Binding of a pleurotolysin ortholog from Pleurotus eryngii to sphingomyelin and cholesterol-rich membrane domains

A mixture of sphingomyelin (SM) and cholesterol (Chol) exhibits a characteristic lipid raft domain of the cell membranes that provides a platform to which various signal molecules as well as virus and bacterial proteins are recruited. Several proteins capable of specifically binding either SM or Chol have been reported. However, proteins that selectively bind to SM/Chol mixtures are less well characterized. In our screening for proteins specifically binding to SM/Chol liposomes, we identified a novel ortholog of Pleurotus ostreatus, pleurotolysin (Ply)A, from the extract of edible mushroom Pleurotus eryngii, named PlyA2. Enhanced green fluorescent protein (EGFP)-conjugated PlyA2 bound to SM/Chol but not to phosphatidylcholine/Chol liposomes. Cell surface labeling of PlyA2-EGFP was abolished after sphingomyelinase as well as methyl-β-cyclodextrin treatment, removing SM and Chol, respectively, indicating that PlyA2-EGFP specifically binds cell surface SM/Chol rafts. Tryptophan to alanine point mutation of PlyA2 revealed the importance of C-terminal tryptophan residues for SM/Chol binding. Our results indicate that PlyA2-EGFP is a novel protein probe to label SM/Chol lipid domains both in cell and model membranes.     

Involvement of AQP6 in the Mercury-Sensitive Osmotic Lysis of Rat Parotid Secretory Granules

In secretory granules and vesicles, membrane transporters have been predicted to permeate water molecules, ions and/or small solutes to swell the granules and promote membrane fusion. We have previously demonstrated that aquaporin-6 (AQP6), a water channel protein, which permeates anions, is localized in rat parotid secretory granules (Matsuki-Fukushima et al., Cell Tissue Res 332:73–80, 2008). Because the localization of AQP6 in other organs is restricted to cytosolic vesicles, the native function or functions of AQP6 in vivo has not been well determined. To characterize the channel property in granule membranes, the solute permeation-induced lysis of purified secretory granules is a useful marker. To analyze the role of AQP6 in secretory granule membranes, we used Hg²⁺, which is known to activate AQP6, and investigated the characteristics of solute permeability in rat parotid secretory granule lysis induced by Hg²⁺ (Hg lysis). The kinetics of osmotic secretory granule lysis in an iso-osmotic KCl solution was monitored by the decay of optical density at 540 nm using a spectrophotometer. Osmotic secretory granule lysis was markedly facilitated in the presence of 0.5–2.0 μM Hg²⁺, concentrations that activate AQP6. The Hg lysis was completely blocked by β-mercaptoethanol which disrupts Hg²⁺-binding, or by removal of chloride ions from the reaction medium. An anion channel blocker, DIDS, which does not affect AQP6, discriminated between DIDS-insensitive and sensitive components in Hg lysis. These results suggest that Hg lysis is required for anion permeability through the protein transporter. Hg lysis depended on anion conductance with a sequence of NO₃ ^? > Br^? > I^? > Cl^? and was facilitated by acidic pH. The anion selectivity for NO₃ ^? and the acidic pH sensitivity were similar to the channel properties of AQP6. Taken together, it is likely that AQP6 permeates halide group anions as a Hg²⁺-sensitive anion channel in rat parotid secretory granules.     

