This document summarizes the engineering and characterization of a soluble cytoplasmic subcomplex (C-MBH) of the membrane-bound respiratory hydrogenase (MBH) from the hyperthermophilic archaeon Pyrococcus furiosus. The researchers engineered a P. furiosus strain that differentially expresses the 14-gene MBH operon, producing a 4-subunit C-MBH complex containing an affinity tag. They purified the C-MBH using affinity chromatography without detergent. The purified C-MBH had catalytic activity and generated hydrogen from the physiological electron donor reduced ferredoxin, optimally at 60°C. This is the first report of engineering and characterizing a soluble
This document summarizes research characterizing the intact 14-subunit respiratory membrane bound [NiFe]-hydrogenase complex (MBH) from the hyperthermophilic archaeon Pyrococcus furiosus. The researchers expressed a His-tagged version of MBH in P. furiosus and purified the intact detergent-solubilized complex using affinity chromatography. The purified MBH complex contained all 14 subunits and exhibited catalytic hydrogen production with physiological electron donors, similar to the membrane-bound form. Small angle X-ray scattering was used to generate a structural model of the purified MBH complex, providing insights into its Z-shaped structure and relationship to other respiratory complexes like Complex I.
The functional complexity of [NiFe] hydrogenases in sulfate reducing bacteria...Tijani Hamzat Ibiyeye
Abstract: Sulfate-reducing bacteria are categories of bacteria and archaea that can obtain energy by oxidizing organic compounds or molecular hydrogen while reducing sulfate to hydrogen sulfide. By analysis, these organisms "respire" sulfate rather than oxygen, a form of anaerobic respiration, the oxidation of hydrogen by the primary
genus of Sulfate Reducing Bacteria (Desulfovibrio, Desulfovibrio desulfuricans) is catalyzed by enzymes called Hydrogenases. Three basic types of hydrogenases have been widely isolated from the primary genus of sulfate-reducing bacteria Desulfobibrio which differ in their structural subunits, metal compositions, physico-chemical characteristics, amino acid sequences, immunological activities, structural gene configuration and their catalytic properties. Broadly, hydrogenases can be considered as ‘iron containing hydrogenases and nickel-containing hydrogenases. The iron-sulfur containing hydrogenase enzyme contains two ferredoxin-type clusters and typical iron-sulfur center believed to be involved in the activation of H2 yet it is the most sensitive domain to CO and NO2−. Even though it is not featured in all species of genus Desulfovibrio. The nickel-(iron-sulfur)-containing hydrogenases, [NiFe] hydrogenase posses two 4Fe-4S centers and one 3Fe-xS cluster in addition to nickel and have been found in all species of Desulfovibrio with strong resistance to CO and NO2- so far investigated. The genes encoding the large and small subunits of a periplasmic and
membrane-bound species of the [NiFe] hydrogenase have been cloned in Escherichia coli and sequenced, however the functional complexity of the hydrogenase system remained unexplored as a result of the metabolic diversity in Desulfovibrio spp. The [NiFe] hydrogenase plays an important role in the energy metabolism of Desulfovibrio spp. Thus,
the expression of the encoded structural genes would be an excellent marker for the metabolic functionalities under specific inducible environment
This document summarizes research on engineering a subcomplex of the hydrogenase enzyme from Pyrococcus furiosus that is capable of producing hydrogen. Key points:
- Researchers successfully produced a heterodimeric form of the cytoplasmic hydrogenase I from P. furiosus (SHI) containing only two of the four subunits in the native enzyme.
- The engineered heterodimer is highly active in producing hydrogen using an artificial electron donor and is thermostable.
- Unlike the native enzyme, the heterodimer directly utilizes reducing power from pyruvate ferredoxin oxidoreductase without needing an intermediate electron carrier.
- This represents a two-enzyme system
This document discusses new roles for bacterial siderophores in metal transport and tolerance. Siderophores are known to strongly chelate iron, but they can also bind other metals. There is evidence that metals other than iron can induce siderophore production, implicating siderophores in homeostasis of multiple metals. Siderophores form stable complexes with various metals, including some heavy metals, and may contribute to bacterial tolerance against metal toxicity. The ability of siderophores to transport both essential and non-essential metals suggests they influence metal migration in the environment.
This document describes research on the heterologous expression of an NADP-dependent [NiFe]-hydrogenase from Pyrococcus furiosus in Escherichia coli. The researchers were able to successfully produce a functional form of the recombinant hydrogenase enzyme by co-expressing 13 P. furiosus genes involved in hydrogenase maturation in E. coli. Remarkably, providing only the four structural genes encoding the hydrogenase subunits and a single protease from P. furiosus was sufficient for the native E. coli maturation machinery to generate functional hydrogenase. This demonstrates that E. coli can assemble a functional hydrogenase from a phylogenetically distant hyperthermophilic organism.
1. The document describes the design and synthesis of novel indole-quinoxaline hybrid molecules to target phosphodiesterase 4 (PDE4).
2. A series of hybrid molecules were designed by connecting an indole moiety to a quinoxaline ring through a linker. Their potential as PDE4 inhibitors was validated through in silico docking studies.
3. The key steps in the synthesis involved coupling an indole derivative containing an iodo group with a propargyl-substituted quinoxaline derivative using a Pd/C-Cu mediated reaction followed by cyclization to form the hybrid molecules. Some of the synthesized compounds showed PDE4 inhibition activity in vitro tests
Investigation of heavy metals content (Cd , Ni and Pb) in the muscle tissue o...Innspub Net
This study has been done to determine the amount of the heavy metals like Cd, Ni, and Pb in the muscle tissue of Hoof benthic fish (Psettodes erumei) and urban pelagic fish (Lethrinus nebulosus) in Qeshm Island in the north of the Persian Gulf. On the whole, the heavy metal content of 30 tissue samples was randomly measured by atomic absorption spectrometer. The mean amount of Cd and Ni in benthic Hoof was significantly greater than that in the Urban pelagic fish while the concentration of pb in urban pelagic fish was higher than that in the Hoof tissue samples (P<0.05). Our results showed that the concentration of the three measured heavy metals in the fishes studied was less than the standard levels proposed by WHO and FAO. Get via original link: http://www.innspub.net/jbes/investigation-of-heavy-metals-content-cd-ni-and-pb-in-the-muscle-tissue-of-two-commercial-fishes-of-the-qeshm-island-persian-gulf/
Studies on the formation of formaldehyde during 2-ethylhexyl 4-(dimethylamino...Maciej Przybyłek
In order to protect the skin from UV radiation, personal care products (PCPS) often contain chemical UV-filters. These compounds can enter the environment causing serious consequences on the water ecosystems. The aim of this study was to examine, the effect of different factors, such as UV light, the presence of NaOCl and H2O2 on the formaldehyde formation during popular UV filter, 2-ethylhexyl 4-(dimethylamino)benzoate (ODPABA) demethylation. The concentration of formaldehyde was determined by VIS spectrophotometry after derivatization. The reaction mixtures were qualitatively analyzed using GC/MS chromatography. The highest concentration of formaldehyde was observed in the case of ODPABA/H2O2/UV reaction mixture. In order to describe two types of demethylation mechanisms, namely, radical and ionic, the experimental results were enriched with Fukui function analysis and thermodynamic calculations. In the case of non-irradiated system containing ODPABA and NaOCl, demethylation reaction probably proceeds via ionic mechanism. As it was established, amino nitrogen atom in the ODPABA molecule is the most susceptible site for the HOCl electrophilic attack, which is the first step of ionic demethylation mechanism. In the case of irradiated mixtures, the reaction is probably radical in nature. The results of thermodynamic calculations showed that abstraction of the hydrogen from N(CH3)2 group is more probable than from 2-ethylhexyl moiety, which indicates higher susceptibility of N(CH3)2 to the oxidation.
This document summarizes research characterizing the intact 14-subunit respiratory membrane bound [NiFe]-hydrogenase complex (MBH) from the hyperthermophilic archaeon Pyrococcus furiosus. The researchers expressed a His-tagged version of MBH in P. furiosus and purified the intact detergent-solubilized complex using affinity chromatography. The purified MBH complex contained all 14 subunits and exhibited catalytic hydrogen production with physiological electron donors, similar to the membrane-bound form. Small angle X-ray scattering was used to generate a structural model of the purified MBH complex, providing insights into its Z-shaped structure and relationship to other respiratory complexes like Complex I.
The functional complexity of [NiFe] hydrogenases in sulfate reducing bacteria...Tijani Hamzat Ibiyeye
Abstract: Sulfate-reducing bacteria are categories of bacteria and archaea that can obtain energy by oxidizing organic compounds or molecular hydrogen while reducing sulfate to hydrogen sulfide. By analysis, these organisms "respire" sulfate rather than oxygen, a form of anaerobic respiration, the oxidation of hydrogen by the primary
genus of Sulfate Reducing Bacteria (Desulfovibrio, Desulfovibrio desulfuricans) is catalyzed by enzymes called Hydrogenases. Three basic types of hydrogenases have been widely isolated from the primary genus of sulfate-reducing bacteria Desulfobibrio which differ in their structural subunits, metal compositions, physico-chemical characteristics, amino acid sequences, immunological activities, structural gene configuration and their catalytic properties. Broadly, hydrogenases can be considered as ‘iron containing hydrogenases and nickel-containing hydrogenases. The iron-sulfur containing hydrogenase enzyme contains two ferredoxin-type clusters and typical iron-sulfur center believed to be involved in the activation of H2 yet it is the most sensitive domain to CO and NO2−. Even though it is not featured in all species of genus Desulfovibrio. The nickel-(iron-sulfur)-containing hydrogenases, [NiFe] hydrogenase posses two 4Fe-4S centers and one 3Fe-xS cluster in addition to nickel and have been found in all species of Desulfovibrio with strong resistance to CO and NO2- so far investigated. The genes encoding the large and small subunits of a periplasmic and
membrane-bound species of the [NiFe] hydrogenase have been cloned in Escherichia coli and sequenced, however the functional complexity of the hydrogenase system remained unexplored as a result of the metabolic diversity in Desulfovibrio spp. The [NiFe] hydrogenase plays an important role in the energy metabolism of Desulfovibrio spp. Thus,
the expression of the encoded structural genes would be an excellent marker for the metabolic functionalities under specific inducible environment
This document summarizes research on engineering a subcomplex of the hydrogenase enzyme from Pyrococcus furiosus that is capable of producing hydrogen. Key points:
- Researchers successfully produced a heterodimeric form of the cytoplasmic hydrogenase I from P. furiosus (SHI) containing only two of the four subunits in the native enzyme.
- The engineered heterodimer is highly active in producing hydrogen using an artificial electron donor and is thermostable.
- Unlike the native enzyme, the heterodimer directly utilizes reducing power from pyruvate ferredoxin oxidoreductase without needing an intermediate electron carrier.
- This represents a two-enzyme system
This document discusses new roles for bacterial siderophores in metal transport and tolerance. Siderophores are known to strongly chelate iron, but they can also bind other metals. There is evidence that metals other than iron can induce siderophore production, implicating siderophores in homeostasis of multiple metals. Siderophores form stable complexes with various metals, including some heavy metals, and may contribute to bacterial tolerance against metal toxicity. The ability of siderophores to transport both essential and non-essential metals suggests they influence metal migration in the environment.
