Doxorubicin (DOX) is an effective anticancer drug, however, side effects such as cognitive impairment and cardiotoxicity have limited its clinical use. Juglanin (JUG) is a flavonoid with excellent antioxidant, anti-inflammatory, neuroprotective and anticancer properties. This study investigated the protective effects of JUG against DOX-induced cognitive decline, oxidative stress and inflammatory response in rats. The rats were orally administrated with JUG or JUG in combination with DOX. After treatment, the animals were subjected to series of behavioral test including Morris water maze, Y-maze and forced swimming tests. After the study, the rats were sacrificed and the level of acetylcholinesterase (AchE), superoxide dismutase (SOD), glutathione (GSH), catalase (CAT), malondialdehyde (MDA), interleukin 6 (IL-6), interleukin 1beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha), caspase 3 and Nuclear factor kappa B (NF-B) were assayed in the brain. Histopathological analysis was also performed on the brain of the rats. JUG significantly protected against DOX-induced cognitive impairment and depressive behaviors. In addition, JUG attenuated altered brain histopathological architecture, reduced oxido-inflammatory responses, acetylcholinesterase and caspase 3 activity in the brain of the treated rats. Collectively, the results suggested that JUG offered neuroprotection against DOX induced Chemobrain via ameliorating oxidative stress and inflammation.
Title: Structure-Based Optimization of Coumestan Derivatives as Polyketide Synthase 13-Thioesterase(Pks13-TE) Inhibitors with Improved hERG Profiles for Mycobacterium tuberculosis Treatment Zhang W, Lun S, Wang SS, Cai YP, Yang F, Tang J, Bishai WR, Yu LF Ref: Journal of Medicinal Chemistry, 65:13240, 2022 : PubMed
Pks13 was identified as a key enzyme involved in the final step of mycolic acid biosynthesis. We previously identified antitubercular coumestans that targeted Pks13-TE, and these compounds exhibited high potency both in vitro and in vivo. However, lead compound 8 presented potential safety concerns because it inhibits the hERG potassium channel in electrophysiology patch-clamp assays (IC(50) = 0.52 microM). By comparing the Pks13-TE-compound 8 complex and the ligand-binding pocket of the hERG ion channel, fluoro-substituted and oxazine-containing coumestans were designed and synthesized. Fluoro-substituted compound 23 and oxazine-containing coumestan 32 showed excellent antitubercular activity against both drug-susceptible and drug-resistant Mtb strains (MIC = 0.0039-0.0078 microg/mL) and exhibited limited hERG inhibition (IC(50) <= 25 microM). Moreover, 32 exhibited improved metabolic stability relative to parent compound 8 while showing favorable bioavailability in mouse models via serum inhibition titration assays.
Title: Impacts of di-(2-ethylhexyl) phthalate on Folsomia candida (Collembola) assessed with a multi-biomarker approach Zheng Y, Zhou K, Tang J, Liu C, Bai J Ref: Ecotoxicology & Environmental Safety, 232:113251, 2022 : PubMed
Di-(2-ethylhexyl) phthalate (DEHP) is extensively used as an additive to produce plastics, but it may damage non-target organisms in soil. In this study, the effects of DEHP on Folsomia candida in terms of survival, reproduction, enzyme activities, and DNA damage were investigated in spiked artificial soil using a multi-biomarker strategy. The 7-day LC(50) (median lethal concentration) and 28-day EC(50) (median effect concentration) values of DEHP were 1256.25 and 19.72 mg a.i. (active ingredient) kg(-1) dry soil, respectively. Biomarkers involved in antioxidant defense including catalase (CAT-catalase), glutathione S-transferases (GST), detoxifying enzymes including acetylcholinesterase (AChE), Cytochrome P450 (CYP450), and peroxidative damage (LPO-lipid peroxide) were also measured (EC(10), EC(20), and EC(50)) after exposure for 2, 4, 7, and 14 days. The Comet assay was also applied to assess the level of genetic damage. The activity of CAT and LPO was drastically enhanced by the highest dose (EC(50)) of DEHP on day two. The activities of GST and AChE in DEHP treatment groups were found to be blocked. In contrast, the activity of CYP450 was significantly enhanced compared to the respective control groups during the first four days of incubation. The Comet assay in F.candida demonstrated that DEHP (EC(50)) could induce DNA damage. The obtained multi-biomarker data were analyzed using an integrated biomarker response (IBR) index, indicating that limited-time exposure triggered higher stress than long-term exposure at low concentrations of DEHP. These results demonstrate that DEHP may cause biochemical and genetic toxicity to F. candida, which illustrated the potential risks of DEHP in the soil environment and might affect soil ecosystem processes. Further studies are necessary to elucidate the toxic mechanisms of DEHP on other non-target organisms in soil.
Title: Whole genome sequencing and analysis of fenvalerate degrading bacteria Citrobacter freundii CD-9 Zhou X, Lei D, Tang J, Wu M, Ye H, Zhang Q Ref: AMB Express, 12:51, 2022 : PubMed
Citrobacter freundii CD-9 is a Gram-negative bacteria sourced from factory sludge that can use fenvalerate as its sole carbon source and has a broad degradation spectrum for pyrethroid pesticides. The whole genome of CD-9 sequenced using Illumina HiSeq PE150 was reported in this study. The CD-9 genome size was 5.33 Mb and the G + C content was 51.55%. A total of 5291 coding genes, 9 5s-rRNA, and 79 tRNA were predicted bioinformatically. 3586 genes annotated to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database that can be involved in 173 metabolic pathways, including various microbial metabolic pathways that degrade exogenous chemicals, especially those that degrade aromatic compounds, and also produce a variety of bioactive substances. Fifty genes related to pyrethroid degradation were identified in the C. freundii CD-9 genome, including 9 dioxygenase, 25 hydrolase, and 16 esterase genes. Notably, RT-qPCR results showed that from the predicted 13 genes related to fenvalerate degradation, the expression of six genes, including esterase, HAD family hydrolase, lipolytic enzyme, and gentisic acid dioxygenase, was induced in the presence of fenvalerate. In this study, the key genes and degradation mechanism of C. freundii CD-9 were analyzed and the results provide scientific evidence to support its application in environmental bioremediation. It can establish application models for different environmental pollution management by constructing genetically engineered bacteria for efficient fenvalerate or developing enzyme formulations that can be industrially produced.
Title: Hyperoside attenuates neuroinflammation, cognitive impairment and oxidative stress via suppressing TNF-alpha/NF-kappaB/caspase-3 signaling in type 2 diabetes rats Chen X, Famurewa AC, Tang J, Olatunde OO, Olatunji OJ Ref: Nutr Neurosci, :1, 2021 : PubMed
OBJECTIVES: Literature findings have instituted the role of hyperglycemia-induced oxidative stress and inflammation in the pathogenesis of cognitive derangement in diabetes mellitus (DM). Hyperoside (HYP) is a flavanone glycoside reported to possess diverse pharmacological benefits such as antioxidant and anti-inflammatory properties. The study explored whether HYP could mitigate DM-induced cognitive dysfunction and further elucidate on potential molecular mechanism in rats. METHODS: Streptozotocin/high-fat diet-induced diabetic rats were treated orally with HYP (50, 200 and 400mg/kg/day) for six consecutive weeks. The blood glucose and serum insulin levels, Morris water maze test, intraperitoneal glucose tolerance test, and brain acetylcholinesterase (AChE) activity were determined. The brain expression of inflammatory nuclear factor-kappa B (NF-kappaB), tumour necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and interleukin-6 (IL-6), as well as superoxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH), total antioxidant capacity (TAC), malondialdehyde (MDA), lipid profile and caspase-3 activity were estimated. RESULTS: DM evoked hyperlipidemia, hypoinsulinemia, cognitive dysfunction by markedly increased AChE and reduction in learning and memory capacity. Brain activities of SOD and CAT, and levels of TAC and GSH were considerably depressed, whereas levels of IL-1beta, IL-6, TNF-alpha, NF-kappaB, caspase-3 and MDA were prominently increased. Interestingly, the HYP treatment dose-dependently abrogated the altered cognitive and biochemical parameters. DISCUSSION: The results suggested that hyperoside prevents DM-induced cognitive dysfunction, neuroinflammation and oxidative stress via antioxidant, anti-inflammatory and antiapoptotic mechanisms in rats.
We previously reported a series of coumestans-a naturally occurring tetracyclic scaffold containing a delta-lactone-that effectively target the thioesterase domain of polyketide synthase 13 (Pks13) in Mycobacterium tuberculosis (Mtb), resulting in superior anti-tuberculosis (TB) activity. Compared to the corresponding 'open-form' ethyl benzofuran-3-carboxylates, the enhanced anti-TB effects seen with the conformationally restricted coumestan series could be attributed to the extra Pi-Pi stacking interactions between the benzene ring of coumestans and the phenyl ring of F1670 residue located in the Pks13-TE binding domain. To further probe this binding feature, novel tetracyclic analogues were synthesized and evaluated for their anti-TB activity against the Mtb strain H(37)Rv. Initial comparison of the 'open-form' analogueues against the tetracyclic counterparts again showed that the latter is superior in terms of anti-TB activity. In particular, the delta-lactam-containing 5H-benzofuro [3,2-c]quinolin-6-ones gave the most promising results. Compound 65 demonstrated potent activity against Mtb H(37)Rv with MIC value between 0.0313 and 0.0625 microg/mL, with high selectivity to Vero cells (64-128 fold). The thermal stability analysis supports the notion that the tetracyclic compounds bind to the Pks13-TE domain as measured by nano DSF, consistent with the observed SAR trends. Compound 65 also showed excellent selectivity against actinobacteria and therefore unlikely to develop potential drug resistance to nonpathogenic bacteria.
