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Shao-Jun Zhao,1,2,* De-Hua Wang,1,2 Yan-Wei Li,1,2 Lei Han,1,2 Xing Xiao,1,2 Min Ma,3,* David Chi-Cheong Wan,4 An Hong,1,2 Yi Ma1,2 1Institute of Biomedicine, Department of Cellular Biology, Jinan University, 2National Engineering Research Center of Genetic Medicine, Key Laboratory of Bioengineering Medicine of Guangdong Province, Jinan University, 3College of traditional Chinese Medicine, Institute of Integrated Traditional Chinese and Western Medicine, Jinan University, Guangdong, 4School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, People’s Republic of China *These authors contributed equally to this work Abstract: A novel neuroendocrine peptide, pituitary adenylate cyclase activating peptide (PACAP), was found to have an important role in carbohydrate or lipid metabolism and was susceptible to dipeptidyl peptidase IV degradation. It can not only mediate glucose-dependent insulin secretion and lower blood glucose by activating VPAC2 receptor, but also raise blood glucose by promoting glucagon production by VPAC1 receptor activation. Therefore, its therapeutic application is restricted by the exceedingly short-acting half-life and the stimulatory function for glycogenolysis. Herein, we generated novel peptide-conjugated selenium nanoparticles (SeNPs; named as SCD), comprising a 32-amino acid PACAP-derived peptide DBAYL that selectively binds to VPAC2, and chitosan-modified SeNPs (SeNPs-CTS, SC) as slow-release carrier. The circulating half-life of SCD is 14.12 h in mice, which is 168.4- and 7.1-fold longer than wild PACAP (~5 min) and DBAYL (~1.98 h), respectively. SCD (10 nmol/L) significantly promotes INS-1 cell proliferation, glucose uptake, insulin secretion, insulin receptor expression and also obviously reduces intracellular reactive oxygen species levels in H2O2-injured INS-1 cells. Furthermore, the biological effects of SCD are stronger than Exendin-4 (a clinically approved drug through

Shao-Jun Zhao,1,2,* De-Hua Wang,1,2 Yan-Wei Li,1,2 Lei Han,1,2 Xing Xiao,1,2 Min Ma,3,* David Chi-Cheong Wan,4 An Hong,1,2 Yi Ma1,2 1Institute of Biomedicine, Department of Cellular Biology, Jinan University, 2National Engineering Research Center of Genetic Medicine, Key Laboratory of Bioengineering Medicine of Guangdong Province, Jinan University, 3College of traditional Chinese Medicine, Institute of Integrated Traditional Chinese and Western Medicine, Jinan University, Guangdong, 4School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, People’s Republic of China *These authors contributed equally to this work Abstract: A novel neuroendocrine peptide, pituitary adenylate cyclase activating peptide (PACAP), was found to have an important role in carbohydrate or lipid metabolism and was susceptible to dipeptidyl peptidase IV degradation. It can not only mediate glucose-dependent insulin secretion and lower blood glucose by activating VPAC2 receptor, but also raise blood glucose by promoting glucagon production by VPAC1 receptor activation. Therefore, its therapeutic application is restricted by the exceedingly short-acting half-life and the stimulatory function for glycogenolysis. Herein, we generated novel peptide-conjugated selenium nanoparticles (SeNPs; named as SCD), comprising a 32-amino acid PACAP-derived peptide DBAYL that selectively binds to VPAC2, and chitosan-modified SeNPs (SeNPs-CTS, SC) as slow-release carrier. The circulating half-life of SCD is 14.12 h in mice, which is 168.4- and 7.1-fold longer than wild PACAP (~5 min) and DBAYL (~1.98 h), respectively. SCD (10 nmol/L) significantly promotes INS-1 cell proliferation, glucose uptake, insulin secretion, insulin receptor expression and also obviously reduces intracellular reactive oxygen species levels in H2O2-injured INS-1 cells. Furthermore, the biological effects of SCD are stronger than Exendin-4 (a clinically approved drug through

The inhibition of fatty acid binding protein 4 (FABP4) by using small molecules could potentially provide therapeutic opportunities for metabolic disorders treatment. According to the results of our in-house virtual screening on the herbal molecules database, this study reports flavonols as an ideal scaffold for FABP4 inhibitors development. Among the popular flavonols examined, we identified hyperoside as a promising FABP4 inhibitor. Identical to the well-known FABP4 inhibitor BMS309403, hyperoside induced lipid accumulation and upregulated peroxisome proliferator-activated receptor γ (PPARγ) protein expression during the adipocyte differentiation process. Furthermore, both PPARγ antagonist and FABP4 overexpression attenuated hyperoside-induced adipogenesis, indicating that hyperoside promoted adipogenesis in adipocytes via the FABP4/PPARγ pathway. We anticipate hyperoside to be a promising, novel FABP4 inhibitor for antidiabetic drug development. © 2015 Elsevier Ltd.

Background Traditionally, molecular docking is primarily employed to screen pure compounds; the top-ranking chemicals are subsequently selected for experimental validation. Unlike synthetic chemicals, most natural products are commercially unavailable. The isolation and purification of each natural product is extremely time-consuming, which has restricted the screening of lead compounds from natural products. Purpose We developed a protocol, Herbalog, to facilitate the identification of bioactive phytochemicals through molecular docking. Methods We wrote a script using Python and Autodock Vina for docking; ligand displacement and adipolysis assays were used to determine the anti-fatty acid binding protein (FABP) 4 activity of bioactive extracts. An ultraperformance liquid chromatography quadrupole time-of-flight mass spectrometry system was applied for identifying major peaks of bioactive extracts. Results Herbalog, a natural product database, contains 5,112 phytochemicals from 197 common herbs and a script that counts the number of hits from docking in each herb and calculates the hit rate of herbs. Herbalog prioritizes herbs according to their hit rates, and top-ranking herb candidates contain a large repertoire of hits. We used Herbalog as a screening tool and identified labdane diterpenoids from Andrographis paniculata as leading candidates of FABP4 inhibitors. Conclusion Herbalog facilitates the discovery of herbs that possess the highest number of inhibitors or activators against target proteins, which reduces the sample preparation time for preliminary validation. © 2016 Elsevier GmbH

Background Traditionally, molecular docking is primarily employed to screen pure compounds; the top-ranking chemicals are subsequently selected for experimental validation. Unlike synthetic chemicals, most natural products are commercially unavailable. The isolation and purification of each natural product is extremely time-consuming, which has restricted the screening of lead compounds from natural products. Purpose We developed a protocol, Herbalog, to facilitate the identification of bioactive phytochemicals through molecular docking. Methods We wrote a script using Python and Autodock Vina for docking; ligand displacement and adipolysis assays were used to determine the anti-fatty acid binding protein (FABP) 4 activity of bioactive extracts. An ultraperformance liquid chromatography quadrupole time-of-flight mass spectrometry system was applied for identifying major peaks of bioactive extracts. Results Herbalog, a natural product database, contains 5,112 phytochemicals from 197 common herbs and a script that counts the number of hits from docking in each herb and calculates the hit rate of herbs. Herbalog prioritizes herbs according to their hit rates, and top-ranking herb candidates contain a large repertoire of hits. We used Herbalog as a screening tool and identified labdane diterpenoids from Andrographis paniculata as leading candidates of FABP4 inhibitors. Conclusion Herbalog facilitates the discovery of herbs that possess the highest number of inhibitors or activators against target proteins, which reduces the sample preparation time for preliminary validation.

