Página 2 dos resultados de 64 itens digitais encontrados em 0.006 segundos

Duplex Scorpion primers in SNP analysis and FRET applications

Solinas, Antonio; Brown, Lynda J.; McKeen, Catherine; Mellor, John M.; Nicol, Jamie; Thelwell, Nicky; Brown, Tom
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
Publicado em 15/10/2001 EN
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267.35496%
Scorpions are fluorogenic PCR primers with a probe element attached at the 5′-end via a PCR stopper. They are used in real-time amplicon-specific detection of PCR products in homogeneous solution. Two different formats are possible, the ‘stem–loop’ format and the ‘duplex’ format. In both cases the probing mechanism is intramolecular. We have shown that duplex Scorpions are efficient probes in real-time PCR. They give a greater fluorescent signal than stem–loop Scorpions due to the vastly increased separation between fluorophore and quencher in the active form. We have demonstrated their use in allelic discrimination at the W1282X locus of the ABCC7 gene and shown that they can be used in assays where fluorescence resonance energy transfer is required.

In Situ Hybridization of Prochlorococcus and Synechococcus (Marine Cyanobacteria) spp. with rRNA-Targeted Peptide Nucleic Acid Probes

Worden, Alexandra Z.; Chisholm, Sallie W.; Binder, Brian J.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /01/2000 EN
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280.85938%
A simple method for whole-cell hybridization using fluorescently labeled rRNA-targeted peptide nucleic acid (PNA) probes was developed for use in marine cyanobacterial picoplankton. In contrast to established protocols, this method is capable of detecting rRNA in Prochlorococcus, the most abundant unicellular marine cyanobacterium. Because the method avoids the use of alcohol fixation, the chlorophyll content of Prochlorococcus cells is preserved, facilitating the identification of these cells in natural samples. PNA probe-conferred fluorescence was measured flow cytometrically and was always significantly higher than that of the negative control probe, with positive/negative ratio varying between 4 and 10, depending on strain and culture growth conditions. Prochlorococcus cells from open ocean samples were detectable with this method. RNase treatment reduced probe-conferred fluorescence to background levels, demonstrating that this signal was in fact related to the presence of rRNA. In another marine cyanobacterium, Synechococcus, in which both PNA and oligonucleotide probes can be used in whole-cell hybridizations, the magnitude of fluorescence from the former was fivefold higher than that from the latter, although the positive/negative ratio was comparable for both probes. In Synechococcus cells growing at a range of growth rates (and thus having different rRNA concentrations per cell)...

Protein-protein interactions: methods for detection and analysis.

Phizicky, E M; Fields, S
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /03/1995 EN
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278.40715%
The function and activity of a protein are often modulated by other proteins with which it interacts. This review is intended as a practical guide to the analysis of such protein-protein interactions. We discuss biochemical methods such as protein affinity chromatography, affinity blotting, coimmunoprecipitation, and cross-linking; molecular biological methods such as protein probing, the two-hybrid system, and phage display: and genetic methods such as the isolation of extragenic suppressors, synthetic mutants, and unlinked noncomplementing mutants. We next describe how binding affinities can be evaluated by techniques including protein affinity chromatography, sedimentation, gel filtration, fluorescence methods, solid-phase sampling of equilibrium solutions, and surface plasmon resonance. Finally, three examples of well-characterized domains involved in multiple protein-protein interactions are examined. The emphasis of the discussion is on variations in the approaches, concerns in evaluating the results, and advantages and disadvantages of the techniques.

Fluorescence correlation microscopy of cells in the presence of autofluorescence.

Brock, R; Hink, M A; Jovin, T M
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /11/1998 EN
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283.71607%
Fluorescence correlation microscopy (FCM), the combination of fluorescence correlation spectroscopy (FCS) and digital microscopy (Brock and Jovin, 1998. Cell. Mol. Biol. 44:847-856), has been implemented for measuring molecular diffusion and association in living cells with explicit consideration of autocorrelations arising from autofluorescence. Autofluorescence excited at 532 nm colocalizes with mitochondria, has flavin-like spectral characteristics, exhibits relaxation times characteristic for the diffusion of high-molecular-weight proteins, and depends on the incubation conditions of the cells. These time- and location-dependent properties preclude the assignment of universal background parameters. The lower limit for detection of microinjected dextran molecules labeled with the carboxymethylindocyanine dye Cy3 was a few thousand molecules per cell, and the diffusion constant of 1.7 x 10(-7) cm2/s agreed well with values measured with other methods. Based on the fluorescence signal per molecule (fpm) and the molecule number derived from autocorrelation analysis, a new method is devised to define intracellular association states. We conclude that FCM is a powerful, noninvasive method for probing molecular interactions in femtoliter volume elements within defined subcellular locations in living cells.

