RESUMO
Histamine dehydrogenase from the gram-negative bacterium Rhizobium sp. 4-9 (HaDHR) is a member of a small family of dehydrogenases containing a covalently attached FMN, and the only member so far identified to date that does not exhibit substrate inhibition. In this study, we present the 2.1 Å resolution crystal structure of HaDHR. This new structure allowed for the identification of the internal electron transfer pathway to abiological ferrocene-based mediators. Alanine 437 was identified as the exit point of electrons from the Fe4S4 cluster. The enzyme was modified with a Ser436Cys mutation to facilitate covalent attachment of a ferrocene moiety. When modified with Fc-maleimide, this new construct demonstrated direct electron transfer from the enzyme to a gold electrode in a histamine concentration-dependent manner without the need for any additional electron mediators.
Assuntos
Elétrons , Rhizobium , Metalocenos , Transporte de Elétrons , OxidantesRESUMO
Demand exists for a nicotine oxidase enzyme with high catalytic efficiency for a variety of applications including the in vivo detection of nicotine, therapeutic enzymatic blockade of nicotine from the CNS, and inactivation of toxic industrial wastes generated in the manufacture of tobacco products. Nicotine oxidase enzymes identified to date suffer from low efficiency, exhibiting either a high kcat or low Km, but not both. Here we present the crystal structure of the (S)-6-hydroxy-nicotine oxidase from Shinella sp HZN7 (NctB), an enzyme that oxidizes (S)-nicotine with a high kcat (>1 s-1), that possesses remarkable structural and sequence similarity to an enzyme with a nanomolar Km for (S)-nicotine, the (S)-nicotine oxidase from Pseudomonas putidia strain S16 (NicA2). Based on a comparison of our NctB structure and the previously published crystal structure of NicA2, we successfully employed a rational design approach to increase the rate of oxidative turnover of the NicA2 enzyme by â¼25% (0.011 s-1 to 0.014 s-1), and reduce the Km of the NctB protein by approximately 34% (940 µM-622 µM). While modest, these results are a step towards engineering a nicotine oxidase with kinetic parameters that fulfill the functional requirements of biosensing, waste remediation, and therapeutic applications.
Assuntos
Proteínas de Bactérias , Nicotina , Proteínas de Bactérias/química , Cinética , Nicotina/metabolismo , Oxirredução , PseudomonasRESUMO
Controlling enzyme orientation and location on surfaces is a critical step for their successful deployment in diverse applications from biosensors to lab-on-a-chip devices. Functional activity of the enzymes on the surface will largely depend on the spatial arrangement and orientation. Solid binding peptides have been proven to offer versatility for immobilization of biomolecules on inorganic materials including metals, oxides, and minerals. Previously, we demonstrated the utility of a gold binding peptide genetically incorporated into the enzyme putrescine oxidase (PutOx-AuBP), enabling self-enzyme assembly on gold substrates. PutOx is an attractive biocatalyst among flavin oxidases, using molecular oxygen as an electron acceptor without requiring a dissociable coenzyme. Here, we explore the selective self-assembly of this enzyme on a range of surfaces using atomic force microscopy (AFM) along with the assessment of functional activity. This work probes the differences in surface coverage, distribution, size, shape, and activity of PutOx-AuBP in comparison to those of native putrescine oxidase (PutOx) on multiple surfaces to provide insight for material-selective enzymatic assembly. Surfaces investigated include metal (templated-stripped gold (TSG)), oxide (native SiO2 on Si(111)), minerals (mica and graphite), and self-assembled monolayers (SAMs) with a range of hydrophobicity and charge. Supported by both the coverage and the dimensions of immobilized enzymes, our results indicate that of the surfaces investigated, material-selective binding takes place with orientation control only for PutOx-AuBP onto the TSG substrate. These differences are consistent with the measurements of surface-bound enzymatic activities. Substrate-dependent differences observed indicate significant variations in enzyme-surface interactions ranging from peptide-directed self-assembly to enzyme aggregation. The implications of this study provide insight for the fabrication of enzymatic patterns directed by self-assembling peptide tags onto localized surface regions. Enabling functional enzyme-based nanoscale materials offers a fascinating path for utilization of sustainable biocatalysts integrated into multiscale devices.
