- The identification of new metallo-β-lactamase inhibitor leads from fragment-based screening
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The emergence of metallo-β-lactamases (MBLs) capable of hydrolysing a broad spectrum of β-lactam antibiotics is particularly concerning for the future treatment of bacterial infections. This work describes the discovery of lead compounds for the development of new inhibitors using a competitive colorimetric assay based on the chromogenic cephalosporin CENTA, and a 500 compound Maybridge library suitable for fragment-based screening. The interactions between identified inhibitory fragments and the active site of the MBL from Klebsiella pneumoniae and Pseudomonas aeruginosa were probed by in silico docking studies.
- Vella, Peter,Hussein, Waleed M.,Leung, Eleanor W.W.,Clayton, Daniel,Ollis, David L.,Miti?, Nata?a,Schenk, Gerhard,McGeary, Ross P.
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- Comparative studies on isolation and characterization of allinase from garlic and onion using PEGylation-A novel method
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Allium plants including garlic (Allium sativum) and onion (Allium cepa) contain an enzyme allinase (E.C-4.4.1.4), which is an enzyme responsible for the production of thio sulphates, a promising therapeutically potential compound. PEG (polyethylene glycol) is used in this study for the first time, to precipitate a protein. Four fold purification was done for both garlic and onion. The protein yield of the garlic was greater than onion shoots. 6.2 mg protein was the yield at the step in garlic, whereas it was 0.6 mg in onion shoots. The total protein content in the garlic is 6.1 g in the 100 g of garlic where as it is 1.6 g in the onion shoots.
- Rathnasamy, Senthilkumar,Auxilia, L. Rufus,Purusothaman
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- Protective effects of piceatannol on methylglyoxal-induced cytotoxicity in MC3T3-E1 osteoblastic cells
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Methylglyoxal (MG) is a reactive α-oxoaldehyde that increases under diabetic conditions and subsequently contributes to the complications associated with this disease. Piceatannol is a naturally occurring analogue of resveratrol that possesses multiple biological functions. The present study investigated the effects of piceatannol on MG-induced cytotoxicity in MC3T3-E1 osteoblastic cells. Piceatannol significantly restored MG-induced reductions in cell viability and reduced lactate dehydrogenase release in MG-treated MC3T3-E1 osteoblastic cells, which suggests that it suppressed MG-induced cytotoxicity. Piceatannol also increased glyoxalase I activity and glutathione levels in MG-treated cells, which indicates that it enhanced the glyoxalase system and thus cellular protection. The present study also showed that piceatannol inhibited the generation of inflammatory cytokines and reactive oxygen species and ameliorated mitochondrial dysfunction induced by MG. Furthermore, piceatannol treatment significantly reduced the levels of endoplasmic reticulum stress and autophagy induced by MG. Therefore, piceatannol could be a potent option for the development of antiglycating agents for the treatment of diabetic osteopathy.
- Suh, Kwang Sik,Chon, Suk,Choi, Eun Mi
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- Entamoeba histolytica thioredoxin reductase: Molecular and functional characterization of its atypical properties
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Background: Entamoeba histolytica, an intestinal protozoan that is the causative agent of amoebiasis, is exposed to elevated amounts of highly toxic reactive oxygen and nitrogen species during tissue invasion. Thioredoxin reductase catalyzes the reversible transfer of reducing equivalents between NADPH and thioredoxin, a small protein that plays key metabolic functions in maintaining the intracellular redox balance. Methods: The present work deals with in vitro steady state kinetic studies aimed to reach a better understanding of the kinetic and structural properties of thioredoxin reductase from E. histolytica (EhTRXR). Results: Our results support that native EhTRXR is a homodimeric covalent protein that is able to catalyze the NAD(P)H-dependent reduction of amoebic thioredoxins and S-nitrosothiols. In addition, the enzyme exhibited NAD(P)H dependent oxidase activity, which generates hydrogen peroxide from molecular oxygen. The enzyme can reduce compounds like methylene blue, quinones, ferricyanide or nitro-derivatives; all alternative substrates displaying a relative high capacity to inhibit disulfide reductase activity of EhTRXR. Conclusions and general significance: Interestingly, EhTRXR exhibited kinetic and structural properties that differ from other low molecular weight TRXR. The TRX system could play an important role in the parasite defense against reactive species. The latter should be critical during the extra intestinal phase of the amoebic infection. So far we know, this is the first in depth characterization of EhTRXR activity and functionality.
- Arias, Diego G.,Regner, Erika L.,Iglesias, Alberto A.,Guerrero, Sergio A.
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- Effect of N-B transition on the microenvironment surrounding 34Cys in human serum albumin
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The effect of pH on the microenvironment surrounding 34Cys in human serum albumin (HSA) has been studied using acrylodan, a Cys-specific fluorescence probe. The reactivity of 34Cys with 5,5'-dithiobis(2-nitro benzoic acid) (DTNB) followed a pseudo-first-order reaction, and the increase in reactivity was dependent on pH and oleate content. Compared with the N- form of HSA-acrylodan conjugate (pH 6.2), the B-form (pH 8.4) has a blue- shifted Em(max) and enhanced fluorescence intensity derived from acrylodan covalently attached to 34Cys suggesting that the exposure around 34Cys in the B-form was less than that in the N-form. The conformational change induced by fatty acid increased the exposure around 34Cys, while that induced by an increase in pH decreased it. Further, since the effect of oleate on the fluorescence of acrylodan was nearly the same for both conformers, the effects of pH and oleate on the microenvironment surrounding 34Cys should be independent and additive. We concluded that the increase of reactivity of 34Cys as a function in increasing pH may well be related to an increase in mercaptide ion content.
- Narazaki, Ryuichi,Maruyama, Toru,Otagiri, Masaki
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- Protein Reduction and Dialysis-Free Work-Up through Phosphines Immobilized on a Magnetic Support: TCEP-Functionalized Carbon-Coated Cobalt Nanoparticles
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Tris(2-carboxyethyl)phosphine (TCEP) is an often-used reducing agent in biochemistry owing to its selectivity towards disulfide bonds. As TCEP causes undesired consecutive side reactions in various analytical methods (e.g., gel electrophoresis, protein labeling), it is usually removed by means of dialysis or gel filtration. Here, an alternative method of separation is presented, namely the immobilization of TCEP on magnetic nanoparticles. This magnetic reagent provides a simple and rapid approach to remove the reducing agent after successful reduction. A reduction capacity of 70 μmol per gram of particles was achieved by using surface-initiated atom transfer polymerization.
- Zwyssig, Adrian,Schneider, Elia M.,Zeltner, Martin,Rebmann, Balder,Zlateski, Vladimir,Grass, Robert N.,Stark, Wendelin J.
