Title: A colorimetric fluorescent probe for the detection of carboxylesterase and carbamate pesticides Feng J, Gong Y, Yang S, Tian H, Sun B Ref: Anal Sci, :, 2023 : PubMed
A colorimetric fluorescent probe (BTCNA) was developed for the determination of carboxylesterase and carbamate pesticides. The probe used naphthalene-benzothiazole as the fluorescent group and naphthyl acetate as the recognition group. The recognition mechanism of BTCNA for carboxylesterase was based on the enzymatic hydrolysis of naphthyl acetate by carboxylesterase (CES). The test paper of the BTCNA gradually changed from light blue to bright yellow with the increase of CES activity. The probe solution gradually changed from light blue to earth-yellow as the carbaryl concentration increased. There was a linear functional relationship between the R*G (red, green) value of the photo and the CES activity. And a linear functional relationship between the carbaryl concentration and the R*G value of the photo was found. Additionally, BTCNA was successfully used to detect the concentration of carbaryl in actual samples. BTCNA is a rapid detection tool for CES activity and carbamate pesticides using a smartphone.
Title: Enzyme catalyzes ester bond synthesis and hydrolysis: The key step for sustainable usage of plastics Lai J, Huang H, Lin M, Xu Y, Li X, Sun B Ref: Front Microbiol, 13:1113705, 2022 : PubMed
Petro-plastic wastes cause serious environmental contamination that require effective solutions. Developing alternatives to petro-plastics and exploring feasible degrading methods are two solving routes. Bio-plastics like polyhydroxyalkanoates (PHAs), polylactic acid (PLA), polycaprolactone (PCL), poly (butylene succinate) (PBS), poly (ethylene furanoate) s (PEFs) and poly (ethylene succinate) (PES) have emerged as promising alternatives. Meanwhile, biodegradation plays important roles in recycling plastics (e.g., bio-plastics PHAs, PLA, PCL, PBS, PEFs and PES) and petro-plastics poly (ethylene terephthalate) (PET) and plasticizers in plastics (e.g., phthalate esters, PAEs). All these bio- and petro-materials show structure similarity by connecting monomers through ester bond. Thus, this review focused on bio-plastics and summarized the sequences and structures of the microbial enzymes catalyzing ester-bond synthesis. Most of these synthetic enzymes belonged to alpha/beta-hydrolases with conserved serine catalytic active site and catalyzed the polymerization of monomers by forming ester bond. For enzymatic plastic degradation, enzymes about PHAs, PBS, PCL, PEFs, PES and PET were discussed, and most of the enzymes also belonged to the alpha/beta hydrolases with a catalytic active residue serine, and nucleophilically attacked the ester bond of substrate to generate the cleavage of plastic backbone. Enzymes hydrolysis of the representative plasticizer PAEs were divided into three types (I, II, and III). Type I enzymes hydrolyzed only one ester-bond of PAEs, type II enzymes catalyzed the ester-bond of mono-ester phthalates, and type III enzymes hydrolyzed di-ester bonds of PAEs. Divergences of catalytic mechanisms among these enzymes were still unclear. This review provided references for producing bio-plastics, and degrading or recycling of bio- and petro-plastics from an enzymatic point of view.
Fatty acid ethyl esters are important flavor chemicals in strong-flavor Baijiu. Monascus purpureus YJX-8 is recognized as an important microorganism for ester synthesis in the fermentation process. Enzyme LIP05 from YJX-8 can efficiently catalyze the synthesis of fatty acid ethyl esters under aqueous phase, but the key catalytic sites affecting esterification were unclear. The present work combined homology modeling, molecular dynamics simulation, molecular docking and site-directed mutation to analyze the catalytic mechanism of LIP05. Protein structure modeling indicated LIP05 belonged to alpha/beta fold hydrolase, contained a lid domain and a core catalytic pocket with conserved catalytic triad Ser150-His215-Asp202, and the oxyanion hole composed of Gly73 and Thr74. Ile30 and Leu37 of the lid domain were found to affect substrate specificity. The Pi-bond stacking between Tyr116 and Tyr149 played an important role in stabilizing the catalytic active center of LIP05. Tyr116 and Ile204 determined the substrate spectrum by composing the substrate-entrance channel. Residues Leu83, Ile204, Ile211 and Leu216 were involved in forming the hydrophobic substrate-binding pocket through steric hindrance and hydrophobic interaction. The catalytic mechanism for esterification in aqueous phase of LIP05 was proposed and provided a reference for clarifying the synthesis of fatty acid ethyl esters during the fermentation process of strong-flavor Baijiu.
