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Static and dynamic bimolecular fluorescence quenching of porphyrin dendrimers in solution

MATOS, Mauricio S.; HOFKENS, Johan; GEHLEN, Marcelo H.
Fonte: SPRINGER/PLENUM PUBLISHERS Publicador: SPRINGER/PLENUM PUBLISHERS
Tipo: Artigo de Revista Científica
ENG
Relevância na Pesquisa
380.85938%
The fluorescence quenching kinetics of two porphyrin dendrimer series (GnTPPH(2) and GnPZn) by different type of quenchers is reported. The microenvironment surrounding the core in GnPZn was probing by core-quencher interactions using benzimidazole. The dependence of quencher binding constant (K(a) ) on generation indicates the presence of a weak interaction between branches and the core of the porphyrin dendrimer. The similar free volume in dendrimers of third and fourth generation suggests that structural collapse in high generations occurs by packing of the dendrimer peripheral layer. Dynamic fluorescence quenching of the porphyrin core by 1,3-dicyanomethylene-2-methyl-2-pentyl-indan (PDCMI) in GnTPPH(2) is a distance dependent electron transfer process with an exponential attenuation factor beta=0.33 angstrom(-1). The quenching by 1,2-dibromobenzene occurs by diffusion process of the quencher toward to the porphyrin core, and its rate constant is practically independent of dendrimer generation.; FAPESP; Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP); Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq); CNPq

Novel Fluorescence Labeling and High-Throughput Assay Technologies for In Vitro Analysis of Protein Interactions

Doi, Nobuhide; Takashima, Hideaki; Kinjo, Masataka; Sakata, Kyoko; Kawahashi, Yuko; Oishi, Yuko; Oyama, Rieko; Miyamoto-Sato, Etsuko; Sawasaki, Tatsuya; Endo, Yaeta; Yanagawa, Hiroshi
Fonte: Cold Spring Harbor Laboratory Press Publicador: Cold Spring Harbor Laboratory Press
Tipo: Artigo de Revista Científica
Publicado em /03/2002 EN
Relevância na Pesquisa
383.7161%
We developed and tested a simple method for fluorescence labeling and interaction analysis of proteins based on a highly efficient in vitro translation system combined with high-throughput technologies such as microarrays and fluorescence cross-correlation spectroscopy (FCCS). By use of puromycin analogs linked to various fluorophores through a deoxycytidylic acid linker, a single fluorophore can be efficiently incorporated into a protein at the carboxyl terminus during in vitro translation. We confirmed that the resulting fluorescently labeled proteins are useful for probing protein–protein and protein–DNA interactions by means of pulldown assay, DNA microarrays, and FCCS in model experiments. These fluorescence assay systems can be easily extended to highly parallel analysis of protein interactions in studies of functional genomics.

Large Bodies of Mycoplasma and L-Form Organisms1

Kang, K. S.; Casida, L. E.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /03/1967 EN
Relevância na Pesquisa
296.69371%
The large bodies of various Mycoplasma and L-form organisms were studied by ultraviolet fluorescence microscopy of preparations stained with various fluorochromes. Primuline and Thioflavine S specifically stained the outer portion or rim of the large bodies, and the fluorescence characteristics of the stained bodies differed from those for other microorganisms and for spheroplasts and protoplasts. Small granular structures similar in size and morphology to minimal reproductive units were observed within some of the large bodies by phase microscopy and by fluorescence microscopy with acridine orange or Coriphosphine O. Micromanipulation probing of the large bodies revealed their elastic nature; many of the large bodies could be subdivided into two or more smaller circular bodies, each retaining the fluorescence staining properties of the parent body. Under these conditions, however, a few of the large bodies were ruptured, leaving the stainable outer boundary area as a stable residual structure. The large bodies were somewhat resistant to various rigorous treatments normally employed to eliminate viability of Mycoplasma and L-form cultures. Structures similr to large bodies were observed in various natural tissues, and structures resembling large bodies in size...