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- Characterization and in vitro functional analysis of thioredoxin glutathione reductase from the liver fluke Opisthorchis viverrini
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The carcinogenic liver fluke Opisthorchis viverrini causes several hepatobiliary diseases including a bile duct cancer-cholangiocarcinoma (CCA), which is a major public health problem in many countries in the Greater Mekong Sub-region. Praziquantel is the main drug against this parasite, however, reduced drug efficacy has been observed in some endemic areas. Therefore, alternative drugs are needed to prepare for praziquantel resistance in the future. The selenoprotein thioredoxin glutathione reductase (TGR) enzyme, which plays a crucial role in cellular redox balance of parasitic flatworms, has been shown as a potential drug target against these parasites. Hence, this study aimed to investigate the TGR of O. viverrini and assess its potential as a drug target. An open reading frame (ORF) that encodes O. viverrini TGR (Ov-TGR) was cloned from an O. viverrini cDNA library and the nucleotide were sequenced. The 1,812 nucleotides of the Ov-TGR full ORF encoded a polypeptide of 603 amino acid residues with a predicted molecular mass of 66 kDa. The putative amino acid sequence shared 55–96.8percent similarities with TGRs from other helminths and mammals. Phylogenetic analysis revealed a close relationship of Ov-TGR with that of other trematodes. The ORF of Ov-TGR was inserted into pABC2 plasmid and transformed into Escherichia coli strain C321.ΔA to facilitate selenocysteine incorporation. The recombinant Ov-TGR (rOv-TGR-SEC) was expressed as a soluble protein and detected as a dimer form in the non-reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Its thioredoxin reductase (TrxR) and glutathione reductase (GR) activities were detected using DTNB, Trx and GSSG substrates with the Michaelis constant (Km) of 292.6 ± 52.3 μM, 8.09 ± 1.91 μM and 13.74 ± 1.2 μM, respectively. The TGR enzyme activities were effectively inhibited by a well-known inhibitor, auranofin in a dose-dependent manner. Moreover, auranofin expressed a lethal toxic effect on both newly excysted juveniles (NEJs) and adult worms of O. viverrini in vitro. Taken together, these results indicated that Ov-TGR is crucial for O. viverrini survival and maybe a potential target for the development of novel agents against opisthorschiasis.
- Chaiyadet, Sujittra,Laha, Thewarach,Laohaviroj, Marut,Plumworasawat, Sirikanya,Prum, Satya,Saichua, Prasert,Sripa, Banchob,Suttiprapa, Sutas,Thanan, Raynoo
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- A scalable synthesis of highly stable and water dispersible Ag 44(SR)30 nanoclusters
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We report the synthesis of atomically monodisperse thiol-protected silver nanoclusters [Ag44(SR)30] m, (SR = 5-mercapto-2-nitrobenzoic acid) in which the product nanocluster is highly stable in contrast to previous preparation methods. The method is one-pot, scalable, and produces nanoclusters that are stable in aqueous solution for at least 9 months at room temperature under ambient conditions, with very little degradation to their unique UV-Vis optical absorption spectrum. The composition, size, and monodispersity were determined by electrospray ionization mass spectrometry and analytical ultracentrifugation. The produced nanoclusters are likely to be in a superatom charge-state of m = 4-, due to the fact that their optical absorption spectrum shares most of the unique features of the intense and broadly absorbing nanoparticles identified as [Ag44(SR) 30]4- by Harkness et al. (Nanoscale, 2012, 4, 4269). A protocol to transfer the nanoclusters to organic solvents is also described. Using the disperse nanoclusters in organic media, we fabricated solid-state films of [Ag44(SR)30]m that retained all the distinct features of the optical absorption spectrum of the nanoclusters in solution. The films were studied by X-ray diffraction and photoelectron spectroscopy in order to investigate their crystallinity, atomic composition and valence band structure. The stability, scalability, and the film fabrication method demonstrated in this work pave the way towards the crystallization of [Ag44(SR)30]m and its full structural determination by single crystal X-ray diffraction. Moreover, due to their unique and attractive optical properties with multiple optical transitions, we anticipate these clusters to find practical applications in light-harvesting, such as photovoltaics and photocatalysis, which have been hindered so far by the instability of previous generations of the cluster.
- Abdulhalim, Lina G.,Ashraf, Sumaira,Katsiev, Khabiboulakh,Kirmani, Ahmad R.,Kothalawala, Nuwan,Anjum, Dalaver H.,Abbas, Sikandar,Amassian, Aram,Stellacci, Francesco,Dass, Amala,Hussain, Irshad,Bakr, Osman M.
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- CENTA as a chromogenic substrate for studying β-lactamases
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CENTA, a chromogenic cephalosporin, is readily hydrolyzed by β-lactamases of all classes except for the Aeromonas hydrophila metalloenzyme. Although it cannot practically be used for the detection of β-lactamase-producing strains on agar plates, it should be quite useful for kinetic studies and the detection of the enzymes in crude extracts and chromatographic fractions.
- Bebrone, Carine,Moali, Catherine,Mahy, Florence,Rival, Sandrine,Docquier, Jean Denis,Rossolini, Gian Maria,Fastrez, Jacques,Pratt, Rex F.,Frere, Jean-Marie,Galleni, Moreno
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- A Dipeptide-Based Hierarchical Nanoarchitecture with Enhanced Catalytic Activity
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Achieving synthetic architectures with simple structures and robust biomimetic catalytic activities remains a great challenge. Herein, we explore a facile supramolecular assembly approach to construct a dipeptide-based hierarchical nanoarchitecture with enhanced enzyme-like catalytic activity. In this nanoarchitecture, nanospheres are put in a chain-like arrangement through coordination-driven directional self-assembly. The reversible transformation of anisotropic nanochains to isotropic nanospheres switches biomimetic activity. Notably, the assembled nanoarchitecture exhibits a high enzyme-like activity and remarkable long-term stability to promote hydroquinone oxidation, superior to the natural counterpart. This work will pave the way to develop reversible and reusable supramolecular biocatalysts with ordered hierarchical structures for accelerating chemical transformations.
- Fei, Jinbo,Li, Junbai,Wang, Chenlei,Wang, Keqing
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- Fluorescence detection of the nitric oxide-induced structural change at the putative nitric oxide sensing segment of TRPC5
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The plausible nitric oxide (NO)-sensing module of TRPC5 was incorporated in a enhanced green fluorescent protein (EGFP) to evaluate its conformational change as an optical response upon the reaction with NO. Two cysteine residues located in the NO-sensing module have been proposed to form a disulfide bond through S-nitrosylation of the thiol group by NO. Modification of the cysteine residues by NO resulted a ratiometric change of EGFP emission through transducing the conformational change of NO-sensing module to the EGFP chromophore. The oxidized form of NO-sensing module fused EGFP changed the intensity of emission spectra upon reduction of the disulfide bond at the NO-reactive module. The NO-sensing module fused EGFP in its reduced form avidly reacted with NO and realized the ratiometric fluorescence intensity changes depending on the formation of disulfide bond. These results support the notion that NO induces a conformational change at the putative NO-sensing segment of TRPC5, and provide a prototype for the genetically encoded cellular NO sensors.