Title: Repressed OsMESL expression triggers reactive oxygen species mediated broad-spectrum disease resistance in rice Hu B, Zhou Y, Zhou Z, Sun B, Zhou F, Yin C, Ma W, Chen H, Lin Y Ref: Plant Biotechnol J, :, 2021 : PubMed
A few reports have indicated that a single gene confer resistance to bacterial blight, sheath blight, and rice blast. In this study, we identified a novel disease resistance mutant gene, methyl esterase-like (osmesl) in rice. Mutant rice with T-DNA insertion displayed significant resistance to bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo), sheath blight caused by Rhizoctonia solani and rice blast caused by Magnaporthe oryzae. Additionally, CRISPR-Cas9 knockout mutants and RNAi lines displayed resistance to these pathogens. Complementary T-DNA mutants demonstrated a phenotype similar to the wild type (WT), thereby indicating that osmesl confers resistance to pathogens. Protein interaction experiments revealed that OsMESL affects reactive oxygen species (ROS) accumulation by interacting with thioredoxin OsTrxm in rice. Moreover, qRT-PCR results showed significantly reduced mRNA levels of multiple ROS scavenging-related genes in osmesl mutants. Nitroblue tetrazolium staining showed that the pathogens cause ROS accumulation, and quantitative detection revealed significantly increased levels of H(2) O(2) in the leaves of osmesl mutants and RNAi lines after infection. The abundance of JA, a hormone associated with disease resistance, was significantly more in osmesl mutants than in WT plants. Overall, these results suggested that osmesl enhances disease resistance to Xoo, R. solani and M. oryzae by modulating the ROS balance.
Off-target drug release and insufficient drug delivery are the main obstacles for effective anticancer chemotherapy. Prodrug-based self-assembled nanoparticles bioactivated under tumor-specific conditions are one of the effective strategies to achieve on-demand drug release and effective tumor accumulation. Herein, stimuli-activable prodrugs are designed yielding smart tumor delivery by combination of the triglyceride-mimic (TG-mimetic) prodrug structure and disulfide bond. Surprisingly, these prodrugs can self-assemble into uniform nanoparticles (NPs) with a high drug loading (over 40%) and accumulate in tumor sites specifically. The super hydrophobic TG structure can act as a gate that senses lipase to selectively control over NP dissociation and affect the glutathione-triggered prodrug activation. In addition, the impacts of the double bonds in the prodrug NPs on parent drug release and the following cytotoxicity, pharmacokinetics, and antitumor efficiency are further demonstrated. Our findings highlight the promising potential of TG-mimetic structure-gated prodrug nanoparticles for tumor-specific drug delivery.
Title: An efficient phthalate ester-degrading Bacillus subtilis: Degradation kinetics, metabolic pathway, and catalytic mechanism of the key enzyme Xu Y, Liu X, Zhao J, Huang H, Wu M, Li X, Li W, Sun X, Sun B Ref: Environ Pollut, 273:116461, 2021 : PubMed
Phthalate ester pollution in the environment and food chain is frequently reported. Microbial treatment is a green and efficient method for solving this problem. The isolation and systematic investigation of microorganisms generally recognized as safe (GRAS) will provide useful resources. A GRAS Bacillus subtilis strain, BJQ0005, was isolated from Baijiu fermentation starter and efficiently degraded phthalate esters (PAEs). The half-lives for di-isobutyl phthalate, di-butyl phthalate and di-(2-ethylhexyl) phthalate were 3.93, 4.28, and 25.49 h, respectively, from the initial amount of 10 mg per 10 mL reaction mixture, which are records using wild-type strains. Genome sequencing and metabolic intermediate analysis generated the whole metabolic pathway. Eighteen enzymes from the alpha/beta hydrolase family were expressed. Enzymes GTW28_09400 and GTW28_13725 were capable of single ester bond hydrolysis of PAEs, while GTW28_17760 hydrolyzed di-ester bonds of PAEs. Using molecular docking, a possible mechanism affecting enzymatic ester bond hydrolysis of mono-butyl phthalate was proposed of GTW28_17760. The carboxyl group generated by the first hydrolysis step interacted with histidine in the catalytic active center, which negatively affected enzymatic hydrolysis. Isolation and systematic investigation of the PAE degradation characteristics of B. subtilis will promote the green and safe treatment of PAEs in the environment and food industry.