BACKGROUND: Postoperative delirium (POD) is a frequent complication after surgery and its occurrence is associated with poor outcomes. The neuropathology of this complication is unclear, but it is important to evaluate relevant biomarkers for postoperative status. The purpose of this study is to explore the relationship between expression levels of cholinergic biomarkers in cerebrospinal fluid (CSF) and the occurrence and development of POD in elderly patients. METHODS: Four hundred and ninety-two elderly patients aged 65 years old or older with elective total hip/knee replacement received combined spinal-epidural anesthesia. Preoperative baseline cognitive function was assessed using the Mini-Mental State Examination (MMSE) before surgery. Each patient was interviewed in post-anesthesia care unit (PACU) and on the first, second, third and seventh (or before discharge) postoperative days. POD was diagnosed using the Confusion Assessment Method (CAM), and POD severity was measured using the Memorial Delirium Assessment Scale (MDAS). Preoperative CSF and plasma choline acetyltransferase (ChAT), acetylcholinesterase (AChE), butyrylcholinesterase (BuChE), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) levels were determined by ELISA. The levels of ChAT, AChE and BuChE activities were determined by spectrophotometry. RESULTS: POD was detected in 11.4% (51/447) of the patients. AChE, BuChE, ChAT, TNF-alpha and IL-6 concentrations in CSF and plasma have higher consistency. In preoperative CSF and preoperative and postoperative plasma, down-regulation of the concentration and activity of AChE and BuChE as well as up-regulation of the concentration and activity of ChAT and the concentrations of IL-6 and TNF-alpha were observed in patients who developed POD, and the decrease in BuChE was the most obvious. Logistic analysis showed the activities of ChAT, AChE and BuChE in CSF were still related to POD after adjusting for related factors such as sex, age, years of education, height, weight, body mass index (BMI), and American Society of Anesthesiologists (ASA) class. Receiver Operating Characteristic (ROC) curve analysis was conducted to determine the Area Under Curve (AUC) of AChE, BuChE and ChAT activity in CSF was 0.679 (P < 0.01), 0.940 (P < 0.01) and 0.819 (P < 0.01) respectively and found that BuChE activity had the most accurate diagnostic value. CONCLUSION: The changes in preoperative activity of AChE, BuChE and ChAT in CSF were associated with the development of POD in elderly patients, and BuChE activity had the greatest diagnostic value, which may be related to central cholinergic degradation. These cholinergic biomarkers might participate in the neuropathology of POD, pending further investigations. TRIAL REGISTRATION: This study was registered at Chictr.org.cn (NO. ChiCTR1900023729 ) June 9th, 2019. (Retrospectively registered).
CONTEXT: Crosstalk through receptor ligand interactions at the maternal-fetal interface is impacted by fetal sex. This affects placentation in the first trimester and differences in outcomes. Sexually dimorphic signaling at early stages of placentation are not defined. OBJECTIVE: Investigate the impact of fetal sex on maternal-fetal crosstalk. DESIGN: Receptors/ligands at the maternal-fetal surface were identified from sexually dimorphic genes between fetal sexes in the first trimester placenta and defined in each cell type using single-cell RNA-Sequencing (scRNA-Seq). SETTING: Academic institution. SAMPLES: Late first trimester (~10-13 weeks) placenta (fetal) and decidua (maternal) from uncomplicated ongoing pregnancies. MAIN OUTCOME MEASURES: Transcriptomic profiling at tissue and single-cell level; immunohistochemistry of select proteins. RESULTS: We identified 91 sexually dimorphic receptor-ligand pairs across the maternal-fetal interface. We examined fetal sex differences in 5 major cell types (trophoblasts, stromal cells, Hofbauer cells, antigen-presenting cells, and endothelial cells). Ligands from the CC family chemokine ligand (CCL) family were most highly representative in females, with their receptors present on the maternal surface. Sexually dimorphic trophoblast transcripts, Mucin-15 (MUC15) and notum, palmitoleoyl-protein carboxylesterase (NOTUM) were also most highly expressed in syncytiotrophoblasts and extra-villous trophoblasts respectively. Gene Ontology (GO) analysis using sexually dimorphic genes in individual cell types identified cytokine mediated signaling pathways to be most representative in female trophoblasts. Upstream analysis demonstrated TGFB1 and estradiol to affect all cell types, but dihydrotestosterone, produced by the male fetus, was an upstream regulator most significant for the trophoblast population. CONCLUSIONS: Maternal-fetal crosstalk exhibits sexual dimorphism during placentation early in gestation.
Title: Ultrasensitive split-type electrochemical sensing platform for sensitive determination of organophosphorus pesticides based on MnO(2) nanoflower-electron mediator as a signal transduction system Sun Y, Xiong P, Tang J, Zeng Z, Tang D Ref: Anal Bioanal Chem, 412:6939, 2020 : PubMed
Organophosphorus pesticides (OPs) are extensively used worldwide as agrochemicals; however, excess use may threaten the health of humans. Thus, it is an urgent need to develop a sensitive method for determination of OPs. Herein, a simple and sensitive split-type electrochemical method was developed by using MnO(2) nanoflower-electron mediator as a signal transduction element. The MnO(2) nanoflower-electron mediator was synthesized and shows an excellent electrochemical signal attributed to the high specific surface area of MnO(2) nanoflower. Meanwhile, the inhibition of OPs on butyrylcholinesterase (BChE) was carried out in the homogeneous system. In the absence of target molecule, a large number of thiocholines (TCh) were yielded from hydrolysis of acetylthiocholine (ATCh) by BChE. The MnO(2) nanoflower was cracked, and subsequently, multiple electron mediator molecules were released from the platform after treated with TCh, thus decreasing the electrochemical response. Furthermore, the inhibition of OPs on BChE resulted in the reduced generation of TCh, thus inducing the recovery of electrochemical signal. Under the optimal experimental, dichlorvos can be detected in a wide range of 10(-6)-10(-10) M, with a detection limit of 3x10(-10) M. Moreover, the assay was successfully used to analyze dichlorvos in cucumber juice and pear juice, showing a great promising potential for detecting organophosphorus pesticides in complex samples. Graphical abstract In this assay, a split-type electrochemical biosensor was proposed for the ultrasensitive determination of organophosphorus pesticides based on the MnO(2) nanoflower-electron mediator as an electrochemical signal component.
Title: Rolling circle amplification promoted magneto-controlled photoelectrochemical biosensor for organophosphorus pesticides based on dissolution of core-shell MnO(2) nanoflower@CdS mediated by butyrylcholinesterase Tang J, Li J, Xiong P, Sun Y, Zeng Z, Tian X, Tang D Ref: Mikrochim Acta, 187:450, 2020 : PubMed
A photoelectrochemical (PEC) aptasensing platform is devised for sensitive detection of an organophosphorus pesticide based on dissolution of core-shell MnO(2) nanoflower@CdS (MnO(2) NF@CdS) by thiocholine (TCh). TCH is produced from the butyrylcholinesterase-acetylthiocholine system, accompanied by target-triggered rolling circle amplification (RCA). The core-shell MnO(2) NF@CdS with excellent PEC performance was synthesized and employed as a photo-sensing platform. The target was detected on a functionalized magnetic probe with the corresponding aptamer. Upon malathion introduction, the aptamer was detached from the magnetic beads, while capture DNA (cDNA, with primer fragment) remained on the beads. The primer fragment in cDNA can trigger the RCA reaction to form a long single-stranded DNA (ssDNA). Furthermore, a large number of butyrylcholinesterase (BChE) were assembled on the long ssDNA strands through the hybridization with the S(2)-Au-BChE probe. Thereafter, TCh generated from hydrolysis of ATCh by BChE can reduce MnO(2) NF (core) to Mn(2+) and release the CdS nanoparticles (shell) from the platform electrode, significantly enhancing the PEC signal. Under optimal conditions, the proposed aptasensor exhibited high sensitivity for malathion with a low detection limit of 0.68 pg mL(-1). Meanwhile, it also presents outstanding specificity, reproducibility, and stability. Importantly, the sensing platform provides a new concept for detection of pesticide. Graphical abstract Herein, this work devised a photoelectrochemical (PEC) aptasensing platform for sensitive detection of organophosphorus pesticide based on dissolution of core-shell MnO(2) nanoflower@CdS (MnO(2) NF@CdS) by the as-produced thiocholine (TCh) from the butyrylcholinesterase-acetylthiocholine system, accompanying with the target-triggered rolling circle amplification (RCA).
Title: GDSL lipase occluded stomatal pore 1 is required for wax biosynthesis and stomatal cuticular ledge formation Tang J, Yang X, Xiao C, Li J, Chen Y, Li R, Li S, Lu S, Hu H Ref: New Phytol, 228:1880, 2020 : PubMed
The plant leaf surface is coated with a waterproof cuticle layer. Cuticle facing the stomatal pore surface needs to be sculpted to form outer cuticular ledge (OCL) after stomatal maturation for efficient gas exchange. Here, we characterized the roles of Arabidopsis GDSL lipase, Occlusion of Stomatal Pore 1 (OSP1), in wax biosynthesis and stomatal OCL formation. OSP1 mutation results in significant reduction in leaf wax synthesis and occlusion of stomata, leading to increased epidermal permeability, decreased transpiration rate, and enhanced drought tolerance. We demonstrated that OSP1 activity is critical for its role in wax biosynthesis and stomatal function. In vitro enzymatic assays demonstrated that OSP1 possesses thioesterase activity, particularly on C22:0 and C26:0 acyl-CoAs. Genetic interaction analyses with CER1 (ECERIFERUM 1), CER3 (ECERIFERUM 3) and MAH1 (Mid-chain Alkane Hydroxylase 1) in wax biosynthesis and stomatal OCL formation showed that OSP1 may act upstream of CER3 in wax biosynthesis, and implicate that wax composition percentage changes and keeping ketones in a lower level play roles, at least partially, in forming stomatal ledges. Our findings provided insights into the molecular mechanism mediating wax biosynthesis and highlighted the link between wax biosynthesis and the process of stomatal OCL formation.
Plant male gametogenesis is a coordinated effort involving both reproductive tissues and sporophytic tissues, in which lipid metabolism plays an essential role. Although GDSL esterases/lipases have been well known as key enzymes for many plant developmental processes and stress responses, their functions in reproductive development remain unclear. Here, we report the identification of a rice male sterile 2 (rms2) mutant in rice (Oryza sativa), which is completely male sterile due to the defects in tapetum degradation, cuticle formation in sporophytic tissues, and impaired exine and central vacuole development in pollen grains. RMS2 was map-based cloned as an endoplasmic reticulum-localized GDSL lipase gene, which is predominantly transcribed during early anther development. In rms2, a three-nucleotides deletion and one base substitution (TTGT to A) occurred within the GDSL domain, which reduced the lipid hydrolase activity of the resulting protein and led to significant changes in the content of 16 lipid components and numerous other metabolites as revealed by a comparative metabolic analysis. Furthermore, RMS2 is directly targeted by male fertility regulators Undeveloped Tapetum 1 (UDT1) and Persistent Tapetal Cell 1 (PTC1) both in vitro and in vivo, suggesting that RMS2 may serve as a key node in the rice male fertility regulatory network. These findings shed light on the function of GDSLs in reproductive development and provide a promising gene resource for hybrid rice breeding.
