Application of methyl parathion hydrolase (MPH) as a labeling enzyme

Methyl parathion hydrolase (MPH) is an enzyme that catalyzes the degradation of methyl parathion, generating a yellow product with specific absorption at 405 nm. The application of MPH as a new labeling enzyme was illustrated in this study. The key advantages of using MPH as a labeling enzyme are as follows: (1) unlike alkaline phosphatase (AP), horseradish peroxidase (HRP), and glucose oxidase (GOD), MPH is rarely found in animal cells, and it therefore produces less background noise; (2) its active form in solution is the monomer, with a molecular weight of 37 kDa; (3) its turnover number is 114.70 ± 13.19 s⁻¹, which is sufficiently high to yield a significant signal for sensitive detection; and (4) its 3D structure is known and its C-terminal that is exposed to the surface can be easily subjected to the construction of genetic engineering monocloning antibody–enzyme fusion for enzyme-linked immunosorbent assay (ELISA). To demonstrate its utility, MPH was ligated to an single-chain variable fragment (scFv), known as A1E, against a white spot syndrome virus (WSSV) with the insertion of a [–(Gly–Ser)₅–] linker peptide. The resulting fusion protein MPH-A1E possessed both the binding specificity of the scFv segment and the catalytic activity of the MPH segment. When MPH-A1E was used as an ELISA reagent, 25 ng purified WSSV was detected; this was similar to the detection sensitivity obtained using A1E scFv and the HRP/Anti-E Tag Conjugate protocol. The fusion protein also recognized the WSSV in 1 μL hemolymph from an infected shrimp and differentiated it from a healthy shrimp. Figure The 3-D structure of MPH. (a) monomer showing C- and N-terminals; (b) the crystal structure of the dimer W. Yang and Y.-F. Zhou contributed equally to this work.     

Urea derivatives of spirocyclic piperidines endowed with antibacterial activity

Antimycobacterial activity of certain ureas as well as spirocyclic piperidines described in the literature prompted us to synthesize and test a series of hybrids of spirocyclic piperidine with ureas. Surprisingly, no activity was detected against Mycobacterium tuberculosis. However, significant antibacterial activity was identified and confirmed against common gram-positive as well as gram-negative bacteria.     

Metabolism of Deuterated erythro‐Dihydroxy Fatty Acids in Saccharomyces cerevisiae: Enantioselective Formation and Characterization of Hydroxylactones

Epoxides of fatty acids are hydrolyzed by epoxide hydrolases (EHs) into dihydroxy fatty acids which are of particular interest in the mammalian leukotriene pathway. In the present report, the analysis of the configuration of dihydroxy fatty acids via their respective hydroxylactones is described. In addition, the biotransformation of (±)‐erythro‐7,8‐ and ‐3,4‐dihydroxy fatty acids in the yeast Saccharomyces cerevisiae was characterized by GC/EI‐MS analysis. Biotransformation of chemically synthesized (±)‐erythro‐7,8‐dihydroxy(7,8‐²H₂)tetradecanoic acid ((±)‐erythro‐ 1 ) in the yeast S. cerevisiae resulted in the formation of 5,6‐dihydroxy(5,6‐²H₂)dodecanoic acid ( 6 ), which was lactonized into (5S,6R)‐6‐hydroxy(5,6‐²H₂)dodecano‐5‐lactone ((5S,6R)‐ 4 ) with 86% ee and into erythro‐5‐hydroxy(5,6‐²H₂)dodecano‐6‐lactone (erythro‐ 8 ). Additionally, the α‐ketols 7‐hydroxy‐8‐oxo(7‐²H₁)tetradecanoic acid ( 9a ) and 8‐hydroxy‐7‐oxo(8‐²H₁)tetradecanoic acid ( 9b ) were detected as intermediates. Further metabolism of 6 led to 3,4‐dihydroxy(3,4‐²H₂)decanoic acid ( 2 ) which was lactonized into 3‐hydroxy(3,4‐²H₂)decano‐4‐lactone ( 5 ) with (3R,4S)‐ 5 =88% ee. Chemical synthesis and incubation of (±)‐erythro‐3,4‐dihydroxy(3,4‐²H₂)decanoic acid ((±)‐erythro‐ 2 ) in yeast led to (3S,4R)‐ 5 with 10% ee. No decano‐4‐lactone was formed from the precursors 1 or 2 by yeast. The enantiomers (3S,4R)‐ and (3R,4S)‐3,4‐dihydroxy(3‐²H₁)nonanoic acid ((3S,4R)‐ and (3R,4S)‐ 3 ) were chemically synthesized and comparably degraded by yeast without formation of nonano‐4‐lactone. The major products of the transformation of (3S,4R)‐ and (3R,4S)‐ 3 were (3S,4R)‐ and (3R,4S)‐3‐hydroxy(3‐²H₁)nonano‐4‐lactones ((3S,4R)‐ and (3R,4S)‐ 7 ), respectively. The enantiomers of the hydroxylactones 4, 5 , and 7 were chemically synthesized and their GC‐elution sequence on Lipodex^® E chiral phase was determined.     