Assuntos
Ouro , Dióxido de Silício , Enzimas Imobilizadas , Oxirredutases atuantes sobre Doadores de Grupo CH-NH , Peptídeos , Propriedades de SuperfícieRESUMO
RNA interference (RNAi) offers the potential to treat disease at the earliest onset by selectively turning off the expression of target genes, such as intracellular oncogenes that drive cancer growth. However, the development of RNAi therapeutics as anti-cancer drugs has been limited by both a lack of efficient and target cell-specific delivery systems and the necessity to overcome numerous intracellular barriers, including serum/lysosomal instability, cell membrane impermeability, and limited endosomal escape. Here, we combine two technologies to achieve posttranscriptional gene silencing in tumor cells: Centyrins, alternative scaffold proteins binding plasma membrane receptors for targeted delivery, and small interfering RNAs (siRNAs), chemically modified for high metabolic stability and potency. An EGFR Centyrin known to internalize in EGFR-positive tumor cells was site-specifically conjugated to a beta-catenin (CTNNb1) siRNA and found to drive potent and specific target knockdown by free uptake in cell culture and in mice inoculated with A431 tumor xenografts (EGFR amplified). The generalizability of this approach was further demonstrated with Centyrins targeting multiple receptors (e.g., BCMA, PSMA, and EpCAM) and siRNAs targeting multiple genes (e.g., CD68, KLKb1, and SSB1). Moreover, by installing multiple conjugation handles, two different siRNAs were fused to a single Centyrin, and the conjugate was shown to simultaneously silence two different targets. Finally, by specifically pairing EpCAM-binding Centyrins that exhibited optimized internalization profiles, we present data showing that an EpCAM Centyrin CTNNb1 siRNA conjugate suppressed tumor cell growth of a colorectal cancer cell line containing an APC mutation but not cells with normal CTNNb1 signaling. Overall, these data demonstrate the potential of Centyrin-siRNA conjugates to target cancer cells and silence oncogenes, paving the way to a new class of anticancer drugs.
Assuntos
Técnicas de Transferência de Genes , Interferência de RNA , RNA Interferente Pequeno/genética , Animais , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Inativação Gênica , Genes erbB-1 , Terapia Genética , Humanos , Ligantes , Camundongos , RNA Mensageiro , RNA Interferente Pequeno/administração & dosagem , Tenascina/genética , Ensaios Antitumorais Modelo de Xenoenxerto , beta Catenina/genéticaRESUMO
Flavin oxidases are valuable biocatalysts for the oxidative synthesis of a wide range of compounds, while at the same time reduce oxygen to hydrogen peroxide. Compared to other redox enzymes, their ability to use molecular oxygen as an electron acceptor offers a relatively simple system that does not require a dissociable coenzyme. As such, they are attractive targets for adaptation as cost-effective biosensor elements. Their functional immobilization on surfaces offers unique opportunities to expand their utilization for a wide range of applications. Genetically engineered peptides have been demonstrated as enablers of the functional assembly of biomolecules at solid material interfaces. Once identified as having a high affinity for the material of interest, these peptides can provide a single step bioassembly process with orientation control, a critical parameter for functional immobilization of the enzymes. In this study, for the first time, we explored the bioassembly of a putrescine oxidase enzyme using a gold binding peptide tag. The enzyme was genetically engineered to incorporate a gold binding peptide with an expectation of an effective display of the peptide tag to interact with the gold surface. In this work, the functional activity and expression were investigated, along with the selectivity of the binding of the peptide-tagged enzyme. The fusion enzyme was characterized using multiple techniques, including protein electrophoresis, enzyme activity, and microscopy and spectroscopic methods, to verify the functional expression of the tagged protein with near-native activity. Binding studies using quartz crystal microbalance (QCM), nanoparticle binding studies, and atomic force microscopy studies were used to address the selectivity of the binding through the peptide tag. Surface binding AFM studies show that the binding was selective for gold. Quartz crystal microbalance studies show a strong increase in the affinity of the peptide-tagged protein over the native enzyme, while activity assays of protein bound to nanoparticles provide evidence that the enzyme retained catalytic activity when immobilized. In addition to showing selectivity, AFM images show significant differences in the height of the molecules when immobilized through the peptide tag compared to immobilization of the native enzyme, indicating differences in orientation of the bound enzyme when attached via the affinity tag. Controlling the orientation of surface-immobilized enzymes would further improve their enzymatic activity and impact diverse applications, including oxidative biocatalysis, biosensors, biochips, and biofuel production.