Characterization of Growing Microorganisms in Soil by Stable Isotope Probing with H218O▿

Schwartz, Egbert
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
384.18152%
A new approach to characterize growing microorganisms in environmental samples based on labeling microbial DNA with H218O is described. To test if sufficient amounts of 18O could be incorporated into DNA to use water as a labeling substrate for stable isotope probing, Escherichia coli DNA was labeled by cultivating bacteria in Luria broth with H218O and labeled DNA was separated from [16O]DNA on a cesium chloride gradient. Soil samples were incubated with H218O for 6, 14, or 21 days, and isopycnic centrifugation of the soil DNA showed the formation of two bands after 6 days and three bands after 14 or 21 days, indicating that 18O can be used in the stable isotope probing of soil samples. DNA extracted from soil incubated for 21 days with H218O was fractionated after isopycnic centrifugation and DNA from 17 subsamples was used in terminal restriction fragment length polymorphism (TRFLP) analysis of bacterial 16S rRNA genes. The TRFLP patterns clustered into three groups that corresponded to the three DNA bands. The fraction of total fluorescence contributed by individual terminal restriction fragments (TRF) to a TRFLP pattern varied across the 17 subsamples so that a TRF was more prominent in only one of the three bands. Labeling soil DNA with H218O allows the identification of newly grown cells. In addition...

Substitution of the use of radioactivity by fluorescence for biochemical studies of RNA

Ying, Bei-Wen; Fourmy, Dominique; Yoshizawa, Satoko
Fonte: Cold Spring Harbor Laboratory Press Publicador: Cold Spring Harbor Laboratory Press
Tipo: Artigo de Revista Científica
Publicado em /11/2007 EN
Relevância na Pesquisa
400.05938%
We present here the use of fluorescent methodologies for structural and functional studies of RNA in place of radioactivity. The methods are highly sensitive and quantitative with the use of an infrared fluorescence imaging system. IRD-700 and IRD-800 labels are used for fluorescence detection. Chemical probing methods are largely used for mapping RNA secondary structure and to monitor ligand interactions and conformational changes involving individual bases of RNA. The new fluorescent primer extension methodology allows simple and fast chemical probing of RNA with high sensitivity. IRD-700 and IRD-800 labeled primers can also be used to monitor protein–RNA interactions by fluorescent mobility shift assays. The speed and ease of these approaches are advantages over prior methods that used hazardous radioisotopes. Structural and biochemical investigations of RNA should benefit from the use of these fluorescent methodologies.

Resolving Genetic Functions within Microbial Populations: In Situ Analyses Using rRNA and mRNA Stable Isotope Probing Coupled with Single-Cell Raman-Fluorescence In Situ Hybridization ▿ †

Huang, Wei E.; Ferguson, Andrew; Singer, Andrew C.; Lawson, Kathryn; Thompson, Ian P.; Kalin, Robert M.; Larkin, Michael J.; Bailey, Mark J.; Whiteley, Andrew S.
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
387.2597%
Prokaryotes represent one-half of the living biomass on Earth, with the vast majority remaining elusive to culture and study within the laboratory. As a result, we lack a basic understanding of the functions that many species perform in the natural world. To address this issue, we developed complementary population and single-cell stable isotope (13C)-linked analyses to determine microbial identity and function in situ. We demonstrated that the use of rRNA/mRNA stable isotope probing (SIP) recovered the key phylogenetic and functional RNAs. This was followed by single-cell physiological analyses of these populations to determine and quantify in situ functions within an aerobic naphthalene-degrading groundwater microbial community. Using these culture-independent approaches, we identified three prokaryote species capable of naphthalene biodegradation within the groundwater system: two taxa were isolated in the laboratory (Pseudomonas fluorescens and Pseudomonas putida), whereas the third eluded culture (an Acidovorax sp.). Using parallel population and single-cell stable isotope technologies, we were able to identify an unculturable Acidovorax sp. which played the key role in naphthalene biodegradation in situ, rather than the culturable naphthalene-biodegrading Pseudomonas sp. isolated from the same groundwater. The Pseudomonas isolates actively degraded naphthalene only at naphthalene concentrations higher than 30 μM. This study demonstrated that unculturable microorganisms could play important roles in biodegradation in the ecosystem. It also showed that the combined RNA SIP-Raman-fluorescence in situ hybridization approach may be a significant tool in resolving ecology...