Phosphate Diester Cleavage Promoted by the Metallomicelles of Ce(III) Complexes of Aza-Crown Ether with Different Numbers of Nitrogen Atoms

The design of artificial hydrolase has attracted extensive attention due to their scientific significance and potential application in the field of gene medicine and molecular biology. This work reports the catalytic activation of two aza-crown ether Ce(III) complexes and their metallomicelles as artificial hydrolase in bis(4-nitrophenyl) phosphate ester (BNPP) hydrolysis. The chemical composition of two complexes was determined by the fluorescence spectra and the mole ratio method for electronic absorption spectra. The bonding effect of BNPP and solubilizing effect of the complexes were proved by a method of fluorescence spectroscopy. The catalytic activity of different catalytic systems in BNPP hydrolysis was measured with UV-vis spectrophotometric method. These catalytic systems showed high catalytic activity for promoting BNPP hydrolysis at the almost physiological conditions. BNPP hydrolysis rate in these catalytic system is about 10⁷- to 10⁹-fold faster than that of the BNPP spontaneous hydrolysis in aqueous solution at the same conditions. The metallomicelle systems exhibited higher catalytic activity compared with the complex solution systems in BNPP hydrolysis, and hexadecyltrimethyl ammonium bromide micelle provides a useful catalytic environment for reaction. The acid effect of the catalytic system is ascribed to the formation of metal-bound hydroxide serving as a better kind of nucleophile.     

Biosensor for direct determination of fenitrothion and EPN using recombinant <Emphasis Type="Italic">Pseudomonas putida</Emphasis> JS444 with surface-expressed organophosphorous hydrolase. 2. Modified carbon paste electrode

A whole cell-based amperometric biosensor for highly selective, sensitive, rapid, and cost-effective determination of the organophosphate pesticides fenitrothion and ethyl p-nitrophenol thiobenzenephosphonate (EPN) is discussed. The biosensor comprised genetically engineered p-nitrophenol (PNP)-degrading bacteria Pseudomonas putida JS444 anchoring and displaying organophosphorous hydrolase (OPH) on its cell surface as biological sensing element and carbon paste electrode as the amperometric transducer. Surface-expressed OPH catalyzed the hydrolysis of organophosphorous pesticides such as fenitrothion and EPN to release PNP and 3-methyl-4-nitrophenol, respectively, which were subsequently degraded by the enzymatic machinery of P. putida JS444 through electrochemically active intermediates to the TCA cycle. The electrooxidization current of the intermediates was measured and correlated to the concentration of organophosphates. Operating at optimum conditions, 0.086 mg dry wt of cell operating at 600 mV of applied potential (vs Ag/AgCl reference) in 50 mM citratephosphate buffer, pH 7.5, with 50 μM CoCl₂ at room temperature, the biosensor measured as low as 1.4 ppb of fenitrothion and 1.6 ppb of EPN. There was no interference from phenolic compounds, carbamate pesticides, triazine herbicides, or organophosphate pesticides without nitrophenyl substituent. The service life of the biosensor and the applicability to lake water were also demonstrated.     

New Disulfiram Derivatives as MAGL-Selective Inhibitors

Monoacylglycerol lipase (MAGL) is a key enzyme in the human endocannabinoid system. It is also the main enzyme responsible for the conversion of 2-arachidonoyl glycerol (2-AG) to arachidonic acid (AA), a precursor of prostaglandin synthesis. The inhibition of MAGL activity would be beneficial for the treatment of a wide range of diseases, such as inflammation, neurodegeneration, metabolic disorders and cancer. Here, the author reports the pharmacological evaluation of new disulfiram derivatives as potent inhibitors of MAGL. These analogues displayed high inhibition selectivity over fatty acid amide hydrolase (FAAH), another endocannabinoid-hydrolyzing enzyme. In particular, compound 2i inhibited MAGL in the low micromolar range. However, it did not show any inhibitory activity against FAAH.     

A new fluorescence probing strategy for the detection of parathion-methyl based on N-doped carbon dots and methyl parathion hydrolase

A new facile fluorescence probing strategy, which was based on N-doped carbon dots(NCDs) and methyl parathion hydrolase(MPH), was developed for the determination of parathion-methyl(PM). The fluorescence intensity of NCDs-MPH system was proportional to PM concentration in the range of 2.38–73.78 mmol/L, with a detection limit of 0.338 mmol/L. Moreover, the present simple and facile method could be used to determine methyl parathion in environmental and agricultural samples successfully.Furthermore, the detection mechanism of this system is inner filter effect and molecular interactions between NCDs and p-nitrophenol, which is the hydrolysis product of PM catalyzed by methyl parathion hydrolase.     

Synthesis and Antiproliferative Activities of OSW‐1 Analogues Bearing 2”‐O‐p‐Acylaminobenzoyl Residues†

OSW‐1 is a well‐known natural saponin with potent antitumor activities. We have designed and prepared a small library of 22 OSW‐1 analogues with a variety of p‐acylamino‐benzoyl groups installed at C2” of the xylose residue, wherein a regioselective (1→3)‐glycosylation of arabinoside 3,4‐diol has been achieved by manipulation of the protecting groups on the imidate donors. Bioassays lead to new structure‐activity relationships as well as two applicable fluorescent probes, which are found to localize to lysosomes in HeLa cells and could be used in further antitumor mechanism studies of OSW‐1 in living cells.     

Synthesis of BCD tricyclic analogues of the novel cardiac glycoside rhodexin A

A concise synthesis of novel cardiac glycoside analogues of rhodexin A, 14 and 24, having the BCD tricyclic system is described. The key constructive step is an inverse-electron demand Diels-Alder reaction of the silyl enol ether 4 and the 2-acetyldiene, 7 and 15.     

人类可溶性环氧化物水解酶抑制剂的研究进展

高血压是现代人的常见疾病.虽然有很多不同机理的降压药在临床使用,但是由于个体的差异性,高血压的治疗越来越倾向于个体化治疗,因此降压药的使用也不仅仅局限于血压的降低.人类可溶性环氧化物水解酶抑制剂最大的优势在于其在降压的同时还具有显著的抗炎作用.详细地阐述了人类可溶性环氧化物水解酶抑制剂从早期的环氧结构类型到第三代脲类结构的发展过程和近期研究进展.