While the role of cholinergic neurotransmission from motoneurons is well established during neuromuscular development, whether it regulates central nervous system development in the spinal cord is unclear. Zebrafish presents a powerful model to investigate how the cholinergic system is set up and evolves during neural circuit formation. In this study, we carried out a detailed spatiotemporal analysis of the cholinergic system in embryonic and larval zebrafish. In 1-day-old embryos, we show that spinal motoneurons express presynaptic cholinergic genes including choline acetyltransferase (chata), vesicular acetylcholine transporters (vachta, vachtb), high-affinity choline transporter (hacta) and acetylcholinesterase (ache), while nicotinic acetylcholine receptor (nAChR) subunits are mainly expressed in interneurons. However, in 3-day-old embryos, we found an unexpected decrease in presynaptic cholinergic transcript expression in a rostral to caudal gradient in the spinal cord, which continued during development. On the contrary, nAChR subunits remained highly expressed throughout the spinal cord. We found that protein and enzymatic activities of presynaptic cholinergic genes were also reduced in the rostral spinal cord. Our work demonstrating that cholinergic genes are initially expressed in the embryonic spinal cord, which is dynamically downregulated during development suggests that cholinergic signaling may play a pivotal role during the formation of intra-spinal locomotor circuit.
Title: Crystal structure and characterization of esterase Est25 mutants reveal improved enantioselectivity toward (S)-ketoprofen ethyl ester Kim J, Seok SH, Hong E, Yoo TH, Seo MD, Ryu Y Ref: Applied Microbiology & Biotechnology, 101:2333, 2017 : PubMed
Esterases comprise a group of enzymes that catalyze the cleavage and synthesis of ester bonds. They are important in biotechnological applications owing to their enantioselectivity, regioselectivity, broad substrate specificity, and the fact that they do not require cofactors. In a previous study, we isolated the esterase Est25 from a metagenomic library. Est25 showed catalytic activity toward the (R,S)-ketoprofen ethyl ester but had low enantioselectivity toward the (S)-ketoprofen ethyl ester. Because (S)-ketoprofen has stronger anti-inflammatory effects and fewer side effects than (R)-ketoprofen, enantioselectivity of this esterase is important. In this study, we generated Est25 mutants with improved enantioselectivity toward the (S)-ketoprofen ethyl ester; improved enantioselectivity of mutants was established by analysis of their crystal structures. The enantioselectivity of mutants was influenced by substitution of Phe72 and Leu255. Substituting these residues changed the size of the binding pocket and the entrance hole that leads to the active site. The enantioselectivity of Est25 (E = 1.1 +/- 0.0) was improved in the mutants F72G (E = 1.9 +/- 0.2), L255W (E = 16.1 +/- 1.1), and F72G/L255W (E = 60.1 +/- 0.5). Finally, characterization of Est25 mutants was performed by determining the optimum reaction conditions, thermostability, effect of additives, and substrate specificity after substituting Phe72 and Leu255.
BACKGROUND: Ginseng (Panax ginseng C.A. Meyer) has been used as a traditional herb in the treatment of many medical disorders. Ginsenosides, which are triterpene derivatives that contain sugar moieties, are the main pharmacological ingredients in ginseng. This study was designed to investigate the effect of ginsenoside Rg3-enriched ginseng extract (Rg3GE) on scopolamine-induced memory impairment in mice. METHODS: Rg3GE (50 and 100 mg/kg) were administered to C57BL/6 mice by oral gavage for 14 days (days 1-14). Memory impairment was induced by scopolamine (1 mg/kg, intraperitoneal injection) for 6 days (days 914). The Morris water maze test was used to assess hippocampus-dependent spatial memory. The effects of scopolamine with or without Rg3GE on acetylcholinesterase and nuclear factor-kappaB (NF-kappaB) in the hippocampus were also examined. RESULTS: Mice with scopolamine treatment alone showed impairments in the acquisition and retention of spatial memory. Mice that received Rg3GE and scopolamine showed no scopolamine-induced impairment in the acquisition of spatial memory. Oral administration of Rg3GE suppressed the scopolamine-mediated increase in acetylcholinesterase activity and stimulation of the NF-kappaB pathway (i.e., phosphorylation of p65) in the hippocampus. CONCLUSION: These findings suggest that Rg3GE may stabilize scopolamine-induced memory deficits through the inhibition of acetylcholinesterase activity and NF-kappaB signaling in the hippocampus.
