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PURPOSE: We developed a method to measure the extracellular and intracellular fluid volumes using the kinetics of uric acid in the bodies of Japanese patients undergoing dialysis. In this research, we aimed to assess the prognosis of vascular events using this uric acid kinetic model method. METHODS: We conducted a retrospective cohort study of 1,298 patients who were undergoing hemodialysis or predilution online hemodiafiltration at the end of December 2019 at 13 institutions in Japan. Information on vascular events was acquired in 2020. Vascular event prognosis was defined as the new incidence of one or more of the following four types of vascular events: myocardial infarction, cerebral infarction, cerebral hemorrhage, or limb amputation. We measured the extracellular fluid volume and intracellular fluid volume after dialysis using the uric acid kinetic model method and determined the association between ECV, ICV, and vascular event risk. RESULTS: A high extracellular volume was substantially linked to an increased risk of vascular events. In addition, while a crude analysis revealed that a high intracellular volume was associated with a low risk of vascular events, this was not statistically significant after multifactorial adjustment. This result was partly affected by the low measurement accuracy of the serum urea nitrogen level used for the intracellular volume calculation. CONCLUSIONS: Extracellular volume calculated using the uric acid kinetic model method is a prognostic factor for vascular events in patients undergoing hemodialysis.
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AIM: The aim of this study was to clarify the prevalence and influencing factors of rear seat belt use among pregnant women. METHODS: Questionnaires were given to 1546 pregnant women who visited obstetrics clinics and hospitals for prenatal checkups from October to December 2013. A total of 1494 pregnant women (96.6%) agreed to participate in this study and completed the questionnaire. RESULTS: Fewer than 20% of the rear-seat passengers 'always' used seat belts before and during pregnancy, whereas a third 'never' used a seat belt before or during pregnancy. There was no significant decrease in seat belt use by rear-seat passengers during compared to before pregnancy. Multivariate analysis revealed that age, knowledge of how to use a seat belt during pregnancy, belief in the compulsory use of a rear seat belt and driver behavioral characteristics before pregnancy were associated with rear seat belt use during pregnancy. CONCLUSIONS: The prevalence of fastening seat belts was substantially low. The provision of information regarding proper seat belt use and its role in protecting the fetus may increase use.
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Conducción de Automóvil/estadística & datos numéricos , Conocimientos, Actitudes y Práctica en Salud , Embarazo/estadística & datos numéricos , Cinturones de Seguridad/estadística & datos numéricos , Adulto , Femenino , Humanos , Japón , PrevalenciaRESUMEN
Processive DNA synthesis by the αεθ core of the Escherichia coli Pol III replicase requires it to be bound to the ß2 clamp via a site in the α polymerase subunit. How the ε proofreading exonuclease subunit influences DNA synthesis by α was not previously understood. In this work, bulk assays of DNA replication were used to uncover a non-proofreading activity of ε. Combination of mutagenesis with biophysical studies and single-molecule leading-strand replication assays traced this activity to a novel ß-binding site in ε that, in conjunction with the site in α, maintains a closed state of the αεθ-ß2 replicase in the polymerization mode of DNA synthesis. The ε-ß interaction, selected during evolution to be weak and thus suited for transient disruption to enable access of alternate polymerases and other clamp binding proteins, therefore makes an important contribution to the network of protein-protein interactions that finely tune stability of the replicase on the DNA template in its various conformational states.