This document describes research on the heterologous expression of an NADP-dependent [NiFe]-hydrogenase from Pyrococcus furiosus in Escherichia coli. The researchers were able to successfully produce a functional form of the recombinant hydrogenase enzyme by co-expressing 13 P. furiosus genes involved in hydrogenase maturation in E. coli. Remarkably, providing only the four structural genes encoding the hydrogenase subunits and a single protease from P. furiosus was sufficient for the native E. coli maturation machinery to generate functional hydrogenase. This demonstrates that E. coli can assemble a functional hydrogenase from a phylogenetically distant hyperthermophilic organism.
1. The document describes the design and synthesis of novel indole-quinoxaline hybrid molecules to target phosphodiesterase 4 (PDE4).
2. A series of hybrid molecules were designed by connecting an indole moiety to a quinoxaline ring through a linker. Their potential as PDE4 inhibitors was validated through in silico docking studies.
3. The key steps in the synthesis involved coupling an indole derivative containing an iodo group with a propargyl-substituted quinoxaline derivative using a Pd/C-Cu mediated reaction followed by cyclization to form the hybrid molecules. Some of the synthesized compounds showed PDE4 inhibition activity in vitro tests
Investigation of heavy metals content (Cd , Ni and Pb) in the muscle tissue o...Innspub Net
This study has been done to determine the amount of the heavy metals like Cd, Ni, and Pb in the muscle tissue of Hoof benthic fish (Psettodes erumei) and urban pelagic fish (Lethrinus nebulosus) in Qeshm Island in the north of the Persian Gulf. On the whole, the heavy metal content of 30 tissue samples was randomly measured by atomic absorption spectrometer. The mean amount of Cd and Ni in benthic Hoof was significantly greater than that in the Urban pelagic fish while the concentration of pb in urban pelagic fish was higher than that in the Hoof tissue samples (P<0.05). Our results showed that the concentration of the three measured heavy metals in the fishes studied was less than the standard levels proposed by WHO and FAO. Get via original link: http://www.innspub.net/jbes/investigation-of-heavy-metals-content-cd-ni-and-pb-in-the-muscle-tissue-of-two-commercial-fishes-of-the-qeshm-island-persian-gulf/
Studies on the formation of formaldehyde during 2-ethylhexyl 4-(dimethylamino...Maciej Przybyłek
In order to protect the skin from UV radiation, personal care products (PCPS) often contain chemical UV-filters. These compounds can enter the environment causing serious consequences on the water ecosystems. The aim of this study was to examine, the effect of different factors, such as UV light, the presence of NaOCl and H2O2 on the formaldehyde formation during popular UV filter, 2-ethylhexyl 4-(dimethylamino)benzoate (ODPABA) demethylation. The concentration of formaldehyde was determined by VIS spectrophotometry after derivatization. The reaction mixtures were qualitatively analyzed using GC/MS chromatography. The highest concentration of formaldehyde was observed in the case of ODPABA/H2O2/UV reaction mixture. In order to describe two types of demethylation mechanisms, namely, radical and ionic, the experimental results were enriched with Fukui function analysis and thermodynamic calculations. In the case of non-irradiated system containing ODPABA and NaOCl, demethylation reaction probably proceeds via ionic mechanism. As it was established, amino nitrogen atom in the ODPABA molecule is the most susceptible site for the HOCl electrophilic attack, which is the first step of ionic demethylation mechanism. In the case of irradiated mixtures, the reaction is probably radical in nature. The results of thermodynamic calculations showed that abstraction of the hydrogen from N(CH3)2 group is more probable than from 2-ethylhexyl moiety, which indicates higher susceptibility of N(CH3)2 to the oxidation.
Metal-organic frameworks (MOFs) are highly porous materials consisting of metal atoms or clusters linked by organic molecules. MOFs have potential applications in hydrogen storage, methane storage, and carbon dioxide capture due to their ability to store gases at high densities through surface bonding. Australian researchers Matthew Hill and David Turner collaborate on using MOFs for gas storage and have achieved several world records. Their research utilizes synchrotron facilities to characterize MOF crystal structures, map their formation, and modify them for improved properties and applications.
This document discusses stable isotope deltas, which are tiny yet robust signatures that can be measured in nature. It explains that two fundamental processes, isotopic fractionation and isotope mixing, are responsible for most stable isotope variations seen in terrestrial systems. Isotopic fractionation occurs through equilibrium or kinetic processes that fractionate isotopes due to small differences in their physical or chemical properties. Isotope mixing models can provide information about processes like 14C abundances in the atmosphere and past ocean isotopic compositions. The document also proposes a new unit called the "urey" to describe isotope deltas in a way that overcomes limitations of traditional units.
Lead Acetate Induced histopathological Changes in Kidney Tissue of RatIOSRJAVS
One of the pollutants that can effect on the life of living animates is lead in different ways; it has toxic problem for human and animals and is gathered in the tissues and guide to harmful effects in their. The current study was carry out to investigate the histological effects caused by lead in the rat. The study was performed on 30 rat, they were divided into 3 groups. The first group was control group that received distilled water. The second groups were orally administered lead acetate 9 mg/l of body weight for 10 days. The third groups were orally administered solution of lead. The rats were anesthetized, the kidney were removed for histological studies. Histological changes which observed in the kidney were fatty degeneration, destruction tubules, and congestion within connective tissue, hemorrhage and infiltration of inflammatory cells. In this study, harmful toxic effects observed in kidney of rats.
This document summarizes metal-organic frameworks (MOFs), including their properties and applications. MOFs are highly porous materials formed by combining metal ions or metal clusters with organic ligands. They have extremely large surface areas, often exceeding 7,000 m2/g. Due to their tunable porous structures, MOFs show promise for applications such as gas storage, carbon capture, catalysis, and luminescence. While MOF research has advanced significantly in recent decades, further developing their potential applications and improving stability remains an active area of research.
This document describes an approach to rapidly generate molecular diversity using photoassisted diversity-oriented synthesis. Linear photoprecursors containing amino ketone and unsaturated pendant groups are assembled using high-yielding coupling reactions. These precursors undergo efficient intramolecular photocyclizations upon irradiation to form polyheterocyclic scaffolds with increased saturation, reduced rotatable bonds, and new structural frameworks. The primary photoproducts contain reactive functional groups that allow for further post-photochemical modifications, growing molecular complexity and accessing elaborated three-dimensional structures. This modular approach aims to generate drug-like molecules with properties known to correlate with clinical success, overcoming limitations of traditional combinatorial chemistry methods.
This thesis studied the metabolic pathways altered in Pseudomonas fluorescens Pf-5 due to hexavalent chromium stress using NMR-based metabolomics. P. fluorescens was exposed to 50 ppm of chromium for 6 and 24 hours. Metabolite extracts were analyzed using NMR and principal component analysis showed distinct metabolic profiles between control and stressed cells. Further analysis identified significant metabolites and probable pathways impacted by chromium stress.
This document summarizes key concepts from chapters 6-8 of a microbiology textbook including:
- The essential nutrients required by microbes including carbon, hydrogen, oxygen, nitrogen, phosphorus, and sulfur.
- The chemical composition of E. coli and what microbes use as energy and carbon sources, categorizing them as autotrophs and heterotrophs.
- The processes of osmosis, diffusion, and endocytosis that transport substances into and out of cells.
- Temperature categories for microbes like psychrophiles, psychrotrophs and how gases like oxygen and carbon dioxide influence microbial growth.
Dr. Shaukat Ali has extensive experience in organic synthesis and heterocyclic chemistry. He received his PhD from Lanzhou University in China, where he conducted research on electrophile-induced reactions to synthesize functionalized quinolines and indoloquinolines. Since 2012, he has worked as an Assistant Professor in Pakistan, where he has published over 20 papers with an h-index of 11. He is proficient in organic reactions and characterization techniques.
The document describes a new flexible synthesis of pyrazoles that allows for varying substituents at the C3 and C5 positions of the pyrazole ring. The synthesis involves coupling protected alkynols with acid chlorides to form alkynyl ketones, which are reacted with hydrazine to install the pyrazole nucleus. Alcohol deprotection and conversion to chlorides provides access to 5-substituted 3-(chloromethyl)- or 3-(2-chloroethyl)pyrazoles. These chlorides can then undergo nucleophilic substitution to generate other polyfunctional pyrazoles. The significance is that substituents at C5 control the steric environment around the pyrazole N-H
Study on Physico-Chemical Parameters of Harsi Reservoir Dabra, Gwalior Distri...ijsrd.com
this study was aimed to estimate physico-chemical characteristic of Harsi reservoir. Harsi reservoir located in Dabra, Gwalior district, Madhya Pradesh is constructed on parwati River. Monthly study in Physico-chemical parameters such as water temperature, depth, transparency, electrical conductivity, turbidity, total dissolved solids, pH, dissolved oxygen, free carbon dioxide, total alkalinity, total hardness, chlorides, sulphates, nitrate, nitrite, phosphate, silicates, ammonia, BOD, COD, calcium, magnesium, sodium, potassium were analyzed from January 2011 to December 2011. The results indicated that Physico-chemical parameters of the water were used for drinking, domestic use, irrigation and pisciculture.
The document summarizes a student research project investigating the design of short peptide amphiphiles that can bind transition metal ions. The student synthesized a peptide called C16-AHLHL3K3 and used various characterization techniques. Results showed the peptide formed beta-pleated sheet fibers under certain conditions and was able to bind transition metals like cobalt. This demonstrates the potential for short peptides to have transition metal binding sites and possibly enzymatic capabilities.
This document summarizes the synthesis of tris(4-((E)-2-nitro-3-(phenylamino)prop-1-enyl)phenyl)amine from a Baylis–Hillman adduct. Specifically, it details the reaction of tris(4-((E)-3-bromo-2-nitroprop-1-enyl)phenyl)amine with aniline to yield the target compound in 95% yield. The synthesis demonstrates the utility of bromo derivatives of Baylis–Hillman adducts as intermediates for generating novel trisubstituted triallylamines. Characterization of the product by NMR spectroscopy is also discussed.
Evidence of heavy metals distribution in placenta in association with residua...Alexander Decker
This study analyzed samples of placenta, kidney, and liver from cattle at an abattoir in Nigeria to investigate the distribution of heavy metals. The study found chromium present in all samples, while cadmium and lead were not detected. Chromium levels were highest in the kidney, followed by the liver and placenta. There was a moderate correlation between chromium levels in the dam's liver and placenta, but a weak correlation with the kidney. The results indicate that heavy metals like chromium can cross the placental barrier from the mother and accumulate in fetal tissues.
International Journal of Pharmaceutical Science Invention (IJPSI)inventionjournals
International Journal of Pharmaceutical Science Invention (IJPSI) is an international journal intended for professionals and researchers in all fields of Pahrmaceutical Science. IJPSI publishes research articles and reviews within the whole field Pharmacy and Pharmaceutical Science, new teaching methods, assessment, validation and the impact of new technologies and it will continue to provide information on the latest trends and developments in this ever-expanding subject. The publications of papers are selected through double peer reviewed to ensure originality, relevance, and readability. The articles published in our journal can be accessed online.
Dr. Raj K. Das is seeking a research position utilizing his skills in chemical industry. He holds a Ph.D. in Organometallic Chemistry from Indian Institute of Technology Kanpur and has extensive experience in synthesis, characterization, and catalytic transformations of inorganic and organometallic compounds. His research has focused on bimetallic cooperative catalysts for C-H bond activation and biomass gasification. He has published 5 papers and presented research at several conferences.
