and formed emulsions and permeabilized the cell membrane, indicating that they have the same features as a surfactant. Consistently with this, surfactants induced responses similar to those of local anesthetics and phenothiazines. Benzethonium chloride, a cationic surfactant, showed a more potent shutdown activity than phenothiazines, whereas SDS, an anionic surfactant, transiently depolarized actin without inhibiting translation initiation, suggesting that a cationic charge in the amphiphile is important to the shutdown of both reactions. The clinical drugs and the cationic surfactants at low concentrations caused shutdown without membrane permeabilization, suggesting that these compounds and stresses activate shutdown, via perturbation rather than disruption of the cell membrane.

It is known that some local anesthetics inhibit the growth of budding yeast cells. To investigate the pathway of local anesthetics’ action, we isolated and characterized mutants that were hyper-sensitive to tetracaine, and at the same time, temperature-sensitive for growth. They were collectively called las (local anesthetic sensitive) mutants. One of the LAS genes, LAS24, was found to be identical to KOG1, which had been independently discovered as a member of the TOR complex 1 (TORC1). Las24p/Kog1p is a widely conserved TOR binding protein containing the NRC domain, HEAT repeats and WD-40 repeats, but its function remains unknown. Like the tor mutants, the las24 mutants were found to have a defect in cell wall integrity and to show sensitivity to rapamycin. Furthermore, Las24p is required not only in TORC1-mediated (rapamycin-sensitive) pathways such as translation initiation control and phosphorylation of Npr1p and Gln3p, but also for the normal distribution of the actin cytoskeleton, which has been regarded as a TORC2-mediated event. Intriguingly, the temperature-sensitivity of the las24 mutant was suppressed by either activation of Tap42/PPase or by down-regulation of the RAS/cAMP pathway. Suppressors of the temperature-sensitivity of the las24-1 mutant were found not to be effective for suppression of the tetracaine-sensitivity of the same mutant. These observations along with the facts that tetracaine and high temperature differentially affected the las24-1 mutant suggest that Las24p/Kog1p is not a target of tetracaine and that the tetracaine-sensitive step may be one of downstream branches of the TORC1 pathway. Consistent with the broad cellular functions exerted by the TOR pathway, we found that Las24p was associated with membranes and was localized at vacuoles, the plasma membrane and small vesicles.