Analyzing Intracellular Binding and Diffusion with Continuous Fluorescence Photobleaching

Wachsmuth, Malte; Weidemann, Thomas; Müller, Gabriele; Hoffmann-Rohrer, Urs W.; Knoch, Tobias A.; Waldeck, Waldemar; Langowski, Jörg
Fonte: Biophysical Society Publicador: Biophysical Society
Tipo: Artigo de Revista Científica
Publicado em /05/2003 EN
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285.65125%
Transport and binding of molecules to specific sites are necessary for the assembly and function of ordered supramolecular structures in cells. For analyzing these processes in vivo, we have developed a confocal fluorescence fluctuation microscope that allows both imaging of the spatial distribution of fluorescent molecules with confocal laser scanning microscopy and probing their mobility at specific positions in the cell with fluorescence correlation spectroscopy and continuous fluorescence photobleaching (CP). Because fluorescence correlation spectroscopy is restricted to rapidly diffusing particles and CP to slower processes, these two methods complement each other. For the analysis of binding-related contributions to mobility we have derived analytical expressions for the temporal behavior of CP curves from which the bound fraction and/or the dissociation rate or residence time at binding sites, respectively, can be obtained. In experiments, we investigated HeLa cells expressing different fluorescent proteins: Although enhanced green fluorescent protein (EGFP) shows high mobility, fusions of histone H2B with the yellow fluorescent protein are incorporated into chromatin, and these nuclei exhibit the presence of a stably bound and a freely diffusing species. Nonpermanent binding was found for mTTF-I...

Resolution of ligand positions by site-directed tryptophan fluorescence in tear lipocalin.

Gasymov, O. K.; Abduragimov, A. R.; Yusifov, T. N.; Glasgow, B. J.
Fonte: Cold Spring Harbor Laboratory Press Publicador: Cold Spring Harbor Laboratory Press
Tipo: Artigo de Revista Científica
Publicado em /02/2000 EN
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283.71607%
The lipocalin superfamily of proteins functions in the binding and transport of a variety of important hydrophobic molecules. Tear lipocalin is a promiscuous lipid binding member of the family and serves as a paradigm to study the molecular determinants of ligand binding. Conserved regions in the lipocalins, such as the G strand and the F-G loop, may play an important role in ligand binding and delivery. We studied structural changes in the G strand of holo- and apo-tear lipocalin using spectroscopic methods including circular dichroism analysis and site-directed tryptophan fluorescence. Apo-tear lipocalin shows the same general structural characteristics as holo-tear lipocalin including alternating periodicity of a beta-strand, orientation of amino acid residues 105, 103, 101, and 99 facing the cavity, and progressive depth in the cavity from residues 105 to 99. For amino acid residues facing the internal aspect of cavity, the presence of a ligand is associated with blue shifted spectra. The collisional rate constants indicate that these residues are not less exposed to solvent in holo-tear lipocalin than in apo-tear lipocalin. Rather the spectral blue shifts may be accounted for by a ligand induced rigidity in holo-TL. Amino acid residues 94 and 95 are consistent with positions in the F-G loop and show greater exposure to solvent in the holo- than the apo-proteins. These findings are consistent with the general hypothesis that the F-G loop in the holo-proteins of the lipocalin family is available for receptor interactions and delivery of ligands to specific targets. Site-directed tryptophan fluorescence was used in combination with a nitroxide spin labeled fatty acid analog to elucidate dynamic ligand interactions with specific amino acid residues. Collisional quenching constants of the nitroxide spin label provide evidence that at least three amino acids of the G strand residues interact with the ligand. Stern-Volmer plots are inconsistent with a ligand that is held in a static position in the calyx...