Assuntos
Técnicas Biossensoriais , Enzimas Imobilizadas , Ouro , Oxirredutases atuantes sobre Doadores de Grupo CH-NH , PeptídeosRESUMO
The enzyme 6-Hydroxy-l-Nicotine oxidase (HLNO) is a flavin-dependent enzyme that catalyzes the first step in the pyridine pathway of oxidation of nicotine as a source of energy and nitrogen in several bacteria. Recombinant Arthrobacter nicotinovorans HLNO also catalyzes oxidation of (s)-nicotine at a low but measurable rate (Fitzpatrick et al., 2016, Biochemistry 55, 697-703). Rational design and bioinformatics approaches, based on the known high-resolution structure of this enzyme (RCSB: 3NG7), were employed to further enhance the catalytic turnover and stability of the enzyme using (S)-nicotine as substrate. The active site residue Tyr311 forms a hydrogen bond with the hydroxyl group of (S)-6-OH-nicotine within the catalytic pocket. Its replacement by a tryptophan residue reduced the kcat for (S)-6-OH-nicotine by more than 6-fold and increased ~1.5-fold. Combining this mutation with two surface mutations that were predicted to enhance enzyme stability, further increased the kcat for nicotine resulting in a comparatively robust oxidation of (s)-nicotine (kcat >1 s-1) at 37 °C, at the same time reducing the specificity for (S)-OH-nicotine (kcat/KM) by more than 100-fold and increasing that for (S)-nicotine by more than 2-fold. Interestingly, adding a maltose-binding protein (MBP) tag onto the N-terminus of HLNO markedly increased the thermal stability of the enzyme, extending the half-life at 37 °C from ~2 h to ~22 h. This effect was due almost entirely to increased FAD retention, an observation that may prove useful to improve flavin retention in other flavin-dependent monoamine oxidases.
Assuntos
Proteínas de Bactérias , Micrococcaceae , Mutação de Sentido Incorreto , Nicotina/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-NH , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Micrococcaceae/enzimologia , Micrococcaceae/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Especificidade por Substrato/genéticaRESUMO
The methionine sulfoxide reductase (Msr) system is known for its function in reducing protein-methionine sulfoxide to methionine. Recently, we showed that one member of the Msr system, MsrA, is involved in the ubiquitination-like process in Archaea. Here, the mammalian MsrA is demonstrated to mediate the ubiquitination of the 14-3-3 zeta protein and to promote the binding of 14-3-3 proteins to alpha synuclein in brain. MsrA was also found to enhance the ubiquitination and phosphorylation of Ser129 of alpha synuclein in brain. Furthermore, we demonstrate that, similarly to the archaeal MsrA, the mammalian MsrA can compete for capturing ubiquitin using the same active site it contains for methionine sulfoxide binding. Based on our previous observations showing that MsrA knockout mice have elevated expression levels of dopamine and 14-3-3 zeta and our current data, we propose that MsrA-dependent 14-3-3 zeta ubiquitination affects the regulation of alpha synuclein degradation and dopamine synthesis in the brain.