Title: Optimization of fermentation conditions for the production of recombinant feruloyl esterase from Burkholderia pyrrocinia B1213 Fan G, Zhu Y, Fu Z, Sun B, Teng C, Yang R, Li X Ref: 3 Biotech, 10:216, 2020 : PubMed
Statistical experimental designs were used to optimize conditions for recombinant Burkholderia pyrrocinia feruloyl esterase (BpFae) production in bacteria under lactose induction. After optimization by single factor design, Plackett-Burman design, steepest ascent design and the response surface method, the optimal conditions for BpFae production were: 6 g/L lactose, pH 5.5, pre-induced period 5 h, 23 degreesC, shaker rotational speed of 240 rpm, medium volume of 50 mL/250 mL, inoculum size 0.2% (v/v), and a post-induced period of 32 h in a Luria-Bertani culture. The produced BpFae activity was 7.43 U/mL, which is 2.92 times higher than that obtained under optimal conditions using IPTG as the inducer. BpFae activity was 4.82 U/mL in a 5 L fermenter under the abovementioned optimal conditions. BpFae produced a small amount of ethyl acetate but had no effect on the synthesis of other important esters in Baijiu. The results underpin further investigations into BpFae characterization and potential applications.
Title: Structure and Catalytic Mechanism of a Pyrethroid Carboxylesterase PytH from Sphingobium faniae JZ-2 Xu D, Gao Y, Sun B, Ran T, Zeng L, He J, Wang W Ref: Applied Environmental Microbiology, :, 2020 : PubMed
Carboxylesterase PytH, isolated from a pyrethroid degrading bacterium Sphingobium faniae JZ-2, could rapidly hydrolyze the ester bond of a wide range of pyrethroid pesticides, including permethrin, fenpropathrin, cypermethrin, fenvalerate, deltamethrin, cyhalothrin and bifenthrin. To elucidate the catalytic mechanism of PytH, here we report the crystal structures of PytH with bifenthrin (BIF) and phenylmethylsulfonyl fluoride (PMSF) and two PytH mutants. Though PytH shares low sequence identity with reported alpha/beta-hydrolase fold proteins, the typical triad catalytic center with Ser-His-Asp triad (Ser78, His230 and Asp202) is present and vital for the hydrolase activity. However, no contact was found between Ser78 and His230 in the structures we solved, which may be due to the fact that the PytH structures we determined are in their inactive or low activity forms. The structure of PytH is composed of a core domain and a lid domain; some hydrophobic amino acid residues surrounding the substrate from both domains form a deeper and wider hydrophobic pocket than its homologous structures. This indicates that the larger hydrophobic pocket makes PytH fit for its larger substrates binding; both lid and core domains are involved in substrate binding and the lid domain induced core domain movement may make the active center correctly positioned with substrates.IMPORTANCE Pyrethroid pesticides are widely applied in agriculture and household, however, extensive use of these pesticides also causes serious environmental and health problems. The hydrolysis of pyrethroids by carboxylesterases is the major pathway of microbial degradation of pyrethroids, but the structure of carboxylesterases and its catalytic mechanism are still unknown. Carboxylesterase PytH from Sphingobium faniae JZ-2 could effectively hydrolyze a wide range of pyrethroid pesticides. The crystal structures of PytH are solved in this study. It showed that it belongs to the alpha/beta-hydrolase fold proteins with typical catalytic Ser-His-Asp triad though PytH has a low sequence identity (about 20%) with them. The special large hydrophobic binding pocket endowed PytH binding bigger pyrethroids family substrates. Our structures shed light on the substrate selectivity and the future application of PytH and deeper the understanding of alpha/beta-hydrolase members.
Title: Discovery and development of a novel short-chain fatty acid ester synthetic biocatalyst under aqueous phase from Monascus purpureus isolated from Baijiu Xu Y, Wang X, Liu X, Li X, Zhang C, Li W, Sun X, Wang W, Sun B Ref: Food Chem, 338:128025, 2020 : PubMed
Short-chain fatty acid esters are important flavor chemicals in Chinese traditional fermented Baijiu. Monascus purpureus was recognized as an important microorganism contributing to ester synthesis. However, the molecular basis for ester synthesis was still lacking. The present work combined genome sequencing, transcriptome sequencing, gene library construction, and enzyme engineering to discover a novel catalyst from M. purpureus (isolated from Baijiu fermentation starter). Enzyme LIP05, belonging to the alpha/beta hydrolase family, was identified to synthesize short-chain fatty acid esters under aqueous phase. After deleting the lid domain of LIP05, the synthesis of ethyl pentanoate, ethyl hexanoate, ethyl octanoate, or ethyl decanoate was achieved. Ethyl octanoate with the highest conversion ratio of 93.7% was obtained with the assistance of ultrasound. The study reveals the molecular basis for synthesizing short-chain fatty acid esters by M. purpureus and will promote the application of the species or the enzyme in food industry.