Facile SNP detection using bifunctional, cross-linking oligonucleotide probes

Peng, Xiaohua; Greenberg, Marc M.
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
EN
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267.35496%
A facile, sensitive method for detecting specific sequences of oligonucleotides was developed. Detection of DNA sequences with single nucleotide discrimination is achieved by combining the selectivity of hybridization with an efficient cross-linking reaction. Readily synthesized bifunctional oligonucleotide probes containing a modified pyrimidine that is capable of forming interstrand cross-links under mild oxidative conditions internally, and biotin at their 5′-termini were used to discriminate between 16-nt long sites in plasmid DNA that differ by a single nucleotide. The target sequence was detected via fluorescence spectroscopy by utilizing conjugates of avidin and horseradish peroxidase in a microtiter plate assay. The method is able to detect as little as 250 fmol of target without using PCR and exhibits single nucleotide discrimination that approaches 200:1. In principle, this method is capable of probing any target sequence containing a 2′-deoxyadenosine.

Development and Application of Flow-Cytometric Techniques for Analyzing and Sorting Endospore-Forming Clostridia▿ †

Tracy, Bryan P.; Gaida, Stefan M.; Papoutsakis, Eleftherios T.
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
EN
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267.35496%
The study of microbial heterogeneity at the single-cell level is a rapidly growing area of research in microbiology and biotechnology due to its significance in pathogenesis, environmental biology, and industrial biotechnologies. However, the tools available for efficiently and precisely probing such heterogeneity are limited for most bacteria. Here we describe the development and application of flow-cytometric (FC) and fluorescence-assisted cell-sorting techniques for the study of endospore-forming bacteria. We show that by combining FC light scattering (LS) with nucleic acid staining, we can discriminate, quantify, and enrich all sporulation-associated morphologies exhibited by the endospore-forming anaerobe Clostridium acetobutylicum. Using FC LS analysis, we quantitatively show that clostridial cultures commonly perform multiple rounds of sporulation and that sporulation is induced earlier by the overexpression of Spo0A, the master regulator of endospore formers. To further demonstrate the power of our approach, we employed FC LS analysis to generate compelling evidence to challenge the long-accepted view in the field that the clostridial cell form is the solvent-forming phenotype.

Probing mechanisms for enzymatic activity enhancement of organophosphorus hydrolase in functionalized mesoporous silica

Chen, Baowei; Lei, Chenghong; Shin, Yongsoon; Liu, Jun
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
283.71607%
We have previously reported that organophosphorus hydrolase (OPH) can be spontaneously entrapped in functionalized mesoporous silica (FMS) with HOOC-as the functional groups and the entrapped OPH in HOOC-FMS showed enhanced enzyme specific activity. This work is to study the mechanisms that why OPH entrapped in FMS displayed the enhanced activity in views of OPH-FMS interactions using spectroscopic methods. The circular dichroism (CD) spectra show that, comparing to the secondary structure of OPH free in solution, OPH in HOOC-FMS displayed increased α-helix/β-strand transition of OPH with increased OPH loading density. The fluorescence emission spectra of Trp residues were used to assess the tertiary structural changes of the enzyme. There was a 42% increase in fluorescence. This is in agreement with the fact that the fluorescence intensity of OPH was increased accompanying with the increased OPH activity when decreasing urea concentrations in solution. The steady-state anisotropy was increased after OPH entrapping in HOOC-FMS comparing to the free OPH in solution, indicating that protein mobility was reduced upon entrapment. The solvent accessibility of Trp residues of OPH was probed by using acrylamide as a collisional quencher. Trp residues of OPH-FMS had less solvent exposure comparing with free OPH in solution due to its electrostatical binding to HOOC-FMS thereby displaying the increased fluorescence intensity. These results suggest the interactions of OPH with HOOC-FMS resulted in the protein immobilization and a favorable conformational change for OPH in the crowded confinement space and accordingly the enhanced activity.

A benchmark for chromatin binding measurements in live cells

Mazza, Davide; Abernathy, Alice; Golob, Nicole; Morisaki, Tatsuya; McNally, James G.
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
276.21723%
Live-cell measurement of protein binding to chromatin allows probing cellular biochemistry in physiological conditions, which are difficult to mimic in vitro. However, different studies have yielded widely discrepant predictions, and so it remains uncertain how to make the measurements accurately. To establish a benchmark we measured binding of the transcription factor p53 to chromatin by three approaches: fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS) and single-molecule tracking (SMT). Using new procedures to analyze the SMT data and to guide the FRAP and FCS analysis, we show how all three approaches yield similar estimates for both the fraction of p53 molecules bound to chromatin (only about 20%) and the residence time of these bound molecules (∼1.8 s). We also apply these procedures to mutants in p53 chromatin binding. Our results support the model that p53 locates specific sites by first binding at sequence-independent sites.

