Title: N-Amidation of Nitrogen-Containing Heterocyclic Compounds: Can We Apply Enzymatic Tools? Yang A, Miao X, Yang L, Xu C, Liu W, Xian M, Zou H Ref: Bioengineering (Basel), 10:, 2023 : PubMed
Amide bond is often seen in value-added nitrogen-containing heterocyclic compounds, which can present promising chemical, biological, and pharmaceutical significance. However, current synthesis methods in the preparation of amide-containing N-heterocyclic compounds have low specificity (large amount of by-products) and efficiency. In this study, we focused on reviewing the feasible enzymes (nitrogen acetyltransferase, carboxylic acid reductase, lipase, and cutinase) for the amidation of N-heterocyclic compounds; summarizing their advantages and weakness in the specific applications; and further predicting candidate enzymes through in silico structure-functional analysis. For future prospects, current enzymes demand further engineering and improving for practical industrial applications and more enzymatic tools need to be explored and developed for a broader range of N-heterocyclic substrates.
Title: Thyroid endocrine disruption and neurotoxicity of gestodene in adult female mosquitofish (Gambusia affinis) Tan J, Liang C, Guo Y, Zou H, Ye J, Hou L, Wang X Ref: Chemosphere, :137594, 2022 : PubMed
The frequent detection of progestins in various aquatic environments and their potential endocrine disruptive effects in fish have attracted increasing attention worldwide. However, data on their effects on thyroid function and neurotoxicity in fish are limited, and the underlying mechanisms remain unclear. Here, the effects of gestodene (GES, a common progestin) on the thyroid endocrine and nervous systems of mosquitofish (Gambusia affinis) were studied. Adult female fish were exposed to GES at environmentally relevant concentrations (4.4-378.7 ng/L) for 60 days. The results showed that exposure to 378.7 ng/L GES caused a significant decrease in fish growth compared with the control and a marked reduction in the total distance traveled (50.6%) and swimming velocity (40.1-61.9%). The triiodothyronine (T3) levels were significantly increased by GES in a dose-dependent manner, whereas those of tetraiodothyronine (T4) were significantly decreased only at the G500 concentration. The acetylcholinesterase (AChE) activity was decreased significantly in the 4.42 ng/L GES treatments, but increased significantly at 378.67 ng/L. In the brain, a strong increase in the transcriptional levels of bdnf, trh, and dio2 was observed in fish after the 378.7 ng/L treatment. In addition, chronic exposure to GES caused colloid depletion with a concentration-dependent manner in the thyroid, and angiectasis, congestion, and vacuolar necrosis in the brain. These findings provide a better understanding of the effects of GES and associated underlying mechanisms in G. affinis.
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: 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.
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.
Spore pigmentation is very common in the fungal kingdom. The best studied pigment in fungi is melanin which coats the surface of single cell spores. What and how pigments function in a fungal species with multiple cell conidia is poorly understood. Here, we identified and deleted a polyketide synthase (PKS) gene PfmaE and showed that it is essential for multicellular conidial pigmentation and development in a plant endophytic fungus, Pestalotiopsis fici. To further characterize the melanin pathway, we utilized an advanced Aspergillus nidulans heterologous system for the expression of the PKS PfmaE and the Pfma gene cluster. By structural elucidation of the pathway metabolite scytalone in A. nidulans, we provided chemical evidence that the Pfma cluster synthesizes DHN melanin. Combining genetic deletion and combinatorial gene expression of Pfma cluster genes, we determined that the putative reductase PfmaG and the PKS are sufficient for the synthesis of scytalone. Feeding scytalone back to the P. fici deltaPfmaE mutant restored pigmentation and multicellular adherence of the conidia. These results cement a growing understanding that pigments are essential not simply for protection of spores from biotic and abiotic stresses but also for spore structural development.