- Mori, Yasuo,Morii, Takashi,Nakata, Eiji,Saimura, Masayuki,Sakaguchi, Reiko,Tajima, Shunsuke
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- Neat and complete: Thiolate-ligand exchange on a silver molecular nanoparticle
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Atomically precise thiolate-protected noble metal molecular nanoparticles are a promising class of model nanomaterials for catalysis, optoelectronics, and the bottom-up assembly of true molecular crystals. However, these applications have not fully materialized due to a lack of ligand exchange strategies that add functionality, but preserve the properties of these remarkable particles. Here we present a method for the rapid (44(SR)30]-4. Only by using this method were we able to preserve the precise nature of the particles and simultaneously replace the native ligands with ligands containing a variety of functional groups. Crucially, as a result of our method we were able to process the particles into smooth thin films, paving the way for their integration into solution-processed devices.
- Abdulhalim, Lina G.,Kothalawala, Nuwan,Sinatra, Lutfan,Dass, Amala,Bakr, Osman M.
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- Biochemical and thermodynamic comparison of the selenocysteine containing and non-containing thioredoxin glutathione reductase of Fasciola gigantica
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The thiol-disulfide redox metabolism in platyhelminth parasites depends entirely on a single selenocysteine (Sec) containing flavoenzyme, thioredoxin glutathione reductase (TGR) that links the classical thioredoxin (Trx) and glutathione (GSH) systems. In the present study, we investigated the catalytic and structural properties of different variants of Fasciola gigantica TGR to understand the role of Sec. The recombinant full-length Sec containing TGR (FgTGRsec), TGR without Sec (FgTGR) and TGRsec without the N-terminal glutaredoxin (Grx) domain (?NTD-FgTGRsec) were purified to homogeneity. Biochemical studies revealed that Sec597 is responsible for higher thioredoxin reductase (TrxR) and glutathione reductase (GR) activity of FgTGRsec. The N-terminal Grx domain was found to positively regulate the DTNB-based TrxR activity of FgTGRsec. The FgTGRsec was highly sensitive to inhibition by auranofin (AF). The structure of FgTGR was modeled, and the inhibitor AF was docked, and binding sites were identified. Unfolding studies suggest that all three proteins are highly cooperative molecules since during GdnHCl-induced denaturation, a monophasic unfolding of the proteins without stabilization of any intermediate is observed. The Cm for GdnHCl induced unfolding of FgTGR was higher than FgTGRsec and ?NTD-FgTGRsec suggesting that FgTGR without Sec was more stable in solution than the other protein variants. The free energy of stabilization for the proteins was also determined. To our knowledge, this is also the first report on unfolding and stability analysis of any TGR.
- Kalita, Parismita,Shukla, Harish,Shukla, Rohit,Tripathi, Timir
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- An Improved Synthesis of CENTA, a Chromogenic Substrate for β-Lactamases
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7-β-Thien-2-yl-acetamido-3-[(4-nitro-3-carboxyphenyl)thiomethyl]-3-cephem-4-carboxylic acid (CENTA) is a yellow chromogenic β-lactamases (BL) substrate. It hydrolyses readily in the presence of all BL and is therefore suitable for kinetic studies, the detection of BL enzymes in crude extracts and chromatographic fractions. CENTA is commercially available at a high price because of the cumbersome synthetic protocol, the only currently available for its preparation. Here we describe a new efficient and improved process for the preparation of CENTA. Starting from the easily available 7-aminocephalosporanic acid (7-ACA) through a three-step synthesis, CENTA was obtained with a 75% overall yield. The newly developed process proceeds through a pivotal intermediate in cephalosporin chemistry, which may be used as starting compound for the development of new cephalosporin derivatives.
- Quotadamo, Antonio,Linciano, Pasquale,Davoli, Paolo,Tondi, Donatella,Costi, Maria Paola,Venturelli, Alberto
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- On the Cleavage of Ellman's Reagent by Dithonite in the Presence of Dioctadecyldimethylammonium Chloride Surfactant Vesicles Laced with Cholesterol
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Ellman's reagent (I) is distributed between the outer aqueous interface and the interior bilayer of DODAC vesicles upon contact when added to presonicated aqueous DODAC suspensions.This distribution also extends to the inner aqueous interface in about 10 min.This suspension reacts with aqueous alkaline sodium dithionite in two kinetic processes.The first process involves the bimolecular cleavage of I at the outer aqueous interface instantaneously.This is followed by a slow reaction of the same due to leakage of the bilayer-embedded substrate to the outer interface.This condition changes, however, when the DODAC vesicles are laced with cholesterol by cosonication of the aqueous solutions.Both Ellman's reagent and the reduced anion II product of cleavage by dithionite now are impermeable to this membrane but the protonated III does penetrate the bilayer.This result is indicated by loss of absorbance at 440 nm concurrent with gain in absorbance at 320 nm upon contact between III and cholesterol-laced vesicles.This result is interpreted in terms of the known permeability changes accompanying the incorporation of cholesterol into the vesicles.Thus Ellman's reaggent (I) can be encapsulated by DODAC vesicles if they contain cholesterol in such a way that they are unreachable by dithionite and lyate species.
- Huffman, Robert W.,McBride, Phil,Brown, David M.
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- Dynamics of the Cleavage of Ellman's Reagent in Surfactant Vesicles
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The cleavage of Ellman's reagent to 2-nitro-5-thiolatobenzoate dianion, 5, by dithionite, sulfite, and thiophenoxide ions and by zwitterion 4, was studied at pH 8 in aqueous solution and in aqueous solutions of hexadecyltrimethylammonium ion micelles or dihexadecyldimethylammonium ion vesicles, 2.Excepting reagent 4, strong micellar and vesicular catalysis of the cleavage reactions was observed.When Ellman's reagent was added to the empty vesicles, followed by the addition of excess cleavage reagent, separate fast and slow reactions were observed.The relative contribution of each process varied with the aging time between the addition of the Ellman's reagent and the cleavage reagent.The results were quantitatively analyzed in terms of reversible substrate distribution between exovesicular (surface) and endovesicular (subsurface) sites.With dithionite and sulfite reagents, rapid cleavage of exovesicular substrate was followed by slow cleavage of endovesicular substrate which had permeated back to the vesicular surface to react with exovesicular dithionite or sulfite.With thiophenoxide, very rapid exovesicular cleavage was followed by slower endovesicular cleavage of substrate by rapidly permeating thiophenoxide (thiophenol).Reagent 4 was not bound by vesicular 2, and the clavage reactions occured only exovesicularly or off the vesicle.