Title: Biodegradation of phthalate esters by Paracoccus kondratievae BJQ0001 isolated from Jiuqu (Baijiu fermentation starter) and identification of the ester bond hydrolysis enzyme Xu Y, Minhazul K, Wang X, Liu X, Li X, Meng Q, Li H, Zhang C, Sun X, Sun B Ref: Environ Pollut, 263:114506, 2020 : PubMed
Phthalate ester (PAE) pollution is an increasing problem globally. Paracoccus kondratievae BJQ0001 was isolated from the fermentation starter of Baijiu and showed an efficient degradation capability toward PAEs. To our poor knowledge, this is the first report of a P. kondratievae strain capable of degrading PAEs. The first complete genome sequence of P. kondratievae was presented without gaps, and composed of two circular chromosomes and one plasmid. The species simultaneously degraded di-methyl phthalate (DMP), di-ethyl phthalate (DEP), di-butyl phthalate (DBP), di-isobutyl phthalate (DIBP) and di-(2-ethylhexyl) phthalate (DEHP), with DMP and DEP as the preferred substrates. The half-life (t(1/2)) of DMP was only 6.34 h with an initial concentration of 200 mg/L. Combined with gene annotation and metabolic intermediate analysis, a metabolic pathway was proposed for the species. Benzoic acid, the intermediate of anaerobic PAE metabolism, was identified in the aerobic degradation process. Two key enzymes for alkyl ester bond hydrolysis were obtained, and belonged to families IV and VI of hydrolases, respectively. These results will promote the investigation of PAE degradation by P. kondratievae, and provide useful information for improving the quality control of food and environmental PAE treatment.
Alzheimer's disease (AD) is a multifaceted and progressive neurodegenerative disease characterized by accumulation of amyloid-beta (Abeta) and deficits of acetylcholine. Accordingly, the intra-/extra-cerebral level of high density lipoprotein (HDL) is crucial on the pathogenesis of AD; and most of all, various HDL-protein subtypes play a double-edged role in AD pathology, of which apolipoprotein A-I (apoA-I) gives protective outcomes. Inspired from "HDL bionics", we proposed biologically reassembled nanodrugs, donepezil-loaded apolipoprotein A-I-reconstituted HDL (rHDL/Do) that concurrently executed dual-missions of Abeta-targeting clearance and acetylcholinesterase (AChE) inhibition in AD therapy. Once prepared, rHDL/Do nanodrug achieved high drug encapsulation efficiency of 90.47%, and mimicked the configurations and properties of natural lipoproteins aiming to significantly enhance BBB penetration and modulate Abeta-induced neuronal damage both in vitro and in vivo. Surface plasmon resonance (SPR) analysis confirmed that rHDL/Do facilitated microglial-mediated Abeta intake and degradation, demonstrating low KD value with Abeta affinity (2.45x10(-8) of Abeta monomer and 2.78x10(-8) of Abeta oligomer). In AD animal models, daily treatment of rHDL/Do efficiently inhibited AChE activity, ameliorated neurologic variation, promoted Abeta clearance, and rescued memory loss at a safe level. The collective findings indicated that the biological nanodrug was provided with the capacities of BBB penetration, Abeta capture and degradation via microglial cells, and cholinergic dysfunction amelioration after controlled donepezil release. In summary, rHDL/Do nanodrugs could offer a promising strategy to synergize both symptom control and disease modification in AD therapy.
Title: Amaryllidaceae alkaloids with new framework types from Zephyranthes candida as potent acetylcholinesterase inhibitors Zhan G, Liu J, Zhou J, Sun B, Aisa HA, Yao G Ref: Eur Journal of Medicinal Chemistry, 127:771, 2017 : PubMed
Three new Amaryllidaceae alkaloids, named zephycandidines I-III (1-3), were isolated from Zephyranthes candida. The structures of 1-3 were elucidated by spectroscopic analyses including HRESIMS, 1H NMR, 13C NMR, DEPT, HSQC, 1H-1H COSY, HMBC, ROESY, and electronic circular dichroism (ECD), as well as ECD calculation. The absolute configuration of 1 was finally established by single crystal X-ray diffraction using Cu Kalpha radiation. Zephycandidines I (1) and III (3) with new framework types represent the first example of 7-phenyl-hexahydroindole and 5,2'-dimethyl-biphenyl-2-ylamine alkaloids, respectively, and their plausible biosynthetic pathway are proposed. Zephycandidine II (2) is the first C3a-phenyl-hexahydroindole type alkaloid isolated from the genus of Zephyranthes. These new alkaloids 1-3 were evaluated for their acetylcholinesterase (AChE) inhibitory activities, and 3 showed potent AChE inhibitory activity with an IC50 value of 8.82 muM, suggesting that the framework of 5,2'-dimethyl-biphenyl-2-ylamine in 3 may be a potential group for the AChE inhibitory activity. The docking studies of 1-3 and galanthamine with AChE revealed that interactions with W286 and Y337 are necessary for the AChE inhibitory activity.
