Pub Date : 2018-01-01DOI: 10.4172/2155-9872.1000411
Eyasu Gebrie Ajebe, Tasew Belete Bahiru
{"title":"Spectrophotometric Determination of Nitrite and Nitrate in Some Selected Vegetables Cultivated in Adami Tulu Judo Kombolicha District Farms, Ethiopia","authors":"Eyasu Gebrie Ajebe, Tasew Belete Bahiru","doi":"10.4172/2155-9872.1000411","DOIUrl":"https://doi.org/10.4172/2155-9872.1000411","url":null,"abstract":"","PeriodicalId":14865,"journal":{"name":"Journal of analytical and bioanalytical techniques","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2018-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"74865641","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2018-01-01DOI: 10.4172/2155-9872.1000402
K. SujeetThakur, S. Eswaran
Chemical cross-linking-mass spectrometry (CX-MS) combined with bioinformatics tools is increasingly being used to analyze large-scale protein–protein interactions. It has gained importance in studies in proteomics, lipidomics, in systems and structural biology. Recently it has gained importance in preparation of homogeneous antibody-drug conjugates, which has been described as “a pinnacle of such targeting efforts.” What makes these approaches exciting is that using the “Click” and Bertozzi protocols in vivo studies can be carried out successfully. Using CX-MS combined with cryo-EM, structures of protein complexes can now be probed at almost molecular resolution (upto 3 A). Chemical crosslinking is useful in materials science, as well. Major advances in both mass spectrometric techniques and bioinformatics tools today allow one to identify cross-linked peptides with highconfidence and with more user-friendly approaches. Crucial to this is the ability to capture intermolecular crosslinking reliably. The use of a new small NHS-aryl azido heterobifunctional cross-linker based is described here, which picks intermolecular crosslinking better. Thus, Lysozyme has been crosslinked successfully as established by the ‘dimeric’ band observed in SDS-PAGE. its tryptic digestion, ‘zip tip’ enrichment, ESI-MS, MS/MS and the data generated analyzed using StavroX 3.6.0.1, a bioinformatics software, especially suited for determining intermolecular crosslinking.
化学交联质谱(CX-MS)结合生物信息学工具越来越多地用于分析大规模蛋白质-蛋白质相互作用。它在蛋白质组学、脂质组学、系统和结构生物学的研究中占有重要地位。最近，它在制备均质抗体-药物偶联物方面变得越来越重要，这被描述为“这种靶向努力的顶峰”。使这些方法令人兴奋的是，使用“Click”和Bertozzi协议可以成功地进行体内研究。使用CX-MS结合冷冻电镜，蛋白质复合物的结构现在可以在几乎分子分辨率(高达3a)下进行探测。化学交联在材料科学中也很有用。今天，质谱技术和生物信息学工具的重大进展使人们能够以高置信度和更友好的方法识别交联肽。对此至关重要的是可靠地捕获分子间交联的能力。本文介绍了一种新型的小的基于nhs -芳基叠氮杂双功能交联剂的使用，它能更好地选择分子间交联。因此，溶菌酶已通过SDS-PAGE上观察到的“二聚体”带成功交联。它的色氨酸消化，' zip tip '富集，ESI-MS, MS/MS和生成的数据分析使用StavroX 3.6.0.1，一个生物信息学软件，特别适合测定分子间交联。
{"title":"ESI-MS and Stavrox 3.6.0.1 Investigations of Crosslinking by an Aryl-Azido-NHS-Heterobifunctional Crosslinker","authors":"K. SujeetThakur, S. Eswaran","doi":"10.4172/2155-9872.1000402","DOIUrl":"https://doi.org/10.4172/2155-9872.1000402","url":null,"abstract":"Chemical cross-linking-mass spectrometry (CX-MS) combined with bioinformatics tools is increasingly being used to analyze large-scale protein–protein interactions. It has gained importance in studies in proteomics, lipidomics, in systems and structural biology. Recently it has gained importance in preparation of homogeneous antibody-drug conjugates, which has been described as “a