Title: Galantamine protects against lipopolysaccharide-induced acute lung injury in rats Li G, Zhou C, Zhou Q, Zou H Ref: Brazilian Journal of Medical & Biological Research, 49:, 2016 : PubMed
Lipopolysaccharide (LPS)-induced endotoxemia triggers the secretion of proinflammatory cytokines and can cause acute lung injury (ALI). The high mobility group box 1 (HMGB1) protein plays an important role as a late mediator of sepsis and ALI. Galantamine (GAL) is a central acetylcholinesterase inhibitor that inhibits the expression of HMGB1. This study evaluated the effects of GAL by measuring levels of inflammatory mediators and observing histopathological features associated with LPS-induced ALI. Sixty 8-10 week old male Sprague-Dawley rats (200-240 g) were randomized into three groups as follows: control group, LPS group (7.5 mg/kg LPS), and LPS+GAL group (5 mg/kg GAL before LPS administration). Histopathological examination of lung specimens obtained 12 h after LPS administration was performed to analyze changes in wet-to-dry (W/D) weight ratio, myeloperoxidase (MPO) activity, and HMGB1 expression level. Additionally, plasma concentrations of tumor necrosis factor-alpha, interleukin-6, and HMGB1 were measured using an enzyme-linked immunosorbent assay at 0 (baseline), 3, 6, 9, and 12 h after LPS administration. Mortality in the three groups was recorded at 72 h. LPS-induced ALI was characterized by distortion of pulmonary architecture and elevation of MPO activity, W/D weight ratio, and levels of pro-inflammatory cytokines, including tumor necrosis factor-alpha, interleukin-6, and HMGB1. Pretreatment with GAL significantly reduced the LPS-induced lung pathological changes, W/D weight ratio, levels of pro-inflammatory cytokines and MPO activity (ANOVA). Moreover, GAL treatment significantly decreased the mortality rate (ANOVA). In conclusion, we demonstrated that GAL exerted a protective effect on LPS-induced ALI in rats.
Title: Structure of Human GIVD Cytosolic Phospholipase A2 Reveals Insights into Substrate Recognition Wang H, Klein MG, Snell G, Lane W, Zou H, Levin I, Li K, Sang BC Ref: Journal of Molecular Biology, 428:2769, 2016 : PubMed
Cytosolic phospholipases A2 (cPLA2s) consist of a family of calcium-sensitive enzymes that function to generate lipid second messengers through hydrolysis of membrane-associated glycerophospholipids. The GIVD cPLA2 (cPLA2delta) is a potential drug target for developing a selective therapeutic agent for the treatment of psoriasis. Here, we present two X-ray structures of human cPLA2delta, capturing an apo state, and in complex with a substrate-like inhibitor. Comparison of the apo and inhibitor-bound structures reveals conformational changes in a flexible cap that allows the substrate to access the relatively buried active site, providing new insight into the mechanism for substrate recognition. The cPLA2delta structure reveals an unexpected second C2 domain that was previously unrecognized from sequence alignments, placing cPLA2delta into the class of membrane-associated proteins that contain a tandem pair of C2 domains. Furthermore, our structures elucidate novel inter-domain interactions and define three potential calcium-binding sites that are likely important for regulation and activation of enzymatic activity. These findings provide novel insights into the molecular mechanisms governing cPLA2's function in signal transduction.
UNLABELLED: Protein phosphorylation is one of the most common post-translational modifications. It plays key roles in regulating diverse biological processes of liver tissues. To better understand the role of protein phosphorylation in liver functions, it is essential to perform in-depth phosphoproteome analysis of human liver. Here, an enzyme assisted reversed-phase-reversed-phase liquid chromatography (RP-RPLC) approach with both RPLC separations operated with optimized acidic mobile phase was developed. High orthogonal separation was achieved by trypsin digestion of the Glu-C generated peptides in the fractions collected from the first RPLC separation. The phosphoproteome coverage was further improved by using two types of instruments, i.e. TripleTOF 5600 and LTQ Orbitrap Velos. A total of 22,446 phosphorylation sites, corresponding to 6526 nonredundant phosphoproteins were finally identified from normal human liver tissues. Of these sites, 15,229 sites were confidently localized with Ascore>/=13. This dataset was the largest phosphoproteome dataset of human liver. It can be a public resource for the liver research community and holds promise for further biology studies. BIOLOGICAL SIGNIFICANCE: The enzyme assisted approach enabled the two RPLC separations operated both with optimized acidic mobile phases. The identifications from TripleTOF 5600 and Orbitrap Velos are highly complementary. The largest phosphoproteome dataset of human liver was generated.
Triacylglycerols are among the most attractive alternative raw materials for biofuel development. Current oil plant-based technologies are limited in terms of triacylglycerol production capacity and rate. These limitations may be circumvented by biotransformation of carbohydrates into lipids; however, our understanding of microbial oleaginicity remains limited. Here we present the results of a multi-omic analysis of Rhodosporidium toruloides, a robust triacylglycerol-producing fungus. The assembly of genome and transcriptome sequencing data reveals a genome of 20.2 Mb containing 8,171 protein-coding genes, the majority of which have multiple introns. Genes including a novel fatty acid synthase are predicted to participate in metabolic pathways absent in non-oleaginous yeasts. Transcriptomic and proteomic data suggest that lipid accumulation under nitrogen-limited conditions correlates with the induction of lipogenesis, nitrogenous compound recycling, macromolecule metabolism and autophagy. The multi-omic map of R. toruloides therefore provides a valuable resource for efforts to rationally engineer lipid-production pathways.
