Plasma ApoE4 Levels Are Lower than ApoE2 and ApoE3 Levels, and Not Associated with Plasma Aß40/42 Ratio as a Biomarker of Amyloid-ß Amyloidosis in Alzheimer's Disease.

BACKGROUND: APOE4 is the strongest risk factor for Alzheimer's disease (AD). However, limited information is currently available on APOE4 and the pathological role of plasma apolipoprotein E (ApoE) 4 remains unclear. OBJECTIVE: The aims of the present study were to measure plasma levels of total ApoE (tE), ApoE2, ApoE3, and ApoE4 using mass spectrometry and elucidate the relationships between plasma ApoE and blood test items. METHODS: We herein examined plasma levels of tE, ApoE2, ApoE3, and ApoE4 in 498 subjects using liquid chromatograph-mass spectrometry (LC-MS/MS). RESULTS: Among 498 subjects, mean age was 60 years and 309 were female. tE levels were distributed as ApoE2/E3 = ApoE2/E4 >ApoE3/E3 = ApoE3/E4 >ApoE4/E4. In the heterozygous group, ApoE isoform levels were distributed as ApoE2 >ApoE3 >ApoE4. ApoE levels were not associated with aging, the plasma amyloid-ß (Aß) 40/42 ratio, or the clinical diagnosis of AD. Total cholesterol levels correlated with the level of each ApoE isoform. ApoE2 levels were associated with renal function, ApoE3 levels with low-density lipoprotein cholesterol and liver function, and ApoE4 levels with triglycerides, high-density lipoprotein cholesterol, body weight, erythropoiesis, and insulin metabolism. CONCLUSION: The present results suggest the potential of LC-MS/MS for the phenotyping and quantitation of plasma ApoE. Plasma ApoE levels are regulated in the order of ApoE2 >ApoE3 >ApoE4 and are associated with lipids and multiple metabolic pathways, but not directly with aging or AD biomarkers. The present results provide insights into the multiple pathways by which peripheral ApoE4 influences the progression of AD and atherosclerosis.

Development of Electrochemical Oxygen Demand Measurement Cells Using a Diamond Electrode.

Electrolytic cells for electrochemical oxygen demand (ECOD) measurements based on total electrolytic decomposition at a boron-doped diamond (BDD) electrode were developed for rapid measurement of organic pollutants at low concentrations. Using improved electrolytic cells designed for efficient mass transfer, the ECOD for 10 µM potassium hydrogen phthalate (theoretical ECOD: 2.3 mg-O2 L-1) was determined in a relatively short electrolysis time. Thus, ECOD measurements using these cells would be useful for estimating organic water pollution in industrial waste and lake water.

Inhibition of MEK1 Signaling Pathway in the Liver Ameliorates Insulin Resistance.

Although mitogen-activated protein kinase kinase (MEK) is a key signaling molecule and a negative regulator of insulin action, it is still uncertain whether MEK can be a therapeutic target for amelioration of insulin resistance (IR) in type 2 diabetes (T2D) in vivo. To clarify whether MEK inhibition improves T2D, we examined the effect of continuous MEK inhibition with two structurally different MEK inhibitors, RO5126766 and RO4987655, in mouse models of T2D. RO5126766 and RO4987655 were administered via dietary admixture. Both compounds decreased blood glucose and improved glucose tolerance in doses sufficient to sustain inhibition of extracellular signal-regulated kinase (ERK)1/2 phosphorylation downstream of MEK in insulin-responsive tissues in db/db mice. A hyperinsulinemic-euglycemic clamp test showed increased glucose infusion rate (GIR) in db/db mice treated with these compounds, and about 60% of the increase was attributed to the inhibition of endogenous glucose production, suggesting that the liver is responsible for the improvement of IR. By means of adenovirus-mediated Mek1 shRNA expression, we confirmed that blood glucose levels are reduced by suppression of MEK1 expression in the liver of db/db mice. Taken together, these results suggested that the MEK signaling pathway could be a novel therapeutic target for novel antidiabetic agents.

Direct determination of chemical oxygen demand by anodic decomposition of organic compounds at a diamond electrode.

Chemical oxygen demand (COD) was measured directly with a simple electrochemical method using a boron-doped diamond (BDD) electrode. By applying a highly positive potential (+2.5 V vs Ag/AgCl) to an aqueous electrolyte containing potassium hydrogen phthalate, glucose, and lactic acid or sodium dodecylbenzenesulfonate using a BDD electrode, an anodic current corresponding to the electrolytic decomposition of these organic compounds was observed. No such current was seen on glassy carbon or platinum electrodes due to a significant background current caused by the oxygen evolution reaction. The electric charge for the anodic current observed at the BDD electrode was found to be consistent with the theoretical charge required for the electrolytic decomposition of the organic compounds to CO2 and was used to calculate COD. This analysis was performed by a simple I-t measurement at constant potential using a BDD electrode, and no calibration was needed. This new simple indicator, "ECOD" (electrochemical oxygen demand), will be useful for continuous monitoring of industrial wastewater with low protein concentrations and on-site instant analysis of natural water with a BDD electrode-based portable ECOD meter.

Role of Src-family kinases in formation of the cortical actin cap at the dorsal cell surface.