pinnacle of such targeting efforts.” What makes these approaches exciting is that using the “Click” and Bertozzi protocols in vivo studies can be carried out successfully. Using CX-MS combined with cryo-EM, structures of protein complexes can now be probed at almost molecular resolution (upto 3 A). Chemical crosslinking is useful in materials science, as well. Major advances in both mass spectrometric techniques and bioinformatics tools today allow one to identify cross-linked peptides with highconfidence and with more user-friendly approaches. Crucial to this is the ability to capture intermolecular crosslinking reliably. The use of a new small NHS-aryl azido heterobifunctional cross-linker based is described here, which picks intermolecular crosslinking better. Thus, Lysozyme has been crosslinked successfully as established by the ‘dimeric’ band observed in SDS-PAGE. its tryptic digestion, ‘zip tip’ enrichment, ESI-MS, MS/MS and the data generated analyzed using StavroX 3.6.0.1, a bioinformatics software, especially suited for determining intermolecular crosslinking.","PeriodicalId":14865,"journal":{"name":"Journal of analytical and bioanalytical techniques","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2018-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"85838002","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2018-01-01DOI: 10.4172/2155-9872.1000412
M. Fanta, Yadava Op
{"title":"Determination of Some Trace Heavy Metals (Pb, Cr, Cd, Mn and Zn) Levels in Iron Ores from Mines in Wollega (Ethiopia) Using Atomic Absorption Spectrometric Technique","authors":"M. Fanta, Yadava Op","doi":"10.4172/2155-9872.1000412","DOIUrl":"https://doi.org/10.4172/2155-9872.1000412","url":null,"abstract":"","PeriodicalId":14865,"journal":{"name":"Journal of analytical and bioanalytical techniques","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2018-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"73096526","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2018-01-01DOI: 10.4172/2155-9872.1000407
Tassew Belete Bahru, Tesfaye Hailemariam Barkea, A. Sali
Pumpkin (Cucurbita pepo L), an herbaceous running plant belonging to family Cucurbitaceae, is one of the natural resources grown in Ethiopia. It is a medium sized plant grown for its fruits, leaves, seeds and flowers are edible. The leaf and fruit produced by C. pepo is the most palatable vegetables in the country. The purpose of the study was to investigate the primary metabolite such as carbohydrate, protein, fat, fiber, moisture and ash in leaf and fruit of three varieties of pumpkin namely; Jarrahdale, Porcelain Doll and Sugar pie. Young leaves and ripened fruits of the varieties of pumpkin were collected from selected districts of Gurage zone. Composite sample was grinded to powder size and air dried before analysis. The results obtained were: Carbohydrate (28.15%)>Protein (26.31%)>Fiber (17.55%)>Ash (10.96%)>Moisture (10.17%) in leaves and Carbohydrate (41.68%)>Moisture (17.33%)>Fiber (16.50%)>Ash (10.95%)>Protein (9.88%)>Fat (3.66%) in fruits of pumpkin were determined using Kjeldahl method (AOAC official method: 920.39, 925.10, 962.09) and APHA 2540. The contents in the plant are high enough and proportional to common vegetables. It is recommendable to enhance the consumption of leaf and fruits of plants.
