Title: Comparison of neuromuscular blockade recovery co-administered with neostigmine and different doses of calcium gluconate: a randomized control trial Choi SR, Kim JH, Lee KH, Park SY Ref: BMC Anesthesiol, 21:93, 2021 : PubMed
BACKGROUND: Calcium increases the probability of transmitter release at the neuromuscular junction. It is not known whether there is a dose-dependent relationship between the dosage of calcium gluconate and the probability of transmitter release for non-depolarizing neuromuscular blockade (NMB) recovery by acetylcholinesterase inhibitors (AchEIs). This study compared the neuromuscular recovery time and the incidence of postoperative residual curarization (PORC) according to the dosage of calcium gluconate co-administered with neostigmine in three patient groups. METHODS: Patients were randomly allocated to a control group, a 5 mg/kg calcium gluconate group (calcium 5 group), or a 10 mg/kg calcium gluconate group (calcium 10 group). In patients with a TOF ratio (TOFr) between 0.2-0.7, 0.04 mg/kg of neostigmine was administered and both 0.2 mg of glycopyrrolate and 0.4 mg of atropine per 1 mg of neostigmine were administered. And additional 5 or 10 mg/kg of calcium gluconate were administrated to the calcium 5 and 10 groups. The primary endpoint was neuromuscular recovery time (the time between reversal and TOFr<=0.9). The secondary endpoints were the incidence of PORC at 5, 10, and 20 min after reversal administration and the train-of-four ratio (TOFr) at each time point. RESULTS: The neuromuscular recovery time was 5.3 min in the control group, 3.9 min in the calcium 5 group, and 4.1 min in the calcium 10 group, respectively (P = 0.004). The incidence of PORC at 5 min after neostigmine administration was 12 in the control group, 4 in the calcium 5 group, and 4 in the calcium 10 group, respectively, with statistical significance (P = 0.014). CONCLUSIONS: The co-administration of calcium gluconate with neostigmine safely promoted early NMB recovery, and the neuromuscular recovery time of the calcium 10 group tended to be more evenly distributed than that of the calcium 5 group. TRIAL REGISTRATION: https://cris.nih.go.kr/cris/index.jsp(KCT0004182 ). Date of registration: August 122,019.
Title: Synthesis and biological evaluation of 1,2,4-triazolidine-3-thiones as potent acetylcholinesterase inhibitors: in vitro and in silico analysis through kinetics, chemoinformatics and computational approaches Mahajan PG, Dige NC, Vanjare BD, Raza H, Hassan M, Seo SY, Kim CH, Lee KH Ref: Mol Divers, 24:1185, 2020 : PubMed
We have designed and synthesized a novel acidic ionic liquid and explored its catalytic efficiency for the synthesis of 1,2,4-triazolidine-3-thione derivatives. A simple reaction between aldehydes and thiosemicarbazide for short time in 60:40 v/v water/ethanol at room temperature offers target 1,2,4-triazolidine-3-thione derivatives. The formation of target compounds is confirmed by NMR, IR and ESI-MS analysis. Pleasingly, synthesized compounds show noteworthy acetylcholinesterase (AChE) inhibitory activity with much lower IC(50) values 0.0269 +/- 0.0021-1.1725 +/- 0.0112 microM than standard Neostigmine methylsulphate. In addition, synthesized 1,2,4-triazolidine-3-thiones exhibits significant free radical scavenging activity as compared to standard vitamin C. The studies on validation of Lipinski's rule through chemoinformatics properties and molecular docking analysis are in support of in vitro analysis. Therefore, overall present study illustrates synthesis of some new 1,2,4-triazolidines-3-thiones which can serve as a template for drug designing such as AChE inhibitors. Herein, we proposed ionic liquid-catalyzed ease of synthetic approach for medicinally important 1,2,4-triazolidine-3-thiones and their bio-evaluations.
We extracted 15 pterosin derivatives from Pteridium aquilinum that inhibited beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) and cholinesterases involved in the pathogenesis of Alzheimer's disease (AD). (2R)-Pterosin B inhibited BACE1, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) with an IC50 of 29.6, 16.2 and 48.1 microM, respectively. The Ki values and binding energies (kcal/mol) between pterosins and BACE1, AChE, and BChE corresponded to the respective IC50 values. (2R)-Pterosin B was a noncompetitive inhibitor against human BACE1 and BChE as well as a mixed-type inhibitor against AChE, binding to the active sites of the corresponding enzymes. Molecular docking simulation of mixed-type and noncompetitive inhibitors for BACE1, AChE, and BChE indicated novel binding site-directed inhibition of the enzymes by pterosins and the structure-activity relationship. (2R)-Pterosin B exhibited a strong BBB permeability with an effective permeability (Pe) of 60.3x10(-6) cm/s on PAMPA-BBB. (2R)-Pterosin B and (2R,3 R)-pteroside C significantly decreased the secretion of Abeta peptides from neuroblastoma cells that overexpressed human beta-amyloid precursor protein at 500 muM. Conclusively, our study suggested that several pterosins are potential scaffolds for multitarget-directed ligands (MTDLs) for AD therapeutics.
