Inhibitory effect of zeatin, isolated from Fiatoua villosa, on acetylcholinesterase activity from PC12 cells

Acetylcholinesterase (AChE) inhibitors, which enhance cholinergic transmission by reducing the enzymatic degradation of acetylcholine, are the only source of the compound that is currently approved for the treatment of Alzheimer's disease (AD). The methanol extract from Fiatoua villosa among 100 traditional edible plants that were tested, showed the most potent inhibitory effect (51%) on acetylcholinesterase in vitro. After the sequential solvent fractionation of the methanol extract of Fiatoua villosa, the active fraction was repeatedly subjected to open-column chromatography on silica gel. From the highest inhibitory fraction, the chloroform fraction (75%) on AChE, the single compound, was obtained by the Sep-Pak Cartridge (C18: reverse phase column). This compound was finally purified by HPLC (micro-bondapack C18 reverse phase column: 19 x 300 mm). According to the electron impact mass spectrometry (EI-MS), we confirmed that the molecular mass was 219 m/z. The structure of this compound was identified as zeatin [2-methyl-4-(1H-purine-6-ylamino)-2-buten-1-ol], one of the derivatives of purine adenine. The concentration that was required for 50% enzyme inhibition (IC50 value) was 1.09 x 10(-4) M. This study demonstrated that the zeatin from Fiatoua villosa appeared to be the most potent AChE inhibitor in AD.     

Functional effects of auxiliary beta4-subunit on rat large-conductance Ca(2+)-activated K(+) channel

Large-conductance calcium-activated potassium (BK(Ca)) channels are composed of the pore-forming alpha-subunit and the auxiliary beta-subunits. The beta4-subunit is dominantly expressed in the mammalian central nervous system. To understand the physiological roles of the beta4-subunit on the BK(Ca) channel alpha-subunit (Slo), we isolated a full-length complementary DNA of rat beta4-subunit (rbeta4), expressed heterolgously in Xenopus oocytes, and investigated the detailed functional effects using electrophysiological means. When expressed together with rat Slo (rSlo), rbeta4 profoundly altered the gating characteristics of the Slo channel. At a given concentration of intracellular Ca(2+), rSlo/rbeta4 channels were more sensitive to transmembrane voltage changes. The activation and deactivation rates of macroscopic currents were decreased in a Ca(2+)-dependent manner. The channel activation by Ca(2+) became more cooperative by the coexpression of rbeta4. Single-channel recordings showed that the increased Hill coefficient for Ca(2+) was due to the changes in the open probability of the rSlo/rbeta4 channel. Single BK(Ca) channels composed of rSlo and rbeta4 also exhibited slower kinetics for steady-state gating compared with rSlo channels. Dwell times of both open and closed events were significantly increased. Because BK(Ca) channels are known to modulate neuroexcitability and the expression of the beta4-subunit is highly concentrated in certain subregions of brain, the electrophysiological properties of individual neurons should be affected profoundly by the expression of this second subunit.     

pH-dependent perturbation of Ras-guanine nucleotide interactions and Ras guanine nucleotide exchange

p21Ras (Ras) proteins cycle between active GTP-bound and inactive GDP-bound states to mediate signal transduction pathways that promote cell growth, differentiation, and apoptosis. To better understand how cellular regulatory factors, such as guanine nucleotide exchange factors (GEFs) and nitric oxide (NO), modulate Ras-guanine nucleotide binding interactions, we have conducted NMR and kinetic studies to investigate the pH dependence of Ras-GDP interactions and Ras-guanine nucleotide exchange (GNE). pH-sensitive amide protons were identified and found to be associated with residues in the switch I (Phe28-Asp30) and switch II (Asp57 and Thr58) regions of Ras. Furthermore, most of the residues that interact with Mg2+ exhibit pH-sensitive amide proton chemical shifts which appear to be coupled to pH-dependent Ras Mg2+ binding and guanine nucleotide binding affinity. These results suggest that perturbation of Mg2+ interactions within the Ras-guanine nucleotide complex is critical for pH-dependent dissociation of guanine nucleotide ligands from Ras. Notably, these same regions undergo conformational changes upon association with the Ras GEF, SOS. In addition, although we have recently shown that addition of NO to Ras in the presence of oxygen produces a Ras thiyl radical intermediate that promotes Ras GNE, we have also postulated that another byproduct of this reaction, a H+, may contribute to NO-mediated GNE. However, the results presented herein suggest that the H+ byproduct of the reaction is unlikely to be involved in the NO-mediated Ras GNE.     