Probing Nuclear Localization Signal-Importin α Binding Equilibria in Living Cells*

Cardarelli, Francesco; Bizzarri, Ranieri; Serresi, Michela; Albertazzi, Lorenzo; Beltram, Fabio
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
380.85938%
The regulated process of protein import into the nucleus of a eukaryotic cell is mediated by specific nuclear localization signals (NLSs) that are recognized by protein-import receptors. In this study, we present fluorescence-based methods to quantitatively address the physicochemical details of NLS recognition by the receptor protein importin α (Impα) in living cells. First, by combining fluorescence recovery after photobleaching measurements and protein-concentration calibration, we quantitatively define nuclear import saturability and afford an affinity value for NLS-Impα binding. Second, by fluorescence lifetime imaging microscopy, we directly monitor the occurrence of NLS-Impα interaction and measure its effective dissociation constant (KD) in the actual cellular environment. Our kinetic and thermodynamic analyses independently indicate that the subsaturation of Impα with the expressed NLS cargo regulates nuclear import rates in living cells, in contrast to what can be predicted on the basis of available in vitro data. Finally, our experiments also provide evidence for the regulation of nuclear import mediated by the intrasteric importin β-binding domain of Impα and yield the first estimate of its autoinhibition energy in living cells.

Accurate Detection of Low Levels of Fluorescence Emission in Autofluorescent Background: Francisella-Infected Macrophage Cells

Davis, Ryan W.; Timlin, Jerilyn A.; Kaiser, Julia N.; Sinclair, Michael B.; Jones, Howland D.T.; Lane, Todd W.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
386.21277%
Cellular autofluorescence, though ubiquitous when imaging cells and tissues, is often assumed to be small in comparison to the signal of interest. Uniform estimates of autofluorescence intensity obtained from separate control specimens are commonly employed to correct for autofluorescence. While these may be sufficient for high signal-to-background applications, improvements in detector and probe technologies and introduction of spectral imaging microscopes have increased the sensitivity of fluorescence imaging methods, exposing the possibility of effectively probing the low signal-to-background regime. With spectral imaging, reliable monitoring of signals near or even below the noise levels of the microscope is possible if compensation for autofluorescence and background signals can be performed accurately. We demonstrate the importance of accurate autofluorescence modeling and the utility of spectral imaging and multivariate analysis methods using a case study focusing on fluorescence confocal spectral imaging of host-pathogen interactions. In this application fluorescent proteins are produced when Francisella novicida invade host macrophage cells. The resulting analyte signal is spectrally overlapped and typically weaker than the cellular autofluorescence. In addition to discussing the advantages of spectral imaging for following pathogen invasion...

Nanoscale thermal probing

Yue, Yanan; Wang, Xinwei
Fonte: CoAction Publishing Publicador: CoAction Publishing
Tipo: Artigo de Revista Científica
Publicado em 12/03/2012 EN
Relevância na Pesquisa
296.60326%
Nanoscale novel devices have raised the demand for nanoscale thermal characterization that is critical for evaluating the device performance and durability. Achieving nanoscale spatial resolution and high accuracy in temperature measurement is very challenging due to the limitation of measurement pathways. In this review, we discuss four methodologies currently developed in nanoscale surface imaging and temperature measurement. To overcome the restriction of the conventional methods, the scanning thermal microscopy technique is widely used. From the perspective of measuring target, the optical feature size method can be applied by using either Raman or fluorescence thermometry. The near-field optical method that measures nanoscale temperature by focusing the optical field to a nano-sized region provides a non-contact and non-destructive way for nanoscale thermal probing. Although the resistance thermometry based on nano-sized thermal sensors is possible for nanoscale thermal probing, significant effort is still needed to reduce the size of the current sensors by using advanced fabrication techniques. At the same time, the development of nanoscale imaging techniques, such as fluorescence imaging, provides a great potential solution to resolve the nanoscale thermal probing problem.