- Moss, Robert A.,Schreck, Ronald P.
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- Enzymatic spectrophotometric method for aflatoxin B detection based on acetylcholinesterase inhibition
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A new method for aflatoxin B (AFB) determination is proposed. The AFB determination is based on acetylcholinesterase (AChE) inhibition, and the AChE residual activity is determined using the colorimetric method (Ellman's method). Cholinesterases (ChEs) from various sources were tested using AFB1 as reference aflatoxin. AChE from electric eel has shown the highest sensitivity to AFB1, and it was chosen for the rest of the work. To select and optimize the analytical procedures, an investigation on the type of AChE inhibition by AFB1 was carried out. The AChE degree of inhibition by AFB1 was independent of the incubation time and the enzyme concentrations, showing the reversibility of the inhibition. This reversibility of the inhibition permits a rapid analysis of AFB1, requiring only 3 min. For the development of the AFB1 assay, the pH, the time of reaction, temperature, and substrate concentration were evaluated and optimized. The linear range of 10-60 ng mL-1 was determined. To evaluate the selectivity of this method, the cross-reactivity with other aflatoxins such as aflatoxin B2, aflatoxin G1, aflatoxin G2, and aflatoxin M1 was investigated. Finally, the suitability of the assay for AFB1 quantification in barley was evaluated. This study shows a new approach to detect anatoxins based on enzyme inhibition and has advantages such as the ease of use, rapidity, and cost effectiveness. Thus, it could find a possible use as a screening method for this type of mycotoxins.
- Arduini, Fabiana,Errico, Ilenia,Amine, Aziz,Micheli, Laura,Palleschi, Giuseppe,Moscone, Danila
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- Molecular recognition of organophosphorus compounds in water and inhibition of their toxicity to acetylcholinesterase
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Molecular tubes with hydrogen bonding donors in their deep hydrophobic cavities are able to selectively bind organophosphorus compounds in water through hydrogen bonding and the hydrophobic effect. They can also be used as a fluorescent sensor for nerve agent simulants and as an inhibitor to reduce the toxicity of paraoxon to acetylcholinesterase.
- Liu, Wei-Er,Chen, Zhao,Yang, Liu-Pan,Au-Yeung, Ho Yu,Jiang, Wei
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- Antioxidant activity and inhibition of human neutrophil oxidative burst mediated by arylpropionic acid non-steroidal anti-inflammatory drugs
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It has been suggested that the anti-inflammatory activity of some non-steroidal anti-inflammatory drugs (NSAIDs) may be partly due to their ability to scavenge reactive oxygen species (ROS) and reactive nitrogen species (RNS), as well as to inhibit the respiratory burst of neutrophils triggered by various activating agents. Therefore, the aim of the present work was to evaluate and compare the potential scavenging activity for an array of ROS (O2.-, H2O2, HO., ROO. and HOCl) and RNS (.NO and ONOO-) using in vitro non-cellular screening systems as well as a cellular screening system (human neutrophil oxidative burst), mediated by the arylpropionic acid derivatives (APAs) NSAIDs ibuprofen, flurbiprofen, fenoprofen, fenbufen, ketoprofen, naproxen and indoprofen. The results obtained in the present work demonstrate that under the present experimental conditions, many of the studied APA NSAIDs showed O2.- scavenging activity (fenbufen≈ flurbiprofen≈indoprofen≈ketoprofen), H2O2 (ketoprofen≈indoprofen≈fenbufen>flurbiprofen>naproxen), HO . (fenoprofen≈ibuprofen>fenbufen≈flurbiprofen> ketoprofen>indoprofen≈naproxen), .NO (indoprofen> naproxen), ONOO- (indoprofen>naproxen>fenoprofen≈flurbiprofen≈ ibuprofen), as well as inhibit myeloperoxidase (MPO) activity (indoprofen) and scavenge human neutrophil derived ROS (ketoprofen>indoprofen >fenbufen>flurbiprofen). The observed effects, if confirmed in vivo, may strongly contribute to the antiinflammatory therapeutical activity observed with these NSAIDs.
- Costa, David,Moutinho, Luis,Lima, Jose Luis Fontes Costa,Fernandes, Eduarda
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- Study of the thiol/disulfide redox systems of the anaerobe Desulfovibrio vulgaris points out pyruvate:ferredoxin oxidoreductase as a new target for thioredoxin
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Sulfate reducers have developed a multifaceted adaptative strategy to survive against oxidative stresses. Along with this oxidative stress response, we recently characterized an elegant reversible disulfide bond-dependent protective mechanism in the pyruvate:ferredoxin oxidoreductase (PFOR) of various Desulfovibrio species. Here, we searched for thiol redox systems involved in this mechanism. Using thiol fluorescent labeling, we show that glutathione is not the major thiol/disulfide balance-controlling compound in four different Desulfovibrio species and that no other plentiful low molecular weight thiol can be detected. Enzymatic analyses of two thioredoxins (Trxs) and three thioredoxin reductases allow us to propose the existence of two independent Trx systems in Desulfovibrio vulgaris Hildenborough (DvH). The TR1/Trx1 system corresponds to the typical bacterial Trx system. We measured a TR1 apparent Km value for Trx1 of 8.9 μM. Moreover, our results showed that activity of TR1 was NADPH-dependent. The second system named TR3/Trx3 corresponds to an unconventional Trx system as TR3 used preferentially NADH (Km for NADPH, 743 μM; Km for NADH, 5.6 μM), and Trx3 was unable to reduce insulin. The Km value of TR3 for Trx3 was 1.12 μM. In vitro experiments demonstrated that the TR1/Trx1 system was the only one able to reactivate the oxygen-protected form of Desulfovibrio africanus PFOR. Moreover, ex vivo pulldown assays using the mutant Trx1C33S as bait allowed us to capture PFOR from the DvH extract. Altogether, these data demonstrate that PFOR is a new target for Trx1, which is probably involved in the protective switch mechanism of the enzyme.
- Pieulle, Laetitia,Stocker, Pierre,Vinay, Manon,Nouailler, Matthieu,Vita, Nicolas,Brasseur, Gael,Garcin, Edwige,Sebban-Kreuzer, Corinne,Dolla, Alain
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- Highly sensitive DNA methylation analysis at CpG resolution by surface-enhanced Raman scattering via ligase chain reaction
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Sensitive and accurate DNA methylation analysis at CpG resolution was demonstrated using surface-enhanced Raman scattering (SERS) via ligase chain reaction (LCR). The method was sensitive to 10% changes in methylation and the accuracy of methylation estimates in cells and serum DNA validated with sequencing. The LCR/SERS approach may have broad applications as an alternative (epi)genetic detection method.