The document is a curriculum vitae for Travis Owen Gurney that details his education, work experience, publications, presentations, and activities. He received a B.A. in Biology from The Ohio State University and has worked in various research roles at the Dorothy M. Davis Heart and Lung Research Institute since 2008. He has over 10 peer-reviewed publications and numerous conference presentations and is currently a Laboratory Manager/Research Associate.
Мутации бактерий на арене с антибиотикамиAnatol Alizar
This document describes experiments using a new device called a MEGA-plate to study the evolution of bacteria in a spatially structured environment. The MEGA-plate consists of a large tray containing agar with gradients of increasing antibiotic concentration. Bacteria are inoculated at one end and spread across the plate through chemotaxis and nutrient depletion. Mutants able to survive in higher antibiotic concentrations can migrate further. Over time, the bacteria evolve increased resistance through sequential adaptive steps of competing lineages. Analysis of isolates from the propagating front found that highly resistant mutants are not always the leaders - they may be trapped behind more sensitive lineages. The MEGA-plate allows direct observation of evolutionary dynamics in a large population under spatial selection pressures.
S. abachi outline of proposed research citationsUniversité Laval
This document discusses food waste globally and focuses on bioactive peptides from fishery by-products like mackerel. It notes that 32% of global food production is lost or wasted in 2009. The proposed research will fractionate and characterize bioactive peptide fractions from mackerel by-products that may modulate glucose uptake and have anti-inflammatory effects in macrophages in vitro. Fractions between 0.2-10 kDa will be isolated using membrane technology and electrodialysis with ultrafiltration membranes to successively fractionate the peptides.
Engineering Hyperthermophilic Archaeon Pyrococcus furiosus to Overproduce Its Cytoplasmic [NiFe]-Hydrogenase
The key points are:
1. Researchers engineered the hyperthermophilic archaeon Pyrococcus furiosus to overproduce its cytoplasmic hydrogenase enzyme through homologous overexpression.
2. They placed the four-gene hydrogenase operon under control of a strong promoter and added an affinity tag to one subunit.
3. The engineered strain overproduced the hydrogenase by almost an order of magnitude compared to the wild-type strain, as measured by enzyme activity and protein yield.
4. This new system will allow further engineering of the hydrogen
This document discusses how content marketing efforts are maturing from experimental to operational. It recommends defining measurable goals and closing the performance loop, using personas and journey maps to understand audiences, bringing content to life with interactive elements, and applying paid media techniques to promote content. The highest performing content marketers treat audience needs as the top priority and use data to optimize their efforts.
Crystal is a compassionate, calm, open-minded and lovable person from Phoenix, Arizona who loves her family, learning, and cooking. She has four siblings and would like to see happiness among loved ones, visit Paris, and find success. Crystal teaches her family while also learning from her parents. She needs love, companionship, and support and gives love, advice, and respect in return. Her fears include bugs, failure, and deception.
Metal-organic frameworks (MOFs) are highly porous materials consisting of metal atoms or clusters linked by organic molecules. MOFs have potential applications in hydrogen storage, methane storage, and carbon dioxide capture due to their ability to store gases at high densities through surface bonding. Australian researchers Matthew Hill and David Turner collaborate on using MOFs for gas storage and have achieved several world records. Their research utilizes synchrotron facilities to characterize MOF crystal structures, map their formation, and modify them for improved properties and applications.
This document discusses stable isotope deltas, which are tiny yet robust signatures that can be measured in nature. It explains that two fundamental processes, isotopic fractionation and isotope mixing, are responsible for most stable isotope variations seen in terrestrial systems. Isotopic fractionation occurs through equilibrium or kinetic processes that fractionate isotopes due to small differences in their physical or chemical properties. Isotope mixing models can provide information about processes like 14C abundances in the atmosphere and past ocean isotopic compositions. The document also proposes a new unit called the "urey" to describe isotope deltas in a way that overcomes limitations of traditional units.
Lead Acetate Induced histopathological Changes in Kidney Tissue of RatIOSRJAVS
One of the pollutants that can effect on the life of living animates is lead in different ways; it has toxic problem for human and animals and is gathered in the tissues and guide to harmful effects in their. The current study was carry out to investigate the histological effects caused by lead in the rat. The study was performed on 30 rat, they were divided into 3 groups. The first group was control group that received distilled water. The second groups were orally administered lead acetate 9 mg/l of body weight for 10 days. The third groups were orally administered solution of lead. The rats were anesthetized, the kidney were removed for histological studies. Histological changes which observed in the kidney were fatty degeneration, destruction tubules, and congestion within connective tissue, hemorrhage and infiltration of inflammatory cells. In this study, harmful toxic effects observed in kidney of rats.
This document summarizes metal-organic frameworks (MOFs), including their properties and applications. MOFs are highly porous materials formed by combining metal ions or metal clusters with organic ligands. They have extremely large surface areas, often exceeding 7,000 m2/g. Due to their tunable porous structures, MOFs show promise for applications such as gas storage, carbon capture, catalysis, and luminescence. While MOF research has advanced significantly in recent decades, further developing their potential applications and improving stability remains an active area of research.
This document describes an approach to rapidly generate molecular diversity using photoassisted diversity-oriented synthesis. Linear photoprecursors containing amino ketone and unsaturated pendant groups are assembled using high-yielding coupling reactions. These precursors undergo efficient intramolecular photocyclizations upon irradiation to form polyheterocyclic scaffolds with increased saturation, reduced rotatable bonds, and new structural frameworks. The primary photoproducts contain reactive functional groups that allow for further post-photochemical modifications, growing molecular complexity and accessing elaborated three-dimensional structures. This modular approach aims to generate drug-like molecules with properties known to correlate with clinical success, overcoming limitations of traditional combinatorial chemistry methods.
This thesis studied the metabolic pathways altered in Pseudomonas fluorescens Pf-5 due to hexavalent chromium stress using NMR-based metabolomics. P. fluorescens was exposed to 50 ppm of chromium for 6 and 24 hours. Metabolite extracts were analyzed using NMR and principal component analysis showed distinct metabolic profiles between control and stressed cells. Further analysis identified significant metabolites and probable pathways impacted by chromium stress.
This document summarizes key concepts from chapters 6-8 of a microbiology textbook including:
- The essential nutrients required by microbes including carbon, hydrogen, oxygen, nitrogen, phosphorus, and sulfur.
- The chemical composition of E. coli and what microbes use as energy and carbon sources, categorizing them as autotrophs and heterotrophs.
- The processes of osmosis, diffusion, and endocytosis that transport substances into and out of cells.
- Temperature categories for microbes like psychrophiles, psychrotrophs and how gases like oxygen and carbon dioxide influence microbial growth.
Dr. Shaukat Ali has extensive experience in organic synthesis and heterocyclic chemistry. He received his PhD from Lanzhou University in China, where he conducted research on electrophile-induced reactions to synthesize functionalized quinolines and indoloquinolines. Since 2012, he has worked as an Assistant Professor in Pakistan, where he has published over 20 papers with an h-index of 11. He is proficient in organic reactions and characterization techniques.
The document describes a new flexible synthesis of pyrazoles that allows for varying substituents at the C3 and C5 positions of the pyrazole ring. The synthesis involves coupling protected alkynols with acid chlorides to form alkynyl ketones, which are reacted with hydrazine to install the pyrazole nucleus. Alcohol deprotection and conversion to chlorides provides access to 5-substituted 3-(chloromethyl)- or 3-(2-chloroethyl)pyrazoles. These chlorides can then undergo nucleophilic substitution to generate other polyfunctional pyrazoles. The significance is that substituents at C5 control the steric environment around the pyrazole N-H
Study on Physico-Chemical Parameters of Harsi Reservoir Dabra, Gwalior Distri...ijsrd.com
this study was aimed to estimate physico-chemical characteristic of Harsi reservoir. Harsi reservoir located in Dabra, Gwalior district, Madhya Pradesh is constructed on parwati River. Monthly study in Physico-chemical parameters such as water temperature, depth, transparency, electrical conductivity, turbidity, total dissolved solids, pH, dissolved oxygen, free carbon dioxide, total alkalinity, total hardness, chlorides, sulphates, nitrate, nitrite, phosphate, silicates, ammonia, BOD, COD, calcium, magnesium, sodium, potassium were analyzed from January 2011 to December 2011. The results indicated that Physico-chemical parameters of the water were used for drinking, domestic use, irrigation and pisciculture.
The document summarizes a student research project investigating the design of short peptide amphiphiles that can bind transition metal ions. The student synthesized a peptide called C16-AHLHL3K3 and used various characterization techniques. Results showed the peptide formed beta-pleated sheet fibers under certain conditions and was able to bind transition metals like cobalt. This demonstrates the potential for short peptides to have transition metal binding sites and possibly enzymatic capabilities.
This document summarizes the synthesis of tris(4-((E)-2-nitro-3-(phenylamino)prop-1-enyl)phenyl)amine from a Baylis–Hillman adduct. Specifically, it details the reaction of tris(4-((E)-3-bromo-2-nitroprop-1-enyl)phenyl)amine with aniline to yield the target compound in 95% yield. The synthesis demonstrates the utility of bromo derivatives of Baylis–Hillman adducts as intermediates for generating novel trisubstituted triallylamines. Characterization of the product by NMR spectroscopy is also discussed.
Evidence of heavy metals distribution in placenta in association with residua...Alexander Decker
This study analyzed samples of placenta, kidney, and liver from cattle at an abattoir in Nigeria to investigate the distribution of heavy metals. The study found chromium present in all samples, while cadmium and lead were not detected. Chromium levels were highest in the kidney, followed by the liver and placenta. There was a moderate correlation between chromium levels in the dam's liver and placenta, but a weak correlation with the kidney. The results indicate that heavy metals like chromium can cross the placental barrier from the mother and accumulate in fetal tissues.
International Journal of Pharmaceutical Science Invention (IJPSI)inventionjournals
International Journal of Pharmaceutical Science Invention (IJPSI) is an international journal intended for professionals and researchers in all fields of Pahrmaceutical Science. IJPSI publishes research articles and reviews within the whole field Pharmacy and Pharmaceutical Science, new teaching methods, assessment, validation and the impact of new technologies and it will continue to provide information on the latest trends and developments in this ever-expanding subject. The publications of papers are selected through double peer reviewed to ensure originality, relevance, and readability. The articles published in our journal can be accessed online.
Dr. Raj K. Das is seeking a research position utilizing his skills in chemical industry. He holds a Ph.D. in Organometallic Chemistry from Indian Institute of Technology Kanpur and has extensive experience in synthesis, characterization, and catalytic transformations of inorganic and organometallic compounds. His research has focused on bimetallic cooperative catalysts for C-H bond activation and biomass gasification. He has published 5 papers and presented research at several conferences.
The document is a curriculum vitae for Travis Owen Gurney that details his education, work experience, publications, presentations, and activities. He received a B.A. in Biology from The Ohio State University and has worked in various research roles at the Dorothy M. Davis Heart and Lung Research Institute since 2008. He has over 10 peer-reviewed publications and numerous conference presentations and is currently a Laboratory Manager/Research Associate.