Determining Serpin Conformational Distributions with Single Molecule Fluorescence

Mushero, Nicole; Gershenson, Anne
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em //2011 EN
Relevância na Pesquisa
291.00467%
Conformational plasticity is key to inhibitory serpin function, and this plasticity gives serpins relatively easy access to alternative, dysfunctional conformations. Thus, a given serpin population may contain both functional and dysfunctional proteins. Single molecule fluorescence (SMF), with its ability to interrogate one fluorescently labeled protein at a time, is a powerful method for elucidating conformational distributions and monitoring how these distributions change over time. SMF and related methods have been particularly valuable for characterizing serpin polymerization. Fluorescence correlation spectroscopy experiments have revealed a second lag phase during in vitro α1-antitrypsin polymerization associated with the formation of smaller oligomers that then condense to form longer polymers [Purkayastha, P., Klemke, J. W., Lavender, S., Oyola, R., Cooperman, B. S., and Gai, F. (2005). Alpha 1-antitrypsin polymerization: A fluorescence correlation spectroscopic study. Biochemistry 44, 2642–2649.]. SMF studies of in vitro neuroserpin polymerization have confirmed that a monomeric intermediate is required for polymer formation while providing a test of proposed polymerization mechanisms [Chiou, A., Hägglöf, P., Orte, A....

Probing the Binding Sites of Antibiotic Drugs Doxorubicin and N-(trifluoroacetyl) Doxorubicin with Human and Bovine Serum Albumins

Agudelo, Daniel; Bourassa, Philippe; Bruneau, Julie; Bérubé, Gervais; Asselin, Éric; Tajmir-Riahi, Heidar-Ali
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 24/08/2012 EN
Relevância na Pesquisa
367.35496%
We located the binding sites of doxorubicin (DOX) and N-(trifluoroacetyl) doxorubicin (FDOX) with bovine serum albumin (BSA) and human serum albumins (HSA) at physiological conditions, using constant protein concentration and various drug contents. FTIR, CD and fluorescence spectroscopic methods as well as molecular modeling were used to analyse drug binding sites, the binding constant and the effect of drug complexation on BSA and HSA stability and conformations. Structural analysis showed that doxorubicin and N-(trifluoroacetyl) doxorubicin bind strongly to BSA and HSA via hydrophilic and hydrophobic contacts with overall binding constants of KDOX-BSA = 7.8 (±0.7)×103 M−1, KFDOX-BSA = 4.8 (±0.5)×103 M−1 and KDOX-HSA = 1.1 (±0.3)×104 M−1, KFDOX-HSA = 8.3 (±0.6)×103 M−1. The number of bound drug molecules per protein is 1.5 (DOX-BSA), 1.3 (FDOX-BSA) 1.5 (DOX-HSA), 0.9 (FDOX-HSA) in these drug-protein complexes. Docking studies showed the participation of several amino acids in drug-protein complexation, which stabilized by H-bonding systems. The order of drug-protein binding is DOX-HSA > FDOX-HSA > DOX-BSA > FDOX>BSA. Drug complexation alters protein conformation by a major reduction of α-helix from 63% (free BSA) to 47–44% (drug-complex) and 57% (free HSA) to 51–40% (drug-complex) inducing a partial protein destabilization. Doxorubicin and its derivative can be transported by BSA and HSA in vitro.