Validation of Response Function Construction and Probing Heterogeneous Protein Hydration by Intrinsic Tryptophan

Qin, Yangzhong; Chang, Chih-Wei; Wang, Lijuan; Zhong, Dongping
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
EN
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276.21723%
Protein solvation dynamics usually occur on multiple time scales with site specificity and the characterization of such heterogeneous dynamics requires a convenient optical probe. We proposed a tryptophan methodology and with site-directed mutagenesis we can use a tryptophan scan to probe any desirable position around protein surfaces. Here, we report our extended solvation model for construction of response functions for probes such as tryptophan with multiple emission peaks and lifetimes. We show our systematic construction procedure and careful analyses of the possible missing percentage of an initial ultrafast component with the established zero-time emission spectrum and limited temporal resolution through two methods of the direct mapping of femtosecond resolved fluorescence spectra (3D FRES) and the constructed FRES (2D) from the fluorescence transients. We unambiguously validate our extended model with reexamination of solvation dynamics (methanol, water and proteins) using conventional dye coumarin, intrinsic tryptophan and cofactor flavin. Using mutant proteins of GB1, we show again the generality of the powerful probe tryptophan for protein hydration (solvation) and the slowdown of the hydration layer dynamics especially at the water-protein interface. These results justify the necessity of our extended solvation model...

Probing enzymatic activity inside living cells using a nanowire-cell “sandwich”assay

Na, Yu-Ran; Kim, So Yeon; Gaublomme, Jellert T.; Shalek, Alex K.; Jorgolli, Marsela; Park, Hongkun; Yang, Eun Gyeong
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
EN
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278.39738%
Developing a detailed understanding of enzyme function in the context of an intracellular signal transduction pathway requires minimally invasive methods for probing enzyme activity in situ. Here, we describe a new method for monitoring enzyme activity in living cells by sandwiching live cells between two vertical silicon nanowire (NW) arrays. Specifically, we use the first NW array to immobilize the cells, and then present enzymatic substrates intracellularly via the second NW array by utilizing the NWs’ ability to penetrate cellular membranes without affecting cells’ viability or function. This strategy, when coupled with fluorescence microscopy and mass spectrometry, enables intracellular examination of protease, phosphatase and protein kinase activities, demonstrating the assay’s potential in uncovering the physiological roles of various enzymes.

Beyond annexin V: fluorescence response of cellular membranes to apoptosis

Demchenko, Alexander P.
Fonte: Springer Netherlands Publicador: Springer Netherlands
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
286.21277%
Dramatic changes in the structure of cell membranes on apoptosis allow easy, sensitive and non-destructive analysis of this process with the application of fluorescence methods. The strong plasma membrane asymmetry is present in living cells, and its loss on apoptosis is commonly detected with the probes interacting strongly and specifically with phosphatidylserine (PS). This phospholipid becomes exposed to the cell surface, and the application of annexin V labeled with fluorescent dye is presently the most popular tool for its detection. Several methods have been suggested recently that offer important advantages over annexin V assay with the ability to study apoptosis by spectroscopy of cell suspensions, flow cytometry and confocal or two-photon microscopy. The PS exposure marks the integrated changes in the outer leaflet of cell membrane that involve electrostatic potential and hydration, and the attempts are being made to provide direct probing of these changes. This review describes the basic mechanisms underlying the loss of membrane asymmetry during apoptosis and discusses, in comparison with the annexin V-binding assay, the novel fluorescence techniques of detecting apoptosis on cellular membrane level. In more detail we describe the detection method based on smart fluorescent dye F2N12S incorporated into outer leaflet of cell membrane and reporting on apoptotic cell transformation by easily detectable change of the spectral distribution of fluorescent emission. It can be adapted to any assay format.