- Wang, Yuling,Wee, Eugene J. H.,Trau, Matt
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- One-step electrochemically deposited gold nanoparticles interface grafted with avidin for acetylcholinesterase biosensor design
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In this study, an interface embedded in situ gold nanoparticles (AuNPs) and biotin in chitosan hydro-gel was constructed by one-step electrochemical deposition in solution containing tetrachloroauric (III) acid, biotin and chitosan. This deposited interface acts as biosensing platforms and provides specific binding sites for avidin, which are further capable of attaching any biotinylated bimolec-ular for biosensor design. Atomic force microscopy (AFM), A.C. impedance and surface plasmon resonance were used to characterize this interface. The immobilized acetylcholinesterase (AChE), as a model, showed excellent activity to its substrate and provided a quantitative measurement of organophosphate pesticide. Under the optimal experimental conditions, the inhibition of dimethoate was proportional to its concentrations in the range of 0.05 to 15 μg mL-1 with detection limit of 0.001 μg mL -1. The simple method showed good fabrication reproducibility and acceptable stability, which provided a new avenue for electrochemical biosensor design.
- Zhang, Weiying,Ding, Jiawang,Qin, Yuehua,Liu, Deli,Du, Dan
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- Novel solid-phase reagents for facile formation of intramolecular disulfide bridges in peptides under mild conditions
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The controlled formation of intramolecular disulfide bridges in peptides, while avoiding unwanted oligomerization, is a significant challenge. Ellman's reagent, 5,5'-dithiobis(2-nitrobenzoic acid), was developed originally in the context of an assay for measuring free thiol concentration under physiological conditions. The present studies demonstrate that this reagent, when bound through two sites to a suitable solid support (PEG-PS, modified Sephadex, or controlled-pore glass), is an effective mild oxidizing reagent that promotes the formation of disulfide bridges. Rates and yields of the reactions were determined as a function of pH, excess of oxidizing reagent, resin loading, and parent support, for the preparation of oxytocin and deaminooxytocin (9 residues, disulfide bridge between residues 1 and 6), somatostatin (14 residues, disulfide bridge between residues 3 and 14), α-conotoxin SI (13 residues, disulfide bridges between residues 2 and 7; 3 and 13), and apamin (18 residues, disulfide bridges between residues 1 and 11; 3 and 15). Cystine dimers of these peptide models formed, if at all, in relatively low amounts. Use of solid-phase Ellman's reagents to oxidize the linear precursors of conotoxin or apamin (tetrathiols) gave as the main products the correctly paired regioisomers. Particular advantages of the overall approach include fast reaction rates over a wide range of pH, from 2.7 to 6.6; easy purification of disulfide-containing products; and the specificity and reusability of the reagents.
- Annis, Ioana,Chen, Lin,Barany, George
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- Characterization of an NADH-dependent persulfide reductase from shewanella loihica PV-4: Implications for the mechanism of sulfur respiration via FAD-dependent enzymes
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The NADH-dependent persulfide reductase (Npsr), a recently discovered member of the PNDOR family of flavoproteins that contains both the canonical flavoprotein reductase domain and a rhodanese domain, is proposed to be involved in the dissimilatory reduction of S0 for Shewanella loihica PV-4. We have previously shown that polysulfide is a substrate for this enzyme, and a recently determined structure of a closely related enzyme (CoADR-Rhod from Bacillus anthracis) suggested the importance of a bound coenzyme A in the mechanism. The work described here shows that the in vivo oxidizing substrates of Npsr are the persulfides of small thiols such as CoA and glutathione. C43S, C531S, and C43,531S mutants were created to determine the role of the flavoprotein domain cysteine (C43) and the rhodanese domain cysteine (C531) in the mechanism. The absolute requirement for C43 in persulfide or DTNB reductase activity shows that this residue is involved in S-S bond breakage. C531 contributes to, but is not required for, catalysis of DTNB reduction, while it is absolutely required for reduction of any persulfide substrates. Titrations of the enzyme with NADH, dithionite, titanium(III), or TCEP demonstrate the presence of a mixed-disulfide between C43 and a tightly bound CoA, and structures of the C43 and C43,531S mutants confirm that this coenzyme A remains tightly bound to the enzyme in the absence of a C43-CoA S-S bond. The structure of Npsr suggests a likely site for binding and reaction with the persulfide substrate on the rhodanese domain. On the basis of kinetic, titration, and structural data, a mechanism for the reduction of persulfides by Npsr is proposed.
- Warner, Megan D.,Lukose, Vinita,Lee, Kyu Hyun,Lopez, Karlo,H. Sazinsky, Matthew,Crane, Edward J.
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- Synthesis, characterization and biological activity of bis[3-ethyl-4-aryl-5-(2-methoxypyridin-5-yl)-1-propyl-1,3-dihydro-2H-imidazol-2-ylidene]gold(i) complexes
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A series of bis[3-ethyl-4-aryl-5-(2-methoxypyridin-5-yl)-1-propyl-1,3-dihydro-2H-imidazol-2-ylidene]gold(i) complexes (2a-f) containing methyl, fluoro or methoxy substituents at various positions in the 4-aryl ring was synthesized and evaluated for their anti-cancer properties in A2780 (wild-type and Cisplatin-resistant) ovarian carcinoma as well as LAMA 84 (imatinib-sensitive and -resistant) and HL-60 leukemia cell lines. The bis-NHC gold(i) complexes were more active compared to their related mono-NHC gold(i) analogues and reduced proliferation and metabolic activity in a low micromolar range. With the exception of2d(3-F), the compounds displayed higher potency than the established drugs Auranofin and Cisplatin. The lack of effects against non-cancerous lung fibroblast SV-80 cells indicated a high selectivity towards tumor cells. All tested complexes generated reactive oxygen species in A2780cis cells; however, the induction of apoptosis was very low. Furthermore, thioredoxin reductase is not the main target of these complexes, because its inhibition pattern did not correlate with their biological activity.
- Gallati, Caroline Marie,Goetzfried, Sina Katharina,Ortmeier, Anna,Sagasser, Jessica,Wurst, Klaus,Hermann, Martin,Baecker, Daniel,Kircher, Brigitte,Gust, Ronald
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Read Online
- An Ultrasmall Fe3O4-Decorated Polydopamine Hybrid Nanozyme Enables Continuous Conversion of Oxygen into Toxic Hydroxyl Radical via GSH-Depleted Cascade Redox Reactions for Intensive Wound Disinfection
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Nanozyme-based chemodynamic therapy (CDT) for fighting bacterial infections faces several major obstacles including low hydrogen peroxide (H2O2) level, over-expressed glutathione (GSH) in infected sites, and inevitable damage to healthy tissue with abundant nonlocalized nanozymes. Herein, a smart ultrasmall Fe3O4-decorated polydopamine (PDA/Fe3O4) hybrid nanozyme is demonstrated that continuously converts oxygen into highly toxic hydroxyl radical (?OH) via GSH-depleted cascade redox reactions for CDT-mediated bacterial elimination and intensive wound disinfection. In this system, photonic hyperthermia of PDA/Fe3O4 nanozymes can not only directly damage bacteria, but also improve the horseradish peroxidase-like activity of Fe3O4 decorated for CDT. Surprisingly, through photothermal-enhanced cascade catalytic reactions, PDA/Fe3O4 nanozymes can consume endogenous GSH for disrupting cellular redox homeostasis and simultaneously provide abundant H2O2 for improving ?OH generation, ultimately enhancing the antibacterial performance of CDT. Such PDA/Fe3O4 can bind with bacterial cells, and reveals excellent antibacterial property against both Staphylococcus aureus and Escherichia coli. Most interestingly, PDA/Fe3O4 nanozymes can be strongly retained in infected sites by an external magnet for localized long-term in vivo CDT and show minimal toxicity to healthy tissues and organs. This work presents an effective strategy to magnetically retain the therapeutic nanozymes in infected sites for highly efficient CDT with good biosafety.