Мутации бактерий на арене с антибиотикамиAnatol Alizar
This document describes experiments using a new device called a MEGA-plate to study the evolution of bacteria in a spatially structured environment. The MEGA-plate consists of a large tray containing agar with gradients of increasing antibiotic concentration. Bacteria are inoculated at one end and spread across the plate through chemotaxis and nutrient depletion. Mutants able to survive in higher antibiotic concentrations can migrate further. Over time, the bacteria evolve increased resistance through sequential adaptive steps of competing lineages. Analysis of isolates from the propagating front found that highly resistant mutants are not always the leaders - they may be trapped behind more sensitive lineages. The MEGA-plate allows direct observation of evolutionary dynamics in a large population under spatial selection pressures.
S. abachi outline of proposed research citationsUniversité Laval
This document discusses food waste globally and focuses on bioactive peptides from fishery by-products like mackerel. It notes that 32% of global food production is lost or wasted in 2009. The proposed research will fractionate and characterize bioactive peptide fractions from mackerel by-products that may modulate glucose uptake and have anti-inflammatory effects in macrophages in vitro. Fractions between 0.2-10 kDa will be isolated using membrane technology and electrodialysis with ultrafiltration membranes to successively fractionate the peptides.
Engineering Hyperthermophilic Archaeon Pyrococcus furiosus to Overproduce Its Cytoplasmic [NiFe]-Hydrogenase
The key points are:
1. Researchers engineered the hyperthermophilic archaeon Pyrococcus furiosus to overproduce its cytoplasmic hydrogenase enzyme through homologous overexpression.
2. They placed the four-gene hydrogenase operon under control of a strong promoter and added an affinity tag to one subunit.
3. The engineered strain overproduced the hydrogenase by almost an order of magnitude compared to the wild-type strain, as measured by enzyme activity and protein yield.
4. This new system will allow further engineering of the hydrogen
This document discusses how content marketing efforts are maturing from experimental to operational. It recommends defining measurable goals and closing the performance loop, using personas and journey maps to understand audiences, bringing content to life with interactive elements, and applying paid media techniques to promote content. The highest performing content marketers treat audience needs as the top priority and use data to optimize their efforts.
Crystal is a compassionate, calm, open-minded and lovable person from Phoenix, Arizona who loves her family, learning, and cooking. She has four siblings and would like to see happiness among loved ones, visit Paris, and find success. Crystal teaches her family while also learning from her parents. She needs love, companionship, and support and gives love, advice, and respect in return. Her fears include bugs, failure, and deception.
This document describes research on improving the purification of a cytoplasmic hydrogenase enzyme (SHI) from the hyperthermophilic archaeon Pyrococcus furiosus. Key points:
1) A polyhistidine tag was added to SHI allowing single-step purification via affinity chromatography, yielding a 50-fold increase in enzyme compared to the native multi-step process.
2) In addition to the full tetrameric SHI enzyme, a trimeric intermediate form lacking the catalytic subunit was also purified. This intermediate retained NADPH oxidation activity, confirming the proposed SHI maturation pathway.
3) Overexpression of the tagged SHI in P. furiosus and its high-yield
1) The study investigated the roles of mannosylglycerate (MG) and di-myo-inositol phosphate (DIP) in stress adaptation in the hyperthermophilic archaeon Pyrococcus furiosus.
2) Genes encoding enzymes in the biosynthetic pathways of MG and DIP were deleted, generating mutants deficient in each solute.
3) Growth and solute accumulation of the mutants and wild type were examined under optimal, heat stress, and osmotic stress conditions.
4) DIP was found to be a suitable replacement for MG during heat stress, but MG and aspartate were less efficient than MG alone during osmotic stress,
Gregory Wayne Clifford provides his personal details and educational and professional qualifications, including certificates in project management, health and safety, drafting, security operations, and commercial fundamentals. He lists over 30 years of experience in electrical distribution design and construction for companies like Ergon Energy, as well as project management experience with the Gladstone Area Water Board. His skills include designing, estimating, and managing various infrastructure projects from simple distribution substations to complex system augmentations.
Five novel psychrophilic bacterial strains were isolated from soil samples collected above the treeline in alpine environments in Mexico and Colorado. Based on phylogenetic and chemotaxonomic analysis, the five strains represent four novel species in the genus Deinococcus. Strain PO-04-20-132 represents Deinococcus radiomollis sp. nov., strain PO-04-19-125 represents D. claudionis sp. nov., strain ME-04-01-32 represents D. altitudinis sp. nov., and strain ME-04-04-52 represents D. alpinitundrae sp. nov. The new species have optimal growth at 10°C and are more sensitive
HPE AppPulse provides tools to help app developers test and monitor their apps across different devices and environments to isolate issues and improve quality. It offers solutions for monitoring battery usage, understanding user transactions, debugging code to the line, and more through products like AppPulse Mobile, Active, and Trace. Developers are under pressure to find issues fast while continuously improving apps, and HPE AppPulse claims to give them the best tools to do so across all device types and permutations of app usage.
Frank Agodi is seeking a position utilizing his 15+ years of experience in technical roles including software testing, quality assurance, and network engineering. He has extensive experience testing networking and telecommunications products at Cisco and other companies. His background includes testing methodologies, troubleshooting, multi-tasking on projects, and working with customers.
This document summarizes biological processes for converting carbon dioxide and hydrogen into liquid fuels and chemicals. It discusses several microorganisms and pathways being developed for this purpose, known as "electrofuels". These include Ralstonia eutropha and Clostridium ljungdahlii, which can fix carbon dioxide via the Calvin-Benson-Bassham cycle or Wood-Ljungdahl pathway respectively. The document also reviews computational analyses of carbon fixation pathways and their relative energetic costs for producing biomass and fuels.
1. The structure of GUN4 from Chlamydomonas reinhardtii was determined to 3.5 Angstrom resolution. GUN4 is involved in chlorophyll biosynthesis and plastid-to-nucleus signaling.
2. The structure consists of six molecules in the asymmetric unit and is predominantly alpha-helical. The structure is similar to previously solved cyanobacterial GUN4 structures, with the main differences being in several variable loops.
3. The conformational dynamics of these loops seen across GUN4 structures provides insight into how GUN4 binds protoporphyrin and magnesium protoporphyrin during chlorophyll biosynthesis.
molecular dynamics simulation of HYD1.compressedCEPHAS MAWERE
This document summarizes a molecular dynamics simulation study of the HYD1 protein from Gibberella moniliformis. The study had four stages: 1) energy minimization to remove clashes, 2) equilibration involving NVT and NPT ensembles, 3) a 5 nanosecond production molecular dynamics simulation, and 4) analysis of the simulation results. Key results included the HYD1 protein remaining stable with a RMSD of around 0.2 nm, greater flexibility at the C-terminal tail, and validation that the simulation reached equilibrium and the correct density. The study provides insight into HYD1's dynamic behavior and stability to better understand its self-assembly mechanism.
Copper catalyzed synthesis of N-Heterocycles containing one M-atomssusercbfc01
This document summarizes copper-catalyzed methods for synthesizing various N-heterocycles containing one nitrogen atom. It discusses the synthesis of pyrrole, pyrrolidine, indole, and indolone derivatives using copper catalysts under mild conditions. Various studies are reviewed that achieve these syntheses with good yields and functional group tolerance. Copper catalysts offer advantages over other metals like palladium in providing cost-effective routes for producing valuable pharmaceutical compounds. Future work may focus on developing more sustainable and efficient copper-catalyzed methods.
The document discusses bio-inspired catalysts for hydrogen production. It begins by noting the importance of hydrogen as an energy carrier and limitations of existing platinum-based catalysts. It then discusses how hydrogenase enzymes provide an efficient model but have limitations as well. Recent research has focused on developing bio-inspired catalysts that incorporate features of the hydrogenase active site and outer coordination sphere to improve catalytic efficiency. Some promising systems discussed include macrocyclic cobalt complexes and nickel bis(diphosphine) complexes containing amino acid groups to mimic the outer coordination sphere, which have shown activity under broader conditions than hydrogenases. Evaluation of catalytic performance focuses on turnover frequency and overpotential.
This document discusses potential medical applications of fullerenes, specifically:
1) Fullerenes have shown antiviral activity against HIV by inhibiting HIV protease and suppressing HIV replication. Derivatives with certain structural configurations are more effective antivirals.
2) Fullerenes act as antioxidants and radical scavengers, properties which can be exploited for medical uses. They also produce reactive oxygen species when exposed to light, allowing use as photosensitizers.
3) Fullerenes can be used as carriers for drug and gene delivery due to their ability to be functionalized and solubilized in physiological media through various chemical modifications. They also show potential as diagnostic tools.
The UsefulChem Project is an open source science initiative that uses blogs and wikis to make the scientific process transparent. It applies this approach to malaria research by synthesizing diketopiperazine derivatives proposed to inhibit an enzyme required by the malaria parasite. Visitors can comment on experimental details, molecules of interest, and synthetic strategies as they are discussed on the blogs. The goal is to facilitate collaboration and problem solving around the project.
Meulepas, 2010, Effect Of Methanogenic Substrates On Anaerobic Oxidation Of M...roelmeulepas
This document describes a study that tested six methanogenic substrates (acetate, formate, methanol, carbon monoxide, hydrogen, and methanethiol) as potential interspecies electron carriers (IECs) in anaerobic oxidation of methane (AOM) coupled to sulfate reduction. The substrates were added to an enrichment culture containing archaea and bacteria involved in AOM and sulfate reduction. The presence of acetate, formate, hydrogen, and carbon monoxide enhanced sulfate reduction but did not inhibit AOM or trigger methanogenesis. Methanol did not enhance sulfate reduction or inhibit AOM. Methanethiol completely inhibited both AOM and sulfate reduction. Based on these results, the tested substrates could not solely support the electron
This document describes an efficient synthetic route to dihydropipercide and pipercide. Key steps include the preparation of methyl 6-oxohexanoate from cyclohexanone via ozonolysis and methanolysis. A Wittig olefination reaction is used to couple methyl 6-oxohexanoate with a phosphonium salt derived from piperonyl bromide. Phenythio radical-induced olefin isomerization is then used to obtain the desired (E)-olefin geometry. Further elaboration allows construction of the targets via a Horner-Emmons reaction and amidation. The described methods provide practical gram-scale syntheses of these biologically active dieneamide natural products.
The document discusses the chemical synthesis of oligonucleotides, which are short fragments of nucleic acids. It describes the early methods of oligonucleotide synthesis using phosphodiesters and phosphotriesters. The current dominant method is the phosphoramidite method, which uses nucleoside phosphoramidites as building blocks. The phosphoramidite method allows for rapid, automated solid-phase synthesis of oligonucleotides up to 200 nucleotides in length. Protecting groups are used to prevent side reactions and are removed upon completion of chain assembly.
Hrustić et al final revised in Biogeosciences 2017Enis Hrustic
This document describes a bioassay used to explore nitrogen and phosphorus limitation in coastal waters. Samples were collected from the Baltic Sea and North Sea and tested in the bioassay. The bioassay involves measuring alkaline phosphatase activity over a matrix of nitrogen and phosphorus additions, allowing determination of primary and secondary nutrient limitation. Tests on Baltic Sea samples indicated nitrogen limitation, while North Sea samples initially showed phosphorus limitation but shifted towards nitrogen limitation with longer incubation times. The nitrogen to phosphorus consumption ratios varied between locations. The bioassay provided information on nutrient limitation but did not detect significant effects of ocean acidification in samples from mesocosm experiments.