Exploiting post-transcriptional regulation to probe RNA structures in vivo via fluorescence

Sowa, Steven W.; Vazquez-Anderson, Jorge; Clark, Chelsea A.; De La Peña, Ricardo; Dunn, Kaitlin; Fung, Emily K.; Khoury, Mark J.; Contreras, Lydia M.
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
509.01008%
While RNA structures have been extensively characterized in vitro, very few techniques exist to probe RNA structures inside cells. Here, we have exploited mechanisms of post-transcriptional regulation to synthesize fluorescence-based probes that assay RNA structures in vivo. Our probing system involves the co-expression of two constructs: (i) a target RNA and (ii) a reporter containing a probe complementary to a region in the target RNA attached to an RBS-sequestering hairpin and fused to a sequence encoding the green fluorescent protein (GFP). When a region of the target RNA is accessible, the area can interact with its complementary probe, resulting in fluorescence. By using this system, we observed varied patterns of structural accessibility along the length of the Tetrahymena group I intron. We performed in vivo DMS footprinting which, along with previous footprinting studies, helped to explain our probing results. Additionally, this novel approach represents a valuable tool to differentiate between RNA variants and to detect structural changes caused by subtle mutations. Our results capture some differences from traditional footprinting assays that could suggest that probing in vivo via oligonucleotide hybridization facilitates the detection of folding intermediates. Importantly...

Thioflavin T as a fluorescence probe for monitoring RNA metabolism at molecular and cellular levels

Sugimoto, Shinya; Arita-Morioka, Ken-ichi; Mizunoe, Yoshimitsu; Yamanaka, Kunitoshi; Ogura, Teru
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
372.70836%
The intrinsically stochastic dynamics of mRNA metabolism have important consequences on gene regulation and non-genetic cell-to-cell variability; however, no generally applicable methods exist for studying such stochastic processes quantitatively. Here, we describe the use of the amyloid-binding probe Thioflavin T (ThT) for monitoring RNA metabolism in vitro and in vivo. ThT fluoresced strongly in complex with bacterial total RNA than with genomic DNA. ThT bound purine oligoribonucleotides preferentially over pyrimidine oligoribonucleotides and oligodeoxyribonucleotides. This property enabled quantitative real-time monitoring of poly(A) synthesis and phosphorolysis by polyribonucleotide phosphorylase in vitro. Cellular analyses, in combination with genetic approaches and the transcription-inhibitor rifampicin treatment, demonstrated that ThT mainly stained mRNA in actively dividing Escherichia coli cells. ThT also facilitated mRNA metabolism profiling at the single-cell level in diverse bacteria. Furthermore, ThT can also be used to visualise transitions between non-persister and persister cell states, a phenomenon of isogenic subpopulations of antibiotic-sensitive bacteria that acquire tolerance to multiple antibiotics due to stochastically induced dormant states. Collectively...

Self-assembly of triblock copolymers in aqueous solution

Urbano, Bruno; Silva, Patricio; Olea, Andrés F.; Fuentes, Irma; Martínez, Francisco
Fonte: Sociedad Chilena de Química Publicador: Sociedad Chilena de Química
Tipo: Artículo de revista
EN
Relevância na Pesquisa
697.2552%
Indexación: Scielo; The aggregation of PE4VP-b-PS-b-PE4VP block copolymers was studied in aqueous solution. Triblock copolymers P4VP-b-PS-b-P4VP were synthesized by sequential anionic polymerization of poly(styrene) and poly(4-vinylpirydine) using sodium naphthalene as a bifunctional initiator. Subsequently, the 4-vinylpyridine units were quaternized with ethyl bromide to obtain cationic PE4VP-b-PS-b-PE4VP block copolymers. Both star and crew-cut micelles were formed. The concentrations at which micelles are formed cmc were determined, by steady-state and time-resolved fluorescence probing methods, as a function of quatemization degree. The results indicate that cmc of crew-cut micelles increases with increasing charge density of the PE4VP blocks. For star micelles there is not a clear dependency of cmc with the percentage of quatemization. The lifetime of pyrene fluorescence and the ratio I1/I3 were determined at concentrations of copolymers well above the cmc, and the results show that the location of pyrene into the micelle changes with the charge density of the micelle corona. The micropolarity sensed by pyrene decreases with increasing quatemization degree. The presence of aggregates was confirmed by transmission electronic microscopy.