NHERF2 specifically interacts with LPA2 receptor and defines the specificity and efficiency of receptor-mediated phospholipase C-beta3 activation

Lysophosphatidic acid (LPA) activates a family of cognate G protein-coupled receptors and is involved in various pathophysiological processes. However, it is not clearly understood how these LPA receptors are specifically coupled to their downstream signaling molecules. This study found that LPA(2), but not the other LPA receptor isoforms, specifically interacts with Na(+)/H(+) exchanger regulatory factor2 (NHERF2). In addition, the interaction between them requires the C-terminal PDZ domain-binding motif of LPA(2) and the second PDZ domain of NHERF2. Moreover, the stable expression of NHERF2 potentiated LPA-induced phospholipase C-beta (PLC-beta) activation, which was markedly attenuated by either a mutation in the PDZ-binding motif of LPA(2) or by the gene silencing of NHERF2. Using its second PDZ domain, NHERF2 was found to indirectly link LPA(2) to PLC-beta3 to form a complex, and the other PLC-beta isozymes were not included in the protein complex. Consistently, LPA(2)-mediated PLC-beta activation was specifically inhibited by the gene silencing of PLC-beta3. In addition, NHERF2 increases LPA-induced ERK activation, which is followed by cyclooxygenase-2 induction via a PLC-dependent pathway. Overall, the results suggest that a ternary complex composed of LPA(2), NHERF2, and PLC-beta3 may play a key role in the LPA(2)-mediated PLC-beta signaling pathway.     

Cloning and expression of a novel esterase gene cpoA from Burkholderia cepacia

AIMS: To screen and clone a novel enzyme with specific activity for the resolution of (R)-beta-acetylmercaptoisobutyrate (RAM) from (R,S)-beta-acetylmercaptoisobutyrate [(R,S)-ester]. METHODS AND RESULTS: A micro-organism that produces a novel esterase was isolated and identified as the bacterium Burkholderia cepacia by using the analysis of cellular fatty acids, Biolog automated microbial identification/characterization system, and 16S rRNA gene sequence analysis. A novel esterase gene was cloned from the chromosomal DNA of B. cepacia and was designated as cpoA. The cpoA encodes a polypeptide of 273 amino acids which shows a strong sequence homology with many bacterial nonhaeme chloroperoxidases. In addition, a typical serine-hydrolase motif, Gly-X-Ser-X-Gly, and the highly conserved catalytic triad, Ser95, Asp224, and His253, were identified in the deduced amino acid sequence of cpoA by multiple sequence alignment. CONCLUSION: The cpoA cloned from B. cepacia encodes a novel esterase which is highly related to the nonhaeme chloroperoxidases. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report that describes the isolation and cloning of a serine esterase gene from B. cepacia, which is useful in the chiral resolution of (R,S)-ester. The cloned gene will allow additional research on the bifunctionality of the enzyme with esterase and chloroperoxidase activity at the structural and functional levels.     

Spread of Neoheterobothrium hirame (Monogenea), a serious pest of olive flounder Paralichthys olivaceus, to Korea

Neoheterobothrium hirame is a large, blood-feeding gill-worm infecting the highly prized olive flounder Paralichthys olivaceus in Japan. There is strong evidence that this worm is the primary cause of anaemia, a common and serious condition causing losses among both wild and cultured olive flounders. N. hirame was first detected in Japanese waters less than a decade ago, and its population then proliferated and spread throughout most of Japan, except Hokkaido. In neighbouring Korea, olive flounder is the most important species of cultured marine fish, and production currently exceeds that in Japan. However, until now, there have been no reports of any monogeneans or anaemia among olive flounders in Korea. Our survey conducted in 2000 of 100 cultured individuals from 4 provinces revealed 2 immature specimens of N. hirame: 1 from a land-based pond-culture system in southern Cheju Island (off the SW coast of Korea) and the other from a floating net cage near Yosu (in the mid-S part of the peninsula). The geographic range of this pathogen may have been enlarged as a result of introduction(s) of infected broodstock from Japan, but this seems unlikely. (The raising of this species in hatcheries developed in Korea in 1985, 7 years before the earliest detection of the worm in Japan.) Low numbers of flounders were also clearly anaemic. This, and the current rarity of N. hirame in Korean farms, appears to favour the hypothesis of a more recent, natural dispersal of the worm, during migrations of infected flounder across the narrow and shallow Tsushima and Korea Straits. Regardless of route of entry, we expect this pathogen will have an impact on Korean flounder fisheries equally serious to that being experienced in Japan.     