Title: Strategies for increasing heterologous expression of a thermostable esterase from Archaeoglobus fulgidus in Escherichia coli Kim J, Kim SI, Hong E, Ryu Y Ref: Protein Expr Purif, 127:98, 2016 : PubMed
Heterologous proteins expressed in bacteria are used for numerous biotechnological applications. Escherichia coli is the most commonly used host for heterologous protein expression because of its many advantages. Researchers have been studying proteins from extremophiles heterologously expressed in E. coli because the proteins of extremophiles are strongly resistant to extreme conditions. In a previous study, a thermostable esterase Est-AF was isolated from Archaeoglobus fulgidus and expressed in E. coli. However, further studies of Est-AF were difficult owing to its low expression levels in E. coli. In this study, we used various strategies, such as changing the expression vector and host strain, codon optimization, and optimization of induction conditions, to increase the expression of Est-AF. Through codon optimization and by changing the vector and host strain, Est-AF expression was increased from 31.50 +/- 0.35 mg/L to 61.75 +/- 0.28 mg/L. The optimized expression system consisted of a codon-optimized Est-AF gene in a pET28a(+)-based expression plasmid in E. coli Rosetta cells. The expression level was further increased by optimizing the induction conditions. The optimized conditions were induction with 0.4 mM isopropyl-b-d-1-thiogalactoside (IPTG) at 37 degrees C for 5 h. Under these conditions, the expression level of Est-AF was increased from 31.5 +/- 0.35 mg/L to 119.52 +/- 0.34 mg/L.
Title: Improved enantioselectivity of thermostable esterase from Archaeoglobus fulgidus toward (S)-ketoprofen ethyl ester by directed evolution and characterization of mutant esterases Kim J, Kim S, Yoon S, Hong E, Ryu Y Ref: Applied Microbiology & Biotechnology, 99:6293, 2015 : PubMed
Thermostable esterases have potential applications in various biotechnology industries because of their resistance to high temperature and organic solvents. In a previous study, we isolated an esterase from Archaeoglobus fulgidus DSM 4304 (Est-AF), which showed high thermostability but low enantioselectivity toward (S)-ketoprofen ethyl ester. (R)-ketoprofenor (S)-ketoprofenis produced by esterase hydrolysis of the ester bond of (R,S)-ketoprofen ethyl ester and (S)-ketoprofen has better pharmaceutical activity and lower side effects than (R)-ketoprofen. Therefore, we have generated mutants of Est-AF that retained high thermostability whilst improving enantioselectivity. A library of Est-AF mutants was created by error-prone polymerase chain reaction, and mutants with improved enantioselectivity were isolated by site-saturation mutagenesis. The regions of Est-AF containing amino acid mutations were analyzed by homology modeling of its three-dimensional structure, and structure-based explanations for the changes in enantioselectivity are proposed. Finally, we isolated two mutants showing improved enantioselectivity over Est-AF (ee% = -16.2 +/- 0.2 and E = 0.7 +/- 0.0): V138G (ee% = 35.9 +/- 1.0 and E = 3.0 +/- 0.1) and V138G/L200R (ee% = 89.2 +/- 0.2 and E = 19.5 +/- 0.5). We also investigated various characteristics of these mutants and found that the mutants showed similar thermostability and resistance to additives or organic solvents to Est-AF, without a significant trade-off between activity and stability.
