Title: Soluble Epoxide Hydrolase Inhibition Protected against Diabetic Cardiomyopathy through Inducing Autophagy and Reducing Apoptosis Relying on Nrf2 Upregulation and Transcription Activation Fang Q, Liu X, Ding J, Zhang Z, Chen G, Du T, Wang Y, Xu R Ref: Oxid Med Cell Longev, 2022:3773415, 2022 : PubMed
BACKGROUND: Many patients with diabetes die from diabetic cardiomyopathy (DCM); however, effective strategies for the prevention or treatment of DCM have not yet been clarified. METHODS: Leptin receptor-deficient (db/db) mice were treated with either the soluble epoxide hydrolase (sEH) inhibitor AUDA or vehicle alone. A virus carrying Nrf2 shRNA was used to manipulate Nrf2 expression in db/db mice. Cardiac structures and functions were analyzed using echocardiography and hemodynamic examinations. Primary cardiomyocytes cultured under high glucose and high fat (HGHF) conditions were used to conduct in vitro loss-of-function assays after culture in the presence or absence of AUDA (1 microM). Fluorescence microscopy-based detection of mCherry-GFP-LC3 was performed to assess autophagic flux. RESULTS: The sEH inhibitor AUDA significantly attenuated ventricular remodeling and ameliorated cardiac dysfunction in db/db mice. Interestingly, AUDA upregulated Nrf2 expression and promoted its nuclear translocation in db/db mice and the HGHF-treated cardiomyocytes. Additionally, AUDA increased autophagy and decreased apoptosis in db/db mice heart. Furthermore, the administration of AUDA promoted autophagic flux and elevated LC3-II protein level in the presence of bafilomycin A1. However, AUDA-induced autophagy was abolished, and the antiapoptotic effect was partially inhibited upon Nrf2 knockdown. CONCLUSION: Our findings suggest that the sEH inhibitor AUDA attenuates cardiac remodeling and dysfunction in DCM via increasing autophagy and reducing apoptosis, which is relevant to activate Nrf2 signaling pathway.
Title: Display of a novel carboxylesterase CarCby on Escherichia coli cell surface for carbaryl pesticide bioremediation Liu Y, Wang X, Nong S, Bai Z, Han N, Wu Q, Huang Z, Ding J Ref: Microb Cell Fact, 21:97, 2022 : PubMed
BACKGROUND: Carbamate pesticides have been widely used in agricultural and forestry pest control. The large-scale use of carbamates has caused severe toxicity in various systems because of their toxic environmental residues. Carbaryl is a representative carbamate pesticide and hydrolase/carboxylesterase is the initial and critical enzyme for its degradation. Whole-cell biocatalysts have become a powerful tool for environmental bioremediation. Here, a whole cell biocatalyst was constructed by displaying a novel carboxylesterase/hydrolase on the surface of Escherichia coli cells for carbaryl bioremediation. RESULTS: The carCby gene, encoding a protein with carbaryl hydrolysis activity was cloned and characterized. Subsequently, CarCby was displayed on the outer membrane of E. coli BL21(DE3) cells using the N-terminus of ice nucleation protein as an anchor. The surface localization of CarCby was confirmed by SDS-PAGE and fluorescence microscopy. The optimal temperature and pH of the engineered E. coli cells were 30 degreesC and 7.5, respectively, using pNPC4 as a substrate. The whole cell biocatalyst exhibited better stability and maintained approximately 8-fold higher specific enzymatic activity than purified CarCby when incubated at 30 degreesC for 120 h. In addition, ~ 100% and 50% of the original activity was retained when incubated with the whole cell biocatalyst at 4 degC and 30 degreesC for 35 days, respectively. However, the purified CarCby lost almost 100% of its activity when incubated at 30 degreesC for 134 h or 37 degreesC for 96 h, respectively. Finally, approximately 30 mg/L of carbaryl was hydrolyzed by 200 U of the engineered E. coli cells in 12 h. CONCLUSIONS: Here, a carbaryl hydrolase-containing surface-displayed system was first constructed, and the whole cell biocatalyst displayed better stability and maintained its catalytic activity. This surface-displayed strategy provides a new solution for the cost-efficient bioremediation of carbaryl and could also have the potential to be used to treat other carbamates in environmental bioremediation.
Endocannabinoid (eCB), 2-arachidonoyl-glycerol (2-AG), the most abundant eCB in the brain, regulates diverse neural functions. Here we linked multiple homozygous loss-of-function mutations in 2-AG synthase diacylglycerol lipase beta (DAGLB) to an early onset autosomal recessive Parkinsonism. DAGLB is the main 2-AG synthase in human and mouse substantia nigra (SN) dopaminergic neurons (DANs). In mice, the SN 2-AG levels were markedly correlated with motor performance during locomotor skill acquisition. Genetic knockdown of Daglb in nigral DANs substantially reduced SN 2-AG levels and impaired locomotor skill learning, particularly the across-session learning. Conversely, pharmacological inhibition of 2-AG degradation increased nigral 2-AG levels, DAN activity and dopamine release and rescued the locomotor skill learning deficits. Together, we demonstrate that DAGLB-deficiency contributes to the pathogenesis of Parkinsonism, reveal the importance of DAGLB-mediated 2-AG biosynthesis in nigral DANs in regulating neuronal activity and dopamine release, and suggest potential benefits of 2-AG augmentation in alleviating Parkinsonism.
Pyrethroids are the third widespread used insecticides globally which have been extensively applied in agricultural or household environments. Due to continuous applications, pyrethroids have been detected both in living cells and environments. The permanent exposure to pyrethroids have caused substantial health risks and ecosystem concerns. In this work, a lambda-cyhalothrin (one kind of pyrethroid insecticides) degrading bacterium Bacillus velezensis sd was isolated and a carboxylesterase gene, CarCB2 was characterized. A whole cell biocatalyst was developed for lambda-cyhalothrin biodegradation by displaying CarCB2 on the surface of Escherichia coli cells. CarCB2 was successfully displayed and functionally expressed on E. coli cells with optimal pH and temperature of 7.5 and 30 degreesC, using p-NPC(4) as substrate, respectively. The whole cell biocatalyst exhibited better stability than the purified CarCB2, and approximately 120%, 60% or 50% of its original activity at 4 degreesC, 30 degreesC or 37 degreesC over a period of 35 d was retained, respectively. No enzymatic activity was detected when incubated the purified CarCB2 at 30 degreesC for 120 h, or 37 degreesC for 72 h, respectively. Additionally, 30 mg/L of lambda-cyhalothrin was degraded in citrate-phosphate buffer by 10 U of the whole cell biocatalyst in 150 min. This work reveals that the whole cell biocatalyst affords a promising approach for efficient biodegradation of lambda-cyhalothrin, and might have the potential to be applied in further environmental bioremediation of other different kinds of pyrethroid insecticides.