- Deng, Le,Gong, Minhui,Hai, Luo,He, Dinggeng,Li, Huan,Li, Yaoyao,Tang, Zifeng,Wang, Zefeng,Xiao, Jiayu
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- Use of pyridazinediones as extracellular cleavable linkers through reversible cysteine conjugation
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Herein we report a retro-Michael deconjugation pathway of thiol-pyridazinedione linked protein bioconjugates to provide a novel cleavable linker technology. We demonstrate that the novel pyridazinedione linker does not suffer from off-Target modification with blood thiols (e.g., glutathione, human serum albumin (HSA)), which is in sharp contrast to an analogous maleimide linker.
- Bahou, Calise,Spears, Richard J.,Aliev, Abil E.,Maruani, Antoine,Fernandez, Marcos,Javaid, Faiza,Szijj, Peter A.,Baker, James R.,Chudasama, Vijay
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supporting information
p. 14829 - 14832
(2019/12/24)
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- Rational design of novel irreversible inhibitors for human arginase
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Parasites have developed a variety of strategies for invading hosts and escaping their immune response. A common mechanism by which parasites escape nitric oxide (NO) toxicity is the activation of host arginase. This activation leads to a depletion of L-arginine, which is the substrate for NO synthase, resulting in lower levels of NO and increased production of polyamines that are necessary for parasite growth and differentiation. For this reason, small molecule inhibitors for arginase show promise as new anti-parasitic chemotherapeutics. However, few arginase inhibitors have been reported. Here, we describe the discovery of novel irreversible arginase inhibitors, and their characterization using biochemical, kinetic, and structural studies. Importantly, we determined the site on human arginase that is labeled by one of the small molecule inhibitors. The tandem mass spectra data show that the inhibitor occupies the enzyme active site and forms a covalent bond with Thr135 of arginase. These findings pave the way for the development of more potent and selective irreversible arginase inhibitors.
- Guo, Xuefeng,Chen, Yiming,Seto, Christopher T.
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supporting information
p. 3939 - 3946
(2018/06/19)
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- Reaction of hydrogen sulfide with disulfide and Sulfenic acid to form the strongly Nucleophilic Persulfide
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Background: Hydrogen sulfide (H2S) modulates physiological processes in mammals. Results: The reactivity of H2S toward disulfides (RSSR) and albumin sulfenic acid (RSOH) to form persulfides (RSSH) was assessed. Conclusion: H2S is less reactive than thiols. Persulfides have enhanced nucleophilicity. Significance: This kinetic study helps rationalize the contribution of the reactions with oxidized thiol derivatives toH2S biology.
- Cuevasanta, Ernesto,Lange, Mike,Bonanata, Jenner,Coiti?o, E. Laura,Ferrer-Sueta, Gerardo,Filipovic, Milos R.,Alvarez, Beatriz
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p. 26866 - 26880
(2015/11/17)
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- Uncatalyzed and copper(II) catalyzed oxidation of glutathione by Co(III)2 bound superoxide complex
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In acid media, glutathione (GSH) is oxidised by metal bound superoxo complex, [(NH3)5Co(III)(μ-O2)Co(III)(NH 3)5]5+ (1) to GSSG. Complex 1 is reduced to its corresponding peroxo complex, [(NH3)5Co(III)(μ-O 2)Co(III)(NH3)5]4+ (2) which readily decomposes in acid media to Co(III), NH4+ and O 2. The oxidation of GSH is profoundly catalyzed by the presence of Cu2+ ion. The observed rate constant ko was found to be proportional to [GSH]2 for both uncatalyzed and catalyzed reaction and for the latter ko was also proportional to [Cu]T 2 ([Cu]T is the analytical concentration of Cu 2+). The rate for uncatalyzed reaction decreases for with increasing ionic strength (I) of the reaction media whereas, ko for catalyzed reaction is invariant of ionic strength (I). Both for the uncatalyzed and catalyzed reaction, ko is proportional to [H+] -3. Under the reaction condition employed, it has been seen that GSH reduces Cu(II) to Cu(I) and forms either mononuclear Cu(I)-GSH or dinuclear (Cu(I)-GSH)2 complex with Cu(I) with a 1:1 composition. Suitable mechanistic pathways for the uncatalyzed and catalyzed reactions through the intermediate formation of mononuclear or dinuclear Cu-GSH complex have been suggested. The activation energy for the uncatalyzed and catalyzed reaction has been calculated as 97.1 ± 5.7 and 29.9 ± 1.2 kJ M-1 respectively.
- Singh, Bula,Das, Ranendu Sekhar,Banerjee, Rupendranath,Mukhopadhyay, Subrata
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- BORONIC AND BORINIC ACID COMPOUNDS AS INHIBITORS OF SULFENIC ACID-CONTAINING PROTEINS
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A boronic or borinic acid compound is used to inhibit the activity of a sulfenic acid-containing protein. Thus, a biologically-active sulfenic acid-containing protein is contacted with an activity-inhibiting effective amount of a boronic or borinic acid compound of Formula (I) or a salt, hydrate or solvate thereof, whose components are disclosed within, and that contact is maintained for a time sufficient to inhibit the biological activity of the protein.
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Page/Page column 67-72
(2014/06/23)
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- Assay platform for clinically relevant metallo-β-lactamases
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Metallo-β-lactamases (MBLs) are a growing threat to the use of almost all clinically used β-lactam antibiotics. The identification of broad-spectrum MBL inhibitors is hampered by the lack of a suitable screening platform, consisting of appropriate substrates and a set of clinically relevant MBLs. We report procedures for the preparation of a set of clinically relevant metallo-β-lactamases (i.e., NDM-1 (New Delhi MBL), IMP-1 (Imipenemase), SPM-1 (Sa?o Paulo MBL), and VIM-2 (Verona integron-encoded MBL)) and the identification of suitable fluorogenic substrates (umbelliferone-derived cephalosporins). The fluorogenic substrates were compared to chromogenic substrates (CENTA, nitrocefin, and imipenem), showing improved sensitivity and kinetic parameters. The efficiency of the fluorogenic substrates was exemplified by inhibitor screening, identifying 4-chloroisoquinolinols as potential pan MBL inhibitors.
