A phytochemical investigation of the fruits of Citrullus colocynthis resulted in the isolation of 21 structurally diverse cucurbitane triterpenoids, including 9 previously undescribed ones, colocynins A-I (1-9). Their absolute configurations were elucidated by means of quantum chemical electronic circular dichroism (ECD) calculations, CD exciton chirality method, and single-crystal X-ray crystallography. Colocynins A-C (1-3) represent the first examples of nonanorcucurbitane-type triterpenoids. An anti-acetylcholinesterase activity assay showed that 6, 10, 13, 18, and 20 exhibited inhibitory activities, with IC(50) values ranging from 5.0 to 21.7 microM. In addition, 18 and 21 showed significant cytotoxicity against PACA, A431, and HepG2 cells, with IC(50) values ranging from 0.042 to 0.60 and 3.6-14.4 microM, respectively.
Polyethylene terephthalate (PET) is among the most extensively produced plastics, but huge amounts of PET wastes that have accumulated in the environment have become a serious threat to the ecosystem. Applying PET hydrolytic enzymes to depolymerize PET is an attractive measure to manage PET pollution, and searching for more effective enzymes is a prerequisite to achieve this goal. A thermostable cutinase that originates from the leaf-branch compost termed ICCG is the most effective PET hydrolase reported so far. Here, we illustrated the crystal structure of ICCG in complex with the PET analogue, mono(2-hydroxyethyl)terephthalic acid, to reveal the enzyme-substrate interaction network. Furthermore, we applied structure-based engineering to modify ICCG and screened for variants that exhibit higher efficacy than the parental enzyme. As a result, several variants with the measured melting temperature approaching 99 C and elevated PET hydrolytic activity were obtained. Finally, crystallographic analyses were performed to reveal the structural stabilization effects mediated by the introduced mutations. These results are of importance in the context of understanding the mechanism of action of the thermostable PET hydrolytic enzyme and shall be beneficial to the development of PET biodegradation platforms.
Poly(ethylene terephthalate) (PET) is the most abundant polyester plastic and a major contributor to plastic pollution. IsPETase, from the PET-assimilating bacterium Ideonella sakaiensis, is a unique PET-hydrolytic enzyme that shares high sequence identity to canonical cutinases, but shows substrate preference towards PET and exhibits higher PET-hydrolytic activity at ambient temperature. Structural analyses suggest that IsPETase harbours a substrate-binding residue, W185, with a wobbling conformation and a highly flexible W185-locating beta6-beta7 loop. Here, we show that these features result from the presence of S214 and I218 in IsPETase, whose equivalents are strictly His and Phe, respectively, in all other homologous enzymes. We found that mutating His/Phe residues to Ser/Ile could enhance the PET-hydrolytic activity of several IsPETase-like enzymes. In conclusion, the Ser/Ile mutations should provide an important strategy to improve the activity of potential PET-hydrolytic enzymes with properties that may be useful for various applications.
Title: Enhancing PET hydrolytic enzyme activity by fusion of the cellulose-binding domain of cellobiohydrolase I from Trichoderma reesei Dai L, Qu Y, Huang JW, Hu Y, Hu H, Li S, Chen CC, Guo RT Ref: J Biotechnol, 334:47, 2021 : PubMed
The large amounts of polyethylene terephthalate (PET) that enter and accumulate in the environment have posed a serious threat to global ecosystems and human health. A PET hydrolase from PET-assimilating bacterium Ideonella sakaiensis (IsPETase) that exhibits superior PET hydrolytic activity at mild conditions is attracting enormous attention in development of plastic biodegrading strategies. In order to enhance the PET hydrolysis capacity of IsPETase, we selected several polymer-binding domains that can adhere to a hydrophobic polymer surface and fused these to a previously engineered IsPETase(S121E/D186H/R280A) (IsPETase(EHA)) variant. We found that fusing a cellulose-binding domain (CBM) of cellobiohydrolase I from Trichoderma reesei onto the C-terminus of IsPETase(EHA) showed a stimulatory effect on enzymatic hydrolysis of PET. Compared to the parental enzyme, IsPETase(EHA)_CBM exhibited 71.5 % and 44.5 % higher hydrolytic activity at 30 degC and 40 degC, respectively. The catalytic activity of IsPETase(EHA)_CBM was increased by 86 % when the protein concentration was increased from 2.5 microg/mL to 20microg/mL. These findings suggest that the fusion of polymer-binding module to IsPETase is a promising strategy to stimulate the enzymatic hydrolysis of PET.