Assuntos
Proteínas 14-3-3/genética , Encéfalo/metabolismo , Metionina Sulfóxido Redutases/genética , Processamento de Proteína Pós-Traducional , Ubiquitina/genética , alfa-Sinucleína/genética , Proteínas 14-3-3/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Ligação Competitiva , Química Encefálica , Dopamina/biossíntese , Lisina/metabolismo , Metionina/análogos & derivados , Metionina/metabolismo , Metionina Sulfóxido Redutases/deficiência , Camundongos , Camundongos Knockout , Fosforilação , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Serina/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , alfa-Sinucleína/metabolismoRESUMO
The wild-type p53-induced phosphatase 1 (WIP1) is a serine/threonine phosphatase that negatively regulates multiple proteins involved in DNA damage response including p53, CHK2, Histone H2AX, and ATM, and it has been shown to be overexpressed or amplified in human cancers including breast and ovarian cancers. We examined WIP1 mRNA levels across multiple tumor types and found the highest levels in breast cancer, leukemia, medulloblastoma and neuroblastoma. Neuroblastoma is an exclusively TP53 wild type tumor at diagnosis and inhibition of p53 is required for tumorigenesis. Neuroblastomas in particular have previously been shown to have 17q amplification, harboring the WIP1 (PPM1D) gene and associated with poor clinical outcome. We therefore sought to determine whether inhibiting WIP1 with a selective antagonist, GSK2830371, can attenuate neuroblastoma cell growth through reactivation of p53 mediated tumor suppression. Neuroblastoma cell lines with wild-type TP53 alleles were highly sensitive to GSK2830371 treatment, while cell lines with mutant TP53 were resistant to GSK2830371. The majority of tested neuroblastoma cell lines with copy number gains of the PPM1D locus were also TP53 wild-type and sensitive to GSK2830371A; in contrast cell lines with no copy gain of PPM1D were mixed in their sensitivity to WIP1 inhibition, with the primary determinant being TP53 mutational status. Since WIP1 is involved in the cellular response to DNA damage and drugs used in neuroblastoma treatment induce apoptosis through DNA damage, we sought to determine whether GSK2830371 could act synergistically with standard of care chemotherapeutics. Treatment of wild-type TP53 neuroblastoma cell lines with both GSK2830371 and either doxorubicin or carboplatin resulted in enhanced cell death, mediated through caspase 3/7 induction, as compared to either agent alone. Our data suggests that WIP1 inhibition represents a novel therapeutic approach to neuroblastoma that could be integrated with current chemotherapeutic approaches.
Assuntos
Aminopiridinas/farmacologia , Dipeptídeos/farmacologia , Mutação , Neuroblastoma/tratamento farmacológico , Fosfoproteínas Fosfatases/antagonistas & inibidores , Alelos , Linhagem Celular Tumoral , Feminino , Loci Gênicos , Humanos , Masculino , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Proteína Fosfatase 2C , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
The spectroscopic, electrochemical and coreactant electrogenerated chemiluminescence (ECL) properties of Ir(ppy)3 (where ppy = 2-phenylpyridine) have been obtained in aqueous buffered (KH2PO4), 50 : 50 (v/v) acetonitrile-aqueous buffered (MeCN-KH2PO4) and 30% trifluoroethanol (TFE) solutions. Tri-n-propylamine was used as the oxidative-reductive ECL coreactant. The photoluminescence (PL) efficiency (Ïem) of Ir(ppy)3 in TFE (Ïem ≈ 0.029) was slightly higher than in 50 : 50 MeCN-KH2PO4 (Ïem ≈ 0.0021) and water (Ïem ≈ 0.00016) compared to a Ru(bpy)32+ standard solution in water (Φem ≈ 0.042). PL and ECL emission spectra were nearly identical in all three solvents, with dual emission maxima at 510 and 530 nm. The similarity between the ECL and PL spectra indicate that the same excited state is probably formed in both experiments. ECL efficiencies (Ïecl) in 30% TFE solution (Ïecl = 0.0098) were higher than aqueous solution (Ïecl = 0.00092) system yet lower than a 50% MeCN-KH2PO4 solution (Ïecl = 0.0091).