Title: Expression and characterization of two glucuronoyl esterases from Thielavia terrestris and their application in enzymatic hydrolysis of corn bran Tang J, Long L, Cao Y, Ding S Ref: Applied Microbiology & Biotechnology, 103:3037, 2019 : PubMed
The thermophilic fungus Thielavia terrestris when cultured on cellulose produces a cocktail of thermal hydrolases with potential application in saccharification of lignocellulosic biomass and other biotechnological areas. Glucuronoyl esterases are considered to play a unique role as accessory enzymes in lignocellulosic material biodegradation by cleaving the covalent ester linkage between 4-O-methyl-D-glucuronic acid (MeGlcA) and lignin in lignin-carbohydrate complexes (LCCs). Two glucuronoyl esterases from T. terrestris named TtGE1 and TtGE2 were expressed in Pichia pastoris. Both esterases displayed features of thermophilic enzymes, with the optimal temperature at 45 degrees C and 55 degrees C. TtGE1 and TtGE2 exhibited activity towards methyl (4-nitrophenyl beta-D-glucopyranosid) uronate (Me-GlcA-pNP) but no catalytic activity to benzyl-D-glucuronate (BnzGlcA), indicating the difference in substrate specificity from previously studied fungal GEs. A substantial increase in the release of monomeric sugars and glucuronic acid from autohydrolysis of corn bran was observed by the supplementing TtGEs into commercial xylanase; the results clearly demonstrated that the TtGEs played a significant role in this degradation process. This research on TtGEs enriches our knowledge of this novel class of fungal GEs. These newly characterized TtGEs could be used as promising accessory enzymes to improve the hydrolysis efficiency of commercial enzymes in saccharification of lignocellulosic materials due to their thermophilic characteristics.
Our group recently reported the identification of novel coumestan derivatives as Mycobacterium tuberculosis ( Mtb) Pks13-thioesterase (TE) domain inhibitors, with mutations observed (D1644G and N1640K) in the generated coumestan-resistant Mtb colonies. Herein, we report a further structure-activity relationships exploration exploiting the available Pks13-TE X-ray co-crystal structure that resulted in the discovery of extremely potent coumestan analogues 48 and 50. These molecules possess excellent anti-tuberculosis activity against both the drug-susceptible (MIC = 0.0039 microg/mL) and drug-resistant Mtb strains (MIC = 0.0078 microg/mL). Moreover, the excellent in vitro activity is translated to the in vivo mouse serum inhibitory titration assay, with administration of coumestan 48 at 100 mg/kg showing an 8-fold higher activity than that of isoniazid or TAM16 given at 10 or 100 mg/kg, respectively. Preliminary ADME-Tox data for the coumestans were promising and, coupled with the practicality of synthesis, warrant further in vivo efficacy assessments of the coumestan derivatives.
Title: Insecticidal and Acetylcholinesterase Inhibition Activity of Veratrum nigrum Alkaloidal Extract against the German Cockroach (Blattella germanica) Cai X, Li Q, Xiao L, Lu H, Tang J, Huang J, Yuan J Ref: J Arthropod Borne Dis, 12:414, 2018 : PubMed
Background: Veratrum nigrum (Liliaceae) is perennial medicinal plant widely used to treat various conditions. To determine its insecticidal properties against the German cockroach (Blattella germanica), several laboratory tests were carried out. Methods: A 4kg dry sample of V. nigrum root was purchased from the medicinal material market in Yunnan Province in 2015, China. In contact toxicity tests, V. nigrum alkaloidal extract was topically applied to the abdomen of cockroaches using a micro-applicator. In vitro acetylcholinesterase (AChE) activity tests were performed using a modified Ellman method. Results: Veratrum nigrum alkaloidal extract was toxic to male adults and 4(th) nymphs cockroaches, with median lethal dose (LD50) values of 14.90mug/insect, 14.21mug/insect for adults and 41.45mug/insect, 39.01mug/insect for 4(th) nymphs after 24h and 48h exposure, respectively. There was a significant difference between adults and nymphs in terms of tolerance to V. nigrum alkaloidal extract. There was no significant difference in mortalities at 24h and 48h, the lethal effect of V. nigrum alkaloidal extract on German cockroach was quick. AChE activity tests showed that V. nigrum alkaloidal extract had an excellent inhibitory effect on AChE: inhibition in the 4(th) nymphs and male adults had 50% inhibiting concentration (IC50) values of 3.56mg/ml and 5.78mg/ml respectively. The inhibitory effect of AChE activity was positively correlated with inhibitory time (0-20min), at a concentration of 1mg/ml, inhibition of nymph and adult AChE activity had 50% inhibiting time (IT50) values of 8.34min and 16.75min, respectively. Conclusion: V. nigrum may be explored as a potential natural insecticide for control of the German cockroach.
Small molecules and antibodies each have advantages and limitations as therapeutics. Here, we present for the first time to our knowledge, the structure-guided design of "chemibodies" as small molecule-antibody hybrids that offer dual recognition of a single target by both a small molecule and an antibody, using DPP-IV enzyme as a proof of concept study. Biochemical characterization demonstrates that the chemibodies present superior DPP-IV inhibition compared to either small molecule or antibody component alone. We validated our design by successfully solving a co-crystal structure of a chemibody in complex with DPP-IV, confirming specific binding of the small molecule portion at the interior catalytic site and the Fab portion at the protein surface. The discovery of chemibodies presents considerable potential for novel therapeutics that harness the power of both small molecule and antibody modalities to achieve superior specificity, potency, and pharmacokinetic properties.
Title: Structure-activity relationship investigation of tertiary amine derivatives of cinnamic acid as acetylcholinesterase and butyrylcholinesterase inhibitors: compared with that of phenylpropionic acid, sorbic acid and hexanoic acid Gao X, Tang J, Liu H, Liu L, Kang L, Chen W Ref: J Enzyme Inhib Med Chem, 33:519, 2018 : PubMed
In the present investigation, 48 new tertiary amine derivatives of cinnamic acid, phenylpropionic acid, sorbic acid and hexanoic acid (4d-6g, 10d-12g, 16d-18g and 22d-24g) were designed, synthesized and evaluated for the effect on AChE and BChE in vitro. The results revealed that the alteration of aminoalkyl types and substituted positions markedly influences the effects in inhibiting AChE. Almost of all cinnamic acid derivatives had the most potent inhibitory activity than that of other acid derivatives with the same aminoalkyl side chain. Unsaturated bond and benzene ring in cinnamic acid scaffold seems important for the inhibitory activity against AChE. Among them, compound 6g revealed the most potent AChE inhibitory activity (IC50 value: 3.64 micromol/L) and highest selectivity over BChE (ratio: 28.6). Enzyme kinetic study showed that it present a mixed-type inhibition against AChE. The molecular docking study suggested that it can bind with the catalytic site and peripheral site of AChE.
Title: Proline-Rich Chaperones Are Compared Computationally and Experimentally for Their Abilities to Facilitate Recombinant Butyrylcholinesterase Tetramerization in CHO Cells Wang Q, Chen CH, Chung CY, Priola J, Chu JH, Tang J, Ulmschneider MB, Betenbaugh MJ Ref: Biotechnol J, 13:e1700479, 2018 : PubMed
Human butyrylcholinesterase (BChE), predominantly tetramers with a residence time of days, offers the potential to scavenge organophosphorus pesticides and chemical warfare agents. Efficient assembly of human BChE into tetramers requires an association with proline-rich peptide chaperones. In this study, the incorporation of different proline-rich peptide chaperones into BChE is investigated computationally and experimentally. First, the authors applied molecular dynamic (MD) simulations to interpret the interactions between proline-rich chaperones with human BChE tetramer domains. The P24 chaperone which contains 24 prolines, promoted the association of BChE tetramer with a 74% simulated helicity of BChE subunits, whereas the control without chaperone and BChE with an 8-proline chaperone (P8) complex exhibited 55.8 and 60.6% predicted helicity, respectively. The interaction of proline-rich chaperones with BChE subunits (B-P) provides a conduit to facilitate the interactions between BChE subunits (B-B) of the complex, which is mainly attributed to hydrophobic interactions and hydrogen-bond binding. Experimental assessment of these two proline-rich chaperones plus a 14-proline chaperone (P14) was performed and confirmed that P24 has superior capability to facilitate recombinant BChE (rBChE) tetramerization with >60% rBChE tetramer in P24-transfected rBChE cells, whereas P14- and P8-transfected rBChE cells had 44 and 33% rBChE tetramer, respectively. The rBChE control had 14% tetramer. Finally, we developed a stable rBChE tetramer expression system in CHO cells by enriching P24 expression in rBChE expressing cells. Overall, our simulations provided a design concept for identifying proline-rich peptides that promote the rBChE tetramerization in CHO cells.
Inhibition of the mycolic acid pathway has proven a viable strategy in antitubercular drug discovery. The AccA3/AccD4/FadD32/Pks13 complex of Mycobacterium tuberculosis constitutes an essential biosynthetic mechanism for mycolic acids. Small molecules targeting the thioesterase domain of Pks13 have been reported, including a benzofuran-based compound whose X-ray cocrystal structure has been very recently solved. Its initial inactivity in a serum inhibition titration (SIT) assay led us to further probe other structurally related benzofurans with the aim to improve their potency and bioavailability. Herein, we report our preliminary structure-activity relationship studies around this scaffold, highlighting a natural product-inspired cyclization strategy to form coumestans that are shown to be active in SIT. Whole genome deep sequencing of the coumestan-resistant mutants confirmed a single nucleotide polymorphism in the pks13 gene responsible for the resistance phenotype, demonstrating the druggability of this target for the development of new antitubercular agents.
Title: Elucidation of pressure-induced lid movement and catalysis behavior of Rhizopus chinensis lipase Chen G, Tang J, Miao M, Jiang B, Jin J, Feng B Ref: Int J Biol Macromol, 103:360, 2017 : PubMed
The changes of lid movement and catalysis behavior of Rhizopus chinensis lipase under high hydrostatic pressure treatment was studied. Molecular dynamics simulation showed that the lipase lid under pressure was partially opened at below 200MPa but got more closed at over 400MPa. The interfacial activation changed little at pressure below 400MPa but became marginal with the pressure increased to 500MPa. The lipase hydrolysis ability by high pressure treatment underwent a course of initial increasing then reducing with maximum activity obtained at 200MPa and 40 degrees C. At moderate given pressure, the pressure treatment lowered the volume between the reactants and the transition state. Km decreased from 0.592 to 0.441mmol/L with pressure increasing from 0.1 to 200MPa. Meanwhile, vmax and Kcat increased 24.2% and 20% respectively. The change trend of reaction kinetic parameters under 400-500MPa was contrary to that under 0.1-200MPa.