Title: Enzymatic degradation of plant biomass and synthetic polymers Chen CC, Dai L, Lixin Ma L, Guo RT Ref: Nature Reviews Chemistry, 4:114, 2020 : PubMed
Plant biomass is an abundant renewable resource on Earth. Microorganisms harvest energy from plant material by means of complex enzymatic systems that efficiently degrade natural polymers. Intriguingly, microorganisms have evolved to exploit these ancient mechanisms to also decompose synthetic plastic polymers. In this Review, we summarize the mechanisms by which they decompose non-starch plant biomass and the six major types of synthetic plastics. We focus on the structural features of the enzymes that contribute to substrate recognition and then describe the catalytic mechanisms of polymer metabolism. An understanding of these natural biocatalysts is valuable if we are to exploit their potential for the degradation of synthetic polymers.
Thraustochytrids are heterotrophic fungus-like protists that can dissolve organic matters with enzymes. Four strains, AP45, ASP1, ASP2, and ASP4, were isolated from the coastal water of Taiwan, and respectively identified as Aurantiochytrium sp., Schizochytrium sp., Parietichytrium sp., and Botryochytrium sp. based on 18S rRNA sequences. Transcriptome datasets of these four strains at days 3-5 were generated using Next Generation Sequencing technology, and screened for enzymes with potential industrial applications. Functional annotations based on KEGG database suggest that many unigenes of all four strains were related to the pathways of industrial enzymes. Most of all four strains contained homologous genes for 15 out of the 17 targeted enzymes, and had extra- and/or intra-cellular enzymatic activities, including urease, asparaginase, lipase, glucosidase, alkaline phosphatase and protease. Complete amino sequences of the first-time identified L-asparaginase and phytase in thraustochytrids were retrieved, and respectively categorized to the Type I and BPPhy families based on phylogenetic relationships, protein structural modeling and active sites. Milligram quantities of highly purified, soluble protein of urease and L-asparaginase were successfully harvested and analyzed for recombinant enzymatic activities. These analytical results highlight the diverse enzymes for wide-range applications in thraustochytrids.
When administrated orally, the vasodilating drug diltiazem can be metabolized into diacetyl diltiazem in the presence of Bacteroides thetaiotaomicron, a human gut microbe. The removal of acetyl group from the parent drug is carried out by the GDSL/SGNH-family hydrolase BT4096. Here the crystal structure of the enzyme was solved by mercury soaking and single-wavelength anomalous diffraction. The protein folds into two parts. The N-terminal part comprises the catalytic domain which is similar to other GDSL/SGNH hydrolases. The flanking C-terminal part is made up of a beta-barrel subdomain and an alpha-helical subdomain. Structural comparison shows that the catalytic domain is most akin to acetyl-xylooligosaccharide esterase and allows a plausible binding mode of diltiazem to be proposed. The beta-barrel subdomain is similar in topology to the immunoglobulin-like domains, including some carbohydrate-binding modules, of various bacterial glycoside hydrolases. Consequently, BT4096 might originally function as an oligosaccharide deacetylase with additional subdomains that could enhance substrate binding, and it acts on diltiazem just by accident.
Title: Two novel lipases purified from rice bran displaying lipolytic and esterification activities Chen CC, Gao GJ, Kao AL, Tsai CT, Tsai ZC Ref: Int J Biol Macromol, 139:298, 2019 : PubMed
In this study, two novel lipases, rice bran lipase 1 (RBL1) and rice bran lipase 2 (RBL2), were first identified in rice (Oryza sativa) bran. Through the purification by ammonium sulfate precipitation, ion-exchange chromatography and size-exclusion chromatography, RBL1 and RBL2 were purified to 36- and 339-fold with the final specific activity of 4.73 and 44.06U/mg, respectively. The purified RBL1 and RBL2 had the molecular weight of 18.8 and 35.5kDa, respectively. The Km values of RBL1 and RBL2 were 0.766 and 0.432mM while catalytic efficiency (kcat/Km) values of RBL1 and RBL2 were 102.4 and 2559.3s(-1)/mM, respectively. RBL1 and RBL2 both showed esterification activity, but had no transesterification activity. In a solvent-free system, RBL1 and RBL2 exhibited degree of esterification (ED) of 4.4% and 6.5%, respectively. These two novel lipases exerted great properties for their potentials in industrial applications. First, RBL1 and RBL2 showed both mild reaction pH of 7.0 and temperature of 35 degrees C and 50 degrees C, respectively. Secondly, they showed great tolerance to several organic solvents and detergents while RBL1 exhibited great pH stability across a very broad range of pH (pH3-12). Lastly and most importantly, RBL1 and RBL2 both exhibited esterification activity better than a commercial lipase, Candida rugosa lipase (CRL), in a solvent-free system. In conclusion, two novel lipases, RBL1 and RBL2, are different from published native lipases in rice bran and may be alternative potential candidates of biocatalysts contributing to the development of diverse industrial application fields.
