Title: The structural and molecular mechanisms of type II PETases: a mini review Duan S, Zhang N, Chao T, Wu Y, Wang M Ref: Biotechnol Lett, :, 2023 : PubMed
The advent of plastics has led to significant advances for humans, although the accompanying pollution has also been a source of concern for countries globally. Consequently, a biological method to effectively degrade polyethylene terephthalate (PET) has been an area of significant scientific interest. Following the report of the highly efficient PET hydrolase from the bacterium Ideonella sakaiensis strain 201-F6 (i.e., IsPETase) in 2016, its structure has been extensively studied, showing that it belongs to the type II PETase group. Unlike type I PETases that include most known cutinases, structural investigations of type II PETases have only been conducted since 2017. Type II PETases are further divided into type IIa and IIb enzymes. Moreover, even less research has been conducted on type IIa plastic-degrading enzymes. Here, we present a review of recent studies of the structure and mechanism of type II PETases, using the known structure of the type IIa PETase PE-H from the marine bacterium Pseudomonas aestusnigri in addition to the type IIb enzyme IsPETase as representatives. These studies have provided new insights into the structural features of type II PETases that exhibit PET catalytic activity. In addition, recent studies investigating the rational design of IsPETases are reviewed and summarized alongside a discussion of controversies surrounding PETase investigations.
Human pancreatic lipase (hPL) is a crucial digestive enzyme responsible for the digestion of dietary lipids in humans, and inhibition of hPL is effective in reducing triglyceride intake, thereby preventing and treating obesity. In this study, a series of fatty acids with different carbon chain lengths were constructed to the fluorophore resorufin based on the substrate preference of hPL. Among them, RLE was found to have the best combination of stability, specificity, sensitivity and reactivity towards hPL. Under physiological conditions, RLE can be rapidly hydrolyzed by hPL and released to resorufin, which triggered approximately 100-fold fluorescence enhancement at 590 nm. RLE was successfully applied for sensing and imaging of endogenous PL in living systems with low cytotoxicity and high imaging resolution. Moreover, a visual high-throughput screening platform was established using RLE, and the inhibitory effects of hundreds of drugs and natural products toward hPL were evaluated. Collectively, this study reports a novel and highly specific enzyme-activatable fluorogenic substrate for hPL that could serve as a powerful tool for monitoring hPL activity in complex biological systems and showcases the potential to explore physiological functions and rapid screening of inhibitors.
Obesity, now widespread all over the world, is frequently associated with several chronic diseases. Human pancreatic lipase (hPL) is a crucial digestive enzyme responsible for the digestion of dietary lipids in humans, and the inhibition of hPL is effective in reducing triglyceride intake and thus preventing and treating obesity. In this work, a practical sequential screening strategy was developed to construct a highly selective near-infrared fluorogenic substrate 7-STCFC for hPL. Under physiological conditions, 7-STCFC can be rapidly hydrolyzed by hPL to form 7-HTCFC, which triggers 254-fold NIR signal enhancement at 670 nm. 7-STCFC was successfully applied for the sensing and imaging of endogenous PL in living systems (including living cells, tissues and organs) with low cytotoxicity and high imaging resolution. Moreover, a high-throughput screening platform was established using 7-STCFC, and the inhibitory effects of 94 kinds of herbs toward hPL were evaluated. Among them, Pu-erh tea stood out with outstanding hPL inhibitory effects, and the inhibitory ingredients and involved inhibitory mechanism were further revealed, which strongly facilitates the discovery of novel anti-obesity agents targeting hPL. Collectively, these findings suggested that our strategy was practical to develop an isoform-specific fluorogenic substrate for a target enzyme, and 7-STCFC was a powerful tool for monitoring PL activity in complex biological systems with value for exploring physiological functions and rapid screening of inhibitors.
Title: Chemical Constituents and Their Biological Activities from Genus Styrax Xia DD, Han XY, Zhang Y, Zhang N Ref: Pharmaceuticals (Basel), 16:, 2023 : PubMed
Plants from the genus Styrax have been extensively used in folk medicines to treat diseases such as skin diseases and peptic ulcers and as an antiseptic and analgesic. Most Styrax species, especially Styrax tonkinensis, which is used as an expectorant, antiseptic, and analgesic in Chinese traditional medicine, could screen resin after external injury. Styrax is also used in folk medicines in Korea to treat sore throat, bronchitis, cough, expectoration, paralysis, laryngitis, and inflammation. Different parts of various Styrax species can be widely employed for ethnopharmacological applications. Moreover, for ethnopharmacological use, these parts of Styrax species can be applied in combination with other folk medicines. Styrax species consist of versatile natural compounds, with some of them exhibiting particularly excellent pharmacological activities, such as cytotoxic, acetylcholinesterase inhibitory, antioxidant, and antifungal activities. Altogether, these exciting results indicate that a comprehensive review of plants belonging to this genus is essential for helping researchers to continuously conduct an in-depth investigation. In this review, the traditional uses, phytochemistry, corresponding pharmacological activities, and structure-activity relationships of different Styrax species are clarified and critically discussed. More insights into potential opportunities for future research are carefully assessed.
Title: Ratiometric fluorescent hydrogel for point-of-care monitoring of organophosphorus pesticide degradation Li H, Zou R, Su C, Zhang N, Wang Q, Zhang Y, Zhang T, Sun C, Yan X Ref: J Hazard Mater, 432:128660, 2022 : PubMed
The residues of organophosphorus pesticides have caused the potential risk in environment and human health, arousing worldwidely great concern. Herein, we fabricated a robust gold nanoclusters/MnO(2) composites-based hydrogel portable kit for accurate monitoring of paraoxon residues and degradation in Chinese cabbages. With the immobilization of gold nanoclusters/MnO(2) composites into a hydrogel, a ratiometric fluorescent signal is generated by catalyzing the oxidation of o-phenylenediamine, which possesses a built-in correction with low background interference. Coupling with acetylcholinesterase catalytic reactions and pesticide inhibition effect, the portable kit can sensitively detect paraoxon residues with a detection limit of 5.0 ng mL(-1). For on-site quantification, the fluorescent color variations of portable kit are converted into digital information that exhibits applicative linear range toward pesticide. Notably, the hydrogel portable kit was successfully applied for precisely monitoring the residue and degradation of paraoxon in Chinese cabbage, providing a potential pathway toward practical point-of-care testing in food safety monitoring.
Title: The dipeptidyl peptidase-4 inhibitor linagliptin ameliorates LPS-induced acute lung injury by maintenance of pulmonary microvascular barrier via activating the Epac1/AKT pathway Zhang N, Tang S, Zhang J, Pei B, Pang T, Sun G Ref: Biomed Pharmacother, 155:113704, 2022 : PubMed
Pulmonary microvascular endothelial cells (PMVECs) barrier dysfunction is a main pathophysiological feature of sepsis-related acute lung injury (ALI). This study aimed to investigate whether the dipeptidyl peptidase (DPP)-4 inhibitor linagliptin could protect against LPS-induced PMVECs barrier disruption and its underlying molecular mechanisms. A classical ALI animal model and LPS-treated PMVECs were applied and all were treated with or without linagliptin. Cellular experiments demonstrated that linagliptin could mitigate LPS-induced PMVECs hyperpermeability and intercellular junction (VE-cadherin, beta-catenin, and ZO-1) disruption in a dose-dependent manner. Correspondingly, it was observed that linagliptin pretreatment distinctly relieved LPS-induced lung injury, oxidative stress, and pulmonary edema in vivo. Furthermore, we found that the inhibition of oxidative stress by linagliptin may be achieved by reversing impaired mitochondrial function. Mechanistically, linagliptin administration promoted the activation of the Epac1 pathway and its downstream AKT pathway, while inhibition of the Epac1/Akt signaling pathway significantly alleviated the above-mentioned protective effect of linagliptin on the PMVECs barrier. Taken together, these data suggest that linagliptin can effectively reserve PMVECs barrier dysfunction and inhibit oxidative stress to protect against ALI via activating the Epac1/AKT signaling pathway, and thus may become a potential clinical therapeutic strategy for ALI.