Title: TPPU Pre-Treatment Rescues Dendritic Spine Loss and Alleviates Depressive Behaviours during the Latent Period in the Lithium Chloride-Pilocarpine-Induced Status Epilepticus Rat Model Peng W, Shen Y, Wang Q, Ding J, Wang X Ref: Brain Sci, 11:, 2021 : PubMed
Epileptogenesis may be responsible for both of recurrent seizures and comorbid depression in epilepsy. Disease-modifying treatments targeting the latent period before spontaneous recurrent seizures may contribute to the remission of seizures and comorbid depression. We hypothesized that pre-treatment with 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU), a soluble epoxide hydrolase (sEH) inhibitor, which has anti-inflammatory and neuroprotective effects might rescue status epilepticus (SE)-induced dendritic spine loss and alleviate depressive behaviours. Rats were either pre-treated with TPPU (0.1 mg/kg/d) intragastrically or with vehicle (40% polyethylene glycol 400) from 7 days before to 7 days after SE that was induced with lithium chloride and pilocarpine intraperitoneally. Rats in the Control group were given saline instead. The forced swim test (FST) was performed on the 8th day after SE to evaluate the depression-like behaviours in rats. The results showed that seizures severity during SE was significantly decreased, and the immobility time during FST was significantly increased through TPPU pre-treatment. Moreover, pre-treatment with TPPU attenuated inflammations including microglial gliosis and the level of proinflammatory cytokine IL-1beta in the hippocampus; in addition, neuronal and dendritic spine loss in the subfields of hippocampus was selectively rescued, and the expression of NR1 subunit of N-methyl-D-aspartate (NMDA) receptor, ERK1/2, CREB, and their phosphorylated forms involved in the dendritic spine development were all significantly increased. We concluded that pre-treatment with TPPU attenuated seizures severity during SE and depressive behaviours during the period of epileptogenesis probably by rescuing dendritic spine loss in the hippocampus.
Title: Ensemble machine learning to evaluate the in vivo acute oral toxicity and in vitro human acetylcholinesterase inhibitory activity of organophosphates Wang L, Ding J, Shi P, Fu L, Pan L, Tian J, Cao D, Jiang H, Ding X Ref: Archives of Toxicology, :, 2021 : PubMed
Organophosphates (OPs) are hazardous chemicals widely used in industry and agriculture. Distribution of their residues in nature causes serious risks to humans, animals, and plants. To reduce hazards from OPs, quantitative structure-activity relationship (QSAR) models for predicting their acute oral toxicity in rats and mice and inhibition constants concerning human acetylcholinesterase were developed according to the bioactivity data of 456 unique OPs. Based on robust, two-dimensional molecular descriptors and quantum chemical descriptors, which accurately reflect OP electronic structures and reactivities, the influences of eight machine-learning algorithms on the prediction performance of the QSAR models were explored, and consensus QSAR models were constructed. Several strict model validation indices and the results of applicability domain evaluations show that the established consensus QSAR models exhibit good robustness, practical prediction abilities, and wide application scopes. Poor correlation was observed between acute oral toxicity at the mammalian level and the inhibition constants at the molecular level, indicating that the acute toxicity of OPs cannot be evaluated only by the experimental data of enzyme inhibitory activity, their toxicokinetic characteristics must also be considered. The constructed QSAR models described herein provide rapid, theoretical assessment of the bioactivity of unstudied or unknown OPs, as well as guidance for making decisions regarding their regulation.
Title: Toxicological effects of nano- and micro-polystyrene plastics on red tilapia: Are larger plastic particles more harmless? Ding J, Huang Y, Liu S, Zhang S, Zou H, Wang Z, Zhu W, Geng J Ref: J Hazard Mater, 396:122693, 2020 : PubMed
Nanoplastics (NPs) and microplastics (MPs) are a heterogeneous class of pollutants with diverse sizes in aquatic environments. To evaluate the hazardous effects of N/MPs with different sizes, the accumulation, oxidative stress, cytochrome P450 (CYP) enzymes, neurotoxicity, and metabolomics changes were investigated in the red tilapia exposed to three sizes of polystyrene (PS) N/MPs (0.3, 5, and 70-90mum). After 14-d exposures, the largest particles (70-90mum) showed the highest accumulation levels in most cases. Exposures to PS-MPs (5 and 70-90mum) caused a more severe oxidative stress in red tilapia than PS-NPs. The activity of CYP3A-related enzyme was obviously inhibited by PS-NPs, whereas the CYP enzymes in the liver may not be sensitive to MP exposures. In the brain, only 5mumPS-MPs significantly inhibited the acetylcholinesterase activity. After exposures, the treatments with 0.3, 5, and 70-90mum N/MPs resulted in 31, 40, and 23 significantly differentially expressed metabolites, respectively, in which the pathway of tyrosine metabolism was significantly affected by all the three PS-N/MP exposures. Overall, the PS particles within the mum size posed more severe stress to red tilapia. Our results suggest that the toxicity of N/MPs may not show a simply monotonic negative correlation with their sizes.
Title: Development of a whole-cell biocatalyst for diisobutyl phthalate degradation by functional display of a carboxylesterase on the surface of Escherichia coli Ding J, Zhou Y, Wang C, Peng Z, Mu Y, Tang X, Huang Z Ref: Microb Cell Fact, 19:114, 2020 : PubMed
BACKGROUND: Phthalic acid esters (PAEs) are widely used as plasticizers or additives during the industrial manufacturing of plastic products. PAEs have been detected in both aquatic and terrestrial environments due to their overuse. Exposure of PAEs results in human health concerns and environmental pollution. Diisobutyl phthalate is one of the main plasticizers in PAEs. Cell surface display of recombinant proteins has become a powerful tool for biotechnology applications. In this current study, a carboxylesterase was displayed on the surface of Escherichia coli cells, for use as whole-cell biocatalyst in diisobutyl phthalate biodegradation. RESULTS: A carboxylesterase-encoding gene (carEW) identified from Bacillus sp. K91, was fused to the N-terminal of ice nucleation protein (inpn) anchor from Pseudomonas syringae and gfp gene, and the fused protein was then cloned into pET-28a(+) vector and was expressed in Escherichia coli BL21(DE3) cells. The surface localization of INPN-CarEW/or INPN-CarEW-GFP fusion protein was confirmed by SDS-PAGE, western blot, proteinase accessibility assay, and green fluorescence measurement. The catalytic activity of the constructed E. coli surface-displayed cells was determined. The cell-surface-displayed CarEW displayed optimal temperature of 45 degrees C and optimal pH of 9.0, using p-NPC2 as substrate. In addition, the whole cell biocatalyst retained ~ 100% and ~ 200% of its original activity per OD600 over a period of 23 days at 45 degrees C and one month at 4 degrees C, exhibiting the better stability than free CarEW. Furthermore, approximately 1.5 mg/ml of DiBP was degraded by 10 U of surface-displayed CarEW cells in 120 min. CONCLUSIONS: This work provides a promising strategy of cost-efficient biodegradation of diisobutyl phthalate for environmental bioremediation by displaying CarEW on the surface of E. coli cells. This approach might also provide a reference in treatment of other different kinds of environmental pollutants by displaying the enzyme of interest on the cell surface of a harmless microorganism.