Title: Induction of Autophagic Death of Human Hepatocellular Carcinoma Cells by Armillaridin from Armillaria mellea Leu YS, Chen YJ, Chen CC, Huang HL Ref: Am J Chin Med, 47:1365, 2019 : PubMed
The honey mushroom, Armillaria mellea, is known to have medicinal qualities and has been used in recent years as a health food and dietary supplement worldwide. In Asia, it is commonly consumed as an herbal medicine, being a key component of the Chinese preparation "Tien-ma". Here, we examined the antitumor effects of armillaridin, a bioactive compound isolated from A. mellea, on human hepatocellular carcinoma (HCC) cells. Armillaridin inhibited the growth of human Huh7, HepG2, and HA22T HCC cells, and its cytotoxicity was confirmed by observations of its induction of mitochondrial transmembrane potential collapse. However, armillaridin treatment did not result in large numbers of cells with fragmented chromosomal DNA, suggesting that apoptosis was not responsible for these effects. We therefore tested for signs of autophagic cell death following armillaridin administration. Armillaridin induced LC3 aggregation in green fluorescent protein-LC3-overexpressing cells. Moreover, flow cytometry and immunoblotting revealed that it increased the number of acridine orange-positive cells and upregulated autophagy-related proteins, respectively. Furthermore, armillaridin cytotoxicity was suppressed by the autophagy inhibitor 3-methyladenine. In summary, our results indicated that armillaridin induces HCC cell death by autophagy, and demonstrated the potential of armillaridin as an antihepatoma agent.
Title: Structural studies reveal the molecular mechanism of PETase Chen CC, Han X, Ko TP, Liu W, Guo RT Ref: Febs J, 285:3717, 2018 : PubMed
Poly(ethylene terephthalate) (PET) is a class of plastic material widely used in modern society, but large amounts of PET waste cause severe environmental problems. Obtained from a PET-consuming bacterium Ideonella sakaiensis, the enzyme PETase exhibits superb hydrolytic activity and substrate preference toward PET. Here, we summarize some recent advances in the crystallographic analysis of PETase. These reports uncover structural features of PETase that are involved in its catalytic activity. In comparison to homologous enzymes, PETase contains an additional disulfide bond as well as an extended beta8-alpha6 loop. More importantly, the crystal structures of PETase in complex with substrate and product analogs provide critical information for understanding the mechanism of action of PETase. In particular, the wobbling conformation of W156 is closely related to the binding of substrate and product. These new findings are of great importance for further in-depth research and engineering development of PETase, and should advance the implementation of plastic biodegradation strategy.
Development of potent biocatalysts for enzymatic detoxification of estrogenic mycotoxin zearalenone (ZEN) and its more toxic derivative alpha-zearalenol (alpha-ZOL) is of great interest. Here, we report the crystal structures of a ZEN-hydrolyzing enzyme from Rhinocladiella mackenziei (RmZHD), including substrate complexes. A molecular mechanism for the distinct activity of RmZHD in hydrolyzing the structurally similar ZEN and alpha-ZOL is then proposed. In addition, structure-based engineering to modify the substrate-binding pocket and improve the RmZHD activity toward alpha-ZOL is presented. These results expand our scope in understanding the catalytic mechanism of ZHD-family enzymes and are of vital importance in further industrial applications.
Title: Using proteomics to discover novel biomarkers for fatty liver development and response to CB1R antagonist treatment in an obese mouse model Chen CC, Lee TY, Kwok CF, Hsu YP, Shih KC, Lin YJ, Ho LT Ref: Proteomics, 17:, 2017 : PubMed
Over activity of cannabinoid receptor type 1 (CB1R) plays a key role in increasing the incidence of obesity-induced non-alcoholic fatty liver disease. Tissue proteome analysis has been applied to investigate the bioinformatics regarding the mode of action and therapeutic mechanism. The aim of this study was to explore the potential pathways altered with CB1R in obesity-induced fatty liver. Male C57BL/6 mice were fed either a standard chow diet (STD) or a high-fat diet (HFD) with or without 1-week treatment of CB1R inverse agonist AM251 at 5 mg/kg. Then, liver tissues were harvested for 2DE analysis and protein profiles were identified by using MALDI-MS. Results showed that eight of significantly altered protein spots at the level of changes > twofold were overlapped among the three groups, naming major urinary protein 1, ATP synthase subunit beta, glucosamine-fructose-6-phosphate aminotransferase 1, zine finger protein 2, s-adenosylmethionine synthase isoform type-1, isocitrate dehydrogenase subunit alpha, epoxide hydrolase 2 and 60S acidic ribosomal protein P0. These identified proteins were involved in glucose/lipid metabolic process, xenobiotic metabolic system, and ATP synthesized process in mitochondria. Based on the findings, we speculated that CB1R blockade might exert its anti-metabolic disorder effect via improvement of mitochondrial function in hepatic steatosis in HFD condition.