Self-assembly of triblock copolymers in aqueous solution; Self-assembly of triblock copolymers in aqueous solution

Fonte: Sociedad Chilena de Química Publicador: Sociedad Chilena de Química
Tipo: Artículo de revista
Relevância na Pesquisa
697.2552%
Resumen: The aggregation of PE4VP-b-PS-b-PE4VP block copolymers was studied in aqueous solution. Triblock copolymers P4VP-b-PS-b-P4VP were synthesized by sequential anionic polymerization of poly(styrene) and poly(4-vinylpirydine) using sodium naphthalene as a bifunctional initiator. Subsequently, the 4-vinylpyridine units were quaternized with ethyl bromide to obtain cationic PE4VP-b-PS-b-PE4VP block copolymers. Both star and crew-cut micelles were formed. The concentrations at which micelles are formed cmc were determined, by steady-state and time-resolved fluorescence probing methods, as a function of quatemization degree. The results indicate that cmc of crew-cut micelles increases with increasing charge density of the PE4VP blocks. For star micelles there is not a clear dependency of cmc with the percentage of quatemization. The lifetime of pyrene fluorescence and the ratio I1/I3 were determined at concentrations of copolymers well above the cmc, and the results show that the location of pyrene into the micelle changes with the charge density of the micelle corona. The micropolarity sensed by pyrene decreases with increasing quatemization degree. The presence of aggregates was confirmed by transmission electronic microscopy.

Synthesis, thermal behavior, and aggregation in aqueous solution of poly(methyl methacrylate)-b-poly(2-hydroxyethyl methacrylate)

ACEVEDO, B.; MARTINEZ, F.; OLEA, A. F.
Fonte: 2014 Sociedad Chilena de Química Publicador: 2014 Sociedad Chilena de Química
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
705.8435%
Indexación: Scielo; ABSTRACT Amphiphilic block copolymers of poly(methyl methacrylate) PMMA and poly(2-hidroxyethyl methacrylate) PHEMA were synthesized by a two-step atom transfer radical polymerization (ATRP). Copolymers with various degrees of polymerization and different relative block sizes were obtained. The structure of the resulting polymers have been characterized and verified by FT-IR and 1H-NMR, molecular weight were determined by size exclusion chromatography analyses. The thermal properties of these polymers were investigated by differential scanning calorimetry DSC and thermogravimetric analysis TGA. The glass transition temperature of mono halogenated PMMA increases from 116 °C to 123 °C with increasing molecular weight, whereas the glass transition temperature of block copolymers depends slightly on polymer structure. The derivatives of TGA curves indicate that thermal degradation occurs in one stage. The self-assembly of PMMA-b-PHEMA in aqueous solution have been investigated by fluorescence probing methods. The critical micelle concentrations are in the range 10-6 - 10-7 M. The micropolarity sensed by pyrene is higher than in aggregates formed by block copolymers based on polystyrene. Keywords: Block copolymers...

Application of Time-Resolved Fluorescence for Direct and Continuous Probing of Release from Polymeric Delivery Vehicles

Viger, Mathieu L.; Sheng, Wangzhong; McFearin, Cathryn L.; Berezin, Mikhail Y.; Almutairi, Adah
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
300.11191%
Though accurately evaluating the kinetics of release is critical for validating newly designed therapeutic carriers for in vivo applications, few methods yet exist for release measurement in real time and without the need for any sample preparation. Many of the current approaches (e.g. chromatographic methods, absorption spectroscopy, or NMR spectroscopy) rely on isolation of the released material from the loaded vehicles, which require additional sample purification and can lead to loss of accuracy when probing fast kinetics of release. In this study we describe the use of time-resolved fluorescence for in situ monitoring of small molecule release kinetics from biodegradable polymeric drug delivery systems. This method relies on the observation that fluorescent reporters being released from polymeric drug delivery systems possess distinct excited-state lifetime components, reflecting their different environments in the particle suspensions, i.e., confined in the polymer matrices or free in the aqueous environment. These distinct lifetimes enable real-time quantitative mapping of the relative concentrations of dye in each population to obtain precise and accurate temporal information on the release profile of particular carrier/payload combinations. We found that fluorescence lifetime better distinguishes subtle differences in release profiles...