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ADN Polimerasa III/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Secuencia de Aminoácidos , Sitios de Unión , Replicación del ADN/genética , Replicación del ADN/fisiología , ADN de Cadena Simple/biosíntesis , ADN de Cadena Simple/metabolismo , Estabilidad de Enzimas/genética , Escherichia coli/genética , Modelos Biológicos , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica/fisiología , Multimerización de Proteína/genética , Multimerización de Proteína/fisiología , Estructura Terciaria de Proteína/fisiología , Homología de Secuencia de AminoácidoRESUMEN
A complex of the three (αεθ) core subunits and the ß2 sliding clamp is responsible for DNA synthesis by Pol III, the Escherichia coli chromosomal DNA replicase. The 1.7 Å crystal structure of a complex between the PHP domain of α (polymerase) and the C-terminal segment of ε (proofreading exonuclease) subunits shows that ε is attached to α at a site far from the polymerase active site. Both α and ε contain clamp-binding motifs (CBMs) that interact simultaneously with ß2 in the polymerization mode of DNA replication by Pol III. Strengthening of both CBMs enables isolation of stable αεθ:ß2 complexes. Nuclear magnetic resonance experiments with reconstituted αεθ:ß2 demonstrate retention of high mobility of a segment of 22 residues in the linker that connects the exonuclease domain of ε with its α-binding segment. In spite of this, small-angle X-ray scattering data show that the isolated complex with strengthened CBMs has a compact, but still flexible, structure. Photo-crosslinking with p-benzoyl-L-phenylalanine incorporated at different sites in the α-PHP domain confirm the conformational variability of the tether. Structural models of the αεθ:ß2 replicase complex with primer-template DNA combine all available structural data.
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ADN Polimerasa III/química , Proteínas de Escherichia coli/química , Exodesoxirribonucleasas/química , Secuencia de Aminoácidos , ADN Polimerasa III/metabolismo , Proteínas de Escherichia coli/metabolismo , Exodesoxirribonucleasas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/química , Pliegue de Proteína , Dominios y Motivos de Interacción de Proteínas , Estructura Terciaria de ProteínaRESUMEN
To explore good anode materials of high safety, high reversible capacity, good cycling, and excellent rate capability, a Li3VO4 microbox with wall thickness of 40 nm was prepared by a one-pot and template-free in situ hydrothermal method. In addition, its composite with graphene nanosheets of about six layers of graphene was achieved. Both of them, especially the Li3VO4/graphene nanosheets composite, show superior electrochemical performance to the formerly reported vanadium-based anode materials. The composite shows a reversible capacity of 223 mAh g(-1) even at 20C (1C = 400 mAh g(-1)). After 500 cycles at 10C there is no evident capacity fading.
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Introduction: Kidney disease of unknown etiology accounts for 1 in 10 adult end-stage renal disease (ESRD) cases worldwide. The aim of this study is to clarify the genetic background of patients with chronic kidney disease (CKD) of unknown etiology who initiated renal replacement therapy (RRT) in adulthood. Methods: This is a multicenter cross-sectional cohort study. Of the 1164 patients who attended 4 dialysis clinics in Japan, we first selected patients who started RRT between the ages of 20 and 49 years. After excluding patients with apparent causes of CKD (e.g., diabetic nephropathy, polycystic kidney disease (PKD) with family history, patients who underwent renal biopsy), 90 patients with CKD of unknown cause were included. The 298 genes associated with CKD were analyzed using capture-based targeted next-generation sequencing. Results: Of the 90 patients, 10 (11.1%) had pathogenic variants in CKD-causing genes and 17 (18.9%) had variant of unknown significance (VUS). Three patients had PKD1 pathogenic variants, and 1 patient had PKD1 and COL4A4 pathogenic variants. In addition, 2 patients were diagnosed with atypical hemolytic uremic syndrome (aHUS) due to C3 or CFHR5. One patient each was diagnosed with Alport syndrome due to COL4A4 and COL4A3 variants, nephronophthisis due to NPHP1 variants, Fabry disease due to GLA variants, and autosomal-dominant tubulointerstitial kidney disease due to UMOD variants. Genetic diagnoses were not concordant with clinical diagnoses, except for patients with PKD1 variant. Conclusion: This largest study on genetic analysis in hemodialysis-dependent adults revealed the presence of undiagnosed inherited kidney diseases.