PET hydrolase (PETase), which hydrolyzes polyethylene terephthalate (PET) into soluble building blocks, provides an attractive avenue for the bioconversion of plastics. Here we present the structures of a novel PETase from the PET-consuming microbe Ideonella sakaiensis in complex with substrate and product analogs. Through structural analyses, mutagenesis, and activity measurements, a substrate-binding mode is proposed, and several features critical for catalysis are elucidated.
Title: Characterization and crystal structure of a novel zearalenone hydrolase from Cladophialophora bantiana Hui R, Hu X, Liu W, Zheng Y, Chen Y, Guo RT, Jin J, Chen CC Ref: Acta Crystallographica F Struct Biol Commun, 73:515, 2017 : PubMed
Zearalenone (ZEN) is a mycotoxin which causes huge economic losses in the food and animal feed industries. The lactonase ZHD101 from Clonostachys rosea, which catalyzes the hydrolytic degradation of ZEN, is the only known ZEN-detoxifying enzyme. Here, a protein homologous to ZHD101, denoted CbZHD, from Cladophialophora batiana was expressed and characterized. Sequence alignment indicates that CbZHD possesses the same catalytic triad and ZEN-interacting residues as found in ZHD101. CbZHD exhibits optimal enzyme activity at 35 degrees C and pH 8, and is sensitive to heat treatment. The crystal structure of apo CbZHD was determined to 1.75 A resolution. The active-site compositions of CbZHD and ZHD101 were analyzed.
Title: Armillaridin induces autophagy-associated cell death in human chronic myelogenous leukemia K562 cells Chang WH, Huang HL, Huang WP, Chen CC, Chen YJ Ref: Tumour Biol, 37:14291, 2016 : PubMed
Armillaridin (AM) is an aromatic ester compound isolated from Armillaria mellea. Treatment with AM markedly reduced the viability of human chronic myelogenous leukemia K562, chronic erythroleukemia HEL 92.1.7, and acute monoblastic leukemia U937 cells, but not normal human monocytes, in a dose- and time-dependent manner. Treatment of K562 cells with AM caused changes characteristic of autophagy. Only a small amount of AM-treated K562 cells exhibited apoptosis. By contrast, AM treatment resulted in extensive apoptotic features in U937 and HEL 92.1.7 cells without evident autophagy. The autophagy of K562 cells induced by AM involved autophagic flux, including autophagosome induction, the processing of autophagosome-lysosome fusion and downregulation of BCL2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3). By bcr-abl knockdown, the growth inhibition of K562 cells caused by AM was partially blocked, suggesting that AM-induced cell death might be a bcr-abl-dependent mode of autophagy-associated cell death. In conclusion, AM is capable of inhibiting growth and inducing autophagy-associated cell death in K562 cells, but not in normal monocytes. It may have potential to be developed as a novel therapeutic agent against leukemia.
Title: Early postnatal treatment with soluble epoxide hydrolase inhibitor or 15-deoxy-Delta(12,14)-prostagandin J2 prevents prenatal dexamethasone and postnatal high saturated fat diet induced programmed hypertension in adult rat offspring Lu PC, Sheen JM, Yu HR, Lin YJ, Chen CC, Tiao MM, Tsai CC, Huang LT, Tain YL Ref: Prostaglandins Other Lipid Mediat, 124:1, 2016 : PubMed
Prenatal dexamethasone (DEX) exposure, postnatal high-fat (HF) intake, and arachidonic acid pathway are closely related to hypertension. We tested whether a soluble epoxide hydrolase (SEH) inhibitor, 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA) or 15-deoxy-Delta(12,14)-prostagandin J2 (15dPGJ2) therapy can rescue programmed hypertension in the DEX+HF two-hit model. Four groups of Sprague Dawley rats were studied: control, DEX+HF, AUDA, and 15dPGJ2. Dexamethasone (0.1mg/kg body weight) was intraperitoneally administered to pregnant rats from gestational day 16-22. Male offspring received high-fat diet (D12331, Research Diets) from weaning to 4 months of age. In AUDA group, mother rats received 25mg/L in drinking water during lactation. In the 15dPGJ2 group, male offspring received 15dPGJ2 1.5mg/kg BW by subcutaneous injection once daily for 1 week after birth. We found postnatal HF diet aggravated prenatal DEX-induced programmed hypertension, which was similarly prevented by early treatment with AUDA or 15dPGJ2. The beneficial effects of AUDA and 15d-PGJ2 therapy include inhibition of SEH, increases of renal angiotensin converting enzyme-2 (ACE2) and angiotensin II type 2 receptor (AT2R) protein levels, and restoration of nitric oxide bioavailability. Better understanding of the impact of arachidonic acid pathway in the two-hit model will help prevent programmed hypertension in children exposed to corticosteroids and postnatal HF intake.