Title: Characterization of EstZY: A new acetylesterase with 7-aminocephalosporanic acid deacetylase activity from Alicyclobacillus tengchongensis Ding J, Zhou Y, Zhu H, Deng M, Gao Y, Yang Y, Huang Z Ref: Int J Biol Macromol, 148:333, 2020 : PubMed
Deacetyl-7-aminocephalosporanic acid (D-7-ACA) is required for producing of many semisynthetic beta-lactam antibiotics; therefore, enzymes capable of converting 7-aminocephalosporanic acid (7-ACA) to D-7-ACA present a valuable resource to the pharmaceutical industry. In the present study, a putative acetylesterase, EstZY, was identified and characterized from a thermophilic bacterium Alicyclobacillus tengchongensis. Sequence alignment showed that EstZY was an acetylesterase which belonged to carbohydrate esterase family 7 (CE7), with substrate preference for short-chain acyl esters p-NPC(2) to p-NPC(8). Maximum enzyme activity was recorded at pH 9.0 and 50 degreesC, where K(m) and V(max) were calculated as 1.9 +/- 0.23 mM and 258 +/- 18.5 microM min(-)(1), respectively. The residues Ser185, Asp274, and His303 were identified as the putative catalytic triad by homology modelling, site-directed mutagenesis and molecular docking. Moreover, EstZY can remove the acetyl group at C3' position of 7-ACA to form D-7-ACA; this is the first report of a 7-ACA deacetylase from CE7 family in A. tengchongensis and may represent a new enzyme with industrial values.
Title: Design, synthesis and evaluation of quinolinone derivatives containing dithiocarbamate moiety as multifunctional AChE inhibitors for the treatment of Alzheimer's disease Fu J, Bao F, Gu M, Liu J, Zhang Z, Ding J, Xie SS Ref: J Enzyme Inhib Med Chem, 35:118, 2020 : PubMed
A series of novel quinolinone derivatives bearing dithiocarbamate moiety were designed and synthesised as multifunctional AChE inhibitors for the treatment of AD. Most of these compounds exhibited strong and clearly selective inhibition to eeAChE. Among them, compound 4c was identified as the most potent inhibitor to both eeAChE and hAChE (IC50 = 0.22 muM for eeAChE; IC50 = 0.16 muM for hAChE), and it was also the best inhibitor to AChE-induced Abeta aggregation (29.02% at 100 muM) and an efficient inhibitor to self-induced Abeta aggregation (30.67% at 25 muM). Kinetic and molecular modelling studies indicated that compound 4c was a mixed-type inhibitor, which could interact simultaneously with the catalytic anionic site (CAS) and the peripheral anionic site (PAS) of AChE. In addition, 4c had good ability to cross the BBB, showed no toxicity on SH-SY5Y neuroblastoma cells and was well tolerated in mice at doses up to 2500 mg/kg (po).
Title: Lipase-catalyzed synthesis of ethyl (R)-2-benzyloxy-2-isopropylhydrogenmalonate: a useful combination of chemical synthesis with enzymatic methods Ding J, Yang Z, Zhao Y, Fang W, Lu Q Ref: Biosci Biotechnol Biochem, :1, 2019 : PubMed
Ethyl (R)-2-benzyloxy-2-isopropylhydrogenmalonate is a key intermediate for the synthesis of the side chain in ergopeptines. In this work, we adopted a method to prepare enantiomerically pure title monoester via immobilized Candida antarctica lipase B (Novozym 435)-catalyzed hydrolysis of the corresponding diester.
Title: Identification and characterization of an acetyl esterase from Paenibacillus sp. XW-6-66 and its novel function in 7-aminocephalosporanic acid deacetylation Ding J, Zhou Y, Zhu H, Deng M, Long L, Yang Y, Wu Q, Huang Z Ref: Biotechnol Lett, 41:1059, 2019 : PubMed
OBJECTIVES: To obtain a new acetyl esterase from Paenibacillus sp. XW-6-66 and apply the enzyme to 7-aminocephalosporanic acid (7-ACA) deacetylation. RESULTS: The acetyl esterase AesZY was identified from Paenibacillus sp. XW-6-66, and its enzymatic properties were investigated. With the putative catalytic triad Ser114-Asp203-His235, AesZY belongs to the Acetyl esterase (Aes) family which is included in the alpha/beta hydrolase superfamily and contains the consensus Gly-X-Ser-X-Gly motif. The maximum activity of AesZY was detected at pH 8.0 and 40 degrees C. AesZY was stable at different pH values ranging from 5.0 to 12.0, and was tolerant to several metal ions. Furthermore, the deacetylation activity of AesZY toward 7-ACA was approximately 7.5 U/mg, and the Kcat/Km value was 2.04 s(-1) mM(-1). CONCLUSIONS: Our results demonstrate the characterization of a new acetyl esterase belonging to the Aes family with potential biotechnological applications.
Title: Novel chromanone-dithiocarbamate hybrids as multifunctional AChE inhibitors with beta-amyloid anti-aggregation properties for the treatment of Alzheimer's disease Jiang N, Ding J, Liu J, Sun X, Zhang Z, Mo Z, Li X, Yin H, Tang W, Xie SS Ref: Bioorg Chem, 89:103027, 2019 : PubMed
By connecting chromanone with dithiocarbamate moieties through flexible linkers, a series of hybrids as novel multifunctional AChE inhibitors have been designed and synthesized. Most of these compounds displayed strong and excellently selective inhibition to eeAChE as well as potent inhibition to self- and AChE-induced Abeta aggregation. Among them, compound 6c showed the best activity to inhibit eeAChE (IC50=0.10muM) and AChE-induced Abeta aggregation (33.02% at 100muM), and could effectively inhibit self-induced Abeta aggregation (38.25% at 25muM). Kinetic analysis and docking study indicated that compound 6c could target both the CAS and PAS, suggesting that it was a dual binding site inhibitor for AChE. Besides, it exhibited good ability to penetrate the BBB and low neurotoxicity in SH-SY5Y cells. More importantly, compound 6c was well tolerated in mice (2500mg/kg, po) and could attenuate the memory impairment in a scopolamine-induced mouse model. Overall, these results highlight 6c as a promising multifunctional agent for treating AD and also demonstrate that the dithiocarbamate is a valid scaffold for design of multifunctional AChE inhibitors.
Title: Anti-inflammatory treatment with a soluble epoxide hydrolase inhibitor attenuates seizures and epilepsy-associated depression in the LiCl-pilocarpine post-status epilepticus rat model Shen Y, Peng W, Chen Q, Hammock BD, Liu J, Li D, Yang J, Ding J, Wang X Ref: Brain Behavior & Immunity, 81:535, 2019 : PubMed
PURPOSE: This study aimed to investigate whether 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU), a soluble epoxide hydrolase inhibitor with anti-inflammatory effects, could alleviate spontaneous recurrent seizures (SRS) and epilepsy-associated depressive behaviours in the lithium chloride (LiCl)-pilocarpine-induced post-status epilepticus (SE) rat model. METHODS: The rats were intraperitoneally (IP) injected with LiCl (127mg/kg) and pilocarpine (40mg/kg) to induce SE. A video surveillance system was used to monitor SRS in the post-SE model for 6weeks (from the onset of the 2nd week to the end of the 7th week after SE induction). TPPU (0.1mg/kg/d) was intragastrically given for 4weeks from the 21st day after SE induction in the SRS+0.1 TPPU group. The SRS+PEG 400 group was given the vehicle (40% polyethylene glycol 400) instead, and the control group was given LiCl and PEG 400 but not pilocarpine. The sucrose preference test (SPT) and forced swim test (FST) were conducted to evaluate the depression-like behaviours of rats. Immunofluorescent staining, enzyme-linked immunosorbent assay, and western blot analysis were performed to measure astrocytic and microglial gliosis, neuronal loss, and levels of soluble epoxide hydrolase (sEH), cytokines [tumour necrosis factor alpha (TNF-alpha), interleukin (IL)-1beta, and IL-6], and cyclic adenosine monophosphate (cAMP)-response element binding protein (CREB). RESULTS: The frequency of SRS was significantly decreased at 6weeks and 7weeks after SE induction in the 0.1TPP U group compared with the SRS+PEG 400 group. The immobility time (IMT) evaluated by FST was significantly decreased, whereas the climbing time (CMT) was increased, and the sucrose preference rate (SPR) evaluated by SPT was in an increasing trend. The levels of sEH, TNF-alpha, IL-1beta, and IL-6 in the hippocampus (Hip) and prefrontal cortex (PFC) were all significantly increased in the SRS+PEG 400 group compared with the control group; neuronal loss, astrogliosis, and microglial activation were also observed. The astrocytic and microglial activation and levels of the pro-inflammatory cytokines in the Hip and PFC were significantly attenuated in the TPPU group compared with the SRS+PEG 400 group; moreover, neuronal loss and the decreased CREB expression were significantly alleviated as well. CONCLUSION: TPPU treatment after SE attenuates SRS and epilepsy-associated depressive behaviours in the LiCl-pilocarpine induced post-SE rat model, and it also exerts anti-inflammatory effects in the brain. Our findings suggest a new therapeutic approach for epilepsy and its comorbidities, especially depression.