Accurate and complete genome sequences are essential in biotechnology to facilitate genome-based cell engineering efforts. The current genome assemblies for Cricetulus griseus, the Chinese hamster, are fragmented and replete with gap sequences and misassemblies, consistent with most short-read-based assemblies. Here, we completely resequenced C. griseus using single molecule real time sequencing and merged this with Illumina-based assemblies. This generated a more contiguous and complete genome assembly than either technology alone, reducing the number of scaffolds by >28-fold, with 90% of the sequence in the 122 longest scaffolds. Most genes are now found in single scaffolds, including up- and downstream regulatory elements, enabling improved study of noncoding regions. With >95% of the gap sequence filled, important Chinese hamster ovary cell mutations have been detected in draft assembly gaps. This new assembly will be an invaluable resource for continued basic and pharmaceutical research.
Title: Facile Synthesis, Crystal Structure, DFT Calculation and Biological Activities of 4-(2-fluorophenyl)-3-(3-methoxybenzyl)-1H-1,2,4-triazol-5 (4H)-one (5) Saleem M, Rafiq M, Jeong YK, Cho DW, Kim CH, Seo SY, Choi CS, Hong SK, Lee KH Ref: Med Chem, 14:451, 2018 : PubMed
BACKGROUND: In the past few decades, design, synthesis, and characterization of novel heterocyclic compounds with auspicious biological profile received the considerable attention of the scientific community. Among them, the small and simple organic molecular backbone like triazole moiety have a broad spectrum of applications in the medicinal as well as diagnostic areas. OBJECTIVE: The objective of present study was synthesis, characterization, and exploration of biological profile of 4-(2-fluorophenyl)-3-(3-methoxybenzyl)-1H-1,2,4-triazole-5(4H)-one (5). The tautomeric interconversion of the molecule was observed by the single crystal XRD and DFT analysis. METHODS: N-(2-fluorophenyl)-2-[2-(3-methoxyphenyl)acetyl]hydrazine carboxamide (4) was synthesized by the condensation of 2-(3-methoxyphenyl)acetohydrazide (3) with 1-fluoro-2- isocyanatobenzene. The dehydrocyclization of compound (4) yielded target compound (5) by refluxing in 2 N aqueous sodium hydroxide solutions. The target molecule was characterized by FTIR, 1H NMR, 13C NMR, single crystal X-ray diffraction analysis and DFT calculation. The enzymatic assay measurements were carried out by using a microplate reader (OPTI Max, Tunable Microplate Reader; Wavelength range: 340-850 nm; for 96-well plates) while DFT calculation was performed by Gaussian 09 package. RESULTS: The XRD result and DFT calculations showed that molecule 5 predominantly exists in thione conformation and crystallized in the triclinic system of P-1 space group. Furthermore, for the practical applicability of synthesized compound 5, the in vitro acetylcholinesterase as well as alpha-glucosidase inhibition activities were performed and found moderate enzyme inhibition potential comparable with that of reference inhibitors. CONCLUSION: This study might be helpful for future design and development of potent enzyme inhibitor to control Alzheimer's as well as diabetic disease. The DFT and single crystal XRD analysis data might be helpful for understanding the mechanism of drug binding and its mode of action.
Title: Knockout of a difficult-to-remove CHO host cell protein, lipoprotein lipase, for improved polysorbate stability in monoclonal antibody formulations Chiu J, Valente KN, Levy NE, Min L, Lenhoff AM, Lee KH Ref: Biotechnol Bioeng, 114:1006, 2017 : PubMed
While the majority of host cell protein (HCP) impurities are effectively removed in typical downstream purification processes, a small population of HCPs are particularly challenging. Previous studies have identified HCPs that are challenging for a variety of reasons. Lipoprotein lipase (LPL)-a Chinese hamster ovary (CHO) HCP that functions to hydrolyze esters in triglycerides-was one of ten HCPs identified in previous studies as being susceptible to retention in downstream processing. LPL may degrade polysorbate 80 (PS-80) and polysorbate 20 (PS-20) in final product formulations due to the structural similarity between polysorbates and triglycerides. In this work, recombinant LPL was found to have enzymatic activity against PS-80 and PS-20 in a range of solution conditions that are typical of mAb formulations. LPL knockout CHO cells were created with CRISPR and TALEN technologies and resulting cell culture harvest fluid demonstrated significantly reduced polysorbate degradation without significant impact on cell viability when compared to wild-type samples. Biotechnol. Bioeng. 2017;114: 1006-1015. (c) 2016 Wiley Periodicals, Inc.