The enzyme ZHD101 from Clonostachys rosea hydrolyzes and deactivates the mycotoxin zearalenone (ZEN) and its zearalenol (ZOL) derivatives. ZHD101 prefers ZEN to ZOL as its substrate, but ZOL, especially the -form, shows higher estrogenic toxicity than ZEN. To enhance alpha-ZOL selectivity, we solved the complex structures of ZHD101 with both ZOLs and modified several lactone-surrounding residues. Among the mutants, V153H maintained activity for ZEN but showed a 3.7-fold increase in specific activity against alpha-ZOL, with an 2.7-fold reduction in substrate affinity but a 5.2-fold higher turnover rate. We then determined two V153H/ZOL complex structures. Here, the alpha-ZOL lactone ring is hydrogen-bonded to the H153 side chain, yielding a larger space for H242 to reconstitute the catalytic triad. In conclusion, structure-based engineering was successfully employed to improve the ZHD101 activity toward the more toxic alpha-ZOL, with great potential in further industrial applications.
Title: Armillaridin, a Honey Medicinal Mushroom, Armillaria mellea (Higher Basidiomycetes) Component, Inhibits Differentiation and Activation of Human Macrophages Liu TP, Chen CC, Shiao PY, Shieh HR, Chen YY, Chen YJ Ref: Int J Med Mushrooms, 17:161, 2015 : PubMed
Armillaridin (AM) is an aromatic ester compound isolated from honey medicinal mushroom, Armillaria mellea, which has anti-cancer potential. This study was designed to examine the effects of AM on differentiation and activation macrophages, the major ontogeny of innate immunity. Macrophages were derived from CD14+ monocytes which were sorted from human peripheral blood mononuclear cells. Cell viability was assessed by trypan blue exclusion test. Cells were stained with Liu's dye for observation of morphology. Expression of surface antigens was examined by flow cytometric analysis. Phagocytosis and generation of reactive oxygen species (ROS), as functional assays, were evaluated by counting engulfed yeasts and DCFH-DA reaction. The viability of macrophages was not significantly reduced by AM. AM at nontoxic concentrations markedly increased cytoplasmic vacuoles. The expression of surface CD14, CD16, CD36, and HLA-DR was suppressed. The phagocytosis function, but not ROS production, of macrophages was inhibited by AM. Armillaridin could inhibit the differentiation and activation of human macrophages. It may have potential to be developed as a biological response modifier for inflammatory diseases.
The mycotoxin zearalenone has been contaminating maize and other grains. It can be hydrolyzed and inactivated by the lactonase ZHD, which belongs to the alpha/beta-hydrolase family. Besides the catalytic core domain, the enzyme comprises an alpha-helical cap domain. Zearalenone differs from other quorum-sensing lactones in its chemical structure. As revealed by the complex structure, the substrate binds into a deep pocket between the core and cap domains, adjacent to the catalytic triad Ser102-His242-Glu126. The enzyme-substrate interactions include three direct hydrogen bonds and several nonpolar contacts. In particular, the Trp183 side chain is engaged in both hydrogen bonding and T-stacking interactions with the benzoate ring. The central role of Trp183 in substrate binding was verified by the mutants W183A, W183H and W183F. Several mutants were also produced to investigate the roles of nearby amino-acid residues. Interestingly, mutants that destabilize the dimer had adverse functional effects on ZHD.