Pimozide is a conventional antipsychotic of the diphenylbutylpiperidine class that has been clinically used for over 30 years. The obvious side effect of this drug is weight gain. However, the mechanism of pimozide-induced weight gain is still unknown. In the present study, we identified pimozide as a novel fatty acid binding protein 4 (FABP4) inhibitor using molecular docking simulation as well as biochemical, characterizations. BMS309403, a well-known FABP4 inhibitor, elevated the basal protein levels of PPAR gamma, therefore stimulating adipogenesis in adipocytes. The present study showed that the inhibitory effect of pimozide on FABP4 promoted adipocyte differentiation with the potency proportional to their propensities for weight gain. These effects in adipogenesis by pimozide may help to explain the Weight gain that is frequently observed in patients treated with pimozide.

Ethnopharmacological relevance: With the prevalent use of highly active antiretroviral therapy (HAART) for AIDS patients since 1996, the mortality of HIV/AIDS patients has been remarkably decreased. With long-term use of HAART, drug resistance and side effects of antiretrovirals have been frequently reported, which not only reduce the efficacy, but also decreases the tolerance of patients. Traditional herbal medicine has become more popular among HIV/AIDS patients as adjuvant therapy to reduce these adverse effects of HAART. SH formula is a Chinese herbal formula consisting of five traditional Chinese herbs including Morus alba L, Glycyrrhiza glabra L, Artemisia capillaris Thumb., Astragalus membranaceus Bge., and Carthamus tinctorius L. SH formula is clinically used for HIV treatment in Thailand. However, the possible pharmacokinetic interactions between these Chinese herbs and antiretroviral drugs have not been well documented. The aim of this study was to investigate the potential herb-drug interaction between SH herbal Chinese formula and the antiretroviral drug atazanavir (ATV); Materials and methods: The combination effect of SH formula and ATV on HIV protease was studied in HIV-1 protease inhibition assay in vitro. The inhibition of SH formula on rat CYP3A2 was assessed by detecting the formation of 1'-OH midazolam from midazolam in rat liver microsomes in vitro. The in vivo pharmacokinetic interaction between SH formula and ATV was investigated by measuring time-dependent plasma concentrations of ATV in male Sprague-Dawley rats with liquid chromatography-mass spectrometry; Results: Through the in vitro HIV-1 protease inhibition assay, combination of SH formula (41.7-166.7 mu g/ml) and ATV (16.7-333 ng/ml) showed additive inhibition on HIV-1 protease activity than SH formula or ATV used alone. In vitro incubation assay indicated that SH formula showed a weak inhibition (IC50=231.2 mu g/ml; Ki=98.2 mu g/ml) on CYP3A2 activity in rat liver microsomes. In vivo p

Ethnopharmacological relevance: With the prevalent use of highly active antiretroviral therapy (HAART) for AIDS patients since 1996, the mortality of HIV/AIDS patients has been remarkably decreased. With long-term use of HAART, drug resistance and side effects of antiretrovirals have been frequently reported, which not only reduce the efficacy, but also decreases the tolerance of patients. Traditional herbal medicine has become more popular among HIV/AIDS patients as adjuvant therapy to reduce these adverse effects of HAART. SH formula is a Chinese herbal formula consisting of five traditional Chinese herbs including Morus alba L, Glycyrrhiza glabra L, Artemisia capillaris Thumb., Astragalus membranaceus Bge., and Carthamus tinctorius L. SH formula is clinically used for HIV treatment in Thailand. However, the possible pharmacokinetic interactions between these Chinese herbs and antiretroviral drugs have not been well documented. The aim of this study was to investigate the potential herb-drug interaction between SH herbal Chinese formula and the antiretroviral drug atazanavir (ATV); Materials and methods: The combination effect of SH formula and ATV on HIV protease was studied in HIV-1 protease inhibition assay in vitro. The inhibition of SH formula on rat CYP3A2 was assessed by detecting the formation of 1'-OH midazolam from midazolam in rat liver microsomes in vitro. The in vivo pharmacokinetic interaction between SH formula and ATV was investigated by measuring time-dependent plasma concentrations of ATV in male Sprague-Dawley rats with liquid chromatography-mass spectrometry; Results: Through the in vitro HIV-1 protease inhibition assay, combination of SH formula (41.7-166.7 mu g/ml) and ATV (16.7-333 ng/ml) showed additive inhibition on HIV-1 protease activity than SH formula or ATV used alone. In vitro incubation assay indicated that SH formula showed a weak inhibition (IC50=231.2 mu g/ml; Ki=98.2 mu g/ml) on CYP3A2 activity in rat liver microsomes. In vivo p

Pimozide is a conventional antipsychotic of the diphenylbutylpiperidine class that has been clinically used for over 30 years. The obvious side effect of this drug is weight gain. However, the mechanism of pimozide-induced weight gain is still unknown. In the present study, we identified pimozide as a novel fatty acid binding protein 4 (FABP4) inhibitor using molecular docking simulation as well as biochemical, characterizations. BMS309403, a well-known FABP4 inhibitor, elevated the basal protein levels of PPAR gamma, therefore stimulating adipogenesis in adipocytes. The present study showed that the inhibitory effect of pimozide on FABP4 promoted adipocyte differentiation with the potency proportional to their propensities for weight gain. These effects in adipogenesis by pimozide may help to explain the Weight gain that is frequently observed in patients treated with pimozide.

