13 results
Search Results
Now showing 1 - 10 of 13
Publication Open Access Aedesin: structure and antimicrobial activity against multidrug resistant bacterial strains.(2014-08-27) Godreuil, Sylvain; Leban, Nadia; Padilla, Andre´; Hamel, Rodolphe; Natthanej Luplertlop; นัฏฐเนศวร์ ลับเลิศลบ; Chauffour, Aure´ lie; Vittecoq, Marion; Hoh, Franc¸ois; Thomas, Fre´de´ ric; Sougakoff, Wladimir; Lionne, Corinne; Yssel, Hans; Misse, Dorothe´e; Misse, Dorothe´e; Mahidol University. Faculty of Tropical Medicine. Department of Microbiology and Immunology.that it is active under physiological conditions encountered in body fluids characterized by ionic salt concentrations. In conclusion, because of its strong lytic activity against multidrug resistant Gram-negative bacterial strains displaying all typesPublication Open Access Combining electrochemical sensors with miniaturized sample preparation for rapid detection in clinical samples(2014-11-21) Natinan Bunyakul; Antje J. Baeumner; Mahidol University. Faculty of Medical Technology. Department of Clinical ChemistryClinical analyses benefit world-wide from rapid and reliable diagnostics tests. New tests are sought with greatest demand not only for new analytes, but also to reduce costs, complexity and lengthy analysis times of current techniques. Among the myriad of possibilities available today to develop new test systems, amperometric biosensors are prominent players-best represented by the ubiquitous amperometric-based glucose sensors. Electrochemical approaches in general require little and often enough only simple hardware components, are rugged and yet provide low limits of detection. They thus offer many of the desirable attributes for point-of-care/point-of-need tests. This review focuses on investigating the important integration of sample preparation with (primarily electrochemical) biosensors. Sample clean up requirements, miniaturized sample preparation strategies, and their potential integration with sensors will be discussed, focusing on clinical sample analyses.Publication Open Access Enzymatic analysis of recombinant Japanese encephalitis virus NS2B(H)-NS3pro protease with fluorogenic model peptide substrates(2012-05) Muhammad Junaid; Chakard Chalayut; Anna Sehgelmeble Torrejon; Chanan Angsuthanasombat; Iryna Shutava; Maris Lapins; Jarl E. S. Wikberg; Gerd Katzenmeier; Mahidol University. Institute of Molecular Biosciences. Laboratory of Molecular and Cellular MicrobiologyBackground Japanese encephalitis virus (JEV), a member of the Flaviviridae family, causes around 68,000 encephalitis cases annually, of which 20–30% are fatal, while 30–50% of the recovered cases develop severe neurological sequelae. Specific antivirals for JEV would be of great importance, particularly in those cases where the infection has become persistent. Being indispensable for flaviviral replication, the NS2B-NS3 protease is a promising target for design of anti-flaviviral inhibitors. Contrary to related flaviviral proteases, the JEV NS2B-NS3 protease is structurally and mechanistically much less characterized. Here we aimed at establishing a straightforward procedure for cloning, expression, purification and biochemical characterization of JEV NS2B(H)-NS3pro protease. Methodology/Principal Findings The full-length sequence of JEV NS2B-NS3 genotype III strain JaOArS 982 was obtained as a synthetic gene. The sequence of NS2B(H)-NS3pro was generated by splicing by overlap extension PCR (SOE-PCR) and cloned into the pTrcHisA vector. Hexahistidine-tagged NS2B(H)-NS3pro, expressed in E. coli as soluble protein, was purified to >95% purity by a single-step immobilized metal affinity chromatography. SDS-PAGE and immunoblotting of the purified enzyme demonstrated NS2B(H)-NS3pro precursor and its autocleavage products, NS3pro and NS2B(H), as 36, 21, and 10 kDa bands, respectively. Kinetic