Rheb (Ras Homologue Enriched in Brain)-dependent Mammalian Target of Rapamycin Complex 1 (mTORC1) Activation Becomes Indispensable for Cardiac Hypertrophic Growth after Early Postnatal Period

Cardiomyocytes proliferate during fetal life but lose their ability to proliferate soon after birth and further increases in cardiac mass are achieved through an increase in cell size or hypertrophy. Mammalian target of rapamycin complex 1 (mTORC1) is critical for cell growth and proliferation. Rheb (Ras homologue enriched in brain) is one of the most important upstream regulators of mTORC1. Here, we attempted to clarify the role of Rheb in the heart using cardiac-specific Rheb-deficient mice (Rheb^−/−). Rheb^−/− mice died from postnatal day 8 to 10. The heart-to-body weight ratio, an index of cardiomyocyte hypertrophy, in Rheb^−/− was lower than that in the control (Rheb^+/+) at postnatal day 8. The cell surface area of cardiomyocytes isolated from the mouse hearts increased from postnatal days 5 to 8 in Rheb^+/+ mice but not in Rheb^−/− mice. Ultrastructural analysis indicated that sarcomere maturation was impaired in Rheb^−/− hearts during the neonatal period. Rheb^−/− hearts exhibited no difference in the phosphorylation level of S6 or 4E-BP1, downstream of mTORC1 at postnatal day 3 but showed attenuation at postnatal day 5 or 8 compared with the control. Polysome analysis revealed that the mRNA translation activity decreased in Rheb^−/− hearts at postnatal day 8. Furthermore, ablation of eukaryotic initiation factor 4E-binding protein 1 in Rheb^−/− mice improved mRNA translation, cardiac hypertrophic growth, sarcomere maturation, and survival. Thus, Rheb-dependent mTORC1 activation becomes essential for cardiomyocyte hypertrophic growth after early postnatal period.     

Omega-3 polyunsaturated fatty acid has an anti-oxidant effect via the Nrf-2/HO-1 pathway in 3T3-L1 adipocytes

Oxidative stress is produced in adipose tissue of obese subjects and has been associated with obesity-related disorders. Recent studies have shown that omega-3 polyunsaturated fatty acid (ω3-PUFA) has beneficial effects in preventing atherosclerotic diseases and insulin resistance in adipose tissue. However, the role of ω3-PUFA on adipocytes has not been elucidated. In this study, 3T3-L1 adipocytes were treated with ω3-PUFA and its metabolites, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), or 4-hydroxy hexenal (4-HHE). ω3-PUFA and its metabolites dose-dependently increased mRNA and protein levels of the anti-oxidative enzyme, heme oxygenase-1 (HO-1); whereas no changes in the well-known anti-oxidant molecules, superoxide dismutase, catalase, and glutathione peroxidase, were observed. Knockdown of nuclear factor erythroid 2-related factor 2 (Nrf-2) significantly reduced EPA, DHA or 4-HHE-induced HO-1 mRNA and protein expression. Also, pretreatment with ω3-PUFA prevented H₂O₂-induced cytotoxicity in a HO-1 dependent manner. In conclusion, treatment with EPA and DHA induced HO-1 through the activation of Nrf-2 and prevented oxidative stress in 3T3-L1 adipocytes. This anti-oxidant defense may be of high therapeutic value for clinical conditions associated with systemic oxidative stress.     

Synthesis and biological evaluation of pyrrolidine derivatives as novel and potent sodium channel blockers for the treatment of ischemic stroke

A novel series of pyrrolidine derivatives as Na⁺ channel blockers was synthesized and evaluated for their inhibitory effects on neuronal Na⁺ channels. Structure–activity relationship (SAR) studies of a pyrrolidine analogue 2 led to the discovery of 5e as a potent Na⁺ channel blocker with a low inhibitory action against human ether-a-go-go-related gene (hERG) channels. Compound 5e showed remarkably neuroprotective activity in a rat transient middle cerebral artery occlusion (MCAO) model, suggesting that 5e would act as a neuroprotectant for ischemic stroke.     

Chemical screening of an inhibitor for gibberellin receptors based on a yeast two-hybrid system

We applied a yeast two-hybrid (Y2H) system to the high-throughput monitoring of two proteins’ interaction, a receptor for phytohormone gibberellin (GA) and its direct signal transducer DELLA. With this system, we screened inhibitors to the interaction. As a result, we discovered a chemical, 3-(2-thienylsulfonyl)pyrazine-2-carbonitrile (TSPC), and we confirmed that TSPC is an inhibitor for GA perception by in vitro and in planta evaluations.     