Ethnopharmacological relevance: With the prevalent use of highly active antiretroviral therapy (HAART) for AIDS patients since 1996, the mortality of HIV/AIDS patients has been remarkably decreased. With long-term use of HAART, drug resistance and side effects of antiretrovirals have been frequently reported, which not only reduce the efficacy, but also decreases the tolerance of patients. Traditional herbal medicine has become more popular among HIV/AIDS patients as adjuvant therapy to reduce these adverse effects of HAART. SH formula is a Chinese herbal formula consisting of five traditional Chinese herbs including Morus alba L, Glycyrrhiza glabra L, Artemisia capillaris Thumb., Astragalus membranaceus Bge., and Carthamus tinctorius L. SH formula is clinically used for HIV treatment in Thailand. However, the possible pharmacokinetic interactions between these Chinese herbs and antiretroviral drugs have not been well documented. The aim of this study was to investigate the potential herb-drug interaction between SH herbal Chinese formula and the antiretroviral drug atazanavir (ATV); Materials and methods: The combination effect of SH formula and ATV on HIV protease was studied in HIV-1 protease inhibition assay in vitro. The inhibition of SH formula on rat CYP3A2 was assessed by detecting the formation of 1'-OH midazolam from midazolam in rat liver microsomes in vitro. The in vivo pharmacokinetic interaction between SH formula and ATV was investigated by measuring time-dependent plasma concentrations of ATV in male Sprague-Dawley rats with liquid chromatography-mass spectrometry; Results: Through the in vitro HIV-1 protease inhibition assay, combination of SH formula (41.7-166.7 mu g/ml) and ATV (16.7-333 ng/ml) showed additive inhibition on HIV-1 protease activity than SH formula or ATV used alone. In vitro incubation assay indicated that SH formula showed a weak inhibition (IC50=231.2 mu g/ml; Ki=98.2 mu g/ml) on CYP3A2 activity in rat liver microsomes. In vivo p

Pimozide is a conventional antipsychotic of the diphenylbutylpiperidine class that has been clinically used for over 30 years. The obvious side effect of this drug is weight gain. However, the mechanism of pimozide-induced weight gain is still unknown. In the present study, we identified pimozide as a novel fatty acid binding protein 4 (FABP4) inhibitor using molecular docking simulation as well as biochemical, characterizations. BMS309403, a well-known FABP4 inhibitor, elevated the basal protein levels of PPAR gamma, therefore stimulating adipogenesis in adipocytes. The present study showed that the inhibitory effect of pimozide on FABP4 promoted adipocyte differentiation with the potency proportional to their propensities for weight gain. These effects in adipogenesis by pimozide may help to explain the Weight gain that is frequently observed in patients treated with pimozide.

A series of 7H-thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives 7a-i were synthesized and evaluated as novel acetylcholinesterase (AChE) inhibitors. All target compounds were evaluated in vitro for the inhibitory activities against AChE via Ellman colorimetric assay. Compound 7c showed an excellent (89.82%) inhibitory activity. The molecular docking studies revealed that 7c, 7d and 7g, with the lateral chain in the para position of the phenyl ring, possessed an optimal docking pose and can perfectly fit into the catalytic active site (CAS) and peripheral anionic site (PAS), simultaneously, and, consequently, exhibited higher inhibitory potency than 7b that bears the same lateral chain as 7g, but in the ortho position of the phenyl ring. © 2014 Elsevier Masson SAS. All rights reserved.

(Chemical Equation Presented) We first identified fluorescein, ketazolam, antrafenine, darifenacin, fosaprepitant, paliperidone, risperidone, pimozide, trovafloxacin, and levofloxacin as inhibitors of fatty acid binding protein 4 using molecular docking screening from FDA-approved drugs. Subsequently, the biochemical characterizations showed that levofloxacin directly inhibited FABP4 activity in both the in vitro ligand displacement assay and cell-based function assay. Furthermore, levo floxacin did not induce adipogenesis in adipocytes, which is the major adverse effect of FABP4 inhibitors.

A series of 7H-thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives 7a-i were synthesized and evaluated as novel acetylcholinesterase (AChE) inhibitors. All target compounds were evaluated in vitro for the inhibitory activities against AChE via Ellman colorimetric assay. Compound 7c showed an excellent (89.82%) inhibitory activity. The molecular docking studies revealed that 7c, 7d and 7g, with the lateral chain in the para position of the phenyl ring, possessed an optimal docking pose and can perfectly fit into the catalytic active site (CAS) and peripheral anionic site (PAS), simultaneously, and, consequently, exhibited higher inhibitory potency than 7b that bears the same lateral chain as 7g, but in the ortho position of the phenyl ring. © 2014 Elsevier Masson SAS. All rights reserved.

(Chemical Equation Presented) We first identified fluorescein, ketazolam, antrafenine, darifenacin, fosaprepitant, paliperidone, risperidone, pimozide, trovafloxacin, and levofloxacin as inhibitors of fatty acid binding protein 4 using molecular docking screening from FDA-approved drugs. Subsequently, the biochemical characterizations showed that levofloxacin directly inhibited FABP4 activity in both the in vitro ligand displacement assay and cell-based function assay. Furthermore, levo floxacin did not induce adipogenesis in adipocytes, which is the major adverse effect of FABP4 inhibitors.

A series of 7H-thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives 7a-i were synthesized and evaluated as novel acetylcholinesterase (AChE) inhibitors. All target compounds were evaluated in vitro for the inhibitory activities against AChE via Ellman colorimetric assay. Compound 7c showed an excellent (89.82%) inhibitory activity. The molecular docking studies revealed that 7c, 7d and 7g, with the lateral chain in the para position of the phenyl ring, possessed an optimal docking pose and can perfectly fit into the catalytic active site (CAS) and peripheral anionic site (PAS), simultaneously, and, consequently, exhibited higher inhibitory potency than 7b that bears the same lateral chain as 7g, but in the ortho position of the phenyl ring. © 2014 Elsevier Masson SAS. All rights reserved.

(Chemical Equation Presented) We first identified fluorescein, ketazolam, antrafenine, darifenacin, fosaprepitant, paliperidone, risperidone, pimozide, trovafloxacin, and levofloxacin as inhibitors of fatty acid binding protein 4 using molecular docking screening from FDA-approved drugs. Subsequently, the biochemical characterizations showed that levofloxacin directly inhibited FABP4 activity in both the in vitro ligand displacement assay and cell-based function assay. Furthermore, levo floxacin did not induce adipogenesis in adipocytes, which is the major adverse effect of FABP4 inhibitors.

