Two novel ent-aromadendrane derivatives, plagiochianin A (1), possessing an unprecedented 2,3:6,7-di- seco-6,8-cyclo-aromadendrane carbon scaffold conjugated with three cyclic acetals, and plagiochianin B (2), an exceptional pyridine type aromadendrane alkaloid, were isolated from the Chinese liverwort Plagiochila duthiana. Their structures were established by comprehensive spectroscopic analysis coupled with single-crystal X-ray diffraction and electronic circular dichroism calculations. A plausible biogenetic pathway of these two compounds is presented, and their acetylcholinesterase inhibitory activities are preliminarily tested using TLC-bioautographic assays.
Title: A Semiautomated Structure-Based Method To Predict Substrates of Enzymes via Molecular Docking: A Case Study with Candida antarctica Lipase B Yao Z, Zhang L, Gao B, Cui D, Wang F, He X, Zhang JZ, Wei D Ref: J Chem Inf Model, 56:1979, 2016 : PubMed
The discovery of unique substrates is important for developing potential applications of enzymes. However, the experimental procedures for substrate identification are laborious, time-consuming, and expensive. Although in silico structure-based approaches show great promise, recent extensive studies have shown that these approaches remain a formidable challenge for current biocomputational methodologies. Here we present an open-source, extensible, and flexible software platform for predicting enzyme substrates called THEMIS, which performs in silico virtual screening for potential catalytic targets of an enzyme on the basis of the enzyme's catalysis mechanism. On the basis of a generalized transition state theory of enzyme catalysis, we introduce a modified docking procedure called "mechanism-based restricted docking" (MBRD) for novel substrate recognition from molecular docking. Comprising a series of utilities written in C/Python, THEMIS automatically executes parallel-computing MBRD tasks and evaluates the results with various molecular mechanics (MM) criteria such as energy, distance, angle, and dihedral angle to help identify desired substrates. Exhaustive sampling and statistical measures were used to improve the robustness and reproducibility of the method. We used Candida antarctica lipase B (CALB) as a test system to demonstrate the effectiveness of our computational prediction of (non)substrates. A novel MM score function for CALB substrate identification derived from the near-attack conformation was used to evaluate the possibility of chemical transformation. A highly positive rate of 93.4% was achieved from a CALB substrate library with 61 known substrates and 35 nonsubstrates, and the screening rate has reached 103 compounds/day (96 CPU cores, 100 samples/compound). The performance shows that the present method is perhaps the first reported scheme to meet the requirement for practical applicability to enzyme studies. An additional study was performed to validate the universality of our method. In this verification we employed two distinct enzymes, nitrilase Nit6803 and SDR Gox2181, where the correct rates of both enzymes exceeded 90%. The source code used will be released under the GNU General Public License (GPLv3) and will be free to download. We believe that the present method will provide new insights into enzyme research and accelerate the development of novel enzyme applications.
Title: Effect of Dietary Cadmium on the Activity of Glutathione S-Transferase and Carboxylesterase in Different Developmental Stages of the Oxya chinensis (Orthoptera: Acridoidea) Zhang YP, Song DN, Wu HH, Yang HM, Zhang JZ, Li LJ, Ma EB, Guo YP Ref: Environ Entomol, 43:171, 2014 : PubMed
Glutathione S-transferases (GSTs) and carboxylesterases (CarEs) play important roles in the detoxification of endogenous and exogenous compounds. In this study, the biochemical effects of dietary cadmium (Cd) on the activities of GST and CarE in different developmental stages of the rice grasshopper Oxya chinensis Thunberg were studied. The results showed that the effects of the Cd concentration and developmental stage on GST activity were statistically significant. GST activity in O. chinensis increased at the highest Cd concentration in most nymphs, suggesting that GST is typically inducible by Cd. However, GST activity was inhibited in adults under Cd stress owing to life-stage-specific physiological characteristics. The results showed that the substrates, developmental stage, and Cd concentration had statistically significant effects on CarE activity. In most studies of CarE activity, the interaction between any two studied factors was statistically significant, although the interaction effects of the substrates, developmental stages, and Cd concentrations were not significant, which implied that the insect physiological condition and the external environmental may affect CarE activity. The results suggest that the insect's life stage and enzyme substrates should be considered when enzyme activity under Cd stress is studied.