Protein-tyrosine phosphorylation is regulated by protein-tyrosine kinases and protein-tyrosine phosphatases (PTPs). Src-family tyrosine kinases (SFKs) participate in the regulation of the actin cytoskeleton. Actin filaments can be accumulated in a cap at the dorsal cell surface, which is called the cortical actin cap. Here, we show that SFKs play an important role in formation of the cortical actin cap. HeLa cells normally exhibit the cortical actin cap, one of the major sites of tyrosine phosphorylation. The cortical actin cap is disrupted by SFK inhibitors or overexpression of the Lyn SH3 domain. Csk-knockout cells form the cortical actin cap when the level of tyrosine phosphorylation is increased by Na(3)VO(4), a PTP inhibitor, and the formation of the cortical actin cap is inhibited by SFK inactivation with re-introduction of Csk. SYF cells lacking SFKs minimally exhibit the cortical actin cap even in the presence of Na(3)VO(4), and transfection with Lyn restores the cortical actin cap in the presence of Na(3)VO(4). Disruption of the cortical actin cap by dominant-negative Cdc42 causes loss of tyrosine phosphorylation at the cell top. These results suggest that SFK(s) is involved in formation of the cortical actin cap, which may serve as a platform of tyrosine phosphorylation signaling.

Differential mitotic activation of endogenous c-Src, c-Yes, and Lyn in HeLa cells.

Src-family tyrosine kinases (SFKs) play an important role in mitosis. Despite overlapping expression of multiple SFK members, little is known about how individual SFK members are activated in M phase. Here, we examined mitotic activation of endogenous c-Src, c-Yes, and Lyn, which are co-expressed in HeLa cells. c-Src, c-Yes, and Lyn were activated at different levels in M phase, and the activation was inhibited by Cdc2 inactivation. Mitotic c-Src and c-Yes exhibited normal- and retarded-electrophoretic-mobility forms on SDS-polyacrylamide gels, whereas Lyn did not show mobility retardation. Like c-Src, the retardation of electrophoretic mobility of c-Yes was caused by Cdc2-mediated phosphorylation. The normal- and retarded-mobility forms of c-Src were comparably activated, but activation of the retarded-mobility form of c-Yes was higher than that of the normal-mobility form of c-Yes. Thus, these results suggest that endogenous c-Src, c-Yes, and Lyn are differentially activated through Cdc2 activation during M phase.

Role of Src-family kinases in formation and trafficking of macropinosomes.

Src-family kinases that localize to the cytoplasmic side of cellular membranes through lipid modification play a role in signaling events including membrane trafficking. Macropinocytosis is an endocytic process for solute uptake by large vesicles called macropinosomes. Although macropinosomes can be visualized following uptake of fluorescent macromolecules, little is known about the dynamics of macropinosomes in living cells. Here, we show that constitutive c-Src expression generates macropinosomes in a kinase-dependent manner. Live-cell imaging of GFP-tagged c-Src (Src-GFP) reveals that c-Src associates with macropinosomes via its N-terminus continuously from their generation at membrane ruffles, through their centripetal trafficking, to fusion with late endosomes and lysosomes. Fluorescence recovery after photobleaching (FRAP) of Src-GFP shows that Src-GFP is rapidly recruited to macropinosomal membranes from the plasma membrane and intracellular organelles through vesicle transport even in the presence of a protein synthesis inhibitor. Furthermore, using a HeLa cell line overexpressing inducible c-Src, we show that following stimulation with epidermal growth factor (EGF), high levels of c-Src kinase activity promote formation of macropinosomes associated with the lysosomal compartment. Unlike c-Src, Lyn and Fyn, which are palmitoylated Src kinases, only minimally induce macropinosomes, although a Lyn mutant in which the palmitoylation site is mutated efficiently induces macropinocytosis. We conclude that kinase activity of nonpalmitoylated Src kinases including c-Src may play an important role in the biogenesis and trafficking of macropinosomes.

Stereochemical course in water addition during LUP1-catalyzed triterpene cyclization.

Arabidopsis thaliana LUP1 (At1g78970) catalyzes the cyclization of oxidosqualene into lupeol and 3beta,20-dihydroxylupane (lupanediol). The stereochemical course of water addition to the lupanyl cation was studied. The X-ray crystal structure of lupanylepoxide 3,5-dinitrobenzoate established the configuration of epoxide as 20S. LiAlD4 reduction of the epoxide enabled the chemical shift assignment of prochiral methyl groups at C20 of lupanediol. Correlation of these methyl groups with biosynthetic lupanediol from [1,2-(13)C(2)] acetate established the stereochemical course of water addition. [reaction: see text].

Identification of beta-amyrin and sophoradiol 24-hydroxylase by expressed sequence tag mining and functional expression assay.

Triterpenes exhibit a wide range of structural diversity produced by a sequence of biosynthetic reactions. Cyclization of oxidosqualene is the initial origin of structural diversity of skeletons in their biosynthesis, and subsequent regio- and stereospecific hydroxylation of the triterpene skeleton produces further structural diversity. The enzymes responsible for this hydroxylation were thought to be cytochrome P450-dependent monooxygenase, although their cloning has not been reported. To mine these hydroxylases from cytochrome P450 genes, five genes (CYP71D8, CYP82A2, CYP82A3, CYP82A4 and CYP93E1) reported to be elicitor-inducible genes in Glycine max expressed sequence tags (EST), were amplified by PCR, and screened for their ability to hydroxylate triterpenes (beta-amyrin or sophoradiol) by heterologous expression in the yeast Saccharomyces cerevisiae. Among them, CYP93E1 transformant showed hydroxylating activity on both substrates. The products were identified as olean-12-ene-3beta,24-diol and soyasapogenol B, respectively, by GC-MS. Co-expression of CYP93E1 and beta-amyrin synthase in S. cerevisiae yielded olean-12-ene-3beta,24-diol. This is the first identification of triterpene hydroxylase cDNA from any plant species. Successful identification of a beta-amyrin and sophoradiol 24-hydroxylase from the inducible family of cytochrome P450 genes suggests that other triterpene hydroxylases belong to this family. In addition, substrate specificity with the obtained P450 hydroxylase indicates the two possible biosynthetic routes from triterpene-monool to triterpene-triol.