Title: Acetylcholinesterase immobilization and characterization, and comparison of the activity of the porous silicon-immobilized enzyme with its free counterpart Saleem M, Rafiq M, Seo SY, Lee KH Ref: Bioscience Reports, 36:, 2016 : PubMed
A successful prescription is presented for acetylcholinesterase physically adsorbed on to a mesoporous silicon surface, with a promising hydrolytic response towards acetylthiocholine iodide. The catalytic behaviour of the immobilized enzyme was assessed by spectrophotometric bioassay using neostigmine methyl sulfate as a standard acetycholinesterase inhibitor. The surface modification was studied through field emission SEM, Fourier transform IR spectroscopy, energy-dispersive X-ray spectroscopy, cathode luminescence and X-ray photoelectron spectroscopy analysis, photoluminescence measurement and spectrophotometric bioassay. The porous silicon-immobilized enzyme not only yielded greater enzyme stability, but also significantly improved the native photoluminescence at room temperature of the bare porous silicon architecture. The results indicated the promising catalytic behaviour of immobilized enzyme compared with that of its free counterpart, with a greater stability, and that it aided reusability and easy separation from the reaction mixture. The porous silicon-immobilized enzyme was found to retain 50% of its activity, promising thermal stability up to 90 degrees C, reusability for up to three cycles, pH stability over a broad pH of 4-9 and a shelf-life of 44 days, with an optimal hydrolytic response towards acetylthiocholine iodide at variable drug concentrations. On the basis of these findings, it was believed that the porous silicon-immobilized enzyme could be exploited as a reusable biocatalyst and for screening of acetylcholinesterase inhibitors from crude plant extracts and synthesized organic compounds. Moreover, the immobilized enzyme could offer a great deal as a viable biocatalyst in bioprocessing for the chemical and pharmaceutical industries, and bioremediation to enhance productivity and robustness.
Title: PET Radioligands for Imaging of Tau Pathology: Current Status Choe YS, Lee KH Ref: Nucl Med Mol Imaging, 49:251, 2015 : PubMed
The incidence of Alzheimer's disease (AD), a progressive neurodegenerative disorder, continues to soar with the rapid growth of the elderly population, thus creating an enormous social and economic burden. Although disease-modifying drugs to treat AD are not yet available, several candidate drugs are in clinical trials. Most of these drugs are expected to be effective at the early stages of the disease, and therefore the early and accurate diagnosis of AD will be a critical factor in efforts to improve the prognosis of patients with AD. This review focuses on lead radioligands developed to date and their preclinical data in order to facilitate the development of tau-specific positron emission tomography radioligands that are of great interest to the scientific community.
Title: ACETYLCHOLINESTERASE INHIBITION ACTIVITY OF SOME QUINOLINYL SUBSTITUTED TRIAZOLOTHIADIAZOLE DERIVATIVES Rafiq M, Abbas Q, Saleem M, Hanif M, Lee KH, Seo SY Ref: Bioorganicheskaia Khimiia, 41:195, 2015 : PubMed
A series of aralkanoic acids was converted into aralkanoic acid hydrazides through their esters formation. The aralkanoic acid hydrazides upon treatment with carbon disulfide and methanolic potassium hydroxide yielded potassium dithiocarbazinate salts, which on refluxing with aqueous hydrazine hydrate yielded 5-aralkyl-4-amino-3-mercapto-1,2,4-triazoles. The target compounds, 3-aralkyl-6-(substitutedquinolinyl)[1,2,4]triazolo[3,4-b][1,3,4]thiadiazoles, were synthesized by condensing various quinolinyl substituted carboxylic acids with 5-aralkyl-4-amino-3-mercapto-1,2,4-triazoles in phosphorus oxychloride. The structures of the newly synthesized triazolothiadiazoles were characterized by IR, 1H NMR, 13C NMR, and elemental analysis studies. The structure of one of the 5-aralkyl-4-amino-3-mercapto-1,2,4-triazoles was unambiguously deduced by single crystal X-ray diffraction analysis. All the synthesized compounds were screened for their acetylcholinesterase inhibition activities. Four of the triazolothiadiazoles exhibited excellent acetylcholinesterase inhibition activities as compared to the reference inhibitor.
Micrometre- and submicrometre-size functionalized beads are frequently used to capture targets of interest from a biological sample for biological characterizations and disease diagnosis. The main challenge of the microbead-based assay is in the immobilization of probe molecules onto the microbead surfaces. In this paper, we report a versatile droplet microfluidics method to fabricate alginate microspheres while simultaneously immobilizing anti-Mycobacterium tuberculosis complex IgY and anti-Escherichia coli IgG antibodies primarily on the porous alginate carriers for specific binding and binding affinity tests. The binding affinity of antibodies is directly measured by fluorescence intensity of stained target bacteria on the microspheres. We demonstrate that the functionalized alginate microspheres yield specificity comparable with an enzyme-linked immunosorbent assay. The high surface area-to-volume ratio of the functionalized porous alginate microspheres improves the detection limit. By using the droplet microfluidics, we can easily modify the size and shape of alginate microspheres, and increase the concentration of functionalized alginate microspheres to further enhance binding kinetics and enable multiplexing.