Objective: To design and synthesize nine 6-arylmethyl-3-(4-alkoxyphenyl)-7H-thiazolo[3, 2-b]-1,2,4-triazin-7-one derivatives based on our previous work and molecular modeling, and to determine the acetylcholinesterase (AChE) inhibitory activity, the structure-activity relationship and the influences of group types on C6 arylmethyl and the side chain containing nitrogen at C3 phenyl on the 7H-thiazolo[3, 2-b]-1, 2, 4-triazin-7-one scaffold. Methods: Benzaldehyde or substituted benzaldehyde was reacted with N-acetoglycine by using Erlenmeyer-Plöchl reaction, hydrolyzation reaction and condensation reaction to obtain 6-arylmethyl-3-(4-hydroxyphenyl)-7H-thiazolo[3, 2-b]-1,2,4-triazin-7-one derivatives. Then the derivatives were transferred to nine target compounds 6-arylmethyl-3-(4-alkoxyphenyl)-7H-thiazolo[3, 2-b]-1,2,4-triazin-7-one derivatives by using Williamson reaction. AChE inhibitory activity was determined by the Ellman colorimetric assay with huperzine-A as the positive control. Results: The structures of all of the target compounds were characterized by mass spectra, infrared spectra and proton NMR. All of the target compounds exhibited inhibitory activities against human AChE in vitro, eight of them exhibited more than 40% inhibition at 10 μmol·L-1. Conclusion: 6-Arylmethyl-3-(4-alkoxyphenyl)-7H-thiazolo[3, 2-b]-1,2,4-triazin-7-one derivatives would be a kind of highly active AChE inhibitors. The inhibitory activities of target compounds with electron-donating group are more powerful than those of target compounds with electron-withdrawing group at C6 arylmethyl on the 7H-thiazolo[3, 2-b]-1, 2, 4-triazin-7-one scaffold.

Acetylcholinesterase (AChE) inhibitors play an important role in the treatment of Alzheimer's disease. To study the effect of the presence of a fluoro group on inhibitory activity against AChE, a series of 3-aryl-6-(4-fluorobenzyl)-7H-thiazolo[3,2]-1,2,4-triazin-7-one derivatives have been synthesized, in which a hydrogen atom has been substituted by a fluorine in the aromatic nucleus of 6-arylmethyl-3-phenyl-7H-thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives, a series of compounds previously reported by us to have inhibitory activity against acetylcholinesterase. From the molecular docking studies, it was found that, in general, a key hydrogen bond of target compounds to the OH of Tyr341 and a close contact with relevant residues in the catalytic site are prerequisites for high inhibitory activity.

A series of 7H-thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives were designed, synthesized and evaluated as dual binding site acetylcholinesterase inhibitors. The target compounds exhibited promising inhibitory activity for AChE. The structure-activity relationships were discussed and their binding conformation and simultaneous interactions mode were further clarified by the molecular docking studies.

隨著個人計算機運算能力的快速發展，虛擬藥物篩檢已被廣泛運用。目前運用於計算機輔助藥物虛擬篩選的化合物數據庫多為人工合成的數據庫，而用於天然產物藥物篩選的數據庫則較少報道。為了加速天然化合物的虛擬篩選，我們建立了包含約8000個天然產物的數據庫。他們中的大多數為傳統中藥。, 為了驗證天然產物數據庫的可用性，其被用於篩選乙酰膽鹼酯酶抑製劑。該數據庫成功地確定了美國藥品監督管理局所批準的乙酰膽鹼酯酶抑製劑，如石杉鹼甲和他克林，表示該天然產物數據庫可以用於藥物虛擬對接篩選。, 除了已知的乙酰膽鹼酯酶抑製劑，十二種植物化學物（大黃酸，大黃素，蘆薈大黃素，大黃酚，花椒毒素，珊瑚菜素，別異歐前胡素，歐前胡素，紫草素，乙酰紫草素，異戊紫草素和β，β-二甲基丙烯酰紫草素）被確定為新的乙酰膽鹼酯酶抑製劑。澱粉樣蛋白聚集可以導致神經細胞死亡；本研究中新發現的乙酰膽鹼酯酶抑製劑乙酰紫草素能夠阻止澱粉樣蛋白的聚集。除此之外，乙酰紫草素及其衍生物可以對抗過氧化氫誘導的神經細胞凋亡。其抗凋亡的活性作用是通過抑制活性氧的產生，以及保護線粒體膜電位的損失所實現的。亞鐵血紅素加氧酶在其神經細胞保護作用中起重要作用。, 趨化因子受體4為跨膜G蛋白偶聯受體（GPCRs）。 CXCR4已被確定為一個新治療以及預防腫瘤轉移的新靶點。本研究利用分子對接篩選，從天然產物數據庫篩選選出CXCR4拮抗劑。通過分子對接和基於細胞的測定，黃芪甲苷，羥基紅花黃色素A和水飛薊賓已被確定為CXCR4拮抗劑。抗轉移的研究表明，黃芪甲苷和水飛薊賓抑制CXCL12誘導乳腺癌細胞的遷移和侵襲。此外，水飛薊賓也抑制CXCL12誘導的人臍靜脈內皮細胞管形成。另一方面，羥基紅花黃色素A對乳腺癌細胞的增殖表現出較強的抑製作用，因此很難進行抗轉移實驗。, With the rapid advances in personal computing power, virtual drug screening has become increasingly popular. While there are numerous databases for synthetic compounds, there are few natural product databases that are specifically for in silico docking studies. To facilitate virtual docking on natural compounds, in-house Natural Products Database has been established, which contains approximately 8,000 naturally occurring chemicals so far. Most of them are documented Traditional Chinese Medicines., In order to validate the usefulness of the database, in silico screening of acetylcholinesterase inhibitors (AChEIs) by virtual docking was performed. The database successfully identified the FDA-approved AChEIs such as huperzine and tacrine, indicating the in-house database is workable for natural products docking screening., Apart from well-known AChE inhibitors, twelve phytochemicals (emodin, aloe-emodin, chrysophanol, rhein, xanthotoxin, phellopterin, alloisoimperatorin, imperatorin, shikonin, acetylshikonin, isovalerylshikonin and β, β-dimethylacrylshikonin) were identified as AChE inhibitors in this study that were not previously reported. Amyloid aggregation leads to toxic species that cause neuronal cell deaths, it was found that the newly identified AChEIs acetylshikonin and shikonin are able to prevent amyloid aggregation. A series of cell-based analysis were conducted for in vitro evaluation of the neuroprotective activities of the newly identified AChEIs. Acetylshikonin and its derivatives was found to prevent apoptotic cell death induced by hydrogen peroxide in human and rat neuronal SH-SY5Y and PC12 cells at 10 μM. Acetylshikonin exhibited the most potent anti-apoptotic activity through inhibition of reactive oxygen species (ROS) generation as well as protection of the loss of mitochondria membrane potential. Furthermore, acetylshikonin upregulates hemooxygenase 1(HO-1) which is a key step mediating its anti-apoptotic activity from oxidative stress in SH-SY5Y cells., The C-X-C chemokine receptor type 4 (CXCR4) belongs to the class A family of seven transmembrane G protein-coupled receptors (GPCRs). CXCR4 has been identified as one of novel target against metastasis. A search for natural CXCR4 antagonists was conducted from natural product database by molecular docking for anti-metastasis study. Astragaloside IV, hydroxy safflower yellow A and silibinin have been identified as novel CXCR4 antagonists by both molecular docking and characterized by various cell-based assays. Anti-metastasis study showed that astragaloside IV and silibinin inhibited CXCL12-induced migration and invasion in breast cancer cells. In addition, silibinin also inhibited CXCL12-induced tube formation in human umbilical vein endothelial cells. On the other hand, hydroxy safflower yellow A exhibited a strong cytotoxicity on breast cancer cell proliferation, which is difficult to conduct anti-metastasis experiments., Detailed summary in vernacular field only., Wang, Yan., Thesis (Ph.D.) Chinese University of Hong Kong, 2013., Includes bibliographical references (leaves 230-250)., s also in Chinese., http://library.cuhk.edu.hk/record=b6115803, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