Title: Accumulation, tissue distribution, and biochemical effects of polystyrene microplastics in the freshwater fish red tilapia (Oreochromis niloticus) Ding J, Zhang S, Razanajatovo RM, Zou H, Zhu W Ref: Environ Pollut, 238:1, 2018 : PubMed
While the presence of microplastics (MPs) in marine environments has been detected worldwide, the importance of MPs pollution in freshwater environments has also been emphasized in recent years. However, the body of knowledge regarding the biological effects of MPs on freshwater organisms is still much more limited than on marine organisms. The aim of the present study was to evaluate the accumulation and tissue distribution of MPs in the freshwater fish red tilapia (Oreochromis niloticus), as well as the biochemical effects of MPs on O. niloticus. During 14 days of exposure to 0.1mum polystyrene-MPs at concentrations of 1, 10, and 100mugL(-1), the MPs concentrations in various tissues of O. niloticus generally increased over time following the order gut>gills>liver approximately brain. Moreover, the acetylcholinesterase (AChE) activity in the fish brain was inhibited by MPs exposure, with a maximum inhibition rate of 37.7%, suggesting the potential neurotoxicity of MPs to freshwater fish. The activities of cytochrome P450 (CYP) enzymes [7-ethoxyresorufin O-deethylase (EROD) and 7-benzyloxy-4-trifluoromethyl-coumarin O-dibenzyloxylase (BFCOD)] in the fish liver exhibited clear temporal variabilities, with significant decreases followed by elevations compared to the control. The alterations of the EROD and BFCOD activities indicate the potential involvement of CYP enzymes for the metabolism of MPs. The activity of antioxidative enzyme superoxide dismutase (SOD) in the liver was significantly induced throughout the exposure period, while the malondialdehyde (MDA) content did not vary with MPs exposure, suggesting that the antioxidative enzymatic system in O. niloticus could prevent oxidative damage. These results highlight the ingestion and accumulation of MPs in different tissues of freshwater fish, which lead to perturbations in fish biological systems and should be considered in environmental risk assessment.
A series of new coumarin-dithiocarbamate hybrids were designed and synthesized as multitarget agents for the treatment of Alzheimer's disease. Most of them showed potent and clearly selective inhibition towards AChE and MAO-B. Among these compounds, compound 8f demonstrated the most potent inhibition to AChE with IC50 values of 0.0068muM and 0.0089muM for eeAChE and hAChE, respectively. Compound 8g was identified as the most potent inhibitor to hMAO-B, and it is also a good and balanced inhibitor to both hAChE and hMAO-B (0.114microM for hAChE; 0.101microM for hMAO-B). Kinetic and molecular modeling studies revealed that 8g was a dual binding site inhibitor for AChE and a competitive inhibitor for MAO-B. Further studies indicated that 8g could penetrate the BBB and exhibit no toxicity on SH-SY5Y neuroblastoma cells. More importantly, 8g did not display any acute toxicity in mice at doses up to 2500mg/kg and could reverse the cognitive dysfunction of scopolamine-induced AD mice. Overall, these results highlighted 8g as a potential multitarget agent for AD treatment and offered a starting point for design of new multitarget AChE/MAO-B inhibitors based on dithiocarbamate scaffold.
Title: Decreased vesicular acetylcholine transporter related to memory deficits in epilepsy: A [(18) F] VAT positron emission tomography brain imaging study Wu XQ, Zhao YN, Ding J, Si Z, Cheng DF, Shi HC, Wang X Ref: Epilepsia, 59:1655, 2018 : PubMed
OBJECTIVE: Vesicular acetylcholine transporter (VAChT) is a rate-limiting factor for synaptic acetylcholine transport. Our study focused on whether [(18) F] VAT, a novel positron emission tomography (PET) tracer, could be used in detecting cognitive deficits in epilepsy. METHODS: Morris water maze test was used to evaluate learning and memory deficits in pilocarpine-induced chronic epilepsy rats 12 weeks after status epilepticus. Interictal [(18) F] VAT PET was performed 13 weeks after status epilepticus to evaluate the level of VAChT in cholinergic pathways compared with [(18) F] fluorodeoxyglucose PET. The association between VAChT levels and memory measures was analyzed. Neuropathological tests were performed. RESULTS: Epileptic rats exhibited significant memory deficits in Morris water maze test. [(18) F] VAT uptake decreased in septum, hippocampus, thalamus, and basal forebrain, and correlated to memory function. Of note, the level of VAChT in basal forebrain significantly decreased, yet no glucose hypometabolism was detected. Immunofluorescence and Western blot demonstrated decreased expression of VAChT in hippocampus and basal forebrain in the epilepsy group, but no change of expression of acetyltransferase or activity of acetylcholinesterase was detected. SIGNIFICANCE: [(18) F] VAT PET is a promising method to test the level of VAChT as a valuable biomarker for memory deficits in pilocarpine-induced chronic epileptic rats.
Title: Sublethal effects of chlorfenapyr on the life table parameters, nutritional physiology and enzymatic properties of Bradysia odoriphaga (Diptera: Sciaridae) Zhao Y, Wang Q, Ding J, Wang Y, Zhang Z, Liu F, Mu W Ref: Pestic Biochem Physiol, 148:93, 2018 : PubMed
Bradysia odoriphaga (Diptera: Sciaridae) is the major pest affecting Chinese chive production. Chlorfenapyr is a halogenated pyrrole-based pro-insecticide that is currently used to control insects and mites on a variety of crops. In the present study, fourth-instar larvae of B. odoriphaga were exposed to chlorfenapyr at LC1, LC20 and LC50 concentrations. The developmental duration of the treated larvae was not significantly different, but fecundity was significantly increased in the LC1 and LC20 treatment groups compared with the control group. The population parameters of the LC1 treatment group were increased significantly, whereas those of the LC50 treatment group were reduced significantly compared with the control. The food consumption by larvae and pupal weight were significantly increased under the LC1 treatment and decreased under the LC50 treatment compared with the control. Moreover, chlorfenapyr decreased the lipid, carbohydrate and trehalose contents significantly, whereas the total protein content was increased compared with the control. Additionally, the activities of protease, lipase and trehalase were significantly decreased. Chlorfenapyr treatment for 24h also induced the activities of glutathione S-transferase (GST), carboxylesterase (CarE) and O-demethylation. The results of this study suggest that low lethal concentrations of chlorfenapyr can affect oviposition, population development, the activities of digestion and detoxification enzymes, and nutrient accumulation in B. odoriphaga. This study provides valuable information for the assessment and rational application of chlorfenapyr for effective control of this pest.