Title: Structure, mechanism, and enantioselectivity shifting of lipase LipK107 with a simple way Zhang L, Gao B, Yuan Z, He X, Yuan YA, Zhang JZ, Wei D Ref: Biochimica & Biophysica Acta, 1844:1183, 2014 : PubMed
Because of the complex mechanisms of enzymatic reactions, no precise and simple method of understanding and controlling the chiral selectivity of enzymes has been developed. However, structure-based rational design is a powerful approach to engineering enzymes with desired catalytic activities. In this work, a simple, structure-based, large-scale in silico design and virtual screening strategy was developed and successfully applied to enzyme engineering. We first performed protein crystallization and X-ray diffraction to determine the structure of lipase LipK107, which is a novel family I.1 lipase displaying activity for both R and S isomers in chiral resolution reactions. The catalytic mechanism of family I.1, which includes LipK107, was ascertained first through comparisons of the sequences and structures of lipases from other families. The binding states of LipK107, including the energy and the conformation of complexes with the R and S enantiomers, have been evaluated by careful biocomputation to figure out the reason for the higher S selectivity. Based on this study, a simple strategy for manipulating the chiral selectivity by modulating a crucial distance in the enzyme-substrate complex and judging virtual mutations in silico is recommended. Then, a novel electrostatic interaction analysis protocol was used to design LipK107 mutants to validate our strategy. Both positive and negative mutations determined using this theoretical protocol have been implemented in wet experiments and were proved to produce the desired enantioselectivity, showing a 176% increase or 50% decrease in enantioselectivity as desired. Because of its accuracy and versatility, the strategy is promising for practical applications.
Title: Mechanisms of organophosphate resistance in a field population of oriental migratory locust, Locusta migratoria manilensis (Meyen) Yang ML, Zhang JZ, Zhu KY, Xuan T, Liu XJ, Guo YP, Ma EB Ref: Archives of Insect Biochemistry & Physiology, 71:3, 2009 : PubMed
The susceptibilities to three organophosphate (OP) insecticides (malathion, chlorpyrifos, and phoxim), responses to three metabolic synergists [triphenyl phosphate (TPP), piperonyl butoxide (PBO), and diethyl maleate (DEM)], activities of major detoxification enzymes [general esterases (ESTs), glutathione S-transferases (GSTs), and cytochrome P450 monooxygenases (P450s)], and sensitivity of the target enzyme acetylcholinesterase (AChE) were compared between a laboratory-susceptible strain (LS) and a field-resistant population (FR) of the oriental migratory locust, Locusta migratoria manilensis (Meyen). The FR was significantly resistant to malathion (57.5-fold), but marginally resistant to chlorpyrifos (5.4) and phoxim (2.9). The malathion resistance of the FR was significantly diminished by TPP (synergism ratio: 16.2) and DEM (3.3), but was unchanged by PBO. In contrast, none of these synergists significantly affected the toxicity of malathion in the LS. Biochemical studies indicated that EST and GST activities in the FR were 2.1- to 3.2-fold and 1.2- to 2.0-fold, respectively, higher than those in the LS, but there was no significant difference in P450 activity between the LS and FR. Furthermore, AChE from the FR showed 4.0-fold higher activity but was 3.2-, 2.2-, and 1.1-fold less sensitive to inhibition by malaoxon, chlorpyrifos-oxon, and phoxim, respectively, than that from the LS. All these results clearly indicated that the observed malathion resistance in the FR was conferred by multiple mechanisms, including increased detoxification by ESTs and GSTs, and increased activity and reduced sensitivity of AChE to OP inhibition.
Title: [Effect of the pre-hospital systematic treatment on prognosis patients of with severe acute organophosphorus pesticide poisoning] Wang WZ, Li YQ, Zhang JZ, Wang L, Ma GY, Cao SQ Ref: Zhonghua Lao Dong Wei Sheng Zhi Ye Bing Za Zhi, 23:371, 2005 : PubMed
OBJECTIVE: To investigate if the duration from poisoning to treatment (no treatment period) is related to the prognosis of patients with severe acute organophosphorus pesticide poisoning (SAOPP). METHODS: One hundred and seventy-four patients with the pre-hospital systematic treatment served as the treatment group while 160 patients going to the hospital by themselves without treatment or rejecting gastrolavage served as the control group. Patients in both groups were treated by gastrolavage, pralidoxime chloride, atropine and other expectant treatment. The duration of no treatment period, death, and severe complication were observed. The time of disappearance of symptoms, the recovery time of acetyl cholinesterase (AChE), atropinization time, atropine dosage, pralidoxime chloride dosage, naloxone dosage, hospitalization days and other targets were also observed. RESULTS: The duration of no treatment period in treatment group [(1.2 +/- 0.3) h] was significantly shorter than that in control group [(2.8 +/- 0.5) h, (P < 0.01)]. The mortality rate in treatment group was 6.32% while that in control group 22.5% (P < 0.01). The incidence of respiratory failure, heart injury, brain injury, atropine poisoning, intermediate syndrome, liver injury in treatment group (12.64%, 5.75%, 8.62%, 1.72%, 4.60%, 5.17% respectively) were lower than those in control group (25.63%, 13.75%, 17.50%, 6.25%, 7.50%, 9.38% respectively, P < 0.05 or P < 0.01). The time of symptoms disappearance, the recovery time of AChE, atropinization time, atropine dosage, pralidoxime chloride dosage, naloxone dosage, hospitalization days in treatment group were significantly superior to those in control group (P < 0.05 or P < 0.01). CONCLUSION: The pre-hospital systematic treatment can improve the prognosis of the patients with SAOPP, which is worth popularizing and using.