{"title":"Investigation of Primary Metabolites in Young Leaf and Fruit of Three Varieties of Pumpkin ( Cucurbita pepo ) from Gurage Zone, Ethiopia","authors":"Tassew Belete Bahru, Tesfaye Hailemariam Barkea, A. Sali","doi":"10.4172/2155-9872.1000407","DOIUrl":"https://doi.org/10.4172/2155-9872.1000407","url":null,"abstract":"Pumpkin (Cucurbita pepo L), an herbaceous running plant belonging to family Cucurbitaceae, is one of the natural resources grown in Ethiopia. It is a medium sized plant grown for its fruits, leaves, seeds and flowers are edible. The leaf and fruit produced by C. pepo is the most palatable vegetables in the country. The purpose of the study was to investigate the primary metabolite such as carbohydrate, protein, fat, fiber, moisture and ash in leaf and fruit of three varieties of pumpkin namely; Jarrahdale, Porcelain Doll and Sugar pie. Young leaves and ripened fruits of the varieties of pumpkin were collected from selected districts of Gurage zone. Composite sample was grinded to powder size and air dried before analysis. The results obtained were: Carbohydrate (28.15%)>Protein (26.31%)>Fiber (17.55%)>Ash (10.96%)>Moisture (10.17%) in leaves and Carbohydrate (41.68%)>Moisture (17.33%)>Fiber (16.50%)>Ash (10.95%)>Protein (9.88%)>Fat (3.66%) in fruits of pumpkin were determined using Kjeldahl method (AOAC official method: 920.39, 925.10, 962.09) and APHA 2540. The contents in the plant are high enough and proportional to common vegetables. It is recommendable to enhance the consumption of leaf and fruits of plants.","PeriodicalId":14865,"journal":{"name":"Journal of analytical and bioanalytical techniques","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2018-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"90115657","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2018-01-01DOI: 10.4172/2155-9872.1000400
C. Fagerquist, William J. Zaragoza
Rationale: Shiga toxin-producing Escherichia coli (STEC) are often subjected to DNA damaging antibiotics during culturing in order to elicit the bacterial SOS response and up-regulation of bacteriophage-encoded proteins including Shiga toxin (Stx). However, such antibiotic exposure and stress may also have effects on protein expression.Methods: Escherichia coli O157:H7 strain EDL933 was grown on Luria-Bertani agar (LBA) supplemented with a sub-inhibitory concentration of ciprofloxacin. Bacterial cells were harvested, suspended in water, gently vortexed and centrifuged. Supernatants were analyzed by MALDI-TOF and nano-LC-ESI-Orbitrap mass spectrometry. A gene knockout was constructed to delete the B-subunit gene from the stx2a operon in the EDL933 strain. Results: We detected the B-subunits of Stx1a and Stx2a and also peaks in close proximity to these B-subunits. The mass difference between these variants and the Stx1a B-subunit are: -43 Da, +16 Da and +54 Da. For Stx2a B-subunit, the mass differences are: -111 Da, -91 Da, -72 Da, -59 Da, -44 Da, -29 Da, -15/-17 Da, +16 Da, +32 Da, +53/54 Da, +106 Da. When the stx2a gene knockout strain was cultured, it revealed the complete absence of the Stx2a B-subunit as well as its associated mass variants suggesting that the variants may be due to amino acid substitutions caused by translational errors. Conclusions: Our results suggest that ciprofloxacin (a fluoroquinolone antibiotic) may cause translational errors in expression of Stx. Incorporation of mistranslated B-subunit sequences into the Stx AB5 holotoxin has the potential to subtly alter its quaternary structure and its binding affinity to surface receptors of eukaryotic cells.