Meroterpenoids are natural products produced from polyketide and terpenoid precursors. A gene targeting system for A. terreus NIH2624 was developed, and a gene cluster for terretonin biosynthesis was characterized. The intermediates and shunt products were isolated from the mutant strains, and a pathway for terretonin biosynthesis is proposed. Analysis of two meroterpenoid pathways corresponding to terretonin in A. terreus and austinol in A. nidulans reveals that they are closely related evolutionarily.
Title: Binding of 2-[18F]fluoro-CP-118,954 to mouse acetylcholinesterase: microPET and ex vivo Cerenkov luminescence imaging studies Kim DH, Choe YS, Choi JY, Lee KH, Kim BT Ref: Nucl Med Biol, 38:541, 2011 : PubMed
Acetylcholinesterase (AChE) has been an important cholinergic factor for the diagnosis of Alzheimer's disease (AD), because of reduced AChE activity in the postmortem brains of AD patients. We previously developed 5,7-dihydro-3-(2-(1-(2-[(18)F]fluorobenzyl)-4-piperidinyl)ethyl)-6H-pyrrolo(3,2,f )-1,2-benzisoxazol-6-one (2-[(18)F]fluoro-CP-118,954) for in vivo studies of AChE in mice. In the present study, we automated the synthesis of 2-[(18)F]fluoro-CP-118,954 for the routine use and evaluated the radioligand by microPET and ex vivo Cerenkov luminescence imaging of mouse AChE. 4-[(18)F]Fluoro-donepezil, another AChE inhibitor, was used for comparison. Automated syntheses of 2-[(18)F]fluoro-CP-118,954 and 4-[(18)F]fluoro-donepezil resulted in high radiochemical yields (25-33% and 30-40%) and high specific activity (27.1-35.4 and 29.7-37.3 GBq/mumol). Brain microPET images of two ICR mice injected with 2-[(18)F]fluoro-CP-118,954 demonstrated high uptake in the striatum (ROI analysis: 5.1 %ID/g for the first 30 min and 4.1 %ID/g for another 30 min), and a blocking study with injection of CP-118,954 into one of the mice at 30 min after radioligand injection led to complete blocking of radioligand uptake in the striatum (ROI analysis: 1.9 %ID/g), whereas (18)F-labeled donepezil did not show specific uptake in the striatum. In another set of experiments, the brain tissues (striatum, parietal cortex, frontal cortex and cerebellum) were excised after brain microPET/CT imaging of mouse injected with 2-[(18)F]fluoro-CP-118,954, and a high striatal uptake was also detected in ex vivo optical and microPET images (ROI analysis: 1.4 %ID/g) and in gamma-counting data (2.1 %ID/g at 50 min post-injection) of the brain tissues. Taken together, these results demonstrated that 2-[(18)F]fluoro-CP-118,954 specifically binds to AChE in mouse brains.
The interaction between fermentation-respiration switch (FrsA) protein and glucose-specific enzyme IIA(Glc) increases glucose fermentation under oxygen-limited conditions. We show that FrsA converts pyruvate to acetaldehyde and carbon dioxide in a cofactor-independent manner and that its pyruvate decarboxylation activity is enhanced by the dephosphorylated form of IIA(Glc) (d-IIA(Glc)). Crystal structures of FrsA and its complex with d-IIA(Glc) revealed residues required for catalysis as well as the structural basis for the activation by d-IIA(Glc).
Vibrio vulnificus is the causative agent of life-threatening septicemia and severe wound infections. Here, we announce the complete annotated genome sequence of V. vulnificus MO6-24/O, isolated from a patient with septicemia. When it is compared with previously known V. vulnificus genomes, the genome of this bacterium shows a unique genetic makeup, including phagelike elements, carbohydrate metabolism-related genes, and the superintegron.
Chinese hamster ovary (CHO)-derived cell lines are the preferred host cells for the production of therapeutic proteins. Here we present a draft genomic sequence of the CHO-K1 ancestral cell line. The assembly comprises 2.45 Gb of genomic sequence, with 24,383 predicted genes. We associate most of the assembled scaffolds with 21 chromosomes isolated by microfluidics to identify chromosomal locations of genes. Furthermore, we investigate genes involved in glycosylation, which affect therapeutic protein quality, and viral susceptibility genes, which are relevant to cell engineering and regulatory concerns. Homologs of most human glycosylation-associated genes are present in the CHO-K1 genome, although 141 of these homologs are not expressed under exponential growth conditions. Many important viral entry genes are also present in the genome but not expressed, which may explain the unusual viral resistance property of CHO cell lines. We discuss how the availability of this genome sequence may facilitate genome-scale science for the optimization of biopharmaceutical protein production.