Drug discovery opportunities where loss-of-function alleles of a target gene link to a disease-relevant phenotype often require an agonism approach to up-regulate or re-establish the activity of the target gene. Antibody therapy is increasingly recognized as a favored drug modality due to multiple desirable pharmacological properties. However, agonistic antibodies that enhance the activities of the target enzymes are rarely developed because the discovery of agonistic antibodies remains elusive. Here we report an innovative scheme of discovery and characterization of human antibodies capable of binding to and agonizing a circulating enzyme lecithin cholesterol acyltransferase (LCAT). Utilizing a modified human LCAT protein with enhanced enzymatic activity as an immunogen, we generated fully human monoclonal antibodies using the XenoMouse(TM) platform. One of the resultant agonistic antibodies, 27C3, binds to and substantially enhances the activity of LCAT from humans and cynomolgus macaques. X-ray crystallographic analysis of the 2.45 A LCAT-27C3 complex shows that 27C3 binding does not induce notable structural changes in LCAT. A single administration of 27C3 to cynomolgus monkeys led to a rapid increase of plasma LCAT enzymatic activity and a 35% increase of the high density lipoprotein cholesterol that was observed up to 32 days after 27C3 administration. Thus, this novel scheme of immunization in conjunction with high throughput screening may represent an effective strategy for discovering agonistic antibodies against other enzyme targets. 27C3 and other agonistic human anti-human LCAT monoclonal antibodies described herein hold potential for therapeutic development for the treatment of dyslipidemia and cardiovascular disease.
Title: Novel ferulic amide derivatives with tertiary amine side chain as acetylcholinesterase and butyrylcholinesterase inhibitors: The influence of carbon spacer length, alkylamine and aromatic group Liu H, Liu L, Gao X, Liu Y, Xu W, He W, Jiang H, Tang J, Fan H, Xia X Ref: Eur Journal of Medicinal Chemistry, 126:810, 2016 : PubMed
Based on our recent investigations on chalcone derivatives as AChE inhibitors, a series of ferulic acid (FA) tertiary amine derivatives similar to chalcone compounds were designed and synthesized. The results of bioactivity evaluation revealed that most of new synthesized compounds had comparable or more potent AChE inhibitory activity than the control drug Rivastigmine. The alteration of carbon chain linking tertiary amine groups and ferulic acid scaffold markedly influenced the inhibition activity against AChE. Among them the inhibitory activity of compound 6d (IC50: 0.71 +/- 0.09 mumol/L) and 6e (IC50: 1.11 +/- 0.17 mumol/L) was equal to 15-fold and 9-fold than that of Rivastigmine against AChE (IC50: 10.54 +/- 0.86 mumol/L), respectively. Moreover, compound 6d shows the highest selectivity for AChE over butyrylcholinesterase(BuChE) (ratio: 18.3). The kinetic study suggested that compound 6d revealed a mixed-type inhibition against AChE. The result of molecular docking showed that compound 6d combines to AChE with three amino acid sites(Trp84, Tyr334 and Trp279), while combines to BuChE with two amino acid sites (Tyr67 and Gly66) in enzyme domains, respectively. Compound 6d might act as a potential agent for the treatment of Alzheimer's diseases (AD).
Title: A new TLC bioautographic assay for qualitative and quantitative estimation of lipase inhibitors Tang J, Zhou J, Tang Q, Wu T, Cheng Z Ref: Phytochem Anal, 27:5, 2016 : PubMed
INTRODUCTION: Lipase inhibitory assays based on TLC bioautography have made recent progress; however, an assay with greater substrate specificity and quantitative capabilities would advance the efficacy of this particular bioassay. OBJECTIVE: To address these limitations, a new TLC bioautographic assay for detecting lipase inhibitors was developed and validated in this study. METHODS: The new TLC bioautographic assay was based on reaction of lipase with beta-naphthyl myristate and the subsequent formation of the purple dye between beta-naphthol and Fast Blue B salt (FBB). The relative lipase inhibitory capacity (RLIC) was determined by a TLC densitometry with fluorescence detection, expressed as orlistat equivalents in millimoles on a per sample weight basis. Six pure compounds and three natural extracts were evaluated for their potential lipase inhibitory activities by this TLC bioautographic assay. RESULTS: The beta-naphthyl myristate as the substrate improved the detection sensitivity and specificity significantly. The limit of detection (LOD) of this assay was 0.01 ng for orlistat, the current treatment for obesity. This assay has acceptable accuracy (92.07-105.39%), intra-day and inter-day precisions [relative standard deviation (RSD), 2.64-4.40%], as well as intra-plate and inter-plate precisions (RSD, 1.8-4.9%). CONCLUSION: The developed method is rapid, simple, stable, and specific for screening and estimation of the potential lipase inhibitors.
Title: A liquid chromatography tandem mass spectrometric method on in vitro nerve agents poisoning characterization and reactivator efficacy evaluation by determination of specific peptide adducts in acetylcholinesterase Yan L, Chen J, Xu B, Guo L, Xie Y, Tang J, Xie J Ref: Journal of Chromatography A, 1450:86, 2016 : PubMed
The terroristic availability of highly toxic nerve agents (NAs) highlights the necessity for a deep understanding of their toxicities and effective medical treatments. A liquid chromatography tandem mass spectrometry (LC-MS/MS) method for a characterization of the NAs poisoning and an evaluation on the efficacy of reactivators in in vitro was developed for the first time. After exposure to sarin or VX and pepsin digestion, the specific peptides of acetylcholinesterase (AChE) in a purified status, i.e. undecapeptide "GESAGAASVGM" in free, unaged, or aged status was identified and quantified. A key termination procedure is focused to make the reaction system "frozen" and precisely "capture" the poisoning, aging and spontaneous reactivation status of AChE, and the abundance of such specific peptides can thus be simultaneously measured. In our established method, as low as 0.72% and 0.84% inhibition level of AChE induced by 0.5nM sarin and VX can be detected from the measurement of peptide adducts, which benefits a confirmation of NAs exposure, especially at extremely low levels. Comparing with conventional colorimetric Ellman assays, our method provides not only enzyme activity and inhibition rate, but also the precise poisoning status of NAs exposed AChE. Based on the full information provided by this method, the efficacy of reactivators, such as HI-6, obidoxime and pralidoxime, in the typical treatment of NAs poisoned AChE in in vitro was further evaluated. Our results showed that this method is a promising tool for the characterization of NAs poisoning and the evaluation of reactivator efficacy.
Title: Development of ESI-MS-based continuous enzymatic assay for real-time monitoring of enzymatic reactions of acetylcholinesterase Fu Q, Tang J, Cui M, Zheng Z, Liu Z, Liu S Ref: Journal of Chromatography B Analyt Technol Biomed Life Sciences, 990:169, 2015 : PubMed
The continuous enzymatic assay based on ESI-MS was developed to real-time monitoring of enzymatic reactions of acetylcholinesterase (AChE). The changes of product concentrations were continuously measured. Calibration curves were established for quantitative calculation. By this method, the Michaelis constant (Km) of acetylcholinesterase was determined to be 70.60+/-0.93muM and Huperzine A as an effective inhibitor of acetylcholinesterase displayed a mixed inhibition with competitive and noncompetitive inhibition behaviors. The half maximal inhibitory concentration (IC50) and inhibition constant (Ki) value of Huperzine A were also calculated as 48.51+/-1.16nM and 26.73+/-0.27nM, respectively. This method provides the rapid and accurate ways to monitor enzyme reactions.
Attention-deficit hyperactive disorder (ADHD) is the most commonly studied and diagnosed psychiatric disorder in children. Methylphenidate (MPH, e.g., Ritalin) has been used to treat ADHD for over 50 years. It is the most commonly prescribed treatment for ADHD, and in the past decade it was the drug most commonly prescribed to teenagers. In addition, MPH has become one of the most widely abused drugs on college campuses. In this study, we examined the effects of MPH on hippocampal synaptic plasticity, which serves as a measurable quantification of memory mechanisms. Field potentials were recorded with permanently implanted electrodes in freely-moving mice to quantify MPH modulation of perforant path synaptic transmission onto granule cells of the dentate gyrus. Our hypothesis was that MPH affects hippocampal synaptic plasticity underlying learning because MPH boosts catecholamine signaling by blocking the dopamine and norepinephrine transporters (DAT and NET respectively). In vitro hippocampal slice experiments indicated MPH enhances perforant path plasticity, and this MPH enhancement arose from action via D1-type dopamine receptors and beta-type adrenergic receptors. Similarly, MPH boosted in vivo initiation of long-term potentiation (LTP). While there was an effect via both dopamine and adrenergic receptors in vivo, LTP induction was more dependent on the MPH-induced action via D1-type dopamine receptors. Under biologically reasonable experimental conditions, MPH enhances hippocampal synaptic plasticity via catecholamine receptors.
Title: Design, synthesis and preliminary structure-activity relationship investigation of nitrogen-containing chalcone derivatives as acetylcholinesterase and butyrylcholinesterase inhibitors: a further study based on Flavokawain B Mannich base derivatives Liu H, Fan H, Gao X, Huang X, Liu X, Liu L, Zhou C, Tang J, Wang Q, Liu W Ref: J Enzyme Inhib Med Chem, :1, 2015 : PubMed
In order to study the structure-activity relationship of Flavokawain B Mannich-based derivatives as acetylcholinesterase (AChE) inhibitors in our recent investigation, 20 new nitrogen-containing chalcone derivatives (4 a-8d) were designed, synthesized, and evaluated for AChE inhibitory activity in vitro. The results suggested that amino alkyl side chain of chalcone dramatically influenced the inhibitory activity against AChE. Among them, compound 6c revealed the strongest AChE inhibitory activity (IC50 value: 0.85 mumol/L) and the highest selectivity against AChE over BuChE (ratio: 35.79). Enzyme kinetic study showed that the inhibition mechanism of compound 6c against AChE was a mixed-type inhibition. The molecular docking assay showed that this compound can both bind with the catalytic site and the peripheral site of AChE.