Anti-genotoxicity of galangin as a cancer chemopreventive agent candidate

Flavonoids are polyphenolic compounds that are present in plants. They have been shown to possess a variety of biological activities at non-toxic concentrations in organisms. Galangin, a member of the flavonol class of flavonoid, is present in high concentrations in medicinal plants (e.g. Alpinia officinarum) and propolis, a natural beehive product. Results from in vitro and in vivo studies indicate that galangin with anti-oxidative and free radical scavenging activities is capable of modulating enzyme activities and suppressing the genotoxicity of chemicals. These activities will be discussed in this review. Based on our review, galangin may be a promising candidate for cancer chemoprevention.     

Protection against ultraviolet B- and C-induced DNA damage and skin carcinogenesis by the flowers of Prunus persica extract

The ethanol extract of the flowers of Prunus persica (Ku-35) (50-200 microg/ml) was found to inhibit UVB- as well as UVC-induced DNA damage measured by the COMET assay in the skin fibroblast cell (NIH/3T3). In addition, Ku-35 inhibited UVB- or UVC-induced lipid peroxidation, especially against UVB-induced peroxidation at higher than 10 microg/ml. We also evaluated the protective effect of Ku-35 against UVB-induced non-melanoma skin cancer in mice. Ku-35 was applied topically before UVB exposure, and its effects on tumor incidence (% of mice with tumors) and tumor multiplicity (number of tumors per mouse) were evaluated. The application of Ku-35 clearly resulted in a delay of tumor development compared to the control. In tumor incidence, 100% mice in the control group and the low dose treatment of Ku-35 had tumors, whereas 94.1% of the mice had tumors after the high dose treatment of Ku-35 at the end of experiment (28 weeks). In tumor multiplicity, low and high treatments of Ku-35 resulted in 25.9 and 53.9% reduction at the end of the experiment (P<0.05, one-way analysis of variance (ANOVA)). The present data indicate that Ku-35 protects against photogenotoxicity in NIH/3T3 fibroblasts. The possible action mechanism of Ku-35 may be through its anti-oxidant activity without pro-oxidant effect. Ku-35 can also show a delay of tumor development against UVB-induced skin carcinogenesis. These results suggest that Ku-35 extract may be useful for protecting UV-induced DNA damage and carcinogenesis when topically applied.     

A novel protein interacts with the Werner's syndrome gene product physically and functionally

Werner's syndrome (WS) is a rare autosomal recessive disorder characterized by premature aging. The gene responsible for WS encodes a protein homologous to Escherichia coli RecQ. Here we describe a novel Werner helicase interacting protein (WHIP), which interacts with the N-terminal portion of Werner protein (WRN), containing the exonuclease domain. WHIP, which shows homology to replication factor C family proteins, is conserved from E. coli to human. Ectopically expressed WHIP and WRN co-localized in granular structures in the nucleus. The functional relationship between WHIP and WRN was indicated by genetic analysis of yeast cells. Disruptants of the SGS1 gene of Saccharomyces cerevisiae, which is the WRN homologue in yeast, show an accelerated aging phenotype and high sensitivity to methyl methanesulfonate as compared with wild-type cells. Disruption of the yeast WHIP (yWHIP) gene in wild-type cells and sgs1 disruptants resulted in slightly accelerated aging and enhancement of the premature aging phenotype of sgs1 disruptants, respectively. In contrast, disruption of the yWHIP gene partially alleviated the sensitivity to methyl methanesulfonate of sgs1 disruptants.