Title: A highly contiguous genome assembly of a polyphagous predatory mite Stratiolaelaps scimitus (Womersley) (Acari: Laelapidae) Yan Y, Zhang N, Liu C, Wu X, Liu K, Yin Z, Zhou X, Xie L Ref: Genome Biol Evol, :, 2021 : PubMed
As a polyphagous soil-dwelling predatory mite, Stratiolaelaps scimitus (Womersley) (Acari: Laelapidae), formerly known as Stratiolaelaps miles (Berlese), is native to the Northern hemisphere and preys on soil invertebrates, including fungus gnats, springtails, thrips nymphs, nematodes, and other species of mites. Already mass-produced and commercialized in North America and Europe, S. scimitus is now introduced in China as a biocontrol agent for field crop. The introduction, however, can lead to unexpected genetic changes within populations of biological control agents, which might decrease the efficacy of pest management or increase the risks to local environments. To better understand the genetic basis of its biology and behavior, we sequenced and assembled the draft genome of S. scimitus using the PacBio Sequel platform II. We generated -150 x (64.81 Gb) PacBio long reads with an average read length of 12.60 kb. Reads longer than 5 kb were assembled into contigs, resulting in the final assembly of 158 contigs with a N50 length of 7.66 Mb, and captured 93.1% of the BUSCO gene set (n = 1,066). We identified 16.39% (69.91 Mb) repetitive elements, 1,686 non-coding RNAs, and 13,305 protein-coding genes, which represented 95.8% BUSCO completeness. Combining analyses of genome family evolution and function enrichment of gene ontology and pathway, a total of 135 families experienced significant expansions, which were mainly involved in digestion, detoxification, immunity and venom. Major expansions of the detoxification enzymes, i.e., P450s and carboxylesterases, suggest a possible genetic mechanism underlying polyphagy and ecological adaptions. Our high-quality genome assembly and annotation provide new insights on the evolutionary biology, soil ecology and biological control for predaceous mites.
Title: Diterpenoid Alkaloids from the Aerial Parts of Aconitum flavum Hand.-Mazz Zhang N, Xia F, Li SY, Nian Y, Wei LX, Xu G Ref: Nat Prod Bioprospect, :, 2021 : PubMed
Sixteen diterpenoid alkaloids (DAs), including six aconitine-type alkaloids (5 and 9 - 13), seven 7,17-seco-aconitine-type alkaloids (1 - 4, 6 - 8), two napelline-type alkaloids (14 and 15) as well as one veatchine-type alkaloid (16), were isolated from the aerial parts of Aconitum flavum Hand.-Mazz. In which, flavumolines A - D (1 - 4) were four new ones, and flavumoline E (5) was reported as natural compound for the first time. Their chemical structures were elucidated by the analysis of extensive spectroscopic data. The inhibitory activities of these isolates on Ca(v)3.1 low voltage-gated Ca(2+) channel, NO production in LPS-activated RAW264.7cells, five human tumor cell lines, as well as acetylcholinesterase (AChE) were tested.
Lipolytic enzymes are essential biocatalysts in food processing as well as pharmaceutical and pesticide industries, catalyzing the cleavage of ester bonds in a variety of acyl chain substrates. Here, we report the crystal structure of an esterase from the deep-sea hydrothermal vent of the East Pacific Rise (EprEst). The X-ray structure of EprEst in complex with the ligand, acetate, has been determined at 2.03 resolution. The structure reveals a unique spatial arrangement and orientation of the helix cap domain and alpha/beta hydrolase domain, which form a substrate pocket with preference for short-chain acyl groups. Molecular docking analysis further demonstrated that the active site pocket could accommodate p-nitrophenyl (pNP) carboxyl ligands of varying lengths (>=6 C atoms), with pNP-butyrate ester predicted to have the highest binding affinity. Additionally, the semirational design was conducted to improve the thermostability of EprEst by enzyme engineering based on the established structure and multiple sequence alignment. A mutation, K114P, introduced in the hinge region of the esterase, which displayed increased thermostability and enzyme activity. Collectively, the structural and functional data obtained herein could be used as basis for further protein engineering to ultimately expand the scope of industrial applications of marine-derived lipolytic enzymes.
Human carboxylesterase 2 (hCES2A), one of the major serine hydrolases distributed in the small intestine, plays a crucial role in hydrolysis of ester-bearing drugs. Accumulating evidence has indicated that hCES2A inhibitor therapy can modulate the pharmacokinetic and toxicological profiles of some important hCES2A-substrate drugs, such as the anticancer agent CPT-11. Herein, a series of indanone-chalcone hybrids are designed and synthesized to find potent and highly selective hCES2A inhibitors. Inhibition assays demonstrated that most indanone-chalcone hybrids displayed strong to moderate hCES2A inhibition activities. Structure-hCES2A inhibition activity relationship studies showed that introduction of a hydroxyl at the C4' site and introduction of an N-alkyl group at the C6 site were beneficial for hCES2A inhibition. Particularly, B7 (an N-alkylated 1-indanone-chalcone hybrid) exhibited the most potent inhibition on hCES2A and excellent specificity (this agent could not inhibit other human esterases including hCES1A and butyrylcholinesterase). Inhibition kinetic analyses demonstrated that B7 potently inhibited hCES2A-mediated FD hydrolysis in a mixed inhibition manner, with a calculated K(i) value of 0.068 muM. Furthermore, B7 was capable of inhibiting intracellular hCES2A in living cells and displayed good metabolic stability. Collectively, our findings show that indanone-chalcone hybrids are good choices for the development of hCES2A inhibitors, while B7 is a promising candidate for the development of novel anti-diarrhea agents to ameliorate irinotecan-induced intestinal toxicity.
Pancreatic lipase (PL), a crucial enzyme responsible for hydrolysis of dietary lipids, has been validated as a key therapeutic target to prevent and treat obesity-associated metabolic disorders. Herein, we report the design, synthesis and biological evaluation of a series of chalcone-like compounds as potent and reversible PL inhibitors. Following two rounds of structural modifications at both A and B rings of a chalcone-like skeleton, structure-PL inhibition relationships of the chalcone-like compounds were studied, while the key substituents that would be beneficial for PL inhibition were revealed. Among all tested chalcone-like compounds, compound B13 (a novel chalcone-like compound bearing two long carbon chains) displayed the most potent PL inhibition activity, with an IC(50) value of 0.33 M. Inhibition kinetic analyses demonstrated that B13 could potently inhibit PL-mediated 4-MUO hydrolysis in a mixed inhibition manner, with the K(i) value of 0.12 M. Molecular docking simulations suggested that B13 could tightly bind on PL at both the catalytic site and a non-catalytic site that was located on the surface of PL, which was consistent with the mixed inhibition mode of this agent. In addition, B13 displayed excellent stability in artificial gastrointestinal fluids and good metabolic stability in human liver preparations. Collectively, our findings suggested that chalcone-like compounds were good choices for design and development of orally administrated PL inhibitors, while B13 could be served as a promising lead compound to develop novel anti-obesity agents via targeting on PL.
Title: Bioguided Isolation and Structure Identification of Acetylcholinesterase Enzyme Inhibitors from Drynariae Rhizome Liu MY, Zeng F, Shen Y, Wang YY, Zhang N, Geng F Ref: J Anal Methods Chem, 2020:2971841, 2020 : PubMed
Drynariae Rhizome, widely distributed in southern China, was clinically used as a traditional treatment for cognitive disfunction, such as Alzheimer's disease (AD). The aim of our work was to evaluate the AChE inhibition activities of extracts of Drynariae Rhizome and pure compounds using a bioguided fractionation procedure. The classical approach for screening potential AChE inhibitors was developed by Ellman. However, the background color of compounds or herb extracts remained uncertain and frequently interfered with the detection of the secondary reaction, thereby easily yielding false positive or false negative results. Here, a high-throughput assay monitoring the transformation of iodized choline from iodized acetylcholine catalyzed by AChE was established based on UPLC-MS/MS. The bioguided fractionation of the extract using this method resulted in the isolation of eight AChE inhibitory flavonoids, including naringenin, eriodictyol, kaempferol, luteolin, astragalin, luteolin-7-O-beta-D-glucoside, naringin, and neoeriocitrin, with the IC50 values of 3.81 +/- 0.21 muM, 7.19 +/- 0.62 muM, 11.09 +/- 1.02 muM, 17.26 +/- 0.23 muM, 18.24 +/- 2.33 muM, 17.13 +/- 1.02 muM, 26.4 +/- 1.17 muM, and 22.49 +/- 1.25 muM. It is assumed that the identified flavonoids contribute to the AChE inhibition activity of Drynariae Rhizome. These results are in agreement with the traditional uses of Drynariae Rhizome for AD.