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Pseudocontact shifts (PCS) from paramagnetic lanthanide ions present powerful long-range structural restraints for structural biology by NMR spectroscopy, but site-specific tagging of proteins with lanthanides remains a challenge, as most of the available lanthanide tags require proteins with single cysteine residues. We show that cyclen-based paramagnetic lanthanide tags can be attached to proteins in a site-specific manner by Cu(I)-catalyzed azide-alkyne cycloaddition to a genetically encoded p-azido-l-phenylalanine residue with a tether that proved sufficiently short and rigid for the observation of PCSs in several proteins. Despite the sterically demanding conditions associated with bulky tags and reactions close to the protein surface, ligation yields consistently above 50% and approaching 100% were obtained with the help of the Cu(I)-stabilizing ligand BTTAA. The yields were high independent of the presence of cysteine residues, thereby avoiding the need for cysteine mutations associated with conventional lanthanide-tagging strategies.
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Elementos de la Serie de los Lantanoides/química , Fenilalanina/análogos & derivados , Proteínas/química , Alquinos/química , Azidas/química , Catálisis , Química Clic , Cobre/química , Ciclamas , Reacción de Cicloadición , Escherichia coli/genética , Compuestos Heterocíclicos/química , Histidina/química , Histidina/genética , Humanos , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Oligopéptidos/química , Oligopéptidos/genética , Fenilalanina/genética , Proteínas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Ubiquitina/química , Ubiquitina/genéticaRESUMEN
PURPOSE: Malnutrition is common among hemodialysis patients. A nutritional index may be important for their prognosis prediction. We examined the use of a modified version of the Controlling Nutritional Status (CONUT) score as a predictor of mortality in hemodialysis patients and investigated whether statin use modified the association. METHODS: We conducted a retrospective cohort study in 1130 adults (mean age 67.7 years, 35.0% women) on maintenance hemodialysis at four outpatient dialysis clinics in Japan. The exposure of interest was modified CONUT scores calculated by one-time data of serum albumin and total cholesterol at baseline, or mean data of these measures using multiple blood test results. The primary outcome was all-cause mortality. We examined the association between modified CONUT groups (low-risk, middle-risk, and high-risk) and mortality using multivariate Cox proportional hazard regression and logistic regression models in overall patients and by statin use. RESULTS: During a median follow-up of 6.6 years, 525 (46.5%) patients died. Analysis using both one-time and mean data showed increased mortality risk in the high-risk modified CONUT score group as compared to the low-risk group (mean data: HR 2.52, 95% CI 1.96, 3.24); when stratified by statin use, the effect appeared stronger among users (mean data: HR 5.84, 95% CI 2.98, 11.44). Strong predictive ability was observed, particularly for mortality risk at 5 year follow-up using mean data (AUC = 0.842). CONCLUSION: Our results suggest that the modified CONUT score may be useful in helping clinicians quickly identify hemodialysis patients with poorer prognosis who may benefit from close monitoring and interventions.
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Inhibidores de Hidroximetilglutaril-CoA Reductasas , Desnutrición , Adulto , Humanos , Femenino , Anciano , Masculino , Estado Nutricional , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , Estudios Retrospectivos , Evaluación Nutricional , Desnutrición/diagnóstico , Desnutrición/etiología , Medición de Riesgo , PronósticoRESUMEN
Using aminoacyl-tRNA synthetase/suppressor tRNA pairs derived from Methanocaldococcus jannaschii, an Escherichia coli cell-free protein production system affords proteins with site-specifically incorporated unnatural amino acids (UAAs) in high yields through the use of optimized amber suppressor tRNA(CUA)(opt) and optimization of reagent concentrations. The efficiency of the cell-free system allows the incorporation of trifluoromethyl-phenylalanine using a polyspecific synthetase evolved previously for p-cyano-phenylalanine, and the incorporation of UAAs at two different sites of the same protein without any re-engineering of the E. coli cells used to make the cell-free extract.
