Rat choline acetyltransferase of the peripheral type differs from that of the common type in intracellular translocation

Choline acetyltransferase (ChAT), the synthesizing enzyme for acetylcholine, has been implicated to involve multiple isoforms of ChAT mRNA in several animals. Since these isoforms are mostly non-coding splice variants, only a homologous ChAT protein of about 68 kDa has been shown to be produced in vivo. Recent evidence indicates the existence of a protein coding splice variant of ChAT mRNA, which lacks exons 6-9 of the rat ChAT gene. The encoded protein was designated ChAT of a peripheral type (pChAT), because of its preferential expression in the peripheral nervous system as confirmed by Western blot and immunohistochemistry. However, functional significance of pChAT is unknown. To obtain a clue to this question, we examined a possible difference in intracellular trafficking between pChAT and the well-known ChAT of the common type (cChAT) using green fluorescent protein (GFP) in living human embryonic kidney cells. Confocal laser scanning microscopy revealed that pChAT-GFP was detectable in the cytoplasm but not in the nucleus, whereas cChAT-GFP was found in both cytoplasm and nucleus. Following treatment with leptomycin B, a nuclear export pathway inhibitor, pChAT-GFP became detectable in both cytoplasm and nucleus, indicating that pChAT can be translocated to the nucleus. In contrast, the leptomycin B treatment did not seem to affect the content of intranuclear cChAT-GFP. After incubation with protein kinase C inhibitors, enhanced accumulation of pChAT-GFP but not cChAT-GFP occurred in the nucleus. These results clearly indicate that pChAT varies from cChAT in intracellular transportation, probably reflecting the difference in physiological roles between pChAT and cChAT.     

Identification of the MHC class I B locus in cynomolgus monkeys

By determining the nucleotide sequences of more than 700 cDNA clones isolated from 16 cynomolgus monkeys, we identified 26 Mafa-B alleles. In addition, nine sequences with similarity to Mamu-I alleles were identified. Since multiple Mafa-B alleles were found in each individual, it was strongly suggested that the cynomolgus MHC class I B locus might be duplicated and that the Mafa-I locus was derived from the B locus by gene duplication, as in the case of the Mamu-I locus of rhesus monkeys.     

Asterosap-induced elevation in intracellular pH is indispensable for ARIS-induced sustained increase in intracellular Ca2+ and following acrosome reaction in starfish spermatozoa

In the starfish, Asterias amurensis, the cooperation of three components of the egg jelly, namely ARIS (acrosome reaction-inducing substance), Co-ARIS and asterosap, is responsible for the induction of acrosome reaction. For the induction, ARIS alone is enough in high-Ca2+ or high-pH seawater, but, besides ARIS, the addition of either Co-ARIS or asterosap is required in normal seawater. Asterosap transiently increased both the intracellular pH (pHi) and Ca2+ ([Ca2+]i), while ARIS slightly elevated the basal level of [Ca2+]i. However, a sustained elevation of [Ca2+]i and acrosome reaction occurred if sperm were simultaneously treated with ARIS and asterosap. EGTA inhibited the sustained [Ca2+]i elevation and acrosome reaction. The sustained [Ca2+]i elevation and acrosome reaction were highly susceptible to SKF96365 and Ni2+, specific blockers of the store-operated Ca2+ channel (SOC). These results suggest that sustained [Ca2+]i elevation, mediated by the SOC-like channel, is a prerequisite for the acrosome reaction. In high-pH seawater, ARIS alone induced a prominent [Ca2+]i increase and acrosome reaction, which were similarly sensitive to SKF96365. The acrosome reaction was effectively induced by ARIS alone when pHi was artificially increased to more than 7.7. Asterosap increased pHi from 7.6 +/- 0.1 to 7.7 +/- 0.1. Furthermore, the sustained [Ca2+]i elevation and acrosome reaction, induced by a combination of ARIS and asterosap, were drastically inhibited by a slight reduction in pHi. Taking these results into account, we suggest that an asterosap-induced pHi elevation is required for triggering the ARIS-induced sustained [Ca2+]i elevation and consequent acrosome reaction.     

Design and development of a fluorescent probe for monitoring hydrogen peroxide using photoinduced electron transfer