Preparation of pyrimido[4,5 b][1,6]naphthyridin-4(1 h)-one derivativeselshimaa eid
This document describes the preparation of pyrimido[4,5-b][1,6]naphthyridin-4(1H)-one derivatives using a zeolite-nanogold catalyst. An efficient one-pot synthesis is developed involving the cyclocondensation of 6-amino-2-thioxo-2,3-dihydropyrimidin-4(1H)-one, aromatic aldehydes, and 1-benzylpiperidin-4-one in ethanol at 80°C. The nanogold catalyst is characterized and found to contain 4-6 nm gold nanoparticles dispersed on zeolite. Several derivatives are synthesized in good yields and characterized. Molecular dock
New Green Synthesis Approaches of Pharmacologically Active Heterocyclic Compo...ijtsrd
Green chemistry is a rapidly developing field providing a proactive avenue for the sustainable development of future science and technology. Green chemistry can be applied to the design of highly efficient, environmentally benign synthetic protocols to deliver life saving medicines, and to accelerate lead optimization processes in drug discovery, while minimizing environmental impact. It also offers enhanced chemical process economics, concomitant with a reduced environmental burden. There are relatively environmentally benign protocols for the synthesis of pharmaceutically active heterocycles that highlight the advantages of using green chemistry, for example, by proceeding under microwave irradiation or in aqueous reaction media. Chandra Prakash Gharu "New Green Synthesis Approaches of Pharmacologically Active Heterocyclic Compounds" Published in International Journal of Trend in Scientific Research and Development (ijtsrd), ISSN: 2456-6470, Volume-6 | Issue-5 , August 2022, URL: https://www.ijtsrd.com/papers/ijtsrd51793.pdf Paper URL: https://www.ijtsrd.com/chemistry/other/51793/new-green-synthesis-approaches-of-pharmacologically-active-heterocyclic-compounds/chandra-prakash-gharu
This document describes the design, synthesis, and evaluation of a series of 1,3-disubstituted pyrrolo[2,3-b]quinoxalines as potential inhibitors of phosphodiesterase 4 (PDE4) and cancer cell growth. A ligand- and phase transfer catalyst-free intramolecular Heck reaction was used to synthesize the target compounds. Some compounds showed significant inhibition of PDE4B and growth inhibition of oral cancer cells in vitro. They also showed acceptable safety profiles in zebrafish embryos, but no apoptosis was observed. The goal was to develop PDE4 inhibitors that do not inhibit luciferase, which could produce false positives in assays.
Chemical synthesis of DNA involves building oligonucleotides from nucleoside phosphoramidite building blocks through a solid phase synthesis process known as the phosphoramidite method. This method involves sequentially coupling the 3’ protected and activated phosphoramidite forms of the nucleosides in the 5’ to 3’ direction to synthesize short DNA strands up to around 200 bases in length. The phosphoramidite method allows for rapid, inexpensive custom synthesis of oligonucleotides which find many uses as probes, primers, and tools for targeted mutagenesis and gene synthesis.
Quantum Mechanical Calculations on Novel Actinide Chelatin.docxamrit47
Quantum Mechanical Calculations on Novel Actinide Chelating Agents
Aisha Mehmood, Maria Benavides, PhD
Department of Natural Sciences
www.uhd.edu/academic/colleges/sciences/ns/
Abstract: Plutonium is produced in significant amounts as the isotope 239Pu during nuclear reactor operations. The use of actinides such as Pu in energy and weapons production has resulted in major environmental and health concerns. In the event of actinide release it is critical to have the means to treat large number of people in a very short time. Chelation therapy is the approach currently used to treat actinide poisoning because it reduces the deposition of actinides in the internal organs. In order to effectively respond and treat actinide poisoning on a massive scale it is crucial to have access to effective, nontoxic chelating agents that can be orally administered, that are easily produced and safely stored at any location. Specific sequestering agents have been designed and synthesized to bind actinides. Our study focuses on determining the structures and molecular properties of four hydroxypyridinone (HOPO)-based sequestering agents 2,2-Dime-3LI(1,2-HOPO), 3LI-(1,2-HOPO), 4LI(1,2-HOPO) and 5LI(1,2-HOPO) which have been shown to strongly and specifically bind plutonium at physiological pH. Our calculations were carried out using density functional theory (DFT) with the B3LYP functional applied in conjunction with three increasingly larger basis sets (3-21G, 6-31G, and 6-311G) to obtain the equilibrium geometries, vibrational frequencies, and IR spectra for all four ligands. The highest occupied molecular orbital (HOMO) – lowest occupied molecular orbital (LUMO) energy gap values for all four compounds are greater than 4 eV suggesting that the ligands are chemically stable. Two hydrogen bonds are observed in each compound which we suspect contribute to their chemical stability. The four compounds exhibit dipole moments ranging between 3 to 4 Debye which indicates they possess polar character. Our computed vibrational frequencies were found in excellent agreement with the experimental frequencies, suggesting our proposed models are good representations of the actual molecular structures.
Introduction: Over the past years concern for harm caused by radiation in human body has increased. Recent disaster such as Fukushima Daiichi nuclear reactor accident in Japan has raised red flags for radiation safety. Such events are not common; however once the damage has happened, it is large and capable of destroying many lives. Nuclear fissions release significant amount of several different actinides that can enter human body through radiation or radioactive substances. All actinides are radioactive and bind with human internal tissue. Chelation therapy has shown positive results for reducing internal human actinide contamination. Plutonium (Pu) is an alpha emitter that is known to have the greatest retention in the human body among actinides. Our studies focuses ...
A STUDY TO EVALUATE THE IN VITRO ANTIMICROBIAL ACTIVITY AND ANTIANDROGENIC E...Dr. Pradeep mitharwal
The present paper deals with synthesis and characterization
of some new chromium (III) Schiff base complexes using microwave irradiation
technique as well as conventional heating. The S∩N donor benzothiazolines, 1-
(2-furanyl) ethanone benzothiazoline (Bzt1N
∩
SH), 1-(2-thienyl) ethanone
benzothiazoline (Bzt2N
∩
SH) and 1-(2-pyridyl) ethanone benzothiazoline
(Bzt3N
∩
SH) were prepared by the condensation of ortho-aminothiophenol with
respective ketones in ethanol.
SiO2@FeSO4 nano composite: A recoverable nano-catalyst for eco-friendly synth...Iranian Chemical Society
Various aldoximes and ketoximes synthesis of corresponding aldehydes and ketones in the presence of SiO2@FeSO4 nano composite as recoverable nano catalyst and NH2OH·HCl. The SiO2@FeSO4 nano composite system was carried out between 10 to 15 min in oil bath (70-80 °C) under solvent-free condition in excellent yields in addition this protocol can be used for industrial scales. This method offers some advantages in term of clean reaction conditions, easy work-up procedure, short reaction time, applied to convert α-diketones to α-diketoximes (as longer than other carbonyl compounds), α,β-unsaturated aldehydes and ketones to corresponding oximes and suppression of any side product. So we think that NH2OH•HCl/SiO2@FeSO4 nano composite system could be considered a new and useful addition to the present methodologies in this area. Structure of products and nano composite elucidation was carried out by 1H NMR, 13C NMR, FT-IR, scanning electron microscopy (SEM).
This document describes a one-pot, three-component reaction to synthesize densely substituted benzofurans from methyl ketones, phenols, and nucleophiles. Specifically, it details:
1) The reaction of phenylglyoxal monohydrate, phenols like sesamol, and indoles produces benzofurans in good to excellent yields with acid catalysis.
2) Replacing phenylglyoxal with methyl ketones and using I2/DMSO allows an in situ generation of arylglyoxal from the methyl ketone for the reaction.
3) Various substituted benzofurans can be synthesized from different methyl ketones, phenols
This document describes a dissertation that examines palladium-catalyzed Heck and Heck/Suzuki domino reactions, as well as applications of microwave heating technology in continuous flow organic synthesis. Specifically, the dissertation includes studies on: 1) developing a ligand-modulated approach to selectively attach aryl groups to a chelating vinyl ether, 2) evaluating a novel microwave applicator for heating solvents and reaction mixtures in continuous flow, 3) developing regioselective oxidative decarboxylative Heck reactions of electron-rich olefins, and 4) a theoretical mechanistic investigation of one such decarboxylative Heck reaction.
Similar to CMBHEngineeringandPurificationPEDS (20)
2. (Vignais and Billoud, 2007). The majority of crystal structures
for [NiFe]-hydrogenases are available for Group 1 hydrogenases
(Volbeda et al., 1995, 2013). Recently, the first crystal structure of a
Group 3 hydrogenase was determined from Methanothermobacter
marburgensis (PDB code: 4OMF) (Vitt et al., 2014).
Group 4 hydrogenases are the least studied of the [NiFe]-
hydrogenases. These hydrogenases are defined as the H2-evolving
energy-conserving membrane-associated hydrogenases (Vignais and
Billoud, 2007). Very little sequence similarity exists between Group
4 hydrogenases and other [NiFe] hydrogenases except for the con-
served residues that bind the [NiFe] catalytic site and its proximal
[4Fe-4S] cluster, indicating a distinct evolutionary history (Hedderich,
2004, 2005; Schut et al., 2013). Group 4 hydrogenases play an im-
portant role in conserving energy by establishing ion gradients across
membranes that can be used to generate ATP. These enzymes contain
at least six subunits and are much more complex than the character-
ized dimeric hydrogenases. The simplest members include the
6-subunit ‘energy-conserving’ hydrogenase (Ech) from the archaeon
Methanosarcina barkeri, which functions in methanogenesis, and
the 7-subunit hydrogenase 3 from Escherichia coli, which oxidizes for-
mate and evolves H2 (Böhm et al., 1990; Sauter et al., 1992; Meuer
et al., 1999; Kurkin et al., 2002). Six subunits conserved within the
Group 4 hydrogenases are homologous to the catalytic core of the ubi-
quitous aerobic respiratory complex NADH quinone oxidoreductase
or Complex I (NuoBCDIHL) (Hedderich, 2004, 2005; Schut et al.,
2013; Marreiros et al., 2013). This conserved homology suggests a
close evolutionary history between Group 4 enzymes and Complex I.
Pyrococcus furiosus is a hyperthermophilic archaeon that grows
optimally at 100°C and contains a complex hydrogenase system
(Fiala and Stetter, 1986; Schut et al., 2013). It grows by fermenting
sugars to acetate, CO2 and H2 and its membrane-bound hydrogenase
(MBH) catalyzes H2 production using reduced ferredoxin (Fd) gener-
ated from sugar oxidation as the electron donor (Schut et al., 2013).
Previous studies of P. furiosus MBH showed that it is encoded by a
14-gene operon (mbhA-N: PF1423-PF1436) (Sapra et al., 2000). Six
of the last seven genes in the operon are homologous to those encoding
the ‘core’ subunits of Complex I (mbhH,J-N) while another eight sub-
units (mbhA-H) are homologous to subunits of the Mrp monovalent
cation/proton antiporter of some mesophilic bacteria (Fig. 1) (Swartz
et al., 2005). The exceptions are mbhI, which does not have homology
to subunits of either Complex I or Mrp, and mbhH, which has hom-
ology to both (Fig. 1). Mrp catalyzes the efflux of monovalent cations,
such as Na+
, K+
and Li+
outward in a coupled reaction that transports
protons inwards. Of the 14 subunits of MBH, only mbhJKLN are pre-
dicted to not encode transmembrane helices (Sapra et al., 2000; Silva
et al., 2000). MbhJ and MbhN are proposed to contain one and two
[4Fe–4S] clusters, respectively, where MbhJ is the equivalent of the
small subunit of the Group 1 dimeric [NiFe]-hydrogenases (Fig. 1).