Assuntos
Acetonitrilas/química , Técnicas Eletroquímicas , Luminescência , Compostos Organometálicos/análise , Trifluoretanol/química , Água/químicaRESUMO
In the search for uranium-based ionic liquids, tris(N,N-dialkyldithiocarbamato)uranylates have been synthesized as salts of the 1-butyl-3-methylimidazolium (C4mim) cation. As dithiocarbamate ligands binding to the UO2(2+) unit, tetra-, penta-, hexa-, and heptamethylenedithiocarbamates, N,N-diethyldithiocarbamate, N-methyl-N-propyldithiocarbamate, N-ethyl-N-propyldithiocarbamate, and N-methyl-N-butyldithiocarbamate have been explored. X-ray single-crystal diffraction allowed unambiguous structural characterization of all compounds except N-methyl-N-butyldithiocarbamate, which is obtained as a glassy material only. In addition, powder X-ray diffraction as well as vibrational and UV/Vis spectroscopy, supported by computational methods, were used to characterize the products. Differential scanning calorimetry was employed to investigate the phase-transition behavior depending on the N,N-dialkyldithiocarbamato ligand with the aim to establish structure-property relationships regarding the ionic liquid formation capability. Compounds with the least symmetric N,N-dialkyldithiocarbamato ligand and hence the least symmetric anions, tris(N-methyl-N-propyldithiocarbamato)uranylate, tris(N-ethyl-N-propyldithiocarbamato)uranylate, and tris(N-methyl-N-butyldithiocarbamato)uranylate, lead to the formation of (room-temperature) ionic liquids, which confirms that low-symmetry ions are indeed suitable to suppress crystallization. These materials combine low melting points, stable complex formation, and hydrophobicity and are therefore excellent candidates for nuclear fuel purification and recovery.
RESUMO
Although therapeutic interventions of signal-transduction cascades with targeted kinase inhibitors are a well-established strategy, drug-discovery efforts to identify targeted phosphatase inhibitors have proven challenging. Herein we report a series of allosteric, small-molecule inhibitors of wild-type p53-induced phosphatase (Wip1), an oncogenic phosphatase common to multiple cancers. Compound binding to Wip1 is dependent on a 'flap' subdomain located near the Wip1 catalytic site that renders Wip1 structurally divergent from other members of the protein phosphatase 2C (PP2C) family and that thereby confers selectivity for Wip1 over other phosphatases. Treatment of tumor cells with the inhibitor GSK2830371 increases phosphorylation of Wip1 substrates and causes growth inhibition in both hematopoietic tumor cell lines and Wip1-amplified breast tumor cells harboring wild-type TP53. Oral administration of Wip1 inhibitors in mice results in expected pharmacodynamic effects and causes inhibition of lymphoma xenograft growth. To our knowledge, GSK2830371 is the first orally active, allosteric inhibitor of Wip1 phosphatase.