Title: Therapeutic and radiosensitizing effects of armillaridin on human esophageal cancer cells Chi CW, Chen CC, Chen YJ Ref: Evid Based Complement Alternat Med, 2013:459271, 2013 : PubMed
Background. Armillaridin (AM) is isolated from Armillaria mellea. We examined the anticancer activity and radiosensitizing effect on human esophageal cancer cells. Methods. Human squamous cell carcinoma (CE81T/VGH and TE-2) and adenocarcinoma (BE-3 and SKGT-4) cell lines were cultured. The MTT assay was used for cell viability. The cell cycle was analyzed using propidium iodide staining. Mitochondrial transmembrane potential was measured by DiOC6(3) staining. The colony formation assay was performed for estimation of the radiation surviving fraction. Human CE81T/VGH xenografts were established for evaluation of therapeutic activity in vivo. Results. AM inhibited the viability of four human esophageal cancer cell lines with an estimated concentration of 50% inhibition (IC50) which was 3.4-6.9 microM. AM induced a hypoploid cell population and morphological alterations typical of apoptosis in cells. This apoptosis induction was accompanied by a reduction of mitochondrial transmembrane potential. AM accumulated cell cycle at G2/M phase and enhanced the radiosensitivity in CE81T/VGH cells. In vivo, AM inhibited the growth of CE81T/VGH xenografts without significant impact on body weight and white blood cell counts. Conclusion. Armillaridin could inhibit growth and enhance radiosensitivity of human esophageal cancer cells. There might be potential to integrate AM with radiotherapy for esophageal cancer treatment.
Title: Efficacy of the predator Mallada basalis (Neuroptera: Chrysopidae) on Tetranychus kanzawai and Panonychus citri (Acari: Tetranychidae) at different predator:prey release ratios Cheng LL, Nechols JR, Margolies DC, Campbell JF, Yang PS, Chen CC, Lu CT Ref: Journal of Asia-Pacific Entomology, 15:142, 2012 : PubMed
We compared population suppression of the phytophagous mites, Tetranychus kanzawai Kishida and Panonychus citri (McGregor), on papaya by second instar larvae of the green lacewing, Mallada basalis (Walker), at various predator:prey release ratios in the laboratory. Initially, we presented M. basalis with mixed age classes of each mite species separately at a density of approximately 30 mites per seedling. After 3 days, predator:prey ratios of 1:30, 1:15, and 1:10 resulted in reductions of T. kanzawai of 66.8%, 82.6%, and 83.3%, respectively, and reductions of P. citri of 41.8%, 75.5%, and 77.2%, respectively. Predation on individual age classes was approximately equal in both species, reinforcing previous findings that this predator does not show a preference among age classes. We next presented M. basalis with mixed populations of the two mite species in which there were equal numbers of each species and the density was as in the single species tests. Total mite reduction with both mite species present was 48.5%, 71.9%, and 74.5% at ratios of 1:30, 1:15, and 1:10, respectively; T. kanzawai was reduced by 50.5%, 77.4%, and 79.5%, respectively, and P. citri was reduced by 44.1%, 60.3%, and 63.2%, respectively. This study suggests that M. basalis has the potential for substantially suppressing populations of both T. kanzawai and P. citri on papaya at a predator:prey ratio of 1:15 or greater. However, evaluation under realistic agricultural settings is needed before specific recommendations about predator release rates can be made.
Acinetobacter baumannii has emerged as a significant nosocomial pathogen worldwide. The increasing trend of carbapenem and fluoroquinolone resistance in A. baumannii severely limits the usage of therapeutic antimicrobial agents. Here we report the genome sequence of a multidrug-resistant A. baumannii strain, TCDC-AB0715, harboring both bla(OXA-23) and bla(OXA-66).
Title: Draft genome sequence of a dominant, multidrug-resistant Neisseria gonorrhoeae strain, TCDC-NG08107, from a sexual group at high risk of acquiring human immunodeficiency virus infection and syphilis Chen CC, Hsia KC, Huang CT, Wong WW, Yen MY, Li LH, Lin KY, Chen KW, Li SY Ref: Journal of Bacteriology, 193:1788, 2011 : PubMed
Neisseria gonorrhoeae infection is the second major cause of sexually transmitted diseases worldwide. Development of resistance to multiple classes of antimicrobials in N. gonorrhoeae has compromised treatment and disease control. Herein, we report the availability of the draft genome sequence of a multidrug-resistant N. gonorrhoeae isolate, TCDC-NG08107, which spread in groups of men who have sex with men (MSM) in Taiwan.