Title: Neuroprotective Effects of Acetylcholinesterase Inhibitory Peptides from Anchovy (Coilia mystus) against Glutamate-Induced Toxicity in PC12 Cells Zhao T, Su G, Wang S, Zhang Q, Zhang J, Zheng L, Sun B, Zhao M Ref: Journal of Agricultural and Food Chemistry, 65:11192, 2017 : PubMed
Ameliorations of cholinergic system dysfunction and oxidative stress in neurodegenerative diseases were main approaches to improve memory disorder. Our previous investigation showed that anchovy protein hydrolysate (APH) could attenuate scopolamine-induced memory deficits in mice by regulating acetylcholinesterase (AChE) activity. Therefore, peptides with AChE inhibitory activity in APH were explored and identified in this study, and their possible neuroprotective mechanisms on glutamate induced apoptosis in PC12 were also elucidated. Two peptides with strong AChE inhibitory capacity were identified as Pro-Ala-Tyr-Cys-Ser (PAYCS) and Cys-Val-Gly-Ser-Tyr (CVGSY) by ultraperformance liquid chromatography coupled with tandem mass spectrometry. The AChE inhibitory was 23.68 +/- 0.97% and 6.08 +/- 0.41%, respectively. Treatment with PAYCS and CVGSY could significantly (p < 0.05) increase cells viability, reduce lactate dehydrogenase release, reactive oxygen species (ROS) production, malondialdehyde content, and the ratio of Bax/Bcl-2 of glutamate-induced apoptosis PC12 cells (82.78 +/- 6.58 and 109.94 +/- 7.16% of control, respectively) as well as increase superoxide dismutase and GSH-px activities. In addition, both the peptides could inhibit Ca(2+) influx but have no effects on mitochondrial membrane potential. Results indicated that AChE inhibitory peptides (PAYCS and CVGSY) possibly protected the PC12 cells against glutamate-induced apoptosis via inhibiting ROS production and Ca(2+) influx. PAYCS and CVGSY might be considered as nutraceuticals for alleviating memory deficits.
Title: Zephycandidine A, the First Naturally Occurring Imidazo[1,2-f]phenanthridine Alkaloid from Zephyranthes candida, Exhibits Significant Anti-tumor and Anti-acetylcholinesterase Activities Zhan G, Qu X, Liu J, Tong Q, Zhou J, Sun B, Yao G Ref: Sci Rep, 6:33990, 2016 : PubMed
Zephycandidine A (1), the first naturally occurring imidazo[1,2-f]phenanthridine alkaloid, was isolated from Zephyranthes candida (Amaryllidaceae). The structure of 1 was elucidated by spectroscopic analyses and NMR calculation, and a plausible biogenetic pathway for zephycandidine A (1) was proposed. Zephycandidine A (1) exhibited significant cytotoxicity against five cancer cell lines with IC50 values ranging from 1.98 to 7.03 muM with selectivity indices as high as 10 when compared to the normal Beas-2B cell. Further studies suggested that zephycandidine A (1) induces apoptosis in leukemia cells by the activation of caspase-3, upregulation of Bax, downregulation of Bcl-2, and degradation of PARP expression. In addition, zephycandidine A (1) showed acetylcholinesterase (AChE) inhibitory activity, and the docking studies of zephycandidine A (1) and galanthamine (2) with AChE revealed that interactions with W286 and Y337 are necessary.
The large yellow croaker Larimichthys crocea (L. crocea) is one of the most economically important marine fish in China and East Asian countries. It also exhibits peculiar behavioral and physiological characteristics, especially sensitive to various environmental stresses, such as hypoxia and air exposure. These traits may render L. crocea a good model for investigating the response mechanisms to environmental stress. To understand the molecular and genetic mechanisms underlying the adaptation and response of L. crocea to environmental stress, we sequenced and assembled the genome of L. crocea using a bacterial artificial chromosome and whole-genome shotgun hierarchical strategy. The final genome assembly was 679 Mb, with a contig N50 of 63.11 kb and a scaffold N50 of 1.03 Mb, containing 25,401 protein-coding genes. Gene families underlying adaptive behaviours, such as vision-related crystallins, olfactory receptors, and auditory sense-related genes, were significantly expanded in the genome of L. crocea relative to those of other vertebrates. Transcriptome analyses of the hypoxia-exposed L. crocea brain revealed new aspects of neuro-endocrine-immune/metabolism regulatory networks that may help the fish to avoid cerebral inflammatory injury and maintain energy balance under hypoxia. Proteomics data demonstrate that skin mucus of the air-exposed L. crocea had a complex composition, with an unexpectedly high number of proteins (3,209), suggesting its multiple protective mechanisms involved in antioxidant functions, oxygen transport, immune defence, and osmotic and ionic regulation. Our results reveal the molecular and genetic basis of fish adaptation and response to hypoxia and air exposure. The data generated by this study will provide valuable resources for the genetic improvement of stress resistance and yield potential in L. crocea.