Assuntos
Aminopiridinas/química , Dipeptídeos/química , Inibidores Enzimáticos/farmacologia , Fosfoproteínas Fosfatases/antagonistas & inibidores , Administração Oral , Regulação Alostérica , Motivos de Aminoácidos , Aminopiridinas/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Domínio Catalítico , Linhagem Celular Tumoral , Dipeptídeos/farmacologia , Modelos Animais de Doenças , Ensaios de Seleção de Medicamentos Antitumorais , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/química , Feminino , Xenoenxertos , Humanos , Camundongos , Camundongos SCID , Modelos Biológicos , Neoplasias , Proteína Fosfatase 2CRESUMO
The E3 ubiquitin ligase activity of the parkin protein is implicated in playing a protective role against neurodegenerative disorders including Parkinson's, Huntington's, and Alzheimer's diseases. Parkin has four zinc-containing domains: RING0, RING1, IBR (in-between ring), and RING2. Mutational analysis of full-length parkin suggests that the C-terminal RING2 domain contains the catalytic core. Here, a catalytically competent recombinant RING2 containing an N-terminal GB1 solubility peptide is described. In cell-free in vitro ubiqitination reactions, the RING2 construct catalyzes the transfer of ubiquitin from the E2 enzyme UbcH7 to the attached GB1 tag. This intramolecular autoubiquitination reaction indicates that (a) ubiquitination by RING2 can occur in the absence of other parkin domains and (b) UbcH7 can interact directly with RING2 to transfer its bound ubiquitin. Mass spectrometry identified sites of mono- and diubiquitin attachment to two surface-exposed lysine residues (Lys24 and Lys39) on the GB1 peptide. The sites of diubiquitination involved Lys11 and Lys48 linkages, which have been identified as general signals for proteasome degradation. Cleaving the linker between the GB1 tag and RING2 resulted in loss of ubiquitination activity, indicating that the substrate must be tethered to RING2 for proper presentation to the active site. Atomic absorption spectrometry and selective mutation of zinc ligands indicated that only one of the two zinc binding sites on RING2, the N-terminal site, needs to be occupied by zinc for expression of ubiquitination activity. This is consistent with the hypothesis that the second, C-terminal, zinc binding site on RING2 has a regulatory rather than a catalytic function.
Assuntos
Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Sítios de Ligação/genética , Humanos , Estrutura Terciária de Proteína , Espectrofotometria Atômica , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Zinco/metabolismoRESUMO
The physiological burden created by heat strain and physical exercise, also called thermal-work strain, was quantified for 10 male Marines (age 21.9 ± 2.3 years, height 180.3 ± 5.2 cm, and weight 85.2 ± 10.8 kg) during three dismounted missions in Helmand Province, Afghanistan. Heart rate (HR) and core body temperature (T core) were recorded every 15 seconds (Equivital EQ-01; Hidalgo, Cambridge, United Kingdom) during periods of light, moderate, and heavy work and used to estimate metabolic rate. Meteorological measures, clothing characteristics, anthropometrics, and estimated metabolic rates were used to predict T core for the same missions during March (spring) and July (summer) conditions. Thermal-work strain was quantified from HR and T core values using the Physiological Strain Index (PSI) developed by Moran et al. July PSI and T core values were predicted and not observed due to lack of access to in-theater warfighters at that time. Our methods quantify and compare the predicted and observed thermal-work strain resulting from environment and worn or carried equipment and illustrate that a small increase in ambient temperature and solar load might result in increased thermal-work strain.
Assuntos
Transtornos de Estresse por Calor/fisiopatologia , Militares , Esforço Físico/fisiologia , Adulto , Campanha Afegã de 2001- , Antropometria , Temperatura Corporal , Vestuário , Frequência Cardíaca , Humanos , Modelos Biológicos , Medicina Naval , Estações do Ano , Estados Unidos , Tempo (Meteorologia) , Adulto JovemRESUMO
We report a study on chemiluminescence-based chemical analyses using luminol molecules covalently attached to 10 nm diameter gold nanoparticles (GNPs). Chemiluminescence (CL) has been systematically studied under two schemes by varying the concentrations of luminol-labeled GNPs and [Fe(CN)6](3-) catalyst, respectively. The CL signal of luminol-labeled GNPs is enhanced by 5 to 10 times compared to the bulk luminol solutions of the same concentration. The log-log plot of the CL signal versus the number of luminol-labeled GNPs suspended in a standard 96-well plate shows two characteristic linear curves with distinct slopes across eight orders of magnitude variation in the GNP quantity (from 1.82 × 10(2) to 1.82 × 10(10) GNPs per well). The detection limit represented by the cross-point of these two curves can reach down to ~6.1 × 10(5) GNPs per well (corresponding to 1.0 × 10(-14) M GNP and 2.4 × 10(-11) M equivalent luminol concentration). The attachment of luminol molecules to GNP nano-carriers allows a large amount of luminol to be placed in a greatly reduced volume (or area) toward developing miniaturized CL sensors. We have demonstrated this by preloading dried luminol-labeled GNPs in homemade microwell arrays (with a volume of ~12 µL per well). A linear log-log curve can be obtained across the full range from 1 × 10(3) to 1 × 10(10) GNPs per microwell. The CL signal was detectable with as few as ~1000 GNPs. We have further applied this microwell method to the detection of highly diluted blood samples, in both intact and lysed forms, which releases Fe(3+)-containing hemoglobin to catalyze luminol CL. The lysed blood sample can be detected even after a 10(8) fold dilution (corresponding to ~0.18 cells per well). This ultrasensitive CL detection method may be readily adapted for developing various miniaturized multiplex biosensors for rapid chemical/biochemical analyses.