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Alanina/análogos & derivados , Aminoacil-ARNt Sintetasas/metabolismo , Escherichia coli/metabolismo , Methanococcus/enzimología , Nitrilos/metabolismo , Fenilalanina/análogos & derivados , Biosíntesis de Proteínas , Alanina/metabolismo , Aminoacil-ARNt Sintetasas/genética , Sistema Libre de Células , Codón sin Sentido , Escherichia coli/genética , Ingeniería Genética , Fenilalanina/metabolismo , ARN de Transferencia/genética , Supresión GenéticaRESUMEN
Transition metal dissolution in cathode active material for Li-based batteries is a critical aspect that limits the cycle life of these devices. Although several approaches have been proposed to tackle this issue, this detrimental process is not yet overcome. Here, benefitting from the knowledge developed in the semiconductor research field, we apply an epitaxial method to construct an atomic wetting layer of LaTMO3 (TM = Ni, Mn) on a LiNi0.5Mn1.5O4 cathode material. Experimental measurements and theoretical analyses confirm a Stranski-Krastanov growth, where the strained wetting layer forms under thermodynamic equilibrium, and it is self-limited to monoatomic thickness due to the competition between the surface energy and the elastic energy. Being atomically thin and crystallographically connected to the spinel host lattices, the LaTMO3 wetting layer offers long-term suppression of the transition metal dissolution from the cathode without impacting its dynamics. As a result, the epitaxially-engineered cathode material enables improved cycling stability (a capacity retention of about 77% after 1000 cycles at 290 mA g-1) when tested in combination with a graphitic carbon anode and a LiPF6-based non-aqueous electrolyte solution.
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Dementia is associated with a high risk of death and hospitalization among patients on hemodialysis (HD). We retrospectively evaluated the prevalence of mild cognitive impairment (MCI) in 421 patients on maintenance HD across nine facilities and investigated whether decreased handgrip strength was associated with decreased cognitive function. The Montreal Cognitive Assessment-Japan (MoCA-J) score and handgrip strength were measured. The mean age was 69.8 ± 11.2 years, and the median dialysis vintage 74.5 (IQR 30-150) months. The median MoCA-J score was 25 (IQR 21-27), and MCI was confirmed in 245 (58.2%) patients. Both the MoCA-J score and MoCA-J executive score were associated with age, history of cerebrovascular disease (CVA), and handgrip strength after adjustments. We found, among patients on HD aged under 70 years with a history of CVA, a handgrip strength < 90% (25.2 kg in males and 16.2 kg in females) correlated with significantly lower MoCA-J scores. A high prevalence of MCI and decreased handgrip strength were observed in patients on HD. Handgrip strength may be useful for the easy detection of MCI. A decrease in handgrip strength would allow for the early detection of MCI, especially among patients on HD aged under 70 years with a history of CVA.
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Disfunción Cognitiva , Fuerza de la Mano , Anciano , Anciano de 80 o más Años , Disfunción Cognitiva/diagnóstico , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pruebas Neuropsicológicas , Prevalencia , Diálisis Renal/efectos adversos , Estudios RetrospectivosRESUMEN
The two-component dengue virus NS2B-NS3 protease (DEN NS2B-NS3pro) is an established drug target, but inhibitor design is hampered by the lack of a crystal structure of the protease in its fully active form. In solution and without inhibitors, the functionally important C-terminal segment of the NS2B cofactor is dissociated from DEN NS3pro ("open state"), necessitating a large structural change to produce the "closed state" thought to underpin activity. We analyzed the fold of DEN NS2B-NS3pro in solution with and without bound inhibitor by nuclear magnetic resonance (NMR) spectroscopy. Multiple paramagnetic lanthanide tags were attached to different sites to generate pseudocontact shifts (PCS). In the face of severe spectral overlap and broadening of many signals by conformational exchange, methods for assignment of (15)N-HSQC cross-peaks included selective mutation, combinatorial isotope labeling, and comparison of experimental PCSs and PCSs back-calculated for a structural model of the closed conformation built by using the structure of the related West Nile virus (WNV) protease as a template. The PCSs show that, in the presence of a positively charged low-molecular weight inhibitor, the enzyme assumes a closed state that is very similar to the closed state previously observed for the WNV protease. Therefore, a model of the protease built on the closed conformation of the WNV protease is a better template for rational drug design than available crystal structures, at least for positively charged inhibitors. To assess the open state, we created a binding site for a Gd(3+) complex and measured paramagnetic relaxation enhancements. The results show that the specific open conformation displayed in the crystal of DEN NS2B-NS3pro is barely populated in solution. The techniques used open an avenue to the fold analysis of proteins that yield poor NMR spectra, as PCSs from multiple sites in combination with model building generate powerful information even from incompletely assigned (15)N-HSQC spectra.