Mechanistic insights into antibiotic action on the ribosome through single-molecule fluorescence imaging

Wang, Leyi; Wasserman, Michael R.; Feldman, Michael B.; Altman, Roger B.; Blanchard, Scott C.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /12/2011 EN
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276.21723%
Single-molecule fluorescence imaging has provided unprecedented access to the dynamics of ribosome function, revealing transient intermediate states that are critical to ribosome activity. Imaging platforms have now been developed that are capable of probing many hundreds of molecules simultaneously at temporal and spatial resolutions approaching the sub-millisecond time and the sub-nanometer scales. These advances enable both steady- and pre-steady state measurements of individual steps in the translation process as well as processive reactions. The data generated using these methods have yielded new, quantitative structural and kinetic insights into ribosomal activity. They have also shed light on the mechanisms of antibiotics targeting the translation apparatus, revealing features of the structure-function relationship that would be difficult to obtain by other means. This review provides an overview of the types of information that can be obtained using such imaging platforms and a blueprint for using the technique to assess how small-molecule antibiotics alter macromolecular functions.

Optogenetic Probing and Manipulation of the Calyx-Type Presynaptic Terminal in the Embryonic Chick Ciliary Ganglion

Egawa, Ryo; Hososhima, Shoko; Hou, Xubin; Katow, Hidetaka; Ishizuka, Toru; Nakamura, Harukazu; Yawo, Hiromu
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 21/03/2013 EN
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278.39738%
The calyx-type synapse of chick ciliary ganglion (CG) has been intensively studied for decades as a model system for the synaptic development, morphology and physiology. Despite recent advances in optogenetics probing and/or manipulation of the elementary steps of the transmitter release such as membrane depolarization and Ca2+ elevation, the current gene-manipulating methods are not suitable for targeting specifically the calyx-type presynaptic terminals. Here, we evaluated a method for manipulating the molecular and functional organization of the presynaptic terminals of this model synapse. We transfected progenitors of the Edinger-Westphal (EW) nucleus neurons with an EGFP expression vector by in ovo electroporation at embryonic day 2 (E2) and examined the CG at E8–14. We found that dozens of the calyx-type presynaptic terminals and axons were selectively labeled with EGFP fluorescence. When a Brainbow construct containing the membrane-tethered fluorescent proteins m-CFP, m-YFP and m-RFP, was introduced together with a Cre expression construct, the color coding of each presynaptic axon facilitated discrimination among inter-tangled projections, particularly during the developmental re-organization period of synaptic connections. With the simultaneous expression of one of the chimeric variants of channelrhodopsins...

Methods for quantifying T cell receptor binding affinities and thermodynamics

Piepenbrink, Kurt H.; Gloor, Brian E.; Armstrong, Kathryn M.; Baker, Brian M.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
EN
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272.70836%
αβ T cell receptors (TCRs) recognize peptide antigens bound and presented by class I or class II major histocompatibility complex (MHC) proteins. Recognition of a peptide/MHC complex is required for initiation and propagation of a cellular immune response, as well as the development and maintenance of the T cell repertoire. Here we discuss methods to quantify the affinities and thermodynamics of interactions between soluble ectodomains of TCRs and their peptide/MHC ligands, focusing on titration calorimetry, surface plasmon resonance, and fluorescence anisotropy. As TCRs typically bind ligand with weak-to-moderate affinities, we focus the discussion on means to enhance the accuracy and precision of low affinity measurements. In addition to further elucidating the biology of the T cell mediated immune response, more reliable low affinity measurements will aid with more probing studies with mutants or altered peptides that can help illuminate the physical underpinnings of how TCRs achieve their remarkable recognition properties.

Shedding Light on Protein Folding Landscapes by Single-molecule Fluorescence

Banerjee, Priya R.; Deniz, Ashok A.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 21/02/2014 EN
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284.3748%
Single-molecule (SM) fluorescence methods have been increasingly instrumental in our current understanding of a number of key aspects of protein folding and aggregation landscapes over the past decade. With the advantage of a model free approach and the power of probing multiple subpopulations and stochastic dynamics directly in a heterogeneous structural ensemble, SM methods have emerged as a principle technique for studying complex systems such as intrinsically disordered proteins (IDPs), globular proteins in the unfolded basin and during folding, and early steps of protein aggregation in amyloidogenesis. This review highlights the application of these methods in investigating the free energy landscapes, folding properties and dynamics of individual protein molecules and their complexes, with an emphasis on inherently flexible systems such as IDPs.