parameters, Km and kcat, for fluorogenic protease model substrates, Boc-GRR-amc, Boc-LRR-amc, Ac-nKRR-amc, Bz-nKRR-amc, Pyr-RTKR-amc and Abz-(R)4SAG-nY-amide, were obtained using inner filter effect correction. The highest catalytic efficiency kcat/Km was found for Pyr-RTKR-amc (kcat/Km: 1962.96±85.0 M−1 s−1) and the lowest for Boc-LRR-amc (kcat/Km: 3.74±0.3 M−1 s−1). JEV NS3pro is inhibited by aprotinin but to a lesser extent than DEN and WNV NS3pro. Conclusions/Significance A simplified procedure for the cloning, overexpression and purification of the NS2B(H)-NS3pro was established which is generally applicable to other flaviviral proteases. Kinetic parameters obtained for a number of model substrates and inhibitors, are useful for the characterization of substrate specificity and eventually for the design of high-throughput assays aimed at antiviral inhibitor discovery.Publication Open Access Responses of primary human nasal epithelial cells to EDIII-DENV stimulation: the first step to intranasal dengue vaccination(2016) Nattika Nantachit; Panya Sunintaboon; Sukathida Ubol; Mahidol University. Faculty of Science. Department of Microbiologyresponses. Results: At tested concentrations, EDIII-D3 TMC NPs not only exerted no detectable toxicity toward HNEpC cultures but also efficiently delivered EDIII-D3 immunogens into HNEpCs. Moreover, HNEpCs quickly and strongly produced proinflammatoryPublication Open Access A time course analysis of the electrophysiological properties of neurons differentiated from human induced Pluripotent Stem Cells (iPSCs)(2014) Pre Deborah; Nestor, Michael W.; Sproul, Andrew A.; Jacob Samson; Koppensteiner Peter; Vorapin Chinchalongporn; Zimmer Matthew; Yamamoto Ai; Noggle, Scott A.; Mahidol University. Institute of Molecular Biosciences. Research Center for NeuroscienceMany protocols have been designed to differentiate human embryonic stem cells (ESCs) and human induced pluripotent stem cells (iPSCs) into neurons. Despite the relevance of electrophysiological properties for proper neuronal function, little is known about the evolution over time of important neuronal electrophysiological parameters in iPSC-derived neurons. Yet, understanding the development of basic electrophysiological characteristics of iPSC-derived neurons is critical for evaluating their usefulness in basic and translational research. Therefore, we analyzed the basic electrophysiological parameters of forebrain neurons differentiated from human iPSCs, from day 31 to day 55 after the initiation of neuronal differentiation. We assayed the developmental progression of various properties, including resting membrane potential, action potential, sodium and potassium channel currents, somatic calcium transients and synaptic activity. During the maturation of iPSC-derived neurons, the resting membrane potential became more negative, the expression of voltage-gated sodium channels increased, the membrane became capable of generating action potentials following adequate depolarization and, at day 48–55, 50% of the cells were capable of firing action potentials in response to a prolonged depolarizing current step, of which 30% produced multiple action potentials. The percentage of cells exhibiting miniature excitatory post-synaptic currents increased over time with a significant increase in their frequency and amplitude. These changes were associated with an increase of Ca2+ transient frequency. Co-culturing iPSC-derived neurons with mouse glial cells enhanced the development of electrophysiological parameters as compared to pure iPSC-derived neuronal cultures. This study demonstrates the importance of properly evaluating the electrophysiological status of the newly generated neurons when using stem cell technology, as electrophysiological properties of iPSC-derived neurons mature over time.Publication Open Access