Mutations in Parkinson disease (PD)3 is a major neurodegenerative disease of the adulthood affecting the extrapyramidal motor system (1). The brains of patients affected with PD are characterized by a loss of neurons in the brainstem monoaminergic neurons, e.g. dopamine neurons in the substantia nigra and noradrenergic neurons in the locus caeruleus, which is accompanied by the deposition of Lewy bodies (LBs) in the cytoplasm of remaining neurons. LBs are characteristic spherical intracytoplasmic inclusions composed of filaments of ϳ7-10 nm in diameter and are pathognomonic for PD and related dementing disorder, dementia with Lewy bodies (DLB) (2). A small percentage of patients inherit PD as an autosomal dominant trait (familial PD; FPD), and missense mutations (3-5) or multiplications (6 -8) of the ␣-synuclein gene have been identified in these families. LBs in the brains of patients with sporadic PD or DLB were shown to be composed of ␣-synuclein (9, 10). Glial cytoplasmic inclusions in the brains of patients with multiple system atrophy, another major sporadic neurodegenerative disease, or dystrophic neurites in Hallervorden-Spatz disease also were shown to be composed of ␣-synuclein, and these neurodegenerative diseases characterized by deposition of ␣-synuclein are collectively designated "synucleinopathies" (2). In vitro studies suggested that ␣-synuclein aggregates and forms filaments that are similar to those seen in PD or DLB brains, and amino acid substitutions linked to familial PD (A53T, A30P, or E46K) have been shown to enhance the aggregation of ␣-synuclein possibly through conformational changes (11-14), implicating deposition of ␣-synuclein in the pathogenesis of synucleinopathies including PD. Taken together with the gene dosage effects of ␣-synuclein in a subset of FPD (i.e. duplication and triplication), transgenic overexpression of ␣-synuclein in neurons would be a rational strategy to model neurodegeneration in PD.A number of transgenic models overexpressing ␣-synuclein using heterologous organisms Drosophila (21,22), Caenorhabditis elegans (23), yeast (24)) have been reported, and some abnormal behavioral or pathological phenotypes have been documented in a subset of these animal models. However, an ideal model in which deposition of ␣-synuclein in dopamine neurons, the most vulnerable subset of neurons in PD, causes behavioral phenotypes that are inherent to the functions of dopamine neurons, has not been established yet. Here we describe a transgenic C. elegans model in which human ␣-synuclein overexpressed specifically in dopamine neurons causes an abnormal phenotype in food-sensing behavior that has been attributed to the function of C. elegans dopamine neurons (25), in a manner dependent on FPD-linked mutations, through reduction of neuronal dopamine.

The fluorogenic derivatization reagents with a positive charge, 4-(dimethylaminoethylaminosulfonyl)-7-chloro-2,1,3-benzoxadiazole (DAABD-Cl) and 7-chloro-2,1,3-benzoxadiazole-4-sulfonylaminoethyltrimethylammonium chloride (TAABD-Cl), are proposed for use in proteomics studies. Following derivatization of protein mixtures with these reagents, a series of standard processes of isolation, digestion, and identification of the proteins were performed utilizing high-performance liquid chromatography-fluorescence detection and tandem mass spectrometry with the probability-based protein identification algorithm. Both DAABD and TAABD derivatives were detected fluorometrically at the femtomole level and showed more than 100-fold improvement in sensitivity compared to the underivatized original compounds with an electrospray ionization ion trap mass spectrometer analysis. The modification of the MASCOT database search system memorized with the fragment information of a DAABD-attached Cys residue allowed the identification of the proteolytic peptide fragments of the derivatized bovine serum albumin (BSA) with an estimated 38% sequence coverage of BSA. Utilizing DAABD-Cl as a derivatization reagent, identification of several proteins was also possible in a soluble extract of Caenorhabditis elegans (10 microg of protein). Consequently, for identification of proteins in the complex matrixes of proteins, DAABD-Cl could be a more appropriate reagent than ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate as reported previously.

Glycan Atlas is a set of glycan maps over the whole body of an organism. The glycan map that includes data of glycan structure and quantity displays micro-heterogeneity of the glycans in a tissue, an organ, or cells. The two-dimensional glycan mapping is widely used for structure analysis of N-linked oligosaccharides on glycoproteins. In this study we developed a comprehensive method for the mapping of both N- and O-glycans with and without sialic acid. The mapping data of 150 standard pyridylaminated glycans were collected. The empirical additivity rule which was proposed in former reports was able to adapt for this extended glycan map. The adapted rule is that the elution time of pyridylamino glycans on high performance liquid chromatography (HPLC) is expected to be the simple sum of the partial elution times assigned to each monosaccharide residue. The comprehensive mapping method developed in this study is a powerful tool for describing the micro-heterogeneity of the glycans. Furthermore, we prepared 42 pyridylamino (PA-) glycans from human serum and were able to draw the map of human serum N- and O-glycans as an initial step of Glycan Atlas editing.