A series of 7H-thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives were designed, synthesized and evaluated as dual binding site acetylcholinesterase inhibitors. The target compounds exhibited promising inhibitory activity for AChE. The structure-activity relationships were discussed and their binding conformation and simultaneous interactions mode were further clarified by the molecular docking studies.

Acetylcholinesterase (AChE) inhibitors play an important role in the treatment of Alzheimer's disease. To study the effect of the presence of a fluoro group on inhibitory activity against AChE, a series of 3-aryl-6-(4-fluorobenzyl)-7H-thiazolo[3,2]-1,2,4-triazin-7-one derivatives have been synthesized, in which a hydrogen atom has been substituted by a fluorine in the aromatic nucleus of 6-arylmethyl-3-phenyl-7H-thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives, a series of compounds previously reported by us to have inhibitory activity against acetylcholinesterase. From the molecular docking studies, it was found that, in general, a key hydrogen bond of target compounds to the OH of Tyr341 and a close contact with relevant residues in the catalytic site are prerequisites for high inhibitory activity.

Acetylcholinesterase (AChE) inhibitors play an important role in the treatment of Alzheimer's disease. To study the effect of the presence of a fluoro group on inhibitory activity against AChE, a series of 3-aryl-6-(4-fluorobenzyl)-7H-thiazolo[3,2]-1,2,4-triazin-7-one derivatives have been synthesized, in which a hydrogen atom has been substituted by a fluorine in the aromatic nucleus of 6-arylmethyl-3-phenyl-7H-thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives, a series of compounds previously reported by us to have inhibitory activity against acetylcholinesterase. From the molecular docking studies, it was found that, in general, a key hydrogen bond of target compounds to the OH of Tyr341 and a close contact with relevant residues in the catalytic site are prerequisites for high inhibitory activity.

A series of 7H-thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives were designed, synthesized and evaluated as dual binding site acetylcholinesterase inhibitors. The target compounds exhibited promising inhibitory activity for AChE. The structure-activity relationships were discussed and their binding conformation and simultaneous interactions mode were further clarified by the molecular docking studies.

Acetylcholinesterase (AChE) inhibitors played an important role in developing a cure for Alzheimer' s disease. In order to study on the influence of modifications at different groups and side chains on the AChE inhibitory ability and the active sites of 7H-thiazolo[3,2-b][1,2,4]triazin-7-one derivatives, fourteen 3,6-diaryl-7H-thiazolo[3,2-b][1,2,4]triazin-7-one derivatives were designed and synthesized. The study of AChE inhibitory activity was carried out using the Ellman colorimetric assay with huperzine-A as the positive control drug. Most of the target compounds exhibited more than 50% inhibition at 10 μM. Some target compounds showed strong inhibition against AChE. The molecular fields analysis and preliminary structureactivity relationships are discussed. © 2010 The Pharmaceutical Society of Korea and Springer Netherlands.

Acetylcholinesterase inhibitors played significant roles in treatment of Alzheimer’s disease. Based on the research foundation of our previous work and molecular modeling, twelve 3,6-diaryl-7H-thiazolo[3,2-b][1,2,4]triazin-7-one derivatives were synthesized and characterized by mass spectra, infrared spectra, NMR and elemental analyses. The study of AChE inhibitory activity was carried out using the Ellman colorimetric assay with huperzine-A as the positive control. All target compounds exhibited more than 40% inhibition at 10 μM. Some target compounds showed good inhibition against AChE. The preliminary structure-activity relationships were the halogen atoms at the phenyl ring at the C6 position, the hydroxy groups and the long side chains at the phenyl ring at the C3 position of the parent nucleus played significant roles in the AChE inhibitory activity of the target compounds.

The docking study on a series of 7H-thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives with acetylcholinesterase has been demonstrated. The synthesis and characterization of a series of 7H-thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives were described. All target compounds were evaluated in vitro for the inhibitory activities against AChE via Ellman colorimetric assay. Most of the target compounds possessed anti-acetylcholinesterase activity. The preliminary structure-activity relationships were discussed. © 2010 Bentham Science Publishers Ltd.

Alzhemier's disease (AD) is a common form of dementia in the ageing population which is characterized by depositions of amyloids and a cholinergic neurotransmission deficit in the brain. Current therapeutic intervention for AD is primarily based on the inhibition of brain acetylcholinesterase (AChE) to restore the brain acetylcholine level. Cryptotanshinone (CT) and dihydrotanshinone (DT) were diterpenoids extracted from Salvia miltiorrhiza Bge. having anti-cholinesterase activity. Here we characterized the inhibition property of these two diterpenoids towards human AChE and butyrylcholinesterase (BChE). Both CT and DT were found to be mixed non-competitive inhibitors for human AChE and an uncompetitive inhibitor for human BChE. The docking analyses of CT and DT into the active sites of both cholinesterases indicate that they interact with the allosteric site inside the active-site gorge mainly by hydrophobic interactions. © 2010 Elsevier Ireland Ltd.