Title: Amino acid, fatty acid, and carbohydrate metabolomic profiles with ginsenoside-induced insecticidal efficacy against Ostrinia furnacalis (Guenee) Liu S, Wang X, Zhang R, Song M, Zhang N, Li W, Wang Y, Xu Y, Zhang L Ref: J Ginseng Res, 44:544, 2020 : PubMed
Background: Previous studies have shown the insecticidal efficacy of ginsenosides. In the present study, we aimed to investigate the metabolic mechanism related to the inhibitory effect of panaxadiol saponins (PDSs) against the Asian corn borer Ostrinia furnacalis (Guenee). Methods: Third instar larvae of O. furnacalis were fed normal diets with different concentrations of PDSs for 4 days. The consumption index, relative growth rate, approximate digestibility, and conversion of ingested and digested food were recorded. A targeted gas chromatography-mass spectrometry assay was performed to detect the profiles of amino acids, fatty acids, and carbohydrates in larvae of O. furnacalis. In addition, the activity of detoxification-related enzymes was determined. Results and Conclusions: PDSs decreased the consumption index, relative growth rate, approximate digestibility, and conversion of ingested and digested food in the 3rd instar larvae of O. furnacalis in a dose-dependent manner. PDSs decreased 15 free amino acids, 16 free fatty acids, and 5 carbohydrates and increased the levels of palmitoleic acid, palmitic acid, and 9-octadecenoic acid in the 3rd instar larvae. The activity of detoxification-related enzymes, such as acetylcholinesterase, glutathione S-transferase, cytochrome P450, carboxylesterase, trehalase, acid phosphatase, and alkaline phosphatase, was reduced in a dose-dependent manner in the 3rd instar larvae exposed to PDSs. These data confirmed the inhibitory effect of PDSs against growth, food utilization, and detoxification in the 3rd instar larvae of O. furnacalis and the potential for using PDSs as an efficient tool for insect pest management for O. furnacalis larvae.
Single-atom nanozymes (SAzymes), as novel nanozymes with atomically dispersed active sites, are of great importance in the de-velopment of nanozymes for their high catalytic activities, the maximum utilization efficiency of metal atoms, and the simple mod-el of active sites. Herein, the peroxidase-like SAzymes with high-concentration Cu sites on carbon nanosheets (Cu-N-C) were syn-thesized through a salt-template strategy. With the densely distributed active Cu atoms (~5.1 wt%), the Cu-N-C SAzymes exhibit remarkable activity to mimic natural peroxidase. Integrating Cu-N-C SAzymes with natural acetylcholinesterase and choline oxi-dase, three-enzyme-based cascade reaction system was constructed for the colorimetric detection of acetylcholine and organo-phosphorus pesticides. This work not only provides a strategy to synthesize SAzymes with abundant active sites but also gives some new insights for robust nanozyme biosensing systems.
Title: Identifying genes for resistant starch, slowly digestible starch, and rapidly digestible starch in rice using genome-wide association studies Zhang N, Wang M, Fu J, Shen Y, Ding Y, Wu D, Shu X, Song W Ref: Genes Genomics, 42:1227, 2020 : PubMed
BACKGROUND: The digestibility of starch is important for the nutritive value of staple food. Although several genes are responsible for resistant starch (RS) and slowly digestible starch (SDS), gaps persist concerning the molecular basis of RS and SDS formation due to the complex genetic mechanisms of starch digestibility. OBJECTIVES: The objective of this study was to identify new genes for starch digestibility in rice and interprete the genetic mechanisms of RS and SDS by GWAS. METHODS: Genome-wide association studies were conducted by associating the RS and SDS phenotypes of 104 re-sequenced rice lines to an SNP dataset of 2,288,867 sites using a compressed mixed linear model. Candidate genes were identified according to the position of the SNPs based on data from the MSU Rice Genome Annotation Project. RESULTS: Seven quantitative trait loci (QTLs) were detected to be associated with the RS content, among which the SNP 6 m1765761 was located on Waxy. Starch branching enzymes IIa (BEIIa) close to QTL qRS-I4 was detected and further identified as a specific candidate gene for RS in INDICA. Two QTLs were associated with SDS, and the LOC_Os09g09360 encoding lipase was identified as a causal gene for SDS. CONCLUSIONS: GWAS is a valid strategy to genetically dissect the formation of starch digestion properties in rice. RS formation in grains is dependent on the rice type; lipid might also contribute to starch digestibility and should be an alternative factor to improve rice starch digestibility.
In cooperative catalysis, the combination of chemo- and biocatalysts to perform one-pot reactions is a powerful tool for the improvement of chemical synthesis. Herein, UiO-66-NH2 was employed to stepwise immobilize Pd nanoparticles (NPs) and Candida antarctica lipase B (CalB) for the fabrication of biohybrid catalysts for cascade reactions. Distinct from traditional materials, UiO-66-NH2 has a robust but tunable structure that can be utilized with a ligand exchange approach to adjust its hydrophobicity, resulting in excellent catalyst dispersity in diverse reaction media. These attractive properties contribute to the formation of MOF-based biohybrid catalysts with high activity and selectivity in the synthesis of benzyl hexanoate from benzaldehyde and ethyl hexanoate. With this proof-of-concept, we reasonably expect that future tailor-made MOFs can combine other catalysts, ranging from chemical to biological catalysts for applications in industry.
Valine-citrulline linkers are commonly used as enzymatically cleavable linkers for antibody-drug conjugates. While stable in human plasma, these linkers are unstable in mouse plasma due to susceptibility to an extracellular carboxylesterase. This instability often triggers premature release of drugs in mouse circulation, presenting a molecular design challenge. Here, we report that an antibody-drug conjugate with glutamic acid-valine-citrulline linkers is responsive to enzymatic drug release but undergoes almost no premature cleavage in mice. We demonstrate that this construct exhibits greater treatment efficacy in mouse tumor models than does a valine-citrulline-based variant. Notably, our antibody-drug conjugate contains long spacers facilitating the protease access to the linker moiety, indicating that our linker assures high in vivo stability despite a high degree of exposure. This technology could add flexibility to antibody-drug conjugate design and help minimize failure rates in pre-clinical studies caused by linker instability.
Fifteen new polycyclic polyprenylated acylphloroglucinols (PPAPs), hyperforatones A-O (1-15), along with 3 structurally related analogues (16-18), were isolated from the stems and leaves of Hypericum perforatum. Their structures and absolute configurations were established by a combination of NMR spectroscopic analyses, experimental and calculated electronic circular dichroism (ECD), modified Mosher's methods, Rh2(OCOCF3)4- and [Mo2(OAc)4]-induced ECD, X-ray crystallography, and the assistance of quantum chemical predictions (QCP) of 13C NMR chemical shifts. Compound 5 was found to be the first PPAP decorated by a rare 2,2,4,4,5-(pentamethyltetrahydrofuran-3-yl)methanol moiety and an oxepane ring. Furthermore, the isolates were screened for their acetylcholinesterase (AChE) and beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitory activities. Compounds 5, 10, 11, and 15 showed desirable AChE inhibitory activities (IC50 6.9-9.2 muM) and simultaneously inhibited BACE1 (at a concentration of 5 muM) with inhibition rates of 50.3%, 34.3%, 47.2%, and 34.6%, respectively. Interestingly, compound 5 showed the most balanced inhibitory activities against both AChE and BACE1 of all the tested compounds, which means that 5 could serve as the first valuable dual-targeted PPAP for the treatment of Alzheimer's disease. Preliminary molecular docking studies of 5 with BACE1 and AChE were also performed.