Title: Characterization of Lignanamides from Hemp (Cannabis sativa L.) Seed and Their Antioxidant and Acetylcholinesterase Inhibitory Activities Yan X, Tang J, Dos Santos Passos C, Nurisso A, Simoes-Pires CA, Ji M, Lou H, Fan P Ref: Journal of Agricultural and Food Chemistry, 63:10611, 2015 : PubMed
Hemp seed is known for its content of fatty acids, proteins, and fiber, which contribute to its nutritional value. Here we studied the secondary metabolites of hemp seed aiming at identifying bioactive compounds that could contribute to its health benefits. This investigation led to the isolation of 4 new lignanamides, cannabisin M (2), cannabisin N (5), cannabisin O (8), and 3,3'-demethyl-heliotropamide (10), together with 10 known lignanamides, among which 4 was identified for the first time from hemp seed. Structures were established on the basis of NMR, HR-MS, UV, and IR as well as by comparison with the literature data. Lignanamides 2, 7, and 9-14 showed good antioxidant activity, among which 7, 10, and 13 also inhibited acetylcholinesterase in vitro. The newly identified compounds in this study add to the diversity of hemp seed composition, and the bioassays implied that hemp seed, with lignanamides as nutrients, may be a good source of bioactive and protective compounds.
Title: Receptor usage and cell entry of bat coronavirus HKU4 provide insight into bat-to-human transmission of MERS coronavirus Yang Y, Du L, Liu C, Wang L, Ma C, Tang J, Baric RS, Jiang S, Li F Ref: Proc Natl Acad Sci U S A, 111:12516, 2014 : PubMed
Middle East respiratory syndrome coronavirus (MERS-CoV) currently spreads in humans and causes approximately 36% fatality in infected patients. Believed to have originated from bats, MERS-CoV is genetically related to bat coronaviruses HKU4 and HKU5. To understand how bat coronaviruses transmit to humans, we investigated the receptor usage and cell entry activity of the virus-surface spike proteins of HKU4 and HKU5. We found that dipeptidyl peptidase 4 (DPP4), the receptor for MERS-CoV, is also the receptor for HKU4, but not HKU5. Despite sharing a common receptor, MERS-CoV and HKU4 spikes demonstrated functional differences. First, whereas MERS-CoV prefers human DPP4 over bat DPP4 as its receptor, HKU4 shows the opposite trend. Second, in the absence of exogenous proteases, both MERS-CoV and HKU4 spikes mediate pseudovirus entry into bat cells, whereas only MERS-CoV spike, but not HKU4 spike, mediates pseudovirus entry into human cells. Thus, MERS-CoV, but not HKU4, has adapted to use human DPP4 and human cellular proteases for efficient human cell entry, contributing to the enhanced pathogenesis of MERS-CoV in humans. These results establish DPP4 as a functional receptor for HKU4 and host cellular proteases as a host range determinant for HKU4. They also suggest that DPP4-recognizing bat coronaviruses threaten human health because of their spikes' capability to adapt to human cells for cross-species transmissions.
Mutations in SHANK3 and large duplications of the region spanning SHANK3 both cause a spectrum of neuropsychiatric disorders, indicating that proper SHANK3 dosage is critical for normal brain function. However, SHANK3 overexpression per se has not been established as a cause of human disorders because 22q13 duplications involve several genes. Here we report that Shank3 transgenic mice modelling a human SHANK3 duplication exhibit manic-like behaviour and seizures consistent with synaptic excitatory/inhibitory imbalance. We also identified two patients with hyperkinetic disorders carrying the smallest SHANK3-spanning duplications reported so far. These findings indicate that SHANK3 overexpression causes a hyperkinetic neuropsychiatric disorder. To probe the mechanism underlying the phenotype, we generated a Shank3 in vivo interactome and found that Shank3 directly interacts with the Arp2/3 complex to increase F-actin levels in Shank3 transgenic mice. The mood-stabilizing drug valproate, but not lithium, rescues the manic-like behaviour of Shank3 transgenic mice raising the possibility that this hyperkinetic disorder has a unique pharmacogenetic profile.
Title: Substantial Neuroprotection Against K(+) Deprivation-Induced Apoptosis in Primary Cerebellar Granule Neurons by Novel Dimer Bis(propyl)-Cognitin Via the Activation of VEGFR-2 Signaling Pathway Hu SQ, Cui W, Xu DP, Mak SH, Tang J, Choi CL, Pang YP, Han YF Ref: CNS Neurosci Ther, 19:764, 2013 : PubMed
BACKGROUND: Neuronal loss via apoptosis in CNS is the fundamental mechanism underlying various neurodegenerative diseases. Compounds with antiapoptotic property might have therapeutic effects for these diseases. In this study, bis(propyl)-cognitin (B3C), a novel dimer that possesses anti-AChE and anti-N-methyl-d-aspartate receptor activities, was investigated for its neuroprotective effect on K(+) deprivation-induced apoptosis in cerebellar granule neurons (CGNs). METHODS: Cerebellar granule neurons were switched to K(+) deprived medium with or without B3C. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assay, fluorescein diacetate (FDA)/propidium iodide (PI) staining, Hoechst staining, and DNA laddering assays were applied to detect cytotoxicity and apoptosis. Additionally, the expression of p-VEGFR-2, p-Akt, p-glycogen synthase kinase 3beta (GSK3beta), and p-extracellular signal-regulated kinase (ERK) was examined in CGNs. RESULTS: Switching CGNs to K(+) deprived medium resulted in remarkable apoptosis, which could be substantially blocked by B3C treatment (IC50 , 0.37 muM). Moreover, a rapid decrease in p-Tyr1054-VEGFR-2 was observed after the switch. B3C significantly reversed the inhibition of p-Tyr1054-VEGFR-2 as well as Akt and ERK pathways. VEGFR-2 inhibitor PTK787/ZK222584, as well as PI3-K inhibitor LY294002 and MEK inhibitor PD98059, each abolished the neuroprotective effect of B3C. CONCLUSIONS: Our results demonstrate that B3C blocks K(+) deprivation-induced apoptosis in CGNs through regulating VEGFR-2/Akt/GSK3beta and VEGFR-2/ERK signaling pathways, providing a molecular insight into the therapeutic potential of B3C for the treatment of neurodegenerative diseases.
Dipeptidyl peptidase IV (DPP-IV) degrades the incretin hormone glucagon-like peptide 1 (GLP-1). Small molecule DPP-IV inhibitors have been used as treatments for type 2 diabetes to improve glucose tolerance. However, each of the marketed small molecule drugs has its own limitation in terms of efficacy and side effects. To search for an alternative strategy of inhibiting DPP-IV activity, we generated a panel of tight binding inhibitory mouse monoclonal antibodies (mAbs) against rat DPP-IV. When tested in vitro, these mAbs partially inhibited the GLP-1 cleavage activity of purified enzyme and rat plasma. To understand the partial inhibition, we solved the co-crystal structure of one of the mAb Fabs (Ab1) in complex with rat DPP-IV. Although Ab1 does not bind at the active site, it partially blocks the side opening, which prevents the large substrates such as GLP-1 from accessing the active site, but not small molecules such as sitagliptin. When Ab1 was tested in vivo, it reduced plasma glucose and increased plasma GLP-1 concentration during an oral glucose tolerance test in rats. Together, we demonstrated the feasibility of using mAbs to inhibit DPP-IV activity and to improve glucose tolerance in a diabetic rat model.
Alzheimer disease is intimately linked to an excess amount of amyloid-beta (Abeta) in the brain. Thus, therapeutic inhibition of Abeta production is an attractive clinical approach to treat this disease. Here we provide the first direct experimental evidence that the treatment of Tg2576 transgenic mice with an inhibitor of beta-secretase, GRL-8234, rescues the age-related cognitive decline. We demonstrated that the injected GRL-8234 effectively enters the brain and rapidly decreases soluble Abeta in the brain of Tg2576 mice. The rescue of cognition, which was observed only after long-term inhibitor treatment ranging from 5 to 7.5 mo, was associated with a decrease of brain amyloid-beta plaque load. We also found no accumulation of amyloid-beta precursor protein after several months of inhibitor treatment. These observations substantiate the idea that Abeta accumulation plays a major role in the cognitive decline of Tg2576 mice and support the concept of Abeta reduction therapy as a treatment of AD.
Uncompetitive N-methyl-d-aspartate (NMDA) receptor antagonists with fast off-rate (UFO) may represent promising drug candidates for various neurodegenerative disorders. In this study, we report that bis(propyl)-cognitin, a novel dimeric acetylcholinesterase inhibitor and gamma-aminobutyric acid subtype A receptor antagonist, is such an antagonist of NMDA receptors. In cultured rat hippocampal neurons, we demonstrated that bis(propyl)-cognitin voltage-dependently, selectively, and moderately inhibited NMDA-activated currents. The inhibitory effects of bis(propyl)-cognitin increased with the rise in NMDA and glycine concentrations. Kinetics analysis showed that the inhibition was of fast onset and offset with an off-rate time constant of 1.9 s. Molecular docking simulations showed moderate hydrophobic interaction between bis(propyl)-cognitin and the MK-801 binding region in the ion channel pore of the NMDA receptor. Bis(propyl)-cognitin was further found to compete with [(3)H]MK-801 with a K(i) value of 0.27 mum, and the mutation of NR1(N616R) significantly reduced its inhibitory potency. Under glutamate-mediated pathological conditions, bis(propyl)-cognitin, in contrast to bis(heptyl)-cognitin, prevented excitotoxicity with increasing effectiveness against escalating levels of glutamate and much more effectively protected against middle cerebral artery occlusion-induced brain damage than did memantine. More interestingly, under NMDA receptor-mediated physiological conditions, bis(propyl)-cognitin enhanced long-term potentiation in hippocampal slices, whereas MK-801 reduced and memantine did not alter this process. These results suggest that bis(propyl)-cognitin is a UFO antagonist of NMDA receptors with moderate affinity, which may provide a pathologically activated therapy for various neurodegenerative disorders associated with NMDA receptor dysregulation.
Title: Addictive nicotine alters local circuit inhibition during the induction of in vivo hippocampal synaptic potentiation Zhang TA, Tang J, Pidoplichko VI, Dani JA Ref: Journal of Neuroscience, 30:6443, 2010 : PubMed
The drug addiction process shares many commonalities with normal learning and memory. Addictive drugs subvert normal synaptic plasticity mechanisms, and the consequent synaptic changes underlie long-lasting modifications in behavior that accrue during the progression from drug use to addiction. Supporting this hypothesis, it was recently shown that nicotine administered to freely moving mice induces long-term synaptic potentiation of the perforant path connection to granule cells of the dentate gyrus. The perforant path carries place and spatial information that links the environment to drug taking. An example of that association is the nicotine-induced synaptic potentiation of the perforant path that was found to underlie nicotine-conditioned place preference. The present study examines the influence of nicotine over local GABAergic inhibition within the dentate gyrus during the drug-induced synaptic potentiation. In vivo recordings from freely moving mice suggested that both feedforward and feedback inhibition onto granules cells were diminished by nicotine during the induction of synaptic potentiation. In vitro brain slice studies indicated that nicotine altered local circuit inhibition within the dentate gyrus leading to disinhibition of granule cells. These changes in local inhibition contributed to nicotine-induced in vivo synaptic potentiation, thus, likely contributed to drug-associated memories. Through this learning process, environmental features become cues that motivate conditioned drug-seeking and drug-taking behaviors.