Alzhemier's disease (AD) is a common form of dementia in the ageing population which is characterized by depositions of amyloids and a cholinergic neurotransmission deficit in the brain. Current therapeutic intervention for AD is primarily based on the inhibition of brain acetylcholinesterase (AChE) to restore the brain acetylcholine level. Cryptotanshinone (CT) and dihydrotanshinone (DT) were diterpenoids extracted from Salvia miltiorrhiza Bge. having anti-cholinesterase activity. Here we characterized the inhibition property of these two diterpenoids towards human AChE and butyrylcholinesterase (BChE). Both CT and DT were found to be mixed non-competitive inhibitors for human AChE and an uncompetitive inhibitor for human BChE. The docking analyses of CT and DT into the active sites of both cholinesterases indicate that they interact with the allosteric site inside the active-site gorge mainly by hydrophobic interactions. © 2010 Elsevier Ireland Ltd.

The docking study on a series of 7H-thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives with acetylcholinesterase has been demonstrated. The synthesis and characterization of a series of 7H-thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives were described. All target compounds were evaluated in vitro for the inhibitory activities against AChE via Ellman colorimetric assay. Most of the target compounds possessed anti-acetylcholinesterase activity. The preliminary structure-activity relationships were discussed. © 2010 Bentham Science Publishers Ltd.

The docking study on a series of 7H-thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives with acetylcholinesterase has been demonstrated. The synthesis and characterization of a series of 7H-thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives were described. All target compounds were evaluated in vitro for the inhibitory activities against AChE via Ellman colorimetric assay. Most of the target compounds possessed anti-acetylcholinesterase activity. The preliminary structure-activity relationships were discussed. © 2010 Bentham Science Publishers Ltd.

Alzhemier's disease (AD) is a common form of dementia in the ageing population which is characterized by depositions of amyloids and a cholinergic neurotransmission deficit in the brain. Current therapeutic intervention for AD is primarily based on the inhibition of brain acetylcholinesterase (AChE) to restore the brain acetylcholine level. Cryptotanshinone (CT) and dihydrotanshinone (DT) were diterpenoids extracted from Salvia miltiorrhiza Bge. having anti-cholinesterase activity. Here we characterized the inhibition property of these two diterpenoids towards human AChE and butyrylcholinesterase (BChE). Both CT and DT were found to be mixed non-competitive inhibitors for human AChE and an uncompetitive inhibitor for human BChE. The docking analyses of CT and DT into the active sites of both cholinesterases indicate that they interact with the allosteric site inside the active-site gorge mainly by hydrophobic interactions. © 2010 Elsevier Ireland Ltd.

Acetylcholinesterase inhibitors are the most frequently prescribed anti-Alzheimer's drugs. A series of 5H-thiazolo[3,2-a]pyrimidine-6-carboxylic acid ethyl ester derivatives as the novel acetylcholinesterase inhibitors was designed based on virtual screening methods. The target compounds were synthesized with Biginelli reaction and Hantzsch-type condensation of dihydropyrimidines with substituted phenacyl chlorides, and were characterized with elemental analysis, IR, MS, 1H NMR, and 13C NMR. The biological evaluation against human acetylcholinesterase in vitro indicated all the target compounds show more than 50% inhibition at 10 μmol/L by means of the Ellman method. The results provide a starting point for the development of novel drags to treat Alzheimer's disease and lay the foundation of searching for improved acetylcholinesterase inhibitors with the novel scaffolds.

The docking study on a novel series of 7H-thiazolo[3,2-b]-1,2,4-triazin-7- one derivatives with acetylcholinesterase from Torpedo californica has demonstrated that the ligands bind to the dualsite of the enzyme. The synthesis and characterization of 7H-thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives was described. The crystal structure of 6-benzyl-3-{4-[2-(1-piperidinyl)-2- oxoethoxy]phenyl}thiazolo[3,2-b]-1,2,4-triazin-7-one has been characterized by X-ray diffraction. All target compounds have been screened for their efficacy as acetylcholinesterase inhibitor. The study of AChE inhibitory activity was carried out using the Ellman colorimetric assay with huperzine-A as a reference against targets. Most of the target compounds exhibit more than 50% inhibition at 10 μM. Some derivatives showed good inhibition against AChE. The preliminary structure-activity relationships were discussed.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressive memory loss and cognitive impairment. It is the most common type of dementia in the ageing population due to a severe loss of cholinergic neurons in selected brain area. At present, acetylcholinesterase inhibitors (AChEI) are the first group of drugs approved by the FDA to treat mild to moderate Alzheimer's disease. Most of these drugs such as huperzine and galanthamine are originally isolated from plants. In this study, the AChE inhibitory activities from extracts of Chinese medicinal herbs that have traditionally been prescribed to treat insomnia and brain function disorders were examined in a 96-well plate assay based on Ellman's method. Both ethanol and aqueous extracts of 26 traditional Chinese medicinal herbs were tested. Inhibitory effects were expressed as the percentage of inhibition. For the herbal extracts that were shown to exert a significant inhibition, dose-dependent inhibitory assays were also performed. Ethanol and aqueous extracts of six herbs were found to have high AChE inhibitory activities in a dose-dependent manner. The IC 50 of these herbal extracts on inhibition of AChE are at around 5-85 μm/ml. The results of this study indicate that there is a great potential to search for novel usage of these medicinal herbs for the treatment of AD. © 2008.

Acetylcholinesterase(AChE) inhibitors are important research topics because of their wide range of associated health implications, especially for the treatment of Alzheimer's disease. The finding of novel AChE inhibitors is presented here. A docking screening model of AChE inhibitor was used to evaluate a series of 5H-thiazolo[3,2-a] pyrimidine derivatives. The virtual screening hits were analyzed in drug likeness and physical-chemical features, therefore were focused to those compounds in the present article. To investigate the relationship between the bioactivities and the structures, 10 target compounds with the 5H-thiazolo[3,2-a]pyrimidine scaffold were synthesized as potential AChE inhibitors, and the pharmacological assay result also conformed, some compounds displayed considerable inhibitory effects with inhibition rates above 50% at 10 μM, and all their core structures are very different from those of known AChE inhibitors. The results demonstrate the effectiveness and validity of the virtual screening approach especially of the docking screening approach, and provide a starting point for the development of novel drugs to treat Alzheimer's disease. © ARKAT USA, Inc.