Probing of Microbial Biofilm Communities for Coadhesion Partners

Ruhl, Stefan; Eidt, Andreas; Melzl, Holger; Reischl, Udo; Cisar, John O.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /11/2014 EN
Relevância na Pesquisa
367.35496%
Investigations of interbacterial adhesion in dental plaque development are currently limited by the lack of a convenient assay to screen the multitude of species present in oral biofilms. To overcome this limitation, we developed a solid-phase fluorescence-based screening method to detect and identify coadhesive partner organisms in mixed-species biofilms. The applicability of this method was demonstrated using coaggregating strains of type 2 fimbrial adhesin-bearing actinomyces and receptor polysaccharide (RPS)-bearing streptococci. Specific adhesin/receptor-mediated coadhesion was detected by overlaying bacterial strains immobilized to a nitrocellulose membrane with a suspended, fluorescein-labeled bacterial partner strain. Coadhesion was comparable regardless of which cell type was labeled and which was immobilized. Formaldehyde treatment of bacteria, either in suspension or immobilized on nitrocellulose, abolished actinomyces type 2 fimbrial adhesin but not streptococcal RPS function, thereby providing a simple method for assigning complementary adhesins and glycan receptors to members of a coadhering pair. The method's broader applicability was shown by overlaying colony lifts of dental plaque biofilm cultures with fluorescein-labeled strains of type 2 fimbriated Actinomyces naeslundii or RPS-bearing Streptococcus oralis. Prominent coadhesion partners included not only streptococci and actinomyces...

SELF-ASSEMBLY OF TRIBLOCK COPOLYMERS IN AQUEOUS SOLUTION

Olea, Andrés F.; Martínez, Francisco; Fuentes, Irma; Silva, Patricio; Urbano, Bruno
Fonte: Sociedad Chilena de Química Publicador: Sociedad Chilena de Química
Tipo: Artículo de revista
EN
Relevância na Pesquisa
376.21723%
The aggregation of PE4VP-b-PS-b-PE4VP block copolymers was studied in aqueous solution. Triblock copolymers P4VP-b-PS-b-P4VP were synthesized by sequential anionic polymerization of poly(styrene) and poly(4-vinylpirydine) using sodium naphthalene as a bifunctional initiator. Subsequently, the 4- vinylpyridine units were quaternized with ethyl bromide to obtain cationic PE4VP-b-PS-b-PE4VP block copolymers. Both star and crew-cut micelles were formed. The concentrations at which micelles are formed cmc were determined, by steady-state and time-resolved fluorescence probing methods, as a function of quaternization degree. The results indicate that cmc of crew-cut micelles increases with increasing charge density of the PE4VP blocks. For star micelles there is not a clear dependency of cmc with the percentage of quaternization. The lifetime of pyrene fluorescence and the ratio I1/I3 were determined at concentrations of copolymers well above the cmc, and the results show that the location of pyrene into the micelle changes with the charge density of the micelle corona. The micropolarity sensed by pyrene decreases with increasing quaternization degree. The presence of aggregates was confirmed by transmission electronic microscopy.; This work was supported by FONDECYT 1070371...

Probing subtle fluorescence dynamics in cellular proteins by streak camera based Fluorescence Lifetime Imaging Microscopy

Krishnan, R. V.; Biener, Eva; Zhang, Jian-Hua; Heckel, Robert; Herman, Brian
Fonte: Universidade Cornell Publicador: Universidade Cornell
Tipo: Artigo de Revista Científica
Publicado em 26/09/2003
Relevância na Pesquisa
383.7161%
We report the cell biological applications of a recently developed multiphoton fluorescence lifetime imaging microscopy system using a streak camera (StreakFLIM). The system was calibrated with standard fluorophore specimens and was shown to have high accuracy and reproducibility. We demonstrate the applicability of this instrument in living cells for measuring the effects of protein targeting and point mutations in the protein sequence which are not obtainable in conventional intensity based fluorescence microscopy methods. We discuss the relevance of such time resolved information in quantitative energy transfer microscopy and in measurement of the parameters characterizing intracellular physiology.