A method to measure catechol- O-methyltransferase (COMT) activity using high performance liquid chromatography-fluorescence detection with norepinephrine (NE) as a natural substrate was optimized for both soluble (S-) and membrane-bound (MB-) COMT activities in rat brain areas, cerebral cortex, cerebellum, hippocampus, brain stem, hypophysis, and hypothalamus. The highest S-COMT activity in Sprague-Dawley rat brain was found in hippocampus. MB-COMT activities in all brain areas were about 3-8 times lower than S-COMT activities. However, considering Vmax/ Km values, specificity constants for NE to S- and MB-COMT contributes mainly to the metabolism of NE in cerebral cortex and cerebellum.

We have previously reported a highly sensitive method for the measurement of catechol-O-methyltransferase (COMT) activities in rat erythrocytes with norepinephrine (NE), an endogenous native substrate, using high-performance liquid chromatography (HPLC)-fluorescence or peroxyoxalate chemiluminescence reaction detection. Applying this method to COMT activities in rat liver and kidney, known to have the highest activities of all organs, the optimum reaction conditions were investigated. Under the optimum conditions, soluble (S)-COMT and membrane-bound (MB)-COMT activities in rat liver, with NE as a substrate, were 2.17 AE 0.33 and 0.16 AE 0.02 nmol/min/mg protein (n = 5), respectively. In rat kidney, S-COMT and MB-COMT activities were 1.81 AE 0.20 and 0.079 AE 0.009 nmol/min/mg protein (n = 5), respectively. Since liver and kidney play important roles in inactivating catecholamines, using the proposed method would yield critical information to delineate the role of metabolism of catecholamines in rat tissues.

Catechol-O-methyltransferase (COMT; EC2.1.1.6) is an enzyme which inactivates the released catecholamines from nerve endings by methylating their catechol moieties using S-adenosyl-L-methionine (SAMe) as a methyl donor. [1][2][3][4] COMT is found in most mammalian tissues, with highest activity in the liver and the kidney. There are two COMT isoforms: in the cytoplasm as soluble COMT (S-COMT) and in association with membranes as membrane-bound COMT (MB-COMT). S-COMT protein is more prevalent than MB-COMT in all tissues in rats. 5)Catecholamines, norepinephrine (NE), dopamine and epinephrine, play important roles in the central nervous system as in the periphery, 6) and in central regions, catecholaminesrelated gene expression was correlated with blood pressure. 7)We have previously reported an assay method for rat brain COMT activities, using NE as an endogenous substrate. 8)The use of the endogenous substrate provides significant information of COMT in vivo as compared with formerly reported method which uses an artificial substrate, 3,4-dihydroxybenzoic acid (DBA). Endogenous NE has higher affinity for COMT than DBA, and our method is more sensitive to measure COMT activity. In this study, COMT activities were evaluated in cerebral cortex, cerebellum, hippocampus, brain stem, hypophysis, and hypothalamus in order to evaluate the role of COMT in blood pressure regulation using spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats. In addition, in order to investigate the contribution of COMT activities to NE metabolism, NE and its 3-Omethyl metabolite, normetanephrine (NMN), concentrations were examined in discrete areas of SHR and WKY rats. MATERIALS AND METHODSReagent NE, NMN, SAMe chloride salt and 4-methoxytyramine (4-MT) were obtained from Sigma (St. Louis, MO, U.S.A.). Ethylenediamine was obtained from Sigma-Aldrich (Milwaukee, WI, U.S.A.). Imidazole and 1,4-dithiothreitol were from Merck (Darmstadt, Germany). Acetonitrile and ethanol, both of HPLC grade, were purchased from Wako Pure Chemicals (Osaka, Japan). All other reagents were of analytical grade.Animals Male WKY rats (22 weeks old, 405-415 g) and SHR (22 weeks old, 330-350 g) were purchased from Charles River Japan Inc. (Kanagawa, Japan), and housed under controlled environment (22-24°C and a 12-h light-dark cycle) with free access to tap water and diet for at least 1 week before study. All animals received animal care in compliance with the National Institute of Health guideline.Preparation of Brain COMT Samples In anesthetized rats with pentobarbital, blood was removed from inferior vena cava, and cerebral cortex, cerebellum, hippocampus, brain stem, hypophysis and hypothalamus were immediately removed and chilled on ice. All further procedures were conducted at 4°C. Each tissue was weighed and then homogenized with four volumes of 50 mmol/l sodium phosphate buffer containing 0.5 mmol/l 1,4-dithiothreitol (pH 7.5). The homogenates were centrifuged at 100000ϫg for 30 min and the supernatants were stored for the determination of S-COMT...

An improved method for proteomics studies, which includes the fluorogenic derivertization of protein mixtures with 7-chloro-4-(dimethylaminoethylaminosulfonyl)-2,1,3-benzoxadiazole (DAABD-Cl), followed by HPLC isolation, enzymatic digestion and identification of the derivatized proteins by HPLC-electrospray ionization (ESI)-MS/MS with the probability-based protein identification algorithm, identified 103 proteins in the soluble extract (10 microg protein) of Caenorhabditis elegans.

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