Title: A low trans margarine fat analog to beef tallow for healthier formulations: Optimization of enzymatic interesterification using soybean oil and fully hydrogenated palm oil Li Y, Zhao J, Xie X, Zhang Z, Zhang N, Wang Y Ref: Food Chem, 255:405, 2018 : PubMed
The health hazard of tallow and partial hydrogenated oils is well known in margarine productions. For this, food manufactures are urged to develop novel alternatives for healthier margarine formulations. The highest interesterification degree acquired with lipase Lipozyme 435 standing out from other catalysts (solid acid, sodium hydroxide and methoxide) was applied to produce low trans margarine fat analogs to beef tallow (BT) with the blend of soybean oil (SO) and fully hydrogenated palm oil (FHPO) in a mass ratio of 4:3. Reaction parameters like enzyme dosage (4.2 wt%), temperature (95 degreeC) and time (245 min) were optimized using the Box-Behnken design. Regarding fatty acid profiles, triacylglycerol species, solid fat content, polymorphism, melting and crystallization behaviors, the resulting interesterified oil was characterized in comparison with BT, FHPO and the SO-FHPO blend so as to prove its potential in formulating low trans fat margarines because of desirable physicochemical properties and polymorphs.
Title: The Herb-Drug Interaction of Clopidogrel and Xuesaitong Dispersible Tablet by Modulation of the Pharmacodynamics and Liver Carboxylesterase 1A Metabolism Ma S, Dai G, Bi X, Gong M, Miao C, Chen H, Gao L, Zhao W, Liu T, Zhang N Ref: Evid Based Complement Alternat Med, 2018:5651989, 2018 : PubMed
Objective: Clopidogrel and Xuesaitong dispersible tablet (XST) have been clinically proven to be effective for treating cardiocerebrovascular disease. The present study was to investigate the herb-drug interaction of Clopidogrel and XST by modulation of the pharmacodynamics and liver Carboxylesterase 1A(CES1A) metabolism. Methods: 30 male SD rats were randomly divided into a control group (equal volumes of saline, 6 rats for mRNA analysis), a clopidogrel group (clopidogrel with dose 30 mg/kg), and a combination group (clopidogrel and XST, with dose 30 and 50 mg/kg respectively, each group continuous administration once daily for 30 days). The clopidogrel and combination group comprised 12 rats, with 6 designated for mRNA analysis and 6 for the pharmacokinetic study. The 2-bromo-3'-methoxyacetophenone- (MPB-) derivatized clopidogrel active thiol metabolite (CAMD) was measured by UHPLC-MS/MS for pharmacokinetics (n=6). The expression of CES1A mRNA was examined with real-time RT-PCR (n=6). Molecular simulation was used to investigate the inhibition effect of XST on the CES1A protein. The CAMD pharmacodynamics and CES1A metabolism were investigated to evaluated the herb-drug interaction. Results: Clopidogrel and XST coadministration appreciably increased the Cmax, AUC, and MRT of CAMD. However, the expression of CES1A mRNA was decreased accordingly. It also indicated that the bioactive components in XST had good interaction with the CES1A metabolism target by molecular simulation. The animal study indicated that clopidogrel and XST coadministration produced significant herb-drug interactions at active CAMD pharmacokinetic and CES1A metabolic enzyme aspect. Conclusion: 30-days dose of coadministration altered hepatic CES1A protein and resulted in reduced plasma levels of active CAMD. both the decreased CES1A mRNA expression and the inhibition on the protein were due to the combination of XST, which accordingly upregulated the pharmacokinetics of plasma active CAMD.
Title: Clinical efficacy and safety of donepezil in the treatment of Alzheimer's disease in Chinese patients Zhang N, Gordon ML Ref: Clin Interv Aging, 13:1963, 2018 : PubMed
Donepezil, an acetylcholinesterase inhibitor (AChEI), has been widely used to treat Alzheimer's disease (AD) in China. However, there are few studies focusing on the efficacy and safety of donepezil in Chinese patients. In this review, we discuss 1) the efficacy of donepezil and its comparison with other AChEIs or memantine, 2) the therapeutic responses to donepezil and its influencing factors, and 3) the safety and tolerability of donepezil in Chinese patients with different stages of AD and amnestic mild cognitive impairment, and further compare the similarities and differences of the results between Chinese studies and previous Western studies that predominantly enrolled Caucasian subjects. We include Chinese clinical trials and other well-designed studies investigating donepezil or using donepezil as a positive control, in which the efficacy and/or safety of donepezil have been analyzed. Based on these studies, donepezil has been shown to be effective and safe in Chinese AD patients and may impact AD biomarkers, such as hippocampal atrophy, Abeta, and tau. In addition, the therapeutic response to donepezil may be influenced by apolipoprotein E or cytochrome P450 2D6 polymorphism.
The new polyprenylated acylphloroglucinol derivatives 1-15 and the known furohyperforin (16) were isolated from the stems and leaves of Hypericum perforatum. Their structures were determined by analyses of NMR and HRESIMS data. Their absolute configurations were elucidated by a combination of electronic circular dichroism (ECD) and Rh2(OCOCF3)4-induced ECD, as well as X-ray diffraction crystallography. The new hyperforatin F (9) contains a unique acetyl functionality at C-1 of the bicyclo[3.3.1]nonane core. Hyperforatins G (10) and H (11) are similarly the first examples of naturally occurring [3.3.1]-type polycyclic prenylated acylphloroglucinols possessing a carbonyl functionality at C-32. The compounds were tested for their acetylcholinesterase (AChE) inhibitory activities and cytotoxic activities against a panel of human tumor cell lines. Compounds 3, 5, 6, 8, and 9 exerted moderate inhibitory activities (IC50 3.98-9.13 muM) against AChE.
Title: Microplastics have a more profound impact than elevated temperatures on the predatory performance, digestion and energy metabolism of an Amazonian cichlid Wen B, Zhang N, Jin SR, Chen ZZ, Gao JZ, Liu Y, Liu HP, Xu Z Ref: Aquat Toxicol, 195:67, 2017 : PubMed
Knowledge on the impacts of microplastics (MPs) pollution on freshwater environments and biota remains limited. Meanwhile, freshwater ecosystems have been threatened by elevated temperatures caused by climate change. To date, no information exists on how MPs-especially under elevated temperature conditions-affect predatory performance, digestive processes and metabolic pathways in freshwater organisms. Here, we examined MPs, elevated temperature and their combined effects on juveniles (0+ group) of an Amazonian cichlid, the discus fish (Symphysodon aequifasciatus). For 30days, fish were exposed to ambient or elevated temperatures (i.e., 28 or 31 degrees C) in the absence or presence of MPs (i.e., 0 or 200mug/L). The following metrics were quantified: MPs accumulation; predatory performance; and biomarkers involved in neurotransmission, digestion and energy production. The results showed that survival rate and body length were not affected by MPs, elevated temperatures or their combination. Elevated temperatures resulted in an increase in MP concentrations in fish bodies. Exposure to MPs decreased the post-exposure predatory performance (PEPP) at ambient temperatures but not at elevated temperatures. Elevated temperatures, however, had no effect on the PEPP but antagonistically interacted with MPs, leading to similar predatory performances under present and future conditions. Acetylcholinesterase (AChE) activity was only affected by MPs and decreased in the presence of MPs, indicating adverse effects in nervous and neuromuscular function and, thus, potentially in predatory performance. Trypsin activity was only influenced by MPs and decreased during exposure to MPs. Elevated temperatures or MPs alone increased the amylase activity but interacted antagonistically. Lipase activity was not influenced by either of the two stressors. In contrast, alkaline phosphatase (ALP) activity was affected by MPs or elevated temperatures alone and decreased with both stressors. Such results indicate deficits in the digestive capabilities of early-stage S. aequifasciatus under elevated temperature conditions and especially during exposure to MPs. Electron transport system (ETS) activity was not influenced by either of the two stressors. Both elevated temperatures and MPs alone increased LDH activity; however, the interaction between the two stressors cancelled activity but was still higher than activity in present conditions. Citrate synthase (CS) activity decreased with elevated temperature but increased during exposure to MPs. Cytochrome c oxidase (COX) activity was only influenced by MPs and increased in the presence of MPs. Thus, S. aequifasciatus juveniles exposed to elevated temperatures and MPs not only relied on anaerobic glycolysis for energy production but also depended on aerobic metabolism in the presence of MPs. Overall, these findings suggested that MPs showed a greater impact than elevated temperatures on the predatory performance, digestion and energy production of S. aequifasciatus. Nevertheless, juvenile survival and growth were minimally impacted, and thus, S. aequifasciatus could cope with near-future temperature increases and MP exposure.