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Virus del Dengue/enzimología , Inhibidores de Proteasas/farmacología , Serina Endopeptidasas/metabolismo , Proteínas no Estructurales Virales/metabolismo , Sitios de Unión/efectos de los fármacos , Modelos Moleculares , Conformación Molecular , Peso Molecular , Inhibidores de Proteasas/química , Pliegue de Proteína/efectos de los fármacos , Serina Endopeptidasas/química , Proteínas no Estructurales Virales/químicaRESUMEN
The bacterial phosphotriesterases catalyze hydrolysis of the pesticide paraoxon with very fast turnover rates and are thought to be near to their evolutionary limit for this activity. To test whether the naturally evolved turnover rate could be improved through the incorporation of unnatural amino acids and to probe the role of peripheral active site residues in nonchemical steps of the catalytic cycle (substrate binding and product release), we replaced the naturally occurring tyrosine amino acid at position 309 with unnatural L-(7-hydroxycoumarin-4-yl)ethylglycine (Hco) and L-(7-methylcoumarin-4-yl)ethylglycine amino acids, as well as leucine, phenylalanine, and tryptophan. Kinetic analysis suggests that the 7-hydroxyl group of Hco, particularly in its deprotonated state, contributes to an increase in the rate-limiting product release step of substrate turnover as a result of its electrostatic repulsion of the negatively charged 4-nitrophenolate product of paraoxon hydrolysis. The 8-11-fold improvement of this already highly efficient catalyst through a single rationally designed mutation using an unnatural amino acid stands in contrast to the difficulty in improving this native activity through screening hundreds of thousands of mutants with natural amino acids. These results demonstrate that designer amino acids provide easy access to new and valuable sequence and functional space for the engineering and evolution of existing enzyme functions.
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Aminoácidos/metabolismo , Paraoxon/metabolismo , Hidrolasas de Triéster Fosfórico/metabolismo , Aminoácidos/química , Biocatálisis , Activación Enzimática , Escherichia coli/enzimología , Concentración de Iones de Hidrógeno , Hidrólisis , Modelos Moleculares , Estructura Molecular , Paraoxon/química , Hidrolasas de Triéster Fosfórico/químicaRESUMEN
In this work, we synthesized micro-mesoporous graphene1-x (MoS2) x with different compositional ratios via co-reduction of graphite oxide and exfoliated MoS2 platelets. We systematically studied the performance of the micro-mesoporous graphene1-x (MoS2) x as anodes in lithium-ion batteries and sodium-ion batteries. The results show that the specific surface areas of the composites decrease with introducing MoS2. The irreversible capacitance, which is related to the formation of solid electrolyte interphases, also decreases. Besides specific surface area, we found that micropores can benefit the lithiation and sodiation. We demonstrated that a specific charge capacity of 1319.02 mA h g-1 can be achieved at the 50th cycle for the graphene½(MoS2)½ anode in lithium-ion batteries. Possible relationships between such a high specific capacity and the micro-mesoporous structure of the graphene1-x (MoS2) x anode are discussed. This work may shed light on a general strategy for the structural design of electrode materials in lithium-ion batteries and sodium-ion batteries.
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Escherichia coli DNA polymerase III holoenzyme is composed of 10 different subunits linked by noncovalent interactions. The polymerase activity resides in the alpha-subunit. The epsilon-subunit, which contains the proofreading exonuclease site within its N-terminal 185 residues, binds to alpha via a segment of 57 additional C-terminal residues, and also to theta, whose function is less well defined. The present study shows that theta greatly enhances the solubility of epsilon during cell-free synthesis. In addition, synthesis of epsilon in the presence of theta and alpha resulted in a soluble ternary complex that could readily be purified and analyzed by NMR spectroscopy. Cell-free synthesis of epsilon from PCR-amplified DNA coupled with site-directed mutagenesis and selective 15N-labeling provided site-specific assignments of NMR resonances of epsilon that were confirmed by lanthanide-induced pseudocontact shifts. The data show that the proofreading domain of epsilon is connected to alpha via a flexible linker peptide comprising over 20 residues. This distinguishes the alpha : epsilon complex from other proofreading polymerases, which have a more rigid multidomain structure.