Synthesis and biological evaluation of novel tryptoline derivatives as indoleamine 2,3-dioxygenase (IDO) inhibitors

Indoleamine 2,3-dioxygenase (IDO) plays a significant role in several disorders such as Alzheimer’s disease, age-related cataracts and tumors. A series of novel tryptoline derivatives were synthesized and evaluated for their inhibitory activity against IDO. Substituted tryptoline derivatives (11a, 11c, 11e, 12b and 12c) were demonstrated to be more potent than known inhibitor MTH-Trp. Suzuki–Miyaura cross-coupling reaction of 11a–d with phenylboronic acid proceeded in high yields. In most cases, C5 and C6 substitutions on the corresponding indole ring were well tolerated. The tryptoline derivative 11c is a promising chemical lead for the discovery of novel IDO inhibitors.     

Properties of New Enzyme Glycerol Oxidase from Aspergillus japonicus AT 008

Purified glycerol oxidase from Aspergillus japonicus AT 008 was homogeneous by ultracentrifugation and acrylamide gel electrophoresis. The molecular weight was determined to be 400,000 by sedimentation equilibrium, and the isoelectric point was found to be 4.9 by isoelectric focusing. The enzyme showed spectral characteristics of a heme protein. The reduced form possessed absorption maxima at 557 and 430 nm and the oxidized one at 557, 530, 420, 280, and 238 nm. The heme in the enzyme was identified as protoheme IX (one mol per mol of enzyme protein).

Glycerol was the best substrate for the enzyme, and the Km value for glycerol was determined to be 10.4 mm. Dihydroxyacetone was oxidized at 59% of that for glycerol, but glycerol 3-phosphate, dihydroxyacetone phosphate, methanol, and ethanol were not oxidized at all. The enzyme had an optimal pH at 7.0 with glycerol as substrate, and the enzymatic activity increased by treatment in alkaline pH. The enzyme was also activated by addition of several divalent metal ions including Zn²⁺, Ni²⁺, and Mg²⁺.     

Changes of Hexosamine and Acetyl Contents during Cultivation of Extracellular Galactosamine-containing Polymer from Aspergillus parasiticus

The reactions of N-acetylchitooligosaccharides with chitinolytic enzyme were analyzed by HPLC using a Tosoh TSK-Gel amide-80 column with 70% acetonitrile as an eluent. We separated α and β anomeric forms of N-acetylchitooligosaccharides, and obtain the following advantages of this HPLC method.

1. We can easily identify the reaction mechanism of chitinolytic enzymes by this method, distinguishing the inverting mechanism showing α anomer formation from the retaining mechanism showing β anomer formation.

2. We can also estimate the cleavage patterns of N-acetylchitooligosaccharides by chitinolytic enzymes by using natural substrates.     

Quantitative Structure-Activity Relationships for Antifungal 3-(3,5-Dichloropheny)-2,4-imidazolidinediones

The antifungal activity of 441-acyl derivatives of 3-(3,5-dichlorophenyl)-2,4-imidazol- idinedione against Botrytis cinerea, and of 10 1-sulfonyl compounds against Aiternaria kikuchi- ana were assayed by the agar medium dilution method. The structure-activity relationships for the substituents of the acyl and sulfonyl moieties were analyzed with such physicochemical parameters as hydrophobic π, inductive electronic σ₁, and steric E′^c_s and B₁ values by multiple regression. The activity of the acyl derivatives against B. cinerea was related parabolically to the hydrophobicity of the substituents. The stronger the electron-donating power, the larger the overall steric bulkiness, and the smaller the minimum width in the direction perpendicular to the bond axis of the substituents, the greater was the activity. The activity of the sulfonyl derivatives against A. kikuciana was related only to the hydrophobicity of the substituents.