Candida rugosa lipase was immobilized on amino-functionalized magnetic supports via cross-linked enzyme aggregates (CLEA) and used to enhance the enzymatic degradation of polycaprolactone (PCL). The maximum amounts of lipase immobilized on the magnetic beads using glutaraldehyde as a coupling agent were determined to be 33.7 mg/g of beads with an 81% recovery of activity after immobilization. Compared to the free enzyme, the immobilized lipase showed the optimum pH at 1 unit higher (pH 8.0) and also retained its enzymatic activity at higher temperatures. There was 62.9% retention of lipase activity after 30 consecutive reuses, indicating its stability and reusability in aqueous media. Moreover, the immobilized lipase maintained more than 80% of its initial activity during 30 days storage period, while the free lipase lost all under same condition. In addition, the immobilized lipase showed a more than 6-fold increase in biodegradability over the free lipase when the immobilized lipase was used to degrade PCL in a batch system. Higher thermal and storage stability, as well as good durability after repeated use of the immobilized lipase CLEA, highlights its potential applicability as large scale continuous systems for the enzymatic degradation of PCL.
Title: Biosynthesis of glycerol carbonate from glycerol by lipase in dimethyl carbonate as the solvent Lee KH, Park CH, Lee EY Ref: Bioprocess Biosyst Eng, 33:1059, 2010 : PubMed
Glycerol carbonate was synthesized from renewable glycerol and dimethyl carbonate using lipase in solvent-free reaction system in which excess dimethyl carbonate played as the reaction medium. A variety of lipases have been tested for their abilities to catalyze transesterification reaction, and Candida antartica lipase B and Novozyme 435 exhibited higher catalytic activities. The silica-coated glycerol with a 1:1 ratio was supplied to prevent two-phase formation between hydrophobic dimethyl carbonate and hydrophilic glycerol. Glycerol carbonate was successfully synthesized with more than 90% conversion from dimethyl carbonate and glycerol with a molar ratio of 10 using Novozyme 435-catalyzed transesterification at 70 degrees C. The Novozyme 435 [5% (w/w) and 20% (w/w)] and silica gel were more than four times recycled with good stability in a repeated batch operation for the solvent-free synthesis of glycerol carbonate.
BACKGROUND/AIMS: Organophosphate poisoning has a high mortality rate. Recently, differences among organophosphorus insecticides in human self-poisoning were reported. This study investigated the prognostic risk factors and the mortality of different organophosphates following acute organophosphate poisoning. METHODS: This retrospective study included 68 patients with acute organophosphate poisoning. We investigated patient survival according to initial parameters, including the initial Acute Physiology and Chronic Health Evaluation (APACHE) II score, serum cholinesterase level, and hemoperfusion and evaluated the mortality according to organophosphate types. RESULTS: Thirteen of the 68 patients died. The agents responsible for mortality were different. The APACHE II score was a significant predictor of mortality (odds ratio [OR], 1.194; p<0.01; 95% confidence interval [CI], 1.089 to 1.309) and respiratory failure (OR, 1.273; p<0.01; 95% CI, 1.122 to 1.444). The mortality was 0% for dichlorvos, malathion, chlorpyrifos and profenofos. However, other organophosphates showed different mortality (16.7% for O-ethyl-O-4-nitrophenyl phenylphosphonothioate, 25% for phenthoate, 37.5% for phosphamidon, 50% for methidathion). The usefulness of hemoperfusion appears to be limited. CONCLUSIONS: The initial APACHE II score is a useful prognostic indicator, and different organophosphates have different mortality.
The effects of soy phytoestrogens on Morris water maze (MWM) performance and neuronal cholinergic enzyme activities and immunoreactivity were studied in ovariectomized (OVX) rats. The rats were assigned to four groups fed control diet (CD), 3.9 mg/kg 17beta-estradiol diet (E2), 263.4 mg/kg soy phytoestrogens diet (SP1), and 526.9 mg/kg soy phytoestrogens diet (SP2). In the MWM task, escape latency and path length were significantly less in the E2 and SP2 groups than in the CD group on the second day. Choline acetyltransferase (ChAT) activity in the cerebral cortex and ChAT immunoreactivity in the diagonal band of Broca were significantly greater in the E2, SP1, and SP2 groups than in the CD group. Acetylcholinesterase activity in the hippocampus in the E2, SP1, and SP2 groups was significantly lower than in the CD group. This study suggests that soy phytoestrogens affect the reference memory and neuronal cholinergic system in OVX rats.