Title: Bioconcentration of the antidepressant fluoxetine and its effects on the physiological and biochemical status in Daphnia magna Ding J, Zou H, Liu Q, Zhang S, Mamitiana Razanajatovo R Ref: Ecotoxicology & Environmental Safety, 142:102, 2017 : PubMed
The aim of this study was to evaluate the bioconcentration potential of fluoxetine and its biological effects in Daphnia magna. After 48h of waterborne exposure, the bioconcentration of fluoxetine in D. magna was determined to be 460.61 and 174.41Lkg-1 for nominal exposure concentrations of 0.5 and 5microgL-1, respectively. Moreover, various biological endpoints, including physiological responses (filtration and ingestion rates), enzymatic biomarkers related to neurotoxicity [acetylcholinesterase (AChE)] and antioxidant defense [superoxide dismutase (SOD)], and an oxidative stress damage marker [malondialdehyde (MDA)], were assessed. Fluoxetine exposure increased the filtration rate of daphnia, while the ingestion rate was not obviously modified. AChE activity was significantly inhibited, highlighting the neurotoxicity of fluoxetine on D. magna. However, with some alterations in the SOD activity and MDA content, no obvious oxidative damage was observed in D. magna exposed to fluoxetine at the tested concentrations. These results indicate that fluoxetine can be accumulated and consequently induce physiological and biochemical perturbations in D. magna.
Title: A Three-Dimensional Origami Paper-Based Device for Potentiometric Biosensing Ding J, Li B, Chen L, Qin W Ref: Angew Chem Int Ed Engl, 55:13033, 2016 : PubMed
Current paper-based potentiometric ion-sensing platforms are planar devices used for clinically relevant ions. These devices, however, have not been designed for the potentiometric biosensing of proteins or small molecule analytes. A three-dimensional origami paper-based device, in which a solid-contact ion-selective electrode is integrated with an all-solid-state reference electrode, is described for the first time. The device is made by impregnation of paper with appropriate bioreceptors and reporting reagents on different zones. By folding and unfolding the paper structures, versatile potentiometric bioassays can be performed. A USB-controlled miniaturized electrochemical detector can be used for simple and in situ measurements. Using butyrylcholinesterase as a model enzyme, the device has been successfully applied to the detection of enzyme activities and organophosphate pesticides involved in the enzymatic system as inhibitors. The proposed 3D origami paper device allows the potentiometric biosensing of proteins and small molecules in a simple, portable, and cost-effective way.
Title: Identification of new binding sites of human transferrin incubated with organophosphorus agents via Q Exactive LC-MS/MS Sun F, Ding J, Yu H, Gao R, Wang H, Pei C Ref: Journal of Chromatography B Analyt Technol Biomed Life Sciences, 1022:256, 2016 : PubMed
Organophosphorus agents (OPs) like sarin, VX, or soman could inhibit acetylcholinesterase activity and cause poisoning. OPs could bind many proteins, such as butyrylcholinesterase and albumin, and the adducts formed could identify the exposure. In this paper, we studied human transferrin, which was one of the proteins that could be labeled by OPs. Pure human transferrin was incubated with an overdose of organophosphorus agents, including sarin, soman, VX, tabun, cyclosarin, ethyl tabun, and propyl tabun, and then additional OPs was removed through dialysis. Trypsin was used to cleave the OP-treated proteins and Q Exactive liquid chromatography tandem mass spectrometry (Q Exactive LC-MS/MS) was used to identify them. The present study set out to accomplish two goals. The first goal was to find a good method for identifying multiple binding sites on a given protein through Q Exactive LC-MS/MS. The second goal was to investigate the labeled peptides when transferrin was incubated with a numerous molar excess of OPs. Results showed that tyrosine, lysine, and serine formed covalent bonds with OPs. Twenty OP-labeled sites were found: ten tyrosine sites (including two reported sites), seven lysine sites, and three serine sites. Characteristic fragments for labeled-tyrosine and labeled-lysine adducts were summarized in detail. In conclusion, the method by Q Exactive LC-MS/MS using in this present work is a good way to diagnose exposure to OPs accurately when the binding sites of OPs are uncertain. Novel modified peptides and the characteristic ions found in this work could help investigators assess exposure to OPs.
Title: Identification and Characterization of a New Alkaline SGNH Hydrolase from a Thermophilic Bacterium Bacillus sp. K91 Yu T, Ding J, Zheng Q, Han N, Yu J, Yang Y, Li J, Mu Y, Wu Q, Huang Z Ref: J Microbiol Biotechnol, 26:730, 2016 : PubMed
est19 is a gene from Bacillus sp. K91 that encodes a new esterase. A comparison of the amino acid sequence showed that Est19 has typical Ser-Gly-Asn-His (SGNH) family motifs and could be grouped into the SGNH hydrolase family. The Est19 protein was functionally cloned, and expressed and purified from Escherichia coli BL21(DE3). The enzyme activity was optimal at 60 degrees C and pH 9.0, and displayed esterase activity towards esters with short-chain acyl esters (C(2)-C(6)). A structural model of Est19 was constructed using phospholipase A1 from Streptomyces albidoflavus NA297 as a template. The structure showed an alpha/beta-hydrolase fold and indicated the presence of the typical catalytic triad Ser49-Asp227-His230, which were further investigated by site-directed mutagenesis. To the best of our knowledge, Est19 is a new member of the SGNH hydrolase family identified from thermophiles, which may be applicable in the industrial production of semisynthetic beta-lactam antibiotics after modification.
Resequencing or reference-based assemblies reveal large parts of the small-scale sequence variation. However, they typically fail to separate such local variation into colinear and rearranged variation, because they usually do not recover the complement of large-scale rearrangements, including transpositions and inversions. Besides the availability of hundreds of genomes of diverse Arabidopsis thaliana accessions, there is so far only one full-length assembled genome: the reference sequence. We have assembled 117 Mb of the A. thaliana Landsberg erecta (Ler) genome into five chromosome-equivalent sequences using a combination of short Illumina reads, long PacBio reads, and linkage information. Whole-genome comparison against the reference sequence revealed 564 transpositions and 47 inversions comprising approximately 3.6 Mb, in addition to 4.1 Mb of nonreference sequence, mostly originating from duplications. Although rearranged regions are not different in local divergence from colinear regions, they are drastically depleted for meiotic recombination in heterozygotes. Using a 1.2-Mb inversion as an example, we show that such rearrangement-mediated reduction of meiotic recombination can lead to genetically isolated haplotypes in the worldwide population of A. thaliana Moreover, we found 105 single-copy genes, which were only present in the reference sequence or the Ler assembly, and 334 single-copy orthologs, which showed an additional copy in only one of the genomes. To our knowledge, this work gives first insights into the degree and type of variation, which will be revealed once complete assemblies will replace resequencing or other reference-dependent methods.