Title: Inactivation of dipeptidyl peptidase IV attenuates the virulence of Streptococcus suis serotype 2 that causes streptococcal toxic shock syndrome Ge J, Feng Y, Ji H, Zhang H, Zheng F, Wang C, Yin Z, Pan X, Tang J Ref: Curr Microbiol, 59:248, 2009 : PubMed
Di-peptidyl peptidase IV (DPP IV), originally recognized as CD26 in eukaryotic cells, is distributed widely in microbial pathogens, including Streptococcus suis (S. suis), an emerging zoonotic agent. However, the role of DPP IV in S. suis virulence remains unclear. Here, we identified a dpp IV homologue from highly invasive isolate of S. suis 2 (SS2) causing streptococcal toxic shock syndrome (STSS). Enzymatic assays reproduced its enzymatic activity of dpp IV protein product as a functional DPP IV, and ELISA analysis demonstrated that SS2 DPP IV can interact with human fibronectin. An isogenic SS2 mutant of dpp IV, Delta dpp IV, was obtained by homologous recombination. Experimental animal infection suggested that an inactivation of dpp IV attenuates greatly its high virulence of Chinese virulent strains of SS2. Functional complementation can restore this defect in SS2 pathogenicity. To our knowledge, it may confirm, for the first time, that DPP IV contributes to SS2 virulence.
Title: The measurement of serum cholinesterase activities by an integration strategy with expanded linear ranges and negligible substrate-activation Liao F, Yang D, Tang J, Yang X, Liu B, Zhao Y, Zhao L, Liao H, Yu M Ref: Clinical Biochemistry, 42:926, 2009 : PubMed
OBJECTIVES: To measure serum cholinesterase (SCHE) with an integration strategy. DESIGN AND METHODS: At 54.0 micromol/L butyrylthiolcholine, SCHE initial rates were calculated with 50.0 micromol/L butyrylthiolcholine and maximal rates via an improved integrated method if substrate consumptions within 5.0 min were over 60%, or were determined by the classical initial rate method. RESULTS: The linear range was from 16 to 1560 nkat/L, and SCHE in clinic sera showed negligible substrate-activation. CONCLUSION: This strategy was effective.
Title: Dopamine enables in vivo synaptic plasticity associated with the addictive drug nicotine Tang J, Dani JA Ref: Neuron, 63:673, 2009 : PubMed
Addictive drugs induce a dopamine signal that contributes to the initiation of addiction, and the dopamine signal influences drug-associated memories that perpetuate drug use. The addiction process shares many commonalities with the synaptic plasticity mechanisms normally attributed to learning and memory. Environmental stimuli repeatedly linked to addictive drugs become learned associations, and those stimuli come to elicit memories or sensations that motivate continued drug use. Applying in vivo recording techniques to freely moving mice, we show that physiologically relevant concentrations of the addictive drug nicotine directly cause in vivo hippocampal synaptic potentiation of the kind that underlies learning and memory. The drug-induced long-term synaptic plasticity required a local hippocampal dopamine signal. Disrupting general dopamine signaling prevented the nicotine-induced synaptic plasticity and conditioned place preference. These results suggest that dopaminergic signaling serves as a functional label of salient events by enabling and scaling synaptic plasticity that underlies drug-induced associative memory.
Title: Poster: Nicotine activates a dopamine signal that enables in vivo synaptic plasticity of the kind that underlies associative memory Tang J, Dani JA Ref: Biochemical Pharmacology, 78:909, 2009 : PubMed
BACKGROUND: Streptococcus suis serotype 2 (SS2) is an important zoonotic pathogen, causing more than 200 cases of severe human infection worldwide, with the hallmarks of meningitis, septicemia, arthritis, etc. Very recently, SS2 has been recognized as an etiological agent for streptococcal toxic shock syndrome (STSS), which was originally associated with Streptococcus pyogenes (GAS) in Streptococci. However, the molecular mechanisms underlying STSS are poorly understood. METHODS AND FINDINGS: To elucidate the genetic determinants of STSS caused by SS2, whole genome sequencing of 3 different Chinese SS2 strains was undertaken. Comparative genomics accompanied by several lines of experiments, including experimental animal infection, PCR assay, and expression analysis, were utilized to further dissect a candidate pathogenicity island (PAI). Here we show, for the first time, a novel molecular insight into Chinese isolates of highly invasive SS2, which caused two large-scale human STSS outbreaks in China. A candidate PAI of approximately 89 kb in length, which is designated 89K and specific for Chinese SS2 virulent isolates, was investigated at the genomic level. It shares the universal properties of PAIs such as distinct GC content, consistent with its pivotal role in STSS and high virulence. CONCLUSIONS: To our knowledge, this is the first PAI candidate from S. suis worldwide. Our finding thus sheds light on STSS triggered by SS2 at the genomic level, facilitates further understanding of its pathogenesis and points to directions of development on some effective strategies to combat highly pathogenic SS2 infections.
BACKGROUND: Because apolipoprotein E (apoE) and lipopoprotein lipase (LPL) polymorphisms interact with each other and with other factors to affect lipid metabolism, we sought to determine their separate and combined effects in association with ischemic vascular disease. METHODS: We performed a case-control study of 816 subjects: 246 acute ischemic stroke patients, 234 acute myocardial infarction patients, and 336 controls. APOE exon 4 and LPL S447X genotypes were determined. RESULTS: APOE epsilon2 and epsilon4 homozygotes were increased in stroke (4.5% vs. 1.0%, p = 0.008), while in myocardial infarction the epsilon4 allele was increased (12.6% vs. 9.5%, p = 0.006) but epsilon2 was decreased (3.7% vs. 12.1%, p = 0.000006). For subjects with either APOE epsilon2 or epsilon4 alleles, LPL X alleles were increased in vascular disease (OR = 2.2, p = 0.01). LPL X alleles displayed opposite tendencies toward association with disease when subjects were divided by sex, smoking, or APOE genotype. Meta-analysis and regression analysis of previous studies supported the sex and smoking dichotomies. CONCLUSION: This is the first report of an association of vascular disease with an interaction of APOE exon 4 and LPL S447X genotypes. Therefore, APOE genotypes and LPL S447X interactions with apoE, sex, and smoking may affect the risk of myocardial infarction and ischemic stroke.
Title: Genetic characterization of the chlorothricin gene cluster as a model for spirotetronate antibiotic biosynthesis Jia XY, Tian ZH, Shao L, Qu XD, Zhao QF, Tang J, Tang GL, Liu W Ref: Chemical Biology, 13:575, 2006 : PubMed
The biosynthetic gene cluster for chlorothricin (CHL) was localized to a 122 kb contiguous DNA from Streptomyces antibioticus DSM 40725, and its involvement in CHL biosynthesis was confirmed by gene inactivation and complementation. Bioinformatic analysis of the sequenced 111.989 kb DNA region revealed 42 open reading frames, 35 of which were defined to constitute the CHL gene cluster. An assembly model for CHL biosynthesis from D-olivose, 2-methoxy-5-chloro-6-methylsalicyclic acid, and chlorothricolide building blocks was proposed. This work represents cloning of a gene cluster for spirotetronate antibiotic biosynthesis and sets the stage to investigate the unusual macrolide biosynthesis including tandem Diels-Alder cyclizations, Baeyer-Villiger oxidation, and incorporation of an enoylpyruvate unit.
An enzyme electrode for the detection of V-type nerve agents, VX (O-ethyl-S-2-diisopropylaminoethyl methylphosphonothioate) and R-VX (O-isobutyl-S-2-diethylaminoethyl methylphosphonothioate), is proposed. The principle of the new biosensor is based on the enzyme-catalyzed hydrolysis of the nerve agents and amperometric detection of the thiol-containing hydrolysis products at carbon nanotube-modified screen-printed electrodes. Demeton-S was used as a nerve agent mimic. 2-(Diethylamino)ethanethiol (DEAET) and 2-(dimethylamino)ethanethiol (DMAET), the thiol-containing hydrolysis product and hydrolysis product mimic of R-VX and VX, respectively, were monitored by exploiting the electrocatalytic activity of carbon nanotubes (CNT). As low as 2 microM DMAET and 0.8 microM DEAET were detected selectively at a low applied potential of 0.5 V vs Ag/AgCl at a CNT-modified mediator-free amperometric electrode. Further, the large surface area and the hydrophobicity of CNT was used to immobilize organophosphorus hydrolase mutant with improved catalytic activity for the hydrolysis of the P-S bond of phosphothiolester neurotoxins including VX and R-VX nerve gases to develop a novel, mediator-free, membrane-free biosensor for V-type nerve agents. The applicability of the biosensor was demonstrated for direct, rapid, and selective detection of V-type nerve agents' mimic demeton-S. The selectivity of the sensor against interferences and application to spiked lake water samples was demonstrated.
Title: The effects of repeated oral exposures to methyl parathion on rat brain cholinesterase and muscarinic receptors during postnatal development Tang J, Carr RL, Chambers JE Ref: Toxicol Sci, 76:400, 2003 : PubMed
Dimethyl phosphorylated cholinesterase (ChE) is known to be more rapidly reactivated, spontaneously, and have a higher aging rate than diethyl phosphorylated ChE. This may result in differences in toxic signs and tolerance development after treatment of juvenile rats with methyl parathion (MPS), a dimethyl phosphorothionate, than after treatment with chlorpyrifos (CPS), a diethyl phosphorothionate. The effects of repeated MPS exposures on brain ChE activity and surface and total muscarinic acetylcholine receptor (mAChR) density were studied in postnatal rats gavaged daily from postnatal day 1 (PND1) through PND 21. Results of this study were compared to our recent report with CPS (Tang et al., 1999, Toxicol. Sci. 51, 265-272). Rats received MPS daily starting at 0.3 mg/kg and increasing gradually to 0.6 mg/kg (for the medium-dosage groups) and then to 0.9 mg/kg (for the high-dosage group). ChE activity was assayed in brain homogenates. Synaptosomal mAChR densities, surface, and total were assayed using 3H-N-methylscopolamine (NMS) and 3H-quinuclidinyl benzilate (QNB), respectively, as ligands. Developmental increases in brain ChE activities and mAChR densities were observed from PND 6 through PND 22. On PND 22, inhibition of ChE activity was observed in the low (26%)-, medium (42%)-, and high (55%)-dosage groups. Significant inhibition was still present on PND 30 (16-24%) and PND 40 (12-14%), which were 9 and 19 days after the last treatment, respectively. Densities of 3H-NMS and 3H-QNB binding sites in treated groups were significantly reduced by PND 22, 1 day following cessation of treatment, and were significantly increased during the recovery period. After MPS exposure, the initial recovery of phosphorylated ChE was more rapid and the density of 3H-NMS binding sites was less readily reduced than following CPS exposure. The lesser effects on surface mAChR may explain why more severe signs appeared after each treatment with the high dosage of MPS than were observed previously with CPS, indicating little or no tolerance had developed to MPS.