Title: Cloning and characterization of a novel thermostable esterase from Bacillus gelatini KACC 12197 Kim J, Deng L, Hong E, Ryu Y Ref: Protein Expr Purif, 116:90, 2015 : PubMed
A novel gene encoding a thermostable esterase (designated as Est-gela) was isolated from the moderate thermophile Bacillus gelatini KACC 12197. The open reading frame of this gene (1170bp) encodes 389 amino acid residues, and the molecular weight of Est-gela is approximately 42kDa. The protein sequence of Est-gela shows similarity with beta-lactamases and esterases (43%). Est-gela contains the Ser-X-X-Lys conserved sequence (Ser58-Met59-Thr60-Lys61) and belongs to family VIII of esterases. We overexpressed Est-gela in Escherichia coli XL1-blue and purified this protein using a His tag. Est-gela showed a strong enzymatic activity toward p-nitrophenyl esters with short acyl chains (C4) and the strongest activity toward p-nitrophenyl butyrate. Est-gela showed an enhanced enzymatic activity at 65-75 degrees C and retained more than 90% of the activity after incubation at 65 degrees C for 180min. These results indicated that Est-gela was thermostable. In addition, Est-gela showed the maximal activity at pH 10. We also evaluated the effects of surfactants and organic solvents. Surfactants were more effective at improving the enzymatic activity than were organic solvents. Finally, Est-gela hydrolyzed (R,S)-ketoprofen ethyl ester (Kcat/Km=5.0+/-0.2s(-1)mM(-1), mean+/-standard error) with enantioselectivity toward (S)-ketoprofen ethyl ester rather than (R)-ketoprofen ethyl ester.
Title: Lithium, phenserine, memantine and pioglitazone reverse memory deficit and restore phospho-GSK3beta decreased in hippocampus in intracerebroventricular streptozotocin induced memory deficit model Ponce-Lopez T, Liy-Salmeron G, Hong E, Meneses A Ref: Brain Research, 1426:73, 2011 : PubMed
Intracerebroventricular (ICV) streptozotocin (STZ) treated rat has been described as a suitable model for sporadic Alzheimer's disease (AD). Central application of STZ has demonstrated behavioral and neurochemical features that resembled those found in human AD. Chronic treatments with antioxidants, acetylcholinesterase (AChE) inhibitors, or improving glucose utilization drugs have reported a beneficial effect in ICV STZ-treated rats. In the present study the post-training administration of a glycogen synthase kinase (GSK3) inhibitor, lithium; antidementia drugs: phenserine and memantine, and insulin sensitizer, pioglitazone on memory function of ICV STZ-rats was assessed. In these same animals the phosphorylated GSK3beta (p-GSK3beta) and total GSK3beta levels were determined, and importantly GSK3beta regulates the tau phosphorylation responsible for neurofibrillary tangle formation in AD. Wistar rats received ICV STZ application (3mg/kg twice) and 2 weeks later short- (STM) and long-term memories (LTM) were assessed in an autoshaping learning task. Animals were sacrificed immediately following the last autoshaping session, their brains removed and dissected. The enzymes were measured in the hippocampus and prefrontal cortex (PFC) by western blot. ICV STZ-treated rats showed a memory deficit and significantly decreased p-GSK3beta levels, while total GSK3beta did not change, in both the hippocampus and PFC. Memory impairment was reversed by lithium (100mg/kg), phenserine (1mg/kg), memantine (5mg/kg) and pioglitazone (30 mg/kg). The p-GSK3beta levels were restored by lithium, phenserine and pioglitazone in the hippocampus, and restored by lithium in the PFC. Memantine produced no changes in p-GSK3beta levels in neither the hippocampus nor PFC. Total GSK3beta levels did not change with either drug. Altogether these results show the beneficial effects of drugs with different mechanisms of actions on memory impairment induced by ICV STZ, and restored p-GSK3beta levels, a kinase key of signaling cascade of insulin receptor.