Cyclic adenosine 3',5'-monophosphate (cAMP)-dependent signalling pathway has been proposed to regulate acetylcholinesterese (AChE) expression in chick muscle; however, its role in mammalian enzyme is not known. We provide several lines of evidence to suggest that the cAMP-mediated AChE expression in myotube is oppositely regulated between avian and mammalian enzymes. Human AChE promoter was tagged with luciferase, namely Hp-Luc, which was transfected into cultured chick myotubes. Application of cAMP and forskolin induced the expression of chick AChE but reduced human AChE promoter-driven luciferase activity. Transfection of cDNAs encoding active mutants of G proteins altered the intracellular cAMP level in myotubes as well as the expression of chick and human AChE. When the constitutively active forms of Activating Transcription Factor-1 (EWS/ATF-1 oncogene) were over expressed in Hp-Luc transfected myotubes, the expression of chick AChE transcript and protein increased from similar to1.8- to similar to2.5-fold, but the luciferase activity was decreased by over 60\%. Overexpression of cAMP-responsive element binding protein (CREB) in Hp-Luc transfected myotubes markedly enhanced the cAMP-mediated AChE expression in up- and downregulated chick and human enzymes, respectively. in addition, CREB bound the CRE sequence of human AChE promoter. Mutation on the CRE site markedly enhanced the expression of the promoter-driven luciferase; however, its response to cAMP inhibition in cultured myotubes was still retained. These findings suggest that a cAMP-dependent pathway is contrasting activation and repression of AChE expression in chick and human muscles.

Cyclic adenosine 3',5'-monophosphate (cAMP)-dependent signalling pathway has been proposed to regulate acetylcholinesterese (AChE) expression in chick muscle; however, its role in mammalian enzyme is not known. We provide several lines of evidence to suggest that the cAMP-mediated AChE expression in myotube is oppositely regulated between avian and mammalian enzymes. Human AChE promoter was tagged with luciferase, namely Hp-Luc, which was transfected into cultured chick myotubes. Application of cAMP and forskolin induced the expression of chick AChE but reduced human AChE promoter-driven luciferase activity. Transfection of cDNAs encoding active mutants of G proteins altered the intracellular cAMP level in myotubes as well as the expression of chick and human AChE. When the constitutively active forms of Activating Transcription Factor-1 (EWS/ATF-1 oncogene) were over expressed in Hp-Luc transfected myotubes, the expression of chick AChE transcript and protein increased from similar to1.8- to similar to2.5-fold, but the luciferase activity was decreased by over 60\%. Overexpression of cAMP-responsive element binding protein (CREB) in Hp-Luc transfected myotubes markedly enhanced the cAMP-mediated AChE expression in up- and downregulated chick and human enzymes, respectively. in addition, CREB bound the CRE sequence of human AChE promoter. Mutation on the CRE site markedly enhanced the expression of the promoter-driven luciferase; however, its response to cAMP inhibition in cultured myotubes was still retained. These findings suggest that a cAMP-dependent pathway is contrasting activation and repression of AChE expression in chick and human muscles.

Cyclic adenosine 3',5'-monophosphate (cAMP)-dependent signalling pathway has been proposed to regulate acetylcholinesterese (AChE) expression in chick muscle; however, its role in mammalian enzyme is not known. We provide several lines of evidence to suggest that the cAMP-mediated AChE expression in myotube is oppositely regulated between avian and mammalian enzymes. Human AChE promoter was tagged with luciferase, namely Hp-Luc, which was transfected into cultured chick myotubes. Application of cAMP and forskolin induced the expression of chick AChE but reduced human AChE promoter-driven luciferase activity. Transfection of cDNAs encoding active mutants of G proteins altered the intracellular cAMP level in myotubes as well as the expression of chick and human AChE. When the constitutively active forms of Activating Transcription Factor-1 (EWS/ATF-1 oncogene) were over expressed in Hp-Luc transfected myotubes, the expression of chick AChE transcript and protein increased from similar to1.8- to similar to2.5-fold, but the luciferase activity was decreased by over 60\%. Overexpression of cAMP-responsive element binding protein (CREB) in Hp-Luc transfected myotubes markedly enhanced the cAMP-mediated AChE expression in up- and downregulated chick and human enzymes, respectively. in addition, CREB bound the CRE sequence of human AChE promoter. Mutation on the CRE site markedly enhanced the expression of the promoter-driven luciferase; however, its response to cAMP inhibition in cultured myotubes was still retained. These findings suggest that a cAMP-dependent pathway is contrasting activation and repression of AChE expression in chick and human muscles.

Parkinson's disease (PD) is a neurodegenerative disease caused by a multitude of environmental, neurochemical, and genetic factors. The gene for human dopamine D4 receptor (DRD4) has been considered as a plausible candidate for the pathogenesis of PD. Different dopamine D4 receptor allelic forms have variable affinity toward certain neuroleptics such as clozapine, suggesting a role for dopamine D4 receptors in neurologic disorders. To test the hypothesis that the DRD4 polymorphism is associated with the susceptibility to Parkinson's disease, we have examined differences in allele frequencies of different DRD4 polymorphisms in 101 Chinese patients with PD and in 105 age-matched control subjects in Hong Kong. The DRD4 gene was analyzed by a non-radioactive polymerase chain reaction-based Southern hybridization with chemiluminescence detection. The number of polymorphic 48 base pair tandem repeats in exon 3 was identified in each study subject. The DRD4 alleles with high frequencies in the control subjects are 4-repeat allele (72.4\%), 2-repeat allele (21.4\%), and 7-repeat allele (3.8\%) which accounted for over 97\% of the total alleles in the elderly Chinese population. The most prevalent genotype in the control subjects is the 4/4 (47.6\%), followed by 4/2 (38.6), 4/7 (7.6\%), and 2/2 (3.0\%). None of the variable number tandem repeat polymorphism showed evidence for genetic association with Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disease caused by a multitude of environmental, neurochemical, and genetic factors. The gene for human dopamine D4 receptor (DRD4) has been considered as a plausible candidate for the pathogenesis of PD. Different dopamine D4 receptor allelic forms have variable affinity toward certain neuroleptics such as clozapine, suggesting a role for dopamine D4 receptors in neurologic disorders. To test the hypothesis that the DRD4 polymorphism is associated with the susceptibility to Parkinson's disease, we have examined differences in allele frequencies of different DRD4 polymorphisms in 101 Chinese patients with PD and in 105 age-matched control subjects in Hong Kong. The DRD4 gene was analyzed by a non-radioactive polymerase chain reaction-based Southern hybridization with chemiluminescence detection. The number of polymorphic 48 base pair tandem repeats in exon 3 was identified in each study subject. The DRD4 alleles with high frequencies in the control subjects are 4-repeat allele (72.4\%), 2-repeat allele (21.4\%), and 7-repeat allele (3.8\%) which accounted for over 97\% of the total alleles in the elderly Chinese population. The most prevalent genotype in the control subjects is the 4/4 (47.6\%), followed by 4/2 (38.6), 4/7 (7.6\%), and 2/2 (3.0\%). None of the variable number tandem repeat polymorphism showed evidence for genetic association with Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disease caused by a multitude of environmental, neurochemical, and genetic factors. The gene for human dopamine D4 receptor (DRD4) has been considered as a plausible candidate for the pathogenesis of PD. Different dopamine D4 receptor allelic forms have variable affinity toward certain neuroleptics such as clozapine, suggesting a role for dopamine D4 receptors in neurologic disorders. To test the hypothesis that the DRD4 polymorphism is associated with the susceptibility to Parkinson's disease, we have examined differences in allele frequencies of different DRD4 polymorphisms in 101 Chinese patients with PD and in 105 age-matched control subjects in Hong Kong. The DRD4 gene was analyzed by a non-radioactive polymerase chain reaction-based Southern hybridization with chemiluminescence detection. The number of polymorphic 48 base pair tandem repeats in exon 3 was identified in each study subject. The DRD4 alleles with high frequencies in the control subjects are 4-repeat allele (72.4\%), 2-repeat allele (21.4\%), and 7-repeat allele (3.8\%) which accounted for over 97\% of the total alleles in the elderly Chinese population. The most prevalent genotype in the control subjects is the 4/4 (47.6\%), followed by 4/2 (38.6), 4/7 (7.6\%), and 2/2 (3.0\%). None of the variable number tandem repeat polymorphism showed evidence for genetic association with Parkinson's disease.