Title: [Protective effects of tacrine and donepezil against staurosporine-induced apoptotic death] Zhang BF, Peng FF, Zhang JZ, Wu DC Ref: Yao Xue Xue Bao, 37:98, 2002 : PubMed
AIM: To study whether tacrine and donepezil can prevent cell apoptosis induced by staurosporine in NG108-15 and Hela cell lines. METHODS: MTT assay was used to examine if staurosporine impairs cell metabolism. Phase-contrast and fluorescence microscope were used to examine cell morphological changes. DNA was isolated and electrophoretically separated on 1% agarose gel to observe if there were DNA fragments. Western blot was made to analyse protein levels of anti-apoptotic Bcl-2 and proapoptotic Bax. RESULTS: NG108-15 cells treated with 0.1 mumol.L-1 staurosporine for 12-24 hours exhibited marked cell death and DNA fragmentation. Pre-treatment with 0.1 mmol.L-1 tacrine provided approximately 40% protective effect and resulted in obvious inhibition or delay of DNA fragmentation. Moreover, NG108-15 cells treated with tacrine became elongated and polarized, and showed longer processes than control cells. Pretreatment with 0.1 mmol.L-1 tacrine significantly increased the expression of Bcl-2 protein level and delayed the staurosporine-induced increase of Bax protein expression. However, donepezil did not show any protective effect on the cell impairment induced by staurosporine in NG108-15 cells. In Hela cells 0.1 mumol.L-1 staurosporine also induced significant cell injury, but pretreatment with tacrine and donepezil did not provide any obvious protective effect against this cell damage. CONCLUSION: Donepezil did not provide obvious protective effect against apoptosis, and protective effects of tacrine might not be mediated through AChE inhibition. Protective effects of tacrine against staurosporine-induced injury might be selective to different cells.
Title: Mechanisms of 5-hydroxytryptamine-induced contraction of isolated rat intrapulmonary bronchi Szarek JL, Zhang JZ, Gruetter CA Ref: Pulmonary Pharmacology, 8:273, 1995 : PubMed
Previous studies in our laboratory and others suggested that activation of 5-HT2 receptors mediates 5-hydroxytryptamine (5-HT)-induced contraction of airway smooth muscle and that this response is dependent in part on endogenous acetylcholine (ACh). The purpose of the present study was to confirm a role for 5-HT2 receptors and endogenous ACh in 5-HT-induced contraction of rat bronchi. In this study, we examined the effects of 5-HT2 receptor antagonists (ketanserin and LY53857), acetylcholinesterase inhibitors (physostigmine and neostigmine), and a muscarinic receptor alkylating agent [propylbenzilylcholine mustard (PBCM)] on contractile responses evoked by 5-HT and the 5-HT2 receptor agonist, alpha-methyl-5-hydroxytryptamine (alpha-Me-5-HT). Concentration-response curves generated in isolated rat intrapulmonary bronchi in response to 5-HT and alpha-Me-5-HT were superimposable. Inhibition of acetylcholinesterase by physostigmine or neostigmine potentiated contractile responses elicited by 5-HT and alpha-Me-5-HT. Alkylation of muscarinic receptors with PBCM decreased maximal responses elicited by 5-HT or alpha-Me-5-HT in a concentration-dependent manner. Maximum contraction attained with exogenous ACh was decreased by PBCM in a concentration-dependent manner and, at the highest concentration evaluated, ACh-induced contractions were abolished. 5-Hydroxytryptamine-induced contraction was inhibited competitively by low concentrations of the 5-HT2-receptor selective antagonist, ketanserin; higher concentrations abolished contractile responses to the amine. The inhibition of 5-HT-induced contractile responses by another 5-HT2-receptor selective antagonist, LY53857, was non-competitive in nature. Together, the results suggest that 5-HT contracts rat airways directly by activating 5-HT2 receptors located on airway smooth muscle and indirectly by activation of 5-HT2 receptors on parasympathetic nerve endings to cause release of ACh. The potential physiological implication of these findings is that 5-HT released in inflammatory conditions such as asthma may play a role in causing bronchoconstriction by releasing ACh or by augmenting release of ACh from activated cholinergic nerves.