Title: Occurrence and risk assessment of organophosphate esters in drinking water from Eastern China Ding J, Shen X, Liu W, Covaci A, Yang F Ref: Sci Total Environ, 538:959, 2015 : PubMed
Organophosphate esters (OPEs) are ubiquitous in the environment and may pose potential health risks to humans. Drinking water is suspected as one possible exposure pathway of OPEs to humans. In this study, we investigated the residues of 9 OPEs in five types of drinking water in Eastern China. The median concentrations of Sigma9OPEs were determined to be 3.99, 4.50, 27.6, 59.2 and 192ng/L in the bottled, well, barreled, direct drinking and tap waters, respectively. Triethyl phosphate (TEP) was the most abundant OPE in the tap water and filtered drinking water with median concentrations of 50.2 and 30.2ng/L, respectively. The mixture of tri(chloropropyl) phosphate (TCPP) and tri(chloroisopropyl) phosphate (TCIPP), named here as TCPP, dominated in the barreled and well water with median concentrations of 8.04 and 2.49ng/L, respectively. The calculated average daily doses of OPEs ranged from 0.14 to 7.07ng/kgbw/day for people consuming the five different types of drinking water. Among the drinking water, the tap water exhibited the highest exposure doses of OPEs. The calculated non-cancer hazard quotients (10(-4)-10(-7)) from OPEs were much lower than the theoretical threshold of risk. The carcinogenic risks posed by TCEP were very low (<10(-7)) for all types of drinking water. The results revealed that there was currently low risk to human health from exposure to OPEs through drinking water in Eastern China.
Title: Properties of a newly identified esterase from Bacillus sp. K91 and its novel function in diisobutyl phthalate degradation Ding J, Wang C, Xie Z, Li J, Yang Y, Mu Y, Tang X, Xu B, Zhou J, Huang Z Ref: PLoS ONE, 10:e0119216, 2015 : PubMed
The widely used plasticizer phthalate esters (PAEs) have become a public concern because of their effects on environmental contamination and toxicity on mammals. However, the biodegradation of PAEs, especially diisobutyl phthalate (DiBP), remains poorly understood. In particular, genes involved in the hydrolysis of these compounds were not conclusively identified. In this study, the CarEW gene, which encodes an enzyme that is capable of hydrolyzing ro-nitrophenyl esters of fatty acids, was cloned from a thermophilic bacterium Bacillus sp. K91 and heterologously expressed in Escherichia coli BL21 using the pEASY-E2 expression system. The enzyme showed a monomeric structure with a molecular mass of approximately 53.76 kDa and pI of 4.88. The enzyme exhibited maximal activity at pH 7.5 and 45 degreesC, with ro-NP butyrate as the best substrate. The enzyme was fairly stable within the pH range from 7.0 to 8.5. High-pressure liquid chromatography (HPLC) and electrospray ionization mass spectrometry (ESI-MS) were employed to detect the catabolic pathway of DiBP. Two intermediate products were identified, and a potential biodegradation pathway was proposed. Altogether, our findings present a novel DiBP degradation enzyme and indicate that the purified enzyme may be a promising candidate for DiBP detoxification and for environmental protection.
Nicotinic acetylcholine receptors (nAChRs) play essential roles in transmitting acetylcholine-mediated neural signals across synapses and neuromuscular junctions, and are also closely linked to various diseases and clinical conditions. Therefore, novel nAChR-specific compounds have great potential for both neuroscience research and clinical applications. Conotoxins, the peptide neurotoxins produced by cone snails, are a rich reservoir of novel ligands that target receptors, ion channels and transporters in the nervous system. From the venom of Conus generalis, we identified a novel dimeric nAChR-inhibiting alphaD-conotoxin GeXXA. By solving the crystal structure and performing structure-guided dissection of this toxin, we demonstrated that the monomeric C-terminal domain of alphaD-GeXXA, GeXXA-CTD, retains inhibitory activity against the alpha9alpha10 nAChR subtype. Furthermore, we identified that His7 of the rat alpha10 nAChR subunit determines the species preference of alphaD-GeXXA, and is probably part of the binding site of this toxin. These results together suggest that alphaD-GeXXA cooperatively binds to two inter-subunit interfaces on the top surface of nAChR, thus allosterically disturbing the opening of the receptor. The novel antagonistic mechanism of alphaD-GeXXA via a new binding site on nAChRs provides a valuable basis for the rational design of new nAChR-targeting compounds.
Title: Bioconcentration, metabolism, and biomarker responses in freshwater fish Carassius auratus exposed to roxithromycin Liu J, Lu G, Wang Y, Yan Z, Yang X, Ding J, Jiang Z Ref: Chemosphere, 99:102, 2014 : PubMed
To investigate the distribution, bioconcentration, metabolism, and biomarker responses of macrolide antibiotic roxithromycin (ROX) in fish, crucian carp (Carassius auratus) were exposed to various concentrations of ROX (4, 20, and 100mugL(-1)) for 20d. The ROX content in different tissues was quantified using UPLC/MS/MS. The liver exhibited the highest ROX concentration followed by the bile, gills, and muscle tissues. After 15d of exposure to different concentrations of ROX, the bioconcentration factors were 2.15-38.0 in the liver, 0.950-20.7 in the bile, 0.0506-19.7 in the gill, and 0.0439-13.8 in the muscle; these results were comparable to the estimated BCF values. The metabolites formed in the bile were identified based on metabolic identification in human bile. Additionally, the biomarkers, including acetylcholinesterase in the brain, as well as 7-ethoxyresorufin O-deethylase and superoxide dismutase in the liver changed significantly after 5, 10, 15, and 20d of exposure (P<0.05). Our results suggest that ROX can accumulate and be metabolized in fish; therefore, interactions between ROX or its metabolites and the biological systems may induce biochemical disturbances in fish.
Title: Tissue distribution, bioconcentration, metabolism, and effects of erythromycin in crucian carp (Carassius auratus) Liu J, Lu G, Ding J, Zhang Z, Wang Y Ref: Sci Total Environ, 490C:914, 2014 : PubMed
In this study, the tissue distribution, bioconcentration, metabolism and biological effects of the macrolide antibiotic erythromycin (ERY) were investigated in fish using crucian carp (Carassius auratus) as a model. Crucian carp were exposed to various concentrations of ERY (4, 20, and 100mug/L) for 28days. The UPLC/MS/MS analysis of both water and tissue provided the bioconcentration of ERY and its metabolites in the fish body. The results from tissue samples showed that a maximum tissue concentration occurred in the muscle and that the bioconcentration factor (BCF) of 72.2 was lower than the theoretical BCF of 90.4 calculated from the octanol-water coefficient of ERY. A significant portion of the absorbed ERY was metabolized via demethylation and dehydration and observed in the form of descladinose in fish. In addition, the relevant biomarkers, including acetylcholinesterase in the brain, as well as 7-ethoxyresorufin O-deethylase and superoxide dismutase in the liver, changed significantly during 28days of exposure (P<0.05). These results clearly indicated that ERY accumulated in fish and that similar metabolites as those observed in mammals were produced, resulting in the biochemical disturbance of biological systems.