Pneumocandins are lipohexapeptides of the echinocandin family that inhibit fungal 1,3-beta-glucan synthase. Most of the pathway steps have been identified previously. However, the lipoinitiation reaction has not yet been experimentally verified. Herein, we investigate the lipoinitiation step of pneumocandin biosynthesis in Glarea lozoyensis and demonstrate that the gene product, GLligase, catalyzes this step. Disruption of GLHYD, a gene encoding a putative type II thioesterase and sitting upstream of the pneumocandin acyl side chain synthase gene, GLPKS4, revealed that GLHYD was necessary for optimal function of GLPKS4 and to attain normal levels of pneumocandin production. Double disruption of GLHYD and GLPKS4 did not affect residual function of the GLligase or GLNRPS4. Mutasynthesis experiments with a gene disruption mutant of GLPKS4 afforded us an opportunity to test the substrate specificity of GLligase in the absence of its native polyketide side chain to diversify pneumocandins with substituted side chains. Feeding alternative side chain precursors yielded acrophiarin and four new pneumocandin congeners with straight C14, C15, and C16 side chains. A comprehensive biological evaluation showed that one compound, pneumocandin I (5), has elevated antifungal activity and similar hemolytic activity compared to pneumocandin B(0), the starting molecule for caspofungin. This study demonstrates that the lipoinitiation mechanism in pneumocandin biosynthesis involves interaction among a highly reducing PKS, a putative type II thioesterase, and an acyl AMP-ligase. A comparison of the SAR among pneumocandins with different-length acyl side chains demonstrated the potential for using GLligase for future engineering of new echinocandin analogues.
Starting from a screening-hit compound, via structure modifications and optimizations, a series of nonfused and nonassembly pyrimidinylthiourea derivatives (2-5) was designed, synthesized, and evaluated as novel multifunctional agents against Alzheimer's disease. Biological activity results demonstrated that compounds 5r and 5t exhibited potent inhibition and excellent selectivity toward acetylcholinesterase (AChE, 5r, IC50 = 0.204 muM, SI > 196; 5t, IC50 = 0.067 muM, SI > 597), specific metal-chelating ability, significant antioxidant effects, modulation of metal-induced Abeta aggregation, inhibition of ROS production by copper redox cycle, low cytotoxicity, and moderate neuroprotection to human neuroblastoma SH-SY5Y cells. Moreover, compound 5r displayed appropriate blood-brain barrier (BBB) permeability both in vitro and in vivo and could improve memory and cognitive function of scopolamine-induced amnesia mice. The multifunctional profiles of 5r and its effectivity in AD mice highlight these structurally distinct pyrimidinylthiourea derivatives as prospective prototypes in the research of innovative multifunctional drugs for Alzheimer's disease.
Title: Production of Diacylglycerol-enriched Oil by Glycerolysis of Soybean Oil using a Bubble Column Reactor in a Solvent-free System Zhang N, Yang X, Fu J, Chen Q, Song Z, Wang Y Ref: J Oleo Sci, 65:207, 2016 : PubMed
In this study, diacylglycerol-enriched soybean oil (DESO) was synthesized through Lipozyme 435-catalyzed glycerolysis of soybean oil (SO) in a solvent-free system using a modified bubble column reactor. The effects of enzyme load, mole ratio of glycerol to soybean oil, reaction temperature, gas flow and reaction time on DAG production were investigated. The selected conditions were established as being enzyme load of 4 wt% (mass of substrates), glycerol/soybean oil mole ratio of 20:1, reaction temperature of 80 degC, gas flow of 10.6 cm/min, and a reaction time of 2.5 h, obtaining the DAG content of 49.4+/-0.5 wt%. The reusability of Lipozyme 435 was evaluated by monitoring the contents of DAG, monoacylglycerol (MAG) and triacylglycerol (TAG) in 10 consecutive runs. After purified by one-step molecular distillation, the DAG content of 63.5+/-0.3 wt% was achieved in DESO. The mole ratio of 1, 3-DAG to 1, 2-DAG was 2:1 and the fatty acid composition had no significant difference from that of soybean oil. However, the thermal properties of DESO and SO had considerable differences. Polymorphic form of DESO were mainly the beta form and minor amounts of the beta' form. Granular aggregation and round-shaped crystals were detected in DESO.
Title: Complete Genome Sequence of Rhodococcus sp. B7740, a Carotenoid-Producing Bacterium Isolated from the Arctic Sea Zhang D, Li L, Zhu S, Zhang N, Yang J, Ma X, Chen J Ref: Genome Announc, 3:, 2015 : PubMed
Rhodococcus sp. B7740 was isolated from Arctic seawater and selected for its capacity to synthesize carotenoids. Here, we report the complete genome sequence of Rhodococcus sp. B7740 to provide the genetic basis for a better understanding of its carotenoid-accumulating capabilities, and we describe the major features of the genome.
Title: Complete Genome Sequence of Paenibacillus polymyxa SQR-21, a Plant Growth-Promoting Rhizobacterium with Antifungal Activity and Rhizosphere Colonization Ability Li S, Yang D, Qiu M, Shao J, Guo R, Shen B, Yin X, Zhang R, Zhang N, Shen Q Ref: Genome Announc, 2:, 2014 : PubMed
Here we report the complete genome sequence of a plant growth-promoting rhizobacterium (PGPR), Paenibacillus polymyxa SQR-21, which consists of one circular chromosome of 5,828,438 bp with 5,024 coding sequences (CDS). The data presented highlight multiple sets of functional genes associated with its plant-beneficial characteristics.
Title: Lab-on-a-drop: biocompatible fluorescent nanoprobes of gold nanoclusters for label-free evaluation of phosphorylation-induced inhibition of acetylcholinesterase activity towards the ultrasensitive detection of pesticide residues Zhang N, Si Y, Sun Z, Li S, Lin Y, Wang H Ref: Analyst, 139:4620, 2014 : PubMed
A simple, sensitive, selective, and "lab-on-a-drop"-based fluorimetric protocol has been proposed using biocompatible fluorescent nanoprobes of gold nanoclusters (AuNCs) for the label-free evaluation of the catalytic activity and phosphorylation of acetylcholinesterase (AChE) under physiologically simulated environments. Protein-stabilized AuNCs were prepared and mixed with acetylthiocholine (ATC) serving as "a drop" of fluorimetric reaction substrate. The AChE-catalyzed hydrolysis of ATC releases thiocholine to cause the aggregation of the AuNCs towards a dramatic decrease in fluorescence intensities, which could be curbed by the phosphorylation-induced inhibition of AChE activity when exposed to organophosphorus compounds (OPs). The reaction procedures and conditions of AChE catalysis and phosphorylation were monitored by fluorimetric measurements and electron microscopy imaging. Moreover, a selective and ultrasensitive fluorimetric assay has been tailored for the detection of pesticide residues using dimethyl-dichloro-vinyl phosphate (DDVP) as an example. Investigation results indicate that the specific catalysis and irreversible OP-induced phosphorylation of AChE, in combination with sensitive fluorimetric outputs could facilitate the detection of total free OPs with high selectivity and sensitivity. A linear concentration of DDVP ranging from 0.032 nM to 20 nM could be obtained with a detection limit of 13.67 pM. Particularly, pesticide residues of DDVP in vegetable samples were quantified down to approximately 36 pM. Such a label-free "lab-on-a drop"-based fluorimetry may promise wide applications for the evaluation of the physiological catalytic activity of various enzymes (i.e., cholinesterase), and especially for monitoring the direct phosphorylation biomarkers of free OPs towards rapid and early warning, and accurate diagnosis of OP exposure.
Title: An acetylcholinesterase antibody-based quartz crystal microbalance for the rapid identification of spinal ventral and dorsal roots Sui T, Ge Y, Liu W, Zhao ZK, Zhang N, Cao X Ref: PLoS ONE, 8:e69049, 2013 : PubMed
Differences in the levels of acetylcholinesterase (AChE) in ventral and dorsal spinal roots can be used to differentiate the spinal nerves. Although many methods are available to assay AChE, a rapid and sensitive method has not been previously developed. Here, we describe an antibody-based quartz crystal microbalance (QCM) assay and its application for the quantification of AChE in the solutions of ventral and dorsal spinal roots. The frequency variation of the QCM device corresponds to the level of AChE over a wide dynamic range (0.5-10 microg/ml), which is comparable to the response range of the ELISA method. The frequency shift caused by the ventral roots is 3-fold greater than that caused by the dorsal roots. The antibody-based QCM sensor was stable across many successive replicate samples, and the method required less than 10 min, including the AChE extraction and analysis steps. This method is a rapid and convenient means for the quantification of AChE in biological samples and may be applicable for distinguishing the ventral and dorsal roots during surgical operations.