Title: Monitoring enzyme reaction and screening enzyme inhibitor based on MALDI-TOF-MS platform with a matrix of oxidized carbon nanotubes Hu L, Jiang G, Xu S, Pan C, Zou H Ref: J Am Soc Mass Spectrom, 17:1616, 2006 : PubMed
A matrix assisted laser desorption/ionization time-of-flight mass spectrometry platform for quantitatively monitoring enzyme activity and screening enzyme inhibitors has been demonstrated. The described method employs a new matrix of oxidized carbon nanotubes. Compared with the traditional fluorescence approach, this label-free method has the advantage of directly identifying the substrates and products in enzymatic reactions. Moreover, the method could be conveniently carried out with any commercial mass spectrometer without modification. We quantitatively monitored the acetylcholinesterase activity and screened acetylcholinesterase inhibitors with a detection rate of about 3.3 s per sample.
Title: [The activity of blood cholinesterase in rats exposed to dimehypo] Wan W, Xu M, Zou H, Lu A, Shen X, Chen Y Ref: Zhonghua Lao Dong Wei Sheng Zhi Ye Bing Za Zhi, 20:416, 2002 : PubMed
OBJECTIVE: To determine whether and to what degree the activity of cholinesterase(ChE) is inhibited by dimehypo at different doses of dimehypo [scientific name: 2-dimethylamine-1,3-bi(sodium hyposulfit)]. METHOD: Rats were dosed with dimehypo or methamidophos orally, and were randomly divided into four subgroups according to the pesticide doses, which were 1/16, 1/8, 1/4 and 1/2 of LD50 respectively(the LD50 of dimethypo and methamidophos is 342 mg/kg and 20 mg/kg respectively). The activity of ChE in blood was determined before and 30 min, 1, 2, 4 and 24 h after exposure. The modified Ellman Method was employed to measure the activity of ChE. RESULT: 1/16 LD50 dose of dimehypo did not affect the activity of ChE. When the dose increased, the activity of ChE decreased accordingly. 1/2 LD50 dose of dimehypo inhibited the activity of ChE by 35.9% compared with that of control group(P < 0.01). In rats dosed with methamidophos, even 1/16 LD50 dose inhibited the activity of ChE by 42.4% compared with that of control group. When the dose of methamidophos increased, the activity of ChE decreased accordingly. 1/2 LD50 dose of methamidophos inhibited the activity of ChE by 52.9%. The activity of ChE in the rats dosed with dimehypo at various doses was significantly lower than that in the rats dosed with corresponding doses of methamidophos(P < 0.01). CONCLUSION: Higher doses of dimehypo may inhibit the activity of ChE. However, as compared with methamidophos, dimehypo is a weaker inhibitor of ChE.
Title: [The activity of blood cholinesterase in rats exposed to dimethypo after drug intervention] Wan W, Xu M, Zou H, Lu A, Shen X, Chen Y Ref: Zhonghua Lao Dong Wei Sheng Zhi Ye Bing Za Zhi, 20:419, 2002 : PubMed
OBJECTIVE: To investigate the activity of ChE in rats poisoned by dimehypo and then treated with pralidoxime methylchloride or unithiol. METHOD: Rats were divided into control group (dimehypo); intervention groups [dimehypo plus pralidoxime methylchloride or dimehypo plus unithiol (sodium dimercaptopropanesulphonate)]. Rats were dosed with 4 different doses of dimehypo: 1/16, 1/8, 1/4 and 1/2 of LD50 respectively(the LD50 of dimehypo is 342 mg/kg). After being poisoned with dimehypo orally, rats were immediately injected intramuscularly with pralidoxime methylchloride or unithiol. The activity of ChE in blood was detected before and 1/2, 1, 2, 4 and 24 h after poisoning in dimehypo and intervention groups. RESULT: The ChE activity of four dose subgroups at 1 h after poisoning were (1.04 +/- 0.21), (0.84 +/- 0.12), (0.71 +/- 0.12), (0.66 +/- 0.07) U/ml respectively; the ChE activity of pralidoxime methylchloride intervention groups were (1.01 +/- 0.18), (1.17 +/- 0.11), (1.01 +/- 0.04), (1.03 +/- 0.12) U/ml respectively; and the ChE activity of unithiol intervention groups were (1.15 +/- 0.15), (1.26 +/- 0.27), (1.08 +/- 0.08), (1.04 +/- 0.12) U/ml respectively. The inhibited ChE in blood was recovered by either treatment with pyraldoxime methylchloride or unithiol. These two drugs had similar effects of recovering the activity of ChE(P > 0.05), but at higher doses(1/4 and 1/2 of LD50) the effects of both were not so good. CONCLUSION: Pralidoxime methylchloride and unithiol could partly recover the activity of ChE inhibited by dimehypo.