MbhKL are equivalent to the large subunit and contain the [NiFe]
active site, with the four Cys residues provided by MbhL.
The evolutionary linkage between respiratory Complex I and
Group 4 [NiFe] hydrogenases has been extensively reviewed (Hedder-
ich, 2004, 2005; Schut et al., 2013). An ‘ancestral group 4 [NiFe] hy-
drogenase’ has been proposed that evolved into the archaeal, bacterial
and eukaryotic (Nuo or Nqo, respectively) Complex I by the shuffling
of two distinct modules—Mrp and Mbh. Moreover, another notion of
a ‘universal adaptor molecule’ has been proposed to understand the
evolution of Nuo or Nqo Complex I and Group 4 hydrogenases
Fig. 1. Comparison of homologous subunits between MBH, Complex I and Mrp antiporter. Nqo numbers are given for each T. thermophilus complex I subunit with
the correlated Nuo letter in parenthesis. Red subunits represent the catalytic subunits and include mbhL and nuoD. Orange subunits represent the Q module and
include mbhJKLN and nuoBCDI. Aqua colored subunits are homologous and include mbhH, nuoLMN, and mrpAD. Blue subunits are homologs to mbhM and
include nuoH. Light green subunits are homologous to mbhG and include mrpC. Green subunits are homologous to mbhABC and include mrpEFG. Gray
subunits are homologous to mbhDEF and include mrpB. Yellow subunits represent the N module of Complex I and include nuoEFG. MbhI is shown in brown
and the dark blue subunits represent nuoAJK. Tan subunits represent two subunits that were observed in the 16-subunit crystal structure of Nqo from
T. thermophiles (Efremov et al., 2010). There is no homology between the three complexes for mbhI, nuoAEFGJK or Nqo15–16. Fdred and Fdox represent
reduced and oxidized ferredoxin, respectively, and QH2 and Q represent reduced and oxidized ubiquionone, respectively.
2 P.M.McTernan et al.
atUniversityofGeorgiaLibraries,SerialsDepartmentonDecember8,2014http://peds.oxfordjournals.org/Downloadedfrom
3. (Batista et al., 2013). The universal adaptor molecule is conceived as a
set of conserved subunits between Group 4 hydrogenases and Com-
plex I and contains four subunits (nuoB, nuoD, nuoH and nuoL).
NuoB and nuoD are analogous to the large and small subunits of
the Group 4 hydrogenases (mbhL, mbhJ) while nuoH (mbhM) is be-
lieved to function like a membrane anchor that links the hydrogenase
module to the membrane, and nuoL (mbhH) is homologous to mrpA
and mrpD from the Mrp monovalent cation/proton antiporter
(Fig. 1). NuoM and nuoN also show homology to mrpA, mrpD and
mbhH (Fig. 1). Close evolutionary relationships have also been ob-
served by analyzing the three-dimensional structure of the hydrophilic
domains of the Nqo Complex I of Thermus thermophilus (PDB 2FUG
and 3M9S) (Sazanov and Hinchliffe, 2006; Efremov et al., 2010).
MBH has significant sequence similarity to the Q-module of Complex
I of T. thermophilus, which includes Nqo4, 5, 6 and 9 (Fig. 1) (Hed-
derich, 2004, 2005). The Q-module has a quinone binding grove in
Nqo4, which is where the [NiFe] site is located in the large subunit
of MBH (MbhL) and the ‘N2’ [FeS] cluster of Nqo6 is analogous to
the proximal [FeS] cluster of small subunit of MBH (MbhJ). Nqo9
shares similarity with MbhN and harbors two [FeS] clusters. Nqo5
is homologous to MbhK and is part of the large subunit.
The best characterized Group 4 hydrogenases are the 6-subunit
Ech from M. barkeri and the 14-subunit MBH from P. furiosus. Bio-
chemical studies of Ech have been described but there is no informa-
tion on its modular structure (Meuer et al., 1999; Kurkin et al., 2002;
Hedderich, 2005; Schut et al., 2013), P. furiosus MBH was recently
affinity tagged and solubilized using detergent to yield an intact and
functional 14-subunit complex and a structural model was obtained
based on small angle X-ray scattering (McTernan et al., 2014). To fur-
ther understand the modular nature of MBH, engineering the 14-gene
operon to generate different forms of the enzyme could have major im-
plications. For example, the generation of subcomplexes of MBH
could give insight into which subunits are essential for catalytic activ-
ity and the generation of a chemical gradient, as well as providing in-
sights into the evolution of the ubiquitous Complex I.
There are limited reports for the successful engineering of ‘minimal’
versions of hydrogenase. This was achieved with the enzyme from a
Ralstonia species but involved dissociation of the native complex rather
than genetic manipulation (Grzeszik et al., 1997). The first successful
example of the engineering of a subcomplex of hydrogenase was accom-
plished using the genetic system established for P. furiosus in order to
make a dimeric version of its heterotetrameric soluble hydrogenase I
(SHI) (Hopkins et al., 2011; Lipscomb et al., 2011). Herein, we have
successfully engineered a strain of P. furiosus that generates a soluble
4-subunit subcomplex of MBH (C-MBH). Previous attempts to purify
the 14-subunit complex led to the purification of subcomplexes of
MBH but these were heterogeneous and difficult to characterize
(Sapra et al., 2000; Silva et al., 2000). In this study, we have taken ad-
vantage of the same genetic system for P. furiosus that has been previ-
ously used to make a dimeric version of SHI and to over-express SHI
(Hopkins et al., 2011; Lipscomb et al., 2011; Chandrayan et al.,
2012). We have engineered a strain of P. furiosus by dividing the native
MBH 14-gene operon into two different transcriptional units and have
incorporated an affinity tag within the operon. This enabled us to purify
a subcomplex of MBH from the cytoplasm (C-MBH) without the use of
detergents. This is the first description of the engineering of a MBH to
generate a soluble, catalytically active enzyme complex.
Materials and methods
Generation of P. furiosus strains expressing affinity
tagged MBH
A competent strain of P. furiosus (COM1) was used to manipulate the
MBH operon (Lipscomb et al., 2011). A one-step marked knock-in
genetic protocol was used in which a polyhistidine (His9) affinity tag
was inserted within the operon at the N-terminus of mbhJ (PF1432)
yielding strain MW0414 (Fig. 2) (McTernan et al., 2014). The knock-
in cassette, which contains the selectable marker and the strong con-
stitutive promoter of the gene encoding the S-layer protein (Pslp) with
an in frame His9 tag, were generated by using overlapping PCR (Hor-
ton et al., 1989). Prime Star HS polymerase premix (Clonetech, USA)
was used to make the knock-in cassette. PyrF was the selectable mark-
er and was placed under the control of the glutamate dehydrogenase
Fig. 2. The genetic strategy used to insert the His9x tag and to overexpress last five genes (mbhJ-N) of the MBH operon relative the first nine genes (mbhA-I). The tag
is inserted at the N-terminus of mbhJ. The abbreviations are: UFR and DFR, upstream and downstream flanking regions (1 kb) of the MBH operon; pyrF, selectable
marker; Pgdh and Pslp, promoters for the gene encoding glutamate dehydrogenase and the S-layer protein of P. furiosus, respectively.
Respiratory hydrogenase cytoplasmic subcomplex 3
atUniversityofGeorgiaLibraries,SerialsDepartmentonDecember8,2014http://peds.oxfordjournals.org/Downloadedfrom
4. promoter (Pgdh). In strain MW0414, the expression of mbhJ and
the subsequent five genes (mbhK-N) was under control of Pslp, while
the first nine genes are under control of the native MBH promoter
(McTernan et al., 2014). Generation of MW0414 required the marker
cassette to be placed in front of mbhJ within the MBH operon (Fig. 2).
The sequence between mbhI and mbhJ is shown in Supplementary
Fig. S1. All transformants were PCR screened for correct insertion
and the PCR product was sequenced (Macrogen, MD).
Protein expression and purification
All purification steps were carried out anaerobically using a Coy an-
aerobic chamber (Coy laboratories; MI, USA). Cells were lysed
using 50 mM EPPS (4-(2-hydroxyethyl)-1-piperazinepropanesulfonic
acid; Sigma-Aldrich, USA), pH 8.0, containing 50 μg/ml deoxyribo-
nuclease I (DNase I; Sigma-Aldrich), and 2 mM dithiothreitol (DTT;
Inalco, Italy) in a 5 : 1 ratio of buffer to cells in an anaerobic chamber
(Coy). After 2 h of incubation at 23°C, cells were passed twice through
a French press at a pressure of 1000 psi. Cytoplasmic extract (S100)
was prepared by centrifugation in a Beckman-Coulter Optima
L-90K ultracentrifuge at 100,000 g for 1 h. Cytoplasmic extract was
loaded on a 5-ml FF His-Trap Ni-NTA column (GE Healthcare,
USA), which was equilibrated with Ni-NTA buffer A (50 mM Tris–
HCl, pH 8.0, containing 400 mM NaCl, 4 mM DTT). The column
was washed with Buffer A and the bound protein was eluted with a
20-column volume gradient from 100% Buffer A to 100% Buffer B
(Buffer A containing 500 mM imidazole). After the Ni-NTA step,
the affinity purified C-MBH was further purified using a Mono-Q col-
umn (Bio-Scale Q2 Column) The column was equilibrated in 50 mM
Tris, pH 8.0 containing 2 mM sodium dithionite (DT). The bound
protein was eluted with a 20 column volume gradient from 100% Buf-
fer A to 100% Buffer B (Buffer A containing 2 M NaCl).
Gel Filtration chromatography
The molecular weight of C-MBH was determined by analyzing the
purified protein on a calibrated Superdex 200 10/300 GL column
equilibrated 50 mM Tris pH 8.0, 400 mM NaCl, 2 mM DT. The col-
umn was calibrated by using these standards: cytochrome c, bovine
serum albumin and thyroglobulin.
Quantitative PCR (Q-PCR) analysis
RNA was isolated using the Absolutely RNA miniprep kit (Agilent
technologies, USA). Turbo DNase (Agilent technologies) was used
to remove DNA contamination. Complementary DNA (cDNA) was
synthesized using a Affinityscript QPCR CDNA synthesis kit (Agilent
technologies). CDNA was analyzed using a Brilliant II SYBR green
QPCR master mix (Agilent technologies) and measured using a
MX3000P instrument (Stratagene, USA). Ct values were normalized
to the internal control pyruvate oxidoreductase (pyruvate oxidore-
ductase, POR, gamma subunit; PF0971).