OBJECTIVES: Alzheimer's disease (AD) is characterized by reduced acetylcholinesterase (AChE) activity in the post-mortem tissues of AD patients. Therefore, AChE has been an attractive target for the diagnosis of AD. In the present study, 5,7-dihydro-3-[2-(1-(phenylmethyl)-4-piperidinyl)ethyl]-6H-pyrrolo[3,2-f]-1,2-ben zisoxazol-6-one (CP-118,954), a potent AChE inhibitor, was labelled with radioiodine and evaluated as an AChE imaging agent for SPECT. METHODS: Radioiodine-labelled CP-118,954 was prepared from CP-144,885 and [(125)I]iodobenzyl bromide, and anti-AChE activities of iodine-substituted CP-118,954 were measured. Metabolism studies were carried out in samples of blood and whole brain of mice injected with 2-[(123)I]iodo-CP-118,954 ((123)I-1). Tissue distribution studies were also performed in mice injected with I-1, and samples of blood, thyroid, stomach, and brain tissue (cerebellum, striatum and cortex) were removed, weighed and counted. RESULTS: Of the ligands, 2-iodo-CP-118,954 exhibited higher binding affinity for AChE (IC50=24 nM) than the other positional isomers. 2-[(125)I]Iodo-CP-118,954 was found to have a lipophilicity (log P=2.1) favouring brain permeability and metabolic stability in mouse brain, but a marginal target (striatum) to non-target (cerebellum) uptake ratio (1.1) in mouse brain. CONCLUSION: This result demonstrates that 2-[(125)I]iodo-CP-118,954 may be unsuitable for AChE imaging. These findings suggest that radioligands suitable for AChE imaging should have not only a specific structure but also a sub-nanomolar to low nanomolar IC50.
Title: Is subnanomolar binding affinity required for the in vivo imaging of acetylcholinesterase? Studies on 18F-labeled G379 Lee SY, Choe YS, Ryu EK, Iimura Y, Choi Y, Lee KH, Kim BT Ref: Nucl Med Biol, 33:91, 2006 : PubMed
Acetylcholinesterase (AChE) is an important cholinergic marker of Alzheimer's disease (AD) and shows reduced activity in postmortem AD brain tissues. 1-(4-Fluorobenzyl)-4-[(5,6-dimethoxy-1-oxoindan-2-fluoro-2-yl)methyl]piperidine (G379, ), an AChE inhibitor with a subnanomolar IC(50) (0.56 nM), was prepared as a (18)F-labeled radioligand ([(18)F]) and evaluated in mice. Metabolism studies of [(18)F] showed no metabolites in the mouse brain. Tissue distribution studies demonstrated its uniform regional distribution in the mouse brain, suggesting that this radioligand is not suitable for the in vivo imaging of AChE. This result along with reports on radiolabeled N-benzylpiperidine lactam benzisoxazole (IC(50) < 1 nM) and other radiolabeled benzylpiperidine derivatives (IC(50) > 1 nM) suggested that a subnanomolar IC(50) may not be the only important factor in determining the suitability of a radioligand for in vivo studies of AChE.
5,7-Dihydro-3-[2-[1-(2-fluorobenzyl)-4-piperidinyl]ethyl]-6H-pyrrolo[3,2,f]-1,2-b enzisoxazol-6-one (2-flouro-CP-118,954; 1), a potent acetylcholinesterase (AChE) inhibitor, was prepared as a radioligand by reductive alkylation of CP-144,885 the debenzylated form of CP 118,954, with 2-[18F]fluorobenzaldehyde. The decay-corrected radiochemical yield was 25-30% and the effective specific activity was 41-53 GBq/micromol. Tissue distribution studies of 2-[18F]fluoro-CP-118,954 ([18F]1) in mice showed that the regional brain distribution correlated well with the known density of AChE in the mouse brain. A high level of uptake in the striatum was also shown at all time points in the olfactory tubercle, which is known to have dopaminergic neurons. Blocking studies showed that radioligand uptake in all brain regions was not altered by either the dopamine receptor antagonists or the sigma receptor agonist. On the other hand, radioligand uptake in both the striatum and the olfactory tubercle was significantly blocked (80%) by ligand 1. The low level of bone uptake over time suggested that [18F]1 underwent little in vivo metabolic defluorination. The lack of metabolite formation in the mouse brain indicated that the regional distribution was attributed to [18F]1. These results demonstrated that [18F]1 binds specifically and selectively to AChE in mice and appears to be a suitable radioligand for the in vivo mapping of AChE.