Title: Rapid eye movement sleep atonia in patients with cognitive impairment Chen PC, Wu D, Chen CC, Chi NF, Kang JH, Hu CJ Ref: Journal of Neurology Sci, 305:34, 2011 : PubMed
Acetylcholine (ACh) plays an important role in cognitive function and muscle atonia in rapid eye movement (REM) sleep. This quantitative study used surface electromyography (sEMG) to investigate changes in muscular activity, which may indicate a deficiency of ACh among patients with cognitive impairment. We recruited 9 controls without dementia, 6 patients with mild cognitive impairment (MCI), and 6 patients with mild Alzheimer's disease (AD). None of the participants had sleep complaints, and all AD patients were receiving cholinesterase inhibitors. Subjects underwent polysomnography (PSG), including sEMG of the leg muscles, which was analyzed using root mean square (rms), mean frequency, and peak frequency. The average rms values during REM sleep among the control, MCI, and AD patients were 0.553 +/- 0.177, 2.886 +/- 2.865, and 0.909 +/- 0.822, respectively. The average peak frequencies during REM sleep in control, MCI, and AD patients were 0.402 +/- 0.758, 30.524 +/- 32.237, and 0.566 +/- 1.153, respectively. The average mean frequencies during REM sleep in control, MCI, and AD patients were 8.849 +/- 6.071, 34.530 +/- 25.564, and 9.553 +/- 6.308, respectively. All rms, mean frequencies, and peak frequencies increased significantly (p<0.05) in MCI patients. A deficiency of ACh may result in an increase of sEMG activity in MCI patients. Because cholinesterase inhibitors are capable of suppressing sEMG activity in AD patients, we speculate that an increase in sEMG activity is associated with a deficiency of Ach, which could be an early indicator of dementia.
Previously, we observed that young-onset hypertension was independently associated with elevated plasma triglyceride(s) (TG) levels to a greater extent than other metabolic risk factors. Thus, focusing on the endophenotype--hypertension combined with elevated TG--we designed a family-based haplotype association study to explore its genetic connection with novel genetic variants of lipoprotein lipase gene (LPL), which encodes a major lipid metabolizing enzyme. Young-onset hypertension probands and their families were recruited, numbering 1,002 individuals from 345 families. Single-nucleotide polymorphism discovery for LPL, linkage disequilibrium (LD) analysis, transmission disequilibrium tests (TDT), bin construction, haplotype TDT association and logistic regression analysis were performed. We found that the CC- haplotype (i) spanning from intron 2 to intron 4 and the ACATT haplotype (ii) spanning from intron 5 to intron 6 were significantly associated with hypertension-related phenotypes: hypertension (ii, P=0.05), elevated TG (i, P=0.01), and hypertension combined with elevated TG (i, P=0.001; ii, P<0.0001), according to TDT. The risk of this hypertension subtype increased with the number of risk haplotypes in the two loci, using logistic regression model after adjusting within-family correlation. The relationships between LPL variants and hypertension-related disorders were also confirmed by an independent association study. Finally, we showed a trend that individuals with homozygous risk haplotypes had decreased LPL expression after a fatty meal, as opposed to those with protective haplotypes. In conclusion, this study strongly suggests that two LPL intronic variants may be associated with development of the hypertension endophenotype with elevated TG.
Much of the material taken into cells by endocytosis is rapidly returned to the plasma membrane by the endocytic recycling pathway. Although recycling is vital for the correct localization of cell membrane receptors and lipids, the molecular mechanisms that regulate recycling are only partially understood. Here we show that in Caenorhabditis elegans endocytic recycling is inhibited by NUM-1A, the nematode Numb homolog. NUM-1AGFP fusion protein is localized to the baso-lateral surfaces of many polarized epithelial cells, including the hypodermis and the intestine. We show that increased NUM-1A levels cause morphological defects in these cells similar to those caused by loss-of-function mutations in rme-1, a positive regulator of recycling in both C. elegans and mammals. We describe the isolation of worms lacking num-1A activity and show that, consistent with a model in which NUM-1A negatively regulates recycling in the intestine, loss of num-1A function bypasses the requirement for RME-1. Genetic epistasis analysis with rab-10, which is required at an early part of the recycling pathway, suggests that loss of num-1A function does not affect the uptake of material by endocytosis but rather inhibits baso-lateral recycling downstream of rab-10.
Title: Altering lipase activity and enantioselectivity in organic media using organo-soluble bases: Implication for rate-limiting proton transfer in acylation step Chen CC, Chen TL, Tsai SW Ref: Biotechnol Bioeng, 94:201, 2006 : PubMed
With the hydrolytic resolution of (R,S)-naproxen 2,2,2-trifluoroethyl esters via a partially purified papaya lipase (PCPL) in water-saturated isooctane as the model system, the enzyme activity, and enantioselectivty is altered by adding a variety of organo-soluble bases that act as either enzyme activators (i.e., TEA, MP, TOA, DPA, PY, and DMA) or enzyme inhibitors (i.e., PDP, DMAP, and PP). Triethylamine (TEA) is selected as the best enzyme activator as 2.24-fold increase of the initial rate for the (S)-ester is obtained when adding 120 mM of the base. By using an expanded Michaelis-Menten mechanism for the acylation step, the kinetic analysis indicates that the proton transfer for the breakdown of tetrahedral intermediates to acyl-enzyme intermediates is the rate-limiting step, or more sensitive than that for the formation of tetrahedral intermediates when the enzyme activators of different pKa are added. However, no correlation for the proton transfers in the acylation step is found when adding the bases acting as enzyme deactivators.