{"title":"Possible Mistranslation of Shiga Toxin from Pathogenic Escherichia coli as Measured by MALDI-TOF and Orbitrap Mass Spectrometry","authors":"C. Fagerquist, William J. Zaragoza","doi":"10.4172/2155-9872.1000400","DOIUrl":"https://doi.org/10.4172/2155-9872.1000400","url":null,"abstract":"Rationale: Shiga toxin-producing Escherichia coli (STEC) are often subjected to DNA damaging antibiotics during culturing in order to elicit the bacterial SOS response and up-regulation of bacteriophage-encoded proteins including Shiga toxin (Stx). However, such antibiotic exposure and stress may also have effects on protein expression.Methods: Escherichia coli O157:H7 strain EDL933 was grown on Luria-Bertani agar (LBA) supplemented with a sub-inhibitory concentration of ciprofloxacin. Bacterial cells were harvested, suspended in water, gently vortexed and centrifuged. Supernatants were analyzed by MALDI-TOF and nano-LC-ESI-Orbitrap mass spectrometry. A gene knockout was constructed to delete the B-subunit gene from the stx2a operon in the EDL933 strain. Results: We detected the B-subunits of Stx1a and Stx2a and also peaks in close proximity to these B-subunits. The mass difference between these variants and the Stx1a B-subunit are: -43 Da, +16 Da and +54 Da. For Stx2a B-subunit, the mass differences are: -111 Da, -91 Da, -72 Da, -59 Da, -44 Da, -29 Da, -15/-17 Da, +16 Da, +32 Da, +53/54 Da, +106 Da. When the stx2a gene knockout strain was cultured, it revealed the complete absence of the Stx2a B-subunit as well as its associated mass variants suggesting that the variants may be due to amino acid substitutions caused by translational errors. Conclusions: Our results suggest that ciprofloxacin (a fluoroquinolone antibiotic) may cause translational errors in expression of Stx. Incorporation of mistranslated B-subunit sequences into the Stx AB5 holotoxin has the potential to subtly alter its quaternary structure and its binding affinity to surface receptors of eukaryotic cells.","PeriodicalId":14865,"journal":{"name":"Journal of analytical and bioanalytical techniques","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2018-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"83055024","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2018-01-01DOI: 10.4172/2155-9872.1000406
Zelalem Tekulu, O. P. Yadav, T. Kebede
During the last few decades, there has been a growing interest in developing alternative energy systems to fossil fuels. Steam reforming of ethanol for producing hydrogen gas is promising since ethanol can readily be obtained through bio-mass fermentation. In the present work, catalytic activity of silica-supported bimetallic Co-Cu composites, for hydrogen production by steam reforming of ethanol, has been studied. A fixed-bed reactor was used to assess the performance of composite catalysts over the temperature range: 300-600°C. Catalyst activity, using varying water:ethanol molar ratio, was evaluated in terms of hydrogen yield and product selectivity. Ethanol conversion, hydrogen yield and selectivity were found to increase on raising water: ethanol molar ratio, catalyst load and the Co:Cu ratio. The catalyst with composition 8% Co/4% Cu/SiO2 at water:ethanol ratio 9:1 and temperature 500°C, gave ethanol conversion as high as 97.2%.
{"title":"Silica-Supported Bimetallic Co-Cu Composite - An Efficient Catalyst for Ethanol Based Hydrogen Production by Steam Reforming Technique","authors":"Zelalem Tekulu, O. P. Yadav, T. Kebede","doi":"10.4172/2155-9872.1000406","DOIUrl":"https://doi.org/10.4172/2155-9872.1000406","url":null,"abstract":"During the last few decades, there has been a growing interest in developing alternative energy systems to fossil fuels. Steam reforming of ethanol for producing hydrogen gas is promising since ethanol can readily be obtained through bio-mass fermentation. In the present work, catalytic activity of silica-supported bimetallic Co-Cu composites, for hydrogen production by steam reforming of ethanol, has been studied. A fixed-bed reactor was used to assess the performance of composite catalysts over the temperature range: 300-600°C. Catalyst activity, using varying water:ethanol molar ratio, was evaluated in terms of hydrogen yield and product selectivity. Ethanol conversion, hydrogen yield and selectivity were found to increase on raising water: ethanol molar ratio, catalyst load and the Co:Cu ratio. The catalyst with composition 8% Co/4% Cu/SiO2 at water:ethanol ratio 9:1 and temperature 500°C, gave ethanol conversion as high as 97.2%.","PeriodicalId":14865,"journal":{"name":"Journal of analytical and bioanalytical techniques","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2018-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"88805010","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2017-12-30DOI: 10.4172/2155-9872.1000393
K. SujeetThakur, S. Eswaran
A new NHS-aryl azido heterobifunctional cross-linker based on an “introverted” carboxylic acid has been used to bring about successful intermolecular cross-linking. As a ‘proof-of-concept’ Lysozyme was incubated with the crosslinker, then photolysed (366 nm, 6 W UV lamp), subjected to SDS-PAGE, excision of the ‘dimer’, trypsin digested and analyzed by ESI-MS and StavroX 3.6.0.1. Previous studies on crosslinking of Lysozyme (SI-I and SIII) using homobifunctional cross-linkers, either no cross-linking was observed or only two crosslinks were detected in the case of BS3, a smaller cross-linker. The heterobifunctional cross-linker described here leads to many more crosslinks, which have been identified by using mass spectrometry (ESI-MS) and StavroX 3.6.0.1, a bioinformatics software, especially suited for identifying intermolecular crosslinking.