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ADN Polimerasa III/química , Proteínas de Escherichia coli/química , Exodesoxirribonucleasas/química , Secuencia de Aminoácidos , Sistema Libre de Células , ADN Polimerasa Dirigida por ADN/química , Datos de Secuencia Molecular , Isótopos de Nitrógeno , Resonancia Magnética Nuclear Biomolecular , Estructura Terciaria de Proteína , Homología de Secuencia de AminoácidoRESUMEN
The high-performance silicon (Si) composite electrodes are being widely developed due to their considerable theoretical capacity. Coating with carbon-based materials is an efficient way to solve the common issues of Si-based materials. Currently, most of the reported strategies are complicated, pollutive, or uneconomic, which hamper their practical applications. Herein, a honeycomb-like Si-based composite was prepared to address these issues via a facile and green reduction approach at room temperature. The pre-anchored Si nanoparticles could be packed and interconnected through a three-dimensional graphene network to further enhance the electrochemical properties of the active materials. As an electrode, this composite shows good rate capabilities upon lithium storage and cycling stability. The continued cycling measurement delivers a -0.049% capacity decay rate per cycle within 600 cycles. A direct comparison further exhibits the obviously improved performance between the as-designed Si-based composite and naked Si, suggesting a potential application of this convenient strategy for other high-performance electrode materials.
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Cell-free protein synthesis provides rapid and economical access to selectively 15N-labelled proteins, greatly facilitating the assignment of 15N-HSQC spectra. While the best yields are usually obtained with buffers containing high concentrations of potassium L-glutamate, preparation of selectively 15N-Glu labelled samples requires non-standard conditions. Among many compounds tested to replace the L-Glu buffer, potassium N-acetyl-L-glutamate and potassium glutarate were found to perform best, delivering high yields for all proteins tested, with preserved selectivity of 15N-Glu labelling. Assessment of amino-transferase activity by combinatorial 15N-labelling revealed that glutarate and N-acetyl-L-glutamate suppress the transfer of the 15N-alpha-amino groups between amino acids less well than the conventional L-Glu buffer. On balance, the glutarate buffer appears most suitable for the preparation of samples containing 15N-L-Glu while the conventional L-Glu buffer is advantageous for all other samples.
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Glutamatos/metabolismo , Glutaratos/metabolismo , Marcaje Isotópico/métodos , Isótopos de Nitrógeno/metabolismo , Proteínas/metabolismo , Aminoácidos/metabolismo , Tampones (Química) , Sistema Libre de Células , Resonancia Magnética Nuclear Biomolecular , Transaminasas/metabolismoRESUMEN
In drug discovery, the occurrence of false positives is a major hurdle in the search for lead compounds that can be developed into drugs. A small-molecular-weight compound that inhibits dengue virus protease at low micromolar levels was identified in a screening campaign. Binding to the enzyme was confirmed by isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR). However, a structure-activity relationship study that ensued did not yield more potent leads. To further characterize the parental compound and its analogues, we developed a high-speed, low-cost, quantitative fluorescence quenching assay. We observed that specific analogues quenched dengue protease fluorescence and showed variation in IC(50) values. In contrast, nonspecifically binding compounds did not quench its fluorescence and showed similar IC(50) values with steep dose-response curves. We validated the assay using single Trp-to-Ala protease mutants and the competitive protease inhibitor aprotinin. Specific compounds detected in the binding assay were further analyzed by competitive ITC, NMR, and surface plasmon resonance, and the assay's utility in comparison with these biophysical methods is discussed. The sensitivity of this assay makes it highly useful for hit finding and validation in drug discovery. Furthermore, the technique can be readily adapted for studying other protein-ligand interactions.