Assuntos
Ouro/química , Medições Luminescentes/métodos , Luminol/química , Nanopartículas Metálicas/química , Animais , OvinosRESUMO
This work provides the first identification of fish glue from a few micrograms of a 17(th) century artwork sample using an adapted proteomics approach. Fish glue has been widely used as a binder in various art objects such as paintings, manuscripts or polychrome objects however its authentication remains particularly challenging. The lack of information on fish species in genomic and proteomic databases represents a major drawback. A supplementary difficulty is provided by the historical sample features, i.e. a few micrograms of a 17(th) century polychrome object with a multilayered structure. SYPRO® Ruby staining was used as a screening technique to probe the presence of proteins in the sample cross-section. Results revealed the presence of several layers containing proteins among which a thin proteinaceous layer located between the silver leaf and the glaze. This thin layer is described as fish glue coating by historical sources but its composition has not been identified yet. The optimized methodology, based on high resolution mass spectrometry and adapted bioinformatic tools, was successfully applied to 50 µg of a polychromy sample and resulted in the identification of several collagen proteins. Extensive interpretation of data generated by tandem mass spectrometry allowed the identification of proteins from different biological origins. In particular, seven peptides specific to fish collagen proteins were identified for the first time proving the presence of fish glue in the sample and corroborating information found in historical texts dealing with the polychromy technique.
Assuntos
Adesivos/análise , Arte , Peixes , Proteômica , Adesivos/química , Sequência de Aminoácidos , Animais , Colágeno/análise , Colágeno/química , Cor , Medicamentos Falsificados , Proteínas de Peixes/análise , Proteínas de Peixes/química , Dados de Sequência Molecular , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/química , Espectrometria de Massas em TandemRESUMO
Core temperature (CT) in combination with heart rate (HR) can be a good indicator of impending heat exhaustion for occupations involving exposure to heat, heavy workloads, and wearing protective clothing. However, continuously measuring CT in an ambulatory environment is difficult. To address this problem we developed a model to estimate the time course of CT using a series of HR measurements as a leading indicator using a Kalman filter. The model was trained using data from 17 volunteers engaged in a 24 h military field exercise (air temperatures 24-36 °C, and 42%-97% relative humidity and CTs ranging from 36.0-40.0 °C). Validation data from laboratory and field studies (N = 83) encompassing various combinations of temperature, hydration, clothing, and acclimation state were examined using the Bland-Altman limits of agreement (LoA) method. We found our model had an overall bias of -0.03 ± 0.32 °C and that 95% of all CT estimates fall within ±0.63 °C (>52 000 total observations). While the model for estimating CT is not a replacement for direct measurement of CT (literature comparisons of esophageal and rectal methods average LoAs of ±0.58 °C) our results suggest it is accurate enough to provide practical indication of thermal work strain for use in the work place.