Genetic sex determination by W and Z chromosomes has developed independently in different groups of organisms. To better understand the evolution of sex chromosomes and the plasticity of sex-determination mechanisms, we sequenced the whole genomes of a male (ZZ) and a female (ZW) half-smooth tongue sole (Cynoglossus semilaevis). In addition to insights into adaptation to a benthic lifestyle, we find that the sex chromosomes of these fish are derived from the same ancestral vertebrate protochromosome as the avian W and Z chromosomes. Notably, the same gene on the Z chromosome, dmrt1, which is the male-determining gene in birds, showed convergent evolution of features that are compatible with a similar function in tongue sole. Comparison of the relatively young tongue sole sex chromosomes with those of mammals and birds identified events that occurred during the early phase of sex-chromosome evolution. Pertinent to the current debate about heterogametic sex-chromosome decay, we find that massive gene loss occurred in the wake of sex-chromosome 'birth'.
Title: Neurotoxicity and Mode of Action of N, N-Diethyl-Meta-Toluamide (DEET) Swale DR, Sun B, Tong F, Bloomquist JR Ref: PLoS ONE, 9:e103713, 2014 : PubMed
Recent studies suggest that N, N-diethyl-meta-toluamide (DEET) is an acetylcholinesterase inhibitor and that this action may result in neurotoxicity and pose a risk to humans from its use as an insect repellent. We investigated the mode of action of DEET neurotoxicity in order to define the specific neuronal targets related to its acute toxicity in insects and mammals. Although toxic to mosquitoes (LD50 ca. 1.5 microg/mg), DEET was a poor acetylcholinesterase inhibitor (<10% inhibition), even at a concentration of 10 mM. IC50 values for DEET against Drosophila melanogaster, Musca domestica, and human acetylcholinesterases were 6-12 mM. Neurophysiological recordings showed that DEET had excitatory effects on the housefly larval central nervous system (EC50: 120 microM), but was over 300-fold less potent than propoxur, a standard anticholinesterase insecticide. Phentolamine, an octopamine receptor antagonist, completely blocked the central neuroexcitation by DEET and octopamine, but was essentially ineffective against hyperexcitation by propoxur and 4-aminopyridine, a potassium channel blocker. DEET was found to illuminate the firefly light organ, a tissue utilizing octopamine as the principal neurotransmitter. Additionally, DEET was shown to increase internal free calcium via the octopamine receptors of Sf21 cells, an effect blocked by phentolamine. DEET also blocked Na+ and K+ channels in patch clamped rat cortical neurons, with IC50 values in the micromolar range. These findings suggest DEET is likely targeting octopaminergic synapses to induce neuroexcitation and toxicity in insects, while acetylcholinesterase in both insects and mammals has low (mM) sensitivity to DEET. The ion channel blocking action of DEET in neurons may contribute to the numbness experienced after inadvertent application to the lips or mouth of humans.