- Van Berkel, Sander S.,Brem, Jürgen,Rydzik, Anna M.,Salimraj, Ramya,Cain, Ricky,Verma, Anil,Owens, Raymond J.,Fishwick, Colin W.G.,Spencer, James,Schofield, Christopher J.
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supporting information
p. 6945 - 6953
(2013/10/01)
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- ANTI-WEAR AGENTS WITH A REDUCED NEUROTOXICITY
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Described is a lubricating composition of an anti-wear agent or a combination of anti-wear agents having a reduced neurotoxicity, the anti-wear agent(s) being selected from triaryl phosphate compounds preferably substituted with one or more linear or branched alkyl group(s) including from 2 to 12 carbon atoms.
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- Photoregulating catalytic activity of cyclodextrin-based artificial glutathione peroxidase by charged azobenzene
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Modulating the activities of enzymes by using an external signal is widely used for many applications. In this paper, it has been performed for the first time that photoregulating the activity of artificial glutathione peroxidase (GPx) by using photocontrolled inclusion-exclusion reaction of the azobenzene with two typical GPx mimics, the telluride β-Cyclodextrin (β-CD) dimmer and the ditelluride β-CD dimmer. The activities of both mimics were differently inhibited by either cationic or anionic azobenzene, owing to the catalytic capacity variance of the telluride moieties of the mimics and the different strength of the electrostatic interactions between the charges of the substrates and the azobenzenes. The inclusion of the anionic azobenzene with the ditelluride β-CD dimmer represented the largest inhibition rate. When the inclusion was irradiated upon UV light, the activity recovered, whereas inhibited again upon visible light. Such process could be repeated many times and a switchable artificial enzyme based on the reaction was proposed. Graphical Abstract: It was performed that photoregulating artificial enzyme by using photocontroled inclusion-exclusion reaction of one 6-ditellurium β-CD dimmer with two anionic azobenzenes. When inclusion underwent the activity of the artificial enzyme was inhibited. After irradiation with UV light, the cis-isomer azobenzene formed, and went away from the cavity left an appropriate cavity for accommodating a substrate, as a result, the activity recovered. After irradiation with visible light, the activity was inhibited over again, owing to that the trans-isomer azobenzene regenerated, and regressed to the inclusion. Such process could be repeated many times.
- Wu, Ping,Xiao, Ruiqing,Zhang, Chunqiu,Zhou, Lipeng,Luo, Quan,Xu, Jiayun,Liu, Junqiu
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experimental part
p. 62 - 67
(2010/11/03)
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- HOMOGENEOUS TIME RESOLVED FLUORESCENCE BASED TEST SYSTEM
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The present invention concerns a fluorescence resonance energy transfer based high throughput test system to measure the formation of the HIV gp41 six-helix bundle. In a first embodiment the current invention relates to a homogeneous time resolved fluorescence-based test system comprising a first helical polypeptide consisting essentially of the sequence of IQN36 (SEQ ID NO:1); a second helical polypeptide consisting essentially of the sequence of C34 (SEQ ID NO: 2) wherein said IQN36 is labeled with a light emitting fluorophore and said C34 is labeled with an ultra-violet excitable fluorophore.
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Page/Page column 35
(2010/12/29)
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- Biotransformation of prasugrel, a novel thienopyridine antiplatelet agent, to the pharmacologically active metabolite
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Prasugrel, a novel thienopyridine antiplatelet agent, undergoes rapid hydrolysis in vivo to a thiolactone, R-95913, which is further converted to its thiol-containing, pharmacologically active metabolite, R-138727, by oxidation via cytochromes P450 (P450). We trapped a sulfenic acid metabolite as a mixed disulfide with 2-nitro-5-thiobenzoic acid in an incubation mixture containing the thiolactone R-95913, expressed CYP3A4, and NADPH. Further experiments investigated one possible mechanism for the conversion of the sulfenic acid to the active thiol metabolite in vitro. A mixed disulfide form of R-138727 with glutathione was found to be a possible precursor of R-138727 in vitro when glutathione was present. The rate constant for the reduction of the glutathione conjugate of R-138727 to R-138727 was increased by addition of human liver cytosol to the human liver microsomes. Thus, one possible mechanism for the ultimate formation of R-138727 in vitro can be through formation of a sulfenic acid mediated by P450s followed possibly by a glutathione conjugation to a mixed disulfide and reduction of the disulfide to the active metabolite R-138727. Copyright
- Hagihara, Katsunobu,Kazui, Miho,Kurihara, Atsushi,Iwabuchi, Haruo,Ishikawa, Minoru,Kobayashi, Hiroyuki,Tanaka, Naoki,Okazaki, Osamu,Farid, Nagy A.,Ikeda, Toshihiko
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experimental part
p. 898 - 904
(2011/03/18)
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- Determination and isolation of a thioesterase from passion fruit (Passiflora edulis Sims) that hydrolyzes volatile thioesters
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Volatile organosulfur compounds (VOSCs) are high impact aroma chemicals characteristic of tropical fruits which are active as both free thiols and the respective thioesters. Using a simple and sensitive colorimetric enzyme assay, a thioesterase activity toward VOSCs has been identified in ripening purple passion fruit (Passiflora edulis Sims). The assay was based on determining the release of free thiols from 2-methyl-3-furanthiol acetate using Ellman's reagent. The major thioesterase in the fruit was found to be a wall-bound protein in the mesocarp. The extracted enzyme activity was purified 150-fold and shown to be associated with a 43 kDa monomeric serine hydrolase which was selectively labeled with a fluorophosphonate suicide probe. MS-MS sequencing identified the thioesterase as a class 13 glycoside hydrolase, most similar to pectin acetylesterase, an enzyme involved in cell wall modifications in the peel of a number of fruit. Our results suggest that cell wall hydrolases in tropical fruit may have additional useful roles in biotransforming VOSCs.
- Tapp, Edward J.,Cummins, Ian,Brassington, David,Edwards, Robert
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experimental part
p. 6623 - 6630
(2010/04/06)
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- A continuous-flow system for high-precision kinetics using small volumes
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A generally applicable continuous-flow kinetic analysis system that gives data of a precision high enough to measure small kinetic isotope effects for enzymatic and nonenzymatic reactions is described. It employs commercially available components that are readily assembled into an apparatus that is easy to use. It operates under laminar flow conditions, which requires that the time between the initiation of the reaction in the mixer and the observation be long enough that molecular diffusion can effect a symmetrization of the concentration profile that results from a thin plug of reagents introduced at the mixer. The analysis of a second-order irreversible reaction under pseudo-first-order conditions is presented. The Yersinia pestis protein tyrosine phosphatase catalyzed hydrolysis of p-nitrophenyl phosphate is characterized with the system, and a proton inventory on kcat is presented.