Organophosphorus (OP) insecticides have the potential to cause behavioral effects in children. This study was designed to determine if repeated oral exposure of preweanling rats to chlorpyrifos would produce behavioral changes at both pre- and postweanling ages. Treatment occurred every second day beginning on post-natal day (PND) 1, and continued through PND 21. The rats received one of the following regimens: a low-dosage (3 mg/kg) from PND 1-21; a medium dosage (mg/kg from PND 1-5, and then 6 mg/kg from PND 7-21; or a high-dosage schedule of 3 mg/kg on PND 1-5, then 6 mg/kg from PND 7-13, and 12 mg/kg from PND 15-21. There were no differences in body weights among the control-, low-, and medium-dosage groups but the high-dosage group had significantly lower body weights on PND 13-21. An open field was used to measure locomotor activity on PND 10, 12, 14, 16, 18, 20, 25, and 30. There were no differences in locomotor activity levels or treatment effects between males and females. On PND 10, 12, 14, 16, 18, and 20 there was no effect on locomotor activity with any dosage. On days 25 and 30, locomotor activity was significantly decreased with the medium- and high-dosage groups. Brain cholinesterase (ChE) inhibition was about 25-38% on PND 25 and 14-34% on PND 30. On PND 25 but not 30, lung and diaphragm ChE and serum butyrylcholinesterase (BChE), with the high-dosage animals, and heart ChE with the medium- and high-dosage groups were significantly inhibited. There was no significant inhibition of skeletal muscle ChE or serum acetylcholinesterase (AChE) on PND 25 and 30. These data suggest that early postnatal chlorpyrifos exposures will depress locomotor activity in juvenile rats, with the effects most pronounced after brain ChE activity has substantially recovered.
In nonneuronal cells, the cell surface protein dystroglycan links the intracellular cytoskeleton (via dystrophin or utrophin) to the extracellular matrix (via laminin, agrin, or perlecan). Impairment of this linkage is instrumental in the pathogenesis of muscular dystrophies. In brain, dystroglycan and dystrophin are expressed on neurons and astrocytes, and some muscular dystrophies cause cognitive dysfunction; however, no extracellular binding partner for neuronal dystroglycan is known. Regular components of the extracellular matrix, such as laminin, agrin, and perlecan, are not abundant in brain except in the perivascular space that is contacted by astrocytes but not by neurons, suggesting that other ligands for neuronal dystroglycan must exist. We have now identified alpha- and beta-neurexins, polymorphic neuron-specific cell surface proteins, as neuronal dystroglycan receptors. The extracellular sequences of alpha- and beta-neurexins are largely composed of laminin-neurexin-sex hormone-binding globulin (LNS)/laminin G domains, which are also found in laminin, agrin, and perlecan, that are dystroglycan ligands. Dystroglycan binds specifically to a subset of the LNS domains of neurexins in a tight interaction that requires glycosylation of dystroglycan and is regulated by alternative splicing of neurexins. Neurexins are receptors for the excitatory neurotoxin alpha-latrotoxin; this toxin competes with dystroglycan for binding, suggesting overlapping binding sites on neurexins for dystroglycan and alpha-latrotoxin. Our data indicate that dystroglycan is a physiological ligand for neurexins and that neurexins' tightly regulated interaction could mediate cell adhesion between brain cells.
Cytosolic phospholipase A2 initiates the biosynthesis of prostaglandins, leukotrienes, and platelet-activating factor (PAF), mediators of the pathophysiology of asthma and arthritis. Here, we report the X-ray crystal structure of human cPLA2 at 2.5 A. cPLA2 consists of an N-terminal calcium-dependent lipid-binding/C2 domain and a catalytic unit whose topology is distinct from that of other lipases. An unusual Ser-Asp dyad located in a deep cleft at the center of a predominantly hydrophobic funnel selectively cleaves arachidonyl phospholipids. The structure reveals a flexible lid that must move to allow substrate access to the active site, thus explaining the interfacial activation of this important lipase.
Title: Changes in rat brain cholinesterase activity and muscarinic receptor density during and after repeated oral exposure to chlorpyrifos in early postnatal development Tang J, Carr RL, Chambers JE Ref: Toxicol Sci, 51:265, 1999 : PubMed
The effects of repeated oral exposures to the organophosphorus insecticide chlorpyrifos (CPS) on brain muscarinic receptor densities, together with cholinesterase (ChE) activity, were studied in early postnatal rats. Initially, the effects on esterases from lactational exposure to CPS were investigated in young rats by administering CPS (100, 150, or 200 mg/kg subcutaneously in corn oil) to dams 1 day postpartum, yielding a significant body burden of CPS in the dams for possible excretion in the milk. Brain ChE inhibition in pups was less severe than in dams, whereas liver carboxylesterase (CbxE) inhibition in pups was at the same level as in dams. Because of the limited brain ChE inhibition obtained following lactation, pups were exposed to CPS directly by gavage, using 3 dosing regimens to yield a dose response. The rats were gavaged with CPS in corn oil on alternate days from postnatal day (PND) 1 through PND 21. Rats in the low-dosage group received 11 treatments at 3 mg/kg, those in the medium-dosage group received 3 treatments at 3 mg/kg and 8 at 6 mg/kg, and those in the high dosage group received 3 treatments at 3 mg/kg, 4 at 6 mg/kg, and 4 at 12 mg/kg. ChE activity in brain homogenates were inhibited significantly by 29% and 63% in the low- and high-dosage groups, respectively, on PND 22 and by 17% in the high dosage group on PND 40. Muscarinic receptor densities in brain synaptosomes were reduced using 3H-N-methylscopolamine (NMS) and 3H-quinuclidinyl benzilate (QNB) as ligands, with the effects more prominent from 3H-NMS. Densities of both ligands recovered to the control level several days after terminating treatment. The results indicate that pups are apparently exposed to only limited amounts of chlorpyrifos and/or its oxon through the milk when dams are exposed to extremely high chlorpyrifos levels. In addition, repeated direct oral exposures of early postnatal rats to CPS will result in persistent brain ChE inhibition and will transiently reduce muscarinic receptor density.
Title: Detoxication of paraoxon by rat liver homogenate and serum carboxylesterases and A-esterases Tang J, Chambers JE Ref: J Biochem Mol Toxicol, 13:261, 1999 : PubMed
Paraoxon, the active metabolite of parathion, can be detoxified through a noncatalytic pathway by carboxylesterases and a catalytic pathway by calcium-dependent A-esterases, producing p-nitrophenol as a common metabolite. The detoxication patterns of carboxylesterases and A-esterases were investigated in vitro in the present study with a high tissue concentration (75 mg/mL rat liver homogenate or 50% rat serum solution) to more closely reflect enzyme concentrations in intact tissues. A final paraoxon concentration of 3.75 microM was used to incubate with liver homogenates or serum solutions for 5 seconds or 3, 5, 15, or 25 minutes; also 0.625, 1.25, 2.5, 3.125, 3.75, or 5.0 microM paraoxon (final concentration) was incubated with liver homogenates or serum solutions for 15 minutes. Phenyl saligenin cyclic phosphate and EDTA were used to inhibit carboxylesterases and A-esterases, respectively. Significant amounts of p-nitrophenol were generated with or without either inhibitor during a 15 minute incubation with paraoxon from low (0.625 microM) to high (5.0 microM) concentrations. The amount of p-nitrophenol generated via carboxylesterase phosphorylation was greater than via A-esterase-mediated hydrolysis in the initial period of incubation or when incubating with a low concentration of paraoxon. Plateau shape curves of p-nitrophenol concentration versus time or paraoxon concentration indicated that carboxylesterase phosphorylation was saturable. When incubated for long time intervals or with high concentrations of paraoxon, more p-nitrophenol was generated via A-esterase-mediated hydrolysis than from carboxylesterase phosphorylation. The ratio of paraoxon concentration to tissue amount used in in vitro assays of this study was equivalent to dosing a rat with toxicologically relevant dosages. These in vitro data suggest that both carboxylesterases and A-esterases detoxify paraoxon in vivo; carboxylesterases may be an important mode of paraoxon detoxication in initial exposures to paraoxon or parathion before they become saturated, whereas A-esterases may contribute to paraoxon detoxication in repeated exposures to paraoxon or parathion because they will not become inhibited and will remain catalytically active unlike the carboxylesterases. The importance of carboxylesterases in detoxication of paraoxon was verified by an in vivo study. In rats pretreated with tri-o-tolyl phosphate, an in vivo carboxylesterase inhibitor, brain acetylcholinesterase was significantly inhibited after intravenous exposure to parathion. No significant inhibition of brain acetylcholinesterase was observed in rats pretreated with corn oil.
Streptokinase, a 47 kDa secreted protein of hemolytic strains of streptococci, is a human plasminogen activator and contains three structural domains linked by flexible loops. We describe here the crystal structure of the isolated streptokinase middle (SKbeta) domain determined at 2.4 A resolution. Among the functionally important structural features is a putative binding site for a kringle domain of plasminogen located at the tip of a fully exposed hairpin loop. The distribution of genetically conserved residues of SKbeta is strongly correlated with their functions. The extensive interface of the SKbeta dimer suggests that such dimers may also exist in solution for free SKbeta.
Title: Anti-acetylcholinesterase antibody in myasthenic syndrome Tang J, Yuan J, Hao H Ref: Chinese Medical Journal, 110:698, 1997 : PubMed
OBJECTIVE To examine the relationship between antibodies to acetylcholinesterase and myasthenic syndrome.
METHODS:
Serum class specific antibodies to acetylcholinesterase and acetylcholine receptor were prepared with solid phase enzyme-linked immunosorbent assay in 51 patients with myasthenic syndrome, 31 controls with other neurological diseases (diseased controls) and 44 normal controls.