{"title":"A New Heterobifunctional Cross-linker Based on an “Introverted” Acid: Mass Spectrometric and Bioinformatics Studies, Analysis of Intermolecular Crosslinking of Proteins","authors":"K. SujeetThakur, S. Eswaran","doi":"10.4172/2155-9872.1000393","DOIUrl":"https://doi.org/10.4172/2155-9872.1000393","url":null,"abstract":"A new NHS-aryl azido heterobifunctional cross-linker based on an “introverted” carboxylic acid has been used to bring about successful intermolecular cross-linking. As a ‘proof-of-concept’ Lysozyme was incubated with the crosslinker, then photolysed (366 nm, 6 W UV lamp), subjected to SDS-PAGE, excision of the ‘dimer’, trypsin digested and analyzed by ESI-MS and StavroX 3.6.0.1. Previous studies on crosslinking of Lysozyme (SI-I and SIII) using homobifunctional cross-linkers, either no cross-linking was observed or only two crosslinks were detected in the case of BS3, a smaller cross-linker. The heterobifunctional cross-linker described here leads to many more crosslinks, which have been identified by using mass spectrometry (ESI-MS) and StavroX 3.6.0.1, a bioinformatics software, especially suited for identifying intermolecular crosslinking.","PeriodicalId":14865,"journal":{"name":"Journal of analytical and bioanalytical techniques","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2017-12-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"91043238","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2017-12-30DOI: 10.4172/2155-9872.1000394
P. NuanCheah, Hendrickx Lpa, S. Borst, Cremers Jwjm, Jansen Ehjm, A. Opperhuizen, R. Talhout
In this study, we compared the pyrolysis products of the three most commonly used tobacco leaves using pyrolysis-gas chromatography coupled with mass spectrometry detection. For tobacco product regulation, it is important to understand the formation of emissions that occurs during smoking of tobacco. To this purpose, we report a simple analytical method to investigate the combustion process of tobacco leaves using a pyrolysis robot coupled to a GC-MS. This method allows for simulation of the tobacco smoking process, by taking one puff (single puff) and more puffs (puff-by-puff), and subsequent determination of the combustion products. We studied the 13 most abundant emissions formed during the pyrolysis of the three most common tobacco varieties: Burley, Virginia and Oriental. Unlike commercially available tobacco products, the tobacco leaves were not treated with any additives, allowing for an assessment of the combustion product of raw tobacco leaves. Our study shows that tobacco, when combusted, produces predominantly tobacco specific alkaloids such as nicotine, hydrocarbons (toluene, xylene and limonene) and other compounds such as acetaldehyde, phenol and furfural. Nicotine is the main chemical produced during pyrolysis followed by β-nicotyrine, acetaldehyde and toluene. For lower molecular weight components such as acetaldehyde, the amounts generally decreased with puff number. On the other hand, nicotine yields in all three tobacco leaves were found to increase with puff number.