A novel fluorescent probe, 7-hydroxy-2-oxo-N-(2-(diphenylphosphino)ethyl)-2H-chromene-3-carboxamide (DPPEA-HC) was developed for use in monitoring hydrogen peroxide (H2O2) production. DPPEA-HC, which consists of a diphenylphosphine moiety and a 7-hydroxycoumarin moiety, reacts with H2O2 to form DPPEA-HC oxide, which is analogous to the reaction of triphenylphosphine with hydroperoxides such as H2O2 to form triphenylphosphine oxide. Photoinduced electron transfer (PET) was applied in the design of DPPEA-HC. Since the diphenylphosphine moiety and the 7-hydroxycoumarin moiety would act as the PET donor and the acceptor, respectively, it would be expected that DPPEA-HC would rationally cancel the PET process via the formation of DPPEA-HC oxide, based on the calculated energy levels of the donor and the acceptor moieties using the B3LYP/6-31G*//AM1 method. The fluorescence intensity of DPPEA-HC increased on the addition of a H2O2 solution in 100 mM sodium phosphate buffer (pH7.4), as predicted from the energy level calculation and a good correlation between increase in the fluorescence of DPPEA-HC and the concentration of H2O2 was observed. DPPEA-HC was also fluoresced by H2O2, which was enzymatically produced in xanthine/xanthine oxidase/superoxide dismutase (XA/XOD/SOD) system. The increase in the fluorescence of DPPEA-HC in the presence of H2O2 immediately ceased on the addition of catalase (CAT), which catalyzes the disproportionation of H2O2. In addition, DPPEA-HC was found to have a much higher selectivity for H2O2 and a greater resistance to autoxidation than 2',7'-dichlorodihydrofluoresein (DCFH). Time-resolved fluorescence measurements of DPPEA-HC and DPPEA-HC oxide confirmed that the fluorescence off/on switching mechanism of DPPEA-HC is based on the PET on/off control.     

Characterization of the oxidation products of BO-653 formed during peroxyl radical-mediated oxidation of human plasma

4,6-Di-tert-butyl-2,3-dihydro-2,2-dipentyl-5-benzofuranol (BO-653) is a novel antioxidant synthesized by theoretical findings and considerations. Here we report on the aqueous peroxyl radical-induced oxidation of human plasma in the presence of BO-653. When BO-653 was given to healthy human subjects at 400 mg twice daily for 28 days, lipids in the resulting plasma were protected from oxidation compared with lipids present in plasma from subjects receiving placebo. Similarly, BO-653 added in vitro at 50 muM inhibited the peroxyl radical-induced accumulation of cholesteryl ester hydroperoxides that occurred in the presence of alpha-tocopherol, although BO-653 did not decrease the rate of consumption of ascorbate, albumin-bound bilirubin, and uric acid. The antioxidant action of in vivo and in vitro added BO-653 was associated with the formation of two major reaction products of BO-653, the structures of which were elucidated by mass spectrometry and nuclear magnetic resonance analyses. The products were identified as stereoisomers of dioxybis(4,6-di-tert.-butyl-2,3,5,7a-tetrahydro-2,2-dipentylbenzofuran-5-one). These dialkylperoxides of BO-653 might be useful markers to assess the antioxidant function of BO-653 in biological systems in vivo.     

Chromatographic resolution of glucosidic compounds, ginsenosides on polyethersulphone membrane, and its application to the quantitative immunoassay for ginseng saponins

A method has been devised for the chromatographic resolution of glucosidic compounds, ginseng saponins, on polyethersulphone (PES) membrane. The method results in good resolution and quantitative immunoassay for ginsenoside Rb1 (G-Rb1), G-Rc, and G-Rd in crude extracts of various ginsengs. The newly established method is simpler and applies for quantitative analysis. Ginsenosides developed by acetonitrile-water-acetic acid solvent system on a PES membrane were directly treated with a NaIO4 solution followed by bovine serum albumin (BSA), resulting in a ginsenoside-BSA conjugate on a PES membrane. Anti-G-Rb1 monoclonal antibody (MAb) was bound, and then a second antibody labeled with peroxidase directed against the first antibody. Finally a substrate reacted to the enzyme and gave staining. The stained membrane was scanned, and spots were analyzed quantitatively using NIH Image software. At least 62.5 ng of G-Rb1, G-Rc, and G-Rd were clearly detectable individually. Three ginsenosides can be analyzed quantitatively between 0.125 and 2.0 microg.     

Utilization of mammalian cells for efficient and reliable evaluation of specificity of antibodies to unravel the cellular function of mKIAA proteins

Complementary DNA (cDNA) clones for human KIAA genes have been isolated as long cDNAs (>4 kb) with unknown functions. To facilitate the functional analysis of these human clones, we have isolated and determined the structures of their respective mouse homologues (mKIAA genes). Furthermore, we have comprehensively raised antibodies against the translated mKIAA proteins in order to establish a platform for their functional analysis. Since the specificity of these antibodies is critical for subsequent analyses of protein function, here we introduce two assays utilizing mammalian cells to improve their evaluation. First, we have established a semi-high-throughput production of C-terminally FLAG epitope-tagged proteins for Western blotting using specially designed mammalian expression vectors. Secondly, we have utilized immunofluorescence staining of mouse cells to analyze the subcellular localization of endogenous mKIAA proteins. Importantly, these methods allow us to detect potential posttranslational modification of the mKIAA/KIAA proteins and to predict their biological function based on their subcellular localization.