Molecular characterization of Plasmodium falciparum uracil-DNA glycosylase and its potential as a new anti-malarial drug target(2014) Thidarat Suksangpleng; Ubolsree Leartsakulpanich; Saengduen Moonsom; Saranya Siribal; Usa Boonyuen; Wright, George E; Porntip Chavalitshewinkoon-Petmitr; Mahidol University. Faculty of Tropical Medicine. Department of Protozoology,Background: Based on resistance of currently used anti-malarials, a new anti-malarial drug target against Plasmodium falciparum is urgently needed. Damaged DNA cannot be transcribed without prior DNA repair; therefore, uracil-DNA glycosylase, playing an important role in base excision repair, may act as a candidate for a new anti-malarial drug target. Methods: Initially, the native PfUDG from parasite crude extract was partially purified using two columns, and the glycosylase activity was monitored. The existence of malarial UDG activity prompted the recombinant expression of PfUDG for further characterization. The PfUDG from chloroquine and pyrimethamine resistant P. falciparum strain K1 was amplified, cloned into the expression vector, and expressed in Escherichia coli. The recombinant PfUDG was analysed by SDS-PAGE and identified by LC-MS/MS. The three dimensional structure was modelled. Biochemical properties were characterized. Inhibitory effects of 12 uracil-derivatives on PfUDG activity were investigated. Inhibition of parasite growth was determined in vitro using SYBR Green I and compared with results from human cytotoxicity tests. Results: The native PfUDG was partially purified with a specific activity of 1,811.7 units/mg (113.2 fold purification). After cloning of 966-bp PCR product, the 40-kDa hexa-histidine tagged PfUDG was expressed and identified. The amino acid sequence of PfUDG showed only 24.8% similarity compared with the human enzyme. The biochemical characteristics of PfUDGs were quite similar. They were inhibited by uracil glycosylase inhibitor protein as found in other organisms. Interestingly, recombinant PfUDG was inhibited by two uracil-derived compounds; 1-methoxyethyl-6-(p-n-octylanilino) uracil (IC50 of 16.75 μM) and 6-(phenylhydrazino)uracil (IC50 of 77.5 μM). Both compounds also inhibited parasite growth with IC50s of 15.6 and 12.8 μM, respectively. Moreover, 1-methoxyethyl-6-(p-n-octylanilino)uracil was not toxic to HepG2 cells, with IC50 of > 160 μM while 6-(phenylhydrazino)uracil exhibited cytoxicity, with IC50 of 27.5 μM. Conclusions: The recombinant PfUDG was expressed, characterized and compared to partially purified native PfUDG. Their characteristics were not significantly different. PfUDG differs from human enzyme in its size and predicted amino acid sequence. Two uracil derivatives inhibited PfUDG and parasite growth; however, only one non-cytotoxic compound was found. Therefore, this selective compound can act as a lead compound for anti-malarial development in the future.Publication Open Access Elucidating the structure-activity relationships of the vasorelaxation and antioxidation properties of thionicotinic acid derivatives(2010) Supaluk Prachayasittikul; Orapin Wongsawatkul; Apilak Worachartcheewan; Chanin Nantasenamat; Somsak Ruchirawat; Virapong PrachayasittikulNicotinic acid, known as vitamin B3, is an effective lipid lowering drug and intense cutaneous vasodilator. This study reports the effect of 2-(1-adamantylthio)nicotinic acid (6) and its amide 7 and nitrile analog 8 on phenylephrine-induced contraction of rat thoracic aorta as well as antioxidative activity. It was found that the tested thionicotinic acid analogs 6-8 exerted maximal vasorelaxation in a dose-dependent manner, but their effects were less than acetylcholine (ACh)-induced nitric oxide (NO) vasorelaxation. The vasorelaxations were reduced, apparently, in both NG-nitro-L-arginine methyl ester (L-NAME) and indomethacin (INDO). Synergistic effects were observed in the presence of L-NAME plus INDO, leading to loss of vasorelaxation of both the ACh and the tested nicotinic acids. Complete loss of the vasorelaxation was noted under removal of endothelial cells. This infers that the vasorelaxations are mediated partially by endothelium-induced NO and prostacyclin. The thionicotinic acid analogs all exhibited antioxidant properties in both 2,2-diphenyl-1-picrylhydrazyl (DPPH) and superoxide dismutase (SOD) assays. Significantly, the thionicotinic acid 6 is the most potent vasorelaxant with ED50 of 21.3 nM and is the most potent antioxidant (as discerned from DPPH assay). Molecular modeling was also used to provide mechanistic insights into the vasorelaxant and antioxidative activities. The findings reveal that the thionicotinic acid analogs are a novel class of vasorelaxant and antioxidant compounds which have potential to be further developed as promising therapeutics.Publication Open Access Altered ATP release and metabolism in dorsal root ganglia of neuropathic rats.(2008) Somsak Mitrirattanakul; Yoshizo Matsuka; Takeshi Ono; Hirotate Iwase; Kevin S Omoto; Ting Cho; Yan Yan N Lam; Bradley Snyder; สมศักดิ์ ไมตรีรัตนะกุล; Igor SpigelmanBackground: Adenosine 5'-triphosphate (ATP) has a ubiquitous role in metabolism and a major role in pain responses after tissue injury. We investigated the changes in basal and KCl-evoked ATP release from rat dorsal root ganglia (DRG) after peripheral neuropathy induction by unilateral sciatic nerve entrapment (SNE). Results: After SNE, rats develop long-lasting decreases in ipsilateral hindpaw withdrawal thresholds to mechanical and thermal stimulation. At 15–21 days after neuropathy induction, excised ipsilateral L4-L5 DRG display significantly elevated basal extracellular ATP levels compared to contralateral or control (naive) DRG. However, KCl-evoked ATP release is no longer observed in ipsilateral DRG. We hypothesized that the differential SNE effects on basal and evoked ATP release could result from the conversion of extracellular ATP to adenosine with subsequent activation of adenosine A1 receptors (A1Rs) on DRG neurons. Adding the selective A1R agonist, 2-chloro-N6-cyclopentyladenosine (100 nM) significantly decreased basal and evoked ATP release in DRG from na?ve rats, indicating functional A1R activation. In DRG ipsilateral to SNE, adding a selective A1R antagonist, 8-cyclopentyl-1,3-dipropylxanthine (30 nM), further increased basal ATP levels and relieved the blockade of KCl-evoked ATP release suggesting that increased A1R activation attenuates evoked ATP release in neurons ipsilateral to SNE. To determine if altered ATP release was a consequence of altered DRG metabolism we compared O2 consumption between control and neuropathic DRG. DRG ipsilateral to SNE consumed O2 at a higher rate than control or contralateral DRG. Conclusion: These data suggest that peripheral nerve entrapment increases DRG metabolism and ATP release, which in turn is modulated by increased A1R activation.Publication Open Access Linalool, derived from Cinnamomum camphora (L.) Presl leaf extracts, possesses molluscicidal activity against Oncomelania hupensis and inhibits infection of Schistosoma japonicum.(2014-08-29) Yang, Fan; Long, Erping; Wen, Juhua; Cao, Lei; Zhu, Chengcheng; Hu, Huanxin; Ruan, Ying; Kamolnetr Okanurak; กมลเนตร โอฆานุรักษ์; Hu, Huiling; Wei, Xiaoxia; Yang, Xiangyun; Wang, Chaofan; Zhang, Limei; Wang, Xiaoying; Ji, Pengyu; Zheng, Huanqin; Wu, Zhongdao; Lv, Zhiyue; Wu, Zhongdao; Lv, Zhiyue; Mahidol University. Faculty of Tropical Medicine. Department of Social and Environmental Medicine.BACKGROUND: Schistosomiasis japonicum remains a considerable economic and public health concern in China, the Philippines and Indonesia. Currently available measures to control the unique intermediate host Oncomelania