Title: Synthesis and evaluation of 5,7-dihydro-3-[2-[1-(4-[18F]-fluorobenzyl)-4-piperidinyl]ethyl]-6H-pyrrolo[3,2-f] -1,2-benzisoxazol-6-one for in vivo mapping of acetylcholinesterase Lee SY, Choe YS, Kim YR, Paik JY, Choi BW, Kim SE, Lee KH, Choi Y, Kim BT Ref: Nucl Med Commun, 25:591, 2004 : PubMed
OBJECTIVES: Acetylcholinesterase (AChE) is an important cholinergic marker for the diagnosis of Alzheimer's disease (AD). A recent study has demonstrated that C-labelled 5,7-dihydro-7-methyl-3-[2-[1-(phenylmethyl)-4-piperidinyl]ethyl]-6H-pyrrolo[3,2-f ]-1,2-benzisoxazol-6-one (CP-126,998) shows promising results. The demethylated form of this ligand (CP-118,954) is a more potent and selective inhibitor than CP-126,998. In this study, therefore, CP-118,954 was labelled with F and evaluated for the in vivo mapping of AChE. METHODS: The 4-fluoro (1). and 2-fluoro (2). derivatives of CP-118,954 were synthesized from 4-methyl-3-nitroanisole in 11 steps. Their in vitro binding affinities to AChE were measured using Ellman's method. The preparation of [F]-1 was carried out by reductive alkylation of the piperidine precursor with 4-[F]-fluorobenzaldehyde, followed by high-performance liquid chromatography (HPLC) purification. In vitro autoradiography was performed by incubating rat brain coronal slices with [F]-1. Tissue distribution studies were performed in mouse brain and the data were expressed as the percentage of the injected dose per gram of tissue (%ID x g). RESULTS: Two fluorine-substituted AChE inhibitors were synthesized and their in vitro binding data showed that the 4-fluoro and 2-fluoro derivatives (1 and 2) had similar or superior binding affinity to that of the unsubstituted ligand, CP-118,954. The F-labelled ligand was synthesized in 20-35% radiochemical yield (EOS) and with high effective specific activity (36-42 GBq x micromol). Autoradiography showed high uptake of [F]-1 in the striatum and this striatal uptake was completely inhibited by the unlabelled ligand 1. Tissue distribution studies demonstrated that high radioactivity was accumulated in the striatum, an AChE-rich region. CONCLUSIONS: This study demonstrates that [F]-1 may hold promise as a radioligand for the in vivo mapping of AChE.
In contrast to our increasingly detailed understanding of how synaptic plasticity provides a cellular substrate for learning and memory, it is less clear how a neuron's voltage-gated ion channels interact with plastic changes in synaptic strength to influence behavior. We find, using generalized and regional knockout mice, that deletion of the HCN1 channel causes profound motor learning and memory deficits in swimming and rotarod tasks. In cerebellar Purkinje cells, which are a key component of the cerebellar circuit for learning of correctly timed movements, HCN1 mediates an inward current that stabilizes the integrative properties of Purkinje cells and ensures that their input-output function is independent of the previous history of their activity. We suggest that this nonsynaptic integrative function of HCN1 is required for accurate decoding of input patterns and thereby enables synaptic plasticity to appropriately influence the performance of motor activity.
The 1,995,275-bp genome of Coxiella burnetii, Nine Mile phase I RSA493, a highly virulent zoonotic pathogen and category B bioterrorism agent, was sequenced by the random shotgun method. This bacterium is an obligate intracellular acidophile that is highly adapted for life within the eukaryotic phagolysosome. Genome analysis revealed many genes with potential roles in adhesion, invasion, intracellular trafficking, host-cell modulation, and detoxification. A previously uncharacterized 13-member family of ankyrin repeat-containing proteins is implicated in the pathogenesis of this organism. Although the lifestyle and parasitic strategies of C. burnetii resemble that of Rickettsiae and Chlamydiae, their genome architectures differ considerably in terms of presence of mobile elements, extent of genome reduction, metabolic capabilities, and transporter profiles. The presence of 83 pseudogenes displays an ongoing process of gene degradation. Unlike other obligate intracellular bacteria, 32 insertion sequences are found dispersed in the chromosome, indicating some plasticity in the C. burnetii genome. These analyses suggest that the obligate intracellular lifestyle of C. burnetii may be a relatively recent innovation.
In vitro metabolism of acetylcholinesterase inhibitors containing 3-[(18)F]fluoromethylbenzyl- ([(18)F]1) and 4-[(18)F]fluorobenzyl-piperidine moieties ([(18)F]2) was studied and compared with the in vivo metabolism. Defluorination of the [(18)F]1 mainly occurred to generate [(18)F]fluoride ion both in vitro and in vivo. In contrast, the [(18)F]2 was converted into an unknown polar metabolite in both metabolism methods and another metabolite, 4-[(18)F]fluorobenzoic acid in vitro. These results demonstrated that the in vitro method can be used to predict the in vivo metabolism of both radiotracers.