Title: Heterologous expression and characterization of a malathion-hydrolyzing carboxylesterase from a thermophilic bacterium, Alicyclobacillus tengchongensis Xie Z, Xu B, Ding J, Liu L, Zhang X, Li J, Huang Z Ref: Biotechnol Lett, 35:1283, 2013 : PubMed
A carboxylesterase gene from thermophilic bacterium, Alicyclobacillus tengchongensis, was cloned and expressed in Escherichia coli BL21 (DE3). The gene coded for a 513 amino acid protein with a calculated molecular mass of 57.82 kDa. The deduced amino acid sequence had structural features highly conserved among serine hydrolases, including Ser204, Glu325, and His415 as a catalytic triad, as well as type-B carboxylesterase serine active site (FGGDPENITIGGQSAG) and type-B carboxylesterase signature 2 (EDCLYLNIWTP). The purified enzyme exhibited optimum activity with beta-naphthyl acetate at 60 degrees C and pH 7 as well as stability at 25 degrees C and pH 7. One unit of the enzyme hydrolyzed 5 mg malathion l(-1) by 50 % within 25 min and 89 % within 100 min. The enzyme strongly degraded malathion and has a potential use for the detoxification of malathion residues.
Relationships between measures of oxidative stress and adaptive antioxidant defenses were studied in red sea bream (Pagrus major, RSB) caged at eight locations along the Fujian coast, China. Oxidative damage in tissues was investigated by measuring concentrations of protein carbonyl (CP) and reactive oxygen species (ROS), lipid peroxidation (LPO), acetyl cholinesterase activity (AChE), concentration of thiol groups and activities of enzymes associated with antioxidant defense. Magnitudes of antioxidant defense parameters were significantly, positively correlated with oxidative stress. Significantly greater ROS, LPO and CP content was observed in most tissues of RSB caged at three locations relative to those at the other locations. RSB caged at one location, Meizhou, exhibited significantly more DNA damage than did RSB caged at other locations. AChE activities in liver, kidney and muscle were statistically different among locations. Multivariate analyses were used to categorize the environmental quality of different locations. The battery of measures of oxidative stress proved to be a useful integrator of oxidative stresses that could be compared among locations.
Title: Functional motor nerve regeneration without motor-sensory specificity following end-to-side neurorrhaphy: an experimental study Yu Q, Lin ZK, Ding J, Wang T, Chi YL, Gao WY Ref: J Hand Surg Am, 36:2010, 2011 : PubMed
PURPOSE: To evaluate the quality of regenerating myelinated axons and motor-sensory specificity in an end-to-side nerve repair model. METHODS: We divided 20 rats into 3 groups: (1) end-to-side neurorrhaphy using the ulnar nerve as donor nerve and the musculocutaneous nerve as recipient nerve; (2) normal control; and (3) transected nerve with the stumps buried. At 5 months, we monitored the grooming test, the electrophysiological response, and the histologic changes in nerve and muscle. RESULTS: Grooming recovered successfully, and electrophysiological investigations revealed that the target muscles had been reinnervated in the end-to-side group. The mean wet weight of the reinnervated biceps brachii muscle was 72% of the normal muscle, and the mean muscle fiber cross-sectional area of the reinnervated muscle was similar to the normal muscle. The implanted musculocutaneous nerve contained varying but satisfactory numbers of axons (end-to-side group: 596 +/- 348 vs normal group: 1,340 +/- 241). Acetylcholinesterase staining revealed a similar percentage of myelinated fibers in the musculocutaneous nerve (39%) and the biceps brachii branch of the musculocutaneous nerve (38%) in the end-to-side group. This was similar to the number of myelinated fibers in the donor ulnar nerve (37%). CONCLUSIONS: The present study confirms that limited but functional reinnervation can occur on the basis of collateral sprouting of intact axons from the ulnar nerve. The motor-sensory specificity is not important.
Title: Potentiometric sensing of butyrylcholinesterase based on in situ generation and detection of substrates Ding J, Qin W Ref: Chem Commun (Camb), :971, 2009 : PubMed
A novel reagentless biosensor has been developed in which the traditional ion selective electrode is used as a controlled-release system for in situ generation and detection of enzyme substrates.
Title: Current-driven ion fluxes of polymeric membrane ion-selective electrode for potentiometric biosensing Ding J, Qin W Ref: Journal of the American Chemical Society, 131:14640, 2009 : PubMed
A polymeric membrane ion-selective electrode (ISE) for rapid potentiometric biosensing has been developed. Butyrylcholinesterase, as a target enzyme, is immobilized on the surface of the polymeric membrane. A macro-command-controlled procedure for switching between the potentiostatic and galvanostatic steps has been designed to provide a current-driven release of the butyrylcholine substrate for in situ biosensing of the enzyme and its inhibitors. This system has the flexibility of trapping substrate ions in the ISE inner filling solution and provides rapid, continuous, and reproducible measurements of enzymes and other bioanalytes involved in enzymatic systems.
Title: In situ electrodeposited nanoparticles for facilitating electron transfer across self-assembled monolayers in biosensor design Du D, Ding J, Cai J, Zhang J, Liu L Ref: Talanta, 74:1337, 2008 : PubMed
Gold nanoparticles (AuNPs) were synthesized in situ and electrodeposited onto Au substrate. The AuNPs modified interface facilitates electron transfer across self-assembled monolayers (SAMs) of 11-mercaptoundecanoic acid (MUA). After activation of surface carboxyl groups with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide, the interface displayed good stability for immobilization of biomolecules. These modification processes were characterized by contact angle measurement, cyclic voltammetry and electrochemical impedance spectra. The immobilized acetylcholinesterase (AChE), as a model, showed excellent activity to its substrate, leading to a stable AChE biosensor. Under the optimal experimental conditions, the inhibition of malathion on AChE biosensor was proportional to its concentration in two ranges, from 0.001 to 0.1 microg mL(-1) and from 0.1 to 25 microg mL(-1), with detection limit of 0.001 microg mL(-1). The simple method showed good reproducibility and acceptable stability, which had potential application in biosensor design.
Multiple myeloma (MM) remains an incurable disease and further development of novel agents is needed. Because constitutive expression of topoisomerase I (TopoI) in MM cells and the efficacy of SN-38, an active metabolite of irinotecan (CPT-11), have been reported, we investigated the therapeutic potential of CPT-11. Of the eight MM cell lines analyzed, four showed 50% inhibitory concentration values of less than 2 microg/mL for CPT-11 and less than 2 ng/mL for SN-38. This efficacy was partly explained by the high expression level of human carboxylesterase-2 (hCE-2) in MM cells. Interestingly, high expression of hCE-2 represented the nature of normal plasma cells, suggesting that hCE-2 could efficiently generate SN-38 within the plasma cells. As expected, higher sensitivity to CPT-11 was observed in hCE-2-overexpressing U266 cells than mock U266 cells. On the other hand, the expression levels of hCE-1, TopoI, UGT1A and ABCG2 did not seem to be associated with the sensitivity of MM cells to CPT-11. In a murine xenograft model inoculated s.c. with RPMI8226 cells, administration of CPT-11 alone significantly reduced the tumor volume. When a combination of CPT-11 and bortezomib was administered, the subcutaneous tumors completely disappeared. Thus, clinical trials on CPT-11 in patients with relapsed or refractory MM are warranted.