Other methods
Hydrogenase assays were performed at 80°C and H2 was measured
using an Agilent Technologies 6850 gas chromatograph. H2 evolution
activity was determined using dithionite-reduced methyl viologen
(MV; Sigma-Aldrich) as the electron donor or P. furiosus ferredoxin
reduced by P. furiosus POR (Chandrayan et al., 2012). The POR-
linked assay contained 100 mM EPPS pH 8.4, 10 mM Na-pyruvate,
0.2 mM coenzyme A, 0.4 mM TPP, 2 mM MgCl2 and 2 mM DTT,
POR (30 μg/ml) and ferredoxin (100 μg/ml). The DT-ferredoxin
assay contained 100 mM EPPS pH 8.4, 5 mM sodium dithionite
and ferredoxin (100 μg/ml). Hydrogen uptake assay was performed
by using methyl viologen as an electron acceptor in vials saturated
with hydrogen. Oxygen sensitivity assays were carried out by exposing
the MBH sample (100 μg/ml) in 50 mM Tris, pH 8.0, containing
400 mM NaCl and 4 mM DTT to air while shaking (30 rpm). Sam-
ples were taken at 0, 2, 4, 8, 16 and 32 h to determine residual hydro-
genase activity. Thermal stability at 90°C was carried out in the same
fashion except that the samples were maintained under anaerobic con-
ditions. The H2 evolution assay at different temperatures was done
using 50 μg of each form of MBH (C-MBH, S-MBH and washed
membranes, WMs).
Purified C-MBH was analyzed by electrophoresis using 4–20%
Tris–glycine NUSEP gels (Bio-Rad, USA). Bands were cut from the
sodium dodecyl–polyacrylamide gel electrophoresis (SDS–PAGE),
digested with trypsin and were analyzed by matrix-assisted laser
desorption-ionization time-of-flight mass spectrometry. Nickel and
iron were measured using an octopole-based ICP-MS (7500ce Agilent
Technologies, Tokyo, Japan)), equipped with a MicroMist nebulizer
(Cvetkovic et al., 2010). X-band (∼9.6 GHz) electron paramagnetic
resonance (EPR) spectroscopy was carried out using a Bruker
ESP-300E EPR spectrometer equipped with an ER-4116 dual-mode
cavity and an Oxford Instruments ESR-9 flow cryostat.
Temperature studies
MW0414 was grown in a 20-l fermenter at 90°C and switched to 72°C
as previously described (Keller et al., 2013). C-MBH was purified as
described above.
Results
Construction of P. furiosus to generate C-MBH
We previously deleted the catalytic subunit from MBH (mbhL) and
showed that a functional complex was required for growth (in the ab-
sence of elemental sulfur) (Schut et al., 2012). In order to obtain a
‘minimal’ subcomplex of MBH, we decided to engineer the native
MBH operon by splitting it into two separate transcriptional units.
The first nine genes (mbhA-I) of the operon would be under the con-
trol of the native MBH promoter, while the last five genes (mbhJ-N)
are under control of the stronger Pslp promoter that controls constitu-
tive expression of the gene encoding the S-layer protein (Fig. 2). At the
same time, an affinity-purification tag (His9) was inserted within the
operon at the N-terminus of mbhJ (the hydrogenase ‘small’ subunit).
We decided to tag the small subunit of MBH as this tag position was
effective for the affinity purification of the intact 14-subunit complex
(S-MBH) (McTernan et al., 2014). Hence, as shown in Fig. 3, in the
engineered strain MW0414, the 14-gene MBH operon is expressed
as two transcripts, mbhA-I and mbhJ-N. If the two protein products
(MbhA-I and MbhJ-N) were able to combine and give a functional
14-subunit complex, then P. furiosus would be able to grow (Schut
et al., 2012). However, due to the relative strengths of the native pro-
moter for MBH (Pmbh) and Pslp, we expected that there would be ex-
cess mbhJ-N transcript relative to mbhA-I. Assuming that the
transcripts were similarly stable (see below) and were translated
with the same efficiency, then MbhJ-N, a potentially soluble form of
the enzyme, should be generated in excess of that which combines with
MbhA-I to generate functional MBH. Moreover, it should be possible
to purify soluble MbhJ-N from the cytoplasmic fraction by its affinity
tag (on MbhJ).
4 P.M.McTernan et al.
atUniversityofGeorgiaLibraries,SerialsDepartmentonDecember8,2014http://peds.oxfordjournals.org/Downloadedfrom
5. Q-PCR analysis
There was no difference in the growth of MW0414 or the parent strain
(data not shown), and the specific activity of WMs from COM1 and
from MW0414 was the same (∼1.0 U/mg). This confirmed that the na-
tive MBH complex in MW0414 was intact and functional despite
being synthesized and assembled from two different subcomplexes en-
coded by two different transcriptional units (Fig. 3). We first used
Quantitative PCR (Q-PCR) analysis to determine if the split MBH
operon was indeed differentially expressed. The results showed that
the mbhJ-N transcript was ∼2-fold higher than that of mbhA-I (Sup-
plementary Fig. S2). Since SHI in the cytoplasm of P. furiosus is a very
active enzyme, it was not expected that a soluble form of MBH would
be detected by an increase in total cytoplasmic hydrogenase activity,
and this proved to be the case. Nevertheless, if an intact soluble
form of MBH was produced in the cytoplasm, it should be possible
to purify it using its affinity tag.
Affinity purification and characterization
of affinity-tagged C-MBH
The cytoplasmic extract of MW0414 was applied to a Ni-NTA col-
umn and after washing with buffer a significant amount of hydrogen-
ase activity was eluted with a histidine gradient. Hence, it appeared
that a soluble His-tagged subcomplex of MBH had been produced,
and this was further purified using a Mono-Q anion exchange column.
A total of 21 mg of protein containing 12 units of hydrogenase activity
(using methyl viologen as the electron donor) eluted from the Ni-NTA
column and 12 mg of protein was obtained from the Mono-Q with no
loss of activity.
From the molecular analysis (Fig. 4), the second transcript should
yield a 5-subunit MbhJ-N complex but MbhM is predicted to be
membrane-associated. Analyses by SDS-PAGE revealed that S-MBH
was tetrameric (MbhJKLN) rather than pentameric (MbhJ-N) and
lacked MbhM. Protein bands corresponding to the calculated molecu-
lar weights for all four subunits were evident on the gel. Note that the
catalytic subunit, MbhL (calculated MWt of 47,903 Da), is predicted
to undergo C-terminal proteolysis during the processing of the [NiFe]-
site with the removal of 47 amino acids. This proved to be the case as
the band on the gel was consistent with the calculated mass of the ma-
ture processed subunit (42,899 Da). The presence of MbhJKLN in
C-MBH was confirmed by liquid chromatography–tandem mass spec-
trometry (LC–MS/MS) analysis from trypsin-digested in-solution
samples (Supplementary Table SI). The protein band slightly smaller
than MbhN in Fig. 4 could not be identified by this approach.
Purified C-MBH was analyzed using a calibrated S-200 gel filtra-
tion column and eluted as a single peak corresponding to a mass of
85 ± 5 kDa (Supplementary Fig. S3). The predicted mass of the
MbhJKLN complex calculated from the deduced amino acid se-
quences is 97 kDa, suggesting that all four subunits are present but
that the subcomplex is not globular in shape. To gain a further under-
standing as to which subunits are associated with C-MBH, we ana-
lyzed the purified protein with both inductively coupled plasma
mass spectrometry and EPR. Purified C-MBH contained both iron
and nickel in a ratio of 14 : 1 (Table I). This result supports the
Fig. 3. Transcription of the engineered MBH operon. (A) Schematic showing the transcribed products of the two different transcriptional units of the engineered MBH
operon. The abbreviations are: Pmbh, the native MBH promoter that controls the expression of mbhA-I; pyrF, selectable marker; Pgdh, promoter for the gene encoding
glutamate dehydrogenase; Pslp, promoter for the S-layer protein that controls the expression of mbhJ-N. Based on Quantitative PCR analysis, mbhJ-N, under the
control of Pslp, were expressed at twice the level of mbhA-I, under the control of the native Pmbh.
Respiratory hydrogenase cytoplasmic subcomplex 5
atUniversityofGeorgiaLibraries,SerialsDepartmentonDecember8,2014http://peds.oxfordjournals.org/Downloadedfrom
6. proposed presence of three [4Fe–4S] clusters in the enzyme (Fig. 4),
which together with the [NiFe] active site should give a predicted Fe :
Ni ratio of 13 : 1. The anaerobically purified protein after reduction
with sodium dithionite exhibited a complex rhombic EPR signal at
6 K that could be attributed to multiple [4Fe–4S]1+
clusters (Supple-
mentary Fig. S4). A schematic of purified C-MBH containing four sub-
units (MbhJKLN) is shown in Fig. 4.
MBH uses reduced ferredoxin as an electron donor in vivo and the
redox protein is proposed to interact with MbhN (Fig. 1) (Schut et al.,
2013). C-MBH also evolves H2 using ferredoxin as the electron donor
at 80°C, both when reduced by sodium dithionite or by the native pyru-
vate ferredoxin oxidoreductase (Table I). The ability of the subcomplex
to catalyze the physiological reaction is also consistent with all the three
[4Fe–4S] clusters being intact and functional in C-MBH. The ratio of
H2 evolution to H2 oxidation by C-MBH using methyl viologen as
the electron carrier was 13 : 1, which is lower than that measured for
the membrane-bound form (26 : 1) but this still demonstrates that this
subcomplex version of MBH prefers to evolve hydrogen rather than
oxidize it. C-MBH and the WM control were similarly insensitive to in-
activation by oxygen, with half-lives under air while shaking of ∼13 h.
Temperature studies of C-MBH
Purified C-MBH was assayed at different temperatures in order to gain
a better understanding of its H2 evolution activity. It was observed that
C-MBH had a different temperature profile in comparison to the form
of MBH present in WMs or as the solubilized, purified 14-subunit
complex (S-MBH) (Fig. 5). With reduced methyl viologen as the elec-
tron donor, C-MBH had maximal activity at 60°C and was inactive at
90°C. In comparison the S-MBH and WM forms of MBH were max-
imally active at or >90°C. Accordingly, H2 evolution by C-MBH using
ferredoxin, the physiological electron donor to MBH, was measured
at 60°C (Table I), and was higher than the activity measured at 80°C.
C-MBH therefore appeared to be less thermostable than MBH in the
WM and this proved to be the case during extended incubation at high
temperature. Purified C-MBH had a half-life at 90°C of 8 h, which
compares with 25 h for MBH in the WM (Table I).
We therefore wondered if the yield of purified C-MBH could be im-
proved if the recombinant P. furiosus strain was grown at a tempera-
ture lower than 90°C, where the enzyme might be unstable and subject
to proteolytic digestion. The MW0414 strain was therefore grown in a
20-liter fermenter at 72°C (Keller et al., 2013). Cytoplasmic extracts
were prepared from cells grown at 90°C and at 72°C and C-MBH
Table I. Characterization of purified C-MBH
Property C-MBH WM
DT-Fd H2 evolution activity (U/mg) at 80°C 0.03 0.05
DT-Fd H2 evolution activity (U/mg) at 60°C 0.06 0.01
POR-Fd H2 evolution activity (U/mg) at 80°C 0.004 0.02
H2 evolution : oxidation activity (MV as electron
carrier) at 80°C
13 : 1 26 : 1
Half-life (t1/2, h) at 90°C under argon 8 h 25 h
Half-life (t1/2, h) at 25°C under air 13 h 13 h
Metal content (Fe : Ni) 14 : 1 19 : 1
Fig. 4. SDS-PAGE of purified C-MBH. Left: MBH subunits identified by LC-MS/MS are labeled with a black arrow. Right: schematic of C-MBH showing location of the
affinity-tag. MbhL harbors the [NiFe] active site, MbhK is part of the large subunit, MbhJ harbors one [4Fe4S] cluster and MbhN harbors two [4Fe4S] clusters. Fdred
and Fdox represent reduced and oxidized ferredoxin, respectively.