{"title":"A New Technique to Determine Emissions of Burley, Virginia andO riental Tobacco Using Single Puff and Puff-By-Puff Pyrolysis","authors":"P. NuanCheah, Hendrickx Lpa, S. Borst, Cremers Jwjm, Jansen Ehjm, A. Opperhuizen, R. Talhout","doi":"10.4172/2155-9872.1000394","DOIUrl":"https://doi.org/10.4172/2155-9872.1000394","url":null,"abstract":"In this study, we compared the pyrolysis products of the three most commonly used tobacco leaves using pyrolysis-gas chromatography coupled with mass spectrometry detection. For tobacco product regulation, it is important to understand the formation of emissions that occurs during smoking of tobacco. To this purpose, we report a simple analytical method to investigate the combustion process of tobacco leaves using a pyrolysis robot coupled to a GC-MS. This method allows for simulation of the tobacco smoking process, by taking one puff (single puff) and more puffs (puff-by-puff), and subsequent determination of the combustion products. We studied the 13 most abundant emissions formed during the pyrolysis of the three most common tobacco varieties: Burley, Virginia and Oriental. Unlike commercially available tobacco products, the tobacco leaves were not treated with any additives, allowing for an assessment of the combustion product of raw tobacco leaves. Our study shows that tobacco, when combusted, produces predominantly tobacco specific alkaloids such as nicotine, hydrocarbons (toluene, xylene and limonene) and other compounds such as acetaldehyde, phenol and furfural. Nicotine is the main chemical produced during pyrolysis followed by β-nicotyrine, acetaldehyde and toluene. For lower molecular weight components such as acetaldehyde, the amounts generally decreased with puff number. On the other hand, nicotine yields in all three tobacco leaves were found to increase with puff number.","PeriodicalId":14865,"journal":{"name":"Journal of analytical and bioanalytical techniques","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2017-12-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"81137494","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2017-12-18DOI: 10.4172/2155-9872.1000390
N. Griesche, V. Pesch, Rohit Nalavade, S. Weber, Ireen König, M. Schölling, Christoph Möhl, Sybille Krau
Traditionally, studies on protein translation rely on systems, in which cells have been lysed prior determination of levels of the protein of interest. However, these assays do not reflect the protein synthesis in living cells in real time, but analyze protein levels after a given incubation time, leading to limitations in results based on experimental parameters. To overcome this problem, we have previously established a Fluorescence recovery after photobleaching (FRAP)-based technique to monitor protein translation in living cells. For this, the protein of interest fused to green fluorescent protein (GFP) is expressed in cell lines. After bleaching the entire cell, the fluorescent signal of the protein of interest is lost, allowing to capture the signal recovery of newly translated GFPtagged protein over time. Here we present two improved versions of this technique using different fluorescent dyes: tFRAP (translational FRAP). For the first improved version of tFRAP we have inserted a second fluorescent dye, red fluorescent protein (RFP), into the same expression vector that drives expression of the protein of interest fused to GFP driven by a second promoter. For the second improved version of tFRAP we have fused our protein of interest to a photo-switchable dye, Dendra2. Both improved versions allow to correct the fluorescence signal intensity of the protein of interest for different transfection rates of individual cells. These two advanced techniques are new powerful tools for quantifying translation rates in living cells and will be useful in future studies on mRNA translation.
{"title":"tFRAP: A FRAP-Based Technique to Monitor Protein Translation in Living Cells","authors":"N. Griesche, V. Pesch, Rohit Nalavade, S. Weber, Ireen König, M. Schölling, Christoph Möhl, Sybille Krau","doi":"10.4172/2155-9872.1000390","DOIUrl":"https://doi.org/10.4172/2155-9872.1000390","url":null,"abstract":"Traditionally, studies on protein translation rely on systems, in which cells have been lysed prior determination of levels of the protein of interest. However, these assays do not reflect the protein synthesis in living cells in real time, but analyze protein levels after a given incubation time, leading to limitations in results based on experimental parameters. To overcome this problem, we have previously established a Fluorescence recovery after photobleaching (FRAP)-based technique to monitor protein translation in living cells. For this, the protein of interest fused to green fluorescent protein (GFP) is expressed in cell lines. After bleaching the entire cell, the fluorescent signal of the protein of interest is lost, allowing