Title: Endogenous serotonin excites striatal cholinergic interneurons via the activation of 5-HT 2C, 5-HT6, and 5-HT7 serotonin receptors: implications for extrapyramidal side effects of serotonin reuptake inhibitors Bonsi P, Cuomo D, Ding J, Sciamanna G, Ulrich S, Tscherter A, Bernardi G, Surmeier DJ, Pisani A Ref: Neuropsychopharmacology, 32:1840, 2007 : PubMed
The striatum is richly innervated by serotonergic afferents from the raphe nucleus. We explored the effects of this input on striatal cholinergic interneurons from rat brain slices, by means of both conventional intracellular and whole-cell patch-clamp recordings. Bath-applied serotonin (5-HT, 3-300 microM), induced a dose-dependent membrane depolarization and increased the rate of spiking. This effect was mimicked by the 5-HT reuptake blockers citalopram and fluvoxamine. In voltage-clamped neurons, 5-HT induced an inward current, whose reversal potential was close to the K(+) equilibrium potential. Accordingly, the involvement of K(+) channels was confirmed either by increasing extracellular K(+) concentration and by blockade of K(+) channels with barium. Single-cell reverse transcriptase-polymerase chain reaction (RT-PCR) profiling demonstrated the presence of 5-HT2C, 5-HT6, and 5-HT7 receptor mRNAs in identified cholinergic interneurons. The depolarization/inward current induced by 5-HT was partially mimicked by the 5-HT2 receptor agonist 2,5-dimethoxy-4-iodoamphetamine and antagonized by both ketanserin and the selective 5-HT2C antagonist RS102221, whereas the selective 5-HT3 and 5-HT4 receptor antagonists tropisetron and RS23597-190 had no effect. The depolarizing response to 5-HT was also reduced by the selective 5-HT6 and 5-HT7 receptor antagonists SB258585 and SB269970, respectively, and mimicked by the 5-HT7 agonist, 5-CT. Accordingly, activation of either 5-HT6 or 5-HT7 receptor induced an inward current. The 5-HT response was attenuated by U73122, blocker of phospholipase C, and by SQ22,536, an inhibitor of adenylyl cyclase. These results suggest that 5-HT released by serotonergic fibers originating in the raphe nuclei has a potent excitatory effect on striatal cholinergic interneurons.
Title: An amperometric acetylthiocholine sensor based on immobilization of acetylcholinesterase on a multiwall carbon nanotube-cross-linked chitosan composite Du D, Huang X, Cai J, Zhang A, Ding J, Chen S Ref: Anal Bioanal Chem, 387:1059, 2007 : PubMed
A simple method has been devised for immobilization of acetylcholinesterase (AChE)--covalent bonding to a multiwall carbon nanotube (MWNT)--cross-linked chitosan composite (CMC)-and a sensitive amperometric sensor for rapid detection of acetylthiocholine (ATCl) has been based on this. Fourier-transform infrared spectroscopy proved that the native structure of the immobilized enzyme was preserved on this chemically clean and homogeneous composite film, because of the excellent biocompatibility and non-toxicity of chitosan. Glutaraldehyde was used as cross-linker to covalently bond the AChE, and efficiently prevented leakage of the enzyme from the film. Because of the inherent conductive properties of the MWNT, the immobilized AChE had greater affinity for ATCl and excellent catalytic effect in the hydrolysis of ATCl, with a K(app)(m) value of 132 micromol L(-1), forming thiocholine, which was then oxidized to produce a detectable and rapid response. Under optimum conditions the amperometric current increased linearly with the increasing concentration of ATCl in the range 2.0-400 micromol L(-1), with a detection limit of 0.10 micromol L(-1). Fabrication reproducibility of the sensor was good and the stability was acceptable. The sensor is a promising new tool for characterization of enzyme inhibitors and for pesticide analysis. Abstract.
Title: Determination of carbaryl pesticide using amperometric acetylcholinesterase sensor formed by electrochemically deposited chitosan Du D, Ding J, Cai J, Zhang A Ref: Colloids Surf B Biointerfaces, 58:145, 2007 : PubMed
A sensitive, fast and cheap sensor for quantitative determination of carbaryl pesticide using amperometric acetylcholinesterase (AChE) sensor based on electrochemically deposited chitosan was reported. From a mildly acidic chitosan solution, a chitosan film is electrochemically deposited on Au electrode surface via a negative voltage bias, leading to a stable AChE sensor. The characteristics of the deposited layer were observed to be dependent upon the deposition time, pH, and the chitosan concentration. Fourier-transform infrared spectra proved that the immobilized enzyme could preserve their native structure due to the excellent biocompatibility and non-toxicity of chitosan. Under the optimal experimental conditions, the carbaryl inhibition on AChE-CHIT/Au was proportional to its concentration in two ranges, from 0.005 to 0.1 microg/ml and 0.5 to 5 microg/ml, with the correlation coefficients of 0.9966 and 0.9982, respectively. The detection limit was 0.003 microg/ml taken as the concentration equivalent to a 10% decrease in signal. The determination of carbaryl in garlic samples obtained from export of farm base showed acceptable accuracy. The developed sensor exhibited good fabrication reproducibility and acceptable stability, which provided a new promising tool for pesticide analysis.
Title: Re-emergence of striatal cholinergic interneurons in movement disorders Pisani A, Bernardi G, Ding J, Surmeier DJ Ref: Trends in Neurosciences, 30:545, 2007 : PubMed
Twenty years ago, striatal cholinergic neurons were central figures in models of basal ganglia function. But since then, they have receded in importance. Recent studies are likely to lead to their re-emergence in our thinking. Cholinergic interneurons have been implicated as key players in the induction of synaptic plasticity and motor learning, as well as in motor dysfunction. In Parkinson's disease and dystonia, diminished striatal dopaminergic signalling leads to increased release of acetylcholine by interneurons, distorting network function and inducing structural changes that undoubtedly contribute to the symptoms. By contrast, in Huntington's disease and progressive supranuclear palsy, there is a fall in striatal cholinergic markers. This review gives an overview of these recent experimental and clinical studies, placing them within the context of the pathogenesis of movement disorders.
Parkinson disease is a neurodegenerative disorder whose symptoms are caused by the loss of dopaminergic neurons innervating the striatum. As striatal dopamine levels fall, striatal acetylcholine release rises, exacerbating motor symptoms. This adaptation is commonly attributed to the loss of interneuronal regulation by inhibitory D(2) dopamine receptors. Our results point to a completely different, new mechanism. After striatal dopamine depletion, D(2) dopamine receptor modulation of calcium (Ca(2+)) channels controlling vesicular acetylcholine release in interneurons was unchanged, but M(4) muscarinic autoreceptor coupling to these same channels was markedly attenuated. This adaptation was attributable to the upregulation of RGS4-an autoreceptor-associated, GTPase-accelerating protein. This specific signaling adaptation extended to a broader loss of autoreceptor control of interneuron spiking. These observations suggest that RGS4-dependent attenuation of interneuronal autoreceptor signaling is a major factor in the elevation of striatal acetylcholine release in Parkinson disease.
Long-term depression (LTD) of the synapse formed between cortical pyramidal neurons and striatal medium spiny neurons is central to many theories of motor plasticity and associative learning. The induction of LTD at this synapse is thought to depend upon D(2) dopamine receptors localized in the postsynaptic membrane. If this were true, LTD should be inducible in neurons from only one of the two projection systems of the striatum. Using transgenic mice in which neurons that contribute to these two systems are labeled, we show that this is not the case. Rather, in both cell types, the D(2) receptor dependence of LTD induction reflects the need to lower M(1) muscarinic receptor activity-a goal accomplished by D(2) receptors on cholinergic interneurons. In addition to reconciling discordant tracts of the striatal literature, these findings point to cholinergic interneurons as key mediators of dopamine-dependent striatal plasticity and learning.