Title: Transgenic CGI-58 expression in macrophages alleviates the atherosclerotic lesion development in ApoE knockout mice Xie P, Zeng X, Xiao J, Sun B, Yang D Ref: Biochimica & Biophysica Acta, 1841:1683, 2014 : PubMed
Comparative Gene Identification-58 (CGI-58), as an adipose triglyceride lipase (ATGL) activator, strongly increases ATGL-mediated triglyceride (TG) catabolism. Previous studies have shown that CGI-58 affects intestinal cholesterol homeostasis independently of ATGL activity. Therefore, we hypothesized that CGI-58 was involved in macrophage cholesterol metabolism and consequently atherosclerotic lesion formation. Here, we generated macrophage-specific CGI-58 transgenic mice (Mac-CGI-58 Tg) using an SRA promoter, which was further mated with ApoE-/- mice to create litters of CGI-58 Tg/ApoE-/- mice. These CGI-58 Tg/ApoE-/- mice exhibited an anti-atherosclerosis phenotype compared with wild type (WT) controls (CGI-58 WT/ApoE-/-), illustrated by less plaque area in aortic roots. Moreover, macrophage-specific CGI-58 overexpression in mice resulted in up-regulated levels of plasma total cholesterol and HDL-cholesterol. Consequently, higher expression levels of PPARa, PPARgamma, LXRalpha, ABCA1, and ABCG1 were detected in macrophages from CGI-58 Tg/ApoE-/- mice compared to CGI-58 WT/ApoE-/- counterparts, which were accompanied by elevated macrophage cholesterol efflux toward HDL and Apo A1. Nevertheless, serum levels of TNF-alpha and IL-6 were reduced by macrophage-specific CGI-58 overexpression. Finally, bone marrow (BM) transplantation experiments further revealed that ApoE-/- mice reconstituted with Mac-CGI-58 Tg BM cells (ApoE-/-/Tg-BM chimera) displayed a significant reduction of atherosclerosis lesions compared with control mice reconstituted with Mac-CGI-58 WT BM cells (ApoE-/-/WT-BM chimera). Collectively, these data strongly suggest that CGI-58 overexpression in macrophages may protect against atherosclerosis development in mice.
Title: Marsupellins A-F, ent-longipinane-type sesquiterpenoids from the Chinese liverwort Marsupella alpine with acetylcholinesterase inhibitory activity Zhang J, Fan P, Zhu R, Li R, Lin Z, Sun B, Zhang C, Zhou J, Lou H Ref: Journal of Natural Products, 77:1031, 2014 : PubMed
Acetylcholinesterase (AChE) inhibitory activity-guided fractionation of the Chinese liverwort Marsupella alpine afforded six new [marsupellins A-F (1-6)] and three known (7-9) ent-longipinane-type sesquiterpenoids. The structures were determined from MS and NMR spectroscopic data, single-crystal X-ray diffraction, and electronic circular dichroism calculations. Compounds 1-9 exhibited moderate to weak AChE inhibitory activity.
Title: Chemical Analog-to-Digital Signal Conversion Based on Robust Threshold Chemistry and Its Evaluation in the Context of Microfluidics-Based Quantitative Assays Huynh T, Sun B, Li L, Nichols KP, Koyner JL, Ismagilov RF Ref: J Am Chem Soc, 135:14775, 2013 : PubMed
In this article, we describe a nonlinear threshold chemistry based on enzymatic inhibition and demonstrate how it can be coupled with microfluidics to convert a chemical concentration (analog input) into patterns of ON or OFF reaction outcomes (chemical digital readout). Quantification of small changes in concentration is needed in a number of assays, such as that for cystatin C, where a 1.5-fold increase in concentration may indicate the presence of acute kidney injury or progression of chronic kidney disease. We developed an analog-to-digital chemical signal conversion that gives visual readout and applied it to an assay for cystatin C as a model target. The threshold chemistry is based on enzymatic inhibition and gives sharper responses with tighter inhibition. The chemistry described here uses acetylcholinesterase (AChE) and produces an unambiguous color change when the input is above a predetermined threshold concentration. An input gives a pattern of ON/OFF responses when subjected to a monotonic sequence of threshold concentrations, revealing the input concentration at the point of transition from OFF to ON outcomes. We demonstrated that this threshold chemistry can detect a 1.30-fold increase in concentration at 22 degrees C and that it is robust to experimental fluctuations: it provided the same output despite changes in temperature (22-34 degrees C) and readout time (10-fold range). We applied this threshold chemistry to diagnostics by coupling it with a traditional sandwich immunoassay for serum cystatin C. Because one quantitative measurement comprises several assays, each with its own threshold concentration, we used a microfluidic SlipChip device to process 12 assays in parallel, detecting a 1.5-fold increase (from 0.64 (49 nM) to 0.96 mg/L (74 nM)) of cystatin C in serum. We also demonstrated applicability to analysis of patient serum samples and the ability to image results using a cell phone camera. This work indicates that combining developments in nonlinear chemistries with microfluidics may lead to development of user-friendly diagnostic assays with simple readouts.