The Pacific oyster Crassostrea gigas belongs to one of the most species-rich but genomically poorly explored phyla, the Mollusca. Here we report the sequencing and assembly of the oyster genome using short reads and a fosmid-pooling strategy, along with transcriptomes of development and stress response and the proteome of the shell. The oyster genome is highly polymorphic and rich in repetitive sequences, with some transposable elements still actively shaping variation. Transcriptome studies reveal an extensive set of genes responding to environmental stress. The expansion of genes coding for heat shock protein 70 and inhibitors of apoptosis is probably central to the oyster's adaptation to sessile life in the highly stressful intertidal zone. Our analyses also show that shell formation in molluscs is more complex than currently understood and involves extensive participation of cells and their exosomes. The oyster genome sequence fills a void in our understanding of the Lophotrochozoa.
Cardiac arrest is a leading cause of death worldwide. While survival rates following sudden cardiac arrest remain relatively low, recent advancements in patient care have begun to increase the proportion of individuals who survive cardiac arrest. However, many of these individuals subsequently develop physiological and psychiatric conditions that likely result from ongoing neuroinflammation and neuronal death. The present study was conducted to better understand the pathophysiological effects of cardiac arrest on neuronal cell death and inflammation, and their modulation by the cholinergic system. Using a well validated model of cardiac arrest, here we show that global cerebral ischemia increases microglial activation, proinflammatory cytokine mRNA expression (interleukin-1beta, interleukin-6, tumor necrosis factor-alpha), and neuronal damage. Cardiac arrest also induces alterations in numerous cellular components of central cholinergic signaling, including a reduction in choline acetyltransferase enzymatic activity and the number of choline acetyltransferase-positive neurons, as well as, reduced acetylcholinesterase and vesicular acetylcholine transporter mRNA. However, treatment with a selective agonist of the alpha7 nicotinic acetylcholine receptor, the primary receptor mediating the cholinergic anti-inflammatory pathway, significantly decreases the neuroinflammation and neuronal damage resulting from cardiac arrest. These data suggest that global cerebral ischemia results in significant declines in central cholinergic signaling, which may in turn diminish the capacity of the cholinergic anti-inflammatory pathway to control inflammation. Furthermore, we provide evidence that pharmacological activation of alpha7 nicotinic acetylcholine receptors provide significant protection against ischemia-related cell death and inflammation within a clinically relevant time frame.
We aimed to evaluate the clinical characteristics of patients with postoperative myasthenia gravis (MG). We retrospectively studied the data of 174 thymoma patients treated between 1990 and 2008 in Xiangya Hospital. Six of 125 patients without preoperative MG (4.8%) developed postoperative MG. The anti-acetylcholine-receptor binding antibody (ARAb) titers were elevated preoperatively in 22 of the 125 patients (17.6%) who did not have preoperative MG (range, 0.5-67.6nmol/L). Four of six patients with postoperative MG had positive ARAb levels preoperatively. Serum titers were exacerbated in all six patients at the onset of postoperative MG. Postoperative MG was responsive to anti-cholinesterase compounds and/or steroids. We concluded that a thymectomy did not prevent postoperative MG. Exacerbated ARAb levels after thymectomy suggested an extrathymic production of ARAb. We suggest that a rise in the ARAb titer might be a risk indictor for post-thymectomy MG.
Title: Enantioselective interaction with acetylcholinesterase of an organophosphate insecticide fenamiphos Wang C, Zhang N, Li L, Zhang Q, Zhao M, Liu W Ref: Chirality, 22:612, 2010 : PubMed
Enantioselectivity in the environmental behavior and ecotoxicity of chiral pesticide is widely observed. However, the investigation of the enantioselective mechanisms remains limited. In this study, we used fenamiphos (FAP), an organophosphorus insecticide, to study enantioselectivity in toxicity to arthropods and the inhibition potential towards acetylcholinesterase (AChE) in the rat pheochromocytoma 12 (PC 12) cell line. Furthermore, we carried out molecular docking to help explain the mechanisms of enantioselective toxicity of FAP. The two enantiomers of FAP were successfully separated and identified as R-(+)-FAP and S-(-)-FAP. Toxicological assays revealed that R-(+)-FAP was 2.4-fold more toxic than S-(-)-FAP to Daphnia magna and approximately threefold more to PC12 cells. Based on molecular docking results, dynamic simulation shows that strong hydrophobic interactions and a key hydrogen bond can only exist between R-(+)-FAP and AChE, which helps explain the preference of R-(+) binding to AChE over that of the S-(-)-enantiomer, and supports our biological results. Our present study considers the impact of stereochemistry on ecotoxicological effects and, ultimately, on development of environmentally safe, insecticidally efficient pesticides.
Title: [Comparison of curative effect of low flow rate plasma exchange combined with hemofiltration for treatment of liver failure] Yang YF, Huang P, Zhang N, Gai XD, Feng XN, Zhong YD, Wang LR, Yang YJ, Zhao W Ref: Zhongguo Wei Zhong Bing Ji Jiu Yi Xue, 21:111, 2009 : PubMed
OBJECTIVE: To investigate the effect of plasma exchange (PE) combined with hemofiltration (HF) on liver failure. METHODS: Seventy-seven inpatients with liver failure admitted during January 2006 to August 2007 were randomly assigned to receive PE combined with HF (PE+HF group, 38 cases), or PE alone (PE group, 39 cases). Forty-one inpatients with liver failure who had not received artificial liver support treatment were assigned to serve as control group. The survival rates and biochemical parameters of three groups were compared. RESULTS: There was no significant difference in biochemical parameters before treatment among three groups. Compared with pre-treatment values, albumin (Alb), cholinesterase (ChE) and prothrombin activity (PTA) of both PE group and PE+HF group were significantly increased after treatment, and total bilirubin (TBIL), alanine transaminase (ALT), aspartate transaminase (AST) of both PE group and PE+HF group were significantly decreased after treatment (P<0.05 or P<0.01). The survival rate of PE group, PE+HF group and control group was 48.7% (19/39), 68.4% (26/38), and 29.3% (12/41) respectively. The survival rate of PE+HF group was significantly higher than that of control group (chi(2)=12.11, P<0.01). The rate of recovery of consciousness of patients with hepatic encephalopathy in PE+HF group was higher than that of PE group (42.8% vs. 0, P<0.05). Compared with PE alone, the result was better when it was combined with HF in correction of electrolyte disturbance and acid-base imbalance (19/23 vs. 0/21, P<0.05). CONCLUSION: Treatment of liver failure by PE combined with HF is safe and effective, and its efficacy is higher than PE alone.
Title: Toxic effects of chlorpromazine on Carassius auratus and its oxidative stress Li T, Zhou Q, Zhang N, Luo Y Ref: J Environ Sci Health B, 43:638, 2008 : PubMed
Under laboratory conditions, ecotoxicological effects of chlorpromazine (CPZ) on freshwater goldfish (Carassius auratus) were examined using the toxic culture experiment. The results showed that the median lethal concentration (LC(50)) of CPZ toxic to Carassius auratus in 24, 48 and 96 h was 1.11, 0.43 and 0.32 mg/L, respectively. Thus, CPZ is an extreme toxicant to goldfish. Furthermore, there were significantly positive correlations between the ecotoxicological effects of CPZ and its concentrations, and the toxicity became higher as the exposure time increased. The activity of superoxide dismutase (SOD) and catalase (CAT) in goldfish livers was significantly influenced by CPZ. At the same exposure time, the activity of SOD reduced first, and increased then, whereas the activity of CAT enhanced first and decreased then. At the same exposure levels of CPZ, the activity of SOD and CAT changed similarly, decreased first, then increased and decreased at last. Within the range of exposure concentrations, the changes in the activity of CAT can more easily reflect the oxidation stress in Carassius auratus by CPZ than those of SOD.