RESULTS:
The positive rate of anti-acetylcholinesterase antibody in patients with myasthenic syndrome was 25%, while the results of the other two control groups were negative. Furthermore, the positive rate in patients without myasthenia gravis was 45%, which was much higher than that (13%) in those with myasthenic gravis.
CONCLUSION:
Anti-acetylcholinesterase antibody may play a role in the pathogenesis of myasthenic syndrome.
Title: The crystal structure of bovine bile salt activated lipase: insights into the bile salt activation mechanism Wang X, Wang CS, Tang J, Dyda F, Zhang XC Ref: Structure, 5:1209, 1997 : PubMed
BACKGROUND:
The intestinally located pancreatic enzyme, bile salt activated lipase (BAL), possesses unique activities for digesting different kinds of lipids. It also differs from other lipases in a requirement of bile salts for activity. A structure-based explanation for these unique properties has not been reached so far due to the absence of a three-dimensional structure.
RESULTS:
The crystal structures of bovine BAL and its complex with taurocholate have been determined at 2.8 A resolution. The overall structure of BAL belongs to the alpha/beta hydrolase fold family. Two bile salt binding sites were found in each BAL molecule within the BAL-taurocholate complex structure. One of these sites is located close to a hairpin loop near the active site. Upon the binding of taurocholate, this loop becomes less mobile and assumes a different conformation. The other bile salt binding site is located remote from the active site. In both structures, BAL forms similar dimers with the active sites facing each other.
CONCLUSIONS:
Bile salts activate BAL by binding to a relatively short ten-residue loop near the active site, and stabilize the loop in an open conformation. Presumably, this conformational change leads to the formation of the substrate-binding site, as suggested from kinetic data. The BAL dimer observed in the crystal structure may also play a functional role under physiological conditions.
Glycosylation positions and oligosaccharide characteristics in the proline-rich, mucin-like, C-terminal region (C-tail) of human milk bile salt-activated lipase (BAL) were studied in order to assess the possible physiological functions of this region. A large-scale purification method has been devised to purify the C-tail fragment from human milk BAL. Chymotryptic, tryptic, and cyanogen bromide cleavages of partially purified BAL and subsequent molecular sieve chromatography yielded 20-30 mg of C-tail fragment from 1 L of human milk. The N-terminal sequence and amino acid composition of the purified C-tail fragment establish that it is derived from residues 528-712 of the enzyme. The O-glycosylated carbohydrates of the C-tail fragment contain fucose, galactose, glucosamine, galactosamine, and neuraminic acid in a molar ratio of 1:3:2:1:0.3, respectively. beta-Elimination reaction revealed that nine threonine residues and less than one serine residue were glycosylated. Edman degradation of C-tail fragment and its pronase subfragment suggest a number of glycosylation sites which are flanked by a consensus motif of PVPP. We suggest that this motif may serve as a signal for O-glycosylation in the C-tail region of BAL. Immunochemical studies indicated that the oligosaccharide chains in the C-tail region of BAL contain Lewis x and Lewis a antigens and, less prominently, sialyl Lewis x and sialyl Lewis a antigens. C-tail fragment was also found to bind jacalin lectin. These observations suggest the possibility that the C-tail region may contribute to adhesive activity in the physiological function of BAL.
Title: Proline-rich domain and glycosylation are not essential for the enzymic activity of bile salt-activated lipase. Kinetic studies of T-BAL, a truncated form of the enzyme, expressed in Escherichia coli Downs D, Xu YY, Tang J, Wang CS Ref: Biochemistry, 33:7979, 1994 : PubMed
We have expressed and purified a truncated recombinant human milk bile salt-activated lipase (T-BAL) from the T7 expression system in Escherichia coli. This T-BAL contains the N-terminal 538 residues of the 722-residue native enzyme. The purified T-BAL, when assayed with PANA (p-nitrophenyl acetate), had a specific activity of 64 +/- 2 units/mg (n = 4), as compared to 52 units/mg for the native enzyme. Because the recombinant T-BAL expressed in E. coli is not glycosylated, these results indicated that the highly glycosylated C-terminal region of BAL is not essential for catalytic function. Heat inactivation patterns of native BAL and T-BAL were found to be similar, further suggesting that the folding of T-BAL is similar to that of the catalytic domain of the native enzyme. With the availability of a sufficient amount of recombinant T-BAL, the specificity and kinetics of T-BAL and native BAL were compared. Fluorescence studies of T-BAL indicated that it has a slightly higher affinity for the monomeric form of taurocholate with a dissociation constant (KA) of 0.32 mM, compared with the reported 0.37 mM for the native enzyme. Further kinetic analysis indicated that there are enzyme specificity changes revealed with the use of PANA and PANB (p-nitrophenyl butyrate) as substrates. When assayed in the presence of taurocholate, T-BAL has a higher turnover rate constant with p-nitrophenyl acetate than with p-nitrophenyl butyrate, which was found to be in contrast to native BAL..
Title: Polysialic acid regulates growth cone behavior during sorting of motor axons in the plexus region Tang J, Rutishauser U, Landmesser L Ref: Neuron, 13:405, 1994 : PubMed
Removal of polysialic acid (PSA) from N-CAM during the time when chick motoneuron axons are segregating into target-specific fascicles at the base of the limb was previously shown to result in motoneuron projection errors. Here, it is established that these errors are associated with altered growth cone behavior in the plexus. In contrast to control embryos, in which individual axons were observed to exhibit dramatic changes in direction and extensive divergence, axonal trajectories following the removal of PSA were relatively straight. To determine whether enhanced axon-axon fasciculation following PSA removal had prevented growth cones from responding appropriately to guidance cues at the base of the limb, we also examined the role of L1, a major mediator of axon-axon fasciculation in this system. Anti-L1 reversed the effects of PSA removal on both growth cone trajectories and projection errors. These results indicate that PSA plays a permissive role, attenuating axon-axon interactions in the plexus and thereby allowing the axonal reorganization that is essential for the formation of specific motoneuron projections.
Title: Reduction of intramuscular nerve branching and synaptogenesis is correlated with decreased motoneuron survival Tang J, Landmesser L Ref: Journal of Neuroscience, 13:3095, 1993 : PubMed
Blockade of neuromuscular activity during the period of naturally occurring cell death increases intramuscular nerve branching, synaptogenesis, and survival of embryonic chicken motoneurons. These results suggested that the enhanced motoneuron survival observed might result not from increased production of a trophic factor by the target, as often suggested, but rather from the enhanced ability of motoneurons to take up this factor via additional branches and/or synapses. Since removal of polysialic acid (PSA) by PSA-specific endoneuraminidase (endo N) during the period of nerve ingrowth and motoneuron cell death had been shown to reduce intramuscular nerve branching by altering adhesive interactions between axons and myotubes, we wished to determine, first, if this resulted in a reduction of synapse formation, and second, if this was correlated with decreased motoneuron survival. When PSA was removed from embryonic day 6 (E6) to E9 by injecting endo N into the limb, the number of synapses in the posterior iliotibialis muscle was greatly reduced both at E6.5, the onset of the cell death period, and at E7.5-E8, the middle of the cell death period. Total motoneuron counts at both E7.5-E8 and E10 (close to the end of cell death period) were significantly decreased compared to controls. These results indicate that a treatment that reduces intramuscular nerve branching and synapse formation also affects motoneuron survival. Furthermore, since removal of PSA in ovo via endo N altered neither the spontaneous embryonic motility nor the activation pattern of motoneurons or muscles, these experiments avoid the complication of activity blockade that may alter motoneuron survival by affecting events other than, or in addition to, intramuscular nerve branching.
The influence of polysialic acid (PSA) on the neural cell adhesion molecule on motoneuron outgrowth and pathway formation was investigated by determining its temporal and spatial pattern of expression and by the effect that its removal had on motoneuron projection patterns. Motoneurons first expressed PSA as their growth cones began to segregate into motoneuron pool-specific groups in the plexus region; furthermore, PSA levels differed between motoneurons projecting to different targets. When PSA was removed during the period of axonal segregation in the plexus region projection errors were common. However, later removal during the process of muscle nerve formation did not result in projection errors. These results suggest that PSA modulates interactions between motoneuron axons and guidance molecules in the plexus region during axonal pathfinding.
The structure and some functional sites of human milk bile salt activated lipase (BAL) were studied by cDNA cloning and chemical analysis of the enzyme. Eighteen cDNA clones of human BAL were identified from lactating human breast cDNA libraries in lambda gt11 and lambda gt10 with antibody and synthetic oligonucleotides as probes. The sequence of four clones was sufficient to construct a 3018-bp BAL cDNA structure. This sequence codes for an open reading frame of 742 amino acid residues. There is a putative signal sequence of 20 residues which is followed by the amino-terminal sequence of BAL, and the mature BAL contains 722 amino acid residues. The cDNA sequence also contains a 678-base 5'-untranslated sequence, a 97-base 3'-untranslated region, and a 14-base poly(A) tail. The sequence of a 1.8-kbp insert of clone G10-4A differs from that of the other cDNA in that it contains a deletion of 198 bases (1966-2163) corresponding to 66 amino acid residues. By use of BAL cDNA as probe, it was found that the major molecular species of BAL mRNA in human mammary gland HBL-100 cells had a size of 2.9 kb and two minor species had sizes of 3.8 and 5.1 kb by Northern blot analyses. The deduced BAL protein structure contains in the carboxyl-terminal region 16 repeating units of 11 amino acids each. The repeating units have the basic structure Pro-Val-Pro-Pro-Thr-Gly-Asp-Ser-Gly-Ala-Pro with only minor substitutions. The amino acid sequence of human BAL is related to that of pancreatic lysophospholipase, cholesterol esterase, cholinesterase, acetylcholinesterase, and thyroglobulin. Ten of the 14 cyanogen bromide fragments of diisopropyl fluorophosphate inhibited human milk BAL were isolated, determined for N-terminal sequences, analyzed for amino sugars, and tested for some functional properties. These chemical studies established that the active site of human milk BAL is located at serine-194, the N-glycosylation site is present at asparagine-187, the O-glycosylation region is in the 16 repeating units near the C-terminus, and the heparin binding domain is in the N-terminal region. We have also determined the location of disulfide bridges as Cys64-Cys80 and Cys246-Cys257. The cyanogen bromide cleavage and the partial sequencing of CNBr peptides also confirmed the location of methionines in the polypeptide chain as well as the deduced cDNA sequence of BAL.