hupensis are frequently associated with severe side effects. Previous studies have demonstrated that linalool-rich extracts from various plants exhibited promising biological activities including cytotoxic, anti-microbial and anti-parasitic properties. METHODS: We identified the components of leaf extracts from Cinnamomum camphora by gas chromatography coupled to mass spectrometry (GC-MS) and investigated molluscicidal and larvicidal effects of linalool against O. hupensis and Schistosoma japonicium. The ultrastructural alterations in gills, salivary gland, stomach and hepatopancreas of snails were observed under the light microscope and transmission electron microscope, and lesions to tegument of cercaria were examined under a light microscope and fluorescence microscope. We then evaluated the effects of linalool on skin penetration and migration of schistosomula and adult survival by measurement of worm burden and egg counts in Balb/C mice infected with linalool-treated cercariae. RESULTS: In the present work, 44 components were identified from the leaf extracts of C. camphora, of which linalool was the most abundant constituent. Linalool exhibited the striking molluscicidal and larvicidal effects with LC50 = 0.25 mg/L for O. hupensis and LC50 = 0.07 mg/L for cercaria of S. japonicium. After exposure to linalool, damage to the gills and hepatopancreas of the snails, and to the tegument and body-tail joint of cercariae was apparent. In addition, linalool markedly reduced the recovered schistosomulum from mouse skin after challenge infection, and therefore decreased the worm burden in infected animals, but not fecundity of female adults of the parasite. CONCLUSIONS: Our findings indicated that linalool might be a novel chemotherapeutic agent against S. japonicium and the snail intermediate host.Publication Open Access การปนเปื้อนของโลหะหนัก โครเมียม แคดเมียม ตะกั่ว ปรอท และสารหนู ในโซ่อีลาสโตเมอร์จัดฟัน(2012-01) ฤดี สุราฤทธิ์; Rudee Surarit; วิทยา วสุเมธารัศมี; Witthaya Wasumaetharatsamee; สมพร เรืองผกา; Somporn Raungpaka; พาสน์ศิริ นิสาลักษณ์; Passiri Nisalak; ภูมิภดา จาวจักรศิริ; Poompada Jaochakarasiri; ฤดี สุราฤทธิ์; Rudee Surarit; มหาวิทยาลัยมหิดล. คณะทันตแพทยศาสตร์. ภาควิชาชีววิทยาช่องปาก; มหาวิทยาลัยมหิดล. คณะทันตแพทยศาสตร์. สาขาทันตกรรมจัดฟัน; มหาวิทยาลัยมหิดล. คณะทันตแพทยศาสตร์. ภาควิชาทันตกรรมจัดฟันวัตุประสงค์: เพื่อหาโลหะหนักได้แก่ แคดเมียม โครเมียม ตะกั่ว ปรอท และสารหนู ที่อาจปนเปื้อนหรือปลดปล่อยจากโซ่อีลาสโตเมอร์ ที่ใช้สำหรับการจัดฟัน วัสดุอุปกรณ์และวิธีการศึกษา: ตัวอย่างที่ใช้ในการศึกษานี้คือโซ่อีลาสโตเมอร์ โครงรูปเปิด 3 สี (สีใส สีเหลือง และสีชมพู) ยี่ห้อ Dynaflex (Dynaflex Company, St Louis, MO, USA) และ Chuang Xin Power chain (PR China) ในการศึกษานี้แบ่งออกเป็น 2 ส่วน คือ ส่วนแรก เป็นการหาปริมาณโลหะหนักที่ปล่อยออกมาจาก โซ่อีลาสโตเมอร์ นำโซ่อีลาสโตเมอร์จำนวน 40 วง มาตัดและแช่ในน้ำลายเทียมเป็นเวลา 28 วัน ทึกตัวอย่างจะผ่านกระบวนการวัฏจักรพีเอช และวัฏจักรความร้อน เพื่อจำลองสภาพให้เสมือนอยู่ในช่องปากเป็นเวลา 28 วัน หลังจากกระบวนการสิ้นสุด นำน้ำลายเทียมไปตรวจหาโลหะหนัก (แคดเมียม, โครเมียม, ตะกั่ว, ปรอท และสารหนู) ด้วยเครื่องอะตอมมิกแอบซอร์บชั่นสเปคโตรโฟโตมิเตอร์ ส่วนที่ 2 เป็นการวิเคราะห์หาปริฒาณโลหะหนักที่เป็นส่วนประกอบในโซ่อีลาสโตเมอร์ โดยวิธีการเผาโดยตรงในเครื่องเฟลมอะตอมมิกแอบซอร์บชั่น สเปคโตร โฟโตมิเตอร์เพื่อดูการปนเปื้อนของโลหะหนัก ตามวิธีมาตรฐานของสมาคมวิชาชีพสหรัฐอเมริกาเพื่อทดสอบและวัสดุ ASTMD4045-06 ผลการศึกษา: พบว่าไม่มีโลหะหนักปลดปล่อยออกมาจากโซ่อีลาสโตเมอร์ จักฟันทั้งสองยี่ห้อในช่วงเวลาทดสอบ 28 วัน โซ่อีลาสโตเมอร์สีใสของทั้ง 2 ยี่ห้อมีปริมาณ แคดเมียม และ ตะกั่ว มากที่สุดในจำนวน 3 สีที่ทำการทดลองปริมาณโลหะหนักที่พบอยู่ในระดับต่ำกว่าที่เสนอแนะไว้โดยองค์กรอนามัยโลกและองค์การอาหารและยา บทสรุป: โซ่อีลาสโตเมอร์จัดฟันของทั้งสองบริษัท คือ Dynaflex และ Chuang Xin Power chain ทั้ง 3 สี มีปริมาณโลหะหนักในระดับต่ำกว่าระดับความปลอดภัย ที่เสนอแนะไว้โดยองค์กรอนามัยโลกและองค์การอาหารและยา