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Calorimetría/métodos , Virus del Dengue/enzimología , Colorantes Fluorescentes/química , Péptido Hidrolasas/metabolismo , Inhibidores de Proteasas/química , Resonancia por Plasmón de Superficie/métodos , Sitios de Unión , Simulación por Computador , Péptido Hidrolasas/química , Inhibidores de Proteasas/farmacología , Serotipificación , Espectrometría de Fluorescencia , Relación Estructura-ActividadRESUMEN
Amoebapore A is a pore-forming protein produced by the pathogenic parasite Entamoeba histolytica, which causes human amoebic dysentery. The pore-forming activity of amoebapore A is regulated by pH-dependent dimerization, a prerequisite for membrane insertion and pore formation. Understanding of these important processes has been hampered by the cytotoxicity of amoebapore A, which prevents the production of this protein in cell-based expression systems. In this study, a protocol for the cell-free production of active recombinant amoebapore A is presented. Protein yields of approximately 500 microg/ml of cell-free reaction were achieved. Recombinant amoebapore A was purified using a three-step procedure. To facilitate the structural characterization of the dimeric and pore forms, we adapted the cell-free system to isotope label amoebapore A for NMR studies. The preliminary assignment of a 2D 1H-15N HSQC spectrum of a uniformly 13C/15N-labeled sample was achieved using a combinatorial selective 15N-labeling approach coupled with available 1H(N) chemical shift data, resulting in the unambiguous assignment of resonances from 55 of the 77 residues. To confirm these results and obtain the full sequence-specific assignments of the 2D 1H-15N HSQC spectrum, a 3D HNCA spectrum was recorded. These assignment data will be used to aid the characterization of amoebapore A dimer formation and membrane insertion.
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Sistema Libre de Células/metabolismo , Entamoeba histolytica/genética , Canales Iónicos/metabolismo , Isótopos de Nitrógeno/metabolismo , Proteínas Protozoarias/metabolismo , Animales , Isótopos de Carbono/química , Isótopos de Carbono/metabolismo , Escherichia coli/química , Canales Iónicos/química , Canales Iónicos/genética , Isótopos de Nitrógeno/química , Resonancia Magnética Nuclear Biomolecular/métodos , Plásmidos/genética , Plásmidos/metabolismo , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
As antenatal environment may influence the development of atopy-predisposing immune response, cord blood cytokine productions may be an important predictor for wheezing. We investigated cord blood cytokines in a prospective birth cohort, intensively monitored for wheezy infant outcome at 1 yr. Cord blood serum samples from 269 children were assayed for interleukin (IL)-1beta, -2, -4 to -8, -10, -12 (p70), -13, and -17, interferon-gamma, tumor necrosis factor-alpha, granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor (G-CSF), monocyte chemotactic protein-1, and macrophage inflammatory protein-1beta. Associations between family histories, antenatal and perinatal factors, cord blood cytokine concentrations, and wheezy infant outcomes (wheezing more than two times) were analyzed. In cord blood sera from 269 children, there were associations between high levels of IL-6, -8 and G-CSF concentrations, and cesarean section. Data at 1 yr were obtained from 213 infants, including 33 wheezy infants. Risk of wheezing was related to gestational age, birth weight, cesarean section, and maternal eczema, but not to bacterial/viral infection during pregnancy, maternal asthma, maternal smoking, or paternal history. High level of cord blood IL-8 concentration had a significant association with wheezy infant outcomes at 1 yr (p = 0.025). By using multivariate logistic regression analysis, birth weight [odds ratio(OR) = 0.998, 95% confidence interval (CI) = 0.997-1.000] and maternal eczema (OR = 5.356, 95% CI = 1.340-21.41), but no other factors, were significant predictors of wheezy infants. Birth weight, gestational age, and maternal history were important risk factors for wheezing in the first year of life. Several cord blood cytokine productions were influenced by cesarean section, and IL-8 may be a predictor for recurrent wheezing at 1 yr.