Assuntos
Temperatura Corporal/fisiologia , Frequência Cardíaca/fisiologia , Aclimatação , Adulto , Algoritmos , Vestuário , Metabolismo Energético/fisiologia , Exercício Físico/fisiologia , Voluntários Saudáveis , Exaustão por Calor/diagnóstico , Exaustão por Calor/fisiopatologia , Humanos , Masculino , Militares , Modelos Biológicos , Reprodutibilidade dos Testes , Fatores de Tempo , Adulto JovemRESUMO
The vast majority of organisms produce ATP by a membrane-bound rotating protein complex, termed F-ATP synthase. In chloroplasts, the corresponding enzyme generates ATP by using a transmembrane proton gradient generated during photosynthesis, a process releasing high amounts of molecular oxygen as a natural byproduct. Due to its chemical properties, oxygen can be reduced incompletely which generates several highly reactive oxygen species (ROS) that are able to oxidize a broad range of biomolecules. In extension to previous studies it could be shown that ROS dramatically decreased ATP synthesis in situ and affected the CF1 portion in vitro. A conserved cluster of three methionines and a cysteine on the chloroplast γ subunit could be identified by mass spectrometry to be oxidized by ROS. Analysis of amino acid substitutions in a hybrid F1 assembly system indicated that these residues were exclusive catalytic targets for hydrogen peroxide and singlet oxygen, although it could be deduced that additional unknown amino acid targets might be involved in the latter reaction. The cluster was tightly integrated in catalytic turnover since mutants varied in MgATPase rates, stimulation by sulfite and chloroplast-specific γ subunit redox-modulation. Some partial disruptions of the cluster by mutagenesis were dominant over others regarding their effects on catalysis and response to ROS.
Assuntos
Trifosfato de Adenosina/química , ATPases de Cloroplastos Translocadoras de Prótons/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Tilacoides/enzimologia , Sequência de Aminoácidos , ATPases de Cloroplastos Translocadoras de Prótons/química , Peróxido de Hidrogênio/metabolismo , Hidrólise , Dados de Sequência Molecular , Subunidades Proteicas , Oxigênio Singlete/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Sulfitos/farmacologiaRESUMO
The reaction of various [Os(L)(2)(L')](2+) complexes (where L and L' are phenanthroline, diphosphine or diarsine ligands) and organic reducing agents after chemical or electrochemical oxidation of the reactants produces an emission of light corresponding to MLCT transitions. In certain instances, the emission was greater than that of [Ru(bipy)(3)](2+), but the relative signals were dependent on many factors, including reagent concentration, mode of oxidation, reducing agent and the sensitivity of the photodetector over the wavelength range.
RESUMO
The adenylyl cyclase (AC) toxin CyaA from Bordetella pertussis constitutes an important virulence factor for the pathogenesis of whooping cough. CyaA is activated by calmodulin (CaM) and compromises host defense by excessive cAMP production. Hence, pharmacological modulation of the CyaA/CaM interaction could constitute a promising approach to treat whooping cough, provided that interactions of endogenous effector proteins with CaM are not affected. As a first step toward this ambitious goal we examined the interactions of CyaA with wild-type CaM and four CaM mutants in which most methionine residues were replaced by leucine residues and studied the effects of the CaM antagonists calmidazolium, trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7). CyaA/CaM interaction was monitored by CaM-dependent fluorescence resonance energy transfer (FRET) between tryptophan residues in CyaA and 2'-(N-methylanthraniloyl)-3'-deoxy-adenosine 5'-triphosphate and catalytic activity. Comparison of the concentration/response curves of CaM and CaM mutants for FRET and catalysis revealed differences, suggesting a two-step activation mechanism of CyaA by CaM. Even in the absence of CaM, calmidazolium inhibited catalysis, and it did so according to a biphasic function. Trifluoperazine and W-7 did not inhibit FRET or catalysis. In contrast to CyaA, some CaM mutants were more efficacious than CaM at activating membranous AC isoform 1. The slope of CyaA activation by CaM was much steeper than of AC1 activation. Collectively, the two-step activation mechanism of CyaA by CaM offers opportunities for pharmacological intervention. The failure of classic CaM inhibitors to interfere with CyaA/CaM interactions and the different interactions of CaM mutants with CyaA and AC1 point to unique CyaA/CaM interactions.