- Zhou, Xianzhi,Medhekar, Rohit,Toney, Michael D.
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p. 3681 - 3687
(2007/10/03)
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- A kinetic approach to characterize the electrostatic environments of thiol groups in proteins
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In this study, we synthesized a zwitterionic DTNB derivative, 5-(2- aminoethyl)-dithio-2-nitrobenzoate (ADNB), and characterized its reactions with several cationic, anionic, and neutral thiols. Reactions with ADNB, unlike those with DTNB, are relatively insensitive to electrostatic environments and ionic strengths. At relatively low ionic strength, rate ratios, k(ADNB)/k(DTNB), varied from 0.22 for reactions with low-molecular- weight cationic thiols to 3.0 for those with low-molecular-weight anionic thiols. A k(ADNB)/k(DTNB) ratio of ~200 for Cys-34 of BSA appears to reflect a very anionic environment. k(ADNB)/k(DTNB) ratios of ~6 and ~1, respectively, for canine and equine serum albumins, which have Glu-82 → Asp and Glu-82 → Ala substitutions suggest Glu-82 is the most important anionic residues affecting the reactivity of Cys-34 in BSA. k(ADNB)/k(DTNB) ratios appear to be useful for characterizing electrostatic environments of thiol groups in proteins.
- Zhang, Hao,Le, Min,Means, Gary E.
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p. 356 - 364
(2007/10/03)
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- Toxin-targeted design for anticancer therapy. I: Synthesis and biological evaluation of new thioimidate heterobifunctional reagents
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In an effort to obtain a more potent and specific immunotoxin for cancer therapy, we designed a series of heterobifunctional linkers characterized by a thioimidate group linked to a S-acetyl thiol (4, 5) or substituted aryldithio group (6-10). These ligands were synthesized by a Pinner-type process from the corresponding nitrile derivatives obtained by thiol- disulphide exchange reaction, reaction with substituted benzene-sulphenyl chloride, or other known procedures. To check the reagent of choice for immunoconjugate preparation, we studied thioldisulphide exchange kinetics between the intermediate nitrile derivatives and cysteine. Among the tested aryldithio derivatives (6-10), we selected ethyl 3-(4-carboxamido- phenyldithio)propionthioimidate (CDPT, 9) for further studies. By analyzing the rate of incorporation of the linkers 4, 5, and 9 in a model immunoglobulin G protein, we found similar results with CDPT 9 and ethyl S- acetyl 3-mercaptopropionthioimidate ester hydrochloride (AMPT, 5) because both reagents showed a linear correlation between the number of introduced thiol groups and factors such as time and protein and reagent concentrations. Comparison of the two acetylthio-derivative ligands 4 and 5 showed that AMPT 5 was more stable toward deacetylation than ethyl S-acetyl 2- mercaptopropionthioimidate ester hydrochloride (AMAT, 4). By comparing the kinetic and biological parameters of seven new thioimidate linkers, we found that two of these (CDPT and AMPT) could be superior ligands for protein- protein conjugation. They offer advantages over the commercially available compounds, such as minimal perturbation of the protein structure, controlled reactivity, and good stability.
- Delprino,Giacomotti,Dosio,Brusa,Ceruti,Grosa,Cattel
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p. 506 - 512
(2007/10/02)
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- Elimination reactions of β-Cyano Thioethers: Evidence for a Carbanion Intermediate and a Change in Rate-Limiting Step
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The addition reactions of thiol anions to form adducts with acrylonitrile (1), 1-chloroacrylonitrile (2), and fumaronitrile (3) and the corresponding elimination reactions were examined in aqueous solution, generally containing 8.3percent Me2SO at 25 deg C.Deuterium exchange into the methanethiol and thiosalicylate adducts of 1 is faster than elimination.Deuterium exchange causes biphasic kinetics for elimination reactions in D2O of the p-nitrothiophenol, but not of the pentafluorothiophenol, adducts 1 and 2.The kinetic solvent deuterium isotope effects of knHOH/knDOD = 2.0 for addition of thiosalicylate to form 3 and 1.1-1.2 for addition of β-mercaptoethanol and thioacetic acid anions to form 1 are smaller than the product discrimination isotope effects of kH/kD = 3.2, 2.8 and 3.2 for these reactions.These differences show that the reactions proceed through a carbanion intermediate that is protonated faster than it expels basic thiol anions.These results exclude a concerted mechanism for addition-elimination with a concurrent, separate exchange reaction.The solvent kinetic deuterium isotope effect is 3.9 for the addition of thionitrobenzoate dianion to form 3.Buffer catalysis of elimination becomes more significant with more acidic leaving groups and is larger for 3 than for 1 with a given leaving group.The results show that the rate-limiting step changes from addition-elimination of the thiol anion to proton transfer with decreasing pKa of the thiol; the same change is favored by addition of CN to the α-position for a given thiol.The effect of the α-CN group is attributed to conjugation with the developing double bond in the transition state for elimination.The Broensted slope is β = 0.90 for rate-limiting deprotonation of the pentafluorothiophenol adduct 3 and Broensted-type plots against the pKa of the leaving group have slopes of β1g = -0.25 and -0.54 for predominantly rate-limiting deprotonation and leaving group expulsion, respectively.
- Fishbein, James C.,Jencks, William P.
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p. 5075 - 5086
(2007/10/02)
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- A KINETIC STUDY OF THE THIOL-DISULFIDE EXCHANGE REACTION BETWEEN AMINOTHIOLS AND 5,5'-DITHIOBIS(2-NITROBENZOIC ACID)
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The rate constants for the thiol-disulfide exchange, kSH, and for the thiolate ion-disulfide exchange, ks-, between biologically relevant aminothiols, i.e., cysteine, cysteamine and penicillamine, and 5,5'-dithiobis(2-nitrobenzoic acid), Ellman's reagent, in aqueous solutions were determined by a spectrophotometric method.The apparent rate constant, k1', increases with increase of pH under the experimental conditions used.The value of ks-, approximately 2x1E5 M-1s-1 except for penicillamine, appears to be independent of the pKa (SH), and kSH is three orders of magnitude smaller than ks-.The thiolate ion-disulfide exchange in penicillamine is one order of magnitude slower than in the other aminothiols.The results of the kinetic study indicate that one of the sulfur atoms of disulfide is attacked by the thiolate anion with subsequent cleavage of the S-S bond.Keywords: Ellman's reagent; thiol-disulfide exchange; rate constant; cysteine; cysteamine; penicillamine; aminothiol
- Ozawa, Toshihiko,Hanaki, Akira
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p. 1101 - 1105
(2007/10/02)
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