Title: Modulation of spindle oscillations by acetylcholine, cholecystokinin and 1S,3R-ACPD in the ferret lateral geniculate and perigeniculate nuclei in vitro Lee KH, McCormick DA Ref: Neuroscience, 77:335, 1997 : PubMed
The transition from sleep to waking is associated with the abolition of spindle waves and the appearance of tonic activity in thalamocortical neurons and thalamic reticular/perigeniculate GABAergic cells. We tested the possibility that changes such as these may arise through modulation of the leak potassium current, IKL, by examining the effects of neurotransmitters known to modulate this current on spindle wave generation in the ferret geniculate slice maintained in vitro. Local application of agents that reduce IKL in thalamocortical neurons, including acetylcholine, DL-muscarine chloride and the glutamate metabotropic receptor agonist 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD), to spontaneously spindling thalamocortical neurons resulted in a 5-10 mV membrane depolarization and the abolition of spindle waves. Local application of 1S,3R-ACPD and cholecystokinin-8-sulfate, both of which reduce IKL, to GABAergic neurons of the perigeniculate nucleus resulted in a 10-20 mV membrane depolarization, appearance of tonic discharge and the abolition of spindle wave generation. Local application of 1S,3R-ACPD and cholecystokinin to the perigeniculate nucleus while recording from thalamocortical neurons resulted in the abolition of spindle wave-associated inhibitory postsynaptic potentials and the occurrence of a continuous barrage of smaller amplitude inhibitory postsynaptic potentials, presumably in response to depolarization and tonic discharge of perigeniculate neurons. These results indicate that modulation of IKL in thalamocortical neurons and perigeniculate neurons is capable of abolishing the generation of spindle waves in thalamic networks. Through the modulation of IKL, ascending and descending activating systems may control the state of the thalamus such that the transition from slow wave sleep to waking is associated with the abolition of slow, synchronized rhythms and the facilitation of a state that is conducive to sensory receptor field analysis, arousal and perception.
Title: Antinociceptive activity of calcitonin and central cholinergic system: behavioural and neurochemical analyses Chen D, Lee KH Ref: Biochemical Pharmacology, 49:1623, 1995 : PubMed
Behavioural and neurochemical analyses were carried out to investigate the relationship between the antinociceptive activity of porcine calcitonin (pCT) and central cholinergic system in mice and rats. Behavioural studies revealed that the antinociceptive activity of pCT encapsulated in sulphatide-containing liposomes injected intravenously into mice was significantly inhibited by atropine sulphate, but not by atropine methylnitrate, and potentiated by physostigmine, but not by neostigmine. Neurochemical studies using rat brain synaptosomes showed that pCT stimulated synaptosomal sodium-dependent high-affinity choline uptake, which was found to be closely associated with acetylcholine (ACh) synthesis (50-60%). This effect was concentration-dependent. In addition, pCT elicited a biphasic effect on ACh release from synaptosomes with an initial brief period of stimulation and subsequent prolonged inhibition. This stimulation was not affected by atropine sulphate, but markedly reduced by incubation in the presence of diltiazem or in a calcium-free medium, indicating that the modulation of ACh release by the peptide may be mediated by calcium fluxes across the synaptosomal membrane independent of cholinergic receptor activation. However, pCT does not affect the activity of synaptosomal acetylcholinesterase. Therefore, the behavioural study in vivo with the neurochemical analysis in vitro suggests that the central cholinergic system may be involved in the antinociceptive activity of calcitonin.
Title: Acetylcholine excites GABAergic neurons of the ferret perigeniculate nucleus through nicotinic receptors Lee KH, McCormick DA Ref: Journal of Neurophysiology, 73:2123, 1995 : PubMed
1. The actions of acetylcholine (ACh) on the GABAergic neurons of the perigeniculate nucleus (PGN) were investigated with the use of extra- and intracellular recording techniques in spontaneously spindling ferret thalamic slices maintained in vitro. 2. Local application of ACh to PGN neurons resulted in rapid depolarization followed by a longer lasting hyperpolarization. Neither of these responses were abolished by blockade of synaptic transmission with tetrodotoxin (TTX) nor with low Ca2+ and elevated Mg2+ solution, indicating that they are direct postsynaptic actions of ACh on PGN cells. Functionally, the rapid depolarizing response could activate both single spike activity, as well as low-threshold Ca2+ spike-mediated bursts. 3. The fast depolarizing response to ACh was selectively blocked by application of the nicotinic antagonist hexamethonium, whereas the slow hyperpolarizing response to ACh was selectively blocked by application of the muscarinic antagonist (-)scopolamine. Application of both hexamethonium and (-)scopolamine blocked the modulation of PGN action-potential firing by ACh. 4. Local application of the nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium (DMPP) resulted in a depolarizing response and an increase in membrane conductance, whereas application of the muscarinic agonist DL-muscarine chloride resulted in a hyperpolarizing response and an increase in membrane conductance. When applied to spontaneously spindling PGN cells, both DMPP and DL-muscarine blocked the occurrence of spindle oscillations. However, only DMPP was associated with depolarization and the generation of single spike activity. 5. These results indicate that the GABAergic cells of the PGN possess postsynaptic nicotinic as well as muscarinic receptors.