Nicotinic acetylcholine (ACh) receptor (nAChR) agonists are potential therapeutic agents for neurological dysfunction. In the present study, the homopentameric mollusk ACh binding protein (AChBP), used as a surrogate for the extracellular ligand-binding domain of the nAChR, was specifically derivatized by the highly potent agonist azidoepibatidine (AzEPI) prepared as a photoaffinity probe and radioligand. One EPI-nitrene photoactivated molecule was incorporated in each subunit interface binding site based on analysis of the intact derivatized protein. Tryptic fragments of the modified AChBP were analyzed by collision-induced dissociation and Edman sequencing of radiolabeled peptides. Each specific EPI-nitrene-modified site involved either Tyr195 of loop C on the principal or (+)-face or Met116 of loop E on the complementary or (-)-face. The two derivatization sites were observed in similar frequency, providing evidence of the reactivity of the azido/nitrene probe substituent and close proximity to both residues. [3H]AzEPI binds to the alpha4beta2 nAChR at a single high-affinity site and photoaffinity-labels only the alpha4 subunit, presumably modifying Tyr225 spatially corresponding to Tyr195 of AChBP. Phe137 of the beta2 nAChR subunit, equivalent to Met116 of AChBP, conceivably lacks sufficient reactivity with the nitrene generated from the probe. The present photoaffinity labeling in a physiologically relevant condition combined with the crystal structure of AChBP allows development of precise structural models for the AzEPI interactions with AChBP and alpha4beta2 nAChR. These findings enabled us to use AChBP as a structural surrogate to define the nAChR agonist site.
Rotenone and deguelin are the major active ingredients and principal components of cuberesin from Lonchocarpus utilis used as a botanical insecticide and piscicide. They are also potent complex I (NADH:ubiquinone oxidoreductase) inhibitors. Rotenone was known earlier, and deguelin is shown here to induce a Parkinson's disease (PD)-like syndrome after subcutaneous treatment of rats by osmotic minipump. Rotenone at 3 mg/kg/day or deguelin at 6 but not 3 mg/kg/day induces degeneration of the nigrostriatal dopaminergic pathway, as shown by reduced tyrosine hydroxylase immunoreactivity with treatments for 5 or 6 days. The neuropathological lesions are associated with a brain level of parent rotenoid of 0.4-1.3 ppm but not with the much smaller brain level of 12abeta-hydroxyrotenoids or other metabolites analyzed by HPLC and LC/MS. We previously established that the hydroxylated metabolites and derivatives of rotenone and deguelin are all less active (i.e., detoxified) as complex I inhibitors relative to the parent rotenoids. The PD-like syndrome induced in rats by rotenone and deguelin is therefore due to the parent compounds rather than metabolites. Deguelin is about half as active as rotenone in inducing the PD-like syndrome in rats and in acute ip LD50 in mice. Rotenone and deguelin are metabolized by human recombinant 3A4 and 2C19 but not five other P450 enzymes. 2C19 is more selective than 3A4 in forming the 12abeta-hydroxyrotenoids. Identified sites of metabolic attack individually or in combination are as follows: 12abeta hydroxylation and 2-O-demethylation of both compounds, oxidation of the rotenone isopropenyl substituent to mono and diol derivatives, and probable oxidation of the deguelin dimethylchromene double bond. These toxicological features must be considered in using rotenone, deguelin, and their analogues as pesticides, candidate radioimaging and cancer chemopreventive agents, and models of PD.
Title: Determination of tributyrin and its metabolite butyrate in Wistar rat plasma samples by gas chromatography/mass spectrometry Su J, Zhang N, Ho PC Ref: Rapid Commun Mass Spectrom, 18:2217, 2004 : PubMed
A gas chromatographic (GC) method with mass spectrometric (MS) detection was developed for the determination of tributyrin and its metabolite butyrate in rat plasma. Following precipitation of plasma protein with acetonitrile, the analytes in the samples were separated on a DB-5ms capillary column with helium as carrier gas. Phenylmethylsulfonyl fluoride (PMSF), an inhibitor for serine proteases, papain and acetylcholinesterase, was found to be essential to inhibit the activity of enzyme(s) responsible for the hydrolysis of tributyrin in both rat and human blood samples. The enzyme inhibitor in 5 mM (final concentration) was added immediately into the blood samples after collection to prevent the hydrolysis. The linear concentration ranges for tributyrin and butyrate were 0.1-2.0 and 1-20 microM, respectively. The coefficients of variation for intra-day and inter-day assays for tributyrin were all <10%, and those for butyrate were also <10%, except for the lowest concentration (1 microM), which was less than 20%. The accuracy of all concentration determinations ranged from 96.0-110.0%. The limit of quantification (LOQ) was 0.1 microM for tributyrin and 1.0 microM for butyrate. This method could detect tributyrin and butyrate simultaneously, and represents an improvement in sensitivity for the detection of tributyrin compared with the previous gas chromatography-flame ionization detection (GC-FID) method.
Title: Alpha-nitro ketone as an electrophile and nucleophile: synthesis of 3-substituted 2-nitromethylenetetrahydrothiophene and -tetrahydrofuran as drosophila nicotinic receptor probes Zhang N, Tomizawa M, Casida JE Ref: J Org Chem, 69:876, 2004 : PubMed
3-(6-Chloropyridin-3-yl)methyl-2-nitromethylenetetrahydrothiophene 2 and -tetrahydrofuran 3 were synthesized through novel approaches using alpha-nitro ketone intermediates as an electrophile and nucleophile, respectively. The 2-nitromethylenetetrahydrothiophene 2 was formed exclusively as the Z-isomer through intramolecular attack by a thiol substituent at the carbonyl group of an alpha-nitro ketone, in which the alpha-nitro ketone served as an electrophile. In contrast, the corresponding 2-nitromethylenetetrahydrofuran 3, not accessible by the above route due to limited stability, was prepared as a mixture of E- and Z-isomers by intramolecular attack of the alpha-nitro ketone enol anion in which the deprotonated alpha-nitro ketone served as a nucleophile. These compounds, together with the corresponding 2-nitromethylenepyrrolidine (1), were used to probe the Drosophila neonicotinoid-nicotinic acetylcholine receptor interaction.
Title: Drosophila nicotinic receptors: evidence for imidacloprid insecticide and alpha-bungarotoxin binding to distinct sites Zhang N, Tomizawa M, Casida JE Ref: Neuroscience Letters, 371:56, 2004 : PubMed
The principal mammalian brain nicotinic acetylcholine receptors (nAChRs) are the alpha-bungarotoxin (alpha-BGT)-insensitive alpha 4 beta 2 and the alpha-BGT-sensitive alpha 7 subtypes assayed with radiolabeled nicotinoids and alpha-BGT, respectively. Drosophila head membranes bind the insecticide radioligand [(3)H]imidacloprid ([(3)H]IMI) and [(3)H]alpha-BGT with K(D) 5.7 and 2.7 nM and B(max) 980 and 1400 fmol/mg protein, respectively. The hypothesis that [(3)H]IMI at 2.5 or 20 nM and [(3)H]alpha-BGT at 1 or 10 nM bind to distinct sites or subtypes is tested by using these radioligands alone and together in simultaneous dual binding experiments. These studies show no interference by one radioligand in the binding of the other one, i.e., independent binding, and that both unlabeled IMI and alpha-BGT give biphasic displacement curves. The pharmacological profiles of [(3)H]IMI and [(3)H]alpha-BGT suggest distinct binding sites for the two radioligands. These findings are consistent with those obtained with hybrid receptors assembled from Drosophila alpha subunits and a vertebrate beta subunit and with immunological and protein biochemical approaches. This study, therefore, provides direct evidence for distinct IMI- and alpha-BGT-sensitive sites or subtypes in Drosophila brain.