Title: Identification of novel esterase from metagenomic library of Yangtze river Wu C, Sun B Ref: J Microbiol Biotechnol, 19:187, 2009 : PubMed
A metagenomic library of surface-water microbes from the Yangtze River in China was constructed, and a novel esterase, designated as EstY, was isolated and characterized. EstY had 423 amino acids with an estimated molecular mass of 44 kDa and pI of 7.28. It hydrolyzed various pnitrophenyl esters (acetate, butyrate, caprate, caprylate, laurate, myristate, and palmitate) and its best substrate was p-nitrophenyl caprate (C8). The optimum pH for EstY activity was 9.0 and the optimum temperature was 50 degrees . Metal ions, such as Mn2+, Co2+, Hg2+, Zn2+, and Fe3+, strongly inhibited the activity of EstY, whereas Mg2+ was required for maximal activity. Activity remained in the presence of 10% alcohol, acetone, isopropanol, and dimethyl sulfoxide, respectively. An analysis of the amino acid sequence deduced from estY revealed that it had 7 closely related lipolytic enzymes. Moreover, a sequence analysis showed that EstY, like its 7 relatives, did not belong to any known lipolytic enzyme family.
Protein palmitoylation is a reversible lipid modification that regulates membrane tethering for key proteins in cell signaling, cancer, neuronal transmission, and membrane trafficking. Palmitoylation has proven to be a difficult study: Specifying consensuses for predicting palmitoylation remain unavailable, and first-example palmitoylation enzymes--i.e., protein acyltransferases (PATs)--were identified only recently. Here, we use a new proteomic methodology that purifies and identifies palmitoylated proteins to characterize the palmitoyl proteome of the yeast Saccharomyces cerevisiae. Thirty-five new palmitoyl proteins are identified, including many SNARE proteins and amino acid permeases as well as many other participants in cellular signaling and membrane trafficking. Analysis of mutant yeast strains defective for members of the DHHC protein family, a putative PAT family, allows a matching of substrate palmitoyl proteins to modifying PATs and reveals the DHHC family to be a family of diverse PAT specificities responsible for most of the palmitoylation within the cell.
Title: Microsomal epoxide hydrolase, endotoxin, and lung function decline in cotton textile workers Hang J, Zhou W, Wang X, Zhang H, Sun B, Dai H, Su L, Christiani DC Ref: American Journal of Respiratory & Critical Care Medicine, 171:165, 2005 : PubMed
Occupational exposure to endotoxin in organic dust may induce lung function decline. Microsomal epoxide hydrolase (mEH) detoxifies reactive oxygen species generated by endotoxin exposure, and polymorphisms of the mEH gene are associated with altered enzyme activity. We investigated the associations between mEH polymorphisms, endotoxin exposure, and lung function decline in a 20-year prospective study of 265 workers exposed to endotoxin and 234 control subjects. mEH Tyr113His and His139Arg polymorphisms were genotyped by the 5' nuclease assay, and data were analyzed using multivariate linear regression models, adjusting for important covariates. Overall, the annual decline rate of FEV1 was 29.47 ml during the 20-year follow-up. Endotoxin exposure was associated with faster lung function decline among genotypes associated with slower enzyme activity: estimates (SE) of annual FEV1 decline rates for endotoxin exposure were -2.33 (2.07), -2.81 (1.66), and -6.73 (2.83) ml for Tyr/Tyr, Tyr/His, and His/His genotype groups, respectively, for the Tyr113His polymorphism; and -1.82 (2.58) and -4.27 (1.33) ml for Arg/Arg + His/Arg and His/His genotypes, respectively, for the His139Arg polymorphism. We conclude that mEH polymorphisms modify the association between occupational endotoxin exposure and longitudinal lung function decline.
Title: [Determination enzyme protein of CK-MB m-AST and ChE by immunological methods and survey of its applying values (abstract)] Kang X, Sun B, Sun S, Hou W, Xie F, Rong M, Sun R Ref: Rinsho Byori, 46:713, 1998 : PubMed
In recent decades, because considerable progress has been made due to rapid developments in basic theory and techniques in molecular biology and immunology, the determination of trace enzyme proteins is not difficult. We measured the serum concentration of Creatine kinase-MB (CK-MB) mitochondria aspartate aminotransferase (m-AST) and cholinesterase (ChE) immunologically and compared these findings with those of an assay of enzyme activity. Purification of enzyme protein and preparation of serum antibodies monoclonal antibodies established the immunological assay methods. Equipment and reagents for enzyme activity test use 7150 Biochemical Analyzer. CK-NAC AST and ChE were produced by trace kits (Australia). CK-MB and m-AST use immunological inhibition method. CK-MB m-AST ChE of protein determination used immunological turbidimetry. The normal group included 150 cases and the 1990 patient group. Results of the two methods did not significantly differ for normal controls, but were significantly different in the patient group. These results demonstrated that the two methods differ, although each may have specific clinical significance. How to evaluate these differences needs to be studied further, but immunological assay uses higher values for clinical diagnosis than enzyme activity assay.