to capture the signal recovery of newly translated GFPtagged protein over time. Here we present two improved versions of this technique using different fluorescent dyes: tFRAP (translational FRAP). For the first improved version of tFRAP we have inserted a second fluorescent dye, red fluorescent protein (RFP), into the same expression vector that drives expression of the protein of interest fused to GFP driven by a second promoter. For the second improved version of tFRAP we have fused our protein of interest to a photo-switchable dye, Dendra2. Both improved versions allow to correct the fluorescence signal intensity of the protein of interest for different transfection rates of individual cells. These two advanced techniques are new powerful tools for quantifying translation rates in living cells and will be useful in future studies on mRNA translation.","PeriodicalId":14865,"journal":{"name":"Journal of analytical and bioanalytical techniques","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2017-12-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"75824409","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2017-11-06DOI: 10.4172/2155-9872.1000387
M. S. Gracia, R. Brandl, E. Haen
The purpose of this study was to evaluate a potential pharmacokinetic interaction between antipsychotics (quetiapine QUE, clozapine CLO and olanzapine OLA) and beta-blockers (metoprolol MET, propranolol PRO, bisoprolol BIS, nebivolol NEB and carvedilol CAR). Antipsychotics and beta-blockers are metabolized by the same cytochrome-P450 (CYP)-isoenzymes, which are inhibited by representatives of both substance classes. Pooled human liver microsomes (HLM) and recombinant CYP isoenzymes were used to determine whether the investigated antipsychotics and beta-blockers inhibit the metabolism of each other. Drug concentrations have been measured by high performance liquid chromatography (HPLC) with ultraviolet (UV) detection. Experiments with HLM showed that the metabolism of QUE as well as CLO slowed down in presence of CAR. There was no significant difference between the metabolism of the antipsychotics alone and the metabolism in combination with BIS, NEB and the two known CYP2D6-inhibitors MET and PRO. Experiments with recombinant CYP2D6 demonstrated an inhibitory effect on the metabolism of QUE and CLO by MET, PRO, NEB and CAR. The results suggest that CAR is also an inhibitor of other CYP enzymes, which are involved in the metabolism of CLO and QUE. It is assumed that CYP2D6 is a minor pathway of the antipsychotics and that the CYP2D6-inhibitory-effect of MET, PRO and NEB is compensated through a higher metabolism rate via the metabolic pathways of the other CYP-isoforms.
{"title":"Lack of Pharmacokinetic Interaction between Antipsychotics (Quetiapine, Clozapine and Olanzapine) and the Beta-Blockers Metoprolol, Propranolol, Bisoprolol and Nebivolol Except between Carvedilol and the Antipsychotics Quetiapine and Clozapine","authors":"M. S. Gracia, R. Brandl, E. Haen","doi":"10.4172/2155-9872.1000387","DOIUrl":"https://doi.org/10.4172/2155-9872.1000387","url":null,"abstract":"The purpose of this study was to evaluate a potential pharmacokinetic interaction between antipsychotics (quetiapine QUE, clozapine CLO and olanzapine OLA) and beta-blockers (metoprolol MET, propranolol PRO, bisoprolol BIS, nebivolol NEB and carvedilol CAR). Antipsychotics and beta-blockers are metabolized by the same cytochrome-P450 (CYP)-isoenzymes, which are inhibited by representatives of both substance classes. Pooled human liver microsomes (HLM) and recombinant CYP isoenzymes were used to determine whether the investigated antipsychotics and beta-blockers inhibit the metabolism of each other. Drug concentrations have been measured by high performance liquid chromatography (HPLC) with ultraviolet (UV) detection. Experiments with HLM showed that the metabolism of QUE as well as CLO slowed down in presence of CAR. There was no significant difference between the metabolism of the antipsychotics alone and the metabolism in combination with BIS, NEB and the two known CYP2D6-inhibitors MET and PRO. Experiments with recombinant CYP2D6 demonstrated an inhibitory effect on the metabolism of QUE and CLO by MET, PRO, NEB and CAR. The results suggest that CAR is also an inhibitor of other CYP enzymes, which are involved in the metabolism of CLO and QUE. It is assumed that CYP2D6 is a minor pathway of the antipsychotics and that the CYP2D6-inhibitory-effect of MET, PRO and NEB is compensated through a higher metabolism rate via the metabolic pathways of the other CYP-isoforms.","PeriodicalId":14865,"journal":{"name":"Journal of analytical and bioanalytical techniques","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2017-11-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"89459028","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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