Endothelins (ETs) are a family of vasoconstrictor and mitogenic peptides originally isolated from the endothelial cells. Three isoforms of ET, namely ET-1, ET-2 and ET-3, are generated from their respective intermediate precursors big ETs through specific endoproteolytic cleavage by endothelin converting enzyme (ECE). Using reverse-transcription polymerase chain reaction (RT-PCR), we have isolated a cDNA encoding for ECE from both the prostatic and epididymal halves of rat vas deferens. In situ hybridization using digoxigenin-labeled ECE cDNA probe demonstrated that ECE mRNA is preferentially localized in the inner longitudinal smooth muscle layer adjacent to submucosa region of rat vas deferens. Both ET-1 and big ET-1 at 30 nM potentiated electrically stimulated contractile response of prostatic vas deferens. Pre-incubation of tissue with a metalloprotease ECE inhibitor phosphoramidon (10 mu M) strongly inhibited the response to big ET-1, but not to ET-1. On the other hand, big ET-1 failed to elicit contractile response of epididymal vas deferens. Phosphoramidon alone did not affect both the basal and electrically stimulated contractile responses in vas deferens. These data indicate that the circulating ET-1 and its immediate precursor big ET-1 could differentially regulate smooth muscle contractions in the prostatic and epididymal vas deferens of the rat.

Endothelins (ETs) are a family of vasoconstrictor and mitogenic peptides originally isolated from the endothelial cells. Three isoforms of ET, namely ET-1, ET-2 and ET-3, are generated from their respective intermediate precursors big ETs through specific endoproteolytic cleavage by endothelin converting enzyme (ECE). Using reverse-transcription polymerase chain reaction (RT-PCR), we have isolated a cDNA encoding for ECE from both the prostatic and epididymal halves of rat vas deferens. In situ hybridization using digoxigenin-labeled ECE cDNA probe demonstrated that ECE mRNA is preferentially localized in the inner longitudinal smooth muscle layer adjacent to submucosa region of rat vas deferens. Both ET-1 and big ET-1 at 30 nM potentiated electrically stimulated contractile response of prostatic vas deferens. Pre-incubation of tissue with a metalloprotease ECE inhibitor phosphoramidon (10 mu M) strongly inhibited the response to big ET-1, but not to ET-1. On the other hand, big ET-1 failed to elicit contractile response of epididymal vas deferens. Phosphoramidon alone did not affect both the basal and electrically stimulated contractile responses in vas deferens. These data indicate that the circulating ET-1 and its immediate precursor big ET-1 could differentially regulate smooth muscle contractions in the prostatic and epididymal vas deferens of the rat.

Endothelins (ETs) are a family of vasoconstrictor and mitogenic peptides originally isolated from the endothelial cells. Three isoforms of ET, namely ET-1, ET-2 and ET-3, are generated from their respective intermediate precursors big ETs through specific endoproteolytic cleavage by endothelin converting enzyme (ECE). Using reverse-transcription polymerase chain reaction (RT-PCR), we have isolated a cDNA encoding for ECE from both the prostatic and epididymal halves of rat vas deferens. In situ hybridization using digoxigenin-labeled ECE cDNA probe demonstrated that ECE mRNA is preferentially localized in the inner longitudinal smooth muscle layer adjacent to submucosa region of rat vas deferens. Both ET-1 and big ET-1 at 30 nM potentiated electrically stimulated contractile response of prostatic vas deferens. Pre-incubation of tissue with a metalloprotease ECE inhibitor phosphoramidon (10 mu M) strongly inhibited the response to big ET-1, but not to ET-1. On the other hand, big ET-1 failed to elicit contractile response of epididymal vas deferens. Phosphoramidon alone did not affect both the basal and electrically stimulated contractile responses in vas deferens. These data indicate that the circulating ET-1 and its immediate precursor big ET-1 could differentially regulate smooth muscle contractions in the prostatic and epididymal vas deferens of the rat.

Again is a synapse-organizing molecule that mediates nerve-induced aggregation of acetylcholine receptors and other postsynaptic components at the developing acid regenerating vertebrate neuromuscular junctions. Several lines of evidence indicate that agrin might play re similar role in directing the organization of postsynaptic specifications of neuron-neuron synapse formation. Here we used immunological methods and polymerase chain reaction to identify the expression of agrin protein and alternatively spliced mRNA isoforms in the culture of rat granule cells. Anti-agrin polyclonal antibody labeled the cultured granule cells and it detected a protein of over 200 kDa in size from the lysate of the cultured cells, Analysis by polymerase chain reaction showed that the granule cells in culture expressed predominantly the B-0 isoform of agrin mRNA. When granule cells were co-cultured with primary chick myotubes, there was no detectable effect on the aggregation of acetylcholine receptors on the surface of the myotubes, These results show that the cerebellar granule cells, similar to motor neurons in vitro, express and secrete agrin but it lacks the acetylcholine receptor aggregating activity.