Probing murine methyltransfease Dnmt3a interactions with benzo[a]pyrene-modified DNA by fluorescence methods

Minero, Antonio S.; Lukashevich, Olga V.; Cherepanova, Natalia A.; Kolbanovskiy, Alexander; Geacintov, Nicholas E.; Gromova, Elizaveta S.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
EN
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287.0489%
The impact of bulky carcinogen-DNA adducts positioned at or near recognition sites (CpG) of eukaryotic DNA methyltransferases on their catalytic activities is poorly understood. In this work we have employed site-specifically modified 30-mer oligodeoxyribonucleotides containing stereoisomeric benzo[a]pyrene diol epoxide (B[a]PDE)-derived guanine (B[a]PDE-N2-dG) or adenine (B[a]PDE-N6-dA) adducts of different conformations as substrates of catalytic domain of murine Dnmt3a (Dnmt3a-CD). The fluorescence of these lesions was used to examine interactions between Dnmt3a-CD and DNA. In B[a]PDE-DNA•Dnmt3a-CD complexes the intensity of fluorescence of the covalently bound B[a]PDE residues is enhanced relative to the protein-free value when the B[a]PDE is positioned in the minor groove ((+)- and (−)-trans-B[a]PDE-N2-dG adducts in the CpG site) and when it is intercalated on the 5′-side of the CpG site ((+)-trans- B[a]PDE-N6-dA adduct). The fluorescence of B[a]PDE-modified DNA•Dnmt3a-CD complexes exhibits only small changes when the B[a]PDE is intercalated with base displacement in (+)- and (−)-cis- B[a]PDE-N2-dG adducts and without base displacement in the (−)-trans- B[a]PDE-N6-dA adduct. The initial rates of methylation were significantly reduced by the minor groove trans-B[a]PDE-N2-dG adducts regardless of their position in the substrate and by the intercalated cis-B[a]PDE-N2-dG adducts within the CpG site. The observed changes in fluorescence and methylation rates are consistent with the flipping of the target cytosine and a catalytic loop motion within the DNA•Dnmt3a-CD complexes. In the presence of the regulatory factor Dnmt3L...

Review of methods to probe single cell metabolism and bioenergetics

Vasdekis, Andreas E.; Stephanopoulos, Gregory
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
EN
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275.51252%
Single cell investigations have enabled unexpected discoveries, such as the existence of biological noise and phenotypic switching in infection, metabolism and treatment. Herein, we review methods that enable such single cell investigations specific to metabolism and bioenergetics. Firstly, we discuss how to isolate and immobilize individuals from a cell suspension, including both permanent and reversible approaches. We also highlight specific advances in microbiology for its implications in metabolic engineering. Methods for probing single cell physiology and metabolism are subsequently reviewed. The primary focus therein is on dynamic and high-content profiling strategies based on label-free and fluorescence microspectroscopy and microscopy. Non-dynamic approaches, such as mass spectrometry and nuclear magnetic resonance, are also briefly discussed.

Improved estimation of postmortem interval with Cardiac Troponin T and improved analytical methods

Memari, Behnoush
Fonte: FIU Digital Commons Publicador: FIU Digital Commons
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
272.70836%
Accurate knowledge of the time since death, or postmortem interval (PMI), has enormous legal, criminological, and psychological impact. In this study, an investigation was made to determine whether the relationship between the degradation of the human cardiac structure protein Cardiac Troponin T and PMI could be used as an indicator of time since death, thus providing a rapid, high resolution, sensitive, and automated methodology for the determination of PMI. ^ The use of Cardiac Troponin T (cTnT), a protein found in heart tissue, as a selective marker for cardiac muscle damage has shown great promise in the determination of PMI. An optimized conventional immunoassay method was developed to quantify intact and fragmented cTnT. A small sample of cardiac tissue, which is less affected than other tissues by external factors, was taken, homogenized, extracted with magnetic microparticles, separated by SDS-PAGE, and visualized with Western blot by probing with monoclonal antibody against cTnT. This step was followed by labeling and available scanners. This conventional immunoassay provides a proper detection and quantitation of cTnT protein in cardiac tissue as a complex matrix; however, this method does not provide the analyst with immediate results. Therefore...