Title: The neonicotinoid electronegative pharmacophore plays the crucial role in the high affinity and selectivity for the Drosophila nicotinic receptor: an anomaly for the nicotinoid cation--pi interaction model Tomizawa M, Zhang N, Durkin KA, Olmstead MM, Casida JE Ref: Biochemistry, 42:7819, 2003 : PubMed
Cation-pi interaction, a prominent feature in agonist recognition by neurotransmitter-gated ion channels, does not apply to the anomalous action of neonicotinoids at the insect nicotinic acetylcholine receptor (nAChR). Insect-selective neonicotinoids have an electronegative pharmacophore (tip) in place of the ammonium or iminium cation of the vertebrate-selective nicotinoids, suggesting topological divergence of the agonist-binding sites in insect and vertebrate nAChRs. This study defines the molecular and electronic basis for the potent and selective interaction of the neonicotinoid electronegative pharmacophore with a unique subsite of the Drosophila but not of the vertebrate alpha4beta2 nAChR. Target site potency and selectivity are retained when the usual neonicotinoid N-nitroimine (=NNO(2)) electronegative tip is replaced with N-nitrosoimine (=NNO) or N-(trifluoroacetyl)imine (=NCOCF(3)) in combination with an imidazolidine, imidazoline, thiazolidine, or thiazoline heterocycle. X-ray crystallography establishes coplanarity between the heterocyclic and imine planes, including the electronegative substituent in the trans configuration. The functional tip is the coplanar oxygen atom of the N-nitrosoimine or the equivalent oxygen of the N-nitroimine. Quantum mechanics in the gas and aqueous phases fully support the conserved coplanarity and projection of the strongly electronegative tip. Further, a bicyclic analogue with a nitro tip in the cis configuration but retaining coplanarity has a high potency, whereas the N-trifluoromethanesulfonylimine (=NSO(2)CF(3)) moiety lacking coplanarity confers very low activity. The coplanar system between the electronegative tip and guanidine-amidine moiety extends the conjugation and facilitates negative charge (delta(-)) flow toward the tip, thereby enhancing interaction with the proposed cationic subsite such as lysine or arginine in the Drosophila nAChR.
Title: Improving tolerance of Candida antarctica lipase B towards irreversible thermal inactivation through directed evolution Zhang N, Suen WC, Windsor W, Xiao L, Madison V, Zaks A Ref: Protein Engineering, 16:599, 2003 : PubMed
To expand the functionality of lipase B from Candida antarctica (CALB) we have used directed evolution to create CALB mutants with improved resistance towards irreversible thermal inactivation. Two mutants, 23G5 and 195F1, were generated with over a 20-fold increase in half-life at 70 degrees C compared with the wild-type CALB (WT-CALB). The increase in half-life was attributed to a lower propensity of the mutants to aggregate in the unfolded state and to an improved refolding. The first generation mutant, 23G5, obtained by error-prone PCR, had two amino acid mutations, V210I and A281E. The second generation mutant, 195F1, derived from 23G5 by error-prone PCR, had one additional mutation, V221D. Amino acid substitutions at positions 221 and 281 were determined to be critical for lipase stability, while the residue at position 210 had only a marginal effect. The catalytic efficiency of the mutants with p-nitrophenyl butyrate and 6,8-difluoro-4-methylumbelliferyl octanoate was also found to be superior to that of WT-CALB.
Title: Novel irreversible butyrylcholinesterase inhibitors: 2-chloro-1-(substituted-phenyl)ethylphosphonic acids Zhang N, Casida JE Ref: Bioorganic & Medicinal Chemistry, 10:1281, 2002 : PubMed
2-Chloroethylphosphonic acid (ethephon) as the dianion phosphorylates butyrylcholinesterase (BChE) at its active site. In contrast, the classical organophosphorus esterase inhibitors include substituted-phenyl dialkylphosphates (e.g., paraoxon) with electron-withdrawing aryl substituents. The chloroethyl and substituted-phenyl moieties are combined in this study as 2-chloro-1-(substituted-phenyl)ethylphosphonic acids (1) to define the structure--activity relationships and mechanism of BChE inhibition by ethephon and its analogues. Phenyl substituents considered are 3- and 4-nitro, 3- and 4-dimethylamino, and 3- and 4-trimethylammonium. Phosphonic acids were synthesized via the corresponding O,O-diethyl phosphonate precursors followed by deprotection with trimethylsilyl bromide. They decompose under basic conditions about 100-fold faster than ethephon to yield the corresponding styrene derivatives. Electron-withdrawing substituents on the phenyl ring decrease the hydrolysis rate while electron-donating substituents increase the rate. The 4-trimethylammonium analogue has the highest affinity (K(i)=180 microM) and potency (IC(50)=19 microM) in first binding reversibly at the substrate site (possibly with stabilization in a dianion--monoanion environment) and then progressively and irreversibly inhibiting the enzyme activity. These observations suggest dissociation of chloride as the first and rate-limiting step both in the hydrolysis and by analogy in phosphorylation of BChE by bound at the active site.
Title: Structural features of azidopyridinyl neonicotinoid probes conferring high affinity and selectivity for mammalian alpha4beta2 and Drosophila nicotinic receptors Zhang N, Tomizawa M, Casida JE Ref: Journal of Medicinal Chemistry, 45:2832, 2002 : PubMed
The higher toxicity of neonicotinoid insecticides such as N-(6-chloropyridin-3-ylmethyl)-2-nitroiminoimidazolidine (imidacloprid) to insects than mammals is due in large part to target site specificity at the corresponding nicotinic acetylcholine receptors (nAChRs). We propose that neonicotinoids with a protonated N-unsubstituted imine or equivalent substituent recognize the anionic subsite of the mammalian alpha4beta2 nAChR whereas the negatively charged (delta(-)) tip of the neonicotinoid insecticides interacts with a putative cationic subsite of the insect nAChR. This hypothesis can be tested by using two photoaffinity probes that differ only in the N-unsubstituted imine vs negatively charged (delta(-)) tip. Synthesis methodology was developed for compounds combining three moieties: pyridin-3-ylmethyl or 6-chloropyridin-3-ylmethyl and their 4- and 5-azido analogues; imidazolidine, 4-imidazoline or 4-thiazoline; and N-unsubstituted imine, nitroimine, cyanoimine, or nitromethylene. Structure-activity studies compared displacement of [(3)H]nicotine binding in mammalian alpha4beta2 nAChR and [(3)H]imidacloprid binding in Drosophila nAChR. Preferred compounds are N-(5-azido-6-chloropyridin-3-ylmethyl) with 2-iminothiazoline for alpha4beta2 (K(i) = 0.47 nM) and with 2-nitroiminothiazoline or 2-nitromethyleneimidazolidine for Drosophila (K(i) = 0.72-3.9 nM).
Title: Phosphoacetylcholinesterase: toxicity of phosphorus oxychloride to mammals and insects that can be attributed to selective phosphorylation of acetylcholinesterase by phosphorodichloridic acid Quistad GB, Zhang N, Sparks SE, Casida JE Ref: Chemical Research in Toxicology, 13:652, 2000 : PubMed
Phosphorus oxychloride (POCl(3)) is an intermediate in the synthesis of many organophosphorus insecticides and chemical warfare nerve gases that are toxic to insects and mammals by inhibition of acetylcholinesterase (AChE) activity. It was therefore surprising to observe that POCl(3), which is hydrolytically unstable, also itself gives poisoning signs in ip-treated mice and fumigant-exposed houseflies similar to those produced by the organophosphorus ester insecticides and chemical warfare agents. In mice, POCl(3) inhibits serum butyrylcholinesterase (BuChE) at a sublethal dose and muscle but not brain AChE at a lethal dose. In houseflies, POCl(3)-induced brain AChE inhibition is correlated with poisoning and the probable cause thereof. POCl(3) in vitro is selective for AChE (IC(50) = 12-36 microM) compared with several other serine hydrolases (BuChE, carboxylesterase, elastase, alpha-chymotrypsin, and thrombin) (IC(50) = 88-2000 microM). With electric eel AChE, methylcarbamoylation of the active site with eserine reversibly protects against subsequent irreversible inhibition by POCl(3). Most importantly, POCl(3)-induced electric eel AChE inhibition prevents postlabeling with [(3)H]diisopropyl phosphorofluoridate; i.e., both compounds phosphorylate at Ser-200 in the catalytic triad. Pyridine-2-aldoxime methiodide does not reactivate POCl(3)-inhibited AChE, consistent with an anionic phosphoserine residue at the esteratic site. The actual phosphorylating agent is formed within seconds from POCl(3) in water, has a half-life of approximately 2 min, and is identified as phosphorodichloridic acid [HOP(O)Cl(2)] by (31)P NMR and derivatization with dimethylamine to HOP(O)(NMe(2))(2). POCl(3) on reaction with water and HOP(O)Cl(2) have the same potency for inhibition of AChE from either electric eel or housefly head as well as the same toxicity for mice. In summary, the acute toxicity of POCl(3) is attributable to hydrolytic activation to HOP(O)Cl(2) that phosphorylates AChE at the active site to form enzymatically inactive [O-phosphoserine]AChE.