Title: Release of acetylcholine by Hon-Chi to raise insulin secretion in Wistar rats Chen CC, Liu IM Ref: Neuroscience Letters, 404:117, 2006 : PubMed
The mandarin Hon-Chi is the red yeast rice fermented with Monascus pilous and Monascus purpureus. The present study is designed to screen the effect of Hon-Chi on plasma glucose and investigate the possible mechanisms. After oral administration into fasting Wistar rats for 90min, Hon-Chi decreased the plasma glucose in a dose-dependent manner. In parallel to the reduction of plasma glucose, an increase of plasma level of insulin or C-peptide was also observed in rats receiving same treatment. Moreover, disruption of synaptic available acetylcholine (ACh) using an inhibitor of choline uptake, hemicholinium-3, or vesicular acetylcholine transport, vesamicol, abolished these actions of Hon-Chi. Also, physostigmine at concentration sufficient to inhibit acetylcholinesterase enhanced the actions of Hon-Chi. Mediation of ACh release from the nerve terminals to enhance insulin secretion by Hon-Chi can thus be considered. Both the plasma glucose lowering action and the raised plasma levels of insulin and C-peptide induced by Hon-Chi were also inhibited by 4-diphenylacetoxy-N-methylpiperdine methiodide (4-DAMP), but not affected by the ganglionic nicotinic antagonist, pentolinium or hexamethonium, indicating the mediation of muscarinic M(3) receptors. The results suggest that Hon-Chi has an ability to raise the release of ACh from nerve terminals, which in turn to stimulate muscarinic M(3) receptors in pancreatic cells and augment the insulin release to result in plasma glucose lowering action. Thus, Hon-Chi seems suitable to employ as the health food for increase of insulin secretion in the prevention of type-2 diabetes.
Title: Implication of substrate-assisted catalysis on improving lipase activity or enantioselectivity in organic solvents Tsai SW, Chen CC, Yang HS, Ng IS, Chen TL Ref: Biochimica & Biophysica Acta, 1764:1424, 2006 : PubMed
In comparison with the biocatalyst engineering and medium engineering approaches, very few examples have been reported on using the substrate engineering approach such as substrate-assisted catalysis (SAC) for naturally occurring or engineered lipases and serine proteases to improve the enzyme activity and enantioselectivity. By employing lipase-catalyzed hydrolysis of (R,S)-naproxen esters in water-saturated isooctane as the model system, we demonstrate the proton shuttle device to the leaving alcohol of the substrate as a new means of SAC to effectively improve the lipase activity or enantioselectivity. The result cannot only provide a strong evidence for the rate-limiting proton transfer for the bond-breaking of tetrahedron intermediate of the acylation step, but also sheds light for performing the hydrolysis, transesterification or aminolysis in organic solvents for the ester substrate that originally lipases cannot catalyze, but now can after introducing the device.
Title: Prediction of the outcome of pull-through surgery for Hirschsprung's disease using acetylcholinesterase activity Huang SF, Chen CC, Lai HS Ref: J Formos Med Assoc, 100:798, 2001 : PubMed
BACKGROUND AND PURPOSE Advances in surgical procedures for the treatment of Hirschsprung's disease have afforded the majority of patients a satisfactory outcome after definitive corrective surgery However some patients continue to have signs of persistent bowel dysfunction despite adequate resection of the aganglionic bowel segment The definite etiology of persistent bowel dysfunction in these patients remains controversial MATERIALS AND METHODS The acetylcholinesterase activity in the proximal resection margin of pull-through surgical specimens collected from September 1992 to June 1998 was evaluated and correlated with clinical outcome RESULTS A total of 24 patients were studied 15 males and 9 females The follow-up period ranged from 1 year 7 months to 7 years 6 months The Soave procedure was performed in 17 cases and the Duhamel in 7 Twenty cases had a good or fair clinical outcome and four 16.7 had a poor outcome There was no mortality Among the 20 patients with a good or fair outcome the acetylcholinesterase activity in the proximal section margins was not increased in 12 mildly increased in seven and moderately increased in one The four cases with poor outcome all had moderately increased acetylcholinesterase activity in at least one specimen The association between moderately increased acetylcholinesterase activity and poor surgical outcome was significant p 0.0005 In contrast there was no association between the surgical method Soave or Duhamel used and clinical outcome p 0.55 CONCLUSION Acetylcholinesterase activity in the proximal resection margins of pull-through surgical specimens is a useful indicator for predicting the postoperative outcome in Hirschsprung's disease