Fig. 5. Temperature profile for C-MBH activity. The H2 evolution activity using
reduced methyl viologen as the electron donor of C-MBH (circular symbols),
WM (square symbols) and solubilized and purified MBH (S-MBH, triangles)
is shown.
6 P.M.McTernan et al.
atUniversityofGeorgiaLibraries,SerialsDepartmentonDecember8,2014http://peds.oxfordjournals.org/Downloadedfrom
7. was purified as described above using two chromatography steps. The
yield of C-MBH from the cells grown at the lower temperature was
more than twice (9.0 mg vs 4.0 mg) that obtained from cells grown
at the higher temperature, indicating that thermal degradation of the
enzyme does occur during growth at 90°C. Primers for Q-PCR were
designed to bind the first gene under control of either Pmbh (PF1423,
mbhA) or Pslp (PF1432, mbhJ) of the MBH operon (Fig. 3). The con-
centration of RNA was 2-fold higher when generated from the Pslp
promoter throughout growth compared with the native MBH pro-
moter and this was independent of the temperature shift from 90 to
72°C (Supplementary Fig. S2).
Discussion
Herein we describe the first successful attempt to engineer a cytoplas-
mic ‘minimal’ catalytically active subcomplex of a membrane-bound
respiratory hydrogenase. We had previously generated a smaller
form of the heterotetrameric cytoplasmic SHI from P. furiosus (Hopkins
et al., 2011). This was achieved by expressing just two of the four genes
that encode the enzyme in a strain in which all four genes had been
deleted. However, it was not possible to express simply part of the
14-gene operon encoding MBH since it impossible to obtain a strain
of P. furiosus lacking a functional MBH. We therefore engineered the
MBH operon by splitting the 14-gene operon into two separate tran-
scriptional units. This approach was risky as it was not known if the
protein products of the two different transcripts would be able to find
each other and assemble into a functional MBH complex (Fig. 3),
which would enable P. furiosus to grow, and if so, would the excess
subunits form a functional complex in the cytoplasm. All of this
proved to be the case, however, as the over-expressed part of the
MBH operon generated a stable cytoplasmic protein with hydrogen-
ase activity, although it contained four (MbhJKLN) rather than the ex-
pected five subunits (MbhM was not present). These results show that,
surprisingly, splitting and disrupting the MBH operon still produced a
functional MBH, and we were able to purify the cytoplasmic subcom-
plex using the engineered affinity tag.
We also expected to see a 1 : 1 ratio of MBH activity in the mem-
brane (from the 14-subunit MBH) and in the cytoplasmic fraction
(from MbhJKLN) based on our Q-PCR data. However, measured
at 80°C using the DT-MV H2 evolution assay, the activity of
C-MBH was only ∼10% of that of S-MBH purified from the same
batch of cells grown at 90°C (McTernan et al., 2014). However, the
activity of C-MBH roughly doubled when assayed at 60°C versus 80°C
(Fig. 5), so the Ni-NTA purified C-MBH represents ∼20% of the
MBH activity present in the membrane in cells grown at 90°C (with
no temperature switch). That this was lower than expected is unlikely
to be due to the five-subunit version of C-MBH (MbhJ-N) remaining
in the membrane (MbhM is predicted to be membrane-associated)
since there was no increase in the hydrogenase activity (measured at
80°C) in the membrane. The lower than expected C-MBH activity
is more likely due to the thermolability of the subcomplex, and this
is supported by the finding that the yield of C-MBH increased
>2-fold when cells were grown at 72°C. The amounts of activity
and protein associated with purified C-MBH doubled when cells
were grown at a lower temperature (90°C then switched to 72°C).
Hence, from cells grown at 72°C we are able to purify the equivalent
of ∼40% of MBH found in the membranes. Either the remainder is
lost due to the low thermal stability of C-MBH or the two transcripts
generated from the MBH operon are not translated with the same
efficiency.
That purified C-MBH was less stable than S-MBH is not unexpect-
ed given that C-MBH lacked 10 partner subunits. What is surprising,
however, is that C-MBH retained the very unusual catalytic preference
of S-MBH (Table I). In in vitro assays using dyes such as methyl violo-
gen as electron carriers, [NiFe]-hydrogenases preferentially oxidize
H2, often by orders of magnitude (Vignais and Billoud, 2007). Pyro-
coccus furiosus MBH is therefore atypical in that it preferentially cat-
alyzes H2 evolution by a ratio of∼26 : 1 in the standard assays
(Table I). It was assumed that this may arise because of the large com-
plex nature of this membrane-bound enzyme where electron transfer is
coupled to both H+
and Na+
pumping, which might account for the
catalytic preference for the physiological reaction of H2 production.
However, the results presented herein show that this is a property of
the cytoplasmic component of MBH that is independent of the ion-
pumping membrane component. Stability assays of C-MBH showed
that it was just as resistant to inactivation by O2 as the native
14-subunit complex, suggesting that the infrastructure of the catalytic
subunit is maintained in the cytoplasmic form when it is dissociated
from the other ten subunits.
Generation of a cytoplasmic catalytic subcomplex of this respiratory
hydrogenase is a significant achievement. In particular, obtaining rela-
tively large amounts of the heterotetrameric soluble complex in the ab-
sence of any detergent greatly facilitates structural analyses of this
representative of the poorly studied Group 4 hydrogenases, with impli-
cations for the evolution of Complex I, and such studies are in progress.
Supplementary data
Supplementary data are available at PEDS online.
Acknowledgements
The authors thank the PAMS Facility at the University of Georgia for the
LC-MS/MS analyses. They also thank the Biofermentation and Expression
Facility at the University of Georgia for growing P. furiosus.
Funding
This work was supported by a grant (DE-FG05-95ER20175) from the Division
of Chemical Sciences, Geosciences and Biosciences, Office of Basic Energy
Sciences of the Department of Energy.
References
Batista,A.P., Marreiros,B.C. and Pereira,M.M. (2013) Biol. Chem., 394,
659–666.
Böhm,R., Sauter,M. and Böck,A. (1990) Mol. Microbiol., 4, 231–243.
Chandrayan,S.K., McTernan,P.M., Hopkins,R.C., Sun,J., Jenney,F.E. and
Adams,M.W.W. (2012) J. Biol. Chem., 287, 3257–3264.
Cvetkovic,A., Menon,A.L., Thorgersen,M.P., et al. (2010) Nature, 466,
779–782.
Efremov,R.G., Baradaran,R. and Sazanov,L.A. (2010) Nature, 465, 441–445.
Fiala,G. and Stetter,K. (1986) Arch. Microbiol., 145, 56–61.
Fontecilla-Camps,J.C. (2009) Met. Ions Life Sci., 6, 151–178.
Friedrich,B., Fritsch,J. and Lenz,O. (2011) Curr. Opin. Biotechnol., 22,
358–364.
Grzeszik,C., Ross,K., Schneider,K., Reh,M. and Schlegel,H.G. (1997) Arch.
Microbiol., 167, 172–176.
Hedderich,R. (2004) J. Bioenerg. Biomembr., 36, 65–75.
Hedderich,R.F.L. (2005) J. Mol. Microbiol. Biotechnol., 10, 92–104.
Hopkins,R.C., Sun,J., Jenney,F.E., Jr., Chandrayan,S.K., McTernan,P.M. and
Adams,M.W.W. (2011) PLOS One, 6, e26569.
Respiratory hydrogenase cytoplasmic subcomplex 7
atUniversityofGeorgiaLibraries,SerialsDepartmentonDecember8,2014http://peds.oxfordjournals.org/Downloadedfrom
8. Horton,R.M., Hunt,H.D., Ho,S.N., Pullen,J.K. and Pease,L.R. (1989) Gene.,
77, 61–68.
Keller,M.W., Schut,G.J., Lipscomb,G.L., et al. (2013) Proc. Natl. Acad. Sci.,
110, 5840–5845.
Kurkin,S., Meuer,J., Koch,J., Hedderich,R. and Albracht,S.P.J. (2002)
Eur. J. Biochem., 269, 6101–6111.
Lee,H.S., Vermaas,W.F. and Rittmann,B.E. (2010) Biotechnol., 28, 262–271.
Lipscomb,G.L., Stirrett,K., Schut,G.J., Yang,F., Jenney,F.E., Jr., Scott,R.A.,
Adams,M.W. and Westpheling,J. (2011) Appl. Environ. Microbiol., 77,
2232–2238.
Marreiros,B.C., Batista,A.P., Duarte,A.M. and Pereira,M.M. (2013) Biochim.
Biophys. Acta., 1827, 198–209.
McTernan,P.M., Chandrayan,S.K., Wu,C.-H., et al. (2014) J. Biol. Chem., 289,
19364–19372.
Meuer,J., Bartoschek,S., Koch,J., Kunkel,A. and Hedderich,R. (1999)
Eur. J. Biochem., 265, 325–335.
Sapra,R., Verhagen,M.F. and Adams,M.W. (2000) J. Bacteriol., 182, 3423–3428.
Sauter,M., Böhm,R. and Böck,A. (1992) Mol. Microbiol., 6, 1523–1532.
Sazanov,L.A. and Hinchliffe,P. (2006) Science, 311, 1430–1436.
Schut,G.J., Nixon,W.J., Lipscomb,G.L., Scott,R.A. and Adams,M.W.W. (2012)
Front. Micro., 3, 1–6.
Schut,G.J., Boyd,E.S., Peters,J.W. and Adams,M.W.W. (2013) FEMS Micro-
biol. Rev., 37, 182–203.
Shafaat,H.S., Rudiger,O., Ogata,H. and Lubitz,W. (2013) Biochim. Biophys.
Acta., 1827, 986–1002.
Silva,P., Van den Ban,E.C.D., Wassink,H., Haaker,H., de Castro,B., Robb,F.T.
and Hagen,W.R. (2000) Eur. J. Biochem., 267, 6541–6551.
Swartz,T.H., Ikewada,S., Ishikawa,O., Ito,M. and Krulwich,T.A. (2005) Extre-
mophiles, 9, 345–354.
Vignais,P. and Billoud,B. (2007) Chem. Rev., 107, 4206–4272.
Vitt,S., Ma,K., Warkentin,E., Moll,J., Pierik,A.J., Shima,S. and Ermler,U.
(2014) J. Mol. Biol., 426, 2813–2826.
Volbeda,A., Charon,M.H., Piras,C., Hatchikian,E.C., Frey,M. and Fontecilla-
Camps,J.C. (1995) Nature, 373, 580–587.
Volbeda,A., Darnault,C., Parkin,A., Sargent,F., Armstrong,F.A. and Fontecilla-
Camps,J.C. (2013) Structure, 21, 184–190.
8 P.M.McTernan et al.
atUniversityofGeorgiaLibraries,SerialsDepartmentonDecember8,2014http://peds.